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A trnL-F cpDNA sequence study of the Condamineeae-Rondeletieae-Sipaneeae complex with implications on the phylogeny of the Rubiaceae¹

²Botanical institute, Göteborg University, P.O. Box 461, SE-405 30 Göteborg, Sweden; ³The Lewis B. and Dorothy Cullman Program for Molecular Systematic Studies, and Institute of Systematic Botany, The New York Botanical Garden, Bronx, New York 10458-5126 USA; and ⁴Natural History Museums and Botanical Garden, University of Oslo, Sars' gate1, N-0562 Oslo, Norway

Received for publication February 2, 2001. Accepted for publication July 19, 2001.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
DNA sequences from the chloroplast trnL-F region of 154 Rubiaceae and 11 outgroup taxa were analyzed cladistically. An emphasis was placed on the tribes Rondeletieae, Sipaneeae, and Condamineeae. Sipaneeae are not close to Rondeletieae and belong in the Ixoroideae. There is no support for a widely distributed Rondeletieae in a broad sense. Instead, Rondeletieae sensu stricto form an almost entirely Antillean clade. Support was found for the separation of Arachnothryx, Rogiera, Roigella, and Suberanthus from Rondeletia. The Guettardeae as well as Gonzalagunia are found close to a complex formed by Arachnothryx, Javorkaea, and Rogiera. Condamineeae, in a strict sense, belongs in the Ixoroideae. A number of Rondeletieae genera should be transferred to Condamineeae or other parts of Ixoroideae. Support is found for an emended tribe Naucleeae, comprising several genera with spherical pseudanthia. For the first time, tribal or subfamilial affiliation based on molecular sequence data is suggested for Allenanthus, Blepharidium, Chione, Coutaportla, Dolichodelphys, Mazaea, Neobertiera, Neoblakea, Phialanthus, Phyllacanthus, Phyllomelia, Schmidtottia, and Suberanthus.

Key Words: cladistics • Condamineeae • Rondeletieae • Rubiaceae • Sipaneeae • systematics • trnL intron • trnL-F spacer

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Although Rubiaceae is one of the largest families of flowering plants, comprising 650 genera and 13 000 species (P. G. Delprete, personal observation), macrosystematics within the family is still debated. One reason for this debate is the tremendous morphological diversity that is found in the family (Robbrecht, 1988 ), a condition that makes it difficult for the phylogenist to find homologous morphological characters for tribal and subfamilial level studies. In 1988, Robbrecht published the last mainly morphological overview of tribal and subfamilial delimitations and relationships in the Rubiaceae. He concluded that the tribes Condamineeae, Rondeletieae, and Sipaneeae should, based on morphology, be regarded as very closely related tribes in subfamily Cinchonoideae.

Shortly after Robbrecht's comprehensive study was published, the first molecular systematic studies of Rubiaceae began to appear (Bremer and Jansen, 1991 ; Bremer, 1992 ; Bremer and Struwe, 1992 ). Since then, molecular techniques have further contributed to the understanding of Rubiaceae systematics from species to family level. In some cases, molecular phylogenies have also reconsidered Robbrecht's apparently stable classification. For example, Robbrecht's (1988) subfamily Antirheoideae was shown to be paraphyletic (Bremer, Andreasen, and Olsson, 1995 ; Bremer and Thulin, 1998 ; Andersson and Rova, 1999 ), a position for the elusive genus Strumpfia was indicated (Bremer et al., 1999 ), support for a close relationship between Condamineeae subtribe Portlandiinae and the tribes Catesbaeeae and Chiococceae (Bremer, 1992 ) was established, and a suggestion to divide the tribes Rondeletieae and Condamineeae between subfamilies Cinchonoideae and Ixoroideae was made, with some representatives of Rondeletieae found to be closely related to Guettardeae in subfamily Cinchonoideae (e.g., Bremer, Andreasen, and Olsson, 1995 ; Andersson and Rova, 1999 ). Further, several molecular systematic studies have focused on particular parts of the family, such as subfamily Ixoroideae (e.g., Andreasen and Bremer, 1996, 2000 ; Andreasen, 1997 ; Andreasen, Baldwin, and Bremer, 1999 ), the Alibertia group (Persson, 2000 ), subfamily Rubioideae (e.g., Ehrendorfer, Manen, and Natali, 1994 ; Andersson and Rova, 1999 ; Bremer and Manen, 2000 ), the Psychotria complex (e.g., Andersson and Rova, 1999 ; Nepokroeff, Bremer, and Sytsma, 1999 ), and the tribe Rubieae (e.g., Manen, Natali, and Ehrendorfer, 1994 ; Manen and Natali, 1995 ; Natali, Manen, and Ehrendorfer, 1995 ; Natali et al., 1996 ). At the same time, several phylogenetic studies using morphological data were performed and showed that Catesbaeeae and Chiococceae were closely related and several Condamineeae genera should be regarded as closely related to Rondeletieae (Delprete, 1996a ).

Through these studies it became apparent that the tribes Condamineeae, Rondeletieae, and Sipaneeae do not form such a tight complex as suggested by Robbrecht (1988) . Rather, circumscriptions and systematic positions of these tribes needed to be reconsidered in relation to the entire family. In other words, what Robbrecht regarded as a question of finding the circumscriptions of the three closely related tribes changed to the task of finding which genera of this "complex" are actually closely related, which are not, and where in the family these tribal fragments have their closest relatives. To answer these questions, we performed a DNA sequence analysis including as many genera as possible from Robbrecht's Condamineeae-Rondeletieae-Sipaneeae complex (from which comparatively few representatives had been included in earlier molecular systematic studies) as well as selected genera from other tribes in the family. This study is based on a cladistic analysis of chloroplast trnL-F intron and spacer DNA sequences from 165 taxa (154 Rubiaceae and 11 outgroup taxa) and a comparison of these results with morphological evidence.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Selection of taxa
When designing the data matrix, our ambition was to get at least one species sequenced from all tribes in the Rubiaceae, based on the Robbrecht (1993) classification. A special effort was also made to select representatives from as many genera as possible from the tribes Rondeletieae, Condamineeae, Sipaneeae, Guettardeae, Calycophylleae, Chiococceae, and Catesbaeeae as circumscribed by Delprete (1996a) . Our goal was initially to include, when possible, at least two species, including the type species, from each genus in these tribes. The Rondeletia complex (Delprete, 1999b ) and Guettarda were considered to be of special interest because of their size and morphological diversity, and these genera were thus sampled more extensively. An attempt was also made to include as many taxa as possible from the "genera incertae sedis" and "tribus incertae sedis" listed by Robbrecht (1993) . Outgroup taxa were sought in families considered close to the Rubiaceae, and two representatives from outside Gentianales (Fraxinus L. and Jasminium L. of the Oleaceae) were also selected (Bremer and Struwe, 1992 ; Struwe, Albert, and Bremer, 1994 ; Bremer, Andreasen, and Olsson, 1995 ; Bremer, 1996b ; De Laet and Smets, 1996 ; Struwe et al., 1998 ). An effort was made to use voucher specimens identified by the authors or identified by specialists in the respective parts of the family and confirmed by the second author. Authors of taxa are given in the text only when mentioned for the first time and when not listed at http://ajbsupp.botany.org/v89 (Appendix 1) or (Appendix 2). An historical summary of the tribal and subfamilial positions of the genera included in this study may be found at http://ajbsupp.botany.org/v89 (Appendix 2).

Extraction, amplification, and sequencing
Total DNA was extracted from leaves or seeds of silica gel dried plant material (Chase and Hills, 1991 [although leaves were usually kept in silica gel for several months]) or from herbarium specimens. Occasionally, fresh material was used. Plant tissue was ground in 1.5–2 mL microcentrifuge tubes either in a FastPrep homogenizer (BIO 101, Vista, California, USA) or by hand with a plastic pestle. Liquid nitrogen was only used when fresh or nonbrittle material was ground manually. A cetyltrimethylammonium bromide (CTAB) buffer was used for lysis together with 2-mercaptoethanol, mainly following Doyle and Doyle (1987) , except that PEG 4000 or PEG 6000 was included (1 g/100 mL buffer). Samples were spun immediately after lysis and cleaned with the Geneclean® III Kit (BIO 101) according to the manufacturer's instructions. The trnL (UAA) intron and the intergenic spacer between the trnL (UAA) 3' exon and the trnF (GAA) gene were chosen for the analysis because they have been shown to evolve more rapidly than a coding region such as rbcL (Gielly and Taberlet, 1994 ). Polymerase chain reaction (PCR) amplifications were performed with the "c," "d," "e," and "f" primers of Taberlet et al. (1991) . DNA extracts made from fresh or silica gel dried material was usually amplified successfully using only the "c" and "f" primers. Extractions from herbarium material, in which DNA was more likely to be degraded, were generally amplified in two parts, i.e., with primer pairs "c" + "d" (amplifying the intron) and "e" + "f" (amplifying the spacer). When first PCR did not yield enough amplified product to perform sequencing, the PCR product was run on a low melting agarose gel, allowing the first PCR product to be cut out and used as template for a second PCR. Amplification products were purified with QIAquick^TM PCR Purification Kit (QIAGEN GmbH, Hilden, Germany) or with Geneclean (BIO 101) according to the manufacturers' instructions. Sequencing was performed on an ABI Prism^TM 377 DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, California, USA) or on an ALFexpress^TM DNA sequencer (Pharmacia Biotech AB, Uppsala, Sweden). Sequencing reactions were done with all four primers (same as for PCR) using the dRhodamine Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit with AmpliTaq DNA polymerase (Perkin-Elmer Applied Biosystems) when using the ABI 377 or Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit with 7-deaza-dGTP (Amersham Life Science, Little Chalfont, UK) when sequencing on the ALFexpress. Reactions to be run on the ABI were cleaned with Sephadex G-50 Fine DNA Grade (Pharmacia Biotech) before loading. Sequences were evaluated using the ABI Prism 377 Collection package (Perkin-Elmer Applied Biosystems) or AM v3.02/ALFwin software (Pharmacia Biotech AB) on the ABI and the ALFexpress, respectively. Editing of the sequences was performed with Sequencher (Gene Codes Corporation, Ann Arbor, Michigan, USA).

