Rediscovery of Malagasy Lathraeocarpa allows determination of its taxonomic position within Rubiaceae

Abstract

Lathraeocarpa acicularis, a small woody Rubiaceae endemic to Madagascar, was rediscovered after more than 50 years. A phylogenetic reconstruction based on four plastid markers (atpB-rbcL, rps16, trnL-trnF, petD) proves that its previous position within the monogeneric tribe Lathraeocarpeae can no longer be supported. Our data clearly show that Lathraeocarpa acicularis has its closest relatives among taxa of the Hedyotis-Oldenlandia group of the herbaceous tribe Spermacoceae sensu lato. The taxon falls within the Pentanopsis clade and is sister to a group comprising the Madagascan genus Gomphocalyx and the Afro-Madagascan genus Phylohydrax. A detailed survey of the morphology and anatomy of the genus based on our recently collected material of Lathraeocarpa acicularis is presented, providing additional arguments for the new taxonomic position.

Full text

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Groeninckx & al. • Taxonomic position of LathraeocarpaTAXON 58 (1) • February 2009: 209–226
INTRODUCTION
Lathraeocarpa Bremek., described by Bremekamp
in 1957, is a genus of Rubiaceae endemic to the south
and south-western part of Madagascar. It comprises two
(sub)shrub species, L. acicularis Bremek. and the type
species L. decaryi Bremek. The generic name refers to the
stipular sheath surrounding the ovary that tears open at
fruit maturity. Within Rubiaceae, the genus is character-
ized by ternate leaves, nearly truncate stipules fused with
the petioles, heterodistyly, calyx lobes that are twice the
number of corolla lobes, a tri- or tetralocular gynoecium
with one basal ovule per locule, a drupaceous fruit, and
pluri-zonocolporate pollen grains (Bremekamp, 1957).
Until recently, Lathraeocarpa was only known from
five herbarium collections, the most recent of which
was collected in 1955. In spite of extensive fieldwork in
Madagascar during the last two decades, and although
the type localities have been revisited (e.g., most recently
by S. Razafimandimbison, Bergius Foundation, Stock-
holm, Sweden and A. Davis, Royal Botanic Gardens, Kew,
U.K.), Lathraeocarpa had never been relocated until we
discovered a population of L. acicularis close to the Parc
National de Tsimanampetsotsa in February 2007.
The presence of raphides, valvate corolla aestiva-
tion, and pluri-zonocolporate pollen grains indicates that
Lathraeocarpa belongs to subfamily Rubioideae, but its
exact position has long been subject to debate. Initially, the
herbarium material of Lathraeocarpa, on which Breme-
kamp (1957) based his description, was thought to belong
to the genus Triainolepis Hook. f. Homolle annotated all
specimens (except Humbert & Capuron 2958) as Triai-
nolepis decaryi Homolle but never published the name.
Bremekamp (1957), however, considered the material to
be too different from Triainolepis and transferred it to
the new genus Lathraeocarpa, which he placed in a tribe
of its own, i.e., Lathraeocarpeae, because of its unique
combination of character states.
Capuron (1973) added the monospecific genus Gom-
phocalyx Baker, another endemic from Madagascar, as a
second genus to Lathraeocarpeae. Piesschaert (2001) even
suggested merging Lathraeocarpa with Gomphocalyx, a
proposal refuted by Dessein & al. (2005a). Gomphocalyx
herniarioides Baker is a procumbent to decumbent annual
or short-lived perennial herb, which in the past was placed
in Spermacoceae sensu stricto (s.str. hereafter) (Schumann,
1891; Robbrecht 1988, 1993). However, recent molecular
studies excluded Gomphocalyx from Sper macoceae s.st r.
and placed it in the Pentanopsis clade (Thulin & Bremer,
2004) of the Hedyotis-Oldenlandia group (Dessein, 2003;
Dessein & al., 2005a; Groeninckx & al., in press), where
it was shown to be sister of the Afro-Madagascan genus
Phylohydrax Puff. Presently, the former tribe Sperma-
coceae s.str. and the Hedyotis-Oldenlandia group are
Rediscovery of Malagasy Lathraeocarpa allows determination of its
taxonomic position within Rubiaceae
Inge Groeninckx1*, Petra de Block2, Franck Rakotonasolo3, Erik Smets1,4 & Steven Dessein2
1 1 Laboratory of Plant Systematics, K.U. Leuven, Kasteelpark Arenberg 31, PO Box 2437, 3001 Leuven,
Belgium. *
inge.groeninckx@bio.kuleuven.be (author for correspondence)
2 2 National Botanic Garden of Belgium, Domein van Bouchout, 1860 Meise, Belgium
3 3 Parc Botanique et Zoologique de Tsimbazaza, B.P. 4096, Tsimbazaza, Antananarivo 101, Madagascar
4 4 National Herbarium of the Netherlands, Leiden University Branch, PO Box 9514, 2300 RA Leiden,
The Netherlands
Lathraeocarpa acicularis, a small woody Rubiaceae endemic to Madagascar, was rediscovered after more
than 50 years. A phylogenetic reconstruction based on four plastid markers (atpB-rbcL, rps16, trnL-trnF, petD)
proves that its previous position within the monogeneric tribe Lathraeocarpeae can no longer be supported.
Our data clearly show that Lathraeocarpa acicularis has its closest relatives among taxa of the Hedyotis-
Oldenlandia group of the herbaceous tribe Spermacoceae sensu lato. The taxon falls within the Pentanopsis
clade and is sister to a group comprising the Madagascan genus Gomphocalyx and the Afro-Madagascan genus
Phylohydrax. A detailed survey of the morphology and anatomy of the genus based on our recently collected
material of Lathraeocarpa acicularis is presented, providing additional arguments for the new taxonomic
position.
KEYWORDS: comparative morphology and anatomy, Lathraeocarpa, Madagascar, molecular phylogeny,
plastid DNA, Rubiaceae

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TAXON 58 (1) • February 2009: 209–226Groeninckx & al. • Taxonomic position of Lathraeocarpa
united forming Spermacoceae sensu lato (s.l. hereafter)
(Robbrecht & Manen, 2006; Groeninckx & al., in press).
If Capuron’s decision (1973) to place Gomphocalyx and
Lathraeocarpa together was correct, Lathraeocarpa too
would be related to Phylohydrax, and should therefore also
be placed in Spermacoceae s.l.
Several morphological characters distinguish Lath-
raeocarpa from Gomphocalyx, some of which might even
point to a n affinit y with Triainolepis. First of all, the (sub)
shrubby habit of Lathraeocarpa is much closer to the
shrubby habit of Triainolepis than to the herbaceous habit
of Gomphocalyx. Furthermore, the pyrene of L. decaryi is
surrounded by eight strands of thin-walled cells, a condi-
tion very similar to that observed in some Triainolepis
species (Bremekamp, 1957; Piesschaert, 2001). Likewise,
Lathraeocarpa and Trianolepis have a plurilocular ovary
and fleshy fruits whereas Gomphocalyx has a bilocular
ovary and dry fruits, which has prompted some authors
(Kårehed & Bremer, 2007) to believe that Lathraeocarpa
should be regarded as a member of the emended tribe
Knoxieae rather than a member of Spermacoceae s.l.
To date, no molecular data have been available for
Lathraeocarpa, so its taxonomic position has remained
controversial (Robbrecht & Manen, 2006). This paper
presents the results of a multidisciplinary study based
on morphological and molecular data illuminating the
relationships of this enigmatic genus.
MATERIALS AND METHODS
Molecular study. —
A few preliminary analyses
of rps16 intron sequences of representatives of the ma-
jor groups within subfamily Rubioideae have shown that
Lathraeocarpa acicularis belongs to Spermacoceae s.l.
We thus used the matrix of Groeninckx & al. (in press)
based on the chloroplast markers atpB-rbcL, rps16 and
trnL-trnF as a basis for an initial analysis presented in
this paper. The chloroplast dataset was enlarged by the
addition of three atpB-rbcL sequences (Gomphocalyx her-
niarioides, Lathraeocarpa acicularis and Pentanopsis
gracilicaulis (Verdc.) Thulin & Bremer), three rps16 se-
quences (Lathraeocarpa acicularis, Oldenlandia biflora
(L.) Lam., Pentanopsis gracilicaulis), and five trnL-F
sequences (Gomphocalyx herniarioides, Lathraeocarpa
acicularis, Oldenlandia rosulata K. Schum., Pentanopsis
gracilicaulis, Phylohydrax carnosa (Hochst.) Puff). Our
dataset included a total of 132 species representing 35 of
the 60 genera within Spermacoceae. To improve reso-
lution, a second, more focused, analysis was performed
with petD included as an extra chloroplast marker using
a smaller set of 25 taxa, including Lathraeocarpa and 21
closely related taxa as well as the pentamerous Dentella
dioeca Airy Shaw, D. repens (L.) J.R. Forst. & G. Forst.
and Pentodon pentandrus (K. Schum. & Thonn.) Vatke
as outgroup taxa. The Appendix lists all taxa included in
this study with author names, voucher information and
GenBank accession numbers.
Methods for DNA extraction, PCR amplification, se-
quencing, sequence assembly, and alignment are as de-
scribed in Groeninckx & al. (in press). The petD region
was amplified with the forward primer PIpetB1365F and
the reverse primer PIpetD738R, as described by Löhne
& Borsch (2005).
Equally weighted maximum parsimony (MP) anal-
yses were performed using Nona 2.0. (Goloboff, 1993)
launched through WinClada 1.00.08 (Nixon, 2002). Par-
simonious informative gaps were coded manually ac-
cording to the conservative ‘simple indel coding’ method
described by Simmons & Ochoterena (2000). The four
plastid regions were first analyzed separately and given
congruence between the individual topologies then com-
bined using a total evidence approach. Heuristic searches
for the shortest trees were performed using the parsimony
ratchet (Nixon, 1999). Ratchet runs of 200 iterations each,
holding 1 tree per iteration and randomly weighting 10%
of the potentially informative characters, were carried out
until the most parsimonious trees (MPTs) were repeat-
edly found. A strict consensus tree was calculated using
the trees obtained in the parsimony ratchet analyses. In
order to evaluate the relative support of the clades, jack-
knife (JS) and bootstrap (BS) analyses were executed us-
ing 1,000 replicates with 100 initial trees holding 1 tree
per random addition, performing TBR (Tree-Bisection
and Reconnection) to hold 1,000 trees and calculating a
consensus on each repetition. Frequency values ( > 65%)
were plotted onto the consensus of the MPTs.
Bayesian inference (BI) analyses were only carried
out for the two combined datasets. A substitution model
was selected for each DNA region with Modeltest 3.7
(Posada & Crandall, 1998) under the Akaike Informa-
tion Criterion (AIC) (Table 1). Indels were not included in
the BI analyses. In the combined analysis a mixed-model
approach was used (Ronquist & Huelsenbeck, 2003). The
combined dataset was partitioned and the same models of
evolution were used on the partitions as selected for the
single analyses. The BI analyses were conducted with Mr-
Bayes 3b4 (Huelsenbeck & Ronquist, 2001) as described
by Groeninckx & al. (in press). We selected a GTR + I + G,
a GTR + G, and a GTR + I substitution model as being
analogous respectively to the TVM + I + G, TVM+G, and
TVM + I models of substitution selected by Modeltest but
not implemented in MrBayes. The TVM model is similar
to the GTR model except for having equal probabilities of
change of transitions. Four Markov chains (one cold, three
heated) starting with a random tree were run simultane-
ously for three million (Analysis 1) and for one million
(Analysis 2) generations, sampling trees at every 1,000th

