Phylogenetic Position of Titanotrichum Oldhamii (Gesneriaceae) Inferred Fromf Our Different Gene Regions

Phylogenetic Position of Titanotrichum oldhamii (Gesneriaceae) Inferred FromF our Different Gene Regions

CHUN-NENG WANG,1,2,3,4 MICHAEL MO¨ LLER,1 and QUENTIN C. B. CRONK1,2,3 1Royal BotanicGarden, 20AIn verleith Row,Edinburgh EH3 5LR,Scotland, UK; 2Institute ofCell and MolecularBiology ,The University ofEdinburgh ,Edinburgh EH9 3JH,Scotland, UK; 3Present address: BotanicalGarden and Centre forPlant Research, University ofBritish Columbia, VancouverV6T 2TL,Canada 4Author forCorrespon dence ([email protected])

CommunicatingEditor: James F .Smith

ABSTRACT. Titanotrichumoldhamii has been variously placed inGesneriace ae orScrophulari aceae, although mostrecent taxonomictreatmentstrea titas amonotypictribe within Gesneriace ae.Inthis study ,wereconstructedabroad-scale phy- logenycontaining Titanotrichum usinggene sequencesfromfour sequence regions(chloropla st trnL-F intronand spacer and atpB-rbcL spacer, nuclear 26S ribosomalDN A, and the low-copy developmentalgene CYCLOIDEA,CYC).The phylogenies inferredfrom eac hindividualdata setand the combinedda ta are congruent inplacing Titanotrichum insideGesneriac eae. The phylogenetic treebased on combinedchloropla stand nuclear DNAsequences grouped Titanotrichum withsubfamilies Gesnerioideae (New World)and Coronantheroideae (SouthP acic and Chile).W ehave isolated CYC, frommost of the species ofGesneriace ae and Scrophulariaceae represented inthis study ,and thisgene phylogenysuggeststhe same placementof Titanotrichum . CYC was found toev olvethreetimes faster than the trnL-F intronand spacer, 3.3 timesfaster than the atpB- rbcL spacer, and eighttimes faster than nuclear 26S rDNA. Althoughthere is considera blephylogen etic informationin this fastev olving gene, analysis isproblema tic because ofhigh lev elsof homopla syand paralogy. Inaddition toa duplication predatinga split between New Worldand OldW orldtaxa ( Gcyc1 vs. Gcyc2),thereare several subsequentlineage-related duplications(mainly within Gcyc1).

The monotypic taxon, Titanotrichumoldhamii nerioideae(N ewWorld) (Burtt and Wiehler 1995).In (Hemsl.) Soler.,is ofuncertain taxonomicafnity ,being Old World species the cotyledons becomeunequal in variously placedin Scrophulariaceae(sens. lat.;W ett- size soon aftergerminati on (anisocotyly) due tothe stein 1891)and Gesneriaceae(Burtt 1962,1977). The extended activityof a meristem atthe baseof the cot- difculty of classifying Titanotrichum arises becauseit yledon, while New World species ofsubfamily Ges- shares several features with bothScrophulariac eae s.l. nerioideaeand Coronantheroideae are allisocotylous, and Gesneriaceae.The species was rst placedin Reh- lacking such persistent meristematicactivity (Burtt mannia (Scrophulariaceaes.l.) as its racemose inores- 1962).Interestingl y, Titanotrichum ,although geograph- cence and showy bell-shaped owers are reminiscent ically Old World, is isocotylous (Wang and Cronk ofScrophularia ceaesuch as Rehmannia and Digitalis 2003). Titanotrichum alsohas someuniquem orpholog- (Hemsley 1895).Later ,in 1909,Solerede rnamed it as icalcharacters, such asbulbil proliferati on in inores- a new genus Titanotrichum in Gesneriaceaebased on cences (Wang and Cronk 2003),not seen in anyother the unilocularov ary (Solereder1909).It w asplaced in Gesneriaceaespecies. the Old World subfamilyCyrtandroid eae on account Toresolve the placement of Titanotrichum , we use ofits superior ovary and geographic distribution(So- anapproach combining molecularevidenc efrom two lereder 1909).Recent taxonomictreatmentsraised it to chloroplast DNA(cpDNA)sequence s, trnL-F intron amonotypic tribe in the Cyrtandroideae because ofits and spacerand atpB-rbcL spacer,the 26Sn uclearri- unique morphology (Wang and Pan1992; Burtt and bosomalD NA(nrDNA)and anucleardev elopmental Wiehler 1995).A recent molecular phylogenetic study gene, CYCLOIDEA (CYC).F or comparisons atthe fam- using chloroplast ndhF gene sequences addressed its ily level or above(as this gene is conservative),26S position within Gesneriaceae; it wasplaced as sister to datahas proven tobe ph ylogeneticallyuseful, partic- the rest ofsubfamily Cyrtandroid eae butwith little ularly forprevious ly unresolvedclades and taxa(e.g., branchsupport (Smith et al.1997a, 1997b). On the oth- Circaeaster ,Oxelman and Lide´n1995;angiosperm phy- er hand, achemotaxonomic study on phenolic acid logeny,Hershkovitzet al.1999). The chloroplast trnL- compoundsgrouped Titanotrichum , Cyrtandromoea , and F intron and spacerand atpB-rbcL spacerhav ebeen Rehmannia into Scrophulariaceae(Kvist and Pedersen successfully used forinferring phylogenies atthe ge- 1986).Sealy (1949)allied Titanotrichum to New World neric and intragenericlev el (Taberlet et al.1991; Go- Gesneriaceaegenera, Isoloma (5Kohleria) and Naegelia lenberg et al.1993; Gielly and Taberlet 1994;Manen et (5Smithiantha ),becausethey possess asimilar habit al.1994), and have alsorecently been used successfully and scaly rhizome.Anisocotyly is probablythe most on Gesneriaceae(M ayer et al.2003). reliable characterto separate Cyrtandroid eae and Ges- CYCLOIDEA belongs toa multigene family,the TCP

