Detecting population structure in a high gene-flow species,Atlantic herring (Clupea harengus): direct,simultaneous evaluation of neutral vs putatively selected loci

In many marine fish species, genetic population structure is typically weak because populations are large, evolutionarily young and have a high potential for gene flow. We tested whether genetic markers influenced by natural selection are more efficient than the presumed neutral genetic markers to detect population structure in Atlantic herring (Clupea harengus), a migratory pelagic species with large effective population sizes. We compared the spatial and temporal patterns of divergence and statistical power of three traditional genetic marker types, microsatellites, allozymes and mitochondrial DNA, with one microsatellite locus, Cpa112, previously shown to be influenced by divergent selection associated with salinity, and one locus located in the major histocompatibility complex class IIA (MHC-IIA) gene, using the same individuals across analyses. Samples were collected in 2002 and 2003 at two locations in the North Sea, one location in the Skagerrak and one location in the low-saline Baltic Sea. Levels of divergence for putatively neutral markers were generally low, with the exception of single outlier locus/sample combinations; microsatellites were the most statistically powerful markers under neutral expectations. We found no evidence of selection acting on the MHC locus. Cpa112, however, was highly divergent in the Baltic samples. Simulations addressing the statistical power for detecting population divergence showed that when using Cpa112 alone, compared with using eight presumed neutral microsatellite loci, sample sizes could be reduced by up to a tenth while still retaining high statistical power. Our results show that the loci influenced by selection can serve as powerful markers for detecting population structure in high gene-flow marine fish species.     

Evidence of microsatellite hitch-hiking selection in Atlantic cod (Gadus morhua L.): implications for inferring population structure in nonmodel organisms

Microsatellites have gained wide application for elucidating population structure in nonmodel organisms. Since they are generally noncoding, neutrality is assumed but rarely tested. In Atlantic cod (Gadus morhua L.), microsatellite studies have revealed highly heterogeneous estimates of genetic differentiation among loci. In particular one locus, Gmo 132, has demonstrated elevated genetic differentiation. We investigated possible hitch-hiking selection at this and other microsatellite loci in Atlantic cod. We employed 11 loci for analysing samples from the Baltic Sea, North Sea, Barents Sea and Newfoundland covering a large part of the species' distributional range. The 'classical' Lewontin-Krakauer test for selection based on variance in estimates of F(ST) and (standardized genetic differentiation) revealed only one significant pairwise test (North Sea-Barents Sea), and the source of the elevated variance could not be ascribed exclusively to Gmo 132. In contrast, different variants of the recently developed ln Rtheta test for selective sweeps at microsatellite loci revealed a high number of significant outcomes of pair-wise tests for Gmo 132. Further, the presence of selection was indicated in at least one other locus. The results suggest that many previous estimates of genetic differentiation in cod based on microsatellites are inflated, and in some cases relationships among populations are obscured by one or more loci being the subject to hitch-hiking selection. Likewise, temporal estimates of effective population sizes in Atlantic cod may be flawed. We recommend, generally, to use a higher number of microsatellite loci to elucidate population structure in marine fishes and other nonmodel species to allow for identification of outlier loci that are subject to selection.     

Allozyme and microsatellite loci provide discordant estimates of population differentiation in the endangered dusky grouper (Epinephelus marginatus) within the Mediterranean Sea

