Time travelling weevils: Unravelling the evolutionary past of Hadramphus using historical and modern DNA

Museum specimens are of increasing importance to the study of ecology, evolution, phylogenetics and conservation biology. In this thesis I explore the use of museum specimens to analyse population changes and declines in the endangered weevil genus Hadramphus over the past 125 years. Molecular phylogenetics aids our understanding of species taxonomy by including the neutral evolutionary history of a species’ genetic code rather than relying on morphological characteristics alone. The phylogeny of the genus Hadramphus was obtained using the mitochondrial gene cytochrome c oxidase subunit I (COI) and the ribosomal nuclear internal transcribe spacer 2 (ITS2). A multi-locus multi-species coalescent approach was used for building a species tree from the gene trees. Hadramphus pittospori was highly divergent from the other three species in the genus, suggesting the current genus is paraphyletic. A study of the phylogeography of Megadromus antarcticus was conducted using COI and ITS2. A Bayesian skyline plot was calculated to determine if the effective population size of M. antarcticus has changed. The population of M. antarcticus showed no decline, and diverged during the Pleistocene. The recent divergence fits with the biogeography of the Canterbury Plains which were heavily affected by the glacial cycles during the Pleistocene. The critically endangered Hadramphus tuberculatus was last sampled in 1922 and presumed extinct until its rediscovery in 2004. After severe range reduction from loss of habitat and the introduction of mammalian predators, the only known remaining population is at Burkes Pass Scenic Reserve. From 2009-2011, the reserve was surveyed for the weevil using visual searches and pitfall traps. Mark recapture was applied to determine population size, movement, and survivability. Analysis of the mark recapture data showed a large year to year variation in the estimated population sizes from 2009 to 2011. The data also showed that the weevil was highly mobile throughout the reserve, covering distances up to 190 metres. DNA from pinned museum specimens of Hadramphus tuberculatus has also been collected so that populations can be compared over time to map the loss of genetic diversity for the species. Non-destructive methods were used to extract DNA from specimens of H. tuberculatus collected from 1890-1922. Primers were designed to amplify short fragments of the COI mitochondrial gene (135 bp). Sequences were aligned and compared to COI sequences obtained from modern samples. Preliminary results suggest variation between modern and historic samples and a loss of nucleotide diversity during the past 100 years. A reduced representation library of genomic DNA for H. tuberculatus and H. spinipennis was built for next generation sequencing (454) to find single nucleotide polymorphism (SNP) for population genetic studies using both historic and modern samples. A method of SNP genotyping using high resolution melting (HRM) for both modern and historical weevil specimens continues to be investigated.