Plutella xylostella L., a destructive pest of Brassica vegetable and oilseed crops worldwide, seasonally colonizes winter canola crops in southern Australia from alternative host plant sources. To investigate movement, we collected 59 P. xylostella populations from canola crops, Brassica vegetable and forage crops and brassicaceous wild host plants throughout southern Australia in 2014 and 2015 and genotyped 833 individuals using RAD-seq for genome-wide analysis. Despite a geographic sampling scale > 3,000 km and a statistically powerful set of 1,032 SNP markers, there was no genetic differentiation among P. xylostella populations irrespective of geographic location, host plant or sampling year, and no evidence for isolation-by-distance. Hierarchical STRUCTURE analysis at K = 2–5 showed nearly uniform ancestry in both years. Cluster analysis showed divergence of a small number of individuals at several locations, possibly reflecting an artefact of sampling related individuals. It is likely that genetic homogeneity within Australian P. xylostella largely reflects the recent colonization history of this species but is maintained through some level of present gene flow. Use of genome-wide neutral markers was uninformative for revealing the seasonal movements of P. xylostella within Australia, but may provide more insight in other global regions where the species has higher genetic diversity.Molecular studies of population structure can reveal insight into the movement patterns of mobile insect pests in agricultural landscapes. The diamondback moth,  Protecting crops from attack by these pests is difficult for pest managers due to the unpredictable nature of seasonal outbreaks, particularly when insecticide resistant genotypes are present5. For mobile insect pests, dispersal among crop and non-crop host plant resources influences both the seasonal dynamics and genetic background of pest populations, with direct consequences for pest management9. Molecular studies of population structure and gene flow can potentially provide insight into patterns of insect movement in agricultural landscapes11.Mobile insect pests regularly colonize annual crops from alternative host plant sourcesPlutella xylostella L., is the most destructive pest of brassicaceous crops worldwide13. It attacks Brassica vegetable crops throughout tropical and temperate regions14 and in recent decades has become a significant pest of canola crops in temperate regions18. The propensity of P. xylostella to evolve insecticide resistance rapidly and a lack of alternative control options has led it to evolve resistance to most pesticides4. Within Australia, P. xylostella has been a major pest of Brassica vegetable crops since the late 1800s19 and a sporadic but damaging pest of canola crops since the 1990s following a dramatic expansion of canola production18. Approximately 3 million hectares of canola is grown annually under a Mediterranean climate in southern Australia20, providing vast host resources for P. xylostella from crop planting in autumn to maturity in late spring. Intermittent outbreaks of P. xylostella in canola during spring cause substantial crop losses21. Commercial Brassica vegetable crops are grown continuously in horticultural areas surrounding the major urban centres in each Australian state, occupying a total area less than 1% of total canola plantings22. Other host plants for P. xylostella include Brassica forage crops grown during spring and summer as stock feed, and a diversity of introduced and native wild brassicaceous species distributed over vast areas and proliferated by rainfall. In Brassica vegetable crops, limited P. xylotella dispersal and intense insecticide use targeting this insect can lead to elevated levels of insecticide resistance in local P. xylostella populations23. In canola-growing areas, the highly seasonal availability of host plants compels P. xylostella to move regularly among crops and other brassicaceous host plants, which tends to homogenise levels of insecticide resistance24. Estimating gene flow among local populations of P. xylostella between host plant types and identifying source populations of P. xylostella that seasonally infest Australian canola are essential to facilitate forecasting of seasonal pest pressure and inform insecticide resistance management19.The diamondback moth, P. xylostella, including allozymes, ISSRs, microsatellites and mtDNA. Plutella xylostella populations from different continents are clearly differentiated26, but a lack of genetic structure among populations within parts of Asia28, the USA30 and Australasia31 implies regular intermixing at an intra-continental level. Many population genetic studies of P. xylostella have been difficult to interpret due to limited sampling32 and few studies have sampled at a sufficient spatiotemporal scale or resolution to investigate movement at a landscape scale. However, two studies successfully identified the seasonal migration pathways of P. xylostella in China through extensive field sampling and analysis of both microsatellite markers and geographic variation in