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Imminent predation risk affects mating behaviours in prey individuals in a multitude of ways that can theoretically impact the strength of sexual selection, as well as its operation on traits. However, empirical studies of the effects of imminent predation risk on sexual selection dynamics are still scarce. Here we explore how perceived predation affects: (1) the relationship between the opportunity for selection and the actual strength of selection on male traits; and (2) which traits contribute to male fitness and the shape of selection on these traits. We simulate two consecutive reproductive episodes, under control conditions and perceived predation risk using experimental populations of Trinidad guppies. The opportunity for selection is higher under predation risk compared to the control condition, but realised selection on traits remains unaffected. Pre- and postcopulatory traits follow complex patterns of nonlinear selection in both conditions. Differences in selection gradients deviate from predictions based on evolutionary and non-lethal effects of predation, the most notable being strong disruptive selection on courtship rate under predation risk. Our results demonstrate that sexual selection is sensitive to imminent predation risk perception and reinforce the notion that both trait-based and variance-based metrics should be employed for an informative quantification.

Life history theory suggests that aging is one of the costs of reproduction. Accordingly, a higher reproductive allocation is expected to increase the deterioration of both the somatic and the germinal lines through enhanced telomere attrition. In most species, males’ reproductive allocation mainly regards traits that increase mating and fertilization success, i.e. sexually selected traits. In the current study, we tested the hypothesis that a higher investment in sexually selected traits is associated with a reduced telomere length in the guppy (Poecilia reticulata), an ectotherm species characterized by strong pre- and postcopulatory sexual selection. We first measured telomere length in both the soma and the sperm over the course of guppy’s lifespan to see if there was any variation in telomere length associated with age. Secondly, we investigated whether a greater expression of pre- and postcopulatory sexually selected traits is linked to shorter telomere length in both the somatic and the sperm germinal lines, and in young and old males. We found that telomeres lengthened with age in the somatic tissue, but there was no age-dependent variation in telomere length in the sperm cells. Telomere length in guppies was significantly and negatively correlated with sperm production in both tissues and life stages considered in this study. Our findings indicate that telomere erosion in male guppies is more strongly associated with their reproductive investment (sperm production) rather than their age, suggesting a trade-off between reproduction and maintenance is occurring at each stage of males’ life in this species.

The environment that parents experience can influence their reproductive output and their offspring’s fitness via parental effects. Perceived predation risk can affect both parent and offspring phenotype, but it remains unclear to what extent offspring behavioral traits are affected when the mother is exposed to predation risk. This is particularly unclear in live-bearing species where maternal effects could occur during embryogenesis. Here, using a half-sib design to control for paternal effects, we experimentally exposed females of a live-bearing fish, the guppy (Poecilia reticulata), to visual predator cues and conspecific alarm cues during their gestation. Females exposed to predation risk cues increased their antipredator behaviors throughout the entire treatment. Offspring of mothers exposed to the predation stimuli exhibited more pronounced exploratory behavior, but did not show any significant differences in their schooling behavior, compared to controls. Thus, while maternally perceived risk affected offspring’s exploration during early stages of life, offspring’s schooling behavior could be influenced more by direct environmental experience rather than via maternal cues. Our results suggest a rather limited role in predator-induced maternal effects on the behavior of juvenile guppies.

Predation risk perception can alter mating behaviours in males and females, but the consequences for sexual selection remain underexplored. We have previously shown that in experimental populations of Trinidadian guppies Poecilia reticulata the opportunity for sexual selection (i.e. the variance in male reproductive fitness) was higher following exposure to a simulated risk of predation than in a no-risk condition. We build upon this result by exploring whether imminent predation risk affects: 1) the relationship between the opportunity for sexual selection and the actual strength of selection on male traits and 2) the traits contributing to male fitness, and he shape of selection on these traits. While predation risk increased the variance in male fitness, realised selection on traits remained unaffected. Pre- and postcopulatory traits follow complex patterns of nonlinear and correlational selection in both treatments. Differences in selection gradients deviate from predictions based on evolutionary responses to predation, the most notable being stronger selection on courtship rate under predation risk. Our results demonstrate that the operation of sexual selection can be altered by perception of an imminent predation risk and reinforce the notion that both trait-based and variance-based metrics should be employed for an informative quantification.

In many species, males can rapidly adjust their ejaculate performance in response to changing levels of sperm competition, an ability that is probably mediated by seminal fluid adaptive plasticity. In the black goby, Gobius niger , territorial males attach viscous ejaculate trails to the nest roof, from which sperm are slowly released into the water during the long-lasting spawning events. Sneaker males release their sperm in the vicinity of the nest, and territorial males try to keep them at a distance by patrolling their territory. We show here that territorial males' ejaculate trails released a higher proportion of their sperm in the presence of a single sneaker, but this proportion decreased when there were three sneakers, an effect that is most likely mediated by a change in the seminal fluid composition. Field observations showed that when multiple sneaking attempts occurred, territorial males spent more time outside the nest, suggesting that ejaculation rate and territory defence are traded-off. Altogether, these results suggest that the adjustment of sperm release from the ejaculate may be strategic, guaranteeing a more continuous concentration of the territorial male's sperm in the nest, although at a lower level, when he is engaged in prolonged territory defence outside the nest.

