Phylogenetic Analysis: How Old are the Parts of Your Body?
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According to the National Academy of Sciences, biology students in the USA are not being adequately prepared for successful futures. Of paramount concern is a lack of sufficient training in quantitative and computational skills, which are needed to compete effectively for an array of educational and occupational opportunities. In this paper, we introduce a classroom exercise that invites students to solve a simple biological problem and illustrates the need for a computer-assisted strategy to arrive at a solution. The exercise invites students to consider the question “How old are the parts of your body?” Some features of the human body are more ancient than others. For example, our bodies have both hair and backbones, but backbones arose much earlier in evolutionary history. Our exercise relies upon MEGA 4.0, a free, visually appealing, and intuitive computer program that allows students to gather DNA or protein sequences from electronic databases, then use them to infer phylogenetic trees. Student-inferred phylogenies are used to explore the relative order in which diverse aspects of the human form evolved. In the process, students are trained to use powerful features of MEGA and encouraged through group discussion to consider additional applications of the technology they have learned. Our lesson plan includes a brief video, a web site with essential terminology and links for further exploration, a hands-on experience using MEGA, and a follow-up discussion.
KeywordsAlignment Bioinformatics Evolution MEGA Phylogeny
Biology, as a discipline, is in a state of continual and rapid flux. What was once a largely qualitative and low-tech branch of science has increasingly become the domain of ornate mathematical models and sophisticated software packages (Steen 2005). Accordingly, two recent studies by the National Academy of Sciences emphasize the need for more extensive and rigorous training of biology students in both quantitative and computational skills (National Research Council 2003, 2005). These studies indicate that students trained in the USA are frequently deficient in these areas when compared with their international counterparts and, consequently, are placed at a competitive disadvantage when pursuing vocational opportunities in the public and private sectors. Hence, there is a compelling need for engaging and empowering educational opportunities for contemporary students of biology, especially at the high school and college levels.
Contemporary students are increasingly engaged in a technology-imbued lifestyle, presenting novel challenges and opportunities to high school and college educators. A challenge is to vie for the attention of students who are exposed to a steady stream of brilliant audio and visual stimulation. A byproduct of a technology-based lifestyle is that the average student has an innate fondness for computer-based technology and is eager to gain expertise and sophistication with its use. Herein lies a promising opportunity. If an introduction to computer-based technology can be coupled to a set of suitable learning objectives, students may engage actively in learning that might not otherwise occur. In addition to providing a means to acquire essential foundational knowledge in any amenable area of biology, an opportunity is also presented to cultivate computational and critical thinking skills called for in the studies conducted by the National Academy.
Recently, a range of computer-based strategies have been developed and implemented by innovative educators, and these approaches have met with considerable success in the classroom. Significant improvement in learning effectiveness was shown in a variety of biological subject areas. For example, successful learning modules have been developed for diffusion and osmosis (Meir et al. 2005), DNA replication (Woods et al. 2008), field biology (Baggott and Rayne 2007), genetics (Calie et al. 2007), macroevolution (Perry et al. 2008), and viral evolution (Rybarczyk 2008a, b). These and other examples illustrate the potential for computational approaches to enhance and invigorate biology education at secondary and postsecondary levels (Syh-Jong 2008). However, to realize these benefits, a variety of potential barriers need to be addressed, notably provision of workable and effective lesson plans as well as training and support for biology instructors (Mueller et al. 2008).
Our strategy is to introduce to students an apparently simple and engaging question through a group discussion that culminates in the formation of a testable hypothesis. Thereafter, students are provided access to a minimal body of foundational knowledge through a stimulating video and a webpage, both of which are freely available to the public. Students are subsequently given an opportunity to work with MEGA 4.0, a computer program that is user-friendly and visually appealing, yet is a professional-grade software application used daily in research laboratories around the world (Tamura et al. 2007; Kumar et al. 2008; http://www.megasoftware.net/). Finally, students are invited to interpret their own results, evaluate the hypothesis they formulated in the preactivity discussion, and brainstorm about additional applications of the technology they have just learned to use.
As groups of organisms diverge and diversify, lineage-specific distinctions arise at both the anatomical and molecular levels (O'Hara 1997). Given sufficient time, a set of characteristics accumulates that can be used to differentiate members of one organismal group from another (Baum et al. 2005; Baum and Offner 2008; Gregory 2008). Although excellent analyses may be conducted with either anatomical or molecular data, the latter offer some distinct advantages for computer-based phylogenetic inference in the classroom. First, molecular data from an enormous array of species, representing the most phylogenetically diverged lineages in the tree of life, are freely and publicly available in web-accessible electronic repositories (e.g., GenBank which is accessible through the National Center for Biotechnology Information (NCBI) website, http://www.ncbi.nlm.nih.gov/). Second, these data can be processed and analyzed for historical content with only a minimal level of background information. In contrast, anatomical data are generally gathered and interpreted by individuals with a considerable amount of organism-specific expertise. Third, a number of engaging and user-friendly software packages are freely available to reconstruct phylogenies from molecular data (e.g., Tamura et al. 2007; Kumar et al. 2008) [NB, in this exercise, students gather sequences of the PaxNEB gene from diverse animal species. PaxNEB encodes an RNA polymerase II elongator protein subunit (for details, see Klenjan et al. 2002), but its function is not relevant to the exercise. It was selected because it evolves at an appropriate rate for resolving relationships among distantly related animals, both in terms of its moderate rate of amino acid substitutions and its low rate of gene duplications].
