Plant Syst. Evol. 230: 173±187 12002)

Nuclear rDNA ITS sequence data used to construct the ®rst phylogeny of )

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 of mostly woody 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 co€ee 1Co€ea 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 . 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. , interpetiolar stipules, sympetalous co- the group, the - 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 , , Lagy- the last ten years, all from the subfamilial level nias, and . 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 dicult group to work with. genera. Morphological characters are discussed and The 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. di€erences found in support are discussed. Consisting of about 600 species 1Robyns Key words: Rubiaceae, , 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 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. 1including Ixoroideae; Calyx and gynoecium with pollen presenter. From Verdcourt 1958) and Antirheoideae 1Robb- J. D. & E. G. Chapman 8939 1UPS). Bar ˆ 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 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 and the large 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). E€ort 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 , ,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 . 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 10xbu€er, 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. at 97 °C followed by 40±50 cycles of 97 °C The strategy when sampling, was to identify all 10 s; 48 °C30s;72°C20s+4°C/cycle, ®nishing type species of all published genera, subgenera, and with 72 °C for 7 min. sections, and to include these in the study when Sequencing reactions were done using the ABI possible. This was done to simplify taxonomic PRISMÒ BigDyeä Terminator Cycle Sequencing conclusions resulting from the analyses, and since Kit 1Applied Biosystems) and Thermo Sequenase the species are described on a morphological basis Dye Terminator Cycle Sequencing Kit 1Amersham and thus should represent a lot of the morpholo- Pharmacia Biotech) using primers ITS2, ITS3, gical variation in the tribe, it is believed that they ITS4, and ITS5 1White et al. 1990). All sequencing also represent a broad sampling in a phylogenetic was performed on an ABIPrism Ò 377 DNA sense. Fifty-three such types were identi®ed and 17 Sequencer 1Applied Biosystems). For the Amer- are included here, 13 of which are types of genera in sham Pharmacia kit each reaction contained 4 ll current use 1Robyns 1928, Bridson 1987b, Verd- mix, 2 ll1lM sequencing primer, 1 ll DMSO, 176 H. Lantz et al.: Phylogeny of Vanguerieae

Table 1. Voucher information and EMBL acession numbers. Voucher information is given for new sequences only. Herbarium abbreviations according to Holmgren et al. 11990) Species Voucher EMBL number Mussaenda erytrophylla AJ224823 Schumach. & Thonn. E. Mey. AJ224842 Polysphaeria parvifolia Hiern Iversen et al. 87279 1UPS) AJ315079 Polysphaeria aethiopica Verdc. Pu€ & Kelbessa 821227-1/5 1UPS) AJ315080 L. AJ224826 Andreasen 36 1UPS) AJ315081 1Burm. f.) Alston 1L.f.) Kuntze Bremer & Bremer 3682 1UPS) AJ315120 Canthium mundianum Cham. & Schltdl. Bremer & Bremer 3704 1UPS) AJ315107 Canthium cf. mundianum Cham. & Schltdl. Bremer & Bremer 3772 1UPS) AJ315106 Cuviera angolensis Welv. ex K. Schum. McPherson 16297 1MO) AJ315088 Fadogia ancylantha Hiern Chapman & Chapman 9109 1UPS) AJ315103 Fadogia elskensii De Wild. Taylor et al. 8318 1UPS) AJ315118 Fadogia tetraquetra K. Krause Bremer & Bremer 3799 1UPS) AJ315099 Fadogia verdcourtii Tennant var. verdcourtii Gereau et al. 6010 1UPS) AJ315116 Fadogiella stigmatoloba 1K. Schum) Robyns Lawton 1318 1S) AJ315100 Hutchinsonia barbata Robyns Adam 20599 1UPS) AJ315102 Keetia zanzibarica 1Klotzsch) Bremer 3069 1UPS) AJ315105 Bridson ssp. zanzibarica 1Sond.) Bridson Bremer 3083 1UPS) AJ315117 Lagynias dryadum 1S. Moore) Robyns Bremer & Bremer 3811 1UPS) AJ315090 Lagynias lasiantha 1Sond.) Bullock Bremer & Bremer 3792 1UPS) AJ315089 tetraphylla 1Hiern) Bremer 3074 1UPS) AJ315083 Robyns ssp. comorensis 1Robyns) Verdc. Multidentia concrescens 1Bullock) Bidgood et al. 845 1K) AJ315086 Bridson & Verdc. Multidentia fanshawei 1Tennant) Bridson Lovett et al. 3311 1K) AJ315087 pygmaeum Robyns Pawek 12335 1BR) AJ315091 armata 1K. Schum.) Robyns Bremer & Bremer 3790 1UPS) AJ315082 nitidum 1Craib.) Wong Ryding 599 1UPS) AJ315108 Psydrax obovata 1Klotzsch Bremer & Bremer 3762 1UPS) AJ315109 ex Eckl. & Zeyh.) Bridson var. obovata Psydrax parvi¯ora 1Afz.) Gilbert & Phillips 9069 1UPS) AJ315110 Bridson ssp. parvi¯ora Psydrax sp. Cult. 1Indonesia, Bogor) AJ315111 zeyheri 1Sond.) Bremer & Bremer 3800 1UPS) AJ315119 Robyns var. Zeyheri Verdc. Pyrostria bibracteata 1Baker) Cavaco Bremer 3036 1UPS) AJ315113 Pyrostria hystrix 1Bremek.) Bridson Bremer & Bremer 3791 1UPS) AJ315114 Pyrostria phyllantoidea 1Baillon) Bridson Taylor et al. 8486 1UPS) AJ315115 Pyrostria sp. Pettersson & Nilsson 752 1UPS) AJ315112 Rytigynia bagshawei 1S. Moore) Borhidi et al. 84439 1UPS) AJ315101 Robyns var. bagshawei Rytigynia bugoyensis 1K. Krause) Verdc. Rwburindore 3536 1UPS) AJ315084 Rytigynia fuscosetulosa Verdc. Frimodt Moller et al. NG117 1K) AJ315097 Rytigynia senegalensis Blume van den Berghen 8746 1BR) AJ315104 H. Lantz et al.: Phylogeny of Vanguerieae 177

