Drift,not selection,shapes toll‐like receptor variation among oceanic island populations

Understanding the relative role of different evolutionary forces in shaping the level and distribution of functional genetic diversity among natural populations is a key issue in evolutionary and conservation biology. To do so accurately genetic data must be analysed in conjunction with an unambiguous understanding of the historical processes that have acted upon the populations. Here, we focused on diversity at toll‐like receptor (TLR) loci, which play a key role in the vertebrate innate immune system and, therefore, are expected to be under pathogen‐mediated selection. We assessed TLR variation within and among 13 island populations (grouped into three archipelagos) of Berthelot's pipit, Anthus berthelotii, for which detailed population history has previously been ascertained. We also compared the variation observed with that found in its widespread sister species, the tawny pipit, Anthus campestris. We found strong evidence for positive selection at specific codons in TLR1LA, TLR3 and TLR4. Despite this, we found that at the allele frequency level, demographic history has played the major role in shaping patterns of TLR variation in Berthelot's pipit. Levels of diversity and differentiation within and across archipelagos at all TLR loci corresponded very closely with neutral microsatellite variation and with the severity of the bottlenecks that occurred during colonization. Our study shows that despite the importance of TLRs in combating pathogens, demography can be the main driver of immune gene variation within and across populations, resulting in patterns of functional variation that can persist over evolutionary timescales.     

Population genetic structure of the bank vole Myodes glareolus within its glacial refugium in peninsular Italy

It is now well established that Southern European peninsulas have been major glacial refugia for temperate species during Pleistocene climatic oscillations. However, substantial environmental changes occurred also within these peninsulas throughout the Pleistocene, raising questions about the role and interplay of various microevolutionary processes in shaping patterns of intraspecific diversity within these areas. Here, we investigate the patterns of genetic variation in the bank vole Myodes glareolus within the Italian peninsula. By using a panel of 13 microsatellite loci, we found more intraspecific variation than expected based on previous assessments. Indeed, both Bayesian and ordination‐based clustering analyses of variation recovered five main geographic/genetic clusters along the peninsula, with three clusters geographically restricted to the southern portion of the peninsula. This clustering is supported by previous evidences of some morphological distinctiveness among these populations. This pattern can be explained by a refugia‐within‐refugia scenario, with the occurrence of multiple sub‐refugia for the bank vole within the Italian peninsula, likely promoted by the major palaeo‐environmental changes which affected forested habitats within this area during the Pleistocene. Moreover, our results support a scenario whereby the high levels of intraspecific diversity observed within major Pleistocene refugia are better explained by dynamic microevolutionary processes occurred within these areas, rather than by long‐term demographic stability of refugial population. Finally, the narrow and isolated distribution of some of the identified lineages suggests the need for future assessments of their conservation and taxonomic status.     

Mito‐nuclear discordance across a recent contact zone for California voles

To examine the processes that maintain genetic diversity among closely related taxa, we investigated the dynamics of introgression across a contact zone between two lineages of California voles (Microtus californicus). We tested the prediction that introgression of nuclear loci would be greater than that for mitochondrial loci, assuming ongoing gene flow across the contact zone. We also predicted that genomic markers would show a mosaic pattern of differentiation across this zone, consistent with genomes that are semi‐permeable. Using mitochondrial cytochrome b sequences and genome‐wide loci developed via ddRAD‐seq, we analyzed genetic variation for 10 vole populations distributed along the central California coast; this transect included populations from within the distributions of both parental lineages as well as the putative contact zone. Our analyses revealed that (1) the two lineages examined are relatively young, having diverged ca. 8.5–54 kya, (2) voles from the contact zone in Santa Barbara County did not include F1 or early generation backcrossed individuals, and (3) there appeared to be little to no recurrent gene flow across the contact zone. Introgression patterns for mitochondrial and nuclear markers were not concordant; only mitochondrial markers revealed evidence of introgression, putatively due to historical hybridization. These differences in genetic signatures are intriguing given that the contact zone occurs in a region of continuous vole habitat, with no evidence of past or present physical barriers. Future studies that examine specific isolating mechanisms, such as microhabitat use and mate choice, will facilitate our understanding of how genetic boundaries are maintained in this system.     

