Proliferation and dissemination of killer meiotic drive loci

Abstract

Killer meiotic drive elements are selfish genetic entities that manipulate the sexual cycle to promote their own inheritance via destructive means. Two broad classes are sperm killers, typical of animals and plants, and spore killers, which are present in ascomycete fungi. Killer meiotic drive systems operate via toxins that destroy or disable meiotic products bearing the alternative allele. To avoid suicidal auto-targeting, cells that bear these selfish elements must either lack the toxin target, or express an antidote. Historically, these systems were presumed to require large non-recombining haplotypes to link multiple functional interacting loci. However, recent advances on fungal spore killers reveal that numerous systems are enacted by single genes, and similar molecular genetic studies in Drosophila pinpoint individual loci that distort gamete sex. Notably, many meiotic drivers duplicate readily, forming gene families that can have complex interactions within and between species, and providing substrates for their rapid functional diversification. Here, we summarize the known families of meiotic drivers in fungi and fruit flies, and highlight shared principles about their evolution and proliferation that promote the spread of these noxious genes.

Introduction

For all sexual organisms with a diploid phase in their life-cycle, regardless of how short it may be, the two alleles of a given gene are expected to segregate at equal frequencies during meiosis. However, the processes that generate meiotic products are vulnerable to manipulation, enabling cheaters to arise [1]. Perhaps the most overt perpetrators are ones that commit sororicide, disabling or killing their siblings to increase their own frequencies among offspring. There are two general processes by which this so-called killer meiotic drive can operate: killer-target or poison-antidote (Figure 1A) [2]. The best-studied systems to date occur within fungi or fruit flies (Figure 1B), and these will be the focus of this review.

Figure 1. Classes of killer meiotic drive systems.

(A) In killer/target systems, the driving locus (red) produces a toxin that targets the non-driving locus (green). This may be at an idiomorphic, syntenic location, or in heteromorphic genomic regions, such as sex chromosomes. During or after meiosis, all nuclei or cells carrying the non-driving allele are destroyed or inactivated. In poison/antidote systems, the driving locus produces a toxin and an antitoxin. All meiotic products are exposed to the toxin, but only the driving locus is immune to the effect, thus only cells carrying the driving locus survive. (B) Fungi and fruit flies differ in whether the haploid or diploid phase is dominant in their life cycles. In fungi, haploid cells or gametes fuse to produce a diploid zygote, immediately followed by meiosis to produce sexual spores that will form the progeny. Spores may have a single haploid nuclear type (monokaryotic; n), or possess two nuclei of different mating types (dikaryotic; n+n). In fruit flies, the diploid germ cells will undergo meiosis to produce haploid gametes. When producing spores or sperm, a number of rounds of mitosis may occur after meiosis to generate numerous cells from the original four meiotic products.

In the killer-target scenario, the driving allele produces a factor that recognizes a target that is unique to cells bearing the sensitive allele (Figure 1A). As killer-target systems require that the killer lacks the target, these drives must occur on distinct haplotypes. Dimorphic sex chromosomes prove to be fertile grounds for such systems to evolve, due to their large disparity in gene and/or repeat content. In particular, male Y chromosomes often harbour only a few genes that determine male-specific functions, and instead are usually replete with transposons and simple repeats. This renders the sex chromosomes largely refractory to recombination. (Analogous considerations exist in ZW systems where females are heterogametic, but we will focus on XY here). These features facilitate the emergence of sperm killer loci on the X chromosome, which can disable Y-bearing sperm. Despite their propensity to evolve on sex chromosomes, killer-target systems can also be autosomal. For example, both Segregation Distorter (SD) from Drosophila melanogaster and the het-s gene from the fungus Podospora anserina are autosomal, and the latter lacks sex chromosomes altogether. While SD is located within a large non-recombining haplotype [3], het-s occupies a small idiomorphic locus consisting of only het-s and its partner, NWD2 [4].

