Experimental support for multiple-locus complementary sex determination in the parasitoid Cotesia vestalis

Despite its fundamental role in development, sex determination is highly diverse among animals. Approximately 20% of all animals are haplodiploid, with haploid males and diploid females. Haplodiploid species exhibit diverse but poorly understood mechanisms of sex determination. Some hymenopteran insect species exhibit single-locus complementary sex determination (sl-CSD), where heterozygosity at a polymorphic sex locus initiates female development. Diploid males are homozygous at the sex locus and represent a genetic load because they are inviable or sterile. Inbreeding depression associated with CSD is therefore expected to select for other modes of sex determination resulting in fewer or no diploid males. Here, we investigate an alternative, heretofore hypothetical, mode of sex determination: multiple-locus CSD (ml-CSD). Under ml-CSD, diploid males are predicted to develop only from zygotes that are homozygous at all sex loci. We show that inbreeding for eight generations in the parasitoid wasp Cotesia vestalis leads to increasing proportions of diploid males, a pattern that is consistent with ml-CSD but not sl-CSD. The proportion of diploid males (0.27 ± 0.036) produced in the first generation of inbreeding (mother–son cross) suggests that two loci are likely involved. We also modeled diploid male production under CSD with three linked loci. Our data visually resemble CSD with linked loci because diploid male production in the second generation was lower than that in the first. To our knowledge, our data provide the first experimental support for ml-CSD.     

Sex determination and inbreeding depression in an ant with regular sib-mating

Haplodiploidy is one of the most widespread mechanisms of sex determination in animals. In many Hymenoptera, including all hitherto investigated social species, diploid individuals, which are heterozygous at the sex locus, develop as females, whereas haploid, hemizygous individuals develop as males (single-locus complementary sex determination, sl-CSD). Inbreeding leads to homozygosity at the sex locus, resulting in the production of diploid males, which are usually sterile and constitute a considerable fitness cost. Nevertheless, regular inbreeding without diploid male production is known from several solitary wasps, suggesting that in these species sex is not determined by sl-CSD but alternative mechanisms. Here, we examine sex determination in an ant with regular inbreeding, Cardiocondyla obscurior. The almost complete absence of diploid males after 10 generations of brother-sister mating in the laboratory documents for the first time the absence of sl-CSD and CSD with two or a few unlinked sex loci in a species of social Hymenoptera. Queens, which mated with a brother, appeared to decrease the number of males in their brood, as expected from the relatedness relationships under inbreeding. In contrast, some colonies began to show signs of an inbreeding depression after several generations of sib-mating, such as shortened queen life span, higher brood mortality, and a shift to more male-biased sex ratios in some colonies, presumably due to lower insemination capability of sperm.     

Complementary Sex Determination in the Parasitic Wasp Diachasmimorpha longicaudata

We studied the sex determination in Diachasmimorpha longicaudata, a parasitoid braconid wasp widely used as biological control agent of fruit pest tephritid flies. We tested the complementary sex determination hypothesis (CSD) known in at least 60 species of Hymenoptera. According to CSD, male or female development depends on the allelic composition of one sex locus (single-locus CSD) or multiple sex loci (multiple-locus CSD). Hemizygote individuals are normal haploid males, and heterozygotes for at least one sex locus are normal diploid females, but homozygotes for all the sex loci are diploid males. In order to force the occurrence of diploid males in D. longicaudata, we established highly inbred lines and examined their offspring using chromosome counting, flow cytometry, and sex ratio analysis. We found that when mother-son crosses were studied, this wasp produced about 20% of diploid males out of the total male progeny. Our results suggest that this parasitoid may represent the second genus with multiple-locus CSD in Hymenoptera. Knowledge about the sex determination system in D. longicaudata is relevant for the improvement of mass rearing protocols of this species. This information also provides the necessary background for further investigations on the underlying molecular mechanisms of sex determination in this species, and a better insight into the evolution of this pathway in Hymenoptera in particular and insects in general.     

