Plant Syst Evol (2010) 285:51–64
DOI 10.1007/s00606-009-0255-8
ORIGINAL ARTICLE
Adaptive radiation in Coffea subgenus Coffea L. (Rubiaceae)
in Africa and Madagascar
François Anthony • Leandro E. C. Diniz •
Marie-Christine Combes • Philippe Lashermes
Received: 31 July 2009 / Accepted: 28 December 2009 / Published online: 5 March 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract Phylogeographic analysis of the Coffea subgenus Coffea was performed using data on plastid DNA
sequences and interpreted in relation to biogeographic data
on African rain forest flora. Parsimony and Bayesian analyses
of trnL-F, trnT-L and atpB-rbcL intergenic spacers from 24
African species revealed two main clades in the Coffea
subgenus Coffea whose distribution overlaps in west equatorial Africa. Comparison of trnL-F sequences obtained
from GenBank for 45 Coffea species from Cameroon,
Madagascar, Grande Comore and the Mascarenes revealed
low divergence between African and Madagascan species,
suggesting a rapid and radial mode of speciation. A chronological history of the dispersal of the Coffea subgenus
Coffea from its centre of origin in Lower Guinea is proposed. No relation was found between phylogenetic topology and the age of emergence of the volcanic islands that
Coffea species have colonised in the Indian Ocean, suggesting dispersal from mainland Africa after the emergence
of the youngest island, Grande Comore, 500,000 years ago.
Additional sequences were obtained from GenBank for 24
species of other Rubiaceae genera, including the Rubia
genus whose origin has been dated from the Upper Miocene.
Estimates of substitution rates suggested that diversification
in Coffea subgenus Coffea occurred about 460,000 years
ago or as recently as the last 100,000 years, depending on
the cpDNA region considered and calibration. The phylogenetic relationships based on plastid sequences confirmed
F. Anthony (&) M.-C. Combes P. Lashermes
IRD, UMR RPB, BP 64501, 34394 Montpellier Cedex 5, France
e-mail: francois.anthony@ird.fr
L. E. C. Diniz
Embrapa Coastal Tablelands, Av. Beira Mar,
3250 Bairro Jardins, Aracaju, SE 49.025-040, Brazil
biogeographic differentiation of coffee species, but they
were not congruent with morphological and biochemical
classifications, or with the capacity to grow in specific
environments. Examples of convergent evolution in the
main clades are given using characters of leaf size, caffeine
content and reproductive mode.
Keywords Africa Biogeography Coffea Evolution
Phylogeny Plastid sequences Rubiaceae
Introduction
Coffeeae tribe belongs to the Ixoroideae monophyletic
subfamily of Rubiaceae family and is close to the tribes
Gardenieae and Pavetteae (Bremer and Jansen 1991; Davis
et al. 2007). The coffee species share the typical coffee
bean morphology, i.e. a groove on the flat side of the seed.
They have been described in two genera, Coffea L. and
Psilanthus Hook. f., which differ in their flower morphology (Leroy 1980; Bridson 1987; Davis et al. 2005). Each
genus has been divided into two subgenera: Coffea subgenus Coffea (95 species), Coffea subgenus Baracoffea
(J.-F. Leroy) J.-F. Leroy (nine species), Psilanthus subgenus Psilanthus (two species) and Psilanthus subgenus
Afrocoffea (Moens) (20 species) (Bridson 1988; Davis
et al. 2005, 2006; Davis and Rakotonasolo 2008). Both
genera occur naturally in tropical Africa; Coffea also
occurs in Madagascar, Grande Comore and the Mascarenes, and Psilanthus in south-east Asia, Oceania and
northern Australia. Research has mainly focused on the
Coffea subgenus Coffea, which comprises the majority of
coffee species, including those of economic importance,
C. arabica L. (65% of world production) and C. canephora
Pierre ex A. Froehner (35%) (more details at www.ico.org).
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52
Coffea subgenus Coffea is represented by 41 species in
Africa, 58 in Madagascar, one in Grande Comore and three
in the Mascarenes, each area having 100% endemicity for
its species (Davis et al. 2006). All species are perennial
woody bushes or trees that differ greatly in morphology,
size and ecological adaptation. They can constitute valuable markers of evolution in the African rain forest since
coffee trees have colonised various types of forest,
including humid evergreen forest, evergreen forest, mixed
evergreen-deciduous forest sometimes seasonally dry,
deciduous forest, savannah woodland, gallery forest,
coastal forest and temporarily flooded riparian forest
(Davis et al. 2006). Except for C. canephora and C. liberica Bull. ex Hiern from West and Central Africa and
C. eugenioides S. Moore from East Africa, coffee species
have a rather restricted distribution, sometimes only a few
square kilometres. Three centres of species diversity have
been identified in Madagascar (mainly in the evergreen
humid forests of eastern Madagascar), Cameroon (14 species) and Tanzania (16 species, mainly in the eastern Arc
Mountain) (Davis et al. 2006). However, many characters
considered in taxonomy are weak and variable, and many
species have not been fully characterized, so it is hard to
draw valid conclusions about their relationships (Bridson
1982; Stoffelen 1998; Davis et al. 2005). All species are
diploid (2n = 2x = 22), except C. arabica, which is tetraploid (2n = 4x = 44) (Charrier and Berthaud 1985).
They are self-incompatible except the tetraploid species
C. arabica and the diploid species C. heterocalyx Stoff.
(Coulibaly et al. 2002) and C. anthonyi Stoff. & F. Anthony
(Stoffelen et al. 2009), which are self-compatible.
Molecular phylogenies of coffee species have been
established based on variations in intergenic spacer
sequences (Lashermes et al. 1996; Cros et al. 1998;
Tesfaye et al. 2007) and introns (Tesfaye et al. 2007) of
plastid DNA, internal transcribed spacer (ITS) sequences
of rDNA (Lashermes et al. 1997) and a combination of four
plastid regions and ITS (Maurin et al. 2007). Low sequence
divergence was found between Coffea and Psilanthus,
indicating that molecular data do not support the recognition of two genera (Lashermes et al. 1997; Cros et al.
