Differential patterns of spatial divergence in microsatellite and allozyme alleles: further evidence for locus‐specific selection in the acorn barnacle,Semibalanus balanoides?

We compared patterns of genetic structure at potentially selected (two allozyme loci) and neutral molecular markers (six microsatellite loci) in the acorn barnacle, Semibalanus balanoides from the Gulf of St. Lawrence. Our results confirmed the presence of a geographical shift in alleles MPI and GPI near the Miramichi River. In contrast, no significant patterns of population differentiation among samples located north and south of the river mouth were detected for four of six microsatellite loci. However, analysis of molecular variance (amova) at individual loci revealed that a significant proportion of the total variance in allele frequencies was partitioned among samples located north and south of the river for both the allozyme and the other two microsatellite loci. The two most common alleles at these microsatellites showed frequencies that were highly correlated (r = 0.65-0.74, P < 0.05) with those of the MPI*2 allele, perhaps because of either physical linkage or epistasis. The two allozyme loci were significantly correlated in barnacles located north of the Miramichi River (r = 0.86, P < 0.05). Overall, our results supported the hypothesis that the broad scale pattern of allozyme allelic shifts is maintained by selection. They also indicated that microsatellites may not always behave in a neutral way and must be used cautiously, especially when evidence for genetic structuring relies on only a few assayed loci.     

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.     

Allelic configuration and polysomic inheritance of highly variable microsatellites in tetraploid gynodioecious Thymus praecox agg.

Polyploidy plays a pivotal role in plant evolution. However, polyploids with polysomic inheritance have hitherto been severely underrepresented in plant population genetic studies, mainly due to a lack of appropriate molecular genetic markers. Here we report the establishment and experimental validation of six fully informative microsatellite markers in tetraploid gynodioecious Thymus praecox agg. Sequence data of 150 microsatellite alleles and their flanking regions revealed high variation, which may be characteristic for polyploids with a reticulate evolutionary history. Understanding the patterns of mutation (indels and substitutions) in microsatellite flanking-sequences was a prerequisite for the development of co-dominant markers for fragment analyses. Allelic segregation patterns among progeny arrays from ten test crosses revealed tetrasomic inheritance in T. praecox agg. No evidence of frequent double reduction was detected. Polymerase chain reaction (PCR) based dosage effects allowed for precise assignment of allelic configuration at all six microsatellite loci. The quantification of allele copy numbers in PCR was verified by comparisons of observed and expected gametic allele frequencies and heterozygosities in test crosses. Our study illustrates how PCR based markers can provide reliable estimates of heterozygosity and, thus, powerful tools for breeding system and population genetic analyses in polyploid organisms.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure

Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre‐eminent system for the study of selective pressures that arise from host–pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population‐genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles.     

Isolation and characterization of microsatellite loci from the endangered Mediterranean sponge Spongia agaricina (Demospongiae: Dictyoceratida)

The abundance of the bath sponge Spongia agaricina has decreased drastically in recent years and it is now considered an endangered species under Annex 3 of Bern and Barcelona conventions. We describe eight microsatellite markers and present data on their allelic variation and utility as high resolution genetic markers. We analyzed 36 individuals from two populations and found that the number of alleles per locus ranged between 1 and 7. Observed heterozygosity ranged from 0 to 0.72. We found deviations from Hardy–Weinberg expectations for some loci. We exclusively detected null alleles for those loci that deviated from Hardy–Weinberg expectations. Also, distributions of allele frequencies differed significantly between the two populations, making them suitable for population genetic analyses.     

Weak selection and protein evolution

The "nearly neutral" theory of molecular evolution proposes that many features of genomes arise from the interaction of three weak evolutionary forces: mutation, genetic drift, and natural selection acting at its limit of efficacy. Such forces generally have little impact on allele frequencies within populations from generation to generation but can have substantial effects on long-term evolution. The evolutionary dynamics of weakly selected mutations are highly sensitive to population size, and near neutrality was initially proposed as an adjustment to the neutral theory to account for general patterns in available protein and DNA variation data. Here, we review the motivation for the nearly neutral theory, discuss the structure of the model and its predictions, and evaluate current empirical support for interactions among weak evolutionary forces in protein evolution. Near neutrality may be a prevalent mode of evolution across a range of functional categories of mutations and taxa. However, multiple evolutionary mechanisms (including adaptive evolution, linked selection, changes in fitness-effect distributions, and weak selection) can often explain the same patterns of genome variation. Strong parameter sensitivity remains a limitation of the nearly neutral model, and we discuss concave fitness functions as a plausible underlying basis for weak selection.     