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Groeninckx & al. • Taxonomic position of LathraeocarpaTAXON 58 (1) • February 2009: 209–226
generation. Of the first sampled trees, 25% were regarded
as ‘burn-in’ and discarded. PAUP* version 4b10 (Swof-
ford, 2002) was used to calculate a 50% majority rule
consensus tree and to report the posterior probabilities for
each clade. Only posterior probabilities (PP) above 0.95
have been considered (Suzuki & al., 2002).
Morphological study. —
Herbarium and alcohol
material of the recently collected specimen of Lathraeo-
carpa acicularis (Madagascar, Toliara, Ankarampona,
zone périphérique du Parc National Tsimanampetsotsa,
Fokontany Ambola [Efoetse], Commune Beheloka, Dis-
trict Toliara II, 24°07′35.8″ S, 43°40′20.4″ E, 04/02/2007,
De Block & al. 2316 [BR, G, K, MO, P, TAN]) was studied
along with all known herbarium collections of L. decaryi
(Madagascar, 18/06/1924, Decary 2803 [holotype P];
Madagascar, Ambovombe, Mahatomotsy, 09/12/1931, De-
cary 9508 [BR, P]; Madagascar, Ambovombe, 02/02/1931,
Decary 8457 [P]; Madagascar, North-East of Cap Sainte
Marie in the direction of Lavanono, 08/03/1955, Humbert
& Capuron 29308 [P] and L. acicularis (Madagascar,
Toliara, dunes of Befanamy, 15/02/1921, Poisson 144 [ho-
lotype P])
Material of L. acicularis preserved in 70% ethanol
was dissected under a Wild M3 stereomicroscope (Wild
Heerbrugg Ltd, Heerbrugg, Switzerland). The dissected
material (flowers, young fruits, leaves, stipules) was
washed repeatedly in 70% ethanol and dehydrated in a
1 : 1 mixture of ethanol and dimethoxymethan (DMM or
formaldehyde-dimethylacetal) for 20 minutes and then in
pure DMM for another 20 minutes. After critical-point
drying (CPD 030, BAL-TEC AG, Balzers, Liechtenstein),
the dried material was mounted on aluminum stubs using
Leit-C and coated with gold (SPI Module Sputter Coater,
Spi Supplies, West Chester, Pennsylvania, U.S.A.) prior
to observation with a JEOL JSM-6360 scanning electron
microscope (SEM; Jeol Ltd, Tokyo, Japan).
Anatomical sections of leaves and young fruits pre-
served in 70% ethanol were made from tissues dehydrated
through a graded ethanol series. Young fruits were embed-
ded in KULZER’s Tech novit 7100 (based on hyd roxyethyl-
methacrylate, HEMA) as detailed by Igersheim (1993).
Leaves were gradually infiltrated with LR white resin (hard
grade) (London Resin) using 1 : 3, 1 : 1, 3 : 1, and 1 : 0 solu-
tions of resin and 100% ethanol for at least 5 hours each.
The leaf s amples were pla ced in closed capsule s filled wit h
fresh resin, and polymerized at 60°C for 48 hours.
Fruit and leaf sections of 2–3 μm in thickness were
made with a rotary microtome (Microm HM 360, Wall-
dorf, Germany) and stained with 0.1% toluidin blue. Pho-
tographs were taken under a Leitz Dialux 20 microscope
(Wetzlar, Germany) equipped with an Olympus DP50
camera (Hamburg, Germany).
Pollen grains from herbarium material were acetoly-
sed according to Reitsma’s (1969) ‘wetting agent’ method.
External features were observed using SEM on grains
that had been suspended in 70% ethanol and left to dry.
Glycerin jelly slides were observed under a light micro-
scope. Untreated pollen grains from anthers preserved in
70% ethanol were also studied following the method of
Halbritter (1998), but using DMM instead of DMP (2,2-di-
methoxypropane). Polar axis length (P) and equatorial
diameter (E) were measured on 20 grains from thrum
(brevistylous) and 20 from pin (longistylous) flowers us-
ing the software programme Carnoy (Schols & al., 2002).
Pollen terminology follows Punt & al. (2007).
Table 1. Substitution models selected with Modeltest 3.7
(Posada & Crandall, 1998) for each DNA region used in
Analysis 1 and for each DNA region used in Analysis 2.
Analysis 1aAnalysis 2b
atpB-rbcL TVM + I + G TVM + G
rps16 GTR + I + G TVM + I
trnL-F TVM + G TVM + G
petD / GTR + G
aatpB-rbcL, rps16, trnL-F
batpB-rbcL, rps16, trnL-F, petD
The models can ignore rate variation or include invariable
sites ( + I), rate variation among sites ( + G), or both ( + I + G).
GTR = General time reversible, TVM = Transversion model.
Table 2. Characteristics of each data matrix used in the phylogenetic analysis in Spermacoceae s.l. (Analysis 1) and the
corresponding tree statistics.
No. of
taxa No. of
characters No. of PI
characters No. of PI
indels No. of
MPT MPT
length CI RI
atpB-rbcL 106 1,292 187 38 4,502 415 0.57 0.85
rps16 111 664 176 22 147 474 0.56 0.83
trnL-trnF 117 914 172 33 678 396 0.61 0.88
Combined 132 2,870 525 93 47,571 1,352 0.54 0.83
CI = Consistency Index (Kluge & Farris, 1969), MPT = Most Parsimonious Tree(s), PI = Potentially-Informative, RI = Retention
Index (Farris, 1989).

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TAXON 58 (1) • February 2009: 209–226Groeninckx & al. • Taxonomic position of Lathraeocarpa
96/96
94/95
84/84
93/95
74/74
98/96
99/98
100/100
87/86
94/94
100/100
91/86
Kadua spp. (16)
Oldenlandia biflora
92/85
99/99
99/99
95/95
100/99 Dentella dioeca
Dentella repens
Pentodon pentandrus
100/100
100/100
100/100
95/95
88/77
Batopedina pulvinellata (outgroup)
Carphalea madagascariensis (outgroup)
Paraknoxia parviflora (outgroup)
100/100
72/69
Pentanopsis clade
Kohautia amatymbica
Kohautia caespitosa
Kohautia coccinea
Kohautia cynanchica
Kohautia subverticillata
Kohautia senegalensis
Manostachya ternifolia
Oldenlandia rosulata
Dibrachionostylus kaessneri
Hedythyrsus spermacocinus
Mitrasacmopsis quadrivalvis
Oldenlandia echinulosa
Oldenlandia echinulosa var. pellicida
Oldenlandia fastigiata
Oldenlandia geophila
Oldenlandia nervosa
99/97
93/87
97/96
Kohautia obtusiloba
Kohautia virgata
Oldenlandia capensis var. capensis
Oldenlandia capensis var. pleiosepala
Oldenlandia nematocaulis
Oldenlandia robinsonii
Oldenlandia taborensis
Oldenlandia wiedemannii
Thecorchus wauensis
Kohautia microcala
Oldenlandia corymbosa
Houstonia caerulea
Houstonia longifolia
Oldenlandia microtheca
Stenaria nigricans
Arcytophyllum spp. (10)
93/92
86/83
Oldenlandia mitrasacmoides
Hedyotis tenelliflora
Oldenlandia galioides
Oldenlandia lancifolia
Synaptantha tillaeacea
88/83
Agathisanthemum bojeri
Agathisanthemum globosum
Lelya osteocarpa
Oldenlandia angolensis
Oldenlandia goreensis
Oldenlandia uniflora
86/87
100/99
98/97
99/99
100/99
Hedyotis spp. (10)
100/100
Amphiasma benguellense
Amphiasma luzuloides
98/99
Oldenlandia affinis
Pentanopsis fragrans
Pentanopsis gracicaulis
100/100 95/96
Conostomium natalense
Oldenlandia herbacea var. goetzei
Oldenlandia herbacea var. herbacea
100/100
Conostomium quadrangulare
Conostomium zoutpansbergense
99/99
99/99
93/95
98/98 Gomphocalyx herniarioides
Phylohydrax carnosa
Phylohydrax madagascariensis
Lathraeocarpa acicularis
100/100
1
Arcytophyllum spp. (10)
Kadua spp. (16)
Hedyotis spp. (10)
Batopedina pulvinellata (outgroup)
Carphalea madagascariensis (outgroup)
Paraknoxia parviflora (outgroup)
Dentella dioeca
Dentella repens
Pentodon pentandrus
Kohautia amatymbica
Kohautia caespitosa
Kohautia coccinea
Kohautia cynanchica
Kohautia subverticillata
Kohautia senegalensis
Manostachya ternifolia
Oldenlandia rosulata 0.93
Gomphocalyx herniarioides
Phylohydrax carnosa
Phylohydrax madagascariensis
Lathraeocarpa acicularis 1
1
11
1
1
1
1
11
1
1
1
11
0.95
1
1
1
1
1
1
1
Agathisanthemum bojeri
Agathisanthemum globosum
Lelya osteocarpa
Oldenlandia angolensis
Oldenlandia goreensis
Oldenlandia uniflora
Dibrachionostylus kaessneri
Oldenlandia echinulosa
Oldenlandia echinulosa var. pellicida
Oldenlandia geophila
Oldenlandia nervosa
1
1
1
1
1
Hedyotis tenelliflora
Oldenlandia galioides
Oldenlandia lancifolia
Synaptantha tillaeacea 1
1
Oldenlandia biflora
Oldenlandia mitrasacmoides
1
1
1
Houstonia caerulea
Houstonia longifolia
Oldenlandia microtheca
Stenaria nigricans
1
1
1
Kohautia microcala
Kohautia obtusiloba
Kohautia virgata 1
Oldenlandia capensis var. capensis
Oldenlandia capensis var. pleiosepala
Oldenlandia nematocaulis
Oldenlandia robinsonii
Oldenlandia taborensis
Oldenlandia wiedemannii
Thecorchus wauensis
Oldenlandia corymbosa
1
1
0.92
1
Amphiasma benguellense
Amphiasma luzuloides
Oldenlandia affinis
Pentanopsis fragrans
Pentanopsis gracicaulis
1
1
1
1
Conostomium natalense
Conostomium quadrangulare
Conostomium zoutpansbergense
Oldenlandia herbacea var. goetzei
Oldenlandia herbacea var. herbacea
Hedythyrsus spermacocinus
Mitrasacmopsis quadrivalvis
Oldenlandia fastigiata

