SAGO PALM 22: 6−12 (2014)

Comparison of species composition under different light
conditions in sago forests
Yoshimasa Kumekawa1,*, Agustinus Murjoko2, Hiroshi Hayakawa1, 3,Makito Mori1, Akira Miyazaki1, Katsura Ito1, Ryo Arakawa1,Tatsuya Fukuda1, Hubertus Matanubun2 and Yoshinori Yamamoto1
1

Faculty of Agriculture, Kochi University, Monobe B200, Nankoku, Kochi 783-8502, Japan2

3

The State University of Papua

National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, 305-8604, Japan*Corresponding author

Key words: chloroplast DNA (cpDNA), flora, sago palm
Introduction

In Indonesia, the sago palm, Metroxylon sagu

Rottb. (Arecaceae), plays an important economic andcultural role, and it has various advantageous featuresof a crop species, as it is economically acceptable,
relatively

sustainable,

andsuitableforenvironmentally sound agro-forestry systems(Yamamoto, 2005). Although natural sago stands arefound in several areas of Indonesia, spread overlowlands, coastal regions, and river deltas, many sago
forests have been destroyed through commercial sago

extraction, irrigation schemes, and conversion for wet
rice production.

Little is known of transitions in the understory of asago palm forest after its initial development. Thesago palm has large pinnate compound leaves with anaverage size of approximately 10 m 2 per leaf

(Nakamura et al., 2009). The shading of these leaves

creates a dark environment in a sago palm forest. Infact, we could observe few species in a sago forest inIndonesia, and many plant species we could find werein the gap around fallen dead trees. From theseobservations, it appears that many plant species would

increase by destroying the sago palm forests as thatwould allow light to reach the floor of the forests.

Sago forests in Indonesia have been destroyed

because of both extreme development projects andcontinuing rubber and oil palm expansions (Okamoto,2000). The variation in solar environments in the sago
forests is considered to affect the growth of many

plant species. It is important to compare the plant
species colonizing the different light conditions
during sago forest development in order to monitor

the invasion of alien plant species that pose a threat tosago conservation. However, most plant species areoften seedlings, and they cannot be determined based
only on morphological characteristics.

DNA barcoding is a technique for characterizing

species by using a short DNA sequence from astandard and predetermined position in the genome(Hebert et al., 2003). Relative to the entire genome,

DNA barcode sequences are very short for identifying
an unknown sample in terms of a preexistingclassification (Kress et al., 2005). Therefore, DNAbarcodes are sometimes used in an effort to identify

unknown species (Koch, 2010). For example, thecytochrome oxidase subunit I (COI) gene inmitochondrial DNA (mtDNA) is emerging as thestandard barcode region for insects and other animals(Hebert et al., 2003); however, the use of the COI

sequence is not appropriate for most species offlowering plants due to the much slower rate of COI

Comparison of species composition under different light conditions in sago forests

7

gene evolution in angiosperms (Kress et al., 2005).

However, some chloroplast DNA (cpDNA) or nuclear

DNA (nrDNA) sequences have a relatively fast

mutation rate, which results in significant variation inthese sequences between and/or within species; thesehave been proposed as potential barcodes (Fukuda etal., 2005; Kress et al., 2005; Kress and Erickson,
2008; Hayakawa et al., 2010; Hayakawa et al., 2011;

Kumekawa et al., 2013). Thus, the aim of our study

was to identify plant species that grow in different
solar environments during transition in theunderstories of sago palm forests using molecularanalyses based on two cpDNA genes, i.e., the rbcL
(ribulose-1,5-bisphosphate carboxylase/oxygenase

large subunit) gene and the trnL (transfer RNAleucine) intron of ferns and angiosperms, respectively.

