Genetic differentiation between the ant Myrmica rubra and its ...

Insect. Soc. (2009) 56:425–437 DOI 10.1007/s00040-009-0042-0

Insectes Sociaux

RESEARCH ARTICLE

Genetic differentiation between the ant Myrmica rubra and its microgynous social parasite K. Vepsa¨laïnen Æ J. R. Ebsen Æ R. Savolainen Æ J. J. Boomsma

Received: 19 March 2009 / Revised: 23 August 2009 / Accepted: 1 September 2009 / Published online: 25 September 2009 Ó Birkhaüser Verlag, Basel/Switzerland 2009

Abstract Hymenopteran inquiline species have been proposed to originate by sympatric speciation through intraspecific social parasitism. One such parasite, Myrmica microrubra, was recently synonymized with its Myrmica rubra host, because comparisons across Europe indicated insufficient genetic differentiation. Here, we use microsatellite markers to study genetic differentiation more precisely in a sample of Finnish M. rubra and its inquilines collected at two localities, supplemented with mitochondrial DNA sequences. The parasite had much lower genetic variation than the host at three of the four loci studied. Genetic differentiation between the host populations was moderate (FST = 0.089), whereas the parasite populations were more strongly subdivided (FST = 0.440). The host and parasite were highly genetically differentiated both across populations (FST = 0.346) and in strict sympatry (0.327, 0.364), a result that remained robust both in a haplotype network and in PCA ordination. Individual assignments of genotypes indicated that gene flow between sympatric host and inquiline populations is reduced by

K. Vepsa¨laïnen and J. R. Ebsen contributed equally. K. Vepsa¨laïnen (&) R. Savolainen Department of Biological and Environmental Sciences, University of Helsinki, P. O. Box 65, 00014 Helsinki, Finland e-mail: [email protected] R. Savolainen e-mail: [email protected] J. R. Ebsen J. J. Boomsma Department of Biology, Centre for Social Evolution, University of Copenhagen, Copenhagen, Denmark e-mail: [email protected] J. J. Boomsma e-mail: [email protected]

about an order of magnitude relative to the gene flow within the morphs. Our results suggest that the parasitic morph of M. rubra may be an incipient species, but it remains unclear to what extent the observed genetic differentiation between host and inquiline is due to possible assortative mating and selection against hybrids or to recurrent bottlenecking and genetic drift. We conclude that an explicitly functional species concept would be unambiguous in treating this inquiline as a full species, as it begets its own kind and maintains its integrity in spite of occasional interbreeding with the host. Keywords Inquiline Microsatellites mtDNA Myrmica microrubra Sympatric speciation

Introduction Many species or even lineages of social insects, in particular of ants, practice some form of social parasitism. They are unable to maintain independent societies and rely instead on exploiting the colonies of other ant species (Ho¨lldobler and Wilson, 1990). The evolution of social parasitism has puzzled naturalists since Darwin (1859), who suggested that the enslavement of Formica fusca and its relatives by F. sanguinea might have evolved as a fortuitous outcome of predatory raiding of pupae. This general hypothesis for the evolution of slavemaking is still valid today, but it took half a century more until Emery (1909) and Wasmann (1909) suggested that close consanguinity is a key factor to facilitate the evolution of social parasitism. This notion, which became known as Emery’s rule, is plausible because relatives tend to be similar in morphology, behaviour, cuticular chemistry, larval development and colony life cycle. More recent empirical studies have

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given considerable support to Emery’s rule in ants, bumblebees and wasps (Wilson, 1971; Pedersen, 1996; Ward, 1996; Carpenter et al., 1993; Schultz et al., 1998; Sanetra and Buschinger, 2000; Smith et al., 2007). Usually the data are consistent with the social parasites being congeneric with or the sister genus of their hosts, which came to be known as the loose version of Emery’s rule. Some examples of the strict version of the rule (that host and parasite are sister species) have, however, also come to light (Savolainen and Vepsa¨laïnen, 2003; Sumner et al., 2004). It has been repeatedly suggested that obligate, usually workerless, social parasites (i.e., inquilines) of ants may evolve through stages of intraspecific parasitism (Wasmann, 1909; Elmes, 1978; Buschinger, 1990; Bourke and Franks, 1991). Ernst Mayr (1993), who was renowned for his scepticism toward sympatric speciation, has stated that ‘‘Up to now the best evidence for sympatric speciation is provided by ants’’, and continued: ‘‘In the few cases that have been well described, the inquiline species seems to be most closely related to the host species and there is no mode of geographic speciation that could produce such a result.’’ The phylogenetic study by Savolainen and Vepsa¨laïnen (2003) has implied that one of the known inquiline–host pairs in European Myrmica ants may be an example of incipient sympatric speciation via intraspecific social parasitism. They found different gradations of social parasitism across the genus, with the inquilines M. karavajevi and M. hirsuta unquestionably being good species with complete lineage sorting of mtDNA sequences, but two samples of M. microrubra being polyphyletic and most similar to their respective M. rubra hosts in England and Finland. This suggested that sympatric gene flow between hosts and parasites may exceed allopatric gene flow among parasite populations, a result consistent with earlier allozyme analyses by Pearson and Child (1980). It remained, however, unclear whether gene flow is substantial enough for M. microrubra to be merely an intraspecific inquiline form of M. rubra without much evolutionary differentiation, or whether gene flow is restricted enough for M. microrubra to be an incipient species with highly viscous populations (Savolainen and Vepsa¨laïnen, 2003). Recently a larger data set of mtDNA sequences across north and central Europe collected by Steiner et al. (2006) confirmed the limited differentiation between host and parasite populations shown by Savolainen and Vepsa¨laïnen (2003) and led the authors to synonymize M. microrubra with M. rubra. To shed more detailed light on the evolutionary status of M. rubra and its inquiline, and on the suitability of these ants for studying mechanisms of sympatric speciation, we estimated gene flow between the inquiline parasite and its host with both nuclear microsatellite and mitochondrial

