Molecular Phylogenetics and Evolution 52 (2009) 879–886 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Molecular phylogenetics and generic assessment in the tribe Morindeae (Rubiaceae–Rubioideae): How to circumscribe Morinda L. to be monophyletic? Sylvain G. Razafimandimbison a,*, Timothy D. McDowell b, David A. Halford c, Birgitta Bremer b a Bergius Foundation, Royal Swedish Academy of Sciences and Botany Department, Stockholm University, SE-10691 Stockholm, Sweden Department of Biological Sciences, Box 70703, East Tennessee State University, Johnson City, TN 37614, USA c Queensland Herbarium, Environmental Protection Agency, Brisbane Botanic Gardens, Mt Coot-tha Road, Toowong, Qld 4066, Australia b a r t i c l e i n f o Article history: Received 13 February 2009 Revised 14 April 2009 Accepted 15 April 2009 Available online 24 April 2009 Keywords: Appunia Coelospermum Gynochthodes Head inflorescences nrETS nrITS Paraphyly Phylogenetic classification Rubiaceae Syncarps trnT-F a b s t r a c t Most of the species of the family Rubiaceae with flowers arranged in head inflorescences are currently classified in three distantly related tribes, Naucleeae (subfamily Cinchonoideae) and Morindeae and Schradereae (subfamily Rubioideae). Within Morindeae the type genus Morinda is traditionally and currently circumscribed based on its head inflorescences and syncarpous fruits (syncarps). These characters are also present in some members of its allied genera, raising doubts about the monophyly of Morinda. We perform Bayesian phylogenetic analyses using combined nrETS/nrITS/trnT-F data for 67 Morindeae taxa and five outgroups from the closely related tribes Mitchelleae and Gaertnereae to rigorously test the monophyly of Morinda as currently delimited and assess the phylogenetic value of head inflorescences and syncarps in Morinda and Morindeae and to evaluate generic relationships and limits in Morindeae. Our analyses demonstrate that head inflorescences and syncarps in Morinda and Morindeae are evolutionarily labile. Morinda is highly paraphyletic, unless the genera Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme are also included. Morindeae comprises four well-supported and morphologically distinct major lineages: Appunia clade, Morinda clade (including Sarcopygme and the lectotype M. royoc), Coelospermum clade (containing Pogonolobus and Morinda reticulata), and Gynochthodes–Morinda clade. Four possible alternatives for revising generic boundaries are presented to establish monophyletic units. We favor the recognition of the four major lineages of Morindeae as separate genera, because this classification reflects the occurrence of a considerable morphological diversity in the tribe and the phylogenetic and taxonomic distinctness of its newly delimited genera. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction A recent molecular phylogenetic study of Razafimandimbison et al. (2008) based on five plastid gene and nrITS regions led to the establishment of new tribal limits for the species-rich Psychotrieae alliance of the subfamily Rubioideae (Rubiaceae or coffee family). These authors recircumscribed the tribe Morindeae in a narrow sense to include only six genera (Appunia Hook.f., Coelospermum Blume, Gynochthodes Blume, Morinda L., Pogonolobus F. Muell., and Siphonandrium K. Schum.). The members of Morindeae can be diagnosed by the following features: massive T-shaped placentae inserted in the middle of the septum with two anatropous ovules per carpel and pyrenes with a single lateral germination slit (Igersheim and Robbrecht, 1993). Some genera traditionally associated with Morindeae are currently classified in the following tribes: Colletoecemateae Rydin & B. Bremer (Colletoecema E.M.A * Corresponding author. Fax: +46 (0)8 165525. E-mail address: sylvain.razafimandimbison@bergianska.se (S.G. Razafimandimbison). 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.04.007 Petit), Lasiantheae B. Bremer & Manen (Lasianthus Jack), Mitchelleae Razafim. & B. Bremer (Damnacanthus C.F. Gaertn. and Mitchella L.), and Prismatomerideae Ruan (Prismatomeris Thw. and its allied genera). The Samoan genus Sarcopygme Setch. & Christoph., classified by Darwin (1979) in Morindeae, was excluded from Morindeae sensu Razafimandimbison et al. (2008) (hereafter called Morindeae) mainly because of its numerous (up to 100) and synchronous flowers with uniovulate locules. Morindeae is a pantropical group of ca. 160 species assigned to six genera whose generic limits are controversial and remain unsettled. Of these genera, the most species-rich genus is Morinda, one of the 24 rubiaceous genera that Linnaeus described in his volume Species Plantarum (Linnaeus, 1753). Linnaeus (1753) included three species (M. citrifolia L., M. royoc L., and M. umbellata L.) in his genus Morinda, which can be characterized by a combination of its head inflorescences and syncarpous fruits (=syncarps or multiple fruits with ovaries fused). Head inflorescences (also known as capitula, Johansson, 1994) in