Delimitation of Malagasy Tribe Coleeae and Implications for Fruit Evolution in Bignoniaceae Inferred from a Chloroplast DNA Phylogeny

Home , Radermachera, Rhigozum, Spathodea

Plant Syst. Evol. 245: 55–67 (2004) DOI 10.1007/s00606-003-0025-y

Delimitation of Malagasy tribe Coleeae and implications for fruit evolution in Bignoniaceae inferred from a chloroplast DNA phylogeny

M. L. Zjhra1, K. J. Sytsma2, and R. G. Olmstead3

1Department of Biology, Georgia Southern University, Statesboro, GA, USA 2Department of Botany, University of Wisconsin, Madison, WI, USA 3Department of Botany, University of Washington, Seattle, WA, USA

Received February 2, 2003; accepted April 23, 2003 Published online: February 23, 2004 Springer-Verlag 2004

Abstract. Coleeae (Bignoniaceae) are a tribe almost the Gondwanan continent, Madagascar was entirely restricted to Madagascar. Coleeae have attached to Africa at present day Somalia, previously been placed in neotropical Crescentieae Kenya and Tanzania until 165 mya (Rabino- due to species with indehiscent fruits, a character witz et al. 1982, 1983). Madagascar was otherwise unusual in Bignoniaceae. A phylogeny completely separated from Africa by ndh trn based on three chloroplast regions ( F, T-L 120 mya (Rabinowitz et al. 1983), but spacer, trnL-F spacer) identifies a monophyletic remained attached to India until 88 mya Coleeae that is endemic to Madagascar and sur- rounding islands of the Indian Ocean (Seychelles, (Storey et al. 1995). During the lower Creta- Comores and Mascarenes). African Kigelia is not a ceous Madagascar traveled to its present member of Coleeae, rather it is more closely related position 400 km off the coast of Mozambique to a subset of African and Southeast Asian species (Fig. 1). The granitic islands of the Seychelles, of Tecomeae. The molecular phylogeny indicates in the Indian Ocean north of Madagascar, that indehiscent fruit have arisen repeatedly in represent fragments of the separation of Mad- Bignoniaceae: in Coleeae, Kigelia and Crescentieae. agascar-India from Africa. In reference to The characteristic fleshy fruits of species of Coleeae Madagascar, Wallace (1895) remarked: ‘‘there likely arose autochthonously in Madagascar. Within is probably no portion of the globe that Coleeae Colea and Ophiocolea are sisters, Phyllar- contains within itself so many and such varied thron Colea Ophiocolea Rho- is sister to + ,and features of interest connected with geograph- docolea is sister to the rest of the tribe. ical distribution.’’ A few taxa of Madagascar’s Key words: Bignoniaceae, biogeography, Coleeae, flora may reflect the original Gondwanan flora Crescentieae, fruit evolution, Kigelia, Madagascar, such as gymnosperms and the phylogenetically ndhF, phylogenetics, trnT-L spacer, trnL-F spacer. basal angiosperms Takhtajania (Winteraceae) (Karol et al. 2000) and Ascarinopsis (Chlo- The large island of Madagascar is in many ranthaceae). The majority of Madagascar’s respects a micro-continent. Originally part of flora has likely arrived via dispersal, either 56 M. L. Zjhra et al.: Phylogenetics of Coleeae

