R E S E A R C H A R T I C L E

Diverse communities ofarbuscularmycorrhizal fungi inhabit siteswithveryhighaltitude inTibetPlateauYongjun Liu1,2, Junxia He1, Guoxi Shi1, Lizhe An1, Maarja Opik3 & Huyuan Feng1

1Key Laboratory of Arid and Grassland Ecology of Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou, China; 2College of Life

Science and Engineering, Northwest University for Nationalities, Lanzhou, China; and 3Department of Botany, Institute of Ecology and Earth Sciences,

University of Tartu, Tartu, Estonia

Correspondence: Huyuan Feng, Key

Laboratory of Arid and Grassland Ecology of

Ministry of Education, School of Life Sciences,

Lanzhou University, Room 322, Lanzhou

730000, China. Tel: 186 931 891 2560;

fax: 186 931 891 2537;

e-mail: fenghy@lzu.edu.cn

Received 24 January 2011; revised 6 June 2011;

accepted 19 June 2011.

Final version published online 18 July 2011.

DOI:10.1111/j.1574-6941.2011.01163.x

Editor: Philippe Lemanceau

Keywords

arbuscular mycorrhiza; Dracocephalum

heterophyllum; pioneer species; cold-

dominated ecosystem; high altitude;

Tibet Plateau.

Abstract

Diversity of arbuscular mycorrhizal fungi (AMF) is well studied in many

ecosystems, but little is known about AMF in cold-dominated regions with very

high altitude. Here, we examined AMF communities associated with two plant

species in the Tibet Plateau. Roots and rhizosphere soils of Dracocephalum

heterophyllum (pioneer species) and Astragalus polycladus (late-successional spe-

cies) were sampled at five sites with altitude from 4500 to 4800 m a.s.l. A total of 21

AMF phylotypes were identified from roots and spores following cloning and

sequencing of 18S rRNA gene, including eight new phylotypes and one new

family-like clade. More AMF phylotypes colonized root samples of D. hetero-

phyllum (5.4� 0.49) than of A. polycladus (1.93� 0.25). Vegetation coverage was

the most important factor influencing AMF community composition in roots.

Globally infrequent phylotype Glo-B2 in Glomus group B was the most dominant

in roots, followed by globally frequent phylotype Glo-A2 related to Glomus

fasciculatum/intraradices group. Our findings suggest that a diverse AMF flora is

present in the Tibet Plateau, comprising both potentially habitat-selective and

generalist fungi.

Introduction

The vast majority of terrestrial plants form mutualistic

associations with arbuscular mycorrhizal fungi (AMF),

which belong to the phylum Glomeromycota with about 200

described species (Smith & Read, 2008). AMF can increase

nutrient uptake as well as improve the stress tolerance of

their hosts (Smith et al., 2003; Aroca et al., 2007). AMF also

exert strong effects on the plant communities (van der

Heijden et al., 1998; Vogelsang et al., 2006). In particular,

the diversity of AMF and plants can be inter-related

(Johnson et al., 2004; Landis et al., 2004). A high number

of AMF taxa has been found in tropical and boreal forests

(Husband et al., 2002; Opik et al., 2008, 2009), whereas the

AMF richness is often low in disturbed and degraded

habitats (Opik et al., 2006; Tian et al., 2009). Even where

richness remains high, environmental factors such as pH

have been shown to determine community composition

even at relatively small spatial scales (Dumbrell et al., 2010).

At a much smaller, within sample site scale, studies indicate

that AMF have different levels of host preference or specifi-

city (e.g. Husband et al., 2002; Vandenkoornhuyse et al.,

2003), suggesting that the host plant is also an important

factor in assembling the AMF community. Thus, it is likely

that the diversity and distribution of AMF are codetermined

by the biotic and abiotic factors, and that the balance

between the two depends on spatial scale and local factors.

Diversity of AMF has been well studied in grasslands,

forests, and some other ecosystems of mainly the temperate

climatic zone (Opik et al., 2010). Little is known about AMF

diversity in regions with very high altitude (above

3500 m a.s.l.; but see Gai et al., 2009 and some studies in

montane sites with altitude below 2000 m a.s.l.: for example

Sykorova et al., 2007; Wu et al., 2007), where the habitats are

often cold-dominated. Kytoviita & Ruotsalainen (2007)

suggested that AMF could not improve nutrient capture of

host plants in low temperature habitats, but this does not

imply that AMF in such habitats cannot persist as plant

symbionts. On the contrary, a high diversity of AMF spores

FEMS Microbiol Ecol 78 (2011) 355–365 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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and phylotypes has been reported in sites with altitude

ranging from 3500 to 5200 m a.s.l. (Gai et al., 2009) and in

a low-Arctic meadow (Pietikainen et al., 2007), respectively.

Nonetheless, it is widely accepted that the major plant–fun-

gal associations in high-altitude and polar regions are

ectomycorrhiza and dark septate endophytes rather than

AM (Gardes & Dahlberg, 1996; Newsham et al., 2009; Gao &

Yang, 2010); this perhaps suggests that most plants in these

regions are not good hosts for AMF, and that AMF should

have a different life-history strategy in these regions. For

example, these fungi would select suitable host plants

(Gardes & Dahlberg, 1996) and the spores might be dor-

mant for a long time (Pietikainen et al., 2007). Even if

diverse AMF communities could be present in the regions

with very high altitude, which AMF species can persist and

which host plants they colonize is still unclear. The evolution

of AMF traits may be highly dependent on abiotic factors

such as soil temperature and hypoxia (Helgason & Fitter,

2009). Specific AMF have been found in extreme habitats

such as geothermal and gypsum soils (Appoloni et al., 2008;

Alguacil et al., 2009).