Morinda sensu Linnaeus (1753) consist of two to many flowers clustered together on a common receptacle. These heads are either solitary (i.e., one cluster of 880 S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886 flowers on a peduncle = single head) or umbel-like; the later are unbranched and comprised of two to many heads. Within each capitulum the number of flowers varies from 2 to 50 but is fairly constant within species. Steyermark (1972) merged the neotropical Morindeae genus Appunia bearing head inflorescences/infructescences composed of free flowers/fruits in Morinda. This taxonomic decision broadened the morphological concept of Morinda and resulted in disagreements among Rubiaceae specialists over its circumscription. For example, Hayden and Dwyer (1969), Johansson (1987), Burger and Taylor (1993), and Lorence (1999) retained Appunia at generic level, while Andersson (1992), Boom and Delprete (2002), and Taylor and Steyermark (2004) included Appunia in Morinda. The occurrence of head inflorescences and syncarpous fruits in some members of its allied genera of Morinda (Coelospermum and Gynochthodes) raises doubts regarding the monophyly of the genus. Morinda currently comprises ca. 130 species of lianescent, arborescent, and suffrutescent plants whose phylogenetic affinities with the other Morindeae genera have never been assessed. The main objective of this study is to reconstruct a robust phylogeny of the tribe Morindeae using combined plastid (trnT-F) and nuclear (nrETS and nrITS) DNA sequence data. The resulting phylogeny will be used to: (1) rigorously test the monophyly of Morinda as presently delimited; (2) evaluate the phylogenetic value of head inflorescences and syncarps traditionally and currently used for circumscribing genera in Morinda and Morindeae; (3) and assess the current generic relationships and limits in Morindeae. 2. Materials and methods 2.1. Taxon sampling We investigated a total of 67 taxa of Morindeae, including Morinda (ca. 41 species), the neotropical genus Appunia (six species), the Australasian genera Coelospermum (six species) and Gynochthodes (four species) and the monotypic New Guinean and northern Australian genus Pogonolobus (one individual). We were unable to identify six New Caledonian Morinda specimens (Morinda sp. 3–7 and 9) using the last taxonomic treatment of Morinda for this region (Johansson, 1994); some of them may represent undescribed new Morinda species. Two Australian Morinda (Morinda sp. 1 and 2) and one Malagasy Morinda (Morinda sp. 8) specimens are undescribed new species. Morinda citrifolia was represented by one individual each of its three varieties, var. bracteata, var. citrifolia, and var. potteri. The Samoan genus Sarcopygme, represented by one individual of its type species, S. pacifica (Reinecke) Setch. & Christoph., was also included in the analyses because Darwin (1979) placed it in Morindeae. No sequenceable material of the New Guinean monotypic genus Siphonandrium was available. Five outgroup taxa were selected on the basis of the molecular phylogenetic study of Razafimandimbison et al. (2008). From the sister tribe Mitchelleae Razafim. & B. Bremer one species of Damnacanthus and one species of Mitchella were used and from the next closest tribe Gaetnereae Bremek. ex S.P. Darwin two species of the paleotropical genus Gaertnera Lam. and one species of the neotropical Pagamea Aubl. were utilized. We investigated a total of 72 taxa for this study (see Table 1). material for all investigated taxa, except Sarcopygme pacifica and isolated following the mini-prep procedure of Saghai-Maroof et al. (1984), as modified by Doyle and Doyle (1987). For S. pacifica total DNA was extracted from a dry young inflorescence (Tronquet 749, P!). Isolated DNA was amplified and sequenced according to the protocols outlined in the following articles: Razafimandimbison et al. (2005) for nrETS, Razafimandimbison and Bremer (2002) for trnT-F, and Razafimandimbison and Bremer (2001) and Razafimandimbison et al. (2004) for nrITS. The primers from these previous studies were used for the nrETS, nrITS, and trnT-F regions. In all PCRs, one reaction was run using water instead of DNA as a negative control to check for contamination. All sequencing reactions were performed using the Big DyeÒ Terminator v3.1 Cycle Sequencing kit and Big DyeÒ Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and sequences were analyzed with the 3100 Genetic Analyzer (Applied Biosystems). 