Ultrasonic Vocalizations of Male Mice Differ among Species and Females Show Assortative Preferences for Male Calls

Male house mice (Mus musculus) emit ultrasonic vocalizations (USVs) during courtship, which attract females, and we aimed to test whether females use these vocalizations for species or subspecies recognition of potential mates. We recorded courtship USVs of males from different Mus species, Mus musculus subspecies, and populations (F1 offspring of wild-caught Mus musculus musculus, Mus musculus domesticus (and F1 hybrid crosses), and Mus spicilegus), and we conducted playback experiments to measure female preferences for male USVs. Male vocalizations contained at least seven distinct syllable types, whose frequency of occurrence varied among species, subspecies, and populations. Detailed analyses of multiple common syllable types indicated that Mus musculus and Mus spicilegus could be discriminated based on spectral and temporal characteristics of their vocalizations, and populations of Mus musculus were also distinctive regardless of the classification model used. Females were able to discriminate USVs from different species, and showed assortative preferences for conspecific males. We found no evidence that females discriminate USVs of males from a different subspecies or separate populations of the same species, even though our spectral analyses identified acoustic features that differ between species, subspecies, and populations of the same species. Our results provide the first comparison of USVs between Mus species or between Mus musculus subspecies, and the first evidence that male USVs potentially facilitate species recognition.     

Subspecies recognition in the house mouse: a study of two populations from the border of a hybrid zone

Mate choice is the outcome of sexual preference for partnerscarrying specific signals. Thus, mating among conspecifics(homogamy) depends on the occurrence of species recognitionsystems. We asked what happens if populations diverge, andwe investigated female sexual preference between two subspeciesof the house mouse in populations from the borders of a hybridzone (Jutland, Denmark). We used choice tests to analyze theoccurrence of recognition signals and to locate these signalsin soiled bedding and urine. Our results show that populationsof the two subspecies can be discriminated on the basis ofurinary signals, suggesting that the latter have diverged. Additionally, these signals seem to have similar features inpopulations of different geographical origins, suggesting thatsubspecific differentiation occurs. This is the first demonstrationthat subspecific recognition through urinary signals occursin the house mouse. However, while Mus musculus domesticusdoes not display a preference, we show that Mus musculus musculusfemales tend to mate with males of the same subspecies. We discuss the different factors that could explain these discrepanciesbetween females of the two taxa: differences in signal perception,evolution at a different pace, or evolution under differentselective pressures in their area of contact. Further, we proposethat the divergence in male signal was at least partly initiatedin allopatry and discuss different evolutionary scenario thatmay explain the patterns observed in Denmark and their relevance to isolation between the two taxa.     

Do changes in gene expression contribute to sexual isolation and reinforcement in the house mouse?

Expression divergence, rather than sequence divergence, has been shown to be important in speciation, particularly in the early stages of divergence of traits involved in reproductive isolation. In the two European subspecies of house mice, Mus musculus musculus and Mus musculus domesticus, earlier studies have demonstrated olfactory‐based assortative mate preference in populations close to their hybrid zone. It has been suggested that this behaviour evolved following the recent secondary contact between the two taxa (~3,000 years ago) in response to selection against hybridization. To test for a role of changes in gene expression in the observed behavioural shift, we conducted a RNA sequencing experiment on mouse vomeronasal organs. Key candidate genes for pheromone‐based subspecies recognition, the vomeronasal receptors, are expressed in these organs. Overall patterns of gene expression varied significantly between samples from the two subspecies, with a large number of differentially expressed genes between the two taxa. In contrast, only ~200 genes were found repeatedly differentially expressed between populations within M. m. musculus that did or did not display assortative mate preferences (close to or more distant from the hybrid zone, respectively), with an overrepresentation of genes belonging to vomeronasal receptor family 2. These receptors are known to play a key role in recognition of chemical cues that handle information about genetic identity. Interestingly, four of five of these differentially expressed receptors belong to the same phylogenetic cluster, suggesting specialization of a group of closely related receptors in the recognition of odorant signals that may allow subspecies recognition and assortative mating.     

