SAGO PALM 26-2: 37−43 (2019)

Phylogenetic Study of Metroxylon Palms in Southeast Asia andOceania Based on 5S nrDNA Spacer Sequence Data
Hiroshi Ehara1,2*, Toshiki Yamamoto3, Tohru Tsuchiya4, Hitoshi Naito5, John L. Dowe6,
Will C. McClatchey7, Takashi Mishima3, Akemi Itaya3, Chitoshi Mizota8,
Yulius B. Pasolon9, Philimon Ala10, Marika Tuiwawa11, Alivereti Naikatini11,
Issac A. Rounds12, Talie Foliga13, Siosinamele Lui13 and Samantha Kwan13
1

2

Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Applied Social System Institute of Asia, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
3

4

International Center for Research and Education in Agriculture, Nagoya University,

Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu 514-8507, Japan

Advanced Science Research Promotion Center, Mie University, 1577 Kurimamachiya-cho, Tsu 514-8507, Japan
5

College of Life Science, Kurashiki University of Science and The Arts, 2640 Nishinoura,
Tsurajima-cho, Kurashiki 712-8505, Japan

6

7

Australian Tropical Herbarium, James Cook University, Cairns, Qld, Australia

Botanical Research Institute of Texas, 1700 University Drive Fort Worth, Texas 76107-3400, USA
8

Faculty of Agriculture, Iwate University, 3-18-1 Ueda, Morioka 020-8500, Japan
9

Faculty of Agriculture, Halu Oleo University, Kendari, Indonesia
1011

Department of Forest, Port Vila, Vanuatu

Institute of Applied Science, The University of the South Pacific, Laucala Campus, Suva, Fiji
12

13

Conservation International, 3 Ma’afu St, Suva, Fiji

Ministry of Natural Resources and Environment, Apia, Samoa* Corresponding author

Keywords: Melanesia, Micronesia, Polynesia, sago palm, specification
Introduction

The genus Metroxylon is distributed from Southeast

Asia (Thailand, Malaysia, Brunei, the Philippines, andIndonesia) to Micronesia, Melanesia (Papua New
Guinea, the Solomon Islands, Vanuatu, and Fiji), andPolynesia (Samoa). It is divided into two sections,

Metroxylon (Eumetroxylon) and Coelococcus (Beccari
1918; Rauwerdink 1986), representing the western

half and the eastern half of this distribution,
respectively (McClatchey 1999). Metroxylon sagu

Rottb. (called the true sago palm: hereafter “sagopalm”) is the only species in the section Metroxylon,

although the monophyly of this section remains
uncertain. The sago palm extends across Southeast

Asia and northwestern Melanesia (Papua New Guinea

and the Solomon Islands). The sago palm produces
the largest amount of starch in the trunk among the
Arecaceae genera, and it is used variously for foodand processed food or floured for noodle making.

This species has long been used as a food similar tobananas and taro (Barrau 1959) and is one of theoldest crops used by human beings since ancient

times (Takamura 1990). In Southeast Asia andMelanesia, the leaves of the sago palm are used formaking roof thatch, and the petioles are used as
construction materials of temporary houses and huts.

Metroxylon amicarum (H. Wendl.) Becc. (Caroline

ivory nut palm (Jones 1995)) of the section

38

H. Ehara et al.

Coelococcus is restricted to Micronesia. The othersupport or refute the phylogeny determined by

and Samoa. Metroxylon salomonense (Warb.) Becc.

were useful for a phylogenetic analysis of the palmfour species are distributed across Melanesia and

Polynesia from the Solomon Islands, Vanuatu to Fiji,(Solomon sago palm) is distributed in the Solomon

Islands (McClatchey 1999) and Vanuatu (Ehara et al.
2003a), and M. vitiense (H. Wendl.) H. Wendl. ex

Benth. & Hook. f. (Fiji sago palm) (Watling 2005) is
distributed in Fiji. Metroxylon warburugii (F. Heim)

Becc. is distributed in Vanuatu, Fiji, and Samoa; andM. paulcoxii McClatchey (M. upoluense Becc.) ismorphology alone. Barrow (1998) showed that DNA

sequences from the nuclear 5S nontranscribed spacer
genus Phoenix. Baker et al. (2000) reported themolecular phylogenetics of Calamus and related rattan

genera based on 5S nrDNA spacer sequence data. The

phylogenetic analysis based on 5S nrDNA spacer

sequence is, therefore, considered to be an available
substitute for the genus Metroxylon, as it belongs to thesame tribe (Calameae) as does the genus Calamus.

