Experimental evaluation of the usefulness of microsatellite DNA for detecting demographic bottlenecks

Evolutionary and conservation biologists often use molecular markers to evaluate whether populations have experienced demographic bottlenecks that resulted in a loss of genetic variation. We evaluated the utility of microsatellites for detection of recent, severe bottlenecks and compared the amounts of genetic diversity lost in bottlenecks of different sizes. In experimental mesocosms, we established replicate populations by releasing 1, 2, 4 or 8 pairs of the western mosquitofish, Gambusia affinis (Poeciliidae). Using eight polymorphic microsatellite loci, we quantified seven indices of genetic diversity or change that have been used to assess the effects of demographic bottlenecks on populations. We compared indices for the experimentally bottlenecked populations to those for the source population and examined differences between populations established with different numbers of founders. Direct count heterozygosity and the proportion of polymorphic loci were not very sensitive to genetic changes that resulted from the experimental bottlenecks. Heterozygosity excess and expected heterozygosity were useful to varying degrees in the detection of bottlenecks. Allelic diversity and temporal variance in allele frequencies were most sensitive to genetic changes that resulted from the bottlenecks, and the temporal variance method was slightly more correlated with bottleneck size than was allelic diversity. Based on comparisons to a previous study with allozymes, heterozygosity, temporal variance in allele frequencies and allelic diversity, but not proportion of polymorphic loci, appear to be more sensitive to demographic bottlenecks when quantified using microsatellites. We found that analysis of eight highly polymorphic loci was sufficient to detect a recent demographic bottleneck and to obtain an estimate of the magnitude of bottleneck severity.     

Selection maintains MHC diversity through a natural population bottleneck

A perceived consequence of a population bottleneck is the erosion of genetic diversity and concomitant reduction in individual fitness and evolutionary potential. Although reduced genetic variation associated with demographic perturbation has been amply demonstrated for neutral molecular markers, the effective management of genetic resources in natural populations is hindered by a lack of understanding of how adaptive genetic variation will respond to population fluctuations, given these are affected by selection as well as drift. Here, we demonstrate that selection counters drift to maintain polymorphism at a major histocompatibility complex (MHC) locus through a population bottleneck in an inbred island population of water voles. Before and after the bottleneck, MHC allele frequencies were close to balancing selection equilibrium but became skewed by drift when the population size was critically low. MHC heterozygosity generally conformed to Hardy-Weinberg expectations except in one generation during the population recovery where there was a significant excess of heterozygous genotypes, which simulations ascribed to strong differential MHC-dependent survival. Low allelic diversity and highly skewed frequency distributions at microsatellite loci indicated potent genetic drift due to a strong founder affect and/or previous population bottlenecks. This study is a real-time examination of the predictions of fundamental evolutionary theory in low genetic diversity situations. The findings highlight that conservation efforts to maintain the genetic health and evolutionary potential of natural populations should consider the genetic basis for fitness-related traits, and how such adaptive genetic diversity will vary in response to both the demographic fluctuations and the effects of selection.     

Comparison of historical bottleneck effects and genetic consequences of re‐introduction in a critically endangered island passerine

Re‐introduction is an important tool for recovering endangered species; however, the magnitude of genetic consequences for re‐introduced populations remains largely unknown, in particular the relative impacts of historical population bottlenecks compared to those induced by conservation management. We characterize 14 microsatellite loci developed for the Seychelles paradise flycatcher and use them to quantify temporal and spatial measures of genetic variation across a 134‐year time frame encompassing a historical bottleneck that reduced the species to ~28 individuals in the 1960s, through the initial stages of recovery and across a second contemporary conservation‐introduction‐induced bottleneck. We then evaluate the relative impacts of the two bottlenecks, and finally apply our findings to inform broader re‐introduction strategy. We find a temporal trend of significant decrease in standard measures of genetic diversity across the historical bottleneck, but only a nonsignificant downward trend in number of alleles across the contemporary bottleneck. However, accounting for the different timescales of the two bottlenecks (~40 historical generations versus <1 contemporary generation), the loss of genetic diversity per generation is greater across the contemporary bottleneck. Historically, the flycatcher population was genetically structured; however, extinction on four of five islands has resulted in a homogeneous contemporary population. We conclude that severe historical bottlenecks can leave a large footprint in terms of sheer quantity of genetic diversity lost. However, severely depleted genetic diversity does not render a species immune to further genetic erosion upon re‐introduction. In some cases, the loss of genetic diversity per generation can, initially at least, be greater across re‐introduction‐induced bottlenecks.     