Fig. 1. Origin of indehiscent fruits in Bignoniaceae according to Gentry (1976). Crescentieae were hypothesized to have been derived from neotropical Tecomeae, with mammal-dispersed, indehiscent fruits evolving from dehiscent fruits. In contrast, Coleeae were derived from Malagasy/African Tecomeae, with baboon- and lemur-dispersal evolving from wind dispersal. Shading indicates distribution of tropical species of Tecomeae long-distance or by way of (now under water) tinent. An additional 16 Malagasy species of oceanic ‘‘stepping stone’’ islands (Gentry 1988, Bignoniaceae belong to tribe Tecomeae. Spe- Schatz 1996). In this respect, Madagascar is cies of Malagasy Coleeae are concentrated in like an oceanic island whereby the majority of the tropical rainforests of Madagascar, with the flora originates via long-distance dispersal. peripheral distribution in Madagascar’s wes- Radiations in species number and/or ecologi- tern dry deciduous forests and the arid spiny cal adaptations such as pollination biology, scrub of the south. Five genera of Malagasy fruit dispersal, and leaf type are common in Coleeae (Rhodocolea, Ophiocolea, Colea, these isolated groups (e.g. Coleeae, Bignonia- Phyllarthron, and Phylloctenium) have been ceae [Gentry 1988]; Adansonia, Bombacaceae delimited on the basis of fruit, flower, and [Baum 1995, Baum et al. 1998]; Dalechampia, leaf characteristics (Perrier de la Baˆ thie Euphorbiaceae [Armbruster et al. 1993]; Didi- 1938a, b). According to Gentry (1976), Rho- ereaceae [Applequist and Wallace 2000]). docolea retains primitive characteristics; after Among biogeographic areas, Madagascar the derivation of Rhodocolea, two lineages of has the second richest flora of Bignoniaceae, more specialized taxa evolved: Ophiocolea/ following South America. The majority of Colea, and Phyllarthron/Phylloctenium. Oph- Malagasy species of Bignoniaceae belong to iocolea gave rise to Colea, and Phyllarthron tribe Coleeae, a group that has been tradi- gave rise to Phylloctenium (Gentry 1976). tionally delimited to include 49 species, of The mostly endemic Malagasy Coleeae which 44 are endemic to Madagascar and are of considerable, and sometimes conten- four to the islands of the Indian Ocean (the tious, evolutionary, biogeographic and sys- Seychelles, Comores, and Mascarenes), and tematic interest. Much of the taxonomic one (monotypic Kigelia) to the African con- history of Coleeae relates to the evolution M. L. Zjhra et al.: Phylogenetics of Coleeae 57 of indehiscent fruits in Bignoniaceae. Species indehiscent fruits with subsequent modifica- of tribe Coleeae are remarkable for their tion in related lineages back to dehiscent diversity of fleshy fruits (in a family charac- fruits with wind dispersed seeds. terized by dehiscent fruits), numerous cauli- Further complicating the phylogenetic florous species, variety of pollination and evolutionary picture of the Malagasy syndromes, diversity of habitats occupied, Coleeae is African Kigelia africana. When and high levels of locally endemic, sympatric Gentry (1976) split Crescentieae and Coleeae, species (Zjhra 1998). Originally, taxa now he placed the monotypic Kigelia in Coleeae. comprising the Coleeae were placed in tribe Kigelia is found throughout the savannas and Crescentieae (de Candolle 1838, 1845; See- gallery forests of Central Africa. Its meter- mann 1860; Baillon 1887, 1888; Perrier de la long, pendulous, baboon-dispersed fruits Bathie 1938a, b), an otherwise neotropical have inspired much folklore and earned it group distributed primarily in Central Amer- the moniker, Sausage Tree. Kigelia shares ica and the Caribbean. The geographically geographic proximity with Coleeae, and disjunct Crescentieae and Coleeae share a unusually large indehiscent fruits and bat number of characteristics otherwise not ob- pollination with Crescentieae. The placement served in the family: spines, phyllodes, simple of Kigelia is important in formulating phylo- leaves, cauliflory, and fleshy indehiscent genetic and biogeographic hypotheses regard- fruits. Gentry (1976) suggested that Crescen- ing the origins of Coleeae, as well as tieae were derived from neotropical Teco- assessment of the phylogenetic status (i.e. meae, with mammal-dispersed, indehiscent homoplasy or homology) of the characters fruits evolving from dehiscent fruits with that appear to be synapomorphic for Coleeae wind dispersed seeds. Coleeae were hypothe- and Crescentieae. sized by Gentry (1976) to have been derived These important systematic, biogeograph- from Malagasy/African Tecomeae, with ba- ic, and evolutionary questions require an boon and lemur dispersal evolving from wind independent, molecular-based phylogenetic dispersal (Fig. 1). Leroy (1978) suggested that framework for their resolution. Only one Malagasy ‘‘Crescentieae’’ originated during previous molecular study has attempted to post-Gondwanan time, and evolved from examine relationships within Bignoniaceae. ancestors (now extinct) common to Africa Spangler and Olmstead (1999), using rbcL and South America. Gentry (1976, 1988) and ndhF sequence variation of 19 genera of argued on the basis of geographic disjunction Bignoniaceae, indicated that Kigelia and that species of Coleeae and Crescentieae Ophiocolea (the only sampled genera of shared similar features due to convergent Coleeae) were sister taxa and related to a evolution rather than common ancestry. complex of Old World and New World Combining Crescentieae and Coleeae as a representatives of Tecomeae and Crescen- single tribe (following de Candolle 1838, tieae. The molecular phylogenetic analysis 1845; Seemann 1860; Baillon 1887, 1888; presented here increases taxon sampling of Perrier de la Baˆ thie 1938a, b) results in a the Malagasy tribe Coleeae and other pre- remarkable disjunct distribution that must be sumed relatives in Bignoniaceae and aims to explained either by long-distance dispersal assess (1) the phylogenetic status of Coleeae, between South America and Madagascar, or in particular its relationship with Crescentieae by complete extinction of all representatives and Kigelia; (2) the evolution of morpholog- of a once more widespread Crescentieae/ ical traits (indehiscent fruits, spines, phyll- Coleeae lineage in Africa, south Asia, and odes) that have variously united and South America. Separating Crescentieae and separated Coleeae, Crescentieae, and Kigelia; Coleeae requires either convergent evolution and (3) the relationships of genera within of indehiscent fruits, or a single origin of Coleeae. Table 1. Accessions of Coleeae and outgroups for which sequences were included Coleeae of Phylogenetics al.: et in Zjhra L. M. phylogenetic analyses. Numbers represent GenBank 58 accession numbers Taxa Voucher 5¢ ndhF3¢ ndhF trnT-L trnL-F Coleeae Colea undescribed sp. 1 ‘‘sytsma’’ Zjhra 917 AY500433 AY500433 AY500394 AY500413 Ophiocolea floribunda (Boj. Ex Lindl.) Zjhra 275 AY500434 AY500395 AY500414 H.Perr. Ambanizana Ophiocolea floribunda (Boj. Ex Lindl.) Schatz 3448 (MO) AF102634 AY500435 H.Perr. Toamisina Ophiocolea floribunda (Boj. Ex Lindl.) van der Werff 13551 AY500436 AY500396 AY500415 H.Perr. Mahajunga. Phyllarthron undescribed sp. 1 ‘‘sahamalaza’’ Zjhra 990 AY500437 AY500397 AY500416 Phyllarthron articulatum Schum. Zjhra 752 AY500438 AY500438 AY500398 AY500417 Phyllarthron undescribed sp. 2 ‘‘vokoanina’’ Zjhra 709 AY500439 AY500399 AY500418 Phyllarthron undescribed sp. 3 ‘‘itremo’’ Malcomber 2846 AY500440 AY500400 AY500419 Phyllarthron antongiliense Capuron Zjhra 707 AY500441 AY500401 AY500420 Rhodocolea undescribed sp. 1 ‘‘lemurflower’’ Zjhra 810 AY500442 AY500402 AY500421 Rhodocolea racemosa (Lam.) H. Perr. Zjhra 943 AY500443 AY500443 AY500422 Rhodocolea nobilis Baill. Zjhra 369 AY500444 AY500403 AY500423 Rhodocolea undescribed sp. 2 ‘‘trunkflower’’ Zjhra 837 AY500445 AY500404 AY500424 Putative Coleeae Kigelia africana Benth. coll. R.C.A. Rica; cult. AF102632 AY500446 AY500405 AY500425 at Waimea Bot. Gard. Living coll. #74s980; no voucher Old World Tecomeae Fernandoa madagascariensis (Baker) A.H. Gentry Phillipson 3940 AY500447 AY500447 AY500426 Markhamia platycalyx Sprague coll. by A. B. Katende; AY500448 AY500448 AY500406 Cult at Waimea Bot Gard.; no voucher Newbouldia laevis Seem. RBG Edinburgh Living AY500449 AY500449 coll. #19671901 Radermachera frondosa Chun & How cult. at Waimea Bot. Gard, AF102638 AY500450 AY500407 AY500427 living coll. #85s277; no voucher Rhigozum madagascariense Drake Malcomber 1138 AY500451 AY500451 M. L. Zjhra et al.: Phylogenetics of Coleeae 59