mtDNA haplotype frequencies28. Inferences from genetic data were corroborated by light trapping33.Various molecular markers have been used to investigate population structure in 19 found no differentiation at six microsatellite loci among 17 populations across Australia and one from New Zealand, despite a sampling scale spanning > 5,000 km. These Australasian populations were clearly differentiated from populations collected in Asia and Africa19. Australian P. xylostella has low genetic diversity consistent with a founder effect35. Present levels of gene flow among Australian P. xylostella populations remain to be resolved because genetic homogeneity could reflect co-ancestry18, and because the statistical power of six microsatellites to detect weak population structure was uncertain. Furthermore, inconsistent patterns of population structure reported among P. xylostella collected from eastern Australia36 may reflect the presence of a cryptic species, Plutella australiana, among analysed samples37.Within Australia, Endersby et al.38 and associated genotyping methods has facilitated genome-wide genetic marker sets and brought unprecedented resolution to questions of population structure40. Restriction-site-associated DNA sequencing (RAD-seq)41 enables sequencing of targeted short regions across the genome, allowing simultaneous discovery and genotyping of single nucleotide polymorphisms (SNPs) in model and non-model species43. The ability to sequence orthologous regions across multiple individuals at high sequencing coverage makes it possible to confidently genotype SNPs and generate high density markers for population genetic studies44. Microsatellites remain popular for population genetic studies due to high polymorphism45, but can be outperformed by large SNP panels in resolving population structure47, with several examples in insects49. RAD-seq has genotyped thousands of SNPs in P. xylostella50 and resolved species-level nuclear divergence between cryptic Australian Plutella species35, suggesting potential for this method to provide insight into the movement patterns of P. xylostella.The revolution in massively parallel sequencing technologiesP. xylostella in Australia. Samples were collected from canola crops, Brassica vegetable crops, Brassica forage crops and wild brassicaceous plant species throughout southern Australia and in two consecutive years to facilitate temporal comparisons. After molecular species identification, P. xylostella individuals were genotyped across genome-wide sites using RAD sequencing for population genetics analysis.Here, we examined whether geographic, host plant-related or temporal population genetic structure exists among geographically distinct populations of Plutella species were collected from different Brassica host plants and locations throughout southern Australia in 2014 and 2015  and most populations had a reasonably balanced sex ratio , host plant types  or seasons . In general, observed heterozygosity was lower than expected as shown by mostly positive Population genetic diversity was estimated using the 590,086 variant and invariant sites. The mean observed heterozygosity per population averaged 0.0092 ± 0.0002 SD  across the 59 populations and showed little variation across populations collected from different years , no pairwise P. xylostella populations collected within a single year. The highest pairwise n = 27 comparisons), indicating allele frequencies in this population were the most divergent from other populations  in 2014  and 3,624 kilometres (Manjimup WA/Bundaberg QLD) in 2015 . Given the vast sampling scale, we expected higher r = 0.1136, p = 0.1316) and 2015  datasets, indicating that P. xylostella populations in close proximity or separated by thousands of kilometres were equally differentiated.Under isolation by distance, geographic and genetic distances should be positively correlatedK, then examined bar plots for several K values to assess hierarchical population structure. In 2014, the data most likely formed two genotypic clusters, with the delta K method and mean likelihood value both producing an optimal at K = 2   were not strongly different from eigenvalues for other PCs, indicating no clear axis of variance in the data, and individuals across different geographic populations clustered together to a high degree Fig. . In both53. Previous molecular studies found a lack of genetic structure19 or inconsistent patterns of genetic structure36 among Australian populations of P. xylostella, and low genetic diversity implied a bottleneck during colonization31. To elucidate the movement patterns of P. xylostella in Australian canola cropping systems, we performed a comprehensive study of genetic structure among P. xylostella populations from crop and non-crop brassicaceous host plants throughout southern Australia. The study design included extensive field sampling to reflect the dispersal ecology of the species, molecular species identification, minimisation of sex bias, and a powerful SNP marker set derived from RAD-seq.In successful invading species, colonizing populations