Phylogenetic Analyses of Basal Angiosperms Based on Nine Plastid, Mitochondrial, and Nuclear Genes Citation Qiu, Yin-Long, Olena Dombrovska, Jungho Lee, Libo Li, Barbara A. Whitlock, Fabiana Bernasconi-Quadroni, Joshua S. Rest, et al. 2005. Phylogenetic analyses of basal angiosperms based on nine plastid, mitochondrial, and nuclear genes. International Journal of Plant Sciences 166(5): 815-842. Published Version http://dx.doi.org/10.1086/431800 Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:2710479 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Int.J.PlantSci. 166(5):815–842.2005. (cid:1)2005byTheUniversityofChicago.Allrightsreserved. 1058-5893/2005/16605-0012$15.00 PHYLOGENETIC ANALYSES OF BASAL ANGIOSPERMS BASED ON NINE PLASTID, MITOCHONDRIAL, AND NUCLEAR GENES Yin-LongQiu,1;*,y,zOlenaDombrovska,*,y,zJunghoLee,2;y,zLiboLi,*,yBarbaraA.Whitlock,3;yFabianaBernasconi-Quadroni,z JoshuaS.Rest,4;*Charles C. Davis,* ThomasBorsch,§Khidir W.Hilu,kSusanne S.Renner,5;#DouglasE. Soltis,** PamelaS.Soltis,yy MichaelJ. Zanis,6;zz JamieJ. Cannone,§§RobinR. Gutell,§§Martyn Powell,kk VincentSavolainen,kkLars W.Chatrou,##and MarkW.Chasekk *DepartmentofEcologyandEvolutionaryBiology,UniversityHerbarium,UniversityofMichigan,AnnArbor,Michigan48109-1048,U.S.A.; yBiologyDepartment,UniversityofMassachusetts,Amherst,Massachusetts01003-5810,U.S.A.;zInstituteofSystematicBotany,University ofZurich,8008Zurich,Switzerland;§Nees-Institutfu¨rBiodiversita¨tderPflanzen,Friedrich-Wilhelms-Universita¨tBonn,Meckenheimer Allee170,53115Bonn,Germany;kBiologyDepartment,VirginiaPolytechnicInstituteandStateUniversity,Blacksburg,Virginia 24061,U.S.A.;#DepartmentofBiology,UniversityofMissouri,St.Louis,Missouri63121-4499,U.S.A.;**Departmentof BotanyandtheGeneticsInstitute,UniversityofFlorida,Gainesville,Florida32611,U.S.A.;yyFloridaMuseumof NaturalHistoryandtheGeneticsInstitute,UniversityofFlorida,Gainesville,Florida32611,U.S.A.;zzSchool ofBiologicalSciences,WashingtonStateUniversity,Pullman,Washington99164,U.S.A.;§§Institutefor CellularandMolecularBiology,andSectionofIntegrativeBiology,UniversityofTexas,Austin,Texas 78712,U.S.A.;kkRoyalBotanicGardens,Kew,Richmond,SurreyTW93DS,UnitedKingdom; and##NationalHerbariumoftheNetherlands,UtrechtUniversity,Heidelberglaan2, 3584CSUtrecht,TheNetherlands DNA sequences of nine genes (plastid: atpB, matK, and rbcL; mitochondrial: atp1, matR, mtSSU, and mtLSU;nuclear:18Sand26SrDNAs)from100speciesofbasalangiospermsandgymnospermswereanalyzed using parsimony, Bayesian, and maximum likelihood methods. All of these analyses support the following consensusofrelationshipsamongbasalangiosperms.First,Amborella,Nymphaeaceae, andAustrobaileyales arestronglysupportedasabasalgradeintheangiospermphylogeny,witheitherAmborellaorAmborellaand Nymphaealesassistertoallotherangiosperms.Anexaminationofnucleotidesubstitutionpatternsofallnine genesruledoutanypossibilityofanalyticalartifactsbecauseofRNAeditingandGC-contentbiasinplacing thesetaxaatthebaseoftheangiospermphylogeny.Second,MagnolialesaresistertoLauralesandPiperales are sister to Canellales. These four orders together constitute the magnoliid clade. Finally, the relationships amongCeratophyllum,Chloranthaceae,monocots,magnoliids,andeudicotsareresolvedindifferentwaysin various analyses, mostly with low support. Our study indicates caution in total evidence approaches in that someofthegenesemployed(e.g.,mtSSU,mtLSU,andnuclear26SrDNA)addedsignalthatconflictedwiththe othergenes inresolving certainparts of thephylogenetic tree. Keywords: basal angiosperms, Amborella, magnoliids, multigene analysis, synapomorphic substitutions, phylogeny. Introduction and Stevenson 1991; Martin and Dowd 1991; Hamby and Zimmer 1992; Taylor and Hickey 1992; Chase et al. 1993; The past 20 years have witnessed significant progress in Qiu et al. 1993, 1999, 2000, 2001; Soltis et al. 1997, 2000; our understanding of the phylogeny of basal angiosperms Nandietal.1998;Hootetal.1999;MathewsandDonoghue fromanalysesofmolecularandnonmoleculardata(Dahlgren 1999,2000;Parkinsonetal.1999;Renner1999;Soltisetal. and Bremer 1985; Donoghue and Doyle 1989; Loconte 1999a; Barkman et al. 2000; Doyle and Endress 2000; Gra- ham and Olmstead 2000b; Savolainen et al. 2000; Nickrent etal.2002;Zanisetal.2002,2003;Borschetal.2003;Hilu 1Authorforcorrespondence;[email protected]. et al. 2003; Lo¨hne and Borsch 2005). Specifically, it has be- 2Current address: School of Biological Sciences, Seoul National come increasingly clear that Amborella, Nymphaeaceae, and University,Shillim,Kwanak,Seoul,SouthKorea151-747. 