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Int. J. Plant Sci. 175(8):000–000. 2014.
䉷2014 by The University of Chicago. All rights reserved.
1058-5893/2014/17508-00XX$15.00 DOI: 10.1086/677648
PHYLOGENETIC ANALYSIS OF THE AFRICAN GENUS GILBERTIODENDRON J. LE
´ONARD
AND RELATED GENERA (LEGUMINOSAE-CAESALPINIOIDEAE-DETARIEAE)
Manuel de la Estrella,
1,
*
,
† Jan J. Wieringa,‡ Barbara Mackinder,§ Xander van der Burgt,§ Juan A. Devesa,† and Anne Bruneau*
*Institut de Recherche En Biologie Ve´ge´tale and De´ partement de Sciences Biologiques, Universite´ de Montre´ al, 4101 Sherbrooke Est, Que´bec
H1X 2B2, Montre´al, Canada; †Departamento de Bota´nica, Ecologı´a y Fisiologı´a Vegetal, Facultad de Ciencias, Campus de Rabanales,
Universidad de Co´rdoba, 14071 Co´ rdoba, Spain; ‡Naturalis Biodiversity Centre, Botany section, Darwinweg 2, 2333 CC Leiden,
The Netherlands; and §Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, United Kingdom
Premise of research.Gilbertiodendron is a genus endemic to Africa with ∼30 species made up of trees
of primary dry-land, riverine, and gallery forests. Recently, the west and central African monotypic genus
Pellegriniodendron was merged into Gilbertiodendron.Gilbertiodendron is one of 17 genera that form the
exclusively African Berlinia clade, and this study presents the findings of a phylogenetic analysis designed to
evaluate the generic limits of Gilbertiodendron and its relationships within the Berlinia clade.
Methodology. To test the monophyly of Gilbertiodendron and its relationships with other genera, we
analyzed nucleotide sequence data from the nuclear ribosomal internal transcribed spacer and the plastid trnL
intron and trnL-F intergenic spacer, using parsimony and Bayesian analyses.
Pivotal results.Gilbertiodendron is recovered as monophyletic, including all the samples previously rec-
ognized as Pellegriniodendron diphyllum.
Conclusions. The placement of Pellegriniodendron in synonymy with Gilbertiodendron is supported by
our results. Our analyses suggest that G. diphyllum is the same taxon on both sides of the Dahomey Gap.
The G. ogoouense complex is a monophyletic group of species that needs a new taxonomic framework and
within which several new species will be described. The phylogenetic framework presentedhere and the ongoing
taxonomic revision should provide the baseline data required for adequate assessment of this group of tree
species, of which only eight have been assessed under the International Union for Conservation of Nature’s
Red List criteria.
Keywords: Didelotia, Fabaceae, ITS, Librevillea,Plagiosiphon,Pellegriniodendron, phylogenetic analyses,
tropical Africa, trnL-trnF.
Introduction
Leguminosae is the third-largest flowering plant family,
made up ∼19,500 species in ∼751 genera, occurring in a great
variety of habitats from rain forests and mangrove swamps to
deserts and temperate zones (Lewis et al. 2005; LPWG 2013a).
The family is traditionally divided into 3 subfamilies—Papil-
ionoideae, Mimosoideae, and Caesalpinioideae—but propos-
als for a new classification currently being discussed will in-
crease that number to 6, 10–12, or even 15 (LPWG 2013b).
The caesalpinioid legumes form the smallest of the three tra-
ditionally recognized subfamilies and includes ∼2,250 species
assigned to 171 genera and four tribes (Lewis et al. 2005;
LPWG 2013a). In terms of species richness, Leguminosae is
the most important angiosperm family in tropical Africa (Le-
brun and Stork 1998). The dominance of Caesalpinioideae
species in tropical Africa was recognized by Letouzey (1968),
who named a specific forest type, the foreˆt biafre´enne.Cae-
salpinioideae species can form large expanses of forests, some
1
Author for correspondence; e-mail: mdelaestrella@gmail.com.
Manuscript received January 2014; revised manuscript received May 2014; elec-
tronically published August xx, 2014.
dominated by a single tree species (e.g., Gilbertiodendron dew-
evrei monodominant forest; Corlett and Primack 2011). About
half of all caesalpinioid genera (82 of 171) belong to the mono-
phyletic tribe Detarieae (Bruneau et al. 2008), which is pan-
tropical in distribution, but the majority of the genera are
confined to Africa and Madagascar (Mackinder 2005). One
consistently reported group within the Detarieae is the African
Berlinia clade (Bruneau et al. 2008), which is made up of 17
genera of medium to large trees. This clade includes a weakly
supported group made up of Didelotia,Plagiosiphon,Librev-
illea, and Gilbertiodendron, sister to a large clade that includes
Anthonotha,Englerodendron,Oddoniodendron (recently re-
viewed by Breteler 2006, 2008, 2010, 2011), Berlinia,Isob-
erlinia,Microberlinia (Mackinder and Pennington 2011), and
the ‘‘Babijt’’ group; the latter, as delimited by Wieringa and
Gervais (2003), includes Brachystegia,Aphanocalyx,Bikinia,
Julbernardia,Icuria, and Tetraberlinia. Many of the tree spe-
cies in the African forests belong to the Berlinia clade, and this
is the most important group of trees in the lowland evergreen
rainforest (Wieringa 1999) and a dominant component of Af-
rican tropical forests (White 1983).
Gilbertiodendron is an endemic African genus with ∼30 spe-
cies and a dominant component in many African forests. All
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species of Gilbertiodendron are trees of primary rain forest
and gallery forest on well-drained or periodically inundated
soil (Le´onard 1957; Mackinder 2005; Estrella et al. 2012a).
The origin of the genus dates from J. Le´onard’s work in the
1950s. Le´onard transferred all African species previously de-
scribed within Macrolobium to four separate genera, three of
which he described as new—Gilbertiodendron (Le´onard
1952), Paramacrolobium (Le´onard 1954), and Pellegriniod-
endron (Le´onard 1955); the fourth was made up of the rein-
stated genus Anthonotha P. Beauv. (Le´onard 1955). Pellegri-
niodendron (one species) was transferred to Gilbertiodendron
following recently published phylogenetic analyses (Bruneau
et al. 2008) and a morphological reevaluation of its status
(Estrella et al. 2012a). A complete taxonomic history of the
genus can be found in Estrella and Devesa (2014). Species
belonging to Gilbertiodendron have leaflets with marginal or
submarginal glands, a pair of bracteoles that encloses the
flower bud, five sepals, and five petals. The lateral and abaxial
petal pairs are reduced, alternate to the sepals and similar to
them. There is a single well-developed adaxial petal, with an
unguiculate base and bilobed apex. The androecium is usually
formed of six staminodes and three stamens fused at the base
on a short, fleshy tube, but there are a few exceptions, such
as G. splendidum, which has nine well-developed stamens. The
ovary, located on a short stipe, develops into a pod with 1–4
longitudinal nerves, the valves twisting when mature to dis-
perse the seeds explosively (Cowan and Polhill 1981; Estrella
et al. 2012a), similar to the ovary described for Tetraberlinia
(van der Burgt 1997).
