Heliconius butterfly clades (H. erato and H. melpomene) to explore patterns of Z chromosome divergence. We show that increased divergence compared to equilibrium expectations can in many cases be explained by demographic change. Among Heliconius erato populations, for instance, population size increase in the ancestral population can explain increased absolute divergence measures on the Z chromosome compared to the autosomes, as a result of increased ancestral Z chromosome genetic diversity. Nonetheless, we do identify increased divergence on the Z chromosome relative to the autosomes in parapatric or sympatric species comparisons that imply postzygotic reproductive barriers. Using simulations, we show that this is consistent with reduced gene flow on the Z chromosome, perhaps due to greater accumulation of incompatibilities. Our work demonstrates the importance of taking demography into account to interpret patterns of divergence on the Z chromosome, but nonetheless provides evidence to support the Z chromosome as a strong barrier to gene flow in incipient Heliconius butterfly species.Sex chromosomes are disproportionately involved in reproductive isolation and adaptation. In support of such a “large‐X” effect, genome scans between recently diverged populations and species pairs often identify distinct patterns of divergence on the sex chromosome compared to autosomes. When measures of divergence between populations are higher on the sex chromosome compared to autosomes, such patterns could be interpreted as evidence for faster divergence on the sex chromosome, that is “faster‐X”, barriers to gene flow on the sex chromosome. However, demographic changes can strongly skew divergence estimates and are not always taken into consideration. We used 224 whole‐genome sequences representing 36 populations from two  For da, the average of the within‐population nucleotide diversity (πS) is used as the estimate of the ancestral nucleotide diversity (πanc). The influence of population size on FST can be seen as resulting from a decrease in the denominator (πT), but not in the numerator (πT and πs change proportionately)FIGURE B1 The effect of population size on the coalescent and measures of diversity and divergence. The branches represent two populations, 1 and 2, that have split at a certain time (grey dashed line). This branching event occurs on two chromosomes that have a different population size, such as the autosomes (grey) and X chromosome (green). The black lines show the coalescent of two alleles in each population. The branches show that the coalescence process is influenced by the split time as well as the population size. Population size affects the nucleotide diversity within each population (π), the total nucleotide diversity . This relationship can be seen with  Wright, . The depm) . As expected, the values FST, dXY and da all increase when migration between populations decreases. FST and dXY are clearly influenced by population size. While for dXY, this results from the variation present at the time of the split, FST does not show a simple linear relationship with population size. Only da represents the net accumulation of differences that can be compared between populations of different sizes, such as the sex chromosomes versus autosomes . A lower effective population size, such as for the X chromosome (green) compared to autosomes (grey), results in higher FST and lower dXY estimates, but has no effect on da under these assumptionsFIGURE B2 Simulated effect of population size differences on divergence measures To show how these different divergence measures perform, we simulated a simplified population split with varying degrees of migration  was found to be Z‐linked.Here, we explore diversity and divergence on the Z chromosome relative to the autosomes among populations of the H. erato clade and 16 populations of the H. melpomene clade. We also use simulations to evaluate the effect that demographic changes have on the estimate of relative rates of divergence on the Z versus the autosomes and demonstrate that in many comparisons, demography can explain much of the observed elevated divergence on the Z relative to the autosomes. However, by taking into account geographic distance or autosomal divergence as a proxy for gene flow, we show that there is evidence for increased divergence on the Z chromosome for species pairs with known postzygotic reproductive barriers. These rates of increased divergence likely reflect reduced admixture on the Z chromosome and provide support for the Z chromosome being a greater barrier to gene flow in some incipient Heliconius butterfly species.The presence of incipient species pairs with different levels of reproductive isolation allows us to examine the relative rate of autosomal and Z chromosomal evolution and the factors that are likely influencing patterns of divergence. We take advantage of a large genomic data set composed of 224 whole genomes representing 20 populations of the 22.1Heliconius erato clade and 115 from the Heliconius melpomene clade , Heliconius erato demophoon , Heliconius erato hydara , Heliconius erato erato , Heliconius erato amalfreda , Heliconius erato notabilis , H. e. etylus , H. e. lativitta , Heliconius erato emma , H. e. favorinus , Heliconius erato phyllis , Heliconius erato venus , Heliconius erato cyrbia , Heliconius erato chestertonii  and H. himera .We used whole‐genome resequenced data of a total of 109 butterflies belonging to the e Figure  and S2. H. melpomene clade samples comprised fourteen colour pattern forms from sixteen localities , H. m. melpomene , H. m. melpomene , H. m. rosina , H. m. malleti , H. m. vulcanus , H. m. plesseni , H. m. aglaope , H. m. amaryllis , and H. m. nanna . Three populations were sampled from the H. timareta clade: H. heurippa , H. t. thelxinoe  and H. t. florencia . Three populations were sampled from the H. cydno clade: H. c. chioneus , H. c. cordula  and H. c. zelinde .The s Figure . Ten pop2.2H. erato and H. melpomene clade samples were aligned to the H. erato v1  and sorted using samtools  using EIGENSTRAT SmartPCA  analyses as implemented in MSMC  calculated as S) calculated as the weighted (w) average of the nucleotide diversity (πi) within each population l and k, asWe first estimated diversity within populations as well as divergence between parapatric and sympatric populations in nonoverlapping 50‐kb windows along the autosomes and Z chromosome using python scripts and egglib  was calculated as the average number of nucleotide differences per site between two DNA sequences in all possible pairs in the sampled population  in the ancestral populations  and positive values indicating a lack of rare alleles  . For the H. erato clade data set, this resulted in a high coverage data set with 322,082 and 15,382 sites on the autosomes and Z chromosome, respectively. For the H. melpomene clade data set, this resulted in 335,636 and 18,623 sites on the autosomes and Z chromosome, respectively. Pairwise dXY values between each individual were used to evaluate the relationship between absolute genetic divergence (dXY) and geographic distance using Mantel's tests  of 0.01 using the coalescent simulator msms  ranging from 0.01 to 100 and at a certain moment backwards in time (t). In a second set of simulations, we considered pairs of populations that were connected through migration (m) ranging from 0 to 1e‐6 and for which migration was reduced with a factor d on the Z chromosome. To compare changes in the nucleotide diversity on autosomes and the Z chromosome, we simulated the Z chromosome as a separate population for which the effective population size was set to three‐quarters that of the autosomal population.In a first set of simulations, we considered one population that underwent a single population size change of a magnitude (Ne), such as the autosomes and the Z chromosome, we expressed time in generations and migration rates as a proportion of the effective population size. Comparable to the Heliconius sampling, we sampled five individuals from each population and ran 300 replicates for each parameter combination. Pseudocode to run the msms command lines are provided in Tables D, nucleotide diversity (π) and dXY were calculated from the simulated sequences using python scripts and egglib   is characterized by population size increase until 200 to 300 KYA, followed by population size decrease and increase again about 40 KYA. Finally, H. heurippa has a similar demographic history as the other H. timareta species, but with a continuous decrease in population size since about 300 KYA.In the A Figure . In contH. melpomene and H. cydno has been estimated between 0.9 and 1.4 MYA ago (~3.6–5.6 million Heliconius generations) , suggesting that the differences observed between populations are largely driven by real demographic change rather than artefacts of hybridization.The PSMC estimates give inference up to about 1 MYA Figure . In cont3.2FST, dXY and da, between incipient species and population pairs of H. erato and H. melpomene , which is only dependent on ancestral diversity and not on diversity within each population. The effect size will depend on the timing and magnitude of the population size change. In our simulations, Z/A diversity ratios ranged from 0.40 to 0.86 under the most extreme simulated population size changes, compared to the expected diversity ratio of 0.75 under equilibrium expectations  diversity ratio will be larger than expected in populations that experienced a recent expansion and smaller than expected in those that experienced a recent contraction Figure b. Theref3.4Heliconius clades, we used the behaviour of Tajima's D as a way to assess likely population size changes within species. Tajima's D is a population genetic measure commonly used to detect whether a locus is evolving neutrally in a population  are correlated with increased nucleotide diversity, whereas positive Tajima's D values (population size decrease) are correlated with reduced nucleotide diversity  all had higher nucleotide diversity (π) values, as well as higher Z/A diversity ratios .To explore how population size changes might have affected Z/A diversity ratios and thus Z/A divergence comparisons within  Tajima, . At a gey Figure . As withp Figure , H. erats Figure . Z/A div Figures  and 7. TH. erato clade as compared to the H. melpomene clade is consistent with the generally greater abundance of H. erato observed in nature /dXY(A) ratios are above the 0.75 ratio that would be expected immediately after the populations split (0.91 ± 0.11) /dXY(A) in these comparisons /dXY(A) ratios among H. melpomene from east of the Andes are closer to the 0.75 /dXY(A) ratios are much lower, consistent with a population size decrease deeper in the ancestry of H. melpomene. Finally, the lower dXY(Z)/dXY(A) ratios in H. cydno–H. timareta comparisons relative to the H. melpomene–H. cydno and H. melpomene–H. timareta comparisons potentially suggest a population contraction of the ancestral population of H. cydno and H. timareta, but after they split from H. melpomene.While changes in population size can have strong effects on measures of sequence divergence, jointly considering patterns of variation on the Z chromosome and autosome can give further insights into the evolutionary history. Among ) Figure  and ther5 Figure . While cH. erato clade, nucleotide diversity as well as Z/A diversity ratios were distinctly higher in populations from east of the Andes and Panama and lower in the H. e. chestertonii and H. himera populations  by comparing population pairs with different levels of absolute migration due to physical isolation, but that otherwise share the same common history. This can be achieved by comparing pairs of populations from the same two species that differ in their extent of geographic isolation. Indeed, previous analyses of sympatric and allopatric populations of H. melpomene, H. cydno and H. timareta, based on shared derived alleles , found evidence of extensive gene flow between the species in sympatry, but with a strong reduction on the Z chromosome  and H. melpomene  populations from east of the Andes. As expected, the correlation between distance and absolute divergence is reduced by geographical barriers, such as when comparing H. erato  and H. melpomene  populations from Panama, east of the Andes and west of the Andes. We also observed a significant trend of increased absolute divergence with distance between populations of H. erato–H. e. chestertonii , H. melpomene–H. cydno  and H. melpomene–H. timareta . This is consistent with gene flow among these species pairs where they are in contact. In contrast, no significant trend between absolute divergence and distance was observed between H. erato–H. himera and H. cydno–H. timareta, suggesting that these species pairs may be more strongly isolated.Among s Figure . This trdXY(Z)/dXY(A)) ratios over short distances, with the ratio decreasing between pairs that are geographically more isolated /dXY(A) ratio increases, and this increase is most pronounced when overall migration rates are high. This relationship can be explained by the absolute difference in effective migration on the Z chromosome compared to the autosomes becoming smaller as overall migration decreases. While overall dXY(Z)/dXY(A) ratios may be influenced by ancestral population size changes, the trend should be independent from population size changes that occurred after the populations split. Our widespread sampling of both Heliconius clades therefore allowed us to test for reduced effective migration on the Z chromosome.Simulations show that if distance is considered a proxy for migration, reduced rates of admixture on the Z chromosome may become apparent as increased Z/A absolute divergence /dXY(A) ratios and its relationship to geographic distance. If rates of admixture between Heliconius populations are similar on the Z chromosome compared to the autosomes, we would not expect any relation between distance and dXY(Z)/dXY(A) ratios. In contrast, we observed increased dXY(Z)/dXY(A) ratios among geographically more closely located population pairs for H. melpomene–H. timareta  and H. melpomene–H. cydno  comparisons /dXY(A) ratios between H. erato–H. e. chestertonii was observed among the geographically closest comparisons, although this was not significant . Finding this trend, however, can be obscured by geographic barriers that would reduce the relation between distance and admixture. For instance, H. e. chestertonii comes into close contact with H. e. venus west of the Andes, but is geographically isolated from relatively closely located H. erato populations east of the Andes. Similarly, PCA of the H. melpomene populations indicates splits between populations east of the Andes, which may reflect additional geographic barriers that do not correlate linearly with distance , H. melpomene–H. cydno  and H. erato–H. e. chestertonii  comparisons, but not in H. timareta–H. cydno and H. erato–H. himera comparisons. This is consistent with crosses showing that the former and not the latter pairs experience hybrid sterility and Haldane's rule  on the W chromosome does contrast with these inferred demographic histories and provides support that the reduced diversity and increased FST measures result from selection /dXY(A) ratios with geographic distance provides opportunities for testing reduced admixture between sex chromosomes in systems for which tree‐based approaches and/or crossing experiments are unfeasible.The disproportionate role of sex chromosomes during speciation has been well documented based on genetic analysis. However, it is less clear how this influences patterns of divergence in natural populations. In Genome assemblies are available on lepbase.org. Sequencing reads are deposited in the Sequence Read Archive (SRA). See Tables S.V.B., M.B., B.A.C., C.D.J. and S.H.M. conceived of the study. S.V.B., M.B. and S.H.M. analyzed the data. S.V.B., M.B., W.O.M., B.A.C., C.D.J. and S.H.M. wrote the paper. R.P. and C.S. provided samples. All authors discussed the results and contributed to the final manuscript. Click here for additional data file.


A similar level of Hd and π was found in Sardinian and Cretan populations, both showing three haplotypes. The different haplotype frequencies and exclusivity detected were in accordance with the limited matrilineal gene flow (STF = 0.07097), probably related to the species reluctance to fly over sea masses. The genetic variability we observe today would therefore be the result of an evolutionary process strongly influenced by isolation leading to the appearance of island variants which deserve to be protected. Furthermore, since nesting sites and food availability are essential elements for colony settlement, we may infer that the island's colonization began when the first domestic animals were transferred by humans during the Neolithic. In conclusion, our research presents a first contribution to the genetic characterization of the griffon vulture populations in the Mediterranean islands of Sardinia, Crete and Cyprus and lays the foundation for conservation and restocking programs.The islands of Sardinia, Crete, and Cyprus are hosting the last native insular griffon populations in the Mediterranean basin. Their states have been evaluated from “vulnerable” to “critically endangered”. The sequence analysis of molecular markers, particularly the mtDNA D-loop region, provides useful information in studying the evolution of closely related taxa and the conservation of endangered species. Therefore, a study of D-loop region sequence was carried out to estimate the genetic diversity and phylogenetic relationship within and among these three populations. Among 84 griffon specimens , we detected four haplotypes including a novel haplotype (HPT-D) that was exclusively found in the Cretan population with a frequency of 6.1%. When considered as a unique population, haplotype diversity (Hd) and nucleotide diversity ( Gyps; G. fulvus) is a vulture species widely distributed from the Mediterranean Basin in the west to the Indian subcontinent in the east. The species breeding range extends over Europe to the Middle East and North Africa including some island populations in the Mediterranean i.e., Majorca, Sardinia, Crete, Cyprus, and the Croatian islets of Cres, Krk, Prvić, and Plavnik. The European population is currently estimated at 32,400-34,400 pairs with the bulk of it residing on the Iberian Peninsula .The European griffon , the rest of them would have had a very low level of exchange with continental griffon populations given their distance from mainland Europe. Because of this geographical isolation and difficulties to cross sea stretches, these griffon populations may have maintained distinctive genetic characteristics compared to the mainland colonies. Le Gouar et al.  investigps genus , 19, andps genus  which poThe aim of the present study was to assess the genetic diversity and phylogenetic relationship within and among native griffon vulture populations hosted on the Mediterranean islands of Sardinia, Crete, and Cyprus by analysing the D-loop region. The species in Crete numbers 250–300 egg-laying pairs or an estimated total population of ca. 1000 individuals and has been evaluated as “vulnerable” . In Cyprn = 44), Crete (n = 33) and Cyprus (n = 7) were collected between January 2016 and December 2018 , while operating in partnership with the Natural History Museum of the University of Crete (Greece) and the Ministry of the Interior of Cyprus (Cyprus). Overall, 84 blood and feather samples of living autochthonous individuals from Sardinia  designed by Johnson et al.  were usepulation .Genomic DNA isolation from blood samples and feathers was performed by using the NucleoSpin Blood kit (MACHEREY-NAGEL) and the NucleoSpin Tissue XS kit (MACHEREY-NAGEL), respectively. Sample quality and DNA concentration were determined via spectrophotometry using an ND-8000 . PCR amplification was conducted using the GeneAmp PCR System 9700 (Applied Biosystems) and the cycling profile was as follows: initial denaturation at 95°C for 5 min, then denaturation, primer annealing, and extension at 95°C for 50 s, 62°C for 50 s and 72°C for 1 min for 35 cycles, followed by a 4 min extension at 72°C. Samples were then cooled and held at 4°C until sequencing. Direct sequencing of the PCR products and processing of raw sequencing data were performed as previously reported in Mereu et al. .G. fulvus sequences (π) and haplotype frequency estimations were calculated as a single population on a 342 bp long fragment encompassing the whole HVS I domain of the mtDNA D-loop region. Calculations of the tests of Fu's Fs and Tajima's D along with the estimation of the coefficient of differentiation (STF) and hierarchical analyses of molecular variance (AMOVAs) were performed in ARLEQUIN 3.5.2 [G. bengalensis with a partial 773 bp long sequence from the D-loop (GB# KJ506786) which was aligned to the griffon sequences then trimmed to contain the same regions. The G. rueppellii, the closest extant species to G. fulvus, was used as additional outgroup. The ML tree was obtained by means of MEGA 7.0.14 [http://tree.bio.ed.ac.uk/software/figtree/). In addition, a haplotype network was inferred by means of the Integer Neighbour-Joining option (IntNJ) of PopART v1.7 (http://popart.otago.ac.nz/index.shtml).The data set encompassed overall 86 equences . All theIN 3.5.2 . The paiIN 3.5.2  with 100A 7.0.14 , which wA 7.0.14  via MarkA 7.0.14  and treeG. fulvus in-group were available, molecular dating was carried out assuming two calibration points (CPs) inferred by molecular analyses performed by Mereu et al. [G. fulvus species appearance (CP1) and the divergence between haplotypes (HPTs) A and B (CP2).Divergence time estimations were calculated using the Bayesian approach implemented in BEAST 1.7.5 . Since nu et al.  on the Dπ) and the average number of nucleotide differences (k) were 0.00176 and 0.606, respectively  already described  and a neectively .The Sardinian and Cretan populations showed a level of genetic diversity characterised by three haplotypes each one .