Advances in the Evolutionary Understanding of MHC Polymorphism.

Proteins encoded by the classical major histocompatibility complex (MHC) genes incite the vertebrate adaptive immune response by presenting peptide antigens on the cell surface. Here, we review mechanisms explaining landmark features of these genes: extreme polymorphism, excess of nonsynonymous changes in peptide-binding domains, and long gene genealogies. Recent studies provide evidence that these features may arise due to pathogens evolving ways to evade immune response guided by the locally common MHC alleles. However, complexities of selection on MHC genes are simultaneously being revealed that need to be incorporated into existing theory. These include pathogen-driven selection for antigen-binding breadth and expansion of the MHC gene family, associated autoimmunity trade-offs, hitchhiking of deleterious mutations linked to the MHC, geographic subdivision, and adaptive introgression.

The quest for good genes: Epigamic traits, fitness, MHC and multilocus heterozygosity in the guppy.

The 'good genes' hypothesis for the evolution of male secondary sexual traits poses that female preferences for such traits are driven by indirect genetic benefits. However, support for the hypothesis remains ambiguous, and, in particular, the genetic basis for the benefits has rarely been investigated. Here, we use seminatural populations of Trinidadian guppies to investigate whether sexually selected traits (orange, black and iridescent colouration, gonopodium length and body size) predict fitness measured as the number of grandoffspring, a metric that integrates across fitness components and sexes. Furthermore, we tested whether two potential sources of genetic benefits-major histocompatibility complex (MHC) genotypes and multilocus heterozygosity (MLH)-are significant predictors of fitness and of the size of sexually selected traits. We found a significant, nonlinear effect of the area of black pigmentation and male body size on the number of grandoffspring, suggesting stabilizing selection on black area, and nonlinear selection favouring small body size. MLH was heritable (h2 = 0.14) and significantly predicted the number of grandoffspring, indicating the potential for genetic benefits based on heterozygosity. We also found support for local heterozygosity effects, which may reflect a noneven distribution of genetic load across the genome. MHC genotype was not significantly associated with any tested fitness component, or with the load of Gyrodactylus parasites. Neither MHC nor MLH was significant predictor of sexually selected traits. Overall, our results highlight the role of heterozygosity in determining fitness, but do not provide support for male sexually selected traits being indicators of genetic quality.

Small-scale genetic structure of populations of the bulb mite Rhizoglyphus robini.

Bulb mites are an economically significant pest of subterranean parts of plants and a versatile laboratory animal. However, the genetic structure of their populations remains unknown. To fill this gap in our knowledge of their biology, we set up a field experiment in which we allowed mites to colonize onion bulbs, and then determined the genetic structure of colonisers based on a panel of microsatellite loci. We found moderate but significant population structure among sites separated by ca. 20 m (FST range 0.03-0.21), with 7% of genetic variance distributed among sites. Allelic richness within some bulbs was nearly as high as that in the total population, suggesting that colonisation of bulbs was not associated with strong population bottlenecks. The significant genetic structure we observed over small spatial scales seems to reflect limited dispersal of mites in soil.

Long term patterns of association between MHC and helminth burdens in the bank vole support Red Queen dynamics.

Major histocompatibility complex (MHC) genes encode proteins crucial for adaptive immunity of vertebrates. Negative frequency-dependent selection (NFDS), resulting from adaptation of parasites to common MHC types, has been hypothesized to maintain high, functionally relevant polymorphism of MHC, but demonstration of this relationship has remained elusive. In particular, differentiation of NFDS from fluctuating selection, resulting from changes in parasite communities in time and space (FS), has proved difficult in short-term studies. Here, we used temporal data, accumulated through long-term monitoring of helminths infecting bank voles (Myodes glareolus), to test specific predictions of NFDS on MHC class II. Data were collected in three, moderately genetically differentiated subpopulations in Poland, which were characterized by some stable spatiotemporal helminth communities but also events indicating introduction of new species and loss of others. We found a complex association between individual MHC diversity and species richness, where intermediate numbers of DRB supertypes correlated with lowest species richness, but the opposite was true for DQB supertypes-arguing against universal selection for immunogenetic optimality. We also showed that particular MHC supertypes explain a portion of the variance in prevalence and abundance of helminths, but this effect was subpopulation-specific, which is consistent with both NFDS and FS. Finally, in line with NFDS, we found that certain helminths that have recently colonized or spread in a given subpopulation, more frequently or intensely infected voles with MHC supertypes that have been common in the recent past. Overall, our results highlight complex spatial and temporal patterns of MHC-parasite associations, the latter being consistent with Red Queen coevolutionary dynamics.

