MICROSPORANGIA, MICROSPORES, MALE GAMETOPHYTES

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1 LAB 06: Seed Plant Synapomorphies Introduction to non-flowering seed plants (Gymnosperms) A seed is a highly modified megasporangium, so seed plants are heterosporous. We will review important differences between heterosporous non-seed plants and seed plants. There are five lineages of extant seed plants, four of which are gymnosperms (seeds not enclosed in a fruit) and one lineage of angiosperms (seeds in a fruit). We will also review some potentially confusing differences in what the terms dioecious and monoecious refer to when applied to homosporous versus heterosporous plants. MICROSPORANGIA, MICROSPORES, MALE GAMETOPHYTES Microsporangia. There is no fundamental difference in the function of microsporangia in heterosporous non-seed and seed plants. The structure of the endosporic male gametophytes and the way they function, however, is drastically different in the two groups of plants. Microspores and Male Gametophytes: In heterosporous, non-seed plants an entire antheridium develops within the microspore wall (review the structure of the Selaginella male gametophyte). It has jacket cells surrounding a substantial number of spermatocytes. The microspore wall eventually cracks open and many sperm are released and swim to the female gametophyte (in dew, rainwater, pond water). Because seed plants are heterosporous, the gametophytes are endosporic just as they are in heterosporous non-seed plants. But the male gametophyte has undergone substantial reduction so that there is no longer any trace of an antheridium. Male gametophytes of seed plants consist of about 2-6 cells. The microspore, with its tiny internal gametophyte, is carried by wind or animals to somatic tissues in the vicinity of the female gametophyte. It germinates there by producing a tubular outgrowth. Two sperm are ultimately released from the male gametophyte and swim or are conveyed to the female gametophyte. In seed plants the movement of sperm to the female gametophyte is independent of water in the environment. The tiny male gametophytes of seed plants are given a new name because they function in a new way. We call them pollen. The transport and arrival of pollen in the vicinity of the female gametophyte is called pollination. A male gametophyte (pollen grain) usually has one to several vegetative cells. It has a single generative cell that produces two sperm. The male gametophyte also has a single tube cell that directs the development of the pollen tube. In some pollen grains, a distal weak spot on the exine (outer wall) of the pollen grain is the site of pollen tube emergence. The proximal side of a pollen grain is the side that was in contact with other microspores at the completion of meiosis (tetrad stage). The distal weak spot where the pollen 66

2 tube emerges is usually seen as an aperture (opening) in the exine. In many angiosperms, there are multiple apertures and a single pollen grain may develop more than one pollen tube. There are two ways in which pollen tubes function. Haustorial pollen tubes have an exclusively nutritive function. They digest the surrounding tissue for a period of weeks or months. The food obtained is absorbed and used to support continued growth of the pollen tube and maintenance of the male gametophyte. Eventually two flagellated, swimming sperm are produced by the generative cell. They are released from the proximal end of the pollen grain - not from the pollen tube itself. They swim to the egg and fertilization is accomplished. Haustorial pollen tubes are found in cycads and Ginkgo. Haustorial pollen tubes represent the ancestral condition in seed plants, indicating that the pollen tube may not have originally evolved as a sperm delivery system. The haustorial process allows pollen grains to be light and easily transported because food storage in the grains is unnecessary; sperm transport likely evolved later as a secondary function. All other seed plants produce siphonogamous pollen tubes. Siphonogamous pollen tubes grow at varying rates and do varying amounts of digestion of the surrounding tissue, but ultimately also serve the function of conveying the sperm to the egg. The sperm are not flagellated and are therefore non-motile. A comparison of male gametophyte structure and function in heterosporous nonseed plants and seed plants. 67

