Volume 94 Number 1 2007 Annals of the Missouri Botanical Garden A PHYLOGENETIC ANALYSIS OF ALYXIEAE (APOCYNACEAE) BASED ON RBCL, MATK, TRNL INTRON, TRNL-F SPACER SEQUENCES, AND MORPHOLOGICAL CHARACTERS1 Mary E. Endress,2 Raymond W. J. M. van der Ham,3 Siwert Nilsson,{ Laure Civeyrel,4 Mark W. Chase,5 Bengt Sennblad,6 Kurt Potgieter,7 Jeffrey Joseph,5 Martyn Powell,5 David Lorence,8 Ylva-Maria Zimmerman,9 and Victor A. Albert10 ABSTRACT Within Rauvolfioideae (Apocynaceae), genera have long been assigned to tribes based mainly on only one or two superficial fruit and seed characters. Taxa with drupaceous fruits were included in Alyxieae. To elucidate relationships within Alyxieae, we analyzed phylogenetically a data set of sequences from four plastid DNA regions (rbcL, matK, trnL intron, and trnL-F intergenic spacer) and a morphological data set for 33 genera of Apocynaceae, including representatives of all genera previously included in Alyxieae and two non-Apocynaceae species. Results of parsimony analysis indicate that Alyxieae as previously delimited are polyphyletic, with most genera falling into two main clades. The Alyxia clade includes seven genera: Alyxia Banks ex R. Br., Lepinia Decne., Lepiniopsis Valeton, Pteralyxia K. Schum., and Condylocarpon Desf. together with Plectaneia Thouars. (earlier included in Plumerieae) and Chilocarpus Blume (earlier included in Chilocarpeae). The Vinca clade includes eight genera: Cabucala Pichon, Petchia Livera, Rauvolfia L., Catharanthus G. Don, Vinca L., Neisosperma Raf., Ochrosia Juss., and Kopsia Blume. Vallesia Ruiz & Pav. and Anechites Griseb. are not related to either clade and come out as sister to Aspidosperma Mart. & Zucc. (Aspidospermeae) and Thevetia L. (Plumerieae), respectively. The fruit and seed 1 We wish to thank the following persons who provided plant material or DNA samples: A. Assi, Paul Berry, F. Billiet, Birgitta Bremer, V. Ferreira, P. Garnock-Jones, P. Kessler, I. Koch, A. Leeuwenberg, S. Liede, D. Neill, R. Omlor, H. Petignat, M. Prévost, G. Romero, A. Specht, S. Tucker, and S. Zona. For technical assistance and photographic help with the pollen contribution sincere thanks are due to Elisabeth Grafström and Magnus Hellbom, Palynological Laboratory, Swedish Museum of Natural History, Stockholm, and Bertie Joan van Heuven and Wim Star, Nationaal Herbarium Nederland, Leiden. This study was supported by a grant from the Helge Ax:son Johnson Foundation and the Swedish Foundation for International Cooperation Research (STINT) to B. Sennblad. 2 Institute of Systematic Botany, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland. mendress@systbot. unizh.ch. 3 Nationaal Herbarium Nederland, P.O. Box 9514, 2300 RA Leiden, The Netherlands. 4 Laboratoire, Dynamique de la Biodiversité, UMR 5172, Université Paul Sabatier, 31062 Toulouse, Cedex 9, France. 5 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond Surrey, TW9 3DS, United Kingdom. 6 Stockholm Bioinformatics Center, Stockholm University, AlbaNova Research Center, SE-10691 Stockholm, Sweden. 7 College of Veterinary Medicine, University of Illinois, Urbana, Illinois 60801, U.S.A. 8 National Tropical Botanical Garden, 3530 Papalina Road, Kalaheo Kauai, Hawaii 96741, U.S.A. 9 Institut für Evolutionsbiologie und Morphologie, Universität Witten/Herdecke, Sockumer Str. 10 D-58448 Witten, Germany. 10 Natural History Museum, University of Oslo, Box 1172 Blindern, NO-0318 Oslo, Norway. { Siwert Nilsson passed away unexpectedly before the manuscript was completed. We lost both an excellent collaborator and a dear friend. We dedicate this paper to him. ANN. MISSOURI BOT. GARD. 94: 1–35. PUBLISHED ON 26 APRIL 2007. 2 Annals of the Missouri Botanical Garden characters previously used to demarcate Alyxieae are homoplasious, as are other morphological characters such as style head structure and syncarpy versus apocarpy. Conversely, pollen morphology, which has not previously played much of a role in tribal delimitation, was shown to be the most useful morphological character for delimiting Alyxieae from other tribes of Rauvolfioideae. Key words: Alyxieae, Apocynaceae, classification, matK, morphology, phylogeny, pollen, rbcL, systematics, trnL, trnL-F. Tribal delimitation in Rauvolfioideae (usually referred to as Plumerioideae in the older literature) has previously been based on fruit and seed characters (Schumann, 1895; Pichon, 1949a, 1949c; Lý, 1986; Leeuwenberg, 1994a). One reason that fruit and seed characters have been so frequently used in classifications and keys is that they are readily observed, permitting easy recognition of many genera. The other main reason for the fruit-based tribal classifications is that the flowers of many Rauvolfioideae tend to be superficially similar. Many are relatively small with a whitish, salverform corolla, and there are few obvious distinguishing floral characters useful for differentiating tribes in this subfamily. The most detailed studies of Rauvolfioideae were those of Pichon (1948a, 1948b, 1949a, 1950b), who published extensively on the family. His classification was a great improvement over its predecessors. Its main weakness was that tribal delimitation was based mainly on a single fruit character. He split the rauvolfioid tribes into two main groups, depending on whether the deeper layers of the mesocarp were fleshy or dry. The group with a fleshy inner mesocarp was divided into five tribes: Carisseae, Ambelanieae, and Macoubeeae comprised those taxa with indehiscent berries and Chilocarpeae and Tabernaemontaneae included those taxa with fruit consisting of dehiscent follicles with arillate seeds. In the group with a dry mesocarp, he recognized three tribes. Two of them are characterized by dehiscent fruits: Alstonieae (Plumerieae sensu Leeuwenberg, 1994a), in which the fruit consists of a pair of follicles, and the monotypic Allamandeae, in which the fruit is a spiny unilocular capsule. The last tribe, Rauvolfieae (Alyxieae sensu Leeuwenberg, 1994a), contained all taxa in which the fruit is an indehiscent drupe with a stony endocarp. It is the relationships within this group that are the focus of this paper. Pichon (1949a) recognized five subtribes within his Rauvolfieae: Rauvolfinae (including Cabucala Pichon, Petchia Livera, Rauvolfia L., and Podochrosia Baill.), Alyxiinae (including Alyxia Banks ex R. Br., Lepinia Decne., and Lepiniopsis Valeton), Ochrosiinae (including only Ochrosia Juss.), Vallesiinae (including Vallesia Ruiz & Pav. and Kopsia Blume), and Condylocarpinae (including Rhipidia Markgr. and Condylocarpon Desf.). Pichon included two genera as incertae sedis: Anechites Griseb. and Notonerium Benth. Notonerium has since been shown to belong to Boraginaceae (Crisp, 1983). In his classification from 1994a, Leeuwenberg maintained Pichon’s (1949a) circumscription of Rauvolfieae as well as the five subtribes included there but gave no insight into the delimitation of the subtribes, stating only that the relatively slight differences between its subtribes are not easily described in a concise way. The only differences between Leeuwenberg’s (1994a) and Pichon’s (1949a) tribal circumscriptions are that Leeuwenberg changed the name of the tribe to Alyxieae, put Podochrosia into synonymy under Rauvolfia and Rhipidia in Condylocarpon (following Fallen, 1983b), and included Anechites in Condylocarpinae, although Fallen (1983a) had suggested earlier that a position closer to taxa previously included in Cerbereae (Cameraria L., Cerbera L., Cerberiopsis Vieill. ex Pancher & Sebert, Thevetia L., and Skytanthus Meyen) was more appropriate. More recently, Leeuwenberg (1997) put Cabucala into synonymy under Petchia. In addition to the taxa mentioned above, there are four other genera characterized by drupaceous fruits: Cerbera, Thevetia, Cerberiopsis, and Cameraria. These, together with Skytanthus, with follicular fruits, were split out of Rauvolfioideae and treated by Pichon (1948b) as a separate subfamily, Cerberoideae. The characters he used for delimitation of this subfamily are ambiguous. Leeuwenberg (1994a) recognized the group as defined by Pichon, but at the tribal level as Cerbereae. Morphological studies by Fallen (1985) suggested a close relationship between Cerbereae and Allamanda L., the sole genus placed in Allamandeae by Pichon (1949a) and Leeuwenberg (1994a). Studies based on molecular or combined morphological and molecular data (Endress et al., 1996; Sennblad & Bremer, 1996, 2000, 2002) indicated that the genera previously included in Cerbereae do form a natural group (see Potgieter & Albert, 2001, for a different opinion) and that they are only a part of a larger group that includes not only Allamanda, but also Plumeria L. (usually included in the Plumerieae: Rauvolfioideae). An analysis of Cerbereae is not the aim of this study, although some representatives from that tribe are included in our analyses. Using fruit characters to delimit tribes in Rauvolfioideae is appealing because it allows taxa to be easily categorized and keys to be constructed. Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 3 However, other characters of these taxa do not indicate the same patterns of relationships. Phylogenetic analyses of mainly molecular data have shown that these fruit- and seed-based classifications are considerably more artificial than previously suspected. An rbcL analysis by Sennblad and Bremer (1996) indicated that Catharanthus G. Don (with dry dehiscent follicles and included in Plumerieae) was more closely related to taxa previously placed in Alyxieae sensu Leeuwenberg (1994a) than to other Plumerieae. In larger studies (Sennblad, 1997; Sennblad & Bremer, 2000, 2002), Catharanthus and Vinca L. formed a well-supported clade together with Rauvolfia, Ochrosia, and Kopsia, which have fleshy drupes. In the same study, Chilocarpus Blume, which has always been thought to be most closely related to Carisseae, formed a strongly supported clade with Alyxia and Lepinia (Alyxieae), confirming results of a strongly supported Chilocarpus–Alyxieae clade reported previously by Civeyrel (1996) and van der Ham et al. (2001). Pichon (1949a) already realized that Geissospermum Allemão (with indehiscent fruits and seeds embedded in pulp) is probably the nearest relative of Aspidosperma Mart. & Zucc. (with dry dehiscent follicles and wind-dispersed seeds with a diaphanous wing), a position supported by Potgieter and Albert (2001) and Simões et al. (2007). In addition, Potgieter and Albert (2001) found that Vallesia (with drupaceous fruits and seeds embedded in juicy pulp) is closely related to Haplophyton A. DC. (with dry dehiscent follicles and wind-dispersed comose seeds). Such results indicate that fruit characters in Apocynaceae are evolutionarily plastic in response to selective pressures for adaptations associated with wind or animal dispersal. The most recent classifications of Apocynaceae s.l. (Endress & Bruyns, 2000; Sennblad & Bremer, 2002) attempted to rectify some of these anomalies. In the classification of Endress and Bruyns (2000), Anechites was moved to a newly defined Plumerieae (including Cerbereae sensu Leeuwenberg (1994a) as well as Allamanda), a position suggested by Fallen (1983a), and Vallesia was included with Aspidosperma, Geissospermum, and Haplophyton in a newly defined Alstonieae. The remainder of Alyxieae (sensu Leeuwenberg, 1994a) was split into two tribes, Alyxieae and Vinceae, based on molecular results as well as additional morphological characters. Alyxieae sensu Endress and Bruyns (2000) included seven genera: Alyxia, Pteralyxia K. Schum., Lepinia, Lepiniopsis, Plectaneia Thouars, Condylocarpon, and Chilocarpus. Vinceae included Amsonia Walter, Catharanthus, Vinca, Rauvolfia, Petchia, Kopsia, Neisosperma Raf., and Ochrosia. Rhazya Decne. was considered to be synonymous with Amsonia, and Cabucala with Petchia (following Leeuwenberg, 1997). Simões et al. (2007) treated Amsonia as a genus incertae sedis and transferred Laxoplumeria Markgr., Tonduzia Pittier, and Kamettia Kostel. to Vinceae, bringing the total number of genera in the tribe up to 10. The classification of Sennblad and Bremer (2002) proposed a new system that is compatible with traditional Linnaean nomenclature but uses a variant of the definitions used in phylogenetic nomenclature to improve the stability of classifications. Although they do not provide lists of included genera, their definitions of the tribes containing traditional Alyxieae genera are completely congruent with those of Endress and Bruyns (2000). The aim of this study is to cladistically evaluate Alyxieae and Vinceae as circumscribed by Endress and Bruyns (2000) in comparison with previous classifications, to reexamine the usefulness of fruit and seed characters for tribal delimitation within Rauvolfioideae, and to discover new morphological characters that have hitherto received little attention in classification of this subfamily but show phylogenetic potential. MATERIALS AND METHODS TAXON SAMPLING The ingroup taxa were chosen to include representatives of all genera of Alyxieae (sensu Leeuwenberg, 1994a), as well as other putatively related genera. The outgroup taxa are one genus each of Loganiaceae and Gelsemiaceae, which several studies (Bremer & Struwe, 1992; Chase et al., 1993; Savolainen et al., 2000; Soltis et al., 2000) have demonstrated to be closely related to Apocynaceae (Appendices 1, 2). Other more narrowly focused studies on Gentianales (Struwe et al., 1994; Endress et al., 1996; Backlund et al., 2000) have also indicated that Loganiaceae and Gelsemiaceae are the closest families to Apocynaceae. For the morphological analyses, we omitted the outgroups altogether because, in preliminary analyses, one or the other of these genera was embedded in different portions of the ingroup due to obvious parallelisms of certain characters; we arranged the morphological tree with the same group sister to the rest as in the molecular results. FLORAL STRUCTURE Fixed flowers at or near anthesis (only buds were available for Lepiniopsis) were dehydrated in an alcohol-xylene series, embedded in paraplast, cut with a rotary microtome at 10 mm, and stained with safranin and astra blue. For SEM studies, material was critical-point dried and then sputter-coated with gold. 4 Annals of the Missouri Botanical Garden POLLEN MORPHOLOGY Doyle and Doyle (1987). For the latter, DNA samples were purified by ultracentrifugation in CsCl–ethidium bromide gradients (1.55 g/ml). Additional purification using the QIAquick PCR purification kit (Qiagen, Valencia, California) was performed in cases with problematic polymerase chain reaction (PCR) amplification using the manufacturer’s protocol. Doublestranded DNA was amplified with PCR primers for rbcL from Fay et al. (1998); the trnL intron and trnL-F intergenic spacer (hereafter, trnL-F) were amplified using the c and f primers of Taberlet et al. (1991); matK primers were those of Endress et al. (1996) and Johnson and Soltis (1994). Direct sequencing of PCR products was performed using the PCR primers plus internal sequencing primers. For rbcL, the internal primers were those of Fay et al. (1998); for trnL-F, we used the d and e primers of Taberlet et al. (1991); and for matK, we designed two new internal primers: 734F, 59-ATGTATGTGACTACGAATCA-39 and 829R, 59-ACTTTCTATTTTTCCATAGA-39. In a number of cases, we also used the internal sequencing primers as PCR primers to amplify shorter products. For sequencing, we used either the Dye Deoxy Terminator Cycle Sequencing or Big Dye kits of Applied Biosystems (ABI; Warrington, Cheshire, United Kingdom). Sequencing reactions were carried out directly on the cleaned PCR products and run on an ABI 277a automated sequencer at Kew following the manufacturer’s protocols. Pollen material was sampled from the following herbaria: BISH, BR, COL, G, L, P, PTBG, QCA, S, UB, WAG, and Z. Pollen studies were carried out in Leiden and Stockholm. For light microscopy (LM), pollen material was acetolyzed (except for Condylocarpon and Vinca), mounted in glycerine jelly, and sealed with paraffin. Generally, 10 pollen grains were measured for polar axis (P) and equatorial diameter (E). For SEM, pollen was sputter-coated with gold and examined with a JSM 5300 or JSM 6300 scanning electron microscope (JEOL, Tokyo). Frozen sections were made using an Ames Tissue-TEK Cryostat. For transmission electron microscopy (TEM), unacetolyzed material (whole anthers) was embedded in Spurr resin or 3/7 Epon, sectioned with a LKB Ultrotome III or V, poststained with uranylacetate and lead citrate, and examined with a Zeiss 10, a JEOL 100-S, or a JEM 1010. Terminology is according to Punt et al. (1994). OTHER MORPHOLOGICAL AND CHEMICAL CHARACTERS Information on fruit and seeds were taken from observations of herbarium specimens as available. Several fruit and seed characters were taken from the literature, as were data on the presence of laticifers and intraxylary phloem (Solereder, 1892; Schumann, 1895; Valeton, 1895; Degener, 1946; Pichon, 1947a, 1947b, 1948a, 1948b, 1948c, 1948d, 1949a, 1949b, 1949c, 1950a, 1950b, 1950c, 1952; Gensel, 1969; Markgraf, 1971, 1976, 1979; Markgraf & Huber, 1975; Corner, 1976; Conn, 1980; Leeuwenberg & Leenhouts, 1980; Rogers, 1986; Rudjiman, 1986; Pagen, 1987; Metcalf & Chalk, 1989; Rosatti, 1989; Wagner et al., 1990; Sévenet et al., 1994; Forster & Williams, 1996; Omino, 1996; Leeuwenberg, 1997; Sidiyasa, 1998; Lin & Bernardello, 1999). Chemical data were taken from the literature (Johns et al., 1968; Hegnauer, 1970, 1989; Coppen & Cobb, 1983; Kisakürek et al., 1983; Homberger & Hesse, 1984; Bisset, 1987; Endress et al., 1990; Wagner et al., 1990; Zhu et al., 1990; Attaurrahman et al., 1989, 1991; Arambewela & Ranatunge, 1991; Jensen, 1992; Sévenet et al., 1994; Zeches et al., 1995; Kam et al., 1997). MOLECULAR METHODS Nine new sequences of rbcL, 16 of matK, and 11 of the trnL intron and trnL-F intergenic spacer were produced for this study; the other sequences were published previously (Appendix 2). Total DNA was extracted from fresh leaves, silica gel–dried material (Chase & Hills, 1991), or herbarium material using the methods of Saghai-Maroof et al. (1984) or modified CLADISTIC ANALYSES The data matrix comprised four submatrices: each of the three DNA regions plus morphology. The morphological submatrix (Appendix 3) comprises 54 characters from floral, fruit, vegetative, and pollen morphology and phytochemistry (Appendix 4). All analyses were performed using PAUP* 4.0b10 (Swofford, 2002). Heuristic searches were performed with all characters given unit weight (Fitch parsimony; Fitch, 1971), and each submatrix was analyzed separately before their joint combined analysis. Each of the searches used the following settings: 1000 replicates of random taxon entry, the subtree pruning re-grafting (SPR) branch swapping algorithm, and MULTREES on (saving multiple equally parsimonious trees) but holding only 10 trees per replicate. All the shortest trees were then collected and used as starting trees for a search with a 25,000-tree limit. If the tree limit was reached, then swapping was allowed to continue until all 25,000 trees were swapped to completion. Bootstrap percentages (BP) (Felsenstein, 1985) were calculated with 500 replicates on each of the submatrices as well as on the two combined matrices Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 5 (molecular combined and molecular/morphological combined). We used the following settings: SPR branch swapping and MULTREES on, holding only 10 trees per step. All other settings were those of the standard defaults of PAUP* 4.0. This strategy produces results statistically indistinguishable from other, more thorough bootstrap protocols (Salamin et al., 2003). We apply the following arbitrary scale in our discussion: 50%–74%, weakly supported; 75%– 84%, moderately supported; 85%–100%, strongly supported. Alignment for rbcL and matK was a simple matter; the former had no length variation, and the latter had only a few easily characterized insertions/ deletions (indels). For trnL-F, we started with the alignment of Potgieter and Albert (2001) and added the additional taxa needed for this analysis, which required adding a few more insertions; we did adjust their alignment in places following Kelchner (2000). We analyzed the trnL intron and trnL-F intergenic spacer in a single analysis (which can be considered ‘‘non-coding’’ because there is only about a 30 bp region of the trnL exon included); this region, termed trnL-F, is composed of two unrelated parts, but the number of variable sites is the lowest and, even when combined, these produced a highly unresolved strict consensus tree. We do report statistics for these two regions separately (Appendix 2) but consider results only for the two combined. Incongruence of different regions of plastid DNA would be unexpected because recombination is unknown in the generally uniparentally inherited plastid genome. Several tests for combinability have been developed, but we have not used any of them in this paper. Such tests have proven to be unreliable indicators of incongruence (Reeves et al., 2001), so we attach no particular significance to results of these tests but instead prefer to look for cases of strongly supported and incongruent patterns of relationships. Differences in relationships are to be expected with different matrix components simply due to sampling effects where there are too few variable characters to obtain clear patterns. If, however, there are only a few characters in a matrix, such as is the case here with the morphological characters, differentiating between sampling error and incongruent patterns is extremely difficult. We note that the morphologically based estimates of relationships deviate from those based on DNA data, particularly for the Vinceae, but the small number of morphological characters (only 54) does not permit us to say whether the differences between molecular and morphological patterns are evidence of true incongruence. Character state distributions of selected morphological characters were individually mapped onto the total evidence tree (Figs. 10, 11) using MacClade 4.0 (Maddison & Maddison, 2000) to illustrate character evolution and compare the usefulness of characters that have previously been used in delimitation of Alyxieae. RESULTS MORPHOLOGY Analysis of the morphological matrix produced 31 trees in three islands (18, 4, and 9 trees) of equally parsimonious trees, each of 246 steps with a consistency index (CI) of 0.37 and a retention index (RI) of 0.62. A strict consensus tree of all three islands (Fig. 1; numbers below the branches are BPs) shows that the position of several genera is unclear with these morphological data, and the three islands place them in different relative positions to the clades that are consistently resolved in all three islands. The positions of Vallesia antillana Woodson, V. glabra (Cav.) Link, and the Plumeria clade (Allamanda cathartica L., Anechites nerium (Aubl.) Urb., Plumeria rubra L., and Thevetia peruviana (Pers.) K. Schum.) are consistent in all three islands, as is a clade composed of all remaining taxa. Within the last, Acokanthera oblongifolia (Hochst.) Codd, A. oppositifolia (Lam.) Codd, Molongum laxum (Benth.) Pichon, Picralima nitida (Stapf ) T. Durand & H. Durand, Pleiocarpa mutica Benth., Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schult., and T. pandacaqui Lam. occupy different positions in each of the three islands with respect to the two consistently resolved clades: (1) Alstonia scholaris (L.) R. Br. to Rhazya stricta Decne. and (2) Alyxia oblongata Domin and A. ruscifolia R. Br. to Nerium oleander L. (Fig. 1). The other conspicuously differently placed group is that composed of Amsonia ciliata Walter, A. tabernaemontana Walter, Rhazya stricta, and Catharanthus roseus (L.) G. Don + Vinca major L. and V. minor L., which in two of the islands (18 and 9 tree islands) are a clade but in the other island form a grade. Clades that receive moderate to strong BPs are the following: Neisosperma nakaiana (Koidz.) Fosberg & Sachet + Ochrosia coccinea (Teijsm. & Binn.) Miq. (BP 83), Alyxia oblongata and A. ruscifolia + Lepinia marquisensis Lorence & W. L. Wagner, L. solomonensis Hemsl. and L. taitensis Decne. + Lepiniopsis ternatensis Valeton and L. trilocularis Markgr. + Pteralyxia kauaiensis Caum and P. macrocarpa (Hillebr.) K. Schum. (BP 97; the last two genera with BP 96; the Alyxia clade), Kibatalia gitingensis (Elmer) Woodson + Mascarenhasia arborescens A. DC. + Nerium oleander (BP 98; the first two genera with BP 94), Picralima nitida + Pleiocarpa mutica (BP 84), and Allamanda cathartica 6 Annals of the Missouri Botanical Garden Figure 1. Strict consensus tree of the three islands found in the morphological analysis. Numbers below the branches indicate bootstrap percentages greater than 50%. Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae + Anechites nerium + Thevetia peruviana (BP 84). Catharanthus roseus + Vinca major and V. minor nearly reached the moderate level (BP 72). nitida + Pleiocarpa mutica as sister to the Alyxia clade, plus Plectaneia stenophylla (BP 70); and the Vinca clade (BP 65; Cabucala polysperma, Petchia ceylanica, Ochrosia coccinea, Catharanthus roseus, Vinca minor, Neisosperma oppositifolia, and Rauvolfia mannii). ANALYSIS OF RBCL Of the 1398 included positions, 244 (18%) were variable and 146 (10%) were potentially parsimony informative. Analysis produced two islands of equally parsimonious trees, one of 39 trees and the other of 16 trees; they had 479 steps with CI (including uninformative positions) of 0.59 and RI of 0.63. The two islands differ in the relative positions of Neisosperma oppositifolia (Lam.) Fosberg & Sachet and Rauvolfia mannii Stapf and Chilocarpus suaveolens Blume + Condylocarpon guyanense Desf. In island one, the latter form a clade with the Kibatalia gitingensis + Mascarenhasia arborescens + Nerium oleander clade, and Neisosperma oppositifolia and Rauvolfia mannii are unresolved members of the clade including Cabucala polysperma (Scott-Elliott) Pichon + Petchia ceylanica (Wight) Livera + Ochrosia coccinea. In island two, Neisosperma oppositifolia + Rauvolfia mannii are sister to Catharanthus roseus + Vinca minor, and Chilocarpus suaveolens + Condylocarpon guyanense are unresolved. The strict consensus of both islands (Fig. 2) therefore shows these taxa to be unresolved. Supported clades that are also found in the morphological analysis include the following: Allamanda cathartica + Plumeria inodora Jacq. (BP 100) and Anechites nerium + Thevetia peruviana (BP 97; the whole Plumeria clade, BP 62); Alyxia ruscifolia + Lepinia taitensis + Lepiniopsis trilocularis + Pteralyxia kauaiensis (the Alyxia clade; BP 99); Picralima nitida sister to Pleiocarpa mutica (BP 100); Catharanthus roseus + Vinca minor (BP 68); and Kibatalia gitingensis + Mascarenhasia arborescens + Nerium oleander (the Nerium clade; BP 93). Wellsupported clades that are not strongly in conflict with the morphological results include: Aspidosperma triternatum Rojas Acosta as sister to Vallesia antillana (BP 100); Plectaneia stenophylla Jum. as sister to the Alyxia clade (BP 98), within which the topology is the same as in the morphological results; Amsonia tabernaemontana as sister to Rhazya stricta (BP 100); Cabucala polysperma as sister to Petchia ceylanica (BP 100); and Molongum laxum as sister to Tabernaemontana divaricata (BP 95). Chilocarpus suaveolens as sister to Condylocarpon guyanense (BP 85) is contradicted in the morphological results by a weak BP 70 for the latter to be sister to Alyxia clade. Weakly supported results not contradicting relationships produced by morphology are the Plumeria clade (BP 62; Allamanda cathartica, Anechites nerium, Plumeria inodora, and Thevetia peruviana); Picralima 7 ANALYSIS OF MATK The aligned matK matrix contained 1647 bp, of which 561 (34%) were variable and 250 (15%) were potentially parsimony informative. We were unable to amplify the following taxa for matK: Anechites, Lepinia, Lepiniopsis, Ochrosia, and Plectaneia. Analysis produced a single, most parsimonious tree of 970 steps with a CI of 0.73 and an RI of 0.59. Patterns of relationships are nearly identical to those found with rbcL, but in general, BPs are higher than with rbcL or trnL-F (Fig. 3). Patterns that were not observed with rbcL include: Chilocarpus suaveolens + Condylocarpon guyanense (BP 100) as sister (BP 99) to the Alyxia clade (BP 100); Alstonia scholaris strongly supported in an isolated position as sister (BP 93) to all but Aspidosperma triternatum + Vallesia antillana; and Aspidosperma triternatum + Vallesia antillana (BP 82) moderately supported as sister to the rest of the ingroup (BP 97). ANALYSIS OF TRNL-F The aligned trnL-F matrix consisted of 1206 bp (761 bp from the trnL intron and 445 bp from the trnLF spacer). We were unable to amplify the following taxa for trnL-F: Kibatalia G. Don and Lepinia. Analysis produced over 25,000 trees of 460 steps with a CI of 0.78 and an RI of 0.70 (trnL intron: 292 steps with a CI of 0.76 and an RI of 0.69; trnL-F intergenic spacer: 168 steps with a CI of 0.82 and an RI of 0.72) (Fig. 4). Relationships from these two, largely non-coding regions are similar to those estimated from rbcL and matK (Figs. 2, 3). The major noteworthy result (also observed in the matK results, but which received BP , 50) is a strongly supported clade (BP 97) composed of Nerium oleander and Mascarenhasia arborescens of the Nerium clade, observed with morphology, rbcL, and matK, with Acokanthera oppositifolia weakly supported as sister (BP 60) also observed but without support with matK, and Allamanda indet. + Plumeria alba Kunth (BP 95), Anechites nerium, and Thevetia ahouai (L.) A. DC. (the last two unresolved with respect to the Nerium clade). COMBINED MOLECULAR ANALYSES The combined data set produced 46 equally parsimonious trees of 1925 steps with a CI of 0.70 8 Annals of the Missouri Botanical Garden Figure 2. Strict consensus tree of the two islands found with the rbcL data. Numbers below the branches indicate bootstrap percentages greater than 50%. Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 9 Figure 3. The single most parsimonious tree found with the matK data. Numbers above the branches indicate estimated substitutions, ACCTRAN optimization. Numbers below the branches indicate bootstrap percentages greater than 50%. and an RI of 0.62. One of the shortest individual trees is shown in Figure 5, with estimated substitutions (ACCTRAN optimization) indicated above the branches and BPs below; groups not present in all shortest trees are marked with an arrowhead. We show a single tree to illustrate relative levels of genetic divergence. The contribution of each region to this tree was: rbcL, 488 steps (vs. 479 for the rbcL trees); matK, 974 steps (vs. 970 for the matK tree); trnL intron, 293 steps (vs. 292 on the trnL-F trees); and trnL-F intergenic spacer, 170 steps (vs. 168 on the trnL-F trees). Patterns of relationships are much like those in the previous analyses, and BPs are generally higher than in any of the individual analyses. ANALYSIS OF ALL DATA COMBINED The combined data produced a single, most parsimonious tree of 2226 steps with a CI of 0.65 and an RI of 0.60 (Fig. 6). The DNA optimized onto 10 Annals of the Missouri Botanical Garden Figure 4. Strict consensus tree of the 25,000 equally most parsimonious trees found with the trnL-F data. Numbers below the branches indicate bootstrap percentages greater than 50%. this tree is 1926 steps, one step longer than the combined DNA tree; this step is caused by shifting Kopsia fruticosa (Ker. Gawl.) A. DC. and Molongum laxum + Tabernaemontana (two spp., Appendix 2) from an unresolved position with respect to the major clades into positions as a grade with respect to the Vinca clade. Otherwise, relationships are exactly as with the combined molecular data. The morphological data optimized onto the combined trees (ACCTRAN optimization) was 276 steps with a CI of 0.33 and an RI of 0.55, versus 266 steps with a CI of 0.38 and an RI of 0.61 in the morphological analysis. DISCUSSION EVALUATION OF TRADITIONAL CIRCUMSCRIPTIONS OF ALYXIEAE Both the morphological and the molecular analysis indicate that Alyxieae as previously circumscribed are polyphyletic. Of the individual data sets analyzed, the tree based on matK provided the best support, followed by that of trnL-F. BPs in the tree based on rbcL and the morphological data set were low, with much of the tree a polytomy, and the positions of several genera were equivocal. Even then, however, Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 11 Figure 5. One of the most parsimonious trees found with the combined molecular data. Numbers above the branches indicate estimated substitutions, ACCTRAN optimization. Numbers below the branches indicate bootstrap percentages greater than 50%. Groups not present in all 46 shortest trees are noted with an arrowhead. clusters of genera are present. The combined data tree is similar to that produced by the combined molecular data and provides much better support for patterns of relationship already seen in each of the individual trees. Therefore, the remainder of the discussion will be based on the total combined tree (Fig. 6). This tree is not intended to represent relationships within or among tribes other than Alyxieae and Vinceae. All genera traditionally included in Alyxieae are preceded by a dot in Figure 6. Vallesia and Anechites, both included in Alyxieae by Pichon (1949a, 1950b, as Rauvolfieae) and maintained there by Leeuwenberg (1994a), are not closely affiliated with any of the other members of the ingroup. Vallesia is sister to Aspidosperma, a position that supports results of previous phylogenetic studies (Sennblad & Bremer, 2000, 2002; Potgieter & Albert, 2001; Simões et al., 2007). It is unlikely that a close relationship between Vallesia and Aspidosperma would have been predicted based on their floral or fruit structures; they are too plesiomorphic to be of much help, and the small indehiscent drupaceous fruits of the former look very 12 Annals of the Missouri Botanical Garden Figure 6. The single most parsimonious tree found in the total combined analysis of the morphological and molecular data. Numbers above the branches indicate estimated substitutions, ACCTRAN optimization. Numbers below the branches indicate bootstrap percentages greater than 50%. The Alyxia clade and the Vinca clade are indicated with thicker branches and brackets. N 5 Alyxieae (Rauvolfieae in earlier classifications). D 5 taxa not recognized at generic rank by Leeuwenberg (1994a). Tribal names on right follow the classification of Endress and Bruyns (2000). different from the dehiscent follicular fruits of the latter. A close relationship between the two genera is, however, supported by pollen morphology. In both genera the pollen has five or six apertures (as opposed to the usual 3-aperturate condition), which are surrounded by distinctive prominent ridges. The inclusion of Vallesia in Alstonieae sensu Endress and Bruyns (2000) is supported by previous rbcL data (Sennblad & Bremer, 2000, 2002), but not by trnL-F data (Potgieter & Albert, 2001), which included more genera near the base of Apocynaceae. In our analysis, Anechites is sister to Thevetia, grouping with members of Cerbereae of traditional classifications (Cerberoideae of Pichon, 1948b), which confirms results based on morphology (Fallen, 1983a; Alvarado-Cárdenas & Ochoterena, 2007) and earlier rbcL data (Sennblad & Bremer, 2000, 2002) and supports Endress and Bruyns’ (2000) inclusion of Anechites in Plumerieae. The remainder of the genera of Alyxieae fall into two main clades. Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae RELATIONSHIPS WITHIN VINCEAE AND ALYXIEAE the Alyxieae, four, Lepinia, Lepiniopsis, Pteralyxia, and Plectaneia, are island endemics (Leeuwenberg, 1997; Lorence & Wagner, 1997), and Alyxia has its greatest species diversity in the Pacific (Middleton, 2000, 2002). Chilocarpus and Condylocarpon are sister genera that are the sister group to the remainder of Alyxieae (Fig. 6). Because of its syncarpous ovary, Chilocarpus was included in Carisseae (Willughbeieae sensu Endress & Bruyns, 2000) by Schumann (1895, as Arduineae) and the invalid Chilocarpinae by Pichon (1948a). Leeuwenberg (1994a) provided a Latin diagnosis, raised Pichon’s subtribe to tribal level, and in his recent revision of Chilocarpus (Leeuwenberg, 2002) considers Chilocarpeae to fall somewhere between Carisseae and Ambelanieae. Condylocarpon is the only New World member of Alyxieae and exhibits a set of deviating pollen features (tetrads, inaperturate, and reduced exine; Fig. 7) that strongly indicate the pollen to be paedomorphic (i.e., underdeveloped regarding pollen wall features, yet viable). The basically decussate tetrad configuration indicates Condylocarpon pollen to be derived from a 2aperturate rather than from a 3-aperturate ancestor, providing additional support for its present position in Alyxieae (van der Ham et al., 2001). Despite its aberrant, inaperturate, nearly exineless pollen, Condylocarpon resembles Chilocarpus in a number of other morphological aspects, especially the distinctive globose head of the flower buds. The fruits exhibit some superficial similarities; in most species of both genera, they are moniliform and woody, although the gynoecium is syncarpous and dehiscent in Chilocarpus and apocarpous and indehiscent in Condylocarpon (Fallen, 1983b). The second main cluster of Alyxieae in the combined analysis (Fig. 6) is the Vinca clade and includes Cabucala, Petchia, Rauvolfia, Ochrosia, Neisosperma, Kopsia, Catharanthus, and Vinca. All taxa in this clade are characterized by a differentiated style head with a distinct annulus at the base, but this is a plesiomorphic feature and is also found in other tribes (e.g., Alstonia R. Br., Alstonieae; Allamanda, Plumerieae). Similarly, the pollen morphology of this clade is unspecialized (Fig. 8). The occurrence of well-developed colpal and mesocolpial plates due to the presence of distinct supplementary endocolpi (absent in Vinca, weak in Kopsia and several taxa outside the Vinceae: Acokanthera G. Don, Allamanda, Aspidosperma, and Plumeria) is their most discriminating feature. Of the taxa in the Vinca clade, Rauvolfia, Ochrosia, and Kopsia were treated in Schumann’s (1895) classification. He included Rauvolfia in Rauvolfinae, together with nine other genera, none of which shows a close relationship with The first main cluster of Alyxieae in the combined analysis (Fig. 6) is the Alyxia clade and includes Alyxia, Lepinia, Lepiniopsis, and Condylocarpon. In addition, the placement of Chilocarpus, Plectaneia, and Pteralyxia in this clade is well supported. This corroborates the positions of Alyxia, Lepinia, and Chilocarpus in the study by Sennblad and Bremer (2000, 2002) based on rbcL data. Except for Condylocarpon, the members of this group have irregular pollen grains with relatively large porate apertures. The aperture number is usually two; Lepinia and Lepiniopsis have three or four apertures (van der Ham et al., 2001). Within the Alyxia clade, Alyxia, Lepinia, Lepiniopsis, Pteralyxia, and Plectaneia form a clade (Fig. 6). The first three genera correspond to Pichon’s (1949a) and Leeuwenberg’s (1994a) Alyxiinae. Pteralyxia was considered to be a synonym of Alyxia by Pichon (1949a) and a synonym of either Alyxia or Ochrosia by Leeuwenberg (see Gunn et al., 1992 and van der Ham et al., 2001: 169, 187). Plectaneia, in contrast, has previously been included in Plumerieae and has usually been considered to be related to genera such as Gonioma E. Mey., Stephanostegia Baill., and Craspidospermum Bojer ex A. DC. (Alstoniinae of Schumann, 1895; Plectaneiinae of Pichon, 1949a; Craspidosperminae of Leeuwenberg, 1994a), all of which are included in Melodineae in Endress and Bruyns (2000). In terms of pollen morphology, Alyxia, Lepinia, Lepiniopsis, and Pteralyxia form a tight-knit monophyletic group (Fig. 7). The porate apertures can be large (maximum 21–33 mm), their margins are clearly thickened (less clearly also in Chilocarpus), the inner exine surface is granular (also in subfamily Apocynoideae), the inner exine layer (nexine) is completely endexinous, the infratectum is hardly recognizable (being reduced to 6 sparse gaps in the inner ectexine), and a relatively thick tectum is present (also in several other taxa). Lepinia and Lepiniopsis share the presence of an ornamentation consisting of anastomosed verrucae (van der Ham et al., 2001). In the large analysis of trnL-F by Potgieter and Albert (2001), Alyxia, Lepiniopsis, Condylocarpon, and Plectaneia were supported in Alyxieae, whereas Pteralyxia affined with Plumerieae. This is an unlikely position for Pteralyxia considering its distinctive pollen, which is a synapomorphy of Alyxieae and found nowhere else in Apocynaceae (van der Ham et al., 2001). As the same vouchered specimen was used as the source of DNA for both studies, the reason for this discrepancy between our trnL-F results and those of Potgieter and Albert (2001) is most likely due to a mix-up in the laboratory. It is noteworthy that of the seven genera in 13 14 Annals of the Missouri Botanical Garden Figure 7. Apocynaceae, Alyxieae. SEM and TEM images of pollen grains. A–C. Alyxia ruscifolia. —A. Barrel-shaped 2porate pollen grain (orientation unknown) with differently sized pores. —B. Detail of A, showing psilate, perforate ornamentation. —C. TEM section of exine and intine, showing tectum (above), infratectum (gaps), and endexinous inner layer with dark inclusions and dark granules on the inner surface (arrow). D, E. Pteralyxia kauaiensis. —D. Barrel-shaped 2-porate pollen grain (orientation unknown) with differently sized pores. —E. Detail of undulate ornamentation with perforations in the depressions. —F. Pteralyxia macrocarpa. TEM section of exine and intine, showing thick tectum (above), infratectum (commissural line and several small gaps), and endexinous inner layer with small dark inclusions and larger dark surface granules (arrow). G, H. Lepinia solomonensis. —G. Tetraporate pollen grain (orientation unknown) with unequal sides and differently sized pores. —H. Detail of 6 verrucate ornamentation. —I. Lepinia taitensis. TEM section of exine and intine, showing thick tectum (above), infratectum (commissural line and gap), and endexinous inner layer with small dark inclusions and larger (locally stacked) dark surface granules (arrow). J–L. Lepiniopsis ternatensis. —J. 3-porate pollen grain (orientation unknown) with unequal sides and differently sized pores. —K. Detail of J, showing 6 verrucate ornamentation, psilate annulus, and inner surface granules (arrow) inside pore. —L. TEM section of exine and intine, showing thick tectum (above), infratectum (commissural line and sparse gaps), and endexinous inner layer with small dark inclusions and large dark surface granules (arrow). M, O. Chilocarpus denudatus. —M. Psilate to finely fossulate 2-porate pollen grain (orientation unknown). — O. TEM section of exine and intine, showing tectum (above), granular infratectum, and ectexinous inner verrucate layer. N, R. Plectaneia thouarsii. —N. Psilate, perforate 2-porate pollen grain (orientation unknown). —R. TEM section of exine and Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 15 Rauvolfia in this study or other recent analyses (Sennblad & Bremer, 2000, 2002; Potgieter & Albert, 2001; Simões et al., 2007), whereas Ochrosia and Kopsia were included in a different subtribe, Cerbereae (Plumerieae of Endress & Bruyns, 2000). Rauvolfieae as circumscribed by Pichon (1949a) included four subtribes. The Rauvolfinae comprised Cabucala, Petchia, and Rauvolfia, which are supported as monophyletic in the Vinca clade. Ochrosinae contained only Ochrosia (including Neisosperma), and Vallesinae included Vallesia and Kopsia, which are only distantly related here. His other two subtribes, Alyxinae and Condylocarpinae, belong to elements of Alyxia clade in our study. Pichon (1949a) included Anechites as a genus incertae sedis, noting that he did not have sufficient material to place it, but he thought that it most probably belonged with Condylocarpon or in a tribe of its own. The classification of Leeuwenberg (1994a) followed Pichon’s, with the same tribal circumscription of Alyxieae and the same subtribal circumscriptions. The only differences were that Anechites was included as a member of Condylocarpinae and the name of the tribe was changed to Alyxieae. More recently, Leeuwenberg (1997) placed Cabucala in synonymy under Petchia. Although there are some differences in pollen morphology (differing colpus length, tectum thickness and ornamentation, and the presence/ absence of deviating mesocolpium centers) in the species studied, the floral structure of the two genera is nearly identical, and our results do not contradict the synonymy of Cabucala. Neisosperma, which was placed into synonymy under Ochrosia by Pichon (1949a) and maintained there by Leeuwenberg (1994a) and Hendrian (2004), is often considered to be a distinct genus, especially by specialists dealing with species of the Pacific Basin, where both genera have their greatest species density (Fosberg et al., 1977; Markgraf, 1979; Boiteau, 1981; Smith, 1988; Wagner et al., 1990; Forster & Williams, 1996). Macromorphologically, plants of Neisosperma and Ochrosia have a clear resemblance (e.g., trees with whorled leaves, corolla lobe aestivation dextrorsely contort, fruits large, fleshy colorful drupes). Their pollen, however, differs in several aspects (size, ectoaperture shape and margin, endoaperture margin, and ornamentation). In our analyses, inclusion of Neisosperma in Ochrosia is only moderately supported. A detailed study including more species of both genera is needed in order to elucidate their relationship. Also in the Vinca clade (Fig. 6) are two genera conventionally included in Plumerieae: Catharanthus and Vinca (Schumann, 1895 as Alstoniinae; Pichon, 1949a as Alstonieae; Leeuwenberg, 1994a). The pollen of Vinca is peculiar and unique by its indistinct ectoapertures, relatively large endoapertures, and thin exine, which makes it difficult to compare with the pollen of other taxa. Vinca also shows some derived floral characteristics, such as the enlarged spathulate, apical anther appendage, which most likely plays a role in inhibiting desiccation of the secondarily presented pollen (Church, 1908), possibly related to the temperate habitat of this genus. The genera included here in the Vinca clade are the same as those included in Vinceae of Endress and Bruyns (2000), with the exception of Amsonia and Rhazya. These two genera have traditionally been considered to be closely related to Catharanthus and Vinca, and thus conventionally included in Plumerieae (Schumann, 1895 as Alstoniinae). Amsonia, Rhazya, Catharanthus, and Vinca constituted Lochnerinae of Pichon (1949a) and Catharanthaninae of Leeuwenberg (1994a). Flowers, fruits, seeds, and pollen of Rhazya scarcely differ from those of Amsonia (Pichon, 1949a; Nilsson, 1986), which was therefore treated as a synonym of Amsonia by Endress and Bruyns (2000) and not contradicted by our study (Fig. 6). However, Amsonia and Rhazya together group here with neither Alyxieae nor Vinceae. Instead, they are placed as sister to the Plumerieae and Carisseae + Apocynoideae, although bootstrap support for this is less than 50% (Fig. 6). In the study by Potgieter and Albert (2001), Amsonia was in a clade together with Thevetia peruviana, which was included in a large polytomy. Floral structure of Amsonia and Rhazya, including details of the style head, is similar to that of Catharanthus and Vinca and does not agree with the more derived