SystematicBotany (2004),29(1): pp. 188– 198 q Copyright 2004by the AmericanSociety of PlantTaxonomists

Viburnum Phylogeny Basedon Chloroplast trnK Intron and Nuclear Ribosomal ITSDNASequences

MICHAEL J. DONOGHUE,1,4 BRUCE G. BALDWIN,2 JIANHUA LI,3 and RICHARD C. WINKWORTH1 1Department ofEcology and Evolutionary Biology,Yale University,NewHaven, Connecticut06520; 2Jepson Herbarium and DepartmentofIntegrativ eBiology,University ofCalifornia, Ber keley,California94720; 3Arnold Arboretum ofH arvard University,JamaicaPlain, Massachuse tts 02130; 4Author forcorrespond ence ([email protected])

CommunicatingEditor: Jam esF .Smith

ABSTRACT. Phylogenetic analyses ofchloropla st trnK intronand nuclear ribosomalITS DN Asequences yieldsigniŽ cant improvements inour understandingof relationsh ips, character evolution, and biogeographyin Viburnum (Adoxaceae). We conŽrm tha tmostof the ten traditionall yrecognized sectionsare monophyletic. The moststriking exception is Odontotinus , which isdivided into: (i)a purple-fruitedN ew Worldclade withinwhich theLa tinAmerican section Oreinotinus is nested, and (ii)an OldW orld, mostlyred-fruited clade thatisclosely related to V.cylindricum (section Megalotinus ) and the New World, purple-fruited V.acerifolium .Weidentifythree major supra-sectio nal groupings: (i)a clade consistingof the Odonto- tinus-Oreinotinus -Megalotinus complex, the circum-boreal section Opulus,and the Eurasian section Tinus,(ii)a cladecontaining theOld W orldsection Viburnum,theN ew Worldsection Lentago,and, withless support, section Pseudotinus ,and (iii)a clade containing the Asian sections Tomentosa and Solenotinus .Twospecies are notclearly allied toany ofthese supra-sectio nal clades: V.urceolatum ,aTaiwanese/Japanese species traditionallyplaced insection Viburnum, and V.clemensiae , a Bornean species previouslyassigned tosection Solenotinus .The placementofthe root also remainsuncertain, but probably does not fall withinany ofthe three major supra-sectio nal clades. Knowledge ofrelationsh ips withinsections is useful inclarifying historical biogeographyand theev olution ofsterile  owersand fruitcolor .The one case ofcon ict between datasets high- lightsa possible instance ofhomoploi dhybridspecia tion.

Viburnum contains approximately 175species of icstudies (Donoghueand Sytsma 1993;Donoghue and shrubs and small trees distributed around the North- Baldwin 1993;summarized in Baldwin et al.1995) ern Hemisphere(Oersted 1861;Rehder 1908;Hara conŽrm ed many aspectsof the morphologicalresults, 1983;Donoghue 1983a),with signiŽcant extension s butcalled other Žndings into question. In particular, into the mountains ofsoutheast Asia (Kern 1951)and the molecular studies indicated thatthe phylogenetic Latin America (Killip and Smith 1931;M orton 1933; arrangement within section Odontotinus was more Donoghue 1982).Although rather uniform in ower complicatedthan suggested byDonoghue (1983a). and fruitm orphology, Viburnum is well known for Although these earlier studies were usefulin eval- striking variation in several features,including the uating the monophyly ofthe sections, theydid little to presence or absenceof naked budsand ofsterile ow- clarify relationships among and within the sections, or ers around the margins ofthe inorescences. Based on toestablish the position ofthe rootwithin Viburnum. these and other characters(e .g.,endocarp shape,ino- Consequently,ourunderstan ding ofbiogeograph yand rescence form,and leafmorpholo gy) Viburnum has charactere volution in the group has remained rather been subdivided byseveral authors, most commonly rudimentary.The aim ofthe present study wasto as- into ten taxaformally recognized assections (Table1; semble twodirectly comparablem oleculardatasets — one nuclear and one chloroplast—in the hope thatto- Oersted 1861;Hara 1983). gether these would improve ourknowledg eofthe evo- Previous analyses have strongly supported boththe lution and biogeographyof Viburnum. monophyly of Viburnum (Wilkinson 1948;Donoghue 1983b;Donoghue andFriedman 1988;Benk o-Iseppon MATERIALSAND METHODS and Morawetz2000) and its placement within the Dip- sacales(Donoghue 1983b;Donoghue et al.1992; Judd Weobtained nuclear ribosomalITS and chloroplast trnK DNA et al.1994; Backlund and Donoghue 1996;Backlund sequences from43 accessions of Viburnum (42 species and two specimens ofthe widespread V.sargentii ),representingall ofthe and Bremer 1997;Pyck et al.1999; Donoghue et al. sectionsand majorspecies complexes, as well as fromtw ospecies 2001;Bell et al.2001). H owever, phylogeneticrelation- of Sambucus forrooting p urposes. Tissue samples were eithersilica ships within Viburnum havereceived muchless atten- gelpreserved Želd collections orw ere obtained fromherbarium tion. An analysis ofmorphol ogicalcharacters by Don- specimens. Appendix 1provides voucherinformationforeach ac- cession and GenBank numbersfor the sequence s. oghue (1983a)found nine ofthe ten traditionallyrec- DNAExtraction,AmpliŽ cation, and Sequencing. Genomic ognized sections tobe monoph yletic. The exception DNAwas extracted fromdried tissue samples usingeither a ce- was section Odontotinus thatcontained acladecorre- tyltrimethylammoniumbromide (CT AB)protocol modiŽed from Doyleand Doyle(1990), orthe Qiagen D Neasy Plant Kit (La Jolla, sponding tothe Latin American section Oreinotinus . CA). Subsequently,twounpublishe dmolecular phylogenet- The 59 portion ofthe trnK intronw as ampliŽed inreaction vol-

