Journal of Biogeography (J. Biogeogr.) (2010) 37, 520–529 ORIGINAL ARTICLE Origin of the pantropical and nutriceutical Morinda citrifolia L. (Rubiaceae): comments on its distribution range and circumscription Sylvain G. Razafimandimbison1*, Timothy D. McDowell2, David A. Halford3 and Birgitta Bremer1 1 Bergius Foundation, Royal Swedish Academy of Sciences and Botany Department, Stockholm University, SE-10691, Stockholm, Sweden, 2 Department of Biological Sciences, Box 70703, East Tennessee State University, Johnson City, TN 37614, USA, 3Queensland Herbarium, Environmental Protection Agency, Brisbane Botanic Gardens, Mt Coot-tha Road, Toowong, QLD 4066, Australia ABSTRACT Aim Morinda citrifolia L., commercially known as noni or the Indian mulberry plant, is morphologically variable and the only widely distributed member of the pantropical genus Morinda sensu stricto (Rubiaceae). This large distribution has been attributed partly to the ability of the seeds of the large-fruited M. citrifolia L. var. citrifolia L. to be transported by oceanic drifting. This form of M. citrifolia var. citrifolia has been predicted to be the progenitor colonizer of the island endemic Morinda species. Using a phylogenetic approach and large sampling of the widespread, large-fruited M. citrifolia var. citrifolia, we assessed the potential area of origin of M. citrifolia and tested the hypothesis that the large-fruited M. citrifolia var. citrifolia is an ancestral colonizer. Location Tropics. Methods We performed Bayesian analyses of 22 species of the tribe Morindeae (including 11 individuals of the three currently recognized varieties of M. citrifolia) based on combined nrETS, nrITS, rps16 and trnT–F sequence data. Geographic origins of the studied taxa were mapped onto the Bayesian majority rule consensus tree. Results Nine sequenced individuals of M. citrifolia from diverse geographic locations formed a highly supported clade, which was sister to the AustraloMicronesian clade that included M. bracteata var. celebica and M. latibracteata. These sister clades are part of the broader Asian, arborescent Morinda clade. We found no support for the current varietal classification of M. citrifolia. *Correspondence: Sylvain G. Razafimandimbison, Bergius Foundation, Department of Botany, Stockholm University, SE-10691, Stockholm, Sweden. E-mail: sylvain.razafimandimbison@bergianska.se 520 Main conclusions Our analyses suggest a Micronesian origin of M. citrifolia. This implies that the large-fruited M. citrifolia var. citrifolia might well have been present in the Pacific before the arrival of the Micronesian and Polynesian ancestors from Southeast Asia. The wide distribution of this form of M. citrifolia var. citrifolia is attributed partly to the trans-oceanic dispersal of its buoyant seeds, self-pollination and its ability to produce flowers and fruits year-round. The hypothesis that the widespread, large-fruited M. citrifolia var. citrifolia is the progenitor colonizer of the island endemic Morinda species is inconsistent with its derived position within the Asian, arborescent Morinda clade and with the fact that the nine sampled individuals of M. citrifolia form a clade. Keywords Biogeography, DNA sequence data, Indian mulberry, Morinda citrifolia, Morinda s.s., Morindeae, noni, phylogeography, Rubiaceae, taxonomy. www.blackwellpublishing.com/jbi doi:10.1111/j.1365-2699.2009.02229.x ª 2009 Blackwell Publishing Ltd Origin of the noni or Indian mulberry plant INTRODUCTION Molecular markers have been used to assess phylogenetic affinities between cultivated crops and their wild relatives (e.g. Gossypium species, Wendel & Albert, 1992; Solanum species, Hosaka, 1995; Triticum species, Kellogg et al., 1997; Malus species, Robinson et al., 2001; Artocarpus species, Zerega et al., 2004). In a recent molecular phylogenetic study by Razafimandimbison et al. (2009, Clade B in figure 2) Morinda citrifolia L., a major crop in many parts of the Pacific and tropical Asia but also growing naturally across much of the tropics, was nested in Morinda s.s. The three sampled individuals of M. citrifolia (one individual each of the largeand small-fruited forms of M. citrifolia L. var. citrifolia L. and M. citrifolia L. var. potteri O. Degen.) formed a well-supported clade in that analysis. The Australian M. bracteata var. celebica, currently merged in M. citrifolia var. bracteata (Roxb.) Kurz (Merrill, 1923), and the Micronesian M. latibracteata were resolved as sisters, making M. citrifolia paraphyletic. Morinda citrifolia is one of the most well-known and studied species of Rubiaceae (coffee family). The large-fruited M. citrifolia var. citrifolia is of key interest to Drosophila researchers investigating the genetic resistance of one fruit