often exhibit reduced genetic diversity compared to their population of originP. xylostella populations, irrespective of geographic location, host plant, or sampling year. This pattern was temporally stable at seven locations sampled in both 2014 and 2015. Our findings based on SNPs were highly consistent with those based on six microsatellites19. Our SNP-based estimates of global 19. In both studies, > 99% of genetic variance occurred within populations with negligible variance explained by different locations or host plants. There was no evidence for isolation by distance, implying that any two populations, whether separated by distances of only several km or > 3,000 km, may be equally differentiated. Australian P. xylostella forms a single homogeneous population across bi-allelic neutral SNP markers.Genome-wide analysis revealed a distinct lack of genetic structure among Australian K = 2 as the most likely number of genotypic clusters. This is a common result among studies employing the delta K method54, because K = 1 cannot be obtained and because K = 2 often represents the top level of hierarchical population structure. At K = 2, a small number of divergent samples were identified at single geographic locations, including Esperance in 2014 and Southend in 2015. In 2015, at K values 56. Among all populations, Southend had the lowest gene diversity, the fewest private sites and the highest 58. In hierarchical STRUCTURE analysis of 2014 and 2015 samples, additional genotype clusters at successively higher K values occurred in a small number of individuals from single locations as STRUCTURE simply grouped the next most related samples at each hierarchical level. These results highlight the need for caution when samples of related individuals are present, to avoid false inferences of population structure.Cluster analysis confirmed the overall lack of genetic divergence among populations. In both years, STRUCTURE analysis identified P. xylostella and the sympatric species, P. australiana. Although these species can hybridize in laboratory crosses, whether hybridization occurs in the wild is unknown35. Whole genome analysis of 29 Plutella individuals found no evidence for widespread introgression between these two species59. In our study, all Esperance and Southend individuals exhibited levels of heterozygosity across > 550,000 variant and invariant genome-wide loci that were similar to other P. xylostella. We would expect substantially higher heterozygosity if individuals were interspecific hybrids or if DNA samples were contaminated. PCA analysis of P. xylostella from Esperance 2014, Southend 2015 and five other locations, together with five P. australiana populations from Perry et al.35, showed clear species groupings and no evidence of introgressed individuals  and two dominant shared haplotypes35. Reduced diversity across the genome is strong evidence for a bottleneck63. By contrast, a recent global study of P. xylostella whole genomes reported unexpectedly high levels of SNP diversity within populations from the Oceania region, including Australia, New Zealand, Vanuatu and Samoa, compared to populations from putative historical source regions in the Americas, Europe, Africa and Asia64. Whether loci under selection may explain this pattern warrants further study. Plutella xylostella within Australia and New Zealand appears to have been founded by a small number of females derived from an ancestral lineage in southern Asia64.Genetic differentiation indices are likely to over-estimate rates of gene flow, to the extent that genetic uniformity among P. xylostella distribution is maintained by some level of ongoing gene flow. RAD-seq markers revealed weak but significant genetic differentiation among field and laboratory-reared Australian P. xylostella populations  of 72, we visually determined the sex of individual pupae (but not larvae) by examining external genital morphology73 under a dissecting microscope, then male and female individuals were selected to achieve a balanced sex ratio within each population where possible. Genomic DNA was isolated by homogenising whole individuals using a TissueLyser II (Qiagen) followed by two phenol and one chloroform extractions according to Zraket et al.74. DNA was treated with RNase A, then precipitated and resuspended in TE buffer. To distinguish P. xylostella from P. australiana, species identification was performed using a PCR-RFLP assay35 and P. xylostella individuals were retained for analysis.For each population, 16 individuals were sequenced where possible after removing parasitised individuals. To avoid biases due to sex-linked markers41 as described in Perry et al.35. Genomic DNA was quantified using a Qubit 2.0 fluorometer (Invitrogen) and 200ng digested with 10 units of high fidelity Sbf1 in Cutsmart buffer (NEB) for 1 h at 37 °C, then heat inactivated at 80 °C for 20 min. One microlitre of P1 adapter (100nM) with a 6-base molecular identifier (MID)  on agarose gel and purified using a minElute Gel Extraction Kit (Qiagen). Illumina paired-end sequencing was performed across seven lanes using