Distinguishing seasonal androgen responses from male-male androgen responsiveness-revisiting the Challenge Hypothesis

Androgen levels show strong patterns throughout the year in male vertebrates and play an important role in the seasonal modulation of the frequency, intensity and persistence of aggression. The Challenge Hypothesis (Wingfield, J.C., Hegner, R.E., Dufty, A.M., Ball, G.F., 1990. The "Challenge Hypothesis": Theoretical implications for patterns of testosterone secretion, mating systems, and breeding strategies. Am. Nat. 136, 829-846) predicts that seasonal patterns in androgen levels vary as a function of mating system, male-male aggression and paternal care. Although many studies have addressed these predictions, investigators have often assumed that the ratio of the breeding season maximum and breeding baseline concentrations (termed "androgen responsiveness") reflects hormonal responses due to social stimulation. However, increasing evidence suggests that seasonal androgen elevations are not necessarily caused by social interactions between males. Here, we separate the seasonal androgen response (R(seasonal)) and the androgen responsiveness to male-male competition (R(male-male)) to begin to distinguish between different kinds of hormonal responses. We demonstrate that R(seasonal) and R(male-male) are fundamentally different and should be treated as separate variables. Differences are particularly evident in single-brooded male birds that show no increase in plasma androgen levels during simulated territorial intrusions (STIs), even though R(seasonal) is elevated. In multiple-brooded species, STIs typically elicit a rise in androgens. We relate these findings to the natural history of single- and multiple-brooded species and suggest a research approach that could be utilized to increase our understanding of the factors that determine different types of androgen responses. This approach does not only include R(seasonal) and R(male-male), but also the androgen responsiveness to receptive females (R(male-female)) and to non-social environmental cues (R(environmental)), as well as the physiological capacity to produce and secrete androgens (R(potential)). Through such studies, we can begin to better understand how social and environmental factors may lead to differences in androgen responses.     

Paternal investment and the human mating system

Paternal investment has long been considered responsible for the evolution of predominantly monogamous marriage in humans. However, male-male competition resulting in mate-guarding and male coercion could be equally important. In this review, I use a comparative approach to examine the effect of variation in human paternal investment on our mating system. I conclude paternal investment is important but so too is mate-guarding. I propose a model of our mating system incorporating both factors. Variation in the mating system is explained by variation in male resource control and contribution, resulting in ecologically imposed monogamy or polygyny, as predicted by the polygyny threshold model, as well as variation in male-male competition for status, resulting in socially imposed monogamy or polygyny.     

Nonbreeding season pairing behavior and the annual cycle of testosterone in male and female downy woodpeckers, Picoides pubescens

Studies in birds show that testosterone (T) concentrations vary over the annual cycle depending on mating system and life history traits. Socially monogamous species show pairing behavior throughout the year and low levels of male-male aggression and are underrepresented in these studies, yet the function of testosterone could be particularly important for sexual and social interactions occurring outside the breeding season. We measured fecal T concentrations over the annual cycle and the frequency of interactions between male and female downy woodpeckers (Picoides pubescens) from late fall through early spring. We validated the fecal assay by collecting blood in conjunction with a subsample of our fecal samples: fecal T correlated with circulating levels in the blood. The annual peak level of T in males was relatively low and short-lived, similar to that of other bird species with low levels of male-male aggression and high paternal care. The annual cycle of female T resembled the male pattern, and the ratio of male T to female T was close to 1.0. Likewise, the frequency of aggression among females was similar to the frequency among males. Overall, testosterone levels in both sexes were variable, even in winter. In other bird species, sexual behavior during nonbreeding periods correlates with circulating levels of T in males. Based on this observation, we tested the hypothesis that T in winter was positively related to the frequency of interaction between mated downy woodpeckers. The results showed no such relationship. We discuss this finding and further relate the annual cycle of T in both males and females to behaviors that appear to facilitate mate choice and retention of the pair bond during conspecific challenge.     

