Schematic presentation of the twin-ribozyme introns in Allovahlkampfia sp. (Asp.S516) and Naegleria (Nae.S516). (A) Secondary structure diagram of Asp.S516 folded according to previously reported models  with some modifications. Asp.S516 contains two ribozyme domains, AspLC and AspGIR2, and a homing endonuclease gene (I-Asp I HEG). Paired RNA segments are denoted P3 to P15 in AspLC and P1 to P13 in AspGIR2. Nucleotide positions invariant among the Naegleria isolates but different in Allovahlkampfia are indicated in blue. Regions in Asp.S516 that deviate from Nae.S516 are boxed (LC-GIR2 junction; P13). (B) Consensus secondary structure diagram of Nae.S516 based on 13 Naegleria introns . Red circles represent variable positions based on at least one deviating Naegleria intron. Invariant positions are presented as uppercase letters. (C) Alignment of the His-Cys homing endonucleases encoded by Asp.S516 and Nae.S516. Identical residues are boxed, and deletions are indicated by dashes. Functionally important C, H and N residues involved in zinc binding and catalysis are indicated (yellow boxes). The I-Nae I sequence represents a consensus of nine Naegleria HEs (see Additional file 2: Figure S2). Uppercase letter, invariant amino acid position; lowercase letter, conserved amino acid position in at least five of nine sequences; X, non-conserved amino acid position.