The Origin

of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

USING DNA TO EXPLORE LIZARD PHYLOGENY

OVERVIEW
This lesson serves as a supplement to the film Lizards in an Evolutionary Tree. The film focuses on a closely related group of
lizards called anoles (genus Anolis) that live in the Caribbean islands. They have evolved to occupy different niches in the
environment, and researchers are finding surprising insights into evolutionary processes, such as adaptation by natural
selection, convergent evolution, and the formation of new species.
In this activity, students explore the evolutionary relationships of the Caribbean anole lizards. In part 1, students examine
the diverse appearance of the lizards and group them into categories according to different body types. Differences
between the species illustrate adaptive radiation; students can generate hypotheses about possible reasons why different
adaptations have evolved and infer evolutionary relationships among lizards based on their adaptations. In part 2,
mitochondrial DNA sequence data from these same species of lizards are used to generate a phylogenetic tree to reveal
their true evolutionary relationships. The students need to reconcile why the evolutionary tree obtained in part 2 is
different from the original grouping proposed in part 1. Students watch the film in stages at different points in the activity.

KEY CONCEPTS AND LEARNING OBJECTIVES

A. An adaptation is a structure or function that confers greater ability to survive and reproduce in a particular
environment.
B. Adaptive radiation occurs when an ancestral species diversifies into many descendant species that occupy
different environmental niches.
C. Different groups of organisms can independently evolve similar traits by adapting to similar environments or
ecological niches in a phenomenon known as convergent evolution.
D. DNA sequence comparisons among different populations and species allow scientists to determine the
relatedness of different species.
After completing this activity, students should be able to:
Explain how the ecomorphs of anole lizards in the Caribbean are examples of both adaptive radiation and
convergent evolution
Use sequence alignment and phylogeny generating software to compare mitochondrial DNA sequences
Analyze data from a phylogenetic tree and determine whether the data support a hypothesis

CURRICULUM CONNECTIONS
Curriculum

Standards

NGSS (April 2013)

MS-LS1-5, MS-LS4-2, MS-LS4-4, MS-LS4.A, MS-LS4.B, MS-LS4.C, HS-LS4-1, HSLS4-3, HS-LS4-4, HS-LS4-5, HS-LS4.A, HS-LS4.B, HS-LS4.C,

AP (201213)

1.A.1, 1.A.2, 1.A.4, 1.B.2, 1.C.1, 1.C.2, 1.C.3

IB (2015)

4,1, 5.1, 5.2, 5.4, C.1

TEXTBOOK CONNECTIONS
Textbook

Chapter Sections

Miller and Levine, Biology (2010 ed.)

16.1, 16.3, 16.4, 17.2, 17.3, 17.4, 18.2, 19.2

Reece et al., Campbell Biology (9th ed.)

22.2, 22.3, 23.3, 23.4, 24.1, 24.2, 24.4, 26.2, 26.3

www.BioInteractive.org

Published March 2014

Updated April 2015
Page 1 of 9

The Origin
of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

TIME REQUIREMENTS
Allowing time for discussion, and viewing the film, this activity will take two 50-minute periods for an AP biology class.
Allow for more time if you need to cover basics of DNA alignment in greater detail.

PRIOR KNOWLEDGE
Before doing this lesson, students should have a basic understanding of natural selection, evolution, and adaptation;
familiarity with the concept that organisms fill specific niches in their environments; an understanding that DNA sequence
changes during evolution, and closely related species have similar DNA sequences; and a basic understanding of
phylogenetic trees, especially the concept that closely related species are located close together on adjacent or nearby
branches of a phylogenetic tree.

MATERIALS

A set of lizard cards that accompanies this document

Access to a computer that is connected to the internet
HHMI Short Film Lizards in an Evolutionary Tree
(Optional) A computer with the freely available ClustalX program installed.

BACKGROUND SUMMARY
This section provides a brief summary. For more information about the anole lizards and the film, please refer to the InDepth Guide (http://media.hhmi.org/biointeractive/activities/lizard/IDG_Lizards.pdf).
Different Morphologies of Anole Lizards
Figure 1: Caribbean islands are home
to the anoles. The anole species
featured in the film and this activity
live on the islands of Cuba, Jamaica,
Hispaniola (which comprises Haiti and
the Dominican Republic), and Puerto
Rico. (Reproduced with permission
from Losos, J. Lizards in an
Evolutionary Tree. UC Press, 2009).