distributed in Samoa. Metroxylon warburgii isThus, we used DNA sequences from the nontranscribed

consume sago (starch obtained from the trunk of the

distributed in Southeast Asia and Oceania.

sometimes called the Vanuatu sago palm. McClatchey(1998) reported that people on Rotuma in Fijipalm) produced from M. warburugii. In other areas,

Metroxylon palms have been used occasionally. For
example, M. amicarum was used on Moen in

Micronesia until the 1940s, and M. warburgii wasused on Gaua in Vanuatu until at least the 1950s

spacer of 5S nrDNA in this study to investigate
phylogenetic relationships among the Metroxylon palms
Materials and Methods
Plant Materials

Two populations each of M. amicarum (plantmaterials of M. amicarum 1 taken from a palm planted(Ehara et al. 2003a). On Malekula Island in Vanuatu,

and grown in Townsville Botanic Gardens, Australia,

harvest M. vitiense to get the pre-emergent young

Micronesia: 6 ° 54.063’ N, 158 ° 17.115’ E), M.

M. warburgii is sometimes used as an emergency
food. On the other hand, Indo-Fijian people often

leaves around the growing point (apical bud together

with the very young leaf sheathes and leaves) (Ehara
2015a). This harvested part is known as “palmcabbage” and, in cooking, is used similarly to bamboo

after germination from a seed collected on Caroline

Island; M. amicarum 2 harvested on Pohnpei Island,

salomonense (plant materials of M. salomonense 1

taken from Singapore Botanic Gardens in Singapore;M. salomonense 2 harvested on Gaua Island, Vanuatu:

14° 15.907’S, 167° 36.098’E) (Ehara et al. 2003a), M.

shoots. The leaves of Coelococcus palms are also

vitiense (plant materials of M. vitiense 1 taken from aendosperm of M. amicarum and M. warburgii seeds isvitiense 2 harvested on Viti Levu Island, Fiji:

important as building and houseware materials similar
to uses of the sago palm (M. sagu). The hardcalled “palm ivory” and is utilized as material for

craftwork (Ehara 2015c).

There have been reports on the specifications of theMetroxylon section Coelococcus based onmorphological characteristics (McClatchey 1998,
1999). However, few studies of the molecularphylogenetics of the genus Metroxylon, including thesection Coelococcus, exist. McClatchey (2002)

suggested that genetic studies of sections Metroxylon
and Coelococcus should be conducted in order topalm planted and grown in Townsville Botanic Garden
after germination from a seed collected in Fiji; M.
18 ° 13.241’S, 178 ° 5.048’E), M. warburgii (M.
warburgii 1 harvested on Espiritu Santo Island,

Vanuatu: 15 ° 29.277’S, 167 ° 11.621’E (2003a); M.
warburgii 2 harvested on Upolu Island, Samoa:
13° 53.576’S, 171° 34.809’W), and one population of

M. paulcoxii (plant materials of this palm taken on
Upolu Island, Samoa: 13° 54.812’S, 171° 35.829’W)
were used. In addition, two populations of M. sagu

grown in Southeast Sulawesi (3 ° 58.566’ S,
122 ° 26.668’E; the folk variety names of M. sagu 1

Phylogenetic Study of Metroxylon Palms in Southeast Asia and Oceania Based on 5S nrDNA Spacer Sequence Data

Philippines

Federated States ofMicronesia

Thailand

BruneiMalaysia
Singapore39

PhonpeiM. sagu

MarshallIslands

M. amicarum
SE. Sulawesi

PNG
M. salomonense

Indonesia

SolomonIslands

Australia

TownsvilleM. paulcoxii

Gaua
Upolu
Esprit Fiji
Santo
Samoa
VanuatuViti Levu

M. warburgii

M. vitienseFig 1. Distribution of sections Metroxylon and Coelococcus

Section Metroxylon: M. sagu
Section Coelococcus: M. amicarum, M. salomonense, M. warburgii, M. vitiense, and M. paulcoxii