COMPLEMENTARY SEX DETERMINATION IN HYMENOPTERAN PARASITOIDS AND ITS IMPLICATIONS FOR BIOLOGICAL CONTROL

Abstract In haplodiploid Hymenoptera, unfertilized eggs produce haploid males while fertilized eggs lead to diploid females under most circumstances. Diploid males can also be produced from fertilization under a system of sex determination known as complementary sex determination (CSD). Under single-locus CSD, sex is determined by multiple alleles at a single sex locus. Individuals heterozygous at the sex locus are female while hemizygous and homozygous individuals develop as haploid and diploid males, respectively. In multiple-locus CSD, two or more loci, each with two or more alleles, determine sex. Diploid individuals are female if one or more sex loci are heterozygous, while a diploid is male only if homozygous at all sex loci. Diploid males are known to occur in 43 hymenopteran species and single-locus CSD has been demonstrated in 22 of these species. Diploid males are either developmentally inviable or sterile, so their production constitutes a genetic load. Because diploid male production is more likely under inbreeding, CSD is a form of inbreeding depression. It is crucial to preserve the diversity of sex alleles and reduce the loss of genetic variation in biological control. In the parasitoid species with single-locus CSD, certain precautionary procedures can prevent negative effects of single-locus CSD on biological control.     

膜翅目寄生蜂的互补性别决定机制及其在生防上的意义(英文)

在膜翅目中 ,未受精卵形成单倍体的雄蜂 ,而在大多数情况下受精卵将产生双倍体的雌蜂。但是 ,因互补性别决定机制 (CSD)的作用 ,受精卵有时也会产生双倍体雄蜂。这种性别决定机制包括单位点的CSD和多位点的CSD。在单位点的CSD作用下 ,唯一的一个性位点上的多个等位基因决定后代个体的性别。性位点上杂合的个体将是雌性 ,半合或同型结合的个体将分别形成单倍体或双倍体的雄性。在多位点的CSD作用下 ,两个或两个以上的性位点控制后代的性别 ,每个性位点上包含两个或两个以上的等位基因。如果一个或一个以上的性位点是杂合的 ,形成的双倍体后代都是雌性的 ,但若是所有的性位点都为同型合子 ,则将产生双倍体的雄蜂。在膜翅目中 ,目前已知 4 3种具有双倍体雄蜂 ,其中 2 2种发现存在单位点的CSD ,但是多位点的CSD还有待于确认。双倍体的雄性个体或者不能存活 ,或者不育 ,这样的个体形成将对寄生蜂种群的增长带来一定的遗传负担。在生物防治上 ,保护寄生蜂种群的性等位基因的多样性及减少其遗传多异性的损失极其重要。如果利用具有单位点CSD的种类 ,采取一定的措施将可避免由于双倍体雄性的形成所带来的负面影响。     

Life‐history traits of the Whiting polyploid line of the parasitoid Nasonia vitripennis

In hymenopterans, males are normally haploid (1n) and females diploid (2n), but individuals with divergent ploidy levels are frequently found. In species with ‘complementary sex determination’ (CSD), increasing numbers of diploid males that are often infertile or unviable arise from inbreeding, presenting a major impediment to biocontrol breeding. Non‐CSD species, which are common in some parasitoid wasp taxa, do not produce polyploids through inbreeding. Nevertheless, polyploidy also occurs in non‐CSD Hymenoptera. As a first survey on the impacts of inbreeding and polyploidy of non‐CSD species, we investigate life‐history traits of a long‐term laboratory line of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (‘Whiting polyploid line’) in which polyploids of both sexes (diploid males, triploid females) are viable and fertile. Diploid males produce diploid sperm and virgin triploid females produce haploid and diploid eggs. We found that diploid males did not differ from haploid males with respect to body size, progeny size, mate competition, or lifespan. When diploid males were mated to many females (without accounting for mating order), the females produced a relatively high proportion of male offspring, possibly indicating that these males produce less sperm and/or have reduced sperm functionality. In triploid females, parasitization rate and fecundity were reduced and body size was slightly increased, but there was no effect on lifespan. After one generation of outbreeding, lifespan as well as parasitization rate were increased, and a body size difference was no longer apparent. This suggests that outbreeding has an effect on traits observed in an inbred polyploidy background. Overall, these results indicate some phenotypic detriments of non‐CSD polyploids that must be taken into account in breeding.     

