Download Fig. 1. Cross-section of a leaf.

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2006 Bio153 Lab 3:
Sexual Reproduction in Plants and the Coevolution of Plants and Pollinators
Feb 6th - 17th
Please read before coming to lab – you may wish to bring your textbook to lab.
Before you move on to examining reproduction in the land plants, you will look at one type
of structure – the leaf – that has contributed significantly to plants’ successful conquest of
the terrestrial environment. You will be looking at the macroscopic structure of leaves and
other structures in the greenhouse lab, but here you have an opportunity to look at the
internal structure of different leaves under the microscope.
Part I: Leaf Structure: To colonize land and compete with other plants, it was
necessary for plants to be able to grow into large structures that could anchor themselves
in the ground, capture light efficiently, and extract water and nutrients from the soil and
transport it around the body. This has involved the evolution of leaves, stems, (collectively
called the shoot), and roots. It appears that these structures have evolved independently
in different plant lineages – current evidence suggests that roots evolved at least twice and
leaves perhaps six times. (If you are interested in this topic, a good recent reference is:
Friedman, W.E., Moore, R.C. and Purugganan, M.D. 2004. The evolution of land plant
development. American Journal of Botany 91: 1726-41.)
In Lab 2, you saw that simple vascular plants such as Psilotum possess small projections
called prophylls. Prophylls differ from true leaves in that they lack vascular tissue. The two
types of true leaves found vascular plants – microphylls and megaphylls – are thought to have
independent evolutionary origins. Microphylls are found in Lycophyta and contain a single
unbranched strand of vascular tissue. It is hypothesized that microphylls arose from sporangia
(spore-bearing structures). Most other vascular plants have leaves with an elaborately branched
vascular system. These leaves are known as megaphylls. The currently accepted hypothesis is
that megaphylls evolved from branched stems. Originally, most plants branched dichotomously
(the stem apex divided to produce two equal branches). In some Devonian plants the two
branches became unequal, so only one continued the growth of the main axis of the plant, while
the other remained short and spread horizontally. This photosynthetic tissue eventually joined
the parts of the short branch. Primitive megaphylls often show dichotomous branching of the
vascular tissue.
¾ Examine the cross-section of a pear leaf. You will
see that it consists of 4 distinct layers:
Fig. 1. Cross-section of a leaf.
1. Upper epidermis: a single layer of cells with a cuticle
on the outer wall.
2. Palisade parenchyma: a layer of long narrow cells
quite tightly packed and with numerous chloroplasts.
Although not clear from a section there are numerous
narrow air spaces in the palisade so a very high
proportion of the surface of the cells is in contact with
air.
3. Spongy parenchyma: a layer of lobed cells with very
large air spaces. Also with some chloroplasts but
not an important photosynthetic tissue.
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4. Lower epidermis: a single layer of cells with stomata (pores to the exterior) scattered
through them. Stomata are formed by two cells known as guard cells that, due to the
irregular thickening of their walls, are able to open and close the pore by the intake and
loss of water, respectively. Epidermal cells are without chloroplasts, but chloroplasts are
present in guard cells.
¾ On display, examine the cross-section of a leaf of Yucca. This is a genus of the dry
areas of western North America. Most of these are perhaps more accurately described
as semi-deserts, so there is a fairly wide diversity of plants in them. Look for stomata
on the leaf. Look for the cuticle on the epidermis. Compare this with the cuticle of
the pear leaf.
¾ On display, examine the cross-section of a water-lily leaf and compare it with pear.
The leaves of water-lily usually float on the surface of water. Look for differences
between the two types and leaves.
Part II: Life Cycles in Plants: In order to understand the evolutionary progression in
terrestrial plants, it is necessary to familiarize yourself with the different types of life cycles
in plants. Life cycles in animals are relatively simple: usually, a diploid (2n) individual
produces haploid (n) gametes, which fuse to produce a diploid zygote, which develops into
the adult animal. In any organism with sexual reproduction, a generation of haploid cells
alternates with a diploid generation. In most animals, there is no question which
generation is more conspicuous or persistent – the diploid individual is what is evident, and
the gametes are just specialized cells. In plants, however, the situation is not so simple.
