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Botanical Journal of the Linnean Society, 2013, 173, 387406. With 2 gures
Bergius Foundation, The Royal Swedish Academy of Sciences, SE-106 91 Stockholm, Sweden Department of Ecology, Environment and Plant Sciences, Stockholm University, SE-106 91 Stockholm, Sweden
Received 4 April 2012; revised 2 October 2012; accepted for publication 31 January 2013
Subfamily Ixoroideae is one of three major lineages in Rubiaceae, with approximately 4000 species. Previous molecular phylogenetic studies have indicated that many genera and tribes previously placed in other subfamilies are better considered as part of Ixoroideae. However, the internal resolution and clade support have generally been low, and several genera found to be nested in the subfamily do not appear to be associated with any described tribe. In order to resolve the phylogeny and assess the tribal delimitations in the expanded Ixoroideae, phylogenetic reconstructions were performed using Bayesian and parsimony analyses of six plastid DNA regions and a broad sampling of genera from all tribes of the subfamily. In the inferred phylogenetic hypotheses, the tribal relationships were mostly well supported, with Ixoroideae consisting of the Coffeeae and the Vanguerieae alliances as sister groups and a grade comprising Condamineeae, Henriquezieae, Posoquerieae, Retiniphylleae, Sipaneeae and the genus Steenisia. A revised tribal classication, including the description of ve new tribes, Airospermeae, Augusteae, Scyphiphoreae, Steenisieae and Trailliaedoxeae, is provided. 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387406.
INTRODUCTION
With >13 000 species (Davis et al., 2009), Rubiaceae is one of the largest and most diverse families of angiosperms. Well-known representatives include Coffea L., Ixora L. and Gardenia J.Ellis, all part of Ixoroideae, a subfamily comprising about 4000 species of pantropical and subtropical distributions. Ixoroideae, as recognized by Bremekamp (1952, 1966), included taxa characterized by secondary pollen presentation (ixoroid pollination mechanism), a feature that Bremekamp considered to be of high taxonomic value because of its requirement for a combination of characters for functionality. Included were tribes Acranthereae, Chiococceae, Coptosapelteae, Cremasporeae, Gardenieae, Ixoreae and Vanguerieae. Verdcourt (1958), however, considered pollen presentation to be of less importance, and preferred to
*Corresponding author. E-mail: kent@bergianska.se
include Ixoroideae in a wide Cinchonoideae (see also Robbrecht & Manen, 2006). In a comprehensive classication of the family, Robbrecht (1988) restricted Ixoroideae to include genera with contorted corolla lobe aestivation and eshy fruits. In his system, Ixoroideae comprised tribes Cremasporeae, Gardenieae and Ixoreae sensu Bremekamp (the correct name of which would have been Coffeeae; Darwin, 1976), i.e. Aulacocalyceae, Coffeeae, Gardenieae, Octotropideae and Pavetteae. For an overview of the changes in the classication of Ixoroideae, the reader is referred to Andreasen & Bremer (2000). Results of molecular phylogenetic studies have since then indicated many unknown relationships and also, consequently, possible improvements in the classication of Rubiaceae. This includes the expansion of Ixoroideae, as many additional tribes or segregates of tribes and a number of taxa of uncertain taxonomic placement have been shown to be associated with the subfamily. Ixora, the type of the subfamily, was shown
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K. KAINULAINEN ET AL. Kainulainen et al., 2009; Razamandimbison et al., 2011). Subsequent tribal recognition of the lineages represented by these genera has so far only been performed for Aleisanthia and Aleisanthiopsis and Greenea (Aleisanthieae and Greeneeae, respectively; Mouly et al., 2009a). In this study, we analyse data from six combined plastid DNA regions (matK, ndhF, rbcL, the rps16 intron, trnS-G and trnT-F) using Bayesian and parsimony methods of phylogenetic reconstruction to resolve the tribal relationships of the expanded Ixoroideae and, in particular, the relationships of the genera which, in previous studies, have not been associated with any previously described tribes. Following the inferred phylogenetic hypothesis, we also provide a revised tribal classication of the subfamily.
to form a clade with Vanguerieae (Bremer et al., 1999), conrming the position of Vanguerieae in Ixoroideae, as suggested by Bremekamp (1952, 1966), and supporting the exclusion of Ixora from Pavetteae. Andreasen & Bremer (2000) resurrected Ixoreae and provided new circumscription for several other tribes in the subfamily. Many additional taxa have been found to be associated with Ixoroideae sensu Robbrecht (1988), including Bertiera Aubl. (Bremer, Andreasen & Olsson, 1995), Mussaendeae and Sabiceeae, (Bremer & Thulin, 1998), Alberteae and Scyphiphora C.F.Gaertn. (Bremer et al., 1999), Condamineeae and many genera formerly placed in Cinchoneae or Rondeletieae (e.g. Bremer et al., 1999; Rova et al., 2002; Delprete & Corts-B., 2004; Kainulainen et al., 2010). Crossopteryx Fenzl, part of Ixoroideae sensu Bremekamp (included in Coptosapelteae), but considered part of Cinchoneae by Robbrecht (1988), has also been found to be nested in Ixoroideae (Razamandimbison & Bremer, 2001), as have Mussendopsis (Razamandimbison & Bremer, 2001), Hekistocarpa Hook.f. (Dessein et al., 2001), Gleasonia Standl., Retiniphyllum Humb. & Bonpl. and Sipaneeae (Rova et al., 2002; Delprete & Corts-B., 2004), Boholia Merr. and Steenisia Bakh.f. (Bremer & Eriksson, 2009), Airosperma K.Schum. & Lauterb. (Kainulainen et al., 2009), Greeniopsis Merr. (Alejandro et al., 2010), Glionnetia Tirveng., Jackiopsis Ridsdale and Trailliaedoxa W.W.Sm. & Forrest (Razamandimbison et al., 2011). Although the expanded Ixoroideae is well supported by molecular data, internal resolution and clade support, especially among the early divergent clades, have generally been low. Ixoroideae, as widely delimited according to recent molecular phylogenetic studies, includes all tribes of Robbrechts (1988) narrow circumscription of the subfamily, whereas tribes Acranthereae, Chiococceae and Coptosapelteae are not part of Ixoroideae as suggested by Bremekamp (1966). Chiococceae has been shown to be part of Cinchonoideae (Bremer et al., 1995), and Acranthera Arn. ex Meisn. and Coptosapelta Korth. appear to represent a separate, early divergent clade in the family (Rydin et al., 2009). The expanded Ixoroideae is morphologically diverse and can no longer be easily characterized morphologically. Molecular phylogenetic studies have also shown that some tribes of the subfamily are not monophyletic (i.e. Gardenieae including Aulacocalyceae, and Pavetteae; Andreasen & Bremer, 2000; Persson, 2000; Bremer & Eriksson, 2009) and that several genera found to be nested in Ixoroideae do not appear to be associated with any described tribe, i.e. Airosperma, Aleisanthia Ridl., Aleisanthiopsis Tange, Augusta Pohl, Boholia, Glionnetia, Greenea Wight & Arn., Scyphiphora, Steenisia, Trailliaedoxa and Wendlandia Bartl. ex DC. (Rova et al., 2002; Bremer & Eriksson, 2009;
2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387406
PHYLOGENY OF IXOROIDEAE which in Wendlandia was considered to be doubtful in the last comprehensive revision of the genus by Cowan (1932). We were unsuccessful in obtaining DNA sequences from any Steenisia spp. other than S. pleurocarpa (Airy Shaw) Bakh.f. An overview of the sampling and voucher specimens is given in the Appendix, and a few species representing the different tribes of Ixoroideae are shown in Figure 1.
389
DNA
Three protein coding genes (matK, ndhF and rbcL) and three noncoding regions [the rps16 intron, the trnS-G spacer including the trnG intron (trnSGCUtrnGUUC-trnGUUC) and the trnT-F region (trnTUGUtrnLUAA-trnLUAA-trnFGAA)] of the plastid DNA, previously shown to be phylogenetically informative in Rubiaceae (Andersson & Rova, 1999; Bremer et al., 1999; Razamandimbison & Bremer, 2002; Rova et al., 2002; Andersson & Antonelli, 2005; Kainulainen et al., 2009), were used in this study. DNA was extracted from leaf material using a MiniBeadbeater 3110BX (BioSpec), following the protocol of Doyle & Dickson (1987), and was subsequently puried using the QIAquick PCR purication kit according to the manufacturers instructions (Qiagen). PCR amplications were performed using standard PCR settings and the following primers: matK1198F and matK2053R (Andersson & Antonelli, 2005) for matK; 2F, 1000R, 720F, 1700R, 1320F and 2280R (Rydin, Razamandimbison & Bremer, 2008) for ndhF; rbcL5F, rbcL_Z895F, rbcL_Z895R and rbcL3R (Bremer et al., 2002) for rbcL; rsp16_2F (Bremer et al., 2002) and rpsR2 (Oxelman, Lidn & Berglund, 1997) for the rps16 intron; and cF, eF, dR, fR (Taberlet et al., 1991), A1F, IR (Bremer et al., 2002), 820R, 940F, 1250F, 1880F and 2670R (Rydin et al., 2008) for trnT-F. The trnS-G region was amplied using the primers and protocols of Shaw et al. (2005). Sequences were assembled using the Staden package v1.5.3 (Staden, 1996). Sequences new to this study (324) were deposited in GenBank (Appendix). Additional sequences (361) were obtained from GenBank (for references, see Appendix).
