Regional Specialization of Sarcodes sanguinea () on a Single Fungal Symbiont from the ellenae (Rhizopogonaceae) Species Complex Author(s): Annette M. Kretzer, Martin I. Bidartondo, Lisa C. Grubisha, Joseph W. Spatafora, Timothy M. Szaro and Thomas D. Bruns Source: American Journal of Botany, Vol. 87, No. 12 (Dec., 2000), pp. 1778-1782 Published by: Botanical Society of America, Inc. Stable URL: http://www.jstor.org/stable/2656828 Accessed: 12-12-2015 01:10 UTC

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This content downloaded from 160.36.178.25 on Sat, 12 Dec 2015 01:10:22 UTC All use subject to JSTOR Terms and Conditions AmericanJournal of Botany87(12): 1778-1782. 2000.

REGIONAL SPECIALIZATION OF SARCODES SANGUINEA (ERICACEAE) ON A SINGLE FUNGAL SYMBIONT FROM THE RHIZOPOGON ELLENAE (RHIZOPOGONACEAE) SPECIES COMPLEX'

ANNETTE M. KRETZER,2'5'6 MARTIN I. BIDARTONDO,3'5 LISA C. GRUBISHA,4 JOSEPH W. SPATAFORA,2 TIMOTHY M. SZARO,4 AND THOMAS D. BRUNS4

2OregonState University, Department of Botanyand PlantPathology, 2082 CordleyHall, Corvallis,Oregon 97331-2902 USA; 3Universityof Californiaat Berkeley,Department of EnvironmentalScience, Policy and Management,111 KoshlandHall, Berkeley, 94720-3102 USA; and 4Universityof Californiaat Berkeley,Department of Plantand MicrobialBiology, 111 KoshlandHall, Berkeley,California 94720-3102 USA

We have sampledthe mycorrhizalroots of 76 snow (Sarcodes san?guinea,, Ericaceae) in two areas of the SierraNevada of Californiathat are - 180 km apart.To identifythe fungal symbionts associated with these plants, we firstanalyzed restrictionfragment length polymorphisms (RFLPs) of the internaltranscribed spacer region (ITS) of the fungalnuclear ribosomal repeat.Fungal ITS-RFLPs were successfullyproduced from 57 of the 76 plantssampled, and all symbiontsshared the same DNA fragmentpattern. The morphologyof S. sanguineamycorrhizae was consistentwith that expected from a Rhizopogonspecies in section Amnylopogon.To confirmand refinethis identification, a total of six fungalITS sequenceswere determined from S. sanguineamy- corrhizae.These sequenceswere analyzedtogether with eight existing and eightnewly determined ITS sequencesfrom Rhizopogon sectionAnmylopogon. The newlydetermined sequences include an ITS sequencefrom the fungal symbiont of pine drops( andromedea,Monotropoideae, Ericaceae), a plantthat was previouslyreported to be exclusivelyassociated with the Rhizopogon subcaerulescensgroup. When these sequences were analyzed together, the Sarcodes symbionts grouped tightly with several collections of R. ellenae includingthe holotype,one collectionof R. idahoensis,and one collectionof R. semireticulatus.A differentlineage comprised collections of R. subgelatinosus, R. subcaerulescens, anothercollection of R. semireticulatus, and the Pterospora symbiont. We concludethat S. sanguineaassociates exclusively with a singlespecies in theR. ellenae speciescomplex throughout our sampling range.These resultsindicate a muchhigher level of specificityin S. sanguineathan was previouslyreported and confirmthe emerging patternthat nonphotosynthetic, monotropoid plants generally associate very specifically with a narrowrange of ectomycorrhizalfungi.

Key words: monotropoidmycorrhizae; Pterospora andromedea; Rhizopogon; Sarcodes san?guinea; specificity(host).

