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.

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 / Ingroup
Creodont / Wolf / Cat / Bear / Hyena
Auditory bullae / Incomplete / Complete / Complete / Complete / Complete
Stance / Plantigrade / Digitigrade / Digitigrade / Plantigrade / Digitigrade
# digits front
foot / 5 / 5 / 5 / 5 / 4
# digits hind foot / 5 / 4 / 4 / 5 / 5
Claws / Non-retractile / Non-retractile / Retractile / Non-retractile / Non-retractile
Alisphenoid
canal / Absent / Present / Absent / Present / Absent
Rostrum / Long / Long / Short / Long / Long
Carnasial / Well developed / Well developed / Well developed / Poorly developed / Well developed
Last molar / Large / Large / Small / Large / Small
# lower molars / 3 / 3 / 1 / 3 / 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 / Taxa
Outgroup / Ingroup
Creodont / Wolf / Cat / Bear / Hyena
Auditory bullae / incomplete
primitive / complete
derived / complete
derived / complete
derived / complete
derived
Stance / plantigrade
primitive / digitigrade
derived / digitigrade
derived / plantigrade
primitive / digitigrade
derived
# digits front
foot / 5
primitive / 5
primitive / 5
primitive / 5
primitive / 4
derived
# digits hind foot / 5
primitive / 4
derived / 4
derived / 5
primitive / 5
primitive
Claws / non-retractile
primitive / non-retractile
primitive / retractile
derived / non-retractile
primitive / non-retractile
primitive
Alisphenoid
canal / absent
primitive / present
derived / absent
primitive / present
derived / absent
primitive
Rostrum / long
primitive / long
primitive / short
derived / long
primitive / long
primitive
Carnasial / well developed
primitive / well developed
primitive / well developed
primitive / poorly developed
derived / well developed
primitive
Last molar / large
primitive / large
primitive / small
derived / large
primitive / small
derived
# lower molars / 3
primitive / 3
primitive / 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.

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