1

Mechanisms influencing the arbuscular mycorrhizal community in a tropical altitudinal

gradient

João Henrique de Azevedo Xavier1, Etiene Silva Coutinho

1, G. Wilson Fernandes

1,2.

1 Ecologia Evolutiva & Biodiversidade/DBG, CP 486, ICB/Universidade Federal de Minas

Gerais, 31270-901 Belo Horizonte, MG, Brazil.

2 Department of Biology, Stanford University, Stanford CA 94395 USA. Email:

gw.fernandes@gmail.com, + 1 650 864 2910.

Abstract

The ecosystem with the highest studied richness of arbuscular mycorrhizal

fungi (AMF) is that of the rupestrian grasslands, in Brazil. The climatic,

edaphic and vegetation factors influencing this AMF community composition,

species richness and spore density in an elevational gradient are further

explored to understand the mechanisms shaping this community. This study

also focuses on AMF dissimilarity among sites and beta diversity nestedness

and turnover components. AMF species richness was negatively correlated

with soil nutrients, supporting that plants associate with AMF more often in

habitats with low nutrient availability like the rupestrian grasslands. There was

a high AMF community dissimilarity caused by sharp species replacement

originated by the variation in the abiotic and biotic mechanisms. This finding

indicates that preserving small parts of this patchy ecosystem is not enough to

preserve its fungal diversity because sites largely differ in composition.

Key Words: altitudinal gradient; arbuscular mycorrhizal fungi; beta-diversity; climate;

community ecology; conservation; rupestrian grasslands; soil; turnover; vegetation.

2

Introduction

The arbuscular mycorrhizal fungi (AMF) (phylum Glomeromycota) are obligate

biotrophs of plants’ roots, forming associations with approximately 74% of the land plants

species (Smith & Read 2008; Brundrett 2009). With fossil record dating back to over 460

million years (Redecker et al. 2000), AMF have played an essential role in the origin and

diversification of land plants (Pirozynski & Malloch 1975; Helgason & Fitter 2005) and

collaborate to the maintenance of ecosystem functions in most land ecosystems (Wang 2006).

The symbiosis between AMF and plants provides increased nutrient uptake and improved

tolerance of drought and pathogens to the host plant, at the expense of carbon synthesized by

the plant (Smith & Read 2008; Kiers et al. 2011).

At the ecosystem level, AMF enhance plant diversity, productivity and ecosystem

variability (Klironomos & Hart 2002; Wagg et al., 2014). AMF also provide a variety of

ecosystem functions and services such as nutrient cycling, soil stability, and carbon

sequestration in soils in multiple scales, acting as a negative feedback to global change

(Newsham et al. 1995; Miller & Jastrow 2000; Staddon et al. 2002; Rillig 2004a; Johnson et

al. 2006). But, despite their prevalence in the environment and ecological importance, much

remains unknown about their diversity patterns (Davison et al. 2015), especially in tropical

ecosystems (Heijden et al. 2015). The understanding of these patterns can alter significantly

the conservation and management practices in megadiverse tropical countries.

The factors influencing AMF communities vary across different scales. In a global

scale, environmental factors such as soil moisture and temperature are as important as spatial

and plant community variables in their influence on AM fungal communities (Kivlin et al.

2011; Davison et al. 2015). Studies in smaller scales revealed the influence of the vegetation

and edaphic characteristics (specially, phosphorus content and texture) in AMF composition

and spore density (eg. Bever 2002ab; Helgason et al. 2002; Husband et al. 2002;

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Vandenkoornhuyse et al. 2002b; 2003; Chaudhary et al. 2008; Yang et al. 2012). Climate

factors can also affect AMF community structure (chaudhary et al. 2008), markedly Rainfall

(eg. Johnson et al. 2013; Tchabi et al. 2008; Lovelock et al. 2003) and temperature (eg. Rillig

et al. 2002, Staddon et al. 2002, Hawkes et al. 2008), usually with positive effects on AMF

spore density and species richness.

The highest studied AMF richness and spore density in the world is found in the

rupestrian grasslands, a montane, grassy-shrubby, fire-prone vegetation mosaic with rocky

outcrops (Coutinho et al. 2015; Carvalho et al. 2012; Fernandes et al. 2016). Rupestrian

grasslands are old, climatically buffered, infertile landscapes (Ocbils) (Hopper 2009; Hopper

et al. 2015; Silveira et al. 2016), ecosystems with high biodiversity that present great

conservation and restoration challenges (e.g. Fernandes et al. 2014). In an altitudinal gradient

in this ecosystem, 51 species of AMF were recorded by Coutinho et al. (2015), with 14

possibly new species to science and nine species being reported for the first time to occur

Brazil. In the same ecosystem, but in a single elevation, Carvalho et al. (2012) reported 49

species of AMF, with four potentially new, indicating the importance of the rupestrian

grasslands for the preservation of AMF's biodiversity.

