Evolution at two time frames: Polymorphisms from an ancient singular divergence event fuel contemporary parallel evolution
[['Adaptation to local environmental conditions may lead to the evolution of distinct ecotypes and , ultimately , new species [1 , 2] .', 'Under prolonged periods of geographical isolation , the absence of gene flow allows populations to accumulate new alleles by mutation and build-up genome-wide differences in the frequency of these alleles [3 , 4] .', 'However , increasing evidence demonstrates that ecological divergence may occur surprisingly fast and even in absence of a physical barrier [5–8] .', 'As new beneficial mutations are unlikely to accumulate rapidly , these cases of fast adaptation likely involve selection on standing genetic variation , i . e . genetic variation that was present in the ancestral population before divergence took place [9–11] .', 'Characterizing the origin and factors that maintain standing genetic variation is important as it can help understand the rate and direction of genetic adaptation to rapid environmental change [10 , 12 , 13] .', 'Populations that have recently and repeatedly adapted to similar ecological conditions ( i . e . parallel adaptation ) hold the promise to identify the loci and alleles involved in ecological divergence [14–16] .', 'However , the origin of the alleles that allow populations to repeatedly adapt to the alternative environment generally remains poorly characterized and different evolutionary scenarios can be proposed [17] .', 'A first scenario comprises repeated adaptation through independent de novo mutations that occur within the alternative environment ( Figs 1A and S1 ) .', 'Alternatively , several scenarios describe repeated adaptation from standing genetic variation ( Figs 1B–1D and S2–S4 ) .', 'In a second scenario , mutations originate as rare neutral or mildly deleterious alleles within the ancestral population and are repeatedly selected when populations become exposed to the alternative environmental condition ( Figs 1B and S2 ) [18] .', 'In a third scenario , the derived alleles initially evolve within a single isolated population that is exposed to the alternative environment and later disperse to come repeatedly into secondary contact with the ancestral ecotype ( Figs 1C and S3 ) .', 'Similarly , in a fourth scenario , the derived alleles evolve in isolation , but secondary contact and admixture with the ancestral population may then result in polymorphism at these adaptive loci .', 'These polymorphisms can then provide the raw genetic material for repeated and rapid evolution when populations later face similar environmental conditions ( Figs 1D and S4 ) [19–21] .', 'This latter scenario is distinct in that rapid and repeated ecological divergence results from evolution at two different time frames , in the sense that contemporary adaptation is based on alleles that evolved during an ancient divergence in geographic isolation .', 'It should be possible to discriminate amongst the alternative scenarios that describe the origin of standing genetic variation by integrating patterns of pairwise differentiation with properties of the gene genealogies at multiple unlinked loci as well as models that describe the demographic history of the populations [22] .', 'If alleles involved in adaptation evolved through independent mutations , they are expected to occur at different loci or at random along the genealogy within a single locus ( Figs 1A and S1 ) .', 'Therefore , they will not be identical-by-descent , because adaptive de novo mutations can occur on different haplotypes in different geographic regions .', 'Alternatively , if ecological differentiation is based on alleles that are present as standing genetic variation in the ancestral population , the derived alleles are expected to be identical-by-descent , but their evolutionary history may differ strongly at unlinked selected loci ( Figs 1B and S2 ) .', 'Next , if adaptive alleles evolved initially within an isolated population and later came into repeated secondary contact with the ancestral ecotype , the initial evolution of the entire ecotype has a singular evolutionary origin and a shared divergence pattern is expected across unlinked selected loci ( Figs 1C and S3 ) .', 'Gene-flow at secondary contact may in this scenario swamp the initial neutral genetic differences and only genomic regions involved in adaptive divergence are expected to withstand the homogenizing effect of gene flow .', 'However , a