Multiple Data Sources Show That the Ant Myrmica Microrubra Is Not a Separate Species but an Alternate Reproductive Morph of Myrmica Rubra

doi:10.1111/j.1420-9101.2005.01053.x

No sympatric speciation here: multiple data sources show that the ant Myrmica microrubra is not a separate species but an alternate reproductive morph of Myrmica rubra

F. M. STEINER*, , ,B.C.SCHLICK-STEINER*, , ,H.KONRAD, K. MODER,à E. CHRISTIAN,* B. SEIFERT,§ R. H. CROZIER,– C. STAUFFER & A. BUSCHINGER** *Department of Integrative Biology, Institute of Zoology, Boku, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria Department of Forest & Soil Sciences, Institute of Forest Entomology, Forest Pathology & Forest Protection, Boku, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria àDepartment of Spatial-, Landscape- & Infrastructure-Sciences, Institute of Mathematics & Applied Statistics, Boku, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria §Staatliches Museum fu¨ r Naturkunde Go¨rlitz, Go¨rlitz, Germany –School of Tropical Biology, James Cook University, Townsville, Australia **Reinheim, Germany

Keywords: Abstract ants; No aspect of speciation is as controversial as the view that new species can gene ﬂow; evolve sympatrically, among populations in close physical contact. Social internal transcribed spacers; parasitism has been suggested to yield necessary disruptive selection for microsatellites; sympatric speciation. Recently, mitochondrial DNA phylogeography has mitochondrial DNA; shown that the ant Myrmica microrubra is closely related to its host, Myrmica morphometrics; rubra, leading to the suggestion that sympatric speciation has occurred. We Myrmica microrubra; investigated the relationships between the two ant forms using mitochondrial probability analysis; and nuclear DNA sequences, microsatellite genotyping and morphometrics. queen size dimorphism; Molecular phylogenetic and population structure analyses showed that sympatric speciation. M. microrubra does not evolve separately to its host but rather shares a gene pool with it. Probability analysis showed that mitochondrial DNA data previously adduced in favour of sympatric speciation do not in fact do so. Morphometrically, M. microrubra is most readily interpreted as a miniature queen form of M. rubra, not a separate species. Myrmica microrubra is not an example of speciation. The large (typical M. rubra) and small (M. microrubra) queen forms are alternative reproductive strategies of the same species. Myrmica microrubra Seifert 1993 is consequently synonymized here with M. rubra Linnaeus, 1758.

most evolutionists. Proponents, however, gathered argu- Introduction ments over the following decades (reviewed by Bush, Allopatric speciation, the divergence of populations with 1975) and in the past 15 years attempts to outline completely separate geographical ranges (Mayr, 1963), conditions under which sympatric speciation could actu- was a dogma of speciation theory over recent decades. ally take place have withstood counterarguments (Bush, Speciation among sympatric populations had already 1994; Johannesson, 2001; Via, 2001). Sympatric speci- been suggested by Darwin (1859) but was criticized by ation has become a hot topic in evolutionary biology Mayr (1963) and thereafter considered unrealistic by (Berlocher, 2003; Coyne & Orr, 2004). Social parasitism, especially the relationship of workerless social parasites (‘inquilines’) to their hosts, is regarded as one of the Correspondence: F. M. Steiner, Department of Integrative Biology, Institute of Zoology, Boku, University of Natural Resources and Applied Life conditions favouring this mode of speciation (Buschinger, Sciences, Vienna, Gregor-Mendel-Strasse 33, A-1180 Vienna, Austria. 1965,1990; Elmes, 1978; Pearson, 1981; Bourke & Franks, Tel.: + 43 1 47654 3200; fax: + 43 1 47654 3203; 1991). However, notes of caution have been sounded e-mail: [email protected] repeatedly for this scenario (Alloway, 1980; Ho¨ lldobler & Authors contributed equally to this work Wilson, 1990; Carpenter et al., 1993; Agosti, 1994;

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Choudhary et al., 1994; Ward, 1996) and indisputable evaluation of genetic variation, nuclear markers should model organisms for sympatric speciation in general are be analyzed in addition to mtDNA (Caterino et al., 2000; still lacking (Schluter, 2001; Coyne & Orr, 2004). The Beltrań et al., 2002; Lin & Danforth, 2004) – congruence radiation of the order Hymenoptera includes a profuse of mtDNA and other data supports evolutionary hypo- array of social parasites, but most of the more recent case theses much more strongly than any of these alone (e.g. studies (Choudhary et al., 1994; Baur et al., 1996; Lowe & Wiens & Reeder, 1997; Wetterer et al., 1998; Feldhaar Crozier, 1997; Sanetra & Buschinger, 2000; Danforth, et al., 2003; Wiens et al., 2003). We employ a multidis- 2002; Parker & Rissing, 2002; Schwarz et al., 2003; Janda ciplinary approach combining morphometrics, mtDNA et al., 2004; Sumner et al., 2004) could not exclude and ITS sequences, and three microsatellite loci to alternative interpretations involving allopatric differenti- evaluate the two hypotheses. ation. Savolainen & Vepsa¨laïnen (2003) presented two cases Material and methods of sympatric speciation of socially parasitic ants from their hosts, one of these being Myrmica microrubra. Samples Mitochondrial DNA sequences of both M. microrubra and its host Myrmica rubra segregated by regions (Finland We collected 116 M. microrubra and 107 M. rubra vs. England) but the authors concluded that ‘M. microru- specimens from all over Europe (49 localities in 9 bra presumably is an incipient species’ and that different countries: Table S2, supplementary web material). All populations are still ‘undergoing lineage sorting, the analyses were performed at the population level because mtDNA not yet having coalesced. Alternatively, M. rubra partly lives in huge supercolonies (Seppa¨ & M. microrubra may evolve in parallel in different regions’. Pamilo, 1995; Walin et al., 2001; A. Tartally, pers. comm; Doubts, however, remained due to a low sample size and own observations), i.e. in ‘unicolonial’ societies without the plausibility of alternative interpretations (hinted at colony limits (Wilson, 1971; Crozier & Pamilo, 1996). by Savolainen & Vepsa¨laïnen, 2003 and explicitly out- lined by Berlocher, 2003 and Coyne & Orr, 2004). In Morphometry particular, the finding that M. microrubra and M. rubra mtDNA haplotypes from the same locality are more Morphometric analyses were carried out on 141 gynes phylogenetically related than they are to haplotypes of and 49 males from 47 localities (nine countries), including either entity from other sites fails to distinguish between the entire type series of M. microrubra. Four measurements the occurrence of two species as against polymorphism were made (Table S3, supplementary web material). within one species, because only one specimen of each Dry-mounted specimens were fixed onto a pin-holding form was sampled per site (Savolainen & Vepsa¨laïnen, goniometer. A Wild M10 stereomicroscope with a 2003). Additional doubts pertain to the nature of 1.6 · planapochromatic lens and a cross-scaled ocular M. microrubra as a social parasite and M. microrubra might micrometer was used at magnifications of 50–320 ·. Gyne instead be the miniature queen (microgyne) form of and male data were subjected separately to discriminant M. rubra (Buschinger, 1997). The morphology, repro- analysis using the software package SPSS. ductive biology and physiology of M. microrubra have been intensively characterized (Pearson, 1981; Elmes, Mitochondrial DNA 1973,1976; Pearson & Child, 1980; Brian, 1986; Cammaerts et al., 1986,1987; Elmes & Brian, 1991). Twenty-two specimens from six localities were selected Seifert (1993) formally described the ant as separate from the morphometrics material (Table S2). For DNA species. Today M. microrubra is known throughout most extraction the gaster was taken, allowing subsequent of Europe (17 states, Table S1 in the supplementary web morphometric analyses. DNA extractions and PCR material) and we posit that M. microrubra can be found with a touchdown program followed the protocols of wherever M. rubra occurs (B. Seifert, in press). F. M. Steiner, B. C. Schlick-Steiner, J. C. Trager, Here we revisit the status of M. microrubra and K. Moder, M. Sanetra, E. Christian & C. Stauffer (in M. rubra using an expanded data set involving more press), except for cytochrome b apoenzyme (Cytb), where samples from a wide geographical range. We evaluate we used the conditions of Savolainen & Vepsa¨laïnen two hypotheses: (A) M. microrubra is a separate species (2003). Mitochondrial cytochrome oxidase subunit I from M. rubra, the 2-species hypothesis and (B) (COI), cytochrome oxidase subunit II (COII) and Cytb M. microrubra is a microgyne morph of M. rubra and primers were those of Savolainen & Vepsa¨laïnen (2003). the forms share a common gene pool, the 1-species PCR products were purified (QIAquick PCR purification hypothesis. The practical issue of delimiting species kit, Qiagen, Hilden, Germany), sequenced in both boundaries is increasingly recognized to be of central directions using the Big Dye termination reaction importance to evolutionary biology (e.g. Wiens, 1999; chemistry (Applied Biosystems, Foster City, CA, USA), Wiens & Servedio, 2000; Sites & Marshall, 2003; Dayrat, and analyzed with an ABI 377 automated sequencer 2005; Hendrixson & Bond, 2005). For a profound (Applied Biosystems).

