J. Jpn. Bot. 86: 15–25 (2011)

Origin of Hawaiian Endemic Species of Canavalia (Fabaceae)
from Sea-Dispersed Species Revealed by Chloroplast andNuclear DNA Sequences
Mohammad Vatanparasta, Koji Takayamaa,†, Mario S. Sousab, Yoichi
Tateishic and Tadashi Kajitaa,*aDepartment of Biology, Graduate School of Science, Chiba University,
1-33, Yayoi, Inage, Chiba, 263-8522 JAPAN;b
Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México,
Apartado Postal 70-367, 04510 México, D. F., MÉXICO;cFaculty of Education, University of the Ryukyus,
1, Senbaru, Nishihara, Okinawa, 903-0129 JAPAN;†
Present address: Department of Plant Systematics and Evolution,
Institute of Botany, University of Vienna. Rennweg 14, A-1030 Wien, AUSTRIA*Corresponding author: tkaji@faculty.chiba-u.jp(Accepted on July 22, 2010)
To reveal the origin of the Hawaiian endemic Canavalia species, phylogenetic
analyses of chloroplast DNA (cpDNA) and internal transcribed spacers (ITS) of nuclear
ribosomal DNA (nrDNA) sequences were performed. Phylogenetic analyses of 6 cpDNA
regions (6386 bp) and of nrDNA ITS (708 bp) for all 6 species of the Hawaiian endemic
subgenus Maunaloa together with samples from the other 3 subgenera of Canavalia
suggested that subgenus Maunaloa is monophyletic and more closely related to subgenus
Canavalia than to other subgenera. Phylogenetic analyses of multiple haplotypes of the
nrDNA ITS suggested that the Hawaiian endemic species of Canavalia originated from asea-dispersed species of subgenus Canavalia, possibly Canavalia rosea (Sw.) DC., which
is a pantropical species whose seeds are spread by sea drift. A single origin for subgenus

Maunaloa might be also suggested.

Key words: Canavalia, chloroplast DNA, Hawaiian Islands, nrDNA ITS, phylogeny,
seed dispersal.

Approximately 90% of native plant
species of the Hawaiian Islands are endemic(Baldwin and Wagner 2010). This represents
an extraordinary example of high endemism inoceanic islands and has attracted the interest of
many botanists. Many studies have focused on
the origin, speciation, and adaptive radiationof the endemic species in the Hawaiian Islands(reviewed in Baldwin and Wagner 2010). As the
Hawaiian Islands lie several thousand km fromthe nearest continents, high dispersibility would
be one of the key factors for the ancestors ofendemic species to achieve successful migration
to the Hawaiian Islands. According to the mostrecent review on studies of Hawaiian endemic
plant species, Baldwin and Wagner (2010) havesuggested that the temperate and boreal regionsof North America are important sources of
Hawaiian flora. This proposal is well supported
by molecular phylogenetic evidence (Vargas et

—15—

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Fig. 1. Distribution range of Canavalia rosea and subgenus Maunaloa (drawn based on Sauer 1964). Plant creeping onbeach (top left) and seed (top right) of C. rosea.

al. 1998, Lindqvist and Albert 2002). Among thespecies originating from North America, only
a few examples (Gossypium (Malvaceae) andJacquemontia (Convolvulaceae)) have seeds
that are dispersed by the sea. In the case of theother species, the seeds are dispersed by birds orwind. Although sea dispersal is one of the mostimportant modes of seed dispersal for shaping
the littoral flora of oceanic islands, detailed
studies on Hawaiian plants are lacking.
The genus Canavalia Adans. is a good model
for investigations of the origin of Hawaiian
endemic species from sea dispersed species.
The genus Canavalia is distributed in the tropics
and subtropics all over the world. According
to the latest taxonomic revision, this genus isfurther divided into four subgenera, namely,
Catodonia (seven species), Wenderothia (16species), Canavalia (23 species), and Maunaloa(six species) (Sauer 1964, St. John 1970, Schrire2005). Species of subgenera Catodonia andWenderothia are distributed mostly in the New
World. Species of subgenus Canavalia are foundin both the Old and New World, including somecrop species. Species of subgenus Maunaloa
are endemic to the Hawaiian Islands. Sauer(1964) studied the morphological differences
and similarities among Canavalia species andreported that the most primitive subgenus isprobably Wenderothia, and subgenera Catodonia
and Canavalia probably originated from thissubgenus. He also proposed that subgenus
Maunaloa would be originated from subgenus
Canavalia because of the presence of apantropical species, Canavalia rosea (Sw.) DC.,in the subgenus.
Canavalia rosea is a typical member of theplant group known as the “pantropical plants
with sea-drifted seeds” (Takayama et al. 2008).
The members of this plant group are distributedin littoral areas of the tropics and subtropics allover the world and their main mode of seeddispersal is sea dispersal (Fig. 1). In addition
to C. rosea, some species of the subgenus
Canavalia also use sea dispersal to spreadtheir seeds. Canavalia cathartica Thouars is
distributed over Indo-West Pacific regions, C.
lineata (Thunb.) DC. is found in South EastFebruary 2011

