Botanical Studies (2008) 49: 253-260.
*
Corresponding author: E-mail: why@scbg.ac.cn; Tel: +86-
20-37252981; Fax: +86-20-37252981.
INTRODUCTION
Biological invasion is a pervasive and costly environ-
mental problem (Vitousek et al., 1996; Kennedy et al.,
2002; Perrings et al., 2005; Pimentel et al., 2005). Invasion
is defined as the establishment of a species after human-
mediated movement beyond its natural range or natural
zone of potential dispersal and it is distinct from coloniza-
tion that is often viewed as natural range expansion (Lee,
2002). Biological invasions are like natural experiments,
but their processes are far more rapid than those in coloni -
zation (Sakai et al., 2001). For an introduced plant species
to become invasive, it must be able to reproduce, even
in initially small populations (van Kleunen and Johnson,
2005), and its rate of spread is influenced by the mode of
reproduction, the reproductive system and potential for
recombination, particularly if continuous adaptation is a
prerequisite for the invasion process (Sakai et al., 2001).
Mikania micrantha H.B.K. (Asteraceae) is a fast-grow -
ing perennial creeping vine native to Central and South
America (Wirjahar, 1976; Holm et al., 1977). It has many-
branched stems and reproduces easily through both sexual
and vegetative reproduction (Swarmy and Ramakrishnan,
1987; Zhang et al., 2004). It entered South China after
1910, and since the 1980s it has spread and invaded widely
(Zhang et al., 2004). In recent years, this notorious weed
has caused severe damage to many ecosystems and local
economies in Guangdong Province, China and elsewhere
in the world (Deng et al., 2004; Yang et al, 2005; Lian et
al., 2006; Song et al., 2007). Therefore, it has been listed
as one of the 100 worst invasive alien species on earth
(Lowe et al., 2001) and as one of the top 10 worst weeds
in the world (Holm et al., 1977).
Secondary pollen presentation is the developmental
relocation of pollen from the anthers onto another floral
organ which then functions as the pollen presenting organ
for pollination (Howell et al., 1993). It is a reproductive
strategy promoting outbreeding and it occurs in five mono -
cotyledon and 20 dicotyledon families and many publica-
tions discuss it in the angiosperms in general (Howell et
al., 1993; Ladd and Donaldson, 1993; Yeo, 1993; Ladd,
1994). Secondary pollen presentation is a widespread phe-
nomenon that characterizes the family Asteraceae nearly
Secondary pollen presentation and style morphology in
the invasive weed Mikania micrantha in South China
Lan HONG
1, 2
, Hao SHEN
1
, Wanhui YE
1,
*, Honglin CAO
1
, and Zhangming WANG
1
1
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, P.R. China
2
Graduate School of the Chinese Academy of Sciences, Beijing 100049, P.R. China
(Received March 8, 2007; Accepted April 3, 2008)
ABSTRACT.
Mikania micrantha H.B.K. is a successful invasive weed in many parts of the world. Its
reproductive biology, specifically, floral functional morphology, growth and behavior of the style during
anthesis, and style morphology, was studied in an open M. micrantha population in South China during the
flowering seasons of 2004 and 2005. Floral biology was studied in detail by examining florets at different
developmental stages under a dissecting microscope and a scanning electron microscope. Stigma receptivity
and pollen viability was determined by MTT [3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-2H-tetrazolium bromide]
staining technique. The results show that M. micrantha is protandrous and has a secondary pollen presentation
system which characterizes the family Asteraceae. Typically, the flowering period is 6 days and can be divided
into six floral stages (A, B, C, D, E and F) based on style morphology and behaviour. At the beginning of
anthesis, the style bends to break and enter the tube formed by five fused anthers. Later, the style protrudes
the anther tube and moves the pollen out from the anther. During anthesis, two partially overlapping phases,
functionally male phase and functionally female phase, can be distinguished by MTT tests: the former is from
stage B to D, and the latter is stage E. The style has two style branches in its terminal part. The sweeping
hairs, which act as pollen presenter, are located on the tips and on the upper parts of the style branches form-
ing developed stylar appendages. The stigmatic papillae are separated into two ventro-marginal bands along
each style branch in symmetric arrangement. The bending behavior of the style and the sweeping hairs play an
important role in presenting pollen to pollinator.
Keywords: Invasive plant; Mikania micrantha; Secondary pollen presentation; Sweeping hairs.
