Genetic differentiation among the Maculinea species (Lepidoptera: Lycaenidae) in eastern Central Europe

The present study aimed to analyse the level of genetic variation in the eastern Central European (Slovenia, Hungary, and Romania/Transylvania) populations of the Large Blues ( Maculinea ) to analyse the pattern of differentiation both between and within the species. One objective was to compare the level of differentiation between the two disputed species ( Maculinea alcon and Maculinea rebeli ) with that among the other species. Imagos were collected from 23 localities in eastern Central Europe in 2002. Enzyme polymorphism was analysed using polyacrylamide gel electrophoresis. Fourteen enzyme loci were studied in all samples. In the analysis of the data, F -statistics and Nei's genetic distances were calculated and a dendrogram (unweighted pair group method with arithmetic mean) was constructed on the basis of the distance matrix. A multivariate analysis of variance was performed to study the pattern of genetic differentiation among the samples. Principal component analysis analysis was also carried out using the allele frequency data of the samples. Our results indicated that the large blues are generally less polymorphic than other European lycaenid butterflies studied. At the same time, the level of genetic differentiation was high, even among local populations within the species. A low level of genetic variation within the populations coupled with strong differentiation among them implies the effect of genetic drift. Strong genetic differentiation of four Maculinea species ( M. alcon , Maculinea teleius, Maculinea nausithous , and Maculinea arion ) was confirmed. Significant differentiation was not found between M. alcon and M. rebeli .  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 11–21.     

Genetic population structure of the large blue butterfly Maculinea alcon in Denmark

We have investigated the genetic population structure within and the genetic differentiation between local populations of the large blue butterfly Maculinea alcon throughout the Jutland peninsula. Samples were collected as eggs on foodplants (Gentiana pneumonanthe), and reared to 4th instar caterpillars in the laboratory. A significant excess of homozygotes was found for all the investigated allozyme loci in most of the populations. A North-South cline was observed for the allele frequencies at some of the loci and for several linkage groups. Because some of the allele frequency clines were parallel to clines in adult morphological variation, we interpret our results as evidence for the co-existence of at least two gene pools within the Danish Maculinea alcon populations. Multilocus electrophoretic data revealed highly positive but variable FST values, which under this scenario would reflect varying frequencies of the Maculinea gene pools across the local populations. The significantly positive FIS values indicate that these gene pools are at least partly reproductively isolated (Wahlund effect). The co-occurrence of several Maculinea alcon gene pools on many local sites in Jutland is of great importance for conservation of the fragmented Maculinea populations. Our results show that there is probably more Maculinea biodiversity to conserve than was previously thought, and suggests that extant populations are more fragmented and vulnerable than counts of flying adults or eggs on foodplants indicate.     

Microsatellite markers for the large blue butterflies Maculinea nausithous and Maculinea alcon (Lepidoptera: Lycaenidae) and their amplification in other Maculinea species

We developed microsatellite markers for Maculinea nausithous and Maculinea alcon, two of five species of endangered large blue butterflies found in Europe. Two separate microsatellite libraries were constructed. Eleven markers were developed for M. nausithous and one for M. alcon. The primers were tested on both species as well as on the three other European Maculinea species. The number of alleles per locus ranged from two to 14. These markers will be useful tools for population genetic studies of Maculinea species.     

Basal hatching by Maculinea butterfly eggs: a consequence of advanced myrmecophily?

Larvae of Maculinea alcon and M. rebeli are unique among Holarctic Lycaenidae in hatching through the base of the eggshell and emerging on the opposite side of the leaf to the egg. This occurs because the exposed upper surfaces of their eggs have exceptionally thick shells. Other species of Maculinea have normal or unusually thin-shelled eggs. It is suggested that both types of Maculinea eggshell have evolved to reduce mortalities from parasitoids and predators. Maculinea alcon and M. rebeli are forced to lay in exposed places due to the structure of their foodplants, and protect their eggs with thick shells. The other three species hide their eggs deep within the inflorescences of their foodplants; M. nausithous and M. teleius require thin flexible shells to achieve this. These adaptations are necessary because Maculinea eggs often occur at much higher densities on their initial foodplants and are hence more vulnerable—than is the case with other Lycaenidae. This is a consequence of myrmecophily. Maculinea population size is regulated more by the status of the ant host than by that of the initial foodplant. Large Maculinea populations, and high densities of eggs, occur on the many sites where the ant is abundant but the foodplant is scarce. Maculinea alcon and M. rebeli eggs occur at the highest densities, and need the greatest protection, due to the advanced form of myrmecophily found in these species. Whereas M. anon, M. teleius and M. nausithous larvae are predators of ant brood, M. alcon and M. rebeli feed mainly at a lower trophic level on ant regurgitations. This leads to roughly seven times more M. alcon and M. rebeli adults emerging per ant nest, and to a similar increase in egg densities per foodplant.     

