Overdrive is essential for targeted sperm elimination by Segregation Distorter

Abstract

Intra-genomic conflict driven by selfish chromosomes is a powerful force that shapes the evolution of genomes and species. In the male germline, many selfish chromosomes bias transmission in their own favor by eliminating spermatids bearing the competing homologous chromosomes. However, the mechanisms of targeted gamete elimination remain mysterious. Here, we show that Overdrive (Ovd), a gene required for both segregation distortion and male sterility in Drosophila pseudoobscura hybrids, is broadly conserved in Dipteran insects but dispensable for viability and fertility. In D. melanogaster, Ovd is required for targeted Responder spermatid elimination after the histone-to-protamine transition in the classical Segregation Distorter system. We propose that Ovd functions as a general spermatid quality checkpoint that is hijacked by independent selfish chromosomes to eliminate competing gametes.

Mendelian segregation is the foundation on which our understanding of genetics and population genetic theory rests. Segregation distorters are selfish chromosomes that violate Mendel’s Laws by over-representing themselves in the mature gamete pool of individuals that carry them (1, 2). In the male germline, distorters act by selectively eliminating spermatozoa that carry their competing homologous chromosomes (3). Although a few segregation distorter genes have been identified, the mechanisms underlying the selective elimination of competing gametes remain poorly understood (4–7).

In Drosophila, some selfish chromosomes act by directly disrupting target chromosome segregation during meiosis (5). In other distorter systems, drive mechanisms involve the elimination of post-meiotic spermatids (8–10). During Drosophila spermiogenesis, groups of interconnected spermatids elongate and condense their nuclei into highly compact, needle-shaped sperm heads (11, 12). This is achieved through a global chromatin remodeling process where almost all histones are progressively replaced first by transition proteins, such as Tpl94D, which are in turn replaced by Sperm Nuclear Basic Proteins (SNBPs), such as the Protamine-like ProtA and ProtB (13, 14). One of the best-described distorter systems is Segregation Distorter (SD) in Drosophila melanogaster (8, 15). In this system, selfish SD chromosomes selectively disrupt the histone-to-protamine transition in spermatid nuclei that carry an SD-sensitive homolog known as Responder (Rsp) (16–18). In SD/Rsp males, Rsp spermatid nuclei fail to condense properly and are eliminated during spermatid individualization. This is a common stage of spermiogenic failure observed in a variety of perturbations including male sterile mutants, high temperature induced sterility, etc. (19, 20). This has led to speculation that a general male germline checkpoint may exist during spermatid individualization (16, 21, 22). However, the mechanisms of elimination of Rsp sperm remain unknown.

Intra-genomic conflict involving segregation distorters provides a leading explanation for the rapid evolution of hybrid male sterility but evidence for this idea remains scarce (23–27). A direct line of evidence connecting intra-genomic conflict to speciation comes from hybrids between two very young subspecies: Drosophila pseudoobscura bogotana (Bogota) and Drosophila pseudoobscura pseudoobscura (USA) (28). F1 hybrid males from crosses between Bogota mothers and USA fathers are nearly sterile but become weakly fertile when aged (29). These aged hybrid males produce nearly all female progeny due to segregation distortion by the Bogota X-chromosome. Although the genetic basis of segregation distortion and male sterility in these hybrids involves a complex genetic architecture, a single gene Overdrive (Ovd) is involved in both phenomena (7, 30). Yet, little is known about the normal function of Ovd within species or its mechanistic role in segregation distortion and male sterility between species.

First, we wanted to understand the origins and patterns of molecular evolution of Ovd. To date the origins of Ovd, we used reciprocal BLAST and synteny analyses to search for orthologs of Ovd across Diptera. Ovd is present in all Drosophilidae and Schizophora species analyzed, but not detected outside of Schizophora (Figure 1A). Based on time-calibrated phylogenies of Dipterans and Schizophora (31, 32), we conclude that Ovd originated at least 60 million years ago in the ancestor of Schizophora and has since been maintained without loss. Hybrid sterility and segregation distortion genes tend to change rapidly under recurrent positive selection (5, 7, 33, 34). We used PAML (Phylogenetic Analysis by Maximum Likelihood) (35) to search for signatures of recurrent positive selection in Ovd in the melanogaster and obscura clades. Surprisingly, we did not observe high d_N/d_s values for Ovd, nor did any individual lineage show accelerated accumulation of nonsynonymous substitutions (Figure 1B). Models of molecular evolution allowing for positive selection were not a significantly better fit than those that excluded it (p = 0.972 for melanogaster group species and p = 0.999 for obscura group species, likelihood ratio test). No individual residues within Ovd showed signatures of positive selection. Despite being involved in both segregation distortion and hybrid sterility, Ovd is surprisingly conserved and shows no signs of accelerated evolution.

Figure 1: Overdrive has been maintained without loss since its origin at least 65 million years ago and does not evolve rapidly under positive selection.

