INSECT PHYLOGENOMICS. Response to Comment on "Phylogenomics resolves the timing and pattern of insect evolution".

Tong et al. comment on the accuracy of the dating analysis presented in our work on the phylogeny of insects and provide a reanalysis of our data. They replace log-normal priors with uniform priors and add a "roachoid" fossil as a calibration point. Although the reanalysis provides an interesting alternative viewpoint, we maintain that our choices were appropriate.

Genetic and developmental basis of F2 hybrid breakdown in Nasonia parasitoid wasps.

Speciation is responsible for the vast diversity of life, and hybrid inviability, by reducing gene flow between populations, is a major contributor to this process. In the parasitoid wasp genus Nasonia, F2 hybrid males of Nasonia vitripennis and Nasonia giraulti experience an increased larval mortality rate relative to the parental species. Previous studies indicated that this increase of mortality is a consequence of incompatibilities between multiple nuclear loci and cytoplasmic factors of the parental species, but could only explain ∼40% of the mortality rate in hybrids with N. giraulti cytoplasm. Here we report a locus on chromosome 5 that can explain the remaining mortality in this cross. We show that hybrid larvae that carry the incompatible allele on chromosome 5 halt growth early in their development and that ∼98% die before they reach adulthood. On the basis of these new findings, we identified a nuclear-encoded OXPHOS gene as a strong candidate for being causally involved in the observed hybrid breakdown, suggesting that the incompatible mitochondrial locus is one of the six mitochondrial-encoded NADH genes. By identifying both genetic and physiological mechanisms that reduce gene flow between species, our results provide valuable and novel insights into the evolutionary dynamics of speciation.

Hybrid incompatibilities in the parasitic wasp genus Nasonia: negative effects of hemizygosity and the identification of transmission ratio distortion loci.

The occurrence of hybrid incompatibilities forms an important stage during the evolution of reproductive isolation. In early stages of speciation, males and females often respond differently to hybridization. Haldane's rule states that the heterogametic sex suffers more from hybridization than the homogametic sex. Although haplodiploid reproduction (haploid males, diploid females) does not involve sex chromosomes, sex-specific incompatibilities are predicted to be prevalent in haplodiploid species. Here, we evaluate the effect of sex/ploidy level on hybrid incompatibilities and locate genomic regions that cause increased mortality rates in hybrid males of the haplodiploid wasps Nasonia vitripennis and Nasonia longicornis. Our data show that diploid F(1) hybrid females suffer less from hybridization than haploid F(2) hybrid males. The latter not only suffer from an increased mortality rate, but also from behavioural and spermatogenic sterility. Genetic mapping in recombinant F(2) male hybrids revealed that the observed hybrid mortality is most likely due to a disruption of cytonuclear interactions. As these sex-specific hybrid incompatibilities follow predictions based on Haldane's rule, our data accentuate the need to broaden the view of Haldane's rule to include species with haplodiploid sex determination, consistent with Haldane's original definition.

Genetics of cuticular hydrocarbon differences between males of the parasitoid wasps Nasonia giraulti and Nasonia vitripennis.

Many insects rely on cuticular hydrocarbons (CHCs) as major recognition signals between individuals. Previous research on the genetics of CHCs has focused on Drosophila in which the roles of three desaturases and one elongase were highlighted. Comparable studies in other insect taxa have not been conducted so far. Here, we explore the genetics of CHCs in hybrids of the jewel wasps Nasonia giraulti and Nasonia vitripennis. We analyzed the CHC profiles of pure strain and of F(2) hybrid males using gas chromatography coupled with mass spectrometry and distinguished 54 peaks, of which we identified 52 as straight-chain, monounsaturated, or methyl-branched CHCs. The latter compound class proved to be particularly abundant and diverse in Nasonia. Quantitative trait locus (QTL) analysis suggests fixed genetic differences between the two strains in 42 of the 54 studied traits, making Nasonia a promising genetic model for identifying genes involved in CHC biosynthesis. QTL for methyl-branched CHCs partly clustered in genomic regions with high recombination rate: a possible indication for pleiotropic genes that control their biosynthesis, which is largely unexplored so far. Finally, we identified and mapped genes in the Nasonia genome with high similarity to genes that have been implicated in alkene biosynthesis in Drosophila and discuss those that match in their position with predicted QTL for alkenes.

The distribution of microsatellites in the Nasonia parasitoid wasp genome.

Microsatellites are important molecular markers used in numerous genetic contexts. Despite this widespread use, the evolutionary processes governing microsatellite distribution and diversity remain controversial. Here, we present results on the distribution of microsatellites of three species in the parasitic wasp genus Nasonia generated by an in silico data-mining approach. Our results show that the overall microsatellite density in Nasonia is comparable to that of the honey bee, but much higher than in eight non-Hymenopteran arthropods. Across the Nasonia vitripennis genome, microsatellite density varied both within and amongst chromosomes. In contrast to other taxa, dinucleotides are the most abundant repeat type in all four species of Hymenoptera studied. Whether the differences between the Hymenoptera and other taxa are of functional significance remains to be determined.