Journal of Japanese Botany Vol. 86 No.1

Asia, and C. sericea A. Gray is distributed in theSouth Pacific. The other species of Canavalia
whose seeds are subjected to sea dispersal is C.
bonariensis Lindley of the subgenus Catodonia.
Other species of the genus Canavalia do nothave sea-drifted seeds and their main mode of
seed dispersal is mechanical (with the seeds
being thrown by dehiscent pods) or gravity(Sauer 1964). Given the distribution ranges ofthe species and their modes of seed dispersal,
Sauer (1964) and Carlquist (1966) suggested
that the endemic species of the Hawaiian
Islands might have originated from species thatreached the Hawaiian Islands by sea dispersal.
A good candidate species would plausibly be
the pantropical species, Canavalia rosea, as it
has native distribution in Hawaii according to aherbarium specimen (Rock s.n., ca. 1910, BISH)
and has the widest distribution range among seadispersed species in the genus. However, thishypothesis has never been tested using modern
molecular phylogenetic approaches.
To test the hypothesis about the origin of
Hawaiian endemic species of Canavalia, we
employed both nuclear and chloroplast DNA(cpDNA) markers. These markers have beensuccessfully used to study the origin of Hawaiian
endemic species in other plant groups (reviewed
in Baldwin and Wagner 2010). Although a
recent phylogenetic study on subtribe Diocleinae
based on nuclear ribosomal DNA (nrDNA)
ITS sequences (Varela et al. 2004) showed thatsubgenus Canavalia is a sister to the subgenus
Catodonia, the taxon sampling of the study
was not sufficient to reveal the phylogenetic
relationships between the four subgenera and theorigin of Hawaiian endemic species. We studied
all species of the subgenus Maunaloa together
with multiple samples of C. rosea collected fromits wide distribution range. Other species thathave sea-drifted seeds and representative speciesof subgenera Wenderothia and Catodonia werealso included in the study.

17

Materials and Methods
Taxon sampling
A total of 18 species of Canavalia wereused in this study. Data of four species were
only for nrDNA ITS analyses and obtained
from GenBank (Table 1). Species from all
4 subgenera according to the subgenericdelimitation of Sauer (1964) were included(Table 1). Multiple accessions were sampled forfive taxa, C. rosea (18), C. lineata (2), C. sericea(2), C. hawaiiensis Degener & al. (2) and C.
galeata Gaudich. (2). Dioclea reflexa Hook. f., aspecies from the genus Dioclea was used as an
outgroup, based on the molecular phylogenetic
tree of Varela et. al. (2004). Voucher specimensof the newly collected samples in this study were
deposited in the herbarium of University of the
Ryukyus (RYU). Some samples were collectedfrom specimens preserved in the herbaria ofJardim Botânico do Rio de Janeiro (JBRJ),
Universidad National Autonoma de Mexico(MEXU), and Bishop Museum (BISH).
DNA extraction, PCR and sequencing
Genomic DNA was extracted from driedleaves or seeds using the method of Doyleand Doyle (1987). The concentration of crude
DNA extract was measured with a GeneQuant
100 electrophotometer (GE Healthcare, Life
Sciences). For cpDNA sequences, after an initial
screening of 15 cpDNA candidate regions (Shawet al. 2007), six regions including intergenic
spacers (IGS) and introns were chosen. The
cpDNA regions and PCR primers used in thisstudy were for atpB-rbcL IGS (Hodges andArnold 1994), ndhD-ndhE (Xu et al. 2000),
trnH-psbA IGS (Hamilton 1999), rps16 intron(Oxelman et al. 1997), trnD-trnT (Demesure
et al. 1995) and trnK, which includes thematK gene (Hu et al. 2000, Lavin et al. 2000,
Wojciechowski et al. 2004). Polymerase chain
reactions (PCR) were performed in reaction
volumes of 10–25 µL containing 1.25 units
ExTaq (TaKaRa), and 0.2 mM dNTPs., 10×
PCR buffer contains 1.5 mM MgCl2, 0.5–1 µM

