Evolution, 58(5), 2004, pp. 1064±1072

VARIABLE QUEEN NUMBER IN COLONIES: NO IMPACT ON QUEEN TURNOVER, INBREEDING, AND POPULATION GENETIC DIFFERENTIATION IN THE ANT SELYSI

MICHEL CHAPUISAT,1 SAMUEL BOCHERENS, AND HERVEÂ ROSSET Department of Ecology and Evolution, Biology Building, University of Lausanne, 1015 Lausanne, Switzerland 1E-mail: [email protected]

Abstract. Variation in queen number alters the genetic structure of social colonies, which in turn affects patterns of kin-selected con¯ict and cooperation. Theory suggests that shifts from single- to multiple-queen colonies are often associated with other changes in the breeding system, such as higher queen turnover, more local mating, and restricted dispersal. These changes may restrict gene ¯ow between the two types of colonies and it has been suggested that this might ultimately lead to sympatric speciation. We performed a detailed microsatellite analysis of a large population of the ant Formica selysi, which revealed extensive variation in social structure, with 71 colonies headed by a single queen and 41 by multiple queens. This polymorphism in social structure appeared stable over time, since little change in the number of queens per colony was detected over a ®ve-year period. Apart from queen number, single- and multiple-queen colonies had very similar breeding systems. Queen turnover was absent or very low in both types of colonies. Single- and multiple-queen colonies exhibited very small but signi®cant levels of inbreeding, which indicates a slight deviation from random mating at a local scale and suggests that a small proportion of queens mate with related males. For both types of colonies, there was very little genetic structuring above the level of the nest, with no sign of isolation by distance. These similarities in the breeding systems were associated with a complete lack of genetic differentiation between single- and multiple-queen colonies, which provides no support for the hy- pothesis that change in queen number leads to restricted gene ¯ow between social forms. Overall, this study suggests that the higher rates of queen turnover, local mating, and population structuring that are often associated with multiple- queen colonies do not appear when single- and multiple-queen colonies still coexist within the same population, but build up over time in populations consisting mostly of multiple-queen colonies.

Key words. Breeding system, dispersal, genetic structure, microsatellites, relatedness, social evolution, social struc- ture.

Received June 13, 2003. Accepted January 28, 2004.

