Kretzer, A., Li, Y., Szaro, T.M., Bruns, T.D. 1996

Mycological Society of America

Internal Transcribed Spacer Sequences from 38 Recognized Species of Suillus sensu lato: Phylogenetic and Taxonomic Implications Author(s): Annette Kretzer, Yunan Li, Timothy Szaro and Thomas D. Bruns Reviewed work(s): Source: Mycologia, Vol. 88, No. 5 (Sep. - Oct., 1996), pp. 776-785 Published by: Mycological Society of America Stable URL: http://www.jstor.org/stable/3760972 . Accessed: 10/12/2012 12:54

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This content downloaded by the authorized user from 192.168.52.70 on Mon, 10 Dec 2012 12:54:17 PM All use subject to JSTOR Terms and Conditions Mycologia,88(5), 1996, pp. 776-785. © 1996 by The New YorkBotanical Garden, Bronx,NY 10458-5126

Internaltranscribed spacer sequences from38 recognizedspecies of Suillus sensu lato: Phylogeneticand taxonomicimplications

AnnetteKretzer the patternof ectomycorrhizalhost-symbiont evolu- Yunan Li tion and potentialcospeciation. TimothySzaro Suillus sensu lato includes organisms sometimes Thomas D. Bruns also classifiedas Boletinusor Fuscoboletinus.Delinea- Departmentof ESPM, Universityof Californiaat tion of Suillus sensu strictofrom the related genera Berkeley,108 Hilgard Hall, Berkeley,California Boletinusand Fuscoboletinushas remained controver- 94720-3110 sial (Pomerleau and Smith,1962; Smithand Thiers, 1971; Pegler and Young, 1981; Singer,1986; Sutara, 1987). Boletinusis a ratherold name and the genus Abstract: Internal transcribedspacer regionsof the has traditionallybeen recognizedby its radiallyelon- nuclear ribosomalrepeat have been sequenced from gated pores (boletinoid hymenophore) (Kalchbren- 47 isolatesbelonging to 38 recognizedspecies of Suil- ner, 1867). Other charactersthat have been used to lus sensu lato. The sequences have been analyzedfor differentiateBoletinus from Suillus are the presence phylogeneticand taxonomic implicationsusing par- of clamp connections,a sterilestipe surfacewithout simonyas well as distance methods. Based on these glandulardots and the absence of fasciculateencrust- data, the genera Boletinus and Fuscoboletinus,that are ed pleurocystidia.Delineations based on these char- often recognized within Suillus sensu lato, are not acters are, however,inconsistent (Singer, 1962). Fi- Isolates of Suillus derived monophyletic. granulatus nally,Singer (1986) restrictedthe genus to threespe- North America or and Asia are fromeither Europe cies (B. cavipes,B. palusterand B. asiaticus) which and seem to at least two dif- polyphyletic represent are characterizedby the presence of clamp connec- ferentspecies. Our data also suggestthat within Suil- tions in theirfruitbodies. It is well known,however, lus sensu associationswith Larix are lato, mycorrhizal that species of Suillus sensu strictoare also able to and host to Pinus and primitive changes Pseudotsuga develop clamp connections in mycelium cultures have occurred once. seem to only Changes among (Hibsch, 1961; Pantidou and Groves, 1966). Fusco- host of the same to be more species genus appear boletinuson the other hand is a comparablyrecent the most clade withinSuil- frequent.Finally, primitive segregatein whichPomerleau and Smith(1962) have lus sensu lato seems to be formed with by organisms grouped togetherall Suillus or Boletinusspecies that a boletinoid strongly hymenophore. give a vinacious to purple brownspore print. Words: Boletales, ITS se- Key Basidiomycota, In the presentstudy, we have determinedinternal quences, phylogeny,taxonomy transcribedspacer (ITS) sequences from47 isolates belonging to 38 differentspecies of Suillus sensu lato. We have used those data to performphyloge- INTRODUCTION netic analysesand to clarifythe confusedstate of taxonomy. The genus Suillus sensu lato (Basidiomycota,Bole- tales) comprises approximately70-80 species that produce epigeous mushroomswith tubular hymen- MATERIALS AND METHODS ophores. Its closestrelatives are Rhizopogon(and other hypogeous fungi closely related to Rhizopogon; Fungal specimensand DNA extraction.-Allfungal Bruns et al., 1989), and the Gomphidiaceae (Bruns specimens used in this studyare listed in TABLE I. and Szaro, 1992); collectively,these organismshave Crude DNA extractswere prepared by eitherof two been referredto as the suilloid radiationin the Bol- methods, a CHELEX method (Taylor and Swann, etales (Bruns and Szaro, 1992). Withfew exceptions, 1994) or a modified CTAB method (Gardes and Suillus and other membersof the suilloid group are Bruns,1993). The formerwas preferablyused forex- ectomycorrhizalassociates of the Pinaceae. Due to tractionsfrom fresh mycelia, and the latterfor ex- the fairlyhigh degree of host specificityespecially tractions from dried fruitbodycollections. Either withinSuillus sensu lato, detailed knowledgeof the method yielded crude extracts with unquantified phylogenyprovides a unique opportunityto examine DNA contents.From these extracts,a range of dilu-