Therefore, we predict that AMF species and their traits in

regions with very high altitude may be different from those

in ecosystems with more moderate abiotic conditions. To

better understand the biodiversity of AMF and its determi-

nants in the regions with very high altitude, we chose to

study AMF associated with two plant species with different

life-history strategies in the Tibet Plateau of China (mean

altitude over 4500 m a.s.l.). We used molecular techniques to

describe AMF communities to address the following ques-

tions: (1) what is the AMF diversity in the roots of a pioneer

and a late-successional plant species in the central Tibet

Plateau? (2) Which environmental factors determine the

richness and community composition of AMF associated

with these two plant species? (3) Are the AMF taxa in this

region different from those in other ecosystems?

Materials and methods

Study sites and sampling

This study was conducted in the central Tibet Plateau, where

the average altitude is above 4500 m a.s.l. and the mean

annual temperature ranges between � 3 and � 7 1C. Five

study sites were chosen along the Qinghai-Tibet highway

from Xidatan to Amdo, in a line running approximately

north-east to south-west (Table 1, Supporting information,

Fig. S1). In each site, a pioneer plant species Dracocephalum

heterophyllum (Lamiaceae) was dominant in the region

nearby the highway (disturbed once due to the construction

of highway 30 years ago, o 50 m from highway), whereas

the natural plant communities (late-successional stage;

4 60 m from highway) were dominated by Cyperaceae,

Caryophyllaceae or leguminous plants such as Astragalus

polycladus (Table 1). Because the plants of Cyperaceae

and Caryophyllaceae are traditionally regarded as little

AM-dependent (Wang & Qiu, 2006; Smith & Read, 2008),

we chose a leguminous species, A. polycladus, as the

Table 1. Sampling sites and their vegetation details

Early-successional plant community Late-successional plant community

Study site

Latitude,

longitude

Altitude

(m a.s.l.)

Vegetation

coverage

Species

richness

Dominant plant

species (coverage)

Vegetation

coverage

Species

richness

Dominant plant

species (coverage)

Xidatan (X)� 351 420N

941 070E

4524 c. 30% 2 D. heterophyllum (c.

25%), Aster alpinus

(c.5%)

c. 45% 10 Androsace tapete (c.15%),

Astragalus polycladus

(c.10%), Arenaria kansuensis

(c.8%)w

Kunlunshan

(K)

351 370N

941 040E

4746 c. 70% 4 D. heterophyllum (c.55%),

Avena fatua (c. 5%),

Poa sp. (c. 5%)

c. 65% 9 Kobresia pygmaea (c.40%)w,

Astragalus polycladus (c.10%)

Wudaoliang

(W)

351 140N

931 050E

4568 c. 30% 2 D. heterophyllum

(c. 20%), Saussurea

wellbyl (c.10%)

c. 15% 1 Astragalus polycladus (c.15%)

Tongtianhe

(T)

331 480N

921 180E

4603 c. 25% 1 D. heterophyllum (c. 25%) c. 65% 10 K. pygmaea (c.30%)w, Stipa

aliena (c.10%), Astragalus

polycladus (c.8%)

Amdo (A) 321 100N

911 410E

4773 c. 75% 6 D. heterophyllum (c.50%),

Potentilla bifurca (c.7%),

Elymus nutans (c.5%)

c. 75% 11 K. pygmaea (c.35%)w,

Potentilla parvifolia (c.8%),

Arenaria kansuensis (c.5%)w,

Astragalus polycladus (c.4%)

�Site codes are shown in parentheses.wPotentially non-AM species of Caryophyllaceae or Cyperaceae (Smith & Read, 2008).

FEMS Microbiol Ecol 78 (2011) 355–365c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

356 Y. Liu et al.

representative of late-successional plant species. Before

sampling, the vegetation coverage, plant species coverage

and species richness were estimated on three plots of 1� 1 m

at each plant community using Sutherland’s method

(Sutherland, 1996). Vegetation coverage and plant species

richness varied in different sites, with low vegetation cover-

age in sites of Xidatan and Wudaoliang due to serious

permafrost degradation and rodent damage. More details

of the sampling sites are presented in Table 1.

Sampling was conducted in the beginning of August 2008.

In each site, three individuals of D. heterophyllum and

A. polycladus were randomly sampled (30 samples in total:

2 plant species� 3 plant individuals� 5 sampling sites).

Plants together with their rhizosphere soil were carefully

excavated and placed in plastic bags. Roots were separated

from each plant individual, washed with tap water and cut

into approximately 1-cm-long fragments. Due to the limited

quantity of roots, we randomly picked about 10 root

fragments from samples of the same plant species and

pooled these in order to verify the AM status of the study

plant species following the method of McGonigle et al.

(1990). The remaining roots (30 samples) were stored at

� 20 1C until DNA extraction. Soils (30 samples) were air-

dried and used for AMF spore extraction and soil chemical

analyses.

Spore extraction and soil chemical analysis

AMF spores were extracted from 30 soil samples of 100 g by

wet sieving (a pair of sieves: 750 and 38-mm mesh) and

sucrose centrifugation (Brundrett et al., 1994), and the spore

densities were counted. Spores from soil samples of the same

plant species in each site were pooled (resulting in 10 pooled

spore samples) and then grouped according to spore mor-

phology and color using a dissecting microscope. Spore

number of each morphotype in each pooled sample was

counted, and the spore morphotypes were further identified

using the molecular method as described below.

