TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) Phylogenetic structure and clade circumscriptions in the Gardenieae complex (Rubiaceae) Arnaud Mouly,1,2 Kent Kainulainen,1 Claes Persson,3 Aaron P. Davis,4 Khoon Meng Wong,5 Sylvain G. Razafimandimbison1 & Birgitta Bremer1 1 The Bergius Foundation, The Royal Swedish Academy of Sciences, and Department of Ecology, Environment and Plant Sciences, Stockholm University, 106 91, Stockholm, Sweden 2 Université de Franche-Comté—CNRS UMR 6249 Chrono-environnement, ESPE de Franche-Comté, 16 Route de Gray, 25030 Besançon cedex, France 3 Department of Biological and Environmental Sciences, University of Gothenburg, P.O. Box 461, 405 30, Göteborg, Sweden 4 Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, U.K. 5 Singapore Botanic Gardens, 1 Cluny Road, Singapore 259569 Author for correspondence: Arnaud Mouly, arnaud.mouly@univ-fcomte.fr DOI http://dx.doi.org/10.12705/634.4 Abstract In this study we investigate the large and diverse Rubiaceae-Gardenieae and closely related tribes Bertiereae, Coffeeae, Cremasporeae, Octotropideae, and Pavetteae. Some of the tribes or groups have been shown to be monophyletic and strongly supported, but the phylogeny of this large complex is still far from being satisfactorily elucidated particularly for Gardenieae, both in terms of intertribal relationships as well as tribal delimitations. We reconstruct the phylogeny of the complex using an extensive sampling of 108 genera and five plastid DNA regions. Phylogenetic relationships demonstrate that Gardenieae sensu Andreasen & Bremer is polyphyletic, as Burchellia, Didymosalpinx, Monosalpinx, and Mantalania are closer to OctotropideaeCremasporeae. In addition, Pavetteae and the investigated members of Aulacocalyceae are nested in a supported but partially unresolved Gardenieae-Pavetteae clade. Within this clade, several strongly supported groups are resolved: an Aidia group, an Alibertia group, a Gardenia group, Pavetteae including Pelagodendron, a Porterandia group, a Randia group, a Rothmannia group (including Aulacocalyx and Heinsenia), a Sherbournia group, and the two isolated genera Massularia and Schumanniophyton. The latter genus presented a high rate of genetic substitutions, which resulted in perturbations of the phylogenetic reconstruction. A revised tribal circumscription is given for Gardenieae, the Alibertia group is recognized at tribal level as an emended Cordiereae, and a new tribe, Sherbournieae, is described to accommodate the members of the Sherbournia group. Keywords Aulacocalyceae; Cordiereae; Gardenieae; mapping; molecular phylogeny; Octotropideae; Pavetteae; radiation; Rubiaceae; Sherbournieae Supplementary Material Electronic Supplement (Fig. S1) and alignment are available in the Supplementary Data section of the online version of this article at http://www.ingentaconnect.com/content/iapt/tax INTRODUCTION Rubiaceae is the fourth-largest flowering plant family (Davis & al., 2009), although only a few species are well known and commonly used, such as coffee and the fragrant ornamental ixoras and gardenias. Coffea L., Ixora L., and Gardenia J.Ellis (respectively) belong to the same Rubiaceae lineage, Ixoroideae, a subfamily with a long and intricate taxonomic history and an unsatisfactorily resolved phylogeny, despite numerous recent studies (e.g., Persson, 1996, 2000a; Andreasen & Bremer, 2000; Rova & al., 2002; Robbrecht & Manen, 2006; Bremer & Eriksson, 2009; Razafimandimbison & al., 2011; Kainulainen & al., 2013). Within Ixoroideae, tribe Gardenieae is a large and morphologically diverse group of ca. 100 genera (Fig. 1), whose circumscription has long been controversial (e.g., Candolle, 1830; Schumann, 1891; Verdcourt, 1958; Bremekamp, 1966; Robbrecht & Puff, 1986; Andreasen & Bremer, 1996, 2000; Robbrecht & Manen, 2006; see Table 1). Candolle (1830) described Gardenieae to accommodate Rubiaceae genera with indehiscent fleshy bilocular (rarely unilocular) fruits and “remarkable” flowers (the author meant large flowers) with or without pedicel. The original circumscription (Candolle, 1830) included 28 genera: Sarcocephalus Afzel. ex R.Br., Zuccarinia Blume, Schradera Vahl (as Lucinaea DC.), Burchellia R.Br., Amaioua Aubl., Mussaenda L. (incl. Menestoria DC.), Kutchubea Fisch. ex DC., Isertia Schreb. (as Cassupa DC.), Aidia Lour. (as Gynopachis Blume, Cupia DC., and Stylocoryna Cav.), Tocoyena Aubl., Posoqueria Aubl., Oxyanthus DC., Genipa L., Gardenia, Randia L. (incl. Oxyceros and Euclinia), Chapelieria A.Rich., Heinsia DC., Timonius DC. (as Received: 28 Dec 2012 | returned for first revision: 10 Jul 2013 | last revision received: 27 May 2014 | accepted: 27 May 2014 | not published online ahead of inclusion in print and online issues || © International Association for Plant Taxonomy (IAPT) 2014 Version of Record (identical to print version). 801 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) TAXON 63 (4) • August 2014: 801–818 Fig. 1. Flowers of Gardenieae and related groups. Gardenieae: A, Gardenia taitensis DC., cultivated in Tahiti Botanical Garden (credit: C. Ollier, IRD Papeete); B, Gardenia thunbergia L.f., cultivated in Lisbon Botanical Garden (credit: A. Mouly); C, Duperrea pavettifolia, cultivated in Stockholm Bergius Botanical Garden (credit: K. Kainulainen); D, Euclinia longiflora, cultivated in Tahiti Botanical Garden (credit: A. Mouly); E, Catunaregam spinosa, Vietnam, forest (credit. K. Kainulainen); Sherbournieae: F, Mitriostigma axillare, cultivated in Stockholm Bergius Botanical Garden (credit: K. Kainulainen); G, Oxyanthus speciosus, cultivated in Leiden Hortus Botanicus (credit: A. Mouly); Cordiereae: H, Mitriostigma axillare, cultivated in Stockholm Bergius Botanical Garden (credit: K. Kainulainen); Octotropideae s.l.: I, Burchellia bubalina, cultivated in Stockholm Bergius Botanical Garden (credit: K. Kainulainen); J Dydimosalpinx norea (Swynn.) Keay, cultivated in Stockholm Bergius Botanical Garden (credit: K. Kainulainen) 802 Version of Record (identical to print version). TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) Helospora DC.), Hippotis Ruiz & Pav., Bertiera Aubl. (incl. Pomatium DC.), Pouchetia A.Rich. ex DC., Tarenna Gaertn., Rondeletia L. (as Petesia P.Browne), Coccocypselum P.Browne, Fernelia Comm. ex Lam., Hypobathrum Blume (as Petunga DC.), Hoffmannia Sw. (incl. Higginsia Pers.), and Catesbaea L. Candolle (1830) described another tribe, Cordiereae, for the two genera Cordiera A.Rich ex DC. and Tricalysia A.Rich. ex DC. characterized according to him by multilocular baccate fruits. Since then, several specialists have worked on the group and proposed different classifications. Among them, Schumann (1891) transferred Cordiereae to Gardenieae as an infratribal group. Robbrecht & Puff (1986) published a comprehensive morphological study of Gardenieae based on several Rubiaceae systematic treatments by Schumann (1891), Verdcourt (1958), Bremekamp (1966), and their own observations and proposed a new classification (Table 1). Tribe Gardenieae was subdivided into two groups, subtribe Diplosporinae with ca. 20 genera and subtribe Gardeniinae including ca. 60 genera. Tribe Aulacocalyceae was described by Robbrecht & Puff (1986) to accommodate several genera previously placed in Gardenieae (i.e., Aulacocalyx Hook.f., Heinsenia K.Schum., Himalrandia T.Yamaz., and Alleizettella Pit.), as these authors considered these genera to be more closely affiliated to subfamily Cinchonoideae of Rubiaceae. The study by Robbrecht & Puff (1986) served as a starting point for subsequent phylogenetic studies dealing with Gardenieae by, e.g., Andreasen & Bremer (1996) and Persson, (1996, 2000a), as reviewed by Bremer (2009). The study of subfamily Ixoroideae by Andreasen & Bremer (1996), based on morphological and molecular (rbcL) data, indicated that subtribe Diplosporineae and Posoqueria should be excluded from Gardenieae. Andreasen & Bremer’s (2000) extended phylogenetic study proposed the return of Heinsenia to Gardenieae from Aulacocalyceae, and the inclusion of Duperrea Pierre ex Pit. in Gardenieae. Persson (1996), in a phylogenetic study of Gardenieae s.str., analysed 70 morphological and anatomical characters for 81 taxa, and found support for a clade grouping genera with pollen grains in tetrads and an Alibertia A.Rich. ex DC. group. In a subsequent molecular phylogenetic study, Persson (2000a) resolved a core Gardenieae group (excluding subtribe Diplosporinae, Burchellia, Didymosalpinx Keay, and Schumanniophyton Harms), which included a Table 1. Summary of generic placement in the Gardenieae complex according to recent morphological or molecular studies, and proposed new circumscription of tribes. Robbrecht & Puff, 1986: morpho-anatomy Robbrecht, 1988, emend. in 1993: morpho-anatomy Andreasen & Bremer, 2000: phylogeny Robbrecht & Manen, 2006: phylogeny Agouticarpa C.H.Perss. This study: phylogeny Cordiereae Aidia Lour. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Alibertia A.Rich. ex DC. Gardenieae Gardenieae Gardenieae Gardenieae Cordiereae Gardenieae Cordiereae Amaioua Aubl. Aoranthe Somers Isertieae ? Argocoffeopsis Lebrun Gardenieae Gardenieae Atractocarpus Schltr. & K.Krause Gardenieae ? Gardenieae Gardenieae Gardenieae Coffeeae Coffeeae Gardenieae Gardenieae Atractogyne Pierre Gardenieae Gardenieae Gardenieae Sherbournieae Aulacocalyx Hook.f. Aulacocalyceae Aulacocalyceae Gardenieae Gardenieae Belonophora Hook.f. Aulacocalyceae ? Coffeeae Coffeeae Gardenieae Gardenieae Gardenieae Gardenieae Bertiera Aubl. Gardenieae Coffeeae Coffeeae Botryarrhena Ducke Retiniphylleae ? Benkara Adans. Aulacocalyceae ? Bertiereae Cordiereae Brachytome Hook.f. Gardenieae Gardenieae Gardenieae Gardenieae Brenania Keay Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Bungarimba K.M.Wong Burchellia R.Br. Gardenieae Calochone Keay Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Calycosiphonia Pierre ex. Robbr. Gardenieae Gardenieae Gardenieae Gardenieae Coffeeae Canephora Juss. Hypobathreae Octotropideae Octotropideae Canthiopsis Seem. Casasia A.Rich Pavetteae Gardenieae Gardenieae Gardenieae Version of Record (identical to print version). Gardenieae Gardenieae 803 TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) Table 1. Continued. Robbrecht & Puff, 1986: morpho-anatomy Robbrecht, 1988, emend. in 1993: morpho-anatomy Andreasen & Bremer, 2000: phylogeny Robbrecht & Manen, 2006: phylogeny This study: phylogeny Catunaregam Wolf Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Ceriscoides (Hook.f) Tirveng. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Chapelieria A.Rich Hypobathreae Octotropideae Cladoceras Bremek. Pavetteae Pavetteae Octotropideae Pavetteae Pavetteae Coddia Verdc. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Coffea L. Coffeeae Coffeeae Coffeeae Coffeeae Coffeeae Pavetteae Pavetteae Octotropideae Octotropideae Coptosperma Hook.f. Pavetteae Cordiera A.Rich. ex DC. Cremaspora Benth. Cordiereae Gardenieae Gardenieae Cremasporeae Deccania Tirveng. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Dictyandra Welw. ex Benth. & Hook.f Pavetteae Pavetteae Pavetteae Pavetteae Pavetteae Didymosalpinx Keay Gardenieae Gardenieae Gardenieae Dioecrescis Tirveng. Gardenieae Gardenieae Gardenieae Gardenieae Discospermum Dalzell Gardenieae Coffeeae Coffeeae Coffeeae Duperrea Pierre ex Pit. Pavetteae Gardenieae Gardenieae Gardenieae Durioa L.f. Gardenieae Gardenieae Gardenieae Gardenieae Cordiereae Euclinia Salisb. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Fagerlindia Tirveng. Gardenieae Gardenieae Feretia Delile Hypobathreae Octotropideae Octotropideae Octotropideae Fernelia Comm. ex Lam. Hypobathreae Octotropideae Octotropideae Octotropideae Flagenium Baill. Hypobathreae Octotropideae Galiniera Delile Hypobathreae Gallienia Dubard & Dop Gardenieae Octotropideae Octotropideae Octotropideae Octotropideae Gardenia J.Ellis Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Genipa L. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Cordiereae Heinsenia K.Schum. Aulacocalyceae Aulacocalyceae Gardenieae Hyperacanthus E.Mey. ex Bridson Gardenieae Gardenieae Gardenieae Hypobathrum Blume Hypobathreae Octotropideae Hyptianthera Wight & Arn. Hypobathreae Octotropideae Octotropideae Jovetia Guédès Hypobathreae Octotropideae Octotropideae Glossostipula Lorence Kailarsenia Tirveng. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Hypobathreae Octotropideae Kutchubaea Fisch. ex DC. Gardenieae Gardenieae Lamprothamnus Hiern Hypobathreae Octotropideae Gardenieae Octotropideae Kochummenia K.M.Wong Kraussia Harv. Gardenieae Gardenieae Gardenieae Octotropideae Octotropideae Gardenieae Cordiereae Octotropideae Gardenieae Lemyrea (A.Chev.) A.Chev. & Beille Octotropideae ? Pavetteae Pavetteae Octotropideae Pavetteae Pavetteae Pavetteae Gardenieae Gardenieae Macrosphyra Hook.f. Gardenieae Gardenieae Gardenieae Mantalania R.Cap. ex J.-F.Leroy Gardenieae Gardenieae Gardenieae Massularia (K.Schum.) Hoyle Gardenieae Melanopsidium Colla 804 Gardenieae Gardenieae Larsenaikia Tirveng. Leptactina Hook.f. Octotropideae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Cordiereae Version of Record (identical to print version). TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) Table 1. Continued. Robbrecht & Puff, 1986: morpho-anatomy Robbrecht, 1988, emend. in 1993: morpho-anatomy Andreasen & Bremer, 2000: phylogeny Robbrecht & Manen, 2006: phylogeny This study: phylogeny Gardenieae Sherbournieae Mitriostigma Hochst. Gardenieae Gardenieae Gardenieae Monosalpinx N.Hallé Gardenieae Gardenieae Gardenieae Morelia A.Rich. ex DC. Gardenieae Neofranciella Guillaumin Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Oligocodon Keay Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Oxyanthus DC. Gardenieae Gardenieae Gardenieae Gardenieae Sherbournieae Oxyceros Lour. Gardenieae Gardenieae Gardenieae Gardenieae Paracephaelis Baill. Pavetteae ? Paragenipa Baill. Hypobathreae Octotropideae Pavetta L. Pavetteae Pavetteae Pavetteae Pelagodendron Seem. Gardenieae Gardenieae Petitiocodon Robbr. Gardenieae Gardenieae Gardenieae Gardenieae Pleiocoryne Rauschert Gardenieae Gardenieae Gardenieae Polysphaeria Hiern Hypobathreae Octotropideae Hypobathreae Octotropideae Gardenieae Porterandia Ridl. Preussiodora Keay Gardenieae Gardenieae Ramosmania Tirveng. Hypobathreae Octotropideae Randia L. Gardenieae Gardenieae Octotropideae Octotropideae Pavetteae Pavetteae Pavetteae Octotropideae Phellocalyx Bridson Pouchetia A.Rich. ex DC. Gardenieae Gardenieae Octotropideae Gardenieae Gardenieae Gardenieae Gardenieae Octotropideae Octotropideae Gardenieae Gardenieae Octotropideae Octotropideae Gardenieae Gardenieae Cordiereae Robbrechtia De Block Pavetteae Rosenbergiodendron Fagerl. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Rubovietnamia Tirveng. Rutidea DC. Octotropideae Gardenieae Riodocea Delprete Rothmannia Thunb. Gardenieae Pavetteae Gardenieae Gardenieae Gardenieae Pavetteae Schizenterospermum Homolle ex Arènes Pavetteae Pavetteae Pavetteae Pavetteae ? Pavetteae Pavetteae Schumanniophyton Harms Gardenieae Gardenieae Gardenieae Sericanthe Robbr. Gardenieae Gardenieae Coffeeae Gardenieae ? Coffeeae Coffeeae Sherbournia G.Don Gardenieae Gardenieae Gardenieae Sherbournieae Sphinctanthus Benth. Gardenieae Gardenieae Gardenieae Gardenieae Stachyarrhena Hook.f. Gardenieae Gardenieae Gardenieae Cordiereae Stenosepala C.H.Perss. Cordiereae Sukunia A.C.Smith Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Tamilnadia Tirveng. Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Tarenna Gaertn. Pavetteae Pavetteae Pavetteae Pavetteae Pavetteae Tarennoidea Tirveng. & Sastre Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Gardenieae Tennantia Verdc. Pavetteae Pavetteae Pavetteae Tocoyena Aubl. Gardenieae Gardenieae Gardenieae Tricalysia A.Rich. ex DC. Gardenieae Trukia Kaneh. Pavetteae Gardenieae Coffeeae Coffeeae Coffeeae Gardenieae Gardenieae Gardenieae Gardenieae Coffeeae Coffeeae Vidalasia Tirveng. Xantonnea Pierre ex Pit. Gardenieae Hypobathreae Octotropideae Version of Record (identical to print version). 805 TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) “Gardenia clade”, a “Randia clade” and an Alibertia group (for a complete circumscription of clades, see Persson, 2000a: fig. 2). In the most recent large-scale phylogenetic study of Rubiaceae, Bremer & Eriksson (2009) included 534 taxa from 329 genera, of which over 100 taxa were from Gardenieae and relatives. Using the Bayesian (MCMC) method, they found no support for a monophyletic Gardenieae, but good support for several “Gardenieae” groups that correspond to extended circumscriptions of groups presented above: an “Alibertia clade” (Alibertia, Amaioua, Borojoa Cuatrec., Duroia L.f., Genipa, Glossostipula Lorence, Kutchubaea including Ibetralia Bremek., Melanopsidium Colla, Stachyarrhena Hook.f., Stenosepala C.H.Perss.; cf. Andreasen & Bremer, 1996; Persson, 2000a, b), a “Randia clade” (Calochone Keay, Casasia A.Rich., Euclinia Salisb., Oligocodon Keay, Macrosphyra Hook.f., Preussiodora Keay, Randia, Rosenbergiodendron Fagerl.), a “Gardenia clade” (Aoranthe Somers p.p., Ceriscoides (Hook.f.) Tirveng., Coddia Verdc., Gardenia, Genipa, Kailarsenia Tirveng.; cf. Persson, 2000a), and an “Aidia clade” (Aidia, Benkara Adans., Hyperacanthus E.Mey. ex Bridson, Oxyceros Lour., Randia p.p., Sphinctanthus Benth.). Schumanniophyton was resolved as the most basal lineage of the Gardenieae in Bremer & Eriksson (2009). Previous phylogenetic studies have indicated the paraphyletic status of Gardenieae with respect to the previously recognized tribe Aulacocalyceae. Even though other major groups (including tribes) have been resolved as monophyletic, e.g., Octotropideae: Andreasen & Bremer (2000), Tosh & al. (2008), Alejandro & al. (2011); Coffeeae and Bertiereae: Davis & al. (2007), Tosh & al. (2009), the circumscriptions of Gardenieae and its relatives remain unsettled. Moreover, many Gardenieae genera have so far not been included in any molecular phylogenetic studies (see Table 1). Most of them are small genera and often narrow endemics, segregated from the traditionally larger Gardenia, Genipa, Randia, or Rothmannia Thunb. The lack of phylogenetic resolution in previous phylogenetic studies within the Gardenieae complex (which is here delimited as the clade shown in Bremer & Eriksson, 2009: fig. 4 and including Cremasporeae, “Gardenieae”, Octotropideae, and Pavetteae) despite the use of several phylogenetic markers (rbcL, rps16, trnLF, ITS) is very intriguing. The low resolution could be explained by missing data, lack of variation in the markers used, or data incongruence (as with Rubiaceae-Ixoroideae: see Mouly & al., 2009). To establish a monophyletic Gardenieae and elucidate the placement of satellite genera such as Burchellia, we consequently investigated an extensive sampling of relevant genera within Gardenieae and related tribes. Using five plastid gene regions (rbcL, ndhF, trnTF, rps16, atpB-rbcL), we performed phylogenetic analyses of combined data in order to obtain a well-resolved and supported tree topology for the Gardenieae complex. MATERIALS AND METHODS Taxon sampling and molecular regions. — We included 137 accessions of Gardenieae and its relatives (the Gardenieae complex) which formed the ingroup (Appendix 1). Attention was given to numerous small genera that are poorly known 806 and have not previously been included in molecular phylogenetic studies, 21 of which were newly sequenced in the present study. When possible we selected type species. We were unable to extract sequenceable DNA for eight genera currently placed in Gardenieae: Adenorandia C.Vermoesen, Aidiopsis Tirveng., Alleizettella Pit., Fosbergia Tirveng., Himalrandia T.Yamaz., Pitardella Tirveng., Pseudaidia Tirveng., and Sulitia Merr. Outgroup taxa were mainly representatives of tribes of Ixoroideae (including 18 taxa), plus two taxa of subfamily Cinchonoideae, and one species of Luculia Sweet. We utilized five regions in the chloroplast genome, rbcL, ndhF, trnTF, rps16, and atpB-rbcL as data sources, as they have been shown to provide numerous informative characters within Rubiaceae (e.g., Razafimandimbison & al., 2008; Rydin & al., 2008, 2009). The primers used are listed in Rydin & al. (2008: table 2). DNA was extracted, amplified and sequenced using procedures previously described (Kårehed & Bremer, 2007; Rydin & al., 2008, 2009). Alignment and phylogenetic reconstruction. — Sequences were aligned using Se-Al v.2.0 (Rambaut, 1996) and subsequently manually adjusted with MEGA5 (Tamura & al., 2011). Bayesian analyses were performed in MrBayes v.3.1.2p (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003; Altekar & al., 2004). For each single-marker dataset or partition, the best performing evolutionary models were identified under the Akaike information criterion (AIC; Akaike, 1973). We performed these calculations with the software MrAIC v.1.4.3 (Nylander, 2004). Ambiguous regions of the alignments were excluded from the datasets in the analyses. Each marker was used as one partition, but trnTF was decomposed into three partitions, one for the trnT-trnL spacer, one for the trnL intron, and the third for the trnL-trnF spacer. Selected models were: (1) GTR + G for the trnT-trnL, trnL-trnF, rps16, and atpB-rbcL partitions; (2) GTR + I for the trnL partition; and (3) GTR + I + G for the ndhF and rbcL partitions. Insertion-deletions were mostly autapomorphic, and were not coded. The single-marker analyses were run for 107 generations, with a sample frequency of 1000 and using four parallel chains. A flat Dirichlet prior probability (all values are 1.0) was selected for the substitution rates (revmatpr) and the nucleotide frequencies (statefreqpr). The analyses of the combined dataset were done using the same settings, but with an extended number of generations (30 × 106). Partitions were unlinked so that each partition was allowed to have its own set of parameters. Due to an apparent high level of substitutions for Schumanniophyton, we also performed a combined data analysis without Schumanniophyton to test the effect of the genus on the inference of relationships and the estimation of posterior probabilities. Parsimony analyses were conducted using PAUP* v.4.0b8b (Swofford, 2002). The maximum parsimony (MP) trees were found by heuristic search, tree bisection-reconnection (TBR) branch swapping, using 1 × 104 replicates of random stepwise addition, with the MULTREES on. Characters were given equal weight, gaps were treated as missing data, and only phylogenetically informative indels were coded. Consistency index (CI; Kluge & Farris, 1969) and retention index (RI; Farris, 1989) were calculated to estimate homoplasy. To assess relative support for clades, bootstrap values (BS) were estimated from 1 × 104 replicates, with MULTREES off, TBR branch swapping, Version of Record (identical to print version). TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) and five random addition sequences. Groups characterized by bootstrap support of more than 85% were regarded as strongly supported. Evolution of morphological and functional traits. — Four morphological and functional features previously considered taxonomically important for recognizing infratribal groups in Gardenieae, i.e., pollen grain type and aperture type, exotesta type, and breeding system (Robbrecht & Puff, 1986; Persson, 2000a), were selected in order to see how these correspond to the phylogeny. The information was taken from earlier publications, mainly from Robbrecht & Puff (1986) and Persson (1993, 1996). The mapping was done using the software RASP v.2.0b (Yu & al., 2010, 2011), with one of the topologies inferred from the Bayesian analysis, reduced to a single representative per genus, limited to Gardenieae and Octotropideae, and using Coffea as outgroup. RESULTS Data information. — The complete cpDNA dataset consisted of 6471 characters (of 7977 aligned sites) of which 2003 were from ndhF, 1551 from trnTF, 803 from the rps16, 702 from atpB-rbcL, and 1402 from rbcL. The combined dataset contained 2524 informative characters (ca. 39%). The ndhF gene alone contained a large part of these informative characters (809; vs. 689 for trnTF, 456 for atpB-rbcL, 323 for rps16, and 247 for rbcL). Only 56% of the total of informative characters was informative in the ingroup. Two short inversions were detected within the datasets, a 4-bp segment (corresponding to positions 55472 to 55475 in the