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ORIGINALARTICLE

Alien plants associate with widespreadgeneralist arbuscular mycorrhizal fungaltaxa: evidence from a continental-scalestudy using massively parallel454 sequencing

Mari Moora1*, Silje Berger2�, John Davison1, Maarja Opik1, Riccardo

Bommarco3, Helge Bruelheide4, Ingolf Kuhn5, William E. Kunin6, Madis

Metsis7,8, Agnes Rortais9�, Alo Vanatoa10, Elise Vanatoa1, Jane C. Stout11,

Merilin Truusa7,8, Catrin Westphal12§, Martin Zobel1 and

Gian-Reto Walther13

1Department of Botany, Institute of Ecology

and Earth Sciences, University of Tartu, 40 Lai

St., 51005 Tartu, Estonia, 2Institute of

Geobotany, University of Hannover,

Nienburger Strasse 17, 30167 Hannover,

Germany, 3Department of Ecology, Swedish

University of Agricultural Sciences, SE-75007

Uppsala, Sweden, 4Institute of Biology/

Geobotany and Botanical Garden, Martin

Luther University Halle Wittenberg, Am

Kirchtor 1, 06108 Halle, Germany, 5UFZ,

Helmholtz Centre for Environmental Research

– UFZ, Department of Community Ecology,

Theodor-Lieser-Strasse 4, 06120 Halle,

Germany, 6Earth & Biosphere Institute, IICB,

Faculty of Biological Sciences, University of

Leeds, Leeds LS2 9JT, UK, 7Tallinn University

of Technology, Centre for Biology of Integrated

Systems, Akadeemia tee 15A, Tallinn 12618,

Estonia, 8BiotaP LLC, Akadeemia tee 15,

Tallinn 12618, Estonia, 9Laboratoire Evolution

Genomes et Speciation, CNRS, 91190 Gif-sur-

Yvette, France, 10Institute of Agricultural and

Environmental Sciences, Estonian University of

Life Sciences, Fr.R. Kreutzwaldi 5, Tartu

51014, Estonia, 11School of Natural Sciences,

Trinity College Dublin, Dublin 2, Ireland,12Department of Animal Ecology I, Working

Group Animal Population Ecology, University

of Bayreuth, 95440 Bayreuth, Germany,13Department of Plant Ecology, University of

Bayreuth, 95440 Bayreuth, Germany

*Correspondence: Mari Moora, Institute of

Ecology and Earth Sciences, University of

Tartu, 40 Lai St., 51005 Tartu, Estonia.

E-mail: [email protected]

ABSTRACT

Aim The biogeography of arbuscular mycorrhizal (AM) fungi is poorly

understood, and consequently the potential of AM fungi to determine plant

distribution has been largely overlooked. We aimed to describe AM fungal

communities associating with a single host-plant species across a wide geographical

area, including the plant’s native, invasive and experimentally introduced ranges.

We hypothesized that an alien AM plant associates primarily with the

geographically widespread generalist AM fungal taxa present in a novel range.

Location Europe, China.

Methods We transplanted the palm Trachycarpus fortunei into nine European

sites where it does not occur as a native species, into one site where it is

naturalized (Switzerland), and into one glasshouse site. We harvested plant roots

after two seasons. In addition, we sampled palms at three sites in the plant’s

native range (China). Roots were subjected to DNA extraction, polymerase chain

reaction (PCR) and 454 sequencing of AM fungal sequences. We analysed fungal

communities with non-metric multidimensional scaling (NMDS) ordination and

cluster analysis and studied the frequency of geographically widespread fungal

taxa with log-linear analysis. We compared fungal communities in the roots of

the palm with those in resident plants at one site in the introduced range

(Estonia) where natural AM fungal communities had previously been studied.

Results We recorded a total of 73 AM fungal taxa. AM fungal communities in

the native and introduced ranges differed from one another, while those in the

invasive range contained taxa present in both other ranges. Geographically

widespread AM fungal taxa were over-represented in palm roots in all regions,

but especially in the introduced range. At the Estonian site, the palm was

colonized by the same community of widespread AM fungal taxa as associate with

resident habitat-generalist plants; by contrast, resident forest-specialist plants

were colonized by a diverse community of widespread and other AM fungal taxa.

Main conclusions AM fungal communities in the native, invasive and

experimentally introduced ranges varied in taxonomic composition and

richness, but they shared a pool of geographically widespread, non-host-specific

taxa that might support the invasion of a generalist alien plant. Our dataset

provides the first geographical overview of AM taxon distributions obtained using

a single host-plant species.

Journal of Biogeography (J. Biogeogr.) (2011) 38, 1305–1317

ª 2011 Blackwell Publishing Ltd http://wileyonlinelibrary.com/journal/jbi 1305doi:10.1111/j.1365-2699.2011.02478.x

INTRODUCTION

Most studies of the distribution and abundance patterns of

species focus on conspicuous macro-organisms, while micro-

bial communities such as those of arbuscular mycorrhizal

(AM) fungi remain relatively understudied (Chaudhary et al.,

2008; Dumbrell et al., 2010). AM fungi, which belong to the

phylum Glomeromycota (Schussler et al., 2001), are obligate

plant-root endosymbionts and associate with more than 80%

of all vascular plant species (Smith & Read, 2008). Through

this mutualism, AM fungi gain all of their carbon from a host

plant while delivering to the plant a range of benefits, including

improved nutrient acquisition. The prevalence and potential

importance of these interactions suggest that AM fungi play a

key role in determining the distribution and abundance of

plant species, and consequently in shaping terrestrial ecosys-

tems (Fitter, 2005). Despite this, the cryptic lifestyle of AM

fungi means that large-scale data on their distribution and

abundance have remained scarce (Fitter, 2005; Opik et al.,

2006, 2010). Consequently, the development of specific

hypotheses regarding the role of AM fungi in influencing

plant distribution and structuring plant communities has been

impeded (Bever et al., 2010).

Our limited understanding of the distribution of AM

fungi has hindered the development of theory concerning the

role played by microbial organisms in shaping plant

invasions. Direct and indirect mechanisms by which plant

and soil microbial community interactions can influence the

invasiveness of alien plant species have only recently been

considered (Mitchell et al., 2006; Reinhart & Callaway, 2006;

van der Putten et al., 2007; Rodrıguez-Echeverrıa, 2010). AM

fungi associate with the majority of plant species, are globally

distributed and are generally believed to exhibit low host

specificity (Smith & Read, 2008). As such, they may appear

unlikely candidates to explain the invasive dominance of

certain alien plants (Richardson et al., 2000). Indeed, using

an experimental approach, Klironomos (2002) found that

local AM fungi generally had a similar effect on native and

invasive plant species. On this basis, it seems reasonable to

conclude that AM fungi do not play a major role in either

facilitating or hindering plant invasions. However, recent

studies have proposed scenarios whereby AM fungi play a

more significant role in plant invasions (Pringle et al., 2009;

Shah et al., 2009). As AM fungal taxa exhibit host-specific

growth responses (Helgason et al., 2002; Bever, 2003) and

invoke differential growth responses in host-plant species

(Klironomos, 2003; Moora et al., 2004a,b), there is some

potential for new combinations of alien species and resident

AM fungi to yield strong mutualistic interactions (Reinhart

& Callaway, 2006). However, to the best of our knowledge, it

has not been demonstrated that alien plants establish new

soil-borne mutualisms that specifically lead to dominance

and the competitive exclusion of native species. Another way

in which invasive plants may influence the composition and

density of resident AM fungal communities is by failing to

promote local AM fungi to the same extent as native species

do (Hoffman & Mitchell, 1986; Mummey & Rillig, 2006;

Hausmann & Hawkes, 2009; Vogelsang & Bever, 2009), or by

inhibiting them via root exudates (Callaway et al., 2008). As

host plants can shape distinctive mycorrhizal communities

even when presented with the same AM fungal inocula

(Uibopuu et al., 2009), invasive plants seem likely to

produce altered AM fungal communities under any of the

scenarios mentioned above (Zhang et al., 2010). Thus, it

seems likely that an invader may contribute to a decrease in

AM fungal density and/or to a change in local AM fungal

diversity, which could detrimentally affect resident plant

communities.

