COEXISTENCE OF THE SOCIAL TYPES: GENETIC POPULATION STRUCTURE IN THE ANT FORMICA EXSECTA

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1 Evolution, 58(11), 2004, pp COEXISTENCE OF THE SOCIAL TYPES: GENETIC POPULATION STRUCTURE IN THE ANT FORMICA EXSECTA PERTTU SEPPÄ, 1,2,3,4 NICLAS GYLLENSTRAND, 1,5 JUKKA CORANDER, 6,7 AND PEKKA PAMILO 1,2,8 1 Department of Conservation Biology and Genetics, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18 D, SE Uppsala, Sweden 2 Department of Biology, Box 3000, FIN University of Oulu, Finland 3 Ecology and Evolutionary Biology Unit, Department of Biological and Environmental Sciences, Biocenter 3, PO Box 65, FIN University of Helsinki, Finland 4 perttu.seppa@helsinki.fi 5 niclas.gyllenstrand@ebc.uu.se 6 Department of Mathematics and Statistics, PO Box 68, FIN University of Helsinki, Finland 7 jukka.corander@helsinki.fi Abstract. The ant Formica exsecta has two types of colonies that exist in sympatry but usually as separate subpopulations: colonies with simple social organization and single queens (M type) or colonial networks with multiple queens (P type). We used both nuclear (DNA microsatellites) and mitochondrial markers to study the transition between the social types, and the contribution of males and females in gene flow within and between the types. Our results showed that the social types had different spatial genetic structures. The M subpopulations formed a fairly uniform population, whereas the P subpopulations were, on average, more differentiated from each other than from the nearby M subpopulations and could have been locally established from the M-type colonies, followed by philopatric behavior and restricted emigration of females. Thus, the relationship between the two social types resembles that of source (M type) and sink (P type) populations. The comparison of mitochondrial ( ST ) and nuclear (F ST ) differentiation indicates that the dispersal rate of males is four to five times larger than that of females both among the P-type subpopulations and between the social types. Our results suggest that evolution toward complex social organization can have an important effect on genetic population structure through changes in dispersal behavior associated with different sociogenetic organizations. Key words. Ants, DNA microsatellites, Formica, gene flow, inbreeding, mitochondrial DNA. Social evolution has brought about changes in the breeding and dispersal biology of individuals, which can lead to profound changes in the genetic structure of populations (e.g., Crozier and Pamilo 1996). The transition from solitary to social life fundamentally changes the spatial movement of individuals because they become dependent on their social group and in the case of social insects, even on the physical nest structure. The further transition from simple to more complex social organization in ants changes the dispersal behavior and colony-founding strategy of females, often making them philopatric. As a consequence, population genetic structure in biparentally (nuclear) and uniparentally (e.g., mtdna) inherited parts of the genome become different (Avise 1994; Prugnolle and de Meeus 2002). Therefore it is essential to use both nuclear and mitochondrial markers to explore how the transition between the social forms takes place and how complex social organization affects genetic population structure and to obtain a complete picture of gene flow patterns. In ants, a suite of social and behavioral characters divides social organization into two basic types (Rosengren and Pamilo 1983; Keller 1991). In the M type, colonies are simple single-queen families (monogyny), which inhabit a single nest site (monodomy). Females usually take part in nuptial flights and afterward found new nests independently or by temporarily parasitizing other nests. P-type colonies are genetically more complex with multiple queens (polygyny) and 8 Present address: Department of Biology, University of Oulu, Finland; pekka.pamilo@oulu.fi The Society for the Study of Evolution. All rights reserved. Received May 10, Accepted August 23, colonies may be spread to more than one nest site (polydomy). Females in the P type are commonly recruited back to their natal nests as new queens (secondary polygyny, Hölldobler and Wilson 1977) and new nests are also often founded dependently by budding, that is, by formation of daughter nests close to the parental one (Rosengren and Pamilo 1983; Keller 1991; Ross and Keller 1995). The association between the social type and female dispersal leads to different expectations about both spatial distribution of genetic variation and breeding system (e.g., Seppä and Pamilo 1995; Ross 2001). A prototype of the M-type dispersal is a nuptial flight, where both sexes gather from large areas to mate (Hölldobler and Wilson 1990). This should promote strong gene flow and uniform distribution of allele frequencies in large areas. Mating by relatives is highly unlikely in this system, and no inbreeding is typically found (Crozier and Pamilo 1996). In contrast, secondary polygyny and dependent colony founding by budding in the P type is expected to restrict gene flow and increase genetic differentiation. In the P type, females may also mate locally with nestmates or with males from adjacent nests (e.g., Formica paralugubris, Chapuisat and Keller 1999). This mating system increases inbreeding and also promotes geographical differentiation, otherwise dispersal of males tends to homogenize the nuclear gene pool even when females are philopatric. In this work, we studied the genetic composition of populations in the socially polymorphic ant Formica exsecta, which has proved a fruitful species for studying various aspects of social evolution (e.g., Sundström et al. 1996; Brown

