Independent hybrid populations of Formica polyctena X rufa wood ants (Hymenoptera: Formicidae) abound under conditions of forest fragmentation

ORI GIN AL PA PER

Independent hybrid populations of Formica polyctena Xrufa wood ants (Hymenoptera: Formicidae) aboundunder conditions of forest fragmentation

Bernhard Seifert • Jonna Kulmuni • Pekka Pamilo

Received: 8 October 2009 / Accepted: 15 February 2010 / Published online: 2 March 2010� Springer Science+Business Media B.V. 2010

Abstract Combined genetic and morphological data indicate frequent hybridisation

between the wood ants Formica polyctena Forster 1850 and F. rufa Linnaeus 1761 in

Central Europe. The genetic and morphological traits give a concordant picture of

hybridisation with a strong correlation between the genotypic admixture proportions at 19

microsatellite loci and the first vectors of a principal component analysis (P \ 0.001) and

of a 3-class discriminant analysis (P \ 0.001) of 15 quantitative morphological characters.

This integrative approach enabled a grouping into F. polyctena, the hybrid and rufa.

Genetic differentiation between the hybrid and F. rufa is significantly larger than between

the hybrid and polyctena, indicating gene flow mainly between the latter entities. A sug-

gested gene flow bias towards F. polyctena agrees with differential queen acceptance and

mating behaviour. Both genetic and phenotypic colony parameters indicate predominance

of monogyny in F. rufa but of polygyny in polyctena and the hybrid. Hybrids are inter-

mediate between the parental species in body size, diagnostic morphological characters,

monogyny frequency, size of nest population, nest diameter and infestation rate with

epizootic fungi. The three entities respond differently to woodland fragmentation. Hybrids

are significantly more abundant in forests with a coherent area\300 ha than in woodland

above this size. Regions with high hybrid frequency in Germany—the Eastern Oberlausitz

(23%) and the Baltic Sea islands Darss, Hiddensee and Rugen (28%)—are characterised by

a fragmented woodland structure whereas regions with low hybrid frequency—Branden-

burg and the lower Erzgebirge (3.4%)—have clearly larger and more coherent forest

Electronic supplementary material The online version of this article (doi:10.1007/s10682-010-9371-8)contains supplementary material, which is available to authorized users.

B. Seifert (&)Senckenberg Museum of Natural History Gorlitz, Am Museum 1, 02826 Gorlitz, Germanye-mail: [email protected]

J. KulmuniDepartment of Biology and Biocenter, University of Oulu, Box 3000, 90014 Oulu, Finland

P. PamiloDepartment of Biosciences, University of Helsinki, Box 65, 00014 Helsinki, Finland

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Evol Ecol (2010) 24:1219–1237DOI 10.1007/s10682-010-9371-8

http://dx.doi.org/10.1007/s10682-010-9371-8

systems. Data from other European countries indicate habitat fragmentation to be a

facilitating factor but no essential precondition for interspecific hybridisation in these ants.

Hybrids are hypothesised to have selective advantage in fragmented systems because of

combining the main reproductive and dispersal strategies of the parental species.

Keywords Interspecific hybrids � Habitat fragmentation � Microsatellites �Morphometry � Integrative taxonomy

Introduction

The wood ant species Formica polyctena Forster 1850 and Formica rufa Linnaeus 1761

are important elements of temperate forest ecosystems of the West Palaearctic. They are

considered to give protection against a number of pest insects in natural and secondary,

managed forests (reviewed by Otto 1967) and are a symbol and main target of nature

conservation in many countries of Europe. Yarrow (1955) and Betrem (1960) considered

F. polyctena and F. rufa as clearly different species and this view had been generally

adopted for 35 years. The situation changed when Seifert (1991) published a compre-

hensive study on external morphology and biological parameters of 430 nests collected in

different regions of Central, East and North Europe. In addition to the typical F. polyctenaand F. rufa, he found a third entity which was intermediate in each investigated phenotypic

or biological character: body size, eight size-corrected pilosity characters, monogyny

frequency, size of nest populations, diameter of nest mounds and infestation rate with

epizootic fungi. He concluded that the third entity was a fertile hybrid between

F. polyctena and F. rufa.In Germany, the hybrid was particularly abundant in a landscape not poor in woodland

but with a high degree of forest fragmentation: as many as 27.4% of 212 investigated nests

were identified as hybrids in eastern Oberlausitz. In contrast, the hybrid was rather rare

(only 6.6% of 218 investigated nests) in regions with large, coherent forests. Attempting to

explain this finding, Hofener et al. (1996) simulated wood ant populations in fragmented

versus coherent woodland systems—parameters considered were distribution of Servifor-mica host nests, dispersal of colonies and queens, number and growth of worker and queen

population, mating and colony founding behaviour of queens, intranidal queen dominance

and acceptance, mortality of colonies and alates, exchange of nest populations within

polycalic systems and territorial fight. They argued that the hybrid can display a selective

advantage in fragmented woodland systems by combining the potency of F. rufa for

dispersal flight and single-queen socially parasitic colony foundation with the potency of

F. polyctena for shifting to polygyny, propagation by colony-fission and building up

locally dominant polydomous colonies.

Eighteen years later, the existence of a hybrid population remains to be the most

probable explanation for eastern Oberlausitz, but the alternative hypothesis that the

intermediate entity represents a morph of F. rufa with no hybridisation cannot be fully

excluded—the more as there has been no genetic analysis so far. In this paper, we want to

overcome this deficiency and present an investigation of nuclear DNA markers (micro-

satellites) in combination with a refined analysis of external morphology. We focus this

combined genetic and morphological approach on the ‘‘hot spot’’ of hybridisation in the

eastern Oberlausitz but will also apply the morphology-based method to describe the

situation in Europe as a whole. Furthermore we will reinvestigate the data of Seifert (1991)

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with more advanced data processing methods and integrate these and the current data in the

analysis of forest fragmentation with the aim to confirm Seifert’s hypothesis of higher

hybrid abundance in fragmented forest systems.