Balancing selection maintains sex determining alleles in multiple‐locus complementary sex determination

Hymenopteran species in which sex is determined through a haplo‐diploid mechanism known as complementary sex determination (CSD) are vulnerable to a unique form of inbreeding depression. Diploids heterozygous at one or more CSD loci develop into females but diploids homozygous at all loci develop into diploid males, which are generally sterile or inviable. Species with multiple polymorphic CSD loci (ml‐CSD) may have lower rates of diploid male production than species with a single CSD locus (sl‐CSD), but it is not clear if polymorphism is consistently maintained at all loci. Here, we assess the rate of diploid male production in a population of Cotesia rubecula, a two‐locus CSD parasitoid wasp species, approximately 20 years after the population was introduced for biological control. We show that diploid male production dropped from 8–13% in 2005 and 2006 to 3–4% by 2015. We also show from experimental crosses that the population maintained polymorphism at both CSD loci in 2015. We use theory and simulations to show that balancing selection on all CSD alleles promotes polymorphism at several loci in ml‐CSD populations. Our study supports the hypothesis that ml‐CSD populations have lower diploid male production and are more likely to persist than comparable sl‐CSD populations.     

Single-locus complementary sex determination absent in Heterospilus prosopidis (Hymenoptera: Braconidae)

In the haplodiploid Hymenoptera, haploid males arise from unfertilized eggs, receiving a single set of maternal chromosomes while diploid females arise from fertilized eggs and receive both maternal and paternal chromosomes. Under single-locus complementary sex determination (sl-CSD), sex is determined by multiple alleles at a single locus. Sex locus heterozygotes develop as females, while hemizygous and homozygous eggs develop as haploid and diploid males, respectively. Diploid males, which are inviable or sterile in almost all cases studied, are therefore produced in high frequency under inbreeding or in populations with low sex allele diversity. CSD is considered to be the ancestral form of sex determination within the Hymenoptera because members of the most basal taxa have CSD while some of the more derived groups have other mechanisms of sex determination that produce the haplo-diploid pattern without penalizing inbreeding. In this study, we investigated sex determination in Heterospilus prosopidis Viereck, a parasitoid from a relatively primitive subfamily of the Braconidae, a hymenopteran family having species with and without CSD. By comparing sex ratio and mortality patterns produced by inbred and outbred females, we were able to rule out sl-CSD as a sex determination mechanism in this species. The absence of sl-CSD in H. prosopidis was unexpected given its basal phylogenetic position in the Braconidae. This and other recent studies suggest that sex determination systems in the Hymenoptera may be evolutionary labile.     

Microsatellite DNA analysis reveals low diploid male production in a communal bee with inbreeding

The mechanism of sex determination assumed widespread in parthenogenetically arrhen-otokous Hymenoptera is that of single locus complementary sex determination (CSD). Functionally sterile diploid males are produced under CSD and generate a genetic load, the cost of which increases with inbreeding. We quantify diploid male production (DMP, proportion of diploid individuals that are male) using a morphological criterion (adult fresh weight) and genetical (microsatellite DNA) markers in a communal, sexually size-dimorphic bee, Andrma scotica , which inbreeds. Male genotypes suggested a DMP of 0.003. The inbreeding coefficient, f , was significandy positive (+ 0.165), equivalent to 44% of matings being among full sibs (predicted DMP of 0.11). We hypothesize three non-mutually exclusive explanations to account for the large difference between the low observed (in males) and high expected (derived fromy f for females) DMP: (i) multilocus CSD, (ii) 'sex allele signalling' tied to mate selection, and (iii) sperm selection within mated females. The costs of inbreeding through DMP are apparendy low in A. scotica .     