Coffea arabica L. plastid genome (GenBank accession number, EF044213; Samson et al., 2007). An AT-rich region in the trnS-G spacer, corresponding to positions 91849203 of the C. arabica plastid genome, was excluded from the analyses because of difficulties in nding an unambiguous alignment. The combined dataset consisted of 10 165 characters, 10 003 of which were included in the analyses. Methods of phylogenetic reconstruction included maximum parsimony (MP) and Bayesian inference (BI; Yang & Rannala, 1997), using the programs PAUP* v4.0b10 (Swofford, 2002) and MrBayes v3.1.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003), respectively. Gaps were treated as missing data. MP analyses were performed using heuristic searches with the tree bisectionreconnection (TBR) branch swapping algorithm, Multrees on, 1000 random sequence addition replicates and a maximum of 10 trees saved per replicate. Clade support was estimated using 1000 bootstrap replicates, with three random addition replicates per replicate. For the BI analyses, the protein coding and noncoding data were analysed as separate partitions with unlinked model parameter estimates (except topology). Substitution models suggested as best t to the data under the corrected Akaike information criterion (AICc), as implemented in MrAIC v1.4.4 (Nylander, 2004; a script dependent on the program PHYML v3.0; Guindon & Gascuel, 2003), were used for each partition (the GTR + G + I model for both datasets). The analysis comprised two runs of four chains each and was monitored for 107 generations, with every 1000th generation being sampled. The chain heating parameter was 0.10. The initial 25% of the sampled trees were considered as burn-in and excluded from the consensus. The effective sample sizes (ESSs) of the model parameters were checked using the program Tracer v1.5 (Rambaut & Drummond, 2007), in order to ensure an ESS > 200, as recommended by Drummond et al. (2007) for the adequate representation of the posterior probability (PP).
RESULTS
The combined dataset contained 10 003 DNA characters, 2410 of which were potentially phylogenetically informative in the MP analysis. Analyses of the coding and noncoding datasets separately (data not shown) showed no incongruences with bootstrap support (BS) values 70% or PPs 0.95, with the exception of the position of Randia L., which formed a sister group to Euclinia Salisb. (PP, 0.99; BS, 73%) in the coding dataset, but was resolved as sister group to a clade comprising Rosenbergiodendron Fagerl. and Tocoyena Aubl. in the noncoding dataset (PP, 0.95; BS, 84%). The inferred phylogenetic hypotheses of the MP and BI
DATA
ANALYSES
The sequences were preliminarily aligned using Clustal W (default settings; Thompson, Higgins & Gibson, 1994), as implemented in BioEdit (Hall, 1999), and then adjusted manually. Sequence inversions were dealt with by separating the alternative sequence versions in the alignment. Inversions in the included sequences occurred in regions corresponding to positions 54175430 (rps16), 90829113, 9164 9169, 93309335 and 97149723 (trnS-G) of the
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H G
K J
Figure 1. Selected taxa of Ixoroideae: A, Posoqueria longiora; B, Schizomussaenda henryi; C, Bremeria pervillei; D, Sabicea diversifolia; E, Steenisia pleurocarpa; F, Scyphiphora hydrophylacea; G, Glionnetia sericea; H, Ixora borboniae; I, Augusta rivalis; J, Wendlandia tinctoria; K, Bertiera rufa; L, Pavetta sp.; M, Mitriostigma axillare; N, Rosenbergiodendron densiorum (image credits: AD, G, H, KN, by Kent Kainulainen; E, F, J, Christian Puff, Faculty Centre of Biodiversity, University of Vienna; used with permission; I, by Indiana Coronado, Tropicos, botanical information system at the Missouri Botanical Garden http://www.tropicos.org, MBG, licensed under a Creative Commons by-nc-nd 3.0 licence).
2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387406
PHYLOGENY OF IXOROIDEAE analyses were overall mostly congruent, well resolved and strongly supported. Figure 2 shows the majority rule consensus tree from the BI analyses of the combined data, including the PPs of the clades and the clade BS values of the MP analysis. The clades corresponding to the Coffeeae and Vanguerieae alliances, respectively (cf. Razamandimbison et al., 2011), were resolved as sister groups (the core Ixoroideae; PP, 1.00; BS, 100%). The Coffeeae alliance comprised the tribes Alberteae, Bertiereae, Coffeeae, Gardenieae, Octotropideae and Pavetteae and the genera Airosperma, Augusta, Boholia and Wendlandia. Gardenieae did not appear to be monophyletic, because Burchellia R.Br. was resolved as sister group to Octotropideae (PP, 1.00; BS, 64%), and Mitriostigma Hochst. and Oxyanthus DC. formed a sister group to Pavetteae, although with weak support (PP, 0.80; BS, 54%). The GardenieaePavetteae clade was resolved as sister group to the Burchellia Octotropideae clade (PP, 1.00; BS, 65%), and a clade comprising Bertiereae and Coffeeae (PP, 1.00; BS, 98%), in turn, formed a sister group to this clade (PP, 1.00; BS, 100%). Strongly supported successive sister groups were Alberteae (PP, 1.00; BS, 85%), an AugustaWendlandia clade (PP, 1.00; BS, 100%) and an AirospermaBoholia clade (PP, 1.00; BS 100%). In the Vanguerieae alliance, Aleisanthieae formed a sister group to Ixoreae (PP, 1.00; BS, 100%) and this clade was, in turn, sister to Greeneeae (PP, 1.00; BS, 100%). Relationships between the Aleisanthieae GreeneeaeIxoreae clade, Vanguerieae and Glionnetia were unresolved. Although strongly supported by the BI analyses (PP, 1.00), the support for this trichotomy was low in the MP analysis (< 50%). Trailliaedoxa was resolved as sister to the clade formed by this trichotomy. However, this clade also had low support (PP, 0.83; BS, 54%). Successive sister groups were Scyphiphora (PP, 1.00; BS, 87%), Jackiopsis (PP, 1.00; BS, 97%) and Crossopteryx (PP, 1.00; BS, 91%). A grade of successive sister clades to core Ixoroideae comprised, in turn, Retiniphylleae (PP, 1.00; BS, 100%), Steenisia (PP, 1.00; BS, 100%) and a clade comprising the sister tribes Mussaendeae and Sabiceeae (PP, 0.98; BS, 54%). The Mussaendeae Sabiceeae clade was well supported (PP, 1.00; BS, 96%). A CondamineeaeHenriquezieaePosoquerieae Sipaneeae clade was resolved as the earliest diverging clade in relation to the rest of the subfamily. However, the support for this clade was low (PP, 0.79; BS, 72%). Subfamily Ixoroideae received strong support (PP, 1.00; BS, 100%).
