Fraxinus excelsior L.) in its northern range compared to its central range in Europe. We used four chloroplast and six nuclear microsatellites to screen 42 populations (1099 trees), half of which corresponded to newly sampled populations in the northern range and half of which represented reference populations from the central range obtained from previously studies. We found that northern range populations of common ash have the same chloroplast haplotypes as south-eastern European populations, suggesting that colonization of the northern range took place along a single migration route, a result confirmed by the structure at the nuclear microsatellites. Along this route, diversity strongly decreased only in the northern range, concomitantly with increasing population differentiation and complex population substructures, a pattern consistent with a leading edge colonization model. Our study highlights that while diversity is maintained in the central range of common ash due to broad colonizing fronts and high levels of gene flow, it profoundly decreases in the northern range, where colonization was unidirectional and probably involved repeated founder events and population fluctuations. Currently, common ash is threatened by ash dieback, and our results on northern populations will be valuable for developing gene conservation strategies.During post glacial colonization, loss of genetic diversity due to leading edge effects may be attenuated in forest trees because of their prolonged juvenile phase, allowing many migrants to reach the colonizing front before populations become reproductive. The northern range margins of temperate tree taxa in Europe are particularly suitable to study the genetic processes that follow colonization because they have been little affected by northern refugia. Here we examined how post glacial range dynamics have shaped the genetic structure of common ash ( The present-day distribution of temperate and boreal tree taxa is largely a result of multiple range changes driven by the Quaternary glaciations . In EuroFor trees, it has been predicted that the leading edge effect during colonization may be less pronounced because of their prolonged juvenile phase. Many migrants have time to reach the newly establishing populations by long-distance dispersal before the first colonizers reach a reproductive age and genetic diversity may thus be maintained , 13. TreMost prior studies on postglacial colonization dynamics of temperate forest tree taxa focused on the southern and central range parts of species because those are the regions from which species expanded in the postglacial and where they are mainly distributed today , 23–25. Fraxinus excelsior L., Oleaceae) is a temperate tree species with a wide distribution across Europe. Its distribution ranges from the Mediterranean and the Caspian Sea in the South to mid-Norway (63.5° N) in the North, and from the Atlantic coast in the West into continental Russia in the East [Common ash , rethe East , 33, 34)the East , 36–38).the East . In thisCommon ash displays genetically distinct chloroplast (plastid) DNA lineages, which are maternally inherited , and refHymenoscyphus fraxineus  presumably originating from Asia [The aim of this study was to investigate the population history and the genetic structure of northern European common ash populations in relation to central European populations. We collected new population samples from the northern range and analysed them together with reference samples from the central European range available from previously published genetic studies , 41.We from Asia –47. The rom Asia ). Thus aA total of 42 common ash population samples (1099 trees) were analysed covering 16 European countries throughout the species’ distribution range , S1 Fig.https://www.fylkesmannen.no. According to the Norwegian Outdoor Recreation Act, everyone can freely roam and pick flowers, berries, nuts and fungi in the forests. As we picked five leaves from the trees, no additional permission from landowners was necessary.Due to ash dieback, common ash in Norway was in 2010 considered near threatened and in 2015 vulnerable (Norwegian Red List for Species ). PermisTo enable direct comparison of the genetic constitution of populations in the northern range with southern and central European populations, a set of 25 populations (529 trees) from other parts of Europe was included in the study . Seven oet al. [et al. [et al. [For the newly sampled populations, DNA was extracted from dried leaf material using the DNeasy 96 Plant Kit (Qiagen) following the manufacturer’s instructions. We assayed four chloroplast DNA microsatellites  developed by Weising & Gardner  and six et al.  and one  [et al. . We used [et al. . All samet al. [et al. [Amplified fragments were separated on an ABI3130 automatic sequencer (Applied Biosystems), fragment sizes and genotypes were determined using GeneMapper Software v. 4.1 (Applied Biosystems by Life Technologies). The coding of the plastid haplotypes followed Heuertz et al.  and Suth [et al.  (Table 1FIS estimates (inbreeding coefficient) across loci for each population calculated in SPAGeDi v. 1.4 [FIS using the Bayesian procedure implemented in INEST v. 2.0 [FIS is 0) using the Bayesian procedure based on the Deviance Information Criterion (DIC) implemented in INEST.Linkage disequilibrium was tested in GENEPOP v. 4.2.2  for all i v. 1.4 . Criticai v. 1.4 . The prei v. 1.4  using FRi v. 1.4 . As someT v. 2.0 , which hT v. 2.0 . StatistAr), observed and expected gene diversity  [Genetic diversity was measured for each locus and population as allelic richness (ctively)  using FSctively) . Privatectively) . AllelicFST [RST, respectively. The significance of genetic differentiation among pairs of populations was tested by 1000 permutations of genotypes among samples. We also calculated standardized measures of population differentiation, Hedrick’s G’ST and Jost’s D, correcting for differences in diversity and heterozygosity using 1000 bootstrap replicates in SMOGD [FST values using the excluding null allele (ENA) method by Chapuis and Estoup [Population differentiation was calculated in ARLEQUIN v. 3.5.1.2.  based onFST  and RST,in SMOGD . We checd Estoup  in FREENK) from 1 to 30, finding the highest possible maximum number of clusters for K = 15. Then we used the CAR  model as described in [K unobserved parental populations. We used the default parameters, with a burn-in of 10 000 and run length of 20 000 iterations, for K = 1 to K = 15 with ten replicates per K value. To determine the K with the best fit to the genetic data, average Deviation Information Criterion (DIC) values were plotted against K. For the STRUCTURE analysis we used the admixture model with correlated allele frequencies, both with and without using location as prior information (LOCPRIOR). Burn-in was set to 500 000 and run length was 500 000 iterations for K = 1 to K = 10 with ten replicates per K value. We used STRUCTURE HARVESTER Web v. 0.6.94 [K. Similarity coefficients across runs and the average matrices of individual membership proportions were estimated using CLUMP v. 1.1.2 [Population genetic structure was analysed employing both spatially informed and non-spatial Bayesian clustering approaches in TESS v. 2.3  and STRUribed in , assumin. 0.6.94  to assesv. 1.1.2 . Clusterv. 1.1.2 . In addiv. 1.1.2 , 76. ComHE, Ar, FISINEST, latitude and longitude. The correlations were calculated for three different groups of populations: i) all populations representing the entire distribution range of common ash, ii) populations where ancestry was larger than 0.6 in TESS group 2, consisting of most Northern and Eastern European populations representing the putative migration route of common ash from south-eastern Europe to Norway  and the natural logarithm of geographical distances was tested with a Mantel test [To test whether the genetic differentiation followed an isolation by distance (IBD) model, the significance of the correlation between tel test  using ARtel test .In total, 12 plastid haplotypes were detected . HaplotyFIS) was observed in 28 populations after sequential Bonferroni corrections (FISINEST) remained significantly different from zero in only nine populations  after sequential Bonferroni correction. All loci were considered to be genetically independent for further analysis. The single locus tests for HW genotypic proportions within populations showed significant departure in 68 out of 252 cases after Bonferroni correction (p ≤0.05), the significant values corresponded mainly to loci Femsatl8 and Femsatl16. Significant multilocus deviation from HW proportions  of 0.686–0.920 and allelic richness (Ar) of 5.417–12.567  and RST = 0.052 (SD = 0.015), both values significantly greater than zero  and three populations on the Norwegian northwest coast; 14Mjo (average pairwise FST = 0.091), 12Ron (average pairwise FST = 0.083) and 13Asa (average pairwise FST = 0.063).Basic diversity and differentiation characteristics for each of the microsatellite loci are given in han zero . After E 0–0.190 . The higK = 4  increased progressively as of K = 5 . Severalof K = 5 . The proof K = 5 . As in TFST values.The two first axes of the PCoA explained 21% and 12% of the variation, respectively . The disAr and latitude as well as between HE and latitude . Within TESS group 2, no significant correlations were observed between Ar, HE and latitude. In all groups, Ar and HE were strongly correlated. No significant correlations were found between genetic diversity and longitude, but FISINEST was positively correlated with longitude both in Europe overall and in TESS group 2, but not in Norway alone  negative correlation between latitude , Fig 4. ay alone . The pat , nor within TESS group 2 . Within Norway, there was a weak though significant IBD pattern . No consistent evidence for bottlenecks  was found in any of the populations. Fifteen populations showed significant heterozygote excess in the Wilcoxon test, either under the infinite allele model or the two phase model , and in southern Sweden 8 cal kyr. BP (0.5–1% pollen threshold) [Fraxinus seeds along with other species into Scandinavia. As only haplotype H01 dispersed to Sweden and Norway, it may indicate that H02 and the other haplotypes found in Denmark arrived after the connection to Sweden was broken. Denmark is thus not the source of colonization to Sweden and Norway, which is in contrast to what has been found in e.g. oak [In south-eastern Europe, pollen diagrams indicate a late glacial presence of common ash in the Balkan peninsula –82. Pollet al. , migratiet al. . A contareshold) , 82. In reshold) . This mae.g. oak . Two of e.g. oak , 82 and Fraxinus pollen (0.5–1%) was detected in the Trøndelag region in the early Holocene .Our nuclear microsatellite analysis suggests that the northernmost population in Norway, Trøndelag (15Hind), constitutes a separate genetic group with very low levels of genetic diversity. Its genetic peculiarity is probably due to a shift in allele frequencies, either because of a founder event following recent long-distance colonization, or owing to drift following long-term persistence of a small ash population in the region. The slightly elevated private allelic richness in the population may also have contributed to its genetic partition, although it is still lower than the mean value plus standard deviation  for all the populations. Interestingly, l kyr BP , 82). Po cordata . FraxinuFST values .