DNA (mtDNA) markers. This approach gives a better resolution than the allozyme study by Pearson and Child (1980) and more direct insight into the dynamics of reproductive isolation than the approach followed by Steiner et al. (2006). To allow a simultaneous analysis of sympatric and allopatric gene flow, we sampled hosts and inquilines from two distinct localities. We used our samples and genetic data to test three alternative hypotheses: (1) Host and inquiline share the same gene pool locally, but differences between sympatric host and inquiline populations are small relative to differences between allopatric populations of both host and inquiline, indicating that all populations belong to a single randomly mating species. (2) Host and inquiline are completely reproductively isolated, as we would expect for two distinct non-hybridizing species. (3) Sympatric hosts and inquilines form distinct populations with limited gene flow between them. We provide multiple lines of evidence in favour of the third hypothesis and discuss whether this differentiation can be explained by recurrent population bottlenecks and genetic drift on an ecological time scale, or whether at least some evolutionary divergence between the inquiline and its host has taken place.

Materials and methods Nomenclature and ant samples Among myrmecologists, the social-parasitic morph of M. rubra has been known as the microgyne (small queen) form, whereas the host with larger queens has been referred to as the macrogyne (normal) form (Elmes, 1991). After Seifert (1993) raised the microgyne morph to the species level, the name M. microrubra became rapidly established, despite doubts about its justification among many specialists. The recent degradation of the inquiline’s taxonomic status by Steiner et al. (2006) implies that in the text below we shall let ‘‘inquiline’’ stand for Seifert’s M. microrubra (i.e., the microgyne morph of M. rubra), and ‘‘host’’ for the macrogyne morph of M. rubra. We collected M. rubra hosts and their inquilines from mixed nests in two populations (Tva¨rminne and Viikki) in southern Finland, separated by 110 km. The 13 sampled nests in the Tva¨rminne population were at least 10 m apart and occurred under moss or in grass tussocks in an area of ca. 5,000 m2 covered with dense, relatively young birch and alder forest. The 14 sampled nests in the Viikki population occurred likewise under moss or in tussocks, but also between the roots of trees in patches of open birch forests. The Viikki nests came from three sites (P, M, L) approximately 0.9, 1.6 and 2.1 km away from each other, and isolated by water and extensive reed beds. In two of

Gene flow between an inquiline and its host

these localities, we sampled multiple nests from areas of 10 9 100 m2 (P) and 20 9 100 m2 (M), whereas only a single nest was sampled at locality L (in between P and M). We collected host workers, winged inquiline females (gynes) and males. The workers were not as ideal as samples of macrogyne queens would have been, but we preferred this solution over having too few or no samples at all, as large queens were rarely observed in numbers. The complication of having to use worker samples is that inquilines may, under field conditions, occasionally produce workers (Pearson, 1981). We tried to minimize possible bias by selecting the largest workers for genetic analysis, although the documented size difference between the inquiline and host queens (Elmes, 1976; Seifert, 1993) need not apply to workers. All ant samples were stored in 99.5% ethanol for genetic analyses. The genotyped individuals of this study are kept in the private collection of R. Savolainen (University of Helsinki, Finland). Microsatellite markers For each nest we extracted DNA from the forelegs of five inquiline gynes and five host workers using a 5% Chelex solution (Walsh et al., 1991). Likewise, we extracted DNA from 33 males from Tva¨rminne and 16 males from Viikki. The ants were kept as vouchers. We tested 13 published microsatellite primer sets developed for M. tahoensis (Evans, 1993), M. punctiventris (Herbers and Mouser, 1998) and M. scabrinodis (Heinrich et al., 2003; Zeisset et al., 2005) on both morphs of M. rubra and found that four of them were sufficiently variable: Myrt4 (Evans, 1993), MP67 (Herbers and Mouser, 1998), Msca50 (Heinrich et al., 2003) and Msc7 (Zeisset et al., 2005). We carried out PCRs in 26 ll volumes containing 0.5 lM of each primer, 100 lM of each dNTP, 2.5 mM MgCl2, 19 GeneAmp PCR Gold buffer (15 mM Tris–HCl, pH 8.0, 50 mM KCl; Applied Biosystems), 0.75 units of AmpliTaq Gold (Applied Biosystems) and 2 ll DNA. The PCR program conditions were: initial denaturing at 95°C for 5 min followed by 30–35 cycles of denaturing at 95°C for 40 s, annealing at 50°C (MP67, Msca50, Msc7) and 60°C (Myrt4) for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 15 min. We visualized and sized the PCR products on an Applied Biosystem 377 DNA sequencer using the internal size-standard (ROX 500 Genesize) and subsequently analysed the data with GENESCAN v. 3.1 and GENOTYPER v. 2.0. Mitochondrial DNA We extracted mitochondrial DNA from one M. rubra worker and one inquiline female per nest, using the Nucleo-Spin Tissue kit (Macherey-Nagel). These were a