Recent findings show that while the composition of local

AM fungal communities can be very variable in nature

(Dumbrell et al., 2010), some AM fungal taxa are ubiquitous

(Opik et al., 2006, 2010). Moreover, widely distributed AM

fungi tend to associate with a wide range of host plants,

including habitat-generalist plant species, while less common

AM fungi are more likely to associate with host-plant species

that occupy a narrower ecological (Opik et al., 2009) and

correspondingly also geographical (Opik et al., 2010) range. As

successful plant invaders are frequently generalists (e.g. Kuhn

et al., 2004; Kuster et al., 2008), we hypothesize that invasive

AM plants are likely to be generalist hosts that associate

primarily with the widely distributed generalist AM fungal taxa

present in any novel range. In this way, invasive AM plants

might have the potential to accelerate a homogenization of the

biosphere, to the extent that a limited set of AM fungal species

Keywords

Arbuscular mycorrhizal fungi, biotic invasion, China, Europe, forest ecosystem,

fungal diversity, Glomeromycota, host specificity, soil microbial community,

Trachycarpus fortunei.

�Present address: Silje Berger, Norwegian

Coastal Administration, Department for Emer-

gency Response, Moloveien 7, 3187 Horten,

Norway.�Present address: Agnes Rortais, Emerging Risks

Unit, EFSA, Largo N. Palli 5/A, 43100 Parma,

Italy.§Present address: Catrin Westphal, Agroecology,

Department of Crop Science, Georg-August-

University Gottingen, Waldweg 26, 37073 Got-

tingen, Germany.

M. Moora et al.

1306 Journal of Biogeography 38, 1305–1317ª 2011 Blackwell Publishing Ltd

becomes widespread and dominant over several continents, to

the detriment of biodiversity.

In this study we use a high-throughput pyrosequencing

methodology – 454 SequencingTM (454 Life Sciences, Bran-

ford, CT, USA) of the small subunit ribosomal RNA (SSU

rRNA) gene – to describe AM fungal communities associated

with the roots of an alien AM plant species, Trachycarpus

fortunei (Hook.) Wendl. (Arecaceae). Originating from China

and introduced into Europe as an ornamental species, this

palm has successfully colonized deciduous forests and has

established a vigorous population in the southern foothills of

the Alps (Walther, 2003).

First, we use experimentally introduced T. fortunei plants as

‘bait plants’ (sensu Opik et al., 2003; Sykorova et al., 2007) to

characterize AM fungal communities over a wide geographical

area outside the native range of the host plant (nine sites in

seven European countries, Walther & Berger, 2010). This could

indicate whether native AM fungi associate with the alien host

plant and might therefore be considered as potential mediators

of the invasion process.

Second, applying a biogeographical approach (Hierro et al.,

2005), we compare the AM fungal communities associated

with T. fortunei in its experimentally introduced range with

those in its native range in both China and Switzerland in

order to determine whether AM fungal community patterns in

the respective ranges might be correlated with the invasive

success of T. fortunei. In particular, we focus on the

geographical range of AM fungal taxa. We predict that the

AM fungi partnering the alien palm should predominantly be

those with a known wide geographical range.

Third, we compare the AM fungal communities in the roots

of the experimentally introduced host with those in resident

plant species at one of the sites in the introduced range

(Estonia) where AM fungal communities associated with

different host-plant ecological groups (habitat-specialist and

habitat-generalist plant species) have previously been described

(Opik et al., 2009). We predict that the AM fungi partnering

the alien palm should also be locally present in the roots of a

wide range of hosts.

MATERIALS AND METHODS

Target plant species

Trachycarpus fortunei occurs naturally in Southeast Asia, but

the species is grown ornamentally in many temperate and

subtropical regions outside its native range (Walther et al.,

2007). Regeneration from cultivated palms has led to the

establishment of naturalized T. fortunei populations in the

southern foothills of the Alps (Walther, 2003). The rapid

expansion of T. fortunei into the semi-natural forests of

Central Europe has been driven by changes in winter

temperature and growing season length, and seems likely to

continue as the climate warms further (Walther et al., 2007).

Trachycarpus fortunei forms associations with AM fungi,

making it a suitable model for investigating the presence and

composition of AM fungal communities colonizing invasive

plant roots. Nomenclature of vascular plants follows Flora

Europaea (Tutin et al., 2001).

Sowing experiment

Experimental introduction of T. fortunei seeds was performed

at nine sites, located in several biogeographical subregions

across Europe, where the palm does not already occur as a

native. The sites constituted part of the Field Site Network

(Table 1, Hammen et al., 2010), which was established within

the framework of the European FP6 project ALARM (Settele

et al., 2005). A further sowing experiment was performed near

Locarno, Switzerland, where T. fortunei has been naturalized

since the 1970s (Walther et al., 2007). To provide a reference

against which to compare the germination and seedling

establishment success of the study plant in field conditions,

seeds were also sown in a greenhouse at the botanical gardens,

University of Hannover, Germany, using regular compost soil.

All the experimental sites and native range sampling sites

used in this study were located in forest vegetation, and all on

mesic soil with gleic texture. We thus expect that geographically

driven variation in AM fungal communities between sites is

considerably larger than variation resulting from local environ-

mental conditions such as soil nutrients and moisture content.

Seeds were sown following the same protocol at all sites.

Within each site, three 0.5 · 0.5 m plots (5–30 m apart, except

for the Serbian site, where the maximum distance between

plots was 300 m) were established in an area of woodland

(canopy cover c. 50%). In order to facilitate the germination

and establishment of T. fortunei and to avoid a competitive

effect from resident plants, soil was removed from each plot to

a depth of 15 cm; large roots, stones and other coarse materials

were removed, and soil returned to half of the plot, to one side

of the diagonal. The other half was filled with locally available

sterile commercial potting soil to account for potential edaphic

variation across sites. Seeds of T. fortunei used at all sites were

collected from two palm individuals in Locarno (Switzerland)

in February 2006. Twenty-five seeds were sown on each soil

type (50 seeds per plot) at the beginning of the 2006 growing

season in the majority of experimental sites, including the

greenhouse. In Austria and Serbia, the experiment started in

the spring of 2007. All plants were harvested in the autumn of

2007. Seedlings had 1–3 leaves at the time of harvesting.

Root sampling

The entire root systems of up to 12 experimental plants (six

each from natural and potting soil) were collected from each

plot, depending on the number of plants surviving at the end

of the experiment. Final sample sizes varied between 4 and 34

root samples per site (Table 1). In the cases of Austria and

Germany3 (Barterode), only plants from potting soil were

available at the end of the experiment. Samples from China

were collected in October 2008 from three localities where the

target plant grows naturally (Table 1). At each locality, 10

Invasive plants associate with ubiquitous arbuscular mychorrhizal fungi

Journal of Biogeography 38, 1305–1317 1307ª 2011 Blackwell Publishing Ltd

randomly selected T. fortunei seedlings were harvested, and

c. 4 g of root mass was collected per individual. Root sampling

and storage protocol followed Opik et al. (2008). All root

samples were sent to the University of Tartu (Estonia) for

DNA extraction.

