PHYLOGENY OF THE HERBACEOUS TRIBE SPERMACOCEAE (RUBIACEAE) BASED ON PLASTID DNA DATA1 Inge Groeninckx,2 Steven Dessein,2,3 Helga Ochoterena,4 Claes Persson,5 Timothy J. Motley,6 Jesper Kårehed,7 Birgitta Bremer,7 Suzy Huysmans,2 and Erik Smets2,8 ABSTRACT In its current circumscription, the herbaceous tribe Spermacoceae s.l. (Rubiaceae, Rubioideae) unites the former tribes Spermacoceae s. str., Manettieae, and the Hedyotis–Oldenlandia group. Within Spermacoceae, and particularly within the Hedyotis–Oldenlandia group, the generic delimitations are problematic. Up until now, molecular studies have focused on specific taxonomic problems within the tribe. This study is the first to address phylogenetic relationships within Spermacoceae from a tribal perspective. Sequences of three plastid markers (atpB-rbcL, rps16, and trnL-trnF) were analyzed separately as well as combined using parsimony and Bayesian approaches. Our results support the expanded tribe Spermacoceae as monophyletic. The former tribe Spermacoceae s. str. forms a monophyletic clade nested within the Hedyotis–Oldenlandia group. Several genera formerly recognized within the Hedyotis–Oldenlandia group are supported as monophyletic (Amphiasma Bremek., Arcytophyllum Willd. ex Schult. & Schult. f., Dentella J. R. Forst. & G. Forst., Kadua Cham. & Schltdl., and Phylohydrax Puff), while others appear to be paraphyletic (e.g., Agathisanthemum Klotzsch), biphyletic (Kohautia Cham. & Schltdl.), or polyphyletic (Hedyotis L. and Oldenlandia L. sensu Bremekamp). Morphological investigations of the taxa are ongoing in order to find support for the many new clades and relationships detected. This study provides a phylogenetic hypothesis with broad sampling across the major lineages of Spermacoceae that can be used to guide future species-level and generic studies. Key words: atpB-rbcL, Hedyotis–Oldenlandia group, Rubiaceae, molecular phylogeny, plastid DNA, rps16, Spermacoceae, trnL-trnF. The systematic relationships of the Rubiaceae herbaceous representatives are still unclear at the species and genus levels (Robbrecht & Manen, 2006). Even the higher-level classification in tribes has been the subject of debate. In the last comprehensive classification based on morphology (Robbrecht, 1988, 1993), most herbaceous representatives were assigned to one of the following tribes: Anthospermeae, Argostemmateae, Coccocypseleae, Hedyotideae, Knoxieae, Rubieae, Sipaneeae, Spermacoceae, and Theligoneae. Among these, the Spermacoceae as traditionally delimited (Hooker, 1873; Bremekamp, 1952, 1966; Verdcourt, 1958; Robbrecht, 1988, 1993), referred to in this paper as Spermacoceae s. str., are characterized by the presence of raphides, fimbriate stipules, uniovulate locules, seeds with an apparent adaxial groove, and the frequent occurrence of pluriaperturate pollen grains. However, molecular data show Spermacoceae s. str. to be deeply nested within the Hedyotideae, making the latter tribe paraphyletic (Bremer, 1996; Andersson & Rova, 1999; Bremer & Manen, 2000; Dessein et al., 2005a). Therefore, Bremer (1996) and later Bremer and Manen (2000) proposed a wider definition for Spermacoceae, in which the former tribes Spermacoceae s. str., Hedyotideae, Manettieae, Knoxieae, and Triainolepideae are merged. 1 We thank the organizers of the Third International Rubiaceae Conference and the editors of this volume for the invitation to participate in the proceedings. We acknowledge the technical assistance of Anja Vandeperre from the Laboratory of Plant Systematics, Katholieke Universiteit Leuven. Frank van Caekenberghe from the National Botanic Garden of Belgium is thanked for providing lovely photographs of Spermacoceae. This research was supported by grants from the Fund for Scientific Research–Flanders (FWO, G.0250.05 and G.0268.04). Inge Groeninckx holds a Ph.D. research grant from the Fund for Scientific Research–Flanders. 2 Laboratory of Plant Systematics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, P.O. Box 2437, BE-3001 Leuven, Belgium. Author for correspondence: inge.groeninckx@bio.kuleuven.be. 3 National Botanic Garden of Belgium, Domein van Bouchout, BE-1860 Meise, Belgium. 4 Instituto de Biologia, Universidad Nacional Autónoma de México, Apdo. Postal 70-367, 04510, Mexico. 5 Department of Plant and Environmental Sciences, Göteborg University, P.O. Box 461, SE-405 30 Göteborg, Sweden. 6 Department of Biological Sciences, Old Dominion University, 110 Mills Godwin Building, 45th Street, Norfolk, Virginia 23529-0266, U.S.A. 7 Bergius Foundation, Royal Swedish Academy of Sciences and Botany Department, Stockholm University, SE-106 91 Stockholm, Sweden. 8 National Herbarium of the Netherlands, Leiden University Branch, P.O. Box 9514, NL-2300 RA Leiden, The Netherlands. doi: 10.3417/2006201 ANN. MISSOURI BOT. GARD. 96: 109–132. PUBLISHED ON 23 APRIL 2009. 110 Annals of the Missouri Botanical Garden Based on rps16 intron data, Andersson and Rova (1999) also found that Hedyotideae is paraphyletic relative to Spermacoceae s. str. They did not accept the wide delimitation for Spermacoceae as proposed by Bremer (1996), but suggested an emended tribe Knoxieae that included a few genera of Hedyotideae (i.e., Otiophora Zucc., Otomeria Benth., and Pentas Benth.) as a more prudent taxonomic approach to handle the information from molecular-based analyses. The latter view was followed by Dessein (2003), who preferred to recognize an emended tribe Knoxieae (including Knoxieae s. str., Triainolepideae, Otiophora, the Pentas group of Hedyotideae fide Dessein et al. [2000], and Carphalea Juss.) as a sister group of Spermacoceae (including Spermacoceae s. str., Manettieae, and most of Hedyotideae). Robbrecht and Manen (2006), based on a supertree analysis of the family, came to a similar conclusion and likewise recognized Knoxieae s.l. and Spermacoceae s.l. The monophyly of the former tribe has also been confirmed by a subsequent molecular study by Kårehed and Bremer (2007). In their taxonomic conspectus, Robbrecht and Manen (2006) listed 33 genera of Spermacoceae s.l. for which molecular sequence data are available. Based on morphological data, we recognize 31 of these 33 genera and consider that the tribe should include 30 additional genera; these are listed in Table 1. For each genus, the number of species, the distribution, and the position in Robbrecht’s classification of 1988 are given. Spermacoceae s.l. forms a primarily herbaceous lineage that is generally characterized by fimbriate stipules and 4-merous flowers. Floral characters (Fig. 1), as well as seeds and fruits, are highly variable. Morphologically, three main groups can be identified within Spermacoceae s.l. The first, the Hedyotis–Oldenlandia group, is characterized by multiovulate locules and comprises the large genera Hedyotis L. and Oldenlandia L. and their presumed relatives. Most of these taxa were formerly placed in the tribe Hedyotideae. The generic delimitations of the Hedyotis–Oldenlandia group have been the subject of controversy for many years. The main issue is whether most species of the complex should be lumped into Hedyotis (advocated by inter alia Merrill & Metcalf, 1946; Wagner et al., 1989; Fosberg & Sachet, 1991; Dutta & Deb, 2004) or whether many small genera should be recognized in addition to a narrow circumscription of Hedyotis and Oldenlandia (supported for African taxa by Bremekamp, 1952; for Neotropical taxa by Terrell et al., 1986; Terrell, 1991, 2001a, b, c; and for Asian taxa by Terrell & Robinson, 2003). The second well-marked group within Spermacoceae s.l. is Spermacoceae s. str., which is character- ized by uniovulate locules. According to Dessein (2003), this group contains 19 genera of which Spermacoce L. is by far the largest with an estimated 275 species. Within Spermacoceae s. str., controversy has focused on the delimitation of its nominal genus Spermacoce. The main question is whether Spermacoce should be limited to species with the same type of fruit dehiscence as S. tenuior L., the type species of the genus. In this species, fruits open asymmetrically, resulting in one closed and one open fruit part. If this narrow delimitation for Spermacoce (referred to as Spermacoce s. str.) is accepted, most other species in the tribe Spermacoceae s. str. must be included in Borreria G. Mey. A third well-defined group within Spermacoceae s.l. comprises only two American genera, Bouvardia Salisb. and Manettia Mutis ex L. Bremekamp (1952) considered Bouvardia closely related to Heterophyllaea Hook. f., Hindsia Benth. ex Lindl., and Lecanosperma Rusby. Robbrecht (1988) placed these genera together with inter alia Manettia in a group with uncertain affinities, because their winged seeds suggest a relation to Cinchoneae, while the presence of raphides indicates a relation to Hedyotideae. In the classification of Bremer and Manen (2000), only Bouvardia and Manettia belong to Spermacoceae s.l., because Hindsia and Heterophyllaea (including Lecanosperma) are included in Coussareeae. Manettia is similar to Bouvardia in many characters, but its winding shoots and corneous endosperm separate it from Bouvardia, which is erect and has fleshy endosperm. These differences were the basis for Bremekamp (1934) to place Manettia in its own tribe, Manettieae. Until now, molecular studies within Spermacoceae s.l. have focused on particular taxonomic problems, such as the circumscription and biogeography of Arcytophyllum Willd. ex Schult. & Schult. f. (Andersson et al., 2002), the generic status of Houstonia L. (Church, 2003), the delimitation of Pentanopsis Rendle, the affinities of Phylohydrax Puff (Thulin & Bremer, 2004), and the taxonomic position of Gomphocalyx Baker (Dessein et al., 2005a). In the present paper, we aim to present a phylogenetic hypothesis of Spermacoceae s.l. based on the analysis of three plastid markers (atpB-rbcL, rps16, and trnL-trnF) with the broadest sampling to date. More specifically, we want to address the following questions: (1) Is Spermacoceae s.l. as circumscribed by Robbrecht and Manen (2006) monophyletic? (2) What are the relationships