Gilbertiodendron is a genus that is important both econom-
ically (Burkill 1995), with species used for timber or traditional
medicine, and ecologically, with species growing gregariously,
forming large forest stands (Poorter et al. 2004; Estrella et al.
2012c; van der Burgt et al. 2012; Estrella and Devesa 2014).
The highest concentration in both number of species and mor-
phological variation is found in the Guineo-Congolian region,
particularly in Gabon, which is a high-diversity area for le-
gumes (Sosef et al. 2006; Estrella et al. 2012b). At least 18
species of Gilbertiodendron have been reported in this area,
with 10 species considered endemic or near endemic (i.e., spe-
cies for which it was estimated that 85% of the distribution
area falls within Gabon; Sosef et al. 2006). Several additional
species new to science are to be described during the ongoing
taxonomic revision of the genus (van der Burgt et al., forth-
coming).
Although Gilbertiodendron plays a central role in many Af-
rican forest areas, as noted by Le´onard (1957), the advance-
ment of taxonomic knowledge and species delimitation within
the genus has been hampered by a lack of adequate material
for study, especially fruiting specimens. Recently, new collec-
tions have become available, resulting in, for example, the
recognition of many new species (Estrella et al. 2012c; van der
Burgt et al. 2012, forthcoming; Estrella and Devesa 2014), but
even now there is a lack of good material for many taxa,
preventing adequate descriptions (Estrella and Devesa 2014).
With the addition of recent collections, the objectives of the
current study are to complement and pursue the ongoing effort
of the past few years on the taxonomy of this genus (Estrella
et al. 2012a, 2012c; van der Burgt et al. 2012; Estrella and
Devesa 2014) with a densely sampled species-level phyloge-
netic analysis in order to identify species groups within Gil-
bertiodendron and to resolve relationships among genera con-
sidered closely related to Gilbertiodendron (Bruneau et al.
2008). More specifically, the aims of the present study are (1)
to test the monophyly of Gilbertiodendron as currently cir-
cumscribed, in particular to determine whether the inclusion
of Pellegriniodendron within Gilbertiodendron based on mor-
phological characters is supported by the phylogenetic results,
and (2) to assess the phylogenetic relationships of Gilbertio-
dendron with the other genera of the Berlinia clade. To address
these issues, we sequenced and analyzed the plastid trnL intron
and trnL-F spacer and the nuclear ribosomal internal tran-
scribed spacers (ITS) in Gilbertiodendron and related genera.
Material and Methods
Taxon Sampling
A total of 85 accessions, representing 28 species of Gil-
bertiodendron (including 10 accessions of G. diphyllum), 9
accessions representing 4 of the 5 recognized Plagiosiphon spe-
cies, 7 accessions representing 4 of 11 Didelotia species, and
1 accession of Librevillea klainei (monotypic genus) were sam-
pled. This is the widest sampling of Gilbertiodendron,Pla-
giosiphon, and Didelotia assembled to date for phylogenetic
analysis (the appendix provides voucher information). To test
the monophyly of Gilbertiodendron and examine relationships
among Detarieae genera, particularly in the Berlinia clade, we
sampled 10 other Berlinia clade genera and 5 representative
genera of the Brownea clade (sensu Bruneau et al. 2008), in-
cluding Macrolobium, which in the past has been considered
a close relative of Gilbertiodendron (see appendix for refer-
ences). Barnebydendron riedelii (Tul.) J. H. Kirkbr. and Schotia
latifolia Jacq. were included as outgroup taxa to root the trees
(Bruneau et al. 2008). Samples collected in the field were pre-
served in silica gel, and other samples were obtained from dried
herbarium specimens.
Molecular Methods
DNA extraction of herbarium and silica gel–dried material
was done using a modified protocol from Ky et al. (2000)
rescaled for a total 3 mL of nucleic extraction buffer (15 mM
Tris, 2 mM EDTA, 80 mm KCl, 20 mM NaCl, 2% b-mer-
captoethanol, PPVP 2%, 0.5% Trixon-X100), and the pellet
was re-covered in 2 mL of lysis buffer pH 8 (0.1 M Tris, 0.02
M EDTA, 1.25 M NaCl, MATAB 4%).
The polymerase chain reaction (PCR) amplification mix in
reaction volumes of 50 mL contained four units of Taq DNA
polymerase, 1#Taq DNA polymerase buffer with 1.5 mmol
MgCl
2
(New England Biolabs, Pickering, Ontario, Canada),
200 mmol/L of each dNTP (Fermentas, Burlington, Ontario,
Canada), 3 mmol/L of each primer, and 50–100 ng of genomic
DNA. For samples that were difficult to amplify, BSA (0.1 mg/
mL; New England BioLabs, Ipswich, MA), Tween 20 (0.03%;
J.-T. Baker, Phillipsburg, NJ), and pure DMSO (4%; Fisher
Scientific, Ottawa, Ontario, Canada) were also added to the
mix.
To maximize the yield of PCR products for the trnL and
trnL-F regions, a nested PCR method was used, as described
by Sinou et al. (2009), with the primer pairs “c”-“f,” and then
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Table 1
Sequence Characteristics, Parsimony and Bayesian Analysis Statistics, and Choice of Evolutionary Model for Each of the Matrices and
Subsets of the Matrices Analyzed in the Phylogenetic Analysis of the Genus Gilbertiodendron and Related Genera
ITS trnL intron trnL-F spacer Combined
No. sequences 127 110 92 127
Aligned length (bp) 1084 966 629 2679
Indels 286 68 72 426
Excluded characters (%) 71 (6.6) 106 (11) 0 177 (6.6)
Variable characters (%) 825 (63.5) 242 (26.7) 217 (31) 1284 (44.2)
Parsimony informative characters (%) 450 (34.6) 99 (10.9) 76 (10.8) 619 (21.3)
CI, RI, length .50, .75, 2227 .82, .90, 326 .78, .85, 303 .55, .75 2955
% GC content 44.5 20.9 27.2 31.9
Evolutionary model, AIC GTR⫹I⫹GGTR⫹I⫹GGTR⫹I⫹GGTR⫹I⫹G
Note. CI pconfidence index, RI pretention index, AIC pAkaike Information Criterion.
the plastid trnL (UAA) intron and the spacer between trnL
(UAA) and trnF (GAA) were, respectively, amplified and se-
quenced with the primer pairs “c”-“d” and “e”-“f,” as de-
scribed in Taberlet et al. (1991). For the ITS region, amplifi-
cations were performed with the “AB101” and “AB102”
primers (Sun et al. 1994; Douzery et al. 1999). Conditions for
the amplification of the trnL-F region were as follows: 5 min
of initial denaturation at 95⬚C, followed by 30 cycles of 30 s
at 95⬚C, 45 s at 50⬚C, and 90 s at 72⬚C, with a final step of
7 min at 72⬚C. For the ITS region, PCR amplification condi-
tions were 4 min of initial denaturation at 94⬚C, followed by
32 cycles of 30 s at 94⬚C, 45 s at 53⬚C, and 90 s at 72⬚C,
with a final step of 7 min at 72⬚C.
