Comparative analysis of worker reproduction and policing in eusocial hymenoptera supports relatedness theory

In many bees, wasps, and ants, workers police each other in order to prevent individual workers from selfishly producing their own male offspring. Although several factors can selectively favor worker policing, genetic relatedness is considered to be of special importance. In particular, kin selection theory predicts that worker policing should be more common in species where workers are more related to the queen's sons than to other workers' sons. Here we provide strong novel support for this theory based on a comparative analysis of policing and male parentage in 109 species of ants, bees, and wasps. First, an analysis of behavioral data confirms that worker policing occurs more frequently in species where workers are more related to the queen's sons than to other workers' sons. Second, an analysis of male parentage shows that a significantly higher percentage of the males are workers' sons in species where the workers are more related to other workers' sons. Both conclusions also hold if data are analyzed using phylogenetically independent contrasts. Although our analysis provides strong overall support for the theory that relatedness affects kin conflict over male parentage, there is also significant residual variation. Several factors that may explain this variation are discussed.     

Sex allocation conflict in ants: when the queen rules

Insect societies are paramount examples of cooperation, yet they also harbor internal conflicts whose resolution depends on the power of the opponents. The male-haploid, female-diploid sex-determining system of ants causes workers to be more related to sisters than to brothers, whereas queens are equally related to daughters and sons. Workers should thus allocate more resources to females than to males, while queens should favor an equal investment in each sex. Female-biased sex allocation and manipulation of the sex ratio during brood development suggest that workers prevail in many ant species. Here, we show that queens of Formica selysi strongly influenced colony sex allocation by biasing the sex ratio of their eggs. Most colonies specialized in the production of a single sex. Queens in female-specialist colonies laid a high proportion of diploid eggs, whereas queens in male-specialist colonies laid almost exclusively haploid eggs, which constrains worker manipulation. However, the change in sex ratio between the egg and pupae stages suggests that workers eliminated some male brood, and the population sex-investment ratio was between the queens' and workers' equilibria. Altogether, these data provide evidence for an ongoing conflict between queens and workers, with a prominent influence of queens as a result of their control of egg sex ratio.     

Queen-worker conflicts over male production and sex allocation in a primitively eusocial wasp

Abstract In a colony headed by a single monandrous foundress, theories predict that conflicts between a queen and her workers over both sex ratio and male production should be intense. If production of males by workers is a function of colony size, this should affect sex ratios, but few studies have examined how queens and workers resolve both conflicts simultaneously. We conducted field and laboratory studies to test whether sex-ratio variation can be explained by conflict over male production between queen and workers in the primitively eusocial wasp Polistes chinensis antennalis.
Worker oviposition rate increased more rapidly with colony size than did queen oviposition. Allozyme and micro-satellite markers revealed that the mean frequency of workers' sons among male adults in queen-right colonies was 0.39 ± 0.08 SE (n = 22). Genetic relatedness among female nestmates was high (0.654–0.796), showing that colonies usually had a single, monandrous queen. The mean sex allocation ratio (male investment/male and gyne investments) of 46 queen-right colonies was 0.47 ± 0.02, and for 25 orphaned colonies was 0.86 ± 0.04. The observed sex allocation ratio was likely to be under queen control. For queen-right colonies, the larger colonies invested more in males and produced reproductives protandrously and/or simultaneously, whereas the smaller colonies invested more in females and produced reproductives protogynously. Instead of positive relationships between colony size and worker oviposition rate, the frequency of workers' sons within queen-right colonies did not increase with colony size. These results suggest that queens control colony investment, even though they allow worker oviposition in queen-right colonies. Eggs laid by workers may be policed by the queen and/or fellow workers. Worker oviposition did not influence the outcome of sex allocation ratio as a straightforward function of colony size.     

