1 Genetics, Relatedness and

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1 01Insect Evolutionary 25/1/05 15:45 Page 1 1 Genetics, Relatedness and Social Behaviour in Insect Societies ANDREW F.G. BOURKE Institute of Zoology, Zoological Society of London, London, UK 1. Introduction Kin selection theory occupies a dominant position in the study of social evolution. Although its meaning and potential uses were slow to be appreciated following its publication (as inclusive fitness theory) by W.D. Hamilton in the early 1960s (Hamilton, 1963, 1964), since the early to mid-1970s, following the publication of work by Wilson (1971), Hamilton (1972), Alexander (1974), West-Eberhard (1975) and especially Trivers (1974) and Trivers and Hare (1976), it has formed the basic theoretical tool with which investigators have attempted to unpick the complexities of social behaviour. The essence of kin selection is that the evolution of social interactions between conspecific individuals, whether cooperative or competitive, should be influenced by their genetic relatedness. This is because, from the viewpoint of genes for social behaviour, relatedness measures the value of other individuals as a route to future generations. This insight is formalized as Hamilton s rule, which specifies the conditions for the spread of a gene for social behaviour as a function of the relatedness of the interactants and the numbers of offspring gained or lost by them (benefits and costs) as a consequence of the behaviour (Hamilton, 1963, 1964). It is worth re-emphasizing the very fundamental nature of kin selection theory. Two conditions are needed. First, there must be genetic variation for social behaviour. Second, interacting conspecifics must express social behaviour non-randomly with respect to relatedness. If these two conditions are met, then kin selection is the relevant theory to apply to achieve an understanding of how natural selection will affect the genes in question and hence how the evolution of the relevant form of social behaviour will proceed. In short, kin selection is the genetic theory of natural selection (Fisher, 1930) logically extended to cover social phenomena (e.g. Grafen, 1985; Frank, 1998). This means that demonstrating the efficacy of kin selection in one context helps strengthen the case for the validity of the kin selection approach in all potential contexts, Royal Entomological Society Insect Evolutionary Ecology (eds M. Fellowes, G. Holloway and J. Rolff) 1

2 01Insect Evolutionary 25/1/05 15:45 Page 2 2 A.F.G. Bourke because the basis of the underlying theory is the same. This is the point missed in a recent critique of the application of kin selection theory to social insects (Alonso and Schuck-Paim, 2002). The fundamental nature of kin selection theory also implies that it is potentially universally applicable. This is why the theory has, rightly, been applied far beyond its traditional province to help explain such phenomena as the evolution of multicellularity (Maynard Smith and Szathmáry, 1995; Michod and Roze, 2001) and the evolution of social behaviour in microorganisms (Crespi, 2001). In sum, kin selection is the basis of any general theory of the evolution of conflict and cooperation in all organisms at all levels in the biological hierarchy (Maynard Smith and Szathmáry, 1995; Keller, 1999; Queller, 2000; Michod and Roze, 2001). Studies of social insects, and in particular of the social Hymenoptera, have been at the forefront of the application and testing of kin selection theory. Overall, such studies have been notably successful in verifying the predictions of the theory and so helping us understand the basis of social evolution in insects (Bourke and Franks, 1995; Crozier and Pamilo, 1996; Choe and Crespi, 1997) and, by extension, in other organisms. They have also been successful in the sense that they have led to the discovery of new and important social phenomena (e.g. worker policing of other workers eggs in honey bees: Ratnieks and Visscher, 1989; selective male-killing by worker ants: Sundström et al., 1996). However, given that 40 years have passed since kin selection theory was first proposed, and that in this period scores of studies on social insects of its predictions and explanatory power have been published, a student new to the field, and even researchers who are growing old in it, might legitimately ask themselves, What remains to be done?. In fact, the field remains dynamic and active, and this is for several reasons. One is that rapid progress in molecular biology holds out the prospect of achieving an integrated understanding of social evolution at the molecular-genetic and behavioural levels. Another is that new contexts in which kin selection theory can be applied in social insects, and hence new a priori predictions derived from the theory continue to be identified. Finally, there are sufficient cases where the theory s predictions are not fulfilled to create a need to conduct multiple tests across several taxa and for the theory s predictions then to be assessed using a rigorous, comparative approach. For these reasons, this seems an appropriate moment both to look backwards and consider the status of the field to date, and to look forwards and speculate as to how it might develop. The present essay is an attempt at these tasks. I first review the evidence for kin selection in the social Hymenoptera (strictly, the eusocial Hymenoptera, i.e. those having a reproductive division of labour between queens and workers) and I then consider the directions that the field might take in future. 2. Evidence for kin selection in social insects The chief predictions of kin selection theory concern effects of relatedness on social behaviour. Of course the theory also makes predictions about individualand group-level benefits and costs, but because these quantities have generally

