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.     

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.     

Being abundant is not enough: a decrease in effective population size over eight generations in a Norwegian population of the seaweed, Fucus serratus

The brown alga Fucus serratus is a key foundation species on rocky intertidal shores of northern Europe. We sampled the same population off the coast of southern Norway in 2000 and 2008, and using 26 microsatellite loci, we estimated the changes in genetic diversity and effective population size (Ne). The unexpectedly low Ne (73-386) and Ne/N ratio (10-3-10-4), in combination with a significant decrease (14%) in allelic richness over the 8-year period, suggests an increased local extinction risk. If small Ne proves to be a common feature of F. serratus, then being abundant may not be enough for the species to weather future environmental changes.     

Mutant alleles of small effect are primarily responsible for the loss of fitness with slow inbreeding in Drosophila melanogaster.

Multilocus simulation is used to identify genetic models that can account for the observed rates of inbreeding and fitness decline in laboratory populations of Drosophila melanogaster. The experimental populations were maintained under crowded conditions for approximately 200 generations at a harmonic mean population size of Nh approximately 65-70. With a simulated population size of N = 50, and a mean selective disadvantage of homozygotes at individual loci approximately 1-2% or less, it is demonstrated that the mean effective population size over a 200-generation period may be considerably greater than N, with a ratio matching the experimental estimate of Ne/Nh approximately 1.4. The buildup of associative overdominance at electrophoretic marker loci is largely responsible for the stability of gene frequencies and the observed reduction in the rate of inbreeding, with apparent selection coefficients in favor of the heterozygote at neutral marker loci increasing rapidly over the first N generations of inbreeding to values approximately 5-10%. The observed decline in fitness under competitive conditions in populations of size approximately 50 in D. melanogaster therefore primarily results from mutant alleles with mean effects on fitness as homozygotes of sm < or = 0.02. Models with deleterious recessive mutants at the background loci require that the mean selection coefficient against heterozygotes is at most hsm approximately 0.002, with a minimum mutation rate for a single Drosophila autosome 100 cM in length estimated to be in the range 0.05-0.25, assuming an exponential distribution of s. A typical chromosome would be expected to carry at least 100-200 such mutant alleles contributing to the decline in competitive fitness with slow inbreeding.     

Effective population size of steelhead trout: influence of variance in reproductive success, hatchery programs, and genetic compensation between life-history forms

The effective population size is influenced by many biological factors in natural populations. To evaluate their relative importance, we estimated the effective number of breeders per year (Nb) and effective population size per generation (Ne) in anadromous steelhead trout (Oncorhynchus mykiss) in the Hood River, Oregon (USA). Using demographic data and genetic parentage analysis on an almost complete sample of all adults that returned to the river over 15 years (>15,000 individuals), we estimated Nb for 13 run years and Ne for three entire generations. The results are as follows: (i) the ratio of Ne to the estimated census population size (N) was 0.17-0.40, with large variance in reproductive success among individuals being the primary cause of the reduction in Ne/N; (ii) fish from a traditional hatchery program (Htrad: nonlocal, multiple generations in a hatchery) had negative effects on Nb, not only by reducing mean reproductive success but also by increasing variance in reproductive success among breeding parents, whereas no sign of such effects was found in fish from supplementation hatchery programs (Hsupp: local, single generation in a hatchery); and (iii) Nb was relatively stable among run years, despite the widely fluctuating annual run sizes of anadromous adults. We found high levels of reproductive contribution of nonanadromous parents to anadromous offspring when anadromous run size is small, suggesting a genetic compensation between life-history forms (anadromous and nonanadromous). This is the first study showing that reproductive interaction between different life-history forms can buffer the genetic impact of fluctuating census size on Ne.     

