EXERCISE 5

Stickleback Evolution

A Laboratory Report Presented to the

Biology Department, Xavier

University Cagayan de Oro City

In Partial Fulfillment

Of the Requirements for the Course

Evolution Lab (BIO 111L)

By:

Mangao, Noreen Claire P.

Pulmano, Jarena Joy
Saburao, Therese T.

Instructor:

Mr. Reggie Ladera

April 12, 2022

 INTRODUCTION

The Gasterosteus aculeatus is a tiny, streamlined torpedo fish that measures at least 3-8
centimeters in length. It is characterized by its three sharp spines, which could occasionally be 2 or
4, that extends from on the back forward of its dorsal fin; this dorsal fin has a broad shape with
10-14 soft rays. This organism is commonly found in marine, brackish and coastal freshwater
habitats in the northern hemisphere, specifically in the boreal and temperate regions. Although they
are prevalent across Northern America to Southern California, they are still considered as native to
the Lake Ontario basin, underneath theNiagara Falls (Dettloff & Sturtevant, 2022). Moreover, this
organism is under the following classification: Kingdom Animalia, Phylum: Chordata, Class
Actinopterygii, Order Gasterosteiformes, Family Gasterosteidae, Genus Gasterosteus, and Species:
G. aculeatus. Its common name, threespine stickleback, is actually derived from its morphological
features of possessing the previously mentioned 3 sharp spines found on the back.

This organism is significantly used as a model organism in studying evolution since it

allows better understanding of adaptation and ecological diversification and is said to uncover the
underlying secrets in evolution. Since G. aculeatus is said to exhibit genetic variations that are
close in comparison to the genetic variations in human populations, this makes them good model
organisms to study the evolution of human populations given the fact that they share some common
ecological characteristics.

The G. aculeatus has a wide range of morphological variation, however it has two known
distinct varieties which are the a form with anadromous existence and the other only inhabiting
freshwater. The anadromous form lives in the ocean and feeds on plankton and only returns to
freshwater to breed, while the other form lives solely in freshwaters of different varieties. The
forms that inhabit the freshwater environment have two morphological variations as well,
depending on which part of the habitat it occupies.

The G. aculeatus that inhibits the surface water area has a limnetic form, in which they
exhibit slender bodies alongside their narrow mouth, big eyes, and long snouts. On the other hand,
those that inhabit the ocean floor have benthic forms, in which they possess small eyes with a wide
arch. Those G. aculeatus that have a benthic form have 30 or even more lateral but bony plates on
every side of its body as well as on its pelvic girdle. Unlike the limnetic form, their pelvic fin is
reduced to a sharp spine and a small ray, while their rakes are long and slender with approximately
17 on the first arch.These two types of fish formation are also related to the feeding-ecology of this
organism. The narrow mouth of the limnetic form is suited for its way of feeding which is water
feeding, while the mouthparts of the benthic forms are also suited for its way of feeding which is
bottom-feeding and raking. (Leonard & Boake, 2010).

In this exercise, the students were tasked to perform the laboratory activity on the threespine
stickleback fish using different techniques to examine the morphological features and phenotypic
diversity of the fish, specifically the forms and structures of their pelvic morphology, coming from
two different water source, the bear paw lake and frog lake.
OBJECTIVES

● To perform the stickleback simulation to assess the phenotypic diversity among stickleback
populations, mainly on the structure of the pelvis.
● To graph the stickleback pelvic phenotype totals generated simulation.
● To examine the fossilized fish specimens in the simulation to identify its skeletal structures.
● To use statistical data analysis to measure confidence in conclusions from the population
data.
● To determine the rate of change of a trait over time based on data from the fossil record.

MATERIALS AND METHODS

Material

1. Laptop or PC
2. Internet access

General Procedure

The virtual laboratory of Stickleback Evolution is divided into 3 experiments, each having
three components, as well as data analysis. It was accessed and executed by navigating the
simulation website https://media.hhmi.org/biointeractive/vlabs/stickleback2/index.html.

Experiment 1: Analysis of Stickleback Fish from Lakes

1. The initial step of the experiment was to use alcohol, Alizarin Red staining solution, and
destaining solution to stain the fish samples from Bear Paw Lake and Frog Lake.

2. The stickleback samples were sieved out of the alcohol-filled jar in which they were kept. The
alcohol was placed in a separate jar and the Alizarin Red staining solution was poured into the jar
containing the fish samples. The jar sat for 3-6 hours, subsequently, the staining solution was then
dumped into the garbage container in the fume hood.

