TAXON 61 (6) • December 2012: 1251–1268
Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
Delimitation of the genus Margaritopsis (Rubiaceae) in the Asian,
Australasian and Pacific region, based on molecular phylogenetic
inference and morphology
Laure Barrabé,1,2 Sven Buerki,3 Arnaud Mouly,4 Aaron P. Davis,3 Jérôme Munzinger5 & Laurent Maggia1,2
1
2
3
4
5
CIRAD, UMR AGAP, 98800 Noumea, New Caledonia, France
IAC, BP 18239, 98857 Noumea sud, New Caledonia, France
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, U.K.
Université de Franche-Comté, UMR CNRS 6249 Chrono-Environnement, 16 route de Gray, 25030 Besançon cedex, France
IRD, UMR AMAP, Laboratoire de Botanique et d’Écologie Végétale Appliquées, Herbarium NOU, 98848 Nouméa, NouvelleCalédonie; and IRD, UMR AMAP, 34000 Montpellier, France
Authors for correspondence: Laure Barrabé, laure.barrabe@ird.fr; Aaron P. Davis, a.davis@kew.org
Abstract In the past, the circumscription of the large genus Psychotria (Rubiaceae) was difficult, until molecular phylogenetic
studies revealed its considerable paraphyly, enabling the delimitation of its major lineages and the grouping of related genera,
and most notably the separation of Psychotria and its relatives (former Psychotrieae) into two tribes: Psychotrieae and Palicoureeae. The genus Margaritopsis, which is included in Palicoureeae, encompasses 27 Neotropical species, and in previous studies
these have been shown to be close relatives of a group of eight Psychotria species that occur over a large region extending from
South-East Asia to tropical South Pacific through Malesia (= the AMP region, defined as including South-East Asia, Malesia,
tropical Australia, Melanesia, Micronesia, New Caledonia and Polynesia). A molecular phylogenetic study, using one nuclear
DNA region (ITS) and four plastid DNA regions (ndhF, rps16, trnH-psbA, trnT-F), is undertaken in order to test the placement of 17 AMP Psychotria species within Palicoureeae. The phylogenetic results show that they form a monophyletic clade
(= clade G), which also includes the monotypic Fijian genus Readea and Hodgkinsonia frutescens from Australia. Clade G is
embedded in a well-supported grade with five Neotropical representatives of Margaritopsis. A morphological survey based on
twenty characters, with the potential to circumscribe generic entities, shows that the monophyly of clade G is supported by a
character combination that is similar to species of Neotropical Margaritopsis, confirming their inclusion in this genus. Taxonomic and nomenclatural work on these species is required to formalize nomenclatural implications. Based on the same set of
morphological characters, but in absence of molecular data, 28 other Psychotria species from the AMP region were detected
as likely candidates for inclusion in Margaritopsis, allowing the estimation of species richness of clade G to be a minimum of
47 species. Within clade G, five well-supported subclades and a Readea lineage are delimited and each of these is generally
supported by unique morphological features. Phylogenetic topologies reveal biogeographical patterns, including a main route
of dispersal from western to eastern parts of the AMP region, with subsequent dispersals between archipelagos in the region.
Keywords biogeography; big genera; Malesia; Margaritopsis; Palicoureeae; phylogeny; Psychotria; South-East Asia;
tropical South Pacific
Supplementary Material Appendix 2 is available in the Electronic Supplement to the online version of this article (http://
www.ingentaconnect.com/content/iapt/tax).
INTRODUCTION
The pantropical genus Psychotria L. is by far the largest
genus of Rubiaceae (tribe Psychotrieae Cham. & Schltdl.) with
ca. 1800 species (Davis & al., 2009). It is also in the top five
largest angiosperm genera and is probably the world’s largest
predominantely woody genus (Frodin, 2004). Due to its size
and apparent lack of defining characters Psychotria is usually
perceived as a ‘botanical nightmare’, especially for those requiring species identifications. Its immense diversity, spanning
the whole tropics, coupled with the difficulty in ascertaining
useful morphological characters (Piesschaert, 2001) has confounded Rubiaceae systematists (e.g., Robbrecht, 1988; Taylor,
1996; Davis & al., 2001). These problems are nearly universal
in big genera: their size rendering them difficult, if not impossible, to study in their entirety (Frodin, 2004).
Progress in understanding the delimitation of Psychotria
has been slow but progressive. The broad Psychotria concept was refuted as early as the 1960s in classical morphotaxonomic studies of the Afro-tropical representatives, including the works of Petit (1964, 1966), Robbrecht (1975) and
Verdcourt (1975, 1977). These authors segregated several genera or established new criteria to distinguish related genera,
mainly using fruit characters (Robbrecht & Manen, 2006). As
soon as molecular analyses were applied to Psychotria and
associated genera, it quickly became clear that Psychotria
was conspicuously paraphyletic (Andersson & Rova, 1999;
Nepokroeff & al., 1999; Bremer & Manen, 2000). Foreseeing
Version of Record (identical to print version).
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
taxonomic and nomenclatural implications, Davis & al. (2001)
typified Psychotria with a species from Hong Kong (P. asiatica L.). Andersson (2001) informally recognized two major groups, viz. a ‘Psychotria group’ (including Psychotria
s.str.) and a ‘Palicourea group’ (including Psychotria spp. and
morphologically related genera). This concept was expanded
and elaborated by Anderson (2002a, b), with the addition of
further sampling and morphological data, although in this
case the two major groups were referred to as the ‘Psychotria
complex’ and the ‘Palicourea complex’. Robbrecht & Manen
(2006) then formally recognized two tribes, Psychotrieae and
Palicoureeae Robbr. & Manen. According to Andersson (2001,
2002a) and Robbrecht & Manen (2006) Palicoureeae is set
apart from Psychotrieae by the following morphological characters (characters for Psychotrieae in parentheses): stipules
non-caducous (vs. caducous), preformed germination slits in
pyrene mostly present (vs. mostly absent; except in Pacific
representatives), endosperm non-ruminate (vs. mostly ruminate; except in Pacific representatives), red seed coat pigment
[ethanol soluble] absent (vs. mostly present). This classification
has been adopted (Razafimandimbison & al., 2008), although
some authors prefer adopting a more conservative approach by
recognizing a more inclusive tribe Psychotrieae (Bremer, 2009;
Bremer & Eriksson, 2009).
The aforementioned molecular studies have enabled a more
focused approach for investigating Psychotria, and workers
have started to provide either morphological synapomorphies
or at least a combination of diagnostic characters to support
phylogenetic entities for Psychotria and its major lineages, and
related genera in Psychotrieae (Davis & Bridson, 2001, 2004;
Piesschaert, 2001). Although new insights into the phylogenetic
relationships within and outside Psychotria are acknowledged,
major problems still persist. Significant amongst these is the
considerable problem of identifying Psychotria species in the
Old World that are in fact not members of the Psychotrieae
lineage at all but instead belong to Palicoureeae. For the New
World, progress has been made in resolving generic delimitation within the Palicoureeae clade, and assigning species formerly included in Psychotria into their respective Palicoureeae
genera, including Carapichea Aubl. (Taylor & Zappi, 2006),
Margaritopsis Sauvalle (Taylor, 2005), Notopleura (Hook. f.)
Bremek. (Taylor, 2001), Palicourea Aubl. (Taylor, 1997), and
Rudgea Salisb. (Zappi, 2003; Taylor & Zappi, 2006). Largely
on the basis of molecular studies (Andersson & Rova, 1999;
Nepokroeff & al., 1999; Andersson, 2001, 2002b) ten genera
are assigned to Palicoureeae: Carapichea, Chassalia Comm.
ex Poir., Chazaliella E.M.A. Petit & Verdc., Geophila D. Don,
Hymenocoleus Robbr., Margaritopsis, Notopleura (= Psychotria sect. Notopleura sensu Hooker, 1873), Palicourea s.l.
(including Palicourea s.str. and Psychotria subg. Heteropsychotria sensu Steyermark, 1972), Readea Gillespie and Rudgea. Phylogenetic relationships between these genera remain
poorly understood, and other questions regarding monophyly,
geographical distribution and species richness persist. In addition, important taxonomic treatments have been undertaken
for several Old World Palicoureeae genera, or for the Old World
representatives, including, e.g., Chassalia (Piesschaert, 2001;
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TAXON 61 (6) • December 2012: 1251–1268
Piesschaert & al., 2001), Chazaliella (Verdcourt, 1977), and
Hymenocoleus (Robbrecht, 1975).
Among the New World Palicoureeae genera, Margaritopsis was initially described to accommodate a species endemic to Cuba, M. nudiflora (Griseb.) K. Schum. (initially
M. acuifolia C. Wright), based on the previous but illegitimate
genus Margaris Griseb. (Sauvalle, 1869). This genus was later
expanded to include 27 species that are widespread throughout the Neotropics (Andersson, 2001; Taylor, 2005). However,
Andersson & Rova (1999) found a close relationship between
Margaritopsis, the type species of the African genus Chazaliella (C. abrupta (Hiern) E.M.A. Petit & Verdc.), and the monospecific Fijian genus Readea. Andersson (2001) confirmed the
former relationship based on a single plastid region (rps16), and
enlarged the delimitation of the clade by adding two African
Chazaliella, and one Caribbean Margaritopsis, and, for Psychotria, two South American species, and eight species from
South-East Asia, Malesia and tropical South Pacific islands.
These phylogenetic results are in agreement with the carpological study conducted by Piesschaert (2001) who found a
similar and stable morphology of pyrenes for all species tested
by Andersson (2001). Consequently, Andersson (2001) enlarged
the generic circumscription of Margaritopsis by including the
genera Chazaliella, Readea, and certain Psychotria species (see
above), and estimated its diversity at 50 species. However, he
only published nomenclatural combinations in Margaritopsis
for the type species of Chazaliella and Readea, and included
the characters of these former genera in an amended generic
description of Margaritopsis (Andersson, 2001, 2002c). This
morphological circumscription was largely completed by
Taylor (2005) to accommodate all other Neotropical species
that belong to the genus. Among them Taylor (2005) delineated three informal groups: the ‘Margaritopsis group’, including M. nudiflora, the ‘Chytropsia group’ and the ‘Chazaliella
group’. The actions of Taylor (2005) implicitly raise the question of the inclusion of the African genus Chazaliella within
Margaritopsis. Even though there are similar morphological
features within the Margaritopsis/Chazaliella/Readea group
of Andersson (2001), its internal phylogenetic relationships
still remain poorly resolved. A study currently in preparation
promises new insights into African Chazaliella (O. Lachenaud,
pers. comm.).