Convergent evolution of worker policing by egg eating in the honeybee and common wasp

Mutual policing, where group members suppress each others' reproduction, is hypothesized to be important in the origin and stabilization of biological complexity. Mutual policing among workers in social insects can reduce within-colony conflict. However, there are few examples. We tested for worker policing in the common wasp Vespula vulgaris. Workers rapidly removed worker-laid eggs but left most queen-laid eggs (four out of 120 worker eggs versus 106 out of 120 queen eggs remained after 1h). Ovary dissection (1150 workers from six colonies) revealed that a small but significant number of workers have active ovaries (4%) equivalent to approximately five to 25 workers per colony. Consistent with effective policing of worker reproduction, microsatellite analysis of males (270 individuals from nine colonies) detected no workers' sons. Worker policing by egg eating has convergently evolved in the common wasp and the honeybee suggesting that worker policing may have broad significance in social evolution. Unlike the honeybee, relatedness patterns in V. vulgaris do not explain selection for policing. Genetic analysis (340 workers in 17 nests) revealed that workers are equally related to the queen's and other workers' sons (worker-worker relatedness was 0.51 +/- 0.04, 95% confidence interval). Worker policing in V. vulgaris may be selected due to the colony-level benefit of conflict suppression.     

REPRODUCTIVE CONFLICT IN BUMBLEBEES AND THE EVOLUTION OF WORKER POLICING

Worker policing (mutual repression of reproduction) in the eusocial Hymenoptera represents a leading example of how coercion can facilitate cooperation. The occurrence of worker policing in “primitively” eusocial species with low mating frequencies, which lack relatedness differences conducive to policing, suggests that separate factors may underlie the origin and maintenance of worker policing. We tested this hypothesis by investigating conflict over male parentage in the primitively eusocial, monandrous bumblebee, Bombus terrestris. Using observations, experiments, and microsatellite genotyping, we found that: (a) worker‐ but not queen‐laid male eggs are nearly all eaten (by queens, reproductive, and nonreproductive workers) soon after being laid, so accounting for low observed frequencies of larval and adult worker‐produced males; (b) queen‐ and worker‐laid male eggs have equal viabilities; (c) workers discriminate between queen‐ and worker‐laid eggs using cues on eggs and egg cells that almost certainly originate from queens. The cooccurrence in B. terrestris of these three key elements of “classical” worker policing as found in the highly eusocial, polyandrous honeybees provides novel support for the hypothesis that worker policing can originate in the absence of relatedness differences maintaining it. Worker policing in B. terrestris almost certainly arose via reproductive competition among workers, that is, as “selfish” policing.     

Kin structure and colony male reproduction in the hornet<Emphasis Type="Italic"> Vespa crabro</Emphasis> (Hymenoptera: Vespidae)

We estimated queen mating frequency, genetic relatedness among workers, and worker reproduction in Vespa crabro flavofasciata using microsatellite DNA markers. Of 20 colonies examined, 15 contained queens inseminated by a single male, 3 colonies contained queens inseminated by two males, and 2 colonies contained queens inseminated by three males. The genetic relatedness among workers was estimated to be 0.73±0.003 (mean±SE). For this high relatedness, kin selection theory predicts a potential conflict between queens and workers over male production. To verify whether males are derived from queens or workers, 260 males from 13 colonies were genotyped at four microsatellite loci. We found that all of the males were derived from the queens. This finding was further supported by the fact that only 33 of 2,990 workers dissected had developed ovaries. These workers belonged to 2 of the 20 colonies. There was no relationship between queen mating frequency and worker reproduction, and no workers produced male offspring in any of the colonies. These results suggest that male production dominated by queens in V. crabro flavofasciata is possibly due to worker policing.     

A test of worker policing theory in an advanced eusocial wasp, Vespula rufa

Mutual policing is an important mechanism for maintaining social harmony in group-living organisms. In some ants, bees, and wasps, workers police male eggs laid by other workers in order to maintain the reproductive primacy of the queen. Kin selection theory predicts that multiple mating by the queen is one factor that can selectively favor worker policing. This is because when the queen is mated to multiple males, workers are more closely related to queen's sons than to the sons of other workers. Here we provide an additional test of worker policing theory in Vespinae wasps. We show that the yellowjacket Vespula rufa is characterized by low mating frequency, and that a significant percentage of the males are workers' sons. This supports theoretical predictions for paternities below 2, and contrasts with other Vespula species, in which paternities are higher and few or no adult males are worker produced, probably due to worker policing, which has been shown in one species, Vespula vulgaris. Behavioral observations support the hypothesis that V. rufa has much reduced worker policing compared to other Vespula. In addition, a significant proportion of worker-laid eggs were policed by the queen.     

