Precision and accuracy of divergence time estimates from STR and SNPSTR variation

Inference of intraspecific population divergence patterns typically requires genetic data for molecular markers with relatively high mutation rates. Microsatellites, or short tandem repeat (STR) polymorphisms, have proven informative in many such investigations. These markers are characterized, however, by high levels of homoplasy and varying mutational properties, often leading to inaccurate inference of population divergence. A SNPSTR is a genetic system that consists of an STR polymorphism closely linked (typically < 500 bp) to one or more single-nucleotide polymorphisms (SNPs). SNPSTR systems are characterized by lower levels of homoplasy than are STR loci. Divergence time estimates based on STR variation (on the derived SNP allele background) should, therefore, be more accurate and precise. We use coalescent-based simulations in the context of several models of demographic history to compare divergence time estimates based on SNPSTR haplotype frequencies and STR allele frequencies. We demonstrate that estimates of divergence time based on STR variation on the background of a derived SNP allele are more accurate (3% to 7% bias for SNPSTR versus 11% to 20% bias for STR) and more precise than STR-based estimates, conditional on a recent SNP mutation. These results hold even for models involving complex demographic scenarios with gene flow, population expansion, and population bottlenecks. Varying the timing of the mutation event generating the SNP revealed that estimates of divergence time are sensitive to SNP age, with more recent SNPs giving more accurate and precise estimates of divergence time. However, varying both mutational properties of STR loci and SNP age demonstrated that multiple independent SNPSTR systems provide less biased estimates of divergence time. Furthermore, the combination of estimates based separately on STR and SNPSTR variation provides insight into the age of the derived SNP alleles. In light of our simulations, we interpret estimates from data for human populations.     

Estimating parameters of speciation models based on refined summaries of the joint site-frequency spectrum

Understanding the processes and conditions under which populations diverge to give rise to distinct species is a central question in evolutionary biology. Since recently diverged populations have high levels of shared polymorphisms, it is challenging to distinguish between recent divergence with no (or very low) inter-population gene flow and older splitting events with subsequent gene flow. Recently published methods to infer speciation parameters under the isolation-migration framework are based on summarizing polymorphism data at multiple loci in two species using the joint site-frequency spectrum (JSFS). We have developed two improvements of these methods based on a more extensive use of the JSFS classes of polymorphisms for species with high intra-locus recombination rates. First, using a likelihood based method, we demonstrate that taking into account low-frequency polymorphisms shared between species significantly improves the joint estimation of the divergence time and gene flow between species. Second, we introduce a local linear regression algorithm that considerably reduces the computational time and allows for the estimation of unequal rates of gene flow between species. We also investigate which summary statistics from the JSFS allow the greatest estimation accuracy for divergence time and migration rates for low (around 10) and high (around 100) numbers of loci. Focusing on cases with low numbers of loci and high intra-locus recombination rates we show that our methods for the estimation of divergence time and migration rates are more precise than existing approaches.     

Using molecular markers with high mutation rates to obtain estimates of relative population size and to distinguish the effects of gene flow and mutation: a demonstration using data from endemic Mauritian skinks

We propose a method of analysing genetic data to obtain separate estimates of the size (N(p)) and migration rate (m(p)) for the sampled populations, without precise prior knowledge of mutation rates at each locus ( micro(L)). The effects of migration and mutation can be distinguished because high migration has the effect of reducing genetic differentiation across all loci, whereas a high mutation rate will only affect the locus in question. The method also takes account of any differences between the spectra of immigrant alleles and of new mutant alleles. If the genetic data come from a range of population sizes, and the loci have a range of mutation rates, it is possible to estimate the relative sizes of the different N(p) values, and likewise the m(p) and the micro(L). Microsatellite loci may also be particularly appropriate because loci with a high mutation rate can reach mutation-drift-migration equilibrium more quickly, and because the spectra of mutants arriving in a population can be particularly distinct from the immigrants. We demonstrate this principle using a microsatellite data set from Mauritian skinks. The method identifies low gene flow between a putative new species and populations of its sister species, whereas the differentiation of two other populations is attributed to small population size. These distinct interpretations were not readily apparent from conventional measures of genetic differentiation and gene diversity. When the method is evaluated using simulated data sets, it correctly distinguishes low gene flow from small population size. Loci that are not at mutation-migration-drift equilibrium can distort the parameter estimates slightly. We discuss strategies for detecting and overcoming this effect.     

