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Summer Bio153 Lab 3: Sexual Reproduction in Plants and the
Coevolution of Plants and Pollinators
week of July 19
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,
and 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, the sporophyte produces a single type of spore that gives
rise to one type of gametophyte. This structure bears both the female
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(archegonia) and male (antheridia) sex organs, which produce female (egg)
and male (sperm) gametes.
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.
In Lab 2, you examined reproduction in the non-vascular land plants (the
bryophytes). Today, we will look at reproduction in seedless vascular plants and
the seed plants.
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Seedless vascular plants
1. Primitive vascular plants:
In these primitive vascular plants we begin to 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. Unlike in the
bryophytes, where the sporophyte is completely dependent on the gametophyte,
the sporophyte in clubmosses grows a root and becomes a separate entity.
2. 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”). Unlike the other seedless
vascular plants seen in this lab, Selaginella is heterosporous; its megaspores
and microspores germinate to form separate male and female gametophytes.
Again, 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. Although Selaginella is not included with the true vascular plants
(i.e. the Tracheophyta), heterosporous reproduction in Selaginella represents an
evolutionary step forward. 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.
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Examine a longitudinal section of a Selaginella cone (known as a
strobilus). Note the presence of microspores and megaspores, the
micro- and megasporangia, and the sporophylls (the modified leaves
that bear the sporangia).
3. 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
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supporting several young sporophytes. Reproduction again requires water,
because the motile sperm must swim to the egg.
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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.
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. But in seed
plants, the megaspore gives rise to a megagametophyte that is highly
reduced – and is retained in 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.
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Seed plants
The seed plants include the cycads, ginkgo, gymnosperms, gnetophytes and the
angiosperms. Today, 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 – while early terrestrial plant
fossils date from about 470 million years ago, the oldest evidence of
angiosperms appear in the late Jurassic (around 140 million years ago).
However, after their appearance, they radiated rapidly and now represent more
than 230,000 species – 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. 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.
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The life cycle of angiosperms is 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.
•
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.
•
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. (If you visit a lake in the boreal forest during
spring, the shores will be thickly coated with huge amounts of bright yellow pine
pollen.) 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. Regardless, the gymnosperms and several
modern angiosperms continue to be wind pollinated. To increase the probability
of trapping pollen grains, 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.
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This created selection for the plant to develop mechanisms to attract insects, and
eventually led to the evolution of nectaries (nectar-producing 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”.
Pollinator
Bees
- don’t see red
- see in UV spectrum
- diurnal
- land to feed
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
Butterflies
- good vision
- poor sense of smell
- diurnal
- long proboscis
Moths
- often white (visible at night)
- nocturnal
- strong smell (esp. at night)
- good sense of smell
- plants flat or bent backwards
- hover feeders
Bats
- white
- nocturnal
- musty smelling
- good sense of smell; good vision! - sturdy flowers (accommodate bat’s head)
- large head
Birds
- bright, often red
- esp. hummingbirds
- little smell
- see red better than blue
- long tube
- poor sense of smell
- no landing platform
Flies
- poor vision
- attracted to carrion, feces
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- brownish red, often inconspicuous
- smell like rotting meat, feces.
Look at the flowers on display. What are the probable pollinators in each
case?
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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.
Coevolution experiment
You will work in groups of four to design and conduct an experiment exploring
some of the trade-offs 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
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-
2 coloured tubes (2 different colours)
2 beakers of water (one clear, one coloured)
suction tubes
coloured cardboard
timers
You will work in groups of four in this exercise. During any “run” of the
experiment, one pair of students will be researchers, and one pair will be 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 plantpollinator interaction.
How to set up (Read through before starting):
It is important that the bees don’t know what the researchers are
doing, so no peeking!
Researchers’ Job: Design the experimental array
The Eppendorf tubes represent the nectaries of your “flowers”. Some of these
“flowers” are easy to handle (the pop-top tubes); some are harder (the screw
top tubes). You will supply each flower with some nectar. The clear water
represents low-sucrose nectar, and the coloured water represents high-sucrose
nectar (twice the concentration of the clear nectar).
