An increasing number of phylogenetic analyses are based on the internal transcribed spacer 2 (ITS2). They mainly use the fast evolving sequence for low-level analyses. When considering the highly conserved structure, the same marker could also be used for higher level phylogenies. Furthermore, structural features of the ITS2 allow distinguishing different species from each other. Despite its importance, the correct structure is only rarely found by standard RNA folding algorithms. To overcome this hindrance for a wider application of the ITS2, we have developed a homology modelling approach to predict the structure of RNA and present the results of modelling the ITS2 in the ITS2 Database. Here, we describe the database and the underlying algorithms which allowed us to predict the structure for 86 784 sequences, which is more than 55% of all GenBank entries concerning the ITS2. These are not equally distributed over all genera. There is a substantial amount of genera where the structure of nearly all sequences is predicted whereas for others no structure at all was found despite high sequence coverage. These genera might have evolved an ITS2 structure diverging from the standard one. The current version of the ITS2 Database can be accessed via http://its2.bioapps.biozentrum.uni-wuerzburg.de.
It was shown that compensatory base changes (CBCs) in internal transcribed spacer 2 (ITS2) sequence-structure alignments can be used for distinguishing species. Using the ITS2 Database in combination with 4SALE -a tool for synchronous RNA sequence and secondary structure alignment and editing -in this study we present an in-depth CBC analysis for placozoan ITS2 sequences and their respective secondary structures. This analysis indicates at least two distinct species in Trichoplax (Placozoa) supporting a recently suggested hypothesis, that Placozoa is "no longer a phylum of one".
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