Sperm competition mechanisms,confidence of paternity,and the evolution of paternal care in the golden egg bug (Phyllomorpha laciniata)

Theoretical models predict how paternal effort should vary depending on confidence of paternity and on the trade-offs between present and future reproduction. In this study we examine patterns of sperm precedence in Phyllomorpha laciniata and how confidence of paternity influences the willingness of males to carry eggs. Female golden egg bugs show a flexible pattern of oviposition behavior, which results in some eggs being carried by adults (mainly males) and some being laid on plants, where mortality rates are very high. Adults are more vulnerable to predators when carrying eggs; thus, it has been suggested that males should only accept eggs if there are chances that at least some of the eggs will be their true genetic offspring. We determined the confidence of paternity for naturally occurring individuals and its variation with the time. Paternity of eggs fertilized by the last males to mate with females previously mated in the field has been determined using amplified fragment length polymorphisms (AFLPs). The exclusion probability was 98%, showing that AFLP markers are suitable for paternity assignment. Sperm mixing seems the most likely mechanism of sperm competition, because the last male to copulate with field females sires an average of 43% of the eggs laid during the next five days. More importantly, the proportion of eggs sired does not change significantly during that period. We argue that intermediate levels of paternity can select for paternal care in this system because: (1) benefits of care in terms of offspring survival are very high; (2) males have nothing to gain from decreasing their parental effort in a given reproductive event because sperm mixing makes it difficult for males to reach high paternity levels and males are left with no cues to assess paternity; (3) males cannot chose to care for their offspring exclusively because they can neither discriminate their own eggs, nor can they predict when their own eggs will be produced; and (4) males suffer no loss of further matings with other females when they carry eggs. Thus, our findings do not support the traditional view that paternal investment is expected to arise only in species where confidence of paternity is high. The results suggest that females maximize the chances that several males will accept eggs at different times by promoting a mechanism of sperm mixing that ensures that all males that have copulated with a female have some chance of fathering offspring, that this probability remains constant with time, and that males have no cues as to when their own offspring will be produced.     

Female choice and the relatedness of mates in the guppy (<Emphasis Type="Italic">Poecilia reticulata</Emphasis>)

Several studies suggest that females may offset the costs of genetic incompatibility by exercising pre-copulatory or post-copulatory mate choice to bias paternity toward more compatible males. One source of genetic incompatibility is the degree of relatedness among mates; unrelated males are expected to be genetically more compatible with a female than her relatives. To address this idea, we investigated the potential for inbreeding depression and paternity biasing mechanisms (pre- and post-copulatory) of inbreeding avoidance in the guppy, Poecilia reticulata. Inbreeding resulted in a reduction in offspring number and quality. Females mated to siblings gave birth to significantly fewer offspring compared to females mated to non-siblings and inbred male offspring took longer to reach sexual maturity. There was no evidence of inbreeding avoidance in pre-copulatory behaviors of females or males. Sexual responsiveness of females to courting males and the number of sexual behaviors males directed at females did not decrease as a function of the relatedness of the two individuals. We also tested whether female guppies can use post-copulatory mechanisms to bias sperm usage toward unrelated males by comparing the number of offspring produced by females mated to two of their siblings (SS), two males unrelated to the female (NN), or to one unrelated male and a sibling male (NS). We found that NS females produced a number of offspring not significantly different than what would be expected if fertilization success were halfway between completely outbreeding (NN) and completely inbreeding (SS) females. This suggests that there is no significant improvement in the number of offspring produced by females mating to both related and unrelated males, relative to that which would be expected if sperm from both males were used equally. Our results suggest that female guppies do not discriminate against closely related males or their sperm.     