highly similar genomic pattern could emerge if the derived ecotype evolved in geographic isolation and adaptive alleles were later reintroduced into the source population ( Figs 1D and S4 ) [21 , 23–25] .', 'Therefore , distinguishing scenario S3 from S4 requires additional lines of evidence that demonstrate repeated secondary contact with only gene flow at neutral loci rather than introgression of derived alleles in the ancestral population and subsequent more recent in situ genetic divergence from these introgressed alleles .', 'Populations of the saltmarsh beetle Pogonus chalceus provide an interesting case to study parallel evolution [26] .', 'Pogonus chalceus beetles have adapted to two contrasting habitat types across Atlantic-Europe; tidal and seasonal salt marshes ( Fig 2A ) .', 'Tidal salt-marshes are inundated on an almost daily basis for at most a few hours and are inhabited by P . chalceus individuals that have a relatively small body size , short wings and submergence behavior during inundation .', 'In contrast , salt-marshes that are subject to seasonal inundations that last for several months , harbor P . chalceus individuals with a larger body size , fully developed wings and more frequent dispersal behavior upon inundation [27–29] .', 'Although these ecotypes diverged in multiple traits towards these contrasting hydrological regimes , we mainly refer to them as the short-winged tidal and long-winged seasonal ecotype , respectively , in accordance with previous studies [27–29] .', 'Populations of both ecotypes can be found along the Atlantic coastal region in Europe and often occur in close proximity and even sympatric mosaics ( Fig 2B ) [30] .', 'In the sympatric mosaics , contrasting behavioral adaptations towards the inundation regimes in the tidal and seasonal marshes potentially result in different habitat preference of the ecotypes and may present an incipient reproductive isolating mechanism [29] .', 'Despite evidence that divergence in wing size in this species is polygenic and under strong genetic control [28 , 30 , 31] , previous research based on microsatellite data also revealed very low neutral genetic differentiation between the ecotypes within geographic locations [30] .', 'This suggests either a very recent differentiation and/or high levels of ongoing gene flow between these ecotypes .', 'At least for wing-size , a fast rate of in situ evolution is corroborated by the observation of a clear reduction in wing size in a small isolated tidal marsh that has been colonized by long-winged individuals less than two decades ago ( S1 Supporting Results ) .', 'To infer the origin of the allelic variants that underlie parallel evolution in P . chalceus , we here investigate genomic differentiation in multiple ecologically divergent population pairs and reconstruct the evolutionary history of the alleles underlying ecological divergence .', 'In agreement with an ancient singular divergence event , we find sharing of the genealogical pattern at unlinked loci that show signatures of selection .', 'Moreover , the apparent potential of these beetles to rapidly and repeatedly adapt to the different tidal and seasonal hydrological regimes , is likely fueled by the maintenance of relatively high frequencies of alleles selected in the alternative habitat .', 'These results contribute to our understanding of the mechanisms underlying fast and parallel ecological adaptation and the factors determining the evolutionary potential of populations and species facing changing environments .'], ['We sampled individuals in four population pairs inhabiting geographically close tidal and seasonally inundated habitats in Belgium ( Be; 48 ind . ) , France ( Fr; 48 ind . ) , Portugal ( Po; 16 ind . ) and Spain ( Sp; 16 ind . ) , as well as a tidal marsh population in the UK ( Uk; 8 ind . ) and a seasonally inundated habitat at the Mediterranean coast of France ( Me; 8 ind . ) ( Fig 2A ) .', 'Individuals from the seasonally inundated habitats had significantly longer wings and larger body sizes compared to those from the tidally inundated marshes ( F1 , 117 = 1904 . 4 , P < 0 . 0001 for wing length and F1 , 117 = 162 . 29 , P < 0 . 0001 for body size ) .', 'The degree of divergence in wing length between the two ecotypes varied among the four population pairs ( F3 , 117 = 23 . 11 , P < 0 . 