(A) We searched for orthologs of Ovd in 139 species of Dipterans using reciprocal BLAST and synteny checks. Orthologs of Ovd were detected in all Schizophora species tested but not detected outside of Schizophora. Time-calibrated phylogenies of Schizophora and Dipterans allow time estimates for our phylogeny (31, 32). (B) Cladogram of Ovd sequence evolution across melanogaster (orange) and pseudoobscura (blue) groups with d_N/d_S values for each branch calculated with the PAML package. Ovd does not evolve under recurrent positive selection as denoted by p-values from M7 vs M8 model comparisons.

Because Ovd is a conserved gene and can cause sterility and segregation distortion in Bogota-USA hybrids, we wondered if it is essential for male germline development. We used CRISPR/Cas9-based gene editing to generate null mutants of Ovd in both the USA and Bogota subspecies (Figure 2A). Surprisingly, Ovd-null individuals from either subspecies were fertile. We observed no difference in male fertility, sex-chromosome segregation ratios, or viability between Ovd^Δ and wild-type controls in either subspecies (Figure 2B, 2C, 2D). Ovd thus appears to be dispensable for viability and male germline development in D. pseudoobscura.

Figure 2: Ovd is non-essential in D. pseudoobscura in pure species but removing it restores fertility and normal segregation in interspecies F1 hybrid males.

(A) Schematic showing CRISPR-Cas9 NHEJ knockouts of Ovd where nearly the whole coding region is deleted in USA and Bogota subspecies of D. pseudoobscura. (B) Viability of Ovd^Δ and wild-type crosses in both D. pseudoobscura subspecies. Viability of Ovd^Δ and wild-type individuals are not significantly different (p=0.10 for USA; p=0.056 for Bogota; two sample Student’s t-test) (C) Male fertility of Ovd^Δ and wild-type individuals are not significantly different (p=0.73 for USA; p=0.54 for Bogota; two sample Student’s t-test). (D) Sex-ratio of progeny produced by Ovd^Δ and wild-type males in both D. pseudoobscura subspecies. Progeny sex-ratios between Ovd^Δ and wild-type individuals are not statistically significant (p=0.87 for USA; p=0.33 for Bogota; two sample Student’s t-test). (E) Crossing schematic and fertility of F1 hybrid males produced by crossing either Ovd^Δ or Ovd⁺ Bogota females to USA males. Deleting Ovd restores fertility to otherwise sterile F1 hybrid males (p=0.002; Wilcoxon test).

Previously, we showed that replacing an incompatible Bogota allele of Ovd with a compatible USA allele restores fertility and normal segregation to F1 hybrid males (7). However, it is not clear whether F1 hybrid males are sterile because they lack a USA allele of Ovd that provides an essential function or because the presence of the Bogota allele blocks male germline development. To discriminate between these possibilities, we crossed Bogota females with or without Ovd to USA males to produce F1 hybrid males (Figure 2E). In the control Ovd⁺ cross, all F1 hybrid males were sterile, as expected. In contrast, Ovd^Δ hybrid F1 males were fertile and showed normal sex-chromosome segregation ratios. We conclude that Ovd is not required for normal male germline development in pure species, but the presence of Bogota Ovd can block male germline development in hybrids.

Although Ovd is not an essential gene, its conservation across long evolutionary timescales suggests that it may have an important function. We hypothesized that Ovd may be involved in a putative male germline checkpoint. An Ovd-mediated checkpoint could explain three distinct observations. First, Ovd is dispensable for normal germline development because removing such a checkpoint would have no discernable effect in the absence of perturbation. For example, null mutants of known DNA damage checkpoint genes, such as p53 and Chk-2 kinase, are viable and develop normally, but only show an effect in the presence of DNA damage (36, 37). Second, removing Ovd restores normal segregation in hybrids. If an Ovd-mediated checkpoint specifically arrests only spermatids targeted for elimination, this would cause segregation distortion. Third, removing Ovd restores hybrid male fertility. If an Ovd-mediated checkpoint arrests all spermatids, this would cause sterility (Figure 3A).

Figure 3: Overdrive is non-essential for viability and fertility in D. melanogaster