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This content downloaded by the authorized user from 192.168.52.70 on Mon, 10 Dec 2012 12:54:17 PM All use subject to JSTOR Terms and Conditions KRETZER ET AL.: SUILLUS PHYLOGENY 777 tionswas made in water (usuallyaround 10-2 to 10-3) for processing the raw data. A few sequences were and used for PCR amplification. determinedby conventionaldideoxy terminationre- action with35S labeled ATP as described by Bruns et Polymerasechain reaction.-Reaction mixtureswere al. (1990). made up fromequal volumes of DNA solutionand a master mix containingall the other necessarycom- Phylogeneticanalysis.-Sequences were aligned by vi- ponents.Final concentrationswere: 10 mM Tris/HCl sual estimationusing a matrixcreated in PAUP 3.1.1 pH 8.3, 50 mM KCl, 2.5 mM MgCl2,0.1 mg/mL gel- (Swofford,1993) and a color font.Computer align- atin,200 pIMof each of the fourdeoxyribonucleotide ment was not found to be usefuldue to shorthyper- triphosphates,0.5 JLMof each of two differentprim- variable areas in the ITS-sequences. Obvious inser- ers, 25 U/mL Taq polymerase,and empiricallydeter- tions/deletions(indels) were considered to be infor- mined amounts of templateDNA (see paragraphon mativeand were weightedequal to one or two tran- DNA extractions).Varying sets of primerswere used sition(s) (except fora verylarge insertionpresent in in the reactions. In most cases, use of the primer the outgroupsthat was weightedas four transitions). pairs ITS5/ITS4 or ITS5/ITS4-S (that amplifyboth Technically,this was done as follows:Alignment gaps ITS regions including the 5.8S rDNA) was found to were treated as missing data, but a new character be convenient. From older specimens, however, state "I" was introducedat deletion sites.The weight yields could be significantlyincreased by individual attributedto the proper deletion was taken into ac- amplificationof the two ITS regionswith the primer count by the number of "I"s being introduced.As pairs ITS5/ITS2 and ITS3/ITS4-S. Primersequences faras possible,only gaps withexactly the same length and maps have been published before (White et al., and positionwere coded forwith "I"s introducedat 1990; Gardes and Bruns, 1993) except forITS4-S the preciselythe same position.Phylogenetic analysis was sequence of which is: 5'-CCTCCGCTTATTGA- done using both parsimony (Fitch, 1971) and dis- TATGCTTAAG-3'.PCR reactionswere overlaidwith tance methods (neighbor joining, Saitou and Nei, a drop of mineral oil (Sigma) and subjected to 35 1987). Searches for most parsimonious trees were amplificationcycles on a Techne Thermal Cycler done in PAUP 3.1.1, and neighborjoining was per- (model PHC-2). Conditions for the thermalcycling formedas follows:First a distancematrix was created are describedin Bruns and Gardes (1993). If the first in PAUP 3.1.1 thatwas subsequentlyadjusted manu- amplificationsyielded some but not enough DNA, ally to preciselyfit the output formatof DNADIST, a aliquots of the amplified fragmentwere run on a program of the PHYLIP 3.5 package that computes 1.5% low melt agarose gel, bands were cut out, melt- distancematrixes from sequence data. The advantage ed into TE buffer(10 mM Tris/HCl pH 8.0, 1 mM of using PAUP 3.1.1 over DNADIST for computing EDTA) at 65-70 C, diluted (10-2 in water) and sub- distanceswas that PAUP 3.1.1 recognized the intro- jected to another round of 35 amplificationcycles. duced characterstate "I", when the data typewas not definedto be "DNA". the edited distancema- Sequencing.-For use in sequencing, PCR products Finally, trixwas fed into another of were cleaned fromthe reactionmixture either by suc- NEIGHBOR, program the PHYLIP 3.5 for Boot- cessive dilutionwith H20 and reconcentrationusing package, neighborjoining. were done under the crite- centrifugalfilters (Millipore Ultrafree-MCfilters) or strapanalyses parsimony withthe BIO 101 Geneclean II kit.In the lattercase, rion in PAUP 3.1.1, and values are based on 500 rep- licates. and vin- DNAs were firstrun on an agarose gel (1% normal Gomphidiusglutinosus Chroogomphus icolorhave been chosen as for the agarose, 2% NuSieve agarose (FMC BioProducts), outgroups phylo- because both are bands were cut out and subsequentlycleaned accord- genetic analyses, closelyrelated to Suillus and ing to the kit instructions.Sequencing was done by (Bruns Szaro, 1992). Rhizopogonsubcaeru- Truncocolumellacitrina and the cyclic reaction terminationmethod using fluo- lescens, Gastrosuilluslari- cinuswere also included in the sub- rescence labeled dideoxyribonucleotide triphos- study.Rhizopogon caerulescensand Truncocolumellacitrina have phates. The sequence reactionand the processingof been shown before to be related to Suillus the reaction products for electrophoresiswere per- closely (Bruns et Bruns et Gastrosuilluslaricinus formedfollowing the instructionsfor the sequencing al., 1989; al., 1990). is a recent derivativeof Suillus et kit (PRISM® Ready Reaction DyeDeoxy' Termina- grevillei(Baura al., tor Cycle Sequencing Kit, Perkin-ElmerCorpora- 1992). tion). Electrophoresisand data collectionwere done on an ABI Model 373A DNA Sequencer (Perkin-El- RESULTS mer Corporation) to which a MacintoshQuadra 650 computerwas connected. DNA Sequencing Analysis Complete, two directional sequences of both ITS (version 2.01) and SeqEd (version 1.03) were used regions (1 and 2) were determinedfrom the speci-

