Population Genomics of Parallel Adaptation in Threespine Stickleback using Sequenced RAD Tags
[['Population genetics provides a rich and mathematically rigorous framework for understanding evolutionary processes in natural populations .', 'This theory was built over the last hundred years by modeling the processes of selection , genetic drift , mutation and migration in spatially distributed populations [1]–[6] .', 'The field has concentrated primarily on the dynamics of one or a small number of genetic loci , largely because of methodological limitations .', 'However , genes are not islands , but rather form part of a genomic community , integrated both by physical proximity on chromosomes and by various evolutionary processes [7]–[10] .', 'With technological advances , such as Next Generation Sequencing ( NGS ) [11]–[13] , the emerging field of population genomics now allows us to address evolutionary processes at a genomic scale in natural populations [14]–[20] .', "Population genetic measures like Wright's F statistics [2] , [21] , [22] , traditionally viewed as point estimates , can now be examined as continuous distributions across a genome [23]–[29] .", 'As a result , in addition to estimating genome-wide averages for such statistics , we can identify specific genomic regions that exhibit significantly increased or decreased differentiation among populations , indicating regions that have likely been under strong diversifying or stabilizing natural selection [9] , [30]–[41] .', 'These signatures of selection can then be used to identify candidate pathways , genes and alleles for targeted functional analyses [42]–[47] .', 'An excellent opportunity for this type of population genomics approach exists in the threespine stickleback , Gasterosteus aculeatus [48]–[50] .', 'This small fish is distributed holarctically and inhabits a large number of marine , estuarine and freshwater habitats in Asia , Europe and North America .', 'In many regions replicate extant freshwater stickleback populations have been independently derived from oceanic ancestors when stickleback became isolated postglacially in newly created freshwater habitats [49] , [51] .', 'Population genetic data support this inference , and also indicate that present day oceanic populations can be used as surrogates for stock that gave rise to nearby derived freshwater populations [52]–[64] .', 'Because of the varied selection regimes in novel habitats , derived stickleback populations have quickly evolved along numerous phenotypic axes , leading to significant variation in behavior , life history , and morphology [65]–[75] .', 'Importantly , despite little or no gene flow between them , populations in similar freshwater habitats often evolve in parallel along the same phenotypic trajectories at a variety of local , regional and global scales [59] , [76]–[80] .', 'Because of their extreme diversification some stickleback populations are actually incipient [81]–[83] or completely differentiated species [84]–[88] .', 'Diversification has happened very rapidly , on the order of just a few thousand years [50] , [58] , [60] , [84] , or in a few rare instances in just a few decades [82] , [89] .', 'Thus , the biogeography of stickleback offers an excellent opportunity to examine the developmental genetic and genomic basis of rapid adaptation by comparing ancestral oceanic and derived freshwater populations .', 'Importantly , these population genomic analyses are greatly advanced by a first draft of the stickleback genome , generated from a line derived from one of the populations used in this study ( Bear Paw Lake; Ensembl: http://www . ensembl . org/Gasterosteus_aculeatus/Info/Index ) .', 'Stickleback can be crossed in the laboratory to produce viable offspring and genetic mapping crosses [79] , [90] , [91] which have been used to successfully identify nearly two dozen quantitative trait loci ( QTL; [78] , [79] , [91]–[97] ) .', 'A surprising result of this work is that , at least in some cases , parallel phenotypic evolution is due to different types of parallel genetic changes .', 'The parallel evolution appears to occur mostly through the fixation of alleles of the same genes from the standing genetic variation in oceanic populations [78]–[80] , [93] , [95] , but these alleles may be the product of single [93] or multiple [96] mutational events .', 'Despite these advances in our understanding of evolutionary genetics in natural populations , a fundamental question remains: Are these instances of parallel evolution