Sperm stratification and paternity success in red flour beetles

Abstract. When females are inseminated by multiple males, male paternity success (sperm precedence) is determined by the underlying processes of sperm storage and sperm utilization. Although informative for many questions, two-male sperm competition experiments may offer limited insight into natural mating scenarios when females are likely to mate with several males. In this study, genetic markers in Tribolium castaneum are used to trace paternity for multiple sires, and to determine whether displacement of stored sperm that occurs after a third mating equally affects both previous mates, or if fertilizations are disproportionately lost by the female's most recent mate. For 20 days after triple-matings, first males retain significantly higher paternity success (relative to first male paternity in double-matings) compared with second males. These results demonstrate that when females remate before sperm mixing occurs, sperm stratification results in differential loss of sperm from the most recent mate. This study provides insight into the mechanisms underlying sperm precedence in a promiscuous mating system, and suggests that T. castaneum females could limit paternity success of particular mates by remating with more highly preferred males.     

Intraspecific sperm competition genes enforce post-mating species barriers in Drosophila

Sexual selection and sexual conflict are considered important drivers of speciation, based on both theoretical models and empirical correlations between sexually selected traits and diversification. However, whether reproductive isolation between species evolves directly as a consequence of intrapopulation sexual dynamics remains empirically unresolved, in part because knowledge of the genetic mechanisms (if any) connecting these processes is limited. Here, we provide evidence of a direct mechanistic link between intraspecies sexual selection and reproductive isolation. We examined genes with known roles in intraspecific sperm competition (ISC) in D. melanogaster and assayed their impact on conspecific sperm precedence (CSP). We found that two such genes (Acp36DE and CG9997) contribute to both offensive sperm competition and CSP; null/knockdown lines both had lower competitive ability against D. melanogaster conspecifics and were no longer able to displace heterospecific D. simulans sperm in competitive matings. In comparison, Sex Peptide (Acp70A)—another locus essential for ISC—does not contribute to CSP. These data indicate that two loci important for sperm competitive interactions have an additional role in similar interactions that enforce post-mating reproductive isolation between species, and show that sexual selection and sexual isolation can act on the same molecular targets in a gene-specific manner.     

Remating and sperm displacement in a natural population of Drosophila buzzatii inferred from mother-offspring analysis of microsatellite loci

Prospects for estimation of parameters of models of sperm competition from field data have improved recently with the development of methods that employ multilocus genotype data from brood-structured samples. Sperm competition in Drosophila buzzatii is of special interest because it is possible to directly observe the breeding behaviour of this species in its natural habitat of rotting cactus. Previous laboratory experiments showed that this species exhibits an unusual pattern of frequent remating and sperm partitioning. This paper reports the first attempt to estimate the frequency of female remating and sperm competition in natural populations of D. buzzatii. For the Australian population studied, the mean remating frequency was lower (alpha = 2.12-2.20) than previously estimated in laboratory experiments with the same population, whereas mean sperm displacement (beta = 0.69-0.71) fell within the limits of previous laboratory results. The evolution of the D. buzzatii mating system is discussed.     

Modeling strategic sperm allocation: Tailoring the predictions to the species

Two major challenges exist when empirically testing the predictions of sperm allocation theory. First, the study species must adhere to the assumptions of the model being tested. Unfortunately, the common assumption of sperm allocation models that females mate a maximum of once or twice does not hold for many, if not most, multiply and sequentially mating animals. Second, a model's parameters, which dictate its predictions, must be measured in the study species. Common examples of such parameters, female mating frequency and sperm precedence patterns, are unknown for many species used in empirical tests. Here, we present a broadly applicable model, appropriate for multiply, sequentially mating animals, and test it in three species for which data on all the relevant parameter values are available. The model predicts that relative allocation to virgin females, compared to nonvirgins, depends on the interaction between female mating rate and the sperm precedence pattern: relative allocation to virgins increases with female mating rate under first‐male precedence, while the opposite is true under later‐male precedence. Our model is moderately successful in predicting actual allocation patterns in the three species, including a cricket in which we measured the parameter values and performed an empirical test of allocation.     

