Polymorphic Allozyme Research Articles

Loss of genetic variation in declining populations of Aricia artaxerxes in Northern Hungary

Rapid decline of population size often gives rise to loss of genetic variation and may result in a reduction in fitness. It is, therefore, essential to detect past demographic changes in populations of endangered species. Aricia artaxerxes occurs in two regions of Northern Hungary: Bukk Mts. and the Aggtelek Karst region. The species prefers short-grass habitats, which are subject to succession. Due to climatic differences between the two regions succession is more rapid in the Bukk plateau resulting in the decline and isolation of A. artaxerxes populations there. In contrast, most populations have remained rather large and stable in the Karst region. This situation provides an excellent possibility to compare the genetic composition of stable and declining populations. A. artaxerxes was collected in four populations of the Bukk Mountains and in ten in the Aggtelek Karst region in 2002 and 2005. We analysed 19 polymorphic allozyme loci in all samples. The results revealed an extremely high level of enzyme polymorphism in A. artaxerxes. Most parameters of polymorphism proved to be significantly higher in the Karst region compared to the Bukk. The shape of the distribution of alleles among the frequency classes indicated the effect of bottleneck in three Bukk and one Karst population. Though heterozygote excess was only significant in one Bukk population a tendency was observed for higher values of standardised heterozygote excess in those populations that had a typical bottleneck distribution of alleles. Our results clearly demonstrated that those populations/subpopulations that compose a network with intense migration are able to maintain their genetic diversity in a long run, while the isolated small populations of the Bukk plateau have lost a sizeable part of their variation.

Genetic consequences of fragmentation on populations of the terrestrial orchid Cymbidium goeringii

Population genetics theory predicts that fragmented plant populations do exhibit less genetic diversity, greater genetic divergence, and higher rates of inbreeding than well-preserved, continuous populations. We investigated the effects of habitat fragmentation on levels and distribution of genetic diversity and inbreeding in the terrestrial orchid Cymbidium goeringii on three islands off the south coast of South Korea. We selected two case studies, each containing 10 fragmented and 10 continuous (i.e. undisturbed) populations (40 populations and 1493 individuals in total); for both case studies, fragmentation has been recorded for almost 200years. Using nine polymorphic allozyme loci, we compared genetic diversity and structure between fragmented and continuous populations for each of two case studies. We found that continuous populations harboured significantly greater allelic richness (AR) than fragmented ones. We also found that fragmented populations exhibited two- to threefold higher degree of genetic differentiation (FST) than continuous ones. We did not found, however, significant differences for expected heterozygosity (He) and fixation index (FIS). Fragmentation effects are noticeable in C. goeringii as AR was significantly reduced in fragmented populations. The lack of significant differences for He would be attributable either to insufficient number of generations elapsed from the beginning of fragmentation, to the high density of individuals in undisturbed populations, or to both. The loss of allelic diversity detected in disturbed populations of C. goeringii is still small, and could be recovered if the current moderate levels of gene flow are maintained and no further fragmentation is occurring.

Salinity and depth as structuring factors of cryptic divergence in Moina brachiata (Crustacea: Cladocera)

Microcrustacean taxa in temporary waters are important contributors to aquatic biodiversity on the landscape scale even though much of the diversity at the molecular level is still undiscovered. Cladoceran species other than Daphnia are not frequently targeted in molecular investigations. We used nuclear allozyme polymorphisms as well as DNA sequence variation in mitochondrial 16 S and COI gene regions to reveal patterns of genetic differentiation among populations of a cladoceran species ? Moina brachiata ? that typically inhabits temporary aquatic habitats. Samples originated from 20 temporary to semi-permanent waterbodies in the Hungarian Great Plain of the Pannonian biogeographic region. We observed strong genetic differentiation in the phylogenetic analyses of the concatenated 16 S and COI genes, based on which M. brachiata was found to represent a complex of four cryptic lineages (A, B, C and D) with, however, one of these (lineage D) detected based on only one individual. Regarding the nuclear markers, diagnostic alleles of the PGM and MDH enzyme loci in complete linkage disequilibrium were observed separating the 'B' lineage from the rest. In addition, indirect evidence was provided by the AAT locus, where the AAT1 allele was found to be potentially diagnostic for lineage 'C'. The three phylogenetically defined lineages ('A', 'B', 'C') could be separated from each other along the first canonical axis of a multivariate analysis of occurrence, and this first axis was strongly correlated with depth and salinity of the ponds. There is a strong association between habitat depth and the occurrence of the 'B' lineage. Our results indicate that habitat depth and associated ecological characteristics driven by differences in hydroperiod likely are responsible for the present distribution of the lineages.