Soil total nitrogen, organic carbon and available (Olsen)

phosphorus contents were analyzed using the methods

described previously (Liu et al., 2009). Soil pH was mea-

sured in 1 M KCl (10 g soil in a 50 mL solution).

DNA extraction and amplification

For each root sample, about 30 root fragments (c. l-cm

lengths) were randomly picked and washed four times with

sterilized distilled water. Root DNA was extracted using

Plant DNA Extraction Kit (Tiangen Biotech, China) follow-

ing the manufacturer’s instructions, and then diluted 1 : 10

with ddH2O to be used as PCR template. For spore samples,

up to three clean and healthy-looking spores of each

morphotype per pooled sample were transferred into micro-

centrifuge tubes and vortexed at maximum speed. Spores

were further rinsed four times with sterilized distilled water.

Single spores were transferred into tubes with 10 mL ddH2O,

and crushed with forceps under the dissecting microscope.

A total of 120 single spores were incubated at 65 1C for

30 min and the liquid used as DNA template in PCR.

All root- and spore-derived DNA samples were subjected

to nested PCR. The first PCR reaction was performed with

the universal fungal primers GeoA2 and Geo11 to amplify a

c. 1.8 kb fragment of the 18S rRNA gene (Schwarzott &

Schußler, 2001). PCR reactions were carried out in a final

volume of 25 mL with 2 mL template and 0.5mM of each

primer using the Pfu PCR mastermix system (Tiangen

Biotech) with the following cycling conditions: 94 1C for

120 s; 30� (94 1C for 30 s; 59 1C for 60 s and 72 1C for

150 s); and 72 1C for 600 s. Successful products of the first

amplification were diluted 1 : 100 and 2 mL of this dilution

was used as a template in the second PCR with universal

eukaryotic primer NS31 (Simon et al., 1992) and AMF-

specific primer AML2 (Lee et al., 2008). Most first PCR

products of spores could not be detected on agarose gel, and

thus 1 mL of the first PCR product was used as template for

the second PCR. The NS31-AML2 primer combination was

used because amplification using AML1-AML2 (Lee et al.,

2008) was unsuccessful in our lab. PCR reactions were run

with the same conditions as described above using the

following cycling conditions: 94 1C for 120 s; 30� (94 1C

for 30 s; 58 1C for 60 s and 72 1C for 80 s); and 72 1C for

600 s. All the PCR reactions were run on a GeneAmps PCR

system 2700 (Applied Biosystems). PCR products were

examined on a 1.5% (w/v) agarose gel with ethidium

bromide staining.

Cloning, restriction fragment lengthpolymorphism (RFLP) typing and sequenceanalysis

All root-derived PCR products were used for constructing

clone libraries (30 in total). PCR products of expected

length (c. 560 bp) were purified using the Gel and PCR clear

up System (Promega). After an A-tailing procedure, the

DNA products were ligated into pGEM-T vector (Promega)

and cloned into Escherichia coli DH5a (Tiangen Biotech)

according to the manufacturer’s instructions. For each clone

library, 50 randomly selected white clones were immersed in

30 mL ddH2O and subjected to three cycles of freezing and

thawing for preparation of plasmid templates. Inserts were

reamplified with primers NS31/AML2 with the same PCR

conditions as above and double-digested with the restriction

enzymes HinfI and Hin1II (MBI, Lithuania). Digested

products were examined on a 2.5% (w/v) agarose gel.

One representative of each root-derived RFLP type was

sequenced with primer T7 by Major Biotech Company

(Shanghai, China).

FEMS Microbiol Ecol 78 (2011) 355–365 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

357Diverse AM fungi in Tibet Plateau

The spore-derived PCR products were directly subjected

to RFLP typing as described above. One representative of

each spore-derived RFLP type was cloned as described

above. One confirmed positive clone was sequenced from

each spore-derived clone library with primer T7 as above.

Sequences were edited using the CONTIGEXPRESS module of

VECTOR NTI Suite 6.0 (InforMax Inc., MD), and submitted to

BLAST search against public nucleotide sequence databases

(Altschul et al., 1997). All AMF sequences have been

submitted to GenBank database under the accession num-

bers GU238334–GU238408 and HQ610612–HQ610619.

The representative sequences obtained in this study, the

most closely related sequences from GenBank and the

representative sequences of major families of Glomero-

mycota were used in the phylogenetic analysis. Sequences

were aligned using CLUSTALX (Thompson et al., 1997) and

manually edited using GENEDOC (Nicholas et al., 1997).

Bayesian (GTR1G1I model) and neighbor-joining

(Kimura 2-parameter model with 1000 bootstrap replica-

tions) phylogenetic analyses were performed using MRBAYES

3.1 (Ronquist & Huelsenbeck, 2003) and MEGA 4.0 (Tamura

et al., 2007), respectively. Bayesian analysis was performed

with two independent runs of Markov chain Monte Carlo

with 106 generations (sampling frequency = 10), and a

conservative burn-in of 250 000 generations (25%) was

chosen.

Statistical data analysis

Due to limited identification success of AMF spores, the

molecular spore identification data are presented, but the

community composition of AMF spores was not analyzed

further. Root-colonizing AMF community composition was

analyzed on the basis of number of clones of each phylotype

in a root sample. The effects of host plant and sampling site

(as independent factors) on AMF richness per root sample

and spore density per soil sample were analyzed using two-

way ANOVA. Differences in AMF richness, spore density and

soil variables between samples were tested using Fisher’s

least significant difference at the 5% level after one-way

ANOVA. All above statistical analyses were performed using

the SPSS 13.0 (SPSS Inc., IL). Sampling effort curves of root-

colonizing AMF richness (Mao Tau) were computed using

ESTIMATES 8.0 (Colwell, 2006).