Table of body parts included in preactivity discussion and in the remainder of the exercise
Very large brain
Forelimbs and hindlimbs
How many species are there on earth?
What do the scientists in the video mean by “the tree of life?”
How old do they estimate this tree is?
How does a single branch in the tree of life split to become two branches?
How do novel characteristics arise in the history of a biological lineage?
What are the sources of evidence used to determine the shape of the tree of life?
Why are computers needed to analyze data sets when determining the shape of the tree of life?
After discussing the video, students progress to an exploratory and interactive web site that introduces additional concepts and vocabulary that will be essential to understanding the products of their analyses (http://www.nescent.org/media/NABT/mega_workshop.php). However, no specific activity is assigned for this web site. Instead, students are made aware of its existence and contents, given the URL, and then encouraged to proceed directly to the hands-on activity. The strategy here being that students will consult the web site just when they perceive a need for its contents. We believe this engenders a student-led learning process, in contrast to an instructor-led process, which might be accomplished through a preactivity lecture.
Students then extract historical information from their aligned data matrices and use it to estimate relationships among the species from which the sequences were sampled. First, students are encouraged to visually inspect their alignments to appreciate that some regions are more conserved among sampled sequences while others appear more chaotic and variable. This illustrates the notion that mutations in some regions are not common and provides an opportunity to explore the concept of natural selection, eliminating unfavorable mutations (later in the postactivity discussion).
If you wish to know the evolutionary relationships among members of a group of species, what sources of evidence could you use?
What sorts of information can be extracted from sequence data? Medical? Genetic? Historical?
Why are some regions of the sequence alignments you generated more uniform, while others are more variable?
Why is it that groups like the insects or the mammals resolved as clades in the trees you reconstructed?
Why is it that all the organisms that have hair or vertebral columns resolved as monophyletic groups?
If you had a copy of the MEGA_4U protocol in hand, do you think you could explain to another student how to reconstruct a phylogenetic tree?
Do you think you could explain what a tree you infer says about the evolutionary history of organisms and their traits?
What other questions could be solved using this technology? For example, where did whales come from? Do all aquatic mammals form a clade? Do all warm-blooded or flighted animals form a clade? How many times did eyes evolve? What fish are the closest relatives of land animals? Did red-colored flowers evolve more than once? What bacteria are most closely related to Bacillus anthracis (which causes anthrax) or Clostridum botulinum (which causes botulism) or Treponema pallidum (which causes syphilis)? Are their closest relatives equally dangerous? If not, how could we use this information to benefit humans?
Individual instructors can decide whether they wish to permit their students to develop research projects to apply and build upon the technical skills that they have just acquired. Projects of this type can reinforce students' understanding of the procedures involved and increase their sense of empowerment. To illustrate how one of the above questions could be developed into a student-driven, inquiry-based individual or group project, let us consider the first example, “Where did whales come from?” Each step that is needed in the process is illustrated in the step-by-step guide in the Appendix. Using MEGA, a student can visit the NCBI web site to recover a protein sequence from their favorite whale species (it is best to use protein sequences that are at least 200 amino acids in length to provide a sufficient amount of data for the problem; cytochrome c oxidase subunit I, also known as COXI, works well here). MEGA can then use this sequence to run a BLAST search to probe GenBank for other similar sequences (it is best to use a protein sequence that recovers one or very few sequences per species to simplify tree reconstruction. If too many sequences per species are encountered at this step, select an alternative gene). Some related sequences will come from whale species, while others will come from land mammals (especially cows, deer, hippos, pigs, camels, horses, and other ungulates). Again, these sequences can be used to generate a phylogeny for all of the sampled species (the tree should be rooted with the most distantly related species, in this case the horse). The resultant tree can be inspected to reveal where whales arose (whales form a clade with dolphins and porpoises; this clade is the sister group to hippos, suggesting that hippos and whales had a common ancestor from which they both descend. As the whale–dolphin clade is the only group of ungulates with an exclusively aquatic lifestyle and without legs, this suggests that the loss of legs and the transition to water occurred along the branch leading to whales from the common ancestor of whales and hippos). Other questions listed above could be pursued in a similar manner using the protocol in the Appendix to guide the inquiry process.
Once you have the skills, there is no limit to the intriguing evolutionary questions that you can solve!
The authors wish to thank the College of Letters and Science and the Undergraduate Research Foundation of the University of Wisconsin at Whitewater for financial support and the McNair Foundation and WiscAMP Alliance for financial support of Courtney P. Thompson. We also wish to thank Andi Kornowski and Melissa Schober for technical assistance during the project and the Peabody Museum of Natural History at Yale University for producing the Discovering the Great Tree of Life video. Additionally, we wish to thank the MEGA development team at the Center for Evolutionary Functional Genomics for producing MEGA 4.0. Finally, we thank Kristin Jenkins of the National Evolutionary Synthesis Center for encouragement and insightful advice during the development of this project.
- MEGA webquest. http://www.nescent.org/media/NABT/mega_workshop.php.
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