ORIGINAL PAPER

Variation of arbuscular mycorrhizal fungal communitiesalong an altitudinal gradient in rupestrian grasslands in Brazil

Etiene Silva Coutinho & G. Wilson Fernandes &Ricardo Luís Louro Berbara & Henrique Maia Valério &

Bruno Tomio Goto

Received: 22 October 2014 /Accepted: 3 March 2015# Springer-Verlag Berlin Heidelberg 2015

Abstract Variation in arbuscular mycorrhizal fungi (AMF)communities is described for the first time in rupestrian grass-lands in Brazil along an altitudinal gradient of 700 m (800 to1400 m a.s.l.). Hypotheses tested were that soil propertiesinfluence the variation in AMF communities and that the fre-quency of the most common species of AMF is inverselyinfluenced by the richness of other AMF. Field and laboratorydata were collected on AMF community composition, rich-ness, density, and frequency in the altitudinal gradient, and therelationships with several physical–chemical soil propertiesand altitude were evaluated. Fifty-one species of AMF wererecorded, with 14 species being reported as possibly new to

science and nine species representing new records forBrazil. This single elevation gradient alone contains22 % of the known world diversity of AMF. Soil proper-ties and AMF community density and richness varied sig-nificantly along the elevation (p<0.05). AMF density andrichness were higher at the intermediate altitude, whileAMF species composition differed statistically amongthe altitudes.

Keywords Espinhaçomountains . Fungal diversity .

Mountain ecosystems . Serra do Cipó . Soil characteristics

Introduction

Due to clear variations found in a relatively small area, moun-tains represent natural laboratories for evolutionary and eco-logical studies, occurring in all climatic zones and coveringapproximately 25 % of the planet’s surface. Climatic parame-ters and soil properties change along the altitudinal gradientsfound in mountain regions, especially temperature, precipita-tion, humidity, soil, wind speed, UV-B radiation and shading,and these variations strongly influence the geography andecology of species (e.g., Fernandes & Price 1988; Ramsayand Oxley 1997; Lomolino 2001; Smith et al. 2002; Diazet al. 2003; Malhi et al. 2010). Mountains have also beenregarded as important places where the impacts of climatechange on earth’s biota shall be the strongest (e.g., Kohleret al. 2010; Fischer et al. 2011; Rangwala and Miller 2012).

The Espinhaço mountain range (the Bback bonemountains^), which spans 1200 km (ca. 10° latitude) and is50–200 km wide, is a natural and large watershed divider ofmajor ecological importance in eastern Brazil (Fernandes et al.2014). The most diverse savanna in the world (the Cerrado)

Electronic supplementary material The online version of this article(doi:10.1007/s00572-015-0636-5) contains supplementary material,which is available to authorized users.

E. S. Coutinho :G. W. FernandesICB/Departamento de Biologia Geral, Laboratório de EcologiaEvolutiva & Biodiversidade, Universidade Federal de Minas Gerais,C. Postal 486, 30161-970 Belo Horizonte, MG, Brazil

G. W. Fernandes (*)Department of Biology, Stanford University, Stanford, CA 94305,USAe-mail: gw.fernandes@gmail.com

R. L. L. BerbaraIA/Departamento de Solos, Laboratório de Biologia de Solos,Universidade Federal Rural do Rio de Janeiro,23890-000 Seropédica, RJ, Brazil

H. M. ValérioDepartamento de Biologia Geral, Laboratório de MicrobiologiaAmbiental, Universidade Estadual de Montes Claros, C. Postal 126,39401-089 Montes Claros, MG, Brazil

B. T. GotoCB/Departamento de Botânica e Zoologia, Laboratório de Biologiade Micorrizas, Universidade Federal do Rio Grande do Norte,59072-970 Natal, RN, Brazil

MycorrhizaDOI 10.1007/s00572-015-0636-5

thrives at the occidental side, at the oriental side the vegetationis dominated by the speciose Atlantic rain forest, while thevegetation in the northern portion is the seasonally dry forestcalled Caatinga (Harley 1995). The physiognomy of theEspinhaço Range varies greatly along an altitudinal gradientof 700–2000 m a.s.l. On the other hand, the vegetation isprimarily dominated by rupestrian grasslands. Although therupestrian grasslands are composed primarily of soils thatare quite poor (Benites et al. 2003; Negreiros et al. 2009), theydisplay a high degree of endemism and biodiversity (Giuliettiand Pirani 1988; Lara and Fernandes 1996), possibly withmore than 6000 species of plants. This old growth grasslandformation (see Veldman et al. 2015) is composed of manyeasily distinguishable habitats. These include sandy marshesthat experience periodic flooding during the rainy season andsupport persistent herbaceous vegetation, peat swamps thatremain constantly flooded during the rainy season and aredominated by a continuous herbaceous stratum, rocky out-crops with high proportions of exposed rock andherbaceous/shrub vegetation, the rupestrian grassland itselfwith a soil surface covered by small fragments of quartziterocks, and the cerrado (sensu stricto) in which the dominanceof arboreal/shrub species declines with increasing altitude,transitioning either gradually or swiftly to the rupestrian grass-land (Giulietti et al. 1987; Carvalho et al. 2012; Fernandeset al. 2014).

