Molecular Ecology (2011) doi: 10.1111/j.1365-294X.2011.05392.x

Extensive gene flow over Europe and possible speciationover Eurasia in the ectomycorrhizal basidiomyceteLaccaria amethystina complex

LUCIE VINCENOT,* 1 KAZUHIDE NARA,† CHRISTOPHER STHULTZ,* JESSY LABBE ,‡§

MARIE-PIERRE DUBOIS , * LEHO TEDERSOO,– FRANCIS MARTIN‡ and MARC-ANDRE SELOSSE*

*UMR5175, Centre d’Ecologie Fonctionnelle et Evolutive, 1919 route de Mende, 34293 Montpellier Cedex 5, France,

†Department of Natural Environmental Studies, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8563, Japan,

‡UMR1136, Interactions Arbres ⁄ Microorganismes, INRA-Nancy, 54280 Champenoux, France, §Environmental Sciences

Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6034, USA, –Institute Ecology

and Earth Sciences and The Natural History Museum, University of Tartu, 40 Lai Str., 51005 Tartu, Estonia

Corresponde

E-mail: lucie.1Present addr

Fondazione E

38100 San M

� 2011 Black

Abstract

Biogeographical patterns and large-scale genetic structure have been little studied in

ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM

symbioses. We coupled population genetics and phylogenetic approaches to understand

spatial structure in fungal populations on a continental scale. Using nine microsatellite

markers, we characterized gene flow among 16 populations of the widespread EM

basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened

our scope to two additional populations from Japan (104 km away) and compared them

with European populations through microsatellite markers and multilocus phylogenies,

using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial

ribosomal genes. European L. amethystina populations displayed limited differentiation

(average FST = 0.041) and very weak isolation by distance (IBD). This panmictic European

pattern may result from effective aerial dispersal of spores, high genetic diversity in

populations and mutualistic interactions with multiple hosts that all facilitate migration.

The multilocus phylogeny based on nuclear genes confirmed that Japanese and

European specimens were closely related but clustered on a geographical basis. By

using microsatellite markers, we found that Japanese populations were strongly

differentiated from the European populations (FST = 0.416), more than expected by

extrapolating the European pattern of IBD. Population structure analyses clearly

separated the populations into two clusters, i.e. European and Japanese clusters. We

discuss the possibility of IBD in a continuous population (considering some evidence for

a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia,

making L. amethystina a promising model of intercontinental species for future studies.

Keywords: cryptic speciation, ectomycorrhizal fungi, gene flow, inbreeding, isolation by

distance, microsatellite markers, phylogeography, population structure

Received 11 July 2011; revision received 27 October 2011; accepted 1 November 2011

nce: Lucie Vincenot, Fax: (+39 0461650872);

vincenot@cefe.cnrs.fr

ess: Sustainable Agro-Ecosystems and Bioresources,

dmund Mach-IASMA, via Edmund Mach 1,

ichele all’Adige, Italy

well Publishing Ltd

Introduction

Many fungal species extend over a whole continent, or

even over several continents (Douhan et al. 2007; Queloz

et al. 2011), and a comparative analysis of regional

inventories revealed that 28–63% of macrofungal spe-

cies are shared by at least two continental regions

(Mueller et al. 2007). Hence, they seem to follow the

2 L. VINCENOT ET AL.

classic Beijerinckian paradigm for microbes, ‘everything is

everywhere, but, the environment selects’ (O’Malley 2007).

Indeed, high dispersibility of spores could allow exten-

sive flow of propagules over intra- and intercontinental

distances. However, because morphological observa-

tions used to name fungi may not distinguish closely

related biological species (i.e. groups of organisms capa-

ble of interbreeding), claims based on such surveys

remain questionable. Indeed, molecular phylogenetic

analyses often challenge global and intercontinental

fungal species (Douhan et al. 2011) and suggest that

morphospecies often encompass cryptic biological spe-

cies, which remain hidden because of the limited mor-

phological characters available for fungi (Taylor et al.

2006; Jargeat et al. 2010). Although multigene phyloge-

nies indirectly allow delineation of biological species by

enforcing the Phylogenetic Species Concept (Taylor

et al. 2000; Douhan et al. 2007), it is often difficult to

settle a clear species boundary within quickly evolving

species complexes. In such cases, population genetics

approaches are powerful tools to reveal reproductive

isolation and thus biological species. Unfortunately,

population genetics studies remain limited for fungi as

compared with other eukaryotes (in ISI Web of Knowl-

edge for 2010, 55 papers dealt with ‘population genet-

ics’ or ‘gene flow’ for fungi, compared with 129 for

animals and 379 for plants). Moreover, because most of

these studies were conducted at a local scale, mycolo-

gists recently called for larger-scale population genetics

studies (Taylor et al. 2006; Halling et al. 2008; Douhan

et al. 2011).

Long-distance dispersal (LDD) via recent human

transport or natural aerial dispersal has been well rec-

ognized in pathogenic fungi (Brown & Hovmøller

2002). This leads to large, continental-scale distributions

of plant pathogenic ascomycetes (Dilmaghani et al.

2009) and basidiomycetes (Barres et al. 2008). Large dis-

tribution areas and efficient LDD have also been

reported in saprobic fungi, e.g. the Ganoderma applana-

tum ⁄ G. australe complex from the Southern Hemisphere

(Moncalvo & Buchanan 2008), Schizophyllum commune

(James et al. 1999) or Serpula lacrymans (Kauserud et al.

2007; human dispersion being involved in this case).

However, for ectomycorrhizal (EM) fungi that associate

with forest trees in most temperate and boreal forests,

the picture remains unclear. The structure of EM popu-

lations has mainly been explored at small local scales,

focusing on genet size, inbreeding and diversity (e.g.

Bergemann & Miller 2002; Gherbi et al. 1999; see review

in Douhan et al. 2011). Among a few large-scale studies

on gene flow, Bergemann & Miller (2002) found strong

genetic differentiation between populations of Russula

brevipes situated 1500 km apart in North American for-

ests and questioned their conspecificity. Xu et al. (2008)

detected isolation by distance (IBD) over 1050 km in

Tricholoma matsutake from Southwestern China. In Eur-

ope, significant IBD over 900 km was found in two truf-

fles, Tuber magnatum (i.e. its whole geographical range;

Rubini et al. 2005) and T. melanosporum (Murat et al.

2004). Significant IBD were even seen at smaller scales

for species such as Rhizopogon spp. (over 5 km, Kretzer

et al. 2005) or Cenococcum geophilum (over 250 km, Jany

et al. 2002), but these species, together with Tuber spp.,

do not produce wind-dispersed spores and depend on

animals for spore dispersal. Moreover, population

genetics sometimes revealed sympatric cryptic species,

e.g. in Tricholoma ‘scalpturatum’ (Carriconde et al. 2008;

Jargeat et al. 2010), Amanita muscaria (Geml et al. 2009)

or Cantharellus formosus (Dunham et al. 2003).

However, no EM population genetic study to date

has extended over more than 1500 km (Douhan et al.

2011). At such scales, EM fungi were investigated by

phylogenetic ⁄ phylogeographic approaches, relying on a

limited number of individuals and molecular loci. Some

monophyletic EM taxa with intercontinental distribution

were divided into several units, a result also obtained

for several non-EM fungi (Taylor et al. 2006). For exam-

ple, Wu et al. (2000) found that morphologically indis-

tinguishable Suillus spraguei specimens from China and

North America belonged to a paraphyletic assemblage.

The putatively global Pisolithus tinctorius was split into

geographical species (Martin et al. 2002). Geml et al.

(2006, 2009) also found several geographical clades

within A. muscaria sensu lato. However, the putative

species, phylogenetic clades or geographical groups

resulting from these phylogenetic approaches were

determined without directly assessing the degree of

gene flow and reproductive isolation, which can be

assessed by population genetics approach using neutral

markers.

Our study combines population genetics and phylo-

genetic approaches over Eurasia, using the EM basidio-

mycete Laccaria amethystina as a model. Mature

dikaryotic individuals of L. amethystina arise from

mating without nuclear fusion between two different

haploids (because of two self-incompatibility genes;

Niculita-Hirzel et al. 2008). Haploids are transient and

arise by germination of small wind-dispersed meiotic

spores that are produced in fleshy fruitbodies. The

sequenced genome of the related L. bicolor (Martin et al.

2008) recently offered the first full genomic resources

for an EM basidiomycete. Although populations of

L. bicolor have been studied at local scales (e.g. Selosse

et al. 1997, 1998), this species rarely forms fruitbodies in

natural forests. Conversely, the Eurasian L. amethystina

fruits abundantly and forms locally abundant small

genets in Europe (Gherbi et al. 1999; Fiore-Donno &

Martin 2001) and Japan (Wadud 2007). Roy et al. (2008)

� 2011 Blackwell Publishing Ltd

EURASIAN POPULATI ON GE NETICS OF LACCARIA A METHYSTINA 3

showed it to be multihost and did not detect any signif-

icant genetic differentiation of L. amethystina over

450 km in France, suggesting that only larger-scale

studies could reveal IBD. Conversely, limited portability

of microsatellites between Europe and Japan suggests a

possible differentiation at this distance (Donges et al.

2008; Roy et al. 2008).

Here we tested the null hypotheses that distant popu-

lations of L. amethystina morphospecies form a single

biological species with few or no spatial structure over

Eurasia. To investigate the genetic structure at two

scales (103 and 104 km), we designed new markers at

microsatellite (neutral) and coding (likely non-neutral)

loci. Together with previously defined markers, they

revealed gene flow in L. amethystina all over Europe

but demonstrated strong differentiation between Euro-

pean and Japanese populations, questioning their con-

specificity. Based on these results, we discuss gene flow

and speciation for EM basidiomycete at the continental

scale.

