matK trnLF andITSrespectively Misodendrumangulatum Phil BarilocheRNArgentina G Amico131 BCRUDLN4587DQ787437DQ788707 DQ788697 Misodendrumbrachystachyum DC Bariloche RNArgentina GAmi ID: 268483
Download Pdf The PPT/PDF document "L.NickrentatSIUC)andGenBankaccessionnumb..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
L.NickrentatSIUC)andGenBankaccessionnumbersfor matK , trnL-F ,andITS,respectively. Misodendrumangulatum Phil. Bariloche,RN,Argentina G. Amico131 (BCRU;DLN4587),DQ787437,DQ788707, DQ788697. Misodendrumbrachystachyum DC Bariloche, RN,Argentina G.Amico132 (BCRU;DLN4588),DQ787440, DQ788708,DQ788698. Misodendrumgayanum Tiegh. Villa LaAngosturaNQN,Argentina G.Amico134 (BCRU; DLN4590),DQ787439,DQ788709,DQ788699. Misodendrum linearifolium DC Chillan,Chile G.Amico136 (BCRU; DLN4591),DQ787438,DQ788712,DQ788700. Misodendrum oblongifolium DC Bariloche,RN,Argentina G.Amico137 (BCRU;DLN4592),DQ787442,DQ788710,DQ788701. Mis- odendrumpunctulatum BanksexDC Bariloche,RN,Argen- tina G.Amico139 (BCRU;DLN4593),DQ787443,DQ788711, DQ788702. Misodendrumquadriflorum DC Bariloche,RN, Argentina N.Tercero&G.Amico140 (BCRU;DLN4594), DQ787441,DQ788713,DQ788703. Atkinsonialigustrina (Lindl.)F.Muell. BlueMountains,NSW,Australia Dave Watson (DLN4343),DQ787444,DQ788714. Gaiadendron punctatum G.Don. Monteverde,CostaRica SaraSargent (DLN2729),DQ787445,DQ788715,DQ788704. Nuytsiaflori- bunda (Labill.)R.Br. WA,Australia AdrienneMarkey (DLN2747),DQ787446,DQ788716,DQ788705. Schoepfia schreberi J.F.Gmel . Paloverde,CostaRica. R.Vidal-Russell 21(INB;DLN4915),DQ787447,DQ788717,DQ788706. Schoepfiafragrans Wall. S.Yunnan,MenglaXian,China Tsizhanhuo91417 (MO4252063;DLN5009),DQ788718. Schoepfiavacciniflora Planch.exTriana&Planch. .Chiriqui, Panama G.McPhersonandP.M.Richardson15981 (DLN3069), DQ787448. Schoepfiajasminodora Sieb.&Zucc. previously publishedsequenceAF534681. A PPENDIX 2.Listofcharactersandcharacterstatesusedin themorphologicalanalysis. GeneralCharacters .1.Barkcolor: 0 yellow,1 gray. 2. Stemtexture: 0 warty,1 smooth. 3.Floralbract: 0 scale,1 leafy. 4.Leafshape: 0 linear,1 scalelike,2 lanceolate. 5.Maleflowers: 0 sessile,1 pedicellate. 6. Femaleflowers: 0 sessile,1 pedicellate. 7.Tipof staminode: 0 pubescent,1 glabrous. 8.Swollen haustoriumbase: 0 absent,1 present. 9.Enlargement ofhostbranch: 0 absent,1 present. 10.Positionoffloral buds: 0 aboveleafbuds,1 belowleafbuds. 11.Lignified pith: 0 absent,1 present. 12.Leaves: 0 sessile,1 petiolate. 13.Stemsection: 0 circular,1 quadrangular. 14.Pubescenceoffloralpedicel: 0 absent,1 present. 15. Numberofmaleflowerperbract: 0 one,1 two,2 six. 16.Numberofstamens: 0 two,1 three,2 four,3 six. 17.Inflorescenceshape: 0 compact,1 lax. 18.Positionof bracteoles: 0 atbaseofflowers,1 onthefloralaxis. 19. Numberoffemaleflowersperbract: 0 one,1 two,2 six. 20.Positionoffemaleflowersontheinflorescence: 0 alternate,1 opposite. 21.Staminodebristles: 0 straight, 1 incurvedorclaviform. 22.Stemincrustations: 0 absent, 1 present. 23.Fascicularareas: 0 diffuse,1 onecircle,2 twocircles. 24.Fibersinfascicularareas: 0 absent,1 present. 25.Sclereidsinoutercortex: 0 absent,1 present. 26.Rays: 0 absent,1 present. 27.Vesselpits: 0 laterallyextended,inhelicalbands,1 elliptical,scalar- iformlike,2 circular,alternate. 28.Axialparenchyma: 0 abundant,nonlignified,1 sparse,lignified. 29.Flowers: 0 bisexual,1 unisexual. 30.Fruit: 0 achene,1 samara,2 drupe. 31.Parasitism :0 root,1 aerial. A PPENDIX 3.Datamatrixusedforthemorphologicalanalysisforeachtaxon,charact ersaredescribedinAppendix2.Parenthesisindicateapolymorphicchara cterstate;?indicates unknowncharacterstate. Character12345678910111213141516171819202122232425262728293031 M.angulatum (0,1)010010000101010102000110011101 M.brachystachyum 1112110100010121113011211100101 M.gayanum 0000100000100000001000100120101 M.linearifolium 1110000000000101100100111100101 M.macrolepis 0010?1?0001000001020?0??????101 M.oblongifolium 1112110100010121113011211100101 M.punctulatum 0001101011100000001000110011101 M.quadriflorum 1112101101010111112111000100101 Schoepfiaschreberi 110211n/a0000100n/a210n/an/an/a0110100020 Nuytsiafloribunda 111200n/a0000000n/a311n/an/an/a03101?0010 (0,1) 568 SYSTEMATICBOTANY [Volume32 penetratesintothehostphloemandthenxylemin amanneranalagoustothecorticalstrandsand sinkersofViscaceae(Johnson1889;Tercero-Bu- cardoandKitzberger2004).Inspeciessuchas M. punctulatum ,theparasitecanliveendophytically foruptotwoyearspriortotheemergenceofthe firstshootfromthehostbranch(Tercero-Bucardo andKitzberger2004). Misodendrum wasfirstdescribedbyCandolle (1830)whoplacedthegenuswithinthelarge mistletoefamilyLoranthaceae.In1858,J.G. Agardhnamedthenewfamily,Misodendraceae, placingitbetweenLoranthaceaeandSantalaceae. Possiblyunawareofthiswork,Benthamand Hooker(1880)movedthegenusfromLoranthaceae toSantalaceaebasedonthesimilaritiesofthe carpel,placentationandovules.In1889,Hierony- musclassifiedMisodendraceaeasaseparatefam- ily(betweenLoranthaceaeandSantalaceae)not knowingthatAgardhhadnamedit30years earlier. Thefirstinfragenericclassificationwasthe monographbySkottsberg(1913).Orfila(1978) addedtwonewspeciestoSkottsbergsclassifica- tion,resultingin12speciesintwosubgenera ( Misodendrum and Gymnophyton ).Someyearslater, Rossow(1982)publishedarevisionofthefamily whereheproposedseveralsynonymsthatreduced thenumberofspeciestoeight.Hisclassification, usedinthisstudy,recognizessubgenus Misoden- drum ,whichischaracterizedbywartystemsand twostamens.Thissubgenuscontainssections Misodendrum ( M.punctulatum BanksexDCand M.gayanum Tiegh.)and Heterophyllum Skottsb.( M. angulatum Phil.and M.macrolepis Phil.).Subgenus Angelopogon (Tiegh.)Rossow,characterizedby threestamensandfoliaciousbracts,containsthree sections: Angelopogon (Tiegh.)Skottsb.( M.linear- ifolium DC), Archiphyllum (Tiegh.)Skottsb.( M. brachystachyum DCand M.oblongifolium DC),and Telophyllum (Tiegh.)Skotsb.( M.quadriflorum DC). Zavaroetal.(1997)publishedacladisticanalysis basedon18externalmorphologicalandanatom- icalcharactersthatsupportedRossowsclassifica- tion. Todate,therehasbeenlittlemolecularphylo- geneticworkonMisodendraceae.Placeholdersfor thefamilyhavebeenincludedinbroad-scale phylogeneticstudiesaimedatplacingallfamilies withinSantalales(NickrentandDuff1996;Nickr- entetal.1998;NickrentandMale ´ cot2001).That workindicatedthatMisodendraceaeissisterto Schoepfia (Schoepfiaceae)andthiscladesisterto Loranthaceae.Both Schoepfia andbasalLorantha- ceae(e.g. Nuytsia )arerootparasites,thusaerial parasitismevolvedtwiceindependently,oncein theancestorof Misodendrum andagaininmore derivedLoranthaceae.Usingacalibratedtreefor theorderSantalales(Male ´ cot2002),aerialparasit- ismmusthavearisenfirstin Misodendrum ca. 75mybp.Thisdateisnotincompatiblewiththe fossilhistoryof Nothofagus whichhadalready differentiatedintofoursubgenera(Knappetal. 2005).ThecalibratedSantalalestreealsoindicates thataerialparasitisminLoranthaceaeevolved later,ca.40mybp. Thisstudyhadtwoobjectives:1)totestthe existingtraditionalclassificationbyreconstructing thephylogenyofthespeciesof Misodendrum using chloroplastmarkersandmorphologyand2)to examinetheevolutionofmorphologicalcharacters inthismistletoegenususingthephylogeny inferredfromthemoleculardata. M ATERIALSAND M ETHODS Sampling. Theingroupformolecularanalysesconsisted ofallspeciesofMisodendraceaeexcepttherare Misodendrum macrolepis .ThisspeciesisrestrictedtoasmallareainChile andonlyafewcollectionsoffemaleindividualsexist.All otherspecieswerecollectednearBariloche,Argentina(the northernpartofthedistributionofthegenus).Theoutgroup comprisestwospeciesof Schoepfia (Schoepfiaceae)andthe threerootparasiticandrelictualspeciesofLoranthaceae, Nuytsiafloribunda fromwesternAustralia, Atkinsonialigus- trina fromeasternAustraliaandtheneotropical Gaiadendron punctatum .Accessionandvoucherinformation,aswellas GenBanknumbersforalltaxaused,aregiveninAppendix1. DNAExtractionandSequencing. DNAwasobtained usingastandardCTABmethodfromsilicadriedtissue (Nickrent1994).ThenuclearribosomalDNAinternal transcribedspacer(ITS-1andITS-2)and5.8SrDNAwere amplifiedandsequenced.Excessivelengthvariationinthe ITS-1regionprecludedunambiguousalignment.Analysisof theremainingregionsyieldednoresolution,thusthisdata partitionwasnotusedinthisstudy(sequenceswere depositedwithGenBank).Primersandmethodologyused areavailablefromRVRuponrequest.Thechloroplastspacer trnL - F ,includingtheintronbetweenthe trnL exons,was amplifiedandsequencedusingtheprimersdescribedin Taberletetal.