Coffea arabica L. chloroplast genome GenBank acc. EF044213, Samson & al., 2007) in the atpB-rbcL spacer, and a 8-bp segment (positions 5428 to 5435 in the C. arabica chloroplast genome) in the rps16 intron. These inversions were excluded from the analyses. The genetic distance of Schumanniophyton magnificum (K.Schum.) Harms to other taxa of the Gardenieae complex ranged from 0.021 to 0.035 (0.027 mean), and the terminal branch length of Schumanniophyton corresponded to 90 substitutions (Electr. Suppl.: Fig. S1A). Genetic distances within the Gardenieae complex, excluding Schumanniophyton, ranged from 0.003 to 0.016 (0.009 in mean) and the mean number of substitutions for ingroup branches was 13.3. A 33-bp insertion (position 48422 in the Coffea arabica chloroplast genome) was observed in the three Schumanniophyton magnificum samples for the trnLF intron segment that did not occur in any other genus within the complete dataset. Separate analyses. — Results of individual markers were mostly congruent and incongruences between analyses, when present, were never supported (not shown). Most of these separate analyses contained phylogenetic information to resolve (but rarely support) some of the groups within Gardenieae but not to resolve relationships between these groups. For a summary of resolution and support for groups see Table 2. Of the five markers used, ndhF provided the highest number of resolved and supported phylogenetic relationships. Combined analyses. — The Bayesian analysis of the combined data resulted in a partially supported tree topology (Fig. 2). The MP analyses were congruent with the Bayesian analyses with the exception of Tocoyena associating with Sherbournia G.Don–Mitriostigma Hochst. and Oxyanthus in MP, but without support. Minor discrepancies between the two analyses were never supported. However, most of main clades inferred in the Bayesian analyses were not supported by the bootstrap analysis (Fig. 2). The ingroup was monophyletic and well supported (PP = 1.00). Tribes Coffeeae and Bertiereae were sister to each (PP = 1.00/BS = 59), and in turn sister to the rest of the ingroup (PP = 0.92). The latter comprised two lineages, henceforth called the Octotropideae s.l. clade (PP = 0.95) and the Gardenieae-Pavetteae clade (PP = 1.00), respectively. A number of genera previously placed in Gardenieae were nested in the Octotropideae s.l. clade: Burchellia, Didymosalpinx, Mantalania R.Cap. ex J.-F.Leroy, and Monosalpinx N.Hallé, which together with Galiniera Delile formed a grade basal to the Octotropideae-Cremasporeae clade, although without highly supported relationships. The OctotropideaeCremasporeae clade received good support (PP = 1.00/BS = 88), but was internally poorly resolved. Within this latter clade, a lineage of Malagasy and Seychellois genera (from Gallienia sclerophylla Dubard & Dop to Canephora madagascariensis J.F.Gmel.; PP = 1.00/BS = 52) and a lineage of African–Indian Ocean–Asian genera (from Petitiocodon parviflorum (Keay) Robbr. to Fernelia buxifolia Lam.; PP = 1.00/BS = 90) were resolved. The Gardenieae-Pavetteae clade consisted of a polytomy including (1) the Sherbournia group (PP = 0.96), (2) the Alibertia group (PP = 1.00), (3) the Pavetteae (PP = 1.00/BS = 100), and (4) a large but unsupported clade (PP = 0.85) that consisted of five supported groups, the Gardenia group (PP = 1.00/BS = 60), the Rothmannia group (PP = 1.00/BS = 92), the Aidia group (PP = 1.00), the Randia group (PP = 0.96), and the Porterandia Ridl. group (PP = 0.98), plus two isolated genera: Schumanniophyton as sister to the entire clade, and Massularia (K.Schum.) Hoyle as unsupported sister to the Randia and Porterandia groups. Relationships between these groups were resolved, but not supported by high PP values. The high level of substitutions in Schumanniophyton apparently has an influence on phylogenetic reconstruction, as node support increased considerably when the genus was removed from the analysis (Electr. Suppl.: Fig. S1B). The inclusion or exclusion of the genus did not influence the inferred main relationships within the ingroup, although support for the Octotropideae s.l. clade was reduced (PP = 0.92) with its inclusion. Evolution of morphological and functional traits. — The four features mapped onto the phylogenetic tree (Fig. 3) showed contrasting patterns. The different pollen aggregation types and colpus types appeared homoplastic within the Gardenieae complex. However, pollen grains in monads and colporate apertures seem to be plesiomorphic, and porate grains have evolved several times independently in the lineage associating the Aidia, Porterandia and Randia groups, the Alibertia group, and the Sherbournia group. Tetrads evolved from monads at least three times in Gardenia, the Randia group, and the Sherbournia group. Massularia possesses an autapomorphic state Version of Record (identical to print version). 807 Version of Record (identical to print version). rps16 atpB-rbcL ndhF rbcL combined data Ingroup resolved (incl. Alberta; PP = 0.91) resolved (PP =1.00) resolved (incl. Boholia) resolved (PP = 0.76) resolved (PP = 0.97) resolved (PP = 1.00) Octotropideae s.l. clade resolved (excl. Burchellia & Galiniera) not resolved not resolved not resolved not resolved resolved (PP = 0.95) Gardenieae-Pavetteae clade not resolved not resolved not resolved not resolved not resolved resolved (PP = 1.00) Octotropideae-Cremasporeae clade resolved (PP = 0.95) not resolved resolved (PP = 0.83) resolved (PP = 1.00) not resolved resolved (PP = 1.00) Gardenieae (sensu this study) not resolved resolved (Schumanniophyton excluded; PP = 0.70) not resolved not resolved not resolved resolved (PP = 0.85) Aidia group not resolved unresolved not resolved resolved (PP = 0.94) resolved (PP = 0.78) resolved (PP = 1.00) Alibertia group resolved (PP = 0.84) resolved (PP = 1.00) resolved (PP = 1.00) resolved (PP = 1.00) resolved (PP = 1.00) resolved (PP = 1.00) Gardenia group resolved (PP = 0.70) resolved (PP = 0.82) not resolved resolved (PP = 1.00) resolved (PP = 0.94) resolved (PP = 1.00) Pavetteae resolved (PP = 0.98) resolved (PP = 1.00) resolved (PP = 1.00) resolved (PP = 1.00) not resolved resolved (PP = 1.00) Porterandia group resolved (excl. Brachytome; PP = 0.63) not resolved resolved (excl. Dioecrescis; PP = 0.89) resolved (PP = 0.96) not resolved resolved (PP = 1.00) Randia group resolved (PP = 1.00) resolved (PP = 0.66) resolved (PP = 1.00) resolved (PP = 1.00) resolved (PP = 1.00) resolved (PP = 0.96) Rothmannia group not resolved not resolved resolved (PP= 1.00) resolved (PP = 0.99) not resolved resolved (PP = 0.98) Sherbournia group not resolved not resolved not resolved resolved (PP = 0.99) not resolved resolved (PP = 0.96) TAXON 63 (4) • August 2014: 801–818 trnTF Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) 808 Table 2. Summary of the major clades observed in the combined marker analysis and indication of resolution or lack of resolution of similar clades in each of the individual marker analyses and their posterior probability when resolved. TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) with grains in massulae (polyads). Hermaphrodite flowers were inferred as plesiomorphic with dioecy present in two groups: as a potential synapomorphy of the Alibertia group and in several genera of the Porterandia and Randia groups. The folded testa type appeared to have evolved from an unfolded testa and was characteristic of the Sherbournia group, but was homoplasic as it is also present in Glossostipula. DISCUSSION The ingroup, consisting of the Gardenieae complex plus Coffeeae and Bertiereae, is monophyletic with high support. It comprises three major lineages (Fig. 2): the Bertiereae-Coffeeae clade, the Octotropideae s.l. clade, and the Gardenieae-Pavetteae clade. The Gardenieae-Pavetteae and Octotropideae s.l. clades are sisters, with relatively moderate support (PP = 0.92); this species-rich clade is in turn sister to the Bertiereae-Coffeeae clade. The latter clade (Fig. 2) includes several members of Gardenieae subtribe Diplosporinae, as shown in earlier studies (Andreasen & Bremer, 2000; Persson, 2000a; Davis & al., 2007; Bremer & Eriksson, 2009). As no other genera of Gardenieae sensu Andreasen & Bremer (2000) investigated in this study belong to the Bertiereae-Coffeeae clade, this lineage is not further discussed here. Tribe Gardenieae sensu Robbrecht & Puff (1986, as Gardenieae subtribe Gardeniinae) and Andreasen & Bremer (2000) is polyphyletic as Burchellia, Didymosalpinx, Monosalpinx, and Mantalania are more closely related to Octotropideae-Cremasporeae (Fig. 2), and Pavetteae and the sampled Aulacocalyceae taxa Aulacocalyx jasminiflora Hook.f. continued next page 1.0/100 0.52/1.0/74 1.0/ 100 0.99/61 0.50/1.0/67 1.0/90 0.9/55 0.98/ 60 0.92/- 0.86/75 1.0/96 1.0/99 1.0/88 1.0/55 * 1.0 /52 1.0/66 1.0/100 1.0/- 0.71/- 1.0/100 0.74/1.0 /100 1.0/98 0.95/- * 1.0/100 0.66/0.50/- 0.89/- 0.59/0.82/1.0/1.0/97 1.0/59 * 1.0/1.0/99 1.0/ 100 1.0/68 1.0/51 1.0/52 1.0/100 1.0/55 0.77/78 1.0/100 1.0/99 1.0/85 0.92/87 1.0/98 1.0/70 0.56/0.98/78 1.0/80 1.0/80 1.0/- Fernelia_buxifolia Ramosmania_rodriguesii Feretia_aeruginescens Kraussia_floribunda Kraussia_floribunda Hypobathrum_racemosum Hyptianthera_stricta Pouchetia_baumanniana Lamprothamnus_zanguebaricus Polysphaeria_lanceolata Petitiocodon_parviflorum Canephora_madagascariensis Chapelieria_madagascariensis Lemyrea_krugii Paragenipa_lancifolia Jovetia_humilis Flagenium_sp. Gallienia_sclerophylla Cremaspora_triflora Cremaspora_triflora Didymosalpinx_abbeokutae Didymosalpinx_abbeokutae Monosalpinx_guillaumetii Monosalpinx_guillaumetii Mantalania_sambiranensis Mantalania_sambiranensis Galiniera_saxifraga Galiniera_saxifraga Burchellia_bubalina Belonophora_coriacea Xantonnea_parvifolia Discospermum_polyspermum Sericanthe_andongensis Tricalysia_ovalifolia Argocoffeopsis_scandens Calycosiphonia_spathicalyx Coffea_mannii Coffea_mannii Coffea_arabica Bertiera_guianensis Alberta_magna Augusta_longifolia Lindenia_rivalis Wendlandia_glabrata Boholia_sp Ixora_margaretae Ixora_trilocularis Ixora_coccinea Aleisanthiopsis_distantiflora Greenea_corymbosa Vangueria_madagascariensis Scyphiphora_hydrophylacea Crossopteryx_febrifuga Mussaenda_erythrophylla Sabicea_aspera Posoqueria_latifolia Sipanea_hispida Emmenopterys_henryi Exostema_lineatum Guettarda_speciosa Luculia_gratissima OCTOTROPIDEAE CREMASPOREAE O C T O T R O P I D E A E C L A D E s. l. C O F F E E A E B E R T I E R E A E Fig. 2. Consensus tree (Bayesian MCMC analysis) of the combined cpDNA sequence data; numbers given at branches are Bayesian posterior probabilities and Bootstrap support (PP/BS) for corresponding nodes. Grey boxes indicate the main groups discussed in the text. Terminals in bold italic indicate taxa currently placed in tribe Gardenieae (as Gardeniieae fide Robbrecht & Puff, 1986; corrected by Andreasen & Bremer, 2000). Asterisks indicate main clades discussed in the text. Version of Record (identical to print version). 