Genetic analysis of a documented population bottleneck: introduced Bennett's wallabies (Macropus rufogriseus rufogriseus) in New Zealand

Few bottlenecks of wild populations are sufficiently well-documented to constitute models for testing theories about the impact of bottlenecks on genetic variation, and subsequent population persistence. Relevant details of the Bennett's wallaby (Macropus rufogriseus rufogriseus) introduction into New Zealand were recorded (founder number, source and approximate bottleneck duration) and suggest this may provide a rare opportunity to examine the efficacy of tests designed to detect recent bottlenecks in wild populations. We first assessed the accuracy of historic accounts of the introduction using genetic diversity detected in mitochondrial DNA (mtDNA) and at five microsatellite loci. Phylogenetic analyses of mtDNA D-loop sequence haplotypes were consistent with the reported origin of the founders as Tasmania, rather than one of the Bass Strait islands in which Bennett's wallabies are also found. Microsatellite allele frequencies from the Tasmanian source population were then used to seed bottleneck simulations encompassing varying sizes and numbers of generations, in order to assess the severity of bottleneck consistent with diversity observed in the New Zealand population. The results suggested that the founder number was unlikely to have been as small as the three animals suggested by the account of the introduction. Nonetheless, the bottleneck was probably severe; in the range of three to five pairs of wallabies for one to three generations. It resulted in significantly reduced levels of allelic diversity and heterozygosity relative to the source population. This bottleneck is only detectable under the infinite allele model (IAM) and not under the stepwise mutation model (SMM) or the two-phase model (TPM), and possible explanations for this are discussed.     

Bottlenecks and rescue effects in a fluctuating population of golden-mantled ground squirrels (Spermophilus lateralis)

Mammal species characterized by highly fluctuating populations often maintain genetic diversity in response to frequent demographic bottlenecks, suggesting the ameliorating influence of life history and behavioral factors. Immigration in particular is expected to promote genetic recovery and is hypothesized to be the most likely process maintaining genetic diversity in fluctuating mammal populations. Most demographic bottlenecks have been inferred retrospectively, and direct analysis of a natural population before, during, and after a bottleneck is rare. Using a continuous 10-year dataset detailing the complete demographic and genetic history of a fluctuating population of golden-mantled ground squirrels (Spermophilus lateralis), we analyzed the genetic consequences of a 4-year demographic bottleneck that reduced the population to seven adult squirrels, and we evaluated the potential “rescue effect” of immigration. Analysis of six microsatellite loci revealed that, while a decline in allelic richness was observed during the bottleneck, there was no observed excess of heterozygosity, a characteristic bottleneck signature, and no evidence for heterozygote deficiency during the recovery phase. In addition, we found no evidence for inbreeding depression during or after the bottleneck. By identifying immigrants and analyzing their demographic and genetic contributions, we found that immigration promoted demographic recovery and countered the genetic effects of the bottleneck, especially the loss of allelic richness. Within 3 years both population size and genetic variation had recovered to pre-bottleneck levels, supporting the role of immigration in maintaining genetic variation during bottleneck events in fluctuating populations. Our analyses revealed considerable variation among analytical techniques in their ability to detect genetic bottlenecks, suggesting that caution is warranted when evaluating bottleneck events based on one technique.     

Signatures of demographic bottlenecks in European wolf populations

Monitoring the loss of genetic diversity in wild populations after a bottleneck event is a priority in conservation and management plans. Here, we used diverse molecular markers to search for signatures of demographic bottlenecks in two wolf populations; an isolated population from the Iberian Peninsula and a non-isolated population from European Russia. Autosomal, mtDNA and Y-chromosomal diversity and the effective population size (N_e) were significantly lower in the Iberian population. Neutrality tests using mtDNA sequences, such as R_2, Fu and Li’s F*, Tajima’s D and Fu’s F_s, were positively significant in the Iberian population, suggesting a population decline, but were not significant for the Russian population, likely due to its larger effective population size. However, three tests using autosomal data confirmed the occurrence of the genetic bottleneck in both populations. The M-ratio test was the only one providing significant results for both populations. Given the lack of consistency among the different tests, we recommend using multiple approaches to investigate possible past bottlenecks. The small effective population size (about 50) in the Iberian Peninsula compared to the presumed extant population size could indicate that the bottleneck was more powerful than initially suspected or an overestimation of the current population. The risks associated with small effective population sizes suggest that the genetic change in this population should be closely monitored in the future. On the other hand, the relatively small effective population size for Russian wolves (a few hundred individuals) could indicate some fragmentation, contrary to what is commonly assumed.     

