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PHYLOGENY OF TRICALYSIA
(RUBIACEAE) AND ITS
RELATIONSHIPS WITH ALLIED
GENERA BASED ON PLASTID
DNA DATA: RESURRECTION OF
THE GENUS EMPOGONA
1
James Tosh,
2
Aaron P. Davis,
3
Steven Dessein,
4
Petra De Block,
4
Suzy Huysmans,
2
Mike F. Fay,
3
Erik Smets,
2
,
5
and Elmar Robbrecht
4
ABSTRACT
Recent studies on the circumscription of the tribe Coffeeae (Rubiaceae) revealed a weakly supported clade containing
Tricalysia A. Rich. and the allied genera Argocoffeopsis Lebrun, Calycosiphonia Pierre ex Robbr., Belonophora Hook. f.,
Diplospora DC., Discospermum Dalzell, Nostolachma T. Durand, and Xantonnea Pierre ex Pit. The phylogenetic relationships of
Tricalysia and these allied taxa are investigated further using sequence data from four plastid regions (trnL-F intron and
intergenic spacer, rpL16 intron, accD-psa1 intergenic spacer, and PetD). Our results demonstrate that Tricalysia sensu Robbrecht
is not monophyletic. The genus name Tricalysia should be restricted to taxa from subgenus Tricalysia; subgenus Empogona
(Hook. f.) Robbr. is sister to the genus Diplospora and is recognized at the generic level. The 34 necessary new combinations for
Empogona Hook. f. are provided: E. acidophylla (Robbr.) J. Tosh & Robbr., E. aequatoria (Robbr.) J. Tosh & Robbr., E. africana
(Sim) J. Tosh & Robbr., E. aulacosperma (Robbr.) J. Tosh & Robbr., E. bequaertii (De Wild.) J. Tosh & Robbr., E. bracteata
(Hiern) J. Tosh & Robbr., E. breteleri (Robbr.) J. Tosh & Robbr., E. buxifolia (Hiern) J. Tosh & Robbr. subsp. buxifolia, E.
buxifolia subsp. australis (Robbr.) J. Tosh & Robbr., E. cacondensis (Hiern) J. Tosh & Robbr., E. concolor (N. Halle
´
) J. Tosh &
Robbr., E. coriacea (Sond.) J. Tosh & Robbr., E. crepiniana (De Wild. & T. Durand) J. Tosh & Robbr., E. deightonii (Brenan) J.
Tosh & Robbr., E. discolor (Brenan) J. Tosh & Robbr., E. filiformistipulata (De Wild.) Bremek. subsp. filiformistipulata, E.
filiformistipulata subsp. epipsila (Robbr.) J. Tosh & Robbr., E. glabra (K. Schum.) J. Tosh & Robbr., E. gossweileri (S. Moore) J.
Tosh & Robbr., E. kirkii Hook. f. subsp. junodii (Schinz) J. Tosh & Robbr., E. lanceolata (Sond.) J. Tosh & Robbr., E. macrophylla
(K. Schum.) J. Tosh & Robbr., E. maputenis (Bridson & A. E. van Wyk) J. Tosh & Robbr., E. ngalaensis (Robbr.) J. Tosh &
Robbr., E. nogueirae (Robbr.) J. Tosh & Robbr., E. ovalifolia (Hiern) J. Tosh & Robbr. var. ovalifolia, E. ovalifolia var. glabrata
(Oliv.) J. Tosh & Robbr., E. ovalifolia var. taylorii (S. Moore) J. Tosh & Robbr., E. reflexa (Hutch.) J. Tosh & Robbr. var. reflexa,
E. reflexa var. ivorensis (Robbr.) J. Tosh & Robbr., E. ruandensis (Bremek.) J. Tosh & Robbr., E. somaliensis (Robbr.) J. Tosh &
Robbr., E. talbotii (Wernham) J. Tosh & Robbr., and E. welwitschii (K. Schum.) J. Tosh & Robbr.
Key words: accD-psal, Coffea, coffee, Coffeeae, Empogona, molecular systematics, petD, rpl16, Rubiaceae,
Tricalysia, trnL-F.
The genus Tricalysia A. Rich. is one of the largest
genera of Rubiaceae in Africa and occurs in
continental Africa (ca. 95 species), Madagascar (12
species), and the Comoros (one species). The genus
typically possesses the distinguishing characteristics
of the tribe Coffeeae (Bridson & Verdcourt, 2003;
Davis et al., 2007). These include axillary inflores-
cences paired at the nodes with obvious calyculi,
flowers with left contorted corolla aestivation and a
distinctly 2-lobed style, and relatively small and few-
seeded fleshy fruits. Most Tricalysia species can be
separated readily from other Coffeeae by the presence
of stipules with needlelike awns, truncate to distinctly
lobed calyces, and seeds with a shallow hilum.
Identification of Tricalysia at the species level is
notoriously difficult, as the genus contains a large
number of species across a broad geographic and
ecologic range, often separated by minor and
continuous characters.
In a series of papers, Robbrecht (1978, 1979, 1982,
1983, 1987) conducted a taxonomic revision of
Tricalysia, with later contributions by Ali and
1
James Tosh would like to acknowledge all members of the conservation genetics, molecular systematic, and Rubiaceae
research groups at the Royal Botanic Gardens, Kew, who provided help and support during my research visit in 2006. The
authors would also like to thank the reviewers of the paper for their helpful comments and suggestions. This research was
supported financially by grants from the Fund for Scientific Research–Flanders (FWO, G.0250.05 and G.0268.04).
2
Laboratory of Plant Systematics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, P.O. Box 2437, BE-3001
Leuven, Belgium. Corresponding author: james.tosh@bio.kuleuven.be.
3
Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AE, United Kingdom.
4
National Botanic Garden of Belgium, Domein van Bouchout, BE-1860 Meise, Belgium.
5
National Herbarium of The Netherlands, Leiden University Branch, P.O. Box 9514, NL-2300 RA Leiden, The Netherlands.
doi: 10.3417/2006202
ANN.MISSOURI BOT.GARD. 96: 194–213. PUBLISHED ON 23 APRIL 2009.
Robbrecht (1991) and Ranarivelo-Randriamboavonjy
et al. (2007). Robbrecht (1979, 1982, 1983, 1987)
recognized and revised two subgenera: subgenera
Tricalysia A. Rich with five sections (Probletostemon
(K. Schum.) Robbr., Tricalysia, Rosea (Klotzsch)
Robbr., Ephedranthera Robbr., and an unnamed
Madagascan section) and subgenus Empogona (Hook.
f.) Robbr. with two sections (Empogona Hook. f. and
Kraussiopsis Robbr.). Separation of the two subgenera
in Tricalysia was supported by differences in calyx
lobe morphology, corolla throat pubescence, fruit
color, and the presence/absence of a sterile append-
age on the anther connective.
Empogona Hook. f. was originally recognized at the
generic level by Hooker (1873) based on a single
Zambezian species, E. kirkii Hook. f. Brenan (1947)
reduced the genus Empogona to a section of
Tricalysia, containing six mainly eastern and south-
ern African species. During his revision of Tricalysia
and, in particular, his treatment of subgenus
Empogona, Robbrecht (1979) showed that ca. 20
other tropical African species, many of them with
Guineo-Congolian distribution, also belonged to this
subgenus.
Robbrecht (1978) also investigated the closely
related genus Neorosea N. Halle
´
, consisting of 17
species, many of which were formerly included in
Tricalysia. Two of these 17 species, including the type
species N. jasminiflora (Klotzsch) N. Halle
´
, proved to
be genuine Tricalysia species; a new genus, Ser-
icanthe Robbr., was described to accommodate the
remaining species (Robbrecht, 1978).
The close association between Diplospora DC. and
Discospermum Dalzell with Tricalysia has long been
recognized, with some authors (e.g., Schumann, 1891)
considering Diplospora and Tricalysia to be synony-
mous. Ali and Robbrecht (1991) broadly surveyed
Diplospora and Discospermum and enumerated a
whole suite of characters that could be used to
distinguish these Asian taxa from the closely related
African Tricalysia species. They also justified Diplo-
spora and Discospermum as separate genera on the
basis of fruit morphology.
The most recent taxonomic work on Tricalysia,by
Ranarivelo-Randriamboavonjy et al. (2007), focused
on the unnamed Madagascan section that was alluded
to, but not treated by, Robbrecht (1987). Of the 12
species of Tricalysia occurring in Madagascar, only
one species belongs to subgenus Empogona (T.
ovalifolia Hiern). The other 11 species, characterized
by the presence of unisexual flowers, belong to
subgenus Tricalysia. Ranarivelo-Randriamboavonjy
et al. (2007) observed that the Madagascan taxa could
be accommodated within section Tricalysia were it not
for the presence of unisexual flowers. As a result, they
formally placed these 11 taxa in Androgyne Robbr., a
new section within subgenus Tricalysia.
Recent phylogenetic investigations incorporating
morphological and molecular data sets have enabled us
to improve our understanding of the systematic position
of Tricalysia and its relationships with associated genera
(Andreasen & Bremer, 2000; Persson, 2000; Bridson &
Verdcourt, 2003; Robbrecht & Manen, 2006; Davis et
al., 2007). Andreasen and Bremer (2000) assessed tribal
and generic delimitation in subfamily Ixoroideae using
morphology, plastid and nuclear ribosomal DNA
sequences, and restriction site (restriction fragment
length polymorphism) data. Their results highlighted
the close affinity between Coffea L. and Psilanthus Hook.
f. (Coffeeae s. str.) and several members of the
Gardenieae subtribe Diplosporinae (Diplospora and
Tricalysia), resulting in an expanded circumscription of
the tribe Coffeeae to include Tricalysia, Diplospora,
Discospermum, Sericanthe, Coffea, Psilanthus,and
Bertiera Aubl. Bridson and Verdcourt (2003) further
enlarged and modified the concept of Coffeeae on the
basis of morphology and provisional plastid data
(provided by A. P. Davis, unpublished). In contrast to
the studies of Andreasen and Bremer (2000) and a
broader study of the Rubiaceae (Robbrecht & Manen,
2006), the genus Bertiera was excluded from Coffeeae
and placed in its own tribe, Bertiereae.
Davis et al. (2007) reexamined the circumscription
and phylogeny of Coffeeae and Bertiera using sequence
data from three plastid regions (trnL-F intron and
intergenic spacer, accD-psa1, and rpl16) in combina-
tion with morphological data. Their study confirmed
the placement of Tricalysia and related taxa (Seri-
canthe, Diplospora, and Discospermum) with Coffea and
Psilanthus, and expanded Coffeeae to include six other
genera (Argocoffeopsis Lebrun, Belonophora Hook. f.,
Nostolachma T. Durand, Calycosiphonia Pierre ex
Robbr., and Xantonnea Pierre ex Pit.). However, this
study only surveyed a limited number of Tricalysia
species, all of which belong to subgenus Tricalysia.
