Reconciling Genetic and Demographic Estimators of Effective Population Size in the Anuran Amphibian Bufo Calamita

We used genetic and demographic methods to estimate the variance effective population sizes (N _e) of three populations of natterjack toads Bufo calamita in Britain. This amphibian breeds in temporary pools where survival rates can vary among families. Census population sizes (N) were derived from spawn string counts. Point and coalescent-based maximum likelihood estimates of N _e based on microsatellite allele distributions were similar. N _e/N ratios based on genetic estimates of N _e ranged between 0.02 and 0.20. Mean demographic estimates of N _e were consistently higher (2.7–8.0-fold) than genetic estimates for all three populations when variance in breeding success was evaluated at the point where females no longer influence their progeny. However, discrepancies between genetic and demographic estimators could be removed by using a model that included extra variance in survivorship (above to Poisson expectations) among families. The implications of these results for the estimation of N _e in wild populations are discussed.     

Making sense of genetic estimates of effective population size

The last decade has seen an explosion of interest in use of genetic markers to estimate effective population size, N_e. Effective population size is important both theoretically (N_e is a key parameter in almost every aspect of evolutionary biology) and for practical application (N_e determines rates of genetic drift and loss of genetic variability and modulates the effectiveness of selection, so it is crucial to consider in conservation). As documented by Palstra & Fraser ( 2012 ), most of the recent growth in N_e estimation can be attributed to development or refinement of methods that can use a single sample of individuals (the older temporal method requires at least two samples separated in time). As with other population genetic methods, performance of new N_e estimators is typically evaluated with simulated data for a few scenarios selected by the author(s). Inevitably, these initial evaluations fail to fully consider the consequences of violating simplifying assumptions, such as discrete generations, closed populations of constant size and selective neutrality. Subsequently, many researchers studying natural or captive populations have reported estimates of N_e for multiple methods; often these estimates are congruent, but that is not always the case. Because true N_e is rarely known in these empirical studies, it is difficult to make sense of the results when estimates differ substantially among methods. What is needed is a rigorous, comparative analysis under realistic scenarios for which true N_e is known. Recently, Gilbert & Whitlock ( 2015 ) did just that for both single‐sample and temporal methods under a wide range of migration schemes. In the current issue of Molecular Ecology, Wang ( 2016 ) uses simulations to evaluate performance of four single‐sample N_e estimators. In addition to assessing effects of true N_e, sample size, and number of loci, Wang also evaluated performance under changing abundance, physical linkage and genotyping errors, as well as for some alternative life histories (high rates of selfing; haplodiploids). Wang showed that the sibship frequency (SF) and linkage disequilibrium (LD) methods perform dramatically better than the heterozygote excess and molecular coancestry methods under most scenarios (see Fig. 1, modified from figure 2 in Wang 2016 ), and he also concluded that SF is generally more versatile than LD. This article represents a truly Herculean effort, and results should be of considerable value to researchers interested in applying these methods to real‐world situations.     

When are genetic methods useful for estimating contemporary abundance and detecting population trends?

The utility of microsatellite markers for inferring population size and trend has not been rigorously examined, even though these markers are commonly used to monitor the demography of natural populations. We assessed the ability of a linkage disequilibrium estimator of effective population size (N_e) and a simple capture-recapture estimator of abundance (N) to quantify the size and trend of stable or declining populations (true N = 100–10,000), using simulated Wright–Fisher populations. Neither method accurately or precisely estimated abundance at sample sizes of S = 30 individuals, regardless of true N. However, if larger samples of S = 60 or 120 individuals were collected, these methods provided useful insights into abundance and trends for populations of N = 100–500. At small population sizes (N = 100 or 250), precision of the N_e estimates was improved slightly more by a doubling of loci sampled than by a doubling of individuals sampled. In general, monitoring N_e proved a more robust means of identifying stable and declining populations than monitoring N over most of the parameter space we explored, and performance of the N_e estimator is further enhanced if the N_e/N ratio is low. However, at the largest population size (N = 10,000), N estimation outperformed N_e. Both methods generally required ≥ 5 generations to pass between sampling events to correctly identify population trend.     

