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Research has been conducted on cytoplasmic incompatibility induction in Drosophila sechellia by Wolbachia variants wSh and wSn. The authors have identified the cytoplasmic incompatibility intensities of wSh and wSn in D. sechellia, and they report that the results have not suggested a consistent role of the host species in the cytoplasmic incompatibility induced by wSh.

Research has been conducted on endocellular bacterium Wolbachia which infects nematodes and arthropods. The authors have investigated cytoplasmic incompatibility phenotype, phylogenetic position based on wsp gene and associated mitochondrial haplotype of Wolbachia infections occurring in African populations of Drosophila simulans, and they report the results.

The flower flies (Diptera, Syrphidae) of French Polynesia are revised. A total of nine syrphid species were recorded from the five archipelagos of French Polynesia. Among them are two species new to science, Allograpta jacqi Mengual & Ramage sp. nov. and Melanostoma polynesiotes Mengual & Ramage sp. nov., and a new record for this country, Syritta aenigmatopatria Hardy, 1964. We provide DNA barcodes for all flower fly species of French Polynesia, making the syrphid fauna of this country the first one in the world to be entirely barcoded. New data on biology, flowers visited and some taxonomic notes are provided. An identification key for the species of Syrphidae in French Polynesia is given, as well as an identification key for the species of Melanostoma Schiner, 1860 in the Australasian and Oceanian Regions.

Abstract Background The interaction between the Blue Moon butterfly, Hypolimnas bolina, and Wolbachia has attracted interest because of the high prevalence of male-killing achieved within the species, the ecological consequences of this high prevalence, the intensity of selection on the host to suppress the infection, and the presence of multiple Wolbachia infections inducing different phenotypes. We examined diversity in the co-inherited marker, mtDNA, and the partitioning of this between individuals of different infection status, as a means to investigate the population biology and evolutionary history of the Wolbachia infections. Results Part of the mitochondrial COI gene was sequenced from 298 individuals of known infection status revealing ten different haplotypes. Despite very strong biological evidence that the sample represents a single species, the ten haplotypes did not fall within a monophyletic clade within the Hypolimnas genus, with one haplotype differing by 5% from the other nine. There were strong associations between infection status and mtDNA haplotype. The presence of wBol1 infection in association with strongly divergent haplotypes prompted closer examination of wBol1 genetic variation. This revealed the existence of two cryptic subtypes, wBol1a and wBol1b. The wBol1a infection, by far the most common, was in strict association with the single divergent mtDNA haplotype. The wBol1b infection was found with two haplotypes that were also observed in uninfected specimens. Finally, the wBol2 infection was associated with a large diversity of mtDNA haplotypes, most often shared with uninfected sympatric butterflies. Conclusion This data overall supports the hypothesis that high prevalence of male-killing Wolbachia (wBol1) in H. bolina is associated with very high transmission efficiency rather than regular horizontal transmission. It also suggests this infection has undergone a recent selective sweep and was introduced in this species through introgression. In contrast, the sharing of haplotypes between wBol2-infected and uninfected individuals indicates that this strain is not perfectly transmitted and/or shows a significant level of horizontal transmission.

The accidental endogenization of viral elements within eukaryotic genomes can occasionally provide significant evolutionary benefits, giving rise to their long-term retention, that is, to viral domestication. For instance, in some endoparasitoid wasps (whose immature stages develop inside their hosts), the membrane-fusion property of double-stranded DNA viruses have been repeatedly domesticated following ancestral endogenizations. The endogenized genes provide female wasps with a delivery tool to inject virulence factors that are essential to the developmental success of their offspring. Because all known cases of viral domestication involve endoparasitic wasps, we hypothesized that this lifestyle, relying on a close interaction between individuals, may have promoted the endogenization and domestication of viruses. By analyzing the composition of 124 Hymenoptera genomes, spread over the diversity of this clade and including free-living, ecto, and endoparasitoid species, we tested this hypothesis. Our analysis first revealed that double-stranded DNA viruses, in comparison with other viral genomic structures (ssDNA, dsRNA, ssRNA), are more often endogenized and domesticated (that is, retained by selection) than expected from their estimated abundance in insect viral communities. Second, our analysis indicates that the rate at which dsDNA viruses are endogenized is higher in endoparasitoids than in ectoparasitoids or free-living hymenopterans, which also translates into more frequent events of domestication. Hence, these results are consistent with the hypothesis that the endoparasitoid lifestyle has facilitated the endogenization of dsDNA viruses, in turn, increasing the opportunities of domestications that now play a central role in the biology of many endoparasitoid lineages., Competing Interests: BG, DL, SC, PB, DN, AC, JR, JS, Dd, BB, JV No competing interests declared, (© 2023, Guinet et al.)