0001 ) , with highly divergent wing lengths in Sp and Po , and some overlap in wing lengths in Be and Fr .', 'Based on these clear-cut differences in wing length , we refer to the populations sampled in the tidal or seasonally inundated habitats as belonging to the short-winged ( S ) tidal or long-winged ( L ) seasonal ecotype , respectively .', 'RAD-tag sequences filtered for a minimum coverage of 10 and quality score higher than 20 resulted in 27 , 757 SNPs with an average individual depth of 62 . 9 ( ± 51 std ) .', 'Of these , 10 , 052 SNPs distributed over 1 , 142 RAD-tag loci were present in at least 80% of the individuals and were used in further analysis .', 'Average nucleotide diversity ( π ) at RAD-tags did not differ between ecotypes ( GLMM with RAD-tag ID as random effect: Ecotype effect: F1 , 1977 = 0 . 31 , P = 0 . 6 ) , but differed among population pairs ( Population effect: F3 , 1977 = 11 . 5 , P < 0 . 0001 , S1 Table ) .', 'The most southern populations had a significantly higher nucleotide diversity compared to the northern populations .', 'The difference in nucleotide diversity among population pairs was also consistent among ecotypes ( Population*Ecotype interaction: F3 , 1977 = 2 . 2 , P = 0 . 09 ) .', 'Genetic differentiation ( FST ) among the 10 different populations , varied considerably and ranged from a low ( BeS vs . UkS: FST = 0 . 052 ) to a high degree of differentiation ( PoS vs . MeL: FST = 0 . 37 ) ( S1 Table ) .', 'Principal Coordinate Analysis ( PCoA ) using all SNP data divided samples largely according to ecotype along the first PCo axis , whereas the second PCo axis grouped samples according to geographic location ( Fig 3A ) .', 'When restricting the SNPs to a ‘neutral’ set wherein we excluded RAD-tags containing a SNP with a signature of divergent selection ( see Outlier loci ) , the importance of both axes was reversed with the first axis ordinating populations according to their geographic location rather than by ecotype ( Fig 3B ) .', 'Genetic differentiation increased significantly with increasing geographic distance between the populations ( rS = 0 . 37 , P = 0 . 017 ) and was higher when populations belonged to a different ecotype ( rS = 0 . 33 , P = 0 . 02 ) .', 'For the ‘neutral’ set there was an even stronger effect of geographic distance on genetic differentiation ( rS = 0 . 54 , P = 0 . 002 ) , while the significant ecotype effect disappeared ( rS = 0 . 09 , P = 0 . 2 ) .', 'Bayesian clustering [32] of individuals based on their genotypes supported 8 and 6 genetically distinct populations ( K ) for the ‘total’ and ‘neutral’ SNP set , respectively ( Fig 3C; S1 Fig ) .', 'For the ‘neutral’ SNP set , individuals from the same population pair ( except Sp ) clustered together as a single population , irrespective of their ecotype .', 'We inferred the demographic history of divergence for each population pair using the joint allele frequency spectrum ( JAFS ) as implemented in δaδi [33] .', 'In all four ecotypic population pairs the JAFS showed a pattern wherein most alleles were present in comparable frequencies in both populations ( high density at the diagonal of the JAFS; Fig 4 ) .', 'However , at the same time the JAFS showed an increase in frequency of alleles present at very low frequencies in either one of the two populations but at high frequencies in the opposite population ( high densities towards the upper left and lower right corner of the JAFS; Fig 4 ) .', 'This was particularly the case for the short-winged tidal populations from Fr , Po and Sp , in which we observed a relatively high frequency of alleles that were present in very low frequency in the long-winged seasonal ecotype , but nearly reached fixation in the short-winged tidal ecotype .', 'These patterns contrasted sharply with those present in the JAFS of the within ecotype comparison of geographically separated populations .', 'Here , a lower density of alleles was observed both for alleles that were present in comparable frequencies , as well as for alleles with highly profound frequency differences ( Fig 4 ) .', 'In all four among ecotype pair comparisons , demographic models incorporating gene-flow after the divergence ( IM and SC ) and heterogeneous genomic divergence ( “2M” ) and/or heterogeneous population size ( “hrf” ) were clearly better supported compared to models that did not incorporate these effects .', 'A Secondary Contact ( SC ) model incorporating both heterogeneous gene-flow and population size ( SC2M_hrf ) yielded the best fit for all ecotype comparisons and predicted the observed JAFS reasonably well ( Fig 4 and S2 Fig ) .', 'However , this fit was only marginally better than a Secondary Contact