Interannual variation in effective number of breeders and estimation of effective population size in long‐lived iteroparous lake sturgeon (Acipenser fulvescens)

Quantifying interannual variation in effective adult breeding number (N_b) and relationships between N_b, effective population size (N_e), adult census size (N) and population demographic characteristics are important to predict genetic changes in populations of conservation concern. Such relationships are rarely available for long‐lived iteroparous species like lake sturgeon (Acipenser fulvescens). We estimated annual N_b and generational N_e using genotypes from 12 microsatellite loci for lake sturgeon adults (n = 796) captured during ten spawning seasons and offspring (n = 3925) collected during larval dispersal in a closed population over 8 years. Inbreeding and variance N_b estimated using mean and variance in individual reproductive success derived from genetically identified parentage and using linkage disequilibrium (LD) were similar within and among years (interannual range of N_b across estimators: 41–205). Variance in reproductive success and unequal sex ratios reduced N_b relative to N on average 36.8% and 16.3%, respectively. Interannual variation in N_b/N ratios (0.27–0.86) resulted from stable N and low standardized variance in reproductive success due to high proportions of adults breeding and the species' polygamous mating system, despite a 40‐fold difference in annual larval production across years (437–16 417). Results indicated environmental conditions and features of the species' reproductive ecology interact to affect demographic parameters and N_b/N. Estimates of N_e based on three single‐sample estimators, including LD, approximate Bayesian computation and sibship assignment, were similar to annual estimates of N_b. Findings have important implications concerning applications of genetic monitoring in conservation planning for lake sturgeon and other species with similar life histories and mating systems.     

Considerations for monitoring population trends of colonial waterbirds using the effective number of breeders and census estimates

Detecting trends in population size fluctuations is a major focus in ecology, evolution, and conservation biology. Populations of colonial waterbirds have been monitored using demographic approaches to determine annual census size (N_a). We propose the addition of genetic estimates of the effective number of breeders (N_b) as indirect measures of the risk of loss of genetic diversity to improve the evaluation of demographics and increase the accuracy of trend estimates in breeding colonies. Here, we investigated which methods of the estimation of N_b are more precise under conditions of moderate genetic diversity, limited sample sizes and few microsatellite loci, as often occurs with natural populations. We used the wood stork as a model species and we offered a workflow that researchers can follow for monitoring bird breeding colonies. Our approach started with simulations using five estimators of N_b and the theoretical results were validated with empirical data collected from breeding colonies settled in the Brazilian Pantanal wetland. In parallel, we estimated census size using a corrected method based on counting active nests. Both in simulations and in natural populations, the approximate Bayesian computation (ABC) and sibship assignment (SA) methods yielded more precise estimates than the linkage disequilibrium, heterozygosity excess, and molecular coancestry methods. In particular, the ABC method performed best with few loci and small sample sizes, while the other estimators required larger sample sizes and at least 13 loci to not underestimate N_b. Moreover, according to our N_b/N_a estimates (values were often ≤0.1), the wood stork colonies evaluated could be facing the loss of genetic diversity. We demonstrate that the combination of genetic and census estimates is a useful approach for monitoring natural breeding bird populations. This methodology has been recommended for populations of rare species or with a known history of population decline to support conservation efforts.     

Effects of population characteristics and structure on estimates of effective population size in a house sparrow metapopulation

Effective population size (N_e) is a key parameter to understand evolutionary processes and the viability of endangered populations as it determines the rate of genetic drift and inbreeding. Low N_e can lead to inbreeding depression and reduced population adaptability. In this study, we estimated contemporary N_e using genetic estimators (LDNE, ONeSAMP, MLNE and CoNe) as well as a demographic estimator in a natural insular house sparrow metapopulation. We investigated whether population characteristics (population size, sex ratio, immigration rate, variance in population size and population growth rate) explained variation within and among populations in the ratio of effective to census population size (N_e/N_c). In general, N_e/N_c ratios increased with immigration rates. Genetic N_e was much larger than demographic N_e, probably due to a greater effect of immigration on genetic than demographic processes in local populations. Moreover, although estimates of genetic N_e seemed to track N_c quite well, the genetic N_e‐estimates were often larger than N_c within populations. Estimates of genetic N_e for the metapopulation were however within the expected range (<N_c). Our results suggest that in fragmented populations, even low levels of gene flow may have important consequences for the interpretation of genetic estimates of N_e. Consequently, further studies are needed to understand how N_e estimated in local populations or the total metapopulation relates to actual rates of genetic drift and inbreeding.     