(1991).Thechloroplastgene matK was amplifiedandsequencedwithprimer78f(5 -CAGGAG TATATTTATGCACT)and1420r(5 -TCGAAGTATATA CTTTATTCG). Polymerasechainreactions(PCR)wereperformedinatotal volumeof25 lusing1 3 PCRbuffer(50mMKCl,10mMtris HCl,pH8.3),1.5mMMgCl 2 ,50 MofdNTPs,0.4 Mof eachprimer,ca.1unit Taq DNApolymeraseand1 lof genomicDNAdiluted1:9.Amplificationswerecarriedoutin aGeneAmpsystem9600thermocycler(AppliedBiosystems). For trnL-F reactions,atouchdownprofilewasused:5minat 95 u C,5cyclesof94 u Cfor30sec,52 u Cfor30sec,and72 u Cfor 1min,followedby33cyclesof94 u Cfor30sec,48 u Cfor 30sec,and72 u Cfor1min,withafinalextensionof72 u Cfor 10min.Forthe matK reactions,astepupPCRprofilewas used:5minat95 u C,5cyclesof94 u Cfor1min,46 u Cfor 1min,and72 u Cfor1min,followedby35cyclesof94 u Cfor 30sec,50 u Cfor30sec,and72 u Cfor1min,withafinal extensionof72 u Cfor10min. PCRproductswerepurifiedusingeithertheQIAquickgel extractionkit(QiagenInc.,Valencia,California)ortheEZNA 2007] VIDAL-RUSSELL&NICKRENT:MISODENDRUMPHYLOGENY 561 AMolecularPhylogenyoftheFeatheryMistletoe Misodendrum R OMINA V IDAL -R USSELL 1 andD ANIEL L.N ICKRENT DepartmentofPlantBiology,SouthernIllinoisUniversityCarbondale,C arbondale, Illinois62901-6509,U.S.A. 1 Authorforcorrespondence(romina@siu.edu) CommunicatingEditor:SaraB.Hoot A BSTRACT . Misodendrum compriseseightspeciesofaerialhemiparasitesendemictotemperatefor estsofChile andArgentinathatparasitize Nothofagus .Thismistletoeisuniqueinthatithasfeatherystaminodesonitswind dispersedachenes.Previousclassificationsincludedtwosubgenera, Misodendrum (twosections)and Angelopogon (threesections).Thepresentstudytestedthisclassificationusingtwo chloroplastgenes( trnL-F and matK )and31 morphologicalcharacters.Maximumparsimony,likelihoodandBayesiana nalyseswereperformedforindividual andcombinedpartitions.Resultsfromanalysesoftheseparatepartition sdifferedonlyinthepositionsof M. linearifolium and M.quadriflorum ;however,the2-genetreegavehighersupportfor M.quadriflorum assistertoall otherspecies. Misodendrumbrachystachyum and M.oblongifolium formawellsupportedcladethatissistertoone composedof M.punctulatum,M.gayanum, and M.angulatum .Thesephylogeneticrelationshipsgenerallyagreewith previoustaxonomicclassifications.Subgenus Misodendrum ,characterizedbywartystemsandtwostamens,here resolvesasapolytomy: M.punctulatum,M.gayanum ,and M.angulatum .Subgenus Angelopogon ,characterizedbythe plesiomorphiesthreestamensandfoliaciousbracts,isparaphyleticgiv enourrooting. Misodendrumbrachystachyum and M.oblongifolium (section Archiphyllum )differmorphologicallyonlybythelengthoftheirfruitingstaminodes. K EYWORDS : matK ,parasiticplant,Santalales,SouthAmerica, trnL-F . Misodendrum BanksexDC,thesolegenusof Misodendraceae,compriseseightspeciesofaerial hemiparasiticshrubsendemictotemperateforests from36 u 30 SincentralChileto55 u SonTierradel FuegoIsland,Argentina.Thesemistletoesarehost specific,naturallyparasitizingvariousspeciesof Nothofagus (rarelyparasiticonotherhosts;Skotts- berg1914).Althoughtherangeof Nothofagus extendstootherGondwananlandmassessuchas AustraliaandNewZealand, Misodendrum isre- strictedtotheNewWorld. Misodendrum ischaracterizedbysympodial brancheswithalternateleaves.Plantsaredioe- cious;however,monoeciousindividualsarerarely found,includingsomewithbisexualflowers.The inflorescenceisbasicallyaracemeorspikewith multipleflowers,althoughsometimesreducedto oneortwoflowers.Theinflorescencebractsare similartotheleaves.Flowersareverysmalland dullincolor.Staminateflowerslackaperianthand beartwoorthreestamensthatsurroundacentral nectariferousdisk.Inthecarpellateflowersthe perianthandthebasesofthestaminodesarefused totheovary.Fromthegroovesformedbythe edgesoftheperianthmembers,threestaminodes emergeapproximatelymidwayalongtheovary. Afterfertilization,thesestaminodeswilldevelop intothecharacteristicfeatheryappendagesofthe fruit.Insomespecies,perianthlobesarerecogniz- ableattheapexoftheovary.Thenatureofthese lobeshasnotbeenadequatelyinvestigated,butwe favortheconceptthattheyarepetals(asin Takhtajan1997),wherefusionandreductionhave progressedtosuchadegreethatnoevidenceof acalyx(orcalyculus)exists.Becausetheperianthis fusedtotheovaryfornearlyitsentirelength,we interpretitasepigynous,nothypogynousasstated inOrfila(1978).Becausethestaminodes(orsta- mensinbisexualflowers)arefusedtotheovary onlyatthebase,theycouldbeconsidered epihypogynous.Anectariferousdiskoccursinside thepetallobesandsurroundsashortstyleending inthreestigmas.Theunilocularovaryistricarpel- lateatthebaseandbearsthreeovules,pendulous fromafree-centralplacenta;eachhasanun- differentiatednucellusandintegument.Following ovularabortion,onlyoneseedremainsinthe