3. After that, an amount of destaining solution was poured into the fishes and gently shaken. The
solution was then dumped into the fume hood's waste container.

4. Another portion of the destaining solution was put into the jar and allowed to sit for one hour. It
was then gently shaken before being emptied into the garbage can. This step was repeated two
times.

5. The jar was then rinsed with water, along with the discolored fish inside. The water was then
thrown into the garbage container, then another water was poured into the jar and sat overnight.
After that, the water was emptied into the trash can. This is repeated several times more to
thoroughly rinse the jar.

6. The original 50 percent isopropyl alcohol solution, which had been kept aside, was then poured
into the jar of fish once the fish had been successfully stained.
7. Finally, the stained sample fishes were scored by observing and counting the number of fishes
with a complete, reduced, or absent pelvic structure using the microscope.

8. The entire data collected in each lake was then compared to Dr. Bell's and his colleagues'
findings. Following that, the findings were graphed using a bar graph.

9. The total data was then evaluated by doing a chi-square test in each lake, after which , the
simulation provided a chi-square test result to be compared with the group's answer . Subsequently,
the chi-square test results were then interpreted by the group.

Experiment 2: Analysis of Fossil Stickleback Fish

1. Fossil fish samples were prepared in this experiment through scraping the rock slowly to disclose
the fossil.

2. Subsequently ,the scoring of the pelvic structure of the fossils in layers 2 and 6 of the Nevada
lakebed was obtained by examining them under a microscope. Furthermore, the simulation
produced the data for the additional layers (1, 3, 4, and 5).

3. Then , the gathered data was graphed using a line graph. The obtained data as well as the trends
of the graph were then compared with those of Dr. Bell's .

4. The rate-of-change per thousand years in each layer was determined using the data acquired by
first determining the relative frequency of the layers. This is done by dividing the total number of
sticklebacks studied in each layer by the number of sticklebacks with complete pelvis, which is 20.

5. After then, the older layer's number was subtracted from the more current neighboring layer .
The discrepancies were then divided by three (as each layer is 3,000 years apart).

6. To generate the rate of changes , the results were then submitted into the simulation . The
obtained data were then compared with those of Dr. Bell's . After that , the group interpreted and
discussed the results.

Experiment 3: Analysis of Pelvic Asymmetry

1. Sticklebacks from Bear Paw Lake and Coyote Lake were stained with the same solutions as in
Experiment 1 such as alcohol, Alizarin Red, and destaining solution. To effectively stain the fish,
similar methods and measurements were taken.

2. After that, the fish with left bias which has a bigger pelvic vestige on the left and right bias
which has a larger pelvic vestige on the right were identified and the pelvic asymmetry was scored.
The totals for pelvic asymmetry were then graphed using a bar graph.

3. Using the same approach as in Experiment 1, the obtained data was subjected to a chi-square
test. The findings were then analyzed and compared to those of Dr. Bell's experiment.
Documentation

Figure 5.1. Tutorial 1: Scoring a living fish. Figure 5.2. Experiment 1: Preparation of fish samples.

Figure 5.3. Experiment 1: Scoring fish samples Figure 5.4. Experiment 1: Scoring fish samples from
from Bear Paw Lake. Frog Lake.

Figure 5.5. Experiment 1: Stickleback Fish Figure 5.6.Tutorial 2: Scoring a fossil fish.
Analysis
Figure 5.7.Experiment 2: Fossil preparation. Figure 5.8. Experiment 2: Layer 2 and Layer 5 scoring.

Figure 5.9. Experiment 3: Fish sample preparation. Figure 5.10. Experiment 3: Fish sample scoring
from Bear Paw Lake and Coyote Lake.
RESULTS

A. Living Stickleback Fish Analysis from Lakes

Table 5.1 shows the group’s generated pelvic structure score totals. In Bear Paw lake, among the 20
examined fish samples, 16 have reduced pelvic structure, 4 exhibited the structure’s absence, and none of the
Stickleback fish have complete pelvic structure. In Frog Lake, all of the 20 Stickleback fishes have the
complete phenotype of the trait which have the presence of pelvic girdle and spines. Although not provided
with Dr. Belle and colleagues’ score totals in tabulated form, similarity of the result is manifested in the
given chi-square tables and graphs.

Table 5.1 Generated pelvic structure score totals.