position for the former two genera indicated by the molecular analysis. Similarly, Amsonia and Rhazya occupy an unexpected position in the molecular tree considering their secondary r intine, showing tectum (above), undulate granular infratectum, and ectexinous inner layer with surface verrucae (partly loose?). P, Q, S. Condylocarpon isthmicum. —P. Tetrad almost filling anther locule, showing the four constituent, psilate members in decussate configuration. —Q. TEM section through tetrad almost filling anther locule, showing three of the four constituent members, locally fused internal walls with sparse pores (arrow), and thin, locally thickened, external walls. —S. TEM section through external wall, showing the thin tectum, the poorly defined infratectum (arrow), and the locally thickened ectexinous inner layer. Scale bar 5 10 mm in A, D, G, J, M, N, P, Q; scale bar 5 1 mm in B, C, E, F, H, I, K, L, O, R, S. 16 Annals of the Missouri Botanical Garden Figure 8. Apocynaceae. Vinceae (A–P), Melodineae (Q–S). SEM and TEM images of pollen grains. —A. Petchia ceylanica. Psilate 3-colporate pollen grain in oblique view; colpi short with relatively large endopore. B, C. Cabucala caudata. —B. Inside of tricolporate pollen grain showing two colpal plates, each enclosing an endopore surrounded by a distinct endoannulus; in the center a mesocolpial plate delimited from the colpal plates by supplementary endocolpi (arrows). —C. TEM section through supplementary endocolpus (arrow), colpal plate (left), and mesocolpial plate (right). —D. Kopsia fruticosa. SEM section, showing psilate to scabrate exine stratified into two equally thick strata (tectum above) separated by a thin infratectum. —E. Ochrosia coccinea. Verrucate 3-colporate pollen grain in polar view. F–H. Neisosperma nakaiana. — F. Psilate/finely fossulate tricolporate pollen grain in polar view. —G. TEM section through mesocolpial exine and intine, showing tectum (above), irregular infratectum and foot layer, and spongy endexine (arrow). —H. TEM section through apertural exine, showing tectum (above), irregular infratectum, foot layer, which is much thickened under the colpus (arrow), and spongy endexine. I–K. Rhazya stricta. —I. Psilate, perforate pollen grain in equatorial view; colpi short and wide with relatively large endopores. —J. Inside of pollen grain showing lalongate endopore delimited polewards by horizontally oriented colpal plates; to the left and to the right, psilate mesocolpial plates delimited by wide verrucate zones (supplementary endocolpi). —K. TEM section through exine and intine, showing tectum (above), slightly thinner, granular-reticulate infratectum, and inner layer of the same thickness as tectum. L, M. Vinca minor. (critical-point dried). —L. Psilate 4aperturate pollen grain in polar view. —M. Psilate 4-aperturate pollen grain in equatorial view, showing the indistinct ectoaperture (porous area) in center of depressed oblong zone that is delimited by the endoapertural costae. N–P. Vinca major. —N. TEM section through thin mesocolpial exine and intine, showing tectum (above) with indistinct perforations, distinct Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae chemistry. Both genera contain numerous complex indole alkaloids of the plumerane type (Ganzinger & Hesse, 1976; Kisakürek et al., 1983), whereas in all genera above them in the uppermost clades of the tree and in Alyxieae, indole alkaloids have been lost. In the comparative study by Nilsson (1986), however, pollen of Amsonia and Rhazya was found to be nearly identical but showed no close relationships with pollen of Catharanthus or Cabucala (Vinceae). Because the focus of our study was Alyxieae, this was the only group densely sampled. To better assess the phylogenetic position of Amsonia and Rhazya, additional representatives of other tribes, especially previously unstudied genera of Melodineae sensu Endress and Bruyns (2000), should be included. The unexpected positioning of Amsonia and Rhazya in the combined molecular tree (Fig. 5) away from the rest of Vinceae could be regarded as a case of incongruence with morphology. However, in the morphological analysis, Amsonia and Rhazya did not always appear together with Catharanthus + Vinca, and their position was relatively unclear (e.g., no BP . 50). Furthermore, combining the DNA and morphological data resulted in generally better resolution and higher support in the all-data combined tree relative to the combined DNA data tree, which would not be expected if there were highly incongruent basic patterns in each of them. The exact position of Amsonia/Rhazya is also not clear with the DNA data, except that their exclusion from the clade with the rest of Vinceae is not strongly supported (Figs. 5, 6), which concurs with the results of Simões et al. (2007). Armbruster (1996), Endress (1996), Hufford (1997), and Clausing et al. (2000). Fruit and seed characters are thus particularly unreliable when used alone for determining relationships among genera in Apocynaceae. Examples of these characters include: fruit dehiscence, mesocarp consistency, and seed appendages. It was the use of such simple, single characterbased categories that led to the artificial tribal classifications of Schumann (1895), Pichon (1949a), and Leeuwenberg (1994a). The most reliable characters are likely to be more subtle, and one must be willing to invest some effort to determine what they are. It is also unrealistic to believe that any single character is going to provide a non-homoplasious synapomorphy for any large genus or tribe, but rather it is more reasonable to expect that these groups can be circumscribed by a specific combination of characters. Alyxieae pollen is characterized by large porate apertures (Fig. 7), which are distinct from the usual small aperturate porate grains found in Apocynoideae, completely different from the colporate grains found in Vinceae, and characteristic for all other Rauvolfioideae (Figs. 8, 9). Details of the pollen ectoapertures proved to be the most important morphological characters for defining Alyxieae because the unusual and distinct pollen type is synapomorphic for the tribe. Aperture type is less informative in the other tribes of Rauvolfioideae because, with a few exceptions (e.g., Craspidospermum; Fig. 8), all Rauvolfioideae have colporate pollen grains. Within Alyxieae, aberrant, inaperturate, nearly exineless pollen that remains in tetrads is an autapomorphy for Condylocarpon. Inaperturate pollen is otherwise known in the family only in Secamonoideae and Asclepiadoideae as well as some genera of Periplocoideae that have pollinia (e.g., Finlaysonia Wall., Hemidesmus R. Br.; Schill & Jäkel, 1978; Verhoeven & Venter, 2001). Condylocarpon is also of interest biogeographically, being the only Neotropical member of Alyxieae; all other genera are found in southeastern Asia and the Pacific. Aperture type thus provides a clear distinction between the Alyxieae and Vinceae. Other morphological characteristics that distinguish Alyxieae from Vinceae (but not necessarily SIGNIFICANCE OF MORPHOLOGICAL AND CHEMICAL CHARACTERS As has been demonstrated here and elsewhere (Fallen, 1983a; Sennblad & Bremer, 2000, 2002; Potgieter & Albert, 2001), superficial resemblance of characters such as fruits and seeds of Apocynaceae that are correlated with dispersal mode are extremely labile, and there is a tendency for the repeated independent evolution of certain fruit and seed types. Similar findings for other angiosperm families have been reported by Bremer and Eriksson (1992), 17 r infratectum, and inner layer consisting of foot layer and spongy endexine (arrow). —O. TEM section through endoapertural costa (arrow) in ectoapertural area. —P. TEM section through endoapertural costa outside ectoapertural area, showing continuous tectum (arrow), discontinuous inner exine, and slightly bulging intine. Q–S. Craspidospermum verticillatum. —Q. Rhomboidal tetrad, showing several pores (in adjacent positions) near sutures. —R. TEM section through two adjacent pores of neighboring grains, showing small ectopores and heavily costate endopore (arrow). —S. TEM section through two adjacent pollen grains, showing exine stratigraphy with joint tecta, separate infratecta, and inner exine layers; note Ubisch bodies (arrow). Scale bar 5 10 mm in A, E, F, L, M, Q; scale bar 5 5 mm in I–K; scale bar 5 1 mm in B–D, G, H, N–P, R, S. 18 Annals of the Missouri Botanical Garden Figure 9. Apocynaceae. Aspidospermeae (A, B, D–F), Alstonieae (C), Hunterieae (G–I ), Plumerieae (J), Carisseae (K, L), Malouetieae (Apocynoideae) (M–O). SEM and TEM images of pollen grains. A, B. Aspidosperma parvifolium. —A. Psilate/ fossulate 6-colporate pollen grain in slightly oblique polar view, showing prominent arcus-like ridges surrounding the colpi. — B. SEM section of ridge, showing a thin, psilate, perforate tectum, thick granular infratectum, and thin inner exine layer. —C. Alstonia scholaris. TEM section through exine and intine, showing thick psilate, perforate tectum, granular infratectum, dimerous foot layer, and indistinct spongy endexine (arrow). D–E. Vallesia glabra. —D. Psilate pentacolporate pollen grain in polar view, showing prominent ridges surrounding colpi. —E. Part of fractured pollen grain showing three ‘empty’ ridges and psilate to scabrate inside with elongated, unevenly distributed granular marks (arrow). —F. Vallesia antillana. TEM section through ridge, showing tectum subtended by granular infratectum (arrow) and cavity, and a 6 dimerous inner layer. G, H. Pleiocarpa mutica. —G. Psilate, perforate tricolporate pollen grain in polar view. —H. Scabrate inside of colporate pollen grain showing two apertures (right and extreme left); endopores have polar costae (arrow). —I. Picralima nitida. TEM section through exine and intine; exine consists of relatively thick, undulated tectum, slightly thinner granular-reticulate infratectum subtended by a thin foot layer and thin indistinct endexine (arrow). —J. Anechites nerium. Psilate, finely perforate tricolporate pollen grain in polar view. K, L. Acokanthera oppositifolia. —K. Inside of tricolporate pollen grain showing colpal plates with lalongate endopores (left and right) and system of endocracks (supplementary endocolpi; arrows) delimiting psilate mesocolpial and polar plates. —L. TEM section through supplementary endocolpus (arrow) subtended by thickened intine (left) and mesocolpial exine (right). M–O. Mascarenhasia arborescens. —M. Psilate triporate pollen grain, showing pores with weak annulus (arrow). —N. Exine fragment showing part of psilate, sparsely perforate tectum, inner side beset with diversely Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae from other tribes of Rauvolfioideae) include the simple style head, which is uniformly secretory and receptive and lacks a pollen-trapping annulus at the base (vs. the style head differentiated into distinct morphological and functional zones, with the receptive region located beneath an annulus at the base). Except for Plectaneia, Alyxieae seeds have a tough and usually conspicuously ruminate endosperm (vs. a smooth and soft endosperm in Vinceae). Because tribes within Rauvolfioideae were so artificial in earlier classifications (Schumann, 1895; Pichon, 1949a, 1950b; Leeuwenberg, 1994a), it was impossible to understand the evolution of secondary chemistry in this subfamily. As tribal circumscriptions become more natural, it is now possible to gain a better understanding of the phylogenetic pattern of indole alkaloids and cardenolides. Generally, indole alkaloids are considered to be characteristic for Rauvolfioideae. However, indole alkaloids characterize only five of the tribes included in this study (Alstonieae, Tabernaemontaneae, Vinceae, Hunterieae, and Melodineae), whereas indole alkaloids have not been reported in the other three tribes (Fig. 6). Acokanthera and Carissa L. contain cardenolides. Based on their syncarpous gynoecium, they were conventionally included with indole alkaloid–containing genera in a polyphyletic Carisseae (Leeuwenberg, 1994a). Here and in other phylogenetic studies (Endress et al., 1996; Potgieter & Albert, 2001; Simões et al., 2007), the Carisseae sensu Endress and Bruyns (2000) are placed with other taxa in which indole alkaloids have been lost or replaced by other secondary compounds (Johns et al., 1968; Hegnauer, 1970; Coppen & Cobb, 1983; Kisakürek et al., 1983; Jensen, 1992). All genera of Vinceae contain various complex indole alkaloids (Hegnauer, 1970, 1989; Ganzinger & Hesse, 1976; Kisakürek et al., 1983). In contrast, all genera of Alyxieae studied for secondary chemistry lack indole alkaloids. Alyxia and Lepiniopsis contain coumarins (Johns et al., 1968; Hegnauer, 1970, 1989), and although Lepinia has never been analyzed for secondary compounds, the crushed leaves are known to emit a strong coumarin scent (D. Lorence, pers. comm., 1999). Coumarins are absent from Plectaneia, and no reliable data are available on the secondary chemistry of Pteralyxia, Chilocarpus, and Condylocarpon. CHARACTER EVOLUTION 19 Gynoecium, fruit, and seed morphology are complex and homoplasious in Rauvolfioideae. In this analysis, the plesiomorphic state of the gynoecium is apocarpous, in concurrence with Potgieter and Albert (2001; but see Sennblad & Bremer, 2000, 2002, for an alternative view), and the majority of the genera are apocarpous. Even in the small sample here, almost every clade includes at least one syncarpous genus; similar findings are reported by Simões et al. (2007). The style head is a useful character for distinguishing genera in Apocynaceae (see Fig. 10), but it is structurally complex, making it difficult to break down into meaningful character states for coding morphological characters. The two taxa sister to the rest in this analysis, Vallesia and Aspidosperma, have a simple style head that is vertically undifferentiated. A simple style head is also characteristic for all Alyxieae and for the Hunterieae and Carisseae (Fallen, 1986; Endress et al., 1996; Endress & Bruyns, 2000). A similar type of style head is found in some (but not all) genera of Alstonieae and Plumerieae. Tabernaemontana s.l., as currently circumscribed by Leeuwenberg (1991, 1994b), includes species with a simple, undifferentiated style head, such as the species included in this study, as well as ones with a complex, vertically differentiated style head with distinct functional regions and a broad pollen-trapping flange at the base (e.g., all the New World species). These results indicate that style head specialization has probably evolved in parallel in various clades of Rauvolfioideae, as was suggested by Potgieter and Albert (2001), and that this probably has proceeded in both directions (Fig. 10). Within Alyxieae, Lepinia and Lepiniopsis have a 3to 5-carpellate ovary, which is partially to completely syncarpous, respectively (Endress et al., 1997). A 3to 5-carpellate ovary is otherwise known in Apocynaceae only in Pleiocarpa (Hunterieae). All other genera have two carpels. Fruit type and seed margin are equivocally optimized: Vallesia has naked seeds in a small juicy drupe, whereas Aspidosperma has thick woody follicles and seeds with a diaphanous wing. This is in sharp contrast to the traditional view of the berries of Carisseae (a conglomeration of Willughbeieae, Melodineae, Hunterieae, and Carisseae, sensu Endress & r sized granules, and endoannulus (arrow). —O. TEM section through exine and intine near pore; exine is thickened into an ecto- and endoannulus; innermost exine layer consists of granules or irregular elements that are separated from tectum by granular infratectum; the intine contains numerous radially oriented dark inclusions (arrow). Scale bar 5 10 mm in A, D, G–J; scale bar 5 5 mm in K, M; scale bar 5 1 mm in B, C, E, F, L, N, O. 20 Annals of the Missouri Botanical Garden Figure 10. Evolution of style head body differentiation and pollen aperture type mapped onto the single most parsimonious tree from the combined analysis of morphological and molecular data using ACCTRAN optimization. Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 21 Figure 11. Evolution of fruit type and seed margin mapped onto the single most parsimonious tree from the combined analysis of morphological and molecular data using ACCTRAN optimization. 22 Annals of the Missouri Botanical Garden Bruyns, 2000) as the most unspecialized fruit type in the family. Vinceae are homogeneous with regard to seed margin, with the seeds being unwinged (see Simões et al., 2007, regarding Kamettia and Tonduzia, not included here). In the genera included in our analysis, the seeds are enclosed in an indehiscent drupe. Only Catharanthus and Vinca deviate, in having delicate, papery dehiscent follicles (Fig. 11). Catharanthus and Vinca also have a derived herbaceous habit, whereas the other genera in Vinceae are trees or shrubs. The seed margin is evolutionarily plastic in Alyxieae (Fig. 11). The four morphologically most specialized genera—Alyxia, Lepinia, Lepiniopsis, and Pteralyxia, as well as Condylocarpon—all have distinctive cylindrical seeds that are longitudinally rolled with a deep hilar groove. Plectaneia, in contrast, has flat seeds with a wing at each end. In Chilocarpus the seeds have a small pink corky aril on the funiculus and are presented in an unusual type of leathery dehiscent fruit that splits apart along one or two valves (Leeuwenberg, 2002). Arils are rare in Apocynaceae, otherwise known only in Tabernaemontaneae. Similar types of fruits are known as display fruits in Gesneriaceae (Wiehler, 1983; Smith, 2000) and Melastomataceae (Clausing et al., 2000) or as dehiscent berries in Oleaceae (Lawrence & Green, 1993; Li et al., 2002). Seed margins also vary considerably in Plumerieae (Pichon, 1949b, 1950b as Cerberoideae). In contrast to this diversity in Rauvolfioideae, in Apocynoideae and all other subfamilies, the fruit and seed type is uniform: the fruit is a pair of follicles (rarely postgenitally united) that dehisce to release small, comose seeds. Palynologically, starting from the basic regular 3colporate condition typical in the Rauvolfioideae (Figs. 8, 9; Nilsson, 1986), an entire suite of changes characterizes the derivation of Alyxieae (Fig. 10): pollen grain shape irregular, aperture number mostly two (sometimes three), and ectoapertures porate with thickened margins (Fig. 7). Within the clade, several other characters change: maximum pore diameter is 9 mm in Chilocarpus, 12 mm in Plectaneia, and 21– 33 mm in the subclade including Alyxia, Pteralyxia, Lepinia, and Lepiniopsis. Possibly due to paedomorphosis, Condylocarpon pollen is inaperturate (van der Ham et al., 2001). Together with the larger maximum pore diameter, the Alyxia clade also shows much larger, barrel-shaped (two pores) or depressed (three or four pores) pollen grains, an endexinous inner exine layer with a granular surface, and an indistinct (reduced) infratectum. Within the Alyxia clade, there is a change toward a more heavily sculptured tectum in the subclade including Alyxia, Pteralyxia, Lepinia, and Lepiniopsis, from psilate in Alyxia, via undulate in Pteralyxia, toward 6 verrucate in Lepinia and Lepiniopsis. Pollen of the last two genera mostly has more than two apertures: three (less often two) in Lepiniopsis and three or four in Lepinia (van der Ham et al., 2001). This is not simply a reversal to basic aperture conditions, as the apertures are still irregular or diverse within a single grain regarding size, configuration, and orientation. The functional significance of the remarkable shift in pollen morphology toward and within Alyxieae is not understood. The oldest fossils of the Alyxia pollen type date from the Paleocene of northwestern Borneo (Muller, 1981), which demonstrates the considerable age of the syndrome. CLASSIFICATION The topologies of this study support recognition of Vinceae and Alyxieae sensu Endress and Bruyns (2000), as well as the exclusion of Vallesia and Anechites from either tribe and their placement in Aspidospermeae sensu Simões et al. (2007) and Plumerieae, respectively. Our results do not support inclusion of Amsonia and Rhazya in Vinceae; however, these two genera are not supported in any of the other groups included in this study. The position of Amsonia was also not resolved in the study by Simões et al. (2007). Because the position of Amsonia and Rhazya remains equivocal, these genera are withdrawn from Vinceae and left unplaced for the time being. Although it is unsatisfying to leave them in limbo, a classification should reflect phylogeny, so it seems best to keep them as unplaced genera until more data are available to place them more definitely. Literature Cited Alvarado-Cárdenas, L. & H. Ochoterena. 2007. A phylogenetic analysis of the Cascabela-Thevetia species complex (Plumerieae; Apocynaceae) based on morphology. Ann. Missouri Bot. Gard. 94 (in press). Arambewela, L. S. R. & T. Ranatunge. 1991. Indole alkaloids from Tabernaemontana divaricata. Phytochemistry 30: 1740–741. Attaurrahman, R. A., A. Muzaffar, K. T. D. Desilva & W. S. J. Silva. 1989. Alkaloids of Petchia ceylanica. Phytochemistry 28: 3221–3225. ———, K. Zaman, S. Parveen, S. Habiburrehman, A. Muzaffar, M. I. Choudhary & A. Pervin. 1991. Alkaloids from Rhazya stricta. Phytochemistry 30: 1285–1293. Armbruster, W. S. 1996. Exaptation, adaptation, and homoplasy: Evolution of ecological traits in Dalechampia vines. Pp. 227–243 in M. J. Sanderson & L. Hufford (editors), Homoplasy: The Recurrence of Similarity in Evolution. Academic Press, San Diego. Backlund, M., B. Oxelman & B. Bremer. 2000. Phylogenetic relationships within the Gentianales based on ndhF and rbcL sequences, with particular reference to the Loganiaceae. Amer. J. Bot. 87: 1029–1043. Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 23 Bisset, N. G. 1987. Phytochemistry of Nerium L. Agric. Univ. Wageningen Pap. 87(2): 27–38. Boiteau, P. 1981. Apocynaceae. In A. Aubréville & J.-F. Leroy (editors), Flore de la Nouvelle Calédonie et Dépendances, Vol. 10. Muséum National d’Histoire Naturelle, Paris. Boke, N. H. 1948. Development of the perianth in Vinca rosea L. Amer. J. Bot. 35: 413–423. Bremer, B. & O. Eriksson. 1992. Evolution of fruit characters and dispersal modes in the tropical family Rubiaceae. Biol. J. Linn. Soc. 47: 79–95. ——— & L. Struwe. 1992. Phylogeny of the Rubiaceae and the Loganiaceae: Congruence or conflict between morphological and molecular data? Amer. J. Bot. 79: 1171–1184. Chase, M. W. & H. G. Hills. 1991. Silica gel: An ideal desiccant for preserving field-collected leaves for use in molecular studies. Taxon 40: 215–220. ———, D. E. Soltis, R. G. Olmstead, D. Morgan, D. H. Les, B. D. Mischler, M. R. Duvall, R. A. Price, H. G. Hills, Y.L. Qiu, K. A. Kron, J. H. Rettig, E. Conti, J. D. Palmer, J. R. Manhart, K. J. Sytsma, H. J. Michaels, W. J. Kress, K. G. Karol, W. D. Clark, M. Hedrén, B. S. Gaut, R. K. Jansen, K.-J. Kim, C. F. Wimpee, J. F. Smith, G. R. Furnier, S. H Strauss, Q.-Y. Xiang, G. M. Plunkett, P. S. Soltis, S. M. Swensen, S. E. Williams, P. A. Gadek, C. J. Quinn, L. E. Eguiarte, E. Golenberg, G. H. Learn, Jr, S. W. Graham, S. C. H. Barrett, S. Dayanandan & V. A. Albert. 1993. Phylogenetics of seed plants: An analysis of nucleotide sequences from the plastid gene rbcL. Ann. Missouri Bot. Gard. 80: 528–580. Church, A. H. 1908. Types of Floral Mechanism I. Clarendon Press, Oxford. Civeyrel, L. 1996. Phylogénie des Asclepiadaceae, Approche Palynologique et Moléculaire. Ph.D. Thesis, Université Montpellier II. ———, A. Le Thomas, K. Ferguson & M. W. Chase. 1998. Critical reexamination of palynological characters used to delimit Asclepiadaceae in comparison to molecular phylogeny obtained from plastid matK sequences. Molec. Phylogen. Evol. 9: 517–527. Clausing, G., K. Meyer & S. S. Renner. 2000. Correlations among fruit traits and evolution of different fruits within Melastomataceae. Bot. J. Linn. Soc. 133: 303–326. Conn, B. J. 1980. A taxonomic revision of Geniostoma (Loganiaceae). Blumea 26: 245–364. Coppen, J. J. W. & A. L. Cobb. 1983. The occurrence of iridoids in Plumeria and Allamanda. Phytochemistry 22: 125–128. Corner, E. J. H. 1976. The Seeds of Dicotyledons, Vols. 1 and 2. Cambridge Univ. Press, Cambridge. Crisp, M. D. 1983. Notonerium (Apocynaceae) laid to rest in the Boraginaceae. J. Adelaide Bot. Gard. 6: 189–191. Cronquist, A. 1981. An Integrated System of Classification of Flowering Plants. Columbia Univ. Press, New York. Degener, O. 1946. Apocynaceae and Asclepiadaceae, Families 305 and 306. In O. Degener (editor), Flora Hawaiiensis: The New Illustrated Flora of the Hawaiian Islands, Books 1–4. Publ. priv., Honolulu. Doyle, J. J. & J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. Bot. Soc. Amer. 19: 11–15. El-Ghazaly, G. 1990. Development of pollen grains of Catharanthus roseus (Apocynaceae). Rev. Palaeobot. Palynol. 64: 165–174. Endress, M. E. & P. Bruyns. 2000. A revised classification of the Apocynaceae s.l. Bot. Rev. (Lancaster) 66: 1–56. ———, M. Hesse, S. Nilsson, A. Guggisberg & J.-P. Zhu. 1990. The systematic position of the Holarrheninae (Apocynaceae). Pl. Syst. Evol. 171: 157–185. ———, D. H. Lorence & P. K. Endress. 1997. Structure and development of the gynoecium of Lepinia marquisensis and its systematic position in the Apocynaceae. Allertonia 7: 267–272. ———, B. Sennblad, S. Nilsson, L. Civeyrel, M. W. Chase, S. Huysmans, E. Graftström & B. Bremer. 1996. A phylogenetic analysis of Apocynaceae s. str. and some related taxa in Gentianales: A multidisciplinary approach. Opera Bot. Belg. 7: 59–102. Endress, P. K. 1996. Homoplasy in angiosperm flowers. Pp. 303–325 in M. J. Sanderson & L. Hufford (editors), Homoplasy: The Recurrence of Similarity in Evolution. Academic Press, San Diego. Erbar, C. 1991. Sympetaly—A systematic character? Bot. Jahrb. Syst. 112: 417–451. Ezcurra, C., M. E. Endress & A. J. M. Leeuwenberg. 1992. Apocynaceae. Pp. 1–121 in R. Spichiger & L. Ramella (editors), Flora del Paraguay, Vol. 17. Conservatoire et Jardin botaniques de la Ville de Genève, Geneva; Missouri Botanical Garden, St. Louis. Fallen, M. E. 1983a. A systematic revision of Anechites (Apocynaceae). Brittonia 25: 222–231. ———. 1983b. A taxonomic revision of Condylocarpon (Apocynaceae). Ann. Missouri Bot. Gard. 70: 149–169. ———. 1985. The gynoecial development and systematic position of Allamanda (Apocynaceae). Amer. J. Bot. 72: 572–579. ———. 1986. Floral structure in Apocynaceae: Morphological, functional, and evolutionary aspects. Bot. Jahrb. Syst. 106: 245–286. Fay, M. F., C. Bayer, W. Alverson, A. Y. de Bruijn, S. M. Swensen & M. W. Chase. 1998. Plastid rbcL sequences indicate a close affinity between Diegodendron and Bixa. Taxon 47: 43–50. Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791. Fitch, W. M. 1971. Toward defining the course of evolution: Minimal change for a specific tree topology. Syst. Zool. 20: 406–416. Forster, P. I. & J. B. Williams. 1996. Apocynaceae. Pp. 104– 196 in A. E. Orchard (editor), Flora of Australia, Vol. 18, Gentianales. CSIRO, Melbourne. Fosberg, F. R., P. Boiteau & M.-H. Sachet. 1977. Nomenclature of the Ochrosiinae (Apocynaceae): 2. Synonymy of Ochrosia Juss. and Neisosperma Raf. Adansonia, Ser. 2 17: 23–33. Ganzinger, D. & M. Hesse. 1976. A chemotaxonomic study of the subfamily Pumerioideae of the Apocynaceae. Lloydia 39: 326–349. Gensel, W. H. 1969. A Revision of the Genus Thevetia (Apocynaceae). M.Sc. Thesis, Univ. Connecticut, Storrs. Gunn, C. R., J. H. Wiersema, C. A. Ritchie & J. H. Kirkbride, Jr. 1992. Families and genera of spermatophytes recognized by the Agricultural Research Service. Techn. Bull. U.S.D.A. 1796: 71–73. Haber, W. A. 1984. Pollination by deceit in a mass-flowering tropical tree Plumeria rubra (Apocynaceae). Biotropica 16: 269–275. Hegnauer, R. 1970. Cardenolide und Bufadienolida (5 Cardenolide). Verbreitung und systematische Bedeutung. Pl. Med. (Stuttgart) 19: 137–153. ———. 1989. Chemotaxonomie der Pflanzen 8. Birkhäuser, Basel. 24 Annals of the Missouri Botanical Garden Hendrian. 2004. Revision of Ochrosia (Apocynaceae) in Malesia. Blumea 49: 101–128. Homberger, K. & M. Hesse. 1984. Kopsirachin, ein ungewöhnliches Alkaloid aus der Apocynaceae Kopsia dasyrachis Ridl. Helv. Chim. Acta 67: 237–248. Huang, T. C. 1986. The Apocynaceae of Taiwan 2: A palynological study. Sci. Rep. Tohoku Imp. Univ., Ser. 4, Biol. 39: 75–102. Hufford, L. 1997. The roles of ontogenetic evolution in the origins of floral homoplasies. Int. J. Pl. Sci. 158(Suppl. 6): S65–S80. Jensen, S. R. 1992. Systematic implications of the distribution of iridoids and other chemical compounds in the Loganiaceae and other families of the Asteridae. Ann. Missouri Bot. Gard. 79: 284–302. Johns, S. R., J. A. Lamberton, J. R. Price & A. A. Sioumis. 1968. Identification of coumarins isolated from Lepiniopsis ternatensis (Apocynaceae), Pterocaulon sphacelatum (Compositae), and Melicope melanophloia (Rutaceae). Austral. J. Chem. 21: 3079–3080. Johnson, L. A. & D. E. Soltis. 1994. matK DNA sequences and phylogenetic reconstruction in Saxifragaceae s.s. Syst. Bot. 19: 143–156. Kam, T. S., K. T. Nyeoh, K. M. Sim & K. Yoganathan. 1997. Alkaloids from Alstonia scholaris. Phytochemistry 45: 1303–1305. Kelchner, S. A. 2000. The evolution of non-coding chloroplast DNA and its application in plant systematics. Ann. Missouri Bot. Gard. 87: 482–498. Kisakürek, M. V., A. J. M. Leeuwenberg & M. Hesse. 1983. A chemotaxonomic investigation of the plant families of Apocynaceae, Loganiaceae, and Rubiaceae by their indole alkaloid content. Pp. 211–376 in W. W. Pelletier (editor), Alkaloids: Chemical and Biological Perspectives, Vol. 1. Wiley, New York. Koch, I. 2002. Estudios das Espécies Neotropicais do Gênero Rauvolfia L. (Apocynaceae). Ph.D. Thesis, Universidade Estadual de Campinas. Lawrence, T. J. & P. S. Green. 1993. The anatomy of a dehiscent berry. Kew Bull. 48(1): 53–57. Leeuwenberg, A. J. M. 1991. A Revision of Tabernaemontana, Vol. 1: The Old World Species. Royal Botanic Gardens, Kew. ———. 1994a. Series of revisions of Apocynaceae XXXVIII. Taxa of the Apocynaceae above the genus level. Wageningen Agric. Univ. Pap. 94(3): 45–60. ———. 1994b. A Revision of Tabernaemontana, Vol. 2: The New World Species. Royal Botanic Gardens, Kew. ———. 1997. Series of revisions of Apocynaceae XLIV. Craspidospermum Boj. ex A. DC., Gonioma E. Mey., Mascarenhasia A. DC., Petchia Livera, Plectaneia Thou., and Stephanostegia Baill. Wageningen Agric. Univ. Pap. 97: 1–124. ———. 2002. Series of revisions of Apocynaceae LII. Chilocarpus. Syst. & Geogr. Pl. 72: 127–166. ——— & P. W. Leenhouts. 1980. Taxonomy. Pp. 8–96 in A. J. M. Leeuwenberg (editor), Engler and Prantl’s Die natürlichen Pflanzenfamilien, Fam. Loganiaceae, Vol. 28b(1). Duncker & Humblot, Berlin. Li, J., J. H. Alexander, III & D. Zhang. 2002. Paraphyletic Syringa (Oleaceae): Evidence from sequences of nuclear ribosomal DNA ITS and ETS regions. Syst. Bot. 27: 592–597. Lin, S. & G. Bernardello. 1999. Flower structure and reproductive biology in Aspidosperma quebracho-blanco (Apocynaceae), a tree pollinated by deceit. Int. J. Pl. Sci. 160: 869–878. Lorence, D. H. & W. L. Wagner. 1997. A revision of Lepinia (Apocynaceae), with description of a new species from the Marquesas Islands. Allertonia 7: 254–266. Lý, T. D. 1986. Die Familie Apocynaceae Juss. in Vietnam. Teil 1. Allgemeiner Teil. Feddes Repert. 97: 235–273. Maddison, D. & W. Maddison. 2000. MacClade 4: Analysis of phylogeny and character evolution. Sinauer Associates, Sunderland, Massachusetts. Markgraf, F. 1971. Florae Malesianae Praecursores LI. Apocynaceae I. 5. Chilocarpus. Blumea 19: 156–165. ———. 1976. Apocynaceae. In H. Humbert & J.-F. Leroy (editors), Flore de Madagascar et des Comores. Fam. 169. Muséum National d’Histoire Naturelle, Paris. ———. 1979. Florae Malesianae Praecursores LIX. Apocynaceae V. Ochrosia, Neisosperma. Blumea 25: 233– 247. ——— & K. Huber. 