1998). Enlarging the number of Coffea species and Psilanthus species did not resolve the relationship between the
two genera (Maurin et al. 2007). At species level, a small
number of parsimony-informative characters were found in
molecular studies and the primary clades were weakly
supported in the trees. This was attributed to the recent
origin of the genus Coffea and a radial mode of speciation
(Lashermes et al. 1997; Cros et al. 1998). All the studies
pointed to a correspondence between the main groups of
species and their geographical origin. In Africa, groups of
species were identified in West Africa, West and Central
Africa, East-Central Africa and East Africa (Lashermes
123
F. Anthony et al.
et al. 1997; Cros et al. 1998; Maurin et al. 2007). A lack of
sequence divergence was found in the Madagascan species,
and consequently their position has remained unresolved
(Maurin et al. 2007).
The major objectives of the present study were to (1)
reconstruct the phylogenetic relationships within Coffea
subgenus Coffea using sequence data from non-coding
regions of plastid DNA, (2) determine the relationships of
new species from Central Africa, (3) investigate divergence
times within Coffea subgenus Coffea and (4) propose a
chronological history of coffee radiation using biogeographic data of African flora.
Materials and methods
Species sampling and outgroup selection
Sequences of the intergenic spacers trnL-F, trnT-L and
atpB-rbcL were produced for 24 Coffea subgenus Coffea
species, 2 Psilanthus species and 1 outgroup plant (Table 1).
The sampling scheme covered the biogeographic diversity
of the Coffea subgenus Coffea in Africa as shown by previous studies (Cros et al. 1998; Maurin et al. 2007). All
coffee accessions were collected during IRD (formerly
ORSTOM) missions in Africa (Anthony et al. 2007) and
maintained in greenhouses at the IRD centre in Montpellier.
One species from Cameroon (Anthony et al. 1985) and four
species from Congo (de Namur et al. 1987) could not be
identified and provisional names were used. Gardenia jasminoides J. Ellis was chosen as the outgroup based on previous molecular studies in the Rubiaceae family (Cros et al.
1998; Bremer et al. 1999; Andreasen and Bremer 2000).
Biogeographic groupings
The African coffee species were grouped according to their
biogeographic origin using the terminology of Maurin et al.
(2007): Upper Guinea (UG), Lower Guinea/Congolian
region (LG/C), East-Central Africa (E-CA) and East Africa
(EA). Based on a chorological analysis, Upper Guinea,
Lower Guinea and Congolia were recognised as subcentres of endemism in the Guineo-Congolian Regional
Centre of Endemism (G-C) (White 1979, 1983).
Sequence generation
Total DNA was obtained from fresh leaves using the
method of Lashermes et al. (1993), modified by Paillard
et al. (1996). DNA samples were purified using QIAquick
columns (QIAGEN). Target regions were amplified in
25 ll reactions with approximately 20–25 ng of total
DNA, 19 colorless GoTaq Flexi Buffer [50 mM KCl,
Radiation in coffee
53
Table 1 Accessions used in cpDNA analysis and their geographical distribution according to Davis et al. (2006)
Species
Geographical distribution
GenBank accession number
atpB-rbcL
trnL-trnF
trnT-trnL
Coffea accessions
C. anthonyi Stoff. & F. Anthony
Cameroon, Congo
FJ493346
FJ493323
FJ493373
C. arabica L.
Ethiopia
FJ493347
FJ493319
FJ493374
C. brevipes Hiern
Cameroon, Gabon, Congo, DRC
FJ493348
FJ493320
FJ493375
C. canephora Pierre ex A. Froehner
West, Central and East-Central Africa
FJ493349
FJ493321
FJ493376
C. charrieriana Stoff. & F. Anthony
Cameroon
FJ493350
FJ493322
FJ493377
C. congensis A. Froehner
C. costatifructa Bridson
Cameroon, CAR, Gabon, Congo, DRC
Tanzania
FJ493351
FJ493352
FJ493324
FJ493325
FJ493378
FJ493379
C. eugenioides S. Moore
DRC, Burundi, Rwanda, Uganda,
Sudan, Kenya, Tanzania
FJ493353
FJ493326
FJ493380
C. heterocalyx Stoff.
Cameroon
FJ493354
FJ493327
FJ493381
C. humilis A. Chev.
Liberia, Ivory Coast
FJ493355
FJ493328
FJ493382
C. kapakata (A. Chev.) Bridson
Angola
FJ493356
FJ493329
FJ493383
C. liberica var. dewevrei
(De Wild. & T. Durand) Lebrun
Central Africa, Uganda, Sudan
FJ493357
FJ493330
FJ493384
C. liberica var. liberica Bull. ex Hiern
C. pocsii Bridson
West Africa
Tanzania
FJ493358
FJ493359
FJ493331
FJ493332
FJ493385
FJ493386
C. pseudozanguebariae Bridson
Kenya, Tanzania
FJ493360
FJ493333
FJ493387
C. racemosa Lour.
Mozambique, Zimbabwe, South Africa
FJ493361
FJ493334
FJ493388
C. salvatrix Swynn. & Phillipson
Malawi, Mozambique, Zimbabwe
FJ493362
FJ493335
FJ493389
C. sessiliflora ssp. sessiliflora Bridson
Kenya
FJ493363
FJ493336
FJ493390
C. stenophylla G. Don
Guinea, Sierra Leone, Ivory Coast
FJ493364
FJ493337
FJ493391
Coffea sp. ‘Congo’
Congo
FJ493365
FJ493338
FJ493392
Coffea sp. ‘Mayombe’
Congo
FJ493366
FJ493339
FJ493393
Coffea sp. ‘Ngongo2’
Congo
FJ493367
FJ493340
FJ493394
Coffea sp. ‘Ngongo3’
Congo
FJ493368
FJ493341
FJ493395
Coffea sp. ‘Nkoumbala’
Cameroon
FJ493369
FJ493342
FJ493396
Related genus Psilanthus
P. ebracteolatus Hiern
West and Central Africa
FJ493370
FJ493343
FJ493397
P. mannii Hook. f.
West and Central Africa
FJ493371
FJ493344
FJ493398
FJ493372
FJ493345
FJ493399
Outgroup
Gardenia jasminoides J. Ellis
DRC Democratic Republic of Congo, CAR Central African Republic
10 mM Tris-HCl (pH 9.0 at 25°C) and 0.1% Triton X100],
1.5 mM of MgCl2, 0.2 mM of each dNTP, 0.25 lM of
each primer and 0.75 U of GoTaq DNA polymerase
(Promega). The PCR program consisted of 5 min at 95°C
followed by 34 cycles of 1 min at 94°C, 1 min at 50°C and
1 min at 72°C, and a final extension of 72°C for 8 min. The
primers used are listed in Table 2. Amplified products were
cleaned using the GFX PCR kit (GE Healthcare).