Table 1 1continued) Species Voucher EMBL number Tapiphyllum cinerascens Milne-Redhead 3292 1BR) AJ315096 1Welv. ex Hiern) Robyns var. cinerascens Tapiphyllum velutinum 1Hiern) Robyns Emanuelsson 672 1S) AJ315098 K. Schum. KaÊ rehed & Odhult 161 1UPS) AJ315095 Burch. Blomberg et al. BMP 466 1UPS) AJ315093 Vangueria madagascariensis Gmelin. Bremer 3077 1UPS) AJ224839 Vangueria parvifolia Sond. Bremer & Bremer 3771 1UPS) AJ315092 Vangueriella spinosa Merello et al. 1494 1K) AJ315085 1Schumach & Thonn.) Verdc. lanci¯ora 1Hiern) Robyns Cult. 1Belgium, Meise) AJ315094 and 3±4 ll DNA adding up to a ®nal volume of 10± ITS1, 5 in ITS2). Due to uncertain alignment 11 ll. The reactions for BigDye contained 2 ll mix, 122 characters were excluded, 664 characters 0.4 ll sequencing primer, 0.6±2.6 ll DNA and if were included in the phylogenetic analysis. Of necessary up to 2 lldH2O to a ®nal volume of 5 ll. these, 428 were constant, 62 variable were Ò Sequences were analysed with the Sequencher non-informative and 174 were informative. 1Gene Codes Corporation) software package and Fadogia verdcourtii, F. elskensii, and Pygmaeo- after a preliminary alignment in ClustalW 1Thomp- thamnus zeyheri could not be sequenced for a son et al. 1994) aligned by eye in a text editor. Sequences are available from EMBL 1see Table 1). region 1up to 68 bases) in the 5.8S region, The alignment is available on request to the ®rst missing bases were replaced with N 1N ˆ any author. base). The phylogenetic analysis was performed with In the phylogenetic analysis, 18 equally PAUP* 4.0b5 1Swo€ord 1998). All phylogeneti- parsimonious trees with a length of 630 were cally informative insertion/deletion events 1indels) found 1CI ˆ 0.47 1uninformative characters were given the same weight independent of size. A removed) RI ˆ 0.72). The jackknife and boot- heuristic parsimony search of 100 random addi- strap values di€ered more than expected, tion replicates with TBR and MULTREES on sometimes with as much as twelve percent was conducted. Branch support was calculated units 1Fig. 2). Few nodes are collapsed in the with the jackknife and bootstrap procedures strict consensus 1Fig. 2) and several re- implemented in PAUP*. The jackknife analysis ceive strong support. Canthium and Rytigynia used a deletion frequency of 37%, 10 000 replicates, TBR branch swapping, 5 random are polyphyletic with members appearing in addition sequence replicates, and MULTREES distantly related clades. Vangueria, Tapiphyl- o€. Bootstrap used 10 000 replicates, TBR branch lum, and Fadogia are paraphyletic and the swapping, 5 random addition sequence replicates, monophyly of Psydrax is very weakly sup- and MULTREES o€. Bremer support was calcu- ported. With the sampling here presented, lated with Autodecay 4.0 1Eriksson 1998) using a Multidentia, Lagynias, Keetia, and Pyrostria heuristic search with 100 random addition se- turn out monophyletic with good support. quence replicates. Polysphaeria has a basal position to the Vanguerieae. Results Discussion The sequences varied in length from 611 bases 1Psydrax obovata) to 671 bases 1Fadogiella From the resulting trees it is evident that ITS stigmatoloba). The aligned matrix had 786 indeed is a useful DNA region for phylogenetic positions including 14 indel-characters 19 in studies in Vanguerieae. Several groups with 178 H. Lantz et al.: Phylogeny of Vanguerieae ugopSpiny group Outgroup Mussaenda erythrophylla Ixora coccinea Alberta magna Polysphaeria parvifolia 100 ? 18 Polysphaeria aethiopica 53/59 Canthium coromandelicum 1 Meyna tetraphylla Plectroniella armata Canthium inerme 88/94 Pygmaeothamnus zeyheri 5 54/57 79/87 Rytigynia bugoyensis 1 3 Vangueriella spinosa 96/97 Multidentia concrescens 3 Multidentia fanshawei Cuviera angolensis ** 95/98 100 Lagynias lasiantha 5 4 Lagynias dryadum Pachystigma pygmaeum Vangueria Vangueria parvifolia Vangueria infausta 89/95 52/59 95/96 1 4 Vangueria madagascariensis

2 group 80/89 * <50/56 5 1 Vangueria apiculata Tapiphyllum cinerascens Rytigynia fuscosetulosa 55/65 Tapiphyllum velutinum 2 Fadogia-Rytigynia 86/85 Fadogia elskenii 93/97 1 Fadogia tetraquetra 4 68/74 Fadogiella stigmatoloba 1 100 Fadogia ancylantha 7 100 Fadogia verdcourtii 9 88/93 Rytigynia bagshawei group 3 Hutchinsonia barbata 100 Rytigynia senegalensis 14 97/98 Keetia gueinzii 4 85/93 Keetia zanzibarica 5 100 Canthium cf. mundianum 14 Canthium mundianum 88/93 Psydrax nitidum 66/75 4 Psydrax sp. <50/56 2 Psydrax obovata 2 59/71 Psydrax parviflora 2 Pyrostria sp. 99/100 9 Pyrostria bibracteta 84/92 4 Pyrostria hystrix Pyrostria phyllantoidea Fig.2. Strict consensus tree of the 18 most parsimonious trees. Bootstrap/jackknife values are above the branches, Bremer support below. When bootstrap and jackknife values are equal only one value is noted. Where the bootstrap and jackknife values both are <50 and the Bremer support is one, no support is shown. The large suprageneric groups discussed in the text are marked with brackets, the question-mark 1?) denotes the H. Lantz et al.: Phylogeny of Vanguerieae 179 good support exist, although few of them agree of Canthium, several closely knit groups of with genera as presently conceived. Some