In this study we aim to investigate the Psychotria species that have been suggested as belonging to Palicoureeae
(Andersson, 2001) from the AMP region, which we define here
as: South-East Asia (= South-East Asian mainland), Malesia
(Indonesian archipelago, New Guinea, the Philippines), tropical
Australia and tropical South Pacific islands (Melanesia: Bismarck, Fijian, Solomons and Vanuatu archipelagos; Micronesia; New Caledonia; Polynesia: Austral, Gambier, Marquesas,
Samoan, Society, Tongan and Tuamotu archipelagos). In the
study of Margaritopsis by Andersson (2001) eight species were
examined: one species from China (P. montana Blume), one
from Indonesia (P. straminea Hutch. in C.S. Sargent), two from
New Caledonia (P. collina Labill., P. oleoides (Baill.) Schltr.),
one from Vanuatu (P. aneityensis Guillaumin), and three from
Fiji (P. amoena A.C. Sm., P. archboldiana Fosberg, P. incompta
Version of Record (identical to print version).
Australia
Fiji
1
2
1
1
Monotypic
NA
Clade G
Clade G
Hodgkinsonia F. Muell.
Readea Gillespie
AMP region (see Fig. 4 for distribution
and species richness)
Paraphyletic (monophyletic with inclusion 17
of Hodgkinsonia frutescens and Readea)
Clade G
AMP Psychotria L.
47
Neotropics
27
Africa
NA
5
Neotropical Margaritopsis
Margaritopsis Sauvalle
1
Paraphyletic (possibly monophyletic)
Chazaliella
Chazaliella E.M.A. Petit & Verdc.
20
Neotropics
Africa
12
12
3
Monophyletic
1
Carapichea
Chassalia-Geophila-Hymenocoleus group NA
Hymenocoleus Robbr.
Carapichea Aubl.
Africa, Madagascar, SE Asia, Indonesia
Worldwide
27
120
Chassalia-Geophila-Hymenocoleus group Monophyletic
Geophila D. Don
2
Chassalia-Geophila-Hymenocoleus group Monophyletic
Chassalia Comm. ex Poir.
6
Neotropics
Neotropics
100
120
1
Notopleura-Rudgea group
Rudgea Salisb.
Version of Record (identical to print version).
NA
NA
Notopleura-Rudgea group
Notopleura (Hook. f.) Bremek.
1
Neotropics
Neotropics
310
2
Monophyletic
Species
sampled
2
Monophyletic
Palicourea s.l.
Psychotria L. subg. Heteropsychotria Bremek. Palicourea s.l.
Palicourea Aubl. s.str.
Taxon sampling. — The sampling comprises 56 species,
including sequences of 13 species retrieved from GenBank.
Voucher information and GenBank accession numbers are
given in Appendix 1. The Palicoureeae sampling was centered
on the AMP region, covering the species richness in each biogeographical area as defined by Mueller-Dombois & Fosberg
(1998) and Keppel & al. (2009): South-East Asia (Vietnam),
Malesia (New Guinea), tropical Australia and tropical South
Pacific islands (Fiji, New Caledonia, Samoa, Society archipelago and Vanuatu). In addition to the 17 AMP Psychotria
sampled, at least one species of each Palicoureeae genus (as
enumerated in the introduction and summarized in Table 1) was
included in the sampling. Hodgkinsonia frutescens C.T. White,
which belongs to the bitypic genus Hodgkinsonia F. Muell.,
endemic to tropical Australia, was also included because its
morphology is similar to some Palicoureeae. The samples Psychotria sp. ‘NC’ and P. sp. ‘V’ belong to new undescribed taxa
from New Caledonia and Vanuatu, respectively. Psychotria
sp. ‘FIJ’, P. sp. 1 [VIET] and P. sp. 2 [VIET] were identified
as species of uncertain affinity but constitute distinct species.
The identification of P. sp. [AUST] was not possible because
no herbarium specimen was provided (cultivated sample,
Appendix 1). Extraction of DNA from herbarium specimens
from Indonesia, the Philippines and the Micronesian archipelagos was carried out but failed to provide useful amounts
of DNA, with the exception of one species of Chassalia from
Sumatra. To further confirm the relationships within Palicoureeae, several species of Psychotrieae from the Pacific clade
and the Psychotria s.str. clade as defined by Andersson (2002a)
were sampled. The type species of Psychotria (Davis & al.,
2001; Sohmer & Davis, 2007), P. asiatica, was also included
in this study. Craterispermum Benth. and Prismatomeris Thw.
were chosen as outgroup taxa following Razafimandimbison
& al. (2008) and Rydin & al. (2009).
Phylogenetic status in this study
MATERIALS AND METHODS
Phylogenetic group
A.C. Sm.). However, this sampling does not satisfactorily represent the distribution and species richness of the group in the
AMP region, as there is no sample from Australia, large parts
of South-East Asia, Malesia and other tropical South Pacific
islands. This is not an issue for Hawaii, as all species occurring
in the archipelago belong to a single clade in Psychotrieae (Nepokroeff & al., 1999, 2003). These considerations provide clear
objectives for the present investigation, which are to: (1) construct a phylogenetic framework that will allow us to elucidate
those Psychotria taxa from the AMP region (= AMP Psychotria)
that are closely related to Neotropical Margaritopsis, based on
plastid and nuclear DNA regions; (2) assess the species richness and geographical range of the AMP Psychotria group (i.e.,
belonging to Palicoureeae) based on a morphological survey of
species occurring in this area; and (3) find morphological synapomorphies that support both the monophyly of clades within
the AMP Psychotria group, and the inclusion of these taxa in
Margaritopsis. Finally, we discuss biogeographical patterns that
may have been involved in shaping current species diversity.
Species
estimated Distribution
Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
Table 1. Genera of Palicoureeae and their phylogenetic status based on the data presented in this study. The distribution and number of species per genus are taken from Govaerts & al. (2011) and
this study.
TAXON 61 (6) • December 2012: 1251–1268
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
DNA regions. — Five plastid and nuclear DNA regions,
which have been widely applied to retrieve phylogenetic relationships within Rubiaceae, were sequenced. Four plastid
markers were used: the rps16 intron (Oxelman & al., 1997), the
trnT-F region (including the trnT-trnL and trnL-trnF spacers
and the trnL intron; Taberlet & al., 1991), the ndhF gene (Kim
& Jansen, 1995), and the trnH-psbA spacer (Aldrich & al.,
1988). A single nuclear marker was used: the ITS region, including ITS1, 5.8S and ITS2 (Baldwin & al., 1995). The primers used in polymerase chains reactions were the following:
A1/IR and C/F for trnT-F (Razafimandimbison & Bremer,
2002; Taberlet & al., 1991); trnHGUG/psbA for trnH-psbA (Sang
& al., 1997; Tate & Simpson, 2003); F/2R for rps16 (Oxelman & al., 1997); 2F/1000R, 720F/1700R and 1320F/2280R for
ndhF (Rydin & al., 2008) and P17/26S-82R for ITS (Bolmgren
& Oxelman in Popp & Oxelman, 2001).
PCR protocols and DNA sequencing. — All DNA samples were extracted with the DNAeasy Plant Mini Kit (Qiagen,
Crawley, U.K.) and DNA regions were amplified with the
GoTaq Flexi DNA Polymerase (Promega, Madison, Wisconsin,
U.S.A.). For the ndhF, rps16, trnH-psbA and trnT-F regions,
the mix template included 5 µL of Buffer (5×), 1.5 µL of MgCl2
(25 mM), 0.25 µL of dNTP mix (10 mM of each dNTP), 0.75 µL
of primers (10 µM, forward and reverse), 0.25 µL of polymerase
(5 U/µL), 2 µL of DNA and the volume was adjusted to 25 µL
with water. For the ITS region, the mix template was the same
as for the plastid regions, but with the addition of 0.625 µL of
BSA (10%) and 0.5 µL of DMSO (10%). The following PCR
programs were used for the plastid markers: 3 min at 94°C,
35 cycles of 1 min at 94°C, 1 min at 50°C, 2 min at 72°C, followed by a final extension of 7 min at 72°C, and for the nuclear
marker: 3 min at 94°C, 35 cycles of 30 s at 94°C, 1 min at 50°C,
1 min 30 s at 72°C, followed by a final extension of 7 min at
72°C. All PCR products were purified using DNA purification
columns according to the manufacturer’s protocol (QIAquick
PCR Purification Kit; Qiagen) or using a Sephadex column
purification (Sephadex G-50 Superfine resin, Sigma-Aldrich,
St. Louis, Missouri, U.S.A.). Dideoxy cycle sequencing was
then performed using the chain termination method and ABI
Prism Big Dye version 3.1 reaction kit, following the manufacturer’s protocol, but using 0.5 µL of reaction mix (Applied
Biosystems, Warrington, U.K). The products were prepared for
sequencing using a Sephadex column purification (Sephadex
G-50 Superfine resin, Sigma-Aldrich) and visualised on an ABI
3730 Genetic Analyser, also according to the manufacturer’s
protocol.
Phylogenetic analyses. — The program Sequencher
v.4.10.1 (Gene Codes, Ann Arbor, Michigan, U.S.A.) was used
to assemble complementary strands and verify software basecalling. The regions were initially aligned individually with the
online version of MUSCLE v.3.8.31 (www.ebi.ac.uk/tools/msa/
muscle), and subsequently manually adjusted with MEGA5
(Tamura & al., 2011). Sequences of Palicourea nitidella (Müll.
Arg.) Standl. and Psychotria stachyoides Benth. were sometimes difficult to align for a large part of the trnH-psbA and
the trnT-F regions. As both species are quite divergent from
ingroup species, these parts were replaced with missing data
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TAXON 61 (6) • December 2012: 1251–1268
(trnH-psbA: positions 112–538; trnT-F: 94–429 for Palicourea
nitidella and 94–407 for P. stachyoides). An inversion was
found in some of the trnH-psbA sequences (88–93) and this
portion was therefore not included in the phylogenetic analyses.
The introduction of many indels in the rps16, trnH-psbA and
trnT-F matrices and the occurrence of a large poly A/T region
in the rps16 sequences (positions 399–566) induced problematic homology hypotheses (Shaw & al., 2005). The nrDNA
ITS region has proven useful for reconstructing relationships
of closely related species, due to high levels of polymorphism
(Zarrei & al., 2012). However, the occurrence of multiple substitutions, and/or an incomplete process of concerted evolution
(that normally allowed elimination of pseudogenes), can corrupt the phylogenetic signal and establish inaccurate relationships (Razafimandimbison & al., 2004; Sanderson & Doyle,
1992). To improve the quality of analyses, ambiguous regions
were removed from all DNA alignments by using the BMGE
software v.1.1 (Criscuolo & Gribaldo, 2010), which estimates
entropy for each position and eliminates those for which the
entropic values are too high. The BMGE analyses were set as
follows: PAM250 similarity matrix; sliding window set to 1
(with the exception of the coding ndhF region that was set with
a sliding window of 3); gap cut-off set to 0.75 for most of the
plastid regions and to 0.2 for ndhF and the ITS region (Table 2).