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RESULTS
Molecular evidence. —
For the initial study involv-
ing samples of 132 taxa, sequence data from the aligned
atpB-rbcL, rps16 and trnL-trnF regions were analyzed
separately (results not presented) and in combination us-
ing a total evidence approach (Table 2). Only the results
from the MP and the BI analysis of the combined matrix
are presented (Fig. 1A, B), because they resulted in more
resolved trees. The MP tree and the BI tree are congruent
with those published by Groeninckx & al. (in press). In
this analysis, Lathraeocarpa falls within the Pentanopsis
clade and comes out as sister to a clade comprising Gom-
phocalyx and Phylohydrax (JS = 100, BS = 100, PP = 100).
This well-supported clade comprising Lathraeocarpa,
Gomphocalyx and Phylohydrax (the LGP clade) shares a
common ancestor with Manostachya ternifolia E. Sampaio
Martins and Oldenlandia rosulata (JS = 100, BS = 100,
PP = 0.93). In the BI analysis, the latter two species are
sister to the LGP clade, whereas in the MP analysis their
relationship to the LGP clade is unresolved.
Results of the second more focused analysis of 25 taxa
based on the three markers indicated above plus petD are
presented in Fig. 2A, B and in Tables 3 and 4. The petD
region has never been used previously in phylogenetic
studies of Rubiaceae. The chloroplast region includes the
petB-petD intergenic spacer, the petD 5′ exon and the petD
5′ intron (Löhne & Borsch, 2005). Our study shows that the
amount of sequence variability in this chloroplast marker
is relatively high, suggesting that it may be useful for fur-
ther studies in the family. After exclusion of regions that
could not be aligned with confidence, the total length of
the aligned sequences was 1,190 characters with 106 vari-
able characters, of which 80 were potentially parsimony
informative. Gap coding generated an extra 26 parsimony
informative characters, which means that about 24.5% of
the total potential phylogenetic information within petD is
present as indel characters. This particularly high number
of (phylogenetically informative) indels explains the con-
siderable length variability in the petD marker.
For the second analysis, the pentamerous genera Den-
tella and Pentodon were chosen as outgroup taxa. As in
the larger analysis, Lathraeocarpa comes out as sister to
Gomphocalyx + Phylohydrax (JS = 100, BS = 100, PP = 1),
comprising the LGP clade, which also shares a common
ancestor with Manostachya ternifolia and Oldenlandia
rosulata (JS = 99, BS = 99, PP = 1). In the MP analysis,
Oldenlandia rosulata is placed as sister to the LGP clade,
but without significant jackknife and bootstrap support.
In the BI analysis, a clade comprising Manostachya terni-
folia and Oldenlandia rosulata is sister to the LGP clade,
but the relationship between Manostachya ternifolia and
Oldenlandia rosulata has low Bayesian posterior prob-
ability (PP < 0.90).
87/87
82/83
91/88
94/95
84/84
93/92
74/71
94/94
100/99
85/87
99/99
100/100
87/87 Nesohedyotis arborea
Oldenlandia salzmannii
Oldenlandia tenuis
Arcytophyllum serpyllaceum
Bouvardia glaberrima
Bouvardia ternifolia
Manettia alba
Manettia lygistum
Crusea calocephala
Crusea megalocarpa
Spermacoce flagelliformis
Diodia aulacosperma
Diodella sarmentosa
Hemidiodia ocymifolia
Mitracarpus frigidus
Mitracarpus microspermus
Spermacoce capitata
Spermacoce confusa
Spermacoce erosa
Spermacoce filituba
Spermacoce prostrata
Spermacoce remota
Spermacoce ruelliae
Richardia scabra
Richardia stellaris
Diodia spicata
Emmeorhiza umbellata
Galianthe brasiliensis
Galianthe eupatoriides
Ernodea littoralis
Spermacoce hispida Spermacoce flagelliformis
Hemidiodia ocymifolia
Spermacoce remota
Oldenlandia salzmannii
Oldenlandia tenuis
Arcytophyllum serpyllaceum
Bouvardia glaberrima
Bouvardia ternifolia
Manettia alba
Manettia lygistum
Nesohedyotis arborea
Emmeorhiza umbellata
Diodia spicata
Galianthe brasiliensis
Galianthe eupatoriides
Spermacoce confusa
Crusea calocephala
Crusea megalocarpa
Richardia scabra
Richardia stellaris
Spermacoce capitata
Spermacoce erosa
Spermacoce prostrata
Diodia aulacosperma
Diodella sarmentosa
Mitracarpus frigidus
Mitracarpus microspermus
Spermacoce filituba
Spermacoce ruelliae
Spermacoce hispida
Ernodea littoralis
1
1
1
1
0.99
1
1
1
1
1
11
1
1
0.98
AB
Fig. 1. Results of phylogenetic Analysis 1. A, strict consensus tree of the MPTs from the combined plastid analysis using atpB-rbcL, rps16 and trnL-trnF sequences for
132 taxa (L = 1,952, CI = 0.54, RI = 0.83). Jackknife (left) and bootstrap (right) values (> 65) are indicated above branches; B, bayesian tree based on combined atpB-rbcL,
rps16 and trnL-trnF data. Posterior probabilities > 0.9 are indicated above branches.

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TAXON 58 (1) • February 2009: 209–226Groeninckx & al. • Taxonomic position of Lathraeocarpa
A
1
1
Kohautia amatymbica
Kohautia caespitosa
Kohautia coccinea
Kohautia cynanchica
Kohautia subverticillata
1
1
Kohautia senegalensis
Dentella dioeca
Dentella repens
Pentodon pentandrus
1
1
1
1
Phylohydrax madagascariensis
Phylohydrax carnosa
Gomphocalyx herniarioides
Lathraeocarpa acicularis
Oldenlandia rosulata
1
1
Amphiasma benguellense
Amphiasma luzuloides
Oldenlandia affinis
Pentanopsis fragrans
1
1
Conostomium natalense
Conostomium quadrangulare
Conostomium zoutpansbergense
Oldenlandia herbacea var. goetzei
Oldenlandia herbacea var. herbacea
Manostachya ternifolia
Pentanopsis gracicaulis
B
Kohautia amatymbica
Kohautia caespitosa
Kohautia coccinea
Kohautia cynanchica
Kohautia subverticillata
Kohautia senegalensis
Dentella dioeca
Dentella repens
Pentodon pentandrus
Manostachya ternifolia
Amphiasma benguellense
Amphiasma luzuloides
Oldenlandia affinis
Pentanopsis fragrans
Oldenlandia rosulata
Conostomium natalense
Conostomium quadrangulare
Conostomium zoutpansbergense
Oldenlandia herbacea var. goetzei
Oldenlandia herbacea var. herbacea
100/100
Pentanopsis gracicaulis
100/100
100/100
100/100 99/99
95/94
91/86
Phylohydrax madagascariensis
Phylohydrax carnosa
Gomphocalyx herniarioides
Lathraeocarpa acicularis
99/99 99/99
100/100
99/99
100/99
100/100
100/100
100/100
100/100
78/77 67/72
1
1
1
1
1
0.98
1
Fig. 2. Results of phylogenetic Analysis 2. A, strict consensus tree of the MPTs from the combined plastid analysis using
atpB-rbcL, rps16, trnL-trnF and petD sequences for 25 taxa (L = 540, CI = 0.69, RI = 0.84). Jackknife (left) and bootstrap
(right) values ( > 65) are indicated above branches; B, bayesian tree based on combined atpB-rbcL, rps16, trnL-trnF and
petD data. Posterior probabilities are indicated above branches.
Table 3. Characteristics of atpB-rbcL, rps16, trnL-trnF and petD sequences in the focused analysis of taxa
in the Pentanopsis clade (Analysis 2).
No. of characters
(unaligned) % PI
characters
% PI indels vs. total
no. of characters
(unaligned)
% PI indels vs.
no. of PI characters
(unaligned)
atpB-rbcL 679–734 (av. 698) 8% 2% 22.0%
rps16 466–482 (av. 476) 12% 1% 11.0%
trnL-trnF 327–384 (av. 350) 24% 6% 29.0%
petD 941–994 (av. 964) 11% 3% 24.5%
PI = Potentially Informative.
Table 4. Characteristics of each data matrix and the corresponding tree statistics.
No. of
taxa No. of
characters No. of PI
characters No. of
PI indels No. of
MPT MPT
length CI RI
atpB-rbcL 23 879 58 13 2 83 0.75 0.88
rps16 23 531 56 6 1 92 0.82 0.91
trnL-trnF 26 645 83 21 5 143 0.74 0.88
petD 24 1,216 106 26 2 168 0.77 0.89
Combined 26 3,271 303 66 12 531 0.70 0.84
CI = Consistency Index (Kluge & Farris, 1969), MPT = Most Parsimonious Tree(s), PI = Potentially-Informative, RI = Retention
Index (Farris, 1989).

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Groeninckx & al. • Taxonomic position of LathraeocarpaTAXON 58 (1) • February 2009: 209–226
Individual plastid sequence MP analyses were to-
pologically congruent, except for the sister group of the
LGP clade, which differs depending on the molecular
marker used. As in the combined MP analysis, the trnL-
trnF analysis places Oldenlandia rosulata as sister to
the LGP clade, although with rather moderate support
(JS = 77, BS = 79). The rps16 intron analysis on the other
hand, shows a clade with O. rosulata and M. ternifolia as
sister to the LGP clade, as in the combined BI analysis,
although the sister relationship of these two species lacks
jackknife and bootstrap support. In the petD analysis, both
O. rosulata and M. ternifolia are unresolved. In the atpB-
rbcL analysis, a clade in which M. ternifolia is sister to
Conostomium (Stapf) Cufod. and O. herbacea (L.) Roxb.
is placed as sister to the LGP clade, but without significant
jackknife or bootstrap support.
Morphology and anatomy. —
Since Bremekamp
(1957) originally described Lathraeocarpa, not a single
study has been devoted to the morphology and anatomy of
the ge nus, exc ept for some pol len obser vat ions of L. decaryi
by Dessein & al. (2005a). The collection of L. acicularis
now makes it possible to present a more detailed description
of the genus, expanding the work of Bremekamp (1957) and
Dessein & al. (2005a) with our new (macro- and micro-)
morphological and anatomical observations.
Growth form. – Lathraeocarpa acicularis individuals
are (sub)shrubs up to ca. 25 cm, with woody stems and
well-developed woody taproots (Figs. 3A; 4A, B). In the
protologue, Bremekamp (1957) describes L. decaryi as a
subshrub (“fruticulus”) of 1.2 m or more. However, the
herbarium labels only tell the species is a subshrub, and
give no details about size. In our opinion the plants are
Fig. 3. Line drawing of
Lathraeocarpa acicularis.
A, habit; B, ovary and ca-
lyx surrounded by three
leaves; C, brevistylous
flower; D, longistylous
flower; E, young fruit.