The dusky grouper, Epinephelus marginatus, inhabits coastal reefs in the Mediterranean Sea and Atlantic Ocean. A decline in the abundance of this long-lived protogynous hermaphrodite has led to its listing as an endangered species in the Mediterranean, and heightened management concerns regarding its genetic variability and population substructure. To address these concerns, we analysed genetic variation at seven microsatellite and 28 allozyme loci in dusky groupers sampled from seven areas (for microsatellites) and three areas (for allozymes) in the west-central Mediterranean. Levels of genetic variability were higher for microsatellites than for allozymes (mean H(E) = 0.78 and 0.07, respectively), but similar to those observed in other marine fishes with comparable markers. Both microsatellites and allozymes revealed significant genetic differentiation among all areas analysed with each class of marker, but the magnitude of differentiation revealed by allozymes over three locales (F(ST) = 0.214) was greater than that detected with microsatellites over seven areas, or over the three areas shared with the allozyme analysis (F(ST) = 0.018 and approximately 0, respectively). A large proportion of the allozyme differentiation was due to a single locus (ADA*) possibly influenced by selection, but allozyme differentiation over the three areas was still highly significant (F(ST) = 0.06, P < 0.0001), and the 95% confidence intervals for allozyme and microsatellite F(ST) did not overlap when this locus was excluded. There was no evidence of isolation by distance with either class of markers. Our results lead us to conclude that dusky groupers are not panmictic in the Mediterranean Sea and suggest that they should be managed on a local basis. However, more work is needed to elucidate genetic relationships among populations.     

Genetic variation in the acorn barnacle from allozymes to population genomics

Understanding the patterns of genetic variation within and among populations is a central problem in population and evolutionary genetics. We examine this question in the acorn barnacle, Semibalanus balanoides, in which the allozyme loci Mpi and Gpi have been implicated in balancing selection due to varying selective pressures at different spatial scales. We review the patterns of genetic variation at the Mpi locus, compare this to levels of population differentiation at mtDNA and microsatellites, and place these data in the context of genome-wide variation from high-throughput sequencing of population samples spanning the North Atlantic. Despite considerable geographic variation in the patterns of selection at the Mpi allozyme, this locus shows rather low levels of population differentiation at ecological and trans-oceanic scales (F(ST)?～?5%). Pooled population sequencing was performed on samples from Rhode Island (RI), Maine (ME), and Southwold, England (UK). Analysis of more than 650 million reads identified approximately 335,000 high-quality SNPs in 19 million base pairs of the S. balanoides genome. Much variation is shared across the Atlantic, but there are significant examples of strong population differentiation among samples from RI, ME, and UK. An F(ST) outlier screen of more than 22,000 contigs provided a genome-wide context for interpretation of earlier studies on allozymes, mtDNA, and microsatellites. F(ST) values for allozymes, mtDNA and microsatellites are close to the genome-wide average for random SNPs, with the exception of the trans-Atlantic F(ST) for mtDNA. The majority of F(ST) outliers were unique between individual pairs of populations, but some genes show shared patterns of excess differentiation. These data indicate that gene flow is high, that selection is strong on a subset of genes, and that a variety of genes are experiencing diversifying selection at large spatial scales. This survey of polymorphism in S. balanoides provides a number of genomic tools that promise to make this a powerful model for ecological genomics of the rocky intertidal.     

Comparative allozyme and microsatellite population structure in a narrow endemic plant species, Centaurea corymbosa Pourret (Asteraceae)

Centaurea corymbosa Pourret (Asteraceae) is a narrow endemic species known only from six populations located in a 3-km2 area in the south of France. Earlier field experiments have suggested that pollen and seed dispersal were highly restricted within and among populations. Consistent with the field results, populations were highly differentiated for five allozyme loci and among-population variation fitted an isolation-by-distance model. In the present study, we investigated the genetic structure of C. corymbosa using six microsatellite loci. As with allozymes, microsatellites revealed no within-population structure and a large differentiation among populations. However, allozyme loci were less powerful than microsatellites in detecting the extent of gene flow assessed by assignment tests. The patterns of structuration greatly varied among loci for both types of marker; we suggest that differences in single-locus pattern could mainly be an effect of stochastic variation for allozymes and an effect of variation in mutation rate for microsatellites. In contrast to the multilocus results, the two most polymorphic microsatellite loci did not show any isolation-by-distance pattern. Our results suggest that highly variable loci might not always be the best suited markers to quantify levels of gene flow among populations.     