at individual loci representative of genome-wide patterns of parallel evolution in independently derived freshwater populations ?', 'To address this question we have performed the first analysis of genome-wide patterns of polymorphism and differentiation using densely spaced single-nucleotide polymorphism ( SNP ) markers in replicate derived freshwater and ancestral oceanic stickleback populations .', 'We used a novel and efficient genotyping approach based on Illumina sequencing of libraries of Restriction-site Associated DNA ( RAD ) tags [98] , [99] .', 'Using short sequence reads , this technique provides genotype information on a large number of SNP markers , although it does not provide gametic phase across SNPs in different tags or haplotype sequence information .', 'We use a kernel-smoothing analysis of these SNP genotype data aligned to the reference genome sequence to assess genome-scale patterns .', 'Here we present a population genomic analysis based on several thousand SNPs across the genomes of 100 individuals from five populations .', 'We focus on three freshwater populations which previous evidence suggests are quite young ( less than 10 , 000 years old ) and are independently derived from oceanic ancestral populations , with little or no gene flow directly among them [53] , [55] , [79] .', 'Because of this history , we expect most of the adaptive evolution in the freshwater habitats to be the result of selection on standing genetic variation present in the founding populations .', 'Accordingly , we focus primarily on measures of nucleotide diversity and differentiation in allele frequencies between the derived freshwater populations and two replicate oceanic populations , quantified with the statistic FST [7] , [21] , [22] , [32] , [100] , [101] .', "We further support our inferences with genomic distributions of private allele density and Tajima's D [102] .", 'We have identified numerous genomic regions that are likely under diversifying selection , and a smaller number of regions that appear subject to balancing selection .', 'We find that many of these regions are shared across the independently derived populations , confirming past results on the genetic basis of morphological evolution from laboratory crosses , and also implicating many other previously unidentified genomic regions as adaptively significant .'], ['RAD tag sequencing provided a genome-wide distribution of over 45 , 000 single nucleotide polymorphisms ( SNPs ) that were simultaneously identified , scored , and used in a genome-wide scan of 100 individuals , 20 each from two oceanic and three freshwater stickleback populations ( Figure 1 and Figure 2; Table 1 ) .', 'The published stickleback genome contains 22 , 830 identifiable SbfI restriction sites across the 21 linkage groups and unassembled scaffolds ( Ensembl , assembly Broad S1 ) .', 'Each site is expected to produce at most two RAD tags ( sequence reads in each direction from the restriction site ) , and our sequencing effort recovered a large proportion of the expected RAD tags ( Table S1 ) .', 'The sites were spread evenly throughout the genome ( Figure 3A ) , and on average each tag was sequenced approximately five to ten times in every individual ( Figure 3B ) .', 'This depth of coverage allowed the identification of SNPs and statistical estimation of the diploid genotype for each individual at most nucleotide sites; sites at which coverage was insufficient were not assigned a genotype ( see Methods ) .', 'The overall frequency of SNPs ( Table 1 ) agrees well with previous estimates of nucleotide polymorphism in stickleback populations .', 'From these SNP genotype data we identified significant genetic variation within and across populations , with average genetic diversity ( π ) equal to 0 . 00336 across all populations and 0 . 0020–0 . 0027 within each population ( Table 2 ) .', 'These findings are in rough agreement with previous studies of genetic variation within and among stickleback populations [55] , [57] , [59] , [60] , although they are somewhat reduced .', 'This may be a consequence of the conservative ( and unbiased ) nature with which SNPs are called using our methodology ( see Methods ) , and additional sequencing of these samples may increase the number of SNPs identified .', 'Furthermore , in agreement with the hypothesis that freshwater populations in this region have been derived post-glacially from oceanic populations [49] , [55] , [65] , [79] , global genetic diversity measures are increased only slightly when combining pairs of populations whether they are both oceanic , both freshwater , or one of each ( Table 2 ) .', 'Our data support the hypothesis that oceanic stickleback populations have few barriers to dispersal , relatively large amounts of gene flow , and little population genetic subdivision [55] , [57] , [59] , [60] , [103] , [104] .', 'Rabbit Slough and Resurrection Bay , the two oceanic populations in our study , are the most geographically distant from one another ( >1000 km as the fish swims ) .', 'Despite this distance , the oceanic populations show the least amount of differentiation between them ( FST\u200a=\u200a0 . 