VII. Ferns II: The Polypodiales, Reproductive Features, and the
Heterosporous Aquatic Ferns
In this lab, we will continue our investigation of the Polypodiales with an
examination of the reproductive features of the sporophyte phase and a
careful look at the gametophyte phase. We'll also take a look at three
heterosporous genera of leptosporangiate ferns, Salvinia, Azolla, and
Marsilea.
A. Polypodiales: Reproductive Features of the Sporophyte Phase
Almost all of the Polypodiales have sporangia abaxial on the megaphylls and
organized into small to large groups called sori (singular sorus). The sori
vary in shape and position, and the sporangia vary in structure - these sorts
of variation have been traditionally used to distinguish the major groups
within the Polypodiales.
We will begin by studying the sorus and sporangium of one species Cyrtomium falcatum (the Holly Fern) carefully. Then we will go on to
compare it with other ferns.
1. Look closely at a fertile leaf of one of the
Cyrtomium plants in the lab (Fig. 16). The
sori are abaxial in several rows on the
undersurface of the leaf and protected (at least
when young) by a tiny umbrella-shaped
outgrowth of the leaf called an indusium.
2. Now look at prepared sections of the
Cyrtomium leaf with sori.
Fig. 16
a. Identify the upper and lower leaf epidermis and the spongy and
palisade mesophyll (typical of most leaves). The palisade layer - of
thin, close-packed cells - is adaxial in the leaf; the spongy layer, of
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isodiametric, loosely-packed cells, is abaxial. Stomates should be
visible leading from the spaces among the spongy layer cells out to
the leaf exterior.
DIAGRAM a leaf in cross section (indicate the abaxial side); DRAW a
sector that includes a sorus. Choose a slide that shows a good medial
section through the stalk of the indusium.
b. The sori are on the abaxial side of the leaf, as you can see from
their position relative to the spongy layer.
c. Note that the indusium has a stalk (that is a supporting axis like the
handle of an umbrella) and a flange (that is the protective part,
comparable to the umbrella's shelter). Both the indusium and
sporangia are attached to a little mound of tissue containing tracheids
called the receptacle.
d. The sporangia are of different ages. Some are large and old and
contain mature spores. Others are small and hardly developed. The
mature sporangium has prominent red-staining cells in a single row
that make up what's called an annulus, which responds to drying by
forcing the sporangium open, then snapping it shut, to disperse the
spores. In the genus Cyrtomium, the mature sporangia contain
monolete spores.
Remember that this is a leptosporangiate fern. Even in the younger
sporangia, the sporangium wall is one cell thick, not counting the
tapetum. This kind of sporangium also typically has a small number
of spores, in this case 32, and develops from a single initial, not from
a large group of cells. And each sporangium is borne on a long, thin
stalk.
3. The submarginal sorus and false indusium of Pteris and Pellaea
Pteris, another greenhouse fern, has an entirely different sorus from
Cyrtomium, though the structure of the sporangium is virtually the same.
a. Look at the living plants of Pteris in the lab. Notice that the sorus,
though abaxial, is near the margin (submarginal), and that the only
protection for the sorus is the incurled margin of the leaf - called a
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false indusium.
b. Look at prepared slides of Pellaea, a genus with sori very much like
those you just observed in Pteris. You can see that the indusium is
false; it's only an extension of the leaf margin. Notice the trilete
spores. DIAGRAM the Pteris leaf with sorus in cross section.
B. Polypodiales: Gametophytes and Gametangia
Most leptosporangiate ferns have a surface-growing, thin, green
gametophyte with a meristem located in an apical notch. According to the
classical (but not necessarily common) story of gametophyte development,
the antheridia are produced among the rhizoids soon after spore germination,
and the archegonia are produced near the notch later in development.
1. First, take a living fern gametophyte from the supply and share it with
a lab companion. Mount it in water, with the rhizoids up (ask for help in
telling the rhizoids) on a slide and drop a coverslip on top.
Add a little more water to the side of the coverslip from a dropper bottle
if there is air under it. Take a look under low power of the compound
microscope.
2. First, concentrate on form. One end of the gametophyte is covered by
the hair-like rhizoids, which are water-absorbing cells. These are older
gametophytes, so they have a well-organized notch meristem at the end
way from the rhizoids. Look for the single, wedge-shaped apical initial
cell sitting right in the deepest part of the notch.
The gametophyte is flat and very thin. Between notch and rhizoids is a
thicker central region called the cushion. To either side of the cushion
are thinner regions called the wings.
3. Now search the cushion region near the notch meristem for
archegonia. They look like tiny fingers sticking up from the cushion.
4. Now look around your gametophyte at lower power. You may see
swimming sperm. See if you can locate the source of these sperm in a
small, spherical antheridium near the rhizoid end of the gametophyte. If
some of the sperm have not yet begun to swim, look at them under high
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power to see that they are helical and multiflagellate. If you don't see
antheridia and sperm in your slide, look at someone else's.
5. Finally, look at stained slides of Adiantum gametophytes. In these
slides, you can see the structure of the antheridia and archegonia much
more clearly. Young antheridia, composed of only a few cells, and
mature antheridia, full of spermatozoans, should be visible. Notice that
these antheridia are standing out from the surface of the gametophyte, not
sunken in the cushion - a characteristic of only the Polypodiales among
the ferns. The archegonia have a neck protruding above the surface of
the gametophyte and a dark-staining egg cell hidden beneath in the cells
of the cushion. The younger archegonia have intact neck cells; in the
older archegonia, the neck cells have disintegrated to open a canal into
the egg cell for the spermatozoans.