鸡SNP多样性的比较研究与群体有效规模的估算

以红色原鸡(Red Jungle Fowl, RJF)、丝羽乌骨鸡(Taihe silk Chicken, TS)、隐性白洛克鸡(White Recessive Rock, WRR)为资源群, 在鸡一号染色体的Contig.060226.1上选取了一个200 kb的区域, 比较研究了3个群体的SNP (single nucleotide polymorphism)多样性、估算了鸡的初始群体有效规模大小(effective size of population, Ne)。红色原鸡、丝羽乌骨鸡、隐性白洛克鸡3个群体的平均杂合度分别为0.28533±0.03475、0.32926±0.03919、0.30168±0.04038。显著性检验差异不显著(P=0.2368>0.05)。根据Latter 和Nei的方法对鸡的群体有效规模进行了估算, 鸡的初始群体有效规模大小为20 000～150 000。鸡在驯养的早期阶段经历过严厉的瓶颈效应, 但瓶颈效应对鸡各品种SNP的多样性并未产生显著影响。笔者认为, 鸡在驯养的早期阶段群体有效规模足够大, 品种分化过程中群体迅速扩张, 品种间的广泛杂交(特别是和红色原鸡之间)以及鸡基因组的高重组率等因素是导致家鸡和原鸡以及各家鸡品种间SNP多样性没有显著差别的重要原因。     

Evaluating the effects of historic bottleneck events: an assessment of microsatellite variability in the endangered,North Atlantic right whale

Commercial exploitation reduced the North Atlantic right whale (Eubalaena glacialis) population from c. 12,000 in the 11th century to around 300 by the 21st century. We examine the effect of this population decline on levels of genetic variation at 16 microsatellite loci and contrast levels of variability to that in a closely related species (E. australis). Of the 13 loci developed from the E. glacialis genome, 100% were polymorphic in E. australis. In contrast, nine loci were polymorphic in E. glacialis and four were fixed. Both allelic diversity (A) and heterozygosity (H) were significantly lower in E. glacialis than E. australis (A = 3.2 +/- 2.6 vs. A= 6.9 +/- 3.3, P < 0.001; H= 0.31 +/- 0.25 vs. H= 0.72 +/- 0.23, P < 0.001, respectively). Bottleneck anlayses indicate that the population is in mutation-drift equilibrium and that a genetic bottleneck did not occur during the most recent decline (18th-20th centuries). Nevertheless, low frequency alleles are relatively uncommon in E. glacialis, suggesting that genetic variability has been reduced in this population. Possible origins of low genetic variability are discussed, including the slow but continual erosion of alleles during the 800-year period of decline.     

Genetic variability, population size and reproduction potential in Ligularia sibirica (L.) populations in Estonia

Ligularia sibirica (L.) Cass. (Asteraceae) is a EU Habitats Directive Annex II plant species that has suffered a lot from human-caused major changes in quality and availability of habitats in Estonia. The aim of this study was to find out if the observed decline in population size is reflected in the amount of genetic variation and fertility in remnant populations of this species. AFLP technique was used for that purpose. Genetic diversity within populations was assessed as the percentage of polymorphic loci in a given population and average gene diversity over loci. The degree of genetic differentiation among populations and genetic differentiation between pairs of populations was estimated. The amount of viable seeds per flower stem was compared among populations and between years (2007 and 2008). Average genetic diversity over loci and proportion of polymorphic loci in L. sibirica populations were significantly correlated with population size, suggesting the action of genetic drift and/or inbreeding. No correlation was found between genetic and geographic distances. Natural barriers like forests may have been efficiently preventing seed migration even between geographically closer populations. Results of this study suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species.     

Decreasing genetic diversity in wild and captive populations of endangered Itasenpara bittering (Acheilognathus longipinnis) in the Himi region,central Japan,and recommendations for conservation

Biodiversity is increasingly declining as a result of direct human impact and structural alteration of ecosystems resulting from changes in human life styles. Itasenpara bittering (Acheilognathus longipinnis), which has been maintained in floodplain and paddy fields, is a threatened cyprinid fish endemic to central Japan. To aid in the preservation of this species, information on genetic diversity and demographic variables in wild and captive populations was obtained using microsatellite DNA analysis. Temporal changes in genetic diversity and effective population size (N _e) tended to be relatively stable in the wild Moo River population, although lower values were detected in the wild Busshouji River population, suggesting an extremely high risk of extinction in the latter. Captive populations derived from the Busshouji River population demonstrated significant genetic divergence even among intrapopulational cohorts, suggesting the influence of genetic drift caused by geographic isolation and small population size. Active maintenance of genetic diversity in captive population is a necessary part of conservation programs, as are continuous addition of wild individuals and replacement of individuals among captive populations. In addition, increasing or maintaining suitable floodplain areas and artificial habitats such as paddy fields might contribute to the conservation of genetic diversity in the Itasenpara bittering.     

