Contrasting patterns of mitochondrial and microsatellite population structure in fragmented populations of greater prairie-chickens

Greater prairie-chickens (Tympanuchus cupido pinnatus) were once found throughout the tallgrass prairie of midwestern North America but over the last century these prairies have been lost or fragmented by human land use. As a consequence, many current populations of prairie-chickens have become isolated and small. This fragmentation of populations is expected to lead to reductions in genetic variation as a result of random genetic drift and a decrease in gene flow. As expected, we found that genetic variation at both microsatellite DNA and mitochondrial DNA (mtDNA) markers was reduced in smaller populations, particularly in Wisconsin. There was relatively little range-wide geographical structure (FST) when we examined mtDNA haplotypes but there was a significant positive relationship between genetic (FST) and geographical distance (isolation by distance). In contrast, microsatellite DNA loci revealed significant geographical structure (FST) and a weak effect of isolation by distance throughout the range. These patterns were much stronger when populations with reduced levels of genetic variability (Wisconsin) were removed from the analyses. This suggests that the effects of genetic drift were stronger than gene flow at microsatellite loci, whereas these forces were in range-wide equilibrium at mtDNA markers. These differences between the two molecular markers may be explained by a larger effective population size (Ne) for mtDNA, which is expected in species such as prairie-chickens that have female-biased dispersal and high levels of polygyny. Our results suggest that historic populations of prairie-chickens were once interconnected by gene flow but current populations are now isolated. Thus, maintaining gene flow may be important for the long-term persistence of prairie-chicken populations.     

Census vs. effective population size in chinook salmon: large- and small-scale environmental perturbation effects

Population viability has often been assessed by census of reproducing adults. Recently this method has been called into question and estimation of the effective population size (Ne) proposed as a complementary method to determine population health. We examined genetic diversity in five populations of chinook salmon (Oncorhynchus tshawytscha) from the upper Fraser River watershed (British Columbia, Canada) at 11 microsatellite loci over 20 years using DNA extracted from archived scale samples. We tested for changes in genetic diversity, calculated the ratio of the number of alleles to the range in allele size to give the statistic M, calculated Ne from the temporal change in allele frequency, used the maximum likelihood method to calculate effective population size (NeM), calculated the harmonic mean of population size, and compared these statistics to annual census estimates. Over the last two decades population size has increased in all five populations of chinook examined; however, Ne calculated for each population was low (81-691) and decreasing over the time interval measured. Values of NeM were low, but substantially higher than Ne calculated using the temporal method. The calculated values for M were generally low (M < 0.70), indicating recent population reductions for all five populations. Large-scale historic barriers to migration and development activities do not appear to account for the low values of Ne; however, available spawning area is positively correlated with Ne. Both Ne and M estimates indicate that these populations are potentially susceptible to inbreeding effects and may lack the ability to respond adaptively to stochastic events. Our findings question the practice of relying exclusively on census estimates for interpreting population health and show the importance of determining genetic diversity within populations.     

Demographic and genetic estimates of effective population size (Ne) reveals genetic compensation in steelhead trout

Estimates of effective population size (Ne) are required to predict the impacts of genetic drift and inbreeding on the evolutionary dynamics of populations. How the ratio of Ne to the number of sexually mature adults (N) varies in natural vertebrate populations has not been addressed. We examined the sensitivity of Ne/N to fluctuations of N and determined the major variables responsible for changing the ratio over a period of 17 years in a population of steelhead trout (Oncorhynchus mykiss) from Washington State. Demographic and genetic methods were used to estimate Ne. Genetic estimates of Ne were gained via temporal and linkage disequilibrium methods using data from eight microsatellite loci. DNA for genetic analysis was amplified from archived smolt scales. The Ne/N from 1977 to 1994, estimated using the temporal method, was 0.73 and the comprehensive demographic estimate of Ne/N over the same time period was 0.53. Demographic estimates of Ne indicated that variance in reproductive success had the most substantial impact on reducing Ne in this population, followed by fluctuations in population size. We found increased Ne/N ratios at low N, which we identified as genetic compensation. Combining the information from the demographic and genetic methods of estimating Ne allowed us to determine that a reduction in variance in reproductive success must be responsible for this compensation effect. Understanding genetic compensation in natural populations will be valuable for predicting the effects of changes in N (i.e. periods of high population density and bottlenecks) on the fitness and genetic variation of natural populations.     

