Blackwell Publishing AsiaMelbourne, AustraliaPSBPlant Species Biology0913-557X2006 The Society for the Study of Species BiologyDecember 2006213193199Original
ArticlePOLLINATION OF METAPLEXIS JAPONICAH. TANAKAET AL.

Plant Species Biology (2006) 21, 193–199

doi: 10.1111/j.1442-1984.2006.00165.x

Andromonoecious sex expression of ﬂowers and pollinia
delivery by insects in a Japanese milkweed Metaplexis
japonica (Asclepiadaceae), with special reference to its
ﬂoral morphology
HAJIME TANAKA,* TAKEO HATANO,† NORIKO KANEKO,‡ SUGATAKO KAWACHINO,§ OSAMU KITAMURA,¶YURIKO SUZUKI,†† TAEKO TADA‡‡ and YOSHIHITO YAOI§§*2-18-19 Minami-otsuka, Toshima-ku, Tokyo 170-0005, Japan, †4-13-19 Den-en-choufu, Oota-ku, Tokyo 145-0091, Japan, ‡2-22-15
Nisi-siba, Kanazawa-ku, Yokohama 236-0017, Japan, §4-4-8 Fukazawa, Setagaya-ku, Tokyo 158-0081, Japan, ¶2241-1 Naruse,
Machida, Tokyo 190-0044, Japan, ††3-9-23 Maechi, Urawa-ku, Saitama 330-0053, Japan, ‡‡1-6-5 Komagome, Toshima-ku, Tokyo
170-0003, Japan, and §§1-30-1-212 Minowa-cho, Kohoku-ku, Yokohama 223-0051, Japan

Abstract
Pollination ecological studies of Metaplexis japonica Makino were carried out in central
Japan. We found that the ﬂowers were andromonoecious; the proportion of hermaphrodite ﬂowers was 61%. Unlike other Asclepiadaceae ﬂowers, M. japonica has a unique andoutstanding long protrusion on the pistil end, which looks like a stigma. However, we
conﬁrmed that the true stigma is present on the lateral surface of the style, as it is in other
Asclepiadaceae ﬂowers. Various insects visited the ﬂowers for nectar and the most effective pollinators were scoliid wasps. The pollen/ovule ratio was 21 and the fruit set of thehermaphrodite ﬂowers was 11%.
Keywords: andromonoecy, Asclepiadaceae, ﬂoral morphology, insect pollination, Metaplexis
japonica.
Received 11 January 2006; accepted 25 July 2006

Introduction
The asclepiad ﬂower has attracted the attention of ﬂower
ecologists for a long time because of its peculiarity inhaving a united column of stamens and a pistil and apackage of pollen delivered in the form of a pollinium,
making it a dicot counterpart to the well-known orchid
ﬂower. Since Sprengel (1793) described the detailed ﬂoral
structure and the pollination mechanism of Asclepias species, extensive investigations have been made on ﬂowersof Asclepiadaceae (Müller 1873; Knuth 1899; Proctor &Yeo 1973). More recently, quantitative and morphologicalobservations of the pollen delivery of Asclepias have been
made, and factors affecting the fruit set and a unique lateacting self-incompatibility system have been reported(Wyatt & Broyles 1990, 1994). Asclepiad ﬂowers have two
free ovaries, but in most cases only one of the two develops to a mature fruit, and the total fruit set can be as
low as 0.33–5.0% (Wyatt & Broyles 1990, 1994). Electron

Correspondence: Hajime Tanaka
Email: tanaka.hajime@nifty.com

microscopic studies of Asclepias amplexicaulis revealed
that pollen tubes penetrating the stigmatic surface weretransmitted to one of the two ovaries (Tammy et al. 1990).
Metaplexis japonica Makino is a climbing perennial herbof Asclepiadaceae, native throughout East Asia. It is common in open habitats in central Japan. The ﬂowers aredescribed as hermaphrodite (Murata 1981; Fu et al. 1999),
ﬂowering typically in July to September. In contrast to
well studied Asclepias, however, little is known about themechanisms of its pollination. Scoliid wasps (Tanaka
1984) and honeybees (Tanaka 1990) are reported to visit
M. japonica ﬂowers, but their contribution to pollination
remains uncertain. Recently, Sugiura and Yamazaki (2005)
reported that various moths visited the ﬂowers of
M. japonica after sunset and that seven species of moths
carried out the pollinia on their proboscis, but pollination
by other insects in the daytime has not been carefully
observed. Moreover, little is known about the function of
the ﬂoral morphology unique to M. japonica; an unusual
shape with a long protrusion projecting out of the corolla,
which makes a striking morphological difference from theﬂowers of Asclepias. In the taxonomic literature of Japan© 2006 The Authors
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H . TA N A K A E T A L .