The high degree of endemism and high biodiversity ofplant species in the Espinhaço mountain ridge have oftenbeen associated to the mosaic vegetation formations foundin the slopes, the rugged landscape, microclimatic variations,and properties of several soil types. In a recent study, Carvalhoet al. (2012) reported 49 species of arbuscular mycorrhizalfungi (AMF) in a single elevation dominated by rupestriangrassland habitats, indicating the importance of diversity inthese mountains. Five new species belonging to the generaAcaulospora, Scutellospora, and Glomus were found, andtwo others were reported for the first time in Brazil,Acaulospora colossica and Pacispora dominikii. In this con-text, the main objective of the present study was to determinefor the first time the composition, richness, density, and rela-tive frequency of species of AMF communities along thistropical altitudinal gradient in South America. Hypothesestested were that soil properties influence the variation inAMF communities, and that the frequency of the most com-mon species of AMF is inversely influenced by the richness ofother AMF. It is argued that the most abundant species occupymost of the resources outcompeting the weaker species, withthe frequency of most common species increasing where thenutritional conditions of the soil become unfavorable (Connell1978; Caproni et al. 2003). Although a negative correlation ofspecies richness and altitude has been shown to represent astrong pattern in nature (see Fernandes and Price 1988, 1991;Whitmore 1990; Kumar et al. 2009), mycorrhizal diversity

and activity are strongly influenced by soil properties so thatmycorrhizal richness may not show a negative relationshipwith altitude (Bryant et al. 2008; Fierer et al. 2011).

Materials and methods

The study was conducted in the Serra do Cipó mountain rangein the state of Minas Gerais (SE Brazil; Online Resource 1),located in the southern part of the Espinhaço Range, betweenlatitudes 19°10′ and 19°22′S and near longitude 43°40′ W.The climate of the region is Cwb (mesothermal climate withmild summers and a rainy season in the summer) according tothe Köppen classification, with average temperatures between17.4 and 19.8 °C. The annual precipitation of the region isapproximately 1500 mm, with a dry winter lasting 3 to4 months and a wet period lasting 7 to 8 months (Madeiraand Fernandes 1990). The Serra do Cipó has soils formedpredominantly by quartz and sandstone, which are acidic,sandy, and shallow, with little water retention capacity, nutri-ent-poor, and with high concentrations of aluminum(Negreiros et al. 2009, 2011).

To evaluate the composition of AMF in the soil, one tran-sect was established at each altitude between 800 and1400 m a.s.l. (Online Resource 1), with an interval of100 m. Transects were at least 3 km apart and a global posi-tioning system (GPS) was used. In this complex environment,the rupestrian habitats are found at higher elevations while thecerrado sensu stricto (savanna) is at the lower end of it, andtransition areas of cerrado sensu stricto/rupestrian grasslandoccur at intermediate altitudes (Table 1). Thirteen plots of100 m2 (10 m×10 m) were defined along each transect for atotal of 91 plots (0.91 ha). In February andMarch of 2011, fivesoil samples were collected (a sample of each angle and asample in the central portion) from the surface layer (0–0.2 m in depth) in each plot at each altitude for soil analysis.

Table 1 Geographic positions and habitats of the seven altitudesanalyzed along the altitude gradient in the Serra do Cipó, Brazil

Altitude (m) Coordinates Habitats

S W

800 19° 21′ 36.2′′ 43° 36′ 25.2′′ Cerrado sensu stricto

900 19° 16′ 17.8′′ 43° 36′ 18.1′′ Rocky outcrop and cerradosensu stricto

1000 19° 15′ 50.6′′ 43° 35′ 10.3′′ Rocky outcrop and cerradosensu stricto

1100 19° 13′ 56.5′′ 43° 34′ 34.8′′ Rocky outcrop and cerradosensu stricto

1200 19° 17′ 43.0′′ 43° 33′ 17.4′′ Rocky outcrop

1300 19° 17′ 49.6′′ 43° 35′ 28.2′′ Rupestrian grassland

1400 19° 16′ 59.3′′ 43° 32′ 08.9′′ Rupestrian grassland

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These samples were combined and homogenized tomake up amixed sample for each plot (total n=91).

The soil samples, stored in plastic bags, were divided intotwo parts: one was used to analyze the physical–chemicalproperties of the soil, while the other was used to evaluatethe richness and density of AMF spores. The soil sampleswere air-dried and sieved at 2.0 mm for texture and chemicalanalysis (pH—hydrogen potential, P—phosphorus, K—po-tassium, Ca—calcium, Mg—magnesium, Al—aluminum,H+Al—potential acidez, SB—sum of bases, T—cation ex-change capacity, t—effective cation exchange capacity, V—base saturation, OM—organic matter, Prem—remainingphosphorous, m—aluminum saturation), according to the pro-cedures described by Embrapa (1997).

The soil texture was characterized by separating the particlesinto classes based on size using the pipette method (Embrapa -Empresa Brasileira de Pesquisa Agropecuária 1997). Thismethod is based on the dispersion of the sample with a solutionof 1 mol L−1 NaOH; the sand fraction was separated by wetsieving, and the clay fraction was separated using the sedimen-tation rate. The silt was measured as the difference between thesand and clay masses and the total mass (dag/kg).

The AMF spores were extracted from the soil using the wetsieving technique with 50 g of soil (Gerdemann and Nicolson1963) and 425 and 53 μm sieves, followed by centrifugationin a sucrose gradient (50 %) (Jenkins 1964). Spores werecounted using a stereomicroscope at ×40 magnification todetermine the total density of AMF (density of spores per50 g of soil collected at al l s tudied elevat ions).Subsequently, the spores were separated into morphospeciesaccording to their morphological similarities (color and size)in Petri dishes, transferred to slides, and crushed under a coverslip in a drop of either polyvinyl alcohol lacto-glycerol(PVLG) or 01:01 PVLG+Melzer’s reagent (vol/vol)(Morton et al. 1993) to characterize the spore walls.