Sequencing reactions were performed by Cogenics using
Sanger technology, separately for each strand to obtain
independent forward and reverse sequences. Forward and
reverse fragments were assembled and, in the case of differences, new reactions were performed. All sequences
were deposited in GenBank (Table 1).
Sequence comparisons
Sequences were obtained from GenBank for 45 species of
Coffea subgenus Coffea and 24 species belonging to
other Rubiaceae genera (Table 3). The Coffea species
originated from Cameroon (C. bakossi Cheek & Bridson,
C. mayombensis A. Chev., C. montekupensis Stoff.),
Grande Comore (C. humblotiana Baill.), the Mascarenes
(C. macrocarpa A. Rich., C. mauritiana Lam., C. myrtifolia (A. Rich. ex DC.) J.-F. Leroy) and Madagascar (38
species). The other Rubiaceae accessions were considered
as representative of the subfamilies Ixoroideae, Cinchonoideae and Rubioideae, according to results of previous
studies (Natali et al. 1995; Bremer et al. 1999; Andreasen
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F. Anthony et al.
Table 2 Amplification primers for trnL-F, trnT-L and atpB-rbcL
Intergenic spacer
Primer
Sequence
Reference
trnL-F
Forward (11L)
50 -GGTTCAAGTCCCTCTATCCC-30
Taberlet et al. (1991)
Reverse (14R)
50 -ATTTGAACTGGTGACACGAG-30
Forward (9L)
50 -CATTACAAATGCGATGCTCT-30
trnT-L
atpB-rbcL
0
Reverse (12R)
5 -TCTACCGATTTCGCCATATC-3
Forward (4L)
50 -GAAGTAGTAGGATTGATTCTC-30
Reverse (4R)
50 -TACAGTTGTCCATGTACCAG-30
and Bremer 2000). These subfamilies are generally
recognised as being the three major lineages within Rubiaceae (Rydin et al. 2008).
Phylogenetic analyses
Sequence alignments were initially performed with
CLUSTAL W (Thompson et al. 1994) and manually
adjusted using the MegAlign program of the DNASTAR
package (Lasergene v7.2) without difficulty due to low
levels of nucleotide variation. Sequence divergence (distance) between accessions was calculated by the DNADIST program in PHYLIP (Felsenstein 1995) using the
Kimura-2-parameter model. The data on the intergenic
spacers trnL-F, trnT-L and atpB-rbcL were not analysed
separately because they all exhibited low levels of
sequence divergence. Nucleotide diversity of combined
sequences was estimated for biogeographic regions using
the Arlequin v3.1 software package (Excoffier et al. 2005).
Phylogenetic analyses were conducted using maximum
parsimony (MP) and maximum likelihood (ML) methods
implemented in PAUP* 4.0b10 (Swofford 2001). Parsimony analyses (Swofford et al. 1996) were performed
using the heuristic search method with a random addition
sequence of ten replicates, tree-bisection-reconnection
(TBR) branch swapping, and the MULTREES option. All
nucleotide substitutions were weighted equally. Branch
support was examined in the maximally parsimonious trees
(MPTs) with the bootstrap method (Felsenstein 1985) using
PAUP* 4.0b10. Bootstrap values were calculated from
10,000 replicates with the random addition and heuristic
search option. Gaps were treated either as missing data or
as additional characters. As MP analysis ignores information on branch lengths, we also used maximum likelihood
(ML), which includes an estimation of branch length and
assumes that changes are more likely along long branches
than short ones. The ML heuristic analysis was run for 10
random-addition-sequence replicates with TBR branch
swapping and the HKY85 sequence evolution model.
Bootstraps were calculated using 10,000 replicates.
Bayesian inference of phylogeny was implemented
using MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001;
123
Taberlet et al. (1991)
0
Savolainen et al. (1994)
Ronquist and Huelsenbeck 2003). MrBayes was run with
two simultaneous analyses with four parallel chains in
each, starting with a random tree and sampling one tree for
each 100 generations. The temperature of the chains and
other parameters were left at default value. The program
was run up to 1–3 9 106 Markov Chain Monte Carlo
(MCMC) generations to arrive at the stationary phase
(average standard deviation of split frequencies \0.01).
Divergence time estimation
Divergence time was estimated using two calibration dates:
(1) Colonisation of volcanic islands by Coffea species is
assumed to have followed their emergence from the Indian
Ocean, 8 mya ago for Mauritius (MacDougal and Chauman
1969), 2 mya for Reunion Island (Emerick and Duncan
1982) and 0.5 mya for the Grande Comore (Nougier et al.
1986; Rocha et al. 2005). (2) The origin of the genus Rubia
is assumed to be coincident with the first occurrence of
fossil pollen records, dated from the Upper Miocene
(Muller 1981). Numbers of substitutions per site were
calculated using branch lengths for Rubia in the ML
analysis and the estimated age of this species, and compared
to those found in the Coffea clades.
Results
Coffee sequence characteristics
The trnL-F sequences generated for 26 coffee species
ranged in length from 331 to 356 bp. Aligned sequences
contained two deletions, one 8 bp and one 11 bp in length.
Aligned with the outgroup sequence, the coffee sequences
presented an insertion of 10 bp and a deletion of 1 bp. The
trnL-F matrix was composed of 357 aligned positions, 21
(5.9%) of which were variable, with 7 (2.0%) parsimonyinformative (Table 4). The maximum divergence was 2.4%
between coffee sequences [i.e. C. liberica var. dewevrei
(De Wild & T. Durand) Lebrun-C. stenophylla G. Don] and
3.6% between the outgroup and coffee (i.e. C. stenophylla)
sequences.