Alignment and gap coding
Initial alignment of a few dozen taxa was done with the assembling features included in the Sequencher program. When more taxa were added, numerous or long deletions in some sequences made computer-calculated alignment unwieldy, and the alignment method was then changed to manual editing. When all taxa had been added to the matrix, a preliminary parsimony analysis was performed using PAUP version 3.1.1 (Swofford, 1993 ). Taxa were then rearranged according to the result of the analysis, alignment was modified, and a new run was made. This set of actions was repeated until a stable alignment was believed to be found. Alignment and gap coding followed mainly the criteria listed in Andersson and Rova (1999) . An effort was made to identify repeated regions and arrange indels according to these. When a repeat in which base substitutions had occurred was identified, one of the copies was deleted from the matrix, and an appropriate IUPAC ambiguity code was inserted in the position where bases differed. As an attempt to avoid a capricious alignment, the alignment was evaluated and discussed by several persons before final analysis, and parts of the matrix where doubts existed were excluded. Dolichodelphys and Pseudomussaenda were only successfully sequenced with the "e" and "f" primers; thus, only the spacer region of these genera are included in the analysis. The Randia sequence was compiled from the intron sequence of R. spinifex and the spacer sequence of R. aristeguietae because of a large autapomorphic deletion in the spacer region of R. spinifex. In the sequence of Bikkia and one of the Arachnothryx leucophylla sequences, the intron and the spacer are sequenced from two different collections. The Fraxinus sequence was obtained as two separate sequences (intron and spacer) from GenBank. All sequences are deposited in GenBank, and the final matrix of aligned sequences with indel codings used in the analysis is available at http://ajbsupp.botany.org/(matrix). Data on origin of plant material, vouchers, and GenBank accession numbers are given at http://ajbsupp.botany.org/v89 (Appendix 1).

Cladistic procedures
In order to find a phylogenetic hypothesis, a jackknife search was performed using a test version of the program "Xac" (J. S. Farris, Swedish Museum of Natural History, Stockholm, personal communication); 1000 replicates were run, each of them with 20 random addition sequence replicates and branch swapping.

When jackknife support is discussed below, supports of 81–100% are considered "strong," those of 51–80% "weak," and those of 50% or less "nonsupport."

Morphology
The material available for the molecular analysis directed the selection of taxa for morphological studies. The material studied is listed at http://ajbsupp.botany.org/v89 (Appendix 1). Macromorphology was studied through direct observation of herbarium material, pickled specimens, and/or fresh material. Pollen was acetolyzed and studied with the aid of scanning electron microscopy (SEM), using the same methods as those described by Andersson (1993a) . Observations on the sculpturing of the inside of exotesta cells were made with the aid of SEM as described by Rova and Andersson (1995) . When material was unavailable, literature was consulted to complement the morphological observations.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The final matrix comprised 165 taxa and 1045 characters, of which 480 were informative. The number of coded informative insertions/deletions was 67. Trees and jackknife values resulting from the cladistic search are presented in Figs. 1–7 and discussed below.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Tree showing the main groups from the jackknife analysis of trnL-F sequence data. Numbers below branches indicate jackknife support. Major clades are shown as abbreviations: R denotes the Rubioideae clade; I1, I2, and I3 the three Ixoroideae (I) clades; and C1 to C4 and C5a–b the different Cinchonoideae (C) clades as discussed in the text

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Within Rubiaceae, three main clades (R, I, and C) are found in this analysis, just as in other molecular systematic studies (e.g., Bremer, Andreasen, and Olsson, 1995 ; Andersson and Rova, 1999 ; Bremer et al., 1999 ). These clades are usually compared to three of the four subfamilies presented by Robbrecht (1988, 1993) . The first clade (clade R in Fig. 1) corresponds to the subfamily Rubioideae, the second clade (I) to a recircumscribed and significantly widened "Ixoroideae," and the third clade (C) to the subfamily "Cinchonoideae" sensu stricto (s.s).

The subfamily Antirheoideae, as recognized by Robbrecht (1988) , has no support in the present analysis, nor in any other of the molecular analyses cited above. Most representatives of the tribe Guettardeae (including Antirhea) are found in the vicinity of Arachnothryx and its related genera (clade C5a). Further, Chiococceae and Catesbaeeae (as defined by Delprete, 1996a ) form a monophyletic group (clade C4). According to this study, Cephalanthus (Cephalantheae) is placed in the Naucleeae (clade C3), as also shown by Bremer and Jansen (1991) . Further, this study confirms that Retiniphyllum (Retiniphylleae) and Vanguerieae (represented by Vangueria, Keetia, and Psydrax) are nested within Ixoroideae (clade I2), as previously shown by Bremer, Andreasen, and Olsson (1995) and Andersson and Rova (1999) . Based on rbcL sequences, Bremer (1996b) placed Pentanisia (Knoxieae) in Rubioideae (clade R), and trnL-F data confirms this here.

The basal, and elusive, position of Luculia is consistent with the results of other authors (e.g., Bremer, Andreasen, and Olsson, 1995 ; Bremer et al., 1999). Although there is no doubt that the genus belongs within Rubiaceae, it can not be unambiguously assigned to any subfamily based on trnL and trnL-F data. According to these results, Luculia might better be treated in a subfamily of its own.

The Rubioideae clade (Fig. 2)
The internal structure of the Rubioideae clade (clade R; Fig. 2) is by and large in accordance with other recent and more densely sampled DNA phylogenies of the Rubioideae, such as those of Manen and Natali (1996) , Bremer and Thulin (1998) , Andersson and Rova (1999) , and Bremer and Manen (2000) . The sparse sampling of Rubioideae taxa in the present study, together with a relatively high DNA sequence mutation rate, made alignment of Rubioideae sequences a difficult task, and because of this difficulty, an insertion/deletion complex unique to the Rubioideae had to be removed from the data matrix before analysis.

View larger version (32K): [in this window] [in a new window]   Fig. 2. The Rubioideae clade (corresponds to clade R in Fig. 1 ). Numbers below branches indicate jackknife support

View larger version (31K): [in this window] [in a new window]   Fig. 3. The first Ixoroideae clade (corresponds to clade I1 in Fig. 1 ). Numbers below branches indicate jackknife support

Dolichodelphys was placed in Gardenieae by Robbrecht (1988, 1993) , but it is reported to have capsular fruits (Delprete, 1996a, 1999b ). Therefore, this genus is morphologically similar to the members of the I1 clade and Delprete (1996a, 1999b) accordingly included Dolichodelphys in his widely circumscribed Rondeletieae. There is a weak (50%) support in this study for a clade that comprises Capirona, Chimarrhis, Dolichodelphys, and Warszewiczia and a 61% support for an Alseis-Calycophyllum clade, but these two clades do not necessarily form a monophyletic group based on present data. Of all these genera, Alseis and Dolichodelphys are the only ones reported to lack calycophylls. It is worth noting that Alseis, Calycophyllum, Chimarrhis (Delprete, 1999b ), and Warszewiczia all have receptive stigmas protruding above corolla lobes before the anthers are mature, which supports protogyny in these genera. Protogyny is rare in the Rubiaceae and never before reported outside Anthospermeae (Robbrecht, 1988 , p. 120), so it is possible that this is a character that could be useful for the study of internal subdivisions in the I1 clade.

With further regard to calycophylls, Claßen-Bockhoff (1996) stated that they can also be found in Carphalea (shrubs/trees with two-seeded indehiscent fruits; Puff, 1988 ), Cruckshanksia (herbs/shrubs with few-seeded capsules; Taylor, 1996 ), Morinda (lianas or shrubs/trees with compound fruits composed of connate drupes; Johansson, 1994 ), Neurocalyx (subshrubs with many-seeded, dry, indehiscent fruits; Bremer, 1979 ), and Pentas (herbs/shrubs with many-seeded capsules; Verdcourt, 1976 ). All these genera have been shown to belong in Rubioideae (e.g., Andersson and Rova, 1999 ) and are not included in the present study. Claßen-Bockhoff (1996) also reported calycophylls in the Alberteae (Alberta and Nematostylis, shrubs/trees with 1–2-seeded, dry fruits; Puff, Robbrecht, and Randrianasolo, 1984 ), Jackieae (Jackiopsis, a tree with one-seeded nutlets; Ridsdale, 1979 ), the genera Mussaenda and Pseudomussaenda (discussed together with the I2 clade below), and the two genera Pentaloncha (of unknown tribal position; Robbrecht, 1993 ) and Cosmocalyx (of the tribe Hamelieae, with pterophylls that develop after anthesis; Delprete, 1998 ). Calycophylls are also present in Kerianthera preclara Kirkbr. of the Isertieae (Delprete, 1996b ). It should also be noted that calycophylls are occasionally present in many other genera of the Rubiaceae, such as Hamelia, Rondeletia, Pteridocalyx, and Sipanea (P. G. Delprete, personal observation). In assessing the importance of this character, it is also important to remember that there is little or no evidence that all calycophylls are strictly homologous.

Resolution within the I1 clade is generally very poor, and, apparently, variation in trnL and trnL-F data is not enough to give reasonable resolution. Only the clade formed by Chimarrhis glabriflora Ducke and Ch. hookeri K. Schum. have support >90%. Thus, trnL and trnL-F data manage to unite neither the two Bathysa species nor the Hippotideae genera (Hippotis, Sommera, and Pentagonia), although the latter have earlier (Rova and Andersson, 1995 ) been shown to form a morphologically distinct group.

The second Ixoroideae clade (Fig. 4)
The second Ixoroideae subclade (clade I2; Fig. 4) comprises mainly taxa of the Ixoroids in the strict, traditional sense of Robbrecht (1993) , i.e., Gardenieae (Gardenia, Duroia, Genipa, Randia, Tricalysia, Bertiera, and Didymosalpinx), Coffeeae (Coffea), Octotropideae (Paragenipa), and Pavetteae (Ixora, Pavetta, and Tarenna), together with a number of genera thought to belong to Isertieae (Mussaenda and Pseudomussaenda), Retiniphylleae (Retiniphyllum), Vanguerieae (Keetia, Vangueria, and Psydrax), and Rondeletieae (Greenea, Aleisanthia, Aleisanthiopsis, Wendlandia, and Augusta). The traditional Ixoroideae tribes do not form a monophyletic group. For instance, the Seychellois genus Paragenipa (Octotropideae) is found connected to Didymosalpinx as part of a basally unresolved Gardenieae-Coffeeae-Pavetteae clade. Furthermore, Posoqueria and Dolichodelphys, both included in the Gardenieae by Robbrecht (1993) , are not closely associated with other Gardenieae. The exclusion of Posoqueria from Gardenieae has also been confirmed by morphological data (Persson, 1996 ). The topology of the clade corroborates results from several other studies (see below). A detailed discussion of subfamily Ixoroideae based on other molecular data can be found in Andreasen and Bremer (1996) , Andreasen (1997) , and Andreasen, Baldwin, and Bremer (1999) .

View larger version (31K): [in this window] [in a new window]   Fig. 4. The second Ixoroideae clade (corresponds to clade I2 in Fig. 1 ). Numbers below branches indicate jackknife support

Retiniphyllum and Vanguerieae
Retiniphyllum (tribe Retiniphylleae) is found in an isolated position between the Mussaendeae and the rest of clade I2. It is a neotropical genus with secondary pollen presentation and contorted corolla aestivation, and thus it fits morphologically well into the Ixoroideae s.s. (Robbrecht, 1988 ).

