PHYLOGENY OF TRICALYSIA
(RUBIACEAE) AND ITS
RELATIONSHIPS WITH ALLIED
GENERA BASED ON PLASTID
DNA DATA: RESURRECTION OF
THE GENUS EMPOGONA1
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 Robbrecht4
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. Hallé) 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 inflorescences paired at the nodes with obvious calyculi,
flowers with left contorted corolla aestivation and a
distinctly 2-lobed style, and relatively small and fewseeded 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.
Volume 96, Number 1
2009
Tosh et al.
Phylogeny of Tricalysia
195
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 appendage 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 southern 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. Hallé, 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. Hallé, proved to
be genuine Tricalysia species; a new genus, Sericanthe 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 synonymous. 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 Diplospora 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 combination with morphological data. Their study confirmed
the placement of Tricalysia and related taxa (Sericanthe, 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 circumscribed Coffeeae, with an expanded sampling from
both subgenera of Tricalysia. Given the wealth of
trnL-F, rpl16, and accD-psa1 sequence data already
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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
Tricalysia sect. Empogona Hook. f. 12 spp.
sensu Robbrecht, 1979
Species group
Species
T. discolor group
T. acidophylla Robbr.
T. junodii group
T. junodii (Schinz) Brenan
T. ngalaensis Robbr.
T. ovalifolia Hiern
No known group affiliation within sect.
Empogona
Tricalysia sect. Kraussiopsis Robbr. 15 spp.
sensu Robbrecht, 1979
T. crepiniana group
T. concolor N. Hallé
T. gossweileri S. Moore
T. bequaertii De Wild.
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
Tricalysia sect. Probletostemon (K. Schum.)
Robbr. 4 spp. sensu Robbrecht, 1983
Species
T. anomala E. A. Bruce
T. elliottii (K. Schum.) Hutch. &
Dalziel
Tricalysia sect. Ephedranthera Robbr. 9 spp.
sensu Robbrecht, 1982
T. aciculiflora Robbr.
T. acocantheroides K. Schum.
T. bridsoniana Robbr.
Tricalysia sect. Tricalysia 40 spp. sensu
Robbrecht, 1987
T. angolensis group
T. griseiflora K. Schum.
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
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Tosh et al.
Phylogeny of Tricalysia
197
Table 2. Amplification primers used in this study.
Region
trnL-F
rpl16
accD-psa1
petD
Primer
Primer sequence (59-39)
Forward (c)
Reverse (f)
Forward (71f)
Reverse (1661r)
Reverse (1516r)
Internal forward
Internal reverse
Forward (769 F)
Reverse (75 R)
Forward (1365)
Reverse (738)
CGA AAT CGG TAG ACG CTA CG
AAT TGA ACT GGT GAC ACG AG
GCT ATG CTT AGT GTG TGA CTC GTT G
CGT ACC CAT ATT TTT CCA CCA CGA C
CCC TTC ATT CTT CCT CTA TGT TG
GTA AGA AGT GAT GGG AAC GA
TCG TTC CCA TCA CTT CTT AC
GGA AGT TTG AGC TTT ATG CAA ATG
AGA AGC CAT TGC AAT TGC CGG AAA
TTG ACY CGT TTT TAT AGT TTA C
AAT TTA GCY CTT AAT ACA GG
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.
MATERIALS 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,
Reference
Taberlet et al., 1991
Jordan et al., 1996
Shaw et al., 2005
Davis et al., 2007
Mendenhall, 1994
Löhne & Borsch, 2004
Pennsylvania, U.S.A.) according to the manufacturer’s
instructions in order to remove any potential polymerase chain reaction (PCR) inhibitors.
Amplification of the trnL-F, rpl16, petD, and accDpsa1 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 MgCl2 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 ml of 103 NH4 reaction
buffer (Bioline), 1.5 ml of 50 mM MgCl2, and 2.5 ml of
dNTPs (Promega, Madison, Wisconsin, U.S.A.). All
amplified PCR products were purified using NucleoSpin purification columns following the manufacturer’s protocol.
Cycle sequencing reactions were carried out using
BigDye Terminator Mix version 3.1 (Applied Biosystems, Inc., Warrington, Cheshire, U.K.). The cycle
198
Annals of the
Missouri Botanical Garden
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 sequencing 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.