407 408 SYSTEMATIC BOTANY [Volume 29 family,which comprises axillary meristem identity reading frame(O RF)w ere amplied forour studyusing forw ard primer GcycFS and reverseprimer GcycR (forthe exact amplied genes in Zea mays (TB1),oralsymmetry genes in An- CYC regionsee Mo¨lleret al. 1999). Toamplifyfour different gene tirrhinummajus (CYC)and DNA-binding protein regionsef ciently across distantlyrela ted species inour study,one genes in Oryzasativa (PCF)(Cubaset al.1999a, 1999b; new PCR primerw as designedor existing ones modied from Cubas2002). The gene family encodes putativetran- originalpublica tions. Forthe amplication ofthe complete atpB- rbcL spacer, anew forward primer‘ ABF’(5 9-GGA AACCCC AGA scription factors(Doebley and Lukens 1998).The TB1/ ACC AGA AG-39)was designedand combinedwith the reverse CYC subfamilyis characterisedbytw oconserved re- primer‘ JF5’from Manen et al. (1994). Toobtain the complete trnL- gions: abasichelix-loop- helix TCPdomain and anar- F intronand spacer region,primers ‘ c’and ‘f’from T aberlet et al. (1991) were chosen. Toobtain theupper part ofthe 26S ribosomal ginine-richRdomain (Cubas1999b). Mo ¨ller et al. DNAregion,forw ard primer‘ ITS-3P’located in5.8S (Mo¨llerand (1999)have isolated twoputative paralogues in Ges- Cronk1997) and reverse primer‘ 28S2R’w ere used (Oxelmanand neriaceae (Gcyc1 vs. Gcyc2)from species with different Lide´n1995). Circa 1,200 base pairs (bp)(35%) fromthe 5 9end of ower symmetries, in anattem ptto test their sequence the26S genew ere amplied. 50–100 ngtempla te DNAwas incor- porated in50 mlreactions, containing 1 mMprimer,100 mM each divergencein relation tomorpholo gicalchanges (zy- dNTPs (Roche, USA),2.5mM MgCl 2, and 0.5U Taq polymerase gomorphyvs. actinomorphy).H owever,their results, (Bioline,UK) and 1X Taq buffer(16mM (NH 4)2SO4, 67mM Tris- together with afollow-upstudy (Citerne et al.2000), HCl (pH 8.8), 0.01% Tween20). did not suggest loss offunctional genes in actinomor- Except forthe CYCLOIDEA region(PCR conditions described in Citerneet al. 2000), auniversal PCR prole for trnL-F,atpB-rbcL, phic taxa.They found that Gcyc evolution wasconsis- and 26S was used as follows:3 mina t95 8C, then ve cycles of1 tent with Gesneriaceaeph ylogenies atthe generic and min at 958C, 1 min at 578C, 2 min at 728C, followed by30 cycles triballevels (Mo¨ller et al.1999; Citerne et al.2000). of 45 s at 948C, 45 s at 578C, 2 min at 728C, witha nal extension step at 728Cfor7 minutes.Ampli cation products were checked Since CYC belongs toa multi-copygene family,it is on 1% agarose gelsin 1X TBE bufferand visualized under UV reasonable toexpect that two or more homologues afterethidium bromide staining (0.1 mg/ml).PCR products were would beisolated in eachtaxon studied. Orthologues puried usingQiagen puri cation columns(Qiagen Ltd, Dorking, and paraloguesmay beidenti ed byph ylogenetic Surrey,UK) according tothe manufactu rer’s protocols. Sequencing. Directcycle sequencingwas carried out usingthe analysis (Baum1998; Eisen 1998;Ba um et al.2002). big-dyeterminator ready reaction mix(P erkinElmer Applied Bio- The aim ofthis study is toobtain chloropla st trnL- systemsdivision, Warrington,UK) followingstandard protocols. F,atpB-rbcL, nuclear 26S,and CYCLOIDEA gene se- Sequencingproducts were analysed on an ABIPRISM 3100 au- tomatic DNAsequencer (Applied Biosystems,W arrington,UK). quences from selected taxato in vestigate the phylo- The PCR amplifyingprimers of all gene regionsw ere also used genetic position of Titanotrichum oldhamii , and to in- as sequencingprimers.Several internal primersw ere designedfor vestigate how the Gcyc homologues evolved in these our samples toallow complementarysequence conrma tion. species with respect togene duplicationor extinction These were forthe atpB-rbcL spacer region(atpB-VMF forw ard: 5 9- GAATTC CGC CTW TTTTC ACATCTA-3 9;VM-Rre verse: 5 9- events. Because Titanotrichum has been placedin both TAGATG TGAAAAT AGGCG GAAT -3 9),for 26S (26S-Q1Ffor- Scrophulariaceae s.l. and Gesneriaceae,we sampled a ward: 59-CATTCG ACC CGT CTT GAAA C-3 9;26S-Q1R reverse: large number ofrepresenta tive taxafrom bothfami- 59-TTTC AAG ACGGG TCG AATGG-3 9), and for CYCLOIDEA lies. (Cyc-NFforw ard: 5 9-GCR AGG GCB AGR GAA AGAAC-3 9; Cyc- NR reverse: 5 9-GCACATTTTCTC YYT YGT TCT TTC-3 9). Cloning of CYCLOIDEA Homologues. Toensure ahighdegree MATERIALSAND METHODS ofrecovery ofthe CYCLOIDEA homologuespresent inour selected taxa, including rarercopies, morethan 20 clones were examined PlantMaterial. Plant material fromselected Gesneriaceae, foreach taxon. PCR fragmentsw ere cloned intothe pCR4-TO PO Scrophulariaceae, and Solanaceaespecies was collected either vector, and the recombinantsw ere transformedto TO P10 Chem- fromliving plants cultivated atthe Royal Botanic Garden Edin- ically Competent E. coli followingthe instructionsof the TOPOT A burgh(E), the Instituteof Botany ,Universityof Vienna, orfrom Cloningkit (Invitrogen, P aisley,UK). Plasmid DNAcontaining eld collections.Fortaxa cultivated at Edinburgh,v oucher speci- cloned products were extracted and puried usingthe Qiagen mensw ere deposited inthe herbarium(E). Details are given in Spin Miniprep kit(Qiagen Ltd, Dorking,Surrey ,UK), priorto se- Table 1. Seven taxa representingmajorclades ofScrophulari aceae quencing. s. l. were selected according toOlmstead et al. (2001), plus Pel- Sequence Analysis. Nucleotide sequences ofthe threedifferent tanthera oribunda ofLoganiacea eand 18 taxa ofmajor tribes in gene regions( trnL-F,atpB-rbcL, and 26S) from30 taxa were rst Gesneriaceae followingBurtt and Wiehler(1995). Solanales are sis- aligned usingdefault settingsin CL USTALX (Thompsonet al. terto Lamiales (Albachet al. 2001) and so Schizanthus 3 wiseto- 1997), thenadjusted manually.Since CYC evolved fasterthan the nensis and Nicotiana tabacum were chosen as outgroups. otherthree gene regions,alignment dif culties arose when very DNAExtraction. DNAfromfresh or silica-dried leaves was divergentsequences were present (i.e., fromSolanacea eoutgroups extracted followinga modied CTABprocedure ofDoyle and toScrophular iaceae s.l. and Gesneriaceae). CYC nucleotidese- Doyle(1987). ForD NAcontaining signicant amounts ofsecond- quences were rsttranslated intoamino acid sequences and then ary metabolic compounds(e .g. Epithema benthamii ),an additional aligned inCLUST ALX toaid manual alignmentafterw ard. To phenol purication step was added (Sambrooket al. 1989). align commonmotifs tha tCLUSTALX failed todetect, alignments PCRCondition s. The primersand PCR conditions used inthis were adjusted manually on thebasis ofshared motifsand related studywere adopted fromthe originalp ublications; i.e., chloroplast amino acid groups (similarchemical structure)and nally re-con- trnL-F intronand spacer (Taberlet et al. 1991), chloroplast atpB-rbcL verted intonucleotid esequences. Alignedma triceswere deposited spacer (Manenet al. 1994), partial 26S ribosomalnuclear DNA inTreeBASE (study acc. no. 5 S965). Sequence characteristics(se- (Oxelmanand Lide´n1995) and lessstringent PCR conditions were quence divergence, G/Ccontents, etc., Tables 2, 3) ofeach gene utilized forextensive cloningof partial sequences ofthe nuclear regionw ere obtained withP AUP*, version 4.0b8 (Swofford2001) developmentalgene CYCLOIDEA (Citerneet al. 2000). Around550 and features such as transitionand transversionratios calculated bp (;70%) ofthe down streamregion of the CYCLOIDEA open usingMacClade version 3.01 (Maddisonand Maddison 1992). The 2004] WANGET AL.: PHYLOGENETICPOSITION OF TITANOTRICHUM 409

TABLE 1. Selected species used inthe phylogen yreconstructionin this study ,withspecimen number,origin and GenBank numbers forthe individua lgenesused. The specimen no. isalso used as thev oucher number.Voucher specimens are deposited inthe respective herbaria E, G, orM O.