2 GENETIC POPULATION STRUCTURE IN FORMICA 2463 FIG. 1. Map of the study areas. In Uppsala the light areas represent farmland systems; white, water; darker, forests and urban areas. In Åland the light areas represent water bodies. and Keller 2000, 2002; Liautard and Keller 2001). Earlier studies have found isolation by distance within the P type populations (Pamilo and Rosengren 1984) and inbreeding in the M type (Pamilo and Rosengren 1984; Sundström et al. 2003), the latter being a rare example in monogynous ants. Furthermore, Liautard and Keller (2001) detected strong mitochondrial structuring, and Sundström et al. (2003) weak nuclear genetic structure among local P and M populations, respectively. Here we present a study of a mixed population system, where M- and P-type subpopulations exist in sympatry, and we use both nuclear and mitochondrial markers to assess the genetic population structure and to infer the patterns of gene flow. We addressed several questions, which all explore the consequences of female philopatry on the genetic population structure. First, is gene flow sex biased as suggested in some other ants (Doums et al. 2002; Gyllenstrand and Seppä 2003; Rüppel et al. 2003)? Second, is genetic differentiation stronger among P than M subpopulations as could be expected if female philopatry leads to strong genetic differentiation, especially in mitochondrial genes, while male dispersal could promote gene flow in both social types? Third, are the two social types differentiated from each other when sympatric? The history and relationships of the sympatric colonies representing different social types can be studied by assessing the spatial distribution of allele frequencies, both differences between populations and variation within them. Finally, do the breeding systems differ between the two social forms? Earlier studies have found inbreeding in the M-type populations of F. exsecta (Pamilo and Rosengren 1984; Sundström et al. 2003) and some isolation by distance within the P-type populations (Pamilo and Rosengren 1984). A comparison of the two social forms in the same locality gives an opportunity to examine to what extent the breeding system is specific to the social form. MATERIALS AND METHODS Sampling Worker ants were sampled during from two localities, Uppsala in east-central Sweden (10 sites) and the Åland Islands between Sweden and Finland (11 sites; Fig. 1). The sampling sites are here called subpopulations. Some of them had a dense population confined to small habitat patches with a radius ranging from a few to tens of meters, while nests at other sites were more scattered in continuous habitats. In Uppsala, most sites were located within the same open farmland system, whereas in Åland the landscape was more heterogeneous with forests and water bodies separating sampling sites. The distance between the sampling sites ranged from 0.2 to 9 km in Uppsala and from 2 to 23 km in Åland, and the average distances between the sites were 3.6 and 11 km, respectively (Fig. 1). Both localities had one large aggregation with several hundred nests in a small area, a subset of which was sampled (38 nests in Bågskyttebana, Uppsala; 55 nests in Saltvik, Åland). All the nests (two to eight) were sampled at all the other sites. The total number of nests sampled was 80 in Uppsala and 103 in Åland (Table 1). Laboratory Methods We genotyped five workers from each nest with three to six microsatellite primer pairs: FL20, FL21, and FL29 (Chapuisat 1996) and FE13, FE19, and FE49 (Gyllenstrand et al. 2002). We analyzed the Uppsala samples first, and only the three most variable microsatellites (FL21, FE13, FE49) were used for the Åland samples. DNA was extracted using a Chelex-100 protocol (Thorén et al. 1995), microsatellites were amplified in a radioactive polymerase chain reaction (PCR) by incorporating -P 33 into the PCR products, which were

