Paternity in naturally-occurring Utetheisa ornatrix (Lepidoptera : Arctiidae) as estimated using enzyme polymorphism

Paternity of offspring in natural populations of insects has received little attention due to the difficulties inherent in following females and sampling each of their mates. Here, an existing statistical technique is modified to estimate paternity based on allozyme variation found in the female and her last mate, thus allowing paternity in nature to be studied by collecting copulating pairs of insects. Using this technique, paternity was investigated in naturally-occurring females of the arctiid moth Utetheisa ornatrix. These females mate promiscuously: upon dissection, they were found to contain up to 13 spermatophores. Statistical paternity estimation revealed considerable variation in the share of offspring sired by the female's last mate: approximately 25% of the males sired all the offspring, while another 25% fathered no offspring; the remainder sired at least some offspring. The proportion of offspring sired by the last male did not correlate with latency to oviposition, the extent of previous mating by the female, or male wing length. Male U. ornatrix are known to make substantial nuptial investments during mating, and this study shows that mating males frequently sire few or no offspring. Thus, male moths stand a chance of being cuckolded.     

Prolonged mating in the milkweed leaf beetle Labidomera clivicollis clivicollis (Coleoptera: Chrysomelidae): a test of the “sperm-loading” hypothesis

Summary Female milkweed leaf beetles (Labidomera clivicollis clivicollis) frequently mate with more than one male, and pairs form mating associations which last for up to 42 h in the field. I tested the hypothesis that males remaining with females for long periods of time benefit by numerically overwhelming the sperm of their competitors. Male L.c.clivicollis copulated intermittently with females throughout an 11 hour period in the laboratory. When virgin females were allowed a single copulation, 94.3% of the sperm they received were located in the spermatheca immediately afterward. Males were not sperm-depleted, for they had large numbers of sperm available after one copulation (mean=230,000±43,200); the maximal number of sperm a male transferred to a female in 24 h was 30,500. There was a positive linear relationship between the number of sperm transferred and time up to 24 h after mounting (r ²=0.178, P<0.003). These data suggest that males transfer increasing numbers of sperm throughout a 24-h-period. Mating duration was the most important determinant of paternity when females were placed with one male for 24 h and another male for 6 hours. Females whose first matings were longer showed first male sperm predominance (as determined by starch-gel electrophoresis), while females whose second matings were longer showed last male sperm predominance. In view of these data, it is puzzling that males do not inseminate with large numbers of sperm immediately after mounting the female. It is possible that female refractory behaviors make insemination difficult and favor prolonged mating by male milkweed leaf beetles.     

Factors influencing paternity in multiply mated female red-sided garter snakes and the persistent use of sperm stored over winter

In some species, sperm is stored within the female reproductive tract for months to years, and yet remains viable to fertilize eggs and produce offspring. Female red-sided garter snakes store sperm for over 7 months of winter dormancy. In previous work, we demonstrated that these stored sperm account for an average of 25 % paternity of a litter when the female mates with a male at spring emergence. Here, we tested whether last-male sperm precedence was prevalent when a female mates with two males during the spring. On average, paternity was shared equally among the first (P₁ proportion of paternity of the first male to mate) and second males (P₂) to mate in the spring, and stored sperm (P_ss), but the variance in paternity was high. Thus, last male sperm precedence may diminish when a female has more than two mates. Male size did not affect paternity, but, as the interval between matings increased, P₁ increased at the expense of P_ss. Interestingly, as the second spring male’s copulation duration increased, P₁ also increased at the expense of P₂. This result suggests that female influence over sperm and/or copulatory plug transfer during matings may also affect which male fathers her offspring in response to coercive matings as we assisted females to mate for their second mating. Finally, all females were spring “virgins”; consequently, sperm stored from autumn matings (and/or previous spring matings) remain competitive even when faced with two rivals in sperm competition and is likely the driver of the evolution of sperm longevity.     

