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

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

Genetic estimates of contemporary effective population size: to what time periods do the estimates apply?

Although most genetic estimates of contemporary effective population size (Ne) are based on models that assume Ne is constant, in real populations Ne changes (often dramatically) over time, and estimates (Ne) will be influenced by Ne in specific generations. In such cases, it is important to properly match Ne to the appropriate time periods (for example, in computing Ne/N ratios). Here I consider this problem for semelparous species with two life histories (discrete generations and variable age at maturity--the 'salmon' model), for two different sampling plans, and for estimators based on single samples (linkage disequilibrium, heterozygote excess) and two samples (temporal method). Results include the following. Discrete generations: (i) Temporal samples from generations 0 and t estimate the harmonic mean Ne in generations 0 through t - 1 but do not provide information about Ne in generation t; (ii) Single samples provide an estimate of Ne in the parental generation, not the generation sampled; (iii) single-sample and temporal estimates never provide information about Ne in exactly the same generations; (iv) Recent bottlenecks can downwardly bias estimates based on linkage disequilibrium for several generations. Salmon model: (i) A pair of single-cohort (typically juvenile) samples from years 0 and t provide a temporal estimate of the harmonic mean of the effective numbers of breeders in the two parental years (N b(0) and N b(t)), but adult samples are more difficult to interpret because they are influenced by Nb in a number of previous years; (ii) For single-cohort samples, both one-sample and temporal methods provide estimates of Nb in the same years (contrast with results for discrete generation model); (iii) Residual linkage disequilibrium associated with past population size will not affect single-sample estimates of Nb as much as in the discrete generation model because the disequilibrium diffuses among different years of breeders. These results lead to some general conclusions about genetic estimates of Ne in iteroparous species with overlapping generations and identify areas in need of further research.     

Estimation of effective population size for the long-lived darkblotched rockfish Sebastes crameri

We report the variance effective population size (Ne) in darkblotched rockfish (Sebastes crameri) utilizing the temporal method for overlapping generations, which requires a combination of age-specific demography and genetic information from cohorts. Following calculations of age-specific survival and reproductive success from fishery data, we genotyped a sample (n = 1087) comprised by 6 cohorts (from 1995 to 2000) across 7 microsatellite loci. Our Ne estimate (Ne) plus 95% confidence interval was (Ne) = 9157 [6495-12 215], showing that the breeding population number could be 3-4 orders of magnitude smaller than the census population size (N) = 24 376 210). Our estimates resemble closely those found for fishes with similar life history, suggesting that the small (Ne)/(N) ratio for S. crameri is most likely explained by a combination of high variance in reproductive success among individuals, genetic structure, and demographic perturbations such as historical fishing. Because small (Ne)/(N) ratios have been commonly associated with potential loss of genetic variation, our estimates need careful consideration in rockfish management and conservation.     

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.     

The effects of cyclic dynamics and mating system on the effective size of an island mouflon population

The Haute Island mouflon (Ovis aries) population is isolated on one small (6.5 km2) island of the remote Kerguelen archipelago. Given a promiscuous mating system, a cyclic demography and a strong female-biased sex ratio after population crashes, we expected a low effective population size (Ne). We estimated Ne using demographic and temporal genetic approaches based on genetic information at 25 microsatellite loci from 62 and 58 mouflons sampled in 1988 and 2003, respectively. Genetic Ne estimates were higher than expected, varying between 104 and 250 depending on the methods used. Both demographic and genetic approaches show the Haute Island Ne is buffered against population crashes. The unexpectedly high Ne likely results from the cyclic winter crashes that allow young males to reproduce, limiting the variance of male reproductive success. Based on individual-based simulations, we suggest that despite a strongly female-biased sex ratio, the effects of the mating system on the effective population size more closely resemble random mating or weak polygyny.     

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.     

