(A) The abundance the ATP6 genome declined when co-resident with mt:ND2del1 + mt:CoIT300I at 29°C. After several generations, the flies at 29°C started to die. (B) A combination of a restriction fragment length polymorphism and a restriction site difference reveals the emergence of a recombinant genome. mtDNA isolated from 40 adults from each generation was cut with EcoRI in the presence or absence of XhoI. The schematics show the distribution of the EcoRI and XhoI sites on the whole parental genomes (left) and a detail of the largest EcoRI fragment with the position (purple bar) of a hybridization probe (center). Southern analysis shows single and double cut samples taken at different generations during the selection. Only the two parental bands were detected early, from G0 to G5 (shown for G3). From G6 onward, a third EcoRI fragment appeared that had a length characteristic of the mt:ND2del1 + mt:CoIT300I genome but with a XhoI site. By G7, the ATP6 specific fragment was not detected while a new apparently recombinant genome dominated the population. (C) Detailed maps of three genomes sequenced by PacBio SMRT. Red lines indicate mismatches between ATP6 and mt:ND2del1 + mt:CoIT300I sequences. The ATP6 genome also lacks ∼1.6 kb of the AT-rich region (two type I repeats and two type II repeats, see Figure 2—figure supplement 1A for details). Pink arrows indicate approximate points of exchange with the given range defined by the nearest neighboring polymorphisms (see deposited full sequences in GenBank as KT174472, KT174473 and KT174474). (D) Proposed progression leading to the recombinant. The original ATP6 genome is still displaced, but the newly emerged recombinant competes effectively and persists. By generation 6, the abundance of the recombinant genome is sufficient to complement the temperature-sensitive genome so that some viable flies sustain the line. Over subsequent generations at 29°C, the recombinant genome increases in relative abundance because of selection against the temperature-sensitive genome.