Green Lab
Green Lab
Green Lab
Objectives
1. Be able to explain the character changes necessary to evolve from a Cyanophora‐like organism to a primitive green alga like
Pyramimonas or Platymonas.
2. Learn the very diverse range of body plans that are found in the chlorophyte and charophytes green algae and understand the
form and function of seagrasses. Be able to give examples of each type shown in the laboratory.
3. Understand haplontic sexual reproduction in the freshwater green algae (i.e. charophyceans).
4. Be able to key out unknown marine green algae using your marine key.
5. Be able to explain and/or identify the terms in the text of this laboratory exercise and listed at the end of this section.
Background
There are about 17,000 species of organisms broadly known as green algae and they occur in marine, freshwater and
terrestrial habitats. More specifically, they occur on rocks, soil, other algae and plants, in the plankton, in symbiotic associations with
fungi (i.e. lichens), a wide range of invertebrates, and in or on the hair of various vertebrates. Ecologically, they can be very
important as primary producers in lakes, ponds, rivers, marine wetlands and tropical reefs.
Taken together, all green organisms (including embryophytes) with a double membrane chloroplast are sister to the
rhodophytes and both of these groups have evolved from a Cyanophora‐like ancestor. There is strong support for the hypothesis
that all of these green organisms form a monophyletic group; some of the homologous characters for green organisms are listed
below. Molecular and phenotypic analyses of phylogenetic relationships within green organisms indicates that there are two major
green sister lineages, both of which have produced taxa that have radiated out of the water and on to the land. The first of these is
the chlorophyte lineage that contains a huge range of bauplans and a number of taxa have independently adapted to an epiphytic
existence on leaves and wood well above the ground; several have also lichenized. The second green lineage is being called the
streptophytes for now and consists of a wide range of basal green algae called the charophytes (the usage of this term is much
broader than its traditional application) in combination with the more derived and terrestrial embryophytes. Unlike for rhodophytes,
modern phylogenetic analyses of green algae (chlorophytes & charophytes) are extremely different from traditional classifications
and indicate than the simpler green bauplans, while ancestral, have evolved multiple times.
Green Plant Characters (traditional green algae + land plants)
A 2‐membrane (i.e. primary) chloroplast with chlorophyll a & b
Stacked thylakoids due to the loss of phycobilisomes
Starch is stored inside the chloroplast and is associated with a
pyrenoid if the latter is present
There is a stellate transition zone of microtubules between the
flagellum and basal body
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2
Green organisms with double membrane chloroplasts. Chlorophytes are the left lineage and streptophytes
(charophytes & embryophytes) are the right lineage. From Lewis & McCourt, 2004.
Exercises
An Example of the Ancestral Green Bauplan
Examine the fresh material of the prasinophyte Pyramimonas or Platymonas.
Note that this genus name indicates that the organism has monad bauplan. Locate
the apical pit, flagella, the orange‐red stigma, and pyrenoid and be able to describe
the shape and position of the chloroplast. Let the cells slow down on their own.
Many green monads are still mixotrophic (specifically osmotrophic) because of the
recent heterotrophic ancestry.
Cells of Pyramimonas have four flagella of equal length that arise from an apical
pit. Four lobes of the cell that are visible with the light microscope surround the
pit. These lobes are a very distinctive feature of Pyramimonas and for several
other taxa in this class. There is one parietal cup‐shaped chloroplast that has
sections extending into each lobe at the anterior end of the cell. A pyrenoid at the
posterior end of the cell and a stigma (eyespot) on the side of the cell close to the
anterior end should be visible. Unlike the majority of green algae, taxa in this class
do not have a cell wall. Instead, taxa like Pyramimonas have three layers of organic
scales to the outside of the plasmalemma (not shown in adjacent figure). Pyramimonas
What character changes would be necessary to evolve from a Cyanophora‐like bauplan to prasinophyte bauplan as
exemplified by Pyramimonas?
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Monads
Study the images of Chlamydomonas, which is in the chlorophycean lineage. This highly studied alga
demonstrates many of the features that are homologous for all green organisms. On these images, be able to
locate and know the function of the following: Cell Wall (glycoprotein cover, not fibrillar cellulose), Cell
Membrane, Flagella, Microtubules of the Flagella Axoneme, Pyrenoid, Starch Grains, Chloroplast (Parietal &
cup‐shaped), Thylakoids, Eyespot, Nucleus, Basal Body, and Mitochondria.
Examine the fresh material of the chlorophycean Chlamydomonas and locate the
flagella and pyrenoid.
