Lab 11: Phylogenetic Analysis
Objectives
Become familiar with basic aspects of cladistics, a method for deducing the phylogenetic
relationships among organisms.
Background
Phylogeny refers to the genealogical relationships among species. The process by which most
species arise is called cladogenesis, which literally means, "branching”. In cladogenesis, one
population of a species becomes reproductively isolated from the others. When the population
evolves differences that prevent interbreeding with its relatives, the split becomes irreversible.
One species has become two. Repeated events of cladogenesis produce groups of related
species, which are called clades. Each clade can be traced back to one species that existed in
the past and was the ancestor of all the species in that clade. These evolutionary relationships
are represented as a branching diagram, or cladogram (Figure 1). Each line in a cladogram
represents a lineage, a series of ancestors and descendants through time. Each branch point
(node) represents an event of cladogenesis in the past, when a new branch (new species)
originated.
Species that share a recent common ancestor are
similar to each other, because they have inherited
a shared set of characteristics. For example,
members of the clade that we call mammals
possess fur or hair, regulate a high body
temperature using internal heat (endothermy),
and feed their young from mammary glands.
Figure 1. A cladogram showing
relationships among three species.
These features were inherited from the ancestral
mammal. Frogs lack these characteristics, which
evolved after the last shared ancestor of
mammals and frogs. However, mammals and
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frogs do share characteristics that were present in a more ancient shared ancestor, such as
having vertebrae, possessing lungs, and having four limbs with similar skeletal structure
(Figure 2).
The naming and classification of organisms
is called taxonomy. Each level of
classification is called a taxonomic category,
and each group of species that is classified is
called a taxon (the plural of taxon is taxa).
For example, one taxonomic category is
"genus". Two taxa in this category are the
genus Homo (which includes our species) and
Figure 2. Cladogram of three vertebrates
showing timing of origin of several
charateristics.
the genus Pan (the chimpanzees). The seven
basic taxonomic categories should already be
familiar to you. They are Kingdom, Phylum, Class, Order, Family, Genus, and Species. These
categories are hierarchical, meaning that each taxon includes one or more taxa in the next level
below it.
Biological classification is based upon similarities among organisms. For example, the species
placed within a genus are more similar to each other than they are to those in other genera.
However, there are two very different kinds of similarity among organisms. These are
homology and analogy. Homologies are those similarities that are inherited from a shared
ancestor. For example, all tetrapod vertebrates (amphibians, reptiles, mammals, and birds) have
three long bones in the front limbs, the humerus, radius, and ulna. This characteristic is
homologous because it was present in the earliest tetrapods.
Analogies are similarities that are the result of convergent evolution. In convergent evolution,
two species independently evolve similar characters, rather than inheriting them from a shared
ancestor. An example of analogous similarity is that both birds and bats have wings and fly.
The wings of birds and bats are analogous, not homologous. That is, the last shared ancestor of
birds and bats did not have wings. Instead, both lineages developed wings independently.
Evidence for this fact includes the appearance of bats in the fossil record long after the
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divergence of the lineages that led to birds and mammals, and the very different structure of the
wings in these groups.
Biologists generally agree that the taxonomic classification of organisms should be based on
homology, and not on analogies. Generally, homologies are much more common. Analogies are
usually superficial and can be recognized by careful examination. Homologies extend right to
the DNA, because they are inherited characteristics. We should also note that homologous
structures may be modified for very different functions. For example, comparative anatomy and
paleontology show that the bones of the middle ear in mammals are homologous with bones in
the lower jaws of reptiles. These bones have been modified for very different purposes in these
two lineages, but they are nonetheless homologous.
Systematics is the study of the phylogenetic relationships among organisms. Systematics is not
the same as taxonomy, but the modern classification of organisms is generally based upon
hypotheses about phylogeny. How can we infer phylogeny? The most popular method at
present is called cladistics.