Although it is often expected that adverse environmental conditions depress the expression of condition-dependent sexually selected traits, the full consequences of environmental change for the action of sexual selection, in terms of the opportunity for total sexual selection and patterns of phenotypic selection, are unknown. Here we show that dietary stress in guppies, Poecilia reticulata, reduces the expression of several sexually selected traits and increases the opportunity for total sexual selection (standardized variance in reproductive success) in males. Furthermore, our results show that dietary stress modulates the relative importance of precopulatory (mating success) and postcopulatory (relative fertilization success) sexual selection, and that the form of multivariate sexual selection (linear vs. nonlinear) depends on dietary regime. Overall, our results are consistent with a pattern of heightened directional selection on condition-dependent sexually selected traits under environmental stress, and underscore the importance of sexual selection in shaping adaptation in a changing world., This work was funded by the University of Padova (Progetto CPDA120105 and Assegno di Ricerca BIRD 2017) to A.P. S.C. was supported by a Postgraduate Scholarship through The University of Padova, and F.G.G. by the Spanish Ministry of Economy (CGL2016-76173-P AEI/FEDER, UE). J.P.E. was supported by a sabbatical grant funded by the University of Western Australia.

Males of many species evolved the capability of adjusting their ejaculate phenotype in response to social cues to match the expected mating conditions. When females store sperm for a prolonged time, the expected fitness return of plastic adjustments of ejaculate phenotype may depend on the interval between mating and fertilization. Although prolonged female sperm storage (FSS) increases the opportunity for sperm competition, as a consequence of the longer temporal overlap of ejaculates from several males, it may also create variable selective forces on ejaculate phenotype, for example by exposing trade-offs between sperm velocity and sperm survival. We evaluated the relationship between the plasticity of ejaculate quality and FSS in the guppy, Poecilia reticulata, a polyandrous live-bearing fish in which females store sperm for several months and where stored sperm contribute significantly to a male's lifelong reproductive success. In this species, males respond to the perception of future mating opportunities by increasing the quantity (number) and quality (swimming velocity) of ready-to-use sperm (an anticipatory response called 'sperm priming'). Here we investigated (a) the effect of sperm priming on in vitro sperm viability at stripping and its temporal decline (as an estimate of sperm survival), and (b) the in vivo competitive fertilization success in relation to female sperm storage using artificial insemination. As expected, sperm-primed males produced more numerous and faster sperm, but with a reduced in vitro sperm viability at stripping and after 4 hr, compared with their counterparts. Artificial insemination revealed that the small (nonsignificant) advantage of primed sperm when fertilization immediately follows insemination is reversed when eggs are fertilized by female-stored sperm, weeks after insemination. By suggesting a plastic trade-off between sperm velocity and viability, these results demonstrate that prolonged female sperm storage generates divergent selection pressures on ejaculate phenotype.

The tight connection between immunity and reproduction has been studied for decades. However, basic knowledge at the molecular level of the effect of mating on immune function is still lacking in many taxa. Determining whether and how the immune system is engaged after mating is a crucial step in understanding post-mating mechanisms of reproduction and sexual selection. Here, we study the transcriptional changes in immunity-related genes caused by the ejaculate in the female reproductive tract using a model species for sexual selection studies, the guppy Poecilia reticulata. To study changes triggered by the ejaculate only, rather than caused by mating, we used artificial inseminations to transfer ejaculate into females. We then compared gene expression in the reproductive tract (gonoduct and ovary) of females artificially inseminated either with ejaculate or with a control solution, after 1 hr and after 6 hr. Overall, contact with ejaculate caused short-term changes in the expression of immune-related genes in the female reproductive tract, with a complex pattern of up- and down-regulation of immune-related pathways, but with clear indication of a marked down-regulation of the immune system shortly after ejaculate contact. This suggests a link between immune function and processes occurring between mating and fertilization in this species.

The role of non-gametic components of the ejaculate (seminal fluid) in fertility and sperm competitiveness is now well established. Surprisingly, however, we know far less about female reproductive fluid (FRF) in the context of sexual selection, and insights into male–FRF interactions in the context of sperm competition have only recently emerged. Despite this limited knowledge, evidence from taxonomically diverse species has revealed insights into the effects of FRF on sperm traits that have previously been implicated in studies of sperm competition. Specifically, through the differential effects of FRF on a range of sperm traits, including chemoattraction and alterations in sperm velocity, FRF has been shown to exert positive phenotypic effects on the sperm of males that are preferred as mating partners, or those from the most compatible or genetically diverse males. Despite these tantalizing insights into the putative sexually selected functions of FRF, we largely lack a mechanistic understanding of these processes. Taken together, the evidence presented here highlights the likely ubiquity of FRF-regulated biases in fertilization success across a diverse range of taxa, thus potentially elevating the importance of FRF to other non-gametic components that have so far been studied largely in males.This article is part of the theme issue ‘Fifty years of sperm competition'.