Geographic variation in sperm traits reflects predation risk and natural rates of multiple paternity in the guppy

Guppies (Poecilia reticulata) are models for understanding the interplay between natural and sexual selection. In particular, predation has been implicated as a major force affecting female sexual preferences, male mating tactics and the level of sperm competition. When predation is high, females typically reduce their preferences for showy males and engage more in antipredator behaviours, whereas males exploit these changes by switching from sexual displays to forced matings. These patterns are thought to account for the relatively high levels of multiple paternity in high‐predation populations compared to low‐predation populations. Here, we assess the possible evolutionary consequences of these patterns by asking whether variation in sperm traits reflect differences in predation intensity among four pairs of Trinidadian populations: four that experience relatively low levels of predation from a gape‐limited predator and four that experience relatively high levels of predation from a variety of piscivores. We found that males in high‐predation populations had faster swimming sperm with longer midpieces compared to males in low‐predation populations. However, we found no differences among males in high‐ and low‐predation populations with respect to sperm number, sperm head length, flagellum length and total sperm length.     

Heritability of sperm competition success in the bulb mite,Rhizoglyphus robini

Multiple mating by females has been proposed to function as a form of mate-choice, which implies that males should show heritable variation in sperm-competitive abilities. In this study, repeatability and heritability of sperm competition success was estimated in the bulb mite, Rhizoglyphus robini. Fertilization success of males was estimated in competition with sperm of two other males. Males differed consistently in their sperm competition success, with repeatability estimated at 0.22. The heritability of sperm competition success was estimated using parent-offspring regression, with the mean fertilization success from two matings used as a measure of each male's competitive ability. There was a significant association between the sperm competition success of fathers and sons. Narrow sense heritability (h²) was 0.284. This result supports the hypotheses proposing the multiple mating is selectively maintained in females by enhancing the reproductive success of their progeny.     

Nontransitivity of sperm precedence in Drosophila

Abstract.— Sperm competition is an important component of fitness in Drosophila , but we still do not have a clear understanding of the unit of selection that is relevant to sperm competition. Here we demonstrate that sperm competitive ability is not a property of the sperm haplotype, but rather of the diploid male's genotype. Then we test whether the relative sperm competitive ability of males can be ranked on a linear array or whether competitive ability instead depends on particular pairwise contests among males. Sperm precedence of six chromosome-extracted lines was tested against three different visible marker lines ( cn bw, bw^D , and Cy ), and the rank order of the six lines differed markedly among the mutant lines. Population genetic theory has shown that departures from transitivity of sperm precedence may be important to the maintenance of polymorphism for genes that influence sperm competitive ability. The non-transitivity seen in sperm precedence should theoretically increase the opportunity for polymorphism in genes that influence this phenotype.     

Multiple paternity in reptiles: patterns and processes

The evolution of female promiscuity poses an intriguing problem as benefits of mating with multiple males often have to arise via indirect, genetic, effects. Studies on birds have documented that multiple paternity is common in natural populations but strong evidence for selection via female benefits is lacking. In an attempt to evaluate the evidence more broadly, we review studies of multiple paternity in natural populations of all major groups of nonavian reptiles. Multiple paternity has been documented in all species investigated so far and commonly exists in over 50% of clutches, with particularly high levels in snakes and lizards. Marine turtles and lizards with prolonged pair-bonding have relatively low levels of multiple paternity but levels are nevertheless higher than in many vertebrates with parental care. There is no evidence that high levels of polyandry are driven by direct benefits to females and the evidence that multiple paternity arises from indirect genetic benefits is weak. Instead, we argue that the most parsimonious explanation for patterns of multiple paternity is that it represents the combined effect of mate-encounter frequency and conflict over mating rates between males and females driven by large male benefits and relatively small female costs, with only weak selection via indirect benefits. A crucial step for researchers is to move from correlative approaches to experimental tests of assumptions and predictions of theory under natural settings, using a combination of molecular techniques and behavioural observations.     

