Nathaniel Ralston and Dr. Keith Crandall, Biology
Crustaceans are some of the richest multi-cellular animals in the world in terms of species. Within the crustaceans the decapods are chief in species numbers and diversity with over 15,000. Among these are the very familiar and well-known species, such as crayfish, shrimp, and crabs, although there also are many lesser known groups as well. The current classification tells us that there are approximately 62,000 species of Crustacea in the world among 849 families. Approximately 152 of those families belong to the Decapoda. To add to this, the decapods have been referred to as “the pinnacle of crustacean evolution.” Knowing this, the importance of understanding as much as possible about this order can easily be seen.
There are many different hypotheses that have been proposed by many different researchers to explain the phylogeny, or evolutionary history, of the decapods. However, there is no consensus between these hypotheses. Previously the decapods were divided in two groups: the Natantia (swimmers) and the Reptantia (crawlers). However, other studies have reorganized the Decapoda into even more groups and subdivisions. For this reason the Crandall laboratory has taken part in a study known as the Decapod Tree of Life, which is centered on researching the phylogeny of the Decapoda.
While the Crandall laboratory as a whole took on the task of sequencing approximately 20 genes for comparison across the 152 extant families of decapods, the purpose of my research was to test a set of nuclear PCR primers to obtain the data necessary for reconstructing a robust estimate of evolutionary relationships among the decapods. Previous to commencing this project I aided in sequencing the 28S and 18S genes for several samples. These, however, are mitochondrial markers. These are useful for our research, but there are disadvantages when using them. Mitochondrial markers are known as fast markers, meaning that they have a fast rate of mutation or evolution. This is helpful when studying differences amongst species, but can be less useful when comparing families. Nuclear markers are beneficial in that they have a slower rate of mutation, therefore giving us the ability to not only compare families, but also to view them over a longer period of time. When studying evolutionary history, this is vitally important.
As I stated, my assignment was to work with these nuclear markers. In my work there were three primary nuclear markers with which I worked: elongation factor-1alpha (ef-1), polymerase II, and hemo. Another important nuclear marker that was used, although not by I, was elongation factor-2 (ef-2). These primers were developed previously by a separate group for this particular subclade of crustaceans; for this reason we decided to use them. It was my hope that these nuclear markers would sequence well across different families so as to provide us with a clearer genetic picture of their similarities. This brings us to the methods I used to carry out the research.
The first thing that I did was choose seven prime DNA samples of specimens from seven different families within the order Decapoda. I was careful to verify that the samples I chose had all previously been extracted and sequenced across various genes in order to know that they were rich in DNA. However, all of the samples were low (around 3-20 micro liters each) so I genomiphied (a process that amplifies a small amount of DNA to give the researcher more sample to use) them so that I would have plenty with which to experiment. Once I had enough to use, I began testing the primer ef-1. A PCR of the DNA at a 1 to 4 dilution resulted in multi-banding of all the samples across the entire gene (Fig. 1). This means that the primer was amplifying more than just the ef-1 gene. The next step for testing this part was to perform a gel extraction of only the band that I wanted and to clone that portion. Sequencing the cloned portions of DNA gave me the desired result; however, the cloning procedure is a very time consuming one and because of such is undesirable. Therefore, we decided against using the ef-1 gene.
The next gene I tested was hemo. A PCR of the DNA at a 1 to 10 dilution gave us multi-banding once again of all the samples. Unlike the ef-1 gene, though, this one seemed to give less multi-banding and so with high hopes I tried sequencing them. This only produced an illegible sequence, so we decided to try a PCR gradient to find a better annealing temperature rather than clone. The best temperature was found to be 50 degrees Celsius and so I re-sequenced the samples. The outcome was the same as before, so we cloned the samples to discover that we could find a clean sequence. Since cloning seemed to be the only reliable method to obtain our data, we decided not to use the hemo gene anymore.
Finally, I tested the polymerase II gene. A PCR of the DNA at a 1 to 10 dilution gave me multi-banding of all the samples, but not all the samples worked across the whole gene. After a re-amplification it was illustrated to us that this gene would also be too problematic to use.
Through my research we discovered that new nuclear primers need to be modeled to better suit our purpose. I expect, though, that since these primers worked relatively well across taxa that we will be able to acquire some that will allow us to quickly and effectively collect the necessary data to finish the Decapod Tree of Life.1
Acknowledgement to Alicia Toon, Ph.D, and Rebecca Scholl for their help without which I could not have done this experiment.