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We used two outgroup species, namely M. ruginodis, sequencing a number r of M. rubra specimens can be the closest relative of M. rubra (Savolainen & Vepsa¨laï- calculated by (1/h)r. This holds true for every of the h h nen, 2003) and Manica rubida a close relative of the genus haplotypes and we have to multiply a1 with ð1Þ. The Myrmica (Bolton, 2003). probability that the haplotypes obtained by sequencing a number m of M. microrubra specimens are different from the haplotypes found in M. rubra is given by (1)1/h)m. ITS1 Combining these terms results in the probability for The ITS1 of the nuclear coded rDNA was amplified with finding not more than one haplotype in M. rubra and for the primer pair 5¢-GAACCTGCGGAAGGATC-3¢ and not finding this haplotype in M. microrubra. The probab- 5¢-GTGATCCACCGTTCAGGG-3¢ (Baur et al., 1996). ility a2 that we find exactly two haplotypes when Fourty-three specimens were directly sequenced (Ta- sequencing r can be calculated by (2/h)r, reduced by 2 ble S2). To evaluate the presence of differing copies of ð1Þa1 to address the case of obtaining the same haplotype the ITS1 unit within single specimens, one PCR product twice, already accounted for by a1. As we are interested each of 10 specimens was cloned with the pGem-t easy in all combinations of two haplotypes we have to h vector kit. 1–10 (on average 6) bacterial colonies were multiply a2 with ð2Þ. The probability that the haplotypes picked and sequenced after plasmid extraction. obtained by sequencing a number m of M. microrubra specimens are different from the haplotypes found in M. rubra is then given by (1)2/h)m. The product of all Microsatellites these terms estimates the probability for finding not more The microsatellite loci MS 26, MS 86 and MS 3.62, than two different haplotypes in M. rubra and not finding polymorphic in M. rubra (Azuma et al., 2005), were these haplotypes in M. microrubra. In a similar way we genotyped for 5–17 specimens per locality (mean calculate probabilities for 3, 4,…, n haplotypes. The 7.2 ± 2.3 SD for M. microrubra, 10.0 ± 4.2 for M. rubra, overall probability P of finding no haplotype common to total 86; Tables S2 and S4). We used the amplification M. rubra and M. microrubra then is calculated by eqn (1): conditions and fluorescence detection conditions described by Azuma et al. (2005). Alleles were scored using h h 2 P¼ ðÞ1=h r ð11=hÞmþ ðÞ2=h r a ðÞ12=h m GENESCAN 2.0.1 and GENOTYPER 3.7 (Applied Biosystems) |fflfflffl{zfflfflffl} 1 1 2 |fflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflffl}1 software. a1 a2 h r n n m Phylogenetic reconstruction þþ ðÞn=h an1 a1 ðÞ1n=h n |fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl}1 n1 mtDNA sequences of the two M. rubra and the two an M. microrubra specimens of Savolainen & Vepsa¨laïnen rr