model with heterogenous genomic divergence , but without heterogeneous population size ( SC2M ) for the population pairs Fr and Po and an Isolation-with-Migration model with heterogeneous genomic divergence ( IM2M ) for population pair Sp .', 'We based interpretation of the estimates of the demographic parameters on the SC2M_hrf model for all four ecotypic population pair comparisons .', 'Estimates of the effective population size revealed a distinct pattern in the relative population sizes of the two ecotypes .', 'In the northern population pair Be , the population size of the short-winged tidal ecotype was estimated to be around four to five times larger compared to the population size of the long-winged seasonal ecotype .', 'In contrast , towards more southern latitudes , this pattern was reversed with population sizes of the short-winged ecotype being estimated to be nearly 50 ( Sp ) to 100 ( Po ) times smaller compared to those of the long-winged seasonal ecotype ( Fig 4 ) .', 'Population migration rates ( M ) were strongly related to the ecotypic differences in population size and a general trend was observed of higher migration rates from the ecotype with the largest population size towards the ecotype with the smallest population size .', 'These migration rates were substantial and varied from approximately 1 . 5 gene copies per generation for both Be and Fr up to more than 30 gene copies per generation for Po and Sp , respectively .', 'The estimated proportion of the genome showing restricted gene flow between both ecotypes was comparable among the four population pairs and varied between 27% and 33% of the genome .', 'The reduction in effective migration rate of this part of the genome was stronger for the southern population pairs Po and Sp ( reduction of 97 . 6% and 99% of the neutral migration rate , respectively ) , compared to the northern populations Fr and Be , with a respective reduction of 78% to 31% of the neutral migration rate .', 'Initial divergence times between the ecotypes were estimated between 43 Kya ( Be ) , 64 Kya ( Po ) and more than 100 Kya ( Fr and Sp ) , while the onset of secondary contact was estimated between 1 , 6 Kya ( Be ) to 23 Kya ( Fr ) .', 'The results obtained from the among ecotype pair comparisons were in clear contrast with the within ecotype comparisons ( Be and Fr only ) .', 'Here , models assuming homogeneous genomic divergence were equally well supported as models assuming heterogeneous genomic divergence ( S2 Fig ) .', 'Fit of the IM and SC model , either with or without heterogeneous population size , was comparable for the geographically separated long-winged seasonal populations , while the SC model was better supported for the geographically separated short-winged tidal populations .', 'The estimated migration rates were substantially lower compared to those of the among ecotype comparisons and were estimated to be higher from Fr into Be compared to the opposite direction ( Fig 4 ) .', 'Despite the apparent close genetic relationship of long- and short-winged ecotypes within each geographic population pair ( S1 Table ) , we observed substantial heterogeneity in FST across SNPs ( Fig 5 , S3 Fig ) .', 'A substantial number of SNPs showed FST values that exceeded 0 . 5 in the ecotype comparisons .', 'For some of these SNPs , different alleles even reached almost complete fixation in the different ecotypes .', 'This proportion of SNPs with FST values higher than 0 . 5 increased towards the more southern population pairs ( Be: 2 . 1% , Fr: 4 . 5% , Po: 6 . 3% and Sp: 11 . 1% ) .', 'In contrast , only very few FST values exceeded 0 . 5 when similar ecotypes were compared from different population pairs ( e . g . Be versus Fr; S3 Fig ) .', 'SNPs that were strongly differentiated in one particular population pair were also significantly more differentiated in any of the other population pairs ( 0 . 498 < r < 0 . 66; P all < 0 . 0001; S3 Fig ) , providing support for extensive sharing of highly differentiated SNPs among population pairs .', 'Significant outliers were identified using two approaches; BayeScan [34] to identify outliers from the genome wide background within each population pair and BayEnv2 [35] for associations between SNP allele frequencies and habitat type ( coded as -1 or 1 if tidal or seasonal habitat , respectively ) across all populations .', 'BayeScan identified a total of 512 ( 3 . 2% ) SNPs that were clustered on 109 ( 15% ) assembled RAD-tag loci with stronger differentiation as expected by chance in at least one of the ecotype comparisons ( false discovery rate = 0 . 