Materials and methods Taxa. In order to address the question concerning the monophyly of Coleeae and their relationship to other groups of Bignoniaceae, representatives of Crescentieae, African and New World Teco- meae, Kigelia, and the major lineages within Malagasy Coleeae were sampled. Thirteen species (Table 1) representing four of ﬁve genera of Malagasy Coleeae were selected to include diverse elements within the major genera (Zjhra and Sytsma, unpub. data). The monotypic Kigelia, from mainland Africa, which was included in the otherwise Malagasy Coleeae by Gentry (1976), was sampled along with eight species from seven genera of African, Malagasy, and Asian Teco- meae, two genera of neotropical Crescentieae, and Tabebuia, representing New World Tecomeae (Table 1). These representatives of Tecomeae and Crescentieae have been demonstrated to be among the nearest relatives of Coleeae based on

AY500452 AY500452 AY500408 AY500428 L36416 AY500455AF102627 AY500410 AY500457 AY500430 AY500412 AY500432 phylogenetic analysis of cpDNA data (Spangler and Olmstead 1999, Olmstead et al., unpub. data). Tabebuia, Crescentia,andAmphitecna were used to root the trees in all of the analyses. Designation of these taxa as outgroups, although supported by previous work (Spangler and Olm- stead 1999), does not affect the relationships of Malagasy Coleeae and the African Kigelia, because relationships presented here are robust under any rooting scheme. DNA extraction and amplification. Total DNA Gard., living coll. #78p1079 At MO 50458 (MO) was isolated from silica-gel-preserved leaves and from leaf material removed from herbarium specimens, following a modified mini-preparation protocol (Murray and Thompson 1980, Saghai- Maroof et al. 1984), and using two or three chloroform:isoamyl alcohol extractions. The chloroplast gene ndhF was amplified using the 3¢ end primers 972, 1318, and 2110R and the entire ndhF gene using the additional primers 1074R, -52, and D.C. M.W. Chase 3891 (K) AY500454 AY500454

D.C. Zjhra 68232 AY500453 (Olmstead AY500453 AY500409and AY500429 Sweere 1994, Olmstead and Reeves 1995). The chloroplast DNA non-coding Britton Gentry & Zardini Beauv. cult. at Waimea Bot. regions trnT-trnL and trnL-trnF (comprising the Britton coll. A. Gentry, cult. A.H. Gentry R. Spangler B1 (MO)trnT and AF102624trnF AY500456 exons AY500411 flanking AY500431 the trnL intron) were amplified using the universal primers a, b, e, and f from Taberlet et al. (1991). DNA was amplified in 50 ll reactions containing 1 ll of genomic DNA, 5 ll of 25 mM MgCl, (continued) 5 ll of 10x buffer, 4 ll40lM of BSA, 0.5 ll Tween, 8 ll of 10 mM dNTP cocktail (2 ll each), 1 ll of each 20 lM primer, and 1 ll of Taq Stereospermum euphorioides Stereospermum nematocarpum New World Tecomeae Tabebuia heterophylla Crescentieae Amphitecna apiculata Crescentia portoricensis Table 1 Spathodea campanulata 60 M. L. Zjhra et al.: Phylogenetics of Coleeae