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specimens of M. microrubra and M. rubra we calculated Hochberg, 1997). Linkage disequilibrium between pairs the number needed each of m and r, using eqn (1), to of loci was tested for each locality using GENEPOP 3.2. arrive at probability values of P < 0.05 that, despite no Pairwise FST estimates between sites and the overall FST detection, haplotype overlap exists at a locality. We were computed using ARLEQUIN 2.0 (Schneider et al., visualized these calculations for numbers between two 2000). We used an analysis of molecular variance and ten haplotypes per locality. (AMOVA, Excoffier et al., 1992) to assess the distribution Distance (Neighbor Joining algorithm, NJ, based on of microsatellite and mtDNA variation. A nested three- Tamura-Nei distances) and character (Bayesian analysis level AMOVA was performed by partitioning the total sum using Markov Chain Monte Carlo, MCMC, optimization) of squares into components representing variation across analyses were performed using PAUP* (test Version localities, between M. microrubra and M. rubra of each 4.0b3a; Swofford, 1998) and MRBAYES V.3.1 (Ronquist locality and within M. rubra and M. microrubra of each & Huelsenbeck, 2003). Prior to Bayesian analysis the locality separately, using ARLEQUIN 2.0. To study haplo- general time reversible model (GTR; Tavare´, 1986) with type differentiation, this method also yields analogues to gamma-distributed rate heterogeneity (+G; Yang, 1993) F-statistics (termed U-statistics), for which we used the was chosen using MODELTEST 3.06 (Posada & Crandall, number of pairwise differences between haplotypes as 1998), which uses hierarchical likelihood-ratio tests Euclidean distance measure. Significance was deter- (Huelsenbeck & Rannala, 1997) to determine how well mined by comparing the observed values to a null competing substitution models fit the data. For Bayesian distribution generated by permutation. The significance analysis, nine partitions according to the three codon of the Pearson correlation coefficient between genetic positions and the three single genes (COI, COII, Cytb) were differentiation of microsatellites (FST) and geographical defined within the data set and all model parameters distance was assessed with a Mantel test (IBD; Bohonak, were estimated separately for the single partitions. 2002). 500 000 generations with a sample frequency set to Analysis of multilocus genotypes allows inference of 100 were run twice. As after 250 000 generations genetic ancestry without relying on information about stationarity was achieved (average standard deviation of sampling locations, even when among-population vari- split frequencies below 0.015, dropping to 0.013 towards ance components are small. We used a model-based the end of the run), we used the last 2500 trees of each clustering algorithm implemented in STRUCTURE (Prit- run to compute a majority rule consensus tree assigning chard et al., 2000) that identifies subgroups with distinc- posterior probabilities of tree topology. Tree topology of tive allele frequencies. The program places specimens additional Bayesian analyses with three partitions into K clusters, where K is established in advance but can according to codon positions were identical with topol- be varied between runs of the algorithm. We carried out ogy of analyses using nine partitions, with minor differ- three analyses. The first analysis combined all the data ences in node support values. and used K ¼ 2, to test for the overall existence of two For intraspecific gene genealogies network methods species. The second examined each of the five localities are superior to bifurcating trees, because population separately, with K ¼ 2 for each locality. The third phenomena such as persistent ancestral nodes, multifur- combined all data with K ¼ 5, to test the tendency of cations and reticulations are allowed for (Maynard the specimens to assort into five clusters. All runs used Smith, 1989; Posada & Crandall, 2001). To construct a 100 000 iterations after a burn-in of 900 000. haplotype network based on a 95% plausible parsimoni- ous set for all haplotype connections, and to calculate Results outgroup probabilities for each haplotype, we used the TCS 1.13 software developed by Clement et al. (2000). Morphometry Standard nesting rules were applied to the haplotype network (Templeton & Sing, 1993). The discriminant function D1 based on head width, CW Due to the presence of genetically distinct ITS1 units and mesosoma length, ML (0.015285 CW + 0.0556 ML) within some single specimens, ITS1 sequences could not be resulted in a clear segregation of M. microrubra from subjected to phylogenetic analyses. Frequency of occur- M. rubra (Fig. 1; 100% classified with P ¼ 0.012). For rence of distinct types of ITS1 sequences in the investigated males, using the discriminant function D2 based on scape M. microrubra and M. rubra samples was analyzed. length, SL, head length, CL, head width and mesosoma length (36.740 SL – 34.239 CL + 17.264 CW + 0.983 ML) the clustering was less pronounced (86.0% with Population structure analysis P < 0.05). Hardy–Weinberg proportions were tested for the three microsatellite loci using exact tests at each locality, for Phylogeny M. rubra and M. microrubra separately (GENEPOP 3.2; Raymond & Rousset, 1995). Bonferroni correction for Excluding the noncoding region, the sequence alignment multiple comparisons was applied (Benjamini & of COI, COII and Cytb consisted of 2724 bp. Sequences

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a locality was high. The haplotype network revealed multifurcations (data not shown). Three ITS1 types were detected (I–III, 741–771 bp; Table 2) and the sequences were deposited in GenBank (DQ075936–DQ075938). Two types were very often found within one specimen (7 of the 10 cloned specimens). All types were found in M. microrubra and M. rubra. At one site (locality 6) all three types occurred. No distinct geographical pattern was apparent for the three types.

Population structure Genotyping 86 specimens for the three microsatellite loci Fig. 1 Size variation among gynes in the ants Myrmica microrubra (black, n ¼ 83) and M. rubra (white, n ¼ 58) in terms of head width yielded 11 alleles (Table S4, supplementary web mater- and mesosoma length; measurements in lm. Typical gynes are ial). Alleles were widely distributed geographically. All shown, legs and antennae removed. alleles were found in both M. microrubra and M. rubra, but the alleles present differed between the two ants for individual localities (Table S4). But on average the were deposited in GenBank (DQ074366 – DQ074434). proportion of alleles found at a locality found in both No indels were observed and, within the total of forms was high, 80.0 ± 27.4%. Hardy–Weinberg propor- 26 analyzed specimens substitutions at 35 sites resulted tions were rejected for five site-locus combinations in 18 haplotypes with the largest distance between (P < 0.05), and after a Bonferroni multiple-comparisons sequences being 0.63% per site. The largest distance correction four of these remained significant. The latter between haplotypes for the 11 haplotypes found in did so because of an excess of homozygotes (from FIS ¼ M. microrubra was 0.59% and within the 10 haplotypes 0.238 to FIS ¼ 0.727 for these combinations). Linkage found in M. rubra 0.55%. was not detected for the three loci, and hence they were There were haplotypes shared between M. microrubra assumed to yield independent information. and M. rubra at three localities (sites 3, 5, 6; Fig. 2a). At The overall estimate of FST ¼ 0.144, which measures the remaining four localities no common haplotypes all effects of population substructure combined, indicated were observed. Under the 2-species hypothesis the genetic differentiation at the microsatellite loci. Pairwise probabilities for these results are P ¼ 1.0. The probabil- FST values ranged from 0.036 to 0.359 within ities for finding no common haplotypes under the M. microrubra, from 0.033 to 0.304 within M. rubra, and 1-species hypothesis are given in Table 1. Assuming that from )0.034 to 0.407 for M. microrubra/M. rubra pairs, but all extant haplotypes per locality were detected, the 18 values (40%) were not significant at P < 0.05 (six probabilities for not observing any common haplotype within M. microrubra, two within M. rubra, ten for varied between 0.063 and 0.500. Allowing for the fact M. microrubra/M. rubra). The mean of all pairwise FST that the observation of singletons indicates that there values was 0.172 ± 0.108, and 0.237 ± 0.085 after exclu- were further, undiscovered, haplotypes, and therefore ding nonsignificant values. assuming higher numbers of haplotypes increases the Exploration of isolation by distance effects suggested probability for not finding haplotypes common to the no difference between M. microrubra and M. rubra. The two ants; probabilities varied between 0.656 and 0.900 plot (Fig. 3) displayed three clusters: (i) very small to for 10 haplotypes. But, how many M. microrubra and medium FST values between M. microrubra and M. rubra M. rubra specimens would have to be sampled – without within localities (left); (ii) values in the same range detecting haplotype overlap – for refuting the 1-species representing among-site variation within M. microrubra, hypothesis at a P < 0.05? For the simplest case of within M. rubra and between the two for geographical analysing equal numbers of M. microrubra and M. rubra distances of 20 km (centre); and (iii) the most pro- the numbers range from 3 + 3 specimens in case two nounced range of genetic variation over large geograph- haplotypes exist at a locality to 6 + 6 specimens for 10 ical distances, from very small to very large (right). extant haplotypes (Fig. S2, supplementary web informa- A matrix comparison using Mantel test showed no tion) – numbers exceeding the analyzed sample sizes. significant association between genetic differentiation The phylogenetic trees based on NJ and Bayesian and geographical distance (r ¼ )0.014, P ¼ 0.476); in analysis over the combined mtDNA are presented in addition, no differences existed between pairwise values Fig. 2; the two trees are congruent on a basis of credibility within M. microrubra, within M. rubra, or between the values of > 95. At several localities haplotype diversity two (partial correlation r ¼ )0.002, P ¼ 0.508). within M. microrubra and within M. rubra exceeded 1. In AMOVA revealed that the genetic component of micro- both ants divergence between the different haplotypes of satellite variation within M. microrubra and within