Sequencing was performed with Big Dye Terminator 3.1
chemistry on an ABI 3730xl DNA Analyzer (Applied Biosys-
tems, Carlsbad, CA) at the Genome Quebec facilities (Mon-
treal, Canada). Sequences were assembled and edited with Ge-
neious 4.8.5 (Biomatters, http://www.geneious.com).
Phylogenetic Analyses
Sequence alignment was performed with Geneious 4.8.5
(Biomatters, http://www.geneious.com) using the default pa-
rameters. Alignments were then verified and modified manu-
ally where inconsistencies were found. Nonautapomorphic
simple gaps (indels) were scored as separate presence/absence
characters, following Simmons and Ochoterena (2000), as im-
plemented in SeqState 1.4.1 (Mu¨ller 2005).
Individual matrices (plastid and nuclear markers) were an-
alyzed separately for exploratory purposes, and a concatenated
data matrix (cp⫹nuc) was analyzed to generate the phyloge-
netic tree. The trnL intron matrix included sequences from
110 accessions (19 genera), the trnL-F intergenic spacer matrix
included data from 92 accessions (6 genera), and the ITS ma-
trix included 127 accessions (21 genera).
Parsimony analyses were performed with PAUP*, version
4.0b10 (Swofford 2003). A first heuristic search was per-
formed with 1000 replicates of random addition sequence, tree
bisection-reconnection (TBR) branch-swapping, retaining only
five most parsimonious trees at each replicate. Starting with
the trees kept in memory from this initial analysis, a second
heuristic search was performed with TBR and a limit of
100,000 trees saved. Because this second analysis uses the to-
pologies obtained initially, it permits the investigation of more
optimal topologies than a “one-step” analysis (e.g., Davis et
al. 2004). Branch support was estimated from 5000 bootstrap
replicates under a heuristic strategy with one random addition-
sequence replicate, TBR branch-swapping, and a maximum
number of trees set at 100.
The Bayesian analyses were implemented in a parallel ver-
sion of Mr. Bayes (ver. 3.2.1; Ronquist and Huelsenbeck 2003).
Computations were made on the supercomputer Cottos from
the Universite´ de Montre´ al, managed by Calcul Que´ bec and
Compute Canada. The GTR⫹I⫹Gnucleotide substitution
model was selected using the Akaike Information Criterion
(Akaike 1974) as implemented in ModelTest (ver. 3.7; Posada
and Crandall 1998) for all DNA nucleotide partitions. The
model for the gap partition was set to “restriction data” (F81-
like model), as suggested by Ronquist and Huelsenbeck (2003).
The Bayesian estimation consisted of two independent runs,
each for 50 #10
6
generations, sampling trees and parameters
every thousandth generation. Each run consisted of four si-
multaneous Monte Carlo Markov chains and four swaps per
generation. All sample points prior to reaching stationarity of
the chains were discarded (equivalent to discarding the first
5#10
6
generations as burn-in). Convergence was assessed
by comparing majority-rule consensus trees from the two anal-
yses and using Tracer (ver. 1.5; Rambaut and Drummond
2007) to compare density plots of the estimated parameters
and of the likelihoods from the two analyses. The posterior
probabilities for individual clades were compared for congru-
ence and summarized on a majority-rule consensus.
Results
Sequence Characteristics
Length, number of indels, number of variable characters,
and statistics for the phylogenetic analyses are given in table
1 for each of the regions studied (individual and combined
matrices). The trnL intron was 470–700 bp in length, the trnL-
trnF spacer was ∼450–600 bp, and the ITS region was gen-
erally 780–930 bp in length. A total of 619 (21.3%) parsimony
informative characters were obtained from the combined anal-
ysis (table 1), with a considerably higher proportion from the
ITS region (34.6%) than from the trnL intron (10.9%) or the
trnL-trnF spacer (10.8%).
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Fig. 1 Phylogenetic analysis of the chloroplast trnL intron and trnL-trnF intergenic spacer and the nuclear internal transcribed spacers for Gilbertiodendron and related caesalpinioid genera.
Bayesian majority-rule consensus derived from 45,000 trees kept after reaching stationarity in two independent analyses. Posterior probabilities (mostly above branches) and bootstrap support values
from the parsimony analysis (mostly below branches) are noted. Branches in bold are those supported by a 1.0 posterior probability; clades that are unresolved in the parsimony analysis are indicated
by broken lines. For species represented by two specimens, the collector name and the collection number are indicated after the species name.
DE LA ESTRELLA ET AL.—GILBERTIODENDRON PHYLOGENY (LEGUMINOSAE) 5
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(Fig. 1, continued)
Phylogenetic Analysis
In both the parsimony and Bayesian analyses, the consensus
trees resulting from the analysis of the ITS data alone (not
shown) were better resolved than the consensus trees from the
trnL and trnL-trnF analyses alone (not shown), but the three
analyses yielded topologies with equivalent relationships
among groups. The parsimony analysis of the combined data
set reached the maximum number of trees retained in memory
(length p2955, confidence index p0.55, retention index p
0.75) and yielded a poorly resolved strict consensus tree (see
fig. 1). The Bayesian majority-rule consensus tree of the com-
bined matrix yielded a topology among groups similar to that
obtained from the parsimony analysis of the combined data
but with better resolution (fig. 1, with posterior probabilities
indicated above the branches).
Our analysis places the genera Microberlinia and Oddon-
iodendron (fig. 1a, bootstrap support [BS] p72%, posterior
probability [PP] p1.00) as a sister clade to the other Berlinia
clade genera. The monophyletic Babijt group defined by Wier-
inga and Gervais (2003) is weakly supported as sister to the
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Didelotia species included in our analyses (BS !50%, PP p
0.74). A clade including all accessions of Berlinia,Isoberlinia,
and Anthonotha is placed as sister to Librevillea klainei (BS
!50%, PP p0.99), and this entire clade is moderately sup-
ported (BS p61%, PP p1) as sister to a group that includes
the American Brownea clade sister to the Plagiosiphon species
sampled (BS p60%, PP p0.84) and a clade that constitutes
all the Gilbertiodendron species.
