ISSN 1346−7565

Acta Phytotax. Geobot. 59 (3): 183−193 (2008)

Two Species of Entada in Japan as Evidenced by cpDNA Phylogeny

NORIHISA WAKITA1, YOICHI TATEISHI2, TETSUO OHI-TOMA3, JIN MURATA3
and TADASHI KAJITA1*
1

Department of Biology, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522,
Japan. *tkaji@faculty.chiba-u.jp (author for correspondence); 2Department of Science Education, Faculty of
Education, University of the Ryukyus, Okinawa 903-0129, Japan; 3Botanical Gardens, Graduate School of Science,
The University of Tokyo 112-0001, Japan

The phylogenetic relationships of Entada (Fabaceae) were examined to determine if two distinct
species occur in Japan. Nucleotide sequences of the trnK and trnL introns and the intergenic
spacer of trnL–trnF of cpDNA resolved 19 haplotypes among 39 widely collected samples ofE. phaseoloides-like plants. In the resultant tree, two distinct clades corresponded to two groups
also distinguished by diagnostic seed characters. The clades agree with the expected chorology ofsamples and with the diagnostic morphology from previous taxonomic work. One clade corresponds
to Entada phaseoloides and the other to E. tonkinensis. A distinctive concentration of haplotypes
was observed. Although seeds of Entada phaseoloides are renowned as sea drift seeds, our results do
not suggest frequent gene flow caused by long distance seed dispersal.
Key words: Entada koshunensis, Entada phaseoloides, Entada tonkinensis, match-box bean, seeddispersal

Entada Adans. (Leguminosae: Mimosoideae) consists of about 30 species (Nielsen 1981,
1992, ILDIS 2005) distributed in the tropics andsubtropics. While more than half of the species
in Entada are shrubs or subshrubs with small
pods, some species are lianas with gigantic podsthat can be more than 1 m long. The large woody
seeds of Entada are also known for drifting in seawater. These remarkable seeds are called “matchbox beans” (the common name for Entada phaseoloides (L.) Merr. and E. rheedii Spreng.) or “seahearts” (E. gigas (L.) Fawc. & Rendle), and are
frequently found on beaches far from their origin.
Entada phaseoloides is widely distributed intropical and subtropical areas of the Pacific, fromeastern to southeastern continental Asia, the south
Asian islands, Oceania, and Polynesia. The northeastern limit of the species’ range is Japan (Nielsen
1992, Huang & Ohashi 1993). The wide distribution of Entada phaseoloides is thought to havebeen achieved by long distance dispersal of theseeds. Although Entada phaseoloides has somedistinctive features in its fruits and seeds, considerable taxonomic confusion exists as to its classification in Japan and adjacent areas. While moststudies treat the species of Entada in Japan as E.
phaseoloides, some works have variously treated
it as E. rheedii Spreng., E. parvifolia Merr., or E.

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koshunensis Hayata & Kaneh. Some authors recognize two distinct species (Ho 1985, Huang &Ohashi 1993, Ohashi 2001), but the recognition of
E. rheedii and E. parvifolia in the Ryukyu Islands(Okinawa), Japan, is apparently misleading and E.
koshunensis cannot readily be separated from E.
phaseoloides according to a detailed taxonomic
study that focused on this taxonomic confusion(Tateishi et al. 2008).
Recently, Wakita et al. (2005) surveyed thenumber of leaflet pairs on the second pinnae,
morphological characters of the seeds, and single
strand conformation polymorphism (SSCP) band
patterns of the rps16 intron in chloroplast DNA(cpDNA) for species of Entada in the Ryukyu
Islands. Although the number of pairs of leafleton the second pinnae did not clearly divide thesamples into two groups, the seeds and molecular markers clearly indicated the two groups of
Entada. The first group has a small (‘S’), convex
seed type, 29.4–46.9 mm wide by 32.4–52.7 mm
long with an angular margin. The second group
has a large (‘L’), compressed seed type, 39.5–59.1
mm wide by 45.8–64.7 mm long with a roundedmargin (Wakita et al. 2005). Different SSCP band
patterns, caused by a nucleotide substitution andsome indels in the rps16 intron, were also clearlyobserved in the two groups. Wakita et al. (2005)
considered the first group as corresponding Entada koshunensis and the second to E. phaseoloides
based on seed characteristics, following the criteria of Ho (1985) for distinguishing species of
Entada in Taiwan. Wakita et al. (2005), however,
did not resolve the taxonomic status of these twogroups due to insufficient sampling limited only
to the Ryukyu Islands. A molecular phylogenetic
study using additional samples from a wider geographic area is necessary to clarify the presence of
two species in Japan.
In this paper, we report the results of molecular phylogenetic analyses using samples obtained
from a broad geographical range, including theVol. 59

presumed type locality of Entada phaseoloides
in Indonesia. Key seed shape characters used in
Wakita et al. (2005) were also examined to determine the correspondence between seed charactergroups and molecular phylogenetic groups. The
goal of this study was to reveal the phylogenetic
relationships of Entada phaseoloides and closely
related species, and to determine if indeed two
species of Entada occur in Japan.