main species have now been removed from the genus groups can be seen and the discussion follow- to form genera of their own 1Bridson 1985; ing will be centred around these groups. Since 1986; 1987a, b), leaving a very heterogeneous very few supra-generic groups have been genus. Bridson was fully aware of this, but discussed in the tribe 1the exceptions being avoided raising the probably more homoge- Robyns 1928, Igersheim 1989) and the ones neous subgenera of Canthium to generic rank that have been mentioned rarely comply with ``¼for pragmatic reasons, the need to avoid the groups found here, informal names will be unnecessary name changes in a tribe where the used 1see Fig. 2). The basic taxonomic frame- generic concepts are still unstable being para- work and terminology generally followed is the mount.'' 1Bridson 1992). The consensus tree one used in the Vanguerieae volumes of clearly demonstrates that Canthium is a po- F.T.E.A. 1Verdcourt and Bridson 1991) and lyphyletic assemblage of species, in part due to Flora Zambesiaca 1Bridson 1998). The excep- the historical emphasis on a single character. tion is the use of geofrutex instead of su€rutex, This use of a `cardinal character' has also been following the recommendation of Robbrecht criticised in the family-level classi®cation of 11988). Support values are listed in the text as Rubiaceae where too much importance was 1bootstrap/jackknife/Bremer support). ascribed to the number of ovules per locule Outgroup. The support in the outgroup is 1e.g. Bremer et al. 1995). poor, with the exception of the two Poly- For a long time Rytigynia included only sphaeria species, which group together with one species, R. senegalensis, until Robyns very strong support 1100/100/18). They were 11928) added 67 species. Twenty-two of these included to test if Polysphaeria is nested within were new, the rest were new combinations. Vanguerieae or not 1for phylogenetic relation- Today Rytigynia is thought to have a central ships of other Octotropideae genera, see core that is easily recognised, but also includes Andreasen and Bremer 2000). Although the some satellite groups 1Verdcourt 1987) inter- position of this group is weakly supported mediate between Rytigynia s. str. and other 1<50/<50/1), there is nothing that points to a genera. In this study R. fuscosetulosa and nested position within Vanguerieae. The inter- R. bugoyensis would be considered belonging nal position of Polysphaeria found in an earlier to such groups. study was probably based on a Vanguerieae Spiny group. In the strongly supported 188/ specimen misidenti®ed as a Polysphaeria. 94/5) spiny group, representatives from a wide Canthium and Rytigynia. Before discussing geographic range are found. Canthium coro- the strongly supported groups found, the mandelicum, the type species of Canthium,is polyphyletic nature of Canthium and Rytigynia restricted to southern India and Sri Lanka needs to be addressed. As mentioned earlier, while the other species in the group together Canthium and Vangueria were the ®rst genera cover most of Vanguerieae's African range. described in the tribe, in part based on the Even though they are distributed over a wide number of locules. The character was often area many morphological similarities exist. All given such importance that other information species except Pygmaeothamnus zeyheri have was ignored in favour of it, and this resulted in spines 1modi®ed branches), a character rare in two large heterogeneous groups. In the case other parts of Vanguerieae. In the case of b uncertain tribal placement of Polysphaeria. Type species of genera are marked in bold. The polyphyletic genera Canthium and Rytigynia aremarkedwiththesymbolss 1Canthium)andd 1Rytigynia). The symbols * and ** indicate strongly supported, unnamed groups discussed in the text 180 H. Lantz et al.: Phylogeny of Vanguerieae

C. coromandelicum, Meyna tetraphylla, Plec- having large fruits and a mitriform pollen troniella armata, R. bugoyensis, and Vangueri- presenter; R. bugoyensis lacks these features. ella spinosa, the spines are associated with There are a number of similarities, including brachyblasts 1lateral shoots with extremely lack of a de¯exed ring of hairs in the corolla, short nodes). Canthium inerme has no such the size of the corolla-tube, and a glabrous shoots and Pygmaeothamnus zeyheri di€ers in disk, but these character states can be found in several aspects from the other species. It lacks several other species in the spiny group, spines and brachyblasts, has a woody although not in that combination. To con- and whorled leaves, in contrast to the other clude, the transfer of R. bugoyensis to Vangu- species that are spiny or trees with eriella subgenus Stenosepalae is supported by opposite leaves. the molecular data, but the morphological Canthium coromandelicum shows a strong support is inconclusive. resemblance to Meyna tetraphylla. The only Vangueria group. As in the case of the spiny major di€erence lies in the number of locules group, the Vangueria group is well supported 12 for Canthium, 4±5 for Meyna) and it has 195/96/2), but good internal support is largely been suggested that Meyna perhaps ought to lacking. With the exceptions of Rytigynia and be included in Canthium s. str. 1Bridson 1992). Vangueriopsis, the