Species of Caribbean anoles can be categorized into six groups according to their body characteristics (morphology) and
the ecological niches they occupy. The groups are referred to as ecological morphotypes, or ecomorphs. Table 1 below lists
the six anole ecomorphs found in the Caribbean islands and their body features. The film discusses only four ecomorphs:
the crown-giant, trunk-ground, twig, and grass-bush anoles. In this activity, we have representatives from the trunk-crown,
trunk-ground, twig, and grass-bush anoles, shaded in the table.

Using DNA to Explore Lizard Phylogen

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Updated April 2015

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The Origin
of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

Table 1: Six Ecomorphs of Anole Lizards Found in the Caribbean Islands.

Ecomorph

Body length

Limb length

Toe pad size

Tail length

Color

Habitat

Crown-giant

130-191 mm

Short

Large

Long

Usually green

Trunk-crown

44-84 mm

Short

Very large

Long

Green

Trunk

40-58 mm

Intermediate

Intermediate

Short

Gray

High trunks and

branches
Trunks, branches, and
leaves
Trunks

Twig

41-80 mm

Very short

Small

Short

Gray

Narrow twigs

Trunk-ground

55-79 mm

Long

Intermediate

Long

Brown

Grass-bush

33-51 mm

Long

Intermediate

Very long

Brown

Lower trunk and

ground
Bush and grasses

The body features of the ecomorphs are adaptations to their particular niches. (See Figure 2 for an illustration of the
habitats of different ecomorphs.) In the film, the long-legged trunk-ground anoles are shown to move faster on the ground
than the short-legged twig anoles. The long-legged adaptation helps them not only catch prey on the ground but also
avoid predators. However, on twigs, the twig anoles can move easily with their short legs, while the trunk-ground anoles
are clumsy. The grass-bush anoles are small and have long legs and strikingly long tails that help them balance on thin
branches and blades of grass, and the trunk-crown anoles, with their large toe pads, are adapted to living on leaf surfaces
in the canopy.

Figure 2: Anoles occupy a variety of

ecological niches. Different types of anole
lizards have evolved adaptations that enable
them to be successful in different ecological
nichesdifferent parts of trees, grasses, and
bushes. The figure shows the six ecomorphs
of anole lizards found in the Caribbean
islands in their habitats. (Reproduced with
permission from Losos, J. Lizards in an
Evolutionary Tree. UC Press, 2009).

Convergent Evolution Among Anole Lizards

There are different explanations for why similar ecomorph classes can be found on each of the different islands.
One hypothesis is that each ecomorph evolved once, and the species belonging to the same ecomorph are closely related
to one another. Under this scenario, one possibility is that the islands were contiguous in the past when the ecomorphs
evolved, and subsequent plate tectonics separated the islands. Another possibility is that the ecomorphs all evolved on the
same island, then they all migrated over the narrow channels and established themselves in each of the islands. In these
Using DNA to Explore Lizard Phylogen
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Updated April 2015

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The Origin
of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

scenarios, you would predict that DNA analysis would reveal, for example, that the twig anole species on all of the islands
are more closely related to one another than they are to the grass-bush, trunk-ground, and trunk-crown anoles on the
same island.
An alternative hypothesis is that the ecomorphs evolved repeatedly and independently on each of the islands. A prediction
based on this hypothesis is that the anoles on each island would be more closely related to one another than they would be
to species on other islands belonging to their ecomorph.
When scientists examined DNA sequences from dozens of species of Caribbean anoles, they found that in general, species
on the same island tend to be more closely related to one another than to species with similar body types found on
different islands. This suggests that the same adaptations evolved independently in different anole populations on each of
the islands, an example of convergent evolution.

SUGGESTED PROCEDURE
Students can work individually or in small groups of perhaps between two and four, depending on your classroom. A
student worksheet is provided to guide the activity, but, as explained below, there are points at which you may wish to
stop and conduct a whole group discussion, show the film to the whole class, or explain a concept. Alternatively, you can
interact with each individual student group, depending on the dynamics of your classroom.
Part1: Identifying the Ecomorphs and Exploring Anole Adaptive Radiation
At this point, students have not seen the film Lizards in an Evolutionary Tree yet.
1.

