Concerted evolution of the rDNA ITS-1 in two caddisfly species : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Ecology at Massey University

The ribosomal RNA (rRNA) gene family shows high levels of within-species homogeneity and between-species diversity. This pattern of mutation distribution among gene family members is termed concerted evolution. Molecular drive is a process by which mutations spread through a gene family (homogenization) and through a population (fixation) as a consequence of non- Mendelian DNA transfer within and between chromosomes. The fast evolving internal transcribed spacers (ITS) of the rDNA are considered to be homogenized within species and within genetically isolated populations. However, recent studies question the dogma of homogenization and caution the use of ITS in phylogenetic and population studies. Two presumed sister species (Triplectides obsoleta and T. dolichos) were chosen for an investigation of ITS-1 variation. ITS-specific PCR primers were developed that amplified products homologous to a range of other insects. In a pilot study, variation in a single individual T. obsoleta was found among eight ITS-1 clones but not in ITS-2. Therefore, ITS-1 was chosen for a survey of the nature of the variation, testing the extent of heterogeneity in these species, the types of coevolutionary changes taking place in this region, and its usefulness as a population genetic marker. This survey required an hierarchical approach, sampling multiple ITS-1 copies from within and among individuals. 203 ITS-1 clones were screened for conformational differences by single-strand conformation polymorphism (SSCP) analysis of restriction digested fragments. Some ITS-1 variants were present at a higher frequency than others in all 17 individuals tested. All variants derived from independent PCR reactions were sequenced. The type of DNA sequence variation was very similar in both species though T. dolichos was more variable. A majority of the variation appears to have resulted from replication slippage and the remainder by point mutation. Repeat DNA and an A×T bias were found in the ITS-1; a characteristic of cryptically simple non-coding DNA. Extensive ITS-1 heterogeneity was present and parsimony analysis for both species showed the absence of population-specific variation. ITS-1 variants common in some individuals were rare in others, even within the same population. Neither complete homogenization nor fixation of the ITS-1 occurs in these species. This precludes its use as a population genetic marker in these species and cautions against the assumption of homogenization