Materials and methods

Nest samples

Fifty nest samples (17 of Formica polyctena Forster 1850, 20 of F. rufa Linnaeus 1761, 12

of F. polyctena x rufa; one F. polyctena nest sampled in two years), collected in the eastern

Oberlausitz, Germany within an elliptic area of about 420 km2 were investigated both

morphologically and genetically (SI 1, online appendix).

A total of 618 Formica polyctena et rufa complex samples collected during the years

1962–2008 were evaluated by morphology alone (Table 1). Of these 148 were investigated

with the current system (see below) and 470 samples with the earlier investigation system

of Seifert (1991).

Table 1 Nest samples of the F. polyctena et rufa complex investigated in the exclusively morphology-based approach and determinations derived from this method

Country New data system Old data system

All poly pXr rufa All poly pXr rufa

Austria 1 1

Belgium 5 1 4

Czech Republic 1 1

England and Wales 19 1 16 2 3 2 1

Finland 14 9 1 4 2 1 1

France 1 1

Germany 84 33 17 34 418 154 80 184

Greece 3 3

Poland 4 1 2 1

Russia, Moscow 4 3 1 15 4 9 2

Russia, Voronesh 12 6 5 1

Spain 1

Sweden 2 1 1 19 11 2 6

Switzerland 10 3 5 2

All countries 148 51 45 52 470 176 96 199

‘‘New data system’’ applies to samples collected in 1981–2008 and subject to the current investigationsystem and ‘‘old data system’’ to samples taken in 1962–1990 and subject to the former investigation system(Seifert 1991). Because of the need to increase the number of hybrid samples, hybrid frequencies are muchlarger than those expected for random sampling—with the exception of realistic ratios in Finland andBritain. Acronyms: poly—F. polyctena, pXr—hybrid, rufa—F. rufa. Detailed collecting data are availableon request from the primary author

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Rationale of hybrid identification using the combined approach

The main rationale of hybrid identification included the following steps:

(1) The 3-class discriminant analysis (DA) of quantitative morphological data of the new

data system (see below) was used to form an initial hypothesis on whether the sample

could belong to the F. polyctena or F. rufa species.

(2) The gene pools of the parental species for estimating admixture frequencies were

defined by combining the morphological hypothesis and the Bayesian mixture

analysis of genotypes.

(3) We then compared genetic classifications with morphological data using both

hypothesis-driven (DA) and unsupervised (PCA) approaches to improve hybrid

identification and to re-evaluate conflicting determinations.

DNA extraction and amplification

DNA was extracted from five workers per nest with DNAeasy Tissue Kit (Qiagen) using

the manufacturer’s protocol designed for insects. We genotyped a total of 19 microsatellite

loci per individual. The microsatellite primers used were designed for F. exsecta(Gyllenstrand et al. 2004) FE7, FE13, FE17, FE19, FE37, FE38, FE42, for F. paralugubris(Chapuisat 1996) FL12, FL20, FL29 and for F. yessensis (Hasegawa and Imai 2004) FY3,

FY4, FY5, FY7, FY9, FY10, FY12, FY13, and FY15. Genotypes were assayed by poly-

merase chain reaction with fluorescent labelling using Peltier Thermal Cycler-200-PCR

equipment (MJ Research). Microsatellite loci were amplified by initial round of denaturing

at 94�C for 2 min, followed by 34 cycles of 94�C for 30 s, 48–65�C (depending on primer

pair; SI 2 of online appendix) for 30 s, 72�C for 30 s, with a final step of 72�C for 30 min.

PCR reactions were performed in a reaction volume of 10 ll containing 10 9 Mg2? -free

dyNAzymeTM buffer (Finnzymes), 250 lM of each dNTP, 0.5 lM labelled primer,

0.5 lM unlabelled complementary primer, 0.5 U dyNAzymeTM II DNA polymerase

(Finnzymes),1–3.5 lM MgCl2 (Finnzymes; depending on primer pair; SI 2 of online

appendix),1 ll template (genomic DNA of unknown concentration) and H2O up to final

volume. Genotypes were resolved by capillary electrophoresis (3730 DNA Analyzer,

Applied Biosystems) using 500 LIZ size standard (Applied Biosystems) and scored with

Genemapper v.4.0 (Applied Biosystems).

Analysis of the genotype data

Admixture proportions were estimated for each individual on the basis of their

microsatellite genotypes by using a Bayesian admixture model in the program BAPS

(Corander and Marttinen 2006). We first used the mixture model of BAPS to delineate

genetically homogeneous groups by forcing the number of groups to be three. The

groups represented well morphologically identified F. polyctena, F. rufa and the hybrids.

One nest which was morphologically identified as hybrid but genetically clustered with

F. polyctena was removed from the F. polyctena cluster in order to minimize the

presence of possible hybrids in the two parental gene pools which were then used in the

admixture analysis. Hybridisation was assessed also by using a Bayesian estimation of

parental and hybrid genotypes without any information on the morphological clustering.

This was done using the program NewHybrids (Anderson and Thompson 2002) which

aims to infer recent hybrids (F1, F2, backcrosses) from the genotypic data (the number

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of sweeps was 20,000 for burnIn and 100,000 for the data gathering). The power of the

program NewHybrids to detect possible hybrids was evaluated by computer simulations,

following the recommendations by Vaha and Primmer (2006). The simulations were

carried out by assuming that the morphologically identified F. polyctena and F. rufarepresent the parental gene pools. Simulated genotypes including the two parental species

(30% each), F1 and F2 hybrids, and both types of backcrosses (10% of each category)

were formed and then analysed by NewHybrids. Simulations were repeated ten times.