Major histocompatibility complex class I diversity limits the repertoire of T cell receptors.

Major histocompatibility complex (MHC) genes encode proteins that initiate adaptive immune responses through the presentation of foreign antigens to T cells. The high polymorphism found at these genes, thought to be promoted and maintained by pathogen-mediated selection, contrasts with the limited number of MHC loci found in most vertebrates. Although expressing many diverse MHC genes should broaden the range of detectable pathogens, it has been hypothesized to also cause deletion of larger fractions of self-reactive T cells, leading to a detrimental reduction of the T cell receptor (TCR) repertoire. However, a key prediction of this TCR depletion hypothesis, that the TCR repertoire should be inversely related to the individual MHC diversity, has never been tested. Here, using high-throughput sequencing and advanced sequencing error correction, we provide evidence of such an association in a rodent species with high interindividual variation in the number of expressed MHC molecules, the bank vole (Myodes glareolus). Higher individual diversity of MHC class I, but not class II, was associated with smaller TCR repertoires. Our results thus provide partial support for the TCR depletion model, while also highlighting the complex, potentially MHC class-specific mechanisms by which autoreactivity may trade off against evolutionary expansion of the MHC gene family.

Expansion of frozen hybrids in the guppy ectoparasite, Gyrodactylus turnbulli.

Hybridization is one of the major factors contributing to the emergence of highly successful parasites. Hybrid vigour can play an important role in this process, but subsequent rounds of recombination in the hybrid population may dilute its effects. Increased fitness of hybrids can, however, be frozen by asexual reproduction. Here, we identify invasion of a 'frozen hybrid' genotype in natural populations of Gyrodactylus turnbulli, a facultatively sexual ectoparasitic flatworm that causes significant damage to its fish host. We resequenced genomes of these parasites infecting guppies from six Trinidad and Tobago populations, and found surprisingly high discrepancy in genome-wide nucleotide diversity between islands. The elevated heterozygosity on Tobago is maintained by predominantly clonal reproduction of hybrids formed from two diverged genomes. Hybridization has been followed by spread of the hybrids across the island, implying a selective advantage compared with native genotypes. Our results thus highlight that a single outcrossing event may be independently sufficient to cause pathogen expansion.

Functional immunogenetic variation, rather than local adaptation, predicts ectoparasite infection intensity in a model fish species.

Natural host populations differ in their susceptibility to infection by parasites, and these intrapopulation differences are still an incompletely understood component of host-parasite dynamics. In this study, we used controlled infection experiments with wild-caught guppies (Poecilia reticulata) and their ectoparasite Gyrodactylus turnbulli to investigate the roles of local adaptation and host genetic composition (immunogenetic and neutral) in explaining differences in susceptibility to infection. We found differences between our four study host populations that were consistent between two parasite source populations, with no indication of local adaptation by either host or parasite at two tested spatial scales. Greater values of host population genetic variability metrics broadly aligned with lower population mean infection intensity, with the best alignments associated with major histocompatibility complex (MHC) "supertypes". Controlling for intrapopulation differences and potential inbreeding variance, we found a significant negative relationship between individual-level functional MHC variability and infection: fish carrying more MHC supertypes experienced infections of lower severity, with limited evidence for supertype-specific effects. We conclude that population-level differences in host infection susceptibility probably reflect variation in parasite selective pressure and/or host evolutionary potential, underpinned by functional immunogenetic variation.

Balancing selection versus allele and supertype turnover in MHC class II genes in guppies.

Selection pressure from parasites is thought to be a major force shaping the extreme polymorphism of the major histocompatibility complex (MHC) genes, but the modes and consequences of selection remain unclear. Here, we analyse MHC class II and microsatellite diversity in 16 guppy (Poecilia reticulata) populations from two islands (Trinidad and Tobago) that have been separated for at least 10 ky. Within-population MHC diversity was high, but allele sharing was limited within islands and even lower between islands, suggesting relatively fast turnover of alleles. Allelic lineages strongly supported in phylogenetic analyses tended to be island-specific, suggesting rapid lineage sorting, and an expansion of an allelic lineage private to Tobago was observed. New alleles appear to be generated locally at a detectably high frequency. We did not detect a consistent signature of local adaptation, but FST outlier analysis suggested that balancing selection may be the more general process behind spatial variation in MHC allele frequencies in this system, particularly within Trinidad. We found no evidence for divergent allele advantage within populations, or for decreased genetic structuring of MHC supertypes compared to MHC alleles. The dynamic and complex nature of MHC evolution we observed in guppies, coupled with some evidence for balancing selection shaping MHC allele frequencies, are consistent with Red Queen processes of host-parasite coevolution.

