ANTHR 111 (#2542)

THE VIRTUAL EVOLUTION STICKLEBACK LAB

As you complete each part of the virtual lab, answer the questions below in the space provided.

QUESTIONS
INTRODUCTION
1. Define “model organism.”
A model organism is a species studied to learn about some biological phenomena so that the
results of those studies can be applied to other organisms.

2. How do spines protect ocean stickleback fish?

Large predator fish cannot swallow them.

3. How did ancestral populations of ocean-dwelling fish come to live in freshwater lakes?
They were trapped in the lakes that were formed after the ice sheet melted.

4. Watch the video about pelvic reduction in freshwater stickleback. The loss of stickleback pelvic
spines is similar to the loss of which body parts in some other four-legged vertebrates?
Hind limbs

5. From a researcher’s perspective, what is the benefit of having access to hundreds of postglacial
lakes?
They can study how different natural conditions caused different ways the same species evolved.

6. Watch the video with evolutionary biologist Dr. Michael Bell.

Why is the threespine stickleback a model organism for studies in evolution? (List at least two
reasons.)
Sticklebacks are widespread around the world, so it is easy to collect and study specimens of that
species from different environments, and there are a lot of fossilized sticklebacks available, so it is
possible to study them in diachronic perspective.

7. Watch the video with Dr. David Kingsley explaining hind-limb reduction. Name two other
vertebrate animals whose evolutionary histories included the loss of hind limbs.
Manatees, whales

OVERVIEW
1. Click on the interactive stickleback fish. Describe where its spines are located.
There are three spines on the back and two spines on the each side of the pelvic girdle.

The Virtual Stickleback Evolution Lab Published October 2012

Updated September 2013
www.BioInteractive.org Page 1 of 11
2. What is the difference between marine, sea-run, and freshwater stickleback fish populations? Be
specific.
Marine sticklebacks live and breed in the sea, sea-run sticklebacks live mostly in the sea but breed
in freshwater streams, and freshwater sticklebacks live and breed in freshwater only.

3. Watch the video about the stickleback fish armor.

a. In addition to the spines, what is another component of the ”armor“ of a stickleback fish?
There is a row of plates between the pelvic girdle and the spines on the back.

b. Explain how the stickleback armor protects the fish from some predators.
Stickleback armor prevents sticklebacks from being swallowed by predator fish or birds.

TUTORIAL 1
1. Describe the following structures:
a. Complete pelvis:
Full pelvic girdle with two spines on the each side.

b. Reduced pelvis:
A range of structures from a simplified girdle with no pelvic spines to an oval nub for a
girdle.

c. Absent pelvis:
No pelvic girdle, no spines.

2. Start Tutorial 1. Continue to practice scoring fish until you have mastered the technique; then
proceed to Experiment 1.
EXPERIMENT 1
1. Explain in your own words the overall objective of Experiment 1.
Analyzing and comparing pelvic structures (girdle and spines) of stickleback from two lakes
around Cook Inlet, Alaska in order to determine whether there are significant differences between
them.

2. Click on the link to the map of Alaska, then click on the blue pin “A” on the larger map.
What lake is located between Bear Paw Lake and Frog Lake just to the north?
Prator Lake

3. In a population, what happens to organisms that are better adapted to the environment in
which they live?
 They live longer and reproduce more.

4. In the virtual lab window, complete Part 1: Staining the Fish.

a. Why do you think it is important to empty the used stain, destaining solution, and water under
a fume hood?
To prevent inhaling vapors that can be toxic.

b. How were the fish you will be using in this virtual lab caught?
Using a special trap with conical enterings.

5. In the virtual lab window, proceed to Part 2: Scoring the Fish.

6. Before scoring the fish, watch the short video on Bear Paw and Frog Lakes.
According to Dr. Bell, what is an important difference between Bear Paw Lake and Frog Lake?
Bear Paw is completely enclosed and therefore lacks large predator fish whereas Frog Lake is
connected with the lake system by a small stream which makes it possible for predator fish like
salmon and trout to breed.