The simulated data showed that NewHybrids finds F. rufa with a high precision (effi-

ciency and accuracy [90%) but tends to mix F. polyctena and the hybrids and does not

well separate different types of hybrids from each other. We present the NewHybrids

results by pooling the four hybrid classes and calculating for each individual the overall

probability that it is a hybrid. Furthermore, principal coordinate analysis (program PCO,

Andersson 2003) based on pairwise genetic distances between pairs of nests was used to

examine the genetic relationships among the nests. Genic differentiation between groups

was estimated as FST by using the algorithm of Weir and Cockerham (1984). The

significance of the pairwise estimates was tested by permutation of nests (1,000 times).

Genetic relatedness among worker nest mates was estimated using the program GENREL

(Pamilo 1984).

Morphological data recording

A detailed description of morphological data recording allowing reproducibility is given in

SI 3 of the online appendix. Here we only inform on the basic principles. Analysis of the

individual composition of nest samples was performed to check if intermediate sample

means (=hybrid indication) might be caused by intranidal coexistence of pure parent

phenotypes. The basic unit for the identification of the three entities was nest sample means

because only the same nests but not the same individuals were subject to a parallel

morphological and genetic investigation. An average of six fully dried workers per nest

mounted on a pinned cardboard were evaluated for fifteen numerically scored phenotypic

characters. In order to save working time, sample size was occasionally reduced to three

when prior visual inspection showed a monomorphic worker population but it was

extended to nine to have more accurate sample means in case of polymorphic nest

populations. All measurements and counts were made using a high-performance stereo-

microscope Leica Wild M10 equipped with a 1.6 9 planapochromatic front lens at

160–320 9 magnification. Standard positioning of the specimens was achieved under use

of a pin-holding stage permitting full 3-dimensional rotations.

Seifert (1991) described the characters and data recording used in the earlier morpho-

logical investigation system. The main difference of the 1991 investigation to the current

system was the use of unmounted, ethanol-stored specimens, a smaller and partially

deviating character set but higher average numbers of investigated specimens per nest. This

did not allow pooling the 1991 and current data in a combined morphological analysis.

However, the determination of species with these separate analyses was used in the

analysis of the effects of forest fragmentation.

Removal of allometric variance

In most species groups of Formica, morphological characters are strongly influenced by

allometric growth. In order to make characters directly comparable in synoptic tables, a

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removal of allometric variance (RAV) was performed with the procedure described by

Seifert (2008). RAV was calculated assuming that all individuals have an identical

cephalic size of 1.75 mm. We applied RAV functions in which the collective parameters

were calculated as the arithmetic mean of the species-specific functions of twelve Palae-

arctic Formica rufa group species. Evaluation of scatter plots indicated the use of mon-

ophasic linear RAV functions (online appendix SI 4).

Processing of morphological data

Canonical discriminant analyses (DA) and principal component analyses (PCA) of abso-

lute head size CS and the fourteen RAV-corrected characters were run using the SPSS 10.0

statistical package. A parallel run of an ordinary DA and of a ‘‘leave-one-out cross-

validation’’ DA (LOOCV-DA, Lachenbruch and Mickey 1968, Lesaffre et al. 1989) was

performed to realistically estimate the error rate. The data presented by Seifert and Schultz

(2008) show that the mean of the pessimistic error indication by the LOOCV-DA and of

the optimistic error indication by the ordinary DA—here called integrated error rate—is

close to the true error.

The starting point was a supervised analysis by a DA assuming three classes:

F. polyctena, F. rufa and putative F. polyctena x rufa. If a run of a DA rejected an a priorihypothesis of a sample, the new hypothesis was taken up and iterative runs were performed

until the indicated error rate achieved a minimum. This partially self-organising approach,

in which only the starting situation is set by a hypothesis of the investigator, was applied to

reduce the subjective component and led to changed determinations in a number of

samples. All 148 European samples were included to increase the mean sample size per

entity to a number required for a reasonable application of a DA.

The data of Seifert (1991) were reinvestigated by the same analysis systems but without

RAV corrections.

Estimation of monogyny frequency, nest diameter, nest-population size

and fungal infestation

Monogyny of specific nests was determined by a modification (Seifert 1991) of the

function of Otto (1960) which uses arithmetic mean, skewness and kurtosis of the head

width distribution of 30–170 workers randomly collected from the mound surface.

Nest diameter refers to the perimeter described by the most peripheral nest entrances.

These are either at mound base or within the outer ring of soil ejections. In nests with an

elliptic basal area, the arithmetic mean of the large and small diameter is recorded.

The nest-population size figure A is a dm2-estimate of nest surface covered by ants

during the main activity period from late April to late September under exclusion of

climatic situations with activity depression. As a rule the estimate was performed at air

temperatures at the nest site between 15� and 22�C. A is the estimated per cent ratio of nest

surface covered by ants multiplied with the whole nest surface calculated by geometric

formulae.

The infection with epizootic fungi was recorded during morphological inspection. There

was no species determination by mycologists but it is generally assumed that ants of the

Formica rufa group are parasitised by Aegeritella superficialis Balazy et Wisniewski 1974

and closely related species and to a lesser degree by Erynia sp. (Wisniewski 1976, 1977;

Espadaler and Wisniewski 1987; Espadaler and Monteserın 2003).

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Estimation of woodland fragmentation

Woodland fragmentation is inversely proportional to the mean size of coherent woodland

areas in a given area. Area was considered coherent if the woodland patches were linked by

woodland stripes of at least 15 m width whereas lines of single trees along streets and

highways were not regarded as linking elements. Any woodless agricultural, natural, set-

tlement or industrial area separating forest patches by more than 30 m was considered to be

an isolating structure. Motor highways, railway lines and rivers wider than 15 m were also

considered as fragmenting elements. This method pays attention to structures which rep-

resent likely barriers for ground movements of wood ant nests but not to spots which mated

gynes can reach by dispersal flight. Forest area was estimated by summing up areas of

approximated geometric forms (triangles, parallelograms, ellipses etc.) under use of the air

photographs and ruler tool provided by the Google-Earth� 2009 Tele Atlas.

