Phylogenetic positions of exon gain and loss in Bru-3. The coding sequences of Bru-3 were inferred for 8 Drosophila species from their genomic sequenced available at FlyBase.org . A distance tree was calculated in PAUP 4.0b10  from the coding parts of eleven exons of Bru-3 (exons 2-5, 7, 9-14) shared by twelve Drosophila species. Applying nucleotide BLASTing , we found that exon 6 was lost along the branch leading to the melanogaster group (the top shaded box), whereas exon 8 was de novo created in the obscura group (the bottom shaded box). We verified both events for Drosophila species in bold by amplifying and aligning the orthologous genomic regions that include exons 6 and 8 (supplementary files S1 and S2). In addition cDNA pools from the species in bold, except for D. simulans, were screened for inclusion of conserved exon 6 and 8 in mature mRNA (fig. 2). The status of exon 6 indicated with a circle; a black circle, the exon was identified in cDNA pool; a white circle, the exon was absent. A square was used to indicate the status of exon 8.