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ItemA genetic approach to the conservation of holly leaf grevilleas (Proteaceae)( 2016)The holly leaf grevilleas consist of an informal aggregate of 15 species found in south-eastern Australia. The group exhibits high levels of morphological variation and the most widespread species, Grevillea aquifoilum Lindl., is also the most variable. Most species are restricted endemics and their geographic limits make them vulnerable to the effects of fragmentation and environmental change. In some species, production of viable seed is unknown or has not been confirmed. Identifying factors that contribute to the persistence of species when fecundity is low is of critical importance to their conservation. Here, a phylogenetic analysis is used to clarify the evolutionary relationships among lineages within the holly leaf grevilleas. The lineages identified are then the basis of chapters 4 – 6 that address questions of what constitutes the units of conservation and how clonal plants should be assessed. Analysis of 12 cpDNA regions strongly supported the more southerly distributed holly leaf grevilleas as a monophyletic group comprising four clades (‘G. aquifolium’, ‘G. dryophylla’, ‘G. repens’, ‘G. ilicifolia’). The two northern holly leaf grevillea species, G. renwickiana and G. scortechinii, found in New South Wales and southern Queensland, were not positioned with the southern species but their relationship with outgroup species G. willisii from northeastern Victoria and G. acanthifolia and G. laurifolia from New South Wales could not be ascertained with confidence. Two nuclear regions, PHYA and waxy1, were less variable and not analysed in combination with cpDNA. PHYA was largely uninformative with most species forming a polytomy. Two major variants were identified in waxy1 and consisted of one functional and one non-functional copy based on DNA translation. Minor alleles of functional and non-functional copies were present in some accessions. Using only the functional copy (including multiple alleles when present), the southern ‘G. ilicifolia’ clade, as identified from cpDNA, was clearly differentiated from the northern clade and the remaining species. Within the southern species, those not belonging to the ‘G. ilicifolia’ clade were grouped together but clades identified in the cpDNA phylogeny were not recovered in the waxy1 analysis. Incongruence between the phylogenetic placement of some taxa and current species assignment based on morphology, including apparent paraphyly of G. aquifolium, may indicate an evolutionary history of hybridisation, introgression and incomplete lineage sorting and/or the use of morphological characters that are not lineage-specific. For example, the two subspecies of G. montis-cole differentiated morphologically on the basis of style-length are positioned in different clades and warrant specific rank if supported with nuclear data, and G. steiglitziana is split between two lineages within the southern ‘G. dryophylla’ clade. The phylogenetic placement of G. ‘williamsonii’, a taxon no longer recognised, with sympatric G. aquifolium, coupled with microsatellite analysis supports the current taxonomic view of its synonymy with G. aquifolium. The cpDNA phylogeney also raises questions about the taxonomy of G. microstegia and G. bedggoodiana, taxa that are also positioned with G. aquifolium in the ‘G. aquifolium’ clade. Population genetic analyses of G. infecunda and G. renwickiana found both species to be comprised of a small number of clonal lineages with no evidence of contemporary sexual reproduction. Within species, no clones were found at multiple locations and cpDNA haplotypes were derived from single lineages. In G. infecunda, 38 clonal lineages were identified from a microsatellite analysis of 280 samples from 11 populations. The number of clones present per population ranged from 1 to 11 and clone size varied from a single stem to several hectares. In G. renwickiana, analysis of 197 samples revealed that all but one of seven populations were monoclonal. Clones were distributed over a minimum area of one hectare. Sequencing of microsatellite alleles showed that variation in allele size profiles among clonemates could be interpreted as somatic mutation. The genetic patterns evident in the two species are likely to be the result of a loss of sexual reproduction, due to pollen sterility in G. infecunda and the effects of triploidy in G. renwickiana. For the clonal taxa, G. infecunda and G. renwickiana, lack of sexual reproduction leaves little opportunity for adaptation or migration in response to changing conditions. However, to facilitate the adaptive responses of ecological communities rather than individual species, conservation should encompass obligately clonal species because of their role, albeit finite, in mitigating ecological instability as floras respond to rapidly changing environments.