Sperm competition as a mechanism of female choice in the field cricket,Gryllus bimaculatus

Summary While traditionally viewed as an extension of intermale competition, mechanisms of sperm competition may be used by multiply mating females for mate choice. In the field cricket G. bimaculatus sperm were shown to mix in the spermatheca. The proportion of offspring sired by the second male increased with spermatophore attachment duration and, therefore, the number of sperm transferred. There was no second male advantage for single matings after an initial double mating. However, the proportion ofoffspring sired by the second male increased in proportion to the number of times he mated such that second males mating three times after an initial double mating had the advantage at fertilization. The data suggested that sperm were utilized in proportion to their numerical representation in the spermatheca. The mechanism of sperm precedence may, therefore, be one of sperm dilution. Female G. bimaculatus may control the degree of sperm competition as a mechanism of mate choice. By accepting large quantities of sperm from chosen males they may determine the paternity of their offspring by diluting out the sperm stored from previous matings.     

Positive association between social and extra-pair mating in a polygynous songbird, the dickcissel (Spiza americana)

In polygynous species, males appear to gain additional offspring by pairing with multiple females simultaneously. However, this may not be true if some females copulate outside of the social pair bond. Polygynous males could experience lower paternity because of trade-offs among gaining multiple social mates, guarding fertility with these mates, and pursuing extra-pair matings. Alternatively, polygynous males could simultaneously gain extra social mates and have high paternity, either because of female preferences or because of male competitive attributes. We tested four predictions stemming from these hypotheses in a facultatively polygynous songbird, the dickcissel (Spiza americana). Unlike most previous studies, we found that males with higher social mating success (harem size) also tended to have higher within-pair paternity and that the number of extra-pair young a male sired increased significantly with his social mating success. Females that paired with mated males were not more likely to produce extra-pair young. In contrast, extra-pair paternity was significantly lower in the nests of females whose nesting activity overlapped that of another female on the same territory. This pattern of mating was robust to differences in breeding density. Indeed, breeding density had no effect on either extra-pair mating or on the association between polygyny and paternity. Finally, nest survival increased with harem size. This result, combined with the positive association between polygyny and paternity, contributed to significantly higher realized reproductive success by polygynous male dickcissels.     

Female benefit,male risk: Polyandry in the true armyworm Pseudaletia unipuncta

In Lepidoptera polyandry is common and females may increase their lifetime reproductive output through repeated matings if they acquire essential resources from male ejaculates. However, the paternity of males mating with previously-mated females is far from assured unless sperm precedence is absolute. In this study on the polyandrous armyworm, Pseudaletia unipuncta, we used two strains of male (the black-eyed wild type and a red-eyed homozygous, recessive mutant), mated with red-eyed females, to determine (i) whether male investment has any impact on female reproductive output, and (ii) if females do benefit from multiple matings, to what extent males fertilize the eggs to which they contributed. Multiple mating resulted in a significant increase in both the fecundity and longevity of females. However, the degree of sperm precedence (those eggs fertilized by the second male) varied from 0–100%, but was not affected by either male size or age, or by the duration of copulation. In cases where sperm precedence was <50% (x = 12%) females produced significantly more eggs (1384 versus 940) prior to the second mating than females where sperm precedence was >50% (x = 89%), indicating that the quality of the first mating influenced the fertilization success of the female's second mate.     

Sexual conflict over sperm ejection in monogamous pairs of kittiwakes<Emphasis Type="Italic"> Rissa tridactyla</Emphasis>

Socially monogamous partners suffer conflicting interests concerning various aspects of reproduction such as parental care, copulation and fertilization. Female black-legged kittiwakes commonly eject their mates' sperm immediately following copulations. Because sperm ejection reduces male sperm competitiveness and paternity assurance, males and females have conflicting interests as regards sperm ejection. Males whose mates ejected their sperm at least once remained longer on their mates' backs after the last insemination which apparently prevented the females from ejecting sperm. These results suggest that compelling females to retain their sperm may be a previously unidentified tactic employed by males to assure their paternity. Females tried to prevent their mates from witnessing sperm ejection by ejecting sperm after their mates departed from the nest. Females were more likely to eject sperm when they terminated the copulations by unbalancing the male. The conflict over sperm ejection was related to the ability of the females to end the copulations which covaried with the body mass of their mates. These findings suggest that conflicts in monogamous pairs also exist over the disposition of sperm.Communicated by C.R. Brown     

Ectomised conductors in the golden orb-web spider, Nephila plumipes (Araneoidea): a male adaptation to sexual conflict?

