Nuclear ribosomal pseudogenes resolve a corroborated monophyly of the eucalypt genus Corymbia despite misleading hypotheses at functional ITS paralogs

Divergent paralogs can create both obstacles and opportunities for phylogenetic reconstruction. Phylogenetic relationships among eucalypt genera have been incongruent among datasets in previous studies, where morphological characters supported monophyly of the genus Corymbia, while intergenic spacers of the nuclear ribosomes (ITS) and chloroplast loci (trnL, trnH, psbA) showed Corymbia as either equivocal or paraphyletic. Ribosomal DNA occurs in multiple copies in a genome. We cloned and sequenced the nrITS to investigate if gene duplication was the cause of incongruence among trees in the eucalypts. Three ITS riboforms, two of them widespread, were recovered within some genomes. One of the ITS riboforms recovered a robust phylogeny showing Corymbia as a monophyletic genus, corroborating the evidence from morphology, fossil data, a recent ITS/ETS dataset and microsatellites (SSRs). Compelling evidence suggested that this divergent riboform is a pseudogene, i.e., non-functional paralog: comparatively lower GC content suggesting lower structural stability, deamination-like mutations at potential methylation sites, lack of conserved helices and hairpins and conspicuously lower thermodynamic stability in secondary structures. Phylogenies from the apparently functional riboform retained Corymbia as paraphyletic. We show here that pseudogenes can recover a well-corroborated phylogeny whereas their functional paralogs show misleading hypotheses. We explain that phylogenetic signals may be obscured when functional constraints in ITS necessitate compensatory mutations in the secondary structure helices involved in RNA transcription, whereas pseudogenes mutate under neutrality.