Sustaining genetic variation in a small population: evidence from the Mauritius kestrel

We obtained measures of genetic diversity in 10 kestrel species at a suite of 12 microsatellite loci. We estimated the relative effective size (Ne) of the species using a Markov chain Monte Carlo (MCMC) approach, which jointly estimated the locus specific mutation rates as nuisance parameters. There was surprisingly high genetic diversity found in museum specimens of the Mauritius kestrel. Being an endemic species on a small island, it is known to have a long history of small population size. Conversely, kestrels with a continental distribution had Ne estimates that were only one order of magnitude larger and similar to each other, despite having current population sizes that were between one and three orders of magnitude larger than the Mauritius kestrel. We show how many of the theoretical results describing the effective size of a subdivided population can be captured in terms of three rates which describe the branching pattern of the gene genealogy, and that they are useful in estimating the time to migration-drift and mutation-drift equilibrium. We use this approach to argue that population subdivision has helped retain genetic diversity in the Mauritius kestrel, and that the continental species' genetic diversity has yet to reach equilibrium after the range changes following the last ice age. We draw parallels with Hewitt's observation that genetic variation seems to survive species' range compression and is rather vulnerable to range expansion.     

Small effective population sizes in a widespread selfing species, Lymnaea truncatula (Gastropoda: Pulmonata)

We present here a spatial and temporal population genetic survey of a common freshwater snail, also a predominantly selfing species, Lymnaea truncatula. The rate of genetic diversity loss was quantified by estimating the effective size (Ne) of the snail populations, using two different methods. A temporal survey allowed estimation of a variance effective size of the populations, and a spatial survey allowed the estimation of an inbreeding effective size, from two-locus identity disequilibria estimates. Both methods were consistent and provided low Ne values. Drift due to (i) high amounts of selfing and (ii) fluctuations in population sizes because of temporary habitats, and also selection coupled to genome-wide linkage disequilibria, could explain such reductions in Ne. The loss of genetic diversity appears to be counterbalanced only very partially by low apparent rates of gene flow.     

Genetic characterization of Atlantic sturgeon, <Emphasis Type="Italic">Acipenser oxyrinchus oxyrinchus</Emphasis>, in the Edisto River,South Carolina and identification of genetically discrete fall and spring spawning

Once widely abundant, most subpopulations of the endangered Atlantic sturgeon are now estimated to be only 1–10% of their historical levels. The Edisto River has been sampled for a long period and extensively for juvenile Atlantic sturgeon from separate spring- and fall-spawned cohorts. Our objectives are to characterize the genetic diversity, stability, adaptive potential, and potential genetic structure of Atlantic sturgeon in the Edisto River and to identify any past bottlenecks experienced by this species, as well as to conduct forward simulation modeling of the population under multiple population trajectories. Our results indicate that fall- and spring-spawned Atlantic sturgeon in the Edisto River are genetically distinct (overall \({{F}_{ST}}\)?=?0.092) with little gene flow or admixture between groups, both of which are diverse from a neutral genetic marker standpoint. Genetic diversity of both groups is on the higher end of published population diversity values. A lack of inbreeding and recent bottlenecks also bode well for these two groups of sturgeon, although future projections indicate a loss of allelic richness and genetic diversity even with population stability. Our effective population size estimates are moderate compared to published estimates for other Atlantic sturgeon populations. The most significant finding of our research is the genetic distinctness of the fall- and spring-spawned Atlantic sturgeon in the Edisto River, which may have several important ramifications for management of the species, including re-evaluating the demarcation of distinct population segments.     

