Determinants of paternity in a butterfly

Success in sperm competition is of fundamental importance to males, yet little is known about what factors determine paternity. Theory predicts that males producing high sperm numbers have an advantage in sperm competition. Large spermatophore size (the sperm containing package) also correlates with paternity in some species, but the relative importance of spermatophore size and sperm numbers has remained unexplored. Males of the small white butterfly, Pieris rapae (Lepidoptera: Pieridae), produce large nutritious spermatophores on their first mating. On their second mating, spermatophores are only about half the size of the first, but with almost twice the sperm number. We manipulated male mating history to examine the effect of spermatophore size and sperm numbers on male fertilization success. Overall, paternity shows either first male or, more frequently, second male sperm precedence. Previously mated males have significantly higher fertilization success in competition with males mating for the first time, strongly suggesting that high sperm number is advantageous in sperm competition. Male size also affects paternity with relatively larger males having higher fertilization success. This may indicate that spermatophore size influences paternity, because in virgin males spermatophore size correlates with male size. The paternity of an individual male is also inversely correlated with the mass of his spermatophore remains dissected out of the female. This suggests that females may influence paternity by affecting the rate of spermatophore drainage. Although the possibility of female postcopulatory choice remains to be explored, these results clearly show that males maximize their fertilization success by increasing the number of sperm in their second mating.     

INFERENCE OF SPERM COMPETITION FROM BROODS OF FIELD-CAUGHT DROSOPHILA

In field studies of multiple mating and sperm competition there typically is no experimental control over the number of times that a female mates, the interval between matings, or the genetic identity of multiple fathers contributing to a brood. Irrespective of this complexity, high-resolution molecular markers can be used to assign paternity with considerable confidence. This study employed two highly heterozygous microsatellite loci to assess multiple paternity and sperm displacement in a sample of broods taken from a natural population of Drosophila melanogaster. The large number of alleles present at each of the loci makes it difficult to derive explicit maximum-likelihood estimates for multiple paternity and sperm displacement from brood samples. Monte Carlo simulations were used to estimate maximum-likelihood parameters for the distribution of female remating frequency and the proportion of offspring sired by the second or subsequent mating males. Estimates were made based on genotypes scored at two distinct marker loci because they were found to give statistically homogeneous results. Fitting a Poisson distribution of number of matings, the mean number of males mated by a female was 1.82. The sperm displacement parameter estimated from doubly mated females were 0.79 and 0.86 for the two loci (0.83 for the joint estimate). The overall probability that a multiply mated female will be misclassified as singly mated was only 0.006, which indicates that microsatellites can provide excellent resolution for identifying multiple mating. In addition, microsatellites can be used to generate relatively precise estimates of sperm precedence in brood-structured samples from a natural population.     

SPERM COMPETITION AND THE EVOLUTION OF NUPTIAL FEEDING BEHAVIOR IN THE CRICKET,GRYLLODES SUPPLICANS (WALKER)

The pattern of sperm predominance in doubly mated female crickets, Gryllodes supplicans, was investigated using a radiation-sterility technique. Female G. supplicans made significant use of sperm from both males in fertilizing eggs; overall, first males to mate enjoyed a small advantage, fertilizing about 60% of the offspring produced subsequent to the second mating. The combined use of the sperm of both males in fertilizing eggs occurred soon after the second mating; evidently, mixing of ejaculates within a female's spermatheca does occur. Male G. supplicans provide females with a nuptial gift, the spermatophylax, which influences the time at which a female removes the externally attached sperm-ampulla; this in turn determines the quantity of sperm that is transferred. Moreover, the degree of sperm precedence achieved by a male may be positively related to the time at which the female removes his sperm ampulla. Thus males, by feeding females, ensure not only that a sufficient number of sperm are transferred to fertilize all of a female's eggs, but also may increase the certainty of their paternity. In mating systems in which females control sperm transfer and paternity is influenced by numbers of sperm (i.e., numerical sperm competition), an increase in prezygotic investment in females may be an adaptive male response.     

Paternity Analysis in a Hexapod (Orchesella cincta; Collembola) with Indirect Sperm Transfer

In species where males and females interact during mating, the role of females in sexual selection cannot always be demonstrated unambiguously. Here we present a model system to study female choice for mates. Orchesella cinca is a soil-dwelling hexapod with indirect sperm transfer. Females and males do not interact physically for reproduction. We gave females the choice between spermatophores produced by two different males. Paternity analysis based on microsatellite variation revealed that offspring in one clutch were sired by one male only. Direct observations showed that after a female has taken up a spermatophore, the female's receptivity to further spermatophore uptake seem to end. Our results imply that the female is in full control of paternity.     