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Fig. 2 Phylogeny of the ants Myrmica microrubra and Myrmica rubra. Partial map of Europe showing collection sites of M. microrubra (white numerals on black background) and M. rubra (black on white) sampled for genetic analyses. Phylogenetic trees based on 2724 bp of the combined mitochondrial genes COI, COII and Cytb; node support values >95 given: NJ, Neighbor Joining; MCMC, majority-rule Bayesian consensus.

Table 1 Probabilities of obtaining the observed result of no mtDNA Table 2 Distribution of ITS1 types beween localities and individuals haplotypes in common between forms for four localities, under the of Myrmica microrubra and Myrmica rubra, with the numbers of 1-species hypothesis (i.e. that in fact all haplotypes at a locality occur specimens showing the various combinations given in parentheses. in both forms). Locality ITS 1 type in M. microrubra M. rubra Probabilities under the 1-species 2 n.a. ll(1) hypothesis for HText equalling 3 ll(2) ll(2)

Locality m/r HTobs HTobs 5104 ll(7), ll + lll(1) ll(4) 5 l(1), ll(4) L+ ll(2), ll(1) 1 1/1 2 0.500 0.800 0.900 6 ll(5), ll + lll(1) L(2), l + ll(1) 2 1/2 3 0.440 0.640 0.810 8 ll(2) ll(1), lll(1) 4 4/1 2 0.063 0.448 0.656 10 ll(1) l + ll(2), ll(2) 8 1/2 3 0.444 0.640 0.810 Collection sites are given in Fig. 2a; n.a. ¼ no material available. The numbers of Myrmica microrubra (m)andMyrmica rubra (r) are shown; HTobs is the number of haplotypes observed and HText is the true number under three assumptions. The probabilities are those In stark contrast, AMOVA for mtDNA revealed that from eqn 1 and the collection sites are given in Fig. 2. 45.99% of genetic variation was due to variation within M. microrubra and M. rubra of single localities; this test produced a negative value of )10.24% between M. rubra of single localities, estimated at 82.38% M. microrubra and M. rubra within localities, and (Table 3), accounted for most of the genetic diversity. 64.25% among localities. Negative values for variation Variation between M. microrubra and M. rubra within components indicate a lack of population structuring at localities was 16.56%, variation among localities 1.06%. the respective hierarchical level.

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0.7

0.6 )

ST 0.5 F

0.4 / (1 – ST F 0.3

0.2

0.1 Fig. 4 Estimated population structure of the ants Myrmica microrubra 0 and Myrmica rubra. Each specimen is represented by a thin vertical 02468101214bar, which is partitioned into K shaded segments that represent the –0.1 ln (distance) specimen’s estimated membership fractions in K clusters. Localities are labelled, M. microrubra white numerals on black background, Fig. 3 Isolation by distance analysis: multilocus microsatellite esti- M. rubra black on white. The first row shows the results for the five ) mates of pairwise differentiation. FST/(1 FST) values are plotted localities analyzed separately, with K ¼ 2 for each analysis. The against the logarithm of geographical distances (in m) including all second row shows the result of combining all specimens in one localities; values within Myrmica microrubra black, within Myrmica analysis, with K ¼ 2, but with morph and locality affiliations of the rubra white, between M. microrubra and M. rubra grey; values not specimens still shown. The third row also reports an analysis significant at P < 0.05 marked with – to left of symbols. including all specimens, but with K ¼ 5. The analyses do not assign specimens to either species or localities. Collection sites of the specimens are given in Fig. 2. When localities were dealt with individually, the model-based clustering algorithm did not separate M. microrubra and M. rubra specimens (Fig. 4). Similarly, (2-species hypothesis) or the microgyne form of M. rubra no discrimination was apparent when all specimens were (1-species hypothesis). analyzed together, neither for a clustering according to The combined molecular data are decisive, as we now species identity (K ¼ 2), nor for geographical origin show. The two ants were found to share three of (K ¼ 5). 18 mtDNA haplotypes, at three localities. Although at these three localities additional haplotypes were found in Discussion exclusively one of the two ants, this does not conflict with all haplotypes actually being common to both ants, The morphometric analyses on a larger sample size as the numbers of sequenced specimens were small. confirm Seifert’s (1993) finding that gynes of M. microru- Probability calculations for the four sites where no bra and M. rubra are well separated based on absolute size. common haplotypes were found demonstrate that sam- The morphological separation of males, however, now ple size per locality was too small to refute the 1-species appears to be less clearcut. The morphological situation in hypothesis for these sites at a P < 0.05 (Fig. S2) – this M. microrubra and M. rubra resembles that in ant species likewise holds true for the data presented by Savolainen with dimorphic queens (McInnes & Tschinkel, 1995), e.g. & Vepsa¨laïnen (2003) which were interpreted in favour the case of Tetramorium moravicum where the description of sympatric speciation, i.e. in favour of the 2-species of microgynes as a separate species had to be retracted hypothesis according to our terms. In fact, although for recently (Schlick-Steiner et al., 2005). The morphometric our four localities where no common haplotypes were results alone are thus insufficient to decide whether detected the probabilities for these results under the M. microrubra is a separate, socially parasitic species 2-species hypothesis are P ¼ 1.0, the failure to find any

Table 3 Analyses of Molecular Variance (AMOVA) for microsatellites and mtDNA.