Natural selection is commonly seen not just as an explanation for adaptive evolution, but as the inevitable consequence of "heritable variation in fitness among individuals". Although it remains embedded in biological concepts, such a formalisation makes it tempting to explore whether this precondition may be met not only in life as we know it, but also in other physical systems. This would imply that these systems are subject to natural selection and may perhaps be investigated in a biological framework, where properties are typically examined in light of their putative functions. Here we relate the major questions that were debated during a three-day workshop devoted to discussing whether natural selection may take place in non-living physical systems. We start this report with a brief overview of research fields dealing with "life-like" or "proto-biotic" systems, where mimicking evolution by natural selection in test tubes stands as a major objective. We contend the challenge may be as much conceptual as technical. Taking the problem from a physical angle, we then discuss the framework of dissipative structures. Although life is viewed in this context as a particular case within a larger ensemble of physical phenomena, this approach does not provide general principles from which natural selection can be derived. Turning back to evolutionary biology, we ask to what extent the most general formulations of the necessary conditions or signatures of natural selection may be applicable beyond biology. In our view, such a cross-disciplinary jump is impeded by reliance on individuality as a central yet implicit and loosely defined concept. Overall, these discussions thus lead us to conjecture that understanding, in physico-chemical terms, how individuality emerges and how it can be recognised, will be essential in the search for instances of evolution by natural selection outside of living systems.

New genetic diagnostic approaches have greatly aided efforts to document global biodiversity and improve biosecurity. This is especially true for organismal groups in which species diversity has been underestimated historically due to difficulties associated with sampling, the lack of clear morphological characteristics, and/or limited availability of taxonomic expertise. Among these methods, DNA sequence barcoding (also known as "DNA barcoding") and by extension, meta-barcoding for biological communities, has emerged as one of the most frequently utilized methods for DNA-based species identifications. Unfortunately, the use of DNA barcoding is limited by the availability of complete reference libraries (i.e., a collection of DNA sequences from morphologically identified species), and by the fact that the vast majority of species do not have sequences present in reference databases. Such conditions are critical especially in tropical locations that are simultaneously biodiversity rich and suffer from a lack of exploration and DNA characterization by trained taxonomic specialists. To facilitate efforts to document biodiversity in regions lacking complete reference libraries, we developed a novel statistical approach that categorizes unidentified species as being either likely native or likely nonnative based solely on measures of nucleotide diversity. We demonstrate the utility of this approach by categorizing a large sample of specimens of terrestrial insects and spiders (collected as part of the Moorea BioCode project) using a generalized linear mixed model (GLMM). Using a training data set of known endemic (n = 45) and known introduced species (n = 102), we then estimated the likely native/nonnative status for 4,663 specimens representing an estimated 1,288 species (412 identified species), including both those specimens that were either unidentified or whose endemic/introduced status was uncertain. Using this approach, we were able to increase the number of categorized specimens by a factor of 4.4 (from 794 to 3,497), and the number of categorized species by a factor of 4.8 from (147 to 707) at a rate much greater than chance (77.6% accuracy). The study identifies phylogenetic signatures of both native and nonnative species and suggests several practical applications for this approach including monitoring biodiversity and facilitating biosecurity., (© 2019 by the Ecological Society of America.)

More than any other genome components, Transposable Elements (TEs) have the capacity to move across species barriers through Horizontal Transfer (HT), with substantial evolutionary consequences. Previous large-scale surveys, based on full-genomes comparisons, have revealed the transposition mode as an important predictor of HT rates variation across TE superfamilies. However, host biology could represent another major explanatory factor, one that needs to be investigated through extensive taxonomic sampling. Here we test this hypothesis using a field collection of 460 arthropod species from Tahiti and surrounding islands. Through targeted massive parallel sequencing, we uncover patterns of HT in three widely-distributed TE superfamilies with contrasted modes of transposition. In line with earlier findings, the DNA transposons under study (TC1-Mariner) were found to transfer horizontally at the highest frequency, closely followed by the LTR superfamily (Copia), in contrast with the non-LTR superfamily (Jockey), that mostly diversifies through vertical inheritance and persists longer within genomes. Strikingly, across all superfamilies, we observe a marked excess of HTs in Lepidoptera, an insect order that also commonly hosts baculoviruses, known for their ability to transport host TEs. These results turn the spotlight on baculoviruses as major potential vectors of TEs in arthropods, and further emphasize the importance of non-vertical TE inheritance in genome evolution., Competing Interests: The authors have declared that no competing interests exist.

