Daucus carota (Apiaceae) in two regions of the United States. We employed 15 nuclear simple sequence repeat (SSR) markers and one polymorphic chloroplast marker. Further, we utilized both conventional population genetic metrics along with Shannon diversity indices as the latter have been proposed to be more sensitive to allele frequency changes and differentiation. We found that populations in both regions that were proximal to crop fields showed lower levels of differentiation to the crops than populations that were located farther away. We also found that Shannon measures were more sensitive to differences in both genetic diversity and differentiation in our study. Finally, we found indirect evidence of paternal transmission of chloroplast DNA and accompanying lower than expected levels of chloroplast genetic structure amongst populations as might be expected if chloroplast DNA genes flow through both seed and pollen. Our findings of substantial gene flow for both nuclear and chloroplast markers demonstrate the efficiency of both pollen and seed to transfer genetic information amongst populations of carrot.Studies of gene flow between crops and their wild relatives have implications for both management practices for cultivation and understanding the risk of transgene escape. These types of studies may also yield insight into population dynamics and the evolutionary consequences of gene flow for wild relatives of crop species. Moreover, the comparison of genetic markers with different modes of inheritance, or transmission, such as those of the nuclear and chloroplast genomes, can inform the relative risk of transgene escape via pollen versus seed. Here we investigate patterns of gene flow between crop and wild carrot,  While, natural land managers may wish to minimize gene flow out of agricultural fields into natural, often weedy, populations of closely related species. Moreover, given a genetically modified  crop system, an additional consideration is the risk of escape of transgenes from an agricultural setting to the wild by gene flow through both hybridization, and/or introgression [Cultivated plant species and their sexually-compatible wild relatives often overlap in terms of geographic proximity and phenology . This ovgression –7.Gene flow between non-GM crops and their wild relatives has the potential to increase weediness of wild populations . MorDaucus carota subsp. carota L., (a.k.a. Queen Anne’s Lace). Wild carrot is abundant in temperate regions across the globe and is widely distributed across much of the United States where it is often found along roadsides, abandoned fields, and pastures. Wild carrot is the progenitor of the cultivated carrot, D. carota subsp. sativus, and the two subspecies are sexually compatible [within cultivated carrot fields have also been reported [One particularly weedy crop wild relative is wild carrot, mpatible –26. The mpatible ,28 and impatible ; howevermpatible ,31. Obsereported ,26,32. IIn plants, gene flow among populations can occur through both pollen and seed owing to the differential transmission of nuclear and organellar genomes –36. NuclTansley Review by [paternal leakage, has been observed. From fish to fungi, natural and experimental studies have demonstrated DNA in the offspring originating from the paternal donor (reviewed recently in [ST measures) would tend to approach those of nuclear genomes depending on the degree of paternal leakage. Moreover, because the two genomes  need not leak together, values of population subdivision may be different for gene markers of the two genomes [Given strict uniparental inheritance of organelles , the comparison of gene flow in seed versus pollen is fairly straightforward. However, it is becoming increasingly evident that the rule of maternal inheritance in flowering plants is not always the case (see eview by ). In facently in ). In wilently in ,43. Pate genomes . BearingST and related measures) for assessing gene flow among plant populations [Recently several authors have suggested additional methods  investigate patterns of gene flow between crop and wild carrot across two regions of the United States using both nuclear and chloroplast DNA markers, 2) investigate evidence for chloroplast heteroplasmy in samples of both crop and wild carrot, and 3) compare conventional population genetic metrics of gene flow and genetic diversity to those that make use of species diversity indices of Shannon information.Daucus seed were generally high (> 90%), and seeds were allowed to grow for up to 8 weeks before tissue was collected for DNA purification.The sampling strategy for wild carrot consisted of collecting leaf material or seeds  from populations that varied from less than 1 km from a crop carrot field to more than 2.5 km from a crop carrot field. Carrot is a primarily outcrossing, insect pollinated species that is also capable of self-fertilization ; seed caThe four carrot cultivars  that are grown as a root crop on Nantucket were obtained from Johnny’s Seed Company and Harris Seed Company (information kindly provided by the farmers on Nantucket). These varieties included ‘Purple Haze F1’, ‘Nelson F1’, ‘Nantindo F1’and open-pollinated ‘Scarlet Nantes’. It should be noted that even though carrot is cultivated for the root on Nantucket, farmers of both locations studied here report early bolters (plants flowering in their carrot fields in the first season) and plants occasionally flowering in their fields after harvest the following season (presumably missed during harvest the previous season). The three open-pollinated cultivars  grown in the Olympic Peninsula/Sequim-Dungeness Valley are self-selected lines grown by the farms: OP-1, OP-2, OP-3 (seed kindly provided by the farmers of the Olympic Peninsula). Since crop lines are continuously rotated , these lines were grouped into one population (per region) for the analyses presented here. Also note that carrot growers who grew a crop from the seed sources above, in both Nantucket and the Olympic Peninsula, reported no incidence of wild carrot outcrosses in their seed stocks.Genomic DNA was isolated from a 2 cm portion of leaf tissue obtained from individual leaves using the SQ Omega Biotek Plant Kit  with the addition of 1% PVP and 1% Ascorbic Acid. DNA was quantified and quality assayed using a Nanodrop spectrophotometer . If DNA quality were low, samples were purified using an Omega Biotek E.Z.N.A. Cycle Pure Kit following the manufacturers protocol.Length variation of the region between the trnG and trnS genes (S/G region) of the chloroplast genome was used as a chloroplast genetic marker. PCR amplification of this region was performed using the ‘S’ and ‘G’ primers from Hamilton . The PCRA total of 15 nuclear Simple Sequence Repeat (SSRs or microsatellite) markers previously developed by  were useST [0 = no genetic differentiation among the populations) was determined by performing 1000 permutations. Further, genetic distance amongst individuals was investigated graphically using principal coordinates (PCO) analysis. For this, a standard genetic distance matrix [Measures of genetic diversity and genetic distance were assayed separately for the two regions studied here using the software GenAlEx v. 6.5  for the ST . Statiste matrix  was consstructure [structure, assembles clusters by minimizing intra-cluster Hardy–Weinberg and linkage disequilibrium and, for each individual, the proportion of membership in a given cluster is estimated. Our analysis here did not use prior population information . For each analysis, K = 1–10 population genetic clusters were evaluated with 5 runs per K value. For each run, the initial burn-in period was set to 200,000 with 500,000 MCMC iterations. The optimal number of K clusters was estimated using the delta K method of Evanno et al. [CLUMPP [Greedy algorithm. Finally, the cluster membership was visualized using the software distruct [Population structure based on nuclear loci in each region (“Nantucket” and “Olympic”) was also investigated using the Bayesian, model-based clustering algorithm implemented in the software package tructure . For theo et al.  as impleo et al. . When an [CLUMPP  using thdistruct . Pie chaIn order to estimate contemporary rates of gene flow among populations within regions and as a complement to our historical methods using traditional population genetic analyses, we used the software BayesAss 3.0.4 . As adviST [ST both measure aspects of genetic diversity and differentiation within and among populations. The measure uHe is the expected heterozygosity, or gene diversity, corrected for sample size for a given population and ranges from 0 to 1 with higher values indicating greater population genetic diversity. The measure FST describes the degree of genetic differentiation among populations and also ranges from 0 to 1; when FST is lower, populations do not differ greatly in terms of allelic diversity, and when FST is higher, populations are described as more genetically different from one another. The Shannon measures, Shannon’s H and SHUA are analogous to the population genetic measures uHe and FST respectively: Shannon’s H measures levels of genetic diversity within a population but is not bounded by 1; SHUA  similarly measures the degree of genetic differentiation amongst sampled populations. For measures of uHe and Shannon’s H and FST and SHUA, populations were grouped according to their proximity to crop fields (see description above of collection strategy). A two-factor ANOVA was performed in JMP (v. 9) using crop proximity and locus as factors to investigate significance among diversity measures. A sign test (in JMP) was also performed to assay significance.Traditional population genetic measures of Nei’s  unbiasedST  were comST –46. The ST value as estimated from AMOVA was 0.43 (p < 0.001) with haplotypic frequency showing some degree of structuring across populations (In Nantucket (the crop production sampling area), three haplotypes were recorded across the six populations (no private haplotypes). All populations except Tuckernuck were polymorphic for the chloroplast marker, and unbiased diversity ranged from 0 at Tuckernuck to 0.56 at Polpis Rd demonstrating substantial haplotypic variation across the island . The FSTulations . Two wilST value as estimated from AMOVA was lower than on Nantucket: 0.29 (p < 0.001) with