REPRODUCTIVE BIOLOGY
pg_0002
254
Botanical Studies, Vol. 49, 2008
throughout, and it is usually the terminal style that acts as
the pollen presenter with active pollen placement (Howell
et al., 1993; Ladd, 1994). In Asteraceae, the pollen pre-
senter is sweeping hairs that cover the stigmas, and it can
just be the style branches for the species that lack sweep-
ing hairs (Ladd, 1994). Pollen is presented on the terminal
section of the modified style where it is actively loaded
onto the distal portion of the style as it elongates through
a connate ring of anthers (Howell et al., 1993). Within the
family Asteraceae, based on the arrangement of the sweep-
ing hairs, different types of secondary pollen presentation
have been found: pump mechanism, brushing mechanism,
transitions between both that were found in the Asteroi-
deae to which subfamily the Eupartorieae belong, and a
special one in the basal subfamily Barnadesioideae (Leins
and Erbar, 1990; Yeo, 1993; Erbar and Leins, 1995, 2000;
Leins and Erbar, 2006).
Mikania micrantha has very small, compact florets in
its inflorescences, and its flower morphology and pollen
presentation are nearly unknown. To provide information
on the basic reproductive biology of M. micrantha, we
initiated this study using laboratory and field approaches
in an open population at the field station of South China
Botanical Garden during the flowering seasons of 2004
and 2005 to investigate: (1) floral functional morphology;
(2) growth and behaviour of style during anthesis; and (3)
style morphology.
MATERIALS AND METHODS
Study site
The study was conducted at the field station of South
China Botanical Garden during the flowering seasons
of 2004 and 2005. It is in the suburb of Dongguan City,
Guangdong Province, China. Dongguan (22¢X39¡¦-23¢X
09¡¦ N, 113¢X31¡¦-114¢X15¡¦E) is in a subtropical area and is
located south of the Tropic of Cancer. It has a lower south
subtropical marine monsoon climate. Mikania micrantha
mostly occurs in open habitats in South China, so at the
station we selected a typical population for our studies in
an open field where an artificial Magnolia denudata forest
was cut 10 years ago, and there were almost no other
plants except M. micrantha.
Flowering phenology
Flowering phenology was observed for 12 M. micrantha
individuals per season from September to February in
2004 and 2005 flowering seasons. On each plant, 10
capitula buds of similar size were marked and observed at
0800, 1200, 1500 and 1800 h every day until the flowers
senesced to collect data on the timing and duration of
flowering and fructifying.
Floral biology
We collected at least 80 samples from flower buds
to flowers at anthesis from different capitula in 10 M.
micrantha plants in each season and observed them under
a dissecting microscope (SE-CTV, Olympus, Japan). The
flowers of M. micrantha are co-sexual and we examined
the spatial and temporal arrangement of male and female
sexual parts within the flowers. We used the information
obtained to define six floral morphological stages (A, B,
C, D, E and F) based on style morphology and behaviour
(Figure 1A-F) and use these stages to classify the flowers
we observed during the flowering seasons.
Pollen viability and stigma receptivity
.
In each
flowering season, at least 50 fresh flowers with dehiscing
anthers in different floral stages (Figure 1A-F) were ran-
domly collected from 10 individual M. micrantha plants.
The pollen samples were removed from the anthers and
then immersed in a drop of 20 g L
-1
3-(4,5-dimethylthia-
zol-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sig-
ma M-2128) solution and examined under a microscope
(Rodriguez-Riano and Dafni, 2000). Dark blue staining in-
dicates the pollen is viable. Pollen viability was expressed
as mean percentage of stained pollen grains of more than
six samples. In each sample, we calculated the percentage
number of dark pollens to total number of pollen grains
in each of seven randomly selected visual fields under the
microscope. The number of dark pollens in each sample
was recorded as the average number in the seven visual
fields. Due to the secondary pollen presentation in M.
micrantha (Figure 1A-F), only pollens in anthers were
examined to avoid including exotic pollens. The same
test was used to determine stigma receptivity (Rodriguez-
Riano and Dafni, 2000).
Fine-scale style and pollen morphology
. A scan-
ning electron microscopy (SEM) was used to examine the
style morphology. For each floral stage, 20 pistils of fresh
flowers were dissected and fixed in 4% glutaraldehyde in
0.1 mol L
-1
phosphate buffer at pH 7.4. The fixed samples
were rinsed three times in the same buffer and fixed again
in it with 1% osmium tetroxide added. They were then
dehydrated in a graded alcohol series (30% - 50% - 70%
- 80% - 90% - 100%) and immediately freeze-dried using
a freeze-drying device JFD-310 apparatus (JEOL Ltd.,
Tokyo, Japan). Then, they were mounted directly on metal
stubs using double-sided adhesive tape and sputter-coated
with platinum using JFC-1600 auto fine coater (JEOL Ltd.,
Tokyo, Japan). Morphology of the stigmas was observed
with a JSM-6360LV SEM (JEOL Ltd., Tokyo, Japan) at
15 KV. For each floral stage, pollen grains were also ran-
domly selected and treated as above, and then 20 were
observed under a SEM to study the pollen morphology
(shape, size, and exine).
