Investigation 3: Comparing DNA Sequences to Understand Evolutionary Relationships with BLAST

Introduction
Recently, a team of scientists have discovered a fossil specimen in China after extensive digging. The scientists have sent the fossil to Mr. Wong's Period 7 AP Biology Class to try and place the species on a cladogram to graph its evolutionary relationships. Luckily for our team, small samples of DNA were salavged from the long dead species, unfortunately only 4 small sequences could be saved. The first approach to figuring out where the fossil lies on the tree of life undertaken by Christos, my fellow researcher, and myself was to use morphology to make a hypothesis of where the fossil lies in the evolutionary tree of life. then we will use gene sequence analysis to compare the genes of the organism that were salvaged in the Basic Local Alignment Search Tool, or BLAST, with genes of other, modern organisms. BLAST contains the DNA sequences of many different organisms so we will be able to pinpoint the location that the species lies on the cladogram.

Procedure:
The first step in our procedure is to create a hypothesis of where the species would lie on the evolutionary cladogram based solely on morphology. Looking at the morphology of the fossil, Christos and I agreed that the organism was bony and therefore also has a backbone. The organism also has eyes on each side of its head, so it does not have binocular vision like humans do. Also, the head shape of the organism is reminiscent of a lizard, so perhaps the organism is closely related to reptiles. The long tail that tapers to a point, further supporting evidence that the creature is related to reptiles. The specimen has long legs and short stubby arms, so we can assume that the creature was bipedal, unlike other reptiles. Along with the dark patches along the top of the organism, which may be some sort of feather or proto-feather, the body shape and long legs shows that perhaps the creature is also related to birds. Unfortunately, the researchers cannot see the internal organs of the fossil because soft tissue does not fossilize, so we cannot make any more guesses about the structures of the organism. The organism is picture below.

I believe that the organism belongs in its position on the cladogram below because it does not have fur, yet it is also closely related to reptiles because its appearance is similar to that of reptiles. Also, the creature is obviously a vertebrate, so it must go after the vertebrate branch of the cladogram.

The next step in our procedure is to use the BLAST database and compare the similarity of the genes that we salvaged from the specimen's fossil. To do this, we downloaded the files from Canvas onto a laptop, and then using the "Saved Strategies" link on the homepage of BLAST, which is located at http://blast.ncbi.nlm.nih.gov/Blast.cgi, we uploaded the downloaded files onto the website. Then, after waiting for a few seconds for the website to recognize the file and input all of the information it needed to do a BLAST search, we pressed the "View" button on the webpage. This took us to the official BLAST search page, but the search parameters were all filled out by the "Saved Strategy" that we downloaded. Pressing the "BLAST" button at the bottom of the page activated the search engine and then we waited as the file we uploaded was compared against hundreds of thousands, if not millions, of genes sequenced by different Genome projects

Below are pictures of the 4 different gene sequences' top five matches in the BLAST database. These are genes in other organisms that are similar or even exactly the same as the genes found in the fossil. The more similar the gene is to the gene of another organism, the more closely related the two organisms are.

BLAST of Gene 1

BLAST of Gene 2

BLAST of Gene 3

BLAST of Gene 4

For the first gene tested, the top result for similarity with the new species is the species Gallus gallus, which is a red junglefowl, or a tropical chicken. This agrees with my previous assumption that the new organism is reptilian because chickens and all aves are descended from reptiles. The similarity in genes is 99%.

For the second gene tested, the most similar result from the BLAST was a gene in Drosophilia melanogaster, which is the common fruit fly. This seems to go against my earlier hypothesis that the organism is a vertebrate and related to reptiles because an insect like the fruit fly has no internal bones and predates the evolution of reptiles by many millions of years. Perhaps the organism is more closely related to insects than Christos and I previously thought, but perhaps this is just a fluke and the organism only shares a single gene with the fruit fly. The other two genes will tell whether or not the organism is more reptilian or not. The similarity between the genes is only 92%.

For the third gene tested, the top result was a gene for Taeniopygia guttata ubiquitin-conjugating enzyme E2Q family member 1, which is quite the mouthful. This gene codes for a certain enzyme that is found in the zebra finch, which is another bird. This further supports the earlier hypothesis, as well as my thought that the species has some sort of proto-feather structures on its body. Furthermore, the relationship with the zebra finch distances the new organism from the insect genes it seems to have. The similarity between the genes is 95%.

The fourth and final gene tested was most similar to a gene found in Alligator sinensis mitochondria. Therefore it is a gene that is found in the mitochondrial DNA of the Chinese alligator. This is the final; piece of evidence linking this fossilized creature to reptiles and birds. Also, because it is closely related to the Chinese alligator with a gene similarity of 100% and the specimen was found in China, it must be some close ancestor of the Chinese alligator.

Conclusion
After looking at the BLAST results and discussing the outcomes, Christos and I came to a consensus on where this organism should lie on a cladogram of species, specifically the one we were given. We believe that the organism is a type of reptile that branched out to be the common ancestor for certain species of both birds and alligators. Because of the immense similarity to the Chinese alligator that we discovered, we believe that the organism is more closely related to alligators and crocodiles than birds, even though two of the genes discovered were bird genes with high reates of similarity. Keeping all of this in mind, we placed the organism on the cladogram below.

Our initial hypothesis taking into account simply morphology was somewhat accurate because we had placed the organism on the correct evolutionary branch, but we were not sure of how far down the branch the organism would be. Using both the morphological hypothesis we formed along with the accuracy of the BLAST database searches, we were able to determine to a finer degree where the organism should be placed on the above cladogram.