3 01Insect Evolutionary 25/1/05 15:45 Page 3 Genetics, Relatedness and Social Behaviour 3 been hard to measure in practice, most tests of the theory have focused on expected effects of relatedness. Many other factors are likely to influence social evolution, especially at the proximate level. However, unlike relatedness, none form the basis of so general a theory. The following review is not comprehensive and does not employ rigorous comparative methods controlling for effects of phylogeny in among-species comparisons. Instead, I concentrate on recent studies of sex allocation, male parentage and kin-selected caste conflict in the social Hymenoptera that have added to or improved our knowledge regarding the influence of relatedness on social behaviour. In the process, I highlight current gaps in our knowledge and anomalies and inconsistencies in the data. I do not consider the effects of relatedness on the origin of eusociality. The reason is that, although this issue has long been discussed, there are still relatively few data on facultatively social Hymenoptera that might prove informative in this context (Bourke, 1997b). Neither do I further discuss the evolution of reproductive skew (partitioning of reproduction among multiple-breeder groups), which is an important and currently extremely active area in the field (Keller and Reeve, 1994; Johnstone, 2000). Again, this is because empirical tests in social insects of the predictions of skew models with respect to relatedness are still relatively few (e.g. Field et al., 1998; Reeve et al., 2000; Rüppell et al., 2002; Seppä et al., 2002; Sumner et al., 2002; Hannonen and Sundström, 2003a). In addition, tests of skew models in social insects have been comprehensively reviewed by Reeve and Keller (2001). Recent reviews of the topics and themes considered in the present chapter include those of Chapuisat and Keller (1999), Keller and Chapuisat (1999), Keller and Reeve (1999), Ratnieks et al. (2001), Sundström and Boomsma (2001) and Mehdiabadi et al. (2003). 2.1 Sex allocation Kin selection theory predicts patterns of sex allocation (relative investment in the sexes) at both the population and colony levels in the social Hymenoptera. At the population level, assuming random mating, the sex investment ratio (expressed as the female : male investment ratio) is predicted to equal the relatedness asymmetry of the party controlling sex allocation (with relatedness asymmetry defined as the relatedness to females divided by relatedness to males) (Trivers and Hare, 1976; Boomsma and Grafen, 1990, 1991). In this context, females refers to new queens produced by the colony and males refers to new males produced by the colony; workers, all being female, are excluded because they are (generally) sterile. This statement of kin selection theory as regards sex allocation is essentially Fisher s (1930) sex ratio theory reformulated to incorporate the concept of a party controlling sex allocation that need not be the parent. An implication is that there may be kin-selected conflict over sex allocation, because the relatedness asymmetries of different potential controlling parties (e.g. queens and workers) need not be equal (Trivers and Hare, 1976). For example, in a random-mating population of social Hymenoptera with one singly-mated queen per colony and control of sex allocation by sterile workers, the population sex

4 01Insect Evolutionary 25/1/05 15:45 Page 4 4 A.F.G. Bourke investment ratio is predicted to equal the well-known value of 3:1 females : males, since the workers relatedness asymmetry is 0.75/0.25. But if the queen controls sex allocation, the population sex investment ratio should equal 1:1, which is the queen s relatedness asymmetry (0.5/0.5). The workers relatedness values stem from the haplodiploid genetic system found in the Hymenoptera, in which females derive from fertilized eggs and are diploid and males derive from unfertilized eggs and are haploid, with the result that full sisters are related by 0.75 due to their sharing the same paternal genes, and sisters are related to brothers by 0.25 because males lack paternal genes (Hamilton, 1964). At the colony level, kin selection theory predicts sex ratio variation if workers control sex allocation and if their relatedness asymmetry varies across colonies within populations; for example if some colonies are headed by singlymated queens and others by multiply-mated queens (Boomsma and Grafen, 1990, 1991). Specifically, the theory then predicts that colonies with relatively high relatedness asymmetries should concentrate on female production, because their workers are comparatively more closely related to females, and colonies with relatively low relatedness asymmetries should concentrate on male production, because their workers are comparatively more closely related to males (i.e. Boomsma and Grafen s (1990, 1991) split sex ratio theory). If queens control sex allocation, the theory makes no specific predictions because the factors that cause workers relatedness asymmetry to vary across colonies generally have no effect on queens relatedness asymmetry (Boomsma and Grafen, 1990, 1991) Population-level sex ratio variation Analyses of sex ratio evolution in relation to kin selection have tended to concentrate on social Hymenoptera that reproduce entirely or at least partly by the release of winged sexuals (e.g. many monogynous species, i.e. those having one queen per colony), in contrast to species that reproduce entirely or mainly by colony division (e.g. honey bees, Apis spp.). This is because in the latter set of species competition between related queens to head daughter colonies (local resource competition) is predicted to increase male bias in the population sex investment ratio, but to a degree that is hard to quantify (Pamilo, 1991; Bourke and Franks, 1995). It is worth noting, however, that the general Fisherian and Trivers Hare approach has been notably successful in predicting patterns of population-level sex ratio variation in social Hymenoptera across a wide variety of social and mating systems (Bourke and Franks, 1995; Crozier and Pamilo, 1996). In monogynous ants in which relatedness asymmetry has been measured or strongly inferred using genetic markers, observed sex ratios are significantly female-biased (mean fraction of investment in females = 0.63; Table 1.1). However, although the quantitative fit to the predicted values of the sex investment ratio is frequently remarkably close, on average observed sex investment ratios are significantly lower than sex investment ratios predicted on the basis of population-wide relatedness asymmetries (0.63 versus 0.72; paired t-test, t = 3.91, d.f. = 15, P < 0.01; Table 1.1). The reasons for this pattern,