Radiation in coffee
55
Table 3 Sequences of the intergenic spacers trnL-F, trnT-L and atpB-rbcL obtained from GenBank for 45 species of Coffea subgenus Coffea
and 24 species belonging to other Rubiaceae genera
Species
Origin
trnL-trnF
trnT-trnL
atpB-rbcL
Coffea subgenus Coffea
C. abbayesii J.-F. Leroy
Madagascar
DQ153805
C. andrambovatensis J.-F. Leroy
Madagascar
DQ153879
C. ankaranensis J.-F. Leroy ex A.P. Davis & Rakotonas.
Madagascar
DQ153774
C. arenesiana J.-F. Leroy
Madagascar
DQ153807
C. augagneuri Dubard
Madagascar
DQ153800
C. bakossi Cheek & Bridson
C. bertrandii A. Chev.
Cameroon
Madagascar
DQ153835
DQ153791
C. betamponensis Portères & J.-F. Leroy
Madagascar
DQ153788
C. boiviniana (Baill.) Drake
Madagascar
DQ153793
C. buxifolia A. Chev.
Madagascar
DQ153809
DQ153784
C. coursiana J.-F. Leroy
Madagascar
C. dubardii Jum.
Madagascar
DQ153802
C. farafanganensis J.-F. Leroy
Madagascar
DQ153772
C. heimii J.-F. Leroy
Madagascar
DQ153798
C. homollei J.-F. Leroy
Madagascar
DQ153769
C. humblotiana Baill.
Grande Comore
DQ153778
C. kianjavatensis J.-F. Leroy
Madagascar
DQ153849
C. lancifolia A. Chev.
Madagascar
DQ153770
C. leroyi A.P. Davis
Madagascar
DQ153771
C. liaudii J.-F. Leroy
Madagascar
DQ153801
C. littoralis A.P. Davis
C. macrocarpa A. Rich.
Madagascar
Mauritius
DQ153808
DQ153838
C. mangoroensis Portères
Madagascar
DQ153870
C. manombensis A.P. Davis
Madagascar
DQ153812
C. mauritiana Lam.
Mauritius & Reunion
DQ153836
C. mayombensis A. Chev.
Cameroon
DQ153828
C. mcphersonii A.P. Davis
Madagascar
DQ153790
C. millotii J.-F. Leroy
Madagascar
DQ153776
C. montekupensis Stoff.
Cameroon
DQ153826
C. montis-sacri A.P. Davis
Madagascar
DQ153797
C. moratii J.-F. Leroy
Madagascar
DQ153869
C. myrtifolia (A. Rich. ex DC.) J.-F. Leroy
Mauritius
DQ153844
DQ153794
C. perrieri Drake ex Jum. & H. Perrier
Madagascar
C. pervilleana (Baill.) Drake
Madagascar
DQ153779
C. rakotonasoloi A. Davis
Madagascar
DQ153783
C. ratsimamangae J.-F. Leroy ex A.P. Davis & Rakotonas.
Madagascar
DQ153811
C. resinosa (Hook. f) Radlk.
C. richardii J.-F. Leroy
Madagascar
Madagascar
DQ153795
DQ153782
C. sahafaryensis J.-F. Leroy
Madagascar
DQ153780
C. sakarahae J.-F. Leroy
Madagascar
DQ153806
C. sambavensis J.-F. Leroy ex A.P. Davis & Rakotonas.
Madagascar
DQ153785
C. tetragona Jum. & H. Perrier
Madagascar
DQ153773
C. tsirananae J.-F. Leroy
Madagascar
DQ153810
C. vatovavyensis J.-F. Leroy
Madagascar
DQ153777
C. vianneyi J.-F. Leroy
Madagascar
DQ153803
Other Rubiaceae
Asperula cynanchica L.
DQ662135
DQ662135
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F. Anthony et al.
Table 3 continued
Species
Origin
trnL-trnF
Asperula tinctoria L.
DQ662136
Chione venosa (Sw.) Urban
AF152687
Cinchona pitayensis (Wedd.) Wedd.
AF152684
Cinchona pubescens Vahl
Cosmibuena grandiflora (Ruı́z Lopez & Pav.) Rusby
trnT-trnL
atpB-rbcL
AJ346963
AJ233990
AF152686
Diplospora dubia (Lindl.) Masam.
DQ131725
Exostema caribaeum (Jacq.) Schult.
Exostema purpureum Grisebach
AJ233991
AF152696
Galium baillonii Brandza
X81671
Gardenia volkensii K. Schum.
AF201044
Guettarda speciosa L.
AF152725
Hillia valerii Standl.
Ixora coccinea L.
Ixora finlaysoniana Wall. ex G. Don.
X81683
AJ620117
AJ620117
DQ131744
Ixora parviflora Lam.
X76477
Pavetta abyssinica Fresen.
Pavetta barbertonensis Bremek.
FM207133
AF152668
Rubia cordifolia Hochst. ex A. Rich.
Rubia horrida Thunb. Puff.
DQ131777
DQ662167
DQ662167
Rubia peregrina L.
X76474
Rubia tinctorum L.
Tricalysia cryptocalyx Baker
FJ695421
Tricalysia elliotii (K. Schum.) Hutchinson & Dalziel
The trnT-L sequences ranged from 348 to 511 bp in
length across the coffee species. Two ambiguous regions
containing variable numbers of A and T repeats were
removed because of possible sequencing errors caused by
Taq polymerase stuttering. Five indels ranging from 1 to
172 bp were required to align the coffee sequences. Their
alignment with the outgroup sequence required six additional indels of 1–6 bp. G. jasminoides presented a 32-bp
region with a 7-bp inverted repeat at each end. The trnT-L
matrix was composed of 524 aligned positions, 17 (3.2%)
of which were variable, with three (0.6%) parsimonyinformative (Table 4). The maximum divergence was 0.9%
between coffee sequences [i.e. C. kapakata (A. Chev.)
Bridson-C. pocsii Bridson] and 2.1% between the outgroup
and coffee (three species) sequences.
The length of atpB-rbcL sequences ranged from 684 to
723 bp. A variable region of 2 bp randomly containing A,
T, C or G was found between a 7-bp inverted repeat
sequence. This variable region was not used for subsequent
analyses. Eight indels ranging from 1 to 32 bp were
required to align the coffee sequences. Two additional indels of 1 bp were included in the alignment of the outgroup
sequence. The atpB-rbcL matrix was composed of 757
aligned positions, 27 (3.6%) of which were variable, with
11 (1.5%) parsimony-informative (Table 4). The maximum
123
X76465
AF152669
DQ131791
divergence was 0.7% between coffee sequences (three
pairs of species) and 2.4% between the outgroup and coffee
(i.e. P. mannii) sequences.