Toll‐like receptor 9 protects non‐immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2

Toll‐like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro‐inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium‐transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca²⁺ handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non‐immune cells—including cardiomyocytes—using the same ligand‐receptor system.     

Contrasting patterns of diversity and population differentiation at the innate immunity gene toll-like receptor 2 (TLR2) in two sympatric rodent species

Comparing patterns of diversity and divergence between populations at immune genes and neutral markers can give insights into the nature and geographic scale of parasite-mediated selection. To date, studies investigating such patterns of selection in vertebrates have primarily focused on the acquired branch of the immune system, whereas it remains largely unknown how parasite-mediated selection shapes innate immune genes both within and across vertebrate populations. Here, we present a study on the diversity and population differentiation at the innate immune gene Toll-like receptor 2 (TLR2) across nine populations of yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) in southern Sweden. In yellow-necked mice, TLR2 diversity was very low, as was TLR2 population differentiation compared to neutral loci. In contrast, several TLR2 haplotypes co-occurred at intermediate frequencies within and across bank vole populations, and pronounced isolation by distance between populations was observed. The diversity and differentiation at neutral loci was similar in the two species. These results indicate that parasite-mediated selection has been acting in dramatically different ways on a given immune gene in ecologically similar and sympatric species. Furthermore, the finding of TLR2 population differentiation at a small geographical scale in bank voles highlights that vertebrate innate immune defense may be evolutionarily more dynamic than has previously been appreciated.     

Macroparasites at peripheral sites of infection are major and dynamic modifiers of systemic antimicrobial pattern recognition responses

Immune defences and the maintenance of immunological homeostasis in the face of pathogenic and commensal microbial exposures are channelled by innate antimicrobial pattern recognition receptors (PRRs) such as toll‐like receptors (TLRs). Whilst PRR‐mediated response programmes are the result of long‐term host‐pathogen or host–commensal co‐evolutionary dynamics involving microbes, an additional possibility is that macroparasitic co‐infections may be a significant modifier of such interactions. We demonstrate experimentally that macroparasites (the model gastrointestinal nematode, Heligmosomoides) at peripheral sites of infection cause substantial alteration of the expression and function of TLRs at a systemic level (in cultured splenocytes), predominantly up‐regulating TLR2, TLR4 and TLR9‐mediated cytokine responses at times of high standing worm burdens. We consistently observed such effects in BALB/c and C57BL/6 mice under single‐pulse and trickle exposures to Heligmosomoides larvae and in SWR and CBA mice under single‐pulse exposures. A complementary long‐term survey of TLR2‐mediated tumour necrosis factor‐alpha responses in wild wood mice (Apodemus sylvaticus) was consistent with substantial effects of macroparasites under some environmental conditions. A general pattern, though, was for the associations of macroparasites with TLR function to be temporally dynamic and context‐dependent: varying with different conditions of infection exposure in the field and laboratory and with host genetic strain in the laboratory. These results are compelling evidence that macroparasites are a major and dynamic modifier of systemic innate antimicrobial responsiveness in naturally occurring mammals and thus likely to be an important influence on the interaction between microbial exposures and the immune system.     

Small‐scale spatial variation in infection risk shapes the evolution of a Borrelia resistance gene in wild rodents