(A) A model for a role for Ovd in a germline checkpoint can explain why Ovd is dispensable for male germline development but is required for segregation distortion and male sterility. (B) Schematic for the design of D. melanogaster Ovd knockout. We use CRISPR-Cas9 to replace the coding sequence of Ovd with DsRed, a visible marker. (C) Progeny counts for Ovd^Δ crosses show that it is not essential for viability. Ovd^Δ shows no viability deficit relative to TM3 balancer. The number of progeny of Ovd^Δ /TM3 heterozygote pairs did not deviate significantly from the expected Mendelian ratio of 2:1 Ovd^Δ/TM3:Ovd^Δ/Ovd^Δ (p = 0.08, Cochran-Mantel-Haenszel test). (D) Male fertility for Ovd^Δ. The number of progeny produced per male for wildtype Ovd, heterozygous for Ovd^Δ, and homozygous for Ovd^Δ are not significantly different from each other (one-way ANOVA; p = 0.124) (E) Sex-ratio of progeny produced by males wildtype for Ovd, heterozygous for Ovd^Δ, and homozygous for Ovd^Δ are not significantly differently from each other (one-way ANOVA; p = 0.875). (F) Nondisjunction rates for sex chromosomes and the fourth chromosome in w¹¹¹⁸ and Ovd^Δmales. Sex chromosome nondisjunction frequencies do not differ significantly between the genotypes, nor do fourth chromosome nondisjunction frequencies. (G) k-values for SD/Rsp^ss males with wild-type Ovd, heterozygous Ovd^Δ and homozygous Ovd^Δ for SD-72 and SD-5 chromosomes. Homozygous Ovd^Δ males show reduced strength of distortion (p < 0.05, Three-way ANOVA, Tukey’s Honestly Significant Differences test). (H) Progeny count per male for SD/Rsp^ss males with wild-type Ovd, heterozygous Ovd^Δ and homozygous Ovd^Δ. Homozygous Ovd^Δ males produce more progeny than wild-type Ovd and heterozygous Ovd^Δ males in an SD background (p < 0.05, Three-way ANOVA, Tukey’s Honestly Significant Differences test)

To test its hypothetical male germline checkpoint function, we switched to studying Ovd in Drosophila melanogaster, where more genetic and cytological tools are available. The predicted Ovd protein has a MADF DNA-binding domain and a BESS protein-interaction domain. MADF-BESS domain-containing proteins are involved in diverse processes such as transcriptional regulation and chromatin remodeling (38–40). We first studied the expression and localization pattern of Ovd in testes by introducing an N-terminus GFP-tag at the endogenous locus using CRISPR/Cas9. Testis imaging shows that Ovd is a nuclear protein expressed during spermatogenesis from male germline stem cells through the histone-to-protamine transition stages (Supplemental figure S1). Next, we used CRISPR/Cas9 to delete Ovd in D. melanogaster (Figure 3B, Supplemental figure S2). We observed little difference between male fertility, progeny sex-ratio, viability, or chromosomal non-disjunction rates between Ovd^Δ and wild type controls (Figure 3C, 3D, 3E, 3F). Like in D. pseudoobscura, Ovd is not essential for normal male germline development in D. melanogaster.

If Ovd functions in a male germline checkpoint, then sperm that are normally eliminated in response to perturbations may develop to maturity when Ovd is removed. We turned to the Segregation Distorter (SD) system to perturb male germline development in D. melanogaster. We hypothesized that when Ovd is removed, SD chromosomes would fail to eliminate gametes bearing homologous chromosomes with Rsp satellite repeats. We first measured the strength of distortion by the SD-72 chromosome against Rsp^ss, a supersensitive Rsp chromosome, and observed near-complete distortion as expected (k > 0.99). We then measured the strength of distortion by the same SD-72 chromosome against Rsp^ss in an Ovd^Δ homozygous background. We observed a strong reduction in the strength of distortion by SD-72 in the absence of Ovd (k ∼0.65; Figure 3G). We repeated our measurements with an independently isolated SD chromosome, SD-5 (8). Again, we found a strong reduction in the strength of distortion by SD-5 in an Ovd homozygous null background (Figure 3H). SD thus requires Ovd for full-strength distortion. Together, our results show that Ovd is required for both sex-chromosome distortion in D. pseudoobscura, and autosomal distortion in D. melanogaster.

We then investigated spermiogenesis in SD males in the presence or absence of Ovd. In SD-72/Rsp^ss males, spermiogenesis appears highly disturbed with many abnormal spermatid nuclei being eliminated, a typical phenotype of SD males (16). In contrast, spermiogenesis in SD-72/Rsp^ss males appeared overall normal in an Ovd^Δ homozygous background (Figure 4A). We then examined the histone-to-protamine transition in these males using Tpl94D-RFP and ProtB-GFP transgenes. As previously observed (16), about half spermatid nuclei showed a delay in protamine incorporation and elongation defects in SD males (Figure 4B). These nuclei fail to individualize and are eliminated before their release into the seminal vesicle, the sperm storage organ in males. In contrast, in an Ovd^Δ homozygous background, nearly all spermatids incorporate protamine and elongate properly (Figure 4A). Removing Ovd thus appears to restore proper histone-to-protamine transition and individualization to Rsp sperm even in the presence of SD. To assess the quality of sperm nuclei produced by SD males that lack Ovd, we stained seminal vesicles with an antibody that recognizes double stranded DNA (dsDNA). We have previously shown that this antibody can stain improperly compacted chromatin in mature Rsp sperm nuclei but not properly condensed sperm nuclei which are refractory to immuno-staining (16). Remarkably, we observed a significant increase of anti-dsDNA positive sperm in seminal vesicles of SD/Rsp^ss males lacking Ovd compared to SD/Rsp^ss males with Ovd (30% decondensed vs. 4% decondensed) (Figure 4C, 4D). We conclude that, when Ovd is removed, Rsp spermatids differentiate to maturity despite chromatin condensation defects caused by SD. Because these sperm produce viable Rsp^ss progeny, these condensation defects are not fatal. Taken together, these results indicate that Ovd does not itself cause condensation defects in mature sperm but is instead required for the selective elimination of improperly condensed Rsp spermatids during individualization.

