A Phylogeny of Begonia Using Nuclear Ribosomal Sequence ... et al... · A Phylogeny of Begonia...

671

Systematic Botany (2005) 30(3) pp 671ndash682q Copyright 2005 by the American Society of Plant Taxonomists

A Phylogeny of Begonia Using Nuclear Ribosomal Sequence Data andMorphological Characters

LAURA LOWE FORREST1 MARK HUGHES and PETER M HOLLINGSWORTH

Royal Botanic Garden Edinburgh 20A Inverleith Row Edinburgh EH3 5LR Scotland UK1Corresponding author present address Department of Plant Biology University of Southern Illinois

Carbondale Illinois 62901-5905 (lforrestsiuedu)

Communicating Editor Wendy B Zomlefer

ABSTRACT With ca 1400 species Begonia is one of the largest plant genera To address the evolution of Begonia we haveproduced maximum parsimony cladograms for nuclear large subunit and internal transcribed spacer sequence data incombination with 34 informative morphological characters Data were obtained from 64 species of Begonia the monotypicgenus Hillebrandia and both species of Datisca The resulting phylogenetic hypotheses supported three main clades withinBegonia two internally resolved clades of African plants and one unresolved transcontinental clade containing species fromsouthern Africa America Asia and the Socotran archipelago Morphological characters often support well-resolved molec-ular clades Tepal number in the staminate and carpellate flowers and fruit characters are discussed None of the morpho-logical characters sampled including traditionally emphasized characters in sectional delimitations such as locule numberand number of placental branches provides a basis for subdividing Begonia into easily circumscribed monophyletic groupsImplications for future sectional classifications of Begonia are discussed

Begonia L is one of the largest genera of vascularplants with ca 1400 species divided into 66 sections(Doorenbos et al 1998 Shui et al 2002 Forrest andHollingsworth 2003 de Wilde and Plana 2003) Mostspecies are monoecious perennials although a few areeither dioecous or annual Begonia has a near-pantrop-ical distribution absent only from the Australian trop-ical forests (Richardson 1993) A single species B gran-dis extends the distribution of the genus into the tem-perate zone growing as far north as the Western Hillsnear Beijing (China)

Begoniaceae also includes the monotypic genus Hil-lebrandia Oliver distinguished by a semi-inferior in-completely closed ovary fruits that dehisce betweenthe styles and more numerous more highly differen-tiated sepals and petals It is the only member of thefamily native to the Hawaiian archipelago and is alsothe only known example of a relict plant genus withinthe Hawaiian flora (Clement et al 2004) The New Gui-nean genus Symbegonia Warb characterised by afleshy conical androecium pistillate flowers with aconnate perianth and distinctive anther endothecialcells (without thickening Tebbitt and McIver 1999)was reduced to a section within Begonia by Forrest andHollingsworth (2003) Synapomorphies of the familyinclude an asymmetric leaf shape dry three-wingedfruit and bifid style which are common to most of thespecies and a ring of collar cells below the micropylar-hilar part of the seed present in all species (Boumanand de Lange 1983)

Begonia because of its remarkable species-richnessprovides a model for investigating the processes re-sponsible for the generation of biodiversity and mor-phological innovation in large genera A prerequisiteto understanding such processes is a reliable estimateof phylogenetic relationships Morphological cladistic

analyses of the genus have been undertaken by Tebbitt(1997) Badcock (1998) and Forrest (2001) All pro-duced largely unresolved topologies with high degreesof homoplasy and few synapomorphies Sosef (1994)conducted an analysis limited to the African sectionsLoasibegonia and Scutobegonia This produced a well-re-solved topology suggesting that homoplasy may beless of a problem at the sectional level in Begonia whileacross the entire genus similar morphologies are re-currently evolving in independent lineages Howeverprior to morphology-based analyses of species fromwithin sub-generic groups in Begonia the isolation ofsuitable monophyletic units (which may or may not beequivalent to sections) is imperative and the morpho-logical characters that have been considered thus farare inappropriate for complete resolution

DNA sequence characters have also been used toaddress phylogenetic and biogeographic questionswithin the family Earlier work used the chloroplasttrnC-trnD spacer region (Badcock 1998) SubsequentlyForrest and Hollingsworth (2003) used a combinationof nuclear ribosomal internal transcribed spacer (ITS)58S and large subunit (LSU or 26S) sequences Plana(2003) the chloroplast region trnL and Plana et al(2004) combined ITS and trnL sequences Clements etal (2004) used a combination of sequences from thenuclear ribosomal regions ITS 58S and small subunit(SSU or 18S) and the chloroplast rbcL region

In this study we have expanded the data set fromForrest and Hollingsworth (2003) by sampling morespecies for the internal transcribed spacers (includingthe 58S region) However morphological data are im-portant for considerations of evolution within the ge-nus and to utilize this we have coded 45 morpholog-ical characters to add to the matrix which will provide

672 [Volume 30SYSTEMATIC BOTANY

TABLE 1 List of characters used in the morphological data set for Begonia Numbers in parenthesis indicate proportion () of samplesfor which data were missing and the fit of the characters (ci ri rc) to one of 10000 MPTs from analysis of the combined DNA andmorphological data set

VEGETATIVE CHARACTERS1 Stem tubers 0-absent 1-present (0 0333 0000 0000) 2 Root tubers 0-absent 1-present (43 0500 0000 0000) 3 Bulbils0-absent 1-present (0 1000 1000 1000) 4 Tubercils 0-absent 1-present (14 0500 0000 0000) 5 Caudex 0-absent 1-present (43 1000 00 00) 6 Leaf shape 0-simple 1-compound (00 0500 0500 0250) 7 Attachment of lamina andpetiole 0-basifixed 1-peltate (14 0250 0700 0175) 8 Trichome ring at top of petiole 0-absent 1-present (43 0250 04000100) 9 Stipule edge 0-entire 1-fringed (116 0200 0692 0138) 10 Tomentose hairs 0-absent 1-present (58 0250 02500062) 11 Stellate hairs 0-absent 1-present (87 0500 0900 0450)

SEXUAL CHARACTERS INFLORESCENCE12 Lifestyle 0-perennial 1-one or more generations per year (00 1000 00 00) 13 Inflorescence position 0-axile 1-terminal(101 0667 0500 0333) 14 Sexual separation 0-dioecious 1-monoecious (00 0500 0500 0250) 15 Inflorescence type 0-monochasial cyme 1-dichasial cyme 2-raceme (145 0667 0909 0606) 16 Inflorescence symmetry 0-symmetric 1-asymmetric(304 constant) 17 Flower number per inflorescence 0-less than seventy 1-over one hundred (87 0500 0667 0333) 18Sexual separation 0-staminate and pistillate in same inflorescence interspersed 1-staminate and pistillate in same inflorescencepistillate basal 2-staminate and pistillate on separate inflorescences (203 0400 0667 0267) 19 Flower sizes 0-similar instaminate and pistillate 1-distinctly larger pistillate than staminate (217 1000 00 00) 20 Flower colour (excluding mark-ings) 0-white or pink 1-yellow 2-red 3-orange (58 0500 0700 0350) 21 Flower pattern 0-tepals all one colour 1-bothtepals with similar red veins or patches 2-red veins or patches only on one tepal (333 0400 0700 0280) 22 Scent 0-imperceptible 1-strong (246 0333 0000 0000) 23 Perianth tube 0-absent 1-present (00 1000 00 00)

