Mixed Paternity Research Articles

Selected polyandry: female choice and inter-sexual conflict in a small nocturnal solitary primate (Microcebus murinus)

Sex-specific interests over the maximization of reproductive success lead to an inter-sexual conflict over the optimal mating system in a species. Traditionally, the outcome of this inter-sexual conflict has been studied from the male perspective but it also depends on female mating strategies, such as manipulating the temporal distribution of sexual activity, advertisement, and mate choice. We used a small nocturnal primate, the gray mouse lemur (Microcebus murinus) to determine the relative importance of female mating strategies on the outcome of this conflict in a species where females are solitary during their activity period. We studied their mating behavior over three consecutive annual mating seasons and determined the genetic relationships among more than 300 study animals to quantify individual reproductive success. We found that most females were receptive asynchronously. Females did not exhibit any obvious direct mate choice, probably due to a highly male-biased operational sex ratio and the corresponding costs of choosiness. However, females exercised indirect choice for multiple matings. They mated with 1–7 males up to 11 times during their single night of receptivity. As a result, mixed paternity was common but heavier males sired more offspring, meaning that indirect female choice for superior males cannot be excluded. Females exhibited a mixed mating strategy, avoiding costly direct mate choice but still counteracting male efforts to monopolize mating, successfully increasing genetic variability among offspring. Thus, females had a major influence on the outcome of the inter-sexual conflict despite male monopolization attempts.

Sperm Competition in a Viviparous Fish

We investigated multiple paternity and sperm precedence in the Amarillo fish, Girardinichthys multiradiatus (Goodeidae). We allowed females to mate with two different-sized males consecutively and assessed the paternity of the ensuing broods using allozyme electrophoresis. We presented one-half of the females the larger, and the other half the smaller, male first. Allozyme variation among individuals was low, yielding conservative estimates of multiple paternity. Half the broods were of mixed paternity, but one male always sired more than 70% of the embryos in each brood. The proportion of the brood sired was not related to mating sequence, but when we classified males by relative size, the larger male of each pair usually fathered greater proportions of offspring than the smaller male. This association disappeared when we used the actual size of the males in the analysis. Instead, for any pair of males, the difference in number of offspring sired was correlated to differences in the rate of courtship displays, rather than size differences, suggesting that courtship intensity is a better predictor of paternity than male size. Within a pair, the larger male usually displayed more than the smaller one, but there was no correlation between male size and display rate across all males. Parsimony suggests a correlation between courtship rate and sperm production, but we cannot rule out the possibility that females allocate paternity according to the relative merits of the males.

Parentage in the cooperative breeding system of long-tailed tits, Aegithalos caudatus

In cooperatively breeding birds multiple maternity and paternity of broods is not uncommon, reproduction often being shared among group members as well as with extragroup members. We investigated the extent of extrapair paternity and intraspecific brood parasitism in a population of cooperatively breeding long-tailed tits. Our aim was to determine the frequency and cause of mixed parentage and to investigate whether shared maternity or paternity was associated with decisions made by helpers. Genetic analyses using eight microsatellite loci showed that extrapair paternity was low (2.4–6.9% of nestlings in 16–29% of broods), and that intraspecific brood parasitism was negligible. Mate switching and extrapair copulations were both observed, but mate switching was not responsible for the mixed paternity we recorded. Some extrapair offspring were assigned to males that became helpers at the nest containing their extrapair young, but these males were also close neighbours of the cuckolded males and so were the most likely males to gain extrapair paternity. There was no evidence that the existence of a direct reproductive stake in a brood played an important role in the helping decisions of either male or female helpers. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.

Sperm mixing in the Alpine newt (Triturus alpestris)

In polyandrous females the ultimate stage of cryptic female choice may involve egg–sperm interactions during different phases of fertilization. This form of sperm discrimination is possible only when sperm from different males have simultaneous access to eggs at the site of fertilization. In polyandrous newts of the genus Triturus, eggs are fertilized internally by sperm stored for an extensive period of time in the tubular spermatheca. The extent of sperm mixing, which is a necessary condition for cryptic female choice involving sperm–egg interactions, was studied in doubly mated female Alpine newts, Triturus alpestris. Using an allozyme marker the paternity of offspring sired by the two males was established in both series of larvae reared from eggs produced consecutively over short period of time (ca. 2 h) and batches of eggs collected during longer periods of time (up to 26 days). Significant sperm mixing was unequivocally demonstrated by the mixed paternity of the progeny produced in series. The paternity pattern in batches of eggs collected during longer periods of time showed neither significant predominance of either male in the progeny nor any effects of sperm stratification in the tubules of the spermatheca.