0076; Table 2 ) .', 'In contrast , higher values of FST were observed in pairwise comparisons among freshwater populations and between freshwater and oceanic populations ( 0 . 05–0 . 15 ) , which is generally interpreted as low to moderate amounts of population structuring ( Table 2 ) .', 'The freshwater populations , despite their younger age , are more divergent both from the oceanic ancestral populations and from each other , consistent with our supposition that they represent independent colonizations from the ancestral oceanic population .', 'These results are remarkably similar to results obtained previously from some of these same populations using a small number of microsatellite and mtDNA markers [55] .', 'This combination of large amounts of genetic variation and overall low-to-moderate differentiation between populations , coupled with recent and rapid phenotypic evolution in the freshwater populations , presents an ideal situation for identifying genomic regions that have responded to various kinds of natural selection .', 'To assess genome-wide patterns we examined mean nucleotide diversity ( π ) and heterozygosity ( H ) using a Gaussian kernel smoothing function across each linkage group ( Figure 4 and Figure S1 ) .', 'Although the overall mean diversity and heterozygosity values are 0 . 00336 and 0 . 00187 , respectively , values vary widely across the genome .', 'Nucleotide diversity within genomic regions ranges from 0 . 0003 to over 0 . 01 , whereas heterozygosity values range from 0 . 0001 to 0 . 0083 .', 'This variation in diversity across the genome provides important clues to the evolutionary processes that are maintaining genetic diversity .', 'For example , while expected ( π ) and observed ( H ) heterozygosity largely correspond , they differ at a few genomic regions ( e . g . , on Linkage Group XI ) .', 'Genomic regions that exhibit significantly ( p<10−5 ) low levels of diversity and heterozygosity ( e . g . on LG II and V , Figure 4 and Figure S1 ) may be the result of low mutation rate , low recombination rate , purifying or positive selection that is consistent across populations , or some combination of factors [9] , [36] , [105]–[107] .', 'In contrast , other genomic regions , such as those on LG III and XIII ( Figure 4 ) , show very high levels of both diversity and heterozygosity .', 'The most striking such region , found near the end of LG III , corresponds precisely with a region of reduced differentiation among populations ( Figure 5 ) .', 'This suggests the presence of balancing selection maintaining a common pool of genetic variation at this genomic region within and among populations .', 'To further investigate the pattern of increased genetic variation on LG III , we delineated a region from 14 . 8 to 16 . 1 Mb ( Figure 5; see Methods ) .', 'Within the corresponding 1 . 3-Mb interval in the published stickleback genome are several candidate targets of balancing selection , namely genes implicated in the first line of defense against pathogens [108]: ZEB1 ( ENSGACG00000017648 ) , and two adjacent APOL genes ( ENSGACG00000017778 , ENSGACG00000017779 ) .', 'Supporting the importance of this region in immune response , there are also orthologs of several inflammation pathway genes: LTB4R ( ENSGACG00000017812 ) , SHARPIN ( ENSGACG00000017834 ) , and CEBPD ( ENSGACG00000017927 ) [109]–[111] .', 'The region of significantly elevated nucleotide diversity on LG XIII ( 18 . 1–19 . 1 Mb ) also contains candidate targets of balancing selection including a TRIM14 ( ENSGACG00000014283 ) and three TRIM35 genes ( ENSGACG00000014250 , ENSGACG00000014251 , ENSGACG00000014403 ) .', 'Many members of this large gene family have been implicated in innate immune response ( reviewed in [112] ) , and one gene , TRIM5alpha , bears the signature of balancing selection in primates [113] .', 'The stickleback TRIM cluster on LG XIII provides a second example of balancing selection acting at a TRIM locus .', 'Evidence for balancing selection on Major HistoCompatibility ( MHC ) loci is somewhat weaker .', 'An MHC Class II gene ( ENSGACG00000017967 ) falls nearly 580 kb outside the interval of maximum nucleotide diversity on LG III , although both π and H are moderately elevated at this region as well ( π\u200a=\u200a0 . 