In single-celled algae the usual state is haploid, but 2 haploid cells may fuse to
produce a diploid zygote, which promptly undergoes meiosis to produce haploid daughter
cells. In higher plants, the diploid zygote does not immediately revert to haploidy, but
grows into a diploid organism called a sporophyte. The sporophyte produces haploid
spores by meiosis. Haploid spores develop into the gametophyte, which mitotically
produces haploid gametes. Gamete union produces a diploid embryo, which is depends
on the gametophyte for nourishment and protection. The embryo develops into the
sporophyte, and the cycle starts again. Thus, plants are characterized by the
alternation of haploid and diploid generations.
In all land plants, the gametophyte and sporophyte generations look very different
from each other. Which generation is predominant (i.e. is the most conspicuous or
persistent) varies among groups. For example, in primitive terrestrial plants such as
liverworts and mosses, the gametophyte generation dominates. Thus, the mosses that you
see growing on trees or rocks are haploid; periodically they produce short-lived structures
shaped like little lamp-posts. These are the diploid, spore-producing sporophytes. In
vascular plants, the sporophyte predominates. The frond of a fern is the sporophyte; the
gametophyte is usually a small, inconspicuous structure clinging to the ground. In seed
plants, the gametophyte is further reduced – it simply becomes part of the seed. In
angiosperms (the most evolutionarily advanced plants), the female gametophyte is only 7
cells (one of them an egg cell), and the male gametophyte is only 3 cells (2 of them
sperm). Thus, the major evolutionary trend in reproduction of terrestrial plants is the
reduction of the gametophyte generation, such that the gametophytes of terrestrial
plants have become smaller and simpler. A second evolutionary trend in the terrestrial
plants is the transition from homospory to heterospory. In homosporous plants,
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the sporophyte produces a single type of spore that gives rise to one type of gametophyte.
This structure bears both the female (archegonia) and male (antheridia) sex organs,
which produce female (egg) and male (sperm) gametes.
Fig. 2. Homosporous life cycle.
In heterosporous plants, 2 types of sporangia (spore-bearing structures) give rise to
2 types of spore - microspores that make male gametophytes, and megaspores, that
make female gametophytes.
Fig. 3. Heterosporous life cycle.
In Lab 2, you examined reproduction in the non-vascular land plants (the bryophytes). In
this lab, we will look at reproduction in seedless vascular plants and the seed plants.
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A: Seedless vascular plants
In early vascular plants we see the shift from the dominance of the gametophyte
generation to that of the sporophyte generation. Rather than being a temporary structure
permanently dependent on the gametophyte, the sporophyte eventually emerges as a
separate, independent entity. However, the early vascular plants are still dependent on
water to complete their life cycle – sperm must swim through water to reach the egg and
produce the diploid embryo. A life cycle typical of the primitive vascular plants is observed
in the club mosses (Lycopoda). Lycopodium is homosporous, meaning that meiosis in
the sporangium forms spores that give rise to bisexual gametophytes bearing both
archegonia and antheridia. The bi-flagellated sperm swim to the egg, and early
development of the embryo occurs in the archegonium. The sporophyte in clubmosses
grows a root and becomes an entity separate from the gametophyte.
Selaginella
Sellaginella is the only genus in this group, but includes 700 known species. Most
live in moist places, but a few are desert plants that lie dormant during the driest parts of
the year (such as the “resurrection plant”). Selaginella is heterosporous; its
megaspores and microspores germinate to form separate male and female
gametophytes. Heterosporous reproduction in Selaginella represents an evolutionary
step forward. Selaginella needs water for reproduction, as its sperm must swim to the egg
in the archegonium. The young embryo is nourished by the megagametophyte, but
eventually emerges from the gametophyte and becomes independent. The sporangium is
able to produce micro- or megaspores depending on its nutritional status. The slides of
Selaginella cones that you will examine will show the random distribution of micro- and
megaspores within a cone.
¾ Examine a longitudinal section of a Selaginella cone (known as a
strobilus). Note the presence of microspores and megaspores, the microand megasporangia, and the sporophylls (the modified leaves that bear the
sporangia).
Pterophyta: the ferns
Most ferns are homosporous, bearing spores in sporangia that are often
clustered in sori on the edges or undersides of leaves. The gametophyte resembles a
liverwort, while the sporophyte is usually structurally complex and large. In ferns, the
sporophyte depends upon the gametophyte during the early phases of its development,
and in some species the gametophyte is capable of supporting several young sporophytes.