SPERMATOPHORE SIZE IN BUSHCRICKETS: COMPARATIVE EVIDENCE FOR NUPTIAL GIFTS AS A SPERM PROTECTION DEVICE

During courtship and copulation, males of many insect species provide the female with a nuptial gift of a prey item or synthesized material. These gifts may be explained as a form of paternal investment by increasing female reproductive output, or in terms of mating effort by increasing male fertilization success. These explanations, while not mutually exclusive, are controversial. While experimental studies examine the maintenance of nuptial gifts in single species, comparative studies are required to indicate more general evolutionary trends. Male bushcrickets provide females with a nuptial gift, a spermatophylax, which is transferred to females at mating along with the sperm-containing ampulla. Analysis of comparative data of 28 species of bushcrickets (Orthoptera: Tettigoniidae), reveals that male spermatophore size (spermatophylax and ampulla weight) is positively correlated with female refractory period, which, in turn, correlates with male fertilization success. Moreover, gift size (the spermatophylax) covaries with ejaculate size (the ampulla), which is consistent with the hypothesis that it serves as a sperm protection device. In contrast, there is no significant correlation between any measure of female fecundity and male spermatophylax size. This indicates that the variation in spermatophore size among bushcrickets is better explained by a mating-effort function than a paternal investment function.     

Social modulation of androgen levels in male teleost fish

Androgens are classically thought of as the sex steroids controlling male reproduction. However, in recent years evidence has accumulated showing that androgens can also be affected by the interactions between conspecifics, suggesting reciprocal interactions between androgens and behaviour. These results have been interpreted as an adaptation for individuals to adjust their agonistic motivation and to cope with changes in their social environment. Thus, male-male interactions would stimulate the production of androgens, and the levels of androgens would be a function of the stability of its social environment ['challenge hypothesis', Gen. Comp. Endocrinol. 56 (1984) 417]. Here the available data on social modulation of androgen levels in male teleosts are reviewed and some predictions of the challenge hypothesis are addressed using teleosts as a study model. We investigate the causal link between social status, territoriality and elevated androgen levels and the available evidence suggests that the social environment indeed modulates the endocrine axis of teleosts. The association between higher androgen levels and social rank emerges mainly in periods of social instability. As reported in the avian literature, in teleosts the trade-off between androgens and parental care is indicated by the fact that during the parental phase breeding males decreased their androgen levels. A comparison of androgen responsiveness between teleost species with different mating and parenting systems also reveals that parenting explains the variation observed in androgen responsiveness to a higher degree than the mating strategy. Finally, the adaptive value of social modulation of androgens and some of its evolutionary consequences are discussed.     

Is male plumage reflectance correlated with paternal care in bluethroats?

Although it is now well established that the conspicuous maleplumage colors of many birds have been subject to sexual selectionby female choice, it is still debated whether females matewith colorful males to obtain direct or indirect benefits.In species where males provide substantial parental care, femalesmay obtain direct benefits from mating with the males that are best at providing care. The good parent hypothesis suggeststhat male plumage coloration signals a male's ability to provideparental care. Alternatively, the differential-allocation hypothesissuggests that colorful males reduce their care in responseto increased investment by females mated to attractive males.We tested these hypotheses on the bluethroat (Luscinia s. svecica),a socially monogamous, sexually dichromatic bird, in which males have a colorful throat patch consisting of a structurallyderived blue area surrounding a melanin-based chestnut spot.Male plumage coloration was objectively quantified by use ofreflectance spectrometry. We found no evidence of a relationshipbetween male coloration of either the blue patch or the chestnutspot and the level of paternal care. Nor were there any correlationsbetween male coloration and body size or body condition. Thus, our study does not support the hypothesis that male colorationsignals male parental quality (the good parent hypothesis)or the hypothesis that colorful males reduce their care inresponse to increased investment by females (the differential-allocationhypothesis).     

Social correlates of fecal testosterone in male ursine colobus monkeys (Colobus vellerosus): the effect of male reproductive competition in aseasonal breeders

Male testosterone (T) levels are thought to be linked with the mating system, degree of parental care, and male–male aggression in reproductive contexts (The ‘challenge hypothesis’; Wingfield et al., 1990). In many species though, T increases associated with mating behavior cannot be separated from those associated with male–male aggression. We tested the challenge hypothesis on aseasonally breeding ursine colobus (Colobus vellerosus), where male–male competition is intense outside of mating contexts. Fecal samples (N = 109) were collected from > 27 subadult and adult males in seven groups during 13-months of research in Ghana in 2004–2005. Fecal T (fT) levels were determined by enzyme immunosorbant assays. Behavioral data was collected using focal-animal and ad libitum sampling. The number of receptive females in each group did not positively correlate with male fT. There was a trend for adult males to have higher fT than subadult males; however there was no effect of rank on fT. The level of male–male aggression experienced was positively correlated with fT and individual males showed higher mean fT during ‘challenge’ than during ‘non-challenge’ periods. The number of male incursions experienced positively correlated with fT whereas the number of between-group encounters did not. Males attempt to gain reproductive opportunities during incursions, thus these results support the ‘challenge hypothesis’ in C. vellerosus. Outside of mating contexts, higher male fT levels are associated with increased aggression. Male parental investment in the form of infant defense was associated with increased fT, rather than the decline expected from other forms of paternal care.     

