Phylogeny of matsutake

Phylogenetic relationship and species delimitation of matsutake and allied species based on

multilocus phylogeny and haplotype analyses

Yuko Ota1

Takashi Yamanaka

Department of Forest Microbiology, Forestry and Forest Products Research Institute,

Tsukuba, lbaraki 305-8687, Japan

Hitoshi Murata

Hitoshi Neda

Department of Applied Microbiology and Mushroom Sciences, Forestry and Forest Products

Research Institute, Tsukuba, lbaraki 305-8687, Japan

Akira Ohta

Shiga Forest Research Center, Yasu, Shiga 520-2321, Japan

Masataka Kawai

Nara Forest Research Institute, Takatori, Nara 635-0133, Japan

Miki Konno

Miyagi Prefectural Forestry Technology Institute, Ohira, Miyagi 981-3602, Japan

Chihiro Tanaka

Laboratory of Environmental Mycoscience, Graduate School of Agriculture, Kyoto

University, Kyoto 606-8502, Japan

Abstract: Tricholoma matsutake (S. Ito & S. Imai) Singer and its allied species are referred

to as matsutake worldwide and are the most economically important edible mushrooms in

Japan. They are widely distributed in the northern hemisphere and established an

ectomycorrhizal relationship with conifer and broadleaf trees. To clarify relationships among

T. matsutake and its allies, and to delimit phylogenetic species, we analyzed multilocus

In Press at Mycologia, preliminary version published on June 8, 2012 as doi:10.3852/12-068

Copyright 2012 by The Mycological Society of America.

datasets (ITS, megB1, tef, gpd) with samples that were correctly identified based on

morphological characteristics. Phylogenetic analyses clearly identified four major groups:

matsutake, T. bakamatsutake, T. fulvocastaneum and T. caligatum; the latter three species

were outside the matsutake group. The haplotype analyses and median-joining haplotype

network analyses showed that the matsutake group included four closely related but clearly

distinct taxa (T. matsutake, T. anatolicum, Tricholoma sp. from Mexico and T. magnivelare)

from different geographical regions; these were considered to be distinct phylogenetic

species.

Key words: ectomycorrhizal fungi, median-joining haplotype network

INTRODUCTION

Tricholoma matsutake is the most economically important edible mushroom in Japan.

Although efforts have been made over a long period to establish an artificial cultivation

system, this mushroom so far has remained uncultivable. More than 2000 tons of matsutake

mushroom (T. matsutake and its allied species), accounting for about 97% of Japanese

consumption, are imported annually from China, Korea, Bhutan, Morocco, Turkey, Canada,

Mexico and the United States (Japan Tariff Association 2010). The species delimitation of

authentic T. matsutake has attracted attention.

Tricholoma matsutake and its allied species are ectomycorrhizal fungi belonging to

the genus Tricholoma (Tricholomataceae, Agaricales, Agaricomycetidae, Agaricomycetes)

and are widely distributed in the northern hemisphere. Tricholoma matsutake is associated

generally with conifers and inhabits the Far East, the Himalayan region and Scandinavia

(Hosford et al. 1997, Ogawa 1978). Other conifer-symbiotic matsutake species are T.

anatolicum H.H. Doğan & Intini inhabiting the Mediterranean region (Intini et al. 2003), T.

magnivelare (Peck) Redhead from Canada and the Pacific Northwest of North America to

Mexico (Hosford et al. 1997, Yamada et al. 2010) and T. robustum (Alb. & Schwein.) Ricken

from Europe (Bon 1984) and the Far East (Ito 1959, Ogawa 1978).

Tricholoma bakamatsutake Hongo and T. fulvocastaneum Hongo from the Far East

and the Himalayan region are matsutake-allied species that are symbiotic with broadleaf trees

(Fagaceae) (Hosford et al. 1997, Ogawa 1978). These species often share the habitat of T.

matsutake in mixed forests of conifers and broadleaf trees (Mastushita et al. 2005, Yamanaka

et al. 2011), and these three species closely resemble each other morphologically. A similar

case in North America is the matsutake ally T. caligatum (Viv.) Ricken from the United

States and Mexico, where this fungus is believed to associate with angiosperm hosts, even

though it is found in mixed forests, often in sympatry with T. magnivelare (Hosford et al.

1997).

Despite the economic and ecological importance, the phylogenetic relationships

among matsutake species and their species delimitations remain elusive. A few studies have

applied molecular approaches to the phylogeny of T. matsutake and its allied species. Chapela

and Garbelotto (2004) revealed that T. magnivelare and T. caligatum were polyphyletic based

on 46 ITS sequences and AFLP analyses using matsutake species mainly from coniferous

forests. The phylogenetic positions of T. bakamatsutake and T. fulvocastaneum from

broadleaf trees in Japan were not determined because few isolates were included as outgroups

(Chapela and Garbelotto 2004, Lim et al. 2003, Matsushita et al. 2005). In addition, studies

indicated that a phylogeny based on one locus (the ITS region) gives low resolution.