π = 0.00197. The HPT B was found exclusively in sequences from Sardinian griffon individuals. The 33 Cretan sequences  were distributed among HPTs A (24), C (7) and D (2), the latter exclusively detected in Cretan specimens, with Hd = 0.436 and π = 0.00215. No differences among the 7 Cypriot sequences were found all of them showing the HPT A  distributed among HPTs A (28), B (11) and C (5), with Hd = 0.532 and he HPT A .The composition, frequency and geographic distribution of each haplotype is reported in Neutrality tests based on Fu's Fs and Tajima's D were positive and not significant, indicating a nonexpanding population  among the insular populations investigated  which were found exclusives of Sardinian and Cretan populations, respectively, followed by the distance between HPTs A and D (0.00765 ± 0.00427). The lowest divergence was detected when comparing HPT-A to HPTs B and C (0.00254 ± 0.00239) .G. rueppellii (RUE) homologous sequence was used as an outgroup, along with the G. bengalensis (BEN) sequence that was even used to root the phylogenetic tree. While resulting in similar clustering, ML bootstrap values (data not shown) offered weaker support than BI tree PP to the nodes identifying the main groups retrieved.The Bayesian tree analysis supported by high posterior probability (PP) value for each node (PP ≥ 93) is presented in G. fulvus populations analysed in the present study  of genetic differentiation among the three griffon populations investigated, according to a limited matrilineal gene flow probably caused by the species reluctance to fly over sea masses or cross long sea stretches and it is confirmed by the analysis on haplotypes frequencies and exclusivity . This evidence is consistent with Le Gouar et al. [Since no mtDNA sequences data from the mainland griffon populations are available for comparative analyses, we cannot infer whether the observed structuring comes from an adaptation to the island environment, successive waves of colonization, bottleneck/genetic drift events or from a mix of all these things. Based on nd Clark , we founr et al.  which der et al. .Climatic, geographical and anthropogenic factors should be invoked to explain the observed phylo-geographical pattern of griffon into Mediterranean islands. It is reasonable to suggest two dynamic ways of colonization from mainland griffon populations. The first way could be related to the exploration of new areas by juvenile vultures, which show very high natal dispersion and leave their breeding colonies during the first autumn of life , 32, 33.We could suppose that at an early stage pioneering individuals lacking social information were attracted by key environmental elements which are fundamental for a griffon colony such as optimal nesting sites and sufficient food availability , 35. GriFood availability represents another fundamental element to keep griffon colony into an island , 35. TheBased on our data, we could suppose that after the first colonization events the Cretan and Sardinian populations remained isolated for a long time during which they maintained genetic variability while the Cypriot colony underwent a drastic bottleneck which only the HPT A survived. However, further analyses including museum specimens should be performed to confirm such a hypothesis.G. fulvus native populations living in the Mediterranean islands. Maintenance of genetic diversity has to be a priority goal for genetic management strategies in long-term conservation programs of the present European griffon. Being places where unique variants not found elsewhere may have been preserved, the islands are often historical memories of past events of which there is no longer any evidence.The different expressiveness of the four mtDNA haplotypes among the three populations analysed in the present study could be explained by assuming that islands colonization occurred in different times. In such a context, each wave of new individuals could have replaced or contributed to enrich the pre-existing gene pool. Most probably, a combination of these two possibilities has determined the genetic variability currently observed in the Consequently, special attention should be devoted to both these populations, with the aim to facilitate the development and application of appropriate conservation measures. Theory predicts that geographically isolated populations could undergo a progressive reduction of genetic variation causing a decrease of fitness and limiting their ability to face off environmental changes . Gene fl


Podarcis wall lizards to test whether phenotypic diversity within and divergence across populations follow concordant patterns, and to examine how phenotypic variation responds to genetic and environmental variability across different hierarchical levels of biological organization, in an explicit geographic framework.Understanding how phenotypic variation scales from individuals, through populations, up to species, and how it relates to genetic and environmental factors, is essential for deciphering the evolutionary mechanisms that drive biodiversity. We used two species of We found a general concordance of phenotypic variation across hierarchical levels . However, we also found that within-population diversity does not exhibit a coherent geographic structure for most traits, while among-population divergence does, suggesting that different mechanisms may underlie the generation of diversity at these two levels. Furthermore, the association of phenotypic variation with genetic and environmental factors varied extensively between hierarchical levels and across traits, hampering the identification of simple rules to explain what yields diversity.Our results in some cases comply with general ecological and evolutionary predictions, but in others they are difficult to explain in the geographic framework used, suggesting that habitat characteristics and other regulatory mechanisms may have a more substantial contribution in shaping phenotypic diversity.The online version of this article (10.1186/s12862-018-1237-7) contains supplementary material, which is available to authorized users. Variation is the cornerstone of evolution. It is the raw material for the action of selection and a central component for identifying how phenotypic diversification occurs across all levels of biological organization . The uniunder the null hypothesis of random accumulation of variation. Of course, evolution, and particularly that of the phenotype, rarely occurs under stochastic processes alone. Instead, phenotypic traits are usually also correlated with the environment where organisms live, through effects on physiological, functional and ecological performance ),Podarcis to be informative  and ventral (VSN) scales, femoral pores (FPN), and supraciliary granules (SCGN) Fig. . These tot fixed ). All scP. bocagei) and Additional file P. vaucheri).Based on each of the above sets of traits, we quantified the level of diversity within each population using the mean distance to the population centroid . In addiP. bocagei [To characterize recent population dynamics, we genotyped all individuals examined for morphological analyses for nine microsatellite loci, initially developed for  bocagei . Based o bocagei  genetic  bocagei .In addition, we examined a 623 bp fragment of the NADH dehydrogenase subunit 4 (ND4) mtDNA gene to describe the phylogeographic structure and deeper evolutionary history of the populations under study. Based on these data, we described the genetic distance among populations using Dxy, the average number of nucleotide substitutions per site between populations ; and estA detailed account of laboratory and analytical procedures followed to obtain genetic data, and the corresponding descriptors of population diversity and differentiation, are provided in Additional file R < 0.7), and which are known to be biologically informative for the species under study [http://glam1.gsfc.nasa.gov/; at 250 m of resolution) for the presumed activity period of wall lizards during our sampling periods . Furthermore, to characterize topographic conditions of each locality we derived the slope variable from altitude , using the “slope” function of ArcGIS [To characterize the average climatic environment of the studied populations, and fluctuations to it, we clipped the 19 bioclimatic variables (at 30 arc-seconds of resolution) included in the Worldclim database  to our ser study , 63, incer study ). We obtf ArcGIS . Finallyf ArcGIS . FurtherTo investigate whether the structure of morphological variation was concordant across individuals and across populations of each species, we performed principal components analyses of each multivariate trait block separately , considering a) the matrix of individual observations and b) population means for each trait. We then permuted population-centered residuals of individual observations across populations to examine whether the first principal component of individual morphological variation aligned with that of population divergence, by testing whether the angle θ between these two PC vectors was larger than that expected at random.To visualize the spatial structure of morphological diversity within populations, we spatially interpolated within-population mean distance to centroid for each of the four explored trait sets for each species, using the kriging interpolation method in ArcGIS . A detaiTo evaluate which genetic and environmental factors may contribute in shaping levels of intrapopulation phenotypic diversity, we used Generalized Least Squares (GLS) to fit single-predictor linear models while taking the spatial structure of our sampling into account. Specifically, we estimated GLS regressions with population phenotypic diversity  for each of the morphological traits examined as the response; and each of the genetic diversity and environmental factors described above as predictors. We preferred this simple, pairwise approach, instead of fitting multiple-predictor GLS models, to obtain an overall view of which factors may influence intraspecific phenotypic diversity without confounding statistical interactions among predictors.The most adequate autocorrelation structure for each response-predictor combination was determined by fitting models using a linear, exponential, Gaussian, or rational quadratic structure, and then comparing their fit to the data using Akaike’s Information Criterion (AIC). The model with the lowest AIC for each combination was considered to optimize the expected covariance among observations due to spatial proximity. When GLS models suggested a significant contribution of a genetic or environmental predictor on the level of population diversity for a trait, we examined the strength of such an association by inspecting the Pearson correlation between response and predictor variables.Similar to what we did for intra-population diversity, we used kriging of inter-population phenotypic distances to explore the geographic structure of phenotypic divergence among populations Additional file 2:Results of Generalized Least Squares analysis performed to identify environmental and genetic variables that contribute significantly in explaining differences across populations in levels of intra-populational morphological diversity while taking geographic distance into account. (DOCX 73 kb)Additional file 3:Geographic coordinates in the WGS1984 system, number of individuals sampled per sex  and in total (Ntot), and abbreviation (CODE) for the examined populations. (DOCX 16 kb)Additional file 4:Geometric morphometric procedures for quantifying variation in dorsal head shape, provides a detailed description of all steps involved in obtaining head shape data, including landmark definitions and digitizing, superimposition of landmark coordinates and other pre-processing applied to GM data. (DOCX 13 kb)Additional file 5:P. bocagei, provides the data used for all analyses for this species, after mean-centering by sex, including biometric and scalation variables as described in Methods, and superimposed, sex-mean-centered landmark coordinates (X1 – Y24), as well as the population of origin in accordance with Additional file Sex-corrected individual values for all morphological traits for populations of Additional file 6:P. vaucheri, provides the data used for all analyses for this species, after mean-centering by sex, including biometric and scalation variables as described in Methods, and superimposed, sex-mean-centered landmark coordinates (X1 – Y24), as well as the population of origin in accordance with Additional file Sex-corrected individual values for all morphological traits for populations of Additional file 7:Genetic analyses, provides a detailed account of of laboratory and analytical procedures followed to obtain genetic data, and the corresponding descriptors of population diversity and differentiation. (DOCX 81 kb)Additional file 8:Individual multilocus genotypes for the nine microsatellite loci. Each allele is coded as its length, given in bp. Missing data is represented by “0”. (XLSX 66 kb)Additional file 9:Maxent models for quantifying environmental suitability, provides a description of methods and results related to ENM used to obtain habitat suitability maps for each species. (DOCX 727 kb)