Male genitalia may facilitate sperm protection by acting as a plug that prevents or hinders future matings. The pedipalps (intromittant organs) of males of the orb-web spider, Nephila plumipes, have a conductor with a peculiarly curved ending and a triangular process near the terminal end. The tip of the conductor, including the process, breaks during most matings and remains inside the female genital tract. We explored the possible function of the conductor as a mating plug using the double-mating sterile-male technique. Our data are not consistent with a plug function because males use only one pedipalp in each mating, thus leaving an unobstructed insemination duct available for future matings; conductors of males mating with virgin females are not more likely to break than those of males mating with mated females, and second males show no preference for used or unused spermathecae. In addition, males that inserted their palp in the insemination duct that contained a tip of the conductor from a previous male obtained a share in the paternity of the female's clutch of eggs. Interestingly, the conductor is more likely to break if it is inserted in an unused spermatheca. We argue that several lines of evidence suggest that the conductor breaks as a result of intersexual conflict over the duration of copulation.     

Underwater oviposition in a damselfly (Odonata: Coenagrionidae) favors male vigilance,and multiple mating by females

Summary Female Enallagma hageni oviposit underwater where they are inaccessible to males. I demonstrate that males guard submerged females rather than perch sites, and are behaviorally distinct from lone males at the water. In contrast to lone males, which always attempt to copulate with females presented to them, guarding males exhibit a conditional latency to remating which corresponds closely to the time required by females to oviposit a complete clutch of eggs. By ovipositing underwater, females decrease the risk that their eggs become exposed. Risks associated with submerged oviposition favor both mate guarding, and multiple, within-clutch matings by females. Both guarding mates and lone males rescue females that float on the water surface as the result of improper resurfacing. Such behavior reduces the mortality risk to females from 0.06 to 0.02 per oviposition bout. By remating between bouts, females benefit from the additional vigilance of lone males, who rescue floating females 1.4 times as often as original mates. A second consequence of multiple mating is an increase in the selective advantage of vigilance by mates. Because receptive females become scarce by early afternoon, whereas male density remains high, a male has little (3%) chance of encountering a second receptive female that day. However, he incurs a 42% risk of losing fertilizations if he abandons a mate. For male E. hageni mate guarding functions in the context of both natural and sexual selection. It insures that a mate lives to lay a complete egg clutch in addition to protecting a male's sperm investment.     

Genetic analysis of parentage within experimental populations of a male dimorphic beetle,<Emphasis Type="Italic"> Onthophagus taurus</Emphasis>, using amplified fragment length polymorphism

We used the multilocus DNA fingerprinting technique, amplified fragment length polymorphism, to examine parentage of 902 offspring from eight experimental populations of the dung beetle, Onthophagus taurus. The males of this species exhibit a suit of morphological and behavioural traits that characterise alternative mating tactics. Hornless minor males sneak copulations with females that are guarded by horned major males. Our aims were to provide a prospective assessment of the potential role of frequency dependence in the maintenance of alternative mating tactics, to assess the levels of polyandry, and to determine the patterns of sperm usage by multiply mated females. The average proportion of offspring sired by major and minor males did not co-vary with the relative frequency of each morph present in experimental populations. However, there was some indication that the effective mating frequency (number of females producing offspring sired by a given male) of major and minor males may exhibit frequency dependence. Mating success of both male types declined with increasing numbers of major males. Paternity was positively associated with effective mating frequency. Females produced offspring sired from between one and eight males and, on average, paternity was distributed equally amongst a females mates, regardless of the number of males mated. Differences in fertilisation success among males were not associated with alternative male phenotypes. Neither did a males fertilisation success depend on his genetic dissimilarity with the female. These results are discussed in the context of the evolution of alternative mating strategies, and mechanisms of postcopulatory sexual selection.Communicated by N. Wedell     