Hand out the student worksheet for part one and a set of lizard cards to each group. Let teams of students examine
photographs of 16 species of anole lizards and sort them according to how they appear in the photographs into as
many groups as they want.
Students can use any clues in the photographs to group the lizards as they see fit. They may use features of the
ecomorphs, including color, thickness or length of legs, body shape, tail length, or size. They may even group them by
the habitat pictured, such as branches, tree trunks, or grass.

2.

Allow students time to answer question 1, then ask the teams to explain how they grouped the lizards and their
rationale for the various groupings. If they pick a body feature, ask them to speculate about the advantages or
disadvantages of such a body feature in the environment they occupy.

3.

Watch the first part of the film (up until 09:48, the end of the experiment on a small rocky island).

4.

Allow students time to answer question 2 and revise their groupings if they wish.

5.

Reveal the four ecomorphs. The 16 species represent the following four ecomorphs. Refer to Table1 for more
descriptions of ecomorphs. Also the first page of the lizard picture file can be used as a master reference.
Trunk-crown: green, foot pads
Twig: short legs, pencil-like morphology
Grass-bush: Slender limbs, body, and long thin tail
Trunk-ground: Robust and chunky appearance, with long legs

6.

Allow students time to answer questions 3 - 6. Discuss the body features of the ecomorphs, how they are adapted to
the environment they live in, and the concept of adaptive radiation. For example, the large toe pads of the trunkcrown anoles allow them to adhere to leaves and move about freely among the leaves of the tree canopy. The short
legs of the twig lizards are adaptations to living on thin twigs. Refer to the Different Morphologies of Anole Lizards
section in the background. Discuss question 6, as it leads into Part 2.
Using DNA to Explore Lizard Phylogen
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Updated April 2015

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The Origin
of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

Part 2: Generating a Phylogeny from DNA Sequences and Revealing Evolutionary Relationships of Anoles.
(Optional) If your students have never been exposed to generating and interpreting phylogenies from DNA sequences, you
may want to use the following resources from BioInteractive:
a. Follow the procedure outlined in Introduction to DNA Sequence Alignment Using ClustalX.
http://media.hhmi.org/biointeractive/activities/lizard/Sequence-Alignment-Introduction.pdf
b. Have your students explore the Click and Learn Creating Phylogenetic Trees from DNA Sequences.
http://www.hhmi.org/biointeractive/creating-phylogenetic-trees-dna-sequences
1. Hand out the first two pages of Part 2 of the student worksheet (pages 3-4). Read the introduction as a whole group or
allow teams time to read the introduction and answer question 1. Students can look back at Part 1, question 6.
2. Have students follow the directions on page 4 to download the Anolis sequences text file and then use
www.phylogeny.fr to align the sequences and create a phylogenetic tree.
3. The website will generate a phylogenetic tree that can be saved in a variety of formats. Because of the nature of the
phylogenetic analysis that involves random resampling, each student may get a slightly different tree, but the overall
pattern should be mostly similar.
Figure 3: Example
phylogeny from
www.phylogeny.fr.
Students phylogeny
should be similar, but
there may be small
differences.

The red numbers in the phylogeny result are bootstrapping values, and denote how confident the program is about the
specific branch pattern. The value of 1 indicates high confidence; 0 indicates no confidence. Values less than 0.5 are
generally regarded as uncertain. However, because the purpose of this activity is to illustrate the principle of DNA
sequence comparison, students should not worry about it too much.
4. Hand out pages 5 and 6 of Part 2. Alternatively, you could print out two copies of the students phylogenetic tree per
group of students. Have them use the list of lizard species to color the trees in the following ways:
a. On one copy, color each branch according to the island of origin (Cuba, Hispaniola, Jamaica, or Puerto Rico). If both
branches at a node are the same color, color the root branch and continue left to the next node.
b. On the other copy, color each branch according to the ecomorph similarly.
5.

Allow students time to examine the phylogenetic tree and answer questions 3-7. Discuss as a class if you wish.

6.

Watch the rest of the film.

7.

Allow students time to answer question 8. Discuss as a class.