maturefruit.Thepresenceofnectarydiskson flowersofbothsexes,inconjunctionwithchar- acteristicsofthepollenexine,suggestinsect pollination(Orfila1978),althoughempiricaldata arelacking.IntheLakeVintterareaofArgentina, Orfila(1978)observedmanyadultCantharidae (Coleoptera)onflowering Misodendrum .These beetlesareknowntoeatpetalsandstamensand Orfila(1978)statesthattheycouldbeinvolvedin pollinating Misodendrum . Thefruitisawinddispersedachenewiththree featheryappendagesofvaryinglengthdepending uponthespecies.Theappendagesnotonlykeep thefruitaloftbutalsoaidinattachmenttothehost branch.Upongermination,thegreenhypocotyl withastickyapicalholdfastadherestothehost branch.Theradicledevelopsanhaustoriumthat firstattachestothehostepidermisandthenenters thecortex.Thehaustoriumbranchesoutand SystematicBotany (2007),32(3):pp.560568 # Copyright2007bytheAmericanSocietyofPlantTaxonomists 560 speciesof Misodendrum .Forthegene matK ,the averagedistanceamongspecieswas0.1whereas thevaluebetween M.brachystachym and M. oblongifolium (section Archiphyllum )was0.02.A similardifferencewasseenforthe trnL-F region wherethemeandistanceamong Misodendrum specieswas0.1asopposedto0.03betweenspecies insection Archiphyllum .Moreindividualsofthese twotaxashouldbesampledalongtheirentire geographicrangetofurtherexaminetheirdistinc- tiveness. A CKNOWLEDGEMENTS .TheauthorsthankG.Amicoand N.Terceroforcollectingmaterial;theherbariaSIandMOfor accesstospecimens;andtoS.Sipesforgenerouslyallowing ususeofherautomatedDNAsequencer.Weappreciatethe usefulcommentsprovidedbyS.Hootandoneanonymous reviewerthatimprovedthismanuscript.Financialsupport wasprovidedbytheNationalScienceFoundationtoD.L. Nickrent. L ITERATURE C ITED A GARDH ,J.G.1858. Theoriasystematisplantarum .Lund:C.W. K.Gleerup. B ARLOW ,B.A.1966.ArevisionoftheLoranthaceaeof AustraliaandNewZealand. AustralianJournalofBotany. 14:421499. B ENTHAM ,G.andJ.D.H OOKER .1880.Santalaceae.Pp.217 231in GeneraPlantarum vol.3.London:Reeve. C ANDOLLE ,A.P. DE .1830.Loranthaceae.Pp.277320,670672 in Prodromussystematisnaturalisregnivegetabilis,sive enumeratiocontractaordinum,generum,specierumqueplan- tarumhucusquecognitarum,juxtamethodinaturalisnormas digesta vol.4.Paris,Strasbourg,London:Treuttelet Wu ¨ rtz. C ARLQUIST ,S.1985.WoodandstemanatomyofMisoden- draceae:systematicandecologicalconclusions. Brittonia 37:5875. H ERBERT ,D.A.1919.TheWestAustralianChristmastree Nuytsiafloribunda (TheChristmastree):itsstructureand parasitism. JournaloftheRoyalSocietyofWesternAustralia 5:7288. H UELSENBECK ,J.P.andF.R ONQUIST .2001.MrBayes:Bayesian inferenceofphylogeny. Bioinformatics 17:754755. J OHNSON ,T.1889.Thenursingoftheembryoandsomeother pointsin Myzodendronpunctulatum ,BanksetSol. Annals ofBotany 3:179206. K NAPP ,M.,K.S TOCKLER ,D.H AVELL ,F.D ELSUC ,F.S EBASTIANI , andP.J.L OCKHART .2005.Relaxedmolecularclock providesevidenceforlong-distancedispersalof Notho- fagus (southernbeech). PLoSBiology 3:e14. M ALE ´ COT ,V.2002. Histoire,classificationetphyloge ´ niedes OlacaceaeBrown(Santalales). Ph.D.Dissertation.Paris: LUniversitieParis. M ADDISON ,D.R.andW.P.M ADDISON .2000.MacClade4: Analysisofphylogenyandcharacterevolution,v.4.0. Sunderland:SinauerAssociates. M ETCALFE ,C.R.andL.C HALK .1950. Anatomyofthe dicotyledons:leaves,stem,andwoodinrelationtotaxonomy withnotesoneconomicuses .Oxford:ClaredonPress. N ARAYANA ,R.1958.Morphologicalandembryological studiesinthefamilyLoranthaceae-III. Nuytsiafloribunda (Labill.)R.Br. Phytomorphology 8:306323. N ICKRENT ,D.L.1994.Fromfieldtofilm:rapidsequencing methodsforfieldcollectedplantspecies. BioTechniques 16:470475. andR.J.D UFF .1996.Molecularstudiesofparasitic plantsusingribosomalRNA.Pp.2852in Advancesin ParasiticPlantResearch ,eds.M.T.Moreno,J.I.Cubero, D.Berner,D.Joel,L.J.Musselman,andC.Parker. Co ´ rdoba:JuntadeAndalucia,Direccio ´ nGeneralde Investigacio ´ nAgraria. andV.M ALE ´ COT .2001.Amolecularphylogenyof Santalales.Pp.6974in Proceedingsofthe7th.Interna- tionalParasiticWeedSymposium ,eds.A.Fer,P.Tha- louarn,D.M.Joel,L.J.Musselman,C.Parker,andJ.A.C. Verkleij.Nantes:Faculte ´ desSciences,Universite ´ de Nantes. ,R.J.D UFF ,A.E.C OLWELL ,A.D.W OLFE ,N.D.Y OUNG , K.E.S TEINER ,andC.W. DE P AMPHILIS .1998.Molecular phylogeneticandevolutionarystudiesofparasitic plants.Pp.211241in MolecularSystematicsofPlantsII. DNASequencing ,eds.D.E.Soltis,P.S.Soltis,andJ.J. Doyle.Boston:KluwerAcademicPublishers. N