Social exhibit a great variation in their social struc- older colonies, should favor the acceptance of new daughter ture. In , the number of queens per colony varies among queens into their natal colonies (Nonacs 1988; Pamilo 1991a; , among populations, and even among colonies within Herbers 1993; Bourke and Franks 1995; Keller 1995). In line the same population (Crozier and Pamilo 1996a; Pamilo et with this argument, the habitat saturation hypothesis predicts al. 1997; Ross 2001). These variations in social structure that queen number should increase over time once all poten- affect the genetic diversity within the colony, the relatedness tial nest sites or territories are occupied by ant colonies (Her- among nestmates, and the architecture of potential kin-con- bers 1986; Bourke and Franks 1995; Pedersen and Boomsma ¯icts and kin-cooperation (Bourke and Franks 1995; Crozier 1999a). In addition, members of multiple-queen colonies may and Pamilo 1996a). Hence, understanding the causes, cor- also bene®t from higher growth rates, larger colony size, relates, and dynamics of variation in queen number is fun- increased genetic diversity, increased colony survival, longer damental to gain a better understanding of social evolution. life-span of the colony, and better short-range colonization The causes of variation in queen number are still contro- ability (HoÈlldobler and Wilson 1977; Bourke and Franks versial. Recent work in the ®re ant Solenopsis invicta showed 1995; Keller 1995). that variation in queen number is determined by a genetic The theory predicts that the shift from single- to multiple- polymorphism that makes workers accept or reject additional queen colonies should often be associated with other changes queens (Ross 1997; Keller and Ross 1998; Ross and Keller in queen turnover, mating system, and dispersal (Bourke and 1998; Krieger and Ross 2002). However, in other species Franks 1995; Pamilo et al. 1997; Ross 2001). Usually, single- queen number may be a phenotypically plastic trait strongly queen colonies do not accept replacement queens, and the in¯uenced by ecological factors (West-Eberhard 1989; Ped- life span of the colony matches the life span of the queen. ersen and Boomsma 1999a; Ingram 2002). Whatever the In contrast, multiple queen colonies accept replacement proximate determinants of queen number, the presence of queens and may have high rates of queen turnover (HoÈl- multiple queens in the same colony seems paradoxical, be- ldobler and Wilson 1990; Bourke and Franks 1995; Keller cause it decreases both the relatedness of workers to the brood and Genoud 1997). Single-queen colonies typically produce they raise and the per capita reproductive output of queens queens and males that participate in extensive mating ¯ights (Hamilton 1964; Bourke and Franks 1995). and mate away from the nest. After dispersal, queens found However, various ecological and demographic factors may new colonies independently, with no assistance from work- select for accepting additional queens. Strong ecological con- ers, or by temporarily parasitizing the nests of other ant spe- straints limiting independent colony founding by queens, cies (HoÈlldobler and Wilson 1990). Therefore, single-queen such as nest-site limitation, predation, or competition from colonies are predicted to form large panmictic populations 1064 ᭧ 2004 The Society for the Study of Evolution. All rights reserved. SOCIAL STRUCTURE VARIATION IN AN ANT COLONY 1065 with no inbreeding, no isolation by distance, and minimal mitochondrial markers (RuÈppell et al. 2003). Hence, more genetic structure above the nest level (Bourke and Franks empirical data are needed to assess whether variation in social 1995; Crozier and Pamilo 1996a; Pamilo et al. 1997). In structure is generally associated with genetic differentiation. contrast, queens from multiple-queen colonies often mate The correlates of variation in queen number and the re- close to or in their natal nest. After mating, they stay in their sulting genetic differentiation between social forms have usu- natal nest or form new colonies by budding, a process where- ally been studied by comparing populations with single- or by queens with workers depart from a colony and establish multiple-queen colonies (Pamilo et al. 1997; Ross 2001). This a new colony in the vicinity (HoÈlldobler and Wilson 1990). is a derived situation, and the coexistence of the two colony Overall, local mating and restricted dispersal should result types in the same site better re¯ects the primary situation in in signi®cant de®ciency of heterozygotes, isolation by dis- which multiple-queen colonies evolved from single-queen tance, and genetic structuring above the level of the nest in ones (Keller and Chapuisat 2002). Therefore, it remains un- populations of multiple-queen colonies (Bourke and Franks clear whether a change in the social structure immediately 1995; Crozier and Pamilo 1996a; Pamilo et al. 1997). promotes differences in the mating system, dispersal, and A large body of empirical data con®rms that changes in genetic structure, or whether the shift in the breeding system social structure are often associated with changes in queen develops progressively in isolated populations consisting turnover, mating system, and dispersal. An absence of queen mostly or exclusively of multiple-queen colonies. To resolve turnover coupled with queen life spans over 20 years has this issue, it is important to study within-population variation been documented in several ant species with single-queen in queen number, queen turnover, mating system, and dis- colonies (Pamilo 1991b; Keller and Genoud 1997). In con- persal, as well as genetic differentiation between sympatric trast, short queen life spans are common in multiple-queen social forms. Data on the dynamics of queen number and on colonies (Keller and Genoud 1997), with annual rates of variation in social structure over time are also needed to get queen turnover close to 50% in several species of a clearer picture of how complex social structures appear and ants (SeppaÈ 1994; Evans 1996; Pedersen and Boomsma are maintained. 1999a). Populations with single-queen colonies are usually In this study, we examine the genetic consequences of in Hardy-Weinberg equilibrium and show little genetic dif- variation in social structure within a large population of the ferentiation above the level of the nest, which suggests that ant Formica selysi. Speci®cally, on the basis of microsatellite queens and males disperse widely (Crozier and Pamilo 1996a; data from several generations of workers, we characterize Pamilo et al. 1997; Ross 2001). In contrast, signi®cant de- how the social structure varies among colonies and over time. ®ciencies of heterozygotes and isolation by distance have We then examine whether variation in social structure cor- been detected in several populations with multiple-queen col- relates with differences in queen turnover, mating system, onies. This indicates that effective dispersal of queens and gene ¯ow, and genetic structuring above the nest level. Fi- males is restricted in these populations, which results in mi- nally, we test for genetic differentiation between the single- crogeographical genetic structure but does not necessarily and multiple-queen social forms. imply high rates of close inbreeding (mating between closely related individuals) if queen number is high (Chapuisat et al. MATERIALS AND METHODS 1997; Pamilo et al. 1997; Chapuisat and Keller 1999; Ross 2001). However, exceptions to this general pattern do exist. Study Species and Study Site For example, queen turnover occasionally occurs in monog- ynous (single-queen colonies) populations of ants (Heinze Formica selysi is a pioneer species that colonizes bare and Keller 2000), and signi®cant levels of inbreeding were sandy and stony ground, as well as open steppes. In the Alps, associated with signs of restricted dispersal of females in F. selysi is found in xeric habitat along large rivers, where monogynous populations of Formica exsecta (SundstroÈm et it is locally very abundant. It nests in the soil and feeds on al. 2003). various prey and on honeydew from aphids (Keller Interestingly, differences in mating location, dispersal, and and Zettel 2001a). Formica selysi is well adapted to resist mode of colony founding may restrict gene ¯ow between occasional ¯oods, and nests can survive under water for sev- single- and multiple-queen colonies. Variation in social struc- eral days (Lude et al. 1999). ture may thus result in genetic differentiation between the Our study site is an alluvial plain located along the river social forms, and it has even been suggested that this process RhoÃne between Sierre and Susten in the central Valais, Swit- might lead to sympatric speciation (West-Eberhard 1986; zerland (the ``Rottensand,'' longitude 7Њ36Ј30ЉE, latitude Crozier and Pamilo 1996b; Shoemaker and Ross 1996). A 46Њ18Ј30ЉN, altitude 565 m). The plain is a mosaic of pine recent model also showed that green-beard selection based forest, steppe areas dominated by the grass Stipa pennata, on the social discrimination of tags may lead to reproductive and ¯oodplains of bare sand and gravel. Parts of this habitat isolation (Hochberg et al. 2003). In line with these ideas, are periodically affected by ¯oods from the river RhoÃne. detailed genetic studies of the ®re ant Solenopsis invicta re- Floods occurred in autumn 1993 and 2000, covering large vealed that gene ¯ow between social forms is restricted, re- areas with sand and gravel, and locally causing severe ero- sulting in weak but signi®cant genetic differentiation for mi- sion. Most samples were taken in mature steppes that had crosatellite markers and strong differentiation for mitochon- not been affected by recent ¯oods and where the density of drial DNA (Shoemaker and Ross 1996; Ross et al. 1999). In nests was high (Keller and Zettel 2001b). A few colonies contrast, no differentiation between social forms was detected were also sampled on a river embankment, in open pine forest in Leptothorax rugatulus, based on both microsatellite and or in the high water bed of the river. 1066 MICHEL CHAPUISAT ET AL.

