Stability of genetic structure and effective population size inferred from temporal changes of microsatellite DNA polymorphisms in the land snail Helix aspersa (Gastropoda: Helicidae)

Temporal evolution of genetic variability may have far-reaching consequences for a diverse array of evolutionary processes. Within the polders of the Bay of Mont-Saint-Michel (France), populations of the land snail Helix aspersa are characterized by a metapopulation structure with occasional extinction processes resulting from farming practices. A temporal survey of genetic structure in H . aspersa was carried out using variability at four microsatellite loci, in ten populations sampled two years apart. Levels of within-population genetic variation, as measured by allelic richness, H _e or F _is , did not change over time and similar levels of population differentiation were demonstrated for both sampling years. The extent of genetic differentiation between temporal samples of the same population established (i) a stable structure for six populations, and (ii) substantial genetic changes for four populations. Using classical F -statistics and a maximum likelihood method, estimates of the effective population size ( N _e) illustrated a mixture of stable populations with high N _e, and unstable populations characterized by very small N _e estimates (of 5–11 individuals). Owing to human disturbances, intermittent gene flow and genetic drift are likely to be the predominant evolutionary processes shaping the observed genetic structure. However, the practice of multiple matings and sperm storage is likely to provide a reservoir of variability, minimizing the eroding genetic effects of population size reduction and increasing the effective population size.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 89–102.     

Loss of genetic variation and effective population size of Kirikuchi charr: implications for the management of small, isolated salmonid populations

Small, isolated populations may face extinction due to a combination of inbreeding depression and other threats. Effective population size ( N _e) is one comprehensive measure that allows us to evaluate the genetic status of a population, and to make management decisions regarding genetic viability. We simulated loss of genetic variation and estimated N _e for two small, isolated populations of Kirikuchi charr Salvelinus leucomaenis japonicus , the endangered, southernmost local populations of the genus Salvelinus in the world, using VORTEX, an individual-based stochastic PVA model. Approximately half of the genetic variation was lost over 200 years regardless of census population size and demographic parameters, and N _e estimates were roughly 50 in each of the two populations, suggesting the possibility of inbreeding depression. The target population size of N _e>500, by securing long-term viability, is several times that of the present size of each of the populations studied, and no local habitats maintaining such a target number are considered to exist. The results strongly indicate a need for recovering natural connections and potential gene flow among local populations. However, the impending threat to these populations from non-native charr widely distributed throughout the drainage has prevented the recovery of the connections. Given the small N _e of the two populations, it would be necessary to retain gene flow artificially within or across local populations. This will be true of many other salmonid populations that have been isolated or fragmented recently.     

Population genetic structure and effective population size of ayu (Plecoglossus altivelis), an amphidromous fish

The temporal and spatial population genetic structure of ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae), an amphidromous fish, was examined using analysis of variation at six microsatellite DNA loci. Intracohort genetic diversities, as measured by the number of alleles and heterozygosity, were similar among six cohorts (2001–2006) within a population (Nezugaseki River), with the mean number of alleles per cohort ranging from 11·0 to 12·5 and the expected heterozygosity ranging from 0·74 to 0·77. Intrapopulational genetic diversities were also similar across the three studied populations along the 50 km coast, with the mean number of alleles and the expected heterozygosity ranging from 11·33 to 11·67 and from 0·75 to 0·76, respectively. The authors observed only one significant difference in pair-wise population differentiation ( F _ST-value) between the cohorts within a population and among three populations. Estimates of the effective population size ( N _e) based on maximum-likelihood method yielded small values (ranging from 94·8 to 135·5), whereas census population size ranged from c. 4800 to 24 000. As a result, the ratio of annual effective population sizes to census population size ( N _e/ N ) ranged from 0·004 to 0·023. These estimates of N _e/ N agree more closely with estimates for marine fishes than that of the larger estimates for freshwater fishes. The present study suggests that ayu which is highly fecund and shows low survival during the early life stages is also characterized by having low value of N _e/ N , similar to marine species with a pelagic life cycle.     