Chlamydomonas; f = flagellum, cv =
contractile vacuole, ey = eyespot
(or stigma), c = chloroplast, py =
pyrenoid (from Scagel et al. 1984)
Coenobial Colonies of Monads
A coenobial colony is one in which the shape
and total number of cells in the colony is
genetically fixed during early development. All
of the taxa in this group are colonies of monads
held together by a stiff mucilagenous matrix.
In the diagram of Volvox to the right, the
asexual cycle of these organisms is illustrated in
steps a‐h. After mitosis produces a clone of the
parent colony (called a daughter colony, or
autocolony), the cloned cells invert themselves
(f‐h) in order to get their flagella to the outside
of the colony.
Sexual reproduction is haplontic for coenobial
monads (i‐n). Packets of sperm are made (i‐k)
and after the egg is fertilized (m) a spiny, thick‐
walled zygote is formed (n), which is called a
hypnozygote (also zygospore) because this cell
also functions as a resting, dormant cell – like
the akinete in some cyanobacteria.
From: Van den Hoek et al. (1995)
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Study the images showing different genera of coenobial colonies of monads. Be able to recognize these as
coenobial colonies as well as being able to identify eyespots and differentiated cell types from these images.
Examine the prepared slides of Volvox. One of the slide types shows stained flagella. The other slide types show
daughter colonies for asexual reproduction and primarily young and old hypnozygotes for haplontic sexual
reproduction.
Observe the living colonies of Volvox under the dissecting microscope, which will have daughter colonies.
Coenobial Colonies of Coccoid Cells
D. Scenedesmus
E. Pediastrum
F. Hydrodictyon
c = chloroplast, py = pyrenoid.
Modified from Scagel et al. (1984)
Examine the fresh material of Scenedesmus (above, D).
This taxon is an example of a coccoid colony with coenobia of usually 4 but sometimes 8 or 16 cells. Spines are
often present. Asexual reproduction occurs via autocolony formation in one of the parent cells. The
autocolony is released by enzymatic breakdown of the parent cell wall.
Examine the fresh material of Hydrodictyon (above, F).
This coenobial taxon is also known as ‘water net’ and each sausage‐shaped link in the net is one cell in the
colony. Autocolony formation in Hydrodictyon is a unique accomplishment. Each cell starts off being
uninucleate but a multinucleate condition is achieved by many mitotic divisions (up to 20,000) without
cytokinesis. Each nucleus then forms a cell membrane around it with two flagella. The zoids swim around
inside the parent cell and attach themselves to the inside of the parent cell wall whereupon they lose their
flagella. Each one of these uninucleate cells becomes multinucleate and larger so that all these daughter cells
become connected into a net while still inside the sausage shaped parent cell!! As this process is occurring, the
cell wall of the parent begins to gelatinize until the pressure from the now completed autocolony causes the
parent cell wall to burst. And then the process starts again!
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Multicellular, Filamentous
Examine the fresh material of Stigeoclonium
Stigeoclonium is a uniseriate, branched filament that may also produces hairs when nutrients like phosphate
are limiting. This type of organization has evolved independently many times in the green algae.
Multicellular, Sheets & Tubes
Learn the diagram of isomorphic diplohaplontic reproduction for Ulva, which is one of the most common ulvoid
marine green seaweeds. Ulvoids are primarily marine in distribution and can be very common in estuaries
where they can bloom in response to eutrophication.
Make a cross‐section of the fresh material of Ulva lactuca or Ulva californica and be able to recognize the
distromatic construction of the sheet. If your section comes from a section of the blade that is discolored (often
the edge), you may be able to see the release of zoids (gametes or meiospores, see above image).
Examine the images of tubular (rather than sheet‐like) ulvoids so that you understand what is meant by
tubular. Some of the tubular species of Ulva (formerly species of Enteromorpha) can withstand very low
salinities and so are commonly found in freshwater seeps leading into the high intertidal as well as in brackish
estuarine waters.
Make a cross‐section of Ulva intestinalis to find the tubular morphology. Also, locate rhizoids at the point
where the thallus was attached to the substratum. Would you describe the type of development in Ulva as
parenchymatous or pseudoparenchymatous?
Examine a surface view of the fresh material of Prasiola.
This is a marine and freshwater green alga that forms a very small sheet that you saw in zone 1 during the 356
Zonation Study. It has a reputation for being guanotrophic meaning that it occurs on rocks that are regularly
enriched by bird droppings. Note the very distinctive grid‐like arrangement of cells. Sexual reproduction in this
taxon is not haplo
ntic, diplontic, or diplohaplontic. It has a unique kind of sexual reproduction called somatic sexual reproduction.