809 TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) Newly proposed classification Supported groups 1.0/94 0.64/50 0.63/1.0/99 0.83/1.0/100 0.72/0.85/1.0/100 1.0/73 1.0/100 0.92/1.0 /99 1.0/100 0.56/- 0.98/1.0/97 * 1.0/71 1.0/61 1.0 0.96/63 /99 1.0/68 1.0/98 0.79/- 1.0/100 0.71/0.68/- 1.0/100 0.88/- 0.96/- * 0.93/- 1.0/99 0.73/- 0.54/76 0.59/1.0/63 1.0/89 0.88/60 0.95/- 1.0/- 0.69/- * 1.0/100 1.0/100 0.86/56 1.0 1.0/92 /88 1.0/100 1.0 /98 0.90/- * 1.0/100 1.0/98 1.0 /98 1.0/97 0.85/- 1.0/60 * 0.75/0.88/65 1.0/100 * 1.0/100 0.99/75 0.99/74 1.0/92 1.0/100 * 1.0/100 0.88/56 1.0 /81 1.0/100 0.52/73 0.97/1.0/89 1.0/100 1.0/- 1.0/100 0.99/92 1.0/51 0.75 1.0/- * 0.98/0.62/1.0 /100 0.96/0.54/1.0/100 0.97/75 1.0/66 0.96/1.0/100 * Sukunia_pentagonoides Trukia_carolinensis Bungarimba_ridsdalei Catunaregam_spinosa Deccania_pubescens Atractocarpus_bracteatus Neofranciella_pterocarpon Tarennoidea_wallichii Porterandia_anisophylla Porterandia_anisophylla Duperrea_pavettifolia Rubovietnamia_aristata Vidalasia_fusca Vidalasia_murina Tamilnadia_uliginosa Tamilnadia_uliginosa Dioecrescis_erythroclada Dioecrescis_erythroclada Brachytome_scortechinii Calochone_redingii Macrosphyra_longistyla Euclinia_longiflora Euclinia_longiflora Oligocodon_cunliffeae Pleiocoryne_fernandensis Preussiodora_sulphurea Preussiodora_sulphurea Randia_aculeata Randia_aculeata Casasia_calophylla Rosenbergiodendron_formosum Sphinctanthus_microphyllus Tocoyena_formosa Massularia_acuminata Fagerlindia_sinensis Oxyceros_sp. Oxyceros_longiflorus Fagerlindia_sp. Benkara_malabarica Aoranthe_cladantha Aoranthe_penduliflora Aidia_abeidii Hyperacanthus_amoenus Morelia_senegalensis Morelia_senegalensis Kochummenia_stenopetala Kochummenia_stenopetala Phellocalyx_vollesenii Rothmannia_capensis Heinsenia_diervilleoides Heinsenia_diervilleoides Aulacocalyx_jasminiflora Coddia_rudis Coddia_rudis Kailarsenia_tentaculata Larsenaikia_ochreata Gardenia_jasminoides Brenania_brieyi Ceriscoides_sessiliflora Genipa_americana Schumanniophyton_magnificum Schumanniophyton_magnificum Schumanniophyton_magnificum Canthiopsis_odorata Pelagodendron_vitiense Tarenna_buruensis Tennantia_sennii Paracephaelis_sp. Schizenterospermum_sp. Robbrechtia_grandifolia Coptosperma_nigrescens Rutidea_fuscescens Rutidea_orientalis Cladoceras_subcapitatum Pavetta_indica Leptactina_delagoensis Leptactina_platyphylla Dictyandra_arborescens Alibertia_edulis Alibertia_edulis Cordiera_triflora Melanopsidium_nigrum Agouticarpa_williamsii Amaioua_guianensis Riodocea_pulcherrima Duroia_eriopila Duroia_eriopila Glossostipula_concinna Kutchubaea_semisericea Stenosepala_hirsuta Botryarrhena_venezuelensis Stachyarrhena_heterochroa Mitriostigma_axillare Oxyanthus_speciosus Sherbournia_calycina Atractogyne_gabonii Atractogyne_gabonii PORTERANDIA GROUP RANDIA GROUP AIDIA GROUP ROTHMANNIA GROUP GARDENIA GROUP PAVETTEAE C L A D E GARDENIEAE PAVETTEAE ALIBERTIA GROUP CORDIEREAE SHERBOURNIA GROUP SHERBOURNIEAE continued from previous page 810 G A R D E N I E A E P A V E T T E A E Version of Record (identical to print version). TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) and Heinsenia diervilleoides K.Schum. are nested in a supported but partially unresolved Gardenieae-Pavetteae clade (Fig. 2). Our results are congruent with that of Bremer & Eriksson (2009). Phylogenetic relationships within the Octotropideae s.l. clade The Octotropideae s.l. clade (Fig. 2) contains the sequenced members of tribes Octotropideae and Cremasporeae and four Gardenieae genera (Burchellia, Didymosalpinx, Mantalania, Monosalpinx). Octotropideae s.str. (Andreasen & Bremer, 2000) comprises about 23 genera from the Old World, of which 17 were here included. A close affinity between Cremaspora Benth. and Octotropideae as indicated by previous studies (Persson, 1996; Andreasen & Bremer, 2000; Robbrecht & Manen, 2006; Bremer & Eriksson, 2009) is also supported in our analyses, but we found insufficient resolution to distinguish Cremasporeae and Octotropideae as currently circumscribed. Two Octotropideae clades were retrieved. One includes most of the genera from Madagascar (Canephora Juss., Chapelieria, Lemyrea (A.Chev.) A.Chev. & Beille, Flagenium Baill., Gallienia Dubard & Dop, Jovetia Guédès) together with Paragenipa Baill. from the Seychelles, whereas the other clade includes genera distributed in tropical Africa, Madagascar (only Polysphaeria Hiern, which is African-Malagasy-Seychellois), and tropical Asia. Unfortunately, we were unable to obtain sequences for the type of the tribe, Octotropis Bedd., an endemic Indian genus, as available specimens of this genus are very few and old (Robbrecht & al., 1994). The West African Petitiocodon Robbr. was recently placed in Octotropideae (Tosh & al., 2008), and this placement is supported by our results. The East African Lamprothamnus Hiern was here sequenced for the first time and fell in the Octotropideae, a phylogenetic position corroborating its placement by Robbrecht (1980). The Indo-Chinese Hyptianthera Wight & Arn. was also newly sequenced here, and its placement in Octotropideae supports that by Robbrecht (1980, 1988) based on morphology. Hyptianthera is sister to the tropical Asian Hypobathrum, of which it is sometimes considered a synonym. Villaria Rolfe, recently sequenced by Alejandro & al. (2011), but not included here, was shown to belong to Octotropideae and is associated with Pouchetia. According to Tosh & al. (2008) and the salient features these authors give for Octotropideae, Cremaspora appears very close to Octotropideae, a reason why several authors have already proposed to merge Cremasporeae and Octotropideae (e.g., Robbrecht & Manen, 2006). Burchellia, Didymosalpinx, Mantalania, and Monosalpinx, currently placed in tribe Gardenieae, are resolved closer to the Octotropideae-Cremasporeae group. The exclusion of Burchellia and Didymosalpinx from Gardenieae was suggested by Persson (1996, 2000a), and this result is supported here. Monosalpinx and Mantalania (Hallé, 1968; Leroy, 1973; respectively) were both newly investigated here; and should now also be excluded from Gardenieae (Hallé, 1968; Leroy, 1973; respectively). The African and Malagasy Galiniera is related to the South African Burchellia, rendering Octotropideae sensu Andreasen & Bremer (2000) polyphyletic. The African Monosalpinx and Didymosalpinx share membranaceous polyspermous fruits (Hallé, 1968; Persson, 1996). Mantalania is endemic to Madagascar and morphologically very similar to Pseudomantalania J.-F.Leroy, another Malagasy endemic not successfully sequenced in the present study. An enlargement of Octotropideae Fig. 3. Morphological and functional traits mapping. The tree topology used is one of the topologies inferred from the Bayesian analysis of the combined cpDNA markers. Characteristics coding: pollen grain apertures (black box, porate/pororate; gray box, colporate); pollen grain types (black box, monads; gray box, tetrads; white box, polyads); breeding system (black box, hermaphroditic; gray box, dioecious; white box, monoecious; star, polygamous); exotesta shape (black box, unfolded; gray box, folded). Version of Record (identical to print version). s re tu s er pe em p y t a t n n ys ai ai s gr gr ing sta n n d e lle lle ee ot Po Po Br Ex * * * * * Sukunia pentagonoides Trukia carolinensis Bungarimba ridsdalei Catunaregam spinosa Deccania pubescens Atractocarpus bracteatus Neofranciella pterocarpon Tarennoidea wallichii Porterandia anisophylla Duperrea pavettifolia Rubovietnamia aristata Vidalasia fusca Vidalasia murina Tamilnadia uliginosa Dioecrescis erythroclada Brachytome scortechinii Calochone redingii Macrosphyra longistyla Euclinia longiflora Oligocodon cunliffeae Pleiocoryne fernandensis Preussiodora sulphurea Randia aculeata Casasia calophylla Rosenbergiodendron formosum Sphinctanthus microphyllus Tocoyena formosa Massularia acuminata Fagerlindia sinensis Oxyceros sp. Oxyceros longiflorus Fagerlindia sp. Benkara malabarica Aoranthe cladantha Aoranthe penduliflora Aidia abeidii Hyperacanthus amoenus Morelia senegalensis Kochummenia stenopetala Phellocalyx vollesenii Rothmannia capensis Heinsenia diervilleoides Aulacocalyx jasminiflora Coddia rudis Kailarsenia tentaculata Larsenaikia ochreata Gardenia jasminoides Brenania brieyi Ceriscoides sessiliflora Genipa americana Schumanniophyton magnificum Canthiopsis odorata Pelagodendron vitiense Tarenna buruensis Tennantia sennii Paracephaelis sp. Schizenterospermum sp. Robbrechtia grandifolia Coptosperma nigrescens Rutidea fuscescens Rutidea orientalis Cladoceras subcapitatum Pavetta indica Leptactina delagoensis Leptactina platyphylla Dictyandra arborescens Alibertia edulis Cordiera triflora Melanopsidium nigrum Agouticarpa williamsii Amaioua guianensis Riodocea pulcherrima Duroia eriopila Glossostipula concinna Kutchubaea semisericea Stenosepala hirsuta Botryarrhena venezuelensis Stachyarrhena heterochroa Mitriostigma axillare Oxyanthus speciosus Sherbournia calycina Atractogyne gabonii Fernelia buxifolia Ramosmania rodriguesii Feretia aeruginescens Kraussia floribunda Hypobathrum racemosum Hyptianthera stricta Pouchetia baumanniana Lamprothamnus zanguebaricus Polysphaeria lanceolata Petitiocodon parviflorum Canephora madagascariensis Chapelieria madagascariensis Lemyrea krugii Paragenipa lancifolia Jovetia humilis Flagenium sp. Gallienia sclerophylla Cremaspora triflora Didymosalpinx abbeokutae Monosalpinx guillaumetii Galiniera saxifraga Burchellia bubalina Coffea arabica 811 TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) to include these genera previously placed in Gardenieae would render the tribe very heterogeneous with respect to their very divergent reproductive features, e.g., inflorescence type and position, and fruit type. Phylogenetic relationships within the GardenieaePavetteae clade The members of Pavetteae sensu Andreasen & Bremer (2000) formed a supported clade, which is nested within the Gardenieae complex (Fig. 2). In contrast to Octotropideae s.l., members of this clade are characterized by terminal or pseudoaxillary inflorescences (with the exception of Burchellia). The placement of Aulacocalyx and Heinsenia within Rubiaceae has long been debated, with various systematic positions postulated. They have been: (1) included together or individually in Alberteae (Bremekamp, 1966; only Aulacocalyx in Robbrecht & Manen, 2006); (2) placed in Ixoreae (Verdcourt, 1958); placed in Gardenieae (only Heinsenia in Andreasen & Bremer, 2000); or, (3) in their own tribe Aulacocalyceae (Robbrecht & Puff, 1986). Kainulainen & al. (2009) recently refuted the placement of Aulacocalyx in Alberteae (see also Mouly & al., 2007: fig. 1). Our present data clearly show that Aulacocalyx and Heinsenia are sisters, and are resolved with the Rothmannia group (Fig. 2). The Gardenieae-Pavetteae clade consists of 78 genera that were resolved in supported lineages, of which we discuss the ones indicated in Fig. 2: the Sherbournia group, the Alibertia group, the Pavetteae, Schumanniophyton, the Gardenia group, the Rothmannia group, the Aidia group, Massularia, the Randia group, and Porterandia group. Sherbournia group. — This informal group was resolved by Persson (2000a) and Bremer & Eriksson (2009), including Atractogyne Pierre, Mitriostigma, and Oxyanthus. In the present study it also includes Sherbournia, a relationship that received high support (Fig. 2). These four genera are quite heterogeneous morphologically, although many species are lianescent, particularly in Atractogyne, Sherbournia, and also one species of Oxyanthus; Mitriostigma species are small to medium shrubs, branched or monocaulous (Sonké & al., 2009). Sherbournia has pollen grains in monads, whereas the other three genera all have pollen grains in tetrads (Robbrecht & Puff, 1986; Persson, 1993) (Fig. 3). The corolla shape in this group results from different pollination strategies (Robbrecht & Puff, 1986): in Sherbournia and Atractogyne the corolla tubes are somewhat campanulate and more than 4 cm long, in Oxyanthus the corolla is 4 to 25 cm long and narrow, whereas in Mitriostigma the corolla tube is less than 3 cm long. Anatomical structures of seeds may provide better morphological support to the group than gross morphology alone. The endosperm is entire, with a more or less folded exotesta (Hallé, 1962a; Bridson, 1979; Robbrecht & Puff, 1986) (Fig. 3). Alibertia group. — This informal group was resolved with strong support by Persson (1996, 2000a), and included Agouticarpa C.H.Perss. (as Genipa williamsii Standl.), Alibertia (incl. Borojoa), Amaioua, Duroia, Glossostipula, Kutchubaea, Melanopsidium, Stachyarrhena, and Stenosepala (as species 812 nova). Botryarrhena Ducke was shown to belong to the group by Cortés-Ballén & al. (2009). In the present study we found that also Cordiera and Riodocea Delprete are associated with the Alibertia group, which appears to be restricted to the Neotropics. The group can be characterized morphologically by the combination: dioecy (Fig. 3) usually with sexually dimorphic inflorescences, heteromerous flowers, and pollen grains shed as monads (Fig. 3). Cordiera was initially not placed in Gardenieae by Candolle (1830) because of its multilocular fruits. Multilocular fruits, however, are observed in Alibertia, Stenosepala, and Agouticarpa, whereas the other genera of the group have more typical Gardenia-like fruits (see Bremer & Eriksson, 1992: berries with a dense fleshy mesocarp and a pulpy placenta embedding the seeds). Riodocea (Delprete, 1999) is a