distributed Amanita species,A.muscaria and A.pantherina

Takashi ODA*,Chihiro TANAKA and Mitsuya TSUDA

Laboratory of Environmental Mycoscience ,Graduate School of Agriculture ,Kyoto University ,Kyoto ,606-8502,Japan.E-mail :takashi@kais.kyoto-u.ac.jp

Received 19March 2003;accepted 15May 2004.

The molecular phylogeny and biogeography of two widely distributed Amanita species,A.muscaria and A.pantherina ,were studied based on specimens from diverse localities.Analyses of both a partial sequence of the ITS region of nuclear DNA and a partial sequence of the b -tubulin gene were able to resolve specimens of each species.Analyses revealed a greater divergence of the b -tubulin region than the ITS region.Based on molecular phylogeny of the combination of the ITS and b -tubulin regions,A.muscaria could be separated into at least three groups (Eurasian,Eurasian subalpine,and North American),and A.pantherina could be separated into at least two groups (North American and Eurasian).We hypothesize that the speciation of A.muscaria occurred in Eurasia with subsequent migration to North America via land bridges.However,it is impossible to determine whether A.pantherina moved from Eurasia to North America or vice versa .For both A.muscaria and A.pantherina ,the intracontinental relationships of both Eurasia and North America were closer than the relationships between eastern Asia and eastern North America.

INTRODUCTION

The genus Amanita has a global distribution and is one of the most well-known genera of macrofungi;most species are ectomycorrhizal.Recent molecular phylo-genetic studies of this genus have contributed to the re-examination of its traditional classifications based on morphology (e.g.Weiß,Yang &Oberwinkler 1998,Drehmel,Moncalvo &Vilgalys 1999,Oda,Tanaka &

Tsuda 1999,Gonza

lez et al .2002).However,in these studies,little attention was given to genetic variation within species or to the relationships between the mol-ecular phylogeny and the biogeography of each species or of the genus Amanita as a whole.Recently,molecu-lar phylogenetic studies of macrofungi with regard to biogeography have attracted more attention (e.g.Vilgalys &Sun 1994,Wu et al .2000,Hibbett 2001,Ko et al .2001,Mueller et al .2001).

In this study,we examined the molecular phylogeny and biogeography of two widely distributed Amanita species,A.muscaria and A.pantherina ,which belong to the subgenus Amanita section Amanita.A.muscaria is a large mushroom that is characterized by a red pileus with many volval warts,a bulbose stipe base,and the presence of clamps.This famous toxic species,the ‘fly

agaric,’is widely distributed in Europe,Asia,Africa,Australia,New Zealand,and North,Central,and South America (e.g.Gilbert 1941,Jenkins 1977,1986,

Reid 1980,Imazeki &Hongo 1987,Pe

rez-Silva &Herrera 1991,Reid &Eicker 1991,Ridley 1991,Tulloss,Ovrebo &Halling 1992,Adhikari &Parajuli 1994). A.pantherina ,‘panther cap’,is also a well-known poisonous fungus and is characterized by a brownish pileus with many volval warts,a bulbose stipe base,and the absence of clamps.This species is dis-tributed in Europe,Asia,Africa,and North and Cen-tral America (e.g.Gilbert 1941,Jenkins 1977,1986,

Imazeki &Hongo 1987,Pe

rez-Silva &Herrera 1991,Reid &Eicker 1991,Adhikari &Parajuli 1994,Yang 1997).We conducted a molecular phylogenetic study of specimens of both species from diverse localities based on a partial sequence of the ITS region of nuclear DNA and a partial sequence of the b -tubulin gene.Based on our results,we discuss the biogeography of these two Amanita species.

MATERIALS AND METHODS Specimens examined

The 49specimens that were examined in this study are listed in Table 1.With the exception of Amanita

*Corresponding author.

Mycol.Res.108(8):885–6(August 2004).f The British Mycological Society 885

DOI:10.1017/S0953756204000620Printed in the United Kingdom .

Table1.Details and nucleotide sequence length(bp)of the ITS and b-tubulin regions of49specimens of Amanita species studied.