The male and female perspective in the link between male infant care and mating behaviour in Barbary macaques

Infant care from adult males is unexpected in species with high paternity uncertainty. Still, males of several polygynandrous primates engage in frequent affiliative interactions with infants. Two non‐exclusive hypotheses link male infant care to male mating strategies. The paternal investment hypothesis views infant care as a male strategy to maximize the survival of sired offspring, while the mating effort hypothesis predicts that females reward males who cared for their infant by preferably mating with them. Both hypotheses predict a positive relationship between infant care and matings with a particular female. However, the paternal investment hypothesis predicts that increased matings come before infant care whereas the mating effort hypothesis predicts that infant care precedes an increase in matings. Both hypotheses are usually tested from the perspective of the proportion of matings and care that individual females engage in and receive, rather than from the perspective of the care and mating behaviour of individual males. We tested the relationships between care and mating from both female and male perspectives in Barbary macaques. Mating predicted subsequent care and care predicted subsequent mating when viewed from the male but not the female perspective. Males mainly cared for infants of their main mating partners, but infants were not mainly cared for by their likely father. Males mated more with the mothers of their favourite infants, but females did not mate more with the main caretakers of their infants. We suggest that females do not choose their mating partners based on previous infant care, increasing paternity confusion. Males might try to increase paternal investment by distributing the care according to their own instead of female mating history. Further, males pursue females for mating opportunities based on previous care.     

Fertility Advertisement in Birds: a Means of Inciting Male-male Competition?

Certain loud calls made by female red junglefowl and Lapland longspurs are given most frequently immediately after egg laying, when a copulation should have the highest probability of fertilizing the next egg to be laid. In these species there is considerable male-male interaction for access to fertilizable females, and males are attracted to or follow females making these calls. Based on our interpretation of these calls, we develop a general hypothesis to predict the pattern of occurrence of fertility advertisement both within and among bird species. We hypothesize that certain loud calls given by female birds before and during the egg-laying period are designed to advertise fertility and thereby incite male-male competition. This hypothesis predicts that calls advertising female fertility should most often occur soon after an egg is laid (i.e. during the period of highest fertility) but may also occur at any time during the female's fertilizable period. Such calls are unlikely to be given by females in strictly monogamous species (especially those with long-term pair bonds) because in these species each female usually mates with only a single male and extra-pair copulations are avoided. Although reports of loud female calls associated with copulation are rare in the literature, the 18 examples we found (including junglefowl and longspurs) were predominantly (15/18) in species adopting mating systems other than strict monogamy, and these calls were most commonly and disproportionately reported in multi-male mating systems. This form of “estrus” in birds may be widespread because few species appear to be strictly monogamous, but it will be difficult to study because the period of high fertility for female birds is so short.     

Effect of male body size on sperm precedence in the polyandrous butterfly Pieris napi L. (Lepidoptera: Pieridae)

Male Lepidoptera produce an ejaculate during copulation thatcontains both sperm and accessory gland nutrients and may functionas paternal investment and/or male mating effort Several studieshave examined how ejaculates function as paternal investment,but few have determined the influence of sperm competition onmale investment This study examines the effect of male bodysize on sperm precedence in the polyandrous butterfly Pierisnapi L. We used male body mass as an indicator of the size ofejaculate transferred and found that relative male size hada significant effect on paternity. The offspring of twice-matedfemales showed a low incidence of mixed paternity. Larger malesobtained the majority of fertilizations, and the degree of second-malesperm precedence was influenced by relative body size of matingmales. In general, second mates obtained fewer fertilizationsthe larger the size of the first mate. The interval betweenthe first and second mating was influenced by the size of thefirst male mate Females first mated to small males remated soonerthan females first mated to larger males Our results suggestthat large males may have a selective advantage over small maleswhen both a male's fertilization success and a female's refractoryperiod are influenced by the size of ejaculate transferred.Furthermore, the effect of male body size on the proportionof offspring sired lends support to the hypothesis that spermcompetition has played a major role in the evolution of ejaculatesize.     

