Sex-ratio-distorting Wolbachia causes sex-role reversal in its butterfly host

Sex-role-reversed mating systems in which females compete for males and males may be choosy are usually associated with males investing more than females in offspring. We report that sex-role reversal may also be caused by selfish genetic elements which distort the sex ratio towards females. Some populations of the butterflies Acraea encedon and Acraea encedana are extremely female biased because over 90% of females are infected with a Wolbachia bacterium that is maternally inherited and kills male embryos. Many females in these populations are virgins suggesting that their reproductive success may be limited by access to males. These females form lekking swarms at landmarks in which females exhibit behaviours which we interpret as functioning to solicit matings from males. The hypothesis that female A. encedon swarm in order to mate is supported by the finding that, in release recapture experiments, mated females tend to leave the swarm while unmated females remained. This behaviour is a sex-role-reversed form of a common mating system in insects in which males form lekking swarms at landmarks and compete for females. Female lekking swarms are absent from less female-biased populations and here the butterflies are instead associated with resources in the form of the larval food plant.     

HOW CAN SEX RATIO DISTORTERS REACH EXTREME PREVALENCES? MALE‐KILLING WOLBACHIA ARE NOT SUPPRESSED AND HAVE NEAR‐PERFECT VERTICAL TRANSMISSION EFFICIENCY IN ACRAEA ENCEDON

Abstract.— Maternally transmitted bacteria that kill male hosts early in their development are found in many insects. These parasites typically infect 1–30% of wild females, but in a few species of insects, prevalences exceed 95%. We investigated one such case in the butterfly Acraea encedon , which is infected with a male-killing Wolbachia bacterium. We measured three key parameters that affect the prevalence of the parasite: transmission efficiency, rate of survival of infected males, and the direct cost of infection. We observed that all wild females transmit the bacterium to all their offspring and that all infected males die in wild populations. We were unable to detect any physiological cost to infection in lab culture. These observations explain the high prevalence of the A. encedon male killer, as theory predicts that under these conditions the parasite will spread to fixation. This will occur provided the death of males provides some benefit to the surviving infected females. The problem therefore becomes why the bacterium has not reached fixation and driven the butterfly extinct due to the shortage of males. We therefore investigated whether males choose to mate with uninfected rather than infected females, as this would prevent the bacterium from reaching fixation. We tested this hypothesis in the "lekking swarms" of virgin females found in the most female-biased populations, and were unable to detect any evidence of mate choice. In conclusion, this male killer has spread to high prevalence because it has a high transmission efficiency and low cost, but the factors maintaining uninfected females in the population remain unknown.     

The inheritance and population genetics of sex ratio in the butterfly Acraea encedon

In West and East Africa the butterfly, Acraea encedon , occurs in well-defined populations that are often predominantly female. Breeding the butterfly in the laboratory revealed the presence of an all-female strain, which is inherited directly through the female parent. It is probable that the inheritance is controlled by a Y-linked gene, causing meiotic drive in the Y chromosome, but the possibility of cytoplasmic inheritance has not been ruled out. A simple model for the population genetics of a predominantly female population indicates that such a population should rapidly become extinct due to the spreading of the all-female strain, but in most field populations studied extinction does not occur as quickly as predicted, if at all. Three factors which could enable populations to avoid extinction are investigated: suppressing systems, frequency-dependent mating preference and the sequence of emergence of the sexes in normal broods. No positive evidence has yet been found for the existence of a gene or genes capable of suppressing the sex ratio aberration, and no frequency-dependent mating preference was found, but an argument is presented which shows that the sequence of emergence in normal broods could be partly responsible for the maintenance of stable equilibria in predominantly female populations. Attempts to upset the sex ratio in the normal strain by making crosses between widely separated populations were not successful.     

