Geographic variation in allele frequency of the gamete recognition protein M7 lysin throughout a mosaic blue mussel hybrid zone

Divergence at gamete recognition loci is hypothesized to result in speciation in broadcast spawning invertebrates. Many gamete recognition loci evolve quickly and show patterns of positive selection, yet the advantage of divergence is rarely known. M7 lysin is a sperm protein in the Mytilus edulis species complex that shows evidence of adaptive evolution. This locus is polymorphic with two distinct clades within Mytilus galloprovincialis, one of which, the D clade, shows the strongest signal of positive selection. We tested whether the geographic patterns in allele frequency were consistent with the hypothesis that positive selection on D clade alleles (G _D ) was due to reinforcement. Populations of M. edulis showed little evidence of introgression of G _D alleles, but there was no consistent evidence of reproductive character displacement. The lack of consistent patterns expected of reinforcement suggests that another mechanism is likely responsible for the adaptive divergence of M7 lysin.     

Among- and within-population variation in sperm quality in the simultaneously hermaphroditic land snail Arianta arbustorum

Sperm competition models on the evolution of sperm size assume associations with another sperm quality trait, sperm longevity. Sperm length can also provide an indication of possible mechanisms affecting motility and thus fertilization success. Despite their importance, however, detailed mechanisms of sperm competition at the gamete level are poorly understood. In simultaneously hermaphroditic land snails, sperm traits and cryptic female choice are assumed to be crucial in determining fertilization success. We examined the variation in sperm length and number among individuals from four natural populations of the land snail Arianta arbustorum, a species with multiple mating and long-term sperm storage. We also assessed variation in velocity, motility and longevity of sperm in snails from two of the four populations. Independent of shell size, sperm length differed among populations and, to a minor extent, even among individuals within populations. Mean sperm length of a snail was not correlated with the number of sperm delivered in a spermatophore. The mean sperm velocity (=VCL) did not differ between snails from two populations. However, VCL varied among snails. Percentage motility and longevity of sperm differed between snails from the two populations. No correlations were found between length, velocity, percentage motility and longevity of sperm. To conclude, individual snails differed in sperm quality, and this variation may partly explain the differential fertilization success between A. arbustorum snails. Moreover, our findings did not support the positive association between sperm length and longevity assumed by sperm competition models for internally fertilizing species.     

The relationship between presumed gamete contribution and parental investment in a communally breeding bird

Summary Measures of relative gamete contribution were related to degree of parental investment for both males and females from six groups of communally breeding pukeko (Porphyrio porphyrio). If there is a fitness cost to participation in parental duties, then for males parental investment should relate to degree of confidence of paternity while for females investment should relate to the number of eggs laid. Data were analysed in two ways: (1) by looking at individuals of the same sex within the same group to see if males that had a greater probability of paternity and females that had greater number of eggs in a nest subsequently invested relatively more in parental duties and (2) by pooling the data from the six groups and determining if there was a positive correlation between measures of presumed gamete contribution and amount of parental investment. The results from both types of analyses are variable and give no clear support for a positive relationship between presumed gamete contribution and parental investment even though a possible fitness cost is suggested. Factors which might influence investment are discussed.     

Rapid sperm evolution in the bluethroat (Luscinia svecica) subspecies complex

Spermatozoa are among the most variable animal cell types, and much research is currently directed towards explaining inter- and intraspecific variation in sperm form and function. Recent comparative studies in passerine birds have found associations between the level of sperm competition and both sperm length and sperm velocity. In species with sperm competition, postcopulatory sexual selection may shape the morphology of sperm as adaptations to the female environment. The speed of evolutionary change in sperm morphology at the species level is largely unknown. In this study, we analysed variation in sperm morphology among morphologically distinct and geographically isolated bluethroat subspecies in Europe. Consistent with previous studies, our analyses of mtDNA and nuclear introns suggest recent divergence and lack of lineage sorting among the subspecies. We found significant divergence in total sperm length and in the length of some sperm components (i.e. head and midpiece). There was a significantly positive relationship between pairwise divergences in sperm morphology and mitochondrial DNA, suggesting a role for genetic drift in sperm divergence. The magnitude of sperm length divergence was considerably higher than that in other geographically structured passerines, and even higher than that observed between several pairs of sister species. We hypothesize that the rapid sperm evolution in bluethroats is driven by sperm competition, and that strong postcopulatory sexual selection on sperm traits can lead to rapid speciation through reproductive incompatibilities.     