Vegetative cells of the sheet are diploid until meiosis converts large patches of cells into being vegetative
haploid cells. Thus the whole sheet can be a patchwork of diploid and haploid cells. The haploid cells do not
remain in a vegetative state for long. Some of these cells differentiate into large, non‐flagellated eggs, whereas
others differentiate into flagellated sperm cells (i.e. gametes are oogamous). Large round mitospores are also
produced and cannot be easily distinguished from eggs.
Siphonocladous (cryptically multicellular, multinucleate)
Examine the fresh material of Valonia if it is available.
Siphonous (= coenocytic: unicellular, multinucleate)
Examine the preserved material of Codium (see below figure) and be able to locate the coenocytic construction,
utricles and gametangia if they are present. Make a wet mount of the C. fragile material that has been
preserved in 4% formaldehyde in seawater. This material was carefully rinsed in clean seawater before being
presented to you but you should use the gloves and protective eyewear just to be safe. You do not need to
make a section. Rather, use your tweezers to pull off some of the outer filaments and make a wet mount. The
below figure from Scagel et al. (1984) will help you to locate the utricles and you may also see the dark, club‐
like gametangia.
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Codium fragile.
E. A mature thallus of Codium fragile, which is made up
of one cell (not including gametangia).
F. Utricles and gametangium from the surface of the
Codium thallus. The only time cross walls are formed is
during reproduction.
g = gametangium; the arrow is indicating a utricle.
Members of the siphonous Bryopsidophyceae, to which Codium
belongs, are almost entirely marine in distribution and are
particularly abundant in sub‐tropical and tropical waters.
Members of this class are considered to have haplontic sexual
reproduction by Van Den Hoek et al. (1995), which is in contrast to
previous treatments that have considered algae like Codium to be
diplontic. Codium is an interesting combination of branched,
coenocytic (i.e. 1 cell, many nuclei) filaments with multiaxial
pseudoparenchymatous development. You also saw this type of
development in the florideophytes. There are two species of
Codium in the northeast Pacific. In C. fragile the branched
coenocytic filaments come together to form a dichotomously branched thallus whereas the same type of
filament forms a crust in Codium setchelli.
Examine the other taxa such as Bryopsis and Caulerpa that have been put out to demonstrate the siphonous
morphology. Be sure you can recognize their organization as being coenocytic.
3 Candidates for the Ancestor of Land Plants (Embryophytes)
Revisit the two phylogenetic trees at the beginning of this laboratory section. The remaining taxa in this
laboratory are all from the streptophytes lineage. All of the ‘green algal’ groups in this lineage are freshwater and
they all have haplontic sexual reproduction. Diplohaplontic reproduction does not occur until the evolution of
embryophytes. Recent taxonomic treatments of the ‘green algae’ have regarded all of the streptophyte algal
groups basal to embryophytes as charophytes. However, when the term charophytes (or stonewort) is used in the
older green algal literature, it is only referring to a very specific group of green algae exemplified by Chara.
1. Zygnematophytes – the Conjugate Greens
There are two main groups of zygnematophytes: the unbranched filaments exemplified by Spirogyra, and the
unicellular, coccoid desmids which have a bilateral symmetry (see below figure). Since flagella do not occur in
either group of zygnematophytes, fertilization during haplontic sexual reproduction is accomplished by
conjugation. Haploid protoplasts (i.e. gametes) must move by amoeboid movement from one strain to a
different mating strain and a hypnozygote is formed.
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Filamentous zygnematophytes:
A. Mougeotia
B. Zygnema
C. Spirogyra
Desmids:
D. Staurastrum
E. Microsterias
F. Cosmarium
G. Closterium
Abbreviations: sc = semicell, c = chloroplast, cw = cell wall, n = nucleus, py = pyrenoid.
From Scagel et al. (1984)
Examine the fresh material of Spirogyra (above, C) and locate the spiral‐shaped chloroplast that is regularly
interrupted by pyrenoids.
Also make wet mounts of Zygnema (above, B) and Mougeotia (above, A), which are also in this group of
filamentous conjugate greens. The chloroplast in Zygnema is axial in position and stellate in shape, whereas the
chloroplast in Mougeotia is axial and band‐like (often a twisted band). Be sure you can ID and describe these
different types of chloroplasts.
Mount some of your Mougeotia in IKI in order to see what a positive starch test looks like. The IKI is staining to
black the starch that is located inside the chloroplast. This test may become necessary when you are keying out
a greenish filament that could either be a green alga (thus a + starch test) or greenish filaments that are
actually not chlorophytes (thus a – starch test).