188 2004] DONOGHUEET AL.: VIBURNUMPHYLOGENY 189

TABLE 1. Traditionallyrecognized sectionsof Viburnum (e.g. the fouroligonucl eotide primersdescribed above; forthe ITSlocus Hara 1983), withthe approximate numberof species and gener- the ampliŽcation primersand, when necessary,the primersITS2 alized geographic range foreach. and ITS3(White et al. 1990) were used insequencin g. Initialresults from the ITSlocus suggestedthe presence ofm ul- tiple sequence types insome accessions .Inthese cases PCR frag- Section Lentago—7spp. Eastern NorthAmerica, except V. ela- mentsw ere cloned usinga TOPOT ACloningKit (Invitrogen). tum in Mexico. Between 10 and 15 colonies fromeach cloningreaction were Section Megalotinus —18 spp.Southeast Asia, extendingwest screened bydirect PCR. Screeningused reaction mixturesas de- toIndia and south toIndonesia. scribedfor the ITSlocus and theM13 primers.Thermocyc ling Section Odontotinus —45 spp.Temperate Asia and Eastern conditions were: cell lysisat 98 8Cfor10 minutes,then 25 cycles NorthAmerica, except V.orientale inthe Caucasus Moun- of1 minuteat 95 8C, 1minutea t55 8C, and 1minutea t72 8C (ex- tains. tension),with a Žnal incubationat 72 8Cfor5 minutes.F oreach Section Opulus—5spp. Circumboreal. accession fouror Ž ve positiveclones were puriŽed usingthe QIA- Section Oreinotinus —42 spp. Mexico, Caribbean, Central and prep Spin Miniprep Kit (Qiagen)and sequencedas aboveusing SouthAmerica. the M13 primers. Section Pseudotinus —4spp. Asia, except V.lantanoides in Alignments andPhylogen etic Analyses. Weobtained prelimi- Eastern NorthAmerica. narym ultiple alignmentsof D NAsequences usingthe progressive Section Solenotinus —25 spp. Asia, extendingwest toIndia alignmentprocedure implemented inClustalX (Thompsonet al. and south toIndonesia. 1994). These alignmentsw ere thenvisually inspected and adjusted Section Tinus—9spp.Asia, except V. tinus in Europe. forminor impro vement. Priorto phylogen etic analyses all ambig- Section Tomentosa—2spp.China, Japan. uous and gapped positionsw ere excluded fromthe data matrix. For the trnK intronand ITSdata setsphylogen etic treeswere Section Viburnum—18 spp.Asia, except V. lantana in Europe. inferredusing both maximum parsimony (MP) and maximum likelihood (ML)optimality criteria as implementedin PAUP*4.0b10 (Swofford2001). MPanalyses used heuristic search- es with‘ ‘tree-bisection-reconnection’’(TBR)branch swapping, umes of 25 mLcontaining 1 3 PCR buffer(P erkin-Elmer),6.25 mM zero-lengthbranches collapsed, and all characters equally weight- MgCl , 625 mMeach dNTP (Invitrogen),5% bovine serumalbu- 2 ed. Analysesw ere repeated 100 timeswith RANDO MADDITION. min(v/ v; New England Biolabs), 10 pM each ampliŽcation prim- Todeterminethe best-Ž tting model of sequence evolution forML er, 1UAmpliTaq DNApolymerase (5 U/ mL; Perkin-Elmer)and analyses, ahierarchical seriesof likelihood ratio testsw as per- 10–100 ngof total cellular DNA. Typically the oligonucleotide formedon the MPtree topologies usingthe programPO RN* (Bell primerstrnK -11 and matK510R (Young et al. 1999) were used in 2001). Subsequentheuristic MLtree searc hesused the mostap- ampliŽca tions.H owever, when necessary thesewere combined propriate model(with parameters simultaneouslyestimated via withthe internal primersVIBmatK1F (5 9-TATATTAGTGCCTGA- maximumlikelihood), TBR branch swapping, and collapsed zero- TACGGG-39)and VIBmatK1R (5 9-GATCTATCTAGCTCTAAA- lengthbranches. Analysesw ere repeated 100 timeswith RAN- TATC-39)toamplify the region in tw ooverlappingsections. Ther- DOMADDITION.Bootstrap testsused 1000 replicateswith near- mocyclingconditions for PCR were: initial denaturationat 98 8C estneighbor interchange (NNI)branch swapping; parameters for for3 minutes,35 cycles of1 minuteat 95 8C(denature),1minute thesetests w ere Žxed tov alues estimated on the optimal maxi- at 508C(annealing), and 2minutesat 72 8C(extension),with a Žnal mumlikelihood tree. incubation at 72 8Cfor5 minutes.The ITSlocus was ampliŽed in Wetestedfor incongruen ce between thetwo datasets usingthe 20 mLreactions containing 1 3 Qsolution (Qiagen),1 3 PCR buffer partition homogeneitytest as implemented inP AUP*4.0b10.Phy- (Qiagen),500 mMeach dNTP (Invitrogen),10 pM primerITS-I logenetic analyses ofthe combined data setw ere conducted as (Urbatschet al. 2000), 10 pM primerITS4 (White et al. 1990), 1U described forthe individual data sets. DNApolymerase (5 U/ mL; Qiagen),and 10–100 ngof total cel- lular DNA. The PCR proŽle was similarto tha tdescribed forthe trnK intronbut included a54 8Cannealing temperature and a1 RESULTS minuteextension time. AmpliŽcation products forboth loci were puriŽed usingthe Aligned DNASequences. Statisticsfor the aligned QIAquickPCR PuriŽcation Kit (Qiagen).A utomated sequencingof datamatrices are presented in Table2. The datamatrix PCR fragmentswas performedusing the ABI PRISM BigDye T er- forour chloropla st marker contained little alignment minatorCycle SequencingReady Reaction kitand analyzed using eitheran ABIPrism 377 automated DNAsequencerorMJ Re- ambiguity asthe DNAsequences were highly similar. search BaseStation51. DNAsequencingofthe trnK intron used In contrast,alignment ofsev eral regions within the ITS

TABLE 2. Statisticsfrom the aligned data matrices. Forthe ITS data setit is difŽ cult toconŽ dently infer the exact numberof indels due touncertainty inthe alignment of several regions.