Testes mass in megachiropteran bats varies in accordance with sperm competition theory

Sperm competition is a widespread phenomenon influencing a range of characters, including investment in gonadal tissue. Conspecific proximity is one factor which can influence the risk of sperm competition and hence testicular investment, and decreased confidence of paternity may be one cost of group living. Aspects of female biology may also influence spermatogenic investment and sperm morphology. This study examines the associations between relative testes mass and roost-group size across 17 species of Megachiroptera. Associations between breeding season duration and investment in spermatogenesis are also examined, as are associations between female reproductive tract dimensions and testes mass and dimensions of spermatozoa across all bats. Relative testes mass was significantly positively associated with roost-group size at a species level and after appropriate phylogenetic control (pairwise comparisons and comparison of independent contrasts). There were no significant relationships between breeding season duration and relative testes mass. Across all bats, neither testes mass nor sperm length were significantly related to dimensions of the female tract. The results are discussed in the context of sperm competition. Received: 7 January 1998 / Accepted after revision: 8 August 1998     

Sperm traits in the quacking frog (<Emphasis Type="Italic">Crinia georgiana</Emphasis>), a species with plastic alternative mating tactics

In species where males use alternative reproductive tactics and male phenotypes are confronted with different risks of sperm competition, theory predicts that between-male-type differences in sperm expenditure may evolve. In the frog Crinia georgiana big males can monopolize females, whereas small males often engage in polyandrous matings. Consequently, big males may experience a lower risk of sperm competition than do small males. We tested if the predictions from theoretical models can be applied to the mating system of C. georgiana. Our results showed that small males do not have larger testes relative to their body size compared to their larger counterparts and that the efficiency with which sperm number, size, motility, and longevity are produced by the testes does not differ between small and large males in the predicted way. These results are not in alignment with predictions from a loaded raffle model of sperm competition on sperm expenditure in males with alternative phenotypes. The plasticity in mating tactics used by C. georgiana males and a high intraseasonal variation in male densities may have prevented the evolution of enhanced sperm performance in smaller males. A fair raffle in the sperm competition game played by C. georgiana males could also explain the observed patterns in sperm traits. Future investigations determining the parameters responsible for the deviation from theoretical predictions in this system will test the degree to which current theoretical models can indeed be applied to species with plastic reproductive tactics.     

Experience affects mating behavior, but does not impact parental reproductive allocation in a lizard

Investment into reproduction is influenced by multiple factors and varies substantially between males and females. Theory predicts that males should adjust their ejaculate size or quality in response to variation in female experience or phenotypic quality. In addition, sperm investment by males may also be influenced by their own status and experience. Although such adjustments of male ejaculate size can impact reproductive success (via fertilization success), fitness returns from male sperm investment may be influenced (either limited or facilitated) by the level of maternal investment. To test this hypothesis, we conducted an experiment that simultaneously evaluated the effect of paternal and maternal experience (which incorporates mating status, age, body size, and other related variables) on paternal sperm investment and maternal reproductive allocation in the lizard Anolis sagrei. During staged mating trials, experienced males were more likely to copulate with females, but these individuals were less likely to transfer sperm during mating than were naïve individuals. Maternal experience had no impact on these mating behaviors. In contrast to expectations, experience and phenotypic quality (of both sexes) had no impact on male ejaculate size or quality (proportion of live sperm) or on maternal reproductive investment (in terms of egg size and yolk steroids). These findings were intriguing given the mating system and past evidence for differential maternal investment in relation to sire quality in A. sagrei. The results found in this study highlight the complexity of reproductive investment patterns, and we urge caution when applying general conclusions across populations or taxa.     

Courting virtual rivals increase ejaculate size in sticklebacks (<Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis>)

Sperm competition occurs when sperm of two or more males compete to fertilize a given set of eggs. Game-theory models of sperm competition predict that males, which face an increased risk of sperm competition, will invest more sperm in a mating. In the pair-spawning three-spined stickleback ( Gasterosteus aculeatus), males attempt to steal fertilizations (i.e. to sneak) when they are in the courting phase, but not when they are parental. The behaviour of neighbouring males may therefore indicate the risk of sperm competition. We visually confronted males before spawning with computer animations of the same virtual stickleback showing two different behaviours: either courting (high risk of sperm competition) or brood-caring (low risk of sperm competition). We show that males invest significantly more sperm (absolutely and relatively with respect to available sperm in the testes) after the courting stimulus. The relative investment ratio between the courting and the brood-caring treatment was on average 1.75. Three-spined sticklebacks thus seem to assess the risk of sperm competition by the behaviour of neighbours and to adjust their ejaculate accordingly. This result suggests that the evolutionary force of sperm competition has led to precise mechanisms of future risk assessment.     

