Jpn. J. Trop. Agr. 50 (3):121-126, 2006

Morphology of Pollen Grains Obtained from Dried Specimens of Spiny and

Spineless Sago Palms Grown at Distant Sites in Indonesia

Hiroshi EHARA1*, Madeline M. HARLEY2, William J. BAKER2, John DRANSFIELD2,

Hitoshi NAITO3 and Chitoshi MIZOTA4

1 Graduate School of Bioresources, Mie University2 Herbarium

, Royal Botanic Gardens, Kew3 College of Life Science , Kurashiki University of Science and The Arts4 Faculty of Agriculture , Iwate University

Abstract The morphology of the pollen grains obtained from dried specimens of one spiny and one spineless folk varieties

grown at distant sites, West Papua (Irian Jaya) and West Sumatra, in Indonesia was examined to investigate intra-specific differences in sago palm (Metroxylon sagu Rottb.). The two folk varieties displayed both perfect (hermaphrodite) and male

(staminate) flowers on a single palm. The two flower types did not differ in size in either folk varieties and the gynoecium was reduced to a pistillode in the male flower. Pollen grains were produced from hermaphrodite and staminate flowers in both folk varieties, and were examined by SEM. The pollen grains were equatorially disulcate, i.e. they exhibited two germination apertures on the short equatorial axis of the pollen grains. In vitro, the general shape of the pollen grains was ellipsoid. There were variations in the pollen grain shape from the hermaphrodite and staminate flowers in both folk varieties, presumably due

to the level of contraction or expansion of the pollen grain wall (exine). However, there were no distinct differences in the shape between the pollen grains produced from hermaphrodite or staminate flowers in either folk varieties. In the hermaphrodite and staminate flowers of both folk varieties, the tectum of the pollen grains was psilate (smooth), and often

sparsely perforated. We did not record any significant differences in the exine characteristics or, in the pollen grain shape, in the two folk varieties examined.Key Words: Folk variety, Hermaphrodite, Metroxylon sagu, SEM observation, Staminate flower

Introduction

Sago palm (Metroxylon sagu Rottb.) that is

distributed in Southeast Asia and in areas inhabited by

Melanesian people grows in swampy, alluvial and peaty

soils where almost no other crops can grow without

drainage or soil improvement (Sato et al., 1979; Flach,

1977; Jong, 1995). This palm species stores a large

amount of starch in the trunk, approximately 300kg

(dry wt.) per palm (Ehara, 2005) and has long been

cultivated like banana and taro (Barrau, 1959;

Takamura, 1990) . Sago palm as a staple food continues

to be important in some areas of Southeast Asia and in

areas inhabited by Melanesian people (Ehara et al.,

2000). The carbohydrate (starch) can be further

processed into various basic raw materials for food,

animal feed and for industrial uses. Sago palm is one

of the most important crops for sustainable agriculture

and for rural development in swampy areas of the

tropics.

Received Feb. 2, 2006Accepted Jun. 3, 2006* Corresponding author

Kurimamachiya-cho, Tsu 514-8507, Japan ehara@bio.mie-u.ac.jp

Morphological data are generally useful to identify

plant species and varieties. Sago palm had previously

been classified by the spine character, i.e. absence or

presence of spines and their length and density

(Rauwerdink, 1986) . However, spines occasionally

emerge in seedlings produced from seeds of the

spineless sago palm (Ehara et al., 1998), and Jong

(1995) reported the opposite case, namely that not only

spiny seedlings but also spineless seedlings grew from

seeds of spiny sago palm. Since the identification of

sago palm varieties using only presence or absence of

spines and their characteristics may not be reliable,

attention should be paid to other characteristics. Floral

and pollen grain morphological studies are important

for plant taxonomy, systematics, evolution and phylogeny.

There are several studies on the floral morphology in

the Metroxylon section Coelococcus: Metroxylon warburgii

Heim. (Uhl and Dransfield, 1987) , M. vitiense Benth. et

Hook. (Tomlinson, 1990), and M paulcoxii McClathey

(M. upoluense Becc.) (McClathey, 1998). Rauwerdink

(1986) had examined the pollen grain morphology of

four species in section Coelococcus (M amicarum

Becc., M. vitiense, M salomonense Becc. and M

warburgii) and one species in section Metroxylon

122 Jpn. J. Trop. Agr. 50 (3) 2006

(Eumetroxylon) (M. sagu: section Metroxylon consists of

only M sagu). However, there are few studies on the

floral and pollen grain morphology in section Met roxylon

and information about the pollen grain morphology of

sago palm (M sagu) varieties is very limited. Therefore,

in the present study we examined the morphology of

the pollen grains obtained from dried specimens of one

spiny and one spineless sago palms grown at distant

sites, West Papua (Irian Jaya) and West Sumatra, in

Indonesia to investigate intra-specific differences in

Metroxylon sagu. Besides, morphological characters of

the flowers were also observed.