Materials and methods

Sampling and DNA extraction

Five hundred and nineteen fruitbodies of Laccaria ame-

thystina were collected in the autumn of 2007 and 2008

from 16 European localities, with a maximum distance

of 2900 km between any two sites (Fig. 1a; Table 1). In

Japan, 36 and 35 fruitbodies were collected on Mount

Fuji in 2008 and on Mount Tokachi in 2009, respec-

tively. These two sites are located 960 km apart

(Fig. 1b). We reused 58 fruitbodies of two French popu-

(a)

Fig. 1 The 18 sampling sites in Europe (a), covering a maximal dista

(b), covering a distance of 960 km. (Sco), Kirkhill, Scotland; (Eng), Ne

France; (Bel), Boisfort, Belgium; (FrO), Orry-la-Ville, France; (FrL), Ly

many; (GeGS), Gut Sunder, Germany; (Aus), Rotwald, Austria; (Pol

fstrom, Sweden; (Fin), Ruissalo, Finland; (Est), Jarvselja, Estonia; (JaM

� 2011 Blackwell Publishing Ltd

lations from a previous study (FrO and FrB are respec-

tively populations Ba and Bbq from Roy et al. 2008). In

all, 667 fruitbodies were used (Table 1). As average

genet diameter found in previous studies was £1.2 m

(Gherbi et al. 1999; Fiore-Donno & Martin 2001; Wadud

2007), fruitbodies were collected in £300 · 300 m plots,

at >1.5 m distance intervals to avoid redundant sam-

pling of the same genet. Fruitbodies were dried and

stored at )20 �C upon arrival at the laboratory. DNA

was extracted as in the study by Roy et al. (2008). We

initially intended to collect populations evenly over

Eurasia, but scarce fructifications in Eastern Europe and

Central Asia from 2007 to 2009 prevented sampling.

Markers for population genetics

Eight microsatellite markers and one rDNA intergenic

spacer, IGS1 (Martin et al. 1999), were used to address

population genetic structure of L. amethystina (Table 2).

Four microsatellites (La03, La17, La23, and Ll35) were

previously published by Wadud et al. (2006a,b), and

La171 and IGS1 were designed by Roy et al. (2008;

La115 was not used because of frequent amplification

problems mentioned in the former paper). We used the

L. bicolor genome sequence (Martin et al. 2008) to find

additional microsatellites portable to L. amethystina.

Microsatellite repetition motifs were detected in the

L. bicolor genome (http://genome.jgi-psf.org/Lacbi2/

Lacbi2.home.html) using MAGELLAN 1.1 software as in

Labbe et al. (2008). Primers were designed with the

online tool PRIMER3 (http://frodo.wi.mit.edu/primer3/).

Each candidate locus was also verified by BLAST for

occurrence as a single copy in the L. bicolor genome.

(b)

nce of 2900 km from Spain (Sp) to Finland (Fin), and in Japan

wton Common, England; (Sp), Guipuscoa, Spain; (FrB), Belleme,

on, France; (CH), Eschenbach, Switzerland; GeF), Freising, Ger-

), Łopuch�owka, Poland; (Den), Østerild, Denmark; (Swe), Olo-

F), Mount Fuji, Japan; (JaMT), Mount Tokachi, Japan.

Table 1 Geographical and forest characteristics of the 18 sampled sites

Population Locality Site location n Overstorey trees

Age of the

forest (years)*

Sco Scotland Kirkhill (57�21¢30¢¢N; 2�46¢47¢¢W) 41 Fagus sylvatica, Larix decidua 150–200

Eng England Newtown Common (51�25¢47¢¢N; 1�26¢30¢¢W) 40 Betula pendula, Quercus robur 50–100

Sp Spain Guipuscoa (43�07¢16¢¢N; 2�15¢38¢¢W) 38 Fagus sylvatica 120–150

FrB† France Belleme (48�22¢36¢¢N; 0�33¢35¢¢E) 26 Abies alba 30–80

Bel Belgium Boisfort (50�49¢43¢¢N; 4�18¢26¢¢E) 46 Fagus sylvatica 250

FrO† France Orry-la-Ville (49�07¢56¢¢N; 2�30¢48¢¢E) 32 Carpinus betulus, Quercus robur 20–50

FrL France Lyon (45�45¢04¢¢N; 4�46¢60¢¢E) 40 Quercus robur, Carpinus betulus 25–50

CH Switzerland Eschenbach (47�09¢24¢¢N; 8�12¢53¢¢E) 43 Picea abies 20–80

GeF Germany Freising (48�29¢52¢¢N; 11�34¢22¢¢E) 36 Fagus sylvatica, Picea abies 60–80

GeGS Germany Gut Sunder (52�43¢60¢¢N; 9�49¢00¢¢E) 44 Fagus sylvatica, Pseudotsuga

menziesii

30–40

Aus Austria Rotwald (47�47¢01¢¢N; 15�03¢29¢¢E) 15 Fagus sylvatica, Picea abies >150

Pol Poland Łopuch�owka (50�17¢25¢¢N; 22�58¢13¢¢E) 39 Larix decidua, Fagus sylvatica 80–110

Den Denmark Østerild (57�09¢34¢¢N; 8�43¢53¢¢E) 37 Fagus sylvatica, Picea abies 40–60

Swe Sweden Olofstrom (56�20¢29¢¢N; 14�26¢06¢¢E) 41 Fagus sylvatica 80

Fin Finland Ruissalo (60�31¢03¢¢N; 22�10¢17¢¢E) 32 Quercus petraea, Corylus avellana 200–400

Est Estonia Jarvselja (58�18¢52¢¢N; 27�15¢23¢¢E) 37 Picea abies, Tilia cordata, Populus tremula 80–100

JapMF Japan Mount Fuji (35�20¢24¢¢N; 138�47¢55¢¢E) 36 Salix reinii <300

JapMT Japan Mount Tokachi (43�27¢31¢¢N; 142�38¢62¢¢E) 35 Salix bakko mainly <90‡

n, number of genotyped fruitbodies.

*Age of oldest individuals for the most common tree species.†FrB, FrO: respectively, Ba and Bbq populations from the study by Roy et al. (2008).‡Sparsely established vegetation patches, established after the last big Mount Tokachi eruption in 1926.

4 L. VINCENOT ET AL.

Reproducible amplification was tested with nonfluores-

cent primers with an annealing temperature gradient

from 50 to 65 �C, on a set of four L. bicolor and eight

European L. amethystina specimens. The presence of

single bands of the expected size was checked on aga-

rose gels. Then, polymorphism in L. amethystina was

examined in 32 European specimens of L. amethystina,

with fluorescent-labelled primers for loci that survived

the initial screening. Of 233 microsatellite loci tested,

only three were portable to L. amethystina and appeared

polymorphic enough (Jl1195M13, Jl1209M5, Lv257;

Table 2). For genotyping, microsatellite loci and IGS1

were amplified as in Roy et al. (2008; annealing temper-

atures are in Table 2). Detection of amplified fragments

was carried out on an ABI PRISM 3130 XL Genetic ana-

lyser (Applied Biosystems, Courtaboeuf, France). Frag-

ment sizes were analysed with GENEMAPPER 3.7 (Applied

Biosystems), using default parameters for microsatellite

analysis. Of 667 genotyped specimens (Data S1, Sup-

porting information), 12 failed to be amplified for at

least two loci and were omitted from further analyses.

Data analysis for population genetics

Allelic frequencies, departure from Hardy–Weinberg

equilibrium, expected and observed heterozygosities

(HE, HO) as well as linkage between loci were calculated

with GENEPOP’007 (Rousset 2008). To assess the discrimi-

nation power of the genetic markers, the probability of

random occurrence was calculated for each genotype

based on allelic frequencies considering all available

fruitbodies and assuming independence between loci,

as in the study by Bergemann & Miller (2002). A corre-

spondence factorial analysis (CFA) using multilocus

genotype data was performed with GENETIX4.05.2

(http://kimura.univ-montp2.fr/genetix/) to detect Wa-

hlund effects within each population. The presence of

null alleles was tested for each locus using Microcheck-

er (Van Oosterhout et al. 2004). Wright indices (FIS, FST)

were calculated with GENEPOP’007. Selfing rates (s) were

evaluated based on FIS (2 FIS ⁄ (1 + FIS)) or using RMES

software (David et al. 2007), which estimates s from the

correlation of heterozygosity between different loci,

independently of FIS and of other technical biases such

as the presence of null alleles.

To detect IBD, correlations between genetic distances

(FST ⁄ (1)FST)) and geographical distances were calcu-

lated by use of Mantel tests as implemented in GENE-

POP’007, with 10 000 permutations. Similarly, Mantel tests

were performed to assess the relationship between

genetic distances and each of the following factors: lati-

tude, longitude, altitude, temperature and precipitations

(climatic data from WorldClim; Hijmans et al. 2005).

These Mantel tests were performed over (i) Europe,

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EURASIAN POPULATI ON GE NETICS OF LACCARIA A METHYSTINA 5

� 2011 Blackwell Publishing Ltd

6 L. VINCENOT ET AL.

including 16 European populations, and (ii) Eurasia, by

adding two Japanese populations to the European pop-

ulations. In both cases, we performed CFA on popula-

tions to detect Wahlund effects as described earlier.

Population structure was also analysed by using GENE-

LAND (Guillot et al. 2008) and STRUCTURE 2.2 (Pritchard

et al. 2000, http://pritch.bsd.uchicago.edu/struc-

ture.html) with the number of initial clusters ranging

from K = 1 to 16 (or 18 for the Eurasian level) and

10 000 iterations. For the cluster analysis with STRUCTURE

2.2, we tested four possible a priori parameter sets

(ancestry model with or without admixture, associated

with allelic frequencies correlated or independent) and

found them to have no effect on the number of a poste-

riori detected clusters if K ‡ 2 (not shown). The amount

of genetic variance between European and Japanese

populations was analysed by AMOVA (10 000 permuta-

tions) as implemented in ARLEQUIN 3.5 (Excoffier &

Lischer 2010).