haplotypic frequency showing some degree of structuring across populations with populations on the northern end of the valley harboring a different haplotype frequency than populations farther south and farther from crop fields (In the Olympic Peninsula (the seed production sampling area), six haplotypes were recorded across the eight populations (no private haplotypes). All populations were polymorphic for the chloroplast marker and unbiased diversity ranged from 0.31 in the crops to 0.69 in the Medsker Rd population demonstrating substantial haplotypic variation across the region . The FSTp fields . Thirty-higher single locus values of uHe and Shannon H, though the Shannon H measures appeared more sensitive to these differences , all fifteen nuclear SSRs were polymorphic in all populations, and unbiased multilocus gene diversity ranged from 0.58 on Tuckernuck to 0.73 at Bartlett Farms . MultiloST value as estimated from AMOVA was 0.14 (p < 0.001) with paired population FST values ranging from 0.11 (Polpis Rd to Cliff Rd—both away populations) to 0.30 (Crops to Cliff Rd)  . The 2-dliff Rd)  demonstrtructure  providedST was calculated on the grouping of populations based on proximity in Nantucket (the crop production sampling area), the near to crop populations versus far from crop populations demonstrated slightly lower values: away wilds to crops FST = 0.23, near wilds to crops FST = 0.21. A single locus approach based on proximity also failed to demonstrate any significant differences in away versus near measures of FST, proximity and locus two-factor ANOVA p = NS. However, when Shannon diversity indices were used, the difference between distance measures was greater: away wilds to crops Shannon Hua = 0.21; near wilds to crops Shannon Hua = 0.12, and the single locus approach demonstrated that the Shannon Hua values were lower in near versus away populations, proximity and locus two-factor ANOVA, F = 25.12 p = 0.0002. Contemporary estimates of migration using the software program BayesAss yielded estimates of migration rates (range: 0.068% to 21%) that were relatively similar across populations for Nantucket. The largest rate was the Cliff Rd population as an immigration source for the Polpis Rd population—both populations located away from crop fields , fourteen nuclear SSRs  were polymorphic in all populations, and unbiased multilocus gene diversity ranged from 0.42 in the crops to 0.80 at Hemlock Rd population . MultiloST value as estimated from AMOVA was 0.17 (p < 0.001) with paired population FST values ranging from 0.049 (Hemlock to Medsker—both away populations) to 0.48 (Crops to Fasola—a near population)  . The 2-dulation)  demonstrtructure  providedST was calculated on the grouping of populations based on proximity in Olympic (the seed production sampling area), the near to crop populations versus away to crop populations demonstrated slightly lower values: away wilds to crops FST = 0.27, near wilds to crops FST = 0.22. The single locus approach based on proximity also failed to demonstrate any significant differences in away versus near measures of FST, proximity and locus two-factor ANOVA (p = NS). When Shannon diversity indices were used, the away populations also showed greater divergence than the near populations but not as large as the difference in Nantucket: away wilds to crops Shannon Hua = 0.22; near wilds to crops Shannon Hua = 0.17; yet still the single locus approach demonstrated that the divergence measures of Shannon Hua were lower in near versus away populations, proximity and locus two-factor ANOVA, F = 20.18 p = 0.0006. Contemporary estimates of migration using the software program BayesAss yielded estimates of migration rates (range: 0.058% to 21%) that were relatively similar across populations for Olympic. The largest rate was the Eberle population as an immigration source for the Fasola population—both populations located near crop fields (When Fp fields . Raw datST) as well as methods more traditionally used for assessing ecological species diversity , the results presented here demonstrate a significant level of gene flow among populations both on Nantucket and in the Olympic Peninsula/Sequim-Dungeness Valley. Our results show that while populations do exhibit genetic structuring, some degree of population connectivity among crop and wild carrot populations certainly occurs. Moreover, gene flow appears to occur at a significant rate with respect to both nuclear and cytoplasmic genomes. The measures of nuclear FST reported here  are on par with those of other angiosperm species as reported by Petit et al. [A major goal of this study was to investigate the evidence for gene flow among populations of carrot with particular interest in levels and patterns of gene flow occurring amongst crop and wild populations. Thus, we sampled from wild carrot locales that were within 1km of a crop field and locations that were more than 2.5km away from crop fields in two regions where carrot is grown in the United States . Using both methods that detect historical levels of gene flow .ST and Shannon diversity indices; however, the Shannon measures were more sensitive to these differences . This result aligns with recent suggestions by several authors to make use of both types of measures when investigating population structure and gene flow [ST  populations that were closer to crop fields harbored lower levels of differentiation as compared to populations that were farther. This finding held when using both measures of Fene flow –46 especene flow . Moreoveene flow  suggest dings of ).An additional finding of Campbell et al.  was that1 hybrids) providing evidence for outcrossing with wild carrot when seed for that crop was produced. The situation is quite different in the Olympic Peninsula/Sequim-Dungeness Valley where commercial carrot seed production is an important priority of the region. Both the local farmers and the Clallam County Extension Office have active campaigns against wild carrot due to concerns of contamination of crop seed. “Wanted” posters can be found in the area stating “Queen Anne’s Lace, Wanted Dead”, and the community is advised to cut the umbels of the plants off before they set seed.In both regions, wild carrot is very common and on Nantucket it is regarded as an attractive wild flower that residents cultivate in their own home gardens. The species is also tolerated or ignored on the farm properties where crop carrot is grown with farmers occasionally reporting early bolters and plants with intermediate root morphology  in populations near to and far from crop production fields in future studies may reflect differential selection for those genomic regions relative to random genetic markers.ST = 0.43; Olympic FST = 0.29). Petit et al. [th and 75th quartiles being 0.416 and 0.871 respectively. Our chloroplast differentiation values were either on the extreme low end (for Nantucket) or well outside the range of the quartiles (for Olympic). Other studies have found similar low values for carrot organellar population structure. Ronfort et al. [While measures of nuclear genetic differentiation follow those of other angiosperm species, the measures of population structure based upon the chloroplast marker were much lower than typically found in angiosperm plant populations  than many other angiosperm species such that the chloroplast genome shows significantly lower levels for differentiation as compared to other taxa . Indeed,The action of metapopulation dynamics and frequent extinction and recolonization events may also play a role in determining population genetic structure in carrot populations. Ronfort et al.  point ouand pollen leading to higher levels of cpDNA gene flow and thus lower levels of population genetic differentiation. Occasional paternal inheritance of organellar DNA, sometimes termed “paternal leakage” has been documented in numerous plant species  making heteroplasmy an indirect indicator of paternal leakage in crosses [Daucus [Another possibility is that the chloroplast genome is not transmitted strictly maternally but occasionally moves via pollen as well. Paternal leakage would allow chloroplast genes to move through both seed iewed in ). Patern crosses . Paterna [Daucus . And rec [Daucus  and wild [Daucus . In the Daucus species. And 2) paternal transmission may also be enhanced in species that exhibit gynodioecious mating systems  as well as crop breeding programs that utilize cytoplasmic male sterility (CMS) like carrot [Our findings have implications for crop-wild transgene escape especially that of organellar transgene escape. Two observations from the literature apply to this: 1) paternal leakage of otherwise maternally inherited cytoplasmic genomes is often associated with hybridization events –74. For e carrot ,75. The e carrot  our studS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Panax notoginseng, a traditional Chinese medicinal plant, has been cultivated and domesticated for approximately 400 years, mainly in Yunnan and Guangxi, two provinces in southwest China. This species was named according to cultivated rather than wild individuals, and no wild populations had been found until now. The genetic resources available on farms are important for both breeding practices and resource conservation. In the present study, the recently developed technology RADseq, which is based on next-generation sequencing, was used to analyze the genetic variation and differentiation of P. notoginseng. The nucleotide diversity and heterozygosity results indicated that P. notoginseng had low genetic diversity at both the species and population levels. Almost no genetic differentiation has been detected, and all populations were genetically similar due to strong gene flow and insufficient splitting time. Although the genetic diversity of P. notoginseng was low at both species and population levels, several traditional plantations had relatively high genetic diversity, as revealed by the He and π values and by the private allele numbers. These valuable genetic resources should be protected as soon as possible to facilitate future breeding projects. The possible geographical origin of Sanqi domestication was discussed based on the results of the genetic diversity analysis. Crop species were first domesticated from their wild relatives approximately 10000 years ago  