Dipterocarpus condorensis is an endangered species in the tropical forests of south-eastern Vietnam, both from its over-exploitation and habitat loss. Therefore, knowledge of population genetic diversity and population structure is essential for identifying the species conservation measures. In the present study, we evaluated genetic diversity and population structure using nine microsatellites for 183 individual trees from eight populations, representing the distribution range of D. condorensis in Vietnam. Two clustering approaches  revealed that all studied individuals clustered into three genetic groups, which were related to gene flow across the range of D. condorensis in the lowland tropical forests of south-eastern Vietnam. Limited gene flow was implicated in anthropogenic disturbance. Genetic differentiation among populations was relatively low (the Weir and Cockerham index of 0.122 and the Hedrick index of 0.149) and showed significant differentiation. The genetic variability of the populations was low (HO = 0.298 and HE = 0.324), which suggested the negative effects of habitat degradation and over-exploitation. Our studies also determined that D. condorensis populations can have undergone recent bottlenecks. We recommend conservation activities for this species based on these results.Anthropogenic disturbances in tropical forests often affect the genetic diversity of a species.  Dipterocarpus condorensisis an endangered species in forests of south-eastern Vietnam, both from its over-exploitation and habitat loss. In this study, we evaluated genetic diversity and population structure for eight populations, representing the distribution range of the species. Bayesian analysis and discriminant analysis of principal components revealed three genetic groups, which were related to gene flow among different areas. Limited gene flow was implicated in anthropogenic disturbance. Genetic differentiation was relatively low and showed significant differentiation. The genetic variability of populations was low, which suggested the negative effects of habitat degradation and over-exploitation. Populations may have undergone recent bottlenecks. Habitat degradation and fragmentation are major threats to many plant species, which affect species distribution, population size and genetic variability . The adaDipterocarpus condorensis has been commonly used in making furnitures and building. The species is used as a valuable source of essential oils. It is also widely exploited due to its commercial trade. The dipterocarp can be found in the lowland rainforests of Vietnam. This species is bisexual and pollinated by insects. The flowers are large, actinomorphic and scented. Fruiting appears only once in every 2–3 years, with fruit maturation taking place between the months of April and May. A single-seeded nut with a wing-like calyx makes up the fruit. Seeds are dispersed by wind. Dipterocarpus condorensis is only found in forests on ancient alluvial rocks, granite and basalt rocks along coast provinces of the southern central region from Ba Ria–Vung Tau to Phu Yen. Deforestation, fragmentation and unsustainable management activities such as selective logging have all been detrimental to D. condorensis habitats caused due to the activities of local people and forestry enterprises exploiting the species for its valuable timber and resin. Logging leads to fragmented habitats and low population density. This threatens the long-term survival of species resources. Globally, D. condorensis is classified as CR A1cd, B1 + 2c  markers are powerful tools because of their high polymorphism and codominant inheritance. Various SSR markers have been developed for dipterocarps  receiver. Samples were packed in plastic bags with silica gel in the field and then transferred to the Laboratory Molecular Biology, Institute of Genome Research and stored at −45 °C until DNA extraction.We extracted genomic DNA from the samples using the modified CTAB method proposed by 2O, 2.5 µL of 1× PCR buffer , 1.0 µL of MgCl2 (2.5 mM), 0.5 µL of dNTPs (0.2 mM each), 1.5 µL of genomic DNA (10 ng), 1.0 µL of Taq DNA polymerase at 1.25 units and 0.5 µL of each primer at 10 pmol. The SSR primer pairs were analysed by testing seven primer pairs for Shorea curtisii , under the following thermal cycles: an initial denaturing step at 94 °C for 2 min, followed by 40 cycles consisting of 1 min at 94 °C, 30 s annealing temperature for each primer pair at 54–56 °C and 1 min extension at 72 °C, and 10 min final at 72 °C to complete the extension of any remaining products before holding the samples at 4 °C until they were analysed. The amplification products were separated using a Sequi-Gen®GT DNA electrophoresis system of 6 % polyacrylamide gels in 1× TAE buffer and then visualized by GelRed™ Nucleic Acid Gel Stain. Allele sizes were detected by the Gel-Analyzer software of GenoSens1850  with a 50-bp DNA ladder (Invitrogen) [see].The polymerase chain reaction (PCR) was performed in a 25-µL solution volume containing 17.5 µL of ddHnceolata . A totalNA), number of effective alleles (NE) per locus, number of private alleles (NP), observed (HO) and expected (HE) heterozygosities across loci and populations, proportion of polymorphic loci (PPL) and gene flow (Nm) using GenAlEx v. 6.5 /4. In addition, the fixation index (FIS—inbreeding coefficient) was calculated for each locus and population by FSTAT v. 2.9.3  using INEst  were implemented for K between 1 and 10 at 500 000 Markov Chain Monte Carlo (MCMC) repetitions and at 100 000 burn-in period. To determine the optimal value of K, we used STRUCTURE HARVESTER  was performed from 1 to 20, and the ‘optimal’ clusters were determined based on the Bayesian information criterion. The DAPC was also performed the prior information to analyse for assignment of individuals to populations. The complot function in adegenet was implemented to visualize the genetic clusters. Fourteen first principal components (PCs) (98.5 % of conserved variance) of PCA and seven discriminant eigenvalues were retained using the xvalDAPC function.We calculated the genetic differentiation between populations according to FST  and GʹSTT values  using GeARVESTER  to detecv. 1.1.2  and bar v. 1.1.2 . We alsoD. condorensis. Of these alleles, two alleles were private. The nine SSR loci were polymorphic in D. condorensis (NA) ranged from 2 at two loci to 4 at one locus, an average of 2.9. The effective allele (AE) ranged from 1.2 at two loci to 2.1 at one locus, an average of 1.6. Polymorphism information content values ranged from 0.159 at the dipt1 locus to 0.509 at the dipt2 locus, an average of 0.33. Eight of the nine loci displayed heterozygote deficits under the Hardy–Weinberg equilibrium that indicated inbreeding or the presence of null alleles. Micro-Checker analysis detected null allele frequencies at seven loci (P < 0.05). The observed (HO) and expected (HE) heterozygosity averaged 0.296, ranging from 0.157 at dipt1 to 0.465 at dipt3 and 0.324, ranging from 0.162 at dipt1 to 0.477 at dipt2, respectively. The average fixation index (FIS) over all populations for each locus was 0.071. The studied nine loci had a positive fixation index, indicating an excess of homozygotes and inbreeding. However, among these loci, two loci had significant inbreeding (P < 0.05). The inbreeding coefficient determined for the total populations (FIT) averaged 0.183  in each population was 2.4. The Bung Rieng population had the highest NA value (2.8) and Tan Ha had the lowest NA values (2.0). The effective allele (AE) varied from 1.3 in Ham Minh to 1.9 in Bung Rieng, with an average of 1.6. The private alleles (NP) were found in two populations of Tan Thuan and Bong Trang (HO) value averaged 0.298, ranging from 0.18 in Ham Minh to 0.425 in Bung Rieng. The mean expected heterozygosity (HE) value was 0.324, ranging from 0.202 (Ham Minh) to 0.442 (Bung Rieng). Thus, Ham Minh had the lowest observed and expected heterozygosity, whereas the highest values were in Bung Rieng. The fixation index (FIS) varied from 0.053 (Binh Chau) to 0.166 (Cam Duc), with an average of 0.109, indicating an excess of homozygotes and inbreeding in all populations. The FIS values were significantly positive in three populations of Ham Minh, Cam Duc and Bong Trang. The inbreeding corrected for null alleles based on the individual inbreeding model (FISIIM) varied from 0.02 in Phuoc Thuan to 0.085 in Tan Ha, with an average of 0.031, also indicating homozygote excess. However, this value was low compared to the fixation index FIS. BOTTLENECK analysis showed that a significant heterozygosity deficit was detected in Ham Minh and Bung Rieng. A significant heterozygosity excess was only found in Bung Rieng. This suggests that there is evidence of a recent bottleneck in some studied populations.At the population level, the PPL was high, an average of 94.4 %, ranging from 77.8 % in Phuoc Thuan to 100 % in five populations of Ham Minh, Tan Thuan, Bung Rieng, Binh Chau and Bong Trang. The mean number of alleles per locus (ng Trang . The obsFST) over all D. condorensis populations for each locus varied from 0.046 (dipt8) to 0.291 (dipt7) with an average of 0.122, indicating relatively low differentiation , whereas the lowest appeared between Ham Minh and Cam Duc (0.024). Significant genetic differentiations were found between populations (P < 0.05), except the FST value between Ham Minh and Binh Chau. The average number of migrants (Nm) per generation was 2.589, which was considered high. The lowest Nm value was found at dipt7 (Nm = 0.609) and the highest value was at dipt8 (Nm = 5.132). Analysis of molecular variance was performed to investigate genetic variations among and within populations on the basis of 10 000 permutations. The results showed that 13.36 % of the total genetic variation occurred among populations, and 86.64 % of the population variation was related to the heterozygosity of individuals within populations [see]. The variation among populations was significant (P < 0.0001).Genetic differentiation  was three, with the highest value of delta K (244.5) obtained from STRUCTURE HARVESTER , and between Bong Trang and Phuoc Thuan (FST = 0.029), whereas Bung Rieng was separated from two populations of Bong Trang and Phuoc Thuan. Tan Thuan was differentiated from Tan Ha and Cam Duc. However, Tan Thuan was closer to Bong Trang than Cam Duc and Tan Ha.We evaluated the population structure of the 183 individuals of ARVESTER . At K = function  and B. Tulations . The higDipterocarpus condorensis to provide essential information for conservation actions needed in the future. This is an endangered species with a narrow geographic distribution and small population size; therefore, the genetic diversity is expected to be low compared to species with a wide distribution range and large population size  compared with previously published studies for some other species, such as S. leprosula  across 183 wild trees from eight populations suggests that relatively high heterozygous deficiency may exist within the distribution range of D. condorensis. This suggested the existence of inbreeding in small populations, although the dipterocarp is the predominantly outcrossing species . Similarly, our AMOVA analysis showed low significant variation among D. condorensis populations (P < 0.0001). In previous studies, similar results were reported low genetic differentiation and reflected the high level of gene flow, such as Shorea lumtensis (S. javanica (D. dyeri (D. condorensis is pollinated by insects (bees) and its seeds are dispersed by wind and small mammals (squirrels and bats). The dispersal of pollen grains is mainly depended on insects. Therefore, increasing isolation distance will reduce pollen transmission among populations. Fragmentation can affect the genetic structure of this species. The lowest genetic differentiation (0.024) was found between populations Ham Minh and Binh Chau related to the closest geographic distance. The highest genetic differentiation (0.138) was found between Ham Minh and Phuoc populations, having the longest geographic distance. These suggest that geographic distance may restrict gene flow between these populations. However, no similar findings were found between Phuoc Thuan and Bung Rieng (FST = 0.109), Phuoc Thuan and Cam Duc (FST = 0.1). The analyses of STRUCTURE and DAPC without prior information revealed three major genetic groups for 183 wild D. condorensis trees. One genetic group was composed of two populations Phuoc Than and Bong Trang. The second group was composed of four populations Cam Duc, Ham Minh, Tan Ha and Tan Thuan. The third group included two remaining populations Bung Rieng and Binh Chau. However, the considerable mixing of the three groups in several populations was determined, suggesting that genetic differentiation was relatively low and gene flow was high among these populations. This can also be found in the DAPC analysis with prior information. The mixing of the three groups was found in five populations Tan Ha, Tan Thuan, Bung Rieng, Binh Chau and Bong Trang, while the mixing of the two groups was observed in Cam Duc and Ham Minh populations. No admixture was found in the Phuoc Thuan population. These isolated populations could be related to anthropogenic disturbance. A reduction in the dispersal of pollen grains and seeds could be considered as a barrier to gene flow for D. condorensis species.Our results showed a relatively low genetic differentiation among populations with the D. dyeri . High geD. condorensis based on a sampling strategy involving its natural distribution areas, and determined the low level of overall genetic diversity and the genetic diversity of its populations. Some populations might have suffered from anthropogenic disturbance. The STRUCTURE analysis showed that three genetic groups were consistent with the DAPC with a prior information. Genetic variation mainly existed within D. condorensis populations. Furthermore, private alleles were found in Tan Thuan and Bong Trang populations. These are resources that contribute to the maintenance and evolution of species. Therefore, in developing a conservation strategy for D. condorensis, populations with high genetic diversity and private alleles, such as Bong Trang, Bung Rieng and Tan Thuan should focus on in situ conservation, meanwhile the remaining populations should be collected for ex situ conservation as a germplasm source of the species in the future.In the present study, we investigated the genetic diversity and genetic structure of The following additional information is available in the online version of this article—Dipterocarpus condorensis.Table S1. Collection localities of Dipterocarpus condorensis.Table S2. Nucleotide sequences of the SSR primers, allele size range, genetic diversities for Dipterocarpus condorensis.Table S3. Microsatellite (SSR) raw data for FST) between the eight Dipterocarpus condorensis populations.Table S4. Pairwise genetic differentiation  assigned to each of the three inferred genetic clusters (columns) using the DAPC without prior information.Figure S4. Individuals (rows) assigned to the genetic clusters (columns) based on discriminant functions. Colour shows membership probabilities to each genetic cluster .plac007_suppl_Supplementary_MaterialClick here for additional data file.plac007_suppl_Supplementary_Table_S3Click here for additional data file.


Coregonus lavaretus, is a widely distributed salmonid fish species on mainland Europe, but in Britain it has only seven native populations, all of which are found on the western extremes of the island. The origins and colonization routes of the species into Britain are unknown but likely contributed to contemporary genetic patterns and regional uniqueness. Here, we used up to 25,751 genome‐wide polymorphic loci to reconstruct the history and to discern the demographic and evolutionary forces underpinning divergence between British populations. Overall, we found lower genetic diversity in Scottish populations but high differentiation (FST = 0.433–0.712) from the English/Welsh and other European populations. Differentiation was elevated genome‐wide rather than in particular genomic regions. Demographic modelling supported a postglacial colonization into western Scotland from northern refugia and a separate colonization route for the English/Welsh populations from southern refugia, with these two groups having been separated for more than ca. 50 Ky. We found cyto‐nuclear discordance at a European scale, with the Scottish populations clustering closely with Baltic population in the mtDNA analysis but not in the nuclear data, and with the Norwegian and Alpine populations displaying the same mtDNA haplotype but being distantly related in the nuclear tree. These findings suggest that neutral processes, primarily drift and regionally distinct pre‐glacial evolutionary histories, are important drivers of genomic divergence in British populations of European whitefish. This sheds new light on the establishment of the native British freshwater fauna after the last glacial maximum.Pleistocene glaciations dramatically affected species distribution in regions that were impacted by ice cover and subsequent postglacial range expansion impacted contemporary biodiversity in complex ways. The European whitefish,  Using a dataset of genome‐wide polymorphic loci and population genomics approaches, we reconstructed the evolutionary history and discerned the demographic and evolutionary forces underpinning divergence between British populations of European whitefish. We found a deep divergence between Scottish and English/Welsh populations, with the latter sharing a more recent common ancestor with populations from central Europe. Combinatorial barcoded Illumina adapters were then ligated to PstI‐HF and MspI overhangs. Samples were size selected using a PippinPrep (Sage Science) at a tight target range of 150–300 bp fragments. To enrich for the selected loci, we performed PCR amplification cycles with the following settings: 30 s at 98°C, 9X , 5 min 72°C. After PCR purification, the library was run on a 1.25% agarose gel stained with SYBR Safe (Life Technologies) to remove any adapter dimers and/or fragments outside the selected size range. DNA was excised manually, cleaned and quantified using the Qubit Fluorometer with the dsDNA BR Assay (Life Technologies) to ensure the final library concentration of >1 ng/μl. The library was composed of 102 samples plus three technical replicates and was sequenced on an Illumina NextSeq500 with 75‐bp paired‐end reads at Glasgow Polyomics.DNA was extracted from fish fin clips using the NucleoSpin Tissue kit (Macherey‐Nagel) following the manufacturer's recommendations. The protocol used for the ddRADseq , ‐r 0.8 (minimum proportion of samples in a population required to have a locus), ‐‐max_obs_het 0.6 (maximum observed heterozygosity for a nucleotide at a locus), ‐‐min_maf 0.05  and ‐‐write_single_snp (retain one SNP per locus). This file was then filtered further with vcftools v.0.1.15  to filter sites that were out of HWE within populations, although no such sites were detected. Finally, we excluded a sample from the Baltic region which had more than 90% missing data after filtering. The Russian samples had between 68% and 87% missing data but were retained in the dataset only for the population structure and phylogenetic analyses. Through this process, we identified 25,751 RAD loci that were retained for the analysis (i.e. whitelist loci). Summary statistics of genetic diversity  were calculated by the population module of Stacks. Rarefied allelic richness was calculated in hierfstat v. 0.04 .Multiple approaches were used to assess population structure. For most of these analyses, we used the retained loci but only used one SNP from each (to reduce the effect of linkage), with a total of 91 samples, 25,751 SNPs, and an average of 12.4 (± 5.5) SNPs per 1 Mb; this is referred as the ‘pop‐gen dataset’. First, to evaluate population structure at the individual level, we ran a principal component analysis (PCA) with Jombart,  to run aJombart, , which uTREEMIX v.1.13 . Windows with fewer than five SNPs were excluded as they could be unreliable  . In addition, we also investigated the relationship of European whitefish populations using unrooted phylogenetic networks as implemented in SplitsTree v.4.14.6  was then used to down‐sample each population to seven individuals, to avoid biases due to different sample sizes, and no missing data were allowed. The filtering resulted in a dataset of 42 individuals and 20,510 SNPs. The minor multidimensional site frequency spectra (MSFS) were generated with the script easySFS.py (available at https://github.com/isaacovercast/easySFS). To determine absolute values for divergence times and other inferred parameters, we corrected the number of monomorphic sites in the SFS  for Sanger sequencing in the forward direction for all samples but two, which were sequenced