Single-gene and combined phylogenetic inferences were
carried out employing both maximum parsimony (MP) and
Bayesian Markov chain Monte Carlo (MCMC) analyses. MP
analyses were performed using PAUP* (Swofford, 2002) with
the following settings: random addition sequence (nreps =
10,000 and 20 trees saved per replicate), tree-bisection-reconnection branch swapping, STEEPEST and MULTREES options
in effect, and an unlimited value for MAXTREES. A majorityrule consensus tree was constructed based on the most parsimonious trees. To estimate homoplasy, the consistency index
(CI) and the retention index (RI) were calculated. To evaluate
node support a bootstrap analysis (BS; Felsenstein, 1985) was
performed using PAUP* (Swofford, 2002) with 500 replicates
by using the same parameters as above.
Best-fit models for each DNA region were identified using jModelTest v.0.1.1 (Posada, 2008) based on the Akaike
criterion (see Table 2). In the combined Bayesian MCMC
analysis, the dataset was divided into five partitions and each
locus was allowed to have partition-specific model parameters
(Ronquist & Huelsenbeck, 2003; Nylander & al., 2004). The
single-gene and combined analyses were set as follows: four
Metropolis-coupled Markov chains with an incremental heating
temperature of 0.2 were run for 25 million generations and a
tree was sampled every 1000th generation. The analysis was
repeated three times, starting with random trees, and was run
in MrBayes v.3.1.2 (Ronquist & Huelsenbeck, 2003). Bayesian MCMC analyses were carried out using the facility Bioportal provided by the University of Oslo (www.bioportal.uio
.no, Norway). The MCMC sampling was considered sufficient
when the effective sampling size (ESS) was higher than 200, as
verified in Tracer v.1.5 (Rambaut & Drummond, 2007). After
a burn-in period of 1 × 106 generations per run, the remaining
trees were used to construct a half-compatible consensus tree
Version of Record (identical to print version).
Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
TAXON 61 (6) • December 2012: 1251–1268
(i.e., majority-rule consensus tree from MrBayes) and its associated Bayesian posterior probabilities (PP). A clade with a
PP or a BS value higher than 0.95 and 95%, respectively, was
considered as well supported.
Hypothesis testing and ambiguous phylogenetic placement of Margaritopsis nudiflora. — Preliminary results suggested ambiguous placements for the type species of Margaritopsis (see the Results section below for more details). We failed
to sequence M. nudiflora for the ndhF region. Therefore, to
estimate the influence of missing data on the phylogenetic position of this taxon, we removed this DNA region from the dataset
and conducted subsequent combined Bayesian MCMC analysis with the pruned matrix (= dataset without ndhF). These
analyses were run with the same parameters as those for the
whole dataset (= dataset with ndhF). Furthermore, because we
are aiming at unravelling phylogenetic relationships between
taxa of Margaritopsis and the AMP Psychotria species, two
additional combined constrained Bayesian MCMC analyses for
each dataset (with ndhF and without) were computed following
the approach described by Bone & al. (2012). In the first constrained Bayesian MCMC analyses, the five Neotropical Margaritopsis (including M. nudiflora) were constrained as monophyletic. In the second constrained Bayesian MCMC analyses,
four Neotropical Margaritopsis (M. astrellantha (Wernham)
L. Andersson, M. boliviana (Standl.) C.M. Taylor, M. guianensis (Bremek.) C.M. Taylor, M. kappleri (Miq.) C.M. Taylor) and all AMP Psychotria species plus their relatives were
constrained as constituting a monophyletic group. These constrained analyses were run with the same parameters as those
in the unconstrained combined Bayesian MCMC analyses (see
above). The Shimodaira-Hasegawa test (SH test, Shimodaira
& Hasegawa, 1999) was employed to determine whether the
half-compatible consensus topologies resulting from each constrained Bayesian MCMC analysis were statistically worse
than unconstrained topologies. The SH test was carried out in
PAUP* (Swofford, 2002) using the RELL method with 10,000
bootstrap replicates. Because only one set of model parameters
can be implemented in PAUP*, each combined dataset was
then considered as a single DNA partition. The best-fit model
for each combined dataset was estimated to be GTR + G + I
with jModelTest (Posada, 2008), and these estimated model
parameters were used to set the SH tests.
Examination of herbarium specimens. — To identify morphological synapomorphies supporting monophyletic lineages, a
representative selection of herbarium specimens of the ingroup
taxa (deposited at K, NOU and P) was examined and combined
with field observations by the authors. Type specimens were systematically consulted where available; Appendix 2 (Electronic
Suppl.) provides details on the herbarium specimens examined.
In addition, local taxonomic treatments of Psychotria and associated genera occurring in the AMP region (Samoa: Whistler,
1986; Fiji: Smith & Darwin, 1988; New Guinea and Bismarck
Archipelago: Sohmer, 1988; the Philippines: Sohmer & Davis,
2007) were used. The revisions of Fosberg & al. (1993) and
Chen & Taylor (2011), respectively, for Micronesian and Chinese
Psychotria taxa did not provide enough diagnostic features and
were therefore not incorporated in this study. There is a lack
of taxonomic treatments for South-East Asian and Indonesian
taxa, which resulted in difficulties in assessing species richness and morphological diversity in these areas. Morphological
characters of outgroup taxa were compiled based on available
taxonomic revisions and carpological data (Petit, 1964; Robbrecht, 1975; Verdcourt, 1975, 1977; Taylor, 1997, 2001, 2005,
2010; Nepokroeff & al., 1999; Piesschaert & al., 1999, 2001;
Piesschaert, 2001; Andersson, 2001, 2002b; Zappi, 2003; Taylor
& Zappi, 2006). These characters must be considered cautiously
because they reflect global tendencies within Palicoureeae
genera, although they allow morphological comparison with
Table 2. Characteristics of the DNA partitions and BMGE software settings used in the phylogenetic analyses of tribes Palicoureeae and Psychotrieae.
trnT-F
trnH-psbA
rps16
ndhF
ITS
Total
Cellular compartment
Plastid
Plastid
Plastid
Plastid
Nuclear
Number of taxa
52
39
53
53
58
58
7089
Number of aligned characters
2209
845
1224
2061
750
Similarity matrix
PAM250
PAM250
PAM250
PAM250
PAM250
Sliding windows size
1
1
1
3
1
GAP rate cut-off
0.75
0.75
0.75
0.2
0.2
Number of included characters (A)
1770
324
863
2040
702
5699
Number of variable uninformative characters
173
38
105
166
72
554
Number of parsimony-informative characters (PICs) (B) 231
83
149
245
245
953
Number of constant characters
203
609
1629
385
4192
17
1366
% PICs within separate DNA region (B / A × 100)
13
26
17
12
35
Evolution model
GTR + G
GTR + I + G
SYM + I + G
GTR + I + G
SYM + G
Retention index (RI)
0.906883
0.877301
0.900510
0.922351
0.723106
0.834950
Consistency index (CI)
0.798246
0.782609
0.790323
0.811287
0.464253
0.658811
Length of most parsimonious trees
570
184
372
567
1105
2843
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
Psychotria taxa from the AMP region. Two taxonomic levels
were investigated in the morphological section. Firstly, at the
species level, features that allowed morphological comparisons
between the AMP Psychotria species include: stipule shape,
inflorescence type, corolla aestivation, calyx and corolla shape,
size and texture, fruit shape, and pyrene details. Secondly, at the
generic level, the characters that allowed comparison between
Palicoureeae genera include: general habit, vegetative features
of the stem, leaves and stipules, general features of corolla
(symmetry and colour), fruit colour, and features of pyrenes.
In addition, herbarium specimens of other AMP Palicoureeae
species (not previously included in the ingroup sampling of the
phylogenetic study) were also examined to determine if they
share morphological affinities with the ingroup (Appendix 2
in the Electronic Suppl.). This work allowed us to estimate the
TAXON 61 (6) • December 2012: 1251–1268
putative species richness and endemism of AMP Psychotria
taxa for each biogeographical area of the AMP region. The species names used in this study correspond to those of the World
Checklist of Rubiaceae (Govaerts & al., 2011).
RESULTS
Sequences characteristic. — The trnT-F region and ndhF
gene provide the longest DNA alignment lengths (with 2209 and
2061 base pairs, and 1770 and 2040 included sites, respectively,
after the exclusion of ambiguous sites using BMGE analyses;
Table 2). The combined DNA dataset is composed of 7089 base
pairs and 5699 sites, retained after trimming the single gene
matrices in BMGE (see Table 2 for further statistics). The ITS
1 / 100
1 / 100
1 / 93
1 / 80
1 / 100
1 / 96
0.63 / -
1 / 100
1 / 100
1 / 100
1 / 100 1 / 100
0.52 / -
G
1 / 100
1 / 100
1 / 97
1 / 97
0.61 / 54
0.97 / 54
1 / 99
0.99 / 53
1 / 100
F
1 / 100
1 / 100
1 / 99
E
0.67 / -
1 / 100
D
1 / 100
1 / 100
1 / 99
1 / 56
1 / 98
0.74 / -
1 / 100
C
0.83 / -
0.73 / 56
1 / 90
1 / 92
PALICOUREEAE
B
1 / 60
1 / 97
A
1 / 100
1 / 100
0.95 / -
1 / 100
Pacific Clade
PSYCHOTRIEAE
1 / 100
1 / 100
0.93 / 79
1 / 100
0.94 / 66
Psychotria s.str.
sensu Andersson
1 / 100
1 / 68
1 / 80
1 / 69
Craterispermum sp2
Craterispermum sp3
Prismatomeris albidiflora
Prismatomeris beccariana
Psychotria lepiniana
Psychotria spFIJ
Psychotria carnea 1
Psychotria archboldiana
Psychotria carnea 2
Readea membranacea
Psychotria forsteriana
Psychotria samoana
Psychotria cf trichostoma
Hodgkinsonia frutescens
Psychotria spAUST
Psychotria leptothyrsa
Psychotria aneityensis
Psychotria lyciiflora
Psychotria spNC
Psychotria spV
Psychotria oleoides
Psychotria collina
Psychotria sp1VIET
Psychotria sp2VIET
Margaritopsis nudiflora
Margaritopsis kappleri
Margaritopsis astrellantha
Margaritopsis boliviana
Margaritopsis guianensis
Chazaliella abrupta 1
Chazaliella abrupta 2
Carapichea affinis
Carapichea ligularis
Carapichea ipecacuanha
Geophila repens
Geophila obvallata
Chassalia sp1VIET
Chassalia sp2VIET
Chassalia spSUMA
Chassalia catatii
Chassalia sp2MADAG
Chassalia sp1MADAG
Hymenocoleus hirsutus
Notopleura tapajozensis
Rudgea stipulacea
Palicourea nitidella
Palicourea crocea
Psychotria stachyoides
Psychotria poeppigiana
Amaracarpus nematopodus 1
Amaracarpus nematopodus 2
Hydnophytum cf longistylum
Psychotria cf impercepta
Psychotria schlechteriana
Psychotria grandis
Psychotria asiatica
Psychotria camptopus
Psychotria parkeri
*
clade G
*
*
Neotropical
Margaritopsis
Chazaliella
Carapichea
*
Chassalia /
Geophila /
Hymenocoleus
*
Notopleura /
Rudgea
Palicourea s.l.
