Genetic diversity and multilocus structure in sexual Otiorhynchus alpicola populations (Goleoptera: Gurculionidae)

Otiorhynchus alpicola Boh. (Coleoptera: Curculionidae) is distributed on high mountains of central, southern and south-eastern Europe. On the Balkan peninsula, this species is patchily distributed on mountain peaks at heights over 1800 m. To examine the organization of isoenzyme variation of the ten sexual (diploid) populations, starch gel electrophoresis of enzymes was used. The average heterozygosity over 14 gene loci was 0.11. For diploid O. alpicola, F-statistics were used to assess population heterogeneity and substructuring. The data indicate that, compared with other insects, this high-altitude weevil species is genetically very differentiated (the average F _ST= 0.35). In addition, gene-flow among populations was extremely low (the estimates of N_em from Wright's F _ST and Slatkin private-allele methods were 0.47 and 0.83, respectively). Pairwise linkage disequilibrium (LD) parameters were estimated from zygotic frequencies using Burrows' method. The average rate of significant LD in analysed populations of O. alpicola was about 27%. The pattern of LD over the diploid populations indicates that stochastic factors might be a primary cause of the observed multilocus associations.     

Extra-pair fertilization and effective population size in the song sparrow Melospiza melodia

The concept of effective population size (N_e) is used widely by conservation and evolutionary biologists as an indicator of the genetic state of populations, but its precision and relation to the census population size is often uncertain. Extra-pair fertilizations have the potential to bias estimates of N_e when they affect the number of breeders or their estimated reproductive success tallied from social pedigrees. We tested if the occurrence of extra-pair fertilizations influenced estimates of N_e in a resident population of song sparrows Melospiza melodia using four years of detailed behavioural and genetic data. Estimates of N_e based on social and genetic data were nearly identical and averaged c. 65% of the census population size over four years, despite that 28% of 471 independent young were sired outside of social pairs. Variance in male reproductive success also did not differ between estimates based on social and genetic data, indicating that extra-pair mating had little effect on the distribution of reproductive success in our study population. Our results show that the genetic assignment will not always be necessary to estimate N_e precisely.     

The consequences of the variance-mean rescaling effect on effective population size

The effective population size (N_e), and the ratio between N_e and census population size (N) are often used as measures of population viability. We show that using the harmonic mean of population sizes over time – a common proxy for N_e– has some important evolutionary consequences and implications for conservation management. This stems from the fact that there is no unambiguous relationship between the arithmetic and harmonic means for populations fluctuating in size. As long as the variance of population size increases moderately with increasing arithmetic mean population size, the harmonic mean also increases. However, if the variance of population size increases more rapidly, which existing data often suggest, then the harmonic mean may actually decrease with increasing arithmetic mean. Thus maximizing N may not maximize N_e, but could instead lower the adaptive potential and hence limit the evolutionary response to environmental change. Large census size has the clear advantage of lowering demographic stochasticity, and hence extinction risk, and under certain conditions large census size also minimizes the loss of genetic variation. Consequently, maximising census size has served as a useful dogma in ecology, genetics and conservation. Nonetheless, due to the intricate relationships among N_e, population viability and the properties of population fluctuations, we suggest that this dogma should be taken only as a rule of thumb.     

A TEST AND REVIEW OF THE ROLE OF EFFECTIVE POPULATION SIZE ON EXPERIMENTAL SEXUAL SELECTION PATTERNS

Experimental evolution, particularly experimental sexual selection in which sexual selection strength is manipulated by altering the mating system, is an increasingly popular method for testing evolutionary theory. Concerns have arisen regarding genetic diversity variation across experimental treatments: differences in the number and sex ratio of breeders (effective population size; N_e ) and the potential for genetic hitchhiking, both of which may cause different levels of genetic variation between treatments. Such differences may affect the selection response and confound interpretation of results. Here we use both census-based estimators and molecular marker-based estimates to empirically test how experimental evolution of sexual selection in Drosophila pseudoobscura impacts N_e and autosomal genetic diversity. We also consider effects of treatment on X-linked N_e s, which have previously been ignored. Molecular autosomal marker-based estimators indicate that neither N_e nor genetic diversity differs between treatments experiencing different sexual selection intensities; thus observed evolutionary responses reflect selection rather than any confounding effects of experimental design. Given the increasing number of studies on experimental sexual selection, we also review the census N_e s of other experimental systems, calculate X-linked N_e , and compare how different studies have dealt with the issues of inbreeding, genetic drift, and genetic hitchhiking to help inform future designs.     