2.3. Phylogenetic analyses Sequence fragments were assembled using the Staden package (Staden, 1996). All new sequences have been submitted to GenBank (FJ906973–FJ907161, Table 1). For each DNA sequence region (or marker) all new sequences and published ones taken from GenBank were aligned together using the computer program CLUSTAL-X (Thompson et al., 1997) to produce an initial alignment and manually adjusted using software SeAl v.2.0 (Rambaut, 1996). Insertion/deletion events were inferred by eye and gaps were treated as missing data in the alignments. The aligned matrices were analyzed with Bayesian inference without coded indels. Separate and combined Bayesian analyses of sequence data were performed in MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). For each of the three DNA sets, the best performing nucleotide substitution model was selected using the computer programs MrModeltest 2.0 (Nylander, 2001) and MrAIC ver. 1.4.3 (Nylander, 2004). The best performing evolutionary models were estimated under three different model selection criteria: Akaike information criterion (AIC) (Akaike, 1974), AICc (a second order AIC, necessary for small samples) and the Bayesian information criterion (BIC) (Schwartz, 1978). All combined Bayesian analyses were conducted with four independent Markov chain runs for 5  106 Metropolis-coupled MCMC generations, with tree sampling every 1  103 generations. Trees sampled from the first 2  106 generations were discarded as burn-in (as detected by plotting the log likelihood scores against generation number). We partitioned the combined data sets into two partitions: partition # 1 with GTR + G applied to the nrITS and trnT-F data; and partition # 2 with HKY + G applied to the ETS data. In all analyses, partitions were unlinked so that each partition was allowed to have its own sets of parameters. Flat prior probabilities were specified according to suggestions produced by the software MrAIC (Nylander, 2004). All separate and combined Bayesian analyses were repeated two times using different random starting trees to evaluate the convergence of the likelihood values and posterior probabilities. All saved trees (after excluding burn-ins) from the two independent runs were pooled for a consensus tree. Groups characterized by posterior probabilities over 95% were regarded as strongly supported. 2.2. DNA extraction, amplification, and sequencing 3. Results Sequence data from the (nuclear ribosomal) nrETS, nrITS, and (chloroplast) trnT-F regions, used alone or in combination with other chloroplast markers (e.g., Razafimandimbison et al., 2005, 2008), have recently been proven useful for inferring phylogenetic relationships within Rubiaceae. Total DNA was extracted from leaves dried in silica gel (Chase and Hills, 1991) and/or herbarium 3.1. Phylogenetic analyses A total of 202 sequences were used, of which 189 (ca. 94%) are published here. The combined nrETS/nrITS/trnT-F matrix contained 3654 base pairs (bp), of which 662 bp (ca. 18%) were parsimony 881 S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886 Table 1 List of taxa investigated in this study, voucher information, country origins, and accession numbers. Taxa Voucher information Country origins Appunia brachycalyx (Bremek.) Steyerm. Appunia calycina (Benth.) Sandwith Appunia debilis Sandwith Appunia guatemalensis Donn.Sm. Appunia odontocalyx Sandwith Appunia tenuiflora (Benth.) B.D. Jacks Coelospermum balansanum Baill. Coelospermum crassifolium J.T. Johanss. Coelospermum dasylobum Halford & A.J. Ford Coelospermum monticola Baill. ex Guillaumin Coelospermum paniculatum F. Muell. var. syncarpum J.T. Johanss. Damnacanthus indicus C.F. Gaertn. Gaertnera phyllosepala Baker Gaertnera phyllostachya Baker Gynochthodes coriacea Blume Gynochthodes epiphytica (Rech.) A.C. Sm. & S.P. Darwin Gynochthodes oresbia Halford Gynochthodes sessilis Halford Mitchella repens L. Morinda ammitia Halford & A.J. Ford Morinda angustifolia Roxb. Granville 5443 (BR) McDowell 2413 (US) McDowell 5728 (ETSU) Razafimandimbison et al. (2008) Smith et al. 1350 (US) Hoffmann 966 (US) Mouly 318 (P) Johansson 85 (P) Q7385 (BRI) Razafimandimbison et al. (2008) Q8854 (BRI) French Guyana (France) Guyana Guyana Morinda Morinda Morinda Morinda bracteata Kurz. var. celebica Miq. buchii Urb. bucidifolia A. Gray candollei (Montrouz.) Beauvis. 1 Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda candollei (Montrouz.) Beauvis. 2 candollei (Montrouz.) Beauvis. 3 candollei (Montrouz.) Beauvis. 4 candollei (Montrouz.) Beauvis. 5 canthoides (F. Muell.) Halford & R.J.F. Hend. citrifolia L. var. citrifolia L. (LF) citrifolia L. var. citrifolia L. (SF) citrifolia L. var. potteri O. Degen. Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda Morinda collina Schltr. coreia Buch.-Ham. deplanchei (Hook. f.) Baill. ex K. Schum. elliptica (Hook.) Ridl. geminata DC. glaucescens Schltr. grayi Seem. jasminoides A. Cunn. latibracteata Valeton longiflora 1 G. Don longiflora 2 G. Don lucida A. Gray moaensis Alain mollis A. Gray morindoides (Baker) Milne-Redh. myrtifolia A. Gray neocaledonica (S. Moore) Guillaumin pedunculata Valeton podistra Halford & A.J. Ford reticulata Benth. retusa Poir. royoc L. 1 royoc L. 2 sp. 1 sp. 2 sp. 3 Morinda sp. 4 Morinda sp. 5 Morinda sp. 6 Morinda Morinda Morinda Morinda Morinda Morinda sp. 7 sp. 8 sp. 9 titanophylla E.M.A. Petit umbellata L. 1 umbellata L. 2 Razafimandimbison et al. Razafimandimbison et al. Razafimandimbison et al. Alejandro et al. (2005) Smith 9377 (S) RJ1411 (BRI) PIF28127 (BRI) Ellison 781 (S) Bremer and Bremer 3909 No voucher Peru