Interval Estimation of the Effective Population Size from Heterozygote‐Excess in SNP Markers

The effective population size N_e is an important parameter in population genetics and conservation biology. In recent years, there has been great interest in the use of molecular markers to estimate N_e. Although the point estimates from molecular markers in general suffer from a low reliability, the use of single nucleotide polymorphism (SNP) markers over a wide range of genome is expected to remarkably improve the reliability. In this study, expressions were derived for interval estimates of N_e from one published method, the heterozygote‐excess method, when it is applied to SNP markers. The conditional variance theory is applied to the derivation of a confidence interval for N_e under random union of gametes, monogamy and polygyny. Stochastic simulation shows that the obtained confidence interval is slightly conservative, but fairly useful for practical applications. The result is illustrated with real data on SNP markers in a pig strain.     

Estimating effective population size from linkage disequilibrium: severe bias in small samples

Effective population size (N _e) is a central concept in evolutionary biology and conservation genetics. It predicts rates of loss of neutral genetic variation, fixation of deleterious and favourable alleles, and the increase of inbreeding experienced by a population. A method exists for the estimation of N _e from the observed linkage disequilibrium between unlinked loci in a population sample. While an increasing number of studies have applied this method in natural and managed populations, its reliability has not yet been evaluated. We developed a computer program to calculate this estimator of N _e using the most widely used linkage disequilibrium algorithm and used simulations to show that this estimator is strongly biased when the sample size is small (<‰100) and below the true N _e. This is probably due to the linkage disequilibrium generated by the sampling process itself and the inadequate correction for this phenomenon in the method. Results suggest that N _e estimates derived using this method should be regarded with caution in many cases. To improve the method’s reliability and usefulness we propose a way to determine whether a given sample size exceeds the population N _e and can therefore be used for the computation of an unbiased estimate.     

Small effective population sizes of two remnant ocelot populations (<Emphasis Type="Italic">Leopardus pardalis albescens</Emphasis>) in the United States

Threatened populations are vulnerable to the effects of genetic drift and inbreeding, particularly when gene flow is low and the effective population size is small. Estimates of effective population size (N _e ) provide important information on the status of endangered populations that have experienced severe fragmentation and serve as indicators of genetic viability. Genetic data from microsatellite loci were used to estimate N _e for the 2 remaining populations of the endangered ocelot (Leopardus pardalis albescens) occurring in the United States. Several methods were used to calculate N _e , resulting in estimates ranging from N _e  = 8.0 (95% CI: 3.2–23.1) to 13.9 (95% CI: 7.7–25.1) for the population located at the Laguna Atascosa Wildlife Refuge in Cameron County, Texas. The ocelot population in Willacy County, Texas, had N _e estimates of 2.9 (95% CI: 1.7–5.6) and 3.1 (95% CI: 1.9–13.5), respectively. Estimates of N _e in both populations were below the critical value recommended for short-term viability.     

A genetic demographic analysis of Lake Malawi rock‐dwelling cichlids using spatio‐temporal sampling

We estimated the effective population sizes (N_e) and tested for short‐term temporal demographic stability of populations of two Lake Malawi cichlids: Maylandia benetos, a micro‐endemic, and Maylandia zebra, a widespread species found across the lake. We sampled a total of 351 individuals, genotyped them at 13 microsatellite loci and sequenced their mitochondrial D‐loop to estimate genetic diversity, population structure, demographic history and effective population sizes. At the microsatellite loci, genetic diversity was high in all populations. Yet, genetic diversity was relatively low for the sequence data. Microsatellites yielded mean N_e estimates of 481 individuals (±99 SD) for M. benetos and between 597 (±106.3 SD) and 1524 (±483.9 SD) individuals for local populations of M. zebra. The microsatellite data indicated no deviations from mutation–drift equilibrium. Maylandia zebra was further found to be in migration–drift equilibrium. Temporal fluctuations in allele frequencies were limited across the sampling period for both species. Bayesian Skyline analyses suggested a recent expansion of M. zebra populations in line with lake‐level fluctuations, whereas the demographic history of M. benetos could only be estimated for the very recent past. Divergence time estimates placed the origin of M. benetos within the last 100 ka after the refilling of the lake and suggested that it split off the sympatric M. zebra population. Overall, our data indicate that micro‐endemics and populations in less favourable habitats have smaller N_e, indicating that drift may play an important role driving their divergence. Yet, despite small population sizes, high genetic variation can be maintained.     

