Download 32 LAB 3- VASCULAR PLANT LIFE CYCLES: Lycophytes

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.
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Laboratory Exercise 3
In this lab you will study the synapomorphies of vascular plants by:
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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.
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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.
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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.
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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.
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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.
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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.)
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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.
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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 heartshaped 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.
A
B
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
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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
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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).
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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
s
Figure 5.9: Equisetum strobilus and sporangiophore. A, unexpanded 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
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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.
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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.
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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.
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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.
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Equisetum. Note ribbed stems and scaly leaves. Dissect strobili to find sporangiophores.
Heterosporous (water) ferns.
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