Competitive PCR reveals the complexity of postcopulatory sexual selection in Teleogryllus commodus

The outcome of mate choice depends on complex interactions between males and females both before and after copulation. Although the competition between males for access to mates and premating choice by females are relatively well understood, the nature of interactions between cryptic female choice and male sperm competition within the female reproductive tract is less clear. Understanding the complexity of postcopulatory sexual selection requires an understanding of how anatomy, physiology and behaviour mediate sperm transfer and storage within multiply mated females. Here we use a newly developed molecular technique to directly quantify mixed sperm stores in multiple mating females of the black field cricket, Teleogryllus commodus. In this species, female postcopulatory choice is easily observed and manipulated as females delay the removal of the spermatophore in favour of preferred males. Using twice‐mated females, we find that the proportion of sperm in the spermatheca attributed to the second male to mate with a female (S₂) increases linearly with the time of spermatophore attachment. Moreover, we show that the insemination success of a male increases with its attractiveness and decreases with the size of the female. The effect of male attractiveness in this context suggests a previously unknown episode of mate choice in this species that reinforces the sexual selection imposed by premating choice and conflicts with the outcome of postmating male harassment. Our results provide some of the clearest evidence yet for how sperm transfer and displacement in multiply mated females can lead directly to cryptic female choice, and that three distinct periods of sexual selection operate in black field crickets.     

Last–male sperm precedence in the bushcricket Poecilimon veluchianus (Orthoptera, Tettigonioidea) demonstrated by DNA fingerprinting

Males of the bushcricket Poecilimon veluchianus pass a large spermatophore to the female during mating. The spermatophore is eaten by the female after copulation. Because females mate with several males during their reproductive life, the competition between spermatozoa of different males affects a male's reproductive success. In order to determine the outcome of sperm competition, the paternity of the progeny of double–mated females was established by DNA fingerprinting with the oligonucleotide (GATA)₄. Typical P. veluchianus DNA fingerprints consisted of 15 scoreable fragments per individual. The proportion of bands shared between presumably unrelated bushcrickets was 17%. After the second copulation the second mating male clearly predominated at fertilization. The mean proportion of eggs fertilized by the second male was 90.1%. There was no significant relationship between the level of sperm precedence and the time of ovipositions after the second mating. If female P. veluchianus increase the fitness of their offspring by the incorporation of spermatophore–derived substances in developing eggs, there is little chance for the feeding male to fertilize eggs containing his nutrients, because of the very short mating intervals of females and the observed high level of last–male sperm precedence in this species. Under such conditions the last mating male would fertilize many eggs containing nutrients from a prior male. Because nuptial gifts, like the tettigoniid spermatophore, function only as paternal investment if the donating male's progeny benefit from the gift, a paternal investment function of the P. veluchianus spermatophore seems to be unlikely.     

Impact of cryptic female choice on insemination success: Larger sized and longer copulating male squid ejaculate more,but females influence insemination success by removing spermatangia

In polyandrous mating systems, sperm competition and cryptic female choice (CFC) are well recognized as postcopulatory evolutionary forces. However, it remains challenging to separate CFC from sperm competition and to estimate how much CFC influences insemination success because those processes usually occur inside the female's body. The Japanese pygmy squid, Idiosepius paradoxus, is an ideal species in which to separate CFC from sperm competition because sperm transfer by the male and sperm displacement by the female can be observed directly at an external location on the female's body. Here, we counted the number of spermatangia transferred to, removed from, and remaining on the female body during single copulation episodes. We measured behavioral and morphological characteristics of the male, such as duration of copulation and body size. Although males with larger body size and longer copulation time were capable of transferring larger amounts of sperm, females preferentially eliminated sperm from males with larger body size and shorter copulation time by spermatangia removal; thus, CFC could attenuate sperm precedence by larger males, whereas it reinforces sperm precedence by males with longer copulation time. Genetic paternity analysis revealed that fertilisation success for each male was correlated with remaining sperm volume that is adjusted by females after copulation.     

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.     