Spatial variation in pathogen‐mediated selection is predicted to influence the evolutionary trajectory of host populations and lead to spatial variation in their immunogenetic composition. However, to date few studies have been able to directly link small‐scale spatial variation in infection risk to host immune gene evolution in natural, nonhuman populations. Here, we use a natural rodent–Borrelia system to test for associations between landscape‐level spatial variation in Borrelia infection risk along replicated elevational gradients in the Swiss Alps and Toll‐like receptor 2 (TLR2) evolution, a candidate gene for Borrelia resistance, across bank vole (Myodes glareolus) populations. We found that Borrelia infection risk (i.e., the product of Borrelia prevalence in questing ticks and the average tick load of voles at a sampling site) was spatially variable and significantly negatively associated with elevation. Across sampling sites, Borrelia prevalence in bank voles was significantly positively associated with Borrelia infection risk along the elevational clines. We observed a significant association between naturally occurring TLR2 polymorphisms in hosts and their Borrelia infection status. The TLR2 variant associated with a reduced likelihood of Borrelia infection was most common in rodent populations at lower elevations that face a high Borrelia infection risk, and its frequency changed in accordance with the change in Borrelia infection risk along the elevational clines. These results suggest that small‐scale spatial variation in parasite‐mediated selection affects the immunogenetic composition of natural host populations, providing a striking example that the microbial environment shapes the evolution of the host's immune system in the wild.     

Borrelia burgdorferi sensu lato infection pressure shapes innate immune gene evolution in natural rodent populations across Europe

Although parasite-mediated selection is assumed to be the main driver of immune gene evolution, empirical evidence that parasites induce allele frequency changes at host immune genes in time and/or space remains scarce. Here, I show that the frequency of a protective gene variant of the innate immune receptor Toll-like receptor 2 in natural bank vole (Myodes glareolus) populations is positively associated with the strength of Borrelia burgdorferi sensu lato infection risk across the European continent. Thereby, this study provides rare evidence for the role of spatially variable infection pressures in moulding the vertebrate immune system.     

Distribution of genetic variation underlying adult migration timing in steelhead of the Columbia River basin

Fish migrations are energetically costly, especially when moving between freshwater and saltwater, but are a viable strategy for Pacific salmon and trout (Oncorhynchus spp.) due to the advantageous resources available at various life stages. Anadromous steelhead (O. mykiss) migrate vast distances and exhibit variation for adult migration phenotypes that have a genetic basis at candidate genes known as greb1L and rock1. We examined the distribution of genetic variation at 13 candidate markers spanning greb1L, intergenic, and rock1 regions versus 226 neutral markers for 113 populations (n = 9,471) of steelhead from inland and coastal lineages in the Columbia River. Patterns of population structure with neutral markers reflected genetic similarity by geographic region as demonstrated in previous studies, but candidate markers clustered populations by genetic variation associated with adult migration timing. Mature alleles for late migration had the highest frequency overall in steelhead populations throughout the Columbia River, with only 9 of 113 populations that had a higher frequency of premature alleles for early migration. While a single haplotype block was evident for the coastal lineage, we identified multiple haplotype blocks for the inland lineage. The inland lineage had one haplotype block that corresponded to candidate markers within the greb1L gene and immediately upstream in the intergenic region, and the second block only contained candidate markers from the intergenic region. Haplotype frequencies had similar patterns of geographic distribution as single markers, but there were distinct differences in frequency between the two haplotype blocks for the inland lineage. This may represent multiple recombination events that differed between lineages where phenotypic differences exist between freshwater entry versus arrival timing as indicated by Micheletti et al. (2018a). Redundancy analyses were used to model environmental effects on allelic frequencies of candidate markers, and significant variables were migration distance, temperature, isothermality, and annual precipitation. This study improves our understanding of the spatial distribution of genetic variation underlying adult migration timing in steelhead as well as associated environmental factors and has direct conservation and management implications.     

Niche differentiation and colonization of a novel environment by an asexual parasitic wasp

