A chromosome-level assembly of the seed beetle Callosobruchus maculatus genome with annotation of its repetitive elements.

Callosobruchus maculatus is a major agricultural pest of legume crops worldwide and an established model system in ecology and evolution. Yet, current molecular biological resources for this species are limited. Here, we employ Hi-C sequencing to generate a greatly improved genome assembly and we annotate its repetitive elements in a dedicated in-depth effort where we manually curate and classify the most abundant unclassified repeat subfamilies. We present a scaffolded chromosome-level assembly, which is 1.01 Gb in total length with 86% being contained within the 9 autosomes and the X chromosome. Repetitive sequences accounted for 70% of the total assembly. DNA transposons covered 18% of the genome, with the most abundant superfamily being Tc1-Mariner (9.75% of the genome). This new chromosome-level genome assembly of C. maculatus will enable future genetic and evolutionary studies not only of this important species but of beetles more generally.

Y-Linked Copy Number Polymorphism of Target of Rapamycin Is Associated with Sexual Size Dimorphism in Seed Beetles.

The Y chromosome is theorized to facilitate evolution of sexual dimorphism by accumulating sexually antagonistic loci, but empirical support is scarce. Due to the lack of recombination, Y chromosomes are prone to degenerative processes, which poses a constraint on their adaptive potential. Yet, in the seed beetle, Callosobruchus maculatus segregating Y linked variation affects male body size and thereby sexual size dimorphism (SSD). Here, we assemble C. maculatus sex chromosome sequences and identify molecular differences associated with Y-linked SSD variation. The assembled Y chromosome is largely euchromatic and contains over 400 genes, many of which are ampliconic with a mixed autosomal and X chromosome ancestry. Functional annotation suggests that the Y chromosome plays important roles in males beyond primary reproductive functions. Crucially, we find that, besides an autosomal copy of the gene target of rapamycin (TOR), males carry an additional TOR copy on the Y chromosome. TOR is a conserved regulator of growth across taxa, and our results suggest that a Y-linked TOR provides a male specific opportunity to alter body size. A comparison of Y haplotypes associated with male size difference uncovers a copy number variation for TOR, where the haplotype associated with decreased male size, and thereby increased sexual dimorphism, has two additional TOR copies. This suggests that sexual conflict over growth has been mitigated by autosome to Y translocation of TOR followed by gene duplications. Our results reveal that despite of suppressed recombination, the Y chromosome can harbor adaptive potential as a male-limited supergene.

Sexually antagonistic selection maintains genetic variance when sexual dimorphism evolves.

Genetic variance (VG) in fitness related traits is often unexpectedly high, evoking the question how VG can be maintained in the face of selection. Sexually antagonistic (SA) selection favouring alternative alleles in the sexes is common and predicted to maintain VG, while directional selection should erode it. Both SA and sex-limited directional selection can lead to sex-specific adaptations but how each affect VG when sexual dimorphism evolves remain experimentally untested. Using replicated artificial selection on the seed beetle Callosobruchus maculatus body size we recently demonstrated an increase in size dimorphism under SA and male-limited (ML) selection by 50% and 32%, respectively. Here we test their consequences on genetic variation. We show that SA selection maintained significantly more ancestral, autosomal additive genetic variance than ML selection, while both eroded sex-linked additive variation equally. Ancestral female-specific dominance variance was completely lost under ML, while SA selection consistently sustained it. Further, both forms of selection preserved a high genetic correlation between the sexes (rm,f). These results demonstrate the potential for sexual antagonism to maintain more genetic variance while fuelling sex-specific adaptation in a short evolutionary time scale, and are in line with predicted importance of sex-specific dominance reducing sexual conflict over alternative alleles.

Experimental Life History Evolution Results in Sex-specific Evolution of Gene Expression in Seed Beetles.