Bertiera was excluded from Coffeeae and retained in
Bertiereae, in agreement with Bridson and Verdcourt
(2003), and Gardenieae subtribe Diplosporinae was
placed in synonymy with Coffeeae.
The current investigation uses DNA sequence data
to test the monophyly of Tricalysia as currently
circumscribed and to assess the accuracy of the
subgeneric classification for the genus (Robbrecht,
1979, 1982, 1983, 1987). This is the first molecular
study to include widespread and representative
sampling of Tricalysia. In addition, we reassess the
phylogenetic relationships within the broadly circum-
scribed Coffeeae, with an expanded sampling from
both subgenera of Tricalysia. Given the wealth of
trnL-F, rpl16, and accD-psa1 sequence data already
Volume 96, Number 1 Tosh et al. 195
2009 Phylogeny of Tricalysia
Table 1. Summary of species from Tricalysia subgen. Empogona and subgen. Tricalysia sampled in this study (following
classification of Robbrecht, 1979, 1982, 1983, 1987).
A) Tricalysia subgen. Empogona (ca. 27 spp., Robbrecht, 1979)
Section Species group Species
Tricalysia sect. Empogona Hook. f. 12 spp. T. discolor group T. acidophylla Robbr.
sensu Robbrecht, 1979
T. junodii group T. junodii (Schinz) Brenan
T. ngalaensis Robbr.
T. ovalifolia Hiern
No known group affiliation within sect. T. concolor N. Halle
´
Empogona T. gossweileri S. Moore
Tricalysia sect. Kraussiopsis Robbr. 15 spp. T. crepiniana group T. bequaertii De Wild.
sensu Robbrecht, 1979
T. talbotii (Wernham) Keay
T. ruandensis group T. cacondensis Hiern
T. lanceolata (Sond.) Burtt Davy
T. ruandensis Bremek.
B) Tricalysia subgen. Tricalysia (ca. 75 spp., Robbrecht, 1982, 1983, 1987)
Section Species group Species
Tricalysia sect. Probletostemon (K. Schum.) T. anomala E. A. Bruce
Robbr. 4 spp. sensu Robbrecht, 1983
T. elliottii (K. Schum.) Hutch. &
Dalziel
Tricalysia sect. Ephedranthera Robbr. 9 spp. T. aciculiflora Robbr.
sensu Robbrecht, 1982
T. acocantheroides K. Schum.
T. bridsoniana Robbr.
Tricalysia sect. Tricalysia 40 spp. sensu T. angolensis group T. griseiflora K. Schum.
Robbrecht, 1987
Core group for sect. Tricalysia T. bagshawei S. Moore
T. coriacea (Benth.) Hiern
T. microphylla Hiern
T. okelensis Hiern
T. pallens Hiern
Tricalysia sect. Rosea (Klotzsch) Robbr. 9 spp.
sensu Robbrecht, 1987
T. jasminiflora (Klotzsch) Benth.
& Hook. f. ex Hiern
T. schliebenii Robbr.
Tricalysia sect. Androgyne Robbr. 11 spp. sensu
Ranarivelo-Randriamboavonjy et al., 2007
T. ambrensis Randriamb. & De
Block
T. analamazaotrensis Homolle ex
Randriamb. & De Block
T. cryptocalyx Baker
T. dauphinensis Randriamb. &
De Block
T. leucocarpa (Baill.) Randriamb.
& De Block
T. perrieri Homolle ex
Randriamb. & De Block
196 Annals of the
Missouri Botanical Garden
available for Coffeeae (Davis et al., 2007), we have
focused on these three plastid regions in the current
investigation and included further sequence data from
the plastid region petD.
M
ATERIALS AND METHODS
TAXON SAMPLING
An expanded sampling of Tricalysia, Diplospora,
Discospermum, Sericanthe, and Bertiera was combined
with sequence data generated by Davis et al. (2007).
Tricalysia samples representing both subgenera and
all of the seven sections of the genus (Robbrecht,
1979, 1982, 1983, 1987) were included in the
analyses (Table 1). Representative taxa from Ixoreae,
Octotropideae, and Gardenieae were selected as the
outgroup. A list of the 80 accessions used in the study
is given in Appendix 1.
DNA EXTRACTION, POLYMERASE CHAIN REACTION
AMPLIFICATION, AND SEQUENCING
Most DNA samples were obtained from silica gel
collections or, alternatively, from seed, flower, or leaf
samples taken from herbarium specimens (BR, K,
MO, WAG). A small number of DNA samples were
obtained from fresh leaf material collected from the
living collections of the National Botanic Garden of
Belgium.
For silica gel samples, DNA was isolated using a
modified DNA Mini Extraction Protocol (Royal
Botanic Gardens, Kew [K] protocol). DNA samples
were obtained from herbarium material using the 23
CTAB protocol of Doyle and Doyle (1987), with the
DNA subsequently purified using cesium chloride/
ethidium bromide gradients and concentrated by
dialysis before inclusion in the DNA Bank at K. All
DNA samples were purified using a NucleoSpin
purification column (Macherey-Nagel, Bethlehem,
Pennsylvania, U.S.A.) according to the manufacturer’s
instructions in order to remove any potential poly-
merase chain reaction (PCR) inhibitors.
Amplification of the trnL-F, rpl16, petD, and accD-
psa1 plastid regions was carried out using the primers
listed in Table 2. Amplification of the rpl16 region
was primarily carried out using the forward primer 71f
and the reverse primers 1661r (Jordan et al., 1996)
and 1516R (Shaw et al., 2005), although Coffeeae
specific internal primers designed by K were also
required for certain taxa (Davis et al., 2007).
All PCR and sequencing reactions were performed
using a Perkin Elmer (Waltham, Massachusetts,
U.S.A) GeneAmp 9700 Thermal Cycler machine.
Amplification of trnL-F was carried out using the
following profile: 94uC for 3 min.; 32 cycles of 94uC
for 1 min., 51uC for 1 min., 72uC for 2 min.; and a
final extension of 72uC for 7 min. accD-psa1 and rpl16
were amplified as follows: 94uC for 3 min.; 32 cycles
of 94uC for 1 min., 52uC for 1 min., 72uC for 1 min.
30 sec.; and a final extension of 72uC for 7 min.
Amplification of petD was carried out as follows: 96uC
for 2 min.; 34 cycles of 94uC for 1 min., 50uC for
1 min., 72uC for 1 min. 30 sec.; and a final extension
of 72uC for 10 min.
For the trnL-F, petD, and rpl16 regions, 25
ml PCR
reactions were made using a commercial PCR master
mix (2.5 mM MgCl
2
ReddyMix; ABgene; Epsom,
Surrey, U.K.). accD-psa1 did not amplify successfully
with the commercial master mix, so 25
ml PCR master
mixes were prepared using Biotaq DNA polymerase
(Bioline, London, U.K.), 2.5
mlof103 NH
4
reaction
buffer (Bioline), 1.5
ml of 50 mM MgCl
2
, and 2.5 mlof
dNTPs (Promega, Madison, Wisconsin, U.S.A.). All
amplified PCR products were purified using Nucleo-
Spin purification columns following the manufactur-
er’s protocol.
Cycle sequencing reactions were carried out using
BigDye Terminator Mix version 3.1 (Applied Biosys-
tems, Inc., Warrington, Cheshire, U.K.). The cycle
Table 2. Amplification primers used in this study.
Region Primer Primer sequence (59-39) Reference
trnL-F Forward (c) CGA AAT CGG TAG ACG CTA CG Taberlet et al., 1991
Reverse (f) AAT TGA ACT GGT GAC ACG AG
rpl16 Forward (71f) GCT ATG CTT AGT GTG TGA CTC GTT G Jordan et al., 1996
Reverse (1661r) CGT ACC CAT ATT TTT CCA CCA CGA C
Reverse (1516r) CCC TTC ATT CTT CCT CTA TGT TG Shaw et al., 2005
Internal forward GTA AGA AGT GAT GGG AAC GA Davis et al., 2007
Internal reverse TCG TTC CCA TCA CTT CTT AC
accD-psa1 Forward (769 F) GGA AGT TTG AGC TTT ATG CAA ATG Mendenhall, 1994
Reverse (75 R) AGA AGC CAT TGC AAT TGC CGG AAA
petD Forward (1365) TTG ACY CGT TTT TAT AGT TTA C Lo
¨
hne & Borsch, 2004
Reverse (738) AAT TTA GCY CTT AAT ACA GG
Volume 96, Number 1 Tosh et al. 197
2009 Phylogeny of Tricalysia
sequence reaction consisted of 26 cycles of 10 sec. at
96uC, 5 sec. at 50uC, and 4 min. at 60uC. Cycle
sequencing products were cleaned with the MagneSil
Clean-Up System (Promega) using an automated robot
(Biomek NX S8; Beckman Coulter, High Wycombe,
Buckinghamshire, U.K.). Analysis of cycle sequenc-
ing products was performed using an AB 3730 DNA
Analyzer (Applied Biosystems). In addition, a number
of the trnL-F and petD samples were sent to Macrogen
(Seoul, South Korea) for sequencing.
ALIGNMENT AND GAP CODING
Sequences were assembled and edited using the
Staden software package (Staden et al., 1998). All
sequences were aligned manually in MacClade
(version 4.04, Maddison & Maddison, 2002). Low
levels of sequence variation enabled sequences to be
aligned without difficulty. Regions of ambiguous
alignment, such as the beginning and end of
sequences, were removed. The edited sequences were
analyzed with gaps treated as missing data and
phylogenetically informative indels (insertions and/or
deletions) coded according to the ‘‘simple indel
coding’’ method of Simmons and Ochoterena (2000).
PHYLOGENETIC ANALYSES
Phylogenetic analyses were performed on the four
separate plastid data sets in addition to the combined
four-region plastid matrix.
Maximum parsimony. Heuristic tree searches were
carried out in PAUP* version 4.0b10 (Swofford, 2003)
using 10,000 replicates of random taxon sequence
addition, holding 10 trees at each step, with tree
bisection-reconnection (TBR) branch swapping,
delayed transformation (DELTRAN) optimization,
and MULTREES in effect, and saving no more than
10 trees per replicate. Support values for clades
recovered in the analyses were estimated using
bootstrap analysis (Felsenstein, 1985). One thousand
replicates of simple sequence addition, TBR swap-
ping, and saving 10 trees per replicate were performed
in PAUP*. We interpreted bootstrap values greater
than 85% as being well supported, 75%–84% as
being moderately supported, and 50%–74% as having
low support.
Bayesian inference. Evolutionary models for each
plastid region were selected using Modeltest v3.06
(Posada & Crandall, 1998) under the Akaike
information criterion. In the case of accD-psa1, petD,
and rpl16, the nucleotide substitution model that best
fits the data was HKY + I + G. The HKY + I model
was selected for the trnL-F sequence matrix. The
combined data set was partitioned into five discrete
units. In addition to the four plastid regions, there was
a fifth partition for the phylogenetically informative
indels. The restriction site (binary) model of evolution
was implemented for the indel data, following the
recommendation of Ronquist et al. (2005). Four
independent Bayesian searches, each consisting of
two simultaneous parallel analyses, were carried out
using MrBayes 3.1 (Huelsenbeck & Ronquist, 2001).