3 2464 PERTTU SEPPÄ ET AL. TABLE 1. Characteristics of DNA microsatellite variation in the study populations. n is the sample size (number of nests); N A is the total number of alleles found and H E is mean expected heterozygosity (both means of 100 resampled datasets, note that N A and H E are not comparable between Uppsala and Åland because they are based on six and three loci, respectively; Bomarsund [*] had too many missing data, and the values in the table are actual values of N A and H E ); r SE is worker nestmate relatedness, r* is inbreeding corrected relatedness, N Q is average effective number of queens per nest, F IS is inbreeding coefficient, and F ST is the mean of the pairwise F ST -estimates for the focal population (both means of 100 resampled datasets). The figures in parentheses (X 1,X 2 ) in column F IS are the percentages showing a significant deviation from random mating in 10,000 randomization tests. The first figure (X 1 ) shows the deficiency of heterozygotes (F IS 0), and the second figure (X 2 ) the excess of heterozygotes (F IS 0) according to Hardy-Weinberg equilibrium. n N A H E r SE r* N Q F IS (X 1,X 2 ) F ST Uppsala M populations Flogsta (41, 0) 0.01 HN (11, 0) 0.06 Hågaby (0, 0) 0.06 Uppsala Näs (7, 0) 0.03 P populations Herrhagen (4, 1) 0.06 HN (8, 2) 0.09 Pustanäs (0, 10) 0.14 HN (1, 1) 0.06 HD (0, 1) 0.07 Bågskyttebana (8, 1) 0.07 Åland Bomarsund 5 19* 0.66* (3, 0) 0.11 Åsgårda (7, 0) 0.07 Gölby (36, 0) 0.10 Vivastby (11, 0) 0.05 Bamböle (22, 0) 0.07 Mösundet (34, 0) 0.06 Norrö (0, 0) 0.06 Tjudö (0, 0) 0.16 Nybygget (0, 0) 0.07 Främmanby (0, 0) 0.15 Saltvik (2, 5) 0.08 then separated in 6% denaturing sequencing gels and visualized by autoradiography. We assessed mtdna variation from one to three workers from each nest (total number of individuals 211 in Uppsala, 228 in Åland) by using single-stranded conformational polymorphism (SSCP) method (Orita et al. 1989). Primers were designed from known F. exsecta cytochrome b sequences. The primers used were ExsU2 (5 -ACATACCACAGGCTC ATCAAAC-3 ) and ExsL2 (5 -GGTTGAATATGGATAGG GGTTACTA-5 ), and the sequence corresponds to bases in the Apis mellifera mitochondrial genome NC (Crozier and Crozier 1993). Amplified fragments were separated on 6% nondenaturing PAA gels and visualized by silverstaining using standard protocol (Bassam et al. 1991). The resulting haplotypes were sequenced using primers CB1 and CB2 (Crozier and Crozier 1992). PCR products were purified using the Wizard PCR Prep kit (Promega, Madison, WI) according to manufacturer s instructions. Direct sequencing of the purified double-stranded PCR product was performed using the T7 Sequencing kit (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Sequences were visualized using autoradiography. Data Analysis Social organization We assigned the subpopulations to M and P types based on genetic data. We inferred the number of queens in each nest directly from the worker genotypes (one vs. many) and assessed the average effective queen number within each subpopulation based on genetic relatedness among worker nestmates. Relatedness was estimated following Queller and Goodnight (1989) by using software Relatedness 4.2 (Goodnight 1994). In the analysis, nests were weighted equally, and the estimates were jackknifed over nests to obtain standard errors. If relatedness between nestmate queens equals that among workers (r w w ) and males breeding in the same colony are not related, the effective mean number of queens per colony (N Q ) is: 2 1 r M w w e NQ (1) 3r w w (Pamilo 1993; Seppä 1994), where M e is the effective queen mating frequency. Formica exsecta females have been estimated to mate one to four times with M e 1.26 (calculated as harmonic mean from the data of Sundström et al. 1996). Genetic population structure The estimates of genetic variability are strongly influenced by the sample size. To remove this effect, we estimated microsatellite variability resampling the subpopulations by us-

4 GENETIC POPULATION STRUCTURE IN FORMICA 2465 ing the smallest sample sizes from any subpopulation (three nests in Uppsala and two in Åland). Resampling was done 100 times and the estimates of the number of alleles (N ALL ) and the expected heterozygosity (H E ) were calculated as the means (Table 1). The Bomarsund subpopulation had too many missing data, and it was dropped from the analyses concerning the amount of genetic variability. Haplotype diversity (h) was estimated from mitochondrial data by using Arlequin (Schneider et al. 2000). Statistical tests are affected by relatedness of workers. Therefore, we created new datasets by resampling one individual from each nest for 100 times. These data were used to test the independence of the microsatellite loci employed (linkage equilibrium) and the departure of subpopulations from Hardy-Weinberg equilibrium (by randomizing alleles among individuals 10,000 times) as well as to estimate inbreeding coefficients (F IS ). These analysis were made by using Fstat (Goudet 2001). Resampled data were also used to estimate the amount of nuclear (F ST ) and mitochondrial differentiation ( ST ) following Weir and Cockerham (1984). Differentiation was first calculated as pairwise estimates between all subpopulations, between subpopulations belonging to the same social type, and between subpopulations belonging to the different social types. The overall estimates for each category were obtained by taking then the mean of the pairwise estimates, with standard errors calculated by jackknifing over loci (F ST ) or subpopulations ( ST ). We also calculated the average differentiation of each subpopulation from all other subpopulations by taking the mean of all pairwise