SEXUAL CHARACTERS STAMINATE FLOWER24 Tepal number 0-two tepals 1-four tepals 2-absent (87 0333 0760 0253) 25 Tepal fusion 0-free 1-partly connate (1741000 00 00) 26 Androecium 0-anthers face all directions 1-anthers face upper and lower tepals 2-anthers face uppertepal (217 0333 0600 0200) 27 Stamen number 0-less than ten 1-ten or more (203 0167 0286 0048) 28 Stamen colour0-yellow 1-orange 2-red (246 1000 00 00) 29 Stamen fusion 0-free 1-fused only in the centre 2-fused only at one side3-on a column (all fused) (217 0150 0469 0070)

SEXUAL CHARACTERS CARPELLATE FLOWER30 Tepal number 0-two tepals 1-three tepals 2-four tepals 3-five tepals 4-six tepals 7-absent (159 0353 0686 0242) 31Tepal fusion 0-free 1-two tepals partly fused 2-all tepals partly fused (217 1000 00 00) 32 Style number 0-two styles1-three styles 2-four styles 3-(five-) six (-seven) styles (145 0444 0667 0296) 33 Style colour 0-yellow 1-greenish 2-white3-pink 4-red (289 0600 0000 0000) 34 Style fusion 0-free 1-fused (203 0091 0375 0034) 35 Ovary position 0-inferior1-semi-inferior (00 1000 00 00) 36 Locule number 0-unilocular 1-two locular 2-three locular 3-four locular 4-(five-) six(-seven) locular (87 0455 0647 0294) 37 Placentation 0-parietal 1-septal 2-axile (58 0400 0796 0308) 38 Placentation0-entire 1-bifid with ovules on inner and outer surfaces of placentae 2-bifid with ovules only on outer surfaces of placentae(275 0143 0727 0104) 39 Fruit wing number 0-absent 1-two wings 2-three wings 3-four wings 4-c six wings (coronate)5-one wing (43 0429 0810 0347) 40 Fruit wing symmetry 0-equal to subequal 1-one distinctly larger (333 0100 05260053) 41 Fruit dry or fleshy 0-dry 1-fleshy (116 0143 0625 0089) 42 Fruit orientation at maturity 0-upright 1-pendantto nodding 2-recurved (391 1000 1000 1000) 43 Beaked fruit 0-absent 1-present (29 constant) 44 Dehiscence 0-notbetween styles 1-between styles (00 1000 1000 1000) 45 Bracteole subtending ovary 0-absent 1-two bracteoles 2-threebracteoles (464 0125 0364 0045)

insight into the evolution of morphology within thegroup

MATERIALS AND METHODS

Taxon Sampling and Outgroup Selection Thirty-six African12 American 14 Asian and two Socotran Begonia species weresampled as was the monotypic genus Hillebrandia The outgroupDatisca was chosen based on studies by Swensen et al (19941998) Wagstaff and Dawson (2000) and Clement et al (2004) Au-thorities sectional delimitations and geographic distributions forthe included species are given in Appendix 1 (following Dooren-bos et al 1998) Twenty-nine ITS sequences were newly generatedwhile the sequence for Hillebrandia was obtained from SusanSwensen (Ithaca College Ithaca NY) Details of voucher speci-mens botanical garden accession numbers and GenBank acces-sion numbers for all taxa are also in Appendix 1 A morphologicaldata matrix was compiled for 45 characters (Table 1) These werebased primarily on living plants sampled for the molecular anal-yses While this sampling strategy reduces taxonomic difficulties(eg species delimitation and how much of the variation within aspecies can be represented by a single exemplar sequence) it also

generates a large amount of missing data for example in caseswhere individuals have remained sterile in cultivation or are di-oecious Where recent revisions exist these were therefore usedto augment the matix (eg de Wilde and Arends 1980 Klazengaet al 1994 Sosef 1994 de Wilde 2002 Tebbitt 2003 de Wilde andPlana 2003) The percentage of missing data for each character isgiven in Table 1 Two of the morphological characters (16 and 43)were constant for this set of taxa while one further character (45)had a very high percentage of missing data (464) (Table 1)These were excluded from the analyses

Molecular Methods DNA sequences for 26S and some of theITS region sequences were obtained as part of a previous analysis(Forrest and Hollingsworth 2003) 29 new ITS sequences were gen-erated using the following protocols

DNA was extracted from fresh or silica gel-dried leaves usinga CTAB method modified from Doyle and Doyle (1987) by theinclusion of 01 2-mercaptoethanol and 01 insoluble PVPP tothe 2 3 CTAB buffer Two chloroformisoamyl alcohol extractionsteps were performed after precipitation with freezer-cold isopro-panol and 30 min in wash buffer (76 ethanol 10 mM sodiumacetate) the resultant DNA pellet was re-suspended in 50 ml tris-ethylenediaminetetraacetic acid (TE)

For most taxa ITS was amplified using primers p4 (White et al

2005] 673FORREST ET AL PHYLOGENY OF BEGONIA

TABLE 2 Data set characteristics and parsimony analysis results over a) a combined DNA and morphological topology (one of 10000MPTs) and b) a DNA topology (one of 6210 MPTs) The consistency index is calculated with uninformative characters excluded

Data setIncluded

charactersConstantcharacters

Parsimonyuninformative

characters

Parsimonyinformativecharacters

TreeLength CI RI RC

ITS 613 208 132 273 1814 1808 03799 04016 06817 06860 02995 03331LSU 540 349 117 74 412 414 05255 05217 07079 07034 04854 04791ITS 1 LSU 1153 557 249 347 2226 2222 04007 04016 06849 06860 03320 03331Morphology 42 0 8 34 211 218 03460 03349 06280 06092 02173 02040Combined 1194 557 257 380 2437 2440 03476 03929 06307 06797 02193 03214

1990) and p6 (Sluiman in Forrest and Hollingsworth 2003) Wherethese did not result in a single clean amplification product 17SEand 26SE were used (Sun et al 1994) Polymerase chain reactions(PCR) were carried out in 50 ml reactions containing 15ndash20 nggenomic DNA 13 PCR buffer (16 mM (NH4)2SO4 67 mM Tris-HCl (pH 88) 001 Tween-20) 25 mM MgCl2 02 mM dNTPs02 mM of each primer and 10 units of BioTaq (Bioline) PCR prod-ucts were electrophoresed on 16 agarose gels to confirm thatthe PCR product was single banded Amplification products werepurified using QIAquick PCR purification kits (QIAGEN) follow-ing protocols supplied by the manufacturer PCR amplification ofITS involved a preliminary denaturing step of 948 C for 3 min (948C for 1 min 558 C for 1 min 728 C for 1 min 30 sec) for 28ndash30cycles and a final extension period of 728 C for 5 min

Cycle sequencing was carried out using Amersham Thermose-quenase II dye terminator cycle sequencing kit (2 ml Thermo Se-quenase II reagent premix 025 mM primer 1ndash3 ml template madeup to 5 ml with water) PCR amplification involved 25 cycles (968C for 10 sec 508 C for 5 sec 608 C for 4 min) Both forward andreverse DNA strands were sequenced Sequences were verifiededited and the two strands were assembled using Sequence Nav-igator (Applied Biosystems Inc) on a G4 Macintosh computer Allsequences were deposited in GenBank

Analyses Sequences were initially aligned using CLUSTAL(Thompson et al 1997) with further alignments performed man-ually in PAUP version 40b10 (Swofford 2000) High rates of se-quence divergence in parts of the ITS data set rendered large partsof the alignment ambiguous (eg the 59 end of the ITS-2 region)Although alignment in such regions was unambiguous withinclades there were ambiguities between major clades These re-gions were excluded from the analysis The data matrix is availableat TreeBASE (study accession number S1220 matrix accessionnumbers M2111 M2112)