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TAXON 58 (1) • February 2009: 209–226Groeninckx & al. • Taxonomic position of Lathraeocarpa
much smaller than 1.2 m and probably not much larger
than those of L. acicularis. Both species have woody
stems with grey or brown bark, which are much branched
(Fig. 4A, B). In L. acicularis, the branches are subterete,
greyish-brown, erect-ascending or horizontal and bear
numerous short branchlets (Fig. 4A, B, D) that are ini-
tially densely pubescent, up to 5 cm long but often much
shorter with short internodes, and terminated by a f lower
(Fig. 4C–E).
Leaves and stipules. – The somewhat succulent leaves
on the branchlets of Lathraeocarpa are ternate, sessile and
connate with the stipule base, forming a sheath around the
stem. The leaf-blades are narrowly elliptic, or sometimes
slightly narrowly ovate, 3–14 mm long and 0.3–1.6 mm
wide. Leaves are pubescent on both surfaces or almost
glabrous below, and have revolute margins (Fig. 5A, B).
In both species, there seems to be a great deal of variation
in the density and the length of the trichomes (e.g., very
short hairs in Humbert & Capuron 29308). When fresh,
the lamina are green above and reddish-brown below (Fig.
4C–E). Venation is indistinct, with the veins sunk in the
mesophyll (Fig. 5A, B, D) and only the mid-vein somewhat
prominent in L. decaryi. The leaf apices are mucronate
and the bases are slightly narrowed. In L. acicularis, a
row of colleters is associated with the inner side of the
leaf bases (Fig. 6A). The stipular sheaths are cup-shaped,
0.4–1.4 mm long, pubescent outside, and appear to be
truncate to the naked eye, while under high magnification
a single short stipular tooth is visible (Fig. 5C).
Leaf anatomy. – Leaf sections of Lathraeocarpa acicu-
laris show that both the upper and lower epidermis consist of
a single layer of relative large cells covered by a cuticle (Fig.
5D, H). The leaves are amphistomatic, but with a higher
density of paracytic stomata on the upper surface (Fig. 5A,
B, D). A mesophyll comprised of 2–4 layers of palisade-
like parenchyma forms a ring from the base towards the
rest of the leaf (Fig. 5D, H) and contains raphide idioblasts
(Fig. 5D). Inside the ring of mesophyll, there is a rather
large-celled parenchymatous tissue without chloroplasts,
which presumably serves to store water, surrounding the
median vascular bundle and some smaller lateral bundles
(Fig. 5D). The vascular bundles contain mainly sclerenchy-
matic cells, which we interpret as metaxylem fibres (Fig.
5D, E). Phloem is present on the abaxial and lateral sides
of the vascular bundles (Fig. 5E, F). On the adaxial side
of the metaxylem fibres, a few protoxylem spiral tracheids
can be observed (Fig. 5E, G–I). Both median and lateral
vascular bundles have conspicuous parenchymatic bundle
sheaths (Fig. 5D, E). The parenchymatic bundle sheath from
the central vascular bundle extends to the lower epidermis
disrupting the otherwise equifacial character of the leaves
(Fig. 5D). The central vascular bundle is crescent-shaped
in cross section and has an inconspicuous sclerenchymatic
cap at its abaxial side (Fig. 5E).
Fig. 4. Photographs of Lathraeocarpa acicularis. A–B, growth form; C, flower bud; D, longistylous flower; E, brevistylous
flower. Photographs by Steven Dessein.

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Groeninckx & al. • Taxonomic position of LathraeocarpaTAXON 58 (1) • February 2009: 209–226
Inflorescence. – The inflorescences of Lathraeocarpa
are one-flowered and terminal on lateral branchlets. Pe-
duncles are very short or absent. Each flower is subtended
by a whorl of three leaves (Fig. 3B; 4C–E).
Flowers. – Lathraeocarpa has white, heterodistylous,
4-merous flowers (Fig. 3C, D; 4D, E). The short calyx
tubes are crowned by 8 ciliate calyx lobes (Fig. 3B; 6A)
which are triangular to narrowly triangular and up to 0.6
mm long in L. acicularis (Fig. 6B), and up to 1.5 mm long
in L. decaryi. In L. acicularis the corolla tubes are fun-
nel-shaped, 4.1–6.4 mm long, 0.7–1 mm wide at the base,
and 1.7–2.3 mm wide at the throat (Figs. 3C, D; 6A). The
corolla tubes are covered with relatively long trichomes
on the outer surface (Fig. 6A) and with shorter trichomes
within (Fig. 6C). The corolla lobes are elliptic, 2.2–3.4 mm
long, 2–2.3 mm wide and glabrous on both surfaces, or
papillose inside (Fig. 6A, D). In L. decaryi the corolla tubes
are also funnel-shaped, ca. 7 mm long, 0.4–0.6 mm wide at
the base, 1–1.5 mm wide at the throat, pubescent outside,
and pubescent in the lower half inside. The corolla lobes
are ca. 2 mm long and sparsely pubescent outside.
The four stamens are inserted just below the throat
of the corolla tube. The white anthers are dorsimedifixed,
elliptic in outline, 0.5–1.2 mm long, and dehisce with lon-
gitudinal slits. In brevistylous flowers, the anthers are
exserted for 1.2–2.6 mm (Fig. 3C), whereas in longistylous
flowers they are completely included within the corolla
tube (Fig. 3D).
The ovaries are cup-shaped and up to 1.8 mm long,
densely covered with trichomes, except on the ribs formed
Fig. 5. Leaf and stipule characters of Lathraeocarpa acicularis. A, adaxial leaf surface; B, abaxial leaf surface; C, stipular
sheath with one stipular tooth (arrow); D, transverse leaf section, stomata indicated by arrows; E, detail of the median
vascular leaf bundle; F, detail of abaxial phloem; G, detail of adaxial xylem, protoxylem tracheids indicated by arrows; H,
longitudinal leaf section at xylem level, spiral tracheids indicated by arrows; I, detail of longitudinal section with metaxy-
lem vessels (blue coloured) and spiral tracheids (arrows). Abbreviations: ep, epidermis; me, mesophyll; mx, metaxylem;
p, phloem; pa, parenchyma; px, protoxylem; sc, sclerenchymatic cap; x, xylem.

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TAXON 58 (1) • February 2009: 209–226Groeninckx & al. • Taxonomic position of Lathraeocarpa
Fig. 6. Flower characters of
Lathraeocarpa acicularis.
A, flower bud with one
corolla lobe removed; ring of
colleters associated with the
bracts indicated by arrow;
B, outside surface of calyx
lobe; C, inner surface of co-
rolla tube; D, inner surface of
corolla lobe; E, outside sur-
face of ovary; F, locule with
one ovule basally attached;
G, cylindrical nectary disc.
Fig. 7. Pollen characters of Lathraeocarpa acicu-
laris. A, equatorial view of thrum pollen grain;
B, detail of thrum mesocolpium; C, polar view
of thrum pollen grain; D, detail of thrum apo-
colpium; E, equatorial view of pin pollen grain;
F, detail of pin mesocolpium; G, polar view of
pin pollen grain; H, detail of pin apocolpium.
All pollen grains studied following the modified
method of Halbritter (1998).

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Groeninckx & al. • Taxonomic position of LathraeocarpaTAXON 58 (1) • February 2009: 209–226
by the vascular bundles (Figs. 3B; 6A, E). The ovaries
are basically 4-locular, although flowers with 3-locular
ovaries are sometimes observed. Each locule has a single
ovule inserted at the base of the septum (Fig. 6F). The
nectary disc on top of the ovary, a circular zone surround-
ing the base of the style (Fig. 6G), is covered with para-
cytic stomata and surrounded by a ring of trichomes (Fig.
6G). The white styles have a 3- or 4-lobed papillose stigma
(Fig. 6A). The styles are included within the corolla tube
in brevistylous flowers (Fig. 3C) and exserted for ca. 2–3
mm in longistylous flowers (Fig. 3D).
Pollen. – The pollen grains are 7–8-zonocolporate,
suboblate, with an equatorial diameter of 24–31 μm in
Lathraeocarpa acicularis (Fig. 7A, C, E, G) and 41–50 μm
in L. decaryi. Detailed observat ions of additional feat ures
are only available for L. acicularis. The tectum is hetero-
brochate and varies from reticulate to micro-reticulate
(Fig. 7B, D, F, H). The inner nexine surface is granu-
lar. Differences in pollen structure between brevi- and
longistylous forms are limited. Brevistylous flowers have
slightly larger pollen and an ornate apocolpium (reticu-
late ornamentation consisting of broad, curved muri and
lumina that are often anastomosing) with granules on the
muri (Fig. 7D), whereas longistylous flowers have a (mi-
cro-)reticulate apocolpium without granules on the muri
(Fig. 7H). Moreover, grains of brevistylous flowers have
a better developed double reticulum than those of longi-
stylous flowers (Fig. 7B, F).
Fruits. – The fruits of Lathraeocarpa acicularis are
obconical, 2.2–2.8 mm long, 1.8–2 mm wide, indehis-
cent, and crowned by persistent calyx lobes (Figs. 3E; 8A).
Distinct ribs are visible on the outer surface. The fruits
are beset with long trichomes, which are more sparse on
the ribs (Fig. 8B). The fruit wall comprises three distinct
layers (Fig. 8C–E): a one-layered exocarp with some cells
developed into trichomes; a parenchymatic mesocarp with
raphides; and a thick sclerenchymatic endocarp. The vas-
cular traces are embedded in the mesocarp, close to the
endocarp. Fruits of L. decaryi are similar in shape but
up to 3.5 mm in diameter and sulcate rather than costate.
According to Bremekamp (1957), the fruit wall anatomy
of L. decaryi differs from the one of L. acicularis in the
presence of eight strands of thin-walled cells surround-
ing the pyrene, which are thought to enhance the floating
capacity of the fruits. Seeds could not be studied due to
lack of appropriate material.
Distribution and habitat. —
Collections of Lath-
raeocarpa come from two areas in the southern part of
Madagascar; L. decaryi from Ambovombe and Cap Sainte
Marie (South Madagascar) and L. acicularis from Toliara
(southwest Madagascar). The new locality for L. acicu-
laris is Lac Tsinamampetsotsa (Toliara). Lathraeocarpa
acicularis grows on sandy soils in dunes close to the sea,
whereas L. decaryi seems to be restricted to limestone in
the dry forests and scrublands of the South.
DISCUSSION
Inclusion of Lathraeocarpa acicularis in tribe Sper-
macoceae s.l. is strongly supported by our molecular
data. Seeing the morphological similarities between
Fig. 8. Fruit characters of Lathraeocarpa acicularis. A, young fruit; B, detail of the outside surface of the fruit wall with rib
in the middle; C, cross-section through young fruit; D, cross-section through young fruit; E, detail of the fruit wall. Ab-
breviations: en, endocarp; ex, exocarp; me, mesocarp.