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).
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 swapping, 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
RESULTS
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 polyA 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 Ixoroideae). 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.
80
4415
—
3796
50
352 (7.9)
929
0.816
0.908
8853
80
889
772–822
765
9
74 (8.3)
174
0.822
0.916
9990
No. of taxa
Total length (base pairs)
Sequence length variation
No. of constant characters
No. of phylogenetically informative indels
No. of variable PI characters (% of total characters)
Tree length
Consistency index
Retention index
No. of trees saved
79
1255
737–1061
1075
22
117 (9.3)
283
0.827
0.923
9920
78
1207
995–1068
982
11
116 (9.6)
339
0.814
0.890
1056
67
1064
937–966
974
8
45 (4.2)
123
0.854
0.937
1392
Combined plastid
accD-psa1
Table 3. Characteristics of accD-psa1, rpl16, petD, trnL-F, and combined data sets and tree statistics.
rpl16
petD
trnL-F
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Tosh et al.
Phylogeny of Tricalysia
199
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 consistent 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), Discospermum (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. acidophylla Robbr. (BS 98%, PP 1.00).
The clade of Belonophora and Tricalysia subgen.
Tricalysia is present in both the MP strict consensus
200
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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.
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Phylogeny of Tricalysia
201
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.
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 Tricalysia 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.
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microphylla Hiern, T. schliebenii Robbr., and the
aforementioned Madagascan group together with T.
jasminiflora.
of Davis et al. (2007). First, we did not recover an
Asian clade. Instead, Diplospora formed a wellsupported 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, Discospermum, 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).
DISCUSSION
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 molecular and combined morphological-molecular phylogenies, Tricalysia (subgen. Tricalysia) was placed in a
poorly supported and unresolved clade containing
Sericanthe, Belonophora, and an Asian clade (including 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
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, Diplospora, 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 synapomorphies.
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 connective (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 (Robbrecht, 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
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Tosh et al.
Phylogeny of Tricalysia
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 orientation.
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-Randriamboavonjy et al., 2007). Ali and Robbrecht (1991)
also justified maintaining Diplospora and Discospermum 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, pendulous 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 Aulacocalyceae, although the axillary inflorescences of
Belonophora contrasted with the terminal or subterminal 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
203
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 provisional 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. acocantheroides 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 subdivided 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 representatives of subgenus Tricalysia, species are characterized by the presence of unisexual flowers. There
is weak bootstrap and significant Bayesian support
Discospermum
(ca. 7 spp.)
Bracts and
bracteoles
Corolla length
(mm)
Flower
organization
Merosity
Calyx
free or fused into calyculi
8–15
free or fused into
calyculi
5–10
Corolla throat
Anthers
glabrous to bearded
medifixed; on short
filaments in throat,
exserted
glabrous or hairy
medifixed; on short
filaments in throat,
exserted
Anther
connective
sometimes protruding in very
short triangle
sometimes protruding
in very short
triangle
Placentation
1–3(–6) ovules on a
(3–)5–15 ovules on a hemihemi-circular to
circular to 6 hemi-ellipsoid
6 hemi-ellipsoid
placenta; attached to the
placenta; attached
middle of the septum
to the middle of
the septum
20–30
5–7
Empogona
(29 spp.)
Tricalysia, excluding
sect. Androgyne
(ca. 80 spp.)
free alternate, sect.
fused into calyculi;
Empogona; fused into
free alternate in
calyculi, sect.
sect.
Kraussiopsis
Probletostemon
(6–)8–17
8–50
hermaphroditic
hermaphroditica
Tricalysia sect.