Polymerase (5 units/ll). The PCR profile for Patterns of fruit evolution were examined by cpDNA amplification was 30 seconds at 94C, 1 character overlay of fruit type (fleshy, indehiscent, minute at 50C, 1 minute 30 seconds at 72C for 30 animal dispersed vs. dry, dehiscent, wind dispersed) cycles. PCR products were either directly se- onto representative molecular cladograms in quenced or cleaned with the PCR Product Pre- MacClade 3.05 (Maddison and Maddison 1992). Sequencing kit (Amersham, Chicago, IL U.S.A.) to Because all other Bignoniaceae have dehiscent remove excess dNTPs and primers. Cycle sequenc- fruits with wind dispersed seeds, that state was ing reactions (20 ll) used 5 ll PCR product, 1– considered plesiomorphic. 2 lM primer, and 8 ll Taq Polymerase Master (Promega, Madison, WI U.S.A.). Two primers Results were used to sequence trn regions, a and f. Three ndhF primers (972, 2110R, and 1318) were used for Complete sequences for the more variable 3¢ sequencing the 3¢ end of ndhF; this permitted end of ndhF were obtained for all taxa, sequencing of both strands for the majority of the whereas the more highly conserved 5¢ end ndhF3¢ region. Three additional primers (32, 536, was obtained for all outgroups and one 1318R) were used to sequence the 5¢ end of the representative species of each genus of Cole- ndhF region. Dye primer sequencing, using Amp- eae. Both the trnT-L and trnL-F regions were liTaq (Promega) DNA Polymerase or Big Dye obtained for all accessions of Coleeae, except (Promega) DNA Polymerase, was done on an ABI Prism 377 DNA sequencer. one of the three accessions of Ophiocolea Phylogenetic analysis. Sequences were aligned floribunda (missing both regions) and one of with Sequencher vs. 3.0 (Gene Codes Corporation, four species of Rhodocolea (missing trnT-L Ann Arbor, MI) and adjusted manually using the only). Among the outgroups, all genera were manual alignment editor Se-Al version 2.0a6 sampled with 5¢ ndhF and 3¢ ndhF; with four (Rambaut 2001). Potentially informative gaps of genera missing either trnT-LortrnL-F, respec- unambiguous alignment located in the trn regions tively. The combined dataset included 3183 were scored using the simple gap coding method of nucleotides (nt) of aligned DNA sequence and Simmons and Ochoterena (2000) and added to the 17 binary gap characters. All of the sequences matrix as binary characters (Baum et al. 1994) could be aligned unambiguously, so no regions under equal weighting. The three discontinuous were excluded from analysis. The ndhF sequence regions were concatenated into a single sequences included 2107 nt of aligned se- data set, along with the gap characters, for parsimony analysis using PAUP* (vers. 4.0b10, quence and provided 84 parsimony informa- Swofford 2002). Most parsimonious trees were tive characters (54 in the more variable 3¢ obtained with Fitch parsimony with all changes region). The trnT-L and trnL-F regions were weighted equally using heuristic searches with 1,000 670 and 406 nt long when aligned, respectively, random addition sequences, tree bisection and and provided a total of 42 informative nucle- reconnection (TBR) with MULTREES activated. otide characters and 17 gap characters. The An incongruence length difference (ILD) test 3-way ILD test revealed no significant differ- (Farris et al. 1994) was conducted using the three ence between the sequence regions and random discontinuous regions of sequence as initial parti- partitions of the same size drawn from the tions to test whether they differ significantly from entire data set (p ¼ 0.92). random partitions of the combined data set (imple- Parsimony analysis yielded 882 most-par- mented in PAUP*). The dataset was bootstrapped simonious trees (Fig. 2) of 452 steps each (Felsenstein 1985) with 1000 replicates using TBR swapping to explore the relative strength of (Consistency Index, CI ¼ 0.912; Retention In- branches on the resulting tree. An analysis was dex, RI ¼ 0.897). The Malagasy Coleeae are performed with Kigelia constrained to monophyly monophyletic with strong support (100% with the Malagasy Coleeae to determine how much bootstrap) and they are sister to an Old World longer trees are that find a group consistent with group of Tecomeae. Within the Coleeae, Gentry’s view of the tribe. the sampled members of Rhodocolea are M. L. Zjhra et al.: Phylogenetics of Coleeae 61