The snow ,Sarcodes sanguinea,is the onlymember thatemerged from these studies was thatin contrastto pho- of a monospecificgenus in the Monotropoideae(Ericaceae) tosyntheticmycorrhizal plants, which typically associate with and occursthroughout the of Californiaas well largenumbers of distantlyrelated fungi, most monotropes ap- as in southernOregon, western Nevada, and northernMexico pear to associatewith specific groups of closelyrelated ecto- (Wallace,1975). Like othermembers of theMonotropoideae, mycorrhizalfungi: uniflora was foundassociated it is nonphotosyntheticand mustreceive all of itscarbon from withtaxa in the Russulaceae,while Pterospora andromedea fungalroot symbionts. The rootsof S. sanguineaare coralloid, was foundonly in symbiosiswith the Rhizopogon subcaeru- fleshy,and surroundedby a fungalmantle except for the tips, lescensspecies group.Recently, Lefevre, Carter, and Molina which are uncolonized.Although the importanceof fungal (1998) foundAllotropa virgata to be specificallyassociated root symbiontsfor the nutritionof monotropoidplants has with Tricholomamagnivelare. This patternof specificityis been recognizedfor a long time (e.g., Kamienski,1881; also observedin nonphotosyntheticorchids (Taylor and Bruns, Francke,1934), the fungi involved are just startingto be iden- 1997), and so it appearsthat specific fungal associations are tifiedthrough the use ofmolecular techniques. Cullings, Szaro, thenorm in nonphotosyntheticmycorrhizal plants. and Bruns(1996) sampledseveral monotropoid taxa mostly in Sarcodessanguinea so farappeared to be theonly exception the westernUnited States and used partialsequence analysis to thispattern. Cullings, Szaro, and Bruns(1996) reportedthat as well as oligonucleotideprobing of themitochondrial large it associatedwith at least threedistantly related lineages of 12 indi- subunitrDNA to identifythe fungalsymbionts. The picture fungiwithin the Hymenomycetes. Furthermore, only vidual plantswere sampled;thus the diversityof associates could have been underestimated.None of thesymbionts of S. Manuscriptreceived 24 August1999; revision accepted 17 February2000. sanguineawas identifiedto species level, and only one fell The authorsthank Jim Trappe and his groupfor help in collectingand identifyinghypogeous fungi in the SierraNational Forest, and the Harry withina lineageknown to be ectomycorrhizalat the timeof ThiersHerbarium at San FranciscoState University for collection loans. The publication.For thesereasons we decidedto investigatethe studywas supportedin partby USDA ForestBiology competitive grant num- mycorrhizalassociates of S. sanguineamore closely. ber 9600479 and in partby cooperativeagreement number PSW-94-0023CA MATERIALS AND METHODS withthe USDA ForestService. 5 These authorshave contributedequally to thisstudy. Sampling-Rootballsof S. sanguineawere sampled by digginga hole next 6 Authorfor reprint requests. to a floweringplant and by carefullyfollowing the to locatethe

1778

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TABLE 1. Sarcodes sanguinea rootballssampled.