Sequencing of non-neutral loci for phylogeneticanalysis

Coding (or transcribed) genes were amplified from 35

L. amethystina specimens, i.e. one or two samples for

each investigated population, plus one sample from the

Ukraine and one from Belarus (no exact location was

provided by the samplers). We also included three

specimens of L. bicolor from France and one from Ser-

bia, and a single specimen of each of the following spe-

cies: L. amethysteo-occidentalis, L. laccata, L. anglica

[PAM0090406], L. scotica [PAM0010112], L. proxima

[PAM01102104], L. moelleri [PAM97090101], L. macrocys-

tidiata [PAM99082801], L. oblongispora [PAM01042805]

(numbers between brackets are accessions in the Herbi-

er de Botanique at the Universite de Lille). For these 45

samples, we tentatively sequenced five loci: the partly

transcribed ITS region of nuclear ribosomal DNA, two

transcribed mitochondrial ribosomal genes (for large

and small subunits, LrRNA and SrRNA, respectively),

and two nuclear coding genes for glucose-6-phosphate

dehydrogenase (G6PD) and nitrate reductase (NAR; see

primers in Table 2). PCR conditions included an initial

denaturation at 95 �C for 5 min, followed by 30 cycles

of denaturation at 94 �C for 30 s, annealing (tempera-

tures on Table 2) for 60 s and extension at 72 �C for

45 s, with a final extension at 72 �C for 10 min.

Sequences were obtained from both strands using

the PCR primers (Table 2), and were manually cor-

rected and aligned using the software SEQSCAPE 2.6

(Applied Biosystems). Corrected sequences were depos-

ited in GenBank (accessions GQ406389–GQ406568 and

HQ896282–HQ896286, Data S2, Supporting information).

Phylogenetic analyses

Phylogenetic analyses were conducted using maxi-

mum-likelihood (ML) and Bayesian methods, by use of

PHYML 3.0 (Guindon & Gascuel 2003) and MRBAYES

3.1.2 (Ronquist & Huelsenbeck 2003), respectively.

Related sequences for each locus (ITS, G6PD, NAR and

mitochondrial LrRNA and SrRNA) were retrieved from

UNITE (Abarenkov et al. 2010) and GenBank databases,

which included sequences of L. bicolor genome (http://

genome.jgi-psf.org/Lacbi2/Lacbi2.home.html). The best

evolutionary model was determined by hierarchical

likelihood ratio tests using PAUP*4.0b (Swofford 2002)

and MRMODELEST 2.3 (Nylander 2004). In the Bayesian

analyses, gaps were treated as (i) missing data or (ii)

presence ⁄ absence of indels (only the first option was

used in PHYML). Analyses were first performed on each

locus separately (for all loci, ML and both Bayesian

analyses provided the same topologies, thus only

Bayesian trees generated from alignments without gaps

are presented here). We built a supermatrix concatenat-

ing the informative loci for all samples, but. We

excluded the two mitochondrial loci, which carried

negligible phylogenetic information, and samples for

which not all nuclear sequences were available. This

supermatrix thus concatenated three nuclear sequences

for 28 samples that were analysed in MRBAYES 3, as

above.

Coalescence test

We tested for migration events between European and

Japanese populations on the basis of each nuclear

sequence sets (ITS, NAR and G6PD) using the program

MDIV (Nielsen & Wakeley 2001). MDIV uses a Markov

Chain Monte Carlo coalescent-based approach to esti-

mate the migration rate M ⁄ 2 (i.e. the mean number of

migrants per generation), the scaled divergence time

T = t ⁄ Ne (where t is the divergence time in generations

and Ne, the effective population size) and the scaled

migration rate H = Ne · M. MDIV was run under the

HKY model of sequence evolution. Starting parameters

were adjusted by a first short run (2.106 Markov chain

generations plus 5.105 as a burn-in), and the program

was then run again with longer chains (107 generations,

5.106 as a burn-in).

Results

Genetic diversity and inbreeding within populations

All nine microsatellite markers were polymorphic in

Europe, with 3–12 alleles per locus, each one never

� 2011 Blackwell Publishing Ltd

Ta

ble

3A

llel

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ty,

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26(2

2)5

33

34

37

32

280.

480.

260.

470.

210.

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35B

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(46)

7(2

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92

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(40)

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EURASIAN POPULATI ON GE NETICS OF LACCARIA A METHYSTINA 7

� 2011 Blackwell Publishing Ltd

pu

lati

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s

lF

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0.00

5—

dia

go

nal

,in

gre

y,

FS

Tca

lcu

late

dw

ith

fou

rlo

cisu

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ing

Har

dy

–Wei

nb

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riu

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5%,

P=

0.05

).

8 L. VINCENOT ET AL.

exceeding 0.89 in frequency (Table 3). The allelic rich-

ness differed in the Japanese populations: La03 and

Lv257 were monomorphic (Table 3), La171 failed to

amplify in some fruitbodies, while the six remaining

loci showed 3–10 alleles per locus. We found a high

diversity within populations, with 646 multilocus geno-

types of 655 successfully typed fruitbodies. All popula-

tions showed a tendency to heterozygote deficiency

(Table 3), which was significant in more than half of

the populations for the five loci Jl1195M13, Jl1209M5,

La17, La171 and Ll35. However, the populations did not

significantly deviate from Hardy–Weinberg equilibrium

for the four other loci, La03, La23, Lv257 and IGS1

(which is unlikely to provide null alleles). This discrep-

ancy suggests that null alleles occurred for Jl1195M13,

Jl1209M5, La17, La171 and Ll35. As no null homozygote

was found in these five loci (except for La171 in Japan),

null allele frequencies were probably low, which was

confirmed by Microchecker analysis (not shown). FIS

values were high when using the nine loci (Table 3),

but decreased when the analyses were restricted to the

four loci supporting Hardy–Weinberg equilibrium

(hereafter ‘unbiased loci’): they ranged from )0.09 to

0.32 in Europe and from )0.12 to 0.05 in Japan. Selfing

rates, based on FIS values in the four unbiased loci, var-

ied from null (nonsignificant excess of heterozygotes in

some populations) to 0.49. Using the RMES method, the

estimated selfing rates were lower, but still variable

among populations (0.008–0.267; Table 3). The selfing

rates estimated with RMES did not correlate with those

estimated from FIS values, and none of the selfing rates

correlated with forest age (not shown). After Bonferroni

corrections for multiple comparisons, no significant

linkage disequilibrium between pairs of markers was

found, either within populations in Europe or in Japan,

or over Europe (not shown). Thus, in spite of some

inbreeding, sexual recombination was predominant

within Laccaria amethystina populations.

Tab

le4

Gen

etic

dif

fere

nti

atio

nb

etw

een

pai

rso

fp

o

Po

pu

lati

on

Sco

En

gS

pF

rBB

e

Sco

—0.

024

0.05

70.

053

0

En

g0.

032

—0.

014

0.06

30

Sp

0.07

00.

006

—0.

041

0

FrB

0.04

30.

039

0.07

7—

0

Bel

0.03

00.

007

0.00

30.

041

—

FrO

0.05

10.

013

0.04

3)

0.00

60

FrL

0.03

00.

007

0.00

30.

033

)0

CH

0.03

40.

001

0.02

00.

006

0

GeF

0.1

04

0.02

40.

010

0.07

50

GeG

S0.1

17

0.03

90.

033

0.05

30

Au

s0.

089

0.01

30.

020

0.03

20

Po

l0.

061

0.03

30.

046

0.00

10

Den

0.01

30.

006

0.02

60.

005

0

Sw

e0.

070

0.01

10.

027

0.02

40

Fin

0.1

35

0.08

20.

044

0.1

74

0

Est

0.07

20.

001

0.00

10.

053

0

Jap

MF

0.5

57

0.5

35

0.5

42

0.5

58

0

Jap

MT

0.6

02

0.5

99

0.5

93

0.6

16

0

Up

per

dia

go

nal

,F

ST

calc

ula

ted

wit

hn

ine

loci

;lo

wer

sig

nifi

can

td

iffe

ren

tiat

ion

bet

wee

np

op

ula

tio

ns

(CI

9

Population structure over Europe

FST values between pairs of populations in Europe ran-

ged from 0.001 to 0.134, with a mean FST value of 0.041

over all loci (Table 4). Among 120 pairwise FST, 82 were

lower than 0.05, and only five showed a significant dif-

ferentiation (CI: 95% with Bonferroni correction,

a = 0.05). Using the four unbiased loci, the mean FST

value decreased to 0.032, while FST values between

pairs of populations were more variable, ranging from

)0.015 to 0.174 (Table 4). Although FST values for the

Finnish population were relatively high compared with

eight other European populations based on the unbi-

ased loci (Table 4), no genetic structure appeared

among European populations. Congruently, a Mantel

� 2011 Blackwell Publishing Ltd

R² = 0.098

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0 500 1000 1500 2000 2500 3000

Geographic distance (km)

R² = 0.951

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

0 2000 4000 6000 8000 10000

F ST/

(1 –

FST

)F S

T/(1

– F

ST)

Geographic distance (km)

*

(a)

(b)

Fig. 2 Mantel tests showing correlation between genetic and

geographical Euclidian distances (d, in km) for Laccaria amethy-

stina populations. In Europe (a), FST ⁄ (1)FST) = 0.02 + 1.59 ·10)5 d. For European plus Japanese populations (b),

FST ⁄ (1)FST) = )0.05 + 8.38 · 10)5 d. The dashed line with aster-

isk in (b) extrapolates the regression line inferred for European

populations.