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Table 1. List of Canavalia samples used in this study
Subgenus

Taxon

Canavalia

C. rosea (Sw.) DC.

Oceanic
regionIndian Ocean

West Pacific
East PacificWest Atlantic

East Atlantic

C. cathartica Thouars

Pacific

C. lineata (Thunb.) DC.

Pacific

Catodonia
Wenderothia

Outgroup

T. Kajita 010509
T. Kajita 021202

Sri Lanka

Wattala, Negambo

T. Kajita 000729

31S

cloningIndonesia

SumatraT. Kajita 000812

500

directAustralia

Headland Harbour

Y. Tateishi 001123

1

directMarquesas

Taipivai, Nuku Hiva

T. Kajita & Y. Tateishi 000713

31M

direct

Tonga

Sopu

T. Kajita & al. 021024

171

directMexicoSinaloa

T. Kajita & al. 1998-965

965

directPanamaVeracruz

T. Kajita & Y. Tateishi 011104

202

directEcuadorIsla Jambel

T. Kajita 990720

1205

directPanamaCuango, Colon

T. Kajita & Y. Tateishi 011031

109

directPanamaPina, Colon

T. Kajita & Y. Tateishi 011030

24

cloning

Costa Rica

Puerto Viejo

T. Kajita 990821

346

cloning

MexicoCoatzcoalcos

T. Kajita & al. 981029

70M

cloningBrazilGaibu Pernanbuco

T. Kajita & al. 991129

70B

cloning

SenegalJoal-Fadiout

T. Kajita 001203

107, 108

cloning

Ghana

Busua beachY. Kita 980808

5G

directAngolaMusul, Luanda

T. Kajita 010505

0cloning

Philippines

PalawanT. Kajita & Y. Tateishi 981210

CH10

cloning

Voucher specimen

nrDNA
ITS**
cloningcloning

TaiwanMaopi Tao

T. Kajita & Y. Tateishi 981212

CL1

directJapan

MiyazakiT. Kajita 020929

74

cloning

Pacific

Hawaii

Maui (Bishop Museum)

T. Kajita & al. 021022

42

directPacific

Tonga

Haashini-Lavengatonga

T. Kajita & al. 021024

138

cloning

C. virosa (Roxb.) Wight & Arn. Indian Ocean

AfricaSeed purchased

M. Vatanparast 090310

S3F8

directC. brasiliensis Benth.

Atlantic

MexicoGeneBank(data from Valera & al. 2004)

AF467034.1-

C. boliviana Piper

Atlantic

BrazilGeneBank(data from Valera & al. 2004)

AY293838.1-

C. hawaiiensis Degener & al.
C. galeata Gaudich.

Pacific
Pacific

Hawaii
HawaiiBishop Museum
Bishop Museum

Stemmermann 6843
Takeuchi 3224

6
7cloningcloning

C. kauaiensis J.D. Sauer

Pacific

HawaiiBishop Museum

Lorence 1987

9

cloning

C. molokaiensis Degener & al.

Pacific

HawaiiBishop Museum

Hughes 43

4cloning

C. napaliensis St. John

Pacific

HawaiiBishop Museum

Perlman 15319

5

cloning

C. pubescens Hook. & Arn.

Pacific

HawaiiBishop Museum

Hobdy 18998

cloning

C. hawaiiensis Degener & al.

Pacific

HawaiiCCRT*

M. Vatanparast 010606001

C6

directC. galeata Gaudich.