Vangueria is basal in a clade comprising Vangueria, Keetia, and Psydrax, all members of Vanguerieae (Robbrecht, 1988, 1993 ). Although stylar pollen presentation is present in the Vanguerieae, as is also predicted from its position in clade I2, it should be noted that corolla aestivation in this tribe is valvate, as opposed to most other genera in the I2 clade (Robbrecht, 1988 ). This close relationship between Vanguerieae and Ixoroideae s.s. has also been reported from restriction site mapping (Bremer and Jansen, 1991 ) and DNA sequence studies (e.g., Bremer, Andreasen, and Olsson, 1995 ; Andreasen, 1997 ).

Aleisanthia, Aleisanthiopsis, Greenea, and Ixora
Aleisanthia and Aleisanthiopsis are found together with Ixora in a clade also including Greenea, the four genera thus forming a sister clade to the Vanguerieae clade. This study supports the conclusions made by Tange (1996b) that Greenea distantiflora Merrill should be removed from Greenea and placed closer to Aleisanthia. Consequently, Tange also transferred G. distantiflora to the new genus Aleisanthiopsis at the same time as he suggested that this new genus might be the sister genus to Aleisanthia. All three genera (Aleisanthia, Aleisanthiopsis, and Greenea) are part of the Indo-Malayan flora (Mabberley, 1993 ; Tange, 1996a, b ), while Ixora (if monophyletic) is a pantropical genus (Bridson and Robbrecht, 1985 ).

Aleisanthia, Aleisanthiopsis (as included in Greenea), and Greenea have traditionally been regarded as Rondeletieae due to their many-seeded, capsular fruits, in spite of their contorted corolla aestivation (Robbrecht, 1988, 1993 ; Tange, 1996b ). The latter character, however, has been considered a rare feature in the Rondeletieae, and it has mostly been used taxonomically to separate these genera from the "valvate, rarely imbricate" Condamineeae (Robbrecht, 1988 ). As mentioned above, of all taxa found in the I2 clade, most are reported to have contorted corolla aestivation. The only exceptions to this are the valvate genera Mussaenda, Pseudomussaenda, Vangueria, and Psydrax. This indicates that valvate corolla aestivation might be plesiomorphic at least in the I2 clade and possibly, because valvate corolla aestivation is the prevailing state in Rubioideae, also in the Rubiaceae as a whole. As will be further discussed below, imbricate corolla aestivation was also found to be predominant among taxa associated with the genus Rondeletia.

Aleisanthia, Aleisanthiopsis, and Ixora all have secondary pollen presentation, in which pollen is deposited onto the outside of the stigmatic lobes before they open and the stigmatic surface becomes receptive (Smith and Darwin, 1988 ; Tange, 1996a, b ; De Block, 1998 ). In Greenea, on the other hand, secondary pollen presentation is lacking (Tange, 1996b ). The fruit in Ixora is a slightly fleshy or coriaceous drupe, usually containing one (by abortion of one ovule) or two thin-walled pyrenes (Bridson, 1988 ; De Block, 1998 ), while both Aleisanthia and Aleisanthiopsis have dry, many-seeded capsules (Tange, 1996a, b ).

Wendlandia and Augusta
Wendlandia and Augusta (sensu Kirkbride, 1997 ) have traditionally been placed in the Rondeletieae (see http://ajbsupp.botany.org/v89 [Appendix 2]), but in this study they form a strongly supported sister group to the Gardenieae-Pavetteae-Coffeeae-Octotropideae clade, i.e., they are well nested among the classical Ixoroideae Rubiaceae. Wendlandia is a genus of >50 species ranging from Southeast Asia to northeastern Africa (Cowan, 1932 ; Robbrecht, 1988 ; Tange, 1996b ), while Augusta (including Lindenia) is a genus of four species of rheophytic shrubs growing in Brazil, Central America, the Fiji Islands, and New Caledonia (Delprete, 1997b ; Kirkbride, 1997 ). In the present study, Lindenia (as Augusta rivalis) and Augusta are found together. This is in accordance with results from morphological data by Kirkbride (1997) and Delprete (1997b) and supports their common view to merge Lindenia into Augusta. Wendlandia and Augusta (including Lindenia) differ in corolla size (significantly larger in Augusta [Cowan, 1932 ; Delprete, 1997b ]), capsule dehiscence (septicidal in Augusta vs. nearly always loculicidal in Wendlandia [Cowan, 1932 ; Darwin, 1976 ]), and seed characteristics (Wendlandia "obscurely winged" vs. nonalate with smooth testa in Augusta [Cowan, 1932 ; Darwin, 1976 ]), but both genera share fruit type (capsule), exserted stigma (Cowan, 1932 ; Darwin, 1976 ), and maybe most interestingly, corolla lobes contorted in bud (Schumann, 1891 ; Darwin, 1976 ; J. H. E. Rova, personal observation), a character not compatible with the taxa of the Rondeletia group as proposed in the present work.

Bertiera
The taxonomic position of the genus Bertiera has long been the cause of debate among Rubiaceae systematists (see Robbrecht, Rohrhofer, and Puff, 1994 , for an historical discussion). In recent years, molecular phylogenies have all placed Bertiera in the Ixoroideae tribe Coffeeae (Andreasen and Bremer, 1996 ; Persson, 1998 ). Although this study is not detailed enough to use for definite tribal affiliations in this part of the family, it clearly shows that Bertiera belongs near to Coffeeae and Pavetteae s.s.

Ixoroideae in conclusion
In conclusion, the I2-clade of this study is in general concordant with the results of Andreasen and Bremer (1996) , Andreasen (1997) , and Andersson and Rova (1999) . Andreasen (1997) , in her combined morphological and molecular analyses of the Ixoroideae, confirmed that the Pavetteae sensu Robbrecht is paraphyletic and should be divided into Ixoreae and Pavetteae s.s. However, none of the traditional Rondeletieae genera (Greenea, Aleisanthia, Aleisanthiopsis, Wendlandia, and Augusta), which in this study are shown to belong to the Ixoroideae, were included in the studies cited above. In order to clarify the tribal affiliations of these genera, an all-embracing phylogenetic study of the Ixoroideae s.s. should be undertaken.

Sipaneeae (Fig. 5)
The first of the I3 subclades, clade I3a in Fig. 5, corresponds to the tribe Sipaneeae (the genera Sipanea and Neobertiera) supplemented by Maguireothamnus and Sipaneopsis, both placed in Rondeletieae by Robbrecht (1993) . Limnosipanea (also Sipaneeae) was not successfully sequenced and thus was not included in the study. Sipaneopsis is a genus of shrubs occurring in the Magdalena-Llanos area of Colombia, Venezuela, and the northern Amazon basin, while Neobertiera consists of a single species of shrubs restricted to the Guyana shield (Andersson, 1992 ). Neobertiera and Sipaneopsis form a clade with strong support, and based on general morphology, they may well be congeneric (J. H. E. Rova, personal observation). Maguireothamnus, a shrub of the tepuis of Venezuela (Steyermark, 1964 ), has thick leaves and a large corolla with a tube length of 10 cm, and thus it is quite different from the small-flowered Sipanea. On the other hand, a number of similarities are found in pollen and exotesta morphology among Sipanea, Maguireothamnus, and Neobertiera (Sipaneopsis was not studied). First, all have an exotesta with sculptured inner tangential and radial walls, the sculpturing in form of more (Limnosipanea, Sipanea, and Maguireothamnus) or less (Neobertiera) prominent warts (J. H. E. Rova, personal observation). Second, pollen is foveolate (sensu Walker and Doyle, 1975 ) at least in some species of Sipanea, Limnosipanea, Maguireothamnus, and Neobertiera (Sipaneopsis not studied), with pits 0.1–0.3 µm in diameter, although the density of tectal pits varies from as many as 4–9 per µm² (in Limnosipanea, Sipanea, and Neobertiera) down to 0.7 per µm² in Maguireothamnus (J. H. E. Rova, personal observation). Third, aestivation is contorted in all genera of the I3a clade except in Sipaneopsis, where both imbricate and contorted aestivation was found (J. H. E. Rova, personal observation).

View larger version (22K): [in this window] [in a new window]   Fig. 5. The third Ixoroideae clade (corresponds to clade I3 in Fig. 1 ). Numbers below branches indicate jackknife support. I3a and I3b are names of subclades within the I3 clade, as discussed in the text

Both Posoqueria and Molopanthera share a pollen presentation mechanism, apparently unique in the Rubiaceae, in which pollen of a flower is formed into a ball that is hurled onto the visiting insect by one of the stamens (Müller, 1866 ; Schumann, 1891 ; Hallé, 1967 ; Robbrecht, 1988 ). The mechanism has been verified in Posoqueria in the field (Beach, 1983 ; P. G. Delprete, personal observation), and the similarity to the mechanism in Molopanthera has also been confirmed (P. G. Delprete, personal observation). One plausible explanation of the difference in corolla size (up to 100 times) between Posoqueria and Molopanthera could be a differentiation in pollinator specialization towards hawk-moths in Posoqueria and bees in Molopanthera (P. G. Delprete, personal observation) after the evolution of the staminal pollen presentation complex. Fruit size and morphology have also evolved differently in the two genera, with Posoqueria having fairly large leathery berries of Gardenia type and Molopanthera possessing minute loculicidal capsules.

Posoqueria has imbricate corolla aestivation, while other members of the Gardenieae have contorted aestivation, and the placement of Posoqueria in the Gardenieae has also lately been questioned (Persson, 1996 ; Andreasen, 1997 , and references therein). Both Posoqueria (Persson, 1996 ) and the Henriquezieae, with the exception of Gleasonia (Rogers, 1984 ), have imbricate corollas, and therefore the systematic position of Posoqueria as indicated in this study is more plausible than the traditional view, that it should belong in the Gardenieae (Robbrecht, 1988, 1993 ). By considering Gleasonia aberrant and assuming that Rogers (1984) was correct when he stated the close relationship between Gleasonia, Henriquezia, and Platycarpum, the I3b clade can thus in general be described as having imbricate, zygomorphic corollas, and comprising the tribe Henriquezieae and a tentative new tribe comprising the genera Posoqueria and Molopanthera.

The Cinchonoideae clade
The six subclades of clade C (C1 to C5 and Isertia in Fig. 1) correspond to the subfamily Cinchonoideae in a more strict sense than that of Robbrecht (1988, 1993) but includes also representatives from several tribes that Robbrecht placed in other subfamilies: Hamelieae (Rubioideae), Cephalantheae, Guettardeae, Chiococceae (all three Antirheoideae), and Catesbaeeae (of uncertain position).

The first Cinchonoideae subclade, clade C1, comprises Ladenbergia and Cinchona. Jackknife support for this clade is strong, and the clade corresponds to the tribe Cinchoneae in the restricted sense of Andersson (1995) .