Evolution of mating systems and sexual size dimorphism in North American cyprinids

Mating systems evolve with sexual size dimorphism (SSD) in many animals. Mating systems with males larger than females occur when males compete for female access or guard territories, while mating systems with group mating tend to occur in species where females are the same size or larger than males. In addition to variation in SSD with mating system, sperm competition varies among mating systems in predictable patterns. We examined the evolution of mating systems with SSD and testes mass in 111 North American Cyprinidae fishes using phylogenetic comparative methods. Our results demonstrate that the evolution of mating systems in Cyprinidae fishes is from ancestral taxa that are group spawners with females the same size or larger than males to pair spawning systems where males tend to be larger than females. We used an additive model to predict male and female body size from testes mass and mating system. Only mating system varied predictably with SSD. Our results for analyses of hyperallometry (Rensch’s rule) were that individual species of Cyprinidae can have hyperallometry for SSD, but the pattern is not present across all taxa.     

Sperm competition and ejaculate investment in red squirrels (<Emphasis Type="Italic">Tamiasciurus hudsonicus</Emphasis>)

Sperm competition is widespread in mammals and occurs when ejaculates from two or more males compete within the female’s reproductive tract to fertilize the ova. Enlarged testes are associated with sperm competition because they produce sperm, but the accessory glands produce fluids and proteins that are also important for fertilization success. Sperm morphology can also have consequences for fertilization success because of its influence on sperm motility. Red squirrels engage in multiple mating, and thus sperm competition is likely. Here, we assess levels of multiple paternity in a natural population of red squirrels, test the prediction that testis size is correlated with size of the accessory glands and sperm morphometry, and test the prediction that ejaculate investment is condition-dependent. Five of six litters (83%) showed evidence of multiple paternity, indicating that sperm competition is likely to have occurred. Testis size was correlated with the size of all three accessory glands (prostate, seminal vesicle, epididymides), and there was a generally positive relationship between the size of the accessory glands and sperm length. Sperm morphology showed significant variation in size and shape among individual male squirrels. There was no evidence of condition dependence of testis size or the size of the accessory glands, but sperm midpiece length was negatively related to body condition. Further work should include determining the fitness consequence of variation in sperm morphometry, testis size, and accessory gland size, and determining the effects of variation in ejaculate investment on sperm motility.     

Sperm swimming speed and energetics vary with sperm competition risk in bluegill (<Emphasis Type="Italic">Lepomis macrochirus</Emphasis>)

Under sperm competition, a males fertilization success depends largely on the ejaculate characteristics of competing males. Theoretical models predict that, in external fertilizers, increased risk of sperm competition should result in selection for increased sperm swimming speed. To test this prediction, we studied the behavior of sperm from parental and sneaker male bluegill (Lepomis macrochirus), a fish species characterized by high levels of cuckoldry due to alternative reproductive tactics of males (parentals and cuckolders). Because cuckolders (sneakers and satellites) always spawn in the presence of a parental male, but the reverse is not true, cuckolders experience the greater risk of sperm competition. We show here that the spermatozoa of sneakers have faster initial swimming speeds but shorter periods of motility than the sperm of parental males. Moreover, we show that sperm swimming speeds shortly after activation (when most fertilization occurs) are correlated with starting ATP levels in spermatozoa, suggesting that sperm competition has selected for higher energetic capacity in the sperm of sneakers. Thus, the higher energetic capacity and initial swimming speed of sneaker sperm may explain why, despite having fewer sperm per ejaculate than parentals, sneakers fertilize more eggs than parental males when they compete to fertilize a clutch of eggs.Communicated by L.W. Simmons     

Effects of female mating status on copulation behaviour and sperm expenditure in the freshwater crayfish <Emphasis Type="Italic">Austropotamobius italicus</Emphasis>

Sperm competition models predict that males should adjust their sperm expenditure according to the risk and/or intensity of sperm competition. In this paper, we analysed copulatory behaviour of both sexes and sperm expenditure in relation to female mating status (virgin or mated) in the freshwater crayfish Austropotamobius italicus, a species where males have been reported to feed on and remove sperm laid by other males. The same females were allowed to be inseminated sequentially by two males, and we compared the sexual behaviours of partners between the first (virgin females) and the second mating (mated females). We found that female resistance did not differ between the first and the second mating, nor males refused or took more time to mount a mated female. However, when mating with a mated female, males reached an effective copulation position significantly later. This occurred because second-mating males removed, by eating, all or most spermatophores previously deposited by first males. As removal was often incomplete, this resulted in a larger amount of sperm being deposited on female ventral parts after the second mating, although second males did not allocate more sperm to mated females than first males did. Thus, the peculiar mode of sperm competition, where males remove previously deposited sperm, and the consequent predictable strong last male prevalence in paternity likely led to the observed lack of adjustment of sperm expenditure to female mating status in this species.     

