Phylogeny and classification of the species-rich pantropical showy genus Ixora (Rubiaceae-Ixoreae) with indications of geographical monophyletic units and hybrids

Arnaud Mouly

Ixora predeepii Balan & S.Harikr. and Ixora sivarajiana Pradeep are synonymised under Ixora cuneifolia Roxb. and Ixora notoniana Wall. ex G.Don respectively. Lectotypes are designated for the names Ixora nigricans R.Br. ex Wight & Arn. and Ixora undulata Roxb. ex Sm. A second step lectotype is designated for the name Ixora cuneifolia Roxb.

American Journal of Botany 96(3): 686–706. 2009. PHYLOGENY AND CLASSIFICATION OF THE SPECIES-RICH PANTROPICAL SHOWY GENUS IXORA (RUBIACEAE-IXOREAE) WITH INDICATIONS OF GEOGRAPHICAL MONOPHYLETIC UNITS AND HYBRIDS1 Arnaud Mouly,2,3 Sylvain G. Razafimandimbison,3 Anbar Khodabandeh,3 and Birgitta Bremer3 2UMS 2700 CNRS—USM 0602 MNHN: Taxonomie et Collections, Département de Systématique et Évolution, Muséum national d’Histoire naturelle, 16 rue Buffon 75005 Paris, France; 3 Bergius Foundation, the Royal Swedish Academy of Sciences and Botany Department, Stockholm University, SE-106 91 Stockholm, Sweden Species-rich genera often have various conflicting circumscriptions from independent regional flora treatments. Testing the monophyly of these groups of plants is an important step toward the establishment of a phylogenetic classification. The genus Ixora of the tribe Ixoreae in the subfamily Ixoroideae (coffee family or Rubiaceae) is a species-rich pantropical genus of ca. 500 species. Phylogenetic analyses of Ixoreae based on combined sequence data from one nuclear (nrETS) and two chloroplast (rps16 and trnT-F) markers reveal the paraphyly of Ixora as presently delimited and also show that the tribe can be subdivided into three major clades: the Mascarene/neotropical/Malagasy/African clade, the Pacific clade, and the Asian clade. Given the lack of morphological synapomorphies supporting the different Ixora clades and the morphological consistency of the ingroup taxa, we propose a broad circumscription of Ixora including all its satellite genera: Captaincookia, Doricera, Hitoa, Myonima, Sideroxyloides, Thouarsiora, and Versteegia. The current infrageneric classification of Ixora is not supported. The different Ixora subclades represent geographical units. Nuclear and chloroplast tree topologies were partially incongruent, indicating at least four potential natural hybridization events. Other conflicting positions for the cultivated species are most likely due to anthropogenic hybridization. Key words: hybridization; Ixora; molecular phylogenetics; nrETS; ornamentals; rps16; Rubiaceae; taxonomy; trnT-F. The pantropical Ixora (Appendix S1, A–J, see Supplemental Data with online version of this article) is probably the second largest Rubiaceae genus after Psychotria, with the highest species diversity (at least 200 species) in tropical Asia (De Block, 1998; Puff et al., 2005). Approximately 60 species are restricted to the neotropics and mid-Atlantic islands (Andersson, 1992), 22 species on the Fiji Islands (Smith and Darwin, 1988), ca. 34 species in continental Africa (De Block, 1998), and probably as many in Madagascar (De Block et al., 2007). The genera Pavetta and Ixora were traditionally treated as a single genus since Linnaeus’ (1753) descriptions and until the beginning of the 20th century (Schumann, 1891; Bremekamp, 1934, 1937b). However, Ixora can easily be distinguished from Pavetta by its salverform bifid stigmas and can additionally be characterized by a combination of the following characters: articulate petioles, hermaphroditic flowers, hypocrateriform corollas, two-carpellate ovaries containing a solitary ovule in each carpel locule, and drupaceous fruits (De Block, 1998; Lorence et al., 2007; Fig. 1, E3, E4, E7, E9). Andreasen and Bremer’s (2000) study based on combined data from rbcL sequences and morphological data demonstrated that the two genera are not closely related. Accordingly, the tribe Pavetteae A.Rich. was restricted to Pavetta and its allied genera (e.g., Tarenna Gaertn., Leptactina Hook.f.); plus, the reinstatement of the tribe Ixoreae A.Gray (Gray, 1858) was necessary to accommodate Ixora and its satellite genera (see Table 1). Several monotypic and small genera with apparent affinities to Ixora were described based on one or two autapomorphic characters (see Table 1). The neotropical genus Sideroxyloides Jacq. (Jacquin, 1763; Bentham, 1850) was established on the basis of its caulinary inflorescences. The Pacific Hitoa Nadeaud (Nadeaud, 1899; Fig. 1, E10) and the Malagasy Thouarsiora Homolle ex Arènes (Arènes, 1960), both monotypic genera, The mainly pantropical Rubiaceae (coffee family) is the fourth largest flowering plant family, which comprises many species-rich genera with more than 200 species (e.g., Psychotria L. with ca. 1800 species [Govaerts et al., 2006]; Ixora L. with at least 500 species [De Block, 1998; Mouly and Hoang, 2007]; Oldenlandia L. and Spermacoce Gaertn. each with at least 250 species [Govaerts, et al., 2006]; Pavetta L. and Tarenna Gaertn., each with ca. 200 species [Mabberley, 1997]). Testing the monophyly of these large genera is an important step toward the establishment of a phylogenetic classification. On the other hand, this task has proven to be challenging for at least two reasons: (1) difficulty in obtaining a sufficiently representative sampling of their species for molecular investigations, and (2) lack of modern floras or taxonomic treatments. 1 Manuscript received 10 July 2008; revision accepted 5 December 2008. The authors thank Dr. P. De Block, F. Jacq, Dr. J.-F. Butaud, Dr. J.-Y. Meyer, and Dr. J. Munzinger for providing Ixora material for biomolecular studies; two anonymous reviewers for comments on the manuscript; the authorities of the North and South provinces of New Caledonia and the French Polynesian Government for access to the field and authorization to collect specimens and the tribal authorities who allowed us to work on their territories; the institutions of BR, G, K, L, MO, NOU, P, S, TAN, TEF, and UPS for granting access to herbarium collections and providing material for bio-molecular studies. Financial support was provided by the UMS CNRS 2700 – USM MNHN 0602 “Taxonomie et Collections”, the Department “Systématique et Evolution”, MNHN, Paris; the Missouri Botanical Garden through Dr. G. McPherson for support of fieldwork in New Caledonia; the European Commission’s Research Infrastructure Action via the SYNTHESYS Project (EU) to A.M. to access BR and L; the Swedish Research Council and the Knut and Alice Wallenberg Foundation to B.B. 4 Author for correspondence (e-mail: arnaud@bergianska.se) doi:10.3732/ajb.0800235 686 March 2009] Mouly et al.—Phylogeny of IXORA (Rubiaceae) 687 Fig. 1. Illustrations of morphological features in the tribe Ixoreae commonly used for Ixora satellite genera distinction and infrageneric classification within Ixora. (A) I. yaouhensis, inflorescence with subopposite and nonarticulate axes (typical of subgenus Pavettoides); (B) I. crassipes, inflorescence with opposite and articulate axes (typical of subgenus Ixora); (C) I. cauliflora, caulinary inflorescence; (D) I. collina, inflorescence triflorous, protected by foliaceous bracts (typical of section Phylleilema); (E) flowers (1–5) and fruit (6–11) details: 1, male, Myonima obovata; 2, female, Myonima obovata; 3, hermaphrodite, I. chinensis; 4, hermaphrodite, I. coccinea; 5, hermaphrodite, Captaincookia sp. 1 (typical of Captaincookia); 6, Captaincookia margaretae; 7, section, I. coccinea; 8, section, Versteegia cauliflora; 9, section, I. francii; 10, section, Hitoa mooreensis; 11, section, Myonima obovata. were independently described based on their four-carpellate ovaries. The Mascarene genus Myonima, presently consisting of four species of shrubs, was established based on its short corolla tubes (shorter than lobes), 2–7-carpellate ovaries (Fig. 1 E10), and functional dioecious flowers (Verdcourt, 1989; Fig. 1, E1, E2). The genus was once placed among many others under synonymy of Ixora (Baillon, 1879), but no Rubiaceae systematists seem to have accepted this taxonomic decision. Versteegia Valeton was described to accommodate three New Guinean species with small caulinary flowers and fruits with disciform pyrenes (Valeton, 1911, 1927; Fig. 1, E8). The New Caledonian monotypic genus Captaincookia N.Hallé (Hallé, 1973) accommodates a species with large drooping, caulinary red flowers, infundibuliform corollas, and stigmatic arms permanently erected (Fig. 1, E5; online Appendix S1, K). A more recently described Mascarene genus, Doricera Verdc. American Journal of Botany 688 Table 1. [Vol. 96 Reproductive characters used as diagnostic elements for generic classification in the tribe Ixoreae. Features Ixora Captaincookia Doricera Hitoa Myonima Sideroxyloides Thouarsiora Versteegia Breeding system Inflorescence position Flower orientation Corolla tube/lobe ratio Ovary Pyrenes shape monoecious terminal erect >1 2(−4)-carpellate globose monoecious caulinary drooping >1 2-carpellate globose dioecious terminal erect <1 3-carpellate globose monoecious terminal erect >1 4-carpellate globose dioecious terminal erect <1 2–7-carpellate globose monoecious caulinary erect >1 2-carpellate globose monoecious terminal erect >1 4-carpellate globose monoecious caulinary erect >1 2-carpellate flattened (Verdcourt, 1983), consists of a sole species, which was originally described as Pyrostria Comm. ex Juss. in the tribe Vanguerieae due to its axillary inflorescences. Bridson and Robbrecht (1985) proposed a close relationship between Doricera and Ixora, and the placement of Doricera in Ixoreae was supported by Mouly et al. (2005, in press) and also recently by Davis et al. (2007). Ixora has always been recognized as a well-circumscribed genus (Fosberg, 1937; De Block, 1998) based on flower morphology (e.g., hypocrateriform corolla, contorted bud, and salverform stigma). However, Andreasen and Bremer (2000), also endorsed by Mouly et al. (in press), indicated that the present circumscription of Ixora was not monophyletic. In the latter study, all sequenced representatives of Captaincookia, Doricera, Myonima, and Versteegia were nested within the seven sampled Ixora species (including the type species I. coccinea L.). Bremekamp (1937a, b, 1938, 1940; Table 2) subdivided Ixora into three subgenera including only Malesian species: Ixora, Pavettoides Bremek., and Sathrochlamys Bremek, though these names are applicable to all species. Subgenus Ixora is, in fact, pantropical and is mostly characterized by a combination of the following features: inflorescence axes opposite and articulate (Fig. 1, B), flowers in distinct triads, pedicels of lateral flowers articulate, and bracts and bracteoles well developed (Bremekamp, 1937b). Subgenus Pavettoides, occurring from the Seychelles to India, tropical Asia, Micronesia, and northern Australia (Bremekamp, 1937b; Smith and Darwin, 1988; De Block, 1998), is recognized by its upper inflorescence axes usually subopposite (Fig. 1, A), nonarticulate flowers in less distinct triads, nonarticulate pedicels, and bracts and bracteoles weakly developed (Bremekamp, 1937b). Subgenus Sathrochlamys is restricted to the eastern part of the Malaysian archipelago, northern Borneo, and New Guinea (De Block, 1998) and presents the following characteristics: inflorescences never opposite nor articulate, flowers not in distinct triads, pedicels with nonarticulate, and bracts and bracteoles minute or absent (Bremekamp, 1937b). These subgeneric circumscriptions were not confirmed from Husain and Paul’s (1989) palynological studies based on the Indo-Asian Ixora species. More