Material and Methods

Flowers at the pre-anthesis stage were taken from

herbarium specimens of a spiny folk variety (20

flowers) grown in Fakfak, West Papua (Irian Jaya),

(Baker 881 (K)) and a spineless folk variety `Rumbio'

(30 flowers) grown in Padang, West Sumatra (Edison, Naito and Mizota s.n.) . The term `folk variety' was

used instead of variety in the present report, based on

the conclusion drawn at the Eighth International Sago

Symposium (EISS 2005 held in Jayapura, Indonesia on

4 - 6, August 2005), according to which different types

of sago palm that show variable specific characters and

are locally recognized as varieties or accessions,

should be designated as folk varieties until significant

evidence of specification is gathered. In case of the

spiny folk variety, flowers were collected from the mid-

2nd branch (80cm length) attached to the 1st branch

(1.5m length) in the inflorescence. Flower samples

were collected from the mid-2nd branch attached to

the mid-1st branch that was cut from the middle

position of the inflorescence in the spineless folk variety. The calyx, two petals and three stamens were

removed under a dissecting microscope, to observe

the presence or absence of a gynoecium, then the

flowers were identified as either hermaphrodite or

staminate.

Pollen grains were obtained from hermaphrodite

and staminate flowers of both folk varieties, and were

examined by scanning electron microscopy (SEM).

Samples were prepared according to the acetolysis

protocols for palm pollen used in the Palynology Unit of the Herbarium at the Kew Gardens (Harley, 1990).

Results and Discussion

A flower of the spiny folk variety at anthesis is

shown in Fig. 1. Sago palm flowers of both spiny and

the spineless folk varieties have three sepals and three

petals, as in M, warburgii (Uhl and Dransfield, 1987)

and M vitiense (Tomlinson, 1990). Metroxylon bears

both perfect (hermaphrodite) and male (staminate)

flowers on a single palm (Tomlinson, 1990). Since it

was difficult to identify hermaphrodite or staminate

flowers by the external appearance, the calyx, two

petals and three of the six stamens were removed from the flowers, preserved at the pre-anthesis stage, for

observation. Of the twenty flowers examined for the

spiny folk variety, ten were hermaphrodite and ten

were staminate. In the spineless folk variety, eighteen

flowers were hermaphrodite and twelve were staminate.

Fig. 2 shows the hermaphrodite flowers with removed

calyx, two petals and three stamens. Staminate flowers

with removed calyx, two petals and three stamens are

shown in Fig. 3. According to Tomlinson (1990), the two

flower types do not differ in size and the gynoecium is

reduced to a pistillode in the male flower in the

Metroxylon section Coelococcus. These characteristics

were also observed in each of the folk varieties used in

the current study (cf. Figs. 2 and 3). The component

parts of each flower in both folk varieties were closely similar to those of the species in section Coelococcus

(Uhl and Dransfield, 1987; Tomlinson, 1990).

Pollen grain morphology

Pollen grains were produced from both hermaph-

rodite and staminate flowers in both samples of the

studied folk varieties of M sagu. The pollen grains

were equatorially disulcate, i.e. they exhibited two

germination apertures on the short equatorial axis of

the pollen grain (Fig. 4). In vitro the general shape of

the pollen grains was ellipsoid. Our results were in

agreement with the findings of Rauwerdink (1986).

