The social consequences of honey bee polyandry: the effects of kinship on worker interactions within colonies

Anim. Behav., 1987, 35, 255-262

The social consequences of honey bee polyandry: the effects of kinship on worker interactions within colonies P E T E R C. F R U M H O F F * & S T A N L E Y S C H N E I D E R t $

* Ecology Graduate Group, Department of Entomology, University of California, Davis', California 95616, U.S.A. t Animal Behaviour Graduate Group, University of California, Davis, California 95616, U.S.A. Abstract. Honey bees, Apis mellfera L., are polyandrous and several males simultaneously father offspring within a single colony. The relatedness of female colony members therefore varies with their paternity; workers encounter both patrilineal full sister (? = 0.75) and non-patrilineal half-sister (? = 0.25) nestmates. The impact of this intra-colony genetic variation on social grooming and trophallaxis (liquid food exchange) among workers in colonies consisting of two phenotypically-distinctworker patrilines was examined. Workers in these colonies groomed and fed a disproportionately large number of full sisters despite a tendency to encounter a disproportionately large number of half-sisters. Thus, workers actively discriminated between full and half-sisters. This patrilineal discrimination occurred both in colonies with laying queens and in a queenless colony rearing replacement queens. These results suggest that intracolony genetic variation may have a major effect on colony social organization.

The role of kinship in the evolution of insect sociality has been debated since Hamilton (1964) first considered the asymmetries in relatedness among family members that result from haplodiploid sex determination in the Hymenoptera (reviewed in Andersson 1984). Hamilton proposed that the high average relatedness among daughters of a singly-mated queen may have facilitated the evolution of non-reproductive female worker castes. In many highly eusocial species, however, a low average and high variance in relatedness among female nestmates may result from either polygyny (Craig & Crozier 1979; Pearson 1983; Ward 1983; Crozier et al. 1984) or polyandry (Page & Metcalf 1982). Polygyny and polyandry do not necessarily contradict Hamilton's hypothesis as they may have arisen subsequent to the evolution of worker sterility. The intra-colony variance in relatedness they produce, however, allows an assessment of the role of kinship in shaping extant societies. For polygyny and polyandry to have an impact on colony social organization, workers must be able to discriminate between nestmates of different relatedness. Workers of the highly eusocial polyan:~Present address: Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223, U.S.A.

drous honey bee, Apis mellifera L., may use genetically-based cues to distinguish differences in relatedness between queens (Breed 1981; Boch & Morse 1982; Page & Erickson 1986a) and workers (Breed 1983; Getz & Smith 1983; Breed et al. 1985), raising the possibility that such discrimination may operate within colonies. A honey bee queen mates on average with 17 males (Adams et al. 1977) whose sperm mix so that several may simultaneously father offspring within a single colony (Page & Metcalf 1982; Laidlaw & Page 1984). Workers therefore encounter both patrilineal full sister (P=0.75) and, more frequently, non-patrilineal half-sister (?=0.25) nestmates. A number of studies have focused on whether workers attempt to skew the reproductive output of a colony towards full sisters via preferential full sister queen rearing (Breed et al. 1984; Page & Erickson 1984, 1986b; Noonan 1985) and swarming (Getz et al. 1982). They have yielded equivocal results and the significance of the ability of honey bee workers to discriminate between queens of different relatedness remains uncertain. Although rearing queens and producing swarms are contexts in which workers have a direct influence over their inclusive fitness, episodes of queen rearing and swarming are short and infrequent relative to a colony's life-span. Workers

255

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Animal Behaviour, 35, 1

spend the vast majority of their lives in. direct contact with one another, and it is their repeated interactions that underlie our perception of social insect colonies as integrated units (Oster & Wilson 1978; Wilson 1985a). There is some evidence, however, that honey bees discriminate between full and half-sister workers. Getz & Smith (1983) found that queenless workers in laboratory arenas were more likely to bite half-sisters than full sisters, but they leave open the question of whether and how workers may discriminate within colonies. In this study, we consider the influence of intracolony variation in relatedness on two common behavioural interactions of honey bee workers within colonies. We monitored social grooming and trophallaxis (liquid food exchange) among workers in colonies consisting of two phenotypically-distinct patrilines. Grooming consists of pairwise interactions in which one worker (the recipient) stands with her wings apart while another worker (the donor) slowly chews on her thoracic hairs. It may be initiated either by a prospective recipient when she gives a rapid lateral shaking of her abdomen (termed the grooming invitation dance by Milum 1956) or by a prospective donor who may groom several workers in rapid succession. The function of social grooming is largely unknown and has been postulated to entail both cleaning (Milum 1947, 1956) and pheromone transfer (Wilson 1971). During trophallaxis, one worker (the recipient) has her proboscis extended on the base of the mouthparts of another worker (the donor), from whom she receives food. It may also be initiated by either a prospective recipient or a donor. While feeding, the recipient rapidly antennates the donor, and it is likely that both nutrients and pheromones are transferred (Michener 1974). Grooming and trophallaxis may be of considerable importance in regulating the flow of food and pheromones through a colony. Workers tend to groom and feed same-age or younger nestmates (Nixon & Ribbands 1952; Milum 1956; Free 1957; Frumhoff, unpublished data). Moreoever, queenless workers in laboratory groups composed of same-age individuals differ in their tendency to give and receive food, with those tending to receive food having significantly greater ovarian development (Korst & Velthuis 1982). Non-random distribution of grooming and trophallaxis due to variation in relatedness among workers may therefore have a major impact upon colony social organization.

