Download Sample lab exercise using Mimulus Pollination

Food for sex or how to attract a pollinator: co-evolution and
speciation in the monkey-flower (Mimulus)
Introduction
Many plants depend on pollination for their reproductive success. Co-evolution of
a plant with its pollinator is often seen in flowering plants. Flowers provide pollinators
with a sweet, high energy meal (nectar). Pollinators in turn collect the flower pollen on
their body while feeding and transport it to the next flower they feed on, assisting in
dispersal of the male gametes and in fertilization. Pollinators with a strict preference for
one type of flower are therefore responsible for creating a pre-zygotic barrier and
contribute to the reproductive isolation of sympatric flowers. Reproductive isolation
then can lead to speciation events and the formation of closely related sister taxa.
When Charles Darwin collected an orchid in Madagascar with a very long spur (tubular
corolla), Agraecum sesquipedale, he predicted the existence of a moth with a very long
proboscis (moth’s tongue), which would have been needed to pollinate the flower.
"I fear that the reader will be wearied, but I must say a few words on the Angræcum
sesquipedale, of which the large six-rayed flowers, like stars formed of snow-white wax,
have excited the admiration of travellers in Madagascar. A whip-like green nectary of
astonishing length hangs down beneath the labellum. In several flowers sent me by Mr.
Bateman I found the nectaries eleven and a half inches long, with only the lower inch and
a half filled with very sweet nectar. What can be the use, it may be asked, of a nectary of
such disproportional length? We shall, I think, see that the fertilisation of the plant
depends on this length and on nectar being contained only within the lower and
attenuated extremity. It is, however, surprising that any insect should be able to reach the
nectar: our English sphinxes have probosces as long as their bodies: but in Madagascar
there must be moths with probosces capable of extension to a length of between ten and
eleven inches!"
Darwin, 1862
Five years later, Wallace predicted the type of moth that should be found in
Madagascar.
"I have carefully measured the proboscis of a specimen of Macrosila cluentius from
South America in the collection of the British Museum, and find it to be nine inches and a
quarter long! One from tropical Africa (Macrosila morganii) is seven inches and a half.
A species having a proboscis two or three inches longer could reach the nectar in the
largest flowers of Angræcum sesquipedale, whose nectaries vary in length from ten to
fourteen inches. That such a moth exists in Madagascar may be safely predicted; and
naturalists who visit that island should search for it with as much confidence as
astronomers searched for the planet Neptune,--and they will be equally successful!"
1
Wallace, 1867
This Malagasy moth, Xanthopan (Macrosila) morgani, was discovered thirty six years
later.
Xanthopan morgani praedicta and the star orchid Angræcum sesquipedale
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
http://perso.orange.fr/cryptozoo/dossiers/moth.htm
Pre-Laboratory activity
The flowering plants of the genus Bauhinia exhibit a great diversity in flower
form, size, and color. Several Bauhinia species can be found in Venezuela, inhabiting
very different forest ecosystems. Some Bauhinia species are tree-form while others are
liana-form. Studies of the pollination of Bauhinia have shown that some species are bat
pollinated, while others are pollinated by moths, insects or bird.
Seven species of closely related Bauhinia from Venezuela have been studied and
have shown great differences in flower morphology, physiology and nectar production.
Several possible pollinators are present in their community.
After studying the plant morphology and physiology, you are asked to make a prediction
for the type of pollinator most likely associated with each plants.
The following potential pollinators are found in the forests associated with Bauhinia
flowers:
Moths (Sphingidae and Noctuidae): nocturnal. Pollen and nectar are their source of
protein.
Bats (Phyllostomidae): nocturnal, cannot assimilate sucrose, some eat insects and do not
require proteins in nectar.
Diurnal insects: butterflies (Pieridae), wasps and bees. Pollen and nectar are their main
source of protein. Prefer sucrose rich nectar.
Hummingbirds (Trochilidae): Nectar is their source of protein. Prefer sucrose rich nectar.
What do pollinators gain when they visit a flower?
2
What do the flowers gain when they attract a pollinator?
Fill out this table with the possible pollinators. Justify your answer.
