Biology 18 Spring 2008 Lab 2 - Illustrating Evolutionary Relationships Between Organisms: Emperor Penguins and Phylogenetic Trees Pre-Lab Reference Reading: Review pp. 542-556 and pp. 722-737 in Life by Sadava et al., 8th edition, 2006. I. Introduction The ability to think broadly about the living world is rooted in an understanding of life's diversity. However, the large number of ways in which various organisms differ from one another can often mask their similarities. The diversity and unity of living organisms would seem to be contradictory concepts. Recall, though, that these were the major themes in Darwin's great work, in which he asked and answered the following question. Since life is so diverse, are there any unifying themes at all? By looking backwards in time, Darwin showed that the answer to this question is a resounding “Yes!” Darwin proposed both a pattern invoking common descent (as the basis for the unity of life) and a mechanism to explain how the relationships between species became fragmented over time (resulting in diversity). In addition, Darwin himself is responsible for the metaphor that is used today to illustrate this shared descent of living organisms: the Tree of Life (TOL). In fact, the diagram shown below was the only figure from his book On the Origin of Species by Natural Selection. “...great tree of life...with its ever-branching and beautiful ramifications...” (C. Darwin, 1859). Time Æ2 The ATOL (Assembling the Tree of Life) Project represents a collective effort of biologists working on different organisms to understand how the diversity of life fits together. Resolving evolutionary relationships that underlie the TOL is unquestionably one of the most important problems in biology today, for two major reasons. • Because it is relevant to all organisms, with the tree potentially encompassing the 1.75 million living species characterized to date and an estimated tens of millions more to be discovered (not to mention the even larger number of species that existed and are now extinct). • There are major applied consequences of understanding the evolutionary relationships between organisms with respect to human health and the environment. These include the origins and dynamics of disease-causing microbes, or the interactions between species that constitute a rich and self-sustaining ecosystem. Reconstructing an evolutionary tree (i.e., a phylogeny) is a powerful way to answer questions about the evolutionary relatedness of living organisms. In addition, modern tools of molecular biology are greatly advancing our analytical power, particularly in the use of DNA and protein sequence data to investigate the similarity/divergence between species. II. Overview of Activity Millions of people around the world have been captivated by the 2005 documentary movie The March of the Penguins. In this movie, viewers learn about the complex reproductive behaviors of Emperor penguins, which include a 70-mile march (by walking or belly gliding) to an inland breeding ground. (See Appendix for life cycle.) Strong selective pressures have likely been in force to establish reproductive behaviors that differ so greatly between Emperor penguins and other birds, thereby reinforcing the evolutionary independence and integrity of related species. A number of interesting biological questions come to mind while watching The March of the Penguins. For example, 1) What is the closest living relative of Emperor penguins? 2) Are all penguins living today the descendants of the same common ancestor? 3) If so, to which taxonomic group of birds are penguins most closely related? 4) What would the common ancestor of penguins and their closest bird relatives have looked like? 5) What is the evolutionary relationship between penguins (and birds in general) to other classes of vertebrates? To address these and other questions, students will use the National Center for Biotechnology Information (NCBI) Website to reconstruct a phylogeny, which will illustrate the evolutionary relationships between Emperor penguins and other living organisms. Students will use gene sequence databases to do this, and DNA sequence comparisons will be done “behind the scenes” using NCBI’s BLAST search engine. BLAST stands for Basic Local Alignment Search Tool, and “the program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches” (http://www.ncbi.nlm.nih.gov/BLAST/).3 A comparison of DNA or protein sequences from different species can reveal evolutionary relationships. This assumption is based on the idea that the nucleotide or amino acid sequence of a particular gene or the protein it encodes, respectively, mutates at a similar rate in different individuals. However, students should remember that the rate of mutation alone does not determine how fast a DNA sequence will change. The effects of mutation on survival and reproductive success (natural selection), as well as the generation time of the organism, also play important roles in the overall rate of genetic change in a population. To sum up, the overall goal of this lab is for students to examine the evidence held within genetic sequences to determine how living organisms are related to one another and to confirm that they share common ancestors. You will do so by using BLAST to measure the degree of gene sequence similarity between various types of organisms. Each pair of students should go through the exercises below with Emperor penguins, using the worksheet on pp. 17-21 to record your answers to questions (in bold) asked in the text. The completed worksheet is due at the beginning of lab next week.4 III. Procedure 1. Go to the NCBI Home Page at http://www.ncbi.nlm.nih.gov/. This page contains links to dozens of different databases and other resources. Every page that is linked to the main NCBI page has a small icon somewhere in the upper-left corner of the screen. Clicking on this icon will immediately return the computer to the NCBI home page. 2. We will build a phylogenetic tree by using BLAST to compare the degree of sequence similarity in a gene between different individuals. Generally, the more divergent gene sequences are, the more time and evolutionary distance separate the two species. 3. To begin our analysis, we would ideally select a gene that is present within the genome of all the