Detecting Holocene divergence in the anadromous-freshwater three-spined stickleback (Gasterosteus aculeatus) system

The anadromous-freshwater three-spined stickleback (Gasterosteus aculeatus) system allows for inferring the role of adaptation in speciation with a high level of accuracy because the freshwater ecotype has evolved multiple times from a uniform anadromous ancestor. A cause for concern is that independent evolution among drainages is not guaranteed in areas with a poorly resolved glacial history. This is the case for the west European great rivers, whose downstream valleys flanked the southern limit of the late Pleistocene ice sheet. We tested for independent and postglacial colonization of these valleys hypothesizing that the relationships among anadromous and freshwater sticklebacks correspond to a raceme structure. We compared the reduction in plate number accompanying this colonization to the genetic differentiation using 13 allozyme and five microsatellite loci in 350 individuals. Overall microsatellite differentiation (F(ST) = 0.147) was twice as large as allozyme differentiation (F(ST) = 0.066). Although habitat-specific gene flow may mask the ancestral relationships among both ecotypes, levels of microsatellite differentiation supported the hypothesis of raceme-like divergence, reflecting independent colonizations rather than the presence of two distinct evolutionary clades. Under an infinite alleles model and in the absence of gene flow, the observed freshwater divergence might be reached after 440 (microsatellites) to 4500 (allozymes) generations. Hence, the anadromous-freshwater stickleback system most likely diverged postglacially. We conclude that the reduction in plate number in two freshwater basins probably occurred independently, and that its considerable variation among populations is not in agreement with the time since divergence.     

Population structure of the African savannah elephant inferred from mitochondrial control region sequences and nuclear microsatellite loci

Two hundred and thirty-six mitochondrial DNA nucleotide sequences were used in combination with polymorphism at four nuclear microsatellite loci to assess the amount and distribution of genetic variation within and between African savannah elephants. They were sampled from 11 localities in eastern, western and southern Africa. In the total sample, 43 haplotypes were identified and an overall nucleotide diversity of 2.0% was observed. High levels of polymorphism were also observed at the microsatellite loci both at the level of number of alleles and gene diversity. Nine to 14 alleles per locus across populations and 44 alleles in the total sample were found. The gene diversity ranged from 0.51 to 0.72 in the localities studied. An analysis of molecular variance showed significant genetic differentiation between populations within regions and also between regions. The extent of subdivision between populations at the mtDNA control region was approximately twice as high as shown by the microsatellite loci (mtDNA F(ST) = 0.59; microsatellite R(ST) = 0.31). We discuss our results in the light of Pleistocene refugia and attribute the observed pattern to population divergence in allopatry accompanied by a recent population admixture following a recent population expansion.     

Evidence for fine-scale genetic structure and estuarine colonisation in a potential high gene flow marine goby (Pomatoschistus minutus)

Marine fish seem to experience evolutionary processes that are expected to produce genetically homogeneous populations. We have assessed genetic diversity and differentiation in 15 samples of the sand goby Pomatoschistus minutus (Pallas, 1770) (Gobiidae, Teleostei) from four major habitats within the Southern Bight of the North Sea, using seven microsatellite and 13 allozyme loci. Despite its high dispersal potential, microsatellite loci revealed a moderate level of differentiation (overall F(ST)=0.026; overall R(ST)=0.058). Both hierarchical analysis of molecular variance and multivariate analysis revealed significant differentiation (P<0.01) between estuarine, coastal and marine samples with microsatellites, but not with allozymes. Comparison among the different estimators of differentiation (F(ST) and R(ST)) pointed to possible historical events and contemporary habitat fragmentation. Samples were assigned to two breeding units in the estuary and coastal region. Despite this classification, there were indications of a complex and dynamic spatiotemporal structure, which is, most likely, determined by historical events and local oceanic currents.     

Genetic structure and mating system of Euterpe edulis Mart. Populations: a comparative analysis using microsatellite and allozyme markers

A comparative study between microsatellite and allozyme markers was conducted on the genetic structure and mating system in natural populations of Euterpe edulis Mart. Three cohorts, including seedlings, saplings, and adults, were examined in 4 populations using 10 allozyme loci and 10 microsatellite loci. As expected, microsatellite markers had a much higher degree of polymorphism than allozymes, but estimates of multilocus outcrossing rate ( = 1.00), as well as estimates of genetic structure (F(IS), G(ST)), were similar for the 2 sets of markers. Estimates of R(ST), for microsatellites, were higher than those of G(ST), but results of both statistics revealed a close agreement for the genetic structure of the species. This study provides support for the important conclusion that allozymes are still useful and reliable markers to estimate population genetic parameters. Effects of sample size on estimates from hypervariable loci are also discussed in this paper.     