Clade C2 comprises Hamelia, Hoffmannia, Cosmibuena, and Chione. The former two clades have been placed in tribe Hamelieae of subfamily Rubioideae, while Cosmibuena is a Hillieae representative, and Chione is of uncertain tribal position (Robbrecht, 1988, 1993 ). A close relationship between Hamelieae and Hillieae has been proposed based on other chloroplast sequence data sets (Bremer, Andreasen, and Olsson, 1995 ; Andersson and Rova, 1999 ), although morphological analyses of comparable sample size (Rova and Andersson, 1995 ; Andersson, 1996 ) do not suggest that they may be closely related.

Isertieae
One of the Cinchonoideae clades is formed by two Isertia species. Of the traditional Isertieae (sensu Garcia Kirkbride, 1979 ) included in this study (Amphidasya, Gonzalagunia, Isertia, Mussaenda, Mycetia, and Pseudomussaenda), none is found to be associated with Isertia; instead, these genera are distributed among the clades R, I2, and C5a (Fig. 1). This study thus definitely supports the conclusions drawn by Andersson (1996) and Bremer and Thulin (1998) , that Isertieae as traditionally circumscribed is polyphyletic.

Naucleeae (Fig. 1)
The third Cinchonoideae clade, C3, includes the genera Cephalanthus, Sarcocephalus, and Uncaria. All these three genera are characterized by globose pseudanthia. Cephalanthus has historically been placed in various tribes with different circumscriptions (Ridsdale, 1976 ). A morphological comparison between Cephalanthus and Naucleeae in a strict sense was performed by Ridsdale (1976) . In this work, he excluded Mitragyna and Uncaria from Naucleeae because of their type of fruit dehiscence and their pendulous, multi-ovulate placentas. He also stated that a pendulous, solitary ovule is a character common to Cephalanthus and Naucleeae s.s. On the other hand, Ridsdale (1976) reported Cephalanthus, Uncaria, and Mitragyna to have a common phytochemistry and growth form. Robbrecht (1993) treated Cephalanthus in the monogeneric tribe Cephalantheae, Sarcocephalus as a member of Naucleeae, and Uncaria as a member of the Cinchonoideae tribe Coptosapeltae. In the latter case, he followed morphological cladistic results of Andersson and Persson (1991) . Results from the present study contradict both Ridsdale and Robbrecht and are instead consistent with the phylogenetic implications of Bremer, Andreasen, and Olsson (1995) , based on rbcL sequence data, in which all three genera are considered to belong in the Naucleeae. A close relation between Cephalanthus and Naucleeae based on cytological data is also proposed by Kiehn (1995) , who reports that the type of interphase nuclei is similar in Cephalanthus and Naucleeae.

Strumpfia
The Antillean monotypic genus Strumpfia has been known for a long time as an elusive member of the Rubiaceae (http://ajbsupp.botany.org/v89 [Appendix 2]). Albeit carefully studied (Igersheim, 1993 ), morphology has failed to indicate its proper affinities to other rubiaceous taxa, since Strumpfia possesses a combination of plesiomorphic characters and a number of more or less unique morphological features, such as protogyny, a "true" anther tube, and plurilocular pyrenes. Igersheim (1993) thus raised the question of whether putting Strumpfia in a tribe of its own was justifiable. Based on molecular data, Bremer et al. (1999) found Strumpfia to be "close to the Chiococceae s.l.," but the only representatives from that clade that were included in their study were Exostema, Chiococca, and Erithalis. However, the present study confirms that Strumpfia holds an isolated position in the Cinchonoideae, and seemingly should be treated as sister to the C4 clade, although jackknife support for this is weak. Morphological evidence for this affiliation is moreover unclear; whereas members of the C4 clade, e.g., typically have echinate perforate pollen, Strumpfia pollen is verrucose (Igersheim, 1993 ).

Catesbaeeae, Chiococceae, and the "Exostema-group" (Fig. 6)
Clade C4 (Fig. 6) comprises the genera Asemnantha, Badusa, Bikkia, Catesbaea, Ceratopyxis, Chiococca, Coutarea, Cubanola, Erithalis, Exostema, Hintonia, Isidorea, Phialanthus, Phyllacanthus, Portlandia, Schmidtottia, Scolosanthus, and Siemensia. These genera are characterized by imbricate corolla aestivation and echinate-perforate pollen (Delprete, 1996a ; Huysmans et al., 1999; J. H. E. Rova, personal observation). The C4 clade thus corresponds to the tribes Catesbaeeae, Chiococceae, and the "Exostema group" as circumscribed by Delprete (1996a) , although this analysis does not support Delprete's separation of the genera into three groups. However, Siemensia, Asemnantha, Phialanthus, and Ceratopyxis were not included in Delprete's analysis, while Nernstia, Thogsennia, Ceuthocarpus, Morierina, and Placocarpa were not available for the present study. The recognition of any of Delprete's groups with their present delimitations would make the other two groups polyphyletic. Instead, and especially if the "Exostema group" of Delprete is treated as part of his Catesbaeeae, the C4 group of the present study is closer to the Chiococceae as broadly delimited by Bremer (1992) (but see below concerning Phialanthus). Later, Bremer also suggested an inclusion of Catesbaea in the Chiococceae (Bremer, Andreasen, and Olsson, 1995 ), and this is also concordant with the present study. Kiehn (1995) noted that Portlandia and Cubanola could be closely related based on their similarities in chromosome morphology, while Chiococca differs in chromosome structure and is more similar to Exostema. The latter suggestion was based on chromosome appearance and shared unusual basic numbers, x = 13 or 14 (although x = 11 and x = 12 were also reported from Chiococca and Exostema, respectively). An affinity between Portlandia and Cubanola is supported by the present study, but the close relationship between Exostema and Chiococca proposed by Kiehn (1995) was not confirmed by trnL-F sequence data.

View larger version (38K): [in this window] [in a new window]   Fig. 6. The fourth Cinchonoideae clade (corresponds to clade C4 in Fig. 1 ). Numbers below branches indicate jackknife support

Three of the taxa in the C4 group were treated as "incertae sedis" by Robbrecht (1993) , i.e., Hintonia, Phialanthus, and Schmidtottia. In Bremer, Andreasen, and Olsson (1995) , the Mesoamerican genus Hintonia was found at a tricotomy between the Ixoroideae s.l. (sensu lato) and the Cinchonoideae s.s. The present study, as well as the studies of Delprete (1996a) , Bremer (1996a) , and Ochoterena-Booth (2000) , indicate, however, that Hintonia is part of the Catesbaeeae-Chiococceae complex. Bremer's (1992) exclusion of the almost entirely West Indian genus Phialanthus from the Chiococceae is not supported by this study nor by Delprete's (1996a) ; if Chiococceae is defined as broadly as by Bremer (1992) , Phialanthus should definitely be included in this tribe.

Two genera are placed very differently in Delprete's (1996a) and in the present analyses: Chione and Allenanthus. In Delprete's study, they form a clade that is part of the Chiococceae, but in the present study, Chione is found to be basal in the C2 clade together with Cosmibuena, Hamelia, and Hoffmannia, while Allenanthus is part of the C5a clade together with Neoblakea and Machaonia. Delprete (1996a) reported that both Chione and Allenanthus have "echinate" pollen, a synapomorphy of the Chiococceae and Catesbaeeae (both sensu Delprete), and one pendulous ovule per locule that develop into a laterally flattened seed. The latter characters were synapomorphies of the entire Chiococceae in Delprete's study, and they helped to place Chione and Allenanthus within that clade, but a single pendulous ovule is also characteristic of, e.g., Guettardeae (Robbrecht, 1988 , p. 147), which might support the position of Allenanthus in the vicinity of Guettarda in the C5a clade of this study. During the present study, Chione sylvicola was found not to have echinate pollen. A closer study of pollen is needed in order to investigate homologies in pollen morphology between Allenanthus, Chione, the members of the Hoffmannia-Hamelia-Hillia taxon group, and other taxa reported to have "echinate" pollen. Although pollen of Hillia and Cosmibuena is not echinate, at least pollen of Hoffmannia gesnerioides is so, but the spinules of this species are much smaller than those found in the C4 clade (J. H. E. Rova, personal observation).

Guettardeae and Rondeletieae s.s. (Fig. 7)
Clade C5 has only weak jackknife support and includes mainly representatives of the tribes Rondeletieae and Guettardeae. A number of genera that until recently have had a more or less doubtful position are also found in this clade, such as Gonzalagunia, Mazaea (syn. Ariadne and Neomazaea; Delprete, 1999a ), Neoblakea, Phyllomelia, Rachicallis, and Suberanthus.

View larger version (35K): [in this window] [in a new window]   Fig. 7. The Cinchonoideae clades C5a and C5b from Fig. 1 . Numbers below branches indicate jackknife support

Circumscription of Rondeletia
Rondeletia in a broad sense forms an important part of the C5 clade. Circumscription of this genus has been debated for a long time. The main issues in dispute have been whether Arachnothryx, Javorkaea, and Rogiera should be treated as separate genera or just as subgenera of Rondeletia, largely following the "species groups" of Standley (1918) . If treated in its broadest sense, Rondeletia is a genus with >250 species distributed over central and northwestern South America, Mexico, and the Antilles (Andersson, 1992 ). Steyermark (1967) listed a number of diagnostic characters to separate South American Arachnothryx from Rondeletia. Kirkbride (1968) and Lorence (1991) found these characters less useful in Central America. Lorence (1991) argued that Arachnothryx, Javorkaea, Rogiera, and Rondeletia are best treated as subgroups of a broadly defined genus Rondeletia, probably at subgeneric level, because of the minute morphological differences between them. Delprete (1999b) included all these taxa into a Rondeletia complex without inferring any taxonomic significance of the group. Steyermark's treatment of Rondeletia and Arachnothryx as separate genera has been insisted on and further developed by Borhidi and collaborators who, in a number of papers, have defended a segregation of Arachnothryx, Javorkaea, Rogiera, Roigella, and Suberanthus from Rondeletia in "the original Linnean sense" (Borhidi and Fernández, 1981a, b ; Borhidi, 1982, 1989, 1993–1994 ; Borhidi and Járai-Komlódi, 1983 ; Fernández, 1993–1994 ). In these studies, Arachnothryx was characterized by four-merous flowers, an externally arachnoid tomentose or pubescent corolla tube, naked annular disc, and a glabrous corolla throat without faucial ring, i.e., an annular thickening below corolla lobe bases (Borhidi, 1982 ). Javorkaea was distinguished by having a broad, connate stipular ring, a glabrous corolla throat without faucial ring, and corolla lobes pubescent at base inside (Borhidi and Járai-Komlódi, 1983 ). Rogiera was separated from Rondeletia by its five-merous flowers with a bearded corolla throat (Borhidi, 1982 ), and Roigella by a combination of large five- to six-merous flowers, a different placentation, and septicidal capsules (Borhidi and Fernández, 1981a ). Suberanthus was recognized by its septicidal capsules, reticulate pollen, and a coriaceous corolla with thickened, angustate throat (Borhidi and Fernández, 1981b ). Rondeletia would thus be a genus almost entirely restricted to the Antilles, and characterized by pubescent annular disc, four- to five-merous flowers, loculicidal capsules, and glabrous corolla throat with a more or less distinct annular thickening (Fernández, 1993–1994 ).