Spatial and temporal genetic differentiation and effective population size of brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>, L.) in small Danish rivers

The spatial and temporal genetic structure of brown trout populations from three small tributaries of Lake Hald, Denmark, was studied using analysis of variation at eight microsatellite loci. From two of the populations temporal samples were available, separated by up to 13 years (3.7 generations). Significant genetic differentiation was observed among all samples, however, hierarchical analysis of molecular variance (AMOVA) showed that differentiation among populations accounted for a non-significant amount of the genetic differentiation, whereas differentiation among temporal samples within populations was highly significant (0.0244, P<0.001). Estimates of effective population size (N _e) using a maximum-likelihood based implementation of the temporal method, yielded small values (N _e ranging from 33 to 79). When a model was applied that allows for migration among populations, N _e estimates were even lower (24–54), and migration rates were suggested to be high (0.13–0.36). All samples displayed a clear signal of a recent bottleneck, probably stemming from a period of unfavourable conditions due to organic pollution in the 1970–1980’s. By comparison to other estimates of N _e in brown trout, Lake Hald trout represent a system of small populations linked by extensive gene flow, whereas other populations in larger rivers exhibit much higher N _e values and experience lower levels of immigration. We suggest that management considerations for systems like Lake Hald brown trout should focus both on a regional scale and at the level of individual populations, as the future persistence of populations depends both on maintaining individual populations and ensuring sufficient migration links among these populations.     

Multiple estimates of effective population size for monitoring a long‐lived vertebrate: an application to Yellowstone grizzly bears

Effective population size (N_e) is a key parameter for monitoring the genetic health of threatened populations because it reflects a population's evolutionary potential and risk of extinction due to genetic stochasticity. However, its application to wildlife monitoring has been limited because it is difficult to measure in natural populations. The isolated and well‐studied population of grizzly bears (Ursus arctos) in the Greater Yellowstone Ecosystem provides a rare opportunity to examine the usefulness of different N_e estimators for monitoring. We genotyped 729 Yellowstone grizzly bears using 20 microsatellites and applied three single‐sample estimators to examine contemporary trends in generation interval (GI), effective number of breeders (N_b) and N_e during 1982–2007. We also used multisample methods to estimate variance (N_eV) and inbreeding N_e (N_eI). Single‐sample estimates revealed positive trajectories, with over a fourfold increase in N_e (≈100 to 450) and near doubling of the GI (≈8 to 14) from the 1980s to 2000s. N_eV (240–319) and N_eI (256) were comparable with the harmonic mean single‐sample N_e (213) over the time period. Reanalysing historical data, we found N_eV increased from ≈80 in the 1910s–1960s to ≈280 in the contemporary population. The estimated ratio of effective to total census size (N_e/N_c) was stable and high (0.42–0.66) compared to previous brown bear studies. These results support independent demographic evidence for Yellowstone grizzly bear population growth since the 1980s. They further demonstrate how genetic monitoring of N_e can complement demographic‐based monitoring of N_c and vital rates, providing a valuable tool for wildlife managers.     

A century-long genetic record reveals that protist effective population sizes are comparable to those of macroscopic species

Effective population size (N_e) determines the rate of genetic drift and the relative influence of selection over random genetic changes. While free-living protist populations characteristically consist of huge numbers of cells (N), the absence of any estimates of contemporary N_e raises the question whether protist effective population sizes are comparably large. Using microsatellite genotype data of strains derived from revived cysts of the marine dinoflagellate Pentapharsodinium dalei from sections of a sediment record that spanned some 100 years, we present the first estimates of contemporary N_e for a local population in a free-living protist. The estimates of N_e are relatively small, of the order of a few 100 individuals, and thus are similar in magnitude to values of N_e reported for multicellular animals: the implications are that N_e of P. dalei is of many orders of magnitude lower than the number of cells present (N_e/N ∼ 10⁻¹²) and that stochastic genetic processes may be more prevalent in protist populations than previously anticipated.     