These genes are important tools for molecular
identification of land plants (CBOL Plant WorkingGroup, 2009; Taberlet et al., 2007).
Materials and Methods
Plant materials

All plant materials used in this study were collectedfrom one sago palm stand (1'95"S, 132'25"E) in the
Malay Archipelago (Fig. 1). Within the study area, we2.ȱ Eight quadrates
study.
ȱ ȱFig.
ȱȱ ȱ ȱ ȱ of
ȱ A,ȱ B,ȱ Cȱ andȱ D inȱ thisȱȱPhotographed in A- 1 (dark), B-1 (bright),ȱȱ ȱ(dark),ȱ Cȱ (bright),
ȱ B-1(bright),ȱ ȱ
B-2ȱ (bright),B-3
B-2 (bright), D-1 (bright), D-2 (bright) andȱ D-3(dark).
ȱȱȱȱ

forest and represented dark environments; B-1, C, and
D-1 were in an open field and represented brightenvironments (Fig. 2). B-2 and D-2 had low ratios ofadult sago palms with high ratios of the rosette stage(Fig. 2). The conditions in these intermediate quadrats
were relatively bright, and so our study was includedin the “bright environment” category.
DNA analyses

Total DNA was isolated from approximately 200-

Fig. 1. Sampling area of this study.

300 mg of an air-dried leaf using a Plant Genomic

11 quadrats (four in A, three in B, one in C, and three

DNA was resuspended in Tris-EDTA (TE) buffer andselected four localities (A, B, C, and D), constructedin D) of 10 m x 10 m based on the brightness in eacharea (Fig. 2), and collected all plant species withinthem. Among these quadrats, A-1, A-2, A-3, A-4, B-3, and D-3 had high ratios of adult sago palms in aȱȱDNA Mini Kit (Viogene, Sunnyvale, USA) inaccordance with the manufacturer’s protocol. Isolated
stored at -20 ° C until use. DNA amplification bypolymerase chain reaction (PCR) was carried out in a50-μ L reaction volume containing approximately 50
ng of total DNA, 10 mM Tris-HCl buffer (pH 8.3)

ȱ ȱȱ ȱȱȱȱ

Y. Kumekawa et al.

8with 50 mM KCl and 1.5 mM MgCl2, 0.2 mM of eachaligned with the CLUSTAL X program (Thompson etfollowing thermal cycle profile for amplification: 40

Our results were almost identical to previouslydNTP, 1.25 U Taq DNA polymerase (Toyobo, Osaka,Japan), and 0.5 μ M of each primer. We used thecycles of 1.5 min at 94° C, 2 min at 48° C, and 3 min
at 72° C, followed by a final extension step of 15 min
at 72 ° C. We amplified rbcL and the trnL intron ofcpDNA with primers designed by Hasebe et al. (1994)

and Taberlet et al. (1991), respectively. After

amplification, the reaction products were run on a 1%
agarose gel stained with ethidium bromide anddigitally photographed under UV light.

We also confirmed the sequences of these regions.

After amplification, the reaction mixtures weresubjected to electrophoresis on 1% low-melting-temperature agarose gels for the purification of theamplified products. We sequenced the purified
reaction products using a BigDye Terminator Cycle

al., 1997), and ambiguously aligned regions weremanually corrected to minimize the number of indels.

published sequences, and the samples were assigned

to the corresponding species published in the DNA

Data Bank of Japan (DDBJ). In cases where there
were no identical matches to previously publishedsequences, phylogenetic trees were constructed using
our sequences and highly similar ones based on ahomological search of the DDBJ. Our samples wereassigned to the genus that included plant species of asister group to our sequences in the phylogenetic tree
(Fig. 3). For example, some of our sequences weresimilar to those of some species of Ficus (Moraceae);

therefore, different sequences of this genus werenamed Ficus sp. 1, Ficus sp. 2, and so on (Fig. 4).

Sequencing Kit (Applied Biosystems, California,Results and Discussion

with the manufacturer’s instructions. For sequencing,Stenochlaena sp. (Blechnaceae), Ficus sp. 1 (Moraceae),USA) and a Model 3730A automated sequencer

In total, we identified 70 species in 35 families (Table(Applied Biosystems, California, USA) in accordance

1). Six species, i.e., Nephrolepis sp. (Lomariopsidaceae),amplification. Sequences for each region were pre-(Pandanaceae), and Syzygium sp. (Myrtaceae), were

we used the same primers as those used for

Fagraea volubilis (Gentianaceae), Freycinetia sp.

Fig. 3. Phylogenetic tree of Microsorum (Polypodiaceae) and its allied species. Numbers indicate bootstrap
values. Bolded name indicates individual of our study. Parentheses indicate accession numbers.