Before analysis of AMF community composition, raw

data of clone counts were square root transformed in order

to down-weigh the importance of abundant phylotypes. The

dissimilarities of AMF communities among samples were

computed by nonmetric multidimensional scaling (NMDS)

with Bray–Curtis distance using the function ‘isoMDS’ in

the R PACKAGE VEGAN version 1.17–8 (R Development Core

Team, 2010). To explore the relationships between environ-

mental variables and AMF community composition, the

environmental variables (including data of soil properties,

altitude, latitude, longitude, plant species richness and

vegetation coverage; all data except the latitude and long-

itude were square root transformed) were fitted as vectors

onto the NMDS plot using the function ‘envfit’ in the R

PACKAGE VEGAN version 1.17-8 (R Development Core Team,

2010).

We used the online database MaarjAM (http://maarjam.

botany.ut.ee; Opik et al., 2010; status of October 20, 2010) to

compare the AMF phylotypes obtained in this study with

the AMF detected in other ecosystems. Because the

MaarjAM database comprises AMF 18S rRNA gene se-

quences derived from 105 published papers of different

ecosystems, we could determine the detection frequencies

of our phylotypes in different ecosystems. We first used our

sequences to BLAST against the MaarjAM database and

grouped our sequences into the corresponding molecular

virtual taxa with the sequence identity Z98%, and then

searched how many studies had detected the corresponding

molecular virtual taxa.

Results

Spore density and molecular identification ofspores

AMF spore density varied in soil samples, with the highest

and lowest spore density being present in rhizosphere soils

of D. heterophyllum (1007� 77.4 spores per 100 g soil;

mean� SE) and A. polycladus (46.7� 4.1) in Xidatan

(Table 2). Spore density was different between sampling

sites (F = 11.45, Po 0.001), but not between host plant

species (F = 3.14, P = 0.09); site and plant species had a

significant interaction (F = 36.24, Po 0.001).

Amplification of spore-derived DNA was unsuccessful for

most, but not all spore morphotypes (Table 2, Table S1). In

total, 56 out of 120 single spores that were subjected to

molecular analysis (46.7%) were successfully amplified and

subjected to RFLP typing (Table 2). A total of 17 RFLP types

were detected, and one representative of each spore-derived

RFLP type was sequenced. Most spores of the same mor-

photype shared the same RFLP pattern (data not shown), so

that the remaining spores were classified by RFLP typing. A

BLAST search showed that eight sequences belonged to

Glomeromycota (Fig. 1), and nine sequences were related to

Ascomycota, possibly representing sequences of parasitizing

ascomycetes in spores (Hijri et al., 2002).

Molecular identification of AMF in roots

Before molecular analysis, the AM status of the plant species

studied was confirmed by root staining and microscopy,

showing high levels of root AM colonization: 53.5%

and 58.9% of root length was colonized by AMF for

FEMS Microbiol Ecol 78 (2011) 355–365c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

358 Y. Liu et al.

D. heterophyllum and A. polycladus, respectively. Both

arbuscules (12.8%) and vesicles (10.6%) were observed

in the roots of D. heterophyllum; only vesicles (15.7%),

but no arbuscules were observed in the roots of A.

polycladus.

All 30 root samples yielded PCR products of expected

length in the first (c. 1.8 kb) and second PCR reactions

(c. 560 bp). A total of 1500 clones were screened, and 1464

clones containing insert of correct size were submitted to

RFLP typing. One representative clone of each root-derived

RFLP type of each sample group (same plant species in same

site) was sequenced, yielding a total of 117 sequences. The

remaining clones were classified by RFLP typing. BLAST

search showed that 75 sequences had high homology to

members of Glomeromycota, 17 sequences were related to

Ascomycota, one sequence to Basidiomycota, nine sequences

to Metazoa and Cercozoa, and 13 sequences to plants.

Two sequences appeared to be chimeric. Altogether, 822

AMF clones/sequences (56%) were retained in the further

analyses.

Phylogenetic analysis

Both Bayesian and neighbor-joining phylogenetic analyses

of 44 representative AMF sequences from roots and eight

spore-derived sequences revealed 21 sequence groups with

sequence identity Z98% (Fig. 1), hereafter referred to as

phylotypes. Most of the phylotypes had high credibility

values and bootstrap support (4 80%). Seven phylotypes

were related to sequences of described species, six to

uncultured AMF and eight phylotypes (Glo-A1, Glo-A6,

Glo-A8, Aca-1, Clade-1, Clade-2, Clade-3 and Amb-2) were

undescribed previously ( � 97% identity with published

sequences; Fig. 1). Twenty phylotypes belonged to six

families and three orders of Glomeromycota (Fig. 1): nine

Glomeraceae A (Glomus group A), two Glomeraceae B

(Glomus group B), five Diversisporaceae, one Acaulospora-

ceae, one Pacisporaceae and two Ambisporaceae phylotypes.

The remaining phylotype Clade-1 within order Diversispor-

ales could not be placed in the known families and should be

considered as a new family-like clade (Fig. 1). Clade-2 and

Clade-3 were highly divergent from the genus Diversispora,

but were placed with high credibility values in the family

Diversisporaceae (Fig. 1), possibly representing two genera;

Clade-2 and Glomus fulvum (AM418543) belong to the

recently described genus Redeckera (Schußler & Walker,

2010); Clade-3 appears to be a new genus. Phylotypes of

Glomerales were most abundant in roots, representing

approximately 84% of the clones, followed by Diversisporales

(13%) and Archaeosporales (3%). Two phylotypes, Glo-B2

(33% of all AMF clones) and Glo-A2 (30%), were most

abundant in both plant species and all sampling sites.