monotypic genus from Brazil with numerous corolla lobes (12–15 lobes), compared to the typical 5–6 lobes of Gardenieae (Robbrecht & Puff, 1986). Delprete (1999) suggested a close relationship of the genus with Kutchubaea, but from our tree topology, Riodocea is closer to Amaioua and Duroia. Pavetteae. — Pavetteae form a supported clade of 13 genera. The Pacific Pelagodendron Seem., which was merged into Aidia by Puttock (2001), is here placed unambiguously in the Pavetteae, close to Tarenna. A persistent confusion between Pelagodendron and Canthiopsis Seem. has been noted in literature (Smith & Darwin, 1988; Tirvengadum, 1991; Puttock & Quinn, 2000). The distinction has generally been made based on the post-fertilization placental development in the fruit, present in Pelagodendron vitiense Seem. and absent in Canthiopsis, justifying the placement in Gardenieae and in Pavetteae, respectively. The new position of Pelagodendron vitiense renders the Pavetta group non-homogeneous in terms of placental development, but Puttock & Quinn (2000: 196) stressed that in Aidia and Pelagodendron the placental development is limited, so that it does not look like either Gardenieae or Pavetteae. The position of two New Caledonian species also placed in Pelagodendron (Tirvengadum, 1991) remains to be tested. The Fijian Canthiopsis, previously synonymized with Tarenna (Smith & Darwin, 1988), is here confirmed to belong to tribe Pavetteae, in close association with both Pelagodendron and Tarenna. Schumanniophyton. — The genus occurs in tropical West Africa and consists of three species only. According to the “simple” habit and the high synchronicity of growth and flowering of Schumanniophyton problematicum (A.Chev.) Aubrév., the genus has been suggested to have retained archaic features of Rubiaceae and Gardenieae (Hallé, 1962b, 1970). The tree topologies presented in this study (Fig. 3; Electr. Suppl.: Fig. S1A) instead suggest that these characteristics are derived within Rubiaceae, although the position within the GardenieaePavetteae clade is resolved but not well supported. Moreover, both the coding and the non-coding regions of Schumanniophyton cpDNA show numerous autapomorphies and consequently a long branch-length (Electr. Suppl.: Fig. S1A), indicating a higher relative rate of molecular evolution compared to other lineages of the Gardenieae-Pavetteae clade. We found no support for the earlier proposal (e.g., Persson, 1996; Robbrecht & Manen, 2006) to exclude Schumanniophyton from Gardenieae, a result in agreement with Bremer & Eriksson (2009). Version of Record (identical to print version). TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) Some important morphological characteristics clearly relate this genus to Gardenieae. The reduction of a leaf of the terminal leaf-pair supporting an inflorescence, characteristic of Schumanniophyton, is present in all members of Kochummenia K.M.Wong and Rothmannia and is common in the Aidia and the Porterandia groups (Wong, 2004). This form of leaf reduction and terminal flowering ties in with an apparently linear sympodial branch development (often resulting in the apparently “leaf-opposed inflorescences” found remarkable by morphologists) and is not known for Pavetteae. Gardenia group. — An informally named Gardenia clade comprising Aoranthe, Ceriscoides, Gardenia, Genipa, and Kailarsenia was resolved in the study by Persson (2000a). In Bremer & Eriksson (2009) the same group was supported (also including Coddia). The clade was also well supported in our analyses, although Aoranthe (two specimens) is nested within the Aidia group. In addition, the newly investigated genera Brenania Keay and Larsenaikia Tirveng. are also part of the Gardenia group. Larsenaikia is endemic to northern Australia and is sister group to the South East Asian Kailarsenia, the former being a segregate of the latter (Tirvengadum, 1993). The Gardenia group appears very heterogeneous morphologically, and is one of two pantropical lineages together with the Randia group (see also Persson, 2000a; Gustafsson & Persson, 2002). Among the genera present in this group, Gardenia is the only one that produces pollen grains in tetrads (Fig. 3), all the others have monads (Persson, 1993; Dessein & al., 2005). Tetrads are found elsewhere in the Gardenieae-Pavetteae clade, namely in the Randia and the Sherbournia groups (Fig. 3). Rothmannia group. — The Rothmannia group includes five genera, of which Rothmannia is the most widely distributed with ca. 40 species in the Old World. However, the monophyly of the genus has never been tested. In the phylogenetic tree presented here (Fig. 2), representatives of Aulacocalyx and Heinsenia form a clade, which in turn is sister to a clade comprising Phellocalyx Bridson, Rothmannia, and the newly investigated genus Kochummenia. Within this clade Phellocalyx and Rothmannia form a clade that is sister to Kochummenia. Kochummenia was described by Wong (1984) and includes three tropical Asian species, of which one was segregated from Rothmannia based on morphological characters. Indeed, in Kochummenia, the corolla shape is tubular and the lobes are much longer than the tube, a feature unique to that genus (Wong, 1984; Robbrecht & Puff, 1986). In Rothmannia the corolla can be campanulate, infundibular or salverform, with broad lobes shorter than the tube. Phellocalyx (Bridson & al., 1980), an East African monotypic genus, also has longer lobes than tubes, but differs from Kochummenia and Rothmannia by an increased number of corolla lobes (8–11 vs. 5 or rarely 8 in Rothmannia) and a spathiform calyx (vs. lobed). Interestingly, Rothmannia in its current circumscription includes both species with corolla lobes strictly contorted to the left (including African and Asian species) and species with a strict right contortion (exclusively African and Indian Ocean species), Phellocalyx has rightcontorted corolla lobes and Kochummenia has left-contorted corolla lobes. Aulacocalyx and Heinsenia mainly differ from the three other genera of the group by their axial placentation, and their reduced or absent seed-coat (Robbrecht & Puff, 1986). As Kochummenia and several Rothmannia species, Aulacocalyx and Heinsenia species present a left-contorted aestivation. Aidia group. — A group including Aidia, Benkara (as Griffithia), Fagerlindia Tirveng., and Oxyceros was suggested by Robbrecht & Puff (1986) based on morphology (shape and size of fruits, seeds, and exotesta cells). Recently, in Bremer & Eriksson (2009), the group was supported, and included Aidia, Benkara, Oxyceros, Hyperacanthus and few nonNeotropical Randia species (see Randia group below). In the present analysis, the group is highly supported and also includes Aoranthe and Morelia. We found no support for a broad Aidia group, as suggested by Persson (1996) based on morphology, which includes genera such as Bertiera, Atractocarpus Schltr. & K.Krause, and Pelagodendron. Fagerlindia was merged into Benkara (Ridsdale, 2008) based on morphology, but our present molecular data show that Oxyceros is paraphyletic with respect to Fagerlindia, and the species representing Benkara is sister to the Fagerlindia-Oxyceros complex (Fig. 2). We retrieved good support for the segregation of Aoranthe from Porterandia proposed by Somers (1988; see above). In the group, Aidia and Hyperacanthus are most diverse in terms of number of species, with ca. 60 (Ridsdale, 1996) and an estimated ca. 50 species (Rakotonasolo & Davis, 2006), respectively. Hyperacanthus shares the unusual feature of right-contorted aestivation, which in Rubiaceae is only recorded for several Rothmannia species, Phellocalyx (Bridson & al., 1980), and Gardeniopsis Miq. (A.P. Davis, pers. obs.), the last of which was not sampled in this study. Massularia. — This is a tropical African monotypic genus, and its species was previously classified as Gardenia. It differs from the other Gardenieae by having inflorescences with scorpioid terminal axes and pollen grains arranged in massulae (polyads; see Dessein & al., 2005: fig. 3), a feature unique in Gardenieae. It is here resolved as sister lineage to the Randia and the Porterandia groups, although with poor support. Randia group. — This group comprises three distinct lineages, one of which consists of the African genera Calochone, Euclinia, Macrosphyra, Oligocodon, Pleiocoryne Rauschert, and Preussiodora, which is sister to a Neotropical lineage comprising Casasia and Randia (sensu Gustafsson & Persson, 2002). Members of these two lineages of the Randia group have pollen grains in tetrads (Fig. 3). The third lineage contains the Neotropical Rosenbergiodendron, Sphinctanthus, and Tocoyena, all with pollen grains in monads (Persson, 2000a) (Fig. 3). The newly investigated African Pleiocoryne was resolved as sister to another African genus, Oligocodon. As discussed by Persson (1995, 2000a), the genera of his Randia group share smooth exotesta cells (with the exception of Casasia), a feature also present in Pleiocoryne. Porterandia group. — Several of the newly investigated genera, i.e., Brachytome Hook.f., Bungarimba K.M.Wong, Dioecrescis Tirveng., Rubovietnamia Tirveng., Sukunia A.C.Smith, Trukia Kaneh., and Vidalasia Tirveng., appear to be associated with Porterandia. The Porterandia group comprises here 15 genera that are mainly distributed in tropical Asia, Australia, and the Pacific Islands. Robbrecht & Puff (1986) considered Atractocarpus best placed in their Gardeniinae, Version of Record (identical to print version). 813 TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) but noted that it also resembles some members of Pavetteae in a number of characters (pauciflorous terminal inflorescences on lateral branches, often with leafy bracts, and exotesta cells with delicate thickenings). Puttock (2000) reduced Neofranciella Guillaumin, Sukunia, Sulitia, and Trukia to synonymy of Atractocarpus, according to the results of a phylogeny by Puttock & Quinn (2000) based on morphological characters. This broad concept of Atractocarpus was criticized by Wong (2004) when he described Bungarimba following a morphology-based phylogenetic analysis. He showed that Atractocarpus sensu Puttock was paraphyletic, with Porterandia nested within it (Wong, 2004: fig. 2). Our molecular phylogenetic results also question the circumscription of Atractocarpus proposed by Puttock (2000), considering evidence (though not supported) for close relationships of Sukunia, Trukia, and Bungarimba to Catunaregam and Deccania Tirveng. rather than to Atractocarpus and Neofranciella. Our analyses confirm the close relationship of Rubovietnamia to Duperrea recently proposed by Mou & Zhang (2010). Furthermore, the continental Asian Vidalasia described as a close relative of Rubovietnamia by Tirvengadum (1998), was retrieved, in our study, as sister to the clade of Duperrea and Rubovietnamia. Most of the taxa associated with Atractocarpus and Porterandia share polygamous or unisexual flowers (Wong, 2004), a feature relatively rare in the Gardenieae outside the Alibertia group (Fig. 3). The fruits of both Brachytome and Duperrea lack an arilloidal placenta and can be interpreted as drupes for Duperrea, characteristics which have been used to separate Gardenieae and Pavetteae. However, the lack of developed placentas in these genera appears to be autapomorphic within the Porterandia clade. Implications of the phylogenetic results for Gardenieae complex classification The initial delimitation of Gardenieae by Candolle (1830) is unnatural as it includes genera here placed within each of the three clades of the ingroup. The phylogenetic support values for the Octotropideae s.l. clade (Fig. 2; Electr. Suppl.: Fig. S1) do not confer enough confidence to include Burchellia, Mantalania, Didymosalpinx, and Monosalpinx, in the so far welldefined Octotropideae-Cremasporeae group. The GardenieaePavetteae clade members (Fig. 2) also contain considerable morphological diversity, and, when considered a single tribe, would be highly heterogeneous. Two main taxonomic strategies can be considered. One is to accommodate the members of the Gardenieae-Pavetteae clade (Aulacocalyceae, Pavetteae, Gardenieae) in one tribe; the name of this clade would have to be Pavetteae (Dumortier, 1829), which is older than Gardenieae (Candolle, 1830) and Aulacocalyceae (Robbrecht & Puff, 1986). The second option is to recognize the lineage of Massularia and five of the supported groups as tribe Gardenieae (Aulacocalyceae as a synonym). This clade received only moderate support (PP = 0.90) in the Bayesian analysis (Fig. 2), but was well supported in the Bayesian analysis when Schumanniophyton with a high amount of autapomorphic characters was excluded from the dataset (PP = 0.99; Electr. Suppl.: Fig. S1B). 