All-female broods and mimetic polymorphism in Acraea encedon (L.) (Lepidoptera: Acraeidae) in Tanzania

In some populations of the African butterfly, Acraea encedon, there are two kinds of females, one producing offspring in a normal 1:1 sex ratio, the other producing females only; in other populations the sex ratio is apparently normal. All-female broods had hitherto been mainly associated with populations in which field sampling revealed an excess of females. The all-female brood trait is described from a population at Dar es Salaam which field sampling suggested was normal, and this indicates that the trait may be much more widespread and common than had previously been supposed. This discovery also extends the known distribution of the trait across Africa from Sierra Leone to eastern Tanzania. The butterfly is also a polymorphic Müllerian mimic of Danaus chrysippus, which is a highly unusual phenomenon as Müllerian mimicry is almost invariably monomorphic. The relative frequencies of two corresponding colour forms of the two species of butterflies at Dar es Salaam adds support to the hypothesis that they are indeed Mullerian mimics. The results of breeding experiments suggest that the polymorphic forms in Acraea encedon are allelic with dominance.     

Male-killing Wolbachia and mitochondrial DNA: selective sweeps,hybrid introgression and parasite population dynamics

Mitochondrial DNA (mtDNA) sequences are widely used as neutral genetic markers in insects. However, patterns of mtDNA variability are confounded by the spread of maternally transmitted parasites, which are genetically linked to the mitochondria. We have investigated these effects in the butterflies Acraea encedon (which is host to two strains of male-killing Wolbachia bacteria) and A. encedana (which is host to one strain). Within a population, the mitochondria are in linkage disequilibrium with the different male-killers. Furthermore, there has been a recent selective sweep of the mtDNA, which has led to the loss of mitochondrial variation within populations and erased any geographical structure. We also found that one of the male-killers, together with the associated mtDNA, has introgressed from A. encedana into A. encedon within the last 16,000 years. Interestingly, because butterflies are female heterogametic, this will presumably have also led to the introgression of genes on the W sex chromosome. Finally, in A. encedon the mitochondria in uninfected females are unaltered by the spread of the male-killer and have diverse, geographically structured mtDNA. This means we can reject the hypothesis that the male-killer is at a stable equilibrium maintained by imperfect transmission of the bacterium. Instead, some other form of balancing selection may be maintaining uninfected females in the population and preventing the species from going extinct due to a shortage of males.     

Male killing can select for male mate choice: a novel solution to the paradox of the lek

In lekking species, intense directional selection is applied to aspects of the male genotype by female choice. Under conventional quantitative genetics theory, the expectation is that this will lead to a rapid loss in additive genetic variance for the trait in question. However, despite female choice, male variation is maintained and hence it pays females to continue choosing. This has been termed the ''paradox of the lek''. Here we present a theoretical analysis of a putative sex-role-reversed lek in the butterfly Acraea encedon. Sex-role reversal appears to have come about because of infection with a male-killing Wolbachia. The bacterium is highly prevalent in some populations, such that there is a dearth of males. Receptive females form dense aggregations, and it has been suggested that males preferentially select females uninfected with the bacterium. As with more conventional systems, this presents a theoretical problem exactly analogous to the lek paradox, namely what maintains female variation and hence why do males continue to choose? We model the evolution of a male choice gene that allows discrimination between infected and uninfected females, and show that the stable maintenance of both female variation and male choice is likely, so long as males make mistakes when discriminating between females. Furthermore, our model allows the maintenance, in a panmictic population, of a male killer that is perfectly transmitted. This is the first model to allow this result, and may explain the long-term persistence of a male killer in Hypolimnas bolina.     

Polymorphic Müllerian mimicry in a group of African butterflies: a re-assessment of the relationship between Danaus chrysippus, Acraea encedon and Acraea encedana (Lepidoptera: Nymphalidae)

