Spliceosomal twin introns (stwintrons) are introns where any of the three consensus sequences involved in splicing is interrupted by another intron (internal intron). In Aspergillus nidulans, a donor-disrupted stwintron (intron-1) is extant in the transcript encoding a reticulon-like protein. The orthologous transcript of Aspergillus niger can be alternatively spliced; the exon downstream the stwintron could be skipped by excising a sequence that comprises this stwintron, the neighbouring intron-2, and the exon bounded by these. This process involves the use of alternative 3' splice sites for the internal intron, the resulting alternative intervening sequence being a longer 3'-extended stwintron. In 29 species of Onygenales, a multi-step splicing process occurs in the orthologous transcript, in which a complex intervening sequence including the stwintron and neigbouring intron-2, generates by three splicing reactions a "second order intron" which must then be excised with a fourth splicing event. The gene model in two species can be envisaged as one canonical intron (intron-1) evolved from this complex intervening sequence of nested canonical introns found elsewhere in Onygenales. Postulated splicing intermediates were experimentally verified in one or more species. This work illustrates a role of stwintrons in both alternative splicing and the evolution of intron structure.