3 MEGASPORANGIA, MEGASPORES, FEMALE GAMETOPHYTES Megasporangia and megaspores: There are many important differences between heterosporous non-seed and seed plants in the structure and function of megasporangia and megaspores. They are listed below: (1) As a megasporangium begins to develop, other tissues at its base begin to grow upward and eventually entirely surround it except for an opening, the micropyle, at its distal end. There may be a single such coat or integument around the megasporangium or two. Gymnosperms usually have one integument. In angiosperms the ancestral number of integuments is two, but in derived angiosperm families it has been reduced to one. An integumented megasporangium with its internal female gametophyte is known as an ovule. (2) In heterosporous, non-seed plants the number of meiocytes (megasporocytes) that develop within a megasporangium is variable. Thus there may be as few as four megaspores produced per sporangium or there may be many. In seed plants, a megasporangium rarely develops more than one megasporocyte, thus the potential number of megaspores is four. Meiosis usually occurs so that a linear tetrad is formed. The most common developmental pattern is for the three distal meiotic products to degenerate. The surviving single megaspore produces the female gametophyte. Later we will discuss some variations on this theme where bisporic and tetrasporic female gametophytes occur in some taxa. (3) The single megasporocyte is embedded in a solid tissue called the nucellus. The dominant hypothesis with respect to the evolutionary history of the nucellus is that it represents sterile tissue of the megasporangium. (4) The megasporangium of seed plants is indehiscent. In other words, the single megaspore with its internal female gametophyte is not released. This should be contrasted with the behavior of heterosporous non-seed plants in which the megasporangia dehisce and the megaspores, which are not fused to surrounding tissue, are released to the outside world where fertilization of their internal female gametophytes takes place. Because the megasporangia of seed plants are indehiscent, they evolved mechanisms that allow sperm access to the female gametophyte. (5) After fertilization, the integumented megasporangium (ovule) ripens into a seed. The megasporangium stalk has an abscission zone that ultimately breaks down and frees the seed to be dispersed. In non-seed plants, the only dispersal stage is the spore (in addition to vegetative propagules). Female gametophyte. The endosporic female gametophytes are very different in gymnosperms and in angiosperms. In most gymnosperms, the single megaspore grows into a gametophyte that consists of hundreds or thousands of cells and in most cases two or more archegonia differentiate at the micropylar end. In angiosperms, the gametophyte is reduced to a few cells and lacks archegonia. FERTILIZATION, EMBRYO AND SEED DEVELOPMENT In non-seed plants the basic unit of dispersal is the spore (megaspore in the case of heterosporous, non-seed plants). Megaspores do not have a very great range of sizes, at least in comparison to seeds, and all megaspores function more-or-less the same way. Embryos of heterosporous non-seed plants must grow into a mature sporophyte wherever the megaspore containing them happens to fall. Embryos of non-seed plants may grow continuously without undergoing a period of dormancy or may remain quiescent during a period when environmental conditions are unfavorable for growth (winter, dry season, etc.). But prolonged survival is impossible. This should be contrasted with seeds that often contain considerable stored food and may survive for years in a dormant condition. Maturation of a seed involves not only the development of the embryo, but also numerous changes in the integuments as they ripen into a tough, protective seed coat. 68

4 MONOECY AND DIOECY. Cruden & Lloyd (1995) have proposed a common terminology to describe sexual phenotypes and breeding systems in all land plants. The terms monoecy and dioecy necessarily refer to different things in heterosporous and homosporous species. A monoecious species bears both male and female sex organs on the same individual plant (which can be described as bisexual or hermaphroditic), whereas in a dioecious species there are separate male and female plants (individual plants are unisexual). The potential for confusion lies in the fact that in heterosporous plants the "individual" we are referring to is a sporophyte, whereas in homosporous plants the "individual" is a gametophyte. As an example of homosporous plants, consider moss. Mosses and other homosporous plants produce only one kind of sporangium and therefore one kind of sporophyte. The terms monoecious and dioecious in homosporous plants can only refer to gametophytes. A monoecious moss species has archegonia and antheridia on the same gametophyte. A dioecious moss species produces separate male gametophytes (antheridia-bearing) and female gametophytes (archegonia-bearing). In many bryophyte species it has been shown that half of the spores from a particular sporangium produce female gametophytes and half produce male gametophytes. This fact implies that sex determination is a consequence of the segregation of sex chromosomes, as in humans. But not all cases of dioecy result from segregating sex chromosomes. Other genetic sex-determining mechanisms than XY chromosomes are known. It is also well known in other organisms that sex is not always genetically determined; it can be environmentally determined (e.g. in reptiles and fish), although in such a case you would not expect a 1:1 ratio of males to females. For our purposes, the important point is that, in homosporous plants, the terms monoecy and dioecy refer to gametophytes and not sporophytes. Heterosporous plants produce two kinds of sporangia and two kinds of spores. Megaspores develop internal female gametophytes and microspores develop internal male gametophytes. Obviously, it is not possible for eggs and sperm to be produced by the same gametophyte. However, both megasporangia and microsporangia may occur on the same sporophyte or different sporophytes. So the term monoecious refers to species in which a single sporophyte bears both mega and microsporangia. The term dioecious refers to the presence of megasporangia and microsporangia on different individual sporophytes. All extant heterosporous non-seed plant species are monoecious. For example, in Selaginella the strobilus always contains both megasporangia and microsporangia. Seed plants may be either monoecious or dioecious. Monoecy is the most common condition, but dioecy is not uncommon. Pines, for example, are always monoecious, with microsporangia (in pollen cones) and megasporangia (in seed cones) developing on the same tree. Ginkgos, on the other hand are always dioecious, with separate "male" and "female" trees. The terms male and female are in quotation marks to remind you that, technically speaking, the sporophyte is not the sexual stage of the life cycle and cannot be male or female. But biologists routinely ignore that fact and slip into using the terms male and female to describe the sporophyte where dioecy is involved. In flowering plants, flowers are usually bisexual, containing both anthers (which bear microsporangia) and carpels (which bear megasporangia). The term monoecy is usually reserved for the situation in which male organs and female organs are in separate flowers on the same plant. Dioecy is obviously used to describe the situation where male and female flowers occur on separate plants. An example of a dioecious flowering plant is the Holly tree. A female Holly has flowers that bear only female organs and is easily spotted because of the bright red fruits it develops. Male hollies are generally recognizable as mature trees that lack fruit. Cruden, R., and Lloyd, R. (1995). Embryophytes Have Equivalent Sexual Phenotypes and Breeding Systems - Why Not a Common Terminology to Describe Them. Am. J. Bot. 82,