Androgyne
(11 spp.)
fused into
calyculi
5–10
unisexual
((4–))5(–6)
(4–)5–6(–12)
4–7
tube short, lobes well- tube well-developed; tube welldeveloped and
lobes none,
developed,
often overlapping
triangular or
with minute
linear
teeth
glabrous to hairy
hairy
densely beardedb
medifixed; on long
medifixed; on long
medifixed; on
filaments in throat,
filaments in
short
exserted
throat, exserted
filaments in
throat or
sessile,
exserted
protruding in
mostly protruding in short apical
conspicuous
short triangle
appendage
ribbon-like
appendagesc
1–ca. 25 ovules on a
1–12 ovules on a
2–8 ovules on a
hemi-circular to 6
hemi-circular to
hemi-circular
hemi-ellipsoid
6 hemi-ellipsoid
to 6 hemiplacenta; attached to
placenta; attached
ellipsoid
the middle of the
to the middle of
placenta;
septum
the septum
attached to
the middle of
the septum
8–10
5–20
5–9
Belonophora
(5 spp.)
Sericanthe
(ca. 20 spp.)
lower bracts fused into
fused into calyculi
calyculi; upper bracts
free, opposite
(10–)20–30(–40)
(8–)12–25
hermaphroditic;
heterostyly
((4–))5
tube short, lobes welldeveloped
hermaphroditic
glabrous
medifixed; sessile in
tube, included
glabrous or hairy
basifixed; on short
filaments in
throat, exserted
protruding in short
triangle
strongly flattened;
no appendage
2 collateral ovules on
inner face of a
pendulous hemicircular 6 hemiellipsoid placenta;
attached to the apex
of the septum
(1–)2(–5) ovules on
a hemi-circular
6 hemiellipsoid
placenta;
attached to the
apex of the
septum
10–30
10–15
(5–)7–8(–9)
tube welldeveloped, with
minute teeth
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4
tube short, rounded lobes
present or absent
hermaphroditic or
unisexual
4–((–5))
tube short, lobes
mostly triangular
Fruit (mm)
hermaphroditic or unisexual
Diplospora
(ca. 10 spp.)
204
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.
lateral
superior
inferior
inferior
away from septum
Embryo radicle
OTHER RELATIONSHIPS WITHIN COFFEEAE AND THE
RELATIONSHIP TO BERTIERA
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.
entire, ruminate in some spp.
Endosperm
entire or ruminate
massive, mostly surrounding
seeds
Placental
outgrowth
205
(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 characterized 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).
a
entire
entire
mostly none;
massive in some
spp.
entire
massive, surrounding
seeds
sclerified or leathery
Pericarp
mostly none
none, with weak
outgrowths in some
spp.
entire, ruminate in some entire
spp.
inferior
inferior
none
fleshy
fleshy
fleshy
fleshy; rarely
sclerotic, sect.
Probletostemon
mostly none
orange
yellow
red
red, rarely orange
first white, turning
purple, then black
fleshy
turning from yellow
and orange to red
fleshy
purplish black
Fruit color
Belonophora
(5 spp.)
Tricalysia sect.
Androgyne
(11 spp.)
Empogona
(29 spp.)
Diplospora
(ca. 10 spp.)
Discospermum
(ca. 7 spp.)
Table 4. Continued.
Tosh et al.
Phylogeny of Tricalysia
Tricalysia, excluding
sect. Androgyne
(ca. 80 spp.)
Sericanthe
(ca. 20 spp.)
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2009
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. Robbrecht 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,
206
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with increased support values (BS 93%, PP 1.00).
There was also strong support for the sister relationship 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).
analysis. They have a basal position in the clade
corresponding to section Empogona.
TAXONOMIC NOVELTIES RESULTING FROM THE GENERIC
RESURRECTION OF EMPOGONA
An outline of an emended infrageneric classification 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.
OUTLINE 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 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 comprises 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.
CHECKLIST OF SPECIES AND INFRASPECIFIC TAXA,
INCLUDING TAXONOMIC NOVELTIES
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 discolor (Robbrecht, 1979: 292). The group comprises 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
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 modifications of infraspecific status. In his revision,
Robbrecht (1979) used varietal status for all infraspecific taxa recognized. Here we reconsider the
appropriateness of that treatment in applying du
Rietz’s criteria (as cited in Stace, 1991) for distinguishing 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-
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2009
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!).
Tosh et al.
Phylogeny of Tricalysia
207
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!).
2. Empogona aequatoria (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia aequatoria 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!).
(*) 9. Empogona cacondensis (Hiern) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia cacondensis Hiern, Cat. Afr. Pl. (Hiern) 1(2): 467.