The genetic effective size of a metapopulation

The structure of a population over time, space and categories of social and sexual role governs its ability to retain genetic variation in the face of drift. A metapopulation is an extreme form of spatial structure in which loosely coupled local populations 'turnover', that is, suffer extinction followed by recolonization from elsewhere within the metapopulation. These local populations turn over with a characteristic half-life. Based on a simulation model that incorporates both realistic features of population ecology and population genetics, the ability of such a metapopulation to retain genetic variation, which may be defined as proportional to its so-called effective population size, denoted N_e(^meta), can be one to two orders of magnitude lower than the maximum total number of individuals in the system. N_e(meta) depends on the persistence time associated with longevity of local populations (the turnover half-life), the average number of local populations extant in the metapopulation and the gene flow between local populations. Habitat fragmentation, which can create a metapopulation from a formerly continuously distributed species, may have unappreciated large genetic consequences for species impacted by human development.     

Genetic variability in the European bison (Bison bonasus) population from Białowieża forest over 50 years

The aim of this study was to assess potential post-bottleneck temporal genetic differentiation following the reintroduction of the species into the Białowieża Forest. Variability of 12 polymorphic microsatellite markers was analysed for 178 individuals born between 1955 and 2005, divided by birth year into five temporal groups. Low overall allelic richness (AR) per locus (AR = 2.0) and a low overall expected heterozygosity (H_E = 0.28) were observed. The overall F _IS was not significantly different from zero. The mean F _IS values were, however, significantly different from zero for individuals born between 1955 and 1965 ( F _IS = 0.19). A Bayesian computation was used to estimate effective population size (N_e) for each temporal group. We observed relatively small values of N_e ranging from 7.9 to 28.4. The low N_e values confirm that, despite a rapid growth of the bison population following the founder event, N_e increased only slowly.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 801–809.     

Microbial diversity--insights from population genetics

Although many environmental microbial populations are large and genetically diverse, both the level of diversity and the extent to which it is ecologically relevant remain enigmatic. Because the effective (or long-term) population size, N_e, is one of the parameters that determines population genetic diversity, tests and simulations that assume selectively neutral mutations may help to identify the processes that have shaped microbial diversity. Using ecologically important genes, tests of selective neutrality suggest that adaptive as well as non-adaptive types of selection act and that departure from neutrality may be widespread or restricted to small groups of genotypes. Population genetic simulations using population sizes between 10³ and 10⁷ suggest extremely high levels of microbial diversity in environments that sustain large populations. However, census and effective population sizes may differ considerably, and because we know nothing of the evolutionary history of environmental microbial populations, we also have no idea what N_e of environmental populations is. On the one hand, this reflects our ignorance of the microbial world. On the other hand, the tests and simulations illustrate interactions between microbial diversity and microbial population genetics that should inform our thinking in microbial ecology. Because of the different views on microbial diversity across these disciplines, such interactions are crucial if we are to understand the role of genes in microbial communities.     

Assessing population genetic structure and variability with RAPD data: Application to Vaccinium macrocarpon (American Cranberry)

A method for estimating and comparing population genetic variation using random amplified polymorphic DNA (RAPD) profiling is presented. An analysis of molecular variance (AMOVA) is extended to accomodate phenotypic molecular data in diploid populations in Hardy-Weinberg equilibrium or with an assumed degree of selfing. We present a two step strategy: 1) Estimate RAPD site frequencies without preliminary assumptions on the unknown population structure, then perform significance testing for population substructuring. 2) If population structure is evident from the first step, use this data to calculate better estimates for RAPD site frequencies and sub-population variance components. A nonparametric test for the homogeneity of molecular variance (HOMOVA) is also presented. This test was designed to statistically test for differences in intrapopulational molecular variances (heteroscedasticity among populations). These theoretical developments are applied to a RAPD data set in Vaccinium macrocarpon (American cranberry) using small sample sizes, where a gradient of molecular diversity is found between central and marginal populations. The AMOVA and HOMOVA methods provide flexible population analysis tools when using data from RAPD or other DNA methods that provide many polymorphic markers with or without direct allelic data.     