7. In the window on the right, click on the link to read more about random sampling. Why are
random samples, rather than entire populations, used in most research studies?
The entire population is typically too large to count

8. What is one advantage of studying larger-sized samples?

It prevents researchers from incurring in a sampling error.

9. Give an example of sampling bias.

Sampling bias occurs when you take samples from a specific area and fail to take samples from
another area.

10. Complete Part 2 of the lab in the window on the left.

11. Why is it important that the labels included in specimen jars be made of special paper that does
not disintegrate in alcohol over time?
To be sure which samples you are working with.

12. Examine the pelvic score data you just collected. Does the pelvic phenotype differ between Bear
Paw Lake and Frog Lake fish? Explain.
Yes, sticklebacks from Bear Paw Lake have reduced or no pelvic girdle and no pelvic spines while
those from Frog Lake have both pelvic girdle and spines.
13. Complete the graphing exercise as instructed by your teacher. After graphing, verify your data.
How do your data compare to those obtained by Dr. Bell and colleagues?
They completely match.

14. Complete the Experiment 1 Quiz. Take time to read the explanation for each correct choice
provided after each question.
15. Explain why the stickleback fish in Frog Lake are more similar to ocean and sea-run stickleback
than they are to the stickleback fish in Bear Paw Lake.
Because there are large predator fish in Frog Lake whereas in Bear Paw Lake there are not.

16. In addition to predators, what other environmental factors might be responsible for the
differences between Bear Paw Lake and Frog Lake stickleback populations? How would you test
your prediction?
Developing spines could become difficult due to some changes in available food.

17. After completing the quiz, click on Experiment 1 Analysis.

18. For this analysis, what is your null hypothesis?
Sticklebacks with pelvic spines and without them are equally presented in both lakes.
19. Complete the following tables as you perform the chi-square calculations in the lab.

Bear Paw Lake

Phenotype Observed (o) Expected (e) (o-e) (o-e)2 (o-e)2/e
Pelvic spines
0 10 -10 100 10
present
Pelvic spines
20 10 10 100 10
absent

Total = 20 Total = 20 χ2 =20

Frog Lake
Phenotype Observed (o) Expected (e) (o-e) (o-e)2 (o-e)2/e
Pelvic spines
20 10 10 100 10
present
Pelvic spines
0 10 -10 100 10
absent

Total = 20 Total = 20 χ2 =20

Morvoro Lake
Phenotype Observed (o) Expected (e) (o-e) (o-e)2 (o-e)2/e
Pelvic spines
8 10 -2 4 0.4
present
Pelvic spines
12 10 2 4 0.4
absent

Total = 20 Total = 20 χ2 = 0.8

20. For each chi-square calculation, how many degrees of freedom are there? ____1___
How did you arrive at this number?
Degree of freedom is a number of forms of the trait minus 1, so 2-1 = 1.

21. What are the p values for the data from each lake?
a. Bear Paw Lake _0.01______
b. Frog Lake _0.01______
c. Morvoro Lake _0.05_____

22. Do you “reject” or “fail to reject” the null hypothesis for the data from each lake?
a. Bear Paw Lake reject
b. Frog Lake reject
c. Morvoro Lake fail to reject
23. Complete the Chi-Square Analysis Quiz. Take time to read the explanation for each correct
choice provided after each question.
24. Explain what it means to reject the null hypothesis.
This means a statistically significant difference between the observed and expected data has been
shown.

25. Explain what it means if the null hypothesis cannot be rejected.

This means there is no statistical difference between the observed and expected data.

26. Explain the difference between the results of the chi-square calculations for Bear Paw Lake and
Morvoro Lake.
For Bear Paw Lake, the difference between observed and expected data is huge so possibility that
it happened by chance is minimal whereas for Morvoro Lake, the difference is not that big.

27. What do you think might be an explanation for why Morvoro Lake contains fish with and without
pelvic spines?
It can be caused by the fact that there are large predator fish in Morvoro Lake, but their amount is
limited, or their habitat in that lake does not completely match the habitat of sticklebacks.