In our analysis, Gilbertiodendron is strongly supported as
monophyletic (fig. 1b;BSp100%, PP p1.00) including all
accessions of G. diphyllum previously recognized as Pellegri-
niodendron diphyllum. Within Gilbertiodendron (fig. 1b), sev-
eral groups are resolved, but many with low support. The west
tropical African Gilbertiodendron species are resolved into two
clades, the “aylmeri” clade (BS p67%, PP p0.82) and the
“ivorense” clade (!50%, 0.69), both made up of species that
grow in the tropical forest from Guinea to east Ghana (with
the exception of G. demonstrans, which occurs in central Af-
rica). The “dewevrei” clade (BS !50%, PP p0.78), with
species from central Africa, includes G. dewevrei,G. imen-
oense,G. stipulaceum,G. grandistipulatum, and G. mayo-
mbense, as well as unidentified Gilbertiodendron samples. The
strongly supported “ogoouense complex” (BS p96%, PP p
1.00) includes a group of species identified as G. klainei,G.
ogoouense, and G. brachystegioides, along with several new
species to be described. All three samples of G. preussii group
in a moderately supported clade, but those of its close relative,
G. diphyllum, are not monophyletic (fig. 1b) in the combined
plastid and ITS analyses. However, in the ITS analyses alone
(results not shown), G. diphyllum samples form a single clade.
Discussion
Generic Relationships of Gilbertiodendron
In our analyses, Gilbertiodendron is resolved as a mono-
phyletic genus moderately supported as sister to a clade con-
stituted of Plagiosiphon and the American Brownea clade. Pre-
vious phylogenetic studies by Bruneau et al. (2008) placed
Gilbertiodendron in a poorly supported clade with three gen-
era—Didelotia, Librevillea, and Plagiosiphon—but with no
resolution among the four genera. Although we include these
3 genera, as well as 10 other Berlinia clade genera, our analyses
do not support a monophyletic Berlinia clade because of the
nested position of the Brownea clade. However, as in all other
phylogenetic analyses of Detarieae, generic-level relationships
in the Amherstieae clade are poorly resolved (LPWG 2013a).
Nevertheless, Plagiosiphon, a genus of five species of trees and
shrubs that grow gregariously in lowland forests and along
rivers (Mackinder 2005), is here supported as monophyletic
with four of the five species sampled (fig. 1a). Didelotia is also
supported as monophyletic (fig. 1a), but it occurs, albeit with
little support, as sister to the Babijt clade (fig. 1a)ofWieringa
and Gervais (2003). The monospecific genus Librevillea is here
resolved as sister to representatives of three other genera of
the Berlinia group (fig. 1a), a relationship that is supported
only in the Bayesian analysis. Thus, despite better species-level
sampling with more samples per species, relationships of Gil-
bertiodendron within the Amherstieae clade remain unclear.
Gilbertiodendron was included within tribe Amherstieae by
Le´onard (1957) based on the position of the bracteoles at the
upper part of the pedicel. Cowan and Polhill (1981) maintained
Gilbertiodendron within that tribe, but they questioned whether
Amherstieae was monophyletic. Breteler (1995) proposed a
modified classification that recognized Gilbertiodendron (in-
cluding Pellegriniodendron and 21 more genera) within the
newly delimited tribe Macrolobieae. Phylogenetic analysis of
trnL data confirmed the position of Gilbertiodendron inaMa-
crolobieae clade (Bruneau et al. 2001), which largely but not
completely corresponded to Breteler’s (1995) delimitation of
tribe Macrolobieae. Subsequently, this lineage made up of the
majority of the genera (but not Macrolobium) was renamed the
Berlinia clade (Bruneau et al. 2008). Mackinder (2005), in a
synopsis of all previous morphological and molecular studies,
considered both Detarieae and Amherstieae (including the Ber-
linia clade) within the tribe Detarieae sensu lato.
Species Relationships within Gilbertiodendron
Our analyses support Gilbertiodendron as monophyletic,
including all accessions of G. diphyllum previously recognized
as Pellegriniodendron. Several of the characters used by Le´-
onard (1957) and Cowan and Raven (1981) to distinguish
between Pellegriniodendron and Gilbertiodendron were found
to be of weak or no value (Estrella et al. 2012a). Le´onard
(1957) and Cowan and Polhill (1981) characterized Pellegri-
niodendron diphyllum (G. diphyllum) by the presence of sti-
pels on leaflets, but Estrella et al. (2012a) found several mature
specimens of G. unijugum with “stipels” present at the base
of the petiolules and in seedlings of other species. Gilbertio-
dendron diphyllum is an easily identifiable species with a single
normal pair of leaflets and a basal pair that is reduced to small
structures, sometimes referred to as stipels. The presence of
submarginal crateriform glands on the leaflets blades of G.
diphyllum is the only character that can be used to consider
Pellegriniodendron as a segregate genus. Extrafloral nectaries
are common in other genera of the Detarieae, and in some
groups, this feature is an informative taxonomic character for
species-level delimitations (e.g., in Daniellia; Estrella et al.
2010). In the Detarieae, these crateriform glands are usually
present on the leaflet blades, as reported in the Babijt clade
(fig. 1b)inAphanocalyx (Wieringa 1999), but in Gilbertio-
dendron, crateriform glands are found only in G. diphyllum,
which is sister to the Gilbertiodendron clade (fig. 1b). The
remaining Gilbertiodendron species have a different and pos-
sibly autapomorphic type of gland, located on the leaflet mar-
gins and not found in any other Detarieae genera. These mar-
ginal glands have been used as a key character for the easy
identification of the genus (e.g., Aubre´ville 1968). Although
the function and nature of the leaflet margin glands is not clear
(E. Smets, personal communication), their presence may be
related to ants, which have been reported as living in associ-
ation with Gilbertiodendron species (e.g., fig. 1 from Estrella
and Devesa 2014).
The species of Gilbertiodendron endemic to west tropical Af-
rica (Upper Guinea: Senegal to Togo) form two clades, but the
relationship between these two clades is not resolved (fig. 1b).