Our objectives were: (i) to clarify relationships among T. matsutake and its allies,

inferred from multilocus sequence analyses, and (ii) to delimit the phylogenetic species of T.

matsutake and its close relatives. We focused on phylogenetic species delimitation and the

phylogenetic position of three morphologically similar species that are sometimes confused if

mature basidiocarps are not available. All, T. bakamatsutake, T. fulvocastaneum and T.

robustum, are found in Japan

MATERIALS AND METHODS

Fungal materials.—We used samples or isolates that were identified based on voucher specimens and/or DNA

samples authorized by former studies. Fungal materials are provided (TABLE I). T. matsutake was collected from

the Far East (Japan, Korea, China, Russia), the Himalayan region (Bhutan and China) and Scandinavia (Norway,

Finland). The NBRC (NITE Biological Resource Center, Japan) isolates, NBRC6932 and 6933 for T. matsutake,

6942 and 6947 for T. fulvocastaneum, 108266 and 107029 for T. bakamatsutake, were used as reference. Isolate

240211 was from the syntype of T. bakamatsutake (specimen 1283, described by Hongo 1974). We used a

sample of T. caligatum from Italy, where this species originally was described (Viviani 1834). The sample of T.

caligatum from Spain was identified as T. caligatum based on its morphological characteristics. Our isolates of T.

magnivelare were derived from commercial materials that were imported from Canada and were identified as T.

magnivelare based on their morphology. Preliminary analysis of ITS sequences of T. magnivelare, identified by

Lim et al. (2003), (GenBank accession numbers AF527370, AF527368, AF527371; specimens DAVFP25966,

DAVFP25945, DAVFP26220) confirmed that the samples we used were identical to these. The other species

also were identified based on its morphological characteristics in comparison with voucher specimens.

Molecular techniques.—DNA was isolated from voucher specimens and cultured mycelia. Cultures were grown

at 25 C in MMN liquid medium modified by the addition of 1.5% V8 juice, instead of NaCl (Campbell Soup

Co., Camden, New Jersey), or in 10 mL liquid MYG medium (2% [w/v] malt extract, 0.2% [w/v] yeast extract,

2% [w/v] glucose) at 25 C in the dark and harvested 10 d after inoculation. DNA was extracted from frozen

mycelia with a lysis buffer containing hexadecyltrimethylammonium bromide and phenol-chloroform (Murata

et al. 1999, Dobinson et al.1993) or using a DNeasy extraction kit (QIAGEN, Valencia, California) following

the manufacturer's protocol. DNA from dried specimens was extracted from 5 mm2 sections of hymenial layers

with a fungal DNA isolation kit (Omega Bio-Tek Inc. Norcross, Georgia) following the manufacturer's protocol.

We analyzed sequences from four genetic markers consisting of these genetic characteristics: the ITS region of

nuclear ribosomal DNA (ITS), megB1 (a short interspersed nuclear elements [SINE] dimer), the translation

elongation factor 1α (tef) and glyceraldehydes-3-phosphodelhydorogenase (gpd). The megB1 is a single copy

element and originally was identified as a marker specific to Basidiomycota (Babasaki et al. 2007).

The oligonucleotide primers were ITS1-F and ITS4-B for ITS (Gardes and Bruns 1993), EF595F and

EF1160R for tef (Kauserud and Schumacher 2001), GPD-F (Johannesson et al. 2000) and GPD-R′ (Jargeat et al.

2010) for gpd and TS1_fwc2 and TS2_rvc1 (Babasaki et al. 2007) for megB1. Each PCR contained

approximately 10 ng template DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 (0.1) mM of

each primer, 2.5 mM (2 mM) each dNTP and Takara taq or Takara Extaq (0.5 U) (Takara, Otsu, Japan) in 20 mL

total volume. DNA amplification was performed with an Applied Biosystems Perkin-Elmer DNA thermal-cycler

(9800) or BIO-RAD iCycler under these conditions: 5 min at 94 C, followed by 35 cycles of 1 min at 94 C, 30

s at 52–56 C and 30 s at 72 C, with a final extension of 7 min at 72 C. PCR products were purified with

MicroSpin columns and Sephacryl S-300 (GE Healthcare, Piscataway, New Jersey). Direct sequencing of PCR

products was conducted with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied

Biosystems, Foster City, California) with an Applied Biosystems 3100 sequencer.

Phylogenetic analyses.—Representative DNA sequences of amplified fragments deposited in GenBank are

included (TABLE I). We included megB1 and ITS sequence data used in Babasaki et al. (2007), Murata et al.

92002) and Yamada et al. (2010). Sequences were aligned with MAFFT software (Katoh et al. 2002) with the

E-INS-I option and corrected manually in BioEdit (Hall 1999). For each gene fragment, coding and noncoding

regions were assigned by comparison with published data. Sequences were deposited in TreeBASE

(http://purl.org/phylo/treebase/phylows/study/TB2:S12515). Phylogenetic analyses of the aligned sequences

were performed with NJ and ML in MEGA 5.05 (Tamura et al. 2011) and Bayesian analysis using MrBayes

3.1.2 (Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003). The positions where gaps were

present were deleted. The strength of the internal branches of the resulting NJ and ML trees was tested

statistically by bootstrap analysis (Felsenstein 1985) from 1000 bootstrap replications. The NJ tree was used for

the Bayesian analysis, and the best-fit evolutionary model was determined by comparing different evolutionary

models via hLRT (hierarchical likelihood ratio test), using PAUP* 4.0b10 (Swofford 2002) and MrModeltest 2.3