0046 , p<0 . 02; H\u200a=\u200a0 . 0030 , pH 0 . 01 ) .', 'In addition , a 250 kb unassembled genomic contig ( scaffold 131 ) contains a block of six MHC class II genes ( ENSGACG00000000330 , ENSGACG00000000336 , ENSGACG00000000344 , ENSGACG00000000346 , ENSGACG00000000348 , ENSGACG00000000350 ) .', 'Nucleotide diversity ( π\u200a=\u200a0 . 0046 , p<0 . 02 ) , heterozygosity ( H\u200a=\u200a0 . 0030 , pH 0 . 01 ) , and freshwater-oceanic differentiation ( FST\u200a=\u200a0 . 0218 , pH 0 . 05 ) averaged over this scaffold are somewhat consistent with a hypothesis of balancing selection .', 'Profiles of population differentiation across each linkage group are generally consistent with the genome-wide average FST values described above .', 'In agreement with the genome-wide results of little genetic structuring among the oceanic populations , we found no genomic regions that exhibit either significantly elevated or reduced ( p<10−5 ) differentiation between the two oceanic populations ( Figure 6A ) .', 'In contrast , comparisons between the ancestral oceanic and individual derived freshwater populations ( Figure 6B–6D ) exhibit several genomic regions of significant differentiation , with FST>0 . 35 , as do the overall freshwater-oceanic comparison ( Figure 6E ) and the comparison among freshwater populations ( Figure 6F ) .', 'Examining more closely the height and location of peaks in FST across these comparisons , we can discern a set of general patterns to generate hypotheses about the modes of genetic variation and selective forces operating in the adaptation to freshwater , and to identify putative candidate genes .', 'Single linkage groups illustrating examples of these distinctive patterns are shown in Figure 7 and Figure 8 .', 'First , the large majority of genomic regions of elevated FST are shared across the three freshwater populations .', 'This pattern suggests independent , parallel evolution in the form of similar genomic regions responding to directional selection across freshwater populations .', 'Second , some , but not all , of these peaks also appear in the overall oceanic-freshwater comparison ( Figure 6E ) .', 'A striking example of this situation is seen on LG XXI ( Figure 8D ) , where a remarkable consistency in both the levels of FST and the location of peak margins across the three freshwater populations is matched by a large peak in the overall oceanic-freshwater comparison .', 'Nucleotide diversity and heterozygosity are reduced in the freshwater populations in this region as well ( at 5 . 7 Mb , π<0 . 001 , p\u200a=\u200a0 . 0003; H\u200a=\u200a0 . 0006 , p\u200a=\u200a0 . 0003 ) .', 'We delineated the nine most consistent and significant of these peaks ( see specific criteria in Methods ) .', 'These regions occur on six linkage groups ( I , IV , VII , VIII , XI , XXI ) and are highlighted in Figure 7 and Figure 8 .', 'Also plotted in Figure 7 and Figure 8 are all FST values at individual SNPs where population differentiation in the overall oceanic-freshwater comparison is significant at the α\u200a=\u200a10−20 level ( equivalent to p<6 . 85×10−23 ) following false discovery rate correction of individual G-tests ( see Methods ) .', 'These highly significant SNPs largely correspond with the genomic regions of elevated differentiation , indicating that the averaged results from the kernel smoothing analysis are not anomalous .', 'Of the 44 , 841 SNPs in this comparison at which FST and a G-statistic could be calculated , 307 were significant at this level .', 'Of these 307 , 227 occur on these six linkage groups , and 119 of these are within the boundaries of the nine peaks , despite the fact that these nine regions collectively account for just ∼2 . 5 percent of the entire genome .', 'In contrast , some of the genomic regions that show consistent differentiation in all of the individual freshwater populations do not exhibit a peak in the overall oceanic-freshwater comparison .', 'An example of this situation is observed on LG II ( Figure 7B ) , where substantial peaks in each of the individual freshwater comparisons cover the same genomic region but differ slightly in their precise location .', 'Accordingly , we do not observe significant differentiation in the overall comparison , and the freshwater populations are substantially differentiated from each other in this region; in fact , the largest peak in the among-freshwater FST ( FST\u200a=\u200a0 . 