In each Bayesian analysis, four Markov chains (three
heated, one cold) were run simultaneously for
2,000,000 generations, sampling trees every 100
generations. The initial 25% of trees were discarded
as a conservative burn-in. After confirming by eye that
trees generated from separate analyses had consistent
topologies, the ‘‘post-burn-in’’ trees from each analysis
were pooled together, imported into PAUP* version
4.0b10 (Swofford, 2003), and summarized by majority
rule consensus, with values on the tree equating to
posterior probabilities (PP).
R
ESULTS
This study generated 229 sequences, which were
combined with the 75 sequences obtained by Davis et
al. (2007). In total, this study included 79 accD-psa1
sequences (53 newly generated), 80 trnL-F sequences
(54 newly generated), 78 rpl16 sequences (55 newly
generated), and 67 petD sequences (all newly
generated). The rpl16 region proved to be the most
problematic region to amplify, due in part to two poly-
A stretches (one 373 bp from the 59 end, the other
466 bp from the 39 end). As a result, it was often
difficult to obtain sufficient overlap during sequence
assembly. Internal primers, designed specifically for
Coffeeae taxa (Davis et al., 2007), were used to obtain
a complete sequence for rpl16 in problematic taxa.
In general, the amount of genetic variability in all
plastid regions was low (Table 3). A large proportion
of the total genetic variation occurred between the
ingroup (Coffeeae) taxa and outgroup (other Ixoroi-
deae). We observed considerable length variability in
the accD-psa1 region. As with all the plastid regions
investigated, accD-psa1 is particularly AT-rich and
subject to several repeat units, giving rise to a number
of potentially phylogenetically informative indels. In
the case of Tricalysia subgen. Empogona, all taxa
included in the study share a 250 bp deletion in the
accD-psa1 region. Less length variation was observed
in petD, rpl16, and trnL-F. The gross tree topologies of
all four individual analyses were examined by eye and
found to be topologically consistent, and the four data
sets were subsequently combined in all further
analyses.
198 Annals of the
Missouri Botanical Garden
The aligned combined matrix had a total length of
4465 bp. There were 669 variable characters and, of
these, 352 characters were parsimony informative
(7.9% of total number of characters). In total, the
matrix contained 50 parsimony informative indels,
consisting of repeat sequences in addition to insertion/
deletion events. Exclusion of outgroup taxa (Ixoreae,
Gardenieae, Octotropideae, and Bertierieae) revealed
211 parsimony informative characters within Coffeeae.
PHYLOGENETIC RESULTS
The heuristic maximum parsimony (MP) analysis of
the combined plastid data matrix generated 8853 most
parsimonious trees with a length of 929 steps, a
consistency index (CI) of 0.816, and a retention index
(RI) of 0.908. Table 3 summarizes the tree statistics
for the individual and combined analyses.
The topologies of the MP strict consensus tree and
the Bayesian majority rule tree (Fig. 1) were consis-
tent with each other. Figure 2 displays one of the most
parsimonious trees and indicates both bootstrap
support (BS) and branch length. Both MP and
Bayesian analyses reconfirm the monophyly of the
ingroup (BS 99%, PP 1.00). Bertiera, here represented
by its two subgenera, is monophyletic (BS 100%,PP
1.00) and is sister to the ingroup (BS 79%, PP 1.00).
The clade of Coffea and Psilanthus is well
supported (BS 100%, PP 1.00) and is sister to the
remaining ingroup taxa (BS 93%, PP 1.00). There is
also strong support for the clade of Argocoffeopsis and
Calycosiphonia (BS 99%, PP 1.00). The sister
relationship of Calycosiphonia and Argocoffeopsis to
the rest of the ingroup receives weak bootstrap support
(BS 50%), but is supported by a PP of 0.98.
Both MP and Bayesian analyses recovered a clade
including Sericanthe, Diplospora, Discopermum, and
Tricalysia subgen. Empogona. Although there is no
bootstrap support for this clade (BS , 50%), the clade
does receive support in the Bayesian analyses (PP
0.98). Within this clade, there is strong support for the
monophyly of Sericanthe (BS 99%, PP 1.00), Dis-
cospermum (BS 100%, PP 1.00), and the group of
Diplospora and Tricalysia subgen. Empogona (BS
99%, PP 1.00). The monophyly of both Diplospora (BS
90%, PP 1.00) and Tricalysia subgen. Empogona (BS
98%, PP 1.00) is confirmed. Within Tricalysia
subgen. Empogona, two groups receive high levels
of support: the group of T. cacondensis Hiern, T.
lanceolata (Sond.) Burtt Davy, and T. ruandensis
Bremek. (BS 85%, PP 1.00); and the group of T.
junodii (Schinz) Brenan, T. ovalifolia, and T. acid-
ophylla Robbr. (BS 98%, PP 1.00).
The clade of Belonophora and Tricalysia subgen.
Tricalysia is present in both the MP strict consensus
Table 3. Characteristics of accD-psa1, rpl16, petD, trnL-F, and combined data sets and tree statistics.
accD-psa1 rpl16 petD trnL-F Combined plastid
No. of taxa 79 78 67 80 80
Total length (base pairs) 1255 1207 1064 889 4415
Sequence length variation 737–1061 995–1068 937–966 772–822 —
No. of constant characters 1075 982 974 765 3796
No. of phylogenetically informative indels 22 11 8 9 50
No. of variable PI characters (% of total characters) 117 (9.3) 116 (9.6) 45 (4.2) 74 (8.3) 352 (7.9)
Tree length 283 339 123 174 929
Consistency index 0.827 0.814 0.854 0.822 0.816
Retention index 0.923 0.890 0.937 0.916 0.908
No. of trees saved 9920 1056 1392 9990 8853
Volume 96, Number 1 Tosh et al. 199
2009 Phylogeny of Tricalysia
Figure 1. Maximum parsimony strict consensus/Bayesian majority rule consensus tree. Bayesian posterior probabilities
are indicated above branches. Sectional groupings are annotated after species names: AND, Tricalysia sect. Androgyne; EMP,
Tricalysia sect. Empogona; EPH, Tricalysia sect. Ephedranthera; KRA, Tricalysia sect. Kraussiopsis; PRO, Tricalysia sect.
Probletostemon; ROS, Tricalysia sect. Rosea; TRI, Tricalysia sect. Tricalysia. See Table 1 for species authorities
and provenance.
200 Annals of the
Missouri Botanical Garden
tree and the Bayesian majority rule tree, although
there is negligible support for this clade (BS , 50%,
PP 0.82). However, the monophyly of Belonophora (BS
100%, PP 1.00) and Tricalysia subgen. Tricalysia (BS
99%, PP 1.00) is strongly supported. Within Trica-
lysia subgen. Tricalysia, several groups receive strong
support: the group of T. elliottii (K. Schum.) Hutch. &
Dalziel and T. anomala E. A. Bruce (BS 95%,PP
1.00), and a group of predominantly Madagascan taxa
with the inclusion of T. jasminiflora (Klotzsch) Benth.
& Hook. f. ex Hiern (BS 97%, PP 1.00). There is also
moderate bootstrap (BS 75%) and high PP (PP 1.00)
for the clade of T. acocantheroides K. Schum., T.
griseiflora K. Schum., T. bridsoniana Robbr., T.
Figure 2. One of the 8853 most parsimonious trees generated in the maximum parsimony analysis. Bootstrap values .
50% are indicated above branches, and selected branch lengths are indicated below branches. Sectional groupings are
annotated after species names: AND, Tricalysia sect. Androgyne; EMP, Tricalysia sect. Empogona; EPH, Tricalysia sect.
Ephedranthera; KRA, Tricalysia sect. Kraussiopsis; ROS, Tricalysia sect. Rosea; PRO, Tricalysia sect. Probletostemon; TRI,
Tricalysia sect. Tricalysia. See Table 1 for species authorities and provenance.
Volume 96, Number 1 Tosh et al. 201
2009 Phylogeny of Tricalysia
microphylla Hiern, T. schliebenii Robbr., and the
aforementioned Madagascan group together with T.
jasminiflora.
D
ISCUSSION
Previous taxonomic work on Tricalysia has focused
on the use of traditional morphological and anatomical
characters to infer relationships within the genus. In
the most recent classification of the genus, Robbrecht
(1979, 1982, 1983, 1987) subdivided it into two
subgenera and seven sections. Here, for the first time,
we have addressed relationships in this group using
molecular data.
In the current investigation, we obtained sequence
data from four plastid regions for both subgenera and
all seven sections of Tricalysia and generated
estimates of phylogeny using both MP and Bayesian
inference methods. The consensus tree topologies of
both analyses (strict consensus for MP, majority rule
consensus for Bayesian) were consistent. As is often
observed, Bayesian PP were higher than bootstrap
support values for any given node (Huelsenbeck et al.,
2002; Erixon et al., 2003; Randle et al., 2005).
TESTING THE MONOPHYLY OF THE GENUS TRICALYSIA
Our phylogenetic analyses indicate that Tricalysia,
as currently circumscribed, is not monophyletic. The
monophyly of subgenera Tricalysia and Empogona is
confirmed, but they are not sister to each other. This
represents a new, though perhaps unsurprising,
observation, which has implications for the taxonomy
of the group (see below).
Davis et al. (2007) included five species of
Tricalysia in their molecular and morphological
reassessment of the circumscription and phylogeny
of Coffeeae. All five species were representatives of
subgenus Tricalysia. In both their combined molec-
ular and combined morphological-molecular phylog-
enies, Tricalysia (subgen. Tricalysia) was placed in a
poorly supported and unresolved clade containing
Sericanthe, Belonophora, and an Asian clade (includ-
ing Diplospora and Discospermum). The study of Davis
et al. (2007) incorporated molecular data from three
plastid regions (trnL-F, accD-psa1, and rpl16). In our
investigation, we included an additional plastid
region, the group II intron petD. The extra characters
provided by this fourth plastid marker were still not
sufficient to fully elucidate systematic relationships
within the clade containing Tricalysia subgen.
Tricalysia, Sericanthe, Belonophora, Diplospora, and
Discospermum.
The inclusion of taxa from Tricalysia subgen.
Empogona led to results conflicting with the study
of Davis et al. (2007). First, we did not recover an
Asian clade. Instead, Diplospora formed a well-
supported monophyletic group with Tricalysia subgen.
Empogona (BS 99%, PP 1.00). Second, both the MP
strict consensus tree and the Bayesian majority rule
consensus tree indicated sister relationships between
Tricalysia subgen. Tricalysia and Belonophora, and
recovered a clade containing Sericanthe, Discosper-
mum, Diplospora, and Tricalysia subgen. Empogona.
The clade of Belonophora and subgenus Tricalysia
received poor internal support (BS , 50%, PP 0.82),
but there was support for the second clade in the
Bayesian analysis (BS , 50%, PP 0.98).