Solanaceae. Schizanthus 3 wisetonensis :Cultivar UoE (E),cultivated; trnL-F AY423121, atpB-rbcL AY423103, 26S AY423076, CYCLOIDEAChadwick (1997). Nicotianatabacum Linn.: Cultivar UoE (E),cultivated; trnL-F Z00044, atpB-rbcL Z00044, 26S AF479172, CYCLOIDEAChadwick (1997). Scrophulariaceae s.l. Paulowniatomentosa (Thunb.)Steud.: 19892925 (E),Japan; trnL-F AY423122, atpB-rbcL AY423104, 26S AY423079, CYCLOIDEAA Y423141 (Pcyc1); AY423140 (Pcyc2). Scrophulariacanina Linn.: PerretS1.119 (G),Europe; trnL-F AY423123, atpB-rbcL AY423105, 26S AY423080, CYCLOIDEAA Y423138 (Scyc1); AY423139 (Scyc2). Rehmanniaglutinosa Steud.: MMO0152B (E),China; trnL-F AY423124, atpB-rbcL AY423106, 26S AY423081, CYCLOIDEA— . Antirrhinummajus L.: Cultivar UoE (E),cultivated; trnL-F AJ492270, atpB-rbcL AJ490883, 26S AY423077, CYCLOIDEAAF208341 (cyc); AF208494 (dich). Tetranemamexicanum Benth.: 19697819 (E),Mexico; trnL-F AJ492272, atpB-rbcL AJ490884, 26S AY423078, CYCLOIDEAA Y423142 (Tcyc2). Calceolariaarachnoidea Graham: 19912379 (E),Chile; trnL-F AY423126, atpB-rbcL AY423108, 26S AY423083, CYCLOIDEAA Y423143 (Ccyc1); AY423144 (Ccyc2). Jovellanapunctata Ruiz &Par.: 19980599 (E), Chile; trnL-F AY423127, atpB-rbcL AY423109, 26S AY423084, CYCLOIDEAA Y423145 (Ccyc2). Loganiaceae. Peltanthera oribunda Benth.: Hammel 20144 (MO),Peru; trnL-F AY423125, atpB-rbcL AY423107, 26S AY423082, CYCLOIDEA— . Gesneriaceae (tribes): (Titanotricheae) Titanotrichumoldhamii (Hemsl.)Solereder: 19973433 (E),Taiwan; trnL-F AY423129, atpB-rbcL AY423111, 26S AY423085, CYCLOIDEAA Y423150 (Gcyc1). (Beslerieae) Besleria labiosa Hanst.: 19822666 (E),V enezuela; trnL-F AY423128, atpB-rbcL AY423110, 26S AY423086, CYCLO- IDEAA Y423148 (Gcyc1). (Napeantheae) Napeanthusreitzii B.L. Burttex Leeuwenb.: Perret156 (G),Brasil; trnL-F AJ492321, atpB-rbcL AJ493036, 26S AY423087, CYCLOIDEAA Y423149 (Gcyc1). (Coronanthereae) Fieldiaaustralis A.Cunn.: 19696862 (E),Australia; trnL-F AY423130, atpB-rbcL AY423112, 26S AY423088, CYCLOIDEAA Y423151 (Gcyc1E); AY423152 (Gcyc1F). Mitrariacoccinea Cav.:19792696 (E),Chile; trnL-F AY423131, atpB- rbcL AY423113, 26S AY423089, CYCLOIDEAA Y423153 (Gcyc1). (Episcieae) Chrysothemispulchella Dece.: 19802568 (E),cultivated; trnL-F AJ492312, atpB-rbcL AY423115, 26S AY423091, CY- CLOIDEAA Y423154 (Gcyc1). (Gloxinieae) Sinningiaschiffneri Fritsch:19781514 (E),Brasil; trnL-F AJ439745, atpB-rbcL AJ439900, 26S AY423092, CYCLO- IDEAAF208327 (Gcyc1). Kohleriaeriantha (Benth.)Hanst.: 19821486 (E),Ecuador; trnL-F AY423132, atpB-rbcL AY423114, 26S AY423090, CYCLOIDEAA Y423155 (Gcyc1). (Gesnerieae) Gesneriahumilis Linn.: Chautems 1179 (G),Cultivated; trnL-F AJ439821, atpB-rbcL AJ439976, 26S AY423093, CY- CLOIDEAA Y423156 (Gcyc1). (Epithemateae) Rhynchoglossumhologlossum Hayata: Wang 1207 (E),T aiwan; trnL-F AY423133, atpB-rbcL AJ490899, 26S AY423094, CYCLOIDEA— . Epithemataiwanense (C.B.Clark)Li &Kao: Wang 1208 (E),T aiwan; trnL-F AJ492276, atpB- rbcL AY423117, 26S AY423096, CYCLOIDEA— . Epithemabenthamii C.B.Clark: 19972563 (E),Phillipines; trnL-F AY423135, atpB-rbcL AY423118, 26S AY423097, CYCLOIDEAA Y423157 (Gcyc1); AY423146 (Gcyc2). Whytockiasasakii (Hayata) B.L.Burtt: 19991504 (E),Taiwan; trnL-F AY423134, atpB-rbcL AY423116, 26S AY423095, CYCLOIDEA— . (Didymocarpeae) Streptocarpusholstii Engl.: 19592272 (E),Tanzania; trnL-F AJ492304, atpB-rbcL AJ490917, 26S AY423099, CYCLOIDEAAF208338 (Gcyc1A); AF208334 (Gcyc1B). Ramondamyconi (L.)Rchb.: 19821564 (E),Macedonia; trnL-F AJ492301, atpB-rbcL AJ490914, 26S AY423098, CYCLOIDEAAF208323 (Gcyc1); AF208318 (Gcyc2). Didymocarpuscitrinus Ridl.: 19830510 (E),Malaysia; trnL-F AJ492293, atpB-rbcL AJ490906, 26S AY423100, CYCLOIDEAA Y423158 (Gcyc1C); AY423159 (Gcyc1D). (Cyrtandreae) Cyrtandraapiculata C.B.Clake: Cronk& Percy T91 (E),T ahiti; trnL-F AY423136; atpB-rbcL AY423119, 26S AY423101, CYCLOIDEAA Y423160 (Gcyc1); AY423147 (Gcyc2). (Trichosporeae) Loxostigma sp.:19962309 (E),China; trnL-F AY423137, atpB-rbcL AY423120, 26S AY423102, CYCLOIDEA AY423161 (Gcyc1C); AY423162 (Gcyc1D).