Guyana New Caledonia (France) New Caledonia (France) Australia Australia (2008) (2008) (2008) (UPS) AF4789 (BRI) Ekman 2452 (S) Smith 4645 (S) McPherson and Munzinger 701 (UPS) Johansson 15 (S) Mouly 190 (P) Mouly 137 (P) Mouly 140 (P) Q8878 (BRI) McDowell 5742 (ETSU) Lorence 9705 (PTBG) Lorence 9704 (PTBG) Johansson 124 (S) Lorence 9460 (PTBG) Johansson 57 (S) Larsen et al. 41223 (AAU) Gledhill 848 (P) Johansson 81 (S) Smith 1521 (S) Q8836 (BRI) Lorence 8777 (PTBG) Andru 5003 (P) 63PT00539 (P) BR-19733106 Rova et al. 2213 (GB) Degener 15262 (S) Leeuwenberg 2249 (P) Johansson 98 (S) Johansson 54 (S) Lorence 9461 (PTBG) AF4753 (BRI) KRM4638 (BRI) De Block et al. 636 (BR) Leyman 126 (BR) Lorence 8419 (PTBG) Q8853 (BRI) AF3963 (BRI) McPherson and Munzinger 18243 (UPS) Mouly 16 (P) Mouly 310 (P) McPherson and Munzinger 18075 (P) Mouly 399 (P) Kårehed et al. 218 (UPS) Mouly 302 (P) Troupin 10732 (BR) Wambeek and Wanntorp 2622 (S) Q8839 (BRI) Fiji Australia Australia USA Australia Cult. Xishuangbann Trop. Bot. Gard. (China) Australia Haiti Fiji New Caledonia (France) nrETS nrITS trnT-F FJ907038 FJ907039 AM945191a FJ907040 FJ906974 FJ906975 FJ906976 AM945332a FJ906977 FJ907041 FJ907042 FJ907043 AM945194a FJ907045 FJ906978 FJ906979 FJ906980 AM945334a FJ906982 FJ907116 AY514061b AM945200a AM945201a AM945192a FJ907046 FJ907047 FJ907048 FJ907037 FJ907051 FJ907050 AM945335a AM945340a AM945341a AJ847407c FJ906983 FJ906984 FJ906985 FJ906973 FJ906988 FJ906987 FJ907119 FJ907120 FJ907121 FJ907122 FJ907054 FJ907055 FJ907056 FJ907057 FJ906991 FJ906992 FJ906993 FJ906994 FJ907123 FJ907151 FJ907148 FJ907156 FJ907124 FJ907125 FJ907126 FJ907127 FJ907058 FJ907086 FJ907083 FJ907091 FJ907059 FJ907060 FJ907061 FJ907062 FJ906995 FJ907025 FJ907022 FJ907030 FJ906996 FJ906997 FJ906998 FJ906999 FJ907128 FJ907129 FJ907130 FJ907131 FJ907063 FJ907064 FJ907065 FJ907066 FJ907067 FJ907068 FJ907069 FJ907070 FJ907071 FJ907049 FJ907072 FJ907073 FJ907000 FJ907001 FJ907002 FJ907003 FJ907004 FJ907005 FJ907006 FJ907007 FJ907008 FJ906986 FJ907009 FJ907010 FJ907011 FJ907012 FJ907013 FJ907014 FJ907015 FJ907016 FJ907017 FJ906981 FJ907018 FJ907019 FJ907020 FJ906989 FJ906990 FJ907028 FJ907026 FJ907027 FJ907021 FJ907103 FJ907104 FJ907105 FJ907106 FJ907107 FJ907108 FJ907110 FJ907111 FJ907101 FJ907100 FJ907099 FJ907112 FJ907113 FJ907114 FJ907102 New Caledonia (France) New Caledonia (France) New Caledonia (France) New Caledonia (France) Australia Guyana Palau Cult. at Natl. Trop. Bot. Gard. (Hawaii, USA) New Caledonia (France) India New Caledonia (France) Thailand Nigeria New Caledonia (France) Fiji Australia Palau Ivory Coast Ivory Coast Cult. Belgium Botanical Garden Cuba New Caledonia (France) Ivory Coast New Caledonia (France) New Caledonia (France) Palau Australia Australia Madagascar Unknown Florida (USA) Australia Australia New Caledonia (France) FJ907145 FJ907146 FJ907118 FJ907117 FJ907154 FJ907076 FJ907077 FJ907078 FJ907044 FJ907079 FJ907080 FJ907081 FJ907053 FJ907052 FJ907089 New Caledonia (France) New Caledonia (France) New Caledonia (France) FJ907152 FJ907153 FJ907147 FJ907087 FJ907088 FJ907082 New Caledonia (France) Madagascar New Caledonia (France) R.D. of Congo Sri Lanka Australia FJ907150 FJ907147 FJ907155 FJ907157 FJ907158 FJ907160 FJ907085 FJ907084 FJ907090 FJ907092 FJ907093 FJ907094 (continued FJ907132 FJ907133 FJ907134 FJ907135 FJ907115 FJ907136 FJ907137 FJ907138 FJ907139 FJ907140 FJ907141 FJ907142 FJ907143 FJ907144 FJ907109 FJ907074 FJ907075 FJ907024 FJ907023 FJ907029 FJ907031 FJ907032 FJ907034 on next page) 882 S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886 Table 1 (continued) Taxa Voucher information Country origins nrETS nrITS trnT-F Morinda umbellata L. 3 Pagamaea guianensis Aubl. Pogonolobus reticulatus F. Muell. Sarcopygme pacifica (Reinecke) Setch. & Christoph., Takeuchi and Ama 15319 (BR) Razafimandimbison et al. (2008) Q8840 (BRI) Tronchet et al. 222 (P) New Guinea (France) FJ907159 FJ907098 FJ907161 FJ907094 AF333846d FJ907096 FJ907097 FJ907033 AM945342a FJ907035 FJ907036 Australia Samoa (USA) Andersson and Rova (1999). a Razafimandimbison et al. (2008). b Bremer and Manen (2000). c Alejandro et al. (2005). d Malcomber (2002). informative characters (PIC). Of the 662 bp PIC 224 (ca. 34%) were from the nrETS data, 239 (ca. 36%) from the nrITS data, and 207 (ca. 30%) from the trnT-F data. The separate Bayesian analyses of the nrETS, nrITS, and trnT-F data produced Bayesian majority rule consensus trees with similar topologies (not shown). Visual inspection of the trees showed no well-supported conflict between them; accordingly, we merged the sequence data of the three markers for combined analyses. The Bayesian majority rule consensus tree (from 6000 trees) from the combined nrETS/nrITS/trnT-F data was almost fully resolved (Fig. 1). The six sampled Appunia species [including the type species A. tenuiflora (Benth.) B.D. Jacks] formed a well-supported clade (A in Fig. 1; posterior probability or PP = 1.00), which was resolved with high support (PP = 1.00) as sister to a large clade