Botanical Studies (2010) 51: 53-60. MicrobioLOGY Phylogenetic relationships of Antrodia species and related taxa based on analyses of nuclear large subunit ribosomal DNA sequences Zhi-HeYU1,Sheng-HuaWU2,*,Dong-MeiWANG3,andCheng-TauCHEN2 1College of Life Science, Yangtze University, Jingzhou 434025, Hubei, P.R. China 2Department of Botany, National Museum of Natural Science, Taichung 404, Taiwan 3Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, P.R. China (Received March 24, 2008; Accepted July 10, 2009) ABSTRACT. ThisstudyaimedtoevaluaterelationshipsofAntrodia species and related taxa, including the taxonomic status of some Antrodiaspeciesthathavebeentreatedasseparategenera(Amyloporia,Fibroporia, andTaiwanofungus).AcomprehensivephylogeneticstudyofHomobasidiomycetespresentedbyBinderetal. in 2005, was consulted for sampling the taxa used for this analysis. The genera of the “residual” polyporoid cladeandphlebioidcladeoftheHomobasidiomyceteswerechosenasoutgroups,andthegenerabelonging totheAntrodiacladeandcorepolyporoidcladewereselectedasingroup.Phylogeneticanalysesofthisstudy werebasedonsequencedataderivedfromthenuclearlargesubunitribosomalDNA(nuc-LSUrDNA).The analytical methods of maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) wereused.Resultsfromthesedifferentanalysesweregenerallyconsistent.Twomaincladeslackinghigh support were detected in the ingroup. Clade A consisted of taxa of the Antrodiaclade,includingalltwelve studiedAntrodiaspeciesandmembersoffourothergenera:Daedalea,Fomitopsis,Neolentiporus,and Piptoporus.ThetwelveAntrodiaspecieswerenotclusteredintoadistinctsubclade,indicatingthat Antrodia isnotamonophyleticgroup.TwospeciesofFibroporia (belongingtoAntrodia sensu lato),characterized byhavingafruitingbodywitharhizomorphicmargin,clusteredtogetherwithverystrongsupport.Five specieswithamyloidskeletalhyphae,diagnosticofAmyloporia,didnotgrouptogether.Thegenericstatus ofFibroporia,butnotAmyloporia,wassupportedinthisstudy.CladeBconsistedofthegeneraofthecore polyporoidclade.BothspeciesoftherecentlyestablishedgenusTaiwanofungusformedadistinctsubclade, supportingitsgenericstatus. Keywords:Amyloporia; Antrodia; Fibroporia; Fomitopsis; Phylogeny; Polypore; Taiwanofungus. INTRODUCTION A number of genera have been segregated from Antrodiasensu lato.ThegeneraAmyloporiaSingerand AntrodiaP.Karst.isapolyporegenuswithmore FibroporiaParmastoareregardedascongenericwith than40speciescausingbrownrotofwood.Thegeneric Antrodia bysomeauthors(GilbertsonandRyvarden,1986, conceptofAntrodiawasamendedbyGilbertsonand Ryvarden,1991), butthishypothesishasnotbeentested Ryvarden(1986)andissummarizedasfollows:resupinate withmolecularanalyses.Eachgenusonlyaccommodates to effused-reflexed or effused-pileate basidiocarps; dimitic afewspecies.Amyloidskeletalhyphaearediagnosticfor hyphalsystemwithnodose-septatecolorlessgenerative Amyloporia,andthefruitingbodyusuallytastesbitter. hyphae,bearingmostlycolorlessskeletalhyphaethatare FruitingbodiesofFibroporia havearhizomorphicmargin. inamyloidformostspeciesandsomewhatamyloidfora Ryvarden(1991)suggestedthatthesecharactersmightnot few species; without true cystidia; and with smooth, thin- be sufficient for supporting these two genera as separate walledandinamyloidbasidiospores.Althoughearlier fromAntrodiaifmoreconvincingevidencecannotbe studiesindicatedthatthegenusAntrodiasensu lato isnot found. TwootherspeciesonceplacedinAntrodia(Wuet monophyletic,somequestionsregardingtherelationship al., 1997; Chang and Chou, 2004) were recently referred remainunanswered. toanewgenusTaiwanofungus ShengH.Wuetal.on thebasisofmorphological,ecological,andphylogenetic *Correspondingauthor:E-mail:[email protected]. analysesderivedfrom nuc-LSU rDNA (Wu et al., 2004). 54 Botanical Studies, Vol. 51, 2010 Wu et al. (2004) included four species of Antrodia,and together; the former two as sister clades to the latter two. othermoreorlessrelatedgeneraofthepolyporoidclade Antrodiella semisupina(Berk.&M.A.Curtis)Ryvarden intheirmolecularanalysis.Theresultsindicatedthat andBjerkandera adusta(Willd.)P.Karst.belonging Taiwanofunguswasdistanttothetwostudiedspeciesof to the “residual” polyporoid clade and phlebioid clade, Antrodia,whichincludedthegenerictype,andtoother respectively, were used as outgroup taxa. The ingroup genera.Thephylogenetic relationshipsofAntrodiaand consistedofgenerabelongingtotheAntrodiacladeand otherstudiedgeneraweredifficulttointerpretfromthe core polyporoid clade. Details of the studied taxa are resultsobtainedduetoasmallsampling. presentedinTable1. Several phylogenetic analyses of Antrodia were previouslypresentedbyotherresearchers.Ananalysisof DNA extraction, PCR amplification, DNA sevenAntrodiaspeciesandrelatedgeneraconductedby cloning, and