Genetic analysis of differentiation among breeding ponds reveals a candidate gene for local adaptation in Rana arvalis

One of the main questions in evolutionary and conservation biology is how geographical and environmental features of the landscape shape neutral and adaptive genetic variation in natural populations. The identification of genomic polymorphisms that account for adaptive variation can aid in finding candidate loci for local adaptation. Consequently, a comparison of spatial patterns in neutral markers and loci under selection may help disentangle the effects of gene flow, genetic drift and selection at the landscape scale. Many amphibians breed in wetlands, which differ in environmental conditions and in the degree of isolation, enhancing the potential for local adaptation. We used microsatellite markers to measure genetic differentiation among 17 local populations of Rana arvalis breeding in a network of wetlands. We found that locus RC08604 deviated from neutral expectations, suggesting that it is a good candidate for directional selection. We used a genetic network analysis to show that the allele distribution in this locus is correlated with habitat characteristics, whereas this was not the case at neutral markers that displayed a different allele distribution and population network in the study area. The graph approach illustrated the genomic heterogeneity (neutral loci vs. the candidate locus for directional selection) of gene exchange and genetic divergence among populations under directional selection. Limited gene flow between wetlands was only observed at the candidate genomic region under directional selection. RC08604 is partially located inside an up‐regulated thyroid‐hormone receptor (TRβ) gene coordinating the expression of other genes during metamorphosis and appears to be linked with variation in larval life‐history traits found among R. arvalis populations. We suggest that directional selection on genes coding larval life‐history traits is strong enough to maintain the divergence in these genomic regions, reducing the effective recombination of locally adapted alleles but not in other regions of the genome. Integrating this knowledge into conservation plans at the landscape scale will improve the design of management strategies to preserve adaptive genetic diversity in wetland networks.     

Geographic variation of the major histocompatibility complex in Eastern Atlantic grey seals (Halichoerus grypus)

Pathogen-driven balancing selection maintains high genetic diversity in many vertebrates, particularly in the major histocompatibility complex (MHC) immune system gene family, which is often associated with disease susceptibility. In large natural populations where subpopulations face different pathogen pressures, the MHC should show greater genetic differentiation within a species than neutral markers. We examined genetic diversity at the MHC-DQB locus and nine putatively neutral microsatellite markers in grey seals (Halichoerus grypus) from eight United Kingdom (UK) colonies, the Faeroe Islands and Sable Island, Canada. Five DQB alleles were identified in grey seals, which varied in prevalence across the grey seal range. Among the seal colonies, significant differences in DQB allele and haplotype frequencies and in average DQB heterozygosity were observed. Additionally, the DQB gene exhibited greater differentiation among colonies compared with neutral markers, yet a weaker pattern of isolation by distance (IBD). After correcting for the underlying IBD pattern, subpopulations breeding in similar habitats were more similar to one another in DQB allele frequencies than populations breeding in different habitats, but the same did not hold true for microsatellites, suggesting that habitat-specific pathogen pressure influences MHC evolution. Overall, the data are consistent with selection at MHC-DQB loci in grey seals with both varying selective pressures and geographic population structure appearing to influence the DQB genetic composition of breeding colonies.     

Analysis of F ST outliers at allozyme loci in Pacific salmon: implications for natural selection

Natural selection has been invoked to explain the observed geographic distribution of allozyme allele frequencies for a number of teleost species. The effects of selection on allozyme loci in three species of Pacific salmon were tested. A simulation-based approach to estimate the null distribution of population differentiation (F _ST) and test for F _ST outliers was used. This approach showed that a majority of allozyme loci conform to neutral expectations predicted by the simulation model, with relatively few F _ST outliers found. No consistent F _ST outlier loci were found across species. Analysis of population sub-groups based on geography and genetic identity reduced the number of outlier loci for some species, indicating that large geographic groups may include genetically divergent populations and/or that there is geographic heterogeneity in selection pressure upon allozyme loci. Two outlier allozyme loci found in this analysis, lactate dehydrogenase-B and malic enzyme, have been shown to be influenced by selection in other teleost species. This approach is also useful in identifying allozyme loci (or other genetic markers) that meet assumptions for population genetic study.     

