Introduction

 
Young ginkgo trees in late autumn, Dec. 2005, ICU campus Ginkgo biloba L. (previously Salisburia adiantifolia) common names: ginkgo, maidenhair tree, 40 crowns tree; there are many Chinese names; in Japan called icho, and the fruit is called ginnan. When young, pyramidal in shape, with slender upright branches (this is not always the case). When older, more spreading and broader in the crown (also, not always the case). Can grow over 35 metres with stem up to 10 metres in girth. Leaves deciduous, scattered on long shoots, crowded at the apex of short shoots. The short shoots are a very interesting aspect of ginkgo. These grow very slowly, producing a crown of new leaves every year, they also produce the microsporangia and ovules. They will, after a number of years growth (this varies) then produce a long shoot with scattered leaves. Long shoots usually produce a terminal short shoot for a few years following. This ability to produce short shoots may be one reason why the tree takes so long to grow (compared to other gymnosperms), it may also have something to do with why the tree is a great survivor.

Leaves are stalked an are variable in size and shape. Generally they are bi-lobed, without midrib, and irregularly crenate. Veins repeatedly fork and sometimes fuse (anastamose). Ginkgo is dioecious, and sporangia arise with the leaves on the short shoots. Microsporangia are catkins, 3-6 on a shoot, each being a pendulous axis bearing numerous stamens loosely arranged. The stamen is a short stalked knob, with 2-4 anthers which dehisce on the long axis. Macrosporangia are usually borne 1-3, more or less erect on the shoot. Each consists of a long stalk which bears an ovule on either side (sometimes three) below the apex. Ovule sessile, straight, surrounded by a collar at the base, and naked.

Fruit: a drupe-like seed, with orange flesh covering a woody shell. The embryo (sometimes 2 or 3) with 2 or 3 cotyledons. The fruit produce large amounts of butyric acid in the autumn, when they drop, and is a particularly nasty smell!

Elwes and Henry described a number of varieties in their publication in 1906. These were variegata, pendule, macrophylla laciniata, triloba, and fastigiata. I have been able to find none of these in Japan so far, although I have heard that there are two specimens of the pendulous variety in the gardens of government house in New Zealand. However, I have found three varieties in Tsukuba. These are a small yellow-leaved variety, an ordinary green leaved variety, and an intermediate of these two. The "ordinary" variety appears to be the most common in the area and in other places I have visited. There is also a variety that produces very small fruits.

Another interesting phenomenon, which may be a variety, is the hatsuki, or ginkgos which grow fruits on their leaves. These were first mentioned in the Japanese literature over 100 years ago, so I guess they've been well known for longer than that in Japan. They are very rare trees, only two are known in Ibaraki prefecture, and both have been labelled living national treasures. These trees produce fruits and leaves normally as well, and only some leaves acquire fruits. The fruits are sterile. There are no obvious attributes of ginkgo to distinguish the sexes, apart from observing the emergence of the sporangia in the Spring; but as the trees generally don't 'flower' for 50 years, its rather a long time to wait!! It is possible to distinguish the sexes by chromosome morphology, because the female lacks one tiny satellite on one chromosome. To tell the sexes apart seems to be important only in countries other than Japan and China, because of the rather bad smell of the fruit in autumn male trees are preferred. However, the Japanese people certainly don't seem to care and don't mind the smell, and many people collect the nuts for food. I have read that the male trees tend to be more upright, pyramidal, and the females more compact with lower branches and some pendulous, but I have seen the opposite of these attributes. And anyway, it would be better to be able to tell the sex from the seed. Perhaps an antibody is the answer.

Chi-chi (nipples) usually develop on old trees. The ginkgo at Kew (a male) is just starting to develop them now and it is about 200 years old. However, I've seen them quite well developed on a female tree known to be 56 years old. Chi-chi occur singly or in clusters and can take root if they reach the ground. Their function wil be described in another paper.

Distribution and origins

The Ginkgoales has origins to the Permian, some 200-225 million years ago. It is know to have reached its peak in Jurassic times, about 100m yrs ago, and the ginkgo leaf has been essentially unchanged since then. Ginkgo was very widespread, and its fossil leaves have been found just about everywhere, including Australia and NZ. I would like to mention that another species Baiera, has fossil leaves very similar to ginkgo juvenile leaves and those leaves on shoots that arise from the base of the tree.

From this history it is clear that no other plant has a stronger claim to be called a living fossil, a term used by Darwin to designate survivors of the past. This tree was thriving 125 m years ago when dinosaurs still roamed the earth and the genus has remained from that time virtually unchanged. Ginkgo biloba is the sole living member of a once great and dominant race of plants. It is thus a most precious and tenuous link between the present and remote past.

Ginkgo now survives in the wild state only in a remote mountain region in eastern China. However, it is much cultivated throughout the world, especially in China, Korea and Japan. In Japan, very old specimens exist in the old gardens of temples, shrines and castles, and there are some famous trees, for example the tree next to the Hachiman Shrine in Kamakura (south of Tokyo and one of the old capitals) is famous because the assassin of an early Shogun hid under the tree before his infamous deed in 1290. This now enormous tree still stands next to the shrine and produces very large amounts of seed every year.