3Current address: Department of Biology, University of Miami, Austrobaileyales (sensu APG II 2003) represent the earliest- Miami,Florida33124-0421,U.S.A. diverging lineages of extant angiosperms. Furthermore, the 4Current address: Department of Ecology and Evolution, magnoliids (sensu APG II 2003; see Qiu et al. 1993 for a re- UniversityofChicago,Chicago,Illinois60637,U.S.A. view of the history of this term) have been identified as a 5Current address: Department of Biology, Ludwig Maximilians monophyleticgroupinsomeanalyses(Qiuetal.1999,2000; UniversityMunich,Munich,Germany. Zanis et al. 2002, 2003; Hilu et al. 2003), but their mono- 6Current address: Division of Biological Sciences, University of phyly (Savolainen et al. 2000; Soltis et al. 2000) and espe- California,SanDiego,LaJolla,California92093,U.S.A. ciallyrelationshipsamongtheirmemberorders(Magnoliales, ManuscriptreceivedNovember2004;revisedmanuscriptreceivedMay2005. Laurales, Piperales, and Canellales) need further evaluation 815 816 INTERNATIONAL JOURNAL OF PLANTSCIENCES andresolution.Finally,allangiospermsexcludingAmborella, quences were generated by us for mtSSU, matK, and nuclear Nymphaeaceae,andAustrobaileyalescanbedividedintofive 26S rDNA. For the five genes used in Qiu et al. (1999), sev- clades: Ceratophyllum, Chloranthaceae, magnoliids, mono- eral new sequences were produced to fill the missing entries cots,andeudicots(tricolpatessensuJuddandOlmstead2004; inthatmatrix.Theorthologousatp1wasusedtoreplacethe see also Walker and Doyle 1975; Crane 1989; Donoghue copy we obtained earlier from Amborella (Qiu et al. 1999, and Doyle 1989; Doyle and Hotton 1991; Chase et al. 2000), which has been shown to be a xenolog horizontally 1993). Relationships among these five lineages, however, are transferred from an asterid (Barkman et al. 2000; Bergthors- best interpreted as unresolved at present because analyses son et al. 2003). For all nine genes we have taken sequences withdifferenttaxonandcharacter-samplingschemesandphy- from GenBank when appropriate. Detailed source informa- logenetic methods have produced conflicting topologies that tion for all sequences and correction to errors in table A1 of are generally only weakly supported (Barkman et al. 2000; Qiu et al. (2000) are provided in tables 1 and 2. Of all taxa Soltisetal.2000;Zanisetal.2002,2003;Hiluetal.2003). andallgenes,onlyfourtaxahavemissingdatainoneortwo Despiteprogress,moreworkisneededtofurtherclarifyre- genes: Metasequoia (mtSSU and matR), Hortonia (matR), lationships among basal angiosperms. In this study, we add and Dioscorea and Myristica (nu26S) (tables 1, 2). Eight of sequencedataoffournewgenestoafive-genematrixassem- the nine genes (all except mtSSU) were aligned using Clustal bled earlier (Qiu et al. 1999, 2000) and conduct parsimony, X (Thompson et al. 1997). Because of extraordinary length Bayesian, and maximumlikelihood (ML) analyses to address variationinseveralregionsofmtSSU,thisgenewasmanually several issues. First, we attempt to show that placement of aligned with the alignment editor AE2 (developed by T. Amborella, Nymphaeaceae, and Austrobaileyales at the base Macke; Larsen et al. 1993). Although these regions typically of angiosperm phylogeny is free of any analytical artifact. hadminimalsequenceidentitythatcouldnotbealignedbased This is especially important in light of recent analyses of the on sequence alone, they usually had similar structural ele- entire plastid genome sequences of Amborella and Nym- ments that facilitated the alignment of these sequences. In