Molecular Phylogenetics and Evolution 52 (2009) 879–886
Contents lists available at ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Molecular phylogenetics and generic assessment in the tribe Morindeae
(Rubiaceae–Rubioideae): How to circumscribe Morinda L. to be monophyletic?
Sylvain G. Razafimandimbison a,*, Timothy D. McDowell b, David A. Halford c, Birgitta Bremer b
a
Bergius Foundation, Royal Swedish Academy of Sciences and Botany Department, Stockholm University, SE-10691 Stockholm, Sweden
Department of Biological Sciences, Box 70703, East Tennessee State University, Johnson City, TN 37614, USA
c
Queensland Herbarium, Environmental Protection Agency, Brisbane Botanic Gardens, Mt Coot-tha Road, Toowong, Qld 4066, Australia
b
a r t i c l e
i n f o
Article history:
Received 13 February 2009
Revised 14 April 2009
Accepted 15 April 2009
Available online 24 April 2009
Keywords:
Appunia
Coelospermum
Gynochthodes
Head inflorescences
nrETS
nrITS
Paraphyly
Phylogenetic classification
Rubiaceae
Syncarps
trnT-F
a b s t r a c t
Most of the species of the family Rubiaceae with flowers arranged in head inflorescences are currently
classified in three distantly related tribes, Naucleeae (subfamily Cinchonoideae) and Morindeae and Schradereae (subfamily Rubioideae). Within Morindeae the type genus Morinda is traditionally and currently
circumscribed based on its head inflorescences and syncarpous fruits (syncarps). These characters are
also present in some members of its allied genera, raising doubts about the monophyly of Morinda.
We perform Bayesian phylogenetic analyses using combined nrETS/nrITS/trnT-F data for 67 Morindeae
taxa and five outgroups from the closely related tribes Mitchelleae and Gaertnereae to rigorously test
the monophyly of Morinda as currently delimited and assess the phylogenetic value of head inflorescences and syncarps in Morinda and Morindeae and to evaluate generic relationships and limits in Morindeae. Our analyses demonstrate that head inflorescences and syncarps in Morinda and Morindeae are
evolutionarily labile. Morinda is highly paraphyletic, unless the genera Coelospermum, Gynochthodes,
Pogonolobus, and Sarcopygme are also included. Morindeae comprises four well-supported and morphologically distinct major lineages: Appunia clade, Morinda clade (including Sarcopygme and the lectotype
M. royoc), Coelospermum clade (containing Pogonolobus and Morinda reticulata), and Gynochthodes–Morinda clade. Four possible alternatives for revising generic boundaries are presented to establish monophyletic units. We favor the recognition of the four major lineages of Morindeae as separate genera, because
this classification reflects the occurrence of a considerable morphological diversity in the tribe and the
phylogenetic and taxonomic distinctness of its newly delimited genera.
Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction
A recent molecular phylogenetic study of Razafimandimbison
et al. (2008) based on five plastid gene and nrITS regions led to
the establishment of new tribal limits for the species-rich Psychotrieae alliance of the subfamily Rubioideae (Rubiaceae or coffee
family). These authors recircumscribed the tribe Morindeae in a
narrow sense to include only six genera (Appunia Hook.f., Coelospermum Blume, Gynochthodes Blume, Morinda L., Pogonolobus F.
Muell., and Siphonandrium K. Schum.). The members of Morindeae
can be diagnosed by the following features: massive T-shaped placentae inserted in the middle of the septum with two anatropous
ovules per carpel and pyrenes with a single lateral germination slit
(Igersheim and Robbrecht, 1993). Some genera traditionally associated with Morindeae are currently classified in the following
tribes: Colletoecemateae Rydin & B. Bremer (Colletoecema E.M.A
* Corresponding author. Fax: +46 (0)8 165525.
E-mail address: sylvain.razafimandimbison@bergianska.se (S.G. Razafimandimbison).
1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2009.04.007
Petit), Lasiantheae B. Bremer & Manen (Lasianthus Jack), Mitchelleae Razafim. & B. Bremer (Damnacanthus C.F. Gaertn. and Mitchella
L.), and Prismatomerideae Ruan (Prismatomeris Thw. and its allied
genera). The Samoan genus Sarcopygme Setch. & Christoph., classified by Darwin (1979) in Morindeae, was excluded from Morindeae
sensu Razafimandimbison et al. (2008) (hereafter called Morindeae) mainly because of its numerous (up to 100) and synchronous
flowers with uniovulate locules.
Morindeae is a pantropical group of ca. 160 species assigned to
six genera whose generic limits are controversial and remain
unsettled. Of these genera, the most species-rich genus is Morinda,
one of the 24 rubiaceous genera that Linnaeus described in his volume Species Plantarum (Linnaeus, 1753). Linnaeus (1753) included
three species (M. citrifolia L., M. royoc L., and M. umbellata L.) in his
genus Morinda, which can be characterized by a combination of its
head inflorescences and syncarpous fruits (=syncarps or multiple
fruits with ovaries fused). Head inflorescences (also known as
capitula, Johansson, 1994) in Morinda sensu Linnaeus (1753)
consist of two to many flowers clustered together on a common
receptacle. These heads are either solitary (i.e., one cluster of
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flowers on a peduncle = single head) or umbel-like; the later are
unbranched and comprised of two to many heads. Within each
capitulum the number of flowers varies from 2 to 50 but is fairly
constant within species. Steyermark (1972) merged the neotropical Morindeae genus Appunia bearing head inflorescences/infructescences composed of free flowers/fruits in Morinda. This
taxonomic decision broadened the morphological concept of Morinda and resulted in disagreements among Rubiaceae specialists
over its circumscription. For example, Hayden and Dwyer (1969),
Johansson (1987), Burger and Taylor (1993), and Lorence (1999)
retained Appunia at generic level, while Andersson (1992), Boom
and Delprete (2002), and Taylor and Steyermark (2004) included
Appunia in Morinda. The occurrence of head inflorescences and syncarpous fruits in some members of its allied genera of Morinda
(Coelospermum and Gynochthodes) raises doubts regarding the
monophyly of the genus. Morinda currently comprises ca. 130 species of lianescent, arborescent, and suffrutescent plants whose
phylogenetic affinities with the other Morindeae genera have
never been assessed.