No. pop- No. No. symbionts Location Year ulations plants identified Shaver Lake Area "high-density"sited 1995 1 15 15a LakeTahoe 1997 11 9b Basin along McKinley grove roade 1996 3 13 9a along roads no. 168 and 801 1996 6 15 13a near the USFS Dinkey Creek 1997 1 1 lb Shaver Lake work station indicatecountieswhereSarcdesang Area Lake Tahoe Basin 1997 6 21 bOa (+lc) Total 17 76 58 (76%) a Identifiedby RFLP analysis afterdirect amplification of the fungal ITS region fromS. sanguinea roots. b Identifiedby RFLP analysis of fungalcultures derived from S. san- guinea roots. c One symbiontfrom the Lake Tahoe populationswas identifieddi- % rectlyby sequencing of the fungalITS region withoutRFLP analysis. SAW~~~~~~~~~~~~~...''...... K d This site is located at 119007'50" W and 37009'06" N. The spatial distributionof Abies magnificaectomycorrhizae around S. sanguinea rootballs has also been studied at this site and is being reportedin an accompanyingpaper. e Between Dinkey Creek and McKinley Grove. fBetween Shaver Lake and HuntingtonLake, and betweenHunting- ton Lake and Mono Hot Springs. rootball;small pieces of rootswere removed, rinsed in watel;and eitherused forculturing or lyophilizedfor molecular analysis. In the springof 1995, 1996, and 1997, we sampleda totalof 64 S. san- GibcoBRL, GrandIsland, New York,and 2% NuSieve agarosefrom FMC guinearootballs for direct identification ofthe fungal symbiont based on poly- BioProducts,Rockland, Maine, USA) and visualizedwith ethidium bromide. merasechain reaction (PCR) withfungus-specific primers. The plantsorigi- PCR were for a PCR natedfrom 16 populationsin two areas of the SierraNevada of Califomia products prepared sequencing using QlAquick puri- ficationkit (QIAGEN, Valencia,California, USA). Nucleotide were (Table 1, Fig. 1). Plantssampled within a populationwere 5-100 m apart, sequences determined the reactiontermination method fluorescencela- and populationswere 5-180 kmapart. Twelve additional plants were sampled by cyclic using beled reactionsand the in thespring of 1997 at the "high-densitysite" and at one othersite (Table dideoxyribonucleotidetriphosphates. Sequencing pro- were instructionsfor the 1) forcultivation of thefungal symbiont from S. sanguinearoots (and sub- cessingof reactionproducts performedfollowing sequentidentification of thecultured mycelium). ABI PRISM" Dye TerminatorCycle SequencingCore Kit (PE AppliedBios- ystems,Foster City, California, USA). Electrophoresisand data collection Cultivationof thefungal symbiont-Multipleroot tips from 12 different were done on an ABI Model 377 DNA Sequencer(Perkin-Elmer Corpora- S. sanguineaplants were washed, surface sterilized in 30% H202 for10 sec, tion).DNA SequencingAnalysis (version 2.1.2) andSequence Navigator (ver- rinsedin steriledistilled watel; and cut undera dissectingmicroscope into sion 1.0.1) wereused forprocessing the raw data. smallpieces. Pieces of rootthat appeared healthy and well colonizedby the symbiontwere placed onto malt extract glucose agar (Stevens, 1981) thathad Phylogeneticanalysis-Phylogenetic