In polyandrous species males invest significant resources in producing large and high-quality ejaculates. As ejaculates are costly, males are expected to modulate their investment in response to social cues associated with the expected level of sperm competition or mating opportunity, to anticipate future mating conditions. Another consequence of ejaculate costs is that the increase in ejaculate production may be traded against traits linked to mate acquisition. In such cases, the effect of this anticipatory plasticity in ejaculate investment on a male's reproductive success will depend on the balance between postcopulatory benefits and precopulatory costs in the sociosexual context subsequently encountered. Here, we used the guppy, Poecilia reticulata, a live-bearing fish characterized by intense sperm competition, to investigate how mismatches between anticipatory phenotypes and the social conditions subsequently experienced affect male reproductive success. Male guppies kept in visual contact with females, a social stimulus predicting high mating opportunities, increase their sperm production compared to female-deprived males which foresee fewer opportunities to mate, but simultaneously reduce their courtship rate. Using a paired experimental design, we manipulated the male's perceived mating opportunities by housing them in the presence (FP) or in the absence (FA) of females. We then measured male mating and insemination success under two conditions: (1) a female-biased sex ratio, matching expected FP cues; and (2) a male-biased sex ratio, matching expected FA cues. Under a female-biased sex ratio, mating and insemination success were not affected by previous exposure to females, although FP males experienced a lower risk of sperm depletion after several copulations. However, FP males showed a small, but significant, reduction in their mating success under a male-biased sex ratio, when directly competing with an FA male. Males showed significant repeatability in mating and insemination success within a sex ratio context, suggesting that the costs of mismatching anticipatory ejaculate responses are probably low in this species.

This study considered possible sexual dimorphism in the relative lengths of the second, third and fourth digits (digit ratio), in calves. Furthermore, a different length of the bone structures of the third (3D) and of the fourth (4D) digits has been examined as an evolutionary adaptation to locomotion on soft ground. The length of the digital bones of the right fore-limb of 33 females and 15 male calves was measured in vivo using a portable X-ray machine. The vestigial structure of the second digit (2D), and 3D and 4D, from metacarpus to the third phalanx were considered in a mixed model, as well as some ratios between 2D and different parts of 3D or 4D (2D:3D and 2D:4D). A covariate for the mean finger length was considered for digit ratios to control for possible biases due to shape allometry. Shorter first phalanx and trotter were found in 3D than in 4D, and the reverse for the third phalanx. The 2D was significantly shorter in females, as well as the second phalanges of 3D and 4D. Significant sex differences in 2D:3D and 2D:4D were found for some digit parts of 3D and 4D and for the first phalanges of 3D:4D. These ratios were always shorter in females, in contrast to that found in most mammals. The asymmetry between 3D and 4D could mean a functional adaptation for locomotion. Sex differences in 2D:4D and 3D:4D were found, but with a reverse pattern than in most mammal species (malesfemales rather than malesfemales). In this regard digit ratio in calves was similar to that of Old World monkeys. This study is the first investigation of digit ratio in Ungulates, whose limbs differ from the limbs of most mammals, maintaining five digits. The reverse pattern of sex differences (digit ratios: malesfemales) could be due to the peculiar nature of the vestigial dewclaw of 2D and to the hormone patterns acting on this digit during development, but further research is required around this topic.

Sperm competition is taxonomically widespread in animals and is usually associated with large sperm production, being the number of sperm in the competing pool the prime predictor of fertilization success. Despite the strong postcopulatory selection acting directionally on sperm production, its genetic variance is often very high. This can be explained by trade-offs between sperm production and traits associated with mate acquisition or survival, that may contribute to generate an overall stabilizing selection. To investigate this hypothesis, we first artificially selected male guppies (Poecilia reticulata) for high and low sperm production for three generations, while simultaneously removing sexual selection. Then, we interrupted artificial selection and restored sexual selection. Sperm production responded to divergent selection in one generation, and when we restored sexual selection, both high and low lines converged back to the mean sperm production of the original population within two generations, indicating that sperm number is subject to strong stabilizing total sexual selection (i.e., selection acting simultaneously on all traits associated with reproductive success). We discuss the possible mechanisms responsible for the maintenance of high genetic variability in sperm production despite strong selection acting on it.

Producing sperm is costly and males have been selected to strategically adjust their sperm production and/or expenditure according to the fitness return associated with a specific mating. For example, males respond to fluctuations in the mating opportunities by adjusting the number of “ready” sperm. This phenomenon is known as “sperm priming” and is interpreted as a strategy to economize the investment in sperm. The cost and benefits of the sperm priming response, however, are expected to depend on a male’s baseline sperm production (BSP) in the absence of females, because of the different risk of sperm depletion and the nonlinearly increasing costs of sperm production. We tested this prediction in 2 replicated lines of male guppies Poecilia reticulata that were artificially selected for high and low BSP. BSP has a large genetic variance and a high sire heritability in guppies, and males respond to the perceived mating opportunities by increasing the number of “ready” sperm. We investigated whether males with a different BSP differed in their sperm priming response. We found that when the perceived mating opportunities increased, males from low-sperm lines had a stronger sperm priming response than those from high-sperm lines. This result suggests that adaptive plasticity in sperm priming has the potential to evolve in response to different levels of BSP. The comparison between guppy populations with different levels of sperm production would allow to test whether the pattern reported here is also observed at the interpopulation level.