EURASIAN POPULATI ON GE NETICS OF LACCARIA A METHYSTINA 9

test comparing geographical distances and genetic dif-

ferentiation revealed that IBD was only marginally sig-

nificant (R2 = 0.098, P = 0.056; Fig. 2a). Genetic distance

between populations did not significantly correlate with

latitude, longitude, elevation or climatic factors such as

mean annual temperature and precipitations (not

shown). CFA revealed that there was no obvious geo-

graphical pattern among 16 European populations

(Fig. 3a). The cluster analysis with the program STRUC-

TURE suggested that whichever be the prerequisites on

ancestry model and allelic frequencies, a single popula-

tion over Europe was the most likely scenario (Fig. S1,

Supporting information). In addition, five repeated runs

of GENELAND supported the single population hypothesis

with a maximum a posteriori estimate (not shown).

Thus, there was no clear IBD over 2900 km among

European L. amethystina populations.

Population structure between Europe and Japan

The two Japanese populations were genetically similar

to each other (FST = 0.04 on nine loci), suggesting lim-

ited IBD over 960 km in Japan. Whereas only seven of

the 16 European populations harboured private alleles

� 2011 Blackwell Publishing Ltd

(at most four per population, with frequencies ranging

up to 0.02), 14 alleles found in at least one Japanese pop-

ulation did not occur in European populations (Table 3)

and their frequencies ranged up to 0.028. Moreover, null

alleles were frequent at locus La171, with 67% and 72%

of null homozygotes in JapMF and JapMT, respectively,

whereas no null homozygote existed in European popu-

lations. AMOVA on all populations showed that 43.1% of

the molecular variance was explained by the differentia-

tion between Europe and Japan (significant at P < 10)5),

whereas differentiation among the 16 European popula-

tions or among the two Japanese populations accounted

for 2.4% of the variance. The differentiation within pop-

ulations explained 54.5% of the molecular variance. FST

values between pairs of Japanese and European popula-

tions (from 0.374 to 0.443) were always consistently

higher than within Europe or within Japan, and the glo-

bal FST between European and Japanese populations

was 0.416 (based on nine loci) or 0.516 (based on the

four unbiased loci; Table 4). A CFA plot clearly distin-

guished a cluster of Japanese populations and another

of European populations, separated on the first axis,

which explained 69.0% of variance (or 78.5% when

using the four unbiased loci; Fig. 3b). The allelic diver-

sity in the Japanese populations was similar to that in

European populations (37 and 29 vs. 30.5 ± 4.8,

mean ± SD). Both STRUCTURE (Fig. S1, Supporting infor-

mation) and GENELAND (data not shown) analyses distin-

guished two clusters, representing respectively all

European populations and the two Japanese popula-

tions. At the Eurasian scale, the Mantel test revealed a

highly significant correlation between geographical and

genetic distances (R2 = 0.95, P = 3.10)4; Fig. 2b) that was

driven by the extreme values involving the Japanese

populations. Thus, a strong IBD pattern occurred

between European and Japanese populations.

Molecular phylogeny in the Laccaria amethystinapopulations

ITS sequences of Laccaria spp. were obtained from 49

fruitbodies (including 11 non-L. amethystina; GenBank

accession numbers GQ406458–GQ406468). Two data sets

were analysed: one containing all the sequences from

this study plus 29 sequences from the UNITE database

with robust identifications (Fig. 4) and a larger one con-

taining all available Laccaria ITS, by retrieving all

related sequences from GenBank (138 sequences in all;

Fig. S2, Supporting information). Both data sets

revealed two well-supported L. amethystina clades, one

from Japan and the other from Europe (Fig. 4),

although their phylogenetic position was unresolved

because of polytomy with other Laccaria taxa. A North

American L. amethystina isolate was a putative sister to

(a)

(b)

Fig. 3 Correspondence factorial analy-

sis (CFA) on European and Eurasian

populations. For European populations

(a), the two mains axes 1 and 2 of the

CFA explain 16.5% and 14.5% of the

variation, respectively, for European

plus Japanese populations (b), the two

mains axes 1 and 2 of the CFA explain

69.0% and 4.8% of the variation, respec-

tively.

10 L . V I N C ENO T ET AL.

the Japanese clade (with 89% posterior probabilities in

the Bayesian analysis, Fig. S2, Supporting information).

Nucleotide variations within the ITS region were low in

Europe (at least 99.7% identity between sequences,

indels not taken into account), while Japanese sequences

were 97.5% identical to the European consensus besides

a 12-bp deletion and two (2 and 4 bp) insertions

(Fig. S3, Supporting information). The American

sequence was intermediate between European and Japa-

nese sequences for nucleotide substitutions (98.0% and

99.3% identity to the respective consensus, not consid-

ering indels; Fig. S3, Supporting information).

We obtained 40 NAR sequences (including eight non-

L. amethystina; GQ406531–GQ406540) that formed two

well-supported geographical clusters in a Bayesian anal-

ysis (Fig. 5a). European sequences were >98.2% similar

to each other, but differed by at least 4.7% from the

Japanese consensus. We obtained 34 G6PD sequences

(including 10 from non-L. amethystina; GQ406340,

GQ406413–GQ406416 and GQ406418–GQ406421) that, in

a Bayesian analysis, also supported two geographical

clusters (Fig. 5b). The Japanese L. amethystina and

American L. amethysteo-occidentalis clustered with good

statistical support (posterior probability of the clade:

97%). The European and Japanese L. amethystina

sequences differed by at least 15 point nucleotide sub-

stitutions (including four nonsynonymous mutations),

while the Japanese L. amethystina sequences and North

American L. amethysteo-occidentalis differed by eight

substitutions only (including two nonsynonymous

mutations). Mitochondrial genes for small (Fig. S4a,

Supporting information) and large (Fig. S4b, Support-

ing information) ribosomal subunits showed low vari-

ability and did not separate European and Japanese

sequences. A Bayesian analysis of the nuclear superma-

trix concatenating the three nuclear genes for 28 fruit-

bodies showed that the Japanese L. amethystina clade

was sister to the European clade, both of which formed

a monophyletic clade with North American L. amethys-

teo-occidentalis (Fig. 5c).

The distributions of probabilities for coalescent analy-

sis parameters were coherent for the three nuclear

sequence sets (using the program MDIV; not shown). For

each locus, the migration rate between European and

Japanese L. amethystina populations was estimated to 0,

indicating a complete genetic isolation between these

� 2011 Blackwell Publishing Ltd

Fig. 4 Bayesian phylogram of Laccaria spp. based on ITS sequences (81 taxa, 635 characters; gaps were encoded in a matrix of in-

dels). The best-fit nucleotide evolutionary model was GTR + I (I = 0.52). Dashed lines represent subtrees with posterior probabilities

<97%; only probabilities ‡90% are indicated; outgroups are from the Unite database. ‘Laccaria amethystina (Europe)’ consists of all 35

European sequences from this study, plus seven European sequences from Unite (UDB002418 and UDB000158 from Scotland,

UDB001687, UDB001492, UDB000039, UDB000019 and UDB000006 from Denmark). ‘L. amethystina (Japan)’ includes three Japanese

sequences from this study. ‘L. bicolor H82’ is an American L. bicolor sequence (Martin et al. 2008).

EURASIAN POPULATI ON GE NETICS OF LACCARIA AMETHYSTINA 11

� 2011 Blackwell Publishing Ltd

12 L . V I N C ENO T ET AL.

two populations. The scaled divergence time T was esti-

mated between 5.5 and 8.4 generations ⁄ Ne, but the

effective population size Ne cannot be estimated (see

Discussion). Hence, the divergence time between

European and Japanese L. amethystina could not be esti-

mated. Therefore, a clear divergence between the Euro-

pean and Japanese L. amethystina was found for the

three nuclear loci, in spite of the sister positions of these

geographical clades.

Discussion

Reproductive biology of Laccaria amethystina

Nine neutral markers revealed high local genotypic

diversities within populations: 97–100% of the fruitbod-

ies showed unique genotypes in each population, and

differentiation within populations accounted for 54.5%

of the genetic variance observed. Such intrapopulation

diversity has already been reported in Laccaria amethysti-

na (Gherbi et al. 1999; Fiore-Donno & Martin 2001) and

other basidiomycetes with aerial spore dispersal (Carri-

conde et al. 2008; Engh et al. 2010). Only 16 genotypes

were found more than once (they accounted for a total of

35 fruitbodies). Ten of these genotypes (3.0% of all fruit-

bodies) were found in two different populations and one

in three populations (Est, Eng and Den). Their probability

P of occurrence by chance ranged from 1.3 · 10)6 to

5.8 · 10)4. They are unlikely to represent the same clone,

because meiotic spore dispersal is the only way to

migrate into distant populations. Six other genotypes

(2.3% of all fruitbodies) were found at least twice within

a single population and had P £ 5.2 · 10)4. Although re-

sampling of the same genet within a population remains

possible, we favour that they represent different clones

because (i) their P-value is in the range of the genotypes

repeated in different populations, which are different

clones, and (ii) genet size is smaller than the sampling

distance (see Materials and methods). Anyway, as in the

study by Roy et al. 2008, discarding genetically identical

fruitbodies produced the same conclusions (not shown).

Fig. 5 Unrooted Bayesian phylograms for nuclear genes of Laccari

Dashed lines represent subtrees with posterior probabilities <97%; on

ican L. bicolor sequence (Martin et al. 2008). (a) Phylogram based on

evolutionary model was HKY + c (a = 0.36). ‘Laccaria amethystina (E

and ‘L. amethystina (Japan)’ groups the two Japanese sequences (GQ4

(34 taxa, 511 characters). The best-fit nucleotide evolutionary model

consists of groups of 13 European sequences from Belgium, Denmar

raine (GQ406393, GQ406395, GQ406412, GQ406398, GQ406399, G

GQ406411, respectively) and ‘L. amethystina (Japan)’ groups the two

based on a concatenation of the ITS, NAR and G6PD loci (28 taxa, 1

HKY + I + c (I = 0.74; a = 1.37). ‘L. bicolor’ consists of three sequence

thystina (Europe)’ groups 19 European fruitbodies, while ‘L. amethysti

Most populations displayed positive FIS values, and

all loci showed the same patterns within each popula-

tion. There were thus some heterozygote deficiencies, as

reported by Fiore-Donno & Martin (2001), Wadud et al.