This content downloaded by the authorized user from 192.168.52.70 on Mon, 10 Dec 2012 12:54:17 PM All use subject to JSTOR Terms and Conditions 778 MYCOLOGIA

= = = TABLEI. Specimens used in thisstudy: B. Boletinus,C. Chroogomphus,F Fuscoboletinus,G. Gomphidius, Ga. Gastrosuillus,R. = Rhizopogon,S. = Suillus, T = Truncocolumella.Only selected synonymsare given

Collection Geographic Specimen number origin Locationa C. vinicolor(Peck) Miller TDB-1010 USA, California TDB Ga. laricinus(Singer & Both) Thiers EB-2031b USA, New York BM G. glutinosus(Schaeff. ex Fr.) Fr. TDB-935B USA, California TDB R. subcaerulescensSmith F-2882b USA, Colorado MICH S. americanus(Peck) Snell ex Slipp & Snell TDB-581 USA, Michigan MICH S. asiaticus (Sing.) Kretzer& Bruns JV-4850F Finland MICH = B. asiaticusSinger S. bovinus(L. ex Fr.) Kuntze AD-5 Sweden TDB S. bresadolae(Quel.) Gerhold HB-399 Germany HB = S. aeruginascensvar. bresadolae(Quel. in Bres.) Mos. S. brevipes(Peck) Kuntze TDB-834 USA, Michigan TDB S. caerulescensSmith & Thiersc TDB-1028 USA, California TDB S. cavipes(Opat.) Smith& Thiers WJS-618 USA, Idaho SFSU = B. cavipes(Opat.) Kalchbr. TDB-646 USA, Michigan TDB HDT-31407 Switzerland SFSU S. collinitus(Fr.) Kuntze J3.15.2 France MNHN S. cothurnatusSinger NSW-4662 USA, Louisiana MICH S. decipiens(Berk. & Curt.) Kuntze DG-1451 USA, Texas MICH = B. decipiens(Berk. & Curt.) Peck S. glandulosipesSmith & Thiers HDT-8394 USA, California MICH S. granulatus(Fries) Kuntze AD-11 Sweden TDB S. granulatus(Fries) Kuntze TDB-878 USA, Michigan TDB S. c. f granulatus(Fries) Kuntze VC-1042 Nepal VC S. grevillei(Klotzsch) Singer EB-2040Ab USA, New York BM S. grisellus(Peck) Kretzer& Bruns TDB-574 USA, Michigan TDB = F grisellus(Peck) Pomerleau & Smith = B. grisellus Peck S. intermedius(Smith & Thiers) Smith& Thiers ACAD-15271 Canada, Nova Scotia TDBd = S. acidus var. intermediusSmith & Thiers S. lakei (Murrill) Smith& Thiers HDT-44112 USA, California SFSU = B. lakei (Murill) Singer S. laricinus(Berkeley ex Hooker) Kuntzee VC-1231 Nepal VC = S. viscidus(Fr. & Hok) Rauschertin Dorfelt TDB-561 USA, Michigan TDB = S. aeruginascens(Secr.) Snell in Slipp & Snell TDB-576 USA, Michigan TDB = F. aeruginascens(Secr.) Pomerleau & Smith S. luteus(Fries) Gray TDB-571 USA, Michigan MICH S. luteus(Fries) Gray TDB-824 USA, Michigan TDB S. luteus(Fries) Gray HB-348 Germany HB S. neoalbidipesPalm & Stewart TDB-835 USA, Michigan TDB S. ochraceoroseus(Snell) Singer SAR-84-137 USA, Washington MICH = F ochraceoroseus(Snell) Pomerleau & Smith = B. ochraceoroseusSnell S. paluster(Peck) Kretzer& Bruns GT-831015/15 Canada, Ontario DAOM-190075 = B. paluster(Peck) Peck = F paluster(Peck) Pomerleau S. placidus (Bonorden) Singer TDB-725 USA, Michigan MICH S. c. f placidus (Bonorden) Singer VC-1022 Nepal VC S. pseudobrevipesSmith & Thiers TDB-663 USA, Colorado TDB S. punctipes(Peck) Singer TDB-265 USA, Minnesota TDB S. pungensSmith & Thiers TDB-1001 USA, California TDB S. serotinus(Frost) Kretzer& Bruns TJB-6597 USA, New York CORT = F serotinus(Frost) Smith& Thiers S. sibiricus(Singer) Singer VC-1040 Nepal VC S. sinuspaulianus(Pomerleau & Smith) Dick & Snell DAOM-66995 Canada, Quebec DAOM-66995 = F sinuspaulianusPomerleau & Smith

