The maternal transmission of mitochondrial genomes invokes a sex-specific selective sieve, whereby mutations in mitochondrial DNA can only respond to selection acting directly on females []. In theory, this enables male-harming mutations to accumulate in mitochondrial genomes when these same mutations are neutral, beneficial, or only slightly deleterious in their effects on females []. Ultimately, this evolutionary process could result in the evolution of male-specific mitochondrial mutation loads; an idea previously termed Mother’s Curse []. Here, we present evidence that the effects of this process are broader than hitherto realized, and that it has resulted in mutation loads affecting patterns of aging in male, but not female Drosophila melanogaster. Furthermore, our results indicate that the mitochondrial mutation loads affecting male aging generally comprise numerous mutations over multiple sites. Our findings thus suggest that males are subject to dramatic consequences that result from the maternal transmission of mitochondrial genomes. They implicate the diminutive mitochondrial genome as a hotspot for mutations that affect sex-specific patterns of aging, thus promoting the idea that a sex-specific selective sieve in mitochondrial genome evolution is a contributing factor to sexual dimorphism in aging, commonly observed across species [].

Results and Discussion

We present experimental evidence to support the contention that the maternal transmission of mitochondrial genomes has enabled sex-specific mutations to accumulate within them, which affect patterns of aging in males. We used thirteen naturally occurring mitochondrial haplotypes of D. melanogaster from around the globe, expressing each inside a completely isogenic nuclear background, w1118. We then subjected the lines to an aging assay, in both males and females, to screen for genetic variation in longevity and the rate of senescence, and we sequenced the complete protein-coding regions of each of the thirteen mitochondrial haplotypes. All known environmental variables (e.g., food source, larval density, temperature, light, parental effects, age at mating, and mating status) were carefully controlled during the experiment, to minimize all other sources of variation.

1 Frank S.A.

Hurst L.D. Mitochondria and male disease. 2 Gemmell N.J.

Metcalf V.J.

Allendorf F.W. Mother’s curse: the effect of mtDNA on individual fitness and population viability. 3 Innocenti P.

Morrow E.H.

Dowling D.K. Experimental evidence supports a sex-specific selective sieve in mitochondrial genome evolution. 10 Friberg U.

Dowling D.K. No evidence of mitochondrial genetic variation for sperm competition within a population of Drosophila melanogaster. 1 Frank S.A.

Hurst L.D. Mitochondria and male disease. 2 Gemmell N.J.

Metcalf V.J.

Allendorf F.W. Mother’s curse: the effect of mtDNA on individual fitness and population viability. 10 Friberg U.

Dowling D.K. No evidence of mitochondrial genetic variation for sperm competition within a population of Drosophila melanogaster. 11 Dowling D.K.

Friberg U.

Arnqvist G. A comparison of nuclear and cytoplasmic genetic effects on sperm competitiveness and female remating in a seed beetle. 12 Dowling D.K.

Nowostawski A.L.

Arnqvist G. Effects of cytoplasmic genes on sperm viability and sperm morphology in a seed beetle: implications for sperm competition theory?. 109.87 = 16.90, p < 0.0001, X males = 49.21 ± 0.45 days, X females = 61.25 ± 0.55 days) and the rate of senescence (after controlling for frailty as a covariate, i.e., the frailty-corrected rate of senescence F 1,113 = 253.70, p < 0.0001) were each strongly sexual dimorphic across our fly lines. Each of these traits should thus, in principle, be vulnerable to the accumulation of male-specific mitochondrial mutation loads. Under this evolutionary process, it is predicted that the susceptibility of any given trait to the accumulation of male-specific mitochondrial mutation loads will be directly tied to the level of sexual dimorphism exhibited by that trait [] (see Supplemental Information available online). This is because the benefits that males can salvage from relying on female-specific adaptation of mitochondrial DNA (mtDNA) will diminish as the level of sexual dimorphism increases and the intersexual genetic correlation erodes []. It is for this reason that most attention has focused on the vulnerability of the male reproductive tissues and gametes to this process, because these are sex-limited traits []. We found that longevity (t= 16.90, p < 0.0001, X= 49.21 ± 0.45 days, X= 61.25 ± 0.55 days) and the rate of senescence (after controlling for frailty as a covariate, i.e., the frailty-corrected rate of senescence F= 253.70, p < 0.0001) were each strongly sexual dimorphic across our fly lines. Each of these traits should thus, in principle, be vulnerable to the accumulation of male-specific mitochondrial mutation loads.

3 Innocenti P.

Morrow E.H.