Identification of the AMF species was performed at thespecies level based on the morphological characteristics ofspores under a light microscope (×100 to ×400) using litera-ture as described by Schenck and Pérez (1990), data availableat the International Culture Collection of (Vesicular)Arbuscular Mycorrhizal Fungi (INVAM: http://invam.caf.wvi.edu/), species deposited in the Department of PlantPathology, University of Agriculture in Szczecin, Poland(http://www.agro.ar.szczecin.pl/~jblaszkowski), andinformation by Schüβler and Walker available at the sourcehttp://www.amf-phylogeny.com/. Following identification,AMF species richness was evaluated based on the numberof species present in the 50 g of soil and the total number ofAMF species. The frequency of each species (Fi) of the mostcommon between altitudes was calculated for each areaaccording to the equation Fi=Ji/K, where Fi is the frequencyof species I, Ji is the number of samples in which species ioccurred, and K is the total number of soil samples (Brower

et al. 1990). To evaluate the efficiency of the sampling, aspecies accumulation curve was constructed for each elevation.These curves were obtained after 1000 randomizations of thesampling order, and the Jackknife non-parametric estimator ofrichness was computed by the program EstimateS 8.20(Cowell 2006).

Given that the data did not display a normal distribution,data were normalized by log (x+1); a general linearized model(GLM), using polynomial regression, was developed to verifywhether there were correlations between altitude and the den-sity of spores and the number of AMF species, assuming aPoisson distribution of errors (Crawley 2002) and using altitudeas the explanatory variable. The chemical and physical proper-ties of the soil along the altitude gradient were characterizedusing a linear regression analysis with altitude as the indepen-dent variable. The analysis was processed in STATISTICAsoftware version 7.0. A significance level of 0.05 was usedfor all tests. The effect of soil properties on variation in speciescomposition of AMF was evaluated by means of a canonicalcorrespondence analysis (CCA). The significancewas accessedbyMonte Carlo permutation test using the PC-Ord program forWindows, version 5.0 (McCune and Mefford 1997). The re-sponse matrix was the composition of AMF species, and theexplanatory matrix was formed by soil properties. All of thevariables that displayed a low correlation with the axes of or-dination (<0.05) were eliminated from this set of variables.

To test whether the composition of AMF species variedwith altitude, a multivariate analysis was used. A two-dimensional ordination map was constructed using non-metric multidimensional scaling (NMDS). Then, to check forpossible similarities in the composition of AMF species amongthe different altitudes, an analysis of similarity (ANOSIM) testwas used. The ordination was performed using presence–ab-sence data, employing the Raup-Crick index of similarity. Thevalues obtainedwere placed in a similarity matrix, and a clusteranalysis was subsequently performed with pair clusteringusing the Sörensen index. The analysis was performed withthe Past software (Hammer et al. 2001).

Results

Soil properties along the altitude

All of the properties related to the chemistry and texture of thesoils varied significantly along the altitudinal gradient understudy (p<0.05, Table 2). The sampled soils were acidic (4.7 to5.2 pH/H2O) and poor in all of the analyzed macronutrients.The average amount of organic matter in the soils was low(3.51 to 6.90 dag/kg). The mean magnesium content was0.11 cmol/dm3. Calcium level was highest at the highest alti-tude, 1400 m (0.90 cmolc/dm3), and lower at 900, 1000, and1300 m (0.20 cmolc/dm3).

Mycorrhiza

At the lowest altitude (800 m), soil values were higher forphosphorus (2.92 mg/kg), potassium (49.45 mg/kg), sum ofbases (0.53 cmol/dm3), and base saturation (22 %), with a lowpotential acidity (1.95 %). The lowest amount of base saturationwas recorded at the intermediate altitude of 1100 m, which wassimilar to the value at the altitude 1200 m (7 %). However, at1200 m, there were lower values for the remaining phosphorus(24.86 mg/L) and higher values for aluminum saturation (84 %)and cation exchange capacity (6.29 cmol/dm3). At 1300m, therewas a higher value for the remaining phosphorus (39.04 mg/L)and lower values for potassium (15.26 mg/kg), aluminum(1.29 cmol/dm3), sum of bases (0.34 cmol/dm3), effective cationexchange capacity (1.63 cmol/dm3), and cation exchange capac-ity (2.38 cmol/dm3). At the highest altitude (1400m), there werehigher values for aluminum (2.66 cmol/dm3), potential acidity(5.90 cmol/dm3), and effective cation exchange capacity(3.18 cmol/dm3).

With regard to texture, soils were predominantly sandyalong the gradient. The highest amount of fine sand was re-corded at the intermediate altitude of 1100 m (81.12 dag/kg),while the lowest amount was observed at the lowest altitude(800m; 62.77 dag/kg) (Table 2). The coarse sandy texture wasmore prominent at 1300 m (18.14 dag/kg) and less prominentat 900 m (4.18 dag/kg), whereas both silt and clay were foundat higher amounts at 900 m (12.46 and 10.77 dag/kg,

respectively) and in lower amounts at 1300 m (5.00 and3.17 dag/kg, respectively).

The values of hydrogen potential (pH), potassium (K), alu-minum (Al), potential acidity (H+Al), effective cation ex-change capacity (t), aluminum saturation (m), cation exchangecapacity (T), base saturation (V), organic matter (OM), finesand (FS), silt, and clay (CL) were positively correlated withincreasing altitude along the gradient (Table 2).

AMF along the altitude gradient

The total number of spores in soil samples varied from 5095,found at 800 m, to 13,510, found at 1100 m in relation toaltitude, with a total of 58,621 spores. The highest averageof spore density was 1039 spores/50 g at the altitude of1100 m (20.8 spores g−1), while the lowest value at391 spores/50 g (7.7 spores g−1) was recorded at the lowestaltitude (800 m). The correlation of altitude and density ofAMF spores was low (r2=0.17, p<0.05, y=−2247.3626+5.0199x−0.0021x2; Fig. 1a).