Fig. 2. Phylogenetic relationships of Malagasy Coleeae, African Kigelia, Old and New World Tecomeae, and New World Crescentieae based on chloroplast DNA. Shown is one of 822 MP trees; branches that collapse in the strict consensus tree are indicated by an asterisk in place of the bootstrap value. Branch lengths are given above the branch and bootstrapvaluesbelow(novalue¼ <50%). Tribal assignments according to Gentry are givenontheright.ThetreeisrootedusingTabebuia (New World Tecomeae) and New World Crescentieae basedonresultsofSpanglerandOlmstead(1999) 62 M. L. Zjhra et al.: Phylogenetics of Coleeae monophyletic (84%) and are sister to the are not closely related; (2) indehiscent fruits remainder of the tribe, which is weakly sup- and mammal dispersal have evolved three times ported as a clade (55%). Neither Ophiocolea in Bignoniaceae; and (3) Rhodocolea is an early nor Phyllarthron is monophyletic in the strict diverging lineage in Malagasy Coleeae. consensus of all most-parsimonious trees. Circumscription and relationships of Cole- However, both of these genera are monophy- eae. The cpDNA sequence analysis provides letic in 81 of the 882 trees, so monophyly of the strong support for a monophyletic Coleeae established genera of Coleeae cannot be ruled with the exclusion of Kigelia (Fig. 2). Based on out by this analysis. Colea, with only one species this molecular study, we delineate Coleeae as sampled, could not be tested for monophyly. an endemic Malagasy/Indian Ocean clade with Kigelia, from mainland Africa, is part of Kigelia excluded. Gentry (1976) placed African the sister group to Coleeae and forms a Kigelia in Coleeae based on shared characters strongly supported clade with Stereospermum of cauliflory, mammal pollination, and mam- (100% bootstrap support), which is embedded mal dispersal of indehiscent fruit. In light of in an Old World clade of Tecomeae compris- our findings that Kigelia is more closely related ing Stereospermum, Markhamia, Newbouldia to members of Old World Tecomeae, than to and Fernandoa (79%). Constraining all tradi- Malagasy Coleeae, one must assume that these tional Coleeae (i.e. Kigelia and the Malagasy shared characters reflect parallel or convergent Coleeae) to monophyly requires a relaxation of evolution (see below). parsimony by 11 steps. The sister group to this The closest relatives of Coleeae comprise a Old World clade of Tecomeae and Malagasy clade of Old World Tecomeae, including Coleeae is another clade of Old World Teco- Fernandoa, Newbouldia, Markhamia, Stereo- meae comprised of Spathodea, Rhigozum and spermum, and Kigelia that are distributed from Radermachera. tropical Africa to Indochina. This clade of Old Evolution of fruit types (fleshy, indehis- World Tecomeae also includes species from cent, animal dispersed vs. dry, dehiscent, wind Madagascar. A second clade of Old World dispersed) is illustrated in Fig. 3. From the Tecomeae, including Radermachera, Rhi- primitive state of dehiscent fruits with wind gozum, and Spathodea, is sister to the more dispersal, indehiscent and animal dispersed inclusive clade of Coleeae and its sister group. fruits have convergently evolved three separate These results are consistent with studies of the times: (1) in the common ancestor of all entire family with broader taxon sampling, but Malagasy Coleeae in Madagascar, (2) in the less sequence data (Spangler and Olmstead ancestor of African Kigelia, and (3) in the 1999, R. Olmstead, unpubl.). Those studies common ancestor of all New World Crescen- find these three clades to form a monophyletic tieae. These three separate origins of indehis- group and identify a New World clade com- cent fruits are seen with all of the molecular prising tribe Crescentieae and several genera of datasets, regardless of how the characters are Tecomeae, including Tabebuia, as the sister to optimized on the tree (e.g. Accelerated or this Old World clade. Malagasy Coleeae thus Delayed transformation). Dispersers of the appear to be a distinctive and early radiation fruits of Coleeae and Kigelia are lemurs and from a more widely distributed Old World baboons, respectively; dispersers of fruits of Tecomeae. Whereas it is now clear that Teco- New Worlds Crescentieae have not been iden- meae are not monophyletic and a new tribal tified with confidence but are likely mammals. classification of the family is needed, additional sampling of both Old and New World Tecomeae is needed before a comprehensive Discussion classification can be attempted. The results presented here provide three impor- Coleeae are phylogenetically distinct from tant findings: (1) Malagasy Coleeae and Kigelia neotropical Crescentieae, supporting Gentry’s M. L. Zjhra et al.: Phylogenetics of Coleeae 63

Fig. 3. Fruit evolution in Bignoniaceae depicted by character state overlay onto the molecular phylogeny of Coleeae, related Old World Tecomeae, and outgroup New World Tecomeae and Crescentieae. Dehiscent fruits with wind dispersal of seeds are depicted as plesiomorphic as all other Bignoniaceae lack ﬂeshy fruits. Animal dispersed, indehiscent fruits have convergently evolved three separate times – once in the common ancestor of all Malagasy Coleeae in Madagascar, once in the African Kigelia, and once in the common ancestor of all New World Crescentieae. Representative ﬂeshy-fruit types of Colea (lemur dispersed), Kigelia (baboon dispersed), and Amphitecna (monkey-dispersed) and dehiscent-fruit type of Stereospermum (wind-dispersed), are illustrated to the right. Subsequent and independent shifts to water dispersal (not shown) have occurred in at least two genera of Crescentieae. Molecular phylogeny is based on Fig. 2 64 M. L. Zjhra et al.: Phylogenetics of Coleeae

