Background: Long-read sequencing platforms offered by Oxford Nanopore Technologies (ONT) allow native DNA containing epigenetic modifications to be directly sequenced, but can be limited by lower per-base accuracies. A key step post-sequencing is basecalling, the process of converting raw electrical signals produced by the sequencing device into nucleotide sequences. This is challenging as current basecallers are primarily based on mixtures of model species for training. Here we utilise both ONT PromethION and higher accuracy PacBio Sequel II HiFi sequencing on two plants, Phebalium stellatum and Xanthorrhoea johnsonii, to train species-specifc basecaller models with the aim of improving per-base accuracy. We investigate sequencing accuracies achieved by ONT basecallers and assess accuracy gains by training single-species and species-specifc basecaller models. We also evaluate accuracy gains from ONT’s improved fowcells (R10.4, FLO-PRO112) and sequencing kits (SQK-LSK112). For the truth dataset for both model training and accuracy assessment, we developed highly accurate, contiguous diploid reference genomes with PacBio Sequel II HiFi reads.

Results: Basecalling with ONT Guppy 5 and 6 super-accurate gave almost identical results, attaining read accuracies of 91.96% and 94.15%. Guppy’s plant-specifc model gave highly mixed results, attaining read accuracies of 91.47% and 96.18%. Species-specifc basecalling models improved read accuracy, attaining 93.24% and 95.16% read accuracies. R10.4 sequencing kits also improve sequencing accuracy, attaining read accuracies of 95.46% (super-accurate) and 96.87% (species-specifc).

Conclusions: The use of a single mixed-species basecaller model, such as ONT Guppy super-accurate, may be reducing the accuracy of nanopore sequencing, due to conflicting genome biology within the training dataset and study species. Training of single-species and genome-specifc basecaller models improves read accuracy. Studies that aim to do large-scale long-read genotyping would primarily benefit from training their own basecalling models. Such studies could use sequencing accuracy gains and improving bioinformatics tools to improve study outcomes.

This dataset includes molecular data and associated metadata supporting the analysis of Prostanthera volucris (Lamiaceae) as a new, distinct species in the Central Tablelands. Samples of 3 taxa for this study were collected from across New South Wales, Australia.

Analysis code can be found at https://github.com/rpodonnell/ASB_PEC

Closely related species often use the same genes to adapt to similar environments. However, we know little about why such genes possess increased adaptive potential and whether this is conserved across deeper evolutionary lineages. Adaptation to climate presents a natural laboratory to test these ideas, as even distantly related species must contend with similar stresses. Here, we re-analyse genomic data from thousands of individuals from 25 plant species as diverged as lodgepole pine and Arabidopsis (~300Myr). We test for genetic repeatability based on within-species associations between allele frequencies in genes and variation in 21 climate variables. Our results demonstrate signifcant statistical evidence for genetic repeatability across deep time that is not expected under randomness, identifying a suite of 108 gene families (orthogroups) and gene functions that repeatedly drive local adaptation to climate. This set includes many orthogroups with well-known functions in abiotic stress response. Using gene co-expression networks to quantify pleiotropy, we find that orthogroups with stronger evidence for repeatability exhibit greater network centrality and broader expression across tissues (higher pleiotropy), contrary to the 'cost of complexity' theory. These gene families may be important in helping wild and crop species cope with future climate change, representing important candidates for future study.

The Australian and New Guinean everlasting paper daisy genus Xerochrysum, widely grown around the world as Strawflower, Everlasting or Immortelle, was recently revised. The first and type species, Xerochrysum bracteatum, was described in 1803 as Xeranthemum bracteatum from a plant cultivated in the Empress Joséphine's garden at Malmaison. During our project, we learned that Xerochrysum was naturalised in St. Helena, the place of Napoleón Bonaparte's last exile, and is presumed to have been introduced by him. We conducted genetic analyses to test the derivation of the St. Helena plants. Comparisons with some colourful hybrid cultivars and naturally occurring populations in Australia found the St. Helena plants to be most similar to Xerochrysum bracteatum s. str. of the Sydney Basin. The St. Helena plants were without admixture of another species as would be expected if they were more recently escaped ornamental cultivars. Our results support the conclusion that the St. Helena Xerochrysum were introduced during Napoleón's exile, likely as a reminder of his wife's garden.

A taxonomic classification that captures both the evolutionary history and the morphological diversity of a group of organisms lays an essential foundation for studies on their biology, and for better management and conservation of the named species. The urgent importance of science-informed conservation management cannot be overstated amidst the global species extinction crisis, especially in Australia, which is both megadiverse and losing that diversity through species extinctions at a high rate. A comprehensive, integrative taxonomic revision of understudied groups, such as Melichrus R.Br. is a potent means of assuring species are discovered, described, named and ultimately conserved.

