Lycophytes Biodiversity Background the Vascular Plants, Or Tracheophytes, Are a Monophyle

Home , Aglaophyton, Anemia (plant), Angiopteris, Epiphyte, Isoetes, Lycophyte

LAB 3- VASCULAR PLANT LIFE CYCLES: Lycophytes

Biodiversity background The vascular plants, or tracheophytes, are a monophyletic subgroup of the land plants, characterized by: 1-independent, long-lived and branched sporophytes, 2-lignified secondary walls, 3-xylem and phloem (transport/vascular tissue), 4-roots and an endodermis. As you look at the materials in lab, try to identify examples of these four synapomorphies (shared, derived traits).

Phylogenetic tree of vascular plants (includes extinct lineages)

From Cronk, Q. 2009. The molecular organography of plants.

Laboratory Exercise 3 In this lab you will study the synapomorphies of vascular plants by:

- Observing the water-conducting cells of an early vascular fossil - Identifying cell types in a cross-section of a stem of Lycopodium. - Studying members of the earliest lineage of vascular plants, the Lycophytes, including the recently sequenced model lycophyte Selaginella moellendorffii.

Pre-vascular tissue: A cross section of the stem of the sporophyte fossil plant Aglaophyton (Rhynia major) shows that it has an epidermis with stomata, a cortex where some of the cells were associated with a fungus (water and mineral absorption), and a central core of vascular tissue with phloem and xylem-like cells (lacking secondary wall thickenings so they are not considered tracheids). Look at the slide of a cross section of this plant on demonstration and identify the tissues shown below.

Label the Aglaophyton Stem cross-section

Primary vascular arrangement. The arrangement of vascular tissue in the stem and root is referred to as the stele. A summary of stelar types can be found in the cronk book (p. 35). In virtually all tracheophytes, roots have a protostele.

STEM AND ROOT ANATOMY

Locate the following tissues in the cross-section of a Lycopodium Shoot (slide): 1) epidermis, 2) cortex, 3) stele, 4) endodermis, 5) pericycle, 6) protoxylem, 7) metaxylem and 6) phloem region. Label each on the illustration below.

Lycopodium is an early vascular plant that has similar anatomy in stems and roots. This type of stele is typical of most roots, but other vascular plants have a distinct type of stele in stems.

Homosporous Lycophytes- Lycopodium (clubmoss) The most common and conspicuous species in our part of the world are terrestrial plants with long runners, but some local species are also small and grow erect (e.g. Huperzia in field trip site). In the tropics, it is common for Lycopodium species to be epiphytic.

Observe a Lycopodium plant and note the following: Branching. 1. Note the long runner (horizontal stem) with its side branches. The growing point of the runner is at the actively growing end and at the other end older tissues are starting to senesce. 2. Note the dichotomous branching. At most branch points, branching is unequal. One branch is indeterminate and continues as the main axis. The other branch grows more slowly and may be determinate if it produces a strobilus. 3. Geotropic behavior of the indeterminate and (usually) determinate branches is different. The indeterminate branch at each dichotomy grows neither up nor down, but along the ground while the determinate branch tends to show negative geotropism (turns upward).

Leaves. 1. Note the spiral phyllotaxy

2. Note the single vein in each leaf, typical (but not exclusive) of microphylls.

Roots.

1. This plant has only shoot-borne (adventitious) roots. Nonseed plant embryos are not distinctly bipolar. The first root often forms late and may be associated with a leaf. Subsequent roots clearly originate on the shoot. 2. Look at the wiry roots. They branch dichotomously (easier to see in young roots). Dichotomous root branching is unusual, even in non-seed plants.

Strobili. Note that the strobili (cones) form on determinate side branches. Depending on which species you are looking at, the cones may be solitary (one per side branch) or there may be several cones on a branched stalk. In some species, there is a clear distinction between the form of sporophylls (modified leaves associated with sporangia) and vegetative leaves.

Examine a strobilus under the dissecting scope.

How many sporangia per sporophyll? ______

Look for the short stalk of the sporangium. Where is it attached? (axil, leaf surface?)______

How does dehiscence occur (opening of? (by pore? slit?) ______

Examine spores with the compound scope. Use fresh spores from the dissection material. The spores may not wet properly unless you use soapy water in making the slide (in dropper bottle). Put a drop of soapy water on a slide, touch a dissecting needle to the drop and use the wet needle to pick up some spores. Examine with the compound scope.