Data were analysed using the maximum parsimony algorithmin PAUP version 40b10 (Swofford 2000) Analyses were conduct-ed on the combined DNA and morphological matrix and on theDNA sequence characters alone under Fitch parsimony An initialheuristic search involved 1000 random addition replicates withtree bisection and reconnection (TBR) saving no more than 10most parsimonious trees (MPTs) per replicate The tree outputfrom this round of swapping was input as starting topologies intoa second round with a limit of 10000 on the number of treessaved per replicate Bootstrap support (BS) was estimated with1000 replicates using simple addition and saving no more thanfive trees per step

RESULTS

The combined data matrix consisted of 380 parsi-mony-informative characters (Table 2) Maximum par-simony (MP) analysis found 10000 equally parsimo-nious trees of length 2437 (CI503476 RI506307)Thirty-eight clades had over 50 BS support Analysisof the molecular data set alone found 6210 MPTs oflength 2222 (CI504016 RI506860) (2440 when allcharacters were included in the length estimate ie

three steps longer than the combined analysis topolo-gy) Thirty-five clades had over 50 BS support Thestrict consensus trees from both analyses were identi-cal (Fig 1) The topology of one tree from each analysiswas used to calculate the ensemble consistency (CI)and retention (RI) indices and rescaled consistency in-dex (RC) for all the data sets (see Table 2)

Topology In the combined analysis there was 100BS for the Begoniaceae and 65 BS for Begonia Withinthe genus three main clades were resolved (Figs 1 2)1 a small clade of African species from sections Sex-alaria and Rostrobegonia (98 BS) 2 a clade of Africanspecies (sections Cristasemen Chasmophila LoasibegoniaScutobegonia Squamibegonia Tetraphila and Mezierea)(55 BS) which includes all Malagasy endemic speciesin a monophyletic group (100 BS) and 3 a transcon-tinental clade containing southern African species (sec-tion Augustia) species from the Socotran archipelago(section Peltaugustia) and all American and Asian spe-cies (85 BS)

There was no BS support for the relationship ofthese three clades relative to each other and little sup-ported resolution within the third clade

Morphological Characters Character performance(in terms of fit to the combined DNA and morpholog-ical topology) was varied Twelve characters have aconsistency index (ci) of 1 however this includes eightautapomorphic characters Three characters have a re-tention index (ri) of 1 (Table 1)

Three of the characters are potentially correlatedovary locule number style number and wing numberReconstructions of these were made using MacClade(Maddison and Maddison 2001) (Fig 3) Most com-monly the number of locules styles and wings is thesame for example three-locular fruits usually havethree styles and three wings However style loss canoccur without the loss of one locule as in the Asiansection Platycentrum where a rudimentary third loculecan form in plants with only two styles taxa with onlyone locule can have various numbers of styles and thenumber of styles on some species from section Tetra-phila can vary even within the same collection locality(Arends 1992) Furthermore there are numerous ex-amples of wing loss generally in relation to animaldispersal that are not correlated with losses of styles


FIG 1 Strict consensus of 10000 minimum length trees generated by the heuristic search of combined ITS and LSU dataand morphological data (380 parsimony-informative characters) Numbers above the branches are BS values Three numberedclades (as discussed in the text) are marked in circles SOC 5 Socotran Archipelago

or locules All three of these characters are importantin Begonia classification and identification and havetherefore been included separately in these analyses

Branches that are well supported by DNA data alsoare often well supported by morphological charactersFor example the LoasibegoniaScutobegoniaFilicibegoniaclade (100 BS) is supported by 30 unambiguous mo-lecular character changes and two unambiguous mor-phological character changes the LoasibegoniaScuto-begonia clade (99 BS) is supported by 19 unambigu-ous molecular character changes and five unambigu-ous morphological character changes the Tetraphila

Squamibegonia clade (100 BS) is supported by 33unambiguous molecular character changes and six un-ambiguous morphological character changes Howev-er the Madagascan clade (100 BS) is supported by53 unambiguous molecular character changes but nounambiguous morphological character changes (mo-lecular data not shown morphological changes recon-structed in Fig 4)

DISCUSSION

Topological Relationships A geographical ar-rangement of clades is a recurrent feature in Begonia


FIG 2 A single most parsimonious tree out of 10000 min-imum length trees generated by the heuristic search of com-bined ITS LSU and morphological data Three numberedclades (as discussed in the text) are marked in circles

molecular phylogenies Forrest and Hollingsworth(2003) with less sampling also resolved the three ma-jor clades shown in Fig 1 Clement et al (2004) re-solved all included African taxa as monophyletic sisterto an Asian and American clade (BS 60) Howeverthey did not sample from sections Sexalaria Rostrobe-gonia and Augustia the sections that render Africapolyphyletic in our topology Plana (2003) obtained atranscontinental AsianAmerican clade that also in-cludes the African sections Sexalaria Rostrobegonia andAugustia although the position of this clade is not sup-ported among other African clades Plana et al (2004)obtained similar topologies although they obtained62 BS for the RostrobegoniaSexalaria clade as sisterto an AmericanAsianSouth African clade Africa hasbeen suggested as the area of origin for the genus Be-gonia (eg van den Berg 1985) but a hypothesis ofAfrica as basal within the genus is not strongly sup-ported by our results

CLADE 1 (AFRICA) This clade includes section Ros-trobegonia which contains about ten species and themonotypic section Sexalaria which contains only B an-nobonensis (Doorenbos et al 1998) Two of the threespecies included in our analyses B johnstonii and B

annobonensis are reportedly annual (B annobonensiscan have several life cycles in a year but B johnstoniimay be able to function as a perennial M Hughespers obs) Both sections have been previously consid-ered to be close relatives of the southern African sec-tion Augustia or synonymous with it (Doorenbos et al1998) This relationship with Augustia (which isgrouped within our transcontinental Clade 3) is notsupported by this topology supporting the distinctionof these two sections However these Clade 1 plantswith short life-spans compared to most other (peren-nial) Begonia species may exhibit an increased rate ofmolecular evolution and therefore the placement ofthese on our phylogeny may be affected by homologyCertainly the branch subtending this clade is longerthan those for most other taxa (Fig 2)

CLADE 2 (AFRICA AND MADAGASCAR) Within thisclade the Malagasy species form one monophyleticgroup another includes many epiphytic fleshy-fruitedspecies from sections Tetraphila and Squamibegoniawhile the last includes the lsquorefuge begoniasrsquo (Sosef1994) a predominantly yellow-flowered group con-sisting of sections Loasibegonia and Scutobegonia Therelationships within and between these groups as sup-ported by our analyses have been discussed in detailin Forrest and Hollingsworth (2003) Plana (2003) andPlana et al (2004)

CLADE 3 (TRANSCONTINENTAL) The position of thesouthern African species (section Augustia) is unsup-ported within Clade 3 although the strict consensustree places them within a mostly Asian clade Plana(2003) and Plana et al (2004) also include these south-ern African species within a transcontinental cladeagain without support Augustia and Rostrobegonia(Clade 1) have traditionally been separated by thepresence of a trichome ring at the top of the petiole(absent in Augustia present in Rostrobegonia) (Irmscher1961 Doorenbos et al 1998) A similar ring of hairsoccurs in several independent lineages including Bolbia (South America section Knesbeckia) and B luxu-rians (South America section Scheidweilaria) indicatingthat homoplasy is more likely than homology

The two species from the Socotran archipelago (Bsocotrana and B samhaensis section Peltaugustia) nestdeeply within the transcontinental clade (Clade 3) andresolve within an Asianmdashsouthern African clade assister to the Sri Lankan B malabarica in the strict con-sensus tree Like Rostrobegonia Peltaugustia has beenconsidered to be a close relative of section Augustia(Doorenbos et al 1998) where it was placed by Hooker(1881) and as a subsection by Irmscher (1961) it waselevated to sectional status by Barkley (1972) Hooker(1881) also suggested a link between Peltaugustia andsome peltate fleshy-leaved Indian species in sectionReichenheimia (eg B floccifera) Although some of ourmost parsimonious topologies suggest a relationship