Specimen No.a Geographic origin ITS b-tubulin

Accession no.b,c Length(bp)Accesion no.b,c Length(bp)

Section Amanita

A.muscaria FB-30961(CBM)Aomori-shi,Aomori,Japan AB080980695AB0952*4

FB-30962(CBM)Kitakoma-gun,Yamanashi,Japan AB080981695AB0953*4

FB-30963(CBM)Kitakoma-gun,Yamanashi,Japan AB080982695AB0954*4

FB-30976(CBM)Kiso-gun,Nagano,Japan AB081294695AB0955*4

FB-30977(CBM)Ohno-gun,Gifu,Japan AB081295695AB0956*4

FB-30985(CBM)Ohno-gun,Gifu,Japan AB096048*695AB0957*4

FB-30978(CBM)Chino-shi,Nagano,Japan AB081296697AB095858*487

FB-30981(CBM)Chino-shi,Nagano,Japan AB096049*697AB095859*487

FB-30982(CBM)Chino-shi,Nagano,Japan AB096050*697AB095860*487

FB-309(CBM)Gdynia,Poland AB080983696AB0959*486

FB-30965(CBM)Gdansk,Poland AB080984695AB095900*486

M-31452(K)Hampshire,England,UK AB080777695AB095901*486

M-31445(K)Surrey,England,UK AB080778695AB095902*486

M-80048(K)Surrey,England,UK AB080779696AB095903*486

FB-30987(CBM)Queenstown,Otago,New Zealand AB096052*696AB095904*4

45843(NY)Hampshire,Massachusetts,USA AB080788*697AB095884*471

45785(NY)Hampshire,Massachusetts,USA AB0807*697AB095885*471

45840(NY)Lawrence,Massachusetts,USA AB080791*697AB095887*471

45820(NY)Bronx,New York,USA AB080790*698AB095886*471

45863(NY)Mendocino,California,USA AB080787*703AB095883*471

var.formosa45883(NY)Piscataquis,Massachusetts,USA AB080792*697AB095888*471 HDT45060(SFSU)Amador,California,USA AB080795*702AB0951*471

HDT44761(SFSU)Alpine,California,USA AB080794*703AB0950*471

var.alba HDT49100(SFSU)Cascade,Idaho,USA AB080793*701AB0958*471

var.regalis506(O)Dovre,Oppland,Norway AB080780697AB095855*487 1539(O)Gjøvik,Oppland,Norway AB080781697AB095856*487

4220(O)Nordre-Land,Oppland,Norway AB080782697AB095857*487

A.pantherina FB-15998(CBM)Chiba-shi,Chiba,Japan AB080978704AB095867*496

FB-30956(CBM)Sendai-shi,Miyagi,Japan AB080973702AB095870*496

FB-30957(CBM)Ohno-gun,Gifu,Japan AB080974703AB095871*495

FB-30958(CBM)Ohtsu-shi,Shiga,Japan AB080975703AB095872*495

FB-30959(CBM)Kyoto-shi,Kyoto,Japan AB080977703AB095868*496

FB-30960(CBM)Kyoto-shi,Kyoto,Japan AB080976703AB095869*496

TNS-F-4490(TNS)Nagarkot,Kathmandu,Nepal AB096043*704AB095876*496

TNS-F-4491(TNS)Phulchowki,Kathmandu,Nepal AB096044*703AB095877*496

TNS-F-4492(TNS)Phulchowki,Kathmandu,Nepal AB096045*704AB095878*496

M-31408(K)Devon,England,UK AB080774705AB095873*497

M-61495(K)Surrey,England,UK AB096046*706AB095875*497

M-79855(K)West Suffolk,England,UK AB080775706AB095874*496

45927(NY)Santa Barbara,California,USA AB080785*708AB095881*494

45929(NY)San Francisco,California,USA AB080784*708AB095880*492

FB-30984(CBM)Loma Mar,California,USA AB096047*708AB095879*492

A.aff.muscaria FB-30986(CBM)Aomori-shi,Aomori,Japan AB096051*698AB0958*488

A.concentrica FB-24901(CBM)Awa-gun,Chiba,Japan AB080783730AB095849*4

A.ibotengutake FB-30969(CBM)Kyoto-shi,Kyoto,Japan AB080988713AB095848*491

A.melleiceps FB-30953(CBM)Ohita-shi,Ohita,Japan AB015688706AB095854*491

A.rubrovolvata FB-30954(CBM)Ohno-gun,Gifu,Japan AB0156721AB095850*495

A.sinensis FB-30955(CBM)Itoigawa-shi,Niigata,Japan AB080979721AB095861*493 Outgroup

