Tim R Birkhead. American Scientist. Volume 84, Issue 3. May 1996.
The first inklings that this might be the case arose with the earliest stirrings of behavioral ecology in the early 1970s. The traditional view of monogamy was that each partner benefited from mutual cooperation, but behavioral ecology’s focus on individual selection raised the notion that even within a monogamous relationship both males and females should behave selfishly. In a landmark paper published in 1972 Robert Trivers, then at Harvard, predicted that males should attempt to inseminate the females of other males and hence parasitize their paternal care. By doing so, they could increase their reproductive success–and, because sperm were believed to be cheap, at minimal expense. Females on the other hand were predicted to be coy, since they need to copulate only once to ensure that their eggs are fertilized. Moreover, it was thought that infidelity was potentially rather costly for a female since her partner would retaliate by reducing his investment in her offspring. At that time there had been relatively few observations of extra-pair behavior of birds, and those that had been made were explained away as nonadaptive (and in highly anthropomorphic terms)–for example, the males were “sexually dissatisfied at home.”
My own involvement in this field had its beginnings in 1970 during a memorable undergraduate lecture in which we were presented the heady cocktail of Trivers’s ideas and the results of University of Liverpool ecologist Geoff Parker’s studies of multiple mating in the yellow dung fly Scatophaga stercoraria. Parker showed that the sperm competition that results from males copulating with already mated females creates powerful and conflicting selection pressures on males. Male dung flies have a suite of adaptations to help them secure multiple matings and a suite of counter-adaptations to help them avoid being cuckolded. These results hung so elegantly on their theoretical framework, and the whole subject area had such a feel of revolution about it, that I knew at that moment that this was what I wanted to work on. With a long-term interest in ornithology, I specifically wondered if these ideas were applicable to birds.
Monogamy Myths
The first field studies of sperm competition in birds were essentially observational. They sought to determine whether extra-pair copulations take place in monogamous birds and whether paired males attempt to protect their partner from the sexual advances of other males. Both prove true: Males routinely attempt to copulate with the fertile partners of other males, while at the same time remaining very close to their own female when she is fertile to avoid being cuckolded. It seems hard to believe now, but it was not even known in the mid-1970s whether multiple mating by female birds in the wild could result in multiple paternity. While these observational studies were in full swing, the new technique of DNA fingerprinting was developed in the mid-1980s, for the first time enabling researchers to assess parentage unambiguously. Luckily for those of us who study birds, Terry Burke of the University of Leicester and David Parkin of the University of Nottingham were quick to realize the potential of fingerprinting for studies of the mating systems of birds. To date, paternity analyses have been conducted on approximately 100 species of bird, revealing levels of extra-pair paternity that even Trivers and Parker would not have predicted. In some species, such as the superb fairy wren MaIurus cyaenus, more than half of all offspring come from extra-pair matings. True monogamy, with zero extra-pair paternity, is relatively rare but is evident in some seabirds, such as the fulmar petrel Fulmarus glacialis. These studies show that extra-pair copulations do increase the reproductive success of certain males–sometimes substantially. For example, in the purple martin Progne subis, a bird familiar to many North Americans, successful cuckolders can double the number of offspring they father.
But two major questions remained. First, why do females engage in extra-pair copulations? And, second, when they do, what determines which male fathers the offspring?
The first of these questions is one that many behavioral ecologists are currently trying to answer. In the true tradition of behavioral ecology it is a functional question and concerns the adaptive significance of females copulating with more than one male during a single reproductive cycle. Although there are plenty of ideas, so far there are no clear answers. A frequent explanation is that females mate with additional males to ensure fertilization, but there is no evidence for this. The answer currently attracting the most attention, some evidence and not a little controversy is that a female gains better genes for her offspring by performing one or more extra-pair copulations with a male that is of better quality than her partner. We still have a long way to go, however, to demonstrate this unequivocally and in enough species for it to constitute a general explanation for female infidelity.
The second question, concerned with the fertilization success of copulations, however, is one where some clear answers are now emerging. What is more, this is a mechanistic question, concerned not with the evolutionary significance of phenomena but with underlying physiological processes. After a long and productive period in which they focused almost exclusively on functional aspects, behavioral ecologists have started to appreciate that by broadening their horizons to include mechanisms, they can look forward to a highly productive marriage between these complementary levels of explanation. The mechanism of sperm competition in birds provides a nice example of this fruitful approach.
