REPRODUCTIVE ISOLATION AND DISTINCT POPULATION STRUCTURES IN TWO TYPES OF THE FRESHWATER SHRIMP PALAEMON PAUCIDENS

Twenty local populations of the Japanese freshwater shrimp Palaemon paucidens were electrophoretically and morphologically surveyed. Based on the diagnostic distributions of some alleles at Gpi, Mpi, Mdh-1, and Mdh-2, these populations were largely classified into two types (A and B). The A type occurred in lakes, ponds, and rivers, while the B type was observed only in rivers. Average Nei's genetic distance (D) between the two types fell into the subspecies range . The coefficient of gene differentiation, G_ST, varied considerably between the two types. In 12 populations of the A type, with a G_ST value of 0.281, nine pond and lake populations showed a higher G_ST (0.246) than the three river populations (0.151). On the other hand, G_ST was 0.036 for the eight local populations of the B type. The lower rostrum tooth number had a mode of two in type A and three in type B. Type-A populations largely varied in the upper rostrum tooth number and egg size but type B did not. Under laboratory conditions, mating frequently occurred within each type, but not between types. Furthermore, no embryonic development was observed in the few cases of intertype mating. These results indicate that the A and B types had experienced cladogenic separation with pre- or postmating isolation, whereafter the A type, under geographic isolation, underwent genetic and phenotypic differentiation, while the B type, under extensive gene flow, did not undergo differentiation.     

THE MAINTENANCE OF POLYGENIC VARIATION IN FINITE POPULATIONS

Models of the maintenance of genetic variance in a polygenic trait have usually assumed that population size is infinite and that selection is weak. Consequently, they will overestimate the amount of variation maintained in finite populations. I derive approximations for the equilibrium genetic variance, in finite populations under weak stabilizing selection for triallelic loci and for an infinite “rare alleles” model. These are compared to results for neutral characters, to the “Gaussian allelic” model, and to Wright's approximation for a biallelic locus under arbitrary selection pressures. For a variety of parameter values, the three-allele, Gaussian, and Wrightian approximations all converge on the neutral model when population size is small. As expected, far less equilibrium genetic variance can be maintained if effective population size, N, is on the order of a few hundred than if N is infinite. All of the models predict that comparisons among populations with N less than about 10⁴ should show substantial differences in . While it is easier to maintain absolute when alleles interact to yield dominance or overdominance for fitness, less additivity also makes more susceptible to differences in N. I argue that experimental data do not seem to reflect the predicted degree of relationship between N and . This calls into question the ability of mutation-selection balance or simple balancing selection to explain observed . The dependence of on N could be used to test the adequacy of mutation-selection balance models.     

EXTINCTION-RECOLONIZATION DYNAMICS IN THE MYCOPHAGOUS BEETLE PHALACRUS SUBSTRIATUS

The population structure of the mycophagous beetle Phalacrus substriatus is characterized by many small, local populations interconnected by migration over a small spatial scale (10 × 75 m²). Each local P. substriatus population has a relatively short expected persistence time, but persistence of the species occurs due to a balance between frequent local extinctions and recolonizations. This nonequilibrium population structure can have profound effects on how the genetic variation is structured between and within populations. Theoretical models have stated that the genetic differentiation among local populations will be enhanced relative to an island model at equilibrium if the number of colonizers is less than approximately twice the number of migrants among local populations. To study these effects, a set of 50 local P. substriatus populations were surveyed over a four-year period to record any naturally occurring extinctions and recolonizations. The per population colonization and extinction rate were 0.237 and 0.275, respectively. Mark-recapture techniques were used to estimate a number of demographic parameters: local population size (N = 11.1), migration rate , number of colonizers (k = 4.0), and the probability of common origin of colonizers (φ = 0.5). The theoretically predicted level of differentiation among local populations (measured as Wright's F_ST) was 0.070. Genetic data obtained from an electrophoretic survey of seven polymorphic loci gave an estimated degree of differentiation of 0.077. There was thus a good agreement between the empirical results and the theoretical predictions. Young populations had significantly higher levels of differentiation than old, more established populations . The extinction-recolonization dynamics resulted in an overall increase in the genetic differentiation among local populations by c. 40%. The global effective population size was also reduced by c. 55%. The results give clear evidence to how nonequilibrium processes shape the genetic structure of populations.     