Large variations in the ratio of effective breeding and census population sizes between two species of pond-breeding anurans

The viability of wild populations is frequently assessed by monitoring adult census sizes ( N _c). This approach is particularly useful for pond-breeding amphibians, because assemblages during the breeding season are relatively easy to detect and count. However, it is the genetic effective population size ( N _e) or surrogates such as effective breeding population size ( N _b) that are of primary importance for long-term viability. Although N _c estimates of one anuran amphibian ( Bufo bufo ) in Britain were much larger than those of another ( Rana temporaria ) at the same sites, the ratios of N _b to N _c were much smaller in B. bufo than in R. temporaria. These differences were sufficiently great as to reverse the effective size order at one site, such that N _b for R. temporaria was larger than that for B. bufo. Differences in adult sex ratios at breeding sites probably contributed to lower N _b values in B. bufo populations compared with those of R. temporaria . The relationship of N _b to N _c can therefore vary dramatically even between similar species, to the extent that just monitoring N _c can give misleading impressions of relative effective breeding sizes and thus of population viability. It will be increasingly important to estimate N _e or N _b in wildlife populations for assessment of conservation priorities.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 365–372.     

Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Perú. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity ( H _E) of 0.8185 (range of 0.698–0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 × 10⁺¹³ to 1.34 × 10⁺¹⁵ and Δ values ranging from 2.80 × 10⁺¹² to 1.34 × 10⁺¹⁵ with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.     

Leaf stomatal responses to vapour pressure deficit under current and CO2-enriched atmosphere explained by the economics of gas exchange

Using the economics of gas exchange, early studies derived an expression of stomatal conductance ( g ) assuming that water cost per unit carbon is constant as the daily loss of water in transpiration ( f _e) is minimized for a given gain in photosynthesis ( f _c). Other studies reached identical results, yet assumed different forms for the underlying functions and defined the daily cost parameter as carbon cost per unit water. We demonstrated that the solution can be recovered when optimization is formulated at time scales commensurate with the response time of g to environmental stimuli. The optimization theory produced three emergent gas exchange responses that are consistent with observed behaviour: (1) the sensitivity of g to vapour pressure deficit ( D ) is similar to that obtained from a previous synthesis of more than 40 species showing g to scale as 1 −  m  log( D ), where m   ∈  [0.5,0.6], (2) the theory is consistent with the onset of an apparent 'feed-forward' mechanism in g , and (3) the emergent non-linear relationship between the ratio of intercellular to atmospheric [CO₂] ( c _i/ c _a) and D agrees with the results available on this response. We extended the theory to diagnosing experimental results on the sensitivity of g to D under varying c _a.     

Relative contribution of contemporary pollen and seed dispersal to the effective parental size of seedling population of California valley oak (Quercus lobata, Née)

For plant populations, gene movement through pollen and seed dispersal governs the size of local genetic neighbourhoods and shapes the opportunities for natural selection and genetic drift. A critical question is how together these two processes influence the evolutionary dynamics of local populations. To assess the respective contributions of pollen and seed flow, we propose a novel indirect assessment of the separate male and female gametic contributions to total effective parental size ( N _e), based on parental correlations estimated via kinship coefficients, that can be applied to data sets that include unambiguous genotypes for male and female gametic contributions. Using the endemic Californian valley oak ( Quercus lobata ) as our study species, we apply this method to a set of microsatellite genotypes for two distinct ecological sets of naturally recruiting seedlings with acorns attached. We found that the effective numbers of contributing male parents ( N _ep) exceed effective numbers of female parents ( N _em) for seedlings established beneath adult trees ( N _ep = 8.1 and N _em = 1.1), as well as for seedlings established away from adult trees ( N _ep = 15.4 and N _em = 2.7), illustrating that seed dispersal enhances pollen dispersal and increases the effective number of seed sources in open seedling patches. The resulting effective parental size of seedling populations translates into smaller effective numbers of parents for undispersed vs. dispersed seedlings ( N _e = 3.6 and N _e = 6.7, respectively). This study introduces a novel statistic method and provides important new evidence that, on a short-term temporal scale, seed dispersal shapes the local neighbourhood size of new recruits.     

Genetics of the peripheral populations of the alpine bullhead, Cottus poecilopus (Scorpaeniformes, Cottidae) in Poland

The alpine bullhead ( Cottus poecilopus ) in Poland is highly polymorphic ( P for examined populations in the range of 0.0–0.45, mean P  = 0.11, P for species 0.64) in seven out of 11 loci studied ( Pgd-1, Mdh-1, Ck-1, Gpd-1, Gpd-2, Gpi-1, Gpi-2 ). However, they reveal very low heterozygosity (mean H _o = 0.007, mean H _e = 0.019), which may result from population subdivisions, small genetically effective size ( N _e) of some populations and frequent inbreeding, the latter being probably mediated through polygyny. The index of genetic differentiation ( G _ST) for the Polish metapopulation equals 0.48, which is relatively high. By far, the strongest are genetic differences between the Sudeten-Carpathian group and the Hańcza population. Gene flow between the Sudeten-Carpathian group and the Hańcza population is very limited and their divergence may have started as early as the last (Eemian) interglacial period.     