The effect of female polyandry and sperm precedence on the evolution of sexual difference in dispersal timing

Predispersal copulation and unpredictable environment facilitate the evolution of female-biased dispersal in species, where females are functionally monandrous. Females should migrate and reproduce over different habitats to spread their risks due to environmental fluctuation. On the other hand, males do not have to disperse because their risks are spread by their mating partners who produce their offspring in different habitats. However, when females are functionally polyandrous, it is expected that they will not contribute to spreading the male's risk extensively. Therefore, by simulation with the individual based model, the present study evaluated how female polyandry influences the sexual difference in dispersal timing. This model revealed that when females are polyandrous, the timing of female remating and sperm priority patterns have an important influence on the evolution of sex-biased dispersal. Particularly when female remating is not synchronized with dispersal or when last-male sperm precedence does not exist, female-biased dispersal is evolved.     

No evidence that polyandry benefits females in Drosophila melanogaster

Understanding the evolution of polyandry (mating with multiple males) is a major issue in the study of animal breeding systems. We examined the adaptive significance of polyandry in Drosophila melanogaster, a species with well-documented costs of mating in which males generally cannot force copulations. We found no direct fitness advantages of polyandry. Females that mated with multiple males had no greater mean fitness and no different variance in fitness than females that mated repeatedly with the same male. Subcomponents of reproductive success, including fecundity, egg hatch rate, larval viability, and larval development time, also did not differ between polyandrous and monogamous females. Polyandry had no affect on progeny sex ratios, suggesting that polyandry does not function against costly sex-ratio distorters. We also found no evidence that polyandry functions to favor the paternity of males successful in precopulatory sexual selection. Experimentally controlled opportunities for precopulatory sexual selection had no effect on postcopulatory sperm precedence. Although these results were generally negative, they are supported with substantial statistical power and they help narrow the list of evolutionary explanations for polyandry in an important model species.     

Polyandrous females provide sons with more competitive sperm: Support for the sexy‐sperm hypothesis in the rattlebox moth (Utetheisa ornatrix)

Given the costs of multiple mating, why has female polyandry evolved? Utetheisa ornatrix moths are well suited for studying multiple mating in females because females are highly polyandrous over their life span, with each male mate transferring a substantial spermatophore with both genetic and nongenetic material. The accumulation of resources might explain the prevalence of polyandry in this species, but another, not mutually exclusive, possibility is that females mate multiply to increase the probability that their sons will inherit more‐competitive sperm. This latter “sexy‐sperm” hypothesis posits that female multiple mating and male sperm competitiveness coevolve via a Fisherian runaway process. We tested the sexy‐sperm hypothesis by using competitive double matings to compare the sperm competition success of sons of polyandrous versus monandrous females. In accordance with sexy‐sperm theory, we found that in 511 offspring across 17 families, the male whose polyandrous mother mated once with each of three different males sired significantly more of all total offspring (81%) than did the male whose monandrous mother was mated thrice to a single male. Interestingly, sons of polyandrous mothers had a significantly biased sex ratio of their brood toward sons, also in support of the hypothesis.     

Divergent natural selection alters male sperm competition success in Drosophila melanogaster

Sexually selected traits may also be subject to non‐sexual selection. If optimal trait values depend on environmental conditions, then “narrow sense” (i.e., non‐sexual) natural selection can lead to local adaptation, with fitness in a certain environment being highest among individuals selected under that environment. Such adaptation can, in turn, drive ecological speciation via sexual selection. To date, most research on the effect of narrow‐sense natural selection on sexually selected traits has focused on precopulatory measures like mating success. However, postcopulatory traits, such as sperm function, can also be under non‐sexual selection, and have the potential to contribute to population divergence between different environments. Here, we investigate the effects of narrow‐sense natural selection on male postcopulatory success in Drosophila melanogaster. We chose two extreme environments, low oxygen (10%, hypoxic) or high CO₂ (5%, hypercapnic) to detect small effects. We measured the sperm defensive (P1) and offensive (P2) capabilities of selected and control males in the corresponding selection environment and under control conditions. Overall, selection under hypoxia decreased both P1 and P2, while selection under hypercapnia had no effect. Surprisingly, P1 for both selected and control males was higher under both ambient hypoxia and ambient hypercapnia, compared to control conditions, while P2 was lower under hypoxia. We found limited evidence for local adaptation: the positive environmental effect of hypoxia on P1 was greater in hypoxia‐selected males than in controls. We discuss the implications of our findings for the evolution of postcopulatory traits in response to non‐sexual and sexual selection.     

