A Next Generation Sequencing (NGS) approach to investigate paralogy in Xanthostemon (Myrtaceae).

A project undertaken at the Royal Botanic Gardens and Domain Trust, Sydney,
and supervised by Dr Peter Wilson. Research associate: Ms Margaret Heslewood

The genus Xanthostemon (Myrtaceae) comprises at least 48 species, occurring in Australia, Papua New Guinea, Solomon Islands, Indonesia, Philippines and New Caledonia. Xanthostemon is an almost exclusively tropical genus, with only one of the Australian species, X, oppositifolius, occurring in subtropical areas. Many species have spectacular flowers and some are grown as ornamentals. An Australian species, X. chrysanthus, the golden penda, and a red-flowered Philippine species, X. verdugonianus, are the most commonly cultivated species, in Australia and Asia respectively.

The genus is a member of the tribe Xanthostemoneae along with two small, segregates that have been recognised in New Caledonia: Pleurocalyptus and Purpureostemon. New Caledonia is a centre of diversification in the tribe with at least 19 species Xanthostemon occurring there, but Australia has species representing four quite diverse groups based on fruit and placentation structure. Three of the latter groups were considered to be possible generic segregates and an earlier study, using traditional sequencing approaches, was undertaken on the Xanthostemoneae to test both the published and the hypothetical genera. This study was hampered by paralogy due to apparent duplication of a portion of the chloroplast (CP) genome in some Australian Xanthostemon species.

As a result of this paralogy, chloroplast datasets can randomly include real or pseudocopies of some chloroplast loci leading to incongruence between phylogenies derived from chloroplast, nuclear and/or morphological data. This issue must be addressed before accurate taxonomic relationships in the tribe can be resolved. Next Generation Sequencing provides the means to determine the structure of the chloroplast genome and identify the source of this paralogy.

This project aims to:

  1. Identify the taxonomic limits of a gene duplication in the CP genome in some Australian members of Xanthostemon.
  2. Produce annotated genomic sequences for 10 taxa using Illumina Next Generation Sequencing (NGS): 8 Xanthostemon spp. and 2 other Myrtaceae to investigate limits of the duplication outside the tribe.
  3. Using the NGS data that we generate, and other published Myrtaceae genomes (e.g. Bayly et al 2013 for tribe Eucalypteae), we will redesign primers that exclude amplification of paralogues for CP loci.
  4. Using the newly designed primers we will add new CP data for our existing 55-taxon 4 locus dataset and complete the phylogeny of the tribe, resolving species relationships and clarifying the status of the three named genera in the tribe.
  5. Additionally, a more detailed study will add a further 20 specimens representing two variable species, X. umbrosus and X. paradoxus, that might result in the recognition of several new species.

 

Figure 1. Xanthostemon umbrosus sens. lat. – An Australian taxon that shows evidence of paralogy in chloroplast sequences. (Photo: K. Kupsch)

Figure 2. X. arenarius ­– One of the few Australian taxa seemingly lacking the chloroplast duplication. (Photo: K. Kupsch)

Figure 3. X. verdugonianus – a red-flowered species from the Philippines, cultivated, Singapore Botanic Gardens (Photo: P.G. Wilson)

Figure 4. X. macrophyllus – a bullate-leaved species from New Caledonia (Photo: P.G. Wilson)

Figure 5. Purpureostemon ciliatus – one of the small, segregate genera of Xanthostemoneae in New Caledonia (Photo: P.G. Wilson)