EPISTASIS AND THE EVOLUTION OF ADDITIVE GENETIC VARIANCE IN POPULATIONS THAT PASS THROUGH A BOTTLENECK

Traditional models of genetic drift predict a linear decrease in additive genetic variance for populations passing through a bottleneck. This perceived lack of heritable variance limits the scope of founder-effect models of speciation. We produced 55 replicate bottleneck populations maintained at two male-female pairs through four generations of inbreeding (average F = 0.39). These populations were formed from an F₂ intercross of the LG/J and SM/J inbred mouse strains. Two contemporaneous control strains maintained with more than 60 mating pairs per generation were formed from this same source population. The average level of within-strain additive genetic variance for adult body weight was compared between the control and experimental lines. Additive genetic variance for adult body weight within experimental bottleneck strains was significantly higher than expected under an additive genetic model This enhancement of additive genetic variance under inbreeding is likely to be due to epistasis, which retards or reverses the loss of additive genetic variance under inbreeding for adult body weight in this population. Therefore, founder-effect speciation processes may not be constrained by a loss of heritable variance due to population bottlenecks.     

Demographic fluctuations lead to rapid and cyclic shifts in genetic structure among populations of an alpine butterfly,Parnassius smintheus

Many populations, especially in insects, fluctuate in size, and periods of particularly low population size can have strong effects on genetic variation. Effects of demographic bottlenecks on genetic diversity of single populations are widely documented. Effects of bottlenecks on genetic structure among multiple interconnected populations are less studied, as are genetic changes across multiple cycles of demographic collapse and recovery. We take advantage of a long‐term data set comprising demographic, genetic and movement data from a network of populations of the butterfly, Parnassius smintheus, to examine the effects of fluctuating population size on spatial genetic structure. We build on a previous study that documented increased genetic differentiation and loss of spatial genetic patterns (isolation by distance and by intervening forest cover) after a network‐wide bottleneck event. Here, we show that genetic differentiation was reduced again and spatial patterns returned to the system extremely rapidly, within three years (i.e. generations). We also show that a second bottleneck had similar effects to the first, increasing differentiation and erasing spatial patterns. Thus, bottlenecks consistently drive random divergence of allele frequencies among populations in this system, but these effects are rapidly countered by gene flow during demographic recovery. Our results reveal a system in which the relative influence of genetic drift and gene flow continually shift as populations fluctuate in size, leading to cyclic changes in genetic structure. Our results also suggest caution in the interpretation of patterns of spatial genetic structure, and its association with landscape variables, when measured at only a single point in time.     

Disentangling the roles of natural selection and genetic drift in shaping variation at MHC immunity genes

The major histocompatibility complex (MHC) forms an integral component of the vertebrate immune response and, due to strong selection pressures, is one of the most polymorphic regions of the entire genome. Despite over 15 years of research, empirical studies offer highly contradictory explanations of the relative roles of different evolutionary forces, selection and genetic drift, acting on MHC genes during population bottlenecks. Here, we take a meta-analytical approach to quantify the results of studies into the effects of bottlenecks on MHC polymorphism. We show that the consequences of selection acting on MHC loci prior to a bottleneck event, combined with drift during the bottleneck, will result in overall loss of MHC polymorphism that is ～15% greater than loss of neutral genetic diversity. These results are counter to general expectations that selection should maintain MHC polymorphism, but do agree with the results of recent simulation models and at least two empirical studies. Notably, our results suggest that negative frequency-dependent selection could be more important than overdominance for maintaining high MHC polymorphism in pre-bottlenecked populations.     