Plant name, plant form, location
Plant description
Possible pollinators
Bauhinia aculeata, tree form, dry premontane forest
Large, white flower. Open asynchronously at night and
at dusk and dawn. High nectar production both night and
day. Nectar rich in sucrose. Some proteins in nectar.
Bauhinia multinervia, tree form, dry premontane forest
Large, white flower. Open at night. High nectar
production at night. Nectar rich in hexose. Few proteins
in nectar.
Bauhinia pauletia, tree form, dry premontane forest
Large, white flower. Open at night. High nectar
production at night. Nectar rich in hexose. Few proteins
in nectar.
Bauhinia ungulata, tree form, tropical dry forest
Large, white flower, some red flower. Open at night.
High nectar production at night. Nectar rich in hexose.
Few proteins in nectar.
Bauhinia glabra, liana form, dry premontane forest
White, smaller flower with purple lines. Open in
daylight.
Very small nectar production but sticky (concentrated),
rich in sucrose and glucose, contains proteins
Bauhinia guianensis, liana form, humid tropical forest
White or clear yellow smaller flower. Open in daylight.
Small production of concentrated nectar rich in both
sucrose and hexose.
Bauhinia rutilans, liana form found high in the canopy
of very humid tropical forest
Pink or yellow-greenish smaller flower. Open in
daylight. Small production of concentrated nectar
How would the pollinator’s choice of flower influence the morphological and
physiological feature of the flower they visit?
3
What are the evolutionary implication of the relationship between pollinator and
pollinated flower?
Laboratory activities:
The purpose of the laboratory activities is to model the effect of pollinators on the
reproductive success of mimulus flowers. We will explore the effect (s) of gene
mutation affecting coloration and/or nectar production. We will also explore the effect of
pollinator preferences on flower reproductive success.
We can also model the condition(s) that would lead to the reproductive isolation (and
speciation) of Mimulus flowers.
The monkey-flowers (Mimulus) are a group of closely related sister taxa, varying
widely in shape, structure and coloration. Field studies have been conducted on two
sympatric species of Mimulus (Mimulus cardinalis and Mimulus lewisii) to determine the
adaptive value of alternative alleles at a single locus. One of the allele under study (YUP
allele) is an allele controlling the presence or absence of yellow carotenoid pigments in
the petal of the mimulus flower.
Mimulus lewisii flowers possess the dominant YUP allele, which prevents the formation
of carotenoids, so the flower petals only express pink anthocyanin pigments, leading to a
pink flower coloration. Mimulus lewisii also produces low amount of nectar and have
wide flattened petals. The amount of nectar produced is mostly controlled by a single
gene. The allele responsible for high nectar production is dominant.
Mimulus cardinalis flowers possess the recessive yup allele, which allows carotenoid
deposition into the petals, leading to a red coloration of the flower. Mimulus cardinalis
also produces high amount of nectar and have narrow petals.
Exercise 1: Define all the glossary terms and make a concept map relating terms
together.
Glossary: allele, recessive allele, dominant allele, pure-breed, parent generation, F1
generation, F2 generation, pre-zygotic barrier, reproductive isolation, speciation,
sympatric, pollination, pollinator.
Exercise 2:
Determine the genotype and the phenotype of two sympatric species of Mimulus flowers,
using the abbreviation used in the simulation. Both Mimulus species are pure breeding.
The following abbreviations are used in the simulation:
Abbreviation used for the genotype:
Gene controlling pink coloration (dominant): P
4
Gene controlling red coloration (recessive): p
Gene controlling high nectar production: N
Gene controlling low nectar production: n
Abbreviation used for the phenotype:
Pink flower: P
Red flower: r
High nectar production: H
Low nectar production: l
Mimulus cardinalis genotype:
____________________
Mimulus cardinalis phenotype: ____________________
Mimulus lewisii genotype:
____________________
Mimulus lewisii phenotype:
_____________________
Exercise 3:
Mimulus cardinalis and Mimulus lewisii can be found in sympatric locations. What
would be the resulting flower phenotypes and genotypes if we crossed these two purebreed species together?
Genotype (s) of Mimulus cardinalis gametes: ____________________________
Genotype (s) of Mimulus lewisii gametes:
____________________________
Draw the Punnett square of the cross: Mimulus cardinalis X Mimulus lewisii
List all the phenotype (s) and genotype (s) of the F1 generation. Give the ratio for each
genotype and each phenotype.