1898. TYPE: Angola. Rd. from Quipaca to
fortress near Ferão, Oct. 1859, F. Welwitsch
3112 (lectotype, designated by Robbrecht [1979:
320], LISU!; duplicates, BM!, COI!, K!).
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!).
(*) 10. Empogona concolor (N. Hallé) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia concolor N. Hallé, Fl. Gabon 17: 283. 1970. TYPE:
Gabon. Bélinga, mine de fer, 21 July 1966, N.
Hallé & A. Le Thomas 119 (holotype, P!; isotypes,
K!, P!).
4. Empogona aulacosperma (Robbr.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia aulacosperma Robbr., Bull. Jard. Bot. Natl. Belg. 49:
296. 1979. TYPE: [Democratic Republic of the
Congo.] Congo belge. Musenge, 20 Dec. 1958, A.
Léonard 2088 (holotype, BR!; isotypes, EA!, K!,
MO!, WAG!).
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!).
(*) 5. Empogona bequaertii (De Wild.) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia bequaertii 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: Tricalysia buxifolia Hiern, Fl. Trop. Afr. [Oliver et al.]
3: 119. 1877. TYPE: Angola. Ambriz, Nov. 1872,
J. Monteiro s.n. (holotype, K!; isotype, W!).
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. Dewèvre 740
(holotype, BR!; isotype, COI!).
13. Empogona deightonii (Brenan) J. Tosh &
Robbr., comb. nov. Basionym: Tricalysia deightonii 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.) Bremek.
15a. Empogona filiformistipulata (De Wild.) Bremek. subsp. filiformistipulata, Bot. Jahrb. 71:
208
Annals of the
Missouri Botanical Garden
201, 222. 1940. Basionym: Urophyllum filiformistipulatum 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: [Democratic 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!).
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 macrophylla K. Schum., Bot. Jahrb. Syst. 28: 66. 1899.
TYPE: Cameroon. Bipinde, Zenker 1569 (lectotype, 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: Mozambique. Matutuı́ne, 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 ngalaensis 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 nogueirae 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.
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).
(*) 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!).
(*) 18b. Empogona kirkii subsp. junodii (Schinz)
J. Tosh & Robbr., comb. et stat. nov. Basionym:
Empogona junodii Schinz, Mém. Herb. Boiss. 10:
67. 1900. Tricalysia junodii (Schinz) Brenan,
Kew Bull. 2: 60. 1947. TYPE: Mozambique. Baia
de Laurenço Marques (Delagoa Bay), s.d., H.
Junod 311 (holotype, Z!).
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. [Kilimanjaro Exp.] (holotype, K!).
(*) 19. Empogona lanceolata (Sond.) J. Tosh &
Robbr., comb. nov. Basionym: Kraussia lanceolata Sond., Linnaea 23: 53. 1850. Tricalysia
lanceolata (Sond.) Burtt Davy, Ann. Transvaal
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.
Volume 96, Number 1
2009
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 ruandensis Bremek., Bull. Jard. Bot. État 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 somaliensis 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 talbotii Wernham, Cat. Pl. Oban 49. 1913.
Tricalysia talbotii (Wernham) Keay, Bull. Jard.
Bot. État 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 welwitschii K. Schum., Bot. Jahrb. Syst. 23: 449.
1897. TYPE: Angola. Near Ponte do Felix
Simões, Apr. 1855, F. Welwitsch 3106 (holotype, LISU!; duplicates, BM not seen, COI!, K!,
P!).
CONCLUSIONS 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
Tosh et al.
Phylogeny of Tricalysia
209
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 consequence, 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. It seems prudent to defer
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, Ixoraoideae): 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.
210
Annals of the
Missouri Botanical Garden
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. État. 28:
297–298.
Löhne, 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 Chemistry, 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. Rabakonandrianina & 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 subgenus 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. (Rubiaceae). 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 aptBrbcL 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 phylogenetic 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.
accD-psa1
petD
rpl16
trnL-F
Argocoffeopsis eketensis (Wernham) Robbr.
Argocoffeopsis rupestris (Hiern) Robbr. subsp. thonneri (Lebrun) Robbr.