Variation in fecal testosterone levels, inter-male aggression, dominance rank and age during mating and post-mating periods in wild adult male ring-tailed lemurs (Lemur catta)

In primate species exhibiting seasonal reproduction, patterns of testosterone excretion in adult males are variable: in some species, peaks correlate with female receptivity periods and heightened male-male aggression over access to estrous females, in others, neither heightened aggression nor marked elevations in testosterone have been noted. In this study, we examined mean fecal testosterone ( f T) levels and intermale aggression in wild adult male ring-tailed lemurs residing in three groups at Beza Mahafaly Reserve, Madagascar. Results obtained from mating and post-mating season 2003 were compared to test Wingfield et al. [1990. Am Nat 136:829-846] "challenge hypothesis", which predicts a strong positive relationship between male testosterone levels and male-male competition for access to receptive females during breeding season. f T levels and rates of intermale aggression were significantly higher during mating season compared to the post-mating period. Mean f T levels and aggression rates were also higher in the first half of the mating season compared with the second half. Number of males in a group affected rates of intermale agonism, but not mean f T levels. The highest-ranking males in two of the groups exhibited higher mean f T levels than did lower-ranking males, and young males exhibited lower f T levels compared to prime-aged and old males. In the post-mating period, mean male f T levels did not differ between groups, nor were there rank or age effects. Thus, although male testosterone levels rose in relation to mating and heightened male-male aggression, f T levels fell to baseline breeding levels shortly after the early mating period, and to baseline non-breeding levels immediately after mating season had ended, offsetting the high cost of maintaining both high testosterone and high levels of male-male aggression in the early breeding period.     

The evolution of indicator traits for parental quality: the role of maternal and paternal effects

In systems where individuals provide material resources to their mates or offspring, mate choice based on traits that are phenotypically correlated with the quality of resources provided is expected to be adaptive. Several models have explored the evolution of mating preference where there are direct benefits to choice, but few have addressed how a phenotypic correlation can be established between a male indicator trait and the degree of parental investment. We present a model with three quantitative traits: male and female parental investment and a potential male indicator trait. In our model, the expression of the "indicator" trait in offspring is affected by parental investment. These effects are referred to as maternal or paternal effects, or as "indirect genetic effects" when parental investment is heritable. With genetic variation for degree of parental investment, offspring harbor genes for parental investment that are unexpressed before mating but will affect the investment that they provide when expressed. Because the investment received from the parents affects the expression of the indicator trait, there will be a correlation between the genes for parental investment inherited and the degree of expression of the indicator trait in the offspring. The indicator trait is thus an "honest" signal for the degree of paternal investment.     

Testosterone, testes size, and mating success in birds: a comparative study

Reproductive behaviors of vertebrates are often underpinned by temporal patterns of hormone secretion. We investigated interspecific patterns of circulating testosterone in male birds to test the hypothesis that testosterone plays a crucial role in sexual selection as determined by degree of polygyny and extra-pair paternity. We predicted that the evolution of increased levels of polygyny and extra-pair paternity would have resulted in the evolution of increased levels of testosterone to allow males more efficiently to compete for mates. This hypothesis was tested in comparative analyses of 116 species of birds using Generalized Least Squares Models. We assessed the importance of latitudinal distribution, because this can confound the relationship between testosterone and mating success. There were weak positive phylogenetic correlations between measures of testosterone and estimates of mating success at the social level, but this association appeared to be confounded by latitudinal distribution, a significant correlate of testosterone titers. However, we found a significantly positive relationship between peak and residual peak testosterone (which is the peak testosterone level that is controlled for the baseline level) and extra-pair paternity independent of latitude. These results suggest that selection pressures arising from social and sexual mating differently affected testosterone levels with the former being mediated by factors associated with latitudinal distribution. An analysis of residual testes size revealed a positive association between peak and residual testosterone and testes size relative to body size. In a path analysis, we show that relative testis size primarily evolved in association with intense sperm competition and thus high sperm production, and these mechanisms had a secondary impact on blood testosterone levels at a phylogenetic scale. Our results suggest that sperm competition has played an important role in the evolution of reproductive mechanisms in birds.     