Paternity analysis of two male mating tactics in the fiddler crab, Uca mjoebergi

The fiddler crab Uca mjoebergi mates both underground in male-defended burrows and on the surface near female-defended burrows. The reproductive tract of Uca species facilitates last-male precedence, suggesting that males that do not guard-mated females are likely to gain very little paternity if the female re-mates with another male. Here, we test the reproductive success of burrow and surface matings using paternity analysis. We found that 100?% of the females that mated in burrows extruded a clutch of eggs. Furthermore, we show conclusively, for the first time in a fiddler crab species, that last-male sperm precedence results in the majority of the female’s eggs being fertilised by the burrow-mated male. In contrast, surface matings resulted in significantly fewer females extruding eggs (5.6?%). Paternity analysis also revealed that more than half of the clutches from burrow-mated females showed low levels of extra-pair paternity from previous matings. Although multiple matings appear common in U. mjoebergi, burrow-mated males that guard females are guaranteed a successful mating with extremely high rates of assured paternity. Surface matings therefore appear to be an opportunistic tactic that may increase male reproductive success in a highly competitive environment.     

Paternity assurance by “helpful” males: adaptations to sperm competition in burying beetles

Summary Male burying beetles invest parentally by participating in the burial of a carcass and in provisioning and guarding the larvae that come to the carcass. Since most of the females arriving at a carcass have stored fertile sperm within their spermathecae, sperm transferred by such helpful males must compete with other males' ejaculates for the fertilization of the female's eggs. We showed that these males are able to achieve a high level of paternity (mean=92%). The mechanism they employ is a repeat-mating tactic, i.e., the female is mated very frequently shortly before and during oviposition. Repeated matings are essential for a high reliability of paternity, since single copulations result in the fertilization of only a very small proportion of the female's eggs.     

Determinants of paternity success in the spider Pholcus phalangioides (Pholcidae: Araneae): the role of male and female mating behaviour

In double mating experiments, we examined whether and to what extent various male and female behavioural traits influence the course of mating and fertilization success in the cellar spider. In males, we focussed on pre-copulatory behaviour and on the rhythmic twisting movements that the male performs with his pedipalps during copulation. In females, we investigated remating decisions and the effect of female termination of copulation. Second males fertilized a high proportion of the eggs (P ₂: median 89%) despite much shorter second matings, with high variation in relative paternity success. The number of pedipalp movements (PPMs) of either male was a better predictor of paternity than copulation duration. Our results suggest that in second matings, PPMs help to remove sperm from previous males, whereas in first matings a high number of PPMs enhances fertilization success, either due to numerical sperm competition or cryptic female choice. Furthermore, we found a negative male age effect on paternity in second matings, implying that age-related deterioration of spermatozoa may promote variation in fertilization success. Female receptivity decreased significantly in second matings; only 70% of the females remated. Females that accepted a second copulation were found to terminate these much earlier and with higher probability than first matings. This suggests that the intensity of conflict between the sexes is higher in second matings. Increased intensity of sexual conflict may be responsible for stronger selection on male traits, as pre-copulatory behaviour and age only affected male copulatory performance and paternity in second matings. Electronic Publication     

Variation in copulation duration in Mnais pruinosa pruinosa Selys (Odonata: Calopterygidae)

Summary The Japanese calopterygid damselfly Mnais pruinosa pruinosa shows three distinct copulation durations (Siva-Jothy and Tsubaki 1989). A variety of factors which might influence copulation duration were investigated. Results indicate that the temperature in the vicinity of copulating pairs, the rate at which males encountered receptive females, territory quality, the rate of agonistic interactions between males, female gravidity and female willingness to oviposit have no effect on copulation duration. The most significant factor was the location of the site at which males captured their mates. We suggest that under natural conditions the location of the capture site provides males with reliable, indirect information about a female's intention to oviposit: it is important for males of this species to acquire such information since sperm precedence, and therefore reproductive success, is in part dependant on the interval experienced by females between copulation and subsequent oviposition. The results are discussed and it is suggested that the three observed mate-securing tactics of M.p. pruinosa are facultative and information-dependant.     