(2006a) and Roy et al. (2008). This could either reflect (i)

a Wahlund effect, (ii) the presence of null alleles, (iii) a

tendency to autogamy, or (iv) any mix of these explana-

tions. No Wahlund effect was detected in CFAs within

populations (data not shown; as in Roy et al. 2008).

Null or preferentially amplified alleles may occur in our

data set, as shown by null homozygotes for locus La171

in Japan, or by the discrepancy between loci that follow

Hardy–Weinberg equilibrium and those that do not.

However, a tendency to inbreeding persisted even

when considering the four loci that did not significantly

depart from Hardy–Weinberg equilibrium. Most impor-

tantly, this tendency was also observed using a method

insensitive to null alleles (RMES; s = 0.01–0.27).

We thus suggest that some inbreeding produced local

heterozygote deficiencies, as described in this species

(Roy et al. 2008) and other basidiomycetes (Douhan

et al. 2011). Abundant aerial fruiting entails massive

emission of meiotic spores, most of which fall within a

few metres around the fruitbody (Li 2005). This may

favour mating among genetically close siblings, because

the tetrapolar incompatibility system in basidiomycetes

allows mating between some meiotic spores from the

same parent, at a rate of 25% (Niculita-Hirzel et al.

2008). Haploid hyphae generated from meiotic spores

near the parent dikaryon may even undergo dikaryon-

monokaryon mating (=di-mon mating), which is typical

for basidiomycetes, including Laccaria spp. (de La Bas-

tide et al. 1995). The level of inbreeding differed among

populations, but, because there was no correlation with

their age, a founder effect is unlikely to contribute. To

explore kinship relationships between genets at the

local scale, it is necessary to sample local populations

over several years, including small genets that may only

be detected on root tips. Beyond this tendency to

inbreeding, the absence of significant linkage disequilib-

rium between loci provides further evidence that

a spp. including European and Japanese Laccaria amethystina.

ly probabilities ‡90% are indicated. ‘L. bicolor H82’ is an Amer-

the NAR locus (42 taxa, 560 characters). The best-fit nucleotide

urope)’ consists of all 28 European sequences from this study

06524 and HQ896584). (b) Phylogram based on the G6PD locus

was K80 + c (a = 0.31). ‘Laccaria amethystina (Europe) subgroup’

k, Estonia, England, Germany, Poland, Spain, Sweden and Uk-

Q406400, GQ406402, GQ406403, GQ406046, GQ406408 and

Japanese sequences (GQ406417 and HQ896283). (c) Phylogram

706 characters). The best-fit nucleotide evolutionary model was

s of L. bicolor (two from France, one from Serbia). ‘Laccaria ame-

na (Japan)’ groups two Japanese fruitbodies.

� 2011 Blackwell Publishing Ltd

(a)

(b)

(c)

EURASIAN POPULATI ON GE NETICS OF LACCARIA AMETHYSTINA 13

� 2011 Blackwell Publishing Ltd

14 L . V I N C ENO T ET AL.

L. amethystina is mainly outcrossing and that recombi-

nation occurs within populations.

Limited geographical structure over Europe

We found very limited genetic differentiation over Europe

(FST values ranging from 0.001 to 0.134) and no IBD

over 2900 km, extending the lack of genetic structure

reported over 450 km in France (Roy et al. 2008). The

northernmost Finnish population showed higher FST

values compared with some European populations, per-

haps because of its low allelic diversity (Table 3). How-

ever, IBD may not account for this observation, because

it had no private alleles and no significant differentia-

tion from its most distant (Spanish) population. Simi-

larly, the low FST value between the two Japanese

populations that were separated by 960 km indicated

very weak IBD. Applying the equation established for

all European populations (Fig. 2a) to the distance

between the two Japanese populations provides an

expected FST value of 0.034, which is close to the

observed value (0.041). The weak IBD in L. amethystina

within Japan and Europe may result from the same

underlying mechanisms and is also reflected in the low

sequence variation within Europe and within Japan for

the five sequenced nuclear loci. Although Japanese data

are congruent with European tendencies, the discussion

below is limited to Europe, given the limited number of

Japanese populations sampled.

The absence of IBD indicates large panmictic L. ame-

thystina populations over 2900 km, with extensive gene

flow in Europe. Another less likely explanation would

be the retention of an ancient polymorphism, without

loss of ancestral alleles because of a very large effective

population size in every European site. Unfortunately,

Ne of L. amethystina remains to be estimated. Private

alleles, which may exist because of local mutations,

remain scarce in Europe (frequencies from 0.01 to 0.02),

and the dominance of the same common alleles in dif-

ferent populations supports quasi-panmixia over Eur-

ope. The abundant meiotic spore production, allowed

by the abundant fruitbody formation, may allow dis-

persal over hundreds of kilometres of at least some

spores by wind, especially by turbulence (Prospero et al.

2005), contributing to frequent gene flow between popu-

lations over large distances. Alternatively, repeated ser-

ies of short- to medium-distance dispersals may finally

build this pattern. Therefore, the heavy local spore rain

and resulting local inbreeding discussed above may be a

by-product of long-distance spore dispersal.

Although previous studies of EM species extended

over <1500 km, they demonstrated some IBD. EM spe-

cies that fruit hypogeously, or do not sporulate, show

IBD over distances of 10–100 km (Jany et al. 2002; Kret-

zer et al. 2005), while EM species dispersed by airborne

spores usually show IBD over distances of 1000 km:

Xu et al. (2008) showed IBD for Tricholoma matsutake

over 1000 km (FST values were similar to those

observed here, but for at least threefold shorter dis-

tances); Bergemann & Miller (2002) detected high IBD

among Russula brevipes populations separated by

1500 km (FST = 0.43, as between Europe and Japan in

this study). Thus, a panmictic population with gene

flow at this 2900-km scale has never before been

described in EM fungi. However, in the EM Tricholoma

‘scalpturatum’ complex, data from a limited number of

fruitbodies showing homogeneity of ITS sequences

(Jargeat et al. 2010) and Inter Simple Sequence Repeats

(ISSR) patterns (Carriconde et al. 2008) in Europe sug-

gest limited IBD over 2000 km. Some saprotrophic bas-

idiomycetes with air-dispersed spores also have low

IBD over Europe, such as Phlebiopsis gigantea (Vainio

et al. 1998) and Serpula lacrymans (Engh et al. 2010), so

this pattern may be soon discovered in some other EM

basidiomycetes.

Several nonexclusive factors may favour the absence

of IBD in European L. amethystina populations.

Although major examples of dispersal over hundreds of

kilometres for microbial species imply single-step or

gradual wind dispersal of propagules (Brown & Hov-

møller 2002; Prospero et al. 2005), anthropogenic dis-

persal may cover longer distances than airstreams. For

L. amethystina, anthropogenic dispersal (e.g. by way of

nursery trees) seems improbable in Europe, where this

species occurs in ancient natural forests rather than in

nurseries or early successional environments. Spontane-

ous aerial, gradual and multidirectional dispersal of

meiotic spores likely predominates in Europe. Land-

scape barriers hamper aerial gene flows, as shown in

Himalayan T. matsutake populations, where treeless

ridgelines act as barriers to gene flow (Amend et al.

2010). Aerial dispersal of spores throughout Europe

could be favoured by the absence of major geographical

barriers to airstreams (neither high mountain chains nor

sea channels larger than 50 km). However, in assigna-

tion tests (not shown; see Roy et al. 2008), fruitbodies

from small populations from the island of Corsica

(�100 km away from continental Europe) or Central

Europe (separated by the Alps from the study area)

belonged to the same pan-European population. Thus,

the high gene flow may also result from spore dispersal

over hundreds of kilometres.

This may be attributable to L. amethystina biological,

ecological and reproductive traits. Patchiness of host

communities and host specificity often prevent the dis-

persal and establishment of natural fungal populations

(Brown & Hovmøller 2002). For the wood saprotrophic

Datronia caperata, high host specificity exacerbates the

� 2011 Blackwell Publishing Ltd

EURASIAN POPULATI ON GE NETICS OF LACCARIA AMETHYSTINA 15

effect of fragmentation of the habitat, generating IBD

(Parrent et al. 2004). Edman et al. (2004) observed that

the dispersal and germination potential of spores were

affected by habitat fragmentation in Swedish forests for

two wood saprotrophic basidiomycetes, Fomitopsis rosea

and Phlebia centrifuga. The situation is quite different in

L. amethystina, a truly multihost, nonspecific EM species

(Roy et al. 2008): even if EM tree species differ, forests

show discontinuous but regular distribution in North-

ern and Western Europe (1–100% of forest area per

km2; data from European Forest Institute), facilitating

migration. In the comparably multihost R. brevipes,

Bergemann & Miller (2002) showed that the host popu-

lation patchiness is not a barrier to gene flow. Indeed,

we confirmed the large ecological range of L. amethysti-

na, because we found no correlation between popula-

tion differentiation and the age of the forest stand, the

climatic conditions or the geographical position (alti-

tude ⁄ latitude).

