Team #3
Tree of Life Research

Abstract
Constructing a phylogenetic tree using molecular data and bioinformatics techniques was the focus of this research project. The specimens were collected at Island Beach State Park along the New Jersey Shore. Genomic DNA was extracted, the segment of the 18s rDNA gene region was amplified, purified, sequenced, and analyzed to determine relationships among the diverse group of organisms. A phylogenetic tree was constructed that shows relative evolutionary relationships based on similarities in that gene sequence.

Question
Based on the 18S gene region, what are the evolutionary or phylogenetic relationships among the organisms collected at Island Beach State Park in New Jersey.

IMRAD Report: Introduction, Materials & Methods, Results, and Discussion

Digital Notebooks

Introduction

The small-subunit rDNA (SSU rDNA) gene contains regions of sequence divergence. This feature of SSU rDNA is used extensively as a molecular marker to infer the evolutionary relationships among both closely and remotely related taxa (Campbell et al. 1994). SSU rDNA genes of animals usually range from 1800 to 1900 bp, showing small size variations. A wide variety of representative animal species representing different taxa were collected from Island Beach State Park, New Jersey. To study their evolutionary relationships, genomic DNA was isolated, the 18S gene region was amplified using by PCR 18S1F and 18S5R primers. that amplify an approximate 940 bp region in the 18s gene and sequencing the amplified products. We examined the phylogenetic relationships of 14 animal specimens as inferred from 18s rDNA sequences. Hypotheses for the evolution of our representative species were developed within a parsimony framework using the PAUP software program. The monophyly of traditionally recognized major taxonomic groupings was assessed.

Background on Building Phylogenetic Trees

Materials & Methods

Research Process Flow Chart has links to 18S Gene Region, ClustalX Matrix, and the phylogenetic tree.

Specimens were collected on Wednesday, July 16, 2003 at two sites within Island Beach State Park in Seaside Park, New Jersey. Island Beach State Park has designated 3,000 acres of preserved barrier island ecosystems and 10 miles of pure white sand beach that is nestled between the Atlantic Ocean and Barnegat Bay.

Collection Materials: seine net, kick net, and sample tubes

At the Lawrenceville School Laboratory, the team dissected tissue of all the specimens, photographed, labeled, created a key of each specimen for identification purposes, washed, and froze extra tissue samples for future analytic study. We created a digital spreadsheet and coded each organism for DNA analysis

Results

We obtained genomic DNA from all 25 animal specimens collected at Island Beach State Park. PCR amplification using primers 1F and 5R resulted in 19 successful amplified products. Figure 1 shows the sizes of the resulting PCR fragments. The size of the fragment varied. Lane one, the isopod had a longer size fragment than the other specimens. Lane 6 BY21 produced two different size fragments. Fourteen samples were cycle sequenced in a thermocycler to incorporate fluorescent dyes.

These specimens have accession numbers and are in storage at the Ambrose Monell Cyro-Collection at the American Museum of Natural History in New York City.

Figure 1 We ran 1% agarose gels to verify the presence of DNA. Fourteen specimens were PCR amplified with 18S rDNA forward and reverse primers (18S 1F and 18S5R). In the left figure, the larger sized band is approximately 750 bp and the smaller sized band is 600 bp.

Discussion

The initial specimen identifications and the BLAST specimen identifications were similar for most of the samples, however there are some discrepancies. The scraping from the clam shell that was sequenced was identified by Blast search as a hermit crab. In hypothesizing, it is possible that some of the DNA from the lane next to the clam was sequenced instead of the clam. Another possibility because only half of the shell was collected and the group took a scraping from that, is that perhaps the clam shell had hermit crab DNA on it when collected, which was then sequenced. In attempting to identify the specimens collected, the team hypothesized that we had collected a few similar specimens of the isopods, but perhaps of different species. With further analysis, the hypothesis was not supported according to the BLAST results, which both came back with the same scientific name.The group recognized that most of the phylogenic tree created made sense according to our findings. The crabs are all grouped together on the tree (paraphyletic), as are the mollusks, arthropods, and Ostyichithese (bony fish). Some of the specimens were represented by a single species, such as the fly, making it impossible to test the monophylogeny of these groups.

Gel Maps for 12 Wells

PCR Preparation

Gene Cleaning and Sequencing