Microsatellites mtDNA

% of total % of total Source of variation d.f. variation PF-statistics d.f. variation P /-statistics

Between localities 4 1.06 0.3923 FCT ¼ 0.011 6 64.25 <0.0001 /CT ¼ 0.643

Between M. microrubra and M. rubra within localities 5 16.56 <0.0001 FSC ¼ 0.167 7 )10.24 0.0212 /SC ¼ )0.286

Within M. microrubra and M. rubra of single localities 162 82.38 <0.0001 FST ¼ 0.176 12 45.99 0.0004 /ST ¼ 0.540

The P-values give the probability that a random value from 10 000 permutations is more extreme than the observed variance component and F-andU-statistics.

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common haplotype also is entirely compatible with the hypothesis in our case. We here deal with the general 1-species hypothesis (Table 1): given that we obtained up problem of proving the absence of differences. As Wiens to four haplotypes by sequencing five specimens per & Servedio (2000) point out, ‘determining whether locality (locality 5), considerable haplotype diversities per diagnostic characters are truly fixed with certainty is locality may be assumed from sample coverage theory. generally impossible with finite sample sizes’ – we have For example, if there are 10 haplotypes per site to consciously accept a pragmatic approach if taxonomy (a number compatible with the observations), and these shall not be rendered impracticable and in fact, though are equally frequent then the probability of not finding a rarely addressed, in most cases of species delimitation the haplotype in common for our sample sizes are very high tacit acceptance of a null-hypothesis, i.e. of character (from P ¼ 0.656 to P ¼ 0.900) and thus compatible with fixation within the single species, is the basis for the the 1-species hypothesis. The substantial haplotype taxonomic decision (Wiens, 1999). From all current diversity within both M. microrubra and M. rubra at some knowledge, M. microrubra belongs to the same biological localities despite small sample sizes is remarkable itself. species as M. rubra. Myrmica microrubra is consequently This finding helps explain the genetic differences synonymized here with M. rubra and from now on between M. microrubra and M. rubra reported by Savol- termed microgyne of the latter. ainen & Vepsa¨laïnen (2003): the differences within AMOVA allows considerations beyond the question of M. rubra are comparable to those between the two ants, one or two species. AMOVA of the microsatellite data show which is compatible with M. microrubra being a morph of that the largest portion of genetic differentiation is made M. rubra. On the other hand, haplotype diversity up by variation within the populations of M. rubra within localities renders parallel speciation (reviewed consisting of macro- and microgynes, which is likewise by Johannesson, 2001) as inferable from mtDNA data reflected by the lack of association between genetic and (Savolainen & Vepsa¨laïnen, 2003) implausible, since this geographical distance by the Mantel test. Nevertheless, would suggest multiple evolution of M. microrubra within some differentiation at the population level is suggested by localities. The interlaced phylogenetic pattern of the two AMOVA. This is not in conflict with the 1-species hypothesis ants as inferred by all methods of phylogenetic recon- because the gene pools of macro- and microgynes in other struction reveals no indication of M. microrubra evolving ants with dimorphic queens or with social forms differing separately from M. rubra. Like mtDNA, the analysis of in queen number are separated to a similar or even greater ITS1 showed variability within M. microrubra and degree (Ru¨ ppell et al., 2001,2003; Ross et al., 1997; M. rubra, but no inherent differentiation of the two ants Goodisman et al., 2000; Gyllenstrand et al., 2005). Differ- was detected. The microsatellite analysis revealed suffi- ent mating strategies (microgynes inside and macrogynes cient differentiation, within the whole sample and outside the nest; Buschinger, 1997) resulting in assortative between single localities, as measured by the number of mating probably cause a partial separation of the gene extant alleles and by the overall FST value. The finding pools, as claimed for the micro- and macrogyne forms of that all 11 microsatellites alleles are shared by M. M. ruginodis, the closest relative of M. rubra (Elmes, 1991). microrubra and M. rubra, and the fact that pairwise FST However, M. rubra macrogynes can also mate inside the values did not differ significantly across M. microrubra nest (B. Seifert, in press), and microgynes have repeatedly pairs, M. rubra pairs or M. microrubra/M. rubra pairs, been observed to join mating swarms of macrogynes suggests gene flow between the two ants. This is in line (Czechowski et al., 1999; Czechowski & Czechowska, with analyses of enzyme polymorphism in English 2002) despite their decreased flying abilities due to smaller populations (Pearson & Child, 1980) and with micro- mesosoma size (Stille, 1996). satellite analyses of Finnish populations (R. Savolainen, Microgynes of M. rubra are promising study subjects for K. Vepsa¨laïnen, J. Elsen & J. J. Boomsma, pers. comm.). the origin and maintenance of queen dimorphisms, a The results of the model-based clustering algorithm significant problem in the developmental biology of analysis illustrate that neither on a locality-level nor ants. This mechanism could either be exclusively genetic across all localities are the microsatellite patterns suffi- as established for queen polymorphism (Winter & ciently distinct to assign specimens to M. microrubra and Buschinger, 1986), queen-worker dimorphism (Volny & M. rubra. This suggests a substantial connection of the Gordon, 2002), and variation in queen number (Krieger gene pools of the two ants and recent gene flow between & Ross, 2002) in other ants, or could be due to a geographically distant populations. combination of genetic and environmental effects as in In conclusion, we refute the 2-species hypothesis: it is worker polymorphism (e.g. Fraser et al., 2000; Hughes explicitly rejected for three localities, and for the et al., 2003) and in one case of gradual queen size remaining four localities – despite the sample size variation (Bargum et al., 2004). Significant environmen- limitations of our data already noted – the accumulated tal influences might include nutritional and climatic phylogenetic and population genetic evidence renders it conditions, which can influence larval development implausible. Clearly, rejection of the 2-species hypothesis shortly before pupation even when these are brief (Brian, in strict logical terms does not entail acceptance of the 1956). Such short-period influences could affect early 1-species hypothesis, which implicitly serves as null- and late broods differently and this would explain the

