Chloroplast genome analysis of box-ironbark Eucalyptus

Eucalyptus L'Hérit. (Myrtaceae) is a taxonomically complex and highly speciose genus that dominates much of Australia's woody vegetation. However, very little information is available about the molecular biology and chloroplast diversity of certain groups, such as Eucalyptus section Adnataria, which is found in many woodland habitats of eastern Australia. We report four new complete chloroplast genomes of Eucalyptus, including three genomes from species previously lacking any chloroplast reference sequences. Plastomes of E. albens, E. conica, E. crebra and E. melliodora assembled using a de novo approach were shown to be largely identical to each other, and similar in size and structure to previously published chloroplast genomes from Eucalyptus. A total of 132 genes (114 single-copy genes and 18 duplicated genes in the IR regions) were identified, and shown to be highly conserved in terms of gene order, content and organization. Slightly higher divergence in the intergenic spacers was identified through comparative genomic analyses. Chloroplast sequences of 35 additional individuals representing 12 species were assembled using a reference guided approach. Rates of nucleotide substitution varied among the protein coding genes, with 17 genes under possible positive selection, and 29 invariant genes. Phylogenetic analysis of either the whole reconstructed plastome sequences or the individual genes revealed extreme discordance with expected species boundaries or higher-level relationships. Plastome relationships were better predicted by geography than by nuclear DNA or taxonomic relationships, suggesting a substantial influence of gene flow over and above the effects of incomplete lineage sorting. These results provide resources for future research and valuable insights into the prevalence of interspecific gene flow among Eucalyptus species.