RESULTS
Flowering phenology
Flowering and fructifying of M. micrantha occurred
from September to February in both study seasons. The
peak bloom occurred between mid November and mid
December. Fruiting started as early as the end of October,
and almost all fruits had dropped in early February.
pg_0003
HONG et al. ¡X Secondary pollen presentation and style morphology in
Mikania micrantha
255
Floral biology
Floral morphology
.
Mikania micrantha is hermaphro-
ditic and its corymbose inflorescence consists of a 4-flow -
ered tight capitula. Its individual florets are 5.87¡Ó0.46 mm
in length and are actinomorphic. The capitulum is 6.6¡Ó0.50
mm in length and 1.65¡Ó0.63 mm in diameter. Five anthers
attach each other and form a tube, and the stigma and the
style grow through the centre of the tube. The flowers pro -
duce nectar and pollen. There is a green and ringed ovary-
roof nectary which encircles the base of the style (Figure
1A
2
).
Floral stages
. The flowers open at any time of day,
but the majority do so in the morning. Typically, the flow -
ering period is 6 days and can be divided into six floral
stages (A, B, C, D, E and F) based on style morphology
and behaviour (Figure 1). In general, within each capitu-
lum, the central florets flower before the surrounding ones.
During the 24 h before anthesis (stage A), the growing
filament raises the anther tube up to the same level as the
style (Figure 1A). During this time of growth, the anthers
dehisce inwardly and discharge pollen grains inside the
anther tube onto the sweeping hairs of the style. The style
branches are aligned and the stigma is not yet receptive.
During anthesis, two partially overlapping phases can
be distinguished: (1) Male phase: Between about 6.00-8.00
h of day 1, the flower opens and the tip part (see Figure
1C) of the style breaks through the anther tube formed
by five fused anthers, and its lower part (see Figure 1C)
bends inside the corolla tube (stage B, Figure 1B). The
growing style then unthreads through the tube and loosens
the pollen grains which then adhere to the sweeping hairs
of the style branches. The green nectary begins to secrete
and accumulate nectar within corolla tube at stage B and
continues. About 1.5 h later, the lower part of the style
straightens, the style begins to protrude out of the anther
tube (Figure 1C) and the pollen grains are removed from
the anthers by the sweeping hairs (stage C). At this stage,
insect visitors were observed. In the afternoon of day 1,
Figure 1. A-F, Floral stages of Mikania micrantha showing secondary pollen presentation. A
1
, Early floral stage showing flower is
beginning to open; A
2
, Dissected flower in A
1
showing nectary (nc), anther (an) and style (st); B
1
, Flower just opened (the arrow indi-
cates the anther tube); B
2
, Dissected flower in B
1
showing the bending part of the style (arrow); B
3
, Dissected anthers in B
1
showing
the bending part of the style is pushing through the anther tube (arrow); C
1
, The style is out of the anther tube; C
2
, Dissected flower
in C
1
(the arrow shows the empty anthers with few pollen grains); D, Style branches (sb) separate, curved and slightly inflexed, while
the anthers (an) are nearly empty; E
1
, The style branches (sb) expose the stigmatic surfaces (ss) and the anthers (an) are dry, brown
and withering; E
2
, Test result of stigma receptivity showing the stigmatic surface appears dark blue when immersed in a drop of MTT
solution under a dissecting microscope; (F) Senescence stage showing the two style branches bending towards the centre of the floret.
Functionally male phase is from stage B to D, and functionally female phase is stage E. All bars=0.5 mm except E
2
=200 £gm.
pg_0004
256
Botanical Studies, Vol. 49, 2008
the style grows longer until the tip has completely emerged
from the anther tube and presented more pollen attached
on the sweeping hairs of the style. At the end of stage C,
the florets are completely open, and the anthers have re-
leased almost all the pollen. The style and style branches
present the yellow, clumped pollen grains to the inside of
the flower while the style branches are still joined. Insect
visitors appear more frequently than before and remove
pollen. By day 2, the style branches separate with a slight
inward inflexation (Figure 1D) but their lower parts with
stigmatic area located on the inner surface and covered
with receptive papilla cells are still joined (stage D). At the
late male phase, early on the third day after flower opened,
tests of stigma receptivity gave negative results. The flo-
ral scent is present during daylight hours, and it becomes
stronger from stage B to D.
