Analyses of the internal transcribed spacer 2 (ITS2) secondary structures were performed for five morphologically almost identical individuals from Paramacrobiotus (Tardigrada). Individuals were sampled from four different continents and sequenced. Sequences were annotated and their secondary structures predicted. A phylogenetic Profile Neighbor-Joining tree was reconstructed on a combined sequence-structure alignment. The topology obtained is consistent with a tree derived from a distance matrix of compensatory base changes (CBCs). CBCs were calculated between all ITS2 sequence-structure pairs of the global multiple sequence-structure alignment. A CBC represents enough evolutionary distance to distinguish between species with a confidence level of 93%.Therefore the CBC analysis indicates that Paramacrobiotus consists of at least three species distinguishable with molecular evidence.

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