M A T E R I A L S AND M E T H O D S

Colonies

We established five colonies at the Bee Biology Facility at the University of California at Davis from five artificially-inseminated queens. Seven days after emergence in a nursery colony, each queen was inseminated with approximately 1/~1 of semen from each of two drones. Following insemination, each queen was tagged with Liquid Paper on her thorax, wing-clipped to prevent mating flights, caged and introduced into a dequeened fiveframe nucleus colony. Queens were anaesthetized with carbon dioxide during insemination and 24 h after insemination to induce oviposition. They were released uncaged into their colonies within 7 days and began laying within l0 days. The queens and drones used in each mating were chosen so that the two worker patrilines would inherit different cuticle colour patterns. Cuticle colour in female honey bees is governed by a onelocus two-allele major gene and several modifiers. Yellow is dominant to black (Woyke 1978) and cordovan is a recessive modifier which in homozygous form produces a brown phenotype (Rothenbuhler et al. 1968). All three phenotypes occur in natural populations. In colonies 1 and 2, black queens were each inseminated with the semen of one black drone and one yellow drone, producing black and yellow daughters, respectively. In colonies 3-5, cordovan queens were each inseminated with the semen of one black drone and one cordovan drone, producing black and cordovan daughters, respectively. Below, offspring of black drones will be referred to as the dark patriline and offspring of cordovan or yellow drones will be termed the light patriline. The queens in colonies 1 and 2 were full sisters as were the queens in colonies 4 and 5. Black drones used in matings in colonies 1 and 2 were brothers as were the yellow drones used in these matings. All other queens and drones came from colonies in distant apiaries and were presumably of low relatedness to one another. Seven weeks after laying began, all workers in each nucleus colony were daughters of the inseminated queen. The ratio of workers of the two patrilines varied between colonies and over time within colonies (Frumhoff, unpublished data), perhaps due to incomplete sperm mixing (Laidlaw & Page 1984). By visual inspection, each patriline

Frumhoff & Schneider. Honey bee kin discrimination consistently comprised at least 20% of the population of each colony. Each queen and most of her workers (5000-6000 bees) were transferred to a 1.5-frame observation hive (42• • cm). Each hive contained a frame of capped and uncapped brood and a halffi-ame of stored pollen and honey. Workers foraged via a single small entrance and were provided with a 50% sucrose solution ad libitum.

Behavioural Interactions We monitored grooming and trophallaxis among workers in each observation hive. We located worker pairs by a systematic scan of a grid of 4 • 4 cm overlaid on the glass walls of each observation hive. The location of each pair and the patriline and behavioural role (donor or recipient) of each worker were recorded. We excluded interactions lasting less than 5 s since they rarely resulted in effective grooming or food exchange. All colonies were observed under fluorescent lighting during warm clear weather. Colonies 1 and 2 were observed in September-October 1983 and colonies 3-5 were observed in August-September 1984. Scans of each colony were repeated between 0800 and 1600 hours, a period of active foraging, for 1-6 h per day over several consecutive days. The duration of each scan was approximately 10 rain. This is longer than the maximum duration reported for grooming bouts (6-25 rain: Milum 1956) or feeding bouts (2.50 min: Korst & Velthuis 1982), thus minimizing the likelihood of repeated observations of any given pair of interacting workers. Colonies l ~ were queenright (i.e. they contained laying queens). Colony 5 was observed during a temporarily queenless period in which several replacement queen larvae had been reared and were enclosed within sealed queen cells. For each colony, we totalled the number of grooming and feeding interactions observed among each of four possible pairs: dark donor and dark recipient, dark donor and light recipient, light donor and dark recipient and light donor and light recipient. Potentially, workers may interact at random with respect to patriline or may bias interactions towards full or half-sisters. The most direct means of assessing bias would be to ask whether donors of each patriline interact with light and dark recipients in proportion to their actual abundance in the colony. This was not feasible because the tendency to act as donor or recipient

257

and the proportion of workers of each patriline varies among age-classes (Frumhoff, unpublished data) and the ages of workers in these colonies were unknown. We assessed bias by determining whether dark donors and light donors in each colony interacted with significantly different proportions of dark and light recipients. Here, a significant difference could result from discrimination by workers of either one or both patrilines. However, it could also result if workers do not encounter full and half-sisters at random within the colony (e.g. via patrilineal differences in geotaxis).