Clade aylmeri includes the recently published species G. ton-
kolili, which cannot be differentiated vegetatively from G. bil-
ineatum but is considered distinct from this species based on
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Fig. 2 Distribution of the genus Gilbertiodendron in Africa: 1 pwest tropical Africa, 2 pwest-central Africa, 3 pcentral Africa. The
arrow indicates the location of the Dahomey Gap biogeographic area.
floral and fruiting characters (Estrella et al. 2012c). Our analysis
resolves the two confirmed accessions of G. tonkolili as most
closely related to G. aylmeri, with strong support. In the sister
clade, G. bilineatum occurs with a recently described species,
G. jongkindii (Estrella and Devesa 2014), which is morpholog-
ically similar to G. obliquum, a species that is resolved as part
of the ivorense clade, the second clade of west tropical African
species (fig. 1b). Gilbertiodendron jongkindii has a symmetric
leaflet base and sepals that are densely hairy at the margins, two
characters that differentiate it from G. obliquum. In addition to
G. obliquum, the ivorense clade includes four other west tropical
African species—G. ivorense,G. limba,G. robynsianum, and
G. sp. nov. Jongkind 8884—and the central African G. de-
monstrans, but with a broad distribution that extends to Nigeria.
These phylogenetic results support the taxonomic framework
proposed for the western African species proposed by Estrella
and Devesa (2014), who recognize G. tonkolili and G. jong-
kindii as different species (fig. 1b).
All four accessions of G. unijugum form a strongly sup-
ported monophyletic group, weakly resolved as sister to one
of the three specimens of G. mayombense sampled (fig. 1b).
This G. mayombense specimen from the Korup National Park
(Cameroon) may represent a segregate species distinct from
the central African G. mayombense (Angola, Gabon, and D.
R. Congo), which group with the dewevrei clade. A detailed
comparative study of these samples will be done during the
ongoing taxonomic revision.
Of the ∼30 species within Gilbertiodendron, only G. di-
phyllum and G. preussii have a distribution that reaches both
margins of the Dahomey Gap (fig. 2). The Dahomey Gap is
a woodland and wooded grassland region from east Ghana to
Benin (Booth 1958) that results from a climatic anomaly as-
sociated with low precipitation (Salzmann and Hoelzmann
2005). Although the Dahomey Gap area is presently covered
with agricultural land, savannah, and deteriorated dry forest,
it has been suggested to be a barrier to rain forest species
dispersal (Hawthorne and Jongkind 2006), and within it, rem-
nant forest patches are considered biodiversity refugia that
should be conserved (Backe´us 1992; Chaı¨r et al. 2011). This
distribution on both sides of the Dahomey Gap is uncommon
for a Detarieae species (found in only 33 out of the 295 in-
digenous Detarieae species from west, west-central, and central
Africa; Lock 1989), probably as a consequence of the relatively
short and limited maximum dispersal distance of the ballistic
seeds (van der Burgt et al. 2012). Despite this unusual distri-
bution, no significant morphological differences were observed
between specimens from west tropical Africa (Senegal to
Ghana) and those from west-central Africa (Cameroon, Equa-
torial Guinea, and Gabon; Estrella et al. 2012a; Estrella and
Devesa 2014). Both G. diphyllum and G. preussii have been
reported from secondary forests, swampy areas, and near man-
groves, possibly explaining the wide distribution of these two
species beyond the Dahomey Gap, since small pockets of for-
ests along swamps and rivers have been far more widespread
during periods of forest contractions than the poper refuges
of dry-land rain forest. However, all four specimens of G.
diphyllum from west tropical Africa group into a single clade,
distinct from the specimens from west-central Africa (fig. 1b),
indicating the likelihood of a genetic isolation between the two
areas of the distribution of this species.
The dewevrei clade (fig. 1b) is a polytomy that includes
accessions representing at least five species from west-central
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and central Africa, with a broad morphological variability. The
dewevrei clade includes G. grandistipulatum,G. mayombense,
G. imenoense,G. stipulaceum, and G. dewevrei. Gilbertio-
dendron dewevrei forms extensive patches of monodominant
forest (Aubre´ville 1970; van Valkenburg et al. 1998; Peh et al.
2011) of great ecological and conservation importance (Peh et
al. 2011). In studies on monodominant forests of G. dewevrei
in central Africa, Peh et al. (2011) showed that dominance is
not related to soil type but rather to seed-dispersal capacity
or to the ectomycorrhizal status of the species. Other species
of Gilbertiodendron form codominant patches, where they
grow mixed with other species of Caesalpinioideae, as well as
with trees from other families. Van der Burgt et al. (2012)
hypothesized that the codominant patches are the consequence
of the relatively short and strictly limited maximum dispersal
distance of the ballistic seeds typical of most Detarieae, in-
cluding all Gilbertiodendron species.
In the ogoouense complex clade, the unclear position of
samples previously identified either as G. ogoouense,G. bra-
chystegioides,G. klainei,orG. newberyi is in accordance with
the result of the morphological study of the group suggesting
the existence of several new taxa. In ongoing taxonomic re-
visions (van der Burgt et al., forthcoming), taxa of the
ogoouense complex will be divided into several new species
(fig. 1b). A common characteristic of taxa in the complex is
the presence of small flowers and congested inflorescences. The
specimens within this group share the absence of a gland at
the apex of the bracteoles, in contrast to other Gilbertioden-
dron species where the apical glands within bracteoles can be
more than 3 mm in length (Estrella and Devesa 2014).
Ongoing agriculture and clear-cutting of forest leading to
severe habitat loss and degradation have been identified as
well-known threats to Gilbertiodendron populations, and
these continue to contribute to their decline. In order to be
considered for a conservation rank, a species should ideally
be described to reflect evolutionary entities, and the species-
level phylogeny presented here provides some of the back-
ground information needed to establish the adequate assess-
ments. Of the ∼30 species within the genus, only 8 have been
assessed so far under the International Union for Conservation
of Nature (IUCN) Red List criteria. From west tropical Africa
(fig. 2), five species are assessed: G. bilineatum,G. robyn-
sianum, and G. splendidum are classified as vulnerable (VU);
G. limba is considered near threatened; and the recently pub-
lished species G. tonkolili is assessed as critically endangered
(Estrella et al. 2012c; IUCN 2013). This would suggest that
the aylmeri and ivorense clades include the most endangered
groups within the genus, but this may reflect the bias in the
sampling assessment with a greater focus on the west tropical
African flora. For west-central Africa and central Africa (fig.
2), three species were assessed: G. klainei (VU), G. newberyi
(endangered), and G. pachyanthum (VU; van der Burgt et al.
2012; IUCN 2013). None of the other species of Gilbertio-
dendron have been assessed, and of the eight existing assess-
ments, five were made in 1998 and need reevaluation in light
of new data. The new data derived from this phylogenetic
study and the ongoing taxonomic treatment of the genus will
help establish the adequate assessments for the genus, which
includes many timber species currently overexploited in trop-
ical Africa.