The main objective of this study is to reconstruct a robust phylogeny of the tribe Morindeae using combined plastid (trnT-F) and
nuclear (nrETS and nrITS) DNA sequence data. The resulting phylogeny will be used to: (1) rigorously test the monophyly of Morinda as presently delimited; (2) evaluate the phylogenetic value of
head inflorescences and syncarps traditionally and currently used
for circumscribing genera in Morinda and Morindeae; (3) and assess the current generic relationships and limits in Morindeae.
2. Materials and methods
2.1. Taxon sampling
We investigated a total of 67 taxa of Morindeae, including Morinda (ca. 41 species), the neotropical genus Appunia (six species),
the Australasian genera Coelospermum (six species) and Gynochthodes (four species) and the monotypic New Guinean and northern
Australian genus Pogonolobus (one individual). We were unable to
identify six New Caledonian Morinda specimens (Morinda sp. 3–7
and 9) using the last taxonomic treatment of Morinda for this region
(Johansson, 1994); some of them may represent undescribed new
Morinda species. Two Australian Morinda (Morinda sp. 1 and 2)
and one Malagasy Morinda (Morinda sp. 8) specimens are undescribed new species. Morinda citrifolia was represented by one individual each of its three varieties, var. bracteata, var. citrifolia, and var.
potteri. The Samoan genus Sarcopygme, represented by one individual of its type species, S. pacifica (Reinecke) Setch. & Christoph., was
also included in the analyses because Darwin (1979) placed it in
Morindeae. No sequenceable material of the New Guinean monotypic genus Siphonandrium was available. Five outgroup taxa were
selected on the basis of the molecular phylogenetic study of Razafimandimbison et al. (2008). From the sister tribe Mitchelleae Razafim. & B. Bremer one species of Damnacanthus and one species of
Mitchella were used and from the next closest tribe Gaetnereae Bremek. ex S.P. Darwin two species of the paleotropical genus Gaertnera
Lam. and one species of the neotropical Pagamea Aubl. were utilized.
We investigated a total of 72 taxa for this study (see Table 1).
material for all investigated taxa, except Sarcopygme pacifica and
isolated following the mini-prep procedure of Saghai-Maroof
et al. (1984), as modified by Doyle and Doyle (1987). For S. pacifica
total DNA was extracted from a dry young inflorescence (Tronquet
749, P!). Isolated DNA was amplified and sequenced according to
the protocols outlined in the following articles: Razafimandimbison et al. (2005) for nrETS, Razafimandimbison and Bremer
(2002) for trnT-F, and Razafimandimbison and Bremer (2001) and
Razafimandimbison et al. (2004) for nrITS. The primers from these
previous studies were used for the nrETS, nrITS, and trnT-F regions.
In all PCRs, one reaction was run using water instead of DNA as a
negative control to check for contamination. All sequencing reactions were performed using the Big DyeÒ Terminator v3.1 Cycle
Sequencing kit and Big DyeÒ Terminator v1.1 Cycle Sequencing
kit (Applied Biosystems) and sequences were analyzed with the
3100 Genetic Analyzer (Applied Biosystems).
2.3. Phylogenetic analyses
Sequence fragments were assembled using the Staden package
(Staden, 1996). All new sequences have been submitted to
GenBank (FJ906973–FJ907161, Table 1). For each DNA sequence
region (or marker) all new sequences and published ones taken
from GenBank were aligned together using the computer program
CLUSTAL-X (Thompson et al., 1997) to produce an initial alignment
and manually adjusted using software SeAl v.2.0 (Rambaut, 1996).
Insertion/deletion events were inferred by eye and gaps were
treated as missing data in the alignments. The aligned matrices
were analyzed with Bayesian inference without coded indels.
Separate and combined Bayesian analyses of sequence data
were performed in MrBayes 3.1.2 (Huelsenbeck and Ronquist,
2001; Ronquist and Huelsenbeck, 2003). For each of the three
DNA sets, the best performing nucleotide substitution model was
selected using the computer programs MrModeltest 2.0 (Nylander,
2001) and MrAIC ver. 1.4.3 (Nylander, 2004). The best performing
evolutionary models were estimated under three different model
selection criteria: Akaike information criterion (AIC) (Akaike,
1974), AICc (a second order AIC, necessary for small samples)
and the Bayesian information criterion (BIC) (Schwartz, 1978). All
combined Bayesian analyses were conducted with four independent Markov chain runs for 5 106 Metropolis-coupled MCMC
generations, with tree sampling every 1 103 generations. Trees
sampled from the first 2 106 generations were discarded as
burn-in (as detected by plotting the log likelihood scores against
generation number). We partitioned the combined data sets into
two partitions: partition # 1 with GTR + G applied to the nrITS
and trnT-F data; and partition # 2 with HKY + G applied to the
ETS data. In all analyses, partitions were unlinked so that each partition was allowed to have its own sets of parameters. Flat prior
probabilities were specified according to suggestions produced
by the software MrAIC (Nylander, 2004). All separate and combined Bayesian analyses were repeated two times using different
random starting trees to evaluate the convergence of the likelihood
values and posterior probabilities. All saved trees (after excluding
burn-ins) from the two independent runs were pooled for a consensus tree. Groups characterized by posterior probabilities over
95% were regarded as strongly supported.