Other intrinsic features specific to L. amethystina may

also be involved. The high diversity of small genets at a

local scale (up to 14 660 genets ⁄ ha, Fiore-Donno & Mar-

tin 2001) suggests a high Ne which, together with the

repeated settlement of new spores every year (Gherbi

et al. 1999), may limit genetic drift. As an example of the

opposite tendency, Amanita muscaria displays stronger in-

tracontinental structure (Geml et al. 2009) and, although

it has aerial spore dispersal similar to that of L. amethysti-

na, it has a greater genet size and lifespan (Bagley & Orlo-

vich 2004). Lastly, high spore germination potential in

L. amethystina (Ishida et al. 2008), combined with abun-

dant meiotic spore production, may also enhance dis-

persal efficiency and gene flow. In the coming years,

comparison of diverse EM species (differing by host or

ecological specificity, size or lifespan of genets, sporulat-

ing effort, etc.) will clarify the factors determining the

strength of IBD in EM fungi (Douhan et al. 2011). In all,

the lack of major geographical barriers in Europe, as well

as the multihost strategy and population dynamics of

L. amethystina, likely favour gene flow over long dis-

tances and prevent local genetic drift in Europe.

Geographical structure (and possible speciation) overEurasia

Contrasting with the gene flow observed on a 1000-km

scale, neutral and selected molecular markers revealed

a clear divergence between Japanese and European

L. amethystina populations, i.e. over the 10 000 km Eur-

asian scale. Although Japanese and European genets

meet the criterion of ‡97% ITS similarity usually

applied to delineate conspecificity (Hughes et al. 2009),

three nuclear non-neutral loci (ITS, NAR and G6PD)

showed that Japanese haplotypes were not present in

� 2011 Blackwell Publishing Ltd

European populations and vice versa. No differentiation

was found for mitochondrial ribosomal genes: congru-

ently, 99.98% similarity over 1839 bp of mitochondrial

LrDNA was previously reported for American and

European L. bicolor differing by nuclear markers (Selos-

se et al. 1998), suggesting that genes may evolve more

slowly in mitochondria in Laccaria spp. Marked differ-

ences in allelic distributions were also observed for the

putatively neutral microsatellites, with FST reaching

0.42. Limited portabilities of markers from Europe to

Japan and vice versa also point to strong differentiation:

according to Donges et al. (2008), Roy et al. (2008) and

this study, only three of the nine markers designated

for Japanese L. amethystina by Wadud et al. (2006a)

were applicable to European samples (La03, La17, La23),

while other loci (La07, La12 and La14) were not ampli-

fied in European material. Reciprocally, Lv257 and La03

designed for European samples were monomorphic in

Japan, and La171 did not amplify for some Japanese

materials. A similarly low portability of microsatellite

loci from Japanese to American Suillus spraguei popula-

tions was recently described by Burchhardt et al. (2011).

One may a posteriori consider the population genetics

approach of limited relevance over Eurasia: this is in

itself a result of this study. Nevertheless, some micro-

satellite loci were portable and useful in the other geo-

graphical area, so that a population genetics approach

still makes sense for evolutionarily close populations.

Noteworthily, European and Japanese L. amethystina

populations have distinctive ecological traits: European

populations are late-stage EM partners in mature forests

(Gherbi et al. 1999; Fiore-Donno & Martin 2001) and so

far have been uncultivable (F. Le Tacon & M.-A. Selos-

se, unpublished observations); conversely, in Japan,

L. amethystina populations are early to middle succes-

sional, and the fungus is easily cultivable (Nara et al.

2003; Nara 2005; Wadud 2007). Although Japanese

L. amethystina tends to be generalist as well, its fruit-

bodies are often found under Salix spp., a situation very

rare in Europe (K. Nara & M.-A. Selosse, personal

observations). Lastly, preliminary morphological inves-

tigations also suggest some subtle differences (G. Muel-

ler, personal communication).

While not documented for EM species, IBD over Eurasia

is reported among saprotrophic basidiomycetes, such

as Megacollybia platyphylla (Hughes et al. 2007) and

S. lacrymans (Engh et al. 2010). Any recent founder

event is unlikely to explain our data because both

European and Japanese populations are molecularly

diverse and display private alleles; moreover, the

molecular diversity in one population is not nested

within the other, as would be expected if one popula-

tion was derived from the other (Barres et al. 2008). We

thus propose two equally parsimonious explanations:

16 L . V I N C ENO T ET AL.

(i) ancient allopatric speciation (i.e. molecular similari-

ties are plesiomorphic traits) or (ii) gradual variation

because of IBD (i.e. molecular similarities result from

limited gene flow).

Sibling allopatric biological species, which are hardly

or not distinguishable by morphology, have been dem-

onstrated in basidiomycetes within the Southern Hemi-

sphere (e.g. for the saprotrophic Ganoderma spp.,

Moncalvo & Buchanan 2008; or the EM Tylopilus spp.,

Halling et al. 2008), between Europe and North Amer-

ica (e.g. for the saprotrophic Lentinula spp., Hibbett

2001, or the parasitic Melampsora larici-populina, Barres

et al. 2008), and between Asia and North America (e.g.

for the saprotrophic S. lacrymans, Kauserud et al. 2007;

or the EM Suillus spp., Wu et al. 2000). Some globally

distributed EM species have been divided into more or

less regional biological species, such as the EM Pisoli-

thus tinctorius (Martin et al. 2002) and A. muscaria

(Geml et al. 2009), although gene flow still occurs in

some cases (Moncalvo & Buchanan 2008). Although the

definition of biological species in L. amethystina is not

possible from our data, allopatric speciation may occur.

One scenario is that during one or more past ice ages,

individuals from a unique preglacial population colo-

nized separate glacial refugia in Asia and Europe. In

Europe, refugia in Italy are postulated during the last

ice age for Tuber melanosporum (Murat et al. 2004) and

T. magnatum (Rubini et al. 2005), followed by recolon-

ization of more northern regions accompanying the

migration of host trees. At the continental scale, the

ancient populations of L. amethystina may have accu-

mulated divergences either during glacial isolation or to

adapt in different environments in Asia vs. Europe,

with limited gene flow between them after divergence.

This predicts that molecular differences do not accumu-

late gradually with distance over Eurasia, but that some

frontier between the two populations may exist; how-

ever, the story may be more complex, with secondary

hybrid zones or rare LDD events, as in allopatric speci-

ations of other basidiomycetes (Hosaka et al. 2008;

Moncalvo & Buchanan 2008).

The alternative model is a progressive IBD within a

continuously distributed species that may produce a

gradual variation over distances of 10 000 km. Similarly,

in S. lacrymans, the divergent Japanese and European

populations are both connected to a centre of diversity in

continental Asia (Engh et al. 2010). Interestingly, L. ame-

thystina IBD over Eurasia was not congruent with extrap-

olation of IBD over Europe: the slope of IBD over Eurasia

was five times higher than that over Europe (Fig. 2b).

This can be viewed as evidence for two distinct biological

species, but we cannot rule out the possibility that some

biological or geographical barriers may locally limit gene

flow and LDD over Eurasia. The prediction of a more or

less gradual genetic variation between Europe and Asia,

contrasting with the previous scenario of sharp popula-

tion delineation, can be tested in the future because

L. amethystina exists in China and Siberia.

The pattern is stranger when considering the limited

data for North American violet Laccaria species: first, a

North American L. amethystina ITS (EU819476) shows a

mix of Japanese and European traits and clusters with

Japanese sequences (Fig. 4); second, the close phyloge-

netic and genetic relationship between North American

L. amethysteo-occidentalis and L. amethystina deserves

further analysis. While the morphology groups together

Japanese and European L. amethystina and contrasts

them with the American L. amethysteo-occidentalis, G6PD

clusters together L. amethysteo-occidentalis and Japanese

L. amethystina, excluding European L. amethystina. An

appealing hypothesis is that the L. amethystina ⁄ L. ame-

thysteo-occidentalis complex undergoes a ring speciation

over the Northern Hemisphere. Ring species show a

gradual population variation between two divergent

and sympatric forms (Irwin 2009), in this case

L. amethysteo-occidentalis and L. amethystina in North

America. So far, there are few examples of true ring

speciation (Irwin 2009), and none among fungi. More

continuous sampling around the Northern Hemisphere,

especially in North America, will be required to test

this hypothesis for L. amethystina.

Perspectives

Our results support the high local genet diversity and

panmictic pattern with some inbreeding previously

reported for L. amethystina in Europe. Finer estimation

of genet turnover, inbreeding level and genet diversity

may explain gene flow at local scales. As for many

other fungal species, estimates of potential for spore

dispersal (e.g. proportion of spores able to escape and

survive in higher airstreams for LDD) would improve

our understanding of gene flow.

In vitro crossing tests may evaluate how far genetic

divergence reflects reproductive isolations over Eurasia.

Prezygotic isolation can easily be tested in vitro (as

between American and European L. bicolor; Selosse et al.

1997, 1998), but testing postzygotic isolation will require

F1 sporulating fruitbodies (Moncalvo & Buchanan 2008).

So far, although Japanese L. amethystina strains are easy

to cultivate and inoculate on seedlings (producing many

fruitbodies; Nara 2005), such tests have been hampered

by the noncultivability of European L. amethystina.

Further analyses of the L. amethystina complex and

the testing of two hypotheses (allopatric speciation vs.

progressive isolation by distance, if not a ring species)

will benefit from a combination of population genetics

and phylogenetic analyses, as in the present study. As

� 2011 Blackwell Publishing Ltd

EURASIAN POPULATI ON GE NETICS OF LACCARIA AMETHYSTINA 17

only two Japanese populations were investigated, future

studies should include more samples from Asia, to

investigate the isolation by distance between 2900 and

10 000 km. Sampling in America, especially around the

Beringia, which turns out to be of major importance in

EM biogeography (Wu et al. 2000; Geml et al. 2006,

2009), is also required. In a context where too few phy-

logeographic hypotheses are available for EM fungi (as

compared with saprobes; Halling et al. 2008; Douhan

et al. 2011), elucidating the biogeography of violet Lac-

caria spp. would generate a first evolutionary model for

EM fungi over the Northern Hemisphere.