Identifying mechanisms of reproductive isolation is key to understanding speciation. Among the putative mechanisms underlying reproductive isolation, sperm-female interactions (postmating-prezygotic barriers) are arguably the hardest to identify, not least because these are likely to operate at the cellular or molecular level. Yet sperm-female interactions offer great potential to prevent the transfer of genetic information between different populations at the initial stages of speciation. Here we provide a preliminary test for the presence of a putative postmating-prezygotic barrier operating between three populations of Trinidadian guppies (Poecilia reticulata), an internally fertilizing fish that inhabits streams with different levels of connectivity across Trinidad. We experimentally evaluate the effect of female ovarian fluid on sperm velocity (a predictor of competitive fertilization success) according to whether males and females were from the same (native) or different (foreign) populations. Our results reveal the potential for ovarian fluid to act as a postmating-prezygotic barrier between two populations from different drainages, but also that the strength of this barrier is different among populations. This result may explain the previous finding that, in some populations, sperm from native males have precedence over foreign sperm, which could eventually lead to reproductive isolation between these populations. This article is protected by copyright. All rights reserved.

Many fish species are social, and individuals spend most of their lives in shoals, but sociability can vary greatly between species, populations and individuals. Sociability has been largely studied by measuring the time spent by a focal fish in proximity to one or more conspecifics. To control for the behaviour of the stimulus fish, the conspecifics have often been substituted by a mirror, on the assumption that the subject perceives its mirror image as a conspecific. The validity of the mirror test has recently been questioned, at both the behavioural and the molecular level, because of the discrepancy in fish responses when exposed to a mirror image and to a live conspecific. In this study, we compared the sociability scores of a social fish, the guppy, Poecilia reticulata, obtained using live fish or a mirror as a stimulus, to assess the validity of the mirror test. We found that the sociability score assessed using the standard mirror test was not significantly correlated with the sociability assessed using live stimuli. Nevertheless, we observed a positive correlation between the scores of the two tests when the mirror test was performed in a more naturalistic context, controlling for the minimum distance between the stimulus fish and the mirror. Our findings provide evidence of the reliability of the mirror test as a measure of sociability compared to the test using live conspecifics when certain requirements are satisfied.

In species where females mate with more than one male during the same reproductive event, males typically increase the number of sperm produced to boost their fertilization share. Sperm is not limitless, however, and theory predicts that their production will come at the cost of other fitness-related traits, such as body growth or immunocompetence, although these evolutionary trade-offs are notoriously difficult to highlight. To this end, we combined artificial selection for sperm production with a transcriptome analysis using Poecilia reticulata, a fish characterized by intense sperm competition in which the number of sperm transferred during mating is the most important predictor of fertilization success, yet sperm production is highly variable among males. We compared the brain and testes transcriptome in male guppies of lines artificially selected for high and low sperm production by identifying pivotal differentially expressed gene sets that may regulate spermatogenesis and immune function in this species. Despite the small differences in single genes' expression, gene set enrichment analysis showed coordinated gene expression differences associated with several pathways differentially regulated in the two selection lines. High sperm production males showed an upregulation of pathways related to immunosuppression and development of spermatozoa indicating a possible immunological cost of sperm production.

Many animals, from humans to invertebrates, exhibit sexual preference for unfamiliar partners. This may reduce the risk of inbreeding and increase offspring heterozygosity. An alternative less tested hypothesis is that selection for neophilia in other contexts (e.g. exploration of unfamiliar environments) may promote mate preference for unfamiliar partners. We tested whether exploratory tendency covaries with female preference for unfamiliar mates in the guppy, Poecilia reticulata, a species in which exploratory behaviour show significant phenotypic and genetic variation. We measured the exploratory behaviour of females using an unfamiliar environment test. The same females were also tested for preference for an unfamiliar male over a male with which they had spent the previous 24 h. We found that a female's tendency to explore an unfamiliar environment was positively correlated with the strength of her preference for unfamiliar males. Our results support the hypothesis that exploration might play a role in the evolution and maintenance of preference for unfamiliar males via functional pleiotropy.

Female sperm storage (FSS) is taxonomically widespread and often associated with intense sperm competition, yet its consequences on postcopulatory sexual selection (PCSS) are poorly known. Theory predicts that FSS will reduce the strength of PCSS, because sperm characteristics favored before and after FSS may be traded-off, and opportunities for nondirectional PCSS should increase. We explored these questions in the guppy (Poecilia reticulata), by allowing females to mate multiply and by comparing the paternity pattern in two successive broods. Contrary to predictions, the variance in male fertilization success increased after FSS, driven by a change in male paternity share across broods. This change was positively associated with sperm velocity (measured before FSS) but not with the duration of FSS, indirectly suggesting that faster sperm were better in entering female storage organs, rather than in persisting within them. Other male traits, such as male size and orange color, heterozygosity, and relatedness to the female, did not influence paternity after FSS. These results indicate that processes associated with FSS tend to reinforce the strength of PCSS in guppies, rather than weaken it. Further work is necessary to test whether this pattern changes in case of more prolonged FSS.

Fifty years of research on sperm competition has led to a very good understanding of the interspecific variation in sperm production traits. The reasons why this variation is often very large within populations have been less investigated. We suggest that the interaction between fluctuating environmental conditions and polyandry is a key phenomenon explaining such variation. We focus here on imminent predation risk (IPR). IPR impacts significantly several aspects of prey behaviour and reproduction, and it is expected to influence the operation of sexual selection before and after mating. We estimated the effect of IPR on the male opportunity for pre- and postcopulatory sexual selection in guppies ( Poecilia reticulata ), a livebearing fish where females prefer colourful males and mate multiply. We used a repeated-measures design, in which males were allowed to mate with different females either under IPR or in a predator-free condition. We found that IPR increased the total opportunity for sexual selection and reduced the relative contribution of postcopulatory sexual selection to male reproductive success. IPR is inherently variable and our results suggest that interspecific reproductive interference by predators may contribute towards maintaining the variation in sperm production within populations. This article is part of the theme issue ‘Fifty years of sperm competition'.

