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

Genomes have a highly organized architecture (nonrandom organization of functional and nonfunctional genetic elements within chromosomes) that is essential for many biological functions, particularly gene expression and reproduction. Despite the need to conserve genome architecture, a high level of structural variation has been observed within species. As species separate and diverge, genome architecture also diverges, becoming increasingly poorly conserved as divergence time increases. However, within plant genomes, the processes of genome architecture divergence are not well described. Here we use long-read sequencing and de novo assembly of 33 phylogenetically diverse, wild and naturally evolving Eucalyptus species, covering 1–50 million years of diverging genome evolution to measure genome architectural conservation and describe architectural divergence. The investigation of these genomes revealed that following lineage divergence, genome architecture is highly fragmented by rearrangements. As genomes continue to diverge, the accumulation of mutations and the subsequent divergence beyond recognition of rearrangements become the primary driver of genome divergence. The loss of syntenic regions also contribute to genome divergence but at a slower pace than that of rearrangements. We hypothesize that duplications and translocations are potentially the greatest contributors to Eucalyptus genome divergence.

The mulga complex (Acacia aneura and closely related taxa) is a widespread group that is dominant in much of arid Australia. The group is taxonomically difficult, due to a complex interaction of sympatry and putative hybridisation between the major species, geographic variation within species and sympatric variation within A. aneura. Mulga is highly variable in a wide range of vegetative and reproductive characters and it is not unusual to find five or six distinct forms growing side by side. The aim of this project was to gain a better understanding of the relationships among mulga species and A. aneura varieties, as well as the maintenance of this variation. A single site in the Northern Territory, containing A. ayersiana, A. minyura and two varieties of A. aneura, was sampled intensively. Six morphotypes were observed in the field and five were strongly supported by morphometric analysis. Although the mulga complex is generally tetraploid (2n = 52), triploid (2n = 39) and pentaploid (2n = 65) seedlings were produced in the study population. Microsatellite primers developed for A. mangium (sect. Juliflorae) were amplified in individuals of each morphotype, resulting in genetic marker patterns consistent with polyploidy. Genetic and morphometric distances were correlated and differences between morphotypes account for 63% of the total genetic variation (ΦPT = 0.63, P < 0.001). Allele sequences confirmed the presence of genuine heterozygosity and clonality was suggested by the low genotypic diversity and the lack of allele segregation. Seedlings had identical genotypes to the maternal plants and polyembryony was observed in each taxon, consistent with apomictic reproduction. Both variation of the ploidy level and apomixis may restrict gene flow among morphotypes, playing a role in the maintenance of morphological diversity at the study site. The success of the group in arid and semi-arid Australia may also be due, in part, to these factors.

Understanding the exchange of individuals between wildlife populations, particularly those with naturally fragmented habitats, is important for the effective management of these species. This is of particular consequence when the species is of conservation concern, and isolated populations may be lost due to pressures from predation or competition, or catastrophic events such as wildfire. Here we demonstrate the use kinship and population structure analysis to show potential recent movement between colonies in metapopulations of yellow-footed rock-wallaby (Petrogale xanthopus Gray 1854) at two sites in the Grey Range of Queensland, and at four sites in the Gawler Ranges of South Australia. These colonies are also compared to a single colony from the Flinders Ranges, a connected landscape of rock-wallaby habitat. Using reduced representation next-generation sequencing, we acquired and filtered a set of similar to 17,000 single-nucleotide polymorphisms to examine population genetic variation, structure and relationships within populations, and also identify putative migrants. Initial STRUCTURE analysis re-confirmed each population should be considered separately. Tests of population genetic variation identify several colonies appearing to be experiencing genetic erosion, also with low calculated effective population sizes (N_e = 4.5-36.6). Pairwise comparisons of individual relatedness (relatedness coeffiecients; r) implied several contemporary movement events between colonies within both the Gawler and Grey Ranges (r > 0.125), which was then affirmed with tests for putative first generation migrants. These results are of particular note in South Australia, where threat abatement (management of key predators and competitors) may facilitate dispersion. Additionally, in Queensland, colonies are separated by anthropogenic barriers: predator exclusion fencing designed to exclude dingoes (Canis familiaris) from grazing land, which may hinder dispersal. This work highlights the usefulness of population genetics to inform management outcomes in wildlife, in this case, highlighting the need for threatened species management at the landscape level.