Objective: Targeted sequencing of 16S rDNA amplicons is routinely used for microbial community profiling but this method suffers several limitations such as bias affinity of universal primers and short read size. Gene capture by hybridization represents a promising alternative. Here we used a metagenomic extract from the pea aphid Acyrthosiphon pisum to compare the performances of two widely used PCR primer pairs with DNA capture, based on solution hybrid selection., Results: All methods produced an exhaustive description of the 8 bacterial taxa known to be present in this sample. In addition, the methods yielded similar quantitative results, with the number of reads strongly correlating with quantitative PCR controls. Both methods can thus be considered as qualitatively and quantitatively robust on such a sample with low microbial complexity.

Wolbachia bacteria infect about half of all arthropods, with diverse and extreme consequences ranging from sex-ratio distortion and mating incompatibilities to protection against viruses. These phenotypic effects, combined with efficient vertical transmission from mothers to offspring, satisfactorily explain the invasion dynamics of Wolbachia within species. However, beyond the species level, the lack of congruence between the host and symbiont phylogenetic trees indicates that Wolbachia horizontal transfers and extinctions do happen and underlie its global distribution. But how often do they occur? And has the Wolbachia pandemic reached its equilibrium? Here, we address these questions by inferring recent acquisition/loss events from the distribution of Wolbachia lineages across the mitochondrial DNA tree of 3,600 arthropod specimens, spanning 1,100 species from Tahiti and surrounding islands. We show that most events occurred within the last million years, but are likely attributable to individual level variation (e.g., imperfect maternal transmission) rather than population level variation (e.g., Wolbachia extinction). At the population level, we estimate that mitochondria typically accumulate 4.7% substitutions per site during an infected episode, and 7.1% substitutions per site during the uninfected phase. Using a Bayesian time calibration of the mitochondrial tree, these numbers translate into infected and uninfected phases of approximately 7 and 9 million years. Infected species thus lose Wolbachia slightly more often than uninfected species acquire it, supporting the view that its present incidence, estimated here slightly below 0.5, represents an epidemiological equilibrium., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: RAD-seq is a powerful tool, increasingly used in population genomics. However, earlier studies have raised red flags regarding possible biases associated with this technique. In particular, polymorphism on restriction sites results in preferential sampling of closely related haplotypes, so that RAD data tends to underestimate genetic diversity., Results: Here we (1) clarify the theoretical basis of this bias, highlighting the potential confounding effects of population structure and selection, (2) confront predictions to real data from in silico digestion of full genomes and (3) provide a proof of concept toward an ABC-based correction of the RAD-seq bias. Under a neutral and panmictic model, we confirm the previously established relationship between the true polymorphism and its RAD-based estimation, showing a more pronounced bias when polymorphism is high. Using more elaborate models, we show that selection, resulting in heterogeneous levels of polymorphism along the genome, exacerbates the bias and leads to a more pronounced underestimation. On the contrary, spatial genetic structure tends to reduce the bias. We confront the neutral and panmictic model to "ideal" empirical data (in silico RAD-sequencing) using full genomes from natural populations of the fruit fly Drosophila melanogaster and the fungus Shizophyllum commune, harbouring respectively moderate and high genetic diversity. In D. melanogaster, predictions fit the model, but the small difference between the true and RAD polymorphism makes this comparison insensitive to deviations from the model. In the highly polymorphic fungus, the model captures a large part of the bias but makes inaccurate predictions. Accordingly, ABC corrections based on this model improve the estimations, albeit with some imprecisions., Conclusion: The RAD-seq underestimation of genetic diversity associated with polymorphism in restriction sites becomes more pronounced when polymorphism is high. In practice, this means that in many systems where polymorphism does not exceed 2 %, the bias is of minor importance in the face of other sources of uncertainty, such as heterogeneous bases composition or technical artefacts. The neutral panmictic model provides a practical mean to correct the bias through ABC, albeit with some imprecisions. More elaborate ABC methods might integrate additional parameters, such as population structure and selection, but their opposite effects could hinder accurate corrections.

Reduced representation genomics approaches, of which RADseq is currently the most popular form, offer the possibility to produce genome wide data from potentially any species, without previous genomic information. The application of RADseq to highly multiplexed libraries (including numerous specimens, and potentially numerous different species) is however limited by technical constraints. First, the cost of synthesis of Illumina adaptors including molecular identifiers (MIDs) becomes excessive when numerous specimens are to be multiplexed. Second, the necessity to empirically adjust the ratio of adaptors to genomic DNA concentration impedes the high throughput application of RADseq to heterogeneous samples, of variable DNA concentration and quality. In an attempt to solve these problems, we propose here some adjustments regarding the adaptor synthesis. First, we show that the common and unique (MID) parts of adaptors can be synthesized separately and subsequently ligated, which drastically reduces the synthesis cost, and thus allows multiplexing hundreds of specimens. Second, we show that self-ligation of adaptors, which makes the adaptor concentration so critical, can be simply prevented by using unphosphorylated adaptors, which significantly improves the ligation and sequencing yield.