–93.90–93et al.  sought tet al. , 96. In FISINEST). Although some bi-parental inbreeding may be expected [FISINEST values significantly larger than zero in some of the eastern populations (e.g. from Finland (Fin) and Lithuania (Kai2012); FISINEST and longitude within TESS group 2. In Finland, common ash has a very scattered and restricted distribution and microsatellite analysis of four Finnish island populations suggested an almost total interruption of gene flow among populations [FIS estimates have been observed, e.g. in British [FIS values were observed [FIS values among studies are probably partly due to loci and the methods used for calculating FIS as well as actual differences in inbreeding.Based on the Bayesian calculations in INEST , there wexpected , the estexpected , and comexpected ). We obsulations , probabl British , French  British  and Ital British  samples,observed . The difet al. [Ash dieback epidemic is currently threatening common ash at continental scale in Europe . A signiet al.  estimateet al. , 102, thet al. ). Howeveet al. ). Given et al. .Trees are generally considered to be relatively resilient to the effect of small population size and increased isolation due to their longevity, high reproductive capacity, and ability for long distance seed dispersal and pollen flow. In common ash, populations in the central distribution range show high genetic diversity and low genetic differentiation, whereas populations in the northern range margins show lower diversity and increased divergence, most probably as a consequence of a leading edge colonization process towards the north. In the northern range in Norway, common ash is mainly restricted to a narrow nemoral forest belt along the coast and colonization of this range was probably one-dimensional, involving repeated establishments of small and isolated populations from the leading colonizing front followed by drift. The northern populations were, however, not affected by inbreeding. Currently, in common ash a strong selection pressure due to the ash dieback epidemic is on-going. The current low genetic diversity in the scattered northern range populations of common ash may compromise their future distribution.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableThe Wilcoxon test was used under the infinite allele model (IAM), two phase model (TPM) and the stepwise mutation model (SMM).(XLS)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


C. argyrosperma ssp. argyrosperma, known in Mexico as calabaza pipiana, and its wild relative C. argyrosperma ssp. sororia. The main aim of this study was to use molecular data (microsatellites) to assess the levels of genetic variation and genetic differentiation within and among populations of domesticated argyrosperma across its distribution in Mexico in comparison to its wild relative, sororia, and to identify environmental suitability in previously proposed centers of domestication. We analyzed nine unlinked nuclear microsatellite loci to assess levels of diversity and distribution of genetic variation within and among populations in 440 individuals from 19 populations of cultivated landraces of argyrosperma and from six wild populations of sororia, in order to conduct a first systematic analysis of their genetic resources. We also used species distribution models (SDMs) for sororia to identify changes in this wild subspecies’ distribution from the Holocene  to the present, and to assess the presence of suitable environmental conditions in previously proposed domestication sites. Genetic variation was similar among subspecies . Nine argyrosperma populations showed significant levels of inbreeding. Both subspecies are well differentiated, and genetic differentiation (FST) among populations within each subspecies ranged from 0.152 to 0.652. Within argyrosperma we found three genetic groups . We detected low levels of gene flow among populations at a regional scale (<0.01), except for the Yucatan Peninsula, and the northern portion of the Pacific Coast. Our analyses suggested that the Isthmus of Tehuantepec is an effective barrier isolating southern populations. Our SDM results indicate that environmental characteristics in the Balsas-Jalisco region, a potential center of domestication, were suitable for the presence of sororia during the Holocene.Analyses of genetic variation allow understanding the origin, diversification and genetic resources of cultivated plants. Domesticated taxa and their wild relatives are ideal systems for studying genetic processes of plant domestication and their joint is important to evaluate the distribution of their genetic resources. Such is the case of the domesticated subspecies  Domestication is an ideal model to study evolution because it is usually fast and gradual . PopulatSometimes the ancestral wild populations can still be studied along with the domesticated forms and varieties, allowing paired comparisons between populations under different selection processes in the same environment . Also, tCucurbita argyrosperma and Zea mays, gene flow is asymmetric, being more frequent from the wild to the domesticated taxa  exists among wild and domesticated taxa . These tThe study of crop origins has traditionally involved identifying geographic areas of high diversity and sampling populations of wild progenitor species . LinkingCucurbita , with 20 taxa of perennial or annual plants, is native to the Americas. Mexico is considered its center of origin and diversification . C. argyrosperma is currently divided into two subspecies: the domesticated C. argyrosperma ssp. argyrosperma (argyrosperma hereafter) and its wild relative C. argyrosperma ssp. sororia  .Among domesticated cucurbits, esources . It is aesources . This isigure 1) . Both wiigure 1) . These sCucurbita argyrosperma is an important crop in local agriculture systems in Mexico and in other countries in the Americas. It is grown and selected in traditional ways. It is commonly found as a seasonal crop, but irrigation is used in some areas. A large amount of its production is not reported because it is used in subsistence agriculture in Mexico and Central and South America  using isozymes (argyrosperma has less genetic variation (HE = 0.35–0.41) than its wild relative (HE = 0.433), and low levels of genetic differentiation among populations (FST = 0.077). Two studies, one based on isozymes in commercial cultivars  than in other domesticated taxa of the genus, such as C. moschata  and in C. pepo   and higher genetic diversity and heterogeneity among southern populations in the states of Guerrero and Oaxaca. Clearly, there is a lack of population-based genetic diversity analyses that include both the cultivated and wild C. argyrosperma throughout its range.Nevertheless, there are few studies focused on analyzing the genetic resources of cucurbits and covering most of their distributions . Only a isozymes , which fultivars , and anoultivars , found lctively) . Recentlargyrosperma across its distribution in Mexico. We also analyzed populations of its wild relative, sororia, to compare levels of genetic variation and differentiation. Additionally, we estimated the levels of recent gene flow among populations and subspecies, and performed projections of the wild subspecies’ distribution area in the mid-Holocene , in order to identify environmental suitability in previously proposed domestication centers, such as the Balsas-Jalisco region based on archeological records  to assess the levels of genetic variation within and among populations of domesticated  records . We expeus study . In addiargyrosperma and 6 wild populations of sororia representative of the species distribution in Mexico  for 40 days, and young leaves were collected for DNA extraction. DNA extraction was performed using a modified CTAB protocol (argyrosperma and 113 to sororia.We obtained seeds from at least 3 fruits (one fruit from each different plant) from 19 populations of cultivated landraces of protocol . For nucC. pepo , 1 μl of Taq polymerase (PROMEGA) and 10 ng of genomic DNA. Amplification reactions were performed in a Veriti 96-well Thermal cycler (Applied Biosystems) with the following program: 95°C for 5min, followed by 35 cycles of 95°C for 40s, 60°C (Ta) for 40s, 75°C for 55s, and a final step of 72°C for 5min followed by 4°C. To control for possible contamination, we used blank controls for each reaction. All products were verified in 2 % agarose gels and PCR products were sent to the Roy J. Carver Biotechnology Center at the University of Illinois, United States for genotyping1. Electropherograms were analyzed with PeakScanner (Applied Biosystems) to build a matrix with the genotypes of each individual.We amplified 12 of the nuclear microsatellite loci reported by P), allelic richness (A), and the expected (HE) and observed (HO) heterozygosities with the same software, and estimated the inbreeding coefficient (FIS) for each population using Genepop 4.0  based on allele frequencies without considering the population of origin. We performed previous runs to assess the best combination of priors to be used for the analysis and the length of the Markov Chain Monte Carlo (MCMC) chains. Accordingly, we performed a final run with admixture and correlated allele frequencies as priors, and without considering the putative population of origin of each individual. We used a burn-in of 500,000 chains and 1,000,000 MCMC chains, and tested values of K from 1 to 10, and 10 repetitions for each K. The results were run through Structure Harvester v 0.6.93 .To detect barriers to gene flow, we used the Monmonier algorithm  implemensororia. We constructed a database with geographic coordinates of collected and known sororia populations. Points from Central America were downloaded from GBIF3, and 699 points from Mexico were obtained from Salvador Montes-Hernández, for a total of 720 occurrence. This database was purged to eliminate duplicated pixels. In addition, to ensure that all points fell within the species distribution, we estimated Mahalanobis distances using previously selected environmental variables (see below for selection methodology). The points deviating by two standard deviations or more from the mean were mapped, checked, and discarded if they fell outside the species range. We finally retained 273 occurrence points to perform the SDMs  projections into the mid-Holocene  for the wild relative r < 0.85 (∗100), Maximum Temperature of Warmest Month, Precipitation of Wettest Month, Precipitation of Driest Month, Precipitation Seasonality, Precipitation of Warmest Quarter, and Precipitation of Coldest Quarter.To reduce the uncertainty associated with SDMs, it is necessary to select only the more informative and uncorrelated climatic variables . To do sr < 0.85 . Nine bisororia (9.51003°N to 34.18357°N and -116.1351°W to -70.80147°W). MaxEnt was executed using a 20% random test rate, 30 replicates, replicated bootstraps, 1000 maximum iterations and a convergence threshold of 0.00001, with extrapolation and clamping turned off. The distribution model was derived from the average model and evaluated using the score of the area under the curve .For SDM projecting to mid-Holocene climate conditions, we downloaded the layers corresponding to atmospheric-ocean general circulation models (AOGCM) based on the Community Climate System Model CCSM4 , which isororia. To identify areas with suitable environmental conditions for the species under current and past climate conditions, we performed a sum of the SDMs, thus highlighting the areas with potential stability conditions from the mid-Holocene to the present.In order to create a presence/absence map, we used the 95th percentile value of observed sample points as a threshold for the logistic model. This value assumes that up to 5% of the records used for generating the