The high floristic richness and high plant endemism of the rupestrian grasslands is

suffering intense degradation, mostly by opencast mining, annual intentional burnings to

support livestock, wood extraction and invasive species (e.g., Fernandes et al. 2014). These

activities have very often strong influence on soil and vegetation structure and therefore,

could possibly impact AMF’s diversity and the related ecosystem services. A better

understanding of the factors influencing the spatial distribution of these fungi contributes to

the comprehension of the community dynamics and the preservation of the ecosystem

services provided by the taxa by enlightening priority conservation sites and scales

(Chaudhary et al. 2008; Alguacil 2015).

4

In the rupestrian grasslands, previous studies stated that AMF species richness, spore

density and community composition are influenced by the patchy vegetation structure

(Carvalho et al. 2012) and elevation, with higher AMF species richness and spore density at

intermediate altitudes (Coutinho et al. 2015). Yet, the most common species distribution

pattern in mountains is the decline of richness with the rise of elevation, (e.g. Terborgh 1977;

Wolda 1987; Fernandes & Price 1988; 1991). Here, we further investigate the possible

mechanisms originating the AMF's community variation studied by Coutinho et al. (2015) by

focusing on the beta component partitioning, that divides community dissimilarity in the

opposite processes of turnover and nestedness (described as species replacement and species

loss, respectively) (e.g. Baselga 2002), and the influence of a wide group of climatic, edaphic

and vegetation variables. We hypothesized that: i) There will be a high dissimilarity among

sites caused by species replacement (turnover), justifying the high AMF diversity with the

heterogeneity of the rupestrian grasslands. ii) AMF community composition will be

influenced by climatic, soil and vegetation variables, indicating that the high diversity of the

rupestrian grasslands is due to the variation of resources and conditions in this rich mosaic

(Carvalho et al. 2012). iii) Vegetation abundance will enhance AMF's species richness and

spore density, while soil nutrients availability will decrease both (Abbott & Robson 1991).

Methods

The study was conducted in Serra do Cipó, in southern Espinhaço Mountain Range,

southeastern Brazil, at latitude and longitude near to 19°15′S and 43°40′ W. The average

temperature is 19.6 °C, and the annual rainfall is approximately 1500 mm with dry winters

and wet summers (Madeira & Fernandes 1999). The site soils are typical of the ecosystem:

sandy, shallow, acidic and nutrient poor, with high aluminum concentrations (Negreiros et al.

2011).

5

Climatic, soil, vegetation and fungi data were sampled in seven transects separated by

at least 2,5 km each. The transects varied in altitude in intervals of 100 meters a.s.l., spanning

from 800 to 1400m a.s.l. On each transect, thirteen squared plots of 100 m2 (10 m×10 m)

were defined totalizing 91 plots (0.91 ha). Soil was sampled in five points of each 1m2 plot

and homogenized to posterior AMF spores extraction and physical-chemical analysis. 50g of

each soil sample was used for AMF spore extraction with the wet sieving technique

(Gerdemann & Nicolson 1963) and centrifugation in sucrose solution (50%) (Jenkins 1964).

The spores were counted to evaluate the AMF spore density and transferred to slides, where

they were crushed with a drop of polyvinyl alcohol lacto-glycerol and Melzer's reagent for

morphological identification at species level (see Coutinho et al. 2015 for further details). For

soil pH in water, P-Mehlich phosphorous, Al aluminum, sum of bases, and sand percentage

the soil samples were air-dried and sieved at 2.0 mm for texture and chemical analysis as

described by the Brazilian Agricultural Research Corporation (Embrapa 1997). Woody plants

were all identified in each plot and a squared 1m2 plot was defined on each plot for the

identification of all herbaceous and regenerating plants (with diameter at ground height lower

than 1cm) (for further details, see Mota et al. 2016). Climate data (Average annual

temperature, photosynthetically active radiation, accumulated rainfall and soil moisture and

temperature, both at 5 and 20 cm depth) were collected by climatic stations (Onset climatic

stations) at each transect from 2011 to 2014.