among members of Spermacoceae s. str. and genera of the former tribes Hedyotideae and Manettieae? (3) What are the major clades within the Hedyotis–Oldenlandia group? Volume 96, Number 1 2009 Groeninckx et al. Phylogeny of Spermacoceae MATERIAL AND METHODS 1998). Sequences were initially aligned with ClustalX (Thompson et al., 1997) applying the default parameters. Further adjustments of the preliminary aligned data matrices were done manually with MacClade 4.04 (Maddison & Maddison, 2001). Parsimonious informative gaps were coded manually according to the conservative simple indel coding method described by Simmons and Ochoterena (2000). PLANT MATERIAL AND SAMPLING The aim was to obtain a broad sampling covering most of the geographic and taxonomic diversity of Spermacoceae and to enable identification of the principal clades within the tribe. We included a total of 128 species representing 32 of the 61 genera within Spermacoceae. Three taxa belonging to the Knoxieae (Batopedina pulvinellata Robbr., Carphalea madagascariensis Lam., and Pentanisia parviflora Stapf ex Verdc.) were chosen as outgroup following Robbrecht and Manen (2006) and Kårehed and Bremer (2007). For rps16 and trnL-trnF, we used 40 and seven previously published sequences, respectively (Andersson & Rova, 1999; Andersson et al., 2002; Dessein et al., 2005a). Two hundred seventy-two sequences are newly generated (100 atpB-rbcL sequences, 67 rps16 sequences, 105 trnL-trnF sequences) using dried silica and herbarium material. Appendix 1 lists all taxa included in this study with voucher information and GenBank accession numbers. DNA EXTRACTION, POLYMERASE CHAIN REACTION AMPLIFICATION, AND SEQUENCING DNA was extracted from silica-dried and herbarium material using the CTAB method as described by Janssens et al. (2006). Amplification of the atpB-rbcL spacer was done with oligonucleotides two and five as primers (Manen et al., 1994). Specific amplification products could be obtained with a touchdown polymerase chain reaction (PCR) with two cycles with an annealing temperature of 53uC, then 12 cycles with an annealing temperature of 52.5uC declining 0.5uC every cycle, followed by 16 cycles with an annealing temperature of 47uC. The rps16 intron was amplified with the rps16F and rps16R2 primers described by Oxelman et al. (1997). For the trnL-trnF intergenic spacer, we used the primers e and f of Taberlet et al. (1991). Both rps16 and trnL-trnF were amplified using standard PCR techniques with an annealing temperature of 55uC. The PCR reaction mixture was cleaned using a Nucleospin Extraction II Kit (Machery-Nagel, Dren, Germany) according to the manufacturer’s instructions. Sequencing was mostly done on an ABI 310 Genetic Analyzer (Applied Biosystems, Lennik, Belgium). Some PCR products were sequenced by Macrogen (Seoul, South Korea) sequencing facilities. SEQUENCE ASSEMBLY, ALIGNMENT, AND GAP CODING The assembling and editing of sequences were conducted using the Staden Package (Staden et al., 111 PHYLOGENETIC ANALYSES Phylogenetic analyses were conducted using both parsimony (MP) and Bayesian inference (BI). The three plastid regions were first analyzed separately and then combined. Equally weighted MP analyses were performed using Nona 2.0 (Goloboff, 1993) launched through WinClada 1.00.08 (Nixon, 2002). Heuristic searches for the shortest trees were performed using the parsimony ratchet (Nixon, 1999). Ratchet runs of 200 iterations each, holding one tree per iteration and randomly weighting 10% of the potentially informative characters, were carried out until the most parsimonious trees (MPTs) were repeatedly found. A strict consensus tree was calculated using the trees obtained in the parsimony ratchet analyses. In order to evaluate the relative support of the clades, jackknife and bootstrap analyses were executed using 1000 replicates with 10 initial trees holding five trees per random addition, doing tree bisection-reconnection (TBR) to hold 1000 trees, and calculating a consensus on each repetition. Frequency values were plotted onto the consensus of the MPTs. For the BI analyses, a substitution model was selected for each DNA region with Modeltest 3.06 (Posada & Crandall, 1998) under the Akaike Information Criterion (AIC). Modeltest selected the GTR+I+G model of evolution for the atpB-rbcL spacer and the GTR+G model for the two remaining markers. Indels were not included in the BI analyses. In the combined analysis, a mixed-model approach was used (Ronquist & Huelsenbeck, 2003). The combined data were partitioned and the same models of evolution were used on the partitions as selected for the single analyses. The BI analyses were conducted with MrBayes 3b4 (Huelsenbeck & Ronquist, 2001). Four Markov chains (one cold, three heated) starting with a random tree were run simultaneously for one million generations, sampling trees at every 100 generations. The first 2500 sampled trees (25%) were regarded as burn-in and discarded. PAUP* version 4b10 (Swofford, 2002) was used to calculate a 50% majority rule tree and to report the posterior probabilities for each clade. Only posterior probabilities above 0.95 are considered (Suzuki et al., 2002). 112 Annals of the Missouri Botanical Garden Table 1. List of genera associated with Spermacoceae s.l., their distribution, and species number following Govaerts et al. (2006), except when stated otherwise. Genera in boldface were listed by Robbrecht and Manen (2006); other genera are here based on morphological similarities. Synonymous taxa are as given by Robbrecht (1988), except when stated otherwise. Genus Robbrecht, 1988 Agathisanthemum Klotzsch Amphiasma Bremek. Anthospermopsis (K. Schum.) J. H. Kirkbr. Arcytophyllum Willd. ex Schult. & Schult. f. Astiella Jovet Bouvardia Salisb. Bradea* Standl. ex Brade Carterella Terrell Conostomium (Stapf) Cufod. Crusea Cham. & Schltdl. Dentella J. R. Forst & G. Forst. Diacrodon Sprague Dibrachionostylus Bremek. Denscantia E. L. Cabral & Bacigalupo Diodella Small (1) Diodia L. (1) Dolichometra K. Schum. Emmeorhiza Pohl ex Endl. Ernodea Sw. (2) Galianthe Griseb. (3) Gomphocalyx Baker Hedyotis L. Hedythyrsus Bremek. Houstonia L. (4) Hydrophylax L. f. Kadua Cham. & Schltdl. (incl. Gouldia A. Gray and Wiegmannia Meyen) (5) Kohautia Cham. & Schltdl. (6) Lelya Bremek. Leptomischus* Drake Leptoscela Hook. f. Lucya DC. Manettia Mutis ex L. Manostachya Bremek. Micrasepalum Urb. Mitracarpus Zucc. ex Schult. & Schult. f. Mitrasacmopsis Jovet Neanotis W. H. Lewis Neohymenopogon* Bennet Nesohedyotis (Hook. f.) Bremek. Nodocarpaea A. Gray Oldenlandia L. (incl. Eionitis Bremek., Exallage Bremek., and Thecorchus Bremek.) Oldenlandiopsis Terrell & W. H. Lewis Pentanopsis Rendle Pentodon Hochst. Phyllocrater Wernham Phylohydrax Puff Native distribution Hed Hed Spe Hed tropical and S Africa, Comoros tropical and S Africa NE Brazil Mexico to W South America Hed Cin/Hed Hed Hed Hed Spe Hed Spe Hed Spe Spe Spe Hed Spe Spe Spe Spe Hed Hed Hed Spe Spe Madagascar S U.S.A., Mexico to C America SE Brazil Mexico Ethiopia to S Africa Arizona, New Mexico, Mexico to C America tropical and subtropical Asia to SW Pacific Brazil E Tropical Africa E Brazil S U.S.A. to S America S U.S.A. to S America Tanzania S tropical America and Trinidad Florida, Mexico to C America, Caribbean S and C America Madagascar tropical and subtropical Asia to NW Pacific C and E tropical Africa N and C America India, Sri Lanka, Thailand Hawaiian Islands to S Pacific Hed Hed Hed Hed Hed Cin/Hed Hed Spe Spe Africa, Madagascar, and Asia tropical Africa Assam to China NE Brazil Caribbean tropical America C and E tropical Africa Caribbean tropical America, naturalized elsewhere Hed Hed Cin/Hed Hed Spe Hed C and E tropical Africa and Madagascar tropical and subtropical Asia E Himalaya, Tibet, SC China, N Indo-China St. Helena Cuba pantropical Hed Hed Hed tropical and subtropical America Ethiopia to N Kenya tropical and S Africa, Arabian Pen., W Indian Ocean, naturalized in America Borneo coastal Tanzania to S Africa, Madagascar Hed Spe No. of species Sampled 4 7 1 17 yes yes no yes 1 42 5 1 5 14 8 1 1 4 16 5 1 1 4 50 1 ca. 115 2 20 1 28 no yes no no yes yes yes no yes no yes no no yes yes yes yes yes yes yes no yes 31 1 7 1 1 124 3 2 58 yes yes no no no yes yes no yes 1 33 3 1 1 ca. 240 yes no no yes no yes 1 2 2 no yes yes 1 2 no yes Volume 96, Number 1 2009 Groeninckx et al. Phylogeny of Spermacoceae 113 Table 1. Continued. Genus Robbrecht, 1988 No. of species Sampled Native distribution Pleiocraterium Bremek. Polyura* Hook. f. Pseudonesohedyotis Tennant Psyllocarpus Mart. & Zucc. Richardia L. Hed Hed Hed Spe Spe Sacosperma* G. Taylor Schwendenera K. Schum. Spermacoce L. (incl. Borreria G. Mey. and Hemidiodia K. Schum.) (7) Staelia Cham. & Schltdl. Stenaria (Raf.) Terrell Stenotis Terrell Stephanococcus Bremek. Synaptantha Hook. f. Tobagoa Urb. Tortuella Urb. Hed Spe Spe tropical Asia E Himalaya to Assam Tanzania Brazil tropical and subtropical America, naturalized elsewhere W and C tropical Africa Brazil pantropical Spe Hed Hed Hed Hed Spe Spe Mexico and S tropical America C and E U.S.A. to Mexico, Bahamas Mexico (Baja California) WC tropical Africa Australia Panama to Tobago Île de la Tortue (Haiti) Hed, Hedyotideae; Spe, Spermacoceae s. str.; Cin, Cinchoneae. 