ITS trnK intron No. oftaxa sequenced 45 45 Sequence length(bp) 607–619 1137–1146 Alignedlength (bp) 641 1147 No. ofindels see table caption 4 No. ofexcluded sites 85 16 No. ofconstant sites 410 1010 No. ofvaried sites 146 121 No. ofparsimony informative sites 108 75 %GC content range (all sites) 57.9–64.2 32.0–33.1 %GC content mean (all sites) 61.6 32.6 %GC content range (varied sitesonly) 47.3–71.2 42.1–52.1 %GC content mean (varied sitesonly) 61.3 47.5 190 SYSTEMATIC BOTANY [Volume 29

FIG.1. Singleoptimal maximumlikelihood tree obtained fromthe analysis of trnK sequences, showingbootstrap values over 50% (1000 replicates). Names ofthe traditional sections of Viburnum are given on the right. dataset wasconfounde dbythe presence ofoverlap- Analysesof the trnK IntronSequences. MP analy- ping butnon-iden ticalindels. These regions, asw ell as ses resulted in foureq ually parsimonious topologies, allother gapped or ambiguouspositions, were exclud- each140 steps long with aconsistency index (CI)of ed from the analyzed datamatrices. Consequently, 0.907and aretention index (RI)of 0.955. These trees there were no missing cells in ourdata sets. Nucleotide differed only in the placement oftwo species; V. odor- sequencesare deposited in GENBANK (see Appendix atissimum and V.cylindricum were placedeither within 1foraccession numbers);aligned datamatrices and or asbasal branches oftheir respective clades. The ML trees are availablein TreeBASE (study accession num- search used aGTR 1G1Isubstitution model and re- ber S998,matrix accession numbers M1677—M1679). sulted in asingle tree (–lnL 52552.8024;Fig. 1)iden- 2004] DONOGHUEET AL.: VIBURNUMPHYLOGENY 191 ticalin topology tothe strict consensus ofthe fourMP recognized sections and their relationships toone an- trees. Furthermore,bootstrapsupport for clades iden- other,while the ITSregion wasmore informativeabout tiŽed in these twoanalyses wassimilar, providing relationships among species within the sections. This moderate (i.e.greater than 65%bootstrap support) to difference is evident in comparing the tree topologies strong support(i.e . .80%bootstrap support) for man y in Figs. 1and 2;the trnK tree is well-resolved towards ofthe major clades within Viburnum. the baseand poorly resolved distally,whereas the re- Twoparsimonyinformativeindel regions excluded verse is true in the ITStree .This shape contrastis from the analyses are compatiblewith topologies in- paralleled bydifferences in the alignability ofthe se- ferred from nucleotide substitutions. SpeciŽcally , quences (trnK being easier toalign than ITS)and con- members ofsection Pseudotinus (e.g., V.cordifolium , V. sistency indices ( trnK with less homoplasy than ITS). furcatum, and V.lantanoides )share a9-nucleotide dele- As emphasizedbelow,these observations are consis- tion (positions 552–560), and the twooutgroup taxa tent with closer inspection ofthe bootstrapvalues and differ from all Viburnum species bya 5-nucleotide indel the apparentcomplem entarity ofthe datasets when (positions 606–610). The tworemaining indels were combined. single nucleotide deletions; one unique to V. davidii (po- Monophyly of theT raditional Sections. Where our sition 465in the aligned datamatrix) and the other to taxonsam pling allowed aproper test (weincluded Sambucusracem osa (position 377). only one species eachfrom sections Tomentosa and Me- Analysesof theITS Sequences. Initial DNAse- galotinus)most ofthe sections were found tobe m ono- quencing suggested thatse veral Viburnum species phyletic, or non-monophyly wasonly weakly support- might containm ultiple ITSsequence types. Wecloned ed. Analysis ofthe chloroplast dataset alone provided PCRfragm ents forthese species, and obtained se- supportfor the monophylyofsections Pseudotinus quences from fouror Žve clones. Preliminary analyses (65%bootstrap support), Opulus (96%), and Tinus ofthese dataindicated thatin eachcase clones from a (74%).In the combined analysis, bootstrapsupport for given accession formed acladein the ITSph ylogeny. eachof these groups washigher than in either indi- Therefore,the sequence ofa single clone wasused to vidual dataset. Sections Lentago and Oreinotinus are representeachof these species in the datamatrix. paraphyletic in the optimalML tree inferred from the MPsearches recovered 240optimal trees, each297 combined dataset. However, in bothcases the result steps long with CI 50.586 and RI50.785.Although the wasonly weakly supported (53%and 61%bootstrap consensus ofthese topologies (Fig. 2)provides limited support,respectiv ely) and in analyses ofthe individ- resolutionand supportfor higher-l evel relationships ualdata sets the arrangements were unresolved. For within Viburnum,ITSsequence sdo provide support the Latin American section Oreinotinus ,arelationship forthe relationships between species within sections. with the eastern North American species, V. dentatum, The ML analysis, which used aGTR 1Gsubstitution is well supported. However, additional taxonsampling model, again recovered asingle tree (–lnL 5 is required toresolve whether one or more North 2576.17788).H owever,asin the MPresults manyof American species are sister to,or nested within, the the higher-level relationships suggested bythis tree Latin American species. In the caseof section Lentago, were not well supported in bootstrapanalyses. V. nudum is clearly distinct from the remaining species, Combined Analyses. The partition homogeneity butagain more dataare needed toestablish the exact test indicated thatthe twodatasets did not conict sig- phylogenetic arrangement. niŽcantly (P50.10).MP analyses ofthe combined data The three remaining sections—Odontotinus , Vibur- set recovered 72trees of446 steps, with CI 50.675 and num and Solenotinus —are clearly not monophyletic. For RI50.832.U sing aGTR 1G1Isubstitution model the section Odontotinus this result wasanticipated based ML search recovered asingle optimaltopology on earlier analyses (e.g.,Donoghu e1983a;Baldwin et (–lnL55165.09407).Gen erally,this tree simply com- al.1995). In agreement with previous studies we found bines the well-supported clades identiŽed in the trnK that Odontotinus wasdivided into twodistinct clades— and ITSanalyses; however, in most casesbootstrap one containing Old World, red-fruited species and the values forthese clades are higher.Two exceptionsare other New World taxawith purple fruits.Our results discussed in detail below:the relationship between are alsoconsistent with the earlier studies in suggest- Pseudotinus and the Viburnum-Lentago cladeand rela- ing that(i) the Latin American species (section Orein- tionships among the species ofsection Lentago. otinus)are nested within the New World Odontotinus clade,and (ii) V.acerifolium ,apurple-fruitedNew DISCUSSION World species, is more closely related tothe Old World Considered individually,neither the trnK nor ITS red-fruited group.However, ouranalyses alsosuggest datasets conŽdently resolved relationships both apreviously unrecognized relationship between Old among and within the major Viburnum clades. Gener- World Odontotinus and V.cylindricum ,oursole repre- ally, the trnK dataprovided supportfor the previously sentative ofthe primarily southeast-Asian section Me- 192 SYSTEMATIC BOTANY [Volume 29