The total dissimilarity of the AMF community among plots was calculated with the

Sørensen dissimilarity index (Baselga 2002). AMF turnover rates were calculated dividing the

Simpson dissimilarity index by the Sørensen dissimilarity index (Baselga 2002) to evaluate if

the main cause of AMF community variation is turnover or nestedness.

The measured climatic, edaphic and vegetation data were submitted to separated

pairwise correlation tests with Pearson's coefficient. Explanatory variables with higher

6

similarity than 50% were grouped and one was chosen based on AMF's literature to avoid

losing significance due to redundancy (eg. Cohen 1988; Rillig et al. 2002; Staddon et al.

2002; Hawkes et al. 2008; Kivlin 2011).

To understand how the measured biotic and soil factors influence AMF community

composition, a Permutational Multivariate Analysis of Variance Using Distance Matrices

(Permanova) was performed with the Jaccard method and 1000 permutations (Anderson 2001,

McArdle & Anderson 2001). For factors influencing AMF's species richness and spore

density, two different generalized linear models (GLM) were performed using the biotic and

soil factors as explanatory variables and AMF's density and species richness as response

variables. The influence of the abiotic factors on AMF density and richness was measured by

two mixed models because the climatic data (one station per transect, totalizing seven

samples) had to be adapted to the fungal data (13 soil samples per transect, totalizing 91

samples). Altitude was included as the last explanatory variable in all models because, as a

gradient pattern that compresses multiple factors, it can indicate that non measured biotic or

abiotic factors that are correlated with altitude are also influencing the response variable

(Korner 2007). The non-significant explanatory variables (p<0.05) were removed from the

higher to the lower p-value to obtain the minimal adequate model. All analyses were

performed in R statistical software (R Development Core Team 2008) with the vegan package

(Oksanen et al. 2015) for beta diversity partitioning and lme4 (Bates et al. 2015) and nlme

(Pinheiro et al. 2015) packages for mixed models.

7

Results

Soil moist, aluminum concentration and sand percentage increased with altitude.

Photosynthetically active radiation, soil temperature, temperature, soil pH and plant species

richness decreased with altitude. Rainfall, Phosphorus content, sum of bases and vegetation

abundance did not show a clear variation trend with altitude (Figures 1 and 2).

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Figure 1: Effect of altitude in (a) rainfall, (b) soil moist, (c) photosynthetically active

radiation, (d) soil temperature, (e) temperature and (f) soil pH across an altitudinal gradient at

Serra do Cipó, Brazil. Curves with p-values above 0.05 are not represented.

Figure 2: Effect of altitude in (a) phosphorus content, (b) aluminum content, (c) sum of bases,

(d) sand percentage, (e) vegetation abundance and (f) plant species richness across an

altitudinal gradient at Serra do Cipó, Brazil. Curves with p-values above 0.05 are not

represented.

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The correlation test among variables indicated that the rainfall variation along the

altitudinal gradient was significantly different from the other climatic variables. Hence,

rainfall was used as a response variable in the model analysis. Temperature,

photosynthetically active radiation, soil moist and soil temperature were correlated along the

altitudinal gradient. This group was represented by temperature in the model analysis (Table

1). Soil attributes (pH in water, phosphorus, aluminum, sum of bases and sand percentage)

presented distinct patterns of variation among sites (Table 2). The biotic variables (vegetation

abundance and plant species richness) had 48% of similarity. Therefore, all soil and

vegetation variables were kept for model analysis.

Table 1: Percentage of similarity between climatic variables across an altitudinal gradient at

Serra do Cipó, Brazil, according to the pairwise correlation test with Pearson's coefficient.

The minus sign indicates negative correlations.

Rainfall* Soil moista Soil moist

b PAR Soil T

a Soil T

b

Rainfall*

(mm)

100% 40.11% 01.78% (-) 27.31% 29.13% 24.12%

Soil moista

(m3/m3)

40.11% 100% 70.16% 18.14% (-) 24.41% (-) 39.71% (-)

Soil moistb

(m3/m3)

01.78% (-) 70.16% 100% 62.76% (-) 66.15% (-) 74.13% (-)

PAR

(lm/W)

27.31% 18.14% (-) 62.76% (-) 100% 89.14% 81.67%

Soil Ta

(oC)

29.13% 24.41% (-) 66.15% (-) 89.14% 100% 97.76%

Soil Tb

(oC)

24.12% 39.71% (-) 74.13% (-) 81.67% 97.76% 100%

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T(oC) 00.89% 22.42% (-) 64.49% (-) 83.55% 92.00% 89.66%

* One year accumulated

a At 5cm depth.

b At 20cm depth.