Genetic estimates of contemporary effective population size: what can they tell us about the importance of genetic stochasticity for wild population persistence?

Genetic stochasticity due to small population size contributes to population extinction, especially when population fragmentation disrupts gene flow. Estimates of effective population size ( N _e) can therefore be informative about population persistence, but there is a need for an assessment of their consistency and informative relevance. Here we review the body of empirical estimates of N _e for wild populations obtained with the temporal genetic method and published since Frankham's (1995 ) review. Theoretical considerations have identified important sources of bias for this analytical approach, and we use empirical data to investigate the extent of these biases. We find that particularly model selection and sampling require more attention in future studies.
We report a median unbiased N _e estimate of 260 (among 83 studies) and find that this median estimate tends to be smaller for populations of conservation concern, which may therefore be more sensitive to genetic stochasticity. Furthermore, we report a median N _e/ N ratio of 0.14, and find that this ratio may actually be higher for small populations, suggesting changes in biological interactions at low population abundances. We confirm the role of gene flow in countering genetic stochasticity by finding that N _e correlates strongest with neutral genetic metrics when populations can be considered isolated. This underlines the importance of gene flow for the estimation of N _e, and of population connectivity for conservation in general. Reductions in contemporary gene flow due to ongoing habitat fragmentation will likely increase the prevalence of genetic stochasticity, which should therefore remain a focal point in the conservation of biodiversity.     

Comparing direct vs. indirect estimates of gene flow within a population of a scattered tree species

The comparison between historical estimates of gene flow, using variance in allelic frequencies, and contemporary estimates of gene flow, using parentage assignment, is expected to provide insights into ecological and evolutionary processes at work within and among populations. Genetic variation at six microsatellite loci was used to quantify genetic structure in the insect-pollinated, animal-dispersed, low-density tree Sorbus torminalis L. Crantz, and to derive historical estimates of gene flow. The neighbourhood size and root-mean-squared dispersal distance inferred from seedling genotypes ( N _b  = 70 individuals, σ _e  = 417 m) were similar to those inferred from adult genotypes ( N _b  = 114 individuals, σ _e  = 472 m). We also used parentage analyses and a neighbourhood model approach after an evaluation of the statistical properties of this method on simulated data. From our data, we estimated even contributions of seed- and pollen-mediated dispersal to the genetic composition of established seedlings, with both fat-tailed pollen and seed dispersal kernels, and slightly higher mean distance of pollen dispersal (248 m) as compared to seed dispersal (135 m). The resulting contemporary estimate of gene dispersal distance (σ _c  = 211 m) was ∼twofold smaller than the historical estimates. Besides different assumptions and statistical nuances of both approaches, this discrepancy is likely to reflect a recent restriction in the scale of gene flow which requires manager's attention in a context of increasing forest fragmentation.     

The distribution of genetic variation in Norway spruce (Picea abies Karst.) populations in the western Alps

Aim  In order to look for a possible centre of survival for the Norway spruce ( Picea abies Karst.) in the south-western Alps, six natural populations of this area were investigated by means of genetic markers in order to assess the degree and the distribution of genetic diversity within the species.
Location  Western and South-western Alps.
Methods  Populations were genotyped using seven simple sequence repeat (SSR) markers. Basic population genetics parameters were estimated and the amount of genetic differentiation calculated.
Results  A large amount of variability was found (0.59 <  H _e < 0.67); genetic differentiation as measured by F _ST was 0.05, close to other similar studies; no isolation by distance was detected by a Mantel test. Analysis of molecular variance confirmed a high degree of variability within populations and a low degree of variability among populations. Finally, the number of populations from which those observed could have arisen was estimated by Bayesian analysis.
Main conclusions  The results presented here suggest that the present populations derive their genetic make-up from three inferred clusters. The possible existence in this area of a relict/refuge population during the last glaciation is discussed.     