Increased genetic variance after a population bottleneck

A conventional view holds that population bottlenecks cause massive losses of genetic variability, thus endangering the viability of the derived population. Although some alleles that were infrequent in the parent population may be lost new empirical evidence from Drosophila and housefly populations has demonstrated that genetic variance available to selection may actually increase following a single severe bottleneck. Several theoretical models support this view, and suggest that the increase may result from conversion of balanced epistatic variance to additive variance that becomes immediately available to selection. These effects appear to be greatest on the inheritance of quantitative characters, releasing new variance through the disruption of covariance matrices that underlie and interrelate quantitative traits. Thus, character change in adaptation and speciation may, in some instances, be promoted by founder events.     

Do bottlenecks increase additive genetic variance?

Because of anthropogenic factors many populations have been at least temporarily reduced to a very small population size. Such reductions could potentially decrease genetic variation and increase the probability of extinction. Analysis of molecular markers has shown a decrease in genetic variation but in many cases this has not reduced the ability of the population to recover from the bottleneck. This apparent paradox is resolved by a consideration of how population bottlenecks can affect additive genetic variance, the relevant measure of ability to respond to selective factors. A bottleneck has the potential to increase additive genetic variance in a population. This may result in an increase in fitness, particularly in populations of conservation concern that are small and lack genetic variation. Here we present a meta-analysis of experimental tests of this prediction using models designed to fit data that is strictly additive and data that has non-additive components. This analysis shows that additive genetic variance in a dataset dominated by morphological traits increases, on average, after a bottleneck event when the inbreeding coefficient is less than 0.3, but neither of the theoretical models alone can adequately explain this result. Because of our inability at present to predict the results of a population bottleneck in a specific case and the probability of extinction associated with small population size we caution against using bottlenecks to increase genetic variance, and thus the fitness, of endangered populations.     

The role of propagule pressure in the invasion success of bluegill sunfish, Lepomis macrochirus, in Japan

The bluegill sunfish, Lepomis macrochirus, is a widespread exotic species in Japan that is considered to have originated from 15 fish introduced from Guttenberg, Iowa, in 1960. Here, the genetic and phenotypic traits of Japanese populations were examined, together with 11 native populations of the USA using 10 microsatellite markers and six meristic traits. Phylogenetic analysis reconfirmed a single origin of Japanese populations, among which populations established in the 1960s were genetically close to Guttenberg population, keeping high genetic diversity comparable to the ancestral population. In contrast, genetic diversity of later-established populations significantly declined with genetic divergence from the ancestral population. Among the 1960s established populations, that from Lake Biwa showed a significant isolation-by-distance pattern with surrounding populations in which genetic bottlenecks increased with geographical distance from Lake Biwa. Although phenotypic divergence among populations was recognized in both neutral and adaptive traits, P(ST)-F(ST) comparisons showed that it is independent of neutral genetic divergence. Divergent selection was suggested in some populations from reservoirs with unstable habitats, while stabilizing selection was dominant. Accordingly, many Japanese populations of L. macrochirus appear to have derived from Lake Biwa population, expanding their distribution with population bottlenecks. Despite low propagule pressure, the invasion success of L. macrochirus is probably because of its drastic population growth in Lake Biwa shortly after its introduction, together with artificial transplantations. It not only enabled the avoidance of a loss in genetic diversity but also formed a major gene pool that supported local adaptation with high phenotypic plasticity.     

Bias in the prediction of genetic gain due to mass and half-sib selection in random mating populations

The prediction of gains from selection allows the comparison of breeding methods and selection strategies, although these estimates may be biased. The objective of this study was to investigate the extent of such bias in predicting genetic gain. For this, we simulated 10 cycles of a hypothetical breeding program that involved seven traits, three population classes, three experimental conditions and two breeding methods (mass and half-sib selection). Each combination of trait, population, heritability, method and cycle was repeated 10 times. The predicted gains were biased, even when the genetic parameters were estimated without error. Gain from selection in both genders is twice the gain from selection in a single gender only in the absence of dominance. The use of genotypic variance or broad sense heritability in the predictions represented an additional source of bias. Predictions based on additive variance and narrow sense heritability were equivalent, as were predictions based on genotypic variance and broad sense heritability. The predictions based on mass and family selection were suitable for comparing selection strategies, whereas those based on selection within progenies showed the largest bias and lower association with the realized gain.     