Adaptive divergence in a high gene flow environment: Hsc70 variation in the European flounder (Platichthys flesus L.)

Little is known about local adaptations in marine fishes since population genetic surveys in these species have typically not applied genetic markers subject to selection. In this study, we used a candidate gene approach to investigate adaptive population divergence in the European flounder (Platichthys flesus L.) throughout the northeastern Atlantic. We contrasted patterns of genetic variation in a presumably neutral microsatellite baseline to patterns from a heat-shock cognate protein gene, Hsc70. Using two different neutrality tests we found that the microsatellite data set most likely represented a neutral baseline. In contrast, Hsc70 strongly deviated from neutral expectations. Importantly, when estimating standardized levels of population divergence (F(ST)'), we also found a large discrepancy in the patterns of structuring in the two data sets. Thus, samples grouped according to geographical or historical proximity with regards to microsatellites, but according to environmental similarities with regards to Hsc70. The differences between the data sets were particularly pronounced in pairwise comparisons involving populations in the western and central Baltic Sea. For instance, the genetic differentiation between geographically close Baltic Sea and North Sea populations was found to be 0.02 and 0.45 for microsatellites and Hsc70 respectively. Our results strongly suggest adaptive population divergence and indicate local adaptations at the DNA level in a background of high levels of gene flow, typically found in many marine fish species. Furthermore, this study highlights the usefulness of the candidate gene approach for demonstrating local selection in non-model organisms such as most marine fishes.     

Genetic differentiation and local adaptation in the salt-marsh beetle Pogonus chalceus: a comparison between allozyme and microsatellite loci

The genetic structure of Pogonus chalceus from 11 Atlantic and seven Mediterranean Western European populations was analysed. Results from seven allozyme loci were compared to five microsatellites to test the hypothesis that some enzymatic loci undergo selection. Both allozyme and microsatellite results showed that Mediterranean beetle populations are genetically distinct from Atlantic populations. The analysis of the genetic structure showed that FST values derived from all microsatellite loci were much smaller than those obtained from allozymes. The enzymatic locus Idh-1 exhibited a high value compared to the other loci, suggesting that it is non-neutral. The same Idh-1 locus was implicated in differentiation between temporary and stable populations, as followed also from a highly significant relationship between the allele two of this Idh-1 locus and dispersal power population estimates. The 'parallel evolution' model may account for the diversification of locally adapted Pogonus chalceus populations between different microhabitats.     

Functional microsatellite and possible selective sweep in natural populations of the black-chinned tilapia Sarotherodon melanotheron (Teleostei, Cichlidae)

The Prolactin I (PrlI) gene is a key locus involved in fish osmotic regulation. Two microsatellites, PrlI GT and PrlI AC, are present in the promoter region of this gene in tilapias. One of these microsatellite is associated to PrlI gene expression and growth rate. Aquaria reared individuals homozygous for long microsatellite alleles at the PrlI AC locus expressed less PrlI in fresh water than fishes with other genotypes. To assess the occurrence of selection acting on these microsatellites we carried out a survey of their variability compared with four others microsatellites in natural populations. Samples of two tilapia species were studied, Oreochromis niloticus (6 samples) a typically freshwater species inhabiting osmotically stable environments and Sarotherodon melanotheron (5 samples) a brackish water species obliged and able to adapt very rapidly to any salinity changes. For both species the different populations studied exhibited a high amount of differentiation (all F(st) value were statistically significant) at every loci. Only one locus, PrlI AC, was monomorphic for the same allele in all S. melanotheron populations. We have hypothesized that this outlier locus with its unique allele could have experienced a selective sweep that took place in the early stages of the species origin. S. melanotheron represents an example of adaptation (to fluctuant salinity environment) acting indirectly on gene product through its promoter.     