5 Bryophytes Homosporous Non-seed Tracheophytes Homosporous Heterosporous Seed plants Heterosporous Monoecy and dioecy in land plants. REPRODUCTIVE CHARACTERISTICS OF GYMNOSPERMS (1) All gymnosperms have "naked" seeds. In other words, ovules are exposed to the outside world at the time pollination takes place - thus pollen can land directly on or near micropyles. When you see the tightly shut cones of pines or other conifers, you may doubt the truth of the above statement, but at the time of pollination the young cone has cone scales that are separated from each other- leaving a space into which windborne pollen can blow. In gymnosperms that do not have cones, the "naked" aspect of the ovules is much more obvious (e.g. Ginkgo biloba). (2) Most gymnosperm ovules have a single integument (Gnetophytes are an exception). (3) Gymnosperms are wind-pollinated (Cycads and Gnetophytes are a partial exception). In most species wind-blown pollen is trapped by a sticky pollination droplet exuded through the micropyle. Appearance of the droplet often correlates with the breakdown of the nucellar tip, creating a space, which in conjunction with the space between the tip of the nucellus and the integumentary lobes is called the pollen chamber. The way in which pollen is ultimately brought into the pollen chamber varies with species. In some conifer species, pollen is wettable and sinks in the droplet, accumulating at the mouth of the micropyle. As the droplet dries or is metabolically retracted into the micropyle, the pollen is pulled into the pollen chamber. In other species ovules are inverted at the time of pollination, so that the micropyles face downward. Species with inverted ovules invariably have saccate (winged) pollen. Saccate pollen is buoyant and non-wettable and stays on the droplet meniscus while moving upward to the vicinity of the micropyle. As in the previous case, as the droplet dries out and the meniscus recedes into the micropyle, pollen is pulled into the pollen chamber. Some gymnosperms lack pollination droplets, in which case windblown pollen lands directly on the protruding integumentary lobes and germinates there. (4) Most gymnosperm female gametophytes consist of hundreds or thousands of cells and have archegonia (some exceptions). (5) Gymnosperm female gametophytes undergo extensive free-nuclear development before becoming cellular. In other words, the division of the megaspore nucleus is not accompanied by wall formation, likewise the many divisions that follow - so that the megaspore cytoplasm is filled with numerous free nuclei. At some point, wall formation takes place and archegonia differentiate. (6) The early development of the embryo is free nuclear, with some exceptions. The extent of the phenomenon varies widely. For example, in the Cycads there may be over a thousand 70