According to theory, in species in which male variance in reproductive success exceeds that of the females, sons are more costly to produce; females mated with high quality males or those in better condition should produce more sons. In monogamous species, however, the variance in the reproductive success of the two sexes is often similar and mate choice is often mutual, making predictions regarding sex allocation more difficult. In the rock sparrow Petronia petronia, both males and females have a sexually selected yellow patch on the breast, whose size correlates with individual body condition. We investigated whether the brood sex ratio co‐varies with the size of the yellow patch of the father and the mother in a sample of 173 broods (818 chicks) over 8 breeding seasons. While the size of the yellow patch of the mother and the father did not predict per se a deviation from the expected 1:1 sex ratio, brood sex ratios were predicted by the interaction of male and female yellow patch size. This result is surprising, as the ornament is sexually selected by both males and females as an indicator of quality in both sexes and should therefore be inherited by all offspring irrespective of their sex. It indirectly suggests that other sex‐specific traits associated with patch size (e.g. polygyny in males and fecundity in females) may explain the sex allocation bias observed in rock sparrows. Thus, female individual quality alone, as expressed through the size of the yellow patch, was not associated with the biases in sex ratios reported in this study. Our results rather suggest that sex allocation occurs in response to male attractiveness in interaction with female attractiveness. In other words, females tend to preferentially allocate towards the sex of the parent with more developed ornament within the pair., AC was funded by a Juan de la Cierva‐formación postdoctoral grant (FJCI‐2015‐23536; Ministerio de Economía, Industria y Competitividad, MINECO, Spanish Government).

Being flexible in reproductive decisions is highly beneficial in variable environments, but flexibility may also entail costs. Male guppies modify their sperm production and sexual behavior when mating opportunities vary. We investigated lifetime effects of these responses. We show that repeatedly adjusting reproductive strategies has little cost for male guppies, as it does not compromise their condition, their lifespan, or the expression of sexual traits.

In several species, males increase their mating and sperm investment in the presence of unfamiliar females, the so-called Coolidge effect. Such an elevated reproductive effort is expected to be associated with a decreased investment in other costly traits, such as somatic growth and maintenance. How precopulatory, postcopulatory, and somatic investments interact one with each other, however, has been rarely evaluated simultaneously. We used the guppy (Poecilia reticulata), a polyandrous livebearing fish with alternative male mating tactics, to compare these 3 investments between males whose encounter rate with unfamiliar females was experimentally maintained at either high (high mate encounter rate [HER]) or low rate (low mate encounter rate [LER]) for 4 months. At the end of this period, HER males showed an increased sperm production as compared with their LER counterparts. This increment was accompanied with a reduction in the time spent following the female and by a shift from costly courtship displays to gonopodial thrusting, a less expensive coercive mating tactic. This effect may indicate a trade-off between these 2 components of male reproductive investment or may reflect a change in male optimal reproductive strategy associated with different mate encounter rate. In contrast, body growth was positively correlated with the increase in sperm production, and did not differ between experimental groups. Collectively, these results suggest that males did not vary their overall reproductive investment in response to their encounter rate with unfamiliar females, but differed significantly in the amount of resources available individually.

Several factors are involved in determining the outcome of sperm competition. In addition to sperm number, sperm quality and male phenotype, insemination order is often associated with skewed paternity share. Patterns of sperm precedence can be produced by the mechanics of sperm storage and fertilization, or by active processes under male or female control. However, as males and females always interact during copulation, it is difficult to identify the mechanism responsible. The Trinidadian guppy, Poecilia reticulata, is a polyandric species characterized by last-male sperm precedence in natural matings. During such matings, females allow attractive males to inseminate more sperm by controlling copulation duration. We used artificial insemination to clarify the extent to which female control of sperm transfer influences the observed pattern of sperm precedence in this species. This technique allowed us to experimentally manipulate the number of sperm transferred and the timing of insemination. We found a significant first-male fertilization advantage. This advantage, however, declined as the time between insemination and parturition increased. Presumably, the anatomy and the physiology of the female genital tract favour egg fertilization by the first ejaculate inseminated, whereas sperm mixing is likely to be responsible for the reduction in first-male advantage associated with longer insemination?parturition intervals. Our results suggest that the last-male precedence detected after two consecutive natural matings is caused by cryptic female preference for attractive males associated with a female trading-up strategy (i.e. the second male is more frequently more attractive than the first male), rather than by insemination order per se. As the pattern of sperm precedence has important consequences for male reproductive strategies (for example mate guarding and male mate choice copying), unravelling its dynamic represents an important contribution to understanding the sexual behaviour of this model species. Fil: Magris, Martina. Università di Padova; Italia Fil: Cardozo Milanesio, Gabriela Alejandra. Università di Padova; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Santi, Francesco. Royal Holloway University Of London; . Università di Padova; Italia Fil: Devigili, Alessandro. Stockholms Universitet; Suecia. Università di Padova; Italia Fil: Pilastro, Andrea. Università di Padova; Italia