fly species (Drosophila sechellia) capable of eating the ripe fruits, which are toxic to most insects (e.g. Jones, 2005; Kopp et al., 2008). Morinda citrifolia, also an economically important species, commercially known as noni or Indian mulberry, is used in traditional medicine across much of the tropics and is marketed globally. Almost all noni fruits currently used for commercial products are from the large-fruited M. citrifolia var. citrifolia (Nelson & Elevitch, 2006; Scot C. Nelson, pers. comm.). The juice of noni, pressed from the fleshy multiple fruits, has become a popular nutriceutical in the past two decades, with claims made that the drink can treat, cure or prevent a range of diseases (Wang et al., 2002, 2008; ChanBlanco et al., 2006; Deng et al., 2007); however, clinical evidence supporting the medicinal efficacy of noni in humans is currently lacking. In spite of its popularity, the origin and circumscription of M. citrifolia have been controversial and remain equivocal. Morinda citrifolia as presently delimited has a pantropical distribution (Fig. 1), although its occurrence in continental Africa has been controversial, mainly because of Hiern’s (1877, 1898) broad concept of M. citrifolia, which included the African Morinda lucida. The Flora of Tropical East Africa (Verdcourt, 1976) and the Flora Zambesiaca (Verdcourt, 1989) treated M. citrifolia sensu Hiern (1877) as M. lucida; however, this does not rule out the occurrence of the large-fruited M. citrifolia var. citrifolia in continental Africa. In fact, Diane Bridson (Royal Botanic Gardens, Kew) identified one specimen of Morinda (Kuchar 10031, KREMU) collected in 1978 from the upper edge of the sandy beach of Watamu peninsula (Kenya) as M. citrifolia. In addition, Klopper et al.’s (2006) checklist of the flowering plants of sub-Saharan Africa included M. citrifolia. Glen (2002) listed M. citrifolia among the cultivated plants in southern Africa. Based on the evidence presented above we consider the large-fruited M. citrifolia var. citrifolia to be present in continental Africa. This form of M. citrifolia var. citrifolia is pantropical and commonly grows along seashores (Fig. 1); however, it can also thrive on a wide range of habitats: low-elevation lava flows, rocky coasts, salty tide pools, open grasslands and lowlands, gulches and cliffs (e.g. Nelson & Elevitch, 2006). This wide distribution has been attributed to the efficient dispersal of its seeds, which can be transported by oceanic drifting. The seeds remain viable after floating in seawater for several months (Guppy, 1917). The pyrene (seed and the associated endocarp) of this form of M. citrifolia var. citrifolia is fairly large (c. 6 mm long, Nelson & Elevitch, 2006) and has a large air-filled cavity on its ventral surface, with the seed enclosed in its dorsal cavity. The seeds of the large-fruited M. citrifolia var. citrifolia are dispersed inland by fruit bats (e.g. Whittaker & Jones, 1994; Shilton, 1997). It is worth noting that this form of M. citrifolia var. citrifolia has also been introduced in historical times and is now reproduc- Figure 1 Geographical distribution of Morinda citrifolia L. The grey area denotes the current geographical distribution of M. citrifolia as presently delimited and the large-fruited M. citrifolia var. citrifolia; the black dashed line delimits that of M. citrifolia var. bracteata (including M. bracteata var. celebica); the grey line delimits that of M. citrifolia var. potteri; and the black line delimits that of the Micronesian small-fruited M. citrifolia var. citrifolia. Journal of Biogeography 37, 520–529 ª 2009 Blackwell Publishing Ltd 521 S. G. Razafimandimbison et al. ing on its own in the wild (e.g. McClatchey, 2003; Nelson & Elevitch, 2006). The small-fruited M. citrifolia var. citrifolia (known as Micronesian lada) seems confined to Micronesia. Other varieties of M. citrifolia have more restricted distributions, with M. citrifolia var. potteri in the Pacific and M. citrifolia var. bracteata Indo-Australian (Fig. 1). There is no available information regarding the natural habitats and nature of dispersals of the small-fruited M. citrifolia var. citrifolia, M. citrifolia var. bracteata or M. citrifolia var. potteri. There have been conflicting opinions regarding the area of origin of the large-fruited M. citrifolia var. citrifolia (e.g. Morton, 1992; McClatchey, 2003; Nelson & Elevitch, 2006). The prevailing view maintains that this plant originated in Southeast Asia and was dispersed by humans and/or sea currents to the Pacific islands and the rest of the tropics (e.g. Morton, 1992). The Southeast Asian ancestors of the Micronesian and Polynesian people are historically known to have migrated from Southeast Asia to the Pacific