Specific phylotypes (occurred in one host-site combination)

such as Glo-A3, Glo-A4 and Glo-A5 were often rare (Table

S2). A total of seven phylotypes were detected from

spores, but only two of them (Glo-B1 and Div-2) were also

detected in roots (Table 3). Most frequent phylotypes

among spores were not found in roots: Pac-1 was detected

at three sites mostly from rhizospheres of A. polycladus

(Xidatan, Kunlunshan, Amdo) and Div-3 in three sites of

D. heterophyllum (Kunlunshan, Wudaoliang, Amdo), re-

spectively (Table S1).

Table 2. Spore density and success rate of molecular identification of single spores in rhizosphere soils of each plant species per site

Sampling sites Plant species

Spore density

(spores per 100 g soil)

No. of morphotypes in

each pooled sample

No. of analyzed

single spores

No. of successfully

amplified

single spores�No. of AMF

phylotypes

X DH 1007.3�77.4 a 5 13 9 1

AP 46.7�4.1 f 4 10 5 0

K DH 400.7�82.2 c 6 17 5 3

AP 232.7�43.3 d 7 19 6 1

W DH 86.7�5.3 ef 4 7 4 0

AP 786�36.1 b 3 8 4 2

T DH 61.3�12.7 f 4 8 1 0

AP 205.3�48.6 de 5 10 7 1

A DH 213.3�55.5 de 6 13 7 3

AP 203.3�14.7 de 6 15 8 1

�If the second PCR reaction yielded a c. 560-bp product, it was considered as a successful amplification regardless of whether the PCR production was

an AMF or a non-AMF sequence.

Spore densities are shown with mean� SE (n = 3). Significant differences of spore densities between samples were tested using Fisher’s least significant

difference at the 5% level and indicated by different letters. Please note that spore morphotypes were analyzed following pooling of three replicate

samples per site (in total of 10 samples; see Materials and methods). Site codes are as in Table 1. DH, Dracocephalum heterophyllum; AP, Astragalus

polycladus.

FEMS Microbiol Ecol 78 (2011) 355–365 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

359Diverse AM fungi in Tibet Plateau

Fig. 1. Bayesian phylogenetic tree inferred from representative AMF 18S rRNA gene sequences identified in this study and reference sequences from

GenBank. Numbers above and below the branches are credibility values of Bayesian analysis and the bootstrap values of the neighbor-joining analysis,

respectively (values 4 70% are shown). Sequences obtained from roots are shown in bold and coded as follows: the first letter indicates the sampling

site (see the site codes in Table 1), the subsequent two letters indicate the host plant (DH: Dracocephalum heterophyllum; AP: Astragalus polycladus).

Sequences obtained from single spores are shown in bold and coded with spore-RFLP. The GenBank accession numbers of all new sequences are shown

in parentheses. Sequence groups (Glo-A1, etc.) identify distinct clusters of sequences with similarity Z98%.

FEMS Microbiol Ecol 78 (2011) 355–365c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

360 Y. Liu et al.

Search against the MaarjAM database

Thirteen out of 21 AMF phylotypes detected in the present

study could be grouped into the corresponding molecular

virtual taxa of the MaarjAM database (Table 3). Eight

phylotypes showed low similarity (93–97%) to the

sequences of this database and those in GenBank

( � 97%). The most dominant phylotype in the present

study (Glo-B2) has been detected by seven studies in the

MaarjAM database (as VT00056, no related species is

known, Table 3), but it was often rare in these studies (e.g.

Opik et al., 2009). Phylotype Glo-A2, second most domi-

nant in this study, has been earlier detected in 37 studies in

three climatic zones (as VT00113 related to Glomus fascicu-

latum group), followed by Glo-A9 (VT00065 related to

Glomus geosporum group, 15 studies) and Div-2 (VT00062,

14 studies) that were infrequent in our study.

Composition and diversity of AMF communitiesin roots

Sampling effort curves clearly indicate that a large propor-

tion of the total AMF diversity colonizing roots was cap-

tured for both plant species (Fig. 2). Fifteen AMF phylotypes

were detected in the roots of D. heterophyllum, whereas only

five phylotypes were detected in the roots of A. polycladus

(Fig. 2, Table S2). Both host plant (F = 386.29, Po 0.001)

and sampling site (F = 36.07, Po 0.001) showed significant

effects on AMF richness, with significant site and host plant

interaction (F = 28.07, Po 0.001). The mean number of

AMF phylotypes per root sample of D. heterophyllum

(5.4� 0.49; mean� SE) was higher than that of A. poly-

cladus (1.93� 0.25). Higher AMF richness was detected in

D. heterophyllum roots at Amdo (8� 0.58) and Kunlunshan

(7� 0) as compared with the other three sites (Fig. 3). The

AMF richness in A. polycladus roots followed a different

pattern: it was highest in Amdo and Tongtianhe (3� 0),

medium in Xidatan (1.67� 0.3) and lowest in Wudaoliang

Table 3. Number of publications in the MaarjAM database (http://maarjam.botany.ut.ee/, Opik et al., 2010) that have earlier reported the AMF

