MHC variation reflects the bottleneck histories of New Zealand passerines

Most empirical evidence suggests that balancing selection does not counter the effects of genetic drift in shaping postbottleneck major histocompatibility complex (MHC) genetic diversity when population declines are severe or prolonged. However, few studies have been able to include data from historical specimens, or to compare populations/species with different bottleneck histories. In this study, we examined MHC class II B and microsatellite diversity in four New Zealand passerine (songbird) species that experienced moderate to very severe declines. We compared diversity from historical samples (collected c. 1884–1938) to present‐day populations. Using a Bayesian framework, we found that the change in genetic diversity from historical to contemporary samples was affected by three main factors: (i) whether the data were based on MHC or microsatellite markers, (ii) species (as a surrogate for bottleneck severity) and (iii) whether the comparison between historical and contemporary samples was made using historical samples originating from the mainland, or using historical samples originating from islands. The greatest losses in genetic diversity occurred for the most severely bottlenecked species, particularly between historical mainland and contemporary samples. Additionally, where loss of diversity occurred, the change was greater for MHC genes compared to microsatellite loci.     

Population structure and loss of genetic diversity in the endangered white-headed duck, <Emphasis Type="Italic">Oxyura leucocephala</Emphasis>

The white-headed duck is a globally threatened species native to the Palaearctic with a range extending from Spain in the west to the western edge of China in the east. Its populations have become fragmented and undergone major declines in recent decades. To study genetic differences between populations across the range and change in genetic diversity over time, we sequenced a portion of the mitochondrial DNA control region from 67 museum specimens (years 1861–1976) as well as 39 contemporary samples from Spain and seven from Greece (years 1992–2003). In the historical sample, we found a lack of significant genetic structure between populations in different areas. We found evidence that the species experienced a rapid expansion in the past, perhaps from glacial refugia centred around the Mediterranean following the last ice age. In Spain, the population went through a dramatic bottleneck in the 1970s and early 1980s, when only a few dozens individuals remained in the wild. Although population size has since recovered to a few thousand individuals, we found a highly significant loss of mitochondrial haplotype diversity between the historical and contemporary samples. Given ongoing declines in other areas, losses in genetic diversity that may reduce the adaptive potential of white-headed ducks in the future are a continuing concern throughout the geographic range of this species.     

Single base errors in PCR products from avian museum specimens and their effect on estimates of historical genetic diversity

Conservation genetic studies often employ DNA extracts from museum specimens for comparisons with extant populations to monitor temporal changes in genetic diversity. Here, we report on artifact base changes in mitochondrial DNA sequences amplified from relatively recent (≤ 35 years) museum specimens of indigobirds (Vidua spp.). Single base errors were confirmed by replicate sequencing and included both double peaks and artifact substitutions at rates of ∼3 × 10⁻⁴ and ∼1 × 10⁻⁴ per base-pair, respectively, resulting in one or more errors or ambiguities in an 1100 base pair sequence in 21% of 219 samples. Most errors involved C→T changes on the L-strand, presumably due to deamination of cytosine in the template. The error rates encountered here bias comparisons of haplotype number between historical and extant populations, such that the ‘loss’ of artifact haplotypes present in a historical sample could be incorrectly attributed to a population decline or bottleneck. Sequencing errors due to miscoding lesions in template DNA have so far been reported only from ancient and formalin-fixed tissue, but they may also affect relatively recent museum samples, as shown here, and perhaps also non-invasive samples that typically yield low-quality DNA.     

Spatiotemporal Genetic Diversity of Lions Reveals the Influence of Habitat Fragmentation across Africa

Direct comparisons between historical and contemporary populations allow for detecting changes in genetic diversity through time and assessment of the impact of habitat fragmentation. Here, we determined the genetic architecture of both historical and modern lions to document changes in genetic diversity over the last century. We surveyed microsatellite and mitochondrial genome variation from 143 high-quality museum specimens of known provenance, allowing us to directly compare this information with data from several recently published nuclear and mitochondrial studies. Our results provide evidence for male-mediated gene flow and recent isolation of local subpopulations, likely due to habitat fragmentation. Nuclear markers showed a significant decrease in genetic diversity from the historical (H_E = 0.833) to the modern (H_E = 0.796) populations, whereas mitochondrial genetic diversity was maintained (H_d = 0.98 for both). Although the historical population appears to have been panmictic based on nDNA data, hierarchical structure analysis identified four tiers of genetic structure in modern populations and was able to detect most sampling locations. Mitogenome analyses identified four clusters: Southern, Mixed, Eastern, and Western and were consistent between modern and historically sampled haplotypes. Within the last century, habitat fragmentation caused lion subpopulations to become more geographically isolated as human expansion changed the African landscape. This resulted in an increase in fine-scale nuclear genetic structure and loss of genetic diversity as lion subpopulations became more differentiated, whereas mitochondrial structure and diversity were maintained over time.     