TAXONOMIC IMPLICATIONS FOR GENERIC CONCEPTS
The revelation that Tricalysia sensu Robbrecht is
not monophyletic calls for a reconsideration of the
taxonomic delimitation of Tricalysia and closely
related taxa. One taxonomic option would be to widen
the genus Tricalysia to include Belonophora, Diplo-
spora, Discospermum, and Sericanthe. However, these
genera are easily identified (e.g., by the use of a key)
and are so diverse morphologically and anatomically
that consolidating them into one genus does not seem
justified (Table 4). A more logical option would be to
separate these taxa into groups at the generic level,
based on morphological and molecular synapomor-
phies.
Robbrecht (1979) enumerated four potential field
characters that distinguish the subgenera Empogona
and Tricalysia. Taxa of subgenus Empogona are
identified by the presence of distinctly lobed calyces
(vs. short and truncate in subgenus Tricalysia),
densely pubescent corolla throats (vs. glabrous to
sparsely hairy), the presence of a large flattened
sterile appendage protruding from the anther connec-
tive (vs. blunt anthers, occasionally forming a short
triangular appendage), and fruits that turn black at
maturity (vs. red fruits). Robbrecht (1979) considered
recognizing Empogona at the generic rank, but opted
to incorporate it as a subgenus of Tricalysia, given the
similarity in a number of other key characters
(placentation, pollen morphology, fruit and seed
morphology, and seed coat anatomy). This decision
was also pragmatic in terms of taxonomic stability, as
it required the fewest nomenclatural changes (Rob-
brecht, 1979).
The revision of Sericanthe (Robbrecht, 1978) and
the survey of the Asian relatives of Tricalysia (Ali &
Robbrecht, 1991) provided ample morphological and
anatomical evidence to justify the exclusion of these
genera from Tricalysia. The genus Sericanthe is
distinguished from Tricalysia by the presence of
bacterial leaf galls (rare in Rubiaceae), wing-shaped
202 Annals of the
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colleters, and pollen with a verrucate sexine (in
contrast to the reticulate sexine occurring in all other
members of Coffeeae). Davis et al. (2007) also
presented the following synapomorphic characters
for the genus: 7- to 9-merous flowers, distinctly
basifixed anthers, and horizontal micropyle orienta-
tion.
Diplospora and Discospermum consistently have
tetramerous flowers, which occur only rarely in
African Tricalysia, and the flowers of Asian taxa are
smaller than their African counterparts (Ali &
Robbrecht, 1991). In addition, there is a strong
tendency toward unisexual flowers in Asian taxa, a
trait that is absent in all but a few representatives of
Tricalysia confined to Madagascar (Ranarivelo-Ran-
driamboavonjy et al., 2007). Ali and Robbrecht (1991)
also justified maintaining Diplospora and Discosper-
mum as separate genera on the basis of rather
divergent fruit types (small, fleshy, and red fruits in
Diplospora and large, leathery, and purplish black
fruits in Discospermum). The decision to maintain
Diplospora and Discospermum as separate genera is
also supported by our molecular analyses.
The tribal position of Belonophora has been fairly
unstable since its initial description by Hooker
(1873), partly due to the erroneous observation by
Hooker that Belonophora possesses a solitary, pendu-
lous ovule in each of the two locules. Keay (1958)
observed that Belonophora species actually possess
two collateral ovules per locule, on the inner surface
of a pendulous placenta, but he felt it premature to
assign the genus to a new tribe until a more
satisfactory tribal classification within Rubiaceae
had been proposed. Robbrecht and Puff (1986)
tentatively placed Belonophora in the tribe Aulaco-
calyceae, although the axillary inflorescences of
Belonophora contrasted with the terminal or subter-
minal inflorescences possessed by other members of
the tribe. The placement of Belonophora in the tribe
Coffeeae was first proposed by Bridson and Verdcourt
(2003) and later supported by the study of Davis et al.
(2007). The imbricate calyx lobes of Belonophora were
synapomorphic for the genus in the study of Davis et
al. (2007), and the genus is also distinguished from
other members of Coffeeae by the presence of a
superior embryo radicle (Cheek & Dawson, 2000).
In light of evidence from our own molecular
investigation, and in combination with morphological
and anatomical observations reported elsewhere, we
believe it is appropriate and fully justified to
recognize Empogona (sensu Robbrecht, 1979) at
generic rank. The necessary taxonomic changes for
the inclusion of many former Tricalysia species in the
genus Empogona are provided at the end of the
Discussion.
RECOGNITION OF INTRAGENERIC GROUPS IN TRICALYSIA
In addition to testing the monophyly of Tricalysia
sensu Robbrecht, we were able to assess the levels of
support for his sectional groups within the genus. All
seven sections were sampled in our analysis, although
some were better represented. Low levels of genetic
variation between species limited the amount of
resolution between taxa, but there are some provi-
sional findings from this study.
Tricalysia subgen. Tricalysia was subdivided into
five sections by Robbrecht (1982, 1983, 1987).
Tricalysia sect. Probletostemon, here represented in
our molecular study by T. elliottii and T. anomala
(Table 1), was thought to possess many morphological
and anatomical features regarded as primitive for the
group. These included free bracteoles, standard
colleters (Robbrecht, 1988), large pleiomerous flowers
with many ovules per placenta, and large fruits
(Robbrecht, 1983). Our study confirms the monophyly
of section Probletostemon (BS 95%, PP 1.00), but it
remains unresolved in a basal polytomy.
Tricalysia sect. Ephedranthera, here represented by
three species, is characterized by the presence of
anthers that are sessile in the corolla throat and
partly included within the corolla tube (Robbrecht,
1982). The monophyly of this section is not supported
in our investigation. Tricalysia aciculiflora Robbr.
falls within the basal polytomy, whereas T. aco-
cantheroides and T. bridsoniana are situated within
the moderately to well-supported clade (BS 75%,PP
1.00) containing all the remaining taxa of subgenus
Tricalysia.
The other three sections (Tricalysia, Rosea, and
Androgyne) are very similar morphologically. Most
species in subgenus Tricalysia belong to section
Tricalysia, which Robbrecht (1987) further subdivid-
ed into four informal groups. Only two of these
informal groups are included in this investigation. The
core group of taxa within section Tricalysia, here
represented by T. coriacea (Benth.) Hiern and the
weakly supported clade of T. pallens Hiern, T.
okelensis Hiern, and T. bagshawei S. Moore, is
unresolved in the basal polytomy. The group of T.
angolensis A. Rich. ex DC., represented by T.
griseiflora K. Schum., falls within the clade containing
T. bridsoniana, T. microphylla, and representatives
from sections Rosea and Androgyne.
In section Rosea, species differ conspicuously from
those in section Tricalysia due to the presence of a
spathaceous calyx (Robbrecht, 1987). In section
Androgyne, which comprises the Madagascan repre-
sentatives of subgenus Tricalysia, species are char-
acterized by the presence of unisexual flowers. There
is weak bootstrap and significant Bayesian support
Volume 96, Number 1 Tosh et al. 203
2009 Phylogeny of Tricalysia
Table 4. Salient morphological characters of Tricalysia and close relatives. Characters in boldface represent unique features for Empogona. Figures in single parentheses 5 rarely; figures in
double parentheses 5 very rarely.
Discospermum
(ca. 7 spp.)
Diplospora
(ca. 10 spp.)
Empogona
(29 spp.)
Tricalysia, excluding
sect. Androgyne
(ca. 80 spp.)
Tricalysia sect.
Androgyne
(11 spp.)
Belonophora
(5 spp.)
Sericanthe
(ca. 20 spp.)
Bracts and
bracteoles
free or fused into calyculi free or fused into
calyculi
free alternate, sect.
Empogona; fused into
calyculi, sect.
Kraussiopsis
fused into calyculi;
free alternate in
sect.
Probletostemon
fused into
calyculi
lower bracts fused into
calyculi; upper bracts
free, opposite
fused into calyculi
Corolla length
(mm)
8–15 5–10 (6–)8–17 8–50 5–10 (10–)20–30(–40) (8–)12–25
Flower
organization
hermaphroditic or unisexual hermaphroditic or
unisexual
hermaphroditic hermaphroditic
a
unisexual hermaphroditic;
heterostyly
hermaphroditic
Merosity 4 4–((–5)) ((4–))5(–6) (4–)5–6(–12) 4–7 ((4–))5 (5–)7–8(–9)
Calyx tube short, rounded lobes
present or absent
tube short, lobes
mostly triangular
tube short, lobes well-
developed and
often overlapping
tube well-developed;
lobes none,
triangular or
linear
tube well-
developed,
with minute
teeth
tube short, lobes well-
developed
tube well-
developed, with
minute teeth
Corolla throat glabrous to bearded glabrous or hairy densely bearded
b
glabrous to hairy hairy glabrous glabrous or hairy
Anthers medifixed; on short
filaments in throat,
exserted
medifixed; on short
filaments in throat,
exserted
medifixed; on long
filaments in throat,
exserted
medifixed; on long
filaments in
throat, exserted
medifixed; on
short
filaments in
throat or
sessile,
exserted
medifixed; sessile in
tube, included
basifixed; on short
filaments in
throat, exserted
Anther
connective
sometimes protruding in very
short triangle
sometimes protruding
in very short
triangle
protruding in
conspicuous
ribbon-like
appendages
c
mostly protruding in
short triangle
short apical
appendage
protruding in short
triangle
strongly flattened;
no appendage
Placentation (3–)5–15 ovules on a hemi-
circular to 6 hemi-ellipsoid
placenta; attached to the
middle of the septum
1–3(–6) ovules on a
hemi-circular to
6 hemi-ellipsoid
placenta; attached
to the middle of
the septum
1–ca. 25 ovules on a
hemi-circular to 6
hemi-ellipsoid
placenta; attached to
the middle of the
septum
1–12 ovules on a
hemi-circular to
6 hemi-ellipsoid
placenta; attached
to the middle of
the septum
2–8 ovules on a
hemi-circular
to 6 hemi-
ellipsoid
placenta;
attached to
the middle of
the septum
2 collateral ovules on
inner face of a
pendulous hemi-
circular 6 hemi-
ellipsoid placenta;
attached to the apex
of the septum
(1–)2(–5) ovules on
a hemi-circular
6 hemi-
ellipsoid
placenta;
attached to the
apex of the
septum
Fruit (mm) 20–30 5–7 8–10 5–20 5–9 10–15 10–30
204 Annals of the
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(BS 61%, PP 0.98) for a clade containing these two
sections. Tricalysia schliebenii (section Rosea) is sister
to a strongly supported clade (BS 97%, PP 1.00)
containing members of section Androgyne and T.
jasminiflora of section Rosea.
