Determinants of paternity in the milkweed leaf beetle

Summary Males and females of the milkweed leaf beetle mate for up to 2.5 d in the field; copulation is interspersed with mounted courtship behavior and periods of passive riding on the female's back. In 1985, females mated with up to 10 different males in Bridgeport, New York, and matings averaged 0.75±0.04 (SE) d in duration. Sperm utilization patterns are complex in the milkweed leaf beetle. In the laboratory, mating duration, mating order and the duration of the gap between matings affected paternity. The second male's sperm predominated when a female's two consecutive matings were of equal duration (but were long: 45 h each) or when they were intermediate in duration (15 h each) with a 5 day gap between the two matings. The mechanism of sperm predominance appears to be one of time-dependent sperm removal and replacement. Neither relative nor absolute size of males affected the extent to which they superceded the sperm of rivals. Electrophoretic analysis of paternity at 6 polymorphic loci showed that offspring of at least eight out of 11 mating pairs pairs collected in the field at the end of the 1984 breeding season were sired by more than one male. At least seven of the 11 families produced some offspring that were sired by the female's most recent mate in the field and in no case was the collected male completely excluded from paternity. In a greenhouse plot, males climbed off females' backs a mean of 1.1±0.3 h before oviposition. Females did not oviposit with males on their backs and did not oviposit sooner when males were removed than when males were allowed to remain mounted, indicating that males don't keep females from ovipositing until they can achieve sperm predominance. The close concordance between male disembarkment and oviposition suggests that males remain with females beyond the time when they have superceded the sperm of their prior mates, possibly to guard them from rival males. Although the prolonged mating association of the milkweed leaf beetle cannot be separated into discrete copulatory and guarding phases, the results of this study suggest that prolonged mating enables males both to remove and replace the sperm of the females' prior mates with their own sperm and to prevent females from remating prior to oviposition.     

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.     

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.     

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.     

Sperm competition in the mite Caloglyphus berlesei

Summary The sperm utilisation pattern of C. berlesei was investigated using the sterile male method. The last male to mate with a female fertilised most of her eggs. In experiments where females mated with two virgin males, second males fertilised on average 86% of eggs when second matings took place immediately after the first. Last male sperm priority was also confirmed in experiments where females mated sequentially with three males. The proportion of eggs fertilised by the second male decreased to 71% and 56% when the interval between matings was 2 and 6 h, respectively. Males that had opportunities to mate prior to experiments copulated for a shorter time than virgin males, and second males fertilised on average 74% eggs.     

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.     

Sex-specific reproductive behaviours and paternity in free-ranging Barbary macaques (Macaca sylvanus)

In a wide variety of species, male reproductive success is determined by contest for access to females. Among multi-male primate groups, however, factors in addition to male competitive ability may also influence paternity outcome, although their exact nature and force is still largely unclear. Here, we have investigated in a group of free-ranging Barbary macaques whether paternity is determined on the pre- or postcopulatory level and how male competitive ability and female direct mate choice during the female fertile phase are related to male reproductive success. Behavioural observations were combined with faecal hormone analysis for timing of the fertile phase (13 cycles, 8 females) and genetic paternity analysis (n = 12). During the fertile phase, complete monopolisation of females did not occur. Females were consorted for only 49% of observation time, and all females had ejaculatory copulations with several males. Thus, in all cases, paternity was determined on the postcopulatory level. More than 80% of infants were sired by high-ranking males, and this reproductive skew was related to both, male competitive ability and female direct mate choice as high-ranking males spent more time in consort with females than low-ranking males, and females solicited copulations mainly from dominant males. As most ejaculatory copulations were female-initiated, female direct mate choice appeared to have the highest impact on male reproductive success. However, female preference was not directly translated into paternity, as fathers were not preferred over non-fathers in terms of solicitation, consortship and mating behaviour. Collectively, our data show that in the Barbary macaque, both sexes significantly influence male mating success, but that sperm of several males generally compete within the female reproductive tract and that therefore paternity is determined by mechanisms operating at the postcopulatory level.     