6 free nuclei before walls develop, whereas in conifers there may be as few as four free nuclei. Angiosperms undergo cellular embryo development from the outset. (7) Cleavage polyembryony (splitting of one embryo into several) is common. Pollination mechanisms in conifers. A, erect ovule, non-saccate pollen sinks into pollination droplet. B-E inverted ovules. B, saccate pollen penetrates pollination droplet. C, pollination droplet not extruded or absent, pollen floats into ovule in rainwater. D, pollen adheres to papillate cells at tip of micropylar lobes. E, pollen germinates on scale, cone axis or bract-scale, long pollen tube penetrates micropyle. Intro to Cycads and Ginkgo Cycads were much more numerous and widespread 150 million years ago when the climate was wet and warm over the entire earth's surface. Cycads are dioecious, with individual plants forming either pollen cones or seed cones, but never both. Seed cones can be very large, often exceeding 20 kg! In the genus Cycas, ovules are attached along the sides of the petiole-like base of the megasporophylls. In the other genera, the spirally arranged megasporophylls are not leaf like. The dominant hypothesis is that the leaf-like megasporophylls of Cycas represent the ancestral form. Cycad megasporophylls. A, Cycas revoluta; B, Cycas circinnalis; C, Dioon edule; D, Ceratozamia mexicana; E, Zamia floridana. 71

7 Male cones bear spirally arranged microsporophylls that are not at all leaf like. They bear numerous abaxial microsporangia that may be arranged in distinct groups like the sori seen on fern leaves. Cycad microsporophylls. A, Cycas; B, Zamia. Pollination, fertilization and embryo growth. For many years it was assumed that Cycads were wind-pollinated. Interestingly, both male and female cones of virtually all genera have been shown to be strongly thermogenic. Thermogenicity in flowering plants is a process that volatilizes insect-attracting odors. Detailed studies of several Zamia species show that pollination is carried out by beetles that feed on starch-rich tissues of the male cone. Although this food source is absent from the seed cones, the pollen-covered beetles are still attracted to the females by the strong odor emitted by their thermogenic cones. It is now thought that, depending on the species, cycads are either exclusively insect-pollinated or pollinated by a combination of factors. About four to five months following pollination, the proximal end of the pollen grain bursts, releasing the two sperm. They swim through the liquid of the fertilization chamber and penetrate the archegonial necks. A single sperm nucleus fuses with each egg nucleus. Diagram of longitudinal sections of a cycad ovule showing male and female gametophytes at the micropilar end. A, pre-pollination ovule. B, ovule immediately before fertilization, with haustorial pollen tubes. Ginkgo Ginkgo is a dioecious species, producing separate male and female trees. Microsporangiate strobili arise in the axils of short shoot bud scales and leaves. A strobilus consists of a stalk bearing numerous spirally arranged appendages, each of which usually has 2 microsporangia at its tip. Ovules are not found in strobili, but develop in pairs at the tip of stalks which arise in a 72

8 position equivalent to that of the microsporangiate strobili - in the axils of short shoot bud scales and leaves. Short shoots of Ginkgo biloba bearing (A) male strobili or (B) ovules. CONIFERS Conifers and Gnetales Conifer reproductive Morphology (1) Cones are unisexual. A cone may bear microsporangia (pollen sacs) or megasporangia (ovules), but not both. This should be contrasted with heterosporous non-seed plants (e.g. Selaginella) where a strobilus usually contains both micro and megasporangia. Some extinct gymnosperms had bisexual cones. Some extant gymnosperms (Gnetales) have cones that bear both pollen sacs and ovules, but they are not functionally bisexual because the ovules in bisexual cones are sterile. (2) Most species are monoecious, but dioecy is not uncommon. In a monoecious species, pollen and seed cones occur on the same tree. A particular branch may produce only pollen cones one or more years and then switch to seed cones for a period of time and vice versa. (3) Simple vs. compound cones. A cone may be thought of as a reproductive short shoot. In all conifer families, pollen cones are simple, meaning it has a cone axis and one set of appendages (scales or microsporophylls). In the Pinaceae, seed cones are compound. The seed cone axis bears first order appendages (bracts) and second order appendages (ovuliferous scales) in the bract axils. In some families, cones appear to be simple because of bract-scale fusions during ontogeny. A, pollen cones are simple, one set of appendages (microsporophylls) bear abaxial pollen sacs (not shown). B, seed cones are compound, first order bracts and second order ovuliferous scales with ovules (not shown). (4) Ovules may be inverted or erect. In Pinaceae, ovules are attached to the scale surface and are inverted (micropyles face inward toward the cone axis). In other conifer families, ovules may be erect (micropyles face outward away from the cone axis). 73