5 01Insect Evolutionary 25/1/05 15:45 Page 5 Genetics, Relatedness and Social Behaviour 5 which was also detected by Boomsma (1989) and Pamilo (1990), although these studies did not compare observed sex investment ratios with expected values inferred from genetic data, are not fully resolved. They could occur (a) because in populations in a poor habitat, workers cannot fully express their kin-selected interests due to a proximate influence of a lack of resources on female investment (e.g. Nonacs, 1986), or (b) because workers are not fully in control of sex allocation, such that population sex investment ratios represent a compromise between their optimal values and those of queens (e.g. Passera et al., 2001; Reuter and Keller, 2001; Mehdiabadi et al., 2003). However, no theory apart from kin selection theory explains the significant female bias in the population sex investment ratios of monogynous ants. In particular, random mating in all the species in Table 1.1 rules out local mate competition (competition between related males for mates) as a general source of female bias (Alexander and Sherman, 1977). Sex investment ratios in slave-making ants have also been used to support kin selection predictions. In these monogynous social parasites, all brood is reared by slave workers of other species. Trivers and Hare (1976) therefore proposed that the slave-maker queen should control sex allocation because any mechanisms adopted by her to achieve her optimal sex ratio would be met with indifference by the unrelated slaves and, contrary to the case in non-parasitic ants, could not be practically opposed by her workers. The resulting prediction of unbiased (1:1) population sex investment ratios is met in two well-studied species (Epimyrma ravouxi and Harpagoxenus sublaevis: Bourke and Franks, 1995). However, in three other species (H. canadensis, Protomognathus americanus and Leptothorax duloticus), Herbers and Stuart (1998) found six of 11 population sex investment ratios to be significantly different from 1:1, although the mean value across all 11 populations did not differ significantly from 1:1 (mean (95% confidence limits) = 0.51 [ ] as the fraction of investment in females). In P. americanus, Herbers and Stuart (1998) also found a positive association across sites and years between slave-maker sex ratios and those of their free-living host. This suggested that, contrary to Trivers and Hare (1976), slave workers influence slave-maker sex allocation. Herbers and Stuart (1998) concluded that there is ongoing conflict over sex allocation between the slave-maker queen, the slave-maker workers, and the slave workers. In a later study, Foitzik and Herbers (2001) found that P. americanus workers are unusually reproductive, producing an estimated two-thirds of all males. This means that the optimal sex ratios of the slave-maker queen and her workers tend to converge, leading to a reduction in the expected degree of sex-ratio conflict between them (Foitzik and Herbers, 2001). Overall, these phenomena suggest that, at least in some species of slave-making ants, the expression of kinselected conflict over sex allocation is likely to be complicated and hence that population sex investment ratios in these species do not provide the tidy test of kin selection theory that Trivers and Hare (1976) proposed. In bumblebees, male-biased or unbiased (1:1) population sex investment ratios formerly appeared to contradict kin selection theory. However, unbiased population sex investment ratios in this group are likely to be a consequence of lack of worker control of sex allocation (Bourke, 1997a; Brown et al., 2003;

6 Table 1.1. Tests of Trivers and Hare s (1976) kin selection theory for the population sex investment ratio in monogynous ants. Adapted from Bourke (1997b), with additional cases. Population sex investment ratio is expressed as proportion of investment in females. Expected sex investment ratios (assuming worker control) were calculated as measured or inferred workers relatedness asymmetry (expressed as fractions). Lack of inbreeding inferred from Hardy Weinberg equilibrium of genetic markers, or zero queen mate relatedness, or both. In a study of L. tuberum (Pearson et al., 1995, 1997), observed and expected population sex investment ratios differed significantly in four of seven site-years, but the actual values were not tabulated. Population sex investment ratio Inbreeding Number of Species Expected Observed present? colonies Reference Colobopsis nipponicus No Hasegawa (1994) Formica truncorum No 22 Sundström (1994) Lasius niger Van der Have et al. (1988) Population No 125 Population No 26 Population No 50 Lasius niger Population 1, Year No 28 Fjerdingstad et al. (2002) Population 1, Year No 34 Population No 52 Leptothorax nylanderi Year No 174 Year No data 179 Year No data 334 Foitzik et al. (1997), Foitzik and Heinze (2000) Myrmica punctiventris Banschbach and Herbers (1996) Year No 84 Year No 40 Pheidole desertorum No 348 Helms (1999) Pheidole pallidula No 22 Fournier et al. (2002, 2003) Solenopsis invicta No 50 Vargo (1996) Mean (95% confidence limits) 0.72 ( ) 0.63 ( ) 6 A.F.G. Bourke 01Insect Evolutionary 25/1/05 15:45 Page 6

7 01Insect Evolutionary 25/1/05 15:45 Page 7 Genetics, Relatedness and Social Behaviour 7 Duchateau et al., 2004), in which case the theory predicts 1:1 sex investment ratios (Trivers and Hare, 1976). Queens appear to have power over sex allocation in bumblebees because essential reproductive decisions in these annual social insects are made before many of the workers have eclosed (Müller et al., 1992). Male-biased sex investment ratios in bumblebees are apparently a consequence of a protandrous mating system (one in which adult males are produced earlier than females) (Bulmer, 1983; Bourke, 1997a; Beekman and Van Stratum, 1998), which represents a violation of the assumptions of the original kin-selection model. In wasps, population sex investment ratios have rarely been estimated, either because it is hard to divide females unambiguously into young queens (gynes) and workers, or because measuring sexual output over the season in a sufficient sample of colonies is difficult, or both. It seems likely that, in many wasps, complexities in the mating system could also complicate the interpretation of population sex investment ratios (Strassmann and Hughes, 1986; Tsuchida et al., 2003) Colony-level sex ratio variation Split sex ratio theory represents a powerful test of kin selection because it predicts within-population patterns of sex ratio variation. Such a test controls for differences other than those involving kin structure, which potentially confound comparisons between population sex ratios of different populations or species (Chapuisat and Keller, 1999). The theory predicts that, if workers control sex allocation and their relatedness asymmetry varies between colonies, sex ratios should be split and specifically should be relatively female-biased in colonies with high relatedness asymmetry and relatively male-biased in colonies with low relatedness asymmetry. Another, weak prediction of the theory is that, if workers relatedness asymmetry does not vary, sex ratios should not be split. This is a weak prediction because Boomsma and Grafen s (1990, 1991) theory does not rule out other causes of split sex ratios in social Hymenoptera, and indeed such other causes undoubtedly exist (see below). Enumerating cases in which the main prediction can be tested, the theory accounts for split sex ratios in 19 of 25 cases (76%: Table 1.2). This comparison is conservative because independent evidence suggests that in some of the negative cases sex allocation is under the control of queens not workers, so violating an assumption of the theory (e.g. population sex investment ratios at the queen optima in B. hypnorum (Paxton et al., 2001) and in the Lasius niger populations studied by Fjerdingstad et al. (2002)). It also needs noting that, where workers relatedness asymmetry is generated by variations in queen number, the predictions of Boomsma and Grafen s (1990, 1991) theory coincide with those stemming from the idea that monogynous colonies should invest relatively more in females to compensate for male-biased sex allocation by polygynous (multiple-queen) colonies due to local resource competition (Boomsma, 1993; Nonacs, 1993; Bourke and Franks, 1995). This argument does not of course apply where relatedness asymmetry varies due to variation in queen mating frequency alone (Sundström, 1994; Sundström et al., 1996), or where, for example, relatedness