in the reverse direction. Overall, we successfully amplified the mtDNA gene ND1 from the Scotland (n = 17), Wales (n = 5), Baltic (n = 5), Alpine (n = 6) and Norway (n = 10) populations; sequences were not available from the English populations. Contigs were aligned to a reference sequence in Geneious v.7.1.9 using the highest sensitivity method. The reference sequence (GenBank JQ661447) came from an individual from Ringkøbing Fjord in Denmark belonging to the southern European mtDNA haplogroup [as defined in Jacobsen et al.   mtDNA gene (975 bp) was PCR amplified using the primers ND1n et al. ]. In addn et al.  and two 33.1To evaluate the extent to which European whitefish populations evolved independently or underwent significant admixture events, we analysed population structure, differentiation, co‐ancestry and admixture within Britain and at the European level. A PCA demonstrated that the Norwegian population was the most distinct from all other populations, separating along PC1, which explained 22.2% of the total variance Figure . The secFST ranging from 0.546 to 0.661 . At the European level, the Baltic population was the least differentiated from all populations (FST = 0.298–0.502), whereas the population from Norway was the most differentiated (FST = 0.457–0.712)  Table , consistT Figure , again wR, HE, HO and π) followed the same pattern across all populations (Table O = 0.095–0.135), followed by the Alpine and Norwegian populations (HO = 0.140–0.149), and it was intermediate in the English and Welsh populations (HO = 0.157–180), and highest in the Baltic population (HO = 0.244).The four measures of within‐population genetic variation   analyses  Figure  and Admis Figure , which cs Figure . At the n Figure . The clo2 Figure . Howevery Figure .TREEMIX recovered instances of gene flow amongst most populations  and Danish (southern mtDNA haplogroup) was in some cases an order of magnitude higher for mtDNA than from microsatellite data (Hansen et al., Our analysis based on nuclear data does not find a clear distinction between a southern and a northern European whitefish clade, as suggested by mtDNA (Hudson et al., 5We provide molecular evidence of a deep divergence between, and a dual colonization of, Britain by Scottish and Welsh/English populations of European whitefish, which reflects a distinctive pre LGM pattern at the edge of the species distribution relevant to continental recolonization. Previous studies uncovered a similar structuring between the seven British populations (Beaumont et al., The authors have no conflict of interest to declare.MC, CEA and KRE designed the research. MC, CWB, ARDG, IJW, JW, GB, KP and CEA provided biological samples. MC and MB conducted laboratory work with support from KRE. MC conducted the analyses. MC wrote the paper with contributions from KRE and CEA. All authors edited/approved the final manuscript.https://publons.com/publon/10.1111/jeb.13948.The peer review history for this article is available at Supplementary MaterialClick here for additional data file.Table S1Click here for additional data file.


Lonicera oblata. A low level of genetic diversity and a high degree of genetic differentiation among populations were observed based on the SNP data. Both principal component and phylogenetic analyses detected seven clusters, which correspond exactly to the seven geographic populations. Under the optimal K = 7, Admixture suggested the combination of the two small and geographically neighboring populations in the Taihang Mountains, Dongling Mountains, and Lijiazhuang, while the division of the big population of Jiankou Great Wall in the Yan Mountains into two clusters. High population genetic diversity and a large number of private alleles were detected in the four large populations, while low diversity and non-private alleles were observed for the remaining three small populations, implying the importance of these large populations as conservation units in priority. Demographic history inference suggested two drastic contractions of population size events that occurred after the Middle Pleistocene Transition and the Last Glacial Maximum, respectively. Combining our previous ecological niche modeling results with the present genomic data, there was a possible presence of glacial refugia in the Taihang and Yan Mountains, North China. This study provides valuable data for the conservation and management of L. oblata and broadens the understanding of the high biodiversity in the Taihang and Yan Mountains.Characterizing genetic diversity and structure and identifying conservation units are both crucial for the conservation and management of threatened species. The development of high-throughput sequencing technology provides exciting opportunities for conservation genetics. Here, we employed the powerful SuperGBS method to identify 33, 758 high-quality single-nucleotide polymorphisms (SNP) from 134 individuals of a critically endangered montane shrub endemic to North China,  The Earth’s biodiversity consists of approximately 9 million types of organisms , while biodiversity losses have exerted a profound impact on the ecology and society as a whole . A totalAcer mono Maxim. (Quercus mongolia Fisch. ex Ledeb. (Juglans mandshurica Maxim. (Eleutherococcus senticosus (Rupr. and Maxim.) Maxim.  Maxim. , and Sch Bailley ]. North Opisthopappus taihangensis (Ling) Shih and Taihangia rupestris Yuet Li. The YMs lie in the north of the North China Basin with an east longitude from 115° to 119°. The west end of the YMs is connected to the northeast end of THMs. Most mountains in the THMs and YMs are extremely steep, particularly in the THMs. These two great mountains meet in the northwest of Beijing, and the valley between Changping District on the westside and Yanqing District on the northside are considered as the division. Uncovering the genetic composition of species endemic to the THMs and YMs may help us understand the genetic pattern and preservation of biodiversity in North China and can also allow us to further explore the role of these mountains in the survival and dispersal of endemic species.Two great mountains are located in North China, namely, the NNE trending Taihang Mountains (THMs) and the EW trending Yan Mountains (YMs), which harbor the highest seed plant diversity in northern China, as well as a high endemism rate . The THMHigh-throughput sequencing (HTS) technology can rapidly detect thousands of single-nucleotide polymorphisms (SNPs) in a cost- and time-efficient manner on a genome-wide scale. Compared to Sanger sequencing method, which generates limited loci, the large data generated from HTS can provide robust phylogeny, reducing the potential incomplete lineage sorting, and enable us to address a wide range of evolutionary questions. Furthermore, a small sample size per population  can also generate big data in the population genomic study based on the HTS approach . Thus, iLonicera oblata K. S. Hao ex P. S. Hsu and H. J. Wang (Caprifoliaceae) is a critically endangered montane shrub endemic to North China , and some individuals are injured directly by cutoff. The highly threatened condition of L. oblata may be attributed to the small number and size of populations scattered in such a vast geographic range, unique but extremely fragmented habitats, strong human disturbances, and sensitivity to climate change  assess the population structure and genetic diversity across the full range of the species; (ii) infer the genetic signatures of potential refugia during the Quaternary climate oscillations in the THMs and YMs; and (iii) identify conservation units (CU) and provide suggestions for the conservation of this species. Our results provide both a theoretical basis and a valuable genetic database for the conservation planning of L. oblata and contribute to improving our understanding of the preservation and evolution of biodiversity in North China.In this study, we employed the novel approach of RAD sequencing, SuperGBS , on the L. oblata were sampled. Based on the number of individuals, the seven populations can be classified into four large populations  located at the species marginal distribution region and three small populations  located at the central region , polymorphism information content (PIC), nucleotide diversity (π), the effective number of alleles (NE), and Wright’s F statistics [fixation index (FST) and inbreeding coefficient (FIS)] were calculated using the module “population” in Stacks version 2.4, VCFtools version 0.1.16, and genepop version 1.1.4  ranged between 2 and 10, each of which was repeated 10 times. The optimal value of K was determined using cross-validation (CV) error, which has the minimum error in Admixture. Principal component analysis (PCA) was performed by GCTA version 1.26.0 (BS) were calculated with 1,000 ultrafast bootstrap replicates.Evolutionary clusters were identified using Admixture version 1.3.0  with then 1.26.0  to explon 1.26.0  to clariL. oblata was inferred using Stairway Plot 2  were generated, resulting in 594, 664, 390 clean reads (86.82 Gb) and an average of 0.65 Gb per sample . The aveHE, HO, PIC, π, and NE) for the seven populations of L. oblata. PIC values in all seven populations were very low (<0.19), with a value of 0.2364 at the species level. Relatively higher values of genetic diversity were observed among the four large populations  at the marginal regions compared to the three small populations located at the center . The northwest population WTS exhibited the highest values of HE (0.2291), HO (0.2054), PIC (0.1853), π (0.2328), and NE (1.3826) (HE (0.1477), PIC (0.1172), π (0.1625), and NE (1.2563) were determined for BJS, one of the smallest populations. Furthermore, a large number of private alleles were detected in the four large populations , while none were detected in the three small populations.