Figure 4: Ovd is required for selective elimination of Rsp sperm in the presence of SD in D. melanogaster.

(A) Cysts of 64 spermatids in testes from SD-72/Rsp^ss males with and without Ovd stained with DAPI. With Ovd, cysts are disorganized, half spermatid nuclei fail to elongate and degenerate (arrows). Without Ovd, spermatid elongation is restored. Bar: 10 µm (B) The histone-to-protamine transition of SD-72/Rsp^ssmales with and without Ovd. ProtB-GFP and Tpl94D-RFP transgenes are used to visualize protamines and transition proteins, respectively. With Ovd, half of the developing spermatids fail to incorporate protamines and do not elongate properly (arrows). Without Ovd, all sperm undergo the histone-to-protamine transition and elongation appropriately. Bar: 10 µm (C) Seminal vesicles of SD-72/Rsp^ss males with and without Ovd stained with an anti-dsDNA antibody to reveal improperly condensed sperm nuclei. Bar: 50 µm. (D) Quantification of decondensed sperm in seminal vesicles of SD-72/Rsp^ss males with and without Ovd, with w¹¹¹⁸and Ovd^Δ/Ovd^Δ as controls. There is a significant increase in number of anti-dsDNA positive sperm nuclei from SD-72/Rsp^ss; Ovd^Δ/Ovd^Δmales compared to SD-72/Rsp^ss; Ovd⁺/Ovd⁺males (Wilcoxon Test; p < 0.01). Each dot corresponds to a male, decondensed sperm from one seminal vesicle was quantified per male.

Ovd is necessary for sex-chromosome segregation distortion and male sterility in D. pseudoobscura hybrids, and for autosomal segregation distortion in D. melanogaster SD males. Although Ovd is dispensable for normal spermiogenesis, it is evolutionarily conserved and blocks proper histone-to-protamine transition in Rsp spermatids in D. melanogaster SD males. Together, these properties suggest that the normal function of Ovd may involve the elimination of abnormal gametes during male germline development, which may get co-opted by selfish chromosomes.

It is worth noting the relationship between the genomic location of Ovd and its rate of evolutionary change. In D. pseudoobscura, Ovd is located on the distorting Bogota X-chromosome and rapidly accumulates non-synonymous changes in the Bogota lineage (7). In D. melanogaster, where Ovd is unlinked to the distorter, it is not rapidly evolving (Ovd is on the third chromosome, SD is on the second chromosome). Ovd thus evolves in a manner consistent with being locked in an evolutionary arms race in D. pseudoobscura but appears to act as an unwitting accomplice to SD in D. melanogaster. Nevertheless, Ovd is essential for two distant non-orthologous segregation distorters raising the possibility that independent selfish chromosomes may operate through shared mechanisms. Together, our findings open the door to understanding how mechanisms of gamete elimination may be involved in the evolution of selfish chromosomes within species and contribute to hybrid sterility between species.

SUPPLEMENTAL INFORMATION

MATERIALS AND METHODS

Experimental Resources

Plasmids and Primers

Fly Strains

CRISPR knockout of Overdrive in D. pseudoobscura

Overdrive knockout (OvdΔ) flies were generated using CRISPR/Cas9 based non-homologous end joining to create deletions of Ovd in D. pseudoobscura pseudoobscura (USA) and D. pseudoobscura bogotana (Bogota). First, gRNAs 5-8 (see plasmids and primers) were designed using CRISPR Optimal Target Finder (42). gRNAs were then complexed with Alt-R® CRISPR-Cas9 tracrRNA (IDT) and mixed with Alt-R® S.p. Cas9 Nuclease V3 (IDT). USA and Bogota embryos were injected with the complexed gRNAs and Cas9 nuclease.

To verify the loss of Ovd, primers flanking the native Ovd region were designed (SR13F and SR13R). Injected flies that produced short amplicons compared to wildtype controls were isolated. Sanger sequencing was done using the same primers to validate the deletion of Ovd. Flies with large deletions of the Ovd coding sequence were isolated in both subspecies (Figure 2A).

Phenotyping D. pseudoobscura Overdrive knockout flies

For all D. pseudoobscura crosses, three males were crossed to three virgin females. Crosses were allowed to proceed for 7 days, then parents were removed. Progeny were counted at 28 days at 21 °C. Fifteen crosses were performed per genotype.

The viability of our OvdΔ mutations was assayed by comparing progeny counts of crosses with both parents homozygous for OvdΔ to crosses where both parents are wildtype. Two-sample Student’s t-tests were then used to analyze differences in progeny counts between wildtype and OvdΔ crosses for both subspecies, all differences were not significant. To assay male fertility and sex-ratio distortion, we crossed OvdΔ and wildtype males to wildtype females. Progeny counts and sex-ratio were compared between OvdΔ and wildtype crosses and differences were assessed with a two-sample Student’s t-test, all differences were not significant.