Genetic variability and large scale differentiation in two species of littorinid gastropods with planktotrophic development, Littorina littorea (L.) and Melarhaphe (Littorina) neritoides (L.) (Prosobranchia: Littorinacea), with notes on a mass occurrence of M. neritoides in Sweden

A planktotrophic larval development suggests a relatively high gene flow over long distances. Both Littorina littorea and Melarhaphe neritoides have an egg capsule and planktotrophic larvae which taken together are pelagic over a period of 4–8 weeks. In the absence of differential selection one would expect low levels of genetic differentiation over large distances in these two species. In this study of allozyme variation, low levels of differentiation were found over thousands of kilometres (Norway to Spain for L. littorea and Sweden to Greece for M. neritoides). This supports the hypothesis of two species with a high dispersal potential. A second expectation from neutral theory is that effective population size is positively correlated with average levels of genetic variation within species. In light of the generally high densities of local populations of these species and the high interpopulation migration rate, both L. littorea and M. neritoides may be considered as having high N_e s. Contrary to neutral expectation, L. littorea revealed very low levels of heterozygosity over its whole European range (mean H_exp= 0.015, 15 loci), while average heterozygosity of M. neritoides (H_exp= 0.084, 11 loci) was no more than in other littorinid species. This paper also reports the occurrence of M. neritoides on the Swedish west coast in 1988, 89 and 90, and two factors which may have promoted this unusually large invasion are discussed.     

Using molecular markers for pedigree reconstruction of the greater amberjack (Seriola dumerili) in the absence of parental information

Ensuring appropriate levels of genetic diversity in captive populations is essential to avoid inbreeding and loss of rare alleles by genetic drift. Pedigree reconstruction and parentage analysis in the absence of parental genotypes can be a challenging task that relies in the assignment of sibship relationships among the offspring. Here, we used eight highly variable microsatellite markers and three different assignment methods to reconstruct the most likely genotypes of a parental group of wild Seriola dumerili fish based on the genotypes of six cohorts of their offspring, to assess their relative contributions to the offspring. We found that a combination of the four most variable microsatellites was enough to identify the number of parents and their contribution to the offspring, suggesting that the variability of the markers can be more critical than the number of markers. Estimated effective population sizes were lower than the number of breeders and variable among years. The results suggest unequal parental contribution that should be accounted for breeding programs in the future.     

The effect of natural selection on estimates of genetic divergence among populations of the Atlantic salmon

The effect of natural selection on the _mMEP-2 * locus on measures of genetic divergence among Atlantic salmon populations was investigated by examining the pattern of change in the level of genetic differentiation (F_ST) averaged over loci when data on the _mMEP-2 * locus were either included or excluded. The level of F_ST among populations at various geographic scales was estimated from allele frequencies at up to four loci (_s AAT-4 *, IDDH-1 *, IDHP-3 *, and _mMEP-2 *). At smaller geographic scales (within river systems or limited geographic regions) levels of variance in _mMEP-2 * allele frequencies were reduced relative to mean levels. At larger geographic scales (across continents or the species range) variation in _mMEP-2 * allele frequencies was greater than mean levels. These results suggest an a priori hypothesis for the effect of selection on the _mMEP-2 * locus which may be applied in future studies on variation in protein coding or other (e.g. mini- and microsatellite) loci in the Atlantic salmon. It is recommended that estimates of gene flow among populations of the Atlantic salmon based on mean F _ST estimates which include data on the _mMEP-2 * locus should be viewed with caution.     

Genetic variation, its assessment and implications to the conservation of seagrasses

In a study of the widespread Australian endemic seagrass Posidonia australis , allozyme analysis identified a wide range in population genetic structure assessed using the multilocus genotype diversity statistic ( D _G). Values of D _G between zero and one were obtained; however, RAPD analysis generally detected higher levels of diversity, where D _G values were all greater than 0.5 ( D _G = 0.67 – 1). Some populations were allozymically monomorphic using allozyme analysis yet were highly polymorphic using RAPD analysis. The differences observed between methods, particularly among allozymically uniform populations, demonstrate the importance of choosing an appropriate method when assessing genotypic diversity. Different methods may reflect different historical aspects of population processes where allozymes reflect broader-scale gene flow and population establishment and DNA fingerprinting methods such as RAPDs may reflect fine-scale local recruitment events and shorter-term population processes. Using either method alone, particularly in genotypically depauperate organisms such as seagrasses and other clonal organisms, will be problematic in assessing their population genetic potential, a parameter being used by conservation managers to decide upon management strategies in rare and endangered organisms. It is recommended that the impact of disturbance assessed using genotypic diversity measures requires more than one technique to provide the most appropriate information for designing subsequent conservation strategies.     