How do asexual taxa become adapted to a diversity of environments, and how do they persist despite changing environmental conditions? These questions are linked by their mutual focus on the relationship between genetic variation, which is often limited in asexuals, and the ability to respond to environmental variation. Asexual taxa originating from a single ancestor present a unique opportunity to assess rates of phenotypic and genetic change when access to new genetic variation is limited to mutation. Diachasma muliebre is an asexual Hymenopteran wasp that is geographically and genetically isolated from all sexual relatives. D. muliebre attack larvae of the western cherry fruit fly (Rhagoletis indifferens), which in turn feed inside bitter cherry fruit (Prunus emarginata) in August and September. R. indifferens has recently colonized a new host plant with an earlier fruiting phenology (June/July), domesticated sweet cherries (P. avium), and D. muliebre has followed its host into this temporally earlier niche. We tested three hypotheses: 1) that all D. muliebre lineages originate from a single asexual ancestor; 2) that different D. muliebre lineages (as defined by unique mtDNA haplotypes) have differentiated on their ancestral host in an important life‐history trait, eclosion timing; and 3) that early‐eclosing lineages have preferentially colonized the new sweet cherry niche. We find that mitochondrial COI and microsatellite data provide strong support for a single ancestral origin for all lineages. Furthermore, COI sequencing revealed five mitochondrial haplotypes among D. muliebre, and individual wasps possessing one distinctive mitochondrial haplotype (haplotype II) eclosed as reproductive adults significantly earlier than wasps with all other haplotypes. In addition, this early‐eclosing lineage of D. muliebre is one of two lineages that have colonized the P. avium habitat, consistent with the preferential colonization hypothesis. These data suggest that D. muliebre has evolved adaptive phenotypic variation despite limited genetic variation, and that this variation has subsequently allowed an expansion of some wasps into a novel habitat. The D. muliebre system may allow for in‐depth study of adaptation and long‐term persistence of asexual taxa.     

Nuclear genes (but not mitochondrial DNA barcodes) reveal real species: Evidence from the Brenthis fritillary butterflies (Lepidoptera,Nymphalidae)

Discordance between entities revealed by nuclear versus mitochondrial genes is a common phenomenon in evolutionary and taxonomic studies. However, little attention has been paid to analysis of how such discordant entities correspond to traditional species detected through investigation of their morphology, ecology, and distribution. Here, we used one mitochondrial (COI, DNA barcode fragment) and four nuclear (CAD, Ca‐ATPase, arginine kinase, wg) genes to analyze the genetic structure of the taxonomically well‐studied butterfly genus Brenthis (Lepidoptera, Nymphalidae). Analysis of COI revealed multiple diverged allopatric and sympatric mitochondrial lineages within the known Brenthis species hinting at possible presence of unrecognized cryptic species. However, these multiple‐species hypotheses were not supported by further studies of nuclear genes and phenotypic traits. The discovered mitochondrial lineages did not correspond to the clusters revealed by nuclear genes. Simultaneously, we found a complete congruence between (a) traditional species boundaries, (b) clusters recognized by nuclear genes, and (c) clusters identified via cladistic analysis of phenotypic traits (genitalia and wing pattern characters, ecological preferences, and chromosome numbers). We conclude that in case of the genus Brenthis, nuclear genes rather than mtDNA barcodes reveal real species boundaries. Additionally, we suggest to support each DNA barcode‐based taxonomic conclusion by analysis of phased alleles of nuclear genes, avoiding widely used practice of nuclear and mitochondrial genes concatenation without any examination of interaction of these different types of data.     

Gut microbial diversity across a contact zone for California voles: Implications for lineage divergence of hosts and mitonuclear mismatch in the assembly of the mammalian gut microbiome

Gut microbial diversity is thought to reflect the co‐evolution of microbes and their hosts as well as current host‐specific attributes such as genetic background and environmental setting. To explore interactions among these parameters, we characterized variation in gut microbiome composition of California voles (Microtus californicus) across a contact zone between two recently diverged lineages of this species. Because this contact zone contains individuals with mismatched mitochondrial‐nuclear genomes (cybrids), it provides an important opportunity to explore how different components of the genotype contribute to gut microbial diversity. Analyses of bacterial 16S rRNA sequences and joint species distribution modelling revealed that host genotypes and genetic differentiation among host populations together explained more than 50% of microbial community variation across our sampling transect. The ranked importance (most to least) of factors contributing to gut microbial diversity in our study populations were: genome‐wide population differentiation, local environmental conditions, and host genotypes. However, differences in microbial communities among vole populations (β‐diversity) did not follow patterns of lineage divergence (i.e., phylosymbiosis). Instead, among‐population variation was best explained by the spatial distribution of hosts, as expected if the environment is a primary source of gut microbial diversity (i.e., dispersal limitation hypothesis). Across the contact zone, several bacterial taxa differed in relative abundance between the two parental lineages as well as among individuals with mismatched mitochondrial and nuclear genomes. Thus, genetic divergence among host lineages and mitonuclear genomic mismatches may also contribute to microbial diversity by altering interactions between host genomes and gut microbiota (i.e., hologenome speciation hypothesis).     