phylotypes of this study

Clones in

roots/from spores

Molecular

virtual taxon� Related species

Temperate

zone

Subtropical

zone

Tropical

zone

Subarctic

zone Total

Glo-A1 18/- – – – – – – –

Glo-A2 250/- VT00113 Glomus fasciculatum 27 8 2 – 37

Glo-A3 5/- VT00064 Glomus constrictum 5 6 1 – 12

Glo-A4 3/- VT00198 Glomus hoi 6 1 0 – 7

Glo-A5 3/- VT00301 – 0 1 0 – 1

Glo-A6 8/- – – – – – – –

Glo-A7 83/- VT00143 – 2 2 1 – 5

Glo-A8 -/1 – – – – – –

Glo-A9 -/1 VT00065 Glomus geosporum 14 1 0 – 15

Glo-B1 49/1 VT00193 Glomus claroideum, G. etunicatum 11 2 0 – 13

Glo-B2 272/- VT00056 – 3 2 2 – 7

Pac-1 -/7 VT00284 Pacispora scintillans 1 1 0 – 2

Aca-1 7/- – – – – – – –

Clade-1 47/- – – – – – – –

Clade-2 18/- – – – – – – –

Clade-3 11/- – – – – – – –

Div-1 8/- VT00060 Diversispora celata, Glomus eburneum 5 1 1 – 7

Div-2 16/2 VT00062 - 8 5 1 – 14

Div-3 -/4 VT00263 Diversispora spurca 1 0 0 – 1

Amb-1 27/- VT00283 Ambispora fennica 1 0 1 1 3

Amb-2 -/1 – – – – – –

�Codes of molecular virtual taxa (VT) in the MaarjAM database (http://maarjam.botany.ut.ee/, Opik et al., 2010).

Fig. 2. Sampling effort curves (Mao Tau) for AMF phylotypes detected in

roots of Dracocephalum heterophyllum and Astragalus polycladus.

FEMS Microbiol Ecol 78 (2011) 355–365 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

361Diverse AM fungi in Tibet Plateau

and Kunlunshan (1� 0) (Fig. 3). Taken as a whole, the total

number of detected AMF phylotypes in roots was highest in

Amdo (11 phylotypes), and lowest in Wudaoliang and

Tongtianhe (both four, Table S2).

NMDS ordination of AMF communities showed a low

stress level (0.09), indicating good representation of phylo-

type composition. NMDS ordination yielded sample group-

ings according to host plant (Fig. 4). There was also a trend

for samples from each site to cluster together regardless of

host plant, except for Kunlunshan and Amdo samples from

D. heterophyllum grouping together, and A. polycladus

samples grouping with samples from the same host from

other sites (Fig. 4). Ten environmental factors fitted as

vectors onto the NMDS plot showed that eight factors were

significantly correlated with the AMF community (Fig. 4);

of these, vegetation coverage was most strongly related to

the AMF community composition (r2 = 0.539, Po 0.001).

Environmental factors

Soil properties varied among samples (Table S3). In parti-

cular, soil available P content, ranging from 0.59 to

2.2 mg kg�1 (Table S3), was affected by sampling site

(F = 120.69, Po 0.001), but not by host plant species

(F = 2.95, P = 0.101), with significant interaction between

them (F = 19.40, Po 0.001). Soil available P (r =� 0.37,

P = 0.05; Pearson’s correlation) and C/N ratio (r =� 0.44,

P = 0.02) were negatively correlated with the vegetation

coverage. None of soil variables were correlated with altitude

(data not shown). Vegetation coverage showed a positive

relationship with plant richness (r = 0.77, Po 0.001) and

altitude (r = 0.63, Po 0.001), suggesting that the vegetation

coverage was higher in high-altitude sites than in low-

altitude sites of this study.

Discussion

Diverse and novel AMF are present in the TibetPlateau

Our results show that a relatively high AMF diversity was

present in the high-altitude alpine environment of the

central Tibet Plateau. A total of 21 AMF phylotypes (from

roots and spores) detected in five study sites of this study is

close to the number of 23 morphospecies detected in the

southern Tibet Plateau (Gai et al., 2009), where the climate

is less extreme than at the sites of this study. Although

comparison of the morphospecies detected by Gai et al.

(2009) and the phylotypes detected in this study is difficult,

a clear difference is the lack of Scutellospora in our study, but

found by Gai and colleagues. This suggests that there is

variation in AMF community composition among locations

in the central and southern Tibet Plateau, also seen among

sampling sites of this study, indicating even higher numbers

of AMF species in this region. Four to 13 AMF phylotypes

were detected per study site, which is comparable to those

known in temperate natural ecosystems (Opik et al., 2006,

2010). Our results are also consistent with a study showing

that at the level of individual plants, the mean number of

AMF taxa detected from plant roots in the low-arctic

meadow habitat was not lower than that in temperate

ecosystems (Pietikainen et al., 2007).

Eight out of 21 AMF phylotypes detected in this study are

not well affiliated to known AMF species or phylotypes.

Fig. 3. The richness of AMF phylotypes detected in root samples in each

site. Mean richness� SE (n = 3) are shown. Abbreviations of sample sites

are as in Table 1. Bars with different letters are significantly different at

P � 0.05.

–1.0 –0.5 0.0 0.5 1.0

–0.5

0.0

0.5

1.0

NMDS axis-1

NM

DS

axis

-2

XKWTA

Fig. 4. Joint plot of NMDS ordination of AMF communities colonizing

roots of different host plant species and the vectors of significant

(P � 0.05) environmental variables across sites. The length of the arrow

is proportional to the strength of correlation between environmental

variable and community dissimilarities. Filled symbols: Dracocephalum

heterophyllum; open symbols: Astragalus polycladus. Abbreviations of

sample sites are as in Table 1.