How species evolve collectively: implications of gene flow and selection for the spread of advantageous alleles

The traditional view that species are held together through gene flow has been challenged by observations that migration is too restricted among populations of many species to prevent local divergence. However, only very low levels of gene flow are necessary to permit the spread of highly advantageous alleles, providing an alternative means by which low-migration species might be held together. We re-evaluate these arguments given the recent and wide availability of indirect estimates of gene flow. Our literature review of F(ST) values for a broad range of taxa suggests that gene flow in many taxa is considerably greater than suspected from earlier studies and often is sufficiently high to homogenize even neutral alleles. However, there are numerous species from essentially all organismal groups that lack sufficient gene flow to prevent divergence. Crude estimates on the strength of selection on phenotypic traits and effect sizes of quantitative trait loci (QTL) suggest that selection coefficients for leading QTL underlying phenotypic traits may be high enough to permit their rapid spread across populations. Thus, species may evolve collectively at major loci through the spread of favourable alleles, while simultaneously differentiating at other loci due to drift and local selection.     

Joint estimation of contemporary seed and pollen dispersal rates among plant populations

There are few statistical methods for estimating contemporary dispersal among plant populations. A maximum-likelihood procedure is introduced here that uses pre- and post-dispersal population samples of biparentally inherited genetic markers to jointly estimate contemporary seed and pollen immigration rates from a set of discrete external sources into a target population. Monte Carlo simulations indicate that accurate estimates and reliable confidence intervals can be obtained using this method for both pollen and seed migration rates at modest sample sizes (100 parents/population and 100 offspring) when population differentiation is moderate (F(ST) ≥ 0.1), or by increasing pre-dispersal samples (to about 500 parents/population) when genetic divergence is weak (F(ST) = 0.01). The method exhibited low sensitivity to the number of source populations and achieved good accuracy at affordable genetic resolution (10 loci with 10 equifrequent alleles each). Unsampled source populations introduced positive biases in migration rate estimates from sampled sources, although they were minor when the proportion of immigration from the latter was comparatively low. A practical application of the method to a metapopulation of the Australian resprouter shrub Banksia attenuata revealed comparable levels of directional seed and pollen migration among dune groups, and the estimate of seed dispersal was higher than a previous estimate based on conservative assignment tests. The method should be of interest to researchers and managers assessing broad-scale nonequilibrium seed and pollen gene flow dynamics in plants.     

Microsatellites can be misleading: an empirical and simulation study

Abstract. It has been long recognized that highly polymorphic genetic markers can lead to underestimation of divergence between populations when migration is low. Microsatellite loci, which are characterized by extremely high mutation rates, are particularly likely to be affected. Here, we report genetic differentiation estimates in a contact zone between two chromosome races of the common shrew ( Sorex araneus ), based on 10 autosomal microsatellites, a newly developed Y-chromosome microsatellite, and mitochondrial DNA. These results are compared to previous data on proteins and karyotypes. Estimates of genetic differentiation based on F - and R -statistics are much lower for autosomal microsatellites than for all other genetic markers. We show by simulations that this discrepancy stems mainly from the high mutation rate of microsatellite markers for F -statististics and from deviations from a single-step mutation model for R -statistics. The sex-linked genetic markers show that all gene exchange between races is mediated by females. The absence of male-mediated gene flow most likely results from male hybrid sterility.     

Molecular demographic history of the annual sunflowers Helianthus annuus and H. petiolaris--large effective population sizes and rates of long-term gene flow.