Argocoffeopsis scandens (K. Schum.) Lebrun
Belonophora coriacea Hoyle
Belonophora coriacea Hoyle
Belonophora sp. indet.
Bertiera bicarpellata (K. Schum.) N. Hallé
Bertiera breviflora Hiern
Bertiera iturensis K. Krause
Bertiera sp. indet.
Calycosiphonia macrochlamys (K. Schum.) Robbr.
Calycosiphonia macrochlamys (K. Schum.) Robbr.
Calycosiphonia spathicalyx (K. Schum.) Robbr.
Canephora sp. indet.
Coffea homollei J.-F. Leroy
Coffea mangoroensis Portères
Coffea moratii J.-F. Leroy ex A. P. Davis & Rakotonas.
Didymosalpinx norae (Swynn.) Keay
Diplospora dubia (Lindl.) Masam.
Diplospora sp. indet.
Diplospora sp. indet.
Discospermum abnorme (Korth.) S. J. Ali & Robbr.
Discospermum sp. indet.
Doricera trilocularis (Balf. f.) Verdc.
Gardenia thunbergia L. f.
Hyperacanthus microphyllus (K. Schum.) Bridson
Hyperacanthus perrieri (Drake) Rakotonas. & A. P. Davis
Hyperacanthus sp. indet.
Ixora guillotii Hochr.
Psilanthus ebracteolatus Hiern
Psilanthus mannii Hook. f.
Psilanthus semsei Bridson
Polysphaeria sp. indet.
Sericanthe andogensis (Hiern) Robbr.
Sericanthe andogensis (Hiern) Robbr.
Davis 3031 (K), Cameroon
Harris 8168 (K), Central African Republic
Davis 3016 (K), Cameroon
Maurin 5 (K), Cameroon
Maurin 19 (K), Cameroon
Tadjouteu 480 (K), Cameroon
Davis 3051 (K), Cameroon
Van Caekenberghe 41 (BR), Gabon*
Van Caekenberghe 40 (BR), Gabon*
Davis 3017 (K), Cameroon
Davis 3044 (K), Cameroon
Davis 3036 (K), Cameroon
Davis 2925 (K), Tanzania
Davis 2727 (K), Madagascar
Davis 2305 (K), Madagascar
Rakotonasolo 41 (K), Madagascar
Davis 2326 (K), Madagascar
Van Caekenberghe 62 (BR), Zimbabwe*
Van Caekenberghe 49 (BR)a
Bremer 15238 (K), Borneo (Brunei)
Nangkat 15238 (K), Borneo (Brunei)
Sidiyasa 2148 (K), Borneo (Kalimantan)
Ismail 16846 (K), Borneo (Brunei)
Friedmann 2939 (K), Mascarenes (Rodrigues)
Davis et al. 1961-29703 (K), SE Africa
Goyder 5024 (K), Madagascar
Davis 2584 (K), Madagascar
Davis 2586 (K), Madagascar
Tosh et al. 408B (BR), Madagascar
Billiet 53054 (BR), Ivory Coast*
Van Caekenberghe 78 (BR), Ghana*
Kisera 1473 (K), Tanzania
Mvungi 15 (K), Tanzania
Bidgood 3490 (K), Tanzania
Dessein 1097 (BR), Zambia
DQ180497
DQ180496
DQ180498
DQ180499
DQ180500
DQ180501
DQ180502
NA
FM160622
DQ180504
DQ180507
DQ180506
DQ180509
DQ180510
DQ153402
DQ153503
DQ153502
FM160621
AM999388
DQ180511
AM999389
AM999380
AM999390
DQ180513
DQ180514
AM999387
FM160619
FM160620
FM160624
AM999392
FM160623
DQ153395
DQ180517
DQ180522
FM177157
AM999399
NA
AM999400
AM999401
AM999402
AM999403
AM999396
AM999397
AM999398
NA
NA
AM999404
AM999405
NA
NA
AM999406
AM999407
AM999395
AM999408
NA
AM999409
AM999410
AM999411
NA
NA
NA
NA
NA
AM999394
AM999412
AM999413
AM999414
NA
AM999416
AM999415
DQ180531
DQ180532
DQ180533
DQ180534
DQ180535
DQ180536
DQ180537
AM999524
AM999525
DQ180539
DQ180542
DQ180541
DQ180544
AM999523
DQ153651
DQ153752
DQ153751
AM999522
AM999526
DQ180546
AM999527
AM999528
AM999529
DQ180548
DQ180549
AM999520
AM999519
AM999521
AM999518
AM999530
AM999531
DQ153644
DQ180552
DQ180557
AM999532
DQ180566
DQ180567
DQ180568
DQ180569
DQ180570
DQ180571
DQ180572
AM999466
AM999467
DQ180574
DQ180576
DQ180575
DQ180578
DQ180579
DQ153769
DQ153870
DQ153869
AM999465
AM999468
DQ180580
AM999510
AM999469
AM999470
DQ180582
DQ180583
AM999464
AM999462
AM999463
AM999461
AM999471
AM999472
DQ153762
DQ180586
DQ180591
AM999473
211
Voucher
Tosh et al.