e

a

b

c

d

Fig. 1 Japanese milkweed Metaplexis japonica. (a) A hermaphrodite ﬂower, (b) a male ﬂower, (c) a mature fruit, (d) a young fruit and (e)a seed with hairs. Illustrated by S. Kawachino.

and China, this protrusion on the pistil end has been
described as a stigma (Ohwi 1953; Jia & Jia 1955; China
Institute of Botany 1974; Murata 1981; Osada 1981; Fu
et al. 1999). However, other asclepiad ﬂowers lack such a
protrusion and the stigma is present on the lateral surfaceof the style (Müller 1873; Knuth 1899; Proctor & Yeo 1973).
Thus, both the morphological feature of the protrusion
and its role in pollination remain entirely obscure. Therefore, we attempted to elucidate the ﬂoral morphology andpollination biology of M. japonica. As a result, we foundthat M. japonica has male ﬂowers in addition to hermaphrodite ones. In the present paper, we report our ﬁndings
with special reference to the following questions.
12
3
4

Where is the position of the stigma: Is it on the pistil
protrusion or not?
How are the ﬂowers pollinated by insects?
How do male ﬂowers differ from hermaphrodite
ones?
How high are the fruit set and pollen/ovule ratio andis there any difference in these traits between
andromonoecious M. japonica and other asclepiads?

Materials and methods
Overall structures of M. japonica and its ﬂowers are illustrated in Figures 1 and 2. The ﬂowers in racemes have

Fig. 2 A ﬂower of Metaplexis japonica. Corolla lobes of the front
side have been removed. Ah, anther hood; Bp, beak-like protrusion of the pistil end; Fi, ﬁssure; Sl, slit. Illustrated by S.
Kawachino.

ﬁve-lobed, pale violet or white corollas measuring
approximately 1 cm in diameter with a number of whitehairs inside. Follicles are fusiform, 6–10 cm long, andhave hundreds of hairy seeds inside. The ﬂower has two
free, superior ovaries. Two styles unite together in the© 2006 The Authors
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P O L L I N AT I O N O F M E TA P L E X I S J A P O N I C A
upper part and fuse with the outer walls of ﬁve surrounding anthers. Each anther is covered with a cordate membranous hood. Five stamens unite together and surround
two ovaries. Unlike Asclepias, the corona at the bottom of
the stamens is small and the corolla forms a tube.
Observations of ﬂoral characteristics and pollinators,
and experiments examining pollination and pollen germination were carried out from August to October in 2002,
2003 and 2004 in the following locations of central Japan;
Setagaya Ward and Machida City of Tokyo Metropolis,
Yokohama City of Kanagawa Prefecture and Saitama Cityof Saitama Prefecture.

a

Results

Morphology of pollinia and the number of pollen grains
Five anthers have paired pollinia, and two pollinia inadjacent anthers are joined by arms to a clip (corpusculum). A pollinium is yellow-brown, elliptic, and measures approximately 550 µm × 300 µm. A clip is darkbrown and approximately 0.5 mm long, with a narrowvertical crack (Fig. 3a). Pollinia contained irregularshaped pollen grains approximately 50 µm in size
(Fig. 3b). The number of grains per pollinium wasapproximately 800.

b

d

195

c

e

Fig. 3 (a) A pair of pollinia in the Metaplexis japonica ﬂower (stamens in the front have been removed). (b) Pollen grains in a pollinium.
A pollinium was made partially transparent in 10% sucrose solution and examined under a microscope. The number of pollen grains
in a pollinium was determined to be approximately 800 on the basis of this photograph. (c) Upwardly pointed comb-like bristles on theedges of a ﬁssure between the stamens. (d) Germinating pollinium in a stigmatic chamber. The arrow indicates a bundle of pollen tubes.
(e) Longitudinal section of the column of a M. japonica ﬂower. Cl, clip; Ov, ovary and ovule; Pg, germinating pollinium in a stigmaticchamber; Sc, stigmatic chamber; Sl, slit. Photos by O. Kitamura (a), Y. Yaoi (b,c) and H. Tanaka (d). Illustration (e) by S. Kawachino.
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H . TA N A K A E T A L .