5 Bacigalupo & Cabral (1999); (2) 5 Negrón-Ortiz & Hickey (1996); Terrell et al. (2005); (6) 5 Mantell (1985); (7) 5 Dessein (2003). * Tentatively included. (1) RESULTS Sequence data from the aligned atpB-rbcL, rps16, and trnL-F regions were analyzed independently and in a combined analysis (Table 2). Individual plastid sequence analyses were topologically congruent. Therefore, only the results from the MP and BI analysis of the combined matrix are presented (Figs. 2–4). Compared to the topologies of the individual plastid sequence analyses, the combined plastid trees show increased resolution and branch support. The Bayesian tree is somewhat better resolved than the consensus of the MP analysis, but more resolved lineages have low posterior probabilities. Spermacoceae s.l., as delimited in the introduction (Table 1), form a well-supported monophyletic group (jackknife support [JS] 5 100, bootstrap support [BS] 5 100, posterior probability [PP] 5 1), as can be seen in Figure 2. A highly supported pentamerous-flowered clade including Dentella J. R. Forst. & G. Forst. and Pentodon Hochst. (JS 5 100, BS 5 99, PP 5 1) is resolved as sister to the rest of the tribe (Fig. 2). The remaining ingroup taxa are part of a clade that lacks significant jackknife and bootstrap support and has only weak posterior probability (PP 5 0.84). Within this clade, stars with Roman numerals I to III are assigned to the three deeper internal nodes that have reasonable support. These three clades are discussed in the following paragraphs. (3) 5 Cabral (1991); (4) 4 1 1 9 16 no no no no yes 2 1 250–300 no no yes 14 5 7 1 2 1 1 no yes no no yes no no 5 Terrell (1996); (5) 5 Clade I in Figure 2 (JS 5 88, BS 5 77, PP 5 1) includes a Kohautia subg. Kohautia Verdc. clade sister to a clade that includes Pentanopsis and allied genera. This Pentanopsis clade (JS 5 95, BS 5 95, PP 5 1) is similar to that proposed by Thulin and Bremer (2004). However, our larger sampling resulted in a broader circumscription adding Gomphocalyx, Oldenlandia affinis (Roem. & Schult.) DC., O. herbacea (L.) Roxb., and O. rosulata K. Schum. Our results support the monophyly of both Amphiasma Bremek. (JS 5 98, BS 5 98, PP 5 1) and Phylohydrax (JS 5 93, BS 5 95, PP 5 1). In clade II (JS 5 88, BS 5 83, PP 5 1) of Figure 2, all Asian and Micronesian Hedyotis species, except H. tenelliflora Blume, are part of a strongly supported Hedyotis s. str. clade (JS 5 100, BS 5 100, PP 5 1), which is sister to a clade including Agathisanthemum Klotzsch and its allies. This clade of Asian and Micronesian Hedyotis species also includes H. fruticosa L., the type species of the genus. Relationships within this Hedyotis s. str. clade remain mostly unresolved. Within the Agathisanthemum clade, Agathisanthemum is paraphyletic to Lelya osteocarpa Bremek. (JS 5 100, BS 5 99, PP 5 1), both sister to a lineage of African (Oldenlandia angolensis K. Schum. and O. goreensis (DC.) Summerh.) and North American (O. uniflora L.) Oldenlandia species (JS 5 100, BS 5 99, PP 5 1). In the MP consensus, clade II is sister to clade III (Figs. 2A, 3A). However, this sister relationship lacks 114 Annals of the Missouri Botanical Garden significant jackknife and bootstrap support and is not recovered in the BI (Figs. 2B, 3B). Within clade III (Figs. 3, 4), the earlier derived clades lack significant support values in the MP consensus (Figs. 3A, 4A) and are collapsed in the BI (Figs. 3B, 4B). Therefore, relationships between the different subclades of clade III should be interpreted with caution. In the following paragraphs, these subclades are discussed individually. In Figure 3, the monospecific genus Dibrachionostylus Bremek. is sister to a clade of African Oldenlandia species (O. echinulosa K. Schum., O. geophila Bremek., and O. nervosa Hiern). However, this sister relationship lacks significant jackknife and bootstrap support (Fig. 3A) and is not supported by the BI (Fig. 3B). The sister relationship of this clade with respect to Mitrasacmopsis Jovet and its allies also lacks support. Mitrasacmopsis, another monospecific genus in the Hedyotis–Oldenlandia group, is nevertheless highly supported as sister to Hedythyrsus Bremek. (JS 5 99, BS 5 97, PP 5 1), and both are sister to O. fastigiata Bremek. (JS 5 99, BS 5 99, PP 5 1). The genus Kadua Cham. & Schltdl. (including Oldenlandia biflora L.) is resolved as monophyletic with moderate jackknife and bootstrap support but maximum Bayesian posterior probability (JS 5 87, BS 5 86, PP 5 1). The Hawaiian Kadua species are unresolved with respect to the French Polynesian species, K. rapensis F. Br. The genus Kadua shares a most recent common ancestor with all sampled Australian taxa (O. galioides (F. Muell.) F. Muell., O. mitrasacmoides F. Muell., and Synaptantha tillaeacea (F. Muell.) Hook. f.), the Austro-Asian species Hedyotis tenelliflora, and the African species O. lancifolia (Schumach.) DC. (JS 5 91, BS 5 86, PP 5 1). The genus Arcytophyllum is strongly supported as monophyletic by our analysis (JS 5 93, BS 5 92, PP 5 1). It is sister to a clade of North and Central American species of Houstonia, Oldenlandia, and Stenaria (Raf.) Terrell. The Houstonia species plus S. nigricans (Lam.) Terrell form one clade, although without significant support. In Figure 4, Spermacoceae s. str. is nested within the Hedyotis–Oldenlandia group. In the MP consensus (Fig. 4A), it forms a monophyletic lineage (although lacking significant jackknife support and bootstrap support), while in the BI tree (Fig. 4B), Nesohedyotis arborea (Roxb.) Bremek. is nested within the Spermacoceae s. str. clade (although with low PP 5 0.77). In both MP and BI analysis, Spermacoceae s. str. has uncertain relationships with respect to Arcytophyllum serpyllaceum (Schltdl.) Terrell, Bouvardia, Manettia, Nesohedyotis (Hook. f.) Bremek. (Fig. 4A), O. tenuis K. Schum., and O. salzmannii (DC.) Benth. & Hook. f. ex B. D. Jacks. Sister to this polytomy is a clade with species of Kohautia subg. Pachystigma Bremek. and Oldenlandia species, including the type species O. corymbosa L. (JS 5 99, BS 5 98, PP 5 1). Consequently, species of the genus Kohautia Cham. & Schltdl. fall in two wellsupported, not closely related clades, which correspond to the two described subgenera: subgenus Kohautia (JS 5 99, BS 5 99, PP 5 1) and subgenus Pachystigma (JS 5 96, BS 5 96, PP 5 1). DISCUSSION Our analysis corroborates the monophyly of Spermacoceae s.l. (Table 1), a mainly herbaceous assemblage distributed pantropically, with only a few genera penetrating into more temperate regions. The morphological variation is considerable, but the fimbriate stipules and tetramerous flowers are shared by most species. There are no clear morphological synapomorphies that separate Spermacoceae s.l. from its sister tribe, the emended Knoxieae. The main differences are listed in Table 3. Our analyses show several major evolutionary lineages within Spermacoceae s.l. and allow us to evaluate the monophyly of a number of genera. Several genera that have been recognized within the Hedyotis–Oldenlandia group are supported here as monophyletic (Amphiasma, Arcytophyllum, Dentella, Kadua, and Phylohydrax), while others appear to be paraphyletic (e.g., Agathisanthemum), biphyletic (Kohautia), or polyphyletic (Hedyotis and Oldenlandia sensu Bremekamp). These groups are discussed in the following paragraphs. SPERMACOCEAE S. STR. In our analyses, Spermacoceae s. str. is nested within the Hedyotis–Oldenlandia group, which no longer makes it possible to recognize this lineage at a tribal level. Additionally, Spermacoceae s. str. as delimited by Robbrecht (1988) is not corroborated as monophyletic. Both MP and BI analyses show that it is necessary to exclude Gomphocalyx and Phylohydrax for Spermacoceae s. str. to be monophyletic, which is in agreement with Thulin and Bremer (2004) and Dessein et al. (2005a). In the BI analyses, Nesohedyotis arborea, a species previously included in Hedyotideae, is placed within Spermacoceae s. str. as sister to Emmeorhiza umbellata (Spreng.) K. Schum., but lacking significant posterior probability (PP 5 0.67). This position of Nesohedyotis within Spermacoceae s. str. was not recovered in the MP analysis. Because no morphological characters can be found to support Nesohed- Volume 96, Number 1 2009 Groeninckx et al. Phylogeny of Spermacoceae 115 Figure 1. Floral diversity among species of Spermacoceae. —A. Kohautia microcala Bremek. —B. Hedythyrsus spermacocinus (K. Schum.) Bremek. —C. Mitracarpus frigidus (Willd. ex Roem. & Schult.) K. Schum. —D. Spermacoce debilis Benth. —E. Oldenlandia herbacea (L.) Roxb. —F. Gomphocalyx herniarioides Baker. —G. Manostachya ternifolia E. S. Martins. —H. Oldenlandia lancifolia (Schumach.) DC. —I. Phylohydrax madagascariensis (Willd. ex Roem. & Schult.) Puff. —J. Manettia luteorubra (Vell.) Benth. —K. Agathisanthemum globosum (Hochst. ex A. Rich.) Klotzsch. —L. Oldenlandia goreensis (DC.) Summerh. —M. Kohautia coccinea Royle. —N. Oldenlandia biflora L. —O. Kadua acuminata Cham. & Schltdl. —P. Oldenlandia robinsonii Pit. yotis as part of Spermacoceae s. str., we suggest that the difference between the MP and BI analysis could be the result of data sampling artifacts (only rps16 was sequenced for N. arborea), which probably affected the BI more than the MP analysis. With the deeper nodes unresolved or only weakly supported, the relationships within Spermacoceae s. str. remain unclear and should be the subject of further phylogenetic studies including more taxa and/ or characters. Nevertheless, our analyses corroborate 116 Annals of the Missouri Botanical Garden Table 2. Characteristics of each data matrix and the corresponding tree statistics. atpB-rbcL rps16 trnL-trnF Combined No. of taxa No. of char. No. of PI char. No. of PI indels No. of MPT MPT length CI RI 100 105 107 128 1237 705 1053 2995 175 191 184 550 31 20 29 80 1949 1351 343 4782 399 525 423 1385 0.55 0.56 0.62 0.56 0.84 0.82 0.88 0.84 Char, characters; CI, consistency index (Kluge & Farris, 1969); MPT, most parsimonious tree(s); PI, potentially informative; RI, retention index (Farris, 1989). the monophyly of most of the commonly accepted genera within Spermacoceae s. str., notably Crusea Cham. & Schltdl., Mitracarpus Zucc. ex Schult. & Schult. f., and Richardia L., although these were sampled only with a few species. In contrast, the two Galianthe Griseb. sampled species are paraphyletic to Diodia spicata Miq., a species that was recently excluded from Diodia s. str. and transferred to Borreria. If the position of D. spicata is confirmed by further phylogenetic studies, the generic circumscription of Galianthe should be widened to include at least this species. Dessein (2003) already showed that palynological data (7-zonocolporate pollen, long ectocolpi, double reticulum) support a close relation between D. spicata and Galianthe. Diodia L. as traditionally delimited, including species