Recent advances in genomics have enabled researchers to shed light on the evolutionary processes driving human adaptation, by revealing the genetic architectures underlying traits ranging from lactase persistence, to skin pigmentation, to hypoxic response, to arsenic tolerance. Complicating the identification of targets of positive selection in modern human populations is their complex demographic history, characterized by population bottlenecks and expansions, population structure, migration, and admixture. In particular, founder effects and recent strong population size reductions, such as those experienced by the indigenous peoples of the Americas, have severe impacts on genetic variation that can lead to the accumulation of large allele frequency differences between populations due to genetic drift rather than natural selection. While distinguishing the effects of demographic history from selection remains challenging, neglecting neutral processes can lead to the incorrect identification of candidate loci. We here review the recent population genomic insights into the genetic basis of arsenic tolerance in Andean populations, and utilize this example to highlight both the difficulties pertaining to the identification of local adaptations in strongly bottlenecked populations, as well as the importance of controlling for demographic history in selection scans. Arsenic, a naturally occurring element, is acutely toxic to humans. Although low levels of environmental exposure to arsenic are generally considered safe, epidemiological studies indicate that exposure to elevated levels represents an important global public health issue, impacting tens of millions of individuals annually, particularly in developing areas  via the highly toxic intermediate metabolite monomethylarsonic acid (MMA) Fig. , though Unlike these recent exposure events, Andean populations in South America have experienced a long-term exposure to arsenic. The Andeans are composed of several different groups including the Aymara, Atacameño, Quechua, and Colla, who have been living along the highlands and the Atacama Desert region  for thousands of years. Bio-archeological evidence suggests that pre-Columbian populations were likely suffering arsenic poisoning in the Atacama Desert at least 7000 years ago , GSTP1 (glutathione s-transferase pi 1), and PNP (purine nucleoside phosphorylase) have been found to alter metabolite profiles  thus represents a unique natural laboratory to study the genetic underpinnings of any potential adaptation associated with arsenic-rich environments.First considering bio-anthropological evidence, several studies have revealed severe arsenic concentrations in the inner organs, as well as visible skin lesions, in the remains of individuals from the ancient Chinchorro population—an Incan colony that was settled in the Camarones Valley  in these populations, there is an upside. There exist increasingly sophisticated approaches for inferring demographic histories  to these simulated replicates, one may quantify the performance of that statistic  under a population history actually relevant for the empirical application. With this information in hand, and if the results indeed suggest an ability to accurately detect selection for the population in question, genomic scan approaches may proceed. Relatedly, in the example of arsenic tolerance, given mixed evidence of selection on the candidate loci identified via association studies to date, as well as their unusual intermediate frequencies observed in both arsenic-exposed and nonexposed populations . As witAS3MT locus, with previous studies demonstrating its importance for arsenic methylation in rats (Lin et al. Thus, we propose that the way forward for the study of arsenic tolerance will involve more extensive demographic modeling, large-scale simulation studies to identify statistics that may be useful for identifying patterns associated with genetic hitchhiking/polygenic adaptation within the context of the inferred population history, and finally an examination of the correspondence between large-scale association studies and genomic scans in order to identify top candidate loci. With regards to the feasibility of functional studies to similarly validate candidates, it is noteworthy that tractable animal models indeed exist for the study of the


Asparagus cochinchinensis, a medicinally important perennial herb, is in decline due to overharvesting in Korea. Eight A. cochinchinensis populations in Korea and three populations from neighboring countries  were examined using nine nuclear microsatellite loci and three chloroplast microsatellite loci to characterize molecular diversity patterns. The average within-population diversity was limited likely due to long-term bottlenecks observed in all eight populations. High pairwise FST values indicated that the populations have largely diverged, but the divergences were not correlated with geographic distances. Clustering analyses revealed a highly complex spatial structure pattern associated with two ESUs. Approximate Bayesian Computation (ABC) suggests that the two ESUs split about 21,000 BP were independently introduced to Korea approximately 1,800 years ago, and admixed in secondary contact zones. The two ESUs found in our study may have different habitat preferences and growth conditions, implying that the two genetically divergent groups should be considered not only for conservation and management but also for breeding programs in agricultural areas.Range-wide population studies of wide spread species are often associated with complex diversity patterns resulting from genetically divergent evolutionary significant units (ESUs). The compound evolutionary history creating such a pattern of diversity can be inferred through molecular analyses.  The past and recent species range expansions with accompanied demographic changes may result in extreme patterns of population divergence3. For example, quaternary glacial and interglacial oscillations have had major impacts on the evolution of divergent lineages as ice sheets isolated populations in distant refugia4. Allopatric divergence might have further led to establishment of ESUs or cryptic species from a range of glacial refugia5. Populations that experience such dramatic evolutionary events often exhibit complex patterns of genetic diversity. However, caution is warranted because similar genetic patterns can also be drawn from gene flow, selection and random effects in combination with life-history characteristics such as reproduction and dispersal modes6.Population-level genetic studies for species across wide ranges often reveal that the species consist of multiple Evolutionary Significant Units (ESUs) or even harbor a cryptic speciesAsparagus L. , consists of ~120 species including edible crops: A. officinalis & A. albus, and ornamental and/or medicinal herbs: A. asparagoides, A. falcatus, A. setaceus, A. scandens9. Asparagus is an Old World genus with diverse life forms . It is distributed across the Mediterranean, semi-arid and arid environments9. Notably, some species in the genus are endangered, whereas several others pose great threats to biodiversity as invasive weeds10. However, the basic biology as well as the phylogenetic relationships of most species in the genus have remained uncertain, except for the few well-known species listed above11. The limited knowledge on most species in the genus raises great challenges in at least two important aspects: (1) finding proper target species to improve the important crop, garden asparagus 12 and (2) management of notorious invaders  causing biodiversity losses10. Empirical studies of ecological characteristics and evolutionary relationships will provide valuable insights for biodiversity management and enhancement of crop production.One of the most commercially important genera, Asparagus cochinchinensis (Lour.) Merr. is a perennial herb that is widely distributed from temperate  to tropical Asia (Laos and Vietnam)13. This species is one of the closest relatives to the commercially important crop, garden asparagus. The tuberous roots of the plants have been widely used as medicine in many oriental countries16. Although global population trend information is completely lacking for the species16, Korean populations are thought to be in decline due to over-harvesting14. In China, A. cochinchinensis widely occur in thinly forested slopes, roadsides and waste fields13, whereas the species is mainly found near seashores in Korea and Japan17. A recent study revealed that the plants mostly occur along the west coast but not along the east coast in Korea14. The large difference in habitat characteristics and distribution patterns between China and Korea/Japan suggests the possibility of multiple ESUs accompanied by a series of demographic changes for A. cochinchinensis in Korea18.20. Coupled with the traditional population genetics approach, ABC method provides more sophisticated reconstructions of the evolutionary history for a target species.By understanding the distribution of genetic diversity, history of demographic changes, i.e., the introductions, genetic bottlenecks and admixtures can be inferred. An advanced model-based approach, Approximate Bayesian Computation (ABC), offers a powerful tool to investigate genetic data. ABC method is equipped to compare multiple evolutionary hypotheses and estimate the parameters of hypothetical scenarios with uncertainty incorporatedAsparagus cochinchinensis in Korea. Our specific aims are to (1) investigate the overall spatial genetic diversity pattern, (2) identify ESUs and/or cryptic species, and (3) determine the most probable evolutionary scenario that characterized the current diversity pattern of A. cochinchinensis populations in Korea. To address these goals properly, we