Dowling D.K. Experimental evidence supports a sex-specific selective sieve in mitochondrial genome evolution. 12,13.18 = 6.31, p = 0.0011; rate of senescence: F 12,14.25 = 3.71, p = 0.0106), but not in females (longevity: F 12,13.02 = 0.71, p = 0.7205; rate of senescence: F 12,12.91 = 0.50, p = 0.8803, Figure S1; Figure 1 Genetic Variance across Mitochondrial Haplotypes for Male and Female Aging Components Show full caption (A) Male longevity. (B) Female longevity. (C) Male frailty-corrected rate of senescence. (D) Female frailty-corrected rate of senescence. (E) Male age-specific mortality hazards for each mitochondrial haplotype. (F) Female age-specific mortality hazards for each mitochondrial haplotype. In (A)–(D), all data points denote means ± 1 SE. Rate of senescence values in (C) and (D) were calculated by taking the mean of the residuals of a linear regression of β on ln(α), where each data point in the regression represented the β and α score of a cohort of on average 90 flies. Positive values of senescence indicate that the rate of senescence, β, associated with a given mitochondrial haplotype is greater than that expected based on the baseline mortality [ln(α)] value, whereas negative values indicate that that the rate of senescence is less than that expected based on the baseline mortality associated with a given haplotype. ALS denotes the Alstonville mtDNA haplotype; BAR, Barcelona; BRO, Brownsville; DAH, Dahomey; HAW, Hawaii; ISR, Israel; JAP, Japan; MAD, Madang; MYS, Mysore; ORE, Oregon; PUE, Puerto Montt; SWE, Sweden; and ZIM, Zimbabwe. In (E) and (F), the age-specific mortality hazards are calculated as [ln(μx)], which is composed of the two aging components, ln(μx) = ln(α) + βx. Each line represents a distinct mitochondrial haplotype. A second important prediction of this evolutionary process is that it should result in mitochondrial genomes that harbor mutation loads that are more pronounced in males and that these loads can be uncovered by demonstrating greater levels of functional mitochondrial genetic variance in males than in females []. Accordingly, we found significant genetic variance across mitochondrial haplotypes for longevity and the frailty-corrected rate of senescence in males (longevity: F= 6.31, p = 0.0011; rate of senescence: F= 3.71, p = 0.0106), but not in females (longevity: F= 0.71, p = 0.7205; rate of senescence: F= 0.50, p = 0.8803, Figure 1 Table S1 ). This is consistent with the underlying existence of male-specific mitochondrial mutation loads for these traits. Therefore, our results satisfy the core predictions of a sex-specific selective sieve in mitochondrial genome evolution and implicate it in the process of aging.

Figure 2 Associations between Molecular and Phenotypic Divergence across the Mitochondrial Haplotypes Show full caption Shown is the relationship between the number of nonsynonymous nucleotide differences and the differences in male mean longevity (A) and male mean frailty-corrected rates of senescence (B), across all pairwise combinations of mtDNA haplotypes. We then asked whether the male-specific mitochondrial mutation loads affecting patterns of aging more likely comprise a few mutations of major effect or numerous mutations of smaller effect dispersed throughout the mitochondrial genome. We hypothesized that clear support for the latter scenario would come from the existence of a positive association between the nucleotide divergence and phenotypic divergence for male aging, across pairwise combinations of mitochondrial haplotypes. We found exactly that, both for longevity (r = 0.243, p = 0.029, Figure 2 ) and the corrected rate of senescence (r = 0.312, p = 0.007, Figure 2 ). These analyses were based only on the pool of nonsynonymous SNPs, because there was a strong positive correlation between the number of nonsynonymous and synonymous SNPs across mitochondrial haplotypes (r = 0.77, p < 0.001; Supplemental Experimental Procedures Figure S2 ), which suggests that the number of nonsynonymous SNPs found across any given pair of mitochondrial haplotypes correlates with the evolutionary divergence separating these haplotypes. Thus, our analysis indicates that the greater the number of SNPs separating any two mitochondrial haplotypes, the more those haplotypes generally differed in their aging profiles, supporting the idea that there are many mtDNA-encoded loci that affect male longevity and aging. Although our analyses cannot disentangle the relative importance of nonsynonymous versus synonymous SNPs in driving the differences in expression of aging phenotypes observed here, it is plausible that both contribute.

13 Lynch M. Mutation accumulation in nuclear, organelle, and prokaryotic transfer RNA genes. 1 Frank S.A.

Hurst L.D. Mitochondria and male disease. 3 Innocenti P.

Morrow E.H.

Dowling D.K. Experimental evidence supports a sex-specific selective sieve in mitochondrial genome evolution. mf = −0.0081, p = 0.978, corrected rate of senescence: r mf = −0.205, p = 0.481). Nonetheless, the mutations might have had positive effects on other components of female life history (e.g., fecundity and fertility) that went unmeasured in this study, with antagonistic effects on male components of aging. The mitochondrial mutations responsible for the male-specific variance in longevity and aging could in theory accumulate if they were neutral [], nearly neutral [], or positive [] in effect when expressed in females. The lack of any significant mitochondrial genetic variance for female longevity and rates of senescence indicates that the mutations involved exerted only slight effects, if any, on female components of aging. These mutations might then have accrued under mutation-selection balance augmented by drift (if slightly deleterious to females), drift alone (if strictly neutral in females), or under positive selection (if beneficial to females). Although we are unable to distinguish between these three possibilities, it is plausible that all three contributed to the mtDNA-induced effects on males. Our results allow us to rule out the possibility that the mutations involved were consistently strongly sexually antagonistic in their effect on aging (in particular, a scenario whereby mutations that were of larger benefit to females were of larger harm to males). In such a case, we would have expected to have observed mitochondrial genetic variance for female longevity and senescence in the first instance, coupled with negative intersexual correlations for these traits across the mitochondrial haplotypes. This was not found (longevity: r= −0.0081, p = 0.978, corrected rate of senescence: r= −0.205, p = 0.481). Nonetheless, the mutations might have had positive effects on other components of female life history (e.g., fecundity and fertility) that went unmeasured in this study, with antagonistic effects on male components of aging.