Figure 5.11 shows the group’s generated bar graph of Stickleback pelvic score totals. The graph
represents the presence of the complete phenotype in all of the Stickleback fishes inhabiting the Frog Lake
while there is an incomplete phenotype in all the Stickleback fishes inhabiting the Bear Paw Lake in which
they lack spines. The result is consistent with the bar graph generated by Dr. Bell and colleagues shown in
Figure 5.12.

Figure 5.11 Generated bar graph of Stickleback pelvic score totals.

Figure 5.12 Bar graph of Stickleback pelvic phenotype totals generated by Dr. Bell and colleagues.

Table 5.2 shows the calculated chi-square values of pelvic structure scoring among the lakes. The
data for Morovor’s lake scoring is already given; 8 fishes having pelvic spines and 12 fishes with absent
spines. The chi-square value is calculated using the formula 𝒳2 = ∑ ((o – e)2/e). To generate the null
hypothesis, we will initially think that the traits are equally abundant, that nothing acts on it, and the
difference in distribution is due to chance. Our null hypothesis is therefore:

H0= The traits are equally distributed

With this being established, we would expect a value of 10 fishes in each trait and the chi-square
value can now be calculated (see appendix). The degrees of freedom in this case are the traits considered (
absence and presence of spine) - 1, that is 2-1 = 1. With the help of the Critical Values Table, we now
determine the probability (p) value. Since the calculated chi-square value exceeds the value listed in the
extreme right-hand column, we input 0.01 as our probability value. This means that the null hypothesis in
Bear Paw Lake and Frog Lake is rejected and that this decision is highly significant. The differences
therefore in these Lakes, are not due to chance but must be caused by some factors. In Morvoro Lake, since
the probability is greater than the usual criterion of 0.05, we therefore fail to reject the null hypothesis. This
result is similar to that of the values obtained by Dr. Bell and colleagues as shown in Table 5.3.

Table 5.2 Calculated chi-square values of pelvic structure scoring.

Table 5.3 Chi-square value of pelvic structure scoring calculated by Dr. Bell and colleagues.

B. Fossil Stickleback Fish Analysis from Lakes

Table 5.4 shows the group’s generated fossil stickleback pelvic score totals. Only Layer 2 and Layer
5 are examined in the simulation. Among the 20 fossils scored, only 18 fossils possessed the complete pelvic
structure while 2 have reduced structure in Layer 2. In layer 5, 15 fossils have reduced pelvic structure while
the other 5 fossils have no pelvic structure. The data in Layers 1,3,4, and 6 are already provided.

Table 5.4 Generated fossil stickleback pelvic score totals.

Figure 5.13 shows the generated line graph of the results from fossil scoring with regards to pelvic
phenotype totals. Looking at trends of the oldest layer which is layer 1 and the newest layer which is layer
6, the line graph suggests that during the early years, the majority of the Stickleback fishes have pelvic
spines but this changed over 15,000 years as the majority of Stickleback fishes currently doesn’t have the
complete phenotype. The result gathered by the group is consistent with the result gathered by Dr. Bell and
colleagues as shown in Figure 5.14.

Figure 5.13 Generated line graph of fossil Stickleback pelvic phenotype totals.
Figure 5.14 Line graph of fossil Stickleback pelvic phenotype totals generated by Dr. Bell and colleagues.

Table 5.5 Shows the group’s calculated rate-of change according to pelvic phenotype scores in
between layers. The rate was calculated using the relative frequency in each layer by dividing the number of
fishes with complete pelvis by the total number of fossils analyzed. The relative frequencies of the older
layer were then subtracted from the newer layer and the result is divided by 3 (each layer is 3,000 years
apart) to get the rate of change per thousand years (see appendix). The calculated result tells us that there
was a rapid decrease of the phenotype from layer 2 to layer 3 which is equal to 25% of phenotype decrease
per thousand years. It is also evident that during the earliest years, the evolution of pelvic structure occurred
promptly and tended to stop during the recent years. The result gathered by the group is also similar to the
result gathered by Dr. Bell and colleagues except for the decimals as shown in Table 5.6.

Table 5.5 Calculated rate-of-change according to pelvic phenotype scores.

Table 5.6 Dr. Bell and colleagues’ calculated rate-of-change according to their pelvic phenotype scores.