A total of 51 species of AMF were identified along thealtitudinal gradient (Table 3). These species belong to 15 gen-era and 11 families (Acaulosporaceae, Ambisporaceae,Dentiscutataceae, Diversisporaceae, Entrophosporaceae,G i g a s po r a c e a e , G l ome r a c e a e , P a c i s p o r a c e a e ,

Table 2 Comparison of the soil properties along the altitudinal gradient in the rupestrian grassland complex in Serra do Cipó, Brazil

Edaphic variables Altitude (m) Regression

800 900 1000 1100 1200 1300 1400 R2 p

pH (H2O) 5.1 5.0 5.1 4.9 4.7 4.7 4.7 0.207 <0.05

P Me (mg/Kg) 2.92 1.16 1.16 1.15 2.71 1.08 2.38 0.000 >0.05

P re (mg/L) 32.6 30.3 32.81 29.14 24.86 39.04 33.9 0.006 >0.05

K (mg/Kg) 49.45 31.66 30.08 33.06 41.15 15.26 25.06 0.142 <0.05

Ca (cmol/dm3) 0.25 0.2 0.2 0.21 0.21 0.2 0.28 0.004 >0.05

Mg (cmol/dm3) 0.15 0.1 0.1 0.11 0.1 0.1 0.17 0.004 >0.05

Al (cmol/dm3) 1.43 1.69 1.62 1.88 2.48 1.29 2.66 0.13 <0.05

H+Al (cmol/dm3) 1.95 5.09 2.47 5.87 5.87 2.04 5.9 0.107 <0.05

SB (cmol/dm3) 0.53 0.38 0.38 0.4 0.42 0.34 0.52 0.003 >0.05

t (cmol/dm3) 1.97 2.07 2.00 2.27 2.9 1.63 3.18 0.112 <0.05

m (%) 73 78 79 82 84 78 83 0.156 <0.05

T (cmol/dm3) 2.49 5.47 2.85 6.27 6.29 2.38 6.41 0.103 <0.05

V (%) 22 9 15 7 7 15 9 0.377 <0.05

OM (dag/Kg) 3.51 5.04 3.57 5.59 6.9 3.56 6.9 0.163 <0.05

CS (dag/Kg) 17.84 4.18 17.65 4.88 12.8 18.14 9.35 0.00003 >0.05

FS (dag/Kg) 62.77 72.59 67.12 81.12 75.66 73.69 78.49 0.357 <0.05

SILT (dag/Kg) 11.54 12.46 8.15 5.54 6.00 5.00 7.85 0.264 <0.05

CL (dag/Kg) 7.85 10.77 7.08 8.46 5.54 3.17 4.31 0.368 <0.05

pH pH in water, K potassium, P-Mehlich phosphorous, P-rem remaining phosphorous, Ca calcium, Mg magnesium, Al aluminum, H+Al hydrogen+aluminum, SB the sum of bases,V base saturation, t effective cation exchange capacity,m aluminum saturation, Tcation exchange capacity at a pH of 7.0,OM organic matter, CS the proportions of coarse sand (2 to 0.2 mm), FS fine sand (0.2 to 0.05 mm), silt (0.05 to 0.02 mm), CL clay (<0.02 mm), Rregression analysis

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Paraglomeraceae, Racocetraceae, and Scutellosporaceae). Ofthe 51 described species, 32 occurred at the intermediate alti-tude of 1200m (8.23 species/50 g of soil), 32 species occurredat the altitude of 1100 m (9.73 species/50 g of soil), 31 at thealtitude of 1300 m (12.92 species/50 g of soil), 30 at thealtitude of 1000 m (11.15 species/50 g of soil), 27 at 1400 m(7.23 species/50 g of soil), 24 at 900 m (8.61 species/50 g ofsoil), and 23 at 800 m (5.15 species/50 g of soil). Again, thecorrelation of AMF species richness and altitude was relative-ly low (r2=0.29, p<0.05, y=44.8516+0.0967x−4.2033E−5x2; Fig. 1b); however, the greatest number of species wasobserved at the intermediate altitudes, 1100 and 1200 m.

Interaction of AMF species and soils

Among the AMF species found, the most common (11)were present at all altitudes. These were, in order of frequency,the following species: Glomus invermaium (84 %),

Fig. 1 Relationship between the total density of AMF spores (a) and thetotal number of AMF species (b) at seven different altitudes (800–1400 m) in the rupestrian grassland complex, Serra do Cipó, Brazil.The continuous line represents the polymonial regression betweendensity/AMF richness in relation to altitude, while the dotted linerepresents the variation of the polymonial regression

Table 3 Species of AMF along the altitudinal gradient in the rupestriangrassland complex in Serra do Cipó, Brazil

Family/species Altitude (m)