Floral morphology and pollination mechanisms
The pistil has a crown-like capital, the end of which elongates into a long, white protrusion approximately 5–8 mm
long. Its tip is beak-like, two-cleft or entire, and it looks
like a stigma (Figs 1,2). However, the tip has no mucilage
or papilla, and pollinia rarely attached themselves to it.
We examined more than 1000 ﬂowers in natural ﬁelds, butpollinia attached to the protrusion were hardly everfound and germination on it was never observed. We also
attempted to attach pollinia to the tips on approximately
50 occasions, but failed in most cases, and even if thepollinia were artiﬁcially attached they never germinatedthere. Thus, we believe that the tip of the long protrusion
might not be a functional stigma that receives pollinia.
The stamen column has ﬁve V-shaped narrow ﬁssures
between each anther ﬁlament, and at their basal parts ﬁve
vertical slits open into the cavity between the stamens andovaries, leading to the stigmatic chambers just beneaththe capital end of the pistil (Figs 2,3e,4b). These ﬂoral
structures of M. japonica are similar to those reported for
Asclepias. Therefore, we assumed that the slits might be
the entrances used by pollinia to the stigmatic chambers,
as in Asclepias, and tried to insert pollinia artiﬁcially into
the slits.
A thin ﬁlament, as a substitute for the proboscis of
insects, was inserted into the bottom of the corona,
where nectar is pooled, and then the ﬁlament was pulled
up along a ﬁssure. The tip of the ﬁlament was easily
caught by the crack of the clip of pollinia and the pollinia
were carried out from the ﬂower. The ﬁssures had anumber of upwardly pointed comb-like minute bristles
on both edges that became progressively narrower, preventing the backward movement of the ﬁlament (Fig. 3c).
When a ﬁlament carrying pollinia was inserted into a slit
and pulled up along the ﬁssure, the pollinia were cut
away from the arms and left in the cavity of the column(Fig. 4b), and ﬁnally lodged in the stigmatic chambers.
Pollen tubes then emerged from the convex surface ofthe pollinia, which is usually facing the stigma(Fig. 3d,e).

Sex expression of ﬂowers
We noticed that plants of M. japonica have two types of
ﬂowers that are morphologically distinguishable by thesize of stamen columns and ovaries (Fig. 4a–c). We called
them large ﬂowers and small ﬂowers, respectively. Stamen columns of large ﬂowers were approximately
4 mm × 4 mm, with vertical slits opening at their bottoms,
to which pollinia are inserted. Ovaries are 3 mm long andcontain hundreds of ovules. Stamen columns of small
ﬂowers are 3 mm × 3 mm, and the slits at the bottom areclosed. Ovaries are 2 mm long and ovules are immature.

The slits of the larger ﬂowers are open throughout thestages of ﬂowering from bud to fade, whereas those in thesmaller ﬂowers are always closed.
Germination of pollen grains began within 3 h in 10%
sucrose solution at approximately 27°C, without complete
loosening of the pollinia. When examined in Saitama City
on 20 September 2004, 27 out of 44 pairs of pollinia (61%)
germinated within 13 h. Pollinia of both larger andsmaller ﬂowers germinated at a similar rate.
The ability of ovaries to attract pollen tubes was compared between the larger and smaller ﬂowers. Ovaries
from which styles had been cut off were placed on 1%
agar plates that contained 10% sucrose, and pollinia wereplaced at 1–2 mm from the ovaries. The agar plates wereleft to stand at room temperature (approximately 28°C)
for 18 h. As shown in Figure 4d, pollen tubes werestrongly attracted to an ovary of a larger ﬂower, but lessdistinctly to a smaller ﬂower (Fig. 4e). Thus, we concluded that large ﬂowers are hermaphrodite, while small
ﬂowers function as males. Pollinia could easily beremoved from the anthers of both types of ﬂowers at
every stage, so dichogamy was not evident.
The proportion of hermaphrodite ﬂowers was examined in Saitama City on 30 September 2004. Out of 102
ﬂowers, 62 (61%) were hermaphrodite. The number ofovules in 14 ovaries of seven hermaphrodite ﬂowers
was 276–380 (n = 14, 313 ± 30.7 [mean ± standard deviation]). Because hermaphrodite ﬂowers have two ovaries
and 10 pollinia, but male ﬂowers lack ovules, the pollen/ovule ratio of the total ﬂowers was calculated to be
21.0.

Insect visitors
The ﬂowers have a sweet and strong smell, with plentyof nectar at the bottom of the corona, and various insects
frequently visited the ﬂowers in the daytime (Fig. 4f,g).
Table 1 shows the insect visitors observed at four sites incentral Japan from September to November 2004. A number of large insects carried pollinia on their ligula, labial
palp or on the hairs on their legs (Fig. 4h,i). However,some small insects tended to be trapped by the ﬂowers
because their tongues or legs were pinched in the ﬁssures
between the stamens.