REPRODUCTIVE SKEW AND SPLIT SEX RATIOS IN SOCIAL HYMENOPTERA

Abstract I present a model demonstrating that, in social Hymenoptera, split sex allocation can influence the evolution of reproductive partitioning (skew). In a facultatively polygynous population (with one to several queens per colony), workers vary in their relative relatedness to females (relatedness asymmetry). Split sex‐ratio theory predicts that workers in monogynous (single‐queen) colonies should concentrate on female production, as their relatedness asymmetry is relatively high, whereas workers in the polygynous colonies should concentrate on male production, as their relatedness asymmetry is relatively low. By contrast, queens in all colonies value males more highly per capita than they value females, because the worker‐controlled population sex ratio is too female‐biased from the queens' standpoint. Consider a polygynous colony in a facultatively polygynous population of perennial, social Hymenoptera with split sex ratios. A mutant queen achieving reproductive monopoly would gain from increasing her share of offspring but, because the workers would assess her colony as monogynous, would lose from the workers rearing a greater proportion of less‐valuable females from the colony's brood. This sets an upper limit on skew. Therefore, in social Hymenoptera, skew evolution is potentially affected by queen‐worker conflict over sex allocation.     

Mechanisms of social regulation change across colony development in an ant

Background  

Mutual policing is an important mechanism for reducing conflict in cooperative groups. In societies of ants, bees, and wasps, mutual policing of worker reproduction can evolve when workers are more closely related to the queen's sons than to the sons of workers or when the costs of worker reproduction lower the inclusive fitness of workers. During colony growth, relatedness within the colony remains the same, but the costs of worker reproduction may change. The costs of worker reproduction are predicted to be greatest in incipient colonies. If the costs associated with worker reproduction outweigh the individual direct benefits to workers, policing mechanisms as found in larger colonies may be absent in incipient colonies.     

Worker interests and male production in Polistes gallicus, a Mediterranean social wasp

The resolution of social conflict in colonies may accord with the interests of the most numerous party. In social insect colonies with single once-mated queens, workers are more closely related to the workers' sons than they are to the queens' sons. Therefore, they should prefer workers to produce males, against the queen's interests. Workers are capable of producing males as they arise from unfertilized eggs. We found Polistes gallicus to have colonies of single, once-mated queens, as determined by microsatellite genotyping of the workers, so worker interests predict worker male production. In colonies lacking queens, workers produced the males, but not in colonies with original queens. Thus worker interests were expressed only when the queen was gone. The high fraction of missing queens and early end to the colony cycle relative to climate so early in the season is surprising and may indicate a forceful elimination of the queen.     

Male parentage and queen mating frequency in the bumblebee <Emphasis Type="Italic">Bombus ignitus</Emphasis> (Hymenoptera: bombinae)

Kin selection theory predicts conflict between queens and workers in the social insect colony with respect to male production. This conflict arises from the haplodiploid system of sex determination in Hymenoptera that creates relatedness asymmetries in which workers are more closely related to the sons of other workers than to those of the queen. In annual hymenopteran societies that are headed by a single queen, the mating frequency of the queen is the only factor that affects the colony kin structure. Therefore, we examined the mating structure of queens and the parentage of males in a monogynous bumblebee, Bombus ignitus, using DNA microsatellites. In the seven colonies that were studied, B. ignitus queens mated once, thereby leading to the prediction of conflict between the queen and workers regarding male production. In each of the five queen-right colonies, the majority of the males (95%) were produced by the colony’s queen. In contrast, workers produced approximately 47% of all the males in two queenless colonies. These results suggest that male production in B. ignitus is a conflict between queen and workers.     