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TABLEI. Specimens used in thisstudy: B. = Boletinus,C. = Chroogomphus,F = Fuscoboletinus,G. = Gomphidius,Ga. Gastrosuillus,R. = Rhizopogon,S. = Suillus, T = Truncocolumella.Only selected synonymsare given (Cont.)

Collection Geographic Specimen number origin Locationa S. spectabilis(Peck) Kuntze TDB-641 USA, Michigan MICH = F. spectabilis(Peck) Pomerleau & Smith = B. spectabilis(Peck) Murrill S. spraguei(Berk. & Curt.) Kuntze TDB-638b USA, Michigan MICH = S. pictus (Peck) Smith& Thiers = B. pictus (Peck) Peck S. subalutaceus(Smith & Thiers) Smith& Thiers ACAD-15288 Canada, Nova Scotia TDBd = S. acidus var. subalutaceusSmith & Thiers S. subaureus(Peck) Snell in Slipp & Snell TDB-780 USA, Massachusetts TDB S. subluteus(Peck) Snell ex Slipp & Snell IB-13-8/19/72 USA, Michigan MICH S. tomentosus(Kauffmann) Singer, Snell & Dick TDB-661 USA, Colorado MICH S. tridentinus(Bres.) Singer HB-347 Germany HB S. umbonatusDick & Snell TDB-978 USA, California TDB S. variegatus(Swartz ex Fr.) Kuntze HB-325 Germany HB S. weaverae(Smith & Shaffe)Kretzer & Bruns MGW-1992 USA, Minnesota MIN-794257 = F weaveraeSmith & Shaffer T. citrinaZeller TDB-2001 USA, California TDB a BM, BuffaloMuseum, New York;CORT, Herbarium of the State Universityof New York-College at Cortland; DAOM, National MycologicalHerbarium of Canda; HB, H. Besl, Universityof Regensburg,Germany; MIN, Universityof Minnesota Herbarium;MICH, Herbariumof the Universityof Michigan;MNHN, Museum National d'HistoireNaturelle, Paris, France; SFSU, San Francisco State UniversityHerbarium; TDB, T. D. Bruns, UC Berkeley;VC, Van Cotter,Cyanamid Forschung GmbH, Schwabenheim,Germany. b ITS sequences of these specimenshave been published previously(Baura et al., 1992). c The specimen TDB-1028 was identifiedas S. caerulescenswithout differentiating between S. caerulescensSmith & Thiers and S. ponderosusSmith & Thiers which are distinguishedony by a dryversus gelatinous annulus. d Cultureswere obtained fromK. A. Harrison,Acadia University,Nova Scotia. eThe specimens TDB-561 and TDB-576 have been identifiedas S. laricinuswithout differentiating between S. laricinus (Berkeleyex Hooker) Kuntze and S. serotinus(Frost) Kretzer& Bruns, two varietiesthat have been raised to the level of separate species by Smithand Thiers (1971). They differin a bluish-greenversus bluish to dark bluish-grayto fuscouscolor reaction. mens listed in TABLE I. In most cases, the sequences Although a number of branches on the neighbor include the complete 5.8S rRNA gene. They have joining tree are weaklysupported (as can be seen by been submittedto the Genome Sequence Data Base comparison to FIG. 1), it currentlyrepresents our under the accession numbersL54074 to L54121. best phylogeneticestimate. Alignmentof the fullset of sequences was in some Finally,we have assessed intraspecificsequence areas ambiguous. Inclusion or exclusion of these ar- variation and relationshipsbetween closely related eas did not, however,seem to influence the main taxa in a numberof separateanalyses, and the results conclusions drawn fromthe study,nor did differen- are shownin FIG. 3. Trees were constructedfrom the tial weightingof transitionsand transversions(either correspondingsubset of taxa by neighborjoining. As equal or transversionstwice over transitions).Long onlyclosely related species were included in each da- branches essentiallyproved to be well supported in- taset, sequences could be aligned unambiguously, dependent of the treatment,whereas short branches and no areas of ambiguous alignmentneeded to be were unstable. Results presented in FIGS. 1 and 2 are excluded. Transitionsand transversionswere, again, based on unweightedanalyses fromwhich areas of weightedequally. ambiguous alignment have been excluded. Under these conditions, the data matrix comprised 676 DISCUSSION charactersout of which 168 were cladisticallyinfor- mative. FIG. 1 summarizes results from parsimony Molecular phylogeneticanalyses presented in thispa- analysis;only brancheswith more than 50% support per stronglysupport the concept of a genus Suillus by bootstrap analysis are shown. FIG. 2 presents a sensu lato as introducedby Bruns and Palmer (1989) neighborjoining tree derivedfrom the same dataset. which comprisesboth the genera Boletinusand Fus-