1975. Die Entwicklung der ‘‘Nasenfrucht’’ von Kopsia flavida Bl. Bot. Jahrb. Syst. 96: 256–269. Metcalf, C. R. & L. Chalk. 1989. Anatomy of the Dictoyledons, Vol. 2, 2nd. ed. Oxford Univ. Press, Oxford. Middleton, D. J. 1999. Apocynaceae. Pp. 1–153 in T. Santisuk & K. Larsen (editors), Flora of Thailand, Vol. 7(1). Diamond Printing, Bangkok. ———. 2000. Revision of Alyxia (Apocynaceae). Part 1: Asia and Malesia. Blumea 45: 1–146. ———. 2002. Revision of Alyxia (Apocynaceae). Part 2: Pacific Islands and Australia. Blumea 47: 1–93. ———. 2004. A revision of Kopsia (Apocynaceae: Rauvolfioideae). Harvard Pap. Bot. 9: 89–142. Muller, J. 1981. Fossil pollen records of extant angiosperms. Bot. Rev. (Lancaster) 47: 1–145. Nilsson, S. 1986. The significance of pollen morphology in the Apocynaceae. Pp. 359–374 in S. Blackmore & I. K. Ferguson (editors), Pollen and Spores: Form and Function. Academic Press, London. Nishino, E. 1982. Corolla tube formation in six species of Apocynaceae. Bot. Mag. (Tokyo) 95: 1–17. Olmstead, R. G., B. Bremer, K. M. Scott & J. D. Palmer. 1993. A parsimony analysis of the Asteridae sensu lato based on rbcL sequences. Ann. Missouri Bot. Gard. 80: 700–722. Omino, E. 1996. A contribution to the leaf anatomy and taxonomy of Apocynaceae in Africa, Pt. 2. A monograph of Pleiocarpinae (Series of revisions of Apocynaceae XLI). Wageningen Agric. Univ. Pap. 96(1): 81–178. Pagen, F. J. J. 1987. Oleanders. Nerium L. Agric. Univ. Wageningen Pap. 87-2: 1–25. Pichon, M. 1947a. Classification des Apocynacées: II. Genre Rauvolfia. Bull. Soc. Bot. France 94: 31–39. ———. 1947b. Classification des Apocynacées: III. Genre Ochrosia. Bull. Mus. Natl. Hist. Nat., 4 sér. 2, 19: 205–212. ———. 1947c. Classification des Apocynacées: IV. Genre Alstonia et genres voisins. Bull. Mus. Natl. Hist. Nat. 19: 294–301. ———. 1948a. Classification des Apocynacées: I. Carissées et Ambelaniées. Mém. Mus. Natl. Hist. Nat., B, Bot. 24: 111–181. ———. 1948b. Classification des Apocynacées: V. Cerbéroïdées. Notul. Syst. (Paris) 13: 212–229. ———. 1948c. Les caractères du genre Plectaneia (Apocynacées). Notul. Syst. (Paris) 13: 255–257. ———. 1948d. Classification des Apocynacées: XIX. Le rétinacle des Echitoïdées. Bull. Soc. Bot. France 95: 211–216. Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 25 ———. 1949a. Classification des Apocynacées: IX. Rauvolfiées, Alstoniées, Allamandées et Tabernaémontanées. Mém. Mus. Natl. Hist. Nat. 24: 153–251. ———. 1949b. Classification des Apocynacées: XXVII. Détermination des graines de Plumérioidées et de Cerbéroidées. Bull. Mus. Nat. Hist. Natl. 21: 266–269. ———. 1949c. Classification des Apocynacées: XXIX. Le genre Neokeithia. Bull. Mus. Nat. Hist. Natl. 21: 375–377. ———. 1950a. Classification des Apocynacées: XXV. Echitoïdées. Mém. Mus. Natl. Hist. Nat. B. 1: 1–143. ———. 1950b. Classification des Apocynacées: XXVIII. Supplément aux Plumérioïdées. Mém. Mus. Natl. Hist. Nat., B, 1: 145–166. ———. 1950c. Classification des Apocynacées: XXXI. Le fruit des genres Thevetia et Ahouai. Bull. Mus. Nat. Hist. Natl. 22: 291–294. ———. 1952. Classification des Apocynacées: XXXIII. Les sous-tribus des Carissées. Notul. Syst. (Paris) 14: 310–315. Potgieter, K. & V. A. Albert. 2001. Phylogenetic relationships within Apocynaceae s.l. based on trnL intron and trnL-F spacer sequences and propagule characters. Ann. Missouri Bot. Gard. 88: 523–549. Punt, W., S. Blackmore, S. Nilsson & A. Le Thomas. 1994. Glossary of Pollen and Spore Terminology. LPP Contributions Series No. 1. LPP Foundation, Utrecht. Reeves, G., P. Goldblatt, M. W. Chase, P. J. Rudall, M. F. May, A. V. Cox, B. Lejeune & T. Souza-Chies. 2001. Molecular systematics of Iridaceae: Evidence from four plastid DNA regions. Amer. J. Bot. 88: 2074–2087. Rogers, G. K. 1986. The genera of Loganiaceae in the southeastern United States. J. Arnold Arbor. 67: 143– 185. Rosatti, T. J. 1989. The genera of suborder Apocynineae (Apocynaceae and Asclepiadaceae) in the southeastern United States. J. Arnold Arbor. 70: 307–514. Rudjiman. 1986. A revision of Beaumontia Wallich, Kibatalia G. Don, and Vallariopsis Woodson (Apocynaceae). Agric. Univ. Wageningen Pap. 86(5): 1–99. Saghai-Maroof, M. A., K. M. Soliman, R. A. Jorgensen & R. W. Allard. 1984. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. U.S.A. 81: 8014–8018. Salamin, N., M. W. Chase, T. R. Hodkinson & V. Savolainen. 2003. Assessing internal support with large phylogenetic DNA matrices. Molec. Phylogen. Evol. 27: 528–539. Savolainen, V., M. W. Chase, C. M. Morton, S. B. Hoot, D. E. Soltis, C. Bayer, M. F. Fay, A. de Bruijn, S. Sullivan & Y.L. Qiu. 2000. Phylogenetics of flowering plants based upon a combined analysis of plastid atpB and rbcL gene sequences. Syst. Biol. 49: 306–362. Schick, B. 1980. Untersuchungen über die Biotechnik der Apocynaceenblüte. I. Morphologie und Funktion des Narbenkopfes. Flora 172: 394–432. Schill, R. & U. Jäkel. 1978. Beitrag zur Kenntnis der Asclepiadaceen-Pollinarien. Trop. Subtrop. Pflanzenwelt 22: 1–122. Schumann, K. 1895. Apocynaceae and Asclepiadaceae. Pp. 109–306 in A. Engler & K. Prantl (editors), Die natürlichen Pflanzenfamilien, Vol. 4(2). Engelmann, Leipzig. Sennblad, B. 1997. Phylogeny of the Apocynaceae s.l. (Ph.D. Thesis summary). Acta Universitas Uppsaliensis, Comprehensive Summaries Uppsala Dissertations. Faculty of Science and Technology 295, Uppsala. ——— & B. Bremer. 1996. The familial and subfamilial relationships of Apocynaceae and Asclepiadaceae evaluated with rbcL data. Pl. Syst. Evol. 202: 153–175. ——— & ———. 2000. Is there a justification for differential a priori weighting in coding sequences?—A case study from rbcL and Apocynaceae s.l. Syst. Biol. 49: 101–113. ——— & ———. 2002. Classification of Apocynaceae s.l. according to a new approach combining Linnaean and phylogenetic taxonomy. Syst. Biol. 51: 389–409. ———, M. E. Endress & B. Bremer. 1998. Morphology and molecular data in phylogenetic fraternity: The tribe Wrightieae (Apocynaceae) revisited. Amer. J. Bot. 85: 1143–1158. Sévenet, T., L. Allorge, B. David, K. Awang, A. Hamid, A. Hadi, C. Kan-Fan, J. C. Quirion, F. Remy, H. Schaller & L. E. Teo. 1994. A preliminary chemotaxonomic review of Kopsia (Apocynaceae). J. Ethnopharmacol. 41: 147–183. Sidiyasa, K. 1998. Taxonomy, phylogeny, and wood anatomy of Alstonia (Apocynaceae). Blumea, Suppl. 11: 1–230. Simões, A. O., T. Livshultz, E. Conti & M. E. Endress. 2007. Phylogeny and systematics of Rauvolfioideae (Apocynaceae) based on molecular and morphological evidence. Ann. Missouri Bot. Gard. 94 (in press). Smith, A. C. 1988. Flora Vitiensis Nova—A New Flora of Fiji (Spermatophytes Only), Vol. 4. Pacific Tropical Botanical Garden, Lawai, Kauai. Smith, J. F. 2000. A phylogenetic analysis of tribes Beslerieae and Napeantheae (Gesneriaceae) and evolution of fruit types: Parsimony and maximum liklihood analyses of ndhF sequences. Syst. Bot. 25: 72–81. Solereder, H. 1892. Loganiaceae. Pp. 19–50 in A. Engler & K. Prantl (editors), Die natürlichen Pflanzenfamilien, Vol. 4, 2nd ed. Engelmann, Leipzig. Soltis, D. E., P. S. Soltis, M. W. Chase, M. E. Mort, D. C. Albach, M. Zanis, V. Savolainen, W. H. Hahn, S. B. Hoot, M. F. Fay, M. Axtell, S. M. Swensen, L. M. Prince, W. J. Kress, K. C. Nixon & J. S. Farris. 2000. Angiosperm phylogeny inferred from 18S rDNA, rbcL, and atpB sequences. Bot. J. Linn. Soc. 133: 381–461. Stapf, O. 1902. Apocynaceae. Pp. 24–233 in W. T. ThiseltonDyer (editor), Flora of Tropical Africa, Vol. 4. L. Reeve, London. Struwe, L., V. A. Albert & B. Bremer. 1994. Cladistics and family level classification of Gentianales. Cladistics 10: 175–206. Swofford, D. L. 2002. PAUP*: Phylogenetic analysis using parsimony (* and other methods), version 4.0b. Sinauer Associates, Sunderland. Taberlet, P., L. Gielly, G. Pautou & J. Bouvet. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Pl. Molec. Biol. 17: 1105–1109. Valeton, T. 1895. Description d’un nouveau genre appartenant à la famille des Apocynacées. Ann. Jard. Bot. Buitenzorg 12: 249–253; t. I, II. van der Ham, R. W. J. M., Y.-M. Zimmerman, S. Nilsson & A. Igersheim. 2001. Pollen morphology and phylogeny of the Alyxieae (Apocynaceae). Grana 40: 169–191. Verhoeven, R. L. & H. J. T. Venter. 2001. Pollen morphology of the Periplocoideae, Secamonoideae and Asclepiadoideae (Apocynaceae). Ann. Missouri Bot. Gard. 88: 569–582. Wagner, W. L., D. R. Herbst & S. H. Sohmer. 1990. Manual of the Flowering Plants of Hawai’i, Vol. 1. Univ. of Hawaii Press/Bishop Museum Press, Honolulu. 26 Annals of the Missouri Botanical Garden Wiehler, H. 1983. A synopsis of the neotropical Gesneriaceae. Selbyana 6: 1–249. Woodson, R. E. Jr. 1933. Studies in the Apocynaceae IV. The American genera of Echitoideae. Ann. Missouri Bot. Gard. 20: 605–790. ———. 1951. Studies in the Apocynaceae VIII. An interim revision of the genus Aspidosperma Mart. & Zucc. Ann. Missouri Bot. Gard. 38: 119–206. Zeches, M., K. Mesbah, B. Richard, C. Moretti, J. M. Nuzillard & L. Lemenolivier. 1995. Alkaloids from leaves and stems of Vallesia glabra. Pl. Med. (Stuttgart) 61: 89–91. Zhu, J.-P., A. Guggisberg, M. Kalt-Hadamowsky & M. Hesse. 1990. Chemotaxonomic study of the genus Tabernaemontana (Apocynaceae) based on their indole alkaloid content. Pl. Syst. Evol. 172: 13–34. Condylocarpon isthmicum (Vell.) A. DC. Brazil, Koch s.n. (Z) FS; Brazil, Hatschbach 13030 (Z) LM, SEM, TEM Craspidospermum verticillatum Bojer ex A. DC. Madagascar, Civeyrel 1234 (Z) FS, LM, SEM, TEM Kibatalia gitingensis (Elmer) Woodson Philippines, Liede 3268 (Z) FS; Philippines, Wenzel 652 (G) LM, SEM, TEM Kopsia fruticosa (Ker Gawl.) A. DC. Java, Prévost 167 (Z); cult. Victoria, Trinidad, Broadway 5963 (S) LM, SEM, TEM Lepinia marquisensis Lorence & W. L. Wagner Fatu Hiva, Marquesas Islands, Perlman 10271 (BISH, Z) FS Lepinia solomonensis Hemsl. Solomon Islands, BSIP 13496 (L) LM, TEM Lepinia taitensis Decne. Society Islands, Moorea, Perlman et al. 15071 (PTBG, Z) FS; Society Islands, Tahiti, Whistler 4932 (BISH) SEM Lepiniopsis ternatensis Valeton Moluccas, Mochtar 306 (L) LM, SEM; PNH 17362 (L) TEM Lepiniopsis trilocularis Markgr. Palau Islands, Lorence 8265 (PTBG, Z) FS Mascarenhasia arborescens A. DC. cult. Fairchild Bot. Gard., Bird s.n. (Z) FS; Madagascar, Schlieben 8128 (Z) LM, SEM; Madagascar, Capuron 22808-SF (P) LM, SEM, TEM Molongum laxum (Benth.) Pichon Venezuela, Berry 5400 (MO, Z) FS; Colombia, Duntii 36267 (COL) LM, SEM, TEM Neisosperma nakaiana (Koidz.) Fosberg & Sachet cult. Waimea Arboretum, Hawaiian Is., Neill 5291 (Z) FS, LM, SEM, TEM Nerium oleander L. cult. Bot. Gard. Zurich, Fallen s.n. (Z) FS; France, Segal 232 (WAG) LM, TEM; cult. Perpignan, Leeuwenberg 12206 (WAG) LM, SEM, TEM Ochrosia coccinea (Teijsm. & Binn.) Miq. cult. Bogor Bot. Gard., Java, anon. s.n. 30/8/1982 (Z) FS, LM, SEM, TEM Petchia ceylanica (Wight) Livera cult. Bot. Gard. Kaiserslautern, Omlor s.n., Kessler s.n. (Z) FS; Sri Lanka, Wambeck 2510 (S) LM, SEM Picralima nitida (Stapf) T. Durand & H. Durand cult. Bot. Gard. Wageningen, Leeuwenberg 10779 (Z) FS; Zaire, Gille 100 (BR) LM, SEM, TEM Plectaneia stenophylla Jum. cult. Madagascar, Petignat s.n. (Z) FS Plectaneia thouarsii Roem. & Schult. Madagascar, Bernardi 11820 (L) LM, SEM, TEM Pleiocarpa mutica Benth. cult. Bot. Gard. Wageningen, van Setten 415 (WAG, Z) FS; Ivory Coast, Leeuwenberg 12145 (WAG) LM, SEM Plumeria rubra L. cult. Bot. Gard. Zurich, Fallen s.n. (Z) FS; Ghana, Leeuwenberg 11089 (WAG) LM, SEM, TEM Pteralyxia kauaiensis Caum Kauai, Perlman 15456 (Z) FS; Hawaii, Flynn 269 (PTBG) SEM Pteralyxia macrocarpa (Hillebr.) K. Schum. Hawaii, Swezey s.n. (L) LM, TEM Rauvolfia vomitoria Afzel. Ivory Coast, Aké Assi s.n. (Z) FS; Nigeria, Leeuwenberg 11337 (WAG) LM, SEM, TEM; Ivory Coast, Leeuwenberg 12122 (WAG) LM, SEM APPENDIX 1. Voucher specimens used for morphological character assessment in the Alyxieae study. Herbarium acronyms are in parentheses. Specimens used to study floral structure are indicated with FS, those used for light microscopy, scanning electron miscroscopy, and transmission electron microscopy of pollen grains are indicated by LM, SEM, and TEM, respectively, following the herbarium acronym. APOCYNACEAE Acokanthera oblongifolia (Hochst.) Codd cult. Bot. Gard. Wageningen, Kas et al. s.n. (Z) FS Acokanthera oppositifolia (Lam.) Codd South Africa, Bayliss BRI 544 (S) LM, SEM, TEM Allamanda cathartica L. cult. Royal Bot. Gard. Kew, 1983, Fallen s.n. (Z) FS; Gabon, Leeuwenberg 12540 (WAG) LM, SEM, TEM Alstonia scholaris (L.) R. Br. cult. Fairchild Trop. Gard., Gillis 6995 (Z) FS; New Guinea, Schodde 2472 (L) LM, SEM, TEM Alyxia oblongata Domin Australia, Dockrill 835 (L) LM Alyxia ruscifolia R. Br. cult. Montpellier, Civeyrel 1055 (Z) FS; Australia, Clark et al. 1753 (L) SEM, TEM Amsonia ciliata Walter U.S.A., Sasseen s.n. (WAG) LM, SEM, TEM Amsonia tabernaemontana Walter cult. Bot. Gard. Zürich, Endress s.n. (Z) FS Anechites nerium (Aubl.) Urb. Ecuador, Asplund 16471 (Z) FS; Dominican Republic, Ekman 15239 (S) LM, SEM Aspidosperma parvifolium A. DC. Brazil, Ferreira s.n. (Z) FS; Brazil, Heringer 10672 (UB) LM, SEM Cabucala caudata Markgr. Madagascar, Capuron 23701-SF (P) LM, SEM, TEM Cabucala polysperma (Scott-Elliot) Pichon Madagascar, Civeyrel 1281 (Z) FS Catharanthus roseus (L.) G. Don cult. Bot. Gard. Zurich, Endress s.n. (Z) FS; Liberia, Van Harten 29 (WAG) LM, SEM, TEM Chilocarpus denudatus Blume cult. Bot. Gard. Bogor, Burck s.n. (Z) FS; India, Ridsdale 757 (L) LM; Java, Blume s.n. (L) LM; Java, anon. s.n. (S) SEM; Sarawak, Richards 1463 (L) TEM Chilocarpus suaveolens Blume Java, Hochreutiner 2547 (L, Z) FS Condylocarpon guyanense Desf. French Guiana, Sastre 5470 (P, Z) FS Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 27 Rhazya stricta Decne. Yemen, Brunner 31 (Z) FS; Saudi Arabia, Schimper 812 (L) LM, SEM, TEM Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schult. cult. Bot. Gard. Calcutta, anon. s.n. (Z) FS Tabernaemontana pandacaqui Lam. Australia, Alkin s.n. (Z) LM, SEM, TEM Thevetia peruviana (Pers.) K. Schum. cult. Bot. Gard. Zurich, Fallen s.n., (Z) FS; cult. Florida, Gillis 9227 (S) LM, SEM, TEM Vallesia antillana Woodson cult. Fairchild Trop. Gard, Zona s.n. (Z) FS; Florida, Killip 43415 (S) TEM Vallesia glabra (Cav.) Link Galapagos Is., A. & H. Andersen 1009 (QCA) LM, SEM Vinca minor L. cult. Zurich, Fallen s.n. (Z) FS; cult. Schipluiden, Netherlands, De Kort s.n. (L) SEM; cult. Sollentuna, Sweden, Nilsson s.n. (S) SEM Vinca major L. cult. Pijnacker, Netherlands, Van der Ham s.n. (L) LM, TEM GELSEMIACEAE Gelsemium sempervirens (L.) J. St.