05; i . e . on average 4 . 70 outlier SNPs per RAD-tag locus ) .', 'BayEnv2 identified a total of 75 ( 0 . 48% ) SNPs in 32 ( 6 . 3% ) assembled RAD-tag loci having allele frequencies that were strongly associated with the ecotypic divergence across all investigated populations ( log10BF = 4 ) .', 'On average 75% of these SNPs were identified as significant outliers with BayeScan .', 'Despite this general agreement in SNPs that were consistently identified by both approaches , few SNPs that were strongly supported to be outlier SNPs across the entire range ( BayEnv2; log10BF > 4 ) were not significantly differentiated within some regional ecotype comparisons .', 'Conversely , significant outliers at the regional level were sometimes not supported to be outliers across the entire range and , therefore , likely population specific ( S4 Fig and S5 Fig ) .', 'SNPs were mapped to a genome assembly ( S1 Supporting Methods ) , which revealed that SNPs with a high FST value clustered into several unlinked regions that were distributed over a large proportion of the genome ( Fig 5 ) .', 'These regions with outlier SNPs were largely consistent across the different population pairs and are primarily clustered on the first half of LG_1 , across the full length of LG_2 and LG_3 and at the center of LG_4 ( Fig 5 ) .', 'R-squared values between the allele frequencies at outlier loci show a sharp decline with distance between the considered loci within the linkage groups ( S6 Fig ) .', 'This suggests the absence of , at least large , structural chromosomal rearrangements for explaining the observed divergence patterns ( i . e . divergent allele combinations recombine ) .', 'No outlier SNPs were observed on LG_6 to LG_10 .', 'Yet , some more subtle differences could be observed as exemplified by the central region of LG_5 where high genomic differentiation was only observed for the Fr and Sp population pair , but not in the Be and Po population comparison .', 'The nuclear-encoded mitochondrial NADP+-dependent isocitrate dehydrogenase ( mtIdh ) locus , that was previously identified to be strongly associated with the ecotypic divergence [27 , 28 , 30 , 36] , is located approximately in the middle of LG_2 ( scaffold Pchal00589: 148 , 569–150 , 947 , chromosome LG_2: 9 , 802 , 630–9 , 805 , 108 ) .', 'This genomic region includes several other outlier RAD-tag loci and suggests mtIdh may not be a direct target of selection , but rather linked to other divergently selected loci .', 'Haplotype networks and trees of the 1 . 2 kb sequence alignments obtained from RAD-tag loci with an outlier SNP ( BayEnv2 [35]; log10BF > 4 ) show that haplotypes selected in short-winged tidal populations are derived and generally clustered as a strongly supported monophyletic clade of closely related sequences ( clade support level > 0 . 96; Fig 6A and 6B and S5 Fig ) .', 'This clustering supports a singular mutational origin of alleles selected in the short-winged tidal ecotype at each of the investigated outlier tags .', 'These alleles appeared to be derived as they most frequently constituted a subclade within those selected in the long-winged seasonal ecotype ( S7 Fig ) .', 'This is in line with the observation that all other species within the genus Pogonus are long-winged [37] .', 'The average absolute divergence between the differentially selected haplotypes ( dXY = 0 . 011 ± 0 . 0014 ) was about 1 . 65 times higher compared to the average divergence between two randomly chosen haplotypes at these loci ( πtot , outliers = 0 . 0067 ± 0 . 00097 , t-test: P < 0 . 0001 ) and highlights a deep divergence between the alleles that are differentially selected between both ecotypes .', 'Dating the divergence time between these allelic clusters using BEAST [38] and the divergence from P . littoralis as a calibration point ( 620 Kya [36] ) , pointed towards comparable divergence times across outlier loci ( Fig 6B and S5 Fig ) .', 'The divergence time of the alleles selected in the short-winged tidal ecotype ranged between 120 Kya and 280 Kya , with an average of 189 Kya ± 90 Kya and suggests that the divergence took place during the Late Pleistocene .', 'Sequences from more strongly differentiated RAD-tags had a significantly higher Tajima’s D ( pooled across ecotypes within each population pair; F = 57 . 36 , P < 0 . 0001; Fig 6C ) and absolute nucleotide divergence between the ecotypes ( normalized by the divergence from the outgroup P . littoralis = dXY / dXY , P . littoralis , see Methods for details; FST: F = 83 . 