5147 , p<10−7; Figure 6F ) occurs at this region .', 'Both of these patterns are observed together on LG IV .', 'Of the three LG IV peaks highlighted in Figure 7C , the third is most consistent in its height , width , and location across the freshwater populations .', 'It corresponds to the most substantial peak of the three in the overall oceanic-freshwater comparison ( FST\u200a=\u200a0 . 4262 , p<10−7 ) and shows virtually no differentiation among the freshwater populations .', 'In contrast , the second peak and neighboring region to 22 . 5 Mb shows more variation among the freshwater populations and is substantially lower in the overall oceanic-freshwater comparison ( FST\u200a=\u200a0 . 3269 , p<10−7 ) .', 'Finally , there are peaks of differentiation observed in one or two , but not all three , freshwater populations .', 'One example of this is seen at 11 . 5–12 Mb on LG VIII ( Figure 8B ) , where the Mud Lake population exhibits a peak in differentiation ( FST\u200a=\u200a0 . 3092 , p<0 . 02 vs . RS; FST\u200a=\u200a0 . 2737 , p<0 . 01 vs . RB ) that is not observed to the same extent in the other two populations .', 'Correspondingly , there is a peak in differentiation among the freshwater populations at this location .', 'This contrasts with the peak at ∼8 . 3 Mb on the same linkage group , which is consistent across the three populations and also observed in the overall oceanic-freshwater comparison ( FST\u200a=\u200a0 . 3844 , p<10−7 ) , but not present in the comparison among freshwater populations .', 'The interpretation of these peaks of population differentiation as foci of selection is further supported by the genome-wide distributions of other statistics ( Figure 9 ) .', "First , we estimated Tajima's D [102] across the genome in the oceanic populations ( Figure 9A ) .", '( Because of their young age and expected non-equilibrium allele frequency distributions , we did not consider this statistic to be informative in the freshwater populations ) .', 'D is negative overall in the oceanic populations , perhaps as a result of demographic processes affecting the entire genome equally .', 'However , regions of significantly negative D correspond with peaks of freshwater-oceanic differentiation .', 'In addition , we examined the genomic distribution of the density of private alleles–alleles that are found in only a single population or group of populations in a comparison .', 'Overall , the private allele density ( ρ ) is higher in oceanic populations compared to freshwater than vice versa ( Figure S2 ) .', 'This is consistent with the view that the genetic variation in the freshwater populations is largely a sample from the oceanic stock .', 'However , peaks in private allele density in freshwater populations relative to the ocean ( Figure 9B–9D ) correspond well with FST peaks in the freshwater-oceanic comparisons ( with the exception of the peaks on LG I and XI ) .', 'Thus the peaks in FST are largely the result of alleles that we did not detect in the oceanic populations .', 'The hypothesis that these are new mutations in the freshwater populations is rejected by the absence of corresponding peaks in private allele density among the freshwater populations ( Figure 9E–9G ) .', 'Instead , while selection in freshwater has acted on haplotypes that were rare ( and not detected in our samples ) in the oceanic stock , these haplotypes are nonetheless shared among the independently derived freshwater populations .', 'Previous work has shown that freshwater-adapted alleles may persist at a very low frequency in the ocean , low enough that we would not expect to detect many of them in our sample of 40 individuals [74] .', "However , the maintenance of such low-frequency alleles in the ocean by gene flow from freshwater populations , combined with selection against them in the oceanic habitats , could also account for the significantly negative Tajima's D in the ocean at these genomic regions .", 'Exceptions to the pattern described above are found at the FST peaks on LG I and XI .', 'Here , the private allele density in freshwater does not differ significantly from the genome-wide average ( Figure 9B–9D ) , but private allele density in the ocean relative to freshwater is significantly higher ( Figure S2B ) .', 'In addition , π is elevated in oceanic populations at the LG I region ( Figure S1A , S1B , S1C ) .', 'These data suggest the hypothesis that the oceanic environment may be permissive for multiple haplotypes at these genomic regions , of which only a subset have relatively high fitness in freshwater .', 'In contrast , in the region centered at 13 . 