Phylogenetics of Begonia section Gireoudia

Begonia is one of the most species-rich angiosperm genera with c. 1500 species currently identified and a pantropical distribution. Although Begonia are predominantly found in ever wet rain forests, they can also be found in other habitats including dry, desert scrub, and at altitudes from sea level to over 3000 meters. Begonia can also exhibit huge morphological variation between closely related species thus making them an ideal system for the study of the ecology, biogeography and developmental evolution of tropical plants. Previous work carried out at the Royal Botanic Gardens, Edinburgh focused on the phylogenetic framework and biogeographic history of African and Asian Begonias. This work on the Neotropical Begonia Section Gireoudia, aims to complement those studies and to provide a framework for determining how macro- and micro-evolutionary processes have contributed to the high level of diversity in Begonia worldwide. Traditionally used nuclear and chloroplast markers for phylogenetics failed to resolve species level relationships within sect. Gireoudia, therefore an alternative approach using next-generation sequencing technology was developed. A multiplexed, massively parallel sequencing approach was developed to sequence sixteen Begonia chloroplast genomes on the Illumina GAIIx genome analyser in order to identify chloroplast regions with sufficient phylogenetic information to resolve a species-level phylogeny. The lack of a reference chloroplast genome sequence for Begonia led to the development of a new method combining sequence from conserved angiosperm chloroplast genome sequences together with long-range PCR to generate the samples. Eighteen, overlapping long-range PCR amplicons for each Begonia species were used in a multiplexed sequencing reaction on an Illumina GAIIx and the chloroplast sequence reads were assembled using a de novo approach. A selection of potentially, phylogenetically informative regions, determined from the large-scale chloroplast alignment generated during this study, were assessed. Two of these regions were chosen for further phylogenetic analysis and resulted in improved resolution of American Begonia, sect. Gireoudia. This study successfully demonstrates a new innovative approach to that normally taken in traditional molecular systematics. The research presented provides a framework for the development of new molecular markers that are suitable for low-level phylogenetic studies, especially where recent radiations make resolution of species groups difficult, such as Begonia. New sequencing technologies such as those used here will provide powerful new tools for students of molecular evolution, phylogenetics and taxonom