Ribosomal DNA pseudogenes are widespread in the eucalypt group (Myrtaceae): implications for phylogenetic analysis

Pseudogenes from the 18S-5.8S-26S cistron of nuclear ribosomal DNA are reported in the eucalypt group (Myrtaceae), which includes seven genera. Putative pseudogenes are identified by a range of sequence comparisons including: the number of CpG and CpNpG methylation sites, GC content, estimated secondary structure stability of internal transcribed spacer transcripts, the presence of conserved motifs, patterns of sequence relationships and inferred substitution patterns. These comparisons indicate that pseudogenes are widespread, being evident in 'Eucalyptus' (subgenera 'Eucalyptus' and 'Eudesmia'), 'Corymbia' (extracodical sections 'Rufaria, Ochraria and Blakearia'), 'Angophora, Stockwellia quadrifida' and 'Arillastrum gummiferum'. At least six sequences used in previous phylogenetic studies are identified as pseudogenes, and a further 10 pseudogenes are newly sequenced here. Gene trees place pseudogenes in a number of distinct lineages: pseudogenes from Eucalyptus group with other Eucalyptus sequences, those from 'Corymbia' and 'Angophora' group with other 'Corymbia/Angophora' sequences, that from Stockwellia groups with other sequences from the Eucalyptopsis group, and that from 'Arillastrum' is placed as sister to the other included sequence of 'Arillastrum'. Some pseudogenes in Eucalyptus, 'Corymbia' and 'Angophora' represent "deep" ribosomal DNA paralogues that pre-date species differentiation in these groups, and a recombination analysis shows no evidence of recombination between putative pseudogenes and their functional counterparts. The presence of divergent paralogues presents both challenges and opportunities for the reconstruction of eucalypt phylogenies using ribosomal DNA sequences. Phylogenetic data sets should include only orthologous sequences, but different paralogues potentially provide additional, independent, character sets for phylogenetic analyses.