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

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

Quantification of inbreeding due to distant ancestors and its detection using dense single nucleotide polymorphism data

Inbreeding depression, which refers to reduced fitness among offspring of related parents, has traditionally been studied using pedigrees. In practice, pedigree information is difficult to obtain, potentially unreliable, and rarely assessed for inbreeding arising from common ancestors who lived more than a few generations ago. Recently, there has been excitement about using SNP data to estimate inbreeding (F) arising from distant common ancestors in apparently "outbred" populations. Statistical power to detect inbreeding depression using SNP data depends on the actual variation in inbreeding in a population, the accuracy of detecting that with marker data, the effect size, and the sample size. No one has yet investigated what variation in F is expected in SNP data as a function of population size, and it is unclear which estimate of F is optimal for detecting inbreeding depression. In the present study, we use theory, simulated genetic data, and real genetic data to find the optimal estimate of F, to quantify the likely variation in F in populations of various sizes, and to estimate the power to detect inbreeding depression. We find that F estimated from runs of homozygosity (Froh), which reflects shared ancestry of genetic haplotypes, retains variation in even large populations (e.g., SD=0.5% when Ne=10,000) and is likely to be the most powerful method of detecting inbreeding effects from among several alternative estimates of F. However, large samples (e.g., 12,000-65,000) will be required to detect inbreeding depression for likely effect sizes, and so studies using Froh to date have probably been underpowered.     

Geography of genetic processes in the population: genetic migration in Northern Eurasia (Central Asia region)]

Based on the data of the census of 1970, a map of the rates of gene migrations in the rural population of Central Asia and Kazakhstan was drawn. The corrected average value for 35 populations of oblasts (administrative regions) and autonomous republics was m = 0.0606 +/- 0.0102. The estimate obtained from the mapped data was 0.0799 +/- 0.0014, which is 1.2-fold higher than similar values for Europe. The relationship between the rate of gene migration m, the genetically effective population size Ne, and the density of elementary populations in the studied area Pp was analyzed by means of correlation and regression analyses. The following relationship was obtained: m = A/Ne + BPp, where A > 0 and B < 0. This was accounted for by the fact that the migration flow is mainly directed to the less densely populated rural areas. Given that the region was chosen somewhat artificially, studying gene migrations in Central Asia and Kazakhstan as separate historical, geographic, and genetic subregions of northern Eurasia might be a better approach.     

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.     

利用全基因组连锁不平衡估计中国荷斯坦牛有效群体大小

有效群体大小是群体遗传学研究的一个重要内容,有助于我们更清楚地了解群体的遗传变异、进化和复杂性状的遗传机制等。随着高密度SNP标记的出现,越来越多的研究利用SNP标记间连锁不平衡估计有效群体大小。文章采集北京地区中国荷斯坦牛2 093个样本,并利用牛SNP芯片(Illumina BovineSNP50,含5 4001 SNPs)进行基因型测定,估计不同世代中国荷斯坦牛的有效群体大小。质量控制标准设定为SNP检出率0.95,最小等位基因频率>0.05,样本检出率0.95,哈代温伯格平衡检验显著性水平P<0.0001。经过质量控制,共1 968个样本和38 796个SNPs用于连锁不平衡分析。文章选取SNP间距0.1、0.2、0.5、1、2、5、10、15(Mb),估计中国荷斯坦牛在4世代之前有效群体大小。结果表明,中国荷斯坦牛的有效群体呈逐代下降趋势,至4世代前,中国荷斯坦牛平均有效群体为45头左右。     

A pseudo-likelihood method for estimating effective population size from temporally spaced samples

A pseudo maximum likelihood method is proposed to estimate effective population size (Ne) using temporal changes in allele frequencies at multi-allelic loci. The computation is simplified dramatically by (1) approximating the multi-dimensional joint probabilities of all the data by the product of marginal probabilities (hence the name pseudo-likelihood), (2) exploiting the special properties of transition matrix and (3) using a hidden Markov chain algorithm. Simulations show that the pseudo-likelihood method has a similar performance but needs much less computing time and storage compared with the full likelihood method in the case of 3 alleles per locus. Due to computational developments, I was able to assess the performance of the pseudo-likelihood method against the F-statistic method over a wide range of parameters by extensive simulations. It is shown that the pseudo-likelihood method gives more accurate and precise estimates of Ne than the F-statistic method, and the performance difference is mainly due to the presence of rare alleles in the samples. The pseudo-likelihood method is also flexible and can use three or more temporal samples simultaneously to estimate satisfactorily the NeS of each period, or the growth parameters of the population. The accuracy and precision of both methods depend on the ratio of the product of sample size and the number of generations involved to Ne, and the number of independent alleles used. In an application of the pseudo-likelihood method to a large data set of an olive fly population, more precise estimates of Ne are obtained than those from the F-statistic method.     