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subset of the individuals genotyped for microsatellite markers, except for two inquilines from Tva¨rminne and one from Viikki. We sequenced part of the cytochrome c oxidase subunit I (COI) gene using the primers LCO1490 and HCO2198 (Folmer et al., 1994), and the cytochrome b (Cyt b) gene with the primers CB1, CB2 (Jermiin and Crozier, 1994), CB7 and tRs (Tay et al., 1997) following the methods of Savolainen and Vepsa¨laïnen (2003). We compiled and edited the sequences in Sequencer 4.7 (Gene Codes), and deposited them in Genbank under accession numbers: GQ872348–GQ872401 (COI) and GQ872402–GQ872417 (Cyt b). We constructed haplotype networks using the statistical criterion of 95% parsimony (Templeton et al., 1992) as implemented in TCS 1.21 (Clement et al., 2000). Hardy–Weinberg equilibrium and linkage disequilibrium tests, and F-statistics For statistical analyses, we initially subdivided the data according to the most likely a priori population structure consisting of four subsets (populations), one for each site and one for each ant morph. Unless stated otherwise in subsequent elaborations, we will refer to this default subdivision when listing population-specific averages. We used the program MICRO-CHECKER (van Oosterhout et al., 2004) to evaluate the level of allelic scoring errors. We specifically evaluated the presence of null alleles, large allele drop-outs and misinterpretations of stutter bands for each locus by comparing the observed and expected heterozygosities and the size differences between allelic combinations within individuals. We used ARLEQUIN v. 3.11 (Excoffier et al., 2005) to test our tentative populations for Hardy–Weinberg (H–W) equilibrium and linkage disequilibrium, and to calculate the F-statistics. However, when estimates of 95% confidence intervals (CI) for FST values were needed, we used GENETIX v. 4.05 (Belkhir et al., 1996–2004) with 1,000 bootstrap replicates per locus. Finally, GENODIVE (Meirmans and Van Tienderen, 2004) provided the standardised measures of population differentiation estimated by AMOVA (Meirmans, 2006). The measure gives FST values relative to the maximum possible values for the observed amount of within-population diversity (Hedrick, 2005). After doing the basic F-statistics, we did two nested AMOVAs: (1) the two morphs nested within each of the two localities, and (2) the two localities nested within each of the morphs. For exact H–W equilibrium tests, the number of steps in the Markov chain was 2,000,000, and the number of dememorization steps 100,000. For the linkage disequilibrium tests, the number of permutations was 16,000, and the number of initial conditions for the Expectation–Maximization algorithm for multi-locus data (when the gametic phase is not known) was 10. For the


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analyses of molecular variance, the number of permutations was 20,000. Principal component and model-based clustering analyses To investigate multivariate patterns of population structure, we did Principal Component Analyses (PCA) with the program PCAGEN v. 1.2.1. (Goudet, 1999), based on the nest samples of five individuals per ant morph. We ran 1,000 iterations to estimate P values for the proportion of explained variation. In ARLEQUIN, we first did a simple assignment of genotypes to populations by determining the log-likelihood of each individual multi-locus genotype in each population sample, assuming that the individual comes from that population (Excoffier et al., 2005). We then investigated the levels of admixture between the two ant morphs with the model-based clustering methods STRUCTURE v. 2.1. (Pritchard et al., 2000) and BAPS v. 3.1 (Corander et al., 2003). We applied STRUCTURE only to the individuallevel data, but used BAPS at two levels of analysis, one inferring the population-level clusters from the nest-sample data and another to (re)assign individuals to the inferred populations. Both programs estimate population structure through Bayesian inference, implemented by simulations with a Markov Chain Monte-Carlo (MCMC) algorithm (STRUCTURE) or a stochastic optimisation algorithm (BAPS). STRUCTURE estimates the K most likely partitions of the data under assumptions of H–W equilibrium within partitions and no linkage disequilibrium among the genetic markers. We ran several MCMC simulations with varying values for K (the number of populations) to find the value of K that gave the highest posterior probability. To estimate population structure from the overall data, we ran simulations for 500,000 (burn-in 100,000) generations for both models with K varying from 1 to 9 for the entire dataset. We did the runs thrice for each value of K to check the consistency of the estimated probabilities. The Admixture model in STRUCTURE allows exchange among partitions, whereas the No-Admixture model assigns each individual to one partition only. As our simulations under these two models gave similar results, we only report the results of the Admixture model. We estimated admixture coefficients Q between inquiline and host separately for the Tva¨rminne and Viikki data, to avoid effects of isolation by distance and to simplify the population structure for a better estimate of Q. We used BABS to estimate the total number of ‘real’ populations (based on simulation runs on the total data set), i.e. to investigate overall population structure and to obtain an estimate of the admixture coefficient Q between the two morphs in Tva¨rminne and Viikki.

Finally, we evaluated gene flow between the inquiline and host by comparing the admixture coefficients Q obtained from the STRUCTURE simulations with the number of populations (K) fixed at two for Tva¨rminne and four for Viikki. We also used BAPS to analyse admixture at the two sites independently, because BAPS finds the optimal number of populations and can thus be used to check the inferred number of populations used in STRUCTURE. The output files from both programs supplied an individual admixture matrix for each inferred cluster. STRUCTURE also gives a population matrix comparing Q between the original samples and the inferred clusters. We calculated equivalent population matrices from the BAPS output as the average individual admixture within each original sample for each inferred cluster. To compare the overall individual admixtures (Q) of the inquiline and host, we pooled the four Viikki clusters from the BAPS and STRUCTURE analyses into two joint clusters representing host and parasite.