8 01Insect Evolutionary 25/1/05 15:45 Page 8 8 A.F.G. Bourke asymmetry varies independently of queen number in polygynous populations (Evans, 1995; Heinze et al., 2001). Experimental manipulations of relatedness asymmetry that generate the predicted sex ratio shift also provide particularly strong support for the theory (Mueller, 1991). Support for split sex ratio theory suggests that workers are able to assess the type of colony to which they belong (with relatively high or low relatedness asymmetry). Evidence exists that workers can achieve this by assessing heritable variation in the cuticular hydrocarbon profiles of nestmate workers (Boomsma et al., 2003). Mechanisms that workers then use to bias the sex ratio include the selective killing of males (Sundström et al., 1996) or the biasing, during larval development, of the final caste (development as queen or worker) of females (Hammond et al., 2002). From the several cases where there are split sex ratios but no variation in workers relatedness asymmetry (Table 1.2), it is clear that split sex ratios can occur for reasons unconnected with colony kin structure. In some of these cases, there is evidence that queens control sex allocation and use strategies to do so that themselves generate split sex ratios, e.g. Pheidole desertorum (Helms, 1999), Solenopsis invicta (Passera et al., 2001) and Bombus terrestris (Bourke and Ratnieks, 2001) (Table 1.2). For example, in the ant P. desertorum, queens appear unusual in that they can apparently lay worker-biased female eggs, most probably via hormonal effects (Helms, 1999). Half of colonies were found to be male specialists, half were found to be female specialists, the population sex investment ratio was at the queen optimum of 1:1, and colonies did not differ in either productivity or the workers relatedness asymmetry. Helms (1999) proposed that queens achieve control of sex allocation by, in half the colonies, laying only worker-biased eggs and haploid eggs, so forcing the sterile workers to raise males. This means that, in the other half of the colonies, the optimum sex ratio for both workers and the queen is all-females (Pamilo, 1982). The outcome is a queen-preferred population sex investment ratio of 1:1 and a split sex ratio in the absence of any variation in workers (or queens ) relatedness asymmetry. In short, the ESS (evolutionarily stable strategy) for a queen heading a colony is effectively to toss a coin and decide to lay, with a chance of 0.5, either (a) worker-biased female eggs and haploid eggs or (b) non-workerbiased female eggs. Other factors altogether that may contribute to sex ratio splitting include variation in colony productivity coupled with a degree of local mate competition within populations (constant male hypothesis: Frank, 1987; Hasegawa and Yamaguchi, 1995), local resource enhancement (related females cooperate to enhance group productivity: e.g. Cronin and Schwarz, 1997) and, in some polygynous species, the need to replace queens to maintain levels of polygyny (queen replenishment hypothesis: Brown and Keller, 2000, 2002; Brown et al., 2002). 2.2 Male parentage Workers of many species of social Hymenoptera are capable of laying unfertilized haploid eggs that develop into males (Bourke, 1988b; Choe, 1988). Successful worker reproduction may nonetheless fail to occur, either because

