Compensatory base changes illuminate morphologically difficult taxonomy

Compensatory base changes (CBCs) in the ribosomal RNA (rRNA) internal transcribed spacer 2 (ITS2) secondary structures have been used to successfully verify the taxonomy of closely related species. CBCs have never been used to distinguish morphologically indistinct species. Under the hypothesis that CBCs will differentiate species in higher eukaryotes, novel software for CBC analysis was applied to morphologically indistinguishable insect species in the genus Altica. The analysis was species-specific for sympatric Altica beetles collected across four ecoregions and concordant with scanning electron microscopy data. This research shows that mining for CBCs in ITS2 rRNA secondary structures is an effective method for eukaryotic taxon analysis.