A comparison of single‐sample estimators of effective population sizes from genetic marker data

In molecular ecology and conservation genetics studies, the important parameter of effective population size (N_e) is increasingly estimated from a single sample of individuals taken at random from a population and genotyped at a number of marker loci. Several estimators are developed, based on the information of linkage disequilibrium (LD), heterozygote excess (HE), molecular coancestry (MC) and sibship frequency (SF) in marker data. The most popular is the LD estimator, because it is more accurate than HE and MC estimators and is simpler to calculate than SF estimator. However, little is known about the accuracy of LD estimator relative to that of SF and about the robustness of all single‐sample estimators when some simplifying assumptions (e.g. random mating, no linkage, no genotyping errors) are violated. This study fills the gaps and uses extensive simulations to compare the biases and accuracies of the four estimators for different population properties (e.g. bottlenecks, nonrandom mating, haplodiploid), marker properties (e.g. linkage, polymorphisms) and sample properties (e.g. numbers of individuals and markers) and to compare the robustness of the four estimators when marker data are imperfect (with allelic dropouts). Extensive simulations show that SF estimator is more accurate, has a much wider application scope (e.g. suitable to nonrandom mating such as selfing, haplodiploid species, dominant markers) and is more robust (e.g. to the presence of linkage and genotyping errors of markers) than the other estimators. An empirical data set from a Yellowstone grizzly bear population was analysed to demonstrate the use of the SF estimator in practice.     

Post‐fragmentation population structure in a cooperative breeding Afrotropical cloud forest bird: emergence of a source‐sink population network

The impact of demographic parameters on the genetic population structure and viability of organisms is a long‐standing issue in the study of fragmented populations. Demographic and genetic tools are now readily available to estimate census and effective population sizes and migration and gene flow rates with increasing precision. Here we analysed the demography and genetic population structure over a recent 15‐year time span in five remnant populations of Cabanis's greenbul (Phyllastrephus cabanisi), a cooperative breeding bird in a severely fragmented cloud forest habitat. Contrary to our expectation, genetic admixture and effective population sizes slightly increased, rather than decreased between our two sampling periods. In spite of small effective population sizes in tiny forest remnants, none of the populations showed evidence of a recent population bottleneck. Approximate Bayesian modelling, however, suggested that differentiation of the populations coincided at least partially with an episode of habitat fragmentation. The ratio of meta‐N_e to meta‐N_c was relatively low for birds, which is expected for cooperative breeding species, while N_e/N_c ratios strongly varied among local populations. While the overall trend of increasing population sizes and genetic admixture may suggest that Cabanis's greenbuls increasingly cope with fragmentation, the time period over which these trends were documented is rather short relative to the average longevity of tropical species. Furthermore, the critically low N_c in the small forest remnants keep the species prone to demographic and environmental stochasticity, and it remains open if, and to what extent, its cooperative breeding behaviour helps to buffer such effects.     

gesp: A computer program for modelling genetic effective population size,inbreeding and divergence in substructured populations

The genetically effective population size (N_e) is of key importance for quantifying rates of inbreeding and genetic drift and is often used in conservation management to set targets for genetic viability. The concept was developed for single, isolated populations and the mathematical means for analysing the expected N_e in complex, subdivided populations have previously not been available. We recently developed such analytical theory and central parts of that work have now been incorporated into a freely available software tool presented here. gesp (Genetic Effective population size, inbreeding and divergence in Substructured Populations) is R‐based and designed to model short‐ and long‐term patterns of genetic differentiation and effective population size of subdivided populations. The algorithms performed by gesp allow exact computation of global and local inbreeding and eigenvalue effective population size, predictions of genetic divergence among populations (G_ST) as well as departures from random mating (F_IS, F_IT) while varying (i) subpopulation census and effective size, separately or including trend of the global population size, (ii) rate and direction of migration between all pairs of subpopulations, (iii) degree of relatedness and divergence among subpopulations, (iv) ploidy (haploid or diploid) and (v) degree of selfing. Here, we describe gesp and exemplify its use in conservation genetics modelling.     