Invasion success and genetic diversity of introduced populations of guppies Poecilia reticulata in Australia

High genetic diversity is thought to characterize successful invasive species, as the potential to adapt to new environments is enhanced and inbreeding is reduced. In the last century, guppies, Poecilia reticulata, repeatedly invaded streams in Australia and elsewhere. Quantitative genetic studies of one Australian guppy population have demonstrated high additive genetic variation for autosomal and Y-linked morphological traits. The combination of colonization success, high heritability of morphological traits, and the possibility of multiple introductions to Australia raised the prediction that neutral genetic diversity is high in introduced populations of guppies. In this study we examine genetic diversity at nine microsatellite and one mitochondrial locus for seven Australian populations. We used mtDNA haplotypes from the natural range of guppies and from domesticated varieties to identify source populations. There were a minimum of two introductions, but there was no haplotype diversity within Australian populations, suggesting a founder effect. This was supported by microsatellite markers, as allelic diversity and heterozygosity were severely reduced compared to one wild source population, and evidence of recent bottlenecks was found. Between Australian populations little differentiation of microsatellite allele frequencies was detected, suggesting that population admixture has occurred historically, perhaps due to male-biased gene flow followed by bottlenecks. Thus success of invasion of Australia and high additive genetic variance in Australian guppies are not associated with high levels of diversity at molecular loci. This finding is consistent with the release of additive genetic variation by dominance and epistasis following inbreeding, and with disruptive and negative frequency-dependent selection on fitness traits.     

Genetic diversity in European red deer (Cervus elaphus L.): anthropogenic influences on natural populations

Allozyme, microsatellite and mtDNA (RFLP and sequence) data of European red deer populations were examined as to their capability of indicating anthropogenic influences such as the keeping of animals in enclosures, selective hunting for trophies translocation of specimens to improve trophy quality and habitat fragmentation. Deer in enclosures revealed considerable deviations of allele frequencies from isolation-by-distance expectations but no remarkable loss of genetic diversity. Particular allozyme genotypes were associated with antler morphology, and selective hunting was shown to alter allele frequencies in the expected direction. Habitat fragmentation is reflected by various kinds of genetic markers but due to the lack of information on population histories no unequivocal evidence on particular human activities could be obtained.     

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake,the Eastern Massasauga (Sistrurus catenatus)

Genetic diversity is fundamental to maintaining the long‐term viability of populations, yet reduced genetic variation is often associated with small, isolated populations. To examine the relationship between demography and genetic variation, variation at hypervariable loci (e.g., microsatellite DNA loci) is often measured. However, these loci are selectively neutral (or near neutral) and may not accurately reflect genomewide variation. Variation at functional trait loci, such as the major histocompatibility complex (MHC), can provide a better assessment of adaptive genetic variation in fragmented populations. We compared patterns of microsatellite and MHC variation across three Eastern Massasauga (Sistrurus catenatus) populations representing a gradient of demographic histories to assess the relative roles of natural selection and genetic drift. Using 454 deep amplicon sequencing, we identified 24 putatively functional MHC IIB exon 2 alleles belonging to a minimum of six loci. Analysis of synonymous and nonsynonymous substitution rates provided evidence of historical positive selection at the nucleotide level, and Tajima's D provided support for balancing selection in each population. As predicted, estimates of microsatellite allelic richness, observed, heterozygosity, and expected heterozygosity varied among populations in a pattern qualitatively consistent with demographic history and abundance. While MHC allelic richness at the population and individual levels revealed similar trends, MHC nucleotide diversity was unexpectedly high in the smallest population. Overall, these results suggest that genetic variation in the Eastern Massasauga populations in Illinois has been shaped by multiple evolutionary mechanisms. Thus, conservation efforts should consider both neutral and functional genetic variation when managing captive and wild Eastern Massasauga populations.     

A comparison of variability and population structure for major histocompatibility complex and microsatellite loci in California coastal steelhead (Oncorhynchus mykiss Walbaum)

The major histocompatibility complex (MHC) contains genes integral to immune response in vertebrates. MHC genes have been shown to be under selection in a number of vertebrate taxa, making them intriguing for population genetic studies. We have conducted a survey of genetic variation in an MHC class II gene for steelhead trout from 24 sites in coastal California and compared this variation to that observed at 16 presumably neutral microsatellite loci. A high amount of allelic variation was observed at the MHC when compared to previously published studies on other Pacific salmonids. Elevated nonsynonymous substitutions, relative to synonymous substitutions, were detected at the MHC gene, indicating the signature of historical balancing selection. The MHC data were tested for correlations to and deviations from the patterns found with the microsatellite data. Estimates of allelic richness for the MHC gene and for the microsatellites were positively correlated, as were estimates of population differentiation (F(ST)). An analysis for F(ST) outliers indicates that the MHC locus has an elevated F(ST) relative to the neutral expectation, although a significant result was found for only one particular geographical subgroup. Relatively uniform allele frequency distributions were detected in four populations, although this finding may be partially due to recent population bottlenecks. These results indicate that, at the scale studied here, drift and migration play a major role in the observed geographical variability of MHC genes in steelhead, and that contemporary selection is relatively weak and difficult to detect.     