estimates involving the focal population. Isolation by distance was estimated by regressing the pairwise F ST and ST values and (ln) geographic distances using the Mantel test (10,000 permutations) as implemented in Genepop 3.3 (Raymond and Rousset 1995). Finally, we studied details of population substructuring with a Bayesian clustering analysis (software Baps 2.0; Corander et al. 2003, 2004). The method is conditioned on the geographical sampling information about the preassigned groups of individuals and estimates posterior probabilities for all different combinations of subpopulations using either complete enumeration (no. subpopulations 10) or Markov Chain Monte Carlo simulation (no. subpopulations 10; Corander et al. 2003, 2004). We conducted the analysis at both nest and subpopulation levels, that is, by giving either nest or subpopulation identification as priors. RESULTS TABLE 2. Nucleotide differences of the haplotypes detected in 210- bp fragment of cytochrome b. Haplotype Position A G C T A T B A C T A T C G C T A C D G T T A C E A C T T T F G C C A T Molecular Variation The total number of alleles ranged from two to 25 per locus. The three loci used in both localities had a total of 47 alleles in Uppsala and 55 in Åland. The estimates obtained by resampling and equalizing the sample sizes showed the total number of alleles over loci ranging from 13.5 to 18.3 per subpopulation in Uppsala and from 7.1 to 10.4 for the three loci in Åland (Table 1). The heterozygosity ranged from 0.46 to 0.63 in Uppsala and from 0.48 to 0.67 in Åland (Table 1). We found six mitochondrial haplotypes, with a total of five variable nucleotide sites (four transitions, one transversion; Table 2). The number of haplotypes per subpopulation ranged from one to six (Table 3). Only 0.4% of all pairs of microsatellite loci showed a significant linkage disequilibrium (5% level) when tested on resampled datasets, separately in each subpopulation. These were randomly distributed among pairs of loci but concentrated on the two largest subpopulations. The overall linkage disequilibrium across loci was never significant. Thus, all the microsatellite loci seemed to segregate independently and were kept in the statistical analyses. Assignment of Populations to the Social Types Subpopulations were assigned to M and P types (Table 1) based on the number of queens inferred directly from the genotypic arrays of workers (one vs. many), the average effective queen number per nest as estimated from worker relatedness (eq. 1), and the distribution of nests in the field. In Uppsala, subpopulations consisted almost invariably of one social type. The effective queen number was close to one in two subpopulations (Flogsta, HN2), and another two subpopulations (Hågaby, Uppsala Näs) had an effective queen number of 1.3 per nest (Table 1). A single (sometimes) multiply mated queen could explain worker genotypes in all but one of these nests, and they were classified as M type (Table 1). In contrast, when the effective queen number was high (N Q 3.3), the subpopulations were classified as P type (Table 1). The worker genotypes within the nests indicated multiple queens, and only three of 63 nests could be explained with a single queen (requiring three to five matings in these cases). The level of polygyny within the P subpopulations most likely varied among nests and subpopulations, but it is useful to classify them as a single category as the difference from the M type was clear and distinct. Thus, four subpopulations were assigned to M and six to P type in Uppsala, with clear differences in the estimates of relatedness and effective queen number. Furthermore, the nests in the P subpopulations nests were often aggregated suggesting polydomy In Åland, classifying the subpopulations as M or P was not clear. Some subpopulations had a low effective queen number (N Q 1) with worker genotypes always compatible with a single queen, and only the large Saltvik subpopulation could be assigned to the P type without any doubt (N Q 16.9, multiple coexisting haplotypes). Other subpopulations showed intermediate effective queen numbers (Table 1), and the worker genotypes suggested both single and multiple queen nests. So, the subpopulations on Åland were not as-

5 2466 PERTTU SEPPÄ ET AL. TABLE 3. Characteristics of mitochondrial variation in the study populations. n is the sample size (number of nests); h is haplotype diversity, and ST is the mean of the pairwise ST -estimates for the focal population (mean of 100 resampled datasets). Letters in the haplotype column indicate the haplotypes (A F) detected in a nest (one to three individuals per nest) and the multiplications show the number of nests with identical haplotype compositions. n h ST Haplotypes Uppsala M populations Flogsta *A; B; F HN *A Hågaby A; B; C Uppsala Näs *AAA; CCC; DDD P populations Herrhagen AAA; 3*ACC; F HN *AAA; BBB; 2*CCC Pustnäs *AAA HN *AAA; AAF HD CDD; 2*DDD Bågskyttebana ADD; DD; 35*DDD Åland Bomarsund AAA; 2*CCC; 2*DDD Åsgårda *AAA; 2*CCC; FFF Gölby CCC; 2*DDD Vivastby *AAA; 4*CCC Bamböle *BBB; 2*CCC; DDD Mösundet *CCC; CCD; DDD Norrö CC; 