phaea that do not support them as basalmost angiosperms addition,allofthe computer-generatedalignmentswereman- (Goremykin et al. 2003a, 2003b, 2004; but see Soltis and ually adjusted with the MacClade 4.05 (Maddison and Mad- Soltis 2004; Soltis et al. 2004; Stefanovic et al. 2004). Sec- dison 2002) alignment editor. All of the aligned positions ond,weaimtoevaluatethemonophylyofmagnoliidsandto were used in the phylogenetic analyses. We also eliminated resolve the relationships among their members: Magnoliales, the positions in regions with significant length variations in Laurales,Piperales,Canellales.Finally,wewishtoresolvere- the four rDNAs from the phylogenetic analyses of the nine- lationships among Ceratophyllum, Chloranthaceae, magno- gene matrix.These latteranalysesyielded results not substan- liids,monocots,andeudicots. tiallydifferentfromthosepresentedhere(datanotshown). Threeseriesofanalyseswereperformedtoaddressvarious issues. First, two separate matrices were assembled to recon- Material and Methods struct the overall phylogeny of basal angiosperms, one con- sisting of all nine genes and the other of five protein-coding We included 100 terminals from 98 genera, representing genes. The decision to make a separate matrix using the five all major lineages of gymnosperms and basal angiosperms. protein-coding genes was based on the following considera- Acorus and Ceratophyllum were the only two genera for tions:(1)allpositionswithintheproteingenes shouldevolve which two species each were sampled. Only two families of more independently than those of rDNAs, many of which basal angiosperms were not included, Gomortegaceae (Ren- evolve in a coupled fashion due to base pairing in stem re- ner 1999) and Hydnoraceae (Nickrent et al. 2002), because gions in these genes (Soltis and Soltis 1998; Soltis et al. of many missing data entries. Most of the terminals consist 1999b; O. Dombrovska and Y.-L. Qiu, unpublished data); ofsequencesderivedfromasinglespecies(andfrequentlythe (2) the protein-coding genes generally show fewer problems same DNA sample) and occasionally from different species of paralogy and xenology compared to nuclear 18S and 26S of the same genus (tables 1, 2). Eight gymnosperms covering rDNAs, for which nonorthologous copies were occasionally allfourextantlineageswereusedasoutgroups. encountered; and (3) the protein-coding genes are free of The four new genes added in this study are: plastid matK alignment uncertainties compared to two mitochondrial (a group II intron-encoded maturase), mitochondrial SSU rDNAs,whichexhibitextraordinarylengthvariationscaused (small subunit) and LSU (large subunit) rDNAs, and nuclear by insertions and deletions in a few regions. The parsimony, 26SrDNA.Withthefivegenesfromourearlieranalyses(mi- Bayesian, and maximum likelihood (ML) analyses were tochondrial atp1 and matR, plastid atpB and rbcL, and nu- implementedseparatelyonbothmatrices.Toevaluatethein- clear 18S rDNA), the total of nine genes used in this study formativeness of the two nuclear rDNAs further, the five- represents a sampling of a large number of characters from protein-gene matrixwascombined with18Sand26SrDNAs eachofthethreeplantgenomes.Furthermore,thesegenesen- sequentially to form two more matrices. Only parsimony compass diverse functions, including energy metabolism, car- bootstrapanalyseswereconductedonthesetwomatrices. bohydratesynthesis,RNAprocessing,andproteinsynthesis. Second, three separate genome-specific matrices were con- DNA extraction and sequencing methods follow Qiu etal. structed to address whether placement of Amborella, Nym- (2000). All primer sequences used for amplifying and se- phaeaceae, and Austrobaileyales as sisters to all other extant quencing the genes are available from the corresponding au- angiospermsissupportedbydatafromtheplastid,mitochon- thor on request. All sequences of mtLSU were newly drial, and nuclear genomes separately. This type of analy- generatedinthisstudy,whereasapproximatelyhalfofthese- sis has only been conducted occasionally (Mathews and QIU ETAL.