Genetic variation of Abies alba Mill. in Polish part of Sudety Mts.

The genetic structure of silver fir (<em>Abies alba</em> Mill.) was investigated among 8 populations from Sudety Mts. and 2 additional populations from other parts of Poland. Nine enzyme systems controlled by 13 allozyme loci were analyzed using starch gel electrophoresis. The mean expected heterozygosity, ranging from 0.06 to 0.11 and was lower compared to that found in other conifers. The mean genetic distance among all silver fir populations was 0.005. The Sudeten group of populations appeared to be genetically different from the two populations from other parts of Poland, indicating that at least two different centers are responsible for the current distribution of allozyme polymorphism in the tested populations.

Genetic legacy from past panmixia: high genetic variability and low differentiation in disjunct populations of the Eastern Large Heath butterfly

Glacial and interglacial cycles of the Pleistocene have led to severe range fluctuations of many species. These range shifts of the past often are reflected by extant genetic signatures. Retractions of distribution areas often have fostered splits into several small and isolated retreats as remnants of the formerly interconnected range. These processes often go in line with losses of intraspecific diversity. By contrast, large and interconnected distribution ranges mostly sustain high levels of genetic variability. The genetic impact of both scenarios strongly depends on the temporal scale. In the present study, we tested the genetic effects of an assumed long-lasting widespread distribution during glacial periods and more short-term population retractions to mountain archipelagos during warm stages. We analyzed polymorphic allozymes for individuals of the Eastern Large Heath butterfly, Coenonympha rhodopensis, including major parts of its distribution, such as central Italy and the Balkan Peninsula. Our data show extraordinarily high genetic diversity. The only remarkable genetic split is detectable between the central Apennines (Italy) and the Balkan mountain systems. The populations sampled over seven Balkan mountain systems (Jakupica, Shar Planina, Ossogovo, Pirin, Rila, Rhodopes, and Stara Planina) show low genetic differentiation. This low genetic differentiation and high genetic diversity diverges from the genetic structures frequently found in species with disjunct distributions. We therefore hypothesize that the obtained molecular structure is the product of down-slope shift during the last cold stage and subsequent expansion over the lowlands of the Balkan Peninsula. The current mountain restriction most probably occurred with the beginning of the postglacial warming, which is too short a time span to be of evolutionary relevance. Therefore, the recent high genetic diversities and low differentiation may still reflect long-lasting glacial panmixia but not (yet) the recent disjunction. The strong genetic differentiation between the Balkans and Italian Apennines must result from an earlier dispersal process, most probably from the Balkans to Italy. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110;, 281–290.

Geographic variation in Megalorchestia californiana allele frequencies may be caused by winter rather than summer temperatures

There are many allozyme polymorphisms with latitudinal clines, for which selection by climate is one obvious explanation. Both warm summer temperatures and cold winter temper- atures could plausibly impose selection on enzyme polymorphisms, but because summer and win- ter temperatures are usually geographically correlated, there has been little attempt to use pat- terns of geographic variation to discriminate between these possible selective factors. The glucose-6-phosphate isomerase and mannose-6-phosphate isomerase allozyme polymorphisms in the amphipod crustacean Megalorchestia californiana have clines that are strongly associated with mean January air temperature but are only weakly associated with mean August air temper- ature. This suggests that winter temperatures are more likely than summer temperatures to be a selective factor on these polymorphisms.

Genetic variability of tall junipers (Juniperus excelsa Bieb.) on the northern and southern boundaries of their natural distribution

The genetic structure, subdivision, and differentiation of six populations of tall junipers (Juniperus excelsa Bieb.) in the Crimean Mountains and one population in Lebanon were investigated using 18 polymorphic allozyme loci as genetic markers. A high level of genetic variability of J. excelsa was established at the northern and southern boundaries of their natural habitats. The mean values of the main indicators of genetic polymorphism were P99 = 1.000, A = 3.167, HE = 0.370, and HO = 0.405. The subdivision and differentiation of populations were low (FST = 0.032, DN = 0.026), indicating the similarity of their gene pools.

