Recent demographic bottlenecks are not accompanied by a genetic signature in banner-tailed kangaroo rats (Dipodomys spectabilis)

Single-sample methods of bottleneck detection are now routine analyses in studies of wild populations and conservation genetics. Three common approaches to bottleneck detection are the heterozygosity excess, mode-shift, and M-ratio tests. Empirical groundtruthing of these methods is difficult, but their performances are critical for the accurate reconstruction of population demography. We use two banner-tailed kangaroo rat (Dipodomys spectabilis) populations from southeastern Arizona (USA) that are known to have experienced recent demographic reductions to search for genetic bottleneck signals with eight microsatellite loci. Over eight total sample-years, neither population showed a genetic bottleneck signature. M-ratios in both populations were large, stable, and never fell below a critical significance value (Mc). The mode shift test did not detect any distortion of allele frequencies, and tests of heterozygosity excess were not significant in postbottleneck samples when we used standard microsatellite mutation models. The genetic effects of bottlenecks like those experienced by our study populations should be strongly influenced by rates of mutation and migration. We used genetic parentage data to estimate a relatively high mutation rate in D. spectabilis (0.0081 mutants/generation/locus), but mutation alone is unlikely to explain the temporal distribution of rare alleles that we observed. Migration (gene flow) is a more likely explanation, despite prior mark-recapture analysis that estimated very low rates of interpopulation dispersal. We interpret our kangaroo rat data in light of the broader literature and conclude that in natural populations connected by dispersal, demographic bottlenecks may prove difficult to detect using molecular genetic data.     

Genetic variability and bottleneck studies in Zalawadi, Gohilwadi and Surti goat breeds of Gujarat (India) using microsatellites

Indian goat breeds are recognized as an invaluable component of the world's goat genetic resources. Microsatellite pairs were chosen from the list suggested by International Society for Animal Genetics (ISAG) and amplified in two multiplexes (Set-I: 7 microsatellites and Set-II: 11 microsatellites) for automated fluorescence genotyping to assess bottleneck and analyze genetic variability and genetic distances within and between three goat breeds viz. Zalawadi, Gohilwadi and Surti. The observed number of alleles ranged from 4 (Oar JMP-29) to 15 (ILSTS-030 and -034) with a total of 178 alleles and mean of 9.89 alleles across the three breeds. The overall heterozygosity, PIC and Shannon index values were 0.61, 0.60 and 1.50 indicating high genetic diversity. The maximum observed heterozygosity was found in Gohilwadi and minimum in Surti goat breed. The Nei's standard genetic distance was minimum between Zalawadi and Gohilwadi, and maximum between Gohilwadi and Surti. Non-significant heterozygote excess on the basis of IAM, TPM and SMM models, as revealed from Wilcoxon sign-rank tests, along with a normal ‘L’-shaped distribution of mode-shift test, indicated no bottleneck in Zalawadi and Gohilwadi goat populations, whereas mild bottleneck in the recent past for Surti breed. This research on goat genetic diversity in Gujarat state provides valuable information on Zalawadi, Gohilwadi and Surti goat genetic resources, and will assist in developing a national plan for the conservation and utilization of indigenous goat breeds.     

Genetic architecture of two red ruffed lemur (<Emphasis Type="Italic">Varecia rubra</Emphasis>) populations of Masoala National Park

The current range of the red ruffed lemur (Varecia rubra) population is primarily restricted to forests of the Masoala Peninsula on the northeastern coast of Madagascar. Whereas much of the peninsula is protected as Masoala National Park, parts of the forest are at risk from anthropogenic pressures and habitat fragmentation. We sampled 32 individual red ruffed lemur from two sites: Ambatoledama (DAMA), a narrow forest corridor across an area of degraded habitat connecting larger blocks of forest in the northwestern reaches of the park, and Masiaposa (MAS) forest, a largely pristine forest on the lower western side of the peninsula. Population genetic parameters were estimated for these two populations employing 15 microsatellite loci derived from the V. variegata genome. We found that by exceeding the expected heterozygosity at mutation-drift equilibrium, the DAMA population has undergone a recent population bottleneck. Population structure analysis detected individuals harboring genotypic admixture of the DAMA genetic cluster in the MAS population, suggesting a possibility of unilateral gene flow or movement between these populations.     