In the alternative poison-antidote scenario, the driving allele possesses both a toxin and an antidote, while gametes bearing the sensitive alternative allele lack the antidote and are thus killed or inactivated (Figure 1A). Classically, poison-antidote systems were thought to require large non-recombining regions to connect the toxin gene to the antidote gene, as exemplified by the t-haplotype in mice [5]. In these cases, large segregating haplotypes form, usually composed of multiple large-scale inversions that are straightforward to detect through classical genetic mapping. The requirement for non-recombining regions was traditionally viewed as a barrier to the formation of poison-antidote systems. The notion was that if recombination occurred, even rarely, the antidote could become unlinked from the poison, resulting in suicidal activity of the poison and/or the spread of resistance through the population [6]. However, recent genomic approaches have identified several single-gene poison-antidote drives, particularly amongst fungal species. This new perspective on meiotic drive has important implications for their evolution and the impact of these systems on the organisms which carry them, necessitating a revision of prevailing notions surrounding the origin, spread, and persistence of killer meiotic drivers.

Spore killer gene families

Genetic investigations of diverse fungal spore killer systems reveal that most are caused by single genes. Furthermore, in nearly all cases, these killer genes are Spok genes in Podosporamembers of broader gene families. These include the [7,8], the wtf genes in Schizosaccharomyces [9,10], Spk-1 in Neurospora [11] and SKC1 in Fusarium [12]. The Spok and wtf genes are well-studied and share common features (Figure 2A–B). In both systems, highly similar homologs exhibit independent killing activities, suggesting that their underlying mechanisms diverge rapidly. However, some copies retain or acquire epistatic interactions, resulting in hierarchical killing interactions where a given gene may drive against another homolog, but be resistant to drive from said homolog itself [7,13]. Of note, both Spok and wtf families are associated with transposable elements (TEs) or other repetitive genes, providing a potential strategy for their proliferation (Figure 2C–E) [10,14,15]. Additionally, the genes can cross species boundaries, as both Spoks and wtf genes display incongruous phylogenies within their respective clades [7,10].

Figure 2. Genomic proliferation of spore killers in fungal species.

(A–B) Schematics of the spore killing gene structures. (A) In Podospora, the Spok genes code for a single protein with three domains. The second domain is required for spore killing and the third domain is required for resistance. (B) In Schizosaccharomyces, the wtf genes code for alternative transcripts. The full transcript acts as an antidote whereas a truncated version that is missing the first exon functions as the poison. (C) Four of the five fission yeast genomes are replete with wtf genes. Numbers represent the number of wtf genes in the reference strains of each species, including pseudogenized copies. (D–E) Both the Spok and wtf genes have associations with repetitive loci that allow for their translocation and duplication. (D) Some of the Spok genes reside within a massive transposon named Enterprise that mediates their mobilization around the genome. P. anserina possesses a version of Enterprise (called the Spok block) which may carry either Spok3, Spok4, or both, along with a number of other genes with unknown functions. (E) Schizosaccharomyces wtf genes are occasionally found in a “solo” configuration, but are typically flanked by repetitive elements that render them susceptible to ectopic recombination. Initial studies of S. pombe revealed the “with tf solo-LTR transposon” (wtf) configuration, but subsequent studies of other fission yeast genomes reveal other wtf loci flanked by 5S rDNA copies, and sometimes in association with the wag gene. Note that wtf loci can be flanked by many other specific combinations of these elements, examples are shown here.

Less is known about the other killers, Spk-1 and SKC1, which were described more recently. While homologs of both genes appear widespread, though within restricted lineages, killing has only been demonstrated for a single copy in each case [11,12]. Thus, whether these drivers represent true spore killer gene families, or recent co-option of domestic gene families requires further investigation. Nevertheless, they display the same trends with regard to duplications and convoluted phylogenies. These observations lead us to hypothesize that gene family expansion of killer meiotic drivers, driven by associations with TEs, may be common across eukaryotes, and could significantly contribute to genome evolution in numerous organisms.