recently, De Block (1998) observed that some of the character states used by Bremekamp (1937b) were dubious and not always consistent. Within the subgenera, Bremekamp (1937a, b, 1938, 1940) recognized up to 20 sections, also based solely on the Malesian representatives of Ixora: sect. Ixora (ca. 90 species), sect. Brachypus Bremek. (20 species), sect. Chlamydanthus Bremek. (9 species), sect. Erythrocalyx Bremek. (1 species), sect. Octobathrum Bremek. (44 species), and sect. Stenopus Bremek. (1 species) in subgen. Ixora; sect. Raphidanthus Bremek. (12 species), sect. Pavettopsis Bremek. (38 species), sect. Pogonanthus Bremek. (10 species), and sect. Amphorion Bremek. (2 species) in subgen. Pavettoides; and sect. Pseudobandhuca Bremek. (1 species), sect. Gymnocorymbus Bremek. (1 species), sect. Hypsophyllum Bremek. (6 species), and sect. Macro- pus Bremek. (5 species) in subgen. Sathrochlamys. The characteristic features of these sections are generally based on inflorescence and flower organizations (De Block, 1998; Table 2). Bremekamp (1937b) postulated that the African Ixora belonged to sect. Otobactrum; the Indian and Continental Asian Ixora to sections Ixora, Brachypus, Chlamydanthus, Otobactrum, Amphorion, and Pavettopsis; and the Pacific representatives to sect. Pavettopsis. More sections were described in Ixora: sect. Phylleilema A.Gray (Gray, 1858) for Pacific taxa with 3(−10)-flowered inflorescences subtended by narrow foliaceous bracts (Fig. 1, D; online Appendix S1, B, J), sect. Cremixora Baill. for a single species in Madagascar (Baillon, 1880; online Appendix S1, E) with pendulous ovules, sect. Micrixora Hochr. (Hochreutiner, 1908) for species having small corollas, sect. Microthamnus (Homolle ex Arènes) Guédès (Guédès, 1986) for one-flowered Malagasy species, and sect. Vitixora Fosberg (Fosberg, 1942; online Appendix S1, D) for the Fijian species with congested subsessile inflorescences. The Myonima group, when placed under Ixora (Baillon, 1879; Table 2), formed its own section. The infrageneric classifications of Ixora, notably the ones proposed by Bremekamp (1937b), have seriously been questioned by many authors (Corner, 1941; Fosberg, 1942; Husain and Paul, 1989; De Block, 1998) but have never been tested using molecular-based phylogenies. Sequence data from the rps16 and trnT-F chloroplast regions have been used separately and/or in combination with data from the rbcL chloroplast coding gene in Mouly et al. (in press), for the circumscription of Ixoreae. Here, we report phylogenetic analyses using combined data from the nuclear ribosomal external transcribed spacer (nrETS), rps16, and trnT-F to reconstruct a robust phylogeny for many more species of Ixoreae. ETS, used in combination with nrITS, has recently been shown to be useful for assessing phylogenetic relationships in closely related genera of the tribe Naucleeae s.l. in the subfamily Cinchonoideae, Rubiaceae (Razafimandimbison et al., 2005). The resulting phylogeny from our combined data will be used to (1) rigorously test the monophyly of the present circumscription of Ixora, (2) present new generic limits within Ixoreae, if necessary, (3) and test the monophyly of the current infrageneric classifications of Ixora. MATERIALS AND METHODS Taxon sampling—We sequenced 106 specimens (Appendix 1) representing 93 Ixoreae taxa including: 79 Ixora species (including Hitoa, Sideroxyloides, and Thouarsiora), the type species of the monotypic Captaincookia and an undescribed species closely related to it (represented by two specimens from two different populations), the monotypic Doricera, four Myonima representatives, and two Versteegia species. Cyclophyllum ixoroides Guillaumin (Vanguerieae) was also included because it was suspected by Guillaumin (1930) to be closely related to Ixora. Several specimens of Ixora included in the analyses could not be identified to species mainly due to the lack of modern treatments of Ixora for many flora regions. Ixora sp. 5, I. finlaysiana Wall. ex G.Don, I. casei Hance, I. chinensis Mouly et al.—Phylogeny of IXORA (Rubiaceae) March 2009] 689 Synthesis by De Block (1998) of the infrageneric classification of the genus Ixora and characteristics and distributions based on literature and completed in this study. Table 2. Subgenera Sections Morphological characteristics of sections Ixora Ixora syn.: Megalixora Hochr. (1908) syn.: Ixorastrum Bremek. (1937) Type: I. coccinea L. Inflorescences sessile or moderately pedunculate, erect; calyx tubes short but distinct; corollas white, red, orange or yellon; tubes much longer than lobes; stamens much shorter than corolla lobes; anthers with short cells; styles glabrous or pubescent, shortly exserted Ixora Ixora Ixora Ixora Ixora Ixora Ixora Ixora Pavettoides Bremek. Pavettoides Bremek. Pavettoides Bremek. Pavettoides Bremek. Pavettoides Bremek. Sathrochlamys Bremek. Sathrochlamys Bremek. Sathrochlamys Bremek. Distribution of sections Sri Lanka, India, Burma, Laos, Cambodia, Vietnam, Malay Peninsula, Indonesia, New Guinea, and Caroline Islands Inflorescences subsessile to moderately or rarely long pedunculate, India, Burma, Laos, Brachypus Bremek. (1937) erect or drooping; bracts and bracteoles narrow; calyx tubes sometimes Cambodia, Vietnam, and Type: I. cuneifolia Roxb. distinct; corollas white or reddish; stamens as long as corolla lobes; Malay Peninsula anthers with long cells; styles glabrous, exserted portion ± as long as corolla lobes Chlamydanthus Bremek. (1937) Inflorescences subsessile or shortly pedunculate, erect; central flowers India, Burma, Laos, in ultimate triads sessile and ebracteolate; bracts and bracteoles well Cambodia, Vietnam, and Type: I. umbellata developed; bracteoles usually longer than ovaries; corollas white or Malay Peninsula Valeton ex Koord. & Valeton ochre-yellow; tubes 3–6 times longer than the lobes; stamens usually shorter than corolla lobes; anthers with long cells; styles glabrous, exserted portion somewhat shorter than corolla lobes Ovaries with descendant ovules Madagascar and Comoro Cremixora Baill. (1880) Islands Type: I. cremixora Drake Inflorescences subsessile, subcapitate, pauciflorous; bracts and Indonesia Erythrocalyx Bremek. (1937) bracteoles linear, large, red; terminal flowers solitary and ebracteolate; Type: I. curtisii Ridl. calyx tubes reduced, lobes linear, red, (almost) as long as the corolla tube: corollas white, lobes and tube short; stamens much shorter than corolla lobes, anthers with moderately long cells; styles densely pubescent, shortly exserted Flowers small; corolla tube <1.5 cm Madagascar and Seychelles Micrixora Hochr. (1908) Types: I. drakei Hochr., I. humblotii Drake, I. microphylla Drake, I. pudica Baker Corollas small; tubes shorter than lobes; ovaries two- to plurilocular Mascarene Islands Myonima Baill. (1879) Type: Non designatus Inflorescences long pedunculate, multiflorous; bracts and bracteoles Tropical Asia and New Otobactrum Bremek. (1937) narrowly triangular to filiform; corollas white, rarely reddish or Guinea, and Occidental Type: I. palludosa (Bl.) Kurz yellowish; stamens somewhat shorter than corolla lobes or of equal Africa length, anthers with long cells; styles glabrous, exserted portion ± as long as corolla lobes Inflorescences distinctly pedunculate, consisting of 1–7 flowers, at the Philippines and Celebes Stenopus Bremek. (1937) end of axillary short shoots arising on the green part of the branches; Type: I. filipes Valeton lower bracts narrowly triangular, the others wide; bracteoles ovate; corollas white; stamens shorter than corolla lobes, anthers with long cells; styles glabrous Inflorescences shortly or moderately pedunculate, subpaniculate; Tropical Asia and Indonesia Amphorion Bremek. (1937) flowers small; corolla tubes with bearded throat Type: I. brunnescens Kurz Inflorescences subsessiles or shortly pedunculate, trichotomously or Seychelles, Sri Lanka, Pavettopsis Bremek. (1937) pentachotomously corymbose; corolla tubes glabrous or pubescent Andaman, Nicobar Islands, Type: I. blumei Zoll. & Mor. inside but never bearded; stamens equalling corolla lobes in length; India, Malay Peninsula, styles glabrous or pilose in their middle part New Guinea, Melanesia, and Micronesia Inflorescences subsessile or shortly pedunculate; corolla tubes bearded Indonesia, Celebes, Pogonanthus Bremek. (1937) at throat or pubescent in its upper half inside; styles near the middle Muloccas, New Guinea, and Type: I. timorensis Decne. usually densely pilose or rarely glabrous Australia Inflorescences long or very long pedunculate; corolla tubes slender or Indonesia Raphidanthus Bremek. (1937) very slender, never bearded, much longer than corolla lobes; stamens Type: I. capillaris Bremek. frequently much shorter than corolla lobes; styles glabrous Leaves shortly petiolate to subsessile; inflorescences multiflorous, Fiji and New Caledonia Vitixora Fosberg (1942) Types: I. amplexicaulis Gillepsie, strongly congested head-like cymes, sessile between a terminal pair I. coronata A.C.Sm., I. pelagica of leaves (not modified); bracts linear and filiform crowded among the flowers; calyx lobes linear or narrowly triangular Seem., I. somosomaensis Gillepsie Philippines and Moluccas Gymnocorymbus Bremek. (1937) Inflorescences pedunculate; bracts and bracteoles absent; styles glabrous Type: I. paradoxalis Bremek. New Guinea Hypsophyllum Bremek. (1937) Leaves (sub)sessile, base rounded to cordate or subauriculate; Type: I. dolichothyrsa Bremek. inflorescences long pedunculate; bracts filiform and setaceous; calyx lobes and tube equal in length; corollas white or ochre-yellow; styles pubescent in their middle part Leaves petiolate, base acute; inflorescences long pedunculate; basal New Guinea, Philippines, Macropus Bremek. (1937) bracts setaceous, others minute; bracteoles absent; calyces cupuliform; and Moluccas Type: I. whitii S.Moore styles pubescent in their middle part American Journal of Botany 690 Table 2. [Vol. 96 Continued. Subgenera Sections Morphological characteristics of sections Sathrochlamys Bremek. Pseudobandhuca Bremek. (1937) Inflorescences shortly or moderately pedunculate, supported by a pair of sessile leaves not reduced in size; bracts and bracteoles small but Type: I. philippinensis Merr. distinct; corollas white or pink; styles glabrous Inflorescences terminal on main and lateral branches, uniflorous, sessile Microthamnus (Homolle ex but with modified inflorescence-supporting leaves present; calyces Arènes) Guédès (1986) Type: I. reducta Drake ex Guédès truncate or with small triangular lobes Inflorescences pauciflorous (often consisting of three flowers), sessile Phylleilema A.Gray (1858) but modified inflorescence-supporting leaves present, the latter obtusely Types: I. amplifolia A.Gray, I. fragrans (Hook. & Arn.) A.Gray, triangular to ± orbicular, with cordate bases. I. samoensis A.Gray, I. vitiensis A.Gray Lam., and I. pavetta Andr. were all represented in the study by individuals cultivated in botanical gardens (Appendix 1); therefore, they are possibly of hybrid origins. Following Mouly et al. (in press), three suitable outgroup taxa from the tribes Aleisanthieae and Greeneeae (Mouly et al., in press), and Vanguerieae were chosen to root the trees (Appendix 1). DNA extraction, amplification, sequencing, and alignment (Appendix 1)—Total DNA was extracted from dried material preserved in silica gel (Chase and Hillis, 1991) or herbarium specimens following the mini-prep procedure of Saghai-Maroof et al. (1984), as modified by Doyle and Doyle (1987). Extracted DNA was cleaned with a Qia-Quick PCR purification kit (Qiagen, Solna, Sweden). PCR reactions were as follows: 27.25 µL H2O, 5 µL of PCR buffer, 5 µL of