Spontaneous abortion depresses female sexual receptivity in a viviparous arthropod

The ‘trading-up’ hypothesis for polyandry, which posits that females copulate with additional males as a means of improving on the quality of their first mating partner, has been proposed as a general explanation for mixed paternity in socially monogamous birds. As a corollary of this hypothesis, abnormally low reproductive output with a first partner should act as a strong stimulus for females to accept sperm from a new male. To test this prediction in a naturally polyandrous, sperm-storing, viviparous species, we restricted female Cordylochernes scorpioides pseudoscorpions to mating with a single male and then gave them the opportunity to remate with a different male after their first brood of embryos either aborted spontaneously, was experimentally removed, or was successfully carried to term. With complete brood failure presumably providing the strongest possible cue to females of a poor-quality first mate, the trading-up hypothesis would predict that females that aborted their first brood should have been significantly more sexually receptive than females that had produced a successful first brood. Instead, they were significantly less receptive. Furthermore, none of the sexually unreceptive, spontaneously aborting females produced a successful second brood, indicating that C.scorpioides females that copulate with a single male may become locked into cycles of brood failure followed by sexual unreceptivity. Our findings suggest that the negative impact of abortion on female sexual receptivity may place physiological constraints on the ability of viviparous females to use polyandry as a facultative mating strategy for improving on a bad first mate.

Sperm competition and genetic diversity in the agile antechinus (Dasyuridae: Antechinus agilis)

AbstractThe mouse‐like marsupial Antechinus agilis is common to south‐eastern Australia with breeding and life‐history traits being highly synchronous. Mating activity is confined to a 10‐ to 15‐day period in August, at the end of which all males die as a result of a stress‐induced suppression of the immune system. Ovulation occurs at this time and females rely on stored sperm from specialized crypts in the lower isthmus of the oviduct for successful fertilization. Here we report a high incidence of mixed paternity litters, which can be attributed to sperm from multiple males being stored in the isthmic crypts. Data from this study also suggest a possible second male siring advantage in controlled ex situ sperm competition mating trials, irrespective of the delay between the two males being given mating access to the female or of the mating time relative to ovulation. In determining paternity through DNA profiling, population genetic data were obtained that showed significant differences in the genetic heterozygosity between unrelated adults, half siblings and full siblings. We suggest that the isthmic crypts, in addition to storing viable sperm, are capable of releasing a mix of sperm that increases the likelihood of mixed paternity litters. This allows all mating males the opportunity to sire young, increases female reproductive fitness and overall maintains high levels of population genetic heterozygosity in the face of total male mortality annually.

Geographic variation in multiple paternity within natural populations of the guppy (Poecilia reticulata)

Mating can increase an individual's risk of mortality by predation. In response to predation hazards, males in some species court females less often, but alternatively engage in coerced copulations more frequently and females become less selective. Such predator-mediated shifts in mating tactics may result in higher levels of multiple inseminations in females and, thus, in greater frequencies of females with broods of mixed paternity. We tested this hypothesis using two polymorphic microsatellite loci to estimate conservatively multiple paternity in broods of female guppies (Poecilia reticulata) originating from ten natural populations that have evolved under different fish predation regimes in Trinidad. The frequency of broods that were multiply sired was significantly greater on average in populations experiencing high predation pressure compared to populations experiencing a relatively low predation risk. These results suggest that the intensity of male sperm competition covaries geographically with predation pressure in this species and that the local risk of predation mediates the opportunity for sexual selection within populations.

Extrapair paternity and egg hatchability in tree swallows: evidence for the genetic compatibility hypothesis?

Tree swallows (Tachycineta bicolor) show one of the highest levels of extrapair paternity in birds, and there is evidence that females have control over who fathers their offspring. However, it is unclear which benefits female tree swallows obtain from mating with multiple males. Using microsatellite DNA fingerprinting, we studied extrapair paternity in relation to nesting success and male, female, and offspring characteristics. More than 70% of all nests contained extrapair young, and more than half of all offspring were extrapair. Within broods, the extrapair young were often fathered by several males. Despite screening all resident and some floater males, we could identify the biological father of only 21% of all extrapair offspring. All identified extrapair males were close neighbors. Extrapair males did not differ from within-pair males in any of the measured characteristics, except that the former had larger cloacal protuberances than the latter. Extrapair males were equally successful in gaining paternity in their own broods as males that did not father extra young. In nests with mixed paternity, extrapair young did not differ from within-pair young in body size or mass. However, nests with extrapair young had higher hatching success than nests without extrapair young. All examined unhatched eggs were fertilized and thus hatch failure resulted from embryo mortality. The available data are in accordance with the genetic diversity and the genetic compatibility hypothesis, but not with the good genes hypothesis.