Cladistics
Let’s say that we want to deduce the evolutionary relationships of a group of taxa. We will
never know the phylogeny for certain, because we don't have a time machine to go back and
watch them evolve. However, we can make hypotheses about their relationships and test the
hypotheses. Cladistics is a method for testing hypothetical phylogenies by comparing the
characteristics of the taxa. It is not the only method, but it is the most popular one at present.
The fundamental question in a phylogenetic analysis is the following: Which two of a group of
three taxa are more closely related? Only homologous characteristics are useful in answering
the question. But cladistics also recognizes that not all homologies are useful. At this point, we
have to dissect homology into several categories. Let’s assume that we know the phylogeny of
species A, B, and C, and that species A and B share a more recent common ancestor with each
other than with species C.
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The cladogram shown in Figures 1-3 show these relationships. The figures indicate the origin
of a character state with a horizontal line, and a grey line is used to represent the inheritance of
that character state in one or more of the lineages leading to the species. The character state
only originates once, so it is a homology among the species.
The homology in figure 3 is a plesiomorphy– a
character state inherited from the ancestor of all
three taxa. Plesiomorphies do not indicate who
is most closely related. Moreover, if a
plesiomorphy is lost from one taxon, it may be
misleading by making the other two look more
closely related.
Figure 3. Primitive character state
(plesiomorphy).
Figure 4 shows a synapomorphy shared by taxa
A and B. The character state is present in two taxa but not the third, because it evolved after the
other taxon split off. Synapomorphies are useful evidence in deducing phylogeny, because they
result from the fact that A & B share a more recent common ancestor with each other than either
shares with C.
Figure 5 shows an autapomorphy, a
characteristic that evolved in one of the taxa,
after it diverged from the others.
Autoapomorphies are not useful in deducing
phylogeny. In fact, autapomorphies in one
lineage can make the other two appear more
similar to one another. That is, B and C appear
Figure 4. Shared, derived character state
(synapomorphy).
more similar to each other in lacking the
character, even though they are not the most
closely related pair.
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Outgroup comparison
How can we determine whether a character state
is primitive or derived? The commonly used
method is called outgroup comparison. Another
taxon, less closely related to the group being
considered than they are to each other, is
included in the comparison. This taxon is called
Figure 5. Derived character state in one
taxon (autopomorphy).
an outgroup, and the other taxa are called the
ingroup. Cladistic analysis assumes that the
character states of the outgroup are primitive for
the ingroup. Obviously, this assumption may or may not be true, depending on how closely
related the outgroup is to the ingroup. Ideally, the outgroup is a close relative of the ingroup,
only slightly less related to them than they are to each other.
Only character states that are derived and shared among some, but not all, of the ingroup taxa,
are useful. While shared, derived characteristics provide evidence that two species are closely
related, it is possible for two species to independently evolve the same derived characteristic.
Convergent evolution leads to analogous similarity. How do we determine whether the traits we
are studying are homologous or analogous? Phenotypic analogies are usually fairly easy to
recognize, and homologies are more common. Generally, systematists study as many different
characters as possible, avoid obvious analogies, and assume that homologies will dominate the
analysis.
The number of possible phylogenies increases rapidly with the number of taxa. With 3 taxa,
there are only 3 possible phylogenies to decide among. With 4 taxa there are 15 possibilities,
and with 10 taxa there are 34,459,425 possibilities! Computers are used to evaluate cladistic
analyses that include more than a small number of taxa.
We need to determine how well each tree is supported by the character states of the taxa. One
statistic often used for comparing trees is the consistency index (CI). The consistency index
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measures the efficiency of the tree in explaining the distribution of character states. It is
calculated as follows:
the minimum possible number of character state changes
CI =
______________________________________________
the actual number of character state changes
Each derived trait must evolve at least once. Therefore, the minimum possible number of
character state changes is equal to the total number of derived states (i.e. different from the
outgroup). The actual number of character state changes is called the treelength. Treelength is
measured by counting the number of points on a tree where the characters must change state. CI
is a proportion, so it ranges from 0 to 1. The higher the CI, the more efficient the tree is. That
is, the tree demands fewer evolutionary changes. The tree that postulates the fewest changes in
character state is assumed to be the most likely to be true*. As a rule of thumb, CI values of
under 0.6 indicate poor support for the phylogeny; CI values between 0.6 and 0.8 indicate fair
support, and CI values above 0.8 indicate good support.