Hybridization between distinct species may lead to introgression of genes across species boundaries, and this pattern can potentially persist for extended periods as long as selection at some loci or genomic regions prevents thorough mixing of gene pools. However, very few reliable estimates of long-term levels of effective migration are available between hybridizing species throughout their history. Accurate estimates of divergence dates and levels of gene flow require data from multiple unlinked loci as well as an analytical framework that can distinguish between lineage sorting and gene flow and incorporate the effects of demographic changes within each species. Here we use sequence data from 18 anonymous nuclear loci in two broadly sympatric sunflower species, Helianthus annuus and H. petiolaris, analyzed within an "isolation with migration" framework to make genome-wide estimates of the ages of these two species, long-term rates of gene flow between them, and effective population sizes and historical patterns of population growth. Our results indicate that H. annuus and H. petiolaris are approximately one million years old and have exchanged genes at a surprisingly high rate (long-term N(ef)m estimates of approximately 0.5 in each direction), with somewhat higher rates of introgression from H. annuus into H. petiolaris than vice versa. In addition, each species has undergone dramatic population expansion since divergence, and both species have among the highest levels of genetic diversity reported for flowering plants. Our results provide the most comprehensive estimate to date of long-term patterns of gene flow and historical demography in a nonmodel plant system, and they indicate that species integrity can be maintained even in the face of extensive gene flow over a prolonged period.     

MOLECULAR DEMOGRAPHIC HISTORY OF THE ANNUAL SUNFLOWERS HELIANTHUS ANNUUS AND H. PETIOLARIS—LARGE EFFECTIVE POPULATION SIZES AND RATES OF LONG-TERM GENE FLOW

Hybridization between distinct species may lead to introgression of genes across species boundaries, and this pattern can potentially persist for extended periods as long as selection at some loci or genomic regions prevents thorough mixing of gene pools. However, very few reliable estimates of long-term levels of effective migration are available between hybridizing species throughout their history. Accurate estimates of divergence dates and levels of gene flow require data from multiple unlinked loci as well as an analytical framework that can distinguish between lineage sorting and gene flow and incorporate the effects of demographic changes within each species. Here we use sequence data from 18 anonymous nuclear loci in two broadly sympatric sunflower species, Helianthus annuus and H. petiolaris , analyzed within an "isolation with migration" framework to make genome-wide estimates of the ages of these two species, long-term rates of gene flow between them, and effective population sizes and historical patterns of population growth. Our results indicate that H. annuus and H. petiolaris are approximately one million years old and have exchanged genes at a surprisingly high rate (long-term N_efm estimates of approximately 0.5 in each direction), with somewhat higher rates of introgression from H. annuus into H. petiolaris than vice versa. In addition, each species has undergone dramatic population expansion since divergence, and both species have among the highest levels of genetic diversity reported for flowering plants. Our results provide the most comprehensive estimate to date of long-term patterns of gene flow and historical demography in a nonmodel plant system, and they indicate that species integrity can be maintained even in the face of extensive gene flow over a prolonged period.     

Migration rates, frequency-dependent selection and the self-incompatibility locus in Leavenworthia (brassicaceae)

Loci subject to negative frequency-dependent selection are expected to exhibit higher effective migration rates compared to reference loci. Although the number of gene copies transferred between populations by migration is the same for all genes, those subject to negative frequency-dependent selection are more likely to be retained in the immigrant population because rare alleles are selectively favored. So far, evidence for this prediction has been indirect, based on summary statistics rather than on migration rate estimates. Here, we introduce an approximate Bayesian procedure to jointly estimate migration rates at two predefined sets of loci between two populations. We applied the procedure to compare migration rate estimates at the self-incompatibility locus (S-locus) with that at 10 reference loci in two plant species, Leavenworthia alabamica and L. crassa (Brassicaceae). The maximum likelihood estimate for the proportion of migrants (m) was four times higher at the S-locus than at reference loci, but the difference was not statistically significant. Lack of significance might be due to insufficient data, but perhaps also to the recent divergence of the two species (311 ka), because we also show using simulations that the effective migration rate at the S-locus is expected to increase with increasing divergence time. These findings aid in understanding the evolutionary dynamics of negative frequency-dependent selection and they suggest that divergence time should be accounted for when employing migration rates to help detect negative frequency-dependent selection.     

