Many reproductive proteins from diverse taxa evolve rapidly and adaptively. These proteins are typically involved in late stages of reproduction such as sperm development and fertilization, and are more often functional in males than females. Surprisingly, many germline stem cell (GSC) regulatory genes, which are essential for the earliest stages of reproduction, also evolve adaptively in Drosophila. One example is the bag of marbles (bam) gene, which is required for GSC differentiation and germline cyst development in females and for regulating mitotic divisions and entry to spermatocyte differentiation in males. Here we show that the extensive divergence of bam between Drosophila melanogaster and D. simulans affects bam function in females but has no apparent effect in males. We further find that infection with Wolbachia pipientis, an endosymbiotic bacterium that can affect host reproduction through various mechanisms, partially suppresses female sterility caused by bam mutations in D. melanogaster and interacts differentially with bam orthologs from D. melanogaster and D. simulans. We propose that the adaptive evolution of bam has been driven at least in part by the long-term interactions between Drosophila species and Wolbachia. More generally, we suggest that microbial infections of the germline may explain the unexpected pattern of evolution of several GSC regulatory genes.

Coevolution between Drosophila and its endosymbiont Wolbachia pipientis has many intriguing aspects. For example, Drosophila ananassae hosts two forms of W. pipientis genomes: One being the infectious bacterial genome and the other integrated into the host nuclear genome. Here, we characterize the infectious and integrated genomes of W. pipientis infecting D. ananassae (wAna), by genome sequencing 15 strains of D. ananassae that have either the infectious or integrated wAna genomes. Results indicate evolutionarily stable maternal transmission for the infectious wAna genome suggesting a relatively long-term coevolution with its host. In contrast, the integrated wAna genome showed pseudogene-like characteristics accumulating many variants that are predicted to have deleterious effects if present in an infectious bacterial genome. Phylogenomic analysis of sequence variation together with genotyping by polymerase chain reaction of large structural variations indicated several wAna variants among the eight infectious wAna genomes. In contrast, only a single wAna variant was found among the seven integrated wAna genomes examined in lines from Africa, south Asia, and south Pacific islands suggesting that the integration occurred once from a single infectious wAna genome and then spread geographically. Further analysis revealed that for all D. ananassae we examined with the integrated wAna genomes, the majority of the integrated wAna genomic regions is represented in at least two copies suggesting a double integration or single integration followed by an integrated genome duplication. The possible evolutionary mechanism underlying the widespread geographical presence of the duplicate integration of the wAna genome is an intriguing question remaining to be answered.

In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis. Previously, we documented that the W. pipientis variant, wMel, rescued the fertility of the bamBW hypomorphic mutant as a transheterozygote over a bam null. However, bamBW was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying bamBW accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation (bamL255F ) and a new bam null disruption mutation into the w1118 isogenic background. We assess the fertility of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/bamL255F mutant females, each infected individually with ten W. pipientis wMel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. Additionally, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.

In Drosophila melanogaster, a key germline stem cell (GSC) differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between sibling species D. melanogaster and D. simulans, but not in the outgroup species D. ananassae. Here, we test the null hypothesis that bam’s differentiation function is conserved between D. melanogaster and four additional Drosophila species in the melanogaster species group spanning approximately 30 million years of divergence. Surprisingly, we demonstrate that bam is not necessary for oogenesis or spermatogenesis in D. teissieri nor is bam necessary for spermatogenesis in D. ananassae. Remarkably bam function may change on a relatively short time scale. We further report tests of neutral sequence evolution at bam in additional species of Drosophila and find a positive, but not perfect, correlation between evidence for positive selection at bam and its essential role in GSC regulation and fertility for both males and females. Further characterization of bam function in more divergent lineages will be necessary to distinguish between bam's critical gametogenesis role being newly derived in D. melanogaster, D. simulans, D. yakuba, and D. ananassae females or it being basal to the genus and subsequently lost in numerous lineages.