2.2. DNA extraction, amplification, and sequencing
3. Results
Sequence data from the (nuclear ribosomal) nrETS, nrITS, and
(chloroplast) trnT-F regions, used alone or in combination with
other chloroplast markers (e.g., Razafimandimbison et al., 2005,
2008), have recently been proven useful for inferring phylogenetic
relationships within Rubiaceae. Total DNA was extracted from
leaves dried in silica gel (Chase and Hills, 1991) and/or herbarium
3.1. Phylogenetic analyses
A total of 202 sequences were used, of which 189 (ca. 94%) are
published here. The combined nrETS/nrITS/trnT-F matrix contained
3654 base pairs (bp), of which 662 bp (ca. 18%) were parsimony
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S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886
Table 1
List of taxa investigated in this study, voucher information, country origins, and accession numbers.
Taxa
Voucher information
Country origins
Appunia brachycalyx (Bremek.) Steyerm.
Appunia calycina (Benth.) Sandwith
Appunia debilis Sandwith
Appunia guatemalensis Donn.Sm.
Appunia odontocalyx Sandwith
Appunia tenuiflora (Benth.) B.D. Jacks
Coelospermum balansanum Baill.
Coelospermum crassifolium J.T. Johanss.
Coelospermum dasylobum Halford & A.J. Ford
Coelospermum monticola Baill. ex Guillaumin
Coelospermum paniculatum F. Muell. var. syncarpum
J.T. Johanss.
Damnacanthus indicus C.F. Gaertn.
Gaertnera phyllosepala Baker
Gaertnera phyllostachya Baker
Gynochthodes coriacea Blume
Gynochthodes epiphytica (Rech.) A.C. Sm. & S.P. Darwin
Gynochthodes oresbia Halford
Gynochthodes sessilis Halford
Mitchella repens L.
Morinda ammitia Halford & A.J. Ford
Morinda angustifolia Roxb.
Granville 5443 (BR)
McDowell 2413 (US)
McDowell 5728 (ETSU)
Razafimandimbison et al. (2008)
Smith et al. 1350 (US)
Hoffmann 966 (US)
Mouly 318 (P)
Johansson 85 (P)
Q7385 (BRI)
Razafimandimbison et al. (2008)
Q8854 (BRI)
French Guyana (France)
Guyana
Guyana
Morinda
Morinda
Morinda
Morinda
bracteata Kurz. var. celebica Miq.
buchii Urb.
bucidifolia A. Gray
candollei (Montrouz.) Beauvis. 1
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
candollei (Montrouz.) Beauvis. 2
candollei (Montrouz.) Beauvis. 3
candollei (Montrouz.) Beauvis. 4
candollei (Montrouz.) Beauvis. 5
canthoides (F. Muell.) Halford & R.J.F. Hend.
citrifolia L. var. citrifolia L. (LF)
citrifolia L. var. citrifolia L. (SF)
citrifolia L. var. potteri O. Degen.
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
collina Schltr.
coreia Buch.-Ham.
deplanchei (Hook. f.) Baill. ex K. Schum.
elliptica (Hook.) Ridl.
geminata DC.
glaucescens Schltr.
grayi Seem.
jasminoides A. Cunn.
latibracteata Valeton
longiflora 1 G. Don
longiflora 2 G. Don
lucida A. Gray
moaensis Alain
mollis A. Gray
morindoides (Baker) Milne-Redh.
myrtifolia A. Gray
neocaledonica (S. Moore) Guillaumin
pedunculata Valeton
podistra Halford & A.J. Ford
reticulata Benth.
retusa Poir.
royoc L. 1
royoc L. 2
sp. 1
sp. 2
sp. 3
Morinda sp. 4
Morinda sp. 5
Morinda sp. 6
Morinda
Morinda
Morinda
Morinda
Morinda
Morinda
sp. 7
sp. 8
sp. 9
titanophylla E.M.A. Petit
umbellata L. 1
umbellata L. 2
Razafimandimbison et al.
Razafimandimbison et al.
Razafimandimbison et al.
