Fish Phylogenetic Tree Based on Protein Size free essay sample

Fish phylogenetic tree based on protein size Amanda Reed Lab partners: Lecia Redwine, Kyle Hatcher TA: Baneshwar Singh Biology 117, Section 25 Tuesday 10:30 AM Introduction Although tree diagrams have been used since the days of Charles Darwin, biologists have only recently adopted the tree model of evolution to read and interpret phylogenies. One of the reasons for this is the confusion that often arises from using a tree model to describe a phylogeny (Baum, 2008a). Many people interpret the trees to show that different species evolve from one another instead of viewing them as ways to trace the common ancestors between species. All species at the tips of the branches should be thought of as being evolutionarily equal; however, many people misinterpret the trees to show how different organisms evolved from one another (Baum, 2008b). Phylogenetic trees are a great way to represent how evolution led to the differentiation of species. However, to determine how to draw the tree, one must first define what a species is. Unfortunately, there is no black-and-white answer to determining the existence of new species. First, though, one must decide if a new organism is different enough from pre-existing species to constitute a new species or not. One must also have a method to detect new species. According to Ernst Mayr, organisms of the same species are able to reproduce fertile offspring. However, George G. Simpson argued that members of the same species have the same evolutionary history. Today, the two ideas have been combined to create the Biological Species Concept. This is still open for individual interpretation, so scientists, for the most part, have chosen to follow the Phylogenetic Species Concept which uses the most accurate phylogenetic trees available depicting organisms with shared traits to determine if an organism is a new species or not. Currently, the easiest way to depict the most accurate phylogenetic tree without using computer software is to attempt to determine the most parsimonious tree. If organisms have many shared traits, then they can be determined to be of the same species as each other (Hey, 2009). Another key piece of information used in determining phylogenetic trees is the evolutionary rate. Biologists often use the evolutionary rate to calibrate a â€Å"molecular clock†Ã‚  to determine an evolutionary timeline for a species for which we may not have much evolutionary information. This helps to determine missing pieces in a phylogenetic tree, thus allowing us to create a phylogenetic tree when we may not have all of the information. We can presume that the tips of the tree are equally evolved, as well (Ho, 2008). By using a relaxed molecular clock that presumes that the evolutionary rate varies between organisms along with the Phylogenetic Species Concept, one can get an idea of a phylogenetic tree for a set of organisms, based on their traits. The purpose of this lab was to try to determine a phylogenetic tree for six different fish using the proteins each fish contained to determine their placement on the tree. Methods 3 flip top microtubules and 3 screw top microtubules were labeled. 250 microliters of Laemmli sample was added to each flip top microtubule. A small piece of each fish sample was added to its designated flip top microtubule. Each flip top microtubule was then agitated by flicking it approximately 15 times with a fingertip. The samples were then incubated at room temperature to separate and extract the fish proteins. The buffer solutions containing the fish proteins were then transferred into their designated screw top microtubules. These microtubules were then heated for 5 minutes at 95 degrees Celsius to denature the proteins. The samples were then stored at temperatures less than -20 degrees Celsius until the following lab meeting. The next lab meeting, the frozen fish samples and actin and myosin samples were reheated at 95 degrees Celsius to redissolve any precipitated detergent. The gel box was assembled, and the polyacrylamide gel was inserted into the vertical electrophoresis unit. TGS buffer was then added to the electrophoresis unit until it was above the top of the smaller plate on both the inside and the outside of the chamber. The wells were then loaded with the protein samples using a micropipetter with thin gel loading tips. The first well remained empty. The second and sixth wells were loaded with Precision Plus Protein Kaleidoscope prestained standard. The third well was loaded with mahi-mahi protein sample. Well four was loaded with salmon protein sample. The fifth well was loaded with catfish protein sample. Well seven was loaded with sardine protein sample. Well eight was loaded with flounder protein sample. The ninth well was loaded with shark protein sample. Well ten was loaded with actin and myosin standard proteins. After all samples were loaded, the lid was placed on the tank and the leads were inserted into the power supply. The voltage was set to 200 V, and the gel ran for 30 minutes. After the gel was ran, the power supply was disconnected and the lid removed. The buffer was poured out of the electrode assembly, and the gel was removed. The gel plates were pried apart. Then the gel was rinsed three times with water for five minutes to improve the intensity of the protein bands. The water was removed, and 50 milliliters of Bio-Safe Coomassie stain was added. The gels were stained for a minimum of an hour. The stain was then removed and replaced with a large volume of water overnight. Once the gel had been destained and dried, the proteins were then scored. This was done by measuring the distance from the wells to the known bands on the Precision Plus Protein Kaleidoscope and graphing the distances versus the weight in kilodaltons on logarithmic graph paper. A best fit line was drawn using these points. Then the distances were measured from the wells to the bands of the proteins from the fish samples. Using the best fit line drawn on the logarithmic graph paper, the band lengths of the fish proteins were translated to protein weights in kilodaltons. These weights were then used to create a matrix. This matrix was then entered into MacClade to make a phylogenetic tree. The branches of the tree were then moved around so that the most parsimonious tree could be created. The most parsimonious tree, in this case, was determined by the tree with the shortest length. Results The polyacrylamide gel showed bands of proteins based upon their weight. The further away from the gel the protein band occurred, the heavier the protein was. The Precision Plus Protein Kaleidoscope prestained standard and actin and myosin standard ladders were very neatly spaced out, and one could easily detect the presence of different proteins. However, the fish proteins were not so easily discerned, especially since, in most cases, the lighter proteins had less color. Wells 2 and 6 contained Precision Plus Protein Kaleidoscope prestained standard. This formed a neat, ladder-like set of bands which were easily used to determine the weights of the other proteins. Based upon the weight versus the distance from the wells, we found 26 different proteins among the six fish. Both sharks and sardines appear to share few proteins with the other fish, while salmon, catfish, mahi-mahi, and flounder appear to be more closely related, and they share at least a few proteins between themselves. No one protein appeared in all fish. Figures and Tables This is a picture of the gel that we ran. From left to right, the wells were numbered 1 through 10. Well 1 remained empty. Well 2 had 5 microliters of Precision Plus Protein Kaleidoscope prestained standard. Well 3 contained 10 microliters of mahi-mahi protein sample. Well 4 contained 10 microliters of salmon protein sample. Well 5 contained 10 microliters of catfish protein sample. Well 6 contained 5 microliters of Precision Plus Protein Kaleidoscope prestained standard. Well 7 contained 10 microliters of sardine protein sample. Well 8 contained 10 microliters of flounder protein sample. Well 9 contained 10 microliters of shark protein sample. Well 10 contained 10 microliters of actin and myosin standard. This is the matrix created using MacClade. The right column gives the names of each fish sample. The top row gives the weight of each protein in kilodaltons. In the picture of the phylogenetic tree that follows, the protein weights are represented by 1 through 26, with 1 being the heaviest and 26 being the lightest. Where the numbers are indicates that protein was either lost or gained at that point. The tree that was generated had a tree length of 31. According to MacClade, the minimum length was 26; however, we were not able to find a tree with a length shorter than this. Discussion No one protein was shared among all six fish. Sharks and sardines appeared to both be outgroups, based upon our results. The shark could easily be an outgroup since it has more cartilage instead of dense bone. The sardines are less different physically from the other fish, so it is unclear why this fish would also be an outgroup. The tree that was created using MacClade had a length of 31. The minimum length was 26, but we were unable to get any closer to the minimum than 31. The results may have been somewhat skewed because the same proteins may have been measured to be slightly different lengths, causing more proteins to have been found than really existed. More fish may have shared more proteins than what we found.