Analysis of African species
The combined plastid data comprised 1,638 bp of aligned
sequence of 26 coffee species and G. jasminoides as outgroup (Table 4). With gaps treated as missing data, parsimony analysis produced 61 MPTs, with a consistency index
(CI) of 0.942 (0.840 excluding uninformative characters), a
retention index (RI) of 0.941, and a rescaled consistency
index (RC) of 0.887. The topology of the MP analysis of
Coffea species supported two sister clades and several
subclades that were consistent with biogeographic regions
(Fig. 1). Clade I comprised four subclades corresponding to
one species from East Africa (subclade Ia), the remaining
East African species (Ib), C. arabica and two species from
East-Central Africa and Lower Guinea (Ic), and two species
from Upper Guinea (Id). Clade II comprised species
exclusively native to the Guineo-Congolian region. Three
subclades were supported corresponding to three species
from the Lower Guinea/Congolia region, closely related to
C. canephora (subclade IIa), three species from the Lower
Guinea/Congolia region (IIb), and the unidentified species
Radiation in coffee
57
Table 4 Characteristics of the cpDNA regions used in the phylogenetic analyses: number of aligned, variable and parsimony-informative
positions, consistency index (CI), retention index (RI) and rescaled consistency index (RC) of MP analyses
Region
trnL-F
trnT-L
atpB-rbcL
Material
Variable
positions (bp)
Parsimony-informative
positions (bp)
CI
RI
RC
24 Coffea and 2 Psilanthus species
357
21 (5.9%)
7 (2.0%)
Not analysed separately
69 Coffea species
299
24 (8.0%)
8 (2.7%)
1.000
69 Coffea and 13 Rubiaceae species
24 Coffea and 2 Psilanthus species
323
524
103 (31.9%)
17 (3.2%)
62 (19.2%)
3 (0.6%)
24 Coffea and 7 Rubiaceae species
681
305 (44.8%)
249 (36.6%)
24 Coffea and 2 Psilanthus species
757
27 (3.6%)
11 (1.5%)
654
228 (34.9%)
187 (28.6%)
0.910
0.958
0.872
65 (4.0%)
21 (1.3%)
0.948
0.905
0.858
24 Coffea and 12 Rubiaceae species
All combined
Aligned
positions (bp)
24 Coffea and 2 Psilanthus species
1,638
from Congo (IIc). C. charrieriana Stoff. & F. Anthony and
C. liberica var. liberica Bull. ex Hiern were included in
clade II, but they were weakly supported as subclades IId
and IIe respectively. The Psilanthus species formed two
sister clades but their position was weakly supported by
bootstrap values. Conflicts between MPTs lay in the position
of C. liberica var. liberica and the Psilanthus species, which
were placed either in clade II or as sisters to clades I and II.
The topology of the Bayesian majority rule consensus tree
(Fig. 1) was identical to that of the MP analysis, except for
the position of the Psilanthus species, which were grouped
as sister clades of clades I and II in the MP analysis, and of
clade II in the Bayesian analysis. As phylogeny of the
Psilanthus species remained uncertain, P. ebracteolatus and
P. mannii were not included in subsequent analyses. Except
for subclade IId, all clades and subclades were supported by
at least one synapomorphy (Fig. 1). A maximum of four
synapomorphies was observed for subclade Id. Ten Coffea
species were characterised by at least one autapomorphy. By
contrast, the outgroup presented many more specific characters (25). Few indels were present in subclades, but there
were more many in the outgroup.
With gaps treated as new characters, MP analysis yielded 54 MPTs (CI = 0.948, RI = 0.905, RC = 0.858), the
consensus of which resembled the tree found with gaps
treated as missing data (data not shown). Bootstrap values
increased in general, except for C. liberica var. dewevrei,
C. heterocalyx and C. kapakata.
Considering the species grouped in biogeographic
regions, estimates of nucleotide diversity (Nei 1987) were
0.026 ± 0.013 in Lower Guinea and Congolia (13 species),
0.021 ± 0.012 in East-Central Africa (6 species),
0.019 ± 0.012 in East-Central Africa (4 species) and
0.008 ± 0.005 in Upper Guinea (4 species).
Analysis of African and Madagascan species
The trnL-F sequences of African species and of the outgroup were aligned with those of three species from
1.000
1.000
0.869
0.931
0.810
Not analysed separately
0.940
0.967
0.908
Not analysed separately
Central Africa and 42 species from the Madagascar region,
available in GenBank (Table 3). One 1-bp deletion was
required to align the sequences of three Madagascan species [i.e. C. augagneurii Dubard, C. pervilleana (Baill.)
Drake, C. ratsimamangae J.-F. Leroy ex A.P. Davis &
Rakotonas.] and one species from Grande Comore (i.e.
C. humblotiana) with those of the remaining species. The
matrix was composed of 299 aligned positions, 24 (8.0%)
of which were variable, with 8 (2.7%) parsimony-informative. Maximum divergence was 2.5% between coffee
sequences (C. leroyi A.P. Davis-C. liberica var. dewevrei)
and 3.6% between the outgroup and coffee (i.e. C. leroyi,
C. stenophylla) sequences.
With gaps treated as missing data, MP analysis produced
a single MPT (CI = 1.0, RI = 1.0, RC = 1.0), the topology of which was identical to that of the Bayesian analysis
(Fig. 2). All species from the Madagascar region fitted in
clade I. No difference was detected among 10 species
from Madagascar, 2 species from the Mascarenes (i.e.
C. macrocarpa, C. myrtifolia) and those from East Africa.
Twenty-seven species formed a subclade, in which three
species from northern Madagascar (i.e. C. augagneurii,
C. pervilleana, C. ratsimamangae) and one from Grande
Comore (i.e. C. humblotiana) grouped together. The species from Cameroon (i.e. C. bakossi, C. mayombensis,
C. montekupensis) were placed in clade II. Referring to
their distribution, clades I and II were consequently named
the clades A-IO (Africa-Indian Ocean) and G-C (GuineaCongolian) respectively.
