Why nuclear ribosomal DNA spacers (ITS) tell different stories in Quercus

The molecular systematics of Quercus (Fagaceae) was recently assessed by two teams using independently generated ITS sequences. Although the results disagreed in several remarkable features, the phylogenetic trees for either hypothesis were highly supported by bootstrap resampling. We have reanalyzed the ITS sequences used by both teams (eight taxa) to reveal the underlying patterns of this divergence. Within species, conspicuous length and G + C% divergence were evident in most sequence comparisons. In addition, a high rate of substitutions and deletions involving highly conserved motifs in both ITS spacers were present in a set of sequences. This was coupled with a less thermodynamic stability in the RNA structure, lacking conserved hairpins that are putatively involved in the processing of the RNA transcripts. Compelling evidence suggests that the divergent ITS alleles reported by one team are pseudogenes, i.e., nonfunctional paralogous loci. The hypothesis that the contrasting phylogenetic histories drawn from Quercus using ITS data are not strictly related to technical differences between laboratories, but that they have rather been generated from the analysis of paralogous sequences, best reconciles the available data. The risk of incorporating ITS paralogues in plant evolutionary studies which can distortion the phylogenetic signal should caution molecular systematists. Without a detailed inspection of some basic features of the sequence, including the integrity of the conserved motifs and the thermodynamic stability of the secondary structures of the RNA transcripts, errors in evolutionary inferences could be easily overlooked.