Global genetic diversity,lineage distribution,and Wolbachia infection of the alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil (Hypera postica) is a well‐known example of a worldwide‐distributed pest with high genetic variation. Based on the mitochondrial genes, the alfalfa weevil clusters into two main mitochondrial lineages. However, there is no clear picture of the global diversity and distribution of these lineages; neither the drivers of its diversification are known. However, it appears likely that historic demographic events including founder effects played a role. In addition, Wolbachia, a widespread intracellular parasite/symbiont, likely played an important role in the evolution of the species. Wolbachia infection so far was only detected in the Western lineage of H. postica with no information on the infecting strain, its frequency, and its consequences on the genetic diversity of the host. We here used a combination of mitochondrial and nuclear sequences of the host and sequence information on Wolbachia to document the distribution of strains and the degree of infection. The Eastern lineage has a higher genetic diversity and is found in the Mediterranean, the Middle East, Eastern Europe, and eastern America, whereas the less diverse Western lineage is found in Central Europe and the western America. Both lineages are infected with the same common strain of Wolbachia belonging to Supergroup B. Based on neutrality tests, selection tests, and the current distribution and diversification of Wolbachia in H. postica, we suggested the Wolbachia infection did not shape genetic diversity of the host. The introduced populations in the United States are generally genetically less diverse, which is in line with founder effects.     

A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors

Bacterial pathogens often subvert the innate immune system to establish a successful infection. The direct inhibition of downstream components of innate immune pathways is particularly well documented but how bacteria interfere with receptor proximal events is far less well understood. Here, we describe a Toll/interleukin 1 receptor (TIR) domain‐containing protein (PumA) of the multi‐drug resistant Pseudomonas aeruginosa PA7 strain. We found that PumA is essential for virulence and inhibits NF‐κB, a property transferable to non‐PumA strain PA14, suggesting no additional factors are needed for PumA function. The TIR domain is able to interact with the Toll‐like receptor (TLR) adaptors TIRAP and MyD88, as well as the ubiquitin‐associated protein 1 (UBAP1), a component of the endosomal‐sorting complex required for transport I (ESCRT‐I). These interactions are not spatially exclusive as we show UBAP1 can associate with MyD88, enhancing its plasma membrane localization. Combined targeting of UBAP1 and TLR adaptors by PumA impedes both cytokine and TLR receptor signalling, highlighting a novel strategy for innate immune evasion.     

Genetic depletion at adaptive but not neutral loci in an endangered bird species

Many endangered species suffer from the loss of genetic diversity, but some populations may be able to thrive even if genetically depleted. To investigate the underlying genetic processes of population bottlenecks, we apply an innovative approach for assessing genetic diversity in the last known population of the endangered Pale‐headed Brushfinch (Atlapetes pallidiceps) in Ecuador. First, we measure genetic diversity at eleven neutral microsatellite loci and adaptive SNP variation in five Toll‐like receptor (TLR) immune system genes. Bottleneck tests confirm genetic drift as the main force shaping genetic diversity in this species and indicate a 99 % reduction in population size dating back several hundred years. Second, we compare contemporary microsatellite diversity with historic museum samples of A. pallidiceps, finding no change in genetic diversity. Third, we compare genetic diversity in the Pale‐headed Brushfinch with two co‐occurring‐related brushfinch species (Atlapetes latinuchus, Buarremon torquatus), finding a reduction of up to 91% diversity in the immune system genes but not in microsatellites. High TLR diversity is linked to decreased survival probabilities in A. pallidiceps. Low TLR diversity is thus probably an adaptation to the specific selection regime within its currently very restricted distribution (approximately 200 ha), but could severely restrict the adaptive potential of the species in the long run. Our study illustrates the importance of investigating both neutral and adaptive markers to assess the effect of population bottlenecks and for recommending specific management plans in endangered species.     

