Nuclear Rdna ITS Sequence Data Used to Construct the First Phylogeny
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Plant Syst. Evol. 230: 173±187 12002) Nuclear rDNA ITS sequence data used to construct the ®rst phylogeny of Vanguerieae Rubiaceae) H.Lantz 1, K.Andreasen 2,3, and B.Bremer 1 1Department of Systematic Botany, EBC, Uppsala University, Uppsala, Sweden 2Jepson Herbarium and Department of Integrative Biology, University of California, Berkeley, USA 3Present address: Laboratory of Molecular Systematics, Swedish Museum of Natural History, Stockholm, Sweden Received March 14, 2001 Accepted August 1, 2001 Abstract. The morphologically homogenous tribe ceae, a family of mostly woody species distrib- Vanguerieae was investigated phylogenetically us- uted over large parts of the world, but centred ing sequence data from the Internal Transcribed in the tropics. Rubiaceae are perhaps best Spacer 1ITS) region in the nuclear ribosomal DNA. known for coee 1Coea sp.), but also include Sequences from 41 Vanguerieae species represent- many ornamentals and are an important ing 19 genera were produced, and a parsimony constituent of many tropical habitats. The analysis was performed. The phylogenetic analysis main characteristics of the family are opposite has several clades with strong support, among which three new informal groups are discussed, i.e. leaves, interpetiolar stipules, sympetalous co- the Vangueria group, the Fadogia-Rytigynia group rollas and inferior ovaries. While many phy- and the Spiny group. Also found monophyletic logenies for the family have been produced in with strong support are Multidentia, Keetia, Lagy- the last ten years, all from the subfamilial level nias, and Pyrostria. Canthium and Rytigynia are down to genera, no phylogeny has so far been revealed as polyphyletic; Vangueria, Tapiphyllum, produced for Vanguerieae. This might in part and Fadogia are paraphyletic. Results from this ®rst be due to a strong morphological similarity phylogenetic analysis of the tribe clearly demon- between the taxa in the tribe, which has made strate a need for new circumscriptions of several the tribe a very dicult group to work with. genera. Morphological characters are discussed and The taxonomy has in recent years become putative synapomorphies are mentioned. Jackknife, re®ned, but no real consensus has been reached bootstrap, and Bremer support are calculated and on how to delimit the genera. dierences found in support are discussed. Consisting of about 600 species 1Robyns Key words: Rubiaceae, Ixoroideae, Vanguerieae, 1928, Cavaco 1969, Smith and Darwin 1988, ITS, phylogeny, support. Bridson 1998), Vanguerieae are among the larger tribes in the family Rubiaceae. The Although a widespread and common group in major part of the described taxa can be found the paleotropics, Vanguerieae are a tribe that in Africa 1almost all parts south of the has received little attention. They are a mem- Sahara), but some can also be found in ber of the large and important family Rubia- southern Asia, the Paci®c, and Australia. 174 H. Lantz et al.: Phylogeny of Vanguerieae As Vanguerieae are delimited today they form a strongly supported monophyletic group 1Bremer et al. 1995, Andreasen and Bremer 1996). Dumortier ®rst mentioned the tribe in 1829 1as ``Vaugnerieae''; Dumortier 1829). He included not only ``Vaugneria'' but also several other genera that now have been excluded from the tribe. Later authors also had opinions on Vanguerieae that deviate from the present classi®cation 1e.g. Hooker 1873, Hiern 1877), and it was not until 1958 that Verdcourt ®nally delimited the tribe as it is known today 1Verdcourt 1958). Several Fig.1. Tapiphyllum velutinum. a Corolla with one subfamilial placements have been suggested, lobe and corresponding part of wall removed. b e.g. Cinchonoideae 1including Ixoroideae; Calyx and gynoecium with pollen presenter. From Verdcourt 1958) and Antirheoideae 1Robb- J. D. & E. G. Chapman 8939 1UPS). Bar 0.5 cm. recht 1988), but now most studies place it in Drawn by H. Lantz Ixoroideae 1e.g. Bremer et al. 1995, Andersson and Rova 1999). 1Lamarck 1783) and Vangueria 1Jussieu The tribe is easily identi®ed by a combina- 1789). Canthium was partly de®ned by having tion of characters: axillary in¯orescences, val- two locules and Vangueria by ®ve. The number vate aestivation, single pendulous ovules and of locules was the main character used when above all, the apex of the style swollen into a later species were described; species with two pollen presenter serving in secondary pollen locules were placed in Canthium 1or Plectronia, presentation. This way of presenting pollen to an invalid synonym; Hiern 1898) and species the pollinator is a rather common pheno- with more than two locules were placed in menon in the family 1Pu et al. 1996), especially Vangueria. New genera were later described, in the subfamily Ixoroideae of which sometimes including species earlier described Vanguerieae