Female choice in a promiscuous wild guinea pig,the yellow-toothed cavy (<Emphasis Type="Italic">Galea musteloides</Emphasis>)

Promiscuity is traditionally considered to increase only male reproductive success but, more recently, female benefits are also assumed to be the driving force for promiscuous mating. The yellow-toothed cavy (Galea musteloides) is characterised by an extremely high degree of multiple paternity (>80%), and females have greater offspring viability after mating multiple males. However, so far it is not clear whether or not it is the female's decision that leads to mating with more than one male. To elucidate the female's role in bringing about promiscuity, female yellow-toothed cavies were given the choice between four different males each, in a mate-choice apparatus that simultaneously prevented monopolisation and harassment of the females by the males. In 10 of the 12 choice tests, mating occurred. Nine of these ten females actively sought copulations with more than one male, and their mating behaviour was displayed in a way that might have favoured the mixing of sperm. At the same time, they significantly preferred heavier and more frequently courting males. These results show that female yellow-toothed cavies are actively involved in mating with more than one male. Thus, the present study is the first to show that in a species in which females produce more viable offspring as a consequence of polyandrous mating, these females are indeed motivated to actively bring about promiscuity. Nevertheless, females are selective in the choice of their mates. Thus, both sperm competition and direct female choice seem to be important for the increased offspring viability due to promiscuous mating.     

Determinants of mating success in the golden hamster (Mesocricetus auratus) IV. Sperm competition

Summary This study was designed to clarify several aspects of sperm competition in the golden hamster (Mesocricetus auratus). By mating females homozygous for a recessive coat color gene with two males, one homozygous for the dominat gene (agouti) and one homozygous for the recessive (cream), paternity of the resulting young could be determined by day 2 postpartum. When females received 5 consecutive ejaculations from a male of one strain followed by 5 ejaculations from a male of the other strain, there was a significant advantage with respect to litter composition for the male ejaculating first. A mechanism for the first male mating advantage was suggested by the results of an experiment in which the time interval between first and second males was varied. Delays of 30 min between first and second males significantly increased the proportion of young sired by the first male. With a delay of 8 h, the first male sired all of the young. Delays between matings by males of the two strains had greater effects when mating tests began around the time of ovulation, rather than several hours before. Ejaculations in excess of those required for pregnancy initiation were shown to have a two-fold effect. They afforded the first male a measure of protection against displacement by a subsequent male. Conversely, prolonged copulation by the second male resulted in significant displacement of the sperm of a predecessor. The protective effects of multiple ejaculations by the first male were mediated by two mechanisms: the delay in introduction of competing sperm and an increase in the relative concentration of the first male's sperm. Prolonged copulation by the second male also appeared to function through its effect on relative sperm concentrations. Males of these genotypes differed with respect to the relative fertilizing capacity of their cjaculates. When ejaculations from males of the two strains were alternated (thus minimizing mating order effects), agouti males sired over 90% of the young. The time during which mating tests were conducted (relative to the occurrence of ovulation) had no effect on the relative performance of the strains. The effects of prolonged copulation and first male mating advantage demonstrated in this study are consistent with observations of mating behavior under semi-natural conditions. Thus, it appears that patterns of sperm allocation and manipulation, male and female mating strategies, and mating systems have coevolved.     

Sperm competition in deer mice (Peromyscus maniculatus bairdi)

Summary Six experiments were conducted to determine the effects of mode of estrus (cycling versus postpartum) and intervals between matings on sperm competition in deer mice. When there was no lengthy interval between matings by different males, sperm competition matings by different males, sperm competition with females in postpartum estrus was the same as that with females in cyclin estrus (i.e., no male order effect). Imposition of 4 h delays between males did not engender a mating advantage when females were in postpartum estrus. When 4 h intervals were imposed with females in cycling estrus and tests were begun relatively early in the dark phase, there was a secondmale advantage with respect to the number of pups sired. When minimal delays were imposed with females in cycling estrus the time of testing within the dark phase did not affect the pattern of sperm competition, but larger litters were born following matings later in the dark phase. When 4 h delays were imposed with the first male mating late in the dark phase a first-male advantage resulted. It is hypothesized that for females in cycling estrus there is a period of peak fertilizability. There is no straight-forward effect of delays between males per se; rather, effects must be analyzed in relation to the period of peak fertilizability. The timing of insemination appears less critical for postpartum females.     

