The genome of the Wollemi pine, a critically endangered "living fossil" unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

Speciation in the presence of gene flow: population genomics of closely related and diverging Eucalyptus species.

Speciation is a complex process that is fundamental to the origins of biological diversity. While there has been considerable progress in our understanding of speciation, there are still many unanswered questions, especially regarding barriers to gene flow in diverging populations. Eucalyptus is an appropriate system for investigating speciation mechanisms since it comprises species that are rapidly evolving across heterogeneous environments. We examined patterns of genetic variation within and among six closely related Eucalyptus species in subgenus Eucalyptus section Eucalyptus in south-eastern Australia (commonly known as the "green ashes"). We used reduced representation genome sequencing to genotype samples from populations across altitudinal and latitudinal gradients. We found one species, Eucalyptus cunninghamii, to be highly genetically differentiated from the others, and a population of mallees from Mount Banks to be genetically distinct and therefore likely to be a new undescribed species. Only modest levels of differentiation were found between all other species in the study. There was population structure within some species (e.g., E. obstans) corresponding to geographical factors, indicating that vicariance may have played a role in the evolution of the group. Overall, we found that lineages within the green ashes are differentiated to varying extents, from strongly diverged to much earlier stages of the speciation continuum. Furthermore, our results suggest the green ashes represent a group where a range of mechanisms (e.g., reticulate evolution and vicariance) have been operating in concert. These findings not only offer insights into recent speciation mechanisms in Eucalyptus, but also other species complexes.

Evaluating the Impact of Genomic Data and Priors on Bayesian Estimates of the Angiosperm Evolutionary Timescale.

The evolutionary timescale of angiosperms has long been a key question in biology. Molecular estimates of this timescale have shown considerable variation, being influenced by differences in taxon sampling, gene sampling, fossil calibrations, evolutionary models, and choices of priors. Here, we analyze a data set comprising 76 protein-coding genes from the chloroplast genomes of 195 taxa spanning 86 families, including novel genome sequences for 11 taxa, to evaluate the impact of models, priors, and gene sampling on Bayesian estimates of the angiosperm evolutionary timescale. Using a Bayesian relaxed molecular-clock method, with a core set of 35 minimum and two maximum fossil constraints, we estimated that crown angiosperms arose 221 (251-192) Ma during the Triassic. Based on a range of additional sensitivity and subsampling analyses, we found that our date estimates were generally robust to large changes in the parameters of the birth-death tree prior and of the model of rate variation across branches. We found an exception to this when we implemented fossil calibrations in the form of highly informative gamma priors rather than as uniform priors on node ages. Under all other calibration schemes, including trials of seven maximum age constraints, we consistently found that the earliest divergences of angiosperm clades substantially predate the oldest fossils that can be assigned unequivocally to their crown group. Overall, our results and experiments with genome-scale data suggest that reliable estimates of the angiosperm crown age will require increased taxon sampling, significant methodological changes, and new information from the fossil record. [Angiospermae, chloroplast, genome, molecular dating, Triassic.].

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution.

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree.

BACKGROUND: With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. RESULTS: The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. CONCLUSIONS: Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology.

Isolation and characterization of polymorphic microsatellite loci from Tetratheca ericifolia (Elaeocarpaceae).

We identified 11 informative microsatellite loci in Tetratheca ericifolia from an (AG)(n) -enriched library. Using these loci on 32 individuals from two populations (16 individuals from each), we detected an average of 11.3 alleles per locus (range of five to 21, average expected heterozygosity of 0.728), of which 56% were unique to one population or the other. All loci were amplifiable in seven to 12 of a further 12 species of Tetratheca under the same reaction conditions. The markers will be useful tools for evolutionary studies of this Australian endemic group.