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Male cone morphogenesis, pollen development and
pollen dispersal mechanism in
Ginkgo biloba
L.
Y. Lu
1,2,4
, L. Wang
1,3,4
, D. Wang
1
, Y. Wang
1
, M Zhang
1
, B. Jin
1,2
, and P. Chen
1,2,5
1
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, People’s Republic of China
;
2
College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, People’s Republic of China;
and
3
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese
Academy of Sciences, Beijing 100093, People’s Republic of China
.
Received 24 February 2011,
accepted 26 May 2011.
Lu, Y., Wang, L., Wang, D., Wang, Y., Zhang, M., Jin, B. and Chen, P. 2011. Male cone morphogenesis, pollen
development and pollen dispersal mechanism in Ginkgo biloba L. Can. J. Plant Sci. 91: 971981. Ginkgo biloba L. is one of the
oldest gymnosperms. Male cone morphogenesis, pollen development and dispersal are important for successful pollination
and reproduction. In this study, we investigated the development of male cone, pollen and the sporangial wall in detail.
The results indicate that: (1) The primordia of male cones and leaves begin to differentiate in early June and remain open
until the following March. The male cones then mature and release pollen in mid-April. The male cones are drooped and
approximately perpendicular to the leaves during pollination. (2) The microsporocytes develop from the sporogenous cell
and form a tetrahedral tetrad after two simultaneous asymmetrically meioses, then produce a matured four-cell pollen after
three polar mitotic divisions. The matured pollen is hemispheric in shape with a large aperture area and three pollen wall
layers; once released from the microsporangia, the pollen becomes boat-like in shape. (3) The sporangial walls are
eusporangiate and consist of epidermis, endothecium and tapetum. The differentiation of the tapetum occurs separately
from that of the epidermis and endothecium, and originates from the outermost layer of sporogenous cells. The sporangial
walls exhibit shrinkage of the epidermis, fibrous thickening of the endothecium, and enzymic dissolution of the tapetum
during pollen dispersal, which contributes to microsporangia opening. Based on these results, we conclude that there many
unique and primitive characteristics of the development of the male cones, pollen and sporangial wall of G. biloba.In
addition, we also found that the male cones, pollen and sporangial walls have evolved efficient structural and
morphological adaptations to anemophily.
Key words: Ginkgo biloba L., male cone, pollen development, sporangial wall, wind pollination
Lu, Y., Wang, L., Wang, D., Wang, Y., Zhang, M., Jin, B. et Chen, P. 2011. Morphoge´ ne` se du co
ˆne ma
ˆle, de´ veloppement du
pollen et me´ canismes de dispersion chez Ginkgo biloba L. Can. J. Plant Sci. 91: 971981. Ginkgo biloba L. est l’un des plus
anciens gymnospermes. La morphoge
´ne
`se du co
ˆne ma
ˆle, le de
´veloppement du pollen et les me
´canismes de dispersion
jouent un ro
ˆle important dans une pollinisation et une reproduction efficaces. L’e
´tude devait pre
´ciser le de
´veloppement du
co
ˆne ma
ˆle, du pollen et de la paroi des sporanges. Les re
´sultats re
´ve
`lent ce qui suit. (1) L’e
´bauche du co
ˆne ma
ˆle et les
feuilles commencent a
`se diffe
´rencier au de
´but de juin, et l’ouverture persiste jusqu’au mois de mars suivant. Le co
ˆne ma
ˆle
parvient alors a
`maturite
´et libe
`re le pollen a
`la mi-avril. Le co
ˆne ma
ˆle est tombant et a
`peu pre
`s perpendiculaire aux feuilles
au moment de la pollinisation. (2) Les microsporocytes sont issus de cellules sporoge
`nes et forment une te
´trade
te
´trae
´drique apre
`s deux me
´ioses asyme
´triques simultane
´es, puis produisent un grain de pollen a
`quatre cellules apre
`s trois
mitoses polaires. Parvenu a
`maturite
´, le grain de pollen est de forme he
´misphe
´rique, posse
`de une large ouverture et sa paroi
est triple; apre
`s libe
´ration du micro-sporange, le grain adopte la forme d’une embarcation. (3) Les parois du sporange sont
eusporangie
´es et comprennent l’e
´piderme, l’endothe
`que et le tapetum. La diffe
´renciation du tapetum diffe
`re de celle de
l’e
´piderme et de l’endothe
`que, car elle de
´rive de la couche exte
´rieure de cellules sporoge
`nes. Les parois du sporange
illustrent un re
´tre
´cissement de l’e
´piderme, un e
´paississement fibreux de l’endothe
`que et la dissolution du tapetum par des
enzymes au moment de la dispersion du pollen, ce qui facilite l’ouverture du microsporange. D’apre
`s ces re
´sultats, les
auteurs concluent que le de
´veloppement des co
ˆnes ma
ˆles, du pollen et des parois du sporange de G. biloba pre
´sente
beaucoup de particularite
´s uniques et primitives. Les auteurs ont e
´galement constate
´que les co
ˆnes ma
ˆles, le pollen et les
parois du sporange ont vu leur structure et leur morphologie e
´voluer de manie
`re efficace pour s’adapter a
`la fe
´condation
ane
´mophile.
Mots cle
´s: Ginkgo biloba L., co
ˆne ma
ˆle, de
´veloppement du pollen, parois des sporanges, pollinisation par le vent
4
These authors contributed equally to this work
5
Corresponding author: College of Horticulture and Plant
Protection, Yangzhou University, Yangzhou 225009,
China (e-mail: chenpeng@yzu.edu.cn).
Abbreviations: SEM, scanning electron microscopy; TEM,
transmission electron microscopy
Can. J. Plant Sci. (2011) 91: 971981 doi:10.4141/CJPS2010-036 971
Ginkgo biloba is a fascinating gymnosperm, which is a
unique extant species of the order Ginkgoales and is
regarded as a living fossil (Gifford and Foster 1989).
