Lycorma delicatula (White) (Hemiptera: Fulgoridae), is a polyphagous pest originating in China and now widely distributed in Asian countries. This is one of the more serious forestry pests with a broad host range and causes significant economic losses. Molecular comparison has been used to investigate this pest’s origin in China, and recent studies have explored the genetic structure among populations in Korea. However, the population structure of this pest in China remains poorly understood. In this study, 13 microsatellite markers and two mitochondrial markers (from nicotinamide adenine dinucleotid (NADH) dehydrogenase subunit 2 (ND2) and NADH dehydrogenase subunit 6 (ND6) regions) were used to reveal the origins and dispersal of L. delicatula based on a genetic analysis of Chinese populations from eight locations. Results show a low to high level of genetic differentiation among populations and significant genetic differentiation between both two clusters and four clusters. The network and phylogenetic analyses for mitochondrial haplotypes and population structure analyses for microsatellite datasets suggest that there is potential gene flow between geographical populations. The populations from Zhejiang and Fujian provinces may come from the other geographical populations in north China. The populations from Beijing, Henan, and Anhui provinces were regarded as the major source of migrants with a high number of migrants leaving (the effective number of migrants (Nem) = 24.40) and the low number of migrants entering (Nem = 2.05) based on the microsatellite dataset, where significant asymmetrical effective migrants to the other populations were detected by non-overlapping 95% confidence intervals.The spotted lanternfly,  Lycorma delicatula . Th. ThHWE > > 0.05)  ranged 4ND2 and ND6 fragments, respectively. Seventeen ND2 haplotypes and nine ND6 haplotypes were uploaded to GenBank (accession numbers: MK450267–MK450288). In this study, 201 combined mitochondrial sequences were used to detect the genetic variation of mitochondrial DNA. The values of haplotype diversity, nucleotide diversity, and the average number of nucleotide differences of combined ND2 and ND6 were calculated in eight populations  based on microsatellite markers suggested a low to high level of genetic differentiation among populations  (p < 0.0001) and the combined mitochondrial datasets . The PCoA results also show a four-cluster pattern of genetic structure. The first two axes explain 40.94% and 27.11% of the overall variance on the population level .The ulations  64]. Th. ThFST vlineages . Althouglineages , the moslineages . Results 0.0001) . Signifion level . There iThe distribution of haplotypes is shown in FST values between populations ranged from 0.0001 to 0.9729 (p < 0.0001) (p = 0.0293) and the combined mitochondrial datasets . There was no significant IBD pattern between the genetic and geographical distances based on mtDNA .The phylogenetic relationships show that all haplotypes of populations were divided into two clusters: the haplotypes of populations north of the Yangtze River clustered closely, as well as the haplotypes of populations south of the Yangtze River . The FSTo 0.9729 . The pri 0.0001) , and theThere are significant asymmetrical effective migrants of migrants between four clusters divided by the result of STRUCTURE analysis. The effective population size ranged from 0.0194 for cluster 1 to 0.0981 for cluster 2 . The effp > 0.05) (p < 0.05), which may suggest a recent genetic bottleneck. Under TPM and SMM, BJ , HN , and AH  populations showed some evidence of heterozygosity excess, as well as the SD population under TPM (p = 0.021).Any result of a bottleneck was rejected for SX, GS, ZJ, and FJ populations under TPM ( > 0.05) . All popp > 0.05), and the significantly negative values of Tajima’s D and Fu’s Fs suggests a recent demographic expansion for FJ populations  .The McDonald–Kreitman neutrality test indicates no deviation from neutrality  . BJ popuL. delicatula populations in areas south of the Yangtze River to be higher than that in the north populations (Although the most common haplotype (H1) was found mostly in BJ, HN, and AH populations, both microsatellite and mtDNA datasets revealed a lower genetic diversity in these three populations than in other populations. This study also found the genetic diversity of ulations  based onulations . The higL. delicatula populations south of the Yangtze River may come from populations north of the Yangtze River. The haplotypes in Korea and Japan came from the north clade, which is consistent with previous suggestions [The phylogenetic analysis  reveal agestions .FST based on microsatellite and mtDNA datasets show a clear genetic differentiation and segregation among L. delicatula populations in China. There was a high level of genetic differentiation between SX/GS populations (cluster 3) and other populations, as well as ZJ/FJ (cluster 4) populations. These four populations have their private haplotypes and a higher genetic diversity, and all experienced a recent demographic expansion (p > 0.05 under TPM and SMM). Significant asymmetrical effective migrants from cluster 2 (BJ/HN/AH populations) to cluster 3 and cluster 4 were detected by non-overlapping 95% confidence intervals  using mitochondrial markers. L. delicatula was previously known to occur in only Shaanxi, Shandong, and Hebei in the 1930s [L. delicatula spread from the BJ, HN, and AH populations to the other populations in the areas north of the Yangtze River. The phylogenetic relationship of private haplotypes from the GS (H15 and H16), SX (H22), BJ (H6), SD (H18-21), HN (H4), and AH (H17) populations and asymmetrical migrants between clusters reveal that the potential gene flow occurred between six populations.Six populations of he 1930s ,25. Now L. delicatula populations in China shows a significant IBD pattern, further subdivision of genetic structure, and more asymmetrical effective migrants. It means microsatellite markers can provide more information than mitochondrial fragments on population structure, population dynamics, and dispersal patterns. These two markers showed some migrants between different populations. A probable cause is the increase in afforested areas in recent years, which in turn expanded the distribution of the host plants throughout China and increased migrants between populations. Meanwhile, higher genetic diversity and some biological features  all contributed to the successful establishment of L. delicatula in new areas [Compared with the mitochondrial dataset, the microsatellite dataset of the ew areas ,20,24. IL. delicatula populations. There was a high level of genetic differentiation between SX/GS populations and other populations, as well as ZJ/FJ populations. Significant asymmetrical effective migrants from BJ/HN/AH populations to SX/GS populations and ZJ/FJ populations were detected. BJ/HN/AH populations were regarded as the major producer of migrants to the other populations. This study improves our understanding of the origin and the dispersal of L. delicatula. Biological features, together with numerous suitable habitats, may account for this establishment of L. delicatula in new areas. In the recent past, sudden outbreaks of this pest occurred in China, Korea, and Japan, and this pest has also invaded into North America. Future work should pay more attention to the possible invasion pattern of L. delicatula based on microsatellite and mitochondrial markers.A significant genetic differentiation was found among Chinese


Rubus chamaemorus L.), collected from Krkonose Mountains (the Czech Republic), continental Norway and Spitsbergen, was examined using microsatellite analyses (SSR). Among 184 individuals, 162 different genotypes were identified. The overall unbiased gene diversity was high (h^=0.463). A high level of genetic differentiation among populations  indicated restricted gene flow between populations. Using a Bayesian approach, six clusters were found which represented the genetic structure of the studied cloudberry populations. The value of correlation index between genetic and geographical distances (r = .44) indicates that gene flow, even over a long distance, could exist. An exact test of population differentiation showed that Rubus chamaemorus populations from regions  are differentiated although some individuals within populations share common alleles even among regions. These results were confirmed by AMOVA, where the highest level of diversity was found within populations (70.8%). There was no difference between 87 pairs of populations (18.7%) mostly within cloudberry populations from continental Norway and from Spitsbergen. Based on obtained results, it is possible to conclude that Czech and Norwegian cloudberry populations are undergoing differentiation, which preserves unique allele compositions most likely from original populations during the last glaciation period. This knowledge will be important for the creation and continuation of in situ and ex situ conservation of cloudberry populations within these areas.The population structure of cloudberry ( Sp. Pl. 494, 1753; Family: Rosaceae; Figure n = 8x = 56 with the estimated genome size 2.46 pg/2C  Has the differentiation of populations of Rubus chamaemorus already occurred from the Czech Republic, continental Norway, and Spitsbergen after last glacial period? (2) Is there any gene flow among populations? (3) How much genetic diversity is maintained in these naturally fragmented populations of Rubus chamaemorus and what does this mean for its conservation management?Therefore, the main aim of this study was to assess the genetic diversity, differentiation, and structure of isolated populations of 22.1Rubus chamaemorus, including 184 individuals, were sampled in 2015 and 2016 , 0.33 μmol/L of each primer , 1U Tth polymerase , and 50 ng DNA template. The PCR was performed in a Sensoquest Labcycler  under the following conditions: an initial denaturing step of 95°C for 5 min, followed by 35 cycles of 30 s at 95°C, 30 s at primer pair‐specific annealing temperature and then 40 s at 72°C, and finally finished at 72°C for 5 min. The analysis of the PCR products was performed using capillary electrophoresis on the sequencer ABI PRISM 3130 . A multiplexed configuration of four reactions was used in one analysis. The internal size standard LIZ500  was used. The electropherograms were processed using the GeneMapper software .To study  Russell , Graham  Russell  and by C Russell . The PCR2.3Clone identity was determined using multilocus matches for codominant data. The probability of identity  or related (PIsib) individuals by a particular set of loci) were calculated based on the distribution of allele frequencies in population samples using software GENECAP . The support for the phenogram branches was obtained using 2,000 bootstrap resamplings.A matrix of distances between all of the samples was calculated using the simple matching dissimilarity coefficient in the DARwin software . Arlequin software was also used to evaluate the correlation between a matrix of logarithmic geographical distances and a matrix of FST values using a Mantel test with the recommended 10,000 permutations.The divergence statistics were estimated using the hierarchical analysis of molecular variance  were performed using locprior model with admixture and correlated allele frequency  and on the ΔK calculation, which considers the rate of change in the ln P(D) values among successive K runs to account for patterns of dispersal that are not homogeneous among populations  of clusters into which the sample data (X) were fitted with posterior probability Pr (X|K) was estimated using the same model with 1,000,000 Markov chain iterations after a burning period of 100,000 iterations , and the highest was in the continental Norway population from Kvaloya island NN7  to 16 (Ru126b3), with a mean number of alleles per locus of 6.4. The percentage of polymorphic loci ranged from 34.6% for the populations S4 and S7 to 85.2% for the population NN7, with an average of 96.4% across all cloudberry samples. Nei's average gene diversity values ranged from 0.194 in population S7 to 0.450 in population NN4 , of moderate level in populations from the Krkonose Mountains  and the highest in main cloudberry localities from Norway .The level of genetic diversity was the lowest in cloudberry populations from Spitsbergen  were identified among Rubus chamaemorus populations  to 0.555 (K5). The highest values of FST were found in the clusters K3 (0.426) and K5 (0.555) which consisted of cloudberry populations from the Czech Republic and K4 (0.491) which consisted of the populations from Spitsbergen. Figure K = 6 and shows admixtures among populations  but statistically significant (p < .01).The results of the analysis of molecular variance (AMOVA) indicated that 70.8% of the total variation was attributable to differences among individuals within populations, 17.3% was attributable to differences among populations within groups (populations within countries), and only 11.9% was attributable to differences among groups  across all the sampled localities, indicating some level of isolation‐by‐distance. If populations where gene flow over a long distance was noticed  were omitted, the value of the correlation coefficient increased . A model of linear regression was selected for the representation of the relationship between geographical and genetic distances  but when we considered regions  as populations, FST value was 0.19. The estimated gene flow, M = Nm was 0.31, and 1.08 when regions were taken as populations. This indicates that some populations are much more differentiated than others and gene flow is generally restricted but occurs between some populations. Almost all pairwise FST values were significant (p < .05), ranging from −0.08 to 0.50  was found for the pairs of population groups from CR and continental Norway, CR and Spitsbergen, continental Norway and Spitsbergen.Significant differentiation was also found between groups of populations from east and west parts of the Krkonose Mountains , groups of populations from the north and south part of continental Norway , and groups of populations from two localities on Spitsbergen . Based on the pairwise test of genetic differentiation between all cloudberry populations, the null hypothesis that there is no difference between a pair of populations could not be rejected in 87 pairs of populations (18.7%)  similar to that of Rubus glaucus , where populations are more frequently reproduced. The lowest level of genetic diversity was detected in Spitsbergen (h^=0.301) and a moderate level in the Krkonose Mountains, CR (h^=0.432). This high level of genetic diversity is surprising especially in the Krkonose Mountain populations which have been isolated from the main cloudberry growing areas since glacial melting after the end of the Last Glacial period  from other CR cloudberry populations. The second true population involves local populations WK2, WK3, WK5 not differentiated from each other . The third population involves the whole local population EK2 which is distinct from other CR populations . So, cloudberry populations in the Krkonose Mountains came most probably from at least three different genetic populations from the last glacial period (Figures R. chamaemorus populations in Scandinavia or Russia, as the level of glaciation changed in the Quaternary period (Engel et al., According to our results, samples from the Krkonose Mountains are three genetically distinct populations Figures  and 3. TVaccinium uliginosum populations and proposed a possibility of recolonization from long‐distance source populations by means of wind, drifting sea ice, drift wood, or birds. Ehrich et al. (Alsos et al.  obtainedh et al.  studied h et al. . Gene flRubus chamaemorus largely reproduces itself vegetatively. Few seeds are produced in most areas but in a restricted number of localities, seed is produced regularly in fair quantity, although viability is poor (Taylor,  Taylor, . In the  Taylor, . It seem Taylor, . In spit Taylor, b.Rubus chamaemorus from the Ural Mountains or from western Siberia (Alsos et al., R. chamaemorus populations. Fitness of these populations is maintained by intermittent flowering brought about by the occasional opportunity of hybridization between individuals from the same or different populations.The northernmost populations of cloudberry in Spitsbergen showed the lowest level of diversity. They grouped into two genetic groups: one unique to Spitsbergen populations and one shared between Spitsbergen and continental North Norway populations Figure . If the In conclusion, cloudberry populations EK1WK2, WK3, WK5S3, S4, S5, S6, and S7 from the Krkonose Mountains, continental Norway, and Spitsbergen are well differentiated and are likely to represent the original gene pool. In contrast, in populations EK2, WK1, WK4 S1, and S2 there is evidence of regular gene flow and hybridization; therefore, these are not differentiated from populations in continental Norway.Rubus chamaemorus populations from the Krkonose Mountains have a moderate level of genetic variability, which is good for sustainable vitality of such heterogenous populations. They may be less susceptible to climatic fluctuations during changing of climate and other anthropogenic factors. The variability is also important for conservation management of the endangered species in the Czech Republic as it means current management strategies are appropriate along with regular monitoring of populations fitness (Phillips, Asdal, Brehm, Rasmussen, & Maxted, R. chamaemorus in the Krkonose Mountains occur within the first zone of the Krkonose National Park, and thus, they have the highest level of protection. On the other hand, the Spitsbergen populations that are restricted to only several fragmented small sites and have a low level of genetic diversity are more threatened. Moreover, as they multiply mostly vegetatively there is a limited gene flow. The diversity of such limited populations is unique and appropriate in situ and ex situ conservation of those populations will be of a high priority. The whole Spitsbergen archipelago is protected; nevertheless, a special attention on R. chamaemorus sites would be desirable, especially an increase in monitoring. Conservation of R. chamaemorus in the global sense does not mean ensuring the survival of every population, but it is necessary to conserve the widest range of its genetic diversity. It will be necessary to work closely with national experts and conservation managers in the Czech Republic, continental Norway, and Spitsbergen to ensure the range of diversity illustrated here is conserved both in situ and ex situ.None declared.L.L.S., J.P., I.M., and V.H. conceived the ideas and collected samples; L.L.S. conducted genotyping and analyzed data; and all authors contributed to interpretation and writing which was led by L.L.S.All data are in supporting information, and they will be available at time of publication. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Medea SGEs occur naturally in some populations of red flour beetle (Tribolium castaneum) and are expected to increase in frequency within populations and spread among populations. The large‐scale U.S. distributions of Medea‐4 (M4) had been mapped based on samples from 1993 to 1995. We sampled beetles in 2011–2014 and show that the distribution of M4 in the United States is dynamic and has shifted southward. By using a genetic marker of Medea‐1 (M1), we found five unique geographic clusters with high and low M1 frequencies in a pattern not predicted by microsatellite‐based analysis of population structure. Our results indicate the absence of rigid barriers to Medea spread in the United States, so assessment of what factors have limited its current distribution requires further investigation. There is great interest in using synthetic SGEs, including synthetic Medea, to alter or suppress pest populations, but there is concern about unpredicted spread of these SGEs and potential for populations to become resistant to them. The finding of patchy distributions of Medea elements suggests that released synthetic SGEs cannot always be expected to spread uniformly, especially in target species with limited dispersal.Selfish genetic elements (SGEs) are DNA sequences that are transmitted to viable offspring in greater than Mendelian frequencies.  Medea‐4 element distribution over time. The Medea‐4 element in the United States is widespread, but no longer in a latitudinal distribution.This figure compares the  Otherwise, beetles were collected by hand or sifted from the substrate. In cases where visiting the sampling site was not possible, beetles were collected and shipped directly from the site to our laboratory by an extension agent or facility employee.Red flour beetles used to assess the current distribution of Dead beetles were frozen as soon as possible after collection. Live beetles were permitted to mate and oviposit for up to 3 weeks on a mixture of organic whole wheat pastry flour and 5% (by volume) brewer's yeast. These beetles were reared at 22–23°C and approximately 60% relative humidity in a controlled quarantine facility in order to establish cultures from each location; after this period, the originally collected beetles were removed and frozen for later genotyping. Cultures were maintained at the same temperature and humidity in a quarantine facility via periodic subculturing and flour replenishment.1, red flour beetles were also obtained from samples collected between 2004 and 2007. Samples from 2007 were collected in traps containing oil. This oil was removed from the beetles prior to DNA extraction by rinsing the beetles for 5 min in CitriSolv (Fisher Scientific), followed by a wash in double‐distilled water.In an effort to make inferences about an earlier distribution of M2.24 molecular marker at the time of these experiments, the presence of M4 in selected populations was assessed via crosses. Females from the homozygous M4pearl strain were crossed to the non‐Medea GA‐1 strain .Because we lacked a reliable Mn Beeman , surviva4‐heterozygous females and wild‐derived males of unknown M4 status is close to 100% survivorship if the male is actually homozygous for M4, roughly 75% for crosses to males heterozygous for M4, and about 50% offspring survivorship in crosses to males lacking the M4 element. We used this information to categorize the probable genotypes of individual beetles.The expectation for offspring from crosses between known M2.31 element has been fully sequenced, we were able to design M1‐specific primers and genotype via PCR.Because the MForward primer: 5′‐TGGCGATAGTCAAAATCCTTTGTCG‐3′1 reverse: 5′‐TGCCACCTTCACGTAGCCCG‐3′MWild‐type reverse: 5′‐CAGGGCCCCGGAGTATTTTTCC‐3′Genomic DNA was extracted for 25 individual beetles per location  using the Qiagen DNeasy Blood and Tissue Kit. Primers used for amplification were as follows:2, 200 µM dNTPs, 2 µl DNA template, 1 μmol forward primer, 0.5 μmol of each reverse primer, 0.5 U Taq DNA polymerase (Genesee), and ddH2O to 25 µl. Thermal cycling consisted of initial denaturation of DNA template at 95°C for 4 min followed by 40 cycles of 95°C for 30 s, 58°C for 45 s, and 72°C for 75 s, and a final extension step of 72°C for 5 min. Alleles were separated on 2.5% agarose gels infused with ethidium bromide and visualized by ultraviolet (UV) illumination.PCRs were performed in 25 µl volumes, each containing 1× PCR buffer, 3.5 mM MgCl2.41 relied on genotyping via a molecular marker, crosses were performed in select M1‐fixed populations to diagnose whether the M1 sequences amplified during genotyping represented fully functional M1 elements. Females from populations that were genotyped as fixed for M1 were crossed to males from the GA‐1 strain, a laboratory strain devoid of Medea elements. Five presumably M1‐heterozygous females derived from each cross were then paired with GA‐1 males, and eggs from each cross were counted, with the proportion of surviving offspring used to determine whether maternal‐effect lethality had occurred. Survival rates near 100% would indicate the absence of Medea elements, while survival rates near 50% would imply the presence of a Medea element in the source population. Five additional heterozygous females per population were backcrossed to males from their population of origin, and the proportion of surviving offspring  was used as a control.Because our assessment of the distribution of M4 element, three heterozygous females from each population were also crossed to males from the homozygous M4pearl strain. The absence of rescue in these crosses indicated the presence of a functional Medea element other than M4, presumably M1, given that M1 is the only other Medea element that has been found in the United States.To determine whether any maternal‐effect lethality uncovered in these populations could be attributed to the M2.51 distribution  sequence CACGACGTTGTAAAACGAC, and a universal M13 (−29) primer labeled with either IRDye 800 or IRDye 700 was added to the reaction , 0.5 U Taq polymerase (Genesee), and ddH2O to 10 µl. The cycling conditions consisted of an initial denaturation step at 94°C for 4 min, followed by 15 cycles of 94°C for 30 s, 65°C (−1°C/cycle) for 30 s, and 72°C for 1 min and 20 s, and ending with 30 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 45 s.PCR was performed in 10 µl reactions, with each containing 2 µl genomic DNA (approximately 50 ng), 1× PCR buffer, 2 mM MgCl2O, and 10 µl of a formamide stop solution  was added, followed by a denaturing step consisting of 95°C for 5 min. Fragments were separated by electrophoresis on a 6.5% Long Ranger 1× TBE polyacrylamide gel, run on a Li‐Cor 4300 automated DNA sequencer at a constant power of 40 W at 45°C for 1.5 hr. Fragments were sized using a 50–350 bp IRDye 800 or 700 standard . Allele sizes were scored using QuantarPro software (KeyGene). Individuals missing data at more than three loci were removed from further analyses. Because this left population SC‐2 with only 10 individuals, it was excluded from population‐level analyses.The PCR products were diluted with 10 µl ddH2.7p‐value. Significance levels of the tests were adjusted for multiple comparison following standard Bonferroni corrections , where both the "admixture" and "no admixture" models were run, with sampling location used as a prior, and allele frequencies correlated. Five replicates each from K = 1 to K = 15 were run with a burn‐in period of 200,000 steps followed by 200,000 MCMC iterations. The most likely value of K was determined in Structure Harvester  with ENA corrected FST values and another semimatrix of natural log‐transformed kilometer linear distances between sample locations. These analyses were also performed with the original uncorrected FST values, and the results obtained were compared to assess whether there were significant differences.The extent of genetic isolation due to geographic distance was assessed via a Mantel test with 10,000 permutations in Genepop's Isolde program , and largely absent at higher latitudes.While we did not obtain enough archived samples from 2004 to 2007 to make a full, comprehensive assessment of the prior distribution of M1 Table , the daty Beeman , where t1 element in the United States , eastern Alabama/western Georgia (4 locations), and the gulf coast (2 locations).Although the small number of sampled locations does not allow for a thorough comparison of the 2004–2007 distribution with the current‐day distribution, the pattern seen in the 2004–2007 samples is interesting, nonetheless. Our distribution analysis for the 2011–2014 samples revealed five nonoverlapping clusters Figure . The lar3.3FST across all loci and locations ranged from a high of 0.264 (between NE and SC) to a low of 0.002 (between MS and SC). Global per‐locus FST values averaged 0.0669 ; however, when excluding food production sites, a positive correlation between genetic distance and geographic distance remains, but the correlation is no longer statistically significant (p = .094) . Overall, samples from these sites also showed significantly lower allelic richness, lower observed heterozygosity, and lower within‐population genetic diversity. We tested to determine whether there was correlation between latitude and M1 frequency based on a sample of 31 populations. The correlation of 0.240 was not significant. We also tested for a correlation between M1 frequency and level of heterozygosity with the hypothesis that beetle populations with M1 would have lower heterozygosity due to linkage between the invading Medea element and alleles at other loci. We found no correlation (r = −.003).When looking at microsatellite loci at all sample locations, we find a trend toward isolation by distance (IBD) (using corrected ) Figure . Driving1 frequency differences among populations  from the structure analysis of microsatellite data was two, the delta K value (5.06) was quite small , we would expect both elements to persist, provided the introduction frequencies were not extremely low   and fixation could take 2–5 years  that have limited dispersal.Recent advances in molecular biology have enabled the development of synthetic None declared.SAC, FG, and MDL designed the research; MDL provided reagents; SAC performed the research, analyzed the data, and wrote the original draft; SAC, FG, and MDL interpreted the data, and wrote, reviewed, and edited the manuscript. Click here for additional data file.