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TAXON 58 (1) • February 2009: 209–226Groeninckx & al. • Taxonomic position of Lathraeocarpa
L. acicularis and L. decaryi, the type species of the ge-
nus, Bremekamp’s (1957) tribe Lathraeocarpeae can no
longer be recognized. A close relationship between Lath-
raeocarpa and Knoxieae s.l., as proposed by Kårehed &
Bremer (2007), is not supported. As Capuron (1973) sug-
gested, our results confirm that Lathraeocarpa is closely
related to Gomphocalyx. A sister relationship between
Lathraeocarpa and the clade comprising Gomphocalyx
and Phylohydrax, as suggested by our analyses, has not
been proposed previously. Table 5 gives a summary of the
morphological characters of the three genera comprising
the LGP clade. In the following paragraphs, we will dis-
cuss these morphological data in the light of our molecular
evolutionary hypothesis.
Growth form. —
Both Gomphocalyx and Phylo-
hydrax are creeping herbs forming dense mats, whereas
Lathraeocarpa is a (sub)shrub. Within the Pentanopsis
clade most species are annual or perennial herbs, some of
which have a woody base (e.g., Manostachya ternifolia).
The (sub)shrubby habit of Lathraeocarpa is thus excep-
tional within the group, and may be interpreted as a case
of secondary woodiness. Although Rubiaceae are mainly
woody, comprising predominantly shrubs and trees, some
lineages of the family include herbaceous representatives.
Recent molecular studies have shown that most herbaceous
Rubiaceae belong to the subfamily Rubioideae (Robbrecht
& Manen, 2006).
Outside Rubioideae, herbaceousness
only occurs in Sabiceeae, Virectarieae and Sipaneeae.
Within Rubioideae, Jansen & al. (2002) reported strong
indications for cases of secondary woodiness in the tribes
Rubieae and Anthospermeae. Within the predominantly
herbaceous Spermacoceae s.l., other possible cases of sec-
ondary woodiness may be found in the Hawaiian genus
Kadua Cham. & Schltdl., the Asian Hedyotis L. species,
the neotropical genus Arcytophyllum Willd. ex Schult. &
Schult. f., and in some genera of the former tribe Sper-
macoceae s.str. (Diodella Small, Ernodea Sw., Galianthe
Griseb., Spermacoce L.).
Phylohydrax is further characterized by a distinct dif-
ferentiation into longer vegetative and shorter reproduc-
tive stems. This kind of shoot differentiation is not found
in Gomphocalyx or Lathraeocarpa, but does character-
ize several other taxa such as Hydrophylax maritima L. f.
and Diodia vaginalis Benth., both belonging to Spermac-
oceae s.str. (Puff, 1986; Dessein & al., 2005a). The simi-
lar growth form observed in Phylohydrax, Hydrophylax
maritima and Diodia vaginalis can be explained as an
adaptation to similar growth conditions.
Leaves and stipules. —
Lathraeocarpa, Gomphoca-
lyx and Phylohydrax all have amphistomatic leaves (Puff,
1986; Dessein & al., 2005a), an uncommon character in
Rubiaceae (Robbrecht, 1988). A stipular sheath with very
short appendage(s) is also a feature shared by the three
taxa (Puff, 1986; Dessein & al., 2005a). Anatomically the
leaves of Lathraeocarpa correspond best with those of
Phylohydrax (Puff, 1986). Both genera have mesophyll
that is not differentiated into spongy and palisade paren-
chyma as in Gomphocalyx (Dessein & al., 2005a), but
Table 5. Selected morphological and anatomical characters and character states observed in Lathraeocarpa, Gompho-
calyx and Phylohydrax.
Character Lathraeocarpa Gomphocalyx Phylohydrax
Growth form Subshrubs with terminal flow-
ers on short branchlets Procumbent to decumbent
annual or short-lived perennial
herbs
Perennial herbs with differentia-
tion into longer vegetative stems
and short erect flowering stems
Leaf Amphistomatic Amphistomatic Amphistomatic
Leaf mesophyll Not differentiated Differentiated Not differentiated
Heterostyly Present Present Present
Calyx tube Reduced Reduced Well-developed
Number of calyx lobes 8 8 4
Corolla tube Funnel-shaped Narrowly cylindrical Funnel-shaped
Ovule position Attached near base of septum Attached near base of septum Attached near base of septum
Nectary disc Ring-shaped 2-lobed Ring-shaped
Stigma 3- or 4-lobed 2-lobed 2-lobed
Fruit type Drupe Dry and indehiscent Dry and indehiscent
Seed Unknown Obovoid, ellipsoid or pyriform
without ventral groove Ellipsoid without ventral groove
Pollen type Pluri-zonocolporate Pluri-zonocolporate Pluri-zonocolporate
Pollen sexine (Micro-)reticulate with granules (Micro-)reticulate with granules (Micro-)reticulate with granules

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Groeninckx & al. • Taxonomic position of LathraeocarpaTAXON 58 (1) • February 2009: 209–226
instead consists only of palisade-like parenchyma. So far,
undifferentiated mesophyll has not been reported within
Spermacoceae s.l. Results thus support the hypothesis
that the loss of mesophyll differentiation has occurred at
least once within the tribe. The main difference between
Lathraeocarpa and Phylohydrax is the presence of an
extension of parenchymatic cells running from the central
vascular bundle to the lower epidermis in the leaves of
Lathraeocarpa.
Inflorescence. —
Lathraeocarpa is characterized
by terminal inflorescences. The inflorescences of Gom-
phocalyx are terminal initially, but are pushed aside dur-
ing anthesis by the developing shoot in one of the axils,
making them pseudo-axillary (Dessein & al., 2005a). The
inflorescences of Phylohydrax are by contrast, truly ax-
illary. Phylohydrax has single-flowered inflorescences
with subsessile flowers (Puff, 1986) similar to those in
Lathraeocarpa, whereas in Gomphocalyx inflorescences
are few- to many-flowered.
Flowers. —
Based on the four herbarium specimens
in which he only observed f lowers with an exserted style
and included anthers, Bremekamp (1957) concluded that
Lathraeocarpa is most likely to be isostylous. However,
in L. acicularis we clearly observed heterodistyly with
longistylous and brevistylous flowers occurring on dif-
ferent individuals within the population. The collection
Humbert & Capuron 29308, unknown to Bremekamp,
proves that also L. decaryi is truly heterostylous. Hetero-
styly is very common in Rubiaceae, especially in genera
of Psychotrieae and Spermacoceae s.l. ( Robbrecht, 1988).
Gomphocalyx, Phylohydrax and all other taxa in the Pen-
tanopsis clade (except Conostomium) are heterostylous
(Bremekamp, 1952; Puff, 1986; Dessein & al., 2005a). The
sister group of the Pentanopsis clade, Kohautia subgenus
Kohautia, is not heterostylous. Taxa of subgenus Kohau-
tia are characterized by a flower morphology in which
anthers and stigma are always included, with the stigma
well below the anthers or occasionally just touching them
(Mantell, 1985). This monomorphic short-styled condition
is, with exception of a few individuals of Conostomium
(Bremekamp, 1952), unique among the African members
of Spermacoceae. It probably evolved as a modification of
the dimorphic condition (Robbrecht, 1988).
Both Lathraeocarpa and Gomphocalyx have a re-
duced calyx tube with eight calyx lobes, whereas Phy-
lohydrax has a well-developed calyx tube with only four
lobes reduced to colleter-tipped outgrowths. Flowers of
Gomphocalyx have a narrowly cylindrical corolla tube and
a bilobed nectary disc, in contrast with those of Lathraeo-
carpa and Phylohydrax which have funnel-shaped corolla
tubes and an annular nectar disc surrounding the base of
the style (Puff, 1986; Dessein & al., 2005a). The bilobed
nectary disc is an aut apomor phy i n Gomphocalyx, not yet
observed within other taxa of the Pentanopsis clade.
In Gomphocalyx and Phylohydrax the ovary is biloc-
ular, whereas in Lathraeocarpa it is tri- or tetralocular;
the 2-locular condition reported by Capuron (1973) could
not be confirmed. The three taxa all have a single ovule
per locule. Almost all taxa within Spermacoceae s.l. have
pluri-ovulate ovaries (Robbrecht, 1988). Only representa-
tives of the former tribe Spermacoceae s.str. have uni-
ovulate ovaries. In Spermacoceae s.str. the ovules are,
however, attached to the middle of the septum (Dessein,
2003), which is not the case for members of the LGP
clade where the ovules are attached near the base of the
septum (Puff, 1986; Dessein & al., 2005a). Within Sper-
macoceae uni-ovulate ovaries with basal attachment of
the ovules can thus be considered as a synapomorphy
for the LGP clade.
Pollen. —
Lathraeocarpa has pluri-zonocolporate
pollen, as previously reported in Gomphocalyx and Phy-
lohydrax (Puff, 1986; Dessein & al., 2005a). The presence
of this type of pollen was one of the main reasons why the
latter two genera were previously included in Spermac-
oceae s.str., where it is more common than in the rest of
Spermacoceae s.l., in which 3-colporate pollen predomi-
nates (Dessein & al., 2002, 2005b; Dessein, 2003). The
Asian genus Neanotis W.H. Lewis is a notable exception
in having pluri-zonocolporate pollen grains. The genus
also shows a trend towards reduction in the number of
seeds per locule. In mature fruits, only one or two seeds
are present (Lewis, 1966). However, with no molecular se-
quence data available for the genus it would be premature
to hypothesize a close relationship between Neanotis and
the LGP clade. Nevertheless, Neanotis confirms that there
is an evolutionary tendency in Spermacoceae to develop
pluri-aperturate pollen grains and uni-ovulate ovaries.
In contrast to Gomphocalyx and Phylohydrax, pol-
len grains of Lathraeocarpa acicularis have a double
reticulum, a feature that also occurs in the tribes Cocco-
cypseleae (Piesschaert & al., 2000) and Pavetteae (De
Block & Robbrecht, 1998), in the genus Metabolos Blume
(Puff & Igersheim, 1994), and in several other members of
Spermacoceae s.l. (Dessein, 2003; Dessein & al., 2005b;
Groeninckx, 2005; Pire, 1997; Pire & Cabral, 1992).
Within the Pentanopsis clade, a double reticulum has also
been observed in Amphiasma Bremek., Oldenlandia af-
finis (Roem. & Schult.) DC., and O. herbacea (Scheltens,
1998). I n Lathraeocarpa, the double reticulum is better de-
veloped in pollen of brev istylous f lowers tha n in pollen of
longistylous flowers. A similar dimorphism was observed
in the genus Galianthe Griseb. of Spermacoceae s.str.
(Pire & Cabral, 1992) and in several species of Cocco-
cypseleae (Piesschaert & al., 2000). Lathraeocarpa pollen
further differs in having 8–10 relatively long ectocolpi,
whereas Gomphocalyx and Phylohydrax are characterized
by pollen with short colpi (8–10 colpi in Gomphocalyx and
10–12 colpi in Phylohydrax) (Puff, 1986; Dessein & al.,