and M. sagu 2 are Roe and Rui, respectively) were

each primer (0.5 μ L each), 4 μ L of HybriPol DNA

DNA Extraction, Amplification, Cloning, andcycle; denaturing step of 96° C for 1 min, annealing

used. The sampling sites are shown in Fig. 1.
Sequencing

Two leaflets were taken from each population, cut

into small pieces, and freeze-dried or silica-dried(Chase and Harold 1991). Some pieces of small dried

leaflets were ground to obtain about 25 mg ofcomparatively fine powder that was used for DNA

extraction. Total genomic DNA was extracted fromthe dried leaflet tissue using the DNeasy Plant Mini
Kit (QIAGEN). The extracted DNA was cleaned

using the QIAquick PCR Purification Kit (QIAGEN),and the purified product was eluted into 50 μ l ofelution buffer or water. The 5S spacer was
amplified from total genomic DNA using 5S F

(AGTTAAGCTTGCTTGGGCGAGAGTA) and 5S R

(AGTTCTGATGGAATTCGGTGYTKTA) primers.

The primers 5S R and 5S F were designed withreference to published tree sequences (Mitchell andWen 2005) and annual herbaceous sequences (Coxet al. 1992). Fifty-microliter reactions were prepared(5 μ L of 10× HybriPol Buffer provided by Bioline, 50

mM MgCl2 1.5 μ L, 2 mM dNTP (4 μ L) each, 10 μ M

polymerase (Bioline), and 0.5 μ L of template DNA).
The PCR profile used is as follows: 96° C for 2 min, 1
step of 60° C for 1 min, extension step of 72° C for 1
min, 60 cycles; final extension step of 72 ° C for 2
min.

All PCR products were loaded in 1% agarose gelelectrophoresis, and the target band was taken forfurther extraction using the QIAEX II Gel Extraction
Kit (QIAGEN). The extracted products were used forligation at 4° C for 16 hours with the pGEM-T Easy
Vector (Promega). Ligations and transformations

were prepared according to the protocol provided by

the manufacturer. Ten microliters of ligation mixture
was mixed with 100 μl of competent cells (Escherichia
coli XL-1 Blue), incubated on ice for 30 min, heatshocked (42° C for 45 sec), then chilled again on ice
for 3 min. Transformation E. coli cells were then put

into 900 μ l of LB liquid medium and incubated for
30 min to 1hour in a shaking incubator.
Transformed Escherichia coli was spread onto 25

mL agar plates (LB medium, including 50 μ g/mL
ampicillin and 20 μ g/mL 5-bromo-4-chloro-3-

indolyl-β-D-galactopyranoside (X-Gal), 1 mM of40

H. Ehara et al.

Isopropyl β-D-1-thiogalactopyranoside (IPTG)) and

Results and Discussion

Q water and used for colony PCR, being spread ontoresult of sequencing, the insert was confirmed atincubated at 37 ° C overnight. Five white colonies

All of the PCR products using 5S F and 5S R

were selected from each plate and suspended in Milli-

primers were at approximately 500 bp. From theFifty microliters of reaction mixture of colony PCR

salomonense, at 489–512 bp in M. vitiense, at

an agar plate for preparing a master plate.

was prepared as follows: 15 μ l of colony-suspended

499–500 bp in M. amicarum, at 508–509 bp in M.

503–509 in M. warburgii, at 493–510 in M. paulcoxii,

water, 2.5 μ L of 10× HybriPol Buffer, 0.75 μ L of 50

and at 501–514 in M. sagu. The ends of the 5S spacer

DNA polymerase (Bioline). The colony PCR profilein this study were deposited in public DNA

mM MgCl2, 2 mM dNTPs (each), 10 μ M of M13

forward and reverse primers, and 0.25 μ L of HybriPol

was as follows: 96° C for 5 min, 1 cycle; denaturing
step of 96° C for 0.5 min, annealing step of 55° C for

0.5 min, extension step of 72° C for 2 min, 35 cycles;

final extension step of 72 ° C for 2 min. The PCR

products were loaded in 1% agarose gelelectrophoresis to select some colonies that produced

the target product. Some selected colonies were usedfrom the master plates for additional procedures. The

selected colony was suspended in LB medium
including 50 μ g/mL ampicillin and then incubateda t 37 ° C overnight in the shaking incubator.