group con®rms with Igers- The two species group together in the strict heim's `Gattungskomplex Vangueria' 1Igers- consensus tree but the support is much too low heim 1989). This group was based on mostly 153/59/1) for a transfer to be based on the anatomical and embryological characters, but result. Plectroniella armata also shares a very later some doubt was put to the reliability of similar with these two species, i.e., it has the characters 1Bridson 1996). Bridson consid- large spines and brachyblasts, but the genus ered Vangueria, Pachystigma, Lagynias, and has been considered closer to other spiny Tapiphyllum to form a close alliance together South African Canthium species than to Can- with Ancylanthos, Fadogia, and Fadogiella, but thium s. str. 1Bridson 1992). No such species with problematic generic delimitations. are included here, but it is quite likely that they Vangueria, the type genus of the tribe, is too belong to the spiny group, considering that paraphyletic. Grouped with the type species the presence of spines is very rare outside the V. madagascariensis are V. infausta and group. Before a more resolved tree can be Vangueriopsis lanci¯ora. Vangueria infausta is constructed and Canthium s.l. investigated a variable species with a large distribution in further, Plectroniella is best left as it is. eastern and southern Africa thought to be Rytigynia bugoyensis and V. spinosa form very close to V. madagascariensis 1Bridson the only strongly supported 179/87/3) group 1998). Vangueriopsis lanci¯ora is the type within the clade. Rytigynia bugoyensis is a species of Vangueriopsis, a genus that in earlier mainly East to Central African species while circumscriptions was much larger but two V. spinosa is restricted to . Vangu- subgenera were removed to Vangueriella when eriella spinosa is the type species of Vangueri- that genus was erected 1Verdcourt 1987). ella subgenus Stenosepalae and R. bugoyensis is Vangueriopsis is today a small genus of only doubtfully included in Rytigynia 1Bridson four species. There are several similarities 1998). They share a number of morphological between V. infausta and V. lanci¯ora features, and R. bugoyensis has been consid- 1Coates-Palgrave 1991) but the latter exhibits ered for transfer to Vangueriella 1Bridson some rare features including hairy anthers and 1992). However, when the similarities are very long corolla lobes. It is also worth investigated it becomes clear that ®nding any mentioning that Vangueriopsis has two ovary good synapomorphies that could support the locules instead of ®ve, otherwise the much transfer of R. bugoyensis to Vangueriella is more common state in the Vangueria group. very dicult. Vangueriella is partly de®ned by Vangueria apiculata has been suspected to H. Lantz et al.: Phylogeny of Vanguerieae 181 hybridise with V. madagascariensis 1Verdcourt ria and Vangueriella. The position of R. fus- 1981), and in the strict consensus tree, this cosetulosa in this analysis supports a close species is sister to the earlier mentioned species relationship with Vangueria but not with Van- but with very low support 1<50/<50/1). Also gueriella subgenus Stenosepalae. Vangueriella is with low support 1<50/<50/1), Vangueria a diverse genus and it is possible that R. fus- parvifolia is the sister taxon to all four already cosetulosa might be closer related to other mentioned species. Vangueriella species not included in this study. Basal to the Vangueria-Vangueriopsis Lagynias is a genus of ®ve well de®ned clade ®ve other genera are found in the species divided into two subgenera, subgenus Vangueria group. Apart from the two Lagy- Lagynias and subgenus Bembea 1Verdcourt nias species that group together with very 1987). The genus is by its large calyx lobes, lax strong support, the support is negligible. in¯orescences and discolorous leaves easily Tapiphyllum is represented by two species, recognisable. Here two species are included, T. cinerascens 1type species) and T. velutinum. L. lasiantha 1type species) and L. dryadum, Tapiphyllum has been thought to be close both from subgenus Lagynias. The two species to Vangueria 1Bridson 1996) and Vangueria group together with very strong support 1100/ parvifolia was recently transferred from 100/4) in an unresolved basal position in the Tapiphyllum 1Bridson 1998). Tapiphyllum is Vangueria-group. Morphologically the genus mainly recognised by having large calyx lobes is rather isolated apart from some shrubby and velvety indumentum and is considered to Pachystigma species, e.g. P. macrocalyx and be rather vaguely de®ned 1Verdcourt 1981, P. tri¯orum, that are very close to Lagynias Havard and Verdcourt 1987). The genus also and have been considered for transfer to has ties to other genera in the group. Verd- Lagynias 1Bridson 1996). Otherwise the court for example considered Tapiphyllum to above-mentioned combination of characters be ``...no more than a velvety Pachystigma...'' is rare in the tribe. 