GAMETOPHYTES. Gametophytes of most Lycophyte species are subterranean and mycotrophic (mutualistic association with fungi). They are not generally available fresh for study. See demo of preserved gametophytes with attached sporophyte.

Heterosporous Lycophytes- (Sellaginella and Isoetes)

Vegetative Morphology of Selaginella kraussiana. This plant is maintained in our greenhouse where it grows vigorously and regularly escapes as a weed.

Observe the potted plants. As in many species of Lycopodium this species of Selaginella is characterized by anisotomous branching (unequal dichotomies). Pick off a healthy green axis, which has 5 or 6 dichotomies in it. Study with the naked eye and dissecting scope. Note adventitious roots. Use transmitted light (from below) so you can see the vascular traces of main axes, side branches and roots. Illustrate below

Reproductive Morphology of Selaginella. Strobili of S. kraussiana. Strobili are usually numerous on these plants, but the young strobili that you need to dissect are very inconspicuous because the sporophylls resemble vegetative leaves. If you cannot locate them ask your instructor for help!

1. Examine a branch with 4 or 5 dichotomies, using the dissecting scope. Look for young strobili out near the tips of the branches. Larger, older strobili are easier to find, but will have megasporangia that have already dehisced.

2. Using forceps and needles on the dissecting scope stage, pry apart the sporophylls and look for mega- and microsporangia. S. kraussiana usually has a single large megasporangium and numerous microsporangia in each strobilus.

3. Find both young strobili with undehisced megasporangia and older strobili in which the megaspores have been shed.

4. Tease out some megaspores into a drop of water and make a temporary slide mount (do not squash them). Observe their surface features using the compound scope.

5. Microspores are too small to handle separately, but a temporary slide mount can be made of an entire microsporangium. Squash the sporangium under the cover slip so its spores will be released. Note the spines on the spore wall, possibly functioning to help the microspores stick to megaspores. Illustrate your observations in the space below.

Challenge: Search for young Selaginella sporophytes in the pots, they are usually still attached to the (endosporic) gametophyte, as in the picture below. Label: megaspore, young sporophyte.

Observe other Species of Selaginella on Display – look at the pattern of branching, phyllotaxy, and leaf shape and form. Specimens include S. moellendorfii, whose genome was fully sequenced in 2011.

Isoetes (quillwort).

This is an unusual plant, most people would mistake it for a monocot! (but you’ll know better…)

A. Gross Morphology. Look at live plants from the greenhouse.

B. Reproductive structures (slides). The drawings below will help you with interpretation.

1. Isoetes microsporophyll and microsporangium l.s. (Triarch A-223-5)

2. Isoetes megasporophyll and megasporangium l.s. (Triarch A-223-7)

Isoetes plant with microsporophylls and megasporophylls.

VASCULAR PLANT LIFE CYCLES: ferns Introduction

Fern Phylogeny (Cronk, 2010)

LEPTOSPORANGIATE FERNS (“FILICOIDS”). This is the largest group of ferns. You will see many species in the field trip. Sporophyte: Some fern species have above ground vertically oriented stems and are often referred to as tree ferns, although they lack secondary growth and are relatively short compared to arborescent seed plants. Most fern species are characterized by the presence of relatively large, compound leaves (usually called “fronds” and composed of pinnae) and an inconspicuous, often subterranean, rhizome. Ferns with a short, erect rhizome tend to do very little branching and to bear numerous leaves arranged in a tight spiral. An example of the latter is our local sword fern (Polystichum munitum). Species with creeping rhizomes often bear relatively few leaves at each branch tip and internodes are relatively long. The ubiquitous bracken fern (Pteridium aquilinum) has an underground, horizontal rhizome and falls into this category, as does the licorice fern (Polypodium glycyrrhiza) which is a common local epiphyte on maples and alders Sporangia. Leptosporangiate sporangia are generally tiny, delicate, stalked structures with relatively few spores (16-64) that are initiated from a single cell and have an annulus, a specialized group of cells involved in dehiscence. The sequence below shows a basic type of leptosporangium. Note the unevenly thickened cell walls of the annulus. Shrinkage and shape change of the annulus cells causes the sporangium to tear open at a weak point, the stomium. Water loss continues until the tension (negative water potential) inside these cells is so great that air bubbles develop. Sudden appearance and expansion of air bubbles causes the cells of the annulus to quickly expand, in turn causing the capsule to spring back towards the closed position - hurling the spores for some distance (Figure 5.1.)

