Samenvatting

Recent research has shown that many chloroplast markers commonly used for the identification of species in land plants are not useful in the moss genus Campylopus. The only known suitable marker in Campylopus is the nuclear ribosomal ITS1-5.8S-ITS2 region. However, evolutionary patterns of this region seem to be more complicated in Campylopus than in other genera, since the internal transcribed spacers ITS1 and ITS2, and possibly also parts within each spacer, may evolve separately. To improve species delimitation in Campylopus, the aims of this study were to (i) identify several Campylopus specimens from Asia based on ITS sequences (ii) compare phylogenetic patterns between different helices of the secondary structures of the ITS and (iii) develop and test new chloroplast primers. The DNA was isolated using three methods: Kit extraction, CTAB extraction and Kingfisher robot extraction. The extracted DNA of the kit and CTAB extraction was amplified using primers M13-ITS2-5.8F/25R, for the ITS2 region, to determine if these methods showed different results. A 96-well plate was extracted using the Kingfisher robot, these samples were amplified with primers M13-ITS-18F/25R for the complete ITS region. The successfully amplified samples were Sanger sequenced, edited and aligned in an already existing alignment containing various Campylopus species. This alignment was used for the identification of several specimens with the complete ITS region and the analysis of the secondary structure of the ITS2 region. The phylogenetic analysis, based on Bayesian Inference, showed that the identification for most species is difficult with only the information of ITS. After phylogenetic analysis of the four main helices of the secondary structure of ITS2, it was concluded that there is no incongruence between them. For designing new chloroplast primers, the genome of Syntrichia ruralis was used. In total 5 primer sets were designed and tested. These primer sets all showed non-specific bands, even after extended testing, resulting in no new chloroplast primers for the genus Campylopus.