Subsequently, plasmid DNA was isolated using the
PureLink Quick Plasmid Miniprep Kit (Invitrogen).

The isolated plasmid DNA was used as a template (2μ L) for cycle sequencing using ABI PRISM BigDye

Terminator Cycle Sequencing Ver.3 with M13 forwardor M13 reverse primers (1 μ l of 2.5 μ M primer). The
products of the cycle-sequencing reaction wereanalyzed using an ABI PRISM 3100 Genetic Analyzer.
Raw sequence data files were assembled and editedusing the Clustal X 2.0.10 provided by Des Higgins,
the Conway Institute, UCD Dublin.
Cladistic Analysis

Cladistic analysis was conducted using the

PHYLIP (ver. 3.6) software package (Felsentein2001), and an unrooted dendrogram was constructedusing the neighbor-joining (NJ) method. The cluster
dendrogram was drawn using the NJ pot program
developed by Perrière and Gouy.

were readily identified using published data (e.g.,

Udovicic et al., 1995). All of the sequence data used
databases under Accession Numbers LC312133 to

LC312143. The average length of the 5S spacer of thegenus Metroxylon was 301 bp (SD # 12.8, range #288–326).

Figure 2 shows the results of the phylogenetic

analysis using DNA sequence data from thenontranscribed spacer of 5S nrDNA to investigate

phylogenetic relationships among the species ofsections Metroxylon and Coelococcus in the

Metroxylon genus. In the NJ dendrogram from thecladistic analysis, the clade of section Metroxylon wasapparently distant from the species of the section
Coelococcus. Palms belonging to the Calamoideae
subfamily of the Arecaceae, including the genus

Metroxylon, produce fruits that are covered withscales. The Metroxylon sagu produces fruits with 18

rows of longitudinally arranged scales withapproximately 5 cm at the longest diameter (Ehara etal. 1998). The other species in the section
Coelococcus bear large fruits covered with 24 to 28

rows of scales 7 to 11 cm in diameter (Ehara 2015a).
The Metroxylon sagu can be propagated from bothseeds and suckers; however, the germination

percentage of seeds is very low (Ehara et al. 1998).
The Metroxylon sagu produces the largest amount ofstarch (310 kg dry starch per plant), depending on its

higher dry matter percentage in pith (41%) and starchconcentration on the basis of pith dry matter (77%)
among Metroxylon palms (Ehara 2015a). The starchyield, dry matter percentage in pith, and starchconcentration in the Coelococcus palms are 60–160

Phylogenetic Study of Metroxylon Palms in Southeast Asia and Oceania Based on 5S nrDNA Spacer Sequence Data

0.01

41

M. paulocoxii
M. warburgii 2
M. warburgii 1
M. salomonense 1
M. salomonense 2
M. vitiense 2
M. vitiense 1
M. amicarum 2
M. amicarum 1
M. sagu 2
M. sagu 1

Fig 2. NJ dendrogram based on sequence data of 5S nrDNA in the Metroxylon
kg/plant, 16–33%, and 27–49%, respectively, on

amicarum grown in Micronesia was geneticallynot produce suckers. Instead, the germination

amicarum grows lateral inflorescences from leaf axils,

average in each species. Coelococcus palms areapparently different from the M. sagu because they dopercentage of seeds is very high in the section

Coelococcus, and those palms depend for theirpropagation only on seeds (Ehara 2015a). Such a highstarch concentration in the pith of the M. sagu may be

distant from other species in the section Coelococcus.
Regarding inflorescence characteristics, only M.

while the other four species in the section
Coelococcus and M. sagu produce a terminal

racemose inflorescence as well. The lateral
inflorescence of M. amicarum is pleonanthic

related to its propagation style; this species will

(polycarpic); however, the terminal inflorescence ofpalms that depend entirely on seed germination. As

other species in inflorescence development.

mainly depend on vegetative propagation as comparedwith the propagation of other species of Coelococcus
described above, there are distinct differences in

the other species is hapaxanthic (monocarpic). In thismatter, M. amicarum is markedly different from theMetroxylon salomonense and M. vitiense clearly are atmorphological and physiological characteristics

or near the purely hapaxanthic definition, and they are

study, M. sagu folk varieties were located in distantMetroxylon salomonense and M. vitiense were

between M. sagu and Coelococcus palms.