referred to Diodella Small by Bacigalupo and Cabral (1999), is not supported as monophyletic. Also, Spermacoce s.l., including Borreria, is not supported as monophyletic. BOUVARDIA AND MANETTIA Manettia is strongly supported as monophyletic (JS 5 100, BS 5 100, PP 5 1), whereas support for Bouvardia is moderate (JS 5 85, BS 5 87, PP 5 0.99). In accordance with Andersson et al. (2002), Arcytophyllum serpyllaceum is corroborated as sister to Bouvardia. This strongly supported relationship (JS 5 99, BS 5 99, PP 5 1), in combination with the fact that the remaining Arcytophyllum species are strongly supported as a monophyletic and distinct lineage (see below), suggests that at least A. serpyllaceum should be included within Bouvardia. Although Bouvardia is generally considered as a genus of shrubs only, it comprises both subshrubs and perennial herbs (Blackwell, 1968), which makes it possible to fit in A. serpyllaceum. Arcytophyllum serpyllaceum is similar to Bouvardia and different from other Arcytophyllum species in many respects. First, the stipule margin of A. serpyllaceum is not dentate or fimbriate, as in most Arcytophyllum species (Mena, 1990), but consists of a basal sheath and a trullate mucro as in most Bouvardia species (Blackwell, 1968). Second, whereas the seeds of Arcytophyllum are more or less cymbiform (Mena, 1990), those of A. serpyllaceum are discoid with a central hilum as in Bouvardia (Andersson et al., 2002). The major difference between seeds of A. serpyllaceum and Bouvardia is that Bouvardia seeds are winged, whereas those of A. serpyllaceum are not. ARCYTOPHYLLUM–HOUSTONIA CLADE Previous studies based on plastid DNA sequences have shown Arcytophyllum to be monophyletic and closely related to the North American Houstonia (Andersson & Rova, 1999; Andersson et al., 2002). Our analyses support the monophyly of the Neotropical genus Arcytophyllum (JS 5 93, BS 5 92, PP 5 1) only if A. serpyllaceum is excluded from the genus (see above). Sister to Arcytophyllum is a group of North and Central American species presently classified in the genera Houstonia, Oldenlandia, and Stenaria. By having its closest relatives in North America rather than in South America, Arcytophyllum may be one of the few examples within Rubiaceae that has reached the Andes by a southern migration (Andersson et al., 2002). From this perspective, Mesoamerican species like O. microtheca (Cham. & Schltdl.) DC. may represent remnants of stepping-stone populations. The Arcytophyllum–Houstonia clade as defined by our results is thus restricted to the New World. Seeds of Arcytophyllum and Houstonia are generally more or less cymbiform. Our results thus support Schumann’s (1891) grouping of genera with cymbiform seeds. So far, Neanotis W. H. Lewis has not been sequenced, but if seed shape is indeed a good phylogenetic marker, Neanotis could be the closest non-American relative of the Arcytophyllum–Houstonia clade (Lewis, 1966). There has been much discussion about the recognition of Houstonia at the generic level. In a recent molecular study based on ITS and trnL intron data (Church, 2003), Houstonia appeared to be paraphyletic with respect to the North American genus Stenaria. Therefore, Church (2003) suggested that Houstonia and Stenaria are better treated as a single genus. As currently circumscribed (Terrell, 1996), the genus Houstonia is composed of 20 species Volume 96, Number 1 2009 Groeninckx et al. Phylogeny of Spermacoceae 117 Figure 2. —A. Part one of the strict consensus tree of the 4782 MPTs from the combined analysis including atpB-rbcL, rps16, and trnL-trnF sequences (L 5 1385, consistency index [CI] 5 0.56, retention index [RI] 5 0.84). Jackknife (left) and bootstrap (right) values (. 50) are indicated above branches. —B. Part one of the Bayesian tree based on combined atpB-rbcL, rps16, and trnL-trnF data. Posterior probabilities are indicated above branches. The tribe Spermacoceae s.l. starts on Figure 2 (as indicated by an arrow) and continues over to Figures 3 and 4. Stars with Roman numerals I to III are assigned to the three reasonably supported clades within Spermacoceae s.l. 118 Annals of the Missouri Botanical Garden Figure 3. —A. Part two of the strict consensus tree of the 4782 MPTs from the combined analysis including atpB-rbcL, rps16, and trnL-trnF sequences (L 5 1385, CI 5 0.56, RI 5 0.84). Jackknife (left) and bootstrap (right) values (. 50) are indicated above branches. —B. Part two of the Bayesian tree based on combined atpB-rbcL, rps16, and trnL-trnF data. Posterior probabilities are indicated above branches. Roman numeral III is assigned to the deeper internal node with reasonable support. The tribe Spermacoceae s.l. starts on Figure 2 and continues over to Figures 3 and 4. Stars with Roman numerals I to III are assigned to the three reasonably supported clades within Spermacoceae s.l. Volume 96, Number 1 2009 Groeninckx et al. Phylogeny of Spermacoceae 119 Figure 4. —A. Part three of the strict consensus tree of the 4782 MPTs from the combined analysis including atpB-rbcL, rps16, and trnL-trnF sequences (L 5 1385, CI 5 0.56, RI 5 0.84). Jackknife (left) and bootstrap (right) values (. 50) are indicated above branches. —B. Part three of the Bayesian tree based on combined atpB-rbcL, rps16, and trnL-trnF data. Posterior probabilities are indicated above branches. The tribe Spermacoceae s.l. starts on Figure 2 and continues over to Figures 3 and 4. Representatives of the former tribe Spermacoceae s. str. are shown in this portion of the strict consensus tree and the Bayesian tree. 120 Annals of the Missouri Botanical Garden Table 3. Major morphological differences between Knoxieae and Spermacoceae s.l. Knoxieae s.l. Spermacoceae s.l. Merosity Inflorescence Calyx lobes Pollen often 5-merous or derived from the 5-merous state terminal (including pseudoaxillary) often 1 or more calyx lobes enlarged bireticulum not yet reported Exotesta Distribution ITW often slightly thickened paleotropical, centered in Madagascar and continental Africa often 4-merous, rarely 5-merous terminal or axillary rarely enlarged calyx lobes bireticulum common, often associated with heterostyly ITW without thickenings pantropical, with a few taxa reaching outside the tropics ITW, inner tangential wall. restricted to North America. The genus contains both annual and perennial herbs with either heterostylous or homostylous flowers, crateriform seeds, and colporate pollen. Chromosome numbers are variable among species of the genus with x 5 6, 7, 8, or 11. Stenaria, a genus only recently described (Terrell, 2001a), includes five species previously included in the North American Hedyotis. The genus contains only perennial, heterostylous herbs. Due to our incomplete sampling of these two genera, and given that Houstonia forms a polytomy with Stenaria, our results are not conclusive with respect to whether it is best to recognize Stenaria or consider it part of a more broadly delimited Houstonia. A more extensive sampling should focus further on this question. Sister to the Houstonia–Stenaria clade is Oldenlandia microtheca. The prevailing basic chromosome number in Oldenlandia is n 5 9, which occurs in the type species O. corymbosa and in many of the species native to North America, Asia, Africa, and Australia (Lewis, 1965), but not in O. microtheca, which is exceptional in having a chromosome number n 5 11. The same chromosome number is found in Oldenlandiopsis Terrell & W. H. Lewis (Terrell, 1991), not included in this study, and in some Houstonia species (e.g., H. rubra Cav.). Until now, Oldenlandia microtheca and Oldenlandiopsis were never considered to be closely related to Houstonia because of the lack of morphological similarities (Lewis, 1965; Terrell, 1991). Oldenlandiopsis contains only one species, O. callitrichoides (Griseb.) Terrell & W. H. Lewis, previously included in Oldenlandia. This smallleaved, small-flowered, creeping herb is native to the West Indies and southern Mexico. Based on its chromosome number and its distribution, a position of Oldenlandiopsis in the Arcytophyllum–Houstonia clade close to Oldenlandia microtheca seems quite likely. However, seeds of Oldenlandiopsis are noncrateriform and pollen are 8-colporate with a lalongate, slightly crassimarginate endoaperture (Terrell & Lewis, 1990). These types of seeds and pollen are unusual within the Arcytophyllum–Houstonia clade. Plurizonocolporate pollen grains are also exceptional within the rest of the Hedyotis–Oldenlandia group, where the aperture number rarely exceeds five. The Asian genus Neanotis (Lewis, 1966), the Malagasy endemic Gomphocalyx (Dessein et al., 2005a), the Afro-Madagascan Phylohydrax (Puff, 1986), and the West Indian monotypic genus Lucya DC. (Terrell & Lewis, 1990) are notable exceptions within the Hedyotis–Oldenlandia group in having plurizonocolporate pollen grains. Both Gomphocalyx and Phylohydrax belong to the Pentanopsis clade (see below). With no molecular sequence data available for Lucya, Neanotis, and Oldenlandiopsis, it would be premature to hypothesize a close relationship between any of these taxa and the Arcytophyllum–Houstonia clade or the Pentanopsis clade. Nevertheless, considering their distribution, the Caribbean-Mexican genera Lucya and Oldenlandiopis are more likely to fall in the Arcytophyllum–Houstonia clade, whereas the Asian genus Neanotis is more likely to have its closest relatives within the Pentanopsis clade. Two closely related genera from Baja California, Stenotis Terrell (Terrell, 2001b) and Carterella Terrell (Terrell, 1987), may also belong to the Arcytophyllum– Houstonia clade. Like the Mesoamerican species Oldenlandia microtheca, they may represent remnants of stepping-stone populations. The monospecific genus Carterella was described based on Bouvardia alexanderae A. M. Carter. It resembles Bouvardia in having unusually long corolla tubes, but differs from Bouvardia in having wingless seeds and chromosome number n 5 13. The genus Stenotis, on the other hand, includes seven former Hedyotis species endemic to the Baja California peninsula (Terrell, 2001b). These