Results Evaluation of scoring errors in microsatellite allele size Analyses of scoring errors implied the possibility of null alleles at some loci, but the H–W analyses (Table 1) and linkage disequilibrium tests (below) suggested that the deviations from H–W and linkage equilibria were more likely due to some inbreeding and population substructuring than to scoring errors. Because the STRUCTURE analysis indicated that there were two populations of both inquiline and host in Viikki, we tested for linkage disequilibrium across the sites within the a priori defined Viikki population (i.e. M vs. P ? L). After sequential Bonferroni adjustment (Rice, 1989) of the P values (n = 36), linkage disequilibrium was detected only between Myrt4 and Mp67 (P = 0.004). We further found H–W equilibrium or heterozygote deficiency for all loci, except for Msca50 that showed an excess of heterozygotes in both host populations. Msca50 segregated only for two alleles, and its overall fixation index was low (FST = 0.050) relative to the other loci (FST = 0.235– 0.375); moreover, its population-level inbreeding coefficient FIS was -0.254 (P = 1.000) and the overall inbreeding coefficient FIT 0.191 (P = 1.000), whereas for the other loci the coefficients varied from 0.198 to 0.433, and 0.497–0.566, respectively (for all, P = 0.000). Because of the deviating resolution and statistics obtained for Msca50 we report the results for the F-statistics both with and without locus Msca50. Another reason for singling out the Msca50 locus is that the two alleles were separated by only a single tandem repeat, so that the


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reading of banding patterns for this locus was relatively more error-prone than for the other loci. Allelic diversity and differentiation between inquiline and host The inquiline had fewer alleles than the host in both populations for three of the four loci, which was clearly reflected in the allelic richness and heterozygosity statistics (Fig. 1). All pairwise statistical tests for allelic and genotypic differentiation between the two morphs in Tva¨rminne and Viikki (details not shown) indicated significant differentiation between the host and inquiline, and basic F-statistics showed substantial population substructuring and moderate inbreeding. For the four populations pooled, the fixation index FST was 0.251 (95% CI min/max = 0.100/0.353; 4-loci data) and 0.305 (0.235/0.375; 3 loci, without Msca50), and the inbreeding coefficient FIS was 0.157 (-0.127/0.386) and 0.306 (0.198/0.437), respectively. Population-specific FST values (for the 4 a priori defined populations, see ‘‘Materials and methods’’) ranged from 0.250 to 0.256 (4 loci) and 0.304–0.311 (3 loci) (for all, P = 0.000), and population-specific FIS values from 0.127 to 0.182 (4 loci) and 0.257–0.535 (3 loci); all inbreeding coefficients differed statistically significantly from zero (P values 0.000–0.024). Nested AMOVA, partitioning the F-statistics among four levels (Table 2) with localities on the highest level— i.e., with the structure {Tva¨rminne inquilines, Tva¨rminne hosts} {Viikki inquilines, Viikki hosts}—indicated substantial differentiation between the inquiline and the host populations (3-loci FSC = 0.346); the localities (each with an inquiline and a host population in the next level of analysis) showed no differentiation. Grouping by morphs— i.e., {Tva¨rminne inquilines, Viikki inquilines} {Tva¨rminne hosts, Viikki hosts}—also indicated differentiation between the inquiline and the host (3-loci FCT = 0.184), and between the localities (FSC = 0.200) for the inquilines or hosts. To further clarify the sources of differentiation, we calculated pairwise F-statistics (Table 3). The inquiline and the host appeared to be strongly differentiated both

in Tva¨rminne and in Viikki (FST = 0.327 and 0.364, respectively), whereas the two populations of the host were less differentiated (FST = 0.089). Notably, the largest fixation index (FST = 0.440) was between the two inquiline populations. The results based on all four loci were similar to those in Table 3, but the values were systematically smaller than for the three loci, probably because the fourth locus provides very little resolution. The assignment of genotypes to populations with ARLEQUIN moved 32 individuals (12%) from their source population to another one. Nine were assigned from one host population to another and 11 from one inquiline population to the other. Between the inquiline and the host, 12 individuals were assigned to another population than their source: 11 host individuals to the inquiline, but only 1 inquiline to the host. The results were identical for the fourand three-loci assignments, so we proceeded to the more detailed analyses (see below) with four loci data only (unless otherwise stated). Principal component analyses confirmed the differentiation between the four putative populations. Pairwise comparisons in each locality separated the inquiline and host along the first PCA axis, which explained 53.7% of the variation in Tva¨rminne and 62.5% in Viikki. A similar analysis over all samples clustered the inquiline nests by locality, whereas the host samples from Viikki split into two groups, of which one overlapped with the Tva¨rminne samples (Fig. 2). The Viikki population was substructured such that the colonies in the L and P patches were clearly separated from those in the M patch. The deep substructuring of the Viikki populations was also seen in the fixation indices (3 loci): the smallest index was between the hosts in the M versus (P ? L) sites, FST = 0.261 (95% CI = 0.101–0.510) (Table 3) and the largest between the inquiline in the M and the host in the (P ? L) sites, FST = 0.602 (95% CI = 0.586–0.611). As anticipated from the population structure estimates based on FST and PCA, the model-based clustering methods gave K values larger than our tentative a priori subdivision between two localities with a host and inquiline population each. STRUCTURE simulations for the total data produced the highest posterior probability for

Table 1 Tests for Hardy–Weinberg proportions per locus within populations Population

Myrt4

Mp67

Msca50

Msc7

FIS

Tinquiline

HW (0.2)

DEF (\0.001)