(2) Female phase: From day 3 to 5, the style grows
to its full length, its two branches completely open, two
layers of receptive papilla cells are exposed, and anthers
become dry and brown and begin to wither (stage E,
Figure 1E). Nectar secretion and scent emission continue
and frequent insect visits occur. By day 6, flowers enter the
senescence stage (stage F, Figure 1F) when the two style
branches bend towards the center of the floret. From day 7
on, fertilized flowers continue to grow and the green ovary
develops into a plump and black seed, while unfertilized
ovules shrivel and turn yellow.
Pollen viability
. Pollen viability decreases with the
progress of floral stages (Figure 2). At stage A, pollen
has the highest viability at 95.0%, which then decreases
to 86.3% at stage B. At stage C, pollen viability further
decreases to about 78.3%. At the late male phase (stage D),
early on the third day after flower opened, pollen viability
is about 53.8%, while at the early female phase (stage E),
late on the third day, viability of the few remaining pollen
in the anthers is about 34.4%. In the senescence stage
(stage F) the pollen viability decreases to only 9.5%.
Pollen characteristics
.
The pollen grains appeared
globular or sub-globular under SEM (Figure 3A) and they
are 3-colporate. The size of pollen grain is 19.95¡Ó1.96
£gm in the polar axis and 14.67¡Ó0.86 £gm in the longest
equatorial axis. The ornamentation of exine is echinate,
and the length is 1.85¡Ó0.33 £gm.
Style morphology
The style has two style branches in its terminal part
(Figure 3B), and the morphology of one style branch is
shown in Figure 3C. Sweeping hairs were concentrated on
the tips (Figure 3D) and on the upper parts (Figure 3E) of
the style branches forming developed stylar appendages
that are sterile structures extended above stigmatic areas.
Generally, the sweeping hairs gradually increase in length
basipetally, and they act as pollen presenter which is
responsible for removing pollen out of the anther tube
while the terminal section of the style branches grows
through and presenting them. The stigmatic surface is
covered with columnar papilla cells that are very close
to each other. The stigmatic papillae are separated into
two ventro-marginal bands along each style branch in
symmetric arrangement (Figure 3F). MTT test showed that
the stigmatic area was receptive (Figure 1E
2
).
DISCUSSION
Secondary pollen presentation system is widespread
in angiosperms (Howell et al., 1993). It characterizes the
family Asteraceae (Howell et al., 1993), and we found
that M. micrantha is no exception. Our observations show
that M. micrantha is protandrous, and pollen is shed from
the anthers onto the sweeping hairs of the style in the bud
stage (Figure 1A) when the style branches are joined and
the stigmatic surfaces are not receptive despite that the
pollen has the highest viability. This is consistent with
the observations on some other species of Asteraceae
(Porras and Alvarez, 1999; Roitman, 1999; Cerana, 2004;
Grombone-Guaratini et al., 2004). Our results show that
the sweeping hairs are both located around the tip of the
style branch and reaching below the branching of the
style. Thus, M. micrantha presumably has attained a com-
bination of a pump pollen presentation mechanism and
a brushing mechanism, based on the arrangement of the
sweeping hairs for different types of secondary pollen
presentation (Yeo, 1993; Erbar and Leins, 1995; Leins and
Erbar, 1990; 2006).
We observed during early stages (stages B-C) of
anthesis of M. micrantha that part of the style is bended.
This bending makes it possible for the style tip to break
and enter the anther tube sidewise. With the growing of
the style, the bending part straightens. This unbending
process may generate a pushing force which would not
only help the style to protrude out of the anther tube but
also facilitate the friction between the sweeping hairs of
Figure 2. Pollen viabilities in different floral stages (data are
presented as means¡Ó1SE). See Figure 1 for the descriptions of
the floral stages from A to F.
pg_0005
HONG et al. ¡X Secondary pollen presentation and style morphology in
Mikania micrantha
257
the style and the anthers. The pollen grains lodged in the
anthers were loosened and adhered to the sweeping hairs
of the style. Then, they were brought out of the anther tube
with the growing of style. Thus, the bending behaviour
of M. micrantha style and the sweeping hairs of the style
branches may play an important role in presenting pollen
to pollinator.