1Verdcourt 1981). Pachystigma is in this Cuviera and Multidentia belong in a strong- analysis represented by P. pygmaeum,a ly supported, mostly pluri-locular clade geofrutex with a wide distribution ranging together with the Vangueria-group and the from to . This genus is Fadogia-Rytigynia group. Their position with- also thought to be heterogeneous 1Bridson in that clade is however unresolved. Cuviera 1998) with species showing anity to Tapi- has a mainly Guineo-Congolian distribution phyllum, Fadogia, Lagynias, and Vangueria 1Robbrecht 1996), with only a few poorly and it certainly is dicult to ®nd any good known species occurring in East Africa. The morphological support for the genus. Pachy- most obvious feature of the genus is the large, stigma seems to be a genus based more on a often many-¯owered cymes with enlarged lin- lack of characters 1e.g. the velvety indumen- ear-foliose bracts and bracteoles. The calyx tum of Tapiphyllum, the larger fruits and lobes are often similarly enlarged. It is also di€erent in¯orescences of Vangueria) than on common that the base of the pollen presenter is actual de®ning characters. markedly widened, and several species have Rytigynia fuscosetulosa, together with hairy styles 1Halle 1959). All these characters R. saliensis Verdc., are the only member of combined make for one of the most derived Rytigynia subgenus Sali. This subgenus forms genera of the tribe. Cuviera was together with an aberrant group within the genus, di€ering Canthium the only genera that Robyns 11928) from the core group in having long calyx lobes did not look into and many authors have been among other characters. In the description of involved in describing the approximately 20 the species 1Verdcourt 1987, Verdcourt and species now accepted. However, due to the rare Bridson 1991) it is mentioned that they show characters mentioned above, the genus seems anity to not only Rytigynia but also Vangue- better delimited than most other genera in 182 H. Lantz et al.: Phylogeny of Vanguerieae

Vanguerieae. Only one species is included here, on the other hand very small with three species Cuviera angolensis, a central African member each 1Robyns 1928, Bremekamp 1952, Bridson of subgenus Cuviera and although it seems 1998). The same grouping of genera as found clear that it belongs neither in the Vangueria- in the Fadogia±Rytigynia group was suggested group nor in the Fadogia-Rytigynia group its by Igersheim 1Gattungskomplex Rytigynia; precise relationships are unresolved. It is clear, 1989) based on anatomical and embryological however, that it belongs to the pluri-locular characters. Fadogia is represented by four clade. This is in agreement with the fact that species and is paraphyletic 168/74/1) with the with few exceptions Cuviera has more than two type species of Fadogiella nested within. With locules. It has been suggested that the genus few exceptions, Fadogia species have a geo- has anity with Vangueriella 1Verdcourt frutescent habit, whorled leaves, reduced calyx 1987). This is not supported, but since both lobes and three to ®ve locules. Fadogiella genera are represented by only one species stigmatoloba shares all these characters but is each and, as this study shows, no monophyly further characterised by its velvety indumen- can be taken for granted, it is much too early tum, a de®ning character for the genus. The to dismiss the possibility. Fadogia species form two clades. Fadogia Multidentia is a morphologically distinct elskensii is considered very close to the type genus occurring in Tropical Africa. It was species of Fadogia, F. cienkowskii Schweinf., described by Gilli 11973), but received its and groups with F. tetraquetra 186/85/1). Both current circumscription when Bridson 11987a) were included in the same subsection 1section added more species to a ®nal of eleven taxa. Subulatae subsection Imparisepalae) by Ro- Common to most of the included species is their byns 11928) and are considered very close cupular calyx limb 1the connate part of the 1Verdcourt and Bridson 1991). The other calyx), a deeply cleft and strongly ribbed pollen group consists of F. ancylantha and F. verdcour- presenter with a usually tapering base, and tii 1100/100/7), both of which have been thickly woody pyrenes. Included in this study suggested to have an anity with are the type species M. concrescens and Robyns 1Tennant 1968, Verdcourt and Brid- M. fanshawei, the type species of the now son 1991). Temnocalyx is very close to Fadogia rejected Canthium section Granditubum. These but is characterised by having large ¯owers species group together with very strong support with curved corolla-tubes. The character was 196/97/3) and the genus seems monophyletic. however considered too variable and the genus All Multidentia species have two locules and is now monotypic after the transfer of several earlier investigations 1Bridson 1987a, Igersheim species to Fadogia 1Verdcourt 1981). Awaiting 1989) have suggested an anity with other an analysis where the type species of Temno- two-locular genera. Bridson 11987a) suggested calyx and more Fadogia species are included an anity with Vangueriella sect. Vangueriella no recommendation is presented about how to 1not included in this study) and Igersheim deal with the paraphyletic Fadogia. Accom- 11989) included Multidentia in his `Gattungs- modating the Fadogiella species in Fadogia komplex Canthium' together with Canthium, would, however, necessitate only slight chang- , Meyna 1this genus is however es in the description of Fadogia. pluri-locular) and Pygmaeothamnus. Here the As noted above, Rytigynia is