814 This option allows the conservation of tribe Pavetteae as currently delimited (though including Pelagodendron). On the other hand, it necessitates the recognition at tribal level of the two remaining supported groups: the Alibertia group and the Sherbournia group. Under this second scenario, the Alibertia group has to be called Cordiereae, the only tribal level name available, and a new tribe needs to be described for the Sherbournia group. This alternative stabilizes the well-established name Gardenieae and the current circumscription of Pavetteae (plus Pelagodendron vitiense), both firmly used in Rubiaceae literature. The placement of Schumanniophyton is not solved yet. We provide here a new circumscription for Gardenieae (Fig. 1); recognize the Alibertia group at a tribal level, as Cordiereae; and describe a new tribe for the Sherbournia group. Pavetteae, though enlarged to include Pelagodendron, does not differ significantly from the concept proposed by Andreasen & Bremer (2000), and therefore does not require a new circumscription. Sherbournieae Mouly & B.Bremer, tr. nov. – Type: Sherbournia G.Don in Loudon, Encycl. Pl., Suppl. 2: 1303. 1855. Diagnosis: Sherbournieae differ from Gardenieae by their seeds possessing a folded testa (vs. unfolded). Monoecious subshrubs, shrubs, or lianas, with hermaphroditic or gynomonoecious flowers. Stipules interpetiolar, entire. Raphides absent. Inflorescences always pseudo-terminal, pauciflorous to multiflorous. Aestivation contorted to the left. Secondary pollen presentation present. Stigma club-shaped or capitate, stigmatic lobes fused over most of their length. Ovary with 2 carpels, unilocular, placentas parietal with many ovules. Fruit a fleshy berry with seeds imbedded in the pulpy placenta. Seeds without adaxial excavation, exotestal, with secondary thickenings and folded testa. Pollen grains in monads or in tetrads, 3(–4)-aperturate, porate or pororate. Genera included: Atractogyne, Mitriostigma, Oxyanthus, Sherbournia. Cordiereae A.Rich. ex DC. emend. Mouly – Type: Cordiera A.Rich. ex DC., Prodr. 4: 342, 445. 1830. Dioecious shrubs or trees. Stipules interpetiolar, entire. Raphides absent. Inflorescence terminal, solitary flower or rarely pauciflorous inflorescence in females and usually multiflorous in males. Flowers heteromorphic. Aestivation contorted to the left. Secondary pollen presentation absent. Stigma linear in male flowers and fusiform in female flowers, stigmatic lobes divided. Ovary of 2–7 carpels, uni- to plurilocular, placentas axile or parietal with (1–)3 to many ovules. Fruit a berry with more or less dry or fleshy walls (Cordiera). Seeds usually imbedded in a pulpy placenta. Seeds without adaxial excavation, exotestal, with thickenings. Pollen grains in monads, 3(–7)-aperturate, porate, pororate or colporate. Genera included: Agouticarpa, Alibertia, Amaioua, Botryarrhena, Cordiera, Duroia, Glossostipula, Kutchubaea, Melanopsidium, Riodocea, Stachyarrhena, Stenosepala. Gardenieae A.Rich. ex DC., Prodr. 4: 342, 367. 1830 – Type: Gardenia J.Ellis in Philos. Trans. 51: 935. 1761, nom. cons. Version of Record (identical to print version). TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) = Aulacocalyceae Robbr. & C.Puff – Type: Aulacocalyx Hook.f., Hooker’s Icon. Pl. 12: t. 1126. 1873. Hermaphrodite, monoecious or dioecious shrubs, trees, lianas, or rarely (hemi-)epiphytic shrubs. Stipules interpetiolar, entire. Raphides absent. Inflorescences terminal or pseudoaxillary. Aestivation contorted to the left, rarely to the right. Secondary pollen presentation predominantly present. Stigma club-shaped, capitate or linear, stigmatic lobes fused over most of their length (divided lobes rare). Ovary of 2–9(–16) carpels, uni- to plurilocular, placentas axile or parietal with one to many ovules. Fruit a berry with a dense fleshy mesocarp and a pulpy placenta embedding the seeds (known as Gardenia-type) or rarely a drupe (Duperrea). Seeds with or without adaxial excavation, mostly exotestal (seed-coat reduced in Duperrea, and Heinsenia), usually with thickenings. Pollen grains in monads, tetrads, or massulae, 3(–4)-porate, 3–4(–5)-colporate, or rarely pantoporate. Genera included: Aidia, Aoranthe, Atractocarpus, Aulacocalyx, Benkara, Brachytome, Brenania, Bungarimba, Calochone, Casasia, Catunaregam Wolf, Ceriscoides, Coddia, Deccania, Dioecrescis, Duperrea, Euclinia, Fagerlindia, Gardenia, Genipa, Heinsenia, Hyperacanthus, Kailarsenia, Kochummenia, Larsenaikia, Macrosphyra, Massularia, Morelia A.Rich. ex DC., Neofranciella, Oligocodon, Oxyceros, Phellocalyx, Pleiocoryne, Porterandia, Preussiodora, Randia, Rosenbergiodendron, Rothmannia, Rubovietnamia, Sphinctanthus, Sukunia, Tamilnadia, Tarennoidea Tirveng. & Sastre, Tocoyena, Trukia, Vidalasia. Genus possibly associated: Schumanniophyton. Genera previously associated with Gardenieae, based on the above morphological characteristics, but not included in a molecular phylogeny so far: Adenorandia, Aidiopsis, Alleizettella, Fosbergia, Himalrandia, Pseudaidia, Pseudomantalania, Sulitia. Genera excluded: Burchellia, Mantalania, Didymosalpinx, Monosalpinx, and members of Sherbournieae and Cordiereae as listed above. ACKNOWLEDGMENTS The authors wish to thank Anbar Khodabandeh and Linda Lundmark for help with lab work and sequencing; the institutions BR, G, GB, K, L, MO, NOU, NY, P, S, TAN, TEF, and UPS for granting access to herbarium collections and providing material for molecular studies; financial support was provided by the Swedish Research Council and the Knut and Alice Wallenberg Foundation to B. Bremer and the Bergius Foundation for a postdoctoral position for A. Mouly. LITERATURE CITED Akaike, H. 1973. A new look at the statistical model identification. IEEE Trans. Automatic Control AC-19: 716–723. Alejandro, G.D.J., Meve, U., Mouly, A., Thiv, M. & LiedeSchumann, S. 2011. Molecular phylogeny and taxonomic revision of the Philippine endemic Villaria Rolfe (Rubiaceae). Pl. Syst. Evol. 296: 1–20. http://dx.doi.org/10.1007/s00606-011-0472-9 Altekar, G., Dwarkadas, S., Huelsenbeck, J.P. & Ronquist, F. 2004. Parallel metropolis coupled Markov chain Monte Carlo for Bayesian phylogenetic inference. Bioinformatics 20: 407–415. http://dx.doi.org/10.1093/bioinformatics/btg427 Andreasen, K. & Bremer, B. 1996. Phylogeny of the subfamily Ixoroideae (Rubiaceae). Opera Bot. Belg. 7: 119–138. Andreasen, K. & Bremer, B. 2000. Combined phylogenetic analysis in the Rubiaceae-Ixoroideae: Morphology, nuclear and chloroplast DNA data. Amer. J. Bot. 87: 1731–1748. http://dx.doi.org/10.2307/2656750 Bremekamp, C.E.B. 1966. Remarks on the position, the delimitation and the subdivision of the Rubiaceae. Acta Bot. Neerl. 15: 1–33. http://dx.doi.org/10.1111/j.1438-8677.1966.tb00207.x Bremer, B. 2009. A review of molecular phylogenetic studies of Rubiaceae. Ann. Missouri Bot. Gard. 96: 4–26. http://dx.doi.org/10.3417/2006197 Bremer, B. & Eriksson, O. 1992. Evolution of fruit characteristics and dispersal modes in the tropical family Rubiaceae. Biol. J. Linn. Soc. 47: 79–95. http://dx.doi.org/10.1111/j.1095-8312.1992.tb00657.x Bremer, B. & Eriksson, T. 2009. Time tree of Rubiaceae: Phylogeny and dating the family, subfamilies, and tribes. Int. J. Pl. Sci. 170: 766–793. http://dx.doi.org/10.1086/599077 Bridson, D.M. 1979. Studies in Oxyanthus and Mitriostigma (Rubiaceae subfam. Cinchonoideae) for part 2 of ‘Flora of tropical East Africa: Rubiaceae’. Kew Bull. 34: 113–130. http://dx.doi.org/10.2307/4117977 Bridson, D.M., Gasson, P. & Robbrecht, E. 1980. Phellocalyx, a new tropical African genus in the Rubiaceae (Gardenieae). Kew Bull. 35: 315–321. http://dx.doi.org/10.2307/4114576 Candolle, A.P de. 1830. Prodromus systematis naturalis regni vegetabilis, vol. 4. Paris: Treuttel & Würz. http://dx.doi.org/10.5962/bhl.title.286 Cortés-Ballén, R., Delprete, P.G. & Motley, T.J. 2009. Phylogenetic placement of the tribe Retiniphylleae among the subfamily Ixoroideae (Rubiaceae). Ann. Missouri Bot. Gard. 96: 61–67. http://dx.doi.org/10.3417/2006198 Davis, A.P., Chester, M., Maurin, O. & Fay, M. 2007. Searching for the relatives of Coffea (Rubiaceae, Ixoroideae): The circumscription and phylogeny of Coffeeae based on plastid sequence data and morphology. Amer. J. Bot. 94: 313–329. http://dx.doi.org/10.3732/ajb.94.3.313 Davis, A.P., Govaerts, R., Bridson, D.M., Ruhsam, M., Moat, J. & Brummitt, N.A. 2009. A global assessment of distribution, diversity, endemism, and taxonomic effort in the Rubiaceae. Ann. Missouri Bot. Gard. 96: 68–78. http://dx.doi.org/10.3417/2006205 Delprete, P.G. 1999. Riodocea (Rubiaceae), a new genus from Brazilian Atlantic forest. Brittonia 51: 15–23. http://dx.doi.org/10.2307/2666550 Dessein, S., Ochoterena, H., De Block, P., Lens, F., Robbrecht, E., Schols, P., Smets, E., Vinkier, S. & Huysmans, S. 2005. Palynological characters and their phylogenetic signal in Rubiaceae. Bot. Review 71: 354–414. http://dx.doi.org/10.1663/0006-8101(2005)071[0354:PCATPS]2.0 .CO;2 Dumortier, B.C.J. 1829. Analyse des familles de plantes, avec l’indication des principaux genres qui s’y rattachent. Tournay: Casterman. http://dx.doi.org/10.5962/bhl.title.443 Farris, J.S. 1989. The retention index and the rescaled consistency index. Cladistics 5: 417–419. http://dx.doi.org/10.1111/j.1096-0031.1989.tb00573.x Gustafsson, C. & Persson, C. 2002. Phylogenetic relationships among species of the neotropical genus Randia (Rubiaceae, Gardenieae) inferred from molecular and morphological data. Taxon 51: 661– 674. http://dx.doi.org/10.2307/1555021 Hallé, F. 1962a. Biologie et position taxonomique du genre Atractogyne L.Pierre (Rubiaceae). Adansonia, sér. 3, 2: 309–321. Hallé, F. 1962b. Un type d’organisation remarquable: Schumanniophyton Version of Record (identical to print version). 815 TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) problematicum (A.Chev.) Aubr. (Rubiaceae-Gardenieae). Compt. Rend. Hebd. Séances Acad. Sci. 254: 4333–4335. Hallé, N. 1968. Monosalpinx N.Hallé, nouveau genre de Gardeniées d’Afrique, voisin de Didymosalpinx Keay emend. N.Hallé (Rub.). Adansonia, ser. 2, 8: 367–372. Hallé, N. 1970. Flore du Gabon, vol. 17, Famille des Rubiacées (2e partie). Paris: Muséum national d’histoire naturelle. Huelsenbeck, J.P. & Ronquist F. 2001. MrBayes: Bayesian inference of phylogeny. Bioinformatics 17: 754–755. http://dx.doi.org/10.1093/bioinformatics/17.8.754 Kainulainen, K., Mouly, A., Khodabandeh, A. & Bremer, B. 2009. Molecular phylogenetic analysis of the tribe Alberteae (Rubiaceae), with description of a new genus, Razafimandimbisonia. Taxon 58: 757–768. Kainulainen, K., Razafimandimbison, S.G. & Bremer, B. 2013. Phylogenetic relationships and new tribal delimitations in subfamily Ixoroideae (Rubiaceae). Bot. J. Linn. Soc. 173: 387–406. http://dx.doi.org/10.1111/boj.12038 Kårehed, J. & Bremer, B. 2007. The systematics of Knoxieae (Rubiaceae): Molecular data and their taxonomic consequences. Taxon 56: 1051–1076. http://dx.doi.org/10.2307/25065904 Kluge, A. & Farris, J.S. 1969. Quantitative phyletics and the evolution of anurans. Syst. Zool. 18: 1–32. http://dx.doi.org/10.2307/2412407 Leroy, J.F. 1973. Phytomorphologie et phytogéographie.