FIG.2. Strictconsensus of240 maximumparsimony trees obtained fromthe analysis ofITS sequences (branchlengths under ACCTRAN), showingbootstrap values over 50% (1000 replicates). Nodes markedwith asteriskscollapse inthe majorityrule consensus ofthe maximumlikelihood bootstrap trees. Names ofthe traditional sections of Viburnum are given on the right.

galotinus.Additional dataare needed toestablish (56%and 78%bootstrap support, respectivel y) that whether this result reects the relationships of Megal- wasstrongly linked with section Lentago (91% and 94% otinus asa whole,which is notablyheterogen eous with bootstrapsupport in the chloroplast and combined respect toleaf, corolla, stamen, and fruitcharacters analyses, respectively).The exception, V.urceolatum , (Kern 1951;Hara 1983). wasseparated from the remainderofthe section, in- Our analyses indicate thatsection Viburnum is not stead being weakly linked with the Tomentosa-Soleno- monophyletic. In ourchloropla st and the combined tinus clade(55% bootstrap support in trnK analyses). trees fourof the Žverepresentatives formed aclade Although V.urceolatum has traditionallybeen placedin 2004] DONOGHUEET AL.: VIBURNUMPHYLOGENY 193 section Viburnum based on the presence ofnak ed buds clades. In contrast,relationsh ips among the sections and stellate hairs, molecular evidencefor its exclusion were not well resolved in the ITSanalyses. Despite from this section is not surprising given its otherwise their differing levels ofresolution ,increased bootstrap strange combinationofmorpholo gicalcharacters. In supportfor two of the three supra-sectionalclades in particular,the owers of Viburnum urceolatum are the combined analyses implies thatthe nuclearand highly unusualwithin Viburnum asa whole (Hara chloroplastdatasets are largely complementary. 1983):the tubularcorolla has short erect lobes (asin Thelargest claderecovered in allof o ur analyses con- some species ofsection Megalotinus ; e.g., V.cylindricum ) tained the Odontotinus -Oreinotinus -Megalotinus grouping and is often red in color;the usually bright white sta- discussed above,along with sections Opulus and Tinus. mens are exerted from the corolla. V.urceolatum also This cladeis poorly supported in analyses ofth eindi- has adistinctive growth form,producing sympodial vidualda tasets (58%and 56%bootstra psupportfor trnK plagiotropicaxes thatresemble those found in section and ITSanalyses,respectively) butm ore strongly sup- Pseudotinus (Donoghue 1981,1982). Although V. urceo- ported in the combined analysis (86%bootstrap v alue). latum is clearly not directly linked tothe core ofsection Sucha grouping has not previously been recognized, Viburnum,additional dataare needed toestablish its butin retrospect may bediagnosed bysev eral mor- relationships more precisely. phological features.These include the presence oftwo The non-monophyly ofsection Solenotinus was not or more pairs ofbud scales (exceptin V.cylindricum expected. This group has primarily been distinguished and other species of Megalotinus ,which typically have byin orescence structure;in contrastto the umbel-like one pairof scales), leaf margins with prominentteeth inorescences ofall other Viburnum species, members in most species, and the tendency forthe Žrst twosec- of section Solenotinus producedistinctive panicle-like ondary veins tobe clustered near the baseof the leaf inorescences. These species may alsodiffer from oth- blade.Given this lastcharacter it may not besurprising er viburnums in having chromosomenumbers of x58 thatwithin this cladewe Žnd the evolution ofstrongly (asapposed to x59elsewhere in Viburnum; Egolf, tri-nerved leaves (e.g., V.cinnamomifolium and V. davidii 1956,1962) and in lacking glycosides (Norn 1978). in section Tinus)and oftri-lobed leaves (e.g., V. aceri- However, this characterneeds further investigationas folium and V.kansuense within Old World Odontotinus , the original observations relied on only asmall sample and probablyindepen dently in section Opulus). ofspecies. Wesampled six ofthe ca.25 species in this Strongly supported in allthree analyses (92%,86%, group;of these ,Žveformed awell-supported clade and 99%bootstrap support in trnK, ITS,and combined (91%bootstrap support in the combined analysis). The analyses, respectively) is acladecontaining the core of one exception is V.clemensiae from Mt.Kinabalu in Solenotinus ,with paniculatein orescences ,and V. pli- northwestern Borneo (Kern 1951;Beaman et al.2001), catum of section Tomentosa,with umbel-like inores- which despite its paniculatein orescences does not cences. Despite this difference,wenote thatm embers appearclosely related tothe remaining Solenotinus . Al- of the Solenotinus-Tomentosa cladeshare characteristi- though its exactplacement within Viburnum remains callyroun ded teeth on the leafmargins (Donoghue uncertain, this result has implications formorpholo g- and Levin 1986;except where teeth are absent,as in V. icalevoluti on in the group.Most importantly,the dis- odoratissimum )and may alsobe united bychromosom e tribution ofpaniculate in orescences becomes disjunct number.In contrastto most other viburnums, where on the tree,implying thatthis characterev olved inde- x59 (2n518,27, 36, or 72),all Solenotinus species for pendentlyin V.clemensiae and the Solenotinus clade. which chromosome countsare availablehave x58 This assumes thatumbel-lik einorescences are ances- (2n516or 32,or 40in someindividuals of V.odoratis- tral in Viburnum,aview supported bycom parison to simum).Interestingl y,for V. plicatum there are reputable Sambucus where such inorescences are alsoinferred reports ofboth 2 n516 and 2n518(J anaki Ammal tobe ancestral (Eriksson &Donoghue 1997).Further , 1953;Egolf 1956,1962). Egolf (1956)docum ented that the thick, entire leaves cited byK ern (1951)as evidenc e 2n516is the predominant chromosome number in V. ofa close relationship between V.clemensiae and V. odor- plicatum,and the only one found in most ofthe varie- atissimum (which is nested within core Solenotinus ) ties and forms ofthis species. Furthermore,he noted would best beinterprete dashaving evolved indepen- that only 2n516plants produced abundantfruit. On dently in response tosimilar ecologicalcircumstances ; this basisw esuspectthat the loss ofa chromosome bothspecies occurat low er elevations and in more sub- pairmarks the entire Solenotinus -Tomentosa clade, and