Pacific

HawaiiCCRT*

M. Vatanparast 010209001

C7

directdirectC. sericea A. Gray

Maunaloa

South Africa Umdloti
Tanzania
Dar es salaamDNA
sample No.
98
184

Locality

C. parviflora Benth.

Atlantic

BrazilJBRJ Herbarium

H. Braga 390727

0809

C. bonariensis LindleyAtlantic

BrazilGeneBank(data from Valera & al. 2004)

AY293839.1-

C. villosa Benth.
C. grandiflora Benth.

Atlantic
Atlantic

MexicoBrazilMEXU Herbarium
GeneBank

Gabriel 4469
(data from Valera & al. 2004)

23
AY293840.1

direct-C. hirsutissima J. D. Sauer

Atlantic

Dioclea reflexa Hook. f.
Atlantic* Center for Conservation Research and Training, Hawaii.
** Sequence method for nrDNA ITS.

MexicoMEXU Herbarium

A. Delgado 1372

11

directCosta Rica

Gandoca

T. Kajita 070113

13

directof each primer pair, and 20 ng genomic DNA.
The PCR conditions were as follows: 3 minfor initial denaturation at 95 °C, followed by
35 amplification cycles of 1 min denaturation
at 95 °C, 1–2 min annealing at a fragmentspecific temperature, 1–2 min extension at 72°C, and a final 10 min extension at 72 °C. The
PCR products were visualized by performing
0.8% agarose gel electrophoresis with ethidium

bromide staining and amplified DNA was
purified using either GENECLEAN III kit(Qiagen) or ExoSAP-IT (USB Corp., Cleveland,Ohio, USA) according to the manufacturer’s
instructions.
The cycle sequencing reactions were carriedout using the ABI BigDye Terminator version
3.1 Cycle Sequencing Kit (Applied Biosystems)
and sequencing reaction products were purified

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by ethanol precipitation. All DNA sequenceswere determined using an ABI 377 or ABI
3500 DNA sequencer (Applied Biosystems).
For four species (Canavalia brasiliensis
Benth., C. boliviana Piper, C. bonariensis
and C. grandiflora Benth.), the nrDNA ITS
sequences were obtained from GenBank(accession numbers AF467034.1, AY293838.1,
AY293839.1 and AY293840.1, respectively).
For ITS sequences in which direct sequencing
yielded complicated electropherograms,
the TOPO-TA cloning kit (Invitrogen) was
subsequently used to obtain clones according to
the manufacturer’s instructions. Twelve colonies
for each sample were picked, purified andamplified using the TempliPhi DNA Sequencing
Template Amplification Kit (GE Healthcare).
This kit utilizes bacteriophage Phi29 DNA
polymerase and rolling circle amplification(RCA) technology for rapid amplification of
circular template DNA (Polidoros et al. 2006).
The products were then sequenced using the
ABI BigDye Terminator version 3.1 Cycle
Sequencing Kit (Applied Biosystems). All
forward and reverse strands for each sequence
were assembled using Autoassembler version
2.1 (Applied Biosystems) with the default
setting. Sequences were then manually edited
and aligned with Se-Al version 2.0a11 (Rambaut2002). For the ITS sequences, cloned products
were aligned and verified with those obtained
from direct sequencing in this study. A haplotype
data matrix was made by the program DNAspversion 5.10.01 (Librado and Rozas 2009). The
Phi test (Bruen et al. 2006) was employed totest the presence of recombination for cloned
sequences of each sample using the program
SplitsTree version 4.11.3 (Huson and Bryant
2006).
Phylogenetic inferences
Prior to the phylogenetic analyses, we
performed the incongruence length difference(ILD) test (Farris et al. 1994) on the chloroplast
sequences using PAUP* version 4.0b10