Within each of the style branches, the sweeping hairs
gradually increase in length basipetally. Moreover, due
to the echinate ornamentation of the exine, the pollens
can adhere onto the sweeping hairs. Thus, the sweeping
hairs can easily carry the pollens with them out of the
anther tube. Therefore, these morphological and structural
characteristics of the stylar sweeping hairs and pollen
are co-adapted to bring the pollens out effectively. The
sweeping hairs of M. micrantha are much different from
those of Polygalaceae and Fabaceae (Westerkamp, 1999),
Cyphiaceae (Leins and Erbar, 2003), because different
Figure 3. The morphology of pollen and style in Mikania micrantha under SEM. A, Pollen (bar=5 £gm); B, A style gives off two style
branches (sb) (bar=200 £gm); C, Detail of a style branch showing the sterile appendage covered by sweeping hairs (D, E) and the stig-
matic surface (F) (bar=100 £gm); Arrows with figure numbers indicate the approximate position of anatomical sections illustrated in
corresponding figures; D, Sweeping hairs at the tip part of the style branch (bar=20 £gm); E, Sweeping hairs located on the upper part of
the style branch (bar=20 £gm); F, Receptive stigmatic surface consisting of columnar papilla cells (bar=10 £gm).
pg_0006
258
Botanical Studies, Vol. 49, 2008
species display variation in this mechanism, perhaps
reflecting differences in mating systems (Anderson et al.,
2000; Etcheverry et al., 2003; Leins and Erbar, 2003).
As the style grows out of the anther tube, the outside
of the style branches presents pollen for pollination. The
receptive papillate stigmatic surface is hidden between
two appressed style branches, preventing auto-pollination
during the functionally male phase of the floret (Figure
1B-D). Our previous study has suggested that the pollen
grains should be transported from the sweeping hairs of
one plant to the stigmatic surface of another plant, which
has been proven by our pollination treatments indicating
that M. micrantha needs insect pollination (Hong et al.,
2007). During the functionally female phase (Figure 1E),
the style branches separate completely, and two layers of
receptive papillae on the adaxial surfaces become exposed
and are receptive. Thus, the behavior of style determines
the timing of the functionally female phase of the floret.
In some Asteraceae species, maturation of the flower is
marked by the gradual separation of the style branches un-
til they eventually reflex back onto the presenting surface
and auto-pollinate though many species are sporophyti-
cally self-incompatible (Howell et al., 1993).
Our pollen viability results show that pollen viability
began to gradually decrease after flower opened which
results in a temporal separation between pollen presenta-
tion and pollen viability and diminishes the chance of
self-pollination, but some are still viable after the flower
have entered female phase. This indicates that there is an
overlap between the time when pollen is viable and when
the stigma of the same floret is receptive, and thus self-
pollination cannot be totally excluded. This shows that the
receptive stigma is in general temporally separated from
the floret¡¦s own viable pollen, but cannot completely avoid
the probability of autogamy or geitonogamy. However,
our pollination experiments for M. micrantha show that
seed/ovule ratio was 0.56 for open pollination and 0.0034
and 0.0038 for wind pollination and selfing, respectively
(Hong et al., 2007). These indicate that the stigmas almost
completely accept outcross pollens. Thus, although flower
morphology does not fully prevent self-pollination (and
geitonogamy can easily take place), the level of autogamy
is very low. Therefore, some self-incompatibility mecha-
nism seems operative in this species (Hong et al., 2007).
Protandry is prevalent throughout the Asteraceae. In
self-incompatible species protandry can avoid pollen-
stigma interference (Webb, 1985), but this is not fully
achieved in M. micrantha, since some overlap exists be-
tween pollen delivery and receipt, which is a fact in most
Asteraceae species.
In conclusion, the results indicate that M. micrantha
has a system of secondary pollen presentation. During
anthesis, the bending behaviour of the M. micrantha
style and the sweeping hairs of its two branches help the
growing style to move the pollens out of the anthers ef-
ficiently. This style-developmental morphology and its
potential functions would be of great importance to better
understand the evolutionary history of floral structure and
function in angiosperms. The secondary pollen presenta-
tion system and the fine-scale morphology and structure of
style indicate that there is some adaptive advantage to be
derived from their formation.
Acknowledgements. We thank Dr. Simon Hiscock and
Dr. Adrian Brennan for their constructive comments on
the manuscript. This work was supported by the National
Natural Science Foundation of China (No. 30530160),
the Natural Science Foundation of Guangdong Province,
China (No. 05200701), and a Dean Scholarship of the
Chinese Academy of Sciences, China.
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