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absence of microgynes in M. rubra populations in some Benjamini, Y. & Hochberg, Y. 1997. Controlling the false years despite their being abundant in others (A. Bus- discovery rate: A practical and powerful approach to multiple chinger, unpubl. data). The two morphs can thus be seen testing. J. R. Statist. Soc. B 57: 289–300. under the Evolutionarily Stable Strategy framework as Berlocher, S.H. 2003. When houseguests become parasites: conditional strategies (reviewed by Gross, 1996) in that Sympatric speciation in ants. Proc. Natl. Acad. Sci. USA 100: 6896–6898. good conditions enable development as large queens Bohonak, A.J. 2002. IBD (Isolation by Distance): a program for capable of independent colony foundation whereas poor analyses of isolation by distance. J. Heredity 93: 153–154. conditions only allow development as small queens, Bolton, B. 2003. Synopsis and classification of Formicidae. Mem. which must enter established colonies to survive. Amer. Entomol. Inst. 71: 1–370. Our findings highlight the necessity of in depth analysis Bourke, A.F.G. & Franks, N.R. 1991. Alternative adaptations, of promising examples of sympatric speciation, although sympatric speciation and the evolution of parasitic, inquiline we have not addressed the general importance of symp- ants. Biol. J. Linn. Soc. 43: 157–178. atric speciation in the origin of inquilines (Buschinger, Brian, M.V. 1956. Studies of caste differentiation in Myrmica 1965; Elmes, 1978; Pearson, 1981; Buschinger, 1990; rubra L. 4. Controlled larval nutrition. Insect. Soc. 3: 369–394. Bourke & Franks, 1991). Brian, M.V. 1986. Bonding between workers and queens in the ant genus Myrmica. Anim. Behav. 34: 1135–1145. Buschinger, A. 1965. Leptothorax (Mychothorax) kutteri n.sp., eine Acknowledgments sozialparasitische Ameise (Hymenoptera, Formicidae). Insect. Soc. 12: 327–334. BCS is supported by the Theodor Ko¨ rner Foundation Buschinger, A. 1990. Sympatric speciation and radiative evolu- (Arbeiterkammer Vienna), BCS, FMS, HK and CS by tion of socially parasitic ants – Heretic hypotheses and their the Austrian Science Foundation (FWF), RHC by factual background. Z. Zool. Syst. Evolut.-forsch. 28: 241–260. the Australian Research Council. We thank P. Boer, Buschinger, A. 1997. Vorkommen der sozialparasitischen Ame- W. Czechowski, W. Dekoninck, A. Lenoir and M. Pendl for ise Myrmica microrubra in Hessen (Hymenoptera, Formicidae). sample donations; V. Antonova, P. Douwes, G.W. Elmes, Hess. Faunist. Briefe 16: 49–57. X. Espadaler, B. Marko´ , A. Radchenko and A. Tartally for Bush, G.L. 1975. Modes of animal speciation. Annu. Rev. Ecol. Syst. 6: 339–164. distribution data; H. Strelec for help with probability Bush, G.L. 1994. Sympatric speciation in animals: new wine in calculations; J.J. Boomsma, J. Ebsen, R. Savolainen and K. old bottles. Trends Ecol. Evol. 9: 285–288. Vepsa¨laïnen for sharing information on microsatellite Cammaerts, M.-C., Cammaerts, R. & Bruge, H. 1986. analyses; A.H. Porter and an anonymous referee, as Contribution a l’etude de la forme microgyne de Myrmica well as the deciding editor, A.J. Moore, for valuable rubra L. (Hymenoptera, Formicidae). Actes Coll. Insect. Soc. 3: comments on the manuscript; M. Stachowitsch for 211–217. linguistic improvements of an earlier version of the Cammaerts, M.-C., Cammaerts, R. & Bruge, H. 1987. Some manuscript. physiological information on the microgyne form of Myrmica rubra L. (Hymenoptera: Formicidae). Annls. Soc. R. Zool. Belg. 117: 147–158. References Carpenter, J.M., Strassmann, J.E., Queller, D.C., Turillazzi, S. & Cervo, R. 1993. Phylogenetic relationships among paper wasp Agosti, D. 1994. A new inquiline ant (Hymenoptera: Formici- social parasites and their hosts (Hymenoptera: Vespidae; dae) in Cataglyphis and its phylogenetic relationships. J. Nat. Polistinae). Cladistics 9: 129–146. Hist. 28: 913–919. Caterino, M.S., Cho, S. & Sperling, F.A.H. 2000. The current Alloway, T.M. 1980. The origins of slavery in Leptothoracine state of insect molecular systematics: A thriving Tower of ants (Hymenoptera, Formicidae). Am. Nat. 115: 247–261. Babel. Annu. Rev. Entomol. 45: 1–54. Azuma, N., Takahashi, J., Kikuchi, T., Yoshimura, M., Onoyama, Chao, A. & Lee, S-M. 1992. Estimating the number of classes via K. & Higashi, S. 2005. Microsatellite loci for Myrmica kotokui sample coverage. J. Amer. Stat. Assoc. 87: 210–217. and their application in some congeneric ant species dis- Choudhary, M., Strassmann, J.E., Queller, D.C., Turillazzi, S. & tributed in northern Japan. 5: 118–120. Mol. Ecol. Notes Cervo, R. 1994. Social parasites in polistine wasps are Bargum, K., Boomsma, J.J. & Sundstro¨ m, L. 2004. A genetic monophyletic: implications for sympatric speciation. Proc. R. component to size in queens of the ant, Formica truncorum. Soc. Lond. B 257: 31–35. Behav. Ecol. Sociobiol. 57: 9–16. Clement, M., Posada, D. & Crandall, K.A. 2000. TCS: a computer Baur, A., Sanetra, M., Chalwatzis, N., Buschinger, A. & program to estimate gene genealogies. Mol. Ecol. 9: 1657–1659. Zimmermann, F.K. 1996. Sequence comparisons of the Coyne, J.A. & H.A. Orr 2004. Speciation. Sinauer Associates, internal transcribed spacer region of ribosomal genes support Sunderland. close relationships between parasitic ants and their Crozier, R.H. & Pamilo, P. 1996. Evolution of Social Insect Colonies. respective host species (Hymenoptera: Formicidae). Insect. Oxford University Press, New York. Soc. 43: 53–67. Czechowski, W. & Czechowska, W. 2002. Myrmica microrubra Beltrań, M., Jiggins, C.D., Bull, V., Linares, M., Mallet, J., Seifert (Hymenoptera, Formicidae) recorded from Poland for McMillan, W.O. & Bermingham, E. 2002. Phylogenetic the second time. Przeglad Zoologiczny 46: 75–77. discordance at the species boundary: comparative gene Czechowski, W., Woyciechowski, M. & Czechowska, W. 1999. genealogies among rapidly radiating Heliconius butterflies. Myrmica microrubra Seifert, 1993 (Hymenoptera, Formicidae) – Mol. Biol. Evol. 19: 2176–2190.