The analysis of woodland fragmentation was only done for German sites with exact

geographic information. We pooled the old data of Seifert (1991), including 411 samples

collected between 1979 and 1989, with data of the new investigation giving a total of

495 samples. We further added 58 samples which were determined by subjective assess-

ment. Since we morphotyped only few nests from phenotypically unambiguous

F. polyctena supercolonies in larger forests but usually all nests in very small forest

patches, the overall abundance of this species in larger forests is strongly underestimated

by the morphometric data set in Table 1.

The analysis included a total of 127 German forest areas differing in size between 0.01

and 65,000 ha. Three patches within forest areas\100 ha were oversampled because of a

detailed local study: Liebstein West (n = 44 samples), Grossmachnow (n = 21) and

Deutsch-Paulsdorf (n = 17). Considering the fact that the maximum sample number in

patches without oversampling was 15 and in order to reduce a bias of data in regional or

overall analyses, the data from these three patches were given a weighting factor of

w = 15/n (i.e., 0.34, 0.71 and 0.88). The weighting factor in the 124 remaining patches

was 1.0. The chi-square test of independence (X2 test) to check the frequency of the hybrid

against those of the parental species was performed as recommended in Sokal and Rohlf

(1995).

Results

Initial definition of morphospecies

Using the current data pool, a self-organizing, iterative 3-class DA stabilised at an inte-

grated error rate of 1.3% and discriminated 93.2% of the 148 European samples with

posterior probabilities of P [ 0.95 (Fig. 1). The rather high 6.8% proportion of cases with

P \ 0.95 was caused by unclear determinations within the hybrid/parental species tran-

sition zones which are unavoidable if hybrids are fertile and backcross with both parental

species.

Using the data pool of Seifert (1991), an iterative 3-class DA stabilised at an integrated

error rate of 0.7% and discriminated 87.2% of the 470 European samples with posterior

probabilities of P [ 0.95. Compared to the determinations by Seifert (1991), the new type

of analysis resulted in the following changes: 1.5% reduction in F.polyctena, 3.6% increase

in the hybrid and 2.1% reduction in F. rufa. All changes occurred in the transition zone

between the hybrid and the parental species.

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Bayesian clustering of microsatellite genotypes

The nineteen loci had a total of 120 alleles (95 in F. polyctena and 88 in F. rufa). The mean

heterozygosity per locus was 0.53 in F. polyctena, 0.49 in F. rufa and 0.56 in the hybrids.

The parent species had no clear diagnostic allele differences, and the amount of differ-

entiation between them was FST = 0.13 ± 0.033 (P \ 0.001). The distance of putative

hybrids to F. polyctena was FST = 0.05 ± 0.012 (P \ 0.001) and to F. rufa FST = 0.10 ±

0.021 (P \ 0.001).

Genetic clustering of the nests with a Bayesian mixture analysis agreed well with the

morphological identification (Table 2). Sample No 128 was genetically clustered together

with F. polyctena but because it was morphologically identified as hybrid we moved it to

the hybrid group in further analyses. Otherwise, the group which was genetically con-

sidered to be hybrids included also some nests which were morphologically identified as

either F. polyctena or rufa (Table 2). We calculated for each individual two estimates for a

hybrid probability, the admixture proportion with BAPS and the overall hybrid probability

with NewHybrids. The two estimates show a significant correlation (Spearman rank cor-

relation rs = 0.69, df = 245, P \ 0.001). Using the morphological classification of nests,

the median admixture proportions differed significantly from each other between the

groups (Mann–Whitney’s test t?[ 8.3, P \ 0.001).

We compared morphological identification and genetic admixture proportions on the

level of nest means (Table 2; Fig. 2). This bears the risk of a wrong hybrid indication in

case of coexistence of both parent species in the same nest. This risk was found to be very

low. According to morphotyping, there were only 1.3% of 148 nests with mixtures of

parental species in the whole W Palaearctic range and within the 50 genetically typed

samples from the Oberlausitz only nest No 125 matched this condition. It contained 3

F. polyctena, 1 hybrid and 2 F. rufa workers according to morphotyping and 1 F.polyctena, 2 hybrids and 2 F. rufa according to genotyping (note that morpho- and

genotyping used same nests but different individuals!).

Fig. 1 Canonical discriminant analysis of 15 phenotypic characters of worker nest sample means ofFormica polyctena (black rhombs), F. polyctena x rufa (grey triangles) and F. rufa (white squares) of 148samples from the entire European range

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Table 2 Initial morphological grouping by a 3-class DA, genetic grouping (k = 3) and percentage ofF.polyctena alleles determined by Bayesian clustering with BAPS, within-nest relatedness according to theGENREL programme and hybrid probability estimated by NewHybrids in 49 samples of the Formicapolyctena et rufa complex in the eastern Oberlausitz, Saxony, Germany. The percentage of predicted F. rufaalleles is complementary and not given. Data based upon 5 workers per nest sample

No. Site Morphol.group

Geneticgroup

Admixture(% polyctena,BAPS)

Related-ness

Hybrid probability(NewHybrids)