YLANDER ,J.A.A.2004.MrModeltestv.2.0.Computer programdistributedbytheauthor.Uppsala:Evolution- aryBiologyCentre,UppsalaUniversity,Availablefrom: http://www.csit.fsu.edu/ nylander/ O RFILA ,E.N.1978. MisodendraceaedelaArgentinayChile . BuenosAires:Fundacio ´ nEl ´ asyEthelMalamud. P OSADA ,D.andK.A.C RANDALL .1998.Modeltest:testingthe modelofDNAsubstitution. Bioinformatics 14:817818. R AMBAUT ,A.2004.Se-Al,amanualsequencealignment editor,v.2.0a11.Oxford:UniversityofOxford,De- partmentofZoology. R EED ,C.F.1955.Thecomparativemorphologyofthe Olacaceae,Opiliaceae,andOctoknemaceae. Memorias daSociedadeBroteriana 10:2979. R OBERTSON ,K.R.1982.ThegeneraofOlacaceaeinthe southeasternUnitedStates. JournaloftheArnoldArbore- tum. 63:387399. R ONQUIST ,F.andJ.P.H UELSENBECK .2003.MrBayes3: Bayesianphylogeneticinferenceundermixedmodels. Bioinformatics 19:15721574. R OSSOW ,J.A.1982.SinopsisdelasMisodendraceae. Parodiana 1:245270. S KOTTSBERG ,C.1913.BemerkungenzurSystematikder GattungMyzodendron. BotanischeJahrbu ¨ cherfu ¨ rSystem- atikPflanzengeschichteundPflanzengeographie 50:384391. S WOFFORD ,D.L.2003.PAUP*:Phylogeneticanalysisusing parsimony(*andothermethods),v.4.0b10.Sunderland: SinauerAssociates. T ABERLET ,P.,L.G IELLY ,G.P AUTOU ,andJ.B OUVET .1991. Universalprimersforamplificationofthreenon-coding regionsofchloroplastDNA. PlantMolecularBiology 17: 1105. T AKHTAJAN ,A.1997. Diversityandclassificationofflowering plants .NewYork:ColumbiaUniversityPress. T ERCERO -B UCARDO ,N.andT.K ITZBERGER .2004.Caracter ´ sti- casdelestablecimientoehistoriadevidade Misoden- drumpunctulatum (Misodendraceae)unmue ´ rdagode Sudame ´ ricaaustral. RevistaChilenadeHistoriaNatural 77: 509521. W ERTH ,C.R.,W.V.B AIRD ,andL.J.M USSELMAN .1979.Root parasitismin Schoepfia Schreb.(Olacaceae). Biotropica 11: 140143. Z AVARO ,C.A.,J.V.C RISCI ,andJ.J.M ORRONE .1997.Synopsis andcladisticsofthegenus Misodendrum (Misodendra- ceae,Santalales). Fontqueria 48:225239. A PPENDIX 1.Taxaincludedinphylogeneticanalyseswith voucherinformation(inparentheses:herbariumacronym andDNAaccessionnumberfromcollectionmaintainedbyD. 2007] VIDAL-RUSSELL&NICKRENT:MISODENDRUMPHYLOGENY 567 positionofthefemalesflowersontheinflores- cence.Inaddition,whencharacter6(female flowers)isoptimizedunderACCTRAN(Fig.2B), thesetwocladeshavepedicellatefemaleflowers withareversaltosessileflowersinsection Misodendrum (subg. Misodendrum ).Whenopti- mizedunderDELTRAN,pedicellateflowersin section Heterophyllum (subg. Misodendrum )are homoplasiouswiththeonesfoundinsection Archiphyllum (subg. Angelopogon ). Manycharacterstatesaresharedamongsubg. Angelopogon taxa,butthesebecomepleisomorphic giventhatsubg. Angelopogon isparaphyletic. Especially,similaritiesareseenbetweensection Archiphyllum and Telophyllum ,thatsharefour characterstates:1)swollenhaustoria,2)axial bracteoles,3)claviformstaminodebristles,and4) stemincrustations. D ISCUSSION Allspeciesof Misodendrum areverysimilar morphologicallyanduniqueamongaerialpara- siteswiththeirwinddispersedfruits.Molecular datastronglysupportthemonophylyofMisoden- draceae(BS 100,MLBS 100,PP 1.00).The phylogeneticrelationshipsfoundamongspeciesin thisstudygenerallysupportpreviousclassifica- tionsofthefamily.Subgenus Misodendrum is adistinctcladecharacterizedbywartyyellow stemsandtwostamens(Fig.2A),butrelationships amongitscomponentspeciesareunresolved.The rarespecies M.macrolepis wasnotincludedinthis molecularanalysis,butwasplacedassisterto M. angulatum inthemorphologicalanalysisreported hereandintheoneconductedbyZavaroetal. (1997).Inthelatterstudy, M.punctulatum and M. gayanum appearassistertothatclade,butwithlow support(BS 50),whereasourmorphological cladisticanalysisyieldedhighsupport(BS 92) forthisclade.The13additionalcharactersadded inourstudy,specificallythoserelatingtostem anatomy,mayhaveresultedinhigherclade support. Misodendrumgayanum isoneofthemost distinctivespecieswithinthesubgenusbasedon itsstemandwoodanatomy(Fig.2).Specifically,it lacksfibersinfascicularareas,itsaxialparenchy- maisnotlignified,andthevesselpitsarerounded whereasinotherspeciestheyareellipticaland scalariform(Carlquist1985). Misodendrumpunctu- latum appearstobeamorespecializedparasitein thatitsleaveshavebecomereducedtoscales.In addition,itsleavesandstemsaremoreyellowthan otherspecies,probablyindicatingalossofchloro- phyllandconcomitantrelaxationofphotosynthetic