Ginkgo first appeared in the Permian about 280 million
years ago and achieved its maximum diversity during
the Jurassic Period (Zhou and Zheng 2003); it is
currently cultivated widely in Asia and other regions
(Tredici 2007). The reproductive biology of Ginkgo has
received considerable attention since the discovery of
its multiflagellate spermatozoids by Hirase in 1896. To
date, many studies on the anatomical and developmen-
tal aspects during the gametophytic life cycle of Ginkgo
have been reported (Friedman 1987a; Lu et al. 2009;
Wang et al. 2010).
Male cone and pollen morphological characteristics,
which are essential for successful fertilization and
reproduction in G. biloba, have been detailed by several
studies. For example, the male cones arise in the axils of
the inner bud scales (Christianson and Jernstedt 2009).
The microsporangia originate from the outer hypoder-
mal layer, which is similar to the developmental pattern
seen in leptosporangiate ferns (Mundry and Stu
¨tzel
2004). During the different stages of meiosis, the
microsporocyte nucleoids (plastids and mitochondria)
go through a series of regular dynamic changes and
establish an axial polarity (Wolniak 1976; Zhang et al.
1999), which is associated with the microtubules and
g-tubulin arrays (Wolniak 1976; Brown and Lemmon
2005). Upon germination, the microspores go through
three polar mitotic divisions and form mature pollen
(Friedman 1987b; Zhang et al. 1999). Although these
studies highlighted many characteristics that are unique
to the development of male cones and pollen in G.
biloba, few results have been reported on male cones
morphogenesis and no study has been carried out to
investigate the development of sporangial walls.
Therefore, systematic studies on male cone morphogen-
esis and the development pollen and sporangial walls are
essential for our comprehensive understanding of the
reproductive biology of G. biloba.
Ginkgo biloba is dioecious with wind pollination
(anemophily) (Whitehead 1983). The male cones are
catkin-like with spirally arranged microsporophylls
(Mundry and Stu
¨tzel 2004; Liu et al. 2006), and they
are borne on short shoots in the axils of bud scales
(Mundry and Stu
¨tzel 2004; Tredici 2007). Previous
studies on other gymnosperms have shown that archi-
tecture and location of male cones play a significant role
in determining the efficiency of pollen dispersal (Niklas
1985; Di-Giovanni and Kevan 1991). For example, the
pendulous, catkin-like or spike-like male cones are
particularly well-designed for pollen disposal in most
gymnosperms (Niklas 1985). Male cones normally
locate higher than or at least equal to the height of
vegetable structures in some species (such as Picea and
Abies firma) (Niklas 1985), or clustered at the ends of
flexible branches in other species (such as Metasequoia
glyptostroboides) (Sze 1951), which all prefer to reduce
pollen filtration and shed pollen (Niklas 1985;
Di-Giovanni and Kevan 1991). However, the role of
male cones in enhancing the efficiency of pollen
dispersal in G. biloba has not been investigated. To the
best of our knowledge, data on the pollen dispersal
mechanism in gymnosperm are still scarce, especially in
G. biloba. The pollen grains have evolved a number of
structural and morphological traits to adapt to
anemophily (Friedman and Barrett 2008). For instance,
the pollen grains of wind-pollinated species are smaller
and have smoother surface ornamentation than those of
animal-pollinated species (Ackerman 2000). Moreover,
pollen grains of many gymnosperms, such as Pinaceae
and Podocarpaceae, contain 13 sacci (Tomlinson 1994;
Schwendemann et al. 2007), which have significance in
adapting to the aerodynamics of wind pollination and
increase pollen dispersal distance (Schwendemann et al.
2007; Leslie 2008; Doyle 2010). However, the functions
of non-saccate pollen in G. biloba during pollination
have been far less studied than those in saccate pollen
grains.
Therefore, this study was carried out to investigate
pollen dispersal mechanisms and male cone morphogen-
esis and pollen development by scanning electron
microscopy (SEM), transmission electron microscopy
(TEM) and light microscopy, to reveal how the pollen
grains disperse effectively in G. biloba. The phylogeny
of G. biloba is also discussed based on the comparison of
male cone and pollen development among Ginkgo,
Cycas and conifer species. Together, these findings
extend our knowledge on wind pollination mechanisms
and reproductive biology in Ginkgo, and increase our
awareness of pollination and reproductive diversity in
gymnosperms.
MATERIALS AND METHODS
Study Species
Male reproductive structures were studied for 3 yr in
20-yr-old male trees of G. biloba, grown in the Ginkgo
Experimental Station in Yangzhou University, Eastern
China (lat. 32820?N, long. 119830?E). The G. biloba
Experimental Station is about 1.6 km
2
, and has 240
mature plants. Of the 240 mature plants, 120 (50%)
were male and 120 (50%) were female. Five healthy trees
were selected based on the presence of numerous
developing male cones.
Morphological Observations
The male floral and leaf buds are very similar in early
developmental stages and are difficult to differentiate
using morphological features. Consequently, the col-
lected buds were dissected under a Motic SMZ-168
dissecting microscope from June to early March before
male cones were exposed and examined by SEM. Male
floral buds were selected, which consisted of three
developmental stages: early June when male cone
primordia started to form; mid-September when male
972 CANADIAN JOURNAL OF PLANT SCIENCE
cones had formed basely; and over-wintering. The
samples for examining male cones were collected from
five male trees with at least 10 developing buds per tree.
About 20 male floral buds without bract at each devel-
opmental stage were separately fixed in 2.5% glutar-
aldehyde (in 0.2 M phosphate buffer, pH 7.2) and FAA
solution (formalin: acetic acid: 70% alcohol 1: 1: 18)
at 48C until use. The fixed male cone buds in FAA were
rinsed three times in 0.2 M phosphate buffer (15 min
each), dehydrated in a graded ethanol series (30, 50, 80,
90, 95 and 100%, 15 min each), and critical point dried
using liquid CO
2
. The samples were mounted on speci-
men stubs with adhesive tabs. Male cone buds were
examined and imaged with a Hitachi S-4800 SEM at
15.0 kV, after coating with goldpalladium using a
sputter coater (SCD500).