While Steyermark worked on the generic complex in a predominantly South American context, Kirkbride and Lorence focused on the Mexican and Central American taxa, and Borhidi with collaborators studied Rondeletia (s.l.) mostly from a Cuban/Antillean point of view. Possibly, these different ways to approach Rondeletian taxonomy have led to the different ways of handling the generic delimitations of this complex.

In the present study, taxa have been named according to Borhidi's (and collaborators) narrow circumscription of Rondeletia and its segregates, since his view was supported by the phylogenetic analyses. For a complete discussion of the history and taxonomy of the species complex, see Borhidi (1982) , Lorence (1991) , Fernández (1993–1994) , Delprete (1999b) , and references therein.

Arachnothryx, Gonzalagunia, and Javorkaea (Fig. 7)
The Arachnothryx-Gonzalagunia-Javorkaea clade (part of clade C5a in Fig. 7) has a very strong support in all analyses, although it is internally not well resolved. Javorkaea is a monotypic Honduran genus characterized by basally connate stipules, five- to six-merous, slightly zygomorphic flowers, a glabrous corolla throat without annular thickening, and terminal, racemose-cymose inflorescences (Borhidi and Járai-Komlódi, 1983 ). Inflorescences are also terminal in Gonzalagunia and Arachnothryx. In Gonzalagunia, as well as in a number of Arachnothryx species, inflorescences are elongated, spike-like or thyrsiform, but in Arachnothryx they may also be shortly paniculate. In both genera, flowers are normally four-merous and always without faucial ring. If only representatives with elongated inflorescences are considered, the two genera are very similar, and it is sometimes extremely difficult to tell them apart if no fruits are present. While Arachnothryx has septicidal capsules, Gonzalagunia is characterized by spongy, berrylike fruits. However, this may be a doubtful generic character since fruit dimorphism (where both spongy and dry fruits are mixed in the same infrutescence) is reported in Gonzalagunia (Ståhl, 1999 ), and its dry fruits often resemble unripe capsules of Arachnothryx. Because of these overall morphological similarities between the two genera, it is not surprising to find them close together in the cladogram. Bremer and Thulin (1998) noted that rbcL data also place Gonzalagunia near Rondeletia s.l. and Guettarda. The affinity between Arachnothryx and Gonzalagunia is also supported by karyological data: Kiehn (1995) reports that both Arachnothryx and Gonzalagunia are tetraploid with a basic number of x = 9, an unusual, and most probably apomorphic, basic number in the Rubiaceae.

Javorkaea, Arachnothryx, Rogiera, and Gonzalagunia all have three-colporate pollen (as do most genera of the Rubiaceae) with a foveolate, smooth surface (Borhidi and Járai-Komlódi, 1983 ; Andersson, 1996 ; J. H. E. Rova, personal observation). Exotesta cells of Arachnothryx and Gonzalagunia have a secondarily thickened inner tangential wall perforated by large pores. In the studied representatives of Arachnothryx, secondary thickening has an even surface, in contrast to the investigated Gonzalagunia specimens where secondary thickenings are covered with distinct knobs. Exotesta cells of Rogiera cordata have netlike secondary thickenings in the inner tangential walls covered with knobs, in the same manner as in Gonzalagunia.

Guettardeae (Fig. 7)
The present study is the first molecular analysis to include a large number of representatives from both Rondeletia s.l. and the tribe Guettardeae. In this study, the members of the Guettardeae are found in two subclades nested within the C5a clade (Fig. 7). The first comprises Guettarda, Chomelia, Timonius, and Antirhea, and the other comprises Machaonia and Neoblakea. The Guettardeae thus appears to be paraphyletic with regard to Rogiera, Javorkaea, Arachnothryx, and Gonzalagunia. Guettardeae as a whole is pantropical, although almost absent from Africa (Robbrecht, 1988 ). Genera of the Guettardeae are characterized by two- to multi-locular ovaries, single, apically attached ovules, more or less fleshy fruits, and the possession of stylar pollen presentation; inflorescences may be terminal or axillary, and corolla aestivation is reported to be valvate or imbricate (Robbrecht, 1988 ). The uniovulate loculi of the Guettardeae are unique in the C5a clade, but not to the C5 clade as a whole, where one-seeded loculi are also reported from Phyllomelia and Mazaea (Delprete, 1999a ). A phylogenetic affinity between Guettardeae and Rondeletieae has been suggested also by earlier authors, e.g., Bremer, Andreasen, and Olsson (1995) , in whose studies rbcL sequence data resulted in a clade comprising Rogiera, Rachicallis, Guettarda, and Antirhea.

Guettarda forms a strongly supported clade together with Timonius, Antirhea, and Chomelia. The result shows that Guettarda is paraphyletic because Chomelia tenuiflora Benth. forms a clade together with Guettarda crispiflora Vahl. Guettarda is a genus of neotropical and pacific distribution, with 80 species. The highest species diversity is found in the Antilles but one species, Guettarda speciosa L., is found throughout the Pacific and as far west as the Seychelles and the coasts of East Africa in the Indian Ocean (Darwin, 1979 ). The representatives of Guettarda, Timonius, Antirhea, and Chomelia included in this analysis have axillary inflorescences, cupular calyces, and imbricate corolla lobes. Flower merosity ranges between four and seven, and no faucial ring was found in any of the specimens studied. Features of the internal indumentum in Timonius are discussed below in connection with Rogiera.

Neoblakea and Machaonia, both Guettardeae, together with Allenanthus, form a strongly supported but internally unresolved subclade at the base of the C5a clade. Bremer (1992) excluded Allenanthus from the Chiococceae, without suggesting any other position, and Robbrecht (1993) listed the genus as "incertae sedis." Allenanthus has dry fruits bordered by a broad wing, this samaroid unit being an adaptation to wind dispersal. Allenanthus comprises three species restricted to Central America, while Neoblakea is a monotypic Antillean genus, and Machaonia is a genus of 25 species occurring in Mexico and central and northern South America (Andersson, 1992 ). In contrast to the mainly axillary inflorescences of the Guettarda clade, Neoblakea, Machaonia, and Allenanthus are reported to have primarily terminal inflorescences (Liogier, 1963 ; Steyermark, 1974 ; Andersson, 1993b ; J. H. E. Rova, personal observation).

The colporate and foveolate pollen of Arachnothryx, Rogiera, and Gonzalagunia contrasts with the pollen of the Guettardeae, which is more or less uniformly porate (occasionally colporate) and semitectate-perforate (tectate-imperforate in Guettarda speciosa), with relatively long collumellae (Darwin, 1979, 1993, 1994 ; J. H. E. Rova, personal observation), although pollen data are lacking from Neoblakea, Machaonia, and Allenanthus.

Rogiera
The two Rogiera species in clade C5a (R. amoena Planch. and R. cordata (Benth.) Planch.) form a strongly supported subclade (Fig. 7). Rogiera is characterized by large foliaceous stipules and a densely yellow-bearded corolla throat (Borhidi, 1982 ). The results from the present study show Rogiera as polyphyletic, while R. amoena (the type of Rogiera) and R. cordata are found in the C5a clade, and R. suffrutescens (Brandeg.) Borhidi is found as a basal member of the C5b clade. Rogiera suffrutescens differs from the other studied species in stipule morphology (small, narrow ascending stipules in R. suffrutescens vs. larger, broader, and reflexed stipules in R. amoena and R. cordata) and external pubescens of corolla (almost glabrous vs. pubescent).

The genera related to Rondeletia s.s. (Fig. 7)
The C5b clade is composed of the genera Acrosynanthus, Blepharidium, Mazaea, Phyllomelia, Rachicallis, Roigella, Rondeletia s.s., and Suberanthus. With the exception of Blepharidium and Acrosynanthus, corolla throats were consistently glabrous in the examined representatives of this clade. Capsules in this group are generally loculicidal and inflorescences axillary, but there are exceptions in both of these characters. Seeds in members of this clade are angular and wingless or winged with concentric or bipolar wings.

Blepharidium, Rogiera, and Suberanthus
Blepharidium is a genus of two species occurring in Mexico and Guatemala. It was included in Cinchoneae by Robbrecht (1988) and later was transferred to Hillieae by Andersson (1995) , although Andersson noted that "it differs from other members of the tribe in the absence of raphides, in having clasping stipules, corolla with imbricate aestivation, and anthers dorsifixed around the middle." No other taxon from the C5 clade of the present study was included in Andersson's analysis.

The genus Suberanthus was separated from Rondeletia by Borhidi and Fernández (1981b) because of its glabrous annular disc, oblong-pyriform ovary, and roughly reticulate pollen grains. The genus corresponds more or less to the Rondeletia section "Stellatae" of Standley (1918) . Based on several morphological characters (e.g., ascending placenta, winged seeds, and a blackish-purple corolla), Borhidi and Fernández transferred Suberanthus from Rondeletieae to Cinchoneae, even though corolla morphology suggests an affinity to Rondeletia because of its Rondeletia-like annular thickening in the corolla throat. In congruence with the conclusions drawn by Borhidi and Fernández, Suberanthus was tentatively treated as member of the Cinchoneae by Robbrecht (1988, 1993) . According to the results of the present analysis, corolla morphology is more important than Borhidi and Fernández anticipated, as Suberanthus is found to be one of the sister clades to Rondeletia s.s. Suberanthus consist of about half a dozen species restricted to Cuba and Hispañola (Borhidi and Fernández, 1981b ), and the two representatives included in this study form a clade with a very strong jackknife support.

As discussed above in connection to the C5a clade, this study shows Rogiera to be a polyphyletic taxon, recognized morphologically by a densely yellow-barbate corolla throat. Since Rogiera amoena (found in clade C5a) is the type of Rogiera, R. suffrutescens obviously has to be transferred to another genus. It should, however, not be included in Rondeletia s.s. according to the results of the present study, as this would make Rondeletia s.s. paraphyletic.

Mazaea, Phyllomelia, Rachicallis, and Roigella
Roigella was separated from Rondeletia by Borhidi and Fernández (1981a) based on ovary, calyx, and pollen characters. However, already Standley (1918) considered Roigella (as Rondeletia correifolia Griseb.) unique in Rondeletia, which he showed by making it the sole member of his section "Correifoliae." According to the present results, trnL-F data also support the isolated position of Roigella in the close vicinity of Rondeletia s.s.