—BASALANGIOSPERM PHYLOGENY 817 Donoghue 1999, 2000; Graham and Olmstead 2000b; Savo- for the matrix of five protein genes plus two nuclear rDNAs lainenetal.2000;Zanisetal.2002).Arobustunderstanding where 5000 resampling replicates were used) with the same of organismal phylogeny should be based on evidence from tree search procedure as described above except with simple each of the three plant genomes (Qiu and Palmer 1999) ex- taxonadditionandthesteepestdescentoptionon. cept in cases of hybridization and horizontal gene transfer. For Bayesian and ML analyses, the optimal models of se- Only parsimony bootstrap analyses were conducted on these quenceevolutionforthenine-geneandfive-protein-genedata datasets. sets were estimated using ModelTest 3.6 (Posada and Cran- Third, we investigated the types of substitutions that pro- dall 1998) and DT-ModSel (Minin et al. 2003). The general vided phylogenetic signal for identifying Amborella, Nym- time-reversible model (Rodrı´guez et al. 1990) including pa- phaeaceae, and Austrobaileyales as the earliest-diverging rameters for invariant sites and rate variation (GTRþIþG) lineages of extant angiosperms. For an issue as critical as bestfitsbothdatasetsandwasusedtoconducttheanalyses. the rooting of angiosperm phylogeny, merely having high Bayesian analyses were performed using MrBayes version bootstrap numbers from an analysis is not enough to gain 3.0b4 (Huelsenbeck and Ronquist 2001). For the nine-gene confidence in the result (Soltis et al. 2004). Some poorly un- matrix,thedatawerepartitionedaccordingtocodonpositions derstood molecular evolutionary phenomena, such as RNA (first, second, and third, for protein genes only), genomes editing (Steinhauser et al. 1999; Kugita et al. 2003; (plastid, mitochondrial, and nuclear), and gene types within DombrovskaandQiu2004)andGC-contentbias(Steeletal. a genome (rRNAvs. protein genes). For the five-protein-gene 1993), both of which can occur in a genome-wide, lineage- matrix,thedatawerepartitionedaccordingtocodonpositions specific fashion, can generate substitutions that lead to spuri- and genomes. Calculations of likelihood for searches of both ousgroupingsinphylogeneticanalyses.Hence,itisimportant matrices were implemented under the GTRþIþG model of that we understand the types of substitutions that are behind sequence evolution, assuming different stationary nucleotide those high bootstrap percentages. We examined the nine-gene frequencies. The posterior probability (PP) was estimated by matrixvisuallyandidentifiedthesitesthat containapparently samplingtrees fromthe PPdistributionusingMetropolis cou- synapomorphicchangesthatseparategymnosperms-Amborella- pled Markov Chain Monte Carlo methods. Two and four Nymphaeaceae-Austrobaileyales from all other angiosperms. chains of 5,000,000 generations were run for the nine-gene Siteswereclassifiedasapparentlysynapomorphiciftheycon- matrixand five-protein-genematrix, respectively. Chains were tainedthesamenucleotideinatleasttwoofthefourgymno- sampled every 100 generations. Likelihood scores converged sperm lineages (cycads, Ginkgo, conifer II [non-Pinaceae onastablevalueafter500,000generations(theburn-inofthe conifers], and GnetalesþPinaceae; Bowe et al. 2000; Chaw chain),andcalculationsofPPwerebasedonthetreessampled et al. 2000) and at least two of the three basal angiosperm afterthisgeneration. lineages (Amborella, Nymphaeaceae, and Austrobaileyales) Maximum likelihood analyses were performed separately but had a different and generally invariable nucleotide in all on the nine-gene and five-protein-gene data sets using other angiosperms (hence a synapomorphy for euangio- PHYML version 2.4.4 (Guindon and Gascuel 2003) under sperms, sensu Qiu et al. 1999). We then performed both a the optimal model of sequence evolution. For both data sets, most parsimonious tree search and a parsimony bootstrap the GTRþIþG model was implemented with parameter analysis with these sites removed to verify our identification. valuesfortheproportionofinvariantsites(nine-gene¼0:19, These