Robbrecht (1979) recognized two sections within
subgenus Empogona: section Empogona is character-
ized by free bracteoles and distinct non-overlapping
calyx lobes; in contrast, the bracteoles in section
Kraussiopsis are fused to form calyculi, and the calyx
lobes either touch or overlap each other (with the
exception of Tricalysia bequaertii De Wild., where the
calyx lobes are not touching). Tricalysia ngalaensis
Robbr., previously thought to be closely related to T.
junodii (Schinz) Brenan (Robbrecht, 1979), is in an
unresolved position (Figs. 1, 2). There is weak
bootstrap but significant Bayesian support for the
monophyly of section Kraussiopsis (BS 56%, PP 0.99),
and the informal group of T. ruandensis is also well
supported (BS 85%, PP 100). The remaining taxa of
section Empogona are weakly supported (BS 60%,PP
0.96), although the clade of T. junodii, T. ovalifolia,
and T. acidophylla is well supported (BS 98%,PP
1.00).
OTHER RELATIONSHIPS WITHIN COFFEEAE AND THE
RELATIONSHIP TO BERTIERA
The sister relationship of Bertiera and Coffeeae is
recovered with moderate bootstrap and significant
Bayesian support (BS 79%, PP 100), although our
outgroup sampling is not complete. In order to confirm
this result, more extensive sampling of representative
groups within subfamily Ixoroideae is needed. Rob-
brecht and Manen (2006) opted to place Bertiera in
subtribe Bertierinae, sister to Coffeinae, as the
characteristic features of Bertiera differ from those of
Coffeeae. Davis et al. (2007) found only weak bootstrap
support for the sister relationship between Bertiera and
Coffeeae (BS , 40%) based on molecular data alone,
and the sister relationship was not recovered following
the addition of morphological characters in their
combined molecular-morphological analysis. Based
on the decision of Bridson and Verdcourt (2003), they
opted to place Bertiera in the monogeneric tribe
Bertiereae. Whether Bertiera is recognized at the tribal
or subtribal level is still open to debate, but we agree
with Davis et al. (2007: 321) that ‘‘Coffeeae, with the
addition of new genera and the removal of Bertiera,is
both scientifically coherent and practical.’’
In the three-region plastid analysis of Davis et al.
(2007), Coffea and Psilanthus form a well-supported
monophyletic clade supported by a bootstrap of 87%,
and are placed sister to the rest of Coffeeae. This
relationship is recovered in our four-region analysis,
Discospermum
(ca. 7 spp.)
Diplospora
(ca. 10 spp.)
Empogona
(29 spp.)
Tricalysia, excluding
sect. Androgyne
(ca. 80 spp.)
Tricalysia sect.
Androgyne
(11 spp.)
Belonophora
(5 spp.)
Sericanthe
(ca. 20 spp.)
Fruit color purplish black turning from yellow
and orange to red
first white, turning
purple, then black
red, rarely orange red yellow orange
Pericarp sclerified or leathery fleshy fleshy fleshy; rarely
sclerotic, sect.
Probletostemon
fleshy fleshy fleshy
Placental
outgrowth
massive, mostly surrounding
seeds
mostly none none, with weak
outgrowths in some
spp.
mostly none none massive, surrounding
seeds
mostly none;
massive in some
spp.
Endosperm entire, ruminate in some spp. entire or ruminate entire, ruminate in some
spp.
entire entire entire entire
Embryo radicle away from septum inferior inferior inferior inferior superior lateral
a
Heterostyly in section Ephedranthera.
b
Glabrous in a few species, e.g., Empogona concolor.
c
Some species with an inconspicuous appendix, e.g., Empogona welwitschii.
Table 4. Continued.
Volume 96, Number 1 Tosh et al. 205
2009 Phylogeny of Tricalysia
with increased support values (BS 93%, PP 1.00).
There was also strong support for the sister relation-
ship between the well-supported Argocoffeopsis and
Calycosiphonia clade and the remaining ingroup taxa
in our Bayesian analysis (PP 0.98), but weak support
for this relationship in the MP analysis (BS 50%).
This relationship was also recovered in the strict
consensus tree of Davis et al. (2007).
TAXONOMIC NOVELTIES RESULTING FROM THE GENERIC
RESURRECTION OF EMPOGONA
An outline of an emended infrageneric classifica-
tion for Empogona is provided below. It contains a
formal new combination for one of the two sections
recognized. The outline is followed by a checklist of
species, providing all necessary new combinations at
the species level and below.
O
UTLINE OF AN EMENDED CLASSIFICATION FOR EMPOGONA
Empogona Hook. f., Hooker’s Icon. Pl. 11: 72, t.
1091. 1871. TYPE: Empogona kirkii Hook. f.
Tricalysia subgen. Empogona (Hook. f.) Robbr., Bull. Jard.
Bot. Natl. Belg. 49: 259. 1979.
The further synonymy of subgenus Empogona
(Robbrecht, 1979: 259) remains applicable to the
genus Empogona.
Empogona Hook. f. sect. Empogona. Tricalysia
subgen. Empogona (Hook. f.) Robbr. sect.
Empogona (Hook. f.) Brenan.
EMPOGONA KIRKII SPECIES GROUP
This corresponds to the group of Tricalysia junodii
(Robbrecht, 1979: 269). The group comprises the
species numbered 11, 18, 22, and 24 in the checklist
below. The position of Empogona ngalaensis (species
22 below) was not confirmed by our molecular
analysis.
EMPOGONA DISCOLOR SPECIES GROUP
This corresponds to the group of Tricalysia dis-
color (Robbrecht, 1979: 292). The group com-
prises the species numbered 1, 4, 6, and 14 in
the checklist below. The group is only represented
by Empogona acidophylla in the analysis, which
falls in a clade corresponding to the previous species
group.
Section Empogona further comprises the three
species numbered 10, 17, and 25 in the checklist
below. They were considered to be of isolated position
(Robbrecht, 1979: 300). Two of these species (10. E.
concolor and 17. E. gossweileri) are included in the
analysis. They have a basal position in the clade
corresponding to section Empogona.
Empogona sect. Kraussiopsis (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia subgen.
Empogona sect. Kraussiopsis Robbr., Bull. Jard.
Bot. Natl. Belg. 49: 309. 1979. TYPE: Empogona
crepiniana (De Wild. & T. Durand) J. Tosh &
Robbr.
EMPOGONA RUANDENSIS SPECIES GROUP
This corresponds to the group of Tricalysia
ruandensis (Robbrecht, 1979: 310). The group com-
prises the species numbered 8, 9, 19, 26, and 27 in
the checklist below.
EMPOGONA GLABRA SPECIES GROUP
This corresponds to the group of Tricalysia glabra
(Robbrecht, 1979: 292). This small group comprises
only two species, numbers 16 and 23 in the checklist
below.
EMPOGONA CREPINIANA SPECIES GROUP
This corresponds to the group of Tricalysia
crepiniana (Robbrecht, 1979: 329). It is the most
speciose group comprising 11 species, numbered 2, 3,
5, 7, 12, 13, 15, 20, 21, 28, and 29 of the checklist
below.
C
HECKLIST OF SPECIES AND INFRASPECIFIC TAXA,
I
NCLUDING TAXONOMIC NOVELTIES
The list below, ordered alphabetically, enumerates
all known taxa of Empogona, including the four
species (species numbered 3, 7, 21, and 27 below)
treated or described after Robbrecht’s (1979) revision.
The infrageneric assignment of the species is given in
the preceding section of the present paper. Taxa
preceeded by an asterisk (*) were included in the
molecular analysis (see Table 1).
The checklist includes taxonomic novelties for all
species, i.e., 34 new combinations and three modifi-
cations of infraspecific status. In his revision,
Robbrecht (1979) used varietal status for all infra-
specific taxa recognized. Here we reconsider the
appropriateness of that treatment in applying du
Rietz’s criteria (as cited in Stace, 1991) for distin-
guishing subspecies and varieties. Therefore, when
infraspecific taxa are allopatric and differing in
several features, we propose subspecific rather than
varietal status.
(*) 1. Empogona acidophylla (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia acid-
206 Annals of the
Missouri Botanical Garden
ophylla Robbr., Bull. Jard. Bot. Natl. Belg. 49:
292. 1979. TYPE: Tanzania. Eastern Usambaras,
2 mi. E of Sigi railway station, 27 July 1953, R.
B. Drummond & J. H. Hemsley 3490 (holotype,
K!; isotypes, B!, BR!, LISU!, S!).
2. Empogona aequatoria (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia aequa-
toria Robbr., Bull. Jard. Bot. Natl. Belg. 48: 465.
1978. TYPE: [Democratic Republic of the Congo.]
Congo belge. Yangambi, 4 Dec. 1937, J. Louis
6887 (holotype, BR!; isotypes, B!, BR!, C!,
COI!, EA!, HBG!, K!, MO!, P!, PRE!, UPS!,
WAG!).
3. Empogona africana (Sim) J. Tosh & Robbr.,
comb. nov. Basionym: Diplospora africana Sim,
Forest Fl. Cape, 238. 1907. Tricalysia africana
(Sim) Robbr., S. African J. Bot. 51: 331. 1985.
TYPE: South Africa. E Pondoland, Egossa Forest,
Aug. 1899, T. R. Sim 2386 (holotype, NU!).
4. Empogona aulacosperma (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia aula-
cosperma Robbr., Bull. Jard. Bot. Natl. Belg. 49:
296. 1979. TYPE: [Democratic Republic of the
Congo.] Congo belge. Musenge, 20 Dec. 1958, A.
Le
´
onard 2088 (holotype, BR!; isotypes, EA!, K!,
MO!, WAG!).
(*) 5. Empogona bequaertii (De Wild.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia be-
quaertii De Wild., Pl. Bequaert. 3: 157. 1925.
TYPE: [Democratic Republic of the Congo.]
Congo belge. [Kisangani] Stanleyville, Tshopo
River, 25 Feb. 1915, J. Bequaert 6969 (holotype,
BR!).
6. Empogona bracteata (Hiern) J. Tosh & Robbr.,
comb. nov. Basionym: Tricalysia bracteata Hiern,
Fl. Trop. Afr. [Oliver et al.] 3: 120. 1877. TYPE:
[Guinea.] Senegambia. Karkandy, s.d., Heudelot
855 (holotype, K!).
7. Empogona breteleri (Robbr.) J. Tosh & Robbr.,
comb. nov. Basionym: Tricalysia breteleri Robbr.,
Bull. Jard. Bot. Natl. Belg. 51: 166. 1981. TYPE:
Gabon. Moanda–Franceville Km 23, 12 Sep.
1970, F. J. Breteler 6431 (holotype, WAG!;
isotypes, BR!, P!).
8. Empogona buxifolia (Hiern) J. Tosh & Robbr.
8a. Empogona buxifolia (Hiern) J. Tosh & Robbr.
subsp. buxifolia, comb. nov. Basionym: Trica-
lysia buxifolia Hiern, Fl. Trop. Afr. [Oliver et al.]
3: 119. 1877. TYPE: Angola. Ambriz, Nov. 1872,
J. Monteiro s.n. (holotype, K!; isotype, W!).
8b. Empogona buxifolia subsp. australis (Robbr.)