High degree of multiple paternity in the viviparous Shiner Perch, <Emphasis Type="Italic">Cymatogaster aggregata</Emphasis>, a fish with long-term female sperm storage

The Shiner Perch (Cymatogaster aggregata Gibbons) exhibits a viviparous reproductive mode and long-term female sperm storage, two biological features that may predispose this fish species for both intense sperm competition and frequent multiple paternity within broods. To test these hypotheses, we used polymorphic microsatellite markers to identify sires and quantify paternal contributions to the progeny arrays of 27 pregnant females from a natural population of C. aggregata. The number of sires per brood ranged from one to eight (mean 4.6), typically with skewed distributions of fertilization success by the fathers but no correlation between sire number and brood size. The extraordinarily high incidences of multiple paternity in this species probably are due in part to high rates of mate encounter, but selection pressures related to the avoidance of maternal–fetal incompatibility may further have promoted the evolution of polyandrous mating behaviors in this female-pregnant species. Our genetic data are consistent with the hypothesis that viviparity, long-term sperm storage, and extreme polyandry are interrelated reproductive phenomena that should promote the evolution of post-copulatory sperm competition and/or cryptic female choice in these fishes.     

Intraspecific variation of testis size and sperm length in the yellow-pine chipmunk (<Emphasis Type="Italic">Tamias amoenus</Emphasis>): implications for sperm competition and reproductive success

Comparative analyses have found that relative testis size is a strong predictor of the prevalence of sperm competition for many taxa, including mammals, yet underlying this pattern is the assumption that intraspecific variation in testis size is related to individual fitness. Because intraspecific variation in ejaculate investment underlies interspecific patterns, it is critical to understand the causes and consequences of intraspecific variation in ejaculate investment. We examined relationships between ejaculate investment (testis size and sperm length) and reproductive success, body size and condition in the yellow-pine chipmunk (Tamias amoenus), a small ground squirrel in which sperm competition occurs. We examined genetic estimates of male reproductive success from a wild population of yellow-pine chipmunks and determined that males with large testes had higher annual reproductive success than males with small testes. This result provides empirical support for the numerous comparative studies that indicate testis size is associated with the intensity of sperm competition. In addition, males in good condition had relatively larger testes than males in poor condition, but there was no evidence of sperm length being dependent on condition. Finally, contrary to many predictions, males that invested more in sperm production (relatively heavy testes) produced shorter sperm, not longer sperm, than males that invested less.Communicated by P.M. Kappeler     

Increased sperm numbers in the vas deferens of meadow voles, <Emphasis Type="Italic">Microtus pennsylvanicus,</Emphasis> in response to odors of conspecific males

Sperm competition occurs when the sperm of two or more males compete to fertilize the egg/s of a particular female. Males of some species respond to a high risk of sperm competition by increasing the number of sperm in their ejaculates. Males may accomplish such a response by increasing the intensity or duration of contraction of the cauda epididymidis and vas deferens. During emission (first phase of the ejaculatory process), the vas deferens receives sperm from the cauda epididymidis and propels the sperm to the urethra. In this paper, we tested the hypothesis that males exposed to a high risk of sperm competition mobilize larger numbers of sperm from the cauda epididymidis to the vas deferens before initiation of copulatory behavior. This accumulation of sperm in the vas deferens would result in a larger number of sperm in the ejaculate. To test this hypothesis, we exposed male meadow voles, Microtus pennsylvanicus, to either low or high risks of sperm competition using soiled bedding of conspecific individuals. At three different times after this exposure (15, 30, or 60 min), we removed both vasa deferentia and counted the sperm within them. We found a significant increase in sperm numbers in the vas deferens of males after 30 min of being exposed to a high risk of sperm competition. The lower sperm numbers after 15 and 60 min of exposure suggest that the observed response is relatively slow and that sperm mobilized to the vasa deferentia may return to the cauda epididymides if ejaculation does not occur some time after the observed response. Our results indicate that the physiological response that may result in high sperm numbers in the ejaculate in relation to high risk of sperm competition can occur before initiation of copulatory behavior.     