Molecular identification of AM fungi

Molecular analyses

A 20-cm subsample of the root system of each plant was used

for DNA extraction, polymerase chain reaction (PCR) and 454

sequencing, as in Opik et al. (2009). We pooled the extracted

DNA samples from individual plants (using an equal volume

from each individual) for each substrate type and site

combination (i.e. natural and potting soil samples were pooled

separately at each site where samples from both substrates were

available). The resulting 22 sample mixes were subjected to

amplicon isolation and 454 sequencing.

Glomeromycota sequences were amplified from the DNA

mixtures using the SSU rRNA gene primers NS31 and AM1

linked to sequencing primers A and B, respectively. Most data

concerning the natural diversity of AM fungi have been

obtained using this molecular marker (Opik et al., 2010) and

thus can be used for purposes of comparison. The NS31/AM1

primer pair amplifies a c. 550-bp central fragment of SSU

rDNA in most Glomeromycota, but excludes the basal families

Archaeosporaceae and Paraglomaceae (Helgason et al., 1998;

Daniell et al., 2001). In order to identify sequences originating

from different samples, we used a set of 6-bp barcodes

designed following Parameswaran et al. (2007). The barcode

sequences were inserted between the A primer and NS31

primer sequences. Thus, the composite forward primer was 5¢GCCTCCCTCGCGCCATCAG (NNNNNN) TTGGAGGGCA

AGTCTGGTGCC 3¢ and the reverse primer 5¢ GCCTTGC

CAGCCCGCTCAGGTTTCCCGTAAGGCGCCGAA 3¢, where

the A and B primers are underlined, the barcode is indicated by

Ns in parentheses, and the specific primers NS31 and AM1 are

shown in italics (Opik et al., 2009). Sequences that were

included in further analyses had to meet a length criterion of

‡ 160 bp including barcode and primer A sequences, but

otherwise PCRs and quality control of 454 sequencing reads

were performed as in Opik et al. (2009).

Bioinformatic analyses

In order to facilitate the taxonomic assignment of obtained

sequences, we upgraded a pre-existing database of published

Glomeromycota SSU rRNA gene sequences (Opik et al., 2006).

This database (MaarjAM) (Opik et al., 2010) contains repre-

sentative NS31/AM1 sequences from published Glomeromy-

cota sequence-based taxa and known morphospecies. As of 6

February 2010, MaarjAM contained a total of 2044 records that

could be associated with SSU sequence-based taxa (referred to

as ‘virtual taxa’ or VT) (cf. Opik et al., 2009), including 1607

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sequence-based taxa were generated following automatic

sequence alignment using the MAFFT multiple sequence

alignment web service implemented in JalView 2.4 (Clamp

et al., 2004) and neighbour-joining analysis of all MaarjAM

sequences (Milne et al., 2004). A total of 291 virtual taxa were

defined on the basis of bootstrap support and sequence

similarity of ‡ 97%. These criteria produced groupings with

sequence variability similar to those used by several previous

authors (e.g. Helgason et al., 1998; Opik et al., 2008). Follow-

ing the removal of identical sequences, 1210 sequences were

retained in a non-redundant sequence dataset for use in blast

searches (see below).

The 454 sequencing reads were used to query the non-

redundant set of Glomeromycota SSU rRNA gene sequences

from the MaarjAM database using the blast algorithm,

following the same criteria as in Opik et al. (2009). The

virtual taxa that were detected as blast matches are indicated

in Fig. 1 and Appendix S1 in the Supporting Information,

following the virtual taxon classification from Opik et al.

(2009). A sample of sequencing reads including examples from

each encountered AM fungal virtual taxon has been deposited

in GenBank under accession numbers GU198530–GU198746

(217 unique sequences in total; £ 3 sequences per virtual

taxon). An additional blast search against the GenBank non-

redundant nucleotide database was used to detect non-

Glomeromycota sequences in our dataset. All blast search

results were parsed using the ‘tcl blast parser version 038’

(Kozik et al., 2003).

Statistical analyses

Our field observations suggested that the small plots

(0.125 m2) containing potting soil were efficiently colonized

by the roots of local understorey plants during the timeframe

of the experiment. Furthermore, the AM fungal communities

that colonized palm roots in potting soil did not differ from

those in adjacent natural soil plots in terms of species richness

(ANOVA F1,14 = 0.61, P = 0.45) or, even more importantly, in

composition permutational multivariate analysis of variance

[PerMANOVA (Anderson, 2001) F1,12 = 0.18, P = 0.99]. For

these reasons, we pooled the data coming from the two

substrate types at each of the respective eight sites (excluding

CN, DE 1, DE 3 and AT), resulting in 14 sites prior to further

analysis (Table 1).

Similarity between the AM fungal communities present at

the various sites was analysed using cluster analysis and non-

metric multidimensional scaling (NMDS) based on the

presence or absence of fungal virtual taxa (McCune & Grace,

2002). Previously published data on the AM fungal commu-

nities inhabiting native plant species at the Estonian study site

(a 10 · 10 m plot known as plot Z sensu Opik et al., 2008,

2009) allowed us to compare the native fungal community

with that associated with T. fortunei at the same site using

cluster analysis. These analyses and PerMANOVA were

performed using pc-ord for Windows version 5 (MjM

Software, Gleneden Beach, OR, USA).

Several statistical methods are available to estimate the

asymptotic number of taxa present at a site, including

undetected taxa (Chao et al., 2009). For each site we used

Coleman rarefaction analysis to produce AM fungal taxon

accumulation curves and calculated Chao1, jackknife and

bootstrap asymptotic richness estimators using 50 randomiza-

tions without replacement (in EstimateS 8.0.0; Colwell,

2006). All estimators gave very similar results, so we present

only the values of the Chao1 estimator (Table 1).

For the focal regions of Europe and Asia we calculated the

expected number of fungal virtual taxa falling into three

categories of biogeographical distribution using the corre-

sponding proportions of accessions in the MaarjAM data-

base: taxa previously (1) found only in the focal region; (2)

found in the focal region and elsewhere; and (3) not found

in the focal region (Fig. 2 bar A shows these categories for

Europe; Fig. 2 bar D shows these categories for Asia). Chi-

square tests were used to compare these expected values with

the observed number of taxa belonging to each of the three

distribution categories: the introduced range (compared with

the expected number of taxa for Europe); the invasive range

(compared with the expected number of taxa for Europe);

and China (compared with the expected number of taxa for

Asia). Log-linear analyses were performed to test whether the

frequencies of fungal virtual taxa with different known global

distributions differed among the AM fungal communities

colonizing the study plant in the European localities (11

sites, including Switzerland) and in China (three sites).

RESULTS

AM fungal communities in palm roots

The 454 sequencing analysis of the 14 composite samples

(Table 1) yielded a total of 109,884 sequences that had a

length ‡ 160 bp (max 295 bp, median 245 bp) and displayed

the correct tag and primer sequences. A blast search against

the GenBank non-redundant database indicated the presence

of non-Glomeromycotan sequences, with hits to sequences of

fungal taxa in the Ascomycetes and Basidiomycetes in most

cases, and to a lesser extent also to those of other fungi or

plants (data not shown). The putatively non-Glomeromyco-

tan sequences had 23,318 sequence reads (i.e. 21.2% of the

total).

