Secondary loss of an intron in Giardia intestinalis assemblage E represented by strain P15. The relationship among G. intestinalis assemblages A1, A2, B, and E based on Monis et al. [19, 20] is schematically provided on left. On right, we compare the intron and its flanking exon sequences of the ORF AACB02000001-6-305427-304747 (orfB) in strain WS (assemblage A1) and the homologous regions in the ORF AHGT01000002-2-45140-45820 in strain DH (assemblage A2), the ORF ACVC01000101-5-40937-40224 in strain P15 (assemblage E), and the ORF ACGJ01002919-1-19018-19737 in strain GS (assemblage B). Intron regions are shaded. The conserved sequence motifs shared amongst Giardia introns/splintrons are highlighted by asterisks. Gaps are represented by dashes. We here propose that (i) the intron found in orfB in strain WS (this study) can be traced back to the common ancestor of the four assemblages, and then (ii) a secondary loss of the intron occurred after assemblages A1/A2 and E separated from each other (highlighted by open circle).