Genetic diversity, population structure, effective population size and demographic history of the Finnish wolf population

The Finnish wolf population (Canis lupus) was sampled during three different periods (1996-1998, 1999-2001 and 2002-2004), and 118 individuals were genotyped with 10 microsatellite markers. Large genetic variation was found in the population despite a recent demographic bottleneck. No spatial population subdivision was found even though a significant negative relationship between genetic relatedness and geographic distance suggested isolation by distance. Very few individuals did not belong to the local wolf population as determined by assignment analyses, suggesting a low level of immigration in the population. We used the temporal approach and several statistical methods to estimate the variance effective size of the population. All methods gave similar estimates of effective population size, approximately 40 wolves. These estimates were slightly larger than the estimated census size of breeding individuals. A Bayesian model based on Markov chain Monte Carlo simulations indicated strong evidence for a long-term population decline. These results suggest that the contemporary wolf population size is roughly 8% of its historical size, and that the population decline dates back to late 19th century or early 20th century. Despite an increase of over 50% in the census size of the population during the whole study period, there was only weak evidence that the effective population size during the last period was higher than during the first. This may be caused by increased inbreeding, diminished dispersal within the population, and decreased immigration to the population during the last study period.     

Formulation and estimation of the effective size of stage-structured populations in Fritillaria camtschatcensis, a perennial herb with a complex life history

The effective population size (Ne) is formulated based on a stage-structured population model and is estimated for two populations of Fritillaria camtschatcensis (L.) Ker-Gawl. (Liliaceae), a perennial, mainly clonally reproducing herb. Plants in these populations change life-history stages year by year, either upward or downward across three unambiguously identifiable stages: one-leaf, nonflowering; multileaf nonflowering; and multileaf, flowering stages. Plants of all stages produce clonal progeny (bulblets) each year, and death of plants occurs only in the first stage. The populations are nearly at equilibrium in both population size and stage structure. Ne is estimated to be 20-30% of the census population size (N), leading to the prediction that a population size of about 20,000 or more will be needed to conserve the normal level of the gene diversity (Ne > or = 5000). With the current demographic pattern of this species, accelerated growth of the first-stage plants with reduced survival of the second- and third-stage plants will increase both the annual (Ny/N) and generation time (Ne/N) effective sizes of population.     

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.     

Genetic diversity and effective size of the Atlantic salmon Salmo salar L. inhabiting the River Eo (Spain) following a stock collapse

Historic angling records suggest the occurrence of a drastic decline in the River Eo (Spain) Atlantic salmon population size during the past two decades, as a result of overexploitation and habitat deterioration. In recent years, the population has been apparently recovering, and the present study is aimed to report information on the level of genetic diversity and the effective size of the current population as these may have immediate consequences for its conservation. Eighty-six salmon from two temporal groups (1998–1999 and 2004–2006), representing three generations, were genotyped using a panel of eight microsatellites. Inspite of the recent decline in census numbers and the detection of the signs of a population bottleneck, the population exhibits a high level of genetic diversity, similar to that from other populations, and almost unchanged during the period of study [average allelic richness ( A ) = 14·0 and 13·9, and average heterozygosity ( H _e) = 0·843 and 0·851 in 1998–1999 and 2004–2006, respectively]. The effective population size ( N _e) estimated by two different temporal methods showed a consistent value around 80 salmon, whereas the estimates from the linkage disequilibrium (LD) method provided a value around 165 individuals for either sample. The recent growing number of salmon, as indicated by fisheries records, the relatively large estimates of the ratio N _e/ N (with range 0·23–0·44 for the temporal estimates and 0·31–0·59 for the LD estimates) and the high levels of diversity found suggest that the population has not been greatly affected by the historical census declines and can be expected to recover in the future.     

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.     