TABLE 2. Sequence characteristicsof the chloropla st trnL-F intron& spacer, atpB-rbcL spacer, nuclear 26S gene and CYCLOIDEA gene region.

CYCLOIDEA CYCLOIDEA Characteristicparameters trnL-F atpB-rbcL 26S nucleotide amino acid Lengthrange (total),bp ora.a. 614–842 717–1278 1190–1194 462–726 154–234 Lengthrange, bp ora.a. 614–827 717–799 1191–1193 531–702 185–234 (Gesneriaceae) Lengthmean (total),bp ora.a. 801.3 745.1 1191.91 608.8 202.9 Lengthmean, bpora.a. 793.5 743.3 1191.6 635.9 212.0 (Gesneriaceae) Sequence divergence (%) 1.6–9.5 0.8–9.1 0.2–8.0 2.4–26.5 5.5–39.9 (Gesneriaceae) Overall sequence divergence (%) 1.6–17.0 0.8–16.6 0.2–11.2 2.4–49.0 5.5–64.1 G 1 Ccontent mean % 34.6 30.81 58.3 41.1 — 410 SYSTEMATIC BOTANY [Volume 29

TABLE 3. Sequence characteristicsrelev ant forthe phylogene tic analysis. CI 5 ConsistencyIndex;RI 5 Retention Index;RC 5 Rescaled ConsistencyIndex.

combined CYCLOIDEA CYCLOIDEA Statisticaldescription trnL-FatpB-rbcL 26S 3-gene data nucleotide amino acid Alignedlength, bp ora.a. 989 946 1200 3133 1020 340 Numberof excluded sites 63 41 0 102 178 12 Size ofindels 1–35 1–28 1–2 1–35 3–114 1–34 No. ofindels 37 31 10 78 43 38 Percentage ofconstant sites 61.4 62.7 69.1 65.0 30.6 23.8 Percentage ofvariable sites 38.6 37.3 30.9 35.0 69.4 76.2 Percentage ofuninformative sites 16.8 20.9 13.9 17.0 21.4 25.3 Percentage ofinformative sites 21.7 16.5 17.0 18.0 48.0 50.9 Transitions/transversions 0.70 0.89 2.87 — 1.06 — Average numberof steps per character 0.63 0.58 0.69 0.65 2.23 3.32 Numberof MP trees 12 149 198 19 12 324 Lengthof MP trees 584 521 827 1962 1879 1089 CI 0.781 0.793 0.567 0.682 0.572 0.729 RI 0.756 0.729 0.514 0.624 0.542 0.684 RC 0.590 0.578 0.291 0.425 0.310 0.458