Direct selection on allozymes is not required to explain heterogeneity among marker loci across a Mytilus hybrid zone

Unequal differentiation between two types of loci (allozyme and DNA markers) across a Mytilus hybrid zone has recently been claimed as evidence for direct selection on some allozyme loci. We provide here a counter-example: a noncoding DNA locus that exhibits as much differentiation as the incriminated allozymes do. The levels of genetic differentiation varied widely among both allozymes and noncoding DNA markers and no clear difference emerged between the two types of markers. This suggests that the strong interlocus variance in genetic differentiation has been confounded with a discrepancy between marker types as a result of an insufficient and unbalanced locus sampling. Heterogeneity in differentiation among neutral loci can be created by stochastic variance during the allopatric divergence preceding a secondary contact. In hybrid zones, a further source of variance is differential introgression among chromosomal regions after the secondary contact owing to the local influence of selected genes on more or less distant markers. However, the degree of differentiation alone gives no way to distinguish indirect pseudo-selection (a regular and ubiquitous feature of hybrid zones) from direct selection. More generally, we suggest that comparative neutrality tests based on discrepancies among marker types have to be applied with caution when the presence of semi-permeable genetic barriers to gene exchange is suspected.     

Genomic scans detect signatures of selection along a salinity gradient in populations of the intertidal seaweed Fucus serratus on a 12 km scale

Detecting natural selection in wild populations is a central challenge in evolutionary biology and genomic scans are an important means of detecting allele frequencies that deviate from neutral expectations among marker loci. We used nine anonymous and 15 EST-linked microsatellites, 362 AFLP loci, and several neutrality tests, to identify outlier loci when comparing four populations of the seaweed Fucus serratus spaced along a 12km intertidal shore with a steep salinity gradient. Under criteria of at least two significant tests in at least two population pairs, three EST-derived and three anonymous loci revealed putative signatures of selection. Anonymous locus FsB113 was a consistent outlier when comparing least saline to fully marine sites. Locus F37 was an outlier when comparing the least saline to more saline areas, and was annotated as a polyol transporter/putative mannitol transporter - an important sugar-alcohol associated with osmoregulation by brown algae. The remaining loci could not be annotated using six different data bases. Exclusion of microsatellite outlier loci did not change either the degree or direction of differentiation among populations. In one outlier test, the number of AFLP outlier loci increased as the salinity differences between population pairs increased (up to 14); only four outliers were detected with the second test and only one was consistent with both tests. Consistency may be improved with a much more rigorous approach to replication and/or may be dependent upon the class of marker used.     

Structure and level of genetic diversity in various bean types evidenced with microsatellite markers isolated from a genomic enriched library

We have constructed a common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA). After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles were detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection.     

Comparative Assessment of Genetic Variability in the Populations of Endemic and Endangered Yellow Catfish, Horabagrus brachysoma (Teleostei: Horabagridae), Based on Allozyme, RAPD, and Microsatellite Markers

The comparative assessment of genetic diversity using allozymes, random amplified polymorphic DNA (RAPD), and microsatellite markers was conducted in endemic and endangered yellow catfish (Horabagrus brachysoma) sampled from three locations in Western Ghats river systems of India. Among the three markers, microsatellites show more polymorphism, having 100% polymorphic loci, whereas allozymes show the least (56%). In RAPD, 60.5% of fragments were polymorphic. Observed heterozygosity and F(ST) values were very high in microsatellites, compared with the other markers. Microsatellite and RAPD markers reported a higher degree of genetic differentiation than allozymes among the populations depicted by pairwise F(ST)/G(ST), AMOVA, Nei's genetic distance, and UPGMA dendrogram. The three classes of markers demonstrated striking genetic differentiation between pairs of H. brachysoma populations. The data emphasize the need for fishery management, conservation, and rehabilitation of this species.     