Spatial Proximity To test the possibility of patrilineal differences in spatial distribution, we measured the proximity of workers to full and half-sisters in colonies 1~4. Workers were highly aggregated and consistently moving throughout each colony. We therefore assessed proximity by counting the number of light and dark workers within one bee-length radius to randomly chosen workers of each patriline. The vertical surface of each observation hive was divided into four quadrants of equal area and counts were made from photographs of one 15-5• 18.1-cm section of each quadrant. Quadrants 1 and 2 were in the upper half of the colony in a region of honey and pollen storage; quadrants 3 and 4 were in the lower half of the colony in a region of sealed and unsealed brood. Each quadrant was photographed with a Nikon F3 camera, Sunpak Flash Unit and Kodachrome 64 colour slide fihn following periods of behavioural observation. Quadrants were rephotographed at minimum intervals of half an hour to allow substantial movement of workers between succeeding photographs. To obtain an unbiased measure of proximity from each quadrant while excluding repeated counts of individual workers, the following method was employed. Each slide was projected onto a screen of 77"5 x 57 cm with a 7 • 7-cm grid. In the left half of the projection, a dark patriline worker closest to a randomly selected coordinate was designated a focal worker. The number of proximate workers on each patriline whose heads were within one worker-length radius of the focal worker were recorded. A grid coordinate outside of the radius of the focal worker was then randomly selected; a light patriline worker closest to it was

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Animal Behaviour, 35, 1

chosen as focal worker and the n u m b e r of workers of each patriline surrounding her were chosen as above. In the right half of each slide, the same counts were made with a focal light worker chosen first. The number of light and dark workers surrounding focal workers on each patriline were summed over the two halves of each slide and over all the slides of each quadrant.

A)

GROOMING

0,8.

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DARK DONORS

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LIGHT DONORS

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Analysis All grooming and feeding bouts and counts of spatial proximity within each colony were treated as independent events. The summed behavioural and spatial proximity data per colony were analysed by two-way G4ests of independence (Sokal & Rohlf 1981). The degree to which grooming, trophallaxis and spatial proximity were biased towards full or halfsisters was compared across colonies by a test of equality of the logs of the odds-ratios (Armitage 1971). The odds-ratio is a ratio of cross-products of a 2 x 2 contingency table and is scaled from 0 to co (Feinberg 1981). The odds-ratio is thus a measure of association in a 2 x 2 contingency table. Its magnitude is independent of the ratio of light to dark patriline workers within a colony, therefore allowing comparison between colonies containing different proportions of light and dark workers. Behavioural data are summarized below as the proportion of recipients that belonged to the dark patriline given light or dark donors. Spatial proximity data are summarized as the proportion of proximate workers that belonged to the dark patriline given light or dark focal workers.

RESULTS For both grooming and trophallaxis, a greater proportion of recipients in each colony were dark when donors were also dark than when donors were light (Fig. 1). That is, worker interactions were biased towards full sisters in each colony. This full sister bias was significant in three of the four queenright colonies and the queenless colony (colony 5) for grooming and in two of the four queenright colonies for trophallaxis. The bias towards full sister interactions was partial rather than absolute. The degree of bias did not vary significantly between colonies for either grooming or trophallaxis (Table I; test of equality

281

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3

4

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COLONY

Figure 1. Proportion of recipients in each colony that were members of the dark patriline given dark versus light donors. Significant full sister biases are found in four of five colonies for (A) grooming and two of five colonies for (B) trophallaxis (2 x 2 G-test of independence: *P < 0.05, **P < 0.01, ***P < 0.001). Colony descriptions are given in Table I. Numbers at base of histograms are the total number of light and dark recipients.

of the In (odds-ratios)- P > 0'10). There were no marked differences in the degree of bias between the queenless colony and queenright colonies or between colonies containing yellow or cordovan light patriline workers. Moreover, the degree of bias was not consistently greater across colonies for either grooming or trophallaxis. Full sister biased interactions were not a consequence of a clumped association of full sisters within colonies. Rather, workers in colonies I M tended to encounter a disproportionately large number of half-sisters; randomly chosen light workers encountered a slightly greater proportion of dark nestmates in each colony than did randomly chosen dark workers (Fig. 2). This halfsister bias was significant in two of the four colonies and the degree of bias did not vary significantly between colonies (Table I; test of equality of the