Complementary sex determination in the genus Diadegma (Hymenoptera: Ichneumonidae)

In the evolution of sexual reproduction we would expect to see a close association between mating systems and sex determination mechanisms. Such associations are especially evident in the insect order Hymenoptera which shows great diversity with respect to both of these characteristics. The ancestral sex determination mechanism in this order is thought to be single‐locus complementary sex determination (sl‐CSD), which is inbreeding sensitive, and where inbreeding results in the production of sterile diploid males rather than daughters. Presently, however, there is insufficient data to give strong support to the hypothesis that sl‐CSD is truly the ancestral condition in the Hymenoptera, principally because of the difficulty of reliably determining the degree of male ploidy. Here we show that six ichneumonid parasitoids from the polyphyletic genus Diadegma are subject to sl‐CSD, using neuronal cell DNA flow cytometry to distinguish ploidy levels. The presence of sl‐CSD in these six species, together with earlier evidence from the authors for D. chrysostictos, provides considerable support for the notion that sl‐CSD was ancestral in the Aculeata/Ichneumonoidea clade, which contains all eusocial Hymenoptera. Moreover, because flow cytometry discriminates reliably between haploid and diploid males, and is independent of the maternal sex allocation or the need for genetic markers, it has considerable potential for the determination of ploidy more generally.     

The effects of functional response and host abundance fluctuations on genetic rescue in parasitoids with single‐locus sex determination

Many parasitoids have single‐locus complementary sex determination (sl‐CSD), which produces sterile or inviable males when homozygous at the sex determining locus. A previous study theoretically showed that small populations have elevated risks of extinction due to the positive feedback between inbreeding and small population size, referred to as the diploid male vortex. A few modeling studies have suggested that the diploid male vortex may not be as common because balancing selection at sex determining loci tends to maintain high allelic diversity in spatially structured populations. However, the generality of the conclusion is yet uncertain, as they were drawn either from models developed for particular systems or from a general‐purpose competition model. To attest the conclusion, we study several well‐studied host–parasitoid models that incorporate functional response specifying the number of attacked hosts given a host density and derive the conditions for a diploid male vortex in a single population. Then, we develop spatially structured individual‐based versions of the models to include female behavior, diploid male fertility, and temporal fluctuations. The results show that producing a handful of successful offspring per female parasitoid could enable parasitoid persistence when a typical number of CSD alleles are present. The effect of functional response depends on the levels of fluctuations in host abundance, and inviable or partially fertile diploid males and a small increase in dispersal can alleviate the risk of a diploid male vortex. Our work supports the generality of effective genetic rescue in spatially connected parasitoid populations with sl‐CSD. However, under more variable climate, the efficacy of the CSD mechanism may substantially decline.     

Inbreeding depression and purging in a haplodiploid: gender-related effects

Compared with diploid species, haplodiploids suffer less inbreeding depression because male haploidy imposes purifying selection on recessive deleterious alleles. However, alleles of genes only expressed in the diploid females are protected in heterozygous individuals. This leads to the prediction that haplodiploids suffer more from inbreeding effects on life-history traits controlled by genes with female-limited expression. To test this, we used a wild population of the haplodiploid mite Tetranychus urticae. First, negative effects of inbreeding were investigated by comparing maturation rate, juvenile survival, oviposition rate and longevity between lines created by three generations of either outbreeding or mother–son inbreeding. Second, purging through inbreeding was investigated by comparing the intensity of inbreeding depression between outbred families with known inbreeding/outbreeding mating histories. Negative effects of inbreeding and evidence for purging were found for the female trait oviposition rate, but not for juvenile survival and longevity. Both male and female maturation rate were negatively affected by inbreeding, most likely due to maternal effects because inbred offspring of outbred mothers was not affected. These results support the hypothesis that, in haplodiploids inbreeding effects and genetic variation due to deleterious recessive alleles may depend on gender.     

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.     