Figure 5.1: Sporangium structure and dehiscence. A. mature sporangium; B, drying of cells of annulus causes them to shrink, tearing open the sporangium; C, sudden release of tension causes the sporangium to snap forward, throwing out spores.

A defining characteristic of ferns is the presence of sporangia on either the abaxial leaf surface or along the leaf margin. Sporangia usually occur in distinct clusters known as sori (Fig. 5.2 A-C). Sori may be covered by a flap of tissue, the indusium or may be naked. If the indusium grows from the abaxial surface it is considered to be a "true" indusium (Fig. 5.2 A, B). In the case of sori present near the leaf margin, the rolled-under edge of the leaf may function as an indusium, in which case it is usually called a false indusium (Fig. 5.2 C).

Figure 5.2: Sori and indusia. A-B, “true” indusia; C, “false” indusium.

Sporangia may occur on all leaves and leaflets or may be restricted to specialized fertile leaves or leaflets (Fig. 10-8). The shape of sori and position on the leaf are also important characters used in identification of species.

Figure 5.3: Sporangia may be restricted to certain fertile pinnae (A, B) or leaves may be dimorphic (C, D) with fertile and sterile leaves. A, Anemia hirsuta; B, Osmunda claytoniana; C, Blechnum spicant; D, Osmunda cinnamomea.

Gametophytes. The classic fern gametophyte shape is that of a heart. But there are numerous other gametophyte shapes. Not all gametophytes are surface dwelling. Some are subterranean and non-green. It is not uncommon for fern spores to require light for germination. Most developing gametophytes pass through an initially filamentous stage and then shift to mitoses in three dimensions, a shift that may require the presence of light. In flattened, surface-dwelling gametophytes, sex organs develop on the ventral surface. In heart- shaped gametophytes, archegonia are usually located on a thickened pad of tissue behind the notch where the apical meristem (apical cell type) is located. Antheridia tend to occur toward the posterior end of the gametophyte as well as on the wings. As in the case of all other homosporous tracheophytes, fern gametophytes are essentially bisexual, but may initially develop only one set of sex organs under the influence of internal physiological factors or due to environmental factors. A common pattern is protandry, in which antheridia are produced first and archegonia develop later as the gametophyte ages. This serves to promote outcrossing between adjacent gametophytes of different ages. In other species it has been shown that interactions between adjacent gametophytes in a population will determine their sex. The faster-growing, larger gametophytes in a population develop archegonia first and secrete hormones, antheridiogens, which stimulate surrounding smaller gametophytes to produce antheridia, another mechanism that promotes outcrossing. Antheridiogens are chemically similar to giberellins, naturally occurring steroid-like hormones that play a number of roles in plant growth and development. Fern archegonia and antheridia are somewhat variable in structure but conform to the generalized structure of these organs. However, a near universal feature of fern archegonia is the reduction of the neck canal to a single binucleate cell. Embryo development. Normally, only one young sporophyte develops on a gametophyte, regardless of the number of archegonia and fertilization events. As the young sporophyte grows, the gametophyte ceases to grow and eventually undergoes senescence. Fig. 5.4 A shows a developing sporophyte attached to the gametophyte. The sporophyte develops a shoot and primary root (the latter will not function for long). The foot is indicated where the gametophyte remains in contact with the sporophyte (Fig. 5.4B).

Figure 5.4: Development of the young sporophyte of Ceratopteris richardii.

B A

Leptosporangiate Heterosporous Water Ferns The heterosporous ferns are all relatively small, aquatic or semi-aquatic plants. Some are found rooted in shallow water and others are free-floating. Marsileales. Grow in damp soil or in shallow water and are adapted to periodic dryness. (Marsilea, Pilularia, Regnellidium). They all have creeping, branched rhizomes. Small, bean-like

structures, sporocarps, develop on the petioles of some leaves, often near the node (Fig. 5.5). The sporocarps contain both mega- and microsporangia.

sporocarps

Figure 5.5: Marsileales showing growth habit, with leaves, rhizomes and sporocarps.

