Building a Phylogenetic Tree of the Human Ape - BioOneкод для вставки
H O W- T O - D O - I T Building a Phylogenetic Tree of the Human & Ape Superfamily Using DNA-DNA Hybridization Data S ystematics is a significant and dynamic discipline within the field of evolutionary biology whose products provide the foundation for other evolutionary and biological studies (Futuyma, 1998). Like many other biology textbooks, CampbellвЂ™s Biology (Sixth Edition) has an informative section on phylogeny, with diagrams of several phylogenetic trees and a good general discussion of the principle that DNA sequence similarity provides exceptionally powerful insights into speciesвЂ™ relationships. But specifically, how can the genetic difference among a group of related species be determined? How do DNA difference data reveal evolutionary relationships? And, how are phylogenetic trees created? To truly understand the principles of phylogeny, students need to go beyond textbook reading assignments and use real data to develop phylogenetic trees of actual species. Unfortunately, activities for high school students and undergraduates that demonstrate principles of phylogeny are hard CAROLINE MAIER teaches undergraduate ecology courses and is a graduate student in evolutionary biology at Rutgers University, Newark, NJ 07102; e-mail: cmaier@ andromeda.rutgers.edu. 560 THE AMERICAN BIOLOGY TEACHER, VOLUME 66, NO. 8, OCTOBER 2004 CAROLINE ALEXANDRA MAIER to come by and many of those that exist are simulations employing hypothetical species. In this 90-minute activity, students learn how the early technique of DNA-DNA hybridization reveals the genetic difference between species. Then they use actual genetic difference data to create a phylogenetic tree of the human and ape superfamily, Hominoidea. They calibrate their DNA clock and use it to estimate the divergence dates of the various branches on the tree. The activity expands theoretical studies of DNA-DNA hybridization, molecular phylogeny, and DNA clocks by allowing students to use real DNA difference data to understand speciesвЂ™ evolutionary relationships. Since the generated tree shows the relationships among humans and our closest relatives, the activity can also serve as an introduction to a unit on human evolution. This activity addresses multiple National Science Education Standards for grades 9-12: AвЂ”Science as Inquiry (Abilities Necessary to do Scientific Inquiry, Understandings about Scientific Inquiry), CвЂ”Life Science (The Molecular Basis of Heredity, Biological Evolution), and GвЂ”History and Nature of Science (Historical Perspectives). Although developed for advanced high school students and undergraduates in general biology courses, this activity can be adapted for younger students by leaving out the concept-laden section on DNA-DNA hybridization. Instead of generating their own genetic difference table, the students can use the data in completed tables to create phylogenetic trees, turning this into a simpler puzzle-solving activity. Background One of the important aspects of evolution is that it gives a historical context to biological diversity. In the eighteenth century, Carolus Linnaeus organized species into a hierarchy of increasingly inclusive groups based on physical similarity. However, because Linnaeus predated Darwin, his taxonomy did not address familial relationships among species. For example, Linnean classification could recognize the striking physical similarities between pygmy and common chimpanzees, but it could not address why these similarities exist. By recognizing that physical similarity can mirror familial relationships, DarwinвЂ™s Law of Common Descent added a new historical dimension to Linnaean taxonomy and initiated the discipline of systematics (Campbell, 2002). The goal of systematics is to understand the history, or phylogeny, of groups of related species. To do this, systematists compare physical similarities among several species, use the comparisons to infer the speciesвЂ™ evolutionary backgrounds, and express the results in a phylogenetic tree (Diamond, 1992; Campbell, 2002). The earliest systematists compared large-scale morphological characteristics. Current researchers still use these valuable features, but advances in molecular biology now allow them to also include DNA sequence comparisons in their studies (Diamond, 1992; Campbell, 2002). Since DNA is the fundamental unit of inheritance, sequence comparisons provide powerful insights into hereditary relationships. At the time two populations split from their common ancestor, they initially carry nearly-identical pools of DNA sequences inherited from the ancestral species. Over time, independent mutations occur throughout the genomes of the two lineages, and this decreases their genetic similarity. The longer the time since two species diverged from their common ancestor, the greater the genetic difference. Since the average rate of DNA sequence