Phylogenomics reveals extreme gene tree discordance in a lineage of dominant trees: hybridization, introgression, and incomplete lineage sorting blur deep evolutionary relationships despite clear species groupings in Eucalyptus subgenus Eudesmia.

Eucalypts are a large and ecologically important group of plants on the Australian continent, and understanding their evolution is important in understanding evolution of the unique Australian flora. Previous phylogenies using plastome DNA, nuclear-ribosomal DNA, or random genome-wide SNPs, have been confounded by limited genetic sampling or by idiosyncratic biological features of the eucalypts, including widespread plastome introgression. Here we present phylogenetic analyses of Eucalyptus subgenus Eudesmia (22 species from western, northern, central and eastern Australia), in the first study to apply a target-capture sequencing approach using custom, eucalypt-specific baits (of 568 genes) to a lineage of Eucalyptus. Multiple accessions of all species were included, and target-capture data were supplemented by separate analyses of plastome genes (average of 63 genes per sample). Analyses revealed a complex evolutionary history likely shaped by incomplete lineage sorting and hybridization. Gene tree discordance generally increased with phylogenetic depth. Species, or groups of species, toward the tips of the tree are mostly supported, and three major clades are identified, but the branching order of these clades cannot be confirmed with confidence. Multiple approaches to filtering the nuclear dataset, by removing genes or samples, could not reduce gene tree conflict or resolve these relationships. Despite inherent complexities in eucalypt evolution, the custom bait kit devised for this research will be a powerful tool for investigating the evolutionary history of eucalypts more broadly.

A genome resource for Acacia, Australia's largest plant genus.

Acacia (Leguminosae, Caesalpinioideae, mimosoid clade) is the largest and most widespread genus of plants in the Australian flora, occupying and dominating a diverse range of environments, with an equally diverse range of forms. For a genus of its size and importance, Acacia currently has surprisingly few genomic resources. Acacia pycnantha, the golden wattle, is a woody shrub or tree occurring in south-eastern Australia and is the country's floral emblem. To assemble a genome for A. pycnantha, we generated long-read sequences using Oxford Nanopore Technology, 10x Genomics Chromium linked reads, and short-read Illumina sequences, and produced an assembly spanning 814 Mb, with a scaffold N50 of 2.8 Mb, and 98.3% of complete Embryophyta BUSCOs. Genome annotation predicted 47,624 protein-coding genes, with 62.3% of the genome predicted to comprise transposable elements. Evolutionary analyses indicated a shared genome duplication event in the Caesalpinioideae, and conflict in the relationships between Cercis (subfamily Cercidoideae) and subfamilies Caesalpinioideae and Papilionoideae (pea-flowered legumes). Comparative genomics identified a suite of expanded and contracted gene families in A. pycnantha, and these were annotated with both GO terms and KEGG functional categories. One expanded gene family of particular interest is involved in flowering time and may be associated with the characteristic synchronous flowering of Acacia. This genome assembly and annotation will be a valuable resource for all studies involving Acacia, including the evolution, conservation, breeding, invasiveness, and physiology of the genus, and for comparative studies of legumes.

Phylogeny and classification of the Australasian and Indomalayan mimosoid legumes Archidendron and Archidendropsis (Leguminosae, subfamily Caesalpinioideae, mimosoid clade).

The morphologically variable genus Archidendron is the second largest mimosoid legume genus from the Indomalayan-Australasian region, yet it has not been well represented in phylogenetic studies. Phylogenies that have included multiple representatives of Archidendron suggest it may not be monophyletic, and the same applies to Archidendropsis, another understudied genus of the Archidendron clade. The most comprehensive phylogeny of Archidendron and Archidendropsis to date is presented, based on four nuclear markers (ITS, ETS, SHMT and RBPCO). Exemplars from all genera of the wider Archidendron clade are sampled, including representatives of all series within Archidendron and the two subgenera of Archidendropsis. Our results confirm that Archidendron and Archidendropsis are not monophyletic. Within Archidendron, only one series (ser. Ptenopae) is resolved as monophyletic and species of Archidendron are divided into two primarily geographic lineages. One clade is distributed in western Malesia and mainland Asia and includes most representatives of series Clypeariae, while the other is mostly restricted to eastern Malesia and Australia and includes representatives of the seven other series plus two samples of series Clypeariae. No taxonomic changes are made for Archidendron due to the high level of topological uncertainty and the lack of discrete macromorphological characters separating these two lineages. Each of the two subgenera of Archidendropsis is monophyletic but they are not closely related. A new genus endemic to Queensland (Australia), Heliodendron Gill.K. Br. & Bayly, gen. nov., is described for the former Archidendropsissubg.Basaltica, and combinations for its three species are proposed: Heliodendronbasalticum (F. Muell.) Gill.K. Br. & Bayly, comb. nov., Heliodendronthozetianum (F. Muell.) Gill.K. Br. & Bayly, comb. nov., and Heliodendronxanthoxylon (C.T. White & W.D. Francis) Gill.K. Br. & Bayly, comb. nov.