Using DNA to Explore Lizard Phylogen

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The Origin
of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

8.

Students may ask whether there are examples of convergent evolution in groups other than the anoles. Perhaps the
students can be enticed to volunteer if they know anything that looks like a shark. They could list tuna, dolphins,
manatees, and ichthyosaurs. Wings in vertebrates are another classic example: birds, bats, pterosaurs. Reduced or
absent limbs is another: snakes, caecilians, sirens, slow worms, worm lizards.

9.

(Optional) Download the Reference-Phylogeny (http://media.hhmi.org/biointeractive/activities/lizard/ReferencePhylogeny.pdf) document and refer to the phylogenetic tree. This is one of the phylogenies published in a scientific
journal. Find the 16 species used in this activity, and see if their phylogeny differs from the tree generated by the
student groups.
Some reasons for differences: The paper uses many more species, which can generate different patterns; the paper
also uses different calculations to align the sequences and generate phylogenies.

LIST OF LIZARD SPECIES

Species name

Island

Ecomorph

Anolis alutaceus

Cuba

Grass-bush

Anolis angusticeps

Cuba

Twig

Anolis chlorocyanus

Hispaniola

Trunk-crown

Anolis cristatellus

Puerto Rico

Trunk-ground

Anolis cybotes

Hispaniola

Trunk-ground

Anolis evermanni

Puerto Rico

Trunk-crown

Anolis grahami

Jamaica

Trunk-crown

Anolis lineatopus

Jamaica

Trunk-ground

Anolis occultus

Puerto Rico

Twig

Anolis olssoni

Hispaniola

Grass-bush

Anolis ophiolepis

Cuba

Grass-bush

Anolis porcatus

Cuba

Trunk-crown

Anolis pulchellus

Puerto Rico

Grass-bush

Anolis sagrei

Cuba

Trunk-ground

Anolis shelpani

Hispaniola

Twig

Anolis valencienni

Jamaica

Twig

Leiocephalus barahonensis

Outgroup

Using DNA to Explore Lizard Phylogen

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The Origin
of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

ANSWERS
Part1: Identifying Ecomorphs and Exploring Anole Adaptive Radiation
For questions 1,2 and 3, student answers will vary. Its important that they can explain their rationale.
4.

After your teacher explains the various ecomorphs, chose one and explain how the characteristics of that body type
are adaptations to that ecomorphs particular habitat.
Refer to the ecomorph chart (Table 1) in the Background Information section.

5.

How are the different ecomorphs examples of adaptive radiation?

Adaptive radiation occurs when an ancestral species diversifies into many descendant species that occupy different
environmental niches. Each of the different ecomorphs displays physical characteristics that make the organism
suited to the particular niche in which it lives.

6.

Why do you think similar ecomorph classes can be found on many of the different islands in the Caribbean?
Student answers will vary. This answer can be referenced again in Part 2 if you wish.

Part 2: Generating a Phylogeny from DNA Sequences and Revealing Evolutionary Relationships of Anoles.
1. Which hypothesis about anole evolution do you wish to test? Why did you choose that one?
Student answers will vary.
2.

Using the list of lizard species & the phylogenetic trees, color in the phylogenetic trees to get a visual picture of how
the species evolved.
a. Color each branch according to the island of origin (Cuba, Hispaniola, Jamaica, or Puerto Rico). If both branches at a
node are the same color, color the root branch and continue left to the next node. Include a legend.
Figure 4: Phylogeny
colored in according to
geographical distribution.
Red = Cuba, Green =
Jamaica, Blue = Puerto Rico,
Yellow = Hispaniola.
All species from Jamaica
cluster together, and many
species from Puerto Rico
cluster together. Species
from Cuba and Hispaniola.

Using DNA to Explore Lizard Phylogen

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Updated April 2015

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The Origin
of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

b. Color each branch according to the ecomorph similarly. Include a legend.

Figure 5: Phylogeny
colored in according to
ecomorphs.
Red = Trunk-ground,
Green = Trunk-crown,
Blue = Grass-bush,
Yellow = Twig.
Except for two twig species,
none of the ecomorphs
cluster together, meaning
they are not closely related.

3.

What general patterns do you see in the tree?