Oestrous females investigate the unfamiliar male more than the familiar male in both commensal and non-commensal populations of house mice

We studied female preferences for familiar and unfamiliar males. The subjects were laboratory-born house mice: (1) non-commensal Mus musculus domesticus from the eastern part of Syria along the Euphrates River; and (2) commensal M. m. musculus from the Czech Republic. Pair-choice preference tests have revealed that oestrous females of both populations sniffed towards unfamiliar males more than familiar males. In the case of females exhibiting postpartum oestrus, this preference was less pronounced and statistically not significant. Thus, our mice clearly exhibited the behavioural pattern known from commensal populations of polygynous and/or promiscuous M. m. domesticus. We found no inverse tendency to seek proximity to the familiar male that has been previously reported from closely related and presumably monogamous aboriginal mouse Mus spicilegus. We conclude that neither commensal M. m. musculus, nor non-commensal M. m. domesticus, are likely to share a monogamous mating system with mound-building mice.     

Seeking signatures of reinforcement at the genetic level: a hitchhiking mapping and candidate gene approach in the house mouse

Reinforcement is the process by which prezygotic isolation is strengthened as a response to selection against hybridization. Most empirical support for reinforcement comes from the observation of its possible phenotypic signature: an accentuated degree of prezygotic isolation in the hybrid zone as compared to allopatry. Here, we implemented a novel approach to this question by seeking for the signature of reinforcement at the genetic level. In the house mouse, selection against hybrids and enhanced olfactory‐based assortative mate preferences are observed in a hybrid zone between the two European subspecies Mus musculus musculus and M. m. domesticus, suggesting a possible recent reinforcement event. To test for the genetic signature of reinforcing selection and identify genes involved in sexual isolation, we adopted a hitchhiking mapping approach targeting genomic regions containing candidate genes for assortative mating in mice. We densely scanned these genomic regions in hybrid zone and allopatric samples using a large number of fast evolving microsatellite loci that allow the detection of recent selection events. We found a handful of loci showing the expected pattern of significant reduction in variability in populations close to the hybrid zone, showing assortative odour preference in mate choice experiments as compared to populations further away and displaying no such preference. These loci lie close to genes that we pinpoint as testable candidates for further investigation.     

Sexual selection and assortative mating: an experimental test

Mate choice and mate competition can both influence the evolution of sexual isolation between populations. Assortative mating may arise if traits and preferences diverge in step, and, alternatively, mate competition may counteract mating preferences and decrease assortative mating. Here, we examine potential assortative mating between populations of Drosophila pseudoobscura that have experimentally evolved under either increased (‘polyandry’) or decreased (‘monogamy’) sexual selection intensity for 100 generations. These populations have evolved differences in numerous traits, including a male signal and female preference traits. We use a two males: one female design, allowing both mate choice and competition to influence mating outcomes, to test for assortative mating between our populations. Mating latency shows subtle effects of male and female interactions, with females from the monogamous populations appearing reluctant to mate with males from the polyandrous populations. However, males from the polyandrous populations have a significantly higher probability of mating regardless of the female's population. Our results suggest that if populations differ in the intensity of sexual selection, effects on mate competition may overcome mate choice.     

Divergence of odorant signals within and between the two European subspecies of the house mouse

The olfactory acuity of mice allows them to discriminate odorsof conspecifics differing by a few genes. This acuity is usedin habituation procedures where investigation of novel odorsby the mouse can be translated into relative difference or similaritybetween the stimuli. This study adapts these behavioral proceduresto address suprapopulation divergence among urinary odors inthe house mouse. Specifically, we investigate geographical patternsof odor divergence within and between 2 subspecies of the housemouse, Mus musculus musculus and Mus musculus domesticus, whichdiverged in allopatry and met secondarily in Europe where theyhybridize. Based on M. m. musculus perception, our study suggeststhat odors of the 2 subspecies differ in both allopatric andcontact zone populations and that divergence is more markedin the latter. Our earlier studies documented mate preferenceand signal divergence between the 2 subspecies. Hence, we considerthe role of the urinary odors as mating signals. We discusshow signal divergence between the 2 subspecies may relate toreproductive character displacement. This study validates theuse of habituation procedures to reconstruct geographical patternsof odorant signal divergence, providing a strong methodologicalplatform to address reproductive character displacement affectingcryptic mating signals in mammals.     

