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.

Taxa Outgroup Ingroup

Discrete Characteristics

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-retractileAlisphenoid canal

Absent Present Absent Present Absent

Rostrum Long Long Short Long LongCarnasial 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 Taxa

Outgroup Ingroup

Discrete Characteristics

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.

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teps of the Wagner algorithm- GENERAL EQUATION: D (A,B) =∑ | X(A,i) – X(B,i) | 2

tep 1. Calculate the distance of each taxon from the outgroup (note that if the outgroup has the primitiveharacter state for each of the characters used, the distance between a taxon and the outgroup is identical tohe number of derived character states of that taxon).

tep 2. Select the taxon with the least distance (fewest number of derived characters) from the outgroup.

tep 3. Connect the outgroup to the taxon selected with a branch or line.

tep 4. Select the taxon with the next lowest distance from the outgroup.

tep 5. Connect this taxon to the branch constructed in step 3.

tep 6. Determine, through parsimonious inference, the characteristics of a hypothetical ancestor that could ave given rise to both members of the ingroup now attached to the tree.

tep 7. Select the taxon with the next lowest distance from the outgroup.

tep 8. Calculate the distance from this taxon to each branch now on the tree.

tep 9. Connect the taxon with the next lowest distance from the outgroup to the branch with the lowest alculated distance.

tep 10. If taxa remain that have not been connected to the tree, repeat steps 6 through 9 until all taxa are places f the Wagner tree.

ontinuing with the example above, we ave already completed step 1 in making ur character state matrix. Bear has the owest distance from the outgroup, with a otal of only 3 derived characteristics

step 2). Bear is connected to the utgroup with a branch (step 3). It is ften useful to list all of the character tates in this stage of drawing a tree for asy reference (as shown here).

olf has the next lowest distance from the outgroup (step 4), being separated rom the outgroup by a total of 4 derived characteristics. Wolf is connected to he branch connecting bear and the outgroup (step 5) and the character states of he hypothetical ancestor (Ha1) are inferred (step 6). Only the derived haracteristics that both wolf and bear share are assumed to be shared as well by he 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.

Taxa Outgroup Ingroup

Characteristic

amphibian turtle crocodile bird mammal type of egg anamniotic amniotic amniotic amniotic amniotic scales present feathers present hair present red blood cells nucleated nucleated nucleated nucleated anucleate 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

yes yes yes no no

4 chambered heart

no no no yes yes

[Note: a. Temporal fossae are the holes behind the eyes and near the ears of vertebrates through which jaw muscles travel.

Figure 2: Two temporal fossae and the larger eye socket in a diapsid skull of a reptile. The holes form between individual fused bones of the skull (listed).

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 the Australian region lay amniotic eggs. d. Although modern birds do not have teeth, many fossil birds had teeth which were placed 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.

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tyscfeharetesolothsinfotwfoilionro3 he4 heDfro

Figure 3: The human pelvic girdle, showing thelocation of the ilium and ischium.

able 5-- Character State Matrix Taxa

Outgroup Ingroup

aracteristic

amphibian turtle crocodile bird mammal pe of egg ales present athers present ir present d blood cells eth set in ckets2

wer jaw more an single bone

gle temporal ssa

o temporal ssae

um projected ly anteriorly

d-like ischium chambered art

chambered art

ifferences m outgroup

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?