32 Deutsch-Paulsdorf (1996) poly poly 97.0 0.15 0.03




198 Diehsa (2005) poly poly 99.0 0.34 0.03

199 Diehsa (2005) poly poly 99.8 0.31 0.04

200 Diehsa (2005) poly p X r 76.2 0.26

201 Diehsa (2005) poly poly 73.6 0.22 0.71

19 Ebersbach (1996) poly poly 99.0 0.26 0.02

129 Ebersbach (1997) poly p X r 88.6 0.08

1 Girbigsdorf (1996) poly p X r 76.8 0.11

1 Girbigsdorf (2005) poly p X r 69.8 0.30

17 Gorlitz, cemetery (1996) poly poly 92.6 0.21 0.19

18 Gorlitz, cemetery (1996) poly poly 94.8 0.47 0.09

3 Konigshain (1996) poly poly 88.0 0.31 0.45



21 Ludwigsdorf (1996) poly poly 97.0 0.68 0.03

31 Deutsch-Paulsdorf (1996) p X r p X r 80.4 0.02



125 Ebersbach (1997) p X r p X r 36.0 0.48

128 Ebersbach (1997) p X r p X r 73.0 0.20

9 Liebstein West (1996) p X r p X r 59.4 0.36







196 Diehsa (2005) rufa rufa 0.0 0.67 0.00

197 Diehsa (2005) rufa rufa 8.4 0.68 0.02

202 Diehsa (2005) rufa rufa 0.6 0.76 0.11

203 Diehsa (2005) rufa rufa 6.4 0.18 0.28

204 Diehsa (2005) rufa rufa 0.0 0.79 0.00

20 Ebersbach (1996) rufa rufa 4.6 0.59 0.03




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Regarding intranidal relatedness, we refrained from presenting estimates for the hybrid

nests because it is difficult to define the proper reference population. Within-nest relat-

edness was estimated as r = 0.60 (SE = 0.04) in 19 F. rufa nests and r = 0.31

(SE = 0.04) in 14 F. polyctena nests (Table 2)—after removing the effects of inbreeding,

the estimates changed to r* = 0.45 in F. rufa and r* = 0.00 in F. polyctena. The results

indicate that F. rufa nests in the Oberlausitz are largely monogynous or have only a small

number of queens, whereas F. polyctena is almost completely polygynous. This is in line

with monogyny frequencies determined by combining Otto-function estimates with data

from field observations (Seifert 1991). Extracting only the Oberlausitz subsample from

Seifert’s data pool, monogyny frequencies in this region are 78.6% in 70 F. rufa nests,

13.7% in 51 hybrid nests and 4.0% in 75 F. polyctena nests.

Matching of genetic and morphologic data

Genetic clustering based on admixture proportions was highly correlated with morpho-

logical clustering performed with both the hypothesis-driven and explorative approaches.

The 1st canonical vector of a DA of the all-European morphological data set is highly

correlated with the proportion of F. polyctena alleles (r = -0.900, P \ 0.0001, Fig. 2) as

is the 1st factor of a PCA (r = -0.845, P \ 0.0001, data not shown). The picture of strong

correlation of morphology and genetics is only disturbed by sample No 130 which has

44.6% F. polyctena alleles (Fig. 2). This sample is genetically a hybrid both in the mean

and in individual data of all five workers; in contrast all five morphotyped workers are very

clear F. rufa. This mismatch is difficult to explain; we cannot exclude a confusion of

samples. Anyway, a mismatch ratio of only 2% between morphology and genetics is a very

good result for a hybrid scenario with parent species not having private alleles. We decided

to use the morphological determination of sample No 130 in any of the results presented

below.

A principal coordinate analysis (PCO) based on pairwise FST estimates between the

nests treated the genetic data in a very different way and is thus an independent approach.

The first two axes of PCO explained somewhat over 50% of all the genetic variation. The

Table 2 continued

No. Site Morphol.group

Geneticgroup

Admixture(% polyctena,BAPS)

Related-ness

Hybrid probability(NewHybrids)


205 Groditz E (2005) rufa rufa 0.2 0.50 0.01

206 Groditz E (2005) rufa rufa 9.2 0.76 0.04

2 Konigshain (1996) rufa rufa 6.0 0.45 0.02



124 Liebstein South (1997) rufa rufa 0.0 0.72 0.00




130 Liebstein South West (1997) rufa p X r 44.6 0.83

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morphotypes of F. polyctena and F. rufa are well separated along the first axis, and the

hybrid morphotypes are partly intermediate and partly overlap with F. polyctena (Fig. 3).

This confirms the indication by the Bayesian analysis that more than 50% of the hybrid

genomes are from F. polyctena. There are several possible factors which could explain this

asymmetry—first of all selection for genotypes and dilution of F. rufa alleles by

Fig. 2 First canonical vector of a discriminant analysis of 15 quantitative morphological characters (largelyseparating the parental species) plotted against the percentage of F. polyctena alleles obtained from aBayesian admixture analysis of 19 microsatellite loci. The percentage of F. rufa alleles is complementary

Fig. 3 Principal coordinate analysis of pairwise genetic distances between nest samples. The two shownaxes explain a little over 50% of variation found in 19 microsatellite loci. The first axis indicates differencesbetween the species and the 2nd axis differences between the nests within a species. The much largervariation along the second axis in F. rufa is probably resulting from the much higher level of monogyny

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differential mating behaviour. The second axis separates mainly F. rufa nests, probably

because there are stronger genetic differences due to monogyny.

Intermediate position of hybrids in colony parameters

The intermediate position of hybrids is also evident in colony parameters (Table 3). The

hybrids differ significantly from the parental species in the size of nest population, nest

diameter and infestation ratio with epizootic fungi. These characters are largely a conse-

quence of colony structure: growing queen number increases the size of nest populations

and nest diameter while propagation by nest-splitting favours spreading of fungal

infestations.