heterostylous, annual or perennial herbs also have chromosome number x 5 13. According to Terrell (1987, 2001b), Carterella and Stenotis have their closest relatives among the Baja California species of Houstonia (H. mucronata group sensu Terrell et al., 1986). Volume 96, Number 1 2009 Groeninckx et al. Phylogeny of Spermacoceae KADUA which includes the type species H. fruticosa (Sri Lanka). Several authors already considered the genus Hedyotis to be a distinct Asian taxon (Bremekamp, 1952; Hallé, 1966; Terrell, 1975, 1991; Andersson et al., 1999). Hedyotis fruticosa and its Asian relatives are not closely related to the American species of Hedyotis (Houstonia lineage) or to the Polynesian species (Kadua). The Asian and Micronesian Hedyotis species (Hedyotis s. str.) differ from the American and Polynesian ones in their combination of a robust (sometimes shrubby) habit, small beaked and diplophragmous capsules, dorsiventrally compressed seeds with the hilum on a conspicuous central ridge (Terrell & Robinson, 2003), and a high chromosome number (Kiehn, 1986). Our results clearly demonstrate that a broad concept of Hedyotis, merging several genera (Hedyotis s. str., Houstonia, Kadua, Kohautia, Oldenlandia, etc.), as was proposed by several researchers (Fosberg, 1943; Merrill & Metcalf, 1946; Rogers, 1987; Wagner et al., 1989; Fosberg & Sachet, 1991; Dutta & Deb, 2004), is no longer supported. If this is confirmed with further sampling, all North American species now called Hedyotis would require new combinations under other generic names. Pleiocraterium Bremek. (not included in this study) is probably related to the Hedyotis s. str. clade. The genus was described by Bremekamp in 1939, including four species distributed in India, Sri Lanka, and Sumatra. The generic name refers to the numerous cups that are formed by the connate leaf bases. The type species of the genus, P. verticillare (Wall. ex Wight & Arn.) Bremek., was previously included in Hedyotis. However, the genus differs from other Hedyotis s. str. species in having distinctly beaked capsules and parallel-nerved, quaternate leaves. The internodes remain very short, as a result of which the leaf whorls are clustered in rosettes. It will be necessary to wait, however, until molecular data of Pleiocraterium become available before a close relation of the genus to the Asian Hedyotis species is confirmed. Our results support the resurrection of the genus Kadua for the Polynesian Hedyotideae (Hawaiian Islands and French Polynesia: Terrell et al., 2005). This taxonomic change was previously suggested by unpublished molecular data (Motley et al., 1998; Motley, 2003) and by morphological studies of the seed anatomy of the Hawaiian species (Terrell et al., 2005). The genus Kadua was treated as a distinct genus until Fosberg’s (1943) revision of the group. He included the genus within a broadly delimited Hedyotis, except for the fleshy-fruited species, which he treated as Gouldia A. Gray (Fosberg, 1937). Kadua species can, however, easily be distinguished from other Hedyotis species by their salverform, fleshy corollas with appendaged lobes, and by their either tardy, often incomplete septicidal dehiscent capsules or indehiscent drupaceous fruits (Terrell et al., 2005). The genus Kadua currently comprises 28 species; all are indigenous to the Pacific Islands with the majority (21 species) occurring on the Hawaiian Islands (Terrell et al., 2005). Seeds of these Hawaiian Kadua species fall into four groups, described by Terrell et al. (2005). Based on the chloroplast data alone, the relationships within the genus Kadua remain mostly unresolved. Only section Wiegmannia Meyen, W. L. Wagner & Lorence (represented in our sampling by K. cordata Cham. & Schltdl., K. degeneri (Fosberg) W. L. Wagner & Lorence, K. elatior (H. Mann) W. L. Wagner & Lorence, K. flynnii (W. L. Wagner & Lorence) W. L. Wagner & Lorence, K. laxiflora H. Mann, K. littoralis Hillebr., and K. parvula A. Gray) and section Gouldiopsis (Fosberg) W. L. Wagner & Lorence (represented in our sampling by Kadua centranthoides Hook. & Arn. and K. foggiana (Fosberg) W. L. Wagner & Lorence) were recovered. A broader sampling including more Kadua species and more molecular markers is needed to discuss molecular evolution in the light of the seed morphological observations of Terrell et al. (2005). Oldenlandia biflora is sister to the Kadua clade. Its distribution from (sub)tropical Asia to the western Pacific is consistent with the sister relationship to the Polynesian Kadua clade. Our results show that O. biflora can no longer be included within the genus Oldenlandia, but it is necessary to await further studies before transferring it to Kadua or describing a new genus. So far, we have not found morphological characters to support the transfer. HEDYOTIS S. STR. It seems appropriate to restrict the name Hedyotis to the Asian and Micronesian species of the genus, 121 AGATHISANTHEMUM CLADE (CLADE II) The African genus Agathisanthemum is not supported as monophyletic by our analyses. The monotypic African genus Lelya Bremek. is nested within Agathisanthemum, making it paraphyletic as currently circumscribed and suggesting that Lelya should be reduced to Agathisanthemum. This proposal is supported by several palynological characters. Scheltens (1998) showed that Agathisanthemum and Lelya share the same pollen type, characterized by a distinct endocolpus or endocingulum, a mesoporus surrounded by a costa at the inside of the grain (described as a compound ora by Lewis, 1965), and a microreticulate 122 Annals of the Missouri Botanical Garden sexine with granules on the muri facing the lumina (bireticulum). A group of African Oldenlandia species is sister to Agathisanthemum. Two of the three Oldenlandia species, O. angolensis and O. goreensis, belong to Oldenlandia subg. Anotidopsis (Hook. f.) K. Schum. This subgenus, as described by Bremekamp (1952), includes three other putative species of which only O. cephalotes (Hochst.) Kuntze (not included in our sampling) is currently recognized. Subgenus Anotidopsis is distributed in Asia, Australia, and Africa and is characterized by distinctly beaked capsules. The New World taxon O. uniflora is sister to O. angolensis and O. goreensis. More detailed (molecular as well as morphological) studies within the Agathisanthemum clade are needed to evaluate if the three Oldenlandia species, O. angolensis, O. goreensis, and O. uniflora, or the entire Oldenlandia subg. Anotidopsis, are to be transferred to a new genus or if these species are better treated as members of the genus Agathisanthemum. The Asian Hedyotis species are sister to the Agathisanthemum–Oldenlandia clade. This relationship is not unexpected as Bremekamp (1952) already suggested a close relationship between Agathisanthemum and the Asian Hedyotis species (i.e., Hedyotis sect. Diplophragma) based on a similar type of dehiscence of the capsules. Thulin & Bremer, 2004; Dessein et al., 2005a). The close relationship between Gomphocalyx and Phylohydrax is supported by our results and by several morphological characters (amphistomatic leaves, plurizonocolporate pollen, indehiscent fruits, and seeds with a weak, pale exotesta) as shown by Dessein et al. (2005a). Almost all taxa in the Hedyotis–Oldenlandia group have multiovulate ovaries, and the number of pollen apertures rarely exceeds five. The presence of uniovulate ovaries and plurizonocolporate pollen were the main reasons why Gomphocalyx and Phylohydrax were previously included in Spermacoceae s. str., where it is more common than in the rest of the Spermacoceae s.l. tribe, in which 3-colporate pollen predominates (Dessein et al., 2002, 2005b; Dessein, 2003). As mentioned above, the Asian genus Neanotis is a notable exception in having plurizonocolporate pollen grains. The genus also shows a trend toward reduction in the number of seeds per locule. In mature fruits, only one or two seeds are present. However, with no molecular sequence data available for the genus it would be premature to hypothesize a close relationship between Neanotis, Gomphocalyx, and Phylohydrax. A few authors (Capuron, 1973; Piesschaert, 2001) also proposed a close relationship between Gomphocalyx and Lathraeocarpa Bremek., another endemic to Madagascar. Although Lathraeocarpa is not a trailing herb like Gomphocalyx but a (sub)shrub, the two taxa share a calyx with eight lobes, uniovulate ovaries, and plurizonocolporate pollen. The last two characters also support a close relationship between Phylohydrax and Lathraeocarpa. However, several morphological characters distinguish Lathraeocarpa from Gomphocalyx, some of which might even point to an affinity with Triainolepis Hook. f. First, the (sub)shrubby habit of Lathraeocarpa is much more similar to the shrubby habit of Triainolepis than to the herbaceous habit of Gomphocalyx. Second, the pyrene of L. decaryi Bremek. is surrounded by eight strands of thin-walled cells, a condition very similar to that observed in some Triainolepis species (Bremekamp, 1957; Piesschaert, 2001). Likewise, Lathraeocarpa and Triainolepis have a plurilocular ovary and fleshy fruits, whereas Gomphocalyx has a bilocular ovary and dry fruits, which has prompted some authors (Kårehed & Bremer, 2007) to tentatively include Lathraeocarpa in the emended tribe Knoxieae rather than in Spermacoceae s.l. However, we will have to wait until molecular data become available to assess the taxonomic position of Lathraeocarpa with more certainty (Dessein et al., 2005a). Species of Conostomium form a strongly supported clade (JS 5 99, BS 5 99, PP 5 1) together with Oldenlandia herbacea. The type of the genus Conostomium, C. natalense (Hochst.) Bremek., is unre- PENTANOPSIS CLADE Our sampling resulted in a broader concept of the Pentanopsis clade than proposed by Thulin and Bremer (2004). They included Amphiasma, Conostomium (Stapf) Cufod., Manostachya Bremek., Pentanopsis, and Phylohydrax. Oldenlandia affinis was not included in the study of Thulin and Bremer (2004), but it was shown to be closely related to the African genus Amphiasma