islands and brought with them fruits of the large-fruited M. citrifolia var. citrifolia among other essential items (e.g. Whistler, 1985, 1991; Abbott, 1992; Morton, 1992). It is, however, unknown whether or not this form of M. citrifolia var. citrifolia was already present in the Pacific before the arrival of the Micronesian and Polynesian ancestors, a question on which the historical record is silent. On the basis of the morphological-based phylogeny of the New Caledonian Morinda by Johansson (1994), McClatchey (2002, 2003), subsequently endorsed by Nelson & Elevitch (2006, p. 1), postulated that the large-fruited M. citrifolia var. citrifolia ‘evolved in Australia, Borneo or intermediate Indonesia and New Guinea and was subsequently distributed by ocean current or birds, bats, or people to other parts of the Indian, and Pacific Oceans’. Furthermore, McClatchey (2003) predicted that the large-fruited M. citrifolia var. citrifolia is the progenitor colonizer that gave rise to the island endemic Morinda species, given its efficient seed dispersal mechanism. If this is correct, we expect the island endemic species of Morinda to be closely related to populations of the large-fruited M. citrifolia var. citrifolia occurring on their island(s). Taxonomy of Morinda citrifolia varieties Morinda citrifolia has long been regarded as morphologically heterogeneous (Hooker, 1880; Valeton, 1908; McClatchey, 2003; Nelson & Elevitch, 2006; Waki et al., 2008); however, there have been no attempts to test its monophyly with a molecular-based phylogeny. Kurz (1877) was the first to establish infraspecific subdivisions for M. citrifolia. He included the continental Asian species Morinda bracteata in synonymy of M. citrifolia and recognized two varieties: M. citrifolia var. citrifolia and M. citrifolia var. bracteata. Hooker (1880) described M. citrifolia var. elliptica Hook. f., which was later recognized by Ridley (1918) as a separate species [Morinda elliptica (Hook. f.) Ridley]; this species has since been widely accepted by the Rubiaceae systematists (e.g. Puff et al., 2007), consistent with Razafimandimbison et al. 522 (2009). The Asian species Morinda tinctoria Roxb. (= M. coreia Buch.-Ham.) was considered by many Indian botanists to be the wild progenitor of M. citrifolia (Hooker, 1880) and was formally placed by Kuntze (1891) in M. citrifolia as a separate variety, M. citrifolia var. tinctoria (Roxb.) O. Kuntze. This taxonomic decision was refuted by Razafimandimbison et al. (2009). Hiern (1898) merged the African M. lucida in M. citrifolia and recognized it at varietal level, M. citrifolia var. lucida (Benth.) Hiern, also refuted by Razafimandimbison et al. (2009). Furthermore, Domin (1929) described M. citrifolia var. typica Domin, a name currently considered by the International Plant Names Index (IPNI) as invalid because it was ‘presumably meant to be the type variety’ of M. citrifolia. Morinda citrifolia var. potteri was described by Degener (1949) based on a cultivated specimen originally from Fiji. Today there are three varieties widely recognized in M. citrifolia: var. bracteata, var. citrifolia and var. potteri [= M. citrifolia L. f. potteri (O. Degen.) H. St. John; John, 1984]. Morinda citrifolia var. bracteata is easily distinguished from the other varieties by its conspicuous well-developed bracts subtending the inflorescence and its small fruits. Miquel (1857) described the distinct variety M. bracteata var. celebica Miq. based on a Sulawesian specimen, which was later synonymized by Merrill (1923) in M. citrifolia var. bracteata. Valeton (1908) considered M. bracteata var. celebica very distinct from M. citrifolia because of its enlarged calyx lobes but never recognized this entity at species level. Morinda citrifolia var. potteri differs from the other varieties of M. citrifolia in having variegated leaves and small fruits, foetid when ripe. It is worth noting, however, that a variegated form of the large-fruited M. citrifolia var. citrifolia is also found in many botanical gardens. The largefruited M. citrifolia var. citrifolia appears to have evolved selfpollinating flowers from the ancestral condition of (hermaphroditic) outcrossing flowers (but see also Waki et al., 2008); it flowers and produces fruits year-round. We have no information regarding the breeding systems of the small-fruited M. citrifolia var. citrifolia, var. bracteata or var. potteri. Fruits of the three recognized varieties of M. citrifolia are foetid-smelling when mature, although those of the large-fruited M. citrifolia var. citrifolia smell worse than others. The fruits of M. citrifolia are fleshy and generally much larger than those of the remaining species of Morinda s.s. (Razafimandimbison et al., 2009); we