synapomorphic substitutions were finally checked to five-gene-protein¼0:21) and the gamma distribution (nine- determine if they could have been generated by RNA editing gene¼0:43, five-gene-protein¼0:68) as estimated by or GC-content bias. In addition, codon position and type of ModelTest 3.6 and DT-ModSel. The optimal rate of nucleo- change(transitionvs.transversion) werenotedfor thesesub- tide substitution and transition/transversion ratios was esti- stitutions. mated from the data during ML searches. Maximum These last two series of analyses were designed to comple- likelihood support values were similarly estimated from 100 ment the analyses we performed earlier (Qiu et al. 1999, bootstrapreplicatesinPHYML. 2000, 2001), to ensure that the placement of Amborella, Nymphaeaceae, and Austrobaileyales as basal lineages is in- deedbasedonhistoricalsignalrecordedinthemultiplegenes Results from all three plant genomes rather than the result of yet poorly understood analytical artifacts. These analyses are Forthenine-genedataset,whichcontained26,990aligned particularlyrelevantintheongoingdebateoverwhetherAm- nucleotides, two islands with two and four shortest trees borella and Nymphaea are basal angiosperms (Goremykin (length¼51;834 steps; consistency index ½CI(cid:1)¼0:47; reten- etal.2003a,2003b,2004;Soltisetal.2004;SoltisandSoltis tion index ½RI(cid:1)¼0:57) were found 259 and 315 times, re- 2004;Stefanovicetal.2004). spectively, out of 1000 random taxon-addition replicates in Inparsimonysearchesweusedequalweightingforallposi- the parsimony search. One of the six trees is shown (fig. 1), tions and character-state changes using PAUP* 4.0b10 (Swof- with the nodes that are not present in the strict consensus of ford 1998). When searching for the shortest trees, a heuristic allsixtreesindicatedbyasterisks. searchwasconductedusing1000randomtaxon-additionrep- For the five-protein-gene data set, which contained 9351 licates, one tree held at each step during stepwise addition, aligned nucleotides, a single island of two shortest trees TBR branch swapping, steepest descent option off, MulTrees (length¼18;839 steps; CI¼0:42; RI¼0:59) was found in option on, and no upper limit of MaxTrees. For bootstrap all 1000 random taxon-addition replicates in the parsimony analyses, 1000 resampling replicates were performed (except search.Oneofthetwotreesisshown(fig.2),withthenodes Table 1 Vouchers,Contributors,GenBankAccessionNumbers,andReferencesfortheSequencesUsedinThisStudy Familyandspecies mt-SSUrDNA mt-LSUrDNA cp-matK nu-26SrDNA Acoraceae: AcoruscalamusL. Parkinsonetal.1999; Qiu94052;OD/FBQ/YQ FuseandTamura2000; Qiu94052;OD/YQ; AF193976 DQ008817 AB040154 DQ008654 AcorusgramineusSoland. Qiu97131;JL/LL/YQ; Qiu97131;OD/FBQ/YQ; FuseandTamura2000; Kuzoffetal.1998;AF036490 DQ008668 DQ008818 AB040155 Alismataceae: Alismaplantago-aquaticaL. Qiu96177;JL/LL/YQ; Qiu96177;OD/FBQ/YQ; Qiu96177;LL/YQ; DQ008669 DQ008812 DQ008651 AlismacanaliculatumA.Br.&Bouche´ FuseandTamura2000; AB040179 Amborellaceae: AmborellatrichopodaBaill. Parkinsonetal.1999; B.Hallsn,IND,Qiu97123*; Hiluetal.2003;B.Hallsn, Zanisetal.2003;AY095449 AF193987 OD/FBQ/YQDQ008832 BONNAF543721(TB) Annonaceae: AnnonamuricataL. Qiu90031;JL/LL/YQ; Qiu90031;OD/FBQ/YQ; K.W.Hilu,K.Mu¨ller,and Sun&An98130,KUN; DQ008670 DQ008783 T.Borsch,unpublished OD/YQ;DQ008634 manuscript;Borsch3460, BONNAF543722(KH) Canangaodorata(Lam.)Hook.f.&Thomson Chase219,NCU;JL/LL/YQ; Chase219,NCU;OD/FBQ/ Borsch&Lo¨hne3555,BONN Chase219,NCU;OD/YQ; DQ008671 YQ;DQ008784 AY437817 DQ008635 Araceae: 8 1 OrontiumaquaticumL. Qiu97112;JL/LL/YQ; Qiu97112;OD/FBQ/YQ; Hiluetal.2003;Borsch3457, Qiu97112;OD/YQ; 8 DQ008672 DQ008813 BONNAF543744(KH) DQ008652 Spathiphyllum3‘Clevelandii’ Qiu94140;JL/LL/YQ; Qiu94140;OD/FBQ/YQ; DQ008673 DQ008814 Spathiphyllumfloribundum(Lind.