Section Phalloideae

A.pseudoporphyria FB-30951(CBM)Kyoto-shi,Kyoto,Japan AB015702636AB095905*485

a Specimen no.in italic represents a type specimen.

b The nucleotide squence data appear in the DDBJ/EMBL/GenBank nucleotide sequence databases.

c Accession no.with an*means that sequence was determine

d in this study.Others wer

e previously published in Oda et al.(1999)and Oda et al.(2002).

Molecular phylogeny and biogeography of Amanita886Chiba(CBM);Herbarium of Cryptogams,Kunming Institute of Botany,Academia Sinica,Kunming (HKAS);Royal Botanic Gardens,Kew(K);New York Botanical Garden,Bronx,New York(NY); Botanical Museum,Oslo(O);Harry D.Thiers Her-barium,San Francisco State University,San Francisco (SFSU);and the Botany Department,National Science Museum,Tsukuba(TNS).

DNA preparations

A minute slice of dried fruit body(ca10mg)was sus-pended in500m l of extraction buffer(50m M Tris–HCl pH8.0,125m M EDTA,100m M NaCl,2%(w/v) sodium N-dodecanoylsarcosinate,1%(v/v)2-mercap-toethanol).DNA was extracted by the method of Nakada et al.(1994).

PCR amplification

The ITS region was amplified with the primers ITS4 and ITS5(White et al.1990).This region consists of a portion of18S rDNA,ITS1,5.8S rDNA,ITS2,and a portion of28S rDNA(van Nues et al.1994).The re-action mixture contained10pmol of each primer,1.5U of KOD Dash(Toyobo,Osaka),and about10ng of template DNA in a volume of50m l.A thermal cycler (iCycler,Bio-Rad,Hercules)was programmed as follows:initial denaturation,1min at95x C;30cycles of0.5min at95x,0.03min at49x,and0.5min at 72x.For the partial sequence of the b-tubulin gene,we used the newly designed PCR primers B-TUB-1F(5k-YMGNCCNGAYAAYTTYGTNTTYG-3k),B-TUB-1R(5k-TANARNGCYTCRTTRTCDATRCARAA-3k),which were based on data of the b-tubulin gene of basidiomycetes available in the DDBJ/EMBL/Gen-Bank nucleotide sequence databases.The region amplified with this primer set consisted of three exons and two introns.The reaction mixture contained 10pmol of each primer,1.5U of Taq DNA polymerase (Takara,Otsu),and about10ng of template DNA in a volume of50m l.The thermal cycler(iCycler,Bio-Rad) was programmed as follows:initial denaturation, 4min at95x;then30cycles of1min at95x,1min at 48x,and1.5min at72x;andfinal extension at72x for 2min.

Cloning and DNA sequencing

After electrophoresis in a low-melt agarose gel(Sea Plaque GTG Agarose,FMC),the amplified products were excised from the gel.The DNA fragments were cloned into pZErO TM-2(Invitrogen,Carlsbad).To avoid artifact DNA sequences caused by errors in DNA polymerization,at least three recombinants were picked up from a batch of transformants,and the homogeneity or majority of DNA sequences was confirmed.DNA was sequenced with CEQ TM DTCS Quick Start Kit(Beckman Coulter,Fullerton)according to the manufacturer’s recommendations. The sequence primers used were M13-20and M13-RV (Pharmacia Biotech,Piscataway).Terminated samples were electrophoresed on a CEQ TM2000DNA Analysis System(Beckman Coulter),and sequence data were generated.For sequence alignments,the data of the ITS region were used with primers ITS4and ITS5, whereas the data of the b-tubulin region were used without primers B-TUB-1F and B-TUB-1R.The nucleotide sequence data are deposited in the DDBJ/ EMBL/GenBank nucleotide sequence databases.For the ITS region,some of the data had been previously published in Oda et al.(1999)and Oda et al.(2002) (Table1).