Male parentage in army ants

In most social insects workers do not mate, but have retained the ability to produce haploid eggs that can develop into viable male offspring. Under what circumstances this reproductive potential is realized and how the ensuing worker-queen conflict over male production is resolved, is an area of active research in insect sociobiology. Here we present microsatellite data for 176 males from eight colonies of the African army ant Dorylus (Anomma) molestus. Comparison with worker genotypes and inferred queen genotypes from the same colonies show that workers do not or at best very rarely reproduce in the presence of the queen. Queens of D. (A.) molestus are known to be highly multiply mated. This implies that workers are on average more closely related to queen sons than to other workers' sons, so that our results are consistent with predictions from inclusive fitness theory. It remains unknown, however, whether worker sterility is maintained by active worker policing or by self-restraint.     

Reproductive alliances and posthumous fitness enhancement in male ants

Ants provide excellent opportunities for studying the evolutionary aspects of reproductive conflict. Relatedness asymmetries owing to the haplodiploid sex determination of Hymenoptera create substantial fitness incentives for gaining control over sex allocation, often at the expense of the fitness interests of nest-mates. Under worker-controlled split sex ratios either the reproductive interests of the mother queen (when workers male bias the sex ratio) or the father (when workers female bias the sex ratio), but never that of both parents simultaneously, are fulfilled. When workers bias sex ratios according to the frequency of queen mating, males which co-sire a colony have a joint interest in manipulating their daughter workers into rearing a more female-biased sex ratio. Here we show that males of the ant Formica truncorum achieve such manipulation by partial sperm clumping, so that the cohort-specific relatedness asymmetry of the workers in colonies with multiple fathers is higher than the cumulative relatedness asymmetry across worker cohorts. This occurs because a single male fathers the majority of the offspring within a cohort. Colonies with higher average cohort-specific relatedness asymmetry produce more female-biased sex ratios. Posthumously expressed male genes are thus able to oppose the reproductive interests of the genes expressed in queens and the latter apparently lack mechanisms for enforcing full control over sperm mixing and sperm allocation.     

Colony kin structure and male production in Dolichovespula wasps

In annual hymenopteran societies headed by a single outbred queen, paternity (determined by queen mating frequency and sperm use) is the sole variable affecting colony kin structure and is therefore a key predictor of colony reproductive characteristics. Here we investigate paternity and male production in five species of Dolichovespula wasps. Twenty workers from each of 10 colonies of each of five species, 1000 workers in total, were analysed at three DNA microsatellite loci to estimate paternity. To examine the relationship between kin structure and reproductive behaviour, worker ovary activation was assessed by dissection and the maternal origin of adult males was assessed by DNA microsatellites. Effective paternity was low in all species (D. media 1.08, D. maculata 1.0, D. sylvestris 1.15, D. norwegica 1.08 and D. saxonica 1.35), leading to the prediction of queen-worker conflict over male production. In support of this, workers with full-size eggs in their ovaries (four out of five species) and adult males that were workers' sons (all five species) were found in queenright colonies. However, workers were only responsible for a minority of male production (D. media 7.4%, D. maculata 20.9%, D. sylvestris 9.8%, D. norwegica 2.6% and D. saxonica 34.6%) suggesting that the queen maintains considerable reproductive power over the workers. Kin structure and reproductive conflict in Dolichovespula contrast with their sister group Vespula. Dolichovespula is characterized by low paternity, worker reproduction, and queen-worker conflict and Vespula by high paternity, effective worker policing and absence of worker reproduction. The trend revealed by this comparison is as predicted by kin selection theory suggesting that colony kin structure has been pivotal in the evolution of the yellowjacket wasps.     