defined Panax notoginseng(Burkill) F. H. Chen ex C. Y. Wu et K M. Feng, commonly known as “Sanqi” in China, is a diploid (2n = 2x = 24) species[Panax [) speciesthat belo) species. It is a) species. However) species. Phylogees[Panax ,12; howees[Panax .The genees[Panax ,15. Welles[Panax . In addiPanax notoginsengis cultivated in some plantations of the Wenshan Autonomous Prefecture of Yunnan province and the Jingxi County of the Guangxi Zhuang Autonomous Region of China. Cultivated P. notoginseng displays a wide range of morphological diversity, such as white-yellow or dark red tuberous roots, green, dark red or mixed color stems, red or yellow fruits [P. stipuleanatus, as evidenced by ITS sequencing and AFLP polymorphism analysis [P. notoginseng from three populations, and nine sites (1.30%) were variable in 51 accessions sampled from eight populations of P. stipuleanatus. The percentage of AFLP polymorphic sites was 76.9% in P. notoginseng and 96.5% in P. stipuleanatus [P. notoginseng also harbored less DNA variation than did its two cultivated tetraploid relatives, P. ginseng and P. quinquefolius, as revealed by the screening of 36 single copy nuclear loci [P. notoginseng studies mentioned above were not sufficient for fully assessing the genetic structure and diversity, especially at the population level.w fruits . Howeveranalysis . No sequleanatus . P. notoear loci .This typear loci . In addiEcoRI or SbfI, or a combination of two enzymes, and is then sequenced from the restriction sites to yield a vast number of short reads [Stacks [pyRAD can greatly [Next-generation sequencing (NGS) technology provides the opportunity to generate large-scale molecular marker data to study genetic diversity at a much higher resolution. Restriction-site associated DNA (RAD) sequencing is a method based on NGS technology that can create a reduced representation of the genome and identify thousands of genetic markers that are randomly distributed across the target genome. It promises to generate high-resolution population genomic data for model and non-model organisms . For RADrt reads –23. RAD rt reads ,24–29. B [Stacks ,31 and p greatly  facilitaSanqi currently faces severe pathogen pressures, and long-term sustainability projects and associated medical industries will rely on the exploitation of the existing natural genetic diversity. The specific objectives of the present study were to determine the genetic diversity, population divergence and structure at both the species and population levels by using RAD sequencing technology. The generated knowledge would be beneficial to breeding and germplasm conservation efforts of this medicinal crop.The materials included 36 samples from 12 plantations. Twenty-seven accessions were obtained from nine populations that were distributed in five Yunnan counties, and nine accessions were obtained from three populations distributed in the Jingxi county of Guangxi . Three sEcoRI, and 36 RAD sequencing libraries were prepared according to the methods described previously [EcoRI enzyme, P1 adapter/barcode ligation and DNA purification, size selection, P2 adapter ligation and RAD tag amplification. Single-end sequencing was aimed to produce approximately 1,000 Mb raw data for each library using Illumina HiSeq2000. The steps mentioned above were carried out by Majorbio Pharm Technology Co., Ltd., Shanghai. The raw sequence data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence-Read Archive (SRA) database with the accession numbers SRR3123274, SRR3123435—SRR3123442, SRR3123444, SRR3123447 and SRR3123450.Total genomic DNA was extracted from silica gel-dried leaf material using a modified CTAB procedure . The geneviously ,34. In bde novo using the Stacks1.0 pipeline [process_radtags program was used to demultiplex and sort the raw data according to the barcodes used in each sample. During this process, the adapter contamination was filtered out, and the raw reads with low average quality score bases (phred score ≤ 10) were discarded. The clean data for each sample were grouped into loci using ustackswith a stack depth parameter (-m) of 5, a mismatch parameter (-M) of 2, and maximum stacks allowed per locus (—max_locus_stacks) of 3. The loci data of all of the samples were merged into a catalog using cstacks, and then the loci of each sample were matched against the catalog so as to determine the allele status in each sample using sstacks. To evaluate the genetic diversity of P. notoginseng at the species level using populations, we treated all 36 samples as a whole population. To include a locus in this analysis, we required it to be present in at least 67% of the samples. When we evaluated the genetic diversity at the population level, we treated each plantation as a population and required a locus to be present in all individuals (r = 1) in at least six populations (p = 6).The raw data were analyzed pipeline ,35to recH), nucleotide diversity (π) and Wright’s F statistics FIS and FST, were calculated for every SNP using the populations program in Stacks. For bi-allelic SNP markers, π is a useful overall measure of genetic diversity in a population, and the F statistic measures the distribution of genetic variation within and among populations [FIS, which measures the reduction in heterozygosity due to inbreeding [FST for pairwise comparisons of all sampled populations in the present study. We then used these average pairwise FST values to cluster populations by a neighbor-joining method implemented in the Mega6.0 program [Population genetic statistics, including the private allele number, heterozygosity . During this data outputting process, only the first SNP per locus was written in both the Genepop-format and Structure-format outputs to avoid tight linkage SNPs [To analyze the organization of the populations using multilocus genotypic information, the at files –42to anaage SNPs with the Arlequin3.5 software [Genepop-format files into an Arlequin-compatible format.The distribution of genetic variation was analyzed by AMOVA analysis using the software  after coStructure2.3 [populations program. For this analysis, 10000 burn-in steps and 100000 iterations were used, with 10 replicates for each value of K, where K is the number of genotypic groups, which ranged from 1 to 12. Output data were processed in Structure Harvester v0.693 [http://taylor0.biology.ucla.edu/structureHarvester/). The optimal K for each analysis was chosen using the delta K method of Evanno et al. [Structure Harvester. Genetic relationships among the studied individuals were also assessed by principal coordinates analysis (PCoA) in R software package adegenet . based on the Euclidian distances between individual genotypes.Sample assignment analysis was performed using the software cture2.3  on the cr v0.693   at the species level was estimated using the software Genepop4.0 (http://genepop.curtin.edu.au/), and the pairwise Nm values at the population level were measured using the formula Nm = (1—FST) / 4 FST[FST values derived from the populations program.Gene flow  / 4 FST,47 and bIMa [Phylip–format SNP data was output by populations with the parameters p = 1 and r = 24, in which one locus was present in at least 24 individuals was used as the input file after manually editing. Demographic parameters including effective population sizes  and migration rates (m1 and m2) were estimated by 20000000 steps following 200000 burn-in periods. To verify convergence upon the same values, the analysis was repeated three times using the same priors but different seeds in each one of the runs.To estimate the migration rates and effective population size for Guangxi and Yunnan groups as well as ancestral group, IM model was performed using IMa . The Phyprocess_radtags, the clean data derived from each sample ranged from 595Mb to 1880Mb, and most of them were approximately 1000Mb (FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The loci of each sample were produced after clean data processing analysis done using the ustacks-cstacks-sstacks program  was 0.1523, the expected heterozygosity (He) was 0.1554, the nucleotide diversity (π) was 0.159, and the inbreeding coefficient (FIS) was 0.0591. When considering all nucleotide positions, including the non-polymorphic ones, the observed heterozygosity decreased to 0.0016, the expected heterozygosity to 0.0016, the nucleotide diversity to 0.0017, and the inbreeding coefficient to 0.0006.For all loci that were polymorphic in the entire data set at the species level, the observed heterozygosity (Ho), the expected heterozygosity (He), the nucleotide diversity (π) and the private allele numbers. The MT and PL populations had relatively higher genetic diversity than did the other populations. In contrast, the CF population showed the lowest heterozygosity and nucleotide diversity values, and the DH population had the fewest private alleles.The statistics for each population were shown in FIS values were very close to zero  from the Guangxi province grouped into a branch , and using the deltaK approach of Evanno [Structure analysis. These results further supported the hypothesis that no genetic differentiation occurred among Sanqi populations.As a further test of potential population structure, we analyzed these 6418 SNPs using the software cture2.3  with an f Evanno , we founGenepop analyses revealed that the overall number of migrants (Nm) per generation was 1.4. The pairwise population Nm values calculated from Wright’s analysis indicated that the level of gene flow between populations was substantially high, with the largest being 156.0 between the NP and DH populations and the smallest being 12.7 between the GH and DL populations.1) was smaller than that of Yunnan group (θ2), and both were much smaller than that of the ancestral population (θA). The migration rates between the Guangxi and Yunnan groups (m1 and m2) were not significant.The demographic parameters of IMa analysis are shown in π)is known as the average pairwise difference between two DNA sequences, and it is a measure of expected heterozygosity for bi-allelic SNP markers [Panax species and evaluate whether the same ortholog exhibits heterogeneous evolutionary rates in diploid and tetraploid species. The π values of P. notoginseng ranged from zero to 0.0139, with anaverage value of 0.0045,whereas the tetraploid P. ginseng and P. quinquefolius