(Nylander 2004). The GTR+G model for the ITS and the K80 model for the 5.8S region were selected as the

best-fit evolutionary models respectively. The tef data was partitioned into four; the noncoding region and first,

second and third codon positions of the coding region. F81, JC+G, HKY+G and GTR+G were applied as the

best-fit evolutionary models for each part respectively. The gpd data was partitioned into four; the noncoding

region and first, second and third codon positions of the coding region. GTR+I, F81+G, F81+G and HKY+G

were applied as the best-fit evolutionary models for each part respectively. GTR+G was applied to MegB1 data

as the best-fit evolutionary model. Two million generations were used for ITS, 3 000 000 generations for tef and

gpd and 1 000 000 generations for megB1. The combined dataset was run in four chains, and the chains were

sampled every 100th generation. After running the analysis, the standard deviation of the split frequencies was

examined to confirm that it was below 0.01, and the potential scale reduction factor was examined to confirm

that all parameters were close to 1.0 and that the plot of the generations versus the log probability of the data had

reached the stationary phase (was not increasing or decreasing). The first 25% of trees (5000 for ITS, 7500 for

tef, gpd and the combined dataset, and 2500 for megB1) were considered the burn-in and excluded from

construction of the consensus tree. A 50% majority rule consensus cladogram was computed from the remaining

trees.

Two separate phylogenetic analyses were performed. First, analyses using 11 Tricholoma species/taxa

were performed to clarify the outline of phylogeny within Tricholoma. Clitocybe is a genus that appears

consistently near Tricholoma in the phylogenetic arrangement (Matheny et al. 2006, Moncalvo et al. 2002).

However the preliminary analyses with Clitocybe species as outgroup did not show an adequate basal position.

Therefore, we used as outgroup Armillaria species, which are relatively close to species of Tricholoma and

harbor megB1 in genomic regions other than rDNA such as matsutake-producing Tricholoma (Babasaki et al.

2007). In fact, Armillaria species as outgroup conferred reasonable phylogenetic trees with any markers and

analytical methods we have used (GenBank accession numbers AB10761-3, AB10782, AB10881, AB10884,

AB10885, AB10897). Second, limited species close to T. matsutake were analyzed to obtain finer resolution of

their relationships.

Statistical analyses and network methods.—Statistical analyses and network methods were performed on T.

matsutake, T. anatolicum, Tricholoma sp. from Mexico, T. magnivelare, T. bakamatsutake, T. fulvocastaneum

and T. caligatum. To use the information in the heterozygous sequence sites, haplotype datasets were constructed

for both DNA regions, with homozygous sequences and sequences with only one heterozygous position. In a

DNA sequence containing a “Y”, for example, the two resulting sequence haplotypes were included. Estimates

of molecular variation (nucleotide diversity [π] [Nei 1987, equation 10.5], average number of nucleotide

differences [k] [Tajima 1983, equation A3] and nucleotide divergence [K] [Nei 1987, equation 10.21]) were

calculated with DnaSP 5.10.01(Librado and Rozas 2009).

Median-joining haplotype networks were drawn for four regions of haplotype data from T. matsutake,

T. anatolicum, Tricholoma sp. from Mexico and T. magnivelare, using Network 4.516

(http://www.fluxus-engineering.com; Bandelt et al. 1999).

RESULTS

Sequence data.—Complete ITS sequences and parts of two loci for tef and gpd, and a SINE

dimer megB1 were successfully amplified and sequenced from the majority of the isolates,

150 sequences in total (TABLE I). The length of each gene fragment varied (640–667 bp for

ITS, 444–498 bp for megB1, 456–472 bp for tef and 651–668 bp for gpd).

The topologies of the NJ, ML and Bayesian trees, based on each of the four DNA

regions with Armillaria species as outgroup, showed some minor differences, but all trees

showed generally consistent relationships among the strongly supported clades

(SUPPLEMENTARY FIG. 1a–d). The entire ITS2 region was eliminated from the dataset because

it was difficult to align. The gpd data was not combined with the dataset because gpd

sequence data were not obtained from T. robustum. The phylogenetic analyses of the

combined ITS dataset (ITS1, 5.8S), tef and megB1, clearly showed four distinct clades: (i)

the matsutake group, consisting of T. matsutake, T. anatolicum, Tricholoma sp. from Mexico

and T. magnivelare; (ii) the T. bakamatsutake clade; (iii) the T. fulvocastaneum clade; and (iv)

the T. caligatum clade. The latter three were placed outside the matsutake group. Tricholoma

robustum, T. focale, T. japonicum, T. flavovirens and T. portentosum were placed outside

those four clades (FIG.1).

The phylogenetic analysis focusing on the matsutake group (T. matsutake, T.

anatolicum, Tricholoma sp. from Mexico, T. magnivelare), based on combined datasets and

data from ITS (ITS1, 5.8S, ITS 2), megB1, tef and gpd, defined several strongly supported

clades (FIG. 2). The topologies of the NJ, ML and Bayesian trees based on each of the four

DNA regions showed some minor differences, but all trees showed generally consistent

relationships among the strongly supported clades (data not shown). Tricholoma magnivelare

formed a strongly supported clade outside T. matsutake, T. anatolicum and T. sp. from

Mexico (FIG. 2). T. anatolicum and Tricholoma sp. from Mexico formed distinct clades.