model are subject to error. For current and mid-Holocene times, we generated presence/absence maps for Three microsatellites  showed evidence of null alleles and were therefore excluded from further analyses. As expected, there was no evidence of linkage disequilibrium among loci.All loci showed significant deviations from Hardy-Weinberg equilibrium (HWE) in at least one population (Supplementary Table Table 1). We found higher levels of polymorphism per population (p = 0.02) in the cultivated populations of argyrosperma  than in the wild sororia . For argyrosperma, the proportion of polymorphic loci ranged between 0.44 and 0.88. For sororia, the proportion of polymorphic loci ranged between 0.55 and 0.77.We obtained a total of 84 alleles for the nine analyzed loci. At least one locus was monomorphic in each population  showed the highest number of private alleles. Within sororia, populations Soax and SoSin showed the highest number of private alleles. Overall, the populations from Oaxaca showed the highest proportion of private alleles in both subspecies. Allelic richness was similar (p = 0.23) in cultivated argyrosperma (A = 2.786 ± 0.76 SD) and in wild sororia (A = 2.93 ± 1.17 SD). For argyrosperma, rarefied allelic richness ranged from 1.9 in Aut to 1.36 in Dgo (Table 1). For sororia, rarefied allelic richness ranged from 1.8 in four populations to 1.6 in Aut (Table 1).Forty alleles were private to HO = 0.388 and HE = 0.428 in sororia, and HO = 0.36 and HE = 0.410 in argyrosperma; HOp = 0.484; HEp = 0.656). For argyrosperma, genetic diversity (HE) ranged from 0.588 in SinalP to 0.25 in Ek. For sororia, mean genetic diversity (HE) was 0.428, with the highest value in Soax (0.502) and the lowest in Sgro (0.3) (Table 1).Mean observed and expected heterozygosity were similar among subspecies . Within argyrosperma, five populations exhibited heterozygosity excess, while nine showed heterozygote deficiency and the rest were in HWE (Table 1). The highest values for heterozygosity deficiency were found in Chan and SinalP (FIS = 0.39) and Yec (FIS = 0.32) and for heterozygosity excess the lowest values were found in Ek and Ome . Within sororia, two populations exhibited heterozygosity deficiency, while the rest were in HWE (Table 1). The highest FIS value was found in Soax (0.303) (Table 1).Both subspecies showed similar overall inbreeding coefficients . For K = 2 there was a clear genetic differentiation between subspecies , except for the sororia populations SoSin and SoSon, that were more similar to argyrosperma. The Structure barplot for K = 3 shows that within argyrosperma, the populations from the Yucatan peninsula, Chiapas, Veracruz, Michoacán and CCC constituted a cluster, while the populations from northern and central Mexico formed another cluster . Finally, for K = 4 the clusters largely corresponded to subspecies’ geographic distributions . The first cluster consisted of four sororia populations: Schis, Soax, Sgro, Sjal . The other two sororia populations (SoSin and SoSon) were assigned to a second cluster with argyrosperma populations CCC, and SinalP located in northern Mexico . The third cluster consisted of argyrosperma populations from Ek, Mot, Chan, Champ and Pal, from the Yucatan Peninsula and populations from Michoacán (Sah) and Veracruz (Tih) . The fourth cluster was constituted by populations Teh, Mix, Ome, Tla, Aut, and Yec, from the Pacific coast and Durango . The results from this analysis showed some degree of admixture among populations, particularly within argyrosperma , but the populations from sororia had low levels of admixture with the domesticated subspecies.The analysis performed with Structure suggested a value of Figure 5). Four sororia populations were clearly differentiated from argyrosperma, while two populations clustered within argyrosperma. All argyrosperma populations were grouped together, except for Tih from the state of Veracruz, which seemed in this analysis to be well differentiated from the other populations . In this DAPC 97.9% of the variance is explained by 40 PCs. A DAPC analysis considering only argyrosperma populations, except Tih , showed that Mtp and Tehuantepec from the states of Guerrero and Oaxaca, respectively, were well differentiated. Some populations formed very cohesive clusters, i.e., populations from the Yucatan Peninsula and populations from the Pacific Coast. In this second DAPC 94.6% of the variance is explained by 25 PCs.The results from the DAPC analysis were consistent with the results from Structure  than for domesticated argyrosperma . Genetic differentiation among populations (pairwise FST) of argyrosperma was moderate to high (FST = 0.031–0.515), while genetic differentiation among populations of sororia was higher in general (FST = 0.171–0.639). Genetic differentiation among populations of the different subspecies was medium to high  values were moderate (FST = 0.181–0.352).Overall genetic structure was higher for wild FST values  showed two well-defined groups: one group including only sororia populations from different states along the Pacific coast in Mexico , and another group of argyrosperma populations, including the two sororia populations (SoSin and SoSon) mentioned above. Bootstrap values are in general low, as is usually found in intraspecific studies (due to both gene flow and recent common ancestry). Low bootstrap values within argyrosperma could also be due to homoplasy and care should be taken with their interpretation. Nevertheless, the two groups had higher support values (above 50%) .The dendrogram built using pairwise argyrosperma in all genetic structure analyses, and the seeds show an intermediate morphology for size and color , we also performed an AMOVA analysis considering these populations as argyrosperma. This analysis showed that a higher percentage (30.3%) of the genetic variance was allocated between subspecies; most variance was still found within individuals (46.1%), and less among populations within subspecies (21.4%), finally, 2.3% of the genetic variance is found among individuals within populations. On the other hand, an AMOVA analysis considering the partition suggested by the Structure analysis, K = 4, explained 24.5% of the genetic variance among clusters, the variance among populations within clusters was 30.8%, and most of the variance was found within populations (44.6%).An AMOVA that considered each subspecies, explained 20.05% of the genetic variance between subspecies, and most of the variance was found within individuals (50.3%), followed by among populations within subspecies (26.2%), and only 3% of the variance was found among individuals within populations. Given that two putatively wild populations (SoSin and SoSon) were identified as belonging to Figure 8).Mantel tests were significant for both subspecies, indicating spatial structure due to isolation by distance. We found that geographically closer populations are genetically more similar than expected by chance  and a Northern population in San Luis Potosi state, Tan; other analyses strongly suggest that SoSin and SoSon are argyrosperma populations escaped from cultivation. This suggests that gene flow between cultivated and truly wild populations is low.Estimates of recent gene flow suggest that the total proportion of migrants for each population was from 17 to 33% , the northern part of the Sierra Madre Occidental may function as an effective barrier to gene flow, isolating the SinalP and Yec populations. This contrasts with results from the DAPC and structure analyses, where these populations do not seem to be isolated. This can be due to differences in the methodologies, in which Monmonier analysis takes spatial distances into account. For sororia, the southern portion of the Sierra Madre Occidental also functions as an effective barrier to gene flow, and isolates population Sjal . When both subspecies were analyzed together, we observed that the main barrier is located in the region of the Isthmus of Tehuantepec, isolating the populations from the Yucatan Peninsula .The Monmonier analysis indicated that for sororia , showed stability and good support (AUC: 0.96). The SDM projection to the mid-Holocene (∼6000 years ago) suggests that the distribution area of sororia has been more or less stable since domestication . Nevertheless, the analysis also suggests that sororia may have been present in the Yucatan Peninsula during the mid-Holocene and its distribution in the regions of Oaxaca and Guerrero, where the most ancient archeological remains have been found, may have been more continuous than today . In addition, the distribution of sororia in Central America may have been wider and more continuous from Guatemala and Honduras to the northern area of Nicaragua.The SDM for the wild subspecies, C. argyrosperma, covering the cultivated argyrosperma distribution in Mexico, and including populations of the wild sororia distribution in the Pacific Coast from Northern Mexico (Sonora) to Southern Mexico (Chiapas). Our analyses show similar levels of genetic variation in the cultivated populations and in its wild ancestors. Genetic differentiation is higher in wild sororia (FST = 0.492) than in domesticated argyrosperma (FST = 0.264), but this estimate is probably the product of including two escaped populations (SoSin and SoSon) that were misclassified and analyzed as sororia. When we remove these populations, differentiation in wild sororia (FST = 0.243) became even lower than the differentiation found in cultivated argyrosperma. Gene flow at a regional level is associated to movement of pollen by Cucurbita pollinators and to human cultural practices, such as seed exchange among populations . Comparable values of polymorphic loci, allelic richness and genetic diversity among argyrosperma and sororia suggests that the subspecies have had similar effective population sizes, and that the theoretical bottleneck associated with domestication was either mild and/or of short duration, followed by a rapid population expansion . Low levels of allelic richness and genetic diversity in sororia may suggest that this species has undergone one or several bottlenecks due to ecological shifts during the Pleistocene, followed by rapid population expansion, as suggested by Genetic variation was similar to what has been reported for other annual plants in microsatellite studies  and seed exchange, that can reduce inbreeding at the local level. Nevertheless, it is advisable to perform similar analyses in other wild and domesticated cucurbits to gain further insight into the amount of genetic variation present in Cucurbita.Certain aspects of agricultural management, such as seed exchange, may also affect the levels of genetic variation in rosperma  and in C showed the highest levels of genetic variation, while the populations from the Yucatan Peninsula (except Chan) showed the lowest levels of genetic variation. These results, together with the barriers analysis, suggest that the cultivated populations from the Yucatan Peninsula are isolated genetically  in argyrosperma were highly variable (Table 1), with some populations showing heterozygote deficiency (9 populations), as may be expected in a domesticated species, and other populations showing heterozygote excess (5 populations), as previously reported by FIS values found in some cases suggests that seed exchange is frequent at a local level  promoting outbreeding, but at a regional level  gene flow is low. It will be important to conduct detailed ethnobotanic studies in different regions of the country, along with genetic analysis, to test the effect of agricultural management on the genetic variation of this