AP Biology
Teacher’s Guide
Learning Evolution Using Phylogenetic Analysis For more information contact Yulia Newton at
[email protected] Pre-requisite skills: Introduction knowledge of evolution. Required equipment: 1. Students will need to use a computer individually or in groups 2. Students will need an internet access
The purpose of this hands-on practice is to learn how to utilize Bioinformatics tools to help students learn about Evolution. Students learn through an in-class discussion and completing a hands-on worksheet. The lesson unit includes the following components: 1. This document (teacher’s guide) 2. Power point presentation for an in-class discussion 3. Student worksheet to be completed by students in class (we suggest groups of 2 students but individual work is perfectly acceptable as well) There are 5 parts in the student worksheet: 1. Big Picture of the worksheet lists an overview of the steps involved in constructing a phylogenetic tree. It provides students with a big picture of the process. 2. Part A of the worksheet is an exercise of inferring phylogenetic relationship from purely morphological features. 3. Part B is a step-by-step walk through performing a phylogenetic analysis. Beta globin sequences from various specie are used for this part of the worksheet. 4. In part C students work on their own, using what they learned in part B, to analyze SIV and HIV sequences to determine which SIV strand HIV evolved from. GAG protein sequences are used in this part of the worksheet.
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Teacher’s Guide
5. In Part D students perform phylogenetic analysis on various placentals and marsupials to determine relationship of platypus and kangaroo rats to other specie. Students should get the following tree as a part of their analysis.
Big Picture 1. Gather your characteristics based on which you want to compare the desired species/entities. 2. Perform multiple sequence alignment on the selected sequences. 3. Calculate the distance matrix from the multiple sequence alignment. 4. Build a phylogenetic tree. 5. Visualize the tree in graphical output from the text representation of the tree produced by the previous step.
In class discussion and presentation The included power point is optional but is strongly encouraged to be used. Below is a discussion guideline for the following topics: • •
Rooted and un-rooted trees Cladograms vs. Phylograms
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AP Biology
Teacher’s Guide
Start this workshop by talking about evolution. 1. What is evolution? Descent with modification. 2. Talk about the time frames (geological time vs. family genealogy) 3. How can we infer relatedness of species (morphology, fossil record, genetic makeup, protein sequence, etc.)? 4. What are the differences for inferring relatedness between species that still exist and species that are now extinct (Neanderthals, Wooly Mammoth, etc.)? 5. Talk about examples of morphological features (bipedalism, shape of limbs, digits vs. hooves, etc.) 6. Give examples of situations when using morphological features makes it hard to determine evolutionary relationship (Quagga) or produces phylogeny that is wrong (Bankisia, Horseshoe crab). 7. Can you think of any reasons when it is adventageous to use genomic over proteomic sequences and visa versa? a. It makes sense to use genomic sequences when there has not been enough evolutionary divergence between the sequences. For example, when looking at fairly young species or recent evolutionary changes. Oherwise, proteomic sequences are best to use.
Rooted and un-rooted trees Let’s talk about rooted and unrooted trees. This is a very important concept to understand. Computational phylogeny is a discipline that lives in the intersection of Computer Science and Biology. However, there are some concepts that mean different things to a computer scientist and to a biologist. Tree root is one of those concepts. To a computer scientist a root is a special node that sits above other nodes in the tree. To a biologist examining a phylogenetic tree, a root means an evolutionary common ancestor. Below are different examples of drawing a tree:
One has to be careful about whether the tree they are working with is rooted or unrooted. Unrooted trees provide information about evolutionary relatedness. Those entities that are more closely grouped are more closely related than those entities that are not as closely grouped. Rooted trees provide information about the evolutionary ancestry in addition to the evolutionary relatedness. The Page 3 of 10
AP Biology
Teacher’s Guide
same tree could be drawn to look rooted or unrooted. In the example below, A appears to be a root of the left tree and therefore we could infer that A is an evolutionary ancestor of all other nodes. However, if that tree is not truly rooted but only drawn as rooted then our analysis is incorrect. The tree on the right is the same tree as the one on the left, only drawn differently. A is not a root in this tree. When using Bioinformatics tools to produce and draw phylogenetic trees, you have to be extra careful about whether the tree is rooted or unrooted. Usually these details are a part of the manual or the readme file.