Comparative molecular population genetics of the Xdh locus in the cactophilic sibling species Drosophila buzzatii and D. koepferae

The Xdh (rosy) gene is one of the best studied in the Drosophila genus from an evolutionary viewpoint. Here we analyze nucleotide variation in a 1875-bp fragment of the second exon of Xdh in Argentinian populations of the cactophilic D. buzzatii and its sibling D. koepferae. The major electrophoretic alleles of D. buzzatii not only lack diagnostic amino acids in the region studied but also differ on average from each other by four to 13 amino acid changes. Our data also suggest that D. buzzatii populations belonging to different phytogeographic regions are not genetically differentiated, whereas D. koepferae exhibits a significant pattern of population structure. The Xdh region studied is twice as polymorphic in D. buzzatii as in D. koepferae. Differences in historical population size or in recombinational environment between species could account for the differences in the level of nucleotide variation. In both species, the Xdh region exhibits a great number of singletons, which significantly departs from the frequency spectrum expected under neutrality for nonsynonymous sites and also for synonymous sites in D. buzzatii. These excesses of singletons could be the signature of a recent population expansion in D. buzzatii, whereas they may be simply explained as the result of negative selection in D. koepferae.     

Pitfalls in experiments testing predictions from sperm competition theory

As females of many species mate with more than one male, ejaculates often face competition from the sperm of other males. In recent years, numerous papers have been published on theoretical predictions of evolutionary, behavioural and physiological responses to variation in the strength of sperm competition (SC). These theoretical predictions have also been extensively tested. However, although predictions from SC theory are relatively straightforward, extra caution has to be paid in the design of experiments testing them. One difficulty is for example to disentangle immediate and mean SC risk and intensity. Without carefully designed experiments, it is also very easy to simultaneously increase SC risk and the probability of intense SC--a situation for which we currently have no clear predictions, as the theoretical models to date only assume variation in either SC risk or intensity. In this paper, we discuss these and some other pitfalls related to manipulations of SC risk and intensity and suggest how to avoid them.     

Securing paternity: mating plugs in the dwarf spider Oedothorax retusus (Araneae: Erigoninae)

One of the various male strategies to prevent or impede female remating is the production of a mating plug that covers the female genital opening or remains inside of the female genital tract after mating. Such structures have been described for many species in many animal taxa; however, in most cases, we know little or nothing about their specific adaptive value. Our investigations demonstrate that females of the dwarf spider species Oedothorax retusus (Westring, 1851) (Linyphiidae, Erigoninae) exhibit a substance on one or both of her paired genital openings only after copulation. We performed double-mating trials and forced the second male to mate into the previously used or unused spermathecal duct of the female by amputating one of his paired male gonopods (pedipalps). Furthermore, to investigate whether the duration of the first mating has an effect on the size and efficiency of the mating plug, we interrupted first matings after either 1 or 3 min, categorized plug size and recorded mating behaviour of subsequent males. The amount of secretion transferred was larger in long compared to short copulations. A long first copulation successfully prevented subsequent males from mating into the used ducts, whereas mating success after short first matings was similar to matings into unused copulatory ducts of the females. The present study demonstrates that a male O. retusus can prevent a rival from transferring sperm into the same spermatheca by applying a mating plug, but only if he mates for long enough.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 574–583.     