Mutual mate choice by mountain pine beetles: size‐dependence but not size‐assortative mating

1. Mutual mate choice may be rare, occurring when both sexes invest heavily in reproduction, mating opportunities are abundant, and individuals differ in quality. 2. Mountain pine beetles, Dendroctonus ponderosae (Curculionidae: Scolytinae) appear to meet the conditions for mutual mate choice. We introduced males to females in breeding sites and observed the occurrence and speed of a male entering a female's gallery. We tested for consequences of mutual mate choice, namely condition‐dependent choosiness and assortative mating. 3. Males were more likely to enter a female's gallery when the gallery was in a smaller tree with less resin production and when the gallery was larger. Female body size and condition did not influence the probability of entry. Larger males were less likely to enter a gallery than were smaller males, probably because of size‐dependent choosiness rather than physical limitations. 4. Small males took longer to enter galleries of large females than of small females, whereas large males entered as quickly into galleries of large females as small females. This suggests size‐dependent choosiness by females. 5. No assortative mating by body size was detected, probably because males appeared to choose on the basis of female‐associated resources rather than on female traits.     

On the role of sexual selection in ecological divergence: a test of body‐size assortative mating in the eastern newt Notophthalmus viridescens

Speciation processes initiated by divergent selection often fail to complete; yet, how sexual selection is involved in the progress of ecological speciation is rarely understood. Intraspecific body‐size variation affects mate preference and male–male competition, which can consequently lead to assortative mating based on body size. In the present study, we tested the importance of body size difference in the potential of assortative mating between the two eastern newt subspecies, larger Notophthalmus viridescens viridescens and smaller Notophthalmus viridescens dorsalis. Through differential expression of life‐cycle polyphenism, these two subspecies are adapted to contrasting environments, which has likely led to the subspecific body‐size difference. We found that males of both subspecies preferred larger females of N. v. viridescens as mates presumably because of the fecundity advantage of larger females. On the other hand, no evidence of female choice was found. Larger males of N. v. viridescens exhibited greater competitive ability and gained primary access to larger females of their own kind. However, smaller males were able to overcome their inferior competitive ability by interfering with larger males' spermatophore transfer and sneakily mating with larger females. Thus, the subspecific body‐size difference importantly affected sexual selection processes, resulting in nonrandom but not completely assortative mating patterns between the larger and smaller subspecies. Although life‐cycle polyphenism facilitates the intraspecific ecological divergence within N. v. viridescens sexual selection processes, namely smaller males' mate preference for larger females and sexual interference during spermatophore transfer, may be halting completion of the ecological speciation. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 884–897.     

Reinforcement selection acting on the European house mouse hybrid zone

Behavioural isolation may lead to complete speciation when partial postzygotic isolation acts in the presence of divergent‐specific mate‐recognition systems. These conditions exist where Mus musculus musculus and M. m. domesticus come into contact and hybridize. We studied two mate‐recognition signal systems, based on urinary and salivary proteins, across a Central European portion of the mouse hybrid zone. Introgression of the genomic regions responsible for these signals: the major urinary proteins (MUPs) and androgen binding proteins (ABPs), respectively, was compared to introgression at loci assumed to be nearly neutral and those under selection against hybridization. The preference of individuals taken from across the zone regarding these signals was measured in Y mazes, and we develop a model for the analysis of the transition of such traits under reinforcement selection. The strongest assortative preferences were found in males for urine and females for ABP. Clinal analyses confirm nearly neutral introgression of an Abp locus and two loci closely linked to the Abp gene cluster, whereas two markers flanking the Mup gene region reveal unexpected introgression. Geographic change in the preference traits matches our reinforcement selection model significantly better than standard cline models. Our study confirms that behavioural barriers are important components of reproductive isolation between the house mouse subspecies.     