Reproduction again requires water, because the motile sperm must swim to the egg.
¾ Examine a fern and note the sori, which contain the sporangia and the
spores. Make sure that you see gametophytes. You may see young
sporophytes growing on them.
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Fig. 4. Life cycle of a homosporous fern.
The final step in plants’ conquest of land came with the evolution of the seed.
Just as the cleidoic egg allowed reptiles to break free of reproduction in water required
by their amphibian ancestors, the seed protected the plant embryo (the young
sporophyte) from desiccation. All seed-bearing plants are heterosporous: they have
microsporangia that produce male microspores and female megasporangia that
produce female megaspores. In seed plants, the megaspore gives rise to a
megagametophyte that is highly reduced and retained inside the megaspore. The
megaspore, in turn, is retained in the megasporangium; the whole structure is called
the ovule. The ovule is enclosed by sporophyte tissue called the integument. Thus,
the whole, vulnerable gametophyte generation that produces the female gamete is
packaged and protected within the tissues of the sporophyte. The first known seed-like
structure appeared in the late Devonian, around 370 million years ago. The
microspore is chaperoned by parent cells in a package called pollen, which is
delivered to the ovule by wind or a pollinator. This process is analogous to copulation in
land animals. The gametes do not need water to produce a zygote.
B: Seed plants
The seed plants include the cycads, ginkgo, gymnosperms, gnetophytes and the
angiosperms. In this lab, we will focus on the angiosperms.
The Angiosperms – the flowering plants
The flowering plants are the most diverse, specialized and complex of the plants. They
appear relatively late in the fossil record – although early terrestrial plant fossils date
from about 470 million years ago, angiosperms appear in the late Jurassic (around 140
million years ago). After their appearance, they radiated rapidly and now represent
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more than 90% of all plant species. Much of their success is due to their complex
associations with pollinators.
Flowers vary greatly in morphology, but are
made up of a consistent pattern of structures.
In general, there are two groups of structures
(fertile and sterile). The sterile structures
include the sepals (collectively the calyx) and
the petals (collectively the corolla). The
sepals are usually green and support the
corolla; the petals are often brightly coloured
to attract pollinators. Collectively, the calyx and
the corolla are called the perianth.
Fig. 5. Generalized morphology of a flower.
The function of the perianth is to
attract pollinators. The fertile structures are the sexual organs (the genitalia of the
plant). The male components are collectively called the androecium (“house of the
male”); the female, the gynoecium (“house of the female”). The androecium is made
up of a variable number of stamens. Each stamen is made up of a filament bearing
the anther, which produces and releases the pollen. The gynoecium is made up of one
or several carpels. The carpel is a modified leaf that contains the ovule. Each carpel
has an ovary at its base, a tube called the style, and a specialized structure at the top
of the style called the stigma, which is designed to receive the pollen. Flowers that
contain both male and female organs are called perfect; those that contain only the
androecium or gynoecium are called imperfect. Flowers may be single or grouped
together in an inflorescence.
Angiosperms are heterosporous. Microsporangia produce
microgametophytes; megasporangia produce megagametophytes. The
gametophytes are now truly parasites on the sporophyte generation; the male
gametophyte is reduced to just 3 haploid cells: 2 sperm cells plus a cell that forms the
pollen tube. The female gametophyte is just 7 haploid cells, one of which is the egg
cell. The remaining 6 cells contribute to the formation of the seed. The term
angiosperm (“enclosed seed”) reflects a unique character of this group – the enclosed
ovule. The ovule of an angiosperm is the organ that contains the female gametophyte,
and within it, the egg cell. Thus, the egg cell is completely enclosed in layers of tissue.
It is thought that the enclosed ovule evolved as a mechanism to prevent herbivory.
Fertilization occurs via the pollen tube which grows out of the pollen (somewhat like
the hypha of a fungus) and burrows through the style to deliver the sperm nuclei.
¾ Look at the cross-section of the Lilium ovule on display.