tropicalforests than do most Viburnum species. thata pairof metacentri cchromosomes waslater add- Relationships Among theSections. Although some ed in some V. plicatum lineages. uncertainties remain, weŽnd supportfor three supra- Our trnK and combined analyses alsoindicate sectional clades. Itappears that m uchof this resolution strong support(91% and 94%,respectiv ely) fora clade is derived from the trnK dataset, which even when containing the New World section Lentago and the pre- analyzed alone showed supportfor each of these three dominantly Asian section Viburnum (except V. urceo- 194 SYSTEMATIC BOTANY [Volume 29 latum).Furtherm ore,in the trnK analyses there was Within this cladethe sister group relationship be- supportfor this cladebeing sister tosection Pseudotin- tween sections Viburnum and Lentago,asopposed to us (79%bootstrap v alue).Consistent with its generally one between Viburnum and Pseudotinus ,is surprising limited resolution ofhigher-l evel relationships, this re- from the standpoint ofthe evolution ofnaked buds. sult wasnot obtained in analyses ofthe ITSdata alone . Most viburnums havetwoor more pairs ofbud scales. Although the optimaltree from ourML analyses of All members ofsections Viburnum and Pseudotinus (as the combined dataset does indicate adirect relation- well as V.urceolatum ,whose relationships remain un- ship between the Lentago-Viburnum clade and Pseudo- clear;see above)have naked budsand in section Len- tinus,this cladereceived less than 50%support in the tago only asingle pairof bud scales is produced. bootstrapanalyses (Fig. 3).This wasthe only supra- Therefore,the relationships inferred among these sec- sectional relationship toreceive less supportin com- tions imply homoplasy in this character.That is, within bined analyses than in analyses ofthe trnK data alone, this cladenaked budseither evolved twice indepen- indicating adegree ofcon ict betw een the chloroplast dently,or theyevolved only once followed bythe re- and ITSdata concerning the position ofsection Pseu- evolution ofbud scales in section Lentago.The re-evo- dotinus. lution ofbud scales is not analtogether unlikely sce- In retrospect, there may bea number ofmorphol og- nario when differencesin budscale develop ment are icalfeatures thatalso unite sections Lentago, Viburnum considered. Cross (1937,1938) carried outdetailed (except V.urceolatum ), and Pseudotinus .First, in allof studies ofbud scale de velopment in V. opulus and V. these groups the inorescence is substantially pre- ruŽdulum. Viburnum opulus probablyexempliŽ es the formed atthe end ofthe growing season, and therefore standard situation in Viburnum,where two(ormore) over-winters with the owers in awell-developed state. pairs ofbud scales are produced, and commitment to This is obviousin species with naked buds,where the adistinct cataphyll developmental pathwayis evident well-formed inorescence ,densely covered bystellate ata very early stage in budscale ontogen y(Cross hairs, is exposed throughoutthe winter.Although the 1938).In marked contrast,the development ofbud budscales in section Lentago obscurethis feature,the scales in V. ruŽdulum resembles thatof normal foliage well-developed inorescence is evident asa distinct leaves, the main difference being the timing ofgrowth bulge within the bud(Donoghue 1981).Second, the in different zones. SpeciŽcally ,the budscale appears vernationofthe foliage leaves within the resting buds directly comparableto the petiole ofa leaf;growth of (ptyxis)is similar in these three sections. In these the bladeportion is suppressed during scalede velop- groups the young leafblades are involuteand non- ment butit often expands somewhatwhen growth re- overlappingwhere they comeinto contact.In contrast, sumes in the spring (Cross 1937). elsewhere in Viburnum (asin section Odontotinus and Rooting the Viburnum Phylogeny. The exactpo- related lineages) the young leaves are conduplicateand sition ofthe rootwithin Viburnum remains unresolved often plicate,with the blades variously overlapping in these analyses. However,it seems unlikely thatthe one another in bud.This contrastw ascarefully docu- rootlies within any ofthe sections or supra-sectional mented byCross (1937,1938) in his studies of V. ruŽ- clades we have identiŽed, butinstead fallssom ewhere dulum (section Lentago) and V. opulus (section Opulus); between them. One very intriguing possibility is that additional studies are needed tocritically evaluatethe the rootlies between V.clemensiae (traditionallyofsec- distributionofthis featurewithin and among the ma- tion Solenotinus ,butsee above)and the remainderof jor Viburnum lineages. Third, members ofthese three Viburnum.This placement received 57%bootstrap sup- sections may share apeculiarpollen exine morphology port in our trnK analyses (Fig. 1),and wasalso recov- (Donoghue 1985).In most viburnums the exine is ered in the optimalML topology forthe combined semitectate and reticulate,butin sections Viburnum dataset (Fig. 3;although bootstrapsupport was less and Lentago it is intectate and more or less regularly than 50%).A dditional dataare needed toe valuatethis retipilate topilate ,and the pilae are scabrate(pollen and other possible placements ofthe root. type ICofDonoghue 1985).Previousl y,this wasinter- The substantialdivergen cebetw een Viburnum and preted asconverge nce (supported in partby a differ- its nearest relatives presents acontinuing problem. ence in grain shape; Donoghue 1985),but our molec- This is particularlyobvious in ourITS alignments ,but ularresults suggest thatthese traits may instead be large numbers ofnucleotid esubstitutionsare inferred homologous.Interestingl y,asimilar condition is seen between Sambucus and Viburnum in allof our analyses. in V.cordifolium of section Pseudotinus ,although in this Adistant relationship between Viburnum and the re- species thepilae do not appearto be scabrate .Finally, mainder ofthe Adoxaceaeis alsoe vident in broader it seems likely thatthe multicellular,peltatescales analyses ofA doxaceae(Eriksson and Donoghue1997) found on the leaves and elsewhere in section Lentago and the Dipsacales (e.g.,Donoghue et al.2001; Bell et were derived from the stellate trichomescharacteristic al.2001). Our ongoing studies ofthe nuclearlocus ofspecies with naked buds. waxy (Winkworth and Donoghue 2002;unpubl. data), 2004] DONOGHUEET AL.: VIBURNUMPHYLOGENY 195