Symbionts that distort their host's sex ratio by favouring the production and survival of females are common in arthropods. Their presence produces intense Fisherian selection to return the sex ratio to parity, typified by the rapid spread of host 'suppressor' loci that restore male survival/development. In this study, we investigated the genomic impact of a selective event of this kind in the butterfly Hypolimnas bolina. Through linkage mapping, we first identified a genomic region that was necessary for males to survive Wolbachia-induced male-killing. We then investigated the genomic impact of the rapid spread of suppression, which converted the Samoan population of this butterfly from a 100:1 female-biased sex ratio in 2001 to a 1:1 sex ratio by 2006. Models of this process revealed the potential for a chromosome-wide effect. To measure the impact of this episode of selection directly, the pattern of genetic variation before and after the spread of suppression was compared. Changes in allele frequencies were observed over a 25 cM region surrounding the suppressor locus, with a reduction in overall diversity observed at loci that co-segregate with the suppressor. These changes exceeded those expected from drift and occurred alongside the generation of linkage disequilibrium. The presence of novel allelic variants in 2006 suggests that the suppressor was likely to have been introduced via immigration rather than through de novo mutation. In addition, further sampling in 2010 indicated that many of the introduced variants were lost or had declined in frequency since 2006. We hypothesize that this loss may have resulted from a period of purifying selection, removing deleterious material that introgressed during the initial sweep. Our observations of the impact of suppression of sex ratio distorting activity reveal a very wide genomic imprint, reflecting its status as one of the strongest selective forces in nature.

Many insect species harbor Wolbachia bacteria that induce cytoplasmic incompatibility (CI), i.e. embryonic lethality in crosses between infected males and uninfected females, or between males and females carrying incompatible Wolbachia strains. The molecular mechanism of CI remains unknown, but the available data are best interpreted under a modification-rescue model, where a mod function disables the reproductive success of infected males' sperm, unless the eggs are infected and express a compatible resc function. Here we examine the evolution of CI in the mosquito Culex pipiens, harbouring a large number of closely related Wolbachia strains structured in five distinct phylogenetic groups. Specifically, we used a worldwide sample of mosquito lines to assess the hypothesis that genetic divergence should correlate with the divergence of CI properties on a low evolutionary scale. We observed a significant association of Wolbachia genetic divergence with CI patterns. Most Wolbachia strains from the same group were compatible whereas those from different groups were often incompatible. Consistently, we found a strong association between Wolbachia groups and their mod-resc properties. Finally, lines from the same geographical area were rarely incompatible, confirming the conjecture that the spatial distribution of Wolbachia compatibility types should be constrained by selection. This study indicates a clear correlation between Wolbachia genotypes and CI properties, paving the way toward the identification of the molecular basis of CI through comparative genomics.

Numerous insects carry intracellular bacteria that manipulate the insects' reproduction and thus facilitate their own spread. Cytoplasmic incompatibility (CI) is a common form of such manipulation, where a (currently uncharacterized) bacterial modification of male sperm induces the early death of embryos unless the fertilized eggs carry the same bacteria, inherited from the mother. The death of uninfected embryos provides an indirect selective advantage to infected ones, thus enabling the spread of the bacteria. Here we use and expand recently developed algorithms to infer the genetic architecture underlying the complex incompatibility data from the mosquito Culex pipiens. We show that CI requires more genetic determinants than previously believed and that quantitative variation in gene products potentially contributes to the observed CI patterns. In line with population genetic theory of CI, our analysis suggests that toxin factors (those inducing embryo death) are present in fewer copies in the bacterial genomes than antitoxin factors (those ensuring that infected embryos survive). In combination with comparative genomics, our approach will provide helpful guidance to identify the genetic basis of CI and more generally of other toxin/antitoxin systems that can be conceptualized under the same framework.

INFERRING PHYLOGENETIC RELATIONSHIPS BETWEEN CLOSELY RELATED TAXA CAN BE HINDERED BY THREE FACTORS: (1) the lack of informative molecular variation at short evolutionary timescale; (2) the lack of established markers in poorly studied taxa; and (3) the potential phylogenetic conflicts among different genomic regions due to incomplete lineage sorting or introgression. In this context, Restriction site Associated DNA sequencing (RAD-seq) seems promising as this technique can generate sequence data from numerous DNA fragments scattered throughout the genome, from a large number of samples, and without preliminary knowledge on the taxa under study. However, divergence beyond the within-species level will necessarily reduce the number of conserved and non-duplicated restriction sites, and therefore the number of loci usable for phylogenetic inference. Here, we assess the suitability of RAD-seq for phylogeny using a simulated experiment on the 12 Drosophila genomes, with divergence times ranging from 5 to 63 million years. These simulations show that RAD-seq allows the recovery of the known Drosophila phylogeny with strong statistical support, even for relatively ancient nodes. Notably, this conclusion is robust to the potentially confounding effects of sequencing errors, heterozygosity, and low coverage. We further show that clustering RAD-seq data using the BLASTN and SiLiX programs significantly improves the recovery of orthologous RAD loci compared with previously proposed approaches, especially for distantly related species. This study therefore validates the view that RAD sequencing is a powerful tool for phylogenetic inference.