Paternity assurance before and after fertilization by male burying beetles (Nicrophorus quadripunctatus)

Parental care requires a large investment of time and energy. This can reduce future parental survival and opportunities for mating. Because males are usually more uncertain of their parentage with respect to the caring of offspring than are females, the reduction in reproductive success is thought to be greater in males. Therefore, males are under selection to ensure paternity of the offspring for which they care. Males can increase paternity before and after fertilization. Before fertilization, males can increase paternity by increasing their competitive ability for fertilization. After fertilization, males can increase paternity by cannibalizing unrelated offspring. Here, we investigated the stage at which male burying beetles, Nicrophorus quadripunctatus, increase their paternity by evaluating the number of offspring sired by a nursing male in asynchronously hatched broods in relation to hatching time. We found that nursing males assure a very high level of the paternity of hatching offspring. We also found that the paternity of non-nursing and nursing males remained constant across hatching time within a brood, indicating that it is unlikely that filial cannibalism plays a role in increasing the paternity of offspring. We concluded that ensuring paternity before fertilization is more important in increasing the paternity of offspring.     

Male reed buntings do not adjust parental effort in relation to extrapair paternity

Parental effort is considered to be costly; therefore, malesare expected to provide less care to unrelated offspring. Theoreticalmodels suggest that males should either reduce their care tothe entire brood or alternatively distinguish between relatedand unrelated nestlings and direct provisioning to kin whenpaternity is in doubt. Reed buntings (Emberiza schoeniclus)have been found to have high levels of extrapair paternity (EPP,i.e., offspring of a male other than the male attending thenest; 55% of offspring), and males are therefore under strongselection pressure to adjust their parental effort accordingto the proportion of EPP in their brood. In this study, we investigatedwhether male reed buntings exhibit a reduction in paternal care(incubation and provisioning nestlings) in relation to decreasedpaternity. We also assess whether males bias their provisioningtoward kin. We measured incubation time, provisioning rates,and food allocation to individual nestlings using video recordingsat the nests. Microsatellite DNA analysis was used to analyzethe paternity of offspring. In direct contrast to a previousstudy on the same species, our results provided no indicationthat males lowered their effort with decreased paternity. Furthermore,in nests of mixed paternity, males did not bias their provisioningbehavior to kin. It remains to be investigated whether the absenceof a relationship between paternity and paternal care can beascribed to absence of reliable paternity cues or whether thebenefits of reducing paternal care did not outweigh the costsin our study population. We found no evidence that the levelof paternal care affected male survival or offspring mass, suggestingthat both the benefits and costs of any reduction in paternalcare would have been low.     

Sperm competition, alternative mating tactics and context-dependent fertilization success in the burying beetle, Nicrophorus vespilloides

Fertilization success in sperm competition is often determined by laboratory estimates of the proportion of offspring sired by the first (P1) or second (P2) male that mates. However, inferences from such data about how sexual selection acts on male traits in nature may be misleading if fertilization success depends on the biological context in which it is measured. We used the sterile male technique to examine the paternity of the same male in two mating contexts in the burying beetle, Nicrophorus vespilloides, a species where males have alternative mating strategies based on the presence or absence of resources. We found no congruence in the paternity achieved by a given male when mating under different social conditions. P2 estimates were extremely variable under both conditions. Body size was unrelated to success in sperm competition away from a carcass but, most probably through pre-copulatory male-male competition, influenced fertilization success on a carcass. The contribution of sperm competition is therefore dependent on the conditions under which it is measured. We discuss our findings in relation to sperm competition theory and highlight the need to consider biological context in order to link copulation and fertilization success for competing males.     

Egg-hatching benefits gained by polyandrous female locusts are not due to the fertilization advantage of nonsibling males

Several studies suggest that polyandrous females bias paternity in favor of unrelated males to avoid inbreeding depression. Here we tested whether the migratory locust biases sperm usage toward unrelated males by analyzing the paternity of offspring from females mated with either two siblings, or two nonsiblings, or a sibling and a nonsibling in either order. We found that the eggs of females mated only with siblings had decreased hatching success. When females mated with both a nonsibling and a sibling, egg hatchability was significantly increased. Subsequent paternity analyses found no evidence that females could avoid fertilization by sibling males. Therefore, improvement of the hatchability of eggs sired by siblings suggests that rather than biased fertilization by females toward genetically compatible or superior males, male-induced maternal effects or direct effects of male ejaculates might influence the survival of offspring sired by related males.     