VARIATION OF SPERM PRECEDENCE IN THE ROVE BEETLE ALEOCHARA CURTULA (COLEOPTERA: STAPHYLINIDAE)

Abstract In order to quantify the outcome of sperm competition in the mating system of the rove beetle Aleochara curtula we performed three different mating experiment series. Paternity was established by DNA fingerprinting with oligonucleotide probes. We found a precedence of the last male with a proportion of eggs fathered by the last male of 87% (median). Nevertheless, we observed a large variation of P2-values in A. curtula and cases of mixed paternity were documented as well as cases of 100% or 0% paternity of the last male. The variation of P2-values correlates with the efficiency of spermatophore and sperm transfer.

Paternity and paternal care in the polygynandrous Smith's longspur

In species where females copulate with more than one male during a single breeding attempt, males risk investing in offspring that are not their own. In the polygynandrous Smith's longspur (Calcarius pictus), females copulate sequentially with one to three males for each clutch of eggs and most of these males later assist in feeding the young. Using multilocus DNA profiling, we determined that there was mixed paternity in >75% of broods (n=31) but that few offspring (<1% of 114 nestlings) were sired by males outside the polygynandrous group. Male feeding rate increased significantly with the number of young sired, with males siring four nestlings feeding the brood at double the frequency of males siring only a single nestling. However, male Smith's longspurs appear to show a graded adjustment of paternal care in response to paternity only when other males are available to compensate for reduced care: feeding rate did not vary in relation to paternity when only one male provisioned young at the nest. There was no evidence that males could recognise their own offspring within a brood and feed them preferentially. The number of offspring sired by each male was significantly correlated with the number of days spent copulating with the attending female: on average, a male sired one offspring for every 2 days of copulatory access. If males use their access to females to estimate paternity (and thereby decide on their subsequent level of parental investment), a positive relationship is expected between the amount of female access and the subsequent feeding rate to the nestlings. Nonetheless, male feeding effort was only weakly correlated with female access and more study is needed to determine how males estimate their paternity in a brood.

No evidence of sperm selection by female common shrews.

There is currently much interest in the suggestion that females are capable of post-copulatory (or cryptic) choice for male genetic compatibility. Here, I investigate this idea using data from mixed-paternity litters of the common shrew (Sorex araneus). Females of this species are highly promiscuous and, in natural populations, regularly incur costs of inbreeding by mating with close relatives. Selection should therefore favour female ability for sperm selection on the basis of male relatedness. No evidence was found in support of this idea. Relative number of offspring sired within mixed paternity litters was not significantly correlated with genetic similarity of males to the female mated. Relative fertilization success was, however, significantly related to male epididymal sperm counts. I conclude that most variation in relative fertilization success of male common shrews can be explained in terms of sperm competition, and that females of this species may not be capable of sperm selection.

Extrapair paternity in the blue tit (Parus caeruleus) : female choice, male characteristics, and offspring quality

Extrapair paternity is common in many birds, and it is now generally accepted that female choice plays an important role. However, die benefits that females obtain from extrapair paternity are much less dear. To test the hypothesis that females obtain indirect fitness benefits, we studied paternity in a blue tit population over 4 years. Extrapair paternity occurred in 31-47% of all nests and accounted for 11-14% of all offspring. Most males that fathered extrapair young did not lose paternity themselves, males never exchanged paternity, and within nests the extrapair offspring were usually fathered by a single male. Comparisons between males that did and did not lose paternity and pairwise comparisons between the extrapair male(s) and the withln-pair male showed that successful males had longer tarsi and sang on average longer strophes during the dawn chorus. Successful males weighed less (relative to their size) during the nettling stage, but neverthalen they survived better. Male age did not influence their likelihood of losing paternity, but extrapair males were usually older than the witbin-pair male they cuckolded. Within nests with mixed paternity, extrapair young were more likely to survive than within-pair young in cases of partial brood mortality. Our data also suggest that extrapair offspring were more likely to be males. Because extrapair males were usually close neighbors, male quality should be considered relative to the quality of the neighbors. Despite this, we found consistency in female choice over years. Our observations provide support for the hypothesis that female blue tits engage in extrapair copuladons to obtain good genes for their offspring.