RELATIONSHIPS BETWEEN INFERRED LEVELS OF GENE FLOW AND GEOGRAPHIC DISTANCE IN A PHILOPATRIC CORAL,BALANOPHYLLIA ELEGANS

When the dispersal capability of a species is considerably less than its geographic range, genetic differences between populations should increase with the distance separating those populations. This pattern should be most evident in linearly distributed species. The sessile solitary cup coral Balanophyllia elegans lives along nearly the entire Pacific coast of North America, yet its crawling larvae usually settle within 40 cm of their birthplace. In this paper, I document geographic patterns of allozyme differentiation within and among populations of B. elegans and estimate the proportion of observed geographic pattern attributable to gene flow between adjacent populations. Genetic subdivision among localities separated by up to 3000 km was high (F_ST = 0.283, SE = 0.038). Inferred gene flow between pairs of localities (, individuals per generation) correlated inversely with the geographic distance between those localities, consistent with the pattern expected for a species at equilibrium in which gene flow occurred exclusively between adjacent localities. Within localities, patches separated by 4 to 30 m were also significantly subdivided, but genetic differentiation between patches did not vary significantly with the distance separating them. Simulations revealed that the power to detect genetic pattern expected from gene flow between adjacent populations increased with both the number of loci used to infer gene flow and the heterozygosity of those loci. Simulations also verified that when geographic distance poorly approximated the number of steps between populations, reduced major-axis regression more accurately portrayed the structural relationship between gene flow and separation than did ordinary least-squares regression. Attenuation of gene flow with distance explained 15% of the between-locality pattern of genetic differentiation in B. elegans. The remaining variation appeared to be due to neither natural selection nor a recent rangewide recolonization. Loci from the northern sampled localities, however, had fewer alleles than those from the remainder of the range, suggesting these localities had been recolonized recently following Pleistocene cooling.     

EPISTASIS,PLEIOTROPY, AND THE MUTATION LOAD IN SEXUAL AND ASEXUAL POPULATIONS

Mutation may impose a substantial load on populations, which varies according to the reproductive mode of organisms. Over the past years, various authors used adaptive landscape models to predict the long‐term effect of mutation on mean fitness; however, many of these studies assumed very weak mutation rates, so that at most one mutation segregates in the population. In this article, we derive several simple approximations (confirmed by simulations) for the mutation load at high mutation rate (U), using a general model that allows us to play with the number of selected traits (n), the degree of pleiotropy of mutations, and the shape of the fitness function (which affects the average sign and magnitude of epistasis among mutations). When mutations have strong fitness effects, the equilibrium fitness of sexuals and asexuals is close to ; under weaker mutational effects, sexuals reach a different regime where is a simple function of U and of a parameter describing the shape of the fitness function. Contrarily to weak mutation results showing that is an increasing function of population size and a decreasing function of n, these parameters may have opposite effects in sexual populations at high mutation rate.     

Effects of partial selfing on the equilibrium genetic variance,mutation load,and inbreeding depression under stabilizing selection

The mating system of a species is expected to have important effects on its genetic diversity. In this article, we explore the effects of partial selfing on the equilibrium genetic variance V_g, mutation load L, and inbreeding depression δ under stabilizing selection acting on a arbitrary number n of quantitative traits coded by biallelic loci with additive effects. When the ratio is low (where U is the total haploid mutation rate on selected traits) and effective recombination rates are sufficiently high, genetic associations between loci are negligible and the genetic variance, mutation load, and inbreeding depression are well predicted by approximations based on single‐locus models. For higher values of and/or lower effective recombination, moderate genetic associations generated by epistasis tend to increase V_g, L, and δ, this regime being well predicted by approximations including the effects of pairwise associations between loci. For yet higher values of and/or lower effective recombination, a different regime is reached under which the maintenance of coadapted gene complexes reduces V_g, L, and δ. Simulations indicate that the values of V_g, L, and δ are little affected by assumptions regarding the number of possible alleles per locus.     