FIG.3. Singleoptimal maximumlikelihood treeobtained fromthe combineddata set, showingbootstrap values over 50% (1000 replicates). Nodes markedwith asterisks collapse inthe majorityrule consensus ofthe maximumparsimony bootstrap trees. Names tothe rightrefer to traditionally recognized sections. Names ofthe traditional sections of Viburnum are given on the right. which wehavefound tobe duplicated in Viburnum groups. Atthis level the phylogeneticsignal stems pri- and its relatives, may ultimately provide analternativ e marily from the ITSdata set. However,the datasets means ofestablishing the position ofthe root(cf. Sang appearto compleme nt one another,asthe inferred re- et al.1997; Mathews and Donoghue 2000). lationshipsoften show increased bootstrapsupport in Relationships within Sections. In addition toclar- the combined analyses. Here we briey highlight the ifying phylogenetic relationships among major clades, most strongly supported and evolutionarily signiŽcant ourresults provide someresolutionwithin these ofthese results. 196 SYSTEMATIC BOTANY [Volume 29

Within section Opulus the ITSand combined analy- Asecondfruitcolor shift is implied bythe position of ses indicate thatthe North American V. edule, which V.melanocarpum ,which, asthe name implies, alsohas lacks sterile marginal owers, is sister toa strongly dark purple fruits.A dditional shifts may bereq uired, supported cladeconsisting of V. trilobum and V. sargen- dependingon the Žnal placement of V.cylindricum and tii (and presumably alsothe European V. opulus), all of other Megalotinus species, which alsogenerally pro- which producesterile marginal owers. Within the lat- ducepurple fruits atmaturity. ter cladeour data separate the North American V. tri- The only conicting relationships suggested byour lobum from twoaccessions ofthe widespreadAsian chloroplast and nuclear datasets are within section species V.sargentii .Within section Pseudotinus the evo- Lentago.In ITSand combined analyses V.prunifolium , lution ofsterile marginal owers may bem ore com- V. ruŽdulum,and the only Mexicanrepresen tativeof plicated.Although the relationships among V. furcatum this otherwise eastern North American group, V. ela- and V.lantanoides ,bothwith sterile marginal owers, tum,formed awell supported clade(86% and 73% and V.cordifolium ,which lacks such owers, are not bootstrapsupport in ITSand combined analyses, re- well resolved by trnK or ITSdata alone ,ourcombined spectively).In contrast, trnK analyses unite the three analyses seem tosupport a direct link between V. fur- eastern North American species—V. lentago, V. pruni- catum and V.cordifolium (64%bootstrap v alue).T aken folium, and V. ruŽdulum—tothe exclusion of V. elatum atface v aluethis implies thateither sterile owers have (63%bootstrap support). Furthermo re,the trnK anal- evolved twice,or they originated once and were then yses suggest asister group relationship between V. len- lost in V.cordifolium .Geographically this result unites tago and V.prunifolium ,with 64%bootstrap support. twoAsian species (the Japanese V. furcatum and Hi- The different relationships inferred from these two malayan V.cordifolium )tothe exclusion of V.lantanoides datasets may supportthe hypothesis that V.prunifol- from eastern North America. ium originated following hybridizationbetween V. ruf- Several other results are ofbiogeograph icinterest. idulum and V. lentago (Brumbaugh and Guard1956; Viburnum lantana ,the sole European representativeof Rader 1976).Se veral other observations are compatible the otherwise Asian section Viburnum,is closely as- with ahybridization scenario. The distributionof V. sociated with the Asian V.rhytidophyllum (97% boot- prunifolium lies between those ofits putativeparents strapsupport in combined ML analyses). Wenote that (although there is signiŽcant overlap in bothdirec- these twospecies canbe readily hybridized, and were tions). SpeciŽcally , V.prunifolium is distributed across the parents ofa widely planted horticultural form, V. the middle ofthe eastern United States,with the range 3 rhytidophylloides (Egolf 1956).In anycase,this result of V. lentago extending further tothe north and thatof is consistent with anAsian radiation ofsection Vibur- V. ruŽdulum extending further south.Furthermo re,suc- num and its subsequent spread toEurope .In contrast, cessful hybrids are readily obtained between V. lentago V. tinus,the sole European member ofsection Tinus, is and V.prunifolium (Egolf 1956);in deed, this is the pre- sister toa pairof Asian species, V.cinnamomifolium and sumed origin ofthe horticultural form V. 3 jackii (Reh- V. davidii.However, in this casea proper test ofth e der 1920).A ttempts tohybridize these species in other geographic origin ofthe group awaitsthe addition of combinations have not been reported, butw eassume other Asian species, such as V.propinquum , which are thatthese would alsobe successful. Although intro- morphologically more similar to V. tinus. Within the gression in the wild has been suggested (Brumbaugh New World Odontotinus clade,we Žnd supportfor the and Guard1956), w enote thatnatural h ybridization V.raŽnesquianum species complex,which includes V. raf- may belimited bydifferences in owering times (Rad- inesquianum , V.ellipticum , and V. molle (Donoghue er 1976;Donoghue 1980).In addition, since neither of 1982). Viburnum ellipticum ,the only species native to the DNAsequences obtained for V.prunifolium is iden- the west coastof N orth America (extending south into ticalto the corresponding sequence in the putativepar- Northern California),appears to be the sister group of ents, anysuch hybridizationmusteither have predated the twoeastern North American species. sequence divergenceor unsampled variation exists As noted above,the Old World and New World within the parental species. The possibility ofhomo- Odontotinus clades generally differ with respect tofruit ploid hybrid speciation in section Lentago warrants fur- color—Old World species being predominantly red- ther investigation. fruited and New World species purple-fruited. Within ACKNOWLEDGEMENTS .Weare gratefulto Bob Cook and the the Old World Odontotinus clade,however, several re- staffof the Arnold Arboretum for their support and forallowing sults suggest the independent evolution ofpurple us tomake extensive collections on the grounds.K en Sytsmasup- fruits.The position ofthe purple-fruited V.acerifolium , ported thisresearch at theU niversityof W isconsin, and Marty linked in ourcombin ed analyses atthe baseof this Wojciechowskiand MikeSanderson assisted atthe Universityof Viburnum cladewith the red-fruited V.kansuense ,implies anin- Arizona. Lab groups at Harvard and Yale endured many discussionsand provided valuable feedback; Chuck Bell also pro- dependent origin ofpurple fruits. This is especially vided technical support. Weare gratefulto P atrickH erendeen, interesting since V.acerifolium is aNew World species. JamesSmith, Thomas J ones and an anonymous reviewerfor v alu- 2004] DONOGHUEET AL.: VIBURNUMPHYLOGENY 197 able comments.MD is grateful to John Beaman forhis guidance; logenyand phylogenetic taxonomyof Dipsacales ,withspe- especially forthe invita tionto Mt. Kinabalu and forhelping to cial referenceto Sinadoxa and Tetradoxa (Adoxaceae). Harvard collect V.clemensiae . Papers in Botany 6: 459–479. ———, R. G. OLMSTEAD, J. F. SMITH, and J. D. PALMER. 1992. Phy- logenetic relationships ofDipsacales based on rbcL sequenc- LITERATURE CITED es. Annalsof the Missouri Botanical Garden 79: 333–345. DOYLE,J.J. and J.L. D OYLE.1990. Isolationof plant DNAfrom BACKLUND, A. and B. BREMER.1997. Phylogenyofthe Asteridae freshtissue. Focus 12: 13–15. s. str.based on rbcL sequences,withparticular referenceto EGOLF,D.R. 1956. 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Phy- nal of Botany 80(6, supplement):146. logenyof the coneow ersand relatives (Heliantheae:Aster- ———, T. ERIKSSON, P. A. REEVES, and R. G. OLMSTEAD. 2001. Phy- aceae) based on nuclear rDNAinternal transcribedspacer 198 SYSTEMATIC BOTANY [Volume 29