The total number of known Glomeromycota sequences

among the 454 sequencing reads was 65,001 (i.e. 59.2% of all

sequences; ranging between 21% and 87% for each sample;

Table 1). These sequences were assigned to 73 virtual taxa

from the MaarjAM database (see Appendix S1). This number

excludes nine taxa that were represented by a single sequence

and were removed from further analysis. The 73 virtual taxa

belonged to Glomeraceae (i.e. Glomus group A, 55), Acaulos-

poraceae (8), Gigasporaceae (4), Diversisporaceae (3) and

Glomus group B (3) (Fig. 1). Fungal community composition

in the roots of study plants at the various localities is provided

in Fig. 1 and Appendix S1. Two fungal taxa were detected at all



study sites: Glomus sp. (VT 113, related to G. fasciculatum) and

Glomus sp. (VT 115, related to G. vesiculiferum).

Rarefaction analysis suggested that sampling intensity was

sufficient, as fungal taxon accumulation curves reached an

asymptote in most cases (Fig. 3). Richness estimator analysis

showed that the estimated AM fungal taxon richness was

similar to the observed richness for most sites, again indicating

sufficient sampling effort (Table 1). However, the expected

MaarjAM virtual

2EDSRONRF1EDEIHC2NC3NC1NCseicepSpuorG/suneGecnerruccOnoxat AT DE3 SE EEVT 200 EU,AM Glomeraceae Glomus hoiVT 129 EU,AM,AF GlomeraceaeVT 62 EU,AS,AM,AF DiversisporaceaeVT 33 EU AcaulosporaceaeVT 135 EU GlomeraceaeVT 162 EU GlomeraceaeVT 186 EU GlomeraceaeVT 193 EU,AS Glomus group B G. claroideum, lamellosum, luteum, etunicatumVT 56 EU,AM,AF Glomus group BVT 163 EU,AM GlomeraceaeVT 72 EU,AM GlomeraceaeVT 196 EU GlomeraceaeVT 34 AM Acaulosporaceae Acaulospora scrobiculataVT 26 EU AcaulosporaceaeVT 151 AM GlomeraceaeVT 212 AM GlomeraceaeVT 214 EU,AM GlomeraceaeVT 198 EU GlomeraceaeVT 152 AM GlomeraceaeVT 143 EU,AM,AF GlomeraceaeVT 160 EU,AM GlomeraceaeVT 140 EU,AM GlomeraceaeVT 64 EU,AS,AM,AF GlomeraceaeVT 191 EU,AS,AM,AF GlomeraceaeVT 74 EU,AM,OC GlomeraceaeVT 108 EU,AS,AM GlomeraceaeVT 37 EU AcaulosporaceaeVT 199 EU,AS,AM GlomeraceaeVT 145 EU,AM GlomeraceaeVT 115 EU,AS,AM Glomeraceae G. vesiculiferumVT 113 EU,AS,AM Glomeraceae G. fasciculatumVT 49 EU,AS,AM Gigasporaceae Scutellospora dipurpurescensVT 60 EU,AS,AF DiversisporaceaeVT 166 EU,AS,AM,AF,OC GlomeraceaeVT 114 EU,AM Glomeraceae G. intraradicesVT 125 EU,AM GlomeraceaeVT 219 EU,AS,AM,AF GlomeraceaeVT 57 EU,AS,AM Glomus group BVT 187 EU,AM GlomeraceaeVT 275 EU,AM GlomeraceaeVT 52 EU,AM Gigasporaceae S. auriglobaVT 273 EU,AM GlomeraceaeVT 247 EU,AS,AM,AF GlomeraceaeVT 194 EU,AS,AM GlomeraceaeVT 156 EU,AS,AM GlomeraceaeVT 201 AM AcaulosporaceaeVT 89 EU GlomeraceaeVT 259 AS GlomeraceaeVT 224 AS GlomeraceaeVT 24 AM AcaulosporaceaeVT 69 EU,AM,AF GlomeraceaeVT 95 EU,AM,AF GlomeraceaeVT 84 EU,AS,AM GlomeraceaeVT 80 AS GlomeraceaeVT 181 AF GlomeraceaeVT 222 EU,AS,AM GlomeraceaeVT 90 AS Glomeraceae G. manihotisVT 264 CU Glomeraceae G. clarumVT 130 EU,AS,AF GlomeraceaeVT 45 AM,AF AcaulosporaceaeVT 188 EU GlomeraceaeVT 270 AF GlomeraceaeVT 267 AM GlomeraceaeVT 180 AF GlomeraceaeVT 53 AM GlomeraceaeVT 211 AF GlomeraceaeVT 227 AF AcaulosporaceaeVT 124 AM GlomeraceaeVT 39 AS,AM GigasporaceaeVT 122 EU,AM GlomeraceaeVT 255 AS,AM Gigasporaceae S. cerradensis, reticulata, heterogama, dipapillosaVT 54 EU DiversisporaceaeVT 216 EU Glomeraceae

Figure 1 Occurrence of Glomeromycota in the native (CN, grey), invasive (CH, chequer) and introduced (black; see codes in Table 1)

ranges of Trachycarpus fortunei. The ‘Occurrence’ column denotes the previously known range of each virtual taxon: Europe (EU), Asia

(AS), America (AM), Africa (AF), Oceania (OC), spore culture without known geographical origin (CU). Each row corresponds to a

different fungal taxon, with filled cells indicating its presence and open cells its absence at a given site. The table is ordered following the non-

metric multidimensional scaling (NMDS) ordination whereby sites are ranked according to their score on the first ordination axis. The final

NMDS solution had three dimensions, and stress was 9.33, which was significantly lower than for randomized data (Monte Carlo test,

P < 0.02, 250 runs). Collectively, the axes explained 88.7% of the variation in the data, with axes 1 and 2 explaining 35.1% and 28.6%,

respectively.

M. Moora et al.


number of taxa was somewhat higher than observed in Ireland

(12%), Switzerland (13%), China1 (17%) and China2 (28%).

Comparison of native, invasive and experimentally

introduced ranges

Forty-nine AM fungal taxa were recorded from T. fortunei in

its native range in China, 20 taxa in the invasive range in

Switzerland and 46 taxa at the experimentally introduced sites

across Europe. Ten fungal taxa were common to all three

regions, 21 were specific to the native range in China, two were

specific to the invasive range in Switzerland, and 18 were

specific to the introduced range in Europe. Fifty-two taxa were

recorded from Europe (including Switzerland) altogether

(Fig. 1, Appendix S2). Palms grown in the invasive range in

Switzerland and those occurring in the native Chinese range

shared four fungal taxa, none of which occurred elsewhere in

Europe (Fig. 1). Fungal taxon richness at European study sites

was variable, ranging from seven taxa in Estonia to 35 taxa in

Serbia (Table 1, Fig. 1). Most study sites in Europe shared a

common dominant taxon – VT 113 (highest number of

detected sequences per site, Appendix S1). The dominant

taxon in Sweden was VT 219, while that in the greenhouse trial

(DE1) was VT 199. In both sites VT 113 was the second most

abundant taxon. All Chinese sites were dominated by different

AM fungal taxa: VT 166 in CN1, VT 219 in CN2 and VT 130 in

CN3. VT 130 was only recorded in CN3 during this study. The

European dominant VT 113 was present at all Chinese sites,

but was the third most abundant taxon in CN1 and CN3 and

even less abundant in CN2 (Fig. 1, Appendix S1).