The Japanese also have many ginkgo planted in parks and as avenues, and thousands are planted every year. Tsukuba has a number of ginkgo avenues which promise to be quite magnificent in a hundred years. Why is ginkgo so popular in Japan? There are a number of reasons, one or two are my guesses. What I haven't told you so far is that ginkgo is exceptionally beautiful in the autumn, the leaves turn a yellow gold, looking like "golden ducks feet" one of the Chinese names for it. It is also a symbol of longevity, which is perhaps why it is also popular around temples and shrines. The nuts are tasty, usually eaten in an egg custard like dish called chawanmushi. Ginkgo is also a great survivor in polluted cities, growing where other trees fade away, and is exceptionally resistant to fungus and insect attack.

Why study ginkgo?

When ginkgo was first known to European taxonomists, it was regarded as one of the conifers, and included in the Taxaceae. However, in 1895, Hirase, a botanist at Tokyo University, published his discovery that the ovules are fertilized by motile sperm cells conveyed by tubes similar to pollen tubes. This differs radically from all other conifers and flowering plants, which have non-motile male nuclei. Motile sperms are found only in the lower plants, ferns and cycads. This discovery established the unique nature of ginkgo , which was raised to ordinal rank by Engler. Ginkgo is thus the only living species in the order. We should know more about the detailed structure of the motile sperms, how they differ from other motile sperms, and how the flagellar apparatus is put together. Why hasn't all this been done already? Well, getting hold of the sperms to study is a rather difficult business, because they appear only for a very short time, about two hours, and in any particular area of Japan, all the sperms are formed within a ten to 14 day period. Before I tell you more about how we capture these elusive sperms I should spend some time telling you about ginkgo's reproductive cycle.

Reproductive cycle

The series of events that culminates in the production of ripe seeds takes about 14 months. Pollination, maturation of male and female gametophytes, and fertilization occur in one season (April to September in the northern hemisphere) but embryogeny is not completed until the following spring. Trees don't produce flowers for about 50 years, as I mentioned earlier. This is why another name in China for ginkgo is Kung Sun Shu, or Grandfather/grandson tree. When a person plants a tree, only the grandchildren can enjoy the fruit. I've already given you a basic description of the male and female flowers.

Early ontogeny of the male gametophyte

The microstrobili develop during the summer preceding their Spring emergence, and attain considerable size within the bud by autumn. Over the winter they stay in the microsporocyte state, and meiosis and microsporogenesis occur in the following spring. The observation that male trees usually bear masses of catkins the year following a hot summer may bear some relation to this.

Development of the male gametophyte

This development is very similar to that in the cycads except that there are two prothallial cells instead of one. However, the first prothallial cell becomes very reduced and is totally degenerated by the time of spermatozoid formation. After meiosis and formation of the tetrad the pollen has a single androspermal cell, which divides to produce a small first prothallial cell and an androgenous initial. This larger cell divides to form the 2nd prothallial and an antheridial initial. This antheridial divides to form the generative cell and the pollen tube cell. At this stage the pollen are shed (ie a four-celled condition). The pollen is wind borne to a mucilaginous droplet which exudes from the micropyles of the ovules. The drop retracts (or evaporates) bringing the pollen into the pollen chamber where a haustorial pollen tube forms and the final stages of male gametophyte development take place.

Development of the female gametophyte

At the time of pollination, the young ovule consists simply of an elongate megasporangium surrounded by an integument that leaves a micropyle at the tip. Meiosis and megasporogenesis occur at the time of pollination or soon after. The megaspore usually takes from late March to early August to produce the female gametophyte. I don't wish to describe the development of the ovule except that it is characterized by free nuclear divisions, similar to those in cycads and pinus, for example. After this coenocytic stage, in which about 8000 free nuclei are produced, a cellular stage occurs in which all the integuments and archegonia are formed. There are usually two, sometimes three archegonia. After completion of archegonia formation, a peculiar column of gametophyte tissue becomes elevated between them. In late August, the nucellar tissue and adjacent region of megaspore membrane become destroyed, creating an archegonial chamber.

Fertilization

Over the summer the pollen grain germinates and the haustoria end of the pollen tube attaches to the wall of the pollen chamber where it ramifies, sending out numerous fine rhizoid-like processes between the cells of the nucellus. The prothallial end of the pollen tube elongates and hangs freely in the archegonial chamber. The generative cell divides anticlinally, giving rise to a sterile cell and the spermatogenous cell. Fertilization, in the Tokyo area, occurs in late August-early September over 10-14 day period.