C. Pelvic Asymmetry

Table 5.7 shows the group’s generated pelvic asymmetry totals. Among the 20 Stickleback fishes
examined, 18 fishes have pelvic vestige larger on the left side (left bias) and 2 fishes have pelvic vestige
larger on the right side (right bias) in Bear Paw Lake whereas all the examined fishes in Coyote Lake have
left biased pelvic vestige structure. The result is consistent with the result gathered by Dr. Bell and
colleagues as shown in Figure 5.8.

Table 5.7 Generated pelvic asymmetry score totals.

Table 5.8 Pelvic asymmetry score totals generated by Dr. Bell and colleagues.
 Figure 5.15 shows the group’s generated bar graph of Stickleback’s pelvic asymmetry records. In
this graph, it is apparent that in both lakes, the majority of the pelvic vestige structure is larger on the left
side of the fishes compared to their right side.

Figure 5.15 Generated bar graph of Stickleback’s pelvic asymmetry records.

Table 5.9 shows the group’s calculated chi-square values of Stickleback’s pelvic asymmetry records.
The calculation is done using the same formula used to calculate the previous chi-square values which is
𝒳2 = ∑ ((o – e)2/e). The same hypothesis is applied in this experiment in which we would initially think that
the differences in distribution is due to chance, that there are no factors acting on it, and that the traits are
equally distributed. The null hypothesis is therefore:

H0= The traits are equally distributed

Similar expected values are also applied, that is, 10 fishes are expected to be left-biased and 10 are
right biased. The calculation can now proceed (See appendix). The degrees of freedom are the traits
considered (left-bias and right bias) - 1, that is 2-1 =1. The probability (p) is then determined using the
Critical Values Table. The calculated chi-square value again exceeds the value listed in the extreme
right-hand column, we therefore input 0.01 as our probability value. Since 0.01 is lesser than the usual
criterion for rejecting the hypothesis which is 0.05 or 5%, we therefore reject the null hypothesis in both
lakes. For that reason, pelvic asymmetry in Stickleback fishes cannot be reasoned by chance alone and must
be explained by other factors. This result is also in line with the results of Dr. Bell and colleagues shown in
Table 5.10.

Table 5.9 Calculated chi-square values of Stickleback’s pelvic asymmetry records.

Table 5.10 Chi-square values of Stickleback’s pelvic asymmetry records calculated by Dr. Bell and
colleagues.
DISCUSSION

In the early evolutionary developments of organisms, one of the most remarkable events is
the transition of organisms from marine to freshwater habitats. The threespine stickleback
(Gasterosteus aculeatus) fish is the best organism that exhibits this evolutionary progress, given the
fact that this organism has two distinct forms: one with anadromous existence, the limnetic form
and the other one that inhabits freshwater only, which is the benthic form (Dettloff & Sturtevant,
2022). The ones with limnetic form live in the ocean and only return to freshwater to breed; their
spines are not reduced as compared to the benthic ones since this protects them from the predators
in the ocean especially that there are a lot of gigantic fishes that prey on them. These spines serve
as an armor against their predators by flaring out its spines extending from the back to the pelvis,
both dorsal and pelvic spines, to prevent the predator from completely swallowing them.
Furthermore, the ancestral populations of ocean-dwelling fish have transitioned to live in
freshwater lakes during a time where some of the fishes swam to a lake and never got back to the
ocean; another reason was also because they were trapped in these lakes after the ice age ended
during that time, they were not able to escape from the formed lakes allowing their bodies to adapt
to the new environment, and fortunately they got used to it.

The loss of stickleback pelvic spines is akin to losing hind-legs in four-legged vertebrates..
According to studies, alterations in the stickleback pelvis are mostly caused by changes in a gene
regulatory switch, a stretch of noncoding DNA sequence that controls whether genes are switched
on or off. Significantly, when they crossed two freshwater populations that were missing the pelvic
section of the sticklebacks partially or completely, they discovered that the loss of expression was
caused by a change of a CRE at the Pitx1 region ( Sadier ,2016). Consequently, the loss of pelvic
tissues in sticklebacks is caused by the lack of a Pitx1 enhancer. Apparently, the threespine
stickleback is used as a model organism for studies in evolution. In detail , sticklebacks are small
fish with a short generation period. With this researchers can monitor several generations of fish in
a very short period of time, hence, these two qualities make them easy to keep in a lab which is
beneficial for genetic investigations. Apart from that, stickleback fish populations may be found all
around the Northern Hemisphere, in a variety of habitats, allowing researchers to compare
populations and see how they have changed over time in response to various environmental
stressors (Barber , 2013 ) . In addition, stickleback fossils are abundant, allowing researchers to
compare extant populations to their ancestors. Consequently, according to the video on stickleback
armor, the shell is made up of not only spines but also a row of two plates along the organism's
sides. When the shell is acting in a protective role from the predators, the spines stick upwards,
forming a nearly perpendicular angle between the pelvis and the spine. As a result, their predators
have a hard time swallowing them. Furthermore, the existence of the two plates makes it difficult
for predators to eat them.
Experiment 1