To generate OvdΔ Bogota-USA F1 hybrid males, homozygous OvdΔ Bogota females were crossed to USA males. F1 males were then crossed to USA females, and progeny were counted. Differences in fertility between OvdΔ F1 hybrid males and control wildtype F1 hybrid males were analyzed with a Pairwise Wilcoxon Rank Sum test.

CRISPR knockout of Overdrive in D. melanogaster

Overdrive knockout (OvdΔ) flies were generated using CRISPR homology-directed repair to replace Overdrive with 3×P3-dsRed. Knockouts were made in a vasa-Cas9 background (w¹¹¹⁸; PBac{y^+mDint2=vas-Cas9}VK00027). Vasa-Cas9 embryos were injected with four pCFD3-dU6:3gRNA (Addgene, Plasmid #49410) vectors, each containing a gRNA with homology within the Overdrive coding sequence (Plasmids and Primers table). The donor template with 3×P3-dsRed2 was flanked with ∼1000 bp homology arms (Fig. 3A).

To verify the loss of Ovd, we used multiple primer pairs targeting Ovd and the region flanking the insertion site (Plasmids and Primers table; Fig. 3B). The first primer pair (JC115F and JC115R,) flanked Ovd, with the primers targeting approximately 2 kb upstream of the 5’ UTR and downstream of the 3’ UTR. These amplified in both OvdΔ and control w¹¹¹⁸ flies, showing a slightly larger product size in OvdΔ consistent with successful replacement with 3×P3-dsRed (expected product sizes 5577 bp with native Ovd, 6298 bp after allele swap with 3×P3-dsRed, Figure 3B). The second primer pair (CRL13F and CRL13R) respectively targeted the Ovd 5’UTR (approximately 450 bp upstream of the start codon) and Ovd coding sequence (immediately upstream of the stop codon). This primer pair produced amplicons in control w¹¹¹⁸flies but not in OvdΔ flies, indicating loss of Ovd sequence in the knockout flies (Figure 3B). To verify that integration of our 3×P3-dsRed2 reporter construct occurred at the native Ovd locus, we used a third primer pair (JC99F and JC99R) with homology within the dsRed2 coding sequence which only amplified in OvdΔ flies. Sanger sequencing reads from the OvdΔ PCR products confirmed that dsRed successfully replaced Ovd in our OvdΔ flies.

Phenotyping D. melanogaster Overdrive knockout flies

Viability Assay: To assess the effects of Overdrive knockout on viability, we compared the viability of homozygous OvdΔ flies and OvdΔ/TM3, Sb flies. Eight OvdΔ/TM3, Sb males aged 0-3 days were mated on Day 1 with ten virgin females of the same genotype aged 0-3 days. This cross was conducted in 10 biological replicates. Parents were allowed to lay in the original vial for Days 1-4, and transferred to new vials every three days for a total of three times. Progeny in each vial were counted 18 days after the parents were introduced. A Cochran-Mantel-Haenszel test was applied to determine if the progeny deviated from the expected distribution of 2:1 OvdΔ/TM3, Sb heterozygotes to OvdΔ homozygotes.

Sex Ratio Assay: Although Overdrive appears to function as a selfish element in the D. pseudoobscura system that eliminates Y-bearing sperm to drive its own transmission, we wanted to exclude the possibility that Overdrive perturbation instead has a general male-killing effect. We therefore compared the sex ratio of the progeny of OvdΔ homozygote fathers and control w¹¹¹⁸ fathers. Single male flies aged 1-2 days were crossed to three w¹¹¹⁸ virgins aged 0-3 days. Flies were mated for one day, transferred to a new vial, and allowed to lay for three days. Parents were then cleared from the vial. Progeny were counted 14 days after parents were cleared. Total numbers of male and female progeny for each genotype were summed across biological replicates, and differences in male and female progeny numbers between genotypes were evaluated using the Student’s two-sample t-test.

Nondisjunction Assays: For all nondisjunction assays, individual males aged 1-2 days (30 males total per genotype) were crossed with three virgins aged 0-3 days (female genotype was w¹¹¹⁸ for sex chromosome assay; C(4)RM for fourth chromosome assay). Parents were transferred into a new vial after three days and cleared after six days. Progeny were scored and counted on Day 18.

We compared nondisjunction rates between OvdΔ fathers and w¹¹¹⁸ fathers for the sex chromosomes and the fourth chromosome. To measure sex chromosome nondisjunction, we used the DcY(H1) chromosome, which contains a translocation of the dominant eye marker B^Sonto the Y chromosome. H1 chromosomes were introduced into wild-type (w¹¹¹⁸) and OvdΔ/OvdΔ backgrounds. This allowed us to distinguish offspring resulting from nondisjunction events (XO males with wild-type eyes and XXY females with Bar eyes) from normal progeny (XY males with Bar eyes and XX females with wild-type eyes).