New targets acquired: Improving locus recovery from the Angiosperms353 probe set.

PREMISE: Universal target enrichment kits maximize utility across wide evolutionary breadth while minimizing the number of baits required to create a cost-efficient kit. The Angiosperms353 kit has been successfully used to capture loci throughout the angiosperms, but the default target reference file includes sequence information from only 6-18 taxa per locus. Consequently, reads sequenced from on-target DNA molecules may fail to map to references, resulting in fewer on-target reads for assembly, and reducing locus recovery. METHODS: We expanded the Angiosperms353 target file, incorporating sequences from 566 transcriptomes to produce a 'mega353' target file, with each locus represented by 17-373 taxa. This mega353 file is a drop-in replacement for the original Angiosperms353 file in HybPiper analyses. We provide tools to subsample the file based on user-selected taxon groups, and to incorporate other transcriptome or protein-coding gene data sets. RESULTS: Compared to the default Angiosperms353 file, the mega353 file increased the percentage of on-target reads by an average of 32%, increased locus recovery at 75% length by 49%, and increased the total length of the concatenated loci by 29%. DISCUSSION: Increasing the phylogenetic density of the target reference file results in improved recovery of target capture loci. The mega353 file and associated scripts are available at: https://github.com/chrisjackson-pellicle/NewTargets.

Divergent lineages in a semi-arid mallee species, Eucalyptus behriana, correspond to a major geographic break in southeastern Australia.

AIM: To infer relationships between populations of the semi-arid, mallee eucalypt, Eucalyptus behriana, to build hypotheses regarding evolution of major disjunctions in the species' distribution and to expand understanding of the biogeographical history of southeastern Australia. LOCATION: Southeastern Australia. TAXON: Eucalyptus behriana (Myrtaceae, Angiospermae). METHODS: We developed a large dataset of anonymous genomic loci for 97 samples from 11 populations of E. behriana using double digest restriction site-associated DNA sequencing (ddRAD-seq), to determine genetic relationships between the populations. These relationships, along with species distribution models, were used to construct hypotheses regarding environmental processes that have driven fragmentation of the species' distribution. RESULTS: Greatest genetic divergence was between populations on either side of the Lower Murray Basin. Populations west of the Basin showed greater genetic divergence between one another than the eastern populations. The most genetically distinct population in the east (Long Forest) was separated from others by the Great Dividing Range. A close relationship was found between the outlying northernmost population (near West Wyalong) and those in the Victorian Goldfields despite a large disjunction between them. CONCLUSIONS: Patterns of genetic variation are consistent with a history of vicariant differentiation of disjunct populations. We infer that an early disjunction to develop in the species distribution was that across the Lower Murray Basin, an important biogeographical barrier separating many dry sclerophyll plant taxa in southeastern Australia. Additionally, our results suggest that the western populations fragmented earlier than the eastern ones. Fragmentation, both west and east of the Murray Basin, is likely tied to climatic changes associated with glacial-interglacial cycles although it remains possible that major geological events including uplift of the Mount Lofty Ranges and basalt flows in the Newer Volcanics Province also played a role.

Long-read assemblies reveal structural diversity in genomes of organelles - an example with Acacia pycnantha.