MgCl2, 5 µL of 0.1 M tetramethylammonium chloride (TMACl), 4 µL of dNTP, 0.25 µL Taq polymerase, 0.5 µL of each primer, and 0.5 µL of 1% of bovine serum albumin. PCR amplifications, performed in a Eppendorf Mastercycler gradient, started with an initial melting phase of 2 min at 95°C; followed by 35–37 cycles of 30 s at 95°C, 1 min at 50–55°C, and 2 min at 72°C; and ended with a final extension phase of 7 min at 72°C. In all PCR runs, one reaction was run with water instead of DNA as a negative control to check for contamination. The rps16 intron was amplified with the primer pair rpsF/rpsR2 (Oxelman et al., 1997). For half of the species, we repeatedly failed to obtain amplification in one reaction because of a problematic poly A/T at the 3′ end of the intron (Shaw et al., 2005). However, amplification was successful with the internal primer pair rpsF2/rpsR3 (Bremer et al., 2002), but the sequences were 50–70 bp shorter. The entire trnT-F region (including the trnL intron) was amplified in two parts. The trnT-trnL segment was amplified with primer pair A1/I (Razafimandimbison and Bremer, 2002; Bremer et al., 2002). The second segment, trnL-trnF, was amplified with primers C/F (Taberlet et al., 1991). For trnL-F, sequencing reactions were performed using the two external primers C/F and two internal primers D/E (Taberlet et al., 1991) to produce a complete sequence of the entire trnT-F region, with at least partial overlaps. The segment of the noncoding region ETS was amplified and sequenced with primer pair 18S-E/H 5′-CTTGTAGGGTTGGTTGGA-3′ (Baldwin and Markos, 1998; H was designed by H. Lantz, Uppsala University, Sweden, unpublished). For 13 specimens, direct sequencing of purified PCR products consistently produced multiple sequence signals for ETS, indicating the presence of infraindividual polymorphism. Therefore, the PCR products of these species were cloned using a TOPO TA cloning kit (Invitrogen, Paisley, UK). This kit used unpurified PCR-amplified DNA, the TOPO vector, and a vial of One Shot chemically competent Escherichia coli according to the manufacturer’s instructions. Two to four white colonies from the cloning reaction of the concerned species were screened and amplified with the M13F and M13R competent primers that were included in the TOPO TA cloning kit. Their respective purified PCR products were sequenced with the 18S-E/H. All sequencing reactions of the markers were performed with a Big Dye Terminator v3.1 Cycle Sequencing kit and Bid Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Life Technologies, Carlsbad, California, USA) and subsequently analyzed using a 3100 Genetic Analyzer (Applied Biosystems). Distribution of sections Philippines, Moluccas, Celebes, and Borneo Madagascar and New Caledonia From Marquesas as far westward as New Caledonia The rps16, trnT-F, and ETS sequences were assembled using the Staden Package version 1.6.0 beta (Staden, 1996) and the program Sequencher 3.1.1 (Gene Codes Corp., Ann Arbor, Michigan, USA) and edited manually. All sequences were aligned manually following similarity criterion (e.g., Simmons, 2004) with the program Se-Al version 1.0al (Rambaut, 1996) (matrices are in Appendices S1–S6, see Supplemental Data with the online version of this article). Unambiguously aligned insertions and deletions (indels) were then coded as binary characters using 0 and 1 symbols for terminals with sequence and those that have a gap, respectively. Phylogenetic analyses—Separate and combined analyses of the rps16, trnT-F, and ETS matrices (online Appendices S2–5) were performed using Bayesian Markov chain Monte Carlo (MCMC) inference (Yang and Rannala, 1997) as implemented in the program MrBayes version 3.0B (Huelsenbeck and Ronquist, 2001) for partitioned data sets, and maximum parsimony (MP) methodology, as implemented in the program PAUP* version 4.0b8 (Swofford, 2002). The Bayesian approach evaluates the posterior probability (PP) of a tree given the character matrix, i.e., the probability that the tree is correct. Bootstrap support (BS) was calculated for MP analyses. Analysis settings—For each partition, the program MrModeltest 2.0 (Nylander, 2004) was used to choose the model of nucleotide substitution that best fits the data, following Akaike’s information criterion (Akaike, 1974). The selected models were general time reversible (GTR) with among-site substitution rate heterogeneity described by a gamma distribution (Yang, 1994) for rps16 and the trnT-L segment as well as the ribosomal ETS (GTR + G), GTR with a fraction invariant site constraint for the trnL-F part (GTR + I). Partitioned Bayesian analyses were conducted to account for the combination of molecular data, according to the nucleotide evolution models selected before, and a no-common-mechanism model (Tuffley and Steel, 1997) for standard binary characters of gap coding. All analyses were performed with four independent Markov chains run for 3 × 106 Metropolis-coupled MCMC generations, with tree sampling every 103 generations, and burn-in after 500 sampled trees (as detected by plotting the log likelihood scores against generation number). The analyses were run three times using different random starting trees to evaluate the convergence of the likelihood values and posterior clades probabilities (Huelsenbeck et al., 2002). Saved trees from the three independent runs were pooled to build the consensus tree. Groups characterized by a posterior probability over 95% were regarded as strongly supported. Phylogenetic analyses using paralogous sequences may be misinterpreted if the orthology of the nuclear alleles is erroneously assumed. Orthologous markers derive from the same locus, whereas paralogous markers derive from different loci that originated by a DNA region duplication event (Fitch, 1970). Considering that cloned ETS Ixora sequences did not always form exclusive lineages in the preliminary analyses, we selected representatives for combined nr- and cpDNA analyses as follows: one randomly selected ETS clonal sequence for those forming exclusive lineages and one randomly selected per lineage for those not forming exclusives lineages. For each selected ETS clonal sequence, the correspondent cpDNA sequences were duplicated according to the number of ETS → Fig. 2. Ixoreae phylogram of the Bayesian analysis majority rule consensus tree illustrating the position of the clone sequences for each cloned taxon of the ETS sequence analysis. Names of cloned individuals end in “clone” and a letter; clones from the same individual grouping in the same clade are shaded in gray; dashed lines connect clones from the same individuals when they fall into separate clades. March 2009] Mouly et al.—Phylogeny of IXORA (Rubiaceae) 691 692 American Journal of Botany copies used in the combined nr- and cpDNA matrices. The duplication of cpDNA information can affect the support of inferred clades, but this effect was not significant in the present case (e.g., the subclade containing Ixora coccinea obtained PP = 0.66 with duplication of cpDNA information, cf. Fig. 4, and PP = 0.64 without duplication; results of the latter analysis not shown) Separate nuclear and chloroplast data—We initially performed separate analyses of the ETS (online Appendix S2), rps16, and trnT-F data (online Appendix S3). The ETS matrix included the cloned ETS sequences of several species. Parsimony bootstrapping (Felsenstein, 1985; NNI, Multrees off, 104 replicates) and jackkniffing (Farris et al., 1996; NNI, Multrees off, 104replicates) of the rps16 and trnT-F analyses under PAUP* (Swofford, 2002), yielded largely unresolved trees; as result, we conducted a combined rps16/trnT-F chloroplast (cp) analysis, considering the cp-regions are not subject to intergenomic recombination. Visual inspection of the trees shows that some Ixora species had strongly supported conflicting positions between nuclear and chloroplast data. Combined nuclear and chloroplast data—Interpretation of gene trees may result in false species-tree hypotheses (Alvarez and Wendel, 2003; Bailey et al., 2003; Ochieng et al., 2007), notably when different gene trees show conflicting relationships (Mort et al., 2007). Previous knowledge regarding incongruent data sets in Rubiaceae (Bremer et al., 1999; Andreasen and Bremer, 2000) has led to combining data to obtain a more supported hypothesis and maximize congruence among all characters sampled (Nixon and Carpenter, 1996; Razafimandimbison and Bremer, 2002). Combination of nr- and cpDNA has been shown to strengthen the signal masked by homoplasy, exhibiting secondary or additive cpDNA signal (Nixon and Carpenter, 1996; Wenzel and Siddall, 1999), which provides an increase of resolution and support (Mort et al., 2007). Combination of chloroplast markers has been used for Ixoreae with rps16, trnT-F, and rbcL markers (Mouly et al., in press), and combination of chloroplast and nuclear regions for several Rubiaceae groups as for the close relative of Ixoreae, tribe Vanguerieae (Razafimandimbison et al., in press). Combination of data sets has been extensively used to reconstruct phylogenies to increase the number of informative characters, notably in case of taxa showing low substitution rates for investigated markers (Soltis et al., 1998). Soltis et al. (1998: 297–348, Fig. 11.1: 301) also pointed out that in case of incongruent topologies for separate data analyses, combining these conflicting data is possible by pruning taxa. On the basis of the conflicting positions between nr- and cpDNA tree topologies, we conducted two types of combined analyses: one excluding all taxa with conflicting positions and the other excluding the putative hybrid species collected from botanical gardens but including only the nuclear data of the species with detected conflicting positions possibly due to natural hybridization (I. brunonis Wall. ex G.Don, I. kuakuensis S.Moore, I. nematopoda K.Schum., I. nimbana Schnell, I. triantha Volkens, and Versteegia grandifolia Valeton). The combined matrices included one to several clone sequences, according to the different paralogous lineages found (Fig. 1). Consequently, the restricted combined data set analyses comprised 91 terminal units representing 85 specimens (online Appendix S4), while the complete combined data sets analyzed included 98 terminal units representing 91 specimens (online Appendix S5). Character optimizations—Currently, several reproductive characters (e.g., inflorescence, ovary, flower sex) are used in combination for generic recognition within in Ixoreae (Table 1). Information about the taxonomic characters (e.g., seed numbers: two or numerous, position of inflorescences: terminal or caulinary, breeding systems: hermaphroditism or functional dioecy) were observed on herbarium material (BR, G, K, L, MO, NOU, NY, P, PAP, TAN) and in the field or compiled from the literature. To illustrate the character state evolution, we optimized them onto the Bayesian majority consensus tree from the combined nuclear–chloroplast analyses manually using Fitch’s (1971) incremental character optimization. Ixora infrageneric classification mapping—No infrageneric treatment has ever been provided for the entire genus Ixora; however, regional works have presented classification tools along with morphological characteristics (Gray, 1858; Baillon, 1879, 1880; Hochreutiner, 1908; Bremekamp, 1937a, b, 1938, 1940; Fosberg, 1942; Guédès, 1986). The available infrageneric classification, summarized in Table 2 (updated and completed from De Block, 1998), was thought applicable [Vol. 96 to the entire genus. To assess the limits of the Ixora infrageneric classification (Table 2), we assigned to ingroup taxa names of subgenera and sections, according