Background: The endosymbiont Wolbachia pipientis causes diverse and sometimes dramatic phenotypes in its invertebrate hosts. Four Wolbachia strains sequenced to date indicate that the constitution of the genome is dynamic, but these strains are quite divergent and do not allow resolution of genome diversification over shorter time periods. We have sequenced the genome of the strain wBol1-b, found in the butterfly Hypolimnas bolina, which kills the male offspring of infected hosts during embyronic development and is closely related to the non-male-killing strain wPip from Culex pipiens., Results: The genomes of wBol1-b and wPip are similar in genomic organisation, sequence and gene content, but show substantial differences at some rapidly evolving regions of the genome, primarily associated with prophage and repetitive elements. We identified 44 genes in wBol1-b that do not have homologs in any previously sequenced strains, indicating that Wolbachia's non-core genome diversifies rapidly. These wBol1-b specific genes include a number that have been recently horizontally transferred from phylogenetically distant bacterial taxa. We further report a second possible case of horizontal gene transfer from a eukaryote into Wolbachia., Conclusions: Our analyses support the developing view that many endosymbiotic genomes are highly dynamic, and are exposed and receptive to exogenous genetic material from a wide range of sources. These data also suggest either that this bacterial species is particularly permissive for eukaryote-to-prokaryote gene transfers, or that these transfers may be more common than previously believed. The wBol1-b-specific genes we have identified provide candidates for further investigations of the genomic bases of phenotypic differences between closely-related Wolbachia strains.

Background: Wolbachia bacteria have invaded many arthropod species by inducing Cytoplasmic Incompatibility (CI). These symbionts represent fascinating objects of study for evolutionary biologists, but also powerful potential biocontrol agents. Here, we assess the density dynamics of Wolbachia infections in males and females of the mosquito Aedes albopitcus, an important vector of human pathogens, and interpret the results within an evolutionary framework., Methodology/principal Findings: Wolbachia densities were measured in natural populations and in age controlled mosquitoes using quantitative PCR. We show that the density dynamics of the wAlbA Wolbachia strain infecting Aedes albopictus drastically differ between males and females, with a very rapid decay of infection in males only., Conclusions/significance: Theory predicts that Wolbachia and its hosts should cooperate to improve the transmission of infection to offspring, because only infected eggs are protected from the effects of CI. However, incompatible matings effectively lower the fertility of infected males, so that selection acting on the host genome should tend to reduce the expression of CI in males, for example, by reducing infection density in males before sexual maturation. The rapid decay of one Wolbachia infection in Aedes albopictus males, but not in females, is consistent with this prediction. We suggest that the commonly observed reduction in CI intensity with male age reflects a similar evolutionary process. Our results also highlight the importance of monitoring infection density dynamics in both males and females to assess the efficiency of Wolbachia-based control strategies.

Sex ratios are subject to distortion by a range of inherited parasites. Although it has been predicted that the presence of these elements will result in spatial and temporal variation in host sex ratio, testing of this hypothesis has been constrained by availability of historical data. We here determine spatial and temporal variation in sex ratio in a interaction between a butterfly and male-killing Wolbachia bacteria by assaying infection presence in museum specimens, and from this inferring infection prevalence and phenotype in historical populations. Comparison of contemporary and museum samples revealed profound change in four of five populations examined. Two populations become extremely female biased, associated with spread of the male-killer bacterium. One evolved from extremely female biased to a sex ratio near parity, resulting from the infection losing male-killing activity. The final population fluctuated widely in sex ratio, associated with varying frequency of the male killer. We conclude that asynchronous invasion and decline of sex-ratio distorters combines with the evolution of host suppressors to produce a rapidly changing mosaic of sex ratio. As a consequence, the reproductive ecology of the host species is likely to be fundamentally altered over short time scales. Further, the study demonstrates the utility of museum specimens as "silent witnesses" of evolutionary change.