(1.3826) , while tFST = 0.3245) was observed at the species level, along with a low inbreeding coefficient (FIS = 0.0986). The highest genetic differentiation was detected between the most northeast population JK in the YMs and the most southwest population HDL in the THMs (FST = 0.3963), while the value was minimized between the two closest  neighbored populations located at the center of its distributional range, LJZ and DLS . The southernmost population HDL differed greatly from the other THM populations (FST of 0.2626–0.3805). Notably, WTS, one of the four largest populations on the northwest side, exhibited less variation with its neighbors, namely, BJS, DLS, LJZ, and HDL, with FST values varying from 0.2320 to 0.2723   .L. oblata, namely, HDL and LJZ, both underwent drastic decline after the Middle Pleistocene Transition  values were detected at the population and species level, suggesting a low degree of genetic diversity within L. oblata. HE values determined here were markedly lower than other Lonicera species  Maxim.) (HE = 0.3482 in Tetraena mongolica Maxim. (HE = 0.364 in Firmiana danxiaensis H. H. Hsue and H. S. Kiu (L. oblata (L. oblata (FST = 0.3245), implying a long history of independent evolution and less gene flow among populations.The genome-wide data generated by HTS are relatively easier to obtain than traditional markers, and the high number of informative loci provide us not only the solid basis for accurately characterizing the population structure  but alsopulation . In this Maxim.) , as wella Maxim. , HE = 0.. S. Kiu ; Table 1L. oblata may be attributed to the following factors. (i) The extremely low numbers both at the population and individual level. The only seven uneven populations are highly fragmented and scattered at the top of mountains in a broad geographic range, and the limestone-preferred habitat of the species has been disturbed by long-term anthropogenic activities throughout history. The strong genetic differentiation among populations of L. oblata implies that the phenomenon of sky islands, which is significant in southwest China, may also occur in the THMs and the YMs in North China. (ii) The combined side effect of its reproductive system and fruit dispersal characteristics. A mixed mating system of biased outcross and partial self-fertilization is observed in L. oblata, and insects are necessary for pollination . Extending the distributional range of L. oblata over the contribution of birds like Zosterops is difficult. (iii) Seedlings barely survive in the field, which further decreases the genetic exchange between populations. Seedlings were difficult to find in the community during our field investigations, and an obvious inverted triangle population pyramid was observed (unpublished data). Hence, multiple negative effects may contribute to the strong genetic structure of L. oblata, whose destiny may be further challenged by future climate changes  were randomly selected in the field just like the other samples. There were no distinct characteristics  identified for the six samples compared to the remaining samples. JK is strongly exposed to tourism activity compared to the other six populations. The Jiankou Great Wall, a grand section of the Great Wall, attracts thousands of tourists each year. Many L. oblata individuals grow right on the Great Wall and are threatened by tourists during their climb. Some deaths of individuals due to the direct damage from tourists were witnessed during our fieldwork. Future work will include fine-scale sampling and landscape genomic study in order to understand the fine-scale genetic structure within JK.The genetic structure of a population provides further information on the evolution of a species. In this study, a significant phylogeographic structure was identified from the structure, principal component, and phylogeny analyses, suggesting the division of seven groups . However cluster . Such a Prunus   of one population may sometimes be resolved in the lineage of another population, such as cushion willow , grape , Prunus Prunus , and Q. . Wilson . In thisS = 100) . Both thS = 100)  also impL. oblata are genetically isolated. A high level of genetic diversity and plentiful private alleles were detected in the four large populations located in the marginal region of the L. oblata distribution range . Furthermore, pollen limitation and harsh climate conditions during its flowering period restrain its survival and reproduction  JK, JMS, HDL, and WTS populations should be given priority among the seven conservation units, including the construction of mini-reserves for the former three that are not distributed in national parks or nature reserves, and forceful conservation actions should be performed; (iii) the collection and long-term preservation of seeds from multi-populations should be accomplished, with the timely determination of seed germination and artificial propagation techniques; and (iv) as the intersection regions between the north of the THMs and west of the YMs are predicted to provide stable climate conditions in the future  forceful e future , this arL. oblata, a highly threatened montane shrub endemic to North China, whose natural distribution range lies in the contact zone between southern warm-temperate and northern cold-temperate forests. Based on the SuperGBS method, we determined a low level of genetic diversity, a high degree of genetic differentiation, and a strong phylogeographic structure. Sharp population size contraction was suggested, which occurred after the middle Pleistocene Transition and the LGM, respectively. The results from the current genomic data combined with our previous ENM analysis suggest the existence of LGM refugia in the THMs and YMs. Affected by multiple factors relating to geography, climate, and biological and ecological characteristics, the future of L. oblata is challenged by the “nowhere to go” scenario. Four key conservation units were identified for L. oblata, and several conservation suggestions were provided. Further work characterizing the whole genome of L. oblata and a fine-scale landscape genomic study should be performed especially on the JK population with the aim to understand how geographic and ecological factors shape its genetic structure and evolutionary history. Our population genomic study provides valuable information for the conservation and management of L. oblata and contributes to the further understanding of the role of the THMs and YMs in preserving the biodiversity in North China.In this study, we performed a genome-wide population genetic investigation on https://www.ncbi.nlm.nih.gov/genbank/, PRJNA787048.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: X-YM conceptualized, supervised the study, analyzed the data, wrote, and revised the draft. Y-MW, X-LS, LT, F-WL, X-FX, and YN contributed to the fieldwork and collected the materials. Y-MW contributed to the species distribution maps. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Deletion/insertion polymorphism (DIP), as a short insertion/deletion sequence polymorphic genetic marker, has attracted the attention of forensic genetic scientist due to its lack of stutter, short amplicon and abundant ancestral information. In this study, based on a self-developed 43 autosomal deletion/insertion polymorphism (A-DIP) loci panel which could meet the forensic application purposes of individual identification, kinship testing and ancestral inference to some extent, we evaluated the forensic efficiencies of the above three forensic objectives in Chinese Yi, Hani and Miao groups of Yunnan province. The cumulative match probability (CPM) and combined probability of exclusion (CPE) of these three groups were 1.11433E-18, 8.24299E-19, 4.21721E-18; 0.999610217, 0.999629285 and 0.999582084, respectively. Average 96.65% full sibling pairs could be identified from unrelated individual pairs (as likelihood ratios > 1) using this DIP panel, whereas the average false positive rate was 3.69% in three target Yunnan groups. With the biogeographical ancestor prediction models constructed by extreme gradient boosting (XGBoost) and support vector machine (SVM) algorithms, 0.8239  of the unrelated individuals could be correctly divided according to the continental origins based on the 43 A-DIPs which were large frequency distribution differentiations among different continental populations. The present results of principal component analysis (PCA), multidimensional scaling (MDS), neighbor joining (NJ) and maximum likelihood (ML) phylogenetic trees and STRUCTURE analyses indicated that these three Yunnan groups had relatively close genetic distances with East Asian populations. Compare studies . Subsequ studies , and 43  studies , and com studies , 38 X-DI studies , AGCU DI studies , and AGC studies  were dev studies .The combined probability of exclusion (CPE), cumulative match probability (CPM) and total discrimination power (TDP) are three regular parameters to evaluate the efficiencies for individual identification and paternity testing. Both the likelihood ratio (LR) and identity by state (IBS) methods could be used to calculate the values for kinship testing with biallelic markers such as DIP and single nucleotide polymorphism (SNP), and the method of LR has been proven to be more accurate . When anYunnan, located in southwest China, is a multi-ethnic province, in which the Yi, Hani and Miao ethnic groups are three numerous minorities. There are more than 5.0 million Yi, 1.6 million Hani and 1.2 million Miao individuals, and these three groups account for 17.11% of the total population of Yunnan province according to the 2010 sixth population census of China. In addition to China, Yi and Hani individuals are also distributed in Vietnam, Burma and other Southeast Asia countries . AccordiAll 542 fingertip blood samples including 125 Yi, 208 Hani and 209 Miao ethnic individuals were collected from Chinese Yunnan province. All the samples were collected with written informed consents. All volunteers who provided blood samples were informed of the purpose and main content of this research; possible risk and many personal rights protecting matters of the sample collection. This research involving human samples was approved by the ethnic committees of Xi’an Jiaotong University Health Science Center, and the approval number is 2019–1039.A self-developed panel including 43 A-DIP loci and a sex determination locus Amelogenin was used to investigate the genetic diversities of three groups living in Chinese Yunnan province. These 44 loci were evenly designed into four channels labeled with four different dyes, and then 11, 9, 11, 13 loci were labeled by 6-FAM, HEX, TAMRA and ROX, respectively. Additionally, Org 500  was used to label the internal size standard fragments. The amplification for 44 loci was performed on the 9700 Thermal Cycler  according to the recommendation in previous work . The ampR (https://www.r-project.org/) was used to draw the LR distribution maps of two relationships mentioned above. The cumulative match probability (CPM) is calculated as the formula of CPM = PM1 ✕ PM2 ✕ PM3 ✕ … ✕ PMk . The total discrimination power (TDP) value is calculated as the formula of TDP = 1 – (1-DP1) ✕ (1-DP2) ✕ (1-DP3) ✕…✕ (1-DPk) . The combined probability of exclusion (CPE) value is calculated as the formula of CPE = 1 – (1-PE1) ✕ (1-PE2) ✕ (1-PE3) ✕…✕ (1-PEk) .The Hardy-Weinberg equilibrium (HWE) test and pairwise linkage disequilibrium (LD) analysis were conducted using the Arlequin software v3.5 in three Yunnan groups . Allele In) value of population pairwise comparison was conducted among 31 populations with INFOCALC v1.1  was conducted based on the raw individuals’ genotypes by R software. The multidimensional scaling (MDS) analyses were performed by R software based on the Nei’s genetic distance and pairwise FST value, respectively. The neighbor joining (NJ) phylogenetic tree was performed with Mega 10 software based on the pairwise FST values (https://itol.embl.de. The maximum likelihood (ML) phylogenetic tree was conducted among three Yunnan groups and the reference populations using with the TreeMix v1.1  was used to visualize the STRCTURE results based on the individual and population levels.To evaluate the amount of information that these 43 A-DIPs provide about ancestry, the informativeness for assignment  results of Hardy-Weinberg equilibrium tests in three Yunnan groups were shown in p values of LD tests at 43 A-DIPs in the Yi, Hani and Miao groups were all shown in p < 0.0001 = (0.05*2)/(43*42)), all the pairwise loci of 43 A-DIPs were in accordance with the linkage equilibrium.The HWE statuses of the 43 A-DIP loci were used to assess the suitabilities of the 43 loci in Yunnan Yi, Hani and Miao groups. And the The deletion and insertion allelic frequencies of the 43 A-DIP loci in the Yunnan Yi, Hani and Miao groups were shown in The forensic parameters of 43 A-DIP loci including PIC, PM, PD, He, Ho, PE and TPI of Chinese Yunnan Yi, Hani and Miao groups were calculated and presented in −18, 8.24299 E−19 and 4.21721 E−18; the TDP and CPE values of the corresponding groups were 0.99999999999999998886, 0.999999999999999991757 and 0.99999999999999995783; 0.999610217, 0.999629285 and 0.999582084, respectively. Considering all the 3890 individuals from five continents as a whole, the CPM, TDP and CPE values of the 43 A-DIP panel were 1.74469 E−19, 0.999999999999999998255, 0.999585879, respectively. Among the continents, the East Asian populations represented the largest average CPE (0.999820028167545) and TDP values (0.99999999999999995194), and the smallest CPM value (2.37739474441527E-18). Whereas, the smallest CPE (0.998911970139209) and TDP (0.9999999999999931467), and the largest CPM (2.87186303945698E-16) were observed, indicating relative lower forensic individual identification efficiency in African populations.To verify the forensic efficiencies of this panel for the individual identification in three target Yunnan groups and 28 reference populations, the CPM, TDP and CPE were calculated and their values were shown in The effectiveness of kinship testing was also an important index of the individual identification panel. The system efficacies of 43 A-DIP panel for full sibling and half sibling relationships were assessed. Genotypes of 1000 full sibling pairs, 1000 half sibling pairs and 1000 unrelated individual pairs based on 43 A-DIP loci were simulated and used to calculate the LRs of prosecutive hypothesis H1 and defense hypothesis H2. The LR was designed to be the prosecutive hypothesis H1  divided by the defense hypothesis H2 . The distribution curves and probability density distributions of LRs between full sibling pairs and unrelated pairs in these three groups were shown in Even though the 43 A-DIP panel was originally developed for forensic individual identification, it was still found that the allele frequency distributions of several loci were different in several continental populations, which might have the potential to infer ancestry informative.In values among Yunnan Yi, Hani and Miao groups and other reference populations were calculated and shown in In values among three Yunnan groups and African populations, which might be adopted as ancestral informative markers to differentiate African populations and non-African ones. As a contrary, rs33990282, rs3830885 loci in cluster Ⅰ and rs10541072 and rs10589141 in cluster ⅠⅠ exhibited high deletion allelic frequencies in African populations and relatively low deletion frequencies in European, Asian, and American populations, indicating which could be used to differentiate the African and non-African populations. Overall, the 43 A-DIP panel was the potential to infer the individual biogeographic ancestry informative and could be also used to analyze the population genetic differentiations and similarities to some extent.In the clustering heatmap of 43 A-DIP deletion allelic frequencies in three Yunnan groups and 28 reference populations was shown in K = 2, the African ancestral component (orange - cluster 1) and the non - African ancestral component (peacock green - cluster 2) were identified from the African populations and non - African populations. As K = 3 , the purple ancestral component (cluster 3) appeared in European, South Asian, East Asian and American populations, and the European populations could be distinguished from others. When the K values increased from 4 to 5, the proportions of shared ancestral components varied among different continental populations, but no significant substructures were identified within three target Yunnan groups and other East Asia populations.The STRUCTURE analyses on individual  and popuK value of 3, we divided all the 3890 individuals from three target groups and 28 reference populations into three categories which included Africa, East Asia and the other continents. The accuracies and 95% CI values of XGBoost, RF, KVM and decision tree methods in judging the origins of three different biogeographic ancestors  were 0.8239 , 0.8126 , 0.8239  and 0.688 , respectively.Four AI algorithms including XGBoost, RF, KVM, and decision tree were used to predict the biogeographical ancestors based on the 43 A-DIP loci genotypes of three Yunnan groups and 28 reference populations. The confusion matrixes of the testing sets, the accuracies and 95% confidence interval (95% CI) results of four models were shown in Due to the existence of these loci with large differences in allele distributions, the analyses of PCA, MDA and phylogenetic trees (NJ and ML) were performed to gain insight into the genetic structures and genetic relationships among Yunnan Yi, Hani and Miao groups and other continental populations.FST values , and Esan in Nigeria (ESN), whereas the smallest three values were found in Xishuangbanna Dai Chinese (CDX), Ho Chi Minh Kinh (KHV), and Chinese Guangzhou Southern Han (CHS). The YRI, MSL and Gambian in ern Division-Mandinka (GWD) were three populations which showed the three largest pairwise FST values with Yunnan Miao group. The CDX, CHS and YNY populations had the closest genetic distances with Miao group, and those populations were also observed the smallest three pairwise FST values. The Nei’s genetic distances among Yunnan Yi, Hani, Miao and 28 reference populations were in ranges from 0.00564 (YNH) to 0.1281 (MSL), 0.0056 (YNY) to 0.1254 (MSL), and 0.0129 (YNY) to 0.1461 (MSL), respectively. MDS analyses based on the matrices of pairwise FST values and Nei’s genetic distances among 31 populations were shown in Genetic affinities among Yunnan Yi, Hani and Miao groups and 28 reference populations were evaluated with the pairwise  values , Nei’s gistances , MDS  were used to predict biogeographic ancestors based on the 43 A-DIPs genotypes, the XGBoost and KVM showed the highest accuracies, which were all 0.8239 . SVM is mainly used to solve the binary classification and regression problems of small samples (Because of the existence of some loci with large differences in allele frequencies among different continental populations, such as rs33990282, rs10541072, rs10541072, 43 A-DIP loci were the potential of ancestral informative inference. With the optimal  samples . XGboost samples . Compare samples .Nei’s genetic distances between each other, and indicated closer genetic relationships. From the results of pairwise FST genetic distances, other than the close genetic relationships were found among the three studied Yunnan groups, smaller FST values were observed among the CHS, CDX and KHV with Yunnan Yi, Hani and Miao groups, which were consistent with the relatively geographic distributions. In the PCA and MDS plots, the Yunnan Yi, Hani and Miao groups had closer genetic distances with other East Asia populations, and among seven East Asian populations, the Southwestern Chinese populations CDX and CHS were the two close ones. Among the NJ and ML phylogenetic trees, Yunnan Yi, Miao and Hani groups represented closer genetic relationships with CDX, CHS and KHV comparing with other reference populations. And similar conclusion was been verified in other study. For instance, Cui et al. reported the NJ and ML trees based on 39 AIM-DIP panel and concluded that the YNM and YNH groups had the close genetic distances with CDX, CHS and KHV (Population genetic structures and population genetic relationships of three target Yunnan groups and 28 reference populations were also analyzed based on the 43 A-DIP genotypes. The Yi, Hani and Miao groups we collected were all from Yunnan province which are located at the Southwestern China, and were geographically close to Xishuangbanna Dai populations. Three Yunnan groups showed smaller  and KHV .The 43 A-DIP panel showed the relatively high discrimination power of individual identification and played a potential role in the full-sibling testing in Yunnan Yi, Hani and Miao ethnic groups. Additionally, we provided the valuable data of the allele frequency information of 43 relatively high polymorphic DIP loci in three Yunnan groups for further forensic applications. The AI models based on XGBoost and KVM had been verified with higher accuracy for the prediction of biogeographical ancestor based on the 43 DIP genotypes. Through the conjoint analyses of the genotypes of 3348 individuals from 28 reference populations, the Yunnan Yi, Hani, Miao groups had relatively close genetic relationships with KHV, CDX and CHS. In conclusion, the 43 A-DIP panel was a powerful tool for forensic individual identification, full sibling testing and biogeographical ancestral inference in Yunnan Yi, Hani and Miao groups.