FIG 3 Character reconstruction over a single MPT from the combined molecular and morphological analysis A Stylenumber B Locule number C Wing number

between Augustia and Peltaugustia (data not shown)more sequence data are needed to rule out competinghypotheses (eg an association with Reichenheimia)and to provide support for relationships

Both sections Augustia and Peltaugustia include taxa

with perenniating organs for surviving dry seasons InPeltaugustia B samhaensis has an irregular tuber andplentiful bulbils (comprised of reduced scale-likefleshy leaves enclosed in papery bracts) while B so-cotrana lacks tubers but has bulbils crowded around


FIG 4 Optimization of 45 morphological characters on thetotal evidence tree (Fig 2) using the lsquoTrace all changesrsquo optionin MacClade Numbers correspond to character numbers list-ed in Table 1

the base of the stem (Hughes and Miller 2002) In Au-gustia both the widespread B sutherlandii and the Na-tal species B geranioides have underground (probablyroot) tubers while B sutherlandii also has bulbils pro-duced from the leaf axils these are compressed shootsand are composed mostly of a succulent stem In thelight of a possible phylogenetic relationship there maybe some utility in reassessing the homology of theseorgans However the B dregei group has a differentperenniating organ a caudex This does not develop inplants taken from cuttings and so it may be derivedfrom the hypocotyl The seasonally arid habitats thatall these plants inhabit likely result in parallelism andconvergence

The lack of resolution within Clade 3 is due to theDNA regions sequenced Although ITS offers resolu-tion at the species level in Begonia (Forrest 2001) thesection of the alignments that are useful at the tips ofthe tree have been excluded here due to ambiguitiesbetween them and the outgroup taxa and due to am-biguities between the more divergent African cladesThe nuclear ribosomal LSU region on the other handalthough not alignment-ambiguous is not informativeat the species level In order to resolve these nodestogether with the deeper level branching patterns fastevolving coding genes which lack alignment prob-lems should be also be sampled

Morphological Evolution The performance of themorphological characters was extremely varied (Table1) the best characters (in terms of ci ri and rescaledconsistency index [rc]) are the presence of leafy bulbilswhether the inflorescence is a monchasial or dichasialcyme or a raceme and whether dehiscence is betweenthe styles (only in Datisca and Hillebrandia) or not (asin Begonia) Leafy bulbils are only present in the twoSocotran species and monochasial cymes are a syna-pomorphy for a clade containing the African sectionsFilicibegonia Loasibegonia and Scutobegonia

The morphological evolution in the group mirrorsthe molecular evolution in that clades well supportedby molecular data are also often supported by mor-phological changes as for example the LoasibegoniaScutobegoniaFilicibegonia clade and the LoasibegoniaScutobegonia clade Many species within this grouphave relatively large bright yellow flowers and mono-chasial inflorescences and they are all rhizomatousherbs These sections have also been grouped togetheron the basis of seed characteristics (de Lange and Bou-man 1992)

Likewise the TetraphilaSquamibegonia clade hasgood molecular and morphological support It in-cludes most of the fleshy-fruited African Begonia spe-cies and many epiphytes It contains several widelydistributed species not only on mainland Africa butalso to the west of the mainland in the Gulf of Guinea(on the islands of Sao Tome and Principe) and on the


Mascarene Islands to the east of Africa The fleshyfruits of these plants are likely to correlate to bird dis-persal which may explain the high representation ofspecies from this clade on islands

In contrast the Malagasy clade which is stronglysupported by molecular data has no unambiguousmorphological synapomorphies which may explainthe failure of traditional classifications to recognizethis geographical grouping of plants (eg Doorenboset al 1998) This clade also exemplifies the strong geo-graphic structure found within Begonia not always ev-ident from morphological taxonomic treatments

Between the major clades and within the transcon-tinental group (Clade 3) there are few well-supportedrelationships using either molecular or morphologicaldata In general (with the exception of the Malagasyspecies) taxa that are classifiable without moleculardata also form supported clades in the molecular anal-yses (but note that alignment problems are acting onthe amount of molecular information for our transcon-tinental clade) whereas relationships that were contro-versial without molecular data are also poorly sup-ported with it This may relate to the amount of ex-tinction that has acted on each of the clades As Cronk(1989) pointed out speciation produces clades but ex-tinction produces taxa Africa with ca 160 species isspecies-depauperate compared to Asia (ca 660 spe-cies) and America (ca 600 species) and the two Afri-can clades are species depauperate compared with thetranscontinental clade If either antiquity andor cli-matic oscillations have led to a higher extinction of Af-rican species this could explain the increased morpho-logical and molecular discontinuities between Africanclades and therefore the greater ease in resolving theclades from that continent

The performance of morphological characters variesbetween clades For example whether or not a leaf ispeltate only has a ci of 025 but an ri of 070 (Table 1)although peltate leaves are found in many sections itis a consistent character in the African LoasibegoniaScutobegonia clade Similarly staminate flower tepalnumber has a ci of 033 but an ri of 076 (Table 1)

TEPALS The two whorls of tepals in staminate Be-gonia flowers have been distinguished as petals andsepals by several authors (eg de Candolle 1859 1864Irmscher 1925) All Begonia species are considered tohave two sepals in the staminate flower (Badcock1998) so this character could be coded as presence orabsence of petals Since we have not examined the vas-cularisation we decided not to make this assumptionHowever considering the changes between two andfour tepals on our topology (Fig 5a) all transforma-tions consist of reductions from four to two tepals con-sistent with the loss of a whorl (fitting a Dollo evolu-tionary model)

Carpellate flower tepal number is far more variable

than in staminate flowers with seven possible statesabsent in Datisca ten in Hillebrandia within Begonia wehave sampled taxa with two three four five and sixtepals Resolution is not as clear as with staminateflower tepal number for example taxa with six tepals(B socotrana B samhaensis) are derived from a five-tepalled clade (Fig 5b) Most of the main African clade(Clade 2) has taxa with either two or four tepals in thecarpellate flower but B bogneri is an exception with sixtepals derived from a four-tepalled clade

Barabe (1980) looked at the vascularisation of the te-pals of carpellate flowers of B handelii Irmscher (sec-tion Sphenanthera) and suggested that as in the sta-minate flowers these could be differentiated intowhorls of sepals and petals However this has notbeen widely accepted Another factor to consider is te-pal fusion In section Symbegonia the five tepals of thecarpellate flower are fused along their margins into atube (associated with sunbird pollination) The two te-pals (sepals) of the staminate flowers are also partiallyconnate Although non-homologous organs may be ad-nate (eg the stamens to the petals in Scrophulari-aceae) connation of components from two separatewhorls cannot happen and therefore at least in sectionSymbegonia we suggest that the five carpellate tepalsare either petals or sepals but not a combination ofboth

FRUIT Locule number is important in sectionalclassification of Begonia the most common number inthe genus is three although there are species with onetwo four and six locules It has been suggested thatunilocular species with parietal placentation as foundin the African section Mezierea and the Asian sectionCoelocentrum are pleisiomorphic within the genus (Jinand Wang 1994) this matches the condition in DatiscaHowever our data indicate that the unilocular condi-tion has evolved independently through reduction inlocule number in both sections rather than being plei-siomorphic for the genus (Fig 3b) Similarly placentalbranch number has lsquolsquoan important rolersquorsquo in Begonia clas-sification (Doorenbos et al 1998 28) Most Begonia spe-cies have two placental branches in each locule withovules on both sides of both branches However therehave been five independent cases of the loss of one ofthese branches over this topology