Background: The interaction between the Blue Moon butterfly, Hypolimnas bolina, and Wolbachia has attracted interest because of the high prevalence of male-killing achieved within the species, the ecological consequences of this high prevalence, the intensity of selection on the host to suppress the infection, and the presence of multiple Wolbachia infections inducing different phenotypes. We examined diversity in the co-inherited marker, mtDNA, and the partitioning of this between individuals of different infection status, as a means to investigate the population biology and evolutionary history of the Wolbachia infections., Results: Part of the mitochondrial COI gene was sequenced from 298 individuals of known infection status revealing ten different haplotypes. Despite very strong biological evidence that the sample represents a single species, the ten haplotypes did not fall within a monophyletic clade within the Hypolimnas genus, with one haplotype differing by 5% from the other nine. There were strong associations between infection status and mtDNA haplotype. The presence of wBol1 infection in association with strongly divergent haplotypes prompted closer examination of wBol1 genetic variation. This revealed the existence of two cryptic subtypes, wBol1a and wBol1b. The wBol1a infection, by far the most common, was in strict association with the single divergent mtDNA haplotype. The wBol1b infection was found with two haplotypes that were also observed in uninfected specimens. Finally, the wBol2 infection was associated with a large diversity of mtDNA haplotypes, most often shared with uninfected sympatric butterflies., Conclusion: This data overall supports the hypothesis that high prevalence of male-killing Wolbachia (wBol1) in H. bolina is associated with very high transmission efficiency rather than regular horizontal transmission. It also suggests this infection has undergone a recent selective sweep and was introduced in this species through introgression. In contrast, the sharing of haplotypes between wBol2-infected and uninfected individuals indicates that this strain is not perfectly transmitted and/or shows a significant level of horizontal transmission.

Maternally inherited parasites are known to impose a wide variety of reproductive manipulations upon their host. These often produce strong selection on the host to suppress the parasite, resulting in a reduction in the frequency of the parasite. However, in the butterfly Hypolimnas bolina, infected with a Wolbachia bacterium, field data demonstrate that suppression of the male-killing phenotype does not depress parasite frequency. Here we test and verify one hypothesis to explain this apparent paradox-Wolbachia induces a second phenotype, Cytoplasmic Incompatibility (CI), in populations where host suppression has evolved. We further demonstrate that the capacity to induce CI has not evolved de novo, but instead is instantaneously expressed upon the survival of infected males. The significance of these results is threefold: (1) multiple phenotypes can provide Wolbachia with the means to maintain itself in a host following suppression of a single manipulative phenotype; (2) the ability to induce CI can remain hidden in systems in which male-killing is observed, just as the ability to induce male-killing may be obscured in strains exhibiting CI; (3) the evolutionary maintenance of CI in a system in which it is not expressed suggests a functional link with male-killing or other traits under selection.

The ratio of males to females in a species is often considered to be relatively constant, at least over ecological time. Hamilton noted that the spread of "selfish" sex ratio-distorting elements could be rapid and produce a switch to highly biased population sex ratios. Selection against a highly skewed sex ratio should promote the spread of mutations that suppress the sex ratio distortion. We show that in the butterfly Hypolimnas bolina the suppression of sex biases occurs extremely fast, with a switch from a 100:1 population sex ratio to 1:1 occurring in fewer than 10 generations.

Several lineages of maternally inherited symbionts have evolved the ability to kill infected females' sons, a phenomenon known as male-killing. Male-killing varies in its timing, from early (death during embryogenesis) to late (mortality of late larval instars). Following the observation that treatment of male-killer infected adult females Hypolimnas bolina with tetracycline, a bacteriostatic antibiotic, produces a delay in the timing of male death, we hypothesized that early male-killers possess the ability to kill males through bacterial activity outside of embryogenesis. We verified this hypothesis by showing that treatment of surviving larvae with the bacteriocidal antibiotic rifampicin rescues males. This discounted the hypothesis that delayed death occurred due to postponed effects of toxins produced at earlier stages, and thus supported the importance of bacterial activity in the larval phase in delayed male-killing. These results argue against the view that early male-killing is achieved by specifically targeting an early developmental process within the sex determination pathway.

Sex-ratio distorters are found in numerous species and can reach high frequencies within populations. Here, we address the compelling, but poorly tested, hypothesis that the sex ratio bias caused by such elements profoundly alters their host's mating system. We compare aspects of female and male reproductive biology between island populations of the butterfly Hypolimnas bolina that show varying degrees of female bias, because of a male-killing Wolbachia infection. Contrary to expectation, female bias leads to an increase in female mating frequency, up to a point where male mating capacity becomes limiting. We show that increased female mating frequency can be explained as a facultative response to the depleted male mating resources in female biased populations. In other words, this system is one where male-killing bacteria trigger a vicious circle of increasing male fatigue and female promiscuity.