Tricholoma matsutake formed a distinct clade with some strongly supported subclades.

However, there was no clear geographic separation within the T. matsutake subclade (FIG. 2).

Haplotype analyses and genetic diversity.—A few sequences included only one heterozygous

position, and it was possible to identify haplotypes and to generate haplophase datasets, with

48 ITS sequences (11 haplotypes), 43 tef sequences (10 haplotypes), 42 gpd sequences (eight

haplotypes) and 48 megB1 sequences (25 haplotypes). The data for Tricholoma sp. from

Mexico are not included because two haplotypes were not enough for analyses. The most

sequence variation, measured as nucleotide diversity (π) within each haplophase dataset,

appeared in the megB1 data (π = 0.082); there was less in the ITS (π = 0.070), and much less

in the tef (π = 0.054) and gpd (π = 0.052). Within each species, and for each marker,

nucleotide diversity was low, 0–0.009 (TABLE II). Intraspecific nucleotide divergence (K) was

not higher than 0.92% (megB1) and 0–0.43% for ITS, tef, and gpd (TABLE II). In contrast,

interspecific values for π, k and genetic divergence values (K) were always higher than the

intraspecific value of each (TABLE II). The π, k and K values of T. matsutake and T.

anatolicum in each locus were apparently very low, compared with the other combinations,

whereas the π, k and K values of each T. matsutake and the combination of Tmat/Tana were

at least 1.4 times greater megB1 for π, 1.4 times megB1 for K and 2.34 times megB1 for K.

Network analyses.—These, carried out with each ITS, tef, and gpd dataset, detected a clear

segregation among T. magnivelare, Tricholoma sp. from Mexico and T. anatolicum (FIG.

3a–c). The network of megB1 haplotypes showed the clades of T. magnivelare, Tricholoma sp.

from Mexico and T. anatolicum, whereas the haplotypes of T. matsutake were positioned at

the center of these species without clear structure (FIG. 3d).

DISCUSSION

The phylogenetic tree based on the multilocus dataset clearly identified four major clades, the

matsutake group, T. bakamatsutake, T. fulvocastaneum and T. caligatum. The matsutake

group included four distinct taxa from different geographical regions (FIG. 2): T. matsutake

from Asia and Europe, T. anatolicum from Morocco and Turkey, Tricholoma sp. from

Mexico and T. magnivelare from Canada. Within the matsutake group, T. magnivelare was

clearly distinct from T. matsutake, T. anatolicum and Tricholoma sp. from Mexico (FIG. 2).

Such a trend is consistent with that of the distributions of the matsutake-specific

retrotransposon marY1, a retrovirus-like DNA carrying long terminal repeat (LTR)

accumulated in the genome. For example, the copy number of marY1 is the same among

matsutake (i.e. T. matsutake, T. anatolicum, Tricholoma sp. from Mexico and T. magnivelare,

while that is ca. 103-fold less in T. bakamatsutake and T. fulvocastaneum) (Murata and

Babasaki 2005). In addition, interretrotransposon amplified polymorphism targeting marY1 in

T. magnivelare is distinct from those in T. matsutake, T. anatolicum and Tricholoma sp. from

Mexico (Murata et al. 2005). The present study based on neutral mutations, along with the

previous ones based on retrotransposon distribution, strongly suggests that matsutake and

allied species were diverged from their ancestral species followed by further diversification

within the matsutake group and among allied species (T. bakamatsutake, T. fulvocastaneum, T.

caligatum) in the northern hemisphere.

Tricholoma magnivelare was divided into several natural groups and revealed as a

complex by Chapela and Garbelotto (2004). The distribution of T. magnivelare recognized by

Hosford et al. (1997) coincided with the northern coniferous forest belt, running east-west

across Canada, and the temperate conifer forest extending southward along the Appalachian,

Rocky, Cascade and Pacific Coast ranges, tanoak forest in southwestern Oregon, along the

coast of northern California, and the high-elevation pine and fir forests in the mountains of

Mexico (Hosford et al. 1997). Tricholoma magnivelare in this study was a commodity

imported from Canada. The ITS sequences were almost the same as the western North

American (WNA) group associated with mixed forests by Chapela and Garbelotto (2004) and

considered to be authentic T. magnivelare. The Tricholoma sp. from Mexico in our study

appears identical to the T. magnivelare of the MX group identified by Chapela and Garbelotto

(2004), based on ITS sequences.

Tricholoma caligatum also was shown to be polyphyletic (Chapela and Garbelotto

2004). Tricholoma caligatum originally was described from Chiavari, Italy (Viviani 1834). It

was reported as a southern species occurring most commonly around the western

Mediterranean, especially in southern France, eastern and southeastern Spain and adjacent

northwestern Africa, occurring under Pinus pinea, P. halepensis and evergreen species (Bon

1984, Kytövuori 1988). Some morphological heterogeneity was suggested to exist in T.

caligatum in central and southern Europe (Kytövuori 1988). Tricholoma caligatum of the AT

(the Atlas Mountains) group identified by Chapela and Garbelotto (2004) was considered

identical with Tricholoma anatolicum in our study. Further analyses with more collections

from these regions will resolve the phylogenetic relationships and taxonomy of T. caligatum

complex; our analysis may serve as a reference for its revision.