crop (Cultivated species often show high levels of inbreeding . Estimathis crop .argyrosperma and 11 in sororia), while the mean number of private alleles per population was similar within subspecies (1.31 in sororia and 1.21 in argyrosperma).When populations are isolated, genetic drift promotes the random fixation of alleles, thus the number of private alleles among populations can be used as a reference for population connectivity . We founargyrosperma ∼ 8,600 years ago  and Oaxaca (Teh). Tih is geographically distant from other sampled populations, and its private alleles may be present in other populations from the Gulf of Mexico; thus, it is advisable to include more populations from this area in further analyses. The population Teh from Oaxaca is located in the area of the Isthmus of Tehuantepec that has been previously identified as an important barrier for the Mexican biota , further supporting the Isthmus of Tehuantepec as an important biogeographical barrier. Furthermore, seed morphology is distinctive in the populations of argyrosperma of Southeastern Mexico, where seeds show clear gray margins in contrast to the golden color found in northern populations . People in southeastern Mexico have a strong preference for local varieties . In addition, this may suggest that the high number of private alleles in this area may be related to strong selection pressures associated with seed morphology, as has been reported in C. pepo commercial varieties . Within argyrosperma, Structure analyses  show geographically associated groups: (1) a northern group; (2) Yucatan Peninsula; and (3) Pacific coast . It is interesting that these genetic groups roughly correspond to the genetic groups reported by The results from the Structure and DAPC analyses show clear genetic differentiation among subspecies (FST) were variable among populations of both subspecies. Sororia showed similar levels of genetic differentiation  as argyrosperma . In addition, our Mantel test results show that geographically close populations of both subspecies are genetically more similar than expected by chance. Both subspecies have wide distributions  that cover a distance of over 1,000 km, thus promoting genetic differentiation among extreme populations. Genetic differentiation in sororia could also be related to its patchy distribution and the limited movement (∼0.7 km) and local low densities of its main pollinators from the genera Peponapis and Xenoglossa . Our results suggest that these may be escaped populations of argyrosperma, as suggested by argyrosperma are capable of surviving without agricultural management, but that these individuals show a reduction in seed size. Reports suggest that cultivars of C. pepo and C. moschata in Tamaulipas, Mexico are also capable of surviving and producing fruits in semi-wild or in extreme environmental conditions . Many domestication events occurred during this time because of environmental changes and vegetation transitions associated with the end of the Last Glacial Maximum-Holocene, and with the impact of anthropogenic activities , drafting the manuscript, and final approval of the version to be published. HH-R contributed to fieldwork, lab work, molecular and data analyses, and final approval of the version to be published. AV-L contributed to laboratory work, logistics, correcting the manuscript, and final approval of the version to be published. EA-P contributed to laboratory work, logistics and molecular analysis, correcting the manuscript, and final approval of the version to be published. JJ-C contributed to correcting the manuscript and final approval of the version to be published. SM-H project leader, contributed to fieldwork, germplasm collection, generated database for project design, and final approval of the version to be published. RL-S project leader, contributed to logistics and final approval of the version to be published. LE project leader, designed and coordinated the project, logistics, drafted and corrected the manuscript, and final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heilongjiang Province located in northeast China is a multi-ethnic region with people who have lived in cold conditions for several generations. Fatty acids are important to people with cold resistance. CPT1A encodes a protein that imports long-chain fatty acids into the mitochondria for fatty-acid oxidation. FADS is an essential enzyme for the synthesis of long-chain polyunsaturated fatty acids.In the present study, we investigated the distributions of three cold resistance-related SNPs  from six populations that included 1093 individuals who have lived in Heilongjiang Province for at least three generations. The frequencies of rs174570 and rs7115739 were different in our six north minorities compared to the Chinese Dai in Xishuangbanna (CDX) in southern China. All the SNPs in Hezhen were significantly different from other five studied populations. In addition, the genetic distribution of rs174570 in Daur was significantly different from Manchu and Korea, and the frequency of rs7115739 in Ewenki was significantly different from the other populations. The results also showed that the frequencies of the three SNPs in the six minorities were different from those of Greenlandic Inuit and Siberian population.Our results showed the distributions of the three cold resistance-related SNPs from six populations that included 1093 individuals in northern China. Distributions of the allele frequencies for the cold resistance-related SNPs in northern China were statistically different from those in southern China. These data help to establish the DNA genome database for the six populations and fully preserve existing minority genetic information.The online version of this article (10.1186/s12864-018-4524-1) contains supplementary material, which is available to authorized users. Many studies have suggested that there are different genetic structures in different populations due to their complex demographic histories . China iHaving lived in northern China for a long period of time, Daur and other minorities have been subjected to cold climates. Thus, there may be some physiological differences contributing to their cold adaptations. Non-shivering thermogenesis is a physiological mechanism that generates body heat through mechanisms aside from mechanical shivering , and it Carnitine palmitoyltransferase 1 (CPT1) is a key regulatory enzyme that regulates fatty acid oxidation and has three isoforms, CPT1A, CPT1B and CPT1C. The CPT1A gene encodes a protein that imports long-chain fatty acids into the mitochondria for fatty acid oxidation. This mechanism is helpful for maintaining energy homeostasis when carbohydrate intake is insufficient . CPT1B iThe Fatty acid desaturase (FADS) genes, including FADS1, FADS2 and FADS3, code for enzymes that introduce double bonds at specific positions in a fatty acid chain . In humaPrevious study identified a missense mutation  in CPT1A as the most likely causative variant for positive selection in Northeast Siberians, using whole-genome high-coverage sequence data  and haveIn the present study, we aimed to explore the distribution of three SNPs in six ethnic minorities. We hoped that these genetic resources could provide information on the origins of the human race, evolution, migration and other aspects.Six populations from Hezhen, Daur, Manchu, Korean, Mongolian, and Ewenki, totaling 1093 individuals from Heilongjiang Province (around 45°44’N126°39′E), China were included in the study  according to the manufacturer’s protocol. The three cold resistance-related SNPs, rs80356779 G > A in CPT1A, rs7115739 T > G in FADS3 and rs174570 C > T in FADS2, were selected based on published articles . Genotyphttps://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/. The three populations included following individuals: (1) 93 Han Chinese in Beijing (CHB) individuals in northern China (around 39°55’N116°27′E); (2) 103 Chinese Dai in Xishuangbanna (CDX) individuals in southern China (around 22°3’N100°49′E); and (3) 105 Southern Han Chinese in China (CHS) individuals in southern China (around 30°22’N103°26′E).The genotype data of individuals from the three populations in China were downloaded from the 1000 genomes Project at Chi-square test. Fst calculations were performed using POPGENE (version 1.32) to assess the genetic differences among the six populations. Shannon’s information index was used to estimate the genetic diversity and the proportion of genetic variation within and between populations. The genotyping data have been submitted to Treebase under study number 22170 (https://treebase.org/treebase-web/user/summary.html?id=22170). All statistical analyses were performed using the SPSS 23.0 software . A probability (P) value < 0.05 was considered statistically significant.Hardy-Weinberg equilibrium of the controls was calculated using the Chi-square test suggested that the three polymorphisms followed Hardy-Weinberg equilibrium in the overall population (P > 0.05).The allele frequencies of three SNPs are shown in Table P < 0.05). Daur were significantly different with Manchu and Korean regarding rs174570 genetic distribution (P < 0.05). The frequency of the rs7115739 polymorphism in Ewenki was significantly different from the other populations (P < 0.05).As shown in Table P < 0.05). The frequencies of rs174570 ranged from 0.5000 to 0.7049 in six minorities. The frequencies in Korean and Manchu were similar, but different with other four minorities, and the frequencies of rs174570 were different in our six minorities from the CDX, which is the southeast population in this study. In addition, the frequencies of rs7115739 were different in the CDX when compared with most of our six minorities except for Hezhen, and there were strong differences between Hezhen and other populations in northern China, including Daur, Manchu, Korean and CHB (P < 0.05). The allele frequency in Korean was also different from that of other ethnic groups. However, in our six minorities, the A-allele in rs80356779 polymorphism was only detected in Hezhen, with the frequency of 0.1027.We also compared the allele frequency distributions in the current minorities and other populations, including one population located in northern China (CHB) and two located in southern China (CDX and CHS) . We found that the frequencies of our six minorities were similar to CHB, although there were some differences among them. However, when compared with populations located in southern China, great differences were observed. Since the CHB and six minorities are all located in northern China, and CHS and CDX are both located in southern China, the genetic differences between the CHB and six minorities are much smaller than those between the minorities and CHS and CDX. Thus, we presume that the gene may be undergoing strong selective pressures to resist cold and adapt to other environments. Additionally, there were strong differences between the six populations and the Greenlandic Inuit and Siberian population in allele frequencies. The allele frequencies of populations in northern China were in the middle of populations in southern China and Greenlandic Inuit and Siberian population, and there was a tendency to change with the latitude. In addition, Finnish in Finland (FIN) and British in England and Scotland (GBR) who live in northern area with an latitude around 55°N were examined to find the frequencies of rs174570 and rs7115739 polymorphisms in the 1000 Genomes Project. The frequencies of rs174570 in FIN and GBR are 0.2525 and 0.1868, respectively. The frequencies of rs7115739 in FIN and GBR are 0.9495 and 0.9505, respectively. These