Increased hybrid frequency in regions with woodland fragmentation

The distributions of F. polyctena, F. rufa and the hybrids in forests smaller or larger than

300 ha differ from each other (Table 4). The hybrid is found in Germany relatively more

often in small forest patches than F. polyctena (X2 13.82, P \ 0.001) and F. rufa (X2 14.82,

P \ 0.001). The distribution of the parental species is not different under this classification

(X2 0.05, P = 0.459). When classifying the forest patches into ‘‘very small’’ (\5 ha),

‘‘small’’ (5–299 ha) and ‘‘large’’ (300–65000 ha), the distribution of the hybrid differs

Table 3 Biological data of F. polyctena et rufa complex species from entire Germany. Significance testingof per cent ratios by X2 test, of nest diameter and population size by a two-tailed t test; n = number ofinvestigated nests

Monogyny Nest diameter(cm)

Population sizefigure (dm2)

Ascomyceteinfestation

F. polyctena 3.0% (n = 135) 201 ± 123 (n = 95) 66.8 ± 88.6 (n = 102) 26.1% (n = 153)

F. polyctena vs.hybrid

P \ 0.039 P \ 0.001 P \ 0.001 P \ 0.056

Hybrid 11.7% (n = 77) 131 ± 68 (n = 70) 24.2 ± 25.0 (n = 69) 14.9% (n = 74)

Hybrid vs. F. rufa P \ 0.001 P \ 0.001 P \ 0.001 P \ 0.001

F. rufa 81.0% (n = 153) 91 ± 40 (n = 136) 7.1 ± 8.7 (n = 144) 1.7% (n = 177)

Table 4 Frequency of the three entities of the F. polyctena et rufa complex on sampling spots embeddedwithin different areas of coherent woodland given as absolute numbers corrected for oversampling and aswithin-species percentages

Area interval of coherentwoodland (ha)

Mean areasize (ha)

Number ofareas

Hybrid F.polyctena F. rufa

Two forest area classes

[0,300) 60.0 74 58.6 (= 72.7%) 109.1 (= 48.70%) 100.9 (= 47.6%)

[300,65000] 11538 58 22.0 (= 27.3%) 115.0 (= 51.3%) 111.0 (= 52.4%)

Three forest area classes

(0,5) 1.5 27 16.0 (= 19.9%) 19.0 (= 8.5%) 37.0 (= 17.5%)

[5,300) 77.9 47 42.6 (= 52.9%) 90.0 (= 40.2%) 63.9 (= 30.2%)

[300,65000] 1,1538 58 22.0 (= 27.3%) 115.0 (= 51.3%) 111.0 (= 52.4%)

1230 Evol Ecol (2010) 24:1219–1237

123

clearly from both F. polyctena (X2 16.47, P \ 0.001) and F. rufa (X2 16.53, P \ 0.001) but

also the parent species differ (X2 9.95, P = 0.007).

Discussion

The intermediate position of hybrids and their relation to the parental species

Different lines of evidence provide a concordant and convincing indication for hybridisation

between F. rufa and F. polyctena. The clearest argument is the high correlation of mor-

phological clustering by DA or PCA with genetic clustering based on the Bayesian analysis

(Fig. 1). The intermediate genetic position of morphological hybrids is also supported by the

FST values, the PCO (Fig. 3) and by intermediate biological features (Table 3). Furthermore

the morphological hybrids are genetically intermediate at the individual level (significant

differences of individual admixture proportions in Mann–Whitney test). It should be noted

that the parental pools of F. polyctena and F. rufa shared allelic polymorphisms without clear

diagnostic alleles—the genetic differences were thus frequency differences. If there are

different levels of introgression over many generations, it may become difficult to draw clear

boundaries between the hybrid and the parental species. Considering these limitations, the

clear separation of the three entities (Fig. 1) is remarkable.

Hybrids are intermediate when considering all morphological characters but they

approach F. rufa in the two characters nGU and GuHL which most strongly contribute to

the separation of the parent species in a DA (online appendix, SI 5). This has led to the

alternative hypothesis that the hybrid phenotype could represent a morph of F. rufa (one of

the reasons to conduct this study). This hypothesis is clearly rejected by the genetic

analysis. Genetically, hybrids are closer to F. polyctena as indicated by their mean

admixture proportion of 66.3% F. polyctena alleles (different from 50% with t = 4.2,

df = 9, P \ 0.01), by the FST values and by the significant overlap of the hybrid and

F. polyctena cluster in the PCO (Fig. 3). From these data it seems likely that backcrossing

of the hybrid and introgression occurs mainly with the F. polyctena parent. Biased gene

flow towards F. polyctena could be expected on the basis of differential queen acceptance

and mating behaviour of the species (Gosswald 1942, 1981; Seifert 1991) and is also

directly suggested by a microsatellite analysis from Sweden (Gyllenstrand et al. 2004). The

following points seem important in this context:

(a) There is a trend in Formica and in ants in general that workers in polygynous nests

are less aggressive against invading mated queens (e.g., Gosswald 1942; Buschinger

1970; Pisarski 1982; Stuart et al. 1993; Sundstrom 1993, Lecat et al. 2008).

According to Seifert (1991), the frequency of polygynous nests increases in Germany

as a whole from F. rufa (14.1%) to the hybrid (85.7%) and to F. polyctena (97.6%).

Consequently, queen acceptance (and gene flow) should be easier from F. rufa to the

hybrid and to F. polyctena and from the hybrid to F. polyctena.

(b) Gynes of monogynous F. rufa and hybrid nests are larger and physically stronger

than those of polygynous societies (Seifert 1991) and they are supposed to have a

stronger influence on workers in having a more attracting or appeasing pheromonal

system = ‘‘grossere duftliche Dominanz’’ (Gosswald 1981). Both factors give them

a higher fitness during socially parasitic colony foundation in Serviformica but could

also facilitate their adoption in polygynous nests of the F. polyctena et rufa complex

in which weaker gynes are present.

Evol Ecol (2010) 24:1219–1237 1231

123

(c) Gynes and males of the polygynous societies of the hybrid and F. polyctena show a

high frequency of intranidal mating and a reduced tendency to perform a swarming

flight to external mating places (Seifert 1991). In contrast, males and gynes of

monogynous F. rufa nests have a mating flight (Seifert 1991).

