Melissa E. Hardy and Dr. Keith Crandall, Zoology
Phylogenetic trees are an estimate of genealogical relationships among groups of organisms. They are reconstructed by analyzing homologous nucleic acid sequences from different organisms and deriving an evolutionary relationship from the similarity in sequences.
Ribosomal DNA consists of gene clusters tandemly repeated on a chromosome. Each cluster contains three genes, which are separated by two regions of DNA, called the internal transcribed spacers (ITS1 and ITS2). These regions of DNA have the potential to be useful phylogenetic tools, if the tandem repeats have been competely homogenized by concerted evolution. However, the utility of this sequence to resolve evolutionary relationships is invalidated if a single organism contains ITS regions with different base pair sequences (Hillis and Davis; Vogler and DeSalle 1994). In recent years, these ITS regions have been used widely for phylogenetic analysis in a number of different organisms without being adequately tested for the homogenization of the repeated gene clusters (Fritz et al. 1994; Schlotterer et al. 1994).
This study used samples from four populations of three species of crayfish to evaluate whether ITS sequences were a suitable phylogenetic tool. Samples of two populations of Orconectus luteus, one from the Meramec River and one from the Niangua River, were compared to samples from Orconectus punctimanus, which belongs to the same genus but is a different species, and Procambarus clarkii, which belongs to the same family but a different genus.
DNA was first extracted from each sample and amplified using polymerase chain reaction (PCR). Next, the region in question was cloned and re-extracted. However, cloning eventually proved to be an unsatisfactory method because extraction from the cloned sample produced an impure yield. Therefore, another method, PCR with specialized primers, was utilized to purify the ITS regions for sequencing. The regions were then sequenced with an AVI 377 sequencer.
Preliminary results from sequences obtained seemed to indicate that not all ITS regions in a single individual were completely homogenized by concerted evolution. There were minute differences between sequences. Despite the differences, however, the sequences tended to reflect the known phylogeny. Sequences from the two O. luteus populations were most similar to each other, and more similar to O. punctimanus than to P. clarkii. However, because all samples were not analyzed, this project is not complete. More research is needed to further define the role of ITS sequences in resolving phylogenetic relationships.
References
- Fritz, G. N., Conn, J., Cockburn, A., and Seawright, J. (1994). Sequence analysis of the ribosomal DNA internal transcribed spacer 2 from populations of Anopheles nunztovari. Mol. Biol. Evol. 11:406-416.
- Hillis, D. M., and Davis, S. K. (1988). Ribosomal DNA: Intraspecific polymorphism, concerted evolution, and phylogeny reconstruction. Syst. Zool. 37:63-66.
- Schlotterer, C., Hauser, M., Haesler, A., and Tautz, D. (1994). Comparative evolutionary analysis of rDNA ITS regions in Drosophila. Mol. Biol. Evol. 11:513-522.
- Vogler, A. P. and DeSalle, R. (1994). Evolution and phylogenetic content of the ITS-1 region in the tiger beetle Cicindela dorsalis. Mol. Biol. Evol. 11:393-405.