Immunogenetic novelty confers a selective advantage in host-pathogen coevolution.

The major histocompatibility complex (MHC) is crucial to the adaptive immune response of vertebrates and is among the most polymorphic gene families known. Its high diversity is usually attributed to selection imposed by fast-evolving pathogens. Pathogens are thought to evolve to escape recognition by common immune alleles, and, hence, novel MHC alleles, introduced through mutation, recombination, or gene flow, are predicted to give hosts superior resistance. Although this theoretical prediction underpins host-pathogen "Red Queen" coevolution, it has not been demonstrated in the context of natural MHC diversity. Here, we experimentally tested whether novel MHC variants (both alleles and functional "supertypes") increased resistance of guppies (Poecilia reticulata) to a common ectoparasite (Gyrodactylus turnbulli). We used exposure-controlled infection trials with wild-sourced parasites, and Gyrodactylus-naïve host fish that were F2 descendants of crossed wild populations. Hosts carrying MHC variants (alleles or supertypes) that were new to a given parasite population experienced a 35-37% reduction in infection intensity, but the number of MHC variants carried by an individual, analogous to heterozygosity in single-locus systems, was not a significant predictor. Our results provide direct evidence of novel MHC variant advantage, confirming a fundamental mechanism underpinning the exceptional polymorphism of this gene family and highlighting the role of immunogenetic novelty in host-pathogen coevolution.

Mating preferences can drive expansion or contraction of major histocompatibility complex gene family.

Major histocompatibility complex (MHC)-based mating rules can evolve as a way to avoid inbreeding or to increase offspring immune competence. While the role of mating preference in shaping the MHC diversity in vertebrates has been acknowledged, its impact on individual MHC diversity has not been considered. Here, we use computer simulations to investigate how simple mating rules favouring MHC-dissimilar partners affect the evolution of the number of MHC variants in individual genomes, accompanying selection for resistance to parasites. We showed that the effect of such preferences could sometimes be dramatic. If preferences are aimed at avoiding identical alleles, the equilibrium number of MHC alleles is much smaller than under random mating. However, if the mating rule minimizes the ratio of shared to different alleles in partners, MHC number is higher than under random mating. Additionally, our simulations revealed that a negative correlation between the numbers of MHC variants in mated individuals can arise from simple rules of MHC-disassortative mating. Our results reveal unexpected potential of MHC-based mating preferences to drive MHC gene family expansions or contractions and highlight the need to study the mechanistic basis of such preferences, which is currently poorly understood.

Gene duplications, divergence and recombination shape adaptive evolution of the fish ectoparasite Gyrodactylus bullatarudis.

Determining the molecular basis of parasite adaptation to its host is an important component in understanding host-parasite coevolution and the epidemiology of parasitic infections. Here, we investigate short- and long-term adaptive evolution in the eukaryotic parasite Gyrodactylus bullatarudis infecting Caribbean guppies (Poecilia reticulata), by comparing the reference genome of Tobagonian G. bullatarudis with other Platyhelminthes, and by analysing resequenced samples from local Trinidadian populations. At the macroevolutionary timescale, we observed duplication of G-protein and serine proteases genes, which are probably important in host-parasite arms races. Serine protease also showed strong evidence of ongoing, diversifying selection at the microevolutionary timescale. Furthermore, our analyses revealed that a hybridization event, involving two divergent genomes, followed by recombination has dramatically affected the genetic composition of Trinidadian populations. The recombinant genotypes invaded Trinidad and replaced local parasites in all populations. We localized more than 300 genes in regions fixed in local populations for variants of different origin, possibly due to diversifying selection pressure from local host populations. In addition, around 70 genes were localized in regions identified as heterozygous in some, but not all, individuals. This pattern is consistent with a very recent spread of recombinant parasites. Overall, our results are consistent with the idea that recombination between divergent genomes can result in particularly successful parasites.