The role of continental shelf width in determining freshwater phylogeographic patterns in south‐eastern Australian pygmy perches (Teleostei: Percichthyidae)

Biogeographic patterns displayed by obligate freshwater organisms are intimately related to the nature and extent of connectivity between suitable habitats. Two of the more significant barriers to freshwater connections are seawater and major drainage divides. South-eastern Australia provides a contrast between these barriers as it has discrete areas that are likely influenced to a greater or lesser extent by each barrier type. We use continental shelf width as a proxy for the potential degree of river coalescence during low sea levels. Our specific hypothesis is that the degree of phylogeographic divergence between coastal river basins should correspond to the continental shelf width of each region. This predicts that genetic divergences between river basins should be lowest in regions with a wider continental shelf and that regions with similar continental shelf width should have similar genetic divergences. Pygmy perches (Nannoperca australis and Nannoperca 'flindersi') in south-eastern Australia provide an ideal opportunity to test these biogeographic hypotheses. Phylogeographic patterns were examined based on range-wide sampling of 82 populations for cytochrome b and 23 polymorphic allozyme loci. Our results recovered only limited support for our continental shelf width hypothesis, although patterns within Bass clade were largely congruent with reconstructed low sea-level drainage patterns. In addition, we identified several instances of drainage divide crossings, typically associated with low elevational differences. Our results demonstrate high levels of genetic heterogeneity with important conservation implications, especially for declining populations in the Murray-Darling Basin and a highly restricted disjunct population in Ansons River, Tasmania.

Allozyme-Based Genetic Variability Of The Daphnia Atkinsoni-Bolivari Species Complex (Cladocera: Daphniidae) In The Hungarian Great Plain

Allozyme polymorphism investigation was performed with the aim to evaluate some population genetic characteristics of two species, Daphnia atkinsoni Baird, 1859 and Daphnia bolivari Richard, 1888 (Cladocera: Daphniidae), of temporary aquatic habitats in the Hungarian Great Plain. The analysis showed that D. bolivari is not a distinct taxon, but is nested within D. atkinsoni. At the same time, remarkable within-species differentiation was revealed in D. atkinsoni; two presumable cryptic lineages were detected by our enzyme polymorphism investigation. The separation of the lineages is mainly due to the allozyme pattern at the AAT locus. Multivariate analysis of abiotic variables of the sampling sites revealed the relation between the genetic and ecological data, pH being the relevant variable explaining 81% of the genetic variability.

Molecular species boundaries in the phreatoicidean genus Amphisopus (Isopoda : Amphisopidae) and evidence for a new freshwater isopod species from Western Australia

The freshwater isopod genus Amphisopus is one of only two phreatoicidean genera in Western Australia with wide distributions and containing multiple described species. Two species (Amphisopus annectans and A. lintoni) are known from the south-western part of the state. With recent sampling extending the known range of Amphisopus and the recorded possibility of an undescribed species, this study aimed to examine genetic species boundaries and to detect additional species diversity. Isopods were sampled from across the range, and genetic structure was examined using mtDNA sequence data from a COI fragment and data from ten polymorphic allozyme loci. While allozyme data supported the clear separation of the known species, phylogenetic analyses presented three divergent monophyletic, geographically restricted clades occurring in the western, eastern and central parts of the Amphisopus distribution. These corresponded to A. annectans, A. lintoni and an undescribed species, respectively. The latter’s status was supported by its phylogenetic position relative to the known species and by the extent of differentiation observed among other phreatoicidean taxa. As with other taxa from the region, divergence times among these three species supported aridity in the Miocene–Pliocene as a driver of diversification, and suggested the existence of wetter refugial areas in south-western Australia.

Low level of allozyme polymorphism in relict aquatic plants of the Far East Nelumbo komarovii Grossh. and Euryale ferox Salisb.