The main purpose of this section is to conduct a cross-examination of the structures of two
sticklebacks obtained from different parts of Alaska. As a result, the data gathered is combined with
the findings from the lake in order to determine the relationship between the species and their
environments. Particularly , a stickleback with three spines , was found out to have a three pelvic
phenotype such as reduced, nonexistent, and complete . The presence of a full pelvis in a
stickleback suggests that the organism is capable of surviving predators. Consequently, in
comparison to the other types, the full phenotype is more available in the environment. Such that ,
sticklebacks have girdle predators and full pelvic spines , which is defined as a bony protuberance
of the pelvis and typically acts as a protection mechanism from. Not to be missed, the back spines
stick up and the pelvic spine protrudes at almost a perpendicular angle, making it difficult for a
predatory fish to swallow a stickleback.

Dr. Bell further outlines the main variation between the frog lake and Bear Lake in which
the latter does not drain at the surface since it is entirely enclosed. As a result, the predator fish
cannot make their entry into the lake. Conversely, the frog lake has a small stream that connects it
with other water systems. Moreover, the stream acts as an entry for the predators into the water
body. Furthermore , many specialists prefer to employ random samples rather than the entire
population in most of their studies because doing so would take up too much of their time. Besides,
many funds would be needed to study the whole population in the ecosystem (Depersio , 2021).
Hence, the researchers employ the random sampling technique where a few members of the
community are used to represent the entire group. From the samples, it is possible to make an
estimate of the properties of the whole group and draw viable conclusions. Conversely, the
advantage of using a larger sample is that they are more representative of the entire group as
compared to the small specimens.

From the pelvic score data collected, there exists a phenotypic variation for the Frog Lake
and the Bear Paw Lake. Strikingly, the population in the Bear Lake contains mostly stickleback fish
which exhibit no spines whereas that of Frog Lake has pelvic spines . Moreover , despite the fact
that Dr. Belle and colleagues' score totals were not provided in tabular form, similarity of the result
is manifested in the given chi-square tables and graphs. As seen in table 5.1 , the result is consistent
with the bar graph generated by Dr. Bell and colleagues shown in Figure 5.12. Significantly , a
chi-square test is a statistical test used to compare observed results with expected results with a
purpose to determine if a difference between observed data and expected data is due to chance. The
chi-square value is calculated using the formula 𝒳2 = ∑ ((o – e)2/e). In generating the null
hypothesis, it was assumed that the qualities are equally abundant, that nothing influences them,
and that the variation in distribution is due to chance. Thus ,

H0= The traits are equally distributed

In this case, the degrees of freedom are the traits considered (absence and presence of spine)
- 1, or 2-1 = 1. Using the Critical Values Table , the p values for the data from each lake were
obtained. In detail, both Bear Paw lake and frog lake obtains a p value of 0.01 whilst Marvoro
lake got 0.5, respectively . Subsequently , 0.01 was used as the probability value because the
estimated chi-square value is greater than the number stated in the extreme right-hand column. This
means that the null hypothesis in Bear Paw Lake and Frog Lake is rejected and that this decision is
highly significant. The differences therefore in these Lakes, are not due to chance but must be
caused by some factors. Rejecting the null hypothesis insinuates that the difference that exists
between the issues at hand and the anticipated results of half value is more likely not to be realized.
On the other hand , in Morvoro Lake, since the probability is greater than the usual criterion of
0.05, we therefore fail to reject the null hypothesis. This result is similar to that of the values
obtained by Dr. Bell and colleagues as shown in Table 5.3. The difference that existed between the
Morvoro and the Paw Lake is that for the Bear Paw, the difference was rejected. It is evident that
fish with pelvic reduction are more than those with pelvic spines. In conclusion, the Morvoro Lake
has both fish with and without pelvic spines. The finding can be explained that the fish are
undergoing a mutation from the species with many pelvic spines to those without. The vice versa is
still applicable. Moreover, there could have been a predator in the time of colonizing the lake but
the same is no longer available , respectively .

