Phylogenetic Methods and
Vertebrate Phylogeny
Objectives:
1. To become familiar with phylogenetic terminology.
2. To determine evolutionary relationships objectively using standard phylogenetic
methods.
3. To understand the evolutionary relationships among representatives of
terrestrial tetrapods traditionally placed in the classes Reptilia, Aves
(birds), and Mammalia.
I BACKGROUND MATERIAL
There are many phenomena in the world that we can observe and would like to
understand. In particular, most of us want to know why things happen. To approach
any question scientifically, we use a formalized approach, using the initial
observations to formulate testable hypotheses. Testable hypotheses are
explanations of how things happen, that can be tested by collecting more data
either through experimentation or further observation.
Comparison of the data we collect with the predicted outcome forms the step of
evaluation. The new data might result in rejection of our hypothesis: such data
would be contrary to our predictions. If the data do not contradict the
hypothesis, we state that they are supportive. Scientists never speak of “proving”
any hypothesis. Finally, we can use the process of evaluation to revisit the initial
observations, perhaps formulating new questions or hypotheses, a step scientists
refer to as drawing inferences.
The evolutionary relationships among organisms are always going to be
hypothetical: scientists can never go back in time and actually observe the
evolution of a new group. The formulation of an initial hypothesis about
relationships among groups is the process of making observations and constructing
a phylogeny, which is a testable hypothesis. This hypothesis can then be tested by
the collection of additional data.
In this laboratory exercise, you will become familiar with some of the techniques
used by systematic biologists to develop hypotheses about the evolutionary history
of living things. You will score individual specimens from different species of
animals and, based on these physical attributes, decide which groups of animals are
most closely related to each other. At the end of the exercise, we will discuss
what data could be used to test your hypotheses.
A. THE CHALLENGE OF SYSTEMATICS AND REMOTE INFERENCE
(Sys·tem·at·ics n: the study of systems and classification, especially the science
of classifying organisms)
Both theoretical and practical problems make inferring evolutionary history (a
discipline known as systematics) one of the most challenging of the life science
disciplines. Like other disciplines in biology, systematics proceeds through the
experimental cycle, depending on the construction of hypotheses from
observations and the rejection or retention of these hypotheses based on
experimental work. What makes systematics unique is that while these hypotheses
address events in the past, often million of years ago, they are based on
observations made today. This process is known as remote inference, and you will
be employing it in this lab. We shall in all likelihood never see major groups of
organisms evolving over long periods of time, so we must use observations of both
extant organisms and fossils to construct hypotheses concerning the origin of the
major groups.
B. TERMINOLOGY
Taxon n (plural: taxa): Any of the groups to which organisms are assigned
according to the principles of taxonomy, including species, genus, family, order,
class, phylum, etc.
Cladogram n: A branching diagram where the branching is based on the inferred
historical connections between the entities as evidenced by shared derived
characters and the end of each branch represents one species.
Figure 1: Two different
examples of phylogenetic
trees, ranging from the
simple to the complex. The
one above is “rooted” while
the one to the right is not.
Phylogenetic tree n: A branching diagram in which the branching portrays the
hypothesized evolutionary relationships and the sequence of hypothetical
ancestors linking observed taxa.
Character state matrix n: A table of characters where the state of the
character in each taxon is coded as being primitive (usually with a zero) or derived
(usually with a 1).
Ingroup n: The group of organisms in an evolutionary study in which relationships
are determined based on the presence, or lack of shared characteristics, and
comparison to the outgroup. There are usually numerous taxa within the ingroup.
Outgroup n: The taxon least related to any other taxon in an evolutionary study, to
which members of the ingroup are compared. There may be one or multiple
outgroups in a study.
C. SELECTION OF OUTGROUP AND SCORING OF CHARACTERISTICS
The first two steps in deciding how organisms are related to each other is to 1)
identify outgroup(s) and 2) enumerate multiple discrete characteristics that can
be evaluated either as “primitive” or “derived.” Often, when determining
evolutionary relationships amongst numerous taxa, an outgroup that is already
known to be more primitive than the taxa in consideration is selected. If there is
no clear outgroup, the outgroup may be selected from the study organisms as the
taxon with the highest number of of primitive characteristics, after
characteristics have been scored.