COPULATORY BEHAVIOR,GENITAL MORPHOLOGY,AND MALE FERTILIZATION SUCCESS IN WATER STRIDERS

Recent theoretical and empirical interest in postmating processes have generated a need for increasing our understanding of the sources of variance in fertilization success among males. Of particular importance is whether such postmating sexual selection merely reinforces the effects of premating sexual selection or whether other types of male traits are involved. In the current study, we document large intraspecific variation in last male sperm precedence in the water strider Gerris lateralis. Male relative paternity success was repeatable across replicate females, showing that males differ consistently in their ability to achieve fertilizations. By analyzing shape variation in male genital morphology, we were able to demonstrate that the shape of male intromittent genitalia was related to relative paternity success. This is the first direct experimental support for the suggestion that male genitalia evolve by postmating sexual selection. A detailed analysis revealed that different components of male genitalia had different effects, some affecting male ability to achieve sperm precedence and others affecting male ability to avoid sperm precedence by subsequent males. Further, the effects of the shape of the male genitalia on paternity success was in part dependent on female morphology, suggesting that selection on male genitalia will depend on the frequency distribution of female phenotypes. We failed to find any effects of other morphological traits, such as male body size or the degree of asymmetry in leg length, on fertilization success. Although males differed consistently in their copulatory behavior, copulation duration was the only behavioral trait that had any significant effect on paternity.     

Cryptic female choice during spermatophore transfer in Tribolium castaneum (Coleoptera: Tenebrionidae)

Sexual selection in both males and females promotes traits and behaviors that allow control over paternity when female mates with multiple males. Nonetheless, mechanisms of cryptic female choice have been consistently overlooked, due to traditional focus on sperm competition as well as difficulty in distinguishing male vs. female influence over processes occurring during and after mating. The first part of this study describes morphology and transformation of Tribolium castaneum spermatophores inferred from dissecting females immediately after normal or interrupted copulations. T. castaneum males are found to transfer spermatophores as an invaginated tube that everts inside the female bursa and which is filled with sperm during copulation. This sequence of events makes it feasible for females to control the sperm quantity transferred in each spermatophore. Through manipulation of the male phenotypic quality (by starvation) and manipulation of female control over sperm transfer (by killing a subset of females), the second part of this study examines whether females use control over transferred sperm quantity as a cryptic choice mechanism. Fed males transferred significantly more sperm per spermatophore than starved males but only when mating with live females. These results suggest an active differentiation by live females against starved males and provide an evidence for the proposed cryptic female choice mechanism.     

Sexual Conflict Over Spermatophore Attachment in a Nuptially Feeding Cricket

Sexual conflict is implicated in the evolution of nuptial feeding. One function of nuptial gifts lies in mating effort, where the female’s eating of the gift reduces her likelihood of prematurely terminating sperm transfer. We test several ideas regarding sexual conflict in the nuptially feeding wood cricket Nemobius sylvestris. In this cricket, males pass two kinds of spermatophores to females: spermless microspermatophores and larger sperm‐filled macrospermatophores. Females may palpate the males’ forewing secretions as a possible additional form of nuptial feeding. We manipulated male mobility and female feeding regime to examine the effects on spermatophore transfer, macrospermatophore attachment duration, and palpations of the males’ forewings. Neither male confinement nor female feeding regime affected the occurrence of macrospermatophore transfer. Males transferred the macrospermatophore sooner to low‐food females than to high‐food females. Males that were freely mobile (unconfined and sham treatments) had longer macrospermatophore attachment durations than confined males, while female feeding regime did not affect attachment duration. The overall occurrence of female palpation was not significantly affected by female feeding regime. However, high‐food females were more likely to perform short palpations before microspermatophore transfer, while low‐food females were more likely to palpate after macrospermatophore transfer. Sexual conflict is evident in that males appear to guard against premature removal of the sperm‐filled macrospermatophore. Low‐food females appear to facilitate the early transfer of the macrospermatophore, while being more likely to perform relatively long post‐insemination palpations than better‐fed females.     