The mature fruit is commonly dry and papery andBegonia seeds are usually dispersed by wind or rain-splash (de Lange and Bouman 1999) However somespecies have fleshy fruits presumably adapted for zo-ochory Fleshy fruits have evolved five to seven timesover this phylogeny four to six times independentlyin Africa and once in Asia (Fig 5c) Similar adapta-tions to seed dispersal evolving in different cladescauses homoplasy in morphology-based cladistic re-constructions within Begonia as there are a suite ofdisperser-correlated characters (eg fleshiness fruit


FIG 5 Character reconstruction over a single MPT from the combined molecular and morphological analysis A Staminatetepal number B Carpellate tepal number C Fruit dry or fleshy

shape fruit wing absence and presence wing shapeand fruit dehiscence) that can swamp phylogenetic sig-nal if unrecognized

IMPLICATIONS FOR CLASSIFICATION A robust sub-generic framework is helpful for identifying species in

large genera (Cronk 1989) However only two of the34 informative morphological characters are synapo-morphic at the subgeneric level (leaf bulbils and inflo-rescence branching) and they group relatively fewspecies within the crown group We cannot draw con-


clusions about the infrageneric classification of theAmerican and Asian sections due to our limited sam-pling of these taxa Within Africa however three sec-tions require recircumscription Section Mezierea re-solves as polyphyletic and sections Loasibegonia andTetraphila are paraphyletic having sections Scutobegoniaand Squamibegonia respectively nested within themMezierea is traditionally diagnosed by having placen-tation that is topographically parietal for more than60 of the ovary (Klazenga et al 1994) Loasibegonia isseparated from Scutobegonia by the amount of schler-enchymatous sheathing on tertiary leaf veins the ori-entation of the mature fruit and the ovaryfruit shape(Doorenbos et al 1998) while Tetraphila is traditionallydistinguished from Squamibegonia by the absence of aperianth tube in the carpellate flower fruit dehiscenceand the absence of large persistent bracts (Doorenboset al 1998) The characters that distinguish Meziereafrom all other sections and Loasibegonia from Scutobe-gonia are quantitative and were not included in ouranalyses Morphological synapomorphies for redefinedmonophyletic sections will likely be revealed by care-ful study of such qualitative characters in the light ofour molecular phylogeny

ACKNOWLEDGEMENTS This work forms a portion of the firstauthorrsquos PhD for which funding from The M L MacIntyre Be-gonia Research Trust is gratefully acknowledged Molecular stud-ies were also supported by a NERC Glasgow Taxonomy Initiativegrant We would also like to thank the horticulture staff at GBGand E for maintaining the Begonia living collections used in thisstudy M Hollingsworth and A Clark for laboratory assistance DHarris and coworkers at E for assistance in the herbarium D Nick-rent for discussion on petal and sepal fusion and J C VillarrealA M C Tebbitt V Plana P S Herendeen and W B Zomleferfor helpful comments on the manuscript

LITERATURE CITED

ARENDS J C 1992 The biosystematics of Begonia squamulosa Hookf and affiliated species in Tetraphila A DC Wageningen Ag-ricultural University Papers 91-6

BADCOCK Z 1998 A phylogenetic investigation of Begonia L sectionKnesbeckia (Klotzsch) A DC PhD thesis University of Glas-gow

BARKLEY F A 1972 Nomenclatural notes on Begonia Phytologia24 156

BARABE D 1980 The perianth of Begonia flowers The Begonian 47268ndash270

BOUMAN F and A DE LANGE 1983 Structure micromorphologyof Begonia seeds The Begonian 50 70ndash78 91

CANDOLLE A DE 1859 Memoire sur la famille des BegoniaceesAnnales des Sciences Naturelles Botanique serie IV 11 93ndash115

mdashmdashmdash 1864 Ordo CLXXII Begoniaceae Prodromus systematis na-turalis regni vegetabilis 15 1 266ndash408 Paris Victoris Massonet filii

CLEMENT W L M C TEBBITT L L FORREST J E BLAIR LBROUILLET T ERIKSSON and S M SWENSEN 2004 Phyloge-netic position and biogeography of Hillebrandia sandwicensis(Begoniaceae) a rare Hawaiian relict American Journal of Bot-any 91 905ndash917

CRONK Q C B 1989 Measurements of biological and historicalinfluences on plant classifications Taxon 38 357ndash370

DOORENBOS J M S M SOSEF and J J F E DE WILDE 1998 Thesections of Begonia including descriptions keys and specieslists Studies in Begoniaceae VI Wageningen Agricultural Uni-versity Papers 98-2 1ndash266

DOYLE J J and J L DOYLE 1987 A rapid DNA isolation proce-dure for small quantities of fresh leaf material PhytochemicalBulletin 19 11ndash15

FORREST L L 2001 Phylogeny of Begoniaceae PhD thesis Univer-sity of Glasgow

mdashmdashmdash and P M HOLLINGSWORTH 2003 A recircumscription ofBegonia based on nuclear ribosomal sequences Plant System-atics and Evolution 241 193ndash211

HOOKER J D 1881 New Garden Plants Gardenerrsquos Chronicle sn15 8

HUGHES M and A G MILLER 2002 A new endemic species ofBegonia (Begoniaceae) from the Socotra archipelago Edin-burgh Journal of Botany 59 273ndash281

IRMSCHER E 1925 Begoniaceae Pp 548ndash588 in Die NaturlichenPflanzenfamilien 2nd edition vol 21 eds A Engler and KPrantl Leipzig Wilhelm Engelmann

mdashmdashmdash 1961 Monographische revision der Begoniaceen Afrikas1 Sekt Augustia und Rostrobegonia sowie einige neue Sippenaus anderen Sektionen Botanische Jahrbucher fur SystematikPflanzengeschichte und Pflanzengeographie 81 106ndash188

JIN X and F-H WANG 1994 Style and ovary antomy of ChineseBegonia and its taxonomic and evolutionary implications Ca-thaya 6 125ndash144

KLAZENGA N J J F E DE WILDE and R J QUENE 1994 Begoniasect Mezierea (Gaud) Warb a taxonomic revision Bulletin duJardin Botanique National de Belgique 63 263ndash312

LANGE A DE and F BOUMAN 1992 Seed micromorphology of thegenus Begonia in Africa taxonomic and ecological implica-tions Wageningen Agricultural University Papers 91-4 1ndash82

mdashmdashmdash and mdashmdashmdash 1999 Seed micromorphology of Neotropicalbegonias Smithsonian Contributions to Botany 90 WashingtonDC Smithsonian Institute Press

MADDISON D R and W P MADDISON 2001 MacClade analysisof phylogeny and character evolution version 403 Sunder-land Sinauer Associates

PLANA V 2003 Phylogenetic relationships of the Afro-Malagasymembers of the large genus Begonia inferred from trnL intronsequences Systematic Botany 28 693ndash704

mdashmdashmdash A GASCOIGNE L L FORREST D HARRIS and R T PEN-NINGTON 2004 Pleistocene and pre-Pleistocene Begonia spe-ciation in Africa Molecular Phylogenetics and Evolution 31 449ndash461

RICHARDSON I B K 1993 Begoniaceae Pp 113ndash114 in Floweringplants of the world ed V H Heywood London BT BatsfordLtd

SHUI Y-M C-I PENG and C-Y WU 2002 Synopsis of the Chinesespecies of Begonia (Begoniaceae) with a reappraisal of sec-tional delimitation Botanical Bulletin of Academia Sinica 43313ndash327

SOSEF M S M 1994 Refuge begonias taxonomy phylogeny andhistorical biogeography of Begonia sect Loasibegonia and sectScutobegonia in relation to glacial rain forest refuges in Africa(Studies in Begoniaceae 5) Wageningen Agricultural UniversityPapers 1ndash306