consider these large and fleshy compound fruits (syncarps) the potential morphological synapomorphies of M. citrifolia. With respect to the delimitation of M. citrifolia, McClatchey (2003) presented the following three hypotheses. H1: the ‘species comprises more than one variety’; H2: M. citrifolia includes ‘more than one species’; and H3: M. citrifolia represents ‘a single species with limited variation and cannot accurately be divided into separate varieties’. The main objective of this study is to perform a phylogenetic analysis of Morinda s.s. based on combined nrETS, nrITS, rps16 and trnT–F sequence data using a much larger sampling of the large-fruited M. citrifolia var. citrifolia to: (1) assess the potential area of origin of M. citrifolia; and (2) test the hypothesis that the large-fruited M. citrifolia var. citrifolia is Journal of Biogeography 37, 520–529 ª 2009 Blackwell Publishing Ltd Origin of the noni or Indian mulberry plant the progenitor colonizer of the island endemic Morinda species. sampled Appunia guatemalensis and Morinda lucida, as their country origins are not known. MATERIALS AND METHODS RESULTS Taxon sampling Molecular phylogenetic analyses Based on the results of Razafimandimbison et al. (2009) we restricted our analyses mainly to the members of Morinda s.s. Morinda citrifolia var. bracteata was represented by three wild specimens: one with conspicuous bracts subtending the inflorescences (= the type species of M. bracteata) from Thailand, and two with conspicuous enlarged calyx lobes (described as M. bracteata var. celebica) from Australia. Morinda citrifolia var. potteri was represented by one cultivated individual, and M. citrifolia var. citrifolia was represented by five wild specimens of the large-fruited form from Guyana (South America), Hawaii, New Caledonia, New Guinea and Puerto Rico (Caribbean region) and by two specimens of the small-fruited form [one grown from seeds collected in Palau (Micronesia) and currently cultivated at the National Tropical Botanical Garden, Hawaii (Pacific) and the other from Kosrae of the Federated State of Micronesia (Micronesia)]. Two species each of the allied genera Appunia Hook. f., Coelospermum Blume and Gynochthodes Blume were used as outgroup taxa to root the trees. We analysed a total of 32 accessions of Morindeae, including 16 species of Morinda s.s. and 11 individuals of M. citrifolia (including the two individuals of M. bracteata var. celebica) (Table 1). The combined nrETS, nrITS, rps16 and trnT–F matrix contained 4271 base pairs (bp), from which 411 (c. 10%) were parsimony-informative characters. The Bayesian majority rule consensus tree from the combined data shown in Fig. 2 is consistent with Razafimandimbison et al. (2009, Clade B in figure 1), except that Morinda s.s. (including the two African lianescent M. longiflora and M. morindoides) is poorly supported (posterior probability, PP = 0.55). All sampled arborescent Morinda species from the Caribbean islands, continental Africa and continental Asia, Micronesia and Australia, and the Samoan Sarcopygme pacifica Setch. Christoph. formed a well-supported lineage (PP = 1.00) (hereafter called the arborescent Morinda clade). Within the arborescent Morinda clade the sequenced continental Asian and Micronesian Morinda species and the sampled specimens of the three varieties of M. citrifolia together formed a well-supported group (PP = 1.00) (hereafter called the Asian Morinda clade, Fig. 2). Within the Asian Morinda clade, the continental Asian Morinda lineage comprising M. angustifolia and M. scabrida (PP = 1.00) was the first group to branch off, followed by another continental Asian Morinda lineage of M. coreia and M. elliptica (PP = 1.00). The latter group was resolved with weak support (PP = 0.69) as sister to a Morinda lineage including the sequenced Micronesian Morinda species and all sampled M. citrifolia (including M. bracteata var. celebica) from Australia, Guyana, Hawaii, Micronesia (Palau and Federal State of Micronesia), New Caledonia, New Guinea, Puerto Rico and Thailand. The Micronesian M. pedunculata was resolved with high support (PP = 1.00) as sister to a highly supported clade of all sequenced specimens of M. citrifolia, M. bracteata var. celebica and M. latibracteata (PP = 1.00). The two individuals of the Australian M. bracteata var. celebica formed a well-supported clade (PP = 1.00), sister to the Micronesian M. latibracteata (PP = 1.00); this AustraloMicronesian Morinda lineage was in turn sister to a highly supported lineage (PP = 1.00) including one specimen each of M. citrifolia var. bracteata (Thailand) and var. potteri (Hawaii) and five sampled accessions of the large-fruited form of M. citrifolia var. citrifolia (Guyana, Hawaii, New Caledonia, New