*
Fig. 1. Bayesian half-compatible consensus tree of tribes Palicoureeae and Psychotrieae. Asterisks indicate generic type species. Taxa from the
AMP region, Neotropics and Africa are shaded in dark, middle and light grey, respectively. Bayesian posterior probability (PP) and bootstrap
(BS) support values for major clades are indicated and separated by a slash.
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
TAXON 61 (6) • December 2012: 1251–1268
and trnH-psbA regions provide most polymorphic characters,
respectively, with 35% and 26% Parsimony Informative Characters (PICs), whereas ndhF and trnT-F regions yield 12% and
13% of PICs, respectively (Table 2).
Phylogenetic analyses. — The MP and Bayesian MCMC
single-gene phylogenetic topologies yield no incongruence
(with a PP > 0.95 or BS > 95%) and are less resolved than
the combined phylogenetic analyses. Both MP and Bayesian MCMC combined analyses provide no incongruence between topologies (data not shown). Since the Bayesian MCMC
analysis allows us to take into account different models of
evolution across the plastid and nuclear regions, and provides
branch lengths, only this analysis method is discussed hereafter (Fig. 1).
The analyses strongly support the monophyly of a group
composed of all AMP Psychotria and Margaritopsis species
(PP = 1; Figs. 1 & 2). Within Palicoureeae seven clades are
highly supported (clades A to G), all with PP = 1 (Fig. 1). Both
species of Palicourea are embedded in a well-supported Neotropical clade with Psychotria stachyoides and P. poeppigiana
Müll. Arg. (clade A). This clade is placed as sister to all other
Palicoureeae with high support (PP = 1). Clade B is composed
of single species representatives of the Neotropical genera Rudgea and Notopleura. Clade C includes species samples of Chassalia, Geophila, and Hymenocoleus from Africa and the AMP
region (the first two genera are retrieved as monophyletic, PP =
1). Clade D includes Neotropical taxa of Carapichea; clade E is
composed of two African Chazaliella accessions. Four of the
five sampled Neotropical Margaritopsis species form clade F
that is sister to the type species of Margaritopsis (M. nudiflora)
and clade G. Finally, clade G comprises all AMP Psychotria
taxa together with Readea membranacea and Hodgkinsonia
carnea subclade
samoana subclade
leptothyrsa subclade
G
collina subclade
Vietnamese subclade
frutescens. The placement of M. nudiflora as sister to clade G
still remains uncertain (PP = 0.61), but the clade which includes
this species and clades F and G is well supported (PP = 1).
Further details on the phylogenetic placement of M. nudiflora
are given in the following section.
The subdivision of clade G into five well-supported monophyletic subclades is well supported (PP ≥ 0.95; Fig. 2). The
‘Vietnamese’ subclade is composed of Vietnamese Psychotria species (P. sp. 1 [VIET] and P. sp. 2 [VIET], PP = 0.97).
The ‘collina’ subclade (PP = 1) includes all species from New
Caledonia (P. collina, P. lyciiflora (Baill.) Schltr., P. oleoides,
P. sp. ‘NC’) and one taxon from Vanuatu (P. sp. ‘V’, PP = 1).
The ‘leptothyrsa’ subclade (PP = 1) comprises both species
from tropical Australia (Hodgkinsonia frutescens is placed
as sister to P. sp. [AUST]; PP = 1), one species from Vanuatu
(P. aneityensis) and the widespread P. leptothyrsa Miq. (sampled from New Guinea). The ‘Samoana’ subclade (PP = 1) is
composed of one Samoan species (P. samoana K. Schum.),
one species occurring in Fiji, Samoa and Tonga (P. forsteriana
A. Gray), and one species from Vanuatu (P. cf. trichostoma
Merr. & L.M. Perry). Readea membranacea Gillespie endemic
to Fiji is placed as sister to the ‘carnea’ subclade (PP = 1).
This latter subclade (PP = 1) includes species from Fiji and
Tonga (P. archboldiana, P. carnea (G. Forst.) A.C. Sm., P. sp.
‘FIJ’), and the single species from the Society Archipelago,
P. lepiniana (Baill. ex Drake) Drake (PP = 1). The relationships between these five subclades are well supported (PP ≥
0.95) with the exception of the relationship between a clade
combining R. membranacea and the Samoana and carnea and
leptothyrsa subclades (Fig. 2). Psychotria carnea is retrieved
as polyphyletic (PP = 1), even if its two accessions are found
in the same clade (carnea subclade).
Psychotria lepiniana
Psychotria spFIJ
Psychotria carnea 1
Psychotria archboldiana
Psychotria carnea 2
Readea membranacea
Psychotria forsteriana
Psychotria samoana
Psychotria cf trichostoma
Hodgkinsonia frutescens
Psychotria spAUST
Psychotria leptothyrsa
Psychotria aneityensis
Psychotria lyciiflora
Psychotria spNC
Psychotria spV
Psychotria oleoides
Psychotria collina
Psychotria sp1VIET
Psychotria sp2VIET
Margaritopsis nudiflora
SOCI
FIJI
FIJI / TONG
FIJI
FIJI / TONG
FIJI
FIJI / SAMO / TONG
SAMO
VANU
AUST
AUST
BISM / INDO / MICR / PAPU / PHIL / SOLO / WAL
VANU
NCAL
NCAL
VANU
NCAL
NCAL
VIET
VIET
Fig. 2. Bayesian half-compatible consensus tree showing relationships within clade G (Fig. 1). Abbreviations used for geographical distribution:
AUST: tropical Australia, BISM: Bismarck Archipelago, FIJI: Fiji, INDO: Indonesia, MICR: Micronesia, NCAL: New Caledonia, PAPU: New
Guinea, PHIL: the Philippines, SAMO: Samoa, SOCI, Society archipelago, SOLO: Solomons, TONG: Tonga, VANU: Vanuatu, VIET: Vietnam,
WAL: Wallis. Areas where species were sampled for the molecular study are indicated in bold. Bayesian posterior probability (PP) support values
for major clades are indicated for each node.
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
Hypothesis testing and ambiguous phylogenetic placement of Margaritopsis nudiflora. — The placement of Margaritopsis nudiflora in all Bayesian MCMC single-gene analyses
is unresolved or unsupported (PP < 0.95, data not shown). Relationships between M. nudiflora, clade G and the four other
Neotropical Margaritopsis remain uncertain based on the
analysis of each trnH-psbA and rps16 regions. Data provided
by the trnT-F region place M. nudiflora sister to clade G but
with low support (PP = 0.63). The monophyly of the five Neotropical Margaritopsis species is weakly supported (PP = 0.54)
by the ITS region analysis. Phylogenetic topologies resulting
from the unconstrained combined Bayesian MCMC analysis
of the dataset without ndhF yield exactly the same topology as
that with ndhF (data not shown). The phylogenetic placement
of M. nudiflora is the same, as sister to clade G, with weak
support (PP = 0.70). For both datasets (with ndhF or without)
the SH tests, which compared the topology resulting from the
unconstrained analysis with one that enforced the monophyly
of the five Margaritopsis species, are not significant (P > 0.1,
see Table 3). The SH tests that compared the unconstrained
topology with one that constrained the monophyly of the group
which included AMP Psychotria plus their relatives plus four
Margaritopsis species, are also not significant (P > 0.1, see
Table 3).
Morphological studies. — The results of the morphological investigation based on herbarium specimens and taxonomic
treatments are found in Tables 4 and 5. Significant morphological traits for species within clade G (i.e., 17 AMP Psychotria
taxa plus Hodgkinsonia frutescens and Readea membranacea)
were selected at the species level after examination of herbarium specimens (Table 4). Morphological characters generally provide support for the recognition of each of the five
subclades of clade G as well as the Readea lineage (characters
in bold in Table 4). Morphological comparison at the generic
level allows recognition of unique characters (characters in
bold in Table 5), or at least a combination of characters, enabling delimitation of Palicoureeae genera. All of the 19 species
belonging to clade G share the following combination of characters (Table 5): (1) shrubs or small trees; (2) vegetative parts
TAXON 61 (6) • December 2012: 1251–1268
pale yellowish green when dried; (3) branchlets smooth (noncorky), young shoots and young internodes often flattened; (4)
stipules semi-deciduous, entire, usually united at the base or
calyptrate for a large part of their length, becoming indurated
(sometimes inconspicuously for calyptrate species) and their
upper parts falling off through fragmentation (Fig. 3D & G);
(5) inflorescence axes always green (Fig. 3A, B, E, F, H, I); (6)
calyx and corolla actinomorphic, corolla with a limited colour
range (white, creamy to yellow-green) and corolla tube straight
at base (i.e., never swollen; Fig. 3A, B, E, F, H); (7) fruits orange
or red (Fig. 3J–L); (8) pyrenes hemispherical in cross-section,
without a ventral depression or intrusion of the seed-coat; (9)
pyrenes with two basal ventral marginal preformed germination slits (PGSs); (10) endosperm non-ruminate, often with a
small inner central ventral invagination; and (11) seed-coat
lacking a red ethanol-soluble pigment.