3 Mb on LG II , the freshwater populations exhibit high densities of private alleles , both with respect to the oceanic populations and with respect to each other ( Figure 9B–9G ) .', 'These correspond with peaks in FST both between oceanic and freshwater populations and among freshwater populations ( Figure 7B ) .', 'Here different haplotypes have evolved to high frequency among the different freshwater populations .', 'To set our results in the context of previous QTL mapping studies , and to explore a set of putative candidate genes associated with adaptation to freshwater , we focused on the nine peaks highlighted in Figure 6 .', 'Our results are complementary to previous QTL mapping of traits relevant to freshwater adaptation , although direct comparison with QTL results is complicated because many of those previous studies used microsatellite markers placed on a genetic linkage map .', 'The order of those markers on the genetic map does not always correspond with the marker order on the physical map of the stickleback genome ( Ensembl , database version 56 . 1j , assembly Broad S1 ) , leading in some cases to quite large physical distances between QTL-associated markers .', 'Also , some of the previously used microsatellite markers do not appear at all in the genome sequence .', 'Nonetheless , of the nine peaks we identified , the three on LG IV co-occur with previously identified QTL and specific genes [78] , [79] , [93] , [97] , [99] .', 'This includes the gene Ectodysplasin A ( Eda ) , implicated in the loss of the lateral plate phenotype [93] , which occurs within the first peak of population differentiation that we identified on LG IV .', 'An additional three peaks show the possibility of an association with previous QTL: Shapiro et al . [95] identified very broad QTL that overlap large portions of LG IV and VII , including all five peaks we identified on those linkage groups , and Albert and colleagues [97] identified a QTL adjacent to our peak on LG XXI .', 'In addition , evidence for directional selection based on microsatellite markers has been found just adjacent to two of our delineated peaks .', 'One of these occurs at ∼22 . 3 Mb on LG I [103] ( but see reanalysis by [28] ) .', 'The other lies at ∼9 . 5–9 . 8 Mb on LG VIII [104] , just outside the strict delineation of the peak in Figure 8C , but within the broader region in which we detected substantially elevated FST values and highly significant SNPs .', 'Other regions outside the nine most significant peaks also exhibit a correspondence with QTL studies .', 'For example , the peak on LG XII ( Figure 6E ) contains many osteogenesis genes and overlaps a QTL peak for many skeletal characters [97] .', 'In contrast , the region at the distal end of LG VII previously associated with the pelvic structure phenotype , specifically containing the Pitx1 gene [79] , [95] , [99] , did not correspond to elevated levels of divergence in any of our comparisons .', 'To evaluate potential candidate genes , we identified all loci overlapping the boundaries of the nine most consistent peaks ( Table S2 provides the complete list ) .', 'Many genes in these defined intervals are already annotated by name and orthology in the Gasterosteus genome database ( Ensembl , database version 56 . 1j , assembly Broad S1 ) ; the orthology relationships of the remaining genes , those for which no gene name is yet listed , were further analyzed by a BLAST comparison of the predicted protein sequence for each of them against the NCBI protein database .', 'We then assessed the ontological relationships of all protein coding genes in each interval with respect to skeletal biology and to osmoregulation , two axes of the phenotype known to change drastically as stickleback evolve in response to freshwater environments with very different ecological and chemical conditions than the ocean .', 'Table 3 identifies genes for which a strong association with either of these two broad ontological classes is supported in the literature .', 'From the nine annotated peaks , covering a total of 12 . 