had an average π value of 0.0097 and 0.0104, respectively. The π values of these selected genes are not more representative than those from RAD tags when used to describe the genetic diversity of a species. Our study revealed that the π values P. notoginseng estimated using RAD sequencing data were 0.0017 at a species-wide level and 0.0011 ~ 0.0017 at a population level. These data indicated that the nucleotide diversity level of Sanqi was low. Using RAD tags, Xiao [Phoebe zhennan, an endemic and endangered species in China. The π values of this tree species ranged from 0.0010 to 0.0016 among different populations[Glycine max) was 0.0005 ~ 0.0010[P. notoginseng and P. zhennan.Nucleotide diversity , including observed heterozygosity(Ho) and expected heterozygosity(He), is another important statistic in population genetics. Although the heterozygosity values estimated using different DNA markers will vary in plants [P. notoginseng and two relatives, P. ginseng and P. quinquefolius. The He value of P. notoginseng estimated from polymorphic sites using RAD sequencing was 0.1554. The total genetic diversity of wild P. ginseng estimated from allozyme data was low at the species level (He = 0.0230) [P. quinquefolius, the He values were 0.1637 based on RAPD data [Heterozygosity . For culAPD data  and 0.31APD data .Ne)of a population, reflecting the rate at which genetic diversity will be lost, will be reduced by a population bottleneck [1) and Yunnan group (θ2) were both much smaller than that of the ancestral population (θA), which meaning that serious population contraction has occurred in the two distribution areas of Sanqi.The effective size  among Sanqi populations ranged from 0.0016 to 0.0193, which is significantly lower than the average FST values of species with mixed mating systems or with short-lived perennial history [FST < 0.0500, there is almost no differentiation between populations. Furthermore, AMOVA analysis revealed that only 3.47% of the genetic variation occurred between populations and that approximately 96.5% of the genetic variation occurred within populations. The results of the Structure analysis and Principal Coordinates Analysis also supported this conclusion. Wang et al. [P. notoginseng and found no significant differences in saponin concentration among different groups; however, the saponin concentration exhibited great variation among individual samples. This distribution pattern of chemical variation coincided with the pattern of genetic variation revealed in the present study. A mixed NJ tree estimated from 11713 bp further supported this conclusion  [In addition to the NP population, the PL plantation, which has a traditional Sanqi cultivation history, had high genetic diversity. This plantation, located in the Wenshan county of the Yunnan province, is a large planting base for a Sanqi production company. Many individuals of e roots)  have beeP. stipuleanatus, P. zingiberensis, P. japonicus and P. vietnamensis still exist in Yunnan, which could potentially provide genetic resources for the improvement of cultivated Sanqi and the further research of Sanqi origin.The first historical record of Sanqi can be found in the “Compendium of Materia Medica”, a book on Chinese herbal medicine published in 1596. In this book, Sanqi is described to have been discovered in the mountains of west Guangxi. Approximately 150 years later, cultivated Sanqi has been shown to be sold in Wenshan, Yunnan, based on observations recorded in the “Annals of Kaihua Prefecture”, published in 1757. The possible geographical origin of Sanqi domestication is therefore still controversial . PrivateS1 Fig(EPS)Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 Fig(EPS)Click here for additional data file.S4 Fig(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Ancestry inference for an individual can only be as good as the reference populations with allele frequency data on the SNPs being used. If the most relevant ancestral population(s) does not have data available for the SNPs studied, then analyses based on DNA evidence may indicate a quite distantly related population, albeit one among the more closely related of the existing reference populations. We have added reference population allele frequencies for 14 additional population samples  to the 125 population samples previously published for the Kidd Lab 55 AISNP panel. Allele frequencies are now publicly available for all 55 SNPs in ALFRED and FROG-kb for a total of 139 population samples. This Kidd Lab panel of 55 ancestry informative SNPs has been incorporated in commercial kits by both ThermoFisher Scientific and Illumina for massively parallel sequencing. Researchers employing those kits will find the enhanced set of reference populations useful.The online version of this article (doi:10.1007/s00414-016-1524-z) contains supplementary material, which is available to authorized users. Soundararajan et al.  recentlyHere, we report on 14 additional population samples with allele frequencies on all of the 55 AISNPs in the Kidd lab panel [The 14 new populations are listed in Table Every locus-population combination for which individual genotypes were available was tested for Hardy-Weinberg on the assumption that each locus was a codominant di-allelic genetic system. Genotypes were examined to ensure that the alleles on the positive strand have been entered into the database and are used in FROG-kb.K value from 6 to 10, the program was run 20 times with 10,000 burn-ins and 10,000 Markov Chain Monte Carlo (MCMC) iterations.The STRUCTURE  softwareNo significant deviations from Hardy-Weinberg ratios were observed beyond those expected by chance. All of the different typing methods appeared consistent: the same alleles are being detected at the same loci. Allele frequencies for the complete set of 55 SNPs in all 139 population samples are accessible in ALFRED. Additional populations have been studied and reported in scientific publications for some of the SNPs, and thus, some SNPs have frequency data on more than 139 population samples in ALFRED. In FROG-kb, the “Kidd Lab—Set of 55 AISNPs” has complete allele frequency data on all 55 SNPs for all 139 reference population samples. The completeness of the data allows likelihoods and likelihood ratios to be calculated for all of these 139 population samples for any input DNA profile for the 55 AISNPs (or a subset of the SNPs).K = 9. This analysis includes 66 additional populations since the previously reported analysis of only 73 populations [The STRUCTURE analysis result displayed in Fig. ulations . The anaWith nearly twice as many population samples, we can see significant differences when that previously published STRUCTURE result  is compaOur interpretation of the multiple cluster memberships for populations in Central Asia is that this set of AISNPs does not work well to distinguish Central Asian populations from populations to the East, South, and West. While, historically, Central Asia has seen many migrations, it has been inhabited since modern humans first reached this far in their expansion from Africa before they reached East Asia. How much the historical migrations are responsible compared to these simply being intermediate and not well differentiated by these SNPs is a question for future research.In our update on the growing number of reference populations studied for the 55 AISNPs [We continue to work on our own and with our collaborators to study more new populations on the 55 AISNP panel. We also continue to assess other SNPs for ancestry informativeness and whether they can improve refined ancestry inference by modifying the existing panel. We continue to encourage other researchers to consider adding their unique populations to this growing dataset of population samples which are all tested for the same set of ancestry informative SNPs. Similarly, we encourage others with excellent candidate AISNPs to request that we test them on our population samples.These results demonstrate the value of more populations studied for a small number of informative SNPs. To date, no other panel of AISNPs has data on such a large number of populations distributed as widely. That does not mean this panel should be considered a final panel. There are other panels, noted above, that are also included in ALFRED and FROG-kb. We are working to increase reference population coverage of those panels as well so that the most globally informative subset of SNPs can be identified from the union.Supplementary Table S1The 139 population samples in STRUCTURE analyses ordered in bar graph Fig.


Diaphorina citri Kuwayama is a major pest in citrus production, transmitting Candidatus Liberibacter asiaticus. It has spread widely across eastern and southern China. Unfortunately, little is known about the genetic diversity and population structure of D. citri, making pest control difficult. In this study, nine specifically developed SSR markers and three known mitochondrial DNA were used for population genetics study of D. citri using 225 samples collected from all 7 distribution regions in China. Based on the SSR data, D. citri was found highly diverse with a mean observed heterozygosity of 0.50, and three subgroups were structured by host plant: (i) Shatangju, NF mandarin and Ponkan; (ii) Murraya paniculata and Lemon; (iii) Citrus unshiu, Bingtangcheng, Summer orange and Navel. No significant genetic differences were found with mtDNA data. We suggested the host-associated divergence is likely to have occurred very recently. A unimodal distribution of paired differences, the negative and significant Tajima’s D and Fu’s FS parameters among mtDNA suggested a recent demographic expansion. The extensive citrus cultivation and increased suitable living habitat was recommended as a key for this expansion event.The Asian citrus psyllid  Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the most serious pest of citrus worldwide, primarily due to its role as a vector for “Candidatus Liberibacter asiaticus”, the bacterium that causes the highly destructive Asian huanglongbing (HLB) (citrus greening)1. HLB affects all citrus cultivars3. Severely HLB-infected trees often appear stunted, sparsely foliated and die back, losing their economic viability. Ledford4 reported that this disease has slashed US orange production in half over the past decade, and