by Andersson and Rova (1999) and Dessein et al. (2005a). Amphiasma, O. affinis, and Pentanopsis share sessile linear leaves, indistinctly beaked capsules, non-mucilaginous seeds and nonpunctate testa cells (Bremekamp, 1952). However, a detailed study is needed to find more unambiguous morphological characters to support a relation among the three taxa. In the past, Gomphocalyx (a monospecific genus endemic to Madagascar) and Phylohydrax (a genus described in 1986 by Puff to accommodate the East African and Madagascan Hydrophylax L. f. species) were both included in Spermacoceae s. str. based on their uniovulate ovaries and plurizonocolporate pollen grains (Robbrecht, 1988). However, recent molecular studies excluded both genera from Spermacoceae s. str. and suggested that they are closely related to one another and to the Pentanopsis clade (Dessein, 2003; Volume 96, Number 1 2009 Groeninckx et al. Phylogeny of Spermacoceae solved with respect to the other species of Conostomium and to O. herbacea. Both Conostomium and O. herbacea have seeds with coarsely granulate testa cells (Bremekamp, 1952; Dessein, 2003) and pollen that is larger than that of most other genera within the Hedyotis–Oldenlandia group (Bremekamp, 1952; Scheltens, 1998). These characters, however, are homoplasious because granulate testa cells and large pollen grains also occur elsewhere in the Hedyotis– Oldenlandia group. We observed granulate testa cells in Kohautia subg. Pachystigma, O. corymbosa, and O. nematocaulis Bremek., whereas large pollen grains are characteristic of Amphiasma, Gomphocalyx, and Phylohydrax. The most striking feature of Conostomium pollen, namely the short ectocolpi (Scheltens, 1998; Dessein et al., 2005a), is not found in O. herbacea or in most other members of the Pentanopsis clade, but it is reported for Gomphocalyx and Phylohydrax (Dessein et al., 2005a). The last additional species falling in the Pentanopsis clade is Oldenlandia rosulata, an African species named after its basal rosulate leaves. The relationship of O. rosulata to other members of the Pentanopsis clade remains unclear. Despite the strong support for the Pentanopsis clade (JS 5 95, BS 5 95, PP 5 1) in our molecular analyses, the group is not easily morphologically characterized. The only unifying feature for the clade would be what Thulin and Bremer (2004) called basal placentation. Nevertheless, the placentation is not truly basal, but rather axile with the placenta or ovule attached near the base of the septum. Our observations show that this kind of placentation is also found outside the Pentanopsis clade. Moreover, the basal placentation character state is only vaguely defined, and more detailed placentation studies within Spermacoceae s.l. are needed before further conclusions can be drawn about the phylogenetic value of this character. relationship of this monospecific genus to Hedythyrsus and Oldenlandia fastigiata. Our own observations have identified similar placentation types within these taxa. Moreover, Hedythyrsus and Mitrasacmopsis have the same type of capsule dehiscence (loculicidal followed by septicidal dehiscence), seeds with testa cells that show the same undulating radial walls, and pollen with a double reticulum (Groeninckx, 2005). The monospecific genus Dibrachionostylus is sister to a clade of African Oldenlandia species. The genus was separated from Oldenlandia largely on the basis of its capsule dehiscence (both loculicidal and septicidal vs. only loculicidal in Oldenlandia). Bremekamp (1952) closely associated Dibrachionostylus with Agathisanthemum because of their similar fruit dehiscence. However, Dibrachionostylus differs markedly from Agathisanthemum in the pollen aperture morphology (Lewis, 1965). As mentioned above, Agathisanthemum has a distinct ectocolpus, an endocolpus or endocingulum, and a mesoporus surrounded by a costa at the inside of the grain (Lewis, 1965). Pollen grains of Dibrachionostylus are also 3-colporate but do not have a costa on the inside (Lewis, 1965). The apertures of Dibrachionostylus pollen are, therefore, more similar to those of Amphiasma, Oldenlandia, and Pentodon (Lewis, 1965). Nesohedyotis is another monospecific genus previously included in the Hedyotideae. Its only species, N. arborea, shows a superficial resemblance to the East African genus Hedythyrsus; specimens of both taxa turn black when dried, and their leaf shape and inflorescence structure are similar (Bremekamp, 1952). However, our results show that Nesohedyotis is more closely related to the former tribes Spermacoceae s. str. and Manettieae than to members of the Hedyotis–Oldenlandia group. Nesohedyotis has unisexual flowers, which are unusual among Spermacoceae, and, in contrast to Hedythyrsus, its fruits open by a single loculicidal split. Although it is one of the more common endemic species on St. Helena, its small population size and small geographical distribution make Nesohedyotis Endangered (EN) according to IUCN Red List criteria (IUCN, 2001). According to Verdcourt (1976), the monospecific Tanzanian Pseudonesohedyotis Tennant, which is not included in our sampling, is closely related to Nesohedyotis and Hedythyrsus. Pseudonesohedyotis has indeed the same leaf shape and inflorescence structure as the latter two taxa. In habit and distribution, however, it resembles Hedythyrsus more than Nesohedyotis. Both Pseudonesohedyotis and Hedythyrsus are (sub)shrubs, whereas Nesohedyotis is a small tree. Moreover, Pseudonesohedyotis differs from Nesohedyotis in having hermaphroditic flowers. Again, it is necessary to wait until molecular data become available to discuss the taxonomic position of Pseudonesohedyotis with more confidence. MONOSPECIFIC GENERA WITHIN THE HEDYOTIS– OLDENLANDIA GROUP Besides the genus Gomphocalyx of the Pentanopsis clade, the Hedyotis–Oldenlandia group comprises several other monospecific genera. These monospecific genera often have several peculiar characters, making it very difficult to discuss their relationship with other Spermacoceae. In our sampling, for example, the Afro-Madagascan genus Mitrasacmopsis has seeds with undulating radial exotesta cell walls, distinctly stalked placentas with ovules positioned on the periphery of the placental tissue, pollen grains with a double reticulum, and fruits with a conspicuous beak (Groeninckx et al., 2007). Our molecular results suggest a close 123 124 Annals of the Missouri Botanical Garden Based on the presence of an apparently superior ovary, Jovet (1941) originally placed Mitrasacmopsis and Astiella Jovet, another monospecific genus of the Hedyotis–Oldenlandia group endemic to Madagascar (not included in this study), within Loganiaceae– Spigelieae. Members of Rubiaceae are generally characterized by the presence of an inferior ovary. Groeninckx et al. (2007) demonstrated that flowers of Mitrasacmopsis are initially epigynous with inferior ovaries. Expansion of the upper part of the ovary in fruiting stage results in a change in the ovary position of Mitrasacmopsis from basically inferior to secondarily semi-inferior. The same kind of fruit development also most likely occurs in Astiella. In her morphological study of the Rubioideae, Hayden (1968) stated that some genera of Spermacoceae s. str. have semiinferior fruits. According to Robbrecht (1988), this statement is based on the strong expansion of the top of the nectary disc in the fruiting stage. However, we have not observed semi-inferior ovaries within Spermacoceae s. str. Nevertheless, within Spermacoceae s.l. several other taxa, apart from Mitrasacmopsis and Astiella, are characterized by the presence of a beak at fruit stage (Conostomium spp., Hedythyrsus spp., Kohautia spp., Oldenlandia spp.). These beaks are not remnants of the nectary disc and probably originate in a similar way as in Mitrasacmopsis. However, the ovaries of these species do not undergo a remarkable reverse in shape in the fruiting stage as observed in Mitrasacmopsis and Astiella. Based on their fruit shape, Mitrasacmopsis and Astiella seem closely related. However, Jovet (1941) also suggested a close relationship between Astiella and the Asian Anotis DC. species, presently classified in the genus Neanotis (Lewis, 1966). Astiella differs from both genera in having only two calyx lobes, a character that so far has not been observed within the Hedyotis– Oldenlandia group, and uniovulate locules. Molecular sequence data of Astiella will allow us to discover the taxonomic position of the genus in the future. Other monospecific genera of the Hedyotis–Oldenlandia group are Carterella, Dolichometra K. Schum., Lelya, Leptoscela Hook. f., Lucya, Phyllocrater Wernham, Polyura Hook. f., Stephanococcus Bremek., and Oldenlandiopsis. The genera Carterella, Lelya, Lucya, and Oldenlandiopsis were already discussed in previous sections. To date, the taxonomic position of most of these monospecific genera remains controversial because molecular data are lacking. Egypt, and throughout most of Africa south of the Sahara (including Socotra, Cape Verde, and Madagascar). The genus can easily be distinguished from other representatives of the Hedyotis–Oldenlandia group by its unique flower morphology. The anthers and stigma are always included, with the stigma held well below the anthers or occasionally just touching them. This monomorphic short-styled condition is, with the exception of a few individuals of Conostomium, unique among the African members of the former tribe Hedyotideae. For this reason, Kohautia has always been considered a distinct genus (Bremekamp, 1952; Mantell, 1985). Our molecular results, however, show that the two subgenera of Kohautia are not sister clades. Subgenus Kohautia is sister to the Pentanopsis clade, whereas subgenus Pachystigma is sister to an Oldenlandia clade containing the type species O. corymbosa. Despite the unifying floral architecture, there are numerous morphological differences between the two subgenera (Lewis, 1965; Mantell, 1985). The number of stigmatic lobes is the most striking diagnostic character that allows identification of the subgenera even