HW (0.8)

HW (1)

0.143 (0.024)

Thost

DEF (\0.001)

DEF (\0.01)

EXC (\0.005)

DEF (\0.001)

0.179 (\0.001)

Vinquiline

HW (1)

–

HW (0.4)

DEF (\0.001)

0.182 (0.024)

Vhost

DEF (\0.001)

DEF (\0.001)

EXC (\0.002)

HW (\0.05)

0.127 (0.004)

HW No significant deviation from Hardy–Weinberg equilibrium, DEF heterozygote deficiency, EXC heterozygote excess, – not tested (the locus had one allele only). T and V refer to the Tva¨rminne and Viikki populations, respectively. FIS is the population-specific inbreeding coefficient. P values are given in brackets

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Fig. 1 Allele frequencies of the inquiline (upper panel) and the host (lower panel) in Tva¨rminne (black) and Viikki (grey); note different scales on the y-axes. N Number of alleles in each of the four loci

(Tva¨rminne/Viikki), AR average allelic richness (rarefied to the size of the smallest sample), Ho observed heterozygosity and He expected heterozygosity

K = 6. The same analysis with BAPS resulted in seven clusters, with the same overall population structure as that obtained with STRUCTURE, except that a new cluster was added by merging one host nest from Tva¨rminne with another one from Viikki. Both analyses resulted in one cluster per host and one per inquiline in Tva¨rminne, but indicated substructuring of the Viikki population with two host and two inquiline clusters, consistent with the F-statistics (Table 2) and the PCA analysis (Fig. 2), thus confirming the stronger spatial substructuring in Viikki. The model-based admixture

coefficients in STRUCTURE were larger for the host than for the inquiline (Q = 0.114 and 0.195 for the host, and 0.032 and 0.069 for the inquiline in Tva¨rminne and Viikki, respectively), indicating that some workers were produced by inquilines (see also below). Assigning individuals to inquiline and host gene pools The individual assignments by STRUCTURE (Fig. 3) confirmed the above conclusions. In Tva¨rminne the inquiline population was genetically distinct from its host.


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Table 2 F-statistics based on nested AMOVA of four a priori defined populations, i.e. for two sites with two populations each; the results of three loci are without locus Msca50 Hierarchical level

Grouping by locality

Grouping by morph

Four loci

Three loci

Four loci

Three loci

FCT

-0.081 NS

–0.095 NS

0.159**

0.184*

FSC

0.289***

0.346***

0.156***

0.200***

FIS

0.154***

0.303***

0.157***

0.306***

FIT

0.350***

0.501***

0.402***

0.547***

p

p

pp

p m m m m

l

t

t

t

The grouping towards the left has inquiline and host populations nested within each of the sites, whereas the grouping towards the right has sites nested within both inquiline and host. FCT is the betweengroup variance component, FSC the between-populations within groups component, FIS the among-individuals within populations component, and FIT the overall among-individuals term. NS Not significant, P = 0.67, * P = 0.04, ** P = 0.01, *** P = 0.000

The Viikki pattern was Tva¨rminne the inquiline population was genetically distinct from its host. The Viikki pattern was more complicated with both host and inquiline populations being spatially subdivided between sites (P ? L vs. M). We appeared to have misassigned more individuals as host (i.e., workers that had inquiline genes) than as inquiline (a single microgyne, morphologically inquiline queen, but genetically a host), which is consistent with the inquiline producing some workers that cannot be morphologically distinguished from host workers. Admixture analyses (Fig. 4) indicated that in Viikki only 6 of 70 host phenotypes were genetically ambiguous (only one of them significantly). In Tva¨rminne 13 of 65 host phenotypes were genetically ambiguous, 8 significantly ambiguous. In Tva¨rminne, 8 of the 33 genotyped males were heterozygous for one or more loci, whereas in Viikki only 1 of 16 males was heterozygous. Diploid males are anomalies that normally arise after inbreeding increases the likelihood of homozygosity at the sex-determining locus. As diploid

M M T T

T P

T T

M

T

T

T

t M

M

M

t

T

L

P

t t

T

P

t

t M

T

P P

M

t t T t

T

Fig. 2 Ordination by the first two Principal Components of inquiline (lower case) and host (capital letters) genotypes (averages per nest) in Tva¨rminne and Viikki. T/t Tva¨rminne, M/m Viikki site M, L/l Viikki site L, P/p Viikki site P. Inquiline groups are marked with ellipses, except for the single nest from the L patch (lower case l)

males were meant to be females, we included the heterozygous males from Tva¨rminne in a STRUCTURE simulation to evaluate which gene pool they belonged to. Seven of these males were assigned to the inquiline population (Q = 0.796–0.975) and one to the host (Q = 0.65). Mitochondrial DNA haplotypes We obtained part of the COI gene (642 bp) of all parasite– host pairs in both populations. For the Cyt b gene (774 bp) we managed to sequence only eight parasite–host pairs from Viikki. For these, we combined the two gene fragments into one sequence (1,416 bp). We found six haplotypes in COI (Fig. 5a). Two common haplotypes were shared between parasites and their hosts, whereas four haplotypes were morph-specific (2 of them by default as they were only observed once). We also found six haplotypes in the combined gene fragments

Table 3 Population pairwise F-statistics with 95% confidence intervals in parentheses based on three microsatellite loci (Msca50 excluded); upper panel: T = Tva¨rminne, V = Viikki; lower panel: M = Maÿra¨metsa¨ subpopulation in Viikki, P = Pornaistenniemi subpopulation in Viikki (including the single nest in Lammassaari, between M and P patches) Population