In the example below, the outgroup has already been selected (the creodont).
First, discrete characteristics within the taxa are enumerated (see Table 1).
Table 1—Morphological characteristics of four taxa of carnivores using Creodont as an
outgroup.
Discrete
Characteristics
Taxa
Outgroup
Creodont
Wolf
Cat
Bear
Hyena
Auditory bullae
Incomplete
Complete
Complete
Complete
Complete
Stance
Plantigrade
Digitigrade
Digitigrade
Plantigrade
Digitigrade
5
5
5
5
4
5
Non-retractile
Absent
4
Non-retractile
Present
4
Retractile
Absent
5
Non-retractile
Present
5
Non-retractile
Absent
# digits front
foot
# digits hind foot
Claws
Alisphenoid
canal
Rostrum
Carnasial
Last molar
# lower molars
Ingroup
Long
Long
Short
Long
Long
Well developed Well developed Well developed Poorly developed Well developed
Large
3
Large
3
Small
1
Large
3
Small
1
[Note:
a. Auditory bullae are bony coverings of the middle ear cavities.
b. Animals that use a plantigrade stance put weight on their entire foot surface, like
humans, while digitigrades only put weight on the tips of their digits, like dogs.
c. The alisphenoid canal is a canal through the alisphenoid bone of the skull (adjacent to
the temporal fossae) through which blood vessels and nerves pass.
d. The rostrum is a projection similar to a bird’s beak, here referring to the nose, and the
carnassials are “canine teeth.”]
Second, the characteristics of the ingroup taxa are compared to the outgroup to
determine which taxa have a primitive or derived state of each characteristic (see
Table 2). Including the outgroup provides an objective mechanism to differentiate
primitive characters from derived character state. Any character shared
between the outgroup and any taxon in the ingroup is considered primitive.
Table 2—Determination of primitive and derived characteristics
Discrete
Characteristics
Auditory bullae
Stance
# digits front
foot
# digits hind foot
Claws
Alisphenoid
canal
Rostrum
Carnasial
Last molar
# lower molars
Taxa
Outgroup
Creodont
Wolf
Ingroup
incomplete
primitive
plantigrade
primitive
5
primitive
5
primitive
non-retractile
primitive
absent
primitive
long
primitive
well developed
primitive
large
primitive
complete
derived
digitigrade
derived
5
primitive
4
derived
non-retractile
primitive
present
derived
long
primitive
well developed
primitive
large
primitive
3
primitive
3
primitive
Cat
Bear
Hyena
complete
complete
complete
derived
derived
derived
digitigrade
plantigrade
digitigrade
derived
primitive
derived
5
5
4
primitive
primitive
derived
4
5
5
derived
primitive
primitive
retractile
non-retractile
non-retractile
derived
primitive
primitive
absent
present
absent
primitive
derived
primitive
short
long
long
derived
primitive
primitive
well developed poorly developed well developed
primitive
derived
primitive
small
large
small
derived
primitive
derived
1
derived
3
primitive
1
derived
[Note: the “poorly developed” carnassials of the bear is a derived characteristic; just
because a characteristic is less developed does not necessarily mean that it is less
evolutionarily advanced.]
Third, a character state matrix of primitive and derived characters is made (see
Table 3). Once the primitive character states are differentiated from the derived
character states, the data are coded with 0s for the primitive state and 1s for the
derived states. Note that we can now sum the number of derived characteristics
that distinguish each taxon from the outgroup.
Table 3—Character state matrix for 10 morphological characters of the four taxa of
carnivore and the outgroup Credontia. Character polarity determined by outgroup
comparison (Table 2), and total distance from outgroup is summed in the last row of the
table.