Effects of age and repeated mating on male sperm supply and paternity in a parasitoid wasp

Post-copulatory paternity biases after female multiple mating are major constraints on both male and female reproductive systems. The outcome of paternity in certain situations is only controlled directly by male sperm stock. This was tested experimentally in the parasitoid wasp Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae), in which sperm stocks are small (several hundred) and the fertilizing efficiency of stored sperm is high (the ratio of sperm stored/fertilized eggs is about 0.75). Sperm in seminal vesicles and paternity of males of different status (virgin young, virgin old, or young previously mated) were measured after female single and double mating. The amount of sperm in the seminal vesicle differed according to male status (increasing from previously mated males to old males), but there was no difference in sperm stored by females after a single mating. In double mating experiments with two males of different status, paternity increased linearly with the relative amount of sperm in seminal vesicles. Paternity distribution conforms to 'a fair raffle' of sperm from both donors following complete mixing of sperm prior to fertilization. Thus, in a female multiple mating context, male fitness depends principally on their sperm stock, which in turn depends on life history parameters, such as age and previous mating.     

Postcopulatory inbreeding avoidance by female crickets only revealed by molecular markers

Multiple mating is thought to provide an opportunity for females to avoid the costs of genetic incompatibility by postcopulatory selection of compatible sperm haplotypes. Few studies have tested the genetic incompatibility hypothesis directly. Here we experimentally manipulated the compatibility of females with their mates using the gryllid cricket Teleogryllus oceanicus. We recorded the hatching success of eggs laid by females mated with two nonsibling males, two siblings, or one nonsibling male and one sibling. In contrast with two previous studies on crickets that have adopted this approach, the hatching success of eggs did not differ between females mated with two full siblings and females mated with two unrelated males, indicating that embryo viability was not a cost of inbreeding in this species. We assigned paternity to offspring produced by females mated to both a sibling and a nonsibling male using microsatellite markers. As in previous studies of this species, we were unable to detect any difference in the proportion of offspring sired by the 1st and the 2nd male to mate with a female when females were unrelated to their mates. However, in our experimental matings the proportion of offspring sired by the nonsibling male depended on his sequence position. Paternity was biased toward the nonsibling male when he mated first. Our data show that molecular analyses of paternity are essential to detect subtle mechanisms of postcopulatory sexual selection.     

Wild yellow dung fly females may not select sperm based on dung pat microclimate but could nevertheless benefit from polyandry

Molecular techniques have substantially improved our knowledge of postcopulatory sexual selection. Nevertheless, studies examining sperm utilization in natural populations of nonsocial insects are rare, support for sperm selection (biased use of stored sperm, e.g. to match offspring genotypes to prevailing environmental conditions) is elusive, and its relevance within natural populations unknown. We performed an oviposition site choice experiment in the field where female yellow dung flies Scathophaga stercoraria could deposit eggs into three different microenvironments on a dung pat (the east–west ridge, north- or south-exposed side), and genotyped the offspring and sperm remaining in storage after oviposition. Females exhibited plasticity in the number of eggs deposited according to pat age. Additionally, temperature strongly influenced egg placement: the warmer the temperature, the higher the proportion of eggs laid into the north-exposed side of dung. The number of ejaculates in storage differed amongst spermathecae, and females stored sperm from more males than fathered their offspring (2.11 sires vs. 2.84 males within sperm stores). Mean last male paternity was 83.4%, roughly matching previous laboratory estimates. Importantly, we found no evidence that females selectively lay eggs of different genotypes, by biasing paternity towards certain males, depending on offspring’s microclimate. Thus, while we show female choice over number of eggs and where these are deposited, there was no evidence for sperm selection. We further revealed positive effects of multiple mating on total number of offspring and proportion of offspring emerging from the dung. We argue that the integration of field studies and laboratory experiments is essential to promote our understanding of polyandry and cryptic female choice.     