Last-Male Precedence
In many organisms, the second of two males to copulate with a female typically fertilizes most of her eggs. This phenomenon, known as second- (or last-) male sperm precedence, has been reported in insects, crustaceans and birds. A study on chickens conducted in the 1970s reported a particularly strong last-male effect: When females were artificially inseminated just four hours apart with equal numbers of sperm from two genotypes, the second insemination fathered about 77 percent of the offspring–regardless of which genotype’s sperm was inseminated first. The proposed mechanism was that the sperm from the two successive inseminations remained stratified within the female’s sperm store; the last sperm in were the first out and hence fertilized most eggs.
Investigating the mechanism in detail meant following sperm along the obstacle course from insemination to fertilization. After insemination, only 1 percent of the several millions of sperm ejaculated traverse the intensely hostile vagina and enter the female’s sperm-storage tubules. Many of the remaining sperm are ejected with fecal material within minutes of insemination. The sperm that get into the storage tubules remain quiescent and can fertilize eggs up to 30 days later. Over this time, however, sperm leak out of the tubules at a constant rate and are carried up the oviduct to the infundibulum, where fertilization takes place. Fertilization in birds is sequential: Each egg of a clutch is fertilized separately and, in the case of most small birds, at 24-hour intervals. The ovum is shed from the ovary, usually in the early morning, into the infundibulum. There is then a 15- to 30-minute window during which fertilization must take place. After that time the protein-rich albumen is laid down around the ovum, preventing further access by sperm. The ovum spends the next 23 hours in the oviduct having water, membranes and shell added before the fully formed egg is laid early the next morning. Within an hour of laying, the next ovum is shed from the ovary, and the cycle repeats itself. Between the laying of one egg and the fertilization of the next ovum, the infundibulum is replenished with sperm from the sperm-storage tubules.
Last In, First Out
When I started to investigate the mechanisms of sperm competition in birds, I needed a species that would breed readily in captivity–allowing me to control natural matings–but also one that I could observe in the wild to determine whether sperm competition takes place naturally. The zebra finch Taeniopygia guttata was an ideal species. An inhabitant of Australia’s arid regions and an opportunistic breeder, the zebra finch breeds throughout the year in captivity, given the right conditions.
The observations I made of individually marked zebra finches at Richard Zann’s study colony in northern Victoria, Australia, confirmed that extra-pair copulation was frequent. Subsequent DNA fingerprinting, performed in collaboration with Terry Burke and other colleagues, revealed that extra-pair copulations resulted in extra-pair paternity. Despite the technological elegance of DNA fingerprinting, for laboratory work in Sheffield I needed a more rapid technique to assign paternity and decided on the low-tech but tried-and-trusted method of genetic plumage markers. Zebra-finch breeders have developed a number of different color forms whose mode of inheritance is well known. The fawn mutation is sex linked and recessive to the wild (gray) type, which means that when a fawn female copulates with a fawn male they produce only fawn offspring. But when a fawn female is fertilized by a homozygous gray (wild-type) male, they produce only gray-plumaged offspring. The beauty of this method is that when a fawn female mates with a male of each genotype, the paternity of the offspring is obvious as soon as they hatch.
I performed two experiments, both designed to mimic situations that take place naturally in the wild. The first was a mate-switching experiment in which two males replaced each other. Despite each securing a similar number of copulations, the second male fertilized most (75 percent) eggs. This suggested a last-male effect, so the second experiment was designed to test simultaneously for this and to determine the efficacy of a single extra-pair copulation. The first male secured about nine copulations on average, but the second male was extraordinarily successful and fertilized over half the eggs with just a single insemination. These results confirmed a last-male advantage, and demonstrated the potency of a single extra-pair copulation. Since my results were similar to those reported for chickens, I started to wonder about the “last-in, first-out” system as an explanation for last-male precedence. Because the sperm competition experiments I was conducting were extremely time-consuming, however, I realized that I could easily spend years conducting empirical tests to figure out the mechanism of last-male sperm precedence.