frequencies are very similar to those of Northeast Siberians and the Inuit, indicating that rs174570 and rs7115739 polymorphisms are associated with adaptation to cold. The results suggest that different minorities display unique genetic diversity in the nation during its long historical evolution for adapt to the cold environment.In this study, we investigated the diversity of cold resistance-related genes among six minorities from the northeast region of China to explore the hereditary differences among six populations. We selected three SNPs from adipose metabolism-related genes for the analysis. We also compared the allele frequencies between our study cohort and other populations in China. The The heterozygosity statistics for all three SNPs were found to be highly similar among Manchu, Korean, and Mongolian individuals, showing that the differences among the three populations were not significant. However, all the SNPs in the Hezhen population were significantly different from the other five populations. Daur was significantly different from Manchu and Korean with respect to the rs174570 genetic distribution. The frequency of the rs7115739 polymorphism in Ewenki was significantly different from the other populations. The differences may be associated with genetic drift.To measure the genetic differentiation, the Fst values were calculated for each SNP to quantify the differences among the populations. The results showed that the genetic differentiation among the populations was not significant. To assess the relationships among populations, a dendrogram was produced by UPGMA. The dendrogram divided the six populations into two main branches: one comprised of four populations, including Hezhen, Daur, Mongolian and Ewenki, and the other comprised of two populations, Manchu and Korean. The occurrence of this cluster relationship is consistent with a previous published study . AccordiIn the present study, we investigated the distributions of three cold resistance-related SNPs from six populations that included 1093 individuals in northern China. Our results showed, for the first time, that the six minorities in northern China exhibit genetic differences in certain cold resistance-related genes when compared with each other and with southern Chinese populations, especially with CDX. These results highlight that ethnicity is a critical factor in the distribution of allele frequencies. Meanwhile, our study also contributed to the DNA genome database for the six populations.Additional file 1: Figure S1.The location of populations. The six minority populations from Hezhen, Daur, Manchu, Korea, Mongolian and Ewenki are located in Heilongjiang Province (around 45°44’N126°39′E). CHB indicates Han Chinese in Beijing (around39°55’N116°27′E). CHS indicates Southern Han Chinese located in Sichuan province (around 30°22’N103°26′E). CDX indicates Chinese Dai in Xishuangbanna (around 22°3’N100°49′E). Greenlandic Inuit population live in area around 71°43’N42°24’W. Northeast Siberian population live in the northeast of Russia (around 78°50’N112°50′E). (DOCX 109 kb)Additional file 2: Table S1.Table S2 The P values for rs7115739 compared between six minorities in northern China and three populations in southern China. (DOCX 16 kb)The frequencies of three polymorphisms in 11 populations


Egypt, located on the isthmus of Africa, is an ideal region to study historical population dynamics due to its geographic location and documented interactions with ancient civilizations in Africa, Asia and Europe. Particularly, in the first millennium BCE Egypt endured foreign domination leading to growing numbers of foreigners living within its borders possibly contributing genetically to the local population. Here we present 90 mitochondrial genomes as well as genome-wide data sets from three individuals obtained from Egyptian mummies. The samples recovered from Middle Egypt span around 1,300 years of ancient Egyptian history from the New Kingdom to the Roman Period. Our analyses reveal that ancient Egyptians shared more ancestry with Near Easterners than present-day Egyptians, who received additional sub-Saharan admixture in more recent times. This analysis establishes ancient Egyptian mummies as a genetic source to study ancient human history and offers the perspective of deciphering Egypt's past at a genome-wide level. Archaeological and historical records had shown ancient Egypt before and after Ptolemaic and Roman periods to be a hub of human migration and exchange. Here, Schuenemann and colleagues analyse ancient mitochondrial and nuclear DNA to investigate the genetic history of Egypt. Egypt provides a privileged setting for the study of population genetics as a result of its long and involved population history. Owing to its rich natural resources and strategic location on the crossroads of continents, the country had intense, historically documented interactions with important cultural areas in Africa, Asia and Europe ranging from international trade to foreign invasion and rule. Especially from the first millennium BCE onwards, Egypt saw a growing number of foreigners living and working within its borders and was subjected to an almost continuous sequence of foreign domination by Libyans, Assyrians, Kushites, Persians, Greeks, Romans, Arabs, Turks and Brits. The movement of people, goods and ideas throughout Egypt's long history has given rise to an intricate cultural and genetic exchange and entanglement, involving themes that resonate strongly with contemporary discourse on integration and globalizationUntil now the study of Egypt's population history has been largely based on literary and archaeological sources and inferences drawn from genetic diversity in present-day Egyptians. Both approaches have made crucial contributions to the debate but are not without limitations. On the one hand, the interpretation of literary and archaeological sources is often complicated by selective representation and preservation and the fact that markers of foreign identity, such as, for example, Greek or Latin names and ethnics, quickly became ‘status symbols' and were adopted by natives and foreigners alike23Despite their potential to address research questions relating to population migrations, genetic studies of ancient Egyptian mummies and skeletal material remain rare, although research on Egyptian mummies helped to pioneer the field of ancient DNA research with the first reported retrieval of ancient human DNAHere, we provide the first reliable data set obtained from ancient Egyptians using high-throughput DNA sequencing methods and assessing the authenticity of the retrieved ancient DNA via characteristic nucleotide misincorporation patterns13By directly studying ancient DNA from ancient Egyptians, we can test previous hypotheses drawn from analysing modern Egyptian DNA, such as recent admixture from populations with sub-SaharanAll 166 samples from 151 mummified individuals  have traces of gold leaf near the mouth and the cheekbone, which is characteristic for mummies from the Ptolemaic Period onwardsRecently, various studies were conducted on these remains, including a study on ancient Egyptian embalming resins, two ancient DNA studies and an anthropological examination of the macerated crania121819For the DNA analysis we sampled different tissues , macerated and non-macerated, to test for human DNA preservation.We extracted DNA from 151 mummified human remains and prepared double-stranded Illumina libraries with dual barcodes22We tested different tissues for DNA preservation and applied strict criteria for authenticity on the retrieved mitochondrial and nuclear DNA to establish authentic ancient Egyptian DNA. First, DNA extracts from several tissues  from 151 individuals were screened for the presence of human mitochondrial DNA (mtDNA) resulting in a total of 2,157 to 982,165 quality filtered mitochondrial reads per sample, and 11- to 4,236-fold coverage. To estimate, identify and filter out potential contamination we applied the program schmutzi13In order to analyse the nuclear DNA we selected 40 samples with high mtDNA coverage and low mtDNA contamination. Using in solution enrichment for 1.2 million genome-wide SNPsn=44), the Ptolemaic Period (n=27) and the Roman Period (n=19)  and P values >0.1 for the continuity test. Modern Egyptians share this profile but in addition show a marked increase of African mtDNA lineages L0–L4 up to 20%  were grouped into three temporal categories based on their radiocarbon dates , correspd (n=19) . To testNe) from directly radiocarbon-dated mitochondrial genomes using BEASTThe finding of a continuous population through time allowed us to estimate the effective population size (3-statistics3-statistics3-statistics using only the least contaminated sample (with <1% contamination estimate) and find very similar results  as well as modern genetic data from contemporary populations that may not be fully representative of past populations.The archaeological site Abusir el-Meleq was inhabited from at least 3250BCE until about 700CE and was of great religious significance because of its active cult to Osiris, the god of the dead, which made it an attractive burial site for centuries332By comparing ancient individuals from Abusir el-Meleq with modern Egyptian reference populations, we found an influx of sub-Saharan African ancestry after the Roman Period, which corroborates the findings by Henn and colleagues5152The ancient DNA data revealed a high level of affinity between the ancient inhabitants of Abusir el-Meleq and modern populations from the Near East and the Levant. This finding is pertinent in the light of the hypotheses advanced by Pagani and colleagues, who estimated that the average proportion of non-African ancestry in Egyptians was 80% and dated the midpoint of this admixture event to around 750 years agoOur genetic time transect suggests genetic continuity between the Pre-Ptolemaic, Ptolemaic and Roman populations of Abusir el-Meleq, indicating that foreign rule impacted the town's population only to a very limited degree at the genetic level. It is possible that the genetic impact of Greek and Roman immigration was more pronounced in the north-western Delta and the Fayum, where most Greek and Roman settlement concentrated43However, our results revise previous scepticism towards the DNA preservation in ancient Egyptian mummies due to climate conditions or mummification proceduresAll pre-amplifications steps were carried out in clean room facilities dedicated to ancient DNA work at the University of Tübingen. Before the sampling all samples were UV irradiated for 60 min to reduce modern contamination. In addition, the surface of the bone or tissue samples was removed and the teeth were sampled from inside of the tooth pulp. DNA was extracted from 50 mg bone powder for bone or tooth samples, from 100 mg tissue for soft tissue samples, respectively. A silica purification protocol was applied as described in ref. 22Subsequently, the indexed libraries were amplified using 100 μl reactions for each library containing 5 μl library template, 4 units AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen), 1 unit 10 × AccuPrime buffer (containing dNTPs) and 0.3 μM IS5 and IS6 primersFor the nuclear capture two additional libraries for selected 40 samples using 20 μl extract were created as described above with the addition of a UDG treatmentAll samples were enriched for human mitochondrial DNA via bead capture hybridization as detailed elsewhereThe resulting FastQ files have been processed using EAGER v1.92 ref. . To achiin silico analysis has been done in order to authenticate samples and