The function and maintenance of the immune system impose costs that can take the form of energy consumption and/or physiological side effects. These, in turn, can affect the expression of several traits, including traits closely related to fitness. The interaction of the immune system with other life-history traits (e.g. growth and survival) and with precopulatory sexually selected traits (e.g. male ornaments) has been widely studied, whereas little is known about its interplay with postcopulatory sexually selected traits, such as sperm characteristics. We studied the effects of an immune challenge on the expression of precopulatory and postcopulatory traits in the guppy, Poecilia reticulata. We injected two groups of males with a lipopolysaccharide (LPS) solution or a saline solution (control) and compared the effects of the immune system activation on traits’ expression. The results showed a specific negative effect of the LPS injection on sperm traits, whereas precopulatory ornaments were lowered merel...

Parents are expected to strategically partition their limited resources among the current and future progeny in order to maximize their fitness. Since an equal investment in offspring of different reproductive value entails fitness costs, natural selection has promoted the evolution of reliable signals of offspring condition, allowing parents to invest in their progeny accordingly. In birds, mouth and skin colouration are hypothesized to be honest signals of offspring condition, because they are affected by diverse factors. Among these, ectoparasite load has been shown to affect nestling condition, but its influence on visual components of begging is poorly known. We experimentally investigated whether nest ectoparasite removal affected flange and skin reflectance of first- and second-brood European starling (Sturnus vulgaris) nestlings. We also tested whether high reflectance in visual components of begging mirrored other aspects of nestling condition, such as morphological (high stature) and physiological (high haematocrit and immune response) traits, and pre-fledging mortality. Ectoparasite removal did not affect visual components of begging in first or second broods. However, larger nestlings from both broods displayed higher ultraviolet (UV) reflectance of skin and higher flange reflectance in the visible-wavelength region (but lower flange UV reflectance) than their siblings. A higher skin UV reflectance relative to siblings also positively predicted pre-fledging survival within-brood. Therefore, visual components of begging did not mirror ectoparasite infestation in this species. However, they provide parents with reliable information about individual quality, thus affecting resource allocation and promoting survival of the most valuable offspring during the entire breeding season. In species with parental care, natural selection favours the evolution of reliable signals of offspring quality, thus allowing parents to invest in their progeny accordingly. We experimentally show that skin and beak flange colour does not mirror ectoparasite infection in European starling nestlings. However, begging visual signals predict nestling body size and survival until fledging. A seasonal variation in the strength of the association between begging visual signals and nestling condition is also shown, indicating that change ecological conditions can affect the association between different condition-dependent traits.

Traits associated with mating and fertilization success are expected to senesce with age, but limited information is available on their relative rates of senescence. In polyandrous species, male reproductive fitness depends on both mating and fertilization success. Because successful mating is a prerequisite for post-copulatory sexual selection, ejaculate traits are expected to senesce faster than pre-copulatory traits, as pre-copulatory sexual selection is often deemed to be stronger than post-copulatory sexual selection. This pattern has generally been found in the few empirical studies conducted so far. We tested this prediction in the guppy ( Poecilia reticulata ), a livebearing fish characterized by intense sperm competition, by comparing the expression of male sexual traits at two ages (four and nine months). Contrary to prediction, we found that post-copulatory traits senesced at a significantly slower rate than pre-copulatory traits. We also looked at whether early investment in those sexual traits affects longevity, and the interaction between sperm age (duration of sperm storage inside the male) and male age. Our results suggest that the relative senescence rate of pre- and post-copulatory sexual traits may vary among species with different mating systems and ecology.

Sexual selection can operate both before and after mating. For males, selection will favor a suite of traits involved in mate acquisition (precopulatory) and in the competition among ejaculates to fertilize eggs (postcopulatory). Sperm competition theory predicts a trade-off between investment in pre- and postcopulatory traits, thus generating negative correlations between traits that function during both episodes of selection. However, such trade-offs can be difficult to detect because they are typically sensitive to the pool of resources available for allocation to competing functions. Although theory predicts that trade-offs will be more apparent when resources are limiting, there have been few attempts to test this prediction. In our study, we used the freshwater fish, Poecilia reticulata, to examine how both pre- and postcopulatory sexually selected traits simultaneously respond to manipulations of diet quantity by comparing behavioral, ornamental, and ejaculate traits between males assigned at random to either a ad libitum or a restricted food diet. We also explore how the relationships between traits that function during pre- and postcopulatory episodes of selection are affected by these treatments. Our results reveal that diet manipulations influenced the expression of both precopulatory (sexual behavior and ornamentation) and postcopulatory sexually selected traits (sperm viability), reinforcing the importance of resource acquisition in sexual selection. However, our data do not support the hypothesis that males trade off their allocation toward these pre- and postcopulatory sexually selected traits. Instead, changes in the expression of pre- and postcopulatory sexually selected traits appear to occur independently. Key wor ds: dietar y manipulation, Poecilia reticulata, postcopulatory sexual selection, precopulatory sexual selection, trade-offs. [Behav Ecol]