INDIVIDUAL VARIATION IN SPERM COMPETITION SUCCESS OF YELLOW DUNG FLIES,SCATOPHAGA STERCORARIA

Intraspecific variation in the proportion of offspring sired by the second male to mate with a female (P₂) is an aspect of sperm competition that has received little attention. We examined variation in the sperm competition success of individual male dung flies, Scatophaga stercoraria. In unmanipulated matings, copula duration was dependent on male size with smaller males copulating for longer. A principal component analysis was used to generate uncorrelated scores based on a male's size and copula duration. Using these scores demonstrated that P₂ values were dependent both on the relative size and copula durations of competing males. When copula duration was held constant, the success of an individual male increased as his body size, relative to the first male, increased. We interrupted copulations of “large” and “small” second males and fitted the resultant P₂ values to a linear model of sperm competition with unequal ejaculates. The data fit well to a model of sperm displacement in which sperm mix quickly on introduction to the sperm stores. Furthermore, they show that “large” males have a greater rate of sperm displacement than “small” males. The levels of prey availability during testis maturation may influence a male's success in sperm competition although his immediate mating history does not. We show why an understanding of variation in sperm competition success is important for understanding the mechanisms and evolutionary significance of sperm competition.     

Male and female effects on sperm precedence in the giant sperm species <Emphasis Type="Italic">Drosophila bifurca</Emphasis>

Sperm competition is expected to be a driving force in sexual selection. In internally fertilized organisms, it occurs when ejaculates from more than one male are present simultaneously within the female’s reproductive tract. It has been suggested that greater sperm size may improve the competitive ability of sperm, but studies provide contradictory results depending on the species. More recently, the role of females in the evolution of sperm morphology has been pointed out. We investigate here the male and female effects that influence sperm precedence in the giant sperm species, Drosophila bifurca Patterson & Wheeler. Females were mated with two successive males, and the paternity outcomes for both males were analyzed after determining sperm transfer and storage. We found very high values of last male sperm precedence, suggesting a strong interaction between rival sperm. However, the data also indicate high frequencies of removal of the sperm of the first male from the female reproductive tract prior to any interaction with the second male. This implies that successful paternity depends mainly on successful sperm storage. Knowing what happens to the sperm within females appears to be a prerequisite for disentangling post-copulatory sexual interactions between males and females.     

Paternity of offspring in multiply-mated,female crickets: the effect of nuptial food gifts and the advantage of mating first

The spermatophore transferred by male decorated crickets (Gryllodes sigillatus) includes a large gelatinous mass, the spermatophylax, that is consumed by the female after mating. This nuptial gift preoccupies the female while sperm are discharged from the remaining portion of the spermatophore, the sperm ampulla, into her reproductive tract. There is considerable variation in the mass of the spermatophylax, and about half of all males produce spermatophylaxes that are too small to ensure complete sperm transfer. We tested two hypotheses concerning the maintenance of this variation: (i) males trade-off investment in spermatophylaxes against copulation frequency; and (ii) males synthesize the largest spermatophylaxes of which they are physiologically capable. Males synthesizing large and small food gifts were permitted multiple mating opportunities with the same females, and allozyme markers were used to establish the paternity of offspring. There was a significant advantage to those males that mated first irrespective of gift size. This advantage probably arose, in part, because the sperm of first males would have had exclusive access to females'' eggs during the first 24 hours of oviposition, and underscores the benefits of matings with virgin females. The paternity of ''small-gift'' males increased with gift mass, but there was no such increase in ''large-gift'' males. This difference probably stems from the relationship between gift mass and sperm transfer: most of the gifts of the large-gift males would have been above the threshold needed to achieve complete inseminations, whereas those of small-gift males would have been below the threshold. Within mating-order positions, there was no significant difference in the paternity of large-gift and small-gift males, a result seemingly consistent with the ''trade-off'' hypothesis. However, there was no correlation between spermatophylax mass and male mating frequency, so that the mechanism by which small-gift males offset their fertilization disadvantage remains unknown.     

Old‐male paternity advantage is a function of accumulating sperm and last‐male precedence in a butterfly

Old‐male mating advantage has been convincingly demonstrated in Bicyclus anynana butterflies. This intriguing pattern may be explained by two alternative hypotheses: (i) an increased aggressiveness and persistence of older males during courtship, being caused by the older males' low residual reproductive value; and (ii) an active preference of females towards older males what reflects a good genes hypothesis. Against this background, we here investigate postcopulatory sexual selection by double‐mating Bicyclus anynana females to older and younger males, thus allowing for sperm competition and cryptic mate choice, and by genotyping the resulting offspring. Virgin females were mated with a younger virgin (2–3 days old) and afterwards an older virgin male (12–13 days old) or vice versa. Older males had a higher paternity success than younger ones, but only when being the second (=last) mating partner, while paternity success was equal among older and younger males when older males were the first mating partner. Older males produced larger spermatophores with much higher numbers of fertile sperm than younger males. Thus, we found no evidence for cryptic female mate choice. Rather, the findings reported here seem to result from a combination of last‐male precedence and the number of sperm transferred upon mating, both increasing paternity success.     