Sampling TABLE 1. Genetic diversity of microsatellite markers in Formica selysi. The number of alleles (NA), frequency of the most common Adult workers were sampled from 112 nests in spring 2000 allele (FCA) and expected heterozygosity (He) are presented. Allele and 2001. Flat stones were placed over the nests to facilitate frequencies were calculated weighting nests equally. The sample subsequent sampling. Nests were mapped with a global po- size was 1444 individuals from 112 nests. sitioning system (GPS, Garmin, Olathe, KS) and a metric Locus NA FCA H rope for short distances. The sampling area was approxi- e FL12 3 0.59 0.51 mately 1500 m long and 500 m wide. To get more insight FL20 4 0.86 0.24 into spatial differentiation, we sampled nests in four geo- FL21 11 0.44 0.72 graphical sectors separated by 100 to 300 m of habitat slightly FE7 4 0.56 0.59 less suitable for the ants. The distance between sampled nests FE8 2 0.99 0.03 ranged from one to 1458 m. FE16 3 0.58 0.49 FE17 6 0.31 0.75 To estimate whether queen number varies over time in the FE19 2 0.66 0.45 population, the social structure of a small set of nests sampled FE38 10 0.74 0.43 in spring 1996 was compared to the social structure of the larger set of nests collected in 2000 and 2001. The 1996 sample consisted of adult workers from 25 nests located in For each of the 25 nests sampled in 1996, 10 adult workers an area approximately 430 m long and 200 m wide. To es- were genotyped at three microsatellite markers (FL12, FL20, timate yearly changes in social structure within colonies, two and FL21) as described in Chapuisat (1996). types of brood samples were taken from part of the 112 nests in which adults had been collected. First, worker brood (lar- Genetic Data Analysis vae and pupae destined to become workers) was collected The social structure was assessed by measuring relatedness twice from a subsample of 17 nests, in summer 2000 and among nestmate workers with the method of Queller and Good- again in summer 2001. Second, worker brood was sampled night (1989), as implemented in the computer program Relat- once from a subsample of 43 nests in summer 2000 or 2001, edness 5.0.4 (http://www.gsoftnet.us/GSoft.html). Nests were a few months after adult workers had been sampled from the weighted equally. The standard error of the average relatedness same nests. Eggs of Formica are laid in spring and summer, was obtained by jackkni®ng over nests (jackkni®ng over loci and no brood overwinters (Forel 1920; GoÈsswald 1989). The yielded smaller standard errors). The standard error and 95% ®rst new workers emerge in June in our study population, con®dence interval of relatedness within single nests were and Formica workers live for a couple of years, at most determined by jackkni®ng over loci. The social structure of (GoÈsswald 1989). Hence, adult workers sampled in spring nests was further determined by examining the arrays of ge- are at least one year older than the worker brood sampled in notypes and by testing the pedigree relationships with the summer, and samples of adults and brood can be used to likelihood approach implemented in the computer program estimate yearly changes in social structure and genetically Kinship 1.3.1 (Goodnight and Queller 1999; http://www. effective queen turnover (see below). Sampled individuals gsoftnet.us/GSoft.html). The effective number of queens (f) were stored in absolute ethanol until genetic analysis. was estimated from the relatedness among workers (r) with the formula f ϭ 3/(4r). This is the number of unrelated, single- Microsatellite Analysis mated, and equally reproducing queens that in a randomly For each of the 112 nests sampled in 2000 and 2001, eight mating population would produce offspring with genetic di- adult workers and up to 16 worker brood (if any) were ge- versities equal to the one observed in the study colonies. notyped at nine microsatellite markers. DNA was extracted Alternatively, if queens stay in their natal nests and mate by incubating three crushed legs from adults or a small piece with unrelated males, the relatedness among queens is equal of tissue from brood in 250 ␮L of 5% Chelex (Sigma-Aldrich, to the relatedness among workers. Then, assuming a stable Steinheim, Germany) at 95ЊC for 20 min. We used three number of queens, the effective number of related queens is microsatellite markers originally developed for Formica par- estimated as (3 Ϫ r)/(3r) (e.g. Pamilo 1991a). alugubris (FL12, FL20, and FL21; Chapuisat 1996) and six The genetically effective queen turnover (Q) was estimated markers developed for Formica exsecta (FE7, FE8, FE16, with the following formula: FE17, FE19, and FE38; Gyllenstrand et al. 2002). The mark- Q ϭ 1 Ϫ [2r /(r ϩ r )], (1) ers varied greatly in terms of genetic diversity in F. selysi, ab aa bb with a mean of ®ve alleles and a mean expected heterozy- where rab is the relatedness between adult workers and worker gosity of 0.47 (Table 1). Taken together, this panel of nine brood, raa is the relatedness among adult workers, and rbb is markers is a powerful tool to reveal the social structure of the relatedness among worker brood (Evans 1996). This mea- nests and the ®ne-scale genetic structure of the population. sure of genetic turnover is close to zero if the same set of Two additional markers had to be discarded because they queens has produced the adults and the current cohort of were dif®cult to score (FE48; Gyllenstrand et al. 2002) or brood, because in that case the relatedness between cohorts had null alleles (FL29; Chapuisat 1996). Primers were labeled is equal to the relatedness within cohorts. In contrast, Q is with HEX, NED, and 6-FAM ¯uorescent dyes and the am- greater than zero if queens and their mates have been replaced pli®cation products were analyzed on an ABI (Applied Bio- by genetically distinct individuals, because in that case the systems, Foster City, CA) Prism 377 XL DNA sequencer. relatedness between cohorts becomes smaller than the relat- Alleles were scored with the Genescan (ABI Prism) software. edness within cohorts. Con®dence intervals of the genetically SOCIAL STRUCTURE VARIATION IN AN ANT COLONY 1067 effective queen turnover estimates were obtained by boot- strapping over colonies 1000 times. The mating system and genetic differentiation among sec- tors were investigated by applying a multilevel hierarchical F-analysis of variance to the worker genotypes (Weir and Cockerham 1984; Weir 1996). To control for the noninde- pendence of individuals from the same colony, individual workers were nested into their respective colonies, and col- onies were nested into sectors. The within sector (Find-sect) and overall (Find-tot) inbreeding coef®cients re¯ect the de®ciency of heterozygotes due to nonrandom mating within the sectors and within the total population, respectively, and Fsect-tot esti- mates the amount of genetic differentiation (allele frequency differences) among the sectors. The hierarchical analysis was performed with the computer program Genetic Data Analysis 1.0 (Lewis and Zaykin 1999). Con®dence intervals were ob- tained by bootstrapping over loci 5000 times. To detect po- tential differences in breeding systems, each social form (sin- FIG. 1. Distribution of single-nest relatedness estimates (the re- gle- or multiple-queen colonies) was analyzed separately, as latedness among workers within each nest) in a large population of well as together. Formica selysi. The social structure of nests (single- or multiple- queen) was inferred from the arrays of genotypes. We examined whether there was isolation by distance (a pos- itive correlation between geographical distance and genetic dif- ferentiation) in both single- and multiple-queen colonies, across arrays consistent with a single queen, usually mated to a all sectors and per sector. We calculated F for all pairs of ST single male, although double mating of the queen was de- nests, and tested the correlation between this matrix of genetic tected in seven (10%) of those 71 nests. The average relat- differentiation and the matrix of geographical distances in a edness in colonies classi®ed as having a single-queen was Mantel test with 5000 permutations, using Spearman rank cor- 0.732 Ϯ 0.012, in good agreement with theoretical expec- relation coef®cients as implemented in the computer program tations for this social structure (Bourke and Franks 1995). Genepop 3.1a (Raymond and Rousset 1995; http://www. The remaining 41 nests had multiple queens. The average biomed.curtin.edu.au:80/genepop/index.html). We also tested relatedness in those nests was 0.176 Ϯ 0.027. This relatedness whether nests of similar social structure were spatially clustered corresponds to an effective queen number of 4.3 if both the by examining the spatial autocorrelation of single-nest relat- queens and males are unrelated, and of 5.3 if queens are as edness (Legendre and Fortin 1989), using the computer program related as workers. R 3.02 (http://www.fas.umontreal.ca/BIOL/Casgrain/en/labo/ R/index.html). Changes in Social Structure Genetic differentiation between the social forms was tested for in two slightly different hierarchical F-analyses of vari- At the population level, the social structure of the set of ance. First, a three-level analysis was performed, with in- nests sampled in 1996 did not differ signi®cantly from the dividuals nested in colonies, colonies in social forms (single- social structure of the set of nests sampled four years later. or multiple-queen colonies), and social forms in the total In the 1996 sample, the average relatedness among nestmate population. Second, a four-level analysis was performed, with workers was 0.437 Ϯ 0.071. Out of the 25 sampled nests, 12 individuals nested in colonies, colonies in social forms, social (48%) had genotypes consistent with a single queen, and the forms in sectors, and sectors in the total population. The ®rst remaining 13 had multiple queens. Overall, the proportion analysis tests for an overall differentiation between the social of nests with a single queen was lower in the 1996 than in forms, and the second analysis examines whether there is the 2000±2001 sample, but this difference was not signi®cant differentiation between the social forms within the sectors. (Fisher's exact test, P ϭ 0.18). Moreover, the distribution of single-nest relatedness values did not differ signi®cantly be- RESULTS tween the two samples (Mann-Whitney U test, Z ϭϪ1.68, P ϭ 0.09). Social Structure At the colony level, the social structure of nests appeared The 112 nests sampled in 2000 and 2001 varied greatly in stable in 2000 and 2001. Very little yearly change in effective their social structure (Fig. 1). The average relatedness among queen number was detected. Two different analyses were nestmate workers was 0.54 Ϯ 0.03 (mean Ϯ SE). However, performed, one comparing the brood sampled in two con- few nests had an average relatedness close to this population secutive years, and the other comparing the samples of adults mean. The distribution of single-nest relatedness was strongly and brood collected during the same year. The ®rst analysis bimodal, with a peak centered on 0.75 and a peak centered suggests that the effective number of queens did not change on 0.15 (Fig. 1). The social structure of nests was also de- signi®cantly in the 17 nests from which brood was sampled termined by inspecting the arrays of genotypes in each nest during two consecutive years. The average relatedness in the and by testing the likelihood of various pedigree relation- brood was 0.523 Ϯ 0.072 in 2000 and 0.484 Ϯ 0.076 in 2001. ships. Out of the 112 nests sampled, 71 (63%) had genotype The relatedness within single nests did not differ signi®cantly 1068 MICHEL CHAPUISAT ET AL.