polyphyletic. genus groups together with the largely pluri- Included in the Fadogia-Rytigynia group are locular species but in an unresolved position. two Rytigynia species, R. senegalensis and The Fadogia-Rytigynia group contains two R. bagshawei. The type species of Rytigynia, of the larger Vanguerieae-genera, Fadogia and R. senegalensis, has an unresolved position in Rytigynia. Both genera include around 50 the group and R. bagshawei is sister to Hutch- species 1Bridson 1998). The other two genera insonia barbata 188/93/3), the type species of a in the group, Hutchinsonia and Fadogiella, are West African genus with very long and narrow H. Lantz et al.: Phylogeny of Vanguerieae 183 corolla-tubes. Both Rytigynia species are mem- the specimen is a new species, subspecies, or if bers of subgenus Rytigynia. Interestingly, R. the variation is just dependent on growth bagshawei is a two-locular species while both conditions is not known, but it was decided R. senegalensis and H. barbata are pluri- that it remains in the study awaiting a better locular, which further demonstrates the risks determination. The subgenus Afrocanthium is of ascribing too much weight to number of based on a combination of characters of which locules. the deciduous habit, reduced calyx lobes, and Keetia is one of the distinct groups re- absence of a ring of re¯exed hairs in the corolla- moved from the earlier very large Canthium in tube is perhaps of most importance. This connection with the F.T.E.A. 1Verdcourt and analysis clearly separates C. mundianum from Bridson 1991) and Flora Zambesiaca 1Bridson subgenus Canthium and subgenus Lycioserissa, 1998) projects. It shares several characters with but without including the type species of the Psydrax, e.g. style much longer than corolla- subgenus 1C. lactescens Hiern) it is too early to tube, pollen presenter cylindrical and usually decide what to do with the subgenus name longer than wide, and cotyledons oriented Afrocanthium. Canthium mundianum needs to parallel to ventral face of seed, but there are be transferred to another genus, but which also important di€erences 1Bridson 1986). Of cannot be decided from the present data. extra importance is considered the presence of Including the species in Keetia would be very a lid-like area surrounding a central crest on problematic since it lacks many of the mor- the pyrenes, which is lacking in Psydrax. phological synapomorphies of that genus. Keetia gueinzii is the type species of the genus, Psydrax has the widest geographical distri- an extremely widespread species that can be bution of all Vanguerieae genera. It is also the found in almost all of Vanguerieae's continen- largest by far with approximately 100 species tal African range. Keetia zanzibarica occurs on 1Bridson 1998). Thirty-four species occur in large parts of the African east coast. The Africa 1incl. ), the rest are found in strong support from the molecular data 197/ southern Asia, the Paci®c and Australia. 98/4) in combination with the morphological However, several of these have still not been characters gives added weight to the conclu- formally transferred from Canthium 1Bridson sion that Keetia is one of the best-delimited 1998). Of the species here included, Psydrax Vanguerieae genera. sp. and P. nitidum were collected in southeast Canthium subgenus Afrocanthium. Consid- Asia, P. obovata and P. parvi¯ora are from ering the many morphological similarities be- Africa. The specimen Psydrax sp. has been tween Keetia and Psydrax, one would expect impossible to determine to species but as a to ®nd Psydrax as the sister group of Keetia representative of Asian Vanguerieae it was still and not two Canthium specimens as seen in included in the study, and groups together with this study. The two specimens might be repre- the other Asian species, P. nitidum with strong sentatives of the same species, C. mundianum,a support 188/93/4). Psydrax forms a monophy- member of Canthium subgenus Afrocanthium letic group in the strict consensus tree, but with 1Bridson 1992). The vegetative specimen an- very low support 1<50/56/2). The low support notated as Canthium cf. mundianum was col- is somewhat surprising since the genus is very lected in South Africa. Based on domatia and well delimited by morphological characters. other macromorphological characters it seems Several characters are shared with Keetia 1as closely related to C. mundianum, but di€ers in noted above) but Psydrax di€ers in usually having an abnormal shape 1broader, size having re¯exed anthers and a short calyx-limb. up to 8.5 ´ 6.0 cm) and an almost complete Two subgenera have been described 1see Brid- lack of tanniniferous cells 1see Tilney et al. son 1985 for a discussion), subgenus Psydrax is 1988, 1990 for a discussion on tanniniferous the largest and occurs throughout the distri- cells in southern African Vanguerieae). Whether bution range. Subgenus Phallaria consists of 184 H. Lantz et al.: Phylogeny of Vanguerieae lianas or scandent shrubs and is restricted ported 195/98/5; ** in Fig. 2), but morpholog- to continental Tropical Africa. All species ical support remains to be found. represented in this study belong to subgenus Support. There are several methods used Psydrax. for calculating support in phylogenetic trees. Pyrostria. As of