J. Tosh & Robbr., comb. et stat. nov. Basionym:
Tricalysia buxifolia var. australis Robbr., Bull.
Jard. Bot. Natl. Belg. 48: 465. 1978. TYPE:
Angola. Tchivinguiro, 13 Dec. 1961, G. Barbosa
9650 (holotype, LISC!; isotypes, COI!, K!, LUAI!).
(*) 9. Empogona cacondensis (Hiern) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia cacon-
densis Hiern, Cat. Afr. Pl. (Hiern) 1(2): 467.
1898. TYPE: Angola. Rd. from Quipaca to
fortress near Fera
˜
o, Oct. 1859, F. Welwitsch
3112 (lectotype, designated by Robbrecht [1979:
320], LISU!; duplicates, BM!, COI!, K!).
(*) 10. Empogona concolor (N. Halle
´
) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia con-
color N. Halle
´
, Fl. Gabon 17: 283. 1970. TYPE:
Gabon. Be
´
linga, mine de fer, 21 July 1966, N.
Halle
´
& A. Le Thomas 119 (holotype, P!; isotypes,
K!, P!).
11. Empogona coriacea (Sond.) J. Tosh & Robbr.,
comb. nov. Basionym: Kraussia coriacea Sond.,
Linnaea 23: 54. 1850. Tricalysia sonderiana
Hiern, Fl. Trop. Afr. [Oliver et al.] 3: 119. 1877,
replacement for Kraussia coriacea Sond., non
Randia coriacea Benth., Niger Fl. [W. J. Hooker]
387. 1849 [5 Tricalysia coriacea (Benth.)
Hiern]. TYPE: [South Africa. KwaZulu-Natal:]
Natal: Durban, s.d., W. Gueinzius 100 (holotype,
W!; isotypes, BM!, C!, K!, PRE!, S!).
12. Empogona crepiniana (De Wild. & T. Durand)
J. Tosh & Robbr., comb. nov. Basionym:
Tricalysia crepiniana De Wild. & T. Durand,
Ann. Mus. Congo Belg., Bot. ser. 3, 1: 120. 1901.
TYPE: [Democratic Republic of the Congo.]
Wangata, 17 Feb. 1896, A. Dewe
`
vre 740
(holotype, BR!; isotype, COI!).
13. Empogona deightonii (Brenan) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia deight-
onii Brenan, Kew Bull. 8: 112. 1953. TYPE:
Sierra Leone. Jama, 10 Mar. 1948, F. C.
Deighton 4723 (holotype, K!; isotype, P!).
14. Empogona discolor (Brenan) J. Tosh & Robbr.,
comb. nov. Basionym: Tricalysia discolor Brenan,
Kew Bull. 2: 72. 1947. TYPE: [Ghana.] Gold
Coast. Mampong Scarp, Feb. 1933, C. Vigne
2748 (holotype, K!; isotype, MO!).
15. Empogona filiformistipulata (De Wild.) Bre-
mek.
15a. Empogona filiformistipulata (De Wild.) Bre-
mek. subsp. filiformistipulata, Bot. Jahrb. 71:
Volume 96, Number 1 Tosh et al. 207
2009 Phylogeny of Tricalysia
201, 222. 1940. Basionym: Urophyllum filiformi-
stipulatum De Wild., Pl. Bequaert. 3: 211. 1925.
Tricalysia filiformi-stipulata (De Wild.) Brenan,
Kew Bull. 8: 112. 1953. TYPE: [Democratic
Republic of the Congo.] Congo belge. Kisangani,
Tshopo River, 12 Jan. 1915, J. Bequaert 6580
(holotype, BR!; isotype, K not seen).
15b. Empogona filiformistipulata subsp. epipsila
(Robbr.) J. Tosh & Robbr., comb. et stat. nov.
Basionym: Tricalysia filiformistipulata (De
Wild.) Brenan var. epipsila Robbr., Bull. Jard.
Bot. Natl. Belg. 48: 465. 1978. TYPE: [Demo-
cratic Republic of the Congo.] Congo belge.
Yangambi, Feb. 1933, J. Louis 14233 (holotype,
BR!; isotypes, COI!, K!, MO!, P!, WAG!).
16. Empogona glabra (K. Schum.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia glabra
K. Schum., Bot. Jahrb. Syst. 23: 445. 1896.
TYPE: Angola. Catete, Nov. 1856, F. Welwitsch
3117 (holotype, LISU!; isotypes, BM!, C!, COI!,
K!, P!).
(*) 17. Empogona gossweileri (S. Moore) J. Tosh
& Robbr., comb. nov. Basionym: Tricalysia
gossweileri S. Moore, J. Linn. Soc. Bot 37: 305.
1906. TYPE: Angola. Cuanza Norte, Cazengo,
1903, J. Gossweiler 688 (holotype, BM!; isotypes,
K!, P!).
18. Empogona kirkii Hook. f.
18a. Empogona kirkii Hook. f. subsp. kirkii,
Hooker’s Icon. Pl. 11: 72, t. 1091. 1871.
Tricalysia junodii (Schinz) Brenan var. kirkii
(Hook. f.) Robbr., Bull. Jard. Bot. Natl. Belg. 49:
271. 1979. TYPE: Malawi. Cape Maclear, Oct.
1861, J. Kirk s.n. (holotype, K!).
Empogona allenii Stapf is the only species validly
published in the genus Empogona not taken up as a
result of the present study. It is a synonym of the
present taxon (Robbrecht, 1979: 272).
(*) 18b. Empogona kirkii subsp. junodii (Schinz)
J. Tosh & Robbr., comb. et stat. nov. Basionym:
Empogona junodii Schinz, Me
´
m. Herb. Boiss. 10:
67. 1900. Tricalysia junodii (Schinz) Brenan,
Kew Bull. 2: 60. 1947. TYPE: Mozambique. Baia
de Laurenc¸o Marques (Delagoa Bay), s.d., H.
Junod 311 (holotype, Z!).
(*) 19. Empogona lanceolata (Sond.) J. Tosh &
Robbr., comb. nov. Basionym: Kraussia lanceo-
lata Sond., Linnaea 23: 53. 1850. Tricalysia
lanceolata (Sond.) Burtt Davy, Ann. Transvaal
Mus. 3: 122. 1912. TYPE: [South Africa.
KwaZulu-Natal:] Natal: Durban, W. Gueinzius
68 (lectotype, designated by Robbrecht [1979:
313], W!; duplicates, P!, S!).
20. Empogona macrophylla (K. Schum.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia macro-
phylla K. Schum., Bot. Jahrb. Syst. 28: 66. 1899.
TYPE: Cameroon. Bipinde, Zenker 1569 (lecto-
type, designated by Robbrecht [1979: 339],
COI!; duplicates, BM!, BR!, COI!, E!, G!,
GOET!, HBG!, L!, M!, MO!, P!, S!, W!, WAG!).
21. Empogona maputenis (Bridson & A. E. van
Wyk) J. Tosh & Robbr., comb. nov. Basionym:
Tricalysia maputensis Bridson & A. E. van Wyk,
Fl. Zambes. 5(3): 475. 2003. TYPE: Mozambi-
que. Matutuı
´
ne, 8 Aug. 1957, L. A. G. Barbosa
& F. L. de Lemos 7807 (holotype, LISC not
seen).
(*) 22. Empogona ngalaensis (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia nga-
laensis Robbr., Bull. Jard. Bot. Natl. Belg. 49:
277. 1979. TYPE: Malawi. North Ngala, 20 mi.
N of Chilumba, 17 Dec. 1969, J. Pawek 3095
(holotype, K!).
23. Empogona nogueirae (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia no-
gueirae Robbr., Bull. Jard. Bot. Natl. Belg. 48:
466. 1978. TYPE: Angola. Musenge, 14 Oct.
1966, J. B. Teixeira 10701 (holotype, LISC!;
isotype, COI!).
24. Empogona ovalifolia (Hiern) J. Tosh & Robbr.
(*) 24a. Empogona ovalifolia (Hiern) J. Tosh &
Robbr. var. ovalifolia, comb. nov. Basionym:
Tricalysia ovalifolia Hiern, Fl. Trop. Afr. [Oliver
et al.] 3: 119. 1877. TYPE: [Tanzania.] Zanzibar:
s. loc., s.d. [acc. K Sep. 1868], J. Kirk s.n.
(lectotype, designated by Robbrecht [1979: 339],
K!).
24b. Empogona ovalifolia var. glabrata (Oliv.) J.
Tosh & Robbr., comb. nov. Basionym: Empogona
kirkii Hook. f. var. glabrata Oliv., Trans. Linn.
Soc., Bot., 2: 336. 1887. Tricalysia ovalifolia
Hiern var. glabrata (Oliv.) Brenan, Kew Bull. 2:
58. 1947. TYPE: Kenya or Tanzania. 40–60 mi.
from coast, [1884], H. H. Johnston s.n. [Kiliman-
jaro Exp.] (holotype, K!).
24c. Empogona ovalifolia var. taylorii (S. Moore)
J. Tosh & Robbr., comb. nov. Basionym:
Empogona taylorii S. Moore, J. Bot. 63: 145.
1925. Tricalysia ovalifolia Hiern var. taylorii (S.
208 Annals of the
Missouri Botanical Garden
Moore) Brenan, Kew Bull. 2: 59. 1947. TYPE:
Kenya. Giriama, Oct. 1887, W. E. Taylor s.n.
(holotype, BM!).
25. Empogona reflexa (Hutch.) J. Tosh & Robbr.
25a. Empogona reflexa (Hutch.) J. Tosh & Robbr.
var. reflexa, comb. nov. Basionym: Tricalysia
reflexa Hutch., Kew Bull. 1915: 44. 1915. TYPE:
Sierra Leone. Kessewe, 17 Apr. 1913, C. E.
Lane-Poole 131 (holotype, K!).
25b. Empogona reflexa var. ivorensis (Robbr.) J.
Tosh & Robbr., comb. nov. Basionym: Tricalysia
reflexa var. ivorensis Robbr., Bull. Jard. Bot.
Natl. Belg. 48: 466. 1978. TYPE: Ivory Coast.
W of Niapidou, 20 Jan. 1959, A. J. M.
Leeuwenberg 2500 (holotype, WAG!; isotypes,
BR!, K!).
(*) 26. Empogona ruandensis (Bremek.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia ruan-
densis Bremek., Bull. Jard. Bot. E
´
tat Bruxelles
26: 253. 1956. TYPE: [Rwanda.] Mayaga,
Mutema, 19 May 1954, L. Liben 1416 (holotype,
U!; isotypes, BR!, WAG!).
27. Empogona somaliensis (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia soma-
liensis Robbr., Bull. Jard. Bot. Natl. Belg. 56:
149. 1986. TYPE: Somalia. 17 km W of Badade,
30 June 1983, J. B. Gillett, C. F. Hemming, R. M.
Watson & H. Julin 25153 (holotype, K!).