examined genetic diversity patterns over a large geographic scale, including most Korean populations and samples from neighboring countries that might have influence the genetic diversity patterns. The ABC paradigm was applied to test multiple likely hypotheses of the evolutionary history. Population divergences are expected to be low considering the reproductive mode and dispersal capability of the species. However, populations might have been much more differentiated than expected if there are multiple ESUs present. Given the wide range of geographic distribution and the breadth of the habitats, we hypothesize that the distribution of genetic variation will exhibit rather complex patterns, and the complexity will be much higher with the presence of multiple ESUs associated with cryptic diversity. Alternatively, the genetic affinities among populations might reflect environmental and/or geographical differences. However, the spatial and environmental patterns of genetic diversity might not be prominent if there was a series of historical demographic events.Here, we employ population genetics tools as well as ABC methods to unravel the complex evolutionary history of et al. 2017 for detailed information about the marker loci)21 appeared to have null alleles present in all eight populations while null alleles were present in only two populations for the other locus (AC079). A significant departure from Hardy-Weinberg Equilibrium (HWE) was shown in locus 3  and locus 8 . We found that two loci  were linked with the remaining 11 loci in most populations . Because some genetic analyses such as STRUCTURE and DIYABC depend on strong assumptions of marker independence and HWE, we purged four markers  that violated the assumptions from all downstream analyses.Genetically identical clones were very rare in all eight populations (150 genets in 158 samples). We found no scoring error in nuclear microsatellite profiles during the genotyping procedure. Null alleles were present for two loci with low (0.1) to moderate (0.35) frequencies. One of the two loci  and OKN (FIS = 0.31 ± 0.20). We do not present FIS values here because most values were not significantly different from zero. Pairwise FST values were all statistically significant and differed greatly among population pairs; the lowest was 0.097 for the YGW/TPE pair (see Table ST) and geographic distance , observed heterozygosity (Ho) and number of alleles per population (Na) averaged over individuals varied among the populations Table . He rangPT values between population pairs within Korea were low (less than 0.1), whereas those estimated for population pairs between Korean populations and the Chinese population (GND) were much higher  revealed that the populations were genetically more diverged within groups (FSC = 0.25) than across groups  which diverged around LGM and that later each ESU migrated to neighboring regions. Secondly, with the migration , the two ESUs were admixed perhaps through secondary contacts independently in Korea (WND) and Japan (OKN).Population divergence is a strong driving force for species range shift and lineage divergence. 22. A. cochinchinensis is an outcrossing dioecious plant that can clonally propagate through tuberous roots23. Thus, all four mechanisms are likely causes of heterozygote excess. Unfortunately, with the current data set, we cannot explicitly determine the underlying mechanism of the heterozygote excess for the locus. Future study may further examine factors leading to heterozygote excess for certain loci using both molecular tools and breeding experiments with a variety of sample sizes, as in Stoeckel et al.22.We found significant departures from HWE in two loci. Heterozygote deficiency observed in one of the two loci likely derived from the presence of null alleles. In contrast, the other locus that deviated from HWE showed an excess of heterozygotes (average observed heterozygosity = 0.75), yet what caused the heterozygote excess is not clear. A few explanations can be considered: small sexual and self-incompatible populations, over dominance, negative assortative mating and asexual reproductionA. cochinchinensis , selection, and demographic changes) influence the level of genetic diversity26. Since outcrossing plants often show higher within-population genetic diversity25, the lowered genetic variation in our results is rather surprising. Inbreeding and clonal reproduction may negatively influence the level of genetic diversity depending on the dispersal and pollination modes, even in dioecious species28. However, clonal assignment results failed to find significant contribution of clonal reproduction in A. cochinchinensis collected for the study. We only found a significant inbreeding rate in one population, HAM (FIS = 0.34). However, the genetic diversity in HAM was not the lowest among the eight populations 24. In general, outcrossing plants show lower population divergence (e.g. average FST = 0.22 for 71 outcrossing spp.)24, yet A. cochinchinensis populations exhibited high divergence for an outcrossing species. Coupled with outcrossing nature, long distance dispersal is also likely to alleviate population divergence. Although there is a complete lack of numerical data examined for A. cochinchinensis thus far, in several congeneric species the most common dispersal vector, birds, can transport the seeds up to 12 km31. Long distance migration by avian vectors facilitates gene flow among local populations, which genetically homogenizes populations. Despite the dispersal capability and the outcrossing nature of A. cochinchinensis, we found inflated population divergence, somewhat opposite to what was expected. The migration rates estimated among population pairs also supported the limited gene flow for population pairs. For example, the population pair with the largest FST  had very low migration rate . To investigate more closely whether the limited gene flow is associated with geographic distance, we performed a correlation test between genetic divergence and geographic distance (Mantel test).The average pairwise FST values (e.g. FST between HAM and YGW = 0.5) compared to those from population pairs across different nations , i.e., no mutation, no selection and approximately equal population sizes32. To rule out the alternative hypotheses, we examined the spatial population structure among the eight populations through assignment analyses and competed plausible evolutionary hypotheses utilizing the ABC approach.AMOVA results from both maternally inherited cpSSRs and nrSSRs were also rather complex for an outcrossing plant, where most of genetic and haplotype variance derived from the group level and, not from the individual level. The complex patterns of genetic variation and population divergence are consistent with the hypothesis of multiple ESUs. As explained by the hypothesis, past and recent species range expansions of divergent lineages accompanied with demographic changes is likely the causal mechanism of the unusual patterns of population divergence. One can argue that the divergence results are due to stochasticity and/or unrealistic assumptions built into the indirect measure of gene flow  revealed a complex pattern of population structure that does not reflect geographical distances among populations Figs  and 3. Dion Figs  and 3. Oion Figs  and 3. Bion Figs . For exaion Figs ; Fig. S233. The clustering pattern observed in our nrSSR data indicated that A. cochinchinensis in Korean populations likely consists of at least two ESUs with admixture between the two clusters, possibly in secondary contact zones. The clustering results are also congruent with the hypothesis proposed in previous studies based on the distribution and ecological characteristics of the relevant habitats34. In addition, the weak correlation between geographic distance and population divergence from the Mantel test  differed notably from the pattern found in the nrSSR data. Compared to biparental nuclear markers, maternally transmitted chloroplast markers often show greater population divergence with a high level of variance (near zero to complete fixation) due to various factors, including a seed-only migration mode, the absence of meiotic recombination and increased sensitivity to stochasticity36. Moreover, the pattern might result from sampling bias and/or from the extremely limited variation; thus, extreme caution should be taken when interpreting the pattern. Of 15 haplotypes, most were identified by rare alleles and were in very low frequencies. A single haplotype (12) predominantly occurred in most Korean populations, while the Chinese population (GND) was solely assigned to another single haplotype (7). The Taiwanese population (TPE) and one Korean population (NAH) showed the largest haplotype diversity and shared similar haplotypes  demonstrated that lineage separation between two ancestral populations  dated back to LGM . This is a crude estimation to some extent, but it is consistent with the major changes in the distribution of the vegetation during LGM throughout east Asia38. The ABC results also ruled out the stochasticity hypothesis, as our data did not support the scenario with independent divergence from an ancestor with subsequent founding events  and TPE  were independently introduced to the two clusters BOR & HAM (Pop2) and NAH & YGW  largely differ from the top two scenarios . Leaf samples were stored in plastic bags with silica-gel desiccant until DNA extraction. Genomic DNA from dried leaves was isolated using the DNeasy Plant Mini Kit  following the manufacturer’s protocol. We measured the quantity and quality of extracted DNAs in a NanoDrop ND1000  and visualized in 1% agarose-gel electrophoresis. The isolated DNAs then were stored at −20 °C until further use.In the springs of both 2015 and 2016, we collected young leaves of 158 individuals from eight populations (~20 samples from each population) from Korea, China, Japan and Taiwan , 2 μL of 2.5 mM dNTPs, 0.01 μM of each of the forward and reverse primers, 0.1 μL of Ex Taq DNA polymerase (5 units/μL) (TaKaRa Bio), 5–10 ng of template DNA, and distilled water . The PCR cycling conditions were as follows: an initial denaturation step at 98 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 55–57 °C for 1 min, and an extension at 72 °C for 1.5 min, with a final extension step at 72 °C for 10 min. The fluorescently labelled  PCR products were pooled with the size standard GS500LIZ  and the amplified fragments were separated out in an ABI 3730XL automated sequencer . Microsatellite profiles were examined on GeneMarker program v. 2.40 (Softgenetics LLC) with automated scoring. The scoring results were manually checked in the final step. We evaluated presence of null alleles and possible scoring errors using FreeNA43. Additionally, we amplified chloroplast microsatellites (cpSSR) for 50 individual genotypes subsampled from the total of 158 samples genotyped by nuclear microsatellites using three universal primer pairs44 and three primer pairs developed for the allied genus Maianthemum in the family, Asparagaceae45. Of the six primer pairs, three were successfully amplified and polymorphic at the population level. We assayed genetic variation with the three cpSSRs for all 158 genotypes and used the variation for further