Genetic diversity loss in a biodiversity hotspot: ancient DNA quantifies genetic decline and former connectivity in a critically endangered marsupial

The extent of genetic diversity loss and former connectivity between fragmented populations are often unknown factors when studying endangered species. While genetic techniques are commonly applied in extant populations to assess temporal and spatial demographic changes, it is no substitute for directly measuring past diversity using ancient DNA (aDNA). We analysed both mitochondrial DNA (mtDNA) and nuclear microsatellite loci from 64 historical fossil and skin samples of the critically endangered Western Australian woylie (Bettongia penicillata ogilbyi), and compared them with 231 (n = 152 for mtDNA) modern samples. In modern woylie populations 15 mitochondrial control region (CR) haplotypes were identified. Interestingly, mtDNA CR data from only 29 historical samples demonstrated 15 previously unknown haplotypes and detected an extinct divergent clade. Through modelling, we estimated the loss of CR mtDNA diversity to be between 46% and 91% and estimated this to have occurred in the past 2000–4000 years in association with a dramatic population decline. In addition, we obtained near‐complete 11‐loci microsatellite profiles from 21 historical samples. In agreement with the mtDNA data, a number of ‘new’ microsatellite alleles was only detected in the historical populations despite extensive modern sampling, indicating a nuclear genetic diversity loss >20%. Calculations of genetic diversity (heterozygosity and allelic rarefaction) showed that these were significantly higher in the past and that there was a high degree of gene flow across the woylie's historical range. These findings have an immediate impact on how the extant populations are managed and we recommend the implementation of an assisted migration programme to prevent further loss of genetic diversity. Our study demonstrates the value of integrating aDNA data into current‐day conservation strategies.     

Spatial and temporal genetic dynamics of the grasshopper Oedaleus decorus revealed by museum genomics

Analyzing genetic variation through time and space is important to identify key evolutionary and ecological processes in populations. However, using contemporary genetic data to infer the dynamics of genetic diversity may be at risk of a bias, as inferences are performed from a set of extant populations, setting aside unavailable, rare, or now extinct lineages. Here, we took advantage of new developments in next‐generation sequencing to analyze the spatial and temporal genetic dynamics of the grasshopper Oedaleus decorus, a steppic Southwestern‐Palearctic species. We applied a recently developed hybridization capture (hyRAD) protocol that allows retrieving orthologous sequences even from degraded DNA characteristic of museum specimens. We identified single nucleotide polymorphisms in 68 historical and 51 modern samples in order to (i) unravel the spatial genetic structure across part of the species distribution and (ii) assess the loss of genetic diversity over the past century in Swiss populations. Our results revealed (i) the presence of three potential glacial refugia spread across the European continent and converging spatially in the Alpine area. In addition, and despite a limited population sample size, our results indicate (ii) a loss of allelic richness in contemporary Swiss populations compared to historical populations, whereas levels of expected heterozygosities were not significantly different. This observation is compatible with an increase in the bottleneck magnitude experienced by central European populations of O. decorus following human‐mediated land‐use change impacting steppic habitats. Our results confirm that application of hyRAD to museum samples produces valuable information to study genetic processes across time and space.     

Genetic variability and structure of the remnant natural populations of <Emphasis Type="Italic">Cedrus libani</Emphasis> (Pinaceae) of Lebanon

Cedrus libani of Lebanon is a valuable natural resource and the dominant species in its natural ecosystem. Intense and diverse anthropogenic pressures over historical times raised concerns about its genetic vigor and continued survival. Our investigation of the genetic diversity included samples from all remnant natural populations. Assessment of the genetic diversity using random amplified polymorphic DNA markers revealed the persistence of considerable variation distributed within populations with low population differentiation corroborated by Bayesian and analysis of molecular variance estimates (G _ST = 0.07, Φ _ST = 0.09). Individual assignment tests were carried out to investigate measures of gene flow. Inferences concluded that this natural heritage is not currently threatened by inbreeding or by random genetic drift. Correlation studies investigated possible effects of spatial distribution and environmental conditions on genetic structure. A climatic trend corresponding to a temperature–humidity gradient correlated significantly with the level of genetic diversity, while the edaphic variation did not.     

