Molecular Biology and Primate Phylogenetics

Target age or ability group:	High school biology, regular, honors, and advanced placement level.
Class time required:	Two 50-minute class periods. Day 1: Students work on counting amino acid differences between each pair of organisms and complete table #1, compare gross anatomy of selected primates from reference materials supplied by the teacher, and then begin the discussion questions and identification of organisms on the graph. Students then take home the activity to complete the discussion questions and the graph. On Day 2 the students meet in teams to discuss/debate their responses and the labeling of the graph, and then participate in a class discussion guided by the teacher.
Materials and equipment:	Student handout for each student and an overhead of each of the pages of the activity for the teacher to use during review. It is also recommended that the teacher provide references comparing the anatomy of the primates mentioned in this activity (see the student handout, question #3 for the list), a geologic time chart, and any phylogenetic tree that will serve as a good reminder to the students that they are discovering relationships that emphasize ancestry.
Summary of activity:	Working in groups of two, students begin by determining the number of amino acid differences in beta hemoglobin that occur between each of the pairs of organisms featured in the activity. The students do not know the names of the organisms studied until after they complete the amino acid comparisons and have begun the discussion. Students compare each possible pairing of organisms, looking for differences in the sequences, and discovering patterns in the degree of difference. The current view is that the number of differences for a particular molecule is an indicator of how long ago in the past any two organisms may have shared a common ancestor. By example, the relatively few differences between the beta hemoglobin of humans and gorillas is an indicator of a relatively recent common ancestor, whereas, a comparison of chimpanzee and lemur beta hemoglobin indicates a far more distant common ancestor. More striking, but less intuitive, is the observation that chimps, humans, and even monkeys are all approximately equidistant from lemurs. This is because the common ancestor of all anthropoidea diverged from the common ancestor of lemurs at the same time, a very long time ago. Students use the data in table 1 to infer relative points of divergence since a common ancestor and, upon comparing gross anatomy to amino acid sequences for each of the 7 species, infer where each of the seven species belongs on the phylogenetic tree.
Prior knowledge, concepts or vocabulary necessary to complete activity:	Students must have a good working knowledge of the structure and function of DNA. They must also be aware of the growing body of evidence that supports the scientific conclusion that life has a long history and has diversified widely through time. Students should have some exposure to phylogenetic trees and sequences in the fossil record.
Teacher instructions:	Begin by showing several overheads of phylogenetic trees (Strickberger, Evolution is an excellent source). Students will be most interested in phylogenetic trees for dinosaurs and other prehistoric organisms that are now extinct. Ask students to offer an explanation as to how scientists establish such evolutionary relationships. Most of their responses will likely focus on anatomical comparisons and fossil sequences. The teacher should then point out that living organisms also share common ancestry and that these ancestral relationships can be understood, in addition, by using molecular comparisons. The teacher might then put up an overhead (non-phylogenetic, merely comparative anatomy) which includes at least several of the primates mentioned in this activity and ask students why we group these organisms together. Most students will respond that these creatures share certain physical characteristics, especially a set of characteristics that they share with no other organisms classified outside of the order primates. Then ask the students why they share characteristics common to all vertebrates (ancestral characters - distant common ancestors)and characteristics common only to the order primates (derived characters - recent common ancestors). Here the teacher could talk about general patterns in the fossil record. The teacher should then pass out the 2 page student lab sheet along with a book or photocopies of the primates that are used in the activity. Working in 2 groups of 2, each lab station team will proceed through the activity. Each pair should work independently of the other pair, except when they hit a significant problem. The teacher should remind students that what they do not finish in class will need to be done by the student at home. On day 2 the teacher lets the students meet in their 4 person lab teams to discuss their answers. There will no doubt be loud debate and discussion. Allow 20 minutes. Students then present their findings in a discussion facilitated by the teacher (allow 30 minutes). The teacher should have overhead copies of both pages of the activity so that as answers are developed, all students can see the data, graph, and discussion questions/responses.

Acknowledgment: I am grateful to Dr. Martin Nickels, Illinois State University, Normal, Illinois for his collaboration in the development of this module.

Answers to Table #1, Graph #1 and Discussion:

In Graph #1: the "letter" order is G,C,A,B,D,E,F, and the ³name of organism² sequence is lemur, gorilla, human, chimpanzee, gibbon, rhesus monkey, squirrel monkey, and horse (for H). To scale the lower x axis as required in discussion question #9 merely draw a dotted line down from the first vertical split on the graph (at about 29 amino acid dissimilarities) to the lower axis and label that "43"(43 million years ago). Then mark the spot on the lower axis directly below the "0 dissimilarities" from the upper axis as "6" (6 million years ago).

43 - 6 = 37 million years from left to right boundary so each 10 units of amino acid dissimilarity is approximately equivalent to 12 million years. Merely subdivide so that 1 amino acid dissimilarity is equal to about 1.2 million years of elapsed time. Obviously your scale can be extended to the left beyond the 43 million year mark.