19(Swofford 2002) to test combinability of the 6plastid regions. A total of 100 replicates wereused in the ILD test.
Maximum parsimony (MP), maximum
likelihood (ML) and Bayesian inference (BI)were employed for the phylogenetic analyses
using cpDNA and nrDNA ITS sequences.
For each of the sequences, the methods wereconducted separately using the relevant
programs. The MP analyses were conductedusing PAUP* version 4.0b10 (Swofford 2002).
Heuristic search were performed with a treebisection–reconnection (TBR) branch swapping
algorithm and with indels and inversions
removed. All characters were unweighted andunordered. Branch support was evaluated using
the bootstrapping method of Felsenstein (1985)
based on 1000 replicates.
The ML nalysis was performed using theRAxML (Randomized Axelerated Maximum
Likelihood) program, version 7.2.6 (Stamatakis2006) which implements a rapid hill-climbing
algorithm. The analysis was run for the bestscoring ML tree inferences under the GTR+G
model for the both cpDNA and nrDNA ITS
sequences separately, with all indel and inversion
characters removed. Rapid bootstrap analyses
were conducted with 1000 replications using the
GTRCAT (Stamatakis 2006) estimation to assessbranch support.
Bayesian inference was performed
using MrBayes version 3.1.2 (Ronquist andHuelsenbeck 2003). For each region weselected appropriate evolutionary models using
MrModeltest version 2 (Nylander 2004). The
HYK (atpB-rbcL IGS, trnH-psbA IGS andrps16), GTR+I (ndhD-ndhE), HKY+I (trnDtrnT and trnK) and SYM+G (nrDNA ITS)
models were selected as best-fit models basedon the Akaike information criterion (Akaike1974) using the program MrModeltest version
2 (Nylander 2004). Two independent Markov
Chain Monte Carlo (MCMC) analyses withfour simultaneous chains and 2,000,000
generations were run. Trees were sampled for

20

植物研究雑誌

every 100 generations and the first 5000 trees
were discarded as burn-in. The convergenceof MCMC chains was visualized with Tracer
version 1.5 (Rambaut and Drummond 2009)and likelihood scores for the sampled trees were
inspected.
Results
Chloroplast (six regions) and nuclear DNA(ITS) molecular markers
Nucleotide sequences of the cpDNAand nrDNA regions were obtained from 40
specimens used in this study (Table 1; GenBank
accsession no. HQ707397–HQ707580). Someof the cpDNA regions included parts that weredifficult to align, and these were excluded fromanalyses. These regions include a 207 bp indel
in atpB-rbcL IGS, four and two base pairs
inversions in ndhD-ndhE and trnH-psbA IGS
respectively, and all length variations containing
homopolymers. Sequences were determinedusing cloned samples for 19 specimens fromnine taxa (Table 1). The aligned length was 6386
bp for concatenated cpDNA regions and 708 bp
for nrDNA.
Phylogenetic analyses based on cpDNA
sequence data
The results of the ILD test for the six
chloroplast regions detected heterogeneity
among these regions (p value = 0.01). Althoughheterogeneity among regions may be expected
at the 1% significance level, the six chloroplast
regions were combined in the phylogenetic
analyses, as we didn’t find major topologicalconflicts among 6 trees generated from eachregion, and it is said that the ILD test might be
too sensitive (Wiens 1998). The Hawaiian taxa,
particularly, retained a clade in three trees, while
they were placed in a polytomy with samples ofsubgenus Canavalia in the other trees (data notshown).
A total of 21 haplotypes, designated from
C1–C21 were recognized in the aligned
sequences of 6386 bp. MP analysis of the 21

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haplotypes resulted in 15 most parsimonious
trees with a length of 257 steps, a consistency
index (CI) of 0.949 excluding uninformative
characters, and a retention index (RI) of 0.879.
The strict consensus of the 15 most parsimonious
trees is shown in Fig. 2. Both ML and BI
analyses produced topologically identical trees(not shown). Bootstrap values for MP and ML
analyses and posterior probability for BI analysis
are shown in the resultant tree when theyexceed 70% (Fig. 2). Within the genus, threemain monophyletic clades (I, II and III) wererecognized with high branch supports. Clade I
consists of members of the subgenera Catodonia
and Wenderothia. Clade II includes membersof the subgenera Canavalia and Maunaloa.
Clade III includes all Hawaiian endemic speciesof the subgenus Maunaloa. Moreover, withinclade I, the species of subgenus Wenderothia (C.
hirsutissima J. D. Sauer and C. villosa Benth.)make a monophyletic group (Fig. 2). In the 10
haplotypes (C1–C10) observed in subgenus
Canavalia, seven haplotypes (C2–C8) were
exclusive to the C. rosea, in which all but one(C8, Mexico from Pacific region) were from theAtlantic region. The haplotype C1 was shared
among C. rosea, C. cathartica and C. lineatafrom various localities in the Pacific and Indian
oceanic regions. Haplotypes C9 and C10 were
only obtained from C. virosa (Roxb.) Wight &Arn. and C. sericea, respectively. In Clade III, C.
kauaiensis J. D. Sauer (C12) and C. napaliensis
St. John (C13) form a monophyletic group and
C. molokaiensis Degener & al. and C. pubescens
Hook. & Arn. have same sequence haplotype(C14).
Phylogenetic analyses based on ITS sequencedata
The total sequence length of the nrDNA
ITS (ITS1, 5.8S, and ITS2) sequences for 40
samples ranged from 601 bp in C. virosa to 708
bp in Dioclea reflexa (an outgroup species).
Aligned sequences, after excluding all gaps,
yielded 514 bp that retained 35 haplotypes (H1–