Pollination and fruit set
Pollinia of two male and 18 hermaphrodite ﬂowers were
examined in Saitama City on 2 October 2004. Twenty-six
pairs of pollinia (26%) had already been removed fromthese ﬂowers. Ten pollinia were found in seven out of 90
stigmatic chambers of hermaphrodite ﬂowers, and all pollinia in the stigmatic chambers had already germinatedthere.

© 2006 The Authors
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c

b

a

f

e

d

g

197

h

i

Fig. 4 (a) Comparison of the longitudinal sections of hermaphrodite (left) and male (right) Metaplexis japonica ﬂowers. (b) Column of ahermaphrodite and (c) a male ﬂower. Cl, clip; Pa, pollinium in the anther; Pi, pollinium being inserted into the slit; Sl, slit of hermaphrodite ﬂower; Slc, closed slit of male ﬂower. (d) Attraction of pollen tubes by an ovary of a hermaphrodite ﬂower compared with (e) the
activity of a male ﬂower on agar plates. (f) Parnara guttata visiting a M. japonica ﬂower. (g) Megacampsomeris grossa visiting a M. japonica
ﬂower. (h) Lasioglossum occidens with pollinia on its mouthpart. (i) Bombus diversus with many pollinia on its proboscis. Photos by H.
Tanaka (a,f,h), Y. Yaoi (d,e), T. Tada (g,i). Illustrations (b,c) by S. Kawachino.

The numbers of ﬂowers per raceme observed on 24 and
30 September 2004 in Saitama City were 6–40 (n = 25,
19.9 ± 7.5 [mean ± standard deviation]) and 2–37 (n = 26,
16.3 ± 7.9 [mean ± standard deviation]), and the numberof ﬂowers in anthesis was 1–15 and 0–8, respectively. Ten
Plant Species Biology 21, 193–199

young fruits were found on 25 randomly selected
racemes; usually one fruit per raceme, but in two cases a
raceme had two young fruits. Traces of fallen ﬂowers
were 146. From these data, we determined the fruit set
to be 11% for hermaphrodite ﬂowers and 6.5% for total© 2006 The Authors
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H . TA N A K A E T A L .

Table 1 Insect visitors to the ﬂowers of Metaplexis japonica

Insects
Hymenoptera
Megacampsomeris grossa
Campsomeriella annulata
Xylocopa appendiculata circumvolans
Bombus diversus
Lasioglossum occidens
Polistes snelleni
Paper wasp
Vespid wasp
Lasius spathepus
Monomorium intrudens
Diptera
Stomorhina obsoleta
Flies
Lepidoptera
Pseudozizeeria mahaParnara guttata
Pelopidas mathias oberthueri
Gurelca himachala sangaica
Coleoptera
Oxycetonia jucunda
Acanthoscelides obtectus
Beetle

Study
sites

Pollinia
attached

SA, SE
SA
SA
SA
SA
SA
SA
SA
YO
MA

+
+
+
+ or – and c
–
–
c
c

SA
SA

–, c
–, c

SA
SA, SESASA–, c+ or –––

MA, SE
SA
MA

+
+
+

MA, Machida City; SA, Saitama City; SE, Setagaya Ward; YO,
Yokohama City; +, attached; –, not attached; c, insects caught by
the ﬁssures between stamens or by the cracks of clips.

ﬂowers. Among 566 fruits examined in November at Setagaya Ward, Tokyo, in 14 cases (2.5%) a single ﬂower
produced double fruits.

Discussion
In the past, ﬂowers of Asclepiadaceae have been
described as hermaphrodites (Honda & Sakisaka 1930;
Ohwi 1953; Jia & Jia 1955; Murata 1981; Fu et al. 1999), andthe presence of male ﬂowers has not been known. However, our observations and experiment showed that 39%
of M. japonica ﬂowers were morphologically and functionally male. Stamen columns of the male ﬂowers lacked
the slits at the bottom and pollinia could not be inserted
into the stigmatic chambers. Their ovules were always
immature and did not attract pollen tubes. In contrast,
the morphology and germinating activity of the polliniaof the male ﬂowers were normal. To our knowledge,
M. japonica is the ﬁrst case in Asclepiadaceae that has been
conﬁrmed to have both hermaphrodite and male ﬂowers.
In ﬂowers of representative asclepiad genera, such as
Asclepias and Vincetoxicum, pistil ends are covered withcrown-like capitals, but the top surfaces of the capitals are
non-receptive for pollinia. Studies on the pollination

