sign in sign up
<<
Home , Alliance (taxonomy), Anthospermeae, Anthospermum, Arcytophyllum, Argostemma, Bouvardia

TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and

Evolutionary relationships in the alliance () using information from six molecular loci: insights into systematic affinities of Neohymenopogon and Mouretia

Catarina Rydin1, Sylvain G. Razafimandimbison2, Anbar Khodabandeh2 & Birgitta Bremer2

1 University of Zürich, Institute of Systematic Botany, Zollikerstrasse 107, 8008 Zürich, Switzerland. [email protected] (author for correspondence) 2 Bergius Foundation, Royal Swedish Academy of Sciences and Botany Department, Stockholm University, 106 91 Stockholm, Sweden

Several recent phylogenetic studies of Rubiaceae have dealt with enigmatic taxa whose systematic positions have been previously unknown or controversial. We address evolutionary relationships in the Spermacoceae alliance () with special emphasis on the Asian genera Mouretia and Neohymenopogon, here se- quenced for the first time. Both genera belong in the Argostemmateae and have persistent calyx lobes on the in common with and . Other previous uncertainties are resolved with strong support; is sister to Paederieae s.str. and is sister to remaining . Our results further reveal some phylogenetic problems. Danaideae is sister to remaining taxa in the Spermacoceae alliance with high posterior probability, which contradicts results in a recent study. The uncertainty concern- ing evolutionary relationships of and is reinforced, despite a denser taxon sampling in the Spermacoceae alliance compared with earlier studies. We also demonstrate yet another example of the controversial correlation between molecular substitution rate and life history.

KEYWORDS: Anthospermeae, Argostemmateae, Danaideae, Paederieae, rate heterogeneity, Saprosma, Theligonum

the apex of the corolla lobe and the has numerous INTRODUCTION seeds. A thin but leafy bract, “elegantly nerved” under- The large coffee , Rubiaceae, comprises more neath is attached to the node where the peduncles unite than 13,000 (Govaerts & al., 2006), a diversity (Wallich, 1824: 157). Hymenopogon Wall. (Wallich, 1824), which is estimated to have originated in the mid-Cre- comprising three species, was an illegitimate homonym taceous (Bremer & Eriksson, 2009). Rubiaceae display and Bennet (1981) suggested it should be replaced by Neo- a great diversity in growth habit ranging from arbores- hymenopogon. Andersson & Persson (1991) included the cent, suffrutescent, lianescent, epiphytic to herbaceous in a morphological study and found support for habits (Robbrecht, 1988; Bremer & al., 1995). A woody Bremekamp’s (1952, 1966) transfer of Neohymenopogon habit is the predominant life form in the Cin- from to Hedyotideae. Apart from this work, chonoideae and , whereas herbaceous as well short descriptions in floras and other surveys (Wallich, as woody habits occur in Rubioideae (Verdcourt, 1958). 1824; Hooker, 1880; Classen-Bockhoff, 1996) and Puff & Climbers are also relatively common in the family and al.’s more comprehensive flora (2005), we have not found about 250 species are epiphytic (Robbrecht, 1988), but a any published information on Neohymenopogon. In parasitic habit is very unusual, probably unknown. to get a first indication on the systematic affinity of this The epiphytic to terrestrial plant Neohymenopogon genus, we produced and analysed a nrITS sequence and parasiticus (Wallich, 1824; Bennet, 1981), distributed in the preliminary results indicated that Neohymenopogon Bhutan, , Tibet and (Yunnan) (Govaerts & parasiticus belongs in Rubioideae, Rubiaceae, probably al., 2006), was originally described under the name Hyme- in the Spermacoceae alliance (further described below). nopogon parasiticus, as a small branchy , attached Several recent phylogenetic studies of Rubiaceae have parasitically to trees by means of fibrous roots (Wallich, investigated enigmatic taxa whose systematic positions 1824). The interpretation of the plant as a parasite was have been unknown or controversial, e.g., (Nie likely a misconception (see, e.g., Puff & al., 2005), but & al., 2005), Rhopalobrachium (Mouly & al., 2007), Dun- the matter has to our knowledge never been thoroughly nia (Rydin & al., 2008), Schizocolea (Razafimandimbison investigated. The are membranous (but green), the & al., 2008; Rydin & al., 2008), Petitiocodon (Tosh & are pentamerous with the stamina inserted near al., 2008) and Acranthera (Rydin & al., 2009). Another

793 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia TAXON 58 (3) • August 2009: 793–810

genus of uncertain systematic affinity is Mouretia (Pitard, elucidate the evolutionary origin of the genus. Neohyme- 1922), which comprises five species distributed in China, nopogon parasiticus is investigated using material from Laos, Vietnam and (Govaerts & al., 2006). Tange two specimens. Relationships within the -Sper- (1997) made a revision of the genus and described Moure- macoceae are investigated elsewhere (i.e., Kårehed tia as herbs to small , often anisophyllous and with & Bremer, 2007; Kårehed & al., 2008; Groeninckx & al., distylous flowers (Tange, 1997; see also Puff & al., 2005). 2009) and will not be discussed here, but to relevantly ad- Mouretia is currently classified in the tribe Hedyotideae dress the topics of the present paper, we have included 24 (Robbrecht, 1988, 1993), and this was tentatively accepted representatives from the Knoxieae-Spermacoceae clade. by Tange (1997). Tange suggested a possible affinity be- We further included a comprehensive set of terminals tween Mouretia and Mycetia but did not do any formal (68) from remaining Rubioideae, representing 14 tribes. changes of the classifications. Mycetia was at that time All trees were rooted on , the sister group of placed in the tribe Isertieae, all other Rubioideae species (Robbrecht & Manen, 2006; (Robbrecht, 1988, 1993), though Bremekamp (1952, 1966) Rydin & al., 2008; Sonké & al., 2008). had suggested that it belonged in Hedyotideae. Hypoth- We utilised information from six loci: five chloroplast eses on the systematic position of Mouretia have to our regions (rbcL, the rps16 intron, ndhF, atpB-rbcL spacer, knowledge never been tested using phylogenetic analyses. the trnT-L-F region), and the internal transcribed spacer of The Spermacoceae alliance and the aim of the nuclear ribosomal DNA, nrITS (nrITS1, 5.8S, nrITS2). this study. — The Spermacoceae alliance (Bremer & Primer references are given in Table 1. We used informa- Manen, 2000), also known as the Hedyotideae- tion from GenBank if available and we also produced clade (Andersson & Rova, 1999) and supertribe Rubiidi- 252 new sequences for this study (Table 2). GenBank nae (Robbrecht & Manen, 2006), is a well-corroborated accession numbers are given in Appendix. DNA was ex- clade consisting of nearly 3,000 species. It comprises two tracted, amplified and sequenced using standard proce- major subclades (Bremer & Manen, 2000); subclade 1: dures previously described (Kårehed & Bremer, 2007). the tribes Knoxieae and Spermacoceae (Bremer, 1996; Robbrecht & Manen, 2006; Kårehed & Bremer, 2007) (not Table 1. Primers. discussed in the present paper), and subclade 2: Anthos- DNA region Primer name Reference permeae, Argostemmateae, Dunnieae, Paederieae, Puto- rieae, Rubieae and Theligoneae (Bremer & Manen, 2000; rbcL 5′F, 3′R and 427F Bremer & al. (2002) Backlund & al., 2007; Rydin & al., 2008, 2009). Further, rbcL Z895R Zurawski, DNAX the small tribe Danaideae belongs to the Spermacoceae rps16 F and 2R Oxelman & al. (1997) alliance (Bremer & Manen, 2000) but its relationship to nrITS P17 and 26S-82R Popp & Oxelman (2001) the remaining tribes has been inconsistent (results differ nrITS P25 Oxelman (1996) between studies). Here, we investigate evolutionary relationships within nrITS ITSForwRub Rydin & al. (2009) the Spermacoceae alliance with special focus on Neohy- nrITS ITSRevRub Rydin & al. (2009) menopogon and Mouretia. The study is based on infor- ndhF 2F Rydin & al. (2008) mation from six loci and utilises a denser taxon sampling ndhF 1000R Rydin & al. (2008) in the Spermacoceae alliance compared with previous ndhF 720F Rydin & al. (2008) studies. ndhF 1700R Rydin & al. (2008) ndhF 1320F Rydin & al. (2008) ndhF 2280R Rydin & al. (2008) MATERIALS AND METHODS atpB-rbcL spacer rbcL5′R Rydin & al. (2008) Selection of species and laboratory proce- atpB-rbcL spacer atpB5′R Rydin & al. (2008) dures. — We selected 159 taxa from subfamily Rubio- trnT-L-F A1 Bremer & al. (2002) ideae for this study (Appendix). We have included 91 terminals from the ingroup, the Spermacoceae alliance trnT-L-F 940R Rydin & al. (2008) (Bremer & Manen, 2000), representing ten tribes. Due trnT-L-F 820F Rydin & al. (2008) to limited access to material, we were only able to in- trnT-L-F IR Bremer & al. (2002) clude one species each of Mouretia and Neohymenopogon. trnT-L-F 1250F Rydin & al. (2008) The material investigated during the revision of Mouretia trnT-L-F D Taberlet & al. (1991) (Tange, 1997) has unfortunately not been possible to trace trnT-L-F 1880F Rydin & al. (2008) but other material of Mouretia larsenii Tange has been studied and used to produce sequences in a first attempt to trnT-L-F 2670R Rydin & al. (2008)

794 TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia

Sequence fragments were assembled using the Staden package (Staden, 1996). Alignment and phylogenetic reconstruction. — Sequences were aligned visually, using computer pro- GTR + I Γ grams Seaview 2.2 (Galtier & al., 1996) and Se-Al v.2.0 Combined matrix, 2 partitions (Rambaut, 1996). Insertion/deletion events were visually

inferred, following the alignment criteria outlined in c Oxelman & al. (1997). Combined matrix, 6 partitions All matrices were analysed with two approaches: —— —— —— —— Bayesian inference and parsimony. We analysed each gene separately, and we also analysed all gene regions together in a combined dataset. Bayesian analyses were performed nrITS in MrBayes 3.1 (Huelsenbeck & Ronquist, 2001; Ronquist GTR + I Γ GTR + I Γ GTR + I Γ GTR + I Γ GTR + I Γ← & Huelsenbeck, 2003). For each single-gene dataset, the best-performing evolutionary model was identified under several different model selection criteria: Akaike informa- tion criterion (AIC) (Akaike, 1973), AICc, the Bayesian trnT/F GTR + I Γ GTR + I Γ GTR + I Γ GTR + I Γ GTR + I Γ information criterion (BIC) (Schwartz, 1978) and hier- archical likelihood ratio tests (four different hierarchies described in Posada & Crandall, 2001). We performed these calculations in softwares MrAIC ver. 1.4.3 and atpB-rbcL spacer GTR + Γ GTR + Γ GTR + Γ GTR + Γ PHYML (Guindon & Gascuel, 2003; Nylander, 2004a), GTR + Γ and in MrModeltest ver. 2.3 and PAUP* ver. 4.0b10 for Unix (Swofford, 1998; Nylander, 2004b). Prior probabilities were set following outputs from GTR + Γ GTR + Γ GTR + Γ GTR + I Γ MrAIC: a flat Dirichlet prior probability (all values are GTR + Γ 1.0) was selected for the substitution rates (revmatpr) and the nucleotide frequencies (statefreqpr). The prior prob- ability for the shape parameter of the gamma distribution HKY + Γ HKY + Γ HKY + Γ HKY GTR + Γ of rate variation (shapepr) was uniformly distributed in the + Γ HKY

interval (0.1, 50.0). For analyses using a gamma distribu- Γ tion with a proportion of invariable sites, we specified a prior probability for this proportion (pinvarpr), uniformly distributed on the interval (0.0, 1.0). When applicable, the 1,000/10 1,000/10 1,000/10 1,000/10 1,000/10 1,000/10 5,000/10 5,000/10 GTR + I + I + GTR No No No No No No No No rbcL rps16 ndhF GTR + I Γ GTR + I Γ GTR + I Γ GTR + I Γ 1.5 million 1.5 million 1.5 million 1.5 million 1.5 million 1.5 million 5 million 5 million prior for the transition/transversion rate ratio (tratio) was set so as to put equal emphasis on transition/transversion rate ratios above 1.0 and below 1.0 (beta 1.0, 1.0). The combined dataset was partitioned in two ways: (1) six partitions (one for each locus) and (2) two parti-

tions (chloroplast data and nuclear data). Partitions were a a a unlinked so that each partition was allowed to have its

own set of parameters. Five million generations were run b with a sample frequency of 1,000, six parallel chains and the temperature set to 0.15. For single-gene analyses, 1.5 million generations were run with a sample frequency of 1,000, four parallel chains and the temperature set to 0.2. Convergence of the Markov chain was assumed to be reached when the likelihood values and individual parameters were stably fluctuating. At this point, the po- tential scale reduction factor was approaching 1.000 for all parameters and standard deviation of split frequencies for the two parallel runs was < 0.007. Trees sampled from the first two million generations (200,000 generations for hLRTs, hierarchical likelihood ratio tests; calculated in software MrModeltest (Nylander, 2004b). hierarchical likelihood ratio tests; calculated in software MrModeltest (Nylander, hLRTs, AIC, Akaike information criterion; AICc, corrected AIC; BICC, Bayesian information criterion; all alculated in software MrAIC.pl (Nylander, 2004a). (Nylander, AIC; BICC, Bayesian information criterion; all alculated in software MrAIC.pl AICc, corrected Akaike information criterion; AIC, Evolutionary model employed for each data partition are as specified to the left (for single genes). Table 2.Table Data description. Taxon a b c Total number of taxa in matrix (number new sequences)Total number of characters in matrixTotal 155 (32)Number of variable charactersNumber of informative charactersAICc weights Best fitting evolutionary model; 153 (30) 127 (61) 139 (53) 149 (32) 1,402 134 (44) 159 (252) 1,689 393 517 159 (252) 2,223 636 967 1,144 765 1,060 3,513 556 338 893 1,905 1,183 10,864 586 486 10,864 5,591 3,801 5,591 3,801 Employed evolutionary model (AICc weights) single-gene analyses) were discarded as burn-in. AIC weights Best fitting evolutionary model; Best fitting evolutionary model; BIC weights Best fitting evolutionary model; hLRTs Conflicts between Bayesian and parsimony analyses Bootstrap analysis (bootstrap replicates /random sequence additions in each replicate) Bayesian analysis (number of generations run)

795 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia TAXON 58 (3) • August 2009: 793–810

Parsimony analyses were performed for single gene in the six-partition analysis. There were no supported con- datasets, as well as for the combined dataset, in PAUP* flicts between Bayesian and parsimony analyses. ver. 4.0b10 for Unix (Swofford, 1998). Most parsimoni- All nodes presented below received 100% posterior ous trees were calculated using the heuristic search op- probability and ≥ 85% bootstrap support unless otherwise tion, 500 random sequence additions and tree bisection is stated as follows (Bayesian posterior probability/boot- reconnection branch swapping and multrees off. Support strap support). values were obtained by using bootstrap in PAUP*, per- The Spermacoceae alliance was monophyletic with forming 5,000 bootstrap replicates, each with ten random Danaideae being sister clade to remaining species (Fig. sequence additions. For single-gene analyses, 1,000 boot- 1A, B). The genera and Schismatoclada were strap replicates were run. A majority rule consensus tree monophyletic. One Malagasy species, Danais nigra, was was produced from the resulting trees, in which nodes sister to remaining Danais, within which the African with a bootstrap support < 50% were collapsed. species Danais xanthorrhoea was sister to Danais co- morensis. Remaining species of the Spermacoceae alliance (95/–), consist of the sister Knoxieae-Spermacoceae RESULTS and Anthospermeae-Argostemmateae-Dunnieae-Paed- Data and model choice. — The combined matrix erieae-Putorieae-Theligoneae-Rubieae. Within the latter, comprised 10,864 aligned characters. Of the 5,591 vari- Anthospermeae are sister to the other tribes. Anthosper- able characters, 3,801 were phylogenetically informative meae are well resolved; Carpacoce is sister to the remain- (Table 2). The hierarchical likelihood ratio tests (hLRTs ing species: a ((-)) clade and in MrModeltest) and calculations of AIC, AICc, and BIC an ((Opercuclaria-) + (((- weights (performed in MrAIC) for estimation of best ))) clade (100/64). performing nucleotide substitution models sometimes Within the next diverging clade (Argostemmateae), identified different optimal models for a given locus (Ta- Mouretia is sister to the remaining species, followed by ble 2). Bayesian and AIC approaches have been shown Mycetia. Neohymenopogon and Argostemma are sisters to have important advantages over the hLRTs for model (97/70). Mycetia javanica and M. malayana are sister to selection (Posada & Buckley, 2004) and when forced to a poorly supported (71/–) clade ((M. gracilis + (M. cauli- make a choice (ndhF, rps16 intron), we used the model flora + (two unknown species of Mycetia))). (Argostemma identified under the corrected Akaike information crite- bifolium + (A. gracile + (A. brachyantherum–A. psychtrio- rion because AICc is appropriate when the ratio between ides))) are sisters to a clade comprising ((A. elatostemma– sample size and number of parameters is small (n / K < 40; A. hookerii) and (A. geesinkii + (A. yappii + (A. rupestre Burnham & Anderson, 2003: 66), but also for higher ra- + (A. parvifolium var. involucratum–A. parvifolium)))). tios because AICc will then converge to AIC (Posada & Support was less than 100% posterior probability and 85% Buckley, 2004). bootstrap for the (A. yappii + (A. rupestre + (A. parvifo- For the rbcL, trnT-L-F and nrITS data, the general lium var. involucratum–A. parvifolium) clade (85/–), and time r eversible model (GTR; Tavare, 1986) with gamma for the (A. rupestre + (A. parvifolium var. involucratum– distributed rates (Yang, 1993) and a proportion of invari- A. parvifolium) clade (98/–). able sites was selected (GTR + Γ + I). For ndhF and the Argostemmateae + (Dunnieae-Paederieae-Putorieae- atpB-rbcL spacer, GTR + Γ was selected. For the rps16 Theligoneae-Rubieae) are well supported in Bayesian intron the Hasegawa, Kishino and Yano model (Hasegawa analysis (100%) but the node is collapsed in the bootstrap & al., 1985) with gamma distributed rates (HKY + Γ) was analysis. Within the latter clade (64/–), Dunnieae are sister selected (see also Table 2). For the combined analysis with to the other tribes. two partitions (chloroplast data; nuclear data), GTR + Γ + I Within Paederieae-Putorieae-Theligoneae-Rubieae was selected for the chloroplast partition. (100/72), Paederieae s.str. (Backlund & al., 2007) is sister Phylogenetic relationships in the Spermacoceae to the other tribes and Saprosma is well supported as sister alliance: the combined analyses. — The majority rule to remaining Paederieae. The (Theligoneae + (Rubieae- consensus tree from the Bayesian analysis with six data Kelloggia)) clade is poorly supported (58/75). partitions is shown in Fig. 1 with posterior probabilities Single-gene datasets. — Generally, single gene of clades above branches. Bootstrap indices for nodes in analyses (not shown) produced the same topologies as the ingroup (the Spermacoceae alliance) and major diver- those described above, but partly collapsed. There are some gences in the Rubioideae are mapped below branches. Re- deviations and “supported” differences (here arbitrarily de- sults from the two different Bayesian analyses of the com- fined as nodes receiving a Bayesian posterior probability bined dataset (six partitions; two partitions) did not differ, higher than 85%, and/or a bootstrap support higher than but a few more nodes were resolved and/or better supported 50%) are presented here. The differences mainly concern

796 TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia

groups, whose systematic positions are poorly supported species in the Spermacoceae alliance. The relationship is in the combined tree (Dunnieae, Theligoneae). well supported in Bayesian analysis, but collapses in the rps16. In the single-gene rps16 analyses, Theligoneae bootstrap analysis (Fig. 1B) and ndhF data resolve Dan- are sister to a clade containing three species of Putorieae aideae as sister to Knoxieae-Spermacoceae. Thus, there (100/86). Neohymenopogon is sister to Mycetia (99/70). are supported conflicts on the position of Danaideae and ndhF. In the ndhF trees, Danaideae are sister to the the result presented here should probably be considered Knoxieae-Spermacoceae clade (96/57). Dunnieae are sis- uncertain despite a high posterior probability and a much ter to the Anthospermeae-Argostemmateae-Paederieae- denser species representation in Danaideae; we have in- Putorieae-Theligoneae-Rubieae clade (96/-). cluded nine terminals, whereas previous studies used one atpB-rbcL spacer. Theligoneae are part of a para- or two terminals. phyletic Putorieae (98/72). Dunnieae contain a single species, the highly endan- nrITS. Anthospermeae are sister (98/–) to (Theli- gered Dunnia sinensis (Tutcher, 1905), endemic to the goneae + (Rubieae-Putorieae)) (99/–). Neohymenopogon southern Guangdong Province of China (Chen, 1999). It is sister to Mycetia (–/77). is a woody shrub with pentamerous flowers in clusters, subtended by a few showy, petaloid structures, often re- ferred to as enlarged calyx lobes (e.g., Chen, 1999; Ge & al., 2002). New investigations show, however, that they are DISCUSSION modified bracts (C. Taylor, pers. comm.; Chen & Taylor, New insights into evolutionary relationships. — in prep.) and this seems also to be in line with the original The present study reveals several new findings on the description, in which the showy structures are described phylogeny of the Spermacoceae alliance, for example as bract-like lobes near the inflorescence (Tutcher, 1905). within Argostemmateae, Anthospermeae, Danaideae and Dunnieae were sister to the Anthospermeae-Argostem- Paederieae. These results are discussed in detail below. mateae-Paederieae-Rubieae-Theligoneae clade in Rydin We were particularly interested in the phylogenetic posi- & al. (2008) and to the Paederieae-Rubieae-Theligoneae tion of Mouretia and Neohymenopogon, here sequenced clade in Rydin & al. (2009). The studies of Rydin & al. for the first time, and the results show that both genera (2008, 2009) addressed major evolutionary events within belong to Argostemmateae. Neohymenopogon parasiticus the entire family (Rubiaceae) and many nodes within the is sister to Argostemma (Fig. 1B), thus, the type species Spermacoceae alliance were poorly supported, conceiv- of a small genus comprising only three species in South- ably due to limited taxon sampling. However, despite the east Asia (Neohymenopogon) is sister to the large genus much denser taxon sampling within the Spermacoceae Argostemma, which comprises more than 160 species alliance employed here, the position of Dunnieae (sister (Govaerts & al., 2006), with a probable centre of origin to the Paederieae-Putorieae-Rubieae-Theligoneae clade in in tropical Asia. Mouretia, also sequenced here for the the present study; Fig. 1B) is again poorly supported due first time, is strongly supported as sister to remaining to conflicting information in investigated loci. species of Argostemmateae (Fig. 1B). Tange’s (1997) ar- The species of Theligonum are herbs, anisophyllous gumentation for strong similarity between Mouretia and and with unisexual, anemophilous flowers (Bremer & Mycetia is thus supported, even if the clade also comprises Manen, 2000). The genus has an interesting disjunct dis- Argostemma and Neohymenopogon. tribution, with one species in , the Mediterra- Conflicting results. — The results also reveal nean region and the Near East and three species in , several conflicts and potentially difficult phylogenetic China and Japan (one in each region). In recent studies, problems, i.e., regarding Danaideae, Dunnieae and The- Theligoneae are generally resolved as sister to Rubieae ligoneae. The position of Danaideae is perhaps the most (Nie & al., 2005; Backlund & al., 2007; Rydin & al., 2009), striking example. Danaideae comprise the woody climber but often with week support. Moreover, it has generally genus Danais and the arborescent genera Schismatoclada been the same sequences (i.e., those originally produced and , all with heterostylous flowers and capsular by Bremer & al., 1995; Andersson & Rova, 1999) that (Buchner & Puff, 1993; Bremer & Manen, 2000). have been used over again. Based on new data from two In previous studies, Danaideae has been suggested to be additional specimens of Theligonum cynocrambe, we con- sister to the remaining species of the Spermacoceae al- firm its sister relationship to Rubieae. The support is very liance (Bremer & Manen, 2000) or sister to the Anthos- low, however, and some loci support a sister relationship permeae-Argostemmateae-Paederieae-Rubieae-Theligo- between Theligoneae and Putorieae (rps16, atpB-rbcL neae clade (Robbrecht & Manen, 2006) and in a recent spacer) or between between Theligoneae and Putorieae– study (Rydin & al., 2009), Danaideae were sister to the Rubieae (nrITS). Spermacoceae-Knoxieae clade, a result which was very The morphology of Theligonum is in many re- well supported. Here, Danaideae are sister to remaining spects different from that of other Rubiaceae (see, e.g.,

797 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia TAXON 58 (3) • August 2009: 793–810 Urophylleae Lasiantheae alliance Colletoecemateae Coussareeae 1 2 . sp. 2 sp. 1 sp. 2 sp. 1 sp sp. 1 sp. 2 sp. sp. 1 sp. 3 sp. 2 . sp. 1 sp. 2 sp Maschalocorymbus corymbosus paucinervis ellipticum zeylanicus Lerchea bracteata Spiradiclis bifida elmeri Ophiorrhiza mungos strigosus Lasianthus lanceolatus Lasianthus kilimandscharicus Lasianthus pedunculatus Saldinia Saldinia Trichostachys multiflora crocea nudiflora catatii Cremocarpon lantzii formicarum capensis Psychotria holtzii Psychotria amboniana Psychotria schliebenii beccariana hydrangeifolia hymenodon Coccocysepselum hirsutum cordigera Declieuxia fruticosa Schizocolea linderi Schizocolea linderi Prismatomeris albidiflora Prismatomeris Prismatomeris subandina Schradera Schradera guianensis repens indicus Damnacanthus macrophyllus monticolum coriacea Schismatoclada psychotrioides Schismatoclada farahimpensis Schismatoclada capitellatum Xanthophytum borneense Ptilti Danais comorensis Danais xanthorrhea Danais Appunia guatemalensis Amphidasya ambigua phyllosepala citrifolia Gynochthodes Pravinaria leucocarpa Schismatoclada Danais nigra Danais fragrans Schradera Gaertnera phyllostachya Psychotria kirkii Craterispermum laurinum obvallata Craterispermum Trichostachys aurea Trichostachys Coussarea macrophylla condalia Colletoecema dewevrei Pauridiantha symplocoides Urophyllum arboreum Neurocalyx championii 95 76 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 71 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 97 98 100 97 100 56 100 100 100 100 --- 100 71 100 100 100 99 100 100 100 97 100 100 100

798 TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia Spermacoceae alliance 1 2 2 1 3 2 1 ca i 2 3 4 5 1 vat . il sp g sp. 2 sp. 1 arapentas s Neohymenopogon parasiticus Neohymenopogon parasiticus Mycetia ternifolia Manostachya ternifolia Argostemma bifolium Argostemma brachyantherum Argostemma gracile Argostemma psychotrichoides Argostemma elatostemma Argostemma hookeri Argostemma hookeri Argostemma yappii Argostemma rupestre Argostemma parvifolium Anthospermum herbaceum Dunnia sinensis Dunnia sinensis Dunnia sinensis Dunnia sinensis Saprosma fruticosum Saprosma foetens Saprosma ternatum Spermadictyon suaveolens potaninii Serissa foetida bojeriana Paederia majungensis calabrica Plocama hymenostephana Plocama aucheri Plocama pendula Theligonum cynocrambe Theligonum cynocrambe Theligonum cynocrambe Kelloggia chinensis alsinoides tinctoria arvensis hispida aristatum Paratriaina xerophila Dirichletia glaucescens Placopoda virgata platycarpa lanceolata Batopedina pulvinellata Otiophora scabra Otomeria oculata prunelloides caespitosa repens pentandrus Dibrachionostylus kaessneri Mitrasacmopsis quadrivalvis caerulea littoralis remota corymbosa Thecorchus wauensis Carpacoce P Nenax acerosa vaginata Pomax umbellata Normandia neocaledonica Coprosma granadensis Mouretia larsenii Mycetia javanica Mycetia malayana Mycetia gracilis Mycetia cauliflora Mycetia Argostemma geesinkii Argostemma involucratum Paederia sambiranensis Dunnia sinensis album Triainolepis mandrarensis Triainolepis 53 52 56 82 89 97 100 100 100 100 100 100 100 100 100 100 100 99 96 100 100 99 98 100 100 100 100 100 100 100 100 100 100 100 100 100 86 100 100 100 85 71 85 100 100 100 100 100 100 100 100 100 79 100 100 100 100 100 100 58 100 100 100 100 100 100 53 100 100 100 97 100 100 100 100 64 100 100 100 100 95 trnT-L– 100 100 rps16 , rbcL spacer, rbcL - atpB , ndhF and from the nuclear ribosomal ITS. F Posterior probabilities are given above branches. Selected bootstrap values (under parsimony) are mapped on this Bayesian tree (below branches). intron, Fig. 1A. Phylogeny of Rubioideae (overview), estimated using Bayes- ian inference of phylogeny (six-data partitions) based on molecular data regions chloroplast from

799 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia TAXON 58 (3) • August 2009: 793–810

100 Schismatoclada sp. 1 100 100 Schismatoclada psychotrioides 100 97 100 Schismatoclada farahimpensis 89 Schismatoclada sp. 2 100 Danais nigra Danaideae 100 100 Danais comorensis 100 100 99 Danais xanthorrhea 100 100 Danais sp. 2 100 Danais fragrans Parapentas silvatica 100 100 Paratriaina xerophila 100 Triainolepis mandrarensis 100 100 100 Dirichletia glaucescens 92 99 Placopoda virgata 79 96 Knoxia platycarpa Knoxieae --- 84 100 85 97 100 Batopedina pulvinellata 100 75 100 85 Otiophora scabra 100 100 53 Otomeria oculata 63 Pentanisia prunelloides 100 Kohautia caespitosa 100 Manostachya ternifolia 100 100 Dentella repens 100 100 Pentodon pentandrus 53 86 Dibrachionostylus kaessneri 66 79 Mitrasacmopsis quadrivalvis 100 100 Arcytophyllum aristatum Spermacoceae 100 100 97 Houstonia caerulea 87 100 100 96 99 96 95 57 100 Oldenlandia corymbosa --- 100 Thecorchus wauensis Carpacoce sp. 100 100 Phyllis nobla 100 100 Anthospermum herbaceum 100 100 99 Nenax acerosa 100 100 Anthospermeae 100 54 Pomax umbellata 64 100 Normandia neocaledonica 98 100 Coprosma pumila 86 Coprosma granadensis Mouretia larsenii 100 Mycetia javanica 100 100 Mycetia malayana 100 100 71 Mycetia gracilis 100 100 Mycetia cauliflora 97 --- 100 100 100 Mycetia sp. 1 100 100 Mycetia sp. 2 92 100 Neohymenopogon parasiticus 1 100 Neohymenopogon parasiticus 2 100 Argostemma bifolium 97 Argostemma gracile Argostemmateae 70 100 100 100 100 Argostemma brachyantherum 100 97 Argostemma psychotrichoides 98 100 Argostemma elatostemma 100 82 Argostemma hookeri 2 100 100 76 Argostemma hookeri 1 --- 100 100 Argostemma geesinkii 100 85 Argostemma yappii --- 98 Argostemma rupestre --- 100 Argostemma involucratum 100 Argostemma parvifolium Dunnia sinensis 1 100 100 Dunnia sinensis 2 100 100 100 Dunnia sinensis 3 98 100 Dunnia sinensis 4 Dunnieae 100 Dunnia sinensis 5 100 Saprosma fruticosum 52 Saprosma foetens 64 100 ------100 Saprosma ternatum 100 Spermadictyon suaveolens 85 100 100 Leptodermis potaninii Paederieae 100 100 Serissa foetida 91 100 Paederia sambiranensis 100 100 100 Paederia bojeriana 100 72 Paederia majungensis 100 Plocama calabrica 100 Plocama hymenostephana 100 56 100 Plocama aucheri Putorieae 100 77 Plocama pendula 100 100 Theligonum cynocrambe 2 100 89 Theligonum cynocrambe 1 58 77 Theligonum cynocrambe 3 Theligoneae 100 75 Kelloggia galioides 100 84 Kelloggia chinensis 100 Didymaea alsinoides 100 100 59 Rubia tinctoria 100 100 Galium album Rubieae 99 99 Sherardia arvensis 90 Valantia hispida

Fig. 1B. Phylogeny of the Spermacoceae alliance, estimated using Bayesian inference of phylogeny (six-data partitions) based on molecular data from chloroplast regions rbcL, rps16 intron, ndhF, atpB-rbcL spacer, trnT-L-F and from the nuclear ribosomal ITS. Posterior probabilities are given above branches, bootstrap values (under parsimony) are mapped on this Bayesian tree (below branches).

800 TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia

Rutis hauser & al., 1998, for a discussion of this) and the be related to Danais and Schismatoclada based on mor- systematic affinity of the genus has been much debated, phology; Buchner & Puff, 1993) is not investigated in the even its inclusion in Rubiaceae. Robbrecht & Manen present study. (2006) argue, based on investigations in Dessein & al. The 31 recognised species of Danais have an interest- (2005), that pollen morphology supports the relationship ing distribution; the majority of species are from Mada- between Theligoneae and Rubieae. They maintain that gascar, but a few are from and there is one Paederieae and Putorieae (and Kelloggia) have retained species each from the Comoro Islands (D. comorensis) ancestral 3-colpate pollen, whereas evolutionary changes and the African mainland (D. xanthorrhea). Because a have occurred, to 4–8-porate grains in Theligonum and Malagasy species (D. nigra) is sister to remaining Danais to pluricolpate pollen in Rubieae (Robbrecht & Manen, and the African Danais xanthorrhea is nested within the 2006). However, because Kelloggia is sister to Rubieae, genus (sister to D. comorensis from the Comoro Islands), an inferred change to several apertures in the common the topology tentatively indicates dispersal from the is- ancestor of Theligoneae and Rubiaceae requires a reversal lands in the Indian Ocean into the African mainland, but back to 3-colpate pollen in Kelloggia. Additional data, not this hypothesis needs to be further tested including more least from remaining species of Theligonum, are needed species and biogeographical analysis. to further assess relationships and character evolution in Morphology-based conclusions on Anthosper- the Putorieae-Theligoneae-Rubieae clade. meae supported. — Anthospermeae are herbs, shrubs Rate heterogeneity. — We find indications that or small trees with unisexual, wind-pollinated flowers; the the mostly herbaceous clade, the Spermacoceae alliance, fruit can be fleshy or dry and Puff (1982) argued that, e.g., overall has higher substitution rates (longer branches) than fruit characters and pollination biology (dichogamy) are its mostly woody sister clade the Psychotrieae alliance useful for subdivisions of the ca. 200 species of the group. (Fig. 2). Danaideae, here resolved as the earliest diverg- We included representatives from eight of the ing clade within the Spermacoceae alliance, has shorter twelve genera of Anthospermeae (Puff, 1982; Bremer & branch lengths than remaining Spermacoceae alliance, but Manen, 2000) and our results confirm many of the hy- this clade comprises woody species. The statistical sig- potheses established by Puff (1982). Based on morphol- nificance of these results remains to be tested, but several ogy, Puff (1982) subdivided Anthospermeae into three previous studies have indicated significant rate heteroge- biogeographically supported subtribes. Our (Phyllis + neity between annual and perennial lineages (Bousquet (Anthospermum-Nenax)) clade largely corresponds to & al., 1992; Andreasen & Baldwin, 2001), or a (negative) the afrotropical subtribe Anthospermineae sensu Puff correlation between generation time and rates of molecu- (1982), characterised by unisexual and/or protandrous her- lar evolution (Gaut & al., 1992). These results may be maphroditic flowers and dry, often dehiscent fruits (Puff, analogous and coupled to generation time rather than to 1982). We further find a clade consisting of (Normandia a herbaceous versus woody plant body. Bousquet & al. + (Coprosma-Nertera)), which corresponds to Puff’s cir- (1992) found for example no differences between woody cumpacific subtribe Coprosminae, and is characterised and herbaceous perennials. The link between generation by unisexual and/or protogynous flowers and more or time and molecular evolutionary rate variation, including less fleshy fruits (Puff, 1982). In contrast to Anderson its theoretical basis in , has been questioned (Whittle & al. (2001), we further find strong support for Puff’s & Johnston, 2003), but accumulating evidence suggests Australian subtribe Operculariinae, here represented by that rates of evolution is consistently lower in angiosperms Opercularia and Pomax, and characterised by umbel- or with longer generation times, than in angiosperms with head-like inflorescences, unisexual to protogynous her- shorter generation times (Smith & Donoghue, 2008). maphroditic flowers and fruits that open by means of an More details on evolutionary relationships in operculum (Puff, 1982). Danaideae. — The close relationship between Danais The only difference between our results and those of and Schismatoclada suggested by Buchner & Puff (1993) Puff (1982) is that the latter study included Carpacoce was supported by an unpublished rbcL sequence, accord- in subtribe Anthospermineae. In Anderson & al. (2001) ing to Bremer & Manen (2000: 61) and this has later been Carpacoce was sister to Knoxieae and they questioned confirmed by additional molecular data (Rydin & al., the inclusion of Carpacoce in Anthospermeae. However, 2008). However, previous studies investigated only one in Bremer & Eriksson (in press), Carpacoce is included specimen of each genus: the single African species Danais in Anthospermeae (higher-level relationships unresolved) xanthorrhoea and an unknown species of Schismatoclada. and here, the South African (Cape) endemic Carpacoce We have added new information from three additional is strongly supported as sister to remaining species in species of Schismatoclada and we include new Malagasy Anthospermeae, a finding that may be in accordance with and Comoro Island species of Danais. Both genera are Puff’s (1982) view that the genus is distantly related to the monophyletic as investigated here. Payera (suggested to other genera in Anthospermineae.

801 Colletoecema dewevrei Amphidasya ambigua PauridianthaP idi th paucinervisp Pauridiantha symplocoides Urophyllumpyh ll ellipticum lli p ti Maschalocorymbusyyy corymbosus Pravinaria leucocarpa Urophyllumpyhlly arboreum b Neurocalyx zeylanicus NeurocalyxNlhilhyp championii Xanthophytumpy capitellatum p Xanthophytum borneense LeLhbttrcheah bracteata Spiradiclis bifida OphiorrhizaOhip hi elmeri l Ophiorrhiza mungos Lasianthus strigosus LasianthusLithl lanceolatusl Lasianthus kilimandscharicus Lasianthus pedunculatus Saldinia sp.p 1 Saldinia sp.p 2 Trichostachys aurea Trichostachysthy spp. Faramea multiflora CoussareaChl macrophyllapy Cruckshanksia hymenodon Coccocypselum condalia CoccocysepselumClhiyp hirsutum Declieuxia cordigerag Declieuxia fruticosa Schizocolea linderi 1 Schizocolea linderi 2 Craterispermum sp. 1 Craterispermum sp. 2 Craterispermum laurinum Palicourea crocea Psychotria poeppigiana Margaritopsis acutifolia Chassalia catatii Geophila obvallata Cremocarpon lantzii Hydnophytum sp. Psychotria capensis Psychotria kirkii Psychotria holtzii Psychotria amboniana Psychotria schliebenii Prismatomeris beccariana Prismatomeris albidiflora Prismatomeris sp. 1 Prismatomeris sp. 2 Schradera subandina Schradera sp. 1 Schradera sp. 2 Schradera sp. 3 Pagamea guianensis Gaertnera phyllosepala Mostly woody species Gaertnera phyllostachya Mitchella repens Damnacanthus indicus Damnacanthus macrophyllus Appunia guatemalensis Morinda citrifolia Coelospermum monticolum Gynochthodes coriacea Gynochthodes sp. Schismatoclada sp. 1 Schismatoclada psychotrioides Schismatoclada farahimpensis Schismatoclada sp. 2 Danais nigra Danais comorensis Danais xanthorrhea Woody Woody Danais sp. 2 species Danais fragrans Parapentas silvatica Paratriaina xerophila Triainolepis mandrarensis glaucescens Placopoda virgata Knoxia platycarpa Pentas lanceolata Batopedina pulvinellata Otiophora scabra Otomeria oculata Pentanisia prunelloides Kohautia caespitosa Manostachya ternifolia Dentella repens Pentodon pentandrus Dibrachionostylus kaessneri Mitrasacmopsis quadrivalvis Arcytophyllum aristatum Houstonia caerulea Ernodea littoralis Bouvardia ternifolia Spermacoce remota Oldenlandia corymbosa Thecorchus wauensis Carpacoce sp. Phyllis nobla Anthospermum herbaceum Nenax acerosa Opercularia vaginata Pomax umbellata Normandia neocaledonica Coprosma pumila Coprosma granadensis Mouretia larsenii Mycetia javanica Mycetia malayana Mycetia gracilis Mycetia cauliflora Mycetia sp. 1 Mycetia sp. 2 Neohymenopogon parasiticus 1 Neohymenopogon parasiticus 2 Argostemma bifolium Argostemma gracile Argostemma brachyantherum Argostemma psychotrichoides Argostemma elatostemma Argostemma hookeri 2 Argostemma hookeri 1 Argostemma geesinkii Argostemma yappii Argostemma rupestre Argostemma involucratum

Argostemma parvifolium Mostly herbaceous species Dunnia sinensis 1 Dunnia sinensis 2 Dunnia sinensis 3 Dunnia sinensis 4 Dunnia sinensis 5 Saprosma fruticosum Saprosma foetens Saprosma ternatum Spermadictyon suaveolens Leptodermis potaninii Serissa foetida Paederia sambiranensis Paederia bojeriana Paederia majungensis Plocama calabrica Plocama hymenostephana Plocama aucheri Plocama pendula Theligonum cynocrambe 2 Theligonum cynocrambe 1 Theligonum cynocrambe 3 Kelloggia galioides Kelloggia chinensis Didymaea alsinoides Rubia tinctoria Galium album Sherardia arvensis 0.04 Valantia hispida TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia

Saprosma is sister to Paederieae s.str. — Re- 1996). Mouretia, Mycetia and Neohymenopogon are all lationships among species assigned to Paederieae have restricted to . Argostemma has a disjunct, long been debated. Puff (1982) considered Paederieae and palaeotropical distribution; most species occur in tropical Anthospermeae closely related and he transferred insect- Asia, but two are from the African mainland. In the sister pollinated genera from Anthospermeae to Paederieae. clade of Argostemmateae (i.e., Dunnieae-Paederieae-Pu- However, Paederieae was later shown to be paraphyletic torieae-Theligoneae-Rubieae), several genera have a broad (Bremer, 1996; Andersson & Rova, 1999; Bremer & palaeotropical or pantropical distribution (e.g., Paederia, Manen, 2000; Backlund & al., 2007), and Backlund & al. Plocama), sometimes even extending into temperate re- (2007) divided the tribe into a monophyletic Paederieae gions (e.g., Galium, Sherardia). s.str. and a re-recognised Putorieae comprising the mem- Mycetia is sister to the Argostemma-Neohymenopogon bers of the former Paederieae with fruits splitting into two clade (Figs. 1B, 3). The relationship between Mycetia and indehiscent, one-seeded mericarps (Backlund & al., 2007). Argostemma (Neohymenopogon not included) was very Saprosma (included in Paederieae by Puff, 1992; Robbre- well supported in previous studies (e.g., Bremer, 1996; cht, 1993) was weakly supported as sister to a Putorieae- Bremer & Manen, 2000), but only a single specimen from Rubieae-Theligoneae clade (Backlund & al., 2007). each genus was investigated. Here we have included six Our results support relationships among and within species of Mycetia and twelve species of Argostemma, and Paederieae and Putorieae found in Backlund & al. (2007), both genera are monophyletic as represented here. There is however, in contrast to Backlund & al. (2007), we find that little morphological support for the subclade consisting of the Southeast Asian genus Saprosma is strongly supported Mycetia + Argostemma-Neohymenopogon (Fig. 3). A possi- as sister to Paederieae s.str. Saprosma is monophyletic as ble synapomorphy is the shape of the stipules, with a change represented here (S. foetens, S. fruticosum, S. ternatum), from bilobed in Mouretia to entire in remaining species but however, the genus comprises more than 40 species and with variable states in Argostemma (entire, bilobed–cleft- Xiao & Zhu (2007) suggested that one of them (Saprosma fringed or -like; Bremer, 1989; Puff & al., 2005). crassipes) is nested within Lasianthus. The relationship between Argostemma and Neohy- Within remaining Paederieae, Paederia is sister to a menopogon is morphologically manifested in a change clade comprising Spermadictyon, Leptodermis, Serissa, all woody species with their main distribution in Southeast

Asia (Paederia has a pantropical distribution). The close clade relationship between Spermadictyon, Leptodermis and clade Serissa was recognised by Puff (1982), based on mor- ouretia Argostemma Argostemma phology. M Mycetia Neohymenopogonpsychotrioides parvifolium Phylogeny and morphology of Argostemma- 4b 9a 7a 6b 10 1b 6c teae. — Argostemmateae are here shown to comprise 5b Mouretia, Mycetia, Argostemma and Neohymenopo- 7c gon (Figs. 1B, 3). A literature survey, based on Wallich 9b 8 (1824), Hooker (1880), Bremer (1989), Tange (1997), 7b Bremer & Manen (2000) and Puff & al. (2005), reveals 9a 6a 5a that Argostemma, Mouretia, Mycetia and Neohymenopo- 4a 3 2 gon all have hermaphroditic flowers, bilocular ovaries 1a (sometimes 3–5-celled in Mycetia), fruits crowned by a persistent calyx lobe, and many small seeds (dust seeds). Terminal cymes and stamens inserted near the base of Fig. 3. An illustration to the discussion of the evolution of selected characters in Argostemmateae. Argostemma is a the corolla tube are also ancestral in the group, with a large genus and many characters are variable within the ge- change in Neohymenopogon to stamens inserted in the nus. We do not cover the full variation here. 1, ovaries, a) bi- upper part of the corolla. The showy bract, which sub- locular, b) 2–5-celled; 2, fruits crowned by persistent calyx tends the inflorescence is unique to Neohymenopogon lobes; 3, dust seeds; 4, inflorescence, a) terminal cyme, b) within Argostemmateae, but found in some distantly re- umbelliform; 5, stamens, a) inserted near corolla base, b) higher up; 6, fruit, a) fleshy capsule, b) , c) dry capsule; lated rubiaceous tribes (Psychotrieae, Robbrecht, 1988; 7, stipules, a) bi-lobed, b) entire, c) variable shape; 8, isosty- Hymenodictyeae, Razafimandimbison & Bremer, 2006; lous flowers; 9, shoots, a) often anisophyllous, b) isophyl- Dunnieae, Rydin & al., 2008; see also Classen-Bockhoff, lous; 10, showy bract subtending the inflorescence.

◄ Fig. 2. Rate heterogeneity in Rubioideae as indicated by a phylogram based on the Bayesian analysis of the combined dataset. The mostly woody lineages, the Psychotrieae alliance and Danaideae are marked with bold branches. The mostly herbaceous Spermacoceae alliance (except Danaideae) is marked with thin branches. Gray indicates outgroups.

803 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia TAXON 58 (3) • August 2009: 793–810

to isostylous flowers (Fig. 3). Many plants in Rubioideae Neo hymenopogon and Mouretia belong to Argostem- are heterostylous (e.g., Verdcourt, 1958: 227) and so are mateae, Saprosma is sister to Paederieae s.str., Carpa- most species of Mouretia (Tange, 1997) and Mycetia (Puff coce is sister to the remaining species in Anthospermeae. & al., 2005). The Argostemma-Neohymenopogon clade The results further indicate a possible correlation between might be further supported by a change to isophyllous molecular substitution rate and generation time, something shoots from mostly anisophyllous, which appear ancestral which has been questioned and considered controversial in Argostemmateae, and by an epiphytic habit. The latter for plants. characters are variable within the genera though, not least However, our results also reveal conflicts and perhaps in Mycetia and Argostemma. difficult phylogenetic problems. The small tribe Danaid- Fruit characters are variable in Argostemmateae, eae is here sister to the remaining Spermacoceae alliance but the fruit is generally crowned by the persistent ca- with high posterior probability, but the topology conflicts lyx lobes in all genera (occurs also in other rubiaceous with well-supported results in a recent study (Rydin & al., plants). Mouretia fruits are fleshy capsules, which open 2009). Theligoneae and Dunnieae are additional examples by an operculum (Tange, 1997) and the fruits become of small isolated clades, which represent unique lineages aggregated. The fruits of Mycetia are baccate and inde- within the Spermacoceae alliance, also in terms of mor- hiscent, but crowned by small calyx lobes. Argostemma phology (see, e.g., Rutishauser & al., 1998; Rydin & al., has retained the ancestral state with a fleshy capsule, 2008). The uncertainty of their relationships to other tribes which opens by an operculum, but Neohymenopogon has in the group is emphasised in the present study, mainly a capsule that splits into valves. There are no reports of due to conflicting information. fleshiness in the fruits of Neohymenopogon (mature fruits Because of the apparent conflicts between results from were not available to us). different loci in the chloroplast genome, it might conceiv- Our results provide a first attempt to test the monophyly ably be difficult to confidently resolve these phylogenetic of Argostemma, as well as hypotheses on intra-generic re- problems only by analysing larger sets of molecular data. lationships based on morphology, using molecular data. Detailed morphological and histological information from Bremer (1989) studied Argostemma in Borneo and argued reproductive structures (i.e., from serial sectioning and that the species could be divided into at least three major SEM investigations) may allow to discriminate between subclades, of which we have included specimens from two: alternative hypotheses indicated by molecular data. Gross the parvifolium group and the psychotrioides group. morphological characters are, however, less likely to be use- The parvifolium group (Argostemma parvifolium and ful because of the great diversity of life forms, morphology A. elatostemma with a broader distribution in Southeast and ecology among the numerous species of Rubiaceae. Asia, and the Borneo endemics A. rupestre and A. geesinkii plus eight more species not included in the present study) is defined, e.g., by anisophyllous shoots, rotate corollas and a glabrous style (Bremer, 1989). It is, as represented here, TAXONOMIC IMPLICATIONS very well supported by molecular data (Fig. 1B) and further Based on the new results we re-define the tribe Ar- includes three species not investigated in Bremer (1989): gostemmateae to comprise four genera: Argostemma, A. parvifolium var. involucratum, A. yappii and A. hook- Mouretia, Mycetia and Neohymenopogon. Robbrecht eri, all from and Sumatra. The clade appears to (1993) transferred Saprosma to Paederieae based on mor- ancestrally have had a broad distribution in Southeast Asia. phological observations by Puff (1992) but Paederieae sensu The psychotrioides group (Argostemma psychotrio- Robbrecht (1993) comprised genera from the Putorieae- ides, A. gracile, A. brachyantherum and eleven additional Rubieae-Theligoneae clade as well as from Paederieae s.str. species not investigated here) is also very well supported by We re-define Paederieae s.str. to comprise Leptodermis, molecular data (Fig. 1B). Morphologically, it is defined, e.g., Paederia, Saprosma, Serissa and Spermadictyon. by umbelliform inflorescences and the style is often much longer than the stamens (Bremer, 1989). All species of the Family – Rubiaceae Juss. psychotrioides group are restricted to Borneo, but its sister- Subfamily – Rubioideae Verdc. taxon (Argostemma bifolium) is from Peninsular Malaysia. Conclusions. — This study presents new insights Tribe Argostemmateae Bremek. ex Verdc. in Bull. Jard. into the phylogeny of the Spermacoceae alliance (Ru- Bot. État Bruxelles 28: 281. 1958 – Type: Argostemma biaceae). Some genera (Neohymenopogon and Mouretia) Wall. in W. Roxburgh (1824). are sequenced and analysed in a modern cladistic frame- Description: Herbs (mainly), or shrubs to small work for the first time and some previous uncertainties treelets, iso- or anisophyllous. Stipules entire or slightly (e.g., the position of Saprosma and relationships within cleft, rarely leaf-like. Flowers hermaphroditic. Stamens Anthospermeae) are also resolved with strong support; usually inserted at base of the corolla or higher up (in

804 TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia

Neohymenopogon), adnate into an anther cone or free. An- in the Psychotria complex (Rubiaceae, Psychotrieae). Syst. thers open with vertical slits or rarely with pores. Ovary Geogr. Pl. 72: 167–202. 2(–6)-locular. Fruit a capsule (often fleshy) or a berry Andersson, L. & Antonelli, A. 2005. Phylogeny of the tribe (Mycetia), with many small seeds. Cinchoneae (Rubiaceae), its position in Cinchonoideae, and description of a new genus, Ciliosemina. Taxon 54: 17–28. Genera included: Argostemma, Mouretia, Mycetia, Andersson, L. & Persson, C. 1991. Circumscription of the Neohymenopogon. tribe Cinchoneae (Rubiaceae) — a cladistic approach. Pl. Useful studies: Wallich (1824), Hooker (1880), Bremer Syst. Evol. 178: 65–94. (1989), Puff & al., (1995), Tange (1997), Bremer & Manen Andersson, L. & Rova, J.H.E. 1999. The rps16 intron and (2000), Puff & al., (2005), the present study. the phylogeny of Rubioideae (Rubiaceae). Pl. Syst. Evol. 214: 161–186. Tribe Paederieae DC., Prodr. 4: 342, 470. 1830 – Type: Andreasen, K. & Baldwin, B.G. 2001. Unequal evolutionary rates between annual and perennial lineages of checker Paederia L. (1767). mallows (Sidalcea, Malvaceae): evidence from 18S-26S Description: Small shrubs or climbers (Paederia), rDNA internal and external transcribed spacers. Molec. often foetid. Stipules divided (Paederia) or entire. Flow- Biol. Evol. 18: 936–944. ers 4- (Saprosma) to 5-merous, stamens inserted in the Backlund, M., Bremer, B. & Thulin, M. 2007. Paraphyly corolla tube, ovary 2–5-locular, with a single erect ovule of Paederieae, recognition of Putorieae and expansion of in each locule. Fruits dry, dehishent into valves (Lepto- Plocama (Rubiaceae-Rubioideae). Taxon 56: 315–328. Backlund, M., Oxelman, B. & Bremer, B. 2000. Phylogenetic dermis, Paederia, Spermadictyon), or fleshy, indehiscent relationships within the based on ndhF and (Saprosma, Serissa). rbcL sequences, with particular reference to the Logania- Genera included: Leptodermis, Paederia, Saprosma, ceae. Amer. J. Bot. 87: 1029–1043. Serissa, Spermadictyon. Bennet, S.S.R. 1981. Rubiaceae. Indian Forester 107: 436–437. Useful studies: Puff (1982), Robbrecht (1982), Bremer Bousquet, J., Strauss, S.H., Doerksen, A.H. & Price, R.A. & Manen (2000), Robbrecht & Manen (2006), Backlund 1992. Extensive variation in evolutionary rate of rbcL & al., (2007), the present study. gene-sequences among seed plants. Proc. Natl. Acad. Sci. U.S.A. 89: 7844–7848. Bremekamp, C.E.B. 1952. The African species of Oldenlandia L. sensu Hiern et K. Schumann. Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2 48: 1–297. ACKNOWLEDGMENTS Bremekamp, C.E.B. 1966. Remarks on the position, the de- We thank the curators of the herbaria AAU, BR, GB, K, limitation and the subdivision of the Rubiaceae. Acta Bot. P, S, and UPS for access to herbarium material, Arnaud Mouly Neerl. 15: 1–33. Bremer, B. 1989. The genus Argostemma (Rubiaceae-Argostem- (Bergius Foundation, Stockholm University) for providing mate- mateae) in Borneo. Ann. Missouri Bot. Gard. 76: 7–49. rial of Danais comorensis and Mouretia larsenii, Mikael Bylund Bremer, B. 1996. Phylogenetic studies within Rubiaceae and for technical assistance and access to powerful computers and relationships to other families based on molecular data. the reviewers for critical comments on the text. The study was Opera Bot. Belg. 7: 33–50. supported by grants from the Swedish Research Council to C.R. Bremer, B., Andreasen, K. & Olsson, D. 1995. Subfamilial and B.B., and the Knut and Alice Wallenberg Foundation to B.B. and tribal relationships in the Rubiaceae based on rbcL sequence data. Ann. Missouri Bot. Gard. 82: 383–397. Bremer, B., Bremer, K., Heidari, N., Erixon, P., Olmstead, R.G., Anderberg, A.A., Källersjö, M. & Barkhorda- rian, E. 2002. Phylogenetics of based on 3 coding LITERATURE CITED and 3 non-coding chloroplast DNA markers and the utility Akaike, H. 1973. Information theory as an extension of the of non-coding DNA at higher taxonomic levels. Molec. maximum likelihood principle. Pp. 267–281 in: Petrov, Phylog. Evol. 24: 274–301. B.N. & Csaki, F. (eds), Second International Symposium of Bremer, B. & Eriksson, T. 2009. Timetree of Rubiaceae — phy- Information Theory. Akademiai Kiado, Budapest. logeny and dating the family, subfamilies and tribes. Int. Alejandro, G.D., Razafimandimbison, S.G. & Liede- J. Pl. Sci. 170: 766–793. Schumann, S. 2005. Polyphyly of Mussaenda inferred Bremer, B., Jansen, R.K., Oxelman, B., Backlund, M., from ITS and trnT-F data and its implication for generic Lantz, H. & Kim, K. 1999. More characters or more taxa limits in Mussaendeae (Rubiaceae). Amer. J. Bot. 92: for a robust phylogeny–case study from the coffee family 544–557. (Rubiaceae). Syst. Biol. 48: 413–435. Anderson, C.L., Rova, J.H.E. & Andersson, L. 2001. Mo- Bremer, B. & Manen, J.F. 2000. Phylogeny and classification lecular phylogeny of the tribe Anthospermeae (Rubiaceae): of the subfamily Rubioideae (Rubiaceae). Pl. Syst. Evol. systematic and biogeographic implications. Austral. Syst. 225: 43–72. Bot. 14: 231–244. Bremer, B. & Thulin, M. 1998. Collapse of Isertieae, re-estab- Andersson, L. 2001. Margaritopsis (Rubiaceae, Psychotrieae) lishment of Mussaendeae, and a new genus of Sabiceeae is a pantropical genus. Syst. Geogr. Pl. 71: 73–85. (Rubiaceae); phylogenetic relationships based on rbcL Andersson, L. 2002. Relationships and generic circumscription data. Pl. Syst. Evol. 211: 71–92.

805 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia TAXON 58 (3) • August 2009: 793–810

Buchner, R. & Puff, C. 1993. The genus complex Danais- Manen, J.F. & Natali, A. 1995. Comparison of the evolu- Schismatoclada-Payera (Rubiaceae). Characters states, tion of ribulose-1,5-biphosphate carboxylase (rbcL) and generic delimitation and taxonomic position. Adansonia atpB-rbcL noncoding spacer sequences in a recent plant 1–4: 23–74. group, the tribe Rubieae (Rubiaceae). J. Molec. Evol. 41: Burnham, K.P. & Anderson, D.R. 2003. Model Selection and 920–927. Multimodel Inference: A Practical Information-theoretic Manen, J.F., Natali, A. & Ehrendorfer, F. 1994. Phylogeny of Approach, 2nd ed. Springer, New York. Rubiaceae-Rubieae inferred from the sequence of a cpDNA Chen, W.C. 1999. Dunnia Tutch. Pp. 233–235, plate 258 in: Lo, intergene region. Pl. Syst. Evol. 190: 195–211. H.S., (ed.), Flora Reipublicae Popularis Sinicae, vol. 71/2. Mouly, A., Razafimandimbison, S.G., Achille, F., Haever- Science Press, Beijing. [In Chinese.] mans, T. & Bremer, B. 2007. Phylogenetic placement of Church, S.A. 2003. of Houstonia (Ru- Rhopalobrachium fragrans (Rubiaceae): evidence from biaceae): descending aneuploidy and breeding system evo- molecular (rps16 and trnT-F) and morphological data. Syst. lution in the radiation of the lineage across North America. Bot. 32: 872–882. Molec. Phylog. Evol. 27: 223–238. Nakamura, K., Chung, S.W., Kokubugata, G., Denda, T. & Church, S.A. & Taylor, D.R. 2005. Speciation and hybridiza- Yokota, M. 2006. Phylogenetic systematics of the mono- tion among Houstonia (Rubiaceae) species: the influence typic genus Hayataella (Rubiaceae) endemic to Taiwan. of polyploidy on reticulate evolution. Amer. J. Bot. 92: J. Pl. Res. 119: 657–661. 1372–1380. Natali, A., Manen, J.F. & Ehrendorfer, F. 1995. Phylog- Classen-Bockhoff, R. 1996. A survey of -like inflo- eny of the Rubiaceae Rubioideae, in particular the tribe rescences in the Rubiaceae. Opera Bot. Belg. 7: 329–367. Rubieae — evidence from a noncoding chloroplast DNA- Dessein, S., Ochoterena, H., De Block, P., Lens, F., sequence. Ann. Missouri Bot. Gard. 82: 428–439. Robbrecht, E., Schols, P., Smets, E., Vinckier, S. & Nepokroeff, M., Bremer, B. & Sytsma, K.J. 1999. Reorga- Huysmans, S. 2005. Palynological characters and their nization of the genus Psychotria and tribe Psychotrieae phylogenetic signal in Rubiaceae. Bot. Rev. 71: 354–414. (Rubiaceae) inferred from ITS and rbcL sequence data. Galtier, N., Gouy, M. & Gautier, C. 1996. SEAVIEW and Syst. Bot. 24: 5–27. PHYLO_WIN: two graphic tools for sequence align- Nie, Z.L., Wen, J., Sun, H. & Bartholomew, B. 2005. Mono- ment and molecular phylogeny. Comput. Appl. Biosci. 12: phyly of Kelloggia Torrey ex Benth. (Rubiaceae) and evo- 543–548. lution of its intercontinental disjunction between west- Gaut, B.S., Muse, S.V., Clark, W.D. & Clegg, M.T. 1992. ern North America and eastern Asia. Amer. J. Bot. 92: Relative rates of nucleotide substitution at the rbcL locus 642–652. of monocotyledonous plants. J. Molec. Evol. 35: 292–303. Novotny, V., Basset, Y., Miller, S.E., Weiblen, G.D., Bremer, Ge, X.J., Chiang, Y.C., Chou, C.H. & Chiang, T.Y. 2002. B., Cizek, L. & Drozd, P. 2002. Low host specificity of Nested clade analysis of Dunnia sinensis (Rubiaceae), a herbivorous insects in a tropical forest. Nature 416: 841– monotypic genus from China based on organelle DNA 844. sequences. Cons. Genet. 3: 351–362. Nylander, J.A.A. 2004a. MrAIC.pl. Program distributed by the Govaerts, R., Andersson, L., Robbrecht, E., Bridson, D., author, Evolutionary Biology Centre, Uppsala University, Davis, A., Schanzer, I. & Sonke, B. 2006. World Checklist Uppsala. http://www.abc.se/~nylander/. of Rubiaceae. The Board of Trustees of the Royal Botanic Nylander, J.A.A. 2004b. MrModeltest v2. Program distributed Gardens, Kew. by the author, Evolutionary Biology Centre, Uppsala Uni- Groeninckx, I., Dessein, S., Ochoterena, H., Persson, C., versity, Uppsala. http://www.abc.se/~nylander/. Motley, T., Kårehed, J., Bremer, B., Huysmans, S. & Oxelman, B. 1996. RAPD patterns, nrDNA ITS sequences Smets, E. 2009 Phylogeny of the herbaceous tribe Sper- and morphological patterns in Silene section Sedoineae macoceae (Rubiaceae) based on plastid DNA data. Ann. (Caryophyllaceae). Pl. Syst. Evol. 201: 93–116. Missouri Bot. Gard. 96: 109–132. Oxelman, B., Liden, M. & Berglund, D. 1997. Chloroplast Guindon, S. & Gascuel, O. 2003. A simple, fast, and accurate rps16 intron phylogeny of the tribe Sileneae (Caryophyl- algorithm to estimate large phylogenies by maximum like- laceae). Pl. Syst. Evol. 206: 393–410. lihood. Syst. Biol. 52: 696–704. Piesschaert, F., Andersson, L., Jansen, S., Dessein, S., Hasegawa, M., Kishino, H. & Yano, T. 1985. Dating the hu- Robbrecht, E. & Smets, E. 2000. Searching for the man-ape splitting by a molecular clock of mitochondrial taxonomic position of the African genus Colletoecema DNA. J. Molec. Evol. 22: 160–174. (Rubiaceae): morphology and anatomy compared to an Hooker, J.D. 1880. Flora of British . Reeve, London. rps16-intron analysis of the Rubioideae. Canad. J. Bot. 78: Huelsenbeck, J.P. & Ronquist, F.R. 2001. MRBAYES: Bayes- 288–304. ian inference of phylogenetic trees. Bioinformatics 17: Pitard, J. 1922. Rubiaceae. Pp. 71 in: Lecomte, P.H. (ed.), Flore 754–755. Génerale de L’Indo-Chine. Masson et Cie, Paris. Kårehed, J. & Bremer, B. 2007. The systematics of Knoxieae Popp, M. & Oxelman, B. 2001. Inferring the history of the (Rubiaceae) — molecular data and their taxonomic conse- polyploid Silene aegaea (Caryophyllaceae) using plastid quences. Taxon 56: 1051–1076. and homoeologous nuclear DNA sequences. Molec. Phy- Kårehed, J., Groeninckx, I., Dessein, S., Motley, T. & log. Evol. 20: 474–481. Bremer, B. 2008. The phylogenetic utility of chloroplast Posada, D. & Buckley, T.R. 2004. Model selection and model and nuclear DNA markers and the phylogeny of the Ru- averaging in phylogenetics: advantages of Akaike infor- biaceae tribe Spermacoceae. Molec. Phylog. Evol. 49: mation criterion and Bayesian approaches over likelihood 843–866. ratio tests. Syst. Biol. 53: 793–808.

806 TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia

Posada, D. & Crandall, K.A. 2001. Selecting the best-fit model Rare and enigmatic genera (Dunnia, Schizocolea, Col- of nucleotide substitution. Syst. Biol. 50: 580–601. letoecema), sisters to species-rich clades: phylogeny and Puff, C. 1982. The delimitation of the tribe Antospermeae and aspects of conservation biology in the coffee family. Molec. its affinities to the Paederieae (Rubiaceae). Bot. J. Linn. Phylog. Evol. 48: 74–83. Soc. 84: 355–377. Schwartz, G. 1978. Estimating the dimensions of a model. Ann. Puff, C. 1992. On the correct tribal position of Saprosma Bl. Statist. 6: 461–464. (Rubiaceae). Pp. 34 in: Second Flora Malesiana Sympo- Smedmark, J.E.E., Rydin, C., Razafimandimbison, S.G., sium, Yogyakarta 7–12 September 1992. Programme & Khan, S.A., Liede-Schumann, S. & Bremer, B. 2008. Summaries of Papers and Posters. Herbarium Bogoriense, A phylogeny of Urophylleae (Rubiaceae) based on rps16 Bogor. [Abstract.] intron data. Taxon 57: 24–32. Puff, C., Chayamarit, K. & Chamchumroon, V. 2005. Ru- Smith, S.A. & Donoghue, M.J. 2008. Rates of molecular evo- biaceae of Thailand: A Pictorial Guide to Indigenous and lution are linked to life history in flowering plants. Science Cultivated Genera. The Forest Herbarium, National Park, 322: 86–89. Wildlife and Plant Conservation Department, Bangkok. Sonké, B., Dessein, S., Taedoumg, H., Groeninckx, I. & Rob- Puff, C., Igersheim, A., Buchner, R. & Rohrhofer, U. 1995. brecht, E. 2008. A new speices of Colletoecema (Rubia- United stamens of Rubiaceae. Morphology, anatomy; their ceae) from southern Cameroon with a discussion of rela- role in pollination ecology. Ann. Missouri Bot. Gard. 82: tionships among basal Rubioideae. Blumea 53: 533–547. 357–382. Staden, R. 1996. The Staden sequence analysis package. Molec. Rambaut, A. 1996. Se-Al: Sequence Alignment Editor. Avail- Biotechnol. 5: 233–241. able at http://evolve.zoo.ox.ac.uk. Struwe, L., Thiv, M., Kadereit, J.W., Pepper, A.S.-R., Mot- Razafimandimbison, S.G. & Bremer, B. 2006. Taxonomic ley, T.J., White, P.J., Rova, J.H.E., Potgieter, K. & Al- revision of the tribe Hymenodictyeae (Rubiaceae, Cin- bert, V.A. 1998. Saccifolium (Saccifoliaceae), an endemic chonoideae). Bot. J. Linn. Soc. 152: 331–386. of Sierra de la Neblina on the Brazilian-Venezuelan border, Razafimandimbison, S.G., Rydin, C. & Bremer, B. 2008. is related to temperate-alpine lineages of Gentianaceae. Evolution and trends in the Psychotrieae alliance (Rubia- Harvard Pap. Bot. 3: 199–214. ceae) — a rarely reported evolutionary change of many- Swofford, D.L. 1998. PAUP*. Phylogenetic analysis using par- seeded carpels from one-seeded carpels. Molec. Phylog. simony (*and other methods). Sinauer, Sunderland. Evol. 48: 207–223. Taberlet, P., Gielly, L., Pautou, G. & Bouvet, J. 1991. Univer- Robbrecht, E. 1982. Pollen morphology of the tribes Antho- sal primers for amplification of three non-coding regions spermeae and Paederieae (Rubiaceae) in relation to tax- of chloroplast DNA. Pl. Molec. Biol. 17: 1105–1109. onomy. Bull. Jard. Bot. Natl. Belg. 52: 349–366. Tange, C. 1997. A revision of the genus Mouretia (Rubiaceae). Robbrecht, E. 1988. Tropical woody Rubiaceae. Opera Bot. Nord. J. Bot. 17: 123–132. Belg. 1: 1–271. Tavare, S. 1986. Some probabilistic and statistical problems on Robbrecht, E. 1993. Supplement to the 1988 outline of the clas- the analysis of DNA sequences. Pp. 57–86 in: Miura, R.M. sification of the Rubiaceae. Index to genera. Pp. 173–196 (ed.), Some Mathematical Questions in Biology — DNA Se- in: Robbrecht, E. (ed.), Advances in Rubiaceae Macro- quence Analysis. American Mathematical Society, Provi- systematics. Opera Botanica Belgica 6. National Botanic dence. Garden of Belgium, Meise. Thulin, M. & Bremer, B. 2004. Studies in the tribe Sperma- Robbrecht, E. & Manen, J.-F. 2006. The major evolutionary coceae (Rubiaceae-Rubioideae): the circumscriptions of lineages of the coffee family (Rubiaceae, angiosperms). Amphiasma and Pentanopsis and the affinities of Phylo- Combined analysis (nDNA and cpDNA) to infer the posi- hydrax. Pl. Syst. Evol. 247: 233–239. tion of Coptosapelta and Luculia, and supertree construc- Tosh, J., De Block, P., Davis, A.P., Dessein, S., Robbrecht, tion based on rbcL, rps16, trnL-trnF and atpB-rbcL data. E. & Smets, E.F. 2008. The tribal placement of the mono- A new classification in two subfamilies, Cinchonoideae specific tropical African genus Petitiocodon (Rubiaceae) and Rubioideae. Syst. Geogr. Pl. 76: 85–146. based on molecular data and morphology. Blumea 53: Ronquist, F. & Huelsenbeck, J.P. 2003. MrBayes 3: Bayesian 549–565. phylogenetic inference under mixed models. Bioinformat- Tutcher, W.J. 1905. Description of some new species, and notes ics 19: 1572–1574. on other Chinese plants. J. Linn. Soc. Bot. 37: 58–70. Rova, J.H.E., Delprete, P.G., Andersson, L. & Albert, V.A. Verdcourt, B. 1958. Remarks on the classification of the Rubia- 2002. A trnL-F cpDNA sequence study of the Condam- ceae. Bull. Jard. Bot. État Bruxelles 28: 209–281. ineeae-Rondeletieae-Sipaneeae complex with implica- Wallich, N. 1824. Hymenopogon Wall. Pp. 156–158 in: Rox- tions on the phylogeny of the Rubiaceae. Amer. J. Bot. burgh, W. (ed.), Flora Indica. Mission press, Serampore. 89: 145–159. Whittle, C.A. & Johnston, M.O. 2003. Broad-scale analysis Rutishauser, R., Ronse Decraene, L.P., Smets, E. & Men- contradicts the theory that generation time affects mo- doza-Heuer, I. 1998. Theligonum cynocrambe: develop- lecular evolutionary rates in plants. J. Molec. Evol. 56: mental morphology of a peculiar rubiaceous herb. Pl. Syst. 223–233. Evol. 210: 1–24. Xiao, L.Q. & Zhu, H. 2007. Paraphyly and phylogenetic rela- Rydin, C., Kainulainen, K., Razafimandimbison, S.G., tionships in Lasianthus (Rubiaceae) inferred from chloro- Smedmark, J.E.E. & Bremer, B. 2009. Deep divergences plast rps16 data. Bot. Stud. 48: 227–232. in the coffee family and the systematic position of Acran- Yang, Z. 1993. Maximum likelihood estimation of phylogeny thera. Pl. Syst. Evol. 278: 101–123. from DNA sequences when substitution rates differ over Rydin, C., Razafimandimbison, S.G. & Bremer, B. 2008. sites. Molec. Biol. Evol. 10: 1396–1401.

807 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia TAXON 58 (3) • August 2009: 793–810

Appendix. Information on included species and GenBank accessions. Taxon, classification, voucher (if applicable), rbcL, rps16, ndhF, atpB-rbcL spacer, trnT/F, nrITS (– indicates missing data) Amphidasya ambigua (Standl.) Standl., Rub-Urophylleae, –, Y1184407, AF12927113, –, EU14533736, EU14557636, EU14538336; Anthospermum herbaceum L. f., Rub-Anthospermeae, –, X8362301, EU14549636, AJ23628409, AJ23402802, EU14554436, EU14535536; Arcytophyllum aristatum Standl., Rub-Sperma- coceae, Bremer 3371 (UPS), AJ28859502, AF33334810, FJ695282*, FJ695343*, AF33334910, AM18206132; Argostemma bifolium Ridl., Rub-Argostemmateae, B & K Bremer 1797 (S), FJ695220*, –, FJ695283*, FJ695344*, FJ695396*, FJ695428*; Argostemma brachyantherum Stapf, Rub-Argostemmateae, Beaman 8931 (S), FJ695221*, FJ695252*, FJ695284*, FJ695345*, FJ695397*, FJ695429*; Argostemma elatostemma Hook. f., Rub-Argostemmateae, B & K Bremer 1722 (S), FJ695222*, –, –, FJ695346*, –, –; Argostemma geesinkii B. Bremer, Rub-Argostemmateae, Beaman 8935 (S), FJ695223*, FJ695253*, FJ695285*, FJ695347*, FJ695398*, FJ695430*; Argostemma gracile Stapf, Rub-Argostemmateae, Beaman 8885 (S), FJ695224*, FJ695254*, FJ695286*, FJ695348*, FJ695399*, FJ695431*; Argostemma hookeri King Spec. 1, Rub-Argostemmateae, –, Z6878811, EU14549736, EU14541936, AJ23403202, EU14554536, EU14535636; Argostemma hookeri King Spec. 2, Rub-Argostemmateae, Wanntorp 88-27 (S), FJ695225*, FJ695255*, FJ695287*, FJ695349*, FJ695400*, FJ695432*; Argostemma involucratum Hemsl., Rub-Argostemmateae, Wanntorp 3047 (S), FJ695226*, FJ695256*, FJ695288*, FJ695350*, FJ695401*, FJ695433*; Argostemma parvifolium Benn., Rub-Argostemmateae, B & K Bremer 1811 (S), FJ695227*, FJ695257*, FJ695289*, FJ695351*, FJ695402*, FJ695434*; Argostemma psychotrichoides Ridl., Rub-Argostemmateae, B & K Bremer 1744 (S), FJ695228*, FJ695258*, FJ695290*, FJ695352*, –, FJ695435*; Argostemma rupestre Ridl., Rub-Argostemmateae, B & K Bremer 1675 (S), FJ695229*, FJ695259*, –, FJ695353*, –, FJ695436*; Argostemma yappii King, Rub-Argostemmateae, B & K Bremer 1609 (S), FJ695230*, FJ695260*, –, FJ695354*, FJ695403*, FJ695437*; Batopedina pulvinellata Robbr., Rub-Knoxieae, Dessein 264 (BR), AJ28859602, AM11728522, FJ695291*, FJ695355*, AM11734922, AM26698923; Bouvardia ternifolia (Cav.) Schltdl. (synonym: Bouvardia glaberrima Engelm.), Rub-Spermacoceae, Forbes s.n. (S), X8362601, AF00275805, FJ695292*, X7647840, DQ35916503, DQ35888403; Carpacoce sp. Sond., Rub-Anthospermeae, B. Bremer & al. 4365, 4385 (UPS), FJ695231*, FJ695261*, FJ695293*, FJ695356*, FJ695404*, FJ695438*; Chassalia catatii Drake ex Bremek., Rub-Psychotrieae, –, AM94530537, AM94533137, AM94528337, AM94525137, AM94536337, AM94521837; Coccocypse- lum condalia Pers., Rub-Coussareeae, –, AM11721722, EU14549936, EU14542036, EU14532436, EU14554736, EU14535836; Coccocypselum hirsutum Bartl. ex DC., Rub-Coussareeae, –, X8714522, EU14550036, EU14542136, EU14532536, EU14554836, EU14535936; Coelospermum fragrans (Montrouz.) Baill. ex Guillaumin (synonym: Coelospermum monticolum Baill. ex Guillaumin.), Rub-Morindeae, –, AF33164410, AF00143805, AM94525537, AM94522137, AM94533437, AM94519437; Colletoecema dewevrei (De Wild.) E.M.A. Petit, Rub-Colletoecemeae, –, EU14545736, AF12927213, EU14540936, DQ1317136, EU14553236, EU14535336; Coprosma granadensis Mutis ex L. f. (synonym: (Mutis ex L. f.) Druce), Rub-Anthospermeae, –, X8365401, AF00274105, –, –, AF15262306, –; Coprosma pumila Hook. f., Rub-Anthospermeae, Forbes s.n. (S), X8714601, FJ695262*, FJ695294*, –, FJ695405*, FJ695439*; Coussarea hydrangeifolia (Benth.) Benth. & Hook. f. ex Müll.Arg., Rub-Coussareeae, –, EU14546036, EU14550136, EU14542236, EU14532636, EU14554936, EU14536036; Coussarea macrophylla (Mart.) Müll. Arg., Rub-Coussareeae, –, Y1184707, AF00404005, –, –, AF15261206, –; Craterispermum laurinum (Poir.) Benth., Rub-Craterispermeae, –, AM94530037, AM94532537, AM94527637, AM94524337, AM94535637, AM94521237; Craterispermum sp. Benth. Spec. 1, Rub-Craterispermeae, –, AM94529737, –, AM94527337, AM94524137, AM94535337, AM94520937; Craterispermum sp. Benth. Spec. 2, Rub- Craterispermeae, –, AM94529837, AM94531337, AM94527437, AM94524237, AM94535437, AM94521037; Cremocarpon lantzii Bremek., Rub-Psychotrieae, –, AM11722222, AM11729622, –, –, AM11735622, –; Cruckshanksia hymenodon Hook. & Arn., Rub-Coussareeae, –, AJ28859902, EU14550236, –, AJ23400402, EU14555036, –; Damnacanthus indicus C.F. Gaertn., Rub-Mitchelleae, –, Z6879311, AF33164710, AM94525637, AJ23401502, AM94533537, AY51406129; Damnacanthus macrophyllus Siebold ex Miq., Rub-Mitchelleae, –, AM94528537, AM94530837, AM94525737, AM94522237, AM94533637, AM94519537; Danais comorensis Drake, Rub-Danaideae, Mouly 687 (P), FJ695232*, FJ695263*, –, FJ695358*, FJ695406*, –; Danais fragrans (Lam.) Pers., Rub-Dan- aideae, Eriksson & al. 966 (S), FJ695233*, FJ695264*, FJ695295*, FJ695359*, FJ695407*, –; Danais sp. Comm. ex Vent., Rub-Danaideae, Eriksson & al. 1032 (S), FJ695234*, FJ695265*, FJ695296*, FJ695360*, FJ695408*, FJ695440*; Danais nigra Homolle, Rub-Danaideae, Kårehed & al. 254 (UPS), FJ695235*, FJ695266*, FJ695297*, FJ695361*, FJ695409*, FJ695441*; Danais xanthorrhoea (K. Schum.) Bremek., Rub-Danaideae, –, Z6879411, AM11729722, AJ23629309, AJ23401902, DQ66213819, EU14536436; Declieuxia cordigera Mart. & Zucc. ex Schult. & Schult. f., Rub-Coussareeae, –, AM11722422, AM11729822, EU14542336, EU14532736, EU14555136, EU14536136; Declieuxia fruticosa (Willd. ex Roem. & Schult.) Kuntze, Rub-Coussareeae, –, AJ00217712, EU14550336, –, DQ1317216, EU14555236, EU14536236; Dentella repens (L.) J.R. Forst. & G. Forst., Rub-Spermacoceae, –, –, AF33337010, –, –, AF38154027, –; Dibrachionostylus kaessneri (S. Moore) Bremek., Rub-Spermacoceae, –, AJ61621117, AF00276105, –, –, EU14557436, –; Didymaea alsinoides (Cham. & Schltdl.) Standl., Rub-Rubieae, Keller 1901 (CAS), Z6879511, –, FJ695298*, AJ23403602, EU14557036, FJ695442*; Dirichletia glau- cescens Hiern (synonym Carphalea glaucescens (Hiern) Verdc.), Rub-Knoxieae, SMP 215 (UPS), Z6878911, AM11728822, AJ23628709, FJ695357*, AM11735122, AM26699323; Dunnia sinensis Tutcher Spec. 1, Rub-Dunnieae, –, EU14546736, EU14551536, EU14544236, EU14533936, EU14558336, EU14539036; Dunnia sinensis Tutcher Spec. 2, Rub-Dunnieae, –, EU14546836, EU14551636, EU14544336, EU14534036, EU14558436, EU14539136; Dunnia sinensis Tutcher Spec. 3, Rub-Dunnieae, –, EU14546936, EU14551736, EU14544436, EU14534136, EU14558536, EU14539236; Dunnia sinensis Tutcher Spec. 4, Rub-Dunnieae, –, EU14547036, EU14551836, EU14544536, EU14534236, EU14558636, EU14539336; Dunnia sinensis Tutcher Spec. 5, Rub-Dunnieae, –, EU14547136, EU14551936, EU14544636, EU14534336, EU14558736, EU14539436; Ernodea littoralis Sw., Rub-Spermacoceae, –, AJ28860102, AF00276305, –, AJ23402502, –, –; Faramea multiflora A. Rich., Rub-Coussareeae, –, Z6879611, AF00404805, EU14542436, EU14532836, AF10242226, EU14536336; Gae- rtnera phyllosepala Baker, Rub-Gaertnereae, –, AM94528837, AM94531137, AM94526137, AM94522737, AM94534037, AM94520037; Gaertnera phyllostachya Baker, Rub-Gaertnereae, –, AM94528937, AM94531237, AM94526237, AM94522837, AM94534137, AM94520137; Galium album Mill., Rub-Rubieae, Bremer 3321 (UPS), X8109016, AF00405005, FJ695299*, X7645940, –, –; Geophila obvallata Didr., Rub-Psychotrieae, –, AM11722822, AF36984515, AM94525937, –, EU14556936, AM94519637; Gynochthodes sp. Blume, Rub-Morindeae, –, AM94528437, AM94530737, AM94525437, AM94522037, AM94533337, AM94519337; Gynochthodes coriacea Blume, Rub-Morindeae, –, AJ28860302, AM11731122, AM94525337, AM94521937, AJ84740725, AM94519237; Houstonia caerulea L., Rub-Spermacoceae, Bremer s.n. (UPS), AJ28860402, AF33337910, FJ695300*, FJ695362*, AF38152427, DQ01270633; DQ01277433; Hydnophytum for- micarum Jack, Rub-Psychotrieae, –, X8364501, AF00133905, –, X7648040, –, AF03491212; Kelloggia chinensis Franch., Rub-, –, AY57077638, AY57077138, –, AY57076538, –, –; Kelloggia galioides Torr., Rub-, Garyfield & al. 2437 (UPS), DQ66217904, DQ66220304, FJ695301*, AY57076838, DQ66214604, FJ695443*; Knoxia platycarpa Arn., Rub-Knoxieae, Lundqvist 11302 (UPS), AJ28863102, AM26682623, FJ695302*, FJ695363*, AM11736722, AM26700223; Kohautia caespitosa Schnizl., Rub-Spermacoceae, Bremer & al. 42566B (UPS), Z6880011, AM11732422, FJ695303*, FJ695364*, EU14557336, FJ695444*; Lasianthus kilimandscharicus K. Schum., Rub-Lasiantheae, –, AM11723722, AM11732722, EU14542636, EU14533036, DQ66214719, –; Lasianthus lanceo- latus (Griseb.) Urb., Rub-Lasiantheae, –, AM11723822, AF00406205, –, EU14533136, EU14555436, EU14536736; Lasianthus pedunculatus E.A. Bruce, Rub- Lasiantheae, –, Z6880211, EU14550436, EU14542736, AJ23400302, EU14555536, EU14536836; Lasianthus strigosus Wight, Rub-Lasiantheae, –, AM11723922, EU14550536, EU14542836, –, EU14555636, EU14536936; Leptodermis potaninii Batalin, Rub-Paederieae, KA 230 Berkeley (UCBG), AM11724122, DQ66220404, FJ695304*, FJ695365*, DQ66214804, FJ695445*; Lerchea bracteata Valeton, Rub-Ophiorrizeae, –, AJ28861002, EU14550836, EU14543336, AJ23399702, EU14556136, EU14537436; Manostachya ternifolia E.S. Martins, Rub-Spermacoceae, Bamps & Martins 4410 (UPS), AJ61621317, AM11732822, FJ695305*, FJ695366*, EU14557236, FJ695446*; Margaritopsis nudiflora (Griseb.) K. Schum. (synonym: Margaritopsis acuifolia C. Wright, Rub-Psychotrieae, –, AM11724722, AF00134005, –, AM945225, EU14556836, AM94519837; Maschalocorymbus corymbosus (Blume) Bremek., Rub-Urophylleae, –, AJ28861102, AM90061135, –, –, EU14557736, EU14538436; Mitchella repens L., Rub-Mitchelleae, –, Z6880511, AF00144105, AM94525837, AM94522337, AM94533737, AB10353530 AB10353630; Mitrasacmopsis quadrivalvis Jovet, Rub-Spermacoceae, –, AJ61621417, AM11732922, EU14543936, EU14533636, EU14557536,