A Candidate Subspecies Discrimination System Involving a Vomeronasal Receptor Gene with Different Alleles Fixed in M. m. domesticus and M. m. musculus

Assortative mating, a potentially efficient prezygotic reproductive barrier, may prevent loss of genetic potential by avoiding the production of unfit hybrids (i.e., because of hybrid infertility or hybrid breakdown) that occur at regions of secondary contact between incipient species. In the case of the mouse hybrid zone, where two subspecies of Mus musculus (M. m. domesticus and M. m. musculus) meet and exchange genes to a limited extent, assortative mating requires a means of subspecies recognition. We based the work reported here on the hypothesis that, if there is a pheromone sufficiently diverged between M. m. domesticus and M. m. musculus to mediate subspecies recognition, then that process must also require a specific receptor(s), also sufficiently diverged between the subspecies, to receive the signal and elicit an assortative mating response. We studied the mouse V1R genes, which encode a large family of receptors in the vomeronasal organ (VNO), by screening Perlegen SNP data and identified one, Vmn1r67, with 24 fixed SNP differences most of which (15/24) are nonsynonymous nucleotide substitutions between M. m. domesticus and M. m. musculus. We observed substantial linkage disequilibrium (LD) between Vmn1r67 and Abpa27, a mouse salivary androgen-binding protein gene that encodes a proteinaceous pheromone (ABP) capable of mediating assortative mating, perhaps in conjunction with its bound small lipophilic ligand. The LD we observed is likely a case of association rather than residual physical linkage from a very recent selective sweep, because an intervening gene, Vmn1r71, shows significant intra(sub)specific polymorphism but no inter(sub)specific divergence in its nucleotide sequence. We discuss alternative explanations of these observations, for example that Abpa27 and Vmn1r67 are coevolving as signal and receptor to reinforce subspecies hybridization barriers or that the unusually divergent Vmn1r67 allele was not a product of fast positive selection, but was derived from an introgressed allele, possibly from Mus spretus.     

Transgressive segregation in a behavioural trait? Explorative strategies in two house mouse subspecies and their hybrids

Hybrid zones between genetically diverged populations are widespread among animals and plants. Their dynamics usually depend on selection against admixture and dispersal of parental forms in the zone. Although indirect estimates of selection have been the target of many studies, dispersal has been neglected. In this study we carried out open field experiments to test whether males of two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus, differ in their propensity to disperse and in their character of exploration. We tested wild‐caught males and males of two wild‐derived inbred strains. In addition, we examined reciprocal F₁ crosses to test the prediction that these hybrids display intermediate behaviours. We revealed that M. m. musculus males were less hesitant to enter the experimental arena than were M. m. domesticus males, but once inside the arena their movements were more timid. F₁ males differed from both parental strains, with longer latencies to enter the arena, but explored the arena in a similar fashion as the M. m. domesticus males, thus displaying transgressive behavioural phenotypes. These results contribute to our knowledge of behavioural divergence between the mouse subspecies, and add a new facet to the study of speciation. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●●, ●●–●●.     

Does sexual experience affect the strength of male mate choice for high‐quality females in Drosophila melanogaster?

Although females are traditionally thought of as the choosy sex, there is increasing evidence in many species that males will preferentially court or mate with certain females over others when given a choice. In the fruit fly, Drosophila melanogaster, males discriminate between potential mating partners based on a number of female traits, including species, mating history, age, and condition. Interestingly, many of these male preferences are affected by the male''s previous sexual experiences, such that males increase courtship toward types of females that they have previously mated with and decrease courtship toward types of females that have previously rejected them. D. melanogaster males also show courtship and mating preferences for larger females over smaller females, likely because larger females have higher fecundity. It is unknown, however, whether this preference shows behavioral plasticity based on the male''s sexual history as we see for other male preferences. Here, we manipulate the sexual experience of D. melanogaster males and test whether this manipulation has any effect on the strength of male mate choice for large females. We find that sexually inexperienced males have a robust courtship preference for large females that is unaffected by previous experience mating with, or being rejected by, females of differing sizes. Given that female body size is one of the most common targets of male mate choice across insect species, our experiments with D. melanogaster may provide insight into how these preferences develop and evolve.     