Perspective: gene divergence, population divergence, and the variance in coalescence time in phylogeographic studies

Molecular methods as applied to the biogeography of single species (phylogeography) or multiple codistributed species (comparative phylogeography) have been productively and extensively used to elucidate common historical features in the diversification of the Earth's biota. However, only recently have methods for estimating population divergence times or their confidence limits while taking into account the critical effects of genetic polymorphism in ancestral species become available, and earlier methods for doing so are underutilized. We review models that address the crucial distinction between the gene divergence, the parameter that is typically recovered in molecular phylogeographic studies, and the population divergence, which is in most cases the parameter of interest and will almost always postdate the gene divergence. Assuming that population sizes of ancestral species are distributed similarly to those of extant species, we show that phylogeographic studies in vertebrates suggest that divergence of alleles in ancestral species can comprise from less than 10% to over 50% of the total divergence between sister species, suggesting that the problem of ancestral polymorphism in dating population divergence can be substantial. The variance in the number of substitutions (among loci for a given species or among species for a given gene) resulting from the stochastic nature of DNA change is generally smaller than the variance due to substitutions along allelic lines whose coalescence times vary due to genetic drift in the ancestral population. Whereas the former variance can be reduced by further DNA sequencing at a single locus, the latter cannot. Contrary to phylogeographic intuition, dating population divergence times when allelic lines have achieved reciprocal monophyly is in some ways more challenging than when allelic lines have not achieved monophyly, because in the former case critical data on ancestral population size provided by residual ancestral polymorphism is lost. In the former case differences in coalescence time between species pairs can in principle be explained entirely by differences in ancestral population size without resorting to explanations involving differences in divergence time. Furthermore, the confidence limits on population divergence times are severely underestimated when those for number of substitutions per site in the DNA sequences examined are used as a proxy. This uncertainty highlights the importance of multilocus data in estimating population divergence times; multilocus data can in principle distinguish differences in coalescence time (T) resulting from differences in population divergence time and differences in T due to differences in ancestral population sizes and will reduce the confidence limits on the estimates. We analyze the contribution of ancestral population size (theta) to T and the effect of uncertainty in theta on estimates of population divergence (tau) for single loci under reciprocal monophyly using a simple Bayesian extension of Takahata and Satta's and Yang's recent coalescent methods. The confidence limits on tau decrease when the range over which ancestral population size theta is assumed to be distributed decreases and when tau increases; they generally exclude zero when tau/(4Ne) > 1. We also apply a maximum-likelihood method to several single and multilocus data sets. With multilocus data, the criterion for excluding tau = 0 is roughly that l tau/(4Ne) > 1, where l is the number of loci. Our analyses corroborate recent suggestions that increasing the number of loci is critical to decreasing the uncertainty in estimates of population divergence time.     

Global population genetic structure and male-mediated gene flow in the green sea turtle (Chelonia mydas): analysis of microsatellite loci

We assessed the degree of population subdivision among global populations of green sea turtles, Chelonia mydas, using four microsatellite loci. Previously, a single-copy nuclear DNA study indicated significant male-mediated gene flow among populations alternately fixed for different mitochondrial DNA haplotypes and that genetic divergence between populations in the Atlantic and Pacific Oceans was more common than subdivisions among populations within ocean basins. Even so, overall levels of variation at single-copy loci were low and inferences were limited. Here, the markedly more variable microsatellite loci confirm the presence of male-mediated gene flow among populations within ocean basins. This analysis generally confirms the genetic divergence between the Atlantic and Pacific. As with the previous study, phylogenetic analyses of genetic distances based on the microsatellite loci indicate a close genetic relationship among eastern Atlantic and Indian Ocean populations. Unlike the single-copy study, however, the results here cannot be attributed to an artifact of general low variability and likely represent recent or ongoing migration between ocean basins. Sequence analyses of regions flanking the microsatellite repeat reveal considerable amounts of cryptic variation and homoplasy and significantly aid in our understanding of population connectivity. Assessment of the allele frequency distributions indicates that at least some of the loci may not be evolving by the stepwise mutation model.     

