Lab 5: Evolutionary Relationships — Procedure

  1. Forming an Evolutionary Hypothesis
    • NOTE: A cladogram is an important tool for forming an evolutionary hypothesis. A cladogram is a tree-shaped chart used to depict the hypothetical genealogical relationships between species. The tips or leaves of the chart represent specific species and the branches of the tree are different lengths. The different lengths represent the degree of change between each of the species. The common ancestor of all of the species that a specific line branches from is located at a node, where the branches intersect. Species that share a node are called a sister group.
    • To begin, look at the provided blank cladogram and make a hypothesis about where the assigned animals should be placed on the tree. If a blank cladogram has not been provided please download one here.
    • Next, observe the image of the fossilized animal.
    • Using the morphology, or physical characteristics, of the fossil, make a hypothesis about where on your cladogram it should be placed. Add a new line or mark to indicate your proposed position for the fossil.
  2. Comparing Gene Sequences Using BLAST
    • NOTE: Historically, cladograms have been constructed through the comparison of morphology. Now, genetic sequences can also be compared between species of interest to construct cladograms. DNA sequences are not usually preserved in fossils, so for this lab you will use the BLAST database to compare the DNA sequences of living species that are closely related to the fossil to thousands of other living species.
    • Before beginning this lab, your instructor will have downloaded the three preserved gene sequences to a folder on your desktop.
    • Navigate to the BLAST homepage, which the instructor should have left open in your browser, and click on Saved Strategies from the menu at the top of the page.
    • Click Open on the Evolutionary_Relationships_Gene1.txt file.
    • Then, click View to go to the Standard Nucleotide BLAST search page. You should be able to see the DNA sequence in the Query Sequence box at the top-right of the screen.
    • Scroll to the bottom of the screen and click the BLAST button. Note: It will take a few moments to process and analyze the data.
    • When the data has been processed, a graphic summary will appear. NOTE: Your query sequence is represented by the blue line at the top of the box. Each of the lines below the blue line represents another sequence in the BLAST database that matches your query. The length and location of these bars represent where and how much of the sequence match up to the query. The color of the bar represents its score, or how identical the sequence is to the query. Below the graphic summary is the Sequences producing significant alignments list, which contains descriptions of the DNA sequences retrieved from the database that most closely align with the query sentence. These are listed in descending order, from the most similar at the top to the least similar at the bottom. On the right, there are several statistics on how closely related each database sequence is to the experimental sequence. The higher the Max, Total, Query Coverage, and Identity scores are, the more similar the query and retrieved sequences are to each other. Similarly, the lower the E value number, the less likely the sequences match was found by random chance and more significantly the alignment is accurate. An E value of 0 indicates a highly significant match.
    • Click on the name of the description of the most similar alignment listed. This will take you further down the page to the exact DNA sequence alignment between the retrieved sequence and the query sequence.
    • Click on the Sequence ID number. This will open a new tab with more specific information on the retrieved gene sequence.
    • Identify and record the scientific and common names of the organism, which should be listed next to Source.
    • Identify and record the protein that the gene codes for, which should be listed next to Definition.
    • Close the tab and hit Back to return to the Sequences producing significant alignments list.
    • Then, repeat steps 7 – 9 for the next most similar sequences.
    • After collecting several species names, return to the Sequences producing significant alignments list and click Select: All to check all of the boxes next to the names of each listed alignment sequence.
    • Click on Distance of tree results to create a phylogram based on the similarity of all of the BLAST result gene sequences to the query gene sequence. Note: Your query fossil relative sequence will be highlighted in yellow.
    • Record your sequence’s location in relation to the other taxon groups shown in the tree.
    • Finally, return to the Saved Strategies tab and upload the next saved gene sequence.
    • Query the remaining two fossil relative DNA sequences as previously demonstrated (steps 1 – 16).
  3. Data Analysis
    • Based on your gene sequence results, come up with a second hypothesis about where your fossil specimen fit in the original cladogram.
    • Compare this placement with your first hypothesis.
    • Compare your tree with your classmates’. If any of your classmates put the fossil on a different branch, ask them how they came to that decision.