Experimental mate switching in pied flycatchers: male copulatory access and fertilization success

By making female birds pair successively with different males, and analysing the paternity of the eggs laid, it is possible to examine how a male's success in obtaining fertilizations is determined by the timing of his copulatory access to the female. Such an experiment is reported here with pied flycatchers,Ficedula hypoleucaMate switching was induced at different stages within the female’s fertile period by removing the resident male. The paternity of the clutch was analysed by microsatellite DNA typing. Removed males had full paternity in the clutch if they were removed as late as after the second egg was laid (day 1), and they lost all paternity if they were removed more than 1 day before the first egg was fertilized (less than day −2). Male switching during the period day −2 to day 1 always resulted in mixed paternity in the clutch. Males that were paired to the female for only a few (1–3) days during this period achieved on average more than one fertilization per access day, with a peak on day −1. Thus, assuming that the timing of observed pair bonds reflects the timing of each male’s copulatory access to the female, the first eggs in the laying sequence were fertilized by inseminations occurring shortly before the time of fertilization, whereas the last eggs were fertilized by inseminations taking place several days prior to their fertilization. Our documentation of the most important period for copulatory access has some important implications for understanding sexual behaviour in this species.

Last-Male Sperm Priority in a Haplogyne Spider (Araneae: Pholcidae): Correlations between Female Morphology and Patterns of Sperm Usage

In species where the female mates more than once, sperm priority patterns have important implications for the mating behavior of both sexes. The shape of female sperm storage organs may play a role in determining priority patterns, although the extent of this effect has been controversial. Spiders provide an interesting test of this hypothesis because 2 phylogenetically distinct groups differ in the structure of sperm storage organs. In entelegyne spiders, the female sperm-storage organs have separate ducts for sperm entrance and exit. The male inseminates the female through the external fertilization duct. Sperm then travel through a “conduit” and are released from an internal fertilization duct to fertilize eggs. This arrangement has been hypothesized to lead to 1st-male priority. Six species in 3 entelegyne families have been shown to have 1st-male sperm priority with some mixing; 1 entelegyne species, however, shows last-male priority. Haplogyne females, in contrast, have a spermstorage area with only a single opening, which may be more likely to lead to last-male sperm priority because the last sperm to enter the storage area would be likely to be the 1st to leave. Few data are available for this group. We used a balanced sterile-male design to test the sperm priority pattern in females mated once with each of a pair of males of a haplogyne spider, Holocnemus pluchei Scopoli. Egg sacs from 72.5% of females were sired predominantly by 2nd males, 7.5% were predominantly sired by 1st males, and 20% showed mixed paternity ( n = 40); these frequencies differed significantly from random expectations. The average percentage of eggs fathered by the 2nd male, or P 2, was 74%. These data clearly differ from the predominantly 1st-male pattern generally found in entelegynes, thereby supporting the hypothesized relationship between female reproductive morphology and sperm use patterns. Mating behavior of spiders that exhibit 2nd-male priority should differ from that exhibiting 1st-male priority, and evidence from H. pluchei and other haplogynes supports this inference.

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

Male Lepidoptera produce an ejaculate during copulation that contain* both sperm and accessory gland nutrients and may function as paternal investment and/or male mating effort Several studies have examined how ejaculates function-as paternal investment, but few have determined the influence of sperm competition on male investment This study examines the effect of male body size on sperm precedence in the polyandrous butterfly Pitris napi L. We used male body mass as an indicator of die size of ejaculate transferred and found that relative male size had a significant effect on paternity. The offspring of twicemated females showed a low incidence of mixed paternity. Larger males obtained the majority of fertilizations, and the degree of second-male sperm precedence was influenced by relative body size of mating males. In general, second mates obtained fewer fertilizations the larger the size of the first mate. The interval between the first and second mating was influenced by the m* of the first male maty Females first mated to small males remated sooner than females first mated to large rPaW Our results suggest that large males may have a selective advantage over small males when both a male's fertilization success and a female's refractory period are influenced by the size of ejaculate transferred. Furthermore, the effect of male body size on the proportion of offspring sired lends support to the hypothesis that sperm competition has played a major role in the evolution of ejaculate size. Key words: body size, ejaculate size, maring frequency, Pitris napi, polyandry, sperm competition. [BAavEcol 8:511-523 (1997)]

Does long‐term storage of spermatozoa occur in the adder (Vipera berus)?

In the adder (Vipera berus) multiple inseminations during the spring mating season have been demonstrated, with fertilization taking place several weeks after mating activities have ceased. Subsequent sperm competition in the female reproductive tract can result in within‐season mixed paternity. Prolonged sperm storage between mating seasons has been suggested as another mechanism of sperm competition in the adder. Competition between new and old spermatozoa would obstruct efforts to reveal reproductive strategies that correlate observed behaviour to paternity in natural populations. In the present study, controlled breeding experiments with adders were performed and DNA fingerprinting was used to determine paternity among all offspring of a total of 15 litters. The offspring of 11 large females, that had most probably also mated in a previous season, were sired exclusively by enclosure males. This result and earlier empirical data support the hypothesis that long‐term sperm storage, over one or several reproductive cycles, does not occur in the adder.