evolution appears to be uniform and constant in related lineages, DNA divergence acts like a smoothly ticking clock (Sibley & Alquist, 1984). Therefore, difference in DNA sequence can be used to determine the pattern of rela- tive divergence events among a group of related species. This can be depicted as a branching pattern that shows the groupвЂ™s evolutionary relationships. Figure 1 illustrates how DNA difference data are used to develop a phylogenetic tree for a hypothetical group of species. If the actual divergence time of a pair of species in the group is known from the fossil record, the DNA clock can be calibrated to absolute values, so that DNA difference data estimate the date when pairs of species diverged from their common ancestor (Sibley & Alquist, 1984). Figure 1 explains how a hypothetical DNA clock is calibrated. DNA difference data were first applied to taxonomy in the 1970s by Charles Sibley and Jon Alquist. They used the newly developed technique of DNA-DNA hybridization to measure the amount of DNA sequence difference among bird species, and their work in avian taxonomy was pivotal. By the 1980s, Sibley and Alquist shifted their attention to the history of humans and our closest relatives: the two chimpanzee species, gorilla, orangutan, and the two gibbon species (Sibley & Alquist, 1984; Diamond, 1992). They constructed a phylogenetic tree of the human and ape superfamily, Hominoidea, using the Old World monkeys (Cercopithecoidea) as an outgroup. Since the dates at which the Old World monkey and orangutan clades diverged from the main Hominoidea trunk are known from the fossil record, the DNA clock could be calibrated and used to date the divergence times of members of the superfamily (Sibley & Alquist, 1984). In this activity, students learn how DNA-DNA hybridization measures genetic difference, then use Sibley and AlquistвЂ™s genetic distance data to create their own phylogenetic tree of the hominoid superfamily. PHYLOGENETIC TREE 561 Figure 1. The use of genetic difference data in creating and calibrating a phylogenetic tree. When two populations initially diverge from their common ancestor, they carry nearly identical pools of DNA sequences inherited from the common ancestor.After divergence, independent mutations occur continuously throughout the genomes of the two lineages, and this gradually decreases their genetic similarity over time.The longer the time since two species diverged from their common ancestor, the greater the genetic difference (A). In this example, we can assume the lineage leading to modern species Y diverged more recently from those of species W and X than did Z, since species Y is more genetically similar to species W and X than is Z.Since the average rate of DNA sequence evolution appears to be uniform and constant in related lineages, genomic difference data can be used to determine relative divergence events among a group of related species (Sibley & Alquist, 1984).This information can then be used to develop a branching pattern that shows the groupвЂ™s evolutionary relationships.Notice that the principle of constant average rate of DNA sequence evolution means that all branches stemming from a divergence point on the tree (B) have identical numbers of genetic character states, as represented by horizontal slashes.In this example, the lineages of species W, X, and Y all reflect three genetic character states since the time they diverged from their common ancestor, represented on the tree by a solid circle.Dashed arrows trace the evolution of species W, X, and Y.Numbers on each arrow reflect the order of the three character states in each lineage.If the actual divergence times of a pair of species in the group are known from the fossil record, the DNA clock can be calibrated to absolute values.A calibrated clock uses DNA difference data to estimate the actual date when pairs of species diverged from their common ancestor.In the example (B), the hypothetical fossil record shows that species W and Z diverged 8,000 years ago.Since W and Z have experienced four genetic character states since they diverged from their common ancestor, genetic character states occur approximately every 2,000 years within this group.The lineage leading to modern species Y must have diverged from the main trunk of the tree approximately 6,000 years ago since three character states have occurred. (A) Percent Genetic Difference Between Pairs of Hypothetical Species W X Y Z W - 10 30 40 X 10 - 30 40 Y 30 30 - 40 Z 40 40 40 - (B) W X Y 3 3 3 2 2 2 1 Z 1 1 562 THE AMERICAN BIOLOGY TEACHER, VOLUME 66, NO. 8, OCTOBER 2004 They calibrate the DNA clock and use it to estimate the divergence dates of the various branches on the tree. The Activity After a general overview of systematics, I use a simulation activity to introduce the idea of genetic difference, the technique of DNADNA hybridization, and the principles of creating phylogenetic trees from genetic