(*) 28. Empogona talbotii (Wernham) J. Tosh &
Robbr., comb. nov. Basionym: Cremaspora tal-
botii Wernham, Cat. Pl. Oban 49. 1913.
Tricalysia talbotii (Wernham) Keay, Bull. Jard.
Bot. E
´
tat Bruxelles 28: 291. 1958. TYPE:
Nigeria. Southern Nigeria, Oban, 1911, P. A.
Talbot 287 (holotype, BM!; isotype, K!).
29. Empogona welwitschii (K. Schum.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia wel-
witschii K. Schum., Bot. Jahrb. Syst. 23: 449.
1897. TYPE: Angola. Near Ponte do Felix
Simo
˜
es, Apr. 1855, F. Welwitsch 3106 (holo-
type, LISU!; duplicates, BM not seen, COI!, K!,
P!).
C
ONCLUSIONS AND FUTURE DIRECTIONS
We have been able to demonstrate that the two
subgenera comprising the large Afro-Malagasy genus
Tricalysia do not form a monophyletic group and
should be treated as separate genera. Empogona has
been previously recognized at generic rank, and
subsequent authors have considered reviving its
generic status. On the basis of our molecular
evidence, it is now fully justified to revive Empogona
at the generic rank. The Asian genus Diplospora is
sister to Empogona, with both genera forming a
strongly supported monophyletic group. As a conse-
quence, the weakly supported Asian clade reported by
Davis et al. (2007) is not recovered in this
investigation. Further data are still required to fully
elucidate the phylogenetic relationships between
Belonophora, Diplospora, Discospermum, Empogona,
Sericanthe, and Tricalysia. There is increased support
for the placement of a Coffea and Psilanthus clade as
sister to the rest of Coffeeae.
Future work requires the inclusion of nuclear
ribosomal and low copy nuclear DNA sequence data, as
well as expanded taxon sampling, in an effort to improve
resolution between terminal taxa within the genera
Tricalysia and Empogona.Itseemsprudenttodefer
detailed discussion on the biogeography of Tricalysia and
Empogona until we have a broader sampling and a more
resolved phylogenetic hypothesis of both genera.
Literature Cited
Ali, S. J. & E. Robbrecht. 1991. Remarks on the tropical
Asian and Australian taxa included in Diplospora or
Tricalysia (Rubiaceae-Ixoroideae-Gardenieae). Blumea
35: 279–305.
Andreasen, K. & B. Bremer. 2000. Combined phylogenetic
analysis in the Rubiaceae-Ixorideae: Morphology, nuclear
and chloroplast DNA data. Amer. J. Bot. 87: 1731–1748.
Brenan, J. P. M. 1947. Empogona Hook. f. and its relation to
Tricalysia DC. Kew Bull. 1947: 53–63.
Bridson, D. M. & B. Verdcourt. 2003. Rubiaceae. Pp. 379–
720 in G. V. Pope (editor), Flora Zambesiaca, Vol. 5(3).
Royal Botanic Gardens, Kew.
Cheek, M. & S. Dawson. 2000. A synoptic revision of
Belonophora (Rubiaceae). Kew Bull. 55: 63–80.
Davis, A. P., M. Chester, O. Maurin & M. Fay. 2007.
Searching for the relatives of Coffea (Rubiaceae, Ixo-
raoideae): The circumscription and phylogeny of Coffeeae
based on plastid sequence data and morphology. Amer. J.
Bot. 94: 313–329.
Doyle, J. J. & J. L. Doyle. 1987. A rapid DNA isolation
procedure for small quantities of fresh leaf tissue.
Phytochem. Bull. 19: 11–15.
Erixon, P., B. Svennblad, T. Britton & B. Oxelman. 2003.
Reliability of Bayesian posterior probabilities and bootstrap
frequencies in phylogenetics. Syst. Biol. 52: 665–673.
Felsenstein, J. 1985. Phylogenies and the comparative
method. Amer. Naturalist 125: 1–15.
Hooker, J. D. 1873. Rubiaceae. Pp. 7–151 in G. Bentham &
J. D. Hooker (editors), Genera Plantarum, Vol. 2(1). Lovell
Reeve & Co., London.
Huelsenbeck, J. P. & F. Ronquist. 2001. MRBAYES.
Bayesian inference of phylogeny. Bioinformatics 17:
754–755.
———, B. Larget, R. E. Miller & F. Ronquist. 2002.
Potential applications and pitfalls of Bayesian inference of
phylogeny. Syst. Biol. 51: 673–688.
Volume 96, Number 1 Tosh et al. 209
2009 Phylogeny of Tricalysia
Jordan, W. C., M. W. Courtney & J. E. Neigel. 1996. Low
levels of interspecific genetic variation at a rapidly
evolving chloroplast DNA locus in North American
duckweed (Lemnaceae). Amer. J. Bot. 83: 430–439.
Keay, R. W. J. 1958. Notes on Rubiaceae for the Flora of
West Tropical Africa, 2nd ed. Bull. Jard. Bot. E
´
tat. 28:
297–298.
Lo
¨
hne, C. & T. Borsch. 2005. Molecular evolution and
phylogenetic utility of the petD group II intron: A case
study in basal angiosperms. Molec. Biol. Evol. 22:
317–332.
Maddison, D. R. & W. P. Maddison. 2002. MacClade 4:
Analysis of phylogeny and character evolution, version
4.01. Sinauer Associates, Sunderland, Massachusetts.
Mendenhall, M. 1994. Phylogeny of Baptista and Thermopsis
(Leguminosae) as Inferred from Chloroplast DNA and
Nuclear Ribosomal DNA Sequences, Secondary Chemis-
try, and Morphology. Ph.D. Dissertation, University of
Texas, Austin.
Persson, C. 2000. Phylogeny of Gardenieae (Rubiaceae)
based on chloroplast DNA sequences from the rps16 intron
and trnL(UAA)-F(GAA) intergenic spacer. Nord. J. Bot.
20: 257–270.
Posada, D. & K. A. Crandall. 1998. Modeltest: Testing the
model of DNA substitution. Bioinformatics 14: 817–818.
Ranarivelo-Randriamboavonjy, T., E. Robbrecht, E. Raba-
konandrianina & P. De Block. 2007. Revision of the
Malagasy species of the genus Tricalysia (Rubiaceae). Bot.
J. Linn. Soc. 155: 83–126.
Randle, C. P., M. E. Mort & D. J. Crawford. 2005. Bayesian
inference of phylogenetics revisited: Developments and
concerns. Taxon 54: 9–15.
Robbrecht, E. 1978. Sericanthe, a new African genus of
Rubiaceae (Coffeeae). Bull. Jard. Bot. Natl. Belg. 48: 3–78.
———. 1979. The African genus Tricalysia A. Rich.
(Rubiaceae-Coffeeae). 1. A revision of the species of sub-
genus Empogona. Bull. Jard. Bot. Natl. Belg. 49: 239–360.
———. 1982. The African genus Tricalysia A. Rich.
(Rubiaceae-Coffeeae). 2. Ephedranthera, a new section
of subgenus Tricalysia. Bull. Jard. Bot. Natl. Belg. 52:
311–339.
———. 1983. The African genus Tricalysia A. Rich. (Rub-
iaceae). 3. Probletostemon revived as a section of subgenus
Tricalysia. Bull. Jard. Bot. Natl. Belg. 53: 299–320.
———. 1987. The African genus Tricalysia A. Rich.
(Rubiaceae). 4. A revision of the species of section
Tricalysia and section Rosea. Bull. Jard. Bot. Natl. Belg.
57: 39–208.
———. 1988. Tropical woody Rubiaceae. Opera Bot. Belg.
1: 1–271.
——— & C. Puff. 1986. A survey of the Gardenieae
and related tribes (Rubiaceae). Bot. Jahrb. Syst. 108:
63–137.
——— & J.-F. Manen. 2006. The major evolutionary
lineages of the coffee family (Rubiaceae, angiosperms).
Combined analysis (nDNA and cpDNA) to infer the
position of Coptosapelta and Luculia, and supertree
construction based on rbcL, rps16, trnL-trnF and aptB-
rbcL data. A new classification in two subfamilies,
Cinchonoideae and Rubioideae. Syst. & Geogr. Pl. 76:
85–146.
Ronquist, F., J. P. Huelsenbeck & P. van der Mark. 2005.
MrBayes 3.1 manual. ,http://mrbayes.csit.fsu.edu/mb3.
1_manual.pdf., accessed 30 October 2008.
Schumann, K. 1891. Rubiaceae. Pp. 1–156 in A. Engler & K.
Prantl (editors), Die naturlichen Pflanzenfamilien, Vol. 4
(4). Wilhelm Engelmann Verlag, Leipzig.
Shaw, J., E. B. Licky, J. T. Beck, S. B. Farmer, W. Liu, J.
Miller, K. C. Siripun, C. T. Winder, E. E. Schilling & R. L.
Small. 2005. The tortoise and the hare II: Relative utility
of 21 noncoding chloroplast DNA sequences for phyloge-
netic analysis. Amer. J. Bot. 92: 142–166.
Simmons, M. P. & H. Ochoterena. 2000. Gaps as characters
in sequence-based phylogenetic analysis. Syst. Biol. 49:
369–381.
Stace, C. A. 1991. Plant Taxonomy and Biosystematics, 2nd
ed. Edward Arnold, London.
Staden, R., K. Beal & J. Bonfield. 1998. The Staden Package.
Pp. 115–130 in S. Miseners & S. Krawetz (editors),
Computer Methods in Molecular Biology. Humana Press,
New York.
Swofford, D. L. 2003. PAUP* 4.0b10: Phylogenetic Analysis
Using Parsimony (* and other methods). Sinnauer
Associates, Sunderland, Massachusetts.
Taberlet, P., L. Gielly, G. Pautou & J. Bovet. 1991. Universal
primers for amplification of three non-coding regions of
chloroplast DNA. Pl. Molec. Biol. 17: 1005–1109.
210 Annals of the
Missouri Botanical Garden
Appendix 1. Taxon voucher and accession data.