In addition to the 19 species used in the phylogenetic sampling, 28 other Psychotria species from the AMP region were
detected by the study of herbarium specimens and on the basis
of taxonomic treatments (Table 6; Appendix 2 in the Electronic
Suppl.). They are assigned to clade G based on the same set of
morphological characters. Indeed, the diagnostic features of the
19 species of clade G (see above) are shared by these 28 species. This part of the study allows us to estimate the species
richness of clade G to a minimum of 47 species (Table 6; Appendix 2), with the inclusion of the 28 species examined using
morphology alone. We are able to show that each archipelago
and landmass of South-East Asia, Malesia, tropical Australia
and tropical South Pacific possesses at least one species, except
Hawaii (Table 6; Fig. 4). According to our findings archipelagos
and landmasses would at least possess: eight species each for
Fiji and Samoa, seven species for the Bismarck Archipelago,
five species for New Guinea, four species each for New Caledonia and Solomons, three species for Vanuatu, two species
each for the Philippines, Tonga and tropical Australia, and one
species for eastern Polynesia. Probably more than eight species
occur in South-East Asia and Indonesia, and more than one
species in Micronesia (Table 6; Fig. 4), although these estimates
need to be reassessed pending future taxonomic treatments.
Table 3. Results of the Shimodaira-Hasegawa tests for the half-compatible Bayesian consensus trees resulting from analyses of
the dataset with or without ndhF. The best hypothesis is given in bold. ‘5 MAR’ refers to the constraint placed to enforce the
monophyly of the five Neotropical species of Margaritopsis (including M. nudiflora (Griseb.) K. Schum.). ‘4 MAR + clade G’
refers to the constraint that enforced the monophyly of a group which includes AMP Psychotria species plus their relatives
(= clade G) and four Neotropical species of Margaritopsis (M. astrellantha (Wernham) L. Andersson, M. boliviana (Standl.)
C.M. Taylor, M. guianensis (Bremek.) C.M. Taylor and M. kappleri (Miq.) C.M. Taylor).
Hypothesis 1 (H1)
Hypothesis 2 (H2)
−ln L H1
−ln L H2
Diff. −ln L
P value
Unconstrained
Constrained 5 MAR
25253.75206
25254.12317
0.37111
0.3265
Unconstrained
Constrained 4 MAR + clade G
25253.75206
25254.13672
0.38466
0.3176
Constrained 5 MAR
Constrained 4 MAR + clade G
25254.12317
25254.13672
0.01355
0.4181
Unconstrained
Constrained 5 MAR
18308.35663
18308.74398
0.38735
0.3216
Unconstrained
Constrained 4 MAR + clade G
18308.35663
18308.57976
0.22313
0.4912
Constrained 5 MAR
Constrained 4 MAR + clade G
18308.74398
18308.57976
0.16423
0.4804
With ndhF
Without ndhF
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
TAXON 61 (6) • December 2012: 1251–1268
Table 4. Morphological characters of the five subclades of clade G and the Readea lineage that are common (roman letters) and unique (bold).
Subclade / lineage
Vietnamese
collina
leptothyrsa
samoana
Readea
carnea
Stipules
Truncate /
triangular
Truncate /
triangular
Truncate /
triangular
Papyraceous
and calyptrate
Truncate /
triangular
Calyptrate /
lanceolate
Calyx type
Reduced, with
or without small
teeth
Reduced, with
or without small
teeth
Reduced, with
or without small
teeth
Reduced, with
or without small
teeth
Funnel-shaped,
with long
reflexed lobes
Inflated,
enlarged, with
erect lobes
Calyx tube
Not enclosing the
corolla tube
Not enclosing the
corolla tube
Not enclosing the
corolla tube
Not enclosing the
corolla tube
Enclosing the
corolla tube
Not enclosing the
corolla tube
Calyx texture
Chartaceous
Chartaceous
Chartaceous
Chartaceous
Fleshy
Chartaceous
Corolla aestivation
Rounded
Rounded
Rounded
Rounded
Rounded
Reduplicate
(winged)
Corolla type
Reduced, with
short lobes
Reduced, with
short lobes
Reduced to urseolate (bell-like),
with short lobes
Reduced, with
short lobes
Funnel-shaped,
with long
reflexed lobes
Funnel-shaped,
with long lobes
Corolla texture
Chartaceous
Chartaceous
Chartaceous
Chartaceous
Fleshy
Chartaceous
Inflorescence
Compound cyme
Compound cyme
Cyme, verticillate at each node
Panicule / thyrse
Compound cyme
Compound
cyme / fascicule
Fruit shape
Rounded to
ellipsoid
Rounded to
ellipsoid
Rounded to
ellipsoid
Rounded to
ellipsoid
Oblongoid
Ellipsoid
Pyrenes ventral face
Concave
Flat
Flat
Flat
Flat
Flat
DISCUSSION
The results presented here corroborate previous conclusions by Nepokroeff & al. (1999), Andersson & Rova (1999)
and Andersson (2001, 2002b) and support the monophyly of a
group that includes the AMP Psychotria species plus Hodgkinsonia frutescens and Readea membranacea (i.e., clade G), and
that falls in a clade together with Neotropical Margaritopsis
(Fig. 1). The results support the same seven major monophyletic
lineages of Palicoureeae as retrieved by Andersson (2001), and
in addition increases our understanding of the phylogenetic
relationships between and within these lineages on the basis
of additional DNA regions and a broader sampling from the
AMP region (Table 1; Fig. 1). More significantly, the close
relationship of species of clade G with Neotropical Margaritopsis necessitates a re-investigation of the circumscription of
Margaritopsis, based on morphological evidence. However,
the unsupported position of the type species of Margaritopsis (M. nudiflora) does not allow us to draw any systematic
conclusions to be made on the phylogenetic status of Neotropical species of Margaritopsis. Single-gene Bayesian MCMC
analyses and combined analyses, with or without ndhF, did not
resolve the placement of M. nudiflora. Results of the SH tests
(Table 3) did not allow us to either exclude the monophyly of
Neotropical Margaritopsis species or support their paraphyly
(M. nudiflora placed as sister to species of clade G).
Diversity and geographic range of clade G. — The species
richness of clade G (19 species) as ascertained by the molecular investigation reported here is higher than the nine species
previously suggested by Andersson (2001). Furthermore, our
investigation of herbarium specimens allows us to increase
the potential species richness of the lineage, here represented
as clade G, to 47 species. These species are widely distributed
throughout the AMP region, from South-East Asia to eastern Polynesia (Fig. 4), as observed in other Rubiaceae genera (e.g., Ixora L., Mouly & al., 2009; Gynochthodes Blume,
Razafimandimbison & Bremer, 2011). This lineage has colonized all archipelagos and landmasses of the AMP region
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
without exception (Fig. 4), and each of them possesses at least
one of the species we attribute to the lineage. Clade G does not
seem to have radiated significantly, because species richness
does not exceed eight species in each archipelago and tropical
landmass of the AMP region (up to seven species for the Bismarck Archipelago, five for New Guinea, and eight each for
Fiji and for Samoa; Fig. 4; Whistler, 1986; Smith & Darwin,
1988; Sohmer, 1988). Most of the species are endemic to a
specific archipelago (Fig. 4), but some are widely distributed
throughout South-East Asia and Malesia (e.g., P. leptothyrsa,
P. membranifolia Bartl. ex DC., P. montana; Sohmer, 1988;
Sohmer & Davis, 2007). Interestingly, species of clade G are
able to occupy a large range of ecological niches (in tropical to
subtropical ecosystems). They grow on a wide range of geological substrates (limestone, clay, metamorphic rocks, ultramafic
rocks, etc.), in most natural vegetation types and in degraded
and secondary vegetation, and from sea level to high elevations
(pers. obs. of the first author; Whistler, 1986; Sohmer, 1988;
TAXON 61 (6) • December 2012: 1251–1268
Smith & Darwin, 1988; Sohmer & Davis, 2007). The estimation
of species richness provided in this study requires confirmation, as some species remain poorly known (e.g., in South-East
Asia and Indonesia) and cases of morphological convergence
have been widely recorded in Rubiaceae (e.g., in Ixora, Mouly
& al., 2009; and Coffea, Davis & al., 2011). Indeed, a taxonomic
revision for the species of clade G, throughout the whole AMP
region, is urgently required.
Diagnostic features of the internal lineages of clade G. —
Our morphological study at the species level reveals that each
of the five subclades of clade G and the Readea lineage can be
characterized by a diagnostic combination of morphological
features, and generally possesses unique characters that further
support their monophyly (characters in bold in Table 4; see below). The exception is the collina subclade which does not show
any distinctive features (Fig. 3F). In contrast, the Vietnamese
subclade is the only group where the ventral face of the pyrenes
(Table 4) is conspicuously concave. The inflorescences of the
Table 5. Morphological characters of the genera of Palicoureeae. The characters that are similar among all genera are shaded in grey, commonly …
Generic group
Palicourea s.l.
Notopleura
Habit
Shrubs
Shrubs / epiphytes / Shrubs
succulents/ climbers
Shrubs
Vegetative colour when dried
Greyish brown
Greyish silver
Greenish
Greyish brown
Stem indurate, corky
No
No
No
No
Stem flattened
No
No
No
No
Stipule type
Entire or bilobed,
truncate, sheathing
Entire, bilobed or
fimbriate, sheathing, sometimes with
an interpetiolar
outer appendage
Entire or fimbriate, Entire or fimbriate,
generally with
sheathing
an interpetiolar
appendage
Stipules not deciduous or semi-deciduous
Yes
Yes
Yes
Yes
Stipules indurate
No
No
No
No
Stipules becoming fragmented
No
No
No
No
Inflorescence axes coloured
Yes / no
No
No
No
Rudgea
Carapichea
Corolla zygomorphic
Yes / no
No
No
No
Corolla colour
White / brightly
coloured
White, creamy to
yellow-green
White, creamy to
yellow-green
White, creamy to
yellow-green
Corolla base swollen and gibbous
Yes / no
No
No
No
Fruit colour
Variable
Orange / red
Variable
Variable
Pyrene outline shape in transverse section
Hemispherical /
spherical
Hemispherical /
triangular
Hemispherical /
spherical
Hemispherical
Pyrene-coat with ventral depression
Yes / no
No
Yes / no
Yes
Preformed germination slits (PGSs) present
Yes / no
Yes
Yes / no
Yes / no
PGSs presence/absence; location
Lacking / 1–2 on
dorsal ribs (sometimes marginal)
2, basal (marginal /
ventral) + 1 medioventral
Lacking / 2 basal
marginal (+ some
dorsal slits)
Lacking / 1 dorsal
Seed-coat with T- or Y- shaped ventral intrusion
No
No
Yes
No
Seed-coat lacking a red ethanol-soluble pigment (SCP)
Yes
Yes
Yes
Yes
Endosperm non-ruminate
Yes
Yes
Yes
Yes
Endosperm with small inner central ventral invagination
No
No
No
No
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TAXON 61 (6) • December 2012: 1251–1268
leptothyrsa subclade have a verticillate arrangement at each
node (Fig. 3H). The morphology of Hodgkinsonia frutescens
mostly matches that of Psychotria leptothyrsa and P. aneityensis. Unfortunately, the living specimen representing P. sp.
