H. A. Booher
When an unknown fish was found in Lake Cunningham recently, it brought about questions of whether or not speciation had occurred within the last 5,000 years in the lake. By extracting DNA from the unknown fish and four of its closest relatives, and using gel electrophoresis, Polymerase Chain Reaction, DNA sequencing, and a Blast search of the unknown fish’s D-loop, a comparison was made possible between all of the fish. Using this information, it was discovered that the D-loop of the unknown fish shares 91% of its base pairs with Poecilia latipinna, a species of fish that is found throughout the tropics, as well as parts of the United States (Williams et al., 1998). This information suggests that the identity of the unknown fish is in fact, Poecilia latipinna, and that a new species has not evolved in the lake.
In June of this year, some odd-looking fish were caught from Lake Cunningham, located in the Bahamas. Because the lake has not been thoroughly studied for its fish, it is not known whether this is a new species, or something else. The lake already contained a large amount of the species Cyprinodon variegates (Cv), and so it is thought that the unknown species may have diverged from them. The unknown species has also been compared to three other species of fish, thought to maybe be close relatives: Poecilia sphenops (Ps), Xiphophorus helleri (Xh), and Xiphophorus variatus (Xv). For this study, the mitochondrial DNA (mtDNA) was studied. The benefit of using mtDNA is that it changes by mutation ten times faster than the nuclear genome. This makes it possible to sort out phylogenetic associations between closely related species or between different populations of the same species (Campbell et al., 1999). Also, mtDNA is preferable over nuclear DNA because it is haploid, as opposed to diploid. This makes it easier to find differences. The D-loop is used because it is non-coding and therefore doesn’t affect the survival of the species (Castro et al., 1999). There will be more mutations in less amount of time, making this the ideal molecular marker since Lake Cunningham is estimated to be about 5,000 years old. Nuclear DNA does not have as many mutations because it codes for traits that are necessary for survival, such as color. If a mutation did occur, it could be detrimental to the species’ survival (Dos Santos and Buck, 1999).
The most accurate way for comparing nuclear and/or mitochondrial DNA is DNA sequence analysis. This is comparing the actual nucleotide sequences of DNA segments. First a portion of the genome that is appropriate to compare (in this case, the D-loop) is selected. After sequencing, the genomic segments are aligned side by side. Common ancestry is evident if two sequences of the same gene from two species that have diverged very recently are the same or differ in only a few nucleotide bases (Campbell et al., 1999). DNA sequencing is a popular method for determining speciation. Recently, it was used to prove that two populations of the bacteria Trichoderma viride were, in fact, two different species. The base pair sequence differences between T. viride types I and II were similar in their variability to data for other species of the same genus (Liekfeldt et al., 1999). These differences indicate that T. viride type II is a distinct species. So by using the D-loop as a molecular marker in this study, and through DNA sequencing, it was discovered that the unknown fish species is most likely Poecilia latipinna, and that a common ancestry is shared with Cyprinodon variegates, Poecilia sphenops, Xiphophorus helleri, and Xiphophorus variatus.
The first step was to determine a morphological phylogeny based on physical observations of the fish. The five different species were measured, and the color was noted. Also, it was recorded whether or not a gonopodium was present. Based on these initial observations, a morphological phylogeny was constructed. Next, the five fish were used to make a DNA extraction. To do this, the tail was cut off of each fish and a small piece of muscle tissue was isolated so that the skin could be peeled away with forceps. The tail was used because of the abundance of mitochondria in the muscle. The exposed white muscle tissue was then chopped up and transferred to a 1.5 mL Eppendorf tube containing 300 ul of Cell lysis solution. The cell lysis causes the cell to open up at certain places along the mitochondrial membrane, making it easier to get to the DNA. 1.5 ul of Proteinase K solution was then added to the lysate, since it is an enzyme that helps to break down proteins. The solution was then incubated at 55 °C for an hour. Next, 1.5 ul of RNase A Solution were added to two tubes, Ps and L13, to get rid of the RNA. These two samples were inverted and incubated at 65 °C for and hour, since that is the ideal temperature for Proteinase K Solution. After all the samples were cooled to room temperature, 100 ul of Protein Precipitation Solution was added. The tubes were centrifuged for 20 seconds to mix the solution uniformly with the cell lysate. Following this, the samples were centrifuged for three minutes so the precipitate would form a tight protein pellet. Afterwards, the supernatants containing the DNA of each sample were poured into clean 1.5 mL microfuge tubes containing 300 ul of 100% isopropanol, and inverted gently. The tubes were centrifuged for one minute, creating a barely visible white DNA pellet. The supernatant was then poured off, and the remaining pellet was washed with 70% ethanol to pull out excess salts and buffers. The samples were then centrifuged for another minute, and then inverted, drained, and allowed to air dry for 15 minutes. The next step was DNA Hydration. 30 ul of DNA Hydration Solution was added to the sample tubes. The DNA was rehydrated by incubating the sample for one hour at 65 °C. This helped to concentrate the DNA, and it was then stored at 4 °C. This was done to turn the DNA back into a liquid, and also to prevent DNases from chewing up the DNA.