-Hil. U.S.A., Louisiana, Tucker 28771 (Z) FS; South Carolina, Wall. s.n. (S) LM, SEM, TEM LOGANIACEAE Geniostoma rupestre (J. R. Forst. & G. Forst.) var. ligustrifolium (A. Cunn.) B. J. Conn New Zealand, Garnock-Jones s.n. (WELTU, Z) FS; New Zealand, Nilsson NZ 9 (S) LM, SEM, TEM 28 Annals of the Missouri Botanical Garden Appendix 2. Voucher specimens used for molecular analyses and GenBank accession numbers. Taxon APOCYNACEAE Acokanthera oblongifolia (Hochst.) Codd Acokanthera oppositifolia (Lam.) Codd Allamanda cathartica L. GenBank Accession No. Voucher/Literature Citation Endress et al., 1996 Sennblad & Bremer, 1996 Potgieter & Albert, 2001 Endress et al., 1996 Sennblad & Bremer, 1996 Allamanda indet. Potgieter & Albert, 2001 Alstonia boonei De Wild. Potgieter & Albert, 2001 Alstonia scholaris (L.) R. Endress et al., 1996 Br. Sennblad & Bremer, 1996 Alyxia buxifolia R. Br. Potgieter & Albert, 2001 Alyxia ruscifolia R. Br. cult. Montpellier, Civeyrel 1055 (TL) Sennblad & Bremer, 2002 Amsonia tabernaemontana Potgieter & Albert, 2001 Walter cult. Royal Bot. Gard. Kew, Civeyrel 1057 (TL) Anechites nerium (Aubl.) Sennblad & Bremer, 2002 Urb. Aspidosperma quebrachoPotgieter & Albert, 2001 blanco Schltdl. Aspidosperma triternatum cult. Bot. Garden, Meise, Bremer Rojas Acosta 3029 (UPS) Sennblad & Bremer, 2002 Cabucala polysperma Madagascar, Civeyrel 1281 (TL) (Scott-Elliott) Pichon Catharanthus roseus (L.) Potgieter & Albert, 2001 G. Don cult. Stockholm Univ., Bremer 3128 (UPS) Sennblad & Bremer, 1996 Chilocarpus suaveolens Endress et al., 1996 Blume cult. Bot. Gard. Bogor, Chase 1208 Potgieter & Albert, 2001 Condylocarpon amazonicum (Markgr.) Ducke Condylocarpon guyanense French Guiana, M. F. Prevost s.n. Desf. (CAY) Craspidospermum Madagascar, Civeyrel 1234 (TL) verticillatum Bojer ex Sennblad & Bremer, 2002 A. DC. Kibatalia gitingensis Philippines, Liede 3268 (Z) (Elmer) Woodson Sennblad & Bremer, 2002 Kopsia fruticosa cult. Bot. Gard. Meise, Bremer (Ker Gawl.) A. DC. 3033 (UPS) Endress et al., 1996 Sennblad & Bremer, 1996 Lepinia taitensis Decne. Sennblad & Bremer, 2002 Lepiniopsis ternatensis Potgieter & Albert, 2001 Valeton Lepiniopsis trilocularis Palau Islands, Lorence 8265 Markgr. (PTBG) matK rbcL trnL trnL-F AF214302 AF214148 AF214304 AF102374 AF214150 AF214151 AF214306 AF214152 AF214307 AF214153 AM295087 AM295087 AF214319 AF214165 AM295088 AM295088 AF102392 AF214175 AM295089 AM295089 AF214337 AF214183 AM295090 AM295090 AM295091 AM295091 AF214374 AF214220 Z70182 X91758 Z70190 X91759 Z70189 X91760 DQ837536 AJ419731 AM295066 AM295078 AJ419733 AM295077 AM295067 AJ419735 AM295079 AM295068 Z70184 DQ837537 X91757 X92445 AM295080 DQ837538 AJ419743 AM295069 AJ419745 Z70178 X91763 AJ419746 AM295081 Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 29 Appendix 2. Continued. Taxon APOCYNACEAE GenBank Accession No. Voucher/Literature Citation Mascarenhasia arborescens Potgieter & Albert, 2001 A. DC. cult. Wageningen Agric. Univ., nr. 80-16, Setten 625 (WAG) Sennblad et al., 1998 Molongum laxum (Benth.) Potgieter & Albert, 2001 Pichon Endress et al., 1996 Neisosperma nakaiana Potgieter & Albert, 2001 (Koidz.) Fosberg & Sachet Neisosperma oppositifolia cult. Nat. Trop. Bot. Gard., Kauai, Lorence s.n., NTBG (Lam.) Fosberg & 970511 (PTBG) Sachet Nerium oleander L. Potgieter & Albert, 2001 Civeyrel et al., 1998 Sennblad et al., 1998 Ochrosia coccinea Sennblad & Bremer, 2002 (Teijsm. & Binn.) cult. Wageningen Agric. Univ. Miq. nr. 78PTHB 354, van der Laan 389 (WAG) Petchia ceylanica (Wight) cult. Univ. Kaiserslautern, Livera Germany, Omlor s.n. (Z) Picralima nitida (Stapf) T. Potgieter & Albert, 2001 Durand & H. Durand Endress et al. 1996 Sennblad & Bremer, 1996 Plectaneia stenophylla Madagascar, Petignat s.n. (Z) Jum. Potgieter & Albert, 2001 Pleiocarpa mutica Benth. Potgieter & Albert, 2001 cult. Royal Bot. Gard. Kew, Civeyrel 1086 (TL) Sennblad & Bremer, 2002 Plumeria alba Kunth Potgieter & Albert, 2001 Plumeria inodora Jacq. Sennblad & Bremer, 1996 Plumeria rubra L. Endress et al., 1996 Pteralyxia kauaiensis Kauai, Hawaii, Lorence 7768 Caum (PTBG, Z) Rauvolfia mannii Stapf Endress et al., 1996 Sennblad & Bremer, 1996 Rauvolfia serpentina Potgieter & Albert, 2001 Benth. ex Kurz Rhazya stricta Decne. Agosti 29 (Z) Tabernaemontana Potgieter & Albert, 2001 citrifolia L. Endress et al., 1996 Tabernaemontana Sennblad & Bremer, 1996 divaricata (L.) R. Br. ex Roem. & Schult. Thevetia ahouai (L.) A. Potgieter & Albert, 2001 DC. Thevetia peruviana (Pers.) Endress et al., 1996 K. Schum. Sennblad & Bremer, 1996 Vallesia antillana cult. Fairchild Trop. Gard., Woodson Meagher 966 (FTG) Sennblad & Bremer, 2002 Potgieter & Albert, 2001 matK rbcL trnL trnL-F AF214378 AF214224 AF214383 AF214229 AF214385 AF214231 AF214386 AF214232 AM295092 AM295092 AM295093 AM295093 AF214404 AF214250 AF214405 AF214407 AF214251 AF214253 AF214408 AF214254 AM295094 AM295094 AF214415 AF214261 AM295095 AF214431 AM295095 AF214277 AF214435 AF214281 AF214447 AF214293 AM295070 AJ002885 Z70185 AM295071 X91765 AM295082 Z98173 AJ002886 AJ419752 AM295072 AM295083 Z70179 X91766 AM295084 DQ837539 AJ419757 X91767 Z70191 AM295073 AM295085 Z70181 X91769 AM295074 AM295086 Z70187 X91772 Z70188 X91773 AM295075 AJ419767 30 Annals of the Missouri Botanical Garden Appendix 2. Continued. Taxon APOCYNACEAE Vinca minor L. GELSEMIACEAE Gelsemium sempervirens (L.) J. St.-Hil. LOGANIACEAE Geniostoma rupestre J. R. Forst. & G. Forst. GenBank Accession No. Voucher/Literature Citation matK cult. Bot. Gard. Uppsala, Sennblad 230 (UPS) Sennblad & Bremer, 2002 Potgieter & Albert, 2001 AM295076 Endress et al., 1996 Olmstead et al., 1993 cult. Royal Bot. Gard. Kew, Civeyrel 1069 (TL) Z70195 Endress et al., 1996 Wellington, New Zealand, Garnock-Jones 2200 (WELTU) Z70194 rbcL trnL trnL-F AF214449 AF214295 AM295096 AM295096 AM295097 AM295097 AJ419768 L14397 Z68828 1 2 3 4 5 123456789012345678901234567890123456789012345678901234 31 010000000000000010000000000000201101000020100110011001 112000011001100121111012010200200010001020000100101200 012000100000000020110012000300202120000020002010001100 012010000000000010000010101002020100001112210010322000 211000010000000021100012001000202130000020000210011200 1100010110000011201110120100002021???0?0200000001?1240 0110001000000000100100120102002010300000303001000?1201 012000000000000021100010100000001120000020001200011101 210000000000000021102012001001202130001020002200001201 1100100000100000100100120130011000???00112201010211100 1120000000100000100000121010021201???1010????0?1011200 012000100000000020110002000100202130010133001000011100 010002010101110230001012012013101100100023101000311200 0100000001000000211020111000001--130001020002100011101 011000000010000010000111101002020100001122210010322010 011000000010000010000111101002020100001122210010322010 010001010100110230001012012013101100100023101010311000 0100010000011101213100100030003011???00031201010211100 012000000100000021200011100100101120000020002200001001 0120011101000102312100120120131011010001231010?0311000 012000000100000021200010100100101120001021200200001021 0120000000000000211000101000000011???00021001200211000 010001100000000010000011003000300120000021002000101040 1100000000000000100000120102010001???00112010010311100 0100001000000000100001110030003001300000210020001?1100 011000010000000010010002000200202010000020000110101201 0110000000000000100000111010020201???01112210010322000 012000000000000021101010100000001120001020003200011100 211000000000000021100012000100212120000020000210011200 0100010000100000100100100030013211400000200020?0211200 0110010110011011201110011132002020010010200000?0101230 0110000000000000110000111030002--030000030300000011200 210000000000000121102012001001200120001024023001001200 100100010200002-0---0012000200201120000020000000000250 0001020102000000000000020130012011???00023101010101100 Endress et al. Phylogenetic Analysis of Alyxieae Acokanthera oblongifoliaa, A. oppositifoliab Allamanda cathartica Alstonia scholaris Alyxia oblongatab, A. ruscifoliaa,b Amsonia ciliatab, A. tabernaemontanaa Anechites nerium Aspidosperma parvifolium Cabucala caudatab, C. polyspermaa Catharanthus roseus Chilocarpus denudatusa,b, C. suaveolensa Condylocarpon guyanensea, C. isthmicuma,b Craspidospermum verticillatum Kibatalia gitingensis Kopsia fruticosa Lepinia marquisensisa, L. solomonensisb, L. taitensisa,b Lepiniopsis ternatensisb, L. trilocularisa Mascarenhasia arborescens Molongum laxum Neisosperma nakaiana Nerium oleander Ochrosia coccinea Petchia ceylanica Picralima nitida Plectaneia stenophyllaa, P. thouarsiib Pleiocarpa mutica Plumeria rubra Pteralyxia kauaiensisa,b, P. macrocarpab Rauvolfia vomitoria Rhazya stricta Tabernaemontana divaricataa, T. pandacaquib Thevetia peruviana Vallesia antillanaa,b, V. glabrab Vinca majorb, V. minora,b Gelsemium sempervirens Geniostoma rupestre Volume 94, Number 1 2007 APPENDIX 3. Matrix based on the morphological character coding. For some genera more than one species was used for character coding. a 5 species used to code characters 1–37 (the nonpollen characters); b 5 species used to code characters 38–54 (the pollen characters). See Appendices 1 and 4. 32 Annals of the Missouri Botanical Garden APPENDIX 4. Leeuwenberg, 1997). Shorter gaps of some microns in length (visible in microtome serial sections) are also found in Alstonia, Craspidospermum, and Hunterieae, whereas in other genera epidermal remnants are still visible, although there are no distinct gaps. In Endress et al. (1996), epidermal remnants and gaps were treated together as a single character state: corolla incompletely fused. Because, however, the tube may be fused yet still show epidermal remnants, here only the presence of distinct gaps, visible with dissecting microscope or in serial sections, is coded as unfused. 8. Corolla tube mouth: 0 5 constricted; 1 5 not constricted. 9. Infrastaminal appendages: 0 5 absent; 1 5 present. Infrastaminal appendages is a term used by Pichon (1948b) for outgrowths of the lower, congenitally fused part of the corolla tube in the staminal sectors (see Alvarado-Cárdenas & Ochoterena, 2007). They are found mainly in taxa previously included in Cerbereae (e.g., Cerbera, Thevetia, Cerberiopsis). These genera have a long, thin style and a disproportionately large, broad style head. 10. Corolla lobe aestivation: 0 5 sinistrorsely contort; 1 5 dextrorsely contort; 2 5 imbricate. Corolla lobe aestivation is one of the most important morphological characters in Apocynaceae. With a few exceptions, the direction is constant within a genus. In Rauvolfioideae corolla lobes are almost always sinistrorsely contort, whereas in Apocynoideae, corolla lobes are normally dextrorsely contort or, rarely, valvate. The corolla lobes in Kopsia, Ochrosia, and Neisosperma (all Rauvolfioideae) are consistently dextrorsely contort (Hendrian, 2004; Middleton, 2004) and thus an exception to the rule. Alstonia is one of the few genera in the family in which both sinistrorsely as well as dextrorsely contort species occur, and this feature is constant only at the species level. 11. Petals in bud: 0 5 not inflexed; 1 5 inflexed. In most Apocynaceae, contorted petals in bud are spiraled upward into a tip. Petals that are inflexed in bud are, instead, folded downward and spiral into the mouth of the corolla tube and only unfold at anthesis. Inflexed petals is a relatively uncommon condition in Apocynaceae. 12. Corolline corona below petal sinus, behind and/or just above anther: 0 5 absent; 1 5 a compact protruding lobe. All outgrowths in the staminal sector and above the insertion of the anther are interpreted here as a corona. These include the vertical ridges in Molongum Pichon, as well as the protuberances termed suprastaminal appendages by Pichon (1948b) in Thevetia; the fimbriate lobes of Allamanda are also interpreted as a corona (see Endress et al., 1996). 13. Anthers: 0 5 atop filaments that arise from the corolla tube; 1 5 sessile upon enlarged staminal ridges. 14. Lignified guide rails: 0 5 absent; 1 5 present. Lignified guide rails are a specialization of the lateral parts of the anther and have a function in the complex pollination mechanism in Apocynaceae; they are absent in most Rauvolfioideae, but are characteristic for Apocynoideae. It is important to note that lignified guide rails are also characteristic for the majority of Tabernaemontana species (including all of the Neotropical taxa), although absent in the two representative species used in this study and in all of section Ervatamia to which they belong (see Leeuwenberg, 1994b: xv). Thus, for this character, most species of Tabernaemontana would show more affinity morphologically to Molongum than is apparent from the representative species used here (compare with Endress et al., 1996, in which a Neotropical species was used in the morphological analysis). Characters and character states for the morphological matrix used in the cladistic analyses. See Appendix 3. The characters and character states used in this study are based on the exemplar method; only the characteristics of the species used in the analysis are considered in assigning codes. In cases of large genera with a range of states, this is indicated here. 1. Habit: 0 5 trees or shrubs; 1 5 lianas or vines; 2 5 perennial herbs. The species used to represent Alyxia here, A. ruscifolia, is a shrub; the great majority of Alyxia species, however, are lianas. 2. Non-articulated laticifers: 0 5 absent; 1 5 present. Non-articulated laticifers are one of the key characters that distinguish Apocynaceae s.l. from other Gentianales. 3. Phyllotaxis: 0 5 leaves opposite; 1 5 leaves alternate; 2 5 leaves verticillate. Some taxa have leaves that are predominantly verticillate but may have some nodes with only two leaves. These taxa are coded as verticillate here. 4. Stipules: 0 5 absent; 1 5 present. Apocynaceae are here considered to be estipulate in the sense of Cronquist (1981) and Rosatti (1989). The colleters or interpetiolar ridges found in some taxa are not considered to be homologous with true stipules, nor are the adaxial outgrowths at the base of the petiole in Alstonia scholaris (Sidiyasa, 1998). Small bract- or scale-like organs that are found in some species of Rauvolfia have sometimes been called stipules. In a recent revision of the Neotropical species, however, Koch (2002) argued convincingly that these organs are better interpreted as cataphylls. 5. Supernumerary bracteoles: 0 5 absent; 1 5 present. Supernumerary bracteoles are clusters of bracteoles on the pedicel subtending the calyx. These bracteoles often resemble the sepals. 6. Calycine colleters: 0 5 absent; 1 5 several, across the inner face of the sepal (these sometimes coalesced at the base); 2 5 few, mostly in the sepal sinuses. Calycine colleters are a common feature in Gentianales, and their lack or presence and arrangement is often used in Apocynaceae as an aid in distinguishing genera (e.g., Stapf, 1902; Woodson, 1933; Rosatti, 1989: 338–339; Ezcurra et al., 1992: 9–10; Omino, 1996: 87–88; Middleton, 1999, fig. 1). In Alyxia ruscifolia, although colleters are lacking at the base of the sepals themselves, they are well developed in the many supernumerary bracteoles clustered below the calyx. In Endress et al. (1996), Plumeria was coded as having a continuous row of calycine colleters. Detailed examination, however, has shown that they are not homologous to typical calycine colleters in that the entire inner surface of the upper part of the sepal is glandular. Because no other taxon shares this condition in this study, it is non-informative and thus not coded here. 7. Fusion of corolla tube: 0 5 fused just above the level of stamen insertion; 1 5 unfused (with gaps) just above the level of stamen insertion. In Apocynaceae, the lower corolla tube (composed of the united petal and stamen primordia) is congenitally fused; the upper part fuses postgenitally and basipetally, so that the last region to fuse is just above the insertion of the stamens. In some genera, flowers reach anthesis before fusion of the upper corolla is complete, resulting in gaps in the corolla tube (Boke, 1948; Nishino, 1982; Erbar, 1991). These gaps are especially long in Aspidosperma, Geissospermum, Haplophyton, and Stephanostegia, resulting in conspicuous slits that are easily visible with a dissecting microscope (Woodson, 1951; Fallen, 1986; Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 33 15. Anther dehiscence: 0 5 introrse; 1 5 latrorse; 2 5 extrorse. 16. Anther/style head synorganization: 0 5 anthers situated above or below, but not closely synorganized with, the style head; 1 5 anthers at about the same level as, and connivent over and encircling the style head; 2 5 anthers agglutinated to the style head via hair pads and adhesive. Synorganization of the anthers and the style head has always been a key character in Apocynaceae. It is the most important traditional character that separates Apocynoideae (in which the anthers are postgenitally united with the style head) from Rauvolfioideae (in which the anthers are free from the style head). The lack of close synorganization of the anthers and style head in Tabernaemontana divaricata is not typical of the whole genus (as defined by Leeuwenberg, 1991). In all Neotropical species of Tabernaemontana and in some Paleotropical ones as well, the style head and anthers are more closely synorganized and would be coded as character state 1 in this study. 