Sperm competition and evidence of sperm fertilization patterns in the carrion ball-roller beetle Canthon cyanellus cyanellus LeConte (Scarabaeidae: Scarabaeinae)

Field observation and laboratory experiments were conducted to investigate sperm competition and patterns of sperm fertilization under different experimental conditions in the carrion ball-roller beetle Canthon cyanellus cyanellus. Males in nature can mate with females whose spermathecae contains fertile sperm from other males. Sperm precedence was investigated using a visible genetic marker. The progeny of red (homozygous recessive) virgin females mated once with a red male and afterward, once with a green beetle (homozygous dominant) and vice versa, revealed that regardless of its color, the last male to mate fertilized c.a. 50% of the eggs, suggesting strong sperm competition. Males were able to achieve higher levels of paternity (more than 80%) when mated ad libitum with previously mated females, although large amount of variance in paternity does not exclude the possibility of first male sperm precedence or female cryptic choice. These results suggest that repeated mating and sperm replacement are the mechanisms by which last males achieve sperm precedence.     

Inbreeding avoidance in a poeciliid fish (Heterandria formosa)

Polyandry is hypothesized to give females an opportunity to avoid inbreeding through postcopulatory selection mechanisms if precopulatory inbreeding avoidance is not possible, for example, because of forced matings. Here, we report a postcopulatory, prefertilization, inbreeding avoidance mechanism in the least killifish, Heterandria formosa, a species in which males can force matings. Females had 50% less sperm in their oviducts and ovarian cavities after mating with a sibling compared to a mating with a nonsibling male. Neither sex showed inbreeding avoidance in a dichotomous precopulatory mate choice test or during mating trials. Females in this species invest substantially in each offspring after fertilization (matrotrophy), whereas males invest little more than sperm. Based on theory, females should therefore be more likely than males to avoid inbreeding in this species. We suggest that females do this by reducing the amount of sibling sperm in their reproductive system. However, the possibility that males invested more sperm to nonsiblings could not be ruled out. In a fertilization success experiment, the first male to mate with a female sired all the offspring in most cases, even if it was a sibling. However, large females were more likely to carry offspring of multiple males. Possibly female sperm storage sites were filled by the first male, and only large females had space for the second male's sperm.     

Mating system,optimal number of matings,and sperm transfer in the Argentine ant Iridomyrmex humilis

Summary In ants, because males have a finite sperm supply and females mate only at the beginning of their reproductive lives, it is possible to infer which is the limiting sex from a few parameters: the amount of sperm produced by males, the amount of sperm stored by females, and the numerical sex ratio. In the Argentine ant Iridomyrmex humilis mating takes place in the nest. Laboratory experiments and field data showed that the numerical sex ratio is heavily male-biased (10.1:1) and that the maximum number of sperm a female can store is similar to the number of sperm a male possesses. Thus females are the limiting sex in this species. In a set of mating experiments, one queen was presented with 1–20 males. The highest proportion of successful matings occurred when females were presented with two males. There was a significant negative correlation between the amount of sperm queens stored and the number of males present in the mating arena. This relationship most likely resulted from male interference during the copulation process. When several males were present in the arena, the mating pair was frequently disturbed by other males trying to copulate. Newly mated queens collected from the field stored 172,000 ± 76,000 sperm, a quantity most similar to that measured in laboratory mating experiments with a ratio of 5 or 10 males per queen. Because the operational sex ratio in I. humilis is highly male-biased, male interference may also decrease the amount of sperm queens store in the field. In many ants, fewer sperm stored by queens should decrease their reproductive success because they would run out of sperm earlier in their reproductive lifetimes. However, comparison of the amount of sperm present in young and old I. humilis queens collected in the field suggests that most use only a small proportion of their sperm supply during their lifetimes. Males mate once and discharge all their sperm during a single mating. Females may mate with several males but dissection of these males indicated that in most cases only one of them had empty seminal vesicles thus suggesting that a single male is responsible for most of the sperm transfer. Thus caution should be exercised in inferring multiple inseminations, as is frequently done in eusocial insects, from the observation of multiple copulations. Correspondence to: L. Keller     