TABLE 2. Genetically effective rate of queen turnover (Q). The number of nests (N) and the nestmate relatedness among adults (raa), among brood (rbb), and between adults and brood (rab) are presented.

Queen turnover

Colonies Nraa rbb rab Q 95% CI Single-queen 24 0.752 0.756 0.752 0.003 Ϫ0.028; 0.039 Multiple-queen 19 0.231 0.276 0.247 0.025 Ϫ0.127; 0.193 All 43 0.522 0.544 0.529 0.008 Ϫ0.032; 0.056

between the two years when considering all nests (pairwise Mating System and Dispersal t-test, df ϭ 16, t ϭ 1.26, P ϭ 0.23), only single- (df ϭ 8, t A small but signi®cant de®ciency of heterozygotes was ϭ 0.75, P ϭ 0.48) or only multiple-queen nests (pairwise t- detected within sectors (F ϭ 3.6%; Table 3). Interest- test, df ϭ 7, t ϭ 0.98, P ϭ 0.36). For each nest, the con®dence ind-sect ingly, this inbreeding coef®cient was similar for single- and interval of the single-nest relatedness estimate from the 2000 multiple-queen colonies, with small but largely overlapping cohort overlapped broadly with the one from the 2001 cohort. con®dence intervals (Table 3). Overall, the amount of genetic The second analysis suggests that the effective queen num- differentiation among the sectors was very small (F ber did not change signi®cantly in 42 out of the 43 nests in sect-tot ϭ which adults and brood were sampled on the same year. The 0.2%) and not signi®cantly different from zero (Table 3). Single- and multiple-queen colonies showed similarly low average relatedness was 0.522 Ϯ 0.047 for the adult workers and 0.544 Ϯ 0.047 for the worker brood. Overall, single-nest levels of differentiation among sectors, with largely overlap- relatedness of the adults and of the brood did not differ sig- ping con®dence intervals, and upper limits of the 95% con- ni®cantly when considering all nests (pairwise t-test, df ϭ ®dence interval of 1.5% and 1.7%, respectively. However, 42, t ϭϪ0.84, P ϭ 0.41), only single- (df ϭ 23, t ϭϪ0.20, the differentiation among sectors was signi®cant for multiple- P ϭ 0.84), or only multiple-queen nests (df ϭ 18, t ϭϪ0.825, queen colonies only, which might indicate a slightly more P ϭ 0.42). In all but one nest, the con®dence interval of the pronounced structure in this social form (Table 3). Because single-nest relatedness estimate from adults overlapped there was only minimal differentiation among sectors, the broadly with the one from the brood. In the nest where the overall inbreeding coef®cient (Find-tot) was very similar to con®dence intervals did not overlap, ®ve matrilines were de- the one measured within sectors (Find-sect; Table 3). tected in the adults and only one in the brood, which indicates No isolation by distance was detected in the population a reduction in queen number. (Table 4). There was no positive correlation between geo- graphical distance and genetic differentiation between pairs Queen Turnover of nests, whatever the social structure of colonies (single- or multiple-queen) and spatial scale of analysis (within sectors The genetically effective queen turnover was estimated or across all sectors). These results should be considered with from the relatedness within and between cohorts of workers some caution, because FST values between pairs of nests have (Table 2). Overall, queen turnover was only 0.8%, with a large errors. However, when considered together, these data 95% con®dence interval overlapping with zero and reaching suggest that single- and multiple-queen colonies are very an upper limit of 5.6%. In the 24 single-queen colonies an- similar with respect to mating system and gene ¯ow among alyzed, the relatedness within and between cohorts was 0.75, nests. Both show small levels of inbreeding and minimal and the queen turnover was effectively zero. Direct inspection genetic differentiation above the level of the nest, with no of genotypes further con®rmed that no queen replacement sign of isolation by distance. had occurred in those colonies. Surprisingly, the queen turn- over within the 19 multiple-queen colonies analyzed was also Spatial Distribution of Social Forms very low, with an estimate of 2.5%. The 95% con®dence interval overlapped with zero, but reached an upper limit of Nests of similar social structure tended to be clustered, as 19%, which indicates that the queen turnover might be slight- shown by the signi®cant spatial autocorrelation of single-nest ly higher in multiple-queen colonies than in single-queen relatedness. The spatial autocorrelation coef®cient showed ones. However, overall the genetically effective yearly rate an alternation of positive and negative values, which indicates of queen replacement was very small in both types of col- patchiness (Legendre and Fortin 1989). Using 20 equifre- onies. quent classes of distances, the Moran's I autocorrelation co-

TABLE 3. Inbreeding coef®cient within the sectors (Find-sect), genetic differentiation among the sectors (Fsect-tot) and overall de®ciency of heterozygous (Find-tot). The 95% con®dence intervals are given in parentheses, and N is the number of nests.