Outbreeding depression, but no inbreeding depression in haplodiploid Ambrosia beetles with regular sibling mating

In sexual reproduction the genetic similarity or dissimilarity between mates strongly affects offspring fitness. When mating partners are too closely related, increased homozygosity generally causes inbreeding depression, whereas crossing between too distantly related individuals may disrupt local adaptations or coadaptations within the genome and result in outbreeding depression. The optimal degree of inbreeding or outbreeding depends on population structure. A long history of inbreeding is expected to reduce inbreeding depression due to purging of deleterious alleles, and to promote outbreeding depression because of increased genetic variation between lineages. Ambrosia beetles (Xyleborini) are bark beetles with haplodiploid sex determination, strong local mate competition due to regular sibling mating within the natal chamber, and heavily biased sex ratios. We experimentally mated females of Xylosandrus germanus to brothers and unrelated males and measured offspring fitness. Inbred matings did not produce offspring with reduced fitness in any of the examined life-history traits. In contrast, outcrossed offspring suffered from reduced hatching rates. Reduction in inbreeding depression is usually attributed to purging of deleterious alleles, and the absence of inbreeding depression in X. germanus may represent the highest degree of purging of all examined species so far. Outbreeding depression within the same population has previously only been reported from plants. The causes and consequences of our findings are discussed with respect to mating strategies, sex ratios, and speciation in this unusual system.     

Contagious parthenogenesis, automixis, and a sex determination meltdown

Because of the twofold cost of sex, genes conferring asexual reproduction are expected to spread rapidly in sexual populations. However, in reality this simple prediction is often confounded by several complications observed in natural systems. Motivated by recent findings in the Cape honey bee and in the parasitoid wasp Lysiphlebus fabarum, we explore through mathematical models the spread of a recessive, parthenogenesis inducing allele in a haplodiploid population. The focus of these models is on the intricate interactions between the mode of parthenogenesis induction through automixis and complementary sex determination (CSD) systems. These interactions may result in asexual production of diploid male offspring and the spread of the parthenogenesis-inducing allele through these males. We demonstrate that if parthenogenetic females produce a substantial proportion of male offspring, this may prevent the parthenogenesis-inducing allele from spreading. However, this effect is weakened if these diploid males are at least partially fertile. We also predict a degradation of multilocus CSD systems during the spread of parthenogenesis, following which only a single polymorphic CSD locus is maintained. Finally, based on empirical parameter estimates from L. fabarum we predict that male production in parthenogens is unlikely to prevent the eventual loss of sexual reproduction in this system.     

Genetic diversity and complementary sex determination (CSD) in Dolerus aeneus (Hymenoptera, Symphyta): implications for the conservation of an ecologically-important sawfly

Graminivorous sawfly numbers have declined steadily over recent years as a consequence of agricultural intensification. In spite of these declines and the importance of sawflies (Hymenoptera, Symphyta) as a food source for threatened populations of farmland birds, sawfly conservation studies have been restricted to census-based research. This is largely due to certain aspects of the sawfly life-cycle which make adequate sampling of these insects difficult. However, genetic research provides a valuable insight into population structure that cannot be obtained by traditional ecological means, and one which is essential for the development of conservation management strategies. In this study, we investigate the population genetic structure of the graminivorous sawfly Dolerus aeneus, and conduct a preliminary study to determine whether complementary sex determination (CSD), which can produce sterile diploid males under inbreeding conditions, operates in this species. Our research suggests that fragmentation of sawfly habitat as a result of agri-intensification has not yet acted to isolate D. aeneus populations, although some genetic effects are apparent (inbreeding and low diversity compared with other solitary Hymenoptera). In addition, diploid males were detected which may have compromised fertility and could indicate that CSD is operative in D. aeneus. This study highlights the need for further genetic research in sawflies to assess population structure on a UK-wide scale and to assess the prevalence of diploid males in key species. We discuss our findings in wider context of the genetics of Hymenoptera and the conservation and management of farmland biodiversity.     

Triploid females and diploid males: underreported phenomena in <Emphasis Type="Italic">Polistes</Emphasis> wasps?