HiSeq2500 (100 bp) or NextSeq500 (75 bp) at the Australian Genome Research Facility (AGRF). Additionally, 16 individuals from a separate sequencing run as described in Perry et al.50 were included in downstream analysis.Libraries were prepared for restriction-site-associated DNA sequencing (RAD-seq) according to a protocol modified from Baird et al.75. As Nextseq reads had low quality base calls within restriction sites , we opted to remove restriction sites from all reads for downstream analysis. Sequence reads were de-multiplexed using RADtools version 1.2.442 allowing one base MID mismatch, then TRIMMOMATIC v0.3276 was used to remove restriction sites, adapter sequences, a thymine base from reverse reads introduced by the P2 adapter, and quality filter using the ILLUMINACLIP tool with parameters: TRAILING:10 SLIDINGWINDOW:4:15 MINLEN:40. Paired reads were aligned to the P. xylostella reference genome (accession number: GCF_000330985.1) using STAMPY version 1.0.2177 with –baq and –gatkcigarworkaround options and expected substitution rate set to 0.03 to reflect our expectations of sequence divergence from the reference strain. Duplicate reads were removed and individual sample BAM files merged using PICARD version 1.7178. Genotypes were jointly called for all individuals using the Genome Analysis Tool Kit version 3.3-080 HaplotypeCaller tool. We determined that base quality score recalibration using bootstrapped SNP databases was inappropriate for this dataset as it globally reduced quality scores. The variant call set was hard-filtered using VCFtools version 0.1.12a81. After iteratively testing multiple filtering parameter sets, we removed indels and retained confidently called bi-allelic SNPs  was performed using the pegas implementation in R package poppr version 2.7.195. The data were analysed under two hierarchical model structures. In model A, all individuals were analysed together and populations were grouped into sampling years and Brassica host types. In Model B, a temporal analysis was performed for locations sampled in both 2014 and 2015, to investigate whether variance was greater among years within locations or vice versa.To examine population differentiation, a global estimate of 55. Variant data were converted from VCF to STRUCTURE file format using PDGSpider version 2.1.1.282. For all runs, we used a burnin length of K value, where K is the number of genotypic clusters, using a different random seed for each run, assuming the locprior model with correlated allele frequencies and K values, we analysed K-values from 1 to 10 in both years. The optimal value of K was estimated using the delta K method96 implemented in STRUCTURE HARVESTER97 and inspection of the likelihood distribution for each model. Q-matrices were aligned using CLUMPP version 1.1.298 and visualised using DISTRUCT version 1.199. To further explore clustering, we performed individual-based principal components analysis (PCA) separately for 2014 and 2015 datasets using R package adegenet version 2.0.1101, using scaled and centred allele frequencies and imputing missing data by taking the mean of population allele frequencies.Two individual-based clustering approaches were used to investigate population structure. First, Bayesian clustering was implemented in the program STRUCTURE version 2.3.4102. This program allows the user to test the likelihood of loci of detecting genetic differentiation for pre-defined values of The statistical power of the SNP marker set to detect population structure was assessed using POWSIM version 4.1RAD sequences are available from the Sequence Read Archive under accession PRJNA471964.Supplementary Information.


Vaccinium macrocarpon Aiton and 24 populations of Vaccinium oxycoccos L. across much of their native ranges in the US using 32 simple sequence repeat (SSR) markers. We observed high levels of heterozygosity for both species across populations with private alleles ranging from 0 to 26. For V. macrocarpon, we found a total of 613 alleles and high levels of heterozygosity . We also observed high numbers of alleles (881) and levels of heterozygosity  in V. oxycoccos (4x). Our genetic analyses confirmed the field identification of a native population of V. macrocarpon on the Okanogan-Wenatchee National Forest in the state of Washington, far outside the previously reported range for the species. Our results will help to inform efforts of the United States Department of Agriculture Agricultural Research Service (USDA-ARS) and the United States Forest Service (USFS) to conserve the most diverse and unique wild cranberry populations through ex situ preservation of germplasm and in situ conservation in designated sites on National Forests.Knowledge of the genetic diversity in populations of crop wild relatives (CWR) can inform effective strategies for their conservation and facilitate utilization to solve agricultural challenges. Two crop wild relatives of the cultivated cranberry are widely distributed in the US. We studied 21 populations of  Crop wild relatives (CWR), in addition to being important components of ecosystems, are