Analysis of Rubiaceae species
The trnL-F sequences of African and Madagascan species
and of the outgroup were aligned with those of 12 Rubiaceae species belonging to other genera than Coffea, and
available in GenBank (Table 3). Several indels (1–19 bp)
were required to align the new sequences with those of
coffee. The matrix was composed of 323 aligned positions,
103 (31.9%) of which were variable, with 62 (19.2%)
123
58
F. Anthony et al.
Fig. 1 Phylogenies of 24 Coffea subgenus Coffea species and two
Psilanthus species from Africa, using sequences of intergenic spacers
trnL-F, trn T-L and atpB-rbcL, with gaps coded as missing data.
G. jasminoides was used as the outgroup. Substitutions and indels
appearing once are represented by solid boxes and open rhombi
respectively. For geographical groupings of species, see White (1979)
and Maurin et al. (2007). UG Upper Guinea, LG Lower Guinea,
C Congolia, E-CA East-Central Africa, EA East Africa. Left Strict
consensus tree generated by MP analysis (CI = 0.942, RI = 0.941,
RC = 0.887), with bootstrap values ([50%) listed above branches.
Right Bayesian majority rule phylogeny with posterior probabilities
([50%) listed above branches
parsimony-informative. Divergence was found to be high
between Rubia and Coffea (24.5–26.1%) and other Ixoroideae species (23.8–24.6%). By contrast, divergence was
only 1.1–4.3% within the Ixoroideae subfamily.
The HKY85?G model (Hasegawa et al. 1985) was
identified by Modeltest as the best nucleotide substitution
model. Base frequencies were A = 0.341, C = 0.195,
G = 0.108 and T = 0.356, the ti/tv ratio was 0.767, and
the estimated value of the gamma shape parameter was
0.826. ML and NJ analyses produced the same phylogenetic relationships supporting the monophyly of Rubiaceae
(Fig. 3). The tree of the trnL-F sequences showed three
main strongly supported lineages, corresponding to the
subfamilies Rubioideae, Cinchonoideae and Ixoroideae.
The distribution of branch lengths was variable among
branches. Long branches were observed for subfamily
branching while short branches were observed within
subfamilies. The clades A-IO and G-C were closely
grouped with the other Ixoroideae species.
C. macrocarpa and C. myrtifolia), 10 species from Madagascar and the East African species of our study. On the
other hand, the species from Grande Comore (i.e. C. humblotiana) presented a sequence identical to that of three
species from Madagascar. Such similarities among species
from East Africa, Madagascar, Mascarenes and Grande
Comore indicate that dispersal of the Coffea subgenus
Coffea in the Indian Ocean occurred after the emergence of
volcanic islands. Given the age of the youngest island (i.e.
Grande Comore), dispersal of Coffea subgenus Coffea
species from mainland Africa probably occurred during the
last 500,000 years.
Based on the origin of the Rubia genus, substitution
rates estimated in the ML analyses varied from
15.5 9 10-9 subst. per site per year to 99.6 9 10-9 subst.
per site per year (Table 5). The Coffea subgenus Coffea
could thus have diverged about 460,000 years BP or as
recently as the last 100,000 years, depending on the
cpDNA region considered and calibration.
Divergence time
Discussion
The molecular phylogenetic trees generated here and in
previous studies did not show any relation between phylogenetic topology and the age of emergence of the volcanic islands that Coffea species have colonised in the
Indian Ocean. On one hand, no difference was detected in
the trnL-F region among 2 species from Mauritius (i.e.
123
General findings
The present study provided new plastid sequences from
Coffea subgenus Coffea species. The intergenic spacer
trnT-L was sequenced for the first time and new species
Radiation in coffee
59
Fig. 3 Maximum likelihood tree of 69 Coffea subgenus Coffea
species and 13 species belonging to other Rubiaceae genera based on
analysis of trnL-F sequences with gaps coded as missing data.
Numbers indicate bootstrap support values above 50% in 10,000
replicates. Clades A-IO and G-C are identified in Fig. 2
Fig. 2 Single MPT resulting from analysis of trnL-F sequences of 69
Coffea subgenus Coffea species from Africa (27), Madagascar (38),
Grande Comore (1) and the Mascarenes (3), with gaps coded as
missing data (CI = 1.0, RI = 1.0, RC = 1.0). G. jasminoides was
used as the outgroup. Bayesian posterior probabilities are listed above
branches, parsimony bootstrap values below. Clades and subclades
are identified in Fig. 1
were included in the phylogenetic analysis. Non-coding
regions were chosen rather than coding regions because
they are under lower selection pressure and reveal more
divergence among related species (Dixon and Hillis 1993;
Gielly and Taberlet 1994). However, non-coding cpDNA
regions present variable evolutionary rates and bring variable numbers of potentially informative characters (Shaw
et al. 2005; 2007). In coffee, sequences of the intergenic
spacers trnL-F and atpB-rbcL were successfully used in
previous phylogenetic studies (Cros et al. 1998; Maurin
et al. 2007; Tesfaye et al. 2007). The substitutions identified in this study were confirmed by separating forward and
reverse sequencing reactions. Our sequences showed a
nucleotide composition and a transition/transversion rate
similar to those observed for angiosperms in the intergenic
regions trnL-F (Bakker et al. 2000) and atpB-rbcL (Manen
and Natali 1995; Morton and Clegg 1995; Hoot and
Douglas 1998). Sequence divergence was low in the Coffea
subgenus Coffea (B2.4%), as shown in previous studies of
the trnL-F region (Cros et al. 1998), other plastid regions
(trnL-F intron, rpl16 intron and accD-psa1) and the internal transcribed spacer (ITS 1/58S/ITS 2) of nuclear rDNA
(Maurin et al. 2007). Divergence was, however, higher in
this study than in the intergenic spacers (atpB-rbcL,
trnS-G, rpl2-rps19 and rps19-rpl22), introns (atpF, trnG
and trnK) and genes (matK, rpl2, rps19 and rpl22) of
chloroplast genomes sequenced by Tesfaye et al. (2007).
Few parsimony-informative characters were found, only 21
on a 1,638 bp length (1.3%), which explained why the
main branches of phylogenetic trees were supported by a
low number of characters.
