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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.

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.

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.