difference data. Pairs of students each receive an envelope containing homologous 20-base pair DNA fragments from five hypothetical species (Figure 2). The 5'-3' and 3'-5' strands of each fragment are on separate strips of paper, so we begin by reviewing DNA structure and base pairing rules as the students match complementary strands. The students count the number of hydrogen bonds holding each double-stranded fragment together and discover that the fragments from species B and C would denature at a higher temperature than the others since they are held by a greater number of H bonds (Figure 3). I explain that DNA-DNA hybridization was an early method for comparing the similarity between two DNA sequences before sequencing technology was developed. DNA from two species is denatured, brought together, and allowed to condense into a hybrid molecule composed of a single strand from each species. The homologous sequences from the two species are similar, but not identical due to the independent mutations that have occurred since the two lineages split from their common ancestor. Hydrogen bonds do not form when mutations have resulted in the pairing of noncomplementary bases, so hybrid molecules denature at a lower temperature than parental fragments. There is a direct relationship between the genetic difference of two speciesвЂ™ DNA and the amount by which the melting point of the hybrid is reduced, called the melting point depression. A mathematical model converts actual depression values to percent genetic difference between two speciesвЂ™ DNA. So even though DNA sequencing technology was not yet readily available in the 1970s, researchers were using melting point depression to accurately estimate genetic difference among groups of related species. Figure 2. Homologous 20-base pair DNA fragments from five hypothetical species. To use these fragments in the DNA-DNA hybridization simulation, enlarge and copy each speciesвЂ™ fragment on its own color of paper, then cut the 5'-3' and 3'-5' strands apart so that students can form hybrid molecules. Species A Species B Species C Species D Species E 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' G C G A C C C T T T T C A A A G G C C A C G C T G G G A A A A G T T T C C G G T A G G G C C T T T A T C C C G G T C A A T C C C G G A A A T A G G G C C A G T T T A G G C C A T T C T A C C G G G C A A A T C C G G T A A G A T G G C C C G T T T G G C C C A T T A T A C T G G G C A A A C C G G G T A A T A T G A C C C G T T T G G C C C A T T A T A T C G G G C A A A C C G G G T A A T A T A G C C C G T T 3' 5' 3' 5' 3' 5' 3' 5' Students see this direct relationship between the number of H bonds and genetic difference as they collect data on the hybrids formed by the five hypothetical species and fill in Figure 3, written on the board as a large table. Once the students understand how DNA-DNA hybridization generates genetic difference data, I show them how to use the data to create a phylogenetic tree of the five hypothetical species. On the finished tree (Figure 4), as on all phylogenetic trees, time is represented vertically. Modern species are listed along the top of the space delineated by the axes, connected by branches that reach down through the space, and back into time, eventually splitting from the main trunk. Divergence points on the tree represent the common ancestor shared by the splitting lineages, and their vertical placement indicates the relative time the event occurred. Since the genetic difference among species is related to the time since lineages split, it is also represented as a vertical axis in Figure 4. Tracing any two species back to their common ancestor on the branching diagram reveals the percentage by which their DNA sequences differ. The genetic difference table for the five hypothetical species (Figure 3) contains all the information needed to create the branching pattern on a copy of the blank axes (Figure 5). First, students root the tree by identifying the outgroup, a species or group only distantly related to the study species. In the simulation, Species A is the outgroup since its DNA differs from the others by the greatest amount (50%). Students begin creating the diagram by drawing a deep вЂњVвЂќ in the area delineated by the axes, with the divergence point positioned across from a 50% genetic difference point on the vertical axis, as shown in Figure 6. They label the top of the right arm вЂњSpecies A.