sequencing Kim et al. (2001) was based on sequences inferred from Myceliaweretransferredfromagarculturesto100ml the internal transcribed spacer (ITS) region of nuclear liquid medium (2% malt extract) and incubated on a ro- ribosomal DNA. Kim et al. (2003) attempted to assess tary shaker (160 rpm) for 2~3 weeks at room temperature. phylogenetic relationships of six Antrodiaspeciesand DNAwasisolatedfromfreeze-driedorfreshlycul- related taxa based on sequences of the mitochondrial tured mycelia using the Plant Genomic DNA Extraction smallsubunitribosomalDNA(mt-SSU).However,the MiniprepSystem(Viogene,Taiwan)accordingtothe generaincludedintheiranalysiswerehighlydiverse manufacturer’s instructions. The primer pair, LR0R/LR5 phylogenetically,andweredistributedamongalmostall (Moncalvo et al., 2000), was used to amplify the nuc-LSU cladesofHomobasidiomycetes.Nevertheless,bothstudies rDNAregion.PCRconditionsweresetaccordingtothe mentionedaboveshowedthatAntrodiaspeciesdonot manufacturer’s instructions (Viogene). The amplification formamonophyleticclade. products were purified with a PCR-M Clean Up kit (Vio- Kim et al. (2005) evaluated the monophyly of gene),andbothstrandsequenceswereproducedusing Fomitopsis,basedonsequencedataderivedfromnuc- the ABI PRISM BigDye Terminator Cycle Sequencing LSUrDNA.Theirresultsshowedthatthefourstudied Ready Reaction kit on an ABI 3730 DNA sequencer (Ap- Antrodiaspecieswereclusteredtogetherwithtenstudied pliedBiosystems,FosterCity,Calif.).Theprimers,LR0R Fomitopsis species, and both of these genera were and LR5, were used for direct sequencing of the amplified respectively shown to be non-monophyletic. Chiu (2007) fragments.Forthestrainswithintragenomicheterogene- conductedphylogeneticanalysisofnineAntrodiaspp. ity,DNAcloningwasperformedusingayT&Acloning TM andelevenstrainsofA.camphorata,basedonsequences vectorandcompetentECOS 9-5 cells (Yeastern Bio- inferred from ITS nrDNA. In Chiu’s analysis, the ingroup tech,Taiwan).Asinglepositivecolonywaspickedforthe consistedofonlyAntrodiaspp.,andhencehisstudy followingPCRamplificationandDNAsequencing.The chieflyrevealedphylogeneticrelationshipsamongthe consensusdatafromtheforwardandreversesequences studiedAntrodiaspp.andstrains. wereassembledusingSeqWebfromtheGCGWisconsin Theaimsofthisstudyweretofurtherevaluatethe Package (available at http://bioinfo.nhri.org.tw). genericstatusofAntrodia sensu stricto, and of some taxa Sequence alignment and phylogenetic analyses thathavebeentreatedasseparategenera(Amyloporia, Fibroporia,andTaiwanofungus)bysomemycologists, Fifty-six taxa were used, including 17 sequences aswellastheirphylogeneticrelationshipswithrelated newly derived for this study(Table1).Forthetwo polyporegenera.Thephylogeneticanalyseswerebased Taiwanofungus salmoneusstrainswithintragenomic onsequencedataderivedfromnuc-LSU,aregionwidely heterogeneity,onlytherepresentativeclonesequences, adoptedinanalyzingphylogeneticrelationshipsofthe EF036246 and EF036249, were chosen for analysis. Homobasidiomycetesatandabovethegenericlevel. SequenceswerealignedusingClustalX1.83(Thompson etal.,1997)andwereadjustedmanuallyusingBioEdit MATERIALS AND METHODS 7.0.4.1 (Hall, 1999). The optimized data matrix was deposited in TreeBase (Study accession number = S2416, Taxon sampling Matrix accession number = M4581). Three analytical methods were used: maximum parsimony (MP), maximum TwelveAntrodiaspecies,includingthegenerictype, likelihood (ML), and Bayesian inference (BI). Ant.albida (Fr.), and the members of 22 other genera withmoreorlesscloserelationshipstoAntrodia,were TheMPanalysiswasperformedinPAUP*4.0b10 chosenforthisanalysis.Allbelongtothepolyporoid (Swofford, 2002), using heuristic searches with 1000 cladecomposedofacorepolyporoidclade,anAntrodia random taxon stepwise addition sequences, TBR branch clade, a phlebioid clade, and a “residual” polyporoid swapping,andtheMAXTREESsettoautoincrease.All cladewithinHomobasidiomycetesaccordingtothe transformationswereconsideredunorderedandequally comprehensive study of Binder et al. (2005). In that weighted,withgapstreatedasmissingdata.Bootstrap study,thecorepolyporoidcladeandAntrodiaclade,and analysis(HillisandBull,1993)wasperformedwith1000 phlebioid clade and “residual” polyporoid clade clustered replicateswithrandomadditionsequencesforobtaining YU et al. — Phylogenetic relationships of Antrodia 55 Table 1. Taxa used in this study, along with their strain/specimen numbers and GenBank accession numbers. Species Strain/Specimen no. GenBankaccessionno. Amylocystis lapponica HHB-13400-sp. AF518598 Antrodia albida FCUG1396 AY333845 Antrodia albida FCUG1100 AY333846 Amyloporia (Antrodia) carbonica DAOM197828 AF287844 Antrodia heteromorpha FCUG 1244 AY333840 Antrodia juniperina