Alejandro et al. (2005)
Smith 9377 (S)
RJ1411 (BRI)
PIF28127 (BRI)
Ellison 781 (S)
Bremer and Bremer 3909
No voucher
Peru
Guyana
New Caledonia (France)
New Caledonia (France)
Australia
Australia
(2008)
(2008)
(2008)
(UPS)
AF4789 (BRI)
Ekman 2452 (S)
Smith 4645 (S)
McPherson and Munzinger 701
(UPS)
Johansson 15 (S)
Mouly 190 (P)
Mouly 137 (P)
Mouly 140 (P)
Q8878 (BRI)
McDowell 5742 (ETSU)
Lorence 9705 (PTBG)
Lorence 9704 (PTBG)
Johansson 124 (S)
Lorence 9460 (PTBG)
Johansson 57 (S)
Larsen et al. 41223 (AAU)
Gledhill 848 (P)
Johansson 81 (S)
Smith 1521 (S)
Q8836 (BRI)
Lorence 8777 (PTBG)
Andru 5003 (P)
63PT00539 (P)
BR-19733106
Rova et al. 2213 (GB)
Degener 15262 (S)
Leeuwenberg 2249 (P)
Johansson 98 (S)
Johansson 54 (S)
Lorence 9461 (PTBG)
AF4753 (BRI)
KRM4638 (BRI)
De Block et al. 636 (BR)
Leyman 126 (BR)
Lorence 8419 (PTBG)
Q8853 (BRI)
AF3963 (BRI)
McPherson and Munzinger 18243
(UPS)
Mouly 16 (P)
Mouly 310 (P)
McPherson and Munzinger 18075
(P)
Mouly 399 (P)
Kårehed et al. 218 (UPS)
Mouly 302 (P)
Troupin 10732 (BR)
Wambeek and Wanntorp 2622 (S)
Q8839 (BRI)
Fiji
Australia
Australia
USA
Australia
Cult. Xishuangbann Trop. Bot. Gard.
(China)
Australia
Haiti
Fiji
New Caledonia (France)
nrETS
nrITS
trnT-F
FJ907038
FJ907039
AM945191a
FJ907040
FJ906974
FJ906975
FJ906976
AM945332a
FJ906977
FJ907041
FJ907042
FJ907043
AM945194a
FJ907045
FJ906978
FJ906979
FJ906980
AM945334a
FJ906982
FJ907116
AY514061b
AM945200a
AM945201a
AM945192a
FJ907046
FJ907047
FJ907048
FJ907037
FJ907051
FJ907050
AM945335a
AM945340a
AM945341a
AJ847407c
FJ906983
FJ906984
FJ906985
FJ906973
FJ906988
FJ906987
FJ907119
FJ907120
FJ907121
FJ907122
FJ907054
FJ907055
FJ907056
FJ907057
FJ906991
FJ906992
FJ906993
FJ906994
FJ907123
FJ907151
FJ907148
FJ907156
FJ907124
FJ907125
FJ907126
FJ907127
FJ907058
FJ907086
FJ907083
FJ907091
FJ907059
FJ907060
FJ907061
FJ907062
FJ906995
FJ907025
FJ907022
FJ907030
FJ906996
FJ906997
FJ906998
FJ906999
FJ907128
FJ907129
FJ907130
FJ907131
FJ907063
FJ907064
FJ907065
FJ907066
FJ907067
FJ907068
FJ907069
FJ907070
FJ907071
FJ907049
FJ907072
FJ907073
FJ907000
FJ907001
FJ907002
FJ907003
FJ907004
FJ907005
FJ907006
FJ907007
FJ907008
FJ906986
FJ907009
FJ907010
FJ907011
FJ907012
FJ907013
FJ907014
FJ907015
FJ907016
FJ907017
FJ906981
FJ907018
FJ907019
FJ907020
FJ906989
FJ906990
FJ907028
FJ907026
FJ907027
FJ907021
FJ907103
FJ907104
FJ907105
FJ907106
FJ907107
FJ907108
FJ907110
FJ907111
FJ907101
FJ907100
FJ907099
FJ907112
FJ907113
FJ907114
FJ907102
New Caledonia (France)
New Caledonia (France)
New Caledonia (France)
New Caledonia (France)
Australia
Guyana
Palau
Cult. at Natl. Trop. Bot. Gard. (Hawaii,
USA)
New Caledonia (France)
India
New Caledonia (France)
Thailand
Nigeria
New Caledonia (France)
Fiji
Australia
Palau
Ivory Coast
Ivory Coast
Cult. Belgium Botanical Garden
Cuba
New Caledonia (France)
Ivory Coast
New Caledonia (France)
New Caledonia (France)
Palau
Australia
Australia
Madagascar
Unknown
Florida (USA)
Australia
Australia
New Caledonia (France)
FJ907145
FJ907146
FJ907118
FJ907117
FJ907154
FJ907076
FJ907077
FJ907078
FJ907044
FJ907079
FJ907080
FJ907081
FJ907053
FJ907052
FJ907089
New Caledonia (France)
New Caledonia (France)
New Caledonia (France)
FJ907152
FJ907153
FJ907147
FJ907087
FJ907088
FJ907082
New Caledonia (France)
Madagascar
New Caledonia (France)
R.D. of Congo
Sri Lanka
Australia
FJ907150
FJ907147
FJ907155
FJ907157
FJ907158
FJ907160
FJ907085
FJ907084
FJ907090
FJ907092
FJ907093
FJ907094
(continued
FJ907132
FJ907133
FJ907134
FJ907135
FJ907115
FJ907136
FJ907137
FJ907138
FJ907139
FJ907140
FJ907141
FJ907142
FJ907143
FJ907144
FJ907109
FJ907074
FJ907075
FJ907024
FJ907023
FJ907029
FJ907031
FJ907032
FJ907034
on next page)
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Table 1 (continued)
Taxa
Voucher information
Country origins
nrETS
nrITS
trnT-F
Morinda umbellata L. 3
Pagamaea guianensis Aubl.
Pogonolobus reticulatus F. Muell.