present, Pyrostria contains Bootstrap 1Felsenstein 1985) and jackknife about 45 species, but this ®gure is very likely to 1Farris et al. 1996) are probably the two most increase. Pyrostria is centred in Madagascar widely used. These two support measures were and forms the central core of a group of calculated, and settings were chosen that genera, many of which quite easily could be would yield as accurate and similar results as accommodated in Pyrostria 1Bridson 1987b). possible 1Hedges 1992, Farris et al. 1996, Pyrostria s. str. is characterised by large bracts DeBry and Olmstead 2000). The results from that enclose the young in¯orescences, pluri- the analyses are presented in Fig. 2. For locular ovaries, and unisexual ¯owers. The several nodes, the di€erences are substantial, generic concept has, however, been widened ranging up to twelve percent units. Jackknife and the genus now includes both hermaphro- values are usually the highest, and the largest dite and two-locular species. Some ebracteate di€erences are seen at nodes with the lowest groups could also be transferred to Pyrostria support values 1see Mort et al. 2000 for a at a later stage 1Bridson 1987b). Here, three similar result). Although the standard devia- species from the African mainland are includ- tion is larger for lower bootstrap values, and ed, P. hystrix, P. phyllantoidea, and P. bi- partly could explain why the lower values bracteata. All of these have bracts but P. di€er more, the high number of replicates hystrix is a hermaphrodite. Pyrostria sp. is a makes the standard deviation expected very probably undescribed very distinct species small 1Hedges 1992). A completely di€erent collected in Madagascar. It has hairy calyx- kind of support, Bremer support 1Bremer lobes, glabrous small leaves, and domatia 1988), was also added. As a non-statistical present as ciliate pits. The presence of bracts, alternative it provides a basis for interesting unisexual ¯owers, and obscure secondary and comparisons. No strong correlation is seen tertiary nerves de®nitely suggest a position in between the Bremer support and the other Pyrostria. The genus receives very strong support measures, at least for lower support support in this analysis 199/100/9) with the values 1also seen in Nore n and Jondelius 1999). non-Madagascan taxa also forming a strongly It is important to note that bootstrap and supported group 184/92/4). In its current jackknife are di€erent measures of support circumscription it seems clear that Pyrostria sensitive to di€erent parameters, and it is only is a monophyletic group with the presence of under very speci®c circumstances rarely en- bracts as a good diagnostic character. countered in real datasets that the values are Other strongly supported clades. Apart expected to be identical 1Oxelman et al. 1999). from the groups discussed above, there are The phenomenon does, however, need to be some additional strongly supported groups. investigated further. The pluri-locular clade, which includes Mul- Conclusions. Vanguerieae is a tribe in great tidentia, Cuviera, the Vangueria group, and the need of revision. The focus of the revisional Fadogia-Rytigynia group, is one such clade work done so far has usually been on the East 180/89/5; * in Fig. 2). Many species in this African species, and the tribe needs a revision group usually have more than two locules, but on a more global level. This analysis reveals Vangueriopsis lanci¯ora and R. bagshawei do some strongly supported groups, but without not. There are more two-locular species not including more species it is dicult to draw sampled which most likely belong to this any conclusions about the classi®cation. The group. The node uniting the spiny group and situation is in many cases, e.g. the Vangueria the pluri-locular group is also strongly sup- group, so confused that a species by species H. Lantz et al.: Phylogeny of Vanguerieae 185 approach is needed to get a clear picture of Faculty of Science and Technology at Uppsala how to delimit the genera. Even so, the results University for collaboration between the depart- from this analysis do improve our understand- ment of Systematic Botany in Uppsala, Sweden, ing of the tribe. Several of the traditional and the Jepson Herbarium in Berkeley, California. genera are not monophyletic, i.e. Canthium, Funding was also received from the Swedish Rytigynia, Vangueria, Tapiphyllum, and Fado- Natural Science Research Council, the Royal Swedish Academy of Sciences and Helge Ax:son gia. There are also some monophyletic groups Johnsons stiftelse. that receive strong support, including some of the genera currently in use. Multidentia, Lagy- nias, Keetia, and Pyrostria all seem monophy- References letic based on the included species and well delimited also in a morphological sense. Inter- Andersson L., Rova J. H. E. 11999) The rps16 intron and the phylogeny of the esting are the larger supra-general groups 1Rubiaceae). Syst. Evol. 214: 161±186. found that are better supported than many Andreasen K., Baldwin B. G., Bremer B. 11999) genera. The spiny group is especially interest- Phylogenetic utility of the nuclear rDNA ITS ing since it has the added support of a region in subfamily Ixoroideae 1Rubiaceae): morphological synapomorphy, the presence comparisons with cpDNA