in the field. Members of subgenus Kohautia have styles with two thin filiform stigma lobes, whereas Pachystigma is characterized by the presence of only a single, ovoid to cylindrical stigma lobe. Seeds are also different in the two subgenera: subgenus Kohautia seeds are angular-conic to subconic in shape with 5- or 6-angled testa cells, whereas in subgenus Pachystigma the seeds are rounded with wavy and punctate testa cells. Pollen of Kohautia can also be divided into two easily recognizable groups coinciding with the two subgenera (Lewis, 1965). Other differences between the two subgenera are found in floral architecture and chromosome number. Based on these differences, Mantell (1985) hypothesized that the two subgenera may have diverged and developed independently of one another fairly early on and she even tentatively proposed the elevation of the two subgenera to generic rank. At that time, Mantell decided to maintain a widely defined genus Kohautia, mainly for practical reasons. However, our molecular data now clearly support the recognition of two genera. Sampling within the genus still needs to be improved before proposing new generic circumscriptions. KOHAUTIA Kohautia is a genus of 31 species (Mantell, 1985) distributed from the Indian subcontinent through Pakistan, Iran, the Arabian Peninsula, Sinai, eastern OLDENLANDIA Govaerts et al. (2006) currently accept 76 Oldenlandia species from Africa, 155 from Asia and Australia, 23 from America, and eight from the Pacific Islands. However, as documented in previous molecular studies (Bremer, 1996; Andersson & Rova, 1999; Volume 96, Number 1 2009 Groeninckx et al. Phylogeny of Spermacoceae 125 Bremer & Manen, 2000), Oldenlandia is shown to be polyphyletic. Bremekamp (1952) divided the 61 species that he recognized from Africa into 16 subgenera. Our results do not support the majority of these subgenera. Only the subgenus Hymenophyllum Bremek. (Oldenlandia echinulosa and O. nervosa) and subgenus Anotidopsis (O. angolensis and O. goreensis) are corroborated. The type species, Oldenlandia corymbosa, is sister to a clade with the African species O. capensis L. f., O. robinsonii Pit., O. nematocaulis, O. taborensis Bremek., and O. wauensis Schweinf. ex Hiern. The last species, O. wauensis, was segregated by Bremekamp (1952) in a new genus Thecorchus Bremek., which he proposed to be allied with Otomeria of the tribe Knoxieae because of its distinctly elongated capsules and equal number of tetramerous and pentamerous flowers. However, Kårehed and Bremer (2007) showed that Thecorchus is not related to Otomeria but is close to Oldenlandia. Our results, which place Thecorchus in a clade comprised of the type species of Oldenlandia, support the transfer of T. wauensis (Schweinf. ex Hiern) Bremek. back into Oldenlandia. The type species O. corymbosa and O. capensis belong to Bremekamp’s (1952) subgenus Oldenlandia K. Schum. Besides these two species, subgenus Oldenlandia also includes O. fastigiata and O. herbacea. These species are apparently not related to O. corymbosa and its allies. Oldenlandia fastigiata is sister to Hedythyrsus and Mitrasacmopsis, whereas O. herbacea in the Pentanopsis clade is sister to a paraphyletic Conostomium. Bremekamp (1952) already pointed out that O. herbacea differs from the rest of the subgenus by the coarsely granulated walls of the testa cells, the rather large flowers, and the slender corolla tube. The Australian species of Oldenlandia, O. mitrasacmoides and O. galioides, sampled here belong to a clade comprising the Australian Synaptantha tillaeacea, the Austro-Asian O. tenelliflora, the African species O. lancifolia, and the Kadua species (including O. biflora). Oldenlandia mitrasacmoides is sister to the rest of the clade. Synaptantha tillaeacea is sister to a clade with Oldenlandia tenelliflora, O. galioides, and O. lancifolia. Synaptantha Hook. f. may be distinguished from the other genera in the clade by its slightly connate corolla lobes, stamens with filaments attached to both the corolla and the ovary, depressed obconic or ovoid seeds, and half-inferior ovaries (Halford, 1992). In his review of Australian Oldenlandia, Halford (1992) distinguished five groups mostly based on seed morphology. Oldenlandia galioides and O. tenelliflora are placed together in his group one, which is characterized by obconic seeds that are slightly laterally compressed and obtriangular in outline. Oldenlandia mitrasacmoides belongs to his group two, which is characterized by scutelliform seeds that are oblong or broadly elliptic in outline, with the hilum situated on a conspicuous central ridge. The African species O. lancifolia has seeds similar in shape to those of its sister O. galioides (Dessein, 1998). Not all American Oldenlandia species included in our sampling are placed within the Arcytophyllum– Houstonia clade (see discussion above). The remaining South American species of Oldenlandia, O. salzmannii and O. tenuis, form a clade sister to the former tribes Spermaococeae s. str. and Manettieae. Terrell (1990) already reported that O. salzmannii is clearly distinct from Houstonia and Oldenlandia. In contrast to other Oldenlandia species, O. salzmannii does not have the typical oldenlandioid seeds or base number of chromosomes (n 5 15 instead of 9). Moreover, it shares some unusual characters with Oldenlandiopsis: stipules are minute, not more than 0.5 mm long (Oldenlandia stipules are often 2–3 mm long); few stiff hairs occur on the leaves (Oldenlandia species usually have smaller, softer hairs); and it has a creeping habit (which is rare in Oldenlandia, the usual habit being erect to spreading or prostrate). It would be very informative to include Oldenlandiopsis in future studies to investigate its relationship to either O. microtheca (see discussion of the Arcytophyllum–Houstonia clade above) or O. salzmannii. FUTURE RESEARCH PLANS AND CONCLUSIONS Although our analyses found well-supported clades within Spermacoceae s.l., many relationships within and between these clades still remain unresolved. Furthermore, many relationships detected here are contradictory to previous taxonomic treatments and await morphological backup. This study was a multipartner collaboration resulting in a framework for future Spermacoceae research. Further studies will focus on obtaining additional DNA markers (i.e., nuclear DNA data) to provide better resolution within the tribe. Besides improving the character sampling, we also need to balance the taxon sampling by including more Asian and American taxa. In addition, concerted studies will focus on the morphological characterization of monophyletic groups within Spermacoceae. This requires a morphological investigation across taxa to find character support for the many new phylogenetic relationships detected. Literature Cited Andersson, L. & J. H. E. Rova. 1999. 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D. dioeca Airy Shaw atpB-rbcL rps16 intron trnL-trnF Zambia, Dessein et al. 671 (BR) Zambia, Dessein et al. 201 (BR) EU542917* EU542918* EU543018* EU543019* EU543077* EU543078* Angola, Kers 3350 (S) Zambia, Dessein et al. 1167 (BR) EU542919* EU542920* AF002753(1) EU543020* EU543079* EU543080* Ecuador, Hekker & Hekking 10335 (GB) Ecuador, Øllgaard et al. 58395 (NY) unknown, Edwin et al. 3624 (S) Costa Rica, Cronquist 8827 (NY) Peru, Wurdack 1073 (NY) Colombia, Andersson et al. 2195 (GB) Venezuela, Pipoly et al. 6467 (GB) Ecuador, Harling & Andersson 22232 (GB) Mexico, Stafford et al. 203 (MO) Colombia, Andersson et al. 2196 (GB) Ecuador, Ståhl 4481 (GB) – – – – – EU542921* – EU542922* – – EU542923* AF333348(2) AF333350(2) AF333352(2) AF333354(2) AF333356(2) AF002754(1) AF333359(2) AF333362(2) AF333364(2) AF002755(1) AF333366(2) AF333349(2) AF333351(2) AF333353(2) AF333355(2) AF333357(2) EU543081* – AF333363(2) – AF333365(2) EU543082* cult., Forbes s.n. (S) unknown, Van Caekenberghe 264 (cult. at BR) Mexico, Spencer et al. 363 (NY) EU542925* – EU543022* AF002758(1) EU543084* – – – EU642537* South Africa, Dahlstrand 1346 (GB) South Africa, Bremer et al. 4341 (UPS) Ethiopia, Puff & Kelbessa 821222 (UPS) South Africa, Bremer et al. 4331 (UPS) – EU542927* EU542928* EU542929* AF002760(1) – EU543024* – EU543085* – EU543086* EU543087* Guatemala, Gustafsson et al. 215 (GB) Mexico, Pringle 3852 (S) EU542930* EU542931* – EU543025* EU543088* EU543089* – – EU543090* Voucher information Australia, Harwood 1559 (BR) Annals of the Missouri Botanical Garden Agathisanthemum Klotzsch A. bojeri Klotzsch A. globosum (Hochst. ex A. Rich.) Klotzsch Amphiasma Bremek. A. benguellense (Hiern) Bremek. A. luzuloides (K. Schum.) Bremek. Arcytophyllum Willd. ex Schult. & Schult. f. A. aristatum Standl. A. ciliolatum Standl. A. ericoides (Willd. ex Roem. & Schult.) Standl. A. lavarum K. Schum. A. macbridei Standl. A. muticum (Wedd.) Standl. A. nitidum (Kunth) Schltdl. A. rivetii Danguy & Cherm. A. serpyllaceum (Schltdl.) Terrell A. setosum (Ruiz & Pav.) Schltdl. A. thymifolium (Ruiz & Pav.) Standl. Bouvardia Salisb. B. glaberrima Engelm. B. ternifolia (Cav.) Schltdl. 