We distinguished the male floral buds from leaf buds
after male cones emerged in early March. As soon as
male cones were exposed, observations were made every
3 days. When pollen dispersed, samples were examined
twice daily (morning and afternoon) until the pollen had
shed from all male cones. The male cones were photo-
graphed against a background of black cloth at the
Experimental Station using a DSC-H7 Sony digital
camera. One microsporophyll from at least six male
cones from each tree (30 microsporophylls in total) was
collected at irregular intervals from early March to mid-
April and fixed separately in 2.5% glutaraldehyde at
48C until use. When the male cones matured, morpho-
logical index measurements, including length and width
of the male cone and microsporangium, were measured
using digital calipers. We measured 30 male cones
(six from each tree) and 30 microsporangia (one from
the 30 male cones). Averages and standard deviations
were calculated with Microsoft Office Excel 2003.
Anatomical Observations
Microsporogenesis, pollen development, sporangial wall
development and microsporangial opening of G. biloba
were examined using light microscopy or TEM. The
materials fixed in 2.5% glutaraldehyde were rinsed three
times in 0.2 M phosphate buffer (pH 7.2) and post-fixed
with 1% (wt/vol) osmium tetroxide for 6 h at room
temperature. After rinsing three times in phosphate
buffer, the samples were dehydrated through a normal
alcohol series (20, 40, 60, 80, 90, 95 and 100%, 15 min
each), infiltrated in Spurr resin, and hardened at 708C
for 24 h. The samples were sectioned at 700 nm with a
glass knife using a Leica EM UC6 ultramicrotome, and
stained with 0.5% Toluidine Blue O prior to examina-
tion. Observation was performed under a light micro-
scope (Zess Primo Star X-2005), with digital images
captured using a Moticam 2306 CCD and Motic Images
Advanced 3.2 software. The samples embedded in Spurr
resin were sectioned at 70 nm with a diamond knife
using the Leica EM UC6 ultramicrotome, and stained
with 1% uranyl acetate in 70% methanol, and 1% lead
citrate. The matured pollen was examined and imaged
with a Philips Tecnai 12 TEM. Pollen size, aperture area
and pollen wall were measured using Auto CAD 2008
software (Autodesk, Inc.). Male reproductive structure
description and terminology followed those of Owens
et al. (1998) and Fernando et al. (2010).
Dispersal Regulation Observations
The male cones and microsporophylls were collected
during pollen dispersal and kept in the laboratory. The
male cones were photographed against a background of
black cloth every 20 min at room temperature, using a
DSC-H7 Sony digital camera, to observe the process of
pollen dispersal. The microsporophylls were examined
under the Motic SMZ-168 dissecting microscope and
imaged every 2 min using a Nikon Coolpix 4500 camera.
RESULTS
Morphogenesis of Male Cones
To understand the morphogenesis of the male cone in G.
biloba and its functional importance in wind pollination,
the development of male cones was observed for 3 yr.
The results showed that the male floral buds were borne
on the perennial short shoots, and they differentiated
into male cone and leaf primordia at the beginning of
June (Fig. 1a, b). In mid-September, the microsporo-
phyll primordium could be distinguished easily (Fig. 1c),
and each was composed of two tight microsporangial
primordia (Fig. 1d). The male floral buds underwent
dormancy for about 6 mo (Fig. 1d, e) until the following
March, when the male floral buds began to open
(Fig. 1f). The stalks of the microsporangia and main
axes of male cones began to extend rapidly just prior to
early March. Thus, the male cones were pushed out of
the subtending bract (Fig. 1g). Subsequently, male cones
and leaves appeared successively within 2 wk (Fig. 1h, i).
The male cones were situated in axils of bracts, and the
leaves in the center (Fig. 1j, k). By early April, the male
cones tended to droop, and were perpendicular to the
fan-shaped leaves above. At that time, they were green
and the microsporophylls were arranged compactly
around the main axis (Fig. 1l). As male cones matured,
the main axis elongated and separated the microspor-
ophylls, and began to turn yellow (Fig. 1m). By the
middle of April, male cones were yellow and began to
shed pollen (Fig. 1n). A large amount of pollen grains
dispersed within a few days. Mature male cones were
pendulous and catkin-like, and 37 male cones tufted at
the tip of short shoot (Fig. 1m). Each male cone was up
to 2.3790.29 cm long and 0.590.23 cm wide, and
contained 6080 microsporophylls that were spirally
arranged around the main axis (Fig. 1o). Each micro-
sporophyll was usually composed of a sterile extension
and two elliptical microsporangia about 2.7890.49 mm
long and 1.0690.14 mm (Fig. 1p). The phenology of the
male cones is presented in Fig. 2.
LU ET AL. *POLLEN DISPERSAL GINKGO BILOBA L. 973
Microsporogenesis and Pollen Development
Sporogenous cells initiated in September and afterwards
underwent dormancy for 6 mo. During the hibernation
period, the sporogenous cells in irregular polygonal
shape were closely arranged and they had thin walls and
concentrated cytoplasm (Fig. 3a). At that time, several
sporogenous cells started to disintegrate, and provided
spaces and nutrients for the development of other
sporogenous cells. The sporogenous cells separated
from each other (Fig. 3b) and formed polygonal
microspore mother cells (Fig. 3c), in which a large
nucleus with an obvious nucleolus and dense cytoplasm
was found, but vacuoles and callose wall were absent.