This content downloaded by the authorized user from 192.168.52.70 on Mon, 10 Dec 2012 12:54:17 PM All use subject to JSTOR Terms and Conditions * S. weaverae (MGW-1992) S. brevipes (TDB-834) S. luteus (TDB-571) S. pseudobrevipes (TDB-663) S. glandulosipes (HDT-8394) S. neoalbidipes (TDB-835) 0 S. granulatus(AD-11) S. pungens (TDB-1001) S. collinitus(J3.15.2) 0 S. granulatus(VC-1042) S. tomentosus(TDB-661) S. variegatus(HB-325) S. punctipes (TDB-265) S. bovinus (AD-5) S. decipiens (DG- 1451) S. spraguei (TDB-638) 0 S. granulatus(TDB-878) S. placidus (TDB-725) S. americanus (TDB-581) S. sibiricus(VC- 1 040) S. umbonatus (TDB-978) S. intermedius(ACAD- 15271) O S. subalutaceus (ACAD-15288) S. cothurnatus(NSW-4662) 2 S. subluteus (IB-13-8/19/72) S. subaureus (TDB-780) S. caerulescens (TDB- 1028) S. lakei (HDT-44112) *S. sinuspaulianus (DAOM-66995) * S. spectabilis (TDB-641) Ga. laricinus(EB-2031) S, grevillei(EB-2040A) S. tridentinus(HB-347) * S. grisellus(TDB-574) * S. laricinus(TDB-561) E S. asiaticus (JV-4850F) 0 S. cavipes (TDB-646) ENS. paluster (GT-831015/15) m S. ochraceoroseus (SAR-84-137) R. subcaerulescens (F-2882) T. citrina(TDB-2001) C. vinicolor(TDB-1010) G. glutinosus(TDB-953B)