Our population genetic results from five different reference datasets indicated that Shanxi Han shares a closer phylogenetic relationship with northern-neighbors and southern ethnically close groups than with Uyghur and Tibetan. Genome-wide variations revealed that modern northern-Han derived their ancestry from Yakut-related population (25.2%) and She-related population (74.8%). Summarily, the genetic mixing that led to the emergence of a Han Chinese ethnicity occurred at a very early period, probably in Neolithic times, and this mixing involved an ancient Tibeto-Burman population and a local pre-Sinitic population, which may have been linguistically Altaic.Modern East Asians derived from the admixture of aborigines and incoming farmers expanding from Yellow and Yangtze River Basins. Distinct genetic differentiation and subsequent admixture between Northeast Asians and Southeast Asians subsequently evidenced by the mitochondrial DNA, Y-chromosomal variations, and autosomal SNPs. Recently, population geneticists have paid more attention to the genetic polymorphisms and background of southern-Han Chinese and southern native populations. The genetic legacy of northern-Han remains uncharacterized. Thus, we performed this comprehensive population genetic analyses of modern and ancient genetic variations aiming to yield new insight into the formation of modern Han, and the genetic ancestry and phylogenetic relationship of the northern-Han Chinese population. We first genotyped 25 forensic associated markers in 3,089 northern-Han Chinese individuals using the new-generation of the Huaxia Platinum System. And then we performed the first meta-analysis focused on the genetic affinity between Asian Neolithic∼Iron Age ancients and modern northern-Han Chinese by combining mitochondrial variations in 417 ancient individuals from 13 different archeological sites and 812 modern individuals, as well as Y-chromosomal variations in 114 ancient individuals from 12 Neolithic∼Iron Age sites and 2,810 modern subjects. We finally genotyped 643,897 genome-wide nucleotide polymorphisms (SNPs) in 20 Shanxi Han individuals and combined with 1,927 modern humans and 40 Eurasian ancient genomes to explore the genetic structure and admixture of northern-Han Chinese. We addressed genetic legacy, population structure and phylogenetic relationship of northern-Han Chinese  Han Chinese, with a total population size circa 1.4 billion, is the world’s largest ethnic group and dominant ethnicity in China and Singapore. The origin of the Han Chinese population, genetic relationship with adjacent groups and past migratory pattern and admixture history have gained considerable attention from scientists working in the anthropology, linguistics, history, population and forensic genetics . ArchaeoPhysical anthropological investigation of somatometric and nonmetric features revealed that a significant difference exists between northern-Han Chinese and southern-Han Chinese . SubsequSTR, also called as microsatellite, is one of the extraordinary mutated genetic markers, is widely existed human autosomal, X-chromosome and Y-chromosome genomes . This levia high-density genetic markers are unclear. Thus, we genotyped and analyzed 23 autosomal STRs in 3,089 unrelated Han Chinese individuals and 643,897 SNPs in 20 Hans residing in Shanxi Province. Shanxi Province is between 34°34′-40°44′ north latitude and 110°14′-114°33′ east longitude, which stretches about a total area of 156,700 km2 from the Yellow River in the west and south to the Taihang Mountain in the east and the Great Wall in the north. This area is bounded by the Shaanxi, Inner Mongolia, Hebei, Henan, and other Provinces. Archaeological, anthropological and genetic evidence from Hengbei site consistently considered that Han Chinese is originated from Shanxi and neighboring regions, also called the Central Plain  from Yuncheng country in the Shanxi Province, northern China. A total of 20 EDTA anti-coagulated peripheral blood samples were collected for whole-genome SNPs genotyping. All self-declared Han Chinese indigenous subjects are needed to be no intermarriage with people of other ethnic groups and resided in here at least three generations. Human genomic DNA from male samples was extracted using the DNeasy blood and tissue kit (Qiagen) and measured utilizing the NanoDrop-2000  following the manufacturer’s instruction, and DNA from female samples was isolated using the Chelex-100 method. All our data used throughout this study are submitted in the 2O as negative in each batch of PCR amplification and electrophoresis. Infinium Global Screening Array BeadChip of Illumina WeGene V2 Arrays  covering approximately 700k SNPs was used to genotype autosomal SNPs based on the manufacturers’ introduction.We employed Huaxia Platinum Amplification System for multiallelic STR genotyping including 23-autosomal-STRs, two sex-linked inherited Y-InDel (rs2032678) and Amelogenin in a ProFlex PCR System (Thermo Fisher Scientific) in accordance with the manufacturer’s recommendation. A 10 μL reaction volume, which contains 4μL of primer set (the concentration of each locus is different), 4μL of master mix, 0.4 ng of template DNA as well as Prep-N-Go buffer was employed with the standard thermal cycling conditions which comprise an initial step at 95°C for 1 min; followed by 27 cycles of denaturation at 94°C for 3 s, anneal at 59°C for 16 s, and extension at 65°C for 29 s; and following a final extension at 60°C for 5 min and preservation at 10 °C. Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific) was utilized to separate and detect the PCR products using the 36 cm capillary array and POP-4 polymer following the corresponding instruction. A 9.5 μL of deionized Hi-Di formamide and a 0.5 of μL GeneScan 600 LIZ Size Standard v2.0 (Thermo Fisher Scientific) were utilized to mix with the amplified products or allelic ladder before the capillary electrophoresis. Finally, we used the GeneMapper ID-X v.1.4 software to identify and analyze the corresponding allele. We employed the typical control DNA of 9947A human cell line sample (Thermo Fisher Scientific) as positive, and the ddHTo comprehensively dissect the genetic background of Shanxi northern-Han Chinese, we first integrated our raw genotyping data (23 STRs) with previously published genotype data from 11 Chinese populations from five Chinese ethnic groups  . To furtSTR Analysis for Forensics (STRAF) online software  was util3, outgroup-f3 , D admixture-f, qpWave and qpAdm to explore the population history of northern Han under the genetic variations of modern and ancient population using the ADMIXTOOLS . We employed 10-fold cross-validation with the predefined ancestry populations varying from 2 to 19. We conducted the MIXTOOLS . We finaMIXTOOLS .The 23 autosomal-STRs included in the Huaxia Platinum System were amplified from 3,089 northern-Han Chinese individuals residing in the Shanxi Province, which is considered as the origin place of the Han ethnic group . Pairwise Fst genetic distances between Shanxi Han and other eleven Chinese populations were listed in via the first three PCA components. The patterns of the genetic affinity of East Asian populations were in accordance with the linguistic