Phylogenetic relationships
The results of our analysis of African species are congruent
with those previously published based on plastid and ITS
sequences (Lashermes et al. 1997; Cros et al. 1998; Maurin
et al. 2007; Tesfaye et al. 2007). Phylogenetic analyses of
our dataset revealed two lineages in Coffea subgenus
Coffea. Clade A-IO spans the entire geographical range of
Coffea subgenus Coffea while clade G-C is restricted to the
Guineo-Congolian region. Within clades, species were
classified in subclades according to their biogeographic
origin (i.e. EA, E-CA, C, LG, UG). Similar groupings were
found by Maurin et al. (2007) who included 83% of Coffea
123
60
F. Anthony et al.
Table 5 Substitution rate estimated for the Rubia species in the ML analyses, using Upper Miocene start (11.6 mya) and end (5.3 mya) for
calibration, and corresponding divergence time estimated for Coffea subgenus Coffea
Region
Calibration 11.6 mya
(subs per site per year)
Calibration 5.3 mya
(subs per site per year)
Divergence time
estimates (years)
trnL-F
15.5 9 10-9
34.0 9 10-9
158,000–345,000
trnT-L
45.5 9 10
-9
99.6 9 10-9
211,000–461,000
atpB-rbcL
20.0 9 10-9
43.8 9 10-9
48,000–104,000
species in their study, but the main clades were erroneously
named EA-IO (East Africa-Indian Ocean) and LG/C
(Lower Guinea/Congolia). These names did not reflect the
biogeographical origin of studied material since clade
EA-IO included a subclade from Upper Guinea and a
species (i.e. C. anthonyi) from Lower Guinea/Congolia.
Similarly clade LG/C included C. canephora and C. liberica var. liberica which can be found in Upper Guinea.
Our molecular analysis resolved the species from
Cameroon (i.e. C. charrieriana) and Congo (i.e. Coffea sp.
‘Congo’, Coffea sp. ‘Ngongo 3’), studied here for the first
time, to two distinct subclades of clade G-C, thus
increasing known diversity in Lower Guinea. High levels
of similarity were observed in the trnL-F sequences of
Coffea sp. ‘Mayombe’, Coffea sp. ‘Ngongo 2’ and Coffea
sp. ‘Ngongo 3’, all from the south-west of the Mayombe
Mountains in Congo. Moreover, their sequences were
identical to that of C. mayombensis whose distribution
covers west equatorial Africa, from southern Nigeria to
Cabinda, including the Mayombe Mountains (Stoffelen
1998). Such grouping resembled that observed around
C. canephora, a widely distributed species, grouped with
species with limited distribution (i.e. C. congensis
A. Froehner, C. brevipes Hiern, Coffea sp. ‘Nkoumbala’).
This confirmed previous observations on the high level of
endemicity in the Mayombe Mountains (Cusset 1981,
1989).
Centre of origin
Nucleotide diversity was higher in Lower Guinea and
Congolia than in any other biogeographic region, as a
consequence of overlap of clades A-IO and G-C in west
equatorial Africa. This suggests that Lower Guinea could
be the centre of origin of Coffea subgenus Coffea. The
origin may thus not be in Kenya as suggested by a biogeographic analysis (Leroy 1982), but in West-Central
Africa. According to floristic records, Lower Guinea is the
richest sub-centre of endemism of the Guineo-Congolian
Region (White 1979). Diversity in Coffea subgenus Coffea
has, however, been underestimated for a long time as
shown by the case of Cameroon. In the early 1990s, only
5 species were known whereas now 15 species are
123
recognised (Anthony et al. 2006), not including the new
species of this study. Sequence diversity appeared maximal
in west equatorial Africa, suggesting that Lower Guinea
constitutes a major centre of speciation for Coffea subgenus Coffea. This region likely played the role of refuge for
coffee trees during the last arid maximum (18,000 years
BP) and previous arid phases. In Central Africa, a chain of
small refuges has been located near the Atlantic Ocean: in
west and south Cameroon, in the Crystal and Chaillu
Mountains in Gabon and in the Mayombe Mountains in
Congo (Maley 1987, 1996). These areas rich in coffee
species are known to be hotspots of biodiversity (Küper
et al. 2004). Forest patches could also have survived
between refuges and formed forest islands in a grassy sea
(Leal 2004).
Radiation in Coffea subgenus Coffea
The low rate of homoplasy and the low number of characters
supporting the main branches confirmed the hypothesis of a
rapid and radial mode of speciation in Coffea subgenus
Coffea (Lashermes et al. 1997; Cros et al. 1998). Judging
from genetic distances, the origin of Coffea subgenus Coffea
is recent. For example, trnL-F uncorrected pairwise
sequence divergence was only 0–2.4% within Coffea species while that between Coffea and Rubia was 24.5–26.1%.
Another fact in favour of a recent origin of Coffea subgenus
Coffea is the low number of insertions and deletions that
were required for plastid sequence alignment. To align the
trnL-F sequences of 42 Madagascar species with those of 26
African species, only one short deletion (1 bp) was required.
Few indels were also reported for sequence alignment of
cpDNA intergenic spacers (Cros et al. 1998) and introns
(Tesfaye et al. 2007), and none in coding regions (Tesfaye
et al. 2007). Moreover, the sequences of Madagascar species
showed high similarities with those of species from the
surrounding islands and from East Africa, suggesting a
common origin. Biodiversity is, however, considerable in
Madagascar (Myers et al. 2000), in particular for coffee trees
since the region contains 60% of Coffea subgenus Coffea
species (Davis et al. 2006). The majority of Madagascan
species have rather limited distribution (Davis et al. 2006),
which corresponds to radial and rapid speciation.
Radiation in coffee
61
isolating Upper Guinea from Lower Guinea over a distance
of some 200 km in Togo and Benin (Salzmann and
Hoelzmann 2005). This savannah barrier is believed to
have occupied a far larger area during previous drier phases
and to have separated the forest refuges of Ivory CoastGhana and west Cameroon by at least 1,200 km. In
C. canephora, the existence of genetic groups distributed in
Upper Guinea and Central Africa (Berthaud 1986; Dussert
et al. 2003), which are easily distinguishable and hardly
differentiated (Leroy et al. 1993), suggests that colonisation of Upper Guinea by coffee trees occurred before formation of the Dahomey Gap, similarly to what happened
for the shea tree (Fontaine et al. 2004).