Bacteriophages are common viruses infecting prokaryotes. In addition to their deadly effect, phages are also involved in several evolutionary processes of bacteria, such as coding functional proteins potentially beneficial to them, or favoring horizontal gene transfer through transduction. The particular lifestyle of obligatory intracellular bacteria usually protects them from phage infection. However, Wolbachia, an intracellular alpha-proteobacterium, infecting diverse arthropod and nematode species and best known for the reproductive alterations it induces, harbors a phage named WO, which has recently been proven to be lytic. Here, phage infection was checked in 31 Wolbachia strains, which induce 5 different effects in their hosts and infect 25 insect species and 3 nematodes. Only the Wolbachia infecting nematodes and Trichogramma were found devoid of phage infection. All the 25 detected phages were characterized by the DNA sequence of a minor capsid protein gene. Based on all data currently available, phylogenetic analyses show a lack of congruency between Wolbachia or insect and phage WO phylogenies, indicating numerous horizontal transfers of phage among the different Wolbachia strains. The absence of relation between phage phylogeny and the effects induced by Wolbachia suggests that WO is not directly involved in these effects. Implications on phage WO evolution are discussed.

Maternally inherited selfish genetic elements are common in animals . Whereas host genetics and ecology are recognized as factors that may limit the incidence of these parasites , theory suggests one further factor-interference with other selfish elements-that could affect their prevalence . In this paper, we show that spatial heterogeneity in the occurrence of the male-killing Wolbachia wBol1 in the tropical butterfly Hypolimnas bolina is caused by a second infection that can exclude the male-killer. We first provide evidence of a second Wolbachia strain, wBol2, present in most populations that do not carry the male-killer but rare or absent when the male-killer is present. Crossing data indicate that wBol2 in males induces cytoplasmic incompatibility to both uninfected and wBol1-infected females. The wBol2 infection can therefore not only spread through uninfected populations but also resist invasion by wBol1. Thus, we provide empirical support for the hypothesis that the incidence of particular selfish genetic elements can limit the presence of competing types.

Male-killing bacteria are widespread in arthropods, and can profoundly alter the reproductive biology of their host species. Here we detail the first case of complete suppression of a male killer. The nymphalid butterfly Hypolimnas bolina is infected with a strain of the bacterium Wolbachia, wBol1, which kills male host embryos in Polynesian populations, but does not do so in many areas of Southeast Asia, where both males and female adults are naturally infected, and wBol1-infected females produce a 1:1 sex ratio. We demonstrate that absence of male killing by wBol1 is associated with dominant zygotic suppression of the action of the male killer. Simulations demonstrate host suppressors of male-killer action can spread very rapidly, and historical data indicating the presence of male killing in Southeast Asia in the very recent past suggests suppressor spread has been a very recent occurrence. Thus, male killer/host interactions are much more dynamic than previously recognised, with rapid and dramatic loss of the phenotype. Our results also indicate that suppression can render male killers completely quiescent, leading to the conclusion that some species that do not currently express a male killer may have done so in the past, and thus that more species have had their biology affected by these parasites than previously believed., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

In addition to their nuclear genome, the vast majority of eukaryotes harbour cytoplasmic genomes, e.g. in mitochondria or chloroplasts. In the majority of cases, these cytoplasmic genomes are transmitted maternally only, leading to selective pressures divergent from those that act on nuclear genes. In particular, cytoplasmic genes, which reduce the fitness of males that carry them, but have no fitness effect in females, are believed to be selectively neutral. Here, we go a step further and argue that in outbreeding populations (i.e. populations with inbreeding avoidance), 'spiteful' cytoplasmic elements that reduce the number of offspring produced by males are in fact selected for. We study this process by means of a stochastic model, analysing both the probability of spread and the impact that such a spiteful cytotype can have on population dynamics. Our results demonstrate that the probability of spread of the spiteful cytotype can be several times higher in outbreeding than in panmictic populations. Spread and fixation of the spiteful cytotype can lead to different qualitative effects on the population dynamics, including extinction, decreased or increased stable population size. We discuss our results in respect to cytoplasmically induced male infertility and cytoplasmic incompatibility.

Cytoplasmic incompatibility (CI) is a reproductive incompatibility induced by maternally transmitted bacteria of the genera Wolbachia and Cardinium. In the simplest form of CI, offspring from infected males and uninfected females suffer from increased mortality. However, it has been noted that crosses between males and females carrying different strains of infection are often also incompatible. The evolutionary processes leading to the emergence of new CI-compatibility types are still not resolved. Here, we develop a model that extends previous theoretical approaches by including segregation of bacterial strains during transmission as well as a continuum of breeding systems ranging from inbreeding (complete sib mating) to outbreeding (complete sib-mating avoidance). Our results demonstrate that (1) with segregation of strains, evolution is unlikely to lead to new CI types that co-occur as a double infection with the preexisting one, (2) inbreeding substantially hampers the evolution of new CI types, and (3) outbreeding facilitates the evolution of new CI types. Our model also provides a hypothesis on the evolutionary origin of CI.