Guinea and Puerto Rico) and two sequenced individuals of the small-fruited form of M. citrifolia var. citrifolia (Fig. 2). In sum, M. citrifolia as currently delimited is monophyletic only if the Australian M. bracteata var. celebica, currently classified in M. citrifolia var. bracteata, is excluded, consistent with Razafimandimbison et al. (2009). The results do not support the current varietal classification of M. citrifolia. DNA sequencing and phylogenetic analyses DNA extraction, amplification and sequencing were conducted using the methods outlined in Razafimandimbison et al. (2009). We performed a Bayesian analysis based on a combined data set of nrETS, nrITS, rps16 and trnT–F. Three unpublished internal transcribed spacer (ITS) sequences of M. citrifolia from GenBank (AY762832, AY762833 and AY762840) produced from wild individuals from Kosrae (Federal State of Micronesia), Australia and Puerto Rico, respectively, were included in the analysis. We re-identified the voucher specimens from which these sequences were produced as follows: a small-fruited M. citrifolia var. citrifolia (AY762832), a large-fruited M. citrifolia var. citrifolia (AY762833) and M. bracteata var. celebica (AY762840) (see Table 1). The same nucleotide substitution models as utilized in Razafimandimbison et al. (2009) were used for nrETS, nrITS and trnT–F, but the GTR + G model was used for the rps16 data (Nylander, 2004). The settings for the Bayesian analysis were also the same as those used in Razafimandimbison et al. (2009). Country (with the exception of Hawaii and New Caledonia) origins of the terminal taxa were mapped onto the resulting Bayesian majority rule consensus tree; however, we used continental origins for the Journal of Biogeography 37, 520–529 ª 2009 Blackwell Publishing Ltd 523 Taxa Voucher information/publications Country/continental origins nrETS nrITS rps16 trnT-F Appunia debilis Sandwith A. guatemalensis Donn. Sm. Coelospermum balansanum Baill. C. paniculatum F. Muell. var. syncarpum J. T. Johanss. Gynochthodes coriacea Blume G. sessilis Halford Morinda angustifolia Roxb. M. citrifolia var. bracteata (Roxb.) Kurz M. bracteata Roxb. var. celebica Miq. M. bracteata Roxb. var. celebica Miq. 1 M. buchii Urb. M. citrifolia L. var. citrifolia L. (LF) M. citrifolia L. var. citrifolia L. 1 (LF) M. citrifolia L. var. citrifolia L. 2 (LF) M. citrifolia L. var. citrifolia L. 3 (LF) M. citrifolia L. var. citrifolia L. 4 (LF) M. citrifolia L. var. citrifolia L. (SF) Razafimandimbison Razafimandimbison Razafimandimbison Razafimandimbison Guyana Sine loc. (Neotropics) New Caledonia Australia FJ907103** FJ907104** FJ907107** FJ907111** FJ907039** AM945191* FJ907041** FJ907045** GQ463257 AM945306* GQ463258 GQ463259 FJ906974** AM945332* FJ906978** FJ906982** FJ907112** AM117311 GQ463260 GQ463261 GQ463263 GQ463262 AJ847407à FJ906985** FJ906987** GQ463251 FJ906991** GQ463264 FJ906992** GQ463243 GQ463240 GQ463242 FJ907125** GQ463241 AM945192* FJ907048** FJ907050** GQ463245 FJ907054** AY762833 FJ907055** AY762840 GQ463243 GQ463246 GQ463248 FJ907060** GQ463247 GQ461270 GQ463265 GQ463269 GQ463266 GQ463267 GQ463255 GQ463252 GQ463254 FJ906997** GQ463254 M. citrifolia L. var. citrifolia L. 1 (SF) M. citrifolia L. var. potteri O. Degen. Lorence 7933 (PTBT) Lorence 9706 (not 9704 as in Razafimandimbison et al., 2009) (PTBG) Razafimandimbison et al. (2009) Indonesia Australia China Thailand Australia Australia Haiti Puerto Rico New Guinea New Caledonia Hawaii Guyana Cultivated at National Tropical Botanical Garden (Hawaii, Pacific), from seed originally from Palau (Micronesia) Kosrae (Federated State of Micronesia) Cultivated at National Tropical Botanical Garden (Hawaii, Pacific) FJ907127** AY762832 FJ907062** GQ463268 FJ906999** FJ907129** FJ907064** GQ463271 FJ907001** FJ907131** FJ907066** FJ907067** FJ907071** FJ907049** FJ907073** AF002740§ FJ907075** GQ463272 GQ463273 GQ463274 GQ463275 GQ463276 GQ463277 GQ463278 FJ907003** FJ907004 FJ907008** FJ906986 FJ907010 FJ907011** FJ907013** M. coreia Buch.-Ham. Journal of Biogeography 37, 520–529 ª 2009 Blackwell Publishing Ltd M. M. M. M. M. M. M. elliptica (Hook. f.) Ridl. geminata DC. latibracteata Valeton longiflora G. Don lucida A. Gray moaensis Alain morindoides (Baker) Milne-Redh. et et et et al. al. al. al. (2009) (2008) (2009) (2009) Alejandro et al. (2005) Razafimandimbison et al. (2009) Razafimandimbison et al. (2009) Ryding 461 (UPS) Razafimandimbison et al. (2009) P. S. Short 4842 Razafimandimbison et al. (2009) Taylor 11790 (MO) Drodz & Mozem 19981109 (UPS) Mouly 227 (P) Lorence 9704 (PTBG) Razafimandimbison et al. (2009) NTBG 980177 Razafimandimbison Razafimandimbison Razafimandimbison Razafimandimbison Razafimandimbison Razafimandimbison Razafimandimbison