Sperm selection and genetic incompatibility: does relatedness of mates affect male success in sperm competition?

Sperm selection may be said to occur if females influence the relative success of ejaculates competing to fertilize their ova. Most evidence that female animals or their ova are capable of sperm selection relates to male genetic incompatibility, although relatively few studies focus on competition between conspecific males. Here I look for evidence of sperm selection with respect to relatedness of mates. Reduced fitness or inbreeding effects in offspring resulting from copulations between close relatives are well documented. If females are capable of sperm selection, they might therefore be expected to discriminate against the sperm of sibling males during sperm competition. I describe an experimental protocol designed to test for evidence of sperm selection while controlling for inbreeding effects. Using decorated field crickets (Gryllodes supplicans), I found that sibling males achieved lower fertilization success in competition with a male unrelated to the female than in competition with another sibling more frequently than expected by chance, although the mean paternity values did not differ significantly between treatments. The tendancy for sibling males to achieve relatively lower fertilization success in competition with males unrelated to the female could not be explained by the effects of increased ejaculate allocation, female control of sperm transfer or inbreeding. This study therefore provides some evidence in support of the idea that female insects (or their ova) may be capable of selection against sperm on the basis of genetic similarity of conspecific males.     

Factors influencing paternity success in Antechinus agilis: last‐male sperm precedence,timing of mating and genetic compatibility

We describe the patterns of paternity success from laboratory mating experiments conducted in Antechinus agilis, a small size dimorphic carnivorous marsupial (males are larger than females). A previous study found last‐male sperm precedence in this species, but they were unable to sample complete litters, and did not take male size and relatedness into account. We tested whether last‐male sperm precedence regardless of male size still holds for complete litters. We explored the relationship between male mating order, male size, timing of mating and relatedness on paternity success. Females were mated with two males of different size with either the large or the small male first, with 1 day rest between the matings. Matings continued for 6 h. In these controlled conditions male size did not have a strong effect on paternity success, but mating order did. Males mating second sired 69.5% of the offspring. Within first mated males, males that mated closer to ovulation sired more offspring. To a lesser degree, variation appeared also to be caused by differences in genetic compatibility of the female and the male, where high levels of allele‐sharing resulted in lower paternity success.     

Male dominance rank and offspring-initiated affiliative behaviors were not predictors of paternity in a captive group of pigtail macaques (Macaca nemestrina)

Paternity of 16 pigtail macaque offspring was determined using a DNA profile analysis and was based on two independent assays of the genome of each individual using multilocus DNA probes. The offspring were members of a group of 59 pigtail macaques, including 5 adult males, 1 subadult male, and 37 adult and subadult females. Rank was unrelated to paternity as the first ranking male sired 0, the second ranking male sired 3; the third ranking male sired 0, the fourth ranking male sired 8, and the fifth ranking male sired 2 offspring. The subadult male sired 0 offspring. The DNA analysis was effective in excluding possible sires of 3 offspring whose mothers had become pregnant by another male before being introduced to the study males. Subsequent semen evaluation revealed an absence of sperm in the semen of the alpha male, but revealed a sperm count within normal limits in the third ranking male, who also sired no offspring. Behavioral data focusing on male-offspring interactions found that offspring did not preferentially affiliate with their sire and that males did not affiliate with their offspring frequently enough for analysis. Thus, this study of one captive pigtail macaque group demonstrates that: (1) rank was not a predictor of reproductive success; and (2) there was no preferential attraction for one's own offspring by males or one's own sire by offspring.     

DOES GENETIC RELATEDNESS OF MATES INFLUENCE COMPETITIVE FERTILIZATION SUCCESS IN GUPPIES?