TUTORIAL 2
1. What score would you assign to a fossil specimen that has only one pelvic spine visible?
Complete

2. A stickleback fossil may show no signs of pelvic structures. What are possible sources of error
associated with scoring the pelvis of such a fossil as “absent”?
Decay or physical damage before the were fossilized, or distortion of fossils when rocks change
due to natural processes.

3. Start Tutorial 2. Continue scoring the fossil specimens until you are confident in your scoring
abilities; then proceed to Experiment 2. (Most people find that it is more difficult to accurately score
fossils than the preserved fish specimens. You may have to practice on 20 specimens or more before
you feel you have mastered the technique.)

EXPERIMENT 2
1. In your own words describe the overall objective of Experiment 2and explain what the data you
collect will allow you to estimate.
Analyzing stickleback fossils from the Truckee Formation in Nevada dated back to 10 million years
ago. The goal is to estimate the rate of evolution of pelvic structures in this population of
sticklebacks.
2. Watch the short video on the Truckee Formation in Nevada. What does each sedimentary rock
layer of the Truckee Formation represent?
One year.

3. What is one type of information that researchers can gain from studying fossils that they cannot
obtain from living populations?
Rates and patterns of evolutionary change.

4. Begin the experiment in the window on the left. Complete Part 1: Preparing Fossils. (You may
skip this step by clicking on “Skip Part1: Fossil Prep.”)

5. Proceed to Part 2: Scoring Fossils. In this virtual lab, you will graph fossil data from six rock
layers. Approximately how many years apart are any two adjacent samples?
3,000 years

6. You will collect data on pelvic structures using fossils from rock layers 2 and 5. Approximately
how many years of deposition separate these two layers?
9,000 years

7. Which layer is older, 2 or 5? Explain your answer.

Layer 2 is older because the layers are stacked one on top of the other, so the oldest layer is 1
which is at the bottom.

8. Complete Part 2 of the lab in the window on the left. Based on the pelvic phenotypes you
measured, do the fossils in layer 2 differ from those in layer 5? Explain how.
Yes, they differ, the vast majority of samples from layer two do have pelvic spines while none of
samples from layer 5 have them.

9. Complete the graphing exercise as instructed by your teacher. After graphing, verify your data.
How do your data compare to those collected by Dr. Bell and colleagues?
I scored one sample less with reduced pelvis from layer 2 and 1 sample more with absent pelvis
from layer 5.

10. Complete the Experiment 2 Quiz. Take time to read the explanations for each correct choice
provided after each question.
11. What can be inferred about the presence or absence of predatory fish when the Truckee
Formation was a lake? Describe the evidence.
There were no predatory fish because there are no fossils of them, and because sticklebacks
developed reduced or absent pelvis.
12. After completing the quiz, click on Experiment 2 Analysis.
13. Complete the tables below as you perform the rate calculations. (The link to the instructions is
very helpful.)

Relative Frequency of
Number of Fish with a Total Number of Fish
Sample Layer Complete Pelvis Trait in
Complete Pelvis Sampled
Population Sampled
1 20 20 1
2 18 20 0.9
3 3 20 0.15
4 1 20 0.05
5 0 20 0
6 0 20 0

Decrease in Percentage of
Complete Pelvis Trait per
Time
Thousand Years
(Rate of Change)
First 3,000 years
-3%
(Layer 1 to Layer 2)
Next 3,000 years
-25%
(Layer 2 to Layer 3)
Next 3,000 years
-3%
(Layer 3 to Layer 4)
Next 3,000 years
-1.66%
(Layer 4 to Layer 5)
Next 3,000 years
0
(Layer 5 to Layer 6)
Total 15,000 years
100%
(Layer 1 to Layer 6)

14. What does it mean when the rate of change is a negative number?
It means decrease of frequency

15. Complete the Analysis Quiz. Take time to read the explanation for each correct choice
provided after each question.
16. Describe the trend in the data over time.
The amount of sticklebacks without pelvic spines started to decrease 15,000 years ago, it
decreased rapidly between 12,000 and 9,000 years ago, than decreasing was slowing down until
the process was completed.
17. Why is it important to calculate the rate of change over time?
Calculating rates of change in fossil fish can be used to infer some aspects of the evolution of
traits in living fish, which are not accessible for study because of the long time scale of most
evolutionary processes.