further determine the amount of potential contamination on the mitochondrial level. Negative controls were processed in parallel with samples. The former show no substantial mapping rates and suggest that the amount of DNA introduced during laboratory work could be kept on a minimal level. The authenticity of the samples has been further assessed by applying a number of methods and criteria. MapDamage 2.0  in our analysis and used a moderate filtering value that did not change our consensus sequence to undefined positions to a larger extent. We ultimately chose a value of q=20 for filtering with ‘log2fasta', but even more strict filtering with q=40 preserved our haplotyping calls to be consistent. However, filtering even stricter introduced more undefined positions (‘N') due to missing support, potentially hindering sequence-based analysis more dramatically than our frequency-based analysis, which is why we kept a quality threshold of q=20, following cutoffs that other authors have been using, tooAccompanying measures to limit contamination of the libraries in the laboratory work, 2.0 ref.  has beenet al.et al.The non-UDG and UDG treated libraries were enriched by hybridization to probes targeting approximately 1.24 million genomic SNPs as described previouslyFor each of the 40 samples, we sequenced two captured libraries: one with enzymatic damage repair (UDG), one without (non-UDG). For all samples, we used the EAGER pipeline version 1.92.15 ref. , with deThree samples were selected for down-stream analysis: JK2134, JK2888 and JK2911. In all three of these samples, contamination estimates were acceptable, and similar in both UDG and non-UDG libraries as can be seen in https://github.com/stschiff/sequenceTools. The resulting genotypes were merged with data from two other data sets: First, 2,367 modern individuals genotyped on the Affymetrix Human Origins Array34We called genotypes from the UDG treated data for the three individuals by sampling a random read per SNP in the SNP-capture panel, using a custom tool ‘pileupCaller', available at K=16 and we show the results of that run in We used the ADMIXTURE software on the merged data set to cluster ancestry proportions using different numbers of clustersWe performed PCA on the joined data set using the ‘smartpca' software from the Eigensoft package3-statistics of the form f3, where ‘Egyptian' means either ancient and modern Egyptian, and ‘X' runs over all populations in the merged data set. For the plot in et al.3-statistics of the form f3, where X runs over all populations in the merged data set. 3-statistics, where a negative Z score indicates a probable source for admixture.We used the ‘qp3pop' tool from the Admixtools packageSince two of the three selected samples had contamination rate estimates over 5%, we repeated this analysis using only sample JK2911, which has the highest SNP coverage and a contamination estimate of below 1%. The result is shown in n=90) and present-day populations (see n=135) and Ethiopian (n=120) mtDNA sequences from the literature for the respective area in upper Egypt, the El-Hayez oasisFST) based on the full mtDNA of these individuals. FST values were calculated using Arlequin v3.5.2.2  using the Benjamini–Hochberg methodp.adjust function in R 3.2.3 . We split our individuals in three groups  based on the 14C dates obtained from the samples  analysis of the HVR-1 sequences .The genetic distances were calculated in Arlequin v3.5.2.2 using the Tamura and Nei substitution model and a gamma shape value of 0.26, determined to be the best setting for the data using jModelTest v2.1.10. We selected the best parameters suggested by the Akaike and Bayesian information criterion (AIC and BIC).FST valuesFST values in a two-dimensional MDS plot with GNU R 3.2.4 using customized R script embedded in the vegan package  are modern populations from Saudi-Arabia, Kuwait, the United Arab Emirates, Yemen and other Near-East populations, whereas the individuals from another ancient population from Turkey (TRO) show more relatedness to modern North-African and populations from the Levantine. For details on the geographic mapping, see We used the linearized Slatkin's  package . Our ancWe used the 90 mitochondrial genomes obtained in this study, together with 135 modern Egyptian mtDNA genomes from Pagani and colleaguesThe obtained Bayesian SkyGrid plot indicates a fairly stable slightly decreasing effective population size for the studied population over the last 5,000 years . The aveprcomp function for categorical PCA implemented in GNU R 3.2.4 and plotted in a two-dimensional space, displaying the first and second principal components and shown in We performed a PCA to define relationships between our three ancient Egyptian populations based on their haplogroup compositions and modern, present-day populations from Europe, the Near East, West Asia and Africa. To generate the PCA, we divided our haplogroups in the following 20 groups: H, HV, I, J, K, L0, L1, L2, L3, L4, M1, N, R, R0, T, T1, T2, U, W, X and all remaining other haplogroups . The assignment was performed with reads that show a mapping quality of more than 30 only. We derived the haplogroups by identifying the most derived Y chromosomal SNPs in each individual  with the study ID ERP017224.The mapped BAM files for the 90 mitochondrial samples and three nuclear samples are deposited in the European Nucleotide Archive .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Sample NameNuclear capture resultsComparison of mitochondrial and nuclear Haplogroups. As the nuclear results do not have any specific enrichment applied for mitochondrial DNA retrieval, there are cases where the position was either not covered (yellow) or covered with less than 3 reads (red). Matching mutations were marked in green respectively between both captures.Results of the genetic distance computation with Arlequin between 56 populations from Europe, Africa, the middle East, Asia and the Ancient Egyptian metapopulation investigated in this study.Results and details of a population continuity test between our investigated three ancient Egyptian populations and modern populations from Egypt and Ethiopia in the respective region.Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary References


Abramis brama orientalis) is one of the most commercially valuable fish in the Caspian Sea. The aim of this study was to compare levels of genetic polymorphism between wild and farmed Bream populations using seven microsatellite loci. Genetic diversity was investigated by studying samples collected from two regions; Chaboksar and the Artificial Propagation Center of Guilan province. Allele frequency was found to have declined in wild and cultured fish due to inbreeding and genetic drift. Significant population differentiation (Fst) was observed between wild and farmed populations, which could be explained by the low number of alleles in two populations. Significant deviations from the Hardy-Weinberg equilibrium were found at more loci. Beyond the null alleles' hypothesis, heterozygote deficiency may have arisen due to inbreeding. Both populations showed lowest genetic diversity according to the number of alleles and genotypes per each locus. This approach was carried out for the first time and could provide information regarding the genetic variability of farmed and wild abramis brama fish using microsatellite markers. Results could be used for the management and conservation of artificial Bream propagation programs.Bream ( Abramis brama orientalis) is a commercially valuable food fish teleost  of the Caspian Sea. It is also found in the Anzali wetland [Abramis brama, currently bred in the aquaculture industry can be used to select strains for brood stock development and management plans and to improve its genetic diversity by minimizing inbreeding.Bream  to analyze genetic relationships among the samples.Higher levels of allelic variation at microsatellite markers can help address questions related to genetic structure. Microsatellites consist of multiple copies of tandemly arranged simple sequence repeats (SSRs) that range in size from 1 to 6 base pairs , 5. MicrOncorhynchus masou) [Oncorhynchus mykiss) [Salmosalar L.) [Salmo trutta L.) [cyprinus carpio) [Research on the genetic variation of farmed and wild populations has been reported for several species -13. Studs masou) , rainbow mykiss) , Atlantialar L.) , 17, broutta L.)  and the  carpio) , 20. Abramis brama farming and breeding programs, but also for the conservation of genetic resources of this species.The aim of the present study was to evaluate and compare the genetic diversity and differentiation of wild and farmed Caspian Sea Bream using microsatellite loci as genetic markers. The results will have implications not only for future Bream Sample collection and DNA extraction: A total of 60 samples were collected from two stations, Chaboksar costal water and the Artificial Propagation center of Guilan province. Total genomic DNA was extracted using standard phenol/chloroform protocols from fin pectoral tissues [2EDTA, pH: 8.0) overnight at 37°C. After incubation, DNA was isolated by two phenol-chloroform  steps followed by precipitation with cold absolute ethanol. The extracted DNA was then preserved at -20°C until used.  tissues . ApproxiMolecular analysis: In this study, seven microsatellite markers were amplified by polymerase chain reactions (PCR) using the following primers: , expected heterozygosity (He), the number of observed alleles (Na), the number of effective alleles (Ne), Hardy-Weinberg equilibrium (HWE) and Fst values were calculated by Genealex ver.6.5 Software [Software . PopGeneSoftware  and the Software .Abramis brama were investigated at seven microsatellite loci. TheBl2-114primer showed the maximum allele number (16) compared to other primers. No statistically significant difference was observed between the numbers of alleles per each locus  and expected (He) heterozygosity means for all samples were 0.30-1.00 and 0.55-0.90 respectively. In the wild fish samples, the mean for heterozygosity and expected heterozygosity values were 0.664 and 0.845, respectively. In the farmed samples, these values were 0.707 and 0.852, respectively, but the differences were not statistically significant (p>0.05).In this study, the genetic diversity of wild and farmed populations of ch locus . The numST) was moderate with a statistically insignificant FST value of 0.024 between the wild and farmed fish populations; however, RST value was significantly high (0.100) between the two populations. Genetic distances and similarities [is values ranged from 0.029 (the locus Bl1-153) to 0.431 (the locus MFW26) for the two populations.Significant deviations from the Hardy-Weinberg equilibrium (HWE) were found at the loci levels . All sevGenetic diversity is a major topic in aquaculture, especially in fish breeding and artificial propagation activities. Genetic differences between farmed and wild populations must be identified and quantified prior to any aquaculture activation if effective monitoring of gene flow is to occur. It is, therefore, important that adequate amounts of this kind of genetic information be available both for farmed and wild populations . HeterozAbramis brama population may be intensified by bottlenecks and inbreeding. In the current study, the observed heterozygosity for 7 microsatellite loci was lower than the expected heterozygosity in the two populations. It must be noted that observed heterozygosity is not always greater at all loci in wild as compared to farmed populations. The results of this study are similar to those reported for other farmed