Huge populations and old species of Costa Rican and Panamanian dirt frogs inferred from mitochondrial and nuclear gene sequences

Molecular genetic data were used to investigate population sizes and ages of Eleutherodactylus (Anura: Leptodactylidae), a species-rich group of small leaf-litter frogs endemic to Central America. Population genetic structure and divergence was investigated for four closely related species surveyed across nine localities in Costa Rica and Panama. DNA sequence data were collected from a mitochondrial gene (ND2) and a nuclear gene (c-myc). Phylogenetic analyses yielded concordant results between loci, with reciprocal monophyly of mitochondrial DNA haplotypes for all species and of c-myc haplotypes for three of the four species. Estimates of genetic differentiation among populations (FST) based upon mitochondrial data were always higher than nuclear-based FST estimates, even after correcting for the expected fourfold lower effective population size (Ne) of the mitochondrial genome. Comparing within-population variation and the relative mutation rates of the two genes revealed that the Ne of the mitochondrial genome was 15-fold lower than the estimate of the nuclear genome based on c-myc. Nuclear FST estimates were approximately 0 for the most proximal pairs of populations, but ranged from 0.5 to 1.0 for all other pairs, even within the same nominal species. The nuclear locus yielded estimates of Ne within localities on the order of 105. This value is two to three orders of magnitude larger than any previous Ne estimate from frogs, but is nonetheless consistent with published demographic data. Applying a molecular clock model suggested that morphologically indistinguishable populations within one species may be 107 years old. These results demonstrate that even a geologically young and dynamic region of the tropics can support very old lineages that harbour great levels of genetic diversity within populations. The association of high nucleotide diversity within populations, large divergence between populations, and high species diversity is also discussed in light of neutral community models.     

Long-term temporal changes of genetic composition in brown trout (Salmo trutta L.) populations inhabiting an unstable environment

The genetic structure of brown trout (Salmo trutta) populations inhabiting rivers on the island of Bornholm in the Baltic Sea was studied on a spatial and temporal scale. Low water levels in the rivers during the summer period are assumed to have a significant impact on the persistence of local populations, possibly resulting in a metapopulation structure. Extinctions may, however, also be buffered by a remnant strategy, whereby juveniles escape to river outlets during periods of drought. We compared polymorphism at seven microsatellite DNA loci in contemporary and past samples collected from 1944 to 1997. A principal component analysis, a hierarchical gene diversity analysis and assignment tests showed that the genetic composition of populations was not temporally stable, and that temporal genetic differentiation was much stronger than spatial differentiation. Genetic variability was high and stable over time. Effective population sizes (Ne) and migration rate (m) were estimated using a maximum-likelihood-based implementation of the temporal method. Ne estimates were low (ranging from 8.3 to 22.9) and estimates of m were high (between 0.23 and 0.99), in contrast to other Danish trout populations inhabiting larger and more environmentally stable rivers (Ne between 39.2 and 289.9 and m between 0.01 and 0.09). Thus, the observed spatio-temporal patterns of genetic differentiation can be explained by drift in small persisting populations, where levels of genetic variation are maintained by strong gene flow. However, observations of rivers devoid of trout suggested that population turnover also takes place. We suggest that Bornholm trout represent a metapopulation where the genetic structure primarily reflects strong drift and gene flow, combined with occasional extinction-recolonization events.     

Low Genetic Variation in the Heath Hen Prior to Extinction and Implications for the Conservation of Prairie-Chicken Populations

Low genetic variation is often considered to contribute to the extinction of species when they reach small population sizes. In this study we examined the mitochondrial control region from museum specimens of the Heath Hen (Tympanuchus cupido cupido), which went extinct in 1932. Today, the closest living relatives of the Heath Hen, the Greater (T. c. pinnatus), Attwater’s (T. c. attwateri) and Lesser (T. pallidicinctus) Prairie-chicken, are declining throughout most of their range in Midwestern North America, and loss of genetic variation is a likely contributor to their decline. Here we show that 30 years prior to their extinction, Heath Hens had low levels of mitochondrial genetic variation when compared with contemporary populations of prairie-chickens. Furthermore, some current populations of Greater Prairie-chickens are isolated and losing genetic variation due to drift. We estimate that these populations will reach the low levels of genetic variation found in Heath Hens within the next 40 years. Genetic variation and fitness can be restored with translocation of individuals from other populations; however, we also show that choosing an appropriate source population for translocation can be difficult without knowledge of historic population bottlenecks and their effect on genetic structure.     