9 , P < 0 . 0001; Fig 6D ) .', 'The significant relation between FST and absolute divergence ( normalized dXY ) further supports that the observed heterogeneity in genomic divergence between the ecotypes is the result of divergent selection of ancient alleles embedded within a genome that is homogenized between the ecotypes , rather than selection on recently obtained new mutations [39 , 40] .', 'Furthermore , a reduced recombination rate was observed between haplotypes that are divergently selected between long- and short-winged populations ( r2 = 0 . 140 ) compared to the recombination rate observed within populations ( r2 = 0 . 184 ) .', 'We further observed that nucleotide diversity ( π ) of haplotypes associated with the short-winged tidal ecotype was strongly reduced and tended to be nearly seven times lower ( πS = 0 . 0009 ± 0 . 0003 ) compared to those associated with the long-winged seasonal ecotype ( πL = 0 . 0062 ± 0 . 0003 ) ( GLMM with tagID as random effect: Ecotype effect: F1 , 66 = 25 . 12 , P < 0 . 0001; Fig 6E ) .', 'This difference was consistent among the four populations ( Ecotype*Population interaction: F3 , 64 = 0 . 87; P = 0 . 45 ) .', 'In contrast , nucleotide diversity at RAD-tags showing no elevated levels of divergence between the ecotypes was comparable between both ecotypes ( GLMM with RAD-tag as random effect: Ecotype effect: F1 , 599 = 0 . 98 , P < 0 . 3; Fig 6E ) .', 'The nucleotide diversity of the haplotypes associated with the long-winged seasonal ecotype was also comparable to average nucleotide diversity observed at non-outlier loci ( πtot , neutral RAD-tags = 0 . 0057 ± 0 . 0003 ) , showing that only haplotypes associated with the short-winged tidal ecotype have this reduced nucleotide diversity ( Fig 6E ) .', 'Similarly , Tajima’s D of haplotypes associated with the short-winged tidal ecotype was significantly lower compared to those of the long-winged seasonal ecotype ( F1 , 27 = 11 . 7; P = 0 . 002; Fig 6E ) and suggests a recent spread of alleles of the short-winged tidal ecotype along the Atlantic European coast .', 'Here , we quantify the extent to which polymorphism at outlier loci determines the genetic variation of each ecotype to potentially adapt to the alternative environment .', 'More specifically , we calculated how many individuals does one need to sample to capture most of the genetic variants that are selected in the alternative environment ?', 'The outlier analyses revealed that restricted regions within the genome are significantly more diverged as expected by chance and thus likely linked to sites under divergent selection , but generally did not reach fixation in most of the investigated population pairs ( Fig 5 ) .', 'This was also indicated by the reconstruction of the demographic history , which revealed that admixture between the ecotypes also involves genomic islands .', 'Therefore , we calculated the frequency of alleles that are selected for in the alternative habitat at outlier loci for each ecotype .', 'We focused on SNPs whose allele frequencies were strongly associated with the ecotypic divergence across all investigated populations ( BayEnv2[35]; log10BF = 4 ) .', 'If multiple outlier SNPs were situated on the same RAD-tag , only the most strongly supported SNP was selected .', 'Individuals of the long-winged seasonal ecotype contained on average at 10% ( SpL ) to 42% ( BeL ) of the outlier loci at least one allele associated with the alternative , short-winged tidal ecotype .', 'Similarly , individuals from the short-winged tidal ecotype contained alleles associated with the long-winged seasonal ecotype at 10% ( SpS ) to 48% ( BeS ) of the outlier SNPs .', 'Moreover , random sampling of an increasing number of individuals showed a steep increase in the proportion of outlier SNPs with at least one allele associated with the alternative habitat ( Fig 7 ) .', 'For example , a random sample of only eight individuals of the long-winged seasonal ecotype of Be , Fr and Po contained at least one copy of the allele selected in the short-winged tidal ecotype at more than 80% of the outlier loci ( Fig 7A ) .', 'This demonstrates that different individuals from the same population generally carry alleles that are selected in the alternative habitat at different loci and suggests the presence of substantial standing genetic variation in individuals sampled in the seasonally inundated marshes to adapt