Müllerian mimicry, in which there is convergence in coloration between unrelated unpalatable species, should lead to uniformity in appearance, not polymorphism, and so the occurrence in tropical Africa of unrelated species of unpalatable butterflies with corresponding polymorphic colour forms suggested a problem of special evolutionary interest. Field work in Uganda and Sierra Leone in 1964 72 demonstrated a statistical association between the occurrence and relative frequencies of polymorphic forms in Danaus chrysippus (Danainae) and Acraea encedon (Acraeinae) which was deemed as confirmation of a Müllerian relationship between them. There were, however, certain anomalies which at the time remained unresolved. Later, in 1976, it was discovered that what had been called A. encedon is in reality two sibling species. A. encedon and a new one, named as A. encedana. The two differ in the structure of both male and female genitalia and in the coloration and the food-plants of the larvae. The recognition of the additional species has enabled a re-assessment of the polymorphie Müllerian association with D. chrysippus. It emerges that, although there is a close qualitative and quantitative reciprocal mimetic relationship between A. encedana and D. chrysippus, the relationship between A. encedon and D. chrysippus is much weaker, and in places non-exitent. The possible origin of the mimetic polymorphism is discussed in terms of hybridization of previously allopatric and monomorphic populations which have met as a consequence of recent expansions of geographical range in all three species resulting from forest clearance and the spread of savanna-like conditions in previously forested areas.     

Female mate preference based on male orange spot patterns in the feral guppy <Emphasis Type="Italic">Poecilia reticulata</Emphasis> in Japan

It is known that females prefer males with larger and/or brighter orange spots in many populations of the guppy Poecilia reticulata. However, female preference for male orange spots varies among populations and changes within several years when they are introduced into new habitats with different environment. Guppies were introduced into Okinawa, Japan, more than 20 years ago and were subjected to natural and sexual selection for a long period. The female preference for orange spot patterns of males was examined by the dichotomous choice experiment for a feral guppy population of the Hiji River, Okinawa. We chose full-sibling males as a pair of stimulus males that were simultaneously presented to a test female, because sibling males should resemble each other. To create different orange spot patterns between stimulus males, one male of the stimulus male pair was fed carotenoid-supplement food such as algae and another male was fed low-carotenoid food. High-carotenoid-treatment males showed not only brighter coloration of orange spots but also larger spots than other males as a result of this dietary-manipulation. In the dichotomous choice experiment, females preferred the high-carotenoid-treatment males. In addition, logistic regression analysis clarified that brighter coloration of male orange spots was the most important factor for female mate preference. This finding suggests indirect benefits of female preference for male orange spot patterns if the male foraging ability for algae were heritable.     

Wolbachia infection associated with all-female broods in Hypolimnas bolina (Lepidoptera: Nymphalidae): evidence for horizontal transmission of a butterfly male killer

Inherited bacteria that kill male hosts during embryogenesis infect a wide range of insect species. In order to ascertain if there are patterns to host infection, with particular male killing bacteria specialising on particular taxa, we investigated the male killing trait in the butterfly Hypolimnas bolina. All-female broods were first reported in this species in the 1920s. Investigation of this system in the Fiji Islands revealed the causal agent of sex ratio distortion in H. bolina to be a male killing Wolbachia bacterium. This bacterium is identical in wsp and ftsZ sequence to a male killer in the butterfly Acraea encedon in Tanzania, suggesting it has moved between host species, yet retained its phenotype. The prevalence of the Wolbachia was calculated for three different island groups of Fiji, and found to vary significantly across the country. Antibiotics failed to cure either the male killing trait or the Wolbachia infection. The implications of these results are discussed.     

Suppressor genes with gender differences in activity in natural populations of Drosophila robusta: another approach to wild-type

Homozygous or hemizygous expression of an X-linked wing mutant of Drosophila robusta varies from a rudimentary wing that does not reach the tip of the abdomen (called 'club') to forms with full-sized but curled or crumpled wings (called 'curly'). Homozygous club females crossed to flies from natural populations or laboratory stocks derived from wild flies invariably produce significantly less club male progeny than the 100% expected, most of them exhibiting less severe phenotypes: 'curly' forms and wild-type. The male progeny from similar crosses using curly females tend to be predominantly normal. By contrast, the male progeny of outcrossed females homozygous for an X-linked eye colour mutant, vermilion, are all vermilion. The data indicate that natural populations of D. robusta contain suppressors of the wing mutant but not of the eye colour mutant studied. Activity of the suppressors differs by gender: in experiments in which genetic theory expects similar results in the two sexes, males consistently show stronger effects of the suppressors than females.     

