Characterisation of bacteria associated with the root nodules of Hypocalyptus and related genera

Abstract

Until recently, most of the legumes that have been studied in South Africa were known to be nodulated by diverse alpha-rhizobia in the class Alphaproteobacteria. Our knowledge regarding the occurrence of so-called beta-rhizobia were limited and restricted to Aspalathus and Cyclopia species. The aim of this study was, therefore, to explore the diversity and evolution of the root-nodule bacteria of various papilionoid legumes indigenous to southern Africa. By making use of housekeeping gene sequence information, the research presented here showed that all 69 of the bacteria isolated from the root-nodules of species in the genera Hypocalyptus, Virgilia, Podalyria and Cyclopia represented beta-rhizobia in the genus Burkholderia (class Betaproteobacteria). Based on these DNA sequences, the isolates could be assigned to 25 independent lineages that most probably represent distinct species. With the exception of one group that tended to associate with B. tuberum in my phylogenies, the majority of these lineages or species appeared to be new to science as they did not group with any of the known diazotrophic and/or nodulating species. Phylogenetic analyses of the nifH and nodA gene sequences also separated the isolates into a number of groups, but surprisingly the groups recovered with these two gene regions did not match, nor did they match with those inferred using housekeeping gene sequences. In general, there was only one exception where the same group of isolates were recovered from phylogenies inferred for the various loci. These findings thus suggested a significant impact of horizontal gene transfer on the evolutionary histories of the determinants of nodulation and nitrogen-fixation in these bacteria. The phylogenetic groups recovered from the various sequences also did not match those expected based on the host or geographic origin of the isolates. However, isolates from the South African legumes generally appeared to group separate from those isolated in other parts of the world. The distinctness of the South African isolates was most pronounced in the nifH and nodA gene trees, where they formed a well-supported cluster separate from all of isolates associated with Mimosa species elsewhere, which suggest a unique and possibly African origin for the root-nodule bacteria examined in this study. The findings presented in this dissertation thus present an important contribution to our understanding of the diversity and evolution of these bacteria from both a Southern African and a global perspective.