ITS2 sequence–structure analysis in phylogenetics: A how-to manual for molecular systematics

The information that can be obtained from the secondary structure of the nuclear ribosomal internal transcribed spacer 2 (ITS2) is substantial, and yet many studies exploit this information inconsistently or inappropriately. This review introduces a remedy in the form of a flowchart where we detail the steps involved in estimating structure-based phylogenetic trees from ITS2 data. The pipeline described consists of the ITS2 Database, 4SALE, the CBCAnalyzer, and ProfDistS. Based on these tools, we describe how to utilize ITS2 sequence and secondary structure information together with an ITS2 specific scoring matrix and an ITS2 specific substitution model. The phylogenetic results thus obtained have been shown to be more reliable than approaches based on primary sequence data alone. Moreover, compensatory base changes (CBCs) in ITS2 sequence–structure pairs are identified as a possible marker for distinguishing species.