Y. Kumekawa et al.

9species, respectively, of thePoaceae and Cyperaceae families
in bright environments. Therefore,
our data supports the report ofWhittaker (1975), and these plants

could become standard indicatorspecies of bright environmental

conditions during the transition ofsago palm forests. In general,grass, such as Poaceae andCyperaceae, is a dominant species
in the secondary succession of theforest (Robertson and Vitousek,1981) and an early successional
species (Tagawa, 1964), suggestingthat locations C and D1 of ourinvestigation are at the secondary
Fig. 4. Phylogenetic tree of Ficus (Moraceae) and its allied species.

Numbers indicate bootstrap values. Bolded names indicate
individuals of our study. Parentheses indicate accessionnumbers.

found in both bright and dark environments, suggestingthat these plants are not dependent on a specific solar
environment during the development of a sago palm
forest. However, the remaining species inhabited

either bright or dark environments; thus, they areconsidered effective species for distinguishingtransitional stages of sago palm forest succession. We

found 23 species (6 ferns and 17 angiosperms) in darkenvironments and 53 species (10 ferns and 43angiosperms) in bright environments, indicating thatthe number of plant species in our quadrats decreased

from bright to dark environments. This result supports
a general tendency reported by Whittaker (1975), that

it is a general tendency of succession that the numberof species was large in bright environments and smallin dark environments. Among our collected species,
grasses of the Poaceae and Cyperaceae families grewin intermediate and bright quadrats, but none grew indark environments. In fact, Table 1 shows 6 and 8

succession stage (Table 1).
Considering this general tendencyof change reported by Whittaker(1975), our results suggest thatlocations A1 and A3 are late in
the succession of the forest; inparticular, A3 was at the climax stage. Therefore, ourcomparison of floras among these locations

reconfirms the succession scheme of sago palm
forests from the early to the late stages. Sago palm

forests would also be formed in this pattern if

deforestation has taken place in the region. Moreover,
it is very interesting that the succession of sago palm
forests from the early to the late stages of our study

appears to correspond to a seed dispersal mode fromwind to birds and animals, as many wind-dispersal
species live in bright environments while birds andanimals live in dark ones (Prach and Pyšek, 1999;

Fuller and Moral, 2003). Praseeda and Jeyanth (2010)

reported that invasive alien species include many
grasses, climbers, colonizer shrubs, and trees withwind dispersal seeds; therefore, changes in the solar

environment by sago palm deforestation would pose aserious risk to the habitat of native plant species in theregion.

Comparison of species composition under different light conditions in sago forests

10Table.1 The list of species, accession numbers, and locations. Taxonomic arrangement follows the Engler system.
Black circles indicate the collected quadrates.

Taxon

FamilyPteridophyte
Gleicheniaceae
Pteridaceae

Aspleniaceae
Blechnaceae

Dennstaedtiaceae
Lomariopsidaceae
Polypodiaceae
Angiosperm
Monocotyledoneae
Alismataceae
Dioscoreaceae
Pontederiaceae
PoaceaeAraceae
Pandanaceae
CyperaceaeDicotyledoneae
Choripetalae
Moraceae

Urticaceae
Menispermaceae
Euphorbiaceae

Phyllanthaceae
Rutaceae
Meliaceae
Celastraceae
Rhamnaceae
Malvaceae
Melastomataceae
MyrtaceaeȱȱSpecies

location

Accession No.

Dark
A-1 A-2 A-3 A-4 B-3 D-3

Dicranopteris linearis
Acrostichum sp.
Ceratopteris thalictroides
Pityrogramma austroamericana
Pityrogramma sp.
Pteris sp.
Taenitis blechnoides
Asplenium sp.
Artocarpus sp.
Blechnum orientale
Stenochlaena sp.
Pteridium sp.
Nephrolepis sp.
Microsorum sp.