SMALL POPULATION GENETIC VARIABILITY AT LOCI UNDER STABILIZING SELECTION

Genetic variability at a locus under stabilizing selection in a finite population is investigated using analytic methods and computer simulations. Three measures are examined: the number of alleles k, heterozygosity H, and additive genetic variance Vg. A nearly-neutral theory results. The composite parameter S = NV_M/V_s (where N is the population size, V_M the variance of new mutant allelic effects and V_s the weakness of stabilizing selection) figures prominently in the results. The equilibrium heterozygosity is similar to that of strictly neutral theory, H = 4N_μc/ (1 + 4N_μc), except that μ_c = where c is about 0.5. Simulations corroborate except for very low N. Genetic variability attains similar equilibrium values at both a “lone” locus and at an “embedded” locus. This agrees with my earlier work concerning molecular clock rates. These results modify the neutralist interpretation of data concerning genetic variability and genetic distances between populations. Low H values are proportional not to N but to . This may explain the narrow observed range of H among species. Heterozygosities need not be highly correlated to genetic variances. Genetic variances are not highly dependent on population size except in very small populations which are difficult to sample without bias because the smallest populations go extinct the fastest. Nearly neutral evolution will not be easily distinguished from strictly neutral theory under the Hudson-Kreitman-Aguade inter-/intraspecific variation ratio test, since a similar effective mutation rate holds for genetic distances and D = 2μ_ct, where . As with strictly neutral theory, comparisons across loci should show D and H to be positively correlated because of the shared μ_c. But unlike neutral theory, for a given locus, comparisons across species should show D and H to be negatively correlated. There is no obvious threshold of population size below which genetic variability inevitably declines. Extinction depends on both genetic variation and natural selection. Neither theory nor observation presently indicates the measure of genetic variability (k, H, V_G or other) that best indicates vulnerability of a small population to extinction.     

RISK OF POPULATION EXTINCTION FROM FIXATION OF NEW DELETERIOUS MUTATIONS

The fixation of new deleterious mutations is analyzed for a randomly mating population of constant size with no environmental or demographic stochasticity. Mildly deleterious mutations are far more important in causing loss of fitness and eventual extinction than are lethal and semilethal mutations in populations with effective sizes, N_e, larger than a few individuals. If all mildly deleterious mutations have the same selection coefficient, s against heterozygotes and 2s against homozygotes, the mean time to extinction, , is asymptotically proportional to for 4N_es > 1. Nearly neutral mutations pose the greatest risk of extinction for stable populations, because the magnitude of selection coefficient that minimizes is about ? = 0.4/N_e. The influence of variance in selection coefficients among mutations is analyzed assuming a gamma distribution of s, with mean and variance . The mean time to extinction increases with variance in selection coefficients if is near ?, but can decrease greatly if is much larger than ?. For a given coefficient of variation of , the mean time to extinction is asymptotically proportional to for . When s is exponentially distributed, (c = 1) is asymptotically proportional to . These results in conjunction with data on the rate and magnitude of mildly deleterious mutations in Drosophila melanogaster indicate that even moderately large populations, with effective sizes on the order of N_e = 10³, may incur a substantial risk of extinction from the fixation of new mutations.     

Additive genetic variance for lifetime fitness and the capacity for adaptation in an annual plant

The immediate capacity for adaptation under current environmental conditions is directly proportional to the additive genetic variance for fitness, V_A(W). Mean absolute fitness, , is predicted to change at the rate , according to Fisher's Fundamental Theorem of Natural Selection. Despite ample research evaluating degree of local adaptation, direct assessment of V_A(W) and the capacity for ongoing adaptation is exceedingly rare. We estimated V_A(W) and in three pedigreed populations of annual Chamaecrista fasciculata, over three years in the wild. Contrasting with common expectations, we found significant V_A(W) in all populations and years, predicting increased mean fitness in subsequent generations (0.83 to 6.12 seeds per individual). Further, we detected two cases predicting “evolutionary rescue,” where selection on standing V_A(W) was expected to increase fitness of declining populations (< 1.0) to levels consistent with population sustainability and growth. Within populations, inter‐annual differences in genetic expression of fitness were striking. Significant genotype‐by‐year interactions reflected modest correlations between breeding values across years, indicating temporally variable selection at the genotypic level that could contribute to maintaining V_A(W). By directly estimating V_A(W) and total lifetime , our study presents an experimental approach for studies of adaptive capacity in the wild.     