Mobilization of meiotic drive factors in Podospora by large mobile elements

Like many meiotic drive systems, spore killing in Podospora was initially observed in the context of hybrid crosses. Genetic investigations revealed that P. comata possessed the spore killer Spok1, while P. anserina possessed the homolog Spok2 at a non-orthologous position [8]. Spok1 exhibits dominance over Spok2, being able to kill spores carrying it, but is resistant to Spok2 itself, generating a killing hierarchy. Subsequent analyses determined that both Spok genes are present at high frequencies in their respective species, and also uncovered the presence of Spok3 and Spok4 in P. anserina [7,15]. These two new genes show no epistatic interactions with Spok2, or each other, implying that they harbor independent killing functions despite their high sequence similarity. The SPOK proteins are predicted to have tripartite domain organization, where the second domain imparts killing function, and where the third is required for resistance (Figure 2A). How a single protein is able to encode both functions is not fully understood, but the mechanism appears to rely on nuclease activity of the killing domain [7]. Intriguingly, this killing function appears to operate universally and can function in heterologous backgrounds, including in Saccharomyces cerevisiae and Escherichia coli [16].

All four known Spok homologs are associated with TEs. Spok1 and Spok2 are found in small TE islands, whereas Spok3 and Spok4 are located within a giant (110 kb – 247 kb) TE named Enterprise (Figure 2D) [15]. In different strains of P. anserina, Enterprise can be found at four different genomic locations, and carries either Spok3, Spok4, or both [7]. The region within Enterprise where the Spok genes reside appears to be the result of a tandem duplication, implicating Enterprise itself in the proliferation of these genes (Figure 2D). Additionally, a near identical copy of Enterprise, with both Spok3 and Spok4, was found in the closely related species P. pauciseta, suggesting that Enterprise may facilitate the movement of the Spoks between species. Subsequent work has determined that Enterprise is not a unique aberration, but rather represents a single element of a much larger family of TEs, named the Starships [17]. Numerous homologs of the Spoks can be found throughout filamentous fungi [7,8], and some of these occur within other Starships [17], indicating that there is either a deep evolutionary relationship between this gene family and these massive TEs, or parallel evolutionary pressures to conjoin these entities.

Repeat-associated amplification and mobilization of meiotic drivers in fission yeast

Individual Schizosaccharomyces pombe isolates exhibit high sequence similarity, but harbour many rearrangements [18], which in part makes different wild strains incompatible [19]. Consequently, despite a large number of characterized S. pombe strains [20], most laboratory studies utilize a single standard strain. However, hidden inside many rearrangements are potent meiotic drive genes of the wtf family [9,10]. Each gene generates both a diffusible poison and an antidote, encoded by alternative isoforms, an autonomous arrangement that facilitates their capacity to spread (Figure 2B). Moreover, with few exceptions [21], wtf antidotes generally antagonize only their cis-encoded poison [9,14], and highly divergent wtf toxins are effective spore killers [13]. Indeed, wtf genes are rapidly evolving and widely heterogeneous amongst S. pombe isolates [22]. This, in part, explains why different wild S. pombe strains are meiotically incompatible. All progeny will lack expression of the antidote to one (or more) wtf toxins encoded by the different parental genomes, resulting in mutual killing among the drives, and death of the spores. In fact, there may not be any fixed wtf genes in S. pombe; each isolate may potentially contain a unique subset of these meiotic drivers, which only become unleashed upon outcrossing [23].