9 Table 1.2. Summary of tests of Boomsma and Grafen s (1990, 1991) split sex ratio theory (predicting split sex ratios as a function of workers relatedness asymmetry, RA). Adapted from Queller and Strassmann s (1998) review, with additional cases. Sex ratio patterns in species/population fit theory Sex ratio patterns in species/population do not fit theory Workers i.e. sex ratio is split in direction predicted (high-ra colonies i.e. sex ratio is split in direction opposite to that predicted, or sex ratio RA varies produce mainly females, low-ra colonies produce mainly males) variation is uncorrelated with workers RA, or sex ratio is not split Formica exsecta (ant, monogynous population) (Sundström et al., Formica exsecta (ant, polygynous population) (Brown and Keller, 2000) 1996; Sundström and Ratnieks, 1998) F. sanguinea (ant) (Pamilo and Seppä, 1994) F. podzolica (ant) (Deslippe and Savolainen, 1995) Lasius niger (ant) (Fjerdingstad et al., 2002) F. truncorum (ant) (Sundström, 1994) Proformica longiseta (ant) (Fernández-Escudero et al., 2002) Leptothorax acervorum (ant, Reichswald population) (Heinze Bombus hypnorum (bee) (Paxton et al., 2001) et al., 2001) Xylocopa sulcatipes (bee) (Stark, 1992) L. acervorum (ant, Santon population) (Chan et al., 1999; Hammond et al., 2002) L. longispinosus (ant) (Herbers, 1984, 1990) Myrmica ruginodis (ant) (Walin and Seppä, 2001) M. sulcinodis (ant) (Elmes, 1987) M. tahoensis (ant) (Evans, 1995) Rhytidoponera chalybaea (ant) (Ward, 1983) Rhytidoponera confusa (ant) (Ward, 1983) Augochlorella striata (bee) (Mueller, 1991; Mueller et al., 1994) Halictus rubicundus (bee) (Yanega, 1988; Boomsma, 1991) Lasioglossum laevissimum (bee) (Packer and Owen, 1994) Brachygastra mellifica (wasp) (Hastings et al., 1998) Parachartergus colobopterus (wasp) (Queller et al., 1993b) Polybia occidentalis (wasp) (Queller et al., 1993b) P. emaciata (wasp) (Queller et al., 1993b) Protopolybia exigua (wasp) (Queller et al., 1993b) Total 19 6 Workers RA i.e. a split sex ratio is absent i.e. a split sex ratio is present does not vary Colobopsis nipponicus (ant) (Hasegawa, 1994) Leptothorax nylanderi (ant) (Foitzik and Heinze, 2000; L. acervorum (ant, Roydon population) (Chan et al., 1999) Foitzik et al., 2003) Pheidole desertorum (ant) (Helms, 1999) P. pallidula (ant) (Aron et al., 1999; Fournier et al., 2003) Solenopsis invicta (ant, monogynous population) (Passera et al., 2001) B. terrestris (bee) (Duchateau and Velthuis, 1988; Duchateau et al., 2004) Total 2 5 Genetics, Relatedness and Social Behaviour 9 01Insect Evolutionary 25/1/05 15:45 Page 9

10 01Insect Evolutionary 25/1/05 15:45 Page A.F.G. Bourke workers refrain from laying male eggs (self-restraint) or because worker-laid male eggs are destroyed by other workers (worker policing) or by queens (queen policing) (Cole, 1986; Ratnieks, 1988). Under conditions of monogyny and single queen mating, kin selection theory predicts that, other things being equal, workers gain greater inclusive fitness from rearing sons (relatedness, r = 0.5) or the sons of other workers (nephews, r = 0.375) rather than the queen s male offspring (brothers, r = 0.25), whereas the queen gains greater inclusive fitness from the rearing of sons (r = 0.5) rather than workers male offspring (grandsons, r = 0.25) (Hamilton, 1964; Trivers and Hare, 1976). Hence, as with sex allocation, there is a potential kin-selected conflict between queens and workers over male parentage. Changes in colony kin structure due to either multiple mating by queens (polyandry) or polygyny can alter the expected pattern of conflict because they alter relative relatedness values. For example, under monogyny with an effective queen mating frequency greater than two, a focal reproductive worker remains more closely related to its male eggs (r = 0.5) than to those of the queen (r = 0.25), but the average worker becomes more closely related to queen-produced males (r = 0.25) than to the average worker-produced male (0.125 < r < 0.25). Under these conditions, reproductive workers are still predicted to lay male eggs (self-restraint is not favoured), but other workers are predicted to stop these eggs being reared (worker policing is favoured), for example by eating the eggs (Starr, 1984; Woyciechowski and Lomnicki, 1987; Ratnieks, 1988). Tests of these predictions rely on determining both the exact kin structure of colonies and populations and on measuring male parentage accurately. Since both tasks can be achieved with the necessary accuracy using microsatellite genetic markers (Queller et al., 1993a), there has recently been a great increase in the number of studies analysing this issue. To test specifically for worker policing, there is also a need either for measurements of male parentage at both the egg stage and the adult stage, or for detailed behavioural observations or experiments to determine the fate of worker-produced male eggs Interspecific tests In ants, the prediction that species with monogynous colonies and singly-mated queens should be characterized by high levels of adult male production by workers even in colonies with a queen does not appear to be fulfilled; many ants matching or approximating this kin structure have non-reproductive workers, or workers that reproduce only after the queen has died (e.g. Villesen and Boomsma, 2003; reviewed by: Bourke, 1988b; Choe, 1988; Bourke and Franks, 1995). A possible exception, in which high levels of worker reproduction in colonies with a queen might occur, is found in the slave-making ants (Heinze, 1996; Heinze et al., 1997; Foitzik and Herbers, 2001). Increased reproduction by workers in slave-makers has been predicted on the assumptions that these workers are unable (or, it now appears, only partly able) to bias sex allocation in their favour (Bourke, 1988a) and/or that female slavemaker larvae are more able to determine whether they develop as queens or workers (Nonacs and Tobin, 1992).