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Study the image of haplontic sexual reproduction for Spirogyra and be able to point to cells undergoing
fertilization as well as cells that have formed hypnozygotes.
Filamentous zygnematophytes can accomplish conjugation in two ways. In scalariform conjugation, filaments of
opposite mating type become aligned and certain vegetative cells, opposite each other in the two filaments,
become gametangia. Each gametangium produces a papilla toward the other cell and these eventually meet
and fuse so that a conjugation tube (i.e. canal) is formed. This gives a ladder‐like, or scalariform appearance, if a
lot of these conjugation tubes are present. Protoplasts (i.e. gametes) from one ‘gametangium’ move by
amoeboid movement through the tube into the adjacent gametangium where a zygote, and eventually a
hypnozygote, is formed. Meiosis occurs in the germinating hypnozygote and three of the four haploid nuclei
degenerate. Some species undergo lateral conjugation where a tube is formed between adjacent cells within
the same filament.
Which type of conjugation is being demonstrated in the image of Spirogyra?
Examine the prepared slide of Spirogyra and then the fresh material of this taxon and be able to identify the
different states of sexual conjugation.
Study the images of desmids in order to understand their common morphological attributes as well as how
they all divide and sexually reproduce.
Desmids are the second group of zygnematophytes; they are almost always unicellular (some form colonial
chains) with two halves, or semicells, that create bilateral symmetry. The two semicells overlap at the
constriction (isthmus), which is where the nucleus is located.
During cell division, first the nucleus divides and then travels to the opposite pole of each semicell. The two
original semicells then become separate cells via cytokinesis. Each parent semicell then pushes out a new
semicell.
Sexual reproduction in desmids is basically the same as in Spirogyra (i.e. by conjugation), except that in order
for fertilization to occur the semicells of opposite mating strains must open up first in order to fertilization to
occur via amoeboid movement of protoplasts.
Examine the fresh material of Cosmarium and be able to locate the semicells, the position of the nucleus, and
the pyrenoids.
Cosmarium cell contents
Abbreviations: cs = chloroplast
strands, n = nucleus, ns = nuclear
strands, py = pyrenoid.
From Brook (1981)
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Sexual reproduction in
Closterium
Abbreviations: p = papillae,
s = suture lines, v =
vacuoles, z = hypnozygote.
From Brook (1981)
2. Coleochaetales
Study the image of Coleochaete and be able to recognize the pseudoparenchymatous development and the
forming hypnozygotes. Unlike zygnematophyhtes , hypnozygotes are not released from the Coleochaetae disk.
Instead, additional filaments grow around the hypnozygote for added protection. Functionally, this is similar to
the protection provided by the multicellular oogonia in Chara (see below) and the multicellular archegonium in
embryophytes. Sequence data sometimes place Coleochaetae closer to embryophytes than Chara.
Some species of Coleochaetae form disks whereas others are uniseriate, branched filaments. The term
“chaetae” refers to having trichomes that are sheathed at their base by other cells. Those sheaths are not
visible in the image.
Examine the fresh material Coleochaete that will be growing epiphytically on older Elodea leaves. Note how
pseudoparenchymatous development forms a simple disk on the Elodea leaf. See if zygotes are present.
3. Charales – the Stoneworts (the Traditional Charophyceae)
Study the image of Chara and be able to note the following: the oogonium (Queen’s crown), the antheridium
(soccer ball), the whorled arrangement of branches, and the uniaxial pseudoparenchymatous development.
This is a very small freshwater class with about 80 species. Stoneworts receive this common name because they
grow in alkaline ponds where their process for photosynthesis and growth can result in the precipitation of
calcium carbonate into and on their cells walls – hence their stony texture. The stoneworts are the only algae
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with multicellular
reproductive structures.
Both gametangia are
surrounded by shield
cells which are spiral‐
shaped in the case of the
oogonium and lobed in
the case of the
antheridium. In both
Chara and Nitella, the
vegetative plant body
grows by uniaxial
pseudoparenchymatous
development, but in the
case of Chara the main
axis is corticated in a
highly organized fashion,
much the same as what
happens in the red alga
Polysiphonia.
Examine the fresh
material of either Chara
or Nitella in the watch
glass under the dissecting
microscope and be able
to recognize the whorled
node/internode
morphology and the
multicellular gametangia.
Examine the prepared
slide of Chara and locate
both types of
gametangia.