Genetic diversity in the endangered dysploid larkspur Delphinium bolosii and its close diploid relatives in the series Fissa of the Western Mediterranean area

Allozyme diversity was evaluated in four closely-related taxa of the Delphinium series Fissa distributed throughout the Western Mediterranean area. All are considered threatened plants. Delphinium bolosii and Delphinium mansanetianum are narrowly endemic to the Eastern Iberian Peninsula, whereas Delphinium fissum ssp. sordidum is found in a few populations across the Peninsula. Delphinium fissum ssp. fissum is more widely distributed but often in small and isolated populations. In this group, Delphinium bolosii is dysploid (2 n  = 18) whereas the other taxa are diploid (2 n  = 16). A total of 12 populations were surveyed, including all known locations for D. bolosii , D. mansanetianum , and D. fissum ssp. sordidum . Eleven enzyme systems were assayed and 15 loci were resolved. Markedly depauperate values for genetic diversity were obtained for D. mansanetianum ( H _e = 0.013) and D. fissum ssp. sordidum ( H _e = 0.044). The estimates for D. fissum ssp. fissum ( H _e = 0.071) were below the values expected for widespread species. Small population size and marginal distribution have probably contributed to the low variability observed in this group. By contrast, D. bolosii exhibited comparatively larger populations and greater genetic diversity ( H _e = 0.138). We suggest that, apart from population size and local adaptation, genetic diversity during speciation may have been promoted by dysploidy through genomic recombination.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 773–784.     

Allozyme Diversity in Populations of Cymbidium goeringii (Orchidaceae)

Abstract: Using 14 allozyme loci, we investigated levels of genetic diversity within populations, and degree of genetic divergence among 24 populations of Cymbidium goeringii (Orchidaceae) in Korea and Japan. Cymbidium goeringii maintains high levels of genetic diversity both at population (mean expected heterozygosity, H _e = 0.238) and species levels (0.260). Means of H _e found in 24 populations were not significantly different from each other. About 90 % of the total variation in the species is common to all populations (mean G _ST = 0.108). No unique allele was found in any population. The indirect estimate of gene flow based on the mean G _ST was high ( Nm = 2.06). Nei's genetic identities for pairs of populations had high values (mean = 0.974 [SD = 0.013]). The Mantel-Z test showed a significant correlation between genetic distance and geographic distance. However, the mean G _ST value between 17 populations in Korea and seven Japanese populations was relatively low (0.029), even though the land connection between the southern Korean peninsula and southern Japanese archipelagos has not existed since the middle Pleistocene. Large numbers of small seeds of C. goeringii might travel long distances by wind from populations to populations both in Korea and Japan, increasing genetic diversity within populations and maintaining low genetic differentiation among populations.     

Comparison of quantitative and molecular genetic variation of native vs. invasive populations of purple loosestrife (Lythrum salicaria L., Lythraceae)

Study of adaptive evolutionary changes in populations of invasive species can be advanced through the joint application of quantitative and population genetic methods. Using purple loosestrife as a model system, we investigated the relative roles of natural selection, genetic drift and gene flow in the invasive process by contrasting phenotypical and neutral genetic differentiation among native European and invasive North American populations ( Q _ST −  F _ST analysis). Our results indicate that invasive and native populations harbour comparable levels of amplified fragment length polymorphism variation, a pattern consistent with multiple independent introductions from a diverse European gene pool. However, it was observed that the genetic variation reduced during subsequent invasion, perhaps by founder effects and genetic drift. Comparison of genetically based quantitative trait differentiation ( Q _ST) with its expectation under neutrality ( F _ST) revealed no evidence of disruptive selection ( Q _ST >  F _ST) or stabilizing selection ( Q _ST <  F _ST). One exception was found for only one trait (the number of stems) showing significant sign of stabilizing selection across all populations. This suggests that there are difficulties in distinguishing the effects of nonadaptive population processes and natural selection. Multiple introductions of purple loosestrife may have created a genetic mixture from diverse source populations and increased population genetic diversity, but its link to the adaptive differentiation of invasive North American populations needs further research.     

Net Glutamate Release from Astrocytes Is Not Induced by Extracellular Potassium Concentrations Attainable in Brain

Abstract: Elevated extracellular potassium concentration ([K⁺]_e) has been shown to induce reversal of glial Na⁺-dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K⁺]_e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K⁺]_e (by equimolar substitution of K⁺ for Na⁺), and glutamate accumulation was measured by HPLC. With [K⁺]_e elevations to 60 m M , medium glutamate concentrations did not increase during incubation periods of 5–120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50–100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer d -aspartate, and parallel results were obtained; no increase in medium d -aspartate content resulted from [K⁺]_e elevation up to 90 m M , whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K⁺]_e attainable in brain.     