222
TAXON 58 (1) • February 2009: 209–226Groeninckx & al. • Taxonomic position of Lathraeocarpa
2005a). Other members of the Pentanopsis clade, as far
as known, all have pollen with long colpi except for the
African genus Conostomium. The presence of short ec-
tocolpi would support a relationship between the African
genus Conostomium and the Gomphocalyx-Phylohydrax
clade, as suggested by our atpB-rbcL data but without sig-
nificant jackknife or bootstrap values. However, besides
short ectocolpi not a single morphological character is
known by us that would support a close relationship be-
tween Conostomium and the LGP clade. Conostomium is
characterized by multiovulate placentas and 3-zonocolpo-
rate pollen grains. The length of the colpi is very variable
within Spermacoceae but sometimes demarcates genera
or groups of related species, as shown by Dessein & al.
(2002) for the African Spermacoce.
Fruits. —
Fruits of Lathraeocarpa are drupes, and
their fleshiness stands in contrast with the dry fruits of
Gomphocalyx and Phylohydrax. Most Spermacoceae have
dry fruits but fleshy fruits are also observed. In all three
taxa, the fruit wall consists of three distinct layers (exo-,
meso-, endocarp), but transverse sections through young
fruits show that the relative thickness of meso- and endo-
carp, as well as the position of the vascular traces in the
fruit wall, differ among the three taxa. In Phylohydrax
a relatively thick sclerenchymatous endocarp and a thin
parenchymatous mesocarp are present, and the vascu-
lar traces are situated just underneath the exocarp (Puff,
1986). Gomphocalyx, on the other hand, has a relatively
thin endocarp, a more extensive mesocarp, and the vascu-
lar traces lie close to the endocarp (Dessein & al., 2005a).
The fruit wall anatomy of Lathraeocarpa shows an af-
finity with that of both Phylohydrax and Gomphocalyx.
As in Phylohydrax, the endocarp is relatively thick in
comparison to the mesocarp, but the vascular traces are
situated closer to the endocarp as in Gomphocalyx. Fruit
wall anatomical characters support a close relationship
between the three taxa of the LGP clade. However, too
little is known about the fruit wall anatomy of other taxa
within Spermacoceae to assess fruit anatomical synapo-
morphies to the LGP clade.
Morphology of taxa in the LGP clade. —
The op-
timization of morphological characters on the molecular
tree implies that the common ancestor of Lathraeocarpa,
Phylohydrax and Gomphocalyx likely had all character
states shared by the three taxa: amphistomatic leaves
with paracytic stomata, a stipular sheath with very short
appendage(s), heterostyly, one basal ovule per locule, and
pluri-zonocolporate pollen grains. The last two character
states are synapomorphies for the LGP clade with respect
to the Pentanopsis clade. Most taxa of Spermacoceae s.l.
have multi-ovulate ovaries and the number of apertures
of the pollen grains rarely exceeds five. Uni-ovulate ova-
ries and pluri-colporate pollen grains are, however, also
observed in the former tribe Spermacoceae s.str. This
supports the hypothesis that the development towards uni-
ovulate ovaries combined with pluri-aperturate pollen
grains has evolved at least two times within Spermaco-
ceae s.l., and that these character states are the result of
convergent evolution between the LGP clade and Sperma-
coceae s.str. More detailed mor pholog ical and a natomical
research within the Pentanopsis clade and the Sperma-
coceae tribe is needed, however, to assess whether the
amphistomatic leaves and the stipular sheath with short
appendage(s) can also be considered as synapomorphies
for the LGP clade.
In all molecular analyses, except for the individual
atpB-rbcL analysis, the LGP clade shares a common
ancestor with the African taxa Manostachya ternifolia
and Oldenlandia rosulata. These two species are erect
herbs, respectively perennial and annual. Despite the high
support in the combined MP and BI analysis (JS = 100,
BS = 100, PP = 0.93), not a single morphological or ana-
tomical character has so far been found to support a rela-
tionship between these two taxa and the LGP clade.
Taxonomic implications. —
Molecular data are
conclusive to place Lathraeocarpa acicularis within
Spermacoceae s.l. With this new taxonomic position, the
monogeneric tribe Lathraeocarpeae can no longer be rec-
ognized. Relationships discovered within the LGP clade
can be translated into three different classifications: (1) re-
duction of Phylohydrax to the synony my of Gomphocalyx
with Lathraeocarpa as sister, (2) merging the three taxa
of the LGP clade into Gomphocalyx, or (3) recognition of
all three taxa of the LGP clade as distinct genera.
The fusion of Lathraeocarpa and Gomphocalyx as
proposed by Piesschaert (2001) is not corroborated by our
study. Merging Gomphocalyx and Phylohydrax based on
morphological si milarit ies (succulent leaves with a ring of
palisade-like parenchyma, one-flowered inflorescences
with subsessile funnel-shaped flowers and a ring-like nec-
tar disc), can also be rejected when listing all differences
between the two taxa: habit (herb vs. shrub), number of
calyx lobes (four vs. eight), fruit type (dry vs. fleshy), and
pollen ectocolpi (short vs. long). In conclusion, it is clear
that morphology does not provide unambiguous evidence
to merge taxa of the LGP cl ade. Therefore , it is b est to main-
tain three separate genera, especially because each genus
is characterized by a unique set of character states.
Environmental adaptation. —
Several morpho-
logical characters shared by members of the LGP clade
can be regarded as adaptations to a dry habitat. Both
Phylo hydrax and Lathraeocarpa acicularis are maritime
plants; species of Phylohydrax are beach pioneers grow-
ing just above the high water mark, whereas L. acicularis
occurs in dunes close to the sea. Gomphocalyx, on the
other hand, grows further inland on sandy or laterite soils
in dry spiny forests up to 850 m high, but also on dunes
and beaches. The most striking environmental adaptation

223
Groeninckx & al. • Taxonomic position of LathraeocarpaTAXON 58 (1) • February 2009: 209–226
are the amphistomatic leaves, which are somewhat suc-
culent, especially in Lathraeocarpa and Phylohydrax.
The absence of metaxylem vessels and the abundance of
metaxylem fibres in the vascular leaf bundles observed
in L. acicularis can also be inter preted as a n environme n-
tal adaptation to xeric conditions. Water transport in L.
acicularis is most likely to be symplastic with the large
parenchymatic bundle sheat cells functioning as bulliform
cells that absorb and release water to allow the leaf blade
to curl or roll up. T his unusual h igh amount of met axylem
fibres is not found in the allied genera Gomphocalyx and
Phylohydrax, and could thus represent an autapomorphy
for Lathraeocarpa.
CONCLUSIONS
Sequence data from four plastid markers (atpB-rbcL,
rps16, trnL-trnF, petD) strongly support the inclusion of
the Madagascan endemic genus Lathraeocarpa within the
tribe Spermacoceae s.l., sister to Phylohydrax and Gom-
phocalyx. As a consequence, the tribe Lathraeocarpeae
can no longer be recognized and instead, Lathraeocarpa
must be included within Spermacoceae. Some morpho-
logical and anatomical characters support this molecular
evolutionary hypothesis. The clade comprising Lathraeo-
carpa, Gomphocalyx and Phylohydrax is supported by
four apparently independent morphological characters.
All three genera have amphistomatic leaves, a stipular
sheath with very short appendage(s), a single basal ovule
per locule, and pluri-zonocolporate pollen grains.
ACKNOWLEDGEMENTS
We thank P. Baas from the National Herbarium of the Neth-
erlands and F. Lens from the Laboratory of Plant Systematics
for helpful discussions, and A. Fernandez from the National
Botanic Garden of Belgium for the botanical line drawing. We
acknowledge the reviewers for their great effort and effective-
ness in improving the quality of the manuscript. Research in
Madagascar was facilitated by the following Malagasy govern-
mental institutions: Association Nationale pour la Gestion des
Aires Protégées (ANGAP), Ministère des Eaux et Forêts (MEF),
and Parc Botanique et Zoologique de Tsimbazaza (PBZT). We
thank P. Lowry, Head of the Africa & Madagascar Depar tment
of Missouri Botanical Garden, for the opportunity to perform
field work within the framework of MBG’s Madagascar Re-
search and Conservation Program. We also thank the members
of the MBG staff in Madagascar for their hospitality and help.
This research was supported by grants from the Fund for Sci-
entific Research, Flanders (F.W.O., G.0205.05 and G.0268.04).
I. Groeninckx holds a Ph.D. research grant from the F.W.O.
Flanders.
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Appendix. List of taxa used in the phylogenetic analyses with voucher information (geographic origin, collector, collector
number, herbarium), accession numbers and literature citations for previouslz published sequences for the four plastid
markers atpB-rbcL, rps16, trnL-trnF, and petD: (1) = Andersson & Rova, 1999; (2) = Andersson & al., 2002; (3) = Dessein &
al., 2005; (4) = Groeninckx & al., in press. New sequences are marked with *.
Agathisanthemum Klotzsch: A. bojeri Klotzsch, Zambia, Dessein & al. 671 (BR), EU542917(4), EU543018(4), EU543077(4); A.
globosum (Hochst. ex A. Rich.) Klotzsch, Zambia, Dessein & al. 201 (BR), EU542918(4), EU543019(4), EU543078(4). Amphiasma
Bremek.: A. benguellense (Hiern) Bremek., Angola, Kers 3350 (S), EU542919(4), AF002753(1), EU543079(4), EU557680*; A. luzu-
loides (K. Schum.) Bremek., Zambia, Dessein & al. 1167 (BR), EU542920(4), EU543020(4), EU543080(4), EU557681*. Arcytophyl-
lum Willd. ex Schult. & Schult. f.: A. aristatum Standl., Ecuador, Hekker & Hekking 10335 (GB), AF333348(2), AF333349(2); A.
ciliolatum Standl., Ecuador, Øllgaard & al. 58395 (NY), AF333350
(2)
, AF333351
(2)
; A. ericoides (Willd. ex Roem. & Schult.) Standl.,
unknown, Edwin & al. 3624 (S), AF333352(2), AF333353(2); A. lavarum K. Schum., Costa Rica, Cronquist 8827 (NY), AF333354(2),
AF333355(2); A. macbridei Standl., Peru, Wurdack 1073 (NY), AF333356(2), AF333357(2); A. muticum (Wedd.) Standl., Colombia,
Andersson & al. 2195 (GB), EU542921(4), AF002754(1), EU543081(4); A. nitidum (Kunth) Schltdl., Venezuela, Pipoly & al. 6467
(GB), AF333359
(2)
; A. rivetii Danguy & Cherm., Ecuador, Harling & Andersson 22232 (GB), EU542922
(4)
, AF333362
(2)
, AF333363
(2)
;