Geographical variation in host–ant specificity of the parasitic butterfly Maculinea alcon in Denmark

Abstract  1. Maculinea alcon uses three different species of Myrmica host ants along a north–south gradient in Europe. Based on this geographical variation in host ant use, Elmes et al . (1994) suggested that M. alcon might consist of three or more cryptic species or host races, each using a single and different host-ant species.
2. Population-specific differences in allozyme genotypes of M. alcon in Denmark ( Gadeberg & Boomsma, 1997 ) have suggested that genetically differentiated forms may occur in a gradient across Denmark, possibly in relation to the use of different host ants.
3. It was found that two host-ant species are indeed used as hosts in Denmark, but not in a clear-cut north–south gradient. Furthermore, specificity was not complete for many M. alcon populations. Of five populations investigated in detail, one used primarily M. rubra as a host, another exclusively used M. ruginodis , while the other three populations used both ant species. No population in Denmark used M. scabrinodis as a host, although this species was present in the habitat and is known to be a host in central and southern Europe.
4. In terms of number of parasites per nest and number of nests parasitised, M. rubra seems to be a more suitable host in populations where two host species are used simultaneously. Host-ant species has an influence on caterpillar size but this varies geographically. Analyses of pupae did not, however, show size differences between M. alcon raised in M. rubra and M. ruginodis nests.
5. The geographical mosaic of host specificity and demography of M. alcon in Denmark probably reflects the co-evolution of M. alcon with two alternative host species. This system therefore provides an interesting opportunity for studying details of the evolution of parasite specificity and the dynamics of host-race formation.     

Low genetic variability and strong differentiation among isolated populations of the rare steppe grass Stipa capillata L. in Central Europe

Stipa capillata L. (Poaceae) is a rare grassland species in Central Europe that is thought to have once been widespread in post‐glacial times. Such relict species are expected to show low genetic diversity within populations and high genetic differentiation between populations due to bottlenecks, long‐term isolation and ongoing habitat fragmentation. These patterns should be particularly pronounced in selfing species. We analysed patterns of random amplified polymorphic DNA (RAPD) variation in the facultatively cleistogamous S. capillata to examine whether genetic diversity is associated with population size, and to draw initial conclusions on the migration history of this species in Central Europe. We analysed 31 S. capillata populations distributed in northeastern, central and western Germany, Switzerland and Slovakia. Estimates of genetic diversity at the population level were low and not related to population size. Among all populations, extraordinarily high levels of genetic differentiation (amova : φ_ST = 0.86; Bayesian analysis: θ^B = 0.758) and isolation‐by‐distance were detected. Hierarchical amova indicated that most of the variability was partitioned among geographic regions (59%), or among populations between regions when the genetically distinct Slovakian populations were excluded. These findings are supported by results of a multivariate ordination analysis. We also found two different groups in an UPGMA cluster analysis: one that contained the populations from Slovakia, and the other that combined the populations from Germany and Switzerland. Our findings imply that S. capillata is indeed a relict species that experienced strong bottlenecks in Central Europe, enhanced by isolation and selfing. Most likely, populations in Slovakia were not the main genetic source for the post‐glacial colonization of Central Europe.     

Higher productivity at the cost of increased host-specificity when Maculinea butterfly larvae exploit ant colonies through trophallaxis rather than by predation

Abstract .1. Field studies were made of the benefits and costs of two feeding strategies in the genus Maculinea, whose final-instar larvae parasitise Myrmica ant colonies. Maculinea arion is an obligate predator of ant brood, whereas M. rebeli and M. alcon mimic ant larvae and are fed (like cuckoos) directly by the workers.
2. Samples of > 1500 Myrmica nests confirmed laboratory-based predictions that, by feeding at a lower trophic level, many (4.7-fold) more individuals of M. rebeli and M. alcon are supported per ant colony than M. arion.
3. Because of their efficient feeding, cuckoo species often occupied sites where their phytophagous early larval populations coincided to only a small extent (> 10%) with host Myrmica colonies, whereas all sites supporting M. arion had 50–100% of the phytophagous stages within foraging range of the host Myrmica species.
4. Greater host-specificity was identified as another consequence of cuckoo-feeding. The ecological cost of this is discussed .
5. The feeding of other Maculinea species had not been fully described: the data suggest that M. nausithous is a predator of ant brood and confirm that M. teleius is predacious .     