analyses.PCR amplification for 13 microsatellite markers (nrSSR) was conducted to genotype all 158 individuals using primer sets developed by Kim 46. To discriminate true clones from clones produced through sexual reproduction by chance, the probability of identical MLG due to sexual reproduction by chance was calculated using the binomial probability function Psex47. Because there were only a few identical clones, we did not account for clonality in the subsequent analyses. We estimated the genetic diversity parameters; He, Ho, Na and FIS using Arlequin v. 3.5 and GENALEX v. 6.50249. We tested for significant deviation from HWE and LD in 13 microsatellite loci within each population using Fisher’s exact test50 in Arlequin. Bonferroni corrections for multiple comparisons  were applied. Four of thirteen microsatellite markers significantly deviated from HWE and/or were not independent of the remaining markers. Certain genetic analyses, such as STRUCTURE and ABC assume HWE and/or independence among markers. To avoid bias influenced by the number of markers used between different analyses, we screened out those 4 markers from all downstream analyses. We calculated pairwise FST between all population pairs in Arlequin with 1,000 permutations for the significant alpha. We tested for Isolation by Distance based on the pairwise genetic distance (Slatkin’s linearized FST = FST/(1 − FST)) and log-transformed Euclidean distance for all population pairs in GENALEX51. For statistical significance of the correlation coefficient (r), 1,000 random permutations with replacement were used.Given the possibility of clonal propagation, we first tested for clonality to avoid presence of multiple clones within each population. All 158 samples were assigned to genets based on the 13 nrSSRs in GenoDive v. 2.0b23PT), and the analogue of GST for haplotype data  in GENALEX49. We used 1,000 permutations with replacements for the significance tests. The hierarchical distribution of haplotype variation among four regional groups  was evaluated by analysis of molecular variance (AMOVA) implemented in Arlequin48. Given the limited variation found in our haplotype analysis, we examined genetic structures only by plotting the distributions of haplotypes using a median-joining network in Network 5 (http://www.fluxus-engineering. com). Alleles were recoded as size of fragments, where 1-bp size change was given a weight of 152.Haplotype diversity of the cpSSRs was assessed as the total number of haplotypes (Nh), haplotype diversity (Hehap) and pairwise genetic differentiation among populations  on the pairwise Nei’s genetic distance estimated for all 158 individuals using GENALEX. AMOVA was used to partition molecular variance hierarchically within and between clusters defined from both STRUCTURE and PCoA in Arlequin. We compared the estimated values against values calculated from 1,000 resampled data sets for statistical significance.Population structures was examined using a Bayesian model-based clustering approach with the correlated allele frequencies model60, respectively. Because bottleneck events are assumed to reduce the allele number more rapidly than the allele size ranges, the M-ratio test uses the ratio between the number of alleles and the allele size range60. BOTTLENECK detects recent population decline within a few generations by finding a significant excess or deficit of heterozygosity relative to an equilibrium state61. A simulation study revealed that the M-ratio is better suited for identifying a historical bottleneck than BOTTLENECK29. We ran BOTTLENECK under the infinite allele model (IAM) and stepwise mutation model (SMM) with Sign and Wilcoxon’s sign rank tests for statistical significance. We also estimated the proportion of migrants among the eight populations within the recent past (the last few generations) using BAYESASS 3.0.162. We used 10,000,000 MCMC iterations with a 1,000,000 burn-in and, then sampled every 2,000 generations during the process. The default settings were used for the mixing parameters.We examined reduction in population size derived by historical and recent bottlenecks using the Garza-Williamson index  implemented in Arlequin and BOTTLENECK v. 1.2.02A. cochinchinensis in Korea was determined through the ABC model approach implemented in DIYABC v. 2.1.063. For the ABC computation, we used all 12 microsatellite loci by combining nine nrSSRs and three cpSSRs. Using both cytoplasmic and nuclear molecular data would allow more insights into admixture among genetically distinct lineages. We generated nine likely evolutionary scenarios that summarize the divergence and admixture history among the five Korean populations and the three populations of neighboring countries. .The most probable evolutionary history of es. Fig. . We form65, we scaled the mutation rate of the cpSSRs to be low  for the species might have dated back to as early as 21,000 years using a generation time of three years (see Vivian-Smith & Gosper10 for the generation time). To retain a clear signature for the posterior distribution of each parameter, we repeated the DIYABC runs five times with different mutation models and parameter sets, particularly for divergence times (t1 and t2) with the finalized nine scenarios. A simulation of each scenario was performed for 3 × 106 iterations based on neutral coalescence.For the nine nrSSRs, we used the default priors of the mutation rate, whereas the prior values of the mutation rate for the three cpSSRs were changed to achieve better posterior distributions Table . Given mow Table . We usedrs Table . As LGM 63. We determined the demographic parameters of the most probable scenario from 10,000 simulated datasets closest to the observed data. We assessed the goodness-of-fit for each model by the Principal Component Analysis (PCA) approach implemented in DIYABC using by model checking function. The function evaluates the discrepancies between the simulated and the observed data. To determine the level of confidence of the chosen scenario, we estimated Type I and Type II errors using the confidence option in DIYABC with a simulation of 500 datasets. Type I error for scenario 7, the scenario of choice, was estimated as the probability at which scenario 7 is rejected when it is the true scenario. Type II error was calculated as the probability of choosing a scenario when it is not the true scenario.We determined the most probable scenario based on posterior supports of each scenario estimated by two approaches: (1) direct estimates by the frequency of a given scenario with the 500 data sets generating summary statistics that most closely matched the observed data and (2) logistic regressionSupplementary information


From around 4,000 to 2,000 BC the forest-steppe north-western Pontic region was occupied by people who shared a nomadic lifestyle, pastoral economy and barrow burial rituals. It has been shown that these groups, especially those associated with the Yamnaya culture, played an important role in shaping the gene pool of Bronze Age Europeans, which extends into present-day patterns of genetic variation in Europe. Although the genetic impact of these migrations from the forest-steppe Pontic region into central Europe have previously been addressed in several studies, the contribution of mitochondrial lineages to the people associated with the Corded Ware culture in the eastern part of the North European Plain remains contentious. In this study, we present mitochondrial genomes from 23 Late Eneolithic and Bronze Age individuals, including representatives of the north-western Pontic region and the Corded Ware culture from the eastern part of the North European Plain. We identified, for the first time in ancient populations, the rare mitochondrial haplogroup X4 in two Bronze Age Catacomb culture-associated individuals. Genetic similarity analyses show close maternal genetic affinities between populations associated with both eastern and Baltic Corded Ware culture, and the Yamnaya horizon, in contrast to larger genetic differentiation between populations associated with western Corded Ware culture and the Yamnaya horizon. This indicates that females with steppe ancestry contributed to the formation of populations associated with the eastern Corded Ware culture while more local people, likely of Neolithic farmer ancestry, contributed to the formation of populations associated with western Corded Ware culture. During this time, the region was occupied by forest-steppe populations attributed to the Eneolithic (3350–3200 BC)1 and the succeeding Bronze Age groups associated with the Yamnaya - Pit Grave 2, the Catacomb , the Babyno  and the Noua  cultures4. Although there were cultural differences between these populations, they all shared a similar nomadic lifestyle, pastoral economy and barrow burial rituals5. Some of the rounded burial mounds founded by Eneolithic people were reused by the succeeding cultural entities of the Early Bronze Age1, while other kurgans shared a mix of characteristics from both the Late Eneolithic and the Early Bronze Age funeral rites4.The forest-steppe north-western Pontic region of the middle Dniester and Prut interfluve was a place of contact and exchange routes between human populations inhabiting the drainages of the Black and Baltic Seas from around 4,000 to 2,000 BC9, the Yamnaya culture originated in the Volga-Ural interfluve and spread across the Pontic-Caspian steppe between 3,300–2,800 BC. This cultural expansion led to the development of a less homogenous group of cultural entities belonging to the so-called Yamnaya Cultural-Historical Area/Unity11, hereafter reffered to as ‘the Yamnaya horizon’12. People associated with the eastern Yamnaya culture spread across the steppe to the east of Don River. With no settlements identified in this area, they were thought to be more mobile because of their supposed nomadic profile of economy stimulated by environmental conditions of Kuban – North Caspian steppes13. On the other hand, Yamnaya settlements were found more frequently in the forest-steppe Pontic regions, to the west of Don River, probably due to favorable environmental conditions12.According to some researchers14. Archaeological records point to some similarities between the Corded Ware culture and the steppe, including shared practices such as the barrow structures and burial rituals2. Adoption of a herding economy based on mobility through the use of wagons and horses, was also proposed as a common trait associated with both the Yamnaya and Corded Ware cultures12. These observations led some researchers to suggest a possible Yamnaya migration toward the Baltic drainage basin15 or a massive westward expansion of the steppe pastoralist people, representing the “barrow culture”, into the North European Plain16. However, specific burial customs of the Yamnaya people, such as the scarcity of grave goods, the presence of ochre, and the building of specific wooden roof or floor structures, generated opposing arguments emphasizing the significant differences between the Corded Ware and steppe cultures2.One of the most widely debated issues, which emerged in connection to studies on the Yamnaya horizon, was the relationship between the people associated with the Yamnaya and the Central European final Neolithic cultures, in