LAB 2- LIFE CYCLES I: NON-VASCULAR PLANTS Introduction Major Land Plant lineages. Just as multicellular animals can be loosely categorized as invertebrates or vertebrates, similar broad categories can be used with land plants. There are two convenient ways to divide land plants. One way depends on the mode of reproduction. Some land plants, for example mosses and ferns, do not produce seeds. We will refer to them as non-seed plants. Other land plants, for example pine trees and roses, are seed plants. Furthermore, within seed plants, there are flowering plants (also known as angiosperms, e.g. roses), and non-flowering plants (also known as gymnosperms, e.g. pine trees). A second way is to distinguish them on the basis of certain details of form and physiology, rather than by their mode of reproduction. On this basis we can divide them into tracheophytes (vascular plants) and non-vascular plants. Vascular plants are those land plants that you are undoubtedly most familiar with; pine trees, roses, ferns et al. When you look at tracheophytes, you normally see only the sporophyte stage. In tracheophytes, the sporophyte stage is large and complex in comparison to the gametophyte stage. The sporophyte consists of a shoot (stems and leaves) and roots and contains vascular tissue. The vascular tissue of all tracheophyte sporophytes contains water-conducting cells known as tracheids - thus the name for this group. The gametophyte stage of tracheophytes is tiny and usually unseen. It lacks stems, roots, leaves, and vascular tissue. A simple plant body of this type is called a thallus. The fern gametophyte (see Fig. 2.2) is a good example of a thallus. Non-vascular plants are less well known, most people would recognize moss, but liverworts and hornworts are less commonly known. The relative size and complexity of the two stages of the life cycle is very different from the situation described above for tracheophytes. Here, the gametophyte is usually larger and longer-lived than the sporophyte. The sporophyte is always a simple, unbranched axis with a terminal sporangium. Although some mosses possess rudimentary vascular tissue, they lack tracheids. Background: Diversity of non-vascular plants Liverworts: Common in damper regions of western Washington: many of the green carpets you see on rocks and soil or tree trunks and branches are not moss, but leafy liverworts. Thallose liverworts are also abundant, particularly on damp soil. Liverworts are much more diverse in form than mosses. The two extremes in liverwort gametophyte form are leafy (having leaves and stems, resembling moss) and thallose (a flattened, straplike, lobed body, lacking leaves and stems). 20
Leafy liverworts might easily be mistaken for moss by the layperson. Moss gametophytes are radial in symmetry and liverwort gametophytes are typically dorsiventral. Also, liverwort leaves are usually lobed and invariably lack a midrib, whereas moss leaves are very rarely lobed and often have a midrib. There are three main types of liverworts: I. Simple Thalloid Liverworts - Gametophyte thallus flattened (ribbon-like) with little internal anatomical complexity. The apical meristem of the thallus consists of a single apical cell. E.g.: Pellia. II. Leafy Liverworts - There are 7,000, comprising about 2/3 of known liverworts. Stems and leaves superficially resemble moss but, with a little practice, you can easily distinguish them. III. Complex Thalloid Liverworts - Marchantia polymorpha. The thallus of these liverworts is thicker and more complex with considerable internal differentiation. Easily found in western Washington: Conocephalum and Marchantia may be found growing in damp, shaded places on the forest floor. Lunularia grows on damp sidewalks on campus and is a terrible weed in greenhouse pots, spreading from pot to pot by means of gemmae. Sporophytes While liverwort gametophytes are diverse, sporophytes are quite uniform in appearance and very different from those of mosses and hornworts. Unfortunately, unlike moss sporophytes, which persist for months, liverwort sporophytes are ephemeral and over much of the year they are unavailable for identification purposes. Life cycle: Fertilization of the egg by the sperm is followed by development of the zygote, which grows and differentiates into foot, seta and capsule. In contrast to mosses where the seta elongates immediately, in liverworts the seta does not elongate until the sporangium is mature and environmental conditions are favorable for spore dispersal. At maturity, the sporangium (capsule) is spherical and blackish. Inside the capsule, elongate cells differentiate as elaters, with helical cell wall thickenings for spore dispersal, other cells differentiate as meiocytes (cells that will undergo meiosis to produce spores). Capsules usually split open along four lines of dehiscence (Fig. 3.1). Because of the spiral wall thickenings, dehydration of the elaters brings about twisting movements, which aid in separating the spore masses so they can be dispersed. Dehiscence of the capsule is followed by dispersal of the entire spore contents in a short period of time, whereas in mosses spore-dispersal goes on over a period of weeks or months. Figure 3.1: Liverwort sporophyte showing seta and capsule. Setae are thin, white or translucent, and quite weak (left). Liverwort capsules split along the sides, often into 4 parts, to spread the spores (right). 21