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DISCUSSION
In this study, we infer the tribal relationships in Ixoroideae by the phylogenetic reconstruction of
six combined plastid regions. The phylogenetic hypotheses indicate that Ixoroideae can be considered as comprising the Coffeeae alliance, the Vanguerieae alliance and a grade consisting of Retiniphylleae, Steenisia, Mussaendeae with Sabiceeae and a CondamineeaeHenriquezieaePosoquerieae Sipaneeae clade (Fig. 2). Henriquezieae, Posoquerieae and Sipaneeae form a clade, as in the study by Rova et al. (2002). This clade forms a weakly supported sister group to Condamineeae, and the CondamineeaeHenriquezieaePosoquerieaeSipaneeae clade is, in turn, sister group to the remainder of the subfamily. This is congruent with the results of Rydin et al. (2009). However, in the phylogenetic hypotheses of Kainulainen et al. (2009), Condamineeae appeared closer to core Ixoroideae. Further research is needed in order to better understand the relationships of these early divergent lineages. As in previous studies (Rova et al., 2002; Bremer & Eriksson, 2009; Kainulainen et al., 2010), the resolution in Condamineeae was low. In the phylogenetic tree presented, Mussaendeae is resolved as sister group to Sabiceeae with strong support, in agreement with the results of Alejandro, Razamandimbison & Liede-Schumann (2005). As, in their study, Heinsia DC. forms a sister group to the remaining genera of the tribe, and Mussaenda L. forms a clade with Pseudomussaenda Wernham., Schizomussaenda H.L.Li (Fig. 1B) from South-East Asia is here further resolved as sister group to a clade comprising the MalagasyMascarene genera Bremeria Razam. & Alejandro (Fig. 1C) and Landiopsis Capuron ex Bosser. In Sabiceeae, Virectaria Bremek. is resolved as sister group to Tamridaea Thulin & B.Bremer, as in previous studies by Bremer & Thulin (1998) and Khan et al. (2008). These genera, together with Hekistocarpa, have been suggested as constituting a clade recognized at tribal level (Virectarieae s.l., based on molecular data) by Dessein et al. (2001), and the three genera also formed a well-supported clade in the study by Bremer & Eriksson (2009). These relationships are not supported in our study, in which Hekistocarpa instead forms a poorly supported sister group to Sabicea Aubl. However, previously, Hekistocarpa has also been resolved as sister group to the remaining Sabiceeae (Khan et al., 2008; based on nrDNA data). Phylogenetic relationships in this tribe should be investigated further. The position of Retiniphyllum indicated in this study conforms with that rst found in the study by Rova et al. (2002). Corts-B., Delprete & Motley (2009) have subsequently demonstrated the monophyly of the monogeneric Retiniphylleae. The South-East Asian genus Steenisia (Fig. 1E) was segregated from Neurocalyx Hook. (subfamily Rubioideae) by Bakhuizen van den Brink (1952). In a
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Gelsemium sempervirens Logania vaginalis Luculia gratissima Colletoecema dewevrei Ophiorrhiza mungos Cinchona pubescens Rondeletia odorata Guettarda speciosa Cubanola domingensis Cephalanthus occidentalis Nauclea orientalis Henriquezia nitida - HEN Molopanthera paniculata - POS Posoqueria latifolia - POS Posoqueria longiflora - POS Chalepophyllum guyanense - SIP Dendrosipanea spigelioides - SIP Neobertiera gracilis - SIP Sipanea aff. biflora - SIP Sipanea hispida - SIP Pinckneya bracteata - CON Mastixiodendron flavidum - CON Emmenopterys henryi - CON Dioicodendron dioicum - CON Condaminea corymbosa - CON Pogonopus speciousus - CON Ferdinandusa speciosa - CON Simira cordifolia - CON Hippotis triflora - CON Rustia thibaudioides - CON Calycophyllum candidissimum - CON Chimarrhis hookeri - CON Bathysa stipulata - CON Virectaria multiflora - SAB Tamridaea capsulifera - SAB Hekistocarpa minutiflora - SAB Sabicea diversifolia - SAB Sabicea aspera - SAB Sabicea africana - SAB Heinsia crinita - MUS Pseudomussaenda flava - MUS Mussaenda erythrophylla - MUS Mussaenda arcuata - MUS Schizomussaenda henryi - MUS Bremeria landia - MUS Landiopsis capuronii - MUS Steenisia pleurocarpa Retiniphyllum pilosum - RET Crossopteryx febrifuga - CRO Jackiopsis ornata - JAC Scyphiphora hydrophylacea Trailliaedoxa gracilis Glionnetia sericea Greenea oblonga - GRE Aleisanthia rupestris - ALE Aleisanthiopsis distantiflora - ALE Ixora novoguineensis - IXO Ixora margaretae - IXO Ixora coccinea - IXO Ixora ferrea - IXO Ixora borboniae - IXO Ixora trilocularis - IXO Canthium coromandelicum - VAN Rytigynia senegalensis - VAN Fadogia tetraquerta - VAN Vangueria madagascariensis - VAN Multidentia concrescens - VAN Psydrax obovata - VAN Afrocanthium lactescens - VAN Keetia gueinzii - VAN Cyclophyllum deplanchei - VAN Peponidium horridum - VAN Pyrostria hystrix - VAN Boholia nematostylis Airosperma sp. Airosperma psychotrioides Airosperma trichotomum Airosperma vanuense Augusta austrocaledonica Augusta longifolia Augusta rivalis Wendlandia ligustroides Wendlandia formosana Wendlandia tinctoria Wendlandia arabica Wendlandia paniculata Alberta magna - ALB Bertiera guianensis - BER Bertiera longithyrsa - BER Bertiera aethiopica - BER Empogona coriacea - COF Coffea ebracteolata - COF Coffea sessiliflora - COF Coffea arabica - COF Burchellia bubalina - GAR Cremaspora triflora - OCT Paragenipa lancifolia - OCT Hypobathrum racemosum - OCT Fernelia buxifolia - OCT Feretia apodanthera - OCT Oxyanthus speciosus - GAR Mitriostigma axillaris - GAR Pavetta abyssinica - PAV Pavetta indica - PAV Tennantia sennii - PAV Coptosperma supra-axillare - PAV Genipa americana - GAR Gardenia hansemanni - GAR Coddia rudis - GAR Kailarsenia tentaculata - GAR Rothmannia capensis - GAR Aulacocalyx jasminiflora - GAR Atractocarpus balansaeanus - GAR Duperrea pavettifolia - GAR Catunaregam spinosa - GAR Aoranthe penduliflora - GAR Hyperacanthus grevei - GAR Euclinia longiflora - GAR Randia aculeata - GAR Tocoyena pittieri - GAR Rosenbergiodendron densiflorum - GAR TRAILLIAEDOXEAE SCYPHIPHOREAE STEENISIEAE AUGUSTEAE AIROSPERMEAE
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Figure 2. The majority-rule consensus tree from the Bayesian inference analysis of the combined plastid data (ndhF, matK, rbcL, rps16 intron, trnS-G and trnT-F). Posterior probabilities and bootstrap support values from the parsimony analysis are indicated below the clades (a dash indicates bootstrap support of <50%). Phylogram of the tree with the highest marginal likelihood in inset. New tribal names presented in this article are highlighted. ALB, Alberteae; ALE, Aleisanthieae; BER, Bertiereae; COF, Coffeeae; CON, Condamineeae; CRO, Crossopterygeae; GAR, Gardenieae; GRE, Greeneeae; HEN, Henriquezieae; IXO, Ixoreae; JAC, Jackieae; MUS, Mussaendeae; OCT, Octotropideae; PAV, Pavetteae; POS, Posoquerieae; RET, Retiniphylleae; SAB, Sabiceeae; SIP, Sipaneeae; VAN, Vanguerieae.
2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387406
PHYLOGENY OF IXOROIDEAE revision of the genus, Bremer (1984) proposed that it should be transferred from Argostemmateae to Rondeletieae (subfamily Cinchonoideae). However, in the molecular phylogenetic study of Bremer & Eriksson (2009), Steenisia was found nested in Ixoroideae. Steenisia can be readily characterized by a suffrutescent unicaulous habit, rotate corolla and anthers with apical appendages fused to form a hollow cone around the style (in a manner reminiscent of the owers of Solanum L.), characters which are all unusual (the latter unique) in Ixoroideae. A region of the style covered with hairs that probably catch released pollen is usually found level with the pollen sacs (Bremer, 1984; Puff et al., 1995). However, as this region does not appear to extend above the surrounding staminal tube, pollen is not presented in a secondary manner. Instead, the mode of pollination is most likely by buzz pollination (Puff et al., 1995). Steenisia is not associated with any previously described tribe in our analyses and, consequently, the recognition of the Steenisia clade at the tribal level is proposed in the Taxonomic synopsis section.
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THE VANGUERIEAE
ALLIANCE
The Vanguerieae alliance comprises Aleisanthieae, Crossopterygeae, Glionnetia, Greeneeae, Ixoreae, Jackieae, Scyphiphora, Trailliaedoxa and Vanguerieae. The clades resolved in Vanguerieae in this study are congruent with the results of Lantz & Bremer (2004). However, the position of Psydrax Gaertn. is here further resolved in a clade consisting of Afrocanthium (Bridson) Lantz & B.Bremer, Keetia E.Phillips and a clade comprising Cyclophyllum Hook.f., Peponidium (Baill.) Arnes and Pyrostria Comm. ex Juss., representing the dioecious group of Razamandimbison et al. (2009). In agreement with the results of Rova et al. (2002) and Mouly et al. (2009a), Ixoreae forms a sister group to Aleisanthieae, and these are, in turn, sister to Greeneeae. Aleisanthia and Greenea both have traditionally been considered part of Rondeletieae (Hooker, 1873), a tribe characterized by contorted or imbricate corolla aestivation and capsular fruits. Molecular phylogenetic studies have indicated that this is an unnatural group, and Rondeletieae has since been much reduced (see Rova et al., 2002; Manns & Bremer, 2010; Rova, Delprete & Bremer, 2009). Glionnetia (Fig. 1G), an endemic of the Seychelles, has also been suggested as part of Rondeletieae by its author (Tirvengadum, 1984). However, in a recent study by Razamandimbison et al. (2011), Glionnetia was found nested in the Vanguerieae alliance, although with an unresolved position relative to Vanguerieae and the AleisanthieaeGreeneeaeIxoreae clade (Rondeletieae s.s. can consequently be consid-
ered as restricted to the New World; cf. Manns & Bremer, 2010). Despite the inclusion of more sequence data in this study, the phylogenetic position of Glionnetia is still unresolved. As in the study by Razamandimbison et al. (2011), successive sister groups to the Aleisanthieae GlionnetiaGreeneeaeIxoreae clade are Trailliaedoxa, Scyphiphora, Jackieae and Crossopterygeae. The little-studied Trailliaedoxa has hitherto remained without taxonomic placement in Rubiaceae. It occurs in the highlands of southern China, although it has rarely been collected. Trailliaedoxa is an erect shrublet with minute schizocarpic fruits with solitary pendulous ovules. The corolla aestivation is leftcontort. Whether or not secondary pollen presentation occurs is not known. Our results support the recognition of a monogeneric tribe, Trailliaedoxeae (see Taxonomic synopsis section). The mangrove plant Scyphiphora (Fig. 1F) occurs in coastal areas of tropical Asia and the western Pacic (Puff & Rohrhofer, 1993). Scyphiphora has a complex taxonomic history (see Puff & Rohrhofer, 1993; Mouly et al., 2009a), as the highly modied fruits, in particular, which are adapted for sea dispersal, have caused difficulties in assessing its systematic placement. The fruits are indehiscent and corky, with one ascending and one descending ovule per locule (Puff & Rohrhofer, 1993). However, the owers have secondary pollen presentation and leftcontort corolla aestivation, characteristics shared by most of the core Ixoroideae. Andreasen & Bremer (2000) tentatively included the genus in Ixoreae. However, in the studies by Bremer et al. (1999), Bremer & Eriksson (2009) and Corts-B. et al. (2009), Scyphiphora was resolved as sister to the Ixoreae Vanguerieae clade, a position congruent with this study, in which Scyphiphora is resolved as sister to the AleisanthieaeGlionnetiaGreeneeaeIxoreae TrailliaedoxaVanguerieae clade. Consequently, we propose the recognition of tribe Scyphiphoreae (see Taxonomic synopsis section).