Ambrosia artemisiifolia (common ragweed), introduced to Europe in the late 19th century, by resequencing 655 ragweed genomes, including 308 herbarium specimens collected up to 190 years ago. In invasive European populations, we found selection signatures in defense genes and lower prevalence of disease-inducing plant pathogens. Together with temporal changes in population structure associated with introgression from closely related Ambrosia species, escape from specific microbial enemies likely favored the plant’s remarkable success as an invasive species.Invasive species are a key driver of the global biodiversity crisis, but the drivers of invasiveness, including the role of pathogens, remain debated. We investigated the genomic basis of invasiveness in  The invasive success of ragweed in Europe was facilitated by release from enemy microbes and interspecies hybridization. The wide-scale introduction of exotic species to novel ranges around the world can be largely attributed to the 19th century colonial activities of Europeans and to escalating global trade activities since the 20th century , an extraordinarily successful noxious weed that is native to North America with a 200-year history of global introductions  offers some hope for reducing ragweed’s impact in Europe  and western ragweed (Ambrosia psilostachya) . Nearly 50% of these samples come from historical herbarium collections collected between 1830 and 1973. Historical herbarium samples show characteristics of ancient DNA damage: The C to T base misincorporation at the first base ranges from 1.1 to 9.6% , and the endogenous content ranges from 55 to 96%  (table S2). We grouped samples into five populations based on geography and genetic clustering. We found that the main source of the introduced European invasive population is the native-range Mideast population that was previously found to be of admixed origin . After filtering this initial assembly to remove haplotigs, the resulting filtered assembly consisted of 41,017 contigs with a total length of 1280.34 Mbp and an N50 of 101.591 kbp. After HiRise scaffolding with the Chicago sequencing data, the final genome assembly’s length was 1258.37 Mbp, and it was composed of 12,228 scaffolds with a scaffold N50 of 270.6 kbp. The repeat analysis resulted in an annotation of 30.76% of the genome sequence in interspersed repeats. Of the whole genome, 8.01% are long terminal repeat elements, 2.01% are long interspersed nuclear elements, 3.75% are DNA transposable elements, 0.20% are short interspersed nuclear elements, and 16.79% are unclassified repeats. The gene annotation of the repeat-masked genome resulted in 34,066 predicted proteins. The benchmarking universal single-copy orthologs (BUSCO) . Pairwise sequentially Markovian coalescent (PSMC)–based demographic reconstruction of a high-depth NA sample from the East population shows a population decline between 105 and 104 years ago (fig. S4).In the native NA range, samples cluster based on geography in both the principal components analysis (PCA)  and admiulations . There age in NA . The MidK = 9. Furthermore, FST values are higher between Europe and NA South than between Europe and the other three NA populations. On the basis of FST, Europe is almost equally close to the West and East population in both the historical and modern time periods. Europe shows the lowest genetic distance to the Mideast population, which is lower than values between the two time periods within the same populations. In the admixture analysis, more than half of the European samples for both the historical and the modern time periods are assigned to the Mideast population. The fraction of samples stays almost constant over time, with 56.7% in the historical time period and 54.7% in the modern time period. For both the East and the West population, the fraction of European samples assigned to them decreases over time. In historical times, 15.2% of samples were assigned to the East population and 11.4% to the West population. Among modern samples, only 6.5% were assigned to the East population and 7.1% to the West population. Over time, more samples within Europe could not be assigned to any NA population , and several unique genetic clusters are found in modern Europe  analysis (Ne) (table S3). All spatial groups in Europe show signals of introgression from A. trifida, with the highest values found in those that form unique genetic clusters  (fig. S2). In addition, two modern populations (Appeldorn and Bordeaux) show signals of introgression from A. psilostachya (fig. S3). Because of the relatively high genetic differences of geographically close spatial groups in Europe, no isolation-by-distance (IBD) pattern could be found in modern Europe, unlike in the native NA range (fig. S5). In historical Europe, a low P value slightly above the significance threshold (0.051) indicates weak IBD (R2 = 0.3). These changes in the IBD pattern in Europe support our findings of strong genetic changes in Europe over time. For those spatial groups that form unique genetic clusters in Europe, nucleotide diversity decreased over time, and the lowest value (0.0199) is found in modern Innsbruck (table S3). In addition, Tajima’s D substantially increases in these spatial groups over time, and the value changes from negative to positive in two of these (Appeldorn and Innsbruck). In historical Caluire and Bordeaux, a unique genetic cluster is found that is not found in modern times. These spatial groups show lower genetic diversity and have a lower admixture diversity score in the historic time period compared to the contemporary time period. The admixture diversity score of Montpellier also decreases over time, and no samples are assigned to the West population anymore  than in all other populations except historical West (P < 0.05) than in all other populations except historical South and modern East. The lowest heterozygosity in the native range is found in modern East, which significantly differs (P < 0.05) from all other native-range populations except historical South. The effective population size (Ne) is higher in Europe than in any of the NA populations (table S4) and decreases over time. In the native range, the South population has the lowest and the Mideast population the highest Ne. In the native range, Ne increases over time for all but the East population. Tajima’s D is negative in all populations, with the lowest value found in historical Europe, followed by historical East and historical Mideast (table S4), and is generally lower in historical populations than in modern ones. All spatial groups except modern Appeldorn and modern Innsbruck have a negative Tajima’s D (table S3).The highest mean heterozygosity is found in the historical Mideast population and is significantly higher , outlier windows have, on average, a negative H in modern Europe, which is significantly lower than in nonoutlier windows (P < 2.2 × 10−16), while outlier windows in historical Europe and modern NA show a positive Fay and Wu’s H on average   average .FST outlier windows (Z > 50) between historical and modern Europe, 43 of 414 are found within 23 gene regions (table S6). Several of these genes are orthologs of well-characterized genes in A. thaliana. For example, functional analysis of AT1G47740 (PPPDE putative thiol peptidase family protein) in A. thaliana showed that mutants are involved in abiotic stress response evidenced by expression changes in response to cold (down-regulation), oxidation (down-regulation), and osmotic stress (up-regulation) (ABA HYPERSENSITIVE 1 (ABH1)] are early flowering (RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD)] is involved in defense response to abiotic stress and pathogens  enrichment analysis shows 45 enriched GO terms in the comparison of historical and modern Europe (P < 0.05) prevalence in historic Europe, while one is significantly higher (P < 0.05) in historical NA. In the contemporary samples, nine pathogens are absent from Europe and present in NA. Of these, two show the same pattern in the historical time period. Seven pathogens present in modern Europe are absent in modern NA. Of these, three show the same pattern in the historical specimens. In the contemporary time period, 8 pathogens show a significantly higher (P < 0.05) prevalence in Europe and 12 in North America. In general, either more or a significantly higher prevalence (P < 0.05) of Xanthomonas  of Dickeya and Brenneria species is found in Europe. Of the 17 plant pathogen bacteria Xanthomonas species identified in our dataset, only 2 species were significantly more abundant (P < 0.05) in ragweed populations in modern Europe than in modern North America, and 2 more were completely absent from modern Europe (table S5). In the historical period, 8 of these Xanthomonas species were absent from North America, and 12 were absent from Europe. In modern Europe, Dickeya species are found throughout the whole range, while Xanthomonas spp. are more common and Brenneria spp. are restricted to south-eastern Europe , consistent with the enemy escape hypothesis, but here, more validation is needed. This indicates that multiple factors such as admixture of different source populations, hybridization with related species, and escape from pathogens facilitated the success of A. artemisiifolia in Europe.Using the largest collection of conspecific genomes derived from herbarium specimens of any species, we found a remarkable turnover in the genetic structure of introduced A. artemisiifolia populations in both NA and Europe, we found genomic signals of putative divergent selection during range expansion, in combination with previous work  but also any of the multitudes of microbial pathogens known to affect A. artemisiifolia (Xanthomonas), we also detected some taxa only in Europe or at higher prevalence than in North America . Bacterial plant pathogens often have high host specificity but can sometimes live on nonhost plants without causing disease  and reduced-representation genomic data from present-day populations, van Boheemen A. artemisiifolia into Europe, rather than a single introduction and a subsequent spread . Hybridization in the introduced range may mitigate Allee effects, which tend to be particularly strong in self-incompatible species such as A. artemisiifolia  were assigned to the West cluster. According to herbarium data and bibliography, the first introductions to Europe occurred over a short time period of about 10 years from Eastern North America  in heterozygosity levels in Europe compared to the native range and higher Ne than in the native range. Over time, the European population has diverged from the NA population, as fewer samples are assigned to the native genetic clusters, and more genetic clusters unique to Europe have emerged.As a whole, the European population is most closely related to the NA Mideast population, which is of admixed origin  in 2008. From these seeds, the individual plant AA19_3_7 was grown to maturity in a greenhouse at the Department of Botany, University of British Columbia. Fresh leaf tissue was sampled, flash-frozen in liquid nitrogen, and