The Cuban endemic genera Phyllomelia and Mazaea (the latter often referred to Ariadne or Neomazaea) form a monophyletic, although internally unresolved, part of the C5b clade. Jackknife support is very strong for this clade, and the results of the current study are consistent with the conclusions drawn from morphological characters by Delprete (1999a) , that Ariadne shaferi (Standl.) Urb. and Mazaea phialanthoides should be referred to the same genus, Mazaea, and that Phyllomelia is closely related to this genus. However, pollen morphology differs between the two Mazaea species: while pollen of M. phialanthoides is reticulate-foveolate without external ornamentation, pollen of M. shaferi is more foveolate and covered with minute verrucae (J. H. E. Rova, personal observation). For a long time, both Ariadne and Phyllomelia have been treated as of uncertain position in the Rubiaceae (Robbrecht, 1988, 1993 ). An interesting feature of the latter is the possession of peculiar pterophyllous calycophylls and indehiscent, two-seeded fruits (Delprete, 1999a ).

Rachicallis, a monotypic genus present in Mexico and the Caribbean, was originally included in the Rondeletieae by Hooker (1873) , followed by Schumann (1891) , and later implicitly by Bremekamp (1966) (see also http://ajbsupp.botany.org/[Appendix 2]). Robbrecht (1988) transferred, for unclear reasons, Rachicallis to Hedyotideae but later (1993) agreed that this was perhaps dubious. Based on rbcL data, Bremer, Andreasen, and Olsson (1995) found that Rachicallis should be placed near to Rogiera and Guettarda, and a similar result was also found based on rps16 sequence data (Andersson and Rova, 1999 ). The present study strongly confirms the inclusion of Rachicallis in Rondeletieae and tentatively places Rachicallis as sister taxon to Phyllomelia and Mazaea. However, an inclusion of Rachicallis in the same clade as Mazaea and Phyllomelia has no jackknife support.

The Rondeletia s.s. clade (Fig. 7)
The largest of the C5b subclades comprises Rondeletia alaternoides A. Rich., R. inermis (Spreng.) Kr. & Urb., R. intermixta Britt., R. odorata Jacq., R. pilosa Sw., R. portoricensis Kr. & Urb., and an unidentified Rondeletia sp. from Cuba. According to Fernández (1993–1994) , Rondeletia portoricensis is supposed to be closely related to R. americana L., the type of Rondeletia, and this clade may thus be named Rondeletia s.s. All these species possess a swollen faucial ring (the unidentified species not studied) and fit well into the description of Rondeletia in the strict sense of Borhidi and Fernández (1981a) . All species in this clade except R. odorata (see below) are entirely Cuban/Puerto Rican, which supports the view of Rondeletia s.s. as an Antillean genus (Fernández, 1993–1994 ; A. Borhidi, Janos Pannonius University, Pécs, personal communication). Pollen in this group is surprisingly diverse, and the present study cannot confirm the picture of uniformity in pollen morphology put forward by Fernández (1993–1994 , p. 67). During the present study, R. alaternoides was found to have tectate-rugulate pollen covered with minute warts, while R. inermis has reticulate to almost semitectate pollen without supratectal sculpturing, and R. odorata has "typical Rondeletia" tectate foveolate pollen with a smooth surface.

While Gonzalagunia and Arachnothryx are generally heterostylous (Lorence, 1991 ; Ståhl, 1999 ), no heterostyly that could indicate pollen dimorphism has been found in Rondeletia. However, this could be an artifact from poor sampling, and it is thus still an open question if further studies of pollen morphology within Rondeletia s.s. might give more characters for determination of infrageneric relationships. Exotesta morphology within Rondeletieae also seems to be variable. In this study, secondarily thickened inner tangential walls of R. inermis were found to be covered with large pores, while thickenings in R. odorata were in the form of netlike bands. Sample density in the present study is unfortunately too low to test the Rondeletia sections proposed by Fernández (1993–1994) .

Biogeographical correlation (Fig. 8)
While C5a is a predominantly Central American clade, C5b is predominantly Antillean (Fig. 8). Separation of the "mainland" genera Rogiera, Javorkaea, and Arachnothryx from the "Antillean" Rondeletia s.s. is thus both phylogenetically supported and geographically correlated. In clade C5a, a number of species with a wide American distribution (Andersson, 1992 ) are found, such as Rogiera amoena (Central and Northern South America), Gonzalagunia dicocca (widespread in tropical South America), Guettarda crispiflora (northwestern South America and the Antilles), Chomelia tenuiflora (northwestern South America), and Guettarda odorata (the Antilles and the Caribbean coast of Central and South America). The Guettardeae are also widespread in the Pacific and Southeast Asian tropics. On the other hand, the northern Central American species Rogiera suffrutescens and Blepharidium guatemalense Standl. are the only taxa included in clade C5b that are endemic to a region other than the Antilles. Rachicallis is found in the Antilles and in extratropical North America, and Rondeletia odorata in Cuba and southern Central America, but the existence of the latter on the mainland is probably due to human dispersal, since the species is a widely cultivated ornamental (Lorence, 1991 ). When biogeography is discussed based on the results from the present study, it is, however, important to remember that only a fraction of all species of Rondeletia s.l. is represented here. An increased sampling among Mexican/Central American species might show also other mainland taxa to belong in clade C5b. One possible mainland member of clade C5b is Rondeletia panamensis DC., a species endemic to Panama but treated as part of Rondeletia s.s. by Fernández (1993–1994) .

View larger version (36K): [in this window] [in a new window]   Fig. 8. Distribution of taxa from the C5a and C5b clades in Fig. 7 . Numbers indicate number of taxa within each region. This map only includes taxa with restricted distribution, here defined as taxa occurring in no more than two adjacent regions. Guettarda speciosa and Timonius sp., occurring outside the Americas, are not included. Rondeletia odorata is assumed to be introduced by humans in southern Central America (see text) and it is because of this treated as Antillean in the figure. Regions and distribution data as reported by Andersson (1992)

Fruit diversity in clade C5
The C5 clade includes an interesting diversity of fruit types, regarding general morphology, number of seeds per fruit, and modes of dispersal. Capsules and indehiscent dry fruits are the most common, but Gonzalagunia has spongy berries or indehiscent dry fruits (Ståhl, 1999 ), and Guettardeae have mostly drupaceous fruits. In Phyllomelia and Allenanthus, the entire, unopened fruit is wind dispersed. In Phyllomelia, the calyx is expanded to form a flight unit (Delprete, 1999a ), and in Allenanthus the fruit is laterally flattened and surrounded by a ringlike wing perpendicular to the equator of the fruit and originating from the exocarp. Pluriovulate ovaries are predominant in the clade, but Guettardeae, Phyllomelia, and Mazaea include species with one-seeded loculi.

Classification of clade C5
The formal classification of the C5 clade may be approached in several ways. One possibility is to treat the whole clade as one large tribe "Rondeletieae" with a very modified circumscription. Another alternative would be to separate the clade into two tribes, "Rondeletieae" in a narrow sense (C5b) and "Guettardeae" in a broad sense (C5a). However, before any final decisions are made, taxon sampling within the clade should be denser, and morphological characters must be studied in greater depth. All representatives of the C5 clade available for morphological studies (see http://ajbsupp.botany.org/[Appendix 1]) have imbricate corolla aestivation, with the exception of one Guettarda species (G. crispiflora), in which it is valvate-induplicate. Because genera such as Greenea, Aleisanthia, Augusta, and Wendlandia have been shown to have affinities to the Ixoroideae rather than to the Rondeletieae, Rondeletieae may, whatever circumscription is chosen, thus be regarded as almost entirely imbricate.

Conclusions
It is important to include a wide sampling of taxa when performing molecular systematic analyses in the Rubiaceae because relationships within the family are still obscure. Overall relationships within the family are much more complex than previously predicted, as groups usually assumed to be monophyletic or phylogenetically close may prove not to be so. Traditional groups may split into portions belonging to different subfamilies and other unexpected relationships may still be revealed. Great care should be taken when relying only on morphological data, especially when just a limited number of morphological characters are used, or when homologies are not fully understood. On the other hand, corolla aestivation patterns seem to be of taxonomic importance when considered in the perspective of this molecular study. This study shows that the chloroplast trnL-F intron and spacer region works well for inferring phylogeny and higher classification of the Rubiaceae, and, for example, in the case of Strumpfia, it has shown how powerful sequence data can be in finding affinities of taxa where morphology alone has failed to do so (similar experiences founded on rbcL sequence data in Bremer, Andreasen, and Olsson, 1995 ). However, in order to propose a more resolved molecular phylogenetic hypothesis of relationships within the I1 clade, the trnL-F region has turned out to be too slowly evolving to be informative. This study also shows that properly collected, well-kept herbarium specimens still have cardinal scientific value even though studies now range from traditional morphology to DNA sequencing projects. Most DNA extractions used in this study were made from herbarium material, and in the case of Phyllacanthus, DNA for sequencing was obtained from an Ekman collection made in 1923—one of the two collections ever made of this species, which is now possibly extinct.

This study has revealed several new relationships within Rubiaceae, as well as it has corroborated conclusions drawn from other molecular and morphological studies. The most important results can be summarized as follows.

Subfamily-level conclusions
(1) The former Rondeletieae genera Aleisanthia, Aleisanthiopsis, Augusta, Greenea, and Wendlandia are members of the Ixoroideae. (2) Sipaneeae are not close to Rondeletieae but is a tribe of the Ixoroideae. (3) Gleasonia (Henriquezieae) belongs in the subfamily Ixoroideae. (4) Molopanthera and Posoqueria are closely related in spite of the huge difference in corolla size and largely different fruit types. They belong in the Ixoroideae, but not in Gardenieae. (5) Results from other molecular studies are corroborated in that Luculia holds an isolated position basally in the family and that there is no support for subfamily Antirheoideae.

Tribal-level conclusions
(1) There is no support for a broad circumscription of the tribe Rondeletieae. (2) There is support for a tribe Sipaneeae, including at least Maguireothamnus, Neobertiera, and Sipanea. (3) The Guettardeae (sensu Robbrecht, 1988, 1993 ) are paraphyletic and their members are found close to Gonzalagunia and the Rondeletia segregates Arachnothryx, Javorkaea, and Rogiera. (4) There is support for an emended tribe Naucleeae that should include Cephalanthus and Uncaria. (5) Rondeletieae s.s. are almost entirely Antillean.

Generic level conclusions
(1) Geographically correlated molecular support is found for the separation of Arachnothryx, Rogiera, Roigella, and Suberanthus from Rondeletia. (2) Arachnothryx, Gonzalagunia, and Javorkaea are possibly congeneric and need a special study. (3) Rogiera is a polyphyletic genus. (4) Based on morphological data, Neobertiera and Sipaneopsis could well be congeneric, and this is also supported by trnL-F sequence data. (5) Guettarda is paraphyletic.