classification. Southern-Han Chinese populations were well-positioned at the end of the tree and then clustered with Central-Han Chinese population, followed by clustering with northern-Han Chinese populations (Shanxi Han), finally followed by Japanese, Korean and other language family or continental populations  and other geographically adjacent populations  and then combined with modern data from 2,810 individuals. A total of 80.6% variance extracted from 38 populations showed population substructure existed among the modern Han Chinese population. And Hengbei, Jinggouzi, Dashanqian, Mogou, Taojiazhai, and Sanguan ancient populations showed a paternally close relationship with the modern Han Chinese populations f showed that the greater genetic affinity identified between Shanxi Han and Han Chinese populations, subsequently followed by the southern Tai-Kadai and Hmong-Mien speakers, and Western Trans-Himalayan and northern Altaic speakers , where X represents the worldwide populations and Y denotes the Chinese populations from different language families, to explore the status of allele sharing. Our results provided supporting evidence for more shared genetic drift between Shanxi Han and northern-Han or neighboring minorities  to find the potentially admixed ancestral populations. Two hundred sixty-four out of 2,016 pairs were observed with significant negative values  and East-Siberian-related population , like the two ancestry populations observed in Indian   .3f = −0.0018 and Z = −6.662) was observed in the form of f3, we then conducted the qpWave and qpAdm using the She and Yakut as source populations and using Yoruba, San, Papuan and Melanesian as outgroup populations. QpWave results (p = 0.052 for rank1) indicated that Shanxi Han was derived from two ancestral populations. The qpAdm analysis further suggested that Shanxi Han has derived 25.2% Yakut-related ancestry and 74.8% She-related ancestry.To further validate the minimum streams of ancestry populations and evaluate the corresponding admixture proportion, we first performed the TreeMix analyses among 65 worldwide populations  (qpWave to find that the minimum ancient ancestry streams modern northern-Han were 2 (rank1: p = 0.118). QpAdm further showed that DevilsGate Hunter-Gatherer-related population contributed 45% ancestry and Oakaie-related ancient population contributed 55% ancestry to modern northern-Han Chinese.Nineteen ancient populations from Eurasian were employed to explore the genetic admixture history between Shanxi Han. The shared genetic history of all pairs was presented in East Asia is occupied by anatomically modern human 50 kya when they migrated out of Africa. These regions are populated by the hunter–gatherer over 40 kya in the Paleolithic time . Under tA recent large-scale whole-genome variation study covering 11,670 Han Chinese individuals from 24 out of 33 administrative regions was carried out to explore the Han Chinese population genetic structure and genetic ancestry . Their vThe processes of ancient whole-genome DNA studies with the technological innovations of DNA hybridization enrichment and next-generation sequencing has revolutionized the phylogenetic relationship and population history reconstruction in the European, American, Oceanian and even southeast Asians . In EastOur present meta-analysis results from the Neolithic ancient people and modern Han Chinese on the basis of the combined genetic variations of mtDNA and Y-chromosome first showed that the ancient populations from West Liao River Valley sites (Dasanqian and Niuheliang) and Yellow River Valley sites  share considerable similar mitochondrial haplogroup with the modern northern-Han Chinese populations. For Y-chromosome variations, ancient people from the Hengbei site shared the more significant genetic similarity with modern northern-Han Chinese from Shanxi and Heilongjiang provinces, and Dashanqian and Mogou ancient people bear a similar genetic assemblage with modern Taiwan Han people. Mogou site in the Ganqing region adjacent to the central plain is the hometown of Di-Qiang people who are thought as the direct ancestral population of Han Chinese, which is genetically close to the Han Chinese population. Our results reveal a close genetic relationship among Hengbei, Mogou and modern northern-Han Chinese populations. Our findings combined with the archeological, historical and linguistic evidence consistently supported the admixed genetic origin of modern Han Chinese.via ancient DNA. Summarily, consistent with previous linguistic and archaeological evidence, the genetic mixing that led to the emergence of a Han Chinese ethnicity occurred at a very early period, probably in Neolithic times, and this mixing involved an ancient Tibeto-Burman population and a local pre-Sinitic population, which may have been linguistically Altaic. Fine-scale population history reconstruction of north Han from modern and ancient genomes consistently model their ancestral populations deriving from ancestral North East Asian and ancestral South East Asian.In summary, we genotyped 23-autosomal-STRs in 3,089 Shanxi northern-Han Chinese individuals and provided the first batch of allele frequency, forensic and population genetic parameters of northern Han Chinese. Comprehensive worldwide and nationwide population comparisons not only showed that Shanxi harbored a strong similar genetic assemblage with adjacent Han populations but also illustrated that there were apparent genetic distinctions between north-to-south Han Chinese as well as genetic differentiation between populations belonging to different language families, obviously differences observed between Tibetan, Uyghur, and others here. The first meta-analysis based on the mitochondrial and Y-chromosomal genetic variations among ancient and modern Asian populations showed a genetic affinity and genetic continuity between Mogou, Hengbei ancient population and present-day northern-Han Chinese. We also found Neolithic agriculture expansion related Dashanqian and Niuheliang ancient populations are genetically close to modern northern Han. The qpWave/qpAdm modeling further revealed that modern northern Han Chinese carried 74.8% She-related ancestry and 25.2% Yakut-related ancestry. Both Hengbei-associated and Tibetan-related uniparental lineage (D haplogroup) were observed in modern Northern Han Chinese. Besides, approximately 45% DevilsGate-like ancestry, one Tungusic-affiliated Neolithic population, was modeled This study was approved by the Ethics Committee of Zunyi Medical University and corresponding experiments have followed the recommendations of the World Medical Association Declaration of Helsinki. Informed consent was obtained before the sample collection from the participants.GH, PC, and FJ conceived the idea for the study. GH, PC, and FJ performed or supervised wet laboratory work. PC, JW, LL, HG, XZ, HL, LY, GH, MW, YH, GC, and YL analyzed the data. GH wrote and edited the manuscript.This work was supported by grants from the PhD Scientific Research Start-up Fund of Affiliated Hospital of Zunyi Medical University (No. 201501) and the National Natural Science Foundation of China (No. 81401562).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.