This content downloaded by the authorized user from 192.168.52.70 on Mon, 10 Dec 2012 12:54:17 PM All use subject to JSTOR Terms and Conditions KRETZER ET AL.: SUILLUS PHYLOGENY 781 coboletinus.Based on ITS data, neitherBoletinus nor therBoletinus nor Fuscoboletinushas been consistent- Fuscoboletinusas defined by Singer (1986) and Pom- lyrecognized by authorsin the past, most of the spe- erleau and Smith (1962), respectively,represents a cies have at some point alreadybeen included in Suil- monophyleticgroup. Several well supportedbranch- lus. Those species that haven't and that are treated es render Fuscoboletinuspolyphyletic, and the short- or mentioned in thisstudy are transferredbelow. est treesshowing Fuscoboletinus as a monophyleticge- Isolates of Suillus granulatusderived fromeither nus were 33 steps longer than the shortest trees North America (S. granulatusTDB-878) or Europe found overall (731 versus698 steps). Boletinus,on the and Asia (S. granulatusAD-11 and S. c. f granulatus other hand, was found to be paraphyletic,because a VC-1042) were widelyseparated into differentclades stronglysupported branch placed Suillus ochraceoro- by stronglysupported branches. These findingsare seus in the middle of the Boletinusclade. S. ochraceo- in agreement with earlier studies based on mating roseus had originally been described as Boletinus behaviorand the physiologyof mycorrhizaformation ochraceoroseus(Snell and Dick, 1941) but was moved which indicated that isolates fromAmerica and Eu- into Suillusby Singer, when he narrowedthe concept rope (Fries and Neumann, 1990) or America and of Boletinusto those species that had clamp connec- Asia (Jacobsonand Miller,1992) mightrepresent two tions in their fruitbodies(= B./S. asiaticus, B./S. differentspecies. To our knowledge,no comparison cavipes,B./S. paluster) (Singer,1962, 1986). Accord- has so far been made between isolates fromEurope ing to Singer's older and broader genus concept and Asia. Althoughisolates fromSweden and Nepal however,Boletinus as a whole is again not monophy- were more closelyrelated to each other in thisstudy letic but polyphyletic,because it includes S. ochraceo- than either of themwas to NorthAmerican isolates, roseuswith S. spraguei,S. spectabilis,S. grisellus,S. they were still separated by a stronglysupported lakei,S. decipiensand some additional taxa thatwere branch which puts S. collinitusnext to S. granulatus not treated in this study (Singer, 1962). Altogether, fromNepal and makes these twoorganisms paraphy- the only natural clade that seems to correlatefairly letic to the Swedish S. granulatusand others.In ad- well with any concept for the genus Boletinuscom- dition, S. granulatusassociates witha verydifferent prises the species asiaticus, cavipes, paluster,and pine in Sweden than in Nepal (Pinus sylvestrisL. sec- ochraceoroseus.Whether these species are to be kept tion Pinus versus Pinus wallichianaJackson section in a separate genus, seems to be a matterof taste. Strobus).Finally, S. c. f granulatusfrom Nepal also However,we do not supportit forseveral reasons: (i) seems to be morphologicallydistinct from other or- Beside molecular evidence, thereare no morpholog- ganismsbeing classifiedas S. granulatusin thatit has ic or physiologicsynapomorphies known that differ- a less mottledcap and sometimesstains slightly blue entiate thisclade fromSuillus. (ii) Based on our mo- green to blue at the stipe base (Cotter,1987). Over- lecular data, the clade is not strikinglydistant from all, current evidence suggests that there might be Suillus. (iii) In fact,there is no strongevidence for three differenttaxa to be recognizedwithin the Suil- the "Boletinus"clade being basal withinSuillus sensu lus granulatuscomplex, but more isolatesneed to be lato, but a basal position is a necessaryrequirement included in futurestudies to fullyresolve the genetic forboth Suillus and "Boletinus"to be monophyletic. heterogeneitywithin this complex. There are at least Other than Boletinusand Fuscoboletinus,Suillus twomore membersof the S. granulatuscomplex that sensu lato appears to be monophyleticwith the ex- need to be examined: (i) anotherAsian S. granulatus ception of Gastrosuilluslaricinus which is a recent fromKorea which growswith Pinus densifloraSieb. derivativeof Suillus grevillei(Baura et al., 1992) and and Zucc. fromthe section Pinus (in contrastto the probablyother Gastrosuilli(unpublished results).As Nepalian isolate included in this study that grows we are planning more detailed studieson the genus withPinus wallichianafrom the section Strobus);(ii) Gastrosuillus,we will discuss this problem at a later S. granulatus found in the southwesternUnited point. For now,we propose to collapse both the gen- States growingwith Pinus ponderosaLaws. (section era Boletinusand Fuscoboletinusinto Suillus. As nei- Pinus), while other NorthAmerican isolates (includ-

FIG. 1. Phylogenetic analysis of the ITS sequence data: Bootstrap analysis under parsimony criterion showed that most of the major lineages are recognized with reasonable confidence,while relationshipsamong them are not. From the major lineagesit is evidentthat neither Boletinus nor Fuscoboletinus as defined by Singer (1986) and Pomerleauand Smith(1962), respectively,is monophyletic. Furthermore, isolates of S. granulatusderived from either North America (S. granulatusTDB- 878) or Europe(S. granulatusAD-1 1) and Asia (S. c.f granulatusVC-1042) are polyphyleticand do notseem to represent the same species. Branch lengthsare arbitrary.