While it is well established that females prefer to mate with well‐ornamented males, the influence of perceptive and cognitive processes on the expression of female mate choice is still poorly known. It has been suggested that the female perception of a male's attractiveness is not absolute, but depends on the other males with which he is compared that have been previously encountered (comparative evaluation). We investigated whether mate preference in bearded reedlings (Panurus biarmicus) is dependent on or independent of social context in relation to two different traits: beard and tail lengths. Each female had a choice between two to three males with different modifications of beard and tail. For each female, three different experiments were conducted (one binary and two trinary tests). We found that when females are presented with options that vary antagonistically with respect to two ornaments (binary test), some individuals prefer one trait while others the other trait. This indicates that in our bearded reedlings population exists a mate preference polymorphisms. Moreover, we found that the presence of a third stimulus, irrespective of the initial preference, reduced the strength of the initial preference – what we can call a “preference dilution effect.” Our results suggest that the female's choice may be constrained by her cognitive abilities when she is simultaneously presented with several options varying for two uncorrelated traits.

Female sperm storage (FSS) is taxonomically widespread and often associated with intense sperm competition, yet its consequences on postcopulatory sexual selection (PCSS) are poorly known. Theory predicts that FSS will reduce the strength of PCSS, because sperm characteristics favored before and after FSS may be traded-off, and opportunities for nondirectional PCSS should increase. We explored these questions in the guppy (Poecilia reticulata), by allowing females to mate multiply and by comparing the paternity pattern in two successive broods. Contrary to predictions, the variance in male fertilization success increased after FSS, driven by a change in male paternity share across broods. This change was positively associated with sperm velocity (measured before FSS) but not with the duration of FSS, indirectly suggesting that faster sperm were better in entering female storage organs, rather than in persisting within them. Other male traits, such as male size and orange color, heterozygosity, and relatedness to the female, did not influence paternity after FSS. These results indicate that processes associated with FSS tend to reinforce the strength of PCSS in guppies, rather than weaken it. Further work is necessary to test whether this pattern changes in case of more prolonged FSS.

Males of several species have been shown to alter their mate preference in the presence of an eavesdropping rival. This evasive tactic has been interpreted as an attempt by the courting male to drive the attention of the rival away from the preferred female. The fitness return of this deceptive strategy will depend on the costs of cheating for the actor (the displayer) and the benefits for the rival (the bystander) of copying the choice of the displayer. We developed a two-person nonzero sum game between two males that compete for mating with one of two receptive females. Males could assess female quality with a varying level of uncertainty, which was modelled using a Bayesian statistical decision theory approach. We explored the actor and bystander payoffs under different levels of uncertainty in mate assessment and difference in quality between females. We found that when being eavesdropped on is costly (i.e. when females differ largely in quality), males are expected to cheat to reduce the amount of public information that is available to the unintended audience. However, under these circumstances, the value of the public information is low and the bystander is not expected to copy the choice of the actor. Our model suggests that deceptive male choice may evolve only under relatively restricted conditions and suggest that other explanations, such as, for example, a reduction in the risk of precopulatory male–male competition may be more likely. Future theoretical and empirical work will be necessary to test alternative interpretations of the audience effects in male mate choice.

Theory predicts that in polyandrous species males that invest more in traits enhancing postcopulatory success should have less energy to invest in mating acquisition, leading to trade-offs between pre- and post-copulatory episodes of sexual selection. Although such trade-offs are well studied, the potential constraints that postcopulatory sexual selection imposes on alternative mating tactics are rarely considered. In guppies, Poecilia reticulata , individual males can obtain matings by performing energetically costly courtship displays or by using less costly forced copulations. In this study we manipulated a component of social environment (namely, the presence of females) to experimentally elevate sperm production in males. We found that male guppies rapidly compensated by reducing their reliance on courtship in favour of forced matings. As both tactics differ in mating and fertilization success, the consequences of this trade-off may have important ramifications for male reproductive fitness.

In seasonally fluctuating environments, timing of reproduction is a crucial determinant of fitness. Studies of birds show that late breeding attempts generally result in offspring of lower reproductive value, with lower recruitment and long-term survival prospects. Several proximate mechanisms, including a seasonal decline of immune system functioning, may lead to a seasonal decline of offspring fitness. We investigated seasonal variation in offspring quality by subjecting first- and second-brood chicks of a sexually size dimorphic species, the European starling Sturnus vulgaris, to an immune challenge with a bacterial endotoxin (LPS), and evaluated their growth and physiological response in terms of total plasma antioxidant capacity (TAC), concentration of reactive oxygen metabolites and hematocrit. LPS challenge did not affect chick growth or oxidative status. However, hematocrit of second-brood chicks was higher in LPS chicks compared to controls. Body mass halfway through the rearing period (days 8–9 post-hatching), TAC and hematocrit were lower among second- vs. first-brood chicks. Interestingly, sexual dimorphism in body mass at days 8–9 post-hatching markedly differed between broods, first-brood males being 4.7% and second-brood males 22.7% heavier than their sisters, respectively. Pre-fledging mortality occurred among second-brood chicks only and was strongly female-biased. Our findings suggest that starling chicks, even if in poor conditions, are little affected by a bacterial challenge, at least in the short-term. Moreover, our study indicates that sex differences in body size, possibly mediated by sex-specific maternal investment in egg size, may heavily impact on pre-fledging survival in a different way in the course of the breeding season, resulting in sex-specific seasonal decline of offspring fitness. Finally, we suggest that levels of circulating antioxidants should be regarded among the proximate causes of the association between timing of fledging and long-term survival in avian species.