Genotype (s): ____________________________________________________________
5
Phenotype (s) ___________________________________________________________
What would be the resulting flower phenotypes and genotypes of the F2 generation if we
crossed the F1 generation together?
Genotype (s) of Mimulus F1 gametes: ____________________________
Draw the Punnett square of the cross: Mimulus F1 X Mimulus F1
List all the phenotype (s) and genotype (s) of the F2 generation and give the ratio for each
genotype and phenotype.
Genotype (s): ____________________________________________________________
________________________________________________________________________
Phenotype (s) ____________________________________________________________
Compare the phenotypes of the F2 generation with the phenotypes of the F1 and the
parental generation. Explain why some of the F2 phenotypes are different from the parent
phenotypes. Are these new species? Why or why not?
6
A: Mimulus lewisii
B: Mimulus F1 hybrid C: Mimulus cardinalis
D-L: Mimulus F2 hybrids
7
Simulation exercises:
Open the excel file labeled “Mimulus Pollination.xls”. You will first see your starting
population genotype and phenotype table. The frequencies should all be set to 0.
Genotypes PPNN PpNN ppNN PPNn PpNn ppNn PPnn Ppnn ppnn
PH
rH
PH
PH
rH
Pl
Pl
rl
Phenotype PH
0
0
0
0
0
0
0
0
0
Frequency
You will see your pollinator (bee or bird) pollination preference tables. The frequencies
should also all be set to 0.
Pink Red
Bee Preference
Hi nectar
0
0
Lo nectar
0
0
You will also know which pollinator is actively pollinating (Visit table). Each bee visit is
as effective as a bird visit for pollination efficiency. In most of our simulation we will
have an equal number of bee and bird visits, so the setting should be 1 for each, unless
specified.
1
Bee visits
1
Bird visits
The goal of the simulations is to see what will happen to your population of flowers after
20 generations when you modify either the population genotype frequencies, the bee or
bird pollination preferences, or the presence or absence of one or all the pollinators.
Simulation 1: Changes in frequencies of the different flower population (s) in the
presence of two pollinators with fixed preferences.
a) Set up the pollinator preferences to the following settings:
Bee Preference
Hi nectar
Lo nectar
Pink Red
0.01
0
0.98 0.01
Pink Red
Hi nectar
0.01 0.98
Lo nectar
0 0.01
Make sure that all the frequencies add up to 1. If they do not, you will see a red number
on the right of the table.
Bird Preference
Based on the preference setting above, you can determine the pollinator preference.
With these preference settings, which flower (s) will be more likely pollinated by bees
(give the genotype(s) and phenotype(s))?
8
With these preference settings, which flower (s) will be more likely pollinated by birds
(give the genotype(s) and phenotype(s))?
Set up the population table so that the only type of flower present is : Mimulus lewisii
Genotypes PPNN PpNN ppNN PPNn PpNn ppNn PPnn Ppnn ppnn
PH
rH
PH
PH
rH
Pl
Pl
rl
Phenotype PH
0
0
0
0
0
0
1
0
0
Frequency
Make sure that all the frequencies add up to 1. If they do not, you will see a red number
on the right of the table.
After 20 generations, which type of flower (s) are present in the environment?
Predict what would happen if all the birds died? Explain your prediction.
Predict what would happen if all the bees died? Explain your prediction.
Do a simulation to test your prediction.
Did your prediction agree with the simulation results? Why or why not?
If your hypothesis did not agree with the simulation results, what would you need to
change in the simulation settings to have your hypothesis agree with your prediction.
b) A single mutation resulting in a change in the coloration of the flower occurs.
Modify the population frequency to reflect the apparition of this mutation in the
population.
Genotypes
Phenotype
Frequency
PPNN PpNN ppNN PPNn PpNn ppNn PPnn Ppnn ppnn
PH
PH
rH
PH
PH
rH
Pl
Pl
rl
0
0
0
0
0
0 0.98 0.01 0.01
At the beginning of the simulation, which type of flower (s) are present in the
environment (give the ratio)?