800 900 1000 1100 1200 1300 1400

Acaulosporaceae

Acaulospora bireticulata x x x x x x x

A. colossica x x x

A. delicata x x x

A. denticulata x x x x x x x

A. koskei x x x x x x

A. mellea x x x x

A. morrowiae x x x x x x x

A. rugosa x

A. scrobiculata x x x x

A. spinosa x x x x x

Acaulospora sp. 1 x x x x x x

Acaulospora sp. 2 x x x

Acaulospora sp. 3 x

Acaulospora sp. 4 x x

Ambisporaceae

Ambispora appendicula x x x x x x x

A. brasiliensis x x x x x x x

A. callosa x x x x x x

Dentiscutataceae

Dentiscutata biornata x x x x x x

Fuscutata heterogama x

F. rubra x x x x

Diversisporaceae

Diversispora sp. x x x x x x

Entrophosporaceae

Claroideoglomusetunicatum

x x x x x x x

C. lamellosum x x x

Gigasporaceae

Gigaspora decipiens x x x x x x

G. gigantea x

G. margarita x x x x

Glomeraceae

Funneliformisgeosporum

x x x x x x x

F. mosseae x x x x x x x

Funneliformis sp. x x

Glomus clarus x x x x x x

G. diaphanus x x x x x

G. fasciculatus x x x

G. glomerulatum x x x x x x x

G. invermaium x x x x x x x

G. macrocarpum x x x x x x x

G. microaggregatum x x x

G. microcarpum x x x x x x

Glomus sp. 1 x

Glomus sp. 2 x x

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Claroideoglomus etunicatum (68 %), Glomus glomerulatum(66 %), Funneliformis mosseae (57 %), Glomus macrocarpum(55 %), Funneliformis geosporus (42 %), Ambisporabrasiliensis (32 %), Acaulospora morrowiae (28 %),Acaulospora denticulata (24 %), Acaulospora bireticulata(14 %), and Acaulospora appendicula (13 %). Seven specieswere found to be exclusive to one altitude: one species at 800 m(Fuscutata heterogama), two species at 1000 m (Acaulosporarugosa and Pacispora robigina), one species at 1200 m(Glomus sp.3), and three species at the highest altitude,1400 m (Acaulospora sp.3, Gigaspora gigantea, and Glomussp.). The greatest number of identified species over all of thesampled altitudes belonged to the genus Acaulospora (14 spe-cies), followed by the genus Glomus (10 species). The generaAcaulospora and Glomus were found at all altitudes.

Among themost commonAMF species across all altitudes,G. invermaium was the species with the highest frequency atthe altitudes of 800, 900, 1200, 1300, and 1400 m. The fre-quency of G. invermaium, along with F. mosseae, decreasedwith increasing AMF species richness. C. etunicatum wasfound at higher frequency at the altitude of 1100 m, andG. macrocarpum occurred at greater frequency at 1000 m;however, the increase in AMF species richness did not affectthe f requencies of these two species or that ofG. glomerulatum (Fig. 2).

The different altitudes were clearly segregated in the CCAanalysis (Online Resource 2). The eigenvalues of the first twoaxes of the CCA diagram were 0.167 (axis 1) and 0.065 (axis2), explaining 35.6 and 14 %, respectively, of the total

variance of the effect of soil properties on variation in speciescomposition of AMF. The eigenvalues for the two axes werelow, meaning that the gradients are short, with low speciesturnover along the altitudinal gradient. In contrast, the spe-cies–environment correlations were high for both axes:0.998 (axis 1) and 0.863 (axis 2). In addition, the MonteCarlo test indicated that the richness of AMF species wassignificantly correlated with soil attributes (p<0.05). The cor-relations between the soil variables and the first two axes ofordination showed that the variable base saturation were pos-itively correlated with axis 1, whereas negative correlationswere observed with the cation exchange capacity and organicmatter content. Meanwhile, there was a positive correlationwith fine sand along axis 2 (Table 4). Therefore, these vari-ables had the strongest influence on the differentiation amongthe altitudes assigned to the correlation between soil propertiesand AMF species.

In addition to the spatial segregation of the studied alti-tudes, there were differences in the distribution of the analyzedsoil attributes (p<0.05) along the two axes in the CCA anal-ysis (Online Resource 2). It should be noted that axis 1 includ-ed the altitudes (1100, 1200, and 1400 m) that displayed highamounts of fine sand and lower values for phosphorus.Therefore, the combination of these altitudes and the separa-tion with other altitudes can be a function of these variables.At the same time, some plots at these altitudes were clusteredbecause they had a high amount of organic matter in relationto the other plots and to the other altitudes. Axis 2 separatedthe altitude with the highest amount of base saturation (800m)from the other altitudes. The cation exchange capacity vari-able grouped the altitudes of 1100, 1200, and 1400m together,separating them from the other altitudes. In the ordinationdiagram of the plots and environmental variables, the majorityof the plots within the same altitude are found in close phys-ical proximity, exhibiting relatively similar characteristicsamong them. However, some plots are separated from theothers, as they were influenced by the following soil variables:base saturation (V), cation exchange capacity (T), organicmatter (OM), and fine sand (FS) (Online Resource 2).

On the first axis of ordination in the CCA, most of thespecies of the Glomeraceae stood out because they display ahigh correlation with the increase in the amounts of fine sand,such as species of the genus Septoglomus, whereas fine sandwas negatively correlated with species of the generaDiversispora and Fuscutata. The organic matter (OM) andcation exchange capacity (T) displayed high correlations withspecies of the genera Acaulospora , Ambispora ,Claroideoglomus, Dentiscutata, Funneliformis, Gigaspora,andGlomus and negative correlations with species of the gen-era Pacispora, Paraglomus, and Scutellospora. On the secondaxis, base saturation (V) was positively correlated with thespecies of the genera Paraglomus and Scutellospora and neg-atively correlated with the species of the genera Acaulospora,

Table 3 (continued)

Family/species Altitude (m)

800 900 1000 1100 1200 1300 1400

Glomus sp. 3 x

Glomus sp. 4 x x x x x

Glomus sp. 5 x x

Glomus sp. 6 x x x

Septoglomusconstrictum

x x x x

Pacisporaceae

Pacispora sp. x

Paraglomeraceae

Paraglomus occultum x x x x x x

Racocetraceae

Racocetra fulgida x x x x x

Scutellosporaceae

Orbisporapernambucana

x x

Scutellospora calospora x x x x x

S. dispurpurescens x x

Scutellospora sp. x x

Mycorrhiza

Ambispora, Claroideoglomus, Dentiscutata, Funneliformis,Gigaspora, and Glomus.

There was a difference in the composition of speciesamong the altitudes (p<0.05; Table 5) although the NMDS,based on the frequency of species in the samples (generalpresence-absence matrix), showed a low similarity of FMAspecies among the altitudes with respect to species composi-tions (Online Resource 3). The similarity in compositions wasgreatest in the contiguous altitudes, with no significant differ-ence between them (p>0.05; Table 5), where habitat types aresimilar. The altitude of 800 m had a similar AMF speciescomposition to the one found at 900 m, a cerrado sensu stricto(savanna), in which the dominance of arboreal/shrub speciesdeclines with increasing altitude. The altitude of 1200 mdisplayed the greatest similarity in its composition with thealtitudes of 1300 and 1400 m. However, the altitudes of1000 and 1100 m displayed significantly different AMF com-munities composed by cerrado sensu stricto in transition torocky outcrop ecosystems.