&Andre)N.E.Br. Hiluetal.2003;Borsch3408 BONNAF542575 SpathiphyllumwallisiiHort. Zanisetal.2003;AY095473 Aristolochiaceae: AristolochiamacrophyllaLam. Qiu91019;JL/LL/YQ; Qiu91019;OD/FBQ/YQ; Zanisetal.2003;AY095450 DQ008674 DQ008796 AristolochiaPistolochiaL. Hiluetal.2003;Borsch3257, FRAF543724(TB) AsarumcanadenseL. Kuhlmansn,IND,Qiu Kuhlmansn,IND,Qiu Kuhlmansn,IND,Qiu 96018*;JL/LL/YQ; 96018*;OD/FBQ/YQ; 96018*;OD/YQ; DQ008676 DQ008799 DQ008643 AsarumyakusimenseMasam. Hiluetal.2003;Hubersn, BONNAF542571(TB) SarumahenryiOliv. Qiu91018;JL/LL/YQ; Qiu91018;OD/FBQ/YQ; Murataetal.2001;AB060736 Qiu91018;OD/YQ; DQ008677 DQ008800 DQ008644 ThotteatomentosaDingHou Chase1211,K;JL/LL/YQ; Chase1211,K;OD/FBQ/YQ; Murataetal.2001;AB060738 Chase1211,K;OD/YQ; DQ008675 DQ008797 DQ008642 Asparagaceae: AsparagusofficinalisL. Qiu94063;JL/LL/YQ; Qiu94063;OD/FBQ/YQ; Qiu94063;OD/YQ; DQ008678 DQ008807 DQ008646 Asparagusfilicinus J.Yamashitaetal.,unpublished data;AB029805 Atherospermataceae: AtherospermamoschatumLabill. Qiu92007;JL/LL/YQ; Qiu92007;OD/FBQ/YQ; Qiu92007;Kew;AJ966790 Qiu92007;LL/YQ; DQ008679 DQ008775 DQ008628 Daphnandramicrantha(Tul.)Benth. Whalen132,NSW;JL/LL/YQ; Whalen132,NSW;OD/FBQ/ Whalen132,NSW;Kew; Whalen132,NSW;OD/YQ; DQ008680 YQ;DQ008776 AJ966791 DQ008629 DoryphorasassafrasEndl. Qiu98109;JL/LL/YQ; Qiu98109;OD/FBQ/YQ; Hiluetal.2003;Borsch3409, Qiu98109;OD/YQ; DQ008681 DQ008777 BONNAF542568(TB) DQ008630 Austrobaileyaceae: AustrobaileyascandensC.T.White Parkinsonetal.1999; Qiu90030;OD/FBQ/YQ; Hiluetal.2003;Borsch3464 Zanisetal.2003;AY095452 AF193988 DQ008827 BONNAF543726(TB) Berberidaceae: Mahoniabealei(Fortune)Carr. Qiu74;JL/LL/YQ;DQ008682 Qiu74;OD/FBQ/YQ; Qiu74;OD/YQ;DQ008613 DQ008754 Mahoniajaponica Hiluetal.2003;Borsch3405, BONNAF542585(TB) PodophyllumpeltatumL. Qiu92003;JL/LL/YQ; Qiu92003;OD/FBQ/YQ; Hiluetal.2003;Borsch3393, Qiu92003;OD/YQ; DQ008683 DQ008755 BONNAF542586(TB) DQ008614 Buxaceae: BuxussempervirensL. Qiu97057;OD/FBQ/YQ; Qiu97057;Kew;AJ966792 S.Kimetal.,unpublisheddata; DQ008743 AF389243 Buxussp. Parkinsonetal.1999;AF193996 PachysandraprocumbensMichx. Chase207,NCU;JL/LL/YQ; Chase207,NCU;OD/FBQ/ Qiusn,Z(QiuL99028*); DQ008684 YQ;DQ008742 OD/YQ;DQ008607 PachysandraterminalisSieb.&Zucc. Hiluetal.2003;Borsch3407, 8 BONNAF542581(KH) 1 9 Cabombaceae: BraseniaschreberiJ.Gmelin Qiu91031;JL/LL/YQ; Qiu91031;OD/FBQ/YQ; Lesetal.1999;AF092973 Qiu91031;LL/YQ; DQ008685 DQ008830 DQ008661 Cabombasp. Parkinsonetal.1999; Qiu97027;OD/FBQ/YQ; AF193982; DQ008831 CabombacarolinianaA.Gray Lesetal.1999;AF108719 Soltisetal.2003;AF479239 Calycanthaceae: CalycanthusfloridusL. Parkinsonetal.1999;AF193989 Qiu94155;OD/FBQ/YQ; K.W.Hilu,K.Mu¨ller,and DQ008780 T.Borsch,unpublished manuscript;Borsch3455 BONNAF543730(KH) CalycanthusoccidentalisHook.&Arn. Zanisetal.2003;AY095454 Chimonanthuspraecox(L.)Link Qiu9;JL/LL/YQ;DQ008686 Qiu9;OD/FBQ/YQ; Hiluetal.2003;Borsch3396, Qiu9;OD/YQ;DQ008632 DQ008781 BONNAF542569(TB) Canellaceae: Canellawinterana(L.)Gaertn. Qiu90017;JL/LL/YQ; Qiu90017;OD/FBQ/YQ; K.W.Hilu,K.Mu¨ller,and Zanisetal.2003;AY095455 DQ008687 DQ008804 T.Borsch,unpublished manuscript;Borsch3466 BONNAF543731(TB) CinnamodendronekmaniiSleum. Zanoni&Jimenez47067; Zanoni&Jimenez47067; Zanoni&Jimenez47067; Zanoni&Jimenez47067; JL/LL/YQ;DQ008688 OD/FBQ/YQ;DQ008805 Kew;AJ966793 MZ/DES/PSSAY095458 Table 1 (Continued) Familyandspecies mt-SSUrDNA mt-LSUrDNA cp-matK nu-26SrDNA Ceratophyllaceae: CeratophyllumdemersumL. Parkssn,IND,Qiu95003*; Parkssn,IND,Qiu95003*; Parkssn,IND,Qiu95003*; Zanisetal.2003;AY095456 JL/LL/YQ;DQ008689 OD/FBQ/YQ;DQ008766 Kew;AJ966794 CeratophyllumsubmersumL. Qiu98088;JL/LL/YQ; Qiu98088;OD/FBQ/YQ; Qiu98088;Kew;AJ581400 Qiu98088;OD/YQ; DQ008690 DQ008767 DQ008622 Chloranthaceae: AscarinarubricaulisSolms Thien500,NO;JL/LL/YQ; Thien500,NO;OD/FBQ/YQ; Thien500,NOKew; Thien500,NO;OD/YQ; DQ008691 DQ008821 AJ966795 DQ008656 ChloranthusbrachystachysBl. Hiluetal.2003;Borsch3467 BONNAF543733(KH) ChloranthusmultistachysPei K.Wurdack92-0010,NCU; K.Wurdack92-0010,NCU; Zanisetal.2003;AY095457 JL/LL/YQ;DQ008692 OD/FBQ/YQ;DQ008819 HedyosmumarborescensSw. Chase338,NCU;JL/LL/YQ; Chase338,NCU;OD/FBQ/ Chase338,NCU;Kew; DQ008693 YQ;DQ008822 AJ581402 HedyosmumbonplandianumL. Zanisetal.2003;AY095461 SarcandrachloranthoidesGardner Parkinsonetal.1999; Qiu92002;OD/FBQ/YQ; Qiu92002;Kew;AJ966796 