For the Discussion:

1. A and B

2. A and B

3. See above under Graph #1

4. All organisms A-F share a common ancestor with G at the same point in time. Using comparative anatomy to develop phylogenetic trees can be misleading, especially when we have a tendency to want to create hierarchical relationships (like a ladder, with the "best" or "most advanced" at the top). This is a typical and difficult error to overcome. If we place these organisms hierarchically, then we are inferring, in fact, that one type of creature evolved into the next which is a significant error. These organisms are all alive today. The difficulty is in determining when they diverged from a common ancestor. Remember: No scientist has ever said that humans evolved from modern day monkeys. What trained scientists have been saying is that humans and monkeys (and all other life on earth in fact) share a common ancestor somewhere in the distant past. By analogy: You are related to your cousin Donald or Sally, but you are not descended from them.

5. Typically, students will see that horses are more distantly related (they are mammals, but not primates) and predict that the bottom row in table #1 be about high-30s or low-40s. The line of descent would be drawn from the right side of the graph all the way to the left and connect somewhere on the single line of descent that enters from the left.

6. It suggests that humans and chimpanzees share a more recent common ancestor than do horses and zebras. Even though horses and zebras "look" more similar, looks can be deceiving. Check a phylogenetic tree for the "horse" in any good college evolution text. The reason that they look so similar even though a longer period of time has passed since they (as opposed to humans and chimpanzees) shared a common ancestor is that selective pressure has favored a similar body plan for a nearly identical feeding pattern. Both are swift, herding herbivores of dry grasslands.

7. Typically, students will say they were too high in their estimate. They will be confused that organisms A-F are apparently more closely related to a horse than to another primate, the lemur. They should question the validity of the data, but the teacher should comment that the data is accurate. They should then be questioned if this single piece of aberrant data is enough to shake confidence in all of the molecular analysis that has been done. Clearly, one piece of unusual data begs explanation, not total rejection of a fruitful technique of great value.

8. This is perhaps the most difficult of the 10 questions. Students will be expected to draw on their understanding of the structure and function of DNA, RNA, and proteins. The teacher is needed here to help guide the students to a logical plan of investigation that involves examining DNA and RNA nucleotides to investigate whether or not a back mutation has occurred in a triplet that would cause the amino acid product to be the same as a primate in its sequence. This can be best understood by showing the table of amino acid triplet codes. Some amino acids have 1 triplet (methionine which is a start codon) and several that have 6 different triplet codes. The giveaway would be if the organism had the same amino acid (when you might predict it should be different) but has a distinctly different triplet. Here it could be inferred that the organism has more similarities because of one or more chance mutations that changed the amino acid to the same as a long distant evolutionary cousin.

9. The rate is about 1 amino acid mutation per 1.2 million years according to this graph. This rate has little to do with experimentally determined rates. For one, there is a difference between a meaningful rate (changes in DNA that produce a noticeable affect such as a change in the type of amino acid encoded) and there is also a silent rate in which changes in the DNA may have no effect on the final amino acid because it has multiple triplet codons. Both silent and meaningful base changes are being studied. You might want to refer to research being done by Li, Nei, Roychoudhury, or the late Motoo Kimura for additional information. 1

10. No they do not. Chimpanzees and squirrel monkeys last shared a common ancestor approximately 23 mya according to this graph.

Name

Period

Class:

Molecular Biology and Primate Phylogenetics

Studying the nucleotide sequences of DNA, and/or the amino acid sequences of proteins, gives scientists one of a growing number of ways to analyze relationships and infer ancestry for life on earth. The work of molecular biologists has been important in further clarifying and refining our understanding of not only family trees (phylogenies) for all life on earth, but also the possible rate of mutation, selection, and speciation.

Among the first proteins to yield its amino acid sequence was hemoglobin, and it remains today one of the most investigated of all proteins. The basic unit of hemoglobin consists of an iron-containing porphyrin (heme) that can reversibly bind oxygen attached to a globin polypeptide chain that is usually no less than 140 amino acids long. In vertebrates, hemoglobins are usually the primary protein of red blood cells, making them relatively easy to isolate and purify in large quantities. (Strickberger, M. W. Evolution. Jones and Barlett Publishers, 1990)

Purpose

To examine the amino acid sequence differences for a specific protein (beta hemoglobin) in several primate species, and from this infer ancestral relationships.

Procedure Part A: Listed below is a comparison of the 146 amino acid beta chain of the hemoglobin molecule in 7 selected primate species. Notice that the amino acid position numbers may not be continuous. This is because those that are the same for ALL seven species have been left off the chart to save space. Count the number of amino acid differences between each of the possible pairs of organisms using the data below and then enter it in Table #1. Draw a diagonal line from the upper left to lower right of the grid and then cross out all of the squares in the upper right side of the table. No need to duplicate your work. You will enter in row H later.

