Characterisation of ITS2 secondary structures for selected allocreadiid species (Digenea: Plagiorchiida: Gorgoderoidea) and their use in taxonomy and systematics.

Secondary structures of the internal transcribed spacer 2 (ITS2) region of ribosomal RNA (rRNA) were reconstructed for 46 species belonging to 12 genera of the family Allocreadiidae. Most structures exhibited the classical four-domain conformation typical of ITS2, with some notable variants. The C-domains of Bunodera acerinae and Allocreadium sp. 2 were divided into three branches. Structures of Creptotrema funduli and Pseudoparacreptotrema macroacetabulata showed unique conformations, with unusually variable A and B domains. Ten-nucleotide sequence variation at the 5' end of the ITS2 region correlates with phylogenetic relationships within the family. A dichotomous key based on ITS2 secondary-structure features is proposed, providing a novel, annotation-independent tool for species identification. Phylogenetic analysis based on ITS2 data is also presented, but we acknowledge the limitations of this marker for resolving deep relationships and interpret the resulting topology as a hypothesis complementary to 28S-based phylogenies. The presence of regulatory motifs and palindromic elements suggests that ITS2 functions as a primary hub-DNA product that accumulates regulatory information, potentially influencing RNA folding, stability, and transcription efficiency. ITS2 secondary structure analysis, combined with the proposed key, provides a powerful approach for understanding the evolution and systematics of this complex trematode family.