However, according to Schuiling et al. (1993), Tuwan

(1991) observed varietal differences in the pollen grain

shape between three spiny varieties grown in Ambon,

Maluku, Indonesia (differences between spiny folk

varieties in the strict sense according to the

symposium conclusion from EISS 2005). Of the three

varieties (three folk varieties), Tuwan (1991) indicated

that the pollen grain of Tuni was elliptic, while that of

Makanaru was round and that of Ihur triangular. Jong

(1995) examined the pollen grain morphology of sago

palms grown in Sarawak, Malaysia and reported that

the pollen grains were mostly oval and, occasionally,

round in the spineless type, and consisted of a mixture

of oval and triangular forms in the spiny type. According

to him, it remained to be determined whether the

triangular pollen grains were defective or deformed by

Ehara et al.: Morphology of Pollen Grains of Sago Palm 123

dehydration. Our own data demonstrated that the

variable pollen grain shape from the hermaphrodite

and staminate flowers of both folk varieties was due to

the level of contraction or expansion of the pollen grain

wall (exine). Fig. 5 shows, schematically, the appearance

of a fully expanded pollen grain in different planes,

(5A, B, Ci, Cii); the triangular appearance of the pollen

grain in alternative equatorial planes should also be

noted (5B, Civ). Fig. 4 (A-D) and Fig. 6 (A-B) show the

pollen grains of the spiny and spineless folk varieties in a slightly oblique distal polar plane (cf. Fig. 5 Ciii).

There was no distinct difference in shape between the

Fig. 1 Photomicrograph of a flower of the spiny folk

variety at anthesis.

A, anther; Pe, petal; C, calyx.

Fig. 2 Photomicrograph of a hermaphrodite flower of the

spiny (left) and the spineless (right) folk varieties. Pe, petal; A, anther; G, gynoecium (i.e. pistil).

Fig. 3 Photomicrograph of a staminate flower of the spiny

(left) and the spineless (right) folk varieties. Pe, petal; A, anther; Pi, pistillode.

Fig. 4 Electronmicrographs of pollen grains produced from A) hermaphrodite and B)

staminate flowers of the spiny folk variety; C) hermaphrodite and D) staminate flowers

of the spineless folk variety •eRumbio•f (pollen grains in slightly oblique distal polar plane).

124 Jpn. J. Trop. Agr. 50 (3) 2006

Fig. 5 Schematic representation of the appearance of a fully expanded equatorial disulcate pollen grain in different planes. A: fully expanded disulcate pollen grain, equatorial plane, long axis , B: fully expanded disulcate pollen grain, equatorial plane, short axis, Ci: fully expanded disulcate pollen grain , polar plane, distal face, Cii: fully expanded disulcate pollen grain, polar plane, proximal face, Ciii: equatorial disulcate pollen grain, aperture margins infolded, oblique polar plane, distal face, Civ: equatorial

disulcate pollen grain, aperture margins infolded, slightly oblique equatorial plane, long axis, Ei:

position of polar (P) and equatorial (E axes, equatorial disulcate pollen grain, equatorial plane, long axis, Eii: position of polar (P and equatorial (E) axes, equatorial disulcate pollen grain, equatorial

plane, short axis, Eiii: position of equatorial (E) axis, equatorial pollen grain, polar plane, distal face.

Fig. 6 Electronmicrographs of pollen grains produced from a hermaphrodite flower of the

spineless folk variety •eRumbio•f; A) pollen grain with infolded aperture margins, B)

pollen grain with aperture margins expanded (pollen grain in slightly oblique distal

polar plane).

pollen grains produced from hermaphrodite or staminate

flowers in either folk varieties.

The wall (exine) of the palm pollen grains is

generally more resilient than that in most other monocotyledonous plants, hence its high fossilization

potential (Harley and Morley, 1995). Nevertheless,

during dehydration, or due to acetolysis (this study)

the pollen grains tended to collapse. As a result, in

disulcate pollen grains, such as those in Metroxylon

spp., and most rattan palms, the aperture margins

tended to fold inwards (Fig. 4C-D, Fig. 6A). In

acetolysed (or fossilized) specimens, the delicate

Ehara et al.: Morphology of Pollen Grains of Sago Palm 125

membrane covering the aperture (Fig. 4B) was usually

destroyed (Fig. 6B). In an extensive survey of palm

pollen based on light microscopy, Sowunmi (1972) recorded measurements of exine thickness for M

amicarum, M salomonense and M warburgii. She

commented that the exine was notably thicker than

that in the other palm taxa she examined. Ferguson

and Harley (1993) who described the pollen grains of

M amicarum, M sagu, M salomonense and M

upoluense Becc., stated that the exine was very thick.

Harley (1996) described and discussed the incidence

of very thick exines of palm pollen grains. The exines

of M. sagu and M. amicarum were included in her

exine ultrastructure Group 1A: palm species with

outstandingly thick exines. In the same study, the

exine of M vitiense was included in exine ultrastruc-

ture Group 1C, a similarly proportioned but thinner

exine. Interestingly, in the palms, the very thick

Group lA type exine is unusual but systematically

widespread. Apart from subfamily Calamoideae, as in

Metroxylon, this type was observed in a few species of

Daemonorps, a rattan palm, as well as in the

subfamilies Coryhoideae (Pritchardia) and Arecoi-

deae: Arecaceae, subtribe Iguanuriae (Harley, 1996).