For fourth chromosome nondisjunction assays, we mated OvdΔ and w¹¹¹⁸ males with compound-fourth females (C(4)RM, ci¹ ey^R/0). Viable haplo-4 and triplo-4 progeny are produced by normal haplo-4 sperm fertilizing nullo-4 or compound-4 eggs. If a nullo-4 sperm resulting from a male nondisjunction event fertilizes a compound-4 egg, the progeny are recognizable due the recessive markers on the maternal compound chromosome. Nullo-4 zygotes are inviable, and while tetra-4 progeny can also arise from male nondisjunction events, they are undetectable in this assay (43). Fourth chromosome nondisjunction assays were repeated with OvdΔ on a pure w¹¹¹⁸ background and on a Rspⁱ/CyO background, Rspⁱ being a Rsp chromosome insensitive to SD.

For sex chromosome assays, differences between the genotypes in the number of nondisjunction events were assessed with a two-sample Student’s t-test. For fourth chromosome assays, results were analyzed with pairwise two-sample c-tests (using the Poisson distribution) with the Bonferroni false-discovery correction.

Cell Cycle Progression Assay: We tested whether Overdrive knockout affected the response of somatic cells to radiation-induced double-strand breaks. Third instar w¹¹¹⁸ and OvdΔ larvae (aged approx. 4 days) were transferred to a laying plate and X-ray irradiated for 4,000 rads. After 30 minutes of recovery, wing and eye disc tissues were dissected in PBS. Tissues were then fixed for 1 hr in 4% paraformaldehyde solution (Life Biotechnologies, Carlsbad, CA), washed 2×15 minutes in PBS with 0.1% (w/v) sodium deoxycholate and 0.1% (v/v) Triton, and 2×15 minutes in PBX (PBS w/ 0.1% (v/v) Triton). Tissues were incubated overnight at 4°C with rabbit anti-His3S10Phos antibody (abcam, Cambridge, UK), washed 3×15 minutes in PBX, incubated in goat anti-rabbit Alexa 568 secondary (Life Biotechnologies, Carlsbad, CA), washed 3×15 minutes in PBX, stained with 0.1% DAPI, mounted in Vectashield, and sealed with nail polish. Z-stacks of tissues were imaged using a Zeiss LSM 880 Airy Scan confocal microscope. Maximum-intensity Z-projections were generated in Fiji, and the number of phospho-Histone3S10 foci were scored in discs of irradiated and non-irradiated larvae using the CellCounter plugin (44). Differences between the irradiated and non-irradiated treatments, and the OvdΔ larvae and w¹¹¹⁸ controls, were analyzed using Mann-Whitney tests.

Overdrive Molecular Evolution

Drosophila species used for domain structure predictions and PAML: melanogaster, simulans, sechellia, mauritiana, yakuba, erecta, eugracilis, biarmipes, takahashii, ficusphila, elegans, rhopaloa, kikkawai, ananassae, bipectinata (melanogaster group) and pseudoobscura pseudoobscura, pseudoobscura bogotana, persimilis, miranda, lowei, and affinis (obscura group). All analyses were conducted using the reference genome assembly or whole-genome sequencing contig databases publicly available through NCBI.

Ovd domain structure: We aligned orthologous Ovd sequences across the melanogaster and obscura groups, compared their predicted secondary structures, and analyzed their rates of nucleotide substitution. Overdrive orthologs from 15 melanogaster group species and six obscura group species (see Fly Strains section for list of species) were identified using reciprocal best-hits BLAST (tBLASTn and BLASTx). We then aligned these sequences using default settings in the MUSCLE alignment program and generated visualizations of conservation rates using Jalview (45, 46). To investigate the degree of secondary structure conservation between the D. pseudoobscura and D. melanogaster orthologs of Overdrive, we predicted the location of alpha helices and beta sheets for the two Ovd orthologs and the analogous MADF– and BESS-containing proteins Adf1 and Dlip3 using the JNetPRED, JNetHMM, and JNetPSSM models in the JPred software package (47).

PAML: Analysis of Overdrive sequence evolution was conducted according to the method described in Cooper and Phadnis (48). Because we hypothesized that Overdrive could display different evolutionary dynamics in the melanogaster and obscura groups, and to avoid the possibility of synonymous site saturation, we analyzed the 15 melanogaster group species and six obscura group species separately. For each group, we used the Phlyogenetic Analysis by Maximum Likelihood (PAML) package (35). We first used the codeml feature in PAML to estimate d_N/d_S for each branch in the gene tree of each species group. To test for positive selection within each species group, we compared NS sites models M7 (which allows only for purifying selection and neutral evolution) and M8 (which allows a class of sites within the sequence to have d_N/d_S values greater than 1). In the results, we present the p-value of the likelihood-ratio test using the log-likelihood scores for the two models. A p-value under 0.025 (after Bonferroni correction for multiple comparisons) would indicate that the M8 model was a better fit for the sequence data, suggesting a pattern of recurrent positive selection within the coding sequence.