Sarcopygme pacifica (Reinecke) Setch. & Christoph.,
Takeuchi and Ama 15319 (BR)
Razafimandimbison et al. (2008)
Q8840 (BRI)
Tronchet et al. 222 (P)
New Guinea (France)
FJ907159
FJ907098
FJ907161
FJ907094
AF333846d
FJ907096
FJ907097
FJ907033
AM945342a
FJ907035
FJ907036
Australia
Samoa (USA)
Andersson and Rova (1999).
a
Razafimandimbison et al. (2008).
b
Bremer and Manen (2000).
c
Alejandro et al. (2005).
d
Malcomber (2002).
informative characters (PIC). Of the 662 bp PIC 224 (ca. 34%) were
from the nrETS data, 239 (ca. 36%) from the nrITS data, and 207 (ca.
30%) from the trnT-F data. The separate Bayesian analyses of the
nrETS, nrITS, and trnT-F data produced Bayesian majority rule consensus trees with similar topologies (not shown). Visual inspection
of the trees showed no well-supported conflict between them;
accordingly, we merged the sequence data of the three markers
for combined analyses.
The Bayesian majority rule consensus tree (from 6000 trees)
from the combined nrETS/nrITS/trnT-F data was almost fully resolved (Fig. 1). The six sampled Appunia species [including the type
species A. tenuiflora (Benth.) B.D. Jacks] formed a well-supported
clade (A in Fig. 1; posterior probability or PP = 1.00), which was resolved with high support (PP = 1.00) as sister to a large clade containing the rest of the sampled Morindeae taxa. Morinda as
presently delimited was resolved as paraphyletic, because Sarcopygme, Coelospermum, Pogonolobus, and Gynochthodes were all
embedded within the large Morinda clade (Fig. 1). The next lineages to branch off after the Appunia clade (A in Fig. 1) were a largely arborescent Morinda clade (B in Fig. 1; including the only two
African lianescent Morinda species, Sarcopygme pacifica, M. citrifolia, and the lectoptype M. royoc; PP = 0.97) and the Coelospermum
clade (C in Fig. 1; including the Australian and New Guinean Pogonolobus reticulatus F. Muell. and the Australian Morinda reticulata
Benth.; PP = 1.00), respectively. This Coelospermum clade was in
turn resolved as sister to a large lianescent Gynochthodes–Morinda
clade (D in Fig. 1; PP = 1.00), within which all sampled species of
Gynochthodes (including the type species G. coriacea Blume)
formed a well-supported subclade (PP = 1.00) sister to a small Morinda subclade (PP = 1.00). This Gynochthodes subclade (including
three Morinda species, PP = 1.00) was resolved as sister to a large
lianescent Morinda subclade (PP = 1.00). Within the largely arborescent Morinda clade (B in Fig. 1; PP = 0.97) the type species of
the Samoan Sarcopygme, S. pacifica and the African M. titanophylla
formed a weakly supported group (PP = 0.81) and the two varieties
of M. citrifolia (var. citrifolia L. and var. potteri O. Degen.) formed a
well-supported group (PP = 1.00); M. bracteata Kurz. var. celebica
Miq., now merged in M. citrifolia L. var. bracteata Kurz. (Merrill,
1923), and the Micronesian M. latibracteata Val. formed a well-supported group, which in turn was sister to the M. citrifolia var. citrifolia–var. potteri clade.
4. Discussion
4.1. Phylogenetic relationships in Morindeae
4.1.1. Monophyly of Appunia Hook.f.
Appunia, originally described by Hooker (1873), is a neotropical
genus with ca. 16 species of shrubs or small trees (Steyermark,
1967; Govaerts et al., 2006). The generic status of Appunia has been
controversial for the last 40 years (e.g., Steyermark, 1972; Johansson, 1987). Many authors (e.g., Steyermark, 1967; Johansson, 1987;
Burger and Taylor, 1993; Lorence, 1999; Borhidi and Diego-Pérez,
2002) recognized Appunia as separate genus. In subsequent publications Steyermark (1972), Andersson (1992), Boom and Delprete
(2002), Taylor and Steyermark (2004), and more recently Govaerts
et al. (2006) merged the genus in Morinda. In Razafimandimbison
et al. (2008), Appunia, represented only by A. guatemalensis
Donn.Sm., was resolved as sister to a clade containing Morinda
citrifolia, Coelospermum monticola Baill. ex Guillaumin, Gynochthodes coricea, and Gynochthodes sp. This position is further corroborated by this study, which investigates six of 16 Appunia species
(type species A. tenuiflora included), Coelospermum, Gynochthodes,
and Morinda. These analyses support the monophyly of Appunia,
which is distinct from the other Morindeae genera by having a
combination of head inflorescences composed of free flowers,
club-shaped stigmas, and simple, non-syncarpous fruits.
4.1.2. Paraphyly of Morinda L.
Morinda sensu Linnaeus (1753) is non-monophyletic because all
three accessions of M. umbellata are more closely related to Coelospermum sensu Johansson (1988), Gynochthodes and Pogonolobus
than to either M. citrifolia or M. royoc (Fig. 1). Morinda as presently
delimited by a combination of head inflorescences, bifid stigmas,
and syncarps is highly paraphyletic, unless Coelospermum,
Gynochthodes, Pogonolobus, and Sarcopygme are also included
(Fig. 1). This is the first study to demonstrate the paraphyly of
the presently circumscribed Morinda and lability of head
inflorescences and syncarpous fruits in Morinda and Morindeae
(Fig. 1).