Make a DRAWING of an antheridium and an archegonium for your
notebook.
DIAGRAM a gametophyte to show the location of these organs.
C. The Heterosporous Leptosporangiate Ferns: Salvinia, Azolla, and
Marsilea
Within the Polypodiales are two families of aquatic, heterosporous ferns the Salviniaceae and Marsileaceae. These families together form a
distinctive clade within the ferns. Though these families include very few
species altogether, they are excellent material to study in vascular plant
morphology, because they present a series of highly derived characteristics
that we assume to be homologous with features of leaf and stem in the
Polypodiales. What's more, the features are fun to watch in operation.
1. Salviniaceae: Salvinia
a. Node Design and components in Salvinia
i. Look at the living Salvinia plants in the lab. The sungathering leaves are unusually good at repelling water, which
you can prove by dribbling water onto the leaves in the dish.
ii. Once you're done with the dribble experiment, take a few
nodes back to your workplace and investigate the features of
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Salvinia's node.
- First, note the two simple sun-gathering leaves at each node.
Remove a leaf and look at it under the dissecting scope (or put
the whole shoot under the scope) and look at the distinctive
hairs. These hairs look like eggbeaters: if you put some water
on the leaves under the 'scope, the role of the hairs in water
repelling becomes clear.
- Second, note that there is a third structure at each node. You
should be able to guess that it is involved in absorption, but the
favored interpretation is that it's an absorptive leaf, not a root.
Underwater leaves are often highly dissected like this one, and
this structure shares bilateral symmetry and attachment at the
node with Salvinia's more traditional leaves. There are no true
roots in Salvinia.
Make a carefully labeled DIAGRAM of the shoot design of Salvinia.
2. Azollaceae: Azolla
a. Shoot Layout of Azolla
i. Take a bit of stem axes from the supply of material and make
a simple dissection to understand branching, leaf layout, and
features of the node. First, figure out what is branch and what
is leaf. Look for the alternating leaves in two rows and the
branch points in the axis.
ii. Now look at the layout and design of the leaves. Probe until
you demonstrate to yourself that each leaf does have two lobes,
the upper one of which is larger.
iii. Now look for the attachment location of the roots. They
should be arising from the same place as a leaf, which is at a
node. Branches also arise from nodes: check to see if you can
identify the leaf associated with a branch somewhere on your
axis.
iv. Mount a bit of your shoot system on a microscope slide and
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chop at it a bit with a razor so that the leaves are split open into
small shreds. Now add a drop of water or two and a coverslip,
and look at your preparation under the microscope. You are
looking for small colonies of the cyanobacterium Anabaena they look like greyish-green strings of beads. Remember, these
are the prokaryotes that provide fixed nitrogen to Azolla. In
parts of Asia, Azolla is cultivated in rice paddies for "green
manure".
3. Marsileaceae
a. Living Plants
i. Living plants of Marsilea from the greenhouse are in the lab this
week. They look a lot like four-leaf clover, but they are ferns. Look
closely at the plants - and look at the sample of plant pulled form the
pot to see the basic life form. A simple rhizome creeps around in the
pot, giving rise to leaves and roots.
The leaves of Marsilea are made up of a petiole and four pinnae
clustered at the leaf tip. Notice that the leaf venation is free, not
net-like, as you'd expect to see in a flowering plant such as clover.
Another clue that you are in fact looking at a fern is that vernation is
circinate, just as it is elsewhere in the Polypodiales.
ii. Remove a bit of stem from the covered dish of rhizomes. Make
your own freehand transverse section of the stem. Stain with
phloroglucinol. (Don't forget, phloroglucinol burns holes in clothing).
Under the 'scope, look for a ring of cortical cavities, an inner cortical
sclerenchyma ring, and a central vascular cylinder. The vascular
cylinder is a siphonostele with sclerenchyma instead of parenchyma in
the pith.
iii. Check your interpretation of your hand section by comparing it
with the photomicrograph at the bottom of page 56.
b. Sporocarps
In the lab this week, your teaching fellow has set up a dish with a
sporocarp of Marsilea for you to see dehisce and develop. These
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sporocarps, traditionally interpreted as fused pinnae, are highly
drought-resistant. In fact, we have to file a notch in the sporocarp wall in
order to get them to germinate. The actual process you're seeing is the
extrusion of the group of sori, usually about 10 to 15, out of the
sporocarp wall (Fig. 17).
i. Note the dark, hardened sporocarp wall and the veins protruding
from within, especially later in opening.
ii. Long, thin, ivory-colored sori are evident, first tightly clustered and
later stretched out on a gelatinous axis called a sorophore. Look
closely at these sori - along one side are large megasporangia, each
with a single small bump protruding from the sporangium. The entire
rest of the sorus is much smaller microsporangia. The whole group of
sporangia in the sorus is surrounded by a transparent indusium.
DIAGRAM the sorus-bearing axis and a single sorus.
Fig. 17: Marsilea sorophore, expanded in water
c. When you come to lab next week, look at the mature female
gametophyte and embryonic sporophyte that have developed. Mount one
of the gametophytes on a microscope slide and stain it with a bit of
Toluidine Blue stain to see the sperm lake - the gelatinous funnel through
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which the sperm swim to the archegonium - and the helical,
multiflagellate sperm trapped in the gelatin.
Marsilea rhizome, cross section
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