Experience Matters: Females Use Smell to Select Experienced Males for Paternal Care

Mate choice and mating preferences often rely on the information content of signals exchanged between potential partners. In species where a female''s reproduction is the terminal event in life it is to be expected that females choose high quality males and assess males using some honest indicator of male quality. The Nereidid polychaete, Neanthes acuminata, exhibits monogamous pairing and the release of eggs by females terminates her life and larval success relies entirely on a male''s ability to provide paternal care. As such females should have developed reliable, condition-dependent criteria to choose mates to guarantee survival and care for offspring. We show that females actively chose males experienced in fatherhood over others. In the absence of experienced males dominance, as evident from male-male fights, is utilized for mate selection. The preference for experienced males is not affected by previous social interactions between the individuals. We show that the choice of the partner is based on chemical signals demonstrating a ‘scent of experience’ to females providing evidence for the role of chemical signals in sexual selection for paternal care adding to our understanding of the mechanisms regulating condition-dependent mate choice.     

When are good genes good? Variable outcomes of female choice in wax moths

Female lesser wax moths (Achroia grisella) choose males based on characters of their ultrasonic advertisement signals. Because a female''s opportunity to obtain increased somatic benefits by mating with a particular male is limited, we investigated whether females obtain genetic benefits for their offspring via mate choice. Controlled breeding experiments conducted under favourable food and temperature conditions showed that developmental characters are heritable, that sire attractiveness and offspring survivorship are unrelated, but that females mating with attractive signallers produce offspring who mature faster than the offspring of females mating with non-attractive signallers. However, under some unfavourable food or temperature conditions, it is the offspring of females mating with non-attractive males who mature faster; these offspring are heavier as well. Thus, the relationship between male attractiveness and offspring development is not environmentally robust, and support for a good genes model of mate choice in A. grisella is dependent on conditions. These findings suggest genotype–environment interactions and emphasize the necessity of testing sexual selection models under a range of natural environments.     

SALIVARY ANDROGEN-BINDING PROTEIN (ABP) MEDIATES SEXUAL ISOLATION IN MUS MUSCULUS

We wanted to determine whether the microevolution of the mouse salivary androgen-binding protein (ABP) Alpha subunit gene (Abpa) could mediate sexual selection and thereby have a potential role in maintaining gene pool integrity where radiating mouse subspecies make secondary contact. This hypothesis is based upon previous work in this laboratory, which has shown that each subspecies apparently has its own allele and that these alleles have a 25-fold excess of nonsynonymous/synonymous base substitutions compared to an average protein under purifying selection. We provide direct evidence for ABP-assortative mate selection in a laboratory setting: Mus musculus domesticus and M. m. musculus female mice recognize and discriminate between the territories of male mice that essentially differ solely in their Abpa genotype and, when the males are present, the female prefers to mate with the one of her own ABP type. The observation that females could differentiate between the territories of the two males when those mice were absent suggests that the males marked their territories with ABP. In this study, we also detected ABP on the pelts of male mice and in their environment. It is likely that the animals apply the protein to their pelts by licking and that it is then deposited in their surroundings. We suggest that females of the two subspecies are able to discriminate between males of those subspecies on the basis of this protein molecule. Mouse salivary ABP might present a worthwhile system with which to study a prezygotic isolation mechanism in a mammal.     

The meek inherit the earth: less aggressive wild mice are more successful in challenging situations

Numerous studies have shown an association between aggressiveness and several other behavioural traits. For example, more aggressive animals were bold and active explorers tending to form persistent routines whereas less aggressive animals were shy, careful but more flexible. While the former are thought to be more successful under stable conditions the latter should have advantages in more dynamic situations. These differences can apply not only to individuals but also to populations, species or groups of species with important implications to species distributions and speciation rates. Here we utilized the Morris water task (MWT) to investigate how two subspecies, Mus musculus musculus and M. m. domesticus, known to differ in aggressiveness, cope with stressful situations. We found that less aggressive musculus males performed significantly better in solving the MWT than more aggressive domesticus males. This suggests that M. m. musculus is more flexible and could be more successful under stressful and/or dynamic situations typical of dispersal bouts. It seems plausible that this difference may have had an influence on the secondary contact between musculus and domesticus populations in the past and perhaps still can affect the dynamics of the European hybrid zone between the subspecies. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 310–319.     