Zizania palustris, Z. latifolia is a perennial aquatic grass widely distributed in China. Although Z. latifolia has been domesticated and cultivated as an aquatic vegetable for >1000 years, a neo-domestication for grain production needs to be soundly evaluated. In this study, we investigated the seed characteristic variations and genetic structure of 15 Z. latifolia wild populations along a latitudinal gradient in China. Our results showed that Z. latifolia tended to produce relatively larger seeds with lower moisture content and lower investments in seed pericarp at lower latitudes. The width, size, shape, seed-pericarp ratio and relative water content of seeds were significantly associated with climatic variables. The seeds of Z. latifolia showed a relatively low germination percentage and strong dormancy, which might hinder the neo-domestication. In addition, high genetic differentiation had been found among Z. latifolia populations, which could be attributed to isolation by distance. This study offered preliminary information for the utilization and conservation of wild Z. latifolia. It suggested that the wild populations in the middle and lower reaches of the Yangtze River could be good candidates for grain crop domestication due to appropriate seed traits and high genetic diversity. The neo-domestication of wild Z. latifolia requires further researches on the genetic mechanism of the Domestication Syndrome and more works on artificial breeding.Crop wild relatives are not only important genetic resources for crop improvement, but also domestication candidates for selecting new crops. As a close relative of American wild rice  Zizania latifolia is a perennial aquatic grass that has been domesticated as a vegetable in China. As a close relative of American wild rice Z. palustris, this species holds great potential in grain production. We found that Z. latifolia tended to produce larger seeds with lower moisture content and less investments in seed pericarp at lower latitudes. This suggested that the populations in the middle and lower reaches of the Yangtze River could be good candidates for grain crop domestication due to appropriate seed traits and high genetic diversity. The growing global human population results in increasing demands on cultivated plant resources . CultivaZizania is a member of tribe Oryzeae in Poaceae. This genus consists of four species, including Zizania aquatica, Zizania palustris, Zizania texana and Zizania latifolia   varieties  among the main aquatic vegetables cultivated in China  the pattern of the variance in seed characteristics among populations and their correlations with climatic variables; (ii) the baseline morphological and genetic information for the neo-domestication of Z. latifolia.Because the seed traits are important for grain crops, they should be firstly characterized before the neo-domestication. For example, seed size, seed dispersal, seed longevity and dormancy are dramatically divergent between cultivated grain crops and their ancestors . The arcZ. latifolia populations . The resolution of all climatic layers was 2.5 arc-minutes (5 km), to be compatible with the resolution of the locality data. In addition, we generated the water availability and temperature variability at the time of seed released from the plants based on the monthly mean temperature and precipitation, including the Max Temperature of Seed dispersal Month (bio20), Min Temperature of Seed dispersal Month (bio21) and Mean Precipitation of Seed dispersal Month (bio22). Thus, we included all 22 climatic variables to infer the habitat heterogeneity. Given the high degree of cross-correlation between these climatic variables, the principal component analysis (PCA) was used to reduce the dimensionality of the climatic data set.The geographic information for each sampling site was collected using a GPS navigator upon sampling. The environmental layers of 19 bioclimatic variables (bio1–19) and monthly mean temperature and precipitation (1950–2000) were obtained from the WorldClim version 2.0 website (GL), seed (hulled grain) length (SL) and seed width (SW) were measured with a digital caliper. Because the awn is an important accessory structure of seed and can affect the activity of seed dispersal, the awn length (AL) was also measured. From these measurements, the seed size (Ssize = SL × SW), seed shape (Sshape = SL/SW) and awn-length/grain-length ratio (AGL) were calculated. Then, another 100 grains were randomly selected from the mixed seed pool of each population. They were oven-dried and weighed (M100grain). Then, they were hulled and weighed to obtain the weight of 100 seeds (M100seed). This procedure was repeated three times to avoid biases, and the mean value was recorded.Firstly, 100 high-quality grains (plump grains with undamaged awns and hulls) were randomly chosen from each population for trait measurements. The grain length (WC) and seed-pericarp ratio (SPR) were investigated. For each population, 100 mature grains were fully hydrated and weighed (M0). They were oven-dried for 5 days to a constant weight and weighed again (MD). The WC was then estimated [WC = (M0− MD)/MD]. The hulls (the pericarps of caryopses) of these dehydrated grains were removed, and the seeds were weighed (MN). The SPR was simply the seed pericarp proportional to dry seed mass [SPR = (MD− MN)/MD]. These procedures were repeated three times to obtain an average value.In addition, the relative water content (2 (w/v) solution for 5 min and washed five times with distilled water. One hundred treated seeds were chosen from each population and put in a Petri dish containing distilled water for germination, with two replicates per population. The seeds were germinated at 24 °C with a 12-h/12-h (light/dark) photoperiod. Germination was monitored for 30 days, and the number of germinated seeds was recorded each day. The germination percentage for each population was determined as a proportion of the initial number of seeds. A tetrazolium test was performed on the seeds that failed to germinate after 30 days to check their viability. The seeds were cut longitudinally through the embryo, soaked in a 0.15 % (w/v) 2,3,5-triphenyltetrazolium chloride solution for 20 h at 20 °C in the dark, and scored according to the intensity and location of staining.As described by P < 0.05. Principal component analysis was performed to reduce the data set to sets of interrelated variables. Correlation analyses were performed using SPSS v22.0. Multiple regressions were also applied to investigate the associations between the variations of the seed characteristics and climatic variables.A descriptive analysis was performed to characterize the variations of the measured seed traits using SPSS v22.0 . One-wayWe extracted DNA and amplified eight highly polymorphic microsatellite loci (screened from 16 species-specific microsatellite markers) for each sample following the methods described in Z. latifolia, we assumed that the different occurrences of the same multilocus genotype (MLG) within a population were ramets (clonemates). Therefore, a single representative of each MLG (genet) was retained to analyse for genetic variations.Because of the intensive clonal reproduction of [see]. The valley between the first and second peaks was considered a good candidate to use as a threshold (G), Simpson’s diversity index (D) and evenness (E) were calculated by GENODIVE (R = (G − 1)/(N − 1), where N is the number of ramets.Genets were identified using similarity thresholds based on the frequency distribution of pairwise genetic distances between ramets (MLGs)  as implehreshold . TherefoGENODIVE . The closeeFis). The global and pairwise differentiation coefficients Fst were estimated. Partitioning of the total genetic variation within and between populations was further analysed by analysis of molecular variance (AMOVA) with 1000 permutations. The Mantel test was applied to detect the effect of isolation by distance (IBD) on the population structure. The pairwise geographical distance (GGD) matrix and genetic distance [pairwise Fst/(1 − Fst)] were generated, and the Mantel test was performed with 1000 bootstraps.MicroChecker see was usedK (1–15) ancestral populations under the admixture model with correlated allele frequencies, using 200 000 Markov chain Monte Carlo iterations and a burn-in of 100 000 iterations. The optimal number of genetic clusters was determined using ΔK (Q) were permuted with CLUMPP  were separate from the other sites in climate conditions . AccordiAGL (seeSW) ranged from 0.98 mm (HLJYC) to 1.51 mm (JXJX), which was negatively correlated with latitude , and the seed length (SL) ranged from 8.03 mm (HLJYC) to 11.30 mm (LNSY). As expected, the estimated Ssize and Sshape were significantly correlated with latitude  as well. M100grain ranged from 0.83 g (HLJYC) to 1.73 g (JXJX), with a mean value of 1.44 g. The average value of M100seed was 1.13 g, and it ranged from 0.57 g (HJLYC) to 1.50 g (JXJX) among the populations. As a function of M100grain and M100seed, SPR was significantly