Sequence alignment and parsimony analysis

The sequences of each region were aligned using the CLUSTAL W multiple alignment program ver.1.8. (Thompson,Higgins&Gibson1994).Parsimony analyses of the ITS region,the b-tubulin region,and the combination of the two regions were performed with PAUP*version4.0b9(Swofford2002).A heuristic search was conducted with the following conditions: alignment gapmode was newstate,the starting tree was obtained via stepwise addition;addition sequence was simple(reference taxon=FB-30951(CBM));the branching-swapping algorithm was tree-bisection-reconnection(TBR),and the‘MulTrees’option was in effect.Bootstrap analyses were performed using ‘full heuristic’with500replicates.The sequence align-ment has been submitted to TreeBASE,a relational database of phylogenetic information(URL:http:// www.treebase.org/treebase/index.html).

RESULTS

Analysis of the ITS region

The sequences of the ITS region ranged in length from 636to730bp among the49specimens(Table1).As a result of the alignment of these sequences,114charac-ters were parsimony-informative,215variable charac-ters were parsimony-uninformative,and463characters were constant out of792total characters.Based on the aligned sequences,heuristic searches revealed12most parsimonious trees with a length of552,a consistency index(CI)of0.7663,a retention index(RI)of0.8522, and a homoplasy index(HI)of0.2337.One of the most parsimonious trees is shown in Fig.1.For both Ama-nita muscaria and A.pantherina,each parsimonious tree shows the same topology,except the placements within the A.muscaria clade of the UK,Poland,Japan and New Zealand.The placements of the other related species were unstable.

Specimens of A.muscaria were separated into three clades,i.e.,the clade consisting of the UK,Poland, Japan,and New Zealand(bootstrap value=98%);the clade of Norway and Japan(bootstrap value=99%);

T.Oda,C.Tanaka and M.Tsuda887

and the clade of the USA (bootstrap value =99%).Amanita aff.muscaria fell on a branch basal to the three clades of A.muscaria .The USA clade included the sub-clade of the western USA (bootstrap value =98%),which contained 45863(NY),HDT44761(SFSU),HDT49100(SFSU),and HDT45060(SFSU).The clade of the UK,Poland,Japan,and New Zealand and the clade of Norway and Japan had no geographically separated subclades.Specimens of A.pantherina were separated into two clades,the USA clade (bootstrap value =100%)and the clade composed of the UK,Nepal,and Japan (bootstrap value =93%).The connection of the two clades received 72%bootstrap

support.

Fig.1.One of the 12most parsimonious trees based on the sequences of the ITS regions.Amanita pseudoporphyria was

used as the outgroup.Bootstrap values over 50%are indicated at the base of the corresponding clade.For A.muscaria and A.pantherina ,each parsimonious tree shows the same topology,with the exception of the placements within the A.muscaria clade of the UK,Poland,Japan,and New Zealand.Placements of the other related species were unstable.

Molecular phylogeny and biogeography of Amanita 888

Analysis of the b -tubulin region

The b -tubulin region ranged in length from 471to 497bp (Table 1).As a result of the alignment of these sequences,117characters were parsimony-informative,90variable characters were parsimony-uninformative,and 299characters were constant out of 506total characters.Based on these aligned sequences,heuristic searches revealed eight most parsimonious trees with a length of 376,a CI of 0.7420,an RI of 0.9062,and an HI of 0.2580.One of the most parsimonious trees is shown in Fig.2.Except for the placements within the Amanita pantherina clade of the UK,Nepal,and Japan,each parsimonious tree showed the same topology.Specimens of A.muscaria were separated into four clades,i.e.,the clade of Norway and Japan (bootstrap value =74%),the clade of Japan and New Zealand (bootstrap value =66%),the clade of the UK

and

Fig.2.One of eight most parsimonious trees based on the sequences of the b -tubulin region.Amanita pseudoporphyria was used as the outgroup.Bootstrap values over 50%are indicated at the base of the corresponding clade.With the exception of the placements within the A.pantherina clade of the UK,Nepal,and Japan,each parsimonious tree shows the same topology.