Archegonia and antheridia. In over 90% of liverwort species the antheridia and archegonia are found on separate plants (dioecious). Antheridia are tiny stalked spheres located singly or in groups in the axils of leaves - often on special antheridial branches (Fig. 3.2). At maturity, an antheridium consists of a small stalk topped by a spherical body, which has a sterile layer of jacket cells surrounding spermatogenous cells. Biflagellate sperm differentiate and are ultimately released into dew or rainwater. Archegonia are produced in groups at the apex of branches (Fig. 3.3), a specialized sheath, derived from fused leaves, surrounds and protects the archegonia. The archegonia are flask-shaped, consisting of venter and neck, and are fundamentally similar to those found in all archegoniate plants. Although several archegonia may be fertilized, normally only one undergoes development. At the time of seta elongation, the calyptra (enlarged venter) ruptures and is left behind as the sporophyte emerges. Figure 3.2: Longitudinal section showing an antheridial branch with many spherical antheridia enclosed by leaves. Figure 3.3: Longitudinal section through the apex of a liverwort stem showing four archegonia surrounded on either side by a sheath of fused leaves. HORNWORTS Gametophyte. As in the case of thallose liverworts, the gametophytes are dorsiventral and anchored to the substrate by rhizoids. The thallus may have a much-lobed ruffled appearance (Fig. 3.4A) and in some taxa bears a midrib. There is little internal differentiation except for some taxa, in which mucilage cavities develop as cells break down. The nitrogen-fixing cyanobacterium, Nostoc, enters through pores in the ventral surface and develops colonies in the thallus. Antheridia and archegonia are sunken on the dorsal surface (Fig. 3.4B). Gametophytes may be dioecious or monoecious, depending on the species. Cells of both the gametophyte and sporophyte usually contain a single large chloroplast. In most species, the chloroplasts have pyrenoids, a unique feature in land plants, otherwise found only in algae. Sporophyte. The zygote grows into a multicellular structure that differentiates a foot at its base (Fig. 3.4C). However, instead of a seta and capsule as seen in all other bryophytes, the hornwort sporophyte consists of an intercalary meristem which functions for a long period of time, producing a needlelike or hornlike spore-producing region (Fig. 3.4C). Spores are continuously produced by meiosis at the base of the "needle" and are continuously maturing and being released at the tip. Multicellular pseudoelaters with spirally thickened walls resemble those of liverworts. The wall of the sporophyte is photosynthetic when young and often contains stomata. Older portions of the sporophyte turn dark and release spores by splitting open along two lines of 22
dehiscence. The columella is persistent and is easily seen as one of three thread-like structures, the other two being the valves of the sporangium wall. Figure 3.4: Hornwort- Sporophyte (A,C) and Gametophyte (B). with Nostoc Mosses Gametophytes moss gametophytes go through several stages of development, 1) protonema, 2) bud, 3) gametophyte proper. Protonema. In most moss taxa, spore germination (Fig. 3.5) is followed by a prolonged protonemal stage - a period of growth during which time the gametophyte is a highly branched mass of filaments forming apical cells that give rise to buds (the early leafy stage). A single spore can give rise to numerous buds. In liverworts, the protonemal stage is brief and only one gametophyte is initiated from each short filament. In some moss taxa, the protonemal stage persists from year to year and serves as a means of vegetative reproduction. Figure 3.5 23