After 20 generations, which type of flower (s) are present in the environment (give the
ratio)?
Predict what would happen if all the birds died? Explain your prediction.
Predict what would happen if all the bees died? Explain your prediction.
9
Do a simulation to test your prediction.
Did your prediction agree with the simulation results? Why or why not?
If your hypothesis did not agree with the simulation results, what would you need to
change in the simulation settings to have your hypothesis agree.
c) Another mutation resulting in a change in the increase in the production of nectar
occurs.
Predict what would happen if the mutation affecting nectar production occurred in the
pink flower?
In order to run a simulation of this mutation in the pink flower, you need to use the
setting in a) (pink flower only) and set up the frequency at 0.01 for all genotypes
leading to a pink flower-high nectar production phenotype. Remember that all the
frequencies must add to 1. See below for the table settings:
Genotypes
Phenotype
Frequency
PPNN PpNN ppNN PPNn PpNn ppNn PPnn Ppnn ppnn
PH
PH
rH
PH
PH
rH
Pl
Pl
rl
0.01
0.01
0 0.01 0.01
0 0.96
0
0
Predict what would happen if you were to change the frequency of the high nectar gene?
Test your prediction with a new simulation.
Was your prediction correct? Why or why not?
Predict what would happen if the mutation changing nectar production occurred in the
red flower?
10
Test your prediction with a new simulation. How do you need to change your frequency
setting in order to test your prediction?
After 20 generations, which type of flower (s) are present in the environment?
Was your prediction correct? Explain the changes in gene frequencies shown in the
simulation.
Predict what would happen if all the birds died? Explain your prediction.
Predict what would happen if all the bees died? Explain your prediction.
Do a simulation to test your prediction.
Did your prediction agree with the simulation results? Why or why not?
If your prediction did not agree with the simulation results, what would you need to
change in the simulation settings in order to have your hypothesis agree with your
prediction?
What would happen if there was a change in frequency of the different flowers. Make a
prediction and test it with a simulation.
Your prediction:
Your frequency table:
Genotypes PPNN PpNN ppNN PPNn PpNn ppNn PPnn Ppnn ppnn
PH
rH
PH
PH
rH
Pl
Pl
rl
Phenotype PH
Frequency
Resulting population from simulation:
Was your prediction correct or not? Why or why not?
Predict what would happen if all the birds died? Explain your prediction.
11
Predict what would happen if all the bees died? Explain your prediction.
Do a simulation to test your prediction.
Did your prediction agree with the simulation results? Why or why not?
Simulation 2: Changes in frequencies of the pollinator’s preference when all the
frequencies of all genotypes are fixed at the beginning of each simulation
Set up the population table so that all genotypes frequencies are equals
Genotypes PPNN PpNN ppNN PPNn PpNn ppNn PPnn Ppnn ppnn
PH
rH
PH
PH
rH
Pl
Pl
rl
Phenotype PH
Frequency
0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11 0.11
a) Change the pollinator preference settings. Explain in a sentence what your
preference setting means. For example, the bees only pollinate the pink flower
and the bird only pollinate the hi nectar flower. Or any combination of preference
you choose.
Bee Preference
Pink
Red
Pink
Red
Hi nectar
Lo nectar
Bird Preference
Hi nectar
Lo nectar
After changing the preferences settings, predict the type of flowers in the field after
20 generations. Justify your prediction.
Run a simulation of your prediction. Was your prediction correct or not?
Why or why not?
12
b) Change the pollinator preference settings. Explain in a sentence what your
preference setting means. For example, the bees only pollinate the pink flower and
the bird only pollinate the hi nectar flower. Or any combination of preference you
choose.
Bee Preference
Pink
Red
Pink
Red
Hi nectar
Lo nectar
Bird Preference
Hi nectar
Lo nectar
After changing the preferences settings, predict the type of flowers in the field after
20 generations. Justify your prediction.
Run a simulation of your prediction. Was your prediction correct or not?
Why or why not?
c) Change the pollinator preference settings. Explain in a sentence what your
preference setting means. For example, the bees only pollinate the pink flower and
the bird only pollinate the hi nectar flower. Or any combination of preference you
choose.