Spermatophore displacement and male fertilization success in the ground beetle Carabus insulicola

Sperm displacement is an effective male mating tactic of spermcompetition to ensure paternity. However, the effectivenessof sperm displacement may depend on defensive mating tacticsemployed by rival males and also may be affected by females.To test this hypothesis, I conducted double-mating experimentswith microsatellite paternity analyses using the ground beetleCarabus insulicola. First, I examined how often spermatophoredisplacement by the second male occurs. Spermatophore depositionsuccess of the second male was high (85%) as was that of thefirst male (100%), in which 80% of the second males displacedthe first male's spermatophore, suggesting that spermatophoredisplacement is a highly effective offensive tactic of spermcompetition in C. insulicola. Second, I examined the effectsof postcopulatory guarding by the first male and remating intervaland sperm utilization pattern of the female on fertilizationsuccess of the second male. P₂ values were moderate on average(0.30–0.57) and negatively correlated with postcopulatoryguarding duration of the first male. Sperm use by the femalewas temporally nonrandom, such that sperm of the second maletended to be used first, although there was no significant effectof female-remating interval. These results suggest that theeffectiveness of spermatophore displacement depends on postcopulatoryguarding by the first male and the sperm utilization patternof the female. These findings provide empirical evidence forthe conflicting nature of selection for male offensive and defensivetactics in sperm competition and significant contribution offemales therein.     

Female choice, male interference, and sperm precedence in the red-spotted newt

Darwin first identified female choice and male—male competitionas forms of sexual selection resulting in the evolution of conspicuoussexual dimorphism, but it has proven challenging to separatetheir effects. Their effects on sexual selection become evenmore complicated when sperm competition occurs because spermprecedence may be either a form of cryptic female choice ora form of male—male competition. We examined the effectsof tail height on male—male competition and female choiceusing the sexually dimorphic red-spotted newt (Notophthalmusviridescens viridescens). Experiment 1 examined whether maletail height influenced male mating success. Males with deeptails were more successful at mating with females than thosewith shallow tails. Successful, deep-tailed males also were bigger(snout-vent length; SVL) than unsuccessful, shallow-tailed males,but they did not vary in tail length or body condition. Of these,only tail height and tail length are sexually dimorphic traits.Experiment 2 tested the hypothesis that the differential successof males with deeper tails was due to female choice by examiningboth simultaneous female preference for association and sequentialfemale choice. We found no evidence of female choice. When maleswere not competing to mate with females, tail height did notinfluence male mating success. Successful males did not havedifferent SVL and tail lengths than unsuccessful males. Thus,tail height in male red-spotted newts appears to be an intrasexuallyselected secondary sexual characteristic. Experiment 3 usedpaternity exclusion analyses based on molecular genetic markersto examine the effect of sperm precedence on sperm competitionin doubly-mated females. Sperm precedence likely does not havea pervasive and consistent effect on fertilization success becausewe found evidence of first, last, and mixed sperm usage.     

Risk of Sperm Competition Mediates Copulation Duration, but not Paternity, of Male Burying Beetles

Males should increase their investment in ejaculates whenever they are faced with an increased risk of sperm competition. Burying beetles (Nicrophorus vespilloides), insects that breed on small vertebrate carcasses, offer an ideal model with which to examine sperm allocation tactics because females typically mate with many males prior to laying eggs. Males compete directly for control of carcasses, and males losing such contests often become satellites, lurking in the vicinity of the carcass and attempting surreptitious copulations with the resident female. We predicted that both the dominant resident male and the satellite male would increase their sperm allocation in the presence of the other, but that relative to dominant males, satellite males would allocate a greater number of sperm per ejaculate. We employed a repeated-measures design in which two full-sib rival males, differing only in their dominance status, were each mated a single time to a previously-inseminated female under two conditions, once in the absence of their rival and once in the presence of their rival. Satellite males exhibited longer copulation durations than dominant resident males when both males were present on a carcass. Copulation durations of dominant males did not differ in the presence or absence of satellite males. Contrary to expectation, the increased copulation durations of satellite males did not result in a greater share of paternity relative to dominant males. The absence of any discernible effect of increased copulation durations on paternity in satellite males could be due to post-copulatory preferences of females or, alternatively, satellite males may require longer durations of copulation to transfer the same amount of sperm as dominant males.     