DNA microsatellites in domesticated dogs: application in paternity disputes.

Microsatellite sequences, like minisatellites, are a class of polymorphic DNA fragments that are randomly distributed throughout mammalian genome. Although they express significantly lower variation than minisatellites, they have potential to be used in paternity disputes. However, the inherently lower variability together with the more genetically homogeneous nature of pedigree dogs due to the major inbreeding, raised doubts about the effectiveness of microsatellites in canine paternity (paternage) testing. Using about 20 polymorphic canine specific microsatellites on various dog litters demonstrated, that canine microsatellites provide a perfectly adequate basis for assigning paternity in pedigree breeds. Microsatellite paternity tests are more straightforward to perform and interpret than those based on minisatellites (DNA fingerprinting) and require as little as 0.1 ml of blood. The case presented here was a paternity dispute in labradors, where a mixed paternity was suspected.

Female control of copulations to maximize male help: a comparison of polygynandrous alpine accentors, Prunella collaris, and dunnocks, P. modularis

Alpine accentors and dunnocks bred in polygynandrous groups in which two or more unrelated males shared two or more females. In both species, a female solicited actively to both alpha and subordinate males whereas an alpha male attempted to guard the female to monopolize paternity. Females combated the restrictions imposed by alpha male guarding by increasing their solicitation rate to males who had gained less mating access. Males increased their copulation rate in response, but alpha males ignored more of the offers. In both species, even when a female mated with both alpha and beta males she often gained just one male’s help with chick feeding. Under these conditions, alpha male alpine accentors reduced their amount of help with a decreased mating share, whereas beta males did not. In dunnocks, however, neither alpha nor beta males reduced their help provided a critical share of the matings was exceeded. As predicted if females attempted to maximize male help, female alpine accentors preferred to give more matings to the alpha male while female dunnocks preferred alpha and beta equally. There was no evidence for either species that alpha males sired fitter offspring; within broods of mixed paternity, there was no difference in the weights of chicks sired by alpha versus subordinate males. Female dunnocks competed with other females by territory defence whereas female alpine accentors had overlapping ranges and competed directly for male attention, increasing their solicitation rate to the alpha male if other females in the group were fertile. We suggest that the extraordinarily high rates of solicitation by females, refusal by males and copulation rates (up to a thousand per clutch) in the two species are the outcome of sexual conflict over the control of mating.

Confirmation of cooperative polyandry in the Galapagos hawk (Buteo galapagoensis)

Without genetic tests of parentage, descrip- tions of mating systems must be considered hypotheti- cal. Here we confirm the existence of cooperative polyandry in the Galapagos hawk (Buteo galapagoensis) using multilocus minisatellite DNA fingerprinting. In this species, breeding groups consist of one adult female and from one to eight males (the modal number of males is two). In polyandrous groups, all males copulate with the female and participate in the provisioning of the young. DNA samples from 66 individual hawks from ten breeding groups on the island of Santiago revealed mixed paternity in most groups. Multiple paternity was detected in five of six groups that produced two chicks in one breeding attempt (year). In addition, different males sired young in consecutive years in five of six groups in which male group membership was constant. Patterns of paternity suggest that reproductive success was randomly distributed among males within groups, with males apparently having equivalent probabilities of siring each young. Analysis of genetic similarity indi- cates that males within groups were typically not close relatives. These results demonstrate that the mating sys- tem of the Galapagos hawk is polyandrous, with rela- tively egalitarian relations among unrelated males be- longing to the same breeding group.

Female red junglefowl choose to mate with multiple males

Although mixed paternity in broods of young is now known to occur in many species of birds, the question remains, does mixed paternity reflect only a male strategy, or might it not also be a reproductive strategy of females? To address this question female red junglefowl, Gallus gallus, in egg-laying condition were given the opportunity to mate repeatedly with either or both of two males, in the absence of male-male competition. Over the course of five mating trials nearly all females chose to mate with both available males, regardless of the appearance of the males' plumage. Similarly, although females are known to prefer males with larger combs, when the two males possessed combs of strikingly different sizes, most females mated with both individuals. These results suggest that in this species a female reproductive strategy is to obtain sperm from more than one male during production of a clutch of eggs.