225
Groeninckx & al. • Taxonomic position of LathraeocarpaTAXON 58 (1) • February 2009: 209–226
A. serpyllaceum (Schltdl.) Terrell, Mexico, Stafford & al. 203 (MO), AF333364(2); A. setosum (Ruiz & Pav.) Schltdl., Colombia,
Andersson & al. 2196 (GB), AF002755
(1)
, AF333365
(2)
; A. thymifolium (Ruiz & Pav.) Standl., Ecuador, Ståhl 4481 (GB), EU542923
(4)
,
AF333366(2), EU543082(4). Batopedina Verdc. (outgroup Analysis 1): B. pulvinellata Robbrecht, Zambia, Dessein & al. 264 (BR),
EU542924
(4)
, EU543021
(4)
, EU543083
(4)
. Bouvardia Salisb.: B. glaberrima Engelm., cult., Forbes s.n. (S), EU542925
(4)
, EU543022
(4)
,
EU543084(4); B. ternifolia (Cav.) Schltdl., unknown, Van Caekenberghe 264 (cult. at BR), AF002758(1); Mexico, Spencer & al. 363
(NY), EU642537(4). Carphalea Juss. (outgroup Analysis 1): C. madagascariensis Lam., Madagascar, De Block & al. 578 (BR),
EU542926(4), EU543023(4). Conostomium (Stapf) Cufod.: C. natalense (Hochst.) Bremek., South Africa, Dahlstrand 1346 (GB),
AF002760(1), EU543085(4), EU557687*; South Africa, Bremer & al. 4341 (UPS), EU542927(4); C. quadrangulare (Rendle) Cufod.,
Ethiopia, Puff & Kelbessa 821222 (UPS), EU542928(4), EU543024(4), EU543086(4), EU557688*; C. zoutpansbergense (Bremek.)
Bremek., South Africa, Bremer & al. 4331 (UPS), EU542929
(4)
, EU543087
(4)
, EU557689*. Crusea Cham. & Schltdl.: C. calocephala
DC., Guatemala, Gustafsson & al. 215 (GB), EU542930(4), EU543088(4); C. megalocarpa (A. Gray) S. Watson, Mexico, Pringle
3852 (S), EU542931(4), EU543025(4), EU543089(4). Dentella J.R. Forst & G. Forst. (outgroup Analysis 2): D. dioeca Airy Shaw,
Australia, Harwood 1559 (BR), EU543090(4), EU557692*; D. repens (L.) J.R. Forst. & G. Forst., Australia, Andersson 2262 (GB),
EU542932(4), AF333370(2), EU543091(4), EU557693*. Dibrachionostylus Bremek.: D. kaessneri (S. Moore) Bremek., Kenya, Strid
2598 (GB), EU542933
(4)
, AF002761
(1)
. Diodella Small: D. sarmentosa (Sw.) Bacigalupo & Cabral ex Borhidi, French Guiana,
Anderson & al. 2071 (GB), AF002762
(1)
. Diodia L.: D. aulacosperma K. Schum., Kenya, Luke 9029 (UPS), EU542934
(4)
, EU543026
(4)
,
EU543092(4). D. spicata Miq., French Guiana, Anderson & al. 1961 (GB), EU542935(4), EU543027(4), EU543093(4). Emmeorhiza
Pohl ex Endl.: E. umbellata (Spreng.) K. Schum., Trinidad, Hummel 4 (GB), EU542936
(4)
, AY764289
(3)
, EU543094
(4)
. Ernodea Sw.:
E. littoralis Sw., Cuba, Rova & al. 2286 (GB), EU542937(4), AF002763(1), EU543095(4). Galianthe Griseb.: G. brasiliensis (Spreng.)
E.L. Cabral & Bacigalupo, Argentina, Vanni & Radovancick 996 (GB), EU542938(4), AY764290(3), EU543096(4); G. eupatorioides
(Cham. & Schltdl.) E.L. Cabral, Argentina, Schinini & Cristobal 9811 (GB), EU542939
(4)
, EU543028
(4)
, EU543097
(4)
. Gomphocalyx
Baker: G. herniarioides Baker, Madagascar, De Block & al. 569 (BR), AY764291
(3)
; Madagascar, Groeninckx & al. 125 (BR),
EU542940*, EU567466*, EU567461*. Hedyotis L.: H. consanguinea Hance, Hong Kong, Shiu Ying Hu 10821 (S), EU542941(4);
H. fruticosa L., Sri Lanka, Larsson & Pyddoke 22 (S), EU542942(4), EU543098(4); H. korrorensis (Valeton) Hosok., The Caroline
Islands, Fosberg 47697 (S), EU542943
(4)
, EU543099
(4)
; H. lawsoniae Wight, Sri Lanka, Wambeek & Wanntorp 2996 (S), EU542944
(4)
;
H. lessertiana var. lassertiana Thwaites, Sri Lanka, Klackenberg 413 (S), EU542945(4), EU543029(4), EU543100(4); H. lessertiana
var. marginata Thwaites & Trimen, Sri Lanka, Fagerlind 3668 (S), EU542946(4), EU543030(4), EU543101(4); H. macrostegia Stapf.,
Malaysia, Sabah, Wallander 6 (GB), EU542947(4), AF002767(1), EU543102(4); H. quinquenervis Thwaites, Sri Lanka, Bremer & al.
163 (S), EU542948(4), EU543103(4); H. rhinophylla Thwaires ex Trimen, Sri Lanka, Fagerlind 5082 (S), EU542949(4), EU543104(4);
H. swertioides Hook. f., South India, Klackenberg & Lundin 3 (S), EU542950(4), EU543031(4), EU543105(4). Hedythyrsus Bremek.:
H. spermacocinus (K. Schum.) Bremek., Zambia, Dessein & al. 1017 (BR), EU542951(4), EU543032(4), EU543107(4). Hemidiodia
K. Schum.: H. ocymifolia (Willd. ex Roem. & Schult.) K. Schum., French Guiana, Andersson & al. 2040 (GB), EU542952
(4)
,
EU543108(4). Houstonia L.: H. caerulea L., U.S.A., Vincent & Lammers s.n. (GB), EU542953(4), AF333379(2), EU543109(4); H.
longifolia Gaertn., U.S.A., Yatskievych 96-49 (MO), EU542954(4), AF002766(1); U.S.A., Weigend 9963 (NY), EU642536(4). Kadua
Cham. & Schltdl.: K. acuminata Cham. & Schltdl., U.S.A., Hawaii, cult. at BR, EU542955(4), EU543110(4); K. affinis Cham. &
Schltdl., U.S.A., Hawaii, Motley 1733 (NY), EU642523(4), EU642538(4); K. axillaris (Wawra) W.L. Wagner & Lorence, U.S.A.,
Hawaii, Harrison-Gagne s.n. (GB), AF002765(1); U.S.A., Hawaii, Maul, Motley 1724 (NY), EU642524(4), EU642535(4); K. centran-
thoides Hook. & Arn., U.S.A., Hawaii, Skottsberg 6788 (S), EU542956(4), EU543033(4), EU543111(4); K. cordata Cham. & Schltdl.,
cult., Lorence 8021 (PTBG), EU542957(4), AF333376(2), EU543112(4); K. coriacea (J.E. Smith) W.L. Wagner & Lorence, U.S.A.,
Hawaii, Motley 1703 (NY), EU642525(4), EU642539(4); K. degeneri (Fosberg) W.L. Wagner & Lorence, cult., Wood 5062 (PTGB),
EU542958
(4)
, AF333371
(2)
, EU543113
(4)
; K. elatior (H. Mann) W.L. Wagner & Lorence, U.S.A., Hawaii, Kauai, Wagner 6350 (BISH),
EU642526(4), EU642540(4); K. fluviatilis C.N. Forbes, U.S.A., Hawaii, Oahu, Motley 1747 (NY), EU642527(4), EU642541(4); K.
flynnii (W.L. Wagner & Lorence) W.L. Wagner & Lorence, U.S.A., Hawaii, Kauai, Perlman 15631 (BISH), EU642528
(4)
, EU642542
(4)
;
K. foggiana (Fosberg) W.L. Wagner & Lorence, U.S.A., Hawaii, Sparre 27 (S), EU542959(4), EU543114(4); K. fosbergii (W.L. Wag-
ner & D.R. Herbst) W.L. Wagner & Lorence, U.S.A., Hawaii, Oahu, Motley 1677 (NY), EU642529(4), EU642543(4); K. laxiflora H.
Mann, U.S.A., Hawaii, Molokai, Perlman 6647 (BISH), EU642530(4), EU642544(4); K. littoralis Hillebr., U.S.A., Hawaii, Molokai,
Kiehn & Luegmayr 920823 (WU), EU542960
(4)
, EU543034
(4)
, EU543115
(4)
; K. parvula A. Gray, cult., Perlman 12783 (GB),
EU542961(4), AF333375(2), EU543116(4); K. rapensis F. Br., Rapa Is. French Polynesia, Perlman 17953 (NY), EU642531(4),
EU642545(4). Kohautia Cham. & Schltdl.: K. amatymbica Eckl. & Zeyh., South Africa, Bremer & al. 4307 (UPS), EU542962(4),
EU543035
(4)
, EU543117
(4)
, EU557721*; K. caespitosa Schnizl., Zambia, Dessein & al. 432 (BR), EU542963
(4)
, EU543036
(4)
,
EU543118(4), EU557722*; K. coccinea Royle, Zambia, Dessein & al. 751 (BR), EU542964(4), EU543037(4), EU543119(4), EU557723*;
K. cynanchica DC., Zambia, Dessein & al. 469 (BR), EU542965(4), EU543038(4), EU543120(4), EU557724*; K. microcala Bremek.,
Zambia, Dessein & al. 1149 (BR), EU542966
(4)
, EU543039
(4)
, EU543121
(4)
; K. obtusiloba Schnizl., Kenya, Luke 9035 (UPS),
EU542967(4), EU543040(4), EU543122(4); K. senegalensis Cham. & Schltdl., Burkina Faso, Madsen 5940 (NY), EU642546(4); K.
subverticillata (K. Schum.) D. Mantell, Zambia, Dessein & al. 432 (BR), EU542968(4), EU543041(4), EU543123(4), EU557727*; K.
virgata (Willd.) Bremek., Madagascar, De Block & al. 539 (BR), EU542969(4), EU543124(4). Lathraeocarpa Bremek.: L. acicularis
Bremek., Madagascar, De Block & al. 2316 brevistylous (BR), EU642516*, EU642521*, EU642532*, EU642519*; Madagascar,
De Block & al. 2316 longistylous (BR), EU642517*, EU642522*, EU642533*, EU642520*. Leyla Bremek.: L. osteocarpa Bremek.,
Tanzania, Gereau 2513 (BR), EU542970(4), EU543125(4). Manettia Mutis ex L.: M. alba (Aubl.) Wernh., French Guiana, Andersson
& al. 1917 (GB), EU542971
(4)
, AF002768
(1)
; M. lygistum (L.) Sw., Colombia, Andersson & al. 2128 (GB), EU542972
(4)
, AF002769
(1)
,
EU543126(4). Manostachya Bremek.: M. ternifolia E.S. Martins, Zambia, Dessein & al. 265 (BR), EU542973(4), EU543042(4),
EU543127(4), EU557731*. Mitracarpus Zucc. ex Schult. & Schult. f.: M. frigidus (Willd. ex Roem. & Schult.) K. Schum., French
Guiana, Andersson & al. 1995 (GB), EU542974(4)
, AF002770
(1)
, EU543128
(4)
; M. microspermus K. Schum., Guiana, Jansen-Jacobs
Appendix. Continued.