correlated with latitude , with a mean value of 0.22. The populations located at high latitudes showed relatively high SPRs. WC was also associated with latitude , with a mean value of 0.23 and a range from 0.09 (JSBY) to 0.46 (JLDH). The grain length (GL) ranged from 28.98 mm (HLJYC) to 45.84 mm (HBHH). The corresponding awn length (AL) ranged from 16.78 mm (HLJYC) to 28.87 mm (HBHH). AGL showed no significant difference among populations, with a mean value of 0.60, indicating a constant ratio between AL and GL.All seed characteristics varied greatly and showed significant differences among the 15 populations except for [see]. PC1, which explained 50.52 % of the total variance, had its high loadings for SW, Ssize, M100grain and M100seed; Sshape greatly determined PC2, which contributed to 29.07 % of the total variance. A scatterplot of PC1 scores against PC2 scores showed that the morphological variation of the northernmost population HLJYC is distinct [see].A PCA on the seed traits showed that the first two principal components accounted for 79.59 % of the total variance Z. latifolia seeds showed a low germination percentage (11.7 %) and a strong dormancy (40.2 %) [see]. The germination percentage and dormancy varied greatly among populations, but they did not demonstrate associations with any of the other seed traits nor the climatic variables. The lowest germination percentage was observed in the JXDA population (0.5 %) along with the highest dormancy (72.0 %); the highest germination percentage could reach to 30.5 % (AHAQ) with a relatively low dormancy (19.5 %). The seeds from the HLJYC, JLDH, LNSY, SDTZ, JSBY and JXJX populations all showed relatively low germination percentages, but the dormancy differed dramatically among these populations [see]. Notably, the northernmost population, HLJYC, showed an extremely high mortality (95.0 %).In addition, SW, Sshape, Ssize, SPR and WC were significantly correlated with PC1clim [see]. Multiple regression analyses further indicated that SW was negatively affected by Temperature Seasonality (bio4) ; Sshape was positively impacted by the Mean Diurnal Range (bio2) ; Ssize was significantly correlated with the Mean Temperature of Warmest Quarter (bio10) ; SPR decreased with increased Annual Precipitation (bio12) ; and WC was negatively correlated with the Mean Temperature of Driest Quarter (bio9)   .Ge) varied from 1.33 (JXDA) to 26.47 (HLJYC), with an average value of 14.68, and the clonal richness (R) ranged from 0.11 to 0.93, with an average value of 0.65. In general, the Z. latifolia populations showed a high level of clonal diversity (mean D = 0.87). Three populations were found to have extensive clonal reproductions .We found the sign of clonal growth in all sampled populations. However, the extents of clonality differed among populations . OverallZ. latifolia populations showed a relatively high genetic diversity (He = 0.495). Most of the fixation indices (Fis) were significantly larger than 0 and ranged from −0.001 to 0.370, with a mean value of 0.160, indicating heterozygote deficits  was 0.287 [seeand]. A hierarchical AMOVA showed that 75.0 % of the total genetic variation was within populations and 18.5 % between populations (P < 0.01); 6.5 % of the total genetic variation was found between two regions revealed by the PCA on climatic variables (P < 0.01). A Mantel test showed significant evidence for IBD in Z. latifolia  [see].The genetic differentiation coefficient  was divergent from the northern group in Northeast China . The remaining populations  had genetic consanguinity with both groups except for the population of JSSQ , seed size (Ssize), seed shape (Sshape), seed-pericarp ratio (SPR) and relative water content (WC) were significantly correlated with climatic variables  of Z. latifolia was negatively correlated with latitude and positively correlated with the Mean Temperature of Warmest Quarter (bio10). Zizania was reported to originate in North America and then dispersed into eastern Asia via the land bridge, which meant that the distribution range of Z. latifolia had expanded from north to south  seeds , which implied that the seeds at high latitudes would remain in the soil longer before germinating. The increased SPR was likely to be a protective step that ensured the survival of seeds. Moreover, the significant negative correlation between seed-pericarp ratio (SPR) and Annual Precipitation (bio12) found in our study implied that SPR might be an adaptive trait to moisture availability. The clinal changed relative seed water content (WC) among Z. latifolia populations further reinforced the importance to cope with seed desiccation. Theoretically, the length of time needed to desiccate was positively associated with the relative investment in the seed pericarp and water content  seeds . The type) seeds . InvestiZ. latifolia. The extents of germination percentage and dormancy differed greatly among populations, which might be attributed to maternal effects. The studies used seed collected in the wild often cannot exclude the role of maternal effects on trait variation . Only a few populations with relatively small population size showed significant signs of clonality. The extensive clonal growth in these populations might be the result of ‘founder effect’. For example, we consulted the locals about the population history of JXDA and found that a few ancestors had floated down from upstream and colonized here 10 years ago.We did find signatures of clonal growth during our sample collections. However, the clonality of Zizania originated in North America and then dispersed into eastern Asia via the Bering land bridge during the Tertiary . However, Z. latifolia restricted to the middle reaches of the Yangtze River and found a high genetic diversity (He = 0.532) (He = 0.497) was detected in our study. The populations located in the high-latitude areas did not show a higher genetic diversity than those in low-latitude regions. In addition, most of the fixation indices (Fis) were significantly >0, indicating a heterozygote deficit that could be attributed to inbreeding or pollination between ramets.Previous phylogeographic analyses suggested that the genus Z. latifolia, which suggested limited inter-population gene flows at large scale  and have uniform ripening . HoweverIn the meantime, seed shattering and dormancy are the first obstacles need to be overcome. Throughout history, the appearance of non-shattering grains was gradual, at least in barley, wheat and rice. In these crops, the non-shattering phenotype only occurred after an increase in grain size, a trait reflects selections on germination and production . AlthougZ. palustris or even from O. sativa, there is a great chance to domesticate Z. latifolia to a perennial grain crop in China.In fact, the successful domestication of American wild rice could be a good example for us. In the 50 years of domestication, they not only focused on developing varieties with advanced traits such like shattering resistance, increased yield, stem sturdiness, removing seed dormancy, reduced plant height and resistance to fungal, but also generated a system of management practices . By applZ. latifolia populations along a latitudinal gradient. Among the measured seed traits, SW, Sshape, Ssize, SPR and WC were significantly associated with climatic variables. In general, Z. latifolia produces larger seeds with lower moisture content and lower investments in seed pericarp at lower latitudes. The low germination percentage and strong dormancy of seed may be potential obstacles for neo-domestication as a grain crop. A prominent genetic structure was found in Z. latifolia populations, which could be mainly attributed to IBD. This study provided fundamental information on morphological and genetic variations in Z. latifolia populations. Nevertheless, more researches on deciphering the genetic basis of important domestication traits are still needed to promote the neo-domestication of Z. latifolia.There are abundant variations in the seed characteristics among https://osf.io/mdu76/.The measured seed trait data and microsatellite genotype data were deposited in: This study was supported by the National Natural Science Foundation of China (no. 31600293) and the China Postdoctoral Science Foundation Grant (no. 2015M571483).Y.Z., J.C. and J.R. conceived the idea and designed the research project. Y.Z., K.Z. and L.Z. collected the data. Y.Z. performed the data analysis. Y.Z. drafted the initial manuscript with contribution from J.R. and Z.S. All the authors contributed critically to the discussion and edited the manuscript before submission.None declared.The following additional information is available in the online version of this article—Zizania latifolia populations.Zizania latifolia.Zizania latifolia populations.Zizania latifolia populations.Fst) between Zizania latifolia populations.Zizania latifolia.Zizania latifolia.Zizania latifolia populations across a latitudinal gradient.Zizania latifolia populations.Supplemetary MaterialClick here for additional data file.