18. In what way is the change in the complete pelvis phenotype in the fossils from the Nevada
lakebed similar to what might have occurred in Bear Paw Lake from Experiment 1?
All the sticklebacks had pelvic spines in the beginning, and all of them developed reduced or
absent pelvis in the end.

EXPERIMENT 3
1. In your own words describe the overall objective of Experiment 3.
Illustrating how anatomical changes can help us understand genetic mechanisms.

2. What is one function of the Pitx1 gene?

Pitx1 involved in the development of the pelvic girdle and spines of stickleback fish and of the hind
limbs of some other vertebrates.

3. Watch the video segment from the HHMI short film, “Evolving Switches, Evolving Bodies.”
a. What is the relationship between changes in body form, the process of development, and
genes?
Changes of form arises from changes in development which is controlled by genes, so changes in
body form linked to changes in genes.

b. Why did Dr. Kingsley do genetic crosses with stickleback fish?

To map the location of genes that make the difference.

4. In the virtual lab window, click on the blue gloves and perform Part 1: Staining the Fish. You
can skip the staining procedure if you already completed it in Experiment 1.

5. Proceed to Part 2: Scoring Pelvic Asymmetry. Which one of the three pelvic girdle phenotypes is
analyzed in more detail in this experiment?
Reduced pelvis

6. What is the difference between left-biased and right-biased pelvic asymmetry?

Left biased pelvic asymmetry is when the left part of the reduced pelvic girdle is larger than the
right part while right biased pelvic asymmetry is when the right part of the reduced pelvic girdle is
larger than the left part.

7. In the virtual lab window, complete Part 2: Scoring Pelvic Asymmetry.

8. Complete the graphing exercise as instructed by your teacher. After graphing, verify your data.
How do your data compare to those collected by Dr. Bell and colleagues?
My data are exactly the same.

9. Complete the Experiment 3 Quiz. Take time to read the explanation for each correct choice
provided after each question.
10. The pelvic asymmetry observed in stickleback fish from Bear Paw Lake and Coyote Lake is
biased toward which side?
Left.

11. Based on previous research conducted in mice, what does this observed bias among Bear Paw
Lake and Coyote Lake stickleback suggest about the genetic mechanisms of pelvic reduction in
these two populations?
Left-biased pelvic asymmetry appears to be a signature of the loss of function of the Pitx1 gene.

12. After completing the quiz, click on Experiment 3 Analysis.

13. For this analysis, what is the null hypothesis?
Roughly equal numbers of stickleback fish have right or left bias asymmetry for their pelvic
vestiges because the asymmetry is the result of chance.
14. Complete the following tables as you perform the chi-square calculations in the lab.

Bear Paw Lake

Phenotype Observed (o) Expected (e) (o-e) (o-e)2 (o-e)2/e
Left bias 18 10 8 64 6.4

Right bias 2 10 -8 64 6.4

Total = 20 Total = 20 χ2 =12.8

Coyote Lake
Phenotype Observed (o) Expected (e) (o-e) (o-e)2 (o-e)2/e
Left bias 20 10 10 100 10

Right bias 0 10 -10 100 10

Total = 20 Total = 20 χ2 =20

15. For each chi-square calculation, how many degrees of freedom are there? ___1____

16. What are the p values for the data from each lake?
a. Bear Paw Lake 0.01
b. Coyote Lake 0.01

17. Do you “reject” or “fail to reject” your null hypothesis for the data from each lake?
a. Bear Paw Lake reject
b. Coyote Lake reject

18. Complete the Chi-Square Analysis Quiz. Take time to read the explanation for each correct
choice after each question.
19. What does it mean to reject the null hypothesis for Bear Paw and Coyote Lakes?
There could be a biological explanation for why stickleback in these two populations have a pelvic
vestige that is larger on the left than the right side.

20. Complete the Final Quiz. You may print and/or email the Progress Report page to your teacher,
be sure to enter your name in the box in the upper right corner.