T.Oda,C.Tanaka and M.Tsuda 8

Analysis of the combination of the ITS region and the b-tubulin region

The combination of the ITS and b-tubulin regions ranged in length from1121to1219bp.As a result of the alignments of these sequences,231characters were parsimony-informative,305variable characters were parsimony-uninformative,and762characters were con-stant out of1298total characters.Based on the aligned sequences,heuristic searches revealed108most parsi-monious trees with a length of945,a CI of0.7429,an RI of0.8726,and an HI of0.2571.One of the most parsimonious trees is shown in Fig.3.Except for the placements within the A.pantherina subclade of Japan, the A.muscaria subclade of the UK and Poland,and the A.muscaria subclade of the western USA,each parsimonious tree shows the same topology. Specimens of A.muscaria were separated into three clades:(1)the UK,Poland,Japan,and New Zealand collections(bootstrap value=93%);(2)Norway and Japan(bootstrap value=100%);and(3)the USA (bootstrap value=100%).Amanita aff.muscaria fell on a branch basal to the three clades of A.muscaria (bootstrap value=99%).The clade of the UK,Poland, Japan,and New Zealand consisted of two subclades: one of the UK and Poland(bootstrap value=98%), and the other of Japanese and New Zealand collections (bootstrap value=88%).The clade of Norway and Japan consisted of two subclades,one for Norway (bootstrap value=75%)and one for Japan(bootstrap value=91%).In the USA clade,four specimens, HDT49100(SFSU),HDT44761(SFSU),HDT45060 (SFSU),and45863(NY),made up the subclade of the western USA(bootstrap value=98%)and were sep-arate from the otherfive specimens from the eastern USA.Specimens of A.pantherina were separated into two clades,the USA clade(bootstrap value=100%) and the clade of the UK,Nepal,and Japan(bootstrap value=100%).The connection between the two clades received100%bootstrap support.The clade of the UK,Nepal,and Japan consisted of three geo-graphically separated subclades:the UK(bootstrap value=99%),Nepal(bootstrap value=93%),and Japan(bootstrap value=80%).

The combined data analysis yielded results similar to those based on the ITS region analysis,showing three distinct clades of A.muscaria and two distinct clades of A.pantherina.Moreover,in the combined data tree,the node of each clade of the two species received much greater bootstrap support.The combined data analysis supported most of the results produced by the b-tubu-lin analysis,except for the placements of the A.mus-caria clade of the UK and Poland and A.aff.muscaria; in contrast to the b-tubulin region tree,the combined data tree placed the A.muscaria clade of the UK and Poland as a sister clade of the clade of Japan and NZ with93%bootstrap support,and A.aff.muscaria was placed on a branch basal to the three clades of A.muscaria with99%bootstrap support. DISCUSSION

Both the ITS region and the b-tubulin region were able to resolve specimens of each of the two species.In particular,b-tubulin region analysis showed greater divergence than did ITS region analysis.For example, Amanita muscaria specimens from Norway and Japan (506(O),1539(O),4220(O),FB-30978(CBM),FB-30981(CBM),FB-30982(CBM))that were placed in a single clade of the ITS region tree were clearly sep-arated into the subclades of Norway and of Japan in the b-tubulin region tree.Despite the differences among the three analysis trees in the placements of A.muscaria from the UK and Poland and A.aff.muscaria,the combined data analysis revealed distinct clades and subclades of the two species with much higher boot-strap values.Thus,the results provided by the com-bined data are considered reliable.We therefore discuss the molecular phylogeny and biogeography based on the combined data analysis.In Fig.3,the three A.muscaria clades are labelled M-I,M-II,and M-III; and the two A.pantherina clades are P-I and P-II. Tables2–3detail the variable sites in the alignments of the ITS and b-tubulin regions among the clades and subclades of each species.