No evidence that reproductive bumblebee workers reduce the production of new queens

Kin selection theory predicts potential conflict between queen and workers over male parentage in hymenopteran societies headed by one, singly mated queen, because each party is more closely related to its own male offspring. In ‘late-switching’ colonies of the bumblebee Bombus terrestris, i.e. colonies whose queens lay haploid eggs relatively late in the colony cycle, workers start to lay male eggs shortly after the queen lays the female eggs that will develop into new queens. It has been hypothesized that this occurs because workers recognize, via a signal given by the queen instructing female larvae to commence development as queens, that egg laying is now in their kin-selected interest. This hypothesis assumes that aggressive behaviour in egg-laying workers does not substantially reduce the production of new queens, which would decrease the workers' fitness payoff from producing males. We tested the hypothesis that reproductive activity inB. terrestris workers does not reduce the production of new queens. We used microsatellite genotyping to sex eggs and hence to select eight size-matched pairs of ‘late-switching’ colonies from a set of commercial colonies. From one colony of each pair we removed every egg-laying or aggressive worker observed. From the other colony, we simultaneously removed a nonegg-laying, nonaggressive worker. Removed workers were replaced with young workers from separate colonies at equal frequencies within the pair. There was no significant difference in queen productivity between colonies with reduced or normal levels of egg-laying or aggressive workers. Therefore, as predicted, reproductive B. terrestris workers did not significantly reduce the production of new queens.     

Sex ratio conflict and worker production in eusocial hymenoptera

The best known of the conflicts occurring in eusocial Hymenoptera is queen-worker conflict over sex ratio. So far, sex ratio theory has mostly focused on optimal investment in the production of male versus female sexuals, neglecting the investment in workers. Increased investment in workers decreases immediate sexual productivity but increases expected future colony productivity. Thus, an important issue is to determine the queen's and workers' optimal investment in each of the three castes (workers, female sexuals, and male sexuals), taking into account a possible trade-off between production of female sexuals and workers (both castes developing from diploid female eggs). Here, we construct a simple and general kin selection model that allows us to calculate the evolutionarily stable investments in the three castes, while varying the identity of the party controlling resource allocation (relative investment in workers, female sexuals, and male sexuals). Our model shows that queens and workers favor the investment in workers that maximizes lifetime colony productivity of sexual males and females, whatever the colony kin structure. However, worker production is predicted to be at this optimum only if one of the two parties has complete control over resource allocation, a situation that is evolutionarily unstable because it strongly selects the other party to manipulate sex allocation in its favor. Queens are selected to force workers to raise all the males by limiting the number of eggs they lay, whereas workers should respond to egg limitation by raising a greater proportion of the female eggs into sexual females rather than workers as a means to attain a more female-biased sex allocation. This tug-of-war between queens and workers leads to a stable equilibrium where sex allocation is between the queen and worker optima and the investment in workers is below both parties' optimum. Our model further shows that, under most conditions, female larvae are in strong conflict with queens and workers over their developmental fate because they value their own reproduction more than that of siblings. With the help of our model, we also investigate how variation in queen number and number of matings per queen affect the level of conflict between queens, workers, and larvae and ultimately the allocation of resource in the three castes. Finally, we make predictions that allow us to test which party is in control of sex allocation and caste determination.     

Male production in stingless bees: variable outcomes of queen-worker conflict

The genetic structure of social insect colonies is predicted to affect the balance between cooperation and conflict. Stingless bees are of special interest in this respect because they are singly mated relatives of the multiply mated honeybees. Multiple mating is predicted to lead to workers policing each others' male production with the result that virtually all males are produced by the queen, and this prediction is borne out in honey bees. Single mating by the queen, as in stingless bees, causes workers to be more related to each others' sons than to the queen's sons, so they should not police each other. We used microsatellite markers to confirm single mating in eight species of stingless bees and then tested the prediction that workers would produce males. Using a likelihood method, we found some worker male production in six of the eight species, although queens produced some males in all of them. Thus the predicted contrast with honeybees is observed, but not perfectly, perhaps because workers either lack complete control or because of costs of conflict. The data are consistent with the view that there is ongoing conflict over male production. Our method of estimating worker male production appears to be more accurate than exclusion, which sometimes underestimates the proportion of males that are worker produced.     