then stored at −80°C. High–molecular weight (HMW) genomic DNA was extracted using a Cetyltrimethylammoniumbromid (CTAB) protocol  from individual AA19_3_7’s homologous chromosomes. We attempted to remove these haplotigs from the reference assembly to ensure that the length of the reference genome is not falsely inflated using the software PurgeHaplotigs . The separations of Chicago read pairs mapped within draft scaffolds were analyzed by HiRise to produce a likelihood model for genomic distance between read pairs, and the model was used to identify and break putative misjoins, to score prospective joins, and to make joins above a threshold. After scaffolding, shotgun sequences were used to close gaps between contigs.The input de novo assembly and Chicago library reads were provided to HiRise, a software pipeline designed specifically for using proximity ligation data to scaffold genome assemblies  by placing the tissue into aluminum envelopes and then into liquid nitrogen. The frozen tissue samples were ground using a mortar and pestle, and total RNA was purified using commercially prepared TRIzol reagent. The total RNA extracts were visualized using a Bioanalyzer instrument using a total RNA 6000 chip. The Bioanalyzer generates an RNA integrity number (RIN) that assesses the integrity of the total RNA sample, and total RNA extracts passed quality control if the RIN was ≥7.5. The total RNA extracts from each tissue type were sent to the Genome Quebec Innovation Centre at McGill University where each one was prepared into a barcoded and stranded TruSeq mRNA library with an insert size of 165 bp (±10%). The four completed libraries were pooled in equal amounts and sequenced on one lane of an Illumina HiSeq 2000 instrument. The RNA sequencing data were first cleaned using Fastx (Hannon Lab), and then a de novo reference was assembled with all four libraries using Trinity (Ambrosia (ragweeds) were used for homology-based gene prediction. As no training gene models were available for A. artemisiifolia, we used CEGMA , and by directly contacting herbaria. We preferred specimens that were collected before 1940 to represent the initially introduced populations into Europe. For some European regions (Romania and Czech Republic), no or very few herbarium samples from before 1940 were available. To include those regions, we used the oldest available herbarium samples instead (fig. S1). When the collection latitude and longitude coordinates were not available as metadata, this information was inferred from the centroid (as defined by the web tool Google Maps) of the most specific geographic sampling location  described on the herbarium voucher sheet. A complete list of samples included in this study can be found in table S1, along with sample locations and assignment to broader populations (as described below). To evaluate whether specimens in our dataset were misidentified and to test for introgression of some species, we included shotgun sequencing data of 123 samples of other Ambrosia species (hereinafter referred to as outgroup samples), including samples of known hybrids of A. artemisiifolia with other Ambrosia species (table S8).To summarize and select from available For the historical herbarium samples processed for this study, all pre-PCR steps were carried out in a dedicated, positively pressurized ancient DNA laboratory at the NTNU University Museum. DNA was extracted from leaf tissue using the QIAGEN DNeasy Plant Mini Kit according to the manufacturer’s instructions except for the addition of an overnight incubation step with proteinase K, as previously described , and the rest of the reaction volume filled up with molecular-grade water. The PCR was performed with an initial denaturation of 10 min at 95°C and then X cycles of a 30-s denature at 95°C, 1 min of annealing at 60°C, and 45 s of extension at 72°C, followed by a final extension of 5 min at 72°C. The optimal number of PCR cycles X was selected for each sample on the basis of quantitative PCR. Amplified libraries were purified with solid phase reversible immobilization (SPRI) beads  using bwa v0.7.17 mem (http://broadinstitute.github.io/picard) or bammarkduplicates from the biobambam2 v2.0.87 package. MapDamage2  was 1.4× for historical herbarium samples and 2.9× for modern samples.Raw reads were processed with the paleomix v.1.2.13.8 BAM pipeline  to identify possible hybrids and misidentifications in the dataset. On the basis of the PCA, a total of 50 samples were removed (see table S1). In addition, 16 samples were removed because they were either first- or second-degree relatives , 23 samples were removed because of too low sequencing depth (<0.1× after filtering for a MAPQ of 25), and 4 samples were removed because of a possible sample mix-up in the laboratory. The genotype likelihood estimation was repeated on the reduced dataset containing 655 samples.The genotype likelihood estimation was once performed on the whole dataset, including other Ambrosia species was performed. Samples that clustered outside the main A. artemisiifolia group  were excluded from further analyses (fig. S8). Some outgroup samples also clustered with the main A. artemisiifolia cluster. This might be due to misidentifications of outgroup samples, but it is more likely the result of low sequencing depth (<0.1×) in these samples. These outgroup samples were therefore excluded in the consideration of misidentified A. artemisiifolia samples. In total, 47 samples  were excluded: 34 historical European (13%), 10 historical NA (8.5%), and 3 contemporary NA (1.5%) samples (table S1). The PCA was repeated including only the final set of samples. For the admixture and PCA analyses, sites in with linkage disequilibrium (LD) ≥ 0.5 were removed. LD was estimated using Plink v1.90 (K). Ten independent runs with different seeds were performed for each K value. CLUMPAK Distruct .PCangsd v.0.95 . To be able to assign European samples to these native populations, we used admixture proportions for Ambrosia chamissonis and Ambrosia carduacea) and mapped against the A. artemisiifolia reference genome. The sequencing depth after MAPQ 25 filtering for both samples is 3.5×. To generate the ancestral state fasta file, angsd v0.931 . The correlation when downsampling to 0.2× is lower, with R2 < 0.9. Thus, samples with sequencing depths below 0.5× after MAPQ 25 filtering were removed from the analysis to avoid bias due to low sequencing depth. To test whether there are significant differences in heterozygosity between populations, a Mann-Whitney U test was performed in R v3.4.4.For each sample, heterozygosity was estimated over the whole genome. First, the site allele frequency (SAF) was estimated with angsd v0.931 . Four samples from three sampling locations were assigned to either the Mideast or East population but were geographically not located in that population (fig. S6). These samples were excluded from their respective population for all population-based analysis. Samples that are first- or second-degree relatives and those that are misidentifications or possible hybrids based on the PCA analysis including outgroups from the genus Ambrosia were removed. First, the SAF was estimated with angsd using the command line options -minMapQ 25, -minQ 20, -remove_bads 1, -uniqueOnly 1, and -trim 5. The SFS was polarized using the ancestral state estimated from A. chamissonis and A. carduacea.For the Ne, thetas were calculated with the angsd realSFS saf2theta tool for each population, polarized using the ancestral state estimated from A. chamissonis and A. carduacea. The mean of the Watterson estimator across the genome was used to calculate Ne, and the 95% confidence interval of the mean was used to calculate the error of Ne. A mutation rate of 1 × 10−8 substitutions per site per generation [estimate from the closely related species Helianthus annuus  and a younger (1900–1940) spatial group for a higher temporal resolution. To create modern geographic groups, we selected from available modern populations that were collected for previous studies or collected new samples in close proximity to the historical spatial groups. For one spatial group (Hamburg), no modern samples could be found because of successful eradication of the invasive plant. In NA, we chose some additional modern locations where less than four historical samples were available. We used these locations to gain a better resolution.FST between spatial groups was calculated with realSFS from angsd  within angsd , , , and  were considered, where N is a spatial group from North America and E a spatial group from Europe. All possible comparisons with E, N, and H2 being from the same time period and H2 being a NA spatial group, excluding those that themselves showed signs of introgression  were calculated, and the mean value of these was used for plotting. For each spatial group, the admixture diversity score was calculated as in . Samples that were grown from seeds in the greenhouse were excluded from the analysis (see table S1).Reads that did not map against the sourceforge.net/projects/bbmap/) using the default parameters. The unmapped reads were then classified with kraken2 (github.com/jenniferlu717/KrakenTools/). The two kraken reports for paired and collapsed reads for each sample were combined into one report using the combine_kreports.py script from KrakenTools. Kraken-biom v1.0.1 (github.com/smdabdoub/kraken-biom/) was used to combine the results from all samples for subsequent analysis in R v4.0.3.For the metagenomic classification, PCR duplicates were removed with clumpify from the BBMap package (t test.Thereafter, several filters were applied to the dataset to exclude particular taxa from further analysis. Only taxonomic identifications at the species level were used. Species with a relative abundance below 0.05% were removed under the assumption that they are likely false positives. The package decontam (