Acknowledgments
We wish to thank the staff of the cited herbaria for their
support during our visits and for loan of material as well as
Annie Archambault, Marielle Babineau, Iva´n de la Providen-
cia, Fatima El Ayadi, Edeline Gagnon, Carole Sinou, Royce
Steeves, Amy Vandal, and Erin Zimmerman for their help at
the Institut de Recherche En Biologie Ve´ge´tale (IRBV) labs at
Montreal University. Manuel de la Estrella was funded by a
Juan de la Cierva grant (JCI-2009-05243) and visited the IRBV
with a Jose´ Castillejo grant (JC2011-0309), both from the
Spanish Ministry of Education; he visited the K, P, and WAG
herbaria under the FP7-funded Integrated Infrastructure Ini-
tiative SYNTHESYS (grants GB-TAF-1006, FR-TAF 2194,
and NL-TAF 2377, respectively). Work undertaken in this
study was also funded by a grant from the Natural Sciences
and Engineering Research Council of Canada to Anne Bruneau
and a travel award from the Que´bec Centre for Biodiversity
Science to de la Estrella. The operation of the supercomputer
Cottos is funded by the Canada Foundation for Innovation,
NanoQue´bec, Re´ seau de Me´ decine Ge´ne´tique Applique´e, and
the Fonds de Recherche du Que´bec–Nature et Technologies.
We would like to thank the two anonymous reviewers for their
valuable comments and the editor in chief, Patrick S. Heren-
deen, for his comments and help.
Appendix
Specimens Studied, Voucher Information, and Genbank Accession Numbers for the
Phylogenetic Analysis of the Genus Gilbertiodendron and Related Genera
All vouchers are deposited at the indicated herbaria. Sequences are compiled from previous works, indicated by superscript
lowercase letters: (a) Bruneau et al. (2001), (b) Gervais and Bruneau (2002), (c) Fouge`re-Danezan et al. (2007), (d) Redden et
al. (2010), and (e) Mackinder and Pennington (2011). Data are ordered by ITS, trnL intron, and trnL-F spacer. Missing data
are denoted with a dash.
Outgroup
Barnebydendron riedelii (Tul.) J.H. Kirkbr., Brammall s. n., no. 1953-35501(K), AY955777
c
, AF365209
a
, AY958491
c
;Schotia
latifolia Jacq., Bruneau s.n., no 1948-52201 (K), AY955775
c
, AF365124
a
, AY958528
c
.Related genera: Anthonotha macrophylla
P. Beauv., Wieringa 2996 (WAG), AF513653
b
, AF365234
a
,—;Aphanocalyx cynometroides Oliver, Wieringa 2355 (WAG),
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AF513654
b
, AF365244
a
,—;Aphanocalyx microphyllus subsp. compactus (Hutchinson ex Lane-Poole) Wieringa, Breteler 13356
(WAG), AF513662
b
, AF365246
a
,—;Berlinia auriculata Benth., Wieringa 5283 (WAG), HM041834
e
,—,—;Berlinia congolensis
(Baker f.) Keay, Harris 8585 (E), HM041826
e
,—,—;Berlinia grandiflora (Vahl) Hutch. & Dalziel, Harris 2895 (E), HM041821
e
,
—, —; Bikinia durandii (F. Halle´ & Normand) Wieringa, Wieringa 3021 (WAG), AF513676
b
, AY116896
b
,—;Bikinia grisea
Wieringa, Breteler 13334 (WAG), AF513678
b
, AY116897
b
,—;Brachystegia longifolia Benth., Herendeen 21-xii-97-2 (US),
AF513687
b
,—,—;Elizabetha macrostachya Benth., Redden 3714 (US), FJ817516
d
, FJ817559
d
,—;Elizabetha speciosa Ducke,
Rodrigues & Coelha 4850 (US), FJ817521
d
,—,—;Heterostemon impar Spruce ex. Benth., Amaral et al. 229 (K), FJ817524
d
,
FJ817562
d
,—;Heterostemon mazarunensis Sandwith, Redden 3203 (US), FJ817526
d
,—,—;Isoberlinia doka Craib & Stapf,
Jongkind 2552a (WAG), AF513691
b
, AF365220
a
,—;Julbernardia brieyi (De Wild.) Troupin, Wieringa 3348 (WAG), AF513692
b
,
AF365264
a
,—;Macrolobium campestre Huber, Redden 3649 (US), FJ817499
d
, FJ817551
d
,—;Macrolobium gracile Spruce ex.
Benth., Redden 3687 (US), FJ817500
d
, FJ817552
d
,—;Macrolobium multijugum var. multijugum (DC.) Benth., Redden 3700
(US), FJ817502
d
, FJ817554
d
,—;Microberlinia brazzavillensis A. Chev., Wieringa 2516 (WAG), AF513697
b
, AF365222
a
,—;
Oddoniodendron normandii Aubrev., Breteler 12608 (WAG), AF513698
b
, AF365224
a
,—;Paloue riparia Pulle, Redden 1161A
(US), FJ817546
d
, EU361826
d
,—;Paloveopsis emarginata R. S. Cowan, Cid Ferreira et al. 916 (NY), FJ817533
d
, FJ817571
d
,
—; Tetraberlinia bifoliolata (Harms) Hauman, Wieringa 3304 (WAG), AF513700
b
, AF365227
a
,—.Study group: Didelotia
africana Baill., Sosef 2509 (WAG), KJ777183, KJ777375, KJ777285; Didelotia africana Baill., Wieringa 3549 (WAG), KJ777184,
KJ777376, KJ777286; Didelotia letouzeyii Pellegr., Wieringa 5065 (WAG), KJ777185, KJ777377, KJ777287; Didelotia letouzeyii
Pellegr., Wieringa 6120 (WAG), KJ777186, KJ777378, KJ777288; Didelotia letouzeyii Pellegr., Wieringa 6187 (WAG), KJ777187,
KJ777379, KJ777289; Didelotia minutiflora (A. Chev.) J. Le´onard, Wieringa 4819 (WAG), KJ777188, KJ777380, KJ777290;
Didelotia unifoliolata J. Le´onard, Sosef 2517 (WAG), KJ777189, KJ777381, KJ777291; Gilbertiodendron aylmeri (Hutch. &
Dalziel) J. Le´onard, Jongkind 7003 (WAG), KJ777191, KJ777383, KJ777293; Gilbertiodendron aylmeri (Hutch. & Dalziel) J.
Le´onard, van der Burgt 1458 (WAG), KJ777190, KJ777382, KJ777292; Gilbertiodendron bilineatum (Hutch. & Dalziel) J.