Sex investment ratios in eusocial Hymenoptera support inclusive fitness theory

Inclusive fitness theory predicts that sex investment ratios in eusocial Hymenoptera are a function of the relatedness asymmetry (relative relatedness to females and males) of the individuals controlling sex allocation. In monogynous ants (with one queen per colony), assuming worker control, the theory therefore predicts female‐biased sex investment ratios, as found in natural populations. Recently, E.O. Wilson and M.A. Nowak criticized this explanation and presented an alternative hypothesis. The Wilson–Nowak sex ratio hypothesis proposes that, in monogynous ants, there is selection for a 1 : 1 numerical sex ratio to avoid males remaining unmated, which, given queens exceed males in size, results in a female‐biased sex investment ratio. The hypothesis also asserts that, contrary to inclusive fitness theory, queens not workers control sex allocation and queen–worker conflict over sex allocation is absent. Here, I argue that the Wilson–Nowak sex ratio hypothesis is flawed because it contradicts Fisher's sex ratio theory, which shows that selection on sex ratio does not maximize the number of mated offspring and that the sex ratio proposed by the hypothesis is not an equilibrium for the queen. In addition, the hypothesis is not supported by empirical evidence, as it fails to explain ‘split’ (bimodal) sex ratios or data showing queen and worker control and ongoing queen–worker conflict. By contrast, these phenomena match predictions of inclusive fitness theory. Hence, the Wilson–Nowak sex ratio hypothesis fails both as an alternative hypothesis for sex investment ratios in eusocial Hymenoptera and as a critique of inclusive fitness theory.     

Anarchy in the UK: Detailed genetic analysis of worker reproduction in a naturally occurring British anarchistic honeybee,Apis mellifera,colony using DNA microsatellites

Anarchistic behaviour is a very rare phenotype of honeybee colonies. In an anarchistic colony, many workers' sons are reared in the presence of the queen. Anarchy has previously been described in only two Australian colonies. Here we report on a first detailed genetic analysis of a British anarchistic colony. Male pupae were present in great abundance above the queen excluder, which was clearly indicative of extensive worker reproduction and is the hallmark of anarchy. Seventeen microsatellite loci were used to analyse these male pupae, allowing us to address whether all the males were indeed workers' sons, and how many worker patrilines and individual workers produced them. In the sample, 95 of 96 of the males were definitely workers' sons. Given that approximately 1% of workers' sons were genetically indistinguishable from queen's sons, this suggests that workers do not move any queen-laid eggs between the part of the colony where the queen is present to the area above the queen excluder which the queen cannot enter. The colony had 16 patrilines, with an effective number of patrilines of 9.85. The 75 males that could be assigned with certainty to a patriline came from 7 patrilines, with an effective number of 4.21. They were the offspring of at least 19 workers. This is in contrast to the two previously studied Australian naturally occurring anarchist colonies, in which most of the workers' sons were offspring of one patriline. The high number of patrilines producing males leads to a low mean relatedness between laying workers and males of the colony. We discuss the importance of studying such colonies in the understanding of worker policing and its evolution.     

How queen and workers share in male production in the stingless bee Melipona subnitida Ducke (Apidae, Meliponini)

Summary. Potential conflict between the queen and workers over the production of males is expected in stingless bees as a result of the higher relatedness of workers with their sons than with their brothers. This conflict was studied in Melipona subnitida by observing how the queen and the workers share in male production. The oviposition of individual cells was observed in two colonies with individually marked workers for a period of 51 and 40 days respectively. The gender that developed from these cells was then determined. The results revealed that most male production was concentrated in a 2–3-week period, during which laying workers were present. During these weeks, the queens produced twice as many males as all laying workers together. Outside this distinct period, the queens produced an occasional male. A reproductive worker either oviposited before the queen did, in which case she immediately proceeded to close the cell and thus prevented the queen from oviposition, or oviposited and sealed the cell after the queen had laid an egg. When cell construction and oviposition occured on several combs simultaneously, the workers preferentially laid male eggs on the newest combs. We discuss the proximate mechanism and ultimate cause of the way in which queen-worker male production occurred. In conclusion, we argue that overt behavioural conflict, occasionally displayed by reproductive workers of this species, can be of great cost to the colony.Received 27 February 2004; revised 6 September 2004; accepted 1 October 2004.