Consequences of the founder effect in the genetic structure of introduced island coral reef fish populations

Between 1955 and 1961, the Division of Fish and Game of the State of Hawaii (USA) undertook an introduction program which brought 11 species of families Serranidae and Lutjanidae (Pisces) from French Polynesia to the coral reefs off Oahu and Big Island in Hawaii. Only three— Cephalopholis argus, Lutjanus fulvus and Lutjanus kasmira —for which we have records of locations and number offish released, are known to have become established. Comparison of allozyme distribution of C. argus and L. kasmira between individuals collected in Hawaiian and Polynesian populations provided a unique opportunity to estimate the impact of genetic drift and selection processes caused by a founder event. We used temporal variance of allelic frequencies to estimate and validate effective population size within marine fish populations. Despite the fact that only 571 C. argus and 2435 L. kasmira were released, we did not observe major changes in polymorphism and heterozygosity. Using different models to estimate effective population size from temporal variance in allelic frequencies and the number of generations, we estimate that the effective population size is about 1–5% of the total population size. Such reduced effective population size explains why most of the species introduced in Hawaii (8/11) failed to become established. Our results have implications for conservation biology because they emphasize that in spite of the fact that only a few individuals bequeathed their characteristics to subsequent generations, no significant change in genetic diversity was observed; success of introduced species is therefore limited by the number of fish released.     

Estimating contemporary effective population size on the basis of linkage disequilibrium in the face of migration

Effective population size (Ne) is an important genetic parameter because of its relationship to loss of genetic variation, increases in inbreeding, accumulation of mutations, and effectiveness of selection. Like most other genetic approaches that estimate contemporary Ne, the method based on linkage disequilibrium (LD) assumes a closed population and (in the most common applications) randomly recombining loci. We used analytical and numerical methods to evaluate the absolute and relative consequences of two potential violations of the closed-population assumption: (1) mixture LD caused by occurrence of more than one gene pool, which would downwardly bias Ne and (2) reductions in drift LD (and hence upward bias in Ne) caused by an increase in the number of parents responsible for local samples. The LD method is surprisingly robust to equilibrium migration. Effects of mixture LD are small for all values of migration rate (m), and effects of additional parents are also small unless m is high in genetic terms. LD estimates of Ne therefore accurately reflect local (subpopulation) Ne unless m>～5-10%. With higher m, Ne converges on the global (metapopulation) Ne. Two general exceptions were observed. First, equilibrium migration that is rare and hence episodic can occasionally lead to substantial mixture LD, especially when sample size is small. Second, nonequilibrium, pulse migration of strongly divergent individuals can also create strong mixture LD and depress estimates of local Ne. In both cases, assignment tests, Bayesian clustering, and other methods often will allow identification of recent immigrants that strongly influence results. In simulations involving equilibrium migration, the standard LD method performed better than a method designed to jointly estimate Ne and m. The above results assume loci are not physically linked; for tightly linked loci, the LD signal from past migration events can persist for many generations, with consequences for Ne estimates that remain to be evaluated.     

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

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

Predicting the annual effective size of livestock populations

Effective population size (Ne) is an important parameter determining the genetic structure of small populations. In natural populations, the number of adults (N) is usually known and Ne can be estimated on the basis of an assumed ratio Ne/N, usually found to be close to 0.5. In farm animal populations, apart from using pedigrees or genetic marker information, Ne can be estimated from the number N of breeding animals, and a value of 1 is commonly assumed for the ratio Ne/N. The purpose of this paper is to show the relation between effective population size and breeding herd size in livestock species. With overlapping generations, Ne can be predicted knowing the number of individuals entering the population per generation and the variance of family size, the latter being directly related to the survival pattern (or replacement policy) in the breeding herd. Assuming an ideal survivorship leading to a geometric age distribution, it can be shown that the number of breeding animals tends to overestimate effective size, particularly in early-maturing species. The ratio of annual effective size to the number of breeding animals is shown to be equal to [1 + (a- 1)(1 - s)]2/(1 - s2), where a is the age at first offspring and s is the survival rate (including culling) of the parents between successive births. This expression shows to what extent inbreeding may be determined by demography or culling policy independently of the actual herd size. In many situations a fast replacement or an early culling will increase annual effective size. Consequences for the management of small populations are discussed.     