(1976) separation of Coleeae and Crescentieae. related to any extant wind-dispersed taxa. The On the other hand, our results do not support results based on DNA-sequence data support Gentry’s (1976) inclusion of Kigelia in Coleeae. Gentry’s (1988) proposal that a ‘‘unique and Coleeae are separated from neotropical Cres- autochthonous evolutionary switch from wind centieae by numerous Old World genera that dispersal to mammal dispersal’’ occurred in the have been placed in tribe Tecomeae. Gentry common ancestor of Coleeae. However, Gen- (1976, 1988) maintained that characters shared try’s (1988) suggestion that indehiscent fruits by Coleeae and Crescentieae were due to originated prior to the isolation of Madagascar convergence rather than shared ancestry and is not supported by our results. It appears that that Coleeae and Crescentieae were derived indehiscent fruits have arisen three times inde- from separate Tecomeae stock. The many pendently in Bignoniaceae (Fig. 3) and on three characteristics shared by Coleeae and Crescen- different land masses: Madagascar, Africa, and tieae may be the result of both history and Central America (i.e. Coleeae: lemurs; Kigelia: ecology, the product of ‘‘concerted conver- baboons; Crescentieae: mammals; respectively). gence’’ (sensu Givnish and Sytsma 1997, Malagasy Coleeae likely evolved from ancestral Patterson and Givnish 2001). Similar environ- Old World Bignoniaceae with dehiscent, cylin- mental conditions on disparate continents, drical fruits and wind or water-dispersed seeds selecting on the same underlying genetic archi- (Fig. 4). Subsequent shifts from mammal dis- tecture, have apparently produced similar persal to water dispersal have apparently solutions (e.g. spines, phyllodes, cauliflory, occurred several times in New World Crescen- indehiscent fruit). A detailed phylogenetic tieae (Gentry 1983). The remaining genera of the analysis of Crescentieae is underway (S. Grose clade containing Kigelia have dehiscent fruit and R. Olmstead, unpubl.); combined with this with wind-dispersed seeds. The remaining study of Coleeae, a better understanding of the clades related to Coleeae and Crescentieae in extent of adaptive parallels between Crescen- our study are Rhigozum/Spathodea and Rader- tieae and Coleeae should emerge. machera, and New World Tecomeae. Plants in Evolution of indehiscent fruit. Fruit char- these clades have dehiscent fruits with wind- acters are traditionally important in circum- dispersed seeds, as is typical of most Bignonia- scribing tribes in Bignoniaceae. The fruit of ceae (Fig. 3). Bignoniaceae have four main vascular bundles In fact, indehiscent fruits in Bignoniaceae that produce a silique-like fruit with a central are only superficially similar. For example, the replum (Endress 1994). Bignoniaceae tradi- fruits of Crescentieae are baccate with a fleshy tionally have been classified on the basis of cortex and fibrous core, or are a hard-shelled fruit anatomy, with a first division based on pepo with soft pulp (Gentry 1980); fruits of whether the fruits are indehiscent (Coleeae, Kigelia are indehiscent capsules with a corky Crescentieae, Kigelia) or dehiscent (all others). exterior and a woody interior (St. John 1933); Further subdivisions of dehiscent-fruited taxa and fruits of Coleeae are fleshy or fibrous, but are based on dehiscence that is parallel (Big- never hard, woody or corky. Furthermore, both nonieae, Oroxyleae) or perpendicular (Teco- Coleeae and Crescentieae contain members with meae) in relation to the central septum, and and without vestigial seed wings, suggesting a those lacking a septum (Eccremocarpeae with transition from winged to wingless seeds. Kigelia a 2-valved capsule, Tourrettieae with a does not have vestigial seed wings. Research on 4-valved capsule). the evolution and structure of indehiscent fruits Gentry (1983) suggested that mammal dis- in Bignoniaceae (S. Grose and R. Olmstead, persal in Bignoniaceae (which only occurs in pers. comm.) will add greatly to our under- Coleeae, Kigelia, and Crescentieae) evolved standing of fruit homology in Bignoniaceae. from wind dispersal, and that members of tribes Genera within Coleeae. As predicted by with mammal-dispersed fruits are not closely Gentry (1976), Rhodocolea is sister to all other M. L. Zjhra et al.: Phylogenetics of Coleeae 65