Phylogeny of Tricalysia
Taxon
Volume 96, Number 1
2009
Appendix 1. Taxon voucher and accession data.
212
Appendix 1. Continued.
Taxon
Carvalho 4169 (K), Gulf of Guinea Islands (Bioko)
Valkenberg 3160 (WAG), Gabon
Manktelow 91215 (K), Tanzania
Luke 7071 (K), Tanzania
Kindekat 122 (BR), Tanzania
Dessein 1212 (BR), Zambia
Brummit 320 (K), Malawi
De Block 1313 (BR), Madagascar
Tosh et al. 11 (BR), Madagascar
De Block et al. 1874 (BR), Madagascar
Davis 3045 (K), Cameroon
Malaisse 2052 (K), Democratic Republic of the Congo
Walters 942 (MO), Gabon
De Block 389 (BR), Kenya
Dessein 1031 (BR), Zambia
Degreef 95 (BR), Gabon
Dessein 1283 (BR), Zambia
Dessein 1359 (BR), Zambia
De Block 527 (BR), Madagascar
Tosh et al. 322 (BR), Madagascar
De Block 694 (BR), Madagascar
Tosh et al. 349 (BR), Madagascar
Rabevohitra 2115 (K), Madagascar
Jongkind 1806 (K), Ghana
Senterre 4041, Equatorial Guinea
Dessein 1044 (BR), Zambia
Dessein 305 (BR), Zambia
Ayami 42 (K), Malawi
Van Caekenberghe 79 (BR), Zimbabwe*
Bagliss 1519 (K), South Africa
Gautier 2442 (K), Madagascar
Tosh et al. 398 (BR), Madagascar
De Block 405 (BR), Kenya
Bidgood 2966 (K), Tanzania
Schmidt 2139 (K), Ghana
accD-psa1
petD
rpl16
trnL-F
DQ180523
AM999391
AM999345
AM999344
AM999346
AM999347
AM999348
AM999349
AM999350
AM999351
DQ180526
AM999352
AM999353
AM999354
AM999355
AM999356
AM999358
AM999357
AM999359
AM999360
AM999361
AM999362
AM999363
AM999364
AM999365
AM999367
AM999366
AM999368
AM999369
AM999370
AM999371
AM999372
AM999373
AM999374
AM999375
NA
AM999417
AM999419
AM999418
AM999420
AM999421
AM999422
AM999423
AM999424
AM999425
AM999426
AM999427
AM999428
AM999429
AM999430
AM999431
AM999433
AM999432
AM999434
AM999435
AM999436
AM999436
AM999438
AM999439
AM999440
AM999442
AM999441
AM999443
AM999444
AM999445
AM999446
AM999447
AM999448
AM999449
AM999450
NA
AM999533
AM999535
AM999534
AM999536
AM999537
FM160581
FM160582
FM160583
FM160584
DQ180560
FM160585
FM160586
FM160587
FM160588
FM160589
FM160591
FM160590
FM160592
FM160593
FM160594
FM160595
FM160596
FM160597
FM160598
FM160600
FM160599
FM160601
FM160602
FM160603
FM160604
FM160605
FM160606
FM160607
FM160608
DQ180592
AM999511
AM999475
AM999474
AM999512
AM999476
AM999513
AM999477
AM999478
AM999514
DQ180595
AM999479
AM999480
AM999481
AM999482
AM999483
AM999485
AM999484
AM999486
AM999487
AM999488
AM999489
AM999490
AM999491
AM999492
AM999494
AM999493
AM999495
AM999496
AM999497
AM999498
AM999499
AM999500
AM999501
AM999505
Annals of the
Missouri Botanical Garden
Sericanthe jacfelicis (N. Hallé) Robbr.