Qiu92002;OD/YQ; AF193992 DQ008820 DQ008655 Cycadaceae: CycasrevolutaThunb. Chawetal.2000;AB029356 Qiu94051;OD/FBQ/YQ; Qiu94051;OD/YQ; 8 DQ008840 DQ008667 2 0 Cycaspanzhihuaensis Wangetal.2000;AF143440 Cyclanthaceae: CarludovicapalmataRuiz&Pavon Qiu97021;JL/LL/YQ; Qiu97021;OD/FBQ/YQ; Hiluetal.2003;Rothsn, Qiu97021;OD/YQ; DQ008694 DQ008809 BONNAF542578(TB) DQ008648 Degeneriaceae: Degeneriavitensis JMMiller1189-63;JL/LL/YQ; JMMiller1189-63;OD/FBQ/ Azumaetal.,unpublished JMMiller1189-63;OD/YQ; DQ008695 YQ;DQ008787 data;AB055549 DQ008637 Didymelaceae: DidymelesperrieriOlivier Andrianantoanina387,MO; Andrianantoanina387,MO; Andrianantoanina387,MO; Andrianantoanina387,MO; JL/LL/YQ;DQ008696 OD/FBQ/YQ;DQ008744 Kew;AJ581406 OD/YQ;DQ008608 Dioscoreaceae: Dioscoreasp. Qiu94044;JL/LL/YQ; Qiu94044;OD/FBQ/YQ; DQ008697 DQ008806 Dioscoreaalata FuseandTamura2000; AB040208 Eupomatiaceae: EupomatiabennettiiF.Muell. Qiu90022;JL/LL/YQ; Qiu90022;OD/FBQ/YQ; Qiu90022;Kew;AJ966797 Qiu90022;OD/YQ; DQ008698 DQ008785 DQ008636 Eupteleaceae: EupteleapolyandraSieb.&Zucc. Parkssn,IND,Qiu95098*; Parkssn,IND,Qiu95098*; Parkssn,IND,Qiu95098*; Zanisetal.2003;AF389249 JL/LL/YQ;DQ008699 OD/FBQ/YQ;DQ008763 Kew;AJ581413 Fumariaceae: Dicentrasp. Qiu95026;JL/LL/YQ; Qiu95026;OD/FBQ/YQ; Qiu95026;Kew;AJ966798 DQ008700 DQ008764 DicentraeximaTorrey Zanisetal.2003;AF389262 Ginkgoaceae: GinkgobilobaL. Chawetal.2000;AB029355 Qiu94015;OD/FBQ/YQ; Hiluetal.2003;Borsch3469 Qiu94015;OD/YQ; DQ008838 BONNAF543736(KH) DQ008665 Gnetaceae: GnetumgnemonL. Qiu97141;JL/LL/YQ; Qiu97141;OD/FBQ/YQ; Hiluetal.2003;Borsch3470 Kuzoffetal.1998;AF036488 DQ008701 DQ008833 BONNAF542561(KH) Gyrocarpaceae: GyrocarpusamericanaJacq. Chase317,NCU;JL/LL/YQ; Chase317,NCU;OD/FBQ/ Chase317,NCU;Kew; Chase317,NCU;OD/YQ; DQ008702 YQ;DQ008770 AJ581417 DQ008624 Hernandiaceae Hernandianymphaeifolia(Presl)Kub. Zanisetal.2003;AY095462 HernandiaovigeraL. Qiu01007;JL/LL/YQ; Qiu01007;OD/FBQ/YQ; Qiu01007;Kew;AJ966799 DQ008703 DQ008771 Himantandraceae: Galbulimimabelgraveana(F.Muell.)Sprague Weston929,NSW;JL/LL/YQ; Weston929,NSW;OD/FBQ/ Weston929,NSW;Kew; Zanisetal.2003;AY095459 DQ008704 YQ;DQ008788 AF465294 Idiospermaceae: Idiospermumaustraliense(Diels)S.T.Blake Qiu91042;JL/LL/YQ; Qiu91042;OD/FBQ/YQ; Qiu91042;Kew;AJ581425 Qiu91042;OD/YQ; DQ008705 DQ008782 DQ008633 Illiciaceae: IlliciumfloridanumEllis Qiu61;JL/LL/YQ;DQ008706 Qiu61;OD/FBQ/YQ; K.W.Hilu,K.Mu¨ller,and Qiu61;OD/YQ;DQ008659 DQ008825 T.Borsch,unpublished manuscript;Borsch3552, BONNAF543738(TB) 8 Juncaginaceae: 21 TriglochinmaritimaL. Qiu97106;JL/LL/YQ; Qiu97106;OD/FBQ/YQ; Hiluetal.2003Borsch3392 Qiu97106;LL/YQ; DQ008707 DQ008811 BONNAF542566(TB) DQ008650 Lactoridaceae: LactorisfernandezianaPhil. Chase1014,K;JL/LL/YQ; Chase1014,K;OD/FBQ/YQ; L.W.Chatrouetal., Zanisetal.2003;AY095463 DQ008708 DQ008798 unpublisheddata;AF465297 Lardizabalaceae: AkebiaquinataDecne. Qiu91020;JL/LL/YQ; Qiu91020;OD/FBQ/YQ; Hiluetal.2003;Borsch3412 Qiu91020;OD/YQ; DQ008709 DQ008761 BONNAF542587(TB) DQ008619 LardizabalabiternataRuiz&Pavon Qiu97135;JL/LL/YQ; Qiu97135;OD/FBQ/YQ; Qiu97135;TB/KH;AY437809 Qiu97135;OD/YQ; DQ008710 DQ008760 DQ008618 Lauraceae: Cinnamomumcamphora(L.)T.Nees&Eberm. Qiu102;JL/LL/YQ;DQ008711 Qiu102;OD/FBQ/YQ; Qiu102;Kew;AJ966800 Qiu102;OD/YQ;DQ008625 DQ008772 Cryptocaryaalba(Molina)Looser Rohwer2000;AJ247158 CryptocaryameisnerianaFrodin Qiu98048;JL/LL/YQ; Qiu98048;OD/FBQ/YQ; Qiu98048;OD/YQ; DQ008712 DQ008774 DQ008627 LaurusnobilisL. Parkinsonetal.1999; Qiu94209;OD/FBQ/YQ; Qiu94209;Kew;AJ966801 Qiu94209;OD/YQ; AF193990 DQ008773 DQ008626 Magnoliaceae: Liriodendronchinense(Hemsl.)Sarg. Parkinsonetal.1999;AF193993 Qiu28;OD/FBQ/YQ; Zanisetal.2003;AY095464 DQ008786 LiriodendrontulipiferaL. Azumaetal.1999;AB021016 MagnoliadenudataDesr. Zanisetal.2003;AF389256 MagnoliagrandifloraL. Chawetal.2000;AF161089 Table 1 (Continued) Familyandspecies mt-SSUrDNA mt-LSUrDNA cp-matK nu-26SrDNA MagnoliatripetalaL. Qiu3;OD/FBQ/YQ; Azumaetal.1999;AB021001 DQ008741 Menispermaceae: CissampelospareiraL. Chase347,NCU;JL/LL/YQ; Chase347,NCU;OD/FBQ/ Chase347,NCU;Kew; Chase347,NCU;OD/YQ; DQ008713 YQ;DQ008758 AJ966802 DQ008616 Cocculustrilobus(Thunb.)DC Qiu91016;JL/LL/YQ; Qiu91016;OD/FBQ/YQ; Qiu91016;OD/YQ; DQ008714 DQ008759 DQ008617 CocculuslaurifoliusDC. Hiluetal.2003;Borsch3406 BONNAF542588(TB) Monimiaceae: HedycaryaarboreaJ.R.