Diversity and genetic divergence values were always lower within than between T.

matsutake, T. anatolicum and T. magnivelare (TABLE II), supporting the status of these three

phylogenetic taxa. The Tricholoma sp. from Mexico was recognized as distinct in the

multilocus analyses (FIG. 2); however, it is allied to T. matsutake, as the megB1 network

analyses showed. Only two samples were analyzed in our study, so additional data should be

added to enable delimitation of this group.

The great similarity between European and eastern Asian populations of T. matsutake

revealed here is consistent with studies using ITS (Bergius and Danell 2000, Chapela and

Garbelotto 2004, Matsushita et al. 2005, Wan et al. 2012), the V4 domains of the mtSSU

rDNA (Bao et al 2007, Wan et al. 2012), RAPD profiles (Bao et al. 2007) and AFLP variation

(Chapela and Garbelotto 2004). No clear genetic separation between populations was revealed

by our multilocus analyses. Two possible methods could be considered to resolve the genetic

structure of T. matsutake populations. Amend et al. (2010) and Xu et al. (2008) showed

population structure from several geographical regions from southwestern China using SNPs;

these are the most frequently observed differences between DNA sequences obtained from

different individuals or between alleles from within the same individual in diploid or higher

ploidy organisms. They indicated that there was limited but significant genetic differentiation

among geographical populations. A second possible method is retroelement-based genotyping.

Murata et al. (2008) reported that a PCR system targeting retroelement integration sites could

differentiate among individual Asian isolates of T. matsutake, based on their geographical

origins.

The Fagaceae associates Tricholoma bakamatsutake and T. fulvocastaneum were

recognized as separate phylogenetic species, basal to the matsutake group. These are

distributed in a limited area compared with T. matsutake. Tricholoma bakamatsutake is

widespread in Honshu and Hokkaido islands of Japan (Hongo 1974, Ogawa 1978). It also has

been reported from northeastern China and New Guinea (Ogawa 1978). Tricholoma

fulvocastaneum is distributed in Honshu and Kyushu in Japan (Hongo 1960), northern

Thailand (Sanmee et al. 2007) and Laos (Yamanaka et al. 2011), having a more southern

distribution than T. bakamatsutake. Tricholoma robustum, considered to be a closely related

species to T. matsutake in Japan, was clearly distinguished and outside the matsutake group

and its allied species. Our results therefore show that the ITS region is a suitable marker for

identification of eastern Asian species.

ACKNOWLEDGMENTS

We sincerely thank Dr A. Yamada for his kind help and useful suggestions. We also thank Dr P. Roda for

providing specimens for this study. We are grateful for the help of Ms E. Tsutsumi and Ms K. Komaru for

technical assistance. The curators of the following herbaria are appreciated for loan of specimens: TNS, O,

SCM and VLA. This work was financially supported by the Research and Development Projects for Application

in Promoting New Policies in Agriculture, Forestry and Fisheries (No. 21080) and by Institute of Fermentation

Osaka Japan.

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LEGENDS

FIG. 1. ML tree obtained through analyses of combined datasets: ITS (ITS1 and 5.8S), megB1, and tef.

Armillaria spp. were used as the outgroup. Bootstrap values (1000 replicates, BSv) and Bayesian posterior

probabilities (PPs) as ML/NJ/Bayesian posterior probabilities, are shown on branches.

FIG. 2. ML tree obtained through analyses of combined datasets, ITS (ITS1, 5.8S and ITS2), megB1, tef and

gpd, root on midpoint. Bootstrap values (1000 replicates, BSv) and Bayesian posterior probabilities (PPs) as

ML/NJ/Bayesian posterior probabilities are on branches.

FIG. 3. a–d. Unrooted median-joining haplotype network constructed from the sequences of internal

transcribed spacer (ITS) (a), tef (b) , gpd(c) and megB1(d), showing links between the four phylogenetic species;

Tricholoma matsutake, T. anatolicum, T. sp from Mexico and T. magnivelare. Size of the circle is proportional

to haplotype frequency. Small circle depict nodes with nonrecorded, hypothetical haplotypes. Numbers indicate

the numbers of mutations between haplotypes, and value above one is shown.

SUPPLEMENTARY FIG. 1a–d. Phylogenetic trees obtained through separate analyses of the four investigated

DNA regions. a. megB1. b. ITS (ITS1 and 5.8S). c: tef. d. gpd. Armillaria spp. were used as outgroup.

Bootstrap values (1000 replicates, BSv) and Bayesian posterior probabilities (PPs) as ML/NJ/Bayesian posterior

probabilities are shown on branches.

FOOTNOTES

Submitted 29 Feb 2012; accepted for publication 17 May 2012.

1Corresponding author. E-mail: yuota@ffpri.affrc.go.jp

TABLE I. Isolates and specimens used in this study Species Isolate or specimen

numbers

Habitat or host Locality Herbarium

Accession no.