A comparison of recombination frequencies in intraspecific versus interspecific mapping populations of Nasonia.

We present the first intraspecific linkage map for Nasonia vitripennis based on molecular markers. The map consists of 36 new microsatellite markers, extracted from the Nasonia genome sequence, and spans 515 cM. The five inferred linkage groups correspond to the five chromosomes of Nasonia. Comparison of recombination frequencies of the marker intervals spread over the whole genome (N=33 marker intervals) between the intraspecific N. vitripennis map and an interspecific N. vitripennis x N. giraulti map revealed a slightly higher (1.8%) recombination frequency in the intraspecific cross. We further considered an N. vitripennis x N. longicornis map with 29 microsatellite markers spanning 430 cM. Recombination frequencies in the two interspecific crosses differed neither between reciprocal crosses nor between mapping populations of embryos and adults. No major chromosomal rearrangements were found for the analyzed genomic segments. The observed differential F(2) hybrid male mortality has no significant effect on the genome-wide recombination frequency in Nasonia. We conclude that interspecific crosses between the different Nasonia species, a hallmark of Nasonia genetics, are generally suitable for mapping quantitative and qualitative trait loci for species differences.

Contrasting patterns of selective constraints in nuclear-encoded genes of the oxidative phosphorylation pathway in holometabolous insects and their possible role in hybrid breakdown in Nasonia.

The principal energy generating system in animals is the oxidative phosphorylation (OXPHOS) pathway, which depends on the tight interaction of nuclear- and mitochondrial-encoded genes to function properly. Mitochondrial genes accumulate substitutions more quickly than nuclear genes, yet the impact of selection on mitochondrial genes is significantly reduced relative to nuclear genes because of the non-recombining nature of the mitochondrial genome and its predicted smaller effective population size. It has therefore been hypothesized that the nuclear-encoded genes of the OXPHOS pathway are under strong selective pressure to compensate for the accumulation of deleterious nucleotide substitutions in mitochondrial-encoded OXPHOS genes, a process known as compensatory co-adaptation. We evaluated this hypothesis by analyzing nuclear-encoded OXPHOS genes for signatures of positive selection as well as evolutionary constraints at amino acid sites. We considered OXPHOS genes of six holometabolous insects and their orthologs from three Nasonia parasitoid wasps, the hybrids of which suffer from an increased mortality rate caused by cytonuclear genic incompatibilities. Although nuclear OXPHOS genes are typically highly conserved, we found significant evidence for elevated amino acid divergence in 4 of the 59 studied nuclear-encoded OXPHOS genes. We also found that three of these four genes, as well as six other OXPHOS genes, contain amino acid substitutions between Nasonia species at evolutionarily constrained sites. It is possible that these genes account for the reported incompatibility in Nasonia hybrids and their characterization may lead to a better understanding of the role of positive selection in the genetics of speciation.

Hybrid breakdown and mitochondrial dysfunction in hybrids of Nasonia parasitoid wasps.

Male F(2) hybrids of the wasps Nasonia giraulti and Nasonia vitripennis suffer increased mortality during development. Previous studies suggested that the mitochondria may play an important role in this pattern of hybrid breakdown. The mitochondrial genome encodes 13 polypeptides, which are integral subunits of the oxidative phosphorylation enzyme complexes I, III, IV and V. We show that the mitochondrial ATP production rate and the efficacy of the enzyme complexes I, III and IV, but not that of the completely nuclear-encoded complex II, are reduced in F(2) hybrid males of N. giraulti and N. vitripennis. We hypothesize that nuclear-mitochondrial protein interactions in the oxidative phosphorylation pathway are disrupted in these hybrids, reducing energy generation capacity and potentially reducing hybrid fitness. Our results suggest that dysfunctional cytonuclear interactions could represent an under-appreciated post-zygotic isolation mechanism that, due to elevated evolutionary rates of mitochondrial genes, evolves very early in the speciation process.

Species-diagnostic single-nucleotide polymorphism and sequence-tagged site markers for the parasitic wasp genus Nasonia (Hymenoptera: Pteromalidae).

Wasps of the genus Nasonia are important biological control agents of house flies and related filth flies, which are major vectors of human pathogens. Species of Nasonia (Hymenoptera: Pteromalidae) are not easily differentiated from one another by morphological characters, and molecular markers for their reliable identification have been missing so far. Here, we report eight single-nucleotide polymorphism and three sequence-tagged site markers derived from expressed sequenced tag libraries for the two closely related and regionally sympatric species N. giraulti and N. vitripennis. We studied variation of these markers in natural populations of the two species, and we mapped them in the Nasonia genome. The markers are species-diagnostic and evenly spread over all five chromosomes. They are ideal for rapid species identification and hybrid recognition, and they can be used to map economically relevant quantitative trait loci in the Nasonia genome.