Materials and Methods
Entada phaseoloides-like plants were collected from eastern to southeastern Asia and theSouth Pacific (Table 1). Samples from Indonesia and Vanuatu were obtained from individuals cultivated in the Bogor Botanical Garden(Bogor, Indonesia) and the Tsukuba Botanical
Garden (Ibaraki, Japan), respectively (Table 1).
Since plants of Entada are generally very large,
spreading over the forest canopy, distinguishing
individuals was difficult. We collected more than
one sample per population only when individuals could be clearly distinguished. Seed samples
were not obtained from all the populations. Key
seed shape characters of all available seeds wereobserved, and seed type (S or L) was determinedaccording to Wakita et al. (2005).
Leaf samples of the species of Entada
obtained from eastern and southeastern Asia,
Oceania, and the South Pacific were used for amolecular phylogenetic study (Table 1). Entada
gigas (L.) Fawc. & Rendle, E. rheedii, and E.
spiralis Ridley, which belong to the same subsection (Entada subsect. Entada) as E. phaseoloides,
were also included in the phylogenetic analysis.
Outgroups were Entada abyssinica Steud. (fromTanzania) and E. polystachya DC. (from Mexico).
Those species were confirmed as not being in the
ingroup by our preliminary analysis (N. Wakita etal., unpublished data) using published trnL–trnF
sequences of Mimosoideae (Luckow et al. 2000,

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WAKITA & AL.: Entada in Japan by cpDNA Phylogeny2003). A total of 47 samples, including outgroups,
was used for the sequence analysis. Voucher
specimens of the samples used in this study were
deposited in QCNE, TI, TNS, or URO (Table 1).
Prior to genomic DNA extraction, crushed
leaf tissue was washed using HEPES buffer following the methods of Setoguchi & Ohba (1995).
Total genomic DNA was extracted from washedleaf pellets using the methods of Doyle & Doyle(1987). The two regions of cpDNA, the trnL
intron including the trnL–trnF intergenic spacer(IGS; hereafter referred to as trnL–trnF) and thetrnK intron (including matK; hereafter referred toas trnK), were amplified by PCR. The following
oligonucleotide primers were used for PCR amplification and sequencing: c, d, e, and f for trnL–
trnF, designed by Taberlet et al. (1991); trnK-F(forward: 5´-TGGGTTGCTAACTCAACGG-3´)
and trnK-R (reverse: 5´-GGAACTAGTCGGATGGAGT-3´) designed according to the complete
cpDNA sequence data of tobacco (GenBank
accession numbers Z00044 and S54304), matK-10(Kato et al. 1998), matK-AF (Takayama et al.
2005), matK-1777L, and matK-1932R (Hu et al.
2000) for trnK. The PCR reaction mixture contained 2.0 µL of 0.2 mmol/L dNTP solution, 1.0µL of 5 mmol/L of each primer, 0.5 unit of ExTaq
DNA polymerase (TaKaRa Bio, Ohtsu, Shiga,
Japan), 2.5 µL of 10× ExTaq buffer, and 10–30 ng
of genomic DNA in a total volume of 25 µL. PCR
amplification was performed in a PCR Thermal
cycler Dice (TaKaRa Bio). The PCR cycle for
trnL–trnF was 95°C for 1 min; followed by 25
cycles at 95°C for 45 s, 51°C for 1.5 min, and 72°
C for 1.5 min; and a final extension at 72°C for 10
min. For trnK, the settings were 96°C for 1 min;followed by 35 cycles at 96°C for 45 s, 48°C for
1 min, and 72°C for 1.5 min; and a final extension
at 72°C for 15 min. The extension time in both
PCR programs was increased by 2 s every cycle.
Amplified DNA was purified using a GeneClean
III DNA purification Kit (BIO 101, Carlsbad,