Phylogeography of the narrow‐headed vole Lasiopodomys (Stenocranius) gregalis (Cricetidae,Rodentia) inferred from mitochondrial cytochrome b sequences: an echo of Pleistocene prosperity

A species‐wide phylogeographic study of the narrow‐headed vole Lasiopodomys (Stenocranius) gregalis was performed using the mitochondrial (mt) cytochrome b gene. We examined 164 specimens from 50 localities throughout the species distribution range. Phylogeographic pattern clearly demonstrates the division into four major mtDNA lineages with further subdivision. The level of genetic differentiation between them was found to be extremely high even for the species level: about 6–11%. The most striking result of our study is extremely high mutation rate of cytb in L. gregalis. Our estimates suggested its value of 3.1 × 10^?5 that is an order of magnitude higher than previous estimates for Microtus species. The mean estimated time of basal differentiation of the narrow‐headed vole is about 0.8 Mya. This time estimate is congruent with the known paleontological record. The greatest mitochondrial diversity is found in Southern Siberia where all four lineages occur; therewith, three of them are distributed exclusively in that area. The lineage that is distributed in south‐eastern Transbaikalia is the earliest derivate and exhibits the highest genetic divergence from all the others (11%). It is quite probable that with further research, this lineage will turn out to represent a cryptic species. Spatial patterns of genetic variation in populations of the narrow‐headed vole within the largest mt lineage indicate the normal or stepping stone model of dispersal to the north and south‐west from the Altay region in Middle Pleistocene. Both paleontological data and genetic diversity estimates suggest that this species was very successful during most of the Pleistocene, and we propose that climate humidification and wide advance of tree vegetation at the Pleistocene–Holocene boundary promoted range decrease and fragmentation for this typical member of tundra‐steppe faunistic complex. However, we still observe high genetic diversity within isolated fragments of the range.     

Endemic species may have complex histories: within‐refugium phylogeography of an endangered Iberian vole

Glacial refugia protected and promoted biodiversity during the Pleistocene, not only at a broader scale, but also for many endemics that contracted and expanded their ranges within refugial areas. Understanding the evolutionary history of refugial endemics is especially important in the case of endangered species to recognize the origins of their genetic structure and thus produce better informed conservation practices. The Iberian Peninsula is an important European glacial refugium, rich in endemics of conservation concern, including small mammals, such as the Cabrera vole (Microtus cabrerae). This near‐threatened rodent is characterized by an unusual suite of genetic, life history and ecological traits, being restricted to isolated geographic nuclei in fast‐disappearing Mediterranean subhumid herbaceous habitats. To reconstruct the evolutionary history of the Cabrera vole, we studied sequence variation at mitochondrial, autosomal and sex‐linked loci, using invasive and noninvasive samples. Despite low overall mitochondrial and nuclear nucleotide diversities, we observed two main well‐supported mitochondrial lineages, west and east. Phylogeographic modelling in the context of the Cabrera vole's detailed fossil record supports a demographic scenario of isolation of two populations during the Last Glacial Maximum from a single focus in the southern part of the Iberian Peninsula. In addition, our data suggest subsequent divergence within the east, and secondary contact and introgression of the expanding western population, during the late Holocene. This work emphasizes that refugial endemics may have a phylogeographic history as rich as that of more widespread species, and conservation of such endemics includes the preservation of that genetic legacy.     