Using allozyme analysis, genetic variation of two relict aquatic plants from Primorsky krai, Komarov lotus (Neliumbo komarovii Grossh.) and Gorgon plant (Euryale ferox Salisb.), was examined. The absence of allozyme variation in the Primorye populations of Neliumbo komarovii along with low polymorphism level in the population of Euryale ferox (P95 = 7.69; A = 1.07; Ho = 0.072; He = 0.038) was demonstrated. Since the data for the species examined are reported for the first time ever, the pheonotypes and genetic interpretation of the enzyme systems tested are presented. The izoenzyme profiles of N. komarovii were compared with the data reported for N. nucifera from China. The absence ofallozyme variation in N. komarovii, along with extremely low level of variation revealed for E. ferox, is discussed in association with the evolutionary histories of these species, their dispersal after the Pleistocene-Holocene cooling, and survival on this territory in range boundaries.

Diversifying Selection Underlies the Origin of Allozyme Polymorphism at the Phosphoglucose Isomerase Locus in Tigriopus californicus

The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations.

The relative contributions of insect and bird pollinators to outcrossing in an African Protea (Proteaceae)

A useful, but seldom applied, measure of the effectiveness of different pollinators is their contribution to the rate of outcrossing. This measure is particularly useful in facultatively autogamous plants for which seed set cannot be used as a direct measure of pollinator effectiveness. We used selective exclusion experiments to assess the importance of insects for outcrossing in Protea caffra, a facultatively autogamous shrub with scented flowers that are visited frequently by both birds and insects (mainly beetles). Pollen loads on stigmas, pollen tube growth, seed set, seed mass, germination, and early seedling survivorship were similar for vertebrate-excluded and open-pollinated inflorescences. Pollen-supplementation mostly did not increase seed set, revealing resource limitation. Mean multilocus outcrossing rates, estimated using eight polymorphic allozyme loci, were similar for progeny from inflorescences excluded from bird visitors (0.65) and for those visited by both birds and insects (0.59). Wright's fixation indices indicated that the adult population is near Hardy-Weinberg equilibrium but differed markedly for maternal plants (F(IS) = -0.187 ± 0.065) and their early stage progeny (F(IS) = 0.258 ± 0.002). Since seed from self and cross hand-pollinations were equally viable in terms of germination, this discrepancy in F(IS) could be explained by inbreeding depression that occurs between germination and reproductive maturity. Since outcrossing rates were not reduced when birds were excluded, we infer that insects are effective agents of cross pollination in P. caffra. This helps to explain the evolution of traits associated with insect pollination, such as fruity floral scent, in this species.

Low microsatellite variation in Aphanius fasciatus from the Tarquinia Salterns

1 - The Tarquinia Salterns (Latium, central Italy) provided the opportunity to analyse the impact of environmental stress on the genetic structure of the resident population of the killifish Aphanius fasciatus. Indeed, after the salt production ceased in 1997, the salterns have undergone habitat degradation due to lack of maintenance. The ecological restoration carried out from 2003 to 2006 reverted the environmental conditions to those of ten years before. 2 - The temporal variation of the gene pool of the population of A. fasciatus inhabiting the Tarquinia Salterns was investigated using microsatellite markers in samples collected in 1998 and 2003. The results obtained showed a low genetic variability and a genetic homogeneity of the population, which appears not divided in sub-demes. 3 - Microsatellites revealed a surprisingly low level of genetic variability when compared to allozyme data obtained in previous studies. This is likely due to a difference in the time of response of the two markers to environmental degradation. Microsatellites would lose genetic variability earlier and faster because of their usually high polymorphisms. Conversely, allozymes would be more resistant to genetic erosion, being moderately variable markers. 4 - Selection probably contributed in maintaining allozyme polymorphism, while microsatellites, being neutral markers, did not benefit from the action of selection and lost diversity earlier and more rapidly. Accordingly, the population appeared subdivided in distinct demes based on allozyme data but spatially homogeneous following microsatellites results.