The patterns of reproductive timing and senescence vary within and across species owing to differences in reproductive strategies, but our understanding of the molecular underpinnings of such variation is incomplete. This is perhaps particularly true for sex differences. We investigated the evolution of sex-specific gene expression associated with life history divergence in replicated populations of the seed beetle Acanthoscelides obtectus, experimentally evolving under (E)arly or (L)ate life reproduction for >200 generations which has resulted in strongly divergent life histories. We detected 1,646 genes that were differentially expressed in E and L lines, consistent with a highly polygenic basis of life history evolution. Only 30% of differentially expressed genes were similarly affected in males and females. The evolution of long life was associated with significantly reduced sex differences in expression, especially in non-reproductive tissues. The expression differences were overall more pronounced in females, in accordance with their greater phenotypic divergence in lifespan. Functional enrichment analysis revealed differences between E and L beetles in gene categories previously implicated in aging, such as mitochondrial function and defense response. The results show that divergent life history evolution can be associated with profound changes in gene expression that alter the transcriptome in a sex-specific way, highlighting the importance of understanding the mechanisms of aging in each sex.

Concerted evolution of metabolic rate, economics of mating, ecology, and pace of life across seed beetles.

Male-female coevolution has taken different paths among closely related species, but our understanding of the factors that govern its direction is limited. While it is clear that ecological factors, life history, and the economics of reproduction are connected, the divergent links are often obscure. We propose that a complete understanding requires the conceptual integration of metabolic phenotypes. Metabolic rate, a nexus of life history evolution, is constrained by ecological factors and may exert important direct and indirect effects on the evolution of sexual dimorphism. We performed standardized experiments in 12 seed beetle species to gain a rich set of sex-specific measures of metabolic phenotypes, life history traits, and the economics of mating and analyzed our multivariate data using phylogenetic comparative methods. Resting metabolic rate (RMR) showed extensive evolution and evolved more rapidly in males than in females. The evolution of RMR was tightly coupled with a suite of life history traits, describing a pace-of-life syndrome (POLS), with indirect effects on the economics of mating. As predicted, high resource competition was associated with a low RMR and a slow POLS. The cost of mating showed sexually antagonistic coevolution, a hallmark of sexual conflict. The sex-specific costs and benefits of mating were predictably related to ecology, primarily through the evolution of male ejaculate size. Overall, our results support the tenet that resource competition affects metabolic processes that, in turn, have predictable effects on both life history evolution and reproduction, such that ecology shows both direct and indirect effects on male-female coevolution.

Rapid evolution of sexual size dimorphism facilitated by Y-linked genetic variance.

Sexual dimorphism is ubiquitous in nature but its evolution is puzzling given that the mostly shared genome constrains independent evolution in the sexes. Sex differences should result from asymmetries between the sexes in selection or genetic variation but studies investigating both simultaneously are lacking. Here, we combine a quantitative genetic analysis of body size variation, partitioned into autosomal and sex chromosome contributions and ten generations of experimental evolution to dissect the evolution of sexual body size dimorphism in seed beetles (Callosobruchus maculatus) subjected to sexually antagonistic or sex-limited selection. Female additive genetic variance (VA) was primarily linked to autosomes, exhibiting a strong intersexual genetic correlation with males ([Formula: see text] = 0.926), while X- and Y-linked genes further contributed to the male VA and X-linked genes contributed to female dominance variance. Consistent with these estimates, sexual body size dimorphism did not evolve in response to female-limited selection but evolved by 30-50% under male-limited and sexually antagonistic selection. Remarkably, Y-linked variance alone could change dimorphism by 30%, despite the C. maculatus Y chromosome being small and heterochromatic. Our results demonstrate how the potential for sexual dimorphism to evolve depends on both its underlying genetic basis and the nature of sex-specific selection.

Sexual conflict drives micro- and macroevolution of sexual dimorphism in immunity.