The flippant moniker “wtf” actually predated the recognition of these loci as meiotic drivers, and derives from much earlier analysis of transposons in the S. pombe reference genome [24,25]. S. pombe contains only one, modestly-sized, family of active full-length transposons (13 copies of Tf2, a long terminal repeat (LTR) retrotransposon). However, numerous transposon relics can be identified, principally solo LTR sequences derived from LTR recombination within Tf2 elements and members of other currently inactive Tf families [25]. Amongst these, 25 solo LTRs on chromosome III are associated with a family of repetitive sequences that define a novel gene family, the “with TF” transposon (wtf) loci (Figure 2C) [24,25]. This provides a potential clue as to the evolution of wtf genes.

The current model used to explain wtf gene amplification is that the TF sequences provide substrate for ectopic recombination. The numerous copies and their concentration on chromosome III align with other observations of these regions as hotspots of structural rearrangements in S. pombe [26]. Thus, this inherent genomic feature of this species may fuel recurrent birth of drive genes [22]. Other processes, such as gene conversion, may also promote rapid divergence to yield unique meiotic drivers [13]. This scenario is supported by the observation that in the other Schizosaccharomyces species, the wtf genes are associated with the 5S rDNA gene rather than the TF transposon (Figure 2E) [27]. Like solo LTRs, the 5S rRNA gene is small (~130 bp) and interspersed throughout the genome, providing templates for ectopic recombination. Congruently, the 5S rDNA gene has been observed to overlap with structural rearrangements in a number of species [28,29]. Some wtf loci in different fission yeasts are associated with both LTR and 5S rDNA copies, as well as with the wag gene (Figure 2E), suggesting that complex interactions amongst repetitive elements of different classes have fostered and shaped the amplification of selfish wtf genes [27].

Rapid evolutionary dynamics of X-linked meiotic drive loci and their suppressors in Drosophila

Although crosses between simulans-clade species are incompatible due to sterility of hybrid males, the corresponding females are fertile, providing an opportunity for inter-specific gene flow. Due to high synteny and identity of simulans-clade genomes, maintained even with D. melanogaster (Figure 3B), most local regions of D. simulans are complemented by the cognate regions of its sister species. However, such introgression studies revealed that replacement of two autosomal D. simulans intervals caused males to sire mostly female progeny and/or become infertile [30,31]. Because of the apparent connection to sex chromosome distortion, loss of these specific regions of the D. simulans genome were interpreted to de-silence selfish X-linked loci (as opposed to affecting normal factors in male reproduction). These genetic activities were dubbed “Not much yang” (Nmy) and “Too much yin” (Tmy), and together with their inferred targets, defined the so-called “Winters” and “Durham” SR conflict systems, respectively.

Figure 3. Proliferation of Dox family genes amongst Drosophila simulans-clade species.

(A) Phylogenetic relationship of D. melanogaster with the three sim-clade species, D. simulans, D. sechellia and D. mauritiana. (B) The ancestral genes in the X chromosome region shown are syntenic across these four species (blue boxes), but the three sim-clade species bear numerous interspersed copies of known or presumed meiotic drivers of the Dox family. All copies shown in solid boxes encode an HMG box protein (black, green and red according to their subfamily); additional partial copies lack an HMG box (dotted boxes). Note that members of different Dox subfamilies reside at certain syntenic regions, suggesting either recurrent insertion or gene conversion. The Cubn (light blue) and Ptpmeg2 (brown) loci are designated as they have also mobilized within the syntenic region of sim-clade genomes that bears Dox family loci. (C) Nearly all Dox family genes bear flanking copies of 359 satellite repeats, and 359 sequences are located in the ancestral genomic regions. The right-hand example illustrates complex evolution of repeat-associated genes. There are non-cognate insertions of Dox family genes within the syntenic interval, along with an additional unrelated gene insertion (Ptpmeg2). (D) Dox family genes are suppressed by autosomal RNAi loci of the hairpin RNA (hpRNA) class, in particular, the Nmy and Tmy family loci. These have undergone rapid evolution amongst the closely-related sim-clade species, conceivably to match the amplification of Dox family genes. For example, D. sechellia contains four tandem copies of a shortened Tmy-class hpRNA (the mini-Tmy-Complex, or mTmy-C), which are preferentially complementary to the expanded set of UDox genes in D. sechellia (B).