Sexual and ecological selection on a sexual conflict gene.

Sexual selection and conflict can act on genes with important metabolic functions, potentially shaping standing genetic variance in such genes and thus evolutionary potential of populations. Here, using experimental evolution, we show how reproductive competition intensity and thermal environment affect selection on phosphogluconate dehydrogenase (6Pgdh)-a metabolic gene involved in sexual selection and conflict in the bulb mite. The S allele of 6Pgdh increases male success in reproductive competition, but is detrimental to S-bearing males' partners. We found that the rate of the S allele spread increased with the proportion of males in the experimental populations, illustrating that harm to females is more easily compensated for males under more intense sexual competition. Furthermore, we found that under equal sex ratio, the S allele spreads faster at higher temperature. While the direction of selection on 6Pgdh was not reversed in any of the conditions we tested, which would be required for environmental heterogeneity to maintain polymorphism at this locus, our study highlights that ecological and sexual selection can jointly affect selection on important metabolic enzymes.

Evolution of major histocompatibility complex gene copy number.

MHC genes, which code for proteins responsible for presenting pathogen-derived antigens to the host immune system, show remarkable copy-number variation both between and within species. However, the evolutionary forces driving this variation are poorly understood. Here, we use computer simulations to investigate whether evolution of the number of MHC variants in the genome can be shaped by the number of pathogen species the host population encounters (pathogen richness). Our model assumed that while increasing a range of pathogens recognised, expressing additional MHC variants also incurs costs such as an increased risk of autoimmunity. We found that pathogen richness selected for high MHC copy number only when the costs were low. Furthermore, the shape of the association was modified by the rate of pathogen evolution, with faster pathogen mutation rates selecting for increased host MHC copy number, but only when pathogen richness was low to moderate. Thus, taking into account factors other than pathogen richness may help explain wide variation between vertebrate species in the number of MHC genes. Within population, variation in the number of unique MHC variants carried by individuals (INV) was observed under most parameter combinations, except at low pathogen richness. This variance gave rise to positive correlations between INV and host immunocompetence (proportion of pathogens recognised). However, within-population variation in host immunocompetence declined with pathogen richness. Thus, counterintuitively, pathogens can contribute more to genetic variance for host fitness in species exposed to fewer pathogen species, with consequences to predictions from "Hamilton-Zuk" theory of sexual selection.

RNA-Seq analysis of the guppy immune response against Gyrodactylus bullatarudis infection.

Gyrodactylids are ubiquitous ectoparasites of teleost fish, but our understanding of the host immune response against them is fragmentary. Here, we used RNA-Seq to investigate genes involved in the primary response to infection with Gyrodactylus bullatarudis on the skin of guppies, Poecilia reticulata, an important evolutionary model, but also one of the most common fish in the global ornamental trade. Analysis of differentially expressed genes identified several immune-related categories, including IL-17 signalling pathway and Th17 cell differentiation, cytokine-cytokine receptor interaction, chemokine signalling pathway, NOD-like receptor signalling pathway, natural killer cell-mediated cytotoxicity and pathways involved in antigen recognition, processing and presentation. Components of both the innate and the adaptive immune responses play a role in response to gyrodactylid infection. Genes involved in IL-17/Th17 response were particularly enriched among differentially expressed genes, suggesting a significant role for this pathway in fish responses to ectoparasites. Our results revealed a sizable list of genes potentially involved in the teleost-gyrodactylid immune response.

Relative costs and benefits of alternative reproductive phenotypes at different temperatures - genotype-by-environment interactions in a sexually selected trait.

BACKGROUND: The maintenance of considerable genetic variation in sexually selected traits (SSTs) is puzzling given directional selection expected to act on these traits. A possible explanation is the existence of a genotype-by-environment (GxE) interaction for fitness, by which elaborate SSTs are favored in some environments but selected against in others. In the current study, we look for such interactions for fitness-related traits in the bulb mite, a male-dimorphic species with discontinuous expression of a heritable SST in the form of enlarged legs that are used as weapons. RESULTS: We show that evolution at 18 °C resulted in populations with a higher prevalence of this SST compared to evolution at 24 °C. We further demonstrate that temperature modified male reproductive success in a way that was consistent with these changes. There was a genotype-by-environment interaction for reproductive success - at 18 °C the relative reproductive success of armored males competing with unarmored ones was higher than at the moderate temperature of 24 °C. However, male morph did not have interactive effects with temperature with respect to other life history traits (development time and longevity). CONCLUSIONS: A male genotype that is associated with the expression of a SST interacted with temperature in determining male reproductive success. This interaction caused an elaborate SST to evolve in different directions (more or less prevalent) depending on the thermal environment. The implication of this finding is that seasonal temperature fluctuations have the potential to maintain male polymorphism within populations. Furthermore, spatial heterogeneity in thermal conditions may cause differences among populations in SST selection. This could potentially cause selection against male immigrants from populations in different environments and thus strengthen barriers to gene flow.