[AUST] was unavailable and no information is available regarding its morphology. The Samoana subclade representatives
have stipules that are papyraceous and calyptrate (Table 4).
Readea membranacea Gillespie has the following autapomorphies that might explain its previous recognition as a distinct
genus (Gillespie, 1930; Smith & Darwin, 1988): corolla and
calyx fleshy (Fig. 3C); corolla and calyx tubes funnel-shaped
and their lobes long and reflexed (Gillespie, 1930: fig. 49);
calyx tube enclosing the corolla tube completely in bud and
almost completely at anthesis (Fig. 3A; Gillespie, 1930: fig.
49); fruits long and oblongoid (Table 4; Fig. 3L). The carnea
subclade is the most spectacular subclade because it contains
species that possess: stipules calyptrate or lanceolate (Fig. 3G);
aestivation reduplicate (Fig. 3B & E); calyx well developed,
inflated (bowl-shaped) or enlarged (plate-shaped), with erect
lobes; and corolla funnel-shaped with long lobes (Table 4;
Fig. 3B & E). The species are often morphologically similar
and have the same general appearance, and this may explain
difficulties encountered by taxonomists to circumscribe species, as exemplified by the polyphyletic Psychotria carnea
(Fig. 2). The morphological study at the generic level reveals
that species of clade G share a common set of characters (see
the aforementioned list in the Results section and in Table 5),
and these support its monophyly.
Generic relationships between Neotropical Margaritopsis and clade G. — As defined by Andersson (2001, 2002a) and
Robbrecht & Manen (2006), the species of clade G show the
typical combination of characters found in Palicoureeae (characters shaded grey in Table 5). These taxa are closely related
to Neotropical Margaritopsis, as shown by our phylogenetic
analyses, which reveals the inclusion of clade G in Margaritopsis (Fig. 1; Table 5). The generic description of Margaritopsis
… observed within the tribe in roman, and unique or discriminative for one or more genera in bold.
Chassalia
Geophila
Hymenocoleus
Chazaliella
Neotropical
Margaritopsis
Clade G species
Shrubs / trees /
epiphytes / climbers
Creeping herbaceous plants with
stolons
Creeping herbaceous plants with
stolons / herbaceous
Shrubs
Shrubs
Shrubs / small trees
Greenish brown
Greenish brown
Greyish black
Greyish
Pale yellow greenish
Pale yellow greenish
No
No
No
Yes
Yes / no
No
No
No
No
Yes / no
Yes / no
Yes / no
Entire or bilobed, free
or sheathing
Bilobed or entire
Bilobed, with a
membranous
sheath inside
Entire, bilobed or
lacerate
Entire, bilobed or
dentate, sometimes
sheathing
Entire, sheathing or
calyptrate
Yes
Yes
Yes
Yes
Yes
Yes
Yes (corky)
No
No
Yes (corky)
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
No
No
No
No
No
Yes / no
No
No
No
No
No
White / brightly
coloured
White, creamy to
yellow-green
White, creamy to
yellow-green
White, creamy to
yellow-green
White, creamy to
yellow-green
White, creamy to
yellow-green
No
No
No
No
No
No
Red / blue / black
Red / blue / black
Orange / red
Orange / red
Orange / red
Orange / red
Hemispherical /
spherical
Hemispherical
Hemispherical
Hemispherical
Hemispherical
Hemispherical
Yes
No
Yes
No
No
No
Yes
No
Yes
Yes
Yes
Yes
1 basal median dorsal
+ 2 marginal
Lacking
2 basal dorsal + a
funicular pore
2 basal marginal
ventral
2 basal marginal
ventral (+ some
dorsal slits)
2 basal marginal
ventral
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes / no
Yes / no
Yes
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
Table 6. Species that belong to or are attributed to clade G based on molecular evidence and morphological investigation. Type specimens that
were consulted in herbaria are indicated in bold, and those observed as images in roman.
Region
Australia
Fiji
Vanuatu
Fiji
The Philippines
Fiji / Tonga
Samoa
New Caledonia
Bismarck Archipelago
Fiji
Borneo / Java / Sumatera
Java to Lesser Sunda Islands
Fiji / Samoa / Tonga
Bismarck Archipelago, New Guinea
Samoa
New Guinea
Java
Fiji
Society Archipelago
Malesia to West Pacific
New Caledonia
New Guinea, the Philippines
Solomons
Bismarck Archipelago
SE China to tropical Asia / (New Guinea?)
Bismarck Archipelago
New Caledonia
Fiji / Samoa
New Guinea
Indo-China to West Malesia
Bismarck Archipelago
Samoa
Samoa
Bismarck Archipelago, Solomons
Samoa
South China to Vietnam
Solomons / (Vanuatu?)
Samoa
Indo-China
New Caledonia
Vanuatu
Fiji
Name
Hodgkinsonia frutescens C.T. White
Psychotria amoena A.C. Sm.
Psychotria aneityensis Guillaumin
Psychotria archboldiana Fosberg
Psychotria balabacensis Merr.
Psychotria carnea (G. Forst.) A.C. Sm.
Psychotria chlorocalyx K. Schum.
Psychotria collina Labill.
Psychotria damasiana Sohmer
Psychotria evansensis A.C. Sm.
Psychotria expansa Blume
Psychotria extensa Miq.
Psychotria forsteriana A. Gray
Psychotria galorei Sohmer
Psychotria geminodens K. Schum.
Psychotria goodenoughiensis Sohmer
Psychotria horsfieldiana Miq.
Psychotria incompta A.C. Sm.
Psychotria lepiniana (Baill. ex Drake) Drake
Psychotria leptothyrsa Miq.
Psychotria lyciiflora (Baill.) Schltr.
Psychotria membranifolia Bartl. ex DC.
Psychotria merrilliana Sohmer
Psychotria monopedicellata Sohmer
Psychotria montana Blume
Psychotria novohiberiensis Sohmer
Psychotria oleoides (Baill.) Schltr.
Psychotria oncocarpa K. Schum.
Psychotria purariensis Sohmer
Psychotria rostrata Blume
Psychotria saidoriensis Sohmer
Psychotria samoana K. Schum.
Psychotria savaiiensis Rech.
Psychotria schmielei Warb.
Psychotria sclerocarpa Whistler
Psychotria straminea Hutch. in C.S. Sargent
Psychotria trichostoma Merr. & L.M. Perry
Psychotria vaupelii Whistler
Psychotria vietnamensis Ruhsam
Psychotria sp. NC
Psychotria sp. V
Readea membranacea Gillespie
Type
seen
X
X
X
X
X
X
Taxonomic treatment
Smith & Darwin, 1988
Smith & Darwin, 1988
Sohmer & Davis, 2007
Whistler, 1986, Smith & Darwin, 1988
Whistler, 1986
X
Sohmer, 1988
Smith & Darwin, 1988
X
X
X
X
Whistler, 1986, Smith & Darwin, 1988
Sohmer, 1988
Whistler, 1986
Sohmer, 1988
X
X
X
X
Smith & Darwin, 1988
X
Sohmer, 1988
Sohmer, 1988, Sohmer & Davis, 2007
Sohmer, 1988
Sohmer, 1988
X
X
X
X
Sohmer, 1988
X
X
X
X
X
Sohmer, 1988
Whistler, 1986
Whistler, 1986
Sohmer, 1988
Whistler, 1986
X
Sohmer, 1988
Whistler, 1986
Whistler, 1986, Smith, 1988
Sohmer, 1988
X
X
Smith & Darwin, 1988
◄ Fig. 3. Examples of species of the five subclades of clade G and the Readea lineage. A, Readea membranacea Gillespie (Barrabé & al. 1128,
NOU); B, Psychotria sp., carnea subclade (Barrabé 1060, NOU); C, Readea membranacea (Barrabé & al. 1128, NOU); D, Readea membranacea
(Barrabé & al. 1128bis, NOU); E, Psychotria archboldiana Fosberg (Barrabé & al. 1123a, NOU); F, Psychotria lyciiflora (Baill.) Schltr. (Barrabé
& Bariole 747, NOU); G, Psychotria sp., carnea subclade (Barrabé & al. 1126, NOU); H, Hodgkinsonia frutescens C.T. White (Baba & al. 786,
NOU); I, Psychotria forsteriana A. Gray (Barrabé & al. 1065, NOU); J, Psychotria sp., carnea subclade (Barrabé & al. 1123bis, NOU); K, Psychotria forsteriana (Buerki & al. 162, NOU); L, Readea membranacea (Barrabé & al. 1128bis). — Credits: A–G, I–L: L. Barrabé; H: F. Rigault.
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
proposed by Andersson (2001) and Taylor (2005) mostly includes the morphology found in species of clade G (cf. generic
characters shown in Table 5), even though some taxa have features that are unique or rare in Palicoureeae. For instance, the
carnea subclade has winged buds (Fig. 3B & E), a character
shared with several species of Chassalia (Piesschaert, 2001;
Piesschaert & al., 2001). We could not find any morphological
characters that would allow the species of clade G to be separated from Neotropical Margaritopsis (Table 5). Consequently,
there is no reason to consider that the species of clade G constitute a genus distinct from Margaritopsis. On this basis we
TAXON 61 (6) • December 2012: 1251–1268
suggest that the circumscription and characterization of Margaritopsis should be expanded to incorporate the 19 species of
clade G as identified here, and include the 28 additional species
we have attributed to the group (Table 6). The adjustments to
the characterization of Margaritopsis would need to include
the characters identified as unique for each internal group of
clade G (see above; Table 4). We refrain from proposing a new
formal description of the genus in the present study, in lieu
of further taxonomic work, and especially for species from
South-East Asia and Indonesia. If we accept that Margaritopsis is paraphyletic without the inclusion of clade G species, at
A
Neotropical
Margaritopsis
clade G
20 ?