Mating season aggression and fecal testosterone levels in male ring-tailed lemurs (Lemur catta)

The challenge hypothesis (J. C. Wingfield, R. E. Hegner, B. G. Ball, and A. M. Duffy, 1990, Am. Nat. 136, 829-846) proposes that in birds, reptiles, and fish, "the frequency or intensity of reproductive aggression as an effect of T[estosterone] is strongest in situations of social instability, such as during the formation of dominance relationships, the establishment of territorial boundaries, or challenges by a conspecific male for a territory or access to mates" (p. 833). To determine the extension of this hypothesis to mammalian species, we tested predictions of the hypothesis in a nonpaternal, seasonal breeding, prosimian primate (ring-tailed lemurs, Lemur catta). Semi-free-ranging males were studied during periods of social stability (premating period) and instability (mating period). The annual mating season consists of several days during which males fight for access to promiscuous group females as each individually becomes sexually receptive for 1 day. Male rates of aggression were compared to fecal testosterone levels within premating and mating periods. In the premating period male rate of aggression was not significantly correlated with testosterone level. By contrast, during the mating season testosterone and aggression levels were positively and significantly correlated. However, on days just preceding estrus, male rate of aggression was not significantly correlated with testosterone, but on days of estrus, when aggressive challenges peaked sharply, testosterone and aggression were highly positively correlated. These results suggest that the challenge hypothesis applies to mammals as well as to birds, reptiles, and fish. In addition, elevations in testosterone were tightly circumscribed around days of estrus, suggesting a compromise between costs and benefits of elevated testosterone levels.     

Primate sexual swellings as coevolved signal systems

Many female catarrhine primates possess visually conspicuous organs that apparently function to increase the sexual interest of adult male conspecifics around the time the female is ovulating—i.e. sexual swellings. The hypothesized functional benefits for both sexes of these sexual swellings are reviewed (honest signaling; paternity confusion; paternity confidence and paternal investment; protection; incitement of precopulatory male-male competition; and postcopulatory sexual selection), as well as an additional hypothesis that has not yet been applied to this problem (sensory exploitation). Currently available evidence is presented that supports or fails to support each of these hypotheses. Predictions associated with broad groupings of these hypotheses, which could be tested in noninvasive field studies, are then presented. Ecological circumstances are discussed that could have led to differential mating success among female primates, and hence to sexual selection on females and directional evolution of sexual swellings. It is concluded that the available evidence does not support the paternity confidence-paternal investment hypothesis; that the paternity confusion hypothesis lacks empirical support, but could still be viable; and that insufficient data exists at present to rigorously test the other hypotheses. The ecological factors that may have led to differential reproductive success among females as a function of mating frequency or mate choice likewise require further empirical investigation.     

Exogenous testosterone inhibits several forms of male parental behavior and stimulates song in a monogamous songbird: the blue-headed vireo (Vireo solitarius)

Natural variation in circulating testosterone is thought to play a role in creating inter- and intraspecific variation in paternal care in birds. Experimental elevation of plasma testosterone in males has reduced rates of male care in many species. However, a small number of studies suggest that male care is not inhibited by testosterone when there appears to be strong selection for male care. Both genetic monogamy and male incubation are thought to be the result of selection for biparental care, yet the sensitivity of male care to inhibition by T had not been examined in a species with both characteristics. Male blue-headed vireos have been shown to be strictly monogamous and provide extensive parental care, including incubation. Males received subcutaneous implants of testosterone, flutamide (an anti-androgen), or empty controls. Consistent with previous studies, testosterone treatment decreased the relative contribution to incubation by males and increased the average duration that the nest was left unattended during incubation, relative to control and flutamide treatments. The relative and absolute rates of feeding offspring and nest sanitation by T-treated males also decreased relative to controls. Flutamide-treated males incubated eggs and brooded young more than testosterone-treated males, with control males performing at intermediate values. Testosterone-treated males sang more often and at higher rates, and flutamide-treated males sang less often, than controls. These results demonstrate that even in a strictly monogamous songbird with biparental incubation, all forms of paternal care remain sensitive to elevated plasma testosterone, suggesting a shift from parental effort to mating effort.     