comparison ofev olutionary distance between the gene regionsw as estimatesof the proportion ofinv ariable sitesand gammadistri- analysed bycalcula tingpair-wise sequence divergences among bution). taxa usingthe DISTANCE option (average pairwise distance) in Forbranch support ofMaximum Likelihood trees(ML-BS), 500 PAUP*, based on unambiguouslyalignable regionswithout gaps. replicates ofML bootstrap analyses were performed.F or CYCLO- Phylogenetic Analysis. Phylogenetic treesw ere reconstructed IDEA data alone aprotein MLanalysis was performedusing P HY- usingP AUP* 4.0b8. Each gene region( trnL-F,atpB-rbcL, 26S, and LIP3.6 (Felsenstein2002) witha Joneset al. (JTT)model of amino CYC)was rstanalyzed independentlyby heuristic treesearches, acid substitution(J ones et al. 1992), allowing one invariant rate inthe attemptto nd the mostparsimonio us (MP)trees. Toin- plus 4categoriesof gamma distributedra te heterogeneities(the crease the chances ofincluding all islands ofmost parsimonio us parameters were estimated inTREEP UZZLE version5.0, Schmidt trees, 10,000 replicatesof RANDO MADDITIONSEQUENCE were et al. 2002). Branchsupport was obtained fromTREEPUZZ LE (us- rstperformed without swapping, saving all shortesttrees. This ingthe same parameters as above). Bayesian analysis also applies was followed byTBR swapping on all theresulting trees with modelparameters fromM odelTestand was carried out using MULTREES, STEEPESTDESCENT ,branch COLLAPSE (max.)on MrBayesversion 2.01 (Huelsenbeck and Ronquist2001). The anal- and ACCTRAN optimization (Mo¨llerand Cronk1997). Allmost ysiswas run for1,000,000 generationswith trees sampled every parsimonious treeswere summarized instrict consensus treesfor 100 generations.F our exchangeable Markov chains were specied each dataset. Characters and substitutionra te congruencebetween allowing treeconstructio ntoexplore the treespace .The rst1,000 differentgene regionsw as testedusing the partition homogeneity ‘‘burn-in’’phase treesw ere discarded. The resultingtrees were test(incongruen ce lengthdifferences test, ILD)(Farris et al., 1995) summarisedin a 50% majority-ruleconsensus treefor the calcu- inP AUP*. Aparsimonyanalysis on combinedda ta from trnL-F, lationof Bayesian branch support (BA-BS)under PAUP*. atpB-rbcL, and 26S sequences was also conducted as described CYCLOIDEA Gene Evolution. Inorder to inv estigate CYCLO- above and used toreconstruct aspecies treefor the GeneTree IDEA duplicationsin rela tionto the species evolution, the CYCLO- analysis (seebelow). IDEA gene phylogenywas reconciled withthe species treeinferred Branch support formaximum parsimony trees (MP -BS)w as fromthe 3-gene data set( trnL-F,atpB-rbcL, and 26S) using testedusing 10,000 replicatesof bootstrap analyses (Felsenstein GeneTree (Page and Cotton2000). The 3-gene data setwas re- 1985) inP AUP*, setto HEURISTIC search and SIMPLE ADDITION duced byexclusio nofthose taxa forwhich a CYCLOIDEA se- SEQUENCE, withTBR swapping but without MULTREE and quence could notbe obtained. The reduced data matrixw as then STEEPESTD ESCENT.Bremersupport indices (DI)(Bremer 1994) reanalysed as above.The mostlikely CYC gene treewas selected were also calculated usingA utodecay version4.0 (Eriksson1998) inGeneTree byminimising the cost ofthe optimality criteria such toestima te the additional steps forcollapsing individualclades. as deep coalescence,duplication and lossevents between each Descriptivestatisticsfor the measures ofcharacter tinparsimony gene treeand species treecombina tion. analysis, such as the consistencyindex (CI)(Kluge and Farris 1969), retentionindex (RI)(F arris1989), and rescaled consistency index (RC)w ere also calculated.Forthe 26S nrDNAregion,rew- RESULTS eightingto correct fortransition/ transversionbias was also eval- uated but the resultsga veno increase inresolution and further SequenceAnalysis of trnL-F,atpB-rbcL, and 26S. analyses were performedunw eighted. The sequence characteristics ofall sequence matrices Toexplore our data further,Maximum Likelihood (ML)and Bayesian analysis (BA)w ere also performed.F ornucleotid eML are summarized in Tables2 and 3.F or the chloroplast analysis, the best-tted modelof substitution was selected using trnL-F intron and spacerregion, Gesneriaceaespecies ModelTestversion 3.06 (Posada and Crandall 1998). The model showed aslightly higher length variation compared to parameters recommended inM odelTestw ere thenimplemen ted inP AUP* toperform heuristic searches as above. The 3-gene (com- Solanaceaeand Scrophulariaceaes.l. species. But the bined trnL-F,atpB-rbcL, and partial 26S sequence data) MLanalysis mean length and sequence divergenceof Scrophulari- was performedusing a GTR 1I1Gmodel(general time rev ersible aceaespecies washigher than forGesneriacea especies. withestima tesof proportion ofinv ariable sitesand gammadis- The atpB-rbcL spacercontained a479bp A T-rich inser- tribution)selected byAIC (Akaike Information Criterion) in ModelTest. The bestmodel for CYCLOIDEA MLanalysis selected tion (position 424to 903) uniq uein Epithemabenthamii . byAIC in Modeltest was TVM 1I1G(transversionmodelwith The length range ofthe entire atpB-rbcL spacer was 2004] WANGET AL.: PHYLOGENETICPOSITION OF TITANOTRICHUM 411 somewhatsmaller (717–799 bp, excludin gthe insertion clones ofall PCR products, although other copiesmay in Epithemabenthamii ).Both chloroplast datasets pos- not beable to be pick ed upwith non-redundant prim- sessed similar GCcontents (G 1 C5 34.6%and 30.8%, ers. In allN ew World Gesneriaceaespecies ofsubfam- Table2). F or alignment, more than 30indels (inser- ily Gesnerioideae analyzed todate only one copyhas tion/deletion events) were recognized foreach cpD NA been isolated. In the other twosubfamilies (Coron- region (Table3). Despite asimilar length, the trnL-F antheroideae and Cyrtandroideae),tw ohomologues matrix contained ahigher proportion ofinformativ e were found in eachspecies exceptfor Mitrariacoccinea sites (21.7%)compared tothe atpB-rbcL matrix (16.5%), where only one copyw asisolated. Someofthese with- buta similar proportion ofv ariablesites ( ;38%). This in-species homologues were very divergentfrom each difference wasdue toa higher proportion ofautapo- other (i.e.,the uncorrected pairwise distance between morphic sites in the latter. Epithemaben thamiiGcyc2 and Gcyc1 was34%, betw een Approximately 1,192bp of 26S w ere amplied for Cyrtandraapiculata Gcyc2 and Gcyc1 23%).Other pairs eachspecies. Generally the length wasmore conserved were closer toeach other (i.e.,the uncorrected pairwise acrossall taxa studied compared tothe cpDNAfrag- distance between Didymocarpuscitrin us Gcyc1C and ments. Afew,one totw obp,indels were found in the Gcyc1D was11%, betw een Fieldiaaustralis Gcyc1E and expansion segment. Abouta quarter longer than atpB- Gcyc1F 8%).M aximum divergencewithin Gesneri- rbcL, 26Sincluded asimilar proportion ofinformativ e aceaehomologu es was39.9%. Up to two CYC homo- sites (17.0%).During PCRampli cation, different cop- logues were alsoisolated from Calceolariaceaeand ies ofthe 26Sgene from Antirrhinummajus and Titan- Scrophulariaceaespecies with the exception of Tetra- otrichumoldhamii were obtained with different internal nemam exicanum and Jovellanapun ctata . Two Peltanthera sequencing primers. Though these putativepseudo- oribundaCY C homologues were alsoisolated. How- genes were still length conserved, their GCcontents ever, inclusion ofthese copiesresulted in ahigh level were signicantly lower than potentially functional ofhomoplas y(CIof the resulting most parsimonious ones (40.2%vs. 58.3%).Thus they were excluded from trees wasreduced from 0.57to 0.41). The ywere there- analysis. The characteristicsof the nuclear26S gene fore excluded from the phylogeneticanalysis. Wehave were quite different from the chloroplast intron and been unableto am plify CYC homologues from Rhyn- spacerregions, the former possessed ahigher GCcon- choglossumhologlossum , Whytockiasasakii , Epithematai- tent (58.3%),and transition/transversion ratio(2.87) wanense, and Rehmanniaglutin osa ,perhaps becausethe and alow proportion ofv ariablesites (30.9%). primer sites are not conserved. 26SEv olution. In the 26SrDN Aanalysis, wefound PseudoCopiesan dAllelicV ariationof CYCLOIDEA. several sites within the expansion segments (Bultet al. In addition tofun ctionalh omologues, pseudogenes and 1995)that contained homoplasticsubstitution s. Tran- allelic variants were alsoampli ed using the same sitional substitutions are extremely frequent among primer set. Somepseudocopie scontained stopcodons these regions (e.g.,C-T changes havebeen observed 12 inside the ORF (i.e., Calceolariaarachnoidea clone 21 and times more often than other basechanges). On the oth- Gesneriahumilis clone7,data not shown). Asingle base er hand, nucleotide changes were rare in the conserved mutationresulted in apremature stopcodon in Titan- core region. The distinctivenature of26S evoluti on otrichumoldhamii clone 26(data not shown), buta PCR may beresponsibl eforthe low CI(0.567) and RI artefactfrom non-proofreading Taq polymerase cannot (0.514)in our26S MP analysis (Table3). H owever, this beruled out.All apparentpseudogen es were thus ex- problem mainly affectsrelations hips abovefamily lev- cluded from further analyses. Allelic variation could el, and the strict consensus topology wascongruent bediscriminat ed from locusv ariation bycomparing with ourother datasets. Thus we included 26Sdata the pairwise sequence differences. Putatively allelic in ourcombined analysis toincrease the number of clones had upto four n ucleotide changes or three ami- informativesites. no acidchanges, while differencesbetween putative CYCLOIDEA Clones. The amplied length ofthe loci(classi ed based on their position in phylogenetic CYCLOIDEA homologues wasbetw een 462and 726bp, trees) included atleast 17bp changes or nine amino indicating asignicant length redundancy offunction- acidchanges. The pairwise distances between likely al CYCLOIDEA copies(but see below).Only putatively alleles wasless than 0.6%but the distance between the functionalcopies were included in the matrix (pos- nearest homologues was2.4% (i.e ., Gesneriahumilis sessing no premature stopcodon or frameshifts). The Gcyc1 and Kohleriaeriantha Gcyc1 ).Allelic variants were aligned length ofthe CYCLOIDEA matrix was1,020 bp , reduced toone forsimplicity .In exploratory analyses indicativeofthe numerous indels necessary foralign- allallelic variants from asingle taxonalw ays formed ment. sister relationships. Different CYC homologues, upto two copies per PhylogeneticP ositionof Titanotrichumo ldhamii . species, were obtained afterextensive cloning. In Ti- The phylogenyinferred from trnL-F,atpB-rbcL, and 26S tanotrichumoldhamii ,one copywas isolated from all rDNAdatasets, individuallyand combined, resulted 412 SYSTEMATIC BOTANY [Volume 29