With regard to similarity, the formation of two clusterlevels can be observed for the altitudes (Fig. 3). The first level

includes the altitudes of 800, 900, 1000, 1100, 1200, and1300 m, with a similarity of 68 %. The second level includesonly the altitude of 1400 m, which had a similarity of 58 %,with the altitudes of 800, 900, 1100, 1200, and 1300 m. Ingeneral, all altitudes displayed overlaps of less than 58 %,indicating that there is at least a 42 % difference among themin terms of AMF species composition. The analysis indicatedthat the altitudes of 1100 and 1300 m were the most similar,with a similarity level of 81 %, whereas the altitude of 1400 mwas the most dissimilar.

The AMF species accumulation for each altitude (Fig. 4)shows an increase in the number of species as new samplingunits were added; stabilization in the number of species wasdetected only at 1300 m. Even though a tendency towardsstabilization after the tenth sample could be observed at1100 and 900 m, additional sampling would still be necessaryto reach the curve’s asymptote. The AMF richness valuesestimated by the Jackknife method show that the individualaltitudes do not represent the entire richness of AMF, except at1300 m with variations from 83.9 to 100 % of the total esti-mated richness (Table 6).

Discussion

The different altitudes in the study site displayed soil charac-teristics that were similar to those of other rupestrian grasslandareas. Negreiros et al. (2008) reported high soil organic mattercontent (from 2.8 to 9.4 dag/kg) and high saturation by Al3+

and high acidity in Serra do Cipó. The soils from theEspinhaço mountain range exhibit similar properties, withacidic soils, low levels of macronutrients, and high aluminumsaturation due to the geological and geomorphological condi-tions (Dossin et al. 1990). The low availability of nutrients andwater stress caused by climatic conditions have been frequent-ly reported in the quartzite regions of the Serra do Cipó

Fig. 2 Relative frequency of themost common AMF species inrelation to the number of AMFspecies sampled along the altitudegradient in the rupestriangrassland complex, Serra doCipó, Brazil. Species richnessrepresents the number of AMFspecies at each sampled altitude

Table 4 Canonical correspondence analysis through the Monte Carlopermutation test for the rupestrian soil complex in Serra do Cipó, Brazil.The values represent correlations (intraset) between the first twoordination axes and the weighted correlation matrix of theenvironmental variables: base saturation (V), cation exchange capacity(T), organic matter (OM), and fine sand (FS)

Internal correlations Edaphic variables

Edaphic variables Axis 1 Axis 2 V T OM FS

V 0.584 −0.042T −0.475 −0.276 −0.827OM −0.516 −0.424 −0.71 0.85

FS −0.637 0.116 −0.615 0.493 0.411

Mycorrhiza

mountains (Fernandes et al. 2011). The distributions of soilhydrogen potential (pH), potassium (K), aluminum (Al),potential acidity (H+Al), effective cation exchange capac-ity (t), aluminum saturation (m), cation exchange capacity(T), base saturation (V), organic matter (OM), fine sand(FS), silt, and clay (CL) increased with increasing altitudein the present study. However, at higher altitudes, moreacidic soils were found most likely due to the weatheringcaused by a high degree of leaching. Sandy soils have a loworganic matter content, low cation exchange capacity, andtherefore a high base leaching (Ca, Mg, and K). Overall,higher levels of H+Al in soils are reported in soils richer inorganic matter, especially if they display a very low pH(Tomé Junior 1997), as occurred at the higher altitudes inthis study. Thus, the present study confirms that highly

adverse conditions prevail in this mountainous environ-ment and, hence, a strong environmental filter for the flora.

The NMDS showed a low similarity of AMF speciesamong the studied altitudes. The differences in the type of soilat the different altitudes may have influenced the establish-ment of the AMF and species composition of their community(Trufem et al. 1989). However, some altitudes displayed sim-ilar soil conditions, which led to an overlap of their composi-tions. The degree of similarity of the AMF species seems to bestructured according to the altitudinal gradient, which wassupported by the formation of AMF groups, since speciescomposition of AMFwas more similar at intermediate altitudeand dissimilar at extreme altitudes. The grouping pattern in thecluster analysis strongly suggests that the distribution of spe-cies between altitudes may be associated with soil variation.

The soil variables of base saturation, cation exchange ca-pacity, organic matter, and fine sand partially explained thegrouping of the plots and the altitudes, and these variablesinfluenced the distribution of AMF. Altitudes were promptlyseparated based on the amount of sandy soil, base saturation,and cation exchange capacity. However, the remaining vari-ance may be associated with other environmental variablessuch as temperature and humidity. Moreover, the presence orabsence of host plants is important in the occurrence of colo-nization and sporulation of AMF (Cavalcante et al. 2009). Theordination of the species according to the environmental var-iables indicates that the survival of AMF species in habitatsmay depend on the acidity and fertility of the soil. The mosaicof soils is perhaps a very important factor that influences thevariety of habitats found in the rupestrian grasslands and prob-ably the most relevant factor in the spatial distribution of AMFand plant species in this complex tropical mountain ecosys-tem. In spite of being apparently short, the altitudinal gradientin the Espinhaço mountains is known to have strong influenceon plant species composition (Giulietti et al. 1987), as well ason its fauna (e.g., Fernandes & Price 1988). Similar trendshave been reported elsewhere in these mountains (e.g.,Conceição and Giulietti 2002, Conceição and Pirani 2007,Nunes et al. 2008.

