A molecular perspective on a complex polymorphic inversion system with cytological evidence of multiply reused breakpoints

Genome sequence comparison across the Drosophila genus revealed that some fixed inversion breakpoints had been multiply reused at this long timescale. Cytological studies of Drosophila inversion polymorphism had previously shown that, also at this shorter timescale, some breakpoints had been multiply reused. The paucity of molecularly characterized polymorphic inversion breakpoints has so far precluded contrasting whether cytologically shared breakpoints of these relatively young inversions are actually reused at the molecular level. The E chromosome of Drosophila subobscura stands out because it presents several inversion complexes. This is the case of the E 1+2+9+3 arrangement that originated from the ancestral E st arrangement through the sequential accumulation of four inversions (E 1 , E 2 , E 9 and E 3 ) sharing some breakpoints. We recently identified the breakpoints of inversions E 1 and E 2 , which allowed establishing reuse at the molecular level of the cytologically shared breakpoint of these inversions. Here, we identified and sequenced the breakpoints of inversions E 9 and E 3 , because they share breakpoints at sections 58D and 64C with those of inversions E 1 and E 2 . This has allowed establishing that E 9 and E 3 originated through the staggered-break mechanism. Most importantly, sequence comparison has revealed the multiple reuse at the molecular level of the proximal breakpoint (section 58D), which would have been used at least by inversions E 2 , E 9 and E 3 . In contrast, the distal breakpoint (section 64C) might have been only reused once by inversions E 1 and E 2 , because the distal E 3 breakpoint is displaced >70 kb from the other breakpoint limits.