Refrerencesa Other isolate

number

GenBank accession numbers

megB1 ITS tef gpd

Tricholoma matsutake NBRC 6932 Japan - AB563496 AB699626 AB699731 JQ696991

NBRC 6933 Japan - AB563497 AB699627 AB699732 JQ696992

Y1 Pinus densiflora Ibaraki, Japan - a, c NBRC 33136,

ATCC MYA-915

AB298298 AB036890 AB699733 JQ696993

TO-1 P. densiflora forest Nagano, Japan TFM: S-08006 AB699617 AB699628 AB699734 JQ696994

Tm029 P. densiflora Shiga, Japan - a, b, f, g AB298297 AB699629 AB699735 JQ696995

TNS-F-12850 P.densiflora forest Hiroshima, Japan TNS: F-125850 AB538258 AB699630 AB699736 -

Tm-31 P. densiflora Gyeongsangbuk-do,

South Korea

- a, b AB298300 AB699631 AB699737 JQ696996

Tm-09 China - a, b AB298303 AB699632 AB699738 JQ696997

CHHE-3 Heilongjiang, China

(Commodity)

- B AB520960 AB699633 AB699739 JQ696998

CHJI-1 Jilin, China (Commodity) - B AB520961 AB699634 AB699740 JQ696999

CHSI-1 Sichuan, China

(Commodity)

- B AB520962 AB699635 AB699741 JQ697000

CHYU-1 Yunnan, China

(Commodity)

- B AB520963 AB699636 AB699742 JQ697001

BH1 Bhutan - a, b, c AB520959 AB699637 AB699743 JQ697002

NKR05 North Korea (Commodity) - B AB298302 AB699638 AB699744 JQ697003

VLA M19537 Q. mongolica forest Primorskey Territory,

Russia

VLA: M-19537 AB520974 AB699639 AB699745 -

O-85552 Norway O: 85552 AB520980 AB699640 AB699746 JQ697004

O-370282 Norway O: 370282 AB520981 AB712394 AB699747 -

Tn-FIN1 Parkano, Finland Shinsyu Univ. no.

AY-2070925001-1

AB520982 AB699641 AB699748 JQ697005

Tricholoma

anatolicum

S-2-2 Cedrus libani Turkey Shinsyu Univ.

no.S-2-2

AB520967 AB699642 AB699749 JQ697006

S-2-3 Ce. libani Yaya Koru,

Turkey

Shinsyu Univ.

no.S-2-3

AB520968 AB699643 AB699750 JQ697007

S-3-2 Ce. libani Babadag, Turkey Shinsyu Univ.

no.S-3-2

a AB520969 AB699644 AB699751 JQ697008

MC1 unknown Kingdom of Morocco - a, b, c ATCC MYA-929 AB298304 AB699645 AB699752 JQ697009

TM-5 unknown Kingdom of Morocco - a AB520978 AB699646 AB699753 JQ697010

Tricholoma sp. MX1 unknown Mexico (commodity) - a, b, c ATCC MYA-921 AB520966 AB699647 AB699754 JQ697011

TM-4 unknown Mexico - A AB298305 AB036891 AB699755 JQ697012

Tricholoma

magnivelare

CA1 unknown Canada (commodity) TFM: M-L903 a, c AB535196 AB712395 AB699756 JQ697013

TM-10 unknown Canada - a, b, f, g AB298306 AB699648 AB699757 JQ697014

Tp-C3 unknown Canada - ATCC

MYA-930

AB293549 AB036893 AB699758 JQ697015

Tricholoma

bakamatsutake

B2 Q. serrata forest Ibaraki, Japan - AB535197 AB699649 AB699759 JQ697016

CB-Tb2 Forest of Pasania edulis and

Castanopsis sieboldii

Chiba, Japan - a, d NBRC 108266 AB535203 AB699650 AB699760 JQ697017

CB-Tb4 Forest of Pasania edulis and

Castanopsis sieboldii

Chiba, Japan - a, d NBRC 107029 AB535205 AB699651 AB699761 JQ697018

WK-Tb1 Ca. sieboldii forest Wakayama, Japan - AB550677 AB699652 AB699762 JQ697019

TNS-F-11543 Ibaraki, Japan TNS: F-11543 AB538256 AB699653 - -

TNS-F-12866 Quercus forest Yamaguchi, Japan TNS: F-12866 AB538255 AB699654 - -

SF-Tb1 Q. serrata Kyoto, Japan - AB699618 AB699655 AB699763 JQ697020

SF-Tf05 Q. salicina Shiga, Japan - AB699619 AB699656 - JQ697021

240211 Q. serrata Kyoto, Japan syntype no.1283,

Hongo1974

N-3S e AB699620 AB699657 AB699764 JQ697022

SF-Tb09 unknown Hokkaido,Japan

(Commodity)

- AB699621 AB712396 - JQ697023

Tricholoma

fulvocastaneum

NBRC 6942 Japan - IFO 6942 AB563498 AB699658 AB699765 JQ697024

NBRC 6947 Japan - IFO 6947 AB563499 AB699659 AB699766 JQ697025

Tf-AM1 Ca. sieboldii forest Kagoshima, Japan TFM: M-L914 AB538249 AB699660 AB699767 JQ697026