Bee Preference
Pink
Red
Pink
Red
Hi nectar
Lo nectar
Bird Preference
Hi nectar
Lo nectar
13
After changing the preferences settings, predict the type of flowers in the field after
20 generations. Justify your prediction.
Run a simulation of your prediction. Was your prediction correct or not?
Why or why not?
d) Change the pollinator preference settings until you reach a threshold when you
see a change from one type of flower to another type of flower.
How much to you need to change the frequencies in order to see a shift into another
type of flowers.
What is your conclusion:
Simulation 3: Make a prediction from your settings of the starting population and
your settings of the pollinator preference.
Your settings for starting population frequencies.
Genotypes
Phenotype
Frequency
PPNN PpNN ppNN PPNn PpNn ppNn PPnn Ppnn ppnn
PH
PH
rH
PH
PH
rH
Pl
Pl
rl
Your setting for pollination preferences:
Bee Preference
Pink
Red
Pink
Red
Hi nectar
Lo nectar
Bird Preference
Hi nectar
Lo nectar
Your prediction and the justification for your prediction:
14
Results from the simulation:
Conclusion:
Simulation 4: changes in the pollinator assemblage
In all our previous simulation, we either assume that both pollinators were actively
active, or that one of the pollinators was absent.
Change the activity rate of one of the pollinator (for example there could be twice as
more visit from bees than birds, or 100 more visits from the birds or anything you
choose)
Change the pollinator visit ratio:
Bee visits
Bird visits
1
1
Predict and justify what would happen when you change the activity of one of the
pollinator.
Run a simulation of your prediction.
Conclusion:
Post-lab exercises:
Post-lab exercise 1:
Mimulus lewisii mostly occurs at mid- to high elevation in the Sierra Nevada and in the
Rocky Mountain and Cascade Range and Mimulus cardinalis is mostly found at low-to
mid elevation in the Sierra Nevada. Field studies have shown that in a narrow altudinal
zone of the Sierra Nevada, Mimulus lewisii and Mimulus cardinalis are sympatric.
Mimulus lewisii and Mimulus cardinalis are closely related but pollinator preferences
prevent intercrossing between the two populations.
Field observations have shown that hummingbirds nearly always pollinate red-flowered,
high nectar Mimulus cardinalis and pink-flowered, low nectar Mimulus lewisii are nearly
always pollinated by bumblebees (see pictures).
Mimulus cardinalis pollinated by hummingbirds
15
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Mimulus lewisii pollinated by bumblebees
From: www.nsf.gov/.../mimulus_cardinalis_lewisii.jpg
Using what you learned in this laboratory exercise, predict the conditions under which
these two flower species have evolved (pollinator’s preferences, ancestral population).
Your prediction:
Test your prediction:
Your conclusion:
Do the pollinator’s preferences need to be strict?
Could a small population arising from a random mutation lead to speciation of the
ancestral population into two distinct, reproductively isolated species?
Do dominant alleles and recessive alleles affect the selection processes?
16
Post-lab exercise 2
Answers to pre-laboratory questions
B. pauletia and B. multinervia are chiropterophilous (pollinated by bats)
B. glabra and B. guianensis are entomophilous (pollinated by diurnal insects)
B. rutilans is pollinated by hummingbirds but also by some bees and wasps
B aculeata is pollinated by both diurnal and nocturnal insects (moths)
References:
Bradshaw HD. And Schemske DW. 2003. Allele substitution at a flower colour locus
produces a pollinator shift in monkeyflowers. Nature 426: 176-178
Darwin C. 1862. On the various contrivance by which British and foreign orchids are
fertilized by insects. London. John Murray. pp 197-98
Hokche O. and Ramirez N. 1990. Pollination Ecology of Severn species of Bauhinia L.
(Leguminosae: Caesalpinioideae), Annals of the Missouri Botanical Garden, 77 (3): 559572
17
Russel G. 2003. Orchid of South Africa. Yearbook of the South African Orchid Council.
pp 59-65
Schemske DW. And Bradshaw HD. 1999. Pollinator preference and the evolution of
floral traits in monkeyflower (Mimulus). PNAS 96 (21): 11910-11915
Wallace AR. 1867. Creation by Law. Quartely Journal of Science, volume 4 (October
1867)
18