After the microspore mother cells formed, meiosis
occurred, with elongated cells and chromosomes
arranged in the equatorial region (Fig. 3d). The
chromosomes were pulled to opposite ends and
dispersed to the cell plate that formed in the equatorial
region, but there were no additional callose deposits at
the first nuclear division (Fig. 3e). Subsequently, the
second division took place in a plane orthogonal to the
first (Fig. 3f) and a tetrad was formed with a tetrahedral
shape by the deposits of the callose walls (Fig. 3g).
Cytokinesis took place after completion of two divi-
sions, by formation of the intersporal callose walls, and
therefore, microsporogenesis was simultaneous. After
formation of the tetrad, the callosic walls were thickened
asymmetrically and formed the thicker proximal side
and the thinner distal side where the aperture area
developed (Fig. 3h). At the end of microsporogenesis,
the callose wall began to degrade and finally disap-
peared following the release of microspores from the
tetrads (Fig. 3h, i).
Fig. 1. Morphogenesis of male cones in G. biloba. (a) SEM image of the male floral bud. (b) Male cone and leaf primordial within a
floral bud on Jun. 10. (c) Male cones and leaves within a floral bud in mid-September. (d) Each microsporophyll consists of two
microsporangia and one sterile extension. (e) Long shoot with short shoots bearing a male floral bud. (f) Opening bracts of a male
floral bud. (g) Male cones emerging from bud scales in mid-March. (h) Leaves emerging following male cones from floral bud.
(i) The facing of short shoot with outer male cone and inner leaves. (j)(k) Short shoot in late March with five male cones rising in
axils of bracts, and five leaves in the center. (l) Male cones of early April showing pendulous and green in color. (m) Male cones
displaying yellow color, and perpendicular to leaves before pollen dispersal. (n) Pollen dispersal of male cones. (o) Male cone with
several spirally arranged microsporophylls bearing two microsporangia. (p) Microsporophyll in detail consisting of a stalk, sterile
extension and two microsporangia. Le foliage leaf; Lh long shoot; MBmale floral bud; MCmale cone; Prprimordium;
SBsubtending bract; SEsterile extension; Sgmicrosporangium; Sh short shoot; St stalk. Scale bars 1 mm (a, p); 500 mm
(bd); 1 cm (en); 5 mm (o).
Fig. 2. Phenology of male cone development in G. biloba.
974 CANADIAN JOURNAL OF PLANT SCIENCE
After release from the tetrad, the microspores
expanded rapidly and formed a large vacuole in the
distal side, which gradually pushed the nucleus to the
proximal side (Fig. 3i). The free microspore divided into
a large central cell and a small and flattened prothallial
cell after the first asymmetric mitosis (Fig. 3j). Subse-
quently, the large central cell divided into a large
antheridial initial and a second small prothallial cell,
while the first prothallial cell degenerated with only a
few remaining residues (Fig. 3k). Finally, the antheridial
initial cell divided into a tube cell and a generative cell
through the third asymmetric mitosis (Fig. 3l). As a
result, the free microspore formed a mature pollen grain
with one tube cell, one generative cell and two prothal-
lial cells. The entire process was rapid and lasted for
about 25 d (meiosis for 10 d and mitosis for 15 d). The
phenology of microsporogenesis and pollen develop-
ment is presented in Fig. 4.
Morphology of Mature Pollen
The mature pollen in the microsporangia was hemisphe-
rical, and about 23.4591.60 mm in equatorial diameter
and 1691.38 mm in polar axis, with a large aperture
area on the distal side about 1191.56 mm in diameter
(Fig. 5a). The pollen grains had four cells that were
separated from each other by the walls, but plasmodes-
mata were not detected (Fig. 5b). Many organelles such
as plastids, endoplasmic reticulum, mitochondria, Golgi
bodies, vesicle and lipids were observed in the pollen
(Fig. 5c, d), which indicated that the pollen was very
active at that time. The pollen wall consisted of ectexine,
endexine and intine, with different features between the
proximal and distal sides. The ectexine of the proximal
face was 0.6990.05 mm, and characterized by a thin foot
layer, an irregular infratectum with several cavities,
Fig. 3. Microsporogenesis and pollen development in G. biloba. (a) Hibernating sporogenous cells. (b) Separated sporogenous cells
after hibernation. (c) Microspore mother cells with a large nucleus with an obvious nucleolus. (d) Early meiosis I. chromosomes
arranged in equatorial region. (e) Late meiosis I. chromosomes moved to opposite ends. (f) Early meiosis II. (g) Middle meiosis II.
Appearance of a callosic wall and a large nucleus within tetrahedral tetrad. (h) Late meiosis II. Callosic wall degraded generally. (i)
Free microspores with a large nucleus in the proximal side and a large vacuole in the distal side. (j) Formation of a large central cell
and a small first prothallial cell after first mitosis. (k) Formation of a large antheridial initial and a second small prothallial cell after
second mitosis. (l) Formation of matured pollen with a tube cell and a generative cell and two prothallial cells through three mitoses.
AIantheridial initial; CCcentral cell; Chchromosome; CPcell plate; CWcallosic wall; GCgenerative cell; Ms
microspore; MMCmicrospore mother cell; Nnucleus; Nunucleolus; PC1first prothallial cell; PC2second prothallial
cell; SCsporogenous cell; TCtube cell; Tatapetum; TCtube cell; Tetetrad; Vavacuole. Scale bars 30 mm.
Fig. 4. Phenology of microsporogenesis and pollen develop-
ment in G. biloba.
LU ET AL. *POLLEN DISPERSAL GINKGO BILOBA L. 975
a thick tectum, and outermost spinules (8895 nm) (Fig.
5e, f). The ectexine of the distal side just contained a
distinct foot layer and numerous spinules without tectum
and infratectum (Fig. 5h). In the transition from the
proximal side towards the distal side (aperture area), the
ectexine gradually became thinner, while the cavities
between the infratectum and the foot layer became larger
(Fig. 5g). The endexine was approximately uniform in its
thickness, both on the distal and proximal sides, which
was 0.1390.03 mm (Fig. 5e, h). The intine was 0.7390.09
mm on the distal side (Fig. 5e) but 0.14 90.01 mm on the
proximal side (Fig. 5h). After release from the micro-
sporangium, pollen grains dehydrated rapidly and closed
in around the aperture area due to its thinner wall, and
the pollen changed to a boat-like shape (Fig. 5i).