Ovd ortholog search: The following Drosophilidae species genome assemblies were used for ortholog searches of Ovd:

Leucophenga varia, Scaptodrosophila lebanonensis, Chymomyza costata, Lordiphosa mommai, Lordiphosa magnipectinata, Lordiphosa clarofinis, Lordiphosa stackelbergi, Lordiphosa collinella, Drosophila sturtevanti, D. neocordata, D. saltans, D. prosaltans, D. sucinea, D. nebulosa, D. insularis, D. tropicalis, D. willistoni, D. equinozialis, D. paulistorum, D. subobscura, D. guanche, D. bifasciata, D. obscura, D. tristis, D. ambigua, D. lowei, D. miranda, D. pseudoobscura pseudoobscura, D. pseudoobscura bogotana, D. persimilis, D. azteca, D. Athabasca, D. varians, D. ercepeae, D. pseudoananassae, D. malerkotiana, D. parabipectinata, D. bipectinate, D. ananassae, D. oshimai, D. elegans, D. fuyamai, D. kurseongensis, D. carrolli, D. rhopoloa, D. ficusphila, D. biarmipes, D. subpulchrella, D. suzukii, D. takahashii, D. eugracilis, D. melanogaster, D. mauritiana, D. sechellia, D. simulans, D. orena, D. erecta, D. teissieri, D. yakuba, D. santomea, D. pectinifera, D. trapezifrons, D. triauraria, D. Auraria, D. tani, D. rufa, D. lacteicornis, D. asahinai, D. kanapiae, D. kikkawai, D. leontia, D. bocki, D. watanabei, D. punjabiensis, D. truncate, D. mayri, D. jambulina, D. seguyi, D. vulcana, D. bakoue, D. tsacasi, D. nikananu, D. burlai, D. bocqueti, D. busckii, D. repletoides, Zaprionus inermis, Zaprionus kolokinae, Zaprionus tsacasi, Zaprionus ornatus, Zaprionus africanus, Zaprionus indianus, Zaprionus gabonicus, Zaprionus capensis, Zaprionus taronus, Zaprionus davidi, Zaprionus vittiger, Zaprionus lachaisei, Zaprionus nigranus, Zaprionus camerounensis, D. quadrilineata, D. pruinosa, D. immigrans, D. sulfurigaster, D. kepylauana, D. albomicans, D. nasuta, D. cardini, D. dunni, D. arawakana, D. funebris, D. innubila, D. lacertosa, D. robusta, D. melanica, D. littoralis, D. virilis, D. americana, D. novamexicana, D. nannoptera, D. pachea, D. hydei, D. navojoa, D. arizonae, D. mojavensis, Scaptomyza hsui, Scaptomyza polygonia, Scaptomyza graminum, Scaptomyza montana, Scaptomyza flava, Scaptomyza pallida, D. grimshawi, D. murphyi, D. sproati

The following non-Drosophilidae Dipterans species genome assemblies were used for ortholog searches of Ovd:

Aedes albopictus, Aedes aegypti, Volucella bombylans, Syritta pipiens, Xylota sylvarum, Anastrepha ludens, Ceratitis capitata, Teleopsis dalmanni, Musca domestica

When multiple genome assemblies were available, we used NCBI reference genomes. For species without a reference genome, the most recent genome assembly on NCBI genomes as of January 1^st, 2024 was used. To identify orthologs of Ovd, tblastn and reciprocal blastx were performed against each genome listed above. tblastn was done with the following parameters: – gapopen 11 –gapextend 1 –evalue .001. D. melanogaster Ovd (CG6683) was used as a query. When the reciprocal best hit was D. melanogaster Ovd, the hit was called as an ortholog. If no reciprocal best hit was called, synteny checks were performed to validate the loss of Ovd. Absence of Ovd was concluded when both reciprocal best hit and synteny checks indicated that Ovd was not present.

Transmission Distortion and Fertility in an SD Background

To determine whether Overdrive knockouts alter the level of transmission distortion caused by Segregation Distorter (SD) chromosomes, we generated 18 different strains of tester males. These represented every possible combination of three driving (or non-driving) chromosomes (SD-5, SD-72, and a CyO control), two chromosomes targeted (or not) by drive (Rsp^ss and Rspⁱ), and three Overdrive states (wild-type, homozygous knockout, and heterozygotes). We used two different driving chromosomes isolated from natural populations, SD-5 and SD-72. In males, these SD chromosomes drive against Responder-sensitive or – supersensitive chromosomes. While all SD chromosomes carry the same RanGAP duplication, their pattern of rearrangements, strength of drive, and viability relative to wild-type vary, so we used two different SD chromosomes in our experiments. As a control, we used the CyO balancer chromosome, a second chromosome that is inverted and homozygous lethal like the SD chromosomes we used (49) but has no driving properties and is insensitive to drive.

We used two different chromosomes as targets for drive. The first line we used, Rsp^ss, has a high copy number of Responder repeats, rendering it supersensitive to SD. The second line, Rspⁱ, has a deletion of the Rsp locus, making the chromosome insensitive to SD (See Fly Strains table for full genotypes). Finally, we tested three different Overdrive conditions: wild-type, homozygous deletion of Overdrive (OvdΔ/OvdΔ), and heterozygotes (OvdΔ/+).