17. Style apex specialization: 0 5 style apex without secretory epithelium; 1 5 style apex transformed into an enlarged style head with epithelium of the body uniformly secretory and receptive; 2 5 style apex transformed into an enlarged style head with epithelium of the body vertically differentiated with stigmatic region at base; 3 5 style apex transformed into an enlarged style head, with epithelium of the body vertically differentiated, stigmatic zone at base, and radially mechanically interrupted by the adnate anthers. All Apocynaceae are characterized by having the carpel apices forming an enlarged style head with secretory epithelium. The degree and manner of histological differentiation of the style head and the epithelium is variable, with a specific type often characteristic of a particular tribe (Schick, 1980; Fallen, 1986). Although the gynoecium apex in Geniostoma J. R. Forst. & G. Forst. is post-genitally fused and enlarged, it is not covered with a secretory epithelium like that found in Apocynaceae. Instead, on male flowers, enlarged glue-filled irritable hairs with an abscissable tip are found scattered among the more numerous smaller, normal papillae (Endress et al., 1996). Specialized glue hairs like those found in Geniostoma are unknown in Apocynaceae. 18. Style head upper hair wreath: 0 5 absent; 1 5 present. Some style heads have a wreath of longer hairs just below the unfused carpel tips. The main function of the wreath is for pollen deposition and secondary presentation. The flowers are protandrous; shortly before anthesis, the anthers dehisce and shed their pollen toward the center of the flower. If the style head has an upper wreath, the pollen is shed onto this ring of hairs, which plays a role in the complex pollination mechanism of Apocynaceae (Church, 1908; Schick, 1980; Fallen, 1986). 19. Style head base: 0 5 without collar or flange; 1 5 with a distinct, thin collar; 2 5 with a wreath of longer hairs; 3 5 with thick flange. The base of the style head is often equipped with a means of scraping off donor pollen from the proboscis of an insect visitor. The presence (or absence) and type of scraper is often diagnostic of a particular tribe, and thus a useful character in the family. When a scraper is present at the base of the style head, the receptive zone is located beneath it (Schick, 1980; Fallen, 1986). 20. Style head unfused apices: 0 5 small, inconspicuous, less than 1/3 the length of the total style head; 1 5 enlarged, conical and tapering to blunt and clavate, at least 1/3 the length of the total style head. 21. Free disc nectary: 0 5 absent; 1 5 entire, annular; 2 5 two separate lobes. In Apocynaceae, a free nectar disc is often present. Sometimes the nectar disc is adnate to the base of the ovary. Some taxa (especially in Rauvolfioideae) are nectarless and apparently use deceit pollination (Haber, 1984; Lin & Bernardello, 1999). In some cases it is difficult to distinguish whether or not the base of the ovary is nectariferous. For this reason, only the presence versus absence of a distinct nectary disc is coded here. In the large genus Alstonia, this character varies from species to species. The species included in this study, A. scholaris, has a shallow nectar disc. In some other species of Alstonia, a slight thickening can be discerned at the base of the ovary, and in yet others there is no indication of a nectary at all. 22. Ovary: 0 5 2-carpellate; 1 5 3–5-carpellate. Throughout Apocynaceae s.l., the gynoecium is composed of two carpels. The only exceptions are found in Lepinia and Lepiniopsis in the Alyxieae and in Pleiocarpa in Hunterieae (Endress et al., 1997). 23. Placentas: 0 5 lignified or indurated in fruit; 1 5 not lignified or indurated in fruit. 24. Mesocarp consistency: 0 5 fleshy, without fibers; 1 5 fleshy, with fibers; 2 5 dry or woody. 25. Endocarp: 0 5 not forming a stone around the seed; 1 5 lignified or sclerified and forming a stone around the seed. 26. Seeds: 0 5 sessile; 15 funiculate. 27. Seed shape: 0 5 broad, compressed, not folded, mostly circular to ovoid; 1 5 cylindrical, as if longitudinally rolled; 2 5 narrowly fusiform, flattened, with a longitudinal fold; 3 5 irregularly shaped, globular or angular, not flattened, or flattened on one side only, the other side convex. 28. Seed margin: 0 5 with neither flattened edge nor wing; 1 5 with a narrow flattened edge, this sometimes dissected; 2 5 with a well-developed, usually membranous wing(s); 3 5 fimbriate. 29. Seed coma: 0 5 absent; 1 5 present. A coma is a tuft of hairs all arising from a small restricted region at the end(s) of a seed. It is not considered to be homologous to the fimbria that are found around the margin of the seed in, for example, Alstonia. 30. Hilar depression: 0 5 absent; 1 5 an ovate depression, less than 50% the length of the seed; 2 5 a deep, broad furrow, traversing the entire seed length; 3 5 a deep, narrow fissure, traversing 75%–80% of the length of the seed. 31. Hilum shape: 0 5 linear, traversing the length of the seed; 1 5 linear, but shorter than the seed; 2 5 small, circular (punctiform); 3 5 ovate, covering a larger area. 32. Endosperm: 0 5 not ruminate; 1 5 with shallow, irregular tubercules or ruminations; 2 5 with deep longitudinal ruminations. Ruminate endosperm, although relatively rare in Apocynaceae, is characteristic for Tabernaemontana and is also found in several genera of Alyxieae. Chilocarpus is unusual in this aspect in that the genus can be divided into two groups: those with smooth and those with ruminate endosperm (Pichon, 1949c; Markgraf, 1971). The representative species used in the analysis here belongs to the group with smooth endosperm; had a species from the other group been selected, Chilocarpus would fit better with other Alyxieae as to this character. 33. Endosperm: 0 5 tough and corneous to subcartilaginous; 1 5 firm, fleshy or starchy; 2 5 delicate, soft or mealy. There is considerable variation in the thickness of the endosperm. For example, the endosperm of Alyxia, Chilocarpus, Condylocarpon, Lepinia, Lepiniopsis, and Pteralyxia is especially thick and tough (even difficult to cut with a razor blade). In Allamanda, Picralima, Plectaneia, and Pleiocarpa, endosperm is also tough but much thinner, but because no 34 Annals of the Missouri Botanical Garden clear demarcation between ‘‘thick’’ and ‘‘thin’’ could be found, only the consistency of the endosperm is coded. 34. Cotyledon base: 0 5 auriculate; 1 5 not auriculate. Although cotyledons are typically auriculate at the base in Tabernaemontaneae (sensu Leeuwenberg, 1991), this was not the case in the species included here. 35. Secoiridoids and complex indole alkaloids: 0 5 absent; 1 5 secoiridoids present, indole alkaloids absent; 2 5 dominant indole alkaloids present, but only those with non-rearranged secologanin skeleton; 3 5 dominant indole alkaloids present, including those with rearranged secologanin skeleton of the eburnan and/or plumeran type; 4 5 dominant indole alkaloids present, including those with rearranged secologanin part of the ibogan type. 36. Cardenolides: 0 5 absent; 1 5 present. 37. Steroidal alkaloids: 0 5 absent; 1 5 present. 38. Pollen unit: 0 5 monad; 1 5 tetrad. Tetrads are rare in Rauvolfioideae, and of the taxa sampled here they occur only in Condylocarpon and Craspidospermum. 39. Pollen grain: 0 5 small (# 51 mm); 1 5 large ($ 60 mm). Average largest pollen grain size (either the length of the polar axis, P, or the diameter of the equatorial plane, E) varies between 25 and 90 mm. It appears that a relatively large gap exists between 51 and 60 mm and that only Anechites (56 mm) falls between. Coding pollen size either as small (# 51 mm) or as large ($ 60 mm), with one ambiguous case, gives two rather well-separated size classes. 40. Pollen grain shape: 0 5 regular; 1 5 irregular. Pollen grains with a regular shape have a zonoaperturate aperture system with equally spaced apertures on the equator. The polar axis and the equatorial plane can be easily indicated. In pollen grains with an irregular shape, the position/ orientation of the polar axis and the equatorial plane cannot be indicated because there are only one or two porate apertures that are unequally spaced and sized and have an oblique orientation. Irregular pollen grains with three porate apertures have unequally spaced and sized apertures with oblique orientations. 41. Aperture number: 0 5 zero; 1 5 one or two; 2 5 three or four; 3 5 five or more. In Rauvolfioideae, only Condylocarpon has inaperturate pollen (aperture number 5 zero). Two-aperturate pollen (sometimes mixed with 1aperturate) occurs in Alyxia, Chilocarpus, Plectaneia, and Pteralyxia, while 3- and/or 4-aperturate pollen (often mixed within a single sample) is found in most other genera. A few genera have five or more apertures (up to 10 apertures are found in Craspidospermum). In most samples studied, minor percentages of pollen grains with deviating aperture numbers are found, which is a common phenomenon in dicots. The coding given is for the dominant aperture numbers. 42. Ectoapertures: 0 5 long colpi; 1 5 short colpi; 2 5 large pori; 3 5 small pori; 4 5 indistinct. Colpate ectoapertures are either longer than ca. 2/3 (long colpi) or shorter than ca. 1/3 (short colpi) the length of the polar axis (P). Large pori are at least 6 mm; if 6 mm (Chilocarpus, Plectaneia), then they are always accompanied by larger pori (up to 9 and 12 mm, respectively) in the same pollen grain. Small pores are 2–5 mm and do not vary much in size in a single grain. Due to its thin outer exine, Vinca pollen has indistinct ectoapertures. 43. Ectoaperture margin: 0 5 not outwardly thickened; 1 5 outwardly weakly thickened; 2 5 outwardly distinctly thickened; 3 5 with conspicuous arcus-like ridges. The ectoaperture margin is usually not thickened. In genera with large pores, but also in the brachycolpate Molongum, it is distinctly thickened into a well-delimited, protruding margin (aspidate pollen). In the genera with small pores, the ectoaperture margin is not or only weakly thickened. Aspidosperma and Vallesia have conspicuous ridges (massive and partly hollow, respectively) along the colpi joining toward the poles. 44. Endoapertures: 0 5 distinct from and smaller than ectoapertures; 1 5 not distinct from ectoapertures; 2 5 distinct from and larger than ectoapertures. In Alyxia, Lepinia, Lepiniopsis, Plectaneia, and Pteralyxia (all with porate pollen), the endoapertures are not delimited from the ectoapertures (endo- and ectoapertures congruent). In other porate genera, the endopore is distinct by being situated in a differentiated inner exine layer, and also in all other taxa the endo- and ectoapertures are incongruent. In colporate pollen grains, the endoapertures are always smaller than the ectoapertures, except in Vinca, in which the endoapertures (delimited by costae) are larger than the ectoapertures. 45. Endoaperture margin: 0 5 not inwardly thickened; 1 5 with endoannulus; 2 5 with polar costae; 3 5 with lateral costae. This character can only be assessed by using LM and/ or SEM images of the inner pollen-wall surface. An endoannulus is an inward thickening encircling the endoaperture. Polar costae are thickenings at the polar sides of usually lalongate endoapertures. Lateral costae are thickenings at the lateral sides of circular to lalongate endoapertures. 46. Supplementary endocolpi: 0 5 absent (no endoplates recognizable); 1 5 weak (zones of endocracks; endoplates indistinct); 2 5 distinct (endoplates well recognizable). Supplementary endocolpi are narrow (e.g., Cabucula) to wide (e.g., Rhazya) zones along the colpi (one at each side) where the inner exine layer is more or less missing. They may join interaperturally toward the poles. In some genera (e.g., Catharanthus), they seem to have taken over at least some of the function of the ectocolpi, bordering on distinctly thicker intine parts (see El-Ghazaly, 1990, fig. 17). Usually, supplementary endocolpi have a granular inner surface and delimit smooth endoplates in the mesocolpium centers (mesocolpial plates) and under the colpi at the polar sides of the endoapertures (colpal plates). Supplementary endocolpi occur only in colporate genera (not all) and are absent in all porate genera. 47. Intine protrusions: 0 5 absent; 1 5 present. Coding of this character is largely based on data provided by Pichon (1947c, 1948a, b, c, 1950a, b). Protruding intine bulges at the endoapertures occur in both genera with porate pollen and genera with colporate pollen. In some cases, it could be observed (TEM) that the outer zone of a bulge has an intricately channeled structure. In Alyxia, there seems even to be a kind of relatively rigid operculum topping the protrusion (Huang, 1986). 48. Exine: 0 5 not reduced; 1 5 reduced (thin). Condylocarpon and Vinca have a thin exine (0.1–0.6 mm and ca. 0.1 mm, respectively), whereas in the other genera, exine thickness is at least 0.9 mm, but is usually much thicker. 49. Inner exine surface: 0 5 psilate; 1 5 scabrate; 2 5 verrucate; 3 5 granular. This character codes for the inner ornamentation of the exine (nexine surface) and was taken from the inside of the mesocolpia (mesoporia) centers. A scabrate surface has elements smaller than 1 mm. Verrucate and granular elements are larger than 1 mm, the former with a broad base, the latter with a constricted base. 50. Inner exine layer (nexine): 0 5 ectexinous/endexinous (foot layer/endexine); 1 5 ectexinous (endexine absent); 2 5 endexinous (foot layer absent). This character codes for the composition of the inner exine layer (nexine). Volume 94, Number 1 2007 Endress et al. Phylogenetic Analysis of Alyxieae 35 Endexinous parts are indicated by ‘white lines,’ a lamellate structure and/or a 6 spongy aspect. Ectexinous parts are 6 homogeneous. Usually endexinous and ectexinous parts differ in contrast. 51. Infratectum: 0 5 columellate; 1 5 granular, reticulate or irregular; 2 5 not recognizable (commissural line). A columellate condition is found only in Gelsemium. In most other genera, the infratectum is granular, reticulate or irregular, and varying in thickness. In Alyxia, Lepinia, Lepiniopsis, and Pteralyxia, an infratectum is indistinct (sparse gaps in inner exine layer), the contact between ectexine and endexine being largely a commissural line. 52. Tectum: 0 5 thicker than infratectum + inner exine layer; 1 5 equal to infratectum + inner exine layer; 2 5 thinner than infratectum + inner exine layer. This character codes for the thickness of the tectum compared with the rest of the exine (infratectum + inner exine layer). It is also an approximate measure for the relative position of the infratectum. Usually the tectum is well delimited. When the boundary is irregular (e.g., in Plectaneia), the average tectum thickness was measured. 53. Outer exine surface: 0 5 psilate (even to undulate); 1 5 verrucate, with angular anastomosing verrucae; 2 5 verrucate, with 6 circular isolated verrucae; 3 5 microreticulate; 4 5 scabrate; 5 5 striate-reticulate. This character codes for the outer ornamentation of the exine (tectum surface). Most genera have psilate pollen (no protuberances), with an even to undulate, often perforate surface. Lepinia and Lepiniopsis have verrucate pollen with anastomosing verrucae. The other states, except for scabrate exine, occur in single genera. 54. Mesocolpium/mesoporium centers: 0 5 outer surface hardly or not deviating from surrounding exine; 1 5 outer surface clearly deviating from surrounding exine. In about 1/ 4 of the sampled genera with colporate pollen, the outer surfaces of the mesocolpium centers have a different ornamentation compared with the surrounding areas. Usually the mesocolpium centers have a rugulate, microfossulate to verrucate, or a less distinctly perforate ornamentation.