BACKGROUND: Sexual dimorphism in immunity is believed to reflect sex differences in reproductive strategies and trade-offs between competing life history demands. Sexual selection can have major effects on mating rates and sex-specific costs of mating and may thereby influence sex differences in immunity as well as associated host-pathogen dynamics. Yet, experimental evidence linking the mating system to evolved sexual dimorphism in immunity are scarce and the direct effects of mating rate on immunity are not well established. Here, we use transcriptomic analyses, experimental evolution and phylogenetic comparative methods to study the association between the mating system and sexual dimorphism in immunity in seed beetles, where mating causes internal injuries in females. RESULTS: We demonstrate that female phenoloxidase (PO) activity, involved in wound healing and defence against parasitic infections, is elevated relative to males. This difference is accompanied by concomitant sex differences in the expression of genes in the prophenoloxidase activating cascade. We document substantial phenotypic plasticity in female PO activity in response to mating and show that experimental evolution under enforced monogamy (resulting in low remating rates and reduced sexual conflict relative to natural polygamy) rapidly decreases female (but not male) PO activity. Moreover, monogamous females had evolved increased tolerance to bacterial infection unrelated to mating, implying that female responses to costly mating may trade off with other aspects of immune defence, an hypothesis which broadly accords with the documented sex differences in gene expression. Finally, female (but not male) PO activity shows correlated evolution with the perceived harmfulness of male genitalia across 12 species of seed beetles, suggesting that sexual conflict has a significant influence on sexual dimorphisms in immunity in this group of insects. CONCLUSIONS: Our study provides insights into the links between sexual conflict and sexual dimorphism in immunity and suggests that selection pressures moulded by mating interactions can lead to a sex-specific mosaic of immune responses with important implications for host-pathogen dynamics in sexually reproducing organisms.

An experimental test of temperature-dependent selection on mitochondrial haplotypes in Callosobruchus maculatus seed beetles.

Mitochondrial DNA (mtDNA) consists of few but vital maternally inherited genes that interact closely with nuclear genes to produce cellular energy. How important mtDNA polymorphism is for adaptation is still unclear. The assumption in population genetic studies is often that segregating mtDNA variation is selectively neutral. This contrasts with empirical observations of mtDNA haplotypes affecting fitness-related traits and thermal sensitivity, and latitudinal clines in mtDNA haplotype frequencies. Here, we experimentally test whether ambient temperature affects selection on mtDNA variation, and whether such thermal effects are influenced by intergenomic epistasis due to interactions between mitochondrial and nuclear genes, using replicated experimental evolution in Callosobruchus maculatus seed beetle populations seeded with a mixture of different mtDNA haplotypes. We also test for sex-specific consequences of mtDNA evolution on reproductive success, given that mtDNA mutations can have sexually antagonistic fitness effects. Our results demonstrate natural selection on mtDNA haplotypes, with some support for thermal environment influencing mtDNA evolution through mitonuclear epistasis. The changes in male and female reproductive fitness were both aligned with changes in mtDNA haplotype frequencies, suggesting that natural selection on mtDNA is sexually concordant in stressful thermal environments. We discuss the implications of our findings for the evolution of mtDNA.

Female-specific resource limitation does not make the opportunity for selection more female biased.

Competition for limiting resources and stress can magnify variance in fitness and therefore selection. But even in a common environment, the strength of selection can differ across the sexes, as their fitness is often limited by different factors. Indeed, most taxa show stronger selection in males, a bias often ascribed to intense competition for access to mating partners. This sex bias could reverberate on many aspects of evolution, from speed of adaptation to genome evolution. It is unclear, however, whether stronger opportunity for selection in males is a pattern robust to sex-specific stress or resource limitation. We test this in the model species Callosobruchus maculatus by comparing female and male opportunity for selection (i) with and without limitation of quality oviposition sites, and (ii) under delayed age at oviposition. Decreasing the abundance of the resource key to females or increasing their reproductive age was challenging, as shown by a reduction in mean fitness, but opportunity for selection remained stronger in males across all treatments, and even more so when oviposition sites were limiting. This suggests that males remain the more variable sex independent of context, and that the opportunity for selection through males is indirectly affected by female-specific resource limitation.

Experimentally induced intrasexual mating competition and sex-specific evolution in female and male nematodes.

Sexual dimorphism in life history traits and their trade-offs is widespread among sexually reproducing animals and is strongly influenced by the differences in reproductive strategies between the sexes. We investigated how intrasexual competition influenced specific life history traits, important to fitness and their trade-offs in the outcrossing nematode Caenorhabditis remanei. Here, we altered the strength of sex-specific selection through experimental evolution with increased potential for intrasexual competition by skewing the adult sex ratio towards either females or males (1:10 or 10:1) over 30 generations and subsequently measured the phenotypic response to selection in three traits related to fitness: body size, fecundity and tolerance to heat stress. We observed a greater evolutionary change in females than males for body size and peak fitness, suggesting that females may experience stronger net selection and potentially harbour higher amounts of standing genetic variance compared to males. Our study highlights the importance of investigating direct and indirect effects of intrasexual competition in both sexes in order to capture sex-specific responses and understand the evolution of sexual dimorphism in traits expressed by both sexes.