Genetic architecture of sexual and asexual populations of the aphid Rhopalosiphum padi based on allozyme and microsatellite markers

Cyclical parthenogens, including aphids, are attractive models for comparing the genetic outcomes of sexual and asexual reproduction, which determine their respective evolutionary advantages. In this study, we examined how reproductive mode shapes genetic structure of sexual (cyclically parthenogenetic) and asexual (obligately parthenogenetic) populations of the aphid Rhopalosiphum padi by comparing microsatellite and allozyme data sets. Allozymes showed little polymorphism, confirming earlier studies with these markers. In contrast, microsatellite loci were highly polymorphic and showed patterns very discordant from allozyme loci. In particular, microsatellites revealed strong heterozygote excess in asexual populations, whereas allozymes showed heterozygote deficits. Various hypotheses are explored that could account for the conflicting results of these two types of genetic markers. A strong differentiation between reproductive modes was found with both types of markers. Microsatellites indicated that sexual populations have high allelic polymorphism and heterozygote deficits (possibly because of population subdivision, inbreeding or selection). Little geographical differentiation was found among sexual populations confirming the large dispersal ability of this aphid. In contrast, asexual populations showed less allelic polymorphism but high heterozygosity at most loci. Two alternative hypotheses are proposed to explain this heterozygosity excess: allele sequence divergence during long-term asexuality or hybrid origin of asexual lineages. Clonal diversity of asexual lineages of R. padi was substantial suggesting that they could have frozen genetic diversity from the pool of sexual lineages. Several widespread asexual genotypes were found to persist through time, as already seen in other aphid species, a feature seemingly consistent with the general-purpose genotype hypothesis.     

Molecular evidence for historical and recent population size reductions of tiger salamanders (Ambystoma tigrinum) in Yellowstone National Park

Population declines caused by natural and anthropogenic factors can quickly erode genetic diversity in natural populations. In this study, we examined genetic variation within 10 tiger salamander populations across northern Yellowstone National Park in Wyoming and Montana, USA using eight microsatellite loci. We tested for the genetic signature of population decline using heterozygosity excess, shifts in allele frequencies, and low ratios of allelic number to allelic size range (M-ratios). We found different results among the three tests. All 10 populations had low M-ratios, five had shifts in allele frequencies and only two had significant heterozygosity excesses. These results support theoretical expectations of different temporal signatures among bottleneck tests and suggest that both historical fish stocking, recent, sustained drought, and possibly an emerging amphibian disease have contributed to declines in effective population size.     

The evolution of the major histocompatibility complex in upstream versus downstream river populations of the longnose dace

Populations in upstream versus downstream river locations can be exposed to vastly different environmental and ecological conditions and can thus harbor different genetic resources due to selection and neutral processes. An interesting question is how upstream–downstream directionality in rivers affects the evolution of immune response genes. We used next‐generation amplicon sequencing to identify eight alleles of the major histocompatibility complex (MHC) class II β exon 2 in the cyprinid longnose dace (Rhinichthys cataractae) from three rivers in Alberta, upstream and downstream of municipal and agricultural areas along contaminant gradients. We used these data to test for directional and balancing selection on the MHC. We also genotyped microsatellite loci to examine neutral population processes in this system. We found evidence for balancing selection on the MHC in the form of increased nonsynonymous variation relative to neutral expectations, and selection occurred at more amino acid residues upstream than downstream in two rivers. We found this pattern despite no population structure or isolation by distance, based on microsatellite data, at these sites. Overall, our results suggest that MHC evolution is driven by upstream–downstream directionality in fish inhabiting this system.     