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21

C1 C. cathartica Philippines (P)C. lineata Taiwan (P), Japan (P)C. rosea Australia (I), Ecuador (P), Indonesia (I)Marquesas (P), Panama 202 (P), South Africa (I)Sri Lanka (I), Tanzania (I), Tonga (P)C2 C. rosea Senegal 107 (A), Ghana (A)Subg. Canavalia

Mexico (A), Panama 109 (A)C3 C. rosea Angola (A)C4 C. rosea Senegal 108 (A)C5 C. rosea Brazil (A)C6 C. rosea Costa Rica (A)100/100/100 C7 C. rosea Panama 24 (A)II

C8 C. rosea Mexico (P)C9 C. virosa (I)C10 C. sericea Tonga 42,138 (P)C11 C. hawaiiensis 6 (P)C12 C. kauaiensis (P)C13 C. napaliensis (P)100/100/100

88/100/100
74/100/100

C14 C. molokaiensis (P)III Subg. Maunaloa

C. pubescens (P)C15 C. galeata 7 (P)C16 C. galeata C7 (P)C17 C. hawaiiensis C6 (P)C18 C. hirsutissima (A)100/100/100
98/100/100

C19 C. villosa (A)Subg. Wenderothia

I

C20 C. parviflora (A) Subg. Catodonia
C21 Dioclea reflexa (A)5 changes

Fig. 2. The strict consensus tree of 15 equally most parsimonious trees based on 6389 bp of 6 cpDNA regions for Canavalia
species. Tree length = 257, CI = 0.949, RI = 0.879 and RC = 0.835. Branch supports for MP/ML/BI analyses are shown
along branches. Dioclea reflexa is used as an outgroup. Abbreviations in parenthesis. P. Pacific Ocean. A. Atlantic Ocean.
I. Indian Ocean. Numbers following locality names correspond to the DNA sample No. shown in Table 1.

H35). Pairwise sequence divergence values wereless than 3% substitution per site across ingrouptaxa. The Phi test did not identify statisticallysignificant evidence for recombination amongcloned sequences.
The MP analysis of the nrDNA ITS sequence
data resulted in 1169 most parsimonious
trees with a length of 141 steps, a CI of 0.823(excluding uninformative characters) and a RI
of 0.795. The strict consensus of these trees
with accompanying bootstrap values for MP

and ML analyses and posterior probability for
BI analysis of more than 70% are shown in
Fig. 3. Both the ML and BI analyses for the
nrDNA ITS produced topologically identicaltrees (not shown). Contrary to the cpDNA tree,
the subgenus Catodonia was found to be moreclosely related to the clade comprised of samples
from subgenera Canavalia and Maunaloa andsupported by relatively high branch supports(Fig. 3). All samples of Hawaiian endemic taxa
used in this study had 2 alleles for ITS sequence.

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Marquesas (P), Mexico (A), Mexico (P), Panama 24,109 (A),
Senegal 107,108 (A), South Africa (I), Tanzania (I), Panama 202 (P)C. lineata Japan (P), Taiwan (P)C. sericea Tonga 138 (P)Subg. Canavalia

H1 C. rosea Angola (A), Australia (I), Costa Rica (A), Ghana (A), Indonesia (I)C. kauaiensis (P)C. molokaiensis (P)C. napaliensis (P)Subg. Maunaloa

C. hawaiiensis 6, C6 (P)C. pubescens (P)C. galeata 7, C7 (P)H2 C. sericea Tonga 42 (P)C. cathartica Philippines (P)H3 C. rosea Tonga (P)H4 C. rosea Panama 24 (A)H5 C. rosea Costa Rica (A)H6 C. rosea Angola (A), Costa Rica (A)H7 C. rosea Mexico (A), Panama 24 (A)H8 C. rosea Mexico (A), Senegal 107,108 (A), Costa Rica (A)Tanzania (I), South Africa (I), Brazil (A)Subg. Canavalia