Zabelia tyaihyonii (Nakai) Hisauti and H. Hara is a perennial shrub endemic to Republic of Korea that grows naturally in only a very limited region of the dolomite areas of Gangwon-do and Chungcheongbuk-do Provinces in the Republic of Korea. Given its geographical characteristics, it is more vulnerable than more widely distributed species. Despite the need for comprehensive information to support conservation, population genetic information for this species is very scarce. In this study, we analyzed the genetic diversity and population structure of 94 individuals from six populations of Z. tyaihyonii using a genotyping-by-sequencing (GBS) approach to provide important information for proper conservation and management. Our results, based on 3088 single nucleotide polymorphisms (SNPs), showed a mean expected heterozygosity (He) of 0.233, no sign of within-population inbreeding , and a high level of genetic differentiation (STF = 0.170). Analysis of molecular variance (AMOVA) indicated that the principal molecular variance existed within populations (84.5%) rather than among populations (17.0%). We suggested that six management units were proposed for conservation considering Bayesian structure analysis and phylogenetic analysis, and given the various current situations faced by Z. tyaihyonii, it is believed that not only the in situ conservation but also the ex situ conservation should be considered. Zabelia tyaihyonii (Nakai) Hirsute and H. Hara (Taihyun’s abelia) (Abelia by Nakai in 1921 [Zabelia (Rehder) Makino from Abelia into a new genus based on the external morphological and anatomical characteristics suggested in a previous study [ abelia) A belongsus study , so thisAbelia by Nakai in 1921 [Zabelia of the genus Abelia into a new genus, Zabelia (Rehder) Makino, based on the external morphological and anatomical characteristics suggested in a previous study [Zabelia mosanensis (T.H. Chung ex Nakai) Hisauti and Hara, but it is distinguished in that its leaf length is half or less, and hair on the filament is absent [Z. mosanensis as a synonym for Z. tyaihyonii according to the principle of priority [This species, first described as a species of the genus us study . This sps absent . In the priority .Z. tyaihyonii has very high value as garden material because it has gorgeous and fragrant flowers [This species is a very important resource because it is a Korean endemic plant and an indicator species of limestone areas . It is a flowers .Z. tyaihyonii, are particularly vulnerable. It is important to establish a conservation strategy based on ecological conditions and the genetic diversity suitable for each species to prevent extinction and maintain continuous natural populations [Z. tyaihyonii were mainly focused on systematics [Z. tyaihyonii habitats were very thoroughly described in three studies conducted on all native habitats [Since endemic plants are very sensitive to subtle changes in environmental factors, they should be prioritized for management and preservation ,12. Speculations ,14,15. Ptematics ,19,20,21tematics ,22,23. Thabitats ,23. HoweNext-generation sequencing (NGS) technology has transformed modern biology, including population genetics, due to its high throughput and low cost ,24. The Z. tyaihyonii individuals collected from all known natural habitats (six populations)  B. The maAn initial quality check of 663,388,244 reads after sequencing resulted in a matrix of 48,326 SNPs. Initial filtering  resulted in the identification of 31,147 SNPs. A further filtering step  resulted in a total of 4085 SNPs, and the final filtering steps yielded 3088 final SNP loci in 94 genotyped individuals across six sampled locations.Z. tyaihyonii were estimated and are summarized in Na) and number of effective alleles (Ne) were 1.734 and 1.389, respectively. Shannon’s information index (I) was the highest in BS (0.406) and the lowest in SH (0.249), with an average of 0.355. The ranges of observed heterozygosity and expected heterozygosity among the six populations were from 0.209 (SH) to 0.273 (TG) and from 0.170 (SH) to 0.263 (BS), respectively. Additionally, inbreeding coefficient (ISG) was negative in all populations except BS.Genetic diversity indices within the six populations of F-statistics from the analysis also showed that the species discussed in this study was moderately to highly structured, with a value significantly different from zero after 20,000 permutations . Although there was a significant expected reduction in heterozygosity among all individuals , the slightly negative total inbreeding coefficient (ISF = −0.018) was not shown to significantly differ from zero (p > 0.05), indicating little or no inbreeding in the species.The results from the analysis of molecular variance (AMOVA)  revealedSTF) and a high level of historical gene flow (mN) of pairs of populations were detected in Z. tyaihyonii  and the highest gene flow (mN = 3.217) were observed between BS and CW , maximum of means (MaxMeak), median of medians (MedMedK) and maximum of medians (MaxMedK), showed t likely A. Thereft likely B. At K = from CW B.PCoA results are presented in To establish phylogenetic relationships among the 94 genotypes, a cladogram was constructed using the UPGMA technique and 3088 SNPs . The treZ. tyaihyonii has lower genetic diversity (eH = 0.233) than other species of endemic and woody perennials, such as Abeliophyllum distichum (eH = 0.319) [Tetraena mongolica (eH = 0.348) [Rhododendron rex (eH = 0.54) [Z. tyaihyonii can be considered low.The genetic diversity of species reflects their long-term evolution and adaptation, as well as demographic history ,36. High= 0.319) , Tetraen= 0.348)  and Rhod = 0.54) . Given tZ. tyaihyonii, the intensity of vegetative reproduction has a significant effect on the genetic diversity of a population. For example, if the intensity of vegetative reproduction is very high, the number of genets becomes extremely low compared to the number of ramets. At the other extreme, if one or only a few genets form a population, the genetic diversity is very low, and even if the population size is large and seed reproduction occurs, the population may face extinction due to high-intensity inbreeding [ISG value close to or even below zero. Therefore, it can be inferred that the reason for the low genetic diversity of Z. tyaihyonii is probably the effect of genetic drift. Many limestone (including dolomites) areas are destroyed each year due to mining, and roadsides and mountain edges, which this species prefers, are also areas with very high human interference. For these reasons, many populations of this species, and many individuals within each population, would have disappeared in a relatively short period of time.The genetic diversity of plant species is related to their distribution range, population size, life cycle, mating system and gene flow ,40. Of tbreeding ,44,45. TI) = 0.336) of this species was evaluated as relatively high, in contrast to the findings of this study. However, a direct comparison cannot be made because the mean values of genetic parameters may differ depending on the markers used in the analysis [In a previous study based on the inter simple sequence repeat (ISSR) method , the genanalysis . HoweverHe value among the populations of Z. tyaihyonii was observed in SH (eH = 0.170), followed by SC (eH = 0.225) and TK (eH = 0.245). SH is located in the southernmost region of the distribution range of Z. tyaihyonii and is the most geographically isolated of all populations . Additionally, the size of this population was the smallest  among the investigated populations. Additionally, SC is located the farthest north among the Z. tyaihyonii populations and was confirmed to have the second smallest population size (after SH). Therefore, the low genetic diversity in these populations is thought to be due to their small size and repeated founder events [The lowest r events ,47,48.mN) and the genetic differentiation coefficient (STF), are employed to assess the genetic structure of populations, and they are negatively correlated [Nm is divided into three grades, high (≥1.0), medium (0.250–0.99) and low (0.0–0.249), and when Nm > 1 there is gene flow between populations [STF value of the populations is between 0 and 0.05, there is no genetic differentiation among populations; if the STF value is between 0.05 and 0.15, they are moderately differentiated; if the STF value is between 0.15 and 0.25, they are highly differentiated. The results of this study suggested that there was a high level of gene flow between all pairs of populations except those including SH. Additionally, the results showed a moderate level of genetic differentiation between BS, CW, TG and YC, and a high level between a population and SH or SC. Flowers of Z. tyaihyonii are entomophilous (pollinated by insects), and fruits with elongated calyx lobes are dispersed by wind [Two important parameters, gene flow  supported a high level of population differentiation and showed major molecular variance within individuals rather than among populations. In outcrossing and long-lived plants in general, most genetic variation exists within populations, while selfing plants maintain the majority of genetic variation among populations [The AMOVA results . Therefore, more active efforts to protect this population will be needed, such as setting up conservation areas and conducting periodic monitoring.Protection of trailing-edge populations should be prioritized because the prolonged isolation and successful persistence of such groups suggest their evolutionary potential, which can be useful for the long-term conservation of genetic diversity and phylogenetic history of the species ,70. AddiGiven its geographic location at the northernmost limit of the distribution range of the species, the SC population is not recommended as a source of transplants, as previously suggested for SH. SC showed high gene flow with other paired populations except for SH and showed moderate genetic differentiation from BS . This prA recommended conservation strategy for endangered plants, in addition to in situ conservation, is the establishment of ex situ gene banks for each population in both the field and laboratory. Although this species is not listed as an endangered wild plant under legal protection in Korea , it is aZ. tyaihyonii is not an endangered or protected species, plant materials were collected without permission. Distribution information for sampling was confirmed through the literature and specimens from the National Institute of Biological Resources and the Korea National Arboretum. A total of 94 individual plants were collected from six natural populations, and 14 to 22 individuals in each population were sampled at least 5 m apart to minimize the likelihood of sampling clones. During field sampling, the sampled site and altitude were recorded  using 150 bp paired-end sequencing runs.Total genomic DNA was extracted using the DNeasy Plant Mini Kit , following the instructions of the manufacturer. The quality and quantity of extracted DNA were evaluated with a LabChip GX II , and DNA was visualized on a 1% agarose gel. Samples that passed quality and quantity filters were subsequently sent to Xenotype Inc.  for GBS library construction . Using tp value for Hardy–Weinberg equilibrium ≤ 0.01. Then, linkage disequilibrium (LD) (2r > 0.8) was computed using the Plink v. 1.07 software [NGSEP Wizard is a tool that implements all the steps necessary to obtain a population variant file with just one run screen, starting from raw sequencing data . We obtasoftware . The onlsoftware  was usedNa), the number of effective alleles (Ne), Shannon’s information index (I), observed heterozygosity (Ho), expected heterozygosity (He), AMOVA, gene flow (Nm), and PCoA were computed with the GenAlEx 6.5 software [ISG) was calculated by Genodive v.2.0b27 [K) ranging from one to six, and each STRUCTURE run was performed with a burn-in size of 50,000 and 100,000 MCMC iterations with 10 runs per K value. Results were fed into StructureSelector [The number of alleles . The results of this study and our suggestions for conservation will serve as important information for the population conservation and management of this species.A total of 3088 high-density SNPs were employed to estimate the genetic diversity and population structure in 95