Following fertilization, the ovule develops into a seed and the ovary wall forms a
fruit. Thus, a fruit is a fertilized, mature ovary. Fruits have evolved as specialized
structures for seed dispersal. Because plants are not motile, there success depends on
being able to exploit other means to move offspring away from the parent plant to
prevent direct competition between parent and offspring for the same resources. Some
fruits are fleshy; seeds are dispersed after the fruit is eaten and the seeds pass
unharmed through the digestive tract of the frugivore (fruit-eating animal). Dry fruits
are inedible and are dispersed by wind, by clinging to the exterior of animals, by
floating, etc.
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¾ Examine the types of seeds and fruits. What are the probable modes of
dispersal in each?
Pollination
Early angiosperms were probably wind pollinated, which has several disadvantages.
First, vast quantities must be produced to increase the chances of pollen reaching a
conspecific. Second, the chance of inbreeding is high, because most pollen falls close to
the parent plant, possibly landing on a closely-related individual. Wind pollination also
doesn’t work well in moist environments, because moisture weighs down the pollen
grain. To increase the probability of trapping pollen grains that float by on the wind, the
ovule exudes sap. In the transition from wind pollination to insect pollination, insects
were attracted to this sap as a food source. By visiting many plants, they transferred
pollen from one to the other. This created selection for the plant to develop
mechanisms to attract insects, and eventually led to the evolution of nectaries (nectarproducing bodies) and conspicuous flowers. By the Cenozoic, other types of pollinators,
including vertebrates (bats and birds) arrived on the scene, contributing to the diversity
of flowering plants.
Coevolution occurs when 2 species exert selective pressures on each other,
resulting in evolutionary changes in both species. Certain flower morphs have evolved
independently several times in response to the characteristics of the pollinators that visit
them. These morphological types are often referred to as “pollinator syndromes”.
Table 1. Some examples of pollinator syndromes.
Pollinator
Bees
- don’t see red
- see in UV spectrum
- diurnal
- land to feed
Butterflies
- good vision
- poor sense of smell
- diurnal
- long proboscis
Moths
- nocturnal
- good sense of smell
- hover feeders
Bats
- nocturnal
- good sense of smell; good vision!
- large head
Birds
- esp. hummingbirds
- see red better than blue
- poor sense of smell
Flies
- poor vision; attracted to carrion, feces
Flower Type
- usually blue or yellow (bees don’t see red)
- often have UV guides (markings visible in the UV
spectrum)
- sweet scent
- landing platform
- narrow floral tube to accommodate the proboscis
- open in daytime
- often red, odorless
- long, narrow floral tube
- landing platform
- often white (visible at night)
- strong smell (esp. at night)
- plants flat or bent backwards
- white
- musty smelling
- sturdy flowers (accommodate bat’s head)
-
bright, often red
little smell
long tube
no landing platform
brownish red, often inconspicuous
smell like rotting meat, feces
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¾ Look at the flowers on display. What are the probable pollinators in each case?
The relationship between plants and pollinators is mutualistic, in that both
receive a benefit from the interaction. At the simplest level, the benefit to the plant is
directed transfer of pollen, and the benefit to the pollinator is a nutritive reward – nectar
and/or pollen. (There are other rewards that we will discuss in lecture.) The plant
provides the reward to ensure that the pollinator will be motivated to visit another plant
of the same type. However, there are costs to both parties in participating in this
relationship. For the plant, the production of nectar (and extra pollen, if the pollinator is
a bee) is energetically costly. For the pollinator, visiting flowers and handling them
(especially while it is learning how to handle the flower) is also energetically costly.
Both parties are trying to maximize their returns and minimize their energetic
expenditure, which can create conflict: the plant “wants” the pollinator to visit many
plants of the same type; the pollinator would “like” to visit only a few plants that provide
substantial rewards. Thus, the plant must balance the benefits of producing a
substantial reward with the costs (not only is it expensive to produce, but a large reward
may produce satiation, discouraging the pollinator from visiting another flower and
depositing pollen!). This cost/benefit asymmetry can have interesting implications for
the coevolutionary paths of plants and pollinators.
Another consideration is that of specialist and generalist pollinators. A plant
benefits from having a specialist pollinator – if the pollinator only visits plants of a
particular species, pollen is always being delivered to the appropriate type of plant.