Extremely low effective population sizes, genetic structuring and reduced genetic diversity in a threatened bumblebee species, Bombus sylvarum (Hymenoptera: Apidae)

Habitat fragmentation may severely affect survival of social insect populations as the number of nests per population, not the number of individuals, represents population size, hence they may be particularly prone to loss of genetic diversity. Erosion of genetic diversity may be particularly significant among social Hymenoptera such as bumblebees (Bombus spp.), as this group may be susceptible to diploid male production, a suggested direct cost of inbreeding. Here, for the first time, we assess genetic diversity and population structuring of a threatened bumblebee species (Bombus sylvarum) which exists in highly fragmented habitat (rather than oceanic) islands. Effective population sizes, estimated from identified sisterhoods, were very low (range 21-72) suggesting that isolated populations will be vulnerable to loss of genetic variation through drift. Evidence of significant genetic structuring between populations (theta = 0.084) was found, but evidence of a bottleneck was detected in only one population. Comparison across highly fragmented UK populations and a continental population (where this species is more widespread) revealed significant differences in allelic richness attributable to a high degree of genetic diversity in the continental population. While not directly related to population size, this is perhaps explained by the high degree of isolation between UK populations relative to continental populations. We suggest that populations now existing on isolated habitat islands were probably linked by stepping-stone populations prior to recent habitat loss.     

Multilocus patterns of nucleotide polymorphism and demographic change in Taxodium distichum (Cupressaceae) in the lower Mississippi River alluvial valley

? Premise of the Study: Studies of the geographic patterns of genetic variation can give important insights into the past population structure of species. Our study species, Taxodium distichum L. (bald-cypress), prefers riparian and wetland habitats and is widely distributed in southeastern North America and Mexico. We compared the genetic variation of T. distichum with that of its close relative, Cryptomeria japonica, which is endemic to Japan. ? Methods: Nucleotide polymorphisms of T. distichum in the lower Mississippi River alluvial valley, USA, were examined at 10 nuclear loci. ? Key Results: The average nucleotide diversity at silent sites, π(sil), across the 10 loci in T. distichum was higher than that of C. japonica (π(sil) = 0.00732 and 0.00322, respectively). In T. distichum, Tajima's D values were each negative at 9 out of 10 loci, which suggests a recent population expansion. Maximum-likelihood and Bayesian estimations of the exponential population growth rate (g) of T. distichum populations indicated that this species had expanded approximately at the rate of 1.7-1.0 × 10(-6) per year in the past. ? Conclusions: Taxodium distichum had significantly higher nucleotide variation than C. japonica, and its patterns of polymorphism contrasted strikingly with those of the latter, which previously has been inferred to have experienced a reduction in population size.     

The effective population size of Anopheles gambiae in Kenya: implications for population structure

We estimated current and long-term effective population size (Ne) of two Anopheles gambiae (savanna cytotype) populations in Kenya. Temporal variation at nine microsatellite loci in each population sampled 7 and 9 years apart and genetic diversity in each sample were analyzed to answer the following questions. (1) Do bottlenecks occur in Kenyan populations of A. gambiae? (2) How variable are different populations with respect to their current and long-term Ne values? (3) What are the implications of these results on population structure and history? The estimates of Ne of Asembo and Jego were 6,359 and 4,258, respectively, and the lower 95% limits were 2,455 and 1,669, respectively. Thus, despite the typical observation of low density at the village level during the dry season, large populations are maintained annually. Large current Ne is consistent with previous studies showing low differentiation across the continent, especially under Wright's isolation-by-distance model. Current Ne in Asembo was 1.5-fold higher than in Jego, but this difference was not significant. Long-term Ne in Asembo (22,667) was 2.9-fold higher than that in Jego (7,855) based on the stepwise mutation model. The difference between populations was significant at both time points regardless of whether long-term Ne values were calculated based on the stepwise mutation model or the infinite-alleles model. Heterozygosity in Jego declined significantly between 1987 (59%) and 1996 (54%), whereas heterozygosity in Asembo was stable (66%-65%). Despite the relatively high and significant differentiation between Asembo and Jego (FST = 0.072-0.10, RST = 0.037- 0.038), all alleles in Jego were found in Asembo but not vice versa. All of these findings suggest that lower Ne in Jego magnifies differentiation between the two populations. The long-term Ne was biased downward, because its calculation was based on an upper bound estimate of microsatellite mutation rate. Ne values based on mtDNA and allozymes were an order of magnitude higher. Long-term Ne therefore, is probably measured in hundreds of thousands and hence does not support a recent expansion of this species from a small population.