Male courtship preferences demonstrate discrimination against allopatric colour morphs in a cichlid fish

Whether premating isolation is achieved by male‐specific, female‐specific or sex‐independent assortative preferences often depends on the underlying evolutionary processes. Here we test mate preferences of males presented with females of different allopatric colour variants of the cichlid fish Tropheus sp., a Lake Tanganyika endemic with rich geographical colour pattern variation, in which the strength of sexual isolation varies between populations. We conducted two‐way mate choice experiments to compare behaviour of males of a red‐bodied morph (population Moliro) towards females from their own population with behaviour towards females from four allopatric populations at different stages of phylogenetic and phenotypic divergence. Males courted same‐population females significantly more intensely than females of other populations, and reduced their heteromorphic courtship efforts both with increasing genetic and increasing phenotypic distinctness of the females. In particular, females of a closely related red‐bodied population received significantly more courtship than either genetically distinct, similarly coloured females (‘Kirschfleck’ morph) or genetically related, differently coloured females (‘yellow‐blotch’ morph), both of which were courted similarly. Genetically and phenotypically distinct females (Tropheus polli) were not courted at all. Consistent with previous female‐choice experiments, female courtship activity also decreased with increasing genetic distance from the males’ population. Given successful experimental and natural introgression between colour morphs and the pervasive allopatry of related variants, we consider it unlikely that assortative preferences of both sexes were driven by direct selection during periods of secondary contact or, in turn, drove colour pattern differentiation in allopatry. Rather, we suggest that sexual isolation evolved as by‐product of allopatric divergence.     

Intrasexual competition among females and the stabilization of a conspicuous colour polymorphism in a Lake Victoria cichlid fish

The maintenance of colour polymorphisms within populations has been a long-standing interest in evolutionary ecology. African cichlid fish contain some of the most striking known cases of this phenomenon. Intrasexual selection can be negative frequency dependent when males bias aggression towards phenotypically similar rivals, stabilizing male colour polymorphisms. We propose that where females are territorial and competitive, aggression biases in females may also promote coexistence of female morphs. We studied a polymorphic population of the cichlid fish Neochromis omnicaeruleus from Lake Victoria, in which three distinct female colour morphs coexist: one plain brown and two blotched morphs. Using simulated intruder choice tests in the laboratory, we show that wild-caught females of each morph bias aggression towards females of their own morph, suggesting that females of all three morphs may have an advantage when their morph is locally the least abundant. This mechanism may contribute to the establishment and stabilization of colour polymorphisms. Next, by crossing the morphs, we generated sisters belonging to different colour morphs. We find no sign of aggression bias in these sisters, making pleiotropy unlikely to explain the association between colour and aggression bias in wild fish, which is maintained in the face of gene flow. We conclude that female-female aggression may be one important force for stabilizing colour polymorphism in cichlid fish.     

Female soldier beetles display a flexible preference for selectively favored male phenotypes

In Georgia (USA) the soldier beetle, Chauliognathus pennsylvanicus (Coleoptera; Cantharidae), exhibits clinal variation in the length of the spot on its elytron. This suggests that the viability of phenotypes varies by habitat. Evidence of viability selection comes from within-site changes in the spot length distribution across a breeding season. When males with spots of intermediate length became less frequent, they became disproportionately less likely to mate, consistent with either a loss of vigor among remaining males or female rejection of disfavored phenotypes. Persistent, daily courtship by males provides females with the opportunity to track changes in male phenotype frequency and to exercise choice for phenotypes favored under natural selection. A laboratory experiment in which the frequency of one spot morph (long) or the other (short) was increased from 25% to 75% over a period of 30 days revealed that females possess a flexible preference that leads them to prefer whichever spot type has become more common over time. A haploid genetic model demonstrates that a flexible female preference for the locally favored male phenotype can be selected for when different viability alleles, genetically correlated with the male trait, are favored in different habitats that are linked by gene flow. Thus, migration between different kinds of habitat patches of a metapopulation could maintain the variation in male quality. This variation favors female choice for any trait that is directly or indirectly favored by natural selection. Such choice imparts positive frequency-dependent selection that could rapidly fix traits pleiotropically linked to viability. Rapid fixation would cause differentiation between populations of colonizing species as females exercise choice for mates favored under new ecological conditions.     