H9 C. rosea Angola (A)H10 C. rosea Angola (A)H11 C. rosea Brazil (A)H12 C. rosea Brazil (A)100/100/100

H13 C. rosea Costa Rica (A)H14 C. rosea Mexico (A)H15 C. rosea Brazil, Senegal 108 (A), Mexico (A)H16 C. rosea Ecuador (P), Mexico (A), Panama 24 (A), Panama 202 (P), Senegal 107 (A)H17 C. rosea Sri Lanka (I), Tanzania (I)H18 C. rosea Sri Lanka (I), Tanzania (I)H19 C. rosea Sri Lanka (I)H20 C. virosa (I)79/100/66

H21 C. brasiliensis (A), GB
H22 C. boliviana (A), GB
H23 C. galeata 7 (P)H24 C. kauaiensis (P)H25 C. hawaiiensis 6 (P)H26 C. hawaiiensis 6 (P)100/100/100

Subg. Maunaloa

H27 C. molokaiensis (P)H28 C. napaliensis (P)H29 C. pubescens (P)94/100/100

H30 C. bonariensis (A), GB
H31 C. parviflora (A)93/100/100

Subg. Catodonia

H32 C. grandiflora (A), GB
98/100/100

H33 C. villosa (A)Subg. Wenderothia

H34 C. hirsutissima (A)5 changes

H35 Dioclea reflexa (A)Fig. 3. The strict consensus tree of 1169 most parsimonious trees based on 708 bp of nrDNA ITS region for Canavalia
species. Tree length = 141, CI = 0.823, RI = 0.795 and RC = 0.654. Branch supports for MP/ML/BI analyses are shown
along branches. Dioclea reflexa is used as an outgroup. Abbreviations. P. Pacific Ocean. A. Atlantic Ocean. I. Indian
Ocean. GB. GeneBank. Numbers following locality names correspond to the DNA sample No. shown in Table 1.

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One was the haplotype H1 that was also shared
among samples of C. rosea, C. lineata and C.
sericea, and the others were specific to eachspecies (H23–H29). Most of the C. rosea
samples from the Atlantic region had morethan 2 alleles (Fig. 3). Canavalia virosa, C.
brasiliensis and C. boliviana represented distinct
haplotypes.
Discussion
The origin of Hawaiian endemic species
The results of the present study suggest thatthe endemic Hawaiian subgenus of Canavalia
originates from a species that reached the
Hawaiian Islands by sea dispersal, and that the
species would be allied to Canavalia rosea, apantropical species. According to the cpDNAand nrDNA ITS phylogenetic trees, Hawaiian
endemic species (subgenus Maunaloa) are moreclosely related to the subgenus Canavalia than
to the other subgenera (Figs. 2, 3). Although
the ancestral lineage of Hawaiian endemic
Canavalia was not clearly shown, Hawaiian
species share the same haplotype (H1) with C.
rosea, C. lineata and C. sericea, all of whichhave seeds which are spread by sea drift. These
results suggest that the Hawaiian endemic
species were originated from one of the three
species or from an ancestor common to them.
As the haplotype H1 of C. rosea is widelydistributed over the Pacific, Atlantic and Indian
Oceans, we propose that C. rosea is the originof the Hawaiian endemic species. Although
C. sericea and another sea-dispersed species,
C. cathartica, are in the Hawaiian Islands atpresent, they are not native to the islands (Sauer
1988, Whistler 1992, Wagner et al. 1999). On
the other hand, C. rosea was present in the
Hawaii according to a herbarium specimen in
Bishop Museum (Rock s.n., ca. 1910, BISH),
although we have no recent herbarium recordsof the species from Hawaii.
Speciation and Loss of dispersibility in
Hawaiian Canavalia