to morphological characteristics of each group given by previous authorities (Table 2; Appendix 2). These names were then mapped on the tree topology of the complete combined markers analysis to estimate their validity. Observed patterns did not suffer any effect from the potential for hybridization of the genus, considering hybridization did not influence significantly the main Ixora subclades circumscriptions (observed from both separated and combined data sets). RESULTS Phylogenetic analyses— In this section, we provide (1) the sequence characteristics and the outputs of the separate nuclear (Fig. 2) and chloroplast (Fig. 3) analyses, with an emphasis on the detailed results of the ETS analysis including the cloned sequences; (2) an illustration of conflicting positions between these data sets (Figs. 2–4); and (3) the characteristics and relationships information for both the restricted and complete sampling analyses of the combined datasets (Figs. 5, 6). Sequence characteristics (Figs. 2, 3; Tables 3, 4)— Ambiguously aligned sites and indels were excluded from the data sets before analyses (Table 3). These excluded data represented 15.1% of the rps16 nucleotide matrix, 14.0% of the trnT-F matrix, 9.5% of the ETS matrices, and 10.3% of the combined data for both combined analyses. The number of parsimony informative characters (PIC) was very low within the complete combined data set (314 bp), representing ca. 10% of the aligned sequences. The trnT-F matrix contained the longest sequences, and most of the PIC were localized in the noncoding trnT-L segment. However, many substitutions and most of the indels, which were resolved as unambiguous synapomorphies for many clades, were from within the trnL intron. The ETS, comprising about half of the PIC of the complete combined data set, was useful to resolve relationships within the ingroup lineage and to detect incongruence between the nuclear and chloroplast data. The ETS matrix included all sampled cloned sequences of the 13 polymorphic taxa. The pairwise sequence divergences of these cloned sequences varied from 0 to 4.5%, with the exception of Ixora coccinea with 7.9% (see Table 4). Only six of the 13 polymorphic specimens had their sampled ETS cloned sequences forming exclusive lineages (Fig. 2; Table 4): Ixora scheffleri K.Schum. & K.Krause (PP = 1.00), I. aluminicola Steyerm. (PP = 0.97), I. finlaysoniana (PP = 1.00), Ixora sp.5 (PP = 1.00), Doricera trilocularis (Balf.f) Verdc. (PP = 1.00), and Versteegia cauliflora (K.Schum. & Lauterb.) Valeton (PP = 1.00). Separate nuclear and chloroplast analyses— The nuclear and chloroplast trees (Figs. 2, 3) both resolved the presently circumscribed Ixora as paraphyletic and retained the following clades: the Mascarene Myonima-Doricera clade (nr-tree, PP = 1.00; cp-tree, PP = 1.00); the Afro-Malagasy-American Ixora clade (nr-tree, PP = 0.71; cp-tree PP = 1.00); the Pacific clade (nr-tree, PP = 0.67; cp-tree, PP = 0.95), and Versteegia cauliflora was left unresolved and isolated in a large polytomy. Nev→ Fig. 3. Ixoreae phylogram of the Bayesian analysis majority rule consensus tree of the chloroplast (combined rps16 and trnT-F) sequence analysis; posterior probability is indicated for each node. Arrows indicate species with conflicting positions according to nuclear data (Fig. 2); bold lines indicate the main groups named in the figure. March 2009] Mouly et al.—Phylogeny of IXORA (Rubiaceae) 693 694 American Journal of Botany ertheless, there were some topological conflicts between the nr- and cp-trees. The cp-tree (Fig. 3) resolved 12 of the 18 Asian Ixora species in a strongly supported clade; two Ixora species (I. brunonis, I. finlaysoniana) formed a highly supported clade nested but unresolved in the Afro-MalagasyAmerican Ixora clade. The remaining four Ixora species (I. casei, Ixora sp.5, I. chinensis, and I. pavetta) constituted a poorly supported clade left unresolved at the base of the AfroMalagasy-American Ixora clade (Fig. 3). In contrast, the large Asian Ixora clade collapsed in the nr-tree (Fig. 2) and instead split into two groups: one forming 13 Ixora species, including the aforementioned six Ixora species, called the “ornamental” Asian group; and the other containing I. iteophylla Bremek., Ixora sp. 16 and 17, and I. coccinea L. clone B, called Asian clade. The cp-tree (Fig. 3) placed I. kuakuensis in the Pacific Ixora clade, while the nr-tree (Fig. 2) resolved it in the “ornamental” Asian Ixora group. The two ETS cloned sequences of I. coccinea did not form a clade: one nested in the Asian Ixora subclade and the other together with several “ornamental” Asian Ixora. Furthermore, I. nematopoda and I. nimbana formed a strongly supported clade (PP = 1.00) sister to the Afro-Malagasy-neotropical clade in the nr-tree (Fig. 2), whereas they did not form a clade and were embedded within the AfroMalagasy clade in the cp-tree (Fig. 3). Versteegia grandifolia was sister to the Afro-Malagasy-neotropical clade (PP = 0.83) in the nr-tree (Fig. 2) but sister to the Pacific clade (PP = 0.82) in the cp-tree (Fig. 3). Combined nuclear–chloroplast analyses— The restricted combined ETS/rps16/trnT-F data set contained 91 terminal units, and the complete combined data set consisted of 98 terminal units. Each matrix comprised 2770 characters, of which 44 (1.7%) were coded indels. These matrices, respectively, contained 324 and 356 (11.7% and 12.9%) PIC. The relationships did not differ significantly between the two combined analyses or between parsimony (figures available from A. Mouly) and model-based analyses, with the exception of the unplaced Mascarene subclade of Ixora in the parsimony topologies. Several main clades were more strongly supported in the restricted combined analysis (Fig. 5) than in the complete combined analysis (Fig. 6) for both parsimony and modelbased reconstructions. We only present the results from the model-based analyses to simplify the comprehensive results. Both the posterior probabilities values from the restricted and complete analyses are given (PP of Fig. 5; PP of Fig. 6, respectively) for comparable nodes present in both inferred consensus tree topologies. The ingroup taxa were resolved in two lineages, consisting of an Afro-Indian Ocean-neotropical clade (PP = 0.85; PP = 0.64) and an Asian-Pacific clade (PP = 0.50; collapsed). The first ingroup lineage was fully resolved in five well-supported main clades: Afro-Malagasy Ixora clade (PP = 0.99; PP = 1.00), Malagasy clade (PP = 0.97; PP = 0.99), neotropical clade (PP = 1.00; PP = 0.94), I. nigricans, and Mascarene clade (PP = 1.00; PP = 1.00). The Mascarene clade was sister to the remaining Afro-Indian Ocean-neotropical clade. Ixora nigricans R.Br. ex Wight & Arn. was the first lineage to branch off, followed by the neotropical and Malagasy clades; the Malagasy clade was resolved as sister to the Afro-Malagasy clade. The second ingroup lineage consisted of poorly to moderately supported relationships (PP = 0.83; PP = 0.56) of two large sister clades: a well-supported Pacific clade including Pacific Captaincookia and 23 Ixora species (PP = 0.99; PP = 1.00) and a moderately supported Asian clade (PP = 0.92 in [Vol. 96 Fig. 5) including the genus type, Ixora coccinea. The inclusion of taxa with numerous missing data in the complete combined analysis considerably decreased the support for the Asian clade (PP = 0.66 in Fig. 6). Versteegia cauliflora was resolved in the restricted combined analysis as sister to the Asian-Pacific association, with very low support (PP = 0.50 in Fig. 5), but was basal in Ixoreae in a trichotomy with the two main clades in the complete combined analysis (Fig. 6). All sampled Ixora species from French Polynesia formed a wellsupported clade sister to a mostly and strongly supported New Caledonian Ixora clade. In addition, all sampled African, Malagasy, and neotropical Ixora species were more closely related to the sequenced Mascarene Doricera and Myonima species than they were to the sampled Asian and Pacific Ixora species. The type species I. coccinea and the sampled Pacific Ixora were more closely related to Captaincookia and Versteegia grandifolia than they were to the sequenced African, Malagasy, and neotropical Ixora species. Furthermore, Thoursiora littoralis Homolle ex Arènes was nested in the strongly supported Malagasy clade (PP = 0.97; PP = 0.99), while Ixora ferrea (Jacq.) Benth. (= Sideroxyloides) was embedded within the strongly supported neotropical clade (PP = 1.00; PP = 0.94). Within the Mascarene clade, the sampled Myonima representatives formed a monophyletic group (PP = 1.00; PP = 1.00), which was in turn sister to Doricera. Ixora mooreensis (Nadeaud) Fosberg (= Hitoa) was nested in the strongly supported (PP = 1.00; PP = 1.00) French Polynesian Ixora clade in the supported Pacific Ixora clade (PP = 0.99; PP = 1.00). The three specimens of Captaincookia also formed a highly supported monophyletic group (PP = 1.00; PP = 1.00) near the base of the Pacific clade. Three taxa represented by two or three specimens each did not form exclusive lineages: I. brachypoda, I. cremixora, and I. guineensis (Figs. 5, 6; results also observed from separate nrand cpDNA data analyses in Figs. 2, 3). Character optimizations—The character state optimization outputs did not differ between the nrDNA, cpDNA, and combined data sets, respectively, or between the parsimony and model-based analyses (results not shown). On the tree presented in Fig. 6, the optimized reproductive characters currently used for generic recognition within Ixoreae (Table 1) are shown to be homoplastic. The caulinary inflorescences that mainly distinguished Versteegia, Captaincookia, and Sideroxyloides from Ixora with terminal inflorescences corresponded to at least six convergent evolution events. The number of carpels per ovary was also shown to be homoplastic, with the two-carpellate ovaries inferred as plesiomorphic and >2-carpellate ovaries appearing independently three times within the ingroup. A reversal from a >2- to 2-carpellate ovary was inferred within the Mascarenes subclade (Fig. 6) for Myonima violacea. On the other hand, functional dioecy (Fig. 1, E1, E2) evolved just once within Ixoreae (the Mascarene clade), and hermaphroditism is plesiomorphic. Ixora infrageneric classification mapping—According to the assignment of our sampled species to the infrageneric classification of Ixora (Appendix 2) mapped on the combined nrand cp-tree topology (Fig. 7), the two tested subgenera Ixora and Pavettoides were not monophyletic. Section Myonima was monophyletic, and sections Amphorion, Brachypus, and Microthamnus were each represented by a single species. Sections Cremixora, Ixora, Micrixora, Otobactrum, Pavettopsis, and Raphidanthus were not resolved as natural lineages. March 2009] Mouly et al.