226
TAXON 58 (1) • February 2009: 209–226Groeninckx & al. • Taxonomic position of Lathraeocarpa
& al. 4785 (GB), EU542975
(4)
, EU543044
(4)
. Mitrasacmopsis Jovet: M. quadrivalvis Jovet, Zambia, Dessein & al. 1273 (BR),
EU542976
(4)
, EU543045
(4)
, EU543129
(4)
. Nesohedyotis (Hook. f.) Bremek.: N. arborea (Roxb.) Bremek., cult., Chase 2915 (K),
AF003607
(1)
. Oldenlandia L.: O. affinis (Roem. & Schult.) DC., Zambia, Dessein & al. 627 (BR), EU542977
(4)
, EU543046
(4)
,
EU543130(4), EU557734*; O. angolensis K. Schum., Zambia, Dessein & al. 932 (BR), EU542978(4), EU543047(4), EU543131(4); O.
biflora (L.) Lam., Japan, Van Caekenberghe 63 (cult. at BR), EU542979(4), EU543132(4); O. capensis L. f. var. capensis, Zambia,
Dessein & al. 843 (BR), EU542980(4), EU543048(4), EU543133(4); O. capensis L. f. var. pleiosepala Bremek., Tanzania, Kayombe &
al. 4 (BR), EU542981(4), EU543049(4), EU543134(4); O. corymbosa L., Zambia, Dessein & al. 487 (BR), EU542982(4), EU543050(4),
EU543135(4); O. echinulosa K. Schum., Zambia, Dessein & al. 928 (BR), EU542983(4), EU543051(4), EU543136(4); O. echinulosa
K. Schum. var. pellicida (Hiern) Verdc., Tanzania, Kayombo & Kahemela 1993 (BR), EU542984
(4)
, EU543137
(4)
; O. fastigiata
Bremek., Zambia, Dessein & al. 1019 (BR), EU542985(4), EU543052(4), EU543138(4); O. galioides (F. Muell.) F. Muell., Australia,
Harwood 1511 (BR), EU542986
(4)
, EU543053
(4)
, EU543139
(4)
; O. geophila Bremek., Zambia, Dessein & al. 935 (BR), EU542987
(4)
,
EU543054
(4)
, EU543140
(4)
; O. goreensis (DC.) Summerh., Zambia, Dessein & al. 1286 (BR), EU542988
(4)
, EU543055
(4)
, EU543141
(4)
;
O. herbacea (L.) Roxb. var. goetzei (DC.) Summerh., Zambia, Dessein & al. 442 (BR), EU542989(4), EU543056(4), EU543142(4),
EU557746*; O. herbacea (L.) Roxb. var. herbacea, Zambia, Dessein & al. 463 (BR), EU542990
(4)
, EU543057
(4)
, EU543143
(4)
,
EU557747*; O. lancifolia (Schumach.) DC., Zambia, Dessein & al. 1356 (BR), EU542991(4), EU543058(4), EU543144(4); O. mi-
crotheca (Cham. & Schltdl.) DC., Mexico, Frödeström & Hultén 681 (S), EU542992(4), EU543059(4), EU543145(4); O. mitrasacmoides
F. Muell., Australia, Harwood 1516 (BR), EU542993(4), EU543146(4); O. nematocaulis Bremek., Zambia, Dessein & al. 924 (BR),
EU542994(4), EU543060(4); O. nervosa Hiern, Gabon, Andersson & Nilsson 2326 (GB), AF333382(2); O. robinsonii Pit., Zambia,
Dessein & al. 346 (BR), EU543061
(4)
, EU543147
(4)
; O. rosulata K. Schum., Zambia, Dessein & al. 1197 (BR), EU543043
(4)
,
EU567467*, EU567465*; O. salzmannii (DC.) Benth. & Hook. f. ex B.D. Jacks., Brazil, Harley 15514 (UPS), EU542995(4),
AY764294(3), EU543148(4); O. taborensis Bremek., Tanzania, Bidgood & al. 4015 (BR), EU542996(4), EU543149(4); O. tenelliflora
(Blume) Kuntze, Japan, Van Caekenberghe 70 (cult. at BR), EU542997(4), EU543062(4), EU543106(4); O. tenuis K. Schum., Guyana,
Jansen-Jacobs & al. 41 (UPS), EU542998(4), AY764293(3); O. uniflora L., U.S.A., Godfrey 57268 (GB), EU542999(4), AY764295(3),
EU543150(4); O. wiedemannii K. Schum., Kenya, Luke & Luke 8362 (UPS), EU543000(4), EU543063(4), EU543151(4). Paraknoxia
Bremek. (outgroup Analysis 1): P. parviflora (Stapf ex Verdc.) Verdc. ex Bremek., Zambia, Dessein & al. 678 (BR), EU543001(4),
EU543064(4), EU543152(4). Pentanopsis Rendle: P. fragrans Rendle, Ethiopia, Gilbert & al. 7458 (UPS), EU543065(4), EU543153(4),
EU557758*; P. gracilicaulis (Verdc.) Thulin & Bremer, Somalia, Thulin & al. 10512 (UPS), EU567458*, EU567460*, EU567468*.
Pentodon Hochst. (outgroup Analysis 2): P. pentandrus (K. Schum. & Thonn.) Vatke, Zambia, Dessein & al. 598 (BR), EU543002
(4)
,
EU543066
(4)
, EU543154
(4)
, EU557759*. Phylohydrax Puff: P. carnosa (Hochst.) Puff, South Africa, Bremer 3783 (UPS), EU543003
(4)
,
EU543067(4), EU642534*, EU557760*; P. madagascariensis (Willd. ex Roem. & Schult.) Puff, Madagascar, De Block & al. 640
(BR), EU543004(4), AY64292(3), EU543155(4), EU557761*. Richardia L.: R. scabra L., Colombia, Andersson & al. 2073 (GB),
EU543005(4), AF003614(1), EU543156(4); R. stellaris L., Australia, Egerod 85343 (GB), EU543006(4), EU543068(4), EU543157(4).
Spermacoce L.: S. capitata Ruiz & Pav., French Guiana, Andersson 1908 (GB), EU543007
(4)
, EU543069
(4)
, EU543158
(4)
; S. confusa
Rendle ex Gillis, Colombia, Andersson & al. 2074 (GB), AF003619
(1)
; S. erosa Harwood, Australia, Harwood 1148 (BR), EU543008
(4)
,
EU543070(4), EU543159(4); S. filituba (K. Schum.) Verdc., Kenya, Luke 9022 (UPS), EU543009(4), EU543071(4), EU543160(4); S.
flagelliformis Poir., Madagascar, De Block & al. 794 (BR), EU543010
(4)
, EU543072
(4)
, EU543161
(4)
; S. hispida L., Sri Lanka,
Wanntorp & al. 2667 (S), EU543011
(4)
, EU543073
(4)
, EU543162
(4)
; S. prostrata Aubl., Colombia, Andersson & al. 2078 (GB),
EU543012(4), EU543163(4); S. remota (Lam.) Bacigalupo & Cabral, French Guiana, Andersson & al. 2016 (GB), EU543013(4),
EU543164(4); S. ruelliae DC., Gabon, Andersson & Nilsson 2296 (GB), EU543014(4), EU543074(4), EU543165(4). Stenaria (Raf.)
Terrell: S. nigricans (Lam.) Terrell, U.S.A., Yatskievych 96-92 (MO), EU543015
(4)
, AF333373
(2)
, EU543166
(4)
. Synaptantha Hook. f.:
S. tillaeacea (F. Muell.) Hook. f., Australia, Lazarides & Palmer 272 (K), EU543016
(4)
, EU543075
(4)
, EU543167
(4)
. Thecorchus
(Hiern) Bremek.: T. wauensis (Hiern) Bremek., Ethiopia, Friis & al. 2560 (UPS), EU543017(4), EU543076(4), EU543168(4).
Appendix. Continued.