Fig. 4. Origin of indehiscent fruits in Bignoniaceae according to evidence presented here. Fruits of species of Coleeae likely evolved from dehiscent, cylindrical fruits with seeds that were wind- or water-dispersed. It is difficult, however, to opti- mize the biogeographic distribution of the ancestor with confidence based on Fig. 2. Hatching indicates distribution of tropical species of Tecomeae members of tribe Coleeae. Gentry (1976) and resolving power to confirm monophyly for considered Rhodocolea to be the ‘‘basal’’ genus all genera. Results of a Coleeae-wide analysis in Coleeae (he viewed it as an ancestral genus using more variable DNA regions will be giving rise to two other lineages), because it presented elsewhere (Zjhra and Sytsma, un- ‘‘combines all the putatively primitive charac- publ. data) to show species level relationships. ters’’ of the tribe. He also noted that one species has actinomorphic flowers and five The authors thank L. McDade and an anony- stamens, although that is better viewed as a mous reviewer for useful comments on this manuscript, and J. Hutcheon for comments on earlier derived, rather than ancestral, floral morphol- versions of this manuscript. Kandis Elliot created ogy. Species of Rhodocolea are characterized the figures. Specimens, including types, were by large flowers, but otherwise have no evident generously loaned by the Museúm National synapomorphy. Of the remaining genera, d’Histoire Naturelle, Paris, the Royal Botanic Phyllarthron is either sister to the morpholog- Gardens, Kew, and the Missouri Botanical Gar- ically derived genera, Ophiocolea and Colea,or den, St. Louis. Wildlife Conservation Society– forms a paraphyletic grade leading to them. Madagascar and Projet Masoala-Antalaha have Phyllarthron is characterized by phyllodes, not repeatedly and generously provided logistical sup- otherwise found in Coleeae, thus suggesting port in the field. The Hatchwell family has monophyly. Monophyly is consistent with the graciously extended their hospitality in Antalaha. cpDNA evidence, despite the lack of resolution We are indebted to the Direction Generale des in the cpDNA results. Colea and Ophiocolea Eaux et Foreˆ ts and ANGAP for their authoriza- tion to conduct this study. This research was share unilocular anthers and cauliflory as supported in part by a NSF Dissertation Improve- synapomorphies. Colea has furrowed fruits, ment Grant [DEB 9423577] a NSF Postdoctoral whereas Ophiocolea has long, cylindrical fruits. Research Fellowship [DEB 9804155], an American Both of these traits could be derived within this Society of Plant Taxonomists Student Research clade, because Rhodocolea and Phyllarthron Grant, and the University of Wisconsin Davis have smooth, oblong fruits (i.e. neither Fund to MLZ and NSF grants BSR 9107827 and long slender nor furrowed). Our analysis is DEB 9509804 to RGO. This research was in consistent with Gentry’s ideas on evolution in partial fulfillment of the Ph.D requirements of the the group, but has insufficient taxon sampling University of Wisconsin for M. Zjhra. 66 M. L. Zjhra et al.: Phylogenetics of Coleeae