Mortality costs of sexual selection and parental care in natural populations of birds

Abstract Is the cost of reproduction different between males and females? On the one hand, males typically compete intensely for mates, thus sexual selection theory predicts higher cost of reproduction for males in species with intense male‐male competition. On the other hand, care provisioning such as incubating the eggs and raising young may also be costly, thus parental care theory predicts higher mortality for the care‐giving sex, which is often the female. We tested both hypotheses of reproductive costs using phylogenetic comparative analyses of sex‐specific adult mortality rates of 194 bird species across 41 families. First, we show that evolutionary increases in male‐male competition were associated with male‐biased mortalities. This relationship is consistent between two measures of mating competition: social mating system and testis size. Second, as predicted by the parental cost hypothesis, females have significantly higher adult mortalities (mean ± SE, 0.364 ± 0.01) than males (0.328 ± 0.01). However, the mortality cost of parental care was only detectable in males, when the influence of mating competition was statistically controlled. Taken together, our results challenge the traditional explanation of female‐biased avian mortalities, because evolutionary changes in female care were unrelated to changes in mortality bias. The interspecific variation in avian mortality bias, as we show here, is driven by males, specifically via the costs of both mating competition and parental care. We also discuss alternative hypotheses for why most birds exhibit female‐biased mortalities, whereas in mammals male‐biased mortalities predominate.     

Male genotype affects female fitness in a paternally investing species

Male nutrient provisioning is widespread in insects. Females of some species use male-derived nutrients for increased longevity and reproductive output. Despite much research into the consequences of paternal nutrient investment for male and female fitness, the heritability, and therefore the potential of this trait to respond to selection, has rarely been examined. Males of several butterfly species provide the female with nutrients in the spermatophore at mating. Females of the green-veined white butterfly Pieris napi (Lepidoptera: Pieridae) use male donations both for developing eggs (resulting in higher lifetime fecundity of multiply mated females), but also for their somatic maintenance (increasing longevity). Using half-sib, father-son regression and full-sib analyses, I showed that paternal nutrient investment is heritable, both in terms of the absolute but also the relative size of the spermatophore (controlling for body size). Male size and spermatophore size were also genetically correlated. Furthermore, a separate study showed male genotype had a significant effect on female longevity and lifetime fecundity. In contrast, male genotype had no influence on the immediate egg-laying rate of females following mating, suggesting limited scope for male manipulation of immediate female oviposition. These results indicate that females may derive both direct (increased lifetime fecundity and longevity) and indirect (sons with greater reproductive success) fitness benefits from paternal nutrient donations in this species.     

An experimental test of condition-dependent male and female mate choice in zebra finches

In mating systems with social monogamy and obligatory bi-parental care, such as found in many songbird species, male and female fitness depends on the combined parental investment. Hence, both sexes should gain from choosing mates in high rather than low condition. However, theory also predicts that an individual's phenotypic quality can constrain choice, if low condition individuals cannot afford prolonged search efforts and/or face higher risk of rejection. In systems with mutual mate choice, the interaction between male and female condition should thus be a better predictor of choice than either factor in isolation. To address this prediction experimentally, we manipulated male and female condition and subsequently tested male and female mating preferences in zebra finches Taeniopygia guttata, a songbird species with mutual mate choice and obligatory bi-parental care. We experimentally altered phenotypic quality by manipulating the brood size in which the birds were reared. Patterns of association for high- or low-condition individuals of the opposite sex differed for male and female focal birds when tested in an 8-way choice arena. Females showed repeatable condition-assortative preferences for males matching their own rearing background. Male preferences were also repeatable, but not predicted by their own or females' rearing background. In combination with a brief review of the literature on condition-dependent mate choice in the zebra finch we discuss whether the observed sex differences and between-studies differences arise because males and females differ in context sensitivity (e.g. male-male competition suppressing male mating preferences), sampling strategies or susceptibility to rearing conditions (e.g. sex-specific effect on physiology). While a picture emerges that juvenile and current state indeed affect preferences, the development and context-dependency of mutual state-dependent mate choice warrants further study.     

The incidence of sperm displacement in insects: four conjectures, one corroboration

The paper tests, by the comparative method, four hypotheses to explain why the degree of sperm displacement differs in different insect species. It summarizes evidence for 57 species. Here are the four hypotheses: species with mating plugs should show low sperm displacement, and species without plugs high displacement (Boorman & Parker); species with spheroid spermathecas should show low displacement, species with elongate spermathecas high displacement (Walker); species with low paternal investment should show low sperm displacement, species with high paternal investment high displaycement (Gwynne); and species with low natural mating frequencies should show low displacement, species with high mating frequencies high displacement (this paper). The spermathecal shape hypothesis is of limited theoretical plausibility; but the other ideas are theoretically possible. The evidence, if numbers of species are counted, generally fits all four theories; but, in a statistically more rigorous cladistic test, it significantly supports only the mating frequency theory.