FIG.1. Strictconsensus treeof 19 mostparsimonious treesbased on combined trnL-F,atpB-rbcL and partial 26S sequence data. Numbersabove the branches are MPbootstrap values and decay indices. Numbersbelow the branches are Bayesian 50% majorityconsensus and MLbootstrap values (bold).Branches inbold indicate the branches persistingin the MPanalysis of individual datasets. The subfamilyrelationship and cotyledon type are indicated

in similar topologies with respect to Titanotrichum . All cies phylogeny.Parsimonyanalysis ofthe combined 3- analyses indicated thatthe genus belongs toGesneri- gene dataset resulted in 19MP trees. The tree length aceae,and has aposition within the ‘‘NewWorld and was1,962 steps, when 102ambiguous aligned sites out south Pacic Rim clade’’(subfamilies Gesnerioideae of3,133 sites were excluded,with aCIof 0.682, a RI and Coronantheroideae). of0.624, and aRC of0.425 (T able3). In the strict con- Although partition homogeneity tests (ILD)for the sensus topology,Gesneriaceaespecies formed awell- three gene regions ( trnL-F,atpB-rbcL, and 26S)rejected supported monophyletic group including Titanotri- totalcongruence (P 5 0.214),the resulting tree topol- chum (MP-BS599%, DI514 steps, BA5100%, ML- ogy wassimilar in eachindividual dataset. Thus, we BS5100%,Fig. 1).The Gesneriaceaecan further bedi- combined datafrom three genes toreconstruct aspe- vided into twodistinct clades with high branch 2004] WANGET AL.: PHYLOGENETICPOSITION OF TITANOTRICHUM 413 support,basically correspond ing togeographic distribution(e .g.,Old World species [MP-BS 580%, DI52 steps, BA5100%, ML-BS578%]versus New World plus SouthP acic species [MP-BS 562%, DI53 steps, BA5100%, ML-BS555%]),except for the Asiatic Titan- otrichum,which unexpectedlygrouped with the New World and SouthPaci c Rim clade.Within eachclade , the tribalrelationsh ips were partly resolved.Tribe Ep- ithemateae (MP-BS 597%, DI58 steps, BA5100%, ML- BS596%)was grouped assister tothe rest ofthe Old World species (MP-BS 5100%, DI536 steps, BA5100%, ML-BS5100%),which included the tribes Didymocar- peae,Trichosporeae,and Cyrtandreae. Didymocarpus (Didymocarpeae)w asincluded in apolytomy with representatives oftribe Trichosporeae and Cyrtan- dreae.In the New World clade, Titanotrichum and tribes Beslerieaeand Napeantheaeformed abasalpolytomy in New World and SouthP acic Rim clade. Calceolaria , Jovellana, and Peltanthera oribunda (Loganiaceae)ap- peared tobe the closest sister groups toGesneriacea e (MP-BS577%, DI53 steps, BA5100%, ML-BS590%). Other Scrophulariaceae species were sister toall the abovegroups. The topology ofthe ML tree wasidentical tothe single MPtree (datanot shown). This phylogram also FIG.2. Frequencyof character changes (steps/characters) showed thatmost species from the Neotropics and of CYCLOIDEA nucleotides between differentcodon positions SouthPaci c, including Titanotrichum ,had relatively fromone ofthe MPtreesillustrating the differencesof third short branchlengths compared toOld World species, codon changes between TCP 1 Rdomain (A),and the inter- vening regions(B). particularlyin tribe Epithemateae. CYCLOIDEA GenePhylogen y. Itappears that third codon positions in the TCP 1 Rdomain had four times more changes (2.06)than the rst and second logenyalsore vealed that EpithemaGcyc1 was basal to codon positions (0.51and 0.43,respectiv ely) (Fig. 2A). the rest ofthe Old World Gcyc1 homologues while Bes- This ratiow astwice ashigh per characteras the ratio leria Gcyc1 occupiedthis position among the Neotrop- forinterv ening regions (rst codon position: second ical/SouthPaci c Gcyc1 homologues. Consistentwith codon position: third codon position 5 0.67:0.60: 1.05) the topology inferred from the 3-gene analysis, Calce- (Fig. 2B).Thus, the third codon positions ofthe TCP olaria and JovellanaCY CLOIDEA homologues were the and Rdomain were likely tobe saturated and therefore closest toall Gesneriace aehomologu es (ML-BS 574%, were excluded.The reconstructed CYCLOIDEA gene BA599%, DI55 steps). Antirrhinum CYC and DICH to- phylogeny washighly congruent with ourcombined gether with TetranemaT cyc2 formed adistinctive clade 3-gene analysis, despite anegative result in the ILD (MP-BS574%, ML-BS565%, BA5100%, DI56 steps) test (P50.312).The MPanalysis ofn ucleotide datare- sister tothe previous clade.Similar results were ob- sulted in 12m ost parsimonious trees of1,879 steps tained in the ML analysis (datanot shown). (CI5 0.572, RI50.542 and RC50.310,T able3). The The topology ofthe nucleotidephylogeny wasgen- Gesneriaceaehomologu es formed amonophyletic erally alsorecovere dbythe amino acidanalysis, which group (BA580%, DI54steps, Fig. 3). EpithemaGcyc2 , resulted in 324MP trees of1,089 steps (CI 5 0.729, RI5 RamondaGcyc2, and CyrtandraGcy c2 formed asister 0.684 and RC50.458).The protein strict consensus tree group tothe rest ofthe Gcyc sequences (Gcyc1) albeit only differed from the nucleotide topology in that Na- with low branchsupport (D I 5 4 steps, BA551%). peanthusGcyc1 instead of BesleriaGcyc1 was basal to Gcyc1 homologues couldbe further divided into two the NewWorld/SouthPaci c Rim Gcyc1 clade,and the well-supported clades reecting their geographical grouping of Schizanthuscyc2 and Nicotianacyc1 col- distribution; Old World taxa(MP -BS 551%, DI53 lapsed. In general, the bootstrapsupport v alues forthe steps, BA589%)and NewWorld/SouthPaci c taxa protein tree were lower than those forthe nucleotide (BA586%, DI52steps). The only exception wasthe phylogeny.The protein ML analysis resulted in similar Titanotrichum Gcyc1 homologue,anOld World taxon relationships compared tothe protein MPanalysis. thatwas placed within the latter clade.The Gcyc1 phy- ML branchsupport from TREEPUZZLE wasgenerally 414 SYSTEMATIC BOTANY [Volume 29