Experiment 2

In this experiment , the proponents should derive the characteristics of a stickleback fossil
from Nevada's Truckee formation . Surprisingly, the Truckee was estimated to have existed
approximately 10 million years ago. The fossil is considered to represent a fish that lived 15000
years ago in the lake. As a result of the material information gathered during the experiment, the
students will be able to compute the rate of variation of the species' complete phenotype.
Supernumerary, after watching the video on the formation of Truckee Lake, it was found out that
the individual rock strata represent a complete year of deposition. Each year, organisms died and
their remains were deposited on the lake's surface, where they mixed with silt and clay, resulting in
the creation of sedimentary rock rings. With this , researchers can know the long term patterns of
the evolutionary changes in species by studying their fossils. The study cannot be done with the
living organisms but rather has to be done on their ancestry forms. Studying fossils can tell
researchers about long-term patterns (i.e., on a time scale of thousands to millions of years) of
evolutionary change and could allow researchers to calculate the rate of change for certain traits.
Significantly, by studying fossils, researchers can learn about long-term patterns of evolutionary
changes in species (i.e., on a time scale of thousands to millions of years) which allows them to
quantify the rate of change for specific features. Moreover, it is worth noting that the study cannot
be done with the living organisms but rather has to be done on their ancestry forms

Furthermore, the fossil data from six rock layers were placed in a graph with approximately
3,000 years space apart from each other. Therefore, rock layers 2 and 5 are separated with a
9,000-year gap. The rock layers are also numbered from 1 to 6, with 1 as the oldest on the bottom
and 6 as the youngest on the top; numbers 2-4 then follow this same format, making the rock layer
2 older than rock layer 5. It is indicated in the graph which layers are closer to the present time, and
it was also stated in the laboratory simulation that the older layers are placed further down in the
reference. Moreover, after measuring the pelvic phenotypes, it can be observed that the pelvic
spines of the stickleback fossils of layer 2 were more complete, distinguishable, and evident in
comparison to the ones in layer 5 which were more reduced. Although some of the stickleback
fossils found in layer 5 had distinguishable pelvic spines, there were still more of them with
reduced pelvic spines.

In comparing the data obtained by the students in this laboratory simulation from the ones
collected by Dr. Bell and her colleagues, both data basically share the same results, both results are
similar to each other as shown in Table 5.6. It is also important to take note that calculation of the
rate of change over time is essential especially in this laboratory simulation since it tackles how the
pelvic systems of the stickleback changed over time and how different variables have affected the
changes that have happened. In this experiment specifically, the calculation of rate of change over
time has allowed the students to observe how gradual the evolution of this species happened over
time; the results of the calculations are found in Table 5.5.

Experiment 3

In this 3rd experiment of this online laboratory simulation, the students were able to analyze
the reduced pelvis traits of the stickleback fish from the two different populations. The pelvic
asymmetry of the fishes was specifically focused, whether on which side is the pelvis of the fish
larger, left or right. If the pelvic vestige is larger on the left side, it is called left-biased asymmetry,
and if it is larger on the right side, it is called right-biased asymmetry. This left and right bias
asymmetry is the result of the reduction in the pelvis of the stickleback fishes of both populations.
Moreover, as for the results of the experiment, it was observed that the stickleback fishes from Bear
Paw Lake and Coyote Lake are mostly biased toward the left. For the Bear Paw Lake, out of 20, 18
fishes had left-biased pelvic asymmetry and 2 had right; while for the Cayote Lake, all the fishes
examined had all left-biased pelvic asymmetry. Table 5.9 then shows the calculated chi-square
values of Stickleback’s pelvic asymmetry record. The degree of freedom for this calculation are the
traits considered (left-bias and right bias) - 1, that is 2-1 =1. As for the probability (p) value, it was
determined using the critical values table; the p value for both populations is 0.01 since the
calculated chi-square value again exceeds the value listed in the extreme right-hand column of the
table. Furthermore, in comparing the data obtained by the students in this exercise 3 of the
laboratory simulation from the ones collected by Dr. Bell and her colleagues, both data basically
share the same results, both results are very similar to each other.
CONCLUSION