Multi-locus estimates of population structure and migration in a fence lizard hybrid zone

A hybrid zone between two species of lizards in the genus Sceloporus (S. cowlesi and S. tristichus) on the Mogollon Rim in Arizona provides a unique opportunity to study the processes of lineage divergence and merging. This hybrid zone involves complex interactions between 2 morphologically and ecologically divergent subspecies, 3 chromosomal groups, and 4 mitochondrial DNA (mtDNA) clades. The spatial patterns of divergence between morphology, chromosomes and mtDNA are discordant, and determining which of these character types (if any) reflects the underlying population-level lineages that are of interest has remained impeded by character conflict. The focus of this study is to estimate the number of populations interacting in the hybrid zone using multi-locus nuclear data, and to then estimate the migration rates and divergence time between the inferred populations. Multi-locus estimates of population structure and gene flow were obtained from 12 anonymous nuclear loci sequenced for 93 specimens of Sceloporus. Population structure estimates support two populations, and this result is robust to changes to the prior probability distribution used in the Bayesian analysis and the use of spatially-explicit or non-spatial models. A coalescent analysis of population divergence suggests that gene flow is high between the two populations, and that the timing of divergence is restricted to the Pleistocene. The hybrid zone is more accurately described as involving two populations belonging to S. tristichus, and the presence of S. cowlesi mtDNA haplotypes in the hybrid zone is an anomaly resulting from mitochondrial introgression.     

Migration between continents: geographical structure and long-distance gene flow in Porpidia flavicunda (lichen-forming Ascomycota)

Historical and contemporary geographical distribution ranges with their associated gene flow patterns interact to produce the genetic diversity observed today. Often it is not possible to separate out the impacts of historical events, e.g. past fragmentation, and contemporary gene flow, e.g. long-distance dispersal. Porpidia flavicunda is a lichen-forming ascomycete occurring circumpolar in the boreal to arctic zones for which vegetation history suggests that its distribution pattern has stayed broadly the same over the past millennia. DNA-sequence diversity in P. flavicunda can, thus, be expected to predominantly represent geographical population differentiation and its contemporary migration rates. The population sample consists of 110 specimens collected in Northern Québec, Baffin Island, Western Greenland and Northern Scandinavia. DNA-sequence data sets of three nuclear gene fragments (LSU, RPB2 and beta-tubulin) were analysed for genetic diversity within, and differentiation between, geographical regions. Tests of population subdivision employing analyses of molecular variance and exact tests of haplotype frequency distributions showed significant structure between the geographical regions. However, the lack of fixed nucleotide polymorphisms and the wide sharing of identical haplotypes between geographical regions suggest recurrent long-distance gene flow of propagules. Still, the means by which propagules are dispersed remain to be discovered. Inference of migration rates shows that in many cases a sufficiently high amount of migrants is exchanged between geographical regions to prevent drastic population differentiation through genetic drift. The observed haplotype distributions and migration rates point to a gene flow model of isolation by distance.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Diversity and divergence patterns in regulatory genes suggest differential gene flow in recently derived species of the Hawaiian silversword alliance adaptive radiation (Asteraceae)

The impact of gene flow and population size fluctuations in shaping genetic variation during adaptive radiation, at both the genome-wide and gene-specific levels, is very poorly understood. To examine how historical population size and gene flow patterns within and between loci have influenced lineage divergence in the Hawaiian silversword alliance, we have investigated the nucleotide sequence diversity and divergence patterns of four floral regulatory genes (ASAP1-A, ASAP1-B, ASAP3-A, ASAP3-B) and a structural gene (ASCAB9). Levels and patterns of molecular divergence across these five nuclear loci were estimated between two recently derived species (Dubautia ciliolata and Dubautia arborea) which are presumed to be sibling species. This multilocus analysis of genetic variation, haplotype divergence and historical demography indicates that population expansion and differential gene flow occurred subsequent to the divergence of these two lineages. Moreover, contrasting patterns of allele- sharing for regulatory loci vs. a structural locus between these two sibling species indicate alternative histories of genetic variation and partitioning among loci where alleles of the floral regulatory loci are shared primarily from D. arborea to D. ciliolata and alleles of the structural locus are shared in both directions. Taken together, these results suggest that adaptively radiating species can exhibit contrasting allele migration rates among loci such that allele movement at specific loci may supersede genetic divergence caused by drift and that lineage divergence during adaptive radiation can be associated with population expansion.     