species using microsatellites and other molecular markers [S. aurata [The results of the present study indicated that the average number of alleles per locus and the observed heterozygosity in farmed fish were10.714 and 0.707, respectively. For the wild fish, these numbers were 10 and 0.664, respectively, indicating no statistical significance between the farmed and wild populations (p>0.05). Allele loss and heterozygosity in the  markers , 29. For. aurata . Heteroz. aurata .Both populations under study showed deviation from the HWE, since nine out of fourteen (7 loci × 2 populations) possible tests for HWE were statistically significant (P<0.05). Significant deviations from Hardy-Weinberg expectations (HWE) were observed in the two populations . Geneticst values were small  is a suitable criterion to assess population structure and determine the differentiation and genetic similarity between populations . Analysire small . Accordio Wright , Fst valei method [Abramis brama [ In the present study, genetic distances among the two populations were small, that is, genetic distances and genetic similarity between the wild and farmed fish, as computed by the Ni method , were 0.is brama .Abramis brama populations. The decline of genetic variability within the farmed and wild populations could be mainly due to the loss of (rare) alleles rather than reduced heterozygosity, and might have been caused by the relatively small number of breeders maintaining these strains. Such situations are typical for farmed fish due to the generally high fecundity of females. In the long run, this may lead to measurable inbreeding depressions such as reduced vitality and growth rate; therefore, inbreeding should be minimized by increasing the effective population size. According to recommendations by previous researchers [Abramis brama be directed towards larger numbers of wild and farmed populations with wider distributions.The results obtained from microsatellite markers showed high genetic diversity within and low diversity among populations. According to the analysis, it seems that the genetic diversity between farmed and wild fish is not statistically significant. Such situation is recommended for genetic diversity maintenance due to its important ecological roles. The results also indicate a tendency towards a reduction in variability within and between farmed and wild earchers , increas


Plasmodium vivax transmission in Thailand has been substantially reduced over the past 10 years, yet it remains highly endemic along international borders. Understanding the genetic relationship of residual parasite populations can help track the origins of the parasites that are reintroduced into malaria-free regions within the country.P. vivax isolates were genotyped from two western provinces (Tak and Kanchanaburi) and one eastern province (Ubon Ratchathani) of Thailand using 10 microsatellite markers. Genetic diversity was high, but recent clonal expansion was detected in all three provinces. Substantial population structure and genetic differentiation of parasites among provinces suggest limited gene flow among these sites. There was no haplotype sharing among the three sites, and a reduced panel of four microsatellite markers was sufficient to assign the parasites to their provincial origins.A total of 127 P. vivax cases can be reliably determined by genotyping four microsatellite markers, which should be useful for monitoring parasite reintroduction after malaria elimination.Significant parasite genetic differentiation between provinces shows successful interruption of parasite spread within Thailand, but high diversity along international borders implies a substantial parasite population size in these regions. The provincial origin of  P. vivax populations along the Thai-Myanmar and the Thai-Cambodian borders. Genotyping of parasite samples collected after intensified malaria control demonstrated that despite the decline in overall transmission intensity, the genetic diversity of the P. vivax parasites remained high. Parasite populations from three border provinces showed clear genetic separation. This indicates successful interruption of parasite gene flow within Thailand, but suggests frequent parasite migration across international borders. From the analysis of 10 microsatellite markers, we further refined a set of four that are sufficient for distinguishing the provincial origins of these parasites, which should allow tracking of parasite introduction among these provinces.This study presents an updated view of the  Plasmodium vivax remains the most geographically widespread human malaria parasite  using the infinite allele model (IAM), while it was 3–4 fold larger based on the stepwise mutational model (SMM). Significant linkage disequilibrium (LD), suggesting of inbreeding, was observed in Tak  and Kanchanaburi , whereas LD did not reach significance in Ubon Ratchathani .Effective population size (Table 4). The models detected significant deficiency in HE from the mutation-drift equilibrium, indicating events of population size reduction with possible clonal expansion in all areas.To detect whether there were significant changes in the parasite population sizes, we performed BOTTLENECK analysis using SMM  were enough to distinguish more than 96% of all haplotypes (112/116 haplotypes) in the study populations , and >94% in each province. Due to subtle differences in the diversity of the MS markers in different provinces, the optimal set of haplotypes for each province differed slightly (S3 Table). A similar cluster pattern was obtained when performing STRUCURE analysis using these four MS markers as compared with that using all 10 MS markers .We sought to identify a minimum set of markers suited to differentiate parasite populations based on their provinces of origin, and for differentiating parasites within each province. Stepwise removal of MS markers based on their P. vivax diversity and population structure along the Thai-Myanmar and the Thai-Cambodian borders with samples collected after malaria control had been intensified and transmission reduced. Our results clearly show that despite reduction of malaria prevalence in recent years, P. vivax diversity (HE) remained high. There were still ~20% of the isolates containing mixed-strain infections, and MOI was only slightly lower than in studies conducted 15–20 years ago in Thailand 2–1 and Neμ = HE_mean/4 (1-HE_mean), respectively. HE_mean is the average of the expected heterozygosity across all loci, while μ is the mutation rate per generation. Mutation rate values were derived from P. falciparum: 1.59 × 10−4  [The effective population size was calculated using two models, SMM and IAM, following the formula  × 10−5) .Assuming the SMM , we asseS1 TableThe mean number of alleles (A) and allelic richness (B) of each microsatellite in the three populations.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableRemaining haplotypes (% and number) by the stepwise removal approach for all three populations (A), and individual populations from Ubon Ratchathani (B), Tak (C) and Kanchanaburi (D).(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 FigR2 = 0.1993 at p < 0.05.The correlation between genetic and geographic distance were examined by the Mantel rank test in GenAlEx 6.5. Analysis was done pairwise, using isolates within and between provinces. The X-axis represents the pairwise geographic distance and the Y-axis indicates corresponding genetic distance. Positive correlation was shown with (TIF)Click here for additional data file.


Saccharina japonica is a commercially and ecologically important seaweed and is an excellent system for understanding the effects of domestication on marine crops. In this study, we used 19 selected simple sequence repeat (SSR) markers to investigate the influence of domestication on the genetic diversity and structure of S. japonica populations. Wild kelp populations exhibited higher genetic diversity than cultivated populations based on total NA, HE, HO, NP and AR. Discriminant analysis of principal components (DAPC), a neighbour-joining (NJ) tree and STRUCTURE analyses indicated that S. japonica populations could be divided into two groups (a cultivated/introduced group and a wild indigenous group) with significant genetic differentiation (P < 0.0001). Divergent selection, continuous inbreeding and inter-specific hybridization have caused the divergence of these two genetically separate gene pools. The significant genetic differentiation between northern and southern cultivated populations appears to be due to inter-specific hybridization and wild germplasm introduction during the domestication process. In addition, the cultivation of S. japonica has not resulted in any serious genetic disturbance of wild introduced S. japonica populations. An understanding of the genetic diversity and genetic structure of domesticated S. japonica will be necessary for further genetic improvement and effective use of germplasm. Saccharina and Undaria and the red algae Porphyra, Gracilaria and Eucheuma.Approximately 13,000 years ago, humans began to domesticate plants and animals225Plant domestication not only modifies economic and agronomic phenotypes but also leaves a genetic signature that affects both the population structure and the genetic diversity of the domesticated species78910Saccharina japonica (Aresch) Lane, Mayes, Druehl & Saunders is native to cold-temperature coasts along northern Japan, northwestern Korea and Far Eastern Russia and was introduced into China in the 1930sS. japonica is a commercially and ecologically important seaweed that is mainly cultivated in China, Japan and Korea. China, in particular, ranks first in the world for the cultivation of this species with a wet weight production of 5,941,658 t in 2013 . S. japonica was initially cultivated in the north of China using conventional methods  as early as the 1930 s, but modern cultivation started in the 1950s1213141315161314S. japonica populations from Japan or wild Chinese introduced populations were cultivated rather than selected strainsS. japonica, the first variety to be produced being “Haiqing No. 1”14202122Early in the domestication process, wild Undaria pinnatifida, Ulva prolifera and Porphyra yezoensis910S. japonica populations in China and indicated that extensive selection and inbreeding for multiple generations are likely to also have reduced the genetic diversity of cultivated S. japonica populations252627S. japonica that are cultivated at present. In addition, some studies showed that the escaped cultivated kelp from the farms might cross-hybridize with wild populations, resulting in genetic introgression of cultivar genetic material into wild populations3511S. japonica. Estimating the existence and extent of such a gene flow might be helpful for designing breeding practices and conservation strategies.There is evidence that domestication has led to a reduction in genetic diversity for several cultivated seaweeds, including S. japonica populations . The objectives of our study were 1) to examine if Chinese populations after domestication are less genetically diverse than wild indigenous populations, a pattern expected given the known history of the introduction of S. japonica in China, 2) to test the prediction that kelp germplasm introduction and inter-specific hybridization will increase the genetic diversity and enlarge the genetic divergence of cultivated S. japonica populations by introducing the new genetic variations, and 3) to evaluate the genetic diversity of wild introduced populations and investigate whether escapes from cultivated populations influence the