THE COFFEEAE
ALLIANCE
The Coffeeae alliance comprises Airosperma, Augusta, Boholia, Wendlandia and tribes Alberteae, Coffeeae, Gardenieae, Octotropideae and Pavetteae. As in previous molecular studies (Andreasen & Bremer, 1996; Bremer & Eriksson, 2009), Gardenieae is not monophyletic. Mitriostigma (Fig. 1M) and Oxyanthus form a poorly supported sister group to Pavetteae, and Burchellia forms a sister group to Octotropideae. Further research is needed to better understand the phylogenetic relationships in the Gardenieae OctotropideaePavetteae complex. Successive sister groups to this complex are the BertiereaeCoffeeae
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K. KAINULAINEN ET AL. included species from the respective areas form sister groups). One of the Fijian species, A. trichotomum, was originally described by Gillespie (1932) as Abramsia trichotoma. Smith (1945: 108) considered the generic concepts as essentially identical and subsumed Abramsia. This synonymization is supported by the molecular data, because A. trichotomum is nested in Airosperma, forming a sister group to A. vanuense S.P.Darwin (Fig. 2). Secondary pollen presentation is not present in Airosperma (Darwin, 1979) and at least some of the New Guinean species of the genus appear to be functionally dioecious (Darwin, 1980). The status of this character in Boholia is not known. Although having been described as right-contorted or imbricate, the corolla aestivation is left-contorted in both genera (K. Kainulainen, pers. observ.). The fruits are eshy and contain pyrenes, and were interpreted by Robbrecht (1988) as adapted for dispersal by sea currents. However, animal dispersal also seems a possibility considering the vivid colour of the fruits (bluepurple, white or yellow; Darwin, 1980). In this study, as in that of Kainulainen et al. (2009), they form a clade separate from Alberteae; consequently, we propose that this clade should be recognized at the tribal level (see Taxonomic synopsis section).
clade, Alberteae, the AugustaWendlandia clade and the AirospermaBoholia clade. The included representatives of Bertiera form a monophyletic group sister to Coffeeae, a relationship also inferred in other studies (e.g. Bremer et al., 1995; Persson, 2000; Davis et al., 2007; see the latter for a discussion on the delimitation and morphological differentiation of these tribes, which some authors have treated as one, i.e. Andreasen & Bremer, 2000; Robbrecht & Manen, 2006). Augusta and Wendlandia have traditionally (Hooker, 1873) been considered as part of Rondeletieae. However, in the molecular phylogenetic study of Rova et al. (2002), they were resolved as a clade in Ixoroideae. Both genera have widely disjunct distributions. In Augusta, A. longifolia occurs in northeastern and central Brazil (Delprete, 1997), A. rivalis (Fig. 1I) in Central America, A. austrocaledonica in New Caledonia and A. vitiensis (Seem.) J.H.Kirkbr. in Fiji. The last three species have previously been recognized as Lindenia, a name that was reduced to synonymy by Kirkbride (1997). This is supported by our results, because A. rivalis and A. austrocaledonica are paraphyletic with respect to A. longifolia (Fig. 2). Wendlandia (Fig. 1J) has a wide distribution in East Asia and Australasia (centre of diversity in China), with one species, W. ligustroides, in southeastern Turkeynorthern Iraq and one species, W. arabica, in Ethiopia, Somalia and Yemen (see Puff, 1990). Cowan (1932), who provided the last comprehensive revision of the genus, considered the last two species as exceptional, and their inclusion in Wendlandia as doubtful. However, Wendlandia appears to be monophyletic in our phylogenetic analyses, in which W. ligustroides forms a sister group to the remaining included species, whereas W. arabica is nested in the East Asian clade. Secondary pollen presentation appears to be absent in Wendlandia (D. Zhang, South China Botanical Garden, Guangzhou, Guangdong, China, pers. comm.). The status of this character in Augusta is not known. Both genera have left-contorted corolla aestivation. In the Coffeeae alliance, the AugustaWendlandia clade is distinguished by capsular fruits. This clade is not associated with any previously described tribe; consequently, we propose that it should be recognized at tribal level (see Taxonomic synopsis section). Airosperma and Boholia were placed in Alberteae by their respective authors (Schumann & Lauterbach, 1900; Merrill, 1926) based on the presence of solitary pendulous ovules and contorted corolla aestivation. Boholia occurs in the Philippines, but has also been collected on the island of Flores in Indonesia (Schmutz 2691; P; image seen). Airosperma has a disjunct distribution, occurring in New Guinea and the Fiji islands (in the phylogenetic hypothesis, the
TAXONOMIC SYNOPSIS
Following the results of the molecular phylogenetic analyses, we present here a revised tribal classication of Ixoroideae, although we do not address the tribal delimitation in the problematic Gardenieae complex, i.e. Gardenieae, Octotropideae (including Cremasporeae) and Pavetteae, which will be discussed further by A. Mouly et al. (Universit de Franche-Comt, Besanon; unpubl. data). Including these three tribes, Ixoroideae comprises 24 tribes, ve of which are newly and formally described below. Although the molecular support for Ixoroideae is strong, morphologically the members are diverse and not easily characterized; no potential morphological synapomorphies for the subfamily are known at this point. In particular, Condamineeae and Henriquezieae contain morphologically aberrant genera (Rogers, 1984; Kainulainen et al., 2010), some of which, at times, have been classied outside Rubiaceae [e.g. Dialypetalanthus Kuhlm. in Dialypetalanthaceae (Rizzini & Occhioni, 1949) and Henriquezia Spruce ex Benth. and Platycarpum Humb. & Bonpl. in Henriqueziaceae (Bremekamp, 1957)].
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PHYLOGENY OF IXOROIDEAE or rarely mbricate; interpetiolar or rarely intrapetiolar. Flowers usually actinomorphic, protandrous, often with secondary pollen presentation; rarely zygomorphic, heterostylous, dioecious, monoecious or protogynous. Calyces mostly persistent, in several taxa with one or several lobes expanded and showy. Corolla aestivation contorted to left, rarely to right, imbricate, valvate or rarely open. Fruits eshy and indehiscent, dry and indehiscent or capsular, with numerous, few or solitary ovules per locule. Tribe Airospermeae Kainul. & B.Bremer, trib. nov. Type: Airosperma K.Schum. & Lauterb., Fl. Schutzgeb. Sdsee: 565 (1900) Suffrutices, frutices vel arbusculae unicaules. Flores 5-meri in paniculis terminalibus. Calyx persistens. Corolla brevi- vel longi-tubularis, lobis in aestivatione sinistrorsum contortis. Ovarium biloculare, ovulo uno pendulo in quoque loculo. Stylus inclusus vel (longi-) exsertus. Fructus carnosi, pyrenis duabus, in maturitate azureopurpurei, albidi vel lutescentes. Unicaulous subshrubs, shrubs or small trees. Flowers 5-merous in terminal panicles. Calyx persistent. Corolla short- to long-tubular, with left-contorted aestivation. Ovary bilocular, with one pendulous ovule in each locule. Style included to (long-)exserted. Fruits eshy, with two pyrenes, becoming blue purple, whitish or yellowish in maturity. Included genera: Airosperma with six species in Fiji and New Guinea, and the monotypic Boholia from the Philippines and the island of Flores, Indonesia. Tribe Alberteae Hook.f. For descriptions, see Puff et al. (1984), Robbrecht (1988) and Kainulainen et al. (2009). Distribution: South-east Africa and Madagascar. Included genera (here investigated): Alberta. Included based on Kainulainen et al. (2009): Nematostylis Hook.f and Razamandimbisonia Kainul. & B.Bremer. Tribe Aleisanthieae Mouly, J.Florence & B.Bremer For descriptions, see Mouly et al. (2009a) and Alejandro et al. (2010). Distribution: South-East Asia. Included genera (here investigated): Aleisanthia and Aleisanthiopsis.