WK-N10-29 unknown Wakayama, Japan TFM: M-R29 NBRC108269 AB699622 AB699661 AB699768 JQ697027

WK-N20-27 unknown Wakayama, Japan TFM: M-R27 NBRC108270 AB699623 AB699662 AB699769 JQ697028

WK-N30-26 unknown Wakayama, Japan TFM: M-R26 AB699624 AB699663 AB699770 JQ697029

NTfu-3 unknown Nara, Japan TFM: M-R123 AB699625 AB699664 AB699771 JQ697030

Tricholoma caligatum TFM-M-L-915-a Pinus pinea forest Carabria, Italy TFM: M-L-915 AB550663 AB699665 AB699772 JQ697031

SCM B-4194 P. halepensis forest Pals, Spain SCM: B-4194 AB535209 AB699666 - JQ697032

SCM B-5116 Pinus sp. forest Platja Llaga, Spain SCM: B-5116

AB535212

AB699667 AB699773 -

Tricholoma japonicum FK-J1 P. densiflora forest Fukui, Japan - a

AB520977

AB036900 AB699774 JQ697033

Tricholoma robustum KB1 P. densiflora forest Nagano, Japan - d AB520964 AB699668 AB699775 -

TNS-F-12918 Quercus crispula/Q. serrata forest Nagano, Japan TNS: F-12918 AB538255 AB699669 - -

Tricholoma focale SCM B-3113 Pinus sp./A. alba forest Espot, Spain SCM: B-3113 AB535213 AB699670 AB699776 -

Tricholoma

flavovirens

613 P. densiflora forest Ibaraki, Japan - A AB520976 AB036895 AB712398 JQ697034

614 P. densiflora forest Ibaraki, Japan TNS: F-15544 A AB545868 AB712397 AB712399 JQ697035

Tricholoma

portentosum

HOLO-T unknown Italy No. HoloT, personal specimen of D. Pietro Roda AB535207 AB699671 AB712400 -

615 P. densiflora forest Ibaraki, Japan TNS-F-11546 a AB545867 AB699672 AB712411 JQ697036

Armillaria nabsnona NB3 unknown Tottori, Japan TMI: C30984 h AB293545 AB510894 AB510763 JQ697037

Armillaria ostoyae NC8 P. densiflora/Q. serrata forest Aomori, Japan TMI: C31491 h AB293547 AB510897 AB510782 JQ697038

Armillaria gallica NA4 Quercus dentata forest Fukushima, Japan TMI: C31063 h AB293543 AB510881 AB510761 JQ697039

Armillaria cepistipes ND1 unknown Aomori, Japan TMI: C30968 h AB293542 AB510885 AB510792 JQ697040

aReferences: a: Murata et al 1999; b: Murata et al 2008; c：Yamada e al 2010, d: Murata and Babasaki 2005; e: Hongo 1974; f: Murata et al 2005 Mycorrhiza 15 505-512; g: Murata et al. 2005. Mycorrhiza 15:179-186; h: Babasaki et

al.2007

TABLE II. The extent of DNA polymorphism and divergence within and between species for Tricholoma matsutake and allies with ITS, megB1, tef and gpd haplophase

datasets

Number of sequences Number of polymorphic sites Number of fixed differences π k Dx, Dxy (%)

ITS megB1 tef gpd ITS megB1 tef gpd ITS megB1 tef gpd ITS megB1 tef gpd ITS megB1 tef gpd ITS megB1 tef gpd

T. matsutake 18 18 18 15 0.75 4.31 0.00 0.50 0.11 0.92 0.00 0.07 3 20 0 2 0.001 0.009 0.000 0.001

T. anatolicum 5 5 5 5 0.60 1.00 0.00 0.00 0.09 0.21 0.00 0.00 1 2 0 0 0.001 0.002 0.000 0.000

T. sp 2 2 2 2 2.00 5.00 0.00 0.00 0.31 1.00 0.00 0.00 2 5 0 0 0.003 0.010 0.000 0.000

T. magnivelare 3 3 3 3 0.00 1.33 2.00 0.00 0.00 0.28 0.43 0.00 0 2 3 0 0.000 0.003 0.004 0.000

T. bakamatsutake 10 10 6 8 0.53 0.67 0.00 0.00 0.08 0.14 0.00 0.00 1 2 0 0 0.001 0.001 0.000 0.000

T. fulvocastaneum 7 7 7 7 0.86 0.57 0.00 0.00 0.15 0.12 0.00 0.00 3 2 0 0 0.002 0.001 0.000 0.000

T. caligatum 3 3 2 2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0 0 0 0 0.000 0.000 0.000 0.000

Tma/Tana 23 23 23 20 2.55 6.22 1.77 1.96 0.92 2.15 1.08 0.63 9 27 5 6 5 5 5 4 0.004 0.013 0.004 0.003

Tma/Tmag 21 21 21 18 6.06 8.65 3.89 3.95 3.36 5.10 3.23 1.80 24 37 16 14 21 19 13 12 0.009 0.019 0.008 0.006