SUN Y D Z SKINNER G H LANG and S H HULBERT 1994Phylogenetic analysis of sorghum and related taxa using in-ternal transcribed spacers of nuclear ribosomal DNA Theo-retical and Applied Genetics 89 26ndash32

SWENSEN S M J N LUTHI and L H RIESEBERG 1998 Datisca-ceae revisited monophyly and the sequence of breeding sys-tem evolution Systematic Botany 23 157ndash169

mdashmdashmdash B C MULLIN and M W CHASE 1994 Phylogenetic affin-ities of Datiscaceae based on an analysis of nucleotide se-


quences from the plastid rbcL gene Systematic Botany 19 157ndash168

SWOFFORD D L 2000 PAUP Phylogenetic analysis using parsi-mony (and other methods) version 40b10 Sunderland Sin-auer Associates

TEBBITT M C 1997 A systematic investigation of Begonia sectionSphenanthera (Hassk) Benth amp Hook f PhD thesis Univer-sity of Glasgow

mdashmdashmdash 2003 Notes on South Asian Begonia (Begoniaceae) Edin-burgh Journal of Botany 60 1ndash9

mdashmdashmdash and C M MACIVER 1999 The systematic significance ofthe endothecium in Begoniaceae Botanical Journal of the Lin-nean Society 131 203ndash221

THOMPSON J D D G HIGGINS and T J GIBSON 1997 CLUSTALW improving the sensitivity of progressive multiple se-quence alignment through sequence weighting position spe-cific gap penalties and weight matrix choice Nucleic AcidsResearch 22 4673ndash4680

VAN DEN BERG R G 1985 Pollen morphology of the genus Begoniain Africa Agricultural University Wageningen Papers 84-3 5ndash94

WAGSTAFF S J and M I DAWSON 2000 Classification originand patterns of diversification of Corynocarpus (Corynocar-paceae) inferred from DNA sequences Systematic Botany 25134ndash149

WHITE T J T BRUNS S LEE and J TAYLOR 1990 Amplificationand direct sequencing of fungal ribosomal RNA genes forphylogenetics Pp 315ndash322 in PCR protocols a guide to methodsand applications eds M A Innis D H Gelfand J J Sninskyand T J White San Diego Academic Press

DE WILDE J J F E 1985 Begonia section Cristasemen J J de Wildesect nov Agricultural University Wageningen Papers 84 113ndash129

mdashmdashmdash 2002 Begonia section Tetraphila A DC a taxonomic revi-sion Studies in Begoniaceae VII Wageningen Agricultural Uni-versity Papers 2001-1 5ndash258

mdashmdashmdash and J C ARENDS 1980 Begonia section SquamibegoniaWarb A taxonomic revision Miscellaneous Papers Landbouwho-geschool Wageningen 19 377ndash421

mdashmdashmdash and V PLANA 2003 A new section of Begonia (Begoni-aceae) from tropical west central Africa Edinburgh Journal ofBotany 60 121ndash130

APPENDIX 1Taxa included in this study with sectional placements geo-

graphic distributions and GenBank accession numbers Taxonomyfollows Doorenbos et al (1998) Where available botanic gardenaccession numbers and voucher specimen numbers are given E 5Royal Botanic Gardens Edinburgh GBG 5 Glasgow Botanic Gar-den K 5 Royal Botanic Gardens Kew

B aequata A Gray Sect Petermannia (Klotzsch) A DC AsiaPhilippines (Luzon) E 1997 2515 Wilkie et al sn (E) ITSAF485147 LSU AF468135 B angularis Raddi Sect Pritzelia(Klotzsch) A DC America Brazil (Rio de Janeiro Minas Gerais)E 1969 1797 Forrest L L 172 (E) ITS AF485183 LSU AF468130B ankaranensis Humb ex Keraudren amp Bosser Sect QuadrilobariaA DC Africa Madagascar GBG 001 064 97 no voucher availableITS AF485229 LSU AF468123 B annobonensis A DC Sect Sex-alaria A DC Africa Cameroon Principe Sao Tome Pagalu (An-nobon) GBG 007 059 98 Forrest L L 231 (E) ITS AF485243 LSUAF468114 B aspleniifolia Hook f ex A DC Sect Filicibegonia ADC Africa Gabon GBG 001 079 97 Forrest L L 209 (E) ITSAF485227 LSU AF468150

B balansana Gagnep Sect Ignota Asia Vietnam Tebbitt M C65 (BKL) ITS AF485091 LSU AF468146 B bogneri Ziesenh SectErminea A DC Africa Madagascar E 1986 0844 Forrest L L 213(E) ITS AF485235

B capillipes Gilg Sect Tetraphila A DC Africa CameroonEquatorial Guinea Gabon GBG 004 079 97 Forrest L L 216 (E)

ITS AF485238 LSU AF468116 B cinnabarina Hook Sect Eupetalum(Lindl) A DC America Bolivia (Acero Cordillera) GBG 008 00692 Forrest L L 183 (E) ITS AF485199 B convolvulacea (Klotzsch)A DC Sect Wageneria (Klotzsch) A DC America Brazil (CearaBahia Rio de Janeiro) GBG 001 093 79 Forrest L L 168 (E) ITSAF485178 LSU AF468131 B crassirostris Irmsch Sect Sphenanth-era (Hassk) Warb Asia China GBG 007 079 97 Forrest L L 109(E) ITS AF485103 LSU AF468144

B dewildei Sosef Sect Scutobegonia Warb Africa Gabon GBG001 041 97 Forrest L L 208 (E) ITS AF485226 LSU AF468125 Bdipetala R Grah Sect Haagia (Klotzsch) A DC Asia India GBG001 004 86 Forrest L L 239 (E) ITS AF469124 LSU AF468149 Bdregei Otto amp Dietr Sect Augustia (Klotzsch) A DC Africa SouthAfrica GBG 004 026 94 Forrest L L 241 (E) ITS AF469126 Bdregei Otto amp Dietr lsquopartitarsquo Sect Augustia (Klotzsch) A DC Af-rica South Africa GBG 002 036 89 Forrest L L 240 (E) ITSAF469125 B dregei Otto amp Dietr subsp homonyma Steud SectAugustia (Klotzsch) A DC Africa South Africa GBG 006 053 89no voucher available ITS AF469119 B duncan-thomasii Sosef SectLoasibegonia A DC Africa Cameroon GBG 009 079 97 Forrest LL 206 (E) ITS AF485224

B edmondoi Brade Sect Gaerdtia (Klotzsch) A DC AmericaBrazil (Rio de Janeiro) GBG 016 103 83 Forrest L L 196 (E) ITSAF485214 B engleri Gilg Sect Rostrobegonia Warb Africa Tan-zania E 1998 2762 Forrest L L 236 (E) ITS AF485248 LSUAF468113

B floccifera Bedd Sect Reichenheimia (Klotzsch) A DC AsiaIndia GBG 030 099 89 Forrest L L 238 (E) ITS AF469123 LSUAF468142 B francoisii Guillaumin Sect Quadrilobaria A DC Af-rica Madagascar GBG 002 064 97 Forrest L L 212 (E) ITSAF485234 LSU AF468121

B gabonensis J J de Wilde Sect Tetraphila A DC Africa GabonGBG 017 079 97 Forrest L L 230 (E) ITS AF485242 B geranioidesHook f Sect Augustia (Klotzsch) A DC Africa South AfricaGBG 018 079 97 no voucher available ITS AF469120 LSUAF468128 B goegoensis N E Br Sect Reichenheimia (Klotzsch) ADC Asia Indonesia (Sumatra) GBG 011 125 57 Forrest L L 132(E) ITS AF485138 B grandis Dryand subsp holostyla Irmsch SectDiploclinium (Lindl) A DC Asia China E 1998 0035 Forrest LL sn (E) ITS AF485088 LSU AF468145