Morphologically Intermediate form betweenAthous haemorrhoidalisandA. vittatus(Coleoptera: Elateridae): A Case of Hybridization? a Preliminary Study

In the present preliminary study we report an investigation on taxonomic status of individuals displaying diagnostic traits of intermediate values between the morphologically typical forms of Athous haemorrhoidalis (Fabricius 1801) and A. vittatus (Gmelin 1790) species occurring sympatrically. The presence of the “intermediate form” invoked the hypothesis of interspecific hybridization between A. haemorrhoidalis and A. vittatus species. An alternative explanation is that the “intermediate form” comprises individuals that are morphological variants of either of the species, without interspecific gene flow. We used partial sequences of two mitochondrial genes coding for the cytochrome oxidase subunit I (cox1) and 16S ribosomal RNA (rrnL), respectively. Additionally, we examined the variability of five polymorphic allozyme loci: 6Pgd, Idh, Mdh, Pgm and Gpi. Altogether, 28 A. haemorrhoidalis, 7 A. vittatus and 8 “intermediate form” individuals were included into the study. Phylogenetic analyses produced the trees of a consistent topology with high statistical support regardless of the method used. The specimens signified as the “intermediate form” constituted a monophyletic group with individuals of A. vittatus species. Empirical data confirmed that the reproductive isolation between the species studied is complete. No haplotype was shared between A. haemorrhoidalis and A. vittatus in sympatry. Similar results were obtained using the analysis of allozyme data. Especially at Idh and 6Pgd loci, alleles characteristic for A. vittatus and the “intermediate form” were not found in A. haemorrhoidalis, despite the larger sample examined. This suggests that the “intermediate form” is a phenotypic variant of A. vittatus without the signature of introgression.

Broad geographic covariation between floral traits and the mating system in Camissoniopsis cheiranthifolia (Onagraceae): multiple stable mixed mating systems across the species' range?

Plants vary widely in the extent to which seeds are produced via self-fertilization vs. outcrossing, and evolutionary change in the mating system is thought to be accompanied by genetic differentiation in a syndrome of floral traits. We quantified the pattern of variation and covariation in floral traits and the proportion of seeds outcrossed (t) to better understand the evolutionary processes involved in mating system differentiation among and within populations of the short-lived Pacific coastal dune endemic Camissoniopsis cheiranthifolia across its geographic range in western North America. We quantified corolla width and herkogamy, two traits expected to influence the mating system, for 48 populations sampled in the field and for a sub-sample of 29 populations grown from seed in a glasshouse. We also measured several other floral traits for 9-19 populations, estimated t for 16 populations using seven allozyme polymorphisms, and measured the strength of self-incompatibility for nine populations. Floral morphology and self-incompatibility varied widely but non-randomly, such that populations could be assigned to three phenotypically and geographically divergent groups. Populations spanned the full range of outcrossing (t = 0·001-0·992), which covaried with corolla width, herkogamy and floral life span. Outcrossing also correlated with floral morphology within two populations that exhibited exceptional floral variation. Populations of C. cheiranthifolia seem to have differentiated into three modal mating systems: (1) predominant outcrossing associated with self-incompatibility and large flowers; (2) moderate selfing associated with large but self-compatible flowers; and (3) higher but not complete selfing associated with small, autogamous, self-compatible flowers. The transition to complete selfing has not occurred even though the species appears to possess the required genetic capacity. We hypothesize that outcrossing populations in this species have evolved to different stable states of mixed mating.

Using estimates of metal bioavailability in the soil and genetic variation of allozymes to investigate heavy metal tolerance in the earthworm Eisenia fetida (Oligochaeta)

In a recent study, we showed that the earthworm species Eisenia fetida, inhabiting an extremely high metal polluted compost heap on a wine farm, did not have elevated body loads of the metals but exhibited genotoxic tolerance when exposed to Cd in the laboratory ( Voua Otomo and Reinecke, 2010). To unravel the mechanism behind the surprisingly low metal body burdens on one hand and genotoxic tolerance on the other hand, we investigated the estimated bioavailability of these metals (Cu, Zn, Pb and Cd) using sequential extraction methods with CaCl 2 and di-ethylene-triamine-pentaacetic acid (DTPA) and allozyme polymorphism in this field population, a laboratory control as well as a long-term Cd exposed population. The amounts of mobile (extracted with CaCl 2) and mobilizable (extracted with DTPA) metals in relation to the total (extracted with nitric acid) metals were all below 0.05% for all four metals, suggesting low availability for uptake. The low availability of these metals could not be explained by physico-chemical properties of soil but by the phenomenon of aging of the metals. There was no difference in allozyme frequency between metal tolerant and non-metal tolerant populations of E. fetida. This suggested that the tolerance found in earlier studies could be a mere physiological adaptation