Estimating selfing rates from reconstructed pedigrees using multilocus genotype data

Several methods have been developed to estimate the selfing rate of a population from a sample of individuals genotyped for several marker loci. These methods can be based on homozygosity excess (or inbreeding), identity disequilibrium, progeny array (PA) segregation or population assignment incorporating partial selfing. Progeny array-based method is generally the best because it is not subject to some assumptions made by other methods (such as lack of misgenotyping, absence of biparental inbreeding and presence of inbreeding equilibrium), and it can reveal other facets of a mixed-mating system such as patterns of shared paternity. However, in practice, it is often difficult to obtain PAs, especially for animal species. In this study, we propose a method to reconstruct the pedigree of a sample of individuals taken from a monoecious diploid population practicing mixed mating, using multilocus genotypic data. Selfing and outcrossing events are then detected when an individual derives from identical parents and from two distinct parents, respectively. Selfing rate is estimated by the proportion of selfed offspring in the reconstructed pedigree of a sample of individuals. The method enjoys many advantages of the PA method, but without the need of a priori family structure, although such information, if available, can be utilized to improve the inference. Furthermore, the new method accommodates genotyping errors, estimates allele frequencies jointly and is robust to the presence of biparental inbreeding and inbreeding disequilibrium. Both simulated and empirical data were analysed by the new and previous methods to compare their statistical properties and accuracies.     

Double labelling to obtain S phase subpopulations: application to determine cell kinetics of diploid cells in an aneuploid tumour

Abstract. We studied the cell kinetics of the murine mammary carcinoma MCa-K using iododeoxyuridine (IdUrd) and chlorodeoxyuridine (CldUrd) given at different times as independently detectable labels of S phase cells. The presence of IdUrd and CldUrd, and the amount of DNA were measured by three-colour flow cytometry making it possible to define three subpopulations within S phase and to measure the progression through the cell cycle during the time following labelling. In DNA histograms of these subpopulations, the diploid and aneuploid cells (which had a DNA index of 1.7) are essentially completely separated. From appropriate combinations of cells labelled with IdUrd only, CldUrd only, or both, it was possible to construct separate DNA distributions for the labelled diploid and aneuploid cells at the times of administration of each label. The kinetics of the diploid and aneuploid cells could be calculated for individual tumours from these two time points without having to make corrections for the presence of the second population. The diploid and aneuploid populations had indistinguishable S and G₂+ M phase durations, T_S and T_G2+M, of about 9 and 2h; however, the potential doubling time values for the aneuploid and diploid populations were 30.2 and 101.2h respectively.     

Elucidation of the DNA Synthetic Cycle of Entamoeba Spp. Using Flow Cytometry and Mathematical Modeling

ABSTRACT. We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G₁, S, and G₂ phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G₁, S, and G₂ proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 × 10^-14 g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 × 10^-14 g DNA/cell.     

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry

Abstract. Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation ( CV ) from 2 to 16%; (ii) constant measurement CV or CV 's increasing from G₁ to G₂ phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time.
The fractions of cells in the G₁, S, and (G₂+ M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10–20%. However, most methods tended to overestimate G₁ fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.     

Microsatellite analysis of genetic population structure in an endangered salmonid: the coastal cutthroat trout (Oncorhynchus clarki clarki)

The genetic population structure of coastal cutthroat trout ( Oncorhynchus clarki clarki ) in Washington state was investigated by analysis of variation in allele frequencies at six highly polymorphic microsatellite loci for 13 anadromous populations, along with one outgroup population from the Yellowstone subspecies ( O. clarki bouvieri) (mean heterozygosity = 67%; average number of alleles per locus = 24). Tests for genetic differentiation revealed highly significant differences in genotypic frequencies for pairwise comparisons between all populations within geographical regions and overall population subdivision was substantial ( F _ST = 0.121, R _ST = 0.093), with 44.6% and 55.4% of the among-population diversity being attributable to differences between streams ( F _SR = 0.054) and between regions ( F _RT = 0.067), respectively. Analysis of genetic distances and geographical distances did not support a simple model of isolation by distance for these populations. With one exception, neighbour-joining dendrograms from the Cavalli-Sforza and Edwards' chord distances and maximum likelihood algorithms clustered populations by physiogeographic region, although overall bootstrap support was relatively low (53%). Our results suggest that coastal cutthroat trout populations are ultimately structured genetically at the level of individual streams. It appears that the dynamic balance between gene flow and genetic drift in the subspecies favours a high degree of genetic differentiation and population subdivision with the simultaneous maintenance of high heterozygosity levels within local populations. Results are discussed in terms of coastal cutthroat trout ecology along with implications for the designation of evolutionarily significant units pursuant to the US Endangered Species Act of 1973 and analogous conservation units.     