Amanita muscaria

We termed clade M-III,which consisted of specimens from the western and eastern USA,the‘North Amer-ican Group’.Jenkins(1977,1986)examined A.mus-caria from the USA morphologically and delineated six varieties(var.muscaria,var.alba,var.flavivolvata,var. formosa,var.persicina,and var.regalis)which differ mainly in the colour of the pileus and volval remnants. We examined three specimens of A.muscaria var. formosa,which has a yellow pileus,45883(NY),

Molecular phylogeny and biogeography of Amanita0

HDT44761(SFSU),and HDT45060(SFSU);this var-iety did not appear to be monophyletic.Four speci-mens from the western USA,i.e.HDT49100(SFSU)from Idaho and 45863(NY),HDT44761(SFSU),and HDT45060(SFSU)from California,represented one subclade and were separate from the other five spe-cimens from the eastern USA.Therefore,it appears that A.muscaria var.formosa does not reflect its mol-ecular phylogeny and that instead the molecular phy-logeny coincides with the geographical

distribution.

Fig.3.One of the 108most parsimonious trees based on the sequences of the ITS and b -tubulin regions.Amanita

pseudoporphyria was used as the outgroup.Bootstrap values over 50%are indicated at the base of the corresponding clade.With the exception of the placements within the A.pantherina subclade of Japan,the A.muscaria subclade of the UK and Poland,and the A.muscaria subclade of the western USA,each parsimonious tree shows the same topology.

T.Oda,C.Tanaka and M.Tsuda 1

(read from top to bottom).

ITS β-tubulin

1111 1111 222 22222 2222222 444 444 4455 55555 555 666 777

999 1111 3333 000 22222 3444444 666 777 9900 44444 777 777 222 666667777777777888888888 Group Specimen No. 345 1234 5678 345 34567 9012345 456 678 70 01234 678 234 123 56701234567012345678 M-I FB-309(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CG---TGTTT---ATTCTTGTGTC (UK & Poland) FB-30965(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CG---TGTTT---ATTCTTGTGTC M-31452(K) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CG---TGTTT---ATTCTTGTGTC

M-31445(K) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CG---TGTTT---ATTCTTGTGTC

M-80048(K) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CG---TGTTT---ATTCTTGTGTC (Japan & NZ) FB-30961(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CGCCCTGTTT---ATTCCTGTGCC FB-30962(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CGCCCTGTTT---ATTCCTGTGCC

FB-30963(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CGCCCTGTTT---ATTCCTGTGCC

FB-30976(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CGCCCTGTTT---ATTCCTGTGCC

FB-30977(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CGCCCTGTTT---ATTCCTGTGCC

FB-30985(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CGCCCTGTTT---ATTCCTGTGCC

FB-30987(CBM) -AT CTC- CTGT CCT GCT-G TATTT-A TGT TG- T--- T---- ATA -TC T-G CGCCCTGTTT---ATTCCTGTGCC M-II 506(O) -GT CCC- CTGT CTT GTT-G TCTTTTA TGT TA- T--- T---- AGA -GC TTG CGCCCTGTTT---ATTCTT--GTC (Norway) 1539(O) -GT CCC- CTGT CTT GTT-G TCTTTTA TGT TA- T--- T---- AGA -GC TTG CGCCCTGTTT---ATTCTT--GTC 4220(O) -GT CCC- CTGT CTT GTT-G TCTTTTA TGT TA- T--- T---- AGA -GC TTG CGCCCTGTTT---ATTCTT--GTC (Japan) FB-30978(CBM) -GT CCC- CTGT CTT GTT-G TCTTTTA TGT TA- T--- T---- AGA -GC TTG CGCCCTGTTT---ATTCTT--GTC FB-30981(CBM) -GT CCC- CTGT CTT GTT-G TCTTTTA TGT TA- T--- T---- AGA -GC TTG CGCCCTGTTT---ATTCTT--GTC

FB-30982(CBM) -GT CCC- CTGT CTT GTT-G TCTTTTA TGT TA- T--- T---- AGA -GC TTG CGCCCTGTTT---ATTCTT--GTC M-III 45843(NY) -GT CCT- CCGT CCT GTT-G TCTTT-A TCT TA- TGT- T---- AGA -GC T-G C---------------------TC (Eastern USA) 45785(NY) -GT CCT- CCGT CCT GTT-G TCTTT-A TCT TA- TGT- T---- AGA -GC T-G C---------------------TC 45820(NY) -GT CCT- CCGT CCT GTT-G TCTTT-A TCT TA- TGT- T---- AGA -GC T-G C---------------------TC