to tidal marshes .', 'Only for the most southern long-winged seasonal populations ( SpL and MeL ) , outlier loci that are likely linked to alleles associated with the short-winged tidal ecotype are present at lower frequencies and these populations are unlikely to contain the full set of alleles associated with the short-winged ecotype .', 'For FrS , PoS and particularly SpS , long-wing selected alleles accumulated at a much lower rate under random sampling of individuals of the short-winged tidal ecotype ( Fig 7B ) .'], ['The initial evolution of co-adapted alleles at multiple physically unlinked loci is facilitated in geographic isolation [3] .', 'Subsequent admixture of gene pools may then enrich the adaptive genetic variation and allow for subsequent fast and repeated adaptation .', 'In agreement to this , in P . chalceus populations the alleles required to adapt to the alternative environment are found to be maintained in the source population .', 'These loci are expected to be maladaptive within the source population and it is likely both the temporal and spatial repetition of this divergence , combined with relatively high levels of gene flow and range expansion , that maintain these allele frequencies .', 'Glacial cycles , in particular , can be expected to have played an important role in this process .', 'During glacial cycles , episodes of fission and fusion of the different ecotypes may have generated strong opportunities for both the evolution of adaptive genetic variants as well as the maintenance of genetic polymorphisms by admixture [55] .', 'As exemplified by the evolution of the P . chalceus ecotypes , historic selection pressures could therefore play a pivotal role in determining the rate , direction and probability of contemporary adaptation to changing environmental conditions .', 'The proposed mechanism illustrates that the distinction between in-situ divergence and secondary contact is less clear-cut as generally assumed if populations are highly admixed and that , moreover , both processes can be involved at different time frames .', 'An important implication is that this mechanism might reconcile different views on the geography of ecological divergence in which adaptive divergence between closely related populations is either interpreted as primary divergence , and thus the onset of speciation [2] , or the result of secondary introgression after initial ecological divergence in allopatry [22 , 25 , 56] .'], ['Diverged population pairs of P . chalceus were collected from both tidal and seasonal salt marshes extending nearly the entire species range ( Fig 2 ) [37] .', 'We sampled four geographically isolated population pairs ( separated between approximately 450 km and 900 km ) of a tidal and seasonally flooded inland population each .', 'Wing and elytral sizes were measured by means of a calibrated ocular with a stereomicroscope .', 'We further conducted RAD-seq genotyping on two specimens of the long-winged outgroup species P . littoralis , which were sampled in the Axion Delta , Thessaloniki , Greece ( S3 Table ) .', 'DNA was extracted using the DNA extraction NucleoSpin Tissue kit ( Macherey-Nagel GmBH ) .', 'Extracted genomic DNA was normalized to a concentration of 7 . 14 ng/μl and processed into RAD libraries according to Etter et al . ( 2011 ) , using the restriction enzyme SbfI-HF ( NEB ) [57] .', 'Final enrichment was based on 16 PCR cycles .', 'A total of nine RAD libraries including 16 individuals each and , hence , a total of 144 individuals were sequenced paired-end for 100 cycles ( i . e . 100 bp ) in a single lane on an Illumina HiSeq2000 platform according to manufacturer’s instructions .', 'The outgroup P . littoralis specimens were sequenced separately .', 'The raw data was demultiplexed to recover individual samples from the Illumina libraries using the process_radtags module in Stacks v1 . 20 software [58] .', 'Reads were quality filtered when they contained 15 bp windows of mean Phred scores lower than 10 .', 'PCR duplicates were identified as almost ( i . e . allowing for sequencing errors ) identical reverse read sequences and removed , using a custom Perl script [59] .', 'Total DNA was extracted from individuals captured in the canal habitat of the salt marshes in the Guérande region ( France ) , using the DNA extraction NucleoSpin Tissue kit ( Macherey-Nagel GmBH ) .', 'Illumina paired-end ( 100 bp ) and mate-paired ( 49 bp ) libraries were constructed with insert sizes of 200 bp , 500 bp , 800 bp , 2 kb and 5 kb and sequenced on an Illumina HiSeq2000 system according to the manufacturer’s protocol ( Illumina Inc . ) .', 'Adapter contamination in reads was deleted using Cutadapt v1 . 