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.

The critically endangered Wollemia nobilis W.G. Jones, K.D. Hill & J.M. Allen is endemic to Wollemi National Park north of Sydney (Australia). All known wild individuals are restricted to four sites in a single canyon system. W. nobilis can reproduce sexually but at all sites individual clumps can be multi-stemmed from a common base. In the first genetic study of this species, no genetic variation was found across multiple genetic marker types representing hundreds of nuclear loci, indicating this species is characterised by very low genetic variation. In this study we searched for variation across the chloroplast using shotgun sequencing, bioinformatic extraction of chloroplast DNA and variant detection. Six chloroplast single nucleotide polymorphisms were detected, producing three chlorotypes. Chlorotype 1 is found in every individual surveyed at Sites 1 and 3, and in individuals from Sites 2 and 4. Chlorotype 2 – the most distinct chlorotype – was found in two individuals from Site 4. Chlorotype 3 consists of a single difference from Chlorotype 1 and may represent a somaclonal mutant. These findings will guide management and translocation of this critically endangered species. This study provides a practical template that is highly informative and easily applicable to other taxa in similar circumstances.