containing the rest of the sampled Morindeae taxa. Morinda as presently delimited was resolved as paraphyletic, because Sarcopygme, Coelospermum, Pogonolobus, and Gynochthodes were all embedded within the large Morinda clade (Fig. 1). The next lineages to branch off after the Appunia clade (A in Fig. 1) were a largely arborescent Morinda clade (B in Fig. 1; including the only two African lianescent Morinda species, Sarcopygme pacifica, M. citrifolia, and the lectoptype M. royoc; PP = 0.97) and the Coelospermum clade (C in Fig. 1; including the Australian and New Guinean Pogonolobus reticulatus F. Muell. and the Australian Morinda reticulata Benth.; PP = 1.00), respectively. This Coelospermum clade was in turn resolved as sister to a large lianescent Gynochthodes–Morinda clade (D in Fig. 1; PP = 1.00), within which all sampled species of Gynochthodes (including the type species G. coriacea Blume) formed a well-supported subclade (PP = 1.00) sister to a small Morinda subclade (PP = 1.00). This Gynochthodes subclade (including three Morinda species, PP = 1.00) was resolved as sister to a large lianescent Morinda subclade (PP = 1.00). Within the largely arborescent Morinda clade (B in Fig. 1; PP = 0.97) the type species of the Samoan Sarcopygme, S. pacifica and the African M. titanophylla formed a weakly supported group (PP = 0.81) and the two varieties of M. citrifolia (var. citrifolia L. and var. potteri O. Degen.) formed a well-supported group (PP = 1.00); M. bracteata Kurz. var. celebica Miq., now merged in M. citrifolia L. var. bracteata Kurz. (Merrill, 1923), and the Micronesian M. latibracteata Val. formed a well-supported group, which in turn was sister to the M. citrifolia var. citrifolia–var. potteri clade. 4. Discussion 4.1. Phylogenetic relationships in Morindeae 4.1.1. Monophyly of Appunia Hook.f. Appunia, originally described by Hooker (1873), is a neotropical genus with ca. 16 species of shrubs or small trees (Steyermark, 1967; Govaerts et al., 2006). The generic status of Appunia has been controversial for the last 40 years (e.g., Steyermark, 1972; Johansson, 1987). Many authors (e.g., Steyermark, 1967; Johansson, 1987; Burger and Taylor, 1993; Lorence, 1999; Borhidi and Diego-Pérez, 2002) recognized Appunia as separate genus. In subsequent publications Steyermark (1972), Andersson (1992), Boom and Delprete (2002), Taylor and Steyermark (2004), and more recently Govaerts et al. (2006) merged the genus in Morinda. In Razafimandimbison et al. (2008), Appunia, represented only by A. guatemalensis Donn.Sm., was resolved as sister to a clade containing Morinda citrifolia, Coelospermum monticola Baill. ex Guillaumin, Gynochthodes coricea, and Gynochthodes sp. This position is further corroborated by this study, which investigates six of 16 Appunia species (type species A. tenuiflora included), Coelospermum, Gynochthodes, and Morinda. These analyses support the monophyly of Appunia, which is distinct from the other Morindeae genera by having a combination of head inflorescences composed of free flowers, club-shaped stigmas, and simple, non-syncarpous fruits. 4.1.2. Paraphyly of Morinda L. Morinda sensu Linnaeus (1753) is non-monophyletic because all three accessions of M. umbellata are more closely related to Coelospermum sensu Johansson (1988), Gynochthodes and Pogonolobus than to either M. citrifolia or M. royoc (Fig. 1). Morinda as presently delimited by a combination of head inflorescences, bifid stigmas, and syncarps is highly paraphyletic, unless Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme are also included (Fig. 1). This is the first study to demonstrate the paraphyly of the presently circumscribed Morinda and lability of head inflorescences and syncarpous fruits in Morinda and Morindeae (Fig. 1). 4.1.3. Sarcopygme Setch. & Christoph. The Samoan genus Sarcopygme, consisting of five species of small trees, was originally established by Setchell and Christophersen (1935) based on Sarcocephalus pacificus Reinecke (Reinecke, 1898), which belonged to the tribe Naucleeae (Cinchonoideae). The authors (Setchell and Christophersen, 1935) argued that their new genus showed ‘‘a superficial resemblance to Sarcocephalus in the fruiting heads but is different from the latter in its single ovules in each locule of the ovary rather than numerous ovules per cell in Sarcocephalus”. Setchell and Christophersen (1935) postulated that Sarcopygme is most closely related to Morinda but is distinct from the latter by its caducous stipules, large involucral bracts, simultaneous opening of all flowers in the head (i.e., synchronous flowering heads), distinct calyces and club-shaped stigmas. Sarcopygme is additionally distinct in its monocaul trunks and relatively large leaves that are clustered at the apex. Darwin (1979) classified Sarcopygme in Morindeae because of its solitary and erect ovules, raphide crystals, valvate aestivation of corolla lobes, and multiple fruits. On the other hand, Johansson (1987) qualified it as a genus of uncertain taxonomic position. A narrow