45840(NY) -GT CCT- CCGT CCT GTT-G TCTTT-A TCT TA- TGT- T---- AGA -GC T-G C---------------------TC

45883(NY) -GT CCT- CCGT CCT GTT-G TCTTT-A TCT TA- TGT- T---- AGA -GC T-G C---------------------TC (Western USA) 45863(NY) -GT CCT- CCGT CCT GTTTG TCTTT-A TCT TA- TGT- TTAT- AGA -GC T-G C---------------------TC HDT49100(SFSU) -GT CCT- CCAT CCT GTTTG TCTTT-A TCT TA- TGT- TTAT- AGA -GC T-G C---------------------TC

HDT44761(SFSU) -GT CCT- CCGT CCT GTTTG TCTTT-A TCT TA- TGT- TTAT- AGA -GC T-G C---------------------TC

HDT45060(SFSU) -GT CCT- CCGT CCT GTTTG TCTTT-A TCT TA- TGT- TTAT- AGA -GC T-G C---------------------TC Molecular phylogeny and biogeography of Amanita 2

Table 3.Variable sites in the alignment of the ITS and b -tubulin regions among groups of Amanita pantherina .The position numbers of the nucleotides are given (read from top to

bottom).

T.Oda,C.Tanaka and M.Tsuda

3

a brownish pileus and yellowish volva;(3)clade M-II is an Eurasian subalpine group of A.muscaria and is defined by habitat conditions.Jenkins(1986)reported that A.muscaria var.regalis occurred in Alaska(USA). Therefore,it will be necessary to examine A.muscaria var.regalis from other localities to determine whether this group occurs only in Eurasia or in both Eurasia and North America.

Our analysis suggests that at least three groups of A.muscaria exist in the world.At this point,we consider these groups to be the Eurasian group,the Eurasian subalpine group,and the North American group,corresponding to geographical differences.In addition,it is necessary to mention specimen FB-30986 (CBM);this collection looked like A.muscaria,but differed in the yellow stipe.In Japan, A.muscaria commonly appears under Pinaceae and/or Betula species,but FB-30986(CBM)was collected in a Fagus crenata forest.Here,we termed it Amanita aff. muscaria,because we were unable to determine if it was a distinct species or if it belonged to a group of A.muscaria.Amanita pantherina

Because all specimens in clade P-II were collected in Eurasia,we termed this clade the‘Eurasian group’.In contrast,clade P-I consisted of specimens from the western USA(California).Here,it is necessary to mention a specimen of Amanita pantherina var.multi-squamosa from eastern North America(Massachusetts) 342(NY)(not listed in Table1);we were unable to determine the sequence of the b-tubulin region of this specimen,although we did obtain a sequence of the ITS region(accession no.AB103329in DDBJ/EMBL/ GenBank).Based on the ITS region,it appears that this specimen is closer to specimens of clade P-I than to those of clade P-II.Therefore,we assumed that clade P-I probably predominates in both western and eastern North America,and it is reasonable to treat clade P-I as the‘North American group’,rather than the‘west-ern North American group’.

Biogeographical implications

Our molecular phylogenetic analyses suggested that there are three groups of Amanita muscaria(the Eur-asian,Eurasian subalpine,and North American),and two of A.pantherina(Eurasian and North American). However the area sampled for this study represents only part of the distribution of these two species.In particular,specimens from the Southern Hemisphere have remained largely unexamined,except for A.mus-caria from New Zealand.Therefore,while further examination might result in a revised grouping for each species,here we consider the biogeographical impli-cations of our results.

To better understand the biogeographical history of the two species,it is necessary to recount the biogeo-graphical history of theflora of the Northern Hemi-sphere,because both species are ectomycorrhizal associated with host trees(mainly Pinaceae,Fagaceae, and Betulaceae).Thefloral biogeography of the Northern Hemisphere has been well-investigated(e.g. Gray1846,Chaney1947,Raven&Axelrod1974, Wolfe1975,Tiffney1985a,b,Graham1993,Manche-ster1999,Wen1999).Although the explanations for the history offloral changes are controversial,it is generally accepted that important factors include the floristic migration between Eurasia and North America via the Bering and North Atlantic land bridges during the late Cretaceous and the Tertiary Periods,and cli-matic changes during the late Tertiary and Quaternary Periods.In addition,based on a molecular phylogeny of rDNA sequences,Berbee&Taylor(1993)estimated that ectomycorrhizal fungi existed during the early Cretaceous.Considering these data and the molecular phylogeny in this study,we hypothesize that the fol-lowing occurred during the late Cretaceous and the Tertiary.Initially,the ancestral group of A.muscaria existed only in Eurasia.Following climate cooling during the Tertiary,it diversified into one group that