In the NJ dendrogram constructed by the current

positions, such as outgroups of Coelococcus palms.

Besides, the folk varieties of M. sagu used in thisstudy, namely ‘Roe’ and ‘Rui’ in Southeast Sulawesi,
are a spineless and spiny type, respectively. It isbelieved that Southeast Sulawesi is comparable to thefolk variety ‘Molat’ of the spineless type and ‘Tuni’of the spiny type in the Maluku Islands, Indonesia,
respectively.

The section Coelococcus was divided into four

clades in the current dendrogram. Metroxylon

intermediate, tending to be closer to the hapaxanthic
than to the pleonanthic condition (McClatchey 1999).

located next to each other in the dendrogram of thisstudy. McClatchey (2002) pointed out that M.

salomonense and M. vitiense share the characteristicsof having: a) a flattened leaf sheath or petiole spines
on juvenile and sometimes on adult leaves; b)

secondary branches (of inflorescence) that are held
horizontally or pendulously to the ground; and c)rachillae that are held horizontally or, more often,
pendulously to the ground. It was, therefore,
considered that there was a comparatively closeH. Ehara et al.

42

relationship between M. salomonense and M. vitiense.

in agreement with a former study based on asection Coelococcus formed their own clade.

M. amicarum is possibly an aboriginal introduction to

Metroxylon warburgii formed a clade together with

M. paulcoxii in this analysis. The other species of theMetroxylon paulcoxii was reported by McClatchey(1998) as a new species. However, it has been

observed that the branching pattern of M. paulcoxii ismostly to the second order and sometimes to the thirdorder (Ehara 2015a). The clade consisting of M.
warburgii and M. paulcoxii was located next to M.

salomonense

in

this

analysis.

Metroxylon

salomonense also has variation in that its

inflorescence branches to the third order or the second
order even in a single inflorescence (Ehara et al.
2003b). Dowe (1989) reported that the inflorescenceof M. salomonense branches to the second and thirdorders. Considering these reports, M. salomonense

and M. paulcoxii are considered to sharecharacteristics of variation in their branching patterns.

McClatchey et al. (2006) stated that M. paulcoxii ispossibly an aboriginal introduction from the Santa

Cruz Islands (Solomon Islands) via Rotuma (Fiji) as acultivar of M. warburgii. Metroxylon warburgii iscalled ‘niu o lotuma’ in the local language of Samoa,
a name that means “palm from Rotuma.” According

to Whistler (2000), M. warburgii was originallynamed M. upoluense, which implies that it is native tophylogenetic analysis using morphologicalcharacteristics (McClatchey 1999). It is supposed that

Pohnpei from the Santa Cruz Islands (McClatchey
1998, 2002). A supposition about the dispersal routeof the aboriginal introduction of M. warburgii (and M.
paulcoxii) to Samoa from or via Rotuma is also

considered, as above. Moreover, Tucker (1998) notedthat intact M. sagu trunks are not uncommonly

washed ashore in north Queensland, Australia. Dowe
(1989) wrote that for the genus Metroxylon,

distribution through whole plants being dislodged andcarried on the ocean’s currents should be considered.
In the case of M. amicarum, the seeds are

disseminated by gravity or water, and fruits are often

found on the shores of the Caroline Islands, having
been carried by ocean waves and currents from one
island to another or one part of an island to another(McClatchey et al. 2006). As McClatchey (2002)
stated, since people extensively use these palms
throughout their current distribution, it is difficult toestimate which parts of their ranges are natural andwhich have resulted from human introductions. The
results of this study show some understanding toward
the genetic relationship among the Metroxylon palms,
including sections Metroxylon and Coelococcus.

Samoa, which it is not; it probably was introduced to

Genetic studies of the range of variation within

his recent publication that M. paulcoxii and M.

phylogeny of the genus Metroxylon.