27
Chazaliella
47
B
8?/?/2
South-East Asia
Philippines
1?/?/0
2/1/0
7/4/0
4/1/0
Hawaii
3/2/3
Indonesia
Micronesia
Papua New
Guinea
8/6/1
Bismarck
Archipelago
1/1/1
Solomons
Samoa
Vanuatu
5/2/1
Tropical Australia
New Caledonia
Fiji
Tonga
Society archipelago
2/0/0
8/5/5
2/2/2
4/4/4
Fig. 4. A, Geographical distribution and species richness of clade G, Neotropical Margaritopsis and African Chazaliella. B, Species distribution
of clade G in South-East Asia, Malesia, tropical Australia and tropical South Pacific. Areas where the species diversity is known are shaded
in dark grey, where it is unknown in light grey. For each area the numbers indicate total number of species present / endemic species / species
sampled in this study, respectively. Information for total number of species and number of endemics was generated from herbarium studies and
published works (Whistler, 1986; Smith & Darwin, 1988; Sohmer, 1988; Fosberg & al., 1993; Sohmer & Davis, 2007; Chen & Taylor, 2011).
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
least 46 new combinations or new names are required, as most
of these species are presently placed in Psychotria (Table 6).
Readea (Gillespie, 1930) and its type species have already been
placed in Margaritopsis (Andersson, 2001, 2002c). Hodgkinsonia frutescens also needs to be formally placed in Margaritopsis. Phylogenetic analyses have placed the type species of
Hodgkinsonia (H. ovatiflora F. Muell.) in tribe Guettardeae
(Cinchonoideae subfamily; Manns & Bremer, 2010), and consequently this genus will not be synonymized with Margaritopsis. Taxonomic and nomenclatural implications related to
these new findings will be formalized in a future publication
(Barrabé & al., in prep.) once the nomenclatural details have
been carefully checked.
Major biogeographical patterns within clade G. — The
phylogenetic topology suggests that Palicoureeae have colonized the AMP region at least three times in the following
lineages: Chassalia, Geophila, and clade G (Fig. 1). Concerning
the origin and diversification of clade G, three geographical
levels can be examined.
Firstly, at a large geographical scale, the close relationships between a clade of the AMP region and Neotropical species (composed of Margaritopsis taxa; Figs. 1 & 4) requires
complex assumptions. Indeed, considering the shape of our
phylogenetic tree, three hypotheses could be evaluated for the
pantropical distribution of the Margaritopsis/clade G/Chazaliella clade, involving three different main dispersal routes: (1)
East–West long-distance dispersal from the Neotropics to the
AMP region; (2) continuous West–East dispersal from the Neotropics to the AMP region through Africa; (3) dispersal from
Africa to both the Neotropics and the AMP region. Additional
sampling of African Chazaliella and Neotropical Margaritopsis is necessary to test these three hypotheses by placing these
species in future biogeographical analyses, as only 17 species
of AMP Psychotria, Readea membranacea, Hodgkinsonia
frutescens, one of 20 species of African Chazaliella, and five
of 27 Neotropical Margaritopsis have so far been included in
phylogenetic studies. Although uncommon in Rubiaceae, a
similar pattern was observed in the Chiococeeae tribe, which
includes two tropical South Pacific lineages, one consisting of
Morierina Vieill. and Thiollierea Montrouz. (as Bikkia Reinw.
p.p.) and the second associating Badusa A. Gray and Bikkia
p.p., nested within a diversified Neotropical clade (Motley
& al., 2005; Manns & Bremer, 2010). In Augusta Pohl two of
the four species occur in the Neotropics, whereas the two others are found in New Caledonia and in Fiji (Kirkbride, 1997).
Secondly, a biogeographical history can be postulated
from the internal phylogenetic topology of clade G (Fig. 2).
The Vietnamese subclade (from South-East Asia) is placed
as sister to all remaining species of clade G. The grouping of
the carnea and Samoana subclades and Readea membranacea
allows us to delimit a Polynesian and Fijian clade. The collina
subclade (from New Caledonia and Vanuatu) and leptothyrsa
subclade (from Malesia, Melanesia and Australia) are inserted
between these two latter monophyletic groups. This topology
may imply the existence of a dispersal route between mainland
South-East Asia (western part of the AMP region) and tropical
South Pacific islands (eastern part) via Malesia and Melanesia
(middle part), as commonly observed in other plant groups
(Fig. 4; Keppel & al., 2009).
Thirdly, and at a yet finer scale, within each of the subclades of clade G, subsequent dispersals to neighbouring islands seem to have occurred and contributed to the current
geographic distribution shown in Fig. 4. The nested placement
of the Tahitian Psychotria lepiniana within a Fijian/Tongan
grade, composed by the carnea subclade and the Readea lineage, could be the result of dispersal from the western to the central Pacific Ocean (Fig. 2). As species from tropical Australia
are nested within a Malesian/Melanesian grade (with P. aneityensis and P. leptothyrsa), only one colonization event seems
to have occurred in tropical Australia from the Malesian or
the Melanesian area (Fig. 2). The placement of P. sp. ‘V’ from
Vanuatu nested among New Caledonian species in the ‘collina
subclade’ (Fig. 2) is likely to be the result of dispersal from
New Caledonia, a feature also observed in other groups (e.g.,
Geissois Labill. or Schefflera J.R. Forst. & G. Forst.; Pillon,
2011; Plunkett & Lowry, 2012).
PERSPECTIVES
The phylogenetic analyses and morphological framework
presented here provides an improved knowledge for Palicoureeae representatives of the Old World, specifically from the
AMP region and comprising the clade we have labelled as
clade G. It is clear that species of this clade have strong affinities with the Neotropical Margaritopsis and should be merged
with it when adequate nomenclatural work has been done. Different kinds of biogeographical processes seem to have been
involved in shaping the current species richness in the AMP
region for the group, and these deserve to be fully investigated
by expanding taxon sampling, undertaking appropriate analyses, and by testing different biogeographical models (Buerki
& al., 2011; Ronquist & Sanmartin, 2011). Otherwise the combination of morphological homogeneity, the large geographical
range in the AMP region, the high ecological plasticity, and low
local species richness, indicate a special adaptive strategy for
the species of clade G, in which dispersal and establishment is
more important than the ability to radiate significantly in each
archipelago and landmass of the area.
ACKNOWLEDGMENTS
We are grateful to the members of the Laboratoire de Botanique
et d’Ecologie Végétale Appliquées and the herbarium at IRD Nouméa
(NOU), especially Frédéric Rigault, Gilles Dagostini, Williams
Nigote, Tanguy Jaffré, Jacqueline Fambart-Tinel, Michèle Magat and
Céline Grignon. We thank Yumiko Baba and Darren Crayn (Tropical
Australian Herbarium of Cairns, James Cook University), Marika
V. Tuiwawa, Alifereti Naikatini and Fiona Tuiwawa (South Pacific
Regional Herbarium, University of the South Pacific of Suva), Isaac
A. Rounds (Conservation International, Fiji), Christopher Davidson
and Sharon Christoph, Martin Callmander and Pete Lowry (Missouri Botanical Garden, Saint Louis) for their unconditional assistance
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Barrabé & al. • Phylogenetic inference of Asian, Australasian and Paciic Margaritopsis
with Australian and Fijian fieldworks. We thank Clarisse Majorel
and Laure Hannibal (Laboratoire des Symbioses Tropicales Méditerranéennes, IRD Nouméa) for their help in the laboratory; Edith
Kapinos and Laszlo Csiba (Royal Botanic Gardens [RBG], Kew),
Sylvain Razafimandimbison, and Birgitta Bremer (Bergius Foundation, Stockholm) for assisting us with access to DNA samples, and
Yohan Pillon (University of Hawaii at Hilo) for his comments and
advice on this study and an earlier version of the manuscript. We
are also grateful to the currators and the staff of the herbaria of the
Museum National d’Histoire Naturelle of Paris (Jean-Noël Labat †),
and RBG Kew (Nina M. Davies and Sally E. Dawson), for their help
accessing collections. For permission to conduct fieldwork, collect and
export specimens we thank the Direction de l’Environnement of the
Province Sud and the Direction du Développement économique et de
l’Environnement of the Province Nord in New Caledonia, the Department of Environment and Resource Management of the Queensland
Government in Australia, and the Department of Environment of the
Government of Fiji. Material from Santo (Vanuatu) was collected during the Santo2006 expedition. Part of this work was funded through
the BIONEOCAL and ULTRABIO projects supported by a grant from
the French Agence Nationale de la Recherche. We also wish to thank
two anonymous reviewers for providing useful comments that have
improved the manuscript.
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Appendix 1. Sequences produced in this study and those previously published. Taxa, voucher information, and GenBank accession numbers. GenBank
accession numbers are in the following order: ITS, ndhF, rps16, trnH-psbA, trnT-F. Newly generated sequences are indicated with an asterisk. The superscript numbers refer to the publication where the sequences were first published: 1 Nepokroeff & al. (1999), 2Andersson (2001), 3Andersson (2002b), 4 Rydin
& al. (2008), 5 Razafimandimbison & al. (2008), 6Andersson & Rova (1999), 7 Andersson (GenBank, unpub.), 8Andersson & Taylor (GenBank, unpub.).
Amaracarpus nematopodus (F. Muell.) P.I. Forst. 1, Australia, Queensland, Atherton Tableland, Barrabé & al. 1030 (NOU), JX155060*, JX155105*, JX155152*,
JX155192*, JX155011*; 2, Vanuatu, Iles Torres, Hiu, Pillon 1072 (NOU), JX155074*, JX155119*, JX155166*, –, JX155026*. Carapichea affinis (Standl.) L. Andersson, America, –, AF0720211, –, AF3700393, –, –. Carapichea ipecacuanha (Brot.) L. Andersson, America, –, AF0720201, –, AF3700403, –, –. Carapichea ligularis
(Rudge) Delprete, French Guiana, –, AF1493908, –, AF1475672, –, –. Chassalia catatii Drake ex Bremek., Madagascar, Razafimandimbison 480 (UPS), AM9452185,
AM9452835, AM9453315, –, AM9453635. Chassalia sp. 1 MADAG, Madagascar, Antsiranana, Diana, Callmander 307 (K), JX155100*, JX155147*, –, –, JX155054*.
Chassalia sp. 2 MADAG, Madagascar, Antsiranana Province, Montagne d’Ambre, 1030 m, Ramandimbimanana 140 (K), JX155092*, JX155138*, JX155181*, –, –.