128 Appendix 1. List of taxa used in the phylogenetic analyses with voucher information (geographic origin, collector, collector number, herbarium), accession numbers, and literature citations from previously published sequences for the three plastid markers atpB-rbcL, rps16 intron, and trnL-trnF: (1) Andersson & Rova, 1999; (2) Andersson et al., 2002; (3) Dessein et al., 2005a. New sequences are marked with an asterisk. Missing sequences are marked with a dash. Taxon rps16 intron trnL-trnF EU542932* AF333370(2) EU543091* Kenya, Strid 2598 (GB) EU542933* AF002761(1) – Kenya, Luke 9029 (UPS) French Guiana, Anderson et al. 2071 (GB) French Guiana, Anderson et al. 1961 (GB) EU542934* – EU542935* EU543026* AF002762(1) EU543027* EU543092* – EU543093* Trinidad, Hummel s.n. (GB) EU542936* AY764289(3) EU543094* Cuba, Rova et al. 2286 (GB) EU542937* AF002763(1) EU543095* Argentina, Vanni & Radovancick 996 (GB) Argentina, Schinini & Cristobal 9811 (GB) EU542938* EU542939* AY764290(3) EU543028* EU543096* EU543097* – AY764291(3) – Hong Kong, Shiu Ying Hu 10821 (S) Sri Lanka, Larsson & Pyddoke 22 (S) Caroline Islands, Fosberg 47697 (S) Sri Lanka, Wambeek & Wanntorp 2996 (S) Sri Lanka, Klackenberg 413 (S) Sri Lanka, Fagerlind 3668 (S) Malaysia, Sabah, Wallander 6 (GB) Sri Lanka, Bremer et al. 163 (S) Sri Lanka, Fagerlind 5082 (S) South India, Klackenberg & Lundin 03 (S) EU542941* EU542942* EU542943* EU542944* EU542945* EU542946* EU542947* EU542948* EU542949* EU542950* – – – – EU543029* EU543030* AF002767(1) – – EU543031* – EU543098* EU543099* – EU543100* EU543101* EU543102* EU543103* EU543104* EU543105* Zambia, Dessein et al. 1017 (BR) EU542951* EU543032* EU543107* French Guiana, Andersson et al. 2040 (GB) EU542952* – EU543108* U.S.A., Vincent & Lammers s.n. (GB) U.S.A., Yatskievych 96-49 (MO) U.S.A., Weigend 9963 (NY) EU542953* EU542954* – AF333379(2) AF002766(1) – EU543109* – EU642536* Voucher information Madagascar, De Block et al. 569 (BR) 129 atpB-rbcL Australia, Andersson 2262 (GB) Groeninckx et al. Phylogeny of Spermacoceae D. repens (L.) J. R. Forst. & G. Forst. Dibrachionostylus Bremek. D. kaessneri (S. Moore) Bremek. Diodia L. as traditionally delimited D. aulacosperma K. Schum. D. sarmentosa Sw. D. spicata Miq. Emmeorhiza Pohl ex Endl. E. umbellata (Spreng.) K. Schum. Ernodea Sw. E. littoralis Sw. Galianthe Griseb. G. brasiliensis (Spreng.) E. L. Cabral & Bacigalupo G. eupatorioides (Cham. & Schltdl.) E. L. Cabral Gomphocalyx Baker G. herniarioides Baker Hedyotis L. H. consanguinea Hance H. fruticosa L. H. korrorensis (Valeton) Hosok. H. lawsoniae Wight H. lessertiana Arn. var. lassertiana Thwaites H. lessertiana var. marginata Thwaites & Trimen H. macrostegia Stapf H. quinquinervis Thwaites H. rhinophylla Thwaites ex Trimen H. swertioides Hook. f. Hedythyrsus Bremek. H. spermacocinus (K. Schum.) Bremek. Hemidiodia K. Schum. H. ocymifolia (Willd. ex Roem. & Schult.) K. Schum. Houstonia L. H. caerulea L. H. longifolia Gaertn. Volume 96, Number 1 2009 Appendix 1. Continued. 130 Appendix 1. Continued. Taxon Kadua Cham. & Schltdl. K. acuminata Cham. & Schltdl. K. affinis Cham. & Schltdl. K. axillaris (Wawra) W. L. Wagner & Lorence K. K. K. K. K. K. K. atpB-rbcL rps16 intron trnL-trnF U.S.A., Hawaii, cult. at BR U.S.A., Hawaii, Motley 1733 (NY) U.S.A., Hawaii, Harrison-Gagne s.n. (GB) U.S.A., Hawaii, Maui, Motley 1724 (NY) U.S.A., Hawaii, Skottsberg 6788 (S) cult., Lorence 8021 (PTBG) U.S.A., Hawaii, Motley 1703 (NY) cult., Wood 5062 (PTGB) U.S.A., Hawaii, Kauai, Wagner 6350 (BISH) U.S.A., Hawaii, Oahu, Motley 1747 (NY) U.S.A., Hawaii, Kauai, Perlman 15631 (BISH) EU542955* – – – EU542956* EU542957* – EU542958* – – – – EU642523* AF002765(1) EU642524* EU543033* AF333376(2) EU642525* AF333371(2) EU642526* EU642527* EU642528* EU543110* EU642538* – EU642535* EU543111* EU543112* EU642539* EU543113* EU642540* EU642541* EU642542* U.S.A., Hawaii, Sparre 27 (S) U.S.A., Hawaii, Oahu, Motley 1677 (NY) EU542959* – – EU642529* EU543114* EU642543* U.S.A., Hawaii, Molokai, Perlman 6647 (BISH) U.S.A., Hawaii, Molokai, Kiehn & Luegmayr 920823 (WU) cult., Perlman 12783 (GB) Rapa Island, French Polynesia, Perlman 17953 (NY) – EU542960* EU542961* – EU642530* EU543034* AF333375(2) EU642531* EU642544* EU543115* EU543116* EU642545* South Africa, Bremer et al. 4307 (UPS) Zambia, Dessein et al. 432 (BR) Zambia, Dessein et al. 751 (BR) South Africa, Dessein et al. 469 (BR) Zambia, Dessein et al. 1149 (BR) Kenya, Luke 9035 (UPS) Burkina Faso, Madsen 5940 (NY) Zambia, Dessein et al. 470 (BR) Madagascar, De Block et al. 539 (BR) EU542962* EU542963* EU542964* EU542965* EU542966* EU542967* – EU542968* EU542969* EU543035* EU543036* EU543037* EU543038* EU543039* EU543040* – EU543041* – EU543117* EU543118* EU543119* EU543120* EU543121* EU543122* EU642546* EU543123* EU543124* Tanzania, Gereau 2513 (BR) EU542970* – EU543125* French Guiana, Andersson et al. 1917 (GB) Colombia, Andersson et al. 2128 (GB) EU542971* EU542972* AF002768(1) AF002769(1) – EU543126* Annals of the Missouri Botanical Garden centranthoides Hook. & Arn. cordata Cham. & Schltdl. coriacea (J. E. Smith) W. L. Wagner & Lorence degeneri (Fosberg) W. L. Wagner & Lorence elatior (H. Mann) W. L. Wagner & Lorence fluviatilis C. N. Forbes flynnii (W. L. Wagner & Lorence) W. L. Wagner & Lorence K. foggiana (Fosberg) W. L. Wagner & Lorence K. fosbergii (W. L. Wagner & D. R. Herbst) W. L. Wagner & Lorence K. laxiflora H. Mann K. littoralis Hillebr. K. parvula A. Gray K. rapensis F. Br. Kohautia Cham. & Schltdl. K. amatymbica Eckl. & Zeyh. K. caespitosa Schnizl. K. coccinea Royle K. cynanchica DC. K. microcala Bremek. K. obtusiloba (Hiern) Bremek. K. senegalensis Cham. & Schltdl. K. subverticillata (K. Schum.) D. Mantell K. virgata (Willd.) Bremek. Lelya Bremek. L. osteocarpa Bremek. Manettia Mutis ex L. M. alba (Aubl.) Wernham M. lygistum (L.) Sw. Voucher information Taxon rps16 intron trnL-trnF Zambia, Dessein et al. 265 (BR) EU542973* EU543042* EU543127* French Guiana, Andersson et al. 1995 (GB) Guiana, Jansen-Jacobs et al. 4785 (GB) EU542974* EU542975* AF002770(1) EU543044* EU543128* – Zambia, Dessein et al. 1273 (BR) EU542976* EU543045* EU543129* – AF003607(1) – EU542977* EU542978* EU542979* EU542980* EU542981* EU542982* EU542983* EU542984* EU542985* EU542986* EU542987* EU542988* EU542989* EU542990* EU542991* EU542992* EU542993* EU542994* – – – EU542995* EU542996* EU542997* EU542998* EU543046* EU543047* – EU543048* EU543049* EU543050* EU543051* – EU543052* EU543053* EU543054* EU543055* EU543056* EU543057* EU543058* EU543059* – EU543060* AF333382(2) EU543061* EU543043* AY764294(3) – EU543062* AY764293(3) EU543130* EU543131* EU543132* EU543133* EU543134* EU543135* EU543136* EU543137* EU543138* EU543139* EU543140* EU543141* EU543142* EU543143* EU543144* EU543145* EU543146* – – EU543147* – EU543148* EU543149* EU543106* – cult., Chase 2915 (K) Zambia, Dessein et al. 627 (BR) Zambia, Dessein et al. 932 (BR) Japan, Van Caekenberghe 63 (cult. at BR) Zambia, Dessein et al. 843 (BR) Tanzania, Kayombo et al. s.n. (BR) Zambia, Dessein et al. 487 (BR) Zambia, Dessein et al. 928 (BR) Tanzania, Kayombo & Kahemela 1993 (BR) Zambia, Dessein et al. 1019 (BR) Australia, Harwood 1511 (BR) Zambia, Dessein et al. 935 (BR) Zambia, Dessein et al. 1286 (BR) Zambia, Dessein et al. 442 (BR) Zambia, Dessein et al. 463 (BR) Zambia, Dessein et al. 1356 (BR) Mexico, Frödeström & Hultén 681 (S) Australia, Harwood 1516 (BR) Zambia, Dessein et al. 924 (BR) Gabon, Andersson & Nilsson 2326 (GB) Zambia, Dessein et al. 346 (BR) Zambia, Dessein et al. 1197 (BR) Brazil, Harley 15514 (UPS) Tanzania, Bidgood et al. 4015 (BR) Japan, Van Caekenberghe 70 (cult. at BR) Guyana, Jansen-Jacobs et al. 41 (UPS) 131 atpB-rbcL Voucher information Groeninckx et al. Phylogeny of Spermacoceae Manostachya Bremek. M. ternifolia E. S. Martins Mitracarpus Zucc. ex Schult. & Schult. f. M. frigidus (Willd. ex Roem. & Schult.) K. Schum. M. microspermus K. Schum. Mitrasacmopsis Jovet M. quadrivalvis Jovet Nesohedyotis (Hook. f.) Bremek. N. arborea (Roxb.) Bremek. Oldenlandia L. O. affinis (Roem. & Schult.) DC. O. angolensis K. Schum. O. biflora L. O. capensis L. f. var. capensis O. capensis var. pleiosepala Bremek. O. corymbosa L. O. echinulosa K. Schum. O. echinulosa K. Schum. var. pellucida (Hiern) Verdc. O. fastigiata Bremek. O. galioides (F. Muell.) F. Muell. O. geophila Bremek. O. goreensis (DC.) Summerh. O. herbacea (L.) Roxb. var. goetzei Bremek. O. herbacea (L.) Roxb. var. herbacea O. lancifolia (Schumach.) DC. O. microtheca (Cham. & Schltdl.) DC. O. mitrasacmoides F. Muell. O. nematocaulis Bremek. O. nervosa Hiern O. robinsonii Pit. O. rosulata K. Schum. O. salzmannii (DC.) Benth. & Hook. f. ex B. D. Jacks. O. taborensis Bremek. O. tenelliflora (Blume) Kuntze O. tenuis K. Schum. Volume 96, Number 1 2009 Appendix 1. Continued. 132 Appendix 1. Continued. Taxon U.S.A., Godfrey 57268 (GB) Ethiopia, Friis et al. 2560 (UPS) Kenya, Luke & Luke 8362 (UPS) atpB-rbcL rps16 intron trnL-trnF EU542999* EU543017* EU543000* (3) AY764295 EU543076* EU543063* EU543150* EU543168* EU543151* – EU543065* EU543153* Zambia, Dessein et al. 598 (BR) EU543002* EU543066* EU543154* South Africa, Bremer 3783 (UPS) Madagascar, De Block et al. 640 (BR) EU543003* EU543004* EU543067* AY764292(3) – EU543155* Colombia, Andersson et al. 2073 (GB) Australia, Egerod 85343 (GB) EU543005* EU543006* AF003614(1) EU543068* EU543156* EU543157* French Guiana, Andersson 1908 (GB) Colombia, Andersson et al. 2074 (GB) Australia, Harwood 1148 (BR) Madagascar, De Block et al. 794 (BR) Kenya, Luke 9022 (UPS) Sri Lanka, Wanntorp et al. 2667 (S) Colombia, Andersson et al. 2078 (GB) French Guiana, Andersson et al. 2016 (GB) Gabon, Andersson & Nilsson 2296 (GB) EU543007* – EU543008* EU543010* EU543009* EU543011* EU543012* EU543013* EU543014* EU543069* AF003619(1) EU543070* EU543072* EU543071* EU543073* – – EU543074* EU543158* – EU543159* EU543161* EU543160* EU543162* EU543163* EU543164* EU543165* U.S.A., Yatskievych 96-92 (MO) EU543015* AF333373(2) EU543166* Australia, Lazarides & Palmer 272 (K) EU543016* EU543075* EU543167* Zambia, Dessein et al. 264 (BR) EU542924* EU543021* EU543083* Madagascar, De Block et al. 578 (BR) EU542926* EU543023* – Zambia, Dessein et al. 678 (BR) EU543001* EU543064* EU543152* Ethiopia, Gilbert et al. 7458 (UPS) Annals of the Missouri Botanical Garden O. uniflora L. O. wauensis Schweinf. ex Hiern O. wiedemannii K. Schum. Pentanopsis Rendle P. fragrans Rendle Pentodon Hochst. P. pentandrus (K. Schum. & Thonn.) Vatke Phylohydrax Puff P. carnosa (Hochst.) Puff P. madagascariensis (Willd. ex Roem. & Schult.) Puff Richardia L. R. scabra L. R. stellaris (Cham. & Schltdl.) Steud. Spermacoce L. S. capitata Ruiz & Pav. S. confusa Rendle ex Gillis S. erosa Harwood S. flagelliformis Poir. S. filituba (K. Schum.) Verdc. S. hispida L. S. prostrata Aubl. S. remota Lam. S. ruelliae DC. Stenaria (Raf.) Terrell S. nigricans (Lam.) Terrell Synaptantha Hook. f. S. tillaeacea (F. Muell.) Hook. f. OUTGROUP TAXA Batopedina Verdc. B. pulvinellata Robbr. Carphalea Juss. C. madagascariensis Lam. Pentanisia Harv. P. parviflora Stapf ex Verdc. Voucher information