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CA, USA). Purified DNA was directly sequenced
using an ABI PRISM Big Dye Terminator version 3.1 Cycle Sequencing Ready Reaction kit
and an ABI PRISM 377 DNA sequencer (Applied
Biosystems, Foster City, CA, USA). Multiple
sequence alignment was performed with ClustalXver. 1.83 (Thompson et al. 1994) using the default
setting, and the obtained alignment was manually
modified using Se-Al Sequence Alignment Editor
version 2.0a11 (Rambaut 2002). Combined nucleotide sequences were used for further analyses
because the incongruence length difference (ILD)
test implemented in PAUP* ver4.0. detected nosignificant incongruence between the two genetic
regions for phylogenetic analyses.
Maximum-parsimony (MP) and neighborjoining (NJ) analyses were conducted using
PAUP* version 4.0 beta 10 (Swofford 2002).
Branch support was assessed with 1,000 replicates for MP and 10,000 for NJ. A Bayesian
phylogenetic analysis was performed in MrBayes
version 3.1.2 (Ronquist & Huelsenbeck 2003)
with the best-fit model (GTR+I+G) for both oftrnL–trnF and trnK selected by Akaike’s information criterion in MrModeltest 2.2 (Nylander
et al. 2004). Metropolis-coupled Markov chain
Monte Carlo sampling was performed with one
cold chain and three heated chains that were runfor 1,000,000 generations. Trees were sampled
every 100 generations. In total, 10,000 trees wereobtained and the first 2,500 samples from eachrun were discarded as burn-in to ensure the chains
had become stationary. A majority rule consensus
tree was generated to compute posterior probabilities. Insertion/deletion events (indels) were notincluded in the NJ and Bayesian analyses. Indelsof more than two base pairs were treated as binary(0/1) characters for the MP analysis if not caused
by a simple sequence repeat.

(URO)TABLE1. Entada samples used in this study. Clade and haplotype are designated as in Fig. 2. Seed type is according to Wakita et al. (2005). Accession numbers are shown on thefirst line for each haplotype.
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Vol. 59

FIG. 1. Geographic distribution of Entada haplotypes. Haplotypes are designated by name as they appear in Table 1 and Fig. 2.
Circles and triangles indicate the clade to which each haplotype belongs (see Fig. 2). Numbers in parentheses after haplotype
names indicate numbers of individuals. Seed type is shown as S or L according to Wakita et al. (2005). (a)–(c): Distribution
maps. (d): Pictures showing seed types. Scale bar = 1cm.

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WAKITA & AL.: Entada in Japan by cpDNA Phylogeny

189

-shan

FIG. 2. Strict consensus tree of 123 most parsimonious trees of E. phaseoloides-like samples and their allies obtained from trnL–
trnF and trnK sequences (length = 202 steps, CI = 0.861, RI = 0.8607, RC = 0.741). Measures of support are given at eachnode: NJ bootstrap support/MP bootstrap support/Bayesian posterior probability.

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Acta Phytotax. Geobot.

Results
Seed morphology
Using the key character of seed shape reported in Wakita et al. (2005), the seeds were sorted
into one of two groups. The seed type assigned
to all available seeds is shown in Table 1. TypeS seeds were obtained from Japan (Okinawa),
southern Taiwan, Indonesia, Australia, Vanuatu,
and Samoa. Type L seeds were obtained fromJapan (Kagoshima), northern Taiwan, China, andVietnam. The geographic distribution of seedtypes is mapped in Fig. 1.
Molecular analyses
The trnL–trnF sequence was 1,050–1,057
bp long and the trnK sequence was 2,513–2,537
bp long. Using the combined sequences, 19
haplotypes were resolved among the 39 Entada
phaseoloides-like samples. Twenty-four indels
were obtained across the matrix of all haplotypes
and three were coded as potentially phylogenetically informative for parsimony analyses. The
phylogenetic analysis using Entada abyssinica
and E. polystachya as outgroups produced 164
most parsimonious trees of 226 steps, with a consistency index of 0.876, retention index of 0.877,
and rescaled consistency index of 0.769 (Fig. 2).
The NJ and Bayesian tree topologies were nearlyconsistent with the MP tree presented here (Fig. 2).
Entada gigas was sister to another clade, which
was further divided into two major clades. Oneof those clades was composed of Entada rheedii
and E. spiralis (branch support (BS) by bootstrap
or posterior probability of 71 (MP analysis), 85(NJ analysis), and 0.90 (Bayesian analysis); BS ispresented in this order in the following text). The
second clade was composed of all Entada phaseoloides-like samples (BS of 96, 96, and 1.00).
This latter clade was further subdivided into two
clades (Clades I and II), both of which were very

Vol. 59

well supported (BS of 99, 99, and 1.00 for CladeI and 98, 99, and 1.00 for Clade II). Clade I consisted of samples from southern Taiwan, Vanuatu,
Samoa, Australia, and Indonesia, where S-typeseeds were obtained. Clade II consisted of samples from northern Taiwan, Hong Kong, and Vietnam, where L-type seeds were obtained. Samples
collected from the type locality of Entada phaseoloides (Ambon Island, Indonesia) were includedin Clade I.