Support for this scenario came with the finding that an X-linked mutant could bypass the need for Nmy, enabling the identification of the “Distorter on X” (Dox) locus [30]. The genetics indicated that the wildtype function of Dox is to disable Y-bearing sperm, a surprising wildtype function for an endogenous gene that strictly requires suppression by Nmy. While the Dox mechanism was enigmatic, as its functional product was initially unclear, sequence similarity between Dox and Nmy loci, along with a putative inverted repeat structure of Nmy, suggested involvement of homology-dependent RNA silencing [30,32]. Moreover, while Dox was a new gene, it bore homology to another de novo X-linked locus, the “Mother of Dox” (MDox) [32]. Based on synteny relationships, Dox and MDox are recently emerged genes that are not even shared amongst the three simulans-clade species (Figure 3B–C).

Recent application of several molecular genomic approaches provided insights into the evolution and spread of Dox-related loci. First, Nmy proved to belong to a class of rapidly-evolving endogenous siRNA loci termed hairpin RNAs (hpRNAs) [33,34], and it can directly repress Dox and MDox [35]. Second, the introgression region that defined the Tmy SR phenotype contains another de novo hpRNA related to Tmy, thereby connecting the previously distinct Winters and Durham SR systems [35,36]. Finally, the recent availability of highly contiguous genome assemblies [37] revealed novel Dox family genes in D. simulans, along with highly expanded cohorts of X-linked Dox family loci and their autosomal hpRNA suppressors in other simulans-clade species [38,39]. While there are four Dox family loci in D. simulans, there are at least 11 in both D. mauritiana and D. sechellia, which altogether assort into the Dox/MDox, ParaDox (PDox) and UnorthoDox (UDox) subfamilies (Figure 3B–C).

On the suppressor side, D. mauritiana and D. sechellia lack a Tmy ortholog at the regions syntenic to D. simulans Tmy, consistent with prior introgression genetics, but they contain a Tmy homolog elsewhere in their genomes. Moreover, D. sechellia contains an additional cluster of mini Tmy-like hairpins (mTmy-C) that are complementary to an expanding subset UDox genes [38,39] (Figure 3D). Recent studies of D. simulans knockouts of the Tmy and Nmy hpRNAs confirm that they strongly depress Dox/MDox and PDox1/2 loci, respectively, and exhibit marked defects during spermatogenesis [36]. Strikingly, even though Tmy knockouts are completely male sterile, Tmy likely has a root activity in suppressing sex chromosome meiotic drive. Evidence for this notion comes from genetic interactions, revealing that tmy heterozygotes strongly enhance the partial loss of male progeny by nmy knockout fathers at permissive temperature [36].

While the molecular functions of Dox family genes remain to be elucidated in detail, an intriguing finding is that they are derived in part from insect protamine, one of family of sperm nuclear basic proteins (SNBPs) that replace histones to mediate the highly condensed state of the paternal genome in mature sperm [40–42]. This suggests that Dox family loci may interfere with normal packaging of the paternal genome. While this notion remains to be evaluated in molecular detail, it is consistent with testis cytological phenotypes in D. simulans knockouts of the Nmy and Tmy hpRNA loci [36].

Repeat-associated dispersal of meiotic drive loci in Drosophila sim-clade species

Dox family genes have multiplied within a ~1Mb region of the X chromosome, but are interspersed amongst syntenic, ancestrally-conserved, protein-coding genes (Figure 3B). In the initial identification of Dox and MDox, they were noted to be flanked by a 359 bp sequence that is often found clustered in heterochromatic regions of the genome (known as the 359 satellite) [32]. Further inspection of the extended Dox family revealed that nearly all Dox family loci in all three simulans-clade species are similarly flanked by 359 satellites [38,39]. Moreover, the corresponding regions of the D. melanogaster genome, which lacks any Dox genes, harbour 359 satellites at the syntenic sites; the same is true for other simulans-clade regions that do not presently share a Dox family insertion found in its sister species (Figure 3C). These observations suggest that 359 satellite repeats mediate the expansion of dispersed Dox family genes within a restricted genomic locale.