A growing number of studies highlight the nontransitive properties of ejaculates when they are in competition to fertilize a female's eggs. Increasingly, these studies suggest that postcopulatory processes act as a filter against sperm from closely related males or those with similar genotypes, limiting the deleterious effects of inbreeding on offspring fitness. We investigated the potential for such postcopulatory mechanisms of inbreeding avoidance in the guppy (Poecilia reticulata), a promiscuous livebearing fish. We used artificial insemination as a method of delivering to a female the combined ejaculates from a first cousin (relatedness coefficient r = 0.125) and an unrelated male. This method of sperm delivery controls behavioral processes of pre- and postcopulatory female choice, which can bias paternity toward unrelated males. Our genetic analysis revealed no effect of parental relatedness on paternity outcomes. The observed mean paternity share for related males (0.47) and associated variance did not differ significantly from an expected binomial distribution that assumes no biased use of sperm with respect to relatedness (0.5). Although our data provide no evidence for postcopulatory mechanisms of inbreeding avoidance, the ability of female guppies to influence ejaculate transfer and retention offers an alternative and easily testable mechanism of inbreeding avoidance in this species.     

Fertility assurance through extrapair fertilizations and male paternity defense

Extrapair paternity has been observed in many formally monogamous species. Male pursuit of extrapair fertilizations is explained by the advantages of having offspring that receive essential paternal care from other males. Since females are capable of exercising a degree of control over the post-copulatory sperm competition, extrapair paternity cannot persist unless it confers fitness benefits on cuckolding females. Thus, extrapair paternity involves cooperation between mated females and extrapair males. On the other hand, paired males frequently exhibit strategies that minimize their loss of paternity and/or conserve paternal investment if paternity is lost. Hence, extrapair attributes of diverse species and populations reported in the literature are particular solutions of evolutionary games involving gender-specific cuckolding/anti-cuckolding strategies. Here we use methods of evolutionary game theory to study the role of male paternity guarding strategies in situations where females seek extrapair fertilizations for reasons of genetic compatibility and/or in pursuit of genetic diversity for their offspring. Our results indicate that in these circumstances pursuit of extrapair fertilizations is the only evolutionary stable female strategy. Males, on the other hand, have two, mutually exclusive, evolutionary stable strategies: full time pursuit of extrapair fertilizations and a compromise strategy wherein they protect in-pair paternity during their mate's fertile periods and pursue extrapair paternity the rest of the time. The relative merits of these two strategies are determined by the efficiency of male in-pair paternity defense, breeding synchrony, fitness advantages of extrapair over in-pair offspring, and the intensity of competition for extrapair fertilizations from floater males.     

Mating system variation and morph fluctuations in a polymorphic lizard

In polymorphic male painted dragon lizards (Ctenophorus pictus), red males win staged contests for females over yellow males, and yellow males have greater success in staged sperm competition trials than red males. This predicts different reproductive strategies in the wild with red males being more coercive or better mate guarders than yellow males. Yellow males would be expected to sire more offspring per copulation and have a greater proportion of offspring from clutches with mixed paternity. However, here we show using microsatellites that the frequency of mixed paternity in the wild is low (< 20% on average across years), that all morphs on average have the same number of offspring sired per year, and that mating system variation (polyandry vs. monandry) is strongly correlated with perch density on male territories. Furthermore, a logistic regression on male successful vs. unsuccessful mate acquisition showed that red males were under negative selection when they dominated the population, which suggests ongoing frequency dependent selection on male colouration.     

Molecular evidence of post-copulatory inbreeding avoidance in the field cricket Gryllus bimaculatus

Female promiscuity has broad implications for individual behaviour, population genetics and even speciation. In the field cricket Gryllus bimaculatus, females will mate with almost any male presented to them, despite receiving no recorded direct benefits. Previous studies have shown that female crickets can benefit from polyandry through increased hatching success of their eggs. There is evidence that this effect is driven by the potential of polyandrous females to avoid fertilizing eggs with sperm from genetically incompatible males. We provide direct evidence supporting the hypothesis that polyandry is a mechanism to avoid genetic incompatibilities resulting from inbreeding. Using microsatellite markers we examined patterns of paternity in an experiment where each female mated with both a related and an unrelated male in either order. Overall, unrelated males were more successful in gaining paternity than were related males, but this effect was driven by a much greater success of unrelated males when they were the first to mate.     