Maximum-likelihood estimation of migration rates and effective population numbers in two populations using a coalescent approach.

A new method for the estimation of migration rates and effective population sizes is described. It uses a maximum-likelihood framework based on coalescence theory. The parameters are estimated by Metropolis-Hastings importance sampling. In a two-population model this method estimates four parameters: the effective population size and the immigration rate for each population relative to the mutation rate. Summarizing over loci can be done by assuming either that the mutation rate is the same for all loci or that the mutation rates are gamma distributed among loci but the same for all sites of a locus. The estimates are as good as or better than those from an optimized FST-based measure. The program is available on the World Wide Web at http://evolution.genetics. washington.edu/lamarc.html/.     

Ascertainment bias in estimates of average heterozygosity.

Population geneticists work with a nonrandom sample of the human genome. Conventional practice ensures that unusually variable loci are most likely to be discovered and thus included in the sample of loci. Consequently, estimates of average heterozygosity are biased upward. In what follows we describe a model of this bias. When the mutation rate varies among loci, bias is increased. This effect is only moderate, however, so that a model of invariant mutation rates provides a reasonable approximation. Bias is pronounced when estimated heterozygosity is < approximately 35% Consequently, it probably affects estimates from classical polymorphisms as well as from restriction-site polymorphisms. Estimates from short-tandem-repeat polymorphisms have negligible bias, because of their high heterozygosity. Bias should vary not only among categories of polymorphism but also among populations. It should be largest in European populations, since these are the populations in which most polymorphisms were discovered. As this argument predicts, European estimates exceed those of Africa and Asia at systems with large bias. The magnitude of this European excess is consistent with the version of our model in which mutation rates vary across loci.     

Estimating ancestral population sizes and divergence times

This article presents a new method for jointly estimating species divergence times and ancestral population sizes. The method improves on previous ones by explicitly incorporating intragenic recombination, by utilizing orthologous sequence data from closely related species, and by using a maximum-likelihood framework. The latter allows for efficient use of the available information and provides a way of assessing how much confidence we should place in the estimates. I apply the method to recently collected intergenic sequence data from humans and the great apes. The results suggest that the human-chimpanzee ancestral population size was four to seven times larger than the current human effective population size and that the current human effective population size is slightly >10,000. These estimates are similar to previous ones, and they appear relatively insensitive to assumptions about the recombination rates or mutation rates across loci.     

A new Bayesian method to identify the environmental factors that influence recent migration

We present a new multilocus genotype method that makes inferences about recent immigration rates and identifies the environmental factors that are more likely to explain observed gene flow patterns. It also estimates population-specific inbreeding coefficients, allele frequencies, and local population F(ST)'s and performs individual assignments. We generate synthetic data sets to determine the region of the parameter space where our method is and is not able to provide accurate estimates. Our simulation study indicates that reliable results can be obtained when the global level of genetic differentiation (F(ST)) is >1%, the number of loci is only 10, and sample sizes are of the order of 50 individuals per population. We illustrate our method by applying it to Pakistani human data, considering altitude and geographic distance as explanatory factors. Our results suggest that altitude explains better the genetic data than geographic distance. Additionally, they show that southern low-altitude populations have higher migration rates than northern high-altitude ones.     

INDIRECT METHODS TO ESTIMATE GENE FLOW IN CAVE AND SURFACE POPULATIONS OF ANDRONISCUS DENTIGER (ISOPODA: ONISCIDEA)

We estimateci gene flow among several populations of the troglophilic woodlouse Androniscus dentiger from central Italy using allozyme data. Estimates of gene flow were obtained from G_ST, θ, and the private alleles method, after being tested whether the assumption of the population genetic model and the assumption of neutrality of alleles had been met. Hierarchical analysis of gene flow has been used to investigate the geographic scale at which gene exchange can actually occur. Results showed that, independent of the methods, no ongoing gene flow can be detected among populations, even among geographically proximate ones. Genetic drift is likely the main agent shaping the pattern of genetic divergence among these populations. Patterns of past and ongoing gene flow were considered, as surface populations have become genetically isolated more recently than cave populations. In general, all three methods to estimate gene flow provided Nm estimates of the same magnitude.