Molecular phylogeny and biogeography of Amanita4continued living under moderate climatic conditions in Eurasia(Eurasian group),and another adapted to cooler climates.After the climate warmed again,the latter diversified into the group that invaded subalpine regions of Eurasia(Eurasian subalpine group)as a relic,and another group that transferred to North America via the land bridges(North American group).

A.pantherina is divided into at least two groups,the North American group and the Eurasian group.Based on its topology,it is impossible to determine whether A. pantherina moved from Eurasia to North America or vice versa.Based on accounts by Tiffney(1985a,b), during the early Eocene to Miocene,two species could have migrated with their host trees to each continent via the North Atlantic land bridge and/or the Bering land bridge.The genetic variations in and between each group of A.pantherina are greater than those of A.muscaria(Fig.3).Therefore,we assume that the diversification and disjunction of A.muscaria are more recent events than those of A.pantherina.

With regard to theflora of the Northern Hemi-sphere,the disjunction described by Gray(1846)is noteworthy.This author made detailed comparisons among the North Temperateflora,and concluded that theflora of eastern North America and eastern Asia were more similar to each other than to theflora of western North America or Europe.In terms of macro-fungi,Hongo&Yokoyama(1978)suggested a close affinity of mycobiotas of eastern Asia and eastern North America,corresponding to Gray’s theory. Moreover,Hongo&Yokoyama(1978)stated that specific differentiation in the fungi had not advanced as much in the vascular plants,which had differentiated at the specific or even generic levels.Wu&Mueller(1997) reached the same conclusions based on a literature and herbarium survey.However,more recent molecular phylogenetic studies suggest that the biogeographical relationships of macrofungi between eastern North America and eastern Asia may not be as close as in-dicated by morphological data,whereas the molecular data do not refute the hypothesis that a relatively close relationship exists between them(Mueller et al.2001). In this study,we found that in A.muscaria and A. pantherina,the intracontinental relationships of both Eurasia and North America were closer than the re-lationships between eastern Asia and eastern North America.Additional molecular phylogenetic data of other Amanita species and specimens from diverse localities will provide an even better understanding of the biogeographical and evolutionary history of the genus.

ACKNOWLEDGEMENTS

This work was supported in part by Hokuto Foundation,and by a Grant-in-Aid(15380104)for Scientific Research(B)from the Japan Society of the Promotion of Science.For supplying specimens,we thank Roman Kotlowski(Department of Food Chemistry and Technology,Gdansk University of Technology,Gdansk),Toshimitsu Fukiharu(Natural History Museum and Institute,Chiba),Yoko Ando,Masaki Endo,Kyoko Isoda,Susumu Ito,Keiko Kudo, Shinichi Kudo,and Youkin-no-kai.We are also grateful to the following institutions:The Natural History Museum and Institute, Chiba;Herbarium of Cryptogams,Kunming Institute of Botany, Academia Sinica,Kunming;Royal Botanic Gardens,Kew;New York Botanical Garden,Bronx,New York;Botanical Museum, Oslo,Harry D.Thiers Herbarium,San Francisco State University, San Francisco;and Botany Department,National Science Museum, Tsukuba.For helping with the collection of specimens from Nepal, we are grateful to Mahesh K.Adhikari(National Herbarium and Plant Laboratory,Department of Plant Resources,Ministry of Forest and Soil Conservation,Kathmandu,Nepal),who guided us around the forests of Nepal,and Yoshimichi Doi(Department of Botany,National Science Museum,Tsukuba,Japan),who arranged the research trip to Nepal.We also thank Naoki Taniguchi(Kyoto Research Center for Food Hygiene and Technology),who provided support for the DNA sequence analyses.

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Corresponding Editor:D.S.Hibbett

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