0%

20%

40%

60%

80%

100%

Expected Europe Observed EU Observed CH Expected Asia Observed CN

Pro

port

ion

of

AM

fungalta

xa

A DCB E

Figure 2 Comparison of the geographical distribution of Glomeromycota taxa recorded in the introduced (bar B), invasive (bar C)

and native (bar E) ranges of Trachycarpus fortunei with expected values for Europe (bar A) and Asia (bar D) based on accessions in the

MaarjAM database. The proportion of taxa falling into different categories of geographical range are presented in the bars: white – taxa

recorded only in Europe (for bars A, B, C) or only in Asia (for bars D, E); grey – taxa recorded in Europe and elsewhere (A, B, C) or recorded

in Asia and elsewhere (D, E); black – taxa absent in Europe (A, B, C) or absent in Asia (D, E).

0

5

10

15

20

25

30

35

40

1 251 501 751 1001 1251 1501 1751 2001 2251 2501 2751 3001 3251 3501 3751 4001 4251 4501 4751

No. of sequences

No.

of A

MF

taxa

RS

DE2

CN1CN2

CN3EIES

CH

NO

FR

AT

EE

DE3

DE1

Figure 3 Expected arbuscular mycorrhizal

fungal (AMF) taxon accumulation curves

(Coleman rarefaction) along the number of

sequences obtained from the various study

sites in Europe and China. Grey lines indicate

sites from the native range of Trachycarpus

fortunei. Curves are presented up to a maxi-

mum of 5000 sequences. See Table 1 for site

codes and total numbers of sequences.



Cluster analysis of AM fungal communities revealed two

clear groups: fungal communities associating with palm roots

at the European introduced sites on one hand, and those

associating with the roots of plants from native and invasive

ranges on the other (Fig. 4a).

Occurrence of widespread and regionally specific AM

fungal species in palm roots

Palm roots from the native, invasive and introduced ranges all

hosted significantly more geographically widespread (occur-

ring in two or more continents) AM fungal taxa than expected

based on the occurrence of such taxa in regional (Europe or

Asia) and global species pools. The difference was more

pronounced among European (introduced range v2 = 65.42,

d.f. = 2, P < 0.001; invasive range v2 = 25.88, d.f. = 2,

P < 0.001) than among Chinese (v2 = 29.37, d.f. = 2,

P < 0.001) plants (Fig. 2). There were no site-specific differ-

ences in the occurrence of fungal taxa corresponding to the

three biogeographical distribution categories of AM fungi

among the 11 European sites (including introduced and

invasive range; log-linear analysis site · category interaction,

v2 = 14.18, d.f. = 20, P = 0.8) or among the three Chinese

sites (log-linear analysis site · category interaction, v2 = 0.84,

d.f. = 4, P = 0.66). Eight AM fungal taxa were detected for the

first time from Europe, and 27 taxa from Asia in the current

study.

Differences between resident and palm AM fungal

communities

Detailed analysis of a single experimental introduction site

(Koeru, Estonia) revealed that the AM fungal community

associated with the introduced alien plant species T. fortunei

was similar to that associated with habitat-generalist native

plant species (Fig. 4b). In common with these species,

T. fortunei hosted relatively few AM fungal taxa (7), which

also tended to be the most common fungal taxa inhabiting

the roots of indigenous host plants (data from Opik et al.,

2009).

DISCUSSION

Variation in the AM fungal communities associated

with T. fortunei

We recorded a total of 73 AM fungal taxa from 14 study sites.

Because fungal taxon accumulation curves reached an asymp-

tote in most cases, our dataset can be considered as the first

representative Eurasian overview of AM taxon distributions for

a single host-plant species. Although recent work has provided

important insights into variation in AM fungal communities

(Opik et al., 2006; Dumbrell et al., 2010), understanding of

general patterns has clearly been hampered by limited

sampling (Fitter, 2005). Our results reveal considerable

Distance (Objective Function)

Information Remaining (%)

2.2 x 10-2

100

3.4 x 10-1

75

6.6 x 10-1

50

9.9 x 10-1

25

1.3

0

EE

AT

FR

DE3

NO

SE

DE1

DE2

RS

IE

CH

CN1

CN2

CN3

Origin of samples

Europe China Switzerland

Distance (Objective Function)

Information Remaining (%)

1.4 x 10-2

100

1.7 x 10-1

75

3.3 x 10-1

50

4.8 x 10-1

25

6.4 x 10-1

0

Fra ves

Hyp mac

Gal lut

Par qua

Hep nob

Oxa ace

Vio mir

Ger pra

Ver cha

Geu riv

Tra for

Species

habitat generalist forest specialist alien

(b)

(a)

Figure 4 Cluster analysis of arbuscular

mycorrhizal fungal (AMF) community com-

position at study sites in (a) the native,

introduced and invasive ranges of the palm

Trachycarpus fortunei and (b) AM fungal

communities associated with different cate-

gories of plant species at Koeru, Estonia:

habitat generalists, forest specialists and an

introduced species Trachycarpus fortunei (Tra

for). Sørensen index and a group-averaging

linkage method were used. Habitat-generalist

plant species: Fragaria vesca (Fra ves),

Hypericum maculatum (Hyp mac), Geum

rivale (Geu riv), Geranium pratense (Ger pra)

and Veronica chamaedrys (Ver cha). Forest-

specialist species: Galeobdolon luteum [syn.

Lamiastrum galeobdolon, (Gal lut)], Paris

quadrifolia (Par qua), Hepatica nobilis (Hep

nob), Oxalis acetosella (Oxa ace) and Viola

mirabilis (Vio mir).

M. Moora et al.


variation between AM fungal communities in the experimen-

tally introduced European sites, all of which were located in

wooded habitats. The lowest number of AM fungal taxa was

recorded in the roots of palms introduced into sites charac-

terized by coniferous forest and a cold climate (Estonia,

Norway, Austria; Hammen et al., 2010). In contrast to the low

number of AM fungal taxa (7) that we recorded at the Estonian

site, an earlier study recorded 47 taxa for the same site (Opik

et al., 2009). The fact that T. fortunei does not associate with a

greater number of the fungal species present at this site may be

a consequence of the unfavourable environmental conditions.

By contrast, the high diversity of AM fungi recorded at the

Serbian site might in part be attributable to the larger spatial

extent of this site. The Chinese sites also contained a high

diversity of AM fungi, comparable with those of the European

sites situated in broadleaf forest.

Our finding that c. 17% of fungal taxa found in European

samples and c. 57% of those in Chinese samples had never

previously been recorded in those respective regions reflects

the present state of knowledge about the global distribution of

AM fungal taxa. More large-scale studies incorporating

multiple continents, biomes, ecosystems and host-plant species

are required in order to build an understanding of the

biogeography of AM fungi (Fitter, 2005; Chaudhary et al.,

2008; Pringle et al., 2009; Dumbrell et al., 2010; Opik et al.,

2010).

Comparison of AM fungal communities in the novel

and native ranges

The role of AM fungi in plant invasions has been largely

overlooked (Levine et al., 2004; Mitchell et al., 2006), and

information on associations between invasive plant species and

AM fungi is extremely limited (Pringle et al., 2009; Shah et al.,

2009). Our study showed that AM fungal communities in the

palm’s native Chinese range and introduced European ranges

differed, while the composition of communities in the invasive

Swiss range (although represented by only one study site)

contained taxa from both regions. These results lend support

to the argument that successful plant invaders are not limited

by a lack of mutualistic fungi (Richardson et al., 2000); in our

study, the invader seems to have replaced ‘lost’ mutualists

from its native range with new mutualists in the introduced

and invasive ranges.