PAR - Photosynthetically active radiation. T - temperature

Table 2: Percentage of similarity between edaphic variables across an altitudinal gradient at Serra do Cipó, Brazil according to the pairwise correlation test with pearson's coefficient. The

minus sign indicates negative correlations.

pH in water P-melich Aluminum Sum of Bases

pH in water

100% 14.79% (-) 47.24% (-) 15.62% (-)

P-melich*

(mg/dm3)

14.79% (-) 100% 30.36% 49.73% (-)

Aluminum

(cmolc/dm3)

47.24% (-) 30.36% 100% 33.99%

Sum of Bases

(cmolc/dm3)

15.62% (-) 49.73% 33.99% 100%

Sand percentage 20.05% (-) 05.27% (-) 04.75% 16.92%

*Phosphorus measured with the P-Mehlich technique

The AMF community composition dissimilarity was 96% (βsor=0.9596), indicating

that the AMF community in this mountain gradient is very heterogeneous. The main

phenomenon explaining the dissimilarity was turnover, or species replacement (96.53% of

total dissimilarity). AMF community composition was influenced by multiple mechanisms

(temperature, rainfall, vegetation abundance, phosphorus content, altitude, percentage of sand,

and plant species richness), indicating that the variation in these factors alter AMF

composition (Table 3).

The climatic, edaphic and vegetation factors did not influence AMF spore density.

AMF species richness was negatively correlated with phosphorus concentration, the sum of

bases and altitude (Figure 3).

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Table 3: Mechanisms influencing AMF spore density, species richness and community

composition across an altitudinal gradient at Serra do Cipó, Brazil. Bold variables and values

indicate statistical significance.

Response variable Explanatory variables Df F-test result P value

AMF spore density a Vegetation abundance 1 0.0087 0.92597

Test: GLM Plant species richness 1 1.2282 0.27038

p-value: 0.3668 pH 1 3.6411 0.05952

Phosphorus 1 0.0628 0.80257

Aluminum 1 1.4076 0.23889

Bases sum 1 1.1959 0.43023

Sand content 1 1.1695 0.27222

Altitude 1 0.1734 0.33432

AMF richness a Vegetation abundance 1 0.2448 0.62205

Test: GLM Plant species richness 1 1.5126 0.2222

p-value: 0. 0103 pH 1 0.2081 0.6495

Phosphorus 1 6.3525 0.0137

Aluminum 1 0.1470 0.7024

Sum of bases 1 12.6551 0.0006

Sand content 1 0.2889 0.5924

Altitude 1 0.9862 0.3236

AMF richness b Phosphorus 1 6.4873 0.0126

Test: GLM Sum of bases 1 9.9722 0.0022

p-value: 0. 0003 Altitude 1 4.1080 0.0022

AMF spore density a Temperature 1 1.13475 0.3468

Test: Mixed model Accumulated rainfall 1 1.05418 0.3468

p-value: 0.0043 Altitude 1 0.01547 0.9013

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Response variable Explanatory variables Df F-test result P value

AMF richness a Temperature 1 0.40134 0.5608

Test: Mixed model Accumulated rainfall 1 2.65842 0.1783

p-value: 0.0063 Altitude 1 0.43800 0.5099

AMF composition a Temperature 1 4.4818 0.0001

Test: Permanova Accumulated rainfall 1 3.2266 0.0001

Vegetation

abundance

1 2.6852 0.0001

Plant species richness 1 1.5154 0.0709

pH 1 0.9163 0.5624

Phosphorus 1 1.9522 0.0099

Aluminum 1 1.3081 0.1508

Sum of bases 1 1.3081 0.5774

Sand content 1 1.6846 0.0299

Altitude 1 1.5917 0.0499

AMF composition b Temperature 1 4.6156 0.0009

Test: Permanova Accumulated rainfall 1 3.3370 0.0009

Vegetation

abundance

1 2.7307 0.0009

Plant species richness 1 1.5854 0.0469

Phosphorus 1 2.0174 0.0140

Sand content 1 1.6096 0.0390

Altitude 1 1.6878 0.0400

a Complete model.

b Minimal adequate model.

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Figure 3: Effect of the sum of bases (p<0.01, F=9.98, curve=exp(2.92-1.74*x)) (a)

and the phosphorus content (p<0.05, F=6.49, curve=exp(2.34-0.03*x)); (b) in AMF species

richness across an altitudinal gradient at Serra do Cipó, Brazil.