Fitness consequences of threshold trait expression subject to environmental cues.

Most cases of alternative reproductive tactics (ARTs) are thought to represent conditional strategies, whereby high-status males express highly competitive phenotypes, whereas males below a certain status threshold resort to sneaky tactics. The underlying evolutionarily stable strategy (ESS) model assumes that males of high competitive ability achieve higher fitness when expressing the territorial phenotype, whereas the less competitive males are more fit as sneakers, caused by fitness functions for the ARTs having different slopes and intersecting at a threshold value of competitive ability. The model, however, is notoriously difficult to test as it requires access to low-status territorials and high-status sneakers, that rarely occur in nature. Here, we test the conditional ESS in the androdimorphic acarid mite Sancassania berlesei, where large males tend to develop into an armoured, aggressive 'fighter' morph, while small males become unarmoured, non-aggressive 'scramblers'. In addition to body size, male morph is affected by pheromones produced by big populations, with fighters being suppressed in dense colonies. By manipulating pheromone concentration, we obtained high-status scramblers and low-status fighters. We also estimated status- and size-dependent fitness functions for male morphs across a range of population sizes. Fighters had the highest fitness in small populations and their fitness declined with increasing density, whereas the reverse was true for scramblers, providing support for condition-dependent ESS with respect to demography. However, whereas male fitness increased with body size, the fitness functions did not differ significantly between morphs. Thus, although we found evidence for the intersection of morph fitness functions with respect to demography, we did not find such an intersection in relation to male body size. Our results highlight how demography can exert selection pressures shaping the evolution of the conditional strategy in species with ARTs.

Blood parasites shape extreme major histocompatibility complex diversity in a migratory passerine.

Pathogens are one of the main forces driving the evolution and maintenance of the highly polymorphic genes of the vertebrate major histocompatibility complex (MHC). Although MHC proteins are crucial in pathogen recognition, it is still poorly understood how pathogen-mediated selection promotes and maintains MHC diversity, and especially so in host species with highly duplicated MHC genes. Sedge warblers (Acrocephalus schoenobaenus) have highly duplicated MHC genes, and using data from high-throughput MHC genotyping, we were able to investigate to what extent avian malaria parasites explain temporal MHC class I supertype fluctuations in a long-term study population. We investigated infection status and infection intensities of two different strains of Haemoproteus, that is avian malaria parasites that are known to have significant fitness consequences in sedge warblers. We found that prevalence of avian malaria in carriers of specific MHC class I supertypes was a significant predictor of their frequency changes between years. This finding suggests that avian malaria infections partly drive the temporal fluctuations of the MHC class I supertypes. Furthermore, we found that individuals with a large number of different supertypes had higher resistance to avian malaria, but there was no evidence for an optimal MHC class I diversity. Thus, the two studied malaria parasite strains appear to select for a high MHC class I supertype diversity. Such selection may explain the maintenance of the extremely high number of MHC class I gene copies in sedge warblers and possibly also in other passerines where avian malaria is a common disease.

Extreme MHC class I diversity in the sedge warbler (Acrocephalus schoenobaenus); selection patterns and allelic divergence suggest that different genes have different functions.