Organelle genomes are typically represented as single, static, circular molecules. However, there is evidence that the chloroplast genome exists in two structural haplotypes and that the mitochondrial genome can display multiple circular, linear or branching forms. We sequenced and assembled chloroplast and mitochondrial genomes of the Golden Wattle, Acacia pycnantha, using long reads, iterative baiting to extract organelle-only reads, and several assembly algorithms to explore genomic structure. Using a de novo assembly approach agnostic to previous hypotheses about structure, we found that different assemblies revealed contrasting arrangements of genomic segments; a hypothesis supported by mapped reads spanning alternate paths.

Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus Corymbia, family Myrtaceae).

Previous molecular phylogenetic analyses have resolved the Australian bloodwood eucalypt genus Corymbia (~100 species) as either monophyletic or paraphyletic with respect to Angophora (9-10 species). Here we assess relationships of Corymbia and Angophora using a large dataset of chloroplast DNA sequences (121,016 base pairs; from 90 accessions representing 55 Corymbia and 8 Angophora species, plus 33 accessions of related genera), skimmed from high throughput sequencing of genomic DNA, and compare results with new analyses of nuclear ITS sequences (119 accessions) from previous studies. Maximum likelihood and maximum parsimony analyses of cpDNA resolve well supported trees with most nodes having >95% bootstrap support. These trees strongly reject monophyly of Corymbia, its two subgenera (Corymbia and Blakella), most taxonomic sections (Abbreviatae, Maculatae, Naviculares, Septentrionales), and several species. ITS trees weakly indicate paraphyly of Corymbia (bootstrap support <50% for maximum likelihood, and 71% for parsimony), but are highly incongruent with the cpDNA analyses, in that they support monophyly of both subgenera and some taxonomic sections of Corymbia. The striking incongruence between cpDNA trees and both morphological taxonomy and ITS trees is attributed largely to chloroplast introgression between taxa, because of geographic sharing of chloroplast clades across taxonomic groups. Such introgression has been widely inferred in studies of the related genus Eucalyptus. This is the first report of its likely prevalence in Corymbia and Angophora, but this is consistent with previous morphological inferences of hybridisation between species. Our findings (based on continent-wide sampling) highlight a need for more focussed studies to assess the extent of hybridisation and introgression in the evolutionary history of these genera, and that critical testing of the classification of Corymbia and Angophora requires additional sequence data from nuclear genomes.

Microsatellite markers for the endangered root holoparasite Dactylanthus taylorii (Balanophoraceae) from 454 pyrosequencing.

PREMISE OF THE STUDY: Microsatellite loci were isolated and developed as polymorphic markers for the New Zealand endemic root holoparasite Dactylanthus taylorii for use in population and conservation genetics studies. METHODS AND RESULTS: Shotgun 454 pyrosequencing was performed on genomic DNA pooled from three individuals of D. taylorii. From 61709 individual sequence reads, primers for 753 microsatellite loci were developed in silico and 72 of these were tested for consistent amplification and variability. Ten microsatellite loci were found to be polymorphic and consistently scorable when screened in 44 individuals from five geographically distant populations. The number of alleles per locus ranged from four to 16 with an average of 9.7, and average observed heterozygosity per locus was between 0.182 and 0.634. CONCLUSIONS: These polymorphic microsatellite markers establish an important resource for ongoing conservation initiatives and planned population genetic studies of D. taylorii.

Two micro-scale protocols for the isolation of DNA from polysaccharide-rich plant tissue.

The high polysaccharide content of some plant species hinders the successful isolation of their DNA. As an alternative to the macro-extraction methods previously published for polysaccharide-rich plants, we present two techniques (STE/CTAB and HEPES/CTAB), which are performed in microcentrifuge tubes. These protocols are suitable for small amounts of silica gel-preserved plant tissue such as are commonly available from endangered plants. The critical step to remove polysaccharides was performing initial washes in either STE (0.25 M sucrose, 0.03 M Tris, 0.05 M EDTA) or HEPES (2% ß-mercaptoethanol, 0.2% PVP, 0.1 M HEPES, pH 8.0) buffer. Precipitating the DNA at room temperature with isopropanol also aided in decreasing polysaccharide co-precipitation. Of the two protocols we present the STE/CTAB method has the advantages of being more cost-effective and avoiding the use of the hazardous chemical ß-mercaptoethanol.