critical sources of genetic variation for crop improvement programs. These wild relatives often display more genetic variability than their cultivated counterparts and their evolution in the wild is dynamic, driven by interactions with both biotic and abiotic stressors . The useVaccinium macrocarpon Aiton) is a diploid perennial species with a natural distribution ranging from Newfoundland to the southern Appalachian Mountains, and extending west to Minnesota , with two exceptions being O-W-1014 (30-MHLCMOR) and O-W-1136 (36-MBSWA), which clustered with samples collected in the Eastern US as well as cultivars , suggesting that the western V. macrocarpon population is not genetically closely related to known cultivars/native selections.A PCA  analysisPhysalospora vaccinii (Shear) Arx and E. Müll.), can be addressed by traits found in wild relatives of cranberry. In order to confront emerging threats while increasing yield and producing excellent quality fruit, wild genetic resources of cranberry should be evaluated, safeguarded, and made available. These resources may hold the genetic variation needed to overcome current and future challenges to cranberry production.Wild relatives of many crops, including corn, sunflower, wheat, rice, potato, tomato, and banana have been the source of genetic variability and beneficial traits, such as disease resistance and abiotic stress tolerance, for crop improvement . In cranVaccinium macrocarpon and V. oxycoccos overlap in many areas  and Franklin (Early Black x Howes) plus a central cultivar Ben Lear (wild selection), and Stevens  and a V. microcarpum accession from Alaska , Upper Island Lake in the Chequamegon-Nicolet National Forest in Wisconsin (24), and Cranberry Glades site 5 in the Monongahela National Forest in West Virginia (17) , South Prairie in the Gifford Pinchot National Forest, Washington (19) and Little Crater Meadow in the Mount Hood National Forest, Oregon (16) (We observed that for nia (17) . Anothergon (16) . These pgon (16) , Table 4Vaccinium macrocarpon and V. oxycoccos, online herbarium and germplasm records in the USDA National Plant Germplasm System , the Global Biodiversity Information Facility, the Global Crop Wild Relative Occurrence Database, the Genesys plant genetic resources portal, the Botanic Gardens Conservation International PlantSearch database, and the Consortium of Midwest Herbaria were searched for known populations on National Forests throughout the US. Scientific staff at the National Forests provided additional information on locations of populations. Collections from two other sites located near National Forests were also included in the analysis. The collection sites ranged from a small seep (14 m2) supporting the population at the southernmost latitude (5-BBKPNC) to large bogs and fens covering hundreds of thousands of square meters. The elevation of populations ranged from 183 m (11-SPBMI) to 1773 m (5-BBKPNC) for V. macrocarpon and from 114 m (28-QIROPWA) to 1196 m (9-BeRBWV) for V. oxycoccos. In the Eastern US, V. macrocarpon and V. oxycoccos were often found growing together on the same site. In the Western US, all populations were V. oxycoccos, except one that was determined to be V. macrocarpon.Plant materials were collected by ARS and USFS botanists and technicians . In ordeV. macrocarpon and V. oxycoccos were sampled separately. Following the technique utilized by Zalapa et al.  33, 72 °C for 30 min, 4 ∞. PCR products were submitted to the University of Wisconsin-Madison Biotechnology Center for Sanger sequencing both ways (forward and reverse).The forward and reverse sequences obtained from Sanger Sequencing were aligned using a pairwise alignment, using the Geneious Alignment tool . Aligned sequences were then trimmed and a consensus sequence was created in order to perform a multiple alignment of all individuals based on similarity using the Clustal Omega tool found on the Geneious Prime® 2020.0.3 software. Sequences were then batch exported in FASTA format for genetic distance analysis.Sanger sequence reads for the six organellar genes were merged into a single FASTA file for each sample using the R package Biostrings v2.50.2 . SequencV. macrocarpon outside its native range, which could be useful in better understanding the evolution of this complex section of the genus Vaccinium.Our study elucidated the genetic diversity and population structure of wild populations of two species of wild cranberry located mainly in National Forests. The populations are distributed across the US and exhibit unique allelic combinations that could be useful for breeding, especially since cranberry is a relatively new crop that has not been the subject of much artificial selection. These results will contribute to decisions on which populations should be prioritized for conservation action, including site management and collection of germplasm, by the USDA Forest Service and Agricultural Research Service. We were also able to identify a unique population of