Although they are not fernlike in appearance, the young leaves are circinnate and other features, such as details of sporangial structure and vascular anatomy are typical of leptosporangiate ferns.

Gametophytes (Marsilea). Male gametophytes are relatively simple structurally, consisting of two jacketed antheridia each of which produces 16 sperm. The remarkable thing is that the total time from germination of the sporocarp to the release of swimming sperm takes only 10-12 hours. Megaspores and female gametophytes are more complex. The outer megaspore wall is a multi-layered gelatinous structure that swells up and has a complex convoluted structure (Fig. 5.6A). As in other heterosporous non-seed plants, gametophyte development is endosporic. Female gametophyte development produces a large basal cell and an apical mass of smaller cells in which a single archegonium differentiates. The archegonium has a single neck canal cell and a ventral canal cell. Sperm are chemotactically attracted to the megaspore and swim down the liquefied apical portion of the megaspore wall (the "sperm lake" in Fig. 5.6B). Figure 5.6: Female gametophyte. A, showing gelatinous cover; B, sperm lake and large basal cell.

A B

Salviniales. These are true water ferns in the sense that they are normally found free-floating. The two genera, Salvinia and Azolla, are widespread, although the former is mainly tropical in

distribution. Azolla has importance in rice and other agriculture. Colonies of the nitrogen-fixing cyanobacterium, Anabaena azollae, live in a cavity within the upper lobe of the bilobed leaves. The dense growth of Azolla on the water surface in rice paddies contributes so much nitrogen to the system that fertilizer is not required. Azolla is extensively used in rice agriculture in Asia.

MARATTIALES. This group of ferns is entirely tropical in distribution. Angiopteris and Marattia have an aboveground, massive, erect stem and extremely large, pinnate leaves. The stem may be up to two feet in diameter. They have mucilage canals that develop throughout the plant body. You can see these large plants in the greenhouse. Sporangia. The sori are abaxial (underside of leaf) as in many ferns (Fig. 5.7A). There is no indusium. In some genera the sporangia are partially or entirely fused into a synangium. Marattia is particularly interesting because the two rows of sporangia in a sorus are fused into a clam-shaped structure (Fig. 5.7B), which must open before the individual sporangia can dehisce. Gametophytes: surface dwelling and fairly massive, resembling thallose liverworts, associated with an endophytic fungus. Figure 5.7: Marattia pinna with sori (A) and sorus detail (B).

EQUISETOIDS-Horsetails and scouring rushes

Figure 5.8: Growth forms in Equisetum. A, E. hyemale, aerial branches are perennial, mostly unbranched, not all bear strobili; B, E. palustre, aerial branches annual, some with strobili, extensive second order branching; C, E. arvense, separate sterile and fertile aerial branches, both annual.

Sporangia. In most species, strobili appear at the tips of green aerial shoots that do not differ in form from the sterile shoots, but in some species, as shown in Fig. 5.9C, the strobili terminate specialized, non-green shoots that are short-lived. Tight whorls or spirals of sporangiophores are close-packed on young strobili, giving them their characteristic club shape (Fig. 5.9A). As the sporangia mature, the sporangiophores separate from each other into a looser structure (Fig. 5.9B). Each sporangiophore is a peltate, stalked structure, somewhat like an umbrella that sticks out at right angles to the axis of the strobilus. The head of the sporangiophore is a flattened disc with a polygonal shape. It bears around its circumference a number of fingerlike sporangia that project back toward the strobilus axis (Fig. 5.9C).

sporangiophore

dehiscing sporangium

Stalk of sporangiophore

Figure 5.9: Equisetum strobilus and sporangiophore. A, unexpandeds strobilus; B, expanded strobilus shedding spores; C, single sporangiophore showing dehiscing sporangia.