Melichrus R.Br. (Ericaceae: Epacridoideae) is a genus of shrubs endemic to Australia, with a widespread distribution in mainland eastern Australia (four species) and a more geographically constrained presence in south-western Western Australia (three phrase-named taxa). Prior to this study, Melichrus had received very little taxonomic study, and its few treating authors largely disagreed on species delimitation. The eastern Australian clade of Melichrus was last revised in 1958.

This thesis investigated species boundaries in the eastern Australian Melichrus (Chapter 3) and the phylogenetic relationships amongst all species of Melichrus (Chapter 4), leading to a new revised taxonomy based on in-depth morphological and molecular evidence (Chapters 2 & 5).

A thorough morphological dataset was constructed (90 individuals from 68 populations, for 26 characters) for analysis by NMDS ordination with Bayesian Inference cluster modelling (Mclust) and UPGMA hierarchical clustering to identify morphological discontinuities in the genus that could indicate species boundaries. Discontinuities in the morphological analyses were compared to those apparent in ordination and clustering (Principal Component Analysis, SplitsTree Neighbour-Net, STRUCTURE and ConStruct) and statistical analyses (H_E, H_O, F_ISand F_ST) of a DArTseq SNP dataset of 548 samples from 110 populations of Melichrus. This new evidence was interpreted to provide detailed recommendations for a revised species taxonomy of Melichrus (Chapter 3, Table 5).

The first species-level phylogenetic trees for Melichrus were inferred from two independent genomic datasets: a DArTseq SNP dataset of 557 samples and 4,100 high quality SNP markers and a target capture dataset of 34 samples of Melichrus based on the Angiosperms353 bait set. The phylogenetic trees inferred from these complementary datasets were used to test the sister relationship between the eastern and western groups of Melichrus, the monophyly of species limits proposed in Chapter 3, and to gain a deeper understanding of the evolutionary relationships among species of Melichrus. HybPhaser was used to identify the parental lineages of a putative hybrid Melichrus sp. Mareeba (Chapter 4).

I considered all available evidence to revise Melichrus, which included the description of eleven new species, clarification of the boundaries of previously described species, and the rectification of a longstanding nomenclatural misapplication (Chapters 2 & 5). In Chapter 2, two long-standing, APC accepted, phrase-named species were formally named and described as, Melichrus hirsutus J.B.Williams ex H.T.Kenn. & I.Telford and Melichrus gibberagee J.B.Williams ex H.T.Kenn. & J.J.Bruhl. Both species are gazetted as Endangered at the State and Federal levels and were therefore prioritised for description early in this study. Chapter 5 brings together the morphological and molecular evidence produced in Chapters 3 and 4 to describe nine new species. Melichrus urceolatus R.Br. was recircumscribed so that it is no longer paraphyletic and is reliably recognisable by its morphology. The long-standing misapplication of the name Melichrus adpressus A. Cunn. ex DC. was rectified. A dichotomous key to species is included in Chapter 5 with additional resources for identification, including an online key and curated photographs that will be deployed in the Flora of Australia (see Appendix 1).

A revised taxonomic classification for Melichrus is produced in this study on the basis of strong new evidence, rectifying many of the long-standing taxonomic and nomenclatural problems inherent in the status quo taxonomy. The improved understanding of species limits and their phylogenetic relationships establishes a strong foundation for future biological research, including further systematics studies. Through the development of identification aids, the results of this research are now accessible and usable for a wide range of stakeholders, in turn facilitating better conservation outcomes for species of Melichrus in eastern Australia.

This thesis investigated the taxonomy, evolutionary relationships, ploidy-level and genome size in the Australian native paper daisies Xerochrysum, Coronidium and Helichrysum leucopsideum. The origins of the popular ornamental cultivars of X. bracteatum, grown worldwide since the late 1800s, and naturalised plants on the island of St Helena reputedly introduced by Napoleon Bonaparte, were investigated (Chapter 2). Our results revealed the cultivars to be hybrids between X. bracteatum and X. macranthum , with no evidence of X. macranthum in the ancestry of the St Helena island populations, supporting the hypothesis of their introduction by Napoleon.