analyses were performed in PAUP*. been supplementedwith 50 mgchloramphenicol, 50 mg streptomycin,50 mg ITS sequenceswere aligned manually using the PAUP editorand a colorfont. ampicillin,and 1 mg benomylper litre.Fast growingand conidialisolates Sequencesimmediately adjacent to thepriming sites of thesequencing prim- (primarilyMucor) were removedand discarded.After -4 wk of growth, ers were of low qualityin some of the sequences,and the respectiveareas cultureswere transferred to modifiedHagem's without antibiotics. fromthe 18S, 5.8S and 28S genes were excludedfrom the analysis.Within theareas includedin theactive data set,most single-basepair alignment gaps Molecular methods-GenomicDNA was extractedfrom mycorrhizae, wereparsimony uninformative and weretreated as missingdata. Presence or fruitbodies,or myceliumusing the method of Gardesand Bruns(1993). PCR absence of one parsimonyinformative single-bp alignment gap and several reactionscontained 10 mmol/LTris/HCl pH 8.3, 50 mmol/LKCl, 2.5 mmol/ 2-4 bp alignmentgaps was weightedequal to one substitution(=1 step). L MgCl2,0.1 mg/mLgelatin, 200 [Lmol/Lof each of thefour deoxyribonu- Technically,this was done by replacingone dash per alignmentgap witha cleotidetriphosphates, 0.5 [Lmol/Lof each of twodifferent primers, 25 U/mL Taq polymerase,and empiricalamounts of genomicDNA. Reactioncondi- new characterstate "I." The longestalignment gap observedwas 6 bp long, tionswere: denaturing at 94?C for35 sec, annealingat 53?C for55 sec, and and was coded forby insertingthe character state "I" twice,giving it a total polymerizationat 72?C initiallyfor 45 sec but increasingby 4 sec on every weightof two steps.This recodingmethod attempts to conservesome of the cycle (35 cyclestotal). PCR primersused wereeither ITS5 (general)or ITS- informationprovided by alignmentgaps, withoutweighting the insertionor If (fungus-specific)in combinationwith either ITS4 (general)or ITS-4b (ba- deletionof everysingle nucleotide equal to a substitution.This downweight- sidiomycete-specific).For sequencing,the intemalprimers ITS2 and ITS3 ing of the alignmentgaps seemsjustified, because severalof the largerin- wereused as well. For primersequences see Whiteet al. (1990) and Gardes sertions(deletions) were found to representduplications (losses) of two and and Bruns(1993). fournucleotide motifs likely stemming from single insertion (deletion) events. ITS-RFLPs wereproduced as describedbefore (Gardes and Bruns,1996). The alignmentwith the recoded gaps can be viewed at http://ajbsupp. In brief,the internal transcribed spacer (ITS) of thenuclear ribosomal repeat botany.org/v87/kretzer.txt. was amplifiedby PCR and subsequentlydigested with the restriction enzymes Most parsimonioustrees were retrieved through ten heuristic searches with AluI and HinjI; DpnII and CfoIwere also used occasionally.Restriction frag- randomsequence addition. Bootstrap values are based on 500 replicateswith mentswere then separatedon agarose gels (1% ultrapureagarose from fiverandom sequence additions each.