The nucleus of the spermatogenous cell divides without forming an intervening wall (the sperms have no walls) forming two sperms, which quickly develop their spiral motile apparatus. The mature spermatozoid is similar to the cycad sperm, but it is smaller and has only 2.5 turns of the spiral, compared to the cycads 5 or 6. Numerous flagella (10-12,000 in cycads, uncounted in Ginkgo) are attached along the spiral. The pollen tube opens and sperms are released (though they literally squeeze out) into an archegonial chamber liquid, which appears at this stage. As you must realise, the ginkgo sperm doesn't have very far so swim, compared to fern sperm for example. The sperm is very flexible, as we have seen from video shots. When it enters the archegonial neck, the sperm becomes greatly stretched.

Authors disagree as to whether the whole spermatid enters the archegonium, or whether the flagella apparatus is left behind. The nucleus of the spermatozoid separates and contacts the egg nucleus, forming a diploid zygote. It has been suggested that fertilization and embryogenesis may occur either on the tree or on the ground. In our experience at Tsukuba, fertilization occurs on the tree, but as the fruit falls soon after this period, embryogenesis probably occurs on the ground. I'm sure this may differ from region to region and according to the weather. Of course, removing the fruits from the tree doesn't affect fertilization, so it is quite feasible that fertilisation could occur on the ground.

A closer view of sperm cells

First, how to study them. During July the generative cell becomes swollen and divides to produce the spermatogenous cell and a sterile cell, as I have already mentioned. The 2nd prothallial cell quickly enlarges at this stage and is surrounded by the sterile cell. At this stage a blepharoplast develops de novo next to the nucleus, where it divides and one daughter blepharoplast migrates to the other side of the nucleus. At this time also another unusual component of the cell develops, this is a pair of supposedly osmiophilic globules (but not in our specimens) which develop separately and next to the nucleus. These globules are thought to act as chromatin carriers at the head of the sperm before fertilization (Lee, 1955), something that obviously requires some clarification. As these event occur the nucleus becomes elongate and a very distinct nucleolus develops.

Another inclusion, about which nothing is known, is a fibrilogranular inclusion that develops at the prothaliall end of the nucleus. This granule divides as the cell divides, so that each sperm has some part of it. Some authors have reported a similarity of staining (at the light microscope level) between the nucleolus and fibrilogranular body. Certainly there are some similarities at the EM level. At this stage plastids are actively dividing, and it we have managed to preserve the constriction ring (presumably actin) of the dividing plastids. This ring is usually only well preserved after freeze substitution. It is interesting that the plastids are active in division, because this suggest that maternal inheritance doesn't occur in ginkgo, and there would seem little point for the plastids to be dividing otherwise, as there would be no advantage in expiring energy away from the motility system. I'm not sure we can say the same thing for mitochondria. Any comments welcome.

After division, during which the blepharoplasts are associated with the spindle poles, the blepharoplasts break up to form the spiral flagella band. How the spiral is formed we have yet to work out, but multi-layered structures (MLS) structures are quickly established after the break up of the blepharoplast. The spiral band is based on an MLS structure of which little is known, but which is apparently very important phylogenetically, because, at least in the green algae, its presence or absence distinguishes the asymmetrical chlamydomonas types (in which it is absent) and the more advanced asymmetrical types, in which it is present in various forms. The MLS is also present in motile stages of liverworts, Equisetum, and of course the cycads. However, the structure of the MLS varies, being only two-layered in some ferns and (possibly) five layered in ginkgo .

On microtubules

There can be little doubt that the microtubular band functions as an elastic cytoskeleton which maintains the overall shape of the mature gametes. As yet we cannot give any real details of the cytoplasmic events during sperm formation. For example it is clear that during development of the motile apparatus, MLS and nuclear metamorphosis, there are also sequential changes in other cytoplasmic organelles. Jeff Duckett, of the University of London, has written at great length about spermatogenesis in pteridophytes and it would appear there are similarities with ginkgo. Eg investment of plastids by sheets of dilated endoplasmic reticulum, which may represent stages in the formation of microbodies, which are apparently common in pteridophyte sperms. Thus, on very preliminary evidence, there appear to be many similarities between ginkgo sperms and sperms of other plants. However, ginkgo sperms have unique aspects too.

Concluding.....

From Albert Seward 1938, who wrote about the fossil history of ginkgo in Scientific Progress (England) 32:420-440 "Ginkgo has for centuries appealed to the imagination of the Oriental mind: the tree with leaves like Golden Ducks' feet became an object of veneration; a legacy, it might be, from a golden age and as such possessing miraculous power. We, despite our matter of fact Western outlook, pay homage to the sacred tree of the East because its story, written in the sands of time, gives us a vision of enduring life. The maidenhair tree appeals to the historic souls: we see it as an emblem of changelessness, a heritage from worlds of an age too remote for our human intelligence to grasp, a tree which has in its keeping "The secrets of the immeasurable past".

Another quote (from Jeremy Pickett-Heaps)

"the primitive cycads and ginkgo provide beautiful examples of extant intermediates in the evolution of sexual reproduction of the angiosperms and more advanced gymnosperms, having evolved the pollen grain but still utilizing a motile sperm."