An important tool in forming an evolutionary hypothesis is the construction of a cladogram, a tree-shaped chart used to depict the hypothetical genealogical relationships between species. In a cladogram, the tips or leaves of the chart represent specific species, and the branches of the tree are different lengths, to represent the degree of change between each species. The common ancestor of all of the species that a specific line branches from is located at the point where the branches intersect, and is called a node.
Species that share a node are called a sister group. In this first exercise, you have been given a copy of this cladogram. Looking at the groups of animals in the figure, what would be your hypothesis about where the animals should be placed on the tree?
Take time now to fill out the cladogram with your best guess as to where each animal fits. Now, look at this image of a fossilized animal. Based on the morphology, or physical characteristics of the fossil, where would you place this animal on your cladogram?
Add a new line or mark to indicate your proposed position for the fossil. Although historically, cladograms have been constructed through the comparison of morphological similarities and differences between species of interest, another method of creating cladograms is to compare the genetic sequences between species. As DNA sequences are not usually preserved in fossils, in this experiment, we will compare the DNA sequences of living species closely related to our fossil to those of thousands of other living species using the BLAST database.
The results will allow us to place the fossil into the cladogram based on DNA similarity. Before beginning this lab, your instructor will have downloaded the three preserved gene sequences, and these should be stored in a folder on your desktop. Go to the BLAST homepage, which your instructor should have left open in your browser, and click on Saved Strategies from the menu at the top of the page.
Under Upload Search Strategy, click on Choose File to browse your computer for the fossil relative DNA sequences, which should be located in a folder on the desktop. Click Open on the Evolutionary_Relationships_Gene1 dot text file. 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 on the top-right of the screen. Scroll to the bottom of the screen and click the BLAST button. It will take a few moments to process and analyze the data.
When the data has been processed, a graphic summary will appear. Your query sequence is represented by the blue line at the top of the box. Each of the lines beneath it represents another sequence in the BLAST database which matches your query.
The length and location of these bars represent where and how much of the sequences match up to the query. Here, many of the bars match the whole query sequence across its length, while some are missing a little at the beginning of the sequence. The color of the bar represents its score, or how identical the sequences are 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 sequence. The sequences are listed in descending order, from the most similar at the top, to the least similar at the bottom. On the right 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. Likewise, the lower the E value number, the less likely the sequence match was found by random chance, and the more significantly the alignment is accurate. Here, you will notice all E values are zero, meaning the matches are all highly significant.
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. Then, 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, and then the protein that the gene codes for, which should be listed next to DEFINITION. Then, close this tab and hit Back to return to the Sequences producing significant alignments list, and repeat this process to identify and record this same information for the next several most similar sequences.
After collecting several species names, go back 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 tree of results to create a phylogram based on the similarity of all of the BLAST result gene sequences to the query gene sequence. 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 gene sequence, and then query the other two fossil relative DNA sequences as just demonstrated. Based on your gene sequence results, come up with a second hypothesis about where your fossil specimen fits in the original cladogram.
Compare this placement with the first hypothesis. How similar are your DNA and morphology-based trees in their placement of the fossil specimen? What do your hypotheses say about the relative importance of morphology in predicting evolutionary relationships compared to DNA sequences?
Compare your tree with your classmates. How similar are your trees? Ask your classmates who put the fossil on a different branch how they came to that decision.