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r S. weaverae MGW-1992 I * S. brevipesTDB-834 S. luteusTDB-571 - _ *U S. pseudobrevipes TDB-663 s* glandulosipes HDT-8394 L * S. neoalbidipes TDB-835 * S. granulatusAD- 11 * S. pungensTDB- 1001 * S. collinitusJ3.15.2 { *E S, c. f.granulatus VC- 1042 - S. tomentosusTDB-661 subgenus: S. variegatusHB-325 EEI S. punctipesTDB-265 / Pinus S. bovinus AD-5 pine * S, decipiens DG-1451 . L[gStrobus : :S. spragueiTDB-638 S. | r- granulatusTDB-878 EL S. placidus TDB-725 -El S. americanus TDB-581 B S. sibiricusVC- 1 040 * S. umbonatusTDB-978 o - S. intermediusACAD- 15271 * S. subalutaceus ACAD-15288 >. * S. cothurnatusNSW-4662 l * S. subluteus IB-13-8/19/72 0.02 I 3 S, subaureus TDB-780 hardwoods S. caerulescensTDB-1028 2E Douglas-fir : 1S3 S. lakei HDT-44112 a -1 El S. sinuspaulianus DAOM-66995 -'i unknown I S. spectabilisTDB-641 * Ga. laricinusEB-2031 S. grevilleiEB-2040A lZ S. tridentinusHB-347 E S. grisellusTDB-574 . larch LI EB S. laricinusTDB-561 - S. asiaticusJV-4850F ii S. cavipes TDB-646 E-I E S. palusterGT- 15 EL s ochraceoroseusSAR-84-137 R. subcaerulescens F-2882 various hosts in the Pinaceae T. citrinaTDB-2001 Douglas-fir C. vinicolorTDB-1010 pine G. glutinosusTDB-953B Douglas-fir

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A B S. decipiensDG-1451 S. tridentinusHB-347 4 1 Ga. laricinusEB-2031 S. spragueiTD-B638 ! S. grevilleiEB-2040A S. grisellusTDB-574 100 rS. granulatusTDB-878 97- S. laricinus TDB-561 (NorthAmerica) _4L-S~. laricinus TDB-576 (NorthAmerica) S. placidus TDB-725(North America) 95 .r S. bresadolae HB-399(Europe) S. serotinus TJB-6597 (NorthAmerica) S. c. f.placidus VC-1022(Nepal) S. c. f. laricinus VC-1231 (Nepal) 0.02

S. weaveraeMGW-1992 D - S. brevipesTDB-834 S. luteusTDB-571 (North America) S. asiaticusJV-4850F S. luteusHB-348 (Europe) 69fr S. cavipes WJS-618(North America) S. luteusTDB-824 (North America) -S. pseudobrevipes TDB-663 100 S. cavipes TDB-646(North America) rS, glandulosipesHDT-8394 -- S. cavipes HDT-31407(Europe) S. neoalbidipesTDB-835 S. pungensTDB-1 001 100 S. palusterGT- 15 S. granulatusAD-11 - S. collinitusJ3.15.2 S. ochraceoroseusSAR-84-137 0.02 S. granulatusVC-1042 I I! 0.02

FIG. 3. Assessingintraspecific sequence divergence:Bootstrap values are givenfor selected branches of interestonly when above 60%. Horizontalbranch lengthsrepresent mean distances(Swofford, 1993). ing TDB-878) grow with Pinus strobusL. (section Stro- American S. placidus TDB-725 and American S. gran- bus). ulatus TDB-878 tightlytogether and makes the two Besides the S. granulatus problem, minor conflicts S. placidus isolates from either North America or Ne- with current species concepts have been observed in pal paraphyletic instead of monophyletic (see FIG. two more cases both of which involve specimens from 3A). (ii) Finally, the isolate VC-1231 (Nepal) of Suil- Nepal: (i) Suillus c. f placidus from Nepal has been lus laricinus fitsinto the clade of S. laricinus and rel- described as fittingcurrent species descriptions well atives but is paraphyletic to S. laricinus sensu stricto but not completely, although observed differences (FIG. 3B). The species concept for S. laricinus has were not explicitly stated (Cotter, 1987). We found S. been considerably narrowed in the last decades by c. f placidus VC-1022 (Nepal) and S. placidus TDB- elevatingthe formervariants S. bresadolaeand S. ser- 725 (North America) to be fairlyclosely related, but otinusto the level of independent species (Gerhold, separated by a well supported branch which puts 1985, and Smith and Thiers, 1971, respectively).Fu-

FIG. 2. Neighborjoining tree representinga best tree estimate:Within the phylogenyof Suillus sensu lato, mycorrhizal associationswith larches seem to be primitiveand switchesto differenthost genera such as pines, Douglas-fir,or hardwoods seem to have occurred only once. On the other hand, host switchesbetween pines of the differentsubgenera Pinus or Strobusseem to be ratherfrequent. The shown host specificitiesare based on observationsof fruitinghabits both by the authors and other authorities(Smith and Thiers, 1964; Smith and Thiers, 1971; Singer, 1986; Cotter,1987). Horizontal branch lengthsrepresent the mean distances(Swofford, 1993). a Fruitbodieshave been found in mixed coniferforests under Pinus, Abies and Picea.