The close association of Dox family genes with 359 satellites (Figure 3C) is remarkably analogous to the linkage of the wtf genes and either Tf transposons or 5S rDNA (Figure 2E). In both cases, meiotic drive genes are adjacent to small, interspersed, repetitive sequences. As these repetitive sequences are not autonomously transposing elements, in contrast to the presence of Spok genes within Starships (Figure 2D), other mechanisms must be invoked to explain the observed amplifications. One possibility is that flanking repeats mediate excision of intervening sequence bearing the drive genes, followed by re-insertion of circular DNA into nearby sites containing the repeat. This is plausible since extrachromosomal circular DNA bearing satellite sequences were detected in Drosophila, and may modulate dynamic composition of satellite islands [43]. Another possibility is that these repeats promote non-allelic gene conversion and/or ectopic recombination.

Further study is needed to discern the extent to which such strategies underlie the expansion of meiotic drive gene families, although it is clear that their rapid dynamics may partially obscure the original events. For example, many seemingly syntenic insertions of Dox family loci actually encode distinct family members (Figure 3B–C) [38,39]. This situation might arise through either mechanism. There are even instances of permuted flanking sequences between Dox copies, which suggest re-insertion of a Dox-containing circular DNA intermediate that broke within genomic regions beyond the flanking 359 satellites [39]. In addition, non-Dox family genes were observed to insert at some 359 satellite regions, sometimes in concert with Dox family loci (Figure 3B–C). It is currently unknown if these apparent hitchhikers may have functional relevance to drive or not. While much remains to be untangled regarding the strategies of repeat-associated gene proliferation, this is clearly a recurrent strategy for the evolution of meiotic drivers.

Persistence of meiotic drives through time

Simplified models of meiotic drive suggest that individual drivers should go extinct under relatively short time spans. This is due to the fact that two main courses are expected to be followed by a given driver: purging from the population due to deleterious effects, or fixation due to successful drive. As meiotic drivers are only functional in heterozygous crosses or in suppressor mutants, fixation also leads to the disappearance of the drive [44]. There are some situations that can promote persistence of drivers at polymorphic frequencies, such as deleterious recessive effects observed in Monkey flowers [45] and the SD system of D. melanogaster [46]. In organisms with haploid dominant life cycles, such as many fungi, deleterious recessive mutations are restricted to operating during the brief diploid phase, but additional processes, such as frequent selfing or inbreeding, can promote the maintenance of drivers at intermediate frequencies [47]. It is thus of particular interest that both the Spok and wtf gene families show persistence over long timescales.

The wtf genes were previously thought to be specific to S. pombe, but genomic analysis of their closest relatives has revealed that these genes are much older than previously appreciated. Careful assessment revealed that highly diverged homologs of the wtf genes are widespread across fission yeasts that collectively diverged ~120M years ago, including in S. cryophilus (5 genes), S. osmophilus (42 genes) and S. octosporus (83 genes) (Figure 2C). Although divergent at the sequence level, these genes possess active poison and antidote functions, and drive was demonstrated for five wtf genes in S. octosporus [27]. Similarly, the Spok genes from Podospora have numerous, though highly diverged, homologs spread across pezizomycotina, and one copy from F. vanettenii (formerly Nectria haematococca) was shown to possess poison functionality [8]. Fusarium and Podospora have diverged for about 300 million years [48], suggesting an ancient persistence as with the wtf genes. The observation that both the wtf and Spok genes readily duplicate and diverge may be a key feature of their extended endurance. If a selfish gene duplicates and undergoes neofunctionalization before the original copy has an opportunity to reach fixation in a population, this will propagate ongoing cycles of birth and death of the genes.