In general, the phylogenies should show that species from the same island are more closely related than are
species from the same ecomorph group. For example, the three Jamaican species should cluster together, as will
three of the four Puerto Rican species. The Cuban and Hispaniola species also cluster, but the pattern is more
complicated.

4.

Do species from the same ecomorph group together on a branch? Explain.

Different ecomorphs are grouped together on branches. Except for two twig species, none of the ecomorphs
cluster together.

5.

Do species from the same island group together on a branch? Explain.

All species from Jamaica cluster together, and many species from Puerto Rico cluster together. Species from
Cuba and Hispaniola also cluster, but the pattern is more complicated.

6.

Did the data support the hypothesis you chose to test?

Student answers will vary depending upon which hypothesis they chose.

7.

Are ecomorphs are examples of convergent evolution? Explain.

In general, the phylogenies should show that species from the same island are more closely related than are
species from the same ecomorph group. These pieces of evidence suggest that most of the lizards on each island
evolved independently, in a process of convergent evolution, but there are exceptions.
An example of this exception is Anolis sheplani, a Hispaniola twig species, which is closely related to another
twig species from Cuba, Anolis angusticeps, in a branch that is otherwise all Cuban. One could hypothesize that A.
sheplani evolved after Hispaniola was colonized by a twig ecomorph species from Cuba.

8.

Do your results agree with whats shown in the film? What are the similarities and differences?
The results should agree in general with whats shown in the film. The film explains that species on an island are
more closely related to one another than to similar ecomorphs on other islands. Students will likely note that the
Using DNA to Explore Lizard Phylogen
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Updated April 2015

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The Origin
of Species
The Making
of the
Fittest:
TheLizards
Making
Fittest: Tree
in of
an the
Evolutionary
Natural Selection and Adaptation
Natural Selection and Adaptation

TEACHER MATERIALS

data set they were working with presented a more complex set of relationships (not as clean as in the film). The
general picture is the same, but there are some cases (see question 7) that dont fit. Furthermore, the film discusses
four ecomorphs (the crown-giant, trunk-ground, twig, and grass-bush anoles), one of which is different from the
species in this activity.

RELATED CLASSROOM RESOURCES

Other Resources for the film (http://www.hhmi.org/biointeractive/classroom-activities-origin-species-lizardsevolutionary-tree) Check this page to see other resources developed for the film.
Sorting Seashells (http://www.hhmi.org/biointeractive/sorting-seashells): In this Click and Learn, students explore
principles of taxonomy by sorting seashells according to their morphological characteristics and constructing an
evolutionary tree.
Classroom Activities: Biodiversity and Evolutionary Trees (http://www.hhmi.org/biointeractive/classroom-activitiesbiodiversity-and-evolutionary-trees): This is a companion classroom resource to accompany the Click and Learn above. It
also includes a DNA sequence comparison activity of the seashells. In the seashell activity, phylogeny generated by
examining morphology closely aligns with that generated from DNA, so it is a good contrast to the case of the anole lizards
where the two do not agree.
Creating Phylogenetic Trees from DNA Sequences (http://www.hhmi.org/biointeractive/creating-phylogenetic-treesdna-sequences): This Click and Learn explains how DNA sequences can be used to generate such trees, and how to
interpret them.

REFERENCES
Losos, J. (2009) Lizards in an Evolutionary Tree. University of California Press, Berkeley, CA.
Rabosky, D. L., and Glor, R. E. (2010) Equilibrium speciation of dynamics in a model adaptive radiation of island lizards.
PNAS 107, 22178-22183.
ClustalX: Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace,
I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G.. (2007) Clustal W and Clustal X version 2.0.
Bioinformatics, 23, 2947-2948.
Phylogeny.fr: Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, S., Lefort,
V., Lescot, M., Claverie, J.M., Gascuel, O. (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic
Acids Res. 2008 Jul 1;36(Web Server issue):W465-9. Epub 2008 Apr 19.

AUTHORS

Written by Satoshi Amagai, PhD, HHMI

Student Worksheet created by Valerie May, Woodstock Academy, Ann Brokaw, Rocky River High School, Susan
Dodge,consultant
Edited by Laura Bonetta, PhD, Laura Helft, PhD, HHMI; copyedited by Linda Felaco
Reviewed by Jonathan Losos, PhD, Harvard University

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