23

Our phylogenetic analyses using cpDNA
sequences showed that the 6 Hawaiian endemic
species are monophyletic (Fig. 2). This result
suggests that they are descended from a single
species and that the speciation of the six species
occurred after the colonization of their ancestor
in the Hawaiian Islands. None of the species ofthe subgenus Maunaloa have seed buoyancy(Sauer 1964, Carlquist 1966). Loss of seedbuoyancy in oceanic island species is a common
phenomenon which occurred in many species
in Hawaiian endemic plants (Carlquist 1974,
Cody and Overton 1996, Baldwin and Wagner
2010). In the case of Canavalia, it would be
associated with the habitat shift toward inland
regions during the speciation process (Carlquist
1967). Similar habitat shift is also observed in
Hibiscus glaber Matsum. that is an endemic
species in the Bonin Islands and was revealed to
be speciated from a pantropical plants with seadrifted seeds, H. tiliaceus L. (Takayama et al.
2006). It remains unclear why the distribution of
the endemic species of Canavalia is restrictedto specific islands among the Hawaiian island
chain. The endemic species might have speciatedon each island after the speciation of theircommon ancestor that retained seed buoyancy
and therefore had the ability to colonize over the
Hawaiian Islands. A more detailed study using
high resolution markers is needed to reveal
speciation process of the Hawaiian endemic
species of Canavalia.
Phylogenetic relationships among species
Our phylogenetic trees do not clearly show
the species relationships among subgenera
Canavalia and Maunaloa although we usedhighly variable cpDNA regions and nrDNA
ITS (Figs. 2, 3). This is perhaps because thespeciation within and/or among these subgenera
occurred rapidly. This result made it difficult tounderstand the evolutionary history among thespecies of these subgenera. As we used rather
long sequences of cpDNA for phylogenetic
inference, increasing the data length of cpDNA

24

植物研究雑誌

may not improve the resolution of phylogenetic
tree. Given the difficulties in performing
phylogenetic analyses for closely related species,
novel nuclear markers are needed (Scherson
et al. 2005), for example, a combination of
single or low copy nuclear genes, or data fromfragment analyses.
We thank Drs H. C. Lima, M. Bovini, A.
Tidiane Ba, D. Darnaedi, I. Ibrahim, D. Neil,
D. A. Madulid, E. Narvaez, I. Seneviratne, J.
Flora, T. Yahara, A. Soejima, T. Ohi-Toma, K.
Watanabe, Y. Mori, Y. Kita, Y. Abe, Bishop
Museum (USA), JBRJ (Brazil), CNPq (Brazil),
STRI (Panama), OTS (Costa Rica) and MEXU
(Mexico) for their support in field sampling,and A. Yoshinaga from Center for Conservation
Research and Training (CCRT), Hawaii forproviding materials. We also thank Drs Y.
Watano, T. Asakawa, B. Adjie, N. Wakita and A.
Shirai for their useful comments on this study.
This work was supported by JSPS Grants-inAid for Scientific Research, mainly by Nos.
19370032, 16370043 and 14740471 (to T. K.),and for sampling by 14405015 and 12575011 (toY. T.).
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M. Vatanparasta，高山浩司 a,†，M. S. Sousab，立石庸一 c，

梶田 忠 a：分子データから示されたハワイ産ナタマメ属
固有種の起源
ハワイ諸島に固有のナタマメ属の Maunaloa 亜属の起源を明らかにするために，葉緑体 DNA と核リボソーム
DNA の ITS 領域を用いた系統解析を行った．Maunaloa

亜属の 6 種全てと，他の 3 亜属から得られたサンプルを用いて，葉緑体 DNA の 6 領域約 6386 bp と核リボソーム DNA の ITS 領域の約 708 bp の塩基配列を用いて，それぞれ系統解析を行った結果，Maunaloa 亜属は他の2 亜属よりもナタマメ亜属により近縁であることが分かった．ITS 領域について 1 サンプルから得られた複数の配列の系統関係からは，Maunaloa 亜属の種は，ナタマ

メ亜属の中でも海流散布能力を持つ 3 種により近縁であることが示唆された．地理的分布とハプロタイプの分布を考慮すると，ナガミハマナタマメ Canavalia rosea
が Maunaloa 亜属の祖先種であったことが考えられる．
葉緑体 DNA の系統解析から，Maunaloa 亜属の種は単系統であることから，ハワイ諸島における Maunaloa
亜属の 6 種は，共通の祖先から起源したことが示された．
（a 千葉大学大学院理学研究科，
メキシコ自治大学生物学研究所植物学部門，c
琉球大学教育学部理科，†
現住所：ウィーン大学植物科学科植物系統進化学分野）
b