We addressed long-standing taxonomic confusion in the X. bracteatum species complex, and in Coronidium sens. lat ., with morphological and molecular data (Chapter 3 and Chapter 5). Using single nucleotide polymorphism data to corroborate morphological differences, we delimited 12 additional species of Xerochrysum, and four new species in Coronidium sens. lat . The data also supported the recircumscription of X. bicolor to include the recently described X. halmaturorum . The distribution, habitat and conservation status are discussed, and keys to genera and to all species are provided (Chapter 3 and Chapter 5).

Evolutionary relationships in the study group were inferred with molecular data, and potential morphological synapomorphies identified (Chapter 4). The analyses supported the recognition of the new genus, Leucozoma T.L.Collins, for eight taxa previously included in Coronidium. Species-level relationships amongst most entities in Coronidium and Leucozoma were well-supported, though relationships amongst species of Xerochrysum were poorly resolved.

We estimated genome sizes in the study group using flow cytometry, confirmed ploidy level using chromosome counts, and placed whole-genome duplication (WGD) events into a phylogenetic context (Chapter 6). Polyploidy was detected in seven species of Xerochrysum representing all major infrageneric clades, and no instances of WGD were detected in Coronidium, Leucozoma or H. leucopsideum . No larger clades have a polyploid ancestor, suggesting that WGD events did not play a role in the deeper diversification of Xerochrysum. Ploidy level provided additional support for the taxonomic recognition of morphologically distinct, tetraploid populations.

Phebalium calcicola, a multi-stemmed shrub from south-eastern South Australia, is segregated from the P. squamulosum subsp. squamulosum assemblage and described here as new, based on phenetic distinctness, habitat preference and geographical disjunction. It is compared with P. squamulosum subsp. squamulosum s.str. from the Sydney region, as well as the geographically closest populations from Victoria, which are currently referred as P. squamulosum subsp. squamulosum. A recommendation for the conservation status of this species as Critically Endangered is proposed. An amended key to species of Phebalium in South Australia is provided.

Golden everlasting paper daisies in the genus Xerochrysum Tzvelev are iconic Australian native plants grown worldwide. The X. bracteatum species complex has been regarded as taxonomically confusing and in need of revision for over 60 years. We applied morphological and molecular analyses to delimit species, detect common ancestry among populations, and identify putative hybrids in the genus Xerochrysum (Asteraceae: Gnaphalieae). Multiple lines of evidence provided strong support for the recognition of new taxa. Here we describe the following 11 new species: X. andrewiae T.L.Collins & J.J.Bruhl, X. berarngutta T.L.Collins & I.Telford, X. copelandii J.J.Bruhl & I.Telford, X. frutescens J.J.Bruhl & I.Telford, X. gudang T.L.Collins & J.J.Bruhl, X. hispidum T.L.Collins & I.Telford, X. macsweeneyorum T.L.Collins, X. murapan T.L.Collins & I.Telford, X. neoanglicum J.J.Bruhl & I.Telford, X. strictum T.L.Collins, and X. wilsonii T.L.Collins, reinstate Helichrysum banksii A.Cunn. ex DC. (as X. banksii (A.Cunn. ex DC.) T.L.Collins & I.Telford), lectotypify X. banksii and X. papillosum (Labill.) R.J.Bayer, and recircumscribe X. bicolor (Lindl.) R.J.Bayer to include X. halmaturorum Paul G.Wilson and some populations of X. bracteatum sens. lat. from mainland South Australia and Victoria. We also provide revised descriptions of all taxa in the genus, their conservation status, a dichotomous key, tables distinguishing closely related taxa and distribution maps.

Golden everlasting paper daisies (Xerochrysum, Gnaphalieae, Asteraceae) were some of the earliest Australian native plants to be cultivated in Europe. Reputedly a favourite of Napoléon Bonaparte and Empress Joséphine, X. bracteatum is thought to have been introduced to the island of St Helena in the South Atlantic during Napoléon's exile there. Colourful cultivars were developed in the 1850s, and there is a widely held view that these were produced by crossing Xerochrysum with African or Asian Helichrysum spp. Recent molecular phylogenetic analyses and subtribal classification of Gnaphalieae cast doubt on this idea. Using single-nucleotide polymorphism (SNP) data, we looked for evidence of gene flow between modern cultivars, naturalized paper daisies from St Helena and four Xerochrysum spp. recorded in Europe in the 1800s. There was strong support for gene flow between cultivars and X. macranthum. Paper daisies from St Helena were genotypically congruent with X. bracteatum and showed no indications of ancestry from other species or from the cultivars, consistent with the continuous occurrence of naturalized paper daisies introduced by Joséphine and Napoléon. We also present new evidence for the origin of colourful Xerochrysum cultivars and hybridization of congeners in Europe from Australian collections.