When the palm–AM fungal associations in the native,

invasive and introduced ranges were compared in greater

detail, a slightly more complex picture emerged. First, AM

fungal communities from the invasive range in Switzerland

contained four fungal taxa that also occurred in the host’s

native range in China, but not in any of the European

introduced sites. One of these fungal taxa was previously

known only from Central America (Glomus VT 124), while

the other three have been found to have a wider distribution

(Gigaspora VT 39, Scutellospora VT 255 and Glomus VT 122)

(Opik et al., 2010). Limited replication of invaded sampling

sites means that we cannot reliably determine whether this

pattern indicates that T. fortunei favours AM fungal taxa that

have been or are currently common in its native range, but

this topic deserves future attention. AM fungal communities

in the invasive range were also notable for the presence of

two AM fungal taxa, namely Glomus VT 216 and Otospora

VT 54, that were not found at any of the other sites in this

study but which have previously been described only from

Europe. Future study would be needed to determine whether

these taxa are specific and efficient symbionts yielding strong

mutualistic interactions that enhanced the successful estab-

lishment of T. fortunei in its invasive range, in accordance

with Reinhart & Callaway’s (2006) ‘enhanced mutualism

hypothesis’.

When interpreting these results it is important to keep in

mind that, whilst we sampled experimentally grown seedlings

in artificially created gaps in the introduced and invasive

ranges, we sampled naturally growing plants in the native

range. Although disturbed ecosystems have frequently been

characterized by a low diversity of AM fungi (Helgason et al.,

1998; Daniell et al., 2001; Antunes et al., 2009), we propose

that the small-scale soil disturbance applied to facilitate target

plant germination in otherwise intact vegetation did not

significantly decrease the inoculation potential of the resident

AM fungal communities. Root colonization in small gaps from

surrounding intact vegetation can be relatively rapid (Partel &

Wilson, 2002), and the small plots created in the current study

were colonized by the roots of neighbouring plants, along with

their AM fungal symbionts, in as short a time as several

months. With the exception of the Austrian and Serbian sites,

plants were grown for two seasons.

The comparison of AM fungal communities from identically

treated experimental plants over a wide area largely eliminates

any potential host and treatment biases. The data obtained

suggest that the patterns detected in this study were not biased

by the experimental protocol. First, the AM fungal taxon

richness associated with naturally growing Chinese seedlings

and that with experimentally grown seedlings at European

broadleaf forest sites were similar. Second, while there was

considerable overlap (28 taxa) between AM fungal communi-

ties associated with experimentally and naturally grown

seedlings, experimental seedlings in the introduced and

invasive ranges harboured distinct AM fungal communities

despite identical treatment.

Occurrence of widespread versus regional AM fungal

taxa

By studying T. fortunei in its native, invasive and experimen-

tally introduced ranges, we found that this plant species is

capable of hosting a wide range of AM fungal taxa, which is

also the case with foliicolous microfungi (Taylor et al., 2000).

The taxa with previously described distributions spanning

several continents were over-represented in the roots of

T. fortunei in all ranges (native, invasive, experimentally

introduced), but especially in the European introduced sites.

Interestingly, most study sites in Europe shared the same local



dominant taxon: globally distributed Glomus sp. (VT 113,

related to G. fasciculatum); different dominant taxa were

recorded only in the artificial conditions (compost soil) of the

greenhouse study and in Sweden. Meanwhile, each site in the

native range was characterized by a different dominant taxon,

although all were geographically widespread. The results from

the European sites conflict with the findings of Dumbrell et al.

(2010), who found that AM fungal communities exhibited a

strong overdominance of particular AM fungal taxa, but that

the dominant taxon in almost all analysed AM fungal

communities was different. They proposed that the processes

determining the dominance of a particular AM fungal taxon

may be stochastic or determined by local adaptation to soil

chemistry and host-plant communities. On the basis of our

data, it appears that the alien palm might have been able to

reshape the local AM fungal communities when any wide-

spread and otherwise appropriate AM fungal partners hap-

pened to be present. In addition, repeated association with the

same dominant taxon may also indicate that local edaphic

conditions were relatively similar between experimental sites.

By contrast, our finding that each native population of

T. fortunei in China was associated with a different dominant

AM fungal taxon matched Dumbrell et al.’s (2010) findings.

Perhaps the most likely reason for the different patterns

observed in our European data and by Dumbrell et al. (2010)

is the different nature of the sampling. We targeted a single,

experimentally introduced host-plant species in relatively

similar (wooded) habitats and followed the same analytical

protocol at all sites; in contrast, Dumbrell et al. (2010)

analysed data from multiple studies, representing different

ecosystems and host-plant species. Moreover, it is possible that

our experimental approach favoured the proliferation of VT

113, which is related to the widespread G. fasciculatum because

it has been shown that genotypes of this species and related

species (i.e. G. intraradices) are able to colonize a bait plant

rapidly and that this colonization is maintained through time

(Sykorova et al., 2007). However, their abundance is also high

in the roots of natural plants from non-disturbed sites (Opik

et al., 2003, 2008, 2009; Sykorova et al., 2007).

The pattern of association between alien host plant and

particular widespread AM fungal taxa was further reflected in

our observations at the Estonian study site, where extensive

information on the resident AM fungal community has

recently been collected (Opik et al., 2008, 2009). The roots

of the alien palm were primarily colonized by widespread AM

fungal taxa that were also common in the roots of local

habitat-generalist plants, while habitat-specialist plant species

harboured a variety of more specialized taxa in addition to the

widespread AM fungi (Opik et al., 2009). This pattern might

be explained by two possible mechanisms: either generalist

fungi are capable of rapidly colonizing the roots of any new

plant species, or generalist plant species inhabiting a wide

range of habitats favour the proliferation of generalist AM

fungal taxa. If a generalist alien plant allows generalist fungi to

proliferate and suppress more localized or specific fungal taxa,

an alien host plant may indirectly depress the diversity of local

AM fungal communities. Indeed, there is some descriptive

evidence that AM fungal communities in invaded sites differ

from those in adjacent non-invaded sites (Mummey & Rillig,

2006), but the existence of such a mechanism should ideally be

tested using experimental methods (Zhang et al., 2010). Our

ability to manipulate AM fungal communities is, however, still

limited because the majority of these fungal taxa are not

cultivable, and we are currently unable to selectively eliminate

particular non-sporulating AM fungi from natural soils.

CONCLUSIONS

Information about the natural distribution patterns of AM

fungi remains scarce. Our study, using massively parallel 454

sequencing, is the first large-scale survey of natural AM

fungal communities associated with a single host-plant

species in its native, invasive and experimentally introduced

ranges. During the timeframe of this study, the introduced

palm sprouted successfully and was not limited by a lack of

AM fungal symbionts colonizing its roots. The results are in

accordance with our general hypothesis that alien plants,

which are themselves typically generalists, predominantly find

their AM fungal partners among the widely distributed

generalist AM fungal taxa present in a novel habitat. In

particular, AM fungi partnering the alien palm were

predominantly those that had a wide geographical range

and were locally present in the roots of a wide range of hosts.

However, more large-scale studies crossing continents, bio-

mes, ecosystems and host-plant species are required in order

to describe the biogeography of AM fungi adequately. Such

knowledge is essential for understanding the role of soil-

borne symbionts such as AM fungi in plant distribution and

invasion processes. With new-generation sequencing technol-

ogies becoming increasingly available, the practical barriers to

such endeavours are rapidly disappearing.