Summary In hymenopteran species, males are usually haploid and females diploid. However, in species that have complementary sex determination (CSD), diploid males arise when a female produces offspring that are homozygous at the sex-determining locus. Although diploid males are often sterile, in some species they have been shown to produce diploid sperm, thus producing triploid daughters if they mate successfully. Diploid males have been observed in very few species of social wasps, and we know of no published reports of triploid females. In this paper, we review the existing literature on diploid males and triploid females in the Hymenoptera, and report the observation of triploid females in three species of Polistes paper wasps. Although polyploid offspring may be produced parthenogenetically, the more likely scenario is that Polistes wasps have CSD and produce diploid males via homozygosity at the sex-determining locus. Therefore, female triploidy indicates that diploid males do exist in Polistes species where they are presumed to be absent, and are likely to be even more frequent among species that have experienced a genetic bottleneck. We conclude by cautioning against the assumption of a selective advantage to the production of early males, and by discussing the implications of male diploidy and female triploidy for measurement of sex ratio investment and assumptions of reproductive skew theory.Received 5 December 2003; revised 20 March 2004; accepted 19 April 2004.     

Evolution of the sterile caste

Biased sex ratios are expected to affect the conditions of the evolution of worker behaviour in malehaploid populations. In a subsocial, malehaploid species an association to female-biased brood sex ratios favours the evolution of worker behaviour. There are various reasons to expect such an association. The critical worker efficiency threshold required for worker behaviour to evolve is lower when looked at from the viewpoint of the mother than from that of the daughter. In this mother-daughter conflict, the mother can expect some help from her other offspring to resolve the conflict in her favour. In semisocial species the sex ratio biases have a less significant role and the worker behaviour can evolve more easily in malehaploid than in diploid populations. Furthermore, limited dispersal (due to the risks attached to it) is expected to favour worker behaviour in malehaploid but inbreeding in diploid populations. In addition to the genetic mechanisms that determine the critical worker efficiency threshold, it is essential to pay attention to those ecological factors that affect the actual worker efficiency in nature. Several such factors exist but it is not easy to associate them with the common occurrence of eusocial species in the Hymenoptera.     

Impacts of inbreeding on bumblebee colony fitness under field conditions

Background  

Inbreeding and the loss of genetic diversity are known to be significant threats to small, isolated populations. Hymenoptera represent a special case regarding the impact of inbreeding. Haplodiploidy may permit purging of deleterious recessive alleles in haploid males, meaning inbreeding depression is reduced relative to diploid species. In contrast, the impact of inbreeding may be exacerbated in Hymenopteran species that have a single-locus complementary sex determination system, due to the production of sterile or inviable diploid males. We investigated the costs of brother-sister mating in the bumblebee Bombus terrestris. We compared inbred colonies that produced diploid males and inbred colonies that did not produce diploid males with outbred colonies. Mating, hibernation and colony founding took place in the laboratory. Once colonies had produced 15 offspring they were placed in the field and left to forage under natural conditions.     

Population structure, mating system, and sex-determining allele diversity of the parasitoid wasp Habrobracon hebetor

Besides haplo-diploid sex determination, where females develop from fertilized diploid eggs and males from unfertilized haploid eggs, some Hymenoptera have a secondary system called complementary sex determination (CSD). This depends on genotypes of a 'sex locus' with numerous sex-determining alleles. Diploid heterozygotes develop as females, but diploid homozygotes become sterile or nonviable diploid males. Thus, when females share sex-determining alleles with their mates and produce low fitness diploid males, CSD creates a genetic load. The parasitoid wasp Habrobracon hebetor has CSD and displays mating behaviours that lessen CSD load, including mating at aggregations of males and inbreeding avoidance by females. To examine the influence of population structure and the mating system on CSD load, we conducted genetic analyses of an H. hebetor population in Wisconsin. Given the frequency of diploid males, we estimated that the population harboured 10-16 sex-determining alleles. Overall, marker allele frequencies did not differ between subpopulations, but frequencies changed dramatically between years. This reduced estimates of effective size of subpopulations to only N3 approximately 20-50, which probably reflected annual fluctuations of abundance of H. hebetor. We also determined that the mating system is effectively monogamous. Models relating sex-determining allele diversity and the mating system to female productivity showed that inbreeding avoidance always decreased CSD loads, but multiple mating only reduced loads in populations with fewer than five sex-determining alleles. Populations with N3 less than 100 should have fewer sex-determining alleles than we found, but high diversity could be maintained by a combination of frequency-dependent selection and gene flow between populations.