Zola Msiska

Date of Graduation


Document Type



Arbuscular mycorrhizal fungi (AMF) associating with 80% of vascular plant families increase uptake of nutrients and water in plants, help to stabilize soil aggregates and improve soil aeration, water percolation and carbon storage. Phylogeny of AMF based on rDNA sequences is in conflict with morphology-based phylogeny. The aim of the present study was to examine a protein encoding gene, β-tubulin, in order to test conclusions drawn from rDNA sequences and morphological data. Inverse-PCR was used to amplify full-length β-tubulin genes of Gigaspora gigantea and Glomus clarum. The gene in both species is organized into five exons and four introns encoding a 447 amino acid protein. In comparisons with other fungal groups, the amino acid sequence is most similar to that of fungi in Chytridiomycota. A striking difference between AMF β-tubulin genes and those of all other fungi is the absence of any intron before codon 174. Introns positioned at codons 174 and 257 in AMF match the position of introns in β-tubulin genes of some Basidiomycete, Zygomycete and Ascomycete fungi. Introns positioned at codons 350 and 421 are unique to AMF. The number of gene copies was determined by Southern hybridization analysis only for Gi. gigantea, where only a single copy was evident. Based on sequences obtained from the two AMF species above, primers were designed to amplify the 3’ end of the β-tubulin gene from 43 species of AMF by nested-PCR approach. The analyses comprised a representative selection of all families except for Pacisporaceae and Geosiphonaceae. Phylogenetic analyses were conducted on sequences excluding intron regions due to sequence and length polymorphisms. The β-tubulin gene phylogeny was identical to the 18S phylogeny at the family and species level but differed at the order level. Sequencing of multiple clones from three isolates of Scutellospora heterogama suggested that this species contains a single copy of the β-tubulin gene. The divergent sequences found in this species were attributed to intraspecific sequence variation. Species limits were investigated within the genus Gigaspora through the phylogenetic analyses of DNA sequences from portions of the β-tubulin, 28S and 18S rRNA genes. The β-tubulin gene trees indicated the division of Gigaspora species into two independent lineages with high support. The two lineages consisted of the Gi. margarita group (Gi. margarita and Gi. decipiens ) and the Gi. rosea group (Gi. gigantea, Gi. rosea and Gi. albida). Trees generated from the 18S rRNA gene resolved the Gi. margarita group but the Gi. rosea group was not resolved. Trees inferred from the 28S rRNA gene and the combined data set indicated the division of the Gi. margarita group into two non-monophyletic clades. Failure to resolve the Gigaspora phylogeny could be due to insufficient isolate and character sampling and failure to distinguish between paralogous and orthologous sequences.