Genetic drift outweighs natural selection at toll‐like receptor (TLR) immunity loci in a re‐introduced population of a threatened species

During population establishment, genetic drift can be the key driver of changes in genetic diversity, particularly while the population is small. However, natural selection can also play a role in shaping diversity at functionally important loci. We used a well‐studied, re‐introduced population of the threatened Stewart Island robin (N = 722 pedigreed individuals) to determine whether selection shaped genetic diversity at innate immunity toll‐like receptor (TLR) genes, over a 9‐year period of population growth following establishment with 12 genetic founders. We found no evidence for selection operating with respect to TLR diversity on first‐year overwinter survival for the majority of loci, genotypes and alleles studied. However, survival of individuals with TLR4_BE genotype was significantly improved: these birds were less than half as likely to die prior to maturity compared with all other TLR4 genotypes. Furthermore, the population frequency of this genotype, at a two‐fold excess over Hardy–Weinberg expectation, was increased by nonrandom mating. Near‐complete sampling and full pedigree and reproductive data enabled us to eliminate other potential causes of these patterns including inbreeding, year effects, density dependence, selection on animals at earlier life history stages or genome‐level association of the TLR4_E allele with ‘good genes’. However, comparison of observed levels of gene diversity to predictions under simulated genetic drift revealed results consistent with neutral expectations for all loci, including TLR4. Although selection favoured TLR4_BE heterozygotes in this population, these effects were insufficient to outweigh genetic drift. This is the first empirical study to show that genetic drift can overwhelm natural selection in a wild population immediately following establishment.     

Maintaining their genetic distance: Little evidence for introgression between widely hybridizing species of Geum with contrasting mating systems

Within the plant kingdom, many genera contain sister lineages with contrasting outcrossing and inbreeding mating systems that are known to hybridize. The evolutionary fate of these sister lineages is likely to be influenced by the extent to which they exchange genes. We measured gene flow between outcrossing Geum rivale and selfing Geum urbanum, sister species that hybridize in contemporary populations. We generated and used a draft genome of G. urbanum to develop dd‐RAD data scorable in both species. Coalescent analysis of RAD data from allopatric populations indicated that the species diverged 2–3 Mya, and that historical gene flow between them was extremely low (1 migrant every 25 generations). Comparison of genetic divergence between species in sympatry and allopatry, together with an analysis of allele frequencies in potential parental and hybrid populations, provided no evidence of contemporary introgression in sympatric populations. Cluster‐ and species‐specific marker analyses revealed that, apart from four early‐generation hybrids, individuals in sympatric populations fell into two genetically distinct groups that corresponded exactly to their morphological species classification with maximum individual admixture estimates of only 1–3%. However, we did observe joint segregation of four putatively introgressed SNPs across two scaffolds in the G. urbanum population that was associated with significant morphological variation, interpreted as tentative evidence for rare, recent interspecific gene flow. Overall, our results indicate that despite the presence of hybrids in contemporary populations, genetic exchange between G. rivale and G. urbanum has been extremely limited throughout their evolutionary history.     

Complex mitonuclear interactions and metabolic costs of mating in male seed beetles

The lack of evolutionary response to selection on mitochondrial genes through males predicts the evolution of nuclear genetic influence on male‐specific mitochondrial function, for example by gene duplication and evolution of sex‐specific expression of paralogs involved in metabolic pathways. Intergenomic epistasis may therefore be a prevalent feature of the genetic architecture of male‐specific organismal function. Here, we assess the role of mitonuclear genetic variation for male metabolic phenotypes [metabolic rate and respiratory quotient (RQ)] associated with ejaculate renewal, in the seed beetle Callosobruchus maculatus, by assaying lines with crossed combinations of distinct mitochondrial haplotypes and nuclear lineages. We found a significant increase in metabolic rate following mating relative to virgin males. Moreover, processes associated with ejaculate renewal showed variation in metabolic rate that was affected by mitonuclear interactions. Mitochondrial haplotype influenced mating‐related changes in RQ, but this pattern varied over time. Mitonuclear genotype and the energy spent during ejaculate production affected the weight of the ejaculate, but the strength of this effect varied across mitochondrial haplotypes showing that the genetic architecture of male‐specific reproductive function is complex. Our findings unveil hitherto underappreciated metabolic costs of mating and ejaculate renewal, and provide the first empirical demonstration of mitonuclear epistasis on male reproductive metabolic processes.