—Sur l’organisation et le mode de développement d’un très remarquable ensemble naturel de Rubiacées-Gardéniées de Madagascar. Compt. Rend. Hebd. Séances Acad. Sci. Sér. D. 277: 1657–1659. Mou, F.J. & Zhang, D.X. 2010. Rubovietnamia nonggangensis (Rubiaceae), a new species from China. Bot. Stud. 51: 119–126. Mouly, A., Razafimandimbison, S.G., Achille, F., Haevermans, T. & Bremer, B. 2007. Phylogenetic placement of Rhopalobrachium fragrans (Rubiaceae): Evidence from molecular (rps16 and trnT-F) and morphological data. Syst. Bot. 32: 872–882. http://dx.doi.org/10.1600/036364407783390845 Mouly, A., Razafimandimbison, S.G., Khodabandeh, A. & Bremer, B. 2009. Phylogeny and classification of the species-rich pantropical showy genus Ixora (Rubiaceae-Ixoreae) with indications of geographical monophyletic units and hybrids. Amer. J. Bot. 96: 686–706. http://dx.doi.org/10.3732/ajb.0800235 Nylander, J.A.A. 2004. MrModeltest, version 2.0. Program distributed by the author. Evolutionary Biology Centre, Uppsala University. Persson, C. 1993. Pollen morphology of the Gardenieae-Gardeniinae (Rubiaceae). Nordic J. Bot. 13: 561–582. http://dx.doi.org/10.1111/j.1756-1051.1993.tb00101.x Persson, C. 1995. Exotesta morphology of the Gardenieae-Gardeniinae (Rubiaceae). Nordic J. Bot. 15: 285–300. http://dx.doi.org/10.1111/j.1756-1051.1995.tb00155.x Persson, C. 1996. Phylogeny of the Gardenieae (Rubiaceae). Bot. J. Linn. Soc. 121: 91–109. http://dx.doi.org/10.1111/j.1095-8339.1996.tb00746.x Persson, C. 2000a. Phylogeny of Gardenieae (Rubiaceae) based on chloroplast DNA sequences from the rps16 intron and trnL(UAA)F(GAA) intergenic spacer. Nordic J. Bot. 20: 257–269. http://dx.doi.org/10.1111/j.1756-1051.2000.tb00742.x Persson, C. 2000b. Phylogeny of the neotropical Alibertia group (Rubiaceae), with emphasis on the genus Alibertia, inferred from ITS and 5S ribosomal DNA sequences. Amer. J. Bot. 87: 1018–1028. http://dx.doi.org/10.2307/2657002 Puttock, C.F. 2000. Revision of Atractocarpus (Rubiaceae: Gardenieae) in Australia and new combinations for some extra-Australian taxa. Austral. Syst. Bot. 12: 271–309. http://dx.doi.org/10.1071/SB97030 Puttock, C.F. 2001. From everlasting to Aidia Lour. (Rubiaceae: Gardenieae). Malaysian Naturalist 55: 147–179 Puttock, C.F. & Quinn, C.J. 2000. Generic concepts in Australian Gardenieae (Rubiaceae): A cladistic approach. Austral. Syst. Bot. 12: 181–199. http://dx.doi.org/10.1071/SB98001 816 Rakotonasolo, F. & Davis, A.P. 2006. Six species of Madagascan Genipa transferred to Hyperacanthus (Rubiaceae-Gardenieae) and new data on general morphology, placentation and ovary structure in Hyperacanthus. Taxon 55: 387–396. http://dx.doi.org/10.2307/25065586 Rambaut, A. 1996. Se-Al, version 1.dl. Sequence alignment program. ftp://evolve.zo.ox.ac.uk/packages/Se-Al-All0a1.hqx Razafimandimbison, S.G., Rydin, C. & Bremer, B. 2008. Evolution and trends in the Psychotrieae alliance (Rubiaceae): A rarely reported evolutionary change of many-seeded carpels from oneseeded carpels. Molec. Phylogen. Evol. 48: 207–223. http://dx.doi.org/10.1016/j.ympev.2008.03.034 Razafimandimbison, S.G., Kainulainen, K., Wong, K.M., Beaver, K. & Bremer, B. 2011. Molecular support for a basal grade of morphologically distinct, monotypic genera in the species-rich Vanguerieae alliance (Rubiaceae, Ixoroideae): Its systematic and conservation implications Taxon 60: 941–952. Ridsdale, C.E. 1996. A review of Aidia s.l. (Rubiaceae) in southeast Asia and Malesia. Blumea 41: 135–179. Ridsdale, C.E. 2008. Thorny problems in the Rubiaceae: Benkara, Fagerlindia and Oxyceros. Reinwardtia 12: 289–298. Robbrecht, E. 1980. The Hypobathreae (Rubiaceae - Ixoroideae) 1. Delimitation and division of a new tribe. Bull. Jard. Bot. Natl. Belg. 50: 69–77. http://dx.doi.org/10.2307/3667775 Robbrecht, E. 1988. Tropical woody Rubiaceae. Opera Bot. Belg. 1: 1–272. Robbrecht, E. 1993. Supplement to the 1988 outline of the classification of the Rubiaceae. Index to genera. Opera Bot. Belg. 6: 173–196. Robbrecht, E. & Manen, J.-F. 2006. The major evolutionary lineages of the coffee family (Rubiaceae, Angiosperms): Combined analysis (nDNA and cpDNA) to infer the position of Coptosapelta and Luculia, and supertree construction based on rbcL, rps16, trnLtrnF and atpB-rbcL data; A new classification in two subfamilies, Cinchonoideae and Rubioideae. Syst. Geogr. Pl. 76: 85–146. Robbrecht, E. & Puff, C. 1986. A survey of the Gardenieae and related tribes (Rubiaceae). Bot. Jahrb. Syst. 108: 63–137. Robbrecht, E., Bridson, D. & Deb, D.B. 1994. The south Indian genus Octotropis (Rubiaceae). Opera Bot. Belg. 6: 93–100. Ronquist, F. & Huelsenbeck, J.P. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. http://dx.doi.org/10.1093/bioinformatics/btg180 Rova, J.H.E., Delprete, P.G., Andersson, L. & Albert, V.A. 2002. A trnL-F cpDNA sequence study of the Condamineeae-Rondeletieae-Sipaneeae complex with implications on the phylogeny of the Rubiaceae. Amer. J. Bot. 89: 145–159. http://dx.doi.org/10.3732/ajb.89.1.145 Rydin, C., Razafimandimbison, S.G. & Bremer, B. 2008. Rare and enigmatic genera (Dunnia, Schizocolea, Colletoecema), sisters to species-rich clades: Phylogeny and aspects of conservation biology in the coffee family. Molec. Phylogen. Evol. 48: 74–83. http://dx.doi.org/10.1016/j.ympev.2008.04.006 Rydin, C., Kainulainen, K., Razafimandimbison, S.G., Smedmark, J.E.E. & Bremer, B. 2009. Deep divergences in the coffee family and systematic position of Acranthera. Pl. Syst. Evol. 278: 101–123. http://dx.doi.org/10.1007/s00606-008-0138-4 Samson, N., Bausher, M.G., Lee, S.B., Jansen, R.K. & Daniell, H. 2007. The complete nucleotide sequence of the coffee (Coffea arabica L.) chloroplast genome: Organization and implications for biotechnology and phylogenetic relationships amongst angiosperms. Pl. Biotechnol. J. 5: 339–353. http://dx.doi.org/10.1111/j.1467-7652.2007.00245.x Schumann, K. 1891. Rubiaceae. Pp. 1–156 in: Engler, A. & Prantl, K. (eds.), Die natürlichen Pflanzenfamilien, vol. 4(4). Leipzig: Engelmann. http://dx.doi.org/10.5962/bhl.title.4635 Smith, A.C. & Darwin, S.P. 1988. Family 168. Rubiaceae. Pp. 143–362 in: Smith, A.C. (ed.), Flora vitiensis nova: A new Flora of Fiji (Spermatophytes only), vol. 4. Lawai, Kauai, Hawaii: Pacific Tropical Botanical Garden. http://dx.doi.org/10.5962/bhl.title.44033 Version of Record (identical to print version). TAXON 63 (4) • August 2014: 801–818 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) Somers, C. 1988. Aoranthe (Rubiaceae), a new genus to accommodate the African species of Porterandia. Bull. Jard. Bot. Natl. Belg. 58: 47–75. http://dx.doi.org/10.2307/3668401 Sonké, B., Simo, M. & Dessein, S. 2009. Synopis of the genus Mitriostigma (Rubiaceae) with a new monocaulous species from south Cameroon. Nordic J. Bot. 27: 305–312. http://dx.doi.org/10.1111/j.1756-1051.2009.00415.x Swofford, D. L. 2002. PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4.08b. Sunderland, Massachusetts: Sinauer. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molec. Biol. Evol. 28: 2731–2739. http://dx.doi.org/10.1093/molbev/msr121 Tirvengadum, D.D. 1991. Remarques sur le genre Pelagodendron et typification du genre Rhopalobrachium (Rubiaceae). Candollea 46: 251–254. Tirvengadum, D.D. 1993. Larsenaikia, a new genus of the Rubiaceae from Australia. Nordic J. Bot. 13: 175–184. http://dx.doi.org/10.1111/j.1756-1051.1993.tb00034.x Tirvengadum, D.D. 1998. Novelties in Rubiaceae from the limestone flora of southeast Asia. Biogeographica 74: 163–175. Tosh, J., De Block, P., Davis, A.P., Dessein, S., Robbrecht, E. & Smets, E. 2008. The tribal placement of the monospecific tropical African genus Petitiocodon (Rubiaceae) based on molecular data and morphology. Blumea 53: 549–565. http://dx.doi.org/10.3767/000651908X607503 Tosh, J., Davis, A.P., Dessein, S., De Block, P., Huysmans, S., Fay, M.F., Smets, E.F. & Robbrecht, E. 2009. Phylogeny of Tricalysia A.Rich. (Rubiaceae) and its relationships with allied genera based on plastid DNA data: Resurrection of the genus Empogona. Ann. Missouri Bot. Gard. 96: 194–213. http://dx.doi.org/10.3417/2006202 Verdcourt, B. 1958. Remarks on the classification of the Rubiaceae. Bull. Jard. Bot. État Bruxelles 28: 209–290. Wong, K.M. 1984. The genera of Peninsular Malaysian Rubiaceae formerly confused with Randia. Malayan Nat. J. 38: 1–57. Wong, K.M. 2004. Bungarimba (Rubiaceae), a new genus distinguished from Porterandia and other allies. Sandakania 15: 25–54. Yu, Y., Harris, A.J. & He, X.J. 2010. S-DIVA (statistical dispersalvicariance analysis): A tool for inferring biogeographic histories. Molec. Phylogen. Evol. 56: 848–850. http://dx.doi.org/10.1016/j.ympev.2010.04.011 Yu, Y., Harris, A.J. & He, X.J. 2011. RASP (Reconstruct Ancestral State in Phylogenies), version 2.0b. http://mnh.scu.edu.cn/soft/ blog/RASP Appendix 1. Accession numbers for included nucleotide sequences, presenting species name, voucher, and cpDNA markers in the following order: rbcL, rps16, trnTF, ndhF, atpB-rbcL. Agouticarpa williamsii (Standl.) C.H.Perss., Persson & Nodernhäll 441 (GB), KF964807, KF964894, KF965153, KF965008, KF964676. Aidia abeidii S.E.Dawson & Guereau, Luke 8317 (UPS), –, KF964895, KF965154, –, –. Alberta magna E.Mey., Bremer & Bremer 3773 (UPS), Y18708, FM204702, FM207110, KF965009, KF964677. Aleisanthopsis distantiflora (Merr.) Tange, Kessler & al. 41 (P), EU817411, EU817434, EU817453, –, –. Alibertia edulis (A.Rich.) A.Rich. 1, M.K. 880325-1/1 (UPS), KF964808, KF964896, KF965155, KF965010, KF964678; 2, M.K. s.n. (GB ), –, KF964897, KF965156, KF965011, KF964679. Amaioua guianensis Aubl., Persson & al. 2061 (GB), AM117202, AF200976, KF965157, KF965012, KF964680. Aoranthe cladantha (K.Schum.) Somers., McPherson 16283a (P), KF964809, –, KF965158, KF965013, KF964681. Aoranthe penduliflora (K.Schum.) Somers., Iverssen & Steiner 86776 (UPS), Y11845, KF964898, KF965159, KF965014, KF964682. Argocoffeopsis scandens (K.Schum.) Robbr., Davis 3016 (K), –, –, DQ1805668, –, –. Atractocarpus bracteatus Schltr. & K.Krause, Mouly 149 (P), KF964810, KF964899, KF965160, KF965015, KF964683. Atractogyne gabonii Pierre 1, Hladik 2323A (P), KF964811, KF964900, KF965161, KF965016, KF964684; 2, Hladik 2035A (P), KF964812, KF964901, KF965162, KF965017, KF964685. Augusta longifolia (Spreng.) Rehder, Macias 96-348 (NY), –, AF242913, KF965163, –, –. Aulacocalyx jasminiflora Hook.f., Schmidt & al. 1672 (MO), KF964813, KF964902, KF965164, HM164354, KF964686. Belonophora coriacea Hoyle, Maurin 19 (K), –, –, DQ180570, –, –. Benkara malabarica (Lam.) Tirveng., Meebold 13753 (S), AM117207, KF964903, KF965165, KF965018, KF964687. Bertiera guianensis Aubl., Bremer & al. 3363 (UPS), AJ224845, KF964904, KF965166, KF965019, –. Boholia sp., Bicknell 1516A (SUNIV) AM117210, FM204711, HM164312, KF965020, KF964688. Botryarrhena venezuelensis Steyerm., Persson & al. 625 (GB), KF964814, KF964905, KF965167, KF965021, KF964689. Brachytome scortechinii King & Gamble 1, David 136 (P), –, KF964906, KF965168, –, –; 2, Geesink & Santisuk 5154 (P), –, –, –, KF965022, –. Brenania brieyi (De Wild) E.E.A.Petit, Hallé 4214 (P), –, KF964907, KF965169, –, –. Bungarimba ridsdalei K.M.Wong, Wong 2875 (KLU), KF964815, KF964908, KF965170, KF965023, KF964690. Burchellia bubalina (L.f.) Sims, Bremer 3129 (UPS-HORT), Z68833, KF964909, KF965171, KF965024, KF964691. Calochone redingii (De Wild.) Keay 1, Chase 3355 (K), –, AF200986, AF201036, –, –; 2, coll. unknown (K 820315), Z68845, KF964910, KF965172, KF965025, KF964692. Calycosiphonia spathicalyx (K.Schum.) Robbr., Luke 8330 (UPS), AM117211, KF964911, KF965173, KF965026, KF964693. Canephora madagascariensis J.F.Gmel., Razafimandimbison 468 (UPS), KF964816, KF964912, KF965174, KF965027, KF964694. Canthiopsis odorata Seem., Smith 6560 (P), KF964817, KF964913, KF965175, KF965028, KF964695. Casasia calophylla A.Rich., Rova & al. 2259B (S), KF964818, KF964914, KF965177, KF965030, KF964697. Catunaregam spinosa (Thunb.) Tirveng., Luke 8332A (UPS). AM117214, KF964915, KF965178, KF965031, KF964698. Ceriscoides sessiliflora (Wall. ex C.B.Clarke) Tirveng., Maxwell 87-967 (AAU), KF964819, KF964916, KF965179, KF965032, KF964699. Chapelieria madagascariensis A.Rich. ex DC., Pettersson & Nilsson 20 (S), KF964820, KF964917, KF965180, KF965033, KF964700. Cladoceras sucapitum (K.Schum. & K.Krause) Bremek., Luke 8351 (UPS), AM117216, AM117290, KF965181, KF965034, KF964701. Coddia rudis (E.Mey. ex Harv.) Verdc. 1, Bremer 3764 (UPS), AJ286695, KF964918, KF965182, KF965036, KF964703; 2, Bremer & al. 4279 (UPS), KF964821, AM117291, AM117353, KF965035, KF964702. Coffea arabica L., cultivated (UPS), X83631, KF964919, KF965183, KF965037, KF964704. Coffea mannii (Hook.f.) A.P.Davis 1, Andreasen 223 (UPS), –, –, KF965248, KF965117, –; 2, coll. unknown (UPS 71PT000839), KF964872, KF964980, KF965249, KF965118, –. Coptosperma nigrescens Hook.f., Razafimanbimbison 442 (UPS), KF964822, KF964920, KF965184, KF965038, KF964705. Cordiera triflora A.Rich. ex DC., Persson & Gustafsson 302 (GB), KF964823, KF964921, KF965185, KF965039, KF964706. Cremaspora triflora K.Schum. 