Polymorphic growth rates in myrmecophilous insects

A polymorphism in growth rates was recently described affecting the larval development of the myrmecophilous butterfly Maculinea rebeli, spanning different years in a single insect population. The close integration of M. rebeli into the host ant colonies, facilitated by adaptations in behaviour and chemical mimicry, make extended larval development a successful strategy. Here we present additional data for M. rebeli and new data for Maculinea alcon (another cuckoo-feeding lycaenid) and the two myrmecophilous predators Maculinea arion and Microdon mutabilis (Diptera: Syrphidae). As predicted, M. alcon shows the same growth pattern as M. rebeli with a proportion of caterpillars developing in one year and the remainder over two years. This pattern holds in both northern and southern European populations, where M. alcon exploits different species of host. Against expectation, the same bimodal distribution of pre-pupation body weights, indicating one and two year developers, was found for the larvae of M. arion and M. mutabilis. As predators, both species are less closely integrated in their host ant colonies, suggesting that the polymorphism in growth rates is a more general adaptation to a myrmecophilous life style, arrived at by convergent evolution between the Maculinea and Microdon species. For predatory species we suggest that biennialism is an adaptation to the migratory behaviour of the host made possible by the predators' ability to fast over extended periods. We also hypothesize that M. arion represents an ancestral strategy in Maculinea butterflies and that the growth polymorphism might have become genetically fixed in the cuckoo-feeding species.     

Population structure of a large blue butterfly and its specialist parasitoid in a fragmented landscape

Habitat fragmentation may interrupt trophic interactions if herbivores and their specific parasitoids respond differently to decreasing connectivity of populations. Theoretical models predict that species at higher trophic levels are more negatively affected by isolation than lower trophic level species. By combining ecological data with genetic information from microsatellite markers we tested this hypothesis on the butterfly Maculinea nausithous and its specialist hymenopteran parasitoid Neotypus melanocephalus. We assessed the susceptibility of both species to habitat fragmentation by measuring population density, rate of parasitism, overall genetic differentiation (theta(ST)) and allelic richness in a large metapopulation. We also simulated the dynamics of genetic differentiation among local populations to asses the relative effects of migration rate, population size, and haplodiploid (parasitoid) and diploid (host) inheritance on metapopulation persistence. We show that parasitism by N. melanocephalus is less frequent at larger distances to the nearest neighbouring population of M. nausithous hosts, but that host density itself is not affected by isolation. Allelic richness was independent of isolation, but the mean genetic differentiation among local parasitoid populations increased with the distance between these populations. Overall, genetic differentiation in the parasitoid wasp was much greater than in the butterfly host and our simulations indicate that this difference is due to a combination of haplodiploidy and small local population sizes. Our results thus support the hypothesis that Neotypus parasitoid wasps are more sensitive to habitat fragmentation than their Maculinea butterfly hosts.     

The ecology of Myrmica ants in relation to the conservation of Maculinea butterflies

Maculinea butterflies in Europe, and probably most of Asia, are host specific social parasites of various species of Myrmica ants. The latest summary of field data showing the pattern of host specificity by Maculinea is presented. Myrmica ants have been well studied in the laboratory but much less is known about the ecology of their natural populations. While the former is important in understanding the adaptive evolution of Maculinea larval behaviours, the latter is of more practical importance to conservationists charged with the protection of specific populations of Maculinea. The current knowledge of habitat partition, colony growth and colony reproduction within communities of Myrmica ants is summarized in relation to the ecology of Maculinea species. Concepts used in current population simulation models are explained. A key concept is the idea that community structure (both number of species and size and abundance of nests) is controlled by the quantity and quality of suitable nest sites. Some advice is given to conservationists who might need to manipulate Myrmica ant populations in order to maintain a robust population of a Maculinea species. © Rapid Science Ltd. 1998     