AB889467
AB889455
AB889456
AB889401
AB889468
AB889457
AB889469
AB889470
AB889451
AB889435
AB889402
AB889440
AB889466
AB889465

Alisma sp.
Dioscorea alata
Pontederia sp.
Centotheca lappacea
Echinochloa frumentacea
Echinochloa sp.
Paspalum conjugatum
Paspalum sp.
Pa. vaginatum
Epipremnum papuanum
Rhaphidophora sp.
Freycinetia sp.
Cyperus sp.
Eleocharis palustris
Erioscirpus sp.
Fimbristylis miliacea
Fuirena umbellata
Schoenoplectus littoralis
Unidentified1
Unidentified2

AB889427
AB889458
AB889426
AB889442
AB889432
AB889425
AB889406
AB889420
AB889405
AB889417
AB889418
AB889410
AB889399
AB889408
AB889407
AB889428
AB889400
AB889462
AB889409
AB889429

Ficus sp.1
Ficus sp.2
Ficus sp.3
Cypholophus sp.
Stephania sp.
Endospermum moluccanum
Homalanthus populneus
H. nutans
Homalanthus sp.
Unidentified
Melicope elleryana
Chisocheton lasiocarpus
Lophopetalum sp.
Alphitonia sp.1
Alphitonia sp.2
Unidentified
Unidentified
Syzygium sp.

AB889414
AB889450
AB889403
AB889412
AB889461
AB889423
AB889448
AB889449
AB889441
AB889443
AB889416
AB889413
AB889453
AB889446
AB889447
AB889459
AB889460
AB889415

䖃䖃Bright
B-1 B-2 D-1 D-2 C䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃䖃
䖃
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Comparison of species composition under different light conditions in sago forests

Taxon

FamilySpecies

location

Accession No.

Dark
A-1 A-2 A-3 A-4 B-3 D-3Angiosperm

11Bright
B-1 B-2 D-1 D-2C

Sympetalae

Apocynaceae

Asclepiadoideae
Gentianaceae
Rubiaceae

Convolvulaceae
Linderniaceae
Phrymaceae
Plantaginaceae
Asteraceae

Alstonia scholaris
Alstonia sp.
Trachelospermum sp.
Unidentified
Fagraea volubilis
Unidentified
Breonia sp.1
Breonia sp.2
Cinchonoideae sp.
Nauclea orientalis
Ipomoea purpurea
Picria fel-terrae
Torenia sp.
Unidentified
Limnophila aromatica
Limnophila sp.
Blumea sp.
Unidentified

AB889439
AB889421
AB889454
AB889411
AB889437
AB889452
AB889419
AB889422
AB889436
AB889438
AB889433
AB889463AB889434
AB889444
AB889404
AB889430
AB889445
AB889431

Our investigation using molecular identification

was able to identify unknown samples without
diagnostic morphological features in terms of apreexisting classification. For example, Ficus is easy

to recognize using a characteristic feature of milky

sap containing latex (Heywood, 1978); however, it䖃
䖃

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䖃
䖃
䖃

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䖃
䖃
䖃
䖃
䖃
䖃
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ȱ

National Academy of Sciences of the United Statesof America 106: 12794-12797.

Fukuda, T., I. J. Song, T. Nakamura, M. Nakada, A.
Kanno, T. Kameya, H. Yamaji, A. Terabayashi, S.
Takeda, M. Aburada and J. Yokoyama 2005
Natural Medicines 59: 91-94.

was very difficult to recognize each fern without

Fuller, R.N. and R. del Moral 2003 Journal ofsilica accumulation in the aerial parts is one of theKato and K. Iwatsuki 1994 Proceedings of thespores, which are important characters for identifying
species in ferns (Iwatsuki, 1995). Moreover, althoughmost prominent characteristics of Poaceae (Jones andHandreck, 1967), it is difficult to identify infrafamilial

Vegetation Science 14: 637-644.

Hasebe, M., T. Omori, M. Nakazawa, T. Sano, M.

National Academy of Sciences of the United Statesof America 91: 5730-5734.

taxa without flowers and fruits of this family. Our

Hayakawa, H., T. Kobayashi, Y. Minamiya, K. Ito, A.

because this knowledge is needed to understand theHayakawa, H., T. Kobayashi, Y. Minamiya, K. Ito, A.

fields and locations, to clarify the succession of sago

Hebert P. D. N., A. Cywinska, S. L. Ball and J. R.

Acknowledgment

Heywood, V.H. 1978 Flowering plants of the world.

genetic information on different species growing insago palm forests is an important research focus

succession of these important forests. Additionalanalyses should be conducted, including in otherpalm forests.

This research was supported by a research grantfrom The Mitsui & Co., Ltd., Environment Fund.
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