Genetic consequences of polygyny and social structure in an Indian fruit bat, Cynopterus sphinx. II. Variance in male mating success and effective population size

Variance in reproductive success is a primary determinant of genetically effective population size (Ne), and thus has important implications for the role of genetic drift in the evolutionary dynamics of animal taxa characterized by polygynous mating systems. Here we report the results of a study designed to test the hypothesis that polygynous mating results in significantly reduced Ne in an age-structured population. This hypothesis was tested in a natural population of a harem-forming fruit bat, Cynopterus sphinx (Chiroptera: Pteropodidae), in western India. The influence of the mating system on the ratio of variance Ne to adult census number (N) was assessed using a mathematical model designed for age-structured populations that incorporated demographic and genetic data. Male mating success was assessed by means of direct and indirect paternity analysis using 10-locus microsatellite genotypes of adults and progeny from two consecutive breeding periods (n = 431 individually marked bats). Combined results from both analyses were used to infer the effective number of male parents in each breeding period. The relative proportion of successfully reproducing males and the size distribution of paternal sibships comprising each offspring cohort revealed an extremely high within-season variance in male mating success (up to 9.2 times higher than Poisson expectation). The resultant estimate of Ne/N for the C. sphinx study population was 0.42. As a result of polygynous mating, the predicted rate of drift (1/2Ne per generation) was 17.6% higher than expected from a Poisson distribution of male mating success. However, the estimated Ne/N was well within the 0.25-0.75 range expected for age-structured populations under normal demographic conditions. The life-history schedule of C. sphinx is characterized by a disproportionately short sexual maturation period scaled to adult life span. Consequently, the influence of polygynous mating on Ne/N is mitigated by the extensive overlap of generations. In C. sphinx, turnover of breeding males between seasons ensures a broader sampling of the adult male gamete pool than expected from the variance in mating success within a single breeding period.     

Gene trees and species trees – mutual influences and interdependences of population genetics and systematics

Both population genetics and systematics are core disciplines of evolutionary biology. While systematics deals with genealogical relationships among taxa, population genetics has mainly been based on allele frequencies and the distribution of genetic variants whose genealogical relations could for a long time, due mainly to methodological constraints, not be inferred. The advent of mitochondrial DNA analyses and modern sequencing techniques in the 1970s revolutionized evolutionary genetic studies and gave rise to molecular phylogenetics. In the wake of this new development systematic approaches and principles were incorporated into intraspecific studies at the population level, e.g. the concept of monophyly which is used to delineate evolutionarily significant units in conservation biology. A new discipline combining phylogenetic analyses of genetic lineages with their geographic distribution ('phylogeography') was introduced as an explicit synthesis of population genetics and systematics. On the other hand, it has increasingly become obvious that discordances between gene trees and species trees not only result from spurious data sets or methodological flaws in phylogenetic analyses, but that they often reflect real population genetic processes such as lineage sorting or hybridization. These processes have to be taken into account when evaluating the reliability of gene trees to avoid wrong phylogenetic conclusions. The present review focuses on the phenomenon of non-phylogenetic sorting of ancestral polymorphisms, its probability and its consequences for molecular systematics.     

Parasites: proxies for host genealogy and ecology?

Genetic information is used extensively to reconstruct the evolutionary and demographic history of organisms. Recently, it has been suggested that genetic information from some parasites can complement genetic data from their hosts. This approach relies upon the hypothesis that such parasites share a common history with their host. In some cases, parasites provide an additional source of information because parasite data can better reconstruct the common history. Here, we discuss which parasite traits are important in determining their usefulness for analysing host history. The key is the matching of the traits of the parasite (e.g. effective population size, generation time, mutation rate and level of host specificity) with the timescales (phylogenetic, phylogeographic and demographic) that are relevant to the issues of concern in host history.     

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.