Le´onard, Jongkind 5437 (WAG), KJ777193, —, —; Gilbertiodendron bilineatum-tonkolili, Jongkind 8775 (WAG), KJ777192,
KJ777384, KJ777294; Gilbertiodendron brachystegioides (Harms) J. Le´onard, Breteler 14983 (WAG), KJ777194, KJ777385,
—; Gilbertiodendron brachystegioides (Harms) J. Le´onard, van der Burgt 801 (WAG), KJ777195, KJ777386, KJ777295; Gil-
bertiodendron brachystegioides (Harms) J. Le´onard, Wieringa 2242 (WAG), KJ777196, KJ777387, KJ777296; Gilbertiodendron
brachystegioides (Harms) J. Le´onard, Wieringa 6226 (WAG), KJ777197, KJ777388, KJ777297; Gilbertiodendron comb. nov.,
M’Boungou 144 (WAG), KJ777199, KJ777390, KJ777299; Gilbertiodendron comb. nov., M’Boungou 387 (WAG), KJ777200,
KJ777391, —; Gilbertiodendron comb. nov., Wieringa 6070 (WAG), KJ777201, —, —; Gilbertiodendron demonstrans (Baill.)
J. Le´onard, van der Burgt 731 (WAG), KJ777202, KJ777392, KJ777300; Gilbertiodendron demonstrans (Baill.) J. Le´onard,
Wieringa 2202 (WAG), KJ777203, —, KJ777301; Gilbertiodendron demonstrans (Baill.) J. Le´onard, Wieringa 2362 (WAG),
KJ777204, —, —; Gilbertiodendron dewevrei (De Wild.) J. Le´onard, Andel 4045 (WAG), KJ777205, KJ777393, KJ777302;
Gilbertiodendron dewevrei (De Wild.) J. Le´onard, van der Burgt 783 (WAG), KJ777206, KJ777394, KJ777303; Gilbertiodendron
dewevrei (De Wild.) J. Le´onard, van der Burgt 905 (WAG), KJ777207, KJ777395, KJ777304; Gilbertiodendron dewevrei (De
Wild.) J. Le´onard, Wieringa 3552 (WAG), KJ777208, KJ777396, KJ777305; Gilbertiodendron dewevrei (De Wild.) J. Le´onard,
Wieringa 6091 (WAG), KJ777209, KJ777397, KJ777306; Gilbertiodendron dewevrei (De Wild.) J. Le´onard, Wieringa 6261
(WAG), KJ777210, KJ777398, KJ777307; Gilbertiodendron diphyllum (Harms) Estrella & Devesa, Andel 3502 (WAG),
KJ777211, KJ777399, KJ777308; Gilbertiodendron diphyllum (Harms) Estrella & Devesa, Andel 4111 (WAG), KJ777212,
KJ777400, KJ777309; Gilbertiodendron diphyllum (Harms) Estrella & Devesa, Breteler 13402 (WAG), KJ777213, KJ777401,
KJ777310; Gilbertiodendron diphyllum (Harms) Estrella & Devesa, Breteler 13734 (WAG), KJ777214, KJ777402, KJ777311;
Gilbertiodendron diphyllum (Harms) Estrella & Devesa, Jongkind 9311 (WAG), KJ777215, KJ777403, KJ777312; Gilbertio-
dendron diphyllum (Harms) Estrella & Devesa, Louis 2481 (WAG), KJ777216, KJ777404, KJ777313; Gilbertiodendron di-
phyllum (Harms) Estrella & Devesa, Parren 306 (WAG), KJ777217, —, KJ777314; Gilbertiodendron diphyllum (Harms) Estrella
& Devesa, Wieringa 4105 (WAG), KJ777219, KJ777406, KJ777316; Gilbertiodendron diphyllum (Harms) Estrella & Devesa,
Wieringa 6218 (WAG), KJ777220, KJ777407, KJ777317; Gilbertiodendron diphyllum sp. nov., Breteler 10782 (WAG),
KJ777218, KJ777405, KJ777315; Gilbertiodendron grandistipulatum (De Wild.) J. Le´onard, Breteler 10428 (WAG), KJ777221,
KJ777408, KJ777318; Gilbertiodendron grandistipulatum (De Wild.) J. Le´onard, White 1118 (WAG), KJ777222, KJ777409,
KJ777319; Gilbertiodendron grandistipulatum (De Wild.) J. Le
´onard, Wieringa 4687 (WAG), KJ777223, KJ777410, KJ777320;
Gilbertiodendron imenoense (Pellegr.) J. Le´onard, Breteler 9974 (WAG), KJ777224, KJ777411, KJ777321; Gilbertiodendron
ivorense (A. Chev.) J. Le´onard, Jongkind 9210 (WAG), KJ777225, KJ777412, KJ777322; Gilbertiodendron ivorense (A. Chev.)
J. Le´onard, Stoop 19 (WAG), KJ777226, KJ777413, KJ777323; Gilbertiodendron jongkindii Estrella & Devesa, Jongkind 4502
(WAG), KJ777227, KJ777414, KJ777324; Gilbertiodendron jongkindii Estrella & Devesa, Jongkind 9350 (WAG), KJ777228,
KJ777415, KJ777325; Gilbertiodendron klainei (Pierre ex Pellegr.) J. Le´onard, Wieringa 1239 (WAG), KJ777229, KJ777416,
KJ777326; Gilbertiodendron klainei (Pierre ex Pellegr.) J. Le´onard, Wieringa 2443 (WAG), KJ777230, KJ777417, KJ777327;
Gilbertiodendron limba (Scott-Elliot) J. Le´onard, Jongkind 1438 (WAG), KJ777231, KJ777418, KJ777328; Gilbertiodendron
mayombense (Pellegr.) J. Le´onard, Breteler 14147 (WAG), KJ777232, KJ777419, KJ777329; Gilbertiodendron mayombense
(Pellegr.) J. Le´onard, Wieringa 1475 (WAG), KJ777233, —, —; Gilbertiodendron cf. mayombense, van der Burgt 595 (WAG),
KJ777198, KJ777389, KJ777298; Gilbertiodendron newberyi Burgt, van der Burgt 774 (WAG), KJ777234, KJ777420,
KJ777330; Gilbertiodendron newberyi Burgt, van der Burgt 776 (WAG), KJ777235, KJ777421, KJ777331; Gilbertiodendron
obliquum (Stapf) J. Le´onard, Jongkind 9972 (WAG), KJ777236, KJ777422, KJ777332; Gilbertiodendron ogoouense (Pellegr.)