MHC-disassortative mating preferences reversed by cross-fostering.

House mice (Mus musculus domesticus) avoid mating with individuals that are genetically similar at the major histocompatibility complex (MHC). Mice are able recognize MHC-similar individuals through specific odour cues. However, to mate disassortatively for MHC genes, individuals must have a referent, either themselves (self-inspection) or close kin (familial imprinting), with which to compare the MHC identity of potential mates. Although studies on MHC-dependent mating preferences often assume that individuals use self-inspection, laboratory experiments with male mice indicate that they use familial imprinting, i.e. males learn the MHC identity of their family and then avoid mating with females carrying ''familial'' MHC alleles. To determine if female mice use familial imprinting, we cross-fostered wild-derived female mouse pups into MHC-dissimilar families, and then tested if this procedure reversed their mating preferences compared with in-fostered controls. Our observations of the female''s mating behaviour in seminatural social conditions and the genetic typing of their progeny both indicated that females avoided mating with males carrying MHC genes of their foster family, supporting the familial imprinting hypothesis. We show that MHC-dependent familial imprinting potentially provides a more effective mechanism for avoiding kin matings and reducing inbreeding than self-inspection.     

The telocentric tandem repeat at the p-arm is not conserved in Mus musculus subspecies

Mouse chromosomes, with the exception of the Y chromosome, are telocentric. The telomere at the p-arm is separated from the centromere by the tL1 sequence and TLC tandem repeats. A previous report showed that the TLC array was also conserved in other strains of the subgenus Mus. These results suggest that the TLC arrays promote the stable evolutionary maintenance of a telocentric karyotype in the subgenus Mus. In this study, we investigated the degree of conservation of TLC arrays among a variety of wild-derived inbred strains, all of which are descendants of wild mice captured in several areas of the world. Genomic PCR analysis indicates that the sequential order of telomere-tL1 is highly conserved in all strains, whereas tL1-TLC is not. Next, Southern blot analysis of DNAs isolated from a panel of mouse subspecies showed both Mus musculus domesticus and Mus musculus castaneus subspecies possess TLC arrays. Unexpectedly, this repeat appears to be lost in almost all Mus musculus musculus and Mus musculus molossinus subspecies, which show a clear geographic divide. These results indicate that either other unknown sequences were replaced by the TLC repeat or almost all M. m. musculus and M. m. molossinus subspecies do not have any sequence between the telomere and minor satellites. Our observation suggests that the TLC array might be evolutionarily unstable and not essential for murine chromosomal conformation. This is the first example of the subspecies-specific large genome alterations in mice.     

Patterns of morphological evolution in the mandible of the house mouse Mus musculus (Rodentia: Muridae)

The worldwide distributed house mouse, Mus musculus, is subdivided into at least three lineages, Mus musculus musculus, Mus musculus domesticus, and Mus musculus castaneus. The subspecies occur parapatrically in a region considered to be the cradle of the species in Southern Asia (‘central region’), as well as in the rest of the world (‘peripheral region’). The morphological evolution of this species in a phylogeographical context is studied using a landmark‐based approach on mandible morphology of different populations of the three lineages. The morphological variation increases from central to peripheral regions at the population and subspecific levels, confirming a centrifugal sub‐speciation within this species. Furthermore, the outgroup comparison with sister species suggests that M. musculus musculus and populations of all subspecies inhabiting the Iranian plateau have retained a more ancestral mandible morphology, suggesting that this region may represent one of the relevant places of the origin of the species. Mus musculus castaneus, both from central and peripheral regions, is morphologically the most variable and divergent subspecies. Finally, the results obtained in the present study suggest that the independent evolution to commensalism in the three lineages is not accompanied by a convergence detectable on jaw morphology. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 635–647.