Wolbachia-induced cytoplasmic incompatibility (CI) is observed when males bearing the bacterium mate with uninfected females or with females bearing a different Wolbachia variant; in such crosses, paternal chromosomes are lost at the first embryonic mitosis, most often resulting in developmental arrest. The molecular basis of CI is currently unknown, but it is useful to distinguish conceptually the male and female sides of this phenomenon: in males, Wolbachia must do something, before it is shed from maturing sperm, that will disrupt paternal chromosomes functionality [this is usually termed "the modification (mod) function"]; in females, Wolbachia must somehow restore embryonic viability, through what is usually called "the rescue (resc) function." The occurrence of CI in crosses between males and females bearing different Wolbachia variants demonstrates that the mod and resc functions interact in a specific manner: different mod resc pairs make different compatibility types. We are interested in the evolutionary process allowing the diversification of compatibility types. In an earlier model, based on the main assumption that the mod and resc functions can mutate independently, we have shown that compatibility types can evolve through a two-step process, the first involving drift on mod variations and the second involving selection on resc variations. This previous study has highlighted the need for simulation-based models that would include the effects of nondeterministic evolutionary forces. This study is based on a simulation program fulfilling this condition, allowing us to follow the evolution of compatibility types under mutation, drift, and selection. Most importantly, simulations suggest that in the frame of our model, the evolution of compatibility types is likely to be a gradual process, with new compatibility types remaining partially compatible with ancestral ones.

Cytoplasmic incompatibility (CI) is induced in arthropods by the maternally inherited bacterium Wolbachia. When infected males mate with uninfected females or with females bearing a different Wolbachia variant, paternal chromosomes behave abnormally and embryos die. This pattern can be interpreted as resulting from two bacterial effects: One (usually termed mod, for modification) would affect sperm and induce embryo death, unless Wolbachia is also present in the egg, which implies the existence of a second effect, usually termed resc, for rescue. The fact that CI can occur in crosses between males and females infected by different Wolbachia shows that mod and resc interact in a specific manner. In other words, different compatibility types, or mod/resc pairs seem to have diverged from one (or a few) common ancestor(s). We are interested in the process allowing the evolution of mod/resc pairs. Here this question is addressed experimentally after cytoplasmic injection into a single host species (Drosophila simulans) by investigating compatibility relationships between closely related Wolbachia variants naturally evolving in different dipteran hosts: D. simulans, Drosophila melanogaster, and Rhagoletis cerasi. Our results suggest that closely related bacteria can be totally or partially incompatible. The compatibility relationships observed can be explained using a formal description of the mod and resc functions, implying both qualitative and quantitative variations.

In this study, we report data about the presence of Wolbachia in Drosophila yakuba, D. teissieri, and D. santomea. Wolbachia strains were characterized using their wsp gene sequence and cytoplasmic incompatibility assays. All three species were found infected with Wolbachia bacteria closely related to the wAu strain, found so far in D. simulans natural populations, and were unable to induce cytoplasmic incompatibility. We injected wRi, a CI-inducing strain naturally infecting D. simulans, into the three species and the established transinfected lines exhibited high levels of CI, suggesting that absence of CI expression is a property of the Wolbachia strain naturally present or that CI is specifically repressed by the host. We also tested the relationship between the natural infection and wRi and found that it fully rescues the wRi modification. This result was unexpected, considering the significant evolutionary divergence between the two Wolbachia strains.

Wolbachia are endosymbiotic bacteria, widespread in terrestrial Arthropods. They are mainly transmitted vertically, from mothers to offspring and induce various alterations of their hosts' sexuality and reproduction, the most commonly reported phenomenon being Cytoplasmic Incompatibility (CI), observed in Drosophila melanogaster and D. simulans. Basically, CI results in a more or less intense embryonic mortality, occurring in crosses between males infected by Wolbachia and uninfected females. In D. simulans, Wolbachia and CI were observed in 1986. Since then, this host species has become a model system for investigating the polymorphism of Wolbachia infections and CI. In this review we describe the different Wolbachia infections currently known to occur in D. melanogaster and D. simulans. The two species are highly contrasting with regard to symbiotic diversity: while five Wolbachia variants have been described in D. simulans natural populations, D. melanogaster seems to harbor one Wolbachia variant only. Another marked difference between these two Drosophila species is their permissiveness with regard to CI, which seems to be fully expressed in D. simulans but partially or totally repressed in D. melanogaster, demonstrating the involvement of host factors in the control of CI levels. The potential of the two host species regarding the understanding of CI and its evolution is also discussed.