Divergence time
Fig. 4 Reconstruction of the dispersal of Coffea subgenus Coffea
from its centre of origin in Lower Guinea. Actual distribution of
Coffea subgenus Coffea and putative forest refuges during the last
major arid phase (18,000 years BP) (Maley 1996; Roy 1997) are in
grey and black respectively. Biogeographic regions in Africa are
outlined by marks according to White (1979) and Maurin et al.
(2007). UG Upper Guinea, LG Lower Guinea, C Congolia, E-CA
East-Central Africa, EA East Africa
Coffee would have spread radially from the centre of
origin located in Lower Guinea, westwards up to Upper
Guinea and eastwards through Central Africa (Fig. 4).
Dispersal could have benefitted from several putative refuges in the Congo-Zaire Basin (Maley 1996; Colyn et al.
1991), in East Central Africa (Lovett 1993) and in East
Africa (Fjeldså and Lovett 1997; Roy 1997) where montane regions offered a great range of habitats. Colonisation
of Madagascar was doubtless the result of a single dispersal
event from the African mainland, followed by insular
speciation. Such a scenario has been already proposed to
explain speciation in the genera Begonia (Plana et al. 2004)
and Gaertnera (Malcomer 2002) in Madagascar. High
similarity between C. humblotiana from Grande Comore
and three species from north Madagascar (i.e. C. augagneurii, C. pervilleana, C. ratsimamangae) indicate that
Grande Comore was colonised by coffee trees from north
Madagascar (Maurin et al. 2007) or, more likely according
to the geographic position of Grande Comore, in one step
when coffee trees crossed the Mozambique channel.
Lastly, the species from the Mascarenes showed a common
origin with the East African species in our study and with
ten Madagascar species, suggesting rapid colonisation of
Mauritius and Reunion Island from Madagascar.
The Dahomey Gap has recently (ca. 4,000 years BP)
fragmented rain forest in the Guineo-Congolian region,
Radiation observed in Madagascar and the surrounding
islands demonstrated that coffee trees are not remnants of a
putative Cretaceous Gondwana flora (Guillaumet and
Mangenot 1975; Leroy 1978). Their origin is much more
recent than the Gondwana dislocation mentioned by Leroy
(1982) and even more recent than the appearance of the
volcanic islands around Madagascar. Given the age of the
youngest island (i.e. Grande Comore), coffee dispersal
occurred within the last 500,000 years. Sequence comparison between Coffea subgenus Coffea species and Rubia,
whose origin was dated from the Upper Miocene (Muller
1981), enabled us to estimate the divergence time at about
100,000–450,000 years BP. Even though these ages should
be considered as preliminary estimates, radiation in Coffea
subgenus Coffea occurred probably in the second half of
the Middle Pleistocene (780,000–126,000 years BP).
Coffee dispersal could have benefitted from humid conditions during interglacials of the past 200,000 years (Dupont
et al. 2001). The rapidity of colonisation points to the
effective dispersal of coffee seeds, likely by monkeys in
Africa and lemurs in Madagascar. The role of primates in
seed dispersal has already been put forward to explain the
rapid radiation of Aframomum the genus in Africa (Harris
et al. 2000).
Adaptation and speciation
The phylogenetic relationships based on nucleotide
sequences were not congruent with either morphological
and biochemical classifications (Stoffelen 1998; Dussert
et al. 2008) or with the adaptive capacity to grow in
specific environments. For example, only three smallleaved species are known in Central Africa: C. anthonyi
(Stoffelen et al. 2009), C. charrieriana (Stoffelen et al.
2008) and C. kapakata (Chevalier 1947; Bridson 1994). In
our study, the first species was placed in the clade A-IO
while the two others were placed in the clade G-C.
123
62
Another example is the absence of caffeine in coffee
seeds. Two caffeine-free species have been reported in
Africa up to now, C. pseudozanguebariae Bridson (Hamon
et al. 1984) and C. charrieriana (Stoffelen et al. 2008).
They were classified in clades A-IO and G-C respectively.
However, these species occupy very different habitats, the
coastal dry forest on a coral reef substrate close to the
Indian Ocean in the case of C. pseudozanguebariae
(Anthony et al. 1987) and rain forest in west Cameroon in
the case of C. charrieriana (Stoffelen et al. 2008). On the
other hand, all caffeine-free species from Madagascar (e.g.
C. homollei J.-F. Leroy) (Anthony et al. 1993) were
grouped in the clade A-IO together with Madagascan
species containing caffeine (e.g. C. lancifolia A. Chev.)
(Rakotomalala et al. 1992). The absence of caffeine in
seeds and leaves where the component is synthesised does
not appear to be associated with one or more particular
lineages in Coffea subgenus Coffea. Similarly, autofertility
of C. anthonyi (Stoffelen et al. 2009) and C. heterocalyx
(Coulibaly et al. 2002) is a character that appeared independently in clades A-IO and G-C. These characters are
examples of convergent evolution on the scale of equatorial Africa and the islands in the Indian Ocean. Finally,
the high adaptive capacity of Coffea subgenus Coffea
probably originates in variations in gene expression
mechanisms rather than in the nucleotide composition of
the genes themselves.
Accelerated rates of regulatory gene evolution could
accompany rapid morphological diversification in adaptive
radiation (Barrier et al. 2001). Phenotypic plasticity has
been shown to affect plant morphology, anatomy and
physiology (Walbot 1996; Sultan 2000) as well as the
ecological organisation of populations (Miner et al. 2005).
Plasticity of coffee trees could be the key to rapid colonisation of African forests from Guinea to Mozambique and,
farther away, of islands in the Indian Ocean. This would
explain the restricted distribution of the majority of coffee
species and the number of species described up to now
using morphological criteria. Further studies on the evolution of Coffea subgenus Coffea should include regulatory
genes whose divergence could correlate better with phenotypic evolution than molecular evolution did.
Acknowledgments The research was supported by IRD (Institut de
recherche pour le développement, Paris, France) and a postdoctoral
fellowship from the CNPq (Conselho Nacional de Desenvolvimento
Cientı́fico e Tecnológico, Brasilia, Brazil) to L. Diniz. The authors are
grateful to J. Maley for helpful comments on African flora and climate. Two anonymous reviewers are also thanked for constructive
critics.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
123
F. Anthony et al.
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