The genomic footprint of sexual conflict.

Genes with sex-biased expression show a number of unique properties and this has been seen as evidence for conflicting selection pressures in males and females, forming a genetic 'tug-of-war' between the sexes. However, we lack studies of taxa where an understanding of conflicting phenotypic selection in the sexes has been linked with studies of genomic signatures of sexual conflict. Here, we provide such a link. We used an insect where sexual conflict is unusually well understood, the seed beetle Callosobruchus maculatus, to test for molecular genetic signals of sexual conflict across genes with varying degrees of sex-bias in expression. We sequenced, assembled and annotated its genome and performed population resequencing of three divergent populations. Sex-biased genes showed increased levels of genetic diversity and bore a remarkably clear footprint of relaxed purifying selection. Yet, segregating genetic variation was also affected by balancing selection in weakly female-biased genes, while male-biased genes showed signs of overall purifying selection. Female-biased genes contributed disproportionally to shared polymorphism across populations, while male-biased genes, male seminal fluid protein genes and sex-linked genes did not. Genes showing genomic signatures consistent with sexual conflict generally matched life-history phenotypes known to experience sexually antagonistic selection in this species. Our results highlight metabolic and reproductive processes, confirming the key role of general life-history traits in sexual conflict.

Identification of novel ejaculate proteins in a seed beetle and division of labour across male accessory reproductive glands.

The male ejaculate contains a multitude of seminal fluid proteins (SFPs), many of which are key reproductive molecules, as well as sperm. However, the identification of SFPs is notoriously difficult and a detailed understanding of this complex phenotype has only been achieved in a few model species. We employed a recently developed proteomic method involving whole-organism stable isotope labelling coupled with proteomic and transcriptomic analyses to characterize ejaculate proteins in the seed beetle Callosobruchus maculatus. We identified 317 proteins that were transferred to females at mating, and a great majority of these showed signals of secretion and were highly male-biased in expression in the abdomen. These male-derived proteins were enriched with proteins involved in general metabolic and catabolic processes but also with proteolytic enzymes and proteins involved in protection against oxidative stress. Thirty-seven proteins showed significant homology with SFPs previously identified in other insects. However, no less than 92 C. maculatus ejaculate proteins were entirely novel, receiving no significant blast hits and lacking homologs in extant data bases, consistent with a rapid and divergent evolution of SFPs. We used 3D micro-tomography in conjunction with proteomic methods to identify 5 distinct pairs of male accessory reproductive glands and to show that certain ejaculate proteins were only recovered in certain male glands. Finally, we provide a tentative list of 231 candidate female-derived reproductive proteins, some of which are likely important in ejaculate processing and/or sperm storage.

Evolution of sex-specific pace-of-life syndromes: genetic architecture and physiological mechanisms.

Sex differences in life history, physiology, and behavior are nearly ubiquitous across taxa, owing to sex-specific selection that arises from different reproductive strategies of the sexes. The pace-of-life syndrome (POLS) hypothesis predicts that most variation in such traits among individuals, populations, and species falls along a slow-fast pace-of-life continuum. As a result of their different reproductive roles and environment, the sexes also commonly differ in pace-of-life, with important consequences for the evolution of POLS. Here, we outline mechanisms for how males and females can evolve differences in POLS traits and in how such traits can covary differently despite constraints resulting from a shared genome. We review the current knowledge of the genetic basis of POLS traits and suggest candidate genes and pathways for future studies. Pleiotropic effects may govern many of the genetic correlations, but little is still known about the mechanisms involved in trade-offs between current and future reproduction and their integration with behavioral variation. We highlight the importance of metabolic and hormonal pathways in mediating sex differences in POLS traits; however, there is still a shortage of studies that test for sex specificity in molecular effects and their evolutionary causes. Considering whether and how sexual dimorphism evolves in POLS traits provides a more holistic framework to understand how behavioral variation is integrated with life histories and physiology, and we call for studies that focus on examining the sex-specific genetic architecture of this integration.