Chassalia sp. 1 VIET, Vietnam, Hoa Binh Province, Mai Chau District, Pa co, the forest alongate main road from HoaBinh to son La, 942 m, HNK 610 (K), JX155087*,
JX155132*, JX155177*, JX155218*, JX155039*. Chassalia sp. 2 VIET, Vietnam, Quảng Bình Province, Phong Nha-Ke Bang National Park, in forest on the edge of
agricultural land and Highway 1, 255 m, Briggs 114 (K), JX155090*, JX155135*, JX155179*, JX155219*, JX155042*. Chassalia sp. SUMA, Sumatra, Jambi Province,
Batanghari District, SPAS, 50 m, De Kok 1297 (K), JX155088*, JX155133*, JX155178*, –, JX155040*. Chazaliella abrupta (Hiern) E.M.A. Petit & Verdc. 1, Tanzania, Kilombero District, Uzungwa Mountains, Kihanzi Gorge, 800 m, Davis 2924 (K), JX155089*, JX155134*, –, JX155217*, JX155041*; 2, Tanzania, Morogoro
Province, Nguru Mountains, near Mhonda mission, along Chazi River, Bremer 3081 (UPS), JX155101*, JX155148*, –, JX155227*, JX155055*. Craterispermum sp.
2, Madagascar, Razafimandimbison & Ravelonarivo 656 (SPF), AM9452105, AM9452745, AM9453235, –, AM9453545. Craterispermum sp. 3, Madagascar, 24169-SF
(P), AM9452115, AM9452755, AM9453245, –, AM9453555. Geophila obvallata Didr., Africa, –, AM9451965, AM9452595, AF3698452, –, EU1455694. Geophila repens
(L.) I.M. Johnst., Vanuatu, Santo, Pénaorou, Munzinger 3649 (NOU), JX155079*, JX155124*, –, JX155210*, JX155031*. Hodgkinsonia frutescens C.T. White,
Australia, Queensland, Wongabel State Forest, Baba & al. 786 (NOU), JX155061*, JX155106*, JX155153*, JX155194*, JX155012* / JX155013*. Hydnophytum cf.
longistylum Becc., Vanuatu, Espiritu Santo Island, Sanama Province, McPherson 19437 (NOU), JX155078*, JX155123*, JX155170*, JX155209*, JX155030*. Hymenocoleus hirsutus (Benth.) Robbr., Africa, –, AF0720181, –, AF3698487, –, –. Margaritopsis astrellantha (Wernham) L. Andersson, Brazil, Parque Estadual Cristalino,
Zappi 938 (K), JX155096*, JX155142*, JX155185*, JX155225*, JX155047* / JX155048*. Margaritopsis boliviana (Standl.) C.M. Taylor, Brazil, Parque Estadual
Cristalino, Zappi 973 (K), JX155097*, JX155143*, JX155186* / JX155187*, JX155220*, JX155049* / JX155050*. Margaritopsis guianensis (Bremek.) C.M. Taylor,
Brazil, Parque Estadual Cristalino, Zappi 980 (K), JX155098*, JX155144*, JX155188*, –, JX155051* / JX155052*. Margaritopsis kappleri (Miq.) C.M. Taylor,
Brazil, Parque Estadual Cristalino, Zappi 921 (K), JX155095*, JX155141*, JX155184*, JX155224*, JX155046*. Margaritopsis nudiflora (Griseb.) K. Schum. in
H.G.A. Engler & K.A.E. Prantl, Cuba, Oriente Province, Guantánamo, Monte Libanon ad San Fernandez, Ekman 10248 (UPS), AM9451985, –, AF0013406, JX155230*,
EU1455684. Notopleura tapajozensis (Standl.) Bremek., Brazil, Parque Estadual Cristalino, Zappi 869 (K), JX155094*, JX155140*, JX155183*, JX155223*, JX155045*.
Palicourea crocea (Sw.) Schult. in J.J. Roemer & J.A. Schultes, America, Cordiero 2736 (SP), AF1493228, AM9452805, AF1475108, –, AM9453595. Palicourea nitidella
(Müll. Arg.) Standl., Brazil, Parque Estadual Cristalino, Sasaki 1828 (K), JX155093*, JX155139*, JX155182*, JX155222*, JX155044*. Prismatomeris albidiflora
Thwaites, Cambodge, Marie 89 (P), AM9452055, AM9452705, AM9453205, –, AM9453515. Prismatomeris beccariana (Baill. ex K. Schum.) J.T. Johanss., Indonesia,
Ridsdale 2461 (L), AM9452065, AM9452715, AF3316527, –, AM9453525. Psychotria aneityensis Guillaumin, Vanuatu, Iles Torres, Loh, Pillon 1127 (NOU), JX155075*,
JX155120*, JX155167*, JX155207*, JX155027*. Psychotria archboldiana Fosberg, Fiji, Viti Levu, Nadroga Navosa Province, Nadrau district, along the road between
Naqelewai and Monasavu, Buerki & al. 170 (NOU), JX155068*, JX155113*, JX155160*, JX155201*, JX155020*. Psychotria asiatica L., China, Hong-Kong (cultivated), Davis s.n. (K), JX155082*, JX155127*, –, –, JX155034*. Psychotria camptopus Verdc., Cameroon, Hong-Kong (cultivated), Maurin 38 (K), JX155084*,
JX155129*, JX155174*, JX155214*, JX155036*. Psychotria carnea 1 (G. Forst.) A.C. Sm., Fiji, Vanua Levu, Cakaudrove Province, Wailevu District, West of Savu
Savu, Barrabé & Tuiwawa 1113 (NOU), JX155065*, JX155110*, JX155157*, JX155198*, JX155017*. Psychotria carnea 2 (G. Forst.) A.C. Sm., Fiji, Taveuni,
Cakaudrove Province, Somo Somo District, Naibili, road from Somo Somo to hydroelectricdam, Barrabé & al. 1123 (NOU), JX155066*, JX155111*, JX155158*,
JX155199*, JX155018*. Psychotria collina Labill., New Caledonia, Province Nord, Ponérihouen, Aoupinié, Barrabé & al. 698 (NOU), JX155072*, JX155117*,
JX155164*, JX155205*, JX155024*. Psychotria forsteriana A. Gray, Fiji, Viti Levu, Naitasiri Province, Vuna District, Colo-i-Suva Forest Park, Buerki & al. 162
(NOU), JX155067*, JX155112*, JX155159*, JX155200*, JX155019*. Psychotria grandis Sw., Caribbean, Puerto Rico, Municipio Río Grande: El Verde Research
Station, Taylor 11745 (MO), JX155080*, JX155125*, JX155171*, JX155211*, JX155032*. Psychotria cf. impercepta A.C. Sm. & S.P. Darwin, Fiji, Viti Levu, Serua
Province, West of Nabukeleva village, Barrabé & al. 1086 (NOU), JX155063*, JX155108*, JX155155*, JX155196*, JX155015*. Psychotria lepiniana (Baill. ex
Drake) Drake, French Polynesia, Mont Mahutaa, vallée d’Orofero, Orohena, Nadeaud 345 (P), JX155059*, JX155104*, JX155151*, JX155193*, JX155010*. Psychotria
leptothyrsa Miq., New Guinea, Madang Province, Drozd & Molem s.n (CBSF), JX155102*, JX155149*, JX155190*, JX155228*, JX155056*. Psychotria lyciiflora
(Baill.) Schltr., New Caledonia, Province Sud, Sarraméa, Dogny, Barrabé & Bariole 747 (NOU), JX155069*, JX155114*, JX155161*, JX155202*, JX155021*. Psychotria oleoides (Baill.) Schltr., New Caledonia, Province Sud, Dumbéa, Montagne des Sources, Barrabé & Rigault 658 (NOU), JX155076*, JX155121*, JX155168*,
JX155208*, JX155028*. Psychotria parkeri Baker, Madagascar, Toamasina Province, Alaotra-Mangoro Region, Moramanga District, Mantadia National Park, Maurin
132 (K), JX155085*, JX155130*, JX155175*, JX155215*, JX155037*. Psychotria poeppigiana Müll. Arg. in C.F.P.von Martius & auct. suc. (eds.), Ecuador, Bremer
3330 (UPS), AF1494008, AM9452795, AF0027486, –, –. Psychotria samoana K. Schum., Samoa, Savai’i, far inland from Aopo, Bristol 2128 (K), JX155091*, JX155136*
/ JX155137*, JX155180*, JX155221*, JX155043*. Psychotria schlechteriana K. Krause, New Caledonia, Province Nord, Poya, Vallée des Roches d’Adio, Barrabé
& al. 712 (NOU), JX155070*, JX155115*, JX155162*, JX155203*, JX155022*. Psychotria stachyoides Benth., Brazil, Campos do Jordão, Parque Estadual, Trilha
do Rio Sapucai, São Paulo, Pirani & al. 4897 (SPF), JX155103*, JX155150*, JX155191*, JX155229*, JX155057* / JX155058*. Psychotria cf. trichostoma Merr. &
L.M. Perry, Vanuatu, Iles Torres, Hiu, Pillon 1057 (NOU), JX155073*, JX155118*, JX155165*, JX155206*, JX155025*. Psychotria sp. AUST, Australia, Sydney
Botanical Garden (cultivated), Weston 888 (Syd. Bot. Gard. 862980), JX155086*, JX155131*, JX155176*, JX155216*, JX155038*. Psychotria sp. FIJ, Fiji, Viti Levu,
Naitasiri Province, Vuna District, Colo-I-Suva, Forest Park, near Suva, Track between Rangers Station and Waisili Falls, Barrabé 1060 (NOU), JX155062*, JX155107*,
JX155154*, JX155195*, JX155014*. Psychotria sp. NC, New Caledonia, Province Nord, Poya, Boulinda, Munzinger 4963 (NOU), JX155071*, JX155116*, JX155163*,
JX155204*, JX155023*. Psychotria sp. V, Vanuatu, Espiritu Santo Island, Sanama Province, McPherson 19425 (NOU), JX155077*, JX155122*, JX155169*, –,
JX155029*. Psychotria sp. 1 VIET, Vietnam, Dong Nai Province, Tan Phu District, Cat Tien National Park, Davis 4031 (K), JX155081*, JX155126*, JX155172*,
JX155212*, JX155033*. Psychotria sp. 2 VIET, Vietnam, Hoa Binh Province, Mai Chau District, Cat Tien National Park, HNK 614 (K), JX155083*, JX155128*,
JX155173*, JX155213*, JX155035*. Readea membranacea Gillespie, Fiji, Vanua Levu, Cakaudrove Province, Wailevu District, West of Savu Savu, Waisali Rainforest Reserve, Barrabé & al. 1093 (NOU), JX155064*, JX155109*, JX155156*, JX155197*, JX155016*. Rudgea stipulacea (DC.) Steyerm., Brazil, Parque Estadual
Cristalino, Zappi 986 (K), JX155099*, JX155145* / JX155146*, JX155189*, JX155226*, JX155053*.
For Appendix 2, see Electronic Supplement.
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Version of Record (identical to print version).