The effects of genetic drift in experimental evolution

Experimental evolution is characterized by exponential or logistic growth and periodic population bottlenecks. The fate of a rare beneficial mutation in these systems is influenced both by the bottleneck effect and by genetic drift. This paper explores the effects of incorporating genetic drift into models of fixation probability in populations with periodic bottlenecks. To model the inherent stochasticity during the growth phase in these populations, we assume a Poisson distribution of offspring. An analytical solution is developed to calculate the fixation probability and a computer simulation is used to verify the results. We find that the fixation rate of a favourable mutant is significantly lower when genetic drift is considered; fixation probability is reduced by over 25% for realistic experimental protocols. Our method is valid for both weak and strong selection; since very large selection coefficients have been reported in the experimental literature, this is an important improvement over previous results.     

Evidence for demographic bottlenecks and limited gene flow leading to low genetic diversity in a rare thistle

European calcareous grasslands have decreased dramatically in area and number during the last two centuries. As a result, many populations of calcareous grassland species are confined to small and isolated fragments, where their long-term survival is to some extent uncertain. Recently, several restoration projects have been initiated to enlarge the current grassland area in order to maintain the exceptionally high species richness. However, from a genetic point of view, the success of these restoration measures is not necessarily guaranteed, as strong historical decreases in population size and limited gene flow may have led to low genetic diversity through genetic bottlenecks and drift. In this study, we investigated genetic diversity and structure of 16 populations of the calcareous grassland specialist Cirsium acaule in a severely fragmented landscape in south-western Belgium. The overall distribution of this species in the study area was significantly and positively related to patch area, suggesting that small patches do not allow survival of this species. Both allelic richness and genetic diversity were significantly and positively related to population size. Estimation of observed and expected gene diversity provided evidence for population bottlenecks in the history of not less than 31% of all sampled populations. Reconstruction of the historical land use showed that patch area decline in populations that went through a recent bottleneck was significantly larger than that in populations that showed no evidence of a bottleneck. Assignment analyses showed low migration rates, suggesting that replenishment of lost alleles through gene flow is highly unlikely. Overall, our results indicate that in the absence of gene flow strong decreases in calcareous grassland area may have long-lasting effects on genetic diversity of plant populations and may hamper the success of restoration projects that simply aim at restoring initial habitat conditions or enlarging population fragments, as indicated by the fact that none of the recently restored areas has been occupied by C. acaule.     

Immigration and the ephemerality of a natural population bottleneck: evidence from molecular markers.

Population bottlenecks are often invoked to explain low levels of genetic variation in natural populations, yet few studies have documented the direct genetic consequences of known bottlenecks in the wild. Empirical studies of natural population bottlenecks are therefore needed, because key assumptions of theoretical and laboratory studies of bottlenecks may not hold in the wild. Here we present microsatellite data from a severe bottleneck (95% mortality) in an insular population of song sparrows (Melospiza melodia). The major findings of our study are as follows: (i) The bottleneck reduced heterozygosity and allelic diversity nearly to neutral expectations, despite non-random survival of birds with respect to inbreeding and wing length. (ii) All measures of genetic diversity regained pre-bottleneck levels within two to three years of the crash. This rapid recovery was due to low levels of immigration. (iii) The rapid recovery occurred despite a coincident, strong increase in average inbreeding. These results show that immigration at levels that are hard to measure in most field studies can lead to qualitatively very different genetic outcomes from those expected from mutations only. We suggest that future theoretical and empirical work on bottlenecks and metapopulations should address the impact of immigration.     

Long‐term ‘islands’ in the landscape: low gene flow,effective population size and genetic divergence in the shrub Hakea oldfieldii (Proteaceae)