Acknowledgements

We warmly thank Mohammad Bahram, Simon Egli, Andreas

Gminder, Ibai Olariaga Ibarguren, Pavel Kolmakov, Damjan

Krstajic, Thomas Lassøe, Mireille Lenne, Daniel Mousain, Jor-

ma Palmen, Mykola Prydiuk, Stefan Raidl, Franck Richard,

Maria Rudawska, Arne Ryberg, Stuart Skeates, Yutaka Tamai,

Alexander Urban and Sietse Van der Linde for help in sam-

pling, and Sebastien Leclerq for help in screening of microsat-

ellites. We thank Joelle Ronfort, Finn Kjellberg, Patrice David,

Francois Rousset, Noppol Kobmoo, Pierre-Olivier Cheptou and

Mathieu Sauve for discussions on population genetics, as well

as Marc de Dinechin and Pierre-Henri Fabre for help in phylo-

genetic analyses. We also acknowledge Gerhardt Kost, Thomas

Kuyper, Greg Mueller and Pierre-Arthur Moreau for inspiring

discussions on the model taxon and three anonymous referees

for their comments on an earlier version of this manuscript,

and David Marsh for English correction. This work was

funded by the European Commission Network of Excellence

EVOLTREE (to F.M. and M.-A.S.) and the Agence Nationale de

la Recherche (SYSTRUF programme to M.-A.S). L.T. receives

support from ESF grants JD-92, 6606 and FIBIR. Most molecu-

lar data used in this work were produced at the IFR119 ‘Mont-

pellier Environnement Biodiversite’.

References

Abarenkov K, Nilsson RH, Larsson K-H et al. (2010) The UNITE

database for molecular identification of fungi—recent updates

and future perspectives. New Phytologist, 186, 281–285.

Amend A, Garbelotto M, Fang Z, Keeley S (2010) Isolation by

landscape in populations of a prized edible mushroom

Tricholoma matsutake. Conservation Genetics, 11, 795–802.

Bagley SJ, Orlovich DA (2004) Genet size and distribution of

Amanita muscaria in a suburban park, Dunedin, New

Zealand. New Zealand Journal of Botany, 42, 939–947.

Barres B, Halkett F, Dutech C, Andrieux A, Pinon J, Frey P

(2008) Genetic structure of the poplar rust fungus

Melampsora larici-populina: evidence for isolation by distance

in Europe and recent founder effects overseas. Infection,

Genetics and Evolution, 8, 577–587.

Bergemann SE, Miller SL (2002) Size, distribution, and

persistence of genets in local populations of the late-stage

ectomycorrhizal basidiomycete, Russula brevipes. New

Phytologist, 156, 313–320.

� 2011 Blackwell Publishing Ltd

Brown JKM, Hovmøller MS (2002) Epidemiology—aerial

dispersal of pathogens on the global and continental scales

and its impact on plant disease. Science, 297, 537–541.

Burchhardt KM, Rivera Y, Baldwin T, Vanearden D, Kretzer

AM (2011) Analysis of genet size and local gene flow in the

ectomycorrhizal basidiomycete Suillus spraguei (synonym

S. pictus). Mycologia, 103, 722–730.

Carriconde F, Gardes M, Jargeat P, Heilmann-Clausen J,

Mouhamadou B, Gryta H (2008) Population evidence of

cryptic species and geographical structure in the

cosmopolitan ectomycorrhizal fungus, Tricholoma

scalpturatum. Microbial Ecology, 56, 513–524.

David P, Pujol B, Viard F, Castella V, Goudet J (2007) Reliable

selfing rate estimates from imperfect population genetic

data. Molecular Ecology, 16, 2474–2487.

Dilmaghani A, Balesdent MH, Didier JP et al. (2009) The

Leptosphaeria maculans—Leptosphaeria biglobosa species complex

in the American continent. Plant Pathology, 58, 1044–1058.

Donges K, Schlobinski D, Cremer E, Rexer KH, Kost G (2008)

Six newly developed microsatellite markers of Laccaria

amethystina, using an improved CSSR approach. Mycological

Progress, 7, 285–290.

Douhan GW, Huryn KL, Douhan LI (2007) Significant diversity

and potential problems associated with inferring population

structure within the Cenococcum geophilum species complex.

Mycologia, 99, 812–819.

Douhan G, Vincenot L, Gryta H, Selosse MA (2011) Population

genetics of ectomycorrhizal fungi: from current knowledge

to emerging directions. Fungal Biology, 115, 569–597.

Dunham SM, Kretzer A, Pfrender ME (2003) Characterization

of Pacific golden chanterelle (Cantharellus formosus) genet size

using co-dominant microsatellite markers. Molecular Ecology,

12, 1607–1618.

Edman M, Gustafsson M, Stenlid J, Ericson L (2004)

Abundance and viability of fungal spores along a forestry

gradient—responses to habitat loss and fragmentation?

Oikos, 104, 35–42.

Engh IB, Carlsen T, Sætre GP, Hogberg N, Doi S, Kauserud H

(2010) Two invasive populations of the dry rot fungus

Serpula lacrymans show divergent population genetic

structures. Molecular Ecology, 19, 706–715.

Excoffier L, Lischer HEL (2010) Arlequin suite ver. 3.5: a new

series of programs to perform population genetics analyses

under Linux and Windows. Molecular Ecology Resources, 10,

564–567.

Fiore-Donno AM, Martin F (2001) Populations of

ectomycorrhizal Laccaria amethystina and Xerocomus spp.

show contrasting colonization patterns in a mixed forest.

New Phytologist, 152, 533–542.

Gardes M, Bruns TD (1993) ITS primers with enhanced

specificity for basidiomycetes: application to the identification

of mycorrhiza and rusts. Molecular Ecology, 2, 113–118.

Geml J, Laursen GA, O’Neill K, Nusbaum HC, Taylor DL

(2006) Beringian origins and cryptic speciation events in the

fly agaric Amanita muscaria. Molecular Ecology, 15, 225–239.

Geml J, Laursen GA, Timling I et al. (2009) Molecular

phylogenetic biodiversity assessment of arctic and boreal

ectomycorrhizal Lactarius Pers. (Russulales; Basidiomycota)

in Alaska, based on soil and sporocarp DNA. Molecular

Ecology, 18, 2213–2227.

18 L . V I N C ENO T ET AL.

Gherbi H, Delaruelle C, Selosse MA, Martin F (1999) High

genetic diversity in a population of the ectomycorrhizal

basidiomycete Laccaria amethystina in a 150-year-old beech

forest. Molecular Ecology, 12, 2003–2013.

Guillot G, Santos F, Estoup A (2008) Analysing georeferenced

population genetics data with Geneland: a new algorithm to

deal with null alleles and a friendly graphical user interface.

Bioinformatics, 24, 1406–1407.

Guindon S, Gascuel O (2003) A simple, fast and accurate

method to estimate large phylogenies by maximum

likelihood. Systematic Biology, 52, 696–704.

Halling RE, Osmundson TW, Neves MA (2008) Pacific boletes:

implications for biogeographic relationships. Mycological

Research, 112, 437–447.

Hibbett DS (2001) Shiitake mushrooms and molecular clocks:

historical biogeography of Lentinula. Journal of Biogeography,

28, 231–241.

Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A (2005)

Very high resolution interpolated climate surfaces for global

land areas. International Journal of Climatology, 25, 1965–1978.

Hosaka K, Castellano MA, Spatafora JW (2008) Biogeography

of Hysterangiales (Phallomycetidae, Basidiomycota).

Mycological Research, 112, 448–462.

Hughes KW, Petersen RH, Lickey EB (2007) Megacollybia

(Agaricales). Reports of the Tottori Mycological Institute, 45, 1–57.

Hughes KW, Petersen RH, Lickey EB (2009) Using

heterozygosity to estimate a percentage DNA sequence

similarity for environmental species’ delimitation across

basidiomycete fungi. New Phytologist, 182, 795–798.

Irwin DE (2009) Incipient ring speciation revealed by a

migratory divide. Molecular Ecology, 18, 2923–2925.

Ishida TA, Nara K, Tanaka M, Kinoshita A, Hogetsu T (2008)

Germination and infectivity of ectomycorrhizal fungal spores

in relation to their ecological traits during primary

succession. New Phytologist, 180, 491–500.

James TY, Porter D, Hamrick JL, Vilgalys R (1999) Evidence for

limited intercontinental gene flow in the cosmopolitan

mushroom, Schizophyllum commune. Evolution, 53, 1665–1677.

Jany JL, Garbaye J, Martin F (2002) Cenococcum geophilum

populations show a high degree of genetic diversity in beech

forests. New Phytologist, 154, 651–659.

Jargeat P, Martos F, Carriconde F, Gryta H, Moreau P-A,

Gardes M (2010) Phylogenetic species delimitation in

ectomycorrhizal fungi and implications for barcoding: the

case of the Tricholoma scalpturatum complex (Basidiomycota).

Molecular Ecology, 19, 5216–5230.

Kauserud H, Svegarden IB, Sætre GP et al. (2007) Asian origin

and rapid global spread of the destructive dry rot fungus

Serpula lacrymans. Molecular Ecology, 16, 3350–3360.

Kretzer AM, Dunham S, Molina R, Spatafora JW (2005)

Patterns of vegetative growth and gene flow in Rhizopogon

vinicolor and R. vesiculosus (Boletales, Basidiomycota).

Molecular Ecology, 14, 2259–2268.

de La Bastide PY, Kropp BR, Piche R (1995) Vegetative

interactions among mycelia of Laccaria bicolor in pure culture

and in symbiosis with Pinus banksiana. Canadian Journal of

Botany, 73, 1768–1779.