A A Fig 3.6: Examples of moss gametophytes. B More than half of moss species produce separate male and female gametophytes (unisexual) while others produce them on the same gametophyte (bisexual). Sex determination in many mosses is based on an X/Y system of dimorphic chromosomes. Sex organs (antheridia, archegonia) are usually surrounded by highly modified leaves making fertile branches easy to spot. Vegetative reproduction is common by a variety of mechanisms, including gemmae. Sporophytes: Moss sporophytes are superficially similar to liverwort sporophytes, consisting of a seta (stalk) and sporangium (capsule). But the details of structure are very different in the two groups. The moss sporophyte is tough and wiry, persisting for many months after spores are shed. The sporangium in mosses may be closed, may open by vertical slits or may be operculate. In closed sporangia there is no dehiscence mechanism and the spores are released by disintegration of the capsule. In species where the capsule opens by means of vertical slits, the process is similar to that in liverworts. In the vast majority of moss species, dehiscence involves an operculum, a lid that pops off, allowing spores to exit through the mouth of the capsule. In operculate species, the mouth of the capsule is usually surrounded by peristome teeth that play a role in spore dispersal, changes in moisture cause alterations in position of the teeth that open or close the capsule mouth. Elaters are absent in mosses. The majority of moss species have a capsule wall that contains stomata. Stomata are not found elsewhere on the plant and are entirely lacking in liverworts. The sporangium usually contains a central column of sterile tissue, the columella, as in hornworts (not liverworts). In the majority of moss species, a seta is present and begins to elongate before the capsule has developed. The calyptra (archegonial venter) is torn loose and worn as a cover over the tip of the sporophyte as it emerges. 24
Types of Mosses True Mosses I. The majority of moss species are in this group (~9,000 species). The most distinctive feature is the structure of the peristome teeth. Peristome teeth uncurl slowly when the air is dry and curl up again when it is moist, gradually releasing spores (Figure 3.7). Figure 3.7: Peristome teeth closed (left) and open (right). True Mosses II (Hair Cap Mosses). The name hair cap is based on the fact that in some genera, e.g. Polytrichum, the calyptra is covered by a dense mass of hair. Peristome consists of 32 to 64 teeth that are fused to a membrane. Spores must shake out from between the teeth - much like salt from a saltshaker. These mosses have a completely integrated water-conducting system and a well-formed cuticle. This may explain why individuals of Dawsonia can reach a height of 60 cm (about 2 feet). Figure 3.8: Morphology of the Sphagnum sporophyte Peat Mosses. Sphagnum grows in dense aggregations in wet areas generating acidic, nutrient-poor and anoxic bogs. The inability of even decomposers to function in such conditions leads to a thick peat mat. In many parts of the world peat is mined, dried, and burned for fuel. Archegonia and antheridia occur on branch apices. Antheridial branches are often reddish and easy to spot. The seta of the Sphagnum sporophyte is short to nonexistent (Fig. 3.8). The capsule appears to be attached directly to the foot, separated only by a constriction. The sporangium has a short, broad columella and an operculum but no peristome teeth. As in other mosses, the calyptra remains attached to the tip of the capsule until just before dehiscence. Dehiscence of the capsules is explosive. Mature capsules shrink, compressing the gases inside until the pressure is sufficient to blow off the lid and shoot the spores into the air. 25
Laboratory Exercise Goal: Observe, diagram and compare the life cycle stages of three representatives of early land plant lineages that are also emerging as model systems: 1- The liverwort Marchantia polymorpha 2- The moss Physcomitrella patens (Physco, for short) 3- The hornwort Anthoceros Other liverworts and mosses will be available to familiarize you with the diversity within these lineages, use the introduction as a guide for your observations. Go ahead and explore! I. LIVERWORTS: The two main types are thallose and leafy. Thallose liverworts: Observe Marchantia polymorpha. Look for: thallose gametophyte, rhizoids, pores (lab 1), archegoniophores (structures containing archegonia), antheridiophores (structures containing antheridia) and sporophytes (check for spores). Diagram the life cycle of Marchantia based on your observations Examine and illustrate gemmae, structures used for asexual reproduction. Pull one out of a gemmae cup, make a wet mount and look for the apical cell responsible for development of the thallus. 26
Other thallose liverworts: observe Conocephalum, a local genus with cone shaped archegoniophores on the gametophyte. Dissect and find the stalk and the umbrella-shaped cap. Fertilization occurs before the stalk of the archegoniophore elongates, when the archegonia are in contact with the surface of the thallus. Look for club-shaped sporophytes under the cap (mostly black, immature ones may be green). Make a squash preparation of a mature sporangium, look for spores and elaters. Leafy liverworts: Examine the local genus Porella, a common epiphyte on alders or maples. Local species are dioecious (separate male and female). Note lack of midrib and lobed leaves, features used to differentiate them from moss gametophytes. Observe the dorsal (top) surface, noting two rows of leaves. Observe the ventral surface and notice what appear to be 5 rows of leaves. The leaves of the two lateral rows each have a small lobe (like a folded corner of a sheet of paper). This fact plus the presence of a third underleaf produces the appearance of 5 rows of leaves. Make a temporary slide mount of one leaf and observe with the compound scope, noting: absence of a midrib (present in some mosses, never in liverworts) and oil bodies within cells, a liverwort synapomoprhy. Dissect an archegonial branch. Under the dissecting scope, pry apart the surrounding leaves. The oldest may have a plainly visible senescent sporophyte from last spring or may contain one that will expand next spring. The youngest should have archegonia 27