11 01Insect Evolutionary 25/1/05 15:45 Page 11 Genetics, Relatedness and Social Behaviour 11 In bees, broad comparisons of honey bees (Apis spp.), on the one hand, which are characterized by monogyny, high effective queen mating frequencies and low levels of adult male production by workers (e.g. Visscher, 1989; Barron et al., 2001), and stingless bees (Meliponinae) and bumblebees (Bombini), on the other hand, which are characterized by monogyny, low effective mating frequencies and high levels of adult male production by workers (e.g. Peters et al., 1999; Palmer et al., 2002; Tóth et al., 2002a,b; Brown et al., 2003; Paxton et al., 2003), support the predictions of kin selection theory. However, there is considerable unexplained variation in the degree of worker reproduction across and within stingless bee and bumblebee species (e.g. Drumond et al., 2000; Paxton et al., 2001; Brown et al., 2003; Tóth et al., 2003). In addition, worker policing of worker-laid male eggs in honey bees, for which strong evidence exists (Ratnieks and Visscher, 1989; Barron et al., 2001) and which accounts for the lack of adult males derived from workers, has also been found to occur in the Cape honey bee, A. mellifera capensis. This lacks a kin structure predicting such policing because workers reproduce by thelytoky (parthenogenetic production of female eggs) (Pirk et al., 2003). Likewise, worker policing by aggression against reproductive workers occurs in the thelytokous ponerine ant, Platythyrea punctata (Hartmann et al., 2003). Worker policing conceivably occurs in these cases because it reduces the productivity costs of successful worker reproduction (Ratnieks, 1988; Ratnieks and Reeve, 1992; Hartmann et al., 2003; Pirk et al., 2003). This raises the question of whether, in other Apis species, worker policing is driven by relatedness benefits as had previously been assumed, economic benefits, or both. In polistine wasps, adult male production by workers occurs in queenless colonies but is variable in colonies with a queen, with the estimated fraction of worker-produced adult males ranging from around 0% in Polistes bellicosus, P. dorsalis and P. gallicus (Arévalo et al., 1998; Strassmann et al., 2003) to 39% in P. chinensis (Tsuchida et al., 2003), even though in all these cases the colony kin structure predicted workers should be reproductive. In swarm-founding, polygynous wasps, adult male production by workers is rare; a finding in line with expectations from kin selection theory (Hastings et al., 1998; Henshaw et al., 2002). In the monogynous vespine wasps, patterns of male parentage broadly support kin selection predictions. Across ten species, Foster and Ratnieks (2001b) found an association between high effective queen mating frequencies and the absence of adult male production by workers on the one hand (in three species), and low effective mating frequencies and above-zero levels of adult male production by workers on the other hand (in six species). In the remaining species (the hornet Vespa crabro), a low effective mating frequency was coupled with absence of successful worker reproduction, which was later shown to be due to worker policing (Foster et al., 2002). This again suggests that worker policing may occur for its purely economic benefits (Foster et al., 2002). Extensions of kin selection theory predict that, because queen worker conflict over male parentage is strongest in monogynous colonies with a singly-mated queen, kin-selected worker matricide (killing of the mother queen followed by male production) should be likeliest in these conditions (Trivers and Hare, 1976; Ratnieks, 1988; Bourke, 1994). Foster and

12 01Insect Evolutionary 25/1/05 15:45 Page A.F.G. Bourke Ratnieks (2001b) found support for this prediction in vespine wasps by demonstrating a significant negative relationship between the frequency of queenless colonies and the effective mating frequency. In the polistine wasp Polistes gallicus, Strassmann et al. (2003) suggested that the unusually high frequency of queenless nests (74%) also stemmed from worker matricide, since, in these colonies, workers were shown to produce most of the males Intraspecific tests Male parentage among adult males has been genetically investigated in several species with facultative variation in kin structure (e.g. ants: Sundström et al., 1996; Evans, 1998; Herbers and Mouser, 1998; Walin et al., 1998; Foitzik and Heinze, 2000; bees: Paxton et al., 2001; wasps: Arévalo et al., 1998; Hastings et al., 1998; Goodisman et al., 2002; Henshaw et al., 2002). In general, worker production of adult males was found to be absent or rare irrespective of colony kin structure, even when kin selection theory predicted workers to be reproductive. Male parentage has been investigated in detail (genetically or behaviourally) among both male eggs and adult males in two species with facultative variation in kin structure, the wasp Dolichovespula saxonica (Foster and Ratnieks, 2000) and the ant Leptothorax acervorum (Hammond et al., 2003). In D. saxonica, workers under a singly-mated queen laid 70% of male eggs and 70% of adult males reared by the colony were worker-derived. By contrast, workers under a multiply-mated queen laid 25 90% of male eggs and only 7% of adult males were worker-derived. This suggested that, as predicted, workers under a multiply-mated queen facultatively policed worker-laid eggs (Foster and Ratnieks, 2000). However, in L. acervorum, the fraction of workerproduced males was low (2 5%) and did not differ either between eggs and adults or between monogynous colonies and polygynous colonies (Hammond et al., 2003). This was contrary to the predictions of kin selection theory, which predicted high frequencies of worker-laid eggs in both colony types and high frequencies of adult worker-derived males in monogynous colonies. One possible reason for the general lack of success of kin selection theory in intraspecific tests of the male parentage predictions is that, in principle, interactions with sex-ratio splitting may alter the benefits and costs of worker reproduction and policing (Walin et al., 1998; Foster and Ratnieks, 2001a; Hammond et al., 2003). Such effects need considering because species whose facultative variation in colony kin structure makes them likely to be chosen for within-population studies of male parentage are also likely to exhibit split sex ratios. Although these effects were consistent with the absence or rarity of worker reproduction in some cases (Sundström et al., 1996; Walin et al., 1998), in L. acervorum patterns of male parentage were still not consistent with predictions of kin selection theory modified to account for the occurrence of sex-ratio splitting in the study population (Hammond et al., 2003). Another possible reason for the lack of match between kin selection predictions and intraspecific measures of male parentage is that there are unmeasured costs of worker reproduction that select for self-restraint among workers in all types of colony (e.g. Arévalo et al., 1998; Foster et al., 2002; Pirk et al., 2003).