In addition to this, tribal or subfamilial affiliation based on molecular sequence data is for the first time suggested for the following genera: Allenanthus (close to Guettardeae/Rondeletieae), Blepharidium (Rondeletieae), Chione (close to Hamelieae/Hillieae in Cinchonoideae), Coutaportla (Chiococceae), Dolichodelphys (close to Calycophyllum/Condaminea/Hippotis in Ixoroideae), Mazaea (Rondeletieae), Neobertiera (Sipaneeae in Ixoroideae), Neoblakea (close to Guettardeae/Rondeletieae), Phialanthus (Chiococceae-Catesbaeeae complex), Phyllacanthus (Chiococceae-Catesbaeeae complex), Phyllomelia (Rondeletieae), Schmidtottia (Chiococceae-Catesbaeeae complex), and Suberanthus (Rondeletieae).

	FOOTNOTES

 
¹ The authors thank the staff at the New York Botanical Garden, the Botanical Institute in Göteborg, and the American Museum of Natural History in New York; Magnus Lidén and Claes Persson for their comments on the data matrix; Attila Borhidi for help with identification of specimens; Mari Källersjö and James Farris for performing the Xac-run; and Per-Ola Karis, Henrik Lantz, and the anonymous reviewers for valuable comments on the manuscript. In addition to the herbaria listed at http://ajbsupp.botany.org/(Appendix 1), the following institutions, organizations and their staff are acknowledged for their kind help in accessing material for this study: ORSTOM in Cayenne, French Guyana; herbarium HUA, Fundación Inguedé, and Municipio de Coquí in Colombia; the Smithsonian Tropical Research Institute (STRI), INRENARE, and IRHE in Panama; and Instituto de Ecología y Sistemática and Jardin Botanico Nacional in Havana, Cuba. This work was supported by grants from the Lewis B. and Dorothy Cullman program for molecular research to VA, the Swedish Natural Science Research Council (NFR) to LA, and grants to JR from the Royal Swedish Academy of Sciences, Helge Ax:son Johnsons stiftelse, Stiftelsen Wilhelm och Martina Lundgrens vetenskapsfond, Kungliga och Hvitfeldska stipendieinrättningen, Stiftelsen Kulturfonden Botaniskas vänner, Knut och Alice Wallenbergs stiftelse, and Göteborg University. They are all gratefully acknowledged.

⁵ Author for reprint requests (jorova{at}privat.utfors.se ).

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Andersson L. 1992 A provisional checklist of Neotropical Rubiaceae. Scripta Botanica Belgica 1: 1-199

Andersson L. 1993a Pollen characteristics of the tribes Calycophylleae, Cinchoneae, and Hillieae (Rubiaceae). Nordic Journal of Botany 13: 405-417[Web of Science]

Andersson L. 1993b Rubiaceae: introduction. In G. Harling and L. Andersson [eds.], Flora of Ecuador 47: 1-11

Andersson L. 1995 Tribes and genera of the Cinchoneae complex (Rubiaceae). Annals of the Missouri Botanical Garden 82: 409-427[CrossRef][Web of Science]

Andersson L. 1996 Circumscription of the tribe Isertieae (Rubiaceae). Opera Botanica Belgica 7: 139-164

Andersson L. C. Persson 1991 Circumscription of the tribe Cinchoneae (Rubiaceae): a cladistic approach. Plant Systematics and Evolution 178: 65-94[CrossRef][Web of Science]

Andersson L. J. H. E. Rova 1999 The rps16 intron and the phylogeny of the Rubioideae (Rubiaceae). Plant Systematics and Evolution 214: 161-186[CrossRef][Web of Science]

Andreasen K. 1997 Phylogeny of Ixoroideae: molecular systematics within Rubiaceae. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 308: 1-22

Andreasen K. B. G. Baldwin B. Bremer 1999 Phylogenetic utility of the nuclear rDNA ITS region in the subfamily Ixoroideae (Rubiaceae): comparisons with cpDNA rbcL sequence data. Plant Systematics and Evolution 217: 119-135[CrossRef][Web of Science]

Andreasen K. B. Bremer 1996 Phylogeny of the subfamily Ixoroideae (Rubiaceae). Opera Botanica Belgica 7: 119-138

Andreasen K. B. Bremer 2000 Combined phylogenetic analysis in the Rubiaceae-Ixoroideae: morphology, nuclear and chloroplast DNA data. American Journal of Botany 87: 1731-1748[Abstract/Free Full Text]

Beach J. 1983 Posoqueria latifolia. In D. H. Janzen [ed.], Costa Rican natural history, 307–308. University of Chicago Press, Chicago, Illinois, USA

Borhidi A. 1982 Studies in Rondeletieae (Rubiaceae). III. The genera Rogiera and Arachnothryx. Acta Botanica Academiae Scientiarum Hungaricae 28: 65-71

Borhidi A. 1989 Studies on Rondeletieae (Rubiaceae). XI. Critical notes on some central American species of Rondeletia s.l. Acta Botanica Hungarica 35: 309-312

Borhidi A. 1993–1994 [1994] Studies in Rondeletieae. XII. New combinations of Mexican and Central American taxa. Acta Botanica Hungarica 38: 139-142

Borhidi A. M. Z. Fernández 1981a Studies in Rondeletieae (Rubiaceae) I. A new genus: Roigella. Acta Botanica Academiae Scientiarum Hungaricae 27: 309-312[Web of Science]

Borhidi A. M. Z. Fernández 1981b Studies in Rondeletieae (Rubiaceae). II. A new genus: Suberanthus. Acta Botanica Academiae Scientiarum Hungaricae 27: 313-316[Web of Science]

Borhidi A. M. Járai-Komlódi 1983 Studies in Rondeletieae (Rubiaceae). IV. A new genus: Javorkaea. Acta Botanica Hungarica 29: 13-27

Bremekamp C. E. B. 1966 Remarks on the position, the delimitation and the subdivision of the Rubiaceae. Acta Botanica Neerlandica 15: 1-33

Bremer B. 1979 The genus Neurocalyx (Rubiaceae-Argostemmateae) in Ceylon. Botaniska Notiser 132: 399-407[Web of Science]

Bremer B. 1992 Phylogeny of the Rubiaceae (Chiococceae) based on molecular and morphological data: useful approaches for classification and comparative ecology. Annals of the Missouri Botanical Garden 79: 380-387[CrossRef][Web of Science]

Bremer B. 1996a Combined and separate analyses of morphological and molecular data in the plant family Rubiaceae. Cladistics 12: 21-40

Bremer B. 1996b Phylogenetic studies within Rubiaceae and relationships to other families based on molecular data. Opera Botanica Belgica 7: 33-50

Bremer B. K. Andreasen D. Olsson 1995 Subfamilial and tribal relationships in the Rubiaceae based on rbcL sequence data. Annals of the Missouri Botanical Garden 82: 383-397[CrossRef][Web of Science]

Bremer B. R. K. Jansen 1991 Comparative restriction site mapping of the chloroplast DNA implies new phylogenetic relationships within the Rubiaceae. American Journal of Botany 78: 198-213[CrossRef][Web of Science]

Bremer B. R. K. Jansen B. Oxelman M. Backlund H. Lantz K.-J. Kim 1999 More characters or more taxa for a robust phylogeny: case study from the coffee family (Rubiaceae). Systematic Biology 48: 413-435[CrossRef][Web of Science][Medline]

Bremer B. J.-F. Manen 2000 Phylogeny and classification of the subfamily Rubioideae (Rubiaceae). Plant Systematics and Evolution 225: 43-72[CrossRef][Web of Science]

Bremer B. L. Struwe 1992 Phylogeny of the Rubiaceae and the Loganiaceae: congruence or conflict between morphological and molecular data?. American Journal of Botany 79: 1171-1184[CrossRef][Web of Science]

Bremer B. M. Thulin 1998 Collapse of Isertieae, re-establishment of Mussaendeae, and a new genus of Sabiceeae (Rubiaceae): phylogenetic relationships based on rbcL data. Plant Systematics and Evolution 211: 71-92[CrossRef][Web of Science]

Bridson D. M. 1988 Pavetteae. In R. M. Polhill [ed.], Flora of tropical East Africa, Rubiaceae part 2, 581–693. A. A. Balkema, Rotterdam, The Netherlands

Bridson D. M. E. Robbrecht 1985 Further notes on the tribe Pavetteae (Rubiaceae). Bulletin du Jardin botanique national de Belgique 55: 83-115[CrossRef]

Chase M. W. H. Hillis 1991 Silica gel: an ideal material for field preservation of leaf samples for DNA studies. Taxon 40: 215-220[CrossRef][Web of Science]

Claßen-Bockhoff R. 1996 A survey of flower-like inflorescences in the Rubiaceae. Opera Botanica Belgica 7: 329-367

Cowan J. M. 1932 The genus Wendlandia. Notes from the Royal Botanic Garden Edinburgh 16: 233-314

Darwin S. P. 1976 The genus Lindenia (Rubiaceae). Journal of the Arnold Arboretum 57: 426-449[Web of Science]

Darwin S. P. 1979 A synopsis of the indigenous genera of Pacific Rubiaceae. Allertonia 1: 1-44

Darwin S. P. 1993 A revision of Timonius subgenus Timonius (Rubiaceae: Guettardeae). Allertonia 7: 1-39

Darwin S. P. 1994 Systematics of Timonius subgenus Abbottia (Rubiaceae-Guettardeae). Systematic Botany Monographs, vol. 42, 1–86

De Block P. 1998 The African species of Ixora. Opera Botanica Belgica 9: 1-218

De Laet J. E. Smets 1996 A commentary on the circumscription and evolution of the order Gentianales, with special emphasis on the position of the Rubiaceae. Opera Botanica Belgica 7: 11-18

Delprete P. G. 1996a Evaluation of the tribes Chiococceae, Condamineeae and Catesbaeeae (Rubiaceae) based on morphological characters. Opera Botanica Belgica 7: 165-192

Delprete P. G. 1996b Notes on the taxonomic position of the monotypic Brazilian genus Kerianthera (Rubiaceae). Opera Botanica Belgica 7: 271-275

Delprete P. G. 1997a Notes on calycophyllous Rubiaceae. Part II. Morphological comparison of the genera Bathysa and Schizocalyx. Brittonia 49: 480-486[CrossRef][Web of Science]

Delprete P. G. 1997b Revision and typification of Brazilian Augusta (Rubiaceae, Rondeletieae), with ecological observations on the riverine vegetation of the cerrado and Atlantic forests. Brittonia 49: 487-497[CrossRef][Web of Science]

Delprete P. G. 1998 Notes on calycophyllous Rubiaceae. Part III. Systematic position of the monotypic Mexican genus Cosmocalyx and notes on the calycophyll development. Brittonia 50: 309-317[CrossRef][Web of Science]

Delprete P. G. 1999a Morphological and taxonomical comparison of the Cuban endemic taxa Ariadne, Mazaea, Acunaeanthus, Phyllomelia (Rubiaceae, Rondeletieae) and Eosanthe, with one new combination. Brittonia 51: 217-230[CrossRef][Web of Science]

Delprete P. G. 1999b Rondeletieae (Rubiaceae), part I. Flora Neotropica Monograph 77: 1-226