(d) Once a F. rufa queen has been accepted in a polygynous nest of a hybrid or of

F. polyctena, her daughters can mate intranidally in the next year and stay as

reproductives in the nest. Such a scenario seems likely since males of polygynous

societies of the hybrid and F. polyctena have regularly been seen to show excessive

and unselective mating behaviour trying to copulate with any female ant on the

mound including their sterile worker nest mates.

The whole data set presented above is best explained when assuming fertility of hybrids

and their enduring existence independent from the parent species. The strongest direct

indication of this is given by their very strong dominance in some isolated woodland

patches. A good example is the locality Liebstein West. According to the data of Seifert

(1991), hybrids accounted for 85% of the worker population and 76% of the nests within

34 wood ant nests in this isolated 7.5 ha woodland patch in 1987/1988—the remaining ants

belonged to F. rufa while F. polyctena was completely absent. The current morphologic

and genetic analyses of 9 and 7 nest samples, respectively found only hybrids in Liebstein

West (Table 2)—undoubtedly hybrids could not have persisted with such a dominance

over 20 years unless fertile and independent from parent species. From an evolutionary

point of view it would be most interesting to check if the whole population in this patch

consists of hybrids and to estimate the genetic contributions of parental species in the

present gene pool.

Hybrid frequency and habitat fragmentation

The underestimation of F. polyctena abundance in large forests (see section Estimation of

woodland fragmentation) increases our estimates of relative hybrid abundance in unfrag-

mented woodland areas. Nevertheless, the data show a significant increase of hybrid fre-

quency with growing woodland fragmentation (P \ 0.001 both against F. polyctena and

F. rufa) when subdividing the forests in two size classes (below and above 300 ha,

Table 4). A subdivision into three size classes—very small, small and large—also showed

significant differences in the overall hybrid distribution compared to those of the parent

species (P \ 0.001 both against F. polyctena and F. rufa). However, the details should be

more complicated: if isolated forest patches become very small, such patches can only be

colonised by flight-dispersal of queens establishing monogynous nests after accidental

extinction of the resident wood ant population (Seifert 1991; Maki-Petays et al. 2005).

Considering the known monogyny frequencies of the three entities (Table 3), the fre-

quency in very small forest patches should be low for F. polyctena, moderate for the hybrid

and largest for F. rufa. The data in Table 4 support this idea: there is a significant

advantage of the hybrid over F. polyctena in very small compared to large patches

(X2 14.24, P \ 0.001). A significant advantage of F. rufa over F. polyctena exists in very

small compared to small patches (X2 = 9.91, P = 0.002) and in very small compared to

large patches (X2 = 5.17, P = 0.023). However, there is no indication that the hybrid is

less abundant than F. rufa in very small patches. This can be seen as an indication of a

well-developed colonising ability of the hybrids in highly fragmented systems as supposed

by Seifert (1991) and Hofener et al. (1996).

1232 Evol Ecol (2010) 24:1219–1237

123

A very high hybrid abundance (23% out of 291 weighted samples) was found in the

Oberlausitz whereas this proportion was only 6% in 227 weighted samples from outside

this area. The main hybridisation area in the Oberlausitz is characterised by a very patchy

and highly differentiated landscape: there is a big number of rocky hills of very different

size within large areas of fertile agricultural land. As a rule, only these hills are covered by

woodland which causes a high degree of woodland fragmentation (Fig. 4). Another region

in Germany with an increased hybrid frequency (28% within 25 samples) is the landscape

complex of the Baltic Sea islands Darss, Hiddensee and Rugen where forest fragmentation

is comparable to Oberlausitz. In contrast, only 3.4% of hybrids were found within 145

samples from Brandenburg and the lower Erzgebirge where larger forests predominate.

Other regions or localities in Europe with high hybrid frequencies were: England and

Wales (82% within 22 samples, collected between 1975 and 2005), the region of Schlatt/

Kanton Zurich (50% within 10 samples, collected 2005), the forest at the Zvenigorod

Biological Station 56 km W of the centre of Moscow (47% within 19 samples, collected

1985) and the Voronesh Nature Reserve in Russia (42% within 12 samples, collected in

1962 by G.M. Dlussky). England and Wales provide an excellent example of strong

woodland fragmentation over several hundred years. The landscape structure near Schlatt

in Switzerland, however, is ambiguous in respect to our hypothesis: forest area is here

strongly structured (with many protrusions and peninsular areas) but according to our

definition it is contiguous for about 1500 ha and the basic woodland distribution has been

stable for more than 100 years. The situation near Zvenigorod does not support our

hypothesis: there are about 2,400 ha of contiguous, compact forest area and this situation

had been stable during the twentieth century (G. Dlussky, pers. comm. 2009). The exact

position of the collecting site in the Voronesh Nature Reserve is unclear, but the area is

remarkable in that it is the only big forest within a huge area of agricultural steppe. The

Fig. 4 Detail of the woodland structure in the hybridisation zone in the Eastern Oberlausitz. Image widthequals 15.3 km. Woodland area given in blackish grey (= dark green in the online version), settlement areain medium grey, agricultural and grassland area in lighter greys and white. Black (= red in the onlineversion) sections in circle diagrams indicate hybrid frequencies for four patches with hybridfrequencies [ 35%

Evol Ecol (2010) 24:1219–1237 1233

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forest was heavily logged before (and after) it became protected in 1927 (UNESCO—

MAB Bioreserves Directory 2009).