—Phylogeny of IXORA (Rubiaceae) 695 Information for phylogenetic analyses of (A) separate (rps16, trnT-F, ETS) and (B) combined chloroplast (rps16 + trnT-F) or combined (rps16 + trnT-F + ETS) data sets. Table 3. Phylogenetic information A) Separate analyses No. of sequences investigated No. of new sequences Range of sequence lengths (bp) Length of aligned matrices (bp) No. of ambiguous sites and gaps excluded No. of PIC No. of coded indel events Range of G : C within ingroup (mean) Length of MP trees Consistency index Retention index Retention index corrected Phylogenetic information Fig. 4. Schematic comparison of nrDNA (ETS tree of Fig. 2) and cpDNA (combined rps16 and trnT-F of Fig. 3) tree topologies, summarizing species with conflicting positions between data sets. DISCUSSION Following the recent resurrection (Andreasen and Bremer, 2000) and recircumscription (Mouly et al., in press) of tribe Ixoreae, the current study constitutes the first phylogeny of the species-rich genus Ixora based on a large sampling of Ixora and its allies. The present discussion focuses on (1) the composition of the nuclear data set, (2) incongruencies between the nuclear and chloroplast data sets, (3) the circumscriptions and preliminary biogeographical elements of Ixora and its infra-generic classification, and (4) taxonomic implications of the phylogenetic results. ETS data set information (Fig. 2; Table 4)— The ETS region is part of the 18S-26S nrDNA and has a role in the maturation of rRNAs. The clones obtained for each polymorphic species are generally poorly differentiated (ranging from 0 to ca. 4.5%; mean < 2%) within a species, with the exception of I. coccinea with ca. 8% divergence. Despite the low differentiation of paralogs (Table 4), only half of the polymorphic species (Doricera trilocularis, Ixora aluminicola, I. finlaysoniana, I. scheffleri, Ixora sp. 5, and Versteegia cauliflora) form separate groups (Fig. 2; Table 4). The sampled clones of I. tanzaniensis Bridson, I. narcissodora K.Schum. and Ixora sp. 10, Ixora sp. 13, and Myonima sp.1 do not form separate lineages (Fig. 2; Table 4). The observed levels of intraindividual polymorphisms can be explained by different rates of concerted evolution of the ETS region among the species in question. In other words, the presence of divergent ETS paralogs in 13 Ixoreae species indicates that mutation rates are faster than that of concerted evolution; as a result, concerted evolution has not been fast enough to nullify the differences between the paralogs (see also Razafimandimbison et al., 2004). Nonconcerted evolution has been reported for nrITS (e.g., Buckler et al., 1997; Alvarez and Wendel, 2003; Bailey et al., 2003; Harpke and Peterson, 2006; Zheng et al., 2008) but is less documented for the ETS region. Divergent ITS and B) Combined analyses No. of sequences investigated Length of aligned matrices (bp) No. ambiguous sites and gaps excluded No. PIC No. indel events Length of MP trees Consistency index Retention index Retention index corrected rps16 trnT-F ETS 92 80 485–645 762 115 92 80 531–1584 1863 260 112 112 281–411 458 47 53 1 26.8–32.9% (31.5%) 201 0.83 0.88 0.73 95 17 24.6–35.5% (30.6%) 327 0.83 0.90 0.75 166 3 53–60.5% (57.4%) 623 0.59 0.83 0.49 Chloroplast Restricted combined a Complete combined a 95 2625 365 91 3083 421 98 3083 421 148 18 542 0.81 0.88 0.71 266 21 995 0.71 0.84 0.60 275 21 1052 0.69 0.83 0.57 a In restricted combined analysis, data set was restricted to samples for which a potential hybridization was not suspected. For complete combined analysis, all sample specimens were included, except potential anthropogenic hybrids and potential natural hybrids chloroplast information. Notes: bp = base pairs, MP = most parsimonious, parsimony informative characters = PIC. ETS pseudogenes (Razafimandimbison et al., 2004, 2005, respectively) have also been reported from the tribe Naucleeae s.l. in subfamily Cinchonoideae (Rubiaceae). The percentage of GC content in the ingroup ETS sequences ranges from 53 to 60.5% (mean 57.4%) compared to that reported from the genus Neonauclea Merr. in Naucleeae s.sl. (61.4–62%; Razafimandimbison et al., 2005). The large range of GC content in Ixora ETS sequences can be an indicator of the presence of putative nrDNA pseudogenes (e.g., Bailey et al., 2003), characterized by lower GC contents compared to their functional counterparts in the ETS matrix (e.g., Ochieng et al., 2007). For example, the two sampled ETS clonal sequences of I. coccinea may belong to putative nonfunctional nrDNA regions (clones A and B with GC contents of 56 and 57.5%, respectively), but no definitive conclusion can be drawn from the ETS only, according to the nrDNA pseudogene indicators listed in Bailey et al. (2003). On the other hand, each case of individual polymorphism studied here (I. coccinea excepted) represents shallow paralogy that does not interfere significantly in the phylogenetic reconstruction process (Bailey et al., 2003). Plausible causes of the topological conflicts in Ixora and data sets combination—Incongruence between nr- and cp-trees (Figs. 2–4) are evident in our study, as many species have American Journal of Botany 696 Table 4. ETS clonal sequence differences within a specimen, by clone pair; and the phylogenetic status of clones of a specimen within the tree topologies inferred from nuclear data (Fig. 2) and combined data (Fig. 6). Polymorphic taxa Captaincookia sp. 02 Doricera trilocularis Ixora aluminicola Ixora coccinea Ixora finlaysoniana Ixora narcissodora Ixora scheffleri Ixora sp. 05 Ixora sp. 10 Ixora sp. 13 Ixora tanzaniensis Myonima sp. 01 Versteegia cauliflora Genetic distances among ETS clones (%) 0–0.24 0.48 0.24–1.43 7.87 0–1.19 0.24 0.48–1.67 0.24 0.72–3.1 1.06–4.33 0.95–2.15 1.19 0 ETS clonal relation unresolved clustered clustered not clustered clustered unresolved clustered clustered unresolved not clustered not clustered not clustered clustered highly supported conflicting positions: I. nimbana and I. nematopoda in Africa; I. brunonis, I. casei, I. chinensis, I. finlaysoniana, I. pavetta, and Ixora sp. 5 in Asia (as the “ornamental” group in Fig. 4); I. kuakuensis, I. triantha, and Versteegia grandifolia in Pacific Islands. Three main biological processes are known to produce such effects: paralogy, incomplete lineage sorting, and hybridization (Sang and Zhong, 2000; Takahashi et al., 2001; Funk and Omland, 2003; Hudson and Turelli, 2003; Maddison and Knowles, 2006; Baum, 2007). No intraindividual polymorphism is observed in the sampled noncultivated Ixora species; this observation would also favor the hybridization hypothesis because polymorphic alleles would be expected to be more randomly distributed under a scenario of incomplete lineage sorting (Soltis et al., 1998: 273–277). The hypothesis of paralogy is unlikely to explain the observed incongruencies in the results because the ETS cloned sequences from the same individuals of I. scheffleri, I. aluminicola, I. finlaysiana, Ixora sp. 5, Doricera trilocularis, and Vesteegia cauliflora respectively, form separate clades (Fig. 2). Neither the sampled ETS cloned sequences of Ixora sp. 10 nor those of Ixora sp.13 nor those of I. coccinea form a monophyletic group; however, these species are still placed in the two Asian and “ornamental” Asian subclades (Fig. 2). Interestingly, five of the six sampled Asian Ixora species with conflicting positions in nr- and cp-trees (Figs. 2, 3) are commonly cultivated Ixora (see Appendix 1). Some of these cultivated specimens are likely to have hybrid origins, because plant breeders have successfully produced many cultivars of I. casei, I. coccinea, and I. chinensis (Fosberg and Sachet, 1989a, b; Staples and Herbst, 2005). The fact that the sampled I. casei, I. chinensis, I. pavetta, and Ixora sp. 5 form a clade (Fig. 3) may indicate that their maternal parents, which belong to the large neotropical-Afro-Malagasy-Mascarene Ixora lineage, are closely related. The current study clearly demonstrates how important it is for phylogeneticists to know the origin of specimens they include in their studies, notably for cultivated ones. Not considering this origin may lead to misinterpretations of the results (see Fig. 4). For example, phylogeneticists may end [Vol. 96 up performing age estimates for what appears to them to be a case of natural hybridization. Another case of an unnatural hybridization has been observed by Razafimandimbison et al. (unpublished data) during ITS investigations of three cultivated individuals of Mussaenda philippica A.Rich. A well-supported sister-group relation (Fig. 3) between the noncultivated individual of I. brunonis from Thailand and the cultivated individual of I. finlaysoniana does not seem to support an artificial hybrid origin of the latter, because no artificial hybrids have been reported from the latter species; instead, it appears to support the occurrence of natural hybridizations (Arnold, 1997) between two Ixora species, which currently have allopatric distributions but may have grown sympatrically in the past. A larger sampling of the Asian Ixora species is needed to retest the monophyly of the Asian group. If the placement of the noncultivated I. nigricans as sister to the neotropical-AfroMalagasy lineage is correct (Figs. 3, 5), this latter clade could have had an Asian origin. The conflicting positions of the Micronesian species I. triantha within the Pacific clade and that of the Asian I. kuakuensis in the “ornamental” Asian (Figs. 2, 4) and in the Pacific clades (Figs. 2, 3) could represent two independent cases of natural hybridizations. Interspecific hybridization is considered common among plants (Soltis et al., 1998; Hegarty and Hiscock, 2005; Pan et al. 2007), but phylogenetic methods produce only divergently branching hypotheses and thus cannot give a reliable interpretation of the tree topology if an analysis includes hybrids (McDade, 1990), except by using reticulation models. If the hypothesis of hybridization put forward here is true, then relations within Ixora should be inferred from nuclear or chloroplast data alone and gene-tree topologies discussed separately. On the other hand, an inclusion of specimens from probable anthropogenic crossing origin would very much affect the phylogenetic reconstruction and the tree topology. For evolutionary assessments, it is important to solely combine and analyze nuclear and chloroplast data sets if taxa with highly supported conflicting positions due to hybridization are excluded. In the case of Ixora, phylogenetic information appeared to benefit from the data set combination, considering the more resolved and supported phylogenetic hypothesis obtained in combination of data sets (Fig. 5), compared to separate genomes analyses (Figs. 2, 3). The current study concurs with Barber et al. (2007), who favor using a multiple marker approach from both nuclear and plastid genomes for assessing infrageneric relationships and controlling the occurrence of supported incongruencies, notably in the case of large genera like Ixora. Conflicts due to paralogy and/or hybridization observed from our data set do not appear to significantly affect the main subclades circumscriptions or their relations. New circumscriptions of Ixora—The present analyses (Figs. 5, 6) demonstrate that Ixora as currently circumscribed is paraphyletic and that Versteegia is polyphyletic. According to the combined nr- and cpDNA analyses (Figs. 5, 6), the sequenced African, Malagasy, and neotropical Ixora seem more closely related to the Mascarene Doricera and Myonima than they are to the Pacific and Asian Ixora (including the genus type I. coccinea). Plus, Captaincookia is nested within the highly supported → Fig. 5. Ixoreae cladogram of the Bayesian analysis majority rule consensus tree for the restricted sampling (excluding putative hybrids) of combined data sets. The native area of species follows the taxon: AFR, Africa; AS, Asia; CAR, Caribbean; FIJ, Fiji; FP, French Polynesia; MAD, Madagascar; MAL, Malesia; MAS, Mascarenes; MIC, Micronesia; NC, New Caledonia; NEO, neotropics; NG, New Guinea. March 2009] Mouly et al.