BACKGROUND: Recent work suggests that gene duplications may play an important role in the evolution of immunity genes. Passerine birds, and in particular Sylvioidea warblers, have highly duplicated major histocompatibility complex (MHC) genes, which are key in immunity, compared to other vertebrates. However, reasons for this high MHC gene copy number are yet unclear. High-throughput sequencing (HTS) allows MHC genotyping even in individuals with extremely duplicated genes. This HTS data can reveal evidence of selection, which may help to unravel the putative functions of different gene copies, i.e. neofunctionalization. We performed exhaustive genotyping of MHC class I in a Sylvioidea warbler, the sedge warbler, Acrocephalus schoenobaenus, using the Illumina MiSeq technique on individuals from a wild study population. RESULTS: The MHC diversity in 863 genotyped individuals by far exceeds that of any other bird species described to date. A single individual could carry up to 65 different alleles, a large proportion of which are expressed (transcribed). The MHC alleles were of three different lengths differing in evidence of selection, diversity and divergence within our study population. Alleles without any deletions and alleles containing a 6 bp deletion showed characteristics of classical MHC genes, with evidence of multiple sites subject to positive selection and high sequence divergence. In contrast, alleles containing a 3 bp deletion had no sites subject to positive selection and had low divergence. CONCLUSIONS: Our results suggest that sedge warbler MHC alleles that either have no deletion, or contain a 6 bp deletion, encode classical antigen presenting MHC molecules. In contrast, MHC alleles containing a 3 bp deletion may encode molecules with a different function. This study demonstrates that highly duplicated MHC genes can be characterised with HTS and that selection patterns can be useful for revealing neofunctionalization. Importantly, our results highlight the need to consider the putative function of different MHC genes in future studies of MHC in relation to disease resistance and fitness.

Genomic Response to Selection for Predatory Behavior in a Mammalian Model of Adaptive Radiation.

If genetic architectures of various quantitative traits are similar, as studies on model organisms suggest, comparable selection pressures should produce similar molecular patterns for various traits. To test this prediction, we used a laboratory model of vertebrate adaptive radiation to investigate the genetic basis of the response to selection for predatory behavior and compare it with evolution of aerobic capacity reported in an earlier work. After 13 generations of selection, the proportion of bank voles (Myodes [=Clethrionomys] glareolus) showing predatory behavior was five times higher in selected lines than in controls. We analyzed the hippocampus and liver transcriptomes and found repeatable changes in allele frequencies and gene expression. Genes with the largest differences between predatory and control lines are associated with hunger, aggression, biological rhythms, and functioning of the nervous system. Evolution of predatory behavior could be meaningfully compared with evolution of high aerobic capacity, because the experiments and analyses were performed in the same methodological framework. The number of genes that changed expression was much smaller in predatory lines, and allele frequencies changed repeatably in predatory but not in aerobic lines. This suggests that more variants of smaller effects underlie variation in aerobic performance, whereas fewer variants of larger effects underlie variation in predatory behavior. Our results thus contradict the view that comparable selection pressures for different quantitative traits produce similar molecular patterns. Therefore, to gain knowledge about molecular-level response to selection for complex traits, we need to investigate not only multiple replicate populations but also multiple quantitative traits.

Experimental evolution under hyper-promiscuity in Drosophila melanogaster.

BACKGROUND: The number of partners that individuals mate with over their lifetime is a defining feature of mating systems, and variation in mate number is thought to be a major driver of sexual evolution. Although previous research has investigated the evolutionary consequences of reductions in the number of mates, we know little about the costs and benefits of increased numbers of mates. Here, we use a genetic manipulation of mating frequency in Drosophila melanogaster to create a novel, highly promiscuous mating system. We generated D. melanogaster populations in which flies were deficient for the sex peptide receptor (SPR) gene - resulting in SPR- females that mated more frequently - and genetically-matched control populations, and allowed them to evolve for 55 generations. At several time-points during this experimental evolution, we assayed behavioural, morphological and transcriptional reproductive phenotypes expected to evolve in response to increased population mating frequencies. RESULTS: We found that males from the high mating frequency SPR- populations evolved decreased ability to inhibit the receptivity of their mates and decreased copulation duration, in line with predictions of decreased per-mating investment with increased sperm competition. Unexpectedly, SPR- population males also evolved weakly increased sex peptide (SP) gene expression. Males from SPR- populations initially (i.e., before experimental evolution) exhibited more frequent courtship and faster time until mating relative to controls, but over evolutionary time these differences diminished or reversed. CONCLUSIONS: In response to experimentally increased mating frequency, SPR- males evolved behavioural responses consistent with decreased male post-copulatory investment at each mating and decreased overall pre-copulatory performance. The trend towards increased SP gene expression might plausibly relate to functional differences in the two domains of the SP protein. Our study highlights the utility of genetic manipulations of animal social and sexual environments coupled with experimental evolution.