Labbe J, Zhang X, Yin T et al. (2008) A genetic linkage map for

the ectomycorrhizal fungus Laccaria bicolor and its alignment

to the whole-genome sequence assemblies. New Phytologist,

180, 316–328.

Li DW (2005) Release and dispersal of basidiospores from

Amanita muscaria var. alba and their infiltration into a

residence. Mycological Research, 109, 1235–1242.

Martin F, Selosse M-A, Le Tacon F (1999) The nuclear

ribosomal DNA intergenic spacers of the ectomycorrhizal

basidiomycete Laccaria bicolor: structural analysis and

intraspecific polymorphisms. Microbiology, 145, 1605–1611.

Martin F, Diez J, Dell B, Delaruelle C (2002) Phylogeography of

the ectomycorrhizal Pisolithus species as inferred from nuclear

ribosomal DNA ITS sequences. New Phytologist, 153, 345–357.

Martin F, Aerts A, Ahren D et al. (2008) The genome of

Laccaria bicolor provides insights into mycorrhizal symbiosis.

Nature, 452, 88–93.

Moncalvo JM, Buchanan PK (2008) Molecular evidence for

long distance dispersal across the Southern Hemisphere in

the Ganoderma applanatum-australe species complex

(Basidiomycota). Mycological Research, 112, 425–436.

Mueller GM, Schmit JP, Leacock PR et al. (2007) Global

diversity and distribution of macrofungi. Biodiversity and

Conservation, 16, 37–48.

Murat C, Diez J, Luis P et al. (2004) Polymorphism at the

ribosomal DNA ITS and its relation to postglacial re-

colonization routes of the Perigord truffle Tuber

melanosporum. New Phytologist, 164, 401–411.

Nara K (2005) Ectomycorrhizal networks and seedling

establishment during early primary succession. New

Phytologist, 169, 169–178.

Nara K, Nakaya H, Wu B, Zhou Z, Hogetsu T (2003)

Underground primary succession of ectomycorrhizal fungi in

a volcanic desert on Mount Fuji. New Phytologist, 159, 743–746.

Niculita-Hirzel H, Labbe J, Kohler A et al. (2008) Gene

organization of the mating type regions in the

ectomycorrhizal fungus Laccaria bicolor reveals distinct

evolution between the two mating type loci. New Phytologist,

180, 329–342.

Nielsen R, Wakeley JW (2001) Distinguishing migration from

isolation: an MCMC approach. Genetics, 158, 885–896.

Nylander JAA (2004) MrModeltest v2. Program distributed by

the author. Evolutionary Biology Centre, Uppsala University,

Uppsala, Sweden.

O’Malley MA (2007) The nineteenth century roots of

‘everything is everywhere’. Nature Reviews Microbiology, 5,

647–651.

Parrent JL, Garbelotto M, Gilbert GS (2004) Population genetic

structure of the polypore Datronia caperata in fragmented

mangrove forests. Mycological Research, 108, 403–410.

Pritchard JK, Stephens M, Donnelly P (2000) Inference of

population structure using multilocus genotype data.

Genetics, 155, 945–959.

Prospero JM, Blades E, Mathison G, Naidu R (2005)

Interhemispheric transport of viable fungi and bacteria from

Africa to the Caribbean with soil dust. Aerobiologia, 21, 1–19.

Queloz V, Sieber TN, Holdenrieder O, McDonald BA, Grunig CR

(2011) No biogeographical pattern for a root-associated fungal

species complex. Global Ecology and Biogeography, 20, 160–169.

Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian

phylogenetic inference under mixed models. Bioinformatics,

19, 1572–1574.

Rousset F (2008) GENEPOP’007: a complete re-implementation

of the GENEPOP software for Windows and Linux.

Molecular Ecology Resources, 8, 103–106.

� 2011 Blackwell Publishing Ltd

EURASIAN POPULATI ON GE NETICS OF LACCARIA AMETHYSTINA 19

Roy M, Dubois MP, Proffit M, Vincenot L, Desmarais E,

Selosse MA (2008) Evidence from population genetics that

the ectomycorrhizal basidiomycete Laccaria amethystina is an

actual multihost symbiont. Molecular Ecology, 17, 2825–2838.

Rubini A, Paolocci F, Riccioni C, Vendramin GG, Arcioni S

(2005) Genetic and phylogeographic structures of the

symbiotic fungus Tuber magnatum. Applied and Environmental

Microbiology, 71, 6584–6589.

Selosse MA, Martin F, Bouchard D, Le Tacon F (1997)

Temporal persistence and spatial distribution of an

American inoculant strain of the ectomycorrhizal

basidiomycete Laccaria bicolor in French plantations.

Molecular Ecology, 7, 561–574.

Selosse MA, Jacquot D, Bouchard D, Martin F, Le Tacon F

(1998) Survival of an introduced ectomycorrhizal Laccaria

bicolor strain in a European forest plantation monitored by

mitochondrial ribosomal DNA analysis. New Phytologist, 140,

753–761.

Swofford DL (2002) PAUP*: Phylogenetic Analysis Using

Parsimony (*and Other Methods). Version 4.10 b. Sinauer,

Sunderland, Massachusetts, USA.

Taylor JW, Jacobson DJ, Kroken S et al. (2000) Phylogenetic

species recognition and species concepts in fungi. Fungal

Genetics and Biology, 31, 21–32.

Taylor AFS, Hills AE, Simonini G, Both EE, Eberhardt U (2006)

Detection of species within the Xerocomus subtomentosus

complex in Europe using rDNA-ITS sequences. Mycological

Research, 110, 276–287.

Vainio EJ, Korhonena K, Hantulaa J (1998) Genetic variation in

Phlebiopsis gigantea as detected with random amplified

microsatellite (RAMS) markers. Mycological Research, 102,

187–192.

Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P

(2004) MICRO-CHECKER: software for identifying and

correcting genotyping errors in microsatellite data. Molecular

Ecology Notes, 4, 535–538.

Wadud MA (2007) Reproduction ecology of pioneer ectomycorrhizal

fungi, Laccaria amethystina and L. laccata, in the volcanic

desert on Mount Fuji. PhD Thesis, The University of Tokyo,

Tokyo, Japan.

Wadud MA, Lian CL, Nara K, Ishida TA, Hogetsu T (2006a)

Development of microsatellite markers from an ectomycorrhizal

fungus, Laccaria amethystina, by a dual-suppression-PCR

technique. Molecular Ecology Notes, 6, 130–132.

Wadud MA, Lian CL, Nara K, Hogetsu T (2006b) Isolation and

characterization of five microsatellite loci in an

ectomycorrhizal fungus Laccaria laccata. Molecular Ecology

Notes, 6, 700–702.

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and

direct sequencing of fungal ribosomal RNA genes for

phylogenetics. In: PCR Protocols. A Guide to Methods and

Applications (eds. Innis MA, Gelfand DH, Sninsky JJ, White

JJ), pp. 315–322. Academic Press, San Diego, California.

Wu QX, Mueller GM, Lutzoni FM, Huang YQ, Guo SY (2000)

Phylogenetic and biogeographic relationships of Eastern

Asian and Eastern North American disjunct Suillus species

(Fungi) as inferred from nuclear ribosomal RNA ITS

sequences. Molecular Phylogenetics and Evolution, 17, 37–47.

Xu J, Sha T, Li YC, Zhao ZW, Yang ZL (2008) Recombination

and genetic differentiation among natural populations of the

� 2011 Blackwell Publishing Ltd

ectomycorrhizal mushroom Tricholoma matsutake from

southwestern China. Molecular Ecology, 17, 1238–1247.

This work is part of L.V.’s PhD thesis on Laccaria amethystina

population genetics. She did the molecular typing and genetic

analyses, with help from M.-P.D., and J.L., C.S., K.N. and L.T.

helped with sampling and data analysis. L.V. wrote the paper

together with M.-A.S. This project was designed by M.-A.S.

with the help of F.M.

Data accessibility

DNA sequences of microsatellite alleles for loci Jl1195M13,

1209M5, Lv257: GenBank accessions GQ483297–GQ483302

(Data S1).

DNA sequences for multiple gene phylogenies: GenBank acces-

sions GQ406389–GQ406568 plus HQ896282–HQ896286 (Data

S2).

Phylogenetic data for ITS, ITS extended, NAR, G6PD, three

concatenated nuclear loci, LsurRNA, SsurRNA: TreeBASE

Study accessions no.11481, 11482, 11483, 11484, 11485, 11486,

11487.

Data deposited in the Dryad repository: doi: 10.5061/

dryad.21pm7p6n.

Supporting information

Additional supporting information may be found in the online

version of this article.

Fig. S1 Structure diagram of European and Japanese popula-

tions, based on the software STRUCTURE 2.2 for K = 2 clusters.

Fig. S2 Bayesian phylogram of Laccaria spp. using ITS

sequences from this study, Unite and GenBank (138 taxa, 622

characters).

Fig. S3 Alignment of ITS sequences from L. amethystina and

L. amethysteo-occidentalis.

Fig. S4 Unrooted Bayesian phylograms of L. amethystina in

Europe and Japan for two mitochondrial ribosomal genes,

SrRNA (a, 29 taxa, 477 characters) and LrRNA (b, 37 taxa, 410

characters).

Data S1. Microsatellites diploid genotypes for 667 individuals

in 18 L. amethystina populations, 9 microsatellites loci.

Data S2. Genbank accession numbers for sequences of 5 coding

or non-coding loci, on purpose of phylogenetic analysis. Asso-

ciated phylogenetic trees are available on TREEBASE, study

accessions no.11481, 11482, 11483, 11484, 11485, 11486, 11487.

Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting information supplied by the

authors. Any queries (other than missing material) should be

directed to the corresponding author for the article.