activity.Agreaterdependenceoncarbohydrates fromthehostcanalsobeinferredfromthefactthat thisspeciescanexistcompletelyendophyticallyfor thefirsttwoyearsandsometimesuptosixyears afterinfection(Tercero-BucardoandKitzberger 2004).Themorphologicalandphysiologicalsimi- larityof M.punctulatum tothedwarfmistletoes ( Arceuthobium ,Viscaceae)representsanexampleof convergentevolution. Misodendrumquadriflorum isweaklysupported assistertoallotherspeciesinthegenusinthe combinedanalysis,butstronglysowiththe matK dataset.Withthe trnL-F data, Misodendrum linearifolium isweaklysupportedasthefirst divergingtaxon.Anyofthesetopologiesresults inaparaphyleticsubgenus Angelopogon .Compared withsubgenus Misodendrum ,membersofsubgenus Angelopogon havelargerandmoresucculentstems withswollenhaustorialbases(anexceptionis M. linearifolium ).Incontrasttosubgenus Misodendrum , specieswithin Angelopogon differgreatlyintheir woodanatomy(Carlquist1985).Specifically,the vascularbundlesin M.quadriflorum areirregularly oriented,whereassection Archiphyllum ( M.brachys- tachyum and M.oblongifolium )hastwofascicular circles. Misodendrumlinearifolium (section Angelo- pogon )appearstobetheleastspecializedspecies havingonlyonecircleoffascicles.Basedonthe optimizationofcharacterstates,speciespreviously classifiedinsubgenus Angelopogon, shareonly ancestralcharacteristicssuchasgrayandsmooth stems,unlignifiedpith,pubescentfloralshootsand threestamensinthemaleflowers.Thephylogeny inferredbythechloroplastgenome,hererepre- sentedby matK and trnL-F ,showsaparaphyletic subg. Angelopogon .Becauseofthelowsupportand theexaminationofonlyonegenome,thispara- phylyshouldbetestedwithgenesfromthe nucleuspriortoproposingachangeinthe classification. Section Archiphyllum ( M.brachystachyum and M. oblongifolium )wasstronglysupportedasmono- phyleticineveryanalysisconductedinthisstudy andwithallexaminedpartitions.Thesetwotaxa sharethesamestatesforallmorphological charactersexaminedinthisstudy.Theydifferin twocontinuouscharacters,with M.oblongifolium havinglongerstaminodesandahigherdegreeof leafpubescence(Orfila1978;Rossow1982). Mis- odendrumoblongifolium ismoregeographicallyre- strictedthan M.brachystachyum andparasitizes hoststhataregenerallyfoundathigherelevations. Onlytwohostsareknownfor M.oblongifolium ( Nothofaguspumilio and N.dombeyi ),while M. brachystachyum parasitizetheseplusthreemore species( N.antarctica , N.betuloides , N.nitida ). Geneticdistances(HKY85inPAUP*)between thesetwospecieswerethelowestamongall 566 SYSTEMATICBOTANY [Volume32 F IG. 2.Characteroptimization.A.Characterstatechangesoptimizedonthemo leculartree.B.Differentresolutionforthe characterswhereequivocalreconstructionswerefound.Numbersreferto charactersandstatesinAppendix2.Astarnextto thecharacternumberreferstoDELTRANoptimization,theotherstoACCTRA N. 2007] VIDAL-RUSSELL&NICKRENT:MISODENDRUMPHYLOGENY 565 F IG. 1.A.Majorityruleconsensustreeof75,000treesfromBayesiananalysiso fthecombined matK and trnL-F sequences. Branchlengthsarerepresentedbymeanvaluesofthosetrees.Nodalsuppor tisgivenabovethebranchesasbootstrapvalues forparsimony,likelihood,andposteriorprobabilities,respectively. B.Strictconsensusoftwomostparsimonioustrees resultingfromtheanalysisof31morphologicalcharacterswithbootstra psupportaboveeachbranch(1000replicates). 564 SYSTEMATICBOTANY [Volume32 parsimonioustreeswerefound(383steps).The differenceinthesetreeswasthepositionof M. quadriflorum .The trnL-F consensustreestrongly supportedthesisterrelationshipof Misodendrum and Schoepfia .Wheningroupnodesunsupported byatleastonemethodofanalysiswerecollapsed, fourcladesemergedfromapolytomy:1) M. linearifolium ,2) M.quadriflorum ,3) M.brachysta- chyumandM.oblongifolium (BS 100,MLBS 100, PP 1.00),and4) M.angulatum,M.gayanum ,and M.punctulatum (BS 99,MLBS 97,PP 1.00). Misodendrumlinearifolium isweaklysupported(BS 79,MLBS 50,PP 0.52)assistertoallother speciesintheingroup. The matK genedoesnotvarygreatlyinlength amongspeciesof Misodendrum .Duetoamplifica- tionsproblems M.punctulatum ismissing656bp, and S.schreberi ismissing878bpofthisgene. Thereisonein-framedeletionof6bpin M. quadriflorum . Misodendrumlinearifolium shows aframeshiftmutationcausedbyonebasedeletion andthenthereadingframeisrecoveredbyan insertion17basesdownstream.Thisframeshift mutationcausesachangeinsixaminoacids.A tranversionalmodelwithratevariationamong sitesestimatedthroughagammadistributionwith