Propagation of [D1,2]-type spliceosomal twin introns (stwintrons) in <i>Hypoxylaceae</i> and <i>Xylariaceae</i> fungi

[D1,2] stwintrons consist of nested U2 introns, where an internal intron splits the 5'-donor of an external intron between its first and second nucleotide. Almost all stwintrons described to date are of the [D1,2] type, suggesting unique means for their duplication. Sequence-similar [D1,2] stwintrons are typically integrated at new intron positions, specific for one species. Two hundred eighty-eight sequence-similar [D1,2] stwintrons were identified in the genomes of 14 <i>Xylariales</i>. Occasional missplicing was apparent, where almost the entire stwintron was excised as one canonical intron. Near-identical sister stwintrons were identified in <i>Xylaria</i> sp. MSU SB201401 and <i>Xylaria longipes</i> that share near-terminal inverted repeat elements of 10-nt length named 5'-NTIRE-10 and 3'-NTIRE-10, which are complementary as RNA. Complementary NTIRE-10 partners were also present in three <i>Hypoxylaceae</i> species. These NTIRE-10s can form a near-terminal double-stranded RNA stem structure that brings in close proximity the terminal G's of the [D1,2] stwintron and of its alternative misspliced intron. The exact folding of the interior stwintron RNA appears irrelevant. Ten of the stwintrons with complementary NTIRE-10s are present in <i>Xylaria</i> sp. MSU SB201401, implying that [D1,2] stwintron duplication occurs frequently in this species.IMPORTANCESpliceosomal introns are excised from pre-mRNAs by a ribonucleoprotein complex, the U2 spliceosome. Excision does not necessarily occur by one splicing reaction. We had identified and validated intronic sequences in fungi that consist of two nested U2 introns and called them spliceosomal twin introns (stwintrons). In this bioinformatics-based study, we identified and validated almost 300 [D1,2] stwintrons with sequence similarity in the genomes of 14 species in the <i>Xylariales</i> order of <i>Ascomycota</i>. Thirty-seven of them feature small, fully complementary RNA inverted repeat elements. These elements form a near-terminal RNA stem structure, bringing the terminal G's of the stwintron in close proximity. We further demonstrated the existence of an alternative splicing pattern involving one excision event that removes the two constituent introns from the transcript together. This work contributes to the understanding of mechanisms of stwintron gain in fungi.