&G.Forst. Qiu90028;JL/LL/YQ; Qiu90028;OD/FBQ/YQ; Qiu90028;Kew;AJ581436 Qiu90028;LL/YQ; DQ008716 DQ008769 DQ008623 HortoniafloribundaWightexArn. Qiu02002*;JL/LL/YQ; Qiu02002*;OD/FBQ/YQ; Qiu02002*;TB;AY437811 Zanisetal.2003;AF264143 DQ008717 DQ008778 PeumusboldusMolina EdinburghBG19870707; EdinburghBG19870707; Rohwer2000;AJ247183 Zanisetal.2003;AY095466 JL/LL/YQ;DQ008715 OD/FBQ/YQ;DQ008768 Myristicaceae: MauloutchiachapelieriWarb. Schatz3847A,MO;JL/LL/YQ; Schatz3847A,MO;OD/FBQ/ Schatz3847A,MO;TB; Schatz3847A,MO;OD/YQ; DQ008719 YQ;DQ008790 AY437812 DQ008638 MyristicafragransHoutt. Qiu92014;JL/LL/YQ; Qiu92014;OD/FBQ/YQ; Qiu92014;Kew;AJ966803 8 22 DQ008718 DQ008789 Nelumbonaceae: Nelumbolutea(Willd.)Pers. Zanisetal.2003;AF389259 NelumbonuciferaGaertner Parkinsonetal.1999; Qiu91028;OD/FBQ/YQ; K.W.Hilu,K.Mu¨ller,and AF193983 DQ008753 T.Borsch,unpublished manuscript;Borsch& Summers3220, FRAF543740(TB) Nymphaeaceae: Nupharsp. Parkinsonetal.1999; QiuM114;OD/FBQ/YQ; QiuM114;LL/YQ; AF193981 DQ008829 DQ008660 NupharluteaL. Hiluetal.2003;Borsch3337, FRAF543741(TB) Nymphaeasp. Chawetal.2000;AF161091 Qiu91029;OD/FBQ/YQ; Zanisetal.2003;AY095465 DQ008828 NymphaeaodorataAiton Hiluetal.2003;Borsch& Wilde3132,VPI&BONN AF543742(TB) Papaveraceae: SanguinariacanadensisL. Qiu91032;JL/LL/YQ; Qiu91032;OD/FBQ/YQ; Qiu91032;Kew;AJ966804 Qiu91032;OD/YQ; DQ008720 DQ008765 DQ008621 Pinaceae: Pinussp. Qiu94013;JL/LL/YQ; Qiu94013;OD/FBQ/YQ; DQ008721 DQ008835 PinusdouglasianaMartinez L.G.Geadaetal.,unpublished data;AB063520 PinuswallichianaA.B.Jackson I.Capesius,unpublisheddata; AJ271114 Piperaceae: PeperomiagraveolensRauh&Barthlott Hiluetal.2003;Prinslers.n. BONNAF542574(TB) Peperomiaobtusifolia Qiu94135; Qiu94135; Qiu94135; A.Dietr. JL/LL/YQ;DQ008722 OD/FBQ/YQ;DQ008794 LL/YQ;DQ008641 PiperbetleL. Chawetal.2000;AF161088 Qiu91048;OD/FBQ/YQ; Zanisetal.2003;AY095467 DQ008795 PipercrocatumR.&P. Hiluetal.2003;Slottas.n., VPIAF543745(KH) Platanaceae: PlatanusoccidentalisL. Chawetal.2000;AF161090 Qiu94152;OD/FBQ/YQ; K.W.Hiluetal.,unpublished Fishbeinetal.2001;AF274662 DQ008752 data;AF543747 Podocarpaceae: PodocarpuscostalisPresl Chawetal.2000;AF029369 Podocarpusmacrophyllus(Thunb.)Sweet Qiu95006;OD/FBQ/YQ; WangandShu2000; Qiu95006;OD/YQ; DQ008837 AF228111 DQ008664 Potamogetonaceae: PotamogetonberchtoldiiFieber Qiu96063;JL/LL/YQ; Qiu96063;OD/FBQ/YQ; Qiu96063;OD/YQ; DQ008723 DQ008810 DQ008649 PotamogetondistinctusArth.Benn. Tanakaetal.1997;AB002581 Proteaceae: GrevilleabanksiiR.Br. Hiluetal.2003;Borsch3413 8 BONNAF542583(TB) 23 GrevillearobustaCunn.&R.Br. Parkinsonetal.1999; Qiu94087;OD/FBQ/YQ; Qiu94087;OD/YQ; AF193995 DQ008751 DQ008612 PersooniakateraeP.Weston&L.Johnson Weston1120,NSW;JL/LL/YQ; Weston1120,NSW;OD/FBQ/ Weston1120,NSW;TB; Weston1120,NSW;OD/YQ; DQ008724 YQ;DQ008750 AY437813 DQ008611 PetrophilecanescensCunn.exR.Br. Qiu98018;JL/LL/YQ; Qiu98018;OD/FBQ/YQ; Qiu98018;Kew;AJ966805 Qiu98018;OD/YQ; DQ008725 DQ008749 DQ008610 Ranunculaceae: Ranunculussp. Chawetal.2000;AF161093 Qiu95024;OD/FBQ/YQ; DQ008756 RanunculusficariaL. Borsch3554BONN;TB; AY437814 RanunculuskeniensisMilne-Redhead&Turrill Zanisetal.2003;AF389269 XanthorhizasimplicissimaMarshall Qiu91030;JL/LL/YQ; Qiu91030;OD/FBQ/YQ; Hiluetal.2003;Borsch3394 Qiu91030;OD/YQ; DQ008726 DQ008757 BONNAF542567(TB) DQ008615 Sabiaceae: Sabiasp. Qiu91025;JL/LL/YQ; Qiu91025;OD/FBQ/YQ; Qiu91025;Kew;AJ966806 DQ008727 DQ008747 SabiaswinhoeiHemsl. Zanisetal.2003;AF389272 MeliosmasquamulataHance B.Shih3749,HAST;JL/LL/YQ; B.Shih3749,HAST;OD/FBQ/ B.Shih3749,HAST;OD/YQ; DQ008728 YQ;DQ008748 DQ008609 MeliosmaveitchiorumHemsl. Chase2989,K;Kew;AJ581449 Sargentodoxaceae: Sargentodoxacuneata(Oliv.)Rehder&Wilson Pan93001,NCU;JL/LL/YQ; Pan93001,NCU;OD/FBQ/ Pan93001,NCU;Kew; Pan93001,NCU;OD/YQ; DQ008729 YQ;DQ008762 AJ966807 DQ008620