1, Andreasen 51 (UPS), KF964824, KF964922, KF965186, KF965040, KF964707. 2, Andreasen 69 (UPS), Z68856, KF964923, KF965187, KF965041, KF964708. Crossopteryx febrifuga Benth., Bremer 3097 (UPS), AM117223, FM204717, FM207123, KF965042, KF964709. Deccania pubescens (Roth) Tirveng. 1, Fisher s.n. (K), –, AF200991, –, –, –; 2, Tirvengadum s.n. (P), KF964825, KF964923, KF965188, KF965043, KF964710. Dictyandra arborescens Welw. ex Benth. & Hook.f., Sonké 1788 (BR), AJ286708, AM117300, KF965189, KF965044, KF964711. Didymosalpinx abbeokutae (Hiern) Keay 1, Adam 26309 (P), KF964826, KF964925, KF965190, KF965045, KF964712; 2, Verdcourt & Hesse 268 (S), KF964827, KF964926, KF965191, KF965046, KF964713. Dioecrescis erythroclada (Kurz) Tirveng. 1, Wong s.n. (KLU), KF964828, KF964927, KF965192, KF965047, KF964714; 2, Wong s.n. (KLU), –, KF964928, –, KF965048, –. Discospermum polyspermum (Valeton) Ruhsam, Ridsdale IV.E:130 (S), AJ286703, AM117301, EU145538, KF965049, –. Duperrea pavettifolia (Kurz) Pit., Bremer s.n., cult. Bergianska (S), KF964829, KF964929, KF965194, KF965051, KF964715. Duroia eriophila L.f. 1, Persson & al. 1970 (GB), KF964830, KF964930, KF965195, KF965052, KF964716; 2, Persson & al. 2012 (S), KF964831, KF964931, KF965196, KF965053, KF964717. Emmenopterys henryi Oliv., Robbrecht s.n. (UPS), Y18715, FM204719, FM207125, KF965054, KF964718. Euclinia longiflora Salisb. 1, De Block 27 (BR), Z68835, KF964932, AJ847399, KF965055, KF964719; 2, Mouly 346 (P), KF964832, KF964933, KF965197, KF965056, KF964720. Exostema lineatum Roem. & Schult., Andersson 2208 (GB)/McDowell 4353D (UPS), –, AF242944, AJ346924, KF965057, –. Fagerlindia sinensis (Lour.) Tirveng., Gressitt 956 (S), –, –, KF965198, KF965058, KF964721. Fagerlindia sp., ST 1818 (K), KF964833, KF964934, KF965199, KF965059, KF964722. Feretia aeruginescens Stapf., Bremer 3137 (UPS), Z68857, AM117305, EU145539, Version of Record (identical to print version). 817 Mouly & al. • Phylogeny of the Gardenieae complex (Rubiaceae) TAXON 63 (4) • August 2014: 801–818 Appendix 1. Continued. KF965060, KF964723. Fernelia buxifolia Lam., De Block s.n. (BR), AJ286704, AM117306, EU145540, KF965061, KF964724. Flagenium sp., Davis 1210 (K), KF964834, KF964935, KF965200, KF965062, KF964725. Galiniera saxifraga (Hochst.) Bridson. 1, Hylander s.n. (S), KF964835, KF964936, KF965201, KF965063, KF964726; 2, Luke 8289 (S), KF964836, KF964937, KF965202, KF965064, KF964727. Galliena sclerophylla Dubard & Dop., Davis 2192 (K), KF964837, KF964938, KF965203, KF965065, KF964728. Gardenia jasminoides J.Ellis, Bremer 2720 (UPS), AJ286697, KF964939, KF965204, KF965066, KF964729. Genipa americana L., Kiehn, HBV sub RR-420 (WU, UPS), Z68839, KF964940, KF965205, KF965067, KF964730. Glossostipula concinna (Standl.) Lorence, Keller 1901 (CAS), Z68846, KF964941, KF965206, KF965068, KF964731. Greenea corymbosa Voigt., Larsen & al. 44102 (AAU)/Larsen & al. 43140 (P), –, AF242961, EU817461, –, –. Guettarda speciosa L., Rova 2492 (GB), KF964838, GQ852509, –, KF965069, –. Heinsenia diervilleoides K.Schum. 1, Mizray & Pocs 84405 (UPS), KF964839, KF964942, KF965207, KF965070, KF964732; 2, Andreasen 316 (UPS), –, KF964943, KF965208, KF965071, KF964733. Hyperacanthus amoenus (Sims) Bridson, Bremer 3789 (UPS), KF964840, KF964944, KF965209, KF965072, KF964734. Hypobathrum racemosum Kurz, Ridsdale s.n., cult. Bogor Bot. Gard. V.D. 81 (UPS), AJ286705, AM117318, KF965210, KF965073, KF964735. Hyptianthera stricta (Roxb. ex Schult.) Wight & Arn., Van Beusekom 3031 (P), KF964841, KF964945, KF965211, KF965074, KF964736. Ixora coccinea L., Bremer 3104 (S), KF964842, EF205641, EU817464, KF965075, KF964737. Ixora margaretae (N.Hallé) Mouly & B.Bremer, Mouly & Innocente 222 (P), EU817415, EU817436, KF965176, KF965029, KF964696. Ixora trilocularis (Balf.f.) Mouly & B.Bremer, Lesouef 31 (TAN), EU817417, EU817437, KF965193, KF965050, –. Jovetia humilis Guédès, Davis 1197 (K), KF964843, KF964946, KF965212, KF965076, KF964738. Kailarsenia tentaculata (Hook.f.) Tirveng., Larsen 41627 (AAU), KF964844, KF964947, KF965213, KF965077, KF964739. Kochummenia stenopetala (King & Gamble) K.-M.Wong. 1, Low 247 (KLU), KF964845, KF964948, KF965214, KF965078, KF964740; 2, Low 247 bis (KLU), KF964846, KF964949, KF965215, KF965079, KF964741. Kraussia floribunda Harv. 1, Bremer & al. 4280 (UPS), KF964847, KF964950, AM117368, KF965080, KF964742; 2, Kiehn, HBV sub RR-742 (WU), KF964848, AM117325, KF965216, KF965081, KF964743. Kutchubaea semisericea Ducke, Ståhl & al. 4418 (GB), AM117235, KF964951, KF965217, –, KF964744. Lamprothamnus zanguebaricus Hiern, Luke 8335 (S), KF964849, KF964952, KF965218, KF965082, KF964745. Larsenaikia ochreata (F.Muell.) Tirveng., Puttock 722510 (OP), Z68847, KF964953, –, KF965083, –. Lemryrea krugii (A.Chev.) A.Chev., Razafimandimbison & al. 410 (UPS), KF964850, KF964954, KF965219, KF965084, KF964746. Leptactina delagoensis K.Schum., Bremer & al. 4284 (UPS), AM117240, KF964955, AM117370, KF965085, KF964747. Leptactina platyphylla (Hiern) Wernham, Bremer 3064 (UPS), Z68867, KF964956, KF965220, KF965086, KF964748. Lindenia rivalis Benth., McDowell 5017 (ETSU), –, AM117242, HM164194, KF965087, KF964749. Luculia gratissima Sweet, Cult in Univ. Connecticut 870064 (no voucher), EU817429, EU817448, EU817472, KF965088, KF964750. Macrosphyra longistyla Hook.f. 1, Ern 3120 (P), KF964851, –, KF965221, KF965089, KF964751; 2, Bagshawe 1457 (BM), –, AF201004, –, –, –. Mantalania sambiranensis R.Cap. ex J.-F.Leroy 1, Rakotonasolo 815 (K), KF964853, KF964958, KF965223, –, –; 2, Randrianaivo 807 (MO), KF964852, KF964957, KF965222, KF965090, KF964752. Massularia acuminata (G.Don) Bullock ex Hoyle, De Block 7 BR 64-0484 (BR), Z68841, AF201005, KF965224, KF965091, KF964753. Melanopsidium nigrum Colla, Sant’Ane & al. 703 (GB), KF964854, –, KF965225, KF965092, KF964754. Mitriostigma axillare Hochst., Bremer 2705 (UPS), X83650, KF964959, KF965226, KF965093, KF964755. Monosalpinx guillaumetii N.Hallé 1, Aké Assi 9449 (P), –, KF964960, KF965227, KF965094, –; 2, Cremers 822A (P), –, KF964961, KF965228, KF965095, –. Morelia senegalensis A.Rich. ex DC. 1, Ern 2974 (P), KF964855, KF964962, KF965229, KF965096, KF964756; 2, Ervik 335 (GB)/Thomas 1915 (S), KF964856, KF964963, KF965230, KF965097, KF964757. Mussaenda erythrophylla Schumach., Gillis 10838 (FTG), X83652, FJ884628, AJ620116, KF965098, –. Neofranciella pterocarpon Guillaumin, Munzinger 2314 (P), KF964857, KF964964, KF965231, KF965099, KF964758. Oligocodon cunliffeae (Wernham) Keay, coll. unknown 348 (P), KF964858, KF964965, KF965232, KF965100, KF964759. Oxyanthus speciosus DC., Bremer 4348 (S), KF964859, KF964966, KF965233, KF965101, KF964760. Oxyceros longiflorus (Lam.) Yamazaki, Maxwell 78-8 (AAU), –, AF201010, AF201058, –, –. Oxyceros sp., Ridsdale 2235 (L), KF964860, KF964967, KF965234, KF965102, KF964761. Paracephaelis sp., De Block 1174 (BR), KF964861, KF964968, KF965235, KF965103, KF964762. Paragenipa lancifolia (Boj. ex Bak.) Tirveng. & Robbr., Persson 156 (GB), AJ286707, KF964969, KF965236, KF965104, KF964763. Pavetta indica L., Andreasen 202 (UPS), KF964862, KF964970, KF965237, KF965105, KF964764. Pelagodendron vitiense Seem., Smith 9358 (S), KF964863, KF964971, KF965238, KF965106, KF964765. Petitiocodon parviflorum (Keay) Robbr., De Wilde 8032 (P), KF964864, KF964972, KF965239, KF965107, KF964766. Phellocalyx vollesenii Bridson, Luke 9933 (S), KF964865, KF964973, KF965240, KF965108, KF964767. Pleiocoryne fernandensis (Hiern) Rauschert, Hallé 204 (P), –, KF964974, KF965241, KF965109, KF964768. Polysphaeria lanceolata Hiern, Lantz 130 (UPS), KF964867, KF964975, KF965242, KF965110, KF964769. Porterandia anisophylla (Jack. ex Roxb.) Ridl. 1, Zahid 28 (KLU), KF964868, KF964976, KF965243, KF965111, KF964770; 2, Norsham 725 (KLU), KF964869, KF964977, KF965244, KF965112, KF964771. Posoqueria latifolia Roem. & Schult., SU-C-88.10 Bergius Botanic Garden (S), Z68850, FM204728, FM207135, KF965113, KF964772. Pouchetia baumanniana Büttner, Kiehn HBV sub 81-31 (WU), Z68859, AM117336, KF965245, KF965114, KF964773. Preussiodora sulphurea (K.Schum.) Keay 1, De Foresta s.n. (P), KF964870, KF964978, KF965246, KF965115, KF964774; 2, Martin & Ducan 71 (BR), KF964871, KF964979, KF965247, KF965116, KF964775. Ramosmania rodriguesii Tirveng., cult. Kew 881019 & 890925 (K), Z68860, KF964981, KF965250, KF965119, KF964776. Randia aculeata L. 1, Killip 10070 (S), KF964873, KF964983, KF965251, KF965121, KF964778; 2, Meagher 881 (FTG), Z68832, KF964982, HM164334, KF965120, KF964777. Riodocera pulcherrima Delprete. De Souza 245 (NY), KF964874, KF964984, KF965252, KF965122, KF964779. Robbrechtia grandifolia De Block, Karehed & al. 311 (UPS), AM117263, AM117339, AM117383, KF965123, KF964780. Rosenbergiodendron formosum (Jacq.)Fagerl., Mouly & Florence 343 (P), KF964875, KF964985, KF965253, KF965124, KF964781. Rothmannia capensis Thunb., Bremer & al. 4346 (S), AM117266, AM117340, AM117384, KF965125, KF964782. Rubovietnamia aristata Tirveng., Bastien VN352 (P), –, KF964986, KF965254, KF965126, KF964783. Rutidea fuscescens Hiern, Lantz 120 (UPS), KF964876, KF964987, KF965255, KF965127, KF964784. Rutidea orientalis Bridson, Andreasen 68 (S), KF964877, KF964988, KF965256, KF965127, KF964785. Sabicea aspera Aubl., Andersson & al. 1941 (GB)/Andersson & al. 2003 (NY), KF964878, AF004079, AM409143, EU145416, –. Schizenterospermum sp., Davis 2580 (K), KF964879, KF964989, KF965257, KF965129, KF964786. Schumanniophyton magnificum (K.Schum.) Harms. 1, Sembé 64 (P), –, KF964990, KF965258, KF965130, KF964787; 2, Bourobou Bourobou 619 (P), KF964880, KF964991, KF965259, KF965131, KF964788; 3, Robbrecht, Meise 85-0094 (BR), AJ286702, –, KF965260, KF965132, –. Scyphiphora hydrophylacea C.F.Gaertn., Bremer & al. 99 (S), EU817432, EU817450, EU817475, KF965133, KF964789. Sericanthe andongensis (Hiern) Robbr., Bremer & al. 4322 (S), KF964881, KF964992, KF965261, KF965134, –. Sherbournia calycina (G.Don) Hua, Bremer 3132 (UPS), KF964882, KF964993, KF965262, KF965135, KF964790. Sipanea hispida Benth., Irwin & al. 34756 (UPS), EU145458, EU145492, HM164336, KF965136, KF964791. Sphinctanthus microphyllus K.Schum., Persson & Gustavsson 353 (S), KF964883, KF964994, KF965263, KF965137, KF964792. Stachyarrhena heterocroa Standl., Persson & al. 821 (GB), KF964884, KF964995, KF965264, KF965138, KF964793. Stachyarrhena sp., Jansen-Jacobs & al. 4707 (GB), –, AF201021, –, –, –. Stenosepala hirsuta C.H.Perss., Fonnegra & al. 2358 (GB), KF964885, KF964996, KF965265, KF965139, KF964794. Sukunia pentagonoides (Seem.) A.C.Sm., Smith 8376 (S), KF964886, KF964997, KF965266, KF965140, KF964795. Tamildania uliginosa (Retz.) Tirveng. & Sastre 1, Bremer & Bremer 920 (S), KF964888, KF964999, KF965268, KF965142, KF964797. 2,Tirveng. & Sastre, Tirvengadum 2006 (AAU), KF964887, KF964998, KF965267, KF965141, KF964796. Tarenna buruensis Merr., Drozd & Molem s.n. (UPS), AJ318457, AJ320088, KF965269, KF965143, KF964798. Tarennoidea wallichii (Hook.f.) Tirveng. & Sastre, Larsen 43720 (AAU), KF964889, KF965000, KF965270, KF965144, KF964799. Tennantia sennii (Chiov.) Verdc. & Bridson, Abdalla & al. 96/166 (P), KF964890, KF965001, KF965271, KF965145, KF964800. Tocoyena formosa (Cham. & Schltdl.) K.Schum., coll. unknown (P), KF964891, KF965002, KF965272, KF965146, KF964801. Tricalysia ovalifolia Hiern, Bremer C-88-33 (UPS), Z68855, KF965003, KF965273, KF965147, KF964802. Trukia carolinensis (Valeton) Kaneh. & Hatus., Fosberg 60242 (P), –, KF965004, KF965274, KF965148, KF964803. Vangueria madagascariensis J.F.Gmel., Sanders 1798, (FTG), X83670, HM164229, AM117386, KF965149, –. Vidalasia fusca (Craib) Tirveng., Larsen 43277 (P), KF964892, KF965005, KF965275, KF965150, KF964804. Vidalasia murina (Craib) Tirveng., Tirvengadum 2004 (P), KF964893, KF965006, KF965276, KF965151, KF964805. Wendlandia glabrata DC., Low 239 (KLU), –, KF965007, KF965277, –, KF964806. Xantonnea parvifolia (Kuntze) Craib, Cantharanotai 895 (K), –, –, DQ180599, KF965152, –. 818 Version of Record (identical to print version).