Genetic difference in female choice between two guppy populations

The pattern of mating preferences of female guppies, Poecilia reticulata, differs genetically between two Trinidadian populations. Males from the Aripo and Paria rivers of Trinidad differ in the extent and intensity of orange pigment in their colour patterns. The relative area of orange in Paria colour patterns was nearly twice that in Aripo colour patterns. To test the hypothesis that a difference in female mating preferences could have contributed to the difference in colour patterns, patterns of female choice between laboratory-reared Paria and Aripo females were compared. The frequency of sexual responses to male courtship displays was used to measure the average preference of females for individual males. Paria females discriminated significantly between Paria males based upon differences in the extent of orange in the colour pattern. Aripo females showed no evidence of discrimination between Paria males, and thus differed significantly from Paria females in choice behaviour. Neither group of females discriminated between Aripo males. These results are evidence that female choice behaviour can vary genetically within a species and suggest that differences in preferences may have contributed to divergence in colour patterns among guppy populations.     

Population genetic structure of the gynodioecious Thymus vulgaris L. (Labiatae) in southern France

In a self-compatible gynodioecious species, the abundance of female plants (which are obligate outcrossers) relative to hermaphrodites (which may self and outcross) may be a critical factor influencing genetic diversity and population structure. In the gynodioecious Thymus vulgaris L., female frequency varies from 5 to 95%, providing a suitable model to examine this issue. In this study, we use allozyme markers to (1) evaluate the relationship between female frequency, genetic diversity and population structure, (2) determine whether females and hermaphrodites vary in heterozygote deficiency and (3) examine whether other factors such as plant density are related to heterozygote deficiency. Twenty three natural populations, with female frequencies ranging from 11 to 92%, were sampled in and around the St-Martin-de-Londres basin in southern France. Based on four polymorphic allozyme loci, we found no significant correlation between female frequency and heterozygote deficiency. A significant (P < 0.05) F_IS value over loci and over populations of 0.11 was detected. The F_IS value per population showed a significant heterozygote deficiency in 11 of the 23 populations. However, no significant difference between female and hermaphrodite F_IS values was found. A significant heterozygote deficiency only occurred in populations of intermediate density. There was little differentiation among populations (F_ST = 0.038) nor among subpopulations within each population. The significant F_IS values are thus mostly due to inbreeding effects. The lack of a correlation between F_IS values and female frequency may be due to outcrossing in hermaphrodites and/or restoration of male fertility which may occur to a greater extent at low female frequency. The similarity of female and hermaphrodite F_IS values indicates that females may occasion high levels of biparental inbreeding.     

Ecological aspects of polymorphism in an African land snail, Limicolaria martensiana

In Uganda the herbivorous land snail, Limicolaria martensiana occurs in well-defined populations. Samples of snails from 49 populations reveal the existence of five distinct colour forms, the frequency of which varies markedly from population to population. There is a common streaked form that occurs in all populations and a series of pallid forms that may be present or absent. Late Pleistocene fossils from the Western Rift indicate that the polymorphism is at least 8000–10,000 years old. The polymorphism is not correlated with broad geographical components of the environment or with habitat. It is however correlated with population density, and in one population a change in density resulted in a change in the relative frequency of the colour forms. Human activities have probably contributed to the dispersal of the snails to new areas, and because of these the founder effect cannot be ruled out as contributing to the polymorphism.     

Timing of breeding and offspring number covary with plumage colour among Gyrfalcons Falco rusticolus