FST

FIS

FIT

Tinquiline/Thost

0.327 (0.239–0.510)

0.299 (0.215–0.615)

0.528 (0.402–0.811)

Vinquiline/Vhost

0.364 (0.101–0.548)

0.314 (0.182–0.491)

0.564 (0.392–0.630)

Tinquiline/Vinquiline

0.440 (0.084–0.594)

0.356 (0.189–0.536)

0.639 (0.502–0.671)

Thost/Vhost Minquiline/Mhost

0.089 (0.015–0.136) 0.494 (0.034–0.593)

0.292 (0.201–0.395) 0.031 (-0.162–0.655)

0.355 (0.319–0.404) 0.509 (0.222–0.667)

Pinquiline/Phost

0.430 (-0.004–0.572)

0.261 (0.105–0.377)

0.579 (0.375–0.666)

Minquiline/Pinquiline

0.498 (-0.007–0.529)

0.306 (-0.000–0.346)

0.652 (-0.007–0.692)

Mhost/Phost

0.261 (0.101–0.510)

0.143 (0.110–0.227)

0.367 (0.199–0.621)

The standardized FST values, which give FST values relative to the maximum possible values for the observed amount of within-population diversity (Hedrick, 2005), for the four main pairs of populations were almost equal to the non-standardized ones: 0.322, 0.361, 0.433 and 0.087


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Admixture coefficient Q

Tvärminne (K = 2) 1.00

0.50

0.00

Admixture coefficient Q

Viikki (K = 4) M

L

P

L

M

P

1.00

0.50

0.00

Inquiline

Host

Fig. 3 Individual admixture coefficients (Q) obtained from the STRUCTURE simulations, with each bar representing a single individual; the first 50% of the individuals are inquiline gynes, the rest workers. Colours distinguish the subgroups (K) identified by the program. Upper panel Tva¨rminne (K = 2), with individuals assigned

to the host gene pool (red) and individuals assigned to the inquiline (green). Lower panel Viikki (K = 4), with individuals assigned to the host in sites P and L (orange) and site M (yellow) and to the inquiline in sites P and L (light blue) and site M (darker blue)

Fig. 4 STRUCTURE admixture plots for Tva¨rminne (upper panel) and Viikki (lower panel) assuming two gene pools per site (K = 2). The x-axis ranks the individuals according to their Q-score, which represents the proportion of each individual’s genotype inferred to be derived from the inquiline (1) rather than the host (0). Open circles

indicate workers (putative hosts), filled circles gynes (inquilines). Small x letters denote 90% confidence limits and the central rectangle delineates the individuals whose confidence limits overlapped with both 0 and 1 so that they could not be assigned

(Fig. 5b). Of these, only one haplotype was shared between the parasite and its host (nest 4 in Viikki). The small-scale microgeographic separation (L, M, P) is also visible in Fig. 5b.

Discussion In accordance with earlier studies using nuclear markers (Pearson and Child, 1980; Steiner et al., 2006) and mtDNA


Fig. 5 Statistical parsimony network of COI sequence data in both populations (a) and combined COI and Cyt b sequence data in the Viikki population (b). Circles and squares refer to samples from Tva¨rminne and Viikki, respectively, and black and white to inquilines and host (assuming all workers were produced by host), respectively. The numbers refer to nest numbers and the letters L, M, P to subpopulations (see text for details). Each line in between small circles or bigger boxes indicates one mutational step

sequences (Savolainen and Vepsa¨laïnen, 2003; Steiner et al., 2006), our present results rejected the hypothesis of free interbreeding between the M. rubra host and its inquiline (for hypotheses, see ‘‘Introduction’’). Also the hypothesis of complete reproductive isolation between the two morphs is not supported. To prove reproductive isolation among sympatric gene pools, one would need at least a single diagnostic allele at one of the marker loci, i.e., an allele that is present in one group and absent in the other, with homozygotes and heterozygotes at approximate HW proportions in the group where the allele is present. This criterion has been successfully used to identify three cryptic ant species (Boomsma et al., 1990; Schultz et al., 1998, 2002), but would require large sample sizes to avoid under-sampling of rare alleles in controversial cases such as the present one. Also our allele frequency data (Fig. 1) do not warrant the conclusion that the host and inquiline are distinct species with complete reproductive isolation. All assignment tests, including the STRUCTURE analyses (Figs 3, 4), indicated at least some gene flow between host and inquiline in both localities, although these figures may be overestimates as workers cannot be morphologically assigned to morph (Pearson, 1981; Seifert, 2007). Also the large fixation indices (FST [ 0.30; Table 3) between the morphs—twice as high as those obtained by Steiner et al. (2006) on a larger geographical scale—indicate substantial local genetic differentiation between the inquiline and the host, supporting the hypothesis of partial reproductive isolation between the morphs. Likewise, the