By contrast, the genetic conflict involving Dox family loci and their hpRNA suppressors that is currently escalating amongst Drosophila simulans-clade species, are all lacking in the outgroup fruit flies; thus, these systems arose only within the past ~250K years (Figure 3A). Although the large collection of rapidly evolving Dox family loci awaits functional characterization, their divergence even within their major identified domain (the HMG box) hints at functional diversification [38,39]. Their genomic amplification has likely enabled them to sample new variants with distinct drive capacities and/or escape from homology-directed silencing by existing hpRNAs. However, we speculate that Dox systems are not likely to persist, if future lineages are to remain stable. It may be relevant that the driver of the selfish SD system of D. melanogaster, a truncated copy of RanGAP [49], also emerged very recently. SD-RanGAP is absent from other sister Drosophilid species, although this meiotic driver acts upon a pre-existing target, the so-called Responder pericentromeric satellite array [3]. Evidently, these cycles of killer/target meiotic drive systems in different fruit fly species burn out more rapidly than the poison/antidote meiotic drive systems in different fungal species, probably due to the fast turnover of target sequences (Figure 1A). It remains to be seen if this is a coincidence or general principle.

Conclusions

In S. pombe, we are potentially witnessing the consequences of a meiotic drive gene family expanding to the point that sexual reproduction with non-isogenic strains is no longer possible. We may never know whether a shift in mating system from one dominated by outcrossing to one where selfing or inbreeding was common predated the expansion of the wtf genes or if conversely, the runaway amplification of the wtf genes prevented sexual reproduction with non-related individuals. In either case, it seems that the genetic conflict waged between meiotic drive and the host genome has another aspect to it that has been previously overlooked. By allying themselves with repetitive sequences and transposable elements, meiotic drivers may be able to constantly outrun genomic defenses to persist in populations for extended periods of evolutionary time. The high divergence rate of the Spok and wtf genes suggests that individual drivers likely die out, even while the family thrives, leading to complex genomic architectures and distributions.

The dynamics of the HMG-box Dox family loci indicate analogous processes are ongoing within Drosophila simulans-clade species. Dox genes have exploded within the simulans-clade, but were clearly innovated within their recent common ancestor, and thus are evolutionarily young. It seems likely that principles of this system can be broadened. First, while such systems seem to be lacking in D. melanogaster, selfish sex ratio-biasing X chromosomes have been detected across numerous species of flies [50–54]. Although many of the underlying loci remain to be molecularly cloned, they are testament to the male germline as a continual battleground of intragenomic conflict. Second, phylogenomic analyses across Drosophilid species reveal that HMG-box/protamine-like genes exhibit highly variable copy numbers, especially on sex chromosomes [55]. This strongly suggests recurrent recruitment of sperm nuclear basic proteins for meiotic conflicts during male gametogenesis. Third, innovation of meiotic drives need not be limited to this particular family. Indeed, molecular genetic analyses reveal that beyond Dox family loci, other unrelated, recently-evolved, X-linked genes in D. simulans are functionally repressed by hpRNAs [56]. These are potentially additional agents of sex chromosome conflict. Therefore, when individual distorters are lost, they may conceivably be replaced not only by other direct copies, but by other unrelated meiotic drivers. These may comprise parallel, cyclical and/or intertwined genetic battles.

By comparing independently evolved drivers in distantly related systems, we can discern generalizable principles that emerge from these complex systems: (1) Repetitive elements can promote the expansion of meiotic drivers, (2) genes rapidly evolve independent drive functions and (3) species boundaries may be readily crossed by drivers [7,56]. Altogether, these points suggest that the lifespan of meiotic drive families may stretch far beyond that of any individual gene by a considerable degree. Thus, it is time to revisit old conclusions regarding the impact of meiotic drivers on the evolution of organisms and their impact on speciation.