Plumage colour variation exists among Gyrfalcons throughout their Arctic and sub‐Arctic circumpolar distribution, ranging from white through silver and grey to almost black. Although different colour variants coexist within many populations, a few geographical regions, such as northern Greenland, possess a single variant, suggesting that local environments may influence plumage colour variation. In central‐west Greenland (66.5–67.5°N), where multiple colour variants exist, white male Gyrfalcons fathered significantly earlier clutches than grey males. No significant association was observed between female colour and lay date. However, significantly more offspring were produced by both male and female white Gyrfalcons than by grey variants when controlling for lay date, and silver Gyrfalcons produced an intermediate number of offspring for both sexes. This pattern was further supported by breeding plumage colour pairings. Grey females paired with grey males nested significantly later in the season and produced fewer offspring than those paired with white males, whereas no difference in lay date or offspring number was found between white males paired with white or with grey females. The difference in the number of offspring produced at each nest‐site was also inversely correlated with the distance to the nearest neighbouring nest, and grey males nested in closer proximity to other nests compared with white and silver colour variants. These results suggest that factors associated with territory occupancy and timing of breeding may regulate reproductive success differently between colour variants, with directional selection favouring light‐coloured Gyrfalcons and resulting in earlier lay date and a high frequency of white plumage colour variants in this population. Although gene flow exists between our study population and those further north (>75°N), white Gyrfalcons prevail where the breeding season duration is even shorter, suggesting that nesting chronology in combination with genetic drift may play an important role in influencing plumage colour polymorphism among Gyrfalcon populations.     

Sexual selection as a promoter of population divergence in male phenotypic characters: a study on mainland and islet lizard populations

Sexual selection is often viewed as a promoter of population divergence, although some forms of sexual selection could rather hamper divergence. In the present study, we investigated whether sexual selection promotes divergence in sexually‐selected traits. We studied population variation in sexual selection in relation to colour morph and body size in islet and mainland populations of the Skyros wall lizard (Podarcis gaigeae). Females were most likely to mate with orange‐throated males with small body sizes, and male body size and coloration were therefore subject to correlational sexual selection. By contrast, male mating probabilities were not affected by any female phenotypic character. We also found variation in a female resistance trait (escape propensity), with females being more prone to escape when exposed to males from other habitats. Sexual selection could potentially affect the frequencies of throat colour morphs in this species by favouring orange‐throated males of small body size, although there was no evidence of sexual selection for local mates or rare phenotypes. The results obtained in the present study thus do not support a role for sexual selection as a promoter of population divergence in this species. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 374–389.     

High‐prevalence male‐killing Wolbachia in the butterfly Acraea encedana

Inherited bacteria which kill males during early development are widely distributed throughout the insects, but have been little studied outside of a single family of beetles, the Coccinellidae. We have investigated a male‐killing bacterium discovered in the butterfly Acraea encedana. This bacterium belongs to the genus Wolbachia and is identical in wsp gene sequence to a male‐killer in the closely related butterfly A. encedon, suggesting that it has either recently moved between host species or was inherited from a common ancestor of the butterflies. The prevalence of Wolbachia is remarkably high, 95% of females are infected and only 6% of wild caught butterflies are male. Measurements of the vertical transmission efficiency were used to calculate that this high prevalence is the result of infected females producing at least 1.79 times the number of surviving daughters as uninfected females (lower confidence limit is 1.25).     

Life history variation in gametophyte populations of the moss Ceratodon purpureus (Ditrichaceae)

The life cycles of mosses and other bryophytes are unique among land plants in that the haploid gametophyte stage is free-living and the diploid sporophyte stage is ephemeral and completes its development attached to the maternal gametophyte. Despite predictions that populations of haploids might contain low levels of genetic variation, moss populations are characterized by substantial variation at isozyme loci. The extent to which this is indicative of ecologically important life history variation is, however, largely unknown. Gametophyte plants from two populations of the moss Ceratodon purpureus were grown from single-spore isolates in order to assess variation in growth rates, biomass accumulation, and reproductive output. The data were analyzed using a nested analysis of variance, with haploid sib families (gametophytes derived from the same sporophyte) nested within populations. High levels of life history variation were observed within both populations, and the populations differed significantly in both growth and reproductive characteristics. Overall gametophytic sex ratios did not depart significantly from 1:1 within either population, but there was significant variation among families in both populations for progeny sex ratio. Some families produced predominantly male gametophytes, while others yielded predominantly females. Because C. purpureus has a chromosomal mechanism of sex determination, these observations suggest differential (but unpredictable) germination of male and female spores. Life history observations showed that male and female gametophytes are dimorphic in size, maturation rates, and reproductive output.