4*CCC; 2*DDD Tjudö CC; CCC Nybygget CCC; DDD Främmanby *AAA 4*AAA; BEE; CCC; CDE; CFF; Saltvik DD; 8*DDD; 4*DDE; 2*DEE; E; EE; 3*EEE; FFF signed to either social type in our analyses, and the estimate of genetic relatedness was used as a continuous variable to indicate differences in the social organization. Distribution of Molecular Variation in the Study Populations Within the subpopulations, haplotype diversity and microsatellite heterozygosity were positively correlated both in Uppsala (r S 0.64, P 0.047, n 10) and in Åland (r S 0.60, P 0.065, n 10), although the latter correlation was not significant. The levels of diversity within the subpopulations were generally not associated to the social type. The comparison of the M- and P-type subpopulations in Uppsala showed no association between the social type and diversity (Mann-Whitney, U 3.0, P for microsatellites; U 6.5, P 0.24 for mtdna), although the former test was nearly significant for diversity being larger in M than P type. Similarly, the diversity was not associated with the worker relatedness in Åland (r S 0.22, P 0.53 for microsatellites; r S 0.28, P 0.40 for mtdna). Inbreeding coefficients (F IS ) were highly variable across subpopulations, largely because of small sample sizes (Table 1). Ten percent of all tests across loci showed a significant signal. F IS in subpopulations with low worker nestmate relatedness (P type) were relatively close to zero and gave only a few significant signals, with both excesses and deficiencies of heterozygotes. Subpopulations with high relatedness (M type) had generally larger F IS, they gave more often a significant signal, always with F IS 0. As a result, there was a strong positive correlation between relatedness and inbreeding (combined data, r P 0.60, P 0.006, n 19; the subpopulations Gölby and Tjudö inåland had extreme inbreeding values [Table 1], probably because of small sample sizes, and were excluded from this analysis). Spatial Genetic Structure The overall means of F ST and ST showed significant genetic differentiation of subpopulations in both localities (Table 4). Mitochondrial differentiation exceeded nuclear differentiation as expected on the basis of the smaller effective population size. The ratio ST /F ST calculated from the means of pairwise estimates in Uppsala was 4.7 both for all subpopulations and for the P-type subpopulations separately, and 4.3 when calculated between the pairs of M and P subpopulations. Negative estimates of F ST did not allow meaningful calculation of this ratio for the M subpopulations. In Åland the ratio ST /F ST was 1.5 for all subpopulations. In Uppsala, the differentiation was significantly greater among the P subpopulations than among the M subpopulations both in nuclear (Table 4, t 3.86, P 0.005, df 8) and mitochondrial markers (Table 4; t 3.73, P 0.006, df 8). The same trend was seen when calculating separately the mean difference of each subpopulation from the rest (Tables 1, 3). The amount of differentiation was negatively associated with worker relatedness in both nuclear (r S 0.70, P 0.025, n 10) and mitochondrial markers (r S 0.62, P 0.054, n 10), although the latter correlation was not quite significant. The level of differentiation between the P

6 GENETIC POPULATION STRUCTURE IN FORMICA 2467 TABLE 4. Estimates of genetic differentiation (F ST, ST ) and the interpretation as male (N m m m ) and female (N f m f ) gene flow between subpopulations. Values in the table are means and standard errors (jackknifed over loci for F ST, over subpopulations for ST ) from 100 resamplings, n is the number of populations in each category. n.a. refers to values that cannot be calculated with the model used (e.g., negative estimates). F ST SE ST N f m f N m m m (N m m m )/(N f m f ) Uppsala All populations, n M populations, n n.a. n.a. P populations, n Between social types Åland All populations, n n.a. n.a. and M type subpopulations was intermediate compared to differentiation within the social types (Table 4). In Åland, there was no association between the amount of differentiation and worker relatedness (r S 0.13, P 0.71 for nuclear markers; r S 0.49, P 0.12 for haplotypes, n 11). There was no association between pairwise genetic distance and geographical separation of the populations in either nuclear or mitochondrial markers in either area (Mantel test, all P-values 0.17). Thus, the observed genetic differentiation cannot be explained as isolation by distance, but is due to differences in the dispersal patterns of the two social types rather than simple proximity. The Bayesian clustering analysis (Baps, Corander et al. 2003, 2004) was made at two hierarchical levels, at the subpopulation level (with complete enumeration) and at the nest level (MCMC simulations, first 100,000 burn-in iterations followed by 500,000 iterations in the actual analysis). At the subpopulation level, all subpopulations were clustered separately in both study areas (P in both areas 1.0). At the nest level, subpopulation structure in Uppsala with the highest probability (P 0.82) generally pooled nests from the same P subpopulations together, but