In the hermaphrodite and in the staminate

flowers, of both folk varieties, the tectum of the pollen

grains was psilate (smooth), and often sparsely

perforated (Fig. 4, Fig. 6), which is in agreement with

the findings of Rauwerdink (1986) for M. amicarum,

M sagu and M warburgii. Harley (1996) illustrated the

completely psilate pollen grain of M. sagu. Rauwerdink

(1986) reported a reticulate exine for the equatorial faces of M salomonense and M vitiense. Ferguson and

Harley (1993) observed and illustrated an extremely

coarse reticulate exine for M salomonense.

Sago palm formae had previously been classified

by the spine character (Rauwerdink, 1986), while

spines occasionally emerged in the seedlings produced

from the seeds of the spineless sago palm (Ehara et al.,

1998) . In contrast, spineless seedlings were occasionally

produced also from the seeds of the spiny sago palm

(long, 1995). Since the identification of sago palm varieties using only the presence or absence of spines

as a character is not reliable. Other morphological

characters should be used to identify a variety of

formae in sago palm. However, as described above, we

did not record any significant differences in the exine

characteristics or in the pollen grain shape, in the two

folk varieties examined. Variations in the pollen grain

shape were probably due to previous misinterpretation.

Needless to say, from the data based on such a small

sample, we could not draw final and meaningful

conclusions as to whether morphological variations of the

floral parts might become a valuable character set for

distinguishing genetic groups of sago palm. Neverthe-

less, the results obtained so far suggest that there was

no distinct difference in the pollen grain morphology

between the spiny and spineless folk varieties grown at

isolated sites separated by a distance of more than

3500km in a straight line.

Acknowledgments

This article is contribution No. 34 from the

Laboratory of Crop Production and Ecology, Mie

University. We are indebted to Dr. Edison Munaf,

Andalas University for his co-operation in the

collection of the specimens. This study was supported

by the Anglo-Japanese Scientific Exchange Programme

between the Royal Society and the Japan Society for

the Promotion of Science, to which we express our

gratitude.

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(*: in Japanese with English summary, **: in Japanese)

イン ドネシアの遠隔地 に生育す る有刺 ・無刺サ ゴヤシ標本 か ら

採取 した花粉 の形態

江 原 宏1*・Madeline M.HARLEY2・WilliamJ.BAKER2・John DRANSFIELD2・

内 藤 整3・ 溝 田 智 俊4

1三 重大 学大 学院 生物 資源 学研 究科

2Herbarium, Royal Botanic Gardens, Kew

3倉 敷芸 術科 学大 学生 命科 学部

4岩 手大 学農 学部

要 約 サ ゴヤ シ(Metyoxylon sagu Rottb.)の 種 内形 質 変異 を検 討す るため,イ ン ドネ シアの西 パ プ ア(イ リア ンジ ャヤ)に

生育 す る有 刺folk varietyお よび西 ス マ トラに生育 す る無 刺folk varietyの 乾燥 標 本 か ら得 た花 粉 の形 態 を調 査 比較 した.両

folk varietyと も1個 体 に両性 花 と雄 花の両 方 を着 生 し,い ずれ のfolk varietyで も両性 花 と雄花 の サ イズ に大 き な違 い はな く,

雄花 で は雌蕊 は退 化 して い た.両 性花 と雄花 の 両方 と も花 粉 を生 じ,SEM観 察 した ところ,花 粉 粒 は短赤 道軸 上 に2つ の 発

芽孔 を有 す る長楕 円形 で あ った.花 粉形 状 に変異 がみ られ たが,そ れ は花 粉 壁(エ キ シ ン)の 収縮 や膨 張 の程度 の違 い に よ る

もの と考 え られ た.ま た,両folk varietyの 両 性花,雄 花 と も花粉 粒 のテ ク タム は滑 らかで疎 らに穴 が散在 してい た.こ の よ

うに,花 粉 の外壁 形 質,花 粉 の形 状 な どいずれ も両folk variety間 に差 はみ られ ない ことが 明 らか とな った.

キー ワー ド:雄 花,両 性花,SEM観 察,Folk variety, Metroxylon sagu

*Corresponding author

〒514-8507三 重 県 津 市 栗 真 町 屋 町

ehara@bio.mie-u.ac.jp