Phylogenetic analysis of the internal transcribed spacer (ITS) region in Menyanthaceae using predicted secondary structure

Abstract

Sequences of the nuclear internal transcribed spacer (ITS) regions ITS1 and ITS2 have been used widely in molecular phylogenetic studies because of their relatively high variability and facility of amplification. For phylogenetic applications, most researchers use sequence alignments that are based on nucleotide similarity. However, confidence in the alignment often deteriorates at taxonomic levels above genus, due to increasing variability among sequences. Like ribosomal RNA (rRNA) and other RNA molecules, the ITS transcripts consist in part of conserved secondary structures (‘stems’ and ‘loops’) that can be predicted by mathematical algorithm. Researchers have long considered the evolutionary conservation of rRNA secondary structure, but until recently few phylogenetic analyses of the ITS regions specifically incorporated structural data. We outline a novel method by which to derive additional phylogenetic data from ITS secondary structure in order to evaluate support for relationships at higher taxonomic levels. To illustrate the method, we describe an example from the plant family Menyanthaceae. Using predicted ITS secondary structure data, we obtained a well-resolved and moderately supported phylogeny, in which most topological relationships were congruent with the tree constructed using ITS nucleotide sequence data. Furthermore, the explicit encoding of ITS structural data in a phylogenetic framework allowed for the reconstruction of putative ancestral states and structural evolution in the functional but highly variable ITS region.