Tma/Tbaka 28 28 24 23 30.96 19.83 12.13 18.83 10.95 8.20 6.77 5.78 66 54 31 41 64 34 31 39 0.052 0.043 0.026 0.028

Tma/Tful 25 25 25 22 20.05 26.47 14.28 28.98 8.47 12.63 7.33 9.40 50 73 34 65 45 55 34 63 0.036 0.058 0.030 0.043

Tma/Tcal 21 21 20 17 16.99 18.26 5.87 10.37 11.14 13.61 6.80 6.66 67 74 31 47 65 56 31 45 0.029 0.042 0.013 0.015

Tana/Tmag 8 8 8 8 11.79 15.89 7.71 5.36 3.36 6.28 3.01 1.47 22 30 15 10 21 27 12 10 0.018 0.035 0.017 0.008

Tana/Tbaka 15 15 11 13 32.67 20.19 16.36 18.97 11.43 8.99 6.55 5.44 69 44 30 37 67 40 30 37 0.055 0.044 0.036 0.028

Tana/Tful 12 12 12 12 26.33 32.24 18.03 31.29 8.76 13.23 7.33 8.77 51 62 34 59 48 59 34 59 0.047 0.071 0.039 0.046

Tana/Tcal 8 8 7 7 0.07 32.07 16.19 20.48 12.12 13.80 7.33 6.32 72 60 34 43 71 58 34 43 0.065 0.075 0.035 0.030

Tmag/Tbaka 13 13 9 11 26.46 17.39 14.17 17.46 11.43 9.69 6.11 5.88 68 46 29 40 68 43 26 40 0.044 0.038 0.031 0.026

Tmag/Tful 10 10 10 10 22.87 28.09 17.40 28.47 8.58 13.32 7.97 9.06 50 61 38 61 47 59 35 61 0.041 0.063 0.038 0.042

Tmag/Tcal 6 6 5 5 41.40 34.53 23.44 25.80 11.72 13.62 8.19 6.32 69 58 39 43 69 57 36 43 0.070 0.082 0.050 0.038

Tbaka/Tful 17 17 13 15 33.63 28.74 24.77 34.67 11.99 11.87 9.96 9.66 67 57 46 65 63 55 46 65 0.062 0.062 0.054 0.052

Tbaka/Tcal 13 13 8 10 32.23 25.00 17.14 19.20 14.61 14.82 8.65 7.94 83 65 40 54 83 64 40 54 0.057 0.058 0.037 0.028

Tful/Tcal 10 10 9 9 35.00 22.47 17.50 24.50 13.36 11.00 9.62 9.36 76 48 45 63 73 47 45 63 0.063 0.052 0.037 0.036

NRBC6932

NRBC6933

CHJI1

NKR05

CHHE3

Y1

Tm029

TO1

VLAM19537

BH1

CHYU1

TM09

TNSF12850

CHSI1

Tm31

O370282

O85552

TnFIN1

MX1

TM4

S32

S22

TM5

S23

MC1

TpC3

CA1

TM10

SFTb1

240211

B2

CBTb2

CBTb4

WkTb1

NRBC6942

NRBC6947

TfAM1

WKN1029

WKN2027

WKN3026

NTfu3

TFMML915a

SCMB5116

KB1

SCMB3113

FKJ1

613

614

Tmm50porten

615

NB3

NA4

NC8

ND1

Fig. 1

100/100/1.00

77100/1.00

99/100/1.00

99/100/1.00

100/100/1.00

100/100/1.00

100/87/1.00

99/100/1.00

100/100/1.00

100/100/1.00

98/100/1.00

98/100/1.00

100/100/1.00

100/100/1.00

100/100/1.00

HOLOT

Armillaria spp.

T. matsutake

T. sp. from Mexico

T. fulvocastaneum

T. bakamatsutake

T. anatolicum

T. magnivelare

T. caligatum

T. robustum/T.focaleT. japonicum

T. flavovirens

T. portentosum

matsutake”

Fig.2.

NRB

C693

2

NRB

C693

3

Y1

Tm

029

NKR

05

CHH

E3

CHJ

I1

Tm

31

O85

552

TnF

IN1

TO1

Tm

09

BH1 C

HSI1

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U1 S32 S

22 TM

5

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MX1

TM

4

TpC

3 CA1

TM

10

77/77/1.00

85/86/0.99

96/97/1.00

89/89/1.00

99/99/1.00

100/10

0/1.00

100/10

0/1.00

77/77/1.00

Japan

Japan

Japan

Japan

North

Korea

China

China

Japan

Norway

Finland

Japan

Japan

Bhutan

China

ChinaYunn

an

Turkey

Turkey

Morocco

Morocco

Turkey

Mexico

Mexico Canada Canada

Canada

T.matsutake

T.an

atolicum

T.sp.from

Mexico

T.mag

nivelare

T. matsutake

T. sp. fromMexico

T. anatolicum

T. magnivelare

aT. matsutake

T. sp. from Mexico

T. anatolicum

T. magnivelare

T. matsutake

T. sp. fromMexico

T. anatolicum

T. matsutake

T. sp. fromMexico

T. anatolicum

T. magnivelare

b

T. magnivelare

c

d

Figs 3, a d

19

5 2

4

8

6

10

39

9

22

1

9

3

92

1 43