REVIEW: The recent distribution of the European Maculinea species

The genus Maculinea is confined to the Palaearctic region. Four species occur from Central Europe to China and/or Japan, while M. rebeli is probably restricted to Europe. Although within many Western European countries the distribution of the species is well known, there are still many questions. The exact status of some of the species is still unclear and the amount of decline in this century is unknown. In Asia, especially within the nations of the former USSR, the distributions of species are even less well known and as a basis for good research and effective nature conservation, knowledge on these subjects is needed. © Rapid Science Ltd. 1998     

Do assemblages of Coregonus (Teleostei: Salmoniformes) in the Central Alpine region of Europe represent species flocks?

To examine models of evolution for Coregonus from the Central Alpine region of Europe, 20 populations from nine lakes were assessed for variation at six microsatellite DNA loci. Patterns of variation were tested against three evolutionary models: phenotypic plasticity, multiple invasions of lakes by divergent forms, and within-lake radiation of species flocks. All sympatric and all but one allopatric pairs of populations were significantly divergent in allele frequencies. Pairwise F -statistics indicated reduced gene flow among phenotypically divergent sympatric populations. These results reject the hypothesis that within-lake morphological and ecological diversity reflects phenotypic plasticity within a single gene pool. Genetic similarity was higher among forms within lakes than between populations of the same form in different lakes. Among-lake divergence was primarily a product of allele size differences. Mantel tests contrasting patterns of genetic divergence against patterns predicted from the multiple invasions and species flocks models indicated that the latter is the best explanation of the observed genetic variation. Thus, reproductively isolated species diverged within lakes, with similar patterns repeatedly emerging among lakes. While this study argues for a particular mode of evolution in Central Alpine Coregonus , the taxonomy of these forms remains unresolved.     

Intraspecific allozymatic differentiation reveals the glacial refugia and the postglacial expansions of European Erebia medusa (Lepidoptera: Nymphalidae)

Allozyme analysis of Erebia medusa over large regions of Europe revealed a significant population differentiation (F_ST: 0.149 ± 0.016). A UPGMA-analysis showed a division into four major lineages with mean inter-group genetic distances ranging from 0.051 (±0.010) to 0.117 (±0.024). An AMOVA revealed that rather more than two-thirds of the variance between samples was being between these lineages and less than one-third within lineages. An eastern group included the samples from the Czech Republic, Slovakia and north-eastern Hungary. This genetic lineage expressed significantly higher genetic diversity than the other three. A second lineage was formed by the samples from France and Germany. The two samples from western Hungary represent a third delimited lineage and the sample from northern Italy a fourth. We suppose that this genetic differentiation took place during the last ice-age in four disjunct refugia. The genetically more diverse eastern genetic lineage might have evolved in a relatively large refugium in south-eastern Europe. We assume that the other three lineages developed in relatively small relict areas around the Alps. It is likely for the western lineage that its ice-age distribution showed at least one disjunction in late Würm with the consequence of further genetic differentiation. Most probably, the eastern lineage colonized postglacial Central Europe using two alternative routes: one north and one south of the Carpathians. Up to now, neither similar glacial refugia, nor comparable secondary disjunctions in late Würm, are reported for any other animal or plant species.     

Integrative approach revealed contrasting pattern of spatial structuring within urban and rural biotypes of Culex pipiens

Population structure of pests is an important issue when designing management strategies to optimize control measures. In this study, we investigated a spatial pattern of genetic and phenotypic variation within seven urban and within six rural populations of Culex pipiens from Vojvodina Province (Serbia) incorporating landscape genetic methods (using allozyme data) and wing size and shape (using geometric morphometric approach). Comparing rural samples, no strong genetic groupings of individuals were detected. Nevertheless, traditional approaches where individuals are pre‐assigned to populations, including F statistics and amova (analysis of molecular variance), revealed low, but significant genetic differentiation among samples. Similarly, phenotypic data (wing size and shape) indicated some level of heterogeneity among rural samples. Contrary to genetic homogeneity found within rural biotype, the individual‐based structuring characterized urban biotype. Geneland revealed the presence of two genetic clusters within urban group which is in concordance with F_ST and amova results. These results showed that sample from Novi Sad (NS) is a distinct genetic unit, which has been likely resulted in intensive insecticide use over several decades. Furthermore, phenotypic differentiation supported the existence of spatial structuring. Therefore, complementary use of molecular markers and phenotypic traits may be a powerful tool for revealing hidden spatial diversity within Cx. pipiens.     