The next step in this process was running a 1.0% Agarose gel. Each 500 ul Eppendorf tube was labeled with the names of the samples, and to each tube was added 6.0 ul H2O to dilute the sample, 2.0 ul dye to visualize how far the bands moved, and 4.0 ul of the DNA template, making the total volume of each tube 12.0 ul. One tube was labeled “ladder” and to it was added 6.0 ul H2O, 2.0 ul dye, and 2.0 Marker Ladder. The purpose of the ladder was to have a known band of DNA to compare the other samples to, once the gel had been run. Ethidium Bromide was added to the gel because it intercalates into DNA, which allowed the DNA to be visible under UV light, when a picture was taken. 500 mL of 1X TAE (Tris acetate EDTA) Buffer Solution was poured into the gel rig chamber to cover the Agarose gel by ¼ of an inch. The purpose of the EDTA solution was to bind up divalent cations and prevent DNase activity. It also causes the DNA to fluoresce. Each sample was then loaded into a well using a micropipette, and the gel was run. Since DNA runs away from the negative charge, the smaller fragments moved faster and farther along the gel. A picture of the gel was taken, and the results were recorded.
Polymerase Chain Reaction, or PCR, was the next step in the process. First, it was determined that six samples would be amplified, plus a negative control. So, into each of the tubes was added 13.5 ul H2O to dilute, 3.5 ul MgCl2 which is a divalent cation that EDTA binds to and something would use as a cofactor, 2.5 ul 10X Buffer to keep the environment in the tube the same as in the cell, 2.0 ul dNTPs, 1.0 ul each of Primer E and K, so that the D loop would be amplified in both direction, and 0.5 ul Taq Polymerase, an enzyme that uses Magnesium as a cofactor. The reason why Primers E and K were used is that they are considered “conservative” primers, which means that they work well to amplify the d-loop in many different species. 24 ul of this cocktail was added to the six properly labeled 500 ul Eppendorf tubes, then 1 ul of the extracted DNA template was added to its corresponding tube.
PCR consists of three main steps: denaturing, primer annealing, and the final, “polishing” step. First, the tubes were heated to 94 °C for one minute to denature the DNA and separate it into two strands. Next, the tubes were heated to 94 °C for 20 seconds to further denature the DNA, then cooled to 50 °C for 30 seconds for the primer annealing to take place, then heated back up to 72 ° for 45 seconds so that the dntps would start working. Basically, the Taq Polymerase jumped onto the primers and started adding nucleotides. The previous 3 steps were repeated for 30 cycles, to insure that a significant amount of DNA would be amplified. The final, “polishing” step was keeping the tubes at 72 °C for five minutes to make sure the reaction was complete. The tubes were then stored at 4 °C to inhibit further reactions. The above reactions all took place in a thermal cycler, which was programmed for the specific temperatures and times. A gel was run on a 1.0% Agarose gel to make sure that the PCR were successful and that DNA was amplified.
Next, the PCR products were cleaned using a Qiagen Kit. First, 63 ul of Buffer QG were added into six tubes. Then, 21 ul of isopropanol was added to increase the yield of DNA fragments. To bind the DNA, the samples were pipetted onto the QIAquick columns and a vacuum was applied. After the samples passed through the column, the vacuum was shut off. The samples were then washed by adding 0.75 mL of Buffer PE to the QIAquick columns and centrifuging. Finally, the QIAquick columns were transferred to clean 1.5 mL microfuge tubes and centrifuged for one minute to remove any residual Buffer PE. The samples were then transferred to clean 1.5 ml microfuge tubes again. To elute the DNA, 50 ul of H2O was added to the center of the QIAquick membranes and the columns were centrifuged for one minute. The QIAquick system works well for this because of the silica-gel membrane in the columns. DNA absorbs to the silica membrane in the presence of high salt while contaminants pass through the column. Impurities are eliminated and the pure DNA is eluted with water.