Discussion
Presence of two species of Entada in JapanThe two well-defined clades (I and II) in
our phylogenetic tree are clearly consistent withthe two seed type groups (S or L) recognized by
Wakita et al. (2005). Our results clearly indicate
the presence of two species of Entada in Japan.
Entada phaseoloides was originally describedby Merrill (1917). The type specimen (drawn as
Faba marina in tab 4, Herbarium Amboinense 5:
5–8, Rumphius 1747) was obtained from Ambon,
Indonesia. According to some floristic works thattreated this species (Walker 1976, Nielsen 1992),
the diagnostic characters of Entada phaseoloides
are 1–3(or 4) pairs of asymmetric elliptic leaflets
and seeds 3–6 cm long. Entada phaseoloides is
distributed throughout subtropical to tropical areasfrom Southeast Asia to the Pacific islands. This
general characterization is applicable to all samples considered to be Entada phaseoloides-like inthis study. The wide distribution reported thus far
may represent the sum of the two species revealed
here. Our study, however, clearly suggests distinctive distribution areas for the two species. Samples included in Clade I were from Southeast Asia
to the South Pacific, whereas samples in Clade II
was collected only in areas extending from Vietnam to Japan (Fig. 1). Since the sample obtained
from the type locality of Entada phaseoloides(Ambon Island, Indonesia) appears in Clade I,

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WAKITA & AL.: Entada in Japan by cpDNA Phylogeny

we conclude that this clade corresponds to E.
phaseoloides. All available seeds obtained fromsamples in Clade I were of the S type. Thus, thediagnostic seed character of Entada phaseoloides
is a convex seed with an angular margin, and therange of E. phaseoloides encompasses the islandsof eastern and southeastern Asia, a part of Oceania, and Polynesia (Fig. 1).
The second clade (Clade II) was composedof samples from Japan (Amami-Oshima and Yakuislands), northern Taiwan, Hong Kong, and Vietnam. All available seeds from these samples weretype L (Table 1, Fig. 1). The samples comprising Clade II have generally been misidentified inJapan and Taiwan as Entada phaseoloides. After
surveying various taxonomic works from Japan,
Taiwan, China, and Vietnam, the seed morphology of Entada tonkinensis Gagnep. appears to beconsistent with the samples in Clade II. Entada
tonkinensis was originally reported from Vietnam(Gagnepain 1911), and its characteristically large
seeds were recorded as 50 mm wide and 60 mm
long. Clade II contained samples from Vietnam
and the seeds obtained from this clade were 39.5
–59.1 mm wide and 45.8–64.7 mm long, whichis consistent with the seed size reported for E.
tonkinensis. Although the species has not beenreported outside Vietnam, our study suggests a
wider distribution extending to China, Taiwan,
and Japan.
Unlike the above discussion, Wakita et al.
(2005) assigned Entada phaseoloides to the groupwith type L seeds and E. koshunensis to the groupwith type S seeds, based on samples obtained
from the Ryukyu Islands. Their assignment of
Entada phaseoloides to the type L seed group is
not likely correct, as discussed above. Regarding
their assignment of Entada koshunensis to thetype S seed group, our phylogenetic tree does notshow clear evidence to distinguish this species.
Entada koshunensis was originally described by
Hayata (1921) based on a sample collected in