mechanisms of asclepiad ﬂowers have well establishedthat the stigma is present on the lateral surface of the stylebeneath the capital. That is, pollinia germinate in ﬁve
stigmatic chambers, and the stigmatic surfaces in thechambers receive pollen tubes (Müller 1873; Knuth 1899;
Procter & Yeo 1973). These facts were correctly cited insome old Japanese publications (Honda & Sakisaka 1930;
Hayata 1935). Nevertheless, the shape of the beak-like
protrusion in the center of the corolla of M. japonica is sooutstanding and misleading that the protrusion has, ingeneral, been described as a stigma in more recently published Japanese and Chinese articles. For this reason, thetrue stigma in ﬂowers of M. japonica has not been exactly
located to date. A few plants of Asclepiadaceae found in
China, for example Metaplexis hemsleyana and Toxocarpus
villosus, have similar protrusions, although their length isvariable (China Institute of Botany 1974).
Our observations and experiment showed that thestigma-like tip of the protrusion of M. japonica was nonreceptive; pollinia neither attached themselves to it nor
germinated there. Using a pollination experiment andmicroscopic observation, we conﬁrmed that the stigma of
M. japonica was present on the lateral surface of the stylejust beneath the capital, as in Asclepias.
Ikuse (2001) reported that pollinia of M. japonica are
300–320 µm × 510–570 µm, and the pollen grains are 22–70 µm. We determined that the number of pollen grains
per pollinium was approximately 800, and that the number of ovules per ovary was 313. Wyatt et al. (2000)
reported that across four tribes of the Asclepiadaceae, thenumber of pollen grains per pollinium varied from 14 to445, and the number of ovules per ovary varied from 4 to229. The numbers of pollen grains per pollinium andovules per ovary in M. japonica are greater than the range
reported by Wyatt et al. (2000). The proportion of hermaphrodites was 61% of total M. japonica ﬂowers. On thebasis of our data, the pollen/ovule (P/O) ratio of
M. japonica is 21.0. The average P/O ratio in hermaphrodite plants is 3450 (Gruden 2000), but the P/O ratio of
Asclepiadaceae and Orchidaceae, in which pollen grains
are packaged in pollinia, is known to be lower. Gruden(2000) reported that the P/O ratio of 10 Asclepiadaceae
plants ranges from 3.8 to 18.4. Our results showed that
the P/O ratio of M. japonica was slightly higher than theother Asclepiadaceae plants, probably because of thepresence of the male ﬂowers.
Sugiura and Yamazaki (2005) recently reported that thepollinia of M. japonica were transported on the tips of theproboscis of nocturnal moths, and that diurnal insects
were not considered to be important pollinators. However, we observed that many insects visited the ﬂowers in
the daytime and transported the pollinia. Pollinia wereborn by the most frequently visited insects, Megacampsomeris grossa and Campsomeriella annulata, indicating that© 2006 The Authors
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they are effective pollinators. A beetle, Oxycentonia
jucunda, visited frequently, and Bombus diversus, which
was observed once on a ﬂower, also appear to be effective
pollinators. In contrast, attachment of pollinia was rarely
observed on Lepidoptera, such as Parnara guttata andGureluca himachala sangaica, which suckle nectar with along proboscis and appear to be nectar thieves. Small,
powerless insects, such as ants, ﬂies, a bee Lasioglossum
occidens and a butterﬂy Pseudzizeeria maha, often could not
escape from the ﬂowers after their mouthparts or legs
were trapped by the cracks of clips or by the ﬁssures
between the stamens, and these species might disturb thepollination by other insects. Thus, we conclude that
M. japonica is pollinated by relatively large, irregularly
visiting insects.
The fruit set of the total ﬂowers was 6.5%, slightly
higher than the 0.33–5.0% recorded in Asclepias, in which
all ﬂowers are hermaphrodite (Wyatt & Broyles 1990). The
proportion of ﬂowers producing double fruits was 2.5%,
lower than the 5.0–24.4% reported in Asclepias (Wyatt &Broyles 1990).
The present study elucidated several fundamental
aspects of the mechanisms of pollination of M. japonica.
These include the ﬂoral morphology, sex expression of
ﬂowers, insect visitors, P/O ratio and fruit set. However,many problems remain to be solved. The physiological
function of a long and outstanding protrusion on the pistil, the possibility of self-incompatibility, and the sourcesof nectar and smell are interesting areas requiring further
study.

Acknowledgments
We thank Dr Naoto Sugiura, Faculty of Sciences, University of Kumamoto, for the identiﬁcation of Hymenoptera
species and for valuable information about other insects.
We also thank Dr Shoichi Kawano, Professor Emeritus ofKyoto University, for his kind support in the preparationof the manuscript.

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© 2006 The Authors
Journal compilation © 2006 The Society for the Study of Species Biology