The most appropriate definition of an organism is a non-human species that has been
subjected to multiple investigations in order to better comprehend a biological scenario. In this
exercise , the threespine stickleback (Gasterosteus aculeatus) species was utilized which was
characterized by its three sharp spines, which could occasionally be 2 or 4, that extends from on the
back forward of its dorsal fin. The virtual laboratory is divided into 3 experiments, each having
three components, as well as data analysis. It was accessed and executed by navigating the
provided simulation website. In experiment 1 , it was found out that both Bear Paw lake and frog
lake obtains a p value of 0.01 whilst Marvoro lake got 0.5 . With this , it was evident that the
population in Bear Lake contains mostly stickleback fish which exhibit no spines whereas that of
Frog Lake has pelvic spines . Morvoro Lake on the other hand , has both fish with and without
pelvic spines. The finding can be explained that the fish are undergoing a mutation from the species
with many pelvic spines to those without. Subsequently , in experiment 2 , it was discovered that
the number of fish having pelvic spines had decreased. Surprisingly, the first 9000 years exhibits a
dramatic reduction, followed by a more gradual decline until the fish with pelvic spines vanished
after 15000 years. The rate of variation over a given moment was assessed and it was found out
that the happenings in Nevada share a common feature with whatever that occurred in the Bear Paw
lake. The phenotype with reduced pelvic has never been witnessed in the sea- run as well as marine
habitats. More so , the two water bodies exhibited a reduction in the fish with a pelvic spine over
time . Lastly , in experiment 3 , it was observed that the stickleback fishes from Bear Paw Lake and
Coyote Lake are mostly biased toward the left. This tells us that it is possible that there is a
biological factor behind the pelvic asymmetry in the fish found in the populations of the lake. In all,
the results gathered by the group in experiments 1 , 2 ,and 3 showed consistency with the result
gathered by Dr. Bell and colleagues .

The principle of evolution was successfully demonstrated in this experiment. The students
were able to grasp both the idea of evolution and the forces that influence it which is crucial for
understanding the past, predicting the future, and organizing our environment. Without properly
comprehending evolution, nothing in biology can be truly comprehended.
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24145060.

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%20type%20of%20research,indicates%2C%20and%20it%20is%20accurate.

Fuller, P., Detloff, K., & Sturtevant, R. (2022). Gasterosteus aculeatus Linnaeus. U.S. Geological

Survey, Nonindigenous Aquatic Species Database. Retrieved April 11, 2022 from,

https://nas.er.usgs.gov/queries/factsheet.aspx?SpeciesID=702

Leonard, J.E., & Boake, C.R.B. (2010). Microevolution and Macroevolution in Behavior. Encyclopedia of

Animal Behavior, 449-454. https://doi.org/10.1016/B978-0-08-045337-8.00114-5.

Sadier, A. (2016). Regulatory and Coding Changes in Developmental Evolution, Roles of.

Encyclopedia of Evolutionary Biology, 433–440.

https://doi.org/10.1016/b978-0-12-800049-6.00129-
APPENDICE/S

Table 5.12 Calculation of experiment 1: Bear Paw Lake chi-square value.

Trait Observed (o) Expected (e) o-e (o-e)^2 (o-e)^2 /e

Present 0 10 -10 100 10

Absent 20 10 10 100 10

χ 2 = 10 + 10
(sum of the
above) = 20

Table 5.13 Calculation of experiment 1: Frog Lake chi-square value.

Trait Observed (o) Expected (e) o-e (o-e)^2 (o-e)^2 /e

Present 20 10 10 100 10

Absent 0 10 -10 100 10

χ 2 = 10 + 10
(sum of the
above) = 20

Table 5.14 Calculation of experiment 1: Morvoro Lake chi-square value.

Trait Observed (o) Expected (e) o-e (o-e)^2 (o-e)^2 /e

Present 8 10 -2 4 0.4

Absent 12 10 2 4 0.4

χ 2 = 0.4 +
0.4 (sum of
the above) =
0.8
Figure 5.16. Experiment 2 Rate of Change Calculation.

Table 5.15 Calculation of experiment 3: Bear Paw Lake chi-square value.

Trait 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

χ 2 = 6.4 +
6.4 (sum of
the above) =
12.8

Table 5.16 Calculation of experiment 3: Bear Paw Lake chi-square value.

Trait 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

χ 2 = 6.4 +
6.4 (sum of
the above) =
12.8
Table 5.17 Calculation of experiment 3: Coyote Lake chi-square value.

Trait 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

χ 2 = 10 + 10
(sum of the
above) = 20

Figure 5.17 Critical Values Table