Sometimes it is also beneficial to a pollinator to specialize on one type of plant – it may
lessen competition with other pollinators. It has been hypothesized that some plants
have evolved extremely complex morphologies with hard-to-access nectaries specifically
for this purpose: by being difficult to handle, they ensure that only one species of
pollinator (or even a small subset of a species that learn the “trick”) will visit the flower.
In these cases, the reward may be high to motivate these pollinators to specialize
exclusively on this type of flower. Yet again, however, there may be conflict: it may be
advantageous for the pollinator to be more generalist, so it can feed on many types of
flowers without having to travel long distances to find food.
Part III: Coevolution experiment
You will work in groups of four to conduct an experiment exploring some of the tradeoffs involved in the coevolution of plants and pollinators. The purpose of the exercise is
to understand how certain variables affect the costs and benefits involved in this
mutualistic relationship.
Materials:
- Eppendorf tubes: 2 types (flip-top and screw-cap)
- 3 Plexiglas holders for tubes
- 2 coloured tubes (2 different colours)
- 2 beakers of water (one clear, one coloured)
- suction tubes
- coloured cardboard
- timers
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You will work with all the students at your bench in this 2-part exercise. Your TA will
give four students (the researchers) a card explaining the experimental design. Do not
show this to the other four students as they are the subjects (the “bees”!) The
researchers will use the materials above to create an array of “flowers”, and test the
bees’ ability to gather the maximum amount of nectar reward in a set amount of time.
By altering characteristics of the “flowers” and the array, the researchers can
demonstrate some of the important variables affecting the plant-pollinator interaction.
Every student will have a chance to be a researcher and a bee.
It is important that the bees don’t know what the researchers are doing, so
no peeking!
Researchers’ Job:
1. Set the experimental array according to the directions on the card.
2. Record the data for each bee according to the directions on the card.
Bees’ Job: A test bee will have a suction tube, and will be let loose to forage on the
array for 2 minutes. The secret is not to go as fast as possible, but as “smart” as
possible. When visiting a flower, the bee opens the tube, sucks once in the nectary, and
closes the tube before moving on. Once the bee flies back to the hive, it deposits the
high-sucrose nectar in one tube and the low-sucrose nectar in the other. Think like a
bee! These experiments will yield meaningful results only if the “bees” make foraging
decisions that are consistent with the constraints faced by real bees foraging on real
flowers. Like all animals, bees should forage optimally (maximize their energy gain
per unit effort; that is, get the most sucrose for the least amount of flying). Flight is
enormously expensive in terms of energy use, so flying madly all over the array is not a
realistic simulation of bee behaviour – this strategy would quickly lead to death. Also,
bees in real life learn what types of rewards are associated with different types of
flowers – bees learn to avoid a flower type with a consistently small reward of poor
quality.
Assessing the results of the experiment:
1. How successful were the bees? The bees maximize their success by getting the
most sucrose for the fewest costs.
bee success =
energy gain
( flight costs + handling costs )
You will get explicit instructions in lab on how to calculate bee success.
2. How successful were the flowers? A flower type visited twice in succession
represents successful pollen transfer. A visit that is not followed by a visit to the
same flower type constitutes pollen loss and “nectar theft” (the plant gives up
nectar while getting nothing in return). For example, suppose a bee visits flower types
A & B in the pattern:
A–B–B–B–A–A–A–B–B–A–A–B–A
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The score for each flower type is obtained by dividing the number of successive
visits by the total number of visits. In the above example, A is visited 3
successive times out of 7 visits to A flowers, and B is visited 3 successive times out of 6
visits to B flowers:
A: 3 successive visits of 7 total visits = 3/7 = 0.43
A–B–B–B–A–A–A–B–B–A–A–B–A
B: 3 successive visits of 6 total visits = 3/6 = 0.5
Fig. 6. Scoring successive visits to flowers.
In real life, over time, the “balancing point” between the costs and benefits to the
flowers and to the bees will set the parameters of the coevolutionary relationship.
The purpose is to gain some insight into the costs and benefits of different strategies
that could be employed by plants and pollinators. Use these results to help you answer
the assignment questions. The results will be discussed as a class, and we encourage
you to discuss these questions amongst yourselves, but each student must submit their
own answers to the assignment questions.
Lab 3 Assignment (4% of final grade)
The assignment questions will be handed out at the end of class, and your TA will notify
you of the date that it is due. Total marks = 12.