Frumhoff & Schneider." Honey bee kin discrimination Table I. Odds-ratio measures of the degree of bias in patrilineal interactions

Hive

Grooming

1 2 3 4 5 2

2.38 1.51 2-91 1-72 3-46 2-29

(1.46, (0.85, (1.81, (l.05, (1.97, (1-69,

Trophallaxis

3.88) 2.68) 4.68) 2-82) 6.08) 3-10)

1.48 2.70 1.89 1.78 1.73 1.88

Spatial proximity

(0.88, 2.50) (I.55,4"71) (1-01, 3.52) (1.26, 2.52) (0.75, 3.96) (1.54, 2.28)

0.77 0-79 0-65 0.93

(0.60, (0.59, (0.45, (0.70,

Qr

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DARKFOCALWORKERS LIGHTFOCALWORKERS

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0.79 (0.69, 0.89)

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COLONY1

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0-98) 1-07) 0.94) 1.23)

Odds-ratios greater than 1.00 reflect full sister biases; odds-ratios less than 1.00 reflect half-sister biases. Numbers in parentheses are 95% confidence intervals. Colonies 1 4 are queenright; colony 5 is queenless. Colonies 1 and 2 consist of yellow and black patrilines; colonies 3-5 consist of cordovan and black patrilines.

,

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COLONY4

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Figure 3. Proportion of proximate workers per quadrant in colonies 1~4 that were members of the dark patriline given dark versus light focal workers. Numbers at base of histograms are the total number of light and dark proximate workers.

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]45J369 2

DISCUSSION 3

4

COLONY Figure 2. Proportion of proximate workers in colonies 1~4 that were members of the dark patriline given dark versus light focal workers. Significant half-sister biases were found in two of four colonies (2 • 2 G-test of independence: *P < 0.05). Numbers at base of histograms are the total number of light and dark proximate workers.

In (odds-ratios): P>0-10). However, this consistent bias among colonies obscures some variation between quadrants within colonies in the proportion of full and half-sisters encountered (Fig. 3). Half-sister biased encounters were found in all four quadrants in colony 3, three of four quadrants in colonies 1 and 2, and two of four quadrants in colony 4.

The characteristic organization of highly eusocial insect colonies emerges from a network of repeated interactions between individual workers. U p o n cursory examination, workers appear to interact at random within the colony. However, workers generally have behavioural specializations associated with their age and, in some species, physical subcaste, which underlie non-random interactions between nestmates (Free 1957; Wilson 1985b). Here we find that interactions between honey bee workers are also non-random with respect to patriline. Workers consistently g r o o m and feed disproportionately large numbers of full sisters despite a tendency to encounter a disproportionately large number of half-sisters. These biases, therefore, apparently result from active discrimination by workers of one or both patrilines. Recent studies using colonies with similarly marked patri-

260

Animal Behaviour, 35, 1

lines indicate that workers may also preferentially rear full sister queen and worker larvae (Noonan 1985). Because all nestmate workers are reared in a common environment, discrimination between full and half-sisters must be mediated by the recognition of genetically-based cues. Since workers encounter both full and half-sisters upon emergence, they cannot use their nestmates as a source for learning differences in relatedness. Rather, our results and those of Noonan (1985) indicate that workers must either learn their own phenotypes and use them as templates in comparison to nestmates or use recognition alleles (see Blaustein 1983; Holmes & Sherman 1983). This is supported by Getz & Smith's (1986) demonstration that workers raised in isolation can distinguish between full and half-sisters. We consider four hypotheses for why workers discrimate between full and half-sisters. (1) Patrilineal discrimination may be an artefact of the atypical composition of our experimental colonies. The colonies in this study were fully functional, but atypical in two fundamental ways. First, they included only two patrilines, whereas several patrilines coexist in colonies with naturally mated queens. Therefore, workers typically encounter a much lower proportion of full sisters, and we cannot directly assess the extent of discrimination in multiple patriline colonies. There is no a priori reason to suspect, however, that discrimination is less likely to occur in colonies with a greater number of patrilines. Second, patrilines in this study were marked by discrete colour phenotypes while colour patterns do not consistently differ among patrilines in colonies with naturally mated queens. Getz et al. (1982) have suggested that workers marked by the cordovan phenotype may behave differently from non-cordovan workers in ways that could conceivably influence worker interactions. However, we found no consistent difference in the degree of bias in colonies where the light patriline is distinguished by the cordovan or yellow phenotype. Moreover, work in progress indicates that the degree to which workers preferentially interact with full sisters is not constant, but varies with the age-classes of interacting workers and the degree of environmental stress (Frumhoff, unpublished data). Discrimination is thus unlikely to be strictly an artefact of the cuticle colour patterns used to allow us to distinguish between patrilines.