—Phylogeny of IXORA (Rubiaceae) 697 698 American Journal of Botany Pacific Ixora clade; Versteegia cauliflora is sister to the moderately supported Asian-Pacific Ixora clade (PP = 0.83). Our molecular phylogenetic results support the inclusion of Hitoa, Sideroxyloides, and Thouarsiora in Ixora as previously suggested by Fosberg (1937), Bentham (1850), Guédès (1986), respectively, based on morphological data (see Fig. 1, E10, for Hitoa). Based on the evidence presented here, Ixora is in need of a new circumscription. Splitting up Ixora into several small genera is conceivable and necessitates the reinstatement of the existing generic names (e.g., Hitoa, Sideroxyloides, Thouarsiora). On the other hand, we find no obvious features for distinguishing or characterizing the major clades identified by our combined tree (Fig. 5). The recognition of several genera would also imply a restriction of the name Ixora to a single group of Asian species and would thus require hundreds of new combinations. We favor a broad circumscription of Ixora, which comprises Captaincookia, Doricera, Hitoa, Myonima, Sideroxyloides, Thouarsiora, and Versteegia, because this would constitute a relatively homogenous genus and only requires a maximum of 11 new combinations. Our results corroborate Baillon’s (1879) decision to merge Myonima in Ixora because he argued that plurilocular ovaries (Fig. 1, E11) did not constitute a strong character for generic recognition for Myonima. Also, the sexual differentiation of flowers in Myonima and Doricera (Fig. 1, E1, E2; Fig. 6) does not seem to be sufficient for generic separation. Indeed, the Seychellean species Ixora pudica Baker (not included here), supposed to be related to Melanesian and Malesian species (Bremekamp, 1934; Friedmann, 1994), also has unisexual flowers (Friedmann, 1994). Caulinary inflorescences (Fig. 1, C; online Appendix S1, C, K), used among other characters for segregating Captaincookia, Sideroxyloides, and Versteegia from Ixora, have evolved several times in Ixoreae (Fig. 6), also in several Ixora s. s. (Fig. 1, C–E), of which I. cauliflora and I. kuakuensis are included in our study (Fig. 6). Captaincookia mainly differs from Ixora by drooping flowers, infundibuliform corollas (Fig. 1, E5), and permanently erected stigma lobes, but its fruits (Fig. 1, E6) resemble those of the Pacific Ixora representatives. The placement of Versteegia within Ixoreae is still unresolved because V. cauliflora is either sister to all other Ixoreae (Fig. 5) or unresolved at the base of the tribe (Fig. 6), and the position of V. grandifolia is unclear in our study (cf. basal nodes support in Fig. 6). Only the flattened convex pyrene (Valeton, 1911; Fig. 1, E8) differentiates these two latter species from other Ixora (with globose pyrenes; Fig. 1, E7, E9), a characteristic not shared by V. solomonensis Ridsd. (not included in our analyses) with globose pyrenes (Ridsdale et al., 1972). Versteegia is a morphologically poorly consistent genus, not supported as monophyletic by our study (Figs. 2, 6), and cannot be maintained at generic level. The species Cyclophyllum ixoroides is confirmed as a member of this broadly delimited Ixora, but this placement does not necessitate a reassessment of the genus Cyclophyllum in Vanguerieae (Mouly et al., 2007; Mouly and Achille, 2007; Razafimandimbison et al., in press). [Vol. 96 This broad circumscription of Ixora renders the tribe Ixoreae sensu Mouly et al. (in press) monogeneric. Relations and geographic distributions of the major lineages of Ixora—The main diversity of Ixora is recorded from tropical Asia (i.e., India, South East Asia, and Malesia; Bremekamp, 1937b; De Block, 1998). The occurrence of the sister tribe Aleisanthieae in Malesia and the next closely related tribe Greeneeae in the tropical Asia strongly indicates a tropical Asian origin of Ixora and Ixoreae (Mouly et al., in press). The position of tropical Asian Ixora species both at the base of the Ixora tree and in the two main Ixora clades further supports this hypothesis (Fig. 6). Proper analyses to assess the historical biogeography of the newly circumscribed Ixora would benefit from a better geographical coverage of the species sampling. Consequently, the biogeography and the molecular dating of Ixora will be addressed in a separate study. The present analyses (Fig. 5) identify monophyletic groups in Ixora, which largely correspond to the following tropical regions: tropical Asia, Pacific Islands, the neotropics, mainland Africa (including Madagascar), and the Mascarenes. The Malagasy and African Ixora clade appears surprisingly more closely related to neotropical Ixora than to the neighboring Mascarene Islands taxa. The sampled Ixora species are resolved in two large lineages: the Asian-Pacific and Afro-Malagasy-neotropicalMascarene lineages. Within the tropical Asian-Pacific clade, our results support the monophyly of the sampled Pacific Ixora species, which are sister to a large tropical Asian clade. On the other hand, the monophyly of the tropical Asian Ixora is not supported, as the noncultivated Asian I. calycina and I. nigricans, respectively, are nested within the Pacific and Afro-Malagasy-neotropical-Mascarene lineages (Fig. 5). Six Ixora species (I. brunonis, I. casei, I. chinensis, I. finlaysoniana, and I. pavetta) are also nested in the latter lineage in the cp-tree (Fig. 3) but embedded in the Asian clade in the nr-tree (Fig. 2); we argue that the placement of these six species is likely to be a reflection of their hybrid origins due to recent artificial and/or natural crossings. Within the Afro-Malagasy-neotropical-Mascarene lineage, the Mascarene clade is sister to the Ixora nigricans-neotropicalAfro-Malagasy clade (Fig. 6). The neotropical clade is sister to the Afro-Malagasy clade, and a strongly supported Malagasy clade is in turn sister to an Afro-Malagasy group. These two Malagasy groups indicate two independent colonization events of Ixora in Madagascar, of which one seems to have been via a single long-dispersal event from either tropical Asia (if the position of I. nigricans in the combined tree is correct) or Mascarene (if it turns out that tropical Asian Ixora descended from a single common ancestor) and the other via Eastern Africa. Ixora infrageneric classification (Fig. 7)—The monophyly of the current subgeneric classifications (Bremekamp, 1937b; De Block, 1998; Table 2) of Ixora is tested for the first time here, by mapping the subgenera and sections of Ixora onto the combined nr- and cpDNA tree. It demonstrates that the combi→ Fig. 6. Ixoreae cladogram of the Bayesian analysis majority rule consensus tree for the large nuclear and chloroplast combined data sets, species with conflicting positions in Figs. 2 and 3 represented only by their ETS sequences, but excluding cultivated species. The native area of species is abbreviated after the taxon: AFR, Africa; AS, Asia; CAR, Caribbean; FIJ, Fiji; FP, French Polynesia; MAD, Madagascar; MAL, Malesia; MAS, Mascarenes; MIC, Micronesia; NC, New Caledonia; NEO, neotropics; NG, New Guinea. Optimization of characters on the tree topology; number of carpels per ovary: black lines = 2, gray lines = >2; position of inflorescences: solid lines = terminal, dashed lines = caulinary. The black rectangle shows the sole change to functional dioecy. March 2009] Mouly et al.—Phylogeny of IXORA (Rubiaceae) 699 700 American Journal of Botany nations of character states previously used for circumscribing infrageneric groups (Table 2) largely represent unnatural groups, because most of these are not monophyletic. For example, sampled species of the subgenus Ixora, characterized by opposite and articulate inflorescence branchlets (Fig. 1 B), are resolved in two distinct clades of Ixora (Fig. 7). Subgenus Pavettoides, which mostly belongs to the Pacific clade and is characterized by subopposite and not articulate branchlets (Fig. 1, A), is also paraphyletic (see Fig. 7). In contrast with Bremekamp (1937b) who thought that inflorescence structure was important for infrageneric classification, our results show that the paraphyly of tested subgenera (Fig. 1, A, B) renders it not diagnostic at the considered level. We are unable to test here the monophyly of the subgenus Sathrochlamys (few species from Papua New Guinea placed in four sections; Bremekamp, 1937b) because no recent material is available for DNA studies, but subgenus Sathrochlamys has been considered as poorly defined from a morphological ground (De Block, 1998). At the section level, the mapping determined both natural and unnatural taxonomic concepts (Fig. 7). The sole clearly supported section is Myonima sensu Baillon (1879), although the author never clearly characterized or defined this section (Fig. 7). Within Ixora subgen. Ixora, six of the eight described sections are represented in the study (Fig. 6). Three are polyphyletic, namely, sections Ixora, Otobactrum (Bremekamp, 1937b), and Cremixora (Baillon, 1880). Cremixora, a monotypic section described by Baillon (1880), is probably polyphyletic here because the different specimens of I. cremixora included in our analyses (Figs. 5, 6) do not form exclusive lineages. Its diagnostic character, pendulous ovules, is also present in Cyclophyllum ixoroides, representing convergent evolution. Section Micrixora (Hochreutiner, 1908), described for species with flowers bearing minute corollas and here represented by the neotropical I. aluminicola and I. peruviana, is paraphyletic. Section Chlamydanthus cannot be tested here because it is represented by a single species, I. finlaysoniana (Bremekamp, 1937b), excluded from the combined analyses because of its conflicting positions. Within subgenus Pavettoides, four of the six recognized sections are tested (Fig. 7). Sections Pavettopsis, Raphidanthus (Bremekamp, 1937b) and Vitixora (Fosberg, 1942; Smith and Darwin, 1988) are polyphyletic. Section Phylleilema (Gray, 1858; Fosberg, 1937; Smith and Darwin, 1988; De Block, 1998), characterized by 3–10-florous subsessile inflorescences embedded within rounded foliaceous bracts (Fig. 1, D), is paraphyletic. Section Microthamnus was suspected by Guédès (1986) to form a natural lineage of species with solitary flowers embedded in a calycule. Considering the geographical units obtained here, this section solely represented by the New Caledonian I. dzumacensis, may not be supported if the other representatives, from Madagascar (e.g., I. reducta Drake ex Guédès), are demonstrated in the future to belong to the AfroMalagasy clade. According to the mapping of infrageneric names (Fig. 7, Table 2, Appendix 2), many sections are not monophyletic. Most of these infrageneric groups were described to accommodate the Malesian species (Bremekamp, 1937b, 1938), which are poorly represented in our study from lack of available material. Several sections or type species of sections are not sampled here, and consequently, no classification can be proposed for the infrageneric clades. It can still be concluded that several infrageneric taxa of Ixora do not constitute natural groups and that the characters combinations used to recognize them (e.g., length of the inflorescence peduncle, number of flowers per inflorescence, size and hairiness of the corolla, shape of the bracts [Vol. 96 and bracteoles) should be newly evaluated in the light of phylogenetic relationships and evolution in Ixora. An appropriate study of Ixora infrageneric classification would clearly benefit from a complete taxonomic revision of the genus, an investigation of morphology and anatomy of numerous species, and the inclusion of subgenera and types of sections, especially the Malesian ones, in phylogenetic reconstruction. Note on widely distributed and morphologically variable Ixora species— At the species level, the Malagasy-Comorian I. cremixora discussed earlier (Ixora infrageneric classification section) and the African I. brachypoda and I. guineensis, represented in the study by two or three specimens each, do not form exclusive lineages (Figs. 5, 6). Interestingly, these species are largely widespread Ixora. Widely distributed species are known to present an important morphological variability, and the circumscription of I. guineensis has already been questioned based on morphological ground (De Block, 1998: 169). Considering so, and according to our molecular phylogenetic results, we suggest that these species need to be more carefully investigated using both molecular and morphological data. Taxonomic synopsis— The enlargement of the genus Ixora to include Captaincookia, Doricera, Myonima and Versteegia species necessitates taxonomic changes. An updated circumscription of Ixora is here provided, with a list of synonyms and the consequent combinations, including the combination of species previously described for Cyclophyllum. Ixora L., Sp. Pl.: 110 (1753).