Sperm morphology and evidence for sperm competition among parrots

Sperm competition is an important component of post‐copulatory sexual selection that has shaped the evolution of sperm morphology. Previous studies have reported that sperm competition has a concurrently directional and stabilizing effect on sperm size. For example, bird species that show higher levels of extrapair paternity and larger testes (proxies for the intensity of sperm competition) have longer sperm and lower coefficients of variation in sperm length, both within and between males. For this reason, these sperm traits have been proposed as indexes to estimate the level of sperm competition in species for which other measures are not available. The relationship between sperm competition and sperm morphology has been explored mostly for bird species that breed in temperate zones, with the main focus on passerine birds. We measured sperm morphology in 62 parrot species that breed mainly in the tropics and related variation in sperm length to life‐history traits potentially indicative of the level of sperm competition. We showed that sperm length negatively correlated with the within‐male coefficient of variation in sperm length and positively with testes mass. We also showed that sperm is longer in sexually dichromatic and in gregarious species. Our results support the general validity of the hypothesis that sperm competition drives variation in sperm morphology. Our analyses suggest that post‐copulatory sexual selection is also important in tropical species, with more intense sperm competition among sexually dichromatic species and among species that breed at higher densities.     

The evolution of sperm-allocation strategies and the degree of sperm competition

The prevailing viewpoint in the study of sperm competition is that male sperm-allocation strategies evolve in response to the degree of sperm competition an ejaculate can expect to experience within a given mating. If males cannot assess the degree of sperm competition their ejaculate will face and/or they are unable to facultatively adjust sperm investment in response to perceived levels of competition, high sperm allocation (per mating) is predicted to evolve in the context of high sperm competition. An implicit assumption of the framework used to derive this result is that the degree of sperm competition is unaffected by changes in sperm-allocation strategies. We present theory based on an alternative perspective, in which the degree of sperm competition and the sperm-allocation strategy are coupled traits that coevolve together. Our rationale is that the pattern of sperm allocation in the population will, in part, determine the level of sperm competition by affecting the number of ejaculates per female in the population. In this setting, evolution in sperm-allocation strategies is driven by changes in underlying environmental parameters that influence both the degree of sperm competition and sperm allocation. This change in perspective leads to predictions that are qualitatively different from those of previous theory.     

Patterns of sperm precedence are not affected by female mating history in Drosophila melanogaster

In promiscuously mating species, there is strong selection on males to maximize their share of paternity through both defensive and offensive means. This has been most extensively examined using the Drosophila melanogaster model system. In these studies, sperm competition has been examined by mating a virgin female to two consecutive males and then determining the fertilization success of both the first male (defending, P1) and the second male (offending, P2). Recent evidence suggests that male defense may be influenced by female mating history (i.e., virgin versus nonvirgin). Here, by mating females to males with three different genotypes, we show that female mating history does not affect male defensive or offensive abilities in sperm competition. We also show that, although female lifetime fecundity was not correlated with the number of times that she mated, it was reduced by increased exposure to males. These data indicate that measures of P1 and P2 previously reported in D. melanogaster may be robust to the specific mating history of the females used in these studies.     

Sex allocation and investment into pre- and post-copulatory traits in simultaneous hermaphrodites: the role of polyandry and local sperm competition

Sex allocation theory predicts the optimal allocation to male and female reproduction in sexual organisms. In animals, most work on sex allocation has focused on species with separate sexes and our understanding of simultaneous hermaphrodites is patchier. Recent theory predicts that sex allocation in simultaneous hermaphrodites should strongly be affected by post-copulatory sexual selection, while the role of pre-copulatory sexual selection is much less clear. Here, we review sex allocation and sexual selection theory for simultaneous hermaphrodites, and identify several strong and potentially unwarranted assumptions. We then present a model that treats allocation to sexually selected traits as components of sex allocation and explore patterns of allocation when some of these assumptions are relaxed. For example, when investment into a male sexually selected trait leads to skews in sperm competition, causing local sperm competition, this is expected to lead to a reduced allocation to sperm production. We conclude that understanding the evolution of sex allocation in simultaneous hermaphrodites requires detailed knowledge of the different sexual selection processes and their relative importance. However, little is currently known quantitatively about sexual selection in simultaneous hermaphrodites, about what the underlying traits are, and about what drives and constrains their evolution. Future work should therefore aim at quantifying sexual selection and identifying the underlying traits along the pre- to post-copulatory axis.