In the following exercises we’ll examine groups of species, determine their characters and
character states, and devise possible phylogenies. We’ll distinguish primitive and derived traits
using outgroup comparison, and then use the derived traits to deduce the most parsimonious
phylogeny.
*In choosing the most likely of several possible explanations, we are employing the principle of
parsimony, which is also called “Occam’s razor”. The philosopher and logician William of
Occam about 700 years ago argued that, lacking contrary evidence, the simplest explanation is
most likely to be correct. “What can be done with fewer [assumptions] is done in vain with
more.”
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Activity
Procedure 1: Doodlebirds
Let’s start with a simple, hypothetical example. The sketches below represent four hypothetical
species. (OK, I’m not an artist. At least they’re simple!).
Figure 6. Four hypothetical critters for considering
cladistically.
Assume that we know that species #1 is the outgroup (that is, #1 is less closely related to the
other three than they are to each other). There are only 3 possible cladograms for the ingroup,
as follows:
Let’s use a cladistic approach to choose the best tree for the ingroup. First, we examine the
species and select potential characters for our analysis. Then we prepare a table of species and
characters and record the character states of each species.
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Table 1. Character states of the four hypothetical birds (Species #1 is the outgroup).
Legs
Beak
Crest
Tails
Toes
#1
Two
Small
Yes
Two
Plain
#2
Two
Large
Yes
Two
Plain
#3
Two
Large
Yes
Three
Web
#4
Two
Large
No
Three
Web
Next, we examine the characters and decide which will be useful in the analysis. Only character
states that are shared and derived within the ingroup are useful. Derived means that the
character state is different from the outgroup (apomorphic). However, to be useful, the
character state must be derived within the ingroup. That is the character state must be present in
at least two, but not all, members of the ingroup. Which of the characters qualify? Of the 10
character states, only three-tails and web-toes fit the criteria. Be sure you understand why the
other characters do not qualify.
Third, we prepare a similarity matrix. The similarity matrix is a table in which we tally the
number of shared, derived characters for each pair of ingroup species (Table 2).
Table 2. Similarity matrix for the ingroup of hypothetical doodlebirds (Figure 6).
Species
#2
#3
#4
#2
#3
#4
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Sketch your cladogram here:
Species #3 and #4 share the largest number of synapomorphies. Therefore, the best cladogram
is “C”, which postulates a more recent common ancestor for #3 and #4 than for either of them
and #2. The CI for this tree is 1.0 (why?).
Procedure 2: Cladistic classification of dragonflies.
Now we will perform a more extensive cladistic analysis on some real organisms- dragonflies.
Dragonflies are insects in the Order Anisoptera. They are convenient for this exercise because
they have several easily visible morphological characters that we can use for our analysis.
Your instructor will provide you with detailed photographs (top and side views) of six species of
dragonflies. These photos are from the “Digital Dragonfly Museum” of the Texas Agricultural
Experiment Station. We’ll refer to the specimens by their collection numbers. Each specimen
represents a different species. Five of these species (#85, #231, #273, #282, and #358) are from
one suborder. These will be the ingroup. The sixth species (#291) is from a different suborder,
and will be used as the outgroup.
Your objective will be to find the most parsimonious cladogram for the ingroup. You will then
check your results by comparing your cladogram with the accepted classification of these
species.
We will use the characters shown on the next page (Table 3). The possible states for each
character are yes or no.