Genetic Variation Within and Among Metal-Tolerant and Non-Tolerant Populations of Armeria maritima (Mill.) Willd. s.l. (Plumbaginaceae) in Central and Northeast Germany

Abstract: Six metal-tolerant populations and sub-populations of Armeria maritima ssp. halleri, ssp. hornburgensis, and ssp. bottendorfensis and two non-tolerant populations of ssp. elongata in Central and Northeast Germany have been analysed using RAPD markers. The populations show very strong genetic differentiation (Φ_ST = 0.46), corresponding gene flow between them is low (N_em = 0.29). A moderate positive correlation between the matrices of genetic and geographical distances was found between the seven populations and sub-populations of central Germany (r = 0.68, p < 0.001). Calculated parameters of genetic variability are molecular variance, percentage of heterozygosity and percentage of polymorphic loci. A significant correlation between population size and parameters of genetic variability was not recognisable. Genetic structure was investigated by an analysis of molecular variance (AMOVA). The studied populations show strong genetic differentiation. Genetic variation within populations ("normal" as well as metalliferous) is higher (53.9 %) than among them (46.1 %). Six hypotheses of possible genetic relatedness between the studied populations have been tested by AMOVA. A data set structure above the populational level is hardly recognisable. It was impossible to combine the populations to edaphic (tolerant and "non-tolerant") or taxonomic groups. A. maritima ssp. halleri of the north Harz mountains and ssp. hornburgensis are clearly separated from a geographical group containing all other populations (across taxonomic and edaphic boundaries). These results are a further indication for a polyphyletic origin of metal-tolerant populations of A. maritima s.l. by multiple colonizations of metalliferous sites from neighbouring populations on non-metalliferous soil.     

CALCULATING LIMITS TO THE ALLOWABLE HUMAN-CAUSED MORTALITY OF CETACEANS AND PINNIPEDS

A simulation method was developed for identifying populations with levels of human-caused mortality that could lead to depletion, taking into account the uncertainty of available information. A mortality limit (termed the Potential Biological Removal, PBR , under the U. S. Marine Mammal Protection Act) was calculated as the product of a minimum population estimate ( N _MIN), one-half of the maximum net productivity rate ( R _MAX), and a recovery factor ( F _R). Mortality limits were evaluated based on whether at least 95% of the simulated populations met two criteria: (1) that populations starting at the maximum net productivity level ( MNPL ) stayed there or above after 20 yr, and (2) that populations starting at 30% of carrying-capacity ( K ) recovered to at least MNPL after 100 yr. Simulations of populations that experienced mortality equal to the PBR indicated that using approximately the 20th percentile (the lower 60% log-normal confidence limit) of the abundance estimate for N _MIN met the criteria for both cetaceans (assuming R _MAX= 0.04) and pinnipeds (assuming R _MAX= 0.12). Additional simulations that included plausible levels of bias in the available information indicated that using a value of 0.5 for F _R would meet both criteria during these "bias trials." It is concluded that any marine mammal population with an estimate of human-caused mortality that is greater than its PBR has a level of mortality that could lead to the depletion of the population. The simulation methods were also used to show how mortality limits could be calculated to meet conservation goals other than the U. S. goal of maintaining populations above MNPL .     

Population divergence in the amphicarpic species Amphicarpaea edgeworthii Benth. (Fabaceae): microsatellite markers and leaf morphology

Comparative analyses of the genetic differentiation in microsatellite markers ( F _ST) and leaf morphology characters ( Q _ST) of Amphicarpaea edgeworthii Benth. were conducted to gain insight into the roles of random processes and natural selection in the population divergence. Simple sequence repeat analyses on 498 individuals of 19 natural populations demonstrate that a significant genetic differentiation occurs among populations (mean F _ST = 0.578), and A. edgeworthii is a highly self-fertilized species (mean selfing rate s  = 0.989). The distribution pattern of genetic diversity in this species shows that central populations possess high genetic diversity (e.g. population WL with H _E = 0.673 and population JG with H _E = 0.663), whereas peripheral ones have a low H _E as in population JD (0.011). The morphological divergence of leaf shape was estimated by the elliptical Fourier analysis on the data from 11 natural and four common garden populations. Leaf morphology analyses indicate the morphological divergence does not show strong correlation with the genetic differentiation ( R  = 0.260, P  = 0.069). By comparing the 95% confidence interval of Q _ST with that of F _ST, Q _ST values for five out of 12 quantitative traits are significantly higher than the average F _ST value over eight microsatellite loci. The comparison of F _ST and Q _ST suggests that two kinds of traits can be driven by different evolutionary forces, and the population divergence in leaf morphology is shaped by local selections.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 505–516.