4.1.3. Sarcopygme Setch. & Christoph.
The Samoan genus Sarcopygme, consisting of five species of
small trees, was originally established by Setchell and Christophersen (1935) based on Sarcocephalus pacificus Reinecke (Reinecke,
1898), which belonged to the tribe Naucleeae (Cinchonoideae).
The authors (Setchell and Christophersen, 1935) argued that their
new genus showed ‘‘a superficial resemblance to Sarcocephalus in
the fruiting heads but is different from the latter in its single ovules
in each locule of the ovary rather than numerous ovules per cell in
Sarcocephalus”. Setchell and Christophersen (1935) postulated that
Sarcopygme is most closely related to Morinda but is distinct from
the latter by its caducous stipules, large involucral bracts, simultaneous opening of all flowers in the head (i.e., synchronous flowering heads), distinct calyces and club-shaped stigmas. Sarcopygme is
additionally distinct in its monocaul trunks and relatively large
leaves that are clustered at the apex. Darwin (1979) classified Sarcopygme in Morindeae because of its solitary and erect ovules,
raphide crystals, valvate aestivation of corolla lobes, and multiple
fruits. On the other hand, Johansson (1987) qualified it as a genus
of uncertain taxonomic position. A narrow circumscription of Morindeae proposed by Igersheim and Robbrecht (1993), also endorsed
by Razafimandimbison et al. (2008), excluded Sarcopygme from
Morindeae because of its uniovulate locules and unbranched stigmas. The present analyses, however, show that Sarcopygme, represented here by the type species S. pacifica, belongs to Morindeae,
S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886
883
Fig. 1. Bayesian majority rule consensus tree from the combined nrETS/nrITS/trnT-F data of 67 Morindeae taxa and five outgroup taxa from the tribes Gaertnereae and
Mitchelleae. Values above nodes are Bayesian posterior probabilities. The vertical bar delimits the outgroup taxa; GAE = Gaertnereae and MIT = Mitchelleae; LF and SF stand
for the large- and small-fruited Morinda citrifolia var. citrifolia, respectively; brackets delimit the major lineages of Morindeae corresponding to our newly defined genera. Taxa
in boldface are with non-headed inflorescences and taxa with asterisks (*) are without syncarpous fruits (syncarps).
consistent with Setchell and Christophersen’s (1935) decision; the
tree-like S. pacifica is nested in the largely arborescent Morinda
clade (B in Fig. 1; PP = 1.00).
4.1.4. Pogonolobus F. Muell. and paraphyly of Coelospermum Blume
The Australian and New Guinean Pogonolobus was originally described by Mueller (1858) as a monotypic genus based on its arborescent habit and flowers with pubescent corolla lobes, and exserted
anthers. The genus was later merged by Bentham (1867) in Coelospermum [C. reticulatum (F. Muell.) Benth.] based on its exserted anthers, but Johansson (1987) re-established Pogonolobus based on
palynological characters. Coelospermum is a small genus of seven
species of mainly lianescent plants, which are characterized by the
following combination of characters according to Johansson
(1988): terete branches, sheathing stipules, paniculate or corymbiform and puberulous inflorescences, white corollas with both long
and short hairs inside the tube, mostly simple fruits with ovules inserted at the middle of the septum, seeds shortly winged at one
end and pollen grains with large lumina. Like Morinda, the circumscription of Coelospermum is highly controversial. The Australian
Morinda reticulatus was transferred by Baillon (1879) to Coelospermum as Coelospermum decipiens Baill.; however, this decision was
not followed by many Rubiaceae specialists (e.g., Johansson, 1987).
The present analyses demonstrate that Coelospermum sensu Johansson (1988) is paraphyletic, unless the Australian M. reticulata and P.
reticulatus are also included (C in Fig. 1). This finding is consistent
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S.G. Razafimandimbison et al. / Molecular Phylogenetics and Evolution 52 (2009) 879–886
with the decisions of Bentham (1867) and Baillon (1879) to merge
these two species into Coelospermum but inconsistent with Johansson (1987). The broadly delimited Coelospermum (including M. reticulatus and Pogonolobus) can be characterized by its mainly lianescent
habit and terminal inflorescences composed flowers with anthers
well-exserted beyond the corolla lobes. The Coelospermum clade is
deeply nested between the largely arborescent Morinda (B in Fig. 1)
and the Gynochthodes–Morinda (=D in Fig. 1) clades, and these together are sister to the Appunia clade (A in Fig. 1). Within the Coelospermum clade the M. reticulata–Pogonolobus–C. dasylobum subclade
is defined by interpetiolar stipules and hairy corolla lobes.
4.1.5. Monophyly of Gynochthodes Blume
Gynochthodes is a genus of ca. 18 lianescent species with winding stems, which are distributed from continental southeast Asia
south- and eastwards through Malesia to Micronesia, Fiji, and
northern Australia. According to Johansson (1987), the genus can
be distinguished from Morinda and Coelospermum by its stipules
and bracts, which have marginal hairs, its axillary, racemose or
cymose inflorescences with white and shortly pedunculate flowers
in whorls, and flowers with recurved calyx tubes, corollas with
long hairs within the tubes and on the adaxial side of the lobes.
Our analyses strongly support the monophyly of Gynochthodes
(type species G. coriacea included), which is resolved as sister to
a small Morinda subclade of three Morinda species (M. retusa Poir.,
M. ammitia Halford & A.J. Ford, and M. grayi Seem.). This Gynochthodes–Morinda subclade is also deeply nested among the largely
Morinda clade sister to the Appunia clade (Fig. 1).