Development of the Sporangial Wall
Our observations on the developmental process of G.
biloba sporangial wall showed that it was composed of
two to three layers of cells in September (Fig. 6a), and
further differentiated to five to six layers of closely
arranged regular cells during the following 2 mo
(Fig. 6b). In the following February, the cells of the
sporangial wall increased in number and size by means of
anticlinal division (Fig. 6c). Up to early March, the
sporangial wall differentiated into 9 to 11 layers of cells,
namely an outermost epidermis, endothecium of six to
seven layers, and two to three inner tapetum layers. The
starch grains accumulated generally in the cells of
the sporangial wall (Fig. 6d). Until the end of March,
the sporangial wall cells expanded rapidly, and the outer
cells of the endothecium elongated radially (Fig. 6e). In
early April, fibrous thickening was detected in the outer
endothecium wall (Fig. 6f), while starch grains comple-
tely disappeared in the cells of the sporangial wall.
The tapetum originated from the outermost layer of
sporogenous cells and showed tangential stretching and
poor staining at the beginning of March (Fig. 6g).
Fig. 5. Transmission electron microscope images of matured pollen of G. biloba. (a) Hemisphere pollen grain with four cells. (b)
Walls of cells within pollen. (c) Organelles within pollen, such as plastids, endoplasmic reticulum, mitochondria, Golgi bodies and
vesicle. (d) Lipids within pollen. (e) Pollen wall in the proximal face consisting of ectexine, endexine and intine. (f) Spinules on the
surface of the pollen wall in the proximal side. (g) Pollen wall in transition from the proximal side towards the distal side. (h) Pollen
wall in the distal side. (i) The boat-like pollen. Ap aperture area; Ca cavity; CW walls cell; Ec ectexine; Enendexine;
ERendoplasmic reticulum; GBGolgi body; FLfoot layer; GCgenerative cell; Inintine; Infinfratectum; L lipids;
Mmitochondria; Pplastid; PC1first prothallial cell; PC2 second prothallial cell; Sp spinule; Ttectum; TCtube cell;
Vevesicle. Scale bars5mm (a, i); 1 mm(bc, e, gh); 0.5 mm (d); 100 nm (f).
976 CANADIAN JOURNAL OF PLANT SCIENCE
During the microspore mother cell period, the tapetum
exhibited dense cytoplasm and two or three nuclei in a
cell (Fig. 6h). Until the meiosis stage, the tapetum
showed many vesicles and tended to disintegrate
(Fig. 6i). Afterwards, nuclear shrinkage and cell wall
collapse within the tapetum were detected successively at
mitosis (Fig. 6j), and finally, the tapetum completely
disappeared at the mature pollen period (Fig. 6k).
Longitudinal Slit Formation and Pollen Dispersal
Longitudinal slit formation occurred at the early stage
of meiotic division (Fig. 7a). At first, the sporangial wall
kept expanding, except at the sides facing both micro-
sporangia (Fig. 7b). Subsequently, these cells were
trapped and sank, which resulted in formation of the
longitudinal slit by the end of the microspore period
(Fig. 7c, d). During the different pollen dispersal stages,
the endothecium cell walls thickened and the epidermis
rapidly dehydrated, leading to an increase in the
cohesive force within the cells, concentrated mainly on
the longitudinal slit. The epidermis and endothecium
within the longitudinal slit broke first (Fig. 7e) and then
the tapeta opened, owing to further dehydration of the
cells (Fig. 7f). Finally, the sporangial wall bent outwards
along the longitudinal slit (Fig. 7g) and the microspor-
angium dehisced to ballistic pollen dispersal (Fig. 7h).
Moreover, we investigated the regulation of pollen
dispersal in individual microsporangia, male cones and
trees. For one microsporangium, the slit dehisced from
the middle part to the ends (Fig. 7i). The dehiscence
took place from bottom to top for individual male cones
(Fig. 7j), and the male cones opened from exterior to
interior for an entire tree.
DISCUSSION
Significance of Male Reproductive Structures in
Relation to Wind Pollination
The morphology of male cones and their relationship
with their ambient leaves play significant roles in pollen
dispersal in wind-pollinated species (Niklas 1985;
Di-Giovanni and Kevan 1991). Urzay et al. (2009)
pointed out that the catkin-like or long filament anthers
are effective in pollen dispersal due to a stochastic
aeroelastic mechanism. In addition, Niklas (1985) sug-
gested that the spiral male cones are particularly
well-designed for pollen dispersal. In G. biloba, the
male cones are catkin-like with many helical microspor-
ophylls, which are the typical characteristics of wind
Fig. 6. Development of sporangial wall in G. biloba. (a) Sporangial wall with two to three layers in September. (b) Sporangial wall
with five to six layers in December. (c) Sporangial wall with seven to eight layers in February. (d) Sporangial wall with nine to eleven
layers and many starch grains in March. (e) Outer cells of the endothecium stretching in a radial direction. (f) Fibrous thickening in
the endothecium. (g) Tapetum originated from sporogenous tissues in early March. (h) Tapetum showing dense cytoplasm and two
or three nuclei in a cell during microspore mother cell period. (i) Tapetum showing many vesicles and tending to disintegrate. (j)
Tapetum showing nuclear shrinkage and cell wall collapse at mitosis. (k) Tapetum completely disappeared during pollen
maturation. Enendothecium; MMCmicrospore mother cell; Nnucleus; Ppollen; SGstarch grains; STsporogenous
tissues; SWsporangial wall; Tatapetum; Tetetrad. Scale bars50 mm.