(2) Patrilineal discrimination may result from selection favouring effective nestmate recognition and may have no functional basis within the colony. Honey bee workers can distinguish between nestmates and non-nestmates, probably using a combination of genetic and environmental cues (Wilson 1971; Breed 1983). Nestmate recognition may enable workers to prevent non-nestmate foragers from robbing a colony's food stores in periods of low food availability. When a colony fissions, the swarm leaving the mother colony typically establishes a new colony 300-1800 m away (Seeley & Morse 1978), within the foraging range of the mother colony (Gary 1975). Thus, workers in neighbouring colonies may be related, and effective nestmate recognition may require that workers discriminate between nestmates and nonnestmates of varying degrees of relatedness. Patrilineal discrimination could thus result as a byproduct of selection for nestmate recognition. One prediction of this hypothesis is that patrilineal discrimination should be greatest during periods of low food availability. Preliminary experiments suggest that the opposite is true; workers in food-stressed colonies discriminate less than in unstressed colonies (Frumhoff, unpublished data). However, the relationship between discrimination among workers within and between colonies needs to be more fully explored. (3) Patrilineal discrimination may result from colony-level selection. The demographic characteristics of a eusocial insect colony are usually viewed as resulting from natural selection acting upon the colony as a whole (Oster & Wilson 1978; Wilson 1985a). From this perspective, worker behaviour has evolved to increase the energetic efficiency and reproductive success of the colony. Although a colony-level perspective has yielded much insight into the behaviour of honey bee workers (e.g. Seeley 1985), it is not clear how colony-level selection would lead to non-random interactions between worker patrilines. One possibility is that the intra-colony genetic variation resulting from multiple mating allows for a genetic division of labour among workers, yielding nonrandom interactions among patrilines. Workers of a given age-class differ in their tendency to groom nestmates (Winston & Punnett 1982) and in their foraging behaviour (Seeley 1985 and references therein). It is not known whether these differences have a genetic basis or whether they increase colony efficiency. Nonetheless, the possibility that patrili-

Frumhoff & Schneider: Honey bee kin discrimination

neal discrimination results from colony-level selection cannot be ruled out. (4) Patrilineal discrimination may result from competition between workers to preferentially rear more closely related reproductives. Kin selection theory predicts that workers may gain inclusive fitness benefits by preferentially rearing (a) full sister queens and (b) drone offspring of full sister workers in queenless colonies. Colonies rear new queens before swarming and in response to the death or senescence of the mother queen. Workers can distinguish between full and half-sister queen larvae and may preferentially rear full sister queens (Noonan 1985). However, it is not known whether there is any connection between discrimination among queen larvae and discrimination among adult workers. Workers do not always successfully replace the mother queen, leaving a colony permanently queenless. Queenless colonies are ephemeral since new workers cannot be produced; workers become aggressive towards one another and a small proportion develop functional ovaries and lay unfertilized (male) eggs (Velthuis 1985). Preferential feeding of full sisters in these colonies could promote their ovarian development (Korst & Velthuis 1982) and thus entail an investment in the production of more closely related drones. The consequences of variation in relatedness in queenless colonies with laying workers clearly merits further study. A potential link between patrilineal discrimination and patrilineal competition in rearing reproductives does not explain why we find full sister preferences a m o n g workers in queenright colonies. Continuous discrimination may be favoured by the unpredictable occurrence of queen loss. Determining which, if any, of these hypotheses underlies patrilineal discrimination will require a more detailed understanding of the dynamics of worker interactions within colonies. While colonies of highly eusocial insects may function primarily as integrated units, intra-colony genetic variation may underlie a greater complexity in their social organization than has previously been supposed.

ACKNOWLEDGMENTS We thank Cathy Angell, Jayne Baker and M a r k Pretti for assistance in data collection, H u g h Dingle, Jerry Marston, Judy Stamps and Phil Ward for constructive suggestions on several phases of

261

this project. Michael Miller for statistical advice and an anonymous reviewer for helpful comments on the manuscript. The research was supported by a University of California at Davis Jastro-Shields Fellowship, University of California at Davis Graduate Research Award, and a grant from Sigma Xi to P.C.F.

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(Received 4 November 1985; revised 24 January 1986; MS. number: A4651)