Table 5 Results for significant differences in the composition of AMF species along the altitude gradient in the rocky soil complex of the Serra doCipó, Brazil, based on non-metric multidimensional scaling (NMDS)

Altitude (m) 800 900 1000 1100 1200 1300 1400

800 0.118 ns 0.018 0.00001 0.001 0.00001 0.004

900 0.118 ns 0.002 0.012 0.001 0.017 0.014

1000 0.018 0.002 0.00001 0.00001 0.00001 0.00001

1100 0.00001 0.012 0.00001 0.042 0.005 0.031

1200 0.001 0.001 0.00001 0.042 0.141 ns 0.358 ns

1300 0.00001 0.017 0.00001 0.005 0.141 ns 0.159 ns

1400 0.004 0.014 0.00001 0.031 0.358 ns 0.159 ns

ns non-significant differences between the altitudes studied

Fig. 3 Dendrogram of cluster analysis using Sorensen similarity indexbased in the composition of the AMF communities along the altitudinalgradient (m) in Serra do Cipó, Brazil

Mycorrhiza

The occurrence of a higher density of spores at the inter-mediate altitude, 1100 m, may be related to the higheramount of fine sand, high aluminum saturation, and loweramount of phosphorus, leading the AMF to produce a largenumber of propagules at this altitude in an environmentwith low nutrient levels. Smith and Read (1997) reporteda positive response of mycorrhizal fungi spores understress. The highest density of AMF at the altitudes (1100and 1200 m) could have been influenced by the presence ofcommon species of AMF due to it being a soil and vegeta-tion transition area, the cerrado sensu stricto, and rupestriangrassland ecosystems. The lower density of spores at thelowest altitude can be a consequence of the greater stabilitydue to lower species competition in the cerrado sensustricto (Caproni et al. 2003). Furthermore, at this low alti-tude, the highest amount of the nutrients K and P and anincrease in pH were recorded. Generally, the availability ofthese nutrients reflects a lesser colonization of AMF inplant species and lower spore density (Abbott and Robson1991), corroborating the hypothesis that plant species ben-efit from the symbiosis with AMF in low fertility

environments (Berbara et al. 2006; Miranda 2008;Moreira et al. 2010; Lisboa et al. 2014).

The spore density observed in this study (7.7–20.8 spores/g) was greater when compared to the average found in othertropical environments in Brazil. Trufem et al. (1989) reported0.5 spores/g in the soil of the Atlantic Forest of Cardoso Island(Ilha do Cardoso, Brazil), while Souza et al. (2003) reported0.34–8.6 spores/g of the soil in a seasonally dry forest calledCaatinga in northeast Brazil. Lima et al. (2013) found 0.65–4.07 spores/g in eucalyptus plantations, and Gomide et al.(2014) reported 0.26–9.32 spores/g in a semideciduous forest.The density of spores in the present study was also greaterthan the density obtained by Carvalho et al. (2012) (0.18–0.4 spores/g) in the same region. Probably, the majority ofthe plant species in these ecosystems are mycotrophic.Pagano and Scotti (2009) found many roots of native speciescolonized and an average of 1.08 spores/g soil in theirrhizospheric soil.

The diversity of mycorrhizal fungi found in this altitudinalgradient indicates that the rupestrian grasslands constitute asource of diversity of AMF. Carvalho et al. (2012) reportedthe occurrence of 49 AMF species in the rupestrian grasslandecosystems in five different habitats restricted to a single ele-vation (ca. 1100 m a.s.l.) in Serra do Cipó. The numbers ofAMF species in other Brazilian studies are usually lower: 18AMF species in crop areas (Benedetti et al. 2005), 29 speciesin an Atlantic Forest site (Stürmer et al. 2006), 27 species in anAraucaria Forest (Souza et al. 2007), and 14 species in thesand dunes in the Cardoso Island, São Paulo (Trufem et al.1989). The diversity of AMF in Serra do Cipó is also higher incomparison to other studies done elsewhere: 41 AMF speciesin the crops of Terminalia spp. in Ivory Coast (Wilson et al.1994) and 29 AMF species in Costa Rican pasture and forestland (Johnson and Wedin 1997). Lugo et al. (2008) reportedthat the lower elevation sites in an altitudinal gradient studiedin the Puna region of Argentina (3320 and 3870 m a.s.l.) sup-ported a higher richness of AMF and argued that the

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9 10 11 12 13

Num

ber o

f AM

F sp

ecie

s

Plots

800 m

900 m

1000 m

1100 m

1200 m

1300 m

1400 m

Fig. 4 Accumulation curves forAMF species observed at sevendifferent altitudes using theJackknife 1 estimator

Table 6 Richness values observed and estimated by the Jackknifeindex for the AMF species along an altitude gradient in the rocky soilcomplex of the Serra do Cipó

Altitude (m) Obs Est %Est

800 26 31 83.87

900 28 29 96.55

1000 36 38 94.74

1100 37 44 84.1

1200 37 43 86.04

1300 35 35 100

1400 32 36 88.89

Obs observed richness, Est estimated richness, %Est percentage of rich-ness observed in relation to the estimation using the jackknife index

Mycorrhiza

mechanisms responsible for the trend were increasedtemperature and number of potential hosts. Schmidt et al.(2008) also found lower species diversity of AMF in altitudesabove 4270 m (see also Wu et al. 2004, but see Oehl et al.2011). Large variations in spore density between altitudes areprobably due to the properties of the soils, host relations, andthe differential survival strategies of AMF. On the other hand,a detailed knowledge on the geography, dynamics, and pro-cesses that influence the AMF along altitudinal gradients stillrepresent a matter for further study, especially in tropicalmountain environments. A more complete explanation forAMF diversity and geography relies on further studies andhypotheses testing. A positive feedback might exist betweenAMF diversity and the high endemism and diversity of plantspecies in the nutrient-poor soils of these harsh tropical moun-tains (Carvalho et al. 2012), something that has not been testedin this work.