2 Mb , we list 31 candidate genes: 23 candidates for patterning and homeostasis of skeletal traits , 8 candidates for response to osmotic stress and development of osmoregulatory organs , and three candidates with pleiotropic roles in both skeletogensis and osmoregulation .', 'The total numbers of all protein-coding genes within each peak are also listed in Table 3 .', 'The abundance of annotated genes within the nine consistent peaks of differentiation does not appear to be an artifact of the distribution of genes across the genome ( Figure S3 ) .', 'Rather , gene density shows no apparent correlation with the regions of population differentiation that we identified here .', 'Although we focused on the nine significant peaks of differentiation that appear most consistent across freshwater populations , several other regions show strong evidence of selection in derived freshwater populations and contain candidate genes worthy of further study .', 'In particular , large regions of LG IV and LG VII outside the delineated peaks appear to be important in differentiation of freshwater stickleback , and these two linkage groups have been the focus of much previous attention .', 'Intriguingly , duplicate synteny groups containing six genes ( CLINT1 , EBF1 , IL12B , ADRB2 , ABLIM3 and AFAP1L1 ) lie just adjacent to Peak 1 of LG IV and partially overlapping Peak 2 of LG VII .', 'Of these , EBF1 , IL12B and ADRB2 are skeletal trait candidates [114]–[116] .', 'As mentioned above , a region of LG XII previously implicated by QTL analysis also shows a signature of selection here .', 'We provide a list of candidate genes in these additional genomic regions in Table S3 .'], ['Population genomic studies depend on having a very high density of markers that can be scored across many individuals .', 'Depending upon demographic factors such as population size and structure , and the strength and nature of selection [117] , [118] , blocks of linkage disequilibrium ( LD ) can be as small as a few hundred base pairs ( as in flies [105] ) to several dozens of kilobases ( kb ) ( as in dogs [119] ) .', 'For most natural populations , the likely size is on the order of 1 to 100 kb , meaning that tens or hundreds of thousands of markers are required to adequately cover an average-sized genome .', 'Furthermore , population genetic sampling variances occur for single point estimates at each marker , requiring numerous individuals to be analyzed from each group or subpopulation of a study .', 'Illumina-sequenced RAD tags provide a powerful new tool to meet these needs , generating a dense battery of SNP markers that are likely to cover a large proportion of the LD blocks produced by stickleback adaptation , and which can be simultaneously identified and scored across entire genomes .', 'The density of markers that can be scored across individuals using RAD-seq holds promise for association mapping of phenotypic traits in natural populations of other organisms .', 'Although we used the stickleback reference genome sequence for the alignment of RAD tags , this tool can be used for population genomic studies in organisms that do not yet have a sequenced genome .', 'Instead of aligning against a genome , the sequence reads can instead be aligned to one another , with SNPs identified and zygosities scored for individuals in the same manner as we describe here ( Hohenlohe and Cresko unpublished data ) .', 'Although these identified RAD sites are initially unanchored with respect to one another , if scored in an F2 or backcross mapping family , they could be ordered to produce a high-density linkage map .', 'This genetic map could then be used to perform genome scans , as well as to help order a physical map from subsequent genome sequencing projects .', 'Such data may be useful even when a preliminary genome assembly already exists .', 'For instance , our approach revealed that nearly 60 Mb - equivalent to two of the largest chromosomes - of the stickleback genome are segregating alleles and show significant signatures of selection , but have not been incorporated into the existing assembly of 21 linkage groups ( Ensembl , Broad S1 assembly ) .', 'A forthcoming RAD genetic map will help incorporate this nearly 10% of the genome into its proper locations .', 'In sum , RAD sequencing has the potential to combine population genetic and genomic studies with genetic and association mapping in populations of both model and non-model organisms , and in addition can help quickly produce or enhance essential genomic resources for organisms that presently have few .', 'We produced genome-wide