HEMOGLOBIN POLYMORPHISMS IN DEER MICE (PEROMYSCUS MANICULATUS): PHYSIOLOGY OF BETA-GLOBIN VARIANTS AND ALPHA-GLOBIN RECOMBINANTS

Wild populations of deer mice (Peromyscus maniculatus) contain hemoglobin polymorphisms at both alpha-globin (Hba, Hbc) and beta-globin (Hbd) loci. Population gene frequencies of beta-globin variants (d⁰ and d¹ haplotypes) are not correlated with altitude, whereas a¹c¹ alpha-globin haplotypes are fixed in low-altitude populations, and a⁰c⁰ haplotypes reach near fixation at high altitudes. We examined the effects of alpha- and beta-globin variants on blood oxygen affinity and on aerobic performance, measured as maximum oxygen consumption (). Exercise and cold exposure were used to elicit . Experiments were performed at low (340 m) and high (3,800 m) altitude to include the range of oxygen partial pressures encountered by wild deer mice. Beta-globin variants had little effect on blood oxygen affinity or . Oxygen-dissociation curves from a⁰c⁰ and a¹c¹ homozygotes and heterozygotes had similar shapes, but the P₅₀ of a⁰c⁰ homozygotes was significantly lower than that of other genotypes. Mice carrying a¹c¹/a¹c¹ genotypes had the highest at low altitude, but mice with a⁰c⁰/a⁰c⁰ genotypes had the highest at high altitude. Mice carrying rare recombinant alpha-globin haplotypes (a⁰c¹) had lower than nonrecombinant genotypes as a whole but in most cases were not significantly different from nonrecombinant heterozygotes (a⁰c⁰/a¹c¹). We conclude that genetic adaptation to different altitudes was important in the evolution of deer mouse alpha-globin polymorphisms and in the maintenance of linkage disequilibrium in the alpha-globin loci but was not a significant factor in the evolution of beta-globin polymorphisms.     

THE EFFECT OF SOFT SELECTION ON THE VARIABILITY OF A QUANTITATIVE TRAIT

The equilibrium phenotypic variance of a normally distributed quantitative character P under soft selection is studied. This character is assumed to undergo Gaussian stabilizing selection W(p, x) = exp[–(p – x)²/2w²]. The environmentally determined optimum (x) is a normal variable with variance s². A stable equilibrium with is found, so that increases both with increasing environmental heterogeneity and with increasing local intensity of stabilizing selection. It is shown that both genetic and environmental components of the variance are selected until this equilibrium is reached. Habitat selection, supposed to be normal (with variance H²) around the optimum, also increases the value. Nevertheless, relatively intense local stabilizing selection (w < s) and accurate habitat choice (H < s) are required for the initial spread and the evolutionary stability of this habitat selection.     

SEX RATIO AND DENSITY AFFECT SEXUAL SELECTION IN A SEX‐ROLE REVERSED FISH

Understanding how demographic processes influence mating systems is important to decode ecological influences on sexual selection in nature. We manipulated sex ratio and density in experimental populations of the sex‐role reversed pipefish Syngnathus typhle. We quantified sexual selection using the Bateman gradient (), the opportunity for selection (I), and sexual selection (I_s), and the maximum standardized sexual selection differential (). We also measured selection on body length using standardized selection differentials (s′) and mating differentials (m′), and tested whether the observed I and I_s differ from values obtained by simulating random mating. We found that I, I_s, and , but not , were higher for females under female than male bias and the opposite for males, but density did not affect these measures. However, higher density decreased sexual selection (m′ but not s′) on female length, but selection on body length was not affected by sex ratio. Finally, I_s but not I was higher than expected from random mating, and only for females under female bias. This study demonstrates that both sex ratio and density affect sexual selection and that disentangling interrelated demographic processes is essential to a more complete understanding of mating behavior and the evolution of mating systems.     

GENETIC CONSEQUENCES OF OUTCROSSING IN THE CLEISTOGAMOUS ANNUAL,IMPATIENS CAPENSIS. I. POPULATION-GENETIC STRUCTURE

We examined the genetic structure of 11 populations of Impatiens capensis, a cleistogamous annual herb, using starch gel electrophoresis. We sampled both cleistogamous (CL) and chasmogamous (CH) progeny (if present) from maternal parents in each population to infer maternal genotypes and to estimate the extent and pattern of inbreeding within and among populations. Only eight of 31 loci were polymorphic, with one to six (mean = 3.1) loci varying within each population. Mean heterozygosity per individual is quite low (mean = 3.9%) and comparable to highly self-fertilized species. Gene flow is low, and genetic distances do not parallel geographical distances, suggesting a population structure similar to Wright's Island model with drift among the populations. Fixation indexes within populations (f? or F_IS) span the largest range yet reported for a plant species (0.26 to 0.94, mean = 0.57). Further inbreeding results from population substructuring , resulting in a total average inbreeding coefficient (F? or F_IT) of 0.77. Despite these high overall levels of inbreeding, chasmogamy significantly reduces fixation, which may account for the observed greater fitness of CH progeny.     

