School of BioSciences - Theses
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ItemResolving the evolutionary history of the Australian grass trees, Xanthorrhoea (Xanthorrhoeaceae), using multiple high-throughput sequencing techniques( 2016)Xanthorrhoea (Xanthorrhoeaceae subfamily Xanthorrhoeoideae) is an iconic component of the Australian flora, occurring in heathlands, woodlands and dry sclerophyll forests in eastern and south-western Australia. The last revision of the genus in 1986 recognised 28 species and five segregate subspecies. However, Xanthorrhoea taxonomy can be challenging due to limited variation in vegetative morphology; identification relies primarily on continuous characters which vary within and between species. Geographical information is often heavily relied upon for identifications, yet some species have overlapping distributions. A comprehensive molecular phylogeny incorporating all currently described species and infraspecific variants is required to understand relationships between species, biogeographical patterns, and to resolve taxonomic issues in Xanthorrhoea. High throughput sequencing is significantly changing the way plant systematics research is done; the sheer volume of genetic data obtainable at a low cost per base pair allows for greater sample numbers and more thorough investigation of evolutionary relationships. In this thesis, I use multiple high-throughput sequencing methods to generate nuclear and plastid datasets for resolving evolutionary relationships in Xanthorrhoea. Targeted amplicon sequencing was performed on over 150 samples of Xanthorrhoea for eight plastid loci, forming a detailed phylogeographic investigation of a widespread genus across the Australian mesic zone. Three distinct haplotype groups were identified: one mostly in Western Australia; one in south eastern Australia; and one in north eastern Australia. The divergence between eastern and western populations is suggested to be caused by vicariance events associated with the formation of the arid zone. The approximate geographic location of the genetic border between north eastern and south eastern Australian haplotype groups is near the Southern Transition Zone in eastern New South Wales, which has been identified as a region of genetic distinction in several plant and animal lineages. Several Queensland samples had haplotype sequence related to Western Australian samples, which is likely caused by failure of widespread ancestral plastid variation to coalesce within a species or geographical region. Targeted amplicon sequencing of four low copy nuclear genes displayed the feasibility of this method for separating allelic variants in heterozygous samples. Phylogenies based on alignments of the individual phased loci (i.e., including all allelic variants for an individual) indicated incomplete lineage sorting, with allelic variation within species, widespread sharing of alleles between species, and a lack of geographic structure. However, phylogenetic analysis methods that utilised both the allelic variation within a locus and combined data from all four loci resolved phylogenies showing some taxonomic and geographic structure. Analysis of unphased loci (i.e., alleles within an individual not distinguished) showed a complete lack of resolution, with most of the species resolved as a polytomy. While a fully resolved phylogeny of Xanthorrhoea was not obtained, results from this study show the value of considering the allelic variation of nuclear loci, and indicate that incomplete lineage sorting is a major factor affecting accurate resolution of a phylogeny for the genus. Restriction associated DNA sequencing, (RADseq) is a powerful method for obtaining thousands of loci from throughout the genome of an organism. A modified double-digest RADseq method was used to explore relationships among the Western Australian species. Three clades of WA taxa were identified, and relationships between taxa were well-resolved, except for X. preissii and X. brunonis; these two species together formed a well-supported clade, but genetic variation within the clade did not correlate with current species limits as defined by morphological characters. Scanning electron microscope images of leaf micromorphology indicated some morphological variation useful to distinguish clades and some species. The phrase name entity X. sp. Lesueur was supported as a distinct genetic lineage worthy of recognition, and will be published as Xanthorrhoea lateritica1. A PCR based reduced representation library method, using random primer sequence to amplify multiple loci from the genome was developed to resolve the phylogeny of Xanthorrhoea. Over 3000 loci were sequenced and used to resolve the phylogeny of Xanthorrhoea. Rogue samples that occupy multiple positions within a set of bootstrap trees caused a significant reduction in phylogenetic resolution. Removing rogue samples increased phylogenetic resolution, identified seven clades of taxa, and clarified relationships among species. Rogue samples were not related to sequencing or locus filtering issues, and may be due to incomplete lineage sorting or hybridisation. Three clades of eastern Australian species were identified; these clades were supported by leaf surface micromorphology and geography. In all datasets, determining genetic relationships between species or geographical regions was difficult in Xanthorrhoea: this is attributed to evolutionary factors such as incomplete lineage sorting and hybridisation, or low sequence variation, as opposed to methodological issues. Explanations for the effect of these factors in Xanthorrhoea were related to life history traits, including a long generation time, fire stimulated flowering, large population sizes, and recent speciation events associated with environmental changes during the Quaternary. Several taxonomic issues arising from the results of this research are discussed, including taxa requiring new circumscriptions or reappraisal.