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Ten of the 12 S. sanguineaplants from which isolations wereattempted yielded cultures of a relativelyslow-growing, (U NJ CN CJ vi CD Cf) L C D nonsporulating,white fungus that developed yellowish-brown U CU (U (U e U U C U (U CU CU( C/) H H~ a) I- H)a} -H Ha)a colorationin thecenter of theplate over time. The othertwo plantsyielded no fungalcultures other than fast-growing spor- 4)000000000 ulatingtypes, which were considered to be contaminants.ITS- RFLPs derivedfrom the slow-growing cultures were identical to those deriveddirectly from S. sanguinearoots (data not shown). Similarityof thefungal ITS-RFLPs fromS. sanguineamy- 400 bp - corrhizaeto thoseobtained previously from Rhizopogon sub- 300 bp - caerulescens(data notshown) as well as morphologicalchar- acteristics(white mantle staining violaceous with age; occa- 200 bp - believe thatthe S. 1000 bp - g| sional presenceof rhizomorphs)led us to sanguinearoot symbiont might be a taxonin Rhizopogonsec- tionAmylopogon. To addressthis hypothesis, we determined 100 bp - the nucleotidesequences of the fungalITS regionfrom four differentS. sanguinearootballs by directsequencing of PCR- productsobtained with the fungus-specific primer pairs ITS lf/ 1000 bp - 4b or ITSlf/4.In addition,we also sequencedthe ITS region fromtwo fungalcultures that had been isolatedfrom two dif- ferentS. sanguineaplants (for details see Materialsand Meth- ods and Table 2). These sequenceswere alignedwith previ- 500 bp - ouslypublished sequences from a wide rangeof Rhizopogon 400 bp - species(Grubisha, 1998) and provedto be mostsimilar to the ITS sequencesof Rhizopogonellenae AHS66137 and T17476 300 bp - (data not shown).Rhizopogon ellenae is a specieswithin the section (Grubisha,1998). To in- 200 bp - monophyletic Amylopogon creasethe resolution within this section, we sequencedthe ITS regionfrom eight additional taxa given in Table 2. Collections with"SNF" collectionnumbers are Amylopogonfruitbodies 100 bp -*_1 ' collectedin 1994 and 1995 in the "SierraNational Forest" of California(southeast of ShaverLake). To determinegenetic Fig. 2. FungalITS-RFLPs producedfrom Sarcodes sanguinea mycorrhi- diversityamong those collections,we firstproduced ITS- zae withthe PCR primersITS-If and ITS4 and therestriction enzymes Alul RFLPs withthe restriction enzymes Hinfl, AluI and CfoI,and and Hinfi.Only a small selectionof samplesare shownhere; they represent subsequentlysequenced one representativeof every RFLP the tensymbionts from the Lake Tahoe area forwhich we successfullypro- duced ITS-RFLPs. type.Collections with "HDT" collectionnumbers were ob- tainedfrom the Harry Thiers Herbarium at San FranciscoState University.Finally, we also sequencedthe ITS regionfrom RESULTS one fungalsymbiont of Pterospora andromedea. The complete dataset has been posted at the followingwebsite: http:// Althoughall S. sanguineaplants were sampledat compa- ajbsupp.botany.org/v87/lkretzer.txt.It comprises 22 taxa from rable stagesof flowerdevelopment (= beforeseed produc- sectionAmylopogon and 539 characters,of which26 were tion),the rootsamples appeared to fall intotwo distinctage parsimonyinformative. classes. About75% of the root samplesappeared fresh and A heuristicsearch with ten random sequence additions re- lush and were coveredwith a dense whitemycelium except sultedin 16 mostparsimonious trees that were 57 stepslong. forthe tips, which in Sarcodessanguinea usually grow beyond One of thetrees is shownin Fig. 3, and internalbranches that the sheathingmantle. About 25% of the rootsamples, how- werepresent in all 16 treesare highlightedin boldface.Four ever,appeared old, and themantle was reducedto a fewpink lineageswere resolved by the currentanalysis within section patchesleaving the rootswith an overallbrown appearance. Amylopogon:(1) lineage 1 comprisescollections of R. semi- Rootsin thelatter category generally failed to yieldPCR am- reticulatus,R. subgelatinosus,R. subcaerulescens,and the plificationproducts, but occasionallyallowed for culturing of Pterosporasymbiont; (2) lineage 2 comprisescollections of thefungal symbiont. R. ellenae (includingthe holotype), R. idahoensis,a different We were able to directlyamplify fungal ITS regionsfrom collectionof R. semireticulatus,and all of theSarcodes sym- 48 outof 64 S. sanguineaplants (Table 1). Amplificationsuc- bionts;this lineage will be referredto as the "R. ellenae spe- cess and failurewere strongly correlated with the presence or cies complex";(3) lineage3 is madeup by twocollections of absenceof a well-developedmantle as describedabove. Since R. subpurpurascens,one of whichis a paratype;and finally theprimer pair ITS-lfITS4 amplifiesa shorterfragment than (4) an unidentifiedcollection forms a fourthlineage. ITS-lfITS-4b, it was oftensuccessful when the latter failed. When the obtainedPCR productswere digestedwith the re- DISCUSSION strictionenzymes Alul or Hinfi,identical banding patterns were obtainedacross all samples,a selectionof which is About25% of theS. sanguinearoots sampled appeared old shownin Fig. 2. and failedto producePCR productsof thefungal ITS region