II. MOSSES Carefully examine one of the available bigger mosses (e.g. Polytrichum). Notice that the gametophyte is composed of an axis (stem) bearing small leaves (this are not true leaves, the proper term for them is phyllidia ), it may be simple or branched, and there may be rhizoids (root-like filaments) present towards the base of the gametophyte. If your specimen does not have a sporophyte attached, look again for another moss that does. Note that the sporophyte is never branched in these land plants. Use the space below to illustrate and label your gametophyte (stem, leaves and rhizoids) and sporophyte (seta=stalk and capsule=sporangium). Where does meiosis occur in the moss? Is the stalk supporting the capsule (sporangium) haploid (n) or diploid (2n)? Gametangia (sperm and egg -ontaining organs) In Lab. 1 you observed Mnium gametangia as an example of how land plants have an outer covering of sterile cells that protect the developing gametes, refer back to that lab and try observing and hand-sectioning the tips of gametophyes that have not yet produced sporophytes (this is challenging) 28
Sporophyte dissection. Remove plants with mature sporophytes. Identify: calyptra, operculum, peristome teeth, seta. Try sectioning and mounting the sporangium for observation under the microscope. Make a wet mount of spores. Illustrate your observations below. Hygroscopic movement of peristome teeth. Obtain a sporophyte with an open capsule with teeth in good shape. Often older brown capsules work fine. Hold the capsule on the dissecting scope stage. While observing through the eyepieces, breathe on the capsule. The moisture should cause movement of the teeth. If available, observe herbarium specimens of Dawsonia, among the largest known moss gametophytes, reaching 2 feet in height, and with a complex internal structure. SOW PHYSCO SPORES Physcomitrella patens: This moss is used as a model organism for studies of plant evolution, development and physiology. Its entire genome has been sequenced. You will get plates with fully developed gametophytes and sporopohytes that have been growing at 15 o C. Working under sterile conditions in the hood, with flame-sterilized tweezers, transfer sporangia that have spores to plates with agar media. Touch sporangia lightly to 5-6 places on the surface of the medium in a petri dish. Cover and check under the dissecting scope for lightly embedded spores. Place in tray, your TA will place at 25 o C for germination. You will check for germination, protonema, buds and gametophytes during subsequent labs. At the end of the quarter, your culture is yours to keep, take good care of it! 29
PHYSCO LIFE CYCLE Using forceps pick out plants from plates provided. Keep them in a covered petri dish with a little water or moist paper. Study them on the dissecting scope stage keeping them moist at all times. Observe gametophytes and sporophytes. Use wet mounts (slide, specimen, water drop, cover-slip) and observe under the compound scope for more detail. To observe protonema and buds, scoop out a tiny square of agar, remove most of the agar with a dissecting needle, add cover slip, flatten and observe with the compound scope.. Reconstruct and draw the Physco life cycle below 30
III. HORNWORTS (Anthoceros) Observe gross morphology of the gametophyte and sporophyte of a local hornwort. Speculate on the origin of this plant s common name Illustrate below Gametophytes superficially resemble a thallose liverwort. Observe a small piece of thallus in a drop of water on a slide on a dissecting scope stage. Note pores on the ventral surface containing Nostoc (symbiotic Cyanobacteria, seen as dark spots) Sporophytes: Is there a distinct "capsule" (sporangium), as seen in liverworts and mosses? With the naked eye or a hand lens, find the columella (observable as a hairlike strand between the two halves of the sporophyte where it has split open). Make your own wet mount of a fragment of the Anthoceros sporophyte. Note and illustrate: Spores Pseudoelaters (why pseudo?) Stomata (are they also found in liverworts?) A single chloroplast per cell (also true in gametophytic cells) Look at the demo slide of an Anthoceros longitudinal section of the mature sporophyte (CBS B300). 31