(ITS)sequences and chloroplastDN Arestrictionsite da ta. AY265130, trnK AY265176). V.japonicum (Thunb.)Sprengel (sect. Systematic Botany 25: 539–565. Odontotinus )—Cult. US.N at. Arb.56545 (China), Donoghue,v oucher WHITE T. J., T. BRUNS, S. LEE, and J. TAYLOR.1990. AmpliŽca tion lacking (ITSA Y265131 , trnK AY265177). V. jucundum Morton(sect. and direct sequencingoffungal ribosomalRN Agenesfor Oreinotinus )—Chiapas, Mexico, Donoghue 244 (A,YU) (ITS phylogenetics. Pp 315–322 in PCR protocols:aguidetomethods AY265132, trnK AY265178). V.kansuense Batalin (sect. Odontotin- and applications, eds. M.Innis, D .Gelfand, J.Sninsky ,and T. us)—Sichuan Province,China, D.Boufford etal. 27416 (A) (ITS White.San Diego: Academic Press. AY265133, trnK AY265179). V. lantana L. (sect. Viburnum)—Cult. WILKINSON,A. M.1948. Floral anatomyand morphologyofsome Arn.Arb. 1089-60-A (Europe), D & W 26 (ITSA Y265134 , trnK species ofthe genus Viburnum ofthe Caprifoliaceae. American AY265180). V.lantanoides Michaux (sect. Pseudotinus )—Cult. Arn. Journalof Botany 35: 455–465. Arb.155-97-B (EasternN orthAmerica), D & W 2 (ITSA Y265135 , WINKWORTH,R. C. and M.J. D ONOGHUE. 2002. Viburnum phylog- trnK AY265181). V. lentago L. (sect. Lentago)—Cult. Arn.Arb. enybased on granule-bound starch synthasegenes. Botany 18021-A (EasternN orthAmerica), D & W 21 (ITSA Y265136 , trnK 2002 Abstract(h ttp://www.botany2002.org/section12/ AY265182). V.lobophyllum Graebn. (sect. Odontotinus )—Cult. Arn. abstracts/184.shtml). Arb.1875-80-A (China), D & W 25 (ITSA Y265137 , trnK AY265183). YOUNG,N.D., K. E. S TEINER, and C. W. DEPAMPHILIS. 1999. The V.melanocarpum Hsu inChen et al. (sect. Odontotinus )—Cult.Arn. evolution ofparasitism inScrophula riaceae/Orobanchaceae: Arb.386-81-D (China), D & W 12 (ITSA Y265138 , trnK AY265184). plastid gene sequences refutean evolutionarytransitionse- V. molle Michaux (sect. Odontotinus )—Cult. Arn.Arb. 18294-A ries. Annalsof the Missouri Botanical Garden 86: 876–893. (EasternN orthAmerica), D & W 5 (ITSA Y265139 , trnK AY265185). V. nudum L. (sect. Lentago)—Cult. US.N at. Arb.32198-C (Eastern NorthAmerica), Donoghue,voucher lacking (ITSA Y265140 , trnK APPENDIX 1. Accession details and GenBank numbersfor sam- AY265186). V.odoratissimum KerGa wler (sect. Solenotinus )—Cult. pled Viburnum (traditionallyrecognized section inparentheses ) Univ.ofW ashingtonArb. 1404-56 (Japan, Korea, Taiwan, Philip- and outgroup species. AllDonoghue &Winkworth(D & W)col- pines), R. Olmstead 2002-118 (WTU) (ITSA Y265141 , trnK lections are deposited atYale University(YU) and theArnold Ar- AY265187). V. plicatum var. tomentosum Thunb.(Miquel)(sect. To- boretumat Harvard University(A); loca tionsof remaining vouch- mentosa)—Cult. Arn.Arb. 72-98-A (China, Japan, Taiwan), D & W erspecimens are as noted. Ageneralizedgeographic range isgiv - 10 (ITSA Y265143 , trnK AY265189). V.prunifolium L. (sect. Lenta- en inparentheses forspecimens obtained fromcultiv ated plants. go)—Cult. Arn.Arb. 22586-A (EasternN orthAmerica), D & W 13 V.acerifolium L. (sect. Odontotinus )—Cult. Arn.Arb. 1505-67 (ITSA Y265144 , trnK AY265190). V.raŽnesquianum Schultes (sect. (EasternN orthAmerica), D & W 27 (ITSA Y265114 , trnK Odontotinus )—Cult. Arn.Arb. 17974-A (EasternN orthAmerica), AY265160). V. carlesii Hemsley(sect. Viburnum)—Cult. Arn.Arb. D & W 4 (ITSA Y265145 , trnK AY265191). V.rhytidophyllum Hem- 17981-2-A (Japan, Korea), D & W 24 (ITSA Y265115 , trnK sley (sect. Viburnum)—Cult. Arn.Arb. 133-67-A (China), D & W AY265161). V.cinnamomifolium Rehder (sect. Tinus)—Cult. Univ. 8 (ITSA Y265146 , trnK AY265192). V. ruŽdulum Raf. (sect. Lenta- ofW ashingtonArb. 