Positive germline selection of mtDNA Mutations: evidence from the oocyte

Abstract Purifying selection of mtDNA mutations is a vital process that cleanses the mitochondrial genome of detrimental variants that may endanger individuals and populations. A common measure of purifying selection is the increase of average synonymity by reduction the proportion of mostly detrimental non-synonymous mutations. The mechanisms underlying purifying selection are still debated. The Makova group has recently published high-fidelity analysis of mtDNA mutations in individual human oocytes (Arbeithuber et al., 2025). The authors observed a decrease in the proportion of potentially detrimental coding and conservative mutations at higher mutant fractions (MFs) and interpreted this as purifying selection removing detrimental mutations at higher MFs. We noted, however, that, in contrast to what would be expected under purifying selection, the synonymity of oocyte mutations was very low and decreased, rather than increased, at higher MFs. We hypothesized that this inconsistency resulted from non-synonymous mutations being prone to strong positive selection which erroneously made coding mutations appear negatively selected in comparison. In support of our hypothesis, we show that non-coding oocytes mutations indeed are under strong positive selection. To alleviate this setback, we reanalyzed the data using a new metric of intracellular clonal selection and neutral synonymous mutations as the reference. We demonstrated that coding mutations are in fact under prevailing positive selection. This is in line with previous estimates of positive selection in primordial germ cells (PGCs) and in mother-child pairs. Importantly, “prevailing positive selection” does not imply the absence of negative selection. We show that specific types of mutations may be under prevailing purifying selection (e.g., the Co1 gene). Of note, this prevailing positive selection pertains only to the most recent, germline mtDNA mutations which have not been yet inherited into the next generation. Purifying selection steps in as germline mutations proceed to subsequent generations. The implications of these findings and the potential benefits of positive selection of detrimental mtDNA mutations are discussed. Graphical summary Germline mtDNA mutations fuel evolution, shape population genomics, and cause mitochondrial disease. Yet it remains unresolved whether mtDNA selection in the germline is predominantly purifying or positive. A recent high-fidelity single-oocyte study (Arbeithuber et al., 2025) reported purifying(negative) selection on germline mutations at elevated mutant fractions (MFs). However, the low synonymity of oocyte mutations and concerns about using non-coding mutations as a reference for estimating selection prompted us to reanalyze the data. In single cells, mtDNA mutations are subject to genetic drift which randomly expands mtDNA clones. In this context, selection means that some mutant clones expand systematically faster (positive selection), or slower/get lost (purifying selection) than expected by random drift. Figure A. Cumulative proportion curves of relevant types of mutations (color coded). The corresponding selection metrics, 𝒮 0.01 , and Monte-Carlo p-values are shown, analyses for other thresholds: tables 2 and S1 In Figure 1 , datapoints represent clones of mutations ranked by their size and plotted vs. their cumulative contribution to the mutational pool (in reverse order). The resulting cumulative curves represent the collective expansion of clones of mutations of each type. As previously shown by direct simulations (Franco et al., 2025), the slope of the curve qualitatively depicts the relative intensity of clonal expansion. The green curve consists of synonymous (neutral) mutations and thus defines the trajectory of expansion driven by neutral genetic drift. Curves that diverge upward from the neutral green curve imply faster-than-neutral expansion, i.e., positive selection in corresponding mutation types, and those that diverge downward (grey Co1 curve) imply negative selection. To estimate selection, we first defined ‘extent of clonal expansion’, ℰ t ( mutant class ), a measure of overall clonal e xpansion, i.e., the proportion of aggregate fraction of mutants of a particular class in clones that exceeded a specific size (i.e., MF). For example, ℰ 0.01 ( coding ) is the ratio of aggregate mutant fraction of all large clones (MF>0.01) of coding mutations, divided by the aggregate mutant fraction of all coding mutations. ℰ t changes with t , but at each t , it permits us to compare the extent of expansion between different classes of mutations (e.g., coding vs. non-coding). Selection at the intracellular level manifests as an acceleration or deceleration of the expansion (or loss) of mutant clones relative to the expansion expected under random drift, as represented by synonymous mutations. Accordingly, \a measure of clonal selection for a tested mutation class, denoted 𝒮 t (tested) , is defined as the excess clonal expansion in the tested mutations over expansion of synonymous mutations, normalized by the expansion of synonymous mutations: In Fig.1, 𝒮 0.01 and p-values demonstrate: Strong positive selection of noncoding mutations (blue). Positive selection of coding mutations (orange). A higher positive selection in conservative (i.e., more detrimental) coding mutations (red). So, selection may be driven by the detrimental effects of mtDNA mutations. Negative selection in the Co1 gene (grey). Thus, negative/purifying selection does exist, but dominates only in specific small regions (like Co1). Selection in non-coding mutations starts at low mutant fractions, coding at higher mutant fractions. This confirms the prevalence of positive selection and clarifies why Arbeithuber et al. perceived selection as purifying. The authors compared coding to non-coding mutations. The latter are under stronger positive selection than coding mutations. Thus, coding mutations appear to be under relative negative selection, but only in comparison to non-synonymous mutations, not in abso