fourcategories(TVM C )wasselectedfor matK . The matK datasetconsistedof1151aligned positions,ofwhich260wereparsimonyinforma- tive(Table1).Twomostparsimonioustreewere found(632steps)thatdifferedintherelationship amongthespeciesofthemonophyleticsubg. Misodendrum .Thesamefourcladesfoundwith the trnL-F regionwerefoundanalyzing matK. Subgenus Angelopogon wasparaphyletic,and M. quadriflorum washighlysupportedassistertoall species(BS 93,MLBS 88,PP 0.97). Thetopologyofthe matK treewasmoreresolved thantheonefrom trnL-F ,butthetwotreeswere notincongruent.Forthisreason,thetwodatasets wereconcatenated.Thisanalysisyieldedonemost parsimonioustreeof1028steps(Table1;Fig.1A) whichplaced M.quadriflorum assistertoallother taxabutwithlowsupport(BS 63,MLBS 55,PP 0.73).Thesignalforthisrelationshipderives fromthe matK genetree.Allanalysesrecovered section Archiphyllum (subg. Angelopogon )asmono- phyletic.Thissectionissistertosubg. Misoden- drum ,however,thisrelationshipisweaklysup- ported(BS 63,MLBS 54,PP 0.89).Subgenus Misodendrum ismonophyleticwithhighsupport fromallthreeanalyticalmethods(BS 100,MLBS 100,PP 1.00). Thetwomostparsimonous matK treesdiffered fromthe trnL-F treeintherelativepositionsof M. punctulatum , M.gayanum and M.angulatum .Inthe combinedanalysis,parsimonyplaces M.punctula- tum assisterto M.angulatum (aswith matK )with 63%bootstrapsupport,however,thisrelationship collapsestoapolytomywithlikelihoodand Bayesianinference.Thesetaxaarebestviewedas emergingfromapolytomy,especiallygiventhat M.macrolepis ,includedinthisgroupbyother authors,wasnotsampledinthisstudy;itsin- clusionmighthelptoresolvetherelationships amongspeciesofsubg. Misodendrum . Themorphologicalcharacteranalysisyielded twotrees,eachof56steps(Table1).Thetopologies ofthetwotreescapturedsomeofthesamewell- supportedrelationshipsobtainedfromthemolec- ulardata(Fig.1B).Thecladessupportedbyboth methodologiesweresubg. Misodendrum andsec- tion Archiphyllum (subg. Angelopogon ). Misoden- drumquadriflorum issistertosection Archiphyllum withrelativelyhighsupport(BS 86).This topologyisalsofoundwiththe trnL-F dataset butwithlowsupport(BS 52). Misodendrum linearifolium changespositioninthetwomorphol- ogytrees.Itappearseitherassistertosubg. Misodendrum oraspartofsubg. Angelopogon . Analysisoftheconcatenatedmolecularplus morphologydataset,aswithbothdatasets analyzedseparately,didnotresolveamonophylet- icsubg. Angelopogon ,thusthetwosubgeneraare notreciprocallymonophyletic.Inthisanalysis, Misodendrumlinearifolium (subg. Angelopogon )is sistertotheremainingspecies,butwithno support.Asintheotheranalyses,subg. Misoden- drum ishighlysupportedasmonophyletic.Mod- eratesupport(BS 72)isfoundforthesister relationshipbetween M.angulatum and M.macro- lepis (thislatterspecieswasincludedonlyinthe morphologicaldataanalysis);however,therewere nomorphologicalsynapomorphiesforthisclade. Whenthecharacterstatechangesareoptimized onthemoleculartree(with M.macrolepis addedin itsmostlikelypositionbasedonthemorphological analysis),fivemorphologicalsynapomorphiessup- portthemonophylyofsubgenus Misodendrum (Fig.2A).Additionalsynapomorphiesarefound forthiscladewhenoneexploresthedifferent reconstructionsofancestralstatesforthesix charactersforwhichoptimizationwasequivocal. Ifcharacter4(leafshape)isreconstructedunder theDELTRANoption(Fig.2B),thenlinearleaves characterizethesubgenus Misodendrum clade.In thiscase, M.linearifolium acquiredthesameleaf shapeindependently.WithACCTRANreconstruc- tion,threemorecharacters(absenceofsclereids, absenceofrays,andlignifiedparenchyma)char- acterizethisclade(Fig.2B).Section Archiphyllum shareswithsubg. Misodendrum thealternate 2007] VIDAL-RUSSELL&NICKRENT:MISODENDRUMPHYLOGENY 563 purificationkit(OmegaBiotek,Inc.Doraville,Georgia).Cycle sequencingreactionswereperformeddirectlyonthepurified PCRproductsfollowingstandardprotocolsaccompanying theBigDyeterminatorCycleSequencingReadyReactionKit, withAmpliTaqDNAPolymerase(AppliedBiosystems, FosterCity,California)andBetterBuffer(TheGelCompany, SanFrancisco,California).Sequencingreactionswerepuri- fiedwithethanolprecipitationorwithCentri-Sep100spin columnswithSephadex(PrincetonSeparations,Inc.Adel- phia,NewJersey).SequencesweregeneratedusinganABI 377automatedsequencer(AppliedBiosystems). AlignmentandPhylogeneticAnalysis. Sequenceswere alignedbyeyeinSe-Al(Rambaut2004).Maximumparsimo- ny(MP),maximumlikelihood(ML)andBayesianinference (BI)analyseswereperformedforindividualandcombined partitions.Parsimonyandbootstrapanalyses(BS,1000 replications)wereconductedinPAUP*(Swofford2003)with branchandboundsearches.Gapswereconsideredhomol- ogousiftheysharedidenticalboundariesandlength.They werecodedasasubstitutiononlyforthe trnL-F regionifthey weresharedbymorethanonetaxon.For matK ,allgapswere codedasmissing.Thefinaldatamatricesareavailablefrom TreeBASE(studynumberS1728). MaximumlikelihoodanalyseswereconductedinPAUP* (Swofford2003),withamodelofsequenceevolutionselected usingModelTest(PosadaandCrandall1998)foreachgene partitionaswellasforallcombinedpartitions,implementing theAICcriteria.Treeheuristicsearcheswereperformed usingTreeBisectionReconnection(TBR)branchswapping withaNeighborJoining(NJ)startingtree.Bootstrapnodal support(MLBS)wasdeterminedbyanalyzing100replicates, startingeachtimewithaNJtreeandheuristicsearchandthe TBRbranchswappingalgorithm. BayesianinferencewasperformedusingMrBayes3.1.2 (HuelsenbeckandRonquist2001;RonquistandHuelsenbeck 2003)withamodelselectedbyMrModeltest(Nylander2004). Themodelofmolecularevolutionselectedfor matK,trnL-F andbothpartitionscombinedwasthegeneraltimereversible plusagammadistributiontoaccountforvariationamong sites(GTR C ).Twoindependentanalyseswithfourchains eachwereperformedforfivemillionsgenerations.Treesand parametersweresavedevery100generations,producing 50,000treeseachrun.Modelparameterswereestimatedas partoftheanalysis;uniformpriorprobabilitieswere assignedtoallparametersexceptthestatefrequenciesfor whichaDirichletpriordistributionwasassigned.When morethanonepartitionwasanalyzed,parameterestimations wereunlinkedandtherateofthepriorswassettovary,thus allowingpartitionstoevolveatdifferentrates.Theburn-in wasdeterminedbyidentifyingstationarityusingtheln likelihoodscore.Thevariancebetweenrunsinallcaseswas below0.001,thusrunswerecombinedtherebyincreasingthe numberoftreesintheposteriorprobability(PP)distribution. MorphologicalAnalysis. Morphologicaldatawerecol- lectedfromtheavailableliteratureforMisodendraceae (Johnson1889;Skottsberg1913;Orfila1978;Rossow1982; Carlquist1985;Zavaroetal.1997). Schoepfiaschreberi and Nuytsiafloribunda wereusedasoutgroupbyassembling informationfromtheliterature(Herbert1919;Metcalfeand Chalk1950;Reed1955;Narayana1958;Barlow1966;Werthet al.1979;Robertson1982).Amatrixwith31discretecharacters wasconstructed(Appendix2and3)andanalyzedwith unweightedparsimony,treatingallcharactersasunordered. Treesearchandnodalsupport(1000bootstrapreplicates) wereperformedwithaBranchandBoundsearchinPAUP* (Swofford2003).Thismatrixwasconcatenatedwiththe moleculardatasetandwasanalyzedusingthesame parsimonysettingsusedforthemoleculardatasetalone. Morphologicalcharacterstatechangeswereoptimizedonthe moleculartopologyusingMacClade(MaddisonandMaddi- son2000)underbothDELTRANandACCTRANoptions. R ESULTS Forthe trnL-F region,theingroupvariedin lengthfrom620to772basepairs.Therewere48 gapsrangingfrom1to146basepairs;ofthese gaps,10werecodedforparsimonyanalysis.Most ofthelargersizedifferencesweredeletions, whereasthelargestinsertionwasfoundin M. linearifolium, consistingof35bp.Amongboth ingroupandoutgrouptaxa,tandemduplications inatleast17positionswereseenthataveragedsix basepairsinlength.Ambiguousalignmentposi- tionswerepresentinthe trnL-F matrixbutwere generallyconfinedtoingroupandoutgroup comparisons.Whentheoutgroupwasremoved, thealignmentwaslargelyunambiguous.Inclusion andexclusionofambiguoussitesdidnotchange relationshipsamongthetaxa.Mutationsinthe upstreamanddownstreamduplicatesegments involvedsmalllengthmutationsandsubstitutional changes.Themodelofmolecularevolutionselect- edfor trnL-F wasatransitionalmodelwithrate variationamongsitesestimatedthroughagamma distributionwithfourcategories(TIM C ). Thefinal trnL-F datamatrix(withoutgroups included)consistedof1365alignedpositions,of which147wereparsimonyinformative(Table1). Onemostparsimonioustreewasfound(367steps), however,whengapswerecodedthreeequally T ABLE 1.Summaryoftreestatisticsfromparsimonyanalysesofmolecularandmor phologicaldatasets. trnL-FmatKmatK trnL-F Morphology matK trnL-F morphology Alignmentlength136511512516312547 Missingdata%010.254.693.204.72 Variablecharacters27543270731738 Informativecharacters14726040729436 Numberoftrees12221 Treelength3676321015561074 CI0.9100.8470.8560.7140.846 RI0.8690.8360.8270.7090.817 562 SYSTEMATICBOTANY [Volume32