Remember that evolution is a fluid process? Evolutionary changes are slow and settle, when looking at any short periods of time. Usually when building phylogenetic trees, the common ancestor of two currently existing specie is often extinct and no longer exists. Such common ancestor is indicated in a tree as a junction of two branches. Adding an outlier entity/group to our set of characteristics (in our case sequences) allows creating a tree topology that shows the position of the common ancestor for our group of interest. It causes representing the phylogeny of our entities/specie as a subtree, which indicates a grouping together. The outlier sequence is usually completely unrelated to the other sequences in your analysis and will lie on the outside of all the other groups. It will not group together with any other sequences. This technique allows all the sequences of interest to group together in a subtree, by which separating all of them from the outlier entity. Below is an example of using an outlier group. Eubacteria is used as an outlier in the analysis of eukaryotes based on some enzymes. Without the use of an outgroup it would be impossible to infer an relationship to a common ancestor just from the tree topology.
Da-Fei Feng, Glen Cho, and Russell F. Doolittle. Determining divergence times with a protein clock: Update and reevaluation, PNAS, 1997.
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AP Biology Teacher’s Guide A subtree with two or more nodes is called a clade. Nodes within the same clade are more closely related than those in two different clades.
There are many ways to draw a tree Everything is derived from the common ancestor. Many algorithms do not actually give us the root. Part c: what does evolutionary time means (in cladograms, everything is now
Cladograms vs. Phylograms Another important concept we need to tackle is the difference between the cladograms and phylograms. Both of these types of trees can be drawn as rooted and unrooted. However, there is one big difference between cladograms and phylograms. The length of a tree arm (called an “edge” in computer science and mathematics) in phylograms indicates evolutionary time since the last common ancestor while the length of a tree arm in a cladogram is irrelavant and has no special meaning. In phylogram, the longer the edge is the more time has passed since the last common ancestor and very short edges indicate a very young (in evolutionary time) node.
Answers to the worksheet questions: Part A Exercise 1 Quagga and Zebra are more closely related.
Exercise 2 Banksia and Hakea are more closely related.
Exercise 3 Horseshoe crab and Aquatic spider are more closely related.
Exercise 4 Barnacle and Shrimp are more closely related.
Exercise 5 Species that are more closely related may have more similar DNA sequences and, therefore, we can use those sequences. When the species we are comparing are more distantly related then the appropriate genomic sequences might not align well while the protein sequences still show conservation. In that case, it is better to use protein sequences.
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AP Biology
Teacher’s Guide
Part B Exercise 1 Q11
Exercise 3 Q9
Exercise 4 A Students should see a tree that looks similar to this:
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AP Biology Q8 Different.
Teacher’s Guide
Q9 Chimp (displayed as Pan in the tree). Q10 Gorilla. Exercise 4B Students should see trees similar to this:
Q9 Rooted. It says so in the Standard output (Report) field. Q13 Salmon (appears as Salmo in the tree). Q14 Logically, this relationship does not make sense. As was discussed in the in-class presentation and class discussion, we should use an outgroup to fix this. Q22 The root should be drawn on the edge leading to Salmo node. Exercise 4C Students should see a tree similar to this:
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AP Biology Q10 Unrooted. It says so in Standard output (Report) field.
Teacher’s Guide
Q14 The root should be drawn on the edge leading to Salmo node. Q29 The tree produced in step 8 is a phylogram. It tells you how much evolution occurred from the time of speciation between any two given species. In other words, it tells us how closely two species are related. For example, from this tree we can conclude that there has passed more evolutionary time between Salmo (salmon) and Mus (mouse) than between Rattus (rat) and Mus (mouse). The tree produced in step 26 is a cladogram. It tells us how much time has passed since the speciation between the species in the exercise. For example, we can tell that more evolutionary time passed from the time of speciation between Mus (mouse) and Rattus (Rat) than Mus (mouse) and Otolemur (galago). Exercise 5 Students should see trees similar to this:
Q8 Chicken (Gallus) is most related to the mouse and the rat (Mus and Rattus).
Part C Exercise 1 Students should see a tree similar to one of these trees:
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AP Biology
Teacher’s Guide
Q3 HIV is most closely related to SIV Chimp. Part D Students should see a tree similar to one of these trees:
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AP Biology Teacher’s Guide Q7 Platypus is most closely related to other marsupials: Opassum, Quokka, Rock wallaby, Nail tail wallaby, Swamp wallaby). Q8 Kangaroo rats don’t belong to the same immediate clade. They are more distantly related to platypus than the species listed in Q7. Actually, kangaroo rats are not marsupials at all. Q9 Kangaroo rats are more closely related to other species of rats and mice.
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