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TABLE 2. ITS sequences analyzed in this study.

Taxon Collection IDa Collection site Accessionh

Rhizopogon ellenae AHS66137 (holotype) Valley Co., Idaho Grubisha, 1998 R. ellenae T17476 Josephine Co., OR Grubisha, 1998 R. ellenae HDT46517 Sierra Co., CA GBAN-AF224470, GBAN-AF224471 R. idahoensis HDT30140 Sierra Co., CA GBAN-AF224472 R. subcaerulescens F2882 Colorado Baura et al., 1992 R. subgelatinosus T7624 Hood River Co., OR Grubisha, 1998 R. semireticulatus T7899 Josephine Co., OR Grubisha, 1998 R. semnireticulatus T17562 Josephine Co., OR Grubisha, 1998 R. subpurpurascens AHS65669 (paratype) Custer Co., Idaho Grubisha, 1998 R. subpurpurascens T19168 Bonner Co., Idaho Grubisha, 1998 Rhizopogon sp. SNF210B Sierra National Forest, CA GBAN-AF224475, GBAN-AF224476 Rhizopogon sp. SNF32 Sierra National Forest, CA GBAN-AF224480, GBAN-AF224481 Rhizopogon sp. SNF38 Sierra National Forest, CA GBAN-AF224473, GBAN-AF224474 Rhizopogon sp. SNF260 Sierra National Forest, CA GBAN-AF224477 Rhizopogon sp. SNF306 Sierra National Forest, CA GBAN-AF224478, GBAN-AF224479 Sarcodes symbiont Shaver8 Shaver Lake Area, CA GBAN-AF224489 Sarcodes symbiont Shaverl3 Shaver Lake Area, CA GBAN-AF224276 Sarcodes symbiont Tahoe5.2 Lake Tahoe Basin, CA GBAN-AF224486 Sarcodes symbiont Tahoe2.2 Lake Tahoe Basin, CA GBAN-AF224487 Pterospora symbiont Sierra National Forest, CA GBAN-AF224488 Culture (Sarcodes HD1) 355 Shaver Lake Area, CA GBAN-AF224482, GBAN-AF224483 Culture (Sarcodes HD3) 361 Shaver Lake Area, CA GBAN-AF224484, GBAN-AF224485 a "F" collections were made by Dr. R. Fogel, "T" collections by Dr. J. Trappe, "HDT" collections by Dr. H. Thiers, "AHS" collections by Dr. A. Smith, "SNF" collections are located at the last author's laboratory in Berkeley, "HD" collections are from the "high-density site" (see Table 1). b The prefix GBAN- has been added to link the online version of American Journal of Botany to GenBank but is not part of the actual accession number. In the case of single accession numbers, the whole ITS region (consisting of both internal transcribed spacers plus the 5.8S rRNA gene) were submitted as one continuous sequence; in the case of two accession numbers, the sequences of spacers 1 and 2 were submitted individually.

(see Table 1). Luoma (1996, and personalcommunication) has agingroots did exhibitthe pinkishto purplishcolor charac- monitoreda populationof S. sanguineaover 17 yrand reports teristicof agingmycorrhizae formed by sectionAmylopogon. thatmost plants flowered only once duringthis period. How- (2) In a fewcases we wereable to culturethe fungal symbiont ever,in a certainnumber of cases (varyingfrom 0 to 35% fromsuch aging roots, and it exhibitedthe same culturaland betweenyears and averaging12%) an inflorescencewill ap- ITS-RFLP characteristicsas did all the othersymbionts. We pear in the very same locationas in the