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Inclusion based on Alejandro et al. (2010): Greeniopsis, occurring in South-East Asia. Tribe Augusteae Kainul. & B.Bremer, trib. nov. Type: Augusta Pohl, Pl. Bras. Icon. Descr. 2: 1 (1828) Frutices vel arbusculae. Inorescentiae cymae terminales compositae aut raro corymbi. Flores 5-meri. Calyx persistens. Corolla tubulari-infundibuliformis, lobis in aestivatione sinistrorsum contortis. Ovarium biloculare, ovulis numerosis horizontaliter insertis. Stylus exsertus. Fructus capsulares, dehiscentia septicida (Augusta) aut loculicida (Wendlandia). Semina minuta, complanata, testa reticulata. Shrubs to small trees. Inorescences terminal compound cymes or rarely corymbs. Flowers 5-merous. Calyx persistent. Corolla tubular-funnel-shaped, with left-contorted aestivation. Ovary bilocular, with numerous ovules horizontally inserted. Style exserted. Fruits capsular, with septicidal (Augusta) or loculicidal (Wendlandia) dehiscence. Seeds minute, compressed, with reticulate testa. Included genera: Augusta, with four species occurring in Mexico, north-eastern and central Brazil, the Fiji islands and New Caledonia, and Wendlandia (c. 80 spp.), occurring in north-eastern Africa, west Asia, South-East Asia and northern Australia. Tribe Bertiereae Bridson For a description, see Bridson & Verdcourt (2003). Bertiereae as currently dened is monogeneric. Distribution: Tropical South America to southern Mexico, tropical Africa, Madagascar and Mascarenes. Included genus: Bertiera. Tribe Coffeeae DC. For a description, see Davis et al. (2007). Recent taxonomic changes have been made by Tosh et al. (2009; resurrection of Empogona Hook.f.) and Davis et al. (2011; inclusion of Psilanthus Hook.f. in Coffea). Distribution: Tropical Africa, Madagascar, Comores, Mascarenes, Seychelles and tropical Asia to Australia. Included genera (here investigated): Coffea and Empogona. Inclusion based on Davis et al. (2007): Argocoffeopsis Lebrun, Belonophora Hook.f., Calycosiphonia Pierre ex Robbr., Diplospora DC., Discospermum
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K. KAINULAINEN ET AL. Inclusion based on Rogers (1984) and Delprete & Corts-B. (2004): Gleasonia Standl. and Platycarpum. Tribe Ixoreae A.Gray. For descriptions, see Mouly et al. (2009a, b). Ixoreae as currently dened is monogeneric. Distribution: Neotropics, tropical Africa, Madagascar, Comores, Mascarenes, Seychelles, tropical Asia to the Pacic. Included genus: Ixora. Tribe Jackieae Korthals For descriptions, see Ridsdale (1979) and Razamandimbison et al. (2011). Jackieae as currently dened is monogeneric. Distribution: South-East Asia. Included genus: Jackiopsis. Tribe Mussaendeae Hook.f. For descriptions, see Alejandro et al. (2005). Distribution: Tropical Africa, Madagascar, carenes, South-East Asia to the Pacic. Mas-
Dalzell, Nostolachma T.Durand, Sericanthe Robbr., Tricalysia A.Rich. ex DC. and Xantonnea Pierre ex Pit. Tribe Condamineeae Hook.f. For a description, see Kainulainen et al. (2010). Distribution: Neotropics, south-eastern USA (Pinckneya Michx.), South-East Asia (Emmenopterys Oliv.) and the Malesia-Pacic region (Dolicholobium A.Gray, Mastixiodendron Melch. and Mussaendopsis Baill.). Included genera (here investigated): Bathysa C.Presl, Calycophyllum DC., Chimarrhis Jacq., Condaminea DC., Dioicodendron Steyerm., Emmenopterys, Ferdinandusa Pohl, Hippotis Ruiz & Pav., Mastixiodendron, Pinckneya, Pogonopus Klotzsch, Rustia Klotzsch and Simira Aubl. Inclusion based on Kainulainen et al. (2010): Alseis Schott, Bothriospora Hook.f., Capirona Spruce, Dialypetalanthus, Dolichodelphys K.Schum. & K.Krause, Dolicholobium, Elaeagia Wedd., Holtonia Standl., Macbrideina Standl., Macrocnemum P.Browne, Mussaendopsis, Parachimarrhis Ducke, Pentagonia Benth., Picardaea Urb., Schizocalyx Wedd., Semaphyllanthe L.Andersson, Sommera Schltdl., Tammsia H.Karst., Warszewiczia Klotzsch and Wittmackanthus Kuntze. Tribe Crossopterygeae F.White ex Bridson For a description, see Bridson & Verdcourt (2003). Crossopterygeae as currently dened is monogeneric. Distribution: Tropical Africa. Included genus: Crossopteryx. Tribe Greeneeae Mouly, J.Florence & B.Bremer For a description, see Mouly et al. (2009a). Distribution: South-East Asia. Included genera (here investigated): Greenea. Inclusion based on Mouly et al. (2009a): Spathichlamys R.Parker. Tribe Henriquezieae Hook.f. For a description, see Rogers (1984). Distribution: Tropical South America. Included genera (here investigated): Henriquezia.
Included genera (here investigated): Bremeria, Heinsia, Landiopsis, Mussaenda, Pseudomussaenda and Schizomussaenda. Inclusion based on Tange (1994): Neomussaenda Tange. Tribe Posoquerieae Delprete For a description, see Delprete (2009). Distribution: Neotropics. Included genera: Molopanthera Turcz. and Posoqueria Aubl. Tribe Retiniphylleae Hook.f. For a description, see Robbrecht (1988). Retiniphylleae as currently dened is monogeneric. Distribution: Tropical South America. Included genus: Retiniphyllum. Tribe Sabiceeae Bremek. For a description, see Khan et al. (2008). Distribution: Neotropics, Tropical Africa, Madagascar, So Tom, Socotra, and Sri Lanka.