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     

Multiple paternity, sperm storage, and reproductive success of female and male painted turtles (Chrysemys picta) in nature

When females receive no direct benefits from multiple matings, concurrent multiple paternity is often explained by indirect genetic benefits to offspring. To examine such possibilities, we analyzed genetic paternity for 1,272 hatchlings, representing 227 clutches, from a nesting population of painted turtles (Chrysemys picta) on the Mississippi River. Goals were to quantify the incidence and distribution of concurrent multiple paternity across clutches, examine temporal patterns of sperm storage by females, and deduce the extent to which indirect benefits result from polyandrous female behaviors. Blood samples from adult males also allowed us to genetically identify the sires of surveyed clutches and to assess phenotypic variation associated with male fitness. From the genetic data, female and male reproductive success were deduced and then interpreted together with field data to evaluate possible effects of female mating behaviors and sire identity on offspring fitness. We document that more than 30% of the clutches were likely fathered by multiple males, and that presence of multiple paternity was positively correlated with clutch size. Furthermore, the data indicate that the second male to mate typically had high paternity precedence over the first.     

Sperm precedence, mating interval, and a novel mechanism of paternity bias in a beetle (Tenebrio molitor L.)

When females mate with more than one male, the ensuing sperm competition leads to the evolution of male mechanisms that skew paternity. Males of the yellow mealworm beetle (Tenebrio molitor) transfer a spermatophore to females during copulation, but sperm release and storage occur later. We investigated how the interval between two matings with different males affects sperm precedence by varying the interval between the copulations so that the second mating was either: (1) before sperm release from the first spermatophore (<5 min); (2) after sperm release but before spermatophore ejection (15–20 min); (3) after spermatophore ejection but before sperm storage (4 h), or (4) after complete sperm storage (24 h). We collected offspring over a period of 2 weeks and determined paternity by protein electrophoresis. There was second-male sperm precedence in all treatments, but when the interval was <5 min, the second male usually (86% of cases) had complete sperm precedence (i.e., P ₂=1). Investigations into the mechanism of second-male sperm precedence during <5-min mating intervals indicate that sperm release from the first spermatophore is inhibited, a phenomenon which has not been previously documented. Received: 31 January 2000 / Revised: 9 June 2000 / Accepted: 26 August 2000     

Influence of mating order on courtship displays and stored sperm utilization in Hermann's tortoises (Testudo hermanni hermanni)

Sperm storage is widespread in many vertebrate groups, and it is frequently associated with promiscuous mating systems. Chelonian species are one of the most outstanding examples of a promiscuous group capable of long-term sperm storage; specialized structures have evolved within the oviducts of these vertebrates to ensure sperm vitality across reproductive cycles. Thus far, few studies have investigated the factors regulating multiple paternity, sperm usage by females and paternity distribution in successive clutches. This study aimed to investigate the effect of mating order on male mounting and reproductive success in Testudo hermanni hermanni, combining behavioural and genetic data. A series of planned matings were performed, within which experimental females were mated sequentially to two different males under controlled conditions. Observations conducted during experimental matings revealed that courtship displays did not significantly differ between the first and second males to mate with a female. Interactions ending with a mount were characterized by a significantly higher intensity and occurrence of determinate courtship behaviours, for example biting and running after the female. Paternity analysis performed on hatchlings produced from experimental females revealed that 46 % of the clutches were multi-sired. A significant contribution of previous years' partners was still found, thus confirming the long-term sperm storage within the female oviduct in this species. Finally, mating order did not significantly affect the reproductive success of experimental males during the on-going reproductive season.     

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.     