вЂќ The empty left arm of the V is now ready to be expanded into a tree. Creating the tree is much like solving a jigsaw puzzle. When all the species are in the correct place on the tree, it is possible to trace any pair back to their common ancestor and read their genetic 3' 5' difference off the left axis. Students can most easily build the tree by first identifying the two most closely related species on the genetic difference table (D and E), drawing a branch from the main trunk of the tree at a point corresponding to the correct genetic distance between the taxa, and labeling the end of the branch with the name of the appropriate species. More distantly related lineages are then added to the tree until it is complete (Figure 4). As students finish the sample tree, I point out that Species B has the same genetic difference to Species C as it does to Species D and E (30%). Since evolutionary rates are fairly uniform in related lineages, all groups branching from a single point should have approximately equal genetic difference. Slight differences in evolutionary rates and chance events, among other things, can perturb this pattern, but it is widespread enough PHYLOGENETIC TREE 563 work with real data. I introduce this part of the activity by showing students pictures of the common chimpanzee, pygmy chimpanzee, common gibbon, siamang gibbon, gorilla, human, and orangutan. I ask student groups to predict the evolutionary relationships among the species by organizing them into a possible tree. After recognizing the diversity in the studentsвЂ™ ideas, I explain that systematists traditionally argued over the relationships among these species. In an attempt to clarify the phylogeny of this group, Charles Sibley and Jon Alquist used DNA-DNA hybridization to collect genetic difference data on these species. I challenge the groups to use Sibley and AlquistвЂ™s data to create a phylogenetic tree of the hominoid superfamily. Copies of the pair-wise genetic difference data table (Figure 7) and empty axes (Figure 5) provide the necessary information and structure. This tree is more difficult than the sample the students have just completed, so it is best to work in pencil with an eraser nearby. The split to the two species within the main gibbon lineage is perhaps the greatest challenge because divergence within a side branch, although common in nature, is initially unexpected by the students. This might confuse some groups, but with a hint or two these students will solve the problem and end up with the correct final tree (Figure 8) which they can confirm with a relative rate test. Figure 3. The genetic difference and number of hydrogen bonds between homologous 20-base pair DNA fragments from the five hypothetical species used in the DNA-DNA hybridization simulation. Complementary strands from two different species were aligned.To determine percent genetic difference, the number of mismatched bases in the hybrid molecule was divided by the total number of bases in the fragment (20) and the resulting product was multiplied by 100.To determine the number of hydrogen bonds that would hold the hybrid molecule together, two bonds for each A-T pair and three bonds for each C-G pair were summed.The darkly-shaded diagonal line shows the number of hydrogen bonds found in the native, non-hybrid molecules.White boxes below the diagonal show the number of hydrogen bonds holding together each hybrid molecule, while the lightly-shaded boxes above the diagonal show the percent genetic difference between pairs of molecules. Since the bottom left and top right portions of the table are mirror images of each other, it is possible to compare the number of hydrogen bonds between hybrid molecules to their percent genetic difference by looking across the darkly-shaded diagonal line. B C D E A 50 50 50 50 50 B 24 51 30 30 30 C 26 37 51 20 20 D 26 36 40 50 10 E 26 36 40 45 50 % Genetic Difference A Number of Hydrogen Bonds that systematists use this вЂњrelative rate testвЂќ to doublecheck the trees they develop. Now that students have learned to create phylogenetic trees from DNA difference data, they are ready to 0 E D C B A Present 10 Figure 4. Phylogenetic tree of the five hypothetical species. This diagram shows the branching pattern of the lineages leading to modern species A, B, C, D, and E.Tracing any two species back to their branch point allows their genetic difference to be read from the left vertical axis. 20 30 40 50 % Difference in DNA 564 THE AMERICAN BIOLOGY TEACHER, VOLUME 66, NO. 8, OCTOBER 2004 Deep Past Time Figure 5. Blank axes to use when creating the phylogenetic trees of the five hypothetical species (A) and of the Hominoidea superfamily (B). These axes are adapted from the diagram used by Sibley and Alquist (1984) and shown in The Third Chimpanzee (Diamond, 1992). The left axis corresponds to percentage of DNA difference, while the right axis depicts time and is used to calibrate a DNA clock to the tree. An enlarged copy of these axes guides students in creating their simulated phylogenetic tree. This diagram shows how the tree is started using Species A as the outgroup.The tree will be built along the left arm of the V. Notice that the branching point of the tree corresponds to a genetic difference of 50% since Species A differs from the other species by this amount. 