Kate Lessells, then at the University of Sheffield and now at the Netherlands Institute of Ecology, and I took a shortcut and built mathematical models of sperm competition to identify the most plausible mechanisms for last-male precedence. Because more was known at that time about the reproductive biology of the chicken than of the zebra finch, we used information from the chicken studies to test our models. Almost our first finding was that the last-in, first-out system could not account for the 77 percent last-male effect. This stratification model, as we called it, predicted that with increasing time since insemination the first male’s sperm should fertilize more and more offspring, but the empirical data showed that the ratio of offspring remained constant. In addition, we now know that it takes considerably longer than four hours for sperm to occupy the sperm storage tubules, providing further evidence for the implausibility of this mechanism. Our next model, referred to as the passive sperm-loss model, predicted a last-male effect simply because by the time the second insemination took place some sperm from the initial insemination had already died or been lost from the female tract. In other words, the outcome of sperm competition depended entirely on the relative numbers of sperm from each male present at the point of fertilization. We also rejected this model because the rate at which chickens lose sperm from their reproductive tract is much too slow to explain the observed level of last-male sperm precedence. The only model that came anywhere close to providing a plausible explanation for the 77 percent precedence was a displacement model in which sperm from the second insemination displaced sperm from the earlier insemination. The greater the degree of displacement, the greater the last-male advantage.
The next sage was an empirical test of the displacement model. A simple test would be to inseminate equal numbers of sperm four hours apart and show that after the second insemination fewer of the first male’s sperm remained in the female’s sperm-storage tubules. But to do this, one needs to be able to distinguish between the sperm of different males inside the sperm-storage tubules. At that time there was only a single vital fluorescent dye available that would label sperm without obviously affecting their viability. When we inseminated females sequentially with labeled and unlabeled sperm, however, the dyed sperm stained the unlabeled sperm, preventing us from distinguishing between the two. It was assuring but no less frustrating to learn that two other research groups experienced the same problem in trying to resolve this question.
Science is supposed to progress in a logical Popperian fashion, but sometimes it does not work out like that. On coming up against a brick wall, I changed tack and decided to go back to first principles by repeating the sperm-competition experiment using chickens. When we did this and inseminated females twice four hours apart, we were surprised to detect virtually no last-male sperm precedence. Thinking we might have made an error, we repeated the experiment, several times in fact, but on each occasion there was no evidence of a marked last-male precedence. Our results, however, were consistent with the passive sperm-loss model. This then left unresolved the question of why the two sets of sperm-competition experiments on chickens gave such different results. The answer, as I subsequently realized, is that the outcome of sperm competition, in the chicken at least, depends on the time at which the inseminations are made. In the original study these initial insemination was made very close to the time of egg laying, a time we now know results in a relatively low uptake of sperm by the female. Therefore, because the second insemination was performed four hours later, it had a considerable advantage and hence the 77 precent precedence. In our chicken experiments, both inseminations were made at least seven hours after egg laying and hence avoided this particular problem. I have subsequently analyzed published data from other sperm-competition experiments with chickens and turkeys, and these are all consistent with the passive sperm-loss model.
Encouraged by the success of this model, we then examined the results of our zebra finch experiments to see if they too could be explained by the passive loss of sperm from the female tract. To do this, however, we needed two pieces of information: the numbers of sperm inseminated by males and the rate at which sperm are lost or die in the female tract. We devised an empirical method, using a surrogate female, to determine the number of sperm inseminated. Using this technique, we found that the time since the last ejaculation had an important effect on the numbers of sperm transferred. Males that had not copulated for a week or more, referred to as “rested” males, transferred about 8 million sperm. But since they have limited sperm stores and a relatively low rate of sperm production, subsequent ejaculates on the same or the next day were much smaller–about 1 million sperm. In the single extra-pair copulation experiment, we used males that had not copulated for at least one week, so these males would have produced a single ejaculate containing a large number of sperm. The paired male was also rested when the experiment started, so he would also have produced one large initial ejaculate followed by a series of smaller ones. To estimate the ratio of sperm from the two males at the time of fertilization, however, we also had to take into account that sperm can remain in the sperm-storage tubules for up to 13 days, during which time they constantly leak out and can fertilize new eggs.