The effects of mating system on male mate choice in a coral reef fish

Summary We show how mate limitation appears to be critical in determining whether or not males exercise mate choice among available females. Thalassoma bifasciatum is a Caribbean reef fish with two distinct mating patterns: group-spawning and pair-spawning. In both mating systems, female fecundity is variable and size dependent, and female availability is high. However, sperm competition among group-spawning males apparently limits the number of effective matings in which a male may engage, whereas territorial pair-spawning males have little or no such limitation. Group-spawning males should be discriminating in their choice of mates and our data confirm this: there is strong evidence for assortative mating in group-spawns, with more large males joining in mating groups around large females. In contrast, pair-spawning males show no indication of mate preferences, and spawn with all females who arrive at their territories.     

Genetic evidence of promiscuity in Peromyscus leucopus

Summary We collected pregnant female Peromyscus leucopus from natural populations during the summer of 1987 and 1988 and allowed these females to give birth to their field-conceived young in the laboratory. Blood samples were taken by suborbital puncture and phenotypes of five genetic loci (Esterase-1, trasferrin, hemoglobin, albumin and 6-phosphogluconate dehydrogenase) were studied using horizontal starch-gel electrophoresis to detect multiple paternity in single litters. Only esterase-1 was found to be highly polymorphic, with four alleles in samples of both years. One litter out of 29 in 1987 and 6 litters out of 32 in 1988 contained three different paternal alleles and served as direct evidence of multiple paternity in the field. The proportion of females engaging in multiple matings in natural populations of P. leucopus, assuming that all males were involved in every multiple mating, is 25%–100% (mean 68%). Because it is unlikely that all males are involved in every multiple mating, the actual proportion of females engaging in multiple matings should be greater. Offprint requests to: X. Xia at his Canadian address     

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.     

Influence of alternate reproductive tactics and pre- and postcopulatory sexual selection on paternity and offspring performance in a lizard

Sexual selection theory predicts different optima for multiple mating in males and females. We used mating experiments and genetic paternity testing to disentangle pre- and postcopulatory mechanisms of sexual selection and alternate reproductive tactics in the highly promiscuous lizard Eulamprus heatwolei. Both sexes mated multiply: 30–60 % of clutches were sired by two to four fathers, depending on the experiment. Larger males sired more offspring when we allowed male contest competition: 52 % of large males but only 14 % of small males sired at least one offspring. In the absence of male contest competition, females mated promiscuously and there was no large male advantage: 80 % of large males and 90 % of small males sired at least one offspring, and there was no evidence for last-male precedence. Multiple mating did not yield obvious direct or indirect benefits to females. E. heatwolei represents a complex system in which males attempt to improve their fertility success by limiting rivals from access to females and through adopting alternate reproductive tactics. Conversely, females exhibit no obvious precopulatory mate choice but may influence fitness through postcopulatory means by either promoting sperm competition or through cryptic female choice. Our results support the hypothesis that female multiple mating in nonavian reptiles is best explained by the combined effect of mate encounter frequency and high benefits to males but low costs to females.     

Polyandrous female butterflies forage for matings

Butterfly mating systems exhibit great variation and range from strict monandry to strong polyandry. During mating males transfer ejaculates containing both sperm and accessory substances to females. In the polyandrous green-veined white butterfly, Pieris napi (Lepidoptera, Pieridae) these ejaculates average 15% of male body mass, but can represent up to 23% of body mass for individual males. Hence, mating is costly to males, and recently mated males increase copula duration but decrease ejaculate mass transferred to females. Substances transferred to females during mating are later incorporated into female soma and reproductive tissues, and multiply mated female butterflies have higher lifetime fecundity, lay proportionately larger eggs, and live longer compared to once mated females. Here we report that females of P. napi allowed to mate at liberty with recently mated males only (i.e. males that delivered a small ejaculate) increased their lifetime number of matings compared to females allowed to mate with virgin males only (i.e. males that delivered large ejaculates), the former group mating on average 5.1 times (range 2–10) and the latter group mating on average 2.8 times (range 1–4). The lifetime fecundity of the two groups of females did not differ significantly. Because nutrient donation from males is essential for females to realize their potential fecundity, we conclude that females of the polyandrous green-veined white actively forage for matings.