Doyle J. J. J. L. Doyle 1987 A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15

Ehrendorfer F. J.-F. Manen A. Natali 1994 cpDNA intergene sequences corroborate restriction site data for reconstructing Rubiaceae phylogeny. Plant Systematics and Evolution 190: 245-248[CrossRef][Web of Science]

Fernández M. Z. 1993–1994 [1994] Estudio taxonómico del géneroRondeletia L. s.l. (Rubiaceae) en Cuba. Acta Botanica Hungarica 38: 47-138

Garcia Kirkbride M. C. 1979 Review of the neotropical Isertieae. Brittonia 31: 313-332[CrossRef][Web of Science]

Gielly L. P. Taberlet 1994 The use of chloroplast DNA to resolve plant phylogenies: noncoding versus rbcL sequences. Molecular Biology and Evolution 11: 769-777[Abstract]

Hallé F. 1967 Étude biologique et morphologique de la tribu des Gardéniées (Rubiacées). Mémoire ORSTOM 22: 1-146

Hooker J. D. 1873 Ordo LXXXIV, Rubiaceae. In G. Bentham and J. D. Hooker [eds.], Genera plantarum ad exemplaria imprimis in herbariis kewensibus servata defirmata 2: 7–151. Lovell Reeve, London, UK

Huysmans S. E. Robbrecht P. Delprete E. Smets 1999 [2000] Pollen morphological support for the Catesbaeeae-Chiococceae-Exostema-complex (Rubiaceae). Grana 38: 325-338[CrossRef][Web of Science]

Igersheim A. 1993 The character states of the Caribbean monotypic endemic Strumpfia (Rubiaceae). Nordic Journal of Botany 13: 545-559[Web of Science]

Johansson J. T. 1994 The genus Morinda (Morindeae, Rubioideae, Rubiaceae) in New Caledonia: taxonomy and phylogeny. Opera Botanica 122: 1-67

Kiehn M. 1986 Karyologische Untersuchungen und DNA-Messungen an Rubiaceae und ihre Bedeutung für die Systematik dieser Familie. Ph.D. dissertation, University of Vienna, Vienna, Austria

Kiehn M. 1995 Chromosome survey of the Rubiaceae. Annals of the Missouri Botanical Garden 82: 398-408[CrossRef][Web of Science]

Kirkbride J. H., Jr. 1968 A revision of the Panamanian species of Rondeletia (Rubiaceae). Annals of the Missouri Botanical Garden 55: 372-391[CrossRef][Web of Science]

Kirkbride J. H., Jr. 1997 Manipulus rubiacearum, VI. Brittonia 49: 354-379[CrossRef][Web of Science]

Liogier A. 1963 Rubiaceae. Flora de Cuba 5, 13–146. Editorial Universitaria, Universidad de Puerto Rico, Río Piedras, Puerto Rico

Lorence D. H. 1991 New species and combinations in Mexican and Central American Rondeletia (Rubiaceae). Novon 1: 135-157

Mabberley D. J. 1993 The plant-book, a portable dictionary of the higher plants. Cambridge University Press, Cambridge, UK

Manen J. F. A. Natali 1995 Comparison of the evolution of ribulose-1, 5-bisphosphate carboxylase (rbcL) and atpB-rbcL noncoding spacer sequences in a recent plant group, the tribe Rubieae (Rubiaceae). Journal of Molecular Evolution 41: 920-927[Web of Science][Medline]

Manen J. F. A. Natali 1996 The chloroplast atpB-rbcL spacer in Rubiaceae. Opera Botanica Belgica 7: 51-57

Manen J. F. A. Natali F. Ehrendorfer 1994 Phylogeny of Rubiaceae-Rubieae inferred from the sequence of a cpDNA intergene region. Plant Systematics and Evolution 190: 195-211[CrossRef][Web of Science]

Müller F. 1866 Ueber die Befruchtung der Martha (Posoqueria?) fragans. Botanische Zeitung 24: 129-136 with Fig. VI

Natali A. J.-F. Manen F. Ehrendorfer 1995 Phylogeny of the Rubiaceae-Rubioideae, in particular the tribe Rubieae: evidence from a non-coding chloroplast DNA sequence. Annals of the Missouri Botanical Garden 82: 428-439[CrossRef][Web of Science]

Natali A. J.-F. Manen M. Kiehn F. Ehrendorfer 1996 Tribal, generic, and specific relationships in the Rubioideae-Rubieae (Rubiaceae) based on sequence data of the cpDNA intergene spacer. Opera Botanica Belgica 7: 193-203

Nepokroeff M. B. Bremer K. J. Sytsma 1999 Reorganization of the genus Psychotria and tribe Psychotrieae (Rubiaceae) inferred from ITS and rbcL sequence data. Systematic Botany 24: 5-27

Ochoterena-Booth H. 2000 Systematics of Hintonia Bullock and the Portlandia complex (Rubiaceae). Ph.D. dissertation, Cornell University, Ithaca, New York, USA

Persson C. 1996 Phylogeny of the Gardenieae (Rubiaceae). Botanical Journal of the Linnean Society 121: 91-109[CrossRef]

Persson C. 1998 Phylogeny of the tribe Gardenieae (Rubiaceae). Ph.D. dissertation, Göteborg University, Faculty of Natural Sciences, Göteborg, Sweden

Persson C. 2000 Phylogeny of the neotropical Alibertia group (Rubiaceae), with emphasis on the genus Alibertia, inferred from ITS and 5S ribosomal DNA sequences. American Journal of Botany 87: 1018-1028[Abstract/Free Full Text]

Puff C. 1988 Observations on Carphaela Juss. (Rubiaceae, Hedyotideae), with particular reference to the Madagascan taxa and its taxonomic position. Bulletin du Jardin botanique national de Belgique 58: 271-323[CrossRef]

Puff C. A. Igersheim U. Rohrhofer 1993 Pseudomussaenda and Schizomussaenda (Rubiaceae): close allies of Mussaenda. Bulletin du Jardin botanique national de Belgique 62: 35-68[CrossRef]

Puff C. E. Robbrecht V. Randrianasolo 1984 Observations on the SE African-Madagascan genus Alberta and its ally Nematostylis (Rubiaceae, Alberteae), with a survey of the species and a discussion of the taxonomic position. Bulletin du Jardin botanique national de Belgique 54: 293-366[CrossRef]

Ridsdale C. E. 1976 A revision of the tribe Cephalantheae (Rubiaceae). Blumea 23: 177-188

Ridsdale C. E. 1979 Jackiopsis, a new name for Jackia Wall. (Rubiaceae-Jackieae). Blumea 25: 295-296

Robbrecht E. 1988 Tropical woody Rubiaceae. Opera Botanica Belgica 1: 1-272

Robbrecht E. 1993 [1994] Supplement to the 1988 outline of the classification of the Rubiaceae: index to genera. Opera Botanica Belgica 6: 173-196

Robbrecht E. U. Rohrhofer C. Puff 1994 A survey of Bertiera (Rubiaceae), including a discussion of its taxonomic position. Opera Botanica Belgica 6: 101-141

Rogers G. K. 1984 Gleasonia, Henriquezia, and Platycarpum (Rubiaceae). Flora Neotropica Monograph 19: 1-135

Rova J. H. E. L. Andersson 1995 A reevaluation of the tribes Hippotideae and Tammsieae (Rubiaceae). Nordic Journal of Botany 15: 269-284[Web of Science]

Schumann K. 1891 Rubiaceae. In A. Engler and K. Prantl [eds.], Die Natürlichen Pflanzenfamilien, vol. 4(4), 1–156. Engelmann, Liepzig, Germany

Schumann K. 1897 Rubiaceae. In A. Engler and K. Prantl [eds.], Die Natürlichen Pflanzenfamilien, Nachträge [I] zum II–IV: 309–316. Engelmann, Liepzig, Germany

Smith A. C. P. Darwin 1988 Rubiaceae. In A. C. Smith [ed.], Flora vitiensis nova: a new flora of Fiji, vol. 4, 143–376. National Tropical Botanical Garden, Lawai, Kauai, Hawaii, USA

Ståhl B. 1999 Gonzalagunia. In G. Harling and L. Andersson [eds.], Flora of Ecuador 62: 70-101

Standley P. C. 1918 Rondeletia. North American Flora 32: 44-86

Steyermark J. A. 1964 Rubiaceae. In B. Maguire, J. J. Wurdack, and collaborators, The botany of the Guyana highland, Part V Memoirs of the New York Botanical Garden 10: (5)1-278

Steyermark J. A. 1967 Rondeletia and Arachnothryx. In B. Maguire et al. [eds.], The botany of the Guyana highland, part VII. Memoirs of the New York Botanical Garden 17: 241-261

Steyermark J. A. 1974 Rubiaceae. In T. Lasser [ed.], Flora de Venezuela 9: 1–2070. Instituto Botanico, Caracas, Venezuela

Struwe L. V. A. Albert B. Bremer 1994 Cladistics and family level classification of the Gentianales. Cladistics 10: 175-206

Struwe L. M. Thiv J. W. Kadereit A. S.-R. Pepper T. J. Motley P. J. White J. H. E. Rova K. Potgeiter V. A. Albert 1998 Saccifolium (Saccifoliaceae), an endemic of Sierra de la Neblina on the Brazilian-Venezuelan border, is related to a temperate-alpine lineage of Gentianaceae. Harvard Papers in Botany 3: 199-214

Swofford D. L. 1993 PAUP: phylogenetic analysis using parsimony, version 3.1.1. Computer program and documentation distributed by Illinois Natural History Survey, Champaign, Illinois, USA

Taberlet P. L. Gielly G. Pautou J. Bouvet 1991 Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105-1109[CrossRef][Web of Science][Medline]

Tange C. 1996a Studies in SE Asiatic Rondeletieae I: the West Malaysian endemic genus Aleisanthia (Rubiaceae). Nordic Journal of Botany 16: 563-570[Web of Science]

Tange C. 1996b Studies in SE Asiatic Rondeletieae II: Aleisanthiopsis (Rubiaceae), a new genus from Borneo. Nordic Journal of Botany 16: 571-578[Web of Science]

Taylor C. M. 1996 Taxonomic revision of Cruckshanksia and Oreopolus (Rubiaceae, Hedyotideae). Annals of the Missouri Botanical Garden 83: 461-479[CrossRef][Web of Science]

Verdcourt B. 1958 Remarks on the classification of the Rubiaceae. Bulletin du Jardin Botanique de l'État Bruxelles 28: 209-290[CrossRef]

Verdcourt B. 1976 Rubiaceae (part 1). In R. M. Polhill [ed.], Flora of tropical East Africa, 1–415. Crown Agents for Oversea Governments and Administrations, London, UK

Walker J. W. J. A. Doyle 1975 The bases of angiosperm phylogeny: palynology. Annals of the Missouri Botanical Garden 62: 664-723[CrossRef][Web of Science]

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