To ascertain the rate of loss of sperm from the tract, we developed a technique used initially by poultry biologists to predict the fertility of eggs. This consisted of counting the sperm trapped in the layers surrounding the yolk–the perivitelline layers–of laid eggs. At ovulation the ovum (the yolk) is released from the ovary into the infundibulum where a number of sperm are present. At this stage the ovum is surrounded by the inner perivitelline layer, and within minutes this is penetrated by one or more sperm at the germinal disc. One of these sperm then fuses with the egg’s nucleus, thereby combining paternal and maternal DNA. Shortly afterwards the infundibulum starts to secrete the outer perivitelline layer, and as it is laid down around the ovum, it traps the other sperm present in the infundibulum. These sperm can be readily seen and counted in a laid egg–the perivitelline layers are removed from around the yolk, stained with a DNA-specific fluorescent dye and examined microscopically. We counted the number of sperm on successive eggs after a single copulation and found that numbers declined exponentially, indicating that sperm were lost at a constant rate.
Armed with quantitative measures of the number of sperm inseminated and the rate at which they are lost from the female tract, we were able to determine whether they could account for the last-male sperm precedence we recorded in our experiments with zebra finches. Incorporating these values into the passive sperm-loss model, it was reassuring to find that the fit between what was predicted and what we observed was remarkably close. This means that the relative numbers of sperm from different males in the infundibulum at the time of fertilization determines the outcome of sperm competition. Any factor that affects the uptake of sperm by the female h-act will therefore influence a particular male’s chances of fertilizing eggs. The chicken experiments showed that one such factor is the timing of insemination relative to egg laying–inseminations close to egg laying were less successful. Another factor that could affect the proportion of sperm reaching the site of fertilization is the quality of the sperm themselves.
A Question of Quality
Breeders of domestic mammals and birds, and those who work in human in vitro fertilization (IVF) clinics, have known for a long time that the motility of a semen sample is a reasonable indicator of its likelihood of successfully fertilizing ova. Motility, which usually refers to the proportion of sperm that are actually motile, is, however, a relatively crude measure of quality. More recently the ability to measure the actual velocity of individual sperm, using computer-assisted sperm analyses, has provided a much better predictor of fertilizing potential. In our experiments with surrogate females we found that the ejaculated sperm from rested males moved at twice the speed (33.4 micrometers per second) as those from the next ejaculate made just one hour later (17.4 micrometers per second). In addition, a higher proportion of the sperm in a rested male’s ejaculate are morphologically normal than those in subsequent ejaculates. By examining sperm in the male’s sperm store, the seminal glomera, we found that sperm appear to mature and gain the potential for high-speed movement as they travel down the male tract. A similar maturation process takes place in the epididymis of male mammals, but with one important difference: Unlike the zebra finch, male mammals never ejaculate immature sperm. The reason for this difference remains to be resolved. If faster-swimming sperm are more likely to traverse the female’s hostile vagina and enter the sperm-store tubules, it may provide an additional reason why the single copulation from a rested male zebra finch was so successful in fertilizing eggs. We are currently investigating this.
Across the animal kingdom the original view was that sperm competition has been driven primarily by selection on males. It is clear that by safeguarding their own paternity and fertilizing other females, males increase their reproductive output. In contrast, the only benefit socially monogamous females were thought to obtain from copulating with more than one male was an increase in the quality of her offspring. The view that selection operates more intensively on males than on females in terms of sperm competition has been questioned in recent years, and there is now considerable focus on female strategies. Although males of a few species (for example, mallard ducks Anas platyrhynchos) are able to force copulations on females, I the evidence that females generally control extra-pair behavior in birds is incontrovertible. For example, field studies of the purple martin and blue tit Parus caeruleus, both socially monogamous species, reveal that females actively seek and initiate extra-pair copulations with males that are of better quality than their partner. Similarly, studies of zebra finches in captivity also indicate that females prefer males with certain characteristics (for example, with a high SOI rate and a particularly red beak) as extra-pair partners. How they benefit from this behavioral choice of copulation partner is still unknown. We tested the idea that zebra finches with these particular phenotypic traits might also produce fast moving, high-quality sperm but found no evidence for such an effect.