Sexual harassment is a common outcome of sexual conflict over mating rate. A large number of studies have identified several direct costs to females of sexual harassment including energy expenditure and reduced foraging ability. However, the fitness consequences of sexual harassment for descendants have rarely been investigated. Here, we manipulated the level of sexual harassment and mating rate in two groups of female guppies, Poecilia reticulata, a live-bearing fish in which sexual conflict over mating rate is particularly pronounced. Each female was allowed to interact with three males for one day (low sexual harassment, LSH) or for eight days (high sexual harassment, HSH) during each breeding cycle throughout their life. Female lifetime fecundity did not differ between the groups, but we found a strong effect on offspring fitness. HSH females produced (1) daughters with smaller bodies and (2) sons with shorter gonopodia, which were less attractive to females and less successful in coercive matings than their LSH counterparts. Although these results may be influenced by the indirect effects of sex ratio differences between treatments, they suggest that sexual harassment and elevated mating rate can have negative cross-generational fitness effects and more profound evolutionary consequences than currently thought.

As inbreeding is costly, it has been suggested that polyandry may evolve as a means to reduce the negative fitness consequences of mating with genetically related males. While several studies provide support for this hypothesis, evidence of pure post-copulatory mechanisms capable of biasing paternity towards genetically unrelated males is still lacking; yet these are necessary to support inbreeding avoidance models of polyandry evolution. Here we showed, by artificially inseminating a group of female guppies with an equal number of sperm from related (full-sib) and unrelated males, that sperm competition success of the former was 10 per cent lower, on average, than that of the unrelated male. The paternity bias towards unrelated males was not due to differential embryo survival, as the size of the brood produced by control females, which were artificially inseminated with the sperm of a single male, was not influenced by their relatedness with the male. Finally, we collected ovarian fluid (OF) from virgin females. Using computer-assisted sperm analysis, we found that sperm velocity, a predictor of sperm competition success in the guppy, was significantly lower when measured in a solution containing the OF from a sister as compared with that from an unrelated female. Our results suggest that sperm–OF interaction mediates sperm competition bias towards unrelated mates and highlight the role of post-copulatory mechanisms in reducing the cost of mating with relatives in polyandrous females.

Male and female gametes are often embedded in fluids that are produced by gonads and other reproductive tissues. Female reproductive fluids, usually called ovarian fluid (OF), which often constitute a relevant volumetric component of the egg mass, are rich in ions, sugars and proteins, and are involved in several functions, from protecting gametes to facilitating fertilization, and often act as mediators of post-mating sexual selection. Despite their applied and evolutionary importance, we know virtually nothing about the costs of female reproductive fluid production. We investigated the effect of nutritional condition on OF quality by experimentally manipulating the diet of two groups of female guppies (Poecilia reticulata) which were maintained for 20 days either on a restricted diet or had ad libitum access to food. In this species, OF enhances sperm swimming longevity and velocity (a predictor of sperm competition success) and mediates post-copulatory inbreeding avoidance. We found that sperm velocity was significantly lower in the OF of diet-restricted females, indicating that OF quality is dependent on female nutritional condition. Our results demonstrate that OF represents a non-trivial component of female reproductive investment and provides a tool to investigate which OF constituents are involved in modulating OF–sperm interactions and fertilization. Fil: Cardozo Milanesio, Gabriela Alejandra. Universita Di Padova. Dipartimento Di Biología; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Pilastro, Andrea. Universita Di Padova. Dipartimento Di Biología; Italia

Deleterious mutations can accumulate in the germline with age, decreasing the genetic quality of sperm and imposing a cost on female fitness. If these mutations also affect sperm competition ability or sperm production, then females will benefit from polyandry as it incites sperm competition and, consequently, minimizes the mutational load in the offspring. We tested this hypothesis in the guppy (Poecilia reticulata), a species characterized by polyandry and intense sperm competition, by investigating whether age affects post-copulatory male traits and sperm competition success. Females did not discriminate between old and young males in a mate choice experiment. While old males produced longer and slower sperm with larger reserves of strippable sperm, compared to young males, artificial insemination did not reveal any effect of age on sperm competition success. Altogether, these results do not support the hypothesis that polyandry evolved in response to costs associated with mating with old males in the guppy.

Growing evidence suggests that structural feather colours honestly reflect individual quality or body condition but, contrary to pigment-based colours, it is not clear what mechanism links condition to reflectance in structural feather colours. We experimentally accelerated the moult speed of a group of blue tits (Cyanistes caeruleus) by exposing them to a rapidly decreasing photoperiod and compared the spectral characteristics of their structural feather colours with those of control birds. Blue tits were sexually dimorphic on the UV ⁄blue crown and on the white cheek feathers. Moult speed, however, dramatically reduced brightness and the saturation only on the UV ⁄blue crown feathers, whereas structural white on the cheek feathers was basically unaffected by moult speed. Given that the time available for moulting is usually confined to the period between the end of the breeding season and migration or wintering, UV ⁄blue colours, but not structural white, may convey long-term information about an individual’s performance during the previous breeding season. The trade-off between fast moulting and structural colour expression may represent a previously unrecognized selective advantage for early-breeding birds.