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PHYLOGENY OF IXOROIDEAE Included genera: Hekistocarpa, Sabicea, Tamridaea and Virectaria. Tribe Scyphiphoreae Kainul. & B.Bremer, trib. nov. Type: Scyphiphora C.F.Gaertn., Suppl. Carp.: 91 (1806) Frutices vel arbusculae. Flores 4(5)-meri, in thyrsis axillaribus. Calyx tubularis, brevis, persistens, lobis indistinctis. Corolla tubularis, lobis in aestivatione sinistrorsum contortis. Ovarium biloculare, ovulo uno ascendenti et uno descendenti in quoque loculo. Stylus exsertus. Fructus sicci, indehiscentes, porcati, mesocarpio extenso. Shrubs or small trees. Flowers 4(5)-merous, in axillary thyrses. Calyx tubular, short, persistent, with indistinct lobes. Corolla tubular, with leftcontorted aestivation. Ovary bilocular, with one ascending and one descending ovule in each locule. Style exserted. Fruits dry, indehiscent, ridged, with extensive mesocarp. Included genus: The monotypic Scyphiphora, occurring in tropical Asia and the western Pacic. Tribe Sipaneeae Bremek. For a description, see Delprete & Corts-B. (2004). Distribution: Central America and tropical South America. Included genera (here investigated): Chalepophyllum Hook.f., Dendrosipanea Ducke, Neobertiera Wernham and Sipanea Aubl. Inclusion based on Delprete & Corts-B. (2004): Limnosipanea Hook.f., Maguireothamnus Steyerm., Neblinathamnus Steyerm., Pteridocalyx Wernham, Sipaneopsis Steyerm. and Steyermarkia Standl. Tribe Steenisieae Kainul. & B.Bremer, trib. nov. Type: Steenisia Bakh.f., Webbia 8: 381 (1952) Suffrutices unicaules. Flores 45-meri, in thyrsis axillaribus. Calyx persistens, interdum uno lobo post anthesin expanso. Corolla rotata, lobis in aestivatione sinistrorsum contortis. Ovarium biloculare, ovulis numerosis horizontaliter insertis. Stylus curvatus. Antherae appendicibus apicalibus prolongatis, connatis tubumque stamineum stylum cingentem facientibus. Fructus capsulares septicidales. Semina minuta, complanata, testa reticulata granulata. Unicaulous subshrubs. Flowers 45-merous, in axillary thyrses. Calyx persistent, sometimes with one lobe expanding after anthesis. Corolla rotate, with left-contorted aestivation. Ovary bilocular, with numerous ovules horizontally inserted. Style curved. Anthers with extended apical appendages, united and
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forming a tube surrounding the style. Fruits septicidal capsules. Seeds minute, attened, with a reticulate, granulate testa. Included genus: Steenisia, with ve species in Sundaland (Borneo, Natuna Islands and Peninsular Malaysia). Tribe Trailliaedoxeae Kainul. & B.Bremer, trib. nov. Type: Trailliaedoxa W.W.Sm. & Forrest, Notes Roy. Bot. Gard. Edinburgh 10: 74 (1917) Fruticuli erecti. Flores minuti, 5-meri, in cymis terminalibus. Calyx persistens. Corolla infundibuliformis, lobis in aestivatione sinistrorsum contortis. Ovarium biloculare, ovulo uno pendulo in quoque loculo. Stylus exsertus. Fructus schizocarpi. Erect shrublets. Flowers minute, 5-merous, in terminal cymes. Calyx persistent. Corolla funnel-shaped, with left-contorted aestivation. Ovary bilocular, with one pendulous ovule in each locule. Style exserted. Fruits schizocarpous. Included genus: The monotypic Trailliaedoxa, occurring in south-western China (Sichuan, Yunnan). Tribe Vanguerieae A.Rich. ex Dum. For descriptions, see Robbrecht (1988) and Lantz & Bremer (2004). Distribution: Southern Africa, tropical Africa, Madagascar, Comores, Mascarenes, Seychelles, (sub-) tropical Asia to Australia and the Pacic. Included genera (here investigated): Afrocanthium, Canthium Lam., Cyclophyllum, Fadogia Schweinf., Keetia, Multidentia Gilli, Peponidium, Psydrax, Pyrostria, Rytigynia Blume and Vangueria Juss. Inclusion based on Robbrecht (1988), Lantz & Bremer (2004) and Razamandimbison et al. (2009): Bullockia (Bridson) Razam., Lantz & B.Bremer, Cuviera DC., Eriosemopsis Robyns, Everistia S.T.Reynolds & R.J.F.Hend., Fadogiella Robyns, Hutchinsonia Robyns, Perakanthus Robyns ex Ridl., Plectroniella Robyns, Pygmaeothamnus Robyns, Robynsia Hutch., Temnocalyx Robyns ex Ridl., Vangueriella Verdc. and Vangueriopsis Robyns.
CONCLUSIONS
Here, we analysed the tribal relationships and delimitation in Ixoroideae using six plastid DNA regions and Bayesian and parsimony methods of phylogenetic reconstruction. Following the results of this study, Ixoroideae can be considered as being composed of two well-supported lineages, the Coffeeae alliance and the Vanguerieae alliance, and a grade consisting of the
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Andreasen K, Bremer B. 2000. Combined phylogenetic analysis in the RubiaceaeIxoroideae: morphology, nuclear and chloroplast DNA data. American Journal of Botany 87: 17311748. Backlund M, Bremer B, Thulin M. 2007. Paraphyly of Paederieae, recognition of Putorieae and expansion of Plocama (RubiaceaeRubioideae). Taxon 56: 315328. Backlund M, Oxelman B, Bremer B. 2000. Phylogenetic relationships within the Gentianales based on ndhF and rbcL sequences, with particular reference to the Loganiaceae. American Journal of Botany 87: 10291043. Bakhuizen van den Brink RC Jr. 1952. Steenisia a new genus of Malaysian Rubiaceae. Webbia 8: 381382. Bremekamp CEB. 1952. The African species of Oldenlandia L sensu Hiern et K. Schumann. Verhandelingen der Koninklijke Nederlandsche Akademie van Wetenschappen, Afdeeling Natuurkunde, Tweede sectie 18: 1297. Bremekamp CEB. 1957. On the position of Platycarpum Humb. et Bonpl., Henriquezia Spruce and Gleasonia Standl. Acta Botanica Neerlandica 6: 351377. Bremekamp CEB. 1966. Remarks on the position, the delimitation and the subdivision of the Rubiaceae. Acta Botanica Neerlandica 15: 133. Bremer B. 1984. The genus Steenisia (Rubiaceae) and its taxonomic position. Nordic Journal of Botany 4: 333345. Bremer B, Andreasen K, Olsson D. 1995. Subfamilial and tribal relationships in the Rubiaceae based on rbcL sequence data. Annals of the Missouri Botanical Garden 82: 383397. Bremer B, Bremer K, Heidari N, Erixon P, Olmstead RG, Anderberg AA, Kllersj M, Barkhordarian E. 2002. Phylogenetics of asterids based on 3 coding and 3 non-coding chloroplast DNA markers and the utility of non-coding DNA at higher taxonomic levels. Molecular Phylogenetics and Evolution 24: 274301. Bremer B, Eriksson T. 2009. Time tree of Rubiaceae: phylogeny and dating the family, subfamilies, and tribes. International Journal of Plant Sciences 170: 766793. Bremer B, Jansen RK, Oxelman B, Backlund M, Lantz H, Kim KJ. 1999. More characters or more taxa for a robust phylogeny case study from the coffee family (Rubiaceae). Systematic Biology 48: 413435. Bremer B, Thulin M. 1998. Collapse of Isertieae, re-establishment of Mussaendeae, and a new genus of Sabiceeae (Rubiaceae); phylogenetic relationships based on rbcL data. Plant Systematics and Evolution 211: 7192. Bridson DM, Verdcourt B. 2003. Rubiaceae. In: Pope GV, ed. Flora Zambesiaca, vol. 5, part 3. London: Royal Botanic Gardens, Kew, 379720. Corts-B. R, Delprete PG, Motley TJ. 2009. Phylogenetic placement of the tribe Retiniphylleae among the subfamily Ixoroideae (Rubiaceae). Annals of the Missouri Botanical Garden 96: 6167. Cowan JM. 1932. The genus Wendlandia. Notes from the Royal Botanic Garden, Edinburgh 16: 233314. Darwin SP. 1976. Subfamilial, tribal and sub-tribal nomenclature of Rubiaceae. Taxon 25: 595610. Darwin SP. 1979. A synopsis of the indigenous genera of the Pacic Rubiaceae. Allertonia 2: 144.
tribes Condamineeae, Henriquezieae, Mussaendeae, Posoquerieae, Retiniphylleae, Sabiceeae, Sipaneeae and Steenisieae. The Vanguerieae alliance comprises Glionnetia (with unplaced tribal position) and the tribes Aleisanthieae, Crossopterygeae, Greeneeae, Ixoreae, Jackieae, Scyphiphoreae, Trailliaedoxeae and Vanguerieae. The Coffeeae alliance contains Airospermeae, Alberteae, Augusteae, Bertiereae, Coffeeae, Gardenieae, Octotropideae and Pavetteae. Molecular data furthermore support the transfer of Abramsia to Airosperma and Lindenia to Augusta. Future studies should further investigate the phylogenetic positions of Glionnetia and Hekistocarpa and the relationships of the early divergent Condamineeae and HenriquezieaePosoquerieaeSipaneeae clades with respect to the rest of the subfamily.
ACKNOWLEDGEMENTS
We thank the curators of the herbaria AAU, BR, GB, HUH, L, MO, S and UPS for providing loans and/or access to collections. We also thank Anbar Khodabandeh for laboratory assistance, Claes Persson for DNA samples, Christian Puff and the Missouri Botanical Garden for photographs and Dianxiang Zhang for sharing unpublished information on Wendlandia. Per Ola Karis and Philip Oswald checked the Latin diagnoses. Piero Delprete and two anonymous reviewers provided helpful comments on the manuscript. The Knut and Alice Wallenberg Foundation and the Swedish Research Council provided nancial support to BB.