B holtonis A DC Sect Ruizopavonia A DC America ColombiaEcuador GBG 011 129 84 Forrest L L 152 (E) ITS AF485162 LSUAF468129 B horticola Irmsch Sect Tetraphila A DC Africa Con-go Dem Rep Congo Uganda GBG 021 079 97 Forrest L L 219(E) ITS AF485241

B incarnata Link amp Otto Sect Knesbeckia (Klotzsch) A DCAmerica Mexico GBG 011 089 95 Forrest L L 162 (E) ITSAF485173 LSU AF468138 B integerrima Spreng Sect SolanantheraA DC America Brazil (Rio de Janeiro Minas Gerais Sao Paulo)GBG 019 167 95 Forrest L L 149 (E) ITS AF485158 B iucundaIrmsch Sect Chasmophila de Wilde and Plana Africa CongoDem Rep Congo GBG 022 079 97 no voucher available ITSAF485249 LSU AF468127

B johnstonii Oliv ex Hook f Sect Rostrobegonia Warb AfricaKenya Uganda Tanzania E 1999 0653 Forrest L L 235 (E) ITSAF485247

B kisuluana Buttn Sect Tetraphila A DC Africa Nigeria toUganda south to Angola GBG 023 079 97 Forrest L L 215 (E)ITS AF485237

B letouzeyi Sosef Sect Loasibegonia A DC Africa CameroonGabon Congo GBG 026 079 97 Forrest L L 207 (E) ITSAF485225 B lobata Schott Sect Pritzelia (Klotzsch) A DC Amer-ica Brazil (Rio de Janeiro Minas Gerais) GBG 020 167 95 ForrestL L 174 (E) ITS AF485185 LSU AF468134 B longipetiolata GilgSect Tetraphila A DC Africa Nigeria to Dem Rep Congo GBG028 079 97 Forrest L L 233 (E) ITS AF485245 B loranthoides Hookf subsp rhopalocarpa (Warb) J J de Wilde Sect Tetraphila A DCAfrica Cameroon to Dem Rep Congo Sao Tome GBG 001 086


80 Forrest L L 200 (E) ITS AF485218 B luxurians Schweid SectScheidweilaria (Klotzsch) A DC America Brazil (Sao Paulo to Mi-nas Gerais) E 1968 5494 Forrest L L 179 (E) ITS AF485192 LSUAF468132

B madecassa Keraudren Sect Nerviplacentaria A DC AfricaMadagascar GBG 003 064 97 Forrest L L 211 (E) ITS AF485230LSU AF468122 B malabarica Lam non A DC Sect Ignota AsiaIndia Sri Lanka GBG 002 018 96 Forrest L L 288 (E) ITSAF485140 LSU AF468141 B mananjebensis Humb ex Bosser ampKeraudren Sect Quadrilobaria A DC Africa Madagascar GBG004 064 97 no voucher available ITS AF485231 B mannii HookSect Tetraphila A DC Africa West amp Central Africa GBG 008 06780 Forrest L L 217 (E) ITS AF485239 B masoniana Irmsch SectCoelocentrum Irmsch Asia cult Singapore E 1998 0074 ForrestL L 123 (E) ITS AF485123 LSU AF468136 B meyeri-johannisEngl Sect Mezierea (Gaud) Warb Africa east Africa GBG 002041 97 Forrest L L 214 (E) ITS AF485236 LSU AF468118 Bmolleri Warb Sect Tetraphila A DC Africa Sao Tome GBG 038079 97 Forrest L L 218 (E) ITS AF485240 LSU AF468117

B nossibea A DC Sect Quadrilobaria A DC Africa Madagas-car GBG 007 064 97 no voucher available ITS AF485233 LSUAF468120

B obliqua L Sect Begonia America Martinique GBG 005 10591 Forrest L L 160 (E) ITS AF485170 LSU AF468148 B olbiaKerch Sect Knesbeckia (Klotzsch) A DC America Brazil GBG002 117 94 Forrest L L 185 (E) ITS AF485201

B palmata D Don Sect Platycentrum (Klotzsch) A DC AsiaIndia Nepal Burma China E 1998 0059 Forrest L L 24 (E) ITSAF485114 LSU AF468143 B poculifera Hook f Sect Squamibegon-ia Warb Africa Nigeria to Tanzania and Angola E 1992 3143Forrest L L 234 (E) ITS AF485246 LSU AF468115 B potamophilaGilg Sect Loasibegonia A DC Africa Cameroon Gabon CongoGBG 042 097 97 Forrest L L 201 (E) ITS AF485219 B prismato-carpa Hook Sect Loasibegonia A DC Africa Ivory Coast Came-roon Equatorial Guinea E 1972 2267 Forrest L L 202 (E) ITSAF485220

B quadrialata Warb Sect Loasibegonia A DC Africa West andwestern Central Africa GBG 055 079 97 Forrest L L 204 (E) ITSAF485222

B roxburghii A DC Sect Sphenanthera (Hassk) Warb Asia In-

dia Nepal Burma GBG 001 068 98 Forrest L L 103 (E) ITSAF485092 LSU AF468147

B salaziensis (Gaud) Warb Sect Mezierea (Gaud) Warb AfricaReunion Mauritius K 1986 412 no voucher available ITSAF485232 LSU AF468119 B samhaensis M Hughes amp A G MillSect Peltaugustia (Warb) Barkley Socotran Archipelago Samha E1996 2361 Hughes M sn (E) ITS AF469122 B scapigera Hook fSect Loasibegonia A DC Africa Nigeria Cameroon Gabon Con-go GBG 002 057 96 Forrest L L 205 (E) ITS AF485223 LSUAF468126 B scutifolia Hook f Sect Loasibegonia A DC AfricaCameroon Equatorial Guinea Gabon Angola Dem Rep CongoGBG 049 079 97 Forrest L L 203 (E) ITS AF485221 B sericoneuraLiebm Sect Gireoudia (Klotzsch) A DC America Mexico (Chia-pas Oaxaca) GBG 009 124 82 Forrest L L 188 (E) ITS AF485205

B socotrana Hook f Sect Peltaugustia (Warb) Barkley SocotranArchipelago Socotra E 1989 1081 Hughes M sn (E) ITSAF469121 LSU AF468139 B staudtii Gilg Sect Loasibegonia ADC Africa Nigeria Cameroon GBG 052 079 97 Forrest L L 210(E) ITS AF485228 B subscutata J J de Wilde Sect Tetraphila ADC Africa Cameroon to Congo and Dep Rep Congo GBG 153179 97 Forrest L L 232 (E) ITS AF485244 B sutherlandii Hook fSect Augustia (Klotzsch) A DC Africa Dem Rep Congo Tan-zania Zambia Mozambique South Africa E 1971 1552 Forrest LL 197 (E) ITS AF485215 B symsanguinea L L Forrest amp Hollings-worth Sect Symbegonia (Warb) L L Forrest amp HollingsworthAsia New Guinea GBG 003 127 93 Forrest L L 142 (E) ITSAF485151 LSU AF468137

B thomeana C DC Sect Cristasemen J J de Wilde Africa SaoTome GBG 054 079 97 Forrest L L 199 (E) ITS AF485217 LSUAF468124

B violifolia A DC Sect Weilbachia (Klotzsch amp Oersted exKlotzsch) A DC America Mexico (Tabasco) GBG 004 055 87Forrest L L 181 (E) ITS AF485197 LSU AF468140

B sp indet Sect Pritzelia (Klotzsch) A DC America GBG 029023 92 Forrest L L 177 (E) ITS AF485189 LSU AF468133