Colonies NFind-sect Fsect-tot Find-tot Single-queen 71 0.033 (0.003; 0.067) 0.003 (Ϫ0.008; 0.015) 0.032 (0.003; 0.065) Multiple-queen 41 0.046 (0.010; 0.087) 0.010 (0.004; 0.017) 0.044 (0.008; 0.084) All 112 0.036 (0.010; 0.066) 0.002 (Ϫ0.004; 0.007) 0.035 (0.010; 0.064) SOCIAL STRUCTURE VARIATION IN AN ANT COLONY 1069

TABLE 4. Tests of isolation by distance. The number of nests (N), the correlation between the matrices of FST and geographical distances, and the signi®cance of isolation by distance in the Mantel test (P) are presented. Values per sector are separated by slashes. Dash indicates value not available.

Colonies N Matrix correlation P Single-queen all sectors 71 0.01 0.40 Single-queen per sector 1/30/19/21 Ð/Ϫ0.07/0.05/Ϫ0.29 Ð/0.81/0.35/0.86 Multiple-queen all sectors 41 Ϫ0.1 0.87 Multiple-queen per sector 10/12/13/6 Ϫ0.13/Ϫ0.1/Ϫ0.09/Ϫ0.17 0.69/0.69/0.78/0.65 All colonies all sectors 112 Ϫ0.11 0.62 ef®cient was highly positive and signi®cant (I ϭ 0.39, P Ͻ and some had very low relatedness values indicating that they 0.001) for the ®rst class of distances (up to 33 meters), and had many queens. Hence, there is a pronounced polymor- signi®cantly negative and positive values alternated for larger phism in social structure within this population, with nests distance classes. The correlogram was globally signi®cant, containing either one or from several to many queens. Such with ®ve classes conserving signi®cant autocorrelation co- intraspeci®c polymorphism in queen number seems to be ef®cients after a Bonferroni correction. Hence, neighboring common in the Formica, where it occurs both among nests tended to have a similar social structure, and overall (Rosengren et al. 1993; SundstroÈm 1993) and within popu- single- and multiple-queen nests had a tendency to be clus- lations (Goropashnaya et al. 2001). tered in relatively small patches. The proportion of single- The number of queens per colony appeared stable over and multiple-queen colonies also differed signi®cantly time in F. selysi, at both the population and the colony level. among sectors (df ϭ 3, ␹2 ϭ 17.8, P ϭ 0.0005), as did the At the population level, we detected no change in queen distribution of single-nest relatedness (Kruskal-Wallis test, number over a ®ve-year period. More single-queen nests were df ϭ 3, H ϭ 13.8, P ϭ 0.003), which further shows that nests sampled in 2000 and 2001 than in 1996, although this dif- with alternative social structures have a heterogenous spatial ference was not signi®cant and may be entirely due to sam- distribution. pling errors. At the colony level, very little yearly change in queen number was detected. No additional queen was adopted Genetic Differentiation between the Social Forms in the 24 single-queen colonies that were surveyed. The num- ber of queens also appeared stable in the multiple-queen col- There was no genetic differentiation between single- and mul- onies: in the 43 colonies surveyed, the only signi®cant change tiple-queen colonies. In a three-level analysis, with individuals was an apparent loss of queens in one colony. Together, these nested in colonies and colonies in social forms, the coef®cient data do not support the hypothesis that queen number in- of differentiation between social forms (F ) was social form-total creases over time due to increased habitat saturation, at least Ϫ0.005, with a 95% con®dence interval ranging from Ϫ0.007 in the time period investigated. More long-term data are re- to Ϫ0.004. The slightly negative estimate suggests that allele quired to determine whether this apparent stability in queen frequencies tend to be more similar between social forms than number and coexistence of social forms results from diver- expected by chance, a result that might be due to the small sifying selection, weak selection, or selection pressures ¯uc- amount of genetic differentiation among the sectors. Indeed, in tuating over space or time. a four-level analysis that controlled for the effect of genetic So far, no study has followed queen number over long differentiation among sectors by having individuals nested in periods of time, and evidence for an effect of habitat satu- colonies, colonies in social forms and social forms in sectors, ration is mostly indirect. The number of queens per nest the coef®cient of differentiation between social forms within increased along a density gradient in an invading population sectors (F ) was Ϫ0.001, with a 95% con®dence social form-sector of Linepithema humile, which is consistent with the habitat interval ranging from Ϫ0.007 to 0.006. saturation hypothesis (Ingram 2002). Higher queen number per colony in old than in young habitat was also detected in DISCUSSION one population of Myrmica sulcinodis (Pedersen and Booms- Data on the behavioral and genetic correlates of changes ma 1999a) and in two species of Formica ants (SeppaÈ et al. in social structure are needed to obtain a better understanding 1995), but not in three other ant species (SeppaÈ et al. 1995). of social evolution (Bourke and Franks 1995; Ross 2001). In Controlled experiments in which supplementary nest sites this study, a detailed genetic analysis at nine microsatellite were added had variable outcomes. The number of queens markers revealed that the social structure of nests varied per nest decreased when nests were added in a population of greatly within a population of the ant Formica selysi. About Leptothorax longispinosus, as predicted by the habitat satu- two-thirds (63%) of the 112 nests analyzed were headed by ration hypothesis (Herbers 1986). However, the relatedness a single queen, usually mated to a single male, and the rest among workers decreased after nest supplementation in two of the nests (37%) had multiple queens. In these polygynous populations of Myrmica punctiventris, whereas simultaneous- nests, the effective number of queens was 4.3, and the actual ly adding both food and nest sites increased relatedness in number of queens was likely to be much greater than this one of the populations but decreased it in the other (Herbers value if queens were related or if their shares of reproduction and Banschbach 1999; DeHeer et al. 2001). Overall, the im- were unequal (e.g. Chapuisat and Crozier 2001). Few nests pact of habitat saturation on social structure seems to be had relatedness values consistent with having two queens, variable, and probably depends on details of the ecology of 1070 MICHEL CHAPUISAT ET AL. the species, such as nest site availability, food resources, Single- and multiple-queen colonies showed little genetic demographic regulation, and habitat stability at various differentiation above the level of the nest, with no or minimal scales. differentiation among sectors and no sign of isolation by Apart from the difference in queen number, single- and distance. This lack of spatial differentiation suggests that multiple-queen