ACKNOWLEDGEMENTS

This research was funded by the European Commission within

the FP 6 Integrated Project ALARM ‘Assessing LArge scale

environmental Risks for biodiversity with tested Methods’

(GOCE-CT-2003-506675), by the Estonian Science Founda-

tion grants 7371, 7366, 7738, SF0180098s08 (University of

Tartu), an Enterprise Estonia grant EU 27552 (BiotaP LLC), a

Marie Curie European Reintegration Grant within the 7th

European Community Framework Programme (GLOBAM,

PERG03-GA-2008-231034) and by the European Regional

Development Fund (Centre of Excellence FIBIR). M. Metsis

was partially supported by a grant from Tallinn University of

Technology. Bioinformatics analysis was supported by the

BiotaP LLC. Sampling in China by H.B. was financed by BEF-

China, funded by the German Science Foundation (DFG FOR

891/1). H.B. is indebted to the staff of the Gutianshan National

Nature Reserve. We are thankful to the ALARM local field-site

staff for conducting and harvesting the transplantation exper-

iment.

M. Moora et al.


REFERENCES

Anderson, M.J. (2001) A new method for non-parametric

multivariate analysis of variance. Austral Ecology, 26, 32–46.

Antunes, P.M., Koch, A.M., Dunfield, K.E., Hart, M.M.,

Downing, A., Rillig, M.C. & Klironomos, J.N. (2009)

Influence of commercial inoculation with Glomus intrara-

dices on the structure and functioning of an AM fungal

community from an agricultural site. Plant and Soil, 317,

257–266.

Bever, J.D. (2003) Soil community feedback and the coexis-

tence of competitors: conceptual frameworks and empirical

tests. New Phytologist, 157, 465–473.

Bever, J.D., Dickie, I.A., Facelli, E., Facelli, J.M., Klironomos, J.,

Moora, M., Rillig, M.C., Stock, W.D., Tibbett, M. & Zobel,

M. (2010) Rooting theories of plant community ecology in

microbial interactions. Trends in Ecology and Evolution, 25,

468–478.

Callaway, R.M., Cipollini, D., Barto, K., Thelen, G.C., Hallett,

S.G., Prati, D., Stinson, K. & Klironomos, J. (2008) Novel

weapons: invasive plant suppresses fungal mutualists in

America but not in its native Europe. Ecology, 89, 1043–

1055.

Chao, A., Colwell, R.K., Lin, C.W. & Gotelli, N.J. (2009) Suf-

ficient sampling for asymptotic minimum species richness

estimators. Ecology, 90, 1125–1133.

Chaudhary, V.B., Lau, M.K. & Johnson, N.C. (2008) Mac-

roecology of microbes – biogeography of the Glomeromy-

cota. Mycorrhiza (ed. by A. Varma), pp. 529–562. Springer,

Berlin.

Clamp, M., Cuff, J., Searle, S.M. & Barton, G.J. (2004) The

Jalview Java alignment editor. Bioinformatics, 20, 426–427.

Colwell, R.K. (2006) EstimateS: statistical estimation of species

richness and shared species from samples. Version 8.0. User’s

guide and application. Available at: http://viceroy.eeb.

uconn.edu/estimates.

Daniell, T.J., Husband, R., Fitter, A.H. & Young, J.P.W. (2001)

Molecular diversity of arbuscular mycorrhizal fungi colon-

ising arable crops. FEMS Microbiology Ecology, 36, 203–209.

Dumbrell, A.J., Nelson, M., Helgason, T., Dytham, C. & Fitter,

A.H. (2010) Idiosyncrasy and overdominance in the struc-

ture of natural communities of arbuscular mycorrhizal

fungi: is there a role for stochastic processes? Journal of

Ecology, 98, 419–428.

Fitter, A.H. (2005) Darkness visible: reflections on under-

ground ecology. Journal of Ecology, 93, 231–243.

Hammen, V.C., Biesmeier, J.C., Bommarco, R. et al. (2010)

Establishment of a cross-European field site network in the

ALARM project for assessing large-scale changes in biodi-

versity. Environmental Monitoring Assessment, 164, 337–348.

Hausmann, N.T. & Hawkes, C.V. (2009) Plant neighborhood

control of arbuscular mycorrhizal community composition.

New Phytologist, 183, 1188–1200.

Helgason, T., Daniell, T.J., Husband, R., Fitter, A.H. & Young,

J.P.W. (1998) Ploughing up the wood-wide web? Nature,

394, 431.

Helgason, T., Merryweather, J.W., Denison, J., Wilson, P.,

Young, J.P.W. & Fitter, A.H. (2002) Selectivity and func-

tional diversity in arbuscular mycorrhizas of co-occurring

fungi and plants from a temperate deciduous woodland.

Journal of Ecology, 90, 371–384.

Hierro, J.L., Maron, J.L. & Callaway, R.M. (2005) A biogeo-

graphic approach to plant invasions: the importance of

studying exotics in their introduced and native range.


Hoffman, M.T. & Mitchell, D.T. (1986) The root morphology

of some legume spp. in the south-western Cape and the

relationship of vesicular arbuscular mycorrhizas with dry

mass and phosphorus content of Acacia saligna seedlings.

South African Journal of Botany, 52, 316–320.

Klironomos, J.N. (2002) Feedback with soil biota contributes

to plant rarity and invasiveness in communities. Nature,

417, 67–70.

Klironomos, J.N. (2003) Variation in plant response to native

and exotic arbuscular mycorrhizal fungi. Ecology, 84, 2292–

2301.

Kozik, A., Chan, B. & Michemore, B. (2003) BLAST parser,

distance matrix file and protein sequence clustering. How to

obtain a distance matrix file from BLAST search results and

use it for sequence clustering analysis. Available at: http://

cgpdb.ucdavis.edu/BlastParser/Blast_Parser.html.

Kuhn, I., Brandenburg, M. & Klotz, S. (2004) Why do alien

plant species that reproduce in natural habitats occur more

frequently? Diversity and Distributions, 10, 417–425.

Kuster, E.C., Kuhn, I., Bruelheide, H. & Klotz, S. (2008) Trait

interactions help explain plant invasion success in the Ger-

man flora. Journal of Ecology, 96, 860–868.

Levine, J.M., Adler, P.B. & Yelenik, S.G. (2004) A meta-analysis

of biotic resistance to exotic plant invasions. Ecology Letters,

7, 975–989.

McCune, B. & Grace, J.B. (2002) Analysis of ecological com-

munities. MjM Software Design, Gleneden Beach, OR.

Milne, I., Wright, F., Rowe, G., Marshall, D.F., Husmeier, D. &

McGuire, G. (2004) TOPALi: software for automatic iden-

tification of recombinant sequences within DNA multiple

alignments. Bioinformatics, 20, 1806–1807.

Mitchell, C.E., Agrawal, A.A., Bever, J.D., Gilbert, G.S.,

Hufbauer, R.A., Klironomos, J.N., Maron, J.L., Morris,

W.F., Parker, I.M., Power, A.G., Seabloom, E.W., Torchin,

M.E. & Vazquez, D.P. (2006) Biotic interactions and plant

invasions. Ecology Letters, 9, 726–740.

Moora, M., Opik, M., Sen, R. & Zobel, M. (2004a) Native

arbuscular mycorrhizal fungal communities differentially

influence the seedling performance of rare and common

Pulsatilla species. Functional Ecology, 18, 554–562.