rbcL sequence data. of spines. It is much more dicult to ®nd Plant Syst. Evol. 217: 119±135. morphological support for the Vangueria and Andreasen K., Bremer B. 11996) Phylogeny of the the Fadogia-Rytigynia groups and they require subfamily Ixoroideae 1Rubiaceae). Opera Bot. a more complete sampling and careful Belg. 7: 119±138. morphological investigation before any such Andreasen K., Bremer B. 12000) Combined phylo- conclusions can be drawn. Especially the genetic analysis in the Rubiaceae-Ixoroideae: Vangueria group, but also the spiny group, Morphology, nuclear and chloroplast DNA. Amer. J. Bot. 87: 1731±1748. su€er from poor internal support from the ITS Bremekamp C. E. B. 11952) Rubiace es nouvelles data here used. The inclusion of more charac- pour le Congo Belge. Bull. Jard. Bot. E tat. 22: ters, both molecular and morphological, will 97±104. hopefully rectify the situation and such a work Bremekamp C. E. B. 11954) Les sous-familles et les is in progress. The problem of generic delim- tribus des Rubiace es. Huitie me congreÁ s interna- itation in Vanguerieae is problematic, but not tional de botanique, rapports et communica- unsolvable. tions, sect. 2±6. Paris, pp. 113±114. Bremer B., Andreasen K., Olsson D. 11995) Sub- familial and tribal relationships in the Rubiaceae The authors gratefully acknowledge the help of based on rbcL. Ann. Missouri Bot. Gard. 82: the following people; E. Robbrecht and an anon- 383±397. ymous reviewer for comments on the manuscript. Bremer K. 11988) The limits of amino acid B.G. Baldwin and sta€ at the Jepson Herbarium sequence data in angiosperm phylogenetic re- and Department of Integrative Biology, University construction. Evolution 42: 795±803. of California, Berkeley, for all help and use of their Bridson D. M. 11985) The reinstatement of Psydrax facilities; N. Heidari and J. RoÈ nnholm for technical 1Rubiaceae, subfam. Cinchonoideae tribe Van- assistance; J. KaÊ rehed, B. Oxelman, J. Lundberg, guerieae) and a revision of the African species. J. Nylander and others of the Discussion Group at Kew. Bull. 40: 687±725. the Department of Systematic Botany, Uppsala Bridson D. M. 11986) The reinstatement of the University for comments on the manuscript; African genus Keetia 1Rubiaceae subfam. D. Bridson, Kew, for kindly taking the time to Cinchonoideae, tribe Vanguerieae). Kew. Bull. answer all Vanguerieae-questions; E. Robbrecht, 41: 965±994. C. Ridsdale, and the directors of the herbaria listed Bridson D. M. 11987a) The recognition and recir- in Table 1 for supplying plant material. This work cumscription of the African genus Multidentia was made ®nancially possible by a grant from the 1Rubiaceae-Vanguerieae). Kew. Bull. 42: 641±654. 186 H. Lantz et al.: Phylogeny of Vanguerieae

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Robbrecht E. 11994) Supplement to the 1988 acters of the stem in the southern African species outline of the classi®cation of the Rubiaceae. of Canthium s.l. 1Rubiaceae). S. Afr. J. Bot. 54: Opera Bot. Belg. 6 11993): 173±196. 585±595. Robbrecht E. 11996) Generic distribution patterns Verdcourt B. 11958) Remarks on the classi®cation in subsaharan African Rubiaceae 1Angiosper- of the Rubiaceae. Bull. Jard. Bot. E tat. 28: 209± mae). J. Biogeogr. 23: 311±328. 281. Robyns W. 11928) Tentamen Monographiae Van- Verdcourt B. 11981) Notes on African Rubiaceae. guerieae Generumque Anium. Bull. Jard. Bot. Kew. Bull. 36: 493±557. E tat. XI: 1±359. Verdcourt B. 11987) Notes on African Rubiaceae- Smith A. C., Darwin S. P. 11988) Rubiaceae. In: Vanguerieae. Kew. Bull. 42: 123±199. Smith A. C. 1ed.) Flora Vitiensis Nova. SB Verdcourt B., Bridson D. M. 11991) Rubiaceae Printers, Inc., Honolulu, pp. 143±376. 1Part 3). In: Polhill, R. M. 1ed.) Flora of Tropical Swo€ord D. L. 11998) PAUP*. Phylogenetic Anal- East Africa. A. A. Balkema, Rotterdam/Brook- ysis Using Parsimony 1*and other methods). ®eld, pp. 749±956. Version 4. Sinauer Associates, Sunderland, Mas- White T. J., Bruns T., Lee S., Taylor J. 11990) sachusetts. Ampli®cation and direct sequencing of fungal Tennant J. R. 11968) New or interesting Rubiaceae ribosomal RNA genes for phylogenetics. In: Innis from tropical Africa: II. Kew. Bull. 22: 435±444. M., Gelfand D., Sninsky J., White T. 1eds.) PCR Thompson J. D., Higgins D. G., Gibson T. J. Protocols: A guide to methods and applications. 11994) CLUSTAL W: improving the sensitivity Academic Press, San Diego, pp. 315±322. of progressive multiple sequence alignment through sequence weighting, positions-speci®c gap penalties and weight matrix choice. Nucl. Addresses of the authors: Henrik Lantz and Acids Res. 22: 4673±4680. Birgitta Bremer 1E-mail: [email protected], Tilney P. M., Kok P. D. F., van Wyk A. E. 11990) [email protected]) Department of System- The taxonomic signi®cance of anatomical char- atic Botany, Evolutionary Biology Centre, Uppsala acters of the leaf in the southern African species University, Norbyv. 18D, SE-752 36 Uppsala, of Canthium s.l. 1Rubiaceae). S. Afr. J. Bot. 56: Sweden. Katarina Andreasen 1E-mail: katarina.an- 363±382. [email protected]) Laboratory of Molecular Syst- Tilney P. M., van Wyk A. E., Kok P. D. F. 11988) ematics, Swedish Museum of Natural History, Box The taxonomic signi®cance of anatomical char- 50007, SE-104 05 Stockholm, Sweden.