Otherwise, despite the high AMF species richness in ourstudy, the taxonomic variability at the genus level can be con-sidered low, with a significant contribution of only two gen-era, Acaulospora and Glomus. It seems that these genera ex-perienced an intensive speciation in the rupestrian grasslands.The genera Acaulospora and Glomus are the most frequent incultivated areas and in secondary forests (Benedetti et al.2005; Silva et al. 2006), indicating great adaptability to soilsunder different variations in chemical and physical properties(Carrenho et al. 2001). The genus Acaulospora is adapted to amore acidic pH (4.0 to 6.0), whereas the genus Glomus isadapted to basic pH (6.0 to 8.0) (Silveira 1998). The presentstudy corroborates the assertion made by Silveira (1998) forAcaulospora, which occurs at altitudes with a pH between 4.7and 5.1. However, the genus Glomus also proved to beadapted to this pH range in the rupestrian grasslands. Theseobservations reinforce the need for detailed studies on theAMF evolution and species formation in these singularmountains.

The frequency of AMF varied with different altitudes.G. invermaium was the species with the greatest frequencyat 800, 900, 1200, 1300, and 1400 m, followed by the speciesC. etunicatum , G. glomerulatum , F. mosseae , andF. geosporus, suggesting greater tolerance to different soilconditions found at these altitudes. This distribution of AMFspecies, which is apparently influenced by altitude, may be theresult of preferential symbiosis establishment by mycorrhizalhosts. According to Ribeiro and Fernandes (2000), thenutrient-deficient conditions of rupestrian grasslands shouldcompetitively favor only the plant species adapted to theseconditions. According to Giovannetti and Gianinazzi-Pearson (1994) and Souza et al. (2003), the distribution pat-terns of AMF in different environments are strongly influ-enced by the association with several biotic and abiotic factorsand are also related to the survival strategies of these micro-organisms, not evaluated here.

The species found only at one particular altitude were con-sidered to be exclusive to that altitude. The species exclusiveto 1400 m, G. gigantea, had not yet been reported in therupestrian grassland, although it has already been reported inreforested areas (Caproni et al. 2003), dune ecosystems(Trufem et al. 1989), Caatinga (Goto et al. 2010), AtlanticForest (Santos and Carrenho 2011), and dry forest (Paganoet al. 2013), thus exhibiting a broad distribution in Brazil.A. rugosa and P. robigina species, that were exclusive to thealtitude of 1000 m, have already been recorded in therupestrian grasslands in Serra do Cipó (Carvalho et al.2012). Furthermore, the only species exclusive to the altitudeof 800 m, F. heterogama, also observed by Carvalho et al.(2012), has a broad distribution, having been recorded inAraucaria forests (Moreira et al. 2007), in seasonally dry veg-etation (Souza et al. 2003), in riparian forests (Carrenho et al.2001), in native sandy landscapes (Mello et al. 2006), in agri-cultural areas (Nobre et al. 2010), in Caatinga (Goto et al.2010), and in dune areas (Souza et al. 2011).

The species A. brasiliensis, Glomus pellucidum, Glomusaff. verruculosum, and P. dominikii were recorded for the firsttime in the rupestrian grasslands of Serra do Cipó by Carvalhoet al. (2012), although only A. brasiliensis was found in thepresent study among these species. However, additional spe-cies are reported in our study: A. denticulata, Acaulosporacallosa, and G. gigantea. A. denticulata was previously re-ported in the semiarid region (Souza et al. 2003; Goto et al.2010) and agricultural land (Benedetti et al. 2005; Silva et al.2006) in Brazil. Carvalho et al. (2012) reported five potential-ly new species of the genera Acaulospora, Scutellospora, andGlomus to the rupestrian grasslands. They also reported twonew records for Brazil, Acaulospora colossica andP. dominikii. In the present study, six species of Glomus andfour species of Acaulospora are previously undescribed (B. T.Goto, pers inf.). Hence, the number of endemic species of therupestrian grasslands would increase from 6 (Carvalho et al.2012) to 19 species.

In conclusion, the present study shows that the rupestrianmountain environments in tropical Brazil have an astonishingdiversity of AMF, with 22 % of the global species diversity ofthese organisms recorded in a single elevation transect. Soilproperties were important factors associated with the variationin AMF species composition, richness, and density. On theother hand, the distribution of AMF along the altitudinalgradient was higher at intermediate altitudes characterizedby numerous habitat types and the transition of cerrado/rupestrian grassland physiognomies. This rich mosaic ofhabitats is associated to different degrees of harshness drivenby soil nutrient deficiency. The accumulation curves for AMFspecies richness highlighted the importance of sampling atdifferent altitudes and indicate the potential for new speciesto be found with increasing sampling effort. Therefore, it isnecessary to collect samples from additional plots to better

Mycorrhiza

estimate the species richness at each altitude, given that stabi-lization in the number of AMF species was not achieved. Inaddition, this study assessed the occurrence of AMF in a sin-gle seasonal period (wet season) so that the potential for evenmore species to be found in the study area is also augmented.Consequently, it can be argued that the rupestrian grasslandsof Serra do Cipó are indeed a hotspot for AMF biodiversity inthe world and a potential source for new taxa, as well asinteractions, and as such more studies should follow.

Acknowledgments We thank two anonymous reviewers for reading andproviding critiques of this manuscript and the trainees of the Ecology andPlant Propagation Laboratory and BG Souza for their support in the field.We would also like thank the Conselho Nacional de DesenvolvimentoCientífico e Tecnológico, Rede de Ciência e Tecnologia para Conservaçãoe Uso Sustentável do Cerrado - ComCerrado, the Fundação de Amparo àPesquisa do Estado deMinas Gerais, the Coordenação de Aperfeiçoamentode Pessoal deNível Superior, andReservaVellozia for logistic and financialsupport. This study was part of ES Coutinho’s MSc at Unimontes.

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