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PCA ordination (Fig. 2) placed the inquilines and hosts in separate clusters, and the same is true for ordination analysis of cuticular hydrocarbon profiles of the two morphs (Savolainen R., Akino T., Vepsa¨laïnen K., Punttila P., in prep.). The mtDNA data also indicate substantial differentiation between the morphs and the localities (Fig. 5). The smallscale spatial differentiation was especially clear in the most differentiated Viikki population for which we had longer sequences (Fig. 5b) and where the inquiline and host pairs shared the same haplotype in only one nest. Interestingly, this was the only parasitized nest observed to produce microgyne and macrogyne alates simultaneously, although the inquiline queens are known to usually inhibit the production of sexual offspring by host queens (Elmes, 1976; Pearson, 1981). The separate haplotype lineages of inquiline and host in Viikki suggest that mitochondrial introgression between morphs is low relative to nuclear gene introgression, as was shown earlier for two hybridizing waterstrider (Gerridae) species (Abe et al., 2005), although larger mtDNA samples are needed to support this inference statistically. Overall, it remains unclear how much of the inferred gene flow is due to coancestry of the inquiline and its host, and how much reflects actual gene flow in the recent generations. Thus, the observed genetic differentiation among the morphs may reflect long-term reproductive isolation, founder effects, recent population expansion—or assortative mating, suggested by Seifert (2007) to be common in the inquiline—or, most likely, a combination of several of these factors. Species status of the inquiline morph of Myrmica rubra Traditionally, the biological species concept (Mayr, 1963) maintains that species are entities that do not interbreed. Lack of interbreeding has, however, relatively rarely been tested, and when tested, established species pairs have often violated the reproductive isolation criterion. In fact, many traditionally good species have been shown to interbreed and to have substantial introgression without loosing their integrity as species (Sˇvarc, 1969; Arnold, 1997; Machado and Hey, 2003). Being a proper species is therefore not primarily a matter of lack of interbreeding and gene flow, but one of being a morphologically, physiologically, behaviourally and ecologically distinct entity. In the above functional view of species, it rather depends on the species concept applied (e.g., Sˇvarc, 1969; Van Valen, 1976; Templeton, 1989; Howard and Berlocher, 1998; Wheeler and Meier, 2000; Hey, 2001), whether one considers the inquiline of M. rubra to be only a social form of its host, an incipient species, or a fully established species. Applying traditional criteria, the

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marked genetic differentiation between the M. rubra inquiline and host, as quantified by the respective FST values (0.327 and 0.364) in Tva¨rminne and Viikki, exceeds the differentiation between sympatric races of the pea aphid Acyrthosiphon pisum (FST = 0.21; Via, 1999) and is comparable to the gall moth Gnorimoschema gallae (FST = 0.16; Nason et al., 2002), both considered good candidates for incipient species. Moreover, despite ongoing limited gene flow between inquiline and host, another inquiline ant using Ectatomma tuberculatum as host was recently assessed to be a distinct species, based on population-level genetic differentiation between the morphs (microsatellite FST = 0.45; Hora et al., 2005), similar to what we report in the present study. Finally, the wood ants Formica rufa and F. polyctena, which are considered good ‘‘biological’’ species (but see Vepsa¨laïnen and Pisarski, 1981), show relatively weak genetic differentiation (pairwise FST = 0.101–0.179) based on reduced gene flow between populations on a scale of B50 km (Gyllenstrand et al., 2004). A problem with genetic divergence estimates lacking an adequate time window is, however, the difficulty to tell apart long-term, evolutionary differences from differences reflecting gene flow and population dynamics at a shorter ecological time scale and often a smaller spatial scale. In the following, we will further evaluate our results in a more explicitly ecological context. Possible effects of dispersal and colonization on genetic differentiation Seppa¨ and Pamilo (1995) used allozyme markers to compare the population structures of two closely related Myrmica species, M. rubra and M. ruginodis. The populations of M. ruginodis were much less differentiated than those of M. rubra that had an order of magnitude larger fixation indices, i.e., FST & 0.20, both within and among localities (the maximum respective distances between populations for these two species was one and ca. 600 km). Nash et al. (2008) obtained similar results for the same two Myrmica species for Danish populations that were [100 km apart (M. rubra FST = 0.136, M. ruginodis FST = 0.004). Our estimates for M. rubra host populations both between the Tva¨rminne and Viikki populations (FST = 0.089) separated by 110 km, and between the two Viikki subpopulations that were 1–2 km apart (FST = 0.261) are thus in good agreement with these earlier studies. The highly viscous populations that are typical for the host morph of M. rubra suggest that local populations are founded by one or very few individuals. Strong population viscosity and population differentiation are landmarks of the polygyne–polycalic (P–P) life history syndrome of ants that combines poor dispersal ability with long-lived


supercolonies that monopolize entire habitat patches after successful colonization. Many ant species have evolved this life history as an (often plastic) alternative to the monogyne–monocalic (M–M) life history characterized by efficient dispersal but less enduring occupation of saturated habitats with strong competition (Brian and Brian, 1949, 1955; Brian, 1983; Ho¨lldobler and Wilson, 1977; Seppa¨ et al., 1995; Savolainen and Vepsa¨laïnen, 2003). In several Myrmica and Formica species, deep substructuring among populations has been reported (Sundstro¨m, 1993; Seppa¨ and Pamilo, 1995), with the P–P populations of Formica exsecta (FST = 0.72) that are only a few kilometres apart as a good example (Liautard and Keller, 2001). Although we are aware that the FST values based on enzyme data (Sundstro¨m, 1993; Seppa¨ and Pamilo, 1995) or mtDNA data (Liautard and Keller, 2001) are not directly comparable with values obtained from microsatellite data (our present study), the similarity of these FST values broadly confirms that the P–P life history dominates in M. rubra, whereas M. ruginodis seems to have mainly the M–M life history. The small highly polygynous inquiline morph of M. rubra appears to be an even weaker disperser than its host (Buschinger, 1997) and its colony founding is completely dependent on the availability of hosts and the ability of inquilines to successfully infiltrate a host colony—freeliving inquiline morph colonies have never been reported (Seifert, 2007). As expected, the much lower allelic richness in the inquiline than in the host, and the fixation index (FST = 0.440) between the inquilines in Tva¨rminne and Viikki (compared to an FST of 0.089 between the host populations) indicates severe founder effects and small effective population sizes of the inquiline. The mtDNA data (Fig. 5) further indicate that inquiline queen mobility remains very low once a population has been established. Also, if a population has been founded recently from a small colony, considerable linkage disequilibrium—as documented by us for one pair of loci in one population— may be induced by random sampling (Lewontin, 1974). Severe founder bottlenecks are also likely to affect sex determination of social Hymenoptera, as diploid individuals that are homozygous for the sex determination locus (or loci) develop into males rather than females (Wilson, 1971). Only one of the eight diploid males that we found was assigned to the host, which is consistent with considerable erosion of genetic diversity in the inquiline relative to the host. Thus, it seems that the basic life history of M. rubra leads to strong population subdivision even on a small spatial scale and that this tendency is even more extreme in the inquiline. Although knowledge on the ecological dynamics helps to understand the marked genetic differentiation between the two morphs of M. rubra, the origin and