9 (5) Some conifer families have highly reduced seed cones. In the Cephalotaxaceae ("plum yews"), the highly reduced cones bear only one or two ovules, but additional aborted ovules are present and the reduced cone structure is obvious. In Podocarpus the cone is reduced to one to several bracts, often fleshy, with each bract subtending a single ovule that is partially or entirely surrounded by an enlarged ovuliferous scale (the epimatium). (6) The Taxaceae (yews) lack seed cones. In this family, one or two ovules develop terminally on short branches. The base of the ovule develops a fleshy aril. Developmental studies reveal no signs of reduction from a more complex structure and there is no palaeobotanical evidence for a cone-bearing ancestor. However, the wood anatomy and details of reproductive biology are typical of conifers. GNETALES: General features of reproductive morphology Both pollen and seed cones of all three genera are compound. The basic architecture of the cones seems to be fundamentally the same in the three genera. Although the cones of Gnetophytes are unisexual, there is a definite tendency toward bisexuality. In all three genera, sterile ovules may be present in the male cones. Figure 13.2: Diagrammatic representation of gnetophyte cones. The flower-like nature of secondary axes that contain both pollen-bearing structures and sterile ovules can be very striking. However, DNA sequence data from extant gymnosperms indicates that Gnetophytes and angiosperms are not closely related and that their shared morphological characters are the result of convergence, not homology (Hansen et. al. 1999). The Gnetophytes are most likely sister group to the Pinaceae (Burleigh and Mathews, 2004). 74

10 Reproductive morphology of Ephedra. Both wind and insect pollination seem to play a role in Ephedra. In some species male plants produce nonfunctional ovules that produce a sweet pollination droplet that is presumed to play a role in attracting insects to the male plants. Ephedra cones. A-B, pollen cones; C-E, seed; d ovule detail Reproductive morphology of Gnetum. All species of Gnetum are dioecious and most species occur in humid rain forests, a habitat that is unsuitable for wind pollination. Some species are moth-pollinated. (Fused bracts) B C D Male and female cones of Gnetum gnemon. A, microsporangiate strobilus; B, long section through a node showing 4 developing microsporangiate fertile shoots and one abortive ovule; C, megasporangiate strobilus showing ovules and partially developed seeds; D, long section of young ovule. (from Gifford and Foster, 1988) A 75

11 Reproductive morphology of Welwitschia. Welwitschia is dioecious. Cones form at the tips of branched axes that develop in the leaf axils. Welwitschia ovules secrete a droplet with a high sugar content that might be associated with insect pollination, but it might also simply function as a pollen-trapping and germination medium. At present, the limited evidence available suggests that Welwitschia is predominantly wind-pollinated, but that insect pollination also occurs to some extent. Significance of double fertilization in Ephedra and Gnetum. Double fertilization has always been considered a unique event in angiosperms. It is now known with certainty to also occur in Ephedra and Gnetum. In angiosperms, the second fertilization event involves three nuclei in most cases, a sperm nucleus and two nuclei of the female gametophyte. This triploid fusion product develops into a triploid food tissue, the endosperm that nourishes the embryo. In Ephedra and Gnetum, the two sperm entering the female gametophyte each fuse with a single nucleus, producing diploid embryos. Only one embryo matures. The surviving embryo is nourished, as in other gymnosperms, by the food stored outside the archegonium in the other cells of the female gametophyte 76