Genetic structure and population dynamics of a heteroecious plant pathogen Melampsora larici-epitea in short-rotation coppice willow plantations

Complex life strategies are common among plant pathogens belonging to rust fungi ( Uredinales ). The heteroecious willow rust Melampsora larici-epitea produces five spore stages and alternates on larch ( Larix ). To shed light on the epidemiology of this pathogen, amplified fragment length polymorphisms (AFLPs) were used to determine the genetic diversity and genetic structure of rust samples collected from coppice willow ( Salix ) plantations at three UK sites (LA, CA and MC) over three sampling dates (September 2000, July 2001 and September 2001). Of the total of 819 isolates, 465 were unique AFLP phenotypes and there was a shift in genotype diversity between the two seasons (0.67 in 2000 and 0.87–0.89 in 2001). No phenotypes were common between the two seasons within a site, suggesting that the rust did not overwinter as an asexual stage within plantations. A temporal analysis detected large amounts of genetic drift ( F _S = 0.15–0.26) between the two seasons and very small effective population sizes ( N _e  =  2–3) within sites. These results all point to a new colonization of the plantations by the rust in the second season (2001). The F _ST-analogue values were Φ_CT = 0.121, Weir and Cockerham's θ = 0.086 and the Bayesian estimate θ^B = 0.087–0.096. The results suggest that the sources of inoculum were somewhat localized and the same sources were mainly responsible for disease epidemics in LA and CA over the two seasons. The relatively low F _ST-values among sites (0.055–0.13) suggest the existence of significant gene flow among the three sampled sites.     

Genetic diversity pattern of Stipa purpurea populations in the hinterland of Qinghai–Tibet Plateau

Stipa purpurea is among constructive endemic species in alpine meadow and steppe on the Qinghai–Tibet Plateau. To reveal the genetic diversity of this species and its relationship with geographic distribution pattern, we sampled eight populations across a northward transect with an increasing aridity gradient in the hinterland of Qinghai–Tibet Plateau. Their genetic diversity was quantified using eight intersimple sequence repeat (ISSR) primers. We found that S. purpurea had relatively low genetic diversity ( H _e = 0.135) but significant genetic differentiation among populations ( G _st = 0.391), indicating relatively more genetic diversity retained within populations. A Mantel test revealed a significant relationship between genetic and geographic distance in the S. purpurea populations. The genetic diversity tended to decrease with increasing latitude and longitude, while no significant relationship was found between genetic diversity and altitude, suggesting the possible influences of humidity and temperature on genetic diversity of alpine plant. We propose conservation measures for this plant on the plateau.     

Prediction of offspring fitness based on parental genetic diversity in endangered salmonid populations

By simulating all possible offspring genotypic combinations based on pair-wise matings in three vulnerable or endangered populations of salmonids (two Atlantic salmon Salmo salar and one Arctic charr Salvelinus alpinus ), family level estimates of potential offspring genetic diversity were calculated using several commonly used genetic diversity estimators (     ,     and H _EST). By using this approach, the genetic variability of offspring could be predicted accurately based solely on parental genotype data. In addition, significant associations between offspring genetic variability and fitness-related traits were observed in the two Atlantic salmon populations and these associations were consistent between fitness measures and either estimated or observed genetic diversity. This indicates that associations with offspring fitness measures can be predicted without genetic analysis of the offspring themselves.     

Transient Elevation of Interstitial N-Acetylaspartate in Reversible Global Brain Ischemia

Abstract: We evaluated the changes of interstitial N -acetylaspartate (NAA) concentration ([NAA]_e) in rat striatum by microdialysis following transient global ischemia and depolarization. The dialysate NAA concentration ([NAA]_d) values were corrected for the in vivo recovery to obtain [NAA]_e, by the use of [³H]mannitol in the perfusion fluid. During global ischemia the relative loss (RL) of [³H]mannitol decreased to 40% of preischemic values, reflecting the decrease in extracellular volume fraction. During reperfusion RL of [³H]mannitol quickly normalized. The [NAA]_d doubled during transient ischemia, which, after correction for in vivo recovery, corresponds to a fivefold increase in [NAA]_e ( p < 0.05). Reperfusion induced a >10-fold increase of [NAA]_e ( p < 0.01) with subsequent normalization after 45 min. KCI at 100 m M caused a reversible 50% reduction in RL of [³H]mannitol and a three times increase in [NAA]_e ( p < 0.05) but no further increase when normal perfusate was reintroduced. The mechanisms of NAA release from neurons are unknown but may involve the activation of unknown channels/carriers—possibly in relation to a volume regulatory response. The present study shows that the distribution of NAA in brain is dynamically regulated in acute ischemia and suggests that changes of NAA levels could be caused by other means than neuronal loss.     

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.