References Monogr. Syst. Bot. Missouri Bot. Gard. 25: 175– 185. Applequist W. L., Wallace R. S. (2000) Phylogeny Givnish T. J., Sytsma K. J. (1997) Homoplasy in of the Madagascan endemic family Didiereaceae. molecular vs. morphological data: the likelihood Plant Syst. Evol. 221: 157–166. of correct phylogenetic inference. In: Givnish T. Armbruster W. S., Edwards M. E., Hines J. F., J., Sytsma K. J. (eds.) Molecular evolution and Mahunnah R. L. A., Munyenyembe P. (1993) adaptive radiation. Cambridge University Press, Evolution and pollination of Madagascan and New York, pp. 55–101. African Dalechampia (Euphorbiaceae). Res. Karol K. G., Suh Y. B., Schatz G. E., Zimmer Explor. 9: 430–444. E. A. (2000) Molecular evidence for the phylo- Baillon H. (1887) Notes sur les Crescentieés. Bull. genetic position of Takhtajania in the Wintera- Soc. Linn. Paris 1: 678, 680, 683, 688, 690, 695. ceae: inference from nuclear ribosomal and Baillon H. (1888) Histoire des plantes 10. Hachette, chloroplast gene spacer sequences. Ann. Mis- Paris. souri Bot. Gard. 87: 414–432. Baum D. A. (1995) The comparative pollination and Leroy J.-F. (1978) Composition, origin and affin- floral biology of baobabs (Adansonia-Bombaca- ities of the Madagascan vascular flora. Ann. ceae). Ann. Missouri Bot. Gard. 82: 322–348. Missouri Bot. Gard. 65: 535–589. Baum D. A., Small R. L., Wendel J. F. (1998) Maddison W. P., Maddison D. R. (1992) Biogeography and floral evolution of baobabs MacClade vs. 3.05: analysis of phylogeny and (Adansonia, Bombacaceae) as inferred from character evolution. Sinauer Associates, Sunder- multiple data sets. Syst. Biol. 47: 181–207. land, MA, USA. Baum D. A., Sytsma K. J., Hoch, P. C. (1994) A Murray M.G., Thompson W.F. (1980) Rapid phylogenetic analysis of Epilobium (Onagraceae) isolation of high molecular weight plant DNA. based on nuclear ribosomal DNA sequences. Nucleic Acids Res. 8: 4321–4325. Syst. Bot. 19: 363–388. Olmstead R. G., Sweere J. A. (1994) Combin- Candolle A. P. de. (1838) Revue sommaire de la ing data in phylogenetic systematics: an famille des Bignoniaceés. Bibliothe` que Univer- empirical approach using three molecular selle de Gene` ve, Gene` ve. data sets in the Solanaceae. Syst. Biol. 43: 467– Candolle A. P. de. (1845) Prodromus systematis 481. naturalis regni vegetabilis 9. Treuttel et Wu¨rtz, Olmstead R. G., Reeves P.A. (1995) Evidence for Paris. the polyphyly of the Scrophulariaceae based on Endress P. K. (1994) Diversity and evolutionary chloroplast rbcLandndhF sequences. Ann. biology of tropical flowers. Cambridge Univer- Missouri Bot. Gard. 82: 176–193. sity Press, New York. Patterson T. B., Givnish T. J. (2001) Phylogeny, Farris J. S., Ka¨llersjo¨M., Kluge A. G., Bult C. concerted convergence, and phylogenetic niche (1994) Testing significance of incongruence. conservatism in the core Liliales: insights from Cladistics 10: 315–319. rbcLandndhF sequence data. Evolution 56: Felsenstein J. (1985) Confidence limits of phylog- 233–252. enies: an approach using the bootstrap. Evolu- Perrier de la Baˆ thie H. (1938a) Les Bignoniaceés de tion 39: 783–791. la re´gion Malgache. Ann. Mus. Col. Marseille Gentry A. H. (1976) Relationships of the Mada- 46: 1–101. gascar Bignoniaceae: a striking case of conver- Perrier de la Baˆ thie H. (1938b) Bignoniaceés. Flore gent evolution. Plant Syst. Evol. 126: 255–266. de Madagascar 178: 1–91. Museúm National Gentry A. H. (1980) Bignoniaceae. Flora Neotro- d’Histoire Naturelle, Paris. pica. Monograph 25 (Part 1). New York Botan- Rabinowitz P. D., Coffin M. F., Falvey D. (1982) ical Garden, New York. Salt diapirs bordering the continental margin of Gentry A. H. (1983) Dispersal and distribution in northern Kenya and southern Somalia. Science Bignoniaceae. Sonderb. Naturwiss. Vereins 215: 663–664. Hamburg 7: 187–199. Rabinowitz P. D., Coffin M. F., Falvey D. (1983) Gentry A. H. (1988) Distribution and evolution of The separation of Madagascar and Africa. the Madagascar Bignoniaceae. In: Goldblatt P., Science 220: 67–69. Lowrey P. P. (eds.) Studies in African botany. M. L. Zjhra et al.: Phylogenetics of Coleeae 67

Rambaut A. (2001) Sequence alignment editor, of hot spot-related volcanism and the breakup of v2.0a6. http://evolve.zoo.ox.ac.uk/. Madagascar and India. Science 267: 852–855. Saghai-Maroof K. M., Soliman R. A., Jorgensen Swofford D. L. (2002) PAUP*. Phylogenetic anal- A. Allard R. W. (1984) Ribosomal DNA spacer- ysis using parsimony (*and other methods). length polymorphisms in barley: Mendelian Version 4. Sinauer Associates, Sunderland, inheritance, chromosomal location, and popula- MA, USA. tion dynamics. Proc. Natl. Acad. Sci., USA 81: Taberlet P., Gielly L., Pautou G., Bouvet J. (1991) 8014–8018. Universal primers for amplification of three non- Schatz G. E. (1996) Malagasy/Indo-australo-male- coding regions of chloroplast DNA. Plant Mol. sian phytogeographic connections. In: Lourenço Biol. 17: 1105–1109. W. R. (ed.) Biogeógraphie de Madagascar. Wallace A. R. (1895) Island life. MacMillan and ORSTOM, Paris, pp. 73–84. Co., London. Seemann B. (1860) Synopsis Crescentiacearum: an Zjhra M. L. (1998) Phylogenetics, biogeography enumeration of all the Crescentiaceous plants at and pollination ecology of endemic Malagasy present known. Trans. Linn. Soc. London 23: 1– Coleeae (Bignoniaceae). Ph.D. Thesis. Univer- 22. sity of Wisconsin-Madison. Simmons M. P., Ochoterena H. (2000) Gaps as characters in sequence-based phylogenetic analyses. Syst. Biol. 49: 369–381. Spangler R. E., Olmstead R. G. (1999) Phyloge- Addresses of the authors: Michelle L. Zjhra netic analysis of Bignoniaceae based on the ([email protected]), Department of cpDNA gene sequences rbcL and ndhF. Ann. Biology, Georgia Southern University, Statesboro, Missouri Bot. Gard. 86: 33–46. GA 30460, USA; Kenneth J. Sytsma, Department St. John H. (1933) The sausage tree. Paradise of the of Botany, University of Wisconsin, Madison, WI Pacific 46(3): 5–6. 53706, USA; Richard G. Olmstead, Department of Storey M., Mahoney J. J., Saunders A. D., Duncan Botany, University of Washington, Seattle, WA R. A., Kelley S. P., Coffin M. F. (1995) Timing 98195, USA.