12 Laboratory Exercise 06- Seed Plant Synapomorphies and gymnosperm diversity Exercise: The goal of this lab is to observe the main seed plant synapomorphies and representatives of the major lienages of non-flowering seed plants (the gymnosperms : cycads, Ginkgo, gnetales and conifers). You will study in lab and in the greenhouse: 1- Ginkgo seed dissections (a seed is a fertilized ovule) 2- Pollen grains (endosporic male gametophytes, at maturity). 3- Examples of secondary growth (wood). 4- Cycad diversity and reproductive structures. 5- Gnetales diversity and reproductive structures. 6- Conifer diversity and reproductive structures. 1-Ginkgo seed dissection: We may have mature seeds (from trees around Seattle) or immature seeds (picked off the trees in midsummer). If the seeds are mature, you will know immediately by the odor (see demo article giving the organic constituents of Ginkgo gas). Ovules are found on stalks that develop in the axils of short shoot leaves, they occur in pairs. One of the paired ovules usually aborts and can still be seen. Make a diagram below 1. If a stalk is present, gently break it off the seed keeping track of the micropylar end of the ovule (away from the stalk). 2. The ovule integument has 3 layers. Peel off the outer fleshy layer and wash the now stony seed in water. 3. Try to peel off the stony middle layer. You may need a hammer to crack (not crush!) this hard layer so you can remove it. Do not section the seed with a razor - leave it intact. Working under the dissecting scope, find two papery layers beneath the stony middle layer of the integument. 4. The outermost papery layer is the third or innermost layer of the integument. Note that it is fused to the other papery layer (which is the megaspore wall) at one end of the seed. The other end of the seed is the micropylar end. Carefully peel off the papery layer of integument, leaving the megaspore wall intact. 5. Holding the seed upright under the dissecting scope, use a needle to peel away the megaspore wall at the micropylar end, revealing a reddish spot. Immediately under the reddish spot, as you peel, you will discover the "tent pole" (a raised section of the female gametophyte- see diagram below). On either side of the "tent pole", you will see a tiny opening. These are the archegonial necks. What lies inside each archegonium? 77

13 6. Slice the gametophyte longitudinally with a razor through the archegonial necks to reveal the archegonia. Notice that the female gametophyte is green - a unique feature in seed plants. Depending on whether the ovule had been fertilized, you may have cut through an embryo in sectioning the gametophyte. Male reproductive structures of Ginkgo biloba: Examine the male strobili in the (previously frozen) material provided. Note catkin-like strobilus with spirals of paired sporangia ("catkins" are long, thin stalks bearing numerous small, windpollinated reproductive structures). Make a diagram below 2. Pollen Grains Make a wet mount of pollen grains from Ginkgo or one of the conifers. Note the two sac-like structures emerging from the pollen wall, can you think of a function for these? Illustrate a pollen grain below What part of the life cycle does the pollen grain represent? What does it carry? 78

14 3. Secondary Growth Secondary growth is a synapomorphy of seed (and woody) plants that occurs as the result of lateral meristematic activity, and produces an increase in the girth of an organ. The two most common lateral meristems in conifers and woody dicots are the bifacial vascular cambium that produces secondary xylem (wood, to the inside) and secondary phloem (to the outside), and the cork cambium that produces only cork to the outside, or both cork and phelloderm (to the inside). The secondary xylem (wood) of a plant provides a permanent record of vascular cambial activity throughout the life of the plant. By contrast, the secondary phloem is constantly being displaced further-and-further out into the bark, and is eventually sloughed off with the old bark. Wood Examine a block of wood. There are usually three planes in which wood is cut for examination. If the cut is at right angles to the long axis of the stem or root, it is a cross or transverse section. Note the growth rings in the transverse section. A growth ring is the amount of secondary xylem deposited by the vascular cambium during one growing season. The xylem elements formed in the spring are larger than those formed later in the growing season. Note the radiating lines in transverse section. These structures are vascular rays. A longitudinal cut along a radius produces a radial section. A longitudinal slice parallel with a tangent but not along a radius is a tangential section. What is the appearance of the vascular rays in radial section? In tangential section? Can you identify the age of the tree when it was cut down? In which years did the tree grow more? Can you identify the heartwood and the sapwood? How thick is the bark? Can you distinguish the cork? 79