LU ET AL. *POLLEN DISPERSAL GINKGO BILOBA L. 977
pollination. Furthermore, Di-Giovanni and Kevan
(1991) proposed that the vegetation around male
reproductive structures in wind-pollinated species
increases mechanical turbulence, which can overcome
the threshold imposed by molecular adhesion and
effectively release the pollen into the air (Jackson and
Lyford 1999). In this study, we observed that male cones
and leaves were clustered in the fertile short shoots in
Ginkgo, and the male cones were located below the
leaves. Moreover, we noticed a complex relationship
between the male cones and the surrounding leaves. The
pendulous male cones of G. biloba were more or less
perpendicular to the horizontal leaves during pollina-
tion. This positional relationship can decrease the
disturbance of leaves in pollination. However, it is not
known whether the special relationship of male cones
and leaves produces more harmonic atmospheric turbu-
lence to enhance the efficiency of wind pollination.
Unfortunately, we could not determine this from these
data, and this problem needs to be studied further.
Pollen grains of G. biloba also show several traits for
facilitating wind pollination. Schwendemann et al.
(2007) investigated whether the presence of sacci and
the change in shape of the pollen of many conifers could
contribute to a reduction in settling speed and an
increased dispersal distance by adaptation to the aero-
dynamics of wind pollination. Although the pollen
grains in G. biloba do not have sacci, they could
dehydrate rapidly after release from microsporangia,
which could cause the shape of the pollen grains to
change from hemispheric to boat-like (Tekleva et al.
2007). We speculate that two advantages of pollen
dehydration contribute to pollen dispersal. First, the
desiccation of the pollen could reduce its weight and
increase its time floating in the air allowing it to travel
further, as demonstrated by Niklas (1985) and Friedman
and Barret (2009). Second, the evolution of a boat-like
shape might facilitate its carriage on the wind. Addi-
tional aerodynamic models are needed to determine if
these pollen shapes are particularly adapted to wind
pollination. Moreover, Ginkgo pollen is small (30 mm)
and has cavities within the ectexine that are believed to
increase the floating in the air. Although smooth surface
ornamentation is thought to be beneficial for pollen
dispersal in wind-pollinated species (Niklas 1985), many
spinules appear outside the pollen wall in G. biloba.Why
the spinules exist in the pollen of G. biloba and what
roles they play in wind pollination requires further
investigation.
Fig. 7. Longitudinal slit formation and pollen dispersal in G. biloba. (a) Formation of longitudinal slit at the facing sides of both
microsporangia. (b) The trapped and sunken sporangial wall at the longitudinal slit. (c) Longitudinal slit. (d) Longitudinal slit in
detail. (e) The broken epidermis. (f) The opening of the tapeta. (g) Shrunken epidermis and opening longitudinal slit. (h)
Microsporangium dehiscence. (i) Regulation of pollen release for microsporangium. (j) Regulation of pollen dispersal in male cones.
Ddyad; LSlongitudinal slit; Sgmicrosporangium; Ppollen; SW sporangial wall; Th fibrous thickening. Scale bars 200
mm (a,g); 100 mm (b,c); 50 mm(df); 2 mm (h,i); 1 cm (j).
978 CANADIAN JOURNAL OF PLANT SCIENCE
Mechanism of Microsporangial Opening
In wind-pollinated species, pollen grains are dispersed as
soon as microsporangia (anthers) open. Therefore,
dehiscence of microsporangia is essential for wind
pollination (Keijzer 1987). In wind-pollinated angios-
perms, the mechanism of anther opening consists of
complex physiological and physical processes (Keijzer
1999). In Oryza sativa, pollen pressure combined with
the inward bending of the locule walls, as well as the
mechanical outward bending of thickened endothecium,
cause anther opening and pollen launching (Matsui
et al. 1999). Keijzer et al. (1996) have shown that
enzymatic cell wall breakdown leads to the formation
of the dehiscent cavity in maize. These studies have
mainly focused on angiosperms, and little work has been
done on microsporangial opening mechanism of
gymnosperms. In this study, we found in G. biloba
that the cells of the longitudinal slit ruptured first, and
then the sporangial wall was bent outward due to the
combination of shrinkage of the epidermis and thicken-
ing of the endothecium, which led to microsporangium
opening. These results suggest that the longitudinal slit
and the thickened endothecium are a response to
microsporangial opening in G. biloba. In angiosperms,
the enzymic dissolution and swelling pressure of pollen
are important in rupturing the tapetum (Keijzer 1987).
The tapetum of G. biloba displayed active metabolism
during the process of sporangial wall development;
therefore, we propose that the rupture of the tapetum
might be induced by enzymic dissolution. Unfortu-
nately, from our data, we could not determine whether
the swelling pressure of pollen is involved in the rupture
of the tapetum, and further studies are required. In
addition, we demonstrated that opening is sequential
and regular within an individual microsporangium, male
cone or tree in G. biloba. This suggests that the opening
regulation of male cones could prolong their pollination
period and balance the asynchrony during the develop-
ment of male and female trees.
Systematic Significance of Male Reproductive
Structures
During the development of male reproductive struc-
tures, G. biloba displays many unique and primitive
characteristics:
1. Microsporophyll. Mundry and Stu
¨tzel (2004) specu-
lated there is a common ancestor of the microspor-
ophyll in Gnetales, Ginkgoales, Cordaitales and
Coniferales, which displayed simple microsporo-
phyll with a terminal cluster of free microsporangia.
If a leaf-like tip is present after the reduction of the
adaxial microsporangia, the result would be a
conifer microsporophyll (Mundry and Mundry
2001). However, in cycads, the microsporophylls
are pinnate, with synangia on reduced leaflets,
which is the same as the radial synangia groups
seen in some pteridosperm taxa (Mundry and
Stu
¨tzel 2003). In this study, the microsporophyll
of G. biloba displays a sterile extension in the
adaxial position, which is a reduction of the adaxial
sporangia of primitive microsporophyll. Thus, our
results support a close relationship between
Ginkgoales and Cycadales, as was also suggested
by Mundry and Stu
¨tzel (2004).