Sympatric sexual signal divergence among North American Calopteryx damselflies is correlated with increased intra- and interspecific male–male aggression

Divergence of sexual signals in sympatry can arise as a consequence of (1) interspecific competition for resources, (2) selection against maladaptive hybridization, or (3) as a result of selection to reduce the cost of interspecific aggression; termed agonistic character displacement (ACD). Calopterygid damselflies have emerged as a model system for studying the evolution of divergent sexual signals due to the repeated evolution of sympatric species pairs with fully and partially melanized wings. Damselfly wing patterns function during both courtship and territory defense. However, the relative contributions of natural and sexual selection to phenotypic divergence and enhanced isolation in sympatry remain unclear in many cases. Here, we investigated the hypothesis that interference competition, in the form of increased interspecific male–male aggression, drives the evolution of character displacement in sympatry between two species of North American damselflies, Calopteryx aequabilis and Calopteryx maculata, that show no evidence of ecological divergence or ongoing hybridization. In paired behavioral trials, we found that interspecific male aggression related to territory defense varied between site, species, and as a function of the relative abundance of con- vs. hetero-specific males. Specifically, we found that large-spotted C. aequabilis males received increased intra- and interspecific aggression but that aggression against large-spotted males declined during the middle of the flight season when both species were equally abundant. Based on these results, we suggest that ACD leads to enhanced species recognition, and may be a common outcome of the antagonism between interspecific male–male competition and the countervailing force of intraspecific sexual selection favoring increased wing melanization among territorial damselfly species.     

Mechanisms of sperm competition in birds: mathematical models

Summary We built models of sperm competition and compared their predictions with observations from domestic chickens in an attempt to elucidate the mechanism of sperm competition. The models suggest that (1) observed levels of precedence of sperm are not merely a passive consequence of the greater mortality of sperm from earlier inseminations; (2) displacement of existing sperm from sperm storage tubules by incoming sperm explains observed levels of sperm precedence and disappearance from the reproductive tract, even in the absence of recognition of sperm from different inseminations; (3) stratification of sperm within the sperm storage tubules fails as an explanation of observed patterns of sperm precedence and disappearance because long-term levels of sperm precedence as high as those observed can only be predicted in combination with rates of disappearance of sperm much lower than those observed in chickens. The predictions of sperm precedence levels following multiple inseminations also suggest that displacement rather than stratification is the most likely mechanism of sperm competition in chickens. We discuss the consequences of male or female control of sperm competition, non-uniform distribution of sperm between sperm storage tubules, and variation in sperm precedence with time after the last insemination, and suggest ways in which the models may be used in future to aid in the elucidation of mechanisms of sperm competition. Offprint requests to: C.M. Lessells     

Does delayed fertilization facilitate sperm competition in bats?

Reproductive delays between mating and birth may provide a previously unconsidered avenue for post-copulatory sexual selection in mammals. In particular, delayed fertilization could provide an enhanced opportunity for sperm competition by extending the time for ejaculates to interact in the female reproductive tract. We tested the prediction that species with delayed fertilization exhibit greater degrees of sperm competition than those without delays by examining testis volume (a proxy for sperm competition) in 38 species of bats. Examination of fluid-preserved museum specimens of bat species with and without delays revealed that species with delays (in particular those with delayed fertilization) had significantly larger testes than species without them. Although it predicts the presence of delayed fertilization, hibernation did not predict relative testis size. We conclude that, once they evolve, reproductive delays may facilitate sperm competition.     

Patterns of courtship behavior and ejaculate characteristics in male red-winged blackbirds

Sperm competition in birds is likely to have important effects on the behavior and physiology of reproduction in both sexes. For males, such competition should select for large sperm reserves and behavioral adjustment of copulation when reserves are low. We investigated both these possibilities in free-living red-winged blackbirds (Agelaius phoeniceus), a highly polygynous species with apparently strong sperm competition. We found that the recent copulatory behavior of males did not affect the propensity to copulate with a model female. Ejaculates collected from individual males at 1-h intervals showed no evidence of sperm depletion, yet repeated ejaculates collected less than 10 min apart did. Male ejaculate size was significantly larger if it was the first one of the day (i.e., after an overnight rest). The average ejaculate size was 12.5 (±12.5 SD) million sperm. Males captured during the breeding season had an average of 111.7 (±52.8) million sperm stored in their seminal glomera. Because males average a peak copulation rate of six per female per day, in one day a male might utilize all the sperm in his seminal glomera if more than two females on his territory are fertilizable. We hypothesize that polygyny and sperm competition in this species have combined to select for rapid replenishment of the seminal glomera throughout the day, in contrast to other species that have been studied. Testis size and sperm reserves of male red-winged blackbirds are intermediate between monogamous species and species with intense sperm competition. Several possible explanations for this are discussed. Received: 21 October 1997 / Accepted after revision: 15 February 1998