Assessing the Phylogenetic Utility of DNA Barcoding Using the New Zealand Cicada Genus Kikihia

DNA Barcoding (Hebert et al. 2003) has the potential to revolutionize the process of identifying and cataloguing biodiversity; however, significant controversy surrounds some of the proposed applications. In the seven years since DNA barcoding was introduced, the Web of Science records more than 600 studies that have weighed the pros and cons of this procedure. Unfortunately, the scientific community has been unable to come to any consensus on what threshold to use to differentiate species or even whether the barcoding region provides enough information to serve as an accurate species identification tool. The purpose of my thesis is to analyze mitochondrial DNA (mtDNA) barcoding’s potential to identify known species and provide a well-resolved phylogeny for the New Zealand cicada genus Kikihia. In order to do this, I created a phylogenetic tree for species in the genus Kikihia based solely on the barcoding region and compared it to a phylogeny previously created by Marshall et al. (2008) that benefits from information from other mtDNA and nuclear genes as well as species-specific song data. I determined how well the barcoding region delimits species that have been recognized based on morphology and song. In addition, I looked at the effect of sampling on the success of barcoding studies. I analyzed subsets of a larger, more densely sampled dataset for the Kikihia Muta Group to determine which aspects of my sampling strategy led to the most accurate identifications. Since DNA barcoding would by definition have problems in diagnosing hybrid individuals, I studied two species (K. “murihikua” and K. angusta) that are known to hybridize. Individuals that were not obvious hybrids (determined by morphology) were selected for the case study. Phylogenetic analysis of the barcoding region revealed insights into the reasons these two species could not be successfully differentiated using barcoding alone.

A Study of the Molecular Basis of Microvariants at the FGA and D21S11 Loci Used in Forensic DNA Testing

Microvariant alleles, defined as alleles that contain an incomplete repeat unit, often complicate the process of DNA analysis. Understanding the molecular basis of microvariants would help to catalogue results and improve upon the analytical process involved in DNA testing. The first step is to determine the sequence/cause of a microvariant. This was done by sequencing samples that were determined to have a microvariant at the FGA or D21S11 loci. The results indicate that a .2 microvariant at the D21S11 locus is caused by a -TA- dinucleotide partial repeat before the last full TCTA repeat. The .2 microvariant at the FGA locus is caused by a -TT- dinucleotide partial repeat after the fifth full repeat and before the variable CTTT repeat motif. There are several possibilities for the reason the .2 microvariants are all the same at a locus, each of which carry implications on the forensic community. The first possibility is that the microvariants are identical by descent, which means that the microvariant is an old allele that has been passed down through the generations. The second possibility is that the microvariants are identical by state, which would mean that there is a mechanism selecting for these microvariants. Future research studying the flanking regions of these microvariants is proposed to determine which of these possibilities is the actual cause and to learn more about the molecular basis of microvariants.