2. Sporangial Wall. The two main sporangial wall
types (i.e., eusporangiate walls and leptosporangi-
ate walls) are widely distributed throughout the
gymnosperms, probably evolved many times in the
group, and are considered an important taxonomic
characteristic (An et al. 2007). In cycads, the
sporangial wall contains six to seven layers of cells
and is eusporangiate in form (Ouyang et al. 2004;
An et al. 2007). However, in conifers, the spor-
angial wall contains three to five layers of cells and
is leptosporangiate in form (Cao et al. 2001). In the
current study, we found that the development of the
sporangial wall in G. biloba is of the eusporangiate
form. It displays 911 layers of cells consisting of
the epidermis, endothecium and tapetum. The
above results therefore indicate that the develop-
ment of the sporangial wall in G. biloba is similar to
that seen in cycads.
3. Microsporogenesis and Pollen Development. Two
main types of microsporogenesis (i.e., simultaneous
division and successive division) occur in gymnos-
perms. Both simultaneous and successive divisions
are recorded in cycads and conifers (Ouyang et al.
2004; Rudall and Bateman 2007). In Ginkgo, the
tetrahedral tetrads resulted from simultaneous
meiotic divisions. During the different pollen
dispersal stages, gymnosperm pollen grains contain
a variable number of cells after two to five mitotic
divisions (Fernando et al. 2010). In cycads, the
microspore undergoes two asymmetric cell divisions
to produce three cells axially aligned with a
prothallial cell, an antheridial cell and a tube cell
(Ouyang et al. 2004). In conifers, pollen is shed at
the one-cell stage (no division in Taxaceae and
Cephalotaxaceae) (Wang et al. 2008), two-cell stage
(a generative cell and a tube cell) (Chichiricco
`and
Pacini 2008) or the five-cell stage (two or more
prothallial cells, a sterile cell, a generative cell and a
tube cell in Pinaceae and Podocarpaceae)
(Fernando et al. 2005) after several rounds of cell
division. In Ginkgo, the microspore undergoes three
uneven cell divisions to form four cells, including
two prothallial cells, an antheridial cell and a tube
cell in an axial row. In addition, the first prothallial
cell of Ginkgo aborts before pollen dispersal.
Fernando et al. (2010) considered that the evolution
of the pollen showed the reduction of its compo-
nent cells. Moreover, two major innovations
occurred within cycads and Ginkgo, namely the
haustorial pollen tubes and motile sperms, which
represent original groups of gymnosperms.
LU ET AL. *POLLEN DISPERSAL GINKGO BILOBA L. 979
Based on above analyses, we propose that Gink-
goales, cycads and conifers are not homologous and
Ginkgoales may originate from species that are more
ancient than conifers but younger than cycads.
ACKNOWLEDGEMENTS
We thank Dr. Tong Chen of the Institute of Botany, the
Chinese Academy of Sciences, for reviewing our manu-
script, and International Science Editing for correcting
the English. We would also like to thank Xiao-xue
Jiang, Lei Zhang, Hong Li, and Liang Tang for their
help in the laboratory and field. This work was
supported by the Scientific Research Foundation for
high level talents, Yangzhou University (2008) and the
National Science Fund of China (30870436).
Ackerman, J. D. 2000. Abiotic pollen and pollination:
ecological, functional, and evolutionary perspectives. Plant
Syst. Evol. 222: 167185.
An, L. H.,Wang, B. J. and Ji, C. J.2007. Review of
reproductive characters in Ginkgo and Cycads. Acta. Bot.
Boreal-Occident. Sin. 27: 23392345 [in Chinese, English
abstract].
Brown, R. C. and Lemmon, B. E. 2005. g-Tubulin and
microtubule organization during microsporogenesis in Ginko
biloba. J. Plant Res. 118: 121128.
Cao, Y. F., Xin, H., Chu, Q. G., Lu, R. Y. and Tao, S. R. 2001.
Studies on the development of tapetum and middle layer of
microsporangium in Platycladus orientalis. Acta Bot. Boreal-
Occident. Sin. 21: 546550.
Chichiricco` , G. and Pacini, E. 2008. Cupressus arizonica pollen
wall zonation and in vitro hydration. Plant Syst. Evol. 270:
231242.
Christianson, M. L. and Jernstedt, J. A. 2009. Reproductive
short-shoots of Ginkgo biloba: a quantitative analysis
of the disposition of axillary structures. Am. J. Bot. 96:
19571966.
Di-Giovanni, F. and Kevan, P. G. 1991. Factors affecting pollen
dynamics and its importance to pollen contamination: a
review. Can. J. For. Res. 21: 11551170.
Doyle, A. J. 2010. Function and evolution of saccate pollen.
New Phytol. 188:69.
Fernando, D. D., Lazzaro, M. D. and Owens, J. N. 2005.
Growth and development of conifer pollen tubes. Sex Plant
Rep. 18: 149162.
Fernando, D. D., Quinn, C. R., Brenner, E. D. and Owens, J. N.
2010. Male gametophyte development and evolution in extant
gymnosperms. Int. J. Plant Dev. Biol. 4:4763.
Friedman, W. E. 1987a. Growth and development of the male
gametophyte of Ginkgo biloba within the ovule (in vivo). Am.
J. Bot. 74: 17971815.
Friedman, W. E. 1987b. Morphogenesis and experimental
aspects of growth and development of the male gametophyte
of Ginkgo biloba in vitro. Am. J. Bot. 74: 18161830.
Friedman, J. and Barrett, S. C. H. 2008. A phylogenetic
analysis of the evolution of wind pollination in the angio-
sperms. Int. J. Plant Sci. 169:4958.