A paternity advantage for speedy males? Sperm precedence patterns and female re-mating frequencies in a sexually cannibalistic praying mantid

Scramble competition polygyny is expected when females and/or resources are widely dispersed and not easily monopolized by males, or when there is an abundance of mates during an extremely restricted reproductive period. Additional factors such as first male sperm precedence or low female re-mating rate might further explain the propensity of males to engage in scramble competition. The sexually cannibalistic praying mantid Pseudomantis albofimbriata exhibits a polygynous mating system, where females exist in low-density populations and male competition manifests as the race to find females rather than as direct physical fighting. Here, we aim to determine whether there is a paternity advantage for the first-male to mate and/or a low frequency of female re-mating. First, we determined sperm precedence patterns in P. albofimbriata using the sterile male technique. Second, we tested the likelihood of female re-mating in P. albofimbriata by comparing the close-range approach behaviour and frequency of successful mating attempts for males when paired with virgin as opposed to recently mated females, and by comparing the frequency of long-distance male attraction between virgin and mated females. We found no paternity advantage for the first male to mate, rather a second male advantage. Although mated females were not rejected by males when approached from close-range, they were chemically unattractive to males searching from a distance. Since initial mate attraction in many praying mantids, including P. albofimbriata, is mediated via long-distance chemical communication, we believe the latter result is more ecologically relevant and therefore more important. These results suggest that the relatively low frequency of female re-mating observed in P. albofimbriata may be an additional factor driving scramble competition in this system.     

An integrative view of sexual selection in Tribolium flour beetles

Sexual selection is a major force driving the evolution of diverse reproductive traits. This evolutionary process is based on individual reproductive advantages that arise either through intrasexual competition or through intersexual choice and conflict. While classical studies of sexual selection focused mainly on differences in male mating success, more recent work has focused on the differences in paternity share that may arise through sperm competition or cryptic female choice whenever females mate with multiple males. Thus, an integrative view of sexual selection needs to encompass processes that occur not only before copulation (pre-mating), but also during copulation (peri-mating), as well as after copulation (post-mating), all of which can generate differences in reproductive success. By encompassing mechanisms of sexual selection across all of these sequential reproductive stages this review takes an integrative approach to sexual selection in Tribolium flour beetles (Coleoptera: Tenebrionidae), a particularly well-studied and economically important model organism. Tribolium flour beetles colonize patchily distributed grain stores, and juvenile and adult stages share the same food resources. Adults are highly promiscuous and female reproduction is distributed across an adult lifespan lasting approximately 1 year. While Tribolium males produce an aggregation pheromone that attracts both sexes, there appears to be little pre-mating discrimination among potential mates by either sex. However, recent work has revealed several peri-mating and post-mating mechanisms that determine how offspring paternity is apportioned among a female's mates. During mating, Tribolium females reject spermatophore transfer and limit sperm numbers transferred by males with low phenotypic quality. Although there is some conflicting evidence, male copulatory leg-rubbing appears to be associated with overcoming female resistance to insemination and does not influence a male's subsequent paternity share. Evidence suggests that Tribolium beetles have several possible post-mating mechanisms that they may use to bias paternity. Male sperm precedence has been extensively studied in Tribolium spp. and the related Tenebrio molitor, and several factors influencing male paternity share among a female's progeny have been identified. These include oviposition time, inter-mating interval, male strain/genotype, the mating regimen of a male's mother, male starvation, and tapeworm infection. Females exert muscular control over sperm storage, although there is no evidence to date that females use this to differentiate among mates. Females could also influence offspring paternity by re-mating with additional males, and T. castaneum females more readily accept spermatophores when they are re-mating with more attractive males. Additional work is needed to examine the possible roles played by both male and female accessory gland products in determining male paternity share. Sexual selection during pre-mating episodes may be reinforced or counteracted by peri- and post-copulatory selection, and antagonistic coevolution between the sexes may be played out across reproductive stages. In Tribolium, males' olfactory attractiveness is positively correlated with both insemination success and paternity share, suggesting consistent selection across different reproductive stages. Similar studies across sequential reproductive stages are needed in other taxa to provide a more integrative view of sexual selection.