We tested all possible combinations of SD, Rsp, and Ovd alleles. To quantify segregation distortion, we crossed the males to bw¹ virgins. In each cross, 30 males aged 1-7 days were placed singly in vials and each crossed to 3 virgins aged 4-10 days. Parents were flipped into new vials on Day 3 of the cross, and cleared on Day 6. Progeny were counted on Day 18. Since both the Rsp chromosomes we used were marked with bw¹, we were able to determine that brown-eyed progeny had received a Rsp chromosome from their fathers. Quantifying the proportion of progeny with wild-type eyes thus gave us a measure of k, the coefficient of transmission distortion. Differences in k among the treatment groups were modeled with a three-way ANOVA (k ∼ Overdrive * Distorter * Responder) on arcsine-transformed data (since many of the values of k were near or equal to 1) and analyzed with Tukey’s Honestly Significant Differences test.

Immunofluorescence and imaging

Immunostainings in whole mount testes and seminal vesicles were performed as previously described. Briefly, male gonads we dissected in 1X PBS-0.15% Triton (PBS-T), then fixed for 20min at RT in 4% formaldehyde in PBS-T. Then tissues were wash 3 times and mounted in mounting medium containing DAPI for direct observation of DAPI staining and/or native GFP and RFP fluorescence. For antibody stainings, after fixation and washes, gonads were incubated in primary antibody [1:3000 for the mouse anti-dsDNA antibody (Abcam ref# ab27156)] diluted in PBS-T overnight at 4°C. The next day, tissues were washed 3 times in PBS-T and incubated with secondary antibody (1:500 Jackson Immunoresearch) at RT for 2-3 hrs. They were then mounted in mounting medium containing DAPI (2µg/mL). Testes and seminal vesicles were imaged with a LSM800 confocal microscope (CarlZeiss) and images were processed with Fiji software.

Supplemental Figure S1: Overdrive is expressed in the male germline of D. melanogaster from male germline stem cells to canoe stage spermatids undergoing the histone-to-protamine transition.

DNA is shown in cyan (DAPI), Histone2Av-RFP is shown in yellow, Ovd-GFP in magenta.

Supplemental Figure S2: Validation of replacement of Ovd with DsRed.

Primers placed inside of Ovd no longer amplify in Ovd KO, but primers flanking the gene still amplify.

Supplemental Figure S3:

(A) Alignment of Overdrive coding sequence for 15 melanogaster and 5 obscura group species. Darker blue shading signifies a higher degree of amino acid sequence conservation. Yellow bars under the sequences represent the degree of conservation at each residue on a 0-9 scale (* = perfect conservation). Fixed amino acid differences in D. pse. bogotana (relative to D. pse. pseudoobscura) are shaded in red. (B) Predicted secondary structure for Overdrive in D. pse. bogotana and D. melanogaster based on the JNetPRED (top), JNetHMM (middle), and JNetPSSM (bottom) models in the Jpred package. Alpha helices are shown in red and beta sheets are shown in green. Histogram denotes confidence in the secondary structure calls for each residue on a 0-9 scale. Two proteins with MADF DNA-binding domains and BESS protein-protein interaction domains, Adf1 and Dlip3, are shown for comparison. The cyan box denotes the approximate location of the MADF domain in all four proteins, and the magenta box denotes the approximate location of the BESS domain.

Supplemental Figure S4: Overdrive knockout does not affect cell cycle progression in (A) eye discs or (B) wing discs.

Discs from OvdΔ and w¹¹¹⁸ larvae were stained for DNA (cyan) and the mitotic marker H3S10Phos (magenta) after irradiation. Both OvdΔ and w¹¹¹⁸ largely arrest division after radiation, and the number of mitotic foci does not differ significantly between genotypes either before or after irradiation. Points represent biological replicates, and error bars represent standard deviation. ** = p < 0.005, Mann-Whitney test.

Supplemental Figure S5: Expanded comparison of various SD and Rsp chromosomes in wild-type Ovd, heterozygous Ovd^Δ and homozygous Ovd^Δ backgrounds.

(A) Proportion of progeny of tester males inheriting a Segregation Distorter chromosome (SD5 or SD72) or non-driving CyO control. (B) Total number of offspring sired by each tester male. Overdrive knockout appears to restore fertility relative to SD-inactive controls in SD/Rsp^ss backgrounds, even as it reduces fertility in SD/Rspⁱ backgrounds. For both panels, the center bar of boxes represents the median, and whiskers extend up to 1.5× the interquartile range.

ACKNOWLEDGEMENTS

We thank Kent Golic for helpful discussions and feedback in improving this manuscript. We thank Titine Loppin for her continued support. Strains are available upon request. This work was supported by the National Institute of Health grant R01GM141422 to NP and French National Research Agency grant ANR-21-CE13-0037 to BL. We acknowledge the contribution of Lyon SFR Biosciences (UAR3444/CNRS, US8/INSERM, ENS de Lyon, UCBL) imaging facility (PLATIM) and fly food production (Arthrotools).