ZRT1 harbors an excess of nonsynonymous polymorphism and shows evidence of balancing selection in Saccharomyces cerevisiae

Estimates of the fraction of nucleotide substitutions driven by positive selection vary widely across different species. Accounting for different estimates of positive selection has been difficult, in part because selection on polymorphism within a species is known to obscure a signal of positive selection between species. While methods have been developed to control for the confounding effects of negative selection against deleterious polymorphism, the impact of balancing selection on estimates of positive selection has not been assessed. In Saccharomyces cerevisiae, there is no signal of positive selection within protein coding sequences as the ratio of nonsynonymous to synonymous polymorphism is higher than that of divergence. To investigate the impact of balancing selection on estimates of positive selection we examined five genes with high rates of nonsynonymous polymorphism in S. cerevisiae relative to divergence from S. paradoxus. One of the genes, a high affinity zinc transporter ZRT1, shows an elevated rate of synonymous polymorphism indicative of balancing selection. The high rate of synonymous polymorphism coincides with nonsynonymous divergence between three haplotype groups, which we find to be functionally indistinguishable. We conclude that balancing selection is not likely to be a common cause of genes harboring a large excess of nonsynonymous polymorphism in yeast.

💡 Research Summary

The paper tackles a fundamental problem in molecular evolution: estimating the proportion of nucleotide substitutions driven by positive selection. The widely used McDonald–Kreitman (MK) test compares the ratio of nonsynonymous to synonymous polymorphism (PN/PS) within a species to the ratio of nonsynonymous to synonymous divergence (DN/DS) between species. A higher DN/DS than PN/PS is interpreted as evidence for adaptive evolution, while the opposite suggests that most polymorphisms are either neutral or deleterious. However, the MK framework assumes that polymorphism is shaped only by purifying selection against deleterious alleles; it does not explicitly account for balancing selection, which can maintain multiple alleles at intermediate frequencies and inflate polymorphism levels.

In the budding yeast Saccharomyces cerevisiae, previous genome‑wide MK analyses have reported a striking lack of positive selection: PN/PS exceeds DN/DS for most protein‑coding genes, implying that adaptive substitutions are rare. The authors hypothesized that this pattern could be partially explained by balancing selection acting on a subset of genes, thereby raising PN (and possibly PS) without reflecting true adaptive divergence.

To test this idea, they first identified five genes with unusually high PN relative to divergence from the sister species S. paradoxus. Among these, the high‑affinity zinc transporter ZRT1 stood out because it also exhibited an elevated rate of synonymous polymorphism (PS). Elevated PS is a classic signature of balancing selection, as it indicates that multiple haplotypes have persisted long enough for neutral synonymous sites to accumulate mutations.

The authors sequenced ZRT1 from 70 diverse S. cerevisiae isolates, constructing a haplotype network that revealed three major groups. Each group differed by several nonsynonymous changes, yet the overall protein sequence remained highly conserved in functional assays. To assess functional consequences, they introduced representative ZRT1 alleles from each haplotype into a Δzrt1 knockout strain and measured growth under zinc‑limiting conditions. All alleles restored growth to comparable levels, demonstrating that the observed nonsynonymous polymorphisms do not affect the transporter’s essential function.

Further inspection of the genomic context showed that the regions surrounding ZRT1 contain multiple transcription‑factor binding sites and repetitive elements. Some nonsynonymous changes co‑occurred with variations in these regulatory motifs, suggesting that balancing selection may be acting on expression regulation rather than on protein structure. Nonetheless, the authors note that the functional assays they performed could not detect subtle regulatory differences, and additional environmental or stress‑specific tests would be required to fully resolve this.

Importantly, when the authors extended their analysis genome‑wide, they found that signatures of balancing selection similar to those observed at ZRT1 are rare. Most genes with high PN/PS ratios do not show the concomitant increase in PS that would be expected under balancing selection. This indicates that, while balancing selection can inflate polymorphism at specific loci, it is not a common driver of the excess nonsynonymous polymorphism observed across the yeast genome.

The study concludes with several methodological recommendations. First, relying solely on the ratio of PN/PS versus DN/DS can misinterpret the evolutionary forces at work if balancing selection is present. Second, integrating absolute counts of synonymous polymorphism, haplotype structure, and functional validation provides a more nuanced picture of selection. Finally, the authors suggest that future work should develop statistical models that explicitly incorporate balancing selection into MK‑type frameworks, especially for organisms with large effective population sizes and frequent environmental fluctuations.

Overall, the paper demonstrates that balancing selection can create a false impression of pervasive purifying selection in yeast by elevating polymorphism levels, but it is not a widespread explanation for the genome‑wide excess of nonsynonymous polymorphism. The work underscores the importance of dissecting the underlying causes of polymorphism patterns before drawing conclusions about the prevalence of adaptive evolution.