Habitat fragmentation and anthropogenous impact on landscape structure have long

been considered to promote interspecific hybridisation in plants and animals (e.g.,

Anderson 1948; Stebbins 1959, 1980; Pearson 1983; Levin et al. 1996; Seifert 1999;

Simberloff 2008). The conclusion from our wood ant data is that forest fragmentation

facilitates hybridisation but is not a necessary precondition as suggested by the Zvenigorod

example. Hofener et al. (1996) explored plausible selective forces behind this process in

computer simulations but they also stated that better data on swarming, dispersal flights

and intranidal dominance behaviour of queens are urgently needed.

Evolutionary and taxonomic implications

Fertile hybridisation between concordantly recognised species has been reported in many

groups of animals (reviewed by Mallet 2006). Ducks and geese (Anatidae) offer a phe-

notypically and numerically most impressing example. Here, postzygotic isolation is often

completely lacking, even intergeneric hybridisation between dramatically different species

is no exception and 76% of 21 British duck species hybridise in the wild (Millais 1902,

1913; Phillips 1915, 1921; Gillham and Gillham 1996). According to Price and Bouvier

(2002), bird hybrids become infertile after average divergence times of 5 myr (passerines)

and 17 myr (nonpasserines).

In contrast to ducks and geese, wood ants of the genus Formica are morphologically

difficult to identify and hybrid phenotypes are likely to escape our subjective perception.

It is therefore likely that the frequency of hybridisation in these ants may be higher than

reported. In addition to the case reported here, credible examples for interspecific

hybridisation detected by genetic or combined genetic and morphological evidence come

from Formica aquilonia Yarrow 1955 9 F. polyctena (Goropashnaya et al. 2004,

Saapunki et al. 2008), F. paralugubris Seifert 1996 9 F. aquilonia (Bernasconi 2009)

and F. pratensis Retzius 1783 9 F. lugubris Zetterstedt 1838 (Seifert and Goropashnaya

2004). In addition, combined morphological and chorological evidence (unpublished

results of B.Seifert) indicates at least another two cases: hybridisation between

F. lugubris and F. rufa in southern Finland and one case of F. truncorum Fabricius 1804

crossing with an indeterminate species (probably F. rufa) in southern Bavaria. These data

document hybridisation in seven out of the nine (78%) currently recognised European

species.

Even if so many Formica species can hybridise, the frequency of hybrids is generally

small in natural populations. Within 250 carefully examined nest samples of F. pratensisand another 150 samples of F. truncorum from Europe (unpublished results of B.Seifert)

less than 1% were suspected to be F1 hybrids with another species. This figure possibly

gives us an idea of the ‘‘normal’’ hybrid frequency between wood ant species at the

individual level and tells us that the European average of 6–8% in F. polyctena X rufa is

exceptional and a potential taxonomic problem. As fertile hybridisation between

F. polyctena and F. rufa is widespread, we have to consider whether they are good species

or genetically determined ecological races or morphs of the same species with different

social and ecological strategies. The latter solution was chosen by Seifert (1991).

Gyllenstrand et al. (2004) showed that in central Sweden the genetic clusters follow

morphological rather than geographical boundaries, strongly supporting the existence of

two separate gene pools, one of F. polyctena and the other of F. rufa. Therefore, we follow

the practice also adopted by Seifert (1996, 2007) and consider them as separate species.

1234 Evol Ecol (2010) 24:1219–1237

123

Both F. polyctena and F. rufa are geographically widespread from western Europe to

western Siberia, and they are genetically very similar. We found no good diagnostic

ncDNA markers and the mtDNA lineages are incompletely sorted with very low nucleotide

diversity (Goropashnaya et al. 2004). It seems reasonable to conclude that the species have

separated not earlier than during the last glaciation, and it is difficult to predict whether

they continue to become more differentiated or will gradually fuse together through

hybridisation. The distribution of individual admixture estimates in the German

F. polyctena x rufa hybrids and their independent persistence in the form of locally

dominant, isolated populations indicate a high fertility and fitness.

Hybridisation is increasingly understood as an important factor of evolution also in

animals (Mallet 2007). Many instances in ants refer to strategic hybridisation with hybrid

workers who need to be viable but not fertile (Seifert 1999, 2006; Umphrey 2006).

Hybridogenous formation of a new species with maintenance of the parental species is

apparently rare in animals. It seems rewarding to consider the F. polyctena et rufa case also

within this context. The hybrid nests in Germany were spatially clustered and all (or most)

workers showed admixed genomes. These observations suggest that the hybrid population

can be an independently operating and evolving unit. One possible example of such an

evolution is provided by Formica paralugubris Seifert 1996 from the Alps. The external

morphology of F. paralugubris gynes and workers is intermediate between F. lugubris and

F. aquilonia (unpublished data of B.S) while allozymes and mtDNA sequences are very

close to F. aquilonia (Pamilo et al. 1992; Goropashnaya et al. 2004; Bernasconi 2009). It is

tempting to hypothesise that F. paralugubris has rapidly evolved from a single hybrid

supercolony having been isolated during the last Pleistocene in a Nunatak or somewhere in

an Alpine valley. We are currently examining two other cases in which hybridisation

between two Formica species may have led to a formation of a new evolutionary lineage,

putatively a new species between F. lugubris and F. aquilonia and another one between

F. aquilonia and F. polyctena (Bernasconi 2009; Saapunki et al. 2008). It may turn out that

hybridisation could generally have a significant role in ant evolution with supercolonies

acting as large, separately evolving kin groups (Helantera et al. 2009).

Acknowledgments We wish to thank Philip Attewell, Wouter Dekoninck, Dieter Bretz, Gennady Dlussky,Katrin Moller, Rainer Neumeyer and Roland Schultz for providing samples and Riitta Jokela for the laboratoryanalyses. Jaqueline Gitschmann helped with the transformation of air-photographs into a graphics. The workwas supported by grants from the Academy of Finland (1122210 to P.P.).

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Independent hybrid populations of Formica polyctena X rufa wood ants (Hymenoptera: Formicidae) abound under conditions of forest fragmentation