Taxa
Character
Outgroup
wolf
cat
bear
hyena
Auditory bullae
0
1
1
1
1
Stance
0
1
1
0
1
# digits front foot
0
0
0
0
1
# digits hind foot
0
1
1
0
0
Claws
0
0
1
0
0
Alisphenoid canal
0
1
0
1
0
Rostrum
0
0
1
0
0
Carnasial
0
0
0
1
0
Last molar
0
0
1
0
1
# lower molars
0
0
1
0
1
Distance from outgroup
(total)
0
4
7
3
5
D. CONSTRUCTION OF AN EVOLUTIONARY TREE BY USE OF THE
WAGNER ALGORITHM
The Wagner algorithm is used to construct a phylogenetic (Wagner) tree under the
assumption that the tree that requires the smallest number of character changes is
most desirable. This assumption is based on the principle of simplicity or
parsimony. Outlined below are the steps or procedure of the Wagner algorithm.
Steps of the Wagner algorithm- GENERAL EQUATION: D (A,B) =∑ | X(A,i) – X(B,i) |
2
Step 1. Calculate the distance of each taxon from the outgroup (note that if the outgroup has the primitive
character state for each of the characters used, the distance between a taxon and the outgroup is identical to
the number of derived character states of that taxon).
Step 2. Select the taxon with the least distance (fewest number of derived characters) from the outgroup.
Step 3. Connect the outgroup to the taxon selected with a branch or line.
Step 4. Select the taxon with the next lowest distance from the outgroup.
Step 5. Connect this taxon to the branch constructed in step 3.
Step 6. Determine, through parsimonious inference, the characteristics of a hypothetical ancestor that could
have given rise to both members of the ingroup now attached to the tree.
Step 7. Select the taxon with the next lowest distance from the outgroup.
Step 8. Calculate the distance from this taxon to each branch now on the tree.
Step 9. Connect the taxon with the next lowest distance from the outgroup to the branch with the lowest
calculated distance.
Step 10. If taxa remain that have not been connected to the tree, repeat steps 6 through 9 until all taxa are places
of the Wagner tree.
Continuing with the example above, we
have already completed step 1 in making
our character state matrix. Bear has the
lowest distance from the outgroup, with a
total of only 3 derived characteristics
(step 2). Bear is connected to the
outgroup with a branch (step 3). It is
often useful to list all of the character
states in this stage of drawing a tree for
easy reference (as shown here).
Wolf has the next lowest distance from the outgroup (step 4), being separated
from the outgroup by a total of 4 derived characteristics. Wolf is connected to
the branch connecting bear and the outgroup (step 5) and the character states of
the hypothetical ancestor (Ha1) are inferred (step 6). Only the derived
characteristics that both wolf and bear share are assumed to be shared as well by
the hypothetical ancestor.
Hyena is the taxon with the next
lowest distance from the outgroup,
with a distance of 5 (step 7). Now,
we need to calculate the distances
from hyena to branches A, B, and C
of the tree shown (step 8). This is
done by calculating the distance
from hyena to each of the taxa at
either end of a branch, adding these
distances together, and then
subtracting the distance between
the two taxa at either end of the
branch. This sum is then divided by
two. Thus:
Distance from hyena to branch A:
dist (hyena, bear) + dist (hyena, Ha1) – dist (bear, Ha1) /2 = (6 + 5 – 1)/2 = 5
Distance from hyena to branch B:
dist (hyena, wolf) + dist (hyena, Ha1) – dist (wolf, Ha1) /2 = (5 + 5 – 2)/2 = 4
Distance from hyena to branch C:
dist (hyena, Ha1) + dist (hyena, OG) – dist (Ha1, outgroup) /2 = (5 + 5 – 2)/2 = 4
For step 9, we attach hyena to the branch with the lowest distance, but there are
two branches with equal distance, and either could be used in this example: branch
B or branch C. At this point, we need to think about what makes the most sense,
using actual characteristics instead of just coded numbers. If we attach hyena to
branch C, we make the assumption that the hyena would have derived a digitigrade
stance independently of the wolf (since the Ha1 has, hypothetically, a plantagrade
stance). Conversely, we could make the assumption that Ha1 does have a
digitigrade stance and that the hypothetical ancestor to the hyena and Ha1 on
branch C also has a digitigrade stance, but then we make the assumption that the
bear, at some point, lost this adaptation and reverted back to a plantagrade stance.