colonies had very similar breeding systems. gene ¯ow is high enough to homogenize allele frequencies The genetically effective queen turnover was very low in both over small (within sectors) and intermediate (among sectors) social forms, although perhaps slightly higher in multiple spatial scales. It also indicates that neighboring nests rep- than in single-queen colonies. No queen was replaced in the resent independent colonies that do not exchange high num- 24 single-queen colonies surveyed. This ®nding is in agree- bers of workers. However, the absence of marked differen- ment with the common view that queen replacement is very tiation at nuclear markers does not necessarily mean that rare or absent in single-queen colonies of ants (Pamilo 1991b; queens from single- and multiple-queen colonies have iden- Bourke and Franks 1995). Moreover, the rarity of queen turn- tical dispersal strategies. Indeed, the slightly higher level of over in perennial nests of F. selysi suggests that queens have differentiation among sectors in multiple-queen colonies may long life spans that probably match the life span of the colony. indicate a somewhat more restricted dispersal in this social In line with this result, queen life spans greater than 20 years form, and the spatial clustering of nests with similar social have been recorded in Formica exsecta and in other monog- structure suggests that multiple-queen colonies often arise ynous populations of ants (Pamilo 1991b; Keller 1998). More from budding of nearby polygynous colonies. Even if queens surprisingly, the genetically effective queen turnover was from multiple-queen colonies found new colonies by budding also low in multiple-queen colonies, with a mean rate of only and only disperse over short distances, gene ¯ow through 2.5%. This low queen turnover stands in sharp contrast to males may be suf®cient to erase the genetic signature of estimates of genetically effective queen turnover in polyg- colony budding, as has been documented in Rhytidoponera ynous colonies of several species of Myrmica, which ranged metallica (Chapuisat and Crozier 2001) and Leptothorax ru- between 40% and 60% (SeppaÈ 1994; Evans 1996; Pedersen gatulus (RuÈppell et al. 2003). The hypothesis that female and Boomsma 1999a). In Leptothorax acervorum, a high turn- dispersal is restricted in multiple-queen colonies of F. selysi over of queens was also indicated by the elevated frequency might be tested by examining whether there is a much stron- of sexual progeny that was not the offspring of the extant ger local differentiation at mitochondrial markers than at nu- queen (Bourke et al. 1997). The surprisingly low rate of queen clear markers, as has been documented in several polygynous turnover in polygynous colonies of F. selysi suggests that ant populations (Ross 2001; RuÈppell et al. 2003). queens in this type of colony do also have long life spans. Overall, multiple-queen colonies in this population of F. If the number of queens is stable over time, an average queen selysi did not show clear signs of the shift in mating system turnover of 2.5% roughly corresponds to a mean queen life and genetic structuring that is usually associated with the span of 40 years. This estimate is sensitive to errors in the presence of multiple queens in other ant populations (Bourke estimate of queen turnover, and when we use the upper limit and Franks 1995; Ross 2001). Compared to single-queen col- of the 95% con®dence interval of the queen turnover the onies, multiple-queen colonies did not appear to have a higher queen life span estimate reduces to 5.2 years. This minimal level of inbreeding resulting from local mating, nor did they estimate is still higher than queen life span in six other species show signs of restricted dispersal of both sexes and isolation of polygynous ants, which was only 1.6 years on average by distance over small spatial scales. This result contrasts (Keller and Genoud 1997; Keller 1998). The similarly long with the frequent occurrence of local mating, population dif- life span of queens in single- and multiple-queen colonies of ferentiation, and isolation by distance in polygynous ant pop- F. selysi suggests that these queens have been subject to ulations from species belonging to the genera Formica (Pam- similar patterns of extrinsic mortality over evolutionary time, ilo 1982, 1983; SundstroÈm 1993; Chapuisat et al. 1997; Chap- in contrast to the sharp difference observed between monog- uisat and Keller 1999), Myrmica (SeppaÈ and Pamilo 1995; ynous and polygynous species (Keller and Genoud 1997). Pedersen and Boomsma 1999a,b; van der Hammen et al. The mating system of single- and multiple-queen colonies 2002) and Solenopsis (Ross et al. 1997, 1999). also appeared to be very similar. Both types of colonies Importantly, all these populations with pronounced shifts showed a small but signi®cant de®ciency of heterozygotes. in mating and dispersal strategies are predominantly com- This positive inbreeding coef®cient in the absence of micro- posed of colonies with high numbers of queens per nest. In geographical genetic structure suggests that a small propor- contrast, both single- and multiple-queen colonies coexist in tion of the queens mate with closely related males. For ex- the F. selysi population under study. Comparing the two types ample, an inbreeding coef®cient of 0.036 corresponds to ap- of systems strongly suggests that the shift in the mating sys- proximately 13% of sibling matings, given the relation F ϭ tem and genetic structure that is typically associated with D/(4 Ϫ 3D), where D is the frequency of sibling mating multiple queening does not appear when multiple-queen col- (Suzuki and Iwasa 1980). Our ®eld observations of mating onies ®rst emerge and still coexist with single-queen colo- behavior suggest that at least part of the males and females nies, but progressively develops in isolated populations con- mate close to the nest, which may result in mating among sisting mostly or exclusively of multiple-queen colonies. A relatives. We observed small numbers of males and females simple explanation for this pattern would be that ongoing ¯ying to the tops of pine trees close to their colonies. Males gene ¯ow between social forms prevents the emergence of performed patrol ¯ights around the trees and mating took distinct genetic structures when single and multiple-queen place on the trees. Numerous such mating sites occur within colonies exist in the same site. In the F. selysi population the study area (M. Chapuisat, pers. obs.). under study, periodic habitat perturbations by ¯oods may also SOCIAL STRUCTURE VARIATION IN AN ANT COLONY 1071 result in repeated events of extinction and recolonization of Chapuisat, M. 1996. Characterization of microsatellite loci in For- small habitat patches, which might prevent the building up mica lugubris B and their variability in other ant species. Mol. Ecol. 5:599±601. of divergent genetic structures in each social form. Chapuisat, M., and L. Keller. 