0 A Present 10 (A) 0 Figure 6. Rooting the simulated phylogenetic tree of the five hypothetical species. Present 20 10 30 20 40 30 Deep Past 50 % Difference in DNA Time 40 Deep Past 50 % Difference in DNA Time (B) 0 Present 1 2 3 4 5 6 7 8 % Difference in DNA Millions of Years Ago As each group finishes its tree, I give the students the information they need to calibrate the DNA clock. This is a simple matter of relating the two vertical axes of genetic difference and time to each other. I tell the students that the fossil record shows that the Old World monkey and ape lineages, which differ in 7.3% of their DNA, diverged approximately 30 million years ago. The fossil record also indicates that the orangutan lineage branched from the gorilla/chimp lineage approximately 16 million years ago. Orangutans differ genetically from the great apes by approximately 3.7% (Sibley & Alquist, 1984; Diamond, 1992). I challenge the students to calibrate the DNA clock so that it converts the genetic difference between pairs of species into their approximate divergence date. This is easily done by scaling the Years axis on the right side of the tree diagram. Students need only to place 30 on the Year axis across from the corresponding genetic difference of 7.3%, 16 across from the genetic difference of 3.7%, and divide the rest of the axis proportionally (Figure 8). Students then use their calibrated DNA clock to determine the approximate divergence time for the various lineages on the tree. They identify our closest and most distant relatives among the ape species and determine whether the two chimpanzee species are most closely related to gorillas or to humans. Finally, they identify which pair is most closely related: the two gibbon species or humans and chimpanzees. Extension As groups finish their trees, they check their work by reading pages 16-25 of Jared DiamondвЂ™s The Third PHYLOGENETIC TREE 565 Figure 7. Genetic difference matrix of species in the superfamily Hominoidea. This matrix, developed from Sibley and AlquistвЂ™s DNA-DNA hybridization data (Sibley & Alquist, 1984) and used in The Third Chimpanzee (Diamond, 1992) shows the approximate genetic difference, in percent, between the genomes of all pairs of ape and human species. Human Common Chimp Pygmy Chimp Common Gibbon Siamang Gibbon Gorilla Old World Monkeys Orangutan - 1.6 1.6 5.0 5.0 2.3 7.3 3.6 Common Chimp 1.6 - 0.7 5.0 5.0 2.3 7.3 3.6 Pygmy Chimp 1.6 0.7 - 5.0 5.0 2.3 7.3 3.6 Common Gibbon 5.0 5.0 5.0 - 2.2 5.0 7.3 5.0 Siamang Gibbon 5.0 5.0 5.0 2.2 - 5.0 7.3 5.0 Gorilla 2.3 2.3 2.3 5.0 5.0 - 7.3 3.6 Old World Monkeys 7.3 7.3 7.3 7.3 7.3 7.3 - 7.3 Orangutan 3.6 3.6 3.6 5.0 5.0 3.6 7.3 - Human Figure 8. Phylogenetic tree of the superfamily Hominoidae. This diagram, based on Sibley and AlquistвЂ™s DNA-DNA hybridization data and tree (1984) and included in The Third Chimpanzee (Diamond, 1992) shows the branching pattern of human and ape lineages.Tracing any two species back to their branch point allows their genetic difference to be read from the left axis.The right axis calibrates the molecular clock by relating actual divergence dates for the Old World monkey and orangutan clades known from the fossil record to their percentage of genetic difference.The date when any pair of species diverged from their common ancestor can be determined by tracing the two lineages back to their divergence point and reading the divergence date off the right axis. Common Pygmy Common Siamang Old World Chimp Chimp Human Gorilla Orangutan Gibbon Gibbon Monkeys 0 0 1 4 2 8 3 4 5 12 16 20 24 Chimpanzee. This delightful section describes the process of DNA-DNA hybridization and the history of its use by Sibley and Alquist in simple, straightforward, and interesting language that students easily understand. It describes Sibley and AlquistвЂ™s data, shows a diagram of the tree generated by the data, and explains how a clock can be applied to the tree. In addition, it describes the treeвЂ™s implications for human and chimpanzee classification. Having just finished using the actual data to create the tree described by Diamond, students become engrossed in the reading and understand the concepts more thoroughly than they otherwise would. Many students develop a real interest in The Third Chimpanzee and continue reading it outside of class. When this happens, I know the activity has done what I hoped it wouldвЂ”make evolution relevant and interesting to my students. References Campbell, N. A. & Reece, J. B. (2002). Biology (Sixth Edition). San Francisco, CA: Benjamin/Cummings Publishing Company, Inc. Diamond, J. (1992). The Third Chimpanzee. New York City, NY: Harper Collins Publishers, Inc. 6 28 7 Futuyma, Douglas J. (1998). Evolutionary Biology (Third Edition). Sunderland, MA: Sinauer Associates, Inc. 30 8 % Difference in DNA Millions of Years Ago 566 THE AMERICAN BIOLOGY TEACHER, VOLUME 66, NO. 8, OCTOBER 2004 Sibley C.G. & Alquist, J. E. (1984). The phylogeny of hominoid primates, as indicated by DNA-DNA hybridization. Journal of Molecular Evolution, 20(1), 2-15.