particular the Corded Ware culture (dated to 2800–2300 BC)18. Moreover, people associated with the Yamnaya horizon have been shown to be an admixed population with ancestry from Eastern hunter-gatherers and Caucasus hunter-gatherers20. Ancient DNA data indicate that the Neolithic populations from Central Europe already had the ‘Caucasus’ genetic component from the eastern steppes around 2,500 BC. Presence of this genetic component was used as an argument for the expansion of people from the Pontic-Caspian region into the central Europe17. X chromosome sequence data suggest that it was primarily males who participated in these migrations22 and contributed to the formation of the people associated with the Corded Ware culture17. Based on the X chromosome data obtained mainly from western Corded Ware-associated individuals, it was estimated that, for every female, ~4–15 males migrated from the steppe21. Subsequently, the Yamnaya genetic component spread across Bronze Age Europe and West Asia14.Recent ancient DNA (aDNA) studies suggest that the large-scale migration of steppe populations associated with the Yamnaya horizon contributed to the formation of the final Neolithic central European populations24, the contribution of mitochondrial lineages associated with the Yamnaya horizon to the formation of people associated with the Corded Ware culture from the eastern part of the North European Plain, especially from the region of modern Poland, remains contentious. To investigate the maternal relationship between these two groups, we generated complete mitochondrial genomes from the representatives of Late Eneolithic and Early Bronze Age populations from the north-western Pontic region, including Yamnaya groups and individuals associated with the Corded Ware culture from the eastern part of the North European Plain.Although questions concerning the migrations of nomadic people have been addressed by a number of studiesAncient DNA was extracted from the Corded Ware culture individuals excavated in south-eastern Poland N = 12) and Moravia (N = 3). Late Eneolithic (N = 5) and Bronze Age human remains (N = 25) originated from western Ukraine and came from the Yampil barrow cemetery complex located in the north–western region of the Black Sea. Bronze Age individuals were associated with different archaeological cultures, including Yamnaya (N = 14), Catacomb (N = 2), Babyno (N = 4) and Noua (N = 5). The sampling localities are shown in Fig. 2 and Mor25. One extraction and one PCR blank each were set up as negative controls during amplification of the DNA libraries. The libraries were sequenced on Illumina HiSeq2500  or Illumina HiSeq X Ten  at the SNP & SEQ technology platform in Uppsala, Sweden.Ancient DNA was extracted from teeth or petrous bones in a laboratory dedicated to aDNA analyses at the Department of Human Evolutionary Biology, Adam Mickiewicz University in Poznan (AMU), Poland. Molecular methods used for aDNA extraction and construction of Illumina sequencing genome libraries have been previously described26 with minor modifications (see Supplementary Information Text for details). Two rounds of mtDNA enrichment were carried out on 12 libraries that yielded low levels of endogenous mtDNA reads in the initial Illumina shotgun screening  enrichment was prepared from two present day individuals of known haplotypes following(SNIC) through the Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX)27. Illumina sequencing data were processed using a custom analytical pipeline28.Preliminary pipeline computation was undertaken using resources provided by the Swedish National Infrastructure for Computing 29. BWA software package version 0.7.830 was used to map merged reads as single-end reads against the revised Cambridge Reference Sequence (rCRS)32 (GenBank: NC_012920). The ratio of reads mapping to Y and X chromosomes (Ry)  was calculated to assign molecular sex to the individuals sequenced on the Illumina platform33.First, we trimmed residual removed adapters and merged read pairs according tohttp://hannonlab.cshl.edu/fastx_toolkit/) was used to demultiplex sequences generated by the PGM Ion Torrent. Cutadapt v.1.8.134 was then used to remove long (−M 110), short (−m 35), and low-quality sequences (−q 20). The filtered reads were analyzed with FastQC v 0.11.335 using the options described previously36. The sequences were mapped against the rCRS using TMAP v3.4.137. To collapse duplicate sequence reads with identical start and end coordinates (for both PGM and Illumina sequence data) we used FilterUniqueSAMCons.py38. Misincorporation patterns were assessed using mapDamage v2.0.539. For each individual, contamination levels were estimated with the use of schmutzi40 as previously in36. Consensus sequences were built using ANGSD v0.91041. We accepted only reads with a minimum mapping score of 30, a minimum base quality of 20, and a minimum coverage of 3, as in36. Where necessary, comparative published mt genomes were reconstructed from the bam files with the use of the same methods as described above. Mitochondrial haplogroups (mtDNA hgs) were assigned for each individual with the use of HAPLOFIND42, the PhyloTree phylogenetic tree build 1743 and Mitomaster44.FASTX-Toolkit (www.ebi.ac.uk/ena) and NCBI GenBank (www.ncbi.nlm.nih.gov) web databases. All comparative populations used for principal component analysis (PCA), t-Distributed Stochastic Neighbor Embedding (t-SNE), pairwise genetic distances (FST), and AMOVA analysis are described in detail in Supplementary Tables 46. Because of potential maternal kinship between two Catacomb culture-associated individuals from this study, only one of them was used in the PCA and in a comparative Catacomb group. The eastern Yamnaya horizon group consisted of Yamnaya samples from the Samara region in Russia46. The map with archaeological sites from which the studied individuals originated, was generated using QGIS 2.12.247 . All k-means variants can be found in the Supplementary Material Text and Supplementary Fig. We have used a centroid-based clustering approach to examine the PCA results and search for logical clusters within our data. We applied the k-means method (as implemented in Scikit-learn v. 0.18.1 Python package)50 as implemented in Scikit-learn (18.1) Python package.To further explore the relatedness of populations using the mtDNA hg frequencies, we ran the t-Distributed Stochastic Neighbor Embedding (t-SNE) analysisFST values were computed in Arlequin 3.551. In total, 18 populations were used in FST and following AMOVA analyses, consisting only of ancient individuals with complete mtDNA genomes  analysis with the use of Python Scikit-learn 0.18.1 package48.53 to assess the population differentation using complete mt genomes. We ran AMOVA using the Arlequin 3.5 software package (for details see Supplementary Information Text).We have used the traditional method of analysis of molecular variance (AMOVA)Mitochondrial genome sequences were deposited in GenBank under accession numbers MH176332, MH176333, MH17635 - MH176355.Out of the 45 analyzed samples, we successfully obtained 23 mtDNA genomes, belonging to individuals associated with the Corded Ware culture N = 11), and with the Late Eneolithic (N = 3) and Bronze Age (N = 9) from western Pontic region . Individuals associated with the Eneolithic and Yamnaya cultures were assigned to hgs U2e1a1, U5a2b, H2a1 and U5a1i1, U4c1, W3a1, W3a1a, respectively Table . Other Bk value of 5 as the best representation of the data, at the average silhouette of 0.2608)  Figs . Based oThe k-means clustering on t-SNE results was consistent with the PCA results, although the approach to dimension reduction of the t-SNE algorithm is completely different than that of the PCA. Scatterplot of populations colored according to the k-means k = 7 (average silhouette 0.5158) Fig.  represenFST54 values   and Baltic Corded Ware culture  , than to populations associated with the western Corded Ware culture (CWW) . Western and eastern Yamnaya horizon groups also showed close genetic affinity to the Iron Age western Scythians (SCU) . The most distant populations to the Yamnaya horizon groups were western hunter-gatherers (HGW)  and people associated with Linear Pottery culture (LBK) , between eastern hunter-gatherers (HGE) and Baltic hunter-gatherers  , and between western (HGW) and eastern hunter-gatherers (HGE)   Fig. . The Yam05) Fig. .We investigated the within- and between-group variability using an AMOVA analysis. Concentrating on the eastern and western Corded Ware groups, we found the best variability distribution when the individuals associated with the western Corded Ware culture  shared close maternal genetic affinity with individuals associated with the Yamnaya horizon while the genetic differentiation between individuals associated with the western Corded Ware culture (from present-day Germany) and the Yamnaya horizon was more extensive. This decreasing cline of steppe related ancestry from east to west likely reflect the direction of the steppe migration. It also indicates that more people with steppe-related ancestry, likely both females and males, contributed to the formation of the population associated with the eastern Corded Ware culture. Similarly, closer genetic affinity to populations associated with Yamnaya horizon can be observed in Baltic Corded Ware groups, which confirms earlier indications of a direct migrations from the steppe not only to the west but also to the north, into the eastern Baltic region57. Moreover, we have reported mtDNA haplotypes that might be associated with the migration from the steppe and point to genetic continuity in the north Pontic region from Bronze Age until the Iron Age. These haplotypes were assigned to hgs U5, U4, U2 and W3. MtDNA hgs U5a and U4, identified in this study among Yamnaya, Late Eneolithic and Corded Ware culture-associated individuals, have previously been found in high frequencies among northern and eastern hunter-gatherers59. Moreover, they appeared in the north Pontic region in populations associated with Mesolithic (hg U5a)45, Eneolithic (Post-Stog) (hg U4)24, Yamnaya 24, Catacomb (hgs U5 and U5a)24 and Iron Age Scythians (hg U5a)60, suggesting genetic continuity of these particular mtDNA lineages in the Pontic region from, at least, the Bronze Age. Hgs U5a and U4-carrying populations were also present in the eastern steppe, along with individuals from the Yamnaya culture from Samara region17, the Srubnaya23 and the Andronovo from Russia14. Interestingly, hg U4c1 found in the Yamnaya individual (poz224) has so-far been found only in two Bell Beaker- associated individuals61 and one Late Bronze Age individual from Armenia14, which might suggest a steppe origin for hg U4c1. A steppe origin can possibly also be assigned to hg U4a2f, found in one individual (poz282) but not reported in any other ancient populations to date, and to U5a1- the ancestral lineage of U5a1b, reported for individual poz232, which was identified not only in Corded Ware culture-associated population from central and eastern Europe61 but also in representatives of Catacomb culture from the north Pontic region24, Yamnaya from Bulgaria and Russia46, Srubnaya23 and Andronovo62-associated groups. Hg U2e, reported for Late Eneolithic individual (poz090), was also identified in