Taxon Voucher accD-psa1 petD rpl16 trnL-F
Argocoffeopsis eketensis (Wernham) Robbr. Davis 3031 (K), Cameroon DQ180497 AM999399 DQ180531 DQ180566
Argocoffeopsis rupestris (Hiern) Robbr. subsp. thonneri (Lebrun) Robbr. Harris 8168 (K), Central African Republic DQ180496 NA DQ180532 DQ180567
Argocoffeopsis scandens (K. Schum.) Lebrun Davis 3016 (K), Cameroon DQ180498 AM999400 DQ180533 DQ180568
Belonophora coriacea Hoyle Maurin 5 (K), Cameroon DQ180499 AM999401 DQ180534 DQ180569
Belonophora coriacea Hoyle Maurin 19 (K), Cameroon DQ180500 AM999402 DQ180535 DQ180570
Belonophora sp. indet. Tadjouteu 480 (K), Cameroon DQ180501 AM999403 DQ180536 DQ180571
Bertiera bicarpellata (K. Schum.) N. Halle
´
Davis 3051 (K), Cameroon DQ180502 AM999396 DQ180537 DQ180572
Bertiera breviflora Hiern Van Caekenberghe 41 (BR), Gabon* NA AM999397 AM999524 AM999466
Bertiera iturensis K. Krause Van Caekenberghe 40 (BR), Gabon* FM160622 AM999398 AM999525 AM999467
Bertiera sp. indet. Davis 3017 (K), Cameroon DQ180504 NA DQ180539 DQ180574
Calycosiphonia macrochlamys (K. Schum.) Robbr. Davis 3044 (K), Cameroon DQ180507 NA DQ180542 DQ180576
Calycosiphonia macrochlamys (K. Schum.) Robbr. Davis 3036 (K), Cameroon DQ180506 AM999404 DQ180541 DQ180575
Calycosiphonia spathicalyx (K. Schum.) Robbr. Davis 2925 (K), Tanzania DQ180509 AM999405 DQ180544 DQ180578
Canephora sp. indet. Davis 2727 (K), Madagascar DQ180510 NA AM999523 DQ180579
Coffea homollei J.-F. Leroy Davis 2305 (K), Madagascar DQ153402 NA DQ153651 DQ153769
Coffea mangoroensis Porte
`
res Rakotonasolo 41 (K), Madagascar DQ153503 AM999406 DQ153752 DQ153870
Coffea moratii J.-F. Leroy ex A. P. Davis & Rakotonas. Davis 2326 (K), Madagascar DQ153502 AM999407 DQ153751 DQ153869
Didymosalpinx norae (Swynn.) Keay Van Caekenberghe 62 (BR), Zimbabwe* FM160621 AM999395 AM999522 AM999465
Diplospora dubia (Lindl.) Masam. Van Caekenberghe 49 (BR)
a
AM999388 AM999408 AM999526 AM999468
Diplospora sp. indet. Bremer 15238 (K), Borneo (Brunei) DQ180511 NA DQ180546 DQ180580
Diplospora sp. indet. Nangkat 15238 (K), Borneo (Brunei) AM999389 AM999409 AM999527 AM999510
Discospermum abnorme (Korth.) S. J. Ali & Robbr. Sidiyasa 2148 (K), Borneo (Kalimantan) AM999380 AM999410 AM999528 AM999469
Discospermum sp. indet. Ismail 16846 (K), Borneo (Brunei) AM999390 AM999411 AM999529 AM999470
Doricera trilocularis (Balf. f.) Verdc. Friedmann 2939 (K), Mascarenes (Rodrigues) DQ180513 NA DQ180548 DQ180582
Gardenia thunbergia L. f. Davis et al. 1961-29703 (K), SE Africa DQ180514 NA DQ180549 DQ180583
Hyperacanthus microphyllus (K. Schum.) Bridson Goyder 5024 (K), Madagascar AM999387 NA AM999520 AM999464
Hyperacanthus perrieri (Drake) Rakotonas. & A. P. Davis Davis 2584 (K), Madagascar FM160619 NA AM999519 AM999462
Hyperacanthus sp. indet. Davis 2586 (K), Madagascar FM160620 NA AM999521 AM999463
Ixora guillotii Hochr. Tosh et al. 408B (BR), Madagascar FM160624 AM999394 AM999518 AM999461
Psilanthus ebracteolatus Hiern Billiet 53054 (BR), Ivory Coast* AM999392 AM999412 AM999530 AM999471
Psilanthus mannii Hook. f. Van Caekenberghe 78 (BR), Ghana* FM160623 AM999413 AM999531 AM999472
Psilanthus semsei Bridson Kisera 1473 (K), Tanzania DQ153395 AM999414 DQ153644 DQ153762
Polysphaeria sp. indet. Mvungi 15 (K), Tanzania DQ180517 NA DQ180552 DQ180586
Sericanthe andogensis (Hiern) Robbr. Bidgood 3490 (K), Tanzania DQ180522 AM999416 DQ180557 DQ180591
Sericanthe andogensis (Hiern) Robbr. Dessein 1097 (BR), Zambia FM177157 AM999415 AM999532 AM999473
Volume 96, Number 1 Tosh et al. 211
2009 Phylogeny of Tricalysia
Taxon Voucher accD-psa1 petD rpl16 trnL-F
Sericanthe jacfelicis (N. Halle
´
) Robbr. Carvalho 4169 (K), Gulf of Guinea Islands (Bioko) DQ180523 NA NA DQ180592
Sericanthe sp. indet. Valkenberg 3160 (WAG), Gabon AM999391 AM999417 AM999533 AM999511
Tricalysia aciculiflora Robbr. Manktelow 91215 (K), Tanzania AM999345 AM999419 AM999535 AM999475
Tricalysia aciculiflora Robbr. Luke 7071 (K), Tanzania AM999344 AM999418 AM999534 AM999474
Tricalysia acidophylla Robbr. Kindekat 122 (BR), Tanzania AM999346 AM999420 AM999536 AM999512
Tricalysia acocantheroides K. Schum. Dessein 1212 (BR), Zambia AM999347 AM999421 AM999537 AM999476
Tricalysia acocantheroides K. Schum. Brummit 320 (K), Malawi AM999348 AM999422 FM160581 AM999513
Tricalysia ambrensis Randriamb. & De Block De Block 1313 (BR), Madagascar AM999349 AM999423 FM160582 AM999477
Tricalysia analamazaotrensis Homolle ex Randriamb. & De Block Tosh et al. 11 (BR), Madagascar AM999350 AM999424 FM160583 AM999478
Tricalysia analamazaotrensis Homolle ex Randriamb. & De Block De Block et al. 1874 (BR), Madagascar AM999351 AM999425 FM160584 AM999514
Tricalysia anomala E. A. Bruce var. guineensis Robbr. Davis 3045 (K), Cameroon DQ180526 AM999426 DQ180560 DQ180595
Tricalysia bagshawei S. Moore Malaisse 2052 (K), Democratic Republic of the Congo AM999352 AM999427 FM160585 AM999479
Tricalysia bequaertii De Wild. Walters 942 (MO), Gabon AM999353 AM999428 FM160586 AM999480
Tricalysia bridsoniana Robbr. De Block 389 (BR), Kenya AM999354 AM999429 FM160587 AM999481
Tricalysia cacondensis Hiern Dessein 1031 (BR), Zambia AM999355 AM999430 FM160588 AM999482
Tricalysia concolor N. Halle
´
Degreef 95 (BR), Gabon AM999356 AM999431 FM160589 AM999483
Tricalysia coriacea (Benth.) Hiern Dessein 1283 (BR), Zambia AM999358 AM999433 FM160591 AM999485
Tricalysia coriacea (Benth.) Hiern Dessein 1359 (BR), Zambia AM999357 AM999432 FM160590 AM999484
Tricalysia cryptocalyx Baker De Block 527 (BR), Madagascar AM999359 AM999434 FM160592 AM999486
Tricalysia cryptocalyx Baker Tosh et al. 322 (BR), Madagascar AM999360 AM999435 FM160593 AM999487
Tricalysia dauphinensis Randriamb. & De Block De Block 694 (BR), Madagascar AM999361 AM999436 FM160594 AM999488
Tricalysia dauphinensis Randriamb. & De Block Tosh et al. 349 (BR), Madagascar AM999362 AM999436 FM160595 AM999489
Tricalysia dauphinensis Randriamb. & De Block Rabevohitra 2115 (K), Madagascar AM999363 AM999438 FM160596 AM999490
Tricalysia elliottii (K. Schum.) Hutch. & Dalziel Jongkind 1806 (K), Ghana AM999364 AM999439 FM160597 AM999491
Tricalysia gossweileri S. Moore Senterre 4041, Equatorial Guinea AM999365 AM999440 FM160598 AM999492
Tricalysia griseiflora K. Schum. Dessein 1044 (BR), Zambia AM999367 AM999442 FM160600 AM999494
Tricalysia griseiflora K. Schum. Dessein 305 (BR), Zambia AM999366 AM999441 FM160599 AM999493
Tricalysia jasminiflora (Klotzsch) Benth. & Hook. f. ex Hiern Ayami 42 (K), Malawi AM999368 AM999443 FM160601 AM999495
Tricalysia junodii (Schinz) Brenan Van Caekenberghe 79 (BR), Zimbabwe* AM999369 AM999444 FM160602 AM999496
Tricalysia lanceolata (Sond.) Burtt Davy Bagliss 1519 (K), South Africa AM999370 AM999445 FM160603 AM999497
Tricalysia leucocarpa (Baill.) Randriamb. & De Block Gautier 2442 (K), Madagascar AM999371 AM999446 FM160604 AM999498
Tricalysia leucocarpa (Baill.) Randriamb. & De Block Tosh et al. 398 (BR), Madagascar AM999372 AM999447 FM160605 AM999499
Tricalysia microphylla Hiern De Block 405 (BR), Kenya AM999373 AM999448 FM160606 AM999500
Tricalysia ngalaensis Robbr. Bidgood 2966 (K), Tanzania AM999374 AM999449 FM160607 AM999501
Tricalysia okelensis Hiern Schmidt 2139 (K), Ghana AM999375 AM999450 FM160608 AM999505
Appendix 1. Continued.
212 Annals of the
Missouri Botanical Garden
Taxon Voucher accD-psa1 petD rpl16 trnL-F
Tricalysia ovalifolia Hiern De Block et al. 1072 (BR), Madagascar AM999378 AM999453 FM160611 AM999504
Tricalysia ovalifolia Hiern De Block et al. 1090 (BR), Madagascar AM999376 AM999452 FM160609 AM999503
Tricalysia ovalifolia Hiern Butly 309 (K), Tanzania AM999377 AM999451 FM160610 AM999502
Tricalysia pallens Hiern Dessein 1266 (BR), Zambia AM999381 AM999455 FM160613 AM999515
Tricalysia pallens Hiern Dessein 953 (BR), Zambia AM999382 AM999456 FM160614 AM999516
Tricalysia pallens Hiern Adams 831 (K), Liberia AM999379 AM999454 FM160612 AM999506
Tricalysia perrieri Homolle ex Randriamb. & De Block De Block 766 (BR), Madagascar AM999383 AM999457 FM160615 AM999507
Tricalysia ruandensis Bremek. Kuchar 22323 (BR), Tanzania AM999384 AM999458 FM160616 AM999517
Tricalysia schliebenii Robbr. Bidgood 1913 (K), Tanzania AM999385 AM999459 FM160617 AM999508
Tricalysia talbotii (Wernham) Keay Latilo 67674 (K), Nigeria AM999386 AM999460 FM160618 AM999509
* Leaf material and vouchers collected from the living collections of National Botanic Garden of Belgium. Country of origin given in the table.
a
Origin unknown. Living material given to National Botanic Garden of Belgium by Hong Kong Herbarium.
Appendix 1. Continued.
Volume 96, Number 1 Tosh et al. 213
2009 Phylogeny of Tricalysia