Morphology and reproduction of the Charales (stoneworts).
A. Chara, with both gametangia and multiseriate, whorled filaments.
B. Nitella, with uniseriate, whorled filaments.
C. Nitella, close up
D. Chara oogonium
E. Chara antheridium
F. Chara antheridium
G. Chara, young uniseriate stage growing from a hypnozygote
Abbreviations: bs = basal system, cf = enclosing filaments, co = cover cells, cr = crown cells, es = erect
system, nd = nodes, in = internodes, oog = egg cell, r = rhizoids, st = sterile cells.
Modified from Scagel et al. (1984)
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Introduction to Seagrasses – Marine Secondarily Aquatic Plants
From the derived charophytes, skip over about
400 million years of green line evolution (i.e.
bryophytes, seedless vascular plants, gymnosperms, and
early angiosperms) and go right to the monocots. All of
the 55 species of seagrasses are monocots, but none of
the several seagrass families are actually true grasses
(Poaceae). On the west coast of North America, and
elsewhere, taxa in the Zosteraceae, particularly Zostera
marina (eelgrass), are immensely important primary
producers and providers of habitat in estuaries and
embayments like Humboldt Bay.
Although seagrasses come from different
monocot families, their general morphologies and
anatomies are very similar. They all have roots arising
from rhizomes, and the latter have vertical shoots (i.e.
short shoots, turions) coming off of them. Apical
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meristems are located at the apex of every root, rhizome, and vertical shoot (= turions, lateral shoot). Leaves are
often, but not always, strap‐like. There is a continuous system of aerenchyma from the leaves down through the
vertical shoots and rhizomes and into the roots. The function of this aerenchyma is to pump oxygen down to the
anaerobic parts of the plant. In the
case of the Zosteraceae, monoecious
inflorescences born on a spadix are
surrounded by a spathe. Pollen
grains are filiform (i.e. thread‐like) so
that they can get tangled in the
forked stigma and male flowers do
not mature at the same time as
female flowers. Pollen and later the
fruits and seeds are negatively
buoyant.
Exercises for Seagrasses
Examine the material of Zostera
marina (eelgrass) and
Phyllospadix scouleri (surfgrass)
and be able to locate roots,
rhizomes, short shoots, the
sheath, leaves, apical meristems,
and inflorescences.
Make a thin cross section of a
Zostera leaf and compare it to the
prepared slide of the terrestrial
Vaccinium (Blueberry &
huckleberry genus) leaf cross
section. In particular, compare
the following in the two leaves:
thickness of the cuticle,
presence/absence of stomata,
locations of chloroplasts,
mesophyll anatomy and xylem
lignification. Be able to explain
the adaptive significance of the two different leaf anatomies.
Compare the Zostera leaf section you already made to a leaf cross section you now make from Phyllospadix.
How does the morphology and anatomy of Zostera and Phyllospadix leaves differ? Be able to explain the
adaptive significance of the two different leaves.
Compare the Zostera leaf section you already made to transverse sections of a Zostera rhizome and a Zostera
root. Where is the aerenchyma in each of these sections? What is the adaptive significance of the aerenchyma
system?
Identification of Marine Green Algae & Seagrasses
Use the Gabrielson et al. dichotomous key to identify, to species, marine green algae and seagrasses. Start with
taxa that you know before trying to ID a specimen that you are not familiar with.
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Vocabulary for the Green Algae and Node
Oogonium
Seagrasses Paraphyletic
Parenchymatous development
Aerenchyma Parietal chloroplast
Antheridium Prasinophyceans
Apical pit Primary phloem
Autocolony (daughter colony) Primary xylem
Axial chloroplast Pseudoparenchymatous development
Band‐like chloroplast Pyrenoid
Charophytes Rhizoids
Chlorophytes Rhizome
Coenobium Scalariform conjugation
Coenocytic (siphonous) Secondarily aquatic
Coleochaetales Semicells
Colonial chains of coccoid cells Siphonocladous
Conjugation Somatic sexual reproduction (somatic R!)
Cup‐shaped chloroplast Spathe & spadix flower
Desmids Spiral chloroplast
Distromatic Stellate chloroplast
Filiform pollen Stigma (eyespot)
Forked stigma Stoneworts
Gametangia Streptophytes
Hypnozygote Tubular
Inflorescence Turions (lateral shoot)
Internode Uninucleate
Isthmus Utricles
Lateral conjugation Vascular bundles
Monad Whorled determinate branches
Monocot Zosteraceae
Monoecious Zygnematales (conjugate greens)
Monostromatic
Multinucleate
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