ª 2005 THE AUTHORS 19 (2006) 777–787 JOURNAL COMPILATION ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 786 F. M. STEINER ET AL.

an inquiline ant species new to Poland. Fragm. Faun. 42: 123– Krieger, M.J.B. & Ross, K.G. 2002. Identification of a major gene 126. regulating complex social behavior. Science 295: 328–332. Danforth, B. N. 2002. Evolution of sociality in a primitively Lin, C.-P. & Danforth, B.N. 2004. How do insect nuclear and eusocial lineage of bees. Proc. Natl. Acad. Sci. USA 99: 286–290. mitochondrial gene substitution patterns differ? Insights from Darwin, C. 1859. On the Origin of Species by Means of Natural Bayesian analyses of combined datasets. Mol. Phylogenet. Evol. Selection or the Preservation of Favored Races in the Struggle for Life. 30: 686–702. Murray, London. Lowe, R.M. & Crozier, R.H. 1997. The phylogeny of bees of the Dayrat, B. 2005. Towards integrative taxonomy. Biol. J. Linn. Soc. socially parasitic Australian genus Inquilina and their Exoneura 85: 407–415. hosts (Hymenoptera, Anthophoridae). Insect. Soc. 44: 409–414. Elmes, G.W. & Brian, M.V. 1991. The importance of the egg- Maynard Smith, J. 1989. Trees, bundles or nets? Trends Ecol. mass to the activity of normal queens and microgynes of Evol. 4: 302–304. Myrmica rubra L. (Hym. Formicidae). Insect. Soc. 38: 51–62. Mayr, E. 1963. Animal Species and Evolution. Belknap Press, Elmes, G.W. 1973. Miniature queens of the ant Myrmica rubra L. Cambridge, MA. (Hymenoptera, Formicidae). Entomologist 106: 133–136. McInnes, D.A. & Tschinkel, W.R. 1995. Queen dimorphism and Elmes, G.W. 1976. Some observations on the microgyne form of reproductive strategies in the fire ant Solenopsis geminata Myrmica rubra L. (Hymenoptera, Formicidae). Insect. Soc. 23:3– (Hymenoptera: Formicidae). Behav. Ecol. Sociobiol. 36: 367– 22. 375. Elmes, G.W. 1978. A morphometric comparison of three closely Parker, J.D. & Rissing, S.W. 2002. Molecular evidence for the related species of Myrmica (Formicidae), including a new origin of workerless social parasites in the ant genus species from England. Syst. Entomol. 3: 131–145. Pogonomyrmex. Evolution 56: 2017–2028. Elmes, G.W. 1991. Mating strategy and isolation between the Pearson, B. 1981. The electrophoretic determination of Myrmica two forms, macrogyna and microgyna, of Myrmica ruginodis rubra microgynes as a social parasite: possible significance in (Hym. Formicidae). Ecol. Entomol. 16: 411–423. the evolution of ant social parasites. In: Biosystematics of Social Excoffier, L., Smouse, P.E. & Quattro, J.M. 1992. Analysis of Insects (P.E. Howse & J.-L. Cle´ment, eds), pp. 75–84. Academic molecular variance inferred from metric distances among DNA Press, London and New York. haplotypes: application to human mitochondrial DNA restric- Pearson, B. & Child, A.R. 1980. The distribution of an esterase tion sites. Genetics 131: 479–491. polymorphism in macrogynes and microgynes of Myrmica Feldhaar, H., Fiala, B., Gadau, J., Mohamed, M. & Maschwitz, U. rubra Latreille. Evolution 34: 105–109. 2003. Molecular phylogeny of Crematogaster subgenus Decacre- Posada, D. & Crandall, K.A. 1998. Modeltest: testing the model ma ants (Hymenoptera: Formicidae) and the colonization of of DNA substitutions. Bioinformatics 14: 817–818. Macaranga (Euphorbiaceae) trees. Mol. Phylogenet. Evol. 27: Posada, D. & Crandall, K.A. 2001. Intraspecific gene genealogies: 441–452. trees grafting into networks. Trends Ecol. Evol. 16: 37–45. Fraser, V.S., Kaufmann, B., Oldroyd, B.P. & Crozier, R.H. 2000. Pritchard, J.K., Stephens, M. & Donnelly, P. 2000. Inference of Genetic influence on caste in the ant Camponotus consobrinus. population structure using multilocus genotype data. Genetics Behav. Ecol. Sociobiol. 47: 188–194. 155: 945–959. Goodisman, M.A.D., Ross, K.G. & Asmussen, M.A. 2000. A Raymond, M. & Rousset, F. 1995. GENEPOP (version 1.2): formal assessment of gene flow and selection in the fire ant population genetics software for exact tests and ecumenicism. Solenopsis invicta. Evolution 54: 606–616. J. Heredity 86: 248–249. Gross, M.R. 1996. Alternative reproductive strategies and tactics: Ronquist, F. & Huelsenbeck, J.P. 2003. MRBAYES 3: Bayesian diversity within sexes. Trends Ecol. Evol. 11: 92–98. phylogenetic inference under mixed models. Bioinformatics 19: Gyllenstrand, N., Seppa¨, P. & Pamilo, P. 2005. Restricted gene 1572–1574. flow between two social forms in the ant Formica truncorum. Ross, K.G., Krieger, M.J.B., Shoemaker, D.D., Vargo, E.L. & J. Evol. Biol. 18: 978–984. Keller, L. 1997. Hierarchical analysis of genetic structure in Hendrixson, B.E. & Bond, J.E. 2005. Testing species boundaries native fire ant populations: results from three classes of in the Antrodiaetus unicolor complex (Araneae: Mygalomor- molecular markers. Genetics 147: 643–655. phae: Antrodiaetidae): ‘‘paraphyly’’ and cryptic diversity. Mol. Ru¨ ppell, O., Heinze, J. & Ho¨ lldobler, B. 2001. Alternative Phylogenet. Evol. 36: 405–416. reproductive tactics in the queen-size-dimorphic ant Lep- Ho¨ lldobler, B. & Wilson, E.O. 1990. The Ants. The Belknap Press tothorax rugatulus (Emery) and their consequences for genetic of Harvard University Press, Cambridge, MA. population structure. Behav. Ecol. Sociobiol. 50: 189–197. Huelsenbeck, J.P. & Rannala, B. 1997. Phylogenetic methods Ru¨ ppell, O., Stra¨tz, M., Baier, B. & Heinze, J. 2003. Mitochon- come of age: testing hypotheses in an evolutionary context. drial markers in the ant Leptothorax rugatulus reveal the Science 276: 227–232. population genetic consequences of female philopatry at Hughes, W.O.H., Sumner, S., Borm, S.V. & Boomsma, J.J. different hierarchical levels. Mol. Ecol. 12: 795–801. 2003. Worker caste polymorphism has a genetic basis in Sanetra, M. & Buschinger, A. 2000. Phylogenetic relationships Acromyrmex leaf-cutting ants. Proc. Natl. Acad. Sci. USA 100: among social parasites and their hosts in the ant tribe 9394–9397. Tetramoriini (Hymenoptera: Formicidae). Eur. J. Entomol. 97: Janda, M., Folkova´, D. & Zrzavy´, J. 2004. Phylogeny of Lasius 95–117. ants based on mitochondrial DNA and morphology, and the Savolainen, R. & Vepsa¨laïnen, K. 2003. Sympatric speciation evolution of social parasitism in the Lasiini (Hymenoptera: through intraspecific social parasitism. Proc. Natl. Acad. Sci. USA Formicidae). Mol. Phylogen. Evol. 33: 595–614. 100: 7169–7174. Johannesson, K. 2001. Parallel speciation: a key to sympatric Schlick-Steiner, B.C., Steiner, F.M., Sanetra, M., Heller, G., divergence. Trends Ecol. Evol. 16: 148–153. Stauffer, C., Christian, E. & Seifert, B. 2005. Queen size