Samoa in modern times, almost certainly from

Rotuma. Furthermore, Whistler (2009) described inupoluense are synonyms for M. warburgii. The

genetic relationship between M. paulcoxii and M.

warburgii should be studied in detail, employing

populations of each species will be a further subject

that will deepen our comprehension about the
Acknowledgments

This study was supported by JSPS KAKENHI

further analysis from the molecular phylogenetic point

Grant Numbers JP14405035, JP20405020, andIslands, Rotuma, and Vanua Levu of Fiji and Samoa.

the Secretariat of Agriculture, Forestry and Fisheriesof view. Besides, the distribution area of M. warburgii
is very wide, from Vanuatu close to the Solomon

From this analysis, it can be considered that

Coelococcus palms were dispersed by two routes: one
to the north and Micronesia, and the other route to theeast to Polynesia through Melanesia. This is generally

JP15H05245 and by the Global-Scale Issue
International Research Network Project launched by

Research Council, MAFF of Japan, to whom we
express our gratitude.

Phylogenetic Study of Metroxylon Palms in Southeast Asia and Oceania Based on 5S nrDNA Spacer Sequence Data

References

Baker, J. B., Henderson, T. A. and J. Dransfield 2000
Molecular

14:218–231.

Phylogenetics

andEvolution

Barrau, J. 1959 Economic Botany 13:151–159 .
Barrow, S. C. 1998 Kew Bulletin 53:513–575.

Beccari, O. 1918 Annals of the Royal Botanic
Garden, Calcutta 12:156–195.

Chase, M. W. and H. H. Hrold 1991 Taxon
40:215–220.

Cox, A. V., M. D. Bennett and T. A. Dyer 1992
Theoretical and Applied Genetics 83: 684–690.

Dowe, J. L. 1989 Palms of the South-West Pacific.

Palm and Cycad Societies of Australia (Milton)
1–154.

Ehara, H. 2015a Taxonomy. In: The sago palm: thefood and environmental challenges of the 21stCentury (The Society of Sago Palm Studies ed.).
Kyoto University Press (Kyoto) 1–16.

Ehara, H. 2015b Geographical origin, dispersal anddistribution of true sago palm. In: The sago palm:

the food and environmental challenges of the 21stCentury (The Society of Sago Palm Studies ed.).
Kyoto University Press (Kyoto) 17–22.

Ehara, H. 2015c Use of fruit. In: The sago palm: thefood and environmental challenges of the 21stCentury (The Society of Sago Palm Studies ed.).
Kyoto University Press (Kyoto) 304–307.

Ehara, H., C. Komada and O. Morita 1998 Principes
42: 212–217.

Ehara, H., H. Naito, C. Mizota and P. Ala 2003a Sago
Palm 10:64–72

Ehara, H., H. Naito, C. Mizota and P. Ala 2003b Sago
Palm 11:14–17

Jones, D. L. 1995. Palms. Reed Books (Chatswood)
pp.410.

McClatchey, W. C. 1998 Novon 8:252–258

McClatchey, W. C. 1999 Memoirs of The New York
Botanical Garden 83:285–306.

McClatchey, W. C. 2002 Biogeographical observationof Metroxylon section Coelococcus. In: New

Frontiers of sago palm studies (Kainuma, K. et al.

43eds.). Universal Academy Press (Tokyo) 119–126.

McClatchey, W. C., H. I. Manner and C. R. Elevitch

2006 Metroxylon amicarum, M. paulcoxii, M. sagu,
M. salomonense, M. vitiense, and M. warburgii(sago palm). Traditional Tree Initiative—Species

Profiles for Pacific Island Agroforestry(www.traditionaltree.org).

Mitchell, A. and J. Wen 2005 Systematic Botany 30:
872–886.

Rauwerdink, J. B. 1986 Principes 30:165–180

Takamura, T. 1990 Japanese Journal of Tropical
Agriculture 34:51–58. (in Japanese)

Tucker, R. 1988 Palms of sub-equatorial Queensland.

Palm and Cycad Societies of Australia(Townsville) pp.94.

Udovicic, F., G. I. McFadden and P. Y. Ladiges 1995
Molecular Phylogenetics and Evolution 4:247–256.

Watling, D. 2005 Palms of Fiji. Environmental
Consultants Fiji (Suva) pp.192.

Whistler, A. 2000 Plants in Samoan culture. Isle
Botanica (Honolulu) pp.234.

Whistler, A. 2009 Plants of the Canoe people.

National Tropical Botanical Garden (Honolulu)
pp.241.