4.1.6. Siphonandrium K. Schum.
Siphonandrium is a dioecious monotypic genus from New Guinea with scandent habit, umbel- to head-like inflorescences, and
flowers with anthers fused into a tube (Igersheim and Robbrecht,
1993). This latter character is unique within Morindeae and is very
rare in Rubiaceae [but present in e.g., the genera Argostemma Wall.
(Argostemmateae Bremek. ex Verdc.) and Strumpfia Jacq. (Urophylleae Bremek. ex Verdc.)]. No sequenceable material of S. intricatum
K. Schum. is available for this study and therefore its position within the tribe has yet to be investigated. Based on its scandent habit
and dioecious flowers the genus is possibly closely related to the
Gynochthodes–Morinda clade (=D in Fig. 1).
4.2. Generic circumscriptions in Morindeae
The present study clearly indicates that Morinda as presently
delimited is highly paraphyletic, unless Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme are also included. In other
words, head inflorescences and syncarpous fruits are evolutionarily labile in Morinda and Morindeae. Therefore, new generic limits of Morindeae are needed. Below we present four possible
alternatives for revising generic boundaries to establish monophyletic groups.
4.2.1. Alternative # 1
One is to recognize a broad circumscription of Morinda (Appunia, Coelospermum, Gynochthodes, Pogonolobus, Sarcopygme, and
Siphonandrium included) without infrageneric subdivision. Morinda sensu lato can thus be diagnosed by its massive and T-shaped
placentae inserted in the middle of the septum and two anatropous
ovules per carpel (excepting Sarcopygme), although these are not
obvious characters; the genus is additionally characterized by the
frequent occurrence of head inflorescences/infructescences.
(see A–D in Fig. 1) at subgeneric level. Either of these two alternatives would render Morindeae monogeneric and require a total of 29 new combinations and two new names in
Gynochthodes and Pogonolobus. All described species of Appunia
have already been transferred by Steyermark (1972) to Morinda.
The second alternative would require new descriptions of four
new subgenera.
4.2.3. Alternative # 3
The third alternative is to maintain Appunia (A in Fig. 1) as a distinct genus but merge Coelospermum, Gynochthodes, Pogonolobus,
Sarcopygme, and Siphonandrium in Morinda. According to Steyermark (1967), Appunia is mainly distinct from Morinda (Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme not included)
by its head inflorescences composed of free flowers/fruits and
club-shaped stigmas, rather than head inflorescences formed by
flowers with ovaries fully connate and two-branched styles in Morinda. Following this circumscription, however, Morinda is shown to
be paraphyletic in this study. Plus, recognizing Appunia at generic
level would render Morinda a morphologically heterogeneous
genus with no obvious morphological synapomorphy. In other
words, it would make Morinda s.l. (Coelospermum, Gynochthodes,
Pogonolobus and Sarcopygme included) rather difficult to delimit,
as headed and non-headed inflorescences (Gynochthodes and some
Coelospermum species) and club-shaped (Sarcopygme) and twobranched stigmatic lobes [all Morinda sensu Linnaeus (1753)] are
all present. The two stigmatic lobes are further subdivided into
three lobes in some lianescent Morinda species (e.g., M. collina
Schltr., Johansson, 1994: 32).
4.2.4. Alternative # 4
The fourth alternative is to recognize the four major lineages
(A–D in Fig. 1) as separate genera: Appunia; Morinda s.str. (including Sarcopygme, the only two lianescent African species, M. longiflora and M. morindoides, all neotropical tree-like or suffrutescent
Morinda species, and all arborescent and suffrutescent Asian and
African Morinda species, all with large flowers); Coelospermum
(including M. reticulata and Pogonolobus); and Gynochthodes
(including all lianescent Morinda species from Australia, the Pacific,
tropical and subtropical Asia, and Madagascar). For the Gynocthodes–
Morinda clade (=D in Fig. 1) four validly published generic names,
Gynochthodes, Sphaerophora Blume (Blume, 1850), Pogonanthus
Montrouz. (Montrouzier, 1860), and Imantina Hook.f. (Hooker,
1873), are available, with the former genus having priority over the
latter two names. This group can be defined by its lianescent habit,
small flowers with partly exserted anthers and this scenario would
require up to 80 new combinations. Within the Gynochthodes–
Morinda clade (D in Fig. 1) recognizing the sister groups, subclade
Gynochthodes (including M. ammitia, M. grayi, and M. retusa) and
subclade Morinda, as separate genera does not seem an attractive
solution, because there is no obvious character for distinguishing
them.
We favor the fourth of these four alternative realignments, the
recognition of the four major lineages (A–D in Fig. 1) as separate
genera, because this classification reflects the occurrence of a
considerable morphological diversity in Morindeae and the phylogenetic and taxonomic distinctness of its newly delimited genera.
For now we maintain the New Guinean genus Siphonandrium as a
distinct genus. Table 2 summarizes all five accepted genera of
Morindeae and finally, all necessary new combinations will be
published elsewhere.
4.3. Keys to the accepted genera of Morindeae
4.2.2. Alternative # 2
A second scenario is to adopt a broad circumscription of
Morinda and recognize the four well-supported major lineages
Below we present keys that can be used to identify all accepted
genera of Morindeae on the basis of morphological traits.
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Table 2
List of genera accepted here and their synonyms, geographic distributions, and number of species.
Genera accepted in Morindeae
Synonyms
Geographic distributions
Number of species
Appunia Hook.f.
Coelospermum Blume
Gynochthodes Blume
Morinda L.