threatens to destroy the US$3.3-billion industry entirely. To date there is no effective control technique for this disease5, and control of D. citri has been one of three critical components of HLB management in addition to planting pathogen-free nursery stock and removing inoculums by destroying infected trees6.The Asian citrus psyllid, Diaphorina citri was first described in Taiwan in 1907 and currently has spread throughout much of Asia7. In Brazil, D. citri was found in the 1940s8, expanded its range to Florida in the late 1990s9, and is now well established in citrus producing regions of the United States11 as well as Mexico, Belize, Costa Rica, the Caribbean and South America12. The wide distribution of D. citri and probable high genetic diversity make any suppression efforts challenging. Taking China as an example, after a first found in Taiwan in 19076, D. citri spread quickly to the coastal provinces including Guangdong, Fujian and Guangxi, and then to inland provinces such as Hunan, Jiangxi, Yunnan and Sichuan13. During the invasion course, D. citri has adapted itself to a wide range of geographic environments and host species, becoming ubiquitous in almost all citrus growing areas in China15.16. DNA markers such as mtDNA, SSRs, RAPD, AFLP and ESTs have been used as popular marker systems in insect genetics research17. In the last few years, much attention has been drawn to phylogeographic studies of D. citri populations; most of these studies utilized mitochondrial genes  as genetic markers. Studies of D. citri populations with the COI gene in America suggested two independent founder events occurred in South and North America18. Boykin19 found two major haplotype groups with preliminary geographic bias between southwestern and southeastern Asia using the COI gene. Diaphorina citri was reported to have formed two geographically and genetically structured groups (the eastern versus western side of the state of São Paulo) and has undergone a recent population expansion in Brazil based on COI sequences20.Deciphering genetic diversity, population structure, as well as history and adaptation processes is essential for developing effective suppression protocols and sustainable management strategies for insect pests21. Generally, mtDNA is assumed to evolve under neutral or nearly-neutral selection21. However, the Neutral theory is only valid for slow evolving genes yet to reach Maximum Genetic Diversity (MGD) and using the fast evolving part is wrong to analyze phylogy22. Besides, the use of only mtDNA for phylogeographic and population genetic studies is questioned, because within-species diversity does not reflect population size or ecology, and it is difficult to investigate evolutionary questions23. Utilizing both slow evolving and fast evolving molecular markers together can generate a comprehensive picture of both ancient and recent population histories and thus becomes necessary 25. Owing to their rapid evolution, extensive genome distribution and high level of polymorphism, as well as apparent neutrality and co-dominant behavior, Short Simple Repeats (SSR) or microsatellite markers have become a useful marker in studies of ecology, evolution, conservation, and population biology of many insect species27. Compared to the AFLP system, SSR markers are also sufficiently abundant to generate large numbers of markers17 and have been widely used in plants, animals, and microorganisms32. The joint application of mtDNA and SSR markers in the same population will make the results of phylogeography and population genetics analyses more validated and additional genetic information will be gained22. To date, no study has been undertaken in D. citri to investigate the genetic diversity and relationships among populations using different classes of genetic markers simultaneously on the same samples. And, no researcher has tested if mtDNA genes are neutral before using them.As a marker, some of the unique characteristics of mtDNA compared to nuclear DNA are a high rate of nucleotide substitution, maternal inheritance, and little or no recombinationD. citri populations in China using three mtDNA genes  and nine newly developed SSR markers; (2) investigating the factors shaping the population genetic structure of D. citri populations; and (3) investigating the demographic history of D. citri populations in China to propose a hypothesis on the expansion of this pest under China conditions. Through the study, we wish to provide a foundation to improve the monitoring and control of D. citri.This study aimed at (1) investigating the genetic diversity of D. citri, 144 SSR loci were obtained after scanning the whole genome with SSR Hunter 1.3. These motifs contained dinucleotide (57.6%) or trinucleotide (38.9%) motifs. The others were less frequent . Of the 144 potentially amplifiable loci, 50 primer pairs were tested and 20 were selected according to their amplification. Thirteen primer pairs were validated on a panel of samples, i.e., production of specific PCR products of the expected size, and tested for polymorphism. Nine of these 13 loci (69.2%) proved to be polymorphic for all individuals  were identified sporadically in the flanking regions of SSRs in the makers Dci-22 and Dci-26. Since SNPs were not located in the target region of primers and had no affect on the length of amplicons, they would not hamper the amplification of the markers or data analysisHO) of 0.484 ranging from 0.100 (locus Dci-33) to 0.931 (locus Dci-22) (Table p = 0.391). Four of nine loci were found to have deviated from HWE (p < 0.05) after Bonferroni correction in all populations. Further tests  revealed that the deviations from HWE may be caused by heterozygote deficiency  with mean heterozygosity  variable positions in the concatenated sequence  with a transition: trans-version ratio of 1:1. In total, 26 D. citri haplotypes were identified from the concatenated mtDNA in which all populations had low diversity at both the nucleotide (π: 0.00082) and haplotype (Hd: 0.4957) levels.We sequenced three mitochondrial fragments from 225 Diaphorina citri individuals from Shatangju, NF mandarin and Ponkan were clustered in Clade 1; those from Murraya paniculata and Lemon in Clade 2; those from Citrus unshiu, Bingtangcheng, Summer orange, and Navel in Clade 3. There is no evidence of clustering by geographic location suggesting that the phylogenic lineages were mainly associated with the type of host plant regardless of sampling location36.The unrooted NJ phylogram generated from the SSR data indicated a high degree of clustering by host plant species Fig. . Samples37, indicate no significant saturation for all three codon positions . Neither the single gene nor concatenated data sets showed any sign of pervasive homoplasy (CI > 0.5).Saturation analysis results, conducted using DAMBE v.5.3.8Parsimonious analysis of the concatenated mtDNA sequence yielded three equally parsimonious trees, from which a strict consensus tree was constructed. The topology of consensus tree is presented as an unrooted network with scaled branches (data not shown).D. citri mtDNA haplotype sequences, branch lengths in terms of both synonymous (syn) and non-synonymous (nonsyn) sites for each mitochondrial gene were estimated and shown in Fig. Based on the 26 identified D. citri has strong and significant genetic differentiation at the host plant level. In contrast, AMOVA detected 7.83% of variation among locations populations and 92.17% within locations populations  in the overall D. citri populations’ data set was 0.523, with an overall observed heterozygosity (HO) of 0.492. Four host plant populations  showed higher observed heterozygosity than expected; although in many cases this difference was not significant  to 0.591 (Murraya paniculata), and mean HO ranged from 0.377(NF mandarin) to 0.617(Summer Orange) . Pairwise FST values ranged from 0.001 (Navel to Bingtangcheng) to 0.398 (Ponkan to Lemon)  Fig. , and comD. citri  as a measure of population subdivision Fig. . Each ofNucleotide (π) and haplotype (Hd) diversity levels calculated with syn sites was all lower than nonsyn sites as shown in Fig. 2 < 0.01, p ≥ 0.32)  came from variation among populations. Further Mantel tests of isolation by distance revealed no significant relationship between geographical and genetic distances (rD. citri haplotypes. The lowest haplotype diversity (Hd: 0.13333) was detected in Fujian and Guangdong provinces, which seems to contradict the fact that they are among the earliest invaded regions in China. The significantly low diversity level may be due to excessive pesticide use and removal of HLB-infected trees for pest control, which caused a higher citrus distribution disturbance and lower overall D. citri richness38 (Table D. citri area with high haplotype diversity (Hd: 0.61134), possibly hinting at multiple invasive events or that intra- and/or inter-specific hybridization exists39 (Table The strongest association of haplotype diversity with sampling location was seen in Yunnan province (Hd: 0.62299) Table . Yunnan ty Hd: 0.3333 was 39 Table . The strD. citri experienced population expansion events. The haplotype networks, together with the haplotype distribution information  had a relatively high proportion (80%) of shared haplotypes , whereas other populations  had 60% shared haplotypes. This would suggest that the D. citri population from Yunnan might have established relatively early in these areas. More haplotypes (Nh) were found in Jiangxi (JX) and Hunan (HN), suggesting the existence of more relatively suitable habitats in those two regions , pruned hedges of Murraya paniculata are flushing all the year round, which will constitute an extreme psylla reservoir for D. citri40. This would be the reason why the high haplotype frequency of D. citri occurred in Murraya paniculata.In combined analysis with host species, we observed that Murraya paniculata had a relatively high proportion (80%) of shared haplotypes  suggesting that Murraya paniculata, which is a widespread plant, is a much preferred host plant for D and Fu’s Fs when all individuals were tested as a single group (Table D and Fs) and were further confirmed by the Ramos-Onsins and Rozas’ R2 test  expansion. Moreover, the values of D. citri populations size before (θ0 = 0.002) and after (θ1 = 12.415) the demographic expansion of the