circumscription of Morindeae proposed by Igersheim and Robbrecht (1993), also endorsed by Razafimandimbison et al. (2008), excluded Sarcopygme from Morindeae because of its uniovulate locules and unbranched stigmas. The present analyses, however, show that Sarcopygme, represented here by the type species S. pacifica, belongs to Morindeae, S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886 883 Fig. 1. Bayesian majority rule consensus tree from the combined nrETS/nrITS/trnT-F data of 67 Morindeae taxa and five outgroup taxa from the tribes Gaertnereae and Mitchelleae. Values above nodes are Bayesian posterior probabilities. The vertical bar delimits the outgroup taxa; GAE = Gaertnereae and MIT = Mitchelleae; LF and SF stand for the large- and small-fruited Morinda citrifolia var. citrifolia, respectively; brackets delimit the major lineages of Morindeae corresponding to our newly defined genera. Taxa in boldface are with non-headed inflorescences and taxa with asterisks (*) are without syncarpous fruits (syncarps). consistent with Setchell and Christophersen’s (1935) decision; the tree-like S. pacifica is nested in the largely arborescent Morinda clade (B in Fig. 1; PP = 1.00). 4.1.4. Pogonolobus F. Muell. and paraphyly of Coelospermum Blume The Australian and New Guinean Pogonolobus was originally described by Mueller (1858) as a monotypic genus based on its arborescent habit and flowers with pubescent corolla lobes, and exserted anthers. The genus was later merged by Bentham (1867) in Coelospermum [C. reticulatum (F. Muell.) Benth.] based on its exserted anthers, but Johansson (1987) re-established Pogonolobus based on palynological characters. Coelospermum is a small genus of seven species of mainly lianescent plants, which are characterized by the following combination of characters according to Johansson (1988): terete branches, sheathing stipules, paniculate or corymbiform and puberulous inflorescences, white corollas with both long and short hairs inside the tube, mostly simple fruits with ovules inserted at the middle of the septum, seeds shortly winged at one end and pollen grains with large lumina. Like Morinda, the circumscription of Coelospermum is highly controversial. The Australian Morinda reticulatus was transferred by Baillon (1879) to Coelospermum as Coelospermum decipiens Baill.; however, this decision was not followed by many Rubiaceae specialists (e.g., Johansson, 1987). The present analyses demonstrate that Coelospermum sensu Johansson (1988) is paraphyletic, unless the Australian M. reticulata and P. reticulatus are also included (C in Fig. 1). This finding is consistent 884 S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886 with the decisions of Bentham (1867) and Baillon (1879) to merge these two species into Coelospermum but inconsistent with Johansson (1987). The broadly delimited Coelospermum (including M. reticulatus and Pogonolobus) can be characterized by its mainly lianescent habit and terminal inflorescences composed flowers with anthers well-exserted beyond the corolla lobes. The Coelospermum clade is deeply nested between the largely arborescent Morinda (B in Fig. 1) and the Gynochthodes–Morinda (=D in Fig. 1) clades, and these together are sister to the Appunia clade (A in Fig. 1). Within the Coelospermum clade the M. reticulata–Pogonolobus–C. dasylobum subclade is defined by interpetiolar stipules and hairy corolla lobes. 4.1.5. Monophyly of Gynochthodes Blume Gynochthodes is a genus of ca. 18 lianescent species with winding stems, which are distributed from continental southeast Asia south- and eastwards through Malesia to Micronesia, Fiji, and northern Australia. According to Johansson (1987), the genus can be distinguished from Morinda and Coelospermum by its stipules and bracts, which have marginal hairs, its axillary, racemose or cymose inflorescences with white and shortly pedunculate flowers in whorls, and flowers with recurved calyx tubes, corollas with long hairs within the tubes and on the adaxial side of the lobes. Our analyses strongly support the monophyly of Gynochthodes (type species G. coriacea included), which is resolved as sister to a small Morinda subclade of three Morinda species (M. retusa Poir., M. ammitia Halford & A.J. Ford, and M. grayi Seem.). This Gynochthodes–Morinda subclade is also deeply nested among the largely Morinda clade sister to the Appunia clade (Fig. 1). 4.1.6. Siphonandrium K. Schum. Siphonandrium is a dioecious monotypic genus from New Guinea with scandent habit, umbel- to head-like inflorescences, and flowers with anthers fused into a tube (Igersheim and Robbrecht, 1993). This latter character is unique within Morindeae and is very rare in Rubiaceae [but present in e.g., the genera Argostemma Wall. (Argostemmateae Bremek. ex Verdc.) and Strumpfia Jacq. (Urophylleae Bremek. ex Verdc.)]. No sequenceable material of S. intricatum K. Schum. is available for this study and therefore its position within the tribe has yet to be investigated. Based on its scandent habit and dioecious flowers the genus is possibly closely related to the Gynochthodes–Morinda clade (=D in Fig. 1). 