previousyear. We concludethat all S. sanguineaplants that we sampledwere hypothesizethat a small percentageof plantsflower in two associatedwith the same mycorrhizalfungus that is a species subsequentyears and thatthe rootsof thoseplants have an in theR. ellenae speciescomplex. older appearancein the second year.Because those plants Thesefindings are in conflictwith a previousreport by Cull- failed to yield PCR products,direct molecular evidence is ings,Szaro, and Bruns(1996) who foundS. sanguineaasso- missingto supportthe factthat they were colonizedby the ciatedwith fungi from at leastthree different lineages of Hy- samefungal species as werethe younger roots. Two otherlines menomycetes.There are twopossible reasons for this discrep- of evidence,however, suggest that the same funguswas pre- ancy:(1) Cullings,Szaro, and Bruns(1996) includedsamples sent.(1) Remnantpatches of a fungalmantle present on the froma somewhatbroader geographic range in the SierraNe- vada of Californiaand also sampledat leastsome remote and R. subcaerulescensF2882 unmanagedhabitats (K. Cullings,personal communication), {; Pterospora symbiont SNF260 (1) whileall our sampleswere taken in two areasof thesouthern 93 SNF38 "species group 1 R. subgelatinosusT7624 and centralSierra Nevada withina few hundredmetres of R. semireticulatusT7899 roads.Geographic mosaics are a commonfeature of many par- R. ellenae holotypeAHS66137 R. ellenaeT17476 asites; oftenwhat appears to be a generalistat a broadgeo- R. ellenae HDT46517 Sarcodes symbiontTahoe5.2 graphicscale is composedof severalgeographically defined Sarcodes symbiontTahoe2.2 (2) Sarcodes symbiontShaver8 "R. ellenae host-specificpopulations (Thompson, 1994). (2) Cullings,Sza- 99 culture355 (Sarcodes HD1) species complex" ro, and Bruns (1996) may have overestimateddiversity culture361 (Sarcodes HD3) Sarcodes symbiontShaverl3 throughPCR chimerasor may have inadvertentlyamplified SNF21OB 8 8 SNF32 saprobicfungi associated with older roots. In eithercase, how- R. idahoensisHDT30140 R. semireticulatusT17562 ever,S. sanguineacan no longerbe consideredthe generalist 72 r R. subpurpurascensparatype AHS65669 | (3) R. subpurpurascens thatit earlierappeared to be (Cullings,Szaro, and Bruns, R. subpurpurascensT19168 SNF306 | (4) Rhizopogon sp. 1996). Instead,it appearsrestricted to associationswith a sin- 1 step gle species of Rhizopogonwithin at least two largeareas of its rangeand mostof its habitats.This removesS. sanguinea Fig. 3. One of 16 most parsimonioustrees (57 steps long). Branches drawnin boldfacedlines are supportedby all 16 mostparsimonious trees. as theonly current exception to theemerging pattern that non- Numbersindicate support for individualbranches from bootstrap analysis; photosyntheticmycorrhizal plants exhibit high levels of spec- onlybootstrap values >70 are given.The treeis unrooted. ificity.It also changesthe way we view theevolution of spe-