Historical demography of a wild lemur population (Propithecus verreauxi) in southwest Madagascar

The human colonization of Madagascar is associated with the extinction of numerous lemur species. However, the degree to which humans have negatively influenced the historical population dynamics of extant lemur species is not well understood. This study employs genetic and demographic analyses to estimate demographic parameters relating to the historical population dynamics of a wild lemur population, Verreaux’s sifaka (Propithecus verreauxi). The genetic analyses are used to determine whether this population experienced a historically recent (i.e., within the last 2000 years) population bottleneck, as well as to estimate the historical population growth rate and the timing of any changes in population size in the past. In addition, a retrospective demographic analysis is used to determine sources of variation and covariation in the sifaka life cycle and how variation in life-cycle transitions contributes to variation in population growth rate. The genetic analyses indicate that the sifaka population did not experience a recent population bottleneck; however, the historical population growth rate was negative, indicating that the ancestral population size was much larger than the current size. The timing of the ancestral population decline has a point estimate of 2300 years ago, but with large credible intervals: 3611–1736 years ago. This point estimate corresponds with the first evidence for human arrival to Madagascar. Climatic variation has also likely influenced past (and current) population dynamics due to stochastic annual rainfall patterns and climatic desiccation, the latter of which began in southwestern Madagascar around 4000 years ago. Variation in the survival of 2-year-old animals as well as large adult females makes the largest contribution to variation in population growth rate. In the absence of more explicit models pertaining to historical population dynamics, it is difficult to attribute the negative population growth rate of this species solely to a single factor (e.g., hunting, habitat destruction).     

Short tandem repeat based analysis of genetic variability in Kanarese buffalo of South India

The goal of the present study was assessing genetic diversity within Kanarese buffalo, the dual purpose breed of South India. A total of 48 unrelated animals were genotyped at 23 short tandem repeat (STR markers)loci. The total number of observed alleles was 180 with a mean of 7.83 per locus, which varied from 3 to 12 across different loci. The mean observed and expected heterozygosity in South Kanara buffaloes was estimated to be 0.518 and 0.712 respectively. Within population inbreeding estimate (F _IS) was significantly positive in most of the investigated loci which resulted in significant deviation from Hardy-Weinberg equilibrium at 19 of 23 loci analyzed. Evaluation of South Kanara buffalo population for mutation drift equilibrium revealed no significant heterozygosity excess under three different models of evolution viz. infinite alleles model (IAM), step-wise mutation model (SMM) and two phase model (TPM), thus indicating the absence of any recent genetic bottleneck. The results of the present study will help in formulating rational breeding strategies as well as conservation of this important germplasm.     

Genetic structure and diversity of Portunus trituberculatus in Chinese population revealed by microsatellite markers

By using six polymorphic microsatellite loci, the population genetic diversity and structure of 120 individuals of Portunus trituberculatus were examined in five wild populations representing different geographical and ecological ranges along the coast of China. Middle level of genetic diversity of P. trituberculatus was revealed by observed heterozygosity, allele number and Shannon information index. Pairwise population differentiations were found between all pairs of populations except Beihai (BH) and Qingdao (QD) (P < 0.05). The greatest differentiation was detected between Yingkou (YK) and QD populations. It was inconsistent with the result in previous study using mitochondrial control region, which showed no differentiation between QD and YK population. Recent bottleneck was identified in Ningbo (NB) population under TPM and IAM models, as well as Dandong (DD) and QD populations under IAM model. Additionally, compared with YK, QD, DD and BH populations, lower historical effective population size with lower genetic diversity were detected in NB population, which called for urgent assessment of the ecological and economic potential of NB fisheries.     

Assessing flavivirus, lentivirus, and herpesvirus exposure in free-ranging ring-tailed lemurs in southwestern Madagascar

The ring-tailed lemur (Lemur catta) is an endangered species found in southwestern Madagascar, and understanding infectious disease susceptibility is an essential step towards the preservation of wild and captive lemur populations. Lemurs are primates that are widely dispersed throughout the island of Madagascar and may serve as hosts or reservoirs for zoonotic infections. The aim of this study was to determine the prevalence of antibodies to West Nile virus (WNV), simian immunodeficiency virus (SIV), and herpes simplex virus type 1 (HSV-1) in a population of free-ranging ring-tailed lemur from the Beza Mahafaly Special Reserve, Madagascar. Samples were collected from 50 animals during field capture studies in June and July 2004 and assayed for presence of viral antibodies during the 12 mo following collection. Forty-seven of the 50 lemurs sampled had antibodies against WNV detectable by epitope-blocking enzyme-linked immunosorbent assay (ELISA). In addition, 50 of 50 samples had titers against WNV ranging from 80 to > or = 1,280 using plaque reduction neutralization test (PRNT(90)). Ten lemurs had antibodies against lentiviral antigens as determined by Western blot analysis. None of the lemurs had antibodies against HSV-1 using ELISA.     

