We used a chromosome segregation assay modified from Rand et al. (2001) (link) to quantify fitness effects of mitochondrial-nuclear interactions (Figure 2). In this assay, individuals competed in the same vial with siblings carrying a visibly-marked X chromosome (FM6) that confers a Bar eye phenotype. Relative fitness was measured as the egg-to-adult viability of wild-type individuals relative to their FM6-bearing siblings across the 35 mitochondrial-nuclear genotypes (Table 1). Differences between nuclear genotypes in the relative competitive viability of wild-type flies compared to their FM6 siblings result from viability effects of the X chromosome directly competing with FM6, and genetic interactions between the wild-type or FM6 X chromosomes and the autosomes. Differences between mitochondrial-nuclear genotypes in relative competitive viability result from interactions between the mitochondrial genome and the wild-type or FM6 X chromosomes, or more complex interactions between the X chromosome, the autosomes, and the mtDNA. The advantages of this assay are that competing individuals share the same common rearing environment, and genotypes are easily inferred from the Bar phenotype. In males, differences in relative competitive viability arise from the hemizygous effects of X-linked variants in combination with the mitochondrial-nuclear genotype. In females, viability effects of wild-type X-linked variants that are completely dominant to FM6 when combined with particular mitochondrial-nuclear genomes will not be detected in this assay.
We generated strains carrying an FM6 chromosome in both the Ore and Aut nuclear backgrounds. Females of each mitochondrial-nuclear genotype were mated to males carrying the FM6 chromosome in the same autosomal background. For the segregation assay, the resulting heterozygous females (Aut/FM6;Aut;Aut or Ore/FM6;Ore;Ore) were crossed to Aut or Ore males, respectively (Figure 2A). All offspring from these crosses inherit the mtDNA from the initial female. Female offspring were scored as either wild-type homozygotes or Bar heterozygotes, and male offspring were either wild-type or Bar hemizygotes. Relative competitive viability was calculated for each sex in each vial as the number of wild-type offspring divided by one plus the total progeny of that sex emerging from that vial (Haldane 1956 ), and is the measure of relative fitness used throughout.