13 01Insect Evolutionary 25/1/05 15:45 Page 13 Genetics, Relatedness and Social Behaviour 13 Although plausible, this explanation is difficult to test because costs of events that occur infrequently (here, worker egg-laying and the rearing of workerproduced males to adulthood) are hard to measure. 2.3 Caste fate In the social Hymenoptera, as we have seen, the worker caste is entirely female and, like the queen caste, arises from fertilized, diploid eggs. With a few possible exceptions (Julian et al., 2002; Volny and Gordon, 2002; Helms Cahan and Keller, 2003), the queen-worker dichotomy is non-genetic (Wilson, 1971). Caste determination refers to the process by which a totipotent, female individual (that is, one capable of becoming a member of either caste) develops into an adult queen or worker. In species whose queens and workers differ morphologically, the caste fate of an individual female is of paramount importance in determining her options for realizing evolutionary fitness. Queens are specialized for egg-laying. Workers are specialized for helping and either lack ovaries or, if they have them, usually lack a sperm receptacle and are therefore unable to mate and produce diploid offspring (Wilson, 1971; Bourke, 1988b). Given its importance for the final fitness of females, it is not surprising that caste fate is also the focus of potential kin-selected conflict. This idea has been raised and discussed by a number of authors over the past 15 years or so (Ratnieks, 1989; Strassmann, 1989; Nonacs and Tobin, 1992; Ratnieks and Reeve, 1992; Keller and Reeve, 1994; Bourke and Franks, 1995; reviewed by Bourke and Ratnieks, 1999). A context in which potential caste conflict occurs that has proved fruitful for testing the theory was described by Bourke and Ratnieks (1999). This involved the case where workers and queens are reared by colonies simultaneously. In this case, there is no temporal factor (i.e. risk of emerging at an unsuitable time in the colony cycle) preventing female larvae from emergence as queens contrary to the interests of other colony members. In this situation, consider the case in which the workers favour rearing one adult queen and one adult worker from every pair of diploid larvae. Each individual larva would favour her own development as the queen, because each individual larva is more closely related to her own would-be offspring than to the would-be offspring of the other female larva. However, adult workers should be indifferent as to which of the two larvae became the queen, either because they are equally related to them (e.g. under monogyny with a singly-mated queen) or because workers are assumed to be unable to discriminate between larvae on the basis of their relatedness (e.g. Keller, 1997). Hence a potential kin-selected conflict exists, with each larva favouring her own development as a queen in opposition to the interests of the other larva and, if both succeeded in becoming queens, of the workers. Bourke and Ratnieks (1999) defined a general condition for the expression of such conflict. This is that female larvae should have some degree of selfdetermination, namely the power to influence their own caste fate. This condition is necessary because, if self-determination is absent, a female larva s

14 01Insect Evolutionary 25/1/05 15:45 Page A.F.G. Bourke caste fate will be determined by the interests of the adult queens or workers and, hence, even if potential conflict exists between her and these parties over her caste fate, it will not be expressed. In general, workers might be expected to control caste fate through their control of the rearing and nutrition of the brood (Bourke and Ratnieks, 1999). Factors that might facilitate self-determination would then include: (a) a low degree of queen worker size dimorphism (because then just a little extra feeding by a female larva could push her across the size threshold above which she can become a successful queen); and (b) larvae having some practical control over their own nutrition. In the stingless bee genus Melipona, conditions for actual caste conflict are met. Queens and sexuals are reared simultaneously, queen worker size dimorphism is absent (i.e. adult queens and workers are the same size and develop in cells of uniform size), and female larvae have practical power over their own nutrition because they are provisioned by workers with food and the cell in which they develop is then sealed. The theory of kin-selected caste conflict then predicts that more queens should emerge than are favoured by the workers, due to female larvae selfishly developing as queens; it likewise predicts worker actions to correct the excess of queens (Bourke and Ratnieks, 1999). These predictions are fulfilled by Melipona. Queen production in this genus is characterized by the emergence of a vast excess of queens (up to 25% of diploid larvae emerge as queens), which the workers then subject to a largescale cull (Bourke and Ratnieks, 1999; Wenseleers et al., 2003). A factor that probably aggravates this phenomenon is that stingless bees reproduce by colony fission. The colony, which in most species is monogynous, divides into two, with each new colony being headed by a young queen produced in the mother colony. Therefore, only a handful of new queens are required for colony reproduction per fission event, making the unnecessary excess of queens that emerges likely to represent a significant cost to the colony. Trigonine stingless bees (e.g. Trigona), and honey bees (Apis) share with Melipona the simultaneous rearing of queens and workers and reproduction by colony fission. However, in these species there is a relatively large degree of queen worker size dimorphism, associated with the fact that queens develop in cells that are larger than those of workers (Bourke and Ratnieks, 1999). Female larvae in these species therefore lack self-determination, since a female larva s fate is determined by the type of cell she is reared in. Consistent with this, in these species there is no large-scale overproduction of queens and hence no cull of excess queens (Bourke and Ratnieks, 1999; Wenseleers et al., 2003). Therefore, in stingless and honey bees, the theory of caste conflict successfully predicts actual conflict over caste fate between individual females and workers when self-determination is present, and the lack of actual conflict when conditions are the same except that self-determination is absent. The theory of caste conflict has been formalized and expanded by Ratnieks (2001) and Wenseleers et al. (2003). The formal theory incorporates the key point that the fitness payoff from development as queens by selfish female larvae is frequency-dependent. This is because, although each female larva favours her own development as a queen, if all were to succeed in becoming queens the colony as a whole would suffer a large productivity cost through