Based on the assumption that evolving a complex character is more rare than
loosing such a character, the most parsimonious choice is to assume that the
digitigrade stance developed only once; thus, the most parsimonious choice is to
join hyena to branch B (note that this choice is, itself, a testable hypothesis; How
would you test the hypothesis?). The hypothetical ancestor to the hyena and wolf
(Ha2) is digitigrade, and also has an alesphenoid canal, as does the bear, wolf and
Ha1. The hyena, however, does
not have an alesphenoid canal,
meaning that this adaptation
has been lost along this
evolutionary line.
Cat is the final taxon to be
added to the tree with a
distance of 7 from the
outgroup. Now, we need to
calculate the distances from
cat to branches A, B, C, D, and
E on the tree above.
Distance from cat to branch A:
dist (cat, bear) + dist (cat, Ha1) – dist (bear, Ha1) /2 = (8 + 7 – 1)/2 = 7
Distance from cat to branch B:
dist (cat, wolf) + dist (cat, Ha2) – dist (wolf, Ha2) /2 = (5 + 6 – 1)/2 = 5
Distance from cat to branch C:
dist (cat, Ha1) + dist (cat, outgroup) – dist (Ha1, outgroup) /2 = (7 + 7 – 2)/2 = 6
Distance from cat to branch D:
dist (cat, hyena) + dist (cat, Ha2) – dist (hyena, Ha2) /2 = (4 + 6 – 4)/2 = 3
Distance from cat to branch E:
dist (cat, Ha2) + dist (cat, Ha1) – dist (Ha1, Ha2) /2 = (6 + 7 – 1)/2 = 6
Cat is attached to the branch with the lowest distance, branch D in this example,
and the characteristics of Ha3 (the hypothetical ancestor to hyenas and cats) are
assumed.
Finally, trees are
usually redrawn,
keeping them rooted in
the outgroup. Please
note that the trees
shown on this page are
identical hypotheses;
phylogenetic trees can
be swiveled at any joint.
Thus, neither the
horizontal or vertical
“axes” of a tree contain
meaning (although
people often infer that taxa to the right side of the tree and higher up on the tree
are more “advanced”). Instead, in order to infer relationships between taxa shown
on a phylogenitic tree, one has to trace the distance along each branch of the tree.
Wagner tree showing the phylogenetic relationship (hypothesis) of four taxa of Carnivora.
II FORMING HYPOTHESES
1. Make observations about the natural world.
2. Ask questions about those observations.
3. Formulate a reasonable testable hypothesis to explain observations.
4. Create, execute, and replicate experiments testing the hypothesis and
generating results.
5. Analyze results and draw inferences. This stimulates further inquiry. The cycle
begins anew.
Observation: Many vertebrates share characteristics, and yet are remarkably
different in other characteristics. For example, many vertebrates lay eggs
(most reptiles, birds, amphibians), but others don’t (most mammals).
Question: Taking into consideration reptiles, birds, and mammals, which groups are
most related to each other, and which are least related?
Hypothesis: You will use common phylogenetic methods and construct a
phylogenetic tree using the Wagner algorithm as a testable hypothesis. See
background material, section I, and methods, section III.
Record your methods in self-explanatory tables and create neat, legible
phylogenetic trees. Do your trees differ from/ agree with your classmates?
Further tests: be prepared to discuss how you could test the hypotheses you have
developed.
III METHODS
A. Observations
1.
Wander around the laboratory and look at all of the specimens, skulls,
skeletons, and diagrams of amphibians (outgroup), turtles, crocodiles, birds, and
mammals.
2.
Collect data on the characteristics listed in Table 4 for the outgroup and
ingroup taxa. Some of the data are already in the table.
Table 4—Characters of selected vertebrates.