Next, a 1.0% Agarose gel was run again to make sure that the cleaned PCR products were still there. The gel was photographed and checked for the presence or absence of bands and the brightness of the bands was compared to the DNA ladder bands. Next, the concentration of DNA in the tubes containing the cleaned PCR products was determined so that it could be established how much template would be needed to add to the DNA sequencing reaction to get at least 30 ng of DNA template in the reaction. The ingredients in the DNA sequencing reactions that were added to each tube were 4.0 ul of Big Dye Terminator Sequencing juice, 5.0 ul of water, 1.6 ul of Primer E, and 4.4 ul of Template. The ingredients were added in that exact order. The Big Dye juice contains ddNTPs, Big Dye NTPs, Buffer, Salt and Taq Polymerase. The ddNTPs are the nucleotides that will extend the chain, and since they are color-coded (labeled), comparisons between species can be made. Big Dye NTPs are used to stop the sequencing; the base pairs are terminated when a labeled nucleotide jumps on. Once again, the Taq Polymerase is there to add on base pairs. It is important to know that 4.4 ul is the correct amount of template to use because that way, not only short strands of DNA are made. Only one of the PCR amplification primers (Primer E) is used so that it only reads in one direction. If it read in both directions, the laser that is recording the results would not work correctly. Finally, the sequences were determined, and comparisons were made between the four known species of fish, and the unknown species. Based on these results, and how many similarities and differences were between the fish, a molecular phylogeny was constructed. Next, the sequence of the unknown fish was taken and used in a “Blast” search. A Blast search is a way to determine the identity of a DNA sequence, using a search engine that compares the unknown sequence with thousands of other sequences that other scientists have already determined.
Based on physical observations, a morphological phylogeny was constructed (Fig 1.) The results showed that Xiphophorus helleri and Xiphophorus variatus were the two most closely related species. The most recently diverged species from them was Poecilia sphenops. Farther back in their ancestry, the Unknown species diverged. The first fish to diverge from the rest of the group was Cyprinodon variegates. This phylogeny shows that all five fish share a common ancestor.
The first gel run was to see if in fact, mtDNA had been isolated from the five different fish (Fig 2.) A smear of DNA appeared in all of the lanes. The second gel was run after PCR to make sure that the process was successful and the 600 bp fragment of mtDNA was amplified from all five fish in the study (Fig 3.) The results are very faint, but a small band is visible in most of the samples around the 600 band on the marker ladder. Lanes 2, 7 and 8 have no visible bands of DNA in them. The third gel was run before DNA sequencing, after the PCR products were cleaned, to make sure that the products were still there (Fig 4.) All of the lanes showed bands around 600 bp, but lane 6, containing the sample for the pup fish L13 did not show up.
The sequence alignment (Fig 5) shows a comparison in base pairs for the D-loops of all five fish in the study. The D-loop is about 600 bp, but the sequence only shows 486. Using this information, a molecular phylogeny was constructed (Fig 6.) This shows that Poecilia sphenops and the unknown fish are very closely related. Also, Xiphophorus variatus and Xiphophorus helleri are very closely related. Cyprinodon variegates is not closely related to any of the other fish, but all five do share a common ancestor.
The first gel run shows that all lanes contain a smear of “raw” DNA. This means that the DNA was successfully extracted from each fish. The second gel that was run showed that the PCR process was successful and that the 600 bp part of the D-loop was amplified. The negative control was there to make sure that there was no contamination in the DNA, and it worked. Lanes 2, 7 and 8 did not show evidence of DNA in them, but this could be due to the fact that there was not a sufficient amount of DNA in the loaded sample to show up in the photograph.
The third gel was run to make sure that the cleaned PCR products were still there. All the lanes showed evidence of the amplified D-loop, except lane 6, which contained L13, one of the pup fish. This could be due to the fact that the sample was contaminated sometime after the PCR process, or human error occurred while loading the sample into the gel.
The morphological and molecular phylogenies agree with each other, except Poecilia sphenops and the unknown fish are more closely related then had originally been thought. By using the Blast search, it was discovered that the unknown fish shares 91% of its base pairs the species Poecilia latipinna, commonly referred to as mollies, and is also very similar to Poecilia velifera. These fish are known as sailfin mollies, and are named so because of the strikingly large dorsal fin in males (McIvor et al., 1998).
These fish can survive throughout a wide temperature range (McManus and Travis, 1998). In 1998 tests were done with populations from four different locations were the mollies were raised from birth to maturity at temperatures ranging from 23 to 30 ° C. The salinity of the water also varied, being 2, 12 or 20 parts per thousand. The body mass of the mollies was not affected in any of the situations. These results indicate that mollies possess a flexible developmental program with respect to temperature and salinity, and therefore can live across a wide geographical range (Placek and Breden, 1998). There are documented populations of these fish in Northern Florida and Georgia, and all across the south to Texas (Van Fleet et al., 1998). The fact that this species was found in Lake Cunningham is not too unusual. It could have been transported there by humans, or the fish could have found some other way into the lake, since their natural range does overlap with the Bahamas. There is no evidence to suggest that this new fish will be of any threat to Cyprinodon variegatus, even though they will be competing for the same food sources.
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