Non-concerted evolution in ribosomal ITS2 sequence in Cynoglossus zanzibarensis (Pleuronectiformes: Cynoglossidae)

• Three types of ITS2 sequence were found, indicating a non-concerted evolution pattern.
• The specific PCR by adding 8% dimethyl sulfoxide (DMSO) was suggested to enhance the amplification efficiency of long fragment.
• Type B and C sequences were speculated as putative pseudogenes.
• Type C sequences were detected as recombinants between Type A and B through simple crossing over model.

The nuclear ribosomal RNA genes (nrDNA) include three rRNA genes (18S, 5.8S, and 28S) and two internal transcribed spacers (ITS1 and ITS2). The lack of variability in nrDNA has been explained as the result of concerted evolution. In fact, many examples of variation have been discovered, suggesting a non-concerted evolution process. There are few studies on ITS2 in teleostean fishes; particularly no reports on flatfishes. In this study, we focused on ITS2 in Zanzibar tonguesole, Cynoglossus zanzibarensis (Pleuronectiformes: Cynoglossidae). Three types of ITS2 sequences (Type A, B, and C) coexisted and mainly differ in length, nucleotide diversity (π), secondary structure, and minimum free energy. Based on these different features, Type B and C were speculated as the putative pseudogenic ITS2 region. Moreover, Type C sequences were detected as recombinant from crossing over between Type A and B sequences. To our knowledge, this is the first report on ITS2 recombination in animal ribosomal RNA genes. The high degree of ITS2 sequence polymorphism at the intraindividual level supported a non-concerted evolution pattern in the C. zanzibarensis genome. These results provide useful data for further studies on ITS2 in flatfishes and contribute to polymorphism analyses of ribosomal RNA genes in teleostean fishes.