NONSTATIONARY PATTERNS OF ISOLATION‐BY‐DISTANCE: INFERRING MEASURES OF LOCAL GENETIC DIFFERENTIATION WITH BAYESIAN KRIGING

Patterns of isolation‐by‐distance (IBD) arise when population differentiation increases with increasing geographic distances. Patterns of IBD are usually caused by local spatial dispersal, which explains why differences of allele frequencies between populations accumulate with distance. However, spatial variations of demographic parameters such as migration rate or population density can generate nonstationary patterns of IBD where the rate at which genetic differentiation accumulates varies across space. To characterize nonstationary patterns of IBD, we infer local genetic differentiation based on Bayesian kriging. Local genetic differentiation for a sampled population is defined as the average genetic differentiation between the sampled population and fictive neighboring populations. To avoid defining populations in advance, the method can also be applied at the scale of individuals making it relevant for landscape genetics. Inference of local genetic differentiation relies on a matrix of pairwise similarity or dissimilarity between populations or individuals such as matrices of between pairs of populations. Simulation studies show that maps of local genetic differentiation can reveal barriers to gene flow but also other patterns such as continuous variations of gene flow across habitat. The potential of the method is illustrated with two datasets: single nucleotide polymorphisms from human Swedish populations and dominant markers for alpine plant species.     

ADAPTIVE RADIATION AND GENETIC DIFFERENTIATION IN THE HAWAIIAN SILVERSWORD ALLIANCE (COMPOSITAE: MADIINAE)

The Hawaiian silversword alliance consists of the three genera Dubautia, Argyroxiphium, and Wilkesia, and is a classic example of adaptive radiation in an insular setting. Genetic variation and interspecific genetic differentiation based on ten enzyme loci are described for Dubautia and Wilkesia. Genetic identities among species span the range of values expected from interpopulation comparisons within a single species (I = 0.90–1.00) to those typical of interspecific comparisons . Genetic-identity values correspond to biogeographic distribution and morphological distinctiveness, supporting a correlation of increasing genetic distance associated with the time of separation among lineages. It may be inferred that the high genetic identities observed within the Hawaiian Madiinae and other island plant groups are due to limited time spans available for taxa to accumulate new genetic variation through mutation. It appears that species may remain genetically similar (I > 0.90) even after time spans on the order of magnitude of 1,000,000 years.     

THE EFFECT OF INBREEDING IN DIPLOID AND TETRAPLOID POPULATIONS OF EPILOBIUM ANGUSTIFOLIUM (ONAGRACEAE): IMPLICATIONS FOR THE GENETIC BASIS OF INBREEDING DEPRESSION

The partial dominance model for the evolution of inbreeding depression predicts that tetraploids should exhibit less inbreeding depression than their diploid progenitors. We tested this prediction by comparing the magnitude of inbreeding depression in tetraploid and diploid populations of the herbaceous perennial Epilobium angustifolium (Onagraceae). Inbreeding depression was estimated in the greenhouse for three tetraploid and two diploid populations at four life stages. The mating system of a tetraploid population was estimated and compared to a previous estimate for diploids. Tetraploids showed less inbreeding depression than diploids at all life history stages, and these differences were significant for seed-set and cumulative fitness, but not for germination, survival, or plant dry mass at nine weeks. This result suggests that the genetic basis of inbreeding depression may differ among life stages. The primary selfing rate of the tetraploid population was r = 0.43, which is nearly identical to that of a diploid population (r = 0.45), indicating that differences in inbreeding depression between diploids and tetraploids are probably not due to differences in the mating system. Cumulative inbreeding depression, calculated from the four life history stages, was significantly higher for diploids () than for tetraploids (), supporting the partial dominance model of inbreeding depression.     