FEMS Microbiol Ecol 78 (2011) 355–365c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

362 Y. Liu et al.

Among these, Clade-1 and Clade-3 are sufficiently divergent

that they may represent a new family and a new genus

within Diversisporales (Fig. 1). A high proportion of new

AMF taxa detected in this study confirms the view that there

may be many AMF taxa awaiting discovery (Helgason et al.,

2002; Fitter, 2005), in particular in less-studied geographic

regions and biomes (Opik et al., 2010). The most dominant

phylotype (Glo-B2) in this study has been infrequently

detected in other ecosystems (Table 3), suggesting that this

AMF perhaps prefers habitats like those in the present study.

On the other hand, the second most abundant phylotype

was a globally widespread taxon related to G. fasciculatum/

intraradices group (Glo-A2; Table 3; Opik et al., 2006, 2010).

These findings suggest that the AMF communities in cold-

dominated environments in the Tibet Plateau include some

unique taxa and have also some taxa in common with more

moderate habitats. The unique taxa in this system may

include AMF specialized to this extreme habitat with

possibly specific functional properties. For example, Ambis-

pora fennica (Amb-1; Fig. 1), detected in two sites in this

study, may prefer cold habitats because this species was first

isolated from subarctic region in Finland (621300N; Walker

et al., 2007) and has been rarely detected in plant roots in

other climatic regions (Table 3).

AMF richness patterns

Our study system revealed a unique pattern: more AMF

phylotypes colonized roots of early-successional D. hetero-

phyllum in sites disturbed 30 years ago than roots of

A. polycladus in undisturbed vegetation (16 and five phylo-

types, respectively). The same trend was apparent at the level

of individual root samples, whereby the phylotype number

was higher for D. heterophyllum (5.4� 0.49 per sample)

than for A. polycladus (1.93� 0.25). It is generally held that

disturbance affects AMF diversity negatively (e.g. Opik et al.,

2006; Tian et al., 2009). Unfortunately, our study system

does not allow disentangling the effects of disturbance/

successional stage and host plant, because D. heterophyllum

occurs almost exclusively in formerly disturbed vegetation,

while A. polycladus occurs only in late-successional vegeta-

tion. Therefore, we cannot make firm conclusions about the

disturbance effect on the AMF richness. We can still say that

there were abundant arbuscules in the roots of D. hetero-

phyllum, but not A. polycladus, suggesting active symbiosis

in the first case, but not the latter. Spore density and

richness, however, did not differ between vegetation of

different disturbance histories, suggesting that there is no

effect of differing vegetation composition or disturbance

history. At the same time, only two of the AMF phylotypes

were detected both from spores and (rarely) in roots, which

might mean that different factors influence sporulating and

root-colonizing fractions of AMF communities, resulting in

different richness estimates. Clearly, more data are needed,

using both molecular and morphological methods about

ecosystems of different successional stages in this region,

and only then can we fully understand the ecological

importance and traits of AMF in this unique ecosystem.

In fact, natural communities in central Tibet Plateau are

dominated by Kobresia sedges that are known to host diverse

communities of ectomycorrhizal fungi in Himalayan mea-

dows (Gao & Yang, 2010), but can also exhibit AMF

colonization (Gai et al., 2006). Sedges can show quite high

AM colonization levels and positive growth response to

AMF in extreme habitats such as ultramafic soils (Lagrange

et al., 2011). Whether ectomycorrhizal fungi are dominant

over AMF in late-successional plant species in these habitats,

and AMF predominate in plants of early succession, remains

to be clarified in future studies.

We found that vegetation coverage was an important

factor influencing the AMF community composition. The

observed higher AMF richness in sites with higher vegeta-

tion coverage suggests that decrease in vegetation coverage

could decrease AMF diversity (Tian et al., 2009). Also, other

soil factors such as available P and organic C were related

with AMF community composition (Fig. 4), in concordance

with previous studies, showing that abiotic factors may

influence the patterns of AMF communities (Lekberg et al.,

2007; Dumbrell et al., 2010). Nonetheless, it is difficult to

separate environmental selection from effect of host plants

and plant communities on AMF (Schechter & Bruns, 2008),

because these organisms are always codetermined by biotic

and abiotic factors (Bever et al., 2001).

In conclusion, the present study is the first investigation

of the molecular diversity of AMF in the Tibet Plateau of

China. A high AMF diversity with many new taxa detected

in this region suggests that there are unique AMF in cold

ecosystems with very high altitude as well as generalist fungi

common in many types of ecosystems. AMF communities

colonizing roots varied in different plant species, with more

AMF phylotypes inhabiting the roots of pioneer plant

species than late-successional plant, suggesting that the

pioneer plant may be a better host for AMF in the Tibet

Plateau. Because the vegetation area in the Tibet Plateau has

decreased sharply in recent decades due to anthropogenic

disturbances and climate change (Harris, 2010), evaluation

of the AMF diversity patterns as well as the ecological traits

of AMF in this region is necessary if protection and

restoration of degraded regions is to be effective.