The Evolution of Dark Matter in the Mitogenome of Seed Beetles.

Animal mitogenomes are generally thought of as being economic and optimized for rapid replication and transcription. We use long-read sequencing technology to assemble the remarkable mitogenomes of four species of seed beetles. These are the largest circular mitogenomes ever assembled in insects, ranging from 24,496 to 26,613 bp in total length, and are exceptional in that some 40% consists of non-coding DNA. The size expansion is due to two very long intergenic spacers (LIGSs), rich in tandem repeats. The two LIGSs are present in all species but vary greatly in length (114-10,408 bp), show very low sequence similarity, divergent tandem repeat motifs, a very high AT content and concerted length evolution. The LIGSs have been retained for at least some 45 my but must have undergone repeated reductions and expansions, despite strong purifying selection on protein coding mtDNA genes. The LIGSs are located in two intergenic sites where a few recent studies of insects have also reported shorter LIGSs (>200 bp). These sites may represent spaces that tolerate neutral repeat array expansions or, alternatively, the LIGSs may function to allow a more economic translational machinery. Mitochondrial respiration in adult seed beetles is based almost exclusively on fatty acids, which reduces the need for building complex I of the oxidative phosphorylation pathway (NADH dehydrogenase). One possibility is thus that the LIGSs may allow depressed transcription of NAD genes. RNA sequencing showed that LIGSs are partly transcribed and transcriptional profiling suggested that all seven mtDNA NAD genes indeed show low levels of transcription and co-regulation of transcription across sexes and tissues.

Mating Changes Sexually Dimorphic Gene Expression in the Seed Beetle Callosobruchus maculatus.

Sexually dimorphic phenotypes arise largely from sex-specific gene expression, which has mainly been characterized in sexually naïve adults. However, we expect sexual dimorphism in transcription to be dynamic and dependent on factors such as reproductive status. Mating induces many behavioral and physiological changes distinct to each sex and is therefore expected to activate regulatory changes in many sex-biased genes. Here, we first characterized sexual dimorphism in gene expression in Callosobruchus maculatus seed beetles. We then examined how females and males respond to mating and how it affects sex-biased expression, both in sex-limited (abdomen) and sex-shared (head and thorax) tissues. Mating responses were largely sex-specific and, as expected, females showed more genes responding compared with males (∼2,000 vs. ∼300 genes in the abdomen, ∼500 vs. ∼400 in the head and thorax, respectively). Of the sex-biased genes present in virgins, 16% (1,041 genes) in the abdomen and 17% (243 genes) in the head and thorax altered their relative expression between the sexes as a result of mating. Sex-bias status changed in 2% of the genes in the abdomen and 4% in the head and thorax following mating. Mating responses involved de-feminization of females and, to a lesser extent, de-masculinization of males relative to their virgin state: mating decreased rather than increased dimorphic expression of sex-biased genes. The fact that regulatory changes of both types of sex-biased genes occurred in both sexes suggests that male- and female-specific selection is not restricted to male- and female-biased genes, respectively, as is sometimes assumed.

Sex-specific mitonuclear epistasis and the evolution of mitochondrial bioenergetics, ageing, and life history in seed beetles.

The role of mitochondrial DNA for the evolution of life-history traits remains debated. We examined mitonuclear effects on the activity of the multisubunit complex of the electron transport chain (ETC) involved in oxidative phosphorylation (OXPHOS) across lines of the seed beetle Acanthoscelides obtectus selected for a short (E) or a long (L) life for more than >160 generations. We constructed and phenotyped mitonuclear introgression lines, which allowed us to assess the independent effects of the evolutionary history of the nuclear and the mitochondrial genome. The nuclear genome was responsible for the largest share of divergence seen in ageing. However, the mitochondrial genome also had sizeable effects, which were sex-specific and expressed primarily as epistatic interactions with the nuclear genome. The effects of mitonuclear disruption were largely consistent with mitonuclear coadaptation. Variation in ETC activity explained a large proportion of variance in ageing and life-history traits and this multivariate relationship differed somewhat between the sexes. In conclusion, mitonuclear epistasis has played an important role in the laboratory evolution of ETC complex activity, ageing, and life histories and these are closely associated. The mitonuclear architecture of evolved differences in life-history traits and mitochondrial bioenergetics was sex-specific.