Moora, M., Opik, M. & Zobel, M. (2004b) Performance of two

Centaurea species in response to different root-associated

microbial communities and to alterations in nutrient

availability. Annales Botanici Fennici, 41, 263–271.

Mummey, D.L. & Rillig, M.C. (2006) The invasive plant spe-

cies Centaurea maculosa alters arbuscular mycorrhizal fungal

communities in the field. Plant and Soil, 288, 81–90.



Opik, M., Moora, M., Liira, J., Koljalg, U., Zobel, M. & Sen,

R. (2003) Divergent arbuscular mycorrhizal fungal com-

munities colonize roots of Pulsatilla spp. in boreal Scots

pine forest and grassland soils. New Phytologist, 160, 581–

593.

Opik, M., Moora, M., Liira, J. & Zobel, M. (2006) Composi-

tion of root-colonizing arbuscular mycorrhizal fungal

communities in different ecosystems around the globe.


Opik, M., Moora, M., Zobel, M., Saks, U., Wheatley, R.,

Wright, F. & Daniell, T. (2008) High diversity of arbuscular

mycorrhizal fungi in a boreal herb-rich coniferous forest.

New Phytologist, 179, 867–876.

Opik, M., Metsis, M., Daniell, T.J., Zobel, M. & Moora, M.

(2009) Large-scale parallel 454 sequencing reveals host

ecological group specificity of arbuscular mycorrhizal

fungi in a boreonemoral forest. New Phytologist, 184, 424–

437.

Opik, M., Vanatoa, A., Vanatoa, E., Moora, M., Davison, J.,

Kalwij, J.M., Reier, U. & Zobel, M. (2010) The online

database MaarjAM reveals global and ecosystemic distribu-

tion patterns in arbuscular mycorrhizal fungi (Glomer-

omycota). New Phytologist, 188, 223–241.

Parameswaran, P., Jalili, R., Tao, L., Shokralla, S., Gharizadeh,

B., Ronaghi, M. & Fire, A.Z. (2007) A pyrosequencing-tai-

lored nucleotide barcode design unveils opportunities for

large-scale sample multiplexing. Nucleic Acids Research, 35,

e130.

Partel, M. & Wilson, S.D. (2002) Root dynamics and spatial

pattern in prairie and forest. Ecology, 83, 1199–1203.

Pringle, A., Bever, J.D., Gardes, M., Parrent, J.L., Rillig, M.C. &

Klironomos, J.N. (2009) Mycorrhizal symbioses and plant

invasions. Annual Review of Ecology, Evolution, and Sys-

tematics, 40, 699–715.

van der Putten, W.H., Klironomos, J.N. & Wardle, D.A. (2007)

Microbial ecology of biological invasions. ISME Journal, 1,

28–37.

Reinhart, K.O. & Callaway, R.M. (2006) Soil biota and invasive

plants. New Phytologist, 170, 445–457.

Richardson, D.M., Allsopp, N., D’Antonio, C.M., Milton, S.J.

& Rejmanek, M. (2000) Plant invasions – the role of

mutualisms. Biological Review, 75, 65–93.

Rodrıguez-Echeverrıa, S. (2010) Rhizobial hitchhikers from

Down Under: invasional meltdown in a plant–bacteria

mutualism? Journal of Biogeography, 37, 1611–1622.

Schussler, A., Schwarzott, D. & Walker, C. (2001) A new fungal

phylum, the Glomeromycota: phylogeny and evolution.

Mycological Research, 105, 1413–1421.

Settele, J., Hammen, V., Hulme, P. et al. (2005) ALARM:

assessing large-scale environmental risks for biodiversity

with tested methods. Gaia, 14, 69–72.

Shah, M.A., Reshi, Z.A. & Khasa, D.P. (2009) Arbuscular

mycorrhizas: drivers or passengers of alien plant invasion.

The Botanical Review, 75, 397–417.

Smith, S.E. & Read, D.J. (2008) Mycorrhizal symbiosis, 3rd edn.

Academic Press, Amsterdam.

Sykorova, Z., Ineichen, K., Wiemken, A. & Redecker, D. (2007)

The cultivation bias: different communities of arbuscular

mycorrhizal fungi detected in roots from the field, from bait

plants transplanted to the field, and from a greenhouse trap

experiment. Mycorrhiza, 18, 1–14.

Taylor, J.E., Hyde, K.D. & Jones, E.B.G. (2000) The biogeo-

graphical distribution of microfungi associated with three

palm species from tropical and temperate habitats. Journal of

Biogeography, 27, 297–310.

Tutin, T.G., Heywood, V.H., Burges, N.A., Valentine, D.H.,

Walters, S.M. & Webb, D.A. (eds) (2001) Flora Europaea,

2nd edn. Vols 1–5 and CD-ROM Pack. Cambridge Uni-

versity Press, Cambridge.

Uibopuu, A., Moora, M., Saks, U., Daniell, T., Zobel, M. &

Opik, M. (2009) Differential effect of arbuscular mycorrhizal

fungal communities from ecosystems along management

gradient on the growth of forest understorey plant species.

Soil Biology & Biochemistry, 41, 2141–2146.

Vogelsang, K.M. & Bever, J.D. (2009) Mycorrhizal densities

decline in association with nonnative plants and contribute

to plant invasion. Ecology, 90, 399–407.

Walther, G.R. (2003) Are there indigenous palms in Switzer-

land? Botanica Helvetica, 113, 159–180.

Walther, G.R. & Berger, S. (2010) Palms (and other evergreen

broad-leaved species) conquer the North. Atlas of biodiver-

sity risk (ed. by J. Settele, L. Penev, T. Georgiev, R. Grabaum,

V. Grobelnik, V. Hammen, S. Klotz, M. Kotarac and I.

Kuhn), pp. 212–213. Pensoft Publishers, Sofia.

Walther, G.R., Gritti, E.S., Berger, S., Hickler, T., Tang, Z.Y. &

Sykes, M.T. (2007) Palms tracking climate change. Global

Ecology and Biogeography, 16, 801–809.

Zhang, Q., Yang, R.Y., Tang, J.J., Yang, H.S., Hu, S.J. & Chen,

X. (2010) Positive feedback between mycorrhizal fungi and

plants influences plant invasion success and resistance to

invasion. PLoS ONE, 5, e12380.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the

online version of this article:

Appendix S1 Glomeromycota sequences in the roots of

Trachycarpus fortunei at the studied sites.

Appendix S2 Venn diagram showing the number of Glom-

eromycota virtual taxa in the current study that are unique to

and shared between different ranges.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such mate-

rials are peer-reviewed and may be re-organized for online

delivery, but are not copy-edited or typeset. Technical support

issues arising from supporting information (other than

missing files) should be addressed to the authors.

M. Moora et al.


BIOSKETCH

The research programme of M. Moora, J. Davison, M. Opik, A. Vanatoa, E. Vanatoa and M. Zobel addresses local and global

diversity patterns of glomeromycotan fungi using molecular markers. The study was conducted in the ALARM field-site network,

which was established as a platform for integrated research on large-scale risks to biodiversity.

Author contributions: G.R.W., S.B., M.O., M.Z. and M. Moora conceived the research; S.B., R.B., H.B., I.K., W.E.K., A.R., J.C.S, C.W

and M. Moora collected the data; M.O., M. Metsis, E.V. and M.T. performed the molecular analyses; J.D., M.O., A.V., M. Metsis and

M. Moora analysed the data; and M. Moora led the writing.

Editor: Serban Proches



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