western Corded Ware culture-associated individual23 and in succeeding Sintashta14, Potapovka and Andronovo23 groups, suggesting possible genetic continuity of U2e1 in the western part of the north Pontic region.Among the analyzed samples, we identified two Catacomb culture-associated individuals (poz220 and poz221) belonging to hg X4. They are the first ancient individuals assigned to this particular lineage. Haplogroup X4 is rare among present day populations and has been found only in one individual each from Central Europe, Balkans, Anatolia and Armenia17 and later in Únětice and Bell Beaker groups from Germany63, supporting the idea of an eastern European steppe origin of these haplotypes and their contribution to the Yamnaya migration toward the central Europe. The W3a1 lineage was not identified in Neolithic times and, thus, we assume that it appeared in the steppe region for the first time during the Bronze Age. Notably, hgs W1 and W5, which predate the Bronze Age in Europe, were found only in individuals associated with the early Neolithic farmers from Starčevo in Hungary (hg W5)64, early Neolithic farmers from Anatolia (hg W1-T119C)23, and from the Schöningen group (hg W1c)61 and Globular Amphora culture from Poland (hg W5)45.Hgs W3a1 and W3a1a, found in two Yamnaya individuals from this study (poz208 and poz222), were also identified in Yamnaya-associated individuals from the Russia Samara regionThis study is the first to present mitochondrial genome data from the population associated with Corded Ware culture from the south-eastern part of present-day Poland. As this area is geographically close to the steppe region, it provides us with a better picture of the early steppe migration between 3,000 and 2,500 BC. Although our results indicate a contribution of females as well as males to the formation of populations associated with eastern Corded Ware culture, more detailed studies of X chromosome data are needed to clearly resolve female and male migrations, especially between the western Pontic steppe and the eastern part of the North European Plain.Ancient mitochondrial genome data from the western Pontic region and, for the first time, from the south-eastern part of present day Poland, show close genetic affinities between populations associated with the eastern Corded Ware culture and the Yamnaya horizon. This indicates that females had also participated in the migration from the steppe. Furthermore, greater mtDNA differentiation between populations associated with the western Corded Ware culture and the Yamnaya horizon points to an increased contribution of individuals with a maternal Neolithic farmer ancestry with increasing geographic distance from the steppe region, forming the population associated with the western Corded Ware culture. Among the analyzed samples, we identified, for the first time in ancient populations, two Catacomb culture-associated individuals belonging to the now-rare mtDNA hg X4.Supplementary Tables S1-S5Supplementary Information Text


Anolis ortonii and Anolis punctatus, which expanded from Amazonia, presently dominated by warm and wet settings, into the cooler and less rainy Atlantic Forest. To examine whether local adaptation has been constrained by population structure and history, we estimated effective population sizes, divergence times, and gene flow under a coalescent framework. In both species, divergence between Amazonian and Atlantic Forest populations dates back to the mid‐Pleistocene, with subsequent gene flow. We recovered eleven candidate genes involved with metabolism, immunity, development, and cell signaling in A. punctatus and found no loci whose frequency is associated with environmental gradients in A. ortonii. Distinct signatures of adaptation between these species are not associated with historical constraints or distinct climatic space occupancies. Similar patterns of spatial structure between selected and neutral SNPs along the climatic gradient, as supported by patterns of genetic clustering in A. punctatus, may have led to conservative GEAA performance. This study illustrates how tests of local adaptation can benefit from knowledge about species histories to support hypothesis formulation, sampling design, and landscape gradient characterization.Environmental gradients constrain physiological performance and thus species’ ranges, suggesting that species occurrence in diverse environments may be associated with local adaptation. Genome–environment association analyses (GEAA) have become central for studies of local adaptation, yet they are sensitive to the spatial orientation of historical range expansions relative to landscape gradients. To test whether potentially adaptive genotypes occur in varied climates in wide‐ranged species, we implemented GEAA on the basis of genomewide data from the anole lizards  To filter out poor‐quality reads and reduce base‐calling error, we implemented a minimum Phred quality score (=33), minimum sequence coverage (=10×), minimum read length (=70 bp), maximum proportion of heterozygous sites per locus (=0.25), and maximum number of heterozygous individuals per locus (=5) while ensuring that variable sites had no more than two alleles. Following the de‐multiplexing step in Ipyrad, read quality and length were assessed for each sample using FastQC (available at https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). For phylogenetic and genetic clustering analyses, a single SNP was randomly extracted per locus to reduce linkage disequilibrium across sites and to maximize sampling of independent SNP histories. Previous to GEAA and genetic clustering analyses, we used VCFtools v. 0.1.13  within each species separately using sNMF v. 1.2 , we ran preliminary analyses with a = 1, 10, 100, and 1,000; the best entropy scores were obtained with a = 100. Up to 10% missing data were allowed . The resulting datasets were composed of 2,580 unlinked SNPs for A. ortonii and 1,807 for A. punctatus.To approximate the number of populations in pca function of the R package LEA v. 2  based on the SNP data using the 2.3A. punctatus only, for which three populations were recovered; in the case of A. ortonii, where only two populations were found based on the genetic data , we inferred phylogenetic relationships between the genetic clusters (thereafter populations) identified by sNMF. Phylogenetic analyses were performed for A. punctatus using SNAPP  was applied for the θ parameter , using α = 1 and ρ = 0.05 (with mean = 0.05). A gamma prior with α = 2 and ρ = 100 (with mean = 200) was set to the rate of the Yule diversification process (λ) given an expected tree height  of ~0.004 substitutions per site based on recent phylogeographic study that included A. punctatus . Default settings were implemented for the mutation parameters. Tree rooting followed Prates, Xue, et al. , a population tree was inferred for , et al. . To helpA. punctatus.We performed two independent SNAPP runs of five million generations each, sampling every 1,000 steps. After ensuring convergence between runs and stationarity of model parameters in Tracer v. 1.6 .To estimate historical demographic parameters such as gene flow, we used G‐PhoCS v. 1.3  for the θ and τ priors, setting α = 1 and β = 20 (with mean = 0.05). The gamma prior for each migration band, m , was set with α = 1 and β = 2 × 10−7 . To convert mutation rate‐scaled parameter estimates to absolute effective population sizes and divergence times, we assumed an average substitution rate of 2.4 × 10−9 substitutions per site per year, as estimated by a study that included South American anole species . Because temperature and precipitation variables were often correlated (pairwise Pearson correlation coefficients >0.7), we followed best practices for GEAA and implemented a PCA on all variables, using the first principal component as a predictor in GEAA  inferred by sNMF, as recommended by Frichot et al. , and were used in final analyses.To test for associations between environmental predictors and allele frequencies, we used LFMM . These factors are used to modify the baseline null hypothesis in GEAA with the goal of limiting inflation related to population structure and other confounding factors  were adequately controlled for, we corrected association lfmm function of the LEA R package, with 10 independent runs of 50,000 iterations each and 25,000 iterations as burn‐in. A maximum of 10% missing data was allowed. To reduce the potential effects of missing sites in GEAA, we performed haplotype imputation based on the ancestry coefficients estimated by SNMF, using the impute function of the LEA package (as recommended by LFMM developers). Because LFMM makes no assumptions about linkage among SNPs , all SNPs were included in GEAA analyses. After the filtering steps, the genetic dataset was composed of 3,412 SNPs in 2,580 loci for A. ortonii, and 3,249 SNPs in 1,920 loci for A. punctatus.LFMM was implemented using the https://github.com/ivanprates/2018_Anolis_EcolEvol).R and Unix shell scripts used in all analyses are available online through GitHub (2.7https://useast.ensembl.org/Multi/Tools/Blast) to map each sequence against the reference genome of Anolis carolinensis  regions. In the case of mapped loci that correspond to protein‐coding regions, we then examined gene identity and function as summarized by gene ontology terms based on the UniProt database (available at https://www.uniprot.org/uniprot/).LFMM analyses recovered SNPs whose frequencies are linked to environmental gradients  northwards  between Atlantic Forest and Amazonian populations in both s Figure a,b, witha Figure b.3.2A. ortonii differs from the pattern seen in A. punctatus. In A. punctatus, phylogenetic analyses using SNAPP found that the Atlantic Forest population is nested among Amazonian samples , used as a predictor variable in GEAA, explains >70% of the total climatic variation across sampled sites in both species. Positive scores in climate PC1 correspond to lower temperature and precipitation values , suggesting that differences in DNA quality do not account for distinct GEAA results between 3.5A. punctatus, genetic clustering analyses based solely on the candidate SNPs suggest no allele sharing between populations in distinct regions  encodes an enzyme that links the glycolytic and citric acid pathways  encodes a structural component of the extracellular matrix that is involved with skeletal, skin, and eye development through collagen fibril organization  is crucial in immune response by mediating the differentiation of T cells and lymphocyte migration to sites of infection  encodes a transmembrane component of cell‐to‐cell junctional complexes that is essential for intestine development  sampled in a mid‐elevation site (820 m above sea level) in the Guiana Shield, on the Brazil‐Venezuela border, grouped with Atlantic Forest samples in a clustering analysis based solely on candidate SNPs  yields a stricter control of false positives . R and Unix shell scripts used in all analyses are available online through GitHub (https://github.com/ivanprates/2018_Anolis_EcolEvol).All sequence data were deposited in the Sequence Read Archive (accession PRJNA492310). Supplementary tables and figures were deposited in the Dryad Digital Repository ( Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.