Why do female mice mate with multiple males?

Females often show multi-male mating (MMM), but the adaptive functions are unclear. We tested whether female house mice (Mus musculus musculus) show MMM when they can choose their mates without male coercion. We released 32 females into separate enclosures where they could choose to mate with two neighboring males that were restricted to their own territories. We also tested whether females increase MMM when the available males appeared unable to exclude intruders from their territories. To manipulate territorial intrusion, we introduced scent-marked tiles from the neighboring males into males' territories, or we rearranged tiles within males' own territories as a control. Each female was tested in treatment and control conditions and we conducted paternity analyses on the 57 litters produced. We found that 46 % of litters were multiply sired, indicating that multiple paternity is common when females can choose their mates. Intrusion did not increase multiple paternity, though multiple paternity was significantly greater in the first trial when the males were virgins compared to the second trial. Since virgin male mice are highly infanticidal, this finding is consistent with the infanticide avoidance hypothesis. We also found that multiple paternity was higher when competing males showed small differences in their amount of scent marking, suggesting that females reduce MMM when they can detect differences in males' quality. Finally, multiple paternity was associated with increased litter size but only in the intrusion treatment, which suggests that the effect of multiple paternity on offspring number is dependent on male–male interactions.     

Dispersal: an alternative mating tactic conditional on sex ratio and body size

Summary Small male milkweed beetles are less successful at obtaining mates than are larger males. Larger males usually win fights and prevent smaller males from obtaining mates and from choosing larger more fecund females as mates. When sex ratios are male-biased, smaller males are particularly likely to experience these mating disadvantages. It follows that smaller males should be especially responsive to their local competitive environment and behave so as to minimize the mating disadvantages of their smaller size. This paper tests the hypothesis that smaller males disperse from host plant patches with male-biased sex ratios and remain in patches with female-biased sex ratios more readily than larger males.Results show both larger and smaller males disperse from patches with male-biased sex ratios more frequently than from patches with femalebiased sex ratios. As predicted, however, small males are more likely to disperse from patches with male-biased sex ratios and remain in patches with female-biased sex ratios than are larger males.The data also show that smaller males dispersing from patches with male-biased sex ratios obtain more matings than non-dispersing males.For milkweed beetles, moving between patches can be viewed as an alternative mating tactic conditional on male body size and local sex ratio.     

Sperm competition and the level of polyandry in a bushcricket with large nuptial gifts

Variation in paternity due to sperm competition or post-copulatory female choice has a major influence on animal mating system evolution and on the levels of genetic variability in natural populations. However, there are relatively few studies comparing the outcome of sperm-competition experiments in the laboratory with natural variation in polyandry among families from the field. In the bushcricket Ephippiger ephippiger, females mate multiply, and the males provide them with a large, nutritious, and probably expensive, donation at mating. We examined paternity in a series of laboratory matings, where females mated with two males, and amongst a series of families collected from a natural population. In the laboratory, paternity was highly bimodally distributed: 24% of families had offspring fathered by the first male to mate, 68% by the second male (in only 8% was paternity shared). In the field, paternity was more mixed: only 27% of families had a single father, 14% had more than two fathers, whilst 59% had two fathers. While unsuccessful matings may contribute to the highly biased paternity in the laboratory, they cannot fully explain the high incidence of complete P2 families. Nonrandom sperm utilisation is therefore likely. Greater sperm mixing in the field probably results from females mating with more males, but the distribution of paternity also reflects an active process of nonrandom sperm utilisation. Confidence of paternity due to last male advantage may be relatively high in this species, and therefore may have facilitated the evolution of the large spermatophore in E. ephippiger.Communicated by D. Gwynne