This content downloaded by the authorized user from 192.168.52.70 on Mon, 10 Dec 2012 12:54:17 PM All use subject to JSTOR Terms and Conditions 784 MYCOLOGIA ture work including more isolates should show cently:Within the suilloid group, tubes seem to be whetherthis refined species concept is supportedby derivedfrom gills (Bruns and Szaro, 1992). molecular data. If thatis the case, a new species will be needed to accommodate "S. laricinus"from Ne- pal which might also be supported by the unusual TRANSFER OF SPECIES stainingpattern observed on these specimens (Cot- Because the molecularevidence presentedin thispa- ter,1987). per suggeststhat both genera Boletinusand Fuscobol- Intraspecificsequence divergence was less pro- etinusshould be into we nounced in two more cases examined and did not collapsed Suillus, formally propose to transfera number of included in conflictwith currentspecies concepts (FIG. 3C and species this to Suillus: D). Suillus luteus was the least divergentspecies analysis looked at withonly one base differenceeven between isolates from and North America.This find- Europe Suillus asiaticus(Singer) Kretzer& Bruns,comb. nov. ing is in agreementwith the idea of a recent intro- Basionym:Boletinus asiaticus Singer. Rev. Mycol.3. duction of S. luteusinto NorthAmerica. Suillus cav- 164. 1938. ipes formed a divergentbut monophyleticgroup, with the European isolate HDT-31407 being more distantlyrelated to the twoAmerican isolates WJS-618 Suillus grisellus(Peck) Kretzer& Bruns, comb. nov. and TDB-646. Basionym:Boletinus grisellus Peck. Mem. N. Y State Our best tree estimate shown in FIG. 2 suggests Museum4: 169. 1900. some thatare to note evolutionaryaspects interesting Synonym: Fuscoboletinusgrisellus (Peck) Pomer- though theyare not stronglysupported by the data. leau and Smith.Brittonia 14: 168. 1962. First,the patternof host specificitywithin Suillus sensu lato does not seem to have evolved throughco- speciationwith the ectomycorrhizalhosts. In conflict Suillus paluster(Peck) Kretzer& Bruns,comb. nov. withthe idea of cospeciationis primarilythe finding Basionym: Boletuspaluster Peck. Ann. Rept. New that,while Larix is considered to be more recently YorkState Cab. 23: 132. 1872. derived than Pinus (Hart, 1987), larch association Synonyms:Boletinus paluster (Peck) Peck. Bull. seems to be primitivein Suillus,and associationswith N.Y. StateMuseum 2: 78. 1889. pines, Douglas-fir,and hardwoods seem to be de- Boletinelluspaluster (Peck) Murrill.Mycologia 1: 8. rived. Rather than cospeciation, multiplejumps to 1909. new host species and genera seem to have occurred Fuscoboletinus paluster (Peck) Pomerleau and during the evolution of Suillus sensu lato. While Smith.Mycologia 56: 708. 1964. changes in host specificityto closely related hosts seem to be fairlyfrequent events (at leastfive changes between the pine subsectionsPinus and Strobusare Suillus serotinus(Frost) Kretzer& Bruns,comb. nov. implied in FIG. 2), all changes to new host genera Basionym: Boletus serotinus(Frost) Bull. Buffalo seem to be unique eventsin the evolutionof Suillus Soc. Nat. Sci. 2: 100. 1874. sensu lato. Changes in host specificityhave also oc- Synonym: Fuscoboletinusserotinus (Frost) Smith curredin the outgroups,Rhizopogon, Truncocolumella and Thiers. Boletesof Michigan, p. 85. 1971. and the Gomphidiaceae,but are not discussedin this paper. Finally,the mostprimitive clade withinSuillus sensu lato seems to be formed by organismswith a Suillus weaverae (Smith and Schaffer) Kretzer & very pronounced boletinoid hymenophore,that is Bruns,comb. nov. with radiallystrongly elongated pores. It seems that Basionym:Fuscoboletinus weaverae Smith and Schaf- the boletinoid hymenophorerepresents an interme- fer,Mich. Bot. 4: 27. 1965. diate evolutionarystate between the gills found in relatedgenera such as Gomphidiusand Chroogomphus ACKNOWLEDGMENTS and the pores found in Suillus. This is basicallyan old Smith and Thiers idea; (1964) stated: "We find The authorswant to thankDr. Van Cotter, Dr. Anders Dahl- a towarda tendency lamellate typeof hymenophore berg,Dr. HelmutBesl, and Dr. DanielMousain for provid- in Fuscoboletinuspaluster, and it is only a step from ing mycelium cultures, fruitbodycollections, or fungal this group to the Gomphidiaceae,whose species are DNA, Dr. Dennis Desjardin for proofreadingthe manu- trulylamellate." The lines and directionsof evolu- script,and the DFG (Deutsche Forschungsgemeinschaft) tion, however,have startedto become clear only re- forfinancial support.

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