populations were calculated.Population demographic history was reconstructed using three neutrality tests and mismatch distribution analysis from nonsyn sites. Results from neutrality tests showed significant negative values for Tajima’s up Table . The resst Table . This teons Fig.  also sugD. citri in Zhejiang province have experienced a recent expansion event.Also, the applied neutrality tests revealed significant deviations from neutrality for Zhejiang population Table , suggestD. citri populations in China. This result is derived from the SSR marker analyses, while the mtDNA sequences did not detect any significant genetic differentiation for D. citri populations. Furthermore, our results suggest recent population expansions occurred for D. citri populations. These results provide new perspectives for D. citri control, specifically with respect to host-associated structure among D. citri populations.Overall, our present study explores nine polymorphic SSR loci and demonstrates that host-associated differentiation exists among D. citri is the most invasive species on citrus42, there were only a small number of molecular markers  available to study its genetic diversity and population structure35.Even though D. citri genome sequence information43 successfully screened nine polymorphic SSR loci, which were sufficiently variable to allow detailed studies of genetic diversity, LD and population structure. After sequential Bonferroni adjustment44, three loci  in the Chinese D. citri populations significantly deviated from HWE (p < 0.05), most likely due to heterozygote deficiency or population structure within the individuals. These newly developed markers are likely to be a valuable resource for further population genetic studies of D. citri from China and other countries, which could facilitate the conservation and management of D. citri.The SSR primer pairs designed in this study from D. citr with mtDNA like COI gene. De León et al.18 identified 23 haplotypes from populations collected in America, while the worldwide haplotype diversity of D. citri reported by Boykin et al.19 is even lower (only 8 haplotypes). In Brazil, Guidolin et al.20 detected 47 haplotypes using an almost complete sequence of COI gene, indicting a high haplotype diversity (H = 0.839) but still low in nucleotide diversity (π = 0.002). The genetic diversity and tree topology calculated further with nonsyn and syn sites in mtDNA showed a clear pattern of more variation distributed in nonsyn sites (slow evolving gene). Seemingly, neutral processes played a minor role in syn sites (fast evolving gene). Based on MGD thoery, we used nonsyn sites for D. citri phylogenic analysis. Genealogy indicated a limited intraspecific variation in D. citri population from China showing no clear grouping cluster. Different from mtDNA, the results from the SSR markers for the same D. citri populations showed the presence of three host-associated genetically distinct clusters from tree topology and PCoA analysis, which were not related to geographic distance among the D. citri populations. Those SSR data suggested that the populations of D. citri may be experiencing some degree of host plant-associated differentiation. Nevertheless, the phylogenetic trees constructed from the SSR data could be biased towards host plant selection because the SSR markers were derived mostly from fast evolving sites. Cautions are therefore recommended when interpreting the phylogenetic analysis results45.There have been other studies on the genetic diversity of Procambarus Clarkii, Syngnathus typhle and Clupea harengus population genetic studies48. The inconsistent results obtained from the two molecular markers are generally explained by the features of the two kinds of markers. SSRs, with their high mutation rate of 10−2 to 10−6/locus/generation50, can increase the overall resolution to detect genetic differentiation and give clues about recent and contemporary events in the population51. By comparison, the slower mutation rate of the mtDNA sequence marker may provide more of a historic than a recent picture of gene flow52. On the other hand, such results are consistent with the Maximum Genetic Diversity (MGD) hypothesis53. SSRs as the fast evolving sequences are at saturation genetic diversity  that is maintained by stabilizing natural selection53. In our case, host plants provide the selection force. Sequences not compatible with living in a particular plant species would be selected out. Slow evolving sites (mtDNA) are too slow to meet adaptive needs and hence appear neutral within short time frames53.Such discrepancies between different types of markers are also reported in D. citri populations were found to be structured into three subgroups by host plant: (i) Shatangju, NF mandarin and Ponkan; (ii) Murraya paniculata, Lemon; (iii) Citrus unshiu, Bingtangcheng, Summer orange, and Navel. Mechanisms that may contribute to host-associated differentiation of D. citri populations were explored based on the taxonomic, volatile and phenological differences among hosts.In this study, 54, who described 16 species in Citrus, has been the most widely accepted55. Shatangju, NF mandarin, Ponkan and Citrus unshiu all belong to Citrus reticulate, while Lemon belongs to Citrus medical; Bingtangcheng and Summer orange belong to Citrus sinensis. We noticed that the Citrus classification is in accordance with the genetic structure of D. citri populations besides Citrus unshiu. Plants are the site of assembly for D. citri. Population fluctuations of D. citri on citrus and other plants are closely correlated with the rhythm, quantity, and nutritional quality of plant flush because eggs and nymphs are exclusively associated with new growth56. Vassiliou58 found that Lemon  trees flush sporadically throughout the year, providing an important feeding source and refuge for D. citri over winter. Murraya paniculata has a more cold-hardy bud break, advancing flushing by six weeks38. When compared to Citrus sinensis, Citrus reticulate trees are more winter-hardy, letting the autumn plant flush extend to November59. The prolonged flushing stage would extend the lifespan of D. citri. Furthermore, whether or not host plants are available at the early spring and the whole winter poses another high selection pressure on insect populations through effects on diapause61. Variation in diapauses would contribute to the divergence of host-associated populations of insects63, as reported in Rhagoletis pomonella64 and Diatraea saccharalis61. The flushing characteristics of Lemon and Murraya paniculata described above could make diapauses termination be more beneficial to their D. citri populations than populations on other citrus species. Therefore, we suggest that host plant phenological differences may contribute to host-associated differentiation in D. citri populations.Numerous classification systems of Citrus have been formulated, among which that of SwingleD. citri host-associated differentiation. Ehrlich & Raven65 found chemical similarities among unrelated food plants used by related butterflies, and suggested that secondary plant substances played the leading role in determining patterns of utilization. Aroma compounds are important for citrus; each cultivar type generates a different profile of volatiles that contributes to its distinct aroma attributes66. These volatiles provide a critical clue for host plant selection and orientation in D. citri. Linalool was reported to be a major constituent among leaf volatiles in young leaves of most cultivars67. γ-terpinene was a major component in leaf oil of Citrus sinensis69 and thymol ethyl ether was only found in leaf volatiles of Citrus reticulate71. Limonene was predominantly found in Citrus medical (lemon), and β-bisabolene was only identified in Citrus medical leaves72. Interestingly, β-bisabolene was also detected in leaf volatiles of Murraya paniculata oil73. Therefore, such chemical differences among D. citri host plant species could act as an internal driver of locally adapted populations as reported by Ehrlich65.Host plants provide insects not only nutrients but also secondary substances such as volatiles influencing insect behavior. Chemical and nutritional differences existing among host plants may be a reason for 74, and assortative mating could occur between individuals on the same host plant75. Several lines of evidence have suggested that female host preferences exist in D. citri76. Investigation of the location and preference of adult D. citri on Murraya paniculata, Lemon  and Ponkan (Citrus reticulate) found no difference between adult numbers on Murraya paniculata and Lemon, but significantly fewer on Ponkan, suggesting that Murraya paniculata and Lemon are the preferred hosts77. Field observations showed that Citrus medical suffered the heaviest from D. citri, followed by Citrus sinensis, with Citrus reticulate the lightest79. Female D. citri orient to their plant of origin; males orient to the odor of females and co-specific excretions80. This behavior in combination with female oviposition preference behavior likely results in mate choice and limits gene flow between D. citri from different host plant clusters82. In addition, micro-ecological variables such as symbiont composition among different host plants of D. citri may also be an important source of host plant-associated natural selection. Study of the population structure and symbiont community of host-adapted Acyrthosiphon pisum populations showed that the presence of six facultative (secondary) bacterial endosymbionts was nonrandom distributed across aphid genetic clusters and suggested that bacterial endosymbionts affect population divergence and speciation83. More experiments are needed to uncover whether micro-ecological variables exist in host plant-associated D. citri populations.Insects can develop an oviposition preference for the natal plantD. citri populations. The recent population expansion of D. citri and its increasing damage around the world may have resulted from the recent sharp expansion of citrus varieties and growing area, which has provided more food options and ecological environments for D. citri84. Beck & Reese85 proposed that insect survivorship, fecundity, growth rate and activity can be affected by the quantity and quality of hosts. An abundance of hosts in a habitat