Fifteen new microsatellite markers for the reef coral Favia fragum and a new Symbiodinium microsatellite

Sixteen new microsatellite loci were isolated from the Tropical Atlantic coral Favia fragum. One locus amplified with pure zooxanthellae DNA template, revealing a symbiont (Symbiodinium) origin. We genotyped 48 short and 45 tall ecomorphs of F. fragum from the Bocas del Toro region of Panama. For 15 host loci, allelic diversity ranged from three to 23 with an average of 5.75 alleles per locus. Analysis of genotypic data revealed significant heterozygote deficits at all loci and linkage disequilibrium between loci, as did a previous study of the two ecomorphs with allozymes. We found evidence for null alleles at four of the host loci in the form of locus-specific polymerase chain reaction failure; however, extreme inbreeding via self-fertilization is likely to explain the large departures from Hardy-Weinberg equilibrium.     

Characterization of nine polymorphic microsatellite markers in sprat (Sprattus sprattus L.)

Nine polymorphic microsatellites were isolated from sprat (Sprattus sprattus) using a microsatellite enrichment protocol and selective hybridization with a biotinylated (AC)(12) probe. The loci showed different variation patterns in a Baltic Sea population (44 individuals) with mean number of alleles at 12.7 and mean observed heterozygosity at 0.78. These microsatellite loci are expected to be used for taxonomic considerations in sprat, stock differentiation and population genetic analysis.     

Stock composition in North Atlantic populations of whiting using microsatellite markers

A size-selected library constructed from DNA of the whiting Merlangius merlangus was screened. From about 3200 recombinant clones, 43 microsatellite loci were detected. Thirteen were sequenced in full. Primers were designed from the sequence of the flanking regions for six loci and used to test the allelic variability at these loci using the polymerase chain reaction (PCR). In addition, five primer pairs developed for the stickleback and another seven for cod were tested. Only six primer pairs revealed at least three alleles per locus. The three useful loci Gmo2, Mmer- UEAW01 and Mmer- UEAW02, had 14–23 alleles per locus in 370 samples. Estimates of genetic structure (φ) were not statistically significant. However, estimates of genetic differentiation ( F _st) were significantly different from zero. Heterogeneity χ²-analysis of allele frequencies among populations suggested relatively low levels of differentiation among samples. Significantly different allele frequency distributions were found for Borgensfjord and northern and southern North Sea samples for at least one locus, and between the latter samples for Mmer -UEAW02 and Gmo2 . There were significant excesses of homozygotes in all samples, over expectation for randomly mating populations in Hardy-Weinberg equilibrium. The estimated frequencies of null alleles were 14.3%, for Mmer -UEAW01, 10.2% for Mmer- UEAW02 and 11.6% for Gmo2 . This result calls for a careful interpretation of the significance of these microsatellite data.     

The utility of QTL-Linked markers to detect selective sweeps in natural populations--a case study of the EDA gene and a linked marker in threespine stickleback

Sequence polymorphisms in coding genes and variability in quantitative trait loci (QTL)-linked markers can be used to uncover the evolutionary mechanisms of traits involved in adaptive processes. We studied sequence variation in the EDA gene and allelic variation in 18 microsatellites - one of which (Gac4174) is linked with the EDA QTL - in low, partially and completely plated morphs from eight threespine stickleback European populations. The results agree with previous studies in that EDA polymorphism is closely related to plate number variation: EDA sequences grouped populations into low and completely plated morphs, whereas microsatellites failed to do so. Furthermore, partially plated fish were heterozygous with respect to the distinctive EDA alleles for completely and low plated morphs, indicating that completely plated morph alleles are not entirely dominant in controlling the expression of lateral plate number. An examination of population differentiation in plate number with quantitative genetic methods revealed that the degree of differentiation exceeded that expected from genetic drift alone (Q(ST) > F(ST)). Our results support the adaptive genetic differentiation of plate morphs and the view that distinctive EDA gene polymorphism occurs in similar sites across the distribution range of this species. Yet, allele frequency differentiation in the Gac4174 microsatellite locus, informative in experimental crosses for plate number variation, did not differ from that of neutral markers and, was therefore unable to detect the signature of natural selection responsible for population divergence.