In addition to controlling events behaviorally, females that have mated with several males may also have the physiological ability to favor the sperm of one male over another’s. Since fertilization takes place inside the female’s body, it seems reasonable that selection will act particularly strongly to give them he ultimate control. Whether females have the physiological ability to determine paternity in this way, referred to as cryptic choice, remains to be seen, but there are some indications that they might.
One of the most remarkable examples concerns an organism about as unbird like as you can imagine. In the comb-jelly (ctenophore) beroe, several sperm typically penetrate the ovum (a phenomenon referred to as polyspermy, which also occurs in birds), and the egg’s nucleus moves around the cytoplasm visiting each of the sperm nuclei before fusing with one. In birds the potential for female choice is considerable: Over 99 percent of the sperm a male inseminates is rejected by the female. By modifying the degree of rejection only slightly one way or the other, a female could change the odds in the fertilization stakes considerably but whether she has the ability to do this remains to be seen.
All of this raises the perplexing question of why a female should bother to pair up with a male only to go off and mate with another later? Several possible answers have presented themselves.
An attractive possibility is that the extra-pair male helps rear the offspring, but no evidence exists to suggest this is true. A second possibility is that the female gets a valuable item–food, for example–in exchange for services rendered. But this theory also lacks evidence. It has been suggested that females mate with other males when their partner’s sperm count is low, but there is no evidence for this.
So it seems that many scientists are left with a single possible explanation. Females are looking for higher quality males to father their offspring. Why doesn’t the female just choose the highest-quality male to be her mate in the first place? That is not always possible, as the example of migratory birds, such as swallows, shows. Males generally arrive at the breeding grounds a few days before females do. The first females to return have the pick of all the males and generally select the highest quality. Later-returning females have fewer good choices left to them, and the last females to return have the poorest choices. Rather than foregoing her chance to breed at all that season, the late-returning female takes a poor-quality male as her mate. But she doesn’t have to completely forgo the chance to have some higher-quality offspring, and so she engages in extra-pair mating.
It is hard to tell what constitutes high quality for birds. In swallows, it is the length and symmetry of the tail. Studies have shown that females paired to males with short and asymmetric tails seek extra-pair matings from males whose tails a longer and more symmetric.
Studies have also shown that the extra-pair matings are responsible for producing many offspring throughout the animal kingdom. Females of many species routinely copulate with more than one male, but in only a small proportion of species are the processes that determine which males father offspring understood. Just as in birds, in many insects the last male to copulate fertilizes most eggs. In dragonflies and damselflies the mechanism is brutally simple; before inseminating his own sperm, the male scrapes out any previously stored sperm using minute hooks on his penis. In the ghost crab Inachus phalangium, the last male also has precedence, but he uses a different trick. Before transferring sperm to a female, he introduces highly modified seminal fluid that sets hard and seals off previously inseminated sperm at the back of the female’s sperm store. In the majority of invertebrates, however, the mechanisms by which last-male precedence is achieved are more complicated. In the yellow dungfly it is thought that the incoming sperm displace and flush out some of the sperm from previous matings.
Molecular studies of paternity have demonstrated that sperm competition also takes place in mammals–including apparently monogamous species like ourselves. Studies of the inheritance of human disease have revealed a number of mismatches–many of which can be explained only by extra-pair fertilization. None of the paternity data for people have been formally published, but the figures apparently suggest that between one in ten to one in twenty offspring are the result of extra-pair matings. The mechanics of fertilization success in mammals is fundamentally different from that in birds. First, if a female ovulates more than a single ovum, the eggs are fertilized at virtually the same time (not on different days, as in birds). Second, there is no consistent last-male effect. In fact, all combinations of effects occur–sometimes the first male wins, sometimes it is the second male. The reason for this is that in mammals, timing is everything. Unlike birds, the fertilizing lifespan of mammalian sperm is short and they also have to undergo capacitation, the physiological preparation for fertilization. Because ova also remain fertilizable for only a short time, to have a good chance of fertilization, a male must time his copulations so that his sperm have undergone capacitation just at the time the female ovulates and the ovum is ready to be fertilized.