Evaluation of methodology for detection of genetic bottlenecks: inferences from temporally replicated lake trout populations

Statistical methods have been proposed to detect recent bottlenecks on the basis of genetic characterizations of natural populations. In the absence of direct estimates of contemporary or historical population numbers, we tested the indirect M-ratio method based on microsatellite motif size frequency profiles using three historical and three contemporary Great Lakes populations of Salvelinus namaysuch for which severe reductions in population numbers are documented. Simulations employing plausible ranges of empirical population parameter values were used to explore bottleneck likelihood surfaces. We show that single values of the M-ratio are not sufficient to unambiguously infer a bottleneck without knowledge of mutation rates and effective population size (i.e., 4Ne mu or [symbol: see text]). Inferences of the degree of population bottleneck would be best supported if analyses were conducted across plausible ranges of [symbol: see text] and by qualitative comparisons among population samples.     

Molecular evidence for historical and recent population size reductions of tiger salamanders (Ambystoma tigrinum) in Yellowstone National Park

Population declines caused by natural and anthropogenic factors can quickly erode genetic diversity in natural populations. In this study, we examined genetic variation within 10 tiger salamander populations across northern Yellowstone National Park in Wyoming and Montana, USA using eight microsatellite loci. We tested for the genetic signature of population decline using heterozygosity excess, shifts in allele frequencies, and low ratios of allelic number to allelic size range (M-ratios). We found different results among the three tests. All 10 populations had low M-ratios, five had shifts in allele frequencies and only two had significant heterozygosity excesses. These results support theoretical expectations of different temporal signatures among bottleneck tests and suggest that both historical fish stocking, recent, sustained drought, and possibly an emerging amphibian disease have contributed to declines in effective population size.     

Patch size and isolation influence genetic patterns in black-and-white ruffed lemur (Varecia variegata) populations

Land use in Madagascar has resulted in extensive deforestation and forest fragmentation. Endemic species, such as the black-and-white ruffed lemur (Varecia variegata), may be vulnerable to habitat fragmentation due to patchy geographic distributions and sensitivities to forest disturbance. We tested for genetic differentiation among black-and-white ruffed lemur groups in two sites in a large forest patch and three sites in smaller patches. We also investigated the relationship between the genetic diversity of populations and patch configuration (size and isolation), as well as the presence or absence of past genetic bottlenecks. We collected blood (n = 22 individuals) or fecal (n = 33) samples from lemurs and genotyped the extracted DNA for 16 polymorphic microsatellites. Bayesian cluster analysis and F_ST assigned individuals to three populations: Ranomafana (two sites in continuous forest), Kianjavato (two fragments separated by 60 m of non-forest), and Vatovavy (a single fragment, more isolated in time and space). Vatovavy showed significantly lower allelic richness than Ranomafana. Kianjavato also appeared to have lower allelic richness than Ranomafana, though the difference was not significant. Vatovavy was also the only population with a genetic bottleneck indicated under more than one mutation model and a significant F_IS value, showing excess heterozygosity. These results indicate that a small geographic separation may not be sufficient for genetic differentiation of black-and-white ruffed lemur populations and that patch size may influence the rapidity with which genetic diversity is lost following patch isolation.     

Genetic analysis of a documented population bottleneck: introduced Bennett's wallabies (Macropus rufogriseus rufogriseus) in New Zealand

Few bottlenecks of wild populations are sufficiently well-documented to constitute models for testing theories about the impact of bottlenecks on genetic variation, and subsequent population persistence. Relevant details of the Bennett's wallaby (Macropus rufogriseus rufogriseus) introduction into New Zealand were recorded (founder number, source and approximate bottleneck duration) and suggest this may provide a rare opportunity to examine the efficacy of tests designed to detect recent bottlenecks in wild populations. We first assessed the accuracy of historic accounts of the introduction using genetic diversity detected in mitochondrial DNA (mtDNA) and at five microsatellite loci. Phylogenetic analyses of mtDNA D-loop sequence haplotypes were consistent with the reported origin of the founders as Tasmania, rather than one of the Bass Strait islands in which Bennett's wallabies are also found. Microsatellite allele frequencies from the Tasmanian source population were then used to seed bottleneck simulations encompassing varying sizes and numbers of generations, in order to assess the severity of bottleneck consistent with diversity observed in the New Zealand population. The results suggested that the founder number was unlikely to have been as small as the three animals suggested by the account of the introduction. Nonetheless, the bottleneck was probably severe; in the range of three to five pairs of wallabies for one to three generations. It resulted in significantly reduced levels of allelic diversity and heterozygosity relative to the source population. This bottleneck is only detectable under the infinite allele model (IAM) and not under the stepwise mutation model (SMM) or the two-phase model (TPM), and possible explanations for this are discussed.     