Bupleurum euphorbioides in the Baekdudaegan, South Korea, and North Korea could be interglacial refugia, and documented how their rear‐edge populations differ genetically from those of typical temperate species. Phylogeographic analysis and ecological niche modeling (ENM) were used. The genetic structure was analyzed using microsatellite markers and chloroplast DNA sequences. The congener B. longiradiatum was included as a typical temperate plant species. Despite having almost identical life history traits, these congeneric species exhibited contrasting patterns of genetic diversity. ENM revealed an apparent maximum range contraction during the last interglacial. In contrast, its range expanded northward to the Russian Far East (Primorsky) during the Last Glacial Maximum. Thus, we hypothesize that B. euphorbioides retreated to its current refugia during interglacial periods. Unlike populations in the central region, the rear‐edge populations were genetically impoverished and uniform, both within populations and in pooled regional populations. The rear‐edge B. euphorbioides survived at least one past interglacial, contributing to the species’ genetic diversity. We believe that such genetic variation in the cold‐adapted B. euphorbioides gives the species the necessary adaptations to survive an upcoming favorable environment , unless there is artificial environmental change.The high genetic diversity of rear‐edge refugia populations is predicted to have resulted from species repeatedly migrating to low latitudes during glacial periods over the course of Quaternary climate change. However, several recent empirical studies of cold‐tolerant plants revealed the opposite pattern. We investigated whether current habitats of the cold‐adapted and range‐restricted  Bupleurum euphorbioides gives it the necessary adaptations that will enable it to survive an upcoming favorable environment , unless there is artificial environmental change.The study shows a pattern that contrasts with the changes in the distribution range that typical temperate plants experienced in the course of Quaternary climate change. ENM revealed an apparent maximum range contraction during the last interglacial. In contrast, its range expanded northward to Russian Far East (Primorsky) during the Last Glacial Maximum. The rear‐edge populations were genetically impoverished and uniform, both within populations and in pooled regional populations. We believe that such genetic variation in the cold‐adapted  Furthermore, we characterized the genetic characteristics of the rear‐edge populations to assess their contribution to the species’ genetic diversity. Finally, we provide conservation guidelines for the recovery and management of threatened B. euphorbioides populations in the boreal and temperate climatic zones of the Baekdudaegan range.In this study, we examined the possibility that the current habitats of 22.1B. euphorbioides populations in South Korea, representing a significant part of its entire distribution  from the six populations in South Korea.We collected 118 leaf samples from the eight known n Figure . A mean 2.2Total genomic DNA was extracted from the samples using MG Plant Genomic DNA Extraction SV Miniprep Kit  according to the manufacturer's protocol. The extracted DNA was confirmed by electrophoresis, and the concentration was measured using a Nano‐300 micro spectrophotometer, to obtain the same concentration of template DNA in each sample; this was then diluted to 15 ng of DNA for analysis.B. euphorbioides and B. longiradiatum . The allele sizes and peaks were determined via Peak Scanner v. 2 software (Applied Biosystems).For the microsatellite analysis, we used 12 nuclear microsatellite markers, Bul007, Bul008, Bul010, Bul015, Bul016, Bul027, Bul030, Bul032, Bul040, Bul045, Bul049, and Bul050, previously developed for Bupleurum phylogeny  trnS‐F (5′‐CTTTAGTCCACTCAGCCATC‐3′) and trnG‐R (5′‐CCACTAAACTATACCCGCC‐3′); (b) ndhA‐F (5′‐CGCTATTACAGAACCGTACA‐3′) and ndhA‐R (5′‐CCTATGTACAAGAGTTCAGTGA‐3′). Finally, we designed and used three non‐coding chloroplast DNA regions: the rps16 intron , 0.25 µl of Taq polymerase, and sufficient distilled water. Amplification was performed as follows: initial denaturation at 95°C for 10 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 1 min; and a final extension at 72°C for 7 min. The PCR products were visualized on 1% agarose gels dyed with Redsafe , purified using a Wizard SV Gel and PCR Clean‐UP system (Promega), and sequenced using an ABI 3730xl DNA Analyzer (Applied Biosystems). The sequences of the three regions were aligned using the Clustal algorithm , using universal makers widely used in analyzing 2.3B. euphorbioides, we classified the genetic dataset into two subsets , reflecting the geographical distribution based on the pronounced curvature of the Baekdudaegan range. By comparison, in B. longiradiatum, no geographical division was attempted, other than to compare the genetic patterns.Before analyzing the microsatellite data, two individuals identified as homozygous were randomly selected and sequenced by PCR amplification for each locus to account for the presence of null alleles and confirm the accuracy of the markers. