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Molecular Phylogenetics and Evolution 49 (2008) 843–866
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
The phylogenetic utility of chloroplast and nuclear DNA markers
and the phylogeny of the Rubiaceae tribe Spermacoceae
Jesper Kårehed a,*, Inge Groeninckx b, Steven Dessein c, Timothy J. Motley d, Birgitta Bremer a
a
Bergius Foundation, Royal Swedish Academy of Sciences and Botany Department, Stockholm University, SE-106 91 Stockholm, Sweden
Laboratory of Plant Systematics, K.U. Leuven, Leuven, Belgium
c
National Botanic Garden of Belgium, Meise, Belgium
d
Old Dominion University, Department of Biological Sciences, Norfolk, VA, USA
b
a r t i c l e
i n f o
Article history:
Received 31 March 2008
Revised 17 September 2008
Accepted 30 September 2008
Available online 10 October 2008
Keywords:
5S-NTS
Accuracy
atpB-rbcL
Bayesian inference
ETS
ITS
petD
rps16
Phylogenetic utility
Partition metric
Phylogeny
Rubiaceae
Spermacoceae
trnL-F
a b s t r a c t
The phylogenetic utility of chloroplast (atpB-rbcL, petD, rps16, trnL-F) and nuclear (ETS, ITS) DNA regions
was investigated for the tribe Spermacoceae of the coffee family (Rubiaceae). ITS was, despite often raised
cautions of its utility at higher taxonomic levels, shown to provide the highest number of parsimony
informative characters, in partitioned Bayesian analyses it yielded the fewest trees in the 95% credible
set, it resolved the highest proportion of well resolved clades, and was the most accurate region as measured by the partition metric and the proportion of correctly resolved clades (well supported clades
retrieved from a combined analysis regarded as ‘‘true”). For Hedyotis, the nuclear 5S-NTS was shown to
be potentially as useful as ITS, despite its shorter sequence length. The chloroplast region being the most
phylogenetically informative was the petD group II intron.
We also present a phylogeny of Spermacoceae based on a Bayesian analysis of the four chloroplast
regions, ITS, and ETS combined. Spermacoceae are shown to be monophyletic. Clades supported by high
posterior probabilities are discussed, especially in respect to the current generic classification. Notably,
Oldenlandia is polyphyletic, the two subgenera of Kohautia are not sister taxa, and Hedyotis should be
treated in a narrow sense to include only Asian species.
Ó 2008 Elsevier Inc. All rights reserved.
1. Introduction
When inferring phylogenies from molecular data it is important
to use DNA regions with an evolutionary rate suitable for the taxa
under study (Soltis and Soltis, 1998). A slowly evolving region may
provide too little information to recover a fully resolved phylogeny.
On the other hand, regions evolving too quickly will have their
phylogenetic signal masked by homoplasy due to multiple hits,
i.e., aligned nucleotides are the same by chance and not by common ancestry.
In the present paper we will compare the phylogenetic utility of
chloroplast and nuclear DNA sequences for resolving the phylogeny of the species-rich tribe Spermacoceae of the coffee family
(Rubiaceae). The DNA regions (e.g., rbcL, atpB, 18S rDNA) utilized
in the numerous studies synthesized in the high-level classifications of the Angiosperm Phylogeny Group (APG, 1998; APGII,
* Corresponding author. Fax: +46 0 8 16 55 25.
E-mail address: jesper@bergianska.se (J. Kårehed).
1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2008.09.025
2003) would be of limited value in resolving the phylogeny of Spermacoceae, due to their conservative nature. For phylogenetic studies at lower taxonomic levels noncoding chloroplast regions have
been used frequently and successfully. The rationale behind using
noncoding regions is the assumption that they are phylogenetically
more informative because they are under less functional constraints (Gielly and Taberlet, 1994). Shaw et al. (2005) investigated
the relative utility of 21 noncoding chloroplast regions and divided
them into three tiers. They found that five of the regions (rpoB-trnC,
trnD-trnT, trnS-trnfM, trnS-trnG, trnT-L; tier 1) generally have enough phylogenetic signal for studies at the lower taxonomic levels.
Five additional regions (psbM-trnD, rpl16, rps16, trnG, ycf6-psbM;
tier 2) were identified as potentially useful, but less likely to provide full resolution. The remaining regions (3’trnK-matK, 5’rpS12rpL20, matK-5’trnK, psbA-3’trnK, psbB-psbH, rpS4-trnT, trnC-ycf6,
trnH-psbA, trnL, trnL-trnF, trnS-rpS4; tier 3) were all considered to
provide too little phylogenetic information to be recommended.
More recently, Shaw et al. (2007) identified nine newly explored
noncoding chloroplast regions (rpl32-trnL(UAG), trnQ(UUG)-5’rps16,
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3’trnV(UAC)-ndhC, ndhF-rpl32, psbD-trnT(GGU), psbJ-petA, 3’rps16–
5’trnK(UUU), atpI-atpH, and petL-psbE), which provided even higher
levels of variation than the ones identified in their previous study
(Shaw et al., 2005).
Mort et al. (2007) further investigated the utility of the regions
in tier 1 and 2 and compared them to the internal transcribed
spacer (ITS) region (ITS1, 5.8S gene, and ITS2; White et al., 1990;
Baldwin, 1992) of the nuclear ribosomal DNA region. As implied
from its use in numerous studies, ITS is a useful marker for resolving phylogenetic relationships at various taxonomic levels, in particular infrageneric. When analysing ITS caution has to be taken to
avoid problems resulting from concerted evolution on the ribosomal DNA arrays. Concerted evolution may homogenize different
paralogous gene copies in a genome leading to the loss of all but
one of the copies, i.e., different copies may be present in different
organisms by chance and consequently the gene trees and species
trees will not agree (Alvarez and Wendel, 2003). Compared to the
noncoding chloroplast regions tested by Shaw et al. (2005), ITS was
not consistently more informative (Mort et al., 2007). Mort et al.
(2007) raised the concern of how well organismal phylogeny is inferred by ITS data. They concluded that, despite incongruences
with chloroplast data (as measured by the ILD test; Farris et al.,
1995), including ITS data may still result in an increase in both resolution and support suggesting that a phylogenetic signal masked
by homoplasy in chloroplast data could be strengthened by a combined analysis (Nixon and Carpenter, 1996; Wenzel and Siddall,
1999; Mort et al., 2007).
The studies by Shaw et al. (2005, 2007) and Mort et al. (2007)
aimed at identifying molecular markers useful at lower taxonomic
levels across a wide taxonomic range. We want to explore how
useful different markers are for resolving the phylogeny of a large
clade where the taxonomic sample is intended to infer both infrageneric and intrageneric relationships (this terminology is admittedly unfortunate since the current generic classification of
Spermacoceae is known to include both para- and polyphyletic
genera; Groeninckx et al., in press).
We will identify the regions with the highest number of (parsimony) informative characters or the regions providing most resolution or the highest proportion of well resolved clades. We will
also compare how effective different DNA regions are in obtaining
the ‘‘true phylogeny.” To make these comparisons a region should
provide potentially phylogenetic informative characters or produce well supported, resolved phylogenies. It must also provide
accurate topologies, i.e., provide congruent results with other regions and provide data not susceptible to analytical assumptions,
for example long branch attraction. Measures of accuracy often include comparing the number of bipartitions not shared between
topologies. The partition metric, often called the Robinson–Foulds
distance, is twice the number of internal branches that differ in the
number of bipartitions between two topologies (Robinson and
Foulds, 1981; Penny and Hendy, 1985; Steel and Penny, 1993).
Rzhetsky and Nei (1992) adjusted the partition metric to account
for multichotomies. Other methods used to measure how much
deviation exists between two trees are the number of nearestneighbor exchanges needed to transform one tree to the other
(Waterman and Smith, 1978), the number of correctly resolved
taxon bipartitions divided by the number of possible bipartitions
(Hillis, 1995), and comparisons of geometric distances in tree space
between differing topologies (Billera et al., 2001).
We have chosen Spermacoceae, a tribe of the coffee family
(Rubiaceae), to investigate the utility of four chloroplast regions
(atpB-rbcL, rps16, trnL-F, petD) and three nuclear regions (ITS, the
external transcribed spacer, ETS, and the 5S non-transcribed
spacer, 5S-NTS). Rubiaceae are currently classified in two or three
subfamilies and more than 40 tribes (Bremer et al., 1995;
Robbrecht and Manen, 2006; Bremer, in press). Spermacoceae with
over 1000 species have a mainly pantropical distribution, but a few
genera extend into temperate regions. The majority of the species
are herbaceous. Four-merous flowers together with fimbriate stipules characterize the tribe, but these characters are not omnipresent. The delimitations of Spermacoceae have varied from a long
recognized morphologically well-defined tribe (Spermacoceae sensu stricto, s.s.; Hooker, 1873; Bremekamp, 1952, 1966; Verdcourt,
1958; Robbrecht, 1988) to a wider interpretation stemming from
molecular analyses and including the traditionally recognized
tribes Hedyotideae, Knoxieae, Manettieae, and Triainolepideae
(Bremer, 1996; Bremer and Manen, 2000). Here Spermacoceae
are treated to comprise 60 genera and include Manettieae and
most genera of Hedyotideae, but not their probable sister tribe
Knoxieae, which recently is expanded to include Triainolepideae
and a few genera from Hedyotideae (Andersson and Rova, 1999;
Dessein, 2003; Kårehed and Bremer, 2007).
The only previous phylogeny with a global perspective of Spermacoceae (Groeninckx et al., in press) revealed a number of well
supported clades. The relationships between these clades are less
resolved. In addition, the relationships within some of the clades
are not strongly supported. The Spermacoceae sequence data, thus,
provide an interesting mixture of early branching and late branching clades, clades with apparent different rates of evolution, as well
as a combination of well supported clades and unresolved taxa.
The study of Groeninckx et al. (in press) used only chloroplast data
(atpB-rbcL, rps16, trnL-F). Since their study was intended for tribal
relationships, the combination of relatively slower and faster
markers (cf. Shaw et al., 2005) was rational. We chose to investigate the chloroplast region, petD (the petD intron and the petB-petD
spacer; Löhne and Borsch, 2005), which according to Löhne and
Borsch (2005) has less length variation than trnT-F, but has about
the same number of informative characters. The petD region is
comparatively untested in Rubiaceae.
Although used less extensively than ITS, ETS seems to provide
phylogenetic information at about the same magnitude as ITS
(Baldwin and Markos, 1998; Musters et al., 1990). Within Rubiaceae, ETS and ITS have been utilized together at the generic level
by Negrón-Ortiz and Watson (2002) for Erithalis, Markey and de
Lange (2003) for Coprosma, Nepokroeff et al. (2003) for Psychotria,
and Razafimandimbison et al. (2005) for Neonauclea.
5S-NTS (Cox et al., 1992; Sastri et al., 1992) has been even less
used than ETS. For Rubiaceae, 5S-NTS was reported to be about
twice as informative as ITS for the Alibertia group (Persson,
2000). It has also been used for Randia (Gustafsson and Persson,
2002) and most recently for inferring the phylogeny of the Kadua
clade (Motley, in press), one of the well supported clades of Spermacoceae (Groeninckx et al., in press).
The nuclear data were added as faster evolving DNA regions,
which could provide better resolution of relationships within
younger taxa or taxa with slow evolutionary rates, and strengthen
phylogenetic signals masked by homoplasy in the chloroplast data.
We also investigated the extent the nuclear data can resolve early
branching clades of Spermacoceae, where previous chloroplast
studies have failed (Groeninckx et al., in press). This will determine
if the nuclear data only contribute information on closely related
taxa, being too homoplastic at higher levels, or if the nuclear data
also are useful for resolving relationships at somewhat higher taxonomic levels. Topological comparisons of chloroplast-trees to nuclear trees will provide evidence of suitable DNA regions to use for
large, species-rich, tribal and/or family lineages.
In our evaluations of the relative utility of chloroplast (atpBrbcL, rps16, trnL-F, petD) and nuclear (ITS, ETS, 5S-NTS) DNA regions
we want to investigate if the eight well supported clades discussed
by Groeninckx et al. (in press; Section 4.2) remain monophyletic
with the additional data, examine if other clades retrieved in both
studies have increased support with the addition of data, and
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J. Kårehed et al. / Molecular Phylogenetics and Evolution 49 (2008) 843–866
determine if additional data will resolve additional monophyletic
clades. In addition, we will present a total-evidence phylogeny of
Spermacoceae and discuss to what extent the phylogeny agrees
with the current generic classification.
2. Materials and methods
2.1. Taxon sampling and molecular data
We used the chloroplast data set of Groeninckx et al. (in press)
as the basis for our sampling. Their data set included 128 species.
Our chloroplast data set was enlarged by the addition of three
atpB-rbcL sequences (Gomphocalyx hernarioides, Hydrophylax maritima, and Pentanopsis gracilicaulis; the latter two species were not
previously sampled), two rps16 sequences (Oldenlandia biflora, Pentanopsis gracilicaulis), four trnL-F sequences (Gomphocalyx hernarioides, Oldenlandia rosulata, Pentanopsis gracilicaulis, Phylohydrax
carnosa), and petD sequences for the newly sampled Manettia luteorubra and 101 of the species included by Groeninckx et al. (in
press; Appendix).
Before sequencing nuclear data for the entire taxon sample, a
test sample was selected. The test sample included 12 species
(Arcytophyllum thymifolium, Dibrachionostylus kaessneri, Hedyotis
quinquenervis, Kadua littoralis, Kohautia amatymbica, Kohautia virgata, Mitrasacmopsis quadrivalvis, Oldenlandia angolensis, Oldenlandia
echinulosa, Pentodon pentandrus, Spermacoce hispida, and Thecorchus wauensis) representing the major clades recovered by Groeninckx et al. (in press). The sample was used to investigate if
different nuclear regions could be amplified, and if the resulting sequences could be aligned to each other. The nuclear regions investigated were ITS, ETS, and 5S-NTS. Both the ITS and ETS sequences
were straightforward to amplify and possible to align. Therefore,
the two regions were chosen to be sequenced for the larger sample
of Spermacoceae.
In total we sampled 139 taxa for ITS and 98 for ETS (Appendix).
New taxa added to the nuclear data but not present in the taxon
sample of Groeninckx et al. (in press) include: two genera (Diodella
and Psyllocarpus), 13 species (Bouvardia sp. Torres & Torino 3637
(BR), Galianthe sp. Persson & Gustavsson 298 (GB), Hedyotis capitellata, H. effusa, H. megalantha, Kohautia longifolia, K. cf. longifolia,
Oldenlandia cf. galioides, O. monanthos, O. sp. C of Flora Zambesiaca,
Richardia brasiliensis, Spermacoce filifolia, S. verticillata), and one
subspecies Oldenlandia mitrasacmoides subsp. nigricans. Extra individuals for a few taxa already included are also added, either because those sequences were already available (the outgroup taxa)
or, for example, to confirm the placement of a taxon (Amphiasma
luzuloides, Conostomium natalense, Dibrachionostylus kaessneri, Kadua cordata, Oldenlandia corymbosa, O. goreensis, O. herbacea var. goetzei, O. herbacea var. herbacea, O. lancifolia, and Pentodon
pentandrus).
To be able to compare the phylogenetic utility of the different
DNA regions without taking the effect of unequal taxon sampling
into account, we also prepared a reduced taxon sample with those
49 taxa that are present in all separate data sets (referred herein as
the reduced Spermacoceae data set).
The 5S-NTS region was easy to amplify, all test taxa worked. The
PCR (polymerase chain reaction) products resulting in the strongest bands (as measured by the amount of staining with Ethidium
bromide when separated on an agarose gel) were, however, of
quite different lengths. All the test taxa produced a single predominant band, although fainter bands occurred. These might be a
result of unspecific annealing or due to multiple copies. The sequenced PCR products differed from 244 base pairs (bp; Spermacoce hispida) to 899 bp (Kohautia obtusiloba). Homology between
the different bands was, thus, impossible to ascertain. Even the se-
845
quences of the bands of equal lengths, for which homology could
be postulated, were not possible to align.
Since 5S-NTS seemed to be easy to amplify for taxa of the Spermacoceae, but was too variable to be used for the entire tribe, we
wanted instead to test its usefulness for more restricted, well defined clades. For that purpose we chose Hedyotis s.s. (Groeninckx
et al, in press). 5S-NTS was sequenced for nine Hedyotis species
present in the ITS and ETS data sets. This matrix (the Hedyotis data
set) was used to determine how informative and accurate the 5SNTS region is compared to the other two nuclear data sets (see below). For the Hedyotis data set we made no attempts to cut out the
predominant band or clone the PCR product, because the obtained
sequences showed no indications of multiple copies (e.g., multiple
peaks in the chromatograms).
The low copy-number nuclear marker Tpi (Strand et al., 1997)
was also investigated. We were not able to obtain PCR products
for any of the 12 test taxa. Nevertheless, the use of this and other
low copy-number nuclear markers with specifically designed
primers may prove to be valuable in the future for exploring clades
that remain unresolved with other DNA regions.
2.2. Sequencing
DNA was extracted from fresh, silica-gel dried material or herbarium specimens using the CTAB method (Doyle and Doyle,
1987). The petD region was amplified with the forward primer PIpetB1365F and the reverse primer PIpetD738R (Löhne and Borsch,
2005). Polymerase chain reactions and the sequencing of the new
chloroplast data were performed as described by Groeninckx
et al. (in press).
Polymerase chain reactions for the nuclear data sets were run on
an EppendorfÒ MastercyclerÒ gradient (Bergman & Beving
Instrument, Stockholm, Sweden). The 50-ll reactions included 5 ll
reaction buffer, 5 ll MgCl2, 5 ll TMACL (Chevet et al., 1995), 4 ll
DNTP, 0.25 ll Taq (5 U/ll), 0.5 ll 50 primer (20 lM), 0.5 ll 30 primer
(20 lM), 0.5 ll BSA 1%, and 1–2 ll of DNA templates and sterilized
H2O adding up to 50 ll. The amplifications consisted of an initial
denaturation for 1 min at 95 °C, followed by 37 cycles of 1 min at
95 °C, 1 min 30 s at 50 °C, 1 min 30 s at 72 °C (usually +1 s/cycle),
and a final extension phase of 7 min at 72 °C. The PCR products were
purified with the MultiScreenÒ Separations System (Millipore, USA).
The purified products were subsequently sequenced with the BigDyeTM terminator cycle sequencing kit (Applied Biosystems, Stockholm, Sweden) on a GeneAmp PCR System 9700 (Applied
Biosystems) and analyzed on an ABI PRISMÒ 3100 Genetic Analyzer
(Applied Biosystems). Primers used for both PCR and sequence
reactions were for ITS the forward primer P17 and the reverse primer
26S-82R (Popp and Oxelman, 2001) or P25 (Oxelman, 1996). For ETS
the primer pair ETS-Erit-F (Negrón-Ortiz and Watson, 2002) and
18S-E (Baldwin and Markos, 1998) was used. The 5S-NTS region
was amplified using the primers PI and PII (Cox et al., 1992) and attempts to amplify Tpi were performed with primers TPIX4FN and
TPIX6RN (Strand et al., 1997).
2.3. Phylogenetic analyses
All alignments were made by eye. Simple indel coding (Simmons
and Ochoterena, 2000) of insertion/deletion (indel) events was
performed with the computer program SeqState (Müller, 2005).
The indels were subsequently included in the analyses.
Bayesian analyses to estimate the phylogeny of the entire Spermacoceae data set and the reduced Spermacoceae data set were
done for all regions independently, the combined four chloroplast
regions, the combined nuclear ITS and ETS regions, and a total evidence analysis. For the Hedyotis taxon sample the ITS, ETS, and 5SNTS data were analyzed both separately and combined.
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The choice of nucleotide-substitution models for the Bayesian
analyses of the molecular data was determined based on the corrected Akaike criterion (Burnham and Anderson, 2002) calculated
using the computer program MrAIC (Nylander, 2004) in conjunction with the program PhyML (Guindon and Gascuel, 2003). The
general time reversible model with a gamma distribution of substitution rates (GTR + G) was chosen for all the DNA regions of the
Spermacoceae data set, except ETS. For ETS the model (GTR + I + G)
includes a proportion of invariant sites. In the reduced Spermacoceae data set the GTR + G model was used for all regions. The
Hedyotis data set was analysed under the GTR + G model for ETS
and 5S-NTS, and the HKY model (with different substitution rates
for transitions and transversions) was used for ITS. The insertion/
deletion data for all data sets were analyzed under the standard
discrete (morphology) model. In the combined analysis the data
set was partitioned and the partitions were unlinked and, consequently, had their own set of parameters. The Bayesian analyses
were performed using the computer program MRBAYES (v3.1.2;
Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck,
2003). For the Spermacoceae data set the Markov chain was run
for 5,000,000 generations with three additional ‘‘heated” chains
(Metropolis-coupled Markov chain Monte Carlo; Huelsenbeck
et al., 2001). Trees were sampled every 100th generation. Two separate runs were run for all data sets. When the average standard
deviation of split frequencies between the separate runs was
<0.05, it was taken as an indication that the Markov Chains had
converged on the stationary distribution. The first 500,000 generations were, consequently, discarded as a burn-in period for all analyses except for the combined nuclear data and for the combined
chloroplast and nuclear data, for which a burn-in period of
1,000,000 generations was used. The reduced Spermacoceae and
the Hedyotis data sets were run for 1,000,000 generations with a
burn-in period of 100,000 generations and every 100th tree was
sampled.
2.4. Comparison of phylogenetic utility of the seven DNA regions
In order to investigate the phylogenetic utility of the seven DNA
regions we compared the information content, i.e., how much of the
information that could be contributed to sequence length, the number of trees in the 95% credible set of trees, the proportion of well
resolved nodes, and the phylogenetic accuracy of the respective regions. The number of parsimony informative characters excluding
and including indels are shown in Table 1 and Fig. 1(a–c). Linear
regressions of the number of parsimony informative characters
against the aligned length of the data matrices are shown in
Fig. 1(d–f). We also present the 95% credible set of trees (the smallest set of trees whose cumulative posterior probability sum to 0.95;
Felsenstein, 1968; Table 1, Fig. 1g–i). The 95% credible set gives an
indication of the precision of the maximum posterior probability
estimate of the phylogeny (the most probable tree). A DNA region
which produces a 95% credible set including few trees would indicate that the region better discriminates between conflicting topologies than another region which produces a larger 95% credible set.
The size of the 95% credible set could, thus, be seen as an approximation of the strength of the phylogenetic signal in a data set (with
more phylogenetically informative data the likelihood function can
update the prior distribution more strongly).
In Table 2 and Fig. 2(a–c) the proportion of clades resolved with
PP 0.95 divided by the number of possible bipartitions (i.e., number of bipartitions in a fully dichotomous tree, which equals the
number of terminal taxa minus three) are shown. We choose to
compare only clades with PP 0.95, since these clades could be
considered ‘‘true” given the data and the models used in the Bayesian analyses (Huelsenbeck et al., 2002).
As a measure of phylogenetic accuracy we used the partition
metric (PM; Penny and Hendy, 1985) modified to account for multichotomies (Rzhetsky and Nei, 1992). The partition metric ranges
from zero (identical phylogenies) to twice the number of possible
clades resolved (number of taxa minus three). We only compared
phylogenetic accuracy for the reduced Spermacoceae data set
and the Hedyotis data set. Table 2 and Fig. 2(d and e) show the partition metric for the phylogenetic tree from the Bayesian analyses
of each of the DNA regions compared to the phylogenetic tree from
the Bayesian analysis of the combined data set, the latter taken as a
reasonable estimation of the ‘‘true” phylogeny. When calculating
the partition metric only nodes with PP 0.95 were considered.
The calculations were made using the function dist.topo in the R
package APE (Paradis et al., 2004; R Development Core Team,
2007). As another approximation of accuracy, we calculated the
proportion of the number of clades with PP 0.95 in the phylogenetic tree from the separate analysis of a given marker, which also
has a PP 0.95 in the tree from the combined analysis, divided by
the total number of clades with PP 0.95 in the combined tree (i.e.,
‘‘correctly” resolved clades assuming the combined tree to be the
‘‘true” tree; Table 2; Fig. 2f and g).
Table 1
Number of taxa, aligned length of the data matrices, number of parsimony informative characters, and the number of trees in the 95% credible set for the different data sets and
DNA regions. N, number of taxa; Al. l., aligned length; Pars. inf. – indels, number of parsimony informative characters excluding indel characters; Pars. inf. indels, number of
parsimony informative indel characters,% Pars. inf., percentage of the total number of aligned characters that are parsimony informative.
Data set
N
Al. l. (bp)
Pars. inf. - indels
Pars. inf. indels
% Pars. inf.
No. of trees in 95% cred. set
Spermacoceae
atpB-rbcL
rps16
trnL-F
petD
ITS
ETS
103
109
115
102
139
98
1,366
715
1,003
1,799
2,173
1,055
142
153
138
276
452
329
46
26
62
90
308
100
13.8%
25.0%
19.9%
20.3%
35.0%
40.7%
85,420
85,373
85,486
79,697
60,098
66,519
Reduced Spermacoceae
atpB-rbcL
49
rps16
49
trnL-F
49
petD
49
ITS
49
ETS
49
1,366
715
1,003
1,799
2,173
1,055
71
102
88
163
294
253
19
10
17
42
181
76
6.5%
15.7%
10.5%
11.4%
21.9%
31.2%
17,001
17,020
17,066
14,812
7,670
12,410
Hedyotis
ITS
ETS
5S-NTS
424
799
334
33
27
55
1
9
14
8.0%
4.5%
20.7%
3
19
9
9
9
9
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a
d
800
g
90000
700
80000
600
Spermacoceae
70000
500
60000
50000
400
40000
300
30000
200
20000
100
10000
0
0
atpB-rbcL
rps16
trnL-F
petD
No. of parsimony informa tive characters excluding indels
b
ITS
ETS
atpB-rbcL
e
500
Reduced Spermacoceae
trnL-F
petD
ITS
ETS
ITS
ETS
No. of trees in 95 % cred. set
h
450
18000
400
16000
14000
350
12000
300
10000
250
8000
200
6000
150
4000
100
2000
50
0
atpB-rbcL
0
atpB-rbcL
rps16
trnL-F
petD
No. of parsimony informative characters excluding indels
c
rps16
No. of parsimony informa tive indels
ITS
ETS
rps16
trnL-F
petD
No. of trees in 95 % cred. set
No. of parsimony informative indels
f
80
i
20
70
18
60
16
14
Hedyotis
50
12
40
10
8
30
6
20
4
2
10
0
0
ITS
ETS
5S-NTS
ITS
ETS
No. of trees in 95 %
No. of parsimony informative characters excluding indels
5S-NTS
cred. set
No. of parsimony informative indels
Fig. 1. (a–c) Number of parsimony informative characters in: (a) each of the six regions included in the analysis of the Spermacoceae data set, (b) the three nuclear regions
included in the analysis of the reduced Spermacoceae data set, (c) the three nuclear regions included in the analysis of the Hedyotis data set. (d–f) Linear regression of number
of parsimony informative characters (including indels) against aligned length (bp) of: (d) the separate DNA regions of the Spermacoceae data set (black circles = chloroplast
regions, white circles = nuclear regions, dashed line = regression line of the chloroplast data, r2 = 0.70, black line = regression line of all data combined, r2 = 0.61), (e) the
separate DNA regions of the reduced Spermacoceae data set (black circles = chloroplast regions, white circles = nuclear regions, dashed line = regression line of the chloroplast
data, r2 = 0.43, black line = regression line of all data combined, r2 = 0.51), (f) the Hedyotis data set (r2 = 0.37). (g–i) Number of trees in the 95% credible set for: (g) the
Spermacoceae data set, (h) the reduced Spermacoceae data set, (i) the Hedyotis data set.
3. Results
3.1. Phylogenetic utility
In the analysis of the Spermacoceae data the nuclear regions
provide most information (Table 1, Fig. 1a). ITS is the most informative region and provides 1.8 times as many parsimony informative characters as ETS and is more than twice as informative as
petD, the most informative chloroplast region. The petD region is
in turn about twice as informative as the other chloroplast regions.
Linear regressions of the number of parsimony informative
characters (including indels) against the lengths of the separate
matrices for both the chloroplast data separately (r2 = 0.70,
p = 0.161) and in combination with the nuclear data (r2 = 0.61,
p = 0.067) are shown in Fig. 1d. When the nuclear data are added,
less of the information content can be explained by just sequence
length, i.e., the nuclear data are indicated to be more informative
than the chloroplast data for a given sequence length.
A similar pattern is retrieved when comparing the data from the
reduced Spermacoceae data set (Table 1, Fig. 1b). ITS provides 1.4
times as many parsimony informative characters as ETS and 2.3
times as many as petD, the latter region being about twice as informative as the other chloroplast data. The linear regressions of parsimony informative characters relative to aligned length of the
chloroplast and combined data for the reduced Spermacoceae data
set are shown in Fig. 1e. The regressions indicate for this data set,
i.e., with reduced taxon sampling and no missing data, that the
length of the sequences explains less of the information content
(r2 = 0.50, p = 0.289 for the chloroplast and r2 = 0.43, p = 0.155 for
the combined data), than for the entire Spermacoceae data set.
For the Hedyotis data ETS and ITS provide about the same number of informative characters, but the proportion of informative in-
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Table 2
Proportion of clades with posterior probability 0.95 (PP 0.95) divided by the
number of possible bipartitions for the different data sets and DNA regions, the
partition metric (PM), and proportion of correctly resolved clades for the reduced
Spermacoceae and the Hedyotis data sets.
Data set
Clades with PP 0.95/
possible bipartitions
PM (PP 0.95)
Correctly resolved
clades
36.0%
45.3%
39.3%
61.6%
65.7%
63.2%
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
Reduced Spermacoceae
atpB-rbcL
37.0%
rps16
47.8%
trnL-F
41.3%
petD
63.0%
ITS
69.6%
ETS
58.7%
21
20
19
15
12
14
44.7%
55.3%
52.6%
71.1%
81.6%
71.1%
Hedyotis
ITS
ETS
5S-NTS
2
4
1
40.0%
80.0%
80.0%
Spermacoceae
atpB-rbcL
rps16
trnL-F
petD
ITS
ETS
83.3%
50.0%
66.7%
dels is larger for ITS. The 5S-NTS region provides the most parsimony informative characters (Table 1, Fig. 1c). It is about twice
as informative as the other two nuclear regions. A linear regression
of parsimony informative characters against the length of the
matrices is shown in Fig. 1f (r2 = 0.37, p = 0.582). Since the shortest
marker is the most informative, there is a negative correlation between parsimony informative characters and sequence length for
this data set.
For all data sets it seems that there is a variation in the information content among the DNA regions, more variability than can be
explained by sequence length alone. In particular, the nuclear data
are more informative than the chloroplast regions and the 5S-NTS
is the most informative nuclear region despite its short length.
Examination of DNA region variability using the number of
trees in the 95% credible sets (Table 1, Fig. 1g–i) from the separate
analyses of the Spermacoceae and the reduced Spermacoceae data
sets give nearly the same picture as the number of parsimony
informative characters: ITS had the fewest trees, followed by ETS
and petD. The other three chloroplast regions had about the same
number of trees. For the Hedyotis data set the ITS data, not the
5S-NTS, had the fewest number of trees in the 95% credible set.
Additionally, the number of well resolved nodes in the resulting
phylogenies can be an indication of the phylogenetic utility of a
DNA region. Here we consider a node to be well resolved if the posterior probability of that node is 0.95. To account for the different
sized taxon samples in the Spermacoceae data set, we compared
the number of nodes with a posterior probability 0.95 divided
by the maximum number of internal nodes in an unrooted, fully
dichotomous tree (Table 2; Fig. 2). For the Spermacoceae data,
the ITS region resolved the highest proportion of well resolved
nodes (66%; 70% for the reduced data set). The petD region performed comparatively better in this evaluation and resolved almost as many nodes as ETS (62% and 63%, respectively). In the
reduced Spermacoceae data set, petD resolved more nodes than
ETS (63% vs. 59%). The other three chloroplast regions as predicted
retrieved less nodes (36% for atpB-rbcL to 45% for rps16; 37% to 48%
for the reduced data set). For the Hedyotis data set ITS resolved the
highest number of nodes (83%) in spite of the fact that the 5S-NTS
provided the highest number of informative characters.
The most accurate region for the reduced Spermacoceae data
set (Table 2; Fig. 2d) was ITS (PM = 12) followed by ETS
(PM = 14), and the petD region (PM = 15). For the Hedyotis data
set (Table 2; Fig. 2e), 5S-NTS was more accurate than ITS (PM = 1
vs. PM = 2, respectively), which was in turn more accurate than
ETS (PM = 4). When comparing the number of correctly resolved
clades, the same picture emerges (Table 2; Fig. 2f and g). ITS correctly resolved the highest proportion of nodes for the reduced
Spermacoceae data set and is followed by ETS and petD, which performed equally well. For the Hedyotis data set both ITS and 5S-NTS
resolved the same proportion of the clades supported in the combined analysis.
3.2. Phylogenetic analyses
Spermacoceae are monophyletic in all analyses and all independent and combined analyses retrieved a number of well supported
clades (Fig. 3). The separate analyses were naturally less resolved
than the combined. The topologies from the separate analyses,
even if less resolved, agree with the topology of the combined analysis for all clades discussed here. No well supported relationships
in any of the separate analyses are contradicted by the other data.
The separate analyses of the Spermacoceae data will, thus, not be
discussed in detail. Neither will the phylogenies resulting from
the analyses of the reduced Spermacoceae data set, since these
were merely performed to enable us to compare the number of
parsimony informative characters and to calculate the partition
metric on congruent data sets. We regard the combined analysis
as the best estimate of the phylogeny (total evidence) of Spermacoceae and, if not otherwise indicated, it is the result on which we
will focus our discussions (Fig. 3). We also present both the combined and separate analyses of the Hedyotis data set (Fig. 4), because the 5S-NTS was not included in the combined analysis of
the Spermacoceae data set.
Adding the petD data to the previously published chloroplast
data (Groeninckx et al., in press) increased the proportion of well
resolved nodes from 52% to 56%. The addition of nuclear data further increased the proportion to 70%. In fact the nuclear data by
themselves resolved 67% of the possible nodes. The combined analyses showed a marked decrease in the number of trees in the 95%
credible set compared to the separate analysis (33,152 for the
Spermacoceae data set compared to 60,098 for ITS). In the reduced
Spermacoceae data the combined analyses yielded 553 credible
trees as compared to 7670 for ITS. The combined Hedyotis data
set produced three trees as did the ITS analysis. Apart from the difference in resolution between the DNA regions, we do not see any
bias for any of the regions towards better resolving either early or
late branching taxa.
4. Discussion
4.1. Phylogenetic utility
Inferring the phylogeny of a group naturally implies finding one
or several gene regions which are preferably single copy and are
informative. In a phylogenetic study the actual number of potentially informative characters are of interest, i.e., a longer but less
variable region is of more interest than a highly variable but short
region, which despite a high information content per sequenced
base pair may not provide enough characters to resolve the phylogeny. The 21 noncoding chloroplast regions studied by Shaw et al.
(2005) varied in the extent (22–83%) that length per se could explain the variation in number of potentially informative characters.
Mort et al. (2007) found a much lower correlation between length
and information content (5.2% for chloroplast data and 0.9% for
chloroplast plus ITS data), but since they included only rapidly
evolving regions this result is not unanticipated. The linear regres-
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a
0.70
Spermacoceae
0.60
0.50
0.40
0.30
0.20
0.10
0.00
atpB-rbcL
rps16
trnL-F
petD
Proportion of clades
b
ITS
ETS
0.95/possible bipartitions
f
d
0.80
Reduced Spermacoceae
25
0.70
0.60
0.60
20
0.50
0.50
0.40
15
0.40
0.30
10
0.30
0.20
0.20
5
0.10
0.10
0
0.00
atpB-rbcL
rps16
trnL-F
petD
Proportion of clades
c
ITS
0.00
atpB-rbcL
ETS
rps16
trnL-F
petD
ETS
atpB-rbcL
rps16
trnL-F
petD
ITS
ETS
Proportion correctly resolved clades
e
0.90
ITS
PM
0.95/possible bipartitions
g
0.80
0.9
4.5
0.70
0.8
4
0.7
0.60
Hedyotis
0.90
0.80
0.70
3.5
0.50
3
0.40
2.5
0.6
0.5
0.4
2
0.30
1.5
0.3
1
0.2
0.10
0.5
0.1
0.00
0
0.20
ITS
ETS
5S-NTS
0
ITS
ETS
PM
Proportion of clades
0.95/possible bipartitions
5S-NTS
ITS
ETS
5S-NTS
Proportion correctly resolved clades
Fig. 2. (a–c) Proportion of the possible number of clades with a PP 0.95 for: (a) the Spermacoceae data set, (b) the reduced Spermacoceae data set, and (c) the Hedyotis data
set. (d and e) The partition metric (PH-85) for: (d) the reduced Spermacoceae data set, (e) the reduced Hedyotis data set. (f and g) Proportion of correctly resolved clades in:
(f) the reduced Spermacoceae data set, (g) the Hedyotis data set.
sion of the chloroplast regions of our Spermacoceae data (Fig. 1)
showed that more than two-third of the parsimony informative
variation could be explained by the length of the sequences
(r2 = 0.70, p = 0.161). With the inclusion of the nuclear data the correlation decreased (Fig. 1d; r2 = 0.61, p = 0.067). The greater number of informative characters provided by the nuclear data are,
thus, indicative of the nuclear data being more informative than
the chloroplast data and not merely an effect of sequence length.
For the nuclear regions examined using the Hedyotis data set,
where the shortest region provided the highest number of informative characters, the linear regression naturally shows a negative
correlation (Fig. 1f; r2 = 0.37, p = 0.582). These results indicate that
nuclear data generally can be expected to provide more information than chloroplast data (Fig. 1). The highly variable chloroplast
regions identified by Shaw et al. (2005, 2007) should be further explored, especially in cases where gene trees are suspected to differ
from the species tree (Alvarez and Wendel, 2003). However, for
Spermacoceae the chloroplast and nuclear phylogenies are essentially identical (only the position of taxa with low posterior probabilities differ, and this is here interpreted as a lack of
phylogenetic signal and not due to incongruent data sets; see Section 4.2. for details of the Spermacoceae phylogeny). When inves-
tigating more rapidly evolving chloroplast regions with ITS, Mort
et al. (2007) did not find ITS to be either universally the most informative region or the region providing the highest clade support,
although it generally was one of the best choices for resolving their
low taxonomic level phylogenies.
It is noteworthy that the nuclear data not only resolve nodes between closely related taxa, but also support some of the early
branching events in Spermacoceae. Finding relatively fast evolving
DNA regions useful also at higher taxonomic levels (less homoplastic than is often assumed) has also been shown previously in the
Asterids (Bremer et al., 2002), Rubiaceae (Motley et al., 2005),
and basal angiosperms (Müller et al., 2006).
In order to obtain a well supported phylogeny of a group of organisms it is, however, not enough to analyze a high number of potentially phylogenetic informative characters. The included characters
should naturally reflect our best estimate of the true phylogeny.
According to our results, the regions providing the most information are in fact also the most accurate. The proportion of well
resolved nodes is generally also a good approximation, but with
two exceptions. Apparently, the number of parsimony informative
characters and the number of trees in the 95% credible set actually
give a valuable insight into the accuracy of a DNA region, i.e., the
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1
1
1
1
1
clade A
1
.89
1
1
1
1
1
1
1
1
1
1
1
1
.96
1
1
1
1
1
1
1
.91
1
1
1
1
1
1
1
.97
.53
1
1
.99
clade B
.87
.83
1
.99
1
.53
1
1
clade G
1
.89
1
clade D
1
1
1 .53
1
.98
.69
1
1
clade E
.96
1
.52
.60
1
.79
.56
.59
.61
Arcytophyllum-Houstonia clade
F
Agathisanthemum-Hedyotis s.s. clade
clade C
Pentanopsis clade
1
.94
Kohautia
1
.98
Batopedina pulvinellata
Outgroup
Carphalea madagascariensis
Pentanisia parviflora
Kohautia cynanchica
Kohautia subverticillata
Kohautia longifolia
Kohautia amatymbica
Kohautia senegalensis
Kohautia coccinea
Kohautia cf. longifolia
Kohautia caespitosa
Oldenlandia rosulata
Manostachya ternifolia
Gomphocalyx herniarioides
Phylohydrax madagascariensis
Phylohydrax carnosa
Oldenlandia affinis
Pentanopsis gracicaulis
Pentanopsis fragrans
Amphiasma benguellense
Amphiasma luzuloides Tanzania
Amphiasma luzuloides Zambia
Oldenlandia herbacea var. herbacea Dessein et al. 1041
Oldenlandia herbacea var. herbacea Dessein et al. 463
Oldenlandia herbacea var. goetzei Dessein et al. 1218
Oldenlandia herbacea var. goetzei Dessein et al. 442
Conostomium zoutpansbergense
Conostomium quadrangulare
Conostomium natalense Dahlstrand 1346
Conostomium natalense Bremer et al. 4341
Pentodon pentandrus Zambia
Pentodon pentandrus Zanzibar
Dentella repens
Dentella dioeca
Agathisanthemum globosum
Lelya osteocarpa
Agathisanthemum bojeri
Oldenlandia uniflora
Oldenlandia goreensis Dessein et al. 455
Oldenlandia angolensis
Oldenlandia goreensis Richards & Arasululu 25910
Oldenlandia goreensis Dessein et al. 1335
Oldenlandia goreensis Dessein et al. 1286
Hedyotis macrostegia
Hedyotis effusa
Hedyotis consanguinea
Hedyotis megalantha
Hedyotis korrorensis
Hedyotis swertioides
Hedyotis rhinophylla
Hedyotis fruticosa
Hedyotis lawsoniae
Hedyotis quinquenervis
Hedyotis lessertiana var. marginata
Hedyotis lessertiana var. lessertiana
Dibrachionostylus kaessneri Strid 2598
Dibrachionostylus kaessneri Strid 2564
Oldenlandia fastigiata
Mitrasacmopsis quadrivalvis
Hedythyrsus spermacocinus
Oldenlandia nervosa
Oldenlandia geophila
Oldenlandia echinulosa var. pellucida
Oldenlandia echinulosa
Oldenlandia microtheca
Stenaria nigricans
Houstonia longifolia
Houstonia caerulea
Arcytophyllum thymifolium
Arcytophyllum setosum
Arcytophyllum nitidum
Arcytophyllum lavarum
Arcytophyllum ericoides
Arcytophyllum rivetii
Arcytophyllum ciliolatum
Arcytophyllum macbridei
Arcytophyllum muticum
Arcytophyllum aristatum
FIGURE 3B
Fig. 3. (a and b) Phylogenetic tree of the combined analysis with posterior probabilities indicated below the branches.
more potentially phylogenetic informative characters a region provides the better one would expect it to estimate the correct species
phylogeny.
For data sets such as our Spermacoceae tribe data, with a wide
range of taxa representing different taxonomic levels, it seems that
one does not have to be too concerned for nuclear data to be either
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b
.54
1
clade H
1
1
.85
.75
1
1
.77
1
1
1
1
1
1
.95
1
.56
.66
1
1
1
1
1
1
1
1
clade I
1
.91
1
1
.98
.52
.59
1
1
.59
1
1
.73
.97
1
.74
.90
clade J
1
1
.56
1
1
.51
1
1
.90
1
K
.96
.85
.96
1
1
.61
.56
.69
.83
.97
.51
1
L
.96
1
.94
.84
.55
Spermacoce clade
1
Pachystigma
Oldenlandia s.s.
1
1
Kadua
1
Hedyotis capitellata
Oldenlandia mitrasacmoides ssp. trachymenoides
Oldenlandia mitrasacmoides ssp. nigricans
Synaptantha tillaeacea
Oldenlandia tenelliflora
Oldenlandia cf. galioides
Oldenlandia galioides
Oldenlandia lancifolia Dessein et al. 1356
Oldenlandia lancifolia Dessein et al. 1256
Oldenlandia biflora
Kadua affinis
Kadua fosbergii
Kadua axillaris
Kadua fluviatilis
Kadua acuminata
Kadua rapensis
Kadua coriacea
Kadua foggiana
Kadua centranthoides
Kadua parvula
Kadua degeneri
Kadua littoralis
Kadua laxiflora
Kadua cordata
Kadua flynnii
Kadua elatior
Kohautia obtusiloba
Kohautia virgata
Kohautia microcala Dessein et al. 1149
Oldenlandia wiedemannii
Oldenlandia corymbosa Gabon
Oldenlandia corymbosa Australia
Oldenlandia monanthos
Oldenlandia wauensis
Oldenlandia corymbosa Zambia
Oldenlandia sp. C Fl. Zamb.
Oldenlandia taborensis
Oldenlandia nematocaulis
Oldenlandia densa
Oldenlandia capensis var. pleiosepala
Oldenlandia capensis var. capensis
Manettia luteo-rubra
Manettia lygistum
Manettia alba
Arcytophyllum serpyllaceum
Bouvardia sp.
Bouvardia ternifolia
Bouvardia glaberrima
Nesohedyotis arborea
Oldenlandia tenuis
Oldenlandia salzmannii
Galianthe sp.
Galianthe eupatorioides
Galianthe brasiliensis
Diodia spicata
Emmeorhiza umbellata
Crusea megalocarpa
Crusea calocephala
Spermacoce flagelliformis
Spermacoce prostrata
Spermacoce erosa
Richardia stellaris
Richardia brasiliensis
Richardia scabra
Spermacoce confusa
Spermacoce verticillata cult.
Spermacoce verticillata Madagascar
Spermacoce capitata
Spermacoce remota
Spermacoce ocymifolia French Guiana
Spermacoce ocymifolia Ecuador
Mitracarpus microspermus
Mitracarpus frigidus
Psyllocarpus laricoides
Diodella teres
Ernodea littoralis
Spermacoce ruelliae
Spermacoce hispida
Spermacoce filituba
Spermacoce filifolia
Hydrophylax maritima
Diodia sarmentosa
Diodia aulacosperma
Fig. 3 (continued)
too homoplastic to provide a phylogenetic signal or to be indicative
of other gene trees not congruent with the chloroplast tree. Both
ITS and ETS, turn out to be the most useful regions. Perhaps with
a denser sample within some of the smaller well-defined clades
caution must be taken to caveats of nuclear data in general, and
ITS and ETS in particular (see, e.g., Alvarez and Wendel, 2003).
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ETS
Hedyotis effusa
Hedyotis megalantha
1.00
combined
Hedyotis korrorensis
Hedyotis rhinophylla
0.90
Hedyotis fruticosa
Hedyotis effusa
1.00
Hedyotis lawsoniae
Hedyotis lessertiana var. marginata
0.95
Hedyotis lessertiana var. lessertiana
0.94
Hedyotis megalantha
Hedyotis quinquenervis
1.00
Hedyotis korrorensis
ITS
Hedyotis effusa
Hedyotis korrorensis
Hedyotis rhinophylla
1.00
Hedyotis megalantha
Hedyotis rhinophylla
1.00
0.82
Hedyotis lawsoniae
Hedyotis fruticosa
1.00
Hedyotis fruticosa
Hedyotis lessertiana var. lessertiana
0.99
0.99
1.00
0.75
Hedyotis quinquenervis
1.00
Hedyotis lawsoniae
Hedyotis lessertiana var. marginata
Hedyotis lessertiana var. lessertiana
5S-NTS
Hedyotis effusa
1.00
Hedyotis korrorensis
Hedyotis quinquenervis
1.00
Hedyotis megalantha
Hedyotis lessertiana var. marginata
0.95
Hedyotis quinquenervis
1.00
Hedyotis lessertiana var. marginata
Hedyotis lessertiana var. lessertiana
0.95
Hedyotis lawsoniae
0.58
Hedyotis rhinophylla
1.00
Hedyotis fruticosa
Fig. 4. Phylogenetic trees from the combined and separate analyses of the Hedyotis data set with posterior probabilities indicated below the branches.
For example hybridization events between closely related taxa
might become an important issue to account for when studying
lower level relationships of particular clades. The highly variable
5S-NTS region seems, however, promising for such studies. This region is very informative, but should preferably best be used in
combination with some of the fast evolving chloroplast regions recently identified (Shaw et al. 2005, 2007; Mort et al., 2007) to
ascertain that the information it provides does infer the species
phylogeny.
4.2. Phylogeny of Spermacoceae
Our analysis combining nuclear and chloroplast data (Fig. 3)
agrees strongly with the chloroplast phylogeny presented by
Groeninckx et al. (in press). For example, Spermacoceae are monophyletic, Hedyotis and Oldenlandia are polyphyletic, and Kohautia is
biphyletic. Some genera are paraphyletic (e.g., Agathisanthemum
and Spermacoce) and others are monophyletic (e.g., Dentella and
Kadua) in both studies. Groeninckx et al. (in press) specifically discussed eight well supported clades: Kohautia, the Pentanopsis clade,
the Agathisanthemum–Hedyotis s.s. clade, Kadua, the Arcytophyllum-Houstonia clade, Oldenlandia s.s., Pachystigma, and Spermacoceae s.s. These clades are also retrieved in this study. We will
refer to Spermacoceae s.s. as the Spermacoce clade, since we do
not want to imply that the clade could be recognized at tribal level.
If such a tribe is acknowledged several other clades would also
have to be recognized as tribes and we currently do not have the
appropriate knowledge to delimit all such taxa. Furthermore, we
also question the value of a split of Spermacoceae into smaller entities, especially considering that many taxa outside the Spermacoce
clade often have been treated as congeneric (see below).
Our analyses provide several new insights into the phylogeny of
Spermacoceae. Spermacoceae in the combined analysis are resolved as a basal dichotomy. Clade A (PP 1.00) constitutes Kohautia
+ the Pentanopsis clade and includes Conostomium, which is retrieved as monophyletic in contrast to previous results of
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Groeninckx et al. (in press). Clade B was not recovered in the study
by Groeninckx et al. (in press), but it is not supported (PP 0.83).
Clade B constitutes two clades, clades C (PP 0.91) and D (PP
1.00). Clade C contains two subclades, Pentodon + Dentella and their
sister group, the Agathisanthemum–Hedyotis s.s. clade. This sister
relationship is a novel finding. In the analyses by Groeninckx
et al. (in press) Pentodon + Dentella were resolved as the possible
first branching clade of the entire tribe. Clade D constitutes the
remaining taxa of Spermacoceae. The Spermacoce clade is for
the first time retrieved as a well supported monophyletic clade.
The relationships within Spermacoceae will be dealt with in following detailed discussions of each major clade.
4.2.1. Kohautia
Kohautia has 36 species distributed from Africa via the Arabian
Peninsula to India and Australia (Govaerts et al., 2008; number of
species and distribution for genera are in the following taken from
this work, if not otherwise stated). It is well characterized by its
flowers, which always have both anthers and stigmas included
(Bremekamp, 1952). The stigmas are usually situated well below
the anthers, but occasionally they are nearly equal in height. This
flower type is so rare in Rubiaceae that the generic status of Kohautia never has been questioned. Groeninckx et al. (in press), nevertheless, showed that the genus was not monophyletic. Their
result is confirmed by our analyses. The Kohautia species form
two well supported clades corresponding to the two subgenera
of Kohautia, i.e., Kohautia and Pachystigma. Subgenus Kohautia is
the sister group (PP 1.00) of the so-called Pentanopsis clade and
subgenus Pachystigma is well supported as the sister group to Oldenlandia s.s. (PP = 1.00; clade I).
The subgenera Kohautia and Pachystigma are easily distinguished by their stigmas. The former has a style ending with two
filiform stigmas and the latter has a single ovoid or cylindrical stigma (Bremekamp, 1952). Indications from chromosomal and palynological data (Lewis, 1965a) as well as from seed shape
(Mantell, 1985) support the division into two separate clades.
Pachystigma is restricted to Africa and Madagascar, while Kohautia
extends to Tropical Asia and Australia. The two subgenera should
be treated as separate genera. Alternatively, the nine species of
Pachystigma could be included in Oldenlandia s.s., but considering
the distinct flower type we suggest the recognition of a new genus.
4.2.2. The Pentanopsis clade
The Pentanopsis clade as identified by previous chloroplast studies (Thulin and Bremer, 2004; Dessein et al., 2005; Groeninckx
et al., in press) is also supported by our results (PP 1.00). It is an
Afro-Madagascan clade. In addition to Pentanopsis it consists of
Amphiasma, Conostomium, Gomphocalyx, Manostachya, Phylohydrax,
and three species of Oldenlandia.
Pentanopsis itself consists of two species distributed from Ethiopia to Northern Kenya. Pentanopsis gracilicaulis was recently
transferred from Amphiasma. It agrees with Pentanopsis fragrans
in having larger flowers than the species of Amphiasma, persistent,
more or less woody stipules, and four- versus three-colporate pollen grains (Thulin and Bremer, 2004).
Amphiasma, here represented by two of its seven species, is distributed from southern Tanzania to Namibia. Our data together
with morphological support (e.g., tubular stipular sheaths, very
short corolla tube with a hairy throat, flattened seeds, and threecolporate pollen; Bremekamp, 1952) suggest that Amphiasma is
monophyletic (PP 1.00). Oldenlandia affinis has been shown to be
closely related to Amphiasma by Andersson and Rova (1999). Pentanopsis was not included in that study and Oldenlandia affinis is
in fact well supported as the sister group to Pentanopsis (PP 1.00;
this study; Groeninckx et al., in press). A detailed morphological
study should address whether additional Oldenlandia species also
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belong to this clade and if there are morphological characters
shared by Oldenlandia affinis, Pentanopsis, and Amphiasma other
than general characters such as sessile, linear leaves, only indistinctly beaked capsules, and seeds with non-punctate testa cells
(Bremekamp, 1952).
Conostomium is a genus of five species distributed from Ethiopia
to South Africa. It was initially described based on capsules that are
loculicidally dehiscent at the apex. This capsule type is, however,
also observed in other genera of Spermacoceae. Conostomium is
further characterized by its seeds with granulate testa cells and
characteristic pollen grains (Bremekamp, 1952). Other studies
(Lewis, 1965a) have shown that the distinctiveness of the pollen
grains was overemphasized.
Conostomium was not supported as monophyletic by
Groeninckx et al. (in press); Oldenlandia herbacea was nested
within it. In the present analyses we sequenced additional specimens (another specimen of Conostomium natalense, Oldenlandia
herbacea var. goetzei, and O. herbacea var. herbacea, i.e. two of the
three varieties) in order to retest these results. Our data support
Conostomium as monophyletic (PP 0.96) with Oldenlandia herbacea
as its sister group (PP 1.00).
Oldenlandia herbacea is similar to Conostomium in having coarsely granulate testa cells (Bremekamp, 1952; Dessein, 2003) and
comparatively large pollen grains (Bremekamp, 1952; Scheltens,
1998). Bremekamp (1952) placed Oldenlandia herbacea in the subgenus Euoldenlandia (the subgeneric classification of Bremekamp,
1952, only includes African species, but non-African species are
sometimes referred to). He mentioned that together with O. pumila
it differs from the other species of the subgenus by having longpedicellate flowers and granulate testa cells. Oldenlandia pumila
is distributed from India to Java (and is mentioned as introduced
elsewhere; Tanzania and Jamaica according to Bremekamp,
1952). Bremekamp (1952) suggested, consequently, that Oldenlandia herbacea, which is widespread in Africa and present in India
and on Sri Lanka, originated in Asia and spread into Africa. Considering that it is nested within the African Pentanopsis clade, this biogeographic hypothesis seems reversed. Since Oldenlandia herbacea
is related to Conostomium and not to Oldenlandia s.s., it should be
transferred out of Oldenlandia.
Phylohydrax with two species and the monotypic Gomphocalyx
are sister taxa (PP 1.00). They were formerly included in the Spermacoce clade (Robbrecht, 1988) based on their habit, uniovulate
ovary locules, and pluri-colporate pollen grains. Their placement
in the Pentanopsis clade is, however, supported both by their distribution (East Africa and Madagascar) and by several morphological
characters (e.g., heterostylous flowers, filiform stigma lobes, placenta attached at the base of the septum; Thulin and Bremer,
2004; Dessein et al., 2005).
Manostachya and Oldenlandia rosulata (PP 0.89) also belong to
the Pentanopsis clade as the sister group (PP 1.00) to Phylohydrax
and Gomphocalyx. Oldenlandia rosulata occurs in Tropical and
Southern Africa and is closely related to Oldenlandia microcalyx
from Cameroon and Angola, the only other member of Oldenlandia
subgenus Trichopodium (Bremekamp, 1952). Since these two species of Oldenlandia cannot be kept in Oldenlandia a new genus
should possibly be erected.
The three species of Manostachya from Central and Eastern
Tropical Africa are characterized by their testa cells with thick outer walls and a network of ridges on the inside and by large pollen
grains (Bremekamp, 1952). Manostachya notably has a basic chromosome number of x = 11, while most other African Spermacoceae
have x = 9 (Lewis, 1965a). Stephanococcus (not investigated here
due to lack of material) with a single winding species from
Cameroon, Gabon, and D.R. Congo has been considered an isolated
genus, but was suggested as the closest relative of Manostachya
based on similarities in the axillary flower clusters, stipular sheath,
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and in the flattened seeds (Bremekamp, 1952). It might consequently also belong to the Pentanopsis clade.
4.2.3. Clade C
The Pentodon and Dentella clade (PP 1.00) is the sister (PP 0.91)
to the Agathisanthemum–Hedyotis s.s. clade (PP 1.00). This sister
relationship is not found in the chloroplast analyses. Groeninckx
et al. (in press) reported Pentodon + Dentella as the first branching
clade of Spermacoceae (BS < 50%, PP 0.84). According to our chloroplast data the two genera are indicated as the sister group to clade
D, albeit with very low posterior probability (PP 0.56).
Because Pentodon and Dentella are supported as sister taxa in all
independent analyses (PP 1.00), a second individual of Pentodon
pentandrus was sequenced for the nuclear data. It reconfirms the
position and identity of the first individual. Pentodon has two African species, one of which is extending to the Arabian Peninsula and
Madagascar and is also introduced to the New World. Pentodon is
atypical among the Spermacoceae in having five-merous flowers
and peltate placentas with a bilobed apex (Bremekamp, 1952).
Dentella with its eight species (sometimes only one or two recognized; e.g., Ridsdale, 1998) has a wide distribution from Tropical
and Subtropical Asia to the Southwest Pacific. It shares with Pentodon the character five-merous flowers.
4.2.4. The Agathisanthemum–Hedyotis s.s. clade
Agathisanthemum forms a well supported clade (PP 1.00) together with Lelya and three species of Oldenlandia (O. angolensis,
O. goreensis, O. uniflora). This clade is sister to Hedyotis s.s. (PP
1.00). This result supports Bremekamp’s (1952, p. 5) notion that
Agathisanthemum is ‘‘in aspect not unlike the Indian Hedyotis fruticosa L and its nearest allies.”
The four species of the African Agathisanthemum are characterized
by capsules which split into two mericarps. This capsule structure is
similar to Hedyotis s.s., but Agathisanthemum differs from Hedyotis
s.s.by havingmorenumerous,smaller,angularseeds and shorterstigmas (Bremekamp, 1952). Verdcourt (1976) suggested that Agathisanthemum possibly would be better classified as a section of Hedyotis.
Agathisanthemum is, however, not monophyletic (Groeninckx
et al., in press; this study). The monotypic Lelya falls within Agathisanthemum. Both genera have the same pollen aperture type
(Lewis, 1965a). Lelya differs from all other Spermacoceae in having
a thick-walled capsule with a solid beak. This unique capsule was
the reason for the generic status for Lelya, which otherwise resembles Oldenlandia (Bremekamp, 1952). In habit it specifically resembles Oldenlandia goreensis (Bremekamp, 1952), one of the three
Oldenlandia species which constitute the sister group (PP 1.00) to
Agathisanthemum + Lelya (PP 1.00). Two of the three Oldenlandia
species are classified in Oldenlandia subgenus Anotidopsis (O. angolensis, O. goreensis). This subgenus includes other African species
and Asian species that are characterized by distinctly beaked capsules and sheathing stipules with a bifid or bipartite lobe on each
side of the stem (Bremekamp, 1952). The New World taxon
Oldenlandia uniflora (central and eastern North America, Caribbean,
Brazil, Paraguay, N. Argentina), is the sister to O. angolensis and O.
goreensis (PP 1.00). More detailed studies with extended sampling
are needed to evaluate if a new genus should be described to
encompass all or part of subgenus Anotidopsis and O. uniflora or if
these species are better treated as members of Agathisanthemum.
Hedyotis has traditionally been treated either in a narrow sense
(Hedyotis s.s.; Bremekamp, 1952; Hallé, 1966; Terrell, 1975, 1991,
2001c; Andersson et al., 2002) restricted to tropical and subtropical
Asia to the north-western Pacific or has been given a wide circumscription including also American and Polynesian taxa (e.g.,
Fosberg, 1943; Fosberg and Sachet, 1991; Merrill and Metcalf,
1942; Hsienshui et al., 1999; Dutta and Deb, 2004). Hedyotis s.s.
is a well supported clade (PP 1.00). There is no support for a
Hedyotis s.l. (Andersson et al., 2002; Groeninckx et al., in press).
All of the North American species should, thus, be recognized as
Houstonia or other segregate genera (Terrell, 1991, 2001a,b) and
the Polynesian taxa are to be treated as Kadua (Terrell et al.,
2005), as done here and by Govaerts et al. (2008). One of the sampled species currently accepted as Hedyotis (Govaerts et al., 2008)
do not group with the Hedyotis s.s. clade (Hedyotis capitellata; clade
H in Fig. 3b). On the other hand, two Asian species of Oldenlandia,
O. consanguinea and O. effusa, belong to Hedyotis s.s. and consequently the names Hedyotis consanguinea Hance and H. effusa
Hance should be used for the two species.
4.2.5. Clade D
The first branching taxon of clade D (PP 1.00) is the Kenyan
Dibrachionostylus. It is rather distinct within Spermacoceae in
having a style divided into two branches. Bremekamp (1952)
considered the monotypic Dibrachionostylus as a close relative
of Agathisanthemum and he identified additional differences
(e.g., corolla tube glabrous inside, entirely glabrous style, and
non-punctate testa cells). Verdcourt (1976) suggested affinities
also to Hedythyrsus.
Clade D constitutes, apart from Dribrachionostylus, a clade (clade
E; PP 0.96) with two lineages: clade F (PP 0.53) and the remaning
taxa of clade D (Fig. 3b; PP 0.66). Clade F contains Mitrasacmopsis + Hedythyrsus + four species of Oldenlandia (clade G; PP 0.89)
and the Arcytophyllum–Houstonia clade (PP 1.00).
4.2.6. Clade G
The monotypic Mitrasacmopsis from Central and Eastern Tropical Africa and Madagascar was originally placed in Loganiaceae because of its semi-inferior to superior ovary (Jovet, 1941).
Bremekamp (1952) described a new genus Diotocranus within
Hedyotideae, which was later reduced to the synonymy of Mitrasacmopsis. According to Bremekamp (1952), Mitrasacmopsis is morphologically similar to Hedythyrsus in the type of capsule
dehiscence (loculicidal followed by septicidal dehiscence), the
small number of seeds per capsule, and the strongly undulating
walls of the testa cells. They also share beaked capsules (although
the beak is much more pronounced and the capsule base is bilobed
in Mitrasacmopsis) and a similar placentation type (distinctly
stalked placentas with the ovules at a peripheral position of the
placenta; Groeninckx et al., 2007, in press).
Hedythyrsus includes two species with a similar distribution to
Mitrasacmopsis, but is absent from Madagascar. According to our
data Hedythyrsus is sister to Mitrasacmopsis (PP 1.00). The monotypic genera Pseudonesohedyotis and Nesohedyotis are allied to
Hedythyrsus according to Verdcourt (1976) and, consequently, they
probably also belong to this clade. The rps16 sequence of Nesohedyotis (Andersson and Rova, 1999) does according to our analysis,
however, belong to clade J (Fig. 3b). Nesohedyotis with unisexual
flowers is unusual among Spermacoceae. It is endemic to St. Helena. There is, thus, some biogeographical indication that its position in the largely American clade J might be correct. The
hypothesis that the Tanzanian Pseudonesohedyotis with hermaphroditic flowers also belongs to clade J seems less likely from a biogeographical and morphological standpoint and it has more likely
affinities to Hedythyrsus.
The sister group to Mitrasacmopsis and Hedythyrsus is Oldenlandia fastigiata (PP 1.00), which is distributed from Ethiopia to
Mozambique. It has a similar placentation type as the other two
genera (Groeninckx et al., in press), but has testa cells with straight
walls (Bremekamp, 1952). Oldenlandia fastigiata is classified in subgenus Oldenlandia (Bremekamp, 1952), a classification that is
unsupported phylogenetically.
The sister group (PP 0.89) of Mitrasacmopsis, Hedythyrsus, and
Oldenlandia fastigiata is a clade (PP 1.00) of three other species of
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Oldenlandia: O. echinulosa from Tropical Africa, O. geophila from
Zambia, and O. nervosa distributed from West Central Tropical Africa to Angola. Clade G, thus, seems to be an entirely African clade.
Oldenlandia echinulosa and O. nervosa are classified in subgenus
Hymenophyllum and O. geophila in subgenus Orophilum (Bremekamp, 1952). Both subgenera are characterized by distinctly petiolate leaves. Hymenophyllum comprises annuals with rather large
and thin leaves, a short, fimbriate stipular sheath, glabrous style,
and testa cells with undulating walls. It differs from subgenus Orophilum, which includes perennial species with smaller, often leathery leaves, a stipular sheath drawn out into a triangular lobe,
hirtellous style, and testa cells with straight walls (Bremekamp,
1952). The type species of subgenus Orophilum, Oldenlandia
monanthos, belongs to Oldenlandia s.s. (Fig. 3b).
Further studies are needed to properly investigate how to classify
the taxa of clade G. If the current phylogenetic relationships remain
with a denser taxon sampling, three possibilities should be investigated: (1) lumping all taxa into Mitrasacmopsis (generic name with
priority), (2) merging Hedythyrsus and Oldenlandia fastigiata into
Mitrasacmopsis and erecting a new genus for the other species of Oldenlandia, or (3) keeping Mitrasacmopsis and Hedythyrsus as distinct
genera and erecting two new genera to encompass Oldenlandia fastigiata and the other species of Oldenlandia, respectively.
4.2.7. The Arcytophyllum–Houstonia clade
The Arcytopyllum–Houstonia clade is well supported (PP 1.00).
Arcytophyllum with 16 species (excluding A. serpyllaceum; see below) distributed from Mexico to western South America is well
supported as monophyletic (PP 1.00; Andersson et al. 2002). The
sister group of Arcytophyllum is a clade (PP 1.00) comprising Houstonia (PP 0.69) + Stenaria nigricans (PP 0.98), and one species of Oldenlandia (PP 1.00): O. microtheca from Mexico. This clade as well as
the entire Arcytophyllum–Houstonia clade seems to be restricted to
the New World. That Oldenlandia microtheca belongs to the clade
seems reasonable considering both its distribution and the fact
that it has a basic chromosome number of x = 11, which is in contrast to most species of Oldenlandia (Lewis, 1965a), and agrees with
several counts made for species of Houstonia (Lewis, 1962, 1965b).
The relationships within Houstonia, which consists of 30 North and
Central American species, has recently been closer studied by
Church (2003).
Stenaria contains five species distributed from Central and Eastern USA to Mexico and Bahamas. It was previously included in
Hedyotis, but was elevated to generic status by Terrell (2001a). In
the study by Church (2003), Stenaria was nested within Houstonia
and, consequently, suggested to be merged with Houstonia. Two
closely related genera from Baja California, Stenotis (Terrell,
2001b) with seven species and the monotypic Carterella (Terrell,
1987; possibly congeneric with Stenotis; Church, 2003), most likely
also belong to the Arcytophyllum–Houstonia clade.
4.2.8. Clade H; Asian, Australian, and Pacific Spermacoceae
Clade H (Fig. 3b) is a well supported clade (PP 1.00), but its basal
branches are poorly supported. Hedyotis capitellata, distributed
from North Eastern India (Assam) to the Philippines, and the two
Australian species Oldenlandia mitrasacmoides and Synaptantha belong here, but their relationships to the other taxa of clade H are
uncertain.
Synaptantha with two species from Australia might be the sister
(PP 0.75) to the remaining taxa of clade H, the majority of which
have an Australian-Asian-Pacific distribution. Synaptantha has long
been recognised as a separate genus (Hooker, 1873). It differs from
other Spermacoceae by having corolla with the lobes slightly connate, stamens with filaments attached to both the ovary and the
corolla, and semi-inferior ovaries (Halford, 1992). Synaptantha does
not only have a distinct morphology, it also has many autapomor-
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phies in the ITS tree (one of the longest branches in the ITS tree;
not present in the ETS data). The sequence does, however, not seem
to represent a pseudogene since 5.8S is conserved; in a pseudogene
one would expect also 5.8S to have an elevated substitution rate
(Bailey et al., 2003). Synaptantha also seems to have an elevated
rate of evolution in the chloroplast regions. In the trnL-F analysis
it has the longest branch. It also has long branches in the other
three chloroplast regions, but in these regions the rate is not as
conspicuously elevated as for ITS and trnL-F.
The remaining taxa of clade H (PP 0.77) include a clade with
three Oldenlandia species (O. galioides, O. lancifolia, O. tenelliflora
[syn. Scleromitrion tenelliflorum; Hedyotis tenelliflora]; PP 1.00), Kadua (PP 1.00), and its sister species O. biflora (PP 1.00).
Both Oldenlandia galioides, which is distributed from New Guinea to the South West Pacific, and O. tenelliflora, from Tropical
and Subtropical Asia to Northern Queensland, have obconic seeds
(in contrast to, e.g., O. mitrasacmoides, which has scutelliform
seeds; Halford, 1992). Oldenlandia lancifolia has more angulate
seeds (Bremekamp, 1952) and is widespread in Africa and naturalized in Tropical South America. Bremekamp (1952) placed it in the
subgenus Aneurum together with the North American Oldenlandia
boscii and the Asian O. diffusa. One or several new genera should
better be recognised to acknowledge the early branching members
of clade H, since the species clearly do not belong to either Hedyotis
s.s. or Oldenlandia s.s.
Oldenlandia biflora is the sister group to Kadua (PP 1.00) as in the
chloroplast study by Groeninckx et al. (in press). It is distributed
from tropical and subtropical Asia to the West Pacific. That the sister group to the Polynesian Kadua would have such a distribution
seems reasonable. Oldenlandia biflora should be transferred from
Oldenlandia, but whether there is morphological support to include
it in Kadua will have to await further studies.
Kadua with presently 28 species (Terrell et al., 2005; Govaerts
et al., 2008) is monophyletic (PP 1.00). This supports the conclusion from previous molecular and seed anatomical studies that
the Polynesian taxa formerly included in Hedyotideae (as Hedyotis,
Gouldia, or Wiegmania) should be regarded as the separate genus
Kadua (Motley, 2003; Terrell et al., 2005). Motley (in press) has
studied Kadua with a broader sampling and found that Kadua is
monophyletic and subclades for the most part reflect former classifications. This study also showed that the Hawaiian Kadua species
are paraphyletic with respect to the French Polynesian species and
provide strong evidence for migration of a plant lineage out of the
archipelago. The nested position of K. rapensis in the Kadua clade in
this study supports this finding.
4.2.9. Clade I, the true Oldenlandia and Pachystigma
Oldenlandia is clearly polyphyletic. The clade including the type
species, O. corymbosa, is here referred to as Oldenlandia s.s. (PP
1.00). All species of Oldenlandia not belonging to this clade should
be transferred to other genera or assigned to new genera. Some
suggestions are already made above, but we refrain from making
the formal taxonomic changes pending studies with better sampling. The monotypic genus Thecorchus distributed from Ethiopia
to Senegal should, however, no longer be recognized (Kårehed
and Bremer, 2007; Groeninckx et al, in press). It is nested within
Oldenlandia s.s. and the use of the name Thecorchus wauensis
(Schweinf. ex Hiern) Bremek. should be abandoned in favor of its
basionym Oldenlandia wauensis Schweinf. ex Hiern.
As mentioned above, the sister group (PP 1.00) of Oldenlandia
s.s. is Kohautia subgenus Pachystigma (PP 1.00), which should be
recognized as a distinct genus.
4.2.10. Clade J and the origin of the Spermacoce clade
Clade J (PP 1.00) is the sister group (PP 1.00) of clade I. It is almost entirely restricted to the New World. All taxa of clade J, ex-
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cept Nesohedyotis from St. Helena, are from the New World. A
South American origin of the Spermacoce clade as suggested by
Dessein (2003) seems very probable, since the Spermacoce clade
is a predominantly New World taxon. Only Diodia s.l., Hydrophylax,
and Spermacoce s.l. (pantropical) extend into other tropical regions.
Manettia (PP 1.00) and Bouvardia (PP 0.74) are sister genera (PP
0.97). If Arcytophyllum serpyllaceum (rps16 data only) is included
in Bouvardia (PP 1.00) as suggested by Andersson et al. (2002) they
appear monophyletic. Manettia with 124 species from Tropical
America has been recognised as a separate tribe, Manettieae
(Bremekamp, 1934). Based on its winged seeds, Manettieae were
previously placed in the subfamily Cinchonoideae (Schumann,
1891). Manettia was later moved to the Rubioideae and Hedyotideae
because of the presence of raphides (Bremekamp, 1966). Robbrecht
(1988) included both genera in Cinchoneae, but because of the
shared presence of raphides the Hedyotideae were suggested as a
possible alternative placement. Manettia is morphologically similar
to Bouvardia and Bremer (1996) found the two genera to be sister
taxa in Rubioideae based on molecular data. Bouvardia comprises
41 species from Southern USA to Central America. Manettia differs
from Bouvardia mainly in its viney habit and hard endosperm.
Nesohedyotis (rps16 data only) and two Oldenlandia species (O.
salzmanii + O. tenuis; PP 1.00) belong to clade J, but their position as
successive sister taxa to the Spermacoce clade is very weekly
supported.
4.2.11. The Spermacoce clade
The Spermacoce clade, although long recognized as a separate
tribe (Spermacoceae; Berchtold and Presl, 1820; Hooker, 1873;
Bremekamp, 1952, 1966; Verdcourt, 1958; Robbrecht, 1988,
1994), was not recovered as a well supported monophyletic group
in the chloroplast study by Groeninckx et al. (in press). However,
with the addition of nuclear data, the Spermacoce clade is well supported (PP 1.00), provided that Gomphocalyx and Phylohydrax are
excluded. Both genera belong to the Pentanopsis clade (Thulin
and Bremer, 2004; Dessein et al., 2005; see above).
The Spermacoce clade differs from the rest of the tribe in having
ovaries with a single ovule per locule attached near the middle of
the septum and often pluriaperturate pollen grains, in contrast to
few to many ovules per locule and often tricolporate pollen grains.
The genera of the Spermacoce clade are often recognized based on
the type of fruit dehiscence. A detailed overview of the Spermacoce
clade is given by Dessein (2003).
Galianthe (PP 1.00; including Diodia spicata) is the first branching
taxon of the Spermacoce clade. It constitutes 45 species from Mexico
to South America and is characteristic among Spermacoceae s.s. in
having terminal lax inflorescences, heterostylous flowers, and a
stigma with two distinct lobes (Cabral, 1991). Diodia spicata (treated
under the name Spermacoce spicata (Miq.) in ed. by Govaerts et al.,
2008) makes Galianthe paraphyletic. Diodia spicata differs by having
terminal spicate inflorescences with isostylous flowers and fruits
with two cocci separating from the base. Nevertheless, since it approaches Galianthe with its two-armed stigma and 7-zonocolporate
pollen with relatively long colpi and double reticulum, it seems reasonable to include Diodia spicata in Galianthe (Dessein, 2003).
The Central American Crusea, here represented by two of 14 species, is monophyletic (PP 1.00). Emmeorhiza, a South American monotypic genus, is the sister to Crusea (PP 0.90). This relationship was not
found by Groeninckx et al. (in press). The position of Emmeorhiza was
not well supported in their study. In the strict consensus tree of the
parsimony analysis it was the sister to Galianthe + Diodia spicata (JK
and BS <50%) and in the Bayesian inference it was the sister to Nesohedyotis arborea (PP 0.67). The addition of petD data to the other chloroplast data places Emmeorhiza in the same larger clade as in the
combined analysis (clade K; PP 0.96), but as the first branching taxon
(PP 0.96) and not as the sister to Crusea.
Emmeorhiza is somewhat similar in habit to the genus Denscantia.
Both genera are climbers with thyrsoid-like inflorescences with isostylous flowers (Dessein, 2003). Denscantia (first published as Scandentia, but later changed to Denscantia; Cabral and Bacigalupo,
2001a,b) was segregated from the probably closely related Galianthe.
Denscantia has isostylous flowers, lateral fusion of the stipular bases,
and pollen with multiple endoapertures whereas Galianthe often has
heterostylous flowers, stipules fused only with the leaf base or the
petiole, and pollen with a simple endocingulum (Cabral and Bacigalupo, 2001a; Dessein, 2003). Further study is needed to determine if
Denscantia belong to the Crusea + Emmeorhiza clade or if the morphological resemblance to Emmeorhiza is due to convergence.
The genus Spermacoce is not monophyletic in the present study.
Several smaller genera are intermingled with Spermacoce species.
Richardia (PP 1.00) has 16 species from Tropical and Subtropical
America and is naturalized elsewhere. It is a well defined genus
characterized by mainly 3–4-carpellate ovaries and schizocarpous
fruits splitting into indehiscent mericarps (Dessein, 2003).
Spermacoce ocymifolia has been treated as Hemidiodia ocymifolia
in a genus of its own (Schumann, 1888) characterized by its fruits.
They consist of two indehiscent mericarps. At maturity the mericarps are only partially joined by their bases. Hemidiodia was sunk
into Borreria (a genus previously recognized to encompass American Spermacoce) because, apart from the fruit type, they are morphologically similar (Bacigalupo and Cabral, 1996). Our data
show Spermacoce ocymifolia as sister to S. remota (PP 0.83).
Mitracarpus (PP 1.00; represented by two out of the 49 species)
also belongs to the Spermacoce clade as the first branching taxon of
a well supported subclade (clade L; PP 0.96). Mitracarpus is from
Tropical America and is naturalized elsewhere. The genus is distinguished by its cirumscissile opening of the fruits and in having two
large and two small calyx lobes and seeds with an X-shaped ventral groove (Dessein, 2003).
Diodella teres and Psyllocarpus laricoides, two species only represented by nuclear data, are each others closest relatives (PP 1.00)
and the second branching clade (PP 0.94) of clade L. If their sister
group relationship is due only to the present taxon sampling or a
true relationship will have to await further studies. No obvious
morphological characters are shared by the two genera. Psyllocarpus with its nine Brazilian species is distinct from the other genera
of the Spermacoce clade by having a capsule strongly compressed
parallel to the septum.
Govaerts et al. (2008) accepted 30 species in Diodia. In contrast,
Bacigalupo and Cabral (1999) only recognized five species in the
genus. These authors transferred 16 species provisionally to the
genus Diodella. Nine of these are now formally published under
Diodella. Diodella is distributed from Central USA to Tropical America and is also present in Africa. It is characterized by fruits dehiscing
into two, indehiscent, one-seeded mericarps. Dessein (2003) suggests that species assigned to Diodella fall into two groups: one
group is related to Diodella teres, the type species, the other one to
Diodella sarmentosa. This is confirmed by our analysis as Diodella sarmentosa forms a clade (PP 0.84) with Diodia aulacosperma, Ernodea,
Hydrophylax, Spermacoce hispida, S. ruelliae, S. filifolia, and S. fillituba.
The African Diodia aulacosperma (Socotra, S. Somalia to E. Tanzania (including Zanzibar) is not regarded as a member of a restricted Diodia (Bacigalupo and Cabral, 1999; Dessein, 2003).
Ernodea, a genus with nine species distributed from Florida south
to Central America and in the Caribbean Islands, shares some seed
and pollen morphological characters with Diodella sarmentosa
(Dessein, 2003), but are in habit and fruit morphology quite different. Diodella sarmentosa has dry fruits splitting into two mericarps,
while Ernodea has drupaceous fruits, a rare condition in Spermacoceae. Hydrophylax maritima, the sole species in the genus, is a sea
shore plant from India, Sri Lanka, and Thailand. Dessein (2003)
pointed to the strong similarity in habit between Hydrophylax
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and the West African Diodia vaginalis (another of the species probably to be excluded from Diodia) and species of Diodia s.s. However,
pollen grains are very different in these taxa.
Further studies, especially with a larger sampling of Spermacoce
species, will reveal if Spermacoce should be split or if several of the
other genera of the Spermacoce clade should be merged.
Note added in proof
The DNA here sequenced as Spermacoce filifolia was extracted from Spermacoce flagelliformis, De Block et al. 794
(BR). The accession numbers AM939538 and AM933010
consequently refer to sequence data from the latter species.
Acknowledgments
Anbar Khodabandeh has been very helpful in the laboratory.
Sylvain Razafimandimbison and Johan Nylander have contributed
with valuable suggestions and discussions. The study was financed by a research grant to BB from The Swedish Research
Council.
Appendix
List of taxa used in the phylogenetic analyses with voucher information (geographic origin, collector, collector number, herbarium), and
accession numbers. New taxa/specimens not included in by Groeninckx et al. (in press) are indicated in bold. Missing sequences are marked
with. Literature citations for previous published sequences: (1)=Andersson and Rova 1999, (2)=Andersson et al. 2002, (3)=Dessein et al.
2005.
Taxon
Voucher
information
Agathisanthemum Klotzsch
A. bojeri Klotzsch
Zambia: Dessein
et al. 671 (BR)
A. globosum (Hochst.
Zambia: Dessein
ex A. Rich.) Klotzsch et al. 201 (BR)
Amphiasma Bremek.
A. benguellense (Hiern)
Bremek.
A. luzuloides (K.
Schum.) Bremek.
Angola: Kers 3350
(S)
Zambia: Dessein
et al. 1167 (BR)
Tanzania: Iversen
et al. 87694 (UPS)
Arcytophyllum Willd. ex Schult. & Schult. f.
A. aristatum Standl.
Ecuador: Hekker &
Hekking 10335
(GB)
A. ciliolatum Standl.
Unknown: Oligaard
et al. 58395 (NY)
A. ericoides (Willd. ex Unknown: Edwin et
Roem. & Schult.)
al. 3624 (S)
Standl.
A. lavarum K. Schum.
Unknown:
Cronquist 8827
(NY)
A. macbridei Standl.
Unknown:
Wurdack 1073 (NY)
A. muticum (Wedd.)
Colombia:
Standl.
Andersson et al.
2195 (GB)
A. nitidum (Kunth)
Unknown: Pipoly
Schltdl.
et al. 6467 (GB)
A. rivetii Danguy &
Ecuador: Harling &
Cherm.
Andersson 22232
(GB)
A. serpyllaceum
Mexico: Stafford
(Schltdl.) Terrell
et al. 203 (MO)
A. setosum (Ruiz &
Unknown:
Pav.) Schltdl.
Andersson et al.
2196 (GB)
atpB-rbcL
rps16
ITS
ETS
5S-NTS
trnL-trnF
petD
EU542917 EU543018
EU543077
EU557678 AM939424 /
/
EU542918 EU543019
EU543078
EU557679 AM939425 /
/
EU542919 AF002753(1)
EU543079
EU557680 AM939426 AM932918 /
EU542920 EU543020
EU543080
EU557681 AM939428 AM932919 /
/
/
/
/
/
AF333348(2) AF333349(2) /
/
/
/
/
AF333350(2)
AF333351(2) /
/
/
/
/
AF333352(2) AF333353(2) /
/
/
/
/
AF333354(2) AF333355(2) /
/
/
/
/
AF333356(2) AF333357(2) /
/
/
/
EU557682 AM939429 /
/
/
/
/
AM939430 /
/
/
/
/
/
AF333365(2) /
/
/
/
EU542921 AF002754(1)
/
EU543081
AF333359(2) /
EU542922 AF333362(2) AF333363(2) /
/
AF333364(2) /
/
AF002755(1)
AM939427 AM932920 /
/
(continued on next page)
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Appendix Table 1 (continued)
Taxon
Voucher
information
A. thymifolium (Ruiz & Ecuador: Ståhl
Pav.) Standl.
4481 (GB)
Batopedina Verdc. (outgroup)
B. pulvinellata
Zambia: Dessein
Robbrecht
et al. 264 (BR)
D.R. Congo:
Malaisse 7695
(UPS)
Bouvardia Salisb.
B. glaberrima Engelm.
B. ternifolia (Cav.)
Schltdl.
B. sp.
atpB-rbcL
rps16
Crusea Cham. & Schltdl.
C. calocephala DC.
Guatemala:
Gustafsson et al.
215 (GB)
C. megalocarpa (A.
Mexico: Pringle
Gray) S. Watson
3852 (S)
Diodella Small
D. sarmentosa Sw.
French Guiana:
Anderson et al.
2071 (GB)
ETS
5S-NTS
EU542924 EU543021
EU543083
EU557684 /
/
/
/
EU543084
/
EU557685 AM939432 AM932922 /
/
/
/
/
/
/
/
AM266989 /
/
/
/
s.n.
/
/
/
/
/
/
AM939433 AM932923 /
/
EU557686 /
/
/
EU542927 AF002760(1)
EU543085
EU557687 AM939435 AM932925 /
/
/
/
EU542928 EU543024
EU543086
EU557688 AM939436 AM932926 /
EU542929 /
EU543087
EU557689 AM939437 AM932927 /
EU542930 /
EU543088
EU557690 AM939438 AM932928 /
EU542931 EU543025
EU543089
EU557691 AM939439 AM932929 /
EU543090
EU557692 /
EU543091
EU557693 AM939440 AM932930 /
EU542933 AF002761(1)
/
EU557694 AM939442 AM932932 /
/
/
/
/
AM939441 AM932931 /
/
AF002762(1)
/
/
/
/
Dentella J. R. Forst & G. Forst.
D. dioeca Airy Shaw
Australia: Harwood /
/
1559 (BR)
D. repens (L.) J. R. Forst. Australia:
EU542932 AF333370(2)
& G. Forst.
Andersson 2262
(GB)
Dibrachionostylus Bremek.
D. kaessneri (S. Moore) Kenya: Strid 2598
Bremek.
(GB)
Kenya: Strid 2564
(UPS)
ITS
EU557683 AM939431 AM932921 /
Carphalea Juss. (outgroup)
C. madagascariensis
Madagascar: De
EU542926 EU543023
Lam.
Block et al. 578 (BR)
/
/
Madagascar:
Razafimandimbison
524 (UPS)
Conostomium (Stapf) Cufod.
C. natalense (Hochst.) South Africa:
Dahlstrand 1346
Bremek.
(GB)
South Africa:
Bremer et al. 4341
(UPS)
C. quadrangulare
Ethiopia: Puff &
(Rendle) Cufod.
Kelbessa 821222 2/
2 (UPS)
C. zoutpansbergense
South Africa:
(Bremek.) Bremek.
Bremer et al. 4331
(UPS)
petD
EU542923 AF333366(2) EU543082
Cult.: Forbes s.n. (S) EU542925 EU543022
Cult.: S2928 (BR)
/
AF002758(1)
Mexico: Spencer
et al. 363 (NY)
Mexico: Torres &
Torino 3637 (BR)
trnL-trnF
/
/
/
AM266995 /
/
AM939434 AM932924 /
/
/
/
/
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Appendix Table 1 (continued)
Taxon
Voucher
information
atpB-rbcL
rps16
trnL-trnF
petD
ITS
D. teres (Walter)
Small
Madagascar: De
Block et al. 793
(BR)
/
/
/
/
AM939443 AM932933 /
Kenya: Luke 9029
(UPS)
French Guiana:
Anderson et al.
1961 (GB)
EU542934 EU543026
EU543092
EU557695 AM939444 AM932934 /
EU542935 EU543027
EU543093
EU557696 AM939535 AM933008 /
EU542936 AY764289(3) EU543094
EU557697 AM939445 AM932935 /
Cuba: Rova et al.
2286 (GB)
EU542937 AF002763(1)
EU543095
EU557698 AM939446 AM932936 /
Argentina: Vanni &
Radovancick 996
(GB)
Argentina: Schinini
& Cristobal 9811
(GB)
Bolivia: Persson &
Gustavsson 298
(GB)
EU542938 AY764290(3) EU543096
EU557699 AM939447 AM932937 /
EU542939 EU543028
EU543097
EU557700 AM939448 AM932938 /
/
/
/
AM939449 AM932939 /
EU567461
/
/
/
Diodia L. s.l.
D. aulacosperma K.
Schum.
D. spicata Miq.
(Spermaoce spicata
(Miq.) in ed.;
Govaerts et al.,
2008)
Emmeorhiza Pohl ex Endl.
E. umbellata (Spreng.) Trinidad: Hummel
K. Schum.
s.n. (GB)
Ernodea Sw.
E. littoralis Sw.
Galianthe Grieseb.
G. brasiliensis (Spreng.)
E. L. Cabral &
Bacigalupo
G. eupatorioides
(Cham. & Schltdl.)
Cabral
G. sp.
Gomphocalyx Baker
G. herniarioides Baker
Hedyotis L.
H. capitellata Wall.
/
Madagascar: De
EU542940 AY764291(3) EU567466
Block et al. 569 (BR)
Burma: Meebold
17373 (S)
H. consanguinea Hance Hong Kong: Shiu
(syn. Oldenandia
Ying Hu 10821 (S)
consanguinea
(Hance) Kuntze)
H. effusa Hance (syn. China: Tsang
Oldenlandia effusa 21044 (S)
(Hance) Kuntze)
H. fruticosa L.
Sri Lanka: Larsson &
Pyddoke 22 (S)
H. korrorensis
The Caroline
(Valeton) Hosok.
Islands: Fosberg
47697 (S)
H. lawsoniae Wight
Sri Lanka:
Wambeek &
Wanntorp 2996 (S)
H. lessertiana var.
Sri Lanka:
lessertiana Thwaites Klackenberg 413 (S)
H. lessertiana var.
Sri Lanka: Fagerlind
marginata Thwaites 3668 (S)
& Trimen
H. macrostegia Stapf.
Sabah:Wallander 6
(GB)
/
ETS
5S-NTS
/
/
/
AM939452 /
/
EU542941 /
/
EU557701
AM939450
AM931939
/
/
/
AM939491 AM932940 AM931935
EU542942 /
EU543098
EU557702 AM939453 AM932941 AM931929
EU542943 /
EU543099
EU557703 AM939454 AM932942 AM931937
EU542944 /
/
EU557704 AM939455 AM932943 AM931931
EU542945 EU543029
EU543100
EU557705 AM939466 AM932944 AM931932
EU542946 EU543030
EU543101
EU557706 AM939456 AM932945 AM931934
EU542947 AF002767(1)
EU543102
/
/
/
AM942768 /
/
(continued on next page)
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Appendix Table 1 (continued)
Taxon
Voucher
information
H. megalantha Merr.
atpB-rbcL
rps16
trnL-trnF
petD
ITS
ETS
5S-NTS
/
/
/
/
AM939457 AM932946 AM931936
Marianas (Guam):
Andersson 07 (S)
H. quinquenervis
Sri Lanka: Bremer
Thwaites
et al. 163 (S)
H. rhinophylla
Sri Lanka: Fagerlind
Thwaites ex Trimen 5082 (S)
H. swertioides Hook. f. South India:
Klackenberg &
Lundin 03 (S)
EU542948 /
EU543103
EU557707 AM939458 AM932947 AM931933
EU542949 /
EU543104
EU557708 AM939459 AM932948 AM931930
EU542950 EU543031
EU543105
EU557709 AM939460 /
AM931938
Hedythyrsus Bremek.
H. spermacocinus (K.
Schum.) Bremek.
Zambia: Dessein
et al. 1017 (BR)
EU542951 EU543032
EU543107
EU557711 AM939461 AM932950
/
USA: Vincent &
Lammers s.n. (GB)
USA: Yatskievych
96-49 (MO)
USA: Weigend
9963 (NY)
EU542953 AF333379(2) EU543109
EU557713 AM939464 /
/
EU542954 AF002766(1)
/
EU567462 AM939465 /
/
/
s.n.
/
/
/
/
EU567457 /
/
/
/
/
/
EU542955 /
EU543110
EU557714 AM939467 AM932952 /
/
s.n.
s.n.
/
AM942769 /
/
/
AF002765(1)
/
/
/
/
/
/
s.n.
s.n.
/
AM942770 /
/
EU543111
EU557715 AM939468 /
/
EU542957 AF333376(2) EU543112
EU557716
/
/
/
/
/
/
AM939469 /
/
/
s.n.
s.n.
/
AM942771 /
/
Houstonia L.
H. caerulea L.
H. longifolia Gaertn.
Hydrophylax L. f.
H. maritima L. f.
Sri Lanka:
Lundqvist 8945
(UPS)
Kadua Cham. & Schltdl.
K. acuminata Cham. & Hawaii: cult. at BR
Schltdl.
K. affinis Cham. &
Hawaii HI: Motley
Schltdl.
1733 (NY)
K. axillaris (Wawra) W. Hawaii: HarrisonL. Wagner &
Gagne s.n. (GB)
Lorence
Maui HI: Motley
1724 (NY)
K. centranthoides
Hawaii: Skottsberg
6788 (S)
Hook. & Arn.
K. cordata Cham. &
Cult.: Lorence 8021
(PTBG)
Schltdl.
Hawaii HI:
Fagerlind 6863 (S)
K. coriacea (J. E. Smith) Hawaii HI: Motley
W. L. Wagner &
1703 (NY)
Lorence
K. degeneri (Fosberg)
Cult.: Wood 5062
W. L. Wagner &
(PTGB)
Lorence
K. elatior (H. Mann) W. Kauai HI: Wagner
6350 (BISH)
L. Wagner &
Lorence
K. fluviatilis C. N.
Oahu HI: Motley
Forbes
1747 (NY)
K. flynnii (W. L.
Kauai HI: Perlman
Wagner & Lorence) 15631 (BISH)
W. L. Wagner &
Lorence
K. foggiana (Fosberg)
Hawaii: Sparre 27
W. L. Wagner &
(S)
Lorence
/
EU542956 EU543033
EU542958 AF333371(2) EU543113
EU557717 AM939470 AM932953 /
/
s.n.
s.n.
/
AM942772 /
/
/
s.n.
s.n.
/
AM942773 /
/
/
s.n.
s.n.
/
AM942774 /
/
EU543114
EU557718 AM939471 /
/
EU542959 /
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Appendix Table 1 (continued)
Taxon
Voucher
information
atpB-rbcL
rps16
trnL-trnF
petD
ITS
K. fosbergii (W. L.
Wagner & D. R.
Herbst) W. L.
Wagner & Lorence
K. laxiflora H. Mann
Oahu HI: Motley
1677 (NY)
/
s.n.
s.n.
/
AM942775 /
/
Molokai HI:
Perlman 6677
(BISH)
Cult. at WU: Kiehn
& Luegmayr
920823-2/1
Cult.: Perlman
12783 (GB)
Rapa Is.French
Polynesia: Perlman
17953 (NY)
/
s.n.
s.n.
/
AM942776 /
/
EU543115
EU557719 AM939472 AM932954 /
EU542961 AF333375(2) EU543116
EU557720 AM939473 AM932955 /
/
s.n.
/
EU542962 EU543035
EU543117
EU557721 AM939484 AM932956 /
EU542963 EU543036
EU543118
EU557722 AM939474 AM932957 /
EU542964 EU543037
EU543119
EU557723 AM939476 AM932959 /
EU542965 EU543038
EU543120
EU557724 AM939477 AM932960 /
/
/
/
/
AM939478 AM932961 /
/
/
/
/
AM939475 AM932958 /
EU542966 EU543039
EU543121
EU557725 AM939479 AM932962 /
/
/
/
EU542967 EU543040
EU543122
EU557726 AM939481 /
/
/
s.n.
/
/
EU542968 EU543041
EU543123
EU557727 AM939482 AM932964 /
EU542969 /
EU543124
EU557728 AM939483 AM932965 /
EU542970 /
EU543125
EU557729 AM939485 /
/
/
/
EU567463 /
EU543126
EU557730 AM939487 AM932967 /
EU543127
EU557731 /
K. littoralis Hillebr.
K. parvula A. Gray
K. rapensis F. Br.
Kohautia Cham. & Schltdl.
K. amatymbica Eckl. & South Africa:
Zeyh.
Bremer et al. 4307
(UPS)
K. caespitosa Schnizl.
Zambia: Dessein
et al. 432 (BR)
K. coccinea Royle
Zambia: Dessein
et al. 751 (BR)
K. cynanchica DC.
Zambia: Dessein
et al. 469 (BR)
K. longifolia Klotsch
Zambia: Dessein
et al. 462 (BR)
K. cf. longifolia
Zambia: Dessein
Klotsch
et al. 790 (BR)
K. microcala Bremek.
Zambia: Dessein
et al. 1149 (BR)
Zambia: Dessein
et al. 1321 (BR)
K. obtusiloba Schnizl.
Kenya: Luke 9035
(UPS)
K. senegalensis Cham.
Burkina Faso:
& Schltdl.
Madsen 5940 (NY)
K. subverticillata (K.
Zambia: Dessein
Schum.) D. Mantell et al. 432 (BR)
K. virgata (Willd.)
Madagascar: De
Bremek.
Block et al. 539 (BR)
Lelya Bremek.
L. osteocarpa Bremek.
Tanzania: Gereau
2513 (BR)
EU542960 s.n.
s.n.
/
/
Manettia Mutis ex L.
M. alba (Aubl.) Wernh. French Guiana:
EU542971 AF002768(1)
Andersson et al.
1917 (GB)
M. luteorubra (Vell.) Unknown: Bremer /
/
Benth.
2716 (UPS); cult. at
Stockholm
University
M. lygistum (L.) Sw.
Colombia:
EU542972 AF002769(1)
Andersson et al.
2128 (GB)
Manostachya Bremek.
M. ternifolia E.
Zambia: Dessein
Sampaio Martins
et al. 265 (BR)
EU542973 EU543042
/
ETS
/
5S-NTS
/
AM939480 AM932963 /
/
/
/
AM939486 AM932966 /
/
/
AM932968 /
(continued on next page)
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Appendix Table 1 (continued)
Taxon
Voucher
information
ETS
5S-NTS
petD
EU542974 AF002770(1)
EU543128
EU567464 AM939488 /
EU542975 EU543044
/
EU557732 AM939489 AM932969 /
Mitrasacmopsis Jovet
M. quadrivalvis Jovet
Zambia: Dessein
et al. 1273 (BR)
EU542976 EU543045
EU543129
EU557733 AM939490 AM932970 /
Nesohedyotis (Hook. f.) Bremek.
N. arborea (Roxb.)
Cult.: Chase 2915
Bremek.
(K)
/
/
/
EU542977 EU543046
EU543130
EU557734 AM939492 AM932971 /
EU542978 EU543047
EU543131
EU557735 AM939493 AM932972 /
EU542979 EU567459
EU543132
EU557736 AM939494 AM932973 /
EU542980 EU543048
EU543133
EU557737 AM939496 AM932974 /
EU542981 EU543049
EU543134
EU557738 AM939497 AM932975 /
EU542982 EU543050
EU543135
EU557739 AM939502 AM932979 /
/
/
/
/
AM939500 AM932977 /
/
/
/
/
AM939501 AM932978 /
/
EU543061
EU543147
EU557751 AM939503 AM932980 /
EU542983 EU543051
EU543136
EU557740 AM939504 AM932981 /
EU542984 /
EU543137
EU557741 AM939505 AM932982 /
EU542985 EU543052
EU543138
EU557742 AM939506 AM932983 /
EU542986 EU543053
EU543139
EU557743 AM939507 /
/
/
/
/
AM939498 /
/
EU542987 EU543054
EU543140
EU557744 AM939508 /
/
EU542988 EU543055
EU543141
EU557745 AM939510 AM932985 /
/
/
/
/
AM939509 AM932984 /
/
/
/
/
AM939511 /
/
/
/
/
/
AM939495 /
/
Oldenlandia L.
O. affinis (Roem. &
Zambia: Dessein
Schult.) DC.
et al. 627 (BR)
O. angolensis K. Schum. Zambia: Dessein
et al. 932 (BR)
O. biflora (L.) Lam.
Unknown: cult. at
BR
O. capensis L. f. var.
Zambia: Dessein
capensis
et al. 843 (BR)
O. capensis L. f. var.
Tanzania: Kayombe
pleiosepala Bremek. et al. s.n. (BR)
O. corymbosa L.
Zambia: Dessein
et al. 487 (BR)
Australia:
Andersson 2260
(GB)
Gabon: Andersson
& Nilsson 2263
(GB)
O. densa in ed. (O.
Zambia: Dessein
et al. 346 (BR)
robinsonii Verdc.,
nom. illeg.)
O. echinulosa K.
Zambia: Dessein
Schum.
et al. 928 (BR)
O. echinulosa K.
Tanzania: Kayombo
Schum. var.
& Kahemela 1993
pellucida (Hiern)
(BR)
Verdc.
O. fastigiata Bremek.
Zambia: Dessein
et al. 1019 (BR)
O. galioides (F. Muell.) Australia: Harwood
F. Muell.
1511 (BR)
O. cf. galioides (F.
Australia:
Muell.) F. Muell.
Harwood 1519
(BR)
O. geophila Bremek.
Zambia: Dessein
et al. 935 (BR)
O. goreensis (DC.)
Zambia: Dessein
Summerh.
et al. 1286 (BR)
Zambia: Dessein
et al. 455 (BR)
Zambia: Dessein
et al. 1335 (BR)
Tanzania: Richards
& Arasululu 25910
(BR)
rps16
ITS
trnL-trnF
Mitracarpus Zucc. ex Schult. & Schult. f.
M. frigidus (Willd. ex
French Guiana:
Roem. & Schult.) K. Andersson et al.
Schum.
1995 (GB)
M. microspermus K.
Guiana: JansenSchum.
Jacobs et al. 4785
(GB)
atpB-rbcL
AF003607(1)
/
/
/
/
/
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Appendix Table 1 (continued)
Taxon
Voucher
information
atpB-rbcL
O. herbacea (L.) Roxb.
var. goetzei (DC.)
Summerh.
Zambia: Dessein
et al. 442 (BR)
Zambia: Dessein
et al. 1218 (BR)
Zambia: Dessein
et al. 463 (BR)
Zambia: Dessein
et al. 1041 (BR)
Zambia: Dessein
et al. 1356 (BR)
Zambia: Dessein
et al. 1256 (BR)
Mexico:
Frödeström &
Hultén 681 (S)
Australia:
Harwood 1520
(BR)
O. herbacea (L.) Roxb.
var. herbacea
O. lancifolia
(Schumach.) DC.
O. lancifolia
(Schumach.) DC.
O. microtheca (Cham.
& Schltdl.) DC.
O. mitrasacmoides (F.
Muell.) F. Muell.
subsp. nigricans
Halford
O. mitrasacmoides (F.
Muell.) F. Muell.
subsp.
trachymenoides (F.
Muell.) Halford
O. monanthos
(Hochst. ex A.
Rich.) Hiern
O. nematocaulis
Bremek.
O. nervosa Hiern
rps16
Zambia: Dessein
et al. 924 (BR)
Gabon: Andersson
& Nilsson 2326
(GB)
O. rosulata K. Schum.
Zambia: Dessein
et al. 1197 (BR)
O. salzmannii (DC.)
Brazil: Harley
Benth. & Hook. f. ex 15514 (UPS)
B. D. Jacks.
O. sp. C Fl. Zamb.
Zambia: Dessein
et al. 716 (BR)
O. taborensis Bremek. Tanzania: Bidgood
et al. 4015 (BR)
O. tenelliflora (Blume) Unknown: cult. at
Kuntze
BR
O. tenuis K. Schum.
Guyana: JansenJacobs et al. 41
(UPS)
O. uniflora L.
USA: Godfrey
57268 (GB)
O. wauensis Schweinf. Ethiopia: Friis et al.
ex Hiern (syn.
2560 (UPS)
Thecorchus wauensis
(Schweinf. ex
Hiern) Bremek.)
O. wiedemannii K.
Kenya: Luke & Luke
Schum.
8362 (UPS)
Pentanisia (Paraknoxia)
Bremek. (outgroup)
Zambia: Dessein
P. parviflora (Stapf ex
et al. 678 (BR)
Verdc.) Verdc. ex
Bremek.
ETS
5S-NTS
petD
EU542989 EU543056
EU543142
EU557746 AM939550 AM932986 /
/
/
/
EU542990 EU543057
EU543143
EU557747 AM939552 AM932988 /
/
/
/
AM939553 AM932989 /
EU542991 EU543058
EU543144
/
AM939512 AM932990 /
/
/
/
AM939499 AM932976 /
EU542992 EU543059
EU543145
EU557749 AM939513 AM932991 /
/
/
/
EU543146
EU557750 AM939515 AM932992 /
/
/
AM939516 /
/
/
AM939517 AM932994 /
/
/
/
/
Australia: Harwood EU542993 /
1516 (BR)
Ethiopia: Friis
et al. 276 (BR)
ITS
trnL-trnF
/
/
EU542994 EU543060
AM939551 AM932987 /
AM939514 AM932993 /
/
/
AF333382(2) /
XX999999 AM939518 AM932995 /
/
EU543043
EU567465 AM939519 /
EU567467
/
EU542995 AY764294(3) EU543148
EU557752 AM939520 AM932996 /
/
/
/
EU542996 /
EU543149
EU557753 AM939522 /
EU542997 EU543062
EU543106
EU557710 AM939451 AM932949 /
/
AM939549 AM932997 /
/
EU542998 AY764293(3) /
EU557754 AM939523 /
/
EU542999 AY764295(3) EU543150
EU557755 AM939524 /
/
EU543017 EU543076
EU543168
EU557774 AM939548 AM933018 /
EU543000 EU543063
EU543151
EU557756 AM939525 AM933001 /
EU543001 EU543064
EU543152
EU557757 /
/
/
(continued on next page)
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Appendix Table 1 (continued)
Taxon
Pentanopsis Rendle
P. fragrans Rendle
P. gracilicaulis
(Verdc.) Thulin &
B. Bremer
Pentodon Hochst.
P. pentandrus (K.
Schum. & Thonn.)
Vatke
Phylohydrax Puff
P. carnosa (Hochst.)
Puff
P. madagascariensis
(Willd. ex Roem. &
Schult.) Puff
Voucher
information
atpB-rbcL
rps16
trnL-trnF
petD
ITS
Kenya: Verdcourt
2454 (S)
/
/
/
/
AM267020 /
Ethiopia: Gilbert
et al. 7458 (UPS)
Somalia: Thulin
et al. 10512 (UPS)
/
EU543065
EU543153
EU557758 AM939526 AM933002 /
EU567458 EU567460
EU567468
/
Zambia: Dessein
et al. 598 (BR)
EU543002 EU543066
EU543154
EU557759 AM939528 AM933003 /
Zanzibar:
Sundström 2 (GB)
/
/
/
EU543003 EU543067
XX999999
South Africa:
Bremer 3783 (UPS)
Madagascar: De
EU543004 AY764292(3) EU543155
Block et al. 640 (BR)
Psyllocarpus Mart. & Zucc.
P. laricoides Mart. &
Brazil: Andersson
Zucc.
et al. 35750 (UPS)
Richardia L.
R. brasiliensis Gomes
R. scabra L.
R. stellaris L.
Spermacoce L.
S. capitata Ruiz & Pav.
/
Madagascar: De
Block et al. 904
(BR)
Colombia:
Andersson et al.
2073 (GB)
Australia: Egeröd
85343 (GB)
French Guiana:
Andersson 1908
(GB)
S. confusa Rendle ex
Colombia:
Andersson et al.
Gillis
2074 (GB)
S. erosa Harwood
Australia: Harwood
1148 (BR)
S. filifolia (Schumach. Zambia: Dessein
& Thonn.) J.-P.
et al. 881 (BR)
Lebrun & Stork
S. filituba (K. Schum.) Kenya: Luke 9022
Verdc.
(UPS)
S. flagelliformis Poir.
Madagascar: De
Block et al. 794 (BR)
S. hispida L.
Sri Lanka:
Wanntorp et al.
2667 (S)
S. ocymifolia Willd. ex French Guiana:
Roem. & Schult.
Andersson et al.
(Hemidiodia
2040 (GB)
ocymifolia (Willd. ex
Roem. & Schult.) K.
Schum.)
/
ETS
/
5S-NTS
/
/
AM939527 AM933004 /
EU557760 AM939529 /
/
EU557761 AM939530 /
/
/
/
/
/
AM939531 AM933005 /
/
/
/
/
AM939533 AM933007 /
EU543005 AF003614(1)
EU543156
EU557762 AM939532 AM933006 /
EU543006 EU543068
EU543157
EU557763 AM939534 /
/
EU543007 EU543069
EU543158
EU557764 AM939536 /
/
/
/
/
EU543008 EU543070
EU543159
EU557765 AM939537 AM933009 /
/
/
/
EU543009 EU543071
EU543160
EU557766 AM939539 AM933011 /
EU543010 EU543072
EU543161
EU557767 /
EU543011 EU543073
EU543162
EU557768 AM939540 AM933017 /
EU542952 /
EU543108
EU557712 AM939463 /
/
AF003619(1)
/
/
/
AM939538 AM933010 /
/
/
/
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Appendix Table 1 (continued)
Taxon
Voucher
information
Ecuador: Bremer
3340 (UPS)
S. prostrata Aubl.
Colombia:
Andersson et al.
2078 (GB)
S. remota (Lam.)
French Guiana:
Bacigalupo & Cabral Andersson et al.
2016 (GB)
S. ruelliae DC.
Gabon: Andersson
& Nilsson 2296
(GB)
S. verticillata L.
Madagascar: De
Block et al. 632
(BR)
cult. BR; no
voucher
Stenaria (Raf.) Terrell
S. nigricans (Lam.)
USA: Yatskievych
Terrell
96-92 (MO)
Synaptantha Hook. f.
S. tillaeacea (F. Muell.)
Hook. f.
atpB-rbcL
rps16
trnL-trnF
petD
ITS
/
/
/
/
AM939462 AM932951 /
EU543012 /
EU543163
EU557769 AM939541 AM933012 /
EU543013 /
EU543164
EU557770 AM939542 AM933013 /
EU543014 EU543074
EU543165
EU557771 AM939543 AM933014 /
/
/
/
/
AM939544 AM933015 /
/
/
/
/
AM939545 AM933016 /
EU543015 AF333373(2) EU543166
Australia: Lazarides EU543016 EU543075
& Palmer 272 (K)
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