—Type: I. coccinea L. (lectotype designated by Hitchcock and Green, 1929). Schetti Adans., Fam. Pl. 2: 146 (1763), nom. illeg. Sideroxyloides Jacq., Strip. Amer.: 19, t. 175 (1763).—Type: S. ferrum Jacq. Patabea Aubl., Fl. Guiane Fr.: 110, t. 43 (1775).—Type: P. coccinea Aubl. Siderodendrum Schreb., Gen. Pl. ed. 8: 71 (1789), nom. illeg.—Type: S. floribundum Schreb. Myonima Comm. ex A.Juss., Gen. Pl.: 206 (1789).—Type: non designatus. Bemsetia Rafin., Sylv. Tellur.: 12 (1838).—Type: B. paniculata Rafin. Pancheria Montrouz., Mém. Acad. Roy. Sci. Lyon, Sect. Sci. 10: 223 (1860), nom. rej.; Beauvisage, Gen. Montrouz.: 58–60 (1901); Guillaumin and Beauvisage, Species Montrouz.: 22 (1914).—Type: P. collina Montrouz. Charpentiera Vieillard, Bull. Soc. Linn. Normandie 9: 346 (1865); Beauvisage, Gen. Montrouz.: 58 (1901), non Gaudich. (1826), nom. illeg.—Type: C. bracteata Vieill. Hitoa Nadeaud, Journ. Bot. (Morot) 13: 2 (1899); K.Krause, in Engler and Prantl, Nat. Pfl. Fam. Nachtr. 3: 329 (1908).—Type: H. mooreensis Nadeaud. Versteegia Valeton, Nova Guinea 8: 483 (1911), syn. nov.— Type: non designatus. Becheria Ridl., J. Straits Branch Roy. Asiat. Soc. 61: 20 (1912).—Type: B. parviflora Ridl. Thouarsiora Homolle ex Arènes, Not. Syst., Paris, 14: 19 (1961).—Type: T. littoralis Homolle ex Arènes. Captaincookia N.Hallé, Adansonia, n.s., 13: 197 (1973), syn. nov.—Type: C. margaretae N.Hallé. Doricera Verdc., Kew Bull. 37: 554 (1983), syn. nov.— Type: D. trilocularis (Balf.f.) Verdc. March 2009] Mouly et al.—Phylogeny of IXORA (Rubiaceae) Fig. 7. Ixora phylogenetic trees illustrating the different subgenera or sections earlier proposed within Ixora (Appendix 2). The topology used for each case represents the phylogram of the ingroup from the complete combined data set (Fig. 6); branches indicated in bold belong to representatives of the infrageneric taxon indicated below the considered tree. 701 702 American Journal of Botany [Vol. 96 Tsiangia But, H.H.Hsue & P.T.Li, Blumea 31: 311 (1986).— Type: T. hongkongensis (Seem.) But, H.H.Hsue & P.T.Li. here because it is included in the treatment of the revision of Ixora of Madagascar (De Block, in press). 1. Ixora borboniae Mouly & B.Bremer. nom. nov. Type: La Réunion, Commerson & Sonnerat s.n. (Holotype P-LA). Myonima obovata Lam., Illust. 1: 288 (1792), sine nomen; non I. obovata (E.Mey.) Kuntze, Rev. Gen. Pl.: 287 (1891); Myonima myrtifolia Lam., Illust. 1: 288 (1792), sine nomen; non Ixora myrtifolia A.C.Sm., Bull. Bishop Mus., Honolulu, 141: 142 (1936). Note: The specific epithet borboniae is chosen with the reference to a name already used for the species as “? Myonima borboniae” Raeusch., nom. nud. 2. Ixora ixoroides (Guillaumin) Mouly & B.Bremer. comb. nov. Basionym: Cyclophyllum ixoroides Guillaumin, Arch. Bot. Caen, Mém. 5: 22 (1930). Type: Nouvelle-Calédonie, Balade, Vieillard 730 (Holotype P; Isotypes P). Note: This species was placed in the genus Cyclophyllum because of its ovule morphology, despite its extreme resemblance to Ixora species, as noted by Guillaumin (1930). The clear placement of the species in Ixora has been verified by our molecular data. 3. Ixora margaretae (N.Hallé) Mouly & B.Bremer. comb. nov. Basionym: Captaincookia margaretae N.Hallé, Adansonia, sér. 2, 13: 197 (1973). Type: Nouvelle Caledonie, près de Pouembout, forêt basse, fl. & fr., 2 Nov 1971, MacKee 24542 (Holotype, P; Isotype, L, NOU, P). 4. Ixora minor (Valeton) Mouly & B.Bremer. comb. nov. Basionym: Versteegia minor Valeton, in Engler, Bot. Jahrb. 61: 67 (1927), in clavi. Type: Beaufort river, Nova Guinea Neerlandica meridionalis, 80 m, fl. 14 Nov 1912, Pulle 347 (Lectotype BO, hic designatus; Isolectotype L). Note: The species was described in a key, without citation of material. After examination of the few specimens available for the species in L, this collection seemed the most representative of the original specimens. 5. Ixora nitens (Poir.) Mouly & B.Bremer. comb. nov. Basionym: Myrtus nitens Poir. in Lam., Encycl. Suppl. 4: 51 (1816). Myonima nitens (Poir.) Verdc., Kew Bull. 37: 558 (1983). Type: Coll. unknown. s.n. (Holotype Herb. Desf., FI; Photo K). 6. Ixora novoguineensis Mouly & B.Bremer. nom. nov. Replaced name: Psychotria ? cauliflora Laut. & K.Schum., Fl. Schutzgeb. Südsee: 574 (1901); Versteegia cauliflora (Laut. & K.Schum.) Valeton, Nova Guinea 8: 483, table 73 (1911), non Ixora cauliflora Montrouz, Mém. Acad. Lyon, 10: 224 (1860). Type: New Guinea, River Gogol, fr., 8 Nov 1890, Lauterbach 910 (Syntype B); New Guinea, Bismarck-Ebene, 100 m, fr., 8 Jul 1896, Lauterbach 2482 (Syntype B); Bismarck-Gebirge, 30 Jun 1899, Rodatz & Klink 163 (Syntype B). Note: The species needs a new name because the binomial is already occupied. The species epithet was chosen to indicate the islands where the species grows. It is not yet possible to designate a lectotype because we have seen no original material. It is probable that these specimens were destroyed in B, and no duplicate has been seen yet in the other herbaria visited. 7. Ixora ridsdalei Mouly & B.Bremer. nom. nov. Replaced name: Versteegia puberula Ridsdale, Blumea 20: 340 (1972), non Ixora puberula (Hiern) Kuntze, Rev. Gen. Pl. 1: 287 (1891). Type: W. New Guinea, McCluer, Anahasi near Babo, alt. ca 50 m, 15 May1941, Aet (exp. Lundquist) 107 (Holotype BO; Isotype L). Note: The new name is dedicated to the author of the species. 8. Ixora solomonensis (Ridsdale) Mouly & B.Bremer, comb. nov. Basionym: Versteegia solomonensis Ridsdale, Blumea 20: 340 (1972). Type: New Georgia Group, Baga Island, Solomon Islands, fl. 11 Jan 1963, B.S.I.P. (leg. Whitmore) 1364 (Holotype L). 9. Ixora trilocularis (Balf.f.) Mouly & B.Bremer. comb. nov. Basionym: Pyrostria trilocularis Balf.f., J. Linn. Soc. Bot. 16: 14 (1877). Doricera trilocularis (Balf.f.) Verdc., Kew Bull. 37: 555 (1983). Type: Rodrigues, Aug/Dec 1874, fr., Balfour s.n. (Lectotype K, hic designatus; Isolectotypes E, P). Note: The lectotype was chosen among the original material observed from K. 10. Ixora valetoniana Mouly & B.Bremer. nom. nov. Replaced name: Versteegia grandifolia Valeton, Nova Guinea 8: 483, table 73 (1911), non Ixora grandifolia Zoll. & Mor., Syst. Verz.: 65 (1846). Type: New Guinea, Ort, 25 Mar 1908, Branderhorst 320 (Syntype BO); Nova Guinea Neerlandica meridionalis, Versteeg 1039 (Syntype BO). Note: The new name is dedicated to the author of the original description of the species. Because no original material has been seen, it was not possible to lectotypify the species. 11. Ixora vaughanii (Verdc.) Mouly & B.Bremer. comb. nov. Basionym: Myonima vaughanii Verdc., Kew Bull. 37: 558 (1983). Type: Mauritius, Commerson 354 (Holotype P; Isotype L, P). Conclusion— Phylogenetic analyses of a large sample of taxa of Ixoreae for many molecular markers led to interesting results and perspectives for the ornamental, species-rich genus Ixora. The separate analyses of nuclear and chloroplast regions were partially incongruent. At least four natural hybridization events were detected, one or more in Asia, one in Africa, and two in Oceania. Other conflicting positions are most likely due to the inclusion of six specimens of cultivated species suspected to have artificial hybrid origins. The combined data sets provided good resolution and usually strong support for main clades, but considering the hypothesis of hybridizations, one might take into account only tree topologies inferred from nuclear sequences or chloroplast sequences separately. At the generic level, however, the hybridization effect on relationships is low, and both nuclear and chloroplast data sets, separately or combined, concurred to recognize the species-rich genus Ixora, as presently circumscribed, to be poly- or paraphyletic. According to our results, both Captaincookia and Myonima species formed natural lineages within Ixora, and the monotypic Doricera is sister group to the Myonima subclade. The genus Versteegia was polyphyletic because V. cauliflora was unresolved at the first divergence of Ixoreae, while the second species V. grandifolia was included in the Asian-Pacific clade. The results confirmed the previous inclusion of Hitoa, Sideroxyloides and Thouarsiora in Ixora. Cyclophyllum ixoroides, presumed closely related to Ixora based on morphology, was also placed within Ixora. Considering the few distinctive characters between the lineages, we propose a broad circumscription of Ixora including all other Ixoreae representatives: Captaincookia, Doricera, Hitoa, Myonima, Sideroxyloides, Thouarsiora, and Versteegia. The genus enlargement necessitates only 11 combinations or new names in Ixora. Within the newly circumscribed genus, several Ixora infrageneric taxa are shown to be poly- or paraphyletic. However, these latter interpretations may be more affected by the detected hybridization events and need to be assessed carefully for more nuclear data in the future. Molecular phylogenetic studies like the present one are probably the best approach for tackling species-rich, problematic genera and to clarify their circumscriptions. Such phylogenetic studies, including a wide sample of taxa, can be a good start for later global taxonomic treatments. Complementary note: the fourth Myonima species does not need a new name because Ixora parviflora Lam. is the first available name for the species Myonima violacea (Lam.) Verdc., non Ixora violacea Lour. The illegitimate name I. littoralis (Homolle ex Arènes) Guédès non I. littoralis Merr., is not corrected LITERATURE CITED Akaike, H. 1974. A new look at the statistical model identification. IEEE Transactions on Automatic Control 19: 716–723. Alvarez, I., and J. F. Wendel. 2003. Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29: 417–434. Andersson, L. 1992. A provisional checklist of neotropical Rubiaceae. Scripta Botanica Belgica 1: 1–199. 