15 01Insect Evolutionary 25/1/05 15:45 Page 15 Genetics, Relatedness and Social Behaviour 15 lack of workers. Ratnieks (2001) and Wenseleers et al. (2003) derived the ESS frequency of new queens as a function of the relatedness structure of the colony. For example, under conditions of monogyny, single mating of queens and no worker male-production, the predicted frequency of queens is 20% (Ratnieks, 2001; Wenseleers et al., 2003). This is calculated as the ratio, 1 r F : r F + r M, where r F = relatedness to females and r M = relatedness to males; hence, in the present case, r F = 0.75 (for sisters) and r M = 0.25 (for brothers), so the ESS ratio is 0.25:1, which, expressed as a proportion, is 20%. An intuitive explanation for this result is that each female larva weighs up the net gain to herself of becoming a queen (1 unit of fitness, because relatedness to self is 1, minus the cost she inflicts on a sister by denying it queenhood, 0.75), as against the net benefit of becoming a worker and rearing sisters (0.75) and brothers (0.25) (Wenseleers et al., 2003). The mean value from five Melipona species of the frequency of females becoming queens was 21 22% (Ratnieks, 2001). Furthermore, as predicted by the model, this frequency decreases across Melipona species as the fraction of worker-produced males (and hence r M ) rises (Wenseleers and Ratnieks, 2004). The predictions of the ESS caste conflict models of Ratnieks (2001) and Wenseleers et al. (2003) represent unusual examples of quantitative predictions in kin selection theory outside the context of sex allocation, and moreover ones that are confirmed. Strassmann et al. (2002) proposed that a conflict analogous to caste conflict in species with morphological castes occurs in the swarm-founding epiponine wasp Parachartergus colobopterus. In this species, an adult female can opt to be a queen by mating and activating her ovaries. Aggression by workers towards recently emerged potential queens suggested that potential queens were in excess and that workers therefore acted to reduce their frequency (Strassmann et al., 2002; Platt et al., 2004). 3. Future developments The preceding section demonstrates strong but not universal evidence for an effect of relatedness on social evolution and social behaviour in insects as predicted by kin selection theory. The theory is strongly supported by significantly female-biased population sex investment ratios in monogynous ants, split sex ratios occurring as a function of variation in workers relatedness asymmetry, and interspecific patterns of variation in male parentage in bees and vespine wasps. The theory is not supported by interspecific patterns of variation in male parentage in ants and non-vespine wasps or, overall, by intraspecific comparisons in male parentage. Other parts of the current evidence base are supportive but not yet conclusive (e.g. as regards worker reproduction in slave-making ants, worker matricide, and caste conflict). This is not because of inconsistent findings but because of the scarcity of relevant studies. Still other parts of the evidence base are currently equivocal (e.g. as regards sex ratios in slave-making ants and bumblebees). These topics all deserve further investigation in order to determine where the balance of the evidence lies.

16 01Insect Evolutionary 25/1/05 15:45 Page A.F.G. Bourke It seems likely that many anomalies and inconsistencies in the data may in future be resolved by a fuller understanding of (a) which party has power over relevant reproductive decisions, and (b) the economic costs and benefits of selfish manipulations. For example, as we have seen, the issue of how power is monopolized or shared could underpin the fact that population sex investment ratios in monogynous ants fall below the values predicted from workers relatedness asymmetries (e.g. Passera et al., 2001; Reuter and Keller, 2001), the wide variation in population sex investment ratios among slave-making ants (Herbers and Stuart, 1998), and split sex ratio patterns inconsistent with worker control of sex allocation (Helms, 1999; Passera et al., 2001). Likewise, in some cases, costs of worker reproduction and policing may determine, in parallel with or even instead of relatedness differences, the distribution of male parentage and the occurrence of worker policing (e.g. Arévalo et al., 1998; Foster et al., 2002; Hammond et al., 2003; Pirk et al., 2003; Villesen and Boomsma, 2003). Researchers have long recognized the importance of these factors (reviewed by Ratnieks, 1998; Sundström and Boomsma, 2001; Beekman and Ratnieks, 2003; Beekman et al., 2003; Mehdiabadi et al., 2003), but it clearly remains a key task for the future to find appropriate means of analysing and quantifying them. As well as these issues, there are a number of other areas that seem promising for future research in the field, as discussed next. 3.1 Genetic underpinnings The advent of hypervariable microsatellite markers has made possible extremely detailed investigations of kin structure and parentage in social insects, and hence more rigorous tests of kin selection theory (Queller et al., 1993a; Hughes, 1998; Bourke, 2001b). Microsatellites have also brought other technical benefits, such as a relatively rapid method for sexing eggs and so measuring the primary sex ratio (eggs appearing homozygous at many polymorphic loci are haploid, male eggs) (e.g. Ratnieks and Keller, 1998; Passera et al., 2001; Hammond et al., 2002), although other molecular-genetic techniques for this have also been developed (De Menten et al., 2003). Rapid advances in molecular genetics and genomics promise to bring additional benefits to the study of social evolution in insects. A pioneering study of the fire ant Solenopsis invicta by Krieger and Ross (2002) established for the first time in social insects the exact genetic basis of a complex social behaviour. S. invicta is a notorious introduced pest in the USA, where it occurs in a monogynous and a polygynous form. Previous work established that queen number variation (gyny) is controlled by allelic variation at a locus, Gp-9, with two alleles, B and b. Specifically, the b allele, a recessive lethal, acts as a green beard gene. A green beard gene is one whose bearers treat co-bearers favourably and non-bearers unfavourably on the basis of an external label (here, almost certainly a chemical cue associated with the presence of the b allele) (Dawkins, 1976). Heterozygote (Bb) workers, but not BB workers, tolerate multiple queens, but only if these queens bear the b allele; Bb workers detect and kill any BB queens. The result is that the presence or