4 [60] and reads that did not have a matching pair after adaptor filtering were removed .', 'Reads were corrected for sequencing error with SOAPec v2 . 02 [61] , using a k-mer size of 17 and a low frequency cutoff of consecutive k-mer of 3 .', 'Sequencing of the 200 bp , 500 bp , 800 bp , 2 kb and 5 kb insert libraries resulted in a total of ~57 . 7 Gb of sequencing data , of which 56 . 6 Gb was retained after data cleaning ( S4 Table ) .', 'Reads were assembled using SOAPdenovo2 [61] using a k-mer parameter of 47 , which was selected for producing the largest contig and scaffold N50 size after testing a range of k-mer settings between 19 and 71 .', 'The short insert libraries were used for both contig building and scaffolding .', 'The long insert libraries were only used for scaffolding .', 'SOAPdenovo GapCloser v1 . 12 tool [61] was used with default settings to close gaps emerging during scaffolding
['Introduction', 'Results', 'Discussion', 'Methods']
['When environments change , populations may adapt surprisingly fast , repeatedly and even at microgeographic scales .', 'There is increasing evidence that such cases of rapid parallel evolution are fueled by standing genetic variation , but the source of this genetic variation remains poorly understood .', 'In the saltmarsh beetle Pogonus chalceus , short-winged ‘tidal’ and long-winged ‘seasonal’ ecotypes have diverged in response to contrasting hydrological regimes and can be repeatedly found along the Atlantic European coast .', 'By analyzing genomic variation across the beetles’ distribution , we reveal that alleles selected in the tidal ecotype are spread across the genome and evolved during a singular and , likely , geographically isolated divergence event , within the last 190 Kya .', 'Due to subsequent admixture , the ancient and differentially selected alleles are currently polymorphic in most populations across its range , which could potentially allow for the fast evolution of one ecotype from a small number of random individuals , as low as 5 to 15 , from a population of the other ecotype .', 'Our results suggest that cases of fast parallel ecological divergence can be the result of evolution at two different time frames: divergence in the past , followed by repeated selection on the same divergently evolved alleles after admixture .', 'These findings highlight the importance of an ancient and , likely , allopatric divergence event for driving the rate and direction of contemporary fast evolution under gene flow .', 'This mechanism is potentially driven by periods of geographic isolation imposed by large-scale environmental changes such as glacial cycles .']
['Evidence has accumulated that populations may adapt surprisingly fast to changing environments .', 'This rapid , and often parallel , ecological adaptation is presumed to be facilitated when selection acts on preexisting genetic variation .', 'However , the origin of this variation remains to be identified .', 'In our work on genome-wide parallel divergence in a mosaic of two beetle ecotypes , we identify the genomic regions involved in adaptation .', 'We show that the origin of the divergent alleles at different loci can be traced back to a singular , ancient divergence event .', 'This event likely occurred in geographic isolation after which admixture of the diverged populations resulted in a polymorphic population that survived during the most recent glacial maxima .', 'The alleles involved in adaptation to the alternative environments are currently present in much higher frequencies in the populations than generally assumed .', 'Therefore , when habitats become available , the presence of these alleles may result in rapid and parallel ecological differentiation by the reassembly of these ancient alleles .', 'We suggest that this mechanism may be common to examples of parallel evolution and might reconcile different views on the role of geographical isolation in ecological divergence .']
['population genetics', 'genetic mapping', 'parallel evolution', 'paleontology', 'population biology', 'paleogenetics', 'genomics', 'genetic polymorphism', 'comparative genomics', 'evolutionary genetics', 'genetic loci', 'haplotypes', 'heredity', 'earth sciences', 'genetics', 'biology and life sciences', 'computational biology', 'evolutionary biology', 'evolutionary processes']