High allozymic diversity in natural populations of Mycoheterotrophic Orchid Gastrodia elata, an endangered medicinal plant in China

Gastrodia elata Blume, a mycoheterotrophic orchid native to the Far East, is an endangered medicinal plant in China. Genetic variation among 19 natural populations of G. elata was examined in central China by using allozyme polymorphism (16 loci in six enzymatic systems). The species exhibited high level of genetic diversity (P = 56.3%, A = 2.2 and HE = 0.221), which was mainly attributed to its perennial habit and mixed reproduction system (both sexual and asexual). Evident genetic differentiation in G. elata natural populations was suggested by FST = 0.241. AMOVA analysis showed 31.3% of the total molecular variation was attributed to inter-population differentiation. Obvious genetic structure and genetic depauperation of some populations indicate forest fragmentation and over-collection have affected genetic variation of G. elata. A conservation strategy, which is conserving populations with great genetic distinction or high level of genetic variation from four management units, is recommended.

Allozyme Polymorphism and Phylogenetic Relationships in Apis mellifera Subspecies Selectively Reared in Poland and Bulgaria

The genetic variability of honey bee populations of three subspecies selectively reared in Poland (A. m. carnica and A. m. caucasica) and Bulgaria (A. m. macedonica-type rodopica) was studied using isoenzyme analysis of six enzyme systems (MDH-1, ME, EST-3, ALP, PGM and HK) corresponding to 6 loci. All loci were found to be polymorphic in the studied populations. Three alleles were detected at each locus: MHD-1 (MDH65, MDH80 and MDH100), Me (ME90, ME100 and ME106), EST-3 (EST94, EST100 and EST118), ALP (ALP80 ALP90 and ALP100), PGM (PGM80, PGM100 and PGM114) and HK (HK87, HK100 and HK110). The observed and expected heterozygosities (Ho and He) ranged from 0.196 (A. m. macedonica SM) to 0.265 (A. m. carnica MV) and from 0.224 (A. m. macedonica SM) to 0.273 (A. m. carnica GR), respectively. Allele frequencies of all loci were used to estimate Nei's (1972) genetic distance, which was found to range from 0.003 (between A. m. macedonica TR and SM and between A. m. carnica GR and MV populations) to 0.057 (between A. m. macedonica SM and A. m. caucasica populations). The estimated mean F(ST) value from allozyme data was 0.0364. A UPGMA dendrogram was obtained by genetic distance matrix methods; A. m. macedonica (type rodopica), A. m. carnica and A. m. caucasica populations represented different clades.

The genetic dynamics of the rapid and recent colonization of Denmark by Arion lusitanicus (Mollusca, Pulmonata, Arionidae)

We describe the genetic dynamics of the recent establishment of the 'Iberian slug', Arion lusitanicus J. Mabille 1868, in Denmark and compare its population structure to two other members of the 'large Arion complex', Arion ater ater, native to Denmark, and Arion ater rufus, introduced into Denmark in the early 1900s. Assaying allozyme polymorphism at seven enzyme loci, we found that: (1) None of the three taxa reproduce primarily by self-fertilization. Differences among loci and colonies in the pattern of deviation from Hardy-Weinberg equilibrium are most consistent with isolate mixing and perhaps with low amounts of selfing. (2) For both A. lusitanicus and A. a. rufus, gene diversity is lower in Danish colonies than in southern German colonies, implying population bottlenecks in the establishment of Danish colonies. (3) Significant linkage disequilibrium values usually involve the same three loci, viz. PGI, MDH-1 and MDH-2, suggesting physical linkage among these loci. (4) For both A. a. rufus and A. lusitanicus, the overall gene frequencies from Denmark and southern Germany are homogeneous, while variation among colonies within these regions ranges from around 15 to 28% for the three taxa. This indicates strong, local population genetic subdivision but with little restriction to gene flow from possible source areas. The heterogeneity in measures of diversity and differentiation indicates that population structure for all three taxa is dominated by ongoing founder effects, local extinction/colonisation dynamics, and genetic drift processes.