FIG.3. Strictconsensus of12 mostparsimonious treesbased on CYCLOIDEA nucleotide sequence data. The numbersabove the branches are bootstrap values and decay indices. Numbersbelow the branches are Bayesian 50% majorityconsensus and MLbootstrap values (bold).Branches inbold indicate thosepersisting in the nucleotide and protein analysis. The gene abbre- viations of CYC homologuesare accordingly as inT able 1.

low butthe major clades were still supported (datanot pared to26S rD NA,the ratiov aried between different shown).Th ebranches thatappeared in bothprotein and CYC regions. For instance,when only TCPand Rdo- nucleotidestrict consensus trees are in bold(Fig. 3). main sequenceswere included,the rate ofevoluti on of In pairwise sequence comparisons (Fig. 4), CYCLO- CYCLOIDEA wasabout the sameasfor the conserved IDEA apparently evolves ca.three times faster than the core region in 26SrD NA. chloroplast trnL-F intron and spacerregion, ca.3.3 In ourresults, CYCLOIDEA had the highest average times faster than the chloroplast atpB-rbcL spacer, and number ofsteps per characteracross trees compared eight times faster than the nuclear26S rD NAregion tothe other gene regions (2.23for nucleotid edata,T a- used here. Although ourresults indicate that CYCLO- ble 3).The tanglegram reconciling the most likely CYC IDEA has aneight times higher rate ofe volution com- gene evolution tree and the corresponding species phy- 2004] WANGET AL.: PHYLOGENETICPOSITION OF TITANOTRICHUM 415

wassampled in either study,without anyNew World or SouthP acic species included,the conclusionsmust beview ed astentative .This shortcoming wasspeci - callyaddressed in ouranalysis. Our combined 3-gene study and the CYCLOIDEA phylogeny is based on a more extensivesampling, including alltribes ofGes- neriaceae and arepresentative range oftaxa from Scro- phulariales s. l.Our results show unambiguously that Titanotrichum lies within Gesneriaceaeand has strong afnities tothe subfamilies Gesnerioideae and Coron- antheroideae rather than subfamilyCyrtandroid eae (Bayesian supportv alues BA-BS 5100%in 3-gene tree and 99% in CYCLOIDEA tree,Figs. 1,3). UnlikeGesnerioideae and Coronantheroideae, Titan- otrichum is ofOld World distribution(Taiwan,Southern Ryukyu and SEChina). All other Old World Gesneri- aceaeshow anisocotyly involving the persistent growth ofa macro-cotyledon (Jong 1973;J ong and Burtt 1975),but Titanotrichum is isocotylous (Wang and Cronk 2003).A recent report ofanisocotyly in Titano- trichum (Wang et al.2002) appears to refer tocotyledon asymmetry upongerminati on rather than apersistent meristematicactivity.The isocotyly of Titanotrichum is consistent with ourplacem ent ofthe genus with the isocotylous New World Gesneriaceae. Titanotrichum FIG.4. Comparative rate of CYCLOIDEA evolution. Each possesses scaly rhizomes, unusualamong Old World point representsa pairwise distance (gaps excluded) between every two sequences of CYCLOIDEA (xaxis) against the pair- species, which led Sealy (1949)to relate the plantto wise distance between any two sequences of trnL-F, 26S or the New World Gesneriaceaegenera Isoloma (5Kohleria) atpB-rbcL (yaxis). [a]. trnL-F (M ) vs. CYC and 26S rDNA( 1) and Naegelia (5Smithiantha )oftribe Gloxinieae. vs. CYC ; [b]. atpB-rbcL (+ ) vs. CYC. Titanotrichum has ahigh chromosome number (2n540)(Ratter 1963),in common with allSouth P a- cic and Australian taxaof subfamily Coronanth ero- logenyis shown in Fig. 5.T en gene duplicationevents ideae analysed todate (2n 5;74, ;80, ;90 with and twelve losses canbe reconstructe d. 2n574m ost common)(Ratter 1963;Ratter and Prentice 1967).Of the closest allies of Titanotrichum in our anal- DISCUSSION ysis, Besleria has 2n532chrom osomes ( Napeanthus has PhylogeneticP ositionof Titanotrichum . From our not been investigated). In subfamilyGesnerioi deae,the combined 3-gene analysis and the CYCLOIDEA phy- majority have 2n 518,22, 26, 28 (Burtt and Wiehler logenyit becomes clearthat ‘ Gesneriaceaeincluding 1995).F or Old World taxa2n 518,30, 32, 34 are most Titanotrichum ’is awell-supported monophyletic group common numbers. (Figs. 1,3). An inconclusive result wasobtain ed in a Evolution of CYCLOIDEA. The evolution of CY- previous study based on more conserved gene regions, CLOIDEA homologues appearedto be well correlated notablychloropla st atpB and rbcL genes and the nucle- with the species phylogeny,ifduplicatio nevents are ar18S rD NAgene (Soltiset al.2000). Alth ough Soltis taken into account(Fig. 5).Reconciling the 3-gene and et al.(2000) found strong branchsupport for a mono- the CYC tree,ten gene duplicationevents are necessary phyletic Gesneriaceae(jackknife support 5 96%), very (Figs. 3,5). In Gesneriaceae,there is one early dupli- few Gesneriaceaetaxaw ere sampled ( Rhynchoglossum , cationevent leading tothe split between Gcyc1 and Cyrtandra, and Streptocarpus ), and Titanotrichum was Gcyc2. The Gcyc homologues ( Gcyc1)seem tobe de- placedas the sister group tothe rest ofthe family.The rived from one lineage ofthis ancestral duplication same conclusion wasdrawn in astudy ofthe phylog- event (Gcyc1), whereas Gcyc2 apparently has been lost enyofthe asterids (Albachet al.2001). They added a in alllineages butEpithema teae and Cyrtandreae further chloroplast gene region ( ndhF)toperform a4- (Figs. 3,5) or has not been found yet. Within Gcyc1, gene analysis, which placed Titanotrichum as sister to several lineage-related independent duplicationevents the remaining Gesneriaceaewith high branchsupport were alsoobserved. One ofthese duplications seems (bootstrapv alue 5 85%,on p.164,Fig. 1B).H owever, toha ve happened within subfamilyCoronanthe ro- becauseonly afractionof the diversity ofthis family ideae (e.g., FieldiaGcy c1F vs. FieldiaGcyc1E ), which 416 SYSTEMATIC BOTANY [Volume 29

FIG.5. Tanglegram ofreconciled MPtreesbased on combined3-gene data and CYCLOIDEA data, representingthe best combination between 30 CYCLOIDEA treesand 32 3-gene (reduced taxa) species treeselected byGENETREE criteria counting the fewestgene duplication and lossevents. Square blocksindicate nodes where the duplication events occurred. may berelated tothe high polyploidyofthis group.A Thereforethe duplicationpattern of Gcyc in Gesner- second duplicationevent happened prior tothe diver- iaceaefalls into twocategories :lineage related dupli- gence of Loxostigma, and Didymocarpus (Loxostigma and cation(associated with cladogenesis events) and the DidymocarpusGcyc1C vs. Loxostigma and Didymocarpus ancient duplicationpredating the split between New Gcyc1D).A third duplicationarose apparently very re- World and Old World taxa.Lineage-rel ated duplica- cently within the genus Streptocarpus (Streptocarpus tions show on average ca.18.5 % amino aciddiv er- Gcyc1A vs. StreptocarpusGcy c1B ),previouslyreported gence butthe ancient duplicationis associatedwith ca. in Citerneet al.(2000). 39%div ergence. 2004] WANGET AL.: PHYLOGENETICPOSITION OF TITANOTRICHUM 417