et et et et et et et al. al. al. al. al. al. al. (2009) (2009) (2009) (2009) (2009) (2009) (2009) Cultivated at National Tropical Botanical Garden No. 990204 (Hawaii, Pacific), from seed originally from India Thailand Nigeria Palau (Micronesia) Ivory Coast Sine loc. (Africa) Cuba Ivory Coast FJ907116** GQ463239 FJ907119** FJ907120** FJ907135** FJ907115** FJ907137** FJ907138** FJ907140** S. G. Razafimandimbison et al. 524 Table 1 List of taxa investigated in this study, voucher information, country origins and accession numbers. Journal of Biogeography 37, 520–529 ª 2009 Blackwell Publishing Ltd Proujansky & Stern (GenBank *Razafimandimbison et al. (2008); Bremer & Eriksson (2009); àAlejandro et al. (2005); §Andersson & Rova (1999); –Malcomber (2002); **Razafimandimbison et al. (2009); unpublished). GQ463256 FJ907031** FJ907036** GQ463281 GQ463282 GQ463283 GQ463250 F907092** FJ907097** Thailand D.R. of Congo Samoa M. scabrida Craib M. titanophylla E. M. A. Petit Sarcopygme pacifica (Reinecke) Setch. & Christoph. GQ463244 FJ907157** FJ907020** GQ463280 AF333845– M. royoc L. Malcomber (2002); Razafimandimbison et al. (2009) Larsen et al. 43537 (AAU) Razafimandimbison et al. (2009) Tronchet et al. 222 (P) FJ907146** FJ907016** GQ463279 FJ907077** FJ907143** Cultivated at National Tropical Botanical Garden No. 980153 (Hawaii, Pacific) from seed originally from Palau (Micronesia) USA Razafimandimbison et al. (2009) M. pedunculata Valeton nrITS nrETS Country/continental origins Voucher information/publications Taxa Table 1 Continued rps16 trnT-F Origin of the noni or Indian mulberry plant DISCUSSION Phylogenetic affinity and the potential area of origin of Morinda citrifolia All sampled individuals of M. citrifolia are nested within the Asian Morinda clade (Fig. 2). In this clade the two-first lineages to branch off are composed entirely of species from continental Asia (China, India and Thailand). One interpretation of this pattern is that the clade had its origin in continental Asia (Fig. 2). Morinda citrifolia is most closely related to the Micronesian species M. pedunculata and M. latibracteata and Australasian M. bracteata var. celebica rather than to the sequenced continental Asian Morinda species (M. angustifolia, M. scabrida, M. coreia and M. elliptica). Morinda pedunculata, M. latibracteata and the small-fruited M. citrifolia var. citrifolia are all endemics to Micronesia, suggesting a Micronesian origin of M. citrifolia (Fig. 2). This finding does not support the Southeast Asian origin of M. citrifolia and implies that M. citrifolia is likely to have already been present in Micronesia at least before the arrival of the Micronesian ancestors from Southeast Asia over 3000 years ago. Given its efficient seed dispersal mechanism, the large-fruited M. citrifolia var. citrifolia might well have reached Polynesia and the rest of the Pacific before the arrival of the Southeast Asian ancestors of the Polynesian people. The historical record is silent on this, but molecular phylogenetic dating and biogeographical analyses of the tribe Morindeae might provide further evidence (study in progress). The Australian origin of M. citrifolia put forward by McClatchey (2003), based on the sister-group relationship between M. citrifolia and the Australian M. reticulata in Johansson (1994), is not supported by Razafimandimbison et al. (2009), as these two species are consistently resolved into two distinct clades: the former in Morinda sensu Razafimandimbison et al. (2009, Clade B in Fig. 1) and the latter in Coelospermum sensu Razafimandimbison et al. (2009, Clade C in Fig. 1). McClatchey’s (2003) hypothesis is based on the morphological phylogeny of Johansson (1994), which is erroneously rooted with three lianescent Morinda species, M. glomerata, M. grayi and M. myrtifolia, which all belong to a lianescent, derived Gynochthodes (Razafimandimbison et al., 2009; Clade D in Fig. 1). Is the widespread, large-fruited Morinda citrifolia var. citrifolia the progenitor colonizer of island endemic Morinda species? The hypothesis that the widespread, large-fruited M. citrifolia var. citrifolia could have been the progenitor colonizer that gave rise to numerous island endemic Morinda species (McClatchey, 2003) is not supported by our analyses. Morinda citrifolia is derived within the Asian Morinda clade (Fig. 2) and is more closely related to the Southeast Asian, arborescent Morinda species than to the sampled lianescent Morinda species from Australia, the Pacific and Madagascar (now 525 S. G. Razafimandimbison et al. Figure 2 Bayesian majority rule consensus tree of Morinda sensu Razafimandimbison et al. (2009) based on combined nrETS, nrITS, rps16 and trnT–F data from 22 Morindeae taxa. Morinda citrifolia var. citrifolia