Datisca cannabina L Eurasia Crete to NE India E 1984 1126Forrest L L 237 (E) ITS AF485251 LSU AF468151 Datisca glom-erata (Presl) Baill America USA (California) Mexico (Baja Cali-fornia) Liston 767 (RSA) ITS AF485250 LSU AF468112

Hillebrandia sandwicensis Oliv Hawaiian archipelago Brouillet L2960-31 ITS AY429337


TABLE 1 List of characters used in the morphological data set for Begonia Numbers in parenthesis indicate proportion () of samplesfor which data were missing and the fit of the characters (ci ri rc) to one of 10000 MPTs from analysis of the combined DNA andmorphological data set

VEGETATIVE CHARACTERS1 Stem tubers 0-absent 1-present (0 0333 0000 0000) 2 Root tubers 0-absent 1-present (43 0500 0000 0000) 3 Bulbils0-absent 1-present (0 1000 1000 1000) 4 Tubercils 0-absent 1-present (14 0500 0000 0000) 5 Caudex 0-absent 1-present (43 1000 00 00) 6 Leaf shape 0-simple 1-compound (00 0500 0500 0250) 7 Attachment of lamina andpetiole 0-basifixed 1-peltate (14 0250 0700 0175) 8 Trichome ring at top of petiole 0-absent 1-present (43 0250 04000100) 9 Stipule edge 0-entire 1-fringed (116 0200 0692 0138) 10 Tomentose hairs 0-absent 1-present (58 0250 02500062) 11 Stellate hairs 0-absent 1-present (87 0500 0900 0450)

SEXUAL CHARACTERS INFLORESCENCE12 Lifestyle 0-perennial 1-one or more generations per year (00 1000 00 00) 13 Inflorescence position 0-axile 1-terminal(101 0667 0500 0333) 14 Sexual separation 0-dioecious 1-monoecious (00 0500 0500 0250) 15 Inflorescence type 0-monochasial cyme 1-dichasial cyme 2-raceme (145 0667 0909 0606) 16 Inflorescence symmetry 0-symmetric 1-asymmetric(304 constant) 17 Flower number per inflorescence 0-less than seventy 1-over one hundred (87 0500 0667 0333) 18Sexual separation 0-staminate and pistillate in same inflorescence interspersed 1-staminate and pistillate in same inflorescencepistillate basal 2-staminate and pistillate on separate inflorescences (203 0400 0667 0267) 19 Flower sizes 0-similar instaminate and pistillate 1-distinctly larger pistillate than staminate (217 1000 00 00) 20 Flower colour (excluding mark-ings) 0-white or pink 1-yellow 2-red 3-orange (58 0500 0700 0350) 21 Flower pattern 0-tepals all one colour 1-bothtepals with similar red veins or patches 2-red veins or patches only on one tepal (333 0400 0700 0280) 22 Scent 0-imperceptible 1-strong (246 0333 0000 0000) 23 Perianth tube 0-absent 1-present (00 1000 00 00)

SEXUAL CHARACTERS STAMINATE FLOWER24 Tepal number 0-two tepals 1-four tepals 2-absent (87 0333 0760 0253) 25 Tepal fusion 0-free 1-partly connate (1741000 00 00) 26 Androecium 0-anthers face all directions 1-anthers face upper and lower tepals 2-anthers face uppertepal (217 0333 0600 0200) 27 Stamen number 0-less than ten 1-ten or more (203 0167 0286 0048) 28 Stamen colour0-yellow 1-orange 2-red (246 1000 00 00) 29 Stamen fusion 0-free 1-fused only in the centre 2-fused only at one side3-on a column (all fused) (217 0150 0469 0070)

SEXUAL CHARACTERS CARPELLATE FLOWER30 Tepal number 0-two tepals 1-three tepals 2-four tepals 3-five tepals 4-six tepals 7-absent (159 0353 0686 0242) 31Tepal fusion 0-free 1-two tepals partly fused 2-all tepals partly fused (217 1000 00 00) 32 Style number 0-two styles1-three styles 2-four styles 3-(five-) six (-seven) styles (145 0444 0667 0296) 33 Style colour 0-yellow 1-greenish 2-white3-pink 4-red (289 0600 0000 0000) 34 Style fusion 0-free 1-fused (203 0091 0375 0034) 35 Ovary position 0-inferior1-semi-inferior (00 1000 00 00) 36 Locule number 0-unilocular 1-two locular 2-three locular 3-four locular 4-(five-) six(-seven) locular (87 0455 0647 0294) 37 Placentation 0-parietal 1-septal 2-axile (58 0400 0796 0308) 38 Placentation0-entire 1-bifid with ovules on inner and outer surfaces of placentae 2-bifid with ovules only on outer surfaces of placentae(275 0143 0727 0104) 39 Fruit wing number 0-absent 1-two wings 2-three wings 3-four wings 4-c six wings (coronate)5-one wing (43 0429 0810 0347) 40 Fruit wing symmetry 0-equal to subequal 1-one distinctly larger (333 0100 05260053) 41 Fruit dry or fleshy 0-dry 1-fleshy (116 0143 0625 0089) 42 Fruit orientation at maturity 0-upright 1-pendantto nodding 2-recurved (391 1000 1000 1000) 43 Beaked fruit 0-absent 1-present (29 constant) 44 Dehiscence 0-notbetween styles 1-between styles (00 1000 1000 1000) 45 Bracteole subtending ovary 0-absent 1-two bracteoles 2-threebracteoles (464 0125 0364 0045)

insight into the evolution of morphology within thegroup

MATERIALS AND METHODS

Taxon Sampling and Outgroup Selection Thirty-six African12 American 14 Asian and two Socotran Begonia species weresampled as was the monotypic genus Hillebrandia The outgroupDatisca was chosen based on studies by Swensen et al (19941998) Wagstaff and Dawson (2000) and Clement et al (2004) Au-thorities sectional delimitations and geographic distributions forthe included species are given in Appendix 1 (following Dooren-bos et al 1998) Twenty-nine ITS sequences were newly generatedwhile the sequence for Hillebrandia was obtained from SusanSwensen (Ithaca College Ithaca NY) Details of voucher speci-mens botanical garden accession numbers and GenBank acces-sion numbers for all taxa are also in Appendix 1 A morphologicaldata matrix was compiled for 45 characters (Table 1) These werebased primarily on living plants sampled for the molecular anal-yses While this sampling strategy reduces taxonomic difficulties(eg species delimitation and how much of the variation within aspecies can be represented by a single exemplar sequence) it also

generates a large amount of missing data for example in caseswhere individuals have remained sterile in cultivation or are di-oecious Where recent revisions exist these were therefore usedto augment the matix (eg de Wilde and Arends 1980 Klazengaet al 1994 Sosef 1994 de Wilde 2002 Tebbitt 2003 de Wilde andPlana 2003) The percentage of missing data for each character isgiven in Table 1 Two of the morphological characters (16 and 43)were constant for this set of taxa while one further character (45)had a very high percentage of missing data (464) (Table 1)These were excluded from the analyses

Molecular Methods DNA sequences for 26S and some of theITS region sequences were obtained as part of a previous analysis(Forrest and Hollingsworth 2003) 29 new ITS sequences were gen-erated using the following protocols

DNA was extracted from fresh or silica gel-dried leaves usinga CTAB method modified from Doyle and Doyle (1987) by theinclusion of 01 2-mercaptoethanol and 01 insoluble PVPP tothe 2 3 CTAB buffer Two chloroformisoamyl alcohol extractionsteps were performed after precipitation with freezer-cold isopro-panol and 30 min in wash buffer (76 ethanol 10 mM sodiumacetate) the resultant DNA pellet was re-suspended in 50 ml tris-ethylenediaminetetraacetic acid (TE)

For most taxa ITS was amplified using primers p4 (White et al



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