191

the Henchun Peninsula, southern Taiwan. Somestudies distinguish this species from Entada
phaseoloides based on its smaller seeds (ca. 3cm) and greater number of leaflet pairs (Hayata
1921, Ho 1985, Huang & Ohashi 1993), but otherstudies treat it as E. phaseoloides because of its
morphological resemblance (Walker 1976, Huang& Ohashi 1977). In this study, four samples collected from the type locality of Entada koshunensis (Henchun Peninsula, southern Taiwan) had
haplotypes P01 and P03. These haplotypes were
included in Clade I and formed a clade having
weak support with samples from the Ryukyu
Islands (Fig. 2), but no clear morphological orbiogeographical association with this clade wasobserved. Further taxonomic study also supports
the difficulty in distinguishing Entada koshunensis as a separate species (Tateishi et al. 2008).
Geographic distribution of E. phaseoloides and E.
tonkinensis haplotypes
This study revealed the approximate distributions of haplotypes of Entada phaseoloides and
E. tonkinensis (Fig. 1). Both species have verylarge seeds that can drift in seawater, which might
contribute to their population structure. Other seadispersed plants have recently been studied using
molecular markers and evidence of long distancedispersal in haplotype structure has been reported(Takayama et al. 2006, Nettle & Dodd 2007).
For example, Hibiscus tiliaceus L. (Malvaceae)
has a wide distribution throughout the Old World
tropics. Common haplotypes were distributed
throughout the Pacific and Indian ocean areas, andgenetic differentiation among populations wasvery low (Takayama et al. 2006). However, ourresults from the study of Entada in Asia and thePacific region are somewhat contradictory. Basedon the distribution of haplotypes (Fig. 1), few
widespread haplotypes in Entada phaseoloides
are observed and a geographic concentration of
haplotypes appears to exist. Haplotypes P01–

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P09 occur in Japan and Taiwan, haplotypes P11–
P14 in Indonesia, and haplotypes P10 and P15–
P17 in Australia and Samoa (Fig. 1). Although thenumber of samples surveyed was limited, no common haplotypes were found among these regions.
The only case of long distance distribution of acommon haplotype is in Australia and Samoa,
separated by about 5,300 km, for haplotype P17.
The seeds of Entada phaseoloides are viable
for at least 1 year in seawater (Nakanishi 1994);
thus, seeds can be dispersed by ocean currents
over very long distances. The distribution of eachhaplotype, however, was geographically limited(Fig. 1). One possible explanation for this is the
low ability of seeds to establish in new localities
after successful dispersal. Entada phaseoloides israrely seen in littoral areas and is more common
in the canopy of inland forest. Although seeds
frequently reach open sandy beaches, subsequentcolonization may be difficult. Other factors suchas the direction of ocean currents may also resultin the geographically limited haplotype distribution. Further studies using more samples fromvarious localities are necessary to clarify thisissue.
For Entada tonkinensis, Clade II included
only two haplotypes, whereas Clade I of E. phaseoloides contained 17 haplotypes from 23 samples. This perhaps reflects the population size anddistribution ranges of these two species. In CladeII, a common haplotype (T01) was found in eight
populations in five regions (Fig. 2). The distribution of haplotype T01 might have been due to dispersal of seeds from continental Asia northward
to Taiwan and Japan along the island chain, as the
habitat of Entada tonkinensis in Japan and Hong
Kong is on slopes exposed to the sea at 20–150
m. The occurrence in Taiwan, however, is inland
about 15–30 km from the seashore. How this species has successfully colonized an inland forestfrom the seashore on which seeds washed ashore
is still unknown. As the vines of this species growVol. 59

very large, individual growth and seed production
over many years might be one explanation for thecolonization of an inland habitat.
In this study, a phylogenetic analysis using
cpDNA sequences revealed that two distinct
phylogenetic groups of Entada are present inJapan. Morphologically, one clade corresponds
to Entada phaseoloides and the other to E. tonkinensis. Although seeds of Entada phaseoloides
are remarkable examples of sea-drifted seeds, evidence of frequent long distance seed dispersal was
not obtained. Even if the seeds can be dispersed
by ocean currents across very long distances(Ridley 1930), the geographical concentration of
haplotypes suggests that another factor, such asdifficulty in establishment, prevents greater geneflow among regions.
We thank Drs B. Adje, K. Chayamarit, C. Nyomdham, T.
Yukawa, Josph Yip, L. K. C. Chau, N. N. Thin, S. Matsumura, D. Neil, and Bogor Botanical Garden, MEXU,QCNE for their help in obtaining samples. We also
thank Drs Y. Watano, T. Denda, T. Asakawa, K. Takayama for their valuable comments and useful suggestions
during this study. We also express our great thanks to
Drs G. Kenicer and D. Boufford for critically reading
the manuscript. This work was mainly supported by
JSPS KAKENHI No. 16370043 (to T.K.) and 14405015(to Y.T.). Part of the study and sampling were supported
by JSPS KAKENHI for foreign expeditions 14405015
and 12575011 (to Y.T.) and 17255004 to (J.M.). This
work is part of the PhD dissertation of N.W.

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