808 TAXON 58 (3) • August 2009: 793–810 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia

Appendix. Continued. EU14538236; Morinda citrifolia L., Rub-Morindeae, –, AJ31844814, AJ32007814, AJ23630009, AJ23401302, AF15261606, AY76284331; Morinda guatemalen- sis (Donn. Sm.) Steyerm. (synonym: Appunia guatemalensis Donn. Sm.), Rub-Morindeae, –, AJ28859302, AM94530637, AM94525237, AJ23400902, AM94533237, AM94519137; Mouretia larsenii Tange, Rub-Argostemmateae, Beusekom & al. 4743 (P), FJ695236*, FJ695267*, FJ695306*, FJ695367*, FJ695410*, FJ695447*; Mycetia cauliflora Reinw., Rub-Argostemmateae, Larsen & al. 46287 (AAU), FJ695237*, FJ695268*, FJ695307*, FJ695368*, FJ695411*, FJ695448*; Mycetia gracilis Craib, Rub-Argostemmateae, Larsen & al. 46250 (AAU), FJ695238*, FJ695269*, FJ695308*, FJ695369*, FJ695412*, FJ695449*; Mycetia javanica (Blume) Reinw. ex Korth., Rub-Argostemmateae, Larsen & al. 43970 (AAU), FJ695239*, FJ695270*, FJ695309*, FJ695370*, FJ695413*, FJ695450*; Mycetia malayana (G. Don) Craib, Rub-Argostemmateae, Larsen 42486 (UPS), Z6880611, AF00277105, FJ695310*, AJ23403302, AF15262206, –; Mycetia sp. Reinw. Spec. 1, Rub-Argostemmateae, Larsen & al. 44635 (AAU), FJ695240*, FJ695271*, FJ695311*, FJ695371*, FJ695414*, FJ695451*; Mycetia sp. Reinw. Spec. 2, Rub-Argostemmateae, Larsen & al. 43706 (AAU), FJ695241*, FJ695272*, FJ695312*, FJ695372*, FJ695415*, FJ695452*; Nenax acerosa Gaertn., Rub-Anthospermeae, –, –, AF00360605, –, –, –, –; Neohymenopogon parasiticus (Wall.) Bennet Spec. 1, Rub-Argostemmateae, Vidal 5729 (P), FJ695242*, FJ695273*, FJ695313*, FJ695373*, FJ695416*, FJ695453*; Neohymenopogon parasiticus (Wall.) Ben- net Spec. 2, Rub-Argostemmateae, B. Bremer 2743 (UPS), FJ695243*, FJ695274*, FJ695314*, FJ695374*, FJ695417*, –; Neurocalyx championii Benth. ex Thwaites, Rub-Ophiorrizeae, –, EU14546336, EU14550936, EU14543536, –, EU14556336, EU14537636; Neurocalyx zeylanicus Hook., Rub-Ophiorrizeae, –, Z6880711, AM90059435, EU14543436, AJ23399502, EU14556236, EU14537536; Normandia neocaledonica Hook. f., Rub-Anthospermeae, Munzinger 532 (MO), AM11725022, AF25793118, FJ695315*, FJ695375*, EU14554336, AF25793018; Oldenlandia corymbosa L., Rub-Spermacoceae, –, X8365501, AF33338110, AJ13083709, –, AF38153727, AY85405334; Opercularia vaginata Juss., Rub-Anthospermeae, K. Bremer & Gustafsson 25 (UPS), Z6880911, AF25793618, FJ695316*, FJ695376*, FJ695418*, AF25793518; Ophiorrhiza elmeri Merr., Rub-Ophiorrizeae, –, EU14546436, EU14551036, EU14543636, –, EU14556436, EU14537836; Ophiorrhiza mungos L., Rub-Ophiorrizeae, –, X8365601, AF00406405, AJ13083809, –, DQ6621516, EU14537736; Otiophora scabra Zucc., Rub- Knoxieae, Iversen & Martinson 89078 (UPS), FJ695244*, AM26683923, –, DQ13175603, AM26692823, AM26701523; Otomeria oculata S. Moore, Rub- Knoxieae, Puff & al. 82/222-21 (K), AJ28861402, AM26684423, FJ695317*, FJ695377*, AM11737422, AM26701923; Paederia bojeriana (A. Rich. ex DC.) Drake, Rub-Paederieae, Razafimandimbison & H. Bremer 483 (UPS), DQ66218104, DQ66220604, FJ695318*, DQ13175703, DQ66215204, FJ695454*; Pa- ederia majungensis Homolle ex Puff, Rub-Paederieae, Nilsson & al. D152 (UPS), DQ66218404, DQ66220904, FJ695319*, FJ695378*, DQ66215504, –; Paederia sambiranensis Homolle ex Puff, Rub-Paederieae, Kårehed & al. 238 (UPS), DQ66218804, DQ66221304, –, FJ695379*, DQ66215904, –; Pagamea guianensis Aubl., Rub-Gaertnereae, –, AM94529037, AF00274405, AM94526337, AM94522937, AM94534237, AF33384662; Palicourea crocea (Sw.) Schult, Rub-Psychotrieae, –, AM11725322, AF14751020, AM94528037, AM94524737, AM94525937, AF14932220; Parapentas silvatica (K. Schum.) Bremek., Rub- Knoxieae, Bremer 3091 (UPS), X8365701, AM11733222, FJ695320*, AJ23402102, AM11737622, AM26702323; Paratriaina xerophila Bremek., Rub-Knoxieae, Razafimandimbison & Bremer 489 (UPS), AJ2886332, AM26685023, FJ695321*, FJ695380*, AM26693823, AM26702423; Pauridiantha paucinervis (Hiern) Bremek., Rub-Urophylleae, –, Z6881111, AM90060035, AJ23630209, AJ23399802, EU14557836, EU14538536; Pauridiantha symplocoides (S. Moore) Bremek., Rub-Urophylleae, –, AY53850208, AF00406805, EU14544036, EU14533836, AF10246726, EU14538636; Pentanisia prunelloides (Klotzsch) Walp., Rub- Knoxieae, B. Bremer & al. 4316 (UPS), AM11725522, AM26686023, FJ695322*, FJ695381*, AM26694823, AM26703323; Pentas lanceolata (Forssk.) Deflers, Rub-Knoxieae, –, X8365901, AM11733422, AJ23630409, X7647940, AM11737922, AB24727528; Pentodon pentandrus (Schumach. & Thonn.) Vatke, Oesterr., Rub-Spermacoceae, Bremer 3082 (UPS), X8366001, AF00361205, FJ695323*, AJ23402402, FJ695419*, FJ695455*; Phyllis nobla L., Rub-Anthospermeae, K. Bremer 3008 (UPS), Z6881411, AF00361305, FJ695324*, AJ23403102, AY53846808, AF25793918; Placopoda virgata Balf. f., Rub-Knoxieae, Thulin & Gifri 8528 (UPS), Z6881511, AM11733522, FJ695325*, FJ695382*, AM11738222, AM26706423; Plocama aucheri (Guill.) M. Backlund & Thulin, Rub-Pu- torieae, Thulin 9963 (UPS), DQ66217804, DQ66220204, FJ695326*, FJ695383*, DQ66214504, FJ695456*; Plocama calabrica (L. f.) M. Backlund & Thulin, Rub-Putorieae, Jonsell 4216 (UPS), AJ28862002, FJ695275*, FJ695327*, FJ695384*, DQ66216604, FJ695457*; Plocama hymenostephana (Jaub. & Spach) M. Backlund & Thulin, Rub-Putorieae, Thulin 9993 (UPS), DQ66219004, DQ66221504, FJ695328*, FJ695385*, DQ66216304, FJ695458*; Plocama pendula Aiton, Rub-Putorieae, Andreasen 1 (UPS), Z6881601, FJ695276*, FJ695329*, AJ23403502, DQ66216204, FJ695459*; Pomax umbellata (Gaertn.) Sol. ex A. Rich., Rub-Anthospermeae, B. & K. Bremer 3918 (UPS), AM11726022, AF25794118, –, DQ13176703, FJ695420*, AF25794018; Pravinaria leucocarpa Bremek., Rub-Urophylleae, –, AJ28861702, AM90061335, EU14544136, AJ23400102, EU14558036, EU14538836; Prismatomeris albidiflora Thwaites, Rub- Psychotrieae, –, AM94529637, AM94532037, AM94527037, AM94523737, AM94535137, AM94520537; Prismatomeris beccariana (Baill. ex K. Schum.) J.T. Johanss., Rub-Psychotrieae, –, AJ28861802, AF33165210, AM94527137, AM94523837, AM94535237, AM94520637; Prismatomeris sp. Thwaites Spec. 1, Rub- Psychotrieae, –, AM94529237, AM94531637, AM94526637, AM94523337, AM94534737, AM94520237; Prismatomeris sp. Thwaites Spec. 2, Rub-Psychotrieae, –, AM94529337, AM94531737, AM94526737, AM94523437, AM94534837, –-; Psychotria amboniana K. Schum., Rub-Psychotrieae, –, AM94530237, AM94532837, AM94528137, AM94524837, AM94536037, AM94521537; Psychotria capensis (Eckl.) Vatke, Rub-Psychotrieae, –, AM94530137, AM94532637, AM94527737, AM94524537, AM94535737, AM94521337; Psychotria holtzii (K. Schum.) E.M.A. Petit, Rub-Psychotrieae, –, AM94530437, AM94533037, –, AM94525037, AM94536237, AM94521737; Psychotria kirkii Hiern, Rub-Psychotrieae, –, X8366301, AF41072821, AJ23630709, X7648140, AY53846908, AF07203812; Psychotria poeppigiana Müll.Arg., Rub-Psychotrieae, –, Z6881811, AF00274805, AM94527937, AJ23401802, –, AF14940020; Psychotria schlie- benii E.M.A. Petit, Rub-Psychotrieae, –, AM94530337, AM94532937, AM94528237, AM94524937, AM94536137, AM94521637; L., Rub- Rubieae, Bremer 3300 (UPS), X8366601, –, DQ35916703, X7646540, FJ695421*, DQ35888503; Saldinia sp. A. Rich. ex DC. Spec. 1, Rub-Lasiantheae, –, AM11726922, AF12927513, EU14542936, EU14533236, EU14555736, EU14537036; Saldinia sp. A. Rich. ex DC. Spec. 2, Rub-Lasiantheae, –, EU14546136, EU14550636, EU14543036, EU14533336, EU14555836, EU14537136; Saprosma foetens (Wight) K. Schum., Rub-Paederieae, Klackenberg 325 (S), DQ66219304, DQ66221804, –, FJ695386*, DQ66216804, FJ695460*; Saprosma fruticosum Blume, Rub-Paederieae, Ridsdale IV.E.157 (L), DQ66219404, FJ695277*, FJ695330*, FJ695387*, DQ66216904, FJ695461*; Saprosma ternatum (Wall.) Hook. f., Rub-Paederieae, –, –, DQ28264639, –, –, –, –; Schismatoclada fara- himpensis Homolle, Rub-Danaideae, Kårehed & al. 267 (UPS), FJ695245*, FJ695278*, FJ695331*, FJ695388*, FJ695422*, FJ695462*; Schismatoclada sp. Baker Spec. 1, Rub-Danaideae, Razafimandimbison 375 (S), AM11727122, AM11734122, EU14542536, EU14532936, EU14555336, EU14536536; Schisma- toclada sp. Baker Spec. 2, Rub-Danaideae, Razafimandimbison & Ravelonarivo 625 (S), FJ695246*, FJ695279*, FJ695332*, FJ695389*, FJ695423*, FJ695463*; Schismatoclada aff. psychotrioides Baker, Rub-Danaideae, Eriksson & al. 988 (S), FJ695247*, –, FJ695333*, FJ695390*, FJ695424*, FJ695464*; Schizocolea linderi (Hutch. & Dalziel) Bremek. Spec. 1, Rub-Schizocoleeae, Adam 20116 (UPS), AM11727222, EU14549836, FJ695334*, EU14532336, EU14554636, EU14535736; Schizocolea linderi (Hutch. & Dalziel) Bremek. Spec. 2, Rub-Schizocoleeae, Adam 789 (P), AM94528637, AM94530937, FJ695335*, AM94522437, AM94533837, AM94519737; Schradera subandina K. Krause, Rub-Schradereae, –, Y1185907, AM94531337, AM94526437, AJ23401402, AM94534337, –; Schradera sp. Vahl Spec. 1, Rub-Schradereae, ––, –-, AF00361705, –, –, AF15261306, –; Schradera sp. Vahl Spec. 2 , Rub-Schradereae, –, –-, AM94531437, AM94526537, AM94523037, AM94524437, –; Schradera sp. Vahl Spec. 3, Rub-Schradereae, –, –-, AM94531537, –, AM94523137, AM94534537, –; Serissa japonica Thunb. (synonym: Serissa foetida (L. f.) Lam.), Rub-Paederieae, Bremer 2717 (UPS), Z6882211, AF00408105, FJ695336*, AJ23403402, AF15261806, FJ695465*; Sherardia arvensis L., Rub-Rubieae, K. Andreasen 345 (SBT), X8110616, AF00408205, FJ695337*, X7645840, EU14557136, FJ695466*; Spermacoce remota Lam., Rub-Spermacoceae, Bremer 3062 (UPS), Z6882311, –, AJ23630909, –, FJ695425*, FJ695467*; Spermadictyon sua- veolens Roxb., Rub-Paederieae, Bremer 3133 (UPS), Z6882411, DQ66221904, FJ695338*, FJ695391*, DQ66217104, FJ695468*; Spiradiclis bifida Kurz, Rub-Ophiorrizeae, , EU14546536, EU14551136, EU14543736, –, EU14556536, EU14537936; Thecorchus wauensis (Schweinf. ex Hiern) Bremek., Rub- Spermacoceae, Friis & al. 2560 (C), AM11728222, AM26690123, –, FJ695392*, AM26698723, AM26707023; Theligonum cynocrambe L. Spec. 1,

809 Rydin & al. • Systematic affinities of Neohymenopogon and Mouretia TAXON 58 (3) • August 2009: 793–810

Appendix. Continued. Rub-Theligoneae, –, X8366801, AF00408705, –, X8168024, AF15262106, –; Theligonum cynocrambe L. Spec. 2, Rub-Theligoneae, Thor 654 (S), FJ695248*, FJ695280*, FJ695339*, FJ695393*, FJ695426*, FJ695469*; Theligonum cynocrambe L. Spec. 3, Rub-Theligoneae, Reuterswärd & Forsslund 2 (S), FJ695249*, FJ695281*, FJ695340*, –, FJ695427*, FJ695470*; Triainolepis mandrarensis Homolle ex Bremek., Rub-Knoxieae, Razafimandimbison 521 (UPS), FJ695250*, AM26689923, FJ695341*, FJ695394*, AM26698523, AM26706823; Trichostachys aurea Hiern, Rub-Lasiantheae, –, EU14546236, EU14550736, EU14543136, EU14533436, EU14555936, EU14537236; Trichostachys sp. Hook. f., Rub-Lasiantheae, –, AJ28862602, AM90059535, EU14543236, DQ1317926, EU14556036, EU14537336; Urophyllum arboreum (Reinw. ex Blume) Korth., Rub-Urophylleae, –, –, AM90061735, –, DQ13179303, EU14558236, –; Urophyllum ellipticum (Wight) Thwaites, Rub-Urophylleae, –, AJ28862702, AM90061935, –, AJ23400202, EU14558136, EU14538936; Valantia hispida L., Rub-Rubieae, Bremer 3131 (UPS), FJ695251*, AF00409005, FJ695342*, FJ695395*, AM11738522, FJ695471*; Xanthophytum borneense (Valeton) Axelius, Rub-Ophiorrizeae, –, EU14546636, EU14551336, EU14543836, EU14533536, EU14556736, EU14538136; Xanthophytum capitellatum Ridl., Rub-Ophiorrizeae, –, AJ28862802, EU14551236, –, AJ23399602, EU14556636, EU14538029. *Previously unpublished sequence. Published sequences: 01: Bremer & al. (1995). 02: Bremer & Manen (2000). 03: Manen, J.-F. (GenBank unpublished). 04: Backlund & al. (2000). 05: Andersson & Rova (1999). 06: Rova & al. (2002). 07: Bremer & Thulin (1998). 08: Andersson & Antonelli (2005). 09: Bremer & al. (1999). 10: Andersson, L. (GenBank unpublished). 11: Bremer (1996). 12. Nepokroeff & al. (1999). 13: Piesschaert & al. (2000). 14: Novotny & al. (2002). 15: Andersson (2001). 16: Manen & Natali (1995). 17: Thulin & Bremer (2004). 18: Anderson, C.L. & al. (GenBank unpublished). 19: Backlund, M. (GenBank unpublished). 20: Andersson, L. & Taylor, C. (GenBank unpublished). 21: Andersson (2002). 22: Bremer & Eriksson (2009). 23: Kårehed & Bremer (2007). 24: Natali & al. (1995). 25: Alejandro & al. (2005). 26: Struwe & al. (1998). 27: Church (2003). 28: Nakamura & al. (2006). 29: Ding, P. & al. (GenBank unpublished). 30: Yokoyama, J. & al. (GenBank unpublished). 31: Proujansky, A.D. & Stern, D.L. (GenBank unpublished). 32: Wolff, D. & Liede-Schumann, S. (GenBank unpublished). 33: Church & Taylor (2005). 34: Yuan, C.I. (GenBank unpublished). 35: Smedmark & al. (2008). 36: Rydin & al. (2008, 2009). 37: Razafimandimbison & al. (2008). 38: Nie & al. (2005). 39: Xiao L.-Q. & Zhu H. (GenBank unpublished). 40. Manen & al. (1994).

810