Ribosomal ITS sequences and plant phylogenetic inference

One of the most popular sequences for phylogenetic inference at the generic and infrageneric levels in plants is the internal transcribed spacer (ITS) region of the 18S–5.8S–26S nuclear ribosomal cistron. The prominence of this source of nuclear DNA sequence data is underscored by a survey of phylogenetic publications involving comparisons at the genus level or below, which reveals that of 244 papers published over the last five years, 66% included ITS sequence data. Perhaps even more striking is the fact that 34% of all published phylogenetic hypothesis have been based exclusively on ITS sequences. Notwithstanding the many important contributions of ITS sequence data to phylogenetic understanding and knowledge of genome relationships, a number of molecular genetic processes impact ITS sequences in ways that may mislead phylogenetic inference. These molecular genetic processes are reviewed here, drawing attention to both underlying mechanism and phylogenetic implications. Among the most prevalent complications for phylogenetic inference is the existence in many plant genomes of extensive sequence variation, arising from ancient or recent array duplication events, genomic harboring of pseudogenes in various states of decay, and/or incomplete intra- or inter-array homogenization. These phenomena separately and collectively create a network of paralogous sequence relationships potentially confounding accurate phylogenetic reconstruction. Homoplasy is shown to be higher in ITS than in other DNA sequence data sets, most likely because of orthology/paralogy conflation, compensatory base changes, problems in alignment due to indel accumulation, sequencing errors, or some combination of these phenomena. Despite the near-universal usage of ITS sequence data in plant phylogenetic studies, its complex and unpredictable evolutionary behavior reduce its utility for phylogenetic analysis. It is suggested that more robust insights are likely to emerge from the use of single-copy or low-copy nuclear genes.