Historical demography, selection, and coalescence of mitochondrial and nuclear (genes in Prochilodus species of northern South America

Fishes of the genus Prochilodus are ecologically and commercially important, ubiquitous constituents of large river biota in South America. Recent ecologic and demographic studies indicate that these fishes exist in large, stable populations with adult census numbers exceeding one million individuals. Abundance data present a stark contrast to very low levels of genetic diversity (theta) and small effective population sizes (Ne) observed in a mitochondrial (mt) DNA dataset obtained for two species, Prochilodus mariae, and its putative sister taxon, Prochilodus rubrotaeniatus. Both species occupy major river drainages (Orinoco, Essequibo, and Negro) of northeastern South America. Disparity between expectations based on current abundance and life history information and observed genetic data in these lineages could result from historical demographic bottlenecks, or alternatively, natural selection (i.e., a mtDNA selective sweep). To ascertain underlying processes that affect mtDNA diversity in these species we compared theta and Ne estimates obtained from two, unlinked nuclear loci (calmodulin intron-4 and elongation factor-1alpha intron-6) using an approach based on coalescent theory. Genetic diversity and Ne estimated from mtDNA and nuclear sequences were uniformly low in P. rubrotaeniatus from the Rio Negro, suggesting that this population has encountered a historical bottleneck. For all P. mariae populations, theta and Ne based on nuclear sequences were comparable to expectations based on current adult census numbers and were significantly greater than mtDNA estimates, suggesting that a selective mtDNA sweep has occurred in this species. Comparative genetic analysis indicates that a suite of evolutionary processes involving historical demography and natural selection have influenced patterns of genetic variation and speciation in this important Neotropical fish group.     

Survival and habitat use in translocated and resident greater prairie-chickens

Translocation, or the purposeful movement of organisms from one location to another for conservation, is currently being used to bolster populations of the endangered greater prairie-chicken (Tympanuchus cupido). We used radiotelemetry to compare survival between 58 resident birds and 54 newly translocated greater prairie-chickens that were sourced from a location more than 325 km away. Model averaged survival estimates were lower in translocated birds (0.42; 95% CI: 0.17–0.66) than in resident prairie-chickens (0.65; 95% CI: 0.46–0.79) through the breeding season. Habitat, sex and year were each included in at least 1 of the top 4 models, but the model averaged confidence intervals for each parameter encompassed zero. Survival of both resident and translocated prairie-chickens increased throughout the breeding season. Both translocated and resident prairie-chickens selected for core prairie habitat over agriculture, and birds tended to avoid surrounding private grasslands and wooded areas. We suggest that future translocation projects account for reduced survival of translocated birds when determining the appropriate release cohort sizes and sex ratios. We also recommend that future management for greater prairie-chicken habitat focus on the expansion of core protected patches of prairie to promote elevated survival and better chances of conservation success.     

Can effective population size estimates be used to monitor population trends of woodland bats? A case study of Myotis bechsteinii

Molecular approaches to calculate effective population size estimates (Ne) are increasingly used as an alternative to long‐term demographic monitoring of wildlife populations. However, the complex ecology of most long‐lived species and the consequent uncertainties in model assumptions means that effective population size estimates are often imprecise. Although methods exist to incorporate age structure into Ne estimations for long‐lived species with overlapping generations, they are rarely used owing to the lack of relevant information for most wild populations. Here, we performed a case study on an elusive woodland bat, Myotis bechsteinii, to compare the use of the parentage assignment Ne estimator (EPA) with the more commonly used linkage disequilibrium (LD) Ne estimator in detecting long‐term population trends, and assessed the impacts of deploying different overall sample sizes. We used genotypic data from a previously published study, and simulated 48 contrasting demographic scenarios over 150 years using the life history characteristics of this species The LD method strongly outperformed the EPA method. As expected, smaller sample sizes resulted in a reduced ability to detect population trends. Nevertheless, even the smallest sample size tested (n = 30) could detect important changes (60%–80% decline) with the LD method. These results demonstrate that genetic approaches can be an effective way to monitor long‐lived species, such as bats, provided that they are undertaken over multiple decades.     