Temporal genetic samples indicate small effective population size of the endangered yellow-eyed penguin

There is an increasing awareness that the long-term viability of endemic island populations is negatively affected by genetic factors associated with population bottlenecks and/or persistence at small population size. Here we use contemporary samples and historic museum specimens (collected 1888–1938) to estimate the effective population size (N _e) for the endangered yellow-eyed penguin (Megadyptes antipodes) in South Island, New Zealand, and evaluate the genetic concern for this iconic species. The South Island population of M. antipodes—constituting almost half of the species’ census size—is thought to be descended from a small number of founders that reached New Zealand just a few hundred years ago. Despite intensive conservation measures, this population has shown dramatic fluctuations in size over recent decades. We compare estimates of the harmonic mean N _e for this population, obtained using one moment and three likelihood based-temporal methods, including one method that simultaneously estimates migration rate. Evaluation of the N _e estimates reveals a harmonic mean N _e in the low hundreds. Additionally, the inferred low immigration rates (m = 0.003) agree well with contemporary migration rate estimates between the South Island and subantarctic populations of M. antipodes. The low N _e of South Island M. antipodes is likely affected by strong fluctuations in population size, and high variance in reproductive success. These results show that genetic concerns for this population are valid and that the long-term viability of this species may be compromised by reduced adaptive potential.     

Impact of precocious male parr on the effective size of a wild population of Atlantic salmon

1. There is growing evidence that sexually mature but morphologically juvenile males of Atlantic salmon (precocious or mature male parr) actively participate in reproduction and, therefore, in the genetic composition of the populations of this species. The impact of mature male parr on the effective population size (N_e) of such populations has been previously studied under experimental settings, but no studies have been performed directly on natural populations. 2. Continuous monitoring and sampling of all sea returns is possible in the Lérez River (northwest of Spain). From demographic data on variances of reproductive success and genetic data from six microsatellite marker loci we carried out parentage assignment and assessed the impact of male parr on demographic and genetic estimates of N_e in two consecutive years. 3. Our results reveal that: (i) approximately 60% of the total sire paternity is attributable to mature parr; (ii) mature parr decrease the variance of reproductive success of males by a threefold factor and increase the effective population size of males by a 10‐fold factor; (iii) however, they do not substantially affect the variance of reproductive success and the effective size of females; (iv) mature parr increase two‐to threefold the overall effective size of the population but the ratio N_e/N, where N is the population size including or not mature parr in each case, is not affected.     

Contemporary effective population size and predicted maintenance of genetic diversity in the endangered kea (Nestor notabilis)

Population size and the potential for maintenance of genetic diversity are critical information for the monitoring of species of conservation concern. However, direct estimates of population size are not always feasible, making indirect genetic approaches a valuable alternative. We estimated contemporary effective population size (N_e) in the endangered kea (Nestor notabilis) using three different methods. We then inferred the census size (N_C) using published N_e/N_C ratios and modelled the future maintenance of genetic diversity assuming a number of demographic parameters. Short-term N_e was small with a range-wide N_e?N_C was within the range of the current estimate (c. 1000–5000). Forward simulations showed low probability of retaining 90% of rare alleles without immigration. However, the probability of maintaining genetic diversity was high with immigration, juvenile survival of?≥?30%, and an initial sex ratio of c. 0.5–0.6. Despite the low N_e in kea, predator control and/or artificial immigration might be sufficient to maintain the present genetic diversity.     

Genetic substructure and admixture as important factors in linkage disequilibrium‐based estimation of effective number of breeders in recovering wildlife populations

The number of effective breeders (N_b) and effective population size (N_e) are population parameters reflective of evolutionary potential, susceptibility to stochasticity, and viability. We have estimated these parameters using the linkage disequilibrium‐based approach with LDNE through the latest phase of population recovery of the brown bears (Ursus arctos) in Finland (1993–2010; N = 621). This phase of the recovery was recently documented to be associated with major changes in genetic composition. In particular, differentiation between the northern and the southern genetic cluster declined rapidly within 1.5 generations. Based on this, we have studied effects of the changing genetic structure on N_b and N_e, by comparing estimates for whole Finland with the estimates for the two genetic clusters. We expected a potentially strong relationship between estimate sizes and genetic differentiation, which should disappear as the population recovers and clusters merge. Consistent with this, our estimates for whole Finland were lower than the sum of the estimates of the two genetic clusters and both approaches produced similar estimates in the end. Notably, we also found that admixed genotypes strongly increased the estimates. In all analyses, our estimates for N_e were larger than N_b and likely reflective for brown bears of the larger region of Finland and northwestern Russia. Conclusively, we find that neglecting genetic substructure may lead to a massive underestimation of N_b and N_e. Our results also suggest the need for further empirical analysis focusing on individuals with admixed genotypes and their potential high influence on N_b and N_e.