is a single taxon represented by 11 specimens. Values above the nodes are posterior probabilities and numbers below the nodes are numbers of evolutionary steps. LF and SF denote the large- and small-fruited forms of M. citrifolia var. citrifolia, respectively; the bracket delimits the outgroup taxa. AFR, Africa; AUS, Australia; CUB, Cuba; CHI, China; FST, Federated State of Micronesia; GUY, Guyana; HAI, Haiti; HAW, Hawaii; INDI, India; INDO; Indonesia; IVC, Ivory Coast; NEC, New Caledonia; NEG, New Guinea; NET, Neotropics; NIG, Nigeria; PAL, Palau; PUR, Puerto Rico; DRC, Republic Democratic of Congo; SAM, Samoa; and USA, United State of America. members of Gynochthodes sensu Razafimandimbison et al., 2009), where the large-fruited M. citrifolia var. citrifolia is commonly found. In addition, the hypothesis is inconsistent with the fact that the nine sampled individuals of M. citrifolia from diverse geographic locations form a monophyletic group (Fig. 2). Howarth et al. (2003) have reported similar evolutionary patterns in the genus Scaevola (Goodeniaceae), which has two widespread strand species: one occurring throughout the Indian and Pacific regions and the other in tropical America and Africa. Comments on the geographical distribution of the large-fruited Morinda citrifolia var. citrifolia Morinda citrifolia is the only species of Morinda s.s. with a pantropical distribution. The remaining Morinda species are all regional endemics. The wide distribution of the large-fruited M. citrifolia var. citrifolia has been attributed partly to natural dispersal by oceanic floating (seeds remain viable after floating in seawater for several months; Guppy, 1917; Morton, 1992). The acquisition of the buoyant seeds alone is not, however, 526 enough to explain its wide distribution, as the same type of seeds is found in other Morindeae species (e.g. Morinda royoc and Appunia guatemalensis, Hayden & Dwyer, 1969) that have much narrower geographic distributions than M. citrifolia. In fact, the large-fruited M. citrifolia var. citrifolia has the ability to self-pollinate and produce flowers and fruits year-round, allowing it to adapt easily to and colonize a range of new habitats. This form of M. citrifolia var. citrifolia also has been introduced in historical times and is now reproducing in the wild, making it difficult (if not impossible) to know whether or not its natural distribution is much narrower than its current distribution. It has yet to be verified whether or not the fruits of M. citrifolia var. bracteata, M. citrifolia var. potteri and the small-fruited M. citrifolia var. citrifolia also have buoyant seeds. Comments on the circumscription of Morinda citrifolia Our analyses further confirm that Morinda coreia, M. elliptica and M. lucida are not closely related to the presently delimited M. citrifolia, consistent with Razafimandimbison et al. (2009). The present study indicates that the sequenced individual of Journal of Biogeography 37, 520–529 ª 2009 Blackwell Publishing Ltd Origin of the noni or Indian mulberry plant M. citrifolia var. bracteata (with conspicuous bracts subtending the inflorescence) from Thailand and the two sampled individuals of M. bracteata var. celebica from Australia are not closely related to each other. The sister-group relationship between the Australian M. bracteata var. celebica and the Micronesian M. latibracteata is confirmed (27 molecular synapomorphies, PP = 1.00) (Fig. 2). Both taxa have enlarged calyx lobes, although they are relatively smaller in M. bracteata var. celebica. The texture and size of leaves also differ between these two taxa. Merrill (1923) synonymized M. bracteata var. celebica in M. citrifolia var. bracteata sensu Kurz (1877); however, our analysis suggests that it should be excluded from M. citrifolia and recognized at species level in order to make M. citrifolia monophyletic. On the other hand, it would be advisable to sequence accessions of M. bracteata var. celebica from Celebes (now Sulawesi, Indonesia), where the type specimen was originally collected (Miquel, 1857), before making any taxonomic adjustment. Waki et al.’s (2008) cluster analysis based on 58 morphological traits of M. citrifolia showed that M. citrifolia var. citrifolia, M. citrifolia var. bracteata and M. citrifolia var. potteri formed a group. The samples of M. citrifolia var. bracteata investigated in that study match the description of the Australian M. bracteata var. celebica used in this study. The present analyses demonstrate that M. citrifolia is monophyletic only if the Australian M. bracteata var. celebica is excluded. Our analysis reveals for the first time discrepancies between the levels of molecular and morphological variation in M. citrifolia, with the former