wild introduced populations. The long-term objective is to improve selection and breeding and sustainable utilization of S. japonica seed stocks.Using selected microsatellite markers, we conducted a population genetic analysis of 28 A) ranged between 7 for SJ93 and 50 for SJ136  had high frequencies of null alleles (>0.06)  was slightly higher than the corrected FST values , and the pairwise FST per locus without correction was also higher than the pairwise FST with correction (data not shown). High frequencies of null alleles have the potential to influence the estimation of genetic differentiation. Consequently, we excluded these four loci  from this study and reported only the results based on 19 SSRs.A total of 766 individuals from 28 populations  were genor SJ136 . The SSRor SJ136 . The mea (>0.06) . The gloAfter false discovery rate (FDR) correction for multiple tests, linkage disequilibrium (LD) tests for each pair of loci indicated that 171 pairs (3.5%) were significantly in disequilibrium. Given that these loci did not share corresponding disequilibria in all samples, we assumed that none of the loci were physically linked. No consistent pattern of linkage disequilibrium was observed, so the 19 loci were used for all subsequent analyses.S. japonica populations at the population and group levels (NA) varied from 1.79 ± 0.10 for XP (one southern cultivated population from China) to 9.11 ± 1.24 for SA . Allelic richness (AR) based on 20 samples per population was highest (8.19 ± 1.07) in the SA population and lowest (1.78 ± 0.10) in the XP population. There were no private alleles in the cultivated populations, but private alleles existed in all wild introduced/indigenous populations (WI + WR + WJ) except one wild introduced population (YM). The mean observed heterozygosity across loci (HO) ranged from 0.25 ± 0.05 for XP to 0.68 ± 0.04 for HA , and expected heterozygosity across loci (HE) ranged from 0.25 ± 0.04 for XP to 0.71 ± 0.04 for HA.Genetic diversity was evaluated for 28 p levels . At the NA, AR, NP, HO and HE) at the group level (HS = 0.539) was significantly higher . The genetic diversity indices in northern cultivated populations  were higher than in wild introduced populations  and southern cultivated populations   .FIS values showed significant deviation from zero  had significantly positive FIS values, indicating heterozygote deficiency. After FDR correction, the DF2 population showed significant departures from HWE, involving 16 of the 19 loci, all due to heterozygote excess.Recent changes in effective population size were detected based on the Wilcoxon signed rank-test under the infinite allele model (IAM), the stepwise-mutation model (SMM), and the two-phase mutation model (TPM). Eight cultivated populations and two wild introduced populations showed significant heterozygote excess compared to the expected equilibrium in the IAM after FDR correction . The TPMS. japonica populations into two genetic clusters (Da) indicated that the wild indigenous cluster of populations was clearly distinct from the cultivated/introduced cluster (except DF3)  partitioned the clusters . This paclusters . A neighept DF3) . Bayesiaept DF3) . The metept DF3) . A seconept DF3) . Wild inept DF3) . Most ofept DF3) .FST values with and without correction for null alleles showed some slight variation with differences ranging from 0.000 to 0.026 (data not shown). Of the 378 pairwise tests conducted among 28 kelp populations, 370 were highly significant (P < 0.0001), and 6 were significant (P < 0.05) after FDR correction  to 0.695 (XP and EP) . However, pairwise FST values between DF3 and three Japanese populations  varied from 0.152 to 0.240, which indicated moderate genetic differentiation among those populations  compared with other cultivated populations. In addition, two northern cultivated populations (901 and NJ) were highly differentiated from three southern cultivated populations  with FST values ranging from 0.252 to 0.330. When FST values among populations within each group were calculated independently, the highest value was found within the WR group (FST = 0.305) and the lowest value (FST = 0.063) within the WI group (FST = 0.247) was significantly larger than for cultivated populations  (Pairwise rrection . Therefo and EP) . The Chiulations . There wWI group . In addi= 0.199) .ФCT = 0.30; P < 0.001) , while 54.85% of the variation existed within populations. AMOVA results based on northern and southern cultivated populations showed significant Ф-statistics among populations , among populations within groups  and among groups . All variance components were statistically significant  . The genU. pinnatifida, U. prolifera and P. yezoensis910NA, HE, HO, NP, AR and HS in each group indicated that genetic diversity was higher in the Japanese wild populations than in the domesticated kelp populations. This difference may be due to continuous selection having reduced the effective population size and increased genetic drift and hitchhiking during the domestication process832S. japonica in China192627Generally, domestication tends to reduce allelic variation and genetic diversity inducing genetic erosion in cultivated seaweedsS. japonica cultivated populations  . Therefore, we presumed that overdominant selection in the breeding cultivars favoured heterozygote survival and caused the heterozygote excess with hitch-hiking selection. One common explanation for observing heterozygote excess is the self-incompatibility system effectS. japonica was caused by active avoidance of selfing and full-sibling mating.Departures from Hardy–Weinberg expectations due to heterozygosity excess were detected in five  and XP) , possiblS. japonica indigenous populationsIn contrast to cultivated populations, five wild populations showed significant departure from HWE with heterozygote deficiencies. An excess of homozygosity within wild populations can be due to at least three causes: the presence of null alleles, inbreeding and population subdivision. Inbreeding could be common in wild populations, possibly due to the limited dispersal of the kelp gametes, favouring mating within the kelp siblings or close relatives38S. japonica populations. DAPC plots, an NJ tree and STRUCTURE analyses indicated that the clustering of the southern cultivated populations (SC) and the wild introduced populations (WI) was principally driven by these transplantations  had lower genetic diversity than wild indigenous populations (WR + WJ). This was consistent with our previous results that wild introduced populations have lower haplotype and nucleotide diversity than wild indigenous populations1416ntations .S. japonica have not been markedly impacted by gene flow from cultivated populations.The northern cultivated populations (NC) and the wild introduced populations (WI) all grow in the north of China, but they did not cluster together in our analyses. We believe that the northern cultivated populations (NC) are genetically different and diverse as a result of interspecific hybridization and/or multiple introductions of wild indigenous germplasm12202123vs. southern differences), our analyses indicated significant genetic differentiation among these cultivated populations   . The difS. japonica clustered together and were separated from descendants of inter-specific crosses in the NJ tree  that were derived from hybridization between S. japonica and S. longissima clustered togetherS. latissimaMost northern cultivated populations from inter-specific and intra-specific crosses have distinct genetic structure patterns. Indeed, most cultivated populations bred from descendants of  NJ tree . Liu et Based on the STRUCTURE analyses and NJ tree, the northern cultivated population DF3 clustered with wild indigenous populations. We suspected that this cultivar might have been unconsciously or deliberately mixed with wild kelp germplasm by farmers during the breeding or cultivation processes. To verify whether there is contamination and degradation in these DF3 varieties, a detailed study of the DF3 strains cultivated in different farms should be further conducted.S. japonica strains analysed in this study suggest that this cultivated kelp represents a single complex gene pool within which historical movement of germplasm, recent introductions, interspecific hybridizations and human selection are shaping the genetic structure. In our present study, the expected reduced diversity of cultivated populations was confirmed, and genetically distant populations or geographically isolated populations should be used for enhancing diversity and improving productivity. Diverse wild S. japonica resources are essential for the restoration and selection processes necessary for Chinese kelp production. Further genome-wide analysis of the domesticated kelps will provide more information to understand in detail the micro-evolution processes that have occurred during the domestication and thus to improve kelp breeding strategies.In conclusion, the overall genetic structure of the S. japonica populations , including twelve northern cultivated populations (NC), five southern cultivated populations (SC), three wild introduced populations (WI) and eight wild indigenous populations from Russia and Japan (WR and WJ) (2223We sampled 28  and WJ) ; Fig. 1. and WJ) . Six of et al.genemarker v2.2.0 (SoftGenetics).Twenty-three simple sequence repeat (SSR) markers were screened from a list of 76 SSRs previously developed by Li Micro-checker 2.2.3 was used to check for potential genotyping errors caused by stuttering or large allele dropout and the presence of null allelesfreenagenepop 4.2.2qvalue (R Development Core Team 2013). For polymorphism evaluation of each SSR locus, allele numbers (A), expected and observed heterozygosity (HE and HO), and polymorphic information content (PIC) were calculated using popgene 1.3.1NA) and allelic richness (AR) across all loci based on the minimal sample size with the rarefaction method were calculated in fstat 2.9.3.2HE and HO) and numbers of private alleles (NP) were calculated using genalex 6.4149. The significance of departures from Hardy–Weinberg equilibrium, as evidenced by deviation of inbreeding coefficient (FIS) from zero, was tested with 5,000 randomizations using fstat 2.9.3.2pgdspider 2.0.1.0convert 1.3.1bottleneck 1.2.025354Screens for signatures of genetic bottlenecks were carried out for cultivated and wild populations using FST across all loci was investigated with correction for null alleles, using freena with 10,000 bootstrap resampling, to avoid the impact of null alleles on the estimation of genetic differentiationarlequin 3.5.1.3Global and pairwise arlequin 3.5.1.3 to partition genetic variation across nested levels: within populations, between populations within groups, and between groupsAnalysis of molecular variance (AMOVA) was conducted in S. japonica populations, multivariate analyses were carried out using discriminate analyses of principal components (DAPC) using the adegenet 1.3.1 package57Da, and the non-rooted tree was generated using the neighbour-joining (NJ) algorithm with 1,000 bootstrap replicates in poptreefigtree 1.4.2 (available at http://tree.bio.ed.ac.uk/software/figtree/). Bayesian analysis of the population genetic structure with structure 2.3.1 was applied with the admixture model and correlated allele frequencies61structure harvesterdistruct64.To infer genetic clusters in How to cite this article: Zhang, J. et al. Effect of domestication on the genetic diversity and structure of Saccharina japonica populations in China. Sci. Rep.7, 42158; doi: 10.1038/srep42158 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.