1999. Extended family structure in There was no differentiation between single- and multiple- the ant Formica paralugubris: the role of the breeding system. queen colonies of F. selysi. Because the analysis involved Behav. Ecol. Sociobiol. 46:405±412. large numbers of nests and loci, the con®dence intervals of Chapuisat, M., and R. Crozier. 2001. Low relatedness among co- the estimator of differentiation are very small, which ensured operatively breeding workers of the greenhead ant Rhytidopo- nera metallica. J. Evol. Biol. 14:564±573. that even a slight amount of differentiation between the social Chapuisat, M., J. Goudet, and L. Keller. 1997. Microsatellites reveal forms would have been detected with high power. Two non- high population viscosity and limited dispersal in the ant For- mutually exclusive processes may explain this lack of dif- mica paralugubris. Evolution 51:475±482. ferentiation. First, the shifts between single- and multiple- Crozier, R. H., and P. Pamilo. 1996a. Evolution of social insect colonies: sex allocation and kin selection. Oxford Univ. Press, queen colonies may be too recent or too frequent for genetic Oxford, U.K. differentiation to have built up. Second, ongoing gene ¯ow ÐÐÐ. 1996b. Sympatric speciation: one into two will go. Nature between the social forms may be suf®cient to homogenize 383:574±575. allele frequencies at nuclear markers. Signi®cant gene ¯ow DeHeer, C. J., V. L. Backus, and J. M. Herbers. 2001. Sociogenetic between social forms is fully consistent with the fact that responses to ecological variation in the ant Myrmica punctiv- entris are context dependent. Behav. Ecol. Sociobiol. 49: ants in single- and multiple-queen colonies have similar mat- 375±386. ing systems, with low inbreeding coef®cients and no differ- Evans, J. D. 1996. Queen longevity, queen adoption, and posthu- entiation above the level of the nest. Moreover, even a small mous indirect ®tness in the facultatively polygynous ant Myrmica rate of migration between single- and multiple-queen colo- tahoensis. Behav. Ecol. Sociobiol. 39:275±284. nies may be suf®cient to prevent differentiation if the effec- Forel, A. 1920. Les fourmis de la Suisse. Imprimerie coopeÂrative, La Chaux-de-Fond, Switzerland. tive population size of each social form is high (Wright 1943). Goodnight, K. F., and D. C. Queller. 1999. Computer software for Whatever the causes of the lack of differentiation, our study performing likelihood tests of pedigree relationship using ge- does not support the hypothesis that changes in social struc- netic markers. Mol. Ecol. 8:1231±1234. ture lead to genetic differentiation and promote speciation Goropashnaya, A. V., P. SeppaÈ, and P. Pamilo. 2001. Social and when both social forms coexist in the same site. Signi®cant genetic characteristics of geographically isolated populations in the ant Formica cinerea. Mol. Ecol. 10:2807±2818. differentiation between social forms was found in Solenopsis GoÈsswald, K. 1989. Die Waldameise. Band 1. Biologische Grun- invicta. Interestingly, in this species, single- or multiple- dlagen, OÈ kologie und Verhalten. Aula-Verlag, Wiesbaden, Ger- queen colonies exist in distinct populations and the social many. structure is under a simple genetic control that restricts gene Gyllenstrand, N., P. J. Gertsch, and P. Pamilo. 2002. Polymorphic ¯ow between social forms (Shoemaker and Ross 1996; Ross microsatellite DNA markers in the ant Formica exsecta. Mol. Ecol. Notes 2:67±69. et al. 1999; Krieger and Ross 2002). The contrast between Hamilton, W. D. 1964. The genetical evolution of social behaviour. F. selysi and S. invicta suggests that two processes may lead II. J. Theor. Biol. 7:17±52. to genetic differentiation between social forms. First, genetic Heinze, J., and L. Keller. 2000. Alternative reproductive strategies: differentiation may build up over time once the populations a queen perspective in ants. Trends Ecol. Evol. 15:508±512. contain predominantly single- or multiple-queen colonies that Herbers, J. M. 1986. Nest site limitation and facultative polygyny have diverged in their mating and dispersing habits. Second, in the ant Leptothorax longispinosus. Behav. Ecol. Sociobiol. 19: 115±122. differentiation between single- and multiple-queen colonies ÐÐÐ. 1993. Ecological determinants of queen number in ants. may be due to special genetic mechanisms restricting gene Pp. 262±293 in L. Keller, ed. Queen number and sociality in ¯ow, such as green-beard alleles. Hence, differentiation be- insects. Oxford Univ. Press, Oxford, U.K. tween social forms seems unlikely if colony social structure Herbers, J. M., and V. S. Banschbach. 1999. Plasticity of social is a phenotypically plastic trait that varies within populations, organization in a forest ant species. Behav. Ecol. Sociobiol. 45: 451±465. with single- and multiple-queen colonies existing together. Hochberg, M. E., B. Sinervo, and S. P. Brown. 2003. Socially me- diated speciation. Evolution 57:154±158. ACKNOWLEDGMENTS HoÈlldobler, B., and E. O. Wilson. 1977. The number of queens: an important trait in ant evolution. Naturwissenschaften 64:8±15. We thank L. Degen and C. Peter for technical assistance, ÐÐÐ. 1990. The ants. Springer-Verlag, Berlin. N. Gyllenstrand for providing primers ahead of publication; Ingram, K. K. 2002. Plasticity in queen number and social structure J. Goudet for advice on statistics; and R. L. Hammond, L. in the invasive Argentine ant (Linepithema humile). Evolution Keller, J. S. Pedersen, and three anonymous reviewers for 56:2008±2016. Keller, I., and J. Zettel. 2001a. Contribution to the autoecology of comments on the manuscript. This study was supported by Formica selysi Bondroit, 1918 (, Formicidae) in a grants from the Swiss National Science Foundation (31± mature steppe and a newly created alluvial zone at Pfynwald 61934.00 to MC) and from the Herbette Foundation. (Switzerland): I. Feeding ecology. Mitt. Schweiz. Entomol. Ges. 74:183±193. ÐÐÐ. 2001b. Contribution to the autoecology of Formica selysi LITERATURE CITED Bondroit, 1918 (Hymenoptera, Formicidae) in a mature steppe Bourke, A. F. G., and N. R. Franks. 1995. Social evolution in ants. and a newly created alluvial zone at Pfynwald (Switzerland): II. Princeton Univ. Press, Princeton, NJ. Habitat use. Mitt. Schweiz. Entomol. Ges. 74:227±236. Bourke, A. F. G., H. A. A. Green, and M. W. Bruford. 1997. Par- Keller, L. 1995. Social life: the paradox of multiple-queen colonies. entage, reproductive skew and queen turnover in a multiple- Trends Ecol. Evol. 10:355±360. queen ant analysed with microsatellites. Proc. R. Soc. Lond. B ÐÐÐ. 1998. Queen lifespan and colony characteristics in ants and 264:277±283. termites. Insectes Soc. 45:235±246. 1072 MICHEL CHAPUISAT ET AL.