estimates of population diversity and differentiation for five stickleback populations that have been the focus of intense previous research .', 'These data are largely in agreement with previous estimates of genetic diversity for stickleback , and support the view that oceanic stickleback populations have differentiated little from each other due to extensive gene flow over long distances .', 'Each freshwater population exhibits a greater amount of divergence from the oceanic populations and from the other freshwater populations , but the overall amount is generally moderate and in line with previous estimates of population genetic divergence derived from microsatellite markers [55] .', 'Taken together our data support the biogeographic hypothesis that large populations of oceanic stickleback have given rise repeatedly to freshwater populations , which have become phenotypically differentiated on a background of minor neutral population divergence [
['Introduction', 'Results', 'Discussion', 'Methods']
['Next-generation sequencing technology provides novel opportunities for gathering genome-scale sequence data in natural populations , laying the empirical foundation for the evolving field of population genomics .', 'Here we conducted a genome scan of nucleotide diversity and differentiation in natural populations of threespine stickleback ( Gasterosteus aculeatus ) .', 'We used Illumina-sequenced RAD tags to identify and type over 45 , 000 single nucleotide polymorphisms ( SNPs ) in each of 100 individuals from two oceanic and three freshwater populations .', 'Overall estimates of genetic diversity and differentiation among populations confirm the biogeographic hypothesis that large panmictic oceanic populations have repeatedly given rise to phenotypically divergent freshwater populations .', 'Genomic regions exhibiting signatures of both balancing and divergent selection were remarkably consistent across multiple , independently derived populations , indicating that replicate parallel phenotypic evolution in stickleback may be occurring through extensive , parallel genetic evolution at a genome-wide scale .', 'Some of these genomic regions co-localize with previously identified QTL for stickleback phenotypic variation identified using laboratory mapping crosses .', 'In addition , we have identified several novel regions showing parallel differentiation across independent populations .', 'Annotation of these regions revealed numerous genes that are candidates for stickleback phenotypic evolution and will form the basis of future genetic analyses in this and other organisms .', 'This study represents the first high-density SNP–based genome scan of genetic diversity and differentiation for populations of threespine stickleback in the wild .', 'These data illustrate the complementary nature of laboratory crosses and population genomic scans by confirming the adaptive significance of previously identified genomic regions , elucidating the particular evolutionary and demographic history of such regions in natural populations , and identifying new genomic regions and candidate genes of evolutionary significance .']
['Oceanic threespine stickleback have invaded and adapted to freshwater habitats countless times across the northern hemisphere .', 'These freshwater populations have often evolved in similar ways from the ancestral marine stock from which they independently derived .', 'With the exception of a few identified genes , the genetic basis of this remarkable parallel adaptation is unclear .', 'Here we show that the parallel phenotypic evolution is matched by parallel patterns of nucleotide diversity and population differentiation across the genome .', 'We used a novel high-throughput sequence-based genotyping approach to produce the first high density genome-wide scans of threespine stickleback populations and identified several genomic regions indicative of both divergent and balancing selection .', 'Some of these regions have been associated previously with traits important for freshwater adaptation , but others were previously unidentified .', 'Within these genomic regions we identified candidate genes , laying the foundation for further genetic and functional study of key pathways .', 'This research illustrates the complementary nature of laboratory mapping , functional genetics , and population genomics .']
['evolutionary biology/genomics', 'ecology/marine and freshwater ecology', 'computational biology/genomics', 'evolutionary biology/developmental evolution']