Siphonandrium K. Schum.a
Bellynkxia Müll. Arg.
Holostyla Endl.; Merismostigma S. Moore; Olostyla DC.; Pogonolobus F. Muell.
Imantina Hook.f.; Pogonanthus Montrouz.; Sphaerophora Blume; Tetralopha Hook.f.
Appunettia Good; Sarcopygme Setch. & Christoph
Neotropics
Australasia
Australasia and Madagascar
Pantropical
New Guinea
ca. 16
ca. 9
80–100
30–35
1
a
Not included in this study.
1a. Arborescent, rarely suffrutescent (the African M. angolensis
Good and the Haitian M. buchii Urb.) and lianescent [the African
M. longiflora G. Don and M. morindoides (Baker) Milne-Redh.],
large (corolla tubes/corolla lobes > 1) and hermaphroditic
flowers... . .... . .. . .. . .. . ...... . .. . .. . .. . .. . .. . .. . ... . .. . .. . .. . .. . .. . .. . .2.
2a. Flowers free, congested, stigmatic lobes club-shaped, fruits
simple, free ... . .. . .. . ...... . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . ... . ..Appunia.
2b. Flowers fused basally or partly or completely, stigmatic lobes
bifid, fruits syncarpous (syncarps). . .. . ... . .. . .. . .. . .Morinda s.str.
1b. Lianescent, rarely arborescent (the New Guinean and Australian Pogonolobus reticulatus and Hawaiian Morinda trimera
Hillebr.), small (corolla tubes/corolla lobes < 1, except
Gynochthodes sublanceolata Miq. and M. trimera) and polygamous or dioecious flowers. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .3.
3a. Anthers fused into a tube. . .. . .. . .. . .. . .. . .. . .. . .Siphonandrium.
3b. Anthers not fused into a tube.. . .. . .. . .. . .. . .. . ... . ... . ... . ... . ..4.
4a. Inflorescences mostly paniculate, sometimes corymbs,
anthers
well
exserted
beyond
the
corolla
tubes. . .. . .. . .. . .. . .. . .. . .. . .. . ..... . ... . ... . ... . ... . ... . ..Coelospermum.
4b.
Inflorescences
never
paniculate,
anthers
partly
exserted. . .. . .. . .. . .. . .. . ..... . .. . .. . .. . .. . .. . ...... . ... . .Gynochthodes.
It is important to note that seven of the eight described African
species of Morinda s.str. have much larger flowers [corolla tubes
ranging from 12 to 40 mm (rarely 80 mm) long and corolla lobes
varying between 3 and 14 mm (rarely 22 mm) long] than the
species of Appunia and the remaining species of Morinda s.str.
The flowers of Coelospermum as defined here are much larger
[corolla tubes ranging from 3 to 7 mm (rarely 11 mm) long
and corolla lobes varying from 4.5 to 16 mm long] than that of
the newly delimited Gynochthodes [corolla tubes ranging from
0.7 to 5.5 mm long and corolla lobes varying from 1.5 to
11 mm long].
5. Conclusions
The present study demonstrates for the first time that Morinda
as presently delimited is highly paraphyletic, unless Coelospermum,
Gynochthodes, and Sarcopygme are also included. Both head inflorescences and multiple fruits are evolutionarily labile in Morinda
and Morindeae. The tribe Morindeae can be subdivided into four
well-supported major lineages, which can be characterized by a
combination of growth habit, inflorescence type and position, infructescence type, flower size, and breeding systems: Appunia clade,
Morinda clade (including M. royoc, the type species of the genus,
and Sarcopygme), Coelospermum clade (including Pogonolobus and
Morinda reticulata), and Gynochthodes–Morinda clade. Four possible
alternatives for revising generic boundaries are presented to establish monophyletic units. We favor the recognition of the four wellsupported and morphologically distinct major lineages of Morindeae (A–D in Fig. 1) as separate genera, because this classification
reflects the occurrence of a considerable morphological diversity in
the tribe and the phylogenetic and taxonomic distinctness of its
newly delimited genera.
Acknowledgments
The authors thank Dr. Jan Thomas Johansson (Stockholm University, Stockholm, Sweden) for sharing his documentation and
knowledge on Morinda and its allied genera with SGR; Mrs. Anbar
Khodanbadeh and Linda Lundmark for help with sequencing; Dr.
David Lorence (National Tropical Botanical Garden, Hawaii, USA),
Dr. Arnaud Mouly (Bergius Foundation, Stockholm, Sweden), Dr.
Hua Zhu (Xishuangbann Tropical Botanical Garden, the Chinese
Academy of Sciences, Kunning, China), and Dr. Andrew Ford
(CSIRO, Sustainable Ecosystems and Rainforest CRC, Queensland,
Australia) for kindly providing leaf material for this study; the ANGAP (Association Nationale pour Gestion des Aires Protégées) and
MEF (Ministères des Eaux et Forêts) for issuing collecting permits
for SGR; Missouri Botanical Program, Madagascar for logistical support; Lalao Andriamahefarivo (MBG, Madagascar) for arranging
collecting permits for SGR; Désiré Ravelonarivo for being an excellent field assistant in the Marojejy; the following herbaria for
allowing access to their collections: AAU, BR, BRI, ETSU, GB, K, P,
PTBG, S, TAN, TEF, UPS, and US. This work is supported by the
Swedish Research Council to S.R. and B.B. and the Knut and Alice
Wallenberg Foundation to B.B.
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