will increase survival and fecundity and reduce mortality. As Murraya paniculata is the primary wild host for D. citri, the increase in population size of this species would distinctly improve the fecundity and survival capacity of the insect86, and the extensive commercial cultivation of citrus trees in recent years may also have contributed to an expansion of the insect’s territory84. Additionally, the warmer temperatures and increased precipitation not only contribute to D. citri’s physiological activity but also provide suitable living conditions for D. citri in relatively dry seasons in South China, increasing the number of habitats in Mainland China87.Both neutrality tests and unimodal mismatch distributions revealed an expansion of D. citri individuals were collected from 9 host plant types located in 20 citrus production regions across 7 governmental provinces in China . DNA amount per sample was normalized to 30 ng/μL. The polymerase chain reaction (PCR) protocols for SSR markers and mtDNA genes were as follows:Total genomic DNA was extracted from a single intact insect following a modified salt-extraction protocol89 was employed for SSR content. Primer pairs were designed in Software PRIMER v 3.090 with sufficient flanking sequence and tested for amplification and polymorphism using DNA obtained from thirty individuals. The set of individuals was the same as those used for variability testing (see below) and individuals were rotated during initial screening such that a different set of thirty was used for each primer pair. The variability of the selected polymorphic loci was assessed in 225 specimens, and they were deposited in GenBank with the Accession nos.KY053427-KY053435. The conditions and characteristics of the SSR loci are provided in Table 91 with 30 min of final extension at 72 °C. Fragment analyses were run on an ABI 3730 DNA sequencer (Applied Biosystems) and analyzed with GENEMARKER 2.2.0 (Soft Genetics) using GS-500 (LIZ) as a size standard.Based on the entire genome, SSR Hunter 1.3 softwareSequencing of SSR loci was performed for the presence of size homoplasy detection. To isolate the specific bands of SSRs, the PCR products were separated on a 2.5% agarose gel. Only the best amplified bands for each SSR primer combination was collected and purified with QIA quick PCR Purification Kit (Omega). Those DNA fragments were sequenced with the specific forward or reverse SSR primer on an ABI3700 DNA analyser. The sequences obtained were checked for their homology with the SSR source sequence.Three mitochondrial genes ; cytochrome b, Cytb (867 bp); and NADH dehydrogenase subunit 5, ND5 (750 bp)) were amplified and sequenced. Products were amplified by PCR in 25 µL reactions with 1.25 units of AmpliTaq DNA polymerase, 1 × PCR buffer, 2 mM of MgCl2, 0.4 mM of dNTPs and 0.5–1 µM of each primer. A detailed description of the primers used and PCR conditions can be found in the Supporting Information Table . Sequenc92 for further analysis. The genotypic data were used to estimate genetic diversity. Allele dropout and null alleles in the newly developed SSR data were checked using MICROCHECKER v 2.2.393. The observed number of alleles (NA), observed heterozygosity (Ho), expected heterozygosity (He), percentage of polymorphic loci (P), Nei’s genetic diversity (Nei’s), and Nei’s original measure genetic distance (Nei’s D) were calculated with GenAlEx v 6.0 software. Deviations from Hardy-Weinberg equilibrium (HWE) and linkage equilibrium between loci were quantified in GENEPOP v 1.294 within populations. The inbreeding coefficient (FIS) and confidence intervals were calculated in FSTAT v 1.2 with 1000 random permutations95.We employed the Quantity One software  to estimate the band sizes adjusted for the standard molecular weight size marker  and then formatted these size-based data to be used in GenAlEx v 6.096 using the PHYLIP v3.5c97 software. The CONSENSE subroutine within PHYLIP was used to perform 1000 bootstraps of the distance matrix. The resulting trees were visualized using TREEVIEW v 1.6.698.Unrooted neighbor-joining trees were constructed using matrices of chord genetic distanceD. citri populations in China. First, principal coordinate analysis (PCoA) was performed based on the simple matching coefficient using the decenter and eigenvector matrices in GenAlEx v 6.0 with 1000 random permutations. Second, analysis of molecular variance (AMOVA) was performed, where the total genetic variability was subdivided into three variance components, i.e., among clusters (FCT), among populations within clusters (FSC), within populations (FST) using the software Arlequin v 3.5 with 1000 permutations100.Different analyses were conducted to examine the contemporary population structure of 101 and further refined with BioEdit 7.0102. Haplotype diversity (Hd) and nucleotide diversity (π) of total D. citri individuals were calculated as defined by Nei103 in DnaSP v 5.0104 with nonsyn and syn sites separately. Genetic parameters for each population grouped by either location or host, including number of haplotypes (Nh), haplotype diversity (Hd) and nucleotide diversity (π), and number of segregating sites were further analyzed with nonsyn sites in concatenated mtDNA. AMOVA analysis was performed in the same way as described for the SSR data analysis above to test for whether significant population structure exists using the HKY model105. Evidence of isolation by distance among sampling locations in China was obtained by examining the correlations between matrices of pairwise genetic distance (FST) and pairwise physical distance106. Mantel tests107 were used to test the strength of the correlations between the matrices in GenALEx v 6.0.The 225 mtDNA sequences were compiled initially in Clustal-X37. To measure the level of homoplasy, the consistency index (CI) was calculated using PAUP 4.0b10108. Phylogenetic analysis with the maximum parsimony (MP) method was carried out by PAUP* v4.0b10 using 100 replicates, MAXTREES = 200,000, random addition, TBR branch swapping, and MULTREES, with gaps treated as missing data. Support for the monophyletic groups revealed in the maximally parsimonious trees was examined using 1000 bootstrap replicates for the bootstrap (BP) consensus tree. In terms of syn and nonsyn substitutions per site of mtDNA, branch lengths of each mitochondrial gene were estimated with 26 previously defined sequences using a codon-based model of substitution within the codeml application in PAML v4.0109. Tree topologies were visualized using TREEVIEW v 1.6.698. Further, we constructed a haplotype network with nonsyn sites in the concatenated mtDNA to visualize relationships among haplotypes in the program POPART based on a median-joining algorithm110 designed for populations of closely related samples allowing persistent ancestral nodes and multi-furcation in the network as extra information for population genetic structures.Substitution saturation was accessed for the alignments of single genes and concatenated dataset with DAMBE v. 5.3.46D. citri populations’ demographic changes were investigated using three neutrality tests: Tajima’s D111, Fu’s Fs112, and Ramos-Onsins & Rozas’ R2 113. All methods are premised on neutral selection. The neutral theory states that the majority of the genome is variable and neutral. Actually, this assumption is not true22. According to Huang et al.36, the value of the theory is its description for the linear phase, which has been retained by the MGD. Hence, the following neutrality tests of D. citri were from the nonsyn sites. The examination of deviation from neutrality by both D and Fs indices was based on 1000 coalescent simulations, as implemented in Arlequin v 3.5. Significant negative D and Fs values can be interpreted as signatures of population expansion. The R2 statistic of Ramos-Onsins& Rozas, which has more statistical power when population sizes are small, was calculated using a coalescent simulation algorithm implemented in DnaSP, with 1000 simulations.Signatures of To provide other estimates of population size changes, we also examined site mismatch distributions. Contrasting plots of observed and theoretical distributions of site differences yield insight into past population demographics. The expected mismatch distributions under a sudden expansion model were computed in Arlequinv 3.5. The sum of squared deviations (SSD) between observed and expected distributions was used as a measure of fit, and the probability (P) of obtaining a simulated SSD greater than or equal to the expected SSD was computed by randomization. If this probability (P) was >0.05, the expansion model was accepted. Graphical representation was implemented in Arlequin.GenBank accessions of nine polymorphic loci are KY053427-KY053435.Supplementary information


Heliosperma pusillum (Caryophyllaceae) in the south‐eastern Alps.The mosaic distribution of interbreeding taxa with contrasting ecology and morphology offers an opportunity to study microevolutionary dynamics during ecological divergence. We investigate here the evolutionary history of an alpine and a montane ecotype of From six pairs of geographically close populations of the two ecotypes  we obtained a high‐coverage restriction site associated DNA sequencing (RADseq) dataset that was used for demographic inference to test the hypothesis of parallel evolution of the two ecotypes.H. pusillum, uncovering up to five polytopic origins of one ecotype from the other. A complex evolutionary history is evidenced, with local isolation‐with‐migration in two population pairs and intra‐ecotype migration in two others. In all cases, the time of divergence or secondary contact was inferred as postglacial. A metagenomic analysis on exogenous contaminant RAD sequences suggests divergent microbial communities between the ecotypes.The data are consistent with repeated ecological divergence in The lack of shared genomic regions of high divergence across population pairs illustrates the action of drift and/or local selection in shaping genetic divergence across repeated cases of ecological divergence. Nosil Heliosperma pusillum (Caryophyllaceae) in the eastern Alps where two ecotypes, alpine and montane, present a mosaic distribution  produced by restriction site‐associated DNA sequencing (RADseq) of six pairs of geographically close populations of the two