Selective pressures on DNA sequences often result in signatures of departures from neutral evolution that can be captured by the McDonald-Kreitman (MK) test. However, the nature of such selective forces mostly remains unknown to the experimentalists. Here we use the bag of marbles (bam) gene in Drosophila to investigate different types of driving forces behind positive selection. We examine two evolutionary models for bam. The Conflict model originates from a conflict of fitness between Drosophila and Wolbachia that causes reciprocal adaptations in each, resulting in diversifying selection on the bam protein. In the alternative Buffering model, Wolbachia protects bam from deleterious mutations during an infection and thereby allows such mutations to accumulate and even fix in the population. If Wolbachia is subsequently lost from the species, mutations that revert the gene back towards its original biological function become advantageous. We use simulations to show that both models produce signals of positive selection, though the levels of positive selection under the Conflict model are more easily detected by the MK test. By fitting the two models to the empirical divergence of D. melanogaster from an inferred ancestral sequence, we found that the Conflict model reproduced strong signals of positive selection like those observed empirically, while the Buffering model better recapitulated the physicochemical signatures of the amino acid sequence evolution at bam. Our demonstration that the Buffering model can lead to positive selection suggests a novel mechanism that needs to be considered behind observed signals of positive selection on protein coding genes.

The D. melanogaster protein coding gene bag of marbles (bam) plays a key role in early male and female reproduction by forming complexes with partner proteins to promote differentiation in gametogenesis. Like another germline gene, Sex lethal, bam genetically interacts with the endosymbiont Wolbachia, as Wolbachia rescues the reduced fertility of a bam hypomorphic mutant. Here, we explored the specificity of the bam-Wolbachia interaction by generating 22 new bam mutants. We find Wolbachia rescues all six mutants of partially reduced fertility, but none of the four with severely reduced fertility. There is no specificity between the rescue and the known binding regions of bam, suggesting Wolbachia does not interact with one singular bam partner to rescue fertility. We further tested if Wolbachia interacts with bam in a non-specific way, by increasing bam levels or acting upstream in germline stem cells. However, a fertility assessment of a bam RNAi knockdown mutant reveals that Wolbachia rescue is specific to functionally mutant bam alleles and we find no obvious evidence of Wolbachia interaction with germline stem cells in bam mutants.

Selective pressures on DNA sequences often result in departures from neutral evolution that can be captured by the McDonald-Kreitman (MK) test. However, the nature of such selective forces often remains unknown to experimentalists. Amino acid fixations driven by natural selection in protein coding genes are commonly associated with a genetic arms race or changing biological purposes, leading to proteins with new functionality. Here, we evaluate the expectations of population genetic patterns under a buffering mechanism driving selective amino acids to fixation, which is motivated by an observed phenotypic rescue of otherwise deleterious nonsynonymous substitutions at bag of marbles (bam) and Sex lethal (Sxl) in Drosophila melanogaster. These two genes were shown to experience strong episodic bursts of natural selection potentially due to infections of the endosymbiotic bacteria Wolbachia observed among multiple Drosophila species. Using simulations to implement and evaluate the evolutionary dynamics of a Wolbachia buffering model, we demonstrate that selectively fixed amino acid replacements will occur, but that the proportion of adaptive amino acid fixations and the statistical power of the MK test to detect the departure from an equilibrium neutral model are both significantly lower than seen for an arms race/change-in-function model that favors proteins with diversified amino acids. We find that the observed selection pattern at bam in a natural population of D. melanogaster is more consistent with an arms race model than with the buffering model.

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