Discussion

The high dissimilarity among sites reveals that AMF community is very

heterogeneous in this ecosystem. All climatic, edaphic and vegetation factors influence AMF

composition, with the exceptions of the sum of bases, aluminum concentration and soil pH,

indicating that the high diversity of this ecosystem is due to its observable patchy organization

of microhabitats originated by the variation of biotic and abiotic conditions and resources

(Giulietti & Pirani 1988; Vitta 2002; Benites et al. 2003). The main component of the AMF

community dissimilarity was species substitution (turnover), originated by spatial or historical

constraints and environmental sorting (Qian et al. 2005; Baselga 2010). The biodiversity

conservation of a community with this structure requires the conservation of different sites,

not necessarily the richest ones (Wright & Reeves 1992; Baselga 2010).

AMF were thought to be generalists, with only 200 - 300 described species (Öpik

et al. 2010; Schüßler & Walker 2010) and associations with approximately 200,000 of the

land plants species (Kivlin et al. 2011; Öpik et al. 2013). Yet, a crescent number of studies

(a) (b)

14

suggest that species can exhibit a degree of host specificity (Bever et al. 2002b; Hegalson et

al. 2002; Kivlin et al. 2011). In this study, AMF community composition was influenced by

the vegetation abundance and species richness, corroborating that there's a degree of

specificity between AMF and plants. Probably, the high plant diversity and the degree of

endemism of the rupestrian grasslands are important factors shaping AMF community. Yet,

AMF species richness and spore density were not affected by plant species richness or

abundance. It indicates that specificity can be at different levels as family, order, origin or

functional groups (Kivlin et al 2011) or the result of habitat influences in both plants and

AMF (Carvalho et al. 2012).

Plants form mycorrhizal associations with the AMF most beneficial for plant survival,

performance and productivity (Requena et al. 2001; Caravaca et al. 2005; Werner & Kiers

2014) and nutrient-stressed plants tend to release more carbohydrates for AMF associations

then unstressed plants (Sylvia and Neal 1990; Schwab et al. 1991). In this study, AMF species

richness was negatively correlated to the sum of bases and the phosphorus content of the soil,

corroborating that plants associate more frequently and intensively with AMF in low fertility

environments (Moreira et al. 2010; Lisboa et al. 2014) since a significant benefit of the

association is the nutrient acquisition (Smith & read 2008, Heijden et al. 2015), which is less

needed in ecosystems with high soil fertility. Therefore, fertilizing old, climatically buffered,

infertile landscapes as the rupestrian grasslands aiming to their restoration is strongly

discouraged and can possibly hamper the development of a rich AMF community (Hopper

2009; Lambers et al. 2008; 2014b; Barbosa et al. 2010; Hopper et al. 2016; Silveira et al.

2016) and disrupt coadapted mycorrhiza-soil complexes, altering plant and fungal

communities. (Johnson 1993).

Despite the wide AMF spore density variation observed in the samples (minimum of

49 and maximum of 1583), none of the analyzed variables influenced AMF's spore density.

15

Spore density is probably influenced by more seasonal since different species can sporulate in

different periods (Gemma et al.1989), probably due to environmental stimuli. That explains

why annual data did not affect AMF species richness.

Since the sampling of this study was performed only on the wet season, AMF species

richness in this ecosystem is probably underestimated. Also, the identification method of this

study was based on morphological characters, which does not always separate genetically

distinct taxa, resulting in lower richness when compared to molecular identification studies

(Hijri and Sanders 2005), indicating that AMF diversity is even higher in the rupestrian

grasslands.

The intrinsic variation of the rupestrian grasslands is the origin of the many different

environments that make the coexistence of the AMF communities possible in such a small

area. Probably, this phenomenon also affects other organism communities in different scales.

The large AMF community variation originates its richness and indicates that preserving a

small part of this old ecosystem is not enough to preserve unique fungal diversity and the

related ecosystem services.

Acknowledgements

We thank two anonymous reviewers for the valuable comments on earlier versions of

the manuscript, the funding provided by Coordenação de Aperfeiçoamento de Pessoal de

Nível Superior (CAPES), Rede de Ciência e Tecnologia para Conservação e Uso Sustentável

do Cerrado (ComCerrado/CNPq), Peld/CNPq and Fundação de Amparo à Pesquisa do Estado

de Minas Gerais (FAPEMIG) and logistic support provided by Reserva Vellozia. We also

thank Frederico de Siqueira Neves and Marina do Vale Beirão for assistance with the

statistical analyses.

16

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