Population Structure and Stock Identification of Eulachon (Thaleichthys pacificus), an Anadromous Smelt, in the Pacific Northwest

The genetic structure of eulachon (Thaleichthys pacificus) populations was examined in an analysis of variation of 14 microsatellite loci representing approximately 1900 fish from 9 sites between the Columbia River and Cook Inlet, Alaska. Significant genetic differentiation occurred among the putative populations. The mean F_ST for all loci was 0.0046, and there was a significant correlation between population genetic differentiation (F_ST) and geographic distance. Simulated mixed-stock samples comprising populations from different regions suggested that variation at microsatellite loci provided reasonably accurate estimates of stock composition for potential fishery samples. Marine sampling indicated that immature eulachons from different rivers, during the 2 to 3 years of prespawning life in offshore marine waters, do not mix thoroughly. For eulachons captured incidentally in offshore trawl fisheries, there was a clear geographic cline in relative abundance of eulachons from different geographic areas. The sample from northern British Columbia was dominated by northern and central coastal populations of British Columbia, the sample from central British Columbia was composed of eulachons from all regions, and the sample from southern British Columbia was dominated by Columbia River and Fraser River populations. These results have implications for the management of trawl fisheries and conservation of spawning populations in some rivers where abundance is at historically low levels.     

Long-term effective population sizes, temporal stability of genetic composition and potential for local adaptation in anadromous brown trout (Salmo trutta) populations

We examined the long-term temporal (1910s to 1990s) genetic variation at eight microsatellite DNA loci in brown trout (Salmo trutta L) collected from five anadromous populations in Denmark to assess the long-term stability of genetic composition and to estimate effective population sizes (Ne). Contemporary and historical samples consisted of tissue and archived scales, respectively. Pairwise thetaST estimates, a hierarchical analysis of molecular variance (amova) and multidimensional scaling analysis of pairwise genetic distances between samples revealed much closer genetic relationships among temporal samples from the same populations than among samples from different populations. Estimates of Ne, using a likelihood-based implementation of the temporal method, revealed Ne >or= 500 in two of three populations for which we have historical data. A third population in a small (3 km) river showed Ne >or= 300. Assuming a stepping-stone model of gene flow we considered the relative roles of gene flow, random genetic drift and selection to assess the possibilities for local adaptation. The requirements for local adaptation were fulfilled, but only adaptations resulting from strong selection were expected to occur at the level of individual populations. Adaptations resulting from weak selection were more likely to occur on a regional basis, i.e. encompassing several populations. Ne appears to have declined recently in at least one of the studied populations, and the documented recent declines of many other anadromous brown trout populations may affect the persistence of local adaptation.     

High genetic variation in leopards indicates large and long-term stable effective population size

In this paper we employ recently developed statistical and molecular tools to analyse the population history of the Tanzanian leopard (Panthera pardus), a large solitary felid. Because of their solitary lifestyle little is known of their past or present population dynamics. Eighty-one individuals were scored at 18 microsatellite loci. Overall, levels of heterozygosity were high (0.77 +/- 0.03), with a small heterozygote deficiency (0.06 +/- 0.03). Effective population size (Ne) was calculated to be 38 000-48 000. A Ne:N ratio of 0.42 (average from four cat studies) gives a present population size of about 100 000 leopards in Tanzania. Four different bottleneck tests indicated that this population has been large and stable for a minimum of several thousand years. FST values were low and no significant genetic structuring of the population could be detected. This concurs well with the large migration values (Nm) obtained (>3.3 individuals/generation). Our analysis reveals that ecological factors (e.g. disease), which are known to have had major impact on other carnivore populations, are unlikely to have impacted strongly on the population dynamics of Tanzanian leopards. The explanation may be found in their solitary life-style, their often nonconfrontational behaviour toward interspecific competitors, or that any bottlenecks have been of limited size, localized, or too short to have affected genetic variation to any measurable degree. Since the genetic structuring is weak, gene flow is not restricted to within protected areas. Local loss of genetic variation is therefore not of immediate concern.     

Outbreaks, gene flow and effective population size in the migratory locust, Locusta migratoria: a regional-scale comparative survey

The potential effect of population outbreaks on within and between genetic variation of populations in pest species has rarely been assessed. In this study, we compare patterns of genetic variation in different sets of historically frequently outbreaking and rarely outbreaking populations of an agricultural pest of major importance, the migratory locust, Locusta migratoria . We analyse genetic variation within and between 24 populations at 14 microsatellites in Western Europe, where only ancient and low-intensity outbreaks have been reported (non-outbreaking populations), and in Madagascar and Northern China, where frequent and intense outbreak events have been recorded over the last century (outbreaking populations). Our comparative survey shows that (i) the long-term effective population size is similar in outbreaking and non-outbreaking populations, as evidenced by similar estimates of genetic diversity, and (ii) gene flow is substantially larger among outbreaking populations than among non-outbreaking populations, as evidenced by a fourfold to 30-fold difference in F _ST values. We discuss the implications for population dynamics and the consequences for management strategies of the observed patterns of genetic variation in L. migratoria populations with contrasting historical outbreak frequency and extent.     