being relatively low and the latter being high. In addition, we find no molecular support for the current varietal classification of M. citrifolia. The DNA sequence data from nrETS, nrITS, rps16 and trnT–F markers appear to support recognition of a single species with limited molecular sequence variation that cannot accurately be described as more than one variety, consistent with hypothesis H3 of McClatchey (2003). On the other hand, it is possible that with more extensive sampling this widespread species could present greater molecular variation. Similarly, data from low-copy nuclear or microsatellite markers may reveal more structure than the sequence data from the four selected markers. Accordingly, we maintain for now the current varietal classification of M. citrifolia pending further analyses. CONCLUSIONS The present study focuses on the origin of the well-known noni or Indian mulberry plant (Morinda citrifolia) using a phylogenetic approach. Our results confirm the monophyly of M. citrifolia exclusive of the Australian M. bracteata var. celebica and suggest a Micronesian origin of M. citrifolia, which implies that the large-fruited M. citrifolia var. citrifolia is likely to have been present in the Pacific before the arrival of the Micronesian and Polynesian ancestors from Southeast Asia. The wide distribution of the large-fruited M. citrifolia var. citrifolia is attributed partly to the acquisition of its selfpollination and buoyant fruits and to its ability to produce Journal of Biogeography 37, 520–529 ª 2009 Blackwell Publishing Ltd flowers and fruits year-round. We demonstrate that this form of M. citrifolia var. citrifolia could not have been the progenitor colonizer of the island endemic Morinda species. Finally, our findings concerning the phylogenetic relationships of M. citrifolia will allow noni researchers to focus their investigations into medicinal and biochemical properties on closely related species. ACKNOWLEDGEMENTS The authors thank Jan Thomas Johansson for sharing his documentation and knowledge on Morinda and its allied genera with S.G.R.; Anbar Khodanbadeh and Linda Lundmark for help with sequencing; David Lorence (National Tropical Botanical Garden, Hawaii, USA), Arnaud Mouly and Andrew Ford for kindly providing leaf material for this study; Elmar Robbrecht, Estrela Figueiredo and Aaron Davis for providing information on the distribution of M. citrifolia in continental Africa; Louise Shilton for providing information on the seed dispersal of the large-fruited M. citrifolia; Pauline Ladiges, Robert Whittaker and three anonymous referees for providing constructive comments on early versions of the paper; the ANGAP (Association Nationale pour Gestion des Aires Protégées) and MEF (Ministères des Eaux et Forêts) for issuing collecting permits for S.G.R; Missouri Botanical Program, Madagascar for logistical support; Lalao Andriamahefarivo (MBG, Madagascar) for arranging collecting permits for S.G.R; Désiré Ravelonarivo for being an excellent field assistant in the Marojejy; East Tennessee State University Office of Research and Sponsored Programs, the Smithsonian Institution Biological Diversity of the Guianas Program, and the Guyana Environmental Protection Agency and Forestry Department for supporting and facilitating collecting in Guyana by T.D.M; and the following herbaria for allowing access to their collections: BR, K, NY, P, S, TAN, TEF, UPS and US. 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(1991) Polynesian plant introductions. Islands, plants, and Polynesians (ed. by P.A. Cox and S.A. Banack), pp. 41–66. Dioscorides Press, Portland. Whittaker, R.J. & Jones, S.H. (1994) The role of frugivorous bats and birds in the rebuilding of a tropical forest ecosystem, Krakatau, Indonesia. Journal of Biogeography, 21, 689–702. Zerega, N.J.C., Ragone, D. & Motley, T.J. (2004) Complex origins of breadfruit (Artocarpus altilis, Moraceae): implications for human migrations in Oceania. American Journal Botany, 91, 760–766. BIOSKETCH Sylvain G. Razafimandimbison is a senior researcher at the Bergius Foundation of the Royal Swedish Academy of Sciences and uses a variety of methods to study the systematics, biogeography and evolution of Rubiaceae (coffee family). See <http:// www.bergianska.se/forskning_rubiaceae_php> for a comprehensive list of research interests for all members of the Bergius Foundation. Author contributions: S.G.R. analysed the data, conceived the ideas and was responsible for the writing; T.D.M. and D.A.H. provided material and helped with the writing; and B.B. is responsible for the Rubiaceae project at the Bergius Foundation and participated in the writing. Editor: Pauline Ladiges Journal of Biogeography 37, 520–529 ª 2009 Blackwell Publishing Ltd 529