Acknowledgements

We are grateful to Prof. Tuo Chen and Dr Gaosen Zhang

from Cold and Arid Regions Environmental and Engineering

Research Institute, Chinese Academy of Sciences, and

Prof. Shiweng Li from Lanzhou Jiaotong Unviersity for

FEMS Microbiol Ecol 78 (2011) 355–365 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

363Diverse AM fungi in Tibet Plateau

facilitating the sample collection. We owe particular grati-

tude to Dr Thorunn Helgason at University of York for

constructive comments and valuable suggestions on the

manuscript. This research was supported by National Nat-

ural foundation of China (30870438, 40930533), The

Major Project of Cultivating New Varieties of Transgenic

Organisms (2009ZX08009-029B), State Key Laboratory of

Frozen Soil Engineering, Chinese Academy of Sciences

(SKLFSE200901), PhD Programs Foundation of Ministry

of Education of China (2010021111002), Estonian Science

Foundation (7738, SF0180098s08), 7th European Commu-

nity Framework Programme (PERG03-GA-2008-231034)

and the European Regional Development Fund (Centre of

Excellence FIBIR).

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Fig. S1. Geographical location of study sites.

Table S1. Number of spores of AMF phylotypes detected

from rhizospheres of two plant species at five study sites.

Table S2. Number of clones of AMF phylotypes detected in

the roots of two plant species at five study sites.

Table S3. The characteristics of rhizosphere soils of each

plant species in each site.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.

FEMS Microbiol Ecol 78 (2011) 355–365 c� 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

365Diverse AM fungi in Tibet Plateau

Supporting information

Table S1 Number of spores of AMF phylotypes detected from rhizospheres of two plant

species at five study sites. First, number of spores of each morphotype was counted; then at

least one spore per morphotype was subjected to molecular identification. Site codes are as in

Table 1.

Dracocephalum heterophyllum Astragalus polycladus

X K W T A Total X K W T A Total

Glo-A8 - 240 - - - 240 - - - - - 0

Glo-A9 - - - - - 0 - - 26 - - 26

Glo-B1 - 97 - - - 97 - - - - - 0

Div-2 - - - - - 0 - - - 179 - 179

Div-3 16 444 - - 27 487 - - - - - 0

Amb-2 - - - - 12 12 - - - - - 0

Pac-1 - - - - 108 108 - 92 536 - 8 636

Total richness 1 3 0 0 3 5 0 1 2 1 1 3

Table S2 Number of clones of AMF phylotypes detected in the roots of two plant species at

five study sites. Site codes are as in Table 1.

Dracocephalum heterophyllum Astragalus polycladus

X K W T A Total X K W T A Total

Glo-A1 0 0 0 0 0 0 0 0 0 0 18 18

Glo-A2 6 9 109 33 4 161 0 0 46 16 27 89

Glo-A3 0 0 0 0 5 5 0 0 0 0 0 0

Glo-A4 0 0 3 0 0 3 0 0 0 0 0 0

Glo-A5 0 0 0 0 3 3 0 0 0 0 0 0

Glo-A6 0 0 0 8 8 8 0 0 0 0 0 0

Glo-A7 14 0 10 42 0 66 11 0 0 6 0 17

Glo-B1 0 28 0 0 21 49 0 0 0 0 0 0

Glo-B2 74 29 0 17 34 154 40 49 0 29 0 118

Aca-1 0 7 0 0 0 7 0 0 0 0 0 0

Clade-1 0 8 6 0 21 35 0 0 0 0 12 12

Clade-2 0 0 0 0 18 18 0 0 0 0 0 0

Clade-3 11 0 0 0 0 11 0 0 0 0 0 0

Div-1 0 8 0 0 0 8 0 0 0 0 0 0

Div-2 4 7 0 0 2 13 0 0 0 0 0 0

Amb-1 0 20 0 0 7 27 0 0 0 0 0 0

Total clones 109 116 128 100 115 568 51 49 46 51 57 254

Total richness 5 8 4 4 9 16 2 1 1 3 3 5

Table S3 The characteristics of rhizosphere soils of each plant species in each site. Data are

shown with mean ± SE (n=3). Significant differences of each soil variable between samples

were determined using Fisher’s Least Significant Difference at the 5% level after one-way

ANOVA and indicated by different letters. Site codes are as in Table 1. DH: Dracocephalum

heterophyllum; AP: Astragalus polycladus.

Sampling

sites

Plant

species

Total N

(g kg-1)

Organic C

(g kg-1) C/N ratio

Available P

(mg kg-1) pH

X DH 0.72±0.06 c 0.89±0.03 g 1.23±0.06 i 1.32±0.13 d 7.38±0.02 b

AP 0.5±0.02 f 0.79±0.07 h 1.58±0.11 g 1.37±0.05 cd 7.59±0.02 a

K DH 0.53±0.04 e 0.98±0.04 f 1.83±0.09 f 0.59±0.28 g 7.64±0.05 a

AP 0.42±0.03 g 0.96±0.03 f 2.27±0.17 e 0.83±0.2 f 7.56±0.01 a

W DH 0.36±0.02 h 1.75±0.07 c 4.82±0.23 a 2.20±0.11 a 7.57±0.06 a

AP 0.43±0.02 g 1.19±0.15 e 2.76±0.16 c 1.55±0.19 bc 7.38±0.01 b

T DH 0.57±0.09 d 1.85±0.07 b 3.26±0.13 b 0.97±0.17 ef 7.05±0.09 e

AP 0.78±0.05 b 2.02±0.05 a 2.59±0.19 d 1.08±0.08 e 7.31±0.03 bc

A DH 0.87±0.03 a 1.32±0.02 d 1.52±0.04 h 1.29±0.13 cd 7.23±0.02 cd

AP 0.87±0.05 a 1.37±0.05 d 1.57±0.09 g 1.68±0.06 b 7.16±0.06 d

Fig. S1 Geographical location of study sites.