ª 2005 THE AUTHORS 19 (2006) 777–787 JOURNAL COMPILATION ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY Myrmica microrubra: no sympatric speciation 787

dimorphism in the ant Tetramorium moravicum (Hymenoptera, Wiens, J.J., Chippindale, P.T. & Hillis, D.M. 2003. When are Formicidae): Morphometric, molecular genetic and experi- phylogenetic analyses misled by convergence? A case study in mental evidence. Insect. Soc. 52: 186–193. Texas cave salamanders. Syst. Biol. 52: 501–514. Schluter, D. 2001. Ecology and the origin of species. Trends Ecol. Wiens, J.J. & Reeder, T.W. 1997. Phylogeny of the spiny lizards Evol. 16: 372–380. (Sceloporus) based on molecular and morphological evidence. Schneider, S., Roessli, D. & Excoffier, L. 2000. ARLEQUIN VERSION Herpetol. Monogr. 11: 1–101. 2.000: A Software for Population Genetic Analysis. Genetics Wiens, J.J. & Servedio, M.R. 2000. Species delimitation in and Biometry Laboratory, University of Geneva, Geneva. systematics: inferring diagnostic differences between species. Schwarz, M.P., Bull, N.J. & Cooper, S.J.B. 2003. Molecular Proc. R. Soc. Lond. B 267: 631–636. phylogenetics of allodapine bees, with implications for the Wilson, E.O. 1971. The Insect Societies. Harvard University Press, evolution of sociality and progressive rearing. Syst. Biol. 52:1– Cambridge, MA. 14. Winter, U. & Buschinger, A. 1986. Genetically mediated queen Seifert, B. 1993. Taxonomic description of Myrmica microrubra polymorphism and caste determination in the slave-making n.sp. – a social parasitic ant so far known as the microgyne of ant, Harpagoxenus sublaevis (Hymenoptera: Formicidae). Myrmica rubra (L.). Abh. Ber. Naturkundemus. Go¨rlitz 67: 9–12. Entomol. Gener. 11: 125–137. Seifert, B. 2006. Die Ameisen Mittel- und Nordeuropas. Booksagain, Yang, Z. 1993. Maximum-likelihood estimation of phylogeny Pyrbaum. from DNA sequences when substitution rates differ over sites. Seppa¨, P. & Pamilo, P. 1995. Gene flow and population viscosity Mol. Biol. Evol. 10: 1396–1401. in Myrmica ants. Heredity 74: 200–209. Sites, J.W. & Marshall, J.C. 2003. Delimiting species: a Received 12 August 2005; revised 13 October 2005; accepted 17 October Renaissance issue in systematic biology. Trends Ecol. Evol. 18: 2005 462–470. Steiner, F.M., Schlick-Steiner, B.C., Trager, J.C., Moder, K., Sanetra, M., Christian, E. & Stauffer, C. 2006. Tetramorium Supplementary material tsushimae, a new invasive ant in North America. Biol. Invasions. The following supplementary material is available for this Stille, M. 1996. Queen/worker thorax volume ratios and nest- article online: founding strategies in ants. Oecologia 105: 87–93. Sumner, S., Aanen, D. K., Delabie, J. & Boomsma, J.J. 2004. The Fig. S1 Core part of the recursion based Java applet evolution of social parasitism in Acromyrmex leaf-cutting ants: implementing eqn (1). a test of Emery’s rule. Insect. Soc. 51: 37–42. Swofford, D.L. 1998. PAUP*: Phylogenetic Analysis Using Parsimony Fig. S2 Probability P under the 1-species hypothesis to (*and Other Methods). Version 4.0b3. Sinauer, Sunderland, MA. not see haplotypes common to Myrmica microrubra and Tavare´, S. 1986. Some probabilistic and statistical problems on M. rubra when sequencing equal numbers of specimens the analysis of DNA sequences. Lec. Math. Life Sci. 17: 57– of each ant, for numbers between two and ten extant 86. haplotypes per locality, calculated using eqn (1) under Templeton, A.R. & Sing, C.F. 1993. A cladistic analysis of the assumption that all haplotypes are equally frequent phenotypic associations with haplotypes inferred from restric- at a locality and with equal frequency occur in tion endonuclease mapping. IV Nested analyses with clado- gram uncertainty and recombination. Genetics 134: 659–669. M. microrubra and M. rubra. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Table S1 Geographical distribution of the ant Myrmica Higgins, D.G. 1997. The ClustalX windows interface: flexible microrubra. strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 25: 4876–4882. Table S2 Collecting sites and numbers (n) of speci- Via, S. 2001. Sympatric speciation in animals: the ugly duckling mens analyzed of Myrmica microrubra, M. rubra and the grows up. Trends Ecol. Evol. 16: 381–390. outgroup species M. ruginodis, for the different disciplines; Volny, V.P. & Gordon, D.M. 2002. Genetic basis for queen- type and paratype specimens marked *; locality code worker dimorphism in a social insect. Proc. Natl. Acad. Sci. USA 99: 6108–6111. given for sites analyzed genetically. Walin, L., Seppa¨, P. & Sundstro¨ m, L. 2001. Reproductive Table S3 Abbreviations and definitions of the morph- allocation within a polygyne, polydomous colony of the ant ometric characters analyzed in Myrmica microrubra and . 26: 537–546. Myrmica rubra Ecol. Entomol. M. rubra gynes and males. Ward, P.S. 1996. A new workerless social parasite in the ant genus Pseudomyrmex (Hymenoptera: Formicidae), with a dis- Table S4 Genetic diversity of three microsatellite loci cussion of the origin of social parasitism in ants. Syst. Entomol. (MS 26, MS 86, MS 3.62) analyzed in Myrmica microrubra 21: 253–263. and M. rubra from five localities. Wetterer, J.K., Schultz, T.R. & Meier, R. 1998. Phylogeny of fungus-growing ants (Tribe Attini) based on mtDNA sequence This material is available as part of the online article from and morphology. Mol. Phylogenet. Evol. 9: 42–47. http://www.blackwell-synergy.com Wiens, J.J. 1999. Polymorphism in systematics and comparative biology. Annu. Rev. Ecol. Syst. 30: 327–362.

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