Friedman, J. and Barrett, S. C. H. 2009. Wind of change:
new insights on the ecology and evolution of pollination
and mating in wind-pollinated plants. Ann. Bot. 103:
15151527.
Gifford, E. M. and Foster, A. S. 1989. Morphology
and evolution of vascular plants. 3d ed, WH Freeman and
Company, New York, NY.
Hirase, S. 1896. On the spermatazoid of Gingko biloba. Bot.
Mag. 10: 325328 [in Japanese, English abstract].
Jackson, S. T. and Lyford, M. E. 1999. Pollen dispersal models
in quaternary plant ecology: assumptions, parameters, and
prescriptions. Bot. Rev. 65:3975.
Keijzer, C. J. 1987. The processes of anther dehiscence and
pollen dispersal. I. The opening mechanism of longitudinally
dehiscing anthers. New Phytol. 105: 487498.
Keijzer, C. J. 1999. Mechanism of angiosperm anther
dehiscence, a historical review. Pages 5567 in C. Clement,
E. Pacini, and J. C. Audran, eds. Anther and pollen: from
biology to biotechnology. Springer-Verlag, Berlin, Germany.
Keijzer, C. J., Leferink-ten Klooster, H. B. and Reinders, M. C.
1996. The mechanics of the grass flower: anther dehiscence and
pollen shedding in maize. Ann. Bot. 78:1521.
Leslie, A. B. 2008. Interpreting the function of saccate pollen
in ancient conifers and other seed plants. Int. J. Plant Sci. 8:
10381045.
Liu, X. Q., Li, C. S. and Wang, Y. 2006. The pollen cones
of Ginkgo from the early cretaceous of china, and their bearing
on the evolutionary significance. Bot. J. Linn. Soc. 152:
133144.
Lu, Y., Lin, M. M., Wang, L. and Jin, B. 2009. Observation of
pollen germination and pollen tube growth of Ginkgo biloba L.
J. Chinese Electr. Microsc. Soc. 28: 426431 [in Chinese,
English abstract].
Matsui, T., Omasa, K. and Horie, T. 1999. Mechanism
of anther dehiscence in rice (Oryza sativa L.). Ann. Bot. 84:
501506.
Mundry, I. and Mundry, M. 2001. Male cones in Taxaceae
s.l. an example of Wettstein’s pseudanthium concept. Plant
Biol. 3: 405416.
Mundry, M. and Stu
¨tzel, T. 2004. Morphogenesis of leaves
and cones of male short-shoots of Ginkgo biloba L. Flora 199:
437452.
Niklas, K. J. 1985. The aerodynamics of wind pollination. Bot.
Rev. 51: 328386.
Ouyang, H. B.,Li, Y.,Zhang, S. Z.,Li, N. and Wu, H. 2004.
Microsporogenesis of Cycas elongata and its systematic
implication. Acta. Phytotaxon Sin. 42: 500512 [in Chinese,
English abstract].
Owens, J. N., Takaso, T. and Runions, C. J. 1998. Pollination
in conifers. Trends Plant Sci. 3: 478485.
Rudall, P. J. and Bateman, R. M. 2007. Developmental bases
for key innovations in the seed-plant microgametophyte.
Trends Plant Sci. 12: 317326.
Schwendemann, A. B., Wang, G., Mertz, M. L., Mc Williams,
R. T., Thatcher, S. L. and Osborn, J. M. 2007. Aerodynamics
of saccate pollen and its implications for wind pollination.
Am. J. Bot. 94: 13711381.
Sze, H. C.1951. On the cuticular structure of Metasequoia.
Sci. China. 2: 239243 [in Chinese, English abstract].
Tekleva, M. V., Polevova, S. V. and Zavialova, N. E. 2007.
On some peculiarities of sporoderm structure in members
of the Cycadales and Ginkgoales. Paleontol. J. 41:
11621178.
Tomlinson, P. B. 1994. Functional morphology of saccate
pollen in conifers with special reference to Podocarpaceae.
Int. J. Plant Sci. 155: 699715.
980 CANADIAN JOURNAL OF PLANT SCIENCE
Tredici, P. D. 2007. The phenology of sexual reproduction in
Ginkgo biloba: ecological and evolutionary implications.
Bot. Rev. 73: 267278.
Urzay, J., Smith, S. G. L., Thompson, E. and Glover, B. J.
2009. Wind gusts and plant aeroelasticity effects on the
aerodynamics of pollen shedding: a hypothetical turbulence-
initiated wind-pollination mechanism. J. Theor. Biol. 259:
785792.
Wang, B. Y., Su, J. R., Fernando, D. D., Yang, Z. H., Zhang,
Z. J., Chen, X. M. and Zhang, Y. P. 2008. Development of the
male reproductive structures in Taxus yunnanensis. Plant Syst.
Evol. 276:5158.
Wang, L.,Jin, B.,Lu, Y.,Jin, X. X.,Teng, N. J. and Chen, P.
2010. Developmental characteristics of the ovule and its
biological significance in Ginkgo biloba L. J. Beijing Forest
Univ. 32:7985 [in Chinese, English abstract].
Whitehead, D. R. 1983. Wind pollination: some ecological and
evolutionary perspectives. Pages 97108 in L. Real, ed.
Pollination biology. Academic Press, Orlando, FL.
Wolniak, M. S. 1976. Organelle distribution and
apportionment during meiosis in the microsporocyte of Ginkgo
biloba L. Am. J. Bot. 63: 251258.
Zhang, Z. M.,Ji, C. J.,Yang, X. and Lee, C. L. 1999.
Cell divisions during microsporogenesis and development of
the male gametophyte in Ginkgo biloba. Acta. Bot. Sin. 41:
479483 [in Chinese, English abstract].
Zhou, Z. Y. and Zheng, S. L. 2003. The missing link in Ginkgo
evolution. Nature 423: 821822 [in Chinese, English abstract].
LU ET AL. *POLLEN DISPERSAL GINKGO BILOBA L. 981