15 Obtain a slide of pine wood (Pinus). Compare what you see in the slide to what you saw in the block of wood. You can recognize a transverse section from the configuration of the growth rings. Most of the xylem cells look like thick-walled squares or rectangles in this section. These are the tracheids. Extending along radii are elongated cells, which appear to have cell contents. Unlike the dead tracheids, these cells are alive in wood that still functions in conduction. These radially elongated cells make up the vascular rays. Can you determine in which direction in your slide is the center of the stem? Can you see growth rings? In some places you will note circular areas, which are surrounded with thin-walled parenchyma cells. These areas are vertical resin canals, common in many conifers. Obtain one slide from the wood of an angiosperm species. You will notice the wood differs from pine even at first glance. Probably the most conspicuous features in a transverse section are the large perforations or vessels. Each vessel element has perforation plates at each end. In angiosperm wood, the perforation plates are on oblique end walls, and are scalariform. That is to say, they have several bars that dissect the opening or perforation. A vessel is not a cell, but consists of a vertical series of cells with the end walls missing. What would you conclude about the efficiency of such structures in water and mineral conduction? You will notice that in a transverse section, there are many cells with extremely thick walls and very tiny cell cavities. These cells are wood fibers, which serve to strengthen the wood. Did you see wood fibers in pine? Why is pine one of the so-called soft woods, while oak is one of the hard woods? Can you find cells that have cytoplasm and nuclei in them? These are xylem parenchyma cells, which are actually vertical chains of somewhat elongated cells. What cell types found in the wood of an angiosperm are missing from a gymnosperm (pine)? 4. Cycads A. Seed Cones and Megasporophylls. Observe demos and illustrate below: 1. Cycas megasporophylls. Note the resemblance of megasporophylls to compound leaves. Cycas is the only genus that does not produce a seed cone. 2. Dioon megasporophylls. Although Dioon produces a definite cone, note that the megasporophylls are still somewhat leaf like. 80

16 3. Zamia seed cone. Note the shape of the megasporophylls (not at all leaf like). They are peltate like the sporophylls of Equisetum. B. Pollen cones. Observe demos and illustrate below: Zamia pollen cones. a) Whole cones: Observe dried cones and any living cones present on potted plants. b) Microsporophylls: In a petri dish you will find a cone that has been broken up. Pick up one or two microsporophylls to study under the dissecting scope. Note abaxial position and approximate number of microsporangia. 5. Gnetales: This lineage consists of three genera: Ephedra, Gnetum and Welwitschia. The three are morphologically so distinct that no one, at a glance, would suspect they were even remotely related. But detailed study of their vegetative and reproductive structures and processes, as well as DNA sequences, reveals a close relationship. Examine and diagram living representatives of these 3 lineages. 81

17 6. Conifers: Reproductive structures Pollen Cones. Pollen cones are always simple (i.e. have one kind of cone appendage, called microsporophyll). In Deodar cedar (Cedrus deodara, Pine family) both seed and pollen cones form at the apex of spur shoots. At this time of year, Cedrus pollen cones have mostly abscised and are lying on the ground in large numbers. Obtain a detached, mature pollen cone for study. Pick off one or two scales. Observe with the dissecting scope. How many microsporangia per scale? This number is constant throughout the Pinaceae, and highly variable in other conifers. Make a diagram below. Seed Cones. The structure of seed cones varies enormously depending on the conifer family. In the Pinaceae, the compound nature of seed cones is obvious. Recall that in compound cones, the cone axis bears appendages called bracts. Each bract has in its axil or on its adaxial surface an ovuliferous scale. The ovuliferous scales are interpreted to be highly reduced branches. In other conifer families, bract and scale are completely or partially fused. Take a longitudinal section slide of a young Pinus seed cone back to your bench to observe. Note bracts and ovuliferous scales. The diagram to the left will help you interpret the material, you may want to sketch your own as well below. 82

18 Demo of mature cones of various other Pinaceae (Pine, Doug Fir, etc.). The relative sizes of bract and ovuliferous scale may change as the cones grow. Which of the two units (bract or scale) is largest in the mature cone depends on the particular taxon. Both units are quite large and easily seen in Pseudotsuga menziesii (Doug Fir) because the conspicuous 3-pronged bracts stick out from between scales. In Cedrus the bracts are tiny and difficult to find in the mature cone. Diagram a few different cone types below. Female gametophyte of conifers: We will focus on Pinus, the number of archegonia and other details differ from family to family. Pinus mature female gametophyte (on dissecting scope). Label the diagram below, indicate the ploidy of the innermost and outermost structures. Compare to the Ginkgo ovule/seed you dissected. Ploidy Ploidy Pine nut dissection: remove the seed coat (what part of the ovule is it derived from? What is its ploidy level? ). Using a razor blade, make a longitudinal section and observe under the dissecting scope. Identify the embryo with its cotyledons and central vascular strand (stele). Using a dissecting needle, remove the embryo from the seed and separate out the cotyledons (how many? ). Draw your observations beside the above diagram. 83