INBREEDING DEPRESSION IN PARTIALLY SELF-FERTILIZING DECODON VERTICILLATUS (LYTHRACEAE): POPULATION-GENETIC AND EXPERIMENTAL ANALYSES

Inbreeding depression is a major selective force favoring outcrossing in flowering plants. Some self-fertilization, however, should weaken the harmful effects of inbreeding by exposing genetic load to selection. This study examines the maintenance of inbreeding depression in partially self-fertilizing populations of the long-lived, herbaceous wetland plant, Decodon verticillatus (L.) Ell. (Lythraceae). Estimates from ten populations indicate that 30% of offspring are produced through self-fertilization. Population-genetic estimates of inbreeding depression (δ = 1 – relative mean fitness of selfed progeny) based on changes in the inbreeding coefficient for the same ten populations were uniformly high, ranging from 0.49 to 1.79 and averaging 1.11 ± 0.29 SE. Although confidence intervals of individual population estimates were large, estimates were significantly greater than 0 in six populations and greater than 0.5 in four. Inbreeding depression was also estimated by comparing growth, survival, and flowering of experimentally selfed and outcrossed offspring from two of these populations in a 1-yr glasshouse experiment involving three density regimes; after which offspring were transplanted into garden arrays and two field sites and monitored for two consecutive growing seasons. Overall for survival averaged 0.27 ± 0.01 in the glasshouse, 0.33 ± 0.04 in the garden, and 0.46 ± 0.04 in the field. The glasshouse experiment also revealed strong inbreeding depression for growth variables, especially above-soil dry weight ( = 0.42 ± 0.03). The fitness consequences of inbreeding depression for these growth variables approximately doubles if survival to maturity is determined by severe truncation selection. Despite substantial selfing, inbreeding depression appears to be a major selective force favoring the maintenance of outcrossing in D. verticillatus.     

Exploring a Pool‐seq‐only approach for gaining population genomic insights in nonmodel species

Developing genomic insights is challenging in nonmodel species for which resources are often scarce and prohibitively costly. Here, we explore the potential of a recently established approach using Pool‐seq data to generate a de novo genome assembly for mining exons, upon which Pool‐seq data are used to estimate population divergence and diversity. We do this for two pairs of sympatric populations of brown trout (Salmo trutta): one naturally sympatric set of populations and another pair of populations introduced to a common environment. We validate our approach by comparing the results to those from markers previously used to describe the populations (allozymes and individual‐based single nucleotide polymorphisms [SNPs]) and from mapping the Pool‐seq data to a reference genome of the closely related Atlantic salmon (Salmo salar). We find that genomic differentiation (F_ST) between the two introduced populations exceeds that of the naturally sympatric populations (F_ST = 0.13 and 0.03 between the introduced and the naturally sympatric populations, respectively), in concordance with estimates from the previously used SNPs. The same level of population divergence is found for the two genome assemblies, but estimates of average nucleotide diversity differ ( ≈ 0.002 and  ≈ 0.001 when mapping to S. trutta and S. salar, respectively), although the relationships between population values are largely consistent. This discrepancy might be attributed to biases when mapping to a haploid condensed assembly made of highly fragmented read data compared to using a high‐quality reference assembly from a divergent species. We conclude that the Pool‐seq‐only approach can be suitable for detecting and quantifying genome‐wide population differentiation, and for comparing genomic diversity in populations of nonmodel species where reference genomes are lacking.     

Effective size of density‐dependent populations in fluctuating environments

Reliable estimates of effective population size are of central importance in population genetics and evolutionary biology. For populations that fluctuate in size, harmonic mean population size is commonly used as a proxy for (multi‐) generational effective size. This assumes no effects of density dependence on the ratio between effective and actual population size, which limits its potential application. Here, we introduce density dependence on vital rates in a demographic model of variance effective size. We derive an expression for the ratio in a density‐regulated population in a fluctuating environment. We show by simulations that yearly genetic drift is accurately predicted by our model, and not proportional to as assumed by the harmonic mean model, where N is the total population size of mature individuals. We find a negative relationship between and N. For a given N, the ratio depends on variance in reproductive success and the degree of resource limitation acting on the population growth rate. Finally, our model indicate that environmental stochasticity may affect not only through fluctuations in N, but also for a given N at a given time. Our results show that estimates of effective population size must include effects of density dependence and environmental stochasticity.