4.2. Generic circumscriptions in Morindeae The present study clearly indicates that Morinda as presently delimited is highly paraphyletic, unless Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme are also included. In other words, head inflorescences and syncarpous fruits are evolutionarily labile in Morinda and Morindeae. Therefore, new generic limits of Morindeae are needed. Below we present four possible alternatives for revising generic boundaries to establish monophyletic groups. 4.2.1. Alternative # 1 One is to recognize a broad circumscription of Morinda (Appunia, Coelospermum, Gynochthodes, Pogonolobus, Sarcopygme, and Siphonandrium included) without infrageneric subdivision. Morinda sensu lato can thus be diagnosed by its massive and T-shaped placentae inserted in the middle of the septum and two anatropous ovules per carpel (excepting Sarcopygme), although these are not obvious characters; the genus is additionally characterized by the frequent occurrence of head inflorescences/infructescences. (see A–D in Fig. 1) at subgeneric level. Either of these two alternatives would render Morindeae monogeneric and require a total of 29 new combinations and two new names in Gynochthodes and Pogonolobus. All described species of Appunia have already been transferred by Steyermark (1972) to Morinda. The second alternative would require new descriptions of four new subgenera. 4.2.3. Alternative # 3 The third alternative is to maintain Appunia (A in Fig. 1) as a distinct genus but merge Coelospermum, Gynochthodes, Pogonolobus, Sarcopygme, and Siphonandrium in Morinda. According to Steyermark (1967), Appunia is mainly distinct from Morinda (Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme not included) by its head inflorescences composed of free flowers/fruits and club-shaped stigmas, rather than head inflorescences formed by flowers with ovaries fully connate and two-branched styles in Morinda. Following this circumscription, however, Morinda is shown to be paraphyletic in this study. Plus, recognizing Appunia at generic level would render Morinda a morphologically heterogeneous genus with no obvious morphological synapomorphy. In other words, it would make Morinda s.l. (Coelospermum, Gynochthodes, Pogonolobus and Sarcopygme included) rather difficult to delimit, as headed and non-headed inflorescences (Gynochthodes and some Coelospermum species) and club-shaped (Sarcopygme) and twobranched stigmatic lobes [all Morinda sensu Linnaeus (1753)] are all present. The two stigmatic lobes are further subdivided into three lobes in some lianescent Morinda species (e.g., M. collina Schltr., Johansson, 1994: 32). 4.2.4. Alternative # 4 The fourth alternative is to recognize the four major lineages (A–D in Fig. 1) as separate genera: Appunia; Morinda s.str. (including Sarcopygme, the only two lianescent African species, M. longiflora and M. morindoides, all neotropical tree-like or suffrutescent Morinda species, and all arborescent and suffrutescent Asian and African Morinda species, all with large flowers); Coelospermum (including M. reticulata and Pogonolobus); and Gynochthodes (including all lianescent Morinda species from Australia, the Pacific, tropical and subtropical Asia, and Madagascar). For the Gynocthodes– Morinda clade (=D in Fig. 1) four validly published generic names, Gynochthodes, Sphaerophora Blume (Blume, 1850), Pogonanthus Montrouz. (Montrouzier, 1860), and Imantina Hook.f. (Hooker, 1873), are available, with the former genus having priority over the latter two names. This group can be defined by its lianescent habit, small flowers with partly exserted anthers and this scenario would require up to 80 new combinations. Within the Gynochthodes– Morinda clade (D in Fig. 1) recognizing the sister groups, subclade Gynochthodes (including M. ammitia, M. grayi, and M. retusa) and subclade Morinda, as separate genera does not seem an attractive solution, because there is no obvious character for distinguishing them. We favor the fourth of these four alternative realignments, the recognition of the four major lineages (A–D in Fig. 1) as separate genera, because this classification reflects the occurrence of a considerable morphological diversity in Morindeae and the phylogenetic and taxonomic distinctness of its newly delimited genera. For now we maintain the New Guinean genus Siphonandrium as a distinct genus. Table 2 summarizes all five accepted genera of Morindeae and finally, all necessary new combinations will be published elsewhere. 4.3. Keys to the accepted genera of Morindeae 4.2.2. Alternative # 2 A second scenario is to adopt a broad circumscription of Morinda and recognize the four well-supported major lineages Below we present keys that can be used to identify all accepted genera of Morindeae on the basis of morphological traits. 