The amino acidseq uence ofthe TCPand Rdomain facilities. Thismanuscript beneted fromthe expert criticismof P . wasapparently conserved forall taxa w estudied but Herendeen, N.D.Young and J.F .Smith.The PhD studentship of Chun-NengW ang was supported bythe Ministryof Educatio n, anele vated substitution rate atthe third codon posi- Taiwan. tion wasfound in these conserved regions (Fig. 2).The reconstructed CYC nucleotidephylogenywascongru- LITERATURE CITED ent with the amino acidph ylogeny only when the third codon positions ofthe TCPand Rdomain were ex- ALBACH,D.C., P.S.S OLTIS, D. E. SOLTIS, and R. G. OLMSTEAD. cluded. Coding regions beyond these domains were 2001. Phylogenetic analysis ofAsterids based on sequences highly variable,bothin length and amino acidse- offour genes. Annalsof theMissouri Botanical Garden 88: 163– quence.Theycouldeasily bealigned acrossclosely re- 212. BAUM,D.A. 1998. The evolution ofplant development. Current lated taxabut w ere sometimes ambiguousfor taxa Opinion InPlant Biology 1: 79–86. from different families. In addition, Mo¨ller et al.(1999) ———, J. DOEBLEY, V. F. IRISH, and E. M. KRAMER.2002. Response: found that Gcyc evolved in aclock-likemanner in their Missinglinks: the genetic architecture of ower and oral smaller dataset from within Gesneriaceaebut this has diversication. Trends in Plant Science 7: 31–34. not been tested in ourlarger dataset. Judging from BREMER,K. 1994. Branch support and treestability. Cladistics 10: 295–304. ourexperienc ehere,the utility of Gcyc in phylogenetic BULT,C. J., J.A. S WEERE, and E. A. ZIMMER.1995. Crypticsequence studies will bebest atthe genus level or below.Picoet simplicity,nucleotidecompositionbias, and molecular coevo- al.(2002) have even investigated Gcyc sequence varia- lution inthe large subunit ofribosomal D NAinplants: im- tion atthe populationlev el in Ramondamyconi . The use plications forphylogen etic analyses. Annalsof the Missouri Bo- tanical Garden 82: 235–246. of forph ylogeneticpurposes ata higher than ge- Gcyc BURTT,B. L. 1962. Studieson the Gesneriaceae ofthe old world neric level is problematicbecause of alignment dif- XXIV: tentative keysto the tribesand genera. Notesfrom Royal culties and gene duplicationevents. Botanic GardenEdinburgh 24: 205–220. Functional Implicationsfro mthe CYCLOIDEA ———.1977. Classication above thegenus, as exemplied by Homologues. Although only approximately 70%of Gesneriaceae. Plant Systematicsand Evolution Suppl. 1: 97–109. ——— and H. WIEHLER.1995. Classica tionof the family Ges- the ORF of CYCLOIDEA wasisolated in ouranalysis, neriaceae. Gesneriana 1: 1–4. several features indicate thatthese homologues used CHADWICK, M. 1997. Amolecular genetic analysis of oral symmetry. in ouranalysis were functional.All isolated nucleotide PhD Thesis, Universityof East Anglia. sequences couldbe translated into amino acidse- CITERNE, H. L., M. MO¨ LLER,and Q.C. B. C RONK.2000. Diversity ofcycloidea- likegenes in Gesneriace ae inrela tionto oral quences with no premature stopcodons or frame symmetry. Annalsof Botany 86: 167–176. shifts, and allcontained the characteristicTCP and R CUBAS,P.2002. Role ofTCP genesin the e volution ofmorpholog - domain (Cubaset al.1999b). The fewisolated sequenc- ical characters inangiosperms .Pp.247–266 in Developmental es thatcontained stopcodons and frameshifts were genetics and plant evolution, eds. Q.C. B. Cronk,R. M.Bate- believed tobe pseudogen es asthey lacked the Rdo- man, and J.A. Hawkins.London: Taylorand Francis. ———, C. VINCENT, and E. COEN.1999a. Anepigenetic mutation main. In addition,allfunctional homologu es clearly responsiblefor natural variation in oral symmetry. Nature had ahigher substitution rate attheir third codon po- 401: 157–161. sitions compared tothe rst and second position, im- ———, N. LAUTER, J. DOEBLEY, and E. COEN.1999b. The TCP plying thatthe gene is under tight functionalcon- domain: amotiffound inproteins regulating plant growth and development. Plant Journal 18: 215–222. straints, especially in the TCP 1 R domains. DOEBLEY, J. and L. LUKENS.1998. Transcriptional regulatorsand Our analysis clearly places Titanotrichum within theevoluti on ofplant form. ThePlant Cell 10: 1075–1082. Gesneriaceaeand therein with subfamilyGesnerioi- DOYLE J.J. and J.L. D OYLE.1987. Arapid DNAisolationprocedure deae.However, further work is required toestablish forsmall amounts offresh leaf tissue. Phytochemistry Bulletin unambiguously the relationships between Titanotri- 19: 11–15. EISEN,J.A. 1998. Phylogenomics:improving functional predictions chum and its closest allies in tribes Beslerieaeand Na- forunc haracterizedgenesby ev olutionaryanalysis. Genome peantheae.Further molecularand cytologicaldata may Research 8: 163–167. prove usefulin elucidating the precise relationships. A ERIKSSON,T.1998. AutoDecay ver.4.0 (programdistributed by the fully resolved phylogenywould alsoallow a long over- author).Department ofBotany ,StockholmUniv ersity.Stock- holm. due discussion ofthe geographic history ofthe whole FARRIS,J.S. 1989. The retentionindex and homoplasyexcess. Sys- family. tematic Zoology 38: 406–407. ———, M. KA¨ LLERSJO¨ , A. G. KLUGE, and C. BULT.1995. Testing ACKNOWLEDGEMENTS .The authorsthank BengtOxelman, signicance ofincongruen ce. Cladistics 10: 315–319. Uppsala University,forkindly providing primersequences and FELSENSTEIN,J.1985. Condence limitson phylogenetics: an ap- leaf material; DickOlmstead, U niversityof W ashington, foradvice proach usingthe bootstrap . Evolution 39: 783–791. on species selection fromScrophula riaceae; Mathieu Perret,Ge- ———.2002. PHYLIP (PhylogenyInferenceP ackage) version nevaBotanical Garden, Veronica Mayer,Vienna Botanical Garden, 3.6a3. 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