The De Novo Transcriptome and Its Functional Annotation in the Seed Beetle Callosobruchus maculatus.

Despite their unparalleled biodiversity, the genomic resources available for beetles (Coleoptera) remain relatively scarce. We present an integrative and high quality annotated transcriptome of the beetle Callosobruchus maculatus, an important and cosmopolitan agricultural pest as well as an emerging model species in ecology and evolutionary biology. Using Illumina sequencing technology, we sequenced 492 million read pairs generated from 51 samples of different developmental stages (larvae, pupae and adults) of C. maculatus. Reads were de novo assembled using the Trinity software, into a single combined assembly as well as into three separate assemblies based on data from the different developmental stages. The combined assembly generated 218,192 transcripts and 145,883 putative genes. Putative genes were annotated with the Blast2GO software and the Trinotate pipeline. In total, 33,216 putative genes were successfully annotated using Blastx against the Nr (non-redundant) database and 13,382 were assigned to 34,100 Gene Ontology (GO) terms. We classified 5,475 putative genes into Clusters of Orthologous Groups (COG) and 116 metabolic pathways maps were predicted based on the annotation. Our analyses suggested that the transcriptional specificity increases with ontogeny. For example, out of 33,216 annotated putative genes, 51 were only expressed in larvae, 63 only in pupae and 171 only in adults. Our study illustrates the importance of including samples from several developmental stages when the aim is to provide an integrative and high quality annotated transcriptome. Our results will represent an invaluable resource for those working with the ecology, evolution and pest control of C. maculatus, as well for comparative studies of the transcriptomics and genomics of beetles more generally.

Genome size correlates with reproductive fitness in seed beetles.

The ultimate cause of genome size (GS) evolution in eukaryotes remains a major and unresolved puzzle in evolutionary biology. Large-scale comparative studies have failed to find consistent correlations between GS and organismal properties, resulting in the 'C-value paradox'. Current hypotheses for the evolution of GS are based either on the balance between mutational events and drift or on natural selection acting upon standing genetic variation in GS. It is, however, currently very difficult to evaluate the role of selection because within-species studies that relate variation in life-history traits to variation in GS are very rare. Here, we report phylogenetic comparative analyses of GS evolution in seed beetles at two distinct taxonomic scales, which combines replicated estimation of GS with experimental assays of life-history traits and reproductive fitness. GS showed rapid and bidirectional evolution across species, but did not show correlated evolution with any of several indices of the relative importance of genetic drift. Within a single species, GS varied by 4-5% across populations and showed positive correlated evolution with independent estimates of male and female reproductive fitness. Collectively, the phylogenetic pattern of GS diversification across and within species in conjunction with the pattern of correlated evolution between GS and fitness provide novel support for the tenet that natural selection plays a key role in shaping GS evolution.

Mating system variation drives rapid evolution of the female transcriptome in Drosophila pseudoobscura.

Interactions between the sexes are believed to be a potent source of selection on sex-specific evolution. The way in which sexual interactions influence male investment is much studied, but effects on females are more poorly understood. To address this deficiency, we examined gene expression in virgin female Drosophila pseudoobscura following 100 generations of mating system manipulations in which we either elevated polyandry or enforced monandry. Gene expression evolution following mating system manipulation resulted in 14% of the transcriptome of virgin females being altered. Polyandrous females elevated expression of a greater number of genes normally enriched in ovaries and associated with mitosis and meiosis, which might reflect female investment into reproductive functions. Monandrous females showed a greater number of genes normally enriched for expression in somatic tissues, including the head and gut and associated with visual perception and metabolism, respectively. By comparing our data with a previous study of sex differences in gene expression in this species, we found that the majority of the genes that are differentially expressed between females of the selection treatments show female-biased expression in the wild-type population. A striking exception is genes associated with male-specific reproductive tissues (in D. melanogaster), which are upregulated in polyandrous females. Our results provide experimental evidence for a role of sex-specific selection arising from differing sexual interactions with males in promoting rapid evolution of the female transcriptome.