Multiple paternity in Littorina obtusata (Gastropoda, Littorinidae) revealed by microsatellite analyses.

Parental identity for juvenile Littorina obtusata was determined from three egg masses by means of microsatellite DNA markers. Results confirm that the attendant adult female in each case was the dam of the offspring and that at least 4-6 males contributed to each brood. This correlates with our behavioral observations that indicated multiple copulations between the female and several males in each experimental aquarium. A significant number of offspring from each brood were sired by non-sampled males (males that had copulated with females before capture) whose sperm had been stored by the female. This is the first direct evidence of multiple paternity in the Littorinidae. Results are discussed in reference to current theories of sperm competition, male precedence, and cryptic female choice.     

Frass clearing by male pine engraver beetles (Ips pini; Scolytidae): paternal care or paternity assurance

Male parental care and paternity assurance are often associatedwith long-duration pair bonds. The mating system of the pineengraver beetle, Ips pini, includes an association between themale and female that persists for most of the prolonged oppositionperiod. The male beetles remove frass that arnmmlatn as thefemales lay their eggs in die phloem tissue of the host tree.Experiments and field observations were done to test possiblebenefits to males that stay in the galleries removing frasswhile die females are ovipositing. Two hypotheses were thatclearing frass (1) provides some form of care that results inmore offspring being produced and (2) is part of a paternityassurance mechanism. Male removal experiments in the field producedno evidence that male presence significantly influenced anyof five measures of offspring production. Laboratory experimentsin which virgin females were bred reciprocally to sterile andfertile males showed that, while there is no strong patternof last-male pr, last-male paternity does increase over time.Field observations revealed that female pine engravers oftencarry sperm from previous maringi when they solicit entry toa male's breeding gallery. The pattern of paternity and thefemale's sperm storage capacity suggest that males must maintainprolonged mating access to females in order to ensure high paternity.Hence, frass clearing is necessary to maximize paternity     

The unexpected but understandable dynamics of mating,paternity and paternal care in the ocellated wrasse

Although theory generally predicts that males should reduce paternal care in response to cues that predict increased sperm competition and decreased paternity, empirical patterns are equivocal. Some studies have found the predicted decrease in male care with increased sperm competition, while even more studies report no effect of paternity or sperm competition on male care. Here, we report the first example, to our knowledge, of paternal care increasing with the risk and intensity of sperm competition, in the ocellated wrasse (Symphodus ocellatus). Theory also predicts that if paternal care varies and is important to female fitness, female choice among males and male indicators traits of expected paternal care should evolve. Despite a non-random distribution of mating success among nests, we found no evidence for female choice among parental males. Finally, we document the highest published levels of extra-pair paternity for a species with exclusive and obligate male care: genetic paternity analyses revealed cuckoldry at 100 per cent of nests and 28 per cent of all offspring were not sired by the male caring for them. While not predicted by any existing theory, these unexpected reproductive patterns become understandable if we consider how male and female mating and parental care interact simultaneously in this and probably many other species.     

Sperm storage and use in polyandrous females of the globally invasive fruitfly, Ceratitis capitata

The medfly, Ceratitis capitata, is an invasive species in which polyandry, associated with sperm precedence, is a common behaviour in the wild. In this species, characterized by internal fertilization, we disclose how the sperm from two males are stored in the female storage organs and how they are used in terms of paternity outcome. The experiments were designed to furnish comparable and unbiased estimates of sperm numbers and progeny in twice-mated females. Results are incorporated in a model through which it is possible to relate the amount of stored sperm with the progeny of twice-mated females. The results show that polyandrous medfly females conserve equal amounts of sperm from the two males to fertilize their eggs. However, we observed a clear advantage of the second male's sperm in siring progeny, which interestingly decreases in favor of the first male as ovipositions progress. The results enable us to exclude differential sperm mortality and suggest that it is the mechanics governing the storage organs which causes the initial, but decreasing second male sperm precedence during the female reproductive life. These outcomes allow us to correlate sperm use in polyandrous females with the mating strategies and invasiveness of this fly.