nests from the same M populations separately (Table 5). Nests in P subpopulations usually clustering together suggests relative genetic homogeneity of these subpopulations, and consequently, differentiation of P subpopulations from each other. In Åland, the population structure could not be resolved reliably at the nest level (highest P 0.02), probably because data were available only from three loci. TABLE 5. Clustering of individual nests based on the Bayesian clustering analysis (BAPS), the probability of this clustering P Cluster Nests 1 Bågskyttebana: 1 38; HD1: HN4: Pustnäs: HN1: Herrhagen: HN2: 1, 5 7 HN2: 3, Following nests separately: Flogsta: 1 4; HN2: 2; Hågaby: 1 3; Uppsala Näs: 1 5; Herrhagen: 1 DISCUSSION Spatial Genetic Structure: The Effect of Complex Social Organization The picture emerging from both the F ST and ST estimates and the Bayesian clustering in Uppsala shows that the M- type subpopulations formed a fairly uniform population, whereas the P-type subpopulations were well differentiated (Tables 4, 5). Moreover, the P subpopulations were, on average, more differentiated from each other than from the sympatric M subpopulations. This suggests that the P-type subpopulations could be locally established from M-type colonies, followed by philopatric behavior and restricted emigration. Thus, the relationship between the two social types resembles that of source (M type) and sink (P type) populations (Dias 1996). This refers to the female dispersal behavior only, as the P populations could build a large population and dominate a habitat patch. Even weak emigration from such a population would cause a marked immigration pressure in the neighborhood. It should also be noted that not all P-type subpopulations were different from each other. The two subpopulations in Uppsala with the highest level of polygyny (HD1, Bågskyttebana) clustered together based on both microsatellites and haplotypic composition (Table 5). These subpopulations were separated by less than 200 m; one of them could have acted as a source for the other one, or both of them could be remnants of a larger continuous population. Our results are in line with the earlier studies on spatial genetic structure on socially polymorphic ants, including F. exsecta. The studies on F. exsecta have shown low nuclear differentiation between local M subpopulations (F ST 0.09, Pamilo 1983; F ST 0.00, Sundström et al. 2003) and strong mitochondrial differentiation between local P subpopulations ( ST 0.72, Liautard and Keller 2001). Nuclear markers have also shown stronger spatial differentiation in the P type than in the M type within the species Formica truncorum (Sundström 1993) and in a pair of closely related species of Myrmica ants (Seppä and Pamilo 1995). The present study is the first attempt to analyze a situation where both social types occur in sympatry and to distinguish the contributions of males and females in the gene flow. The results show that the social types have different spatial genetic structures, and that the difference was due to restricted female dispersal in the P type (Table 4). Previously, much stronger differenti-

7 2468 PERTTU SEPPÄ ET AL. ation has been shown in mitochondrial compared to nuclear markers among local P populations in several ant species (Ross and Shoemaker 1997; Ross et al. 1997; Doums et al. 2002; Gyllenstrand and Seppä 2003; Rüppel et al. 2003). The use of both nuclear and mitochondrial markers allows the separation of gene flow by males and females. Mitochondrial differentiation is expected to exceed nuclear differentiation because of smaller effective population size in the mitochondrial genome, with the ratio ST /F ST 3 for small values of differentiation and equal dispersal of males and females (see formulae below). Thus, ratios larger than three indicate female philopatry. The mean pairwise estimates between the subpopulations in Uppsala showed the ratio ST / F ST to be 4.7 among the P-type subpopulations and 4.3 between M and P. ST was particularly high in the comparisons including the populations Bågskyttebana and HD1, which were almost fixed for a unique haplotype. An attempt to evaluate the relative strength of gene flow by males and females can be made by applying Wright s (1951) island model as modified for mitochondrial (Ennos 1994) and haplodiploid (Berg et al. 1998) markers. The geographical scale in our study was such that the subpopulations are within possible dispersal distances, but it is likely that gene flow does not follow the strict assumptions of the island model and that the extinction-colonization dynamics also influence the genetic pattern as well. The usefulness of the island model has been questioned recently because of its shortcomings (e.g., Whitlock and McCauley 1999), but it still indicates the relative level of gene flow by the two sexes. At drift-migration equilibrium, the number of female immigrants in a population is expected to be related to ST as N f m f (1 ST )/(2 ST ) (Ennos 1994), where m f is the immigration rate of females and N f the number of females breeding in a population. Similarly, the haplodiploid