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J. Le´onard, Breteler 10258 (WAG), KJ777237, KJ777423, KJ777333; Gilbertiodendron ogoouense (Pellegr.) J. Le´onard, Breteler
11450 (WAG), KJ777238, —, —; Gilbertiodendron ogoouense (Pellegr.) J. Le´onard, Breteler 11524 (WAG), KJ777239,
KJ777424, KJ777334; Gilbertiodendron ogoouense (Pellegr.) J. Le´onard, Schoenmaker 70 (WAG), KJ777242, KJ777427,
KJ777336; Gilbertiodendron ogoouense (Pellegr.) J. Le´onard, van der Burgt 775 (WAG), KJ777240, KJ777425, KJ777335;
Gilbertiodendron ogoouense (Pellegr.) J. Le´onard, Wieringa 1316 (WAG), KJ777243, —, —; Gilbertiodendron ogoouense
(Pellegr.) J. Le´onard, Wieringa 2777 (WAG), KJ777244, KJ777428, KJ777337; Gilbertiodendron ogoouense (Pellegr.) J. Le´onard,
Mpondzou 44 (WAG), KJ777241, KJ777426, —; Gilbertiodendron preussii (Harms) J. Le´onard, Andel 4118 (WAG), KJ777245,
KJ777429, KJ777338; Gilbertiodendron preussii (Harms) J. Le´onard, van der Burgt 794 (WAG), KJ777246, KJ777430,
KJ777339; Gilbertiodendron preussii (Harms) J. Le´onard, Wieringa 1600 (WAG), KJ777247, KJ777431, KJ777340; Gilberti-
odendron robynsianum Aubre´v. & Pellegr., Jongkind 4525 (WAG), KJ777248, KJ777432, KJ777341; Gilbertiodendron robyn-
sianum Aubre´v. & Pellegr., Jongkind 4942 (WAG), KJ777249, KJ777433, KJ777342; Gilbertiodendron robynsianum Aubre´v.
& Pellegr., Jongkind 5644 (WAG), KJ777250, KJ777434, KJ777343; Gilbertiodendron sp., Sosef 2502 (WAG), KJ777261,
KJ777445, KJ777353; Gilbertiodendron sp., Wieringa 1315 (WAG), KJ777262, —, —; Gilbertiodendron sp., Wieringa 3793
(WAG), KJ777263, KJ777446, KJ777354; Gilbertiodendron sp., Wieringa 5253 (WAG), KJ777264, KJ777447, KJ777355;
Gilbertiodendron sp. nov., Jongkind 8884 (WAG (Liberia), KJ777251, KJ777435, KJ777344; Gilbertiodendron sp. nov. A,
Bongou 105 (WAG), KJ777254, KJ777438, —; Gilbertiodendron sp. nov. A, Boungou 415 (WAG), KJ777255, KJ777439,
KJ777347; Gilbertiodendron sp. nov. A, Valkenburg 2635 (WAG), KJ777256, KJ777440, KJ777348; Gilbertiodendron sp. nov.
A, Valkenburg 2636 (WAG), KJ777257, KJ777441, KJ777349; Gilbertiodendron sp. nov. B, Breteler 14250 (WAG), KJ777258,
KJ777442, KJ777350; Gilbertiodendron sp. nov. B, Wieringa 6190 (WAG), KJ777259, KJ777443, KJ777351; Gilbertiodendron
sp. nov. B, Wieringa 6191 (WAG), KJ777260, KJ777444, KJ777352; Gilbertiodendron sp. nov.?, Wieringa 6099 (WAG),
KJ777253, KJ777437, KJ777346; Gilbertiodendron sp. nov., Wieringa 5057 (WAG), KJ777252, KJ777436, KJ777345; Gil-
bertiodendron splendidum (A. Chev. ex Hutch. & Dalziel) J. Le´onard, Jongkind 8782 (WAG), KJ777265, KJ777448, KJ777356;
Gilbertiodendron stipulaceum (Benth.) J. Le´onard, Schoenmaker 313 (WAG), KJ777266, KJ777449, KJ777357; Gilbertiodendron
stipulaceum (Benth.) J. Le´onard, Schoenmaker 385 (WAG), KJ777267, —, —; Gilbertiodendron stipulaceum (Benth.) J. Le´onard,
Wieringa 1644 (WAG), KJ777268, KJ777450, KJ777358; Gilbertiodendron tonkolili Burgt & Estrella, van der Burgt 1423
(WAG), KJ777269, KJ777451, KJ777359; Gilbertiodendron tonkolili Burgt & Estrella, van der Burgt 1457 (WAG), KJ777270,
KJ777452, KJ777360; Gilbertiodendron unijugum (Pellegr.) J. Le´onard, McPherson 15847 (WAG), KJ777272, KJ777454,
KJ777362; Gilbertiodendron unijugum (Pellegr.) J. Le´onard, van der Burgt 73 (WAG), KJ777271, KJ777453, KJ777361; Gil-
bertiodendron unijugum (Pellegr.) J. Le´onard, Wieringa 1075 (WAG), KJ777273, KJ777455, KJ777363; Gilbertiodendron un-
ijugum (Pellegr.) J. Le´onard, Wilde 10974 (WAG), KJ777274, KJ777456, KJ777364; Librevillea klainei (Pierre ex Harms) Hoyle,
Sosef 2505 (WAG), KJ777275, KJ777457, KJ777365; Plagiosiphon emarginatus (Hutch. & Dalziel) J. Le´onard, Wieringa 6067
(WAG), KJ777276, KJ777458, KJ777366; Plagiosiphon gabonensis J. Le´onard, Sosef 2503 (WAG), KJ777277, KJ777459,
KJ777367; Plagiosiphon gabonensis J. Le´onard, Wieringa 4400 (WAG), KJ777278, KJ777460, KJ777368; Plagiosiphon ga-
bonensis J. Le´onard, Wieringa 6137 (WAG), KJ777279, KJ777461, KJ777369; Plagiosiphon longitubus J. Le´onard, Wieringa
5864 (WAG), KJ777280, —, KJ777370; Plagiosiphon longitubus J. Le´onard, Wieringa 5870 (WAG), KJ777281, KJ777462,
KJ777371; Plagiosiphon multijugus J. Le´onard, Andel 4044 (WAG), KJ777282, KJ777463, KJ777372; Plagiosiphon multijugus
J. Le´onard, Wieringa 3813 (WAG), KJ777283, KJ777464, KJ777373; Plagiosiphon sp., Wieringa 4039 (WAG), KJ777284,
KJ777465, KJ777374.
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QUERIES TO THE AUTHOR
q1. I edited your article for clarity and agreement with
journal style, and minor revisions were made with-
out comment. Please review the text and advise
whether any of my changes are unacceptable. Any
layout problems or errors you see will be corrected
after the proof is returned.
q2. Should this be “80 mM KCl”?
q3. Here and in table 1, did spell out L, CI ,and RI
correctly? And, if Lplength, does the value require
a unit of measurement?
q4. If acceptable, we would like to add labels to figures
1a and 1b for ease of reading. They would appear
as labels a and b on the figures themselves. Is this
acceptable?
q5. Please add Cowan and Raven 1981 to the Liter-
ature Cited.
q6. Re. “the poper refuges”: is there a typo here?
q7. Please provide page numbers for Aubre´ville 1968
and 1970.
q8. Please provide updated publication information
for van der Burgt et al, forthcoming.