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Table 3. Possible characters for use in cladistic analysis of dragonflies. Compare these
with your six species and decide whether “Yes” or “No” applies for each character. Characters
that are similar among all six species are not listed (e.g having four wings, or six legs).
However, some of the characters that are listed are not informative, either because they are
uniform within the ingroup or because they are autapomorphies. You will have to recognize
these and leave them out of your similarity matrix.
1. Eyes are
separated
(not
touching at
the midline).
See # 273
top.
7.
Superior
anal
appendages
are longer
than the last
abdominal
segment
(dorsal
view).
See 231 top.
8. Inferior
anal
appendages
are less than
1/3 the
length of
the dorsal
appendages
(side view).
See #85
side.
9. Inferior
anal
appendages
are strongly
hooked
(side view).
See #273
side.
2. Eyes are
confluent at
the midline
for most
their
diameter.
See #85 top.
3. Distance
from
pterostigma
to wing tip
exceeds
length of
pterostigma.
See #231
top.
4. Brace
vein
present.
This is a
cross-vein
set at an
angle
continuous
with the
edge of
pterostigma.
See #282
top.
10. Thorax uniform green color.
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5. M1 and
M2 are
parallel.
These two
wing veins
have been
darkened in
the figure to
make them
evident.
See #231
top.
6.
Bannertail.
The last 4
segments of
the
abdomen
are
expanded
laterally and
are wider
than the
preceding
segments.
See # 273
top.
11. Brown stripes on green thorax.
12. Blue stripes on brown thorax.
13. Uniformly red abdomen.
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Table 4. Character state table for the dragonflies. Examine the six dragonfly species.
Decide whether each of the 13 characters applies (yes) or does not apply (no), and fill out the
table accordingly. Cross our any characters that are not phylogenetically informative, and do
not include them in tabulating your similarity matrix (Table 5).
Species (by
specimen #)
Characters (see table 3)
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10 #11 #12 #13
#291
(outgroup)
#85
#231
#273
#282
#358
Table 5. Similarity matrix for the dragonflies. Examine your character state table and tally
the number of shared and derived character states for each possible pair of species.
#85
#231
#273
#85
#231
#273
#282
#358
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#282
#358
Cladogram:
Fill out the character state table and similarity matrix (Tables 4, 5). Then use the similarity
matrix to deduce the most parsimonious cladogram for the ingroup species. Link those species
and clades that share the most synapomorphies. In this example, there should be only one most
parsimonious tree. It should be fully supported by all the characters. That is, the CI should be
1.0. After you have deduced the best possible cladogram, sketch it in the box provided on the
next page.
Use this space to practice drawing your cladogram:
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Review Questions
1. Be able to construct a cladogram given a character state table (e.g. Table 4).
a. Write out the steps to construct a cladogram:
2. Describe what an outgroup is and for what purpose it is used in cladistic analysis.
3. Are homologies or analogies used to deduce phylogeny? Why?
Terms to Know
Phylogeny
Synapomorphy
Taxonomy
Outgroup
Cladistics
Consistency Index
Cladogenesis
Homology
Systematics
Analogy
Pleisomorphy
Convergent Evolution
Autopomorphy
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Assignment
Sketch your dragonfly cladogram below, indicating the species numbers at the branch ends.
Include the outgroup. Then compare your results to the classification of these species that is
provided. Can you assign each specimen to a family? To a species? Visit the Digital
Dragonfly Museum image gallery (linked to the lab page) and use the names to find the
catalog of each species. Look at the list of “scans” for each of the six species, and find your
specimen number. Then fill in the specimen numbers in the list below, assigning each
specimen to its species. Does your analysis agree with the taxonomic classification?
2. Read the essay titled “The Creation-Evolution Continuum” (linked to the lab page).
After you have read this material, please write a brief (~ 1 page) essay expressing your
personal attitude on this subject. Where do your beliefs fall on the continuum? What
questions do you have on this subject?
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Discussion Notes
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