This content downloaded from 160.36.178.25 on Sat, 12 Dec 2015 01:10:22 UTC All use subject to JSTOR Terms and Conditions 1782 AMERICAN JOURNAL OF BOTANY [Vol. 87 cializationin the Monotropoideae.Initially it appearedthat and S. sanguineaspecialize on two closelyrelated but distinct therewas a gradualshift toward specialization (Cullings, Sza- Rhizopogonspecies fromsection Amylopogon. Since both ro, and Bruns,1996); now thereis no clear evidencefor a monotropoidplant species oftenco-occur within metres of memberof theMonotropoideae that is a truegeneralist. each other,specificity of their fungal associations is apparently ITS sequencescollected and analyzedin this studyhave notgoverned by habitator local availabilitybut rather by di- greatlyrefined our understanding of speciesgroups within sec- rectplant-fungus interaction. tionAmylopogon. We have thereforerevisited the P. andro- medea symbiontthat Cullings, Szaro, and Bruns(1996) iden- LITERATURE CITED tifiedas a species in the "R. subcaerulescensgroup" based CULLINGS,K. W. 1993. Molecularevolution and mycorrhizalecology of the on sequenceanalysis and oligonucleotideprobing of the mi- Monotropoideae.Ph.D. dissertation,University of California,Berkeley, tochondriallarge subunitrDNA. Cullings,Szaro, and Bruns California,USA. (1996) also reporthaving sequenced the ITS regionof six P. , T. M. SZARO,AND T. D. BRUNS. 1996. Evolutionof extremespe- andromedeasymbionts, but only two partialITS sequences cializationwithin a lineageof ectomycorrhizalepiparasites. Nature 379: have been publishedin Cullings(1993). We thereforese- 63-66. quencedthe complete ITS regionof one P. andromedeasym- FRANCKE,H.-L. 1934. Beitragezur Kenntnis der Mykorrhiza von Monotropa hlypopitys L. Analyse und Synthese der Symbiose. Flora (Jena) 129: 1- biontand includedit in thecurrent analysis. Our sequence,as 52. well as Cullings' two partialsequences (data not shown), GARDES,M., AND T. D. BRUNS. 1993. ITS primerswith enhanced specificity groupstightly with species group 1 in thecurrent analysis and forbasidiomycetes application to theidentification of mycorrhizaeand is clearlydistinct from the "R. ellenae species complex"to rusts.Molecular Ecology 2: 113-118. whichthe Sarcodes symbiontbelongs (Fig. 3). However,be- , AND . 1996. Communitystructure of ectomycorrhizalfungi cause Cullings,Szaro, and Bruns (1996) reportRFLP variation in a Piniusmnuricata forest: above- and below-groundviews. Caanadiani Journalof Botany74: 1572-1583. as well as 0.3-2% sequencevariation in theITS regionof P. GRUBISHA,L. 1998. Systematicsof thegenus Rhizopogon inferred from nu- andromedeasymbionts, complete ITS sequences shouldbe clear ribosomalDNA large subunitand internaltranscribed spacer se- generatedfrom the symbionts of moreP. andromedeaplants. quences. Master'sthesis, Oregon State University,Corvallis, Oregon, Althoughour work has contributedto a betterunderstanding USA. of speciesgroups in Rhizopogonsection Amylopogon, taxon- KAMIENSKI,F. 1881. Die Vegetationsorganeder Monotropa hypopitys L. Bo- omy of the groupremains unsettling. Current of tanischeZeitung 29: 458. LEFEVRE, C. K., C. M. CARTER,AND R. MOLINA. 1998. Morphologicaland Rhizopogonis based mostlyon themorphological species con- molecularevidence of specificitybetween Allotropa virgata and Tricho- ceptsof Smithand Zeller (1966), whichoften turn out to be loma magnivelare.Poster presentation at the Second InternationalCon- in conflictwith genetic species concepts.From other studies ferenceon Mycorrhizain Uppsala,Sweden. Program and Abstracts:107. we know for examplethat different paratype collections of LUOMA,D. 1996. Fifteenyears amongst the snowplants. Oral presentation Rhizopogonvinicolor Smith represent two very distinctge- at theFirst International Conference on Mycorrhizaein Berkeley,Cali- netic On the other fornia.Program and Abstracts:79. species (Kretzer,unpublished data). hand, SMITH, A. H., AND S. M. ZELLER. 1966. A preliminaryaccount of theNorth severalcollections representing different species according to Americanspecies of Rhizopogon.Memoirs of theNew YorkBotanical thecurrent morphological species concepts are oftenindistin- Garden14: 1-178. guishableby ITS sequencedata. The latteris also truefor STEVENS, R. B. [ED.] 1981. MycologyGuidebook, 2nd print.University of severalcollections in whatwe call the "Rhizopogonellenae WashingtonPress, Seattle, Washington, USA. speciescomplex." We have chosenthat name, because R. el- TAYLOR, D. L., AND T. D. BRUNS. 1997. Independentspecialized invasion of ectomycorrhizalmutualism by two nonphotosyntheticorchids. Pro- lenae is the only holotypesequence currently known to fall ceedingsof theNational Academy of Sciences,(USA) 94: 4510-4515. withinthis group of taxa thatmay represent one or morere- THOMPSON,J. N. 1994. The coevolutionaryprocess. University of Chicago productivelyisolated species. Press,Chicago, Illinois, USA. In conclusion,this study has shownthat S. sanguineaspe- WALLACE, G. D. 1975. Studiesof theMonotropoideae (Ericaceae): taxono- cializes at leastregionally on a singlefungal symbiont within my and distribution.Wasmnann Journal of Biology33: 1-88. the R. ellenae species complex.Furthermore, based on the WHITE, T. J.,T. D. BRUNS, S. LEE, AND J. W. TAYLOR. 1990. Amplification and directsequencing of fungalribosomal RNA genesfor phylogenetics. analysisof fungalITS sequencesfrom three P. andromedea In M. A. Innis,D. H. Gelfand,J. J. Sninsky,and T. J.White [eds.], PCR plants(two partial sequences are not being shown), the emerg- protocols;a guide to methodsand applications,315-322. Academic ingpicture is thatthe two monotropoid species P. andromedea Press,San Diego, California,USA.

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