ecotypes, our main aim here was to test whether the montane–alpine ecotypic differentiation is the result of a single divergence event or if it originated independently multiple times. We first characterized the observed patterns of genetic divergence, by evaluating the strength of IBD vs IBE among localities, by estimating the amount of admixture between populations, and by assessing the level of parallel genetic divergence between the two ecotypes across localities. We finally assessed the relative probability of three demographic models representing different historical processes behind the observed pattern of divergence: strict isolation, isolation‐with‐migration and secondary contact following a period of allopatric separation.H. pusillum  across the distribution range of the montane ecotype  of the alpine and montane ecotypes of ype Fig. . In all SbfI restriction enzyme  and a protocol adapted from Paun et al. (http://www.vbcf.ac.at/facilities/next-generation-sequencing/) as 100 bp paired‐end reads.Single‐digest RAD libraries were prepared using the n et al.  with modprocess_radtags.pl included in the Stacks1.19 package . Each individual sample was analyzed independently and the abundance of TEs was compared among populations and between species  of 20×, a maximum number of differences between two stacks in a locus in each sample (−M) of seven, and a maximum number of differences among loci to be considered as orthologous across multiple samples (−n) of nine. In this dataset, a bimodal distribution of the GC content across loci was discovered, with one peak below 45% GC and a second one above 60%  the proportion of heterozygous individuals was > 0.65, an arbitrary cut‐off used to reduce the risk of including paralogs; (2) the GC content was > 65%, to further minimize the risk of including bacterial sequences; (3) any of the samples was scored as triallelic, again reducing the risk of including paralogs in the dataset; or (4) it was invariant. We observed an increase in the occurrence of SNPs in the last 10 positions of the reads , individual observed heterozygosity normalized by coverage : expected heterozygosity , ecotype pairs (i.e. six groups) and ecotypes (i.e. two groups).To assess the effect of distance among localities on genetic divergence, we tested for IBD across all populations by comparing matrices of genetic and geographic distances. Nei's genetic distances among localities, including a sample size correction factor Nei, , were eset al.  = 0.01) were considered as local highly divergent loci. The six lists of such loci (one for each ecotype pair) were compared to each other looking for shared highly divergent loci among two or more ecotype pairs. None of these highly divergent loci was shared by five or more ecotype pairs. Using the exact number of screened loci and the number of highly divergent loci scored in each ecotype pair, we simulated (by 1000 randomizations) the expected random distribution of shared loci for each comparison. Observed values were then compared with their expected random distribution. In the case of more overlap between ecotype pairs than expected by chance, we investigated the contribution of each ecotype to this signal by comparing the differentiation within one ecotype to the differentiation within the other ecotype. For this analysis we plotted the joint distribution of between‐localities intra‐ecotype FST . A Bayesian information criterion (BIC) is applied to choose the best k. The Bayesian algorithm implemented in faststructure  using the glPca function in the R package adegenet  strict isolation with no gene flow (SI), (2) isolation‐with‐migration (IM) and (3) secondary contact after allopatry (SC). We used a composite‐likelihood maximization method to estimate the best likelihood by simulating joint site frequency spectra under a continuous‐time Markovian coalescent model in fastsimcoal2  and the mutation rate (μ). Substitution rate is estimated by using an observed site frequency spectrum based on one selected SNP per 95‐bp locus and the resulting value needs to be scaled by two orders of magnitude to represent an average genome‐wide substitution rate. Migration rates in both directions (mam and mma) were estimated in the IM and SC models, time since divergence (Td) of the two ecotypes in SI and IM, and time since secondary contact (Tc) between the two ecotypes in SC. In the last, the time of the initial divergence was fixed to 50 000 generations ago, before the last Würm glaciation peak . We performed a maximum of 60 expectation/conditional maximization (ECM) cycles with each step requiring the generation of 100 000 simulated joint‐spectra. We ran 10 replicates of each model for each ecotype pair. Convergence of ML and parameter estimates was checked and different models were then compared using AIC.The demographic scenario underlying the observed pattern of local divergence was investigated in the four population pairs for which plain local divergence of the two ecotypes has been inferred in our previous analyses . Pairs B and C were excluded from this analysis as it became clear from et al., Na, Nm, mam, mma, μ, and Td or Tc estimated above were used in pseudo‐observed dataset simulations for each ecotype pair. We used π as a summary statistic. For each ecotype pair, we simulated 1000 100‐bp‐long loci on which we estimated the distribution of π calculated on all individuals of both ecotypes (combined π) under the IM and the SC models. The statistical difference between the simulated distribution of π for each model and the observed distribution was assessed by a Kolmogorov–Smirnov two‐samples test.We further tested the goodness‐of‐fit of the combination ‘scenario + posterior estimates of the parameters’ to the data generating pseudo‐observed datasets and comparing them with the observed datasets through summary statistics .We then estimated the 95% confidence intervals of the estimates of the demographic parameters in those models passing both the AIC and the goodness‐of‐fit criteria. For each of the successful models, we used the point estimates of the best‐likelihood run to simulate 100 2D‐SFS, which were then used to re‐estimate the demographic parameters through 30 likelihood‐maximization runs each. These 100 estimates were then used to build the 95% confidence intervals for each parameter in each of the selected models . Raw genomic data are available from the NCBI Short Reads Archive .Data and additional results are available from the Dryad Digital Repository (doi: P = 0.047) in the montane ecotype  million. The proportion of paired‐end reads Blasting to the Viridiplantae Transposable Elements database was very low for all individuals, 0.5–4.1%, with a higher proportion , one bacterial sequence (Borrelia) and one nematode sequence (Ascaridae). These loci were removed from the dataset. We further filtered the final dataset according to the criteria detailed in the Methods section to a total of 1097 variable loci, containing 3401 SNPs and occurring on average in 102 individuals , Watterson's θ and π for each population of the two ecotypes are given in Table Ho ind) comparing the two ecotypes at each locality are shown in Fig. Expected heterozygosity  whereas IBE had no effect on the genetic structure . The three alternative MANOVAs indicated that the most variance is explained by differences between populations, or between ecotype pairs, but not between the two ecotypes .The IBD model Fig.  was conflositan, we found between 79 and 116 loci with an extreme FST (FDR = 0.01) taking into account 500 000 simulations of the joint distribution of FST and He . The same was found in the comparison B vs D and C vs D . At the global level .Analyzing the six ecotype pairs separately with He Fig. a. Ecotypns Notes –S12, excvel Fig. , the diffind.clusters analysis started plateauing after k = 6  and E  ecotype pairs. As the SI model was within four AIC points from the best‐fit model (SC) in E, we generated a pseudo‐observed SI dataset and, after assessing its goodness‐of‐fit as above, we definitely discarded this model . In the remaining ecotype pairs (D and F), the distribution of combined π produced under the two competing models was indistinguishable from the observation  was rejected, whereas the isolation‐with‐migration (IM) and the secondary contact (SC) scenarios were not distinguishable due to too similar AIC scores Table . In all H. pusillum, usually classified as distinct species  in two out of four instances of local divergence of ecotype pairs  between B, C and D, in particular . In the majority of cases, we did not identify more loci with high FST shared among pairs than expected by chance . It is thus very likely that adaptive loci, or neutral loci linked to it, were not covered in our genome scan. In addition, if selection is acting on standing genetic variation present in the ancestral population  and of the genes , probably characterize the evolutionary history of contrasting ecotypes at medium to large geographic scales  should be directed to the Fig. S1 Proportion of transposable elements in the RADseq dataset.Fig. S2 Ecotype‐specific proportion of transposable elements.Fig. S3 RADseq dataset quality control.Fig. S4 Proportion of loci assigned to putative phyllosphere and contaminant taxa.Fig. S5 Analysis of putative phyllosphere and contaminant taxa by ecotype.Fig. S6 Analysis of putative phyllosphere and contaminant taxa by locality.Fig. S7 Minor 2D‐SFS estimated in each ecotype pair.Fig. S8 Individual observed heterozygosity normalized by coverage.Fig. S9 Analysis of isolation‐by‐distance.Fig. S10 Highly divergent loci shared among two ecotype pairs.Fig. S11 Highly divergent loci shared among three ecotype pairs.Fig. S12 Highly divergent loci shared among four ecotype pairs.Fig. S13 Joint distribution of FST between alpine and montane populations.Fig. S14 Bayesian Information Criterion k‐means analysis.Fig. S15 Principal component analysis.Table S1 Sample designTable S2 Microbiome structure and diversityTable S3 Genetic diversityNotes S1 Sampling.Notes S2 Transposable elements.Notes S3 Dataset quality control.Notes S4 Analysis of contamination by leaf microbiome.Notes S5 Custom Python code.Notes S6 Testing alternative demographic scenarios.Notes S7 Summary statistics of genomic diversity.Notes S8 Structure of genetic diversity.Click here for additional data file.