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PHYLOGENY OF IXOROIDEAE
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APPENDIX
Overview of taxon sampling, including GenBank sequence accession numbers and voucher specimens for previously unpublished sequences (indicated with *). Italic sequences originate from a different specimen from that indicated Species
Afrocanthium lactescens (Hiern) Lantz Airosperma psychotrioides K.Schum. & Lauterb. Airosperma sp. Airosperma trichotomum (Gillespie) A.C.Sm. Airosperma vanuense S.P.Darwin Alberta magna E.Mey. Aleisanthia rupestris (Ridl.) Ridl. Aleisanthiopsis distantiora (Merr.) Tange Aoranthe penduliora (K.Schum.) Somers Atractocarpus balansaeanus Guillaumin Augusta austrocaledonica (Brongn.) J.H.Kirkbr. Augusta longifolia (Spreng.) Rehder Augusta rivalis (Benth.) J.H.Kirkbr. Aulacocalyx jasminiora Hook.f. Bathysa stipulata (Vell.) C.Presl Bertiera aethiopica Hiern
Voucher
Luke & Luke 9045 (UPS) Nyman 569 (UPS)
matK
HM119502*
ndhF
HM164346*
rbcL
HM164152*
rps16
HM164189*
trnS-G
HM164234*
trnT-L-F
AJ6201271
HM164347*
HM164302*
Sands 6819 (L) Degener & Ordonez 15542 (S) Smith 8214 (P)
HM119503*
HM164348* HM164349*
HM164190*
HM164235*
HM164303* HM164304*
HM119504*
AM9498452
FM2047002
FM2047402
FM2071082
Tonkin 200 (UPS) Tange 45171 (AAU) Tange 46977 (AAU) Iverssen & Steiner 86776 (UPS)
HM119505* HM119506*
AJ2362823
Y187083 HM164153*
FM2047012 AF2429024
FM2047412 HM164236*
AJ6201181 HM164305*
HM119507*
HM16435028
HM164154*
AF2429034
HM164237*
HM164306*
HM119508*
Y118455
HM164191*
HM164238*
HM164307*
Munzinger & McPherson 635 (MO) Mouly & Innocente 237 (P)
HM119509*
HM164351*
HM164155*
HM164192*
HM164239*
HM164308*
HM119510*
HM164352*
EU8174126
HM164193*
HM164240*
EU8174546
Kirkbride 5295 (BR) McDowell 5017 (ETSU) Schmidt et al. 1672 (MO) Pirani & Bremer 4901 (SPF) Dessein s.n. (BR)
HM119511* HM119512*
HM164353* AM9498462
AM9498422
HM164194*
HM164241* FM2047522
HM164309* FM2071182
HM119513*
HM164354*
EU8174136
EF2056397
FM2047532
EU8174556
FJ9053378 HM119514*
FJ8719478 HM164355*
HM164156* HM164157*
FJ8846378 HM164195*
FJ9483028 HM164242*
FJ9483718 HM164310*
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402
K. KAINULAINEN ET AL.
APPENDIX Continued
Species
Bertiera guianensis Aubl. Bertiera longithyrsa Baker Boholia nematostylis Merr. Bremeria landia (Poir.) Razam. & Alejandro Burchellia bubalina (L.f.) Sims Calycophyllum candidissimum (Vahl) DC. Canthium coromandelicum (Burm.f.) Alston Catunaregam spinosa (Thunb.) Tirveng. Cephalanthus occidentalis L. Chalepophyllum guyanense Hook.f. Chimarrhis hookeri K.Schum. Cinchona pubescens Vahl. Coddia rudis (E.Mey. ex Harv.) Verdc. Coffea arabica L. Coffea ebracteolata (Hiern) Brenan Coffea sessiliora Bridson Colletoecema dewevrei (De Wild.) E.M.A.Petit Condaminea corymbosa (Ruiz & Pav.) DC. Coptosperma supra-axillare (Hemsl.) Degreef Cremaspora triora ssp. conuens (K.Schum.) Verdc. Crossopteryx febrifuga (Afzel. ex G.Don) Benth. Cubanola domingensis (Britton) Aiello
Voucher
Andersson et al. 2029 (GB) Krehed et al. 256 (UPS) Bicknell 1561A (SUNIV) Andriambololonera 37 (MAU) Bremer 3129 (UPS) Sanders 1805 (FTG) Andreasen 36 (UPS) Luke 8332A (UPS) Forbes s.n. (S) Sandwith 1346 (S) Delprete & Verduga 6421 (UPS) McDowell 4613 (DUKE) Bremer 3764 (UPS)
matK
HM119515* HM119516* HM119517*
ndhF
AM9498472 HM164356* AM9498482
rbcL
AJ2248459 HM164158* AM11721011
rps16
AF20098310 HM164196* AM11728611
trnS-G
FM2047542 HM164243* FM2047552
trnT-L-F
FM2071192,33 HM164311* HM164312*
HM119518*
HM164357*
HM164197*
HM164244*
HM164313*
HM119519*
HM164358*
Z6883312
HM164198*
HM164245*
HM164314*
FJ9053408
AJ2362853
X8362713
FM2047122
FM2047562
AJ84739814
HM119520*
HM164359*
Z6885112
HM164199*
HM164246*
AJ84740114
HM119522*
AM11721411
HM164200*
HM164248*
HM164315*
AJ2362883 AJ2362893
HM164249* FJ9483098
Z7019718 HM119524*
AJ23584319 HM164361*
X8363013 AJ28669520
FM2047142 HM164202*
FM2047582 HM164250*
AJ34696317 HM164317*
Pennington & Daza 17436 (MOL) Bremer 3810 (UPS) Andreasen 51 (UPS) Bremer 3097 (UPS) McDowell 4427 (DUKE)
FJ9053478
FJ8719508
HM164161*
FJ8846458
FJ9483128
FJ9483818
HM119527*
HM164363*
AJ28671120
HM164204*
HM164253*
HM164319*
HM119528*
AM9498502
Z6885612
FM2047162
FM2047592
FM2071212
FJ9053298
AM9498512
AM11722311
FM2047172
FM2047602
FM2071232
AY53838615
AM11734511
X8363213
FM2047182
FM2047612
FM2071242
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PHYLOGENY OF IXOROIDEAE
APPENDIX Continued Species
Cyclophyllum deplanchei Hook.f. Dendrosipanea spigelioides Ducke Dioicodendron dioicum (K.Schum. & Krause) Steyerm. Duperrea pavettifolia (Kurz) Pit. Emmenopterys henryi Oliv. Empogona coriacea (Sond.) Tosh & Robbr. Euclinia longiora Salisb. Fadogia tetraquetra K.Schum. & Krause Ferdinandusa speciosa Pohl Feretia apodanthera Delile Fernelia buxifolia Lam. Gardenia hansemannii K.Schum. Gelsemium sempervirens (L.) J.St.-Hil. Genipa americana L. Glionnetia sericea (Baker) Tirveng. Greenea oblonga Craib Guettarda speciosa L. Heinsia crinita (Afzel.) G.Taylor Hekistocarpa minutiora Hook.f. Henriquezia nitida var. macrophylla (Ducke) Steyerm. Hippotis triora Ruiz & Pav. Hyperacanthus grevei Rakotonas. & A.P.Davis
403
Voucher
Mouly & Innocente 228 (P) Prance et al. 16199 (S) Zarucchi & Echeverry 4780 (MO) cult. Bergius Botanic Garden Robbrecht s.n. (UPS) Bremer et al. 4302 (UPS) De Block 27 (BR) Bremer & Bremer 3799 (UPS) Malme 2442 (UPS) Luke 8385 (UPS) Nat. Bot. Gard. Meise, no. 19940025-26 Drozd & Molem 1998-11-13 Bremer 3026 (UPS) Kiehn, HBVsub RR-420 (WU, UPS) Beaver 17 (S) Larsen & Larsen 33451 (P) Persson 141 (GB) McPherson 16188A (MO) Leuwenberg 6393 (BR, K, WAG) Liesner 8531 (MO)
matK
HM119529*
ndhF
rbcL
EU8174166
rps16
EF2056407
trnS-G
HM164254*
trnT-L-F
EF2056317
FJ9053248
FJ8719438
HM164162*
FJ8846278
FJ9482918
FJ9483608
FJ9053498
FJ8719528
HM164163*
FJ8846478
FJ9483148
FJ9483838
HM119531*
HM164365*
AJ28670920
HM164205*
HM164256*
HM164320*
FJ9053608 HM119574*
AJ2362943 HM164401*
Y187153 HM164184*
FM2047192 HM164228*
FM2047622 HM164295*
FM2071252 HM164340*
HM119532* HM119533*
HM164366* HM164367*
Z6883512 AM11722511
HM164206* HM164207*
HM164257* HM164258*
AJ84739914 AJ6201391
HM119537*
AM9498522
AJ31844624
AJ32007724
FM2047632
FM2071262
AJ42932225
AJ01198426
L1439727
AJ43103325
AJ43090825
HM119538*
HM164370*
Z6883912
HM164209*
HM164261*
HM164322*
HM53622328 HM53622928 HM569721* AY53848515 Y118495 AF33236629 EU8174396 AF2429644 HM164210* AF33236729 HM164262* FJ9482898 HM164263* HM164264*
HM164374*
HM164211*
HM164325*
FJ9053658
FJ8719568 HM164375*
HM164165* HM164166*
FJ8846598 HM164212*
FJ9483298 HM164265*
FJ9483978 HM164326*
2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387406
404
K. KAINULAINEN ET AL.
APPENDIX Continued
Species
Hypobathrum racemosum (Roxb.) Kurz Ixora borboniae Mouly & B.Bremer Ixora coccinea L. Ixora ferrea (Jacq.) Benth. Ixora margaretae (N.Hall) Mouly & B.Bremer Ixora novoguineensis Mouly & B.Bremer Ixora trilocularis (Balf.f.) Mouly & B.Bremer Jackiopsis ornata (Wall.) Ridsdale Kailarsenia tentaculata (Hook.f.) Tirveng. Keetia gueinzii (Sond.) Bridson Landiopsis capuronii Capuron ex Bosser Logania vaginalis (Labill.) F.Muell. Luculia gratissima (Wall.) Sweet Mastixiodendron avidum (Seem.) A.C.Sm. Mitriostigma axillare Hochst. Molopanthera paniculata Turcz. Multidentia concrescens (Bullock) Bridson & Verdc. Mussaenda arcuata Poir. Mussaenda erythrophylla Schumach. & Thonn. Nauclea orientalis (L.) L. Neobertiera gracilis Wernh. Ophiorrhiza mungos L.