Gentiana lawrencei var. farreri is endemic to the Qinghai-Tibetan Plateau (QTP). To explore its genetic structure and evolutionary history, the genetic diversity, divergence, and demographics were analyzed in individuals from 31 locations across the QTP using 1 chloroplast marker and 10 nuclear microsatellite loci. High genetic diversity was detected in G. lawrencei var. farreri, and most of the genetic variance was found within populations. Values of FST in G. lawrencei var. farreri from nuclear microsatellite and chloroplast data were 0.1757 and 0.739, respectively. The data indicated the presence of isolation by distance. The southeast edge of the QTP was the main refugium for G. lawrencei var. farreri, and one microrefugium was also detected in the plateau platform of the QTP. Both nuclear microsatellite and chloroplast data indicated that the populations were divided into two geographically structured groups, a southeast group and a northwest group. The current genetic pattern was mainly formed through recolonization from the two independent refugia. Significant melt was detected at the adjacent area of the two geographically structured groups. Approximate Bayesian computation showed that the northwest group had diverged from the southeast group, which then underwent population expansion. Our results suggest that the two-refugia pattern had a significant impact on the genetic structure and evolutionary history of G. lawrencei var. farreri.Understanding the genetic structure and evolutionary history of plants contributes to their conservation and utilization and helps to predict their response to environmental changes. The wildflower and traditional Chinese and Tibetan medicinal plant  As the highest and largest plateau in the world, the Qinghai-Tibetan Plateau (QTP) has experienced drastic environmental and climate changes during the past several million years . The fraSaxifraga  Dumorti , which belongs to section Kudoa (Masamune) Satake and Toyokuni ex Toyokuni and is endemic to the QTP. It lives in alpine meadows and wet meadows with altitude from 2,400 to 4,600 m. Its flower and fruit period is from August to October and pollinators are insects, mostly Bombus, Thripidae, and Formica , 10–30 mature plants were randomly sampled along a single transect. Sampled individuals were at least 20 m apart. For small populations , 25–50% of the plants  were sampled. Voucher specimens were deposited in the herbarium of the College of Life Science, Luoyang Normal University.We sampled 31 populations of ycf1  and was verified in the sampled populations. The ycf1 was then amplified in all of the individuals of G. lawrencei var. farreri. PCRs were performed in 25-μL volumes containing approximately 20 ng of template DNA, 1× PCR Buffer, 1.5 mM MgCl2, 0.25 mM of each dNTP, 0.20 mM of each primer, and 1 unit of Taq DNA polymerase . The PCR cycling profile included an initial step of 5 min at 95°C followed by 35 cycles of denaturation at 94°C for 50 s, 45 s of annealing at 53°C, 20 s at 72°C, and a final extension at 72°C for 6 min. PCR products were sequenced in the reverse direction on an ABI 3730 xl automated capillary sequencer  with BigDye v3.1 (Applied Biosystems).Total genomic DNA was extracted from dry leaves with a Dzup plant genomic DNA extraction kit . Through comparison analysis of chloroplast genomes , one divAll of the samples were genotyped at 10 microsatellite loci , which were amplified and genotyped easily, as described by h), nucleotide diversity (π) indices, FST and a hierarchical AMOVA were performed in ARLEQUIN 3.5  analyses were conducted in PhyML 3.0  using tha Maxim. , G. macrhy Pall.  and G. sdgeworth . The robdgeworth  to eluciG. straminea by lognormal priors with an offset at 5.0 Ma, a mean of 0.7, and a standard deviation of 1.0, as applied by 1 and judged by ESS values (>200). Trees were summarized using TreeAnnotator 1.7.5  were generated in BEAST. The Bayesian MCMC was run for 100,000,000 generations, discarding the initial 10% as a burn-in. Tajima’s D  and Fu’sD , expected heterozygosity (HE), and pairwise FST using ARLEQUIN 3.5 (RS) was calculated using FSTAT 2.9 . Each run started with a burn-in of 100,000 followed by 1 million iterations. The most likely true value of K was determined using the second rate of change in the likelihood distribution  contained populations from the southeast; the second (named NW) contained populations from the northwest. In this study, we tested whether population divergence was followed by changes in effective population size. The ABC analyses were conducted using DIYABC 2.0  a direct estimate, in which the 500 data sets with summary statistics closest to the target values were extracted, and (ii) a polychotomous logistic approach that recovers the 1% closest to the simulated data sets. For the scenario with the highest support, posterior distributions for parameters were estimated using a local linear regression on the closest 1% of the simulated data sets.Demographic history was modeled through coalescent-based approximate Bayesian computation  than the overall average values.All of the samples were amplified at the RS, HO, and HE, were variable across populations  compared to the overall average values. Among the 10 loci, the number of loci/location that deviated from HW at the population level ranged from 0 to 3 with an average of 1.10. Pairwise comparisons among the 10 microsatellite loci revealed no evidence of linkage disequilibrium (P > 0.05). The data from the 10 microsatellite loci used in G. lawrencei var. farreri are summarized in Supplementary Table All 423 of the samples were amplified at 10 nuclear microsatellites. Among all populations, the frequencies of null allele in the 10 microsatellite loci were low (ranged from 0.002 to 0.066) thus all loci were included in following analysis. Overall, population-level indices of genetic diversity, including Na, G. lawrencei var. farreri diverged approximately 4.89 Ma (95% highest posterior density: 1.61–10.77 Ma).The ML trees of the 16 cpDNA haplotypes Figure  supporteG. lawrencei var. farreri, H2 was dominant and appeared in 27 (87.10%) populations. All of the other haplotypes appeared in less than 10 populations. The southeast of the distribution area  contained 8 (50%) haplotypes and YuShu-ChenDuo region  contained 4 haplotypes. SAMOVA results indicated that FCT reached a plateau when K = 2  and YuShu-ChenDuo populations  were 0.374 and 0.111, respectively.In the 16 cpDNA haplotypes detected from K) increased with each K-value and did not plateau . The population pairwise values of FST are listed in Supplementary Table G. lawrencei var. farreri populations .In the Bayesian individual-based clustering analysis based on the microsatellite dataset, the log-likelihood values for the number of clusters  with direct estimation (not shown); there was stronger support with logistic regression, and thus scenario 2 was favored as the most probable Figure . EffectiG. lawrencei var. farreri is divided into two geographically structured groups , which were the ancient haplotypes, occurred in this area as well. The populations situated close to the southeast edge of the QTP  were also highly divergent. Meanwhile, the diversity declined away from the southeast edge of the QTP. These characteristics are well fitted by the refugia hypothesis  was higher than in other areas of the plateau platform. The haplotypes occurring in the plateau platform were observed from single clades in the ML tree except for H12, which occurred in both the YuShu-ChenDuo area and the southeast edge of the QTP. Although the value of Sibiraea  and Rhod Trifida ; these hG. lawrencei var. farreri. Based on the cpDNA data, phylogenetic relationships of haplotypes showed that the southeast and northwest areas were two geographically structured groups  and only favored the scenario in which southeast groups had undergone population expansion after the northwest group had diverged. However, EBSP analysis based on cpDNA indicated that the effective population sizes of the two groups and the whole species were nearly constant during the most recent 200 Kya. Recent population expansions or declines were also detected in very limited areas . The demographic histories of these populations should be explored further.G. lawrencei var. farreri. The main route of recolonization from the southeast edge of the QTP after the most recent glaciation was northward, along the edge of the QTP  to the northern end of the distribution area of G. lawrencei var. farreri. This northward route was described by The genetic dataset indicated recolonization routes for P-CF conceived and designed the experiments, collected the samples, performed the experiments, contributed reagents, materials, analysis tools, and wrote the paper. H-YY contributed reagents, materials, analysis tools, prepared figures and/or tables. Q-WL and H-MC performed the experiments and analyzed the data. S-LC analyzed the data and reviewed drafts of the paper.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.