are a member, and was also used as Canthium or Vangueria, and today 28 as a character to circumscribe the subfamily generic names are in use. For a more complete 1Bremekamp 1954). However, in the case of discussion on the history of Vanguerieae, see Vanguerieae the pollen presenter is represent- Verdcourt 11987). ed by a `stylar head'-complex 1Igersheim In the last 20 years a vast amount of work 1993), a character unique for the tribe 1see on Vanguerieae has been produced for the Fig. 1). The complex consists of both the F.T.E.A. and Flora Zambesiaca projects pollen presenting tissue and of the stigma 1Verdcourt and Bridson 1991, Bridson 1998). lobes and is in most cases globose to cylindri- These publications have greatly helped the cal, often with the style recessed into the understanding of the complicated groupings structure. Furthermore, several anatomical within the tribe. However, very few studies characters support the complex as a unique have attempted to study Vanguerieae as a structure 1Igersheim 1993). However, follow- whole. Only two general works have been ing the example set in Flora of Tropical East produced. The ®rst one is `Tentamen Mono- Africa 1F.T.E.A.; Verdcourt and Bridson graphiae Vangueriae' by Robyns 11928). In 1991) and Flora Zambesiaca 1Bridson 1998), this study Robyns describes many new species the more general term pollen presenter will be and genera but avoids many problems by not used here. dealing with Cuviera and the large genus Of genera later to be included in Canthium, which is perhaps the most problem- Vanguerieae, the two ®rst were Canthium atic one. The other study is the thesis of H. Lantz et al.: Phylogeny of Vanguerieae 175 Igersheim 11989). He investigates morpholog- court and Bridson 1991, Bridson 1998, Reynolds ical and anatomical characters to help with the and Henderson 1999). Eort was also put into generic delimitation of the tribe and also getting species from a wide geographic range. In suggests some groupings of genera. total 41 in-group taxa were sequenced for this study Although the work produced for the 1Table 1) and 19 genera were represented. Authors Floras has helped alleviate the shortcomings of names are included in Table 1, or for species not sequenced, when ®rst mentioned in the text. In of earlier classi®cations, Vanguerieae remain a many cases the availability of good material complicated group of species. Very little is determined what species were sequenced, especially known about phylogenetic relationships with- for the larger genera where more species were in the tribe and the monophyly of several needed. As outgroups Mussaenda, Ixora,and genera is questionable. This study uses se- Alberta were chosen. Ixora and Alberta have been quence data from the Internal Transcribed shown to be closely related to Vanguerieae 1e.g. Spacer 1ITS) region in the nuclear ribosomal Andreasen et al. 1999) but not belonging to the in- DNA to gain insight into these questions and group. Mussaenda, which is basal to all these taxa to identify groups where further studies 1Andreasen and Bremer 2000), was also included in are most needed. There are two reasons why order to orient the trees. Polysphaeria 1Octotrop- ITS was chosen as the DNA region to be ideae, Robbrecht 1994) was included since a sequenced. 11) Vanguerieae are a group of molecular study by J. Rova 1unpubl. thesis) placed an unspeci®ed member of the genus at an internal closely related species, in many cases compa- position in Vanguerieae. rable to large genera, e.g. the Rubiaceae genus DNA was extracted using the CTAB method Psychotria 1Nepokroe et al. 1999) where ITS 1Doyle and Doyle 1987) from silica-dried, fresh or has been shown to be very informative. 12) A herbarium material and cleaned either with Qia- study where ITS was used on the Ixoroideae Quick PCR puri®cation kit 1QIAGEN) or caesium 1Andreasen et al. 1999) also suggested that chloride/ethidium bromide gradient centrifugation. ITS would be useful at lower taxonomic levels The cocktail for PCR ampli®cation was mixed as in the subfamily. follows 125 ll): 2.5 ll 10xbuer, 2.5 ll25mM The aim of this study is to produce a MgCl2, 2.5 ll 10 mM dNTP, 1.25 ll10lM for- phylogeny of the tribe that can be used as ward primer, 1.25 ll10lM reverse primer, 1.25 ll a basis for later taxonomic work. By having a glycerol, 0.1 ll Taq DNA polymerase, 12.65 ll broad taxonomic and geographic sampling it is dH2O, and 1 ll DNA. Several additives were also tried, alone or in combination 1replacing some of hoped that the results will be generally appli- the water): 2.5 ll 6.5 M betaine, 2.5 ll 0.1 M cable and that this will facilitate the ®nding of TMACl 1Chevet et al. 1995) or 0.5 ll BSA. The morphological characters useful for delimiting ampli®cation primers chosen were ITS4 1White genera. et al. 1990) and Leu1 1Andreasen et al. 1999). Reactions were run on a GeneampÒ PCR system Material and methods 9600 or 9700 system 1Applied Biosystems) with 1 min.