Analysis of genetic variability of South American wild rice populations (Oryza glumaepatula) with isozymes and RAPD markers

The knowledge of population structure and genetic diversity of wild relatives of rice is needed to investigate their evolutionary history and potential use in breeding programs. Very little is known about the wild rice species ( Oryza spp.), particularly those that are native to South America. A study using isozyme and RAPD markers was conducted to estimate the level of genetic diversity of four South American wild rice populations ( Oryza glumaepatula ) recently collected in the Amazon forest and western Brazil rivers. F -statistics and genetic diversity parameters calculated from isozyme and RAPD markers indicated high values for inbreeding coefficients and differentiation among the four populations. In agreement with this, a pattern of greater variation between than within populations was observed with both types of markers. These findings were corroborated by an AMOVA analysis, which indicated that a large portion of the total genetic variation was attributed to regional divergence. The partition of the AMOVA analysis among populations showed that most of the genetic diversity was due to differences among populations. This distribution pattern of genetic variation of O. glumaepatula populations is in agreement with the expectation for an autogamous species and provides important baseline data for conservation and collection strategies for this species.     

Genetic distinction of wildcat (Felis silvestris) populations in Europe,and hybridization with domestic cats in Hungary

The genetic integrity and evolutionary persistence of declining wildcat populations are threatened by crossbreeding with widespread free-living domestic cats. Here we use allelic variation at 12 microsatellite loci to describe genetic variation in 336 cats sampled from nine European countries. Cats were identified as European wildcats (Felis silvestris silvestris), Sardinian wildcats (F. s. libyca) and domestic cats (F. s. catus), according to phenotypic traits, geographical locations and independently of any genetic information. Genetic variability was significantly partitioned among taxonomic groups (FST = 0.11; RST = 0.41; P < 0.001) and sampling locations (FST = 0.07; RST = 0.06; P < 0.001), suggesting that wild and domestic cats are subdivided into distinct gene pools in Europe. Multivariate and Bayesian clustering of individual genotypes also showed evidence of distinct cat groups, congruent with current taxonomy, and suggesting geographical population structuring. Admixture analyses identified cryptic hybrids among wildcats in Portugal, Italy and Bulgaria, and evidenced instances of extensive hybridization between wild and domestic cats sampled in Hungary. Cats in Hungary include a composite assemblage of variable phenotypes and genotypes, which, as previously documented in Scotland, might originate from long lasting hybridization and introgression. A number of historical, demographic and ecological conditions can lead to extensive crossbreeding between wild and domestic cats, thus threatening the genetic integrity of wildcat populations in Europe.     

Patterns of genetic and taxonomic differentiation in three Melitaea (subg. Mellicta) species (Lepidoptera, Nymphalidae, Nymphalinae)

In conservation genetics the existence of Evolutionarily Significant Units (ESU) is a crucial question in threatened or vulnerable species. It is of particular concern to determine whether different subspecies or ecotypes of a species can be considered as separate ESUs. Some Melitaea subg. Mellicta species (e.g. Mellicta aurelia, Mellicta britomartis) are declining or scarce in Europe. Therefore, the level of genetic differentiation and pattern of genetic variation were surveyed in three Melitaea (subg. Mellicta) species. Their habitat requirements and food plants partly overlap; accordingly they often co-occur in the same habitat. M. britomartis and M. aurelia have one brood per year in Hungary, while Mellicta athalia has a monovoltine and a bivoltine ecotype. The purpose of the study was to estimate the number of genetically differentiated ESUs among these species in the Carpathian basin. Samples were taken from 5 Hungarian regions and a few samples were collected in Transylvania as well. Enzyme polymorphism was studied using polyacrylamide gel electrophoresis. The structure of genetic variation was analysed by F-statistics, AMOVA, PCA and Bayesian clustering method. UPGMA dendrogram was constructed on the basis of Cavalli-Sforza and Edwards chord distances. The three species are clearly differentiated from each other in all statistical analyses. They are evidently different Evolutionary Significant Units. The two ecotypes of M. athalia, however, do not show any genetic differentiation.     