maintenance of the inquiline life style—and its role in the evolutionary dynamics of the morphs—remain, however, unexplained. Reconstructing details of local gene flow The STRUCTURE program allowed us to make inferences on how restricted gene flow between host and inquiline really is, given what is known about the biology of the two morphs. Estimates by Pearson (1981) for mixed M. rubra populations in southern England indicated that the production of microgynes per inquiline queen is ca. 40 times higher than the production of macrogynes per queen in unparasitized nests, because the inquilines produce almost only reproductives. Of the parasitized colonies studied by Pearson (1981), 73% produced winged microgynes (n = 11), whereas only 13% of the unparasitized colonies produced winged macrogynes (n = 31). The admixture coefficients in Fig. 4 indicate that in Tva¨rminne, 6% of the inquiline alleles are derived from the host gene pool, whereas 10% of the host alleles are derived from the inquiline. The corresponding figures for Viikki are 3.1 and 9.6%. These estimates assume that the mismatches in Fig. 4 are an unbiased estimate of worker production by inquiline queens, but assuming no worker production by inquilines only makes the gene flow estimates across morphs slightly higher. Overall, our results therefore indicate that gene flow between sympatric host and inquiline populations is reduced by about an order of magnitude relative to the gene flow within each of the morphs. Myrmica rubra macrogynes and males leave their nests and aggregate in swarms to mate, whereas inquilines have been inferred to mate in their natal nest (Buschinger, 1997; Seifert, 2007). Such differences in mating behaviour would fit our findings of a stronger structuring of the inquiline populations. At all geographic scales, from the Viikki subpopulations to the largest scale across sites, the FST values between inquiline populations and between inquiline and host populations were systematically larger than those between host populations (Table 3 and the discussion above). This implies that even over short distances the inquiline disperses less effectively than the host, and that newly mated queens of the inquiline tend to be adopted either into their natal or a close-by nest. Sympatric versus allopatric speciation Our present results are unable to resolve whether socially parasitic inquiline ants can or do speciate sympatrically without intermittent allopatric episodes. Clearly, as discussed above, small-scale population subdivision and even larger fixation indices than those found by us in M. rubra are not uncommon within species of ants that follow the

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P–P (polygyne–polycalic) life history. However, by demonstrating that the inquiline social morph of M. rubra can realize about 90% of their reproductive isolation by sympatric divergence in the nests of its host, we maintain that speciation of Hymenopteran social parasites through intraspecific parasitism remains a promising scenario for sympatric speciation. As noted above, the genetic differentiation of the local M. rubra inquiline and its host pairs are equal to or larger than the differentiation in two well-documented candidates for sympatric speciation, the pea aphid and a gall moth (Via, 1999; Nason et al., 2002). Moreover, new models, incorporating simple developmentalgenetic rules allow speciation to happen occasionally even under conditions of strong gene flow (Porter and Johnson, 2002)—a result that matches more traditional modelling and is supported by empirical data (Machado and Hey, 2003; Rieseberg et al., 2003; Emelianov et al., 2004; Niemiller et al., 2008). We thus agree with Mayr (1993) that inquilines living in thriving colonies of closely related host species remain among the most promising test cases for sympatric speciation. Phylogenetic sister-species relations of host and inquiline—as seen in phylogenetic reconstructions and interpreted as outcomes of sympatric speciation—may, however, also be an artefact caused by extinctions of more closely related species during a longer evolutionary history. Studies on the origins of inquiline species through intraspecific social parasitism should therefore consider the age of divergence of lineages, because the risk of misinterpreting a phylogeny increases with divergence time (Smith et al., 2007). This notion underlines the value of evolutionarily young systems such as the one studied here, where the same set of microsatellite markers permits the estimation of extant genetic differentiation. It seems reasonable to assume that the relatively rare ‘‘hybrids’’ between host and inquiline, which do occasionally emerge in a sympatric population with 90% divergence, might be selected against. For example, it is possible that part of the M. rubra workers with inquiline or non-assignable genotypes (Fig. 4) were offspring of hybrid queens. A lower fitness of such queens could then easily maintain selection for assortative mating. Acknowledgments The study was funded by a European Union grant from SYNTHESYS to RS. The authors thank Bo Vest Pedersen, Department of Biology, University of Copenhagen, for endorsing the stay of RS, and Graham Elmes for fruitful discussions on Myrmica bionomics and the origins of inquilinism.

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