885 S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886 Table 2 List of genera accepted here and their synonyms, geographic distributions, and number of species. Genera accepted in Morindeae Synonyms Geographic distributions Number of species Appunia Hook.f. Coelospermum Blume Gynochthodes Blume Morinda L. Siphonandrium K. Schum.a Bellynkxia Müll. Arg. Holostyla Endl.; Merismostigma S. Moore; Olostyla DC.; Pogonolobus F. Muell. Imantina Hook.f.; Pogonanthus Montrouz.; Sphaerophora Blume; Tetralopha Hook.f. Appunettia Good; Sarcopygme Setch. & Christoph Neotropics Australasia Australasia and Madagascar Pantropical New Guinea ca. 16 ca. 9 80–100 30–35 1 a Not included in this study. 1a. Arborescent, rarely suffrutescent (the African M. angolensis Good and the Haitian M. buchii Urb.) and lianescent [the African M. longiflora G. Don and M. morindoides (Baker) Milne-Redh.], large (corolla tubes/corolla lobes > 1) and hermaphroditic flowers... . .... . .. . .. . .. . ...... . .. . .. . .. . .. . .. . .. . ... . .. . .. . .. . .. . .. . .. . .2. 2a. Flowers free, congested, stigmatic lobes club-shaped, fruits simple, free ... . .. . .. . ...... . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . ... . ..Appunia. 2b. Flowers fused basally or partly or completely, stigmatic lobes bifid, fruits syncarpous (syncarps). . .. . ... . .. . .. . .. . .Morinda s.str. 1b. Lianescent, rarely arborescent (the New Guinean and Australian Pogonolobus reticulatus and Hawaiian Morinda trimera Hillebr.), small (corolla tubes/corolla lobes < 1, except Gynochthodes sublanceolata Miq. and M. trimera) and polygamous or dioecious flowers. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .3. 3a. Anthers fused into a tube. . .. . .. . .. . .. . .. . .. . .. . .Siphonandrium. 3b. Anthers not fused into a tube.. . .. . .. . .. . .. . .. . ... . ... . ... . ... . ..4. 4a. Inflorescences mostly paniculate, sometimes corymbs, anthers well exserted beyond the corolla tubes. . .. . .. . .. . .. . .. . .. . .. . .. . ..... . ... . ... . ... . ... . ... . ..Coelospermum. 4b. Inflorescences never paniculate, anthers partly exserted. . .. . .. . .. . .. . .. . ..... . .. . .. . .. . .. . .. . ...... . ... . .Gynochthodes. It is important to note that seven of the eight described African species of Morinda s.str. have much larger flowers [corolla tubes ranging from 12 to 40 mm (rarely 80 mm) long and corolla lobes varying between 3 and 14 mm (rarely 22 mm) long] than the species of Appunia and the remaining species of Morinda s.str. The flowers of Coelospermum as defined here are much larger [corolla tubes ranging from 3 to 7 mm (rarely 11 mm) long and corolla lobes varying from 4.5 to 16 mm long] than that of the newly delimited Gynochthodes [corolla tubes ranging from 0.7 to 5.5 mm long and corolla lobes varying from 1.5 to 11 mm long]. 5. Conclusions The present study demonstrates for the first time that Morinda as presently delimited is highly paraphyletic, unless Coelospermum, Gynochthodes, and Sarcopygme are also included. Both head inflorescences and multiple fruits are evolutionarily labile in Morinda and Morindeae. The tribe Morindeae can be subdivided into four well-supported major lineages, which can be characterized by a combination of growth habit, inflorescence type and position, infructescence type, flower size, and breeding systems: Appunia clade, Morinda clade (including M. royoc, the type species of the genus, and Sarcopygme), Coelospermum clade (including Pogonolobus and Morinda reticulata), and Gynochthodes–Morinda clade. Four possible alternatives for revising generic boundaries are presented to establish monophyletic units. We favor the recognition of the four wellsupported and morphologically distinct major lineages of Morindeae (A–D in Fig. 1) as separate genera, because this classification reflects the occurrence of a considerable morphological diversity in the tribe and the phylogenetic and taxonomic distinctness of its newly delimited genera. Acknowledgments The authors thank Dr. Jan Thomas Johansson (Stockholm University, Stockholm, Sweden) for sharing his documentation and knowledge on Morinda and its allied genera with SGR; Mrs. Anbar Khodanbadeh and Linda Lundmark for help with sequencing; Dr. David Lorence (National Tropical Botanical Garden, Hawaii, USA), Dr. Arnaud Mouly (Bergius Foundation, Stockholm, Sweden), Dr. Hua Zhu (Xishuangbann Tropical Botanical Garden, the Chinese Academy of Sciences, Kunning, China), and Dr. Andrew Ford (CSIRO, Sustainable Ecosystems and Rainforest CRC, Queensland, Australia) for kindly providing leaf material for this study; the ANGAP (Association Nationale pour Gestion des Aires Protégées) and MEF (Ministères des Eaux et Forêts) for issuing collecting permits for SGR; Missouri Botanical Program, Madagascar for logistical support; Lalao Andriamahefarivo (MBG, Madagascar) for arranging collecting permits for SGR; Désiré Ravelonarivo for being an excellent field assistant in the Marojejy; the following herbaria for allowing access to their collections: AAU, BR, BRI, ETSU, GB, K, P, PTBG, S, TAN, TEF, UPS, and US. 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