Amino acid SPECIES position A B C D E F G 1 VAL VAL VAL VAL VAL VAL THR 2 HIS HIS HIS HIS HIS HIS LEU 4 THR THR THR THR THR THR SER 5 PRO PRO PRO PRO PRO GLY ALA 6 GLU GLU GLU GLU GLU ASP GLU 8 LYS LYS LYS LYS LYS LYS ASP 9 SER SER SER SER ASN ALA ALA 10 ALA ALA ALA ALA ALA ALA HIS 12 THR THR THR THR THR ALA THR 13 ALA ALA ALA ALA THR ALA SER 21 ASP ASP ASP ASP ASP GLU GLU 22 GLU GLU GLU GLU LU ASP LYS 33 VAL VAL VAL VAL LEU VAL VAL 50 THR THR THR THR SER THR SER 52 ASP ASP ASP ASP ASP ASP SER 56 GLY GLY GLY GLY GLY ASN SER 69 GLY GLY GLY GLY GLY GLY SER 73 ASP ASP ASP ASP ASP ASP GLU 76 ALA ALA ALA ALA ASN THR HIS 80 ASN ASN ASN ASP ASN ASN ASN 87 THR THR THR LYS GLN GLN GLN 104 ARG ARG LEU ARG LYS ARG LYS 111 VAL VAL VAL VAL VAL VAL SER 112 CYS CYS CYS CYS CYS CYS ALA 113 VAL VAL VAL VAL VAL VAL GLU 114 LEU LEU LEU LEU LEU LEU SER 115 ALA ALA ALA ALA ALA ALA GLU 116 HIS HIS HIS HIS HIS HIS LEU 120 LYS LYS LYS LYS LYS LYS HIS 121 GLU GLU GLU GLU GLU GLU ASP 122 PHE PHE PHE PHE PHE PHE LYS 123 THR THR THR THR THR THR SER 125 PRO PRO PRO GLN GLN GLN ALA 126 VAL VAL VAL VAL VAL LEU VAL 130 TYR TYR TYR TYR TYR TYR PHE

Key ALA = alanine ASN = asparagine ARG = arginine ASP = aspartic acid CYS = cysteine GLN = glutamine GLY = glycine GLU = glutamic acid LYS = lysine PHE = phenylalanine PRO = proline SER = serine TYR = tyrosine THR = threonine VAL = valine

Procedure Part B

The data that you have gathered and entered in Table #1 represents the amount of time (in amino acid substitutions) since one organism has diverged from a common ancestor with another organism. A fewer number of amino acid differences between any two creatures implies that the two organisms share a relatively recent common ancestor, whereas a large number of amino acid differences implies the two organisms share a relatively distant common ancestor. Scientists can take this information to construct a phylogenetic tree that shows the branching patterns of descent. Using the data from Table #1, enter the appropriate letter (A, B, C, D, E, F, or G) in each of the appropriate spaces to the right side of the phylogenetic tree in Graph #1 under "letter." Notice the horizontal time axis on the top of the graph. Then answer the discussion questions below.

Graph #1: Evolutionary tree for seven primate species based on a comparison of amino acid dissimilarities of beta hemoglobin

Discussion

(Answer the essay questions on a separate sheet of paper and attach to this sheet).

1. Which two species have the greatest similarity in the amino acid sequence for this protein? ____ and ____

2. According to the phylogenetic tree which two organisms share the most recent common ancestor? ____ and ____ .

3. After reviewing the basic anatomy of primates from your text and/or references, studying the amino acid differences in Table #1, and viewing the tree above , enter the names of the 7 primates into their appropriate positions at the right under "name of organism." Chose from human, chimpanzee, gorilla, gibbon, rhesus monkey (Old World monkey form), squirrel monkey (New World monkey form) and lemur. (A is human).

4. Why are the organisms A-F all approximately the same number of amino acid differences positions to organism G? How can they possibly be equidistant from the lemur when it appears that a spider monkey looks more like a lemur than does a human? (This data is actually what we would predict)

5. Based on the pattern you see in Table #1 , predict the number of differences for all species (A-G) with a new species H, a horse. Enter your predictions on the last row in Table #1 and draw a line of descent on the tree.

6. At a molecular level, if humans and chimpanzees are about 99% identical, and horses and zebras are about 96% identical, what does this suggest about which pair of organisms is more closely related? Which pair shares a more recent common ancestor? Explain your reasoning.

7. How does your answer in #5 compare with the actual data of approximately 28 amino acid differences with each of the organisms A-F? Should this one piece of anomalous data call into question all of the previous data comparing the primates? Why or why not?

8. How would checking the DNA triplet codon for all of the amino acid sites be a logical first step in determining the source of this aberrant data? (Hint: Look at your triplet codon/anticodon/amino acid tables)

9. By examining evidence from fossils, and molecular and anatomical comparisons, evolutionary biologists have inferred that lemurs and humans share a common ancestor at about 43 million years ago, and that humans and chimpanzees share a common ancestor at about 6 m.y.a . Use this information to completely scale a second x axis at the bottom of the graph to correspond with the upper axis for amino acid mutations. What appears to be the rate of mutation for beta hemoglobin? 1 amino acid mutation per _______ million years.

10. Do chimpanzees and squirrel monkeys share a common ancestor 4 million years ago? Why or why not?

Island Biogeography and Evolution
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