Mature sporangia dehisce along a line of weakness that runs longitudinally down the sporangium on its inner side - facing the sporangiophore stalk. Spore walls have an unusual feature seen in no other land plant group. The outer wall of the spore consists of four spirally wound bands that are attached at a common point to the inner wall (Fig. 5.10A). As spores dry out, the bands unwind and stick out, a process, which may hasten dispersal by separating spores from each other and

allowing the wind to carry them off (Fig. 5.10B). The wall strips are called elaters, the same term used to describe the separate sterile cells in liverwort sporangia that also aid in dehiscence, but are different in origin and structure from Equisetum elaters. Figure 5.10: Equisetum spores and elaters. A, spore with coiled elaters; B, spore with uncoiled elaters.

Equisetum is homosporus, the spores contain functional chloroplasts and thus appear bright green. As in other plants, the presence of green spores is associated with rapid spore germination and a relatively short spore life span. Vegetative reproduction is the dominant mode of propagation in Equisetum; sexual reproduction is relatively inefficient. Gametophytes. Horsetail gametophytes found in the wild are often unisexual. Environmental conditions may favor initial development of just one kind of sex organs, but they often become bisexual as they age. Gametophytes are irregular surface-dwelling, green cushions with few to many erect lobes (Fig. 5.11). The lower surface bears numerous rhizoids, while the upper surface bears the gametangia.

Figure 5.11: Equisetum gametophyte

OPHIOGLOSSOIDS. Botrychium ("Grape fern") and Ophioglossum ("Adder's tongue fern"). Leaves consist of a sterile and a fertile part (Fig. 1). You may see these in the field trip! Unlike the circinnate vernation (fiddlehead) of all other ferns, the young leaves of Ophioglossoids usually are not circinnate. Recent studies have focused on Botrychium because it exhibits secondary growth. Sporangia. The sporangia occupy a marginal position and do not occur in sori. On the unbranched fertile spike of Ophioglossum, a row of sporangia develops on either side of the rachis (Fig. 5.12A). In Botrychium, sporangia protrude and are particularly conspicuous (Fig. 5.12B), leading to the common name "grape" fern. Sporangia dehisce by means of a slit, releasing thousands of spores.

Figure 5.12: Sporangia morphology. A: Ophioglossum. B: Botrychium. A B

Gametophyte: The gametophyte is subterranean and mycotrophic.

PSILOTOIDS Two genera: Psilotum (Whisk Fern) and Tmesipteris, are mainly tropical in distribution, but Psilotum occurs as far north as Florida and Georgia in North America. Psilotum grows as small, highly branched plants that are either terrestrial or epiphytic (Fig 5.13). The absence of roots is a derived condition. Fertile appendages bear synangia on the adaxial surface. In Psilotum, each synangium consists of three sporangia (Fig. 5.13). Gametophytes are subterranean and branched, with endophytic fungi.

Figure 5.13: Psilotum. 1. Highly branched sporophyte. 2. Fertile appendages (branches) with synangia. 3. Enations (arrows). 4. Synangium composed of three sporangia fused into a single unit.

Laboratory Exercise 3- Ferns

You will be observing Ceratopteris gametophytes that you grew from spores during Lab 1. You will look for gametangia and gametes (any embryos, young sporophytes?). We will also have older plates with young sporophytes and older sporophytes from the greenhouse for you to observe. 1) Follow the instructions in your C-Fern manual for Third period (Day 14), pE5-E7. Answer the questions in the manual and make notes of your observations. Make sure to keep you plates warm (under lamps, away from windows) for sperm observations. 2) Based on your observations, reconstruct the life cycle of Ceratopteris in a diagram below.

3) Examine plates containing a “mystery” Ceratopteris mutant. Use the introductory information to guide your investigation (p44). After observing the phenotype of the mutant Ceratopteris gametophytes, write your hypothesis on the type of mutation affecting them below.

4) Examine one each of the following 4 other fern lineages. See introduction to this lab for guidance. Sketch below: general habit and sporangia for each species observed. Wet mounts and squashes of sporangia for more detailed observation are always encouraged. Variety of Leptosporangiate ferns: note different types of sori and indusia.

Marattia ferns (individual frond of Angiopteris). Note size of frond and observe sporangia fused into synangia.

Ophioglossoid ferns. Note fertile and sterile leaf.

Psilotum. Note three sporangia fused into synangia and reduced, scaly leaves.

Equisetum. Note ribbed stems and scaly leaves. Dissect strobili to find sporangiophores.

Heterosporous (water) ferns.