Correlations between observed dispersal capabilities and patterns of genetic differentiation in populations of four aquatic insect species from the Arizona White Mountains, U.S.A.

SUMMARY 1. Dispersal ability is an important ecological factor that can influence population structure. In an attempt to determine the extent that the pattern of genetic differentiation is correlated with dispersal ability in stream-dwelling aquatic insects, we used the amplified fragment length polymorphism (AFLP) technique to characterise genetic variation in four aquatic insect species: Gumaga griseola (Trichoptera: Sericostomatidae), Helicopsyche mexicana (Trichoptera: Helicopsychidae), Psephenus montanus (Coleoptera: Psephenidae) and Ambrysus thermarum (Hemiptera: Naucoridae). Individuals were sampled from several sites within two adjacent catchments in the Arizona White Mountains. In addition to the genetic analyses, a 20-week-long trapping study was used to determine the relative dispersal ability of adults of the four species examined.
2. We obtained hierarchical indicators of genetic differentiation for catchments, sites within catchments and sites across the region examined. Overall, average estimators of genetic differentiation ( F -statistics) were consistent with direct observations of organismal movement, although it was our direct observations on adult insect flight that permitted us to interpret our results correctly. This was because of the fact that a lack of genetic differentiation across watersheds can be interpreted in two ways.
3. In contrast to F -statistics, patterns of genetic isolation by distance for each species more clearly reflected dispersal ability, suggesting that such analytical approaches provide less ambiguous information about the importance of gene flow in the hierarchical partitioning of genetic variation in stream organisms.     

Maculinea rebeli (Hirschke) – a phantom or reality? Novel contribution to a long‐standing debate over the taxonomic status of an enigmatic Lycaenidae butterfly

The main goal of our research was to carry out a comprehensive study on the obligatorily myrmecophilous Maculinea alcon (D enis & S chiffermüller) whose taxonomy has remained highly debated despite the fact that Maculinea V an E ecke species are some of the most intensively studied insects in E urope. We aimed to elucidate the relationship between the different forms exploiting different ecological environments with specific regard to ‘H irschke's rebeli ' (M. rebeli (H irschke)) from the high altitudes of the S tyrian A lps which was studied in this respect for the first time. Molecular analyses including multiple gene sequences, microsatellites and allozymes completed with Wolbachia screening, together with geometric morphometry were applied on various sets of samples. Our results showed that ‘rebeli ’ from the type locality differed strongly from all other samples not only at the highly variable microsatellites, but also at standard genes and clearly represents a well‐separated unit (potentially subspecies) within M. alcon . As all individuals were infected with a single Wolbachia strain, this is possibly an ancient infection with high maternal transmission rate. It also suggests that the separation of the topotypic populations was probably a relatively recent event which could have been promoted by genetic drift or specific conditions at high altitudes, and maintained by restricted gene flow. Both the genetic and the morphometric studies supported the proposition that the geographical origin of the populations has a higher effect on the structure of variation than the food plant use. Moreover, we detected more genetic lineages than could be defined by differential host plant usage.     

Postglacial distribution area expansion of Polyommatus coridon (Lepidoptera: Lycaenidae) from its Ponto-Mediterranean glacial refugium

The genetic population structure of Polyommatus coridon (Poda, 1761) (Chalk-hill blue) was studied by means of allozyme electrophoresis in north-eastern Germany, the Czech Republic, Slovakia and Hungary. All analysed parameters showed high genetic diversity within populations (number of alleles: 2.61; observed and expected heterozygosity: 18.6% and 19.7%, respectively; percentage of polymorphic loci: total: 73.6%, on 95% level: 56.1%), whereas genetic differentiation between populations was comparatively low (F(ST) = 0.028 +/- 0.005 s.d.). Hierarchical variance analysis revealed significant structuring among five regional population clusters. A significant isolation-by-distance structure exists (r = 0.39; P < 0.05). The mean number of alleles per locus declined significantly from south to north and showed a strong correlation with the geographical latitude (r = -0.88, P < 0.0001). We suggest that this reflects the loss of alleles during the postglacial colonisation of eastern Central Europe from an ice-age refugium in the Balkans. A possible scenario for the postglacial expansion process in eastern Central Europe is discussed using these data: coming from the north-western part of the Balkans, P. coridon may have reached the western tip of Hungary, and consecutively colonised eastern Central Europe using two alternative expansion routes.