1392-56 (China), R. Olmstead 2002-120 (WTU) go)—Cult. Arn.Arb. 21418-A (EasternN orthAmerica), D & W 14 (ITSA Y265116 , trnK AY265162). V.clemensiae Kern (sect. Solenotin- (ITSA Y265147 , trnK AY265193). V.sargentii Koehne (sect. Opu- us)—Mt.Kinabalu ,Malaysia, J.Beaman 11781 (K) (ITSA Y265117 , lus)—1:Cult. Arn.Arb. 398-68-B (China, Japan, Korea, Siberia), D trnK AY265163). V.cordifolium Wallich ex DC. (sect. Pseudotinus )— & W 17 (ITSA Y265148 , trnK AY265194); 2:Cult. Arn.Arb. 719-88- Sichuan Province,China, D.Boufford etal. 27388 (A) (ITSA Y265118 , C, D & W 32 (ITSA Y265142 , trnK AY265188). V.sieboldii Miq. trnK AY265164). V.cylindricum Ham. ex D.Don (sect. Megalotin- (sect. Solenotinus )—Cult. Arn.Arb. 616-6-A (Japan), D & W 3 (ITS us)—Yunnan Province,China, D.Boufford etal. 29342 (A) (ITS AY265149, trnK AY265195). V.stenocalyx (Oersted)H emsley(sect. AY265119, trnK AY265165). V. davidii Franchet (sect. Tinus)—Cult. Oreinotinus )—DF ,Mexico, Donoghue127 (A,YU) (ITSA Y265150 , vic. Corvallis, OR, USA(China), Donoghue,v oucherlacking (ITS trnK AY265196). V.suspensum Lindley(sect. Solenotinus )—Cult. AY265120, trnK AY265166). V. dentatum L. (sect. Odontotinus )— Santa Barbara, CA(Japan), D & W 36 (ITSA Y265151 , trnK Cult. Arn.Arb. 5070-1-A (EasternN orthAmerica), D & W 33 (ITS AY265197). V. tinus L. (sect. Tinus)—Cult. Santa Barbara, CA(Eu- AY265121, trnK AY265167). V.dilatatum Thunb. (sect. Odontotin- rope), D & W 35 (ITSA Y265152 , trnK AY265198). V. trilobum Mar- us)—Cult. Arn.Arb. 138-52-A (China, Japan, Korea), D & W 19 shall (sect. Opulus)—Cult. Arn.Arb. 724-68-E (NorthAmerica), D (ITSA Y265122 , trnK AY265168). V. edule (Michaux)Raf. (sect. Opu- & W 22 (ITSA Y265153 , trnK AY265199). V.triphyllum Benth. (sect. lus)—NorthernW isconsin, USA, W.Alverson, voucher lacking (ITS Oreinotinus )—Loja, Ecuador, C. Bell EC-026 (YU) (ITSA Y265154 , AY265123, trnK AY265169). V. elatum Bentham (sect. Lentago)— trnK AY265200). V.urceolatum Sieb.& Zucc. (sect. Viburnum)— Chiapas, Mexico, Donoghue472 (A,YU) (ITSA Y265124 , trnK Cult. US.N at. Arb.40200 (China, Japan, Taiwan), Donoghue, voucher AY265170). V.ellipticum Hooker(sect. Odontotinus )—vic. Corvallis, lacking (ITSA Y265155 , trnK AY265201). V. utile Hemsley(sect. Vi- OR, USA, Donoghue,voucher lacking (ITSA Y265125 , trnK AY265171). burnum)—Cult. US.N at. Arb.(China), Egolf 2336-E (ITSA Y265156 , V. erosum Thunb. (sect. Odontotinus )—Cult. Arn.Arb. 78-90-A trnK AY265202). (China, Japan, Korea), D & W 16 (ITSA Y265126 , trnK AY265172). V.erubescens Wallichex DC. (sect. Solenotinus )—Sichuan Province, Outgroups China, D.Boufford etal. 27190 (A) (ITSA Y265127 , trnK AY265173). V. farreri Stearn(sect. Solenotinus )—Cult. Arn.Arb. 656-89-A (Chi- Sambucus canadensis L.—Cult. MarshBot. Gard., Yale Univ. na), D & W 18 (ITSA Y265128 , trnK AY265174). V. furcatum Blume (NorthAmerica, Mexico, Central America), D & W 37 (ITS (sect. Pseudotinus )—Kyoto Prefecture,Japan, Tsugaru &Takahashi AY265157, trnK AY265203). Sambucus racemosa L.—vic. Stock- 19958 (MO) (ITSA Y265129 , trnK AY265175). V.hartwegii Bentham holm,Sweden (circumboreal), T.Eriksson s.n. (S) (ITSA Y265158 , (sect. Oreinotinus )—Chiapas, Mexico, Donoghue486 (A,YU) (ITS trnK AY265204).