model has at equilibrium the relationship for nuclear markers as N e m e (1 F ST )/(4F ST ). Noting that m e (2m f m m )/3 (Berg et al. 1998) and N e (9N f N m )/(2N f 4N m ) (Wright 1969), and assuming that N f N m,wegetn m m m [(1 F ST )/(2F ST )] [(1 ST )/ ST ]. Because we were interested in estimating the relative contributions of the two sexes in gene flow, we calculated the ratio: N m m m /N f m f [ ST (1 F ST )]/[F ST (1 ST )] 2 (Table 4). This ratio is the same as for diploid organisms (Ennos 1994). This exercise confirms the results already seen from the F ST and ST estimates. Females in the P-type subpopulations are philopatric, and the dispersal rate of males is generally several times larger than that of females in both types (Table 4). We should also note that the value of N f m f among M-type subpopulations is larger than the effective number of females within the subpopulations, which means that it is not a proper estimate of immigrants but simply shows that females disperse widely. Furthermore, mitochondrial markers indicated for some cases (M type in Uppsala; all subpopulations in Åland) a level of female gene flow that alone could explain the spatial homogeneity of nuclear variation. This means that no meaningful value for putative male gene flow could be calculated. Coexistence of the Two Social Types Large P-type aggregations had some rare or unique haplotypes with high frequencies (Table 3), showing that female gene flow from these P-type subpopulations to M type must be rare. Bågskyttebana in Uppsala was almost fixed for the haplotype D (109 of 110 individuals), which was otherwise only found in a nearby P-type subpopulation HD1 (eight of nine individuals) and in one M nest in the whole Uppsala area. Likewise, the haplotype E was unique for Saltvik subpopulation in Åland, with a frequency 27%. Based on the number of nests and the level of polygyny, these two subpopulations included a large majority of reproductive queens in their respective localities. Female dispersal out from these aggregations must be rare or nonexistent, because any female gene flow out from them would spread the unique haplotypes to other subpopulations. It seems likely that the most important difference in social and genetic organization concerning dispersal is not between polygynous (including weakly polygynous) and monogynous societies but between the large supercolonies and the others. Sixteen percent of P nests contained more than one mtdna haplotype, which means that immigrating females have been accepted to the existing P nests at some point. This has been found in some other polygynous ants as well (e.g., Leptothorax acervorum, Stille and Stille 1992, 1993; Iridomyrmex purpureus, Carew et al. 1997), and polygynous Formica colonies have also been shown to readily accept alien queens (Fortelius et al. 1993; Brown et al. 2003). If the immigrating females dispersed after mating, they would also carry the sperm from their mates to the new population, resulting in nuclear gene flow in the same way as if both sexes would disperse before mating. In that sense, female dispersal would also promote the spread of male genes. However, the ratio ST /F ST in Uppsala suggests that gene flow among the P subpopulations and between M and P is carried by males in excess to that by females, so active male dispersal must also occur. It is not possible to infer confidently the direction of male dispersal from the available data. However, the sex ratio in P type subpopulations is generally strongly male biased (Pamilo and Rosengren 1984; Brown and Keller 2002), and the P-type colonies of F. exsecta commonly produce small micraner males that are supposedly good dispersers (Fortelius et al. 1987). These suggest that gene flow by males is mainly from P to M. The relationship between the two social types has some similarities but also differences when compared to the wellstudied system of the introduced populations of the fire ant, Solenopsis invicta (Ross and Shoemaker 1997; Ross et al. 1997, 1999). Both F. exsecta and S. invicta show strong mitochondrial but weak nuclear differentiation among the P- type populations, and weak differentiation in both markers among the M-type populations, which indicates restricted gene flow by females in the P type. The comparison of the levels of differentiation indicates somewhat stronger male gene flow in S. invicta than in F. exsecta in general, and in the P type in particular. The pattern of gene flow between the social types is different in the two species. There is very little female gene flow between the M and P types of S. invicta, because the incompatible phenotype of M females in the locus Gp9 (always BB) prevents their acceptance to P nests and because P females who are not accepted back to P nests only seldom succeed in independent nest founding (Shoemaker and Ross