Significant genetic admixture after reintroduction of peregrine falcon (<Emphasis Type="Italic">Falco peregrinus</Emphasis>) in Southern Scandinavia

The peregrine falcon (Falco peregrinus) population in southern Scandinavia was almost extinct in the 1970’s. A successful reintroduction project was launched in 1974, using captive breeding birds of northern and southern Scandinavian, Finnish and Scottish origin. We examined the genetic structure in the pre-bottleneck population using eleven microsatellite markers and compared the data with the previously genotyped captive breeding population and contemporary wild population. Museum specimens between 53 and 130 years old were analyzed. Despite an apparent loss of historical genetic diversity, the contemporary population shows a relatively high level of genetic variation. Considerable gene introgression from captive breeding stock used to repopulate the former range of southern Scandinavian peregrines may have altered the genetic composition of this population. Both the historical and contemporary northern and southern Scandinavian populations are genetically differentiated. The reintroduction project implemented in the region and the use of non-native genetic stock likely prevented the southern Scandinavian population from extinction and thus helped maintain the level of genetic diversity and prevent inbreeding depression. The population is rapidly increasing in numbers and range and shows no indication of reduced fitness or adaptive capabilities in the wake of the severe bottleneck and the reintroduction.     

Reduced Genetic Diversity and Effective Population Size in an Endangered Atlantic Salmon (Salmo Salar) Population from Maine, USA

Atlantic salmon (Salmo salar) populations in Maine, USA, are listed as a Distinct Population Segment under the U.S. Endangered Species Act due to reduced spawning runs and juvenile densities. Whenever possible, optimal conservation strategies for endangered populations should incorporate both present and historical knowledge of genetic variation. We assayed genetic diversity at seven microsatellite loci and at the mitochondrial ND1 gene in an endangered wild population of Atlantic salmon captured from the Dennys River from 1963 to 2001 using DNA’s extracted from archival scale and tissue samples. We examined temporal trends of genetic diversity, population structure, and effective population size (Ne). Overall temporal trends of diversity and Ne show significant reductions from 1963 to 2001 raising the possibility that current restoration efforts may be impacted by historical loss of diversity potentially critical to adaptation. Although our results suggest genetic stability in this population from 1963 to 1981, significant differentiation was observed for both the 1995 and 2001 samples compared with all other temporal samples. The presence of an ND1 mtDNA haplotype in this population, historically observed only in European and Newfoundland stocks, may represent previously unrecognized local wild diversity or, alternatively, may represent introgression from non-native fish.     

Consequences of a demographic bottleneck on genetic structure and variation in the Scandinavian brown bear

The Scandinavian brown bear went through a major decline in population size approximately 100 years ago, due to intense hunting. After being protected, the population subsequently recovered and today numbers in the thousands. The genetic diversity in the contemporary population has been investigated in considerable detail, and it has been shown that the population consists of several subpopulations that display relatively high levels of genetic variation. However, previous studies have been unable to resolve the degree to which the demographic bottleneck impacted the contemporary genetic structure and diversity. In this study, we used mitochondrial and microsatellite DNA markers from pre‐ and postbottleneck Scandinavian brown bear samples to investigate the effect of the bottleneck. Simulation and multivariate analysis suggested the same genetic structure for the historical and modern samples, which are clustered into three subpopulations in southern, central and northern Scandinavia. However, the southern subpopulation appears to have gone through a marked change in allele frequencies. When comparing the mitochondrial DNA diversity in the whole population, we found a major decline in haplotype numbers across the bottleneck. However, the loss of autosomal genetic diversity was less pronounced, although a significant decline in allelic richness was observed in the southern subpopulation. Approximate Bayesian computations provided clear support for a decline in effective population size during the bottleneck, in both the southern and northern subpopulations. These results have implications for the future management of the Scandinavian brown bear because they indicate a recent loss in genetic diversity and also that the current genetic structure may have been caused by historical ecological processes rather than recent anthropogenic persecution.     

Hybridization ddRAD‐sequencing for population genomics of nonmodel plants using highly degraded historical specimen DNA

Species’ responses at the genetic level are key to understanding the long‐term consequences of anthropogenic global change. Herbaria document such responses, and, with contemporary sampling, provide high‐resolution time‐series of plant evolutionary change. Characterizing genetic diversity is straightforward for model species with small genomes and a reference sequence. For nonmodel species—with small or large genomes—diversity is traditionally assessed using restriction‐enzyme‐based sequencing. However, age‐related DNA damage and fragmentation preclude the use of this approach for ancient herbarium DNA. Here, we combine reduced‐representation sequencing and hybridization‐capture to overcome this challenge and efficiently compare contemporary and historical specimens. Specifically, we describe how homemade DNA baits can be produced from reduced‐representation libraries of fresh samples, and used to efficiently enrich historical libraries for the same fraction of the genome to produce compatible sets of sequence data from both types of material. Applying this approach to both Arabidopsis thaliana and the nonmodel plant Cardamine bulbifera, we discovered polymorphisms de novo in an unbiased, reference‐free manner. We show that the recovered genetic variation recapitulates known genetic diversity in A. thaliana, and recovers geographical origin in both species and over time, independent of bait diversity. Hence, our method enables fast, cost‐efficient, large‐scale integration of contemporary and historical specimens for assessment of genome‐wide genetic trends over time, independent of genome size and presence of a reference genome.     

Genomics and museum specimens

Nearly 25 years ago, Allan Wilson and colleagues isolated DNA sequences from museum specimens of kangaroo rats (Dipodomys panamintinus) and compared these sequences with those from freshly collected animals (Thomas et al. 1990 ). The museum specimens had been collected up to 78 years earlier, so the two samples provided a direct temporal comparison of patterns of genetic variation. This was not the first time DNA sequences had been isolated from preserved material, but it was the first time it had been carried out with a population sample. Population geneticists often try to make inferences about the influence of historical processes such as selection, drift, mutation and migration on patterns of genetic variation in the present. The work of Wilson and colleagues was important in part because it suggested a way in which population geneticists could actually study genetic change in natural populations through time, much the same way that experimentalists can do with artificial populations in the laboratory. Indeed, the work of Thomas et al. ( 1990 ) spawned dozens of studies in which museum specimens were used to compare historical and present‐day genetic diversity (reviewed in Wandeler et al. 2007 ). All of these studies, however, were limited by the same fundamental problem: old DNA is degraded into short fragments. As a consequence, these studies mostly involved PCR amplification of short templates, usually short stretches of mitochondrial DNA or microsatellites. In this issue, Bi et al. ( 2013 ) report a breakthrough that should open the door to studies of genomic variation in museum specimens. They used target enrichment (exon capture) and next‐generation (Illumina) sequencing to compare patterns of genetic variation in historic and present‐day population samples of alpine chipmunks (Tamias alpinus) (Fig. 1). The historic samples came from specimens collected in 1915, so the temporal span of this comparison is nearly 100 years.     

Comparative genetic analyses of historical and contemporary collections highlight contrasting demographic histories for the bumble bees Bombus pensylvanicus and B. impatiens in Illinois

Direct comparison of genetic patterns between museum specimens and contemporary collections can be a powerful approach for detecting recent demographic changes. Using microsatellite markers, we examined historical and contemporary genetic variation from an apparently declining bumble bee species, Bombus pensylvanicus , and from a stable species, Bombus impatiens , in central Illinois. For each species, we genotyped specimens from the Illinois Natural History Survey collected from three populations between 1969–1972 and from a resurvey of the same areas conducted in 2008. Population structure in B . pensylvanicus increased markedly over the last four decades (from θ_ST = 0.001 to 0.027) while no structure was detected in B . impatiens for either time period (θ_ST = –0.006 to –0.003). Changes in genetic diversity were not significant for either species, although small reductions were observed for B . pensylvanicus in all three populations. Coalescent simulations incorporating both contemporary and historical samples suggest that this small change is not surprising for recent population declines, as large reductions in genetic diversity were only apparent under the most severe bottleneck scenarios. These results demonstrate how comparisons of genetic patterns between temporal periods and species can help elucidate potential threats to population health and suggest several strategies that might be useful in the conservation of B . pensylvanicus in the Midwestern USA.     

Genetic variation of the major histocompatibility complex (MHC class II β gene) in the threatened Gila trout, <Emphasis Type="Italic">Oncorhynchus gilae gilae</Emphasis>

Gila trout (Oncorhynchus gilae gilae) was federally protected in 1973 because of severe declines in abundance and geographic range size. At present, four relict genetic lineages of the species remain in mountain streams of New Mexico and Arizona, USA. Management actions aimed at species recovery, including hatchery production and restocking of formerly occupied streams, have been guided by information from non-functional genetic markers. In this study, we investigated genetic variation at exon 2 of the major histocompatibility complex (MHC) class II β gene that is involved in pathogen resistance and thus presumably under natural selection. Phylogenetic analysis revealed trans-species polymorphism and a significantly high ratio of non-synonymous to synonymous amino acid changes consistent with the action of historical balancing selection that maintained diversity at this locus in the past. However, Gila trout exhibited low allelic diversity (five alleles from 142 individuals assayed) compared to some other salmonid fishes, and populations that originated exclusively from hatcheries possessed three or fewer MHC alleles. Comparative analysis of genetic variation at MHC and six presumably neutrally evolving microsatellite loci revealed that genetic drift cannot be rejected as a primary force governing evolution of MHC in contemporary populations of Gila trout. Maintenance of diversity at MHC will require careful implementation of hatchery breeding protocols and continued protection of wild populations to prevent loss of allelic diversity due to drift.     

Museum genomics: low-cost and high-accuracy genetic data from historical specimens

Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome.     

Population genetic structure and evolutionary history of grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis> in the Yangtze River,China

Ample studies have been conducted to investigate the population genetic structure of grass carp Ctenopharyngodon idella in the Yangtze River, China. However, samples from the upper reaches were not included. In this study, we collected samples from the entire river, including three locations in the upper reaches: Yibin, Banan and Yunyang, two locations in the middle reaches: Shishou and Ruichang and one location in the lower reaches: Hanjiang, and sequenced three mitochondrial coding genes (ND5, ND6 and Cytb) and one control region (i.e., the D-loop). Nineteen haplotypes were observed in grass carp of the Yangtze River through the analysis of combined sequence data sets (around 4428 bp). Haplotype diversity indices (0.6000 ∼ 0.9333) and nucleotide diversity indices (0.0002 ∼ 0.0020) demonstrated low genetic diversity in the Yangtze grass carp. The analysis of molecular variance and the fixation index (F _ST = 0.0202) revealed insignificant genetic difference between samples from different reaches. Two monophyletic lineages of haplotypes were identified, with the lineage A experiencing potential expansion events. Along with previous findings, this study provides a better understanding of genetic diversity and variation of grass carp in the Yangtze River and will be served as an important baseline to evaluate the long-term impact of the Three Gorges Dam and other hydroelectric facilities on fish biodiversity.     

Spatial and temporal patterns of genetic diversity in an endangered Hawaiian honeycreeper,the Hawaii Akepa (<Emphasis Type="Italic">Loxops coccineus coccineus</Emphasis>)

As a result of disease, habitat destruction, and other anthropogenic factors, the Hawaii Akepa (Loxops coccineus coccineus) currently occupies <10% of its original range and exists in five disjunct populations, raising concerns about what effect such reduction and fragmentation has had on the connectivity and diversity of Akepa populations. In this study, we used both historical and contemporary samples to assess genetic diversity and structure in this endangered Hawaiian honeycreeper. We generated sequence data from two mtDNA regions (ND2, control region) and two nuclear introns for contemporary samples representing three of the five current populations. We also generated control region sequence data for museum specimens collected over 100 years ago from throughout the historical range of the bird. Results indicate that despite recent declines and fragmentation, genetic diversity has not been lost. We detected a modest level of genetic differentiation, which followed a combined pattern of isolation-by-barriers and isolation-by-distance, across the historical range of Akepa. The similarly low level of differentiation observed between the contemporary populations indicates that not much divergence, if any, has occurred post-fragmentation. Rather, the present structure seen likely reflects the historical pattern of distribution. Ironically, this declining species exhibits the genetic signal of an expanding population, demonstrating that earlier demographic events are outweighing the effects of recent changes in population size, and genetic estimates of N_e, though crude, suggest Hawaii Akepa were at least an order of magnitude more abundant prior to the decline.     

Genetic consequences of trumpeter swan (Cygnus buccinator) reintroductions

Relocation programs are often initiated to restore threatened species to previously occupied portions of their range. A primary challenge of restoration efforts is to translocate individuals in a way that prevents loss of genetic diversity and decreases differentiation relative to source populations—a challenge that becomes increasingly difficult when remnant populations of the species are already genetically depauperate. Trumpeter swans were previously extirpated in the entire eastern half of their range. Physical translocations of birds over the last 70 years have restored the species to portions of its historical range. Despite the long history of management, there has been little monitoring of the genetic outcomes of these restoration attempts. We assessed the consequences of this reintroduction program by comparing patterns of genetic variation at 17 microsatellite loci across four restoration flocks (three wild-released, one captive) and their source populations. We found that a wild-released population established from a single source displayed a trend toward reduced genetic diversity relative to and significant genetic differentiation from its source population, though small founder population effects may also explain this pattern. Wild-released flocks restored from multiple populations maintained source levels of genetic variation and lacked significant differentiation from at least one of their sources. Further, the flock originating from a single source revealed significantly lower levels of genetic variation than those established from multiple sources. The distribution of genetic variation in the captive flock was similar to its source. While the case of trumpeter swans provides evidence that restorations from multiple versus single source populations may better preserve natural levels of genetic diversity, more studies are needed to understand the general applicability of this management strategy.     

Origin and genetic diversity of mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) introduced to Europe

We provide mitochondrial sequence variation of the invasive fish Gambusia holbrooki from 24 European populations, from Portugal to Greece. Phylogeographic structure in Europe was compared with genetic data from native samples (USA) and historical records were reviewed to identify introduction routes. Overall, data agree with records of historical introductions and translocations, and indicate that the most abundant haplotype throughout Europe originated from North Carolina and corresponded to the first introduction in 1921 to Spain, being transferred to Italy in 1922 and to many countries afterwards. Our results also show that at least another independent introduction occurred first in France and subsequently from France to Greece. Haplotypes of G. affinis were not detected in our European sampling effort but historical records and other data suggest that this species was introduced to Italy in 1927 and it might be present. At the continental scale, there is less diversity in Europe than in North America, in agreement with the low number of introduced fish. At the local scale, some European populations gained diversity from multiple introductions and from “de novo” mutations.     

Asymmetric gene flow and the evolutionary maintenance of genetic diversity in small,peripheral Atlantic salmon populations

Small populations may be expected to harbour less genetic variation than large populations, but the relation between census size (N), effective population size (N _e), and genetic diversity is not well understood. We compared microsatellite variation in four small peripheral Atlantic salmon populations from the Iberian peninsula and three larger populations from Scotland to test whether genetic diversity was related to population size. We also examined the historical decline of one Iberian population over a 50-year period using archival scales in order to test whether a marked reduction in abundance was accompanied by a decrease in genetic diversity. Estimates of effective population size (N _e) calculated by three temporal methods were consistently low in Iberian populations, ranging from 12 to 31 individuals per generation considering migration, and from 38 to 175 individuals per generation if they were regarded as closed populations. Corresponding N _e/N ratios varied from 0.02 to 0.04 assuming migration (mean=0.03) and from 0.04 to 0.18 (mean=0.10) assuming closed populations. Population bottlenecks, inferred from the excess of heterozygosity in relation to allelic diversity, were detected in all four Iberian populations, particularly in those year classes derived from a smaller number of returning adults. However, despite their small size and declining status, Iberian populations continue to display relatively high levels of heterozygosity and allelic richness, similar to those found in larger Scottish populations. Furthermore, in the R. Asón no evidence was found for a historical loss of genetic diversity despite a marked decline in abundance during the last five decades. Thus, our results point to two familiar paradigms in salmonid conservation: (1)␣endangered populations can maintain relatively high levels of genetic variation despite their small size, and (2) marked population declines may not necessarily result in a significant loss of genetic diversity. Although there are several explanations for such results, microsatellite data and physical tagging suggest that high levels of dispersal and asymmetric gene flow have probably helped to maintain genetic diversity in these peripheral populations, and thus to avoid the negative consequences of inbreeding.