Keller, L., and M. Genoud. 1997. Extraordinary lifespans in ants: ÐÐÐ. 2001. Molecular ecology of social behaviour: analyses of a test of evolutionary theories of ageing. Nature 389:958±960. breeding systems and genetic structure. Mol. Ecol. 10:265±284. Keller, L., and K. G. Ross. 1998. Sel®sh genes: a green beard in Ross, K. G., and L. Keller. 1998. Genetic control of social orga- the red ®re ant. Nature 394:573±575. nization in an ant. Proc. Natl. Acad. Sci. USA 95:14232±14237. Keller, L., and M. Chapuisat. 2002. and cooperation. Ross, K. G., M. J. B. Krieger, D. D. Shoemaker, E. L. Vargo, and Encyclopedia of Life Sciences. Available at http://www.els.net. L. Keller. 1997. Hierarchical analysis of genetic structure in Nature Publishing Group, London. native ®re ant populations: results from three classes of molec- Krieger, M. J. B., and K. G. Ross. 2002. Identi®cation of a major ular markers. Genetics 147:643±655. gene regulating complex social behavior. Science 295:328±332. Ross, K. G., D. D. Shoemaker, M. J. B. Krieger, C. J. DeHeer, and Legendre, P., and M.-J. Fortin. 1989. Spatial pattern and ecological L. Keller. 1999. Assessing genetic structure with multiple classes of molecular markers: a case study involving the introduced ®re analysis. Vegetatio 80:107±138. ant Solenopsis invicta. Mol. Biol. Evol. 16:525±543. Lewis, P. O., and D. Zaykin. 1999. Genetic data analysis: computer RuÈppell, O., M. Stratz, B. Baier, and J. Heinze. 2003. Mitochondrial program for the analysis of allelic data. Version 1.0 (d12). Avail- markers in the ant Leptothorax rugatulus reveal the population able at http://lewis.eeb.uconn.edu/lewishome/software.html. genetic consequences of female philopatry at different hierar- Lude, A., M. Reich, and H. Plachter. 1999. Life strategies of ants chical levels. Mol. Ecol. 12:795±801. in unpredictable ¯oodplain habitats of Alpine rivers (Hymenop- SeppaÈ, P. 1994. Sociogenetic organization of the ants Myrmica rug- tera: Formicidae). Entomol. Gener. 24:75±91. inodis and Myrmica lobicornis: number, relatedness and longev- Nonacs, P. 1988. Queen number in colonies of social Hymenoptera ity of reproducing individuals. J. Evol. Biol. 7:71±95. as a kin-selected adaptation. Evolution 42:566±580. SeppaÈ, P., and P. Pamilo. 1995. Gene ¯ow and population viscosity Pamilo, P. 1982. Genetic population structure in polygynous For- in Myrmica ants. Heredity 74:200±209. mica ants. Heredity 48:95±106. SeppaÈ, P., L. SundstroÈm, and P. Punttila. 1995. Facultative polygyny ÐÐÐ. 1983. Genetic differentiation within subdivided popula- and habitat succession in boreal ants. Biol. J. Linn. Soc. 56: tions of Formica ants. Evolution 37:1010±1022. 533±551. ÐÐÐ. 1991a. Evolution of colony characteristics in social insects. Shoemaker, D. D., and K. G. Ross. 1996. Effects of social orga- II. Number of reproductive individuals. Am. Nat. 138:412±433. nization on gene ¯ow in the ®re ant Solenopsis invicta. Nature ÐÐÐ. 1991b. Life span of queens in the ant Formica exsecta. 383:613±616. Insectes Soc. 38:111±119. SundstroÈm, L. 1993. Genetical population structure and socioge- netic organisation in Formica truncorum. Behav. Ecol. Sociobiol. Pamilo, P., P. Gertsch, P. ThoreÂn, and P. SeppaÈ. 1997. Molecular 33:345±354. population genetics of social insects. Annu. Rev. Ecol. Syst. 28: SundstroÈm, L., L. Keller, and M. Chapuisat. 2003. Inbreeding and 1±25. sex-biased gene ¯ow in the ant Formica exsecta. Evolution 57: Pedersen, J. S., and J. J. Boomsma. 1999a. Effect of habitat satu- 1552±1561. ration on the number and turnover of queens in the polygynous Suzuki, Y., and Y. Iwasa. 1980. A sex ratio theory of gregarious ant, Myrmica sulcinodis. J. Evol. Biol. 12:903±917. parasitoids. Res. Popul. Ecol. 11:366±382. ÐÐÐ. 1999b. Genetic analysis of colony structure in polydomous van der Hammen, T., J. S. Pedersen, and J. J. Boomsma. 2002. and polygynous ant populations. Biol. J. Linn. Soc. 66:115±144. Convergent development of low-relatedness supercolonies in Queller, D. C., and K. F. Goodnight. 1989. Estimating relatedness Myrmica ants. Heredity 89:83±89. using genetic markers. Evolution 242:258±275. Weir, B. S. 1996. Genetic data analysis II. Sinauer Associates, Raymond, M., and F. Rousset. 1995. Genepop (version 1.2): pop- Sunderland, MA. ulation genetics software for exact tests and ecumenicism. J. Weir, B. S., and C. C. Cockerham. 1984. Estimating F-statistics for Hered. 86:248±249. the analysis of population structure. Evolution 38:1358±1370. Rosengren, R., L. SundstroÈm, and W. Fortelius. 1993. Monogyny West-Eberhard, M. J. 1986. Alternative adaptations, speciation, and and polygyny in Formica ants: the result of alternative dispersal phylogeny. Proc. Natl. Acad. Sci. USA 82:1388±1392. tactics. Pp. 308±333 in L. Keller, ed. Queen number and sociality ÐÐÐ. 1989. Phenotypic plasticity and the origins of diversity. in insects. Oxford Univ. Press, Oxford, U.K. Annu. Rev. Ecol. Syst. 20:249±278. Ross, K. G. 1997. Multilocus evolution in ®re ants: effects of se- Wright, S. 1943. Isolation by distance. Genetics 28:114±138. lection, gene ¯ow and recombination. Genetics 145:961±974. Corresponding Editor: R. Harrison