Sericanthe sp. indet.
Tricalysia aciculiflora Robbr.
Tricalysia aciculiflora Robbr.
Tricalysia acidophylla Robbr.
Tricalysia acocantheroides K. Schum.
Tricalysia acocantheroides K. Schum.
Tricalysia ambrensis Randriamb. & De Block
Tricalysia analamazaotrensis Homolle ex Randriamb. & De Block
Tricalysia analamazaotrensis Homolle ex Randriamb. & De Block
Tricalysia anomala E. A. Bruce var. guineensis Robbr.
Tricalysia bagshawei S. Moore
Tricalysia bequaertii De Wild.
Tricalysia bridsoniana Robbr.
Tricalysia cacondensis Hiern
Tricalysia concolor N. Hallé
Tricalysia coriacea (Benth.) Hiern
Tricalysia coriacea (Benth.) Hiern
Tricalysia cryptocalyx Baker
Tricalysia cryptocalyx Baker
Tricalysia dauphinensis Randriamb. & De Block
Tricalysia dauphinensis Randriamb. & De Block
Tricalysia dauphinensis Randriamb. & De Block
Tricalysia elliottii (K. Schum.) Hutch. & Dalziel
Tricalysia gossweileri S. Moore
Tricalysia griseiflora K. Schum.
Tricalysia griseiflora K. Schum.
Tricalysia jasminiflora (Klotzsch) Benth. & Hook. f. ex Hiern
Tricalysia junodii (Schinz) Brenan
Tricalysia lanceolata (Sond.) Burtt Davy
Tricalysia leucocarpa (Baill.) Randriamb. & De Block
Tricalysia leucocarpa (Baill.) Randriamb. & De Block
Tricalysia microphylla Hiern
Tricalysia ngalaensis Robbr.
Tricalysia okelensis Hiern
Voucher
Volume 96, Number 1
2009
Appendix 1. Continued.
Taxon
ovalifolia Hiern
ovalifolia Hiern
ovalifolia Hiern
pallens Hiern
pallens Hiern
pallens Hiern
perrieri Homolle ex Randriamb. & De Block
ruandensis Bremek.
schliebenii Robbr.
talbotii (Wernham) Keay
De Block et al. 1072 (BR), Madagascar
De Block et al. 1090 (BR), Madagascar
Butly 309 (K), Tanzania
Dessein 1266 (BR), Zambia
Dessein 953 (BR), Zambia
Adams 831 (K), Liberia
De Block 766 (BR), Madagascar
Kuchar 22323 (BR), Tanzania
Bidgood 1913 (K), Tanzania
Latilo 67674 (K), Nigeria
accD-psa1
petD
rpl16
trnL-F
AM999378
AM999376
AM999377
AM999381
AM999382
AM999379
AM999383
AM999384
AM999385
AM999386
AM999453
AM999452
AM999451
AM999455
AM999456
AM999454
AM999457
AM999458
AM999459
AM999460
FM160611
FM160609
FM160610
FM160613
FM160614
FM160612
FM160615
FM160616
FM160617
FM160618
AM999504
AM999503
AM999502
AM999515
AM999516
AM999506
AM999507
AM999517
AM999508
AM999509
* Leaf material and vouchers collected from the living collections of National Botanic Garden of Belgium. Country of origin given in the table.
Origin unknown. Living material given to National Botanic Garden of Belgium by Hong Kong Herbarium.
a
Tosh et al.
Phylogeny of Tricalysia
Tricalysia
Tricalysia
Tricalysia
Tricalysia
Tricalysia
Tricalysia
Tricalysia
Tricalysia
Tricalysia
Tricalysia
Voucher
213