Effective population sizes and temporal stability of genetic structure in Rana pipiens, the northern leopard frog

Although studies of population genetic structure are very common, whether genetic structure is stable over time has been assessed for very few taxa. The question of stability over time is particularly interesting for frogs because it is not clear to what extent frogs exist in dynamic metapopulations with frequent extinction and recolonization, or in stable patches at equilibrium between drift and gene flow. In this study we collected tissue samples from the same five populations of leopard frogs, Rana pipiens, over a 22-30 year time interval (11-15 generations). Genetic structure among the populations was very stable, suggesting that these populations were not undergoing frequent extinction and colonization. We also estimated the effective size of each population from the change in allele frequencies over time. There exist few estimates of effective size for frog populations, but the data available suggest that ranid frogs may have much larger ratios of effective size (Ne) to census size (Nc) than toads (bufonidae). Our results indicate that R. pipiens populations have effective sizes on the order of hundreds to at most a few thousand frogs, and Ne/Nc ratios in the range of 0.1-1.0. These estimates of Ne/Nc are consistent with those estimated for other Rana species. Finally, we compared the results of three temporal methods for estimating Ne. Moment and pseudolikelihood methods that assume a closed population gave the most similar point estimates, although the moment estimates were consistently two to four times larger. Wang and Whitlock's new method that jointly estimates Ne and the rate of immigration into a population (m) gave much smaller estimates of Ne and implausibly large estimates of m. This method requires knowing allele frequencies in the source of immigrants, but was thought to be insensitive to inexact estimates. In our case the method may have failed because we did not know the true source of immigrants for each population. The method may be more sensitive to choice of source frequencies than was previously appreciated, and so should be used with caution if the most likely source of immigrants cannot be identified clearly.     

Small effective population size in the long-toed salamander

The effective population sizes (Ne) of six populations of the long-toed salamander (Ambystoma macrodactylum) from Montana and Idaho, USA were estimated from allozyme data from samples collected in 1978, 1996 and 1997 using the temporal allele frequency method. Five of the six estimates ranged from 23 to 207 (mean = 123 +/- 79); one estimate was indistinguishable from infinity. In order to infer the actual Ne of salamander populations, we compared the frequency distribution of our observed Ne estimates with distributions obtained from simulated populations of known Ne. Our observed Ne estimate distribution was consistent with distributions from simulated populations with Ne values of 10, 25, and 50, suggesting an actual Ne for each of the six salamander populations of less than 100. This Ne estimate agrees with most other Ne estimates for amphibians. We conclude by discussing the conservation implications of small Ne values in amphibians in the context of increasing isolation of populations due to habitat fragmentation.     

Population genetic structure of the African elephant in Uganda based on variation at mitochondrial and nuclear loci: evidence for male-biased gene flow

A drastic decline has occurred in the size of the Uganda elephant population in the last 40 years, exacerbated by two main factors; an increase in the size of the human population and poaching for ivory. One of the attendant consequences of such a decline is a reduction in the amount of genetic diversity in the surviving populations due to increased effects of random genetic drift. Information about the amount of genetic variation within and between the remaining populations is vital for their future conservation and management. The genetic structure of the African elephant in Uganda was examined using nucleotide variation of mitochondrial control region sequences and four nuclear microsatellite loci in 72 individuals from three localities. Eleven mitochondrial DNA (mtDNA) haplotypes were observed, nine of which were geographically localized. We found significant genetic differentiation between the three populations at the mitochondrial locus while three out of the four microsatellite loci differentiated KV and QE, one locus differentiated KV and MF and no loci differentiated MF and QE. Expected heterozygosity at the four loci varied between 0.51 and 0.84 while nucleotide diversity at the mitochondrial locus was 1.4%. Incongruent patterns of genetic variation within and between populations were revealed by the two genetic systems, and we have explained these in terms of the differences in the effective population sizes of the two genomes and male-biased gene flow between populations.     

