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.

The combination of recent advances in both genomic and gene editing technologies have opened up new possibilities for assessing the functional consequences and drivers of positive selection. In Drosophila melanogaster, a key germline stem cell differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between its D. melanogaster and its sibling species D. simulans, but not in the outgroup species D. ananassae. We previously hypothesized that a genetic conflict with the maternally inherited, intracellular bacteria W. pipientis could be driving the adaptive evolution of bam as W. pipientis increases the fertility of a bam partial loss of function mutant. However, we have not been able to further test this hypothesis by assessing bam variation in other Drosophila lineages and their interactions with W. pipientis because bam function has not been examined in non-melanogaster Drosophila species. Since bam is rapidly evolving at the protein level, its function may not be conserved between species, and therefore different evolutionary pressures may be shaping bam in individual lineages. Here, we ask if bam is necessary for GSC daughter differentiation in five Drosophila species in the melanogaster species group that span approximately 15 million years of divergence and show different patterns of nucleotide sequence evolution at bam. We find that bam function is not fully conserved across these species, and that bam function may change on a relatively short time scale. Ultimately, we conclude that a simple gain in function as the germline stem cell differentiation factor alone does not explain our population genetic and functional genetic results we have observed. Our findings provide a foundation on which to explore the evolution of bam as a GSC differentiation factor and its interactions with W. pipientis in specific lineages.

Wolbachia is an intracellular, maternally inherited endosymbiotic bacteria that infects over 65% of insects and manipulates their reproduction for its own transmission. In Drosophila melanogaster, Wolbachia genetically interacts with the adaptively evolving germline stem cell gene bag of marbles (bam). Since Wolbachia must enter the host female germline to propagate, one hypothesis is that Wolbachia and bam are in a genetic conflict for control of oogenesis. In order to understand if Wolbachia could be driving the adaptive evolution of bam, we must understand the nature of the genetic interaction between bam and Wolbachia. Previously, we documented that infection with the wMel strain of Wolbachia rescued the fertility and cytological ovarian defect of a bam hypomorphic mutant. However, this mutant was generated over 20 years ago in an uncontrolled and variable genetic background, and thus we have been unable to perform controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam mutation into the w1118 isogenic background as well as generated a bam null allele in the same background. We confirm that the amino acid replacement results in the bam hypomorphic phenotype previously described when expressed over a bam null allele. We assess the female fertility of wildtype bam, a bam transheterozygous hypomorph/null mutant, and a homozygous bam hypomorphic mutant, each infected individually with 10 diverse Wolbachia variants. Overall, we find that the Wolbachia variants tested here do not generally increase bam+ female fertility, but they do rescue bam hypomorphic defects with variation in the effect size of some variants on female fertility.

Will sex‐specificparasite exposure lead to sexually antagonistic immune trait fitness optima? Despite female stickleback encountering parasites at a higher rate, De Lisle and Bolnick found no evidence of sexual conflict related to their primary immune trait. Both male and female fish suffered high costs of infection and immune response on reproduction. If costs of both immunity and infection are high and concordant, the sexual conflict will not arise despite different parasite exposure rates.