Physiological stress and the maintenance of adaptive genetic variation in a livebearing fish

The importance of genetic variation in evolution is well established. Yet, the mechanisms by which genetic variation—particularly variation in traits under selection—is maintained in natural populations has long been an evolutionary puzzle. Understanding individual variables driving selection and their functional mechanisms is increasingly important in the context of global change and its potential consequences for biodiversity. Here we examined intra-population performance among allelic variants of a pleiotropic locus in response to thermal stress in the variable platyfish, Xiphophorus variatus. The wild-type tailspot allele exhibited significantly lower heat tolerance than all three pattern alleles found in the population, conforming to predictions based on previously observed correlations between temperature and pattern frequencies in the wild. Furthermore, differences between tailspot pattern frequencies in adults and juveniles were broadly consistent with this trend. Thus, it appears that physiological stress and reduced performance of the wild-type allele at higher relative temperatures is a mechanism balancing its frequency in natural populations. Temperature variation and not dissolved oxygen alone, as previously reported, is likely a important abiotic variable contributing to the maintenance of adaptive polymorphism. Furthermore, our findings underscore the potential implications of rising temperatures and physiological stress for levels of genetic variation in natural populations.     

Genetic diversity and differentiation at MHC genes in island populations of tuatara (Sphenodon spp.)

Neutral genetic markers are commonly used to understand the effects of fragmentation and population bottlenecks on genetic variation in threatened species. Although neutral markers are useful for inferring population history, the analysis of functional genes is required to determine the significance of any observed geographical differences in variation. The genes of the major histocompatibility complex (MHC) are well‐known examples of genes of adaptive significance and are particularly relevant to conservation because of their role in pathogen resistance. In this study, we survey diversity at MHC class I loci across a range of tuatara populations. We compare the levels of MHC variation with that observed at neutral microsatellite markers to determine the relative roles of balancing selection, diversifying selection and genetic drift in shaping patterns of MHC variation in isolated populations. In general, levels of MHC variation within tuatara populations are concordant with microsatellite variation. Tuatara populations are highly differentiated at MHC genes, particularly between the northern and Cook Strait regions, and a trend towards diversifying selection across populations was observed. However, overall our results indicate that population bottlenecks and isolation have a larger influence on patterns of MHC variation in tuatara populations than selection.     

Distinguishing adaptive from nonadaptive genetic differentiation: comparison of Q(ST) and F(ST) at two spatial scales

Genetic differentiation in 20 hierarchically sampled populations of wild barley was analyzed with quantitative traits, allozymes and Random Amplified Polymorphic DNAs (RAPDs), and compared for three marker types at two hierarchical levels. Regional subdivision for both molecular markers was much lower than for quantitative traits. For both allozymes and RAPDs, most loci exhibited minor or no regional differentiation, and the relatively high overall estimates of the latter were due to several loci with exceptionally high regional differentiation. The allozyme- and RAPD-specific patterns of differentiation were concordant in general with one another, but not with quantitative trait differentiation. Divergent selection on quantitative traits inferred from very high regional Q(ST) was in full agreement with our previous results obtained from a test of local adaptation and multilevel selection analysis. In contrast, most variation in allozyme and RAPD variation was neutral, although several allozyme loci and RAPD markers were exceptional in their levels of regional differentiation. However, it is not possible to answer the question whether these exceptional loci are directly involved in the response to selection pressure or merely linked to the selected loci. The fact that Q(ST) and F(ST) did not differ at the population scale, that is, within regions, but differed at the regional scale, for which local adaptation has been previously shown, implies that comparison of the level of subdivision in quantitative traits, as compared with molecular markers, is indicative of adaptive population differentiation only when sampling is carried out at the appropriate scale.     

A latitudinal cline in the Chinook salmon (Oncorhynchus tshawytscha) Clock gene: evidence for selection on PolyQ length variants

A critical seasonal event for anadromous Chinook salmon (Oncorhynchus tshawytscha) is the time at which adults migrate from the ocean to breed in freshwater. We investigated whether allelic variation at the circadian rhythm genes, OtsClock1a and OtsClock1b, underlies genetic control of migration timing among 42 populations in North America. We identified eight length variants of the functionally important polyglutamine repeat motif (PolyQ) of OtsClock1b while OtsClock1a PolyQ was highly conserved. We found evidence of a latitudinal cline in average allele length and frequency of the two most common OtsClock1b alleles. The shorter 335 bp allele increases in frequency with decreasing latitude while the longer 359 bp allele increases in frequency at higher latitudes. Comparison to 13 microsatellite loci showed that 335 and 359 bp deviate significantly from neutral expectations. Furthermore, a hierarchical gene diversity analysis based on OtsClock1b PolyQ variation revealed that run timing explains 40.9 per cent of the overall genetic variance among populations. By contrast, an analysis based on 13 microsatellite loci showed that run timing explains only 13.2 per cent of the overall genetic variance. Our findings suggest that length polymorphisms in OtsClock1b PolyQ may be maintained by selection and reflect an adaptation to ecological factors correlated with latitude, such as the seasonally changing day length.