Step 1: Design two types of flowers. Variables that can be altered:
1. difficulty (hard or easy)
2. nectar quality (high or low sucrose)
3. nectar quantity (lots or little nectar)
Assign each of these types of flowers a petal colour (which will be indicated by
coloured paper that you will glue onto the top of the tubes).
Step 2: Decide how many of each type of flower you want, for a total of 18
flowers. (You can have roughly equal numbers of each types of flower, or many
more of one type than the other). Fill the nectaries according to your flower
design. Cut small squares of coloured paper and glue the appropriate colour on
to the top of the tube (it takes a couple of minutes for the glue to dry).
Step 3: While the flowers are drying, decide how you would like to
distribute the 18 flowers among the 3 holders. In the holder on the right-hand,
lower side, place 2 empty coloured Eppendorf tubes. These represent the hive,
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where the bees will deliver their nectar rewards (one for the high-sucrose nectar;
one for the low-sucrose nectar).
hive
Fig. 1. Arrangement of Plexiglas holders for floral array.
Once the researchers have decided on the set up of the array, they will test the
first bee. The second bee should not observe the first bee.
Bees’ Job: Think like a bee!
This experiment 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 may learn to avoid a flower type
with a consistently small reward of poor quality (or one whose nectaries are
consistently empty).
How to forage: a test bee will have a suction tube, and will be let loose to
forage on the array for 1 minute (Tip: 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. Try to suck up
nectar in such a way that the bee can visit either 2 flowers, or suck twice on
the same flower before flying back to the hive. Once the bee flies back to the
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hive, it deposits the high-sucrose nectar in one tube and the low-sucrose nectar
in the other.
Recording Foraging Data:
• One researcher will keep track of amount of each type of nectar
collected (emptying the collection tubes as necessary) and the sequence
of flower types visited (e.g. A-B-A-A-B-B-B-A).
•
One researcher will keep track of the number and the length of flights
around the array. Flights within a holder are considered short; flights on the
diagonal are considered long, and all others are considered “medium”:
medium
long
medium
short
Fig. 2. Types of foraging flights.
•
The second bee should keep time for the first bee, but should not
observe its flight pattern. After the first bee is finished, the second
bee will forage on the same array, but, because flowers will not be
replenished, will run the risk of finding depleted flowers. Record the same
data for the second bee as for the first bee.
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Assessing the results of the experiment:
1. How successful were the bees? The bees maximize their success by
getting the most sucrose for the shortest distance flown. It is not
necessary to be absolutely exact in estimating the amount of sucrose.
Remember that the high sucrose has 2X the concentration of sugar as the lowsucrose nectar; so, for example, approximately 2 tubes of low sucrose plus
approximately ½ tube of high sucrose = 2 + (½ x 2) = approximately 3 tubes of
nectar. To estimate flight costs, short flights = 1, medium = 2 and long = 3; add
up the total values to obtain a measure of total fight costs. Compare the success
of the 2 bees. Did bee #2 do much worse than bee #1? (Don’t worry too much
about exact values – you are trying to understand the factors affecting the
coevolutionary relationship.)
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). The score for each flower type
is obtained by dividing the number of successive visits by the total number of
visits. For example, suppose a bee visits flower types A & B in the pattern A-BA-A-B-B-B-A-A-B-B-A. The score for A is 2/6 = 0.33; the score for B is 3/6 = 0.5.
The larger the score, the more successful the flower type. 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.
Run the experiment as many times as you like, manipulating one or all of the
following variables:
1.
2.
3.
4.
nectar quality
ease of flower handling
spacing and distribution of flowers
relative abundance of flower types
The purpose is not to quantify your results, but 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.
(We encourage you to discuss these questions as a group, but each student
must submit their own answers to the assignment questions.)
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Lab 3 Assignment (5% of final grade)
•
At the end of your lab period, each group of students must submit a
completed data submission form (available online) describing the set-up
and results from one run of the coevolution experiment (worth 4 marks).
The names of each student in the group must be on the data
submission form in order to receive marks!
•
The assignment is due at your next lab. Please complete the
assignment on the assignment form (available online).
The assignment consists of:
1. A drawing of the Selaginella cone (2 marks)
2. Answers to the 3 assignment questions (2 marks each = 6 marks)
The data submission form is worth 4 marks.
Total marks = 12