Voucher
Ridsdale 81
matK
HM119543*
ndhF
AM9498532
rbcL
AJ28670520
rps16
AM11731811
trnS-G
HM164266*
trnT-L-F
FM2071272
Friedmann 3049 (P) Bremer 2719 (UPS) Taylor 11693 (MO) Mouly & Innocente 222 (P) Drozd & Molem 1998-11-13
HM119552*
HM164383*
HM164173*
FJ1506856
HM164275*
FJ1506096
HM119581*
HM164406*
AJ31845924
AJ32009024
HM164300*
EU8174766
Lesouef 31 (TAN)
HM119530*
HM164364*
EU8174176
EU8174376
HM164255*
EU8174576
K. S. Tan s.n. Larsen 41627 (AAU) Bremer et al. 4349 (UPS) Gautier 4533 (MO)
HM119546* HM119547*
HM53621328 HM164378*
HM53623128 HM164327*
HM119548* HM119549*
HM164379* HM164380*
HM164169* HM164170*
HM164214* HM164215*
HM164270* HM164271*
AJ6201431 HM164328*
Bremer 3013 (UPS) CT 870064 Rova & Gustavsson 2429 (GB) Bremer 2705 (UPS) Williams 6861 (S) Bidgood et al. 845 (K)
HM119550*
HM164381* HM164382*
X8365013 HM164172*
HM164216* AF2429814
HM164273* HM164274*
HM119551* FJ9053268
AJ2363013 AJ1308363
Y118545 X8365213
FM2047212 FJ8846288
FM2047672 FJ9482928
FM2071282 AJ6201161
AY53840715 AY53840815
EU14541022 AJ1308383
X8365313 X8365613
HM164276* HM164277*
2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387406
PHYLOGENY OF IXOROIDEAE
APPENDIX Continued Species
Oxyanthus speciosus DC. Paragenipa lancifolia (Bojer ex Baker) Tirveng. & Robbr Pavetta abyssinica Fresen. Pavetta indica L.
405
Voucher
Bremer 4348 (S) Persson 156 (GB)
matK
HM119554* HM119555*
ndhF
HM164384* HM164385*
rbcL
AM11725211 AJ28670720
rps16
AM11733011 AF00406616
trnS-G
HM164278* HM164279*
trnT-L-F
AM11737511 HM164330*35
De Block 6 (BR)
Andreasen 202 (UPS) Peponidium Labat et al. 2236 horridum Arnes (K) Pinckneya bracteata Massey s.n. 15F (Bartram) Raf. Pogonopus speciosus Meier 2548 (GB) (Jacq.) K.Schum. Posoqueria latifolia Andreasen 90 (Rudge) Roem. & (UPS) Schult. Posoqueria cult. Bergius longiora Aubl. Botanic Garden Pseudomussaenda Nissen s.n. (UPS) ava Verdc. Psydrax obovata Bremer & Bremer (Klotzsch ex Eckl. 3762 (UPS) & Zeyh.) Bridson Pyrostria hystrix Bremer & Bremer (Bremek.) Bridson 3791 (UPS) Randia aculeata L. F.G.x.1145 Retiniphyllum Wurdack & pilosum (Spruce Adderley 43270 ex Benth.) Mll. (S) Arg. Rondeletia odorata Bremer & Jacq. Andreasen 3504 (UPS) Rosenbergiodendron Jansen-Jacobs densiorum (K. 3977 (GB) Schum.) Fagerl. Rothmannia Bremer et al. 4346 capensis Thunb. (S) Rustia Delprete 6378 thibaudioides (UPS) (H.Karst.) Delprete Rytigynia van den Berghen senegalensis 8746 (BR) Blume Sabicea africana (P. Wieringa 3591 Beauv.) Hepper (WAG) Sabicea aspera Andersson et al. Aubl. 1941 (GB) Sabicea diversifolia Bremer et al. Pers. 4018-B18 (UPS) Schizomussaenda Puff 961116-1/1 henryi (Hutch.) (WU) X.F.Deng & D.X.Zhang
HM119562* HM119563*
AJ23584519
Y118575
AF2430104
FJ9482908
FJ9483598,36
HM119566*
HM164393*
HM164177*
HM164222*
HM164287*
HM164335*
HM119567* FJ9053858
HM164394* AJ2363103
AM11726611 Y187163
AM11734011 FM2047312
HM164288* FM2047772
AM11738411 FM2071382
HM164178*
HM164223*
HM164289*
AJ6201751
2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387406
406
K. KAINULAINEN ET AL.
APPENDIX Continued
Species
Scyphiphora hydrophyllacea C.F.Gaertn. Simira cordifolia (Hook.f.) Steyerm. Sipanea aff. biora (L.f.) Cham. & Schltdl. Sipanea hispida Benth. ex Wernham Steenisia pleurocarpa (Airy Shaw) Bakh.f. Tamridaea capsulifera (Balf. f.) Thulin & B. Bremer Tennantia sennii (Chiov.) Verdc. & Bridson Tocoyena pittieri (Standl.) Standl. Trailliaedoxa gracilis W.W.Sm. & Forrest Vangueria madagascariensis J.F.Gmel. Wendlandia arabica Deers Wendlandia formosana Cowan Wendlandia ligustroides (Boiss. & Hohen.) Blakelock Wendlandia paniculata (Roxb.) DC. Wendlandia tinctoria (Roxb.) DC. Virectaria multiora (Sm.) Bremek.
Voucher
Bremer et al. 99 (S) Bremer 3361 (UPS) Rova et al. 2005 (GB) Irwin et al. 34756 (UPS) Puff BF 990619-1/4 (WU) Thulin & Gifri 8663 (UPS)
matK
FJ9053278
ndhF
AJ2363113
rbcL
Y187173
rps16
EU8174506
trnS-G
FM2047792
trnT-L-F
EU8174756
FJ9053888 AY53842115
FJ8719708 EU14541322
HM164179* AY53850915
FJ8846778 AF00408516
FJ9483508 FM2047802
FJ9484198 FM2071412,38
EU14541422
EU14545822
EU14549222
HM164336*
FJ9053288
HM164396*
AM11727911
FM2047352
FM2047812
FM2071422
HM164398*
Y118605
FM2047362
FM2047822
FM2071432
Abdalla et al. 96/166 (P) Rova et al. 2369 (GB) Boufford et al. 35041 (MO) Sanders 1798, #62146 (FTG) Thulin et al. 10192 (UPS) Chung 1403 (S) Rechinger 183 (S)
HM119570*
AM9498582
AM9498432
FM2047372
FM2047832
FM2071442
HM119571* HM119573*
AM9498592 HM16440028
HM164181*
FM2047382
FM2047842
FM2071452 HM16433928
HM119575*
AJ1308403
X8367013
HM164229*
FM2047852
AJ6201841
Ridsdale 2179
HM119579*
HM164405*
HM164188*
HM164232*
HM164299*
HM164344*
HM119580*
AM9498602
FM2076492
FM2047392
FM2047862
FM2071472
HM119582*
HM164407*
Y118615
HM164233*
HM164301*
HM164345*
Published sequences: 1Lantz & Bremer (2004); 2Kainulainen et al. (2009); 3Bremer et al. (1999); 4J. H. E. Rova (unpubl. data); 5Bremer & Thulin (1998); 6Mouly et al. (2009a); 7Mouly et al. (2007); 8Kainulainen et al. (2010); 9Andreasen, Baldwin & Bremer (1999); 10Persson (2000); 11Bremer & Eriksson (2009); 12Andreasen & Bremer (1996); 13Bremer et al. (1995); 14Alejandro et al. (2005); 15Andersson & Antonelli (2005); 16Andersson & Rova (1999); 17Razamandimbison & Bremer (2002); 18Endress et al. (1996); 19Backlund, Oxelman & Bremer (2000); 20Andreasen & Bremer (2000); 21Samson et al. (2007); 22Rydin et al. (2008); 23Piesschaert et al. (2000); 24Novotny et al. (2002); 25Bremer et al. (2002); 26Oxelman, Backlund & Bremer (1999); 27Olmstead et al. (1993); 28 Razamandimbison et al. (2011); 29Dessein et al. (2001); 30Rova et al. (2002); 31Backlund, Bremer & Thulin (2007); 32L. Andersson (unpubl. data). Combined sequences: 33with AF152670; 34with AF152725; 35with AF152672; 36with AF152741; 37with AY538475; 38with AF152675; 39with AF152661.
2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387406