Characteristic
type of egg
scales present
feathers present
hair present
red blood cells
teeth set in
sockets
lower jaw more
than single bone
single temporal
fossa
two temporal
fossae
ilium projected
only anteriorly
rod-like ischium
3 chambered
heart
4 chambered
heart
Taxa
Outgroup
amphibian
anamniotic
Ingroup
bird
amniotic
turtle
amniotic
crocodile
amniotic
nucleated
nucleated
nucleated
nucleated
anucleate
yes
yes
yes
no
no
no
no
no
yes
yes
mammal
amniotic
[Note:
a. Temporal fossae are the holes behind the eyes and near the ears of vertebrates
through which jaw muscles travel.
b. The ilium and ischium are bones that are
fused together to make the pelvic girdle (along
with the sacrum and other bones). The
character concerning the projection of the
ilium refers to whether it projects toward the
head (anterior) or toward the tail (posterior).
c. Although most mammals do not lay eggs, the
duckbilled platypus and the spiny echidna of
Figure 2: Two temporal fossae and the
the Australian region lay amniotic eggs.
larger eye socket in a diapsid skull of a
d. Although modern birds do not have teeth,
reptile. The holes form between
many fossil birds had teeth which were placed
individual fused bones of the skull (listed).
in sockets; score birds as having socketed
teeth.]
3.
Using outgroup comparison,
determine which character states
(for each character listed in Table
4) are primitive and which are
derived. Remember that any
character shared between the
outgroup and any taxon of the
ingroup is the primitive character.
4.
Code the primitive
character state with 0 and
derived states with 1 and
construct a character state
matrix in the table below.
Figure 3: The human pelvic girdle, showing the
location of the ilium and ischium.
Table 5-- Character State Matrix
Characteristic
Taxa
Outgroup
amphibian
type of egg
scales present
feathers present
hair present
red blood cells
teeth set in
sockets2
lower jaw more
than single bone
single temporal
fossa
two temporal
fossae
ilium projected
only anteriorly
rod-like ischium
3 chambered
heart
4 chambered
heart
Differences
from outgroup
Ingroup
turtle
crocodile
bird
mammal
5.
Calculate the distance (number of derived characters) between each taxon
of the ingroup and the outgroup. Place this distance in the final column of the
character state matrix (above).
B. Construction of Phylogenetic Tree
These steps are an abbreviated version of the steps described for the Wagner
algorithm (listed in the background material).
1.
Decide which taxon is most primitive (most similar to outgroup) and draw a
line between these taxa.
2.
Connect a third taxon (the next most related to the outgroup) to your
incipient tree, and infer the characteristics of its hypothetical ancestor.
3.
Decide which branch of the tree you should attach the next most related
taxon using the Wagner algorithm.
4.
Define the characteristics of this taxon’s hypothetical ancestor.
5.
Repeat steps 3 and 4 until all taxa have been placed.
If you have some taxa that could be placed in alternative positions, develop
alternative trees.
6.
Redraw your phylogenetic tree(s) neatly and rooted in the outgroup.
IV QUESTIONS
1. Compare your hypothetical trees to other lab groups. What groups of
vertebrates are most closely related to what other groups? What inferences or
evolutionary hypotheses are shown in your evolutionary tree(s)?
2. What data would allow you to test your hypotheses?
3. Do the reptiles (turtles and crocodiles) share a common ancestor that is not
shared with members of any other class (birds or mammals) of vertebrates
examined? What characters are diagnostic of reptiles? What characters are
diagnostic of birds? Of mammals?
4. What characters are inferred to be convergent (i.e. evolved independently along
different evolutionary lineages)?
5. Does your evolutionary tree make any inferences of character reversal (a
derived character state that reverts back to the primitive state)?
6. Although the scoring of characteristics as either derived or primitive is
designed to be as objective as possible, there are some aspects of constructing a
phylogenetic tree that are subjective. For example, do you think that the
determination of the outgroup is objective or subjective? Were some of the
characteristics scored less than clear (e.g.. was it hard to tell whether an organism
had a projecting ilium or not)? Do you think that evolutionary relationships can, in
the end, be determined objectively?
7. What affect does the selection of the outgroup have on the determination of
primitive and derived characters states? If you hadn’t been told to use the
amphibians as your outgroup, do you think that you would have come to the same
conclusion and used them as an outgroup anyway, based on their characteristics?
Are there any characteristics your hypotheses identify as being lost during the
evolution of the taxa in the ingroup?