8 GENETIC POPULATION STRUCTURE IN FORMICA ; Ross and Shoemaker 1997; Keller and Ross 1998; DeHeer et al. 1999). This results in strong differentiation between the social types. In F. exsecta, however, differentiation between the social types was smaller than among the P-type subpopulations. Another difference concerns male gene flow. Male gene flow from P to M is severely limited in S. invicta, because the P nests produce mainly sterile diploid males (e.g., Ross and Shoemaker 1993; Shoemaker and Ross 1996). Possible gene flow between the social types in S. invicta is thus largely restricted to M males mating with P females, with about 80% of P females mating with males from nearby M populations (Ross and Shoemaker 1993; Ross and Keller 1995; Ross 1997), even though a theoretical model including gene flow and selection shows that the genotype frequencies at the Gp9 can be best explained without such gene flow (Goodisman et al. 2000). Previous studies of F. exsecta have found elevated inbreeding in the M-type (Pamilo and Rosengren 1984; Sundström et al. 2003) and random mating in the P-type subpopulations (Pamilo and Rosengren 1984). Our present results showed a similar pattern. Because inbreeding inflates the relatedness estimates as relatedness of nestmate workers depends both on the number of reproductive individuals and the relatedness among the reproductives, we removed the component caused by inbreeding using the approach of Pamilo (1985). These new values (Table 1) did not qualitatively change the assignment of subpopulations to their social type, and the correlation between inbreeding and the remaining relatedness stayed significant (P 0.046). The inbreeding pattern can be explained with alternative dispersal strategies (Sundström et al. 2003) associated with male size polymorphism (Fortelius et al. 1987). The M-type colonies normally produce large macraner males that are considered to be poor dispersers and to specialize in local matings (Pamilo and Rosengren 1983, 1984; Fortelius et al. 1987). In the M type, at least part of the females seem to forego long-range dispersal and start new nests independently in the vicinity of their natal nest, resulting in population viscosity (Sundström et al. 2003). If their mating partners are macraner males from adjacent M nests, the outcome would then be elevated inbreeding. In the P type, female dispersal is also restricted to short range (Liautard and Keller 2001, this work). These females mate either with local males or males dispersing from other populations, but the large effective queen number in the P nests should ensure that close relatives rarely mate. Development of Complex Social Organization In socially polymorphic Formica species, one social type normally strongly dominates within a locality, as observed in F. exsecta (Pamilo and Rosengren 1984; Liautard and Keller 2001; this work), F. truncorum (Sundström 1993; Seppä et al. 1995), F. lugubris (Pamilo et al. 1994; Gyllenstrand and Seppä 2003), and F. cinerea (Goropashnaya et al. 2001; Zhu et al. 2003) but not in F. selysi (Chapuisat et al. 2004). Such purity suggests that the transition between the social types is generally fast. Several hypotheses have been put forward to explain which factors direct the development of a colony and trigger the transition from simple to a more complex social type. First, the two types could represent historically different evolutionary lineages (Wilson 1971). In our study, the local separation of M- and P-type subpopulations can be rather old and stable as shown clearly by the large differences in the distribution of mtdna haplotypes. In both study localities, the largest P-type subpopulations had haplotypes that were rarely or never seen in the neighboring subpopulations. These large nest aggregations must be fairly old, but there is no evidence suggesting that the two social types would represent different evolutionary lineages. Each mtdna haplotype could be connected to at least one other haplotype by a single nucleotide change, and the differences did not suggest any long-term historical separation (Table 2). The average nuclear and mitochondrial differentiation between the social types was lower than among the P-type subpopulations, and the M- and P-type subpopulations shared haplotypes. These are straightforward indications of gene flow between the types. Other hypotheses explaining the development of complex social organization include the local increase of queen number when genetic variation is reduced during a population bottleneck (Tsutsui et al. 2000), perhaps associated with selection (Giraud et al. 2002); incompatibility of M and P types, associated with a single Mendelian locus (e.g., Ross and Keller 1998); and ecological constraints in the form of habitat saturation in stable habitats (e.g., Hölldobler and Wilson 1977; Rosengren and Pamilo 1983), but our results allow only speculation on these. For instance, the relationships between the M and P subpopulations with gene flow to both directions make it unlikely that the difference would be determined by a single locus, as in S. invicta. Finally, to what extent the social types are determined by intrinsic factors (e.g., possible decrease in the diversity of kin recognition cues in the P type) and by extrinsic factors (characteristics of the habitat) remains to be explored. 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