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Taxon; locality; voucher specimen (herbarium code); GenBank accession numbers: ETS (clones); rps16; trnT-F. Aleisanthiopsis distantiflora (Merr.) Tange; Indonesia; Kessler et al. 41 (P); FJ150425; EU817434; EU817453. Captaincookia margaretae N.Hallé; New Caledonia; Mouly A. & Innocente E. 222 (P); FJ150426; EU817436; EU817456. Captaincookia sp. 1; New Caledonia; Munzinger J. & Gateblé G. 2148 (NOU); FJ150427; FJ150613; FJ150537. Captaincookia sp. 2; New Caledonia; Munzinger J. (leg. Létocart) 2182 (NOU); FJ150428, FJ150429, FJ150430, FJ150431; FJ150614; FJ150538. Cyclophyllum ixoroides Guillaumin; New Caledonia; McKee H.S. 33549 (P); NA; FJ150615; FJ150539. Cyclophyllum merrillianum Guillaumin; New Caledonia; Mouly A. 150 (P); FJ150432; FJ150616; FJ150540. Doricera trilocularis (Balf.f.) Verdc.; Rodrigues; Lesouef 31 (TAN); FJ150433, FJ150434; EU817437; EU817457. Greenea corymbosa (Jack) K.Schum.; Thailand; Beusekom et al. 752 (P); FJ150435; EU817438; EU817458. Hitoa mooreensis Nadeaud; Society Is.; Florence s.n. (P); FJ150478; EU817441; EU817462. Ixora aluminicola Steyerm.; French Guyana; Prévost 4160 (P); FJ150436, FJ150437, FJ150438, FJ150439; FJ150617; FJ150541. Ixora amplidentata De Block ined.; Madagascar; Andrianantoanina et al. 292 (P); NA; FJ150618; FJ150542. Ixora bauchiensis Hutch. & Dalziel; Cameroun; Fotius 3047 (P); FJ150440; FJ150619; FJ150543. Ixora brachypoda DC. 1; Gabon; Bradley A.F. et al. 1022 (MO); FJ150441; EU817442; EU817463. Ixora brachypoda DC. 2; Togo; Hakki et al. 297 (P); NA; FJ150620; FJ150544. Ixora brachypoda DC. 3; Gabon; Walters et al. 1437 (MO); FJ150442; FJ150621; FJ150545. Ixora brunonis Wall. ex G.Don; Thailand; Larsen K. et al. 43463 (P); FJ150443; EU817446; EU817470. Ixora calycina Thwaites; Sri Lanka; Tirvengadum et al. 18 (P); NA; FJ150622; FJ150546. Ixora casei Hance; Cultivated Tahiti, French Polynesia; Mouly A. & Florence J. 348 (P); FJ150444; FJ150623; FJ150547. Ixora cauliflora Montrouz.; New Caledonia; Mouly A. & Innocente E. 267 (P); FJ150445; FJ150624; FJ150548. Ixora chinensis Lam.; Cultivated Uppsala, Sweden; no voucher; FJ150446; FJ150625; FJ150549. Ixora coccinea L.; Cultivated Uppsala, Sweden; Bremer B. 2719 (UPS); FJ150447, FJ150448; EF205641; EU817464. Ixora collina (Montrouz.) Beauvis.; New Caledonia; Mouly A. & Innocente E. 236 (P); FJ150449; FJ150626; FJ150550. Ixora comptonii S.Moore; New Caledonia; Munzinger J. 1606 (NOU); FJ150450; FJ150627; FJ150551. Ixora cremixora Drake 1; Madagascar; Leeuwenberg 13879 (P); FJ150451; FJ150628; FJ150552. Ixora cremixora Drake 2; Madagascar; Kårehed J. et al. 219 (UPS); FJ150452; NA; NA. Ixora diversifolia R.Br. ex Kurz; Thailand; Charoenphol et al. 3719 (P); NA; FJ150629; FJ150553. Ixora dzumacensis Guillaumin; New Caledonia; Mouly A. et al. 275 (P); FJ150453; FJ150630; FJ150554. Ixora elegans Gillepsie; Fiji; Smith A.C. 9535 (P); NA; FJ150631; FJ150555. Ixora elliotii De Block ined.; Madagascar; Dumetz 1175 (P); FJ150454; FJ150632; FJ150556. Ixora emirnensis Baker; Madagascar; Lowry P.P II et al. 6018 (MO); FJ150455; FJ150633; FJ150557. Ixora fastigiata (R.G.Good) Bremek.; de Foresta HF1213 (P); NA, NA, FJ150558. Ixora finlaysoniana Wall. & G.Don.; Cultivated Tanzania; Luke G. 9042 (S); FJ150458, FJ150459, FJ150460, FJ150461; EF205643; EU817466. Ixora foliosa Hiern; Cameroun; Onana et al. 566 (P); FJ150462; FJ150635; FJ150560. Ixora francii Schltr. & K.Krause; New Caledonia; Mouly A. & McPherson G. 126 (P); FJ150463; FJ150636; FJ150561. Ixora graciliflora Benth.; French Guyana; De Granville J.J. 1459 (P); FJ150464; FJ150637; NA. Ixora guineensis Benth. 1; Ghana; Gereau R.E. et al. 5601 (MO); FJ150465; EU817443; EU817467. Ixora guineensis Benth. 2; Cameroun; Cheek 7075 (P); FJ150466; FJ150638; NA. Ixora hiernii Scott-Elliot; Sierra Leone; Adam 23101 (P); FJ150467; FJ150639; FJ150562. Ixora hookeri (Oudem.) Bremek.; Cultivated Tahiti, French Polynesia; Mouly A. & Florence J. 342 (P); FJ150468; EU817444; EU817468. Ixora iteophylla Bremek.; Malaysia; Schaller et al. 3932 (P); FJ150469; FJ150640; FJ150563. Ixora javanica (Blume) DC.; Laos; Munzinger J. 119 (P); FJ150519; NA; FJ150602. Ixora jourdanii Mouly & J.Florence; Marquesas Is.; Mouly A. 513 (P); FJ150470; FJ150641; FJ150564. Ixora killipii Standl.; Colombia; Andersson L. et al. 2160 (GB); NA; AF201001; AF152659. Ixora kuakuensis S.Moore; New Caledonia; Munzinger J. 2180 (NOU); FJ150471; FJ150642; FJ150565. Ixora lecardii Guillaumin; New Caledonia; Mouly A. et al. 282 (P); FJ150472; FJ150643; FJ150566. Ixora longiloba Guillaumin; New Caledonia; Mouly A. 165 (P); FJ150474; FJ150644; FJ150567. Ixora marquesensis F.Br.; Marquesas Is.; Mouly A. 504 (P); FJ150475; FJ150645; FJ150568. Ixora minutiflora Hiern; Gabon; Hallé F. et al. 4778 (P); FJ150476; FJ150646; FJ150569. Ixora mocquerysii Aug.DC.; Madagascar; Malcomber S. 2805 (MO); FJ150477; FJ150647; FJ150570. Ixora narcissodora K.Schum.; Kenya; Luke G. 8324 (UPS); FJ150479, FJ150480; FJ150648; FJ150571. Ixora nematopoda K.Schum.; Cameroun; Nemba et al. 332 (P); FJ150481; FJ150649; FJ150572. Ixora nigricans R.Br. ex Wight & Arn.; Thailand; Larsen K. et al. 43037 (P); FJ150482; FJ150650; FJ150573. Ixora nimbana Schnell; Liberia; Adam 26253 (P); FJ150483; FJ150651; FJ150574. Ixora oligantha Schltr. & K.Krause; New Caledonia; Mouly A. et al. 296 (P); FJ150484; FJ150652; FJ150575. Ixora pavetta Andr.; Cultivated Uppsala, Sweden; FTG P 1738 (UPS); FJ150485; FJ150653; FJ150576. Ixora pelagica Seem.; Fiji; Smith A.C. 9288 (P); FJ150486; FJ150654; FJ150577. Ixora peruviana (Spruce ex K.Schum.) Standl.; Peru; Plowman et al. 11541 (P); FJ150487; FJ150655; FJ150578. Ixora platythyrsa Baker; Madagascar; Antilahimena 94 (P); FJ150488; NA; FJ150579. Ixora quadrilocularis Capuron ex De Block, ined.; Madagascar; Capuron R. 23980SF (P); NA; FJ150656; NA. Ixora raiateensis J.W.Moore; Society Is.; Mouly A. et al. 400 (P); FJ150489; FJ150657; FJ150580. Ixora scheffleri K.Schum. & K.Krause; Tanzania; Luke G. 9162 (UPS); FJ150490, FJ150491, FJ150492, FJ150493; FJ150658; FJ150581. Ixora setchellii Fosberg; Society Is.; Mouly A. et al. 352 (P); FJ150494; FJ150659; FJ150582. Ixora siphonantha Oliv. 1; Madagascar; Carlson 45 (P); FJ150495; FJ150660; FJ150583. Ixora siphonantha Oliv. 2; Madagascar; Rabenantoandro et al. 944 (MO); FJ150496; FJ150661; FJ150584. Ixora sp. 01; Madagascar; Kårehed J. et al. 234 (UPS); FJ150497; FJ150662; FJ150585. Ixora sp. 02; Madagascar; Razafimandimbison S.G. et al. 519 (UPS); FJ150498; FJ150663; FJ150586. Ixora sp. 03; Madagascar; Andrianjafy et al. 30 (MO); FJ150499; NA; NA. Ixora sp. 04; Madagascar; RRH 5 (P); FJ150500; FJ150664; FJ150587. Ixora sp. 05; Cultivated Stockholm, Sweden; Mouly s.n. (P); FJ150501, FJ150502; FJ150665; FJ150588. Ixora sp. 06; New Caledonia; Mouly A. & Innocente E. 215 (P); FJ150503; FJ150666; FJ150589. Ixora sp. 07; New Caledonia; Mouly A. & Innocente E. 272 (P); FJ150504; FJ150667; FJ150590. Ixora sp. 08; Society Is.; Florence J. 3936 (P); FJ150505; FJ150668; FJ150591. Ixora sp. 09; New Caledonia; Dagostini G. 859 (NOU); FJ150506; FJ150669; FJ150592. Ixora sp. 10; Asia; Martin 1314 (P); FJ150507, FJ150508, FJ150509; FJ150670; FJ150593. Ixora sp. 11; Thailand; Geesink et al. 7226 (P); FJ150510; FJ150671; FJ150594. Ixora sp. 12; Thailand; Vidal 5771 (P); NA; FJ150672; FJ150595. Ixora sp. 13; Thailand; Vidal 5758B (P); FJ150511, FJ150512, FJ150513, FJ150514; FJ150673; FJ150596. Ixora sp. 14; Cultivated Costa Rica; Nissen 89BIO01344 DN89 (UPS); FJ150515; FJ150674; FJ150597. Ixora sp. 15; Vietnam; Poilane 103 (P); NA; FJ150675; FJ150598. Ixora sp. 16; Brunei; Malcomber S. et al. 2980 (MO); FJ150516; FJ150676; FJ150599. Ixora sp. 17; Thailand; Larsen K. et al. 86KL14 (UPS); FJ150517; FJ150677; FJ150600. Ixora st-johnii Fosb.; Society Is.; Mouly A. 450 (P); FJ150518; FJ150678; FJ150601. Ixora tanzaniensis Bridson; Tanzania; Luke G. 9304 (UPS); FJ150520, FJ150521, FJ150522, FJ150523; EU817447; EU817471. Ixora tenuis De Block; Ghana; Hall et al. 46573 (P); FJ150524; FJ150679; FJ150603. Ixora triantha Volkens; Mariannas Is.; Stone 16019 (P); FJ150525; FJ150680; FJ150604. Ixora trichocalyx Hochr.; Madagascar; Schatz et al. 3515 (P); FJ150526; FJ150681; FJ150605. Ixora vieillardii Guillaumin; New Caledonia; Mouly A. et al. 322 (P); FJ150527; FJ150682; FJ150606. Ixora yaouhensis Schltr.; New Caledonia; Mouly A. 153 (P); FJ150528; FJ150683; FJ150607. Myonima nitens (Poir.) Verdc.; Mascarene Is.; Friedmann F. 2631 (P); FJ150529; FJ150684; FJ150608. Myonima obovata Lam.; Mascarene Is.; Friedmann F. 3049 (P); FJ150530; FJ150685; FJ150609. Myonima sp. American Journal of Botany 706 01; Mascarene Is.; Andrianbololonera S. 47 (TAN); FJ150531, FJ150532; FJ150686; FJ150610. Myonima violacea (Lam.) Verdc.; Mascarene Is.; Lorence D.L. 1526 (P); FJ150533; EU817449; EU817473. Sideroxyloides ferreum Jacq. 1; Carribean Is.; Taylor C. 11693 (UPS); FJ150456; EF205642; EU817465. Sideroxyloides ferreum Jacq. 2; Puerto Rico.; Axelrod et al. 1515 (P); FJ150457; FJ150634; FJ150559. Thouarsiora littoralis Homolle ex Arènes; Madagascar; McPherson G. & Rabenantoandro J. 18287 (MO); FJ150473; EU817445; EU817469. Versteegia cauliflora (K.Schum & Lauterb.) Valeton; Cultivated Bogor, Indonesia; Drodz & Molem s.n. (UPS); FJ150534, FJ150535; EU817451; EU817476. Versteegia grandifolia Valeton; Cultivated Bogor, Indonesia; Ridsdale s.n. (UPS); FJ150536; FJ150687; FJ150611. Appendix 2. A priori assignation of ingroup species to infrageneric groups of Ixora based on literature and morphological limits presented in Table 2, to test infrageneric classifications proposed in the past for Ixora by optimization on the phylogram of the tree topology presented in Fig. 6 (see Fig. 7). Infrageneric group: species list. Subgenus Ixora: Ixora siphonantha Oliv. 1, Ixora siphonantha Oliv. 2, Ixora sp. 03, Ixora sp. 04, Ixora sp. 12, Ixora sp. 13. Section Brachypus: Ixora nigricans R.Br. ex Wight & Arn., Ixora tenuis De Block. Subgenus Pavettoides: Section Chlamydanthus: Ixora finlaysoniana Wall & G.Don. Section Pavettopsis: Ixora elegans Gillepsie, Ixora francii Schltr. & K.Krause, Ixora lecardii Guillaumin, Ixora raiateensis J.W.Moore, Ixora sp. 08, Ixora sp. 09, Ixora vieillardii Guillaumin, Ixora yaouhensis Schltr. Section Cremixora: Ixora cremixora Drake 1, Ixora cremixora Drake 2. Section Amphorion: Ixora calycina Thwaites. Section Ixora: Ixora bauchiensis Hutch. & Dalziel, Ixora brunonis Wall. ex G.Don, Ixora casei Hance, Ixora chinensis Lam., Ixora coccinea L., Ixora elliotii De Block ined., Ixora emirnensis Baker, Ixora graciliflora Benth., Ixora foliosa Hiern, Ixora guineensis Benth. 1, Ixora guineensis Benth. 2, Ixora hiernii Scott-Elliot, Ixora iteophylla Bremek., Ixora javanica (Blume) DC., Ixora killipii Standl., Ixora minutiflora Hiern, Ixora narcissodora K.Schum., Ixora pavetta Andr., Ixora sp. 01, Ixora sp. 02, Ixora sp. 05, Ixora sp. 10, Ixora sp. 11, Ixora sp. 14, Ixora sp. 15, Ixora sp. 16, Ixora sp. 17, Ixora tanzaniensis Bridson, Ixora trichocalyx Hochr. Section Vitixora: Ixora comptonii S.Moore, Ixora longiloba Guillaumin, Ixora pelagica Seem. Section Myonima: Myonima nitens (Poir.) Verdc., Myonima obovata Lam., Myonima violacea (Lam.) Verdc., Myonima sp. 1. Section Phylleilema: Ixora collina (Montrouz.) Beauvis., Ixora jourdanii Mouly & J.Florence, Ixora marquesensis F.Br., Ixora oligantha Schltr. & K.Krause, Ixora setchellii Fosberg, Ixora sp. 06, Ixora sp. 07, Ixora st-johnii Fosb. Section Micrixora: Ixora aluminicola Steyerm., Ixora peruviana (Spruce ex K.Schum.) Standl. Section Otobactrum: Ixora amplidentata De Block ined., Ixora brachypoda DC. 1, Ixora brachypoda DC. 2, Ixora brachypoda DC. 3, Ixora fastigiata (R.G.Good) Bremek., Ixora hookeri (Oudem.) Bremek., Ixora mocquerysii Aug.DC., Ixora nematopoda K.Schum., Ixora nimbana Schnell, Ixora platythyrsa Baker, Ixora scheffleri K.Schum. & K.Krause, Section Raphidanthus: Ixora diversifolia R.Br. ex Kurz, Ixora triantha Volkens. Not assigned to subgeneric group: Section Microthamnus: Ixora dzumacensis Guillaumin. Not assigned to section: Cyclophyllum ixoroides Guillaumin, Captaincookia margaretae N.Hallé, Captaincookia sp. 1, Captaincookia sp. 2, Doricera trilocularis (Balf.f.) Verdc., Hitoa mooreensis Nadeaud, Ixora cauliflora Montrouz., Ixora kuakuensis S.Moore, Ixora quadrilocularis Capuron ex De Block ined., Sideroxyloides ferreum Jacq. 1, Sideroxyloides ferreum Jacq. 2, Thouarsiora littoralis Homolle ex Arènes, Versteegia cauliflora (K.Schum & Lauterb.) Valeton, Versteegia grandifolia Valeton.

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Phylogeny and classification of the species-rich pantropical showy genus Ixora (Rubiaceae-Ixoreae) with indications of geographical monophyletic units and hybrids

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