The genetic structure of disjunct populations is determined by founding genetic properties, demographic processes, gene flow, drift and local selection. We aim to identify the genetic consequences of natural population disjunction at regional and local scales in Hakea oldfieldii using nuclear and plastid markers to investigate long‐term effective population sizes and gene flow, and patterns of diversity and divergence, among populations. Regional divergence was significant as shown by a consistent pattern in principal coordinates, neighbor‐joining and Bayesian analyses, but divergence at the local level was also significant with localized distribution of plastid haplotypes and populations clustering separately in Bayesian analyses. Historical, recent and first‐generation gene flow was low, suggesting that recent habitat fragmentation has not reduced gene migration significantly. Genetic bottlenecks were detected in three populations. Long‐term effective population size was significantly correlated with the number of alleles/locus and observed heterozygosity, but not with census population size, suggesting that the loss of diversity is associated with long‐term changes rather than recent fragmentation. Inbreeding coefficients were significant in only three populations, suggesting that the loss of diversity is linked to drift and bottlenecks associated with demographic processes (local extinction by fires) rather than inbreeding. Historical disjunction as a result of specific ecological requirements, contraction of habitats following drying during the Pleistocene, low gene flow and changes in population size are likely to have been important forces driving divergence through isolation by distance and drift. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 319–334.     

Population bottlenecks increase additive genetic variance but do not break a selection limit in rain forest Drosophila

According to neutral quantitative genetic theory, population bottlenecks are expected to decrease standing levels of additive genetic variance of quantitative traits. However, some empirical and theoretical results suggest that, if nonadditive genetic effects influence the trait, bottlenecks may actually increase additive genetic variance. This has been an important issue in conservation genetics where it has been suggested that small population size might actually experience an increase rather than a decrease in the rate of adaptation. Here we test if bottlenecks can break a selection limit for desiccation resistance in the rain forest-restricted fly Drosophila bunnanda. After one generation of single-pair mating, additive genetic variance for desiccation resistance increased to a significant level, on average higher than for the control lines. Line crosses revealed that both dominance and epistatic effects were responsible for the divergence in desiccation resistance between the original control and a bottlenecked line exhibiting increased additive genetic variance for desiccation resistance. However, when bottlenecked lines were selected for increased desiccation resistance, there was only a small shift in resistance, much less than predicted by the released additive genetic variance. The small selection response in the bottlenecked lines was no greater than that observed in the control lines. Thus bottlenecks might produce a statistically detectable change in additive genetic variance but this change has no impact on the response to selection.     

Microsatellite markers to assess the influence of population size, isolation and demographic change on the genetic structure of the UK butterfly Polyommatus bellargus

Five microsatellite DNA markers were isolated and used to quantify population genetic structure among a subset of UK populations of the Adonis blue (Polyommatus bellargus Rottemburg). Specifically, whether population size, degree of isolation or history of bottlenecking in 1976-1978 can explain current patterns of genetic variation. The butterfly is at its northern range limit in the UK, where it exists as a highly fragmented metapopulation on isolated pockets of calcareous grassland. Most populations were affected by a severe bottleneck in the late 1970s, when a drought caused the host plant (Hippocrepis comosa) to wilt. Mantel tests and spatial autocorrelation analysis indicated a significant effect of isolation by distance among the UK populations, a relationship that broke down at greater geographical scales (> 23.85 km), probably because of large areas of unsuitable habitat presenting barriers to gene flow. Similarly, amova revealed that variation among geographical regions was almost double that observed within regions. Larger populations were found to support significantly higher levels of genetic diversity, suggesting that small populations may lose genetic diversity through drift. If, as in other butterfly species, low genetic diversity increases the probability of population extinction, then these populations are likely to be under threat. Neither isolation nor a history of bottlenecks appeared to influence genetic diversity. The results indicate that adequate population size a crucial factor in the conservation of genetic diversity in P. bellargus in the UK.     

EPISTASIS AS A SOURCE OF INCREASED ADDITIVE GENETIC VARIANCE AT POPULATION BOTTLENECKS

The role of epistasis in evolution and speciation has remained controversial. We use a new parameterization of physiological epistasis to examine the effects of epistasis on levels of additive genetic variance during a population bottleneck. We found that all forms of epistasis increase average additive genetic variance in finite populations derived from initial populations with intermediate allele frequencies. Average additive variance continues to increase over many generations, especially at larger population sizes (N = 32 to 64). Additive-by-additive epistasis is the most potent source of additive genetic variance in this situation, whereas dominance-by-dominance epistasis contributes smaller amounts of additive genetic variance. With additive-by-dominance epistasis, additive genetic variance decreases at a relatively high rate immediately after a population bottleneck, rebounding to higher levels after several generations. Empirical examples of epistasis for murine adult body weight based on measured genotypes are provided illustrating the varying effects of epistasis on additive genetic variance during population bottlenecks.