Rapid loss of MHC class II variation in a bottlenecked population is explained by drift and loss of copy number variation

Population bottlenecks may reduce genetic variation and potentially increase the risk of extinction. Here, we present the first study to use historic samples to analyse loss of variation at the major histocompatibility complex (MHC), which plays a central role in vertebrate disease resistance. Balancing selection acts on the MHC and could moderate the loss of variation expected from drift; however, in a Wisconsin population of greater prairie-chickens (Tympanuchus cupido), the number of MHC class II B alleles per individual declined by 44% following a population bottleneck, compared to a loss of only 8% at microsatellites. Simulations indicate that drift likely reduced MHC variation at the population level, as well as within individuals by reducing the number of gene copies per individual or by fixing the same alleles across multiple loci. These multiple effects of genetic drift on MHC variation could have important implications for immunity and fitness.     

Genetic variation within and among fragmented populations of lesser prairie-chickens (Tympanuchus pallidicinctus)

As a result of recurrent droughts and anthropogenic factors, the range of the lesser prairie-chicken (Tympanuchus pallidicinctus) has contracted by 92% and the population has been reduced by approximately 97% in the past century, resulting in the smallest population size and most restricted geographical distribution of any North American grouse. We examined genetic variation through DNA sequence analysis of 478 base pairs of the mitochondrial genome and by assaying allelic variation at five microsatellite loci from lesser prairie-chickens collected on 20 leks in western Oklahoma and east-central New Mexico. Traditional population genetic analyses indicate that lesser prairie-chickens maintain high levels of genetic variation at both nuclear and mitochondrial loci. Although some genetic structuring among lesser prairie-chicken leks was detected within Oklahoma and New Mexico for both nuclear and mitochondrial loci, high levels of differentiation were detected between Oklahoma and New Mexico populations. Nested-clade analysis of mitochondrial haplotypes revealed that both historic and contemporary processes have influenced patterns of haplotype distributions and that historic processes have most likely led to the level of differentiation found between the Oklahoma and New Mexico populations.     

Microsatellite analysis of population structure in Canadian polar bears

Attempts to study the genetic population structure of large mammals are often hampered by the low levels of genetic variation observed in these species. Polar bears have particularly low levels of genetic variation with the result that their genetic population structure has been intractable. We describe the use of eight hypervariable microsatellite loci to study the genetic relationships between four Canadian polar bear populations: the northern Beaufort Sea, southern Beaufort Sea, western Hudson Bay, and Davis Strait - Labrador Sea. These markers detected considerable genetic variation, with average heterozygosity near 60% within each population. Interpopulation differences in allele frequency distribution were significant between all pairs of populations, including two adjacent populations in the Beaufort Sea. Measures of genetic distance reflect the geographic distribution of populations, but also suggest patterns of gene flow which are not obvious from geography and may reflect movement patterns of these animals. Distribution of variation is sufficiently different between the Beaufort Sea populations and the two more eastern ones that the region of origin for a given sample can be predicted based on its expected genotype frequency using an assignment test. These data indicate that gene flow between local populations is restricted despite the long-distance seasonal movements undertaken by polar bears.     

High levels of variation despite genetic fragmentation in populations of the endangered mountain pygmy-possum, Burramys parvus, in alpine Australia

In endangered mammals, levels of genetic variation are often low and this is accompanied by genetic divergence among populations. The mountain pygmy-possum (Burramys parvus) is an endangered marsupial restricted to the alpine region of Victoria and New South Wales, Australia. By scoring variation at eight microsatellite loci, we found that B. parvus populations exhibit high levels of genetic divergence and fall into three distinct groups from the northern, central and southern areas of the distribution of this species, consistent with previous assessments of mitochondrial DNA variation. F(ST) values between populations from these regions ranged from 0.19 to 0.54. Within the central area, there was further genetic fragmentation, and a linear association between genetic and geographical distance. This pattern is likely to reflect limited dispersal across barriers despite the fact that individual B. parvus can move several kilometres. Levels of genetic variation within populations were high with the exception of a southern population where there was evidence of inbreeding. From a conservation perspective, all three areas where B. parvus are found should be considered as separate gene pools; management of populations within these areas needs to take into account the low gene flow between populations, as well as threats posed by roads, resorts and other developments in the alpine region. The low genetic variability and inbreeding in the southern population is of particular concern given the high levels of variability in other B. parvus populations.