Island colonization and founder effects: the invasion of the Guadeloupe islands by ship rats (Rattus rattus)

The stepwise colonization of islands within an archipelago is typically punctuated by successive founder effects, with each newly founded population being a subsample of the gene pool of the source island. Thus, the genetic signature of successive bottlenecks should be detected when analysing the genetic structure between islands of an archipelago. To test this prediction, we investigated introduced ship rat populations, Rattus rattus (Linnaeus, 1758), in the Guadeloupe Archipelago. Three different methods, commonly named the heterozygosity excess, the mode-shift indicator and the M ratio method, were used to detect bottlenecks from genetic data obtained with eight microsatellite markers on Guadeloupe and two neighbouring islands, Petite-Terre and Fajou. Moreover, a recent eradication failure on Fajou allowed us to test the accuracy of the methods in an 'experimental-like' situation. The results indicate that rats were introduced on Guadeloupe first, which then became the source population for independent secondary colonization of Fajou and Petite-Terre. Moreover, the heterozygosity excess and the mode-shift indicator only detected bottlenecks for the recent colonization of Petite-Terre and the eradication failure on Fajou. However, bottlenecks were detected for all the populations using the M ratio method. This could be interpreted as the remaining signature of the early introduction of the ship rat in the archipelago.     

Effects of anthropogenic disturbance on indri (Indri indri) health in Madagascar

Anthropogenic habitat disturbance impairs ecosystem health by fragmenting forested areas, introducing environmental contamination, and reducing the quality of habitat resources. The effect of this disturbance on wildlife health is of particular concern in Madagascar, one of the world's biodiversity hotspots, where anthropogenic pressures on the environment remain high. Despite the conservation importance of threatened lemur populations in Madagascar, few data exist on the effects of anthropogenic disturbance on lemur health. To examine these impacts, indri (Indri indri) populations were evaluated from two forest reserves that differ in their exposure to anthropogenic disturbance. We compared the health status of 36 indri individuals from two sites: one population from a protected, undisturbed area of lowland evergreen humid forest and the other population from a reserve exposed to frequent tourism and forest degradation. Comparison of indri health parameters between sites suggests an impact of anthropogenic disturbance, including significant differences in leukocyte count and differential, 12 serum parameters, 6 trace minerals, and a higher diversity of parasites, with a significant difference in the presence of the louse, Trichophilopterus babakotophilus. These data suggest that indri living in disturbed forests may experience physiological changes and increased susceptibility to parasitism, which may ultimately impair reproductive success and survival.     

A comparison of salivary pH in sympatric wild lemurs (Lemur catta and Propithecus verreauxi) at Beza Mahafaly Special Reserve, Madagascar

Chemical deterioration of teeth is common among modern humans, and has been suggested for some extinct primates. Dental erosion caused by acidic foods may also obscure microwear signals of mechanical food properties. Ring-tailed lemurs at the Beza Mahafaly Special Reserve (BMSR), Madagascar, display frequent severe tooth wear and subsequent tooth loss. In contrast, sympatric Verreaux's sifaka display far less tooth wear and infrequent tooth loss, despite both species regularly consuming acidic tamarind fruit. We investigated the potential impact of dietary acidity on tooth wear, collecting data on salivary pH from both species, as well as salivary pH from ring-tailed lemurs at Tsimanampesotse National Park, Madagascar. We also collected salivary pH data from ring-tailed lemurs at the Indianapolis Zoo, none of which had eaten for at least 12 hr before data collection. Mean salivary pH for the BMSR ring-tailed lemurs (8.098, n=41, SD=0.550) was significantly more alkaline than Verreaux's sifaka (7.481, n=26, SD=0.458). The mean salivary pH of BMSR (8.098) and Tsimanampesotse (8.080, n=25, SD=0.746) ring-tailed lemurs did not differ significantly. Salivary pH for the Indianapolis Zoo sample (8.125, n=16, SD=0.289) did not differ significantly from either the BMSR or Tsimanampesotse ring-tailed lemurs, but was significantly more alkaline than the BMSR Verreaux's sifaka sample. Regardless of the time between feeding and collection of pH data (from several minutes to nearly 1 hr), salivary pH for each wild lemur was above the "critical" pH of 5.5, below which enamel demineralization occurs. Thus, the high pH of lemur saliva suggests a strong buffering capacity, indicating the impact of acidic foods on dental wear is short-lived, likely having a limited effect. However, tannins in tamarind fruit may increase friction between teeth, thereby increasing attrition and wear in lemurs. These data also suggest that salivary pH varies between lemur species, corresponding to broad dietary categories.     

Genetic consequences of population decline in the European otter (Lutra lutra): an assessment of microsatellite DNA variation in Danish otters from 1883 to 1993

The European otter (Lutra lutra) was common in Denmark until the 1960s, but its present distribution encompasses only a minor part of the country. The aim of this study was to assess whether the recent population decline has resulted in loss of genetic variability and to gain further insight into the dynamics of the population decline. This was done by analysing microsatellite DNA variation in contemporary and historical samples, the latter encompassing DNA samples extracted from museum specimens covering a time-span from the 1880s to the 1960s. Tests for differences in expected heterozygosity and the numbers of alleles in contemporary versus historical samples and a test for detecting population bottlenecks provided few indications of a recent bottleneck and loss of variability. However, a procedure for detecting population expansions and declines, based on the genealogical history of microsatellite alleles, suggested that a drastic long-term population decline has taken place, which could have started more than 2000 years ago, possibly due to ancient anthropogenic pressure. Finally, assignment tests and pairwise F(ST) values suggested weak but statistically significant genetic differentiation between the extant population and historical samples of otters from other regions in Denmark, more likely reflecting differentiation among original populations rather than recent drift.     

Lemurs in a complex landscape: mapping species density in subtropical dry forests of southwestern Madagascar using data at multiple levels

The study of southern dry forest lemurs has been largely restricted to small reserves; yet, the majority of the region's lemur populations reside outside protected areas. Lemur catta and Propithecus verreauxi occupy the same forests but have different dietary preferences. This study assessed L. catta and P. verreauxi population densities across a 3-km dry forest gradient (1,539?ha) in southern Madagascar. The study was designed to allow lemur densities to be related to particular forest types. A particular aim of this study was to collect lemur data in both protected and unprotected areas. Density estimates were calculated using point transect distance sampling in a study area that contained the Beza Mahafaly Special Reserve and the adjacent disturbed forests. The highest densities recorded for each species were in the protected area where the two species were most segregated in their distribution, with L. catta density highest in gallery forest type and P. verreauxi density highest in dry deciduous. Densities of both species varied widely outside the protected area, but P. verreauxi density was more uniform than was L. catta. Results of this study indicate that patterns of lemur density in protected areas are not representative of patterns in disturbed forests; this also suggests that we cannot fully understand the ecological constraints facing primate species by studying them only in protected areas. This research highlights the value of pairing the study of landscape-level patterns of species distribution with both local ground-level ecological interpretations and broad-scale satellite data; information from only one level may give an incomplete view of the community.     

Considering the Influence of Nonadaptive Evolution on Primate Color Vision

Color vision in primates is variable across species, and it represents a rare trait in which the genetic mechanisms underlying phenotypic variation are fairly well-understood. Research on primate color vision has largely focused on adaptive explanations for observed variation, but it remains unclear why some species have trichromatic or polymorphic color vision while others are red-green color blind. Lemurs, in particular, are highly variable. While some species are polymorphic, many closely-related species are strictly dichromatic. We provide the first characterization of color vision in a wild population of red-bellied lemurs (Eulemur rubriventer, Ranomafana National Park, Madagascar) with a sample size (87 individuals; N_{X chromosomes} = 134) large enough to detect even rare variants (0.95 probability of detection at ≥ 3% frequency). By sequencing exon 5 of the X-linked opsin gene we identified opsin spectral sensitivity based on known diagnostic sites and found this population to be dichromatic and monomorphic for a long wavelength allele. Apparent fixation of this long allele is in contrast to previously published accounts of Eulemur species, which exhibit either polymorphic color vision or only the medium wavelength opsin. This unexpected result may represent loss of color vision variation, which could occur through selective processes and/or genetic drift (e.g., genetic bottleneck). To indirectly assess the latter scenario, we genotyped 55 adult red-bellied lemurs at seven variable microsatellite loci and used heterozygosity excess and M-ratio tests to assess if this population may have experienced a recent genetic bottleneck. Results of heterozygosity excess but not M-ratio tests suggest a bottleneck might have occurred in this red-bellied lemur population. Therefore, while selection may also play a role, the unique color vision observed in this population might have been influenced by a recent genetic bottleneck. These results emphasize the need to consider adaptive and nonadaptive mechanisms of color vision evolution in primates.     

Variation in dental wear and tooth loss among known‐aged,older ring‐tailed lemurs (Lemur catta): a comparison between wild and captive individuals

Tooth wear is generally an age‐related phenomenon, often assumed to occur at similar rates within populations of primates and other mammals, and has been suggested as a correlate of reduced offspring survival among wild lemurs. Few long‐term wild studies have combined detailed study of primate behavior and ecology with dental analyses. Here, we present data on dental wear and tooth loss in older (>10 years old) wild and captive ring‐tailed lemurs (Lemur catta). Among older ring‐tailed lemurs at the Beza Mahafaly Special Reserve (BMSR), Madagascar (n=6), the percentage of severe dental wear and tooth loss ranges from 6 to 50%. Among these six individuals, the oldest (19 years old) exhibits the second lowest frequency of tooth loss (14%). The majority of captive lemurs at the Indianapolis Zoo (n=7) are older than the oldest BMSR lemur, yet display significantly less overall tooth wear for 19 of 36 tooth positions, with only two individuals exhibiting antemortem tooth loss. Among the captive lemurs, only one lemur (a nearly 29 year old male) has lost more than one tooth. This individual is only missing anterior teeth, in contrast to lemurs at BMSR, where the majority of lost teeth are postcanine teeth associated with processing specific fallback foods. Postcanine teeth also show significantly more overall wear at BMSR than in the captive sample. At BMSR, degree of severe wear and tooth loss varies in same aged, older individuals, likely reflecting differences in microhabitat, and thus the availability and use of different foods. This pattern becomes apparent before “old age,” as seen in individuals as young as 7 years. Among the four “older” female lemurs at BMSR, severe wear and/or tooth loss do not predict offspring survival. Am. J. Primatol. 72:1026–1037, 2010. © 2010 Wiley‐Liss, Inc.     

Testing for a historical population bottleneck in wild Verreaux's sifaka (Propithecus verreauxi verreauxi) using microsatellite data

The degree to which historical human activities negatively impacted past and present lemur species is a long-standing question in primatology. At present, most evidence addressing this issue comes from archaeology, paleontology, and behavioral studies. Genetic data provide another source of evidence. In this study, six microsatellite loci, genotyped on more than 360 wild Verreaux's sifaka, are used in order to test the hypothesis that this population experienced a population bottleneck in the last 2000 years. Excess heterozygosity is compared with the heterozygosity expected under mutation-drift equilibrium in order to test for the genetic signature of a rapid population contraction in the past. The results indicate that the sifaka population did not experience a population bottleneck. Various methodological and conceptual implications of this result are discussed.     

Genetic impoverishment of the last black grouse (Tetrao tetrix) population in the Netherlands: detectable only with a reference from the past

We have studied a small isolated population of black grouse (Tetrao tetrix) in the Netherlands to examine the impact of isolation and reduction in numbers on genetic diversity. We compared the genetic diversity in the last extant Dutch population with Dutch museum samples and three other black grouse populations (from England, Austria and Norway, respectively) representing isolated and continuous populations. We found significantly lower allelic richness, observed and expected heterozygosities in the present Dutch population compared to the continuous populations (Austria and Norway) and also to the historical Dutch population. However, using a bottleneck test on each population, signs of heterozygosity excess were only found in the likewise isolated English population despite that strong genetic drift was evident in the present Dutch population in comparison to the reference populations, as assessed both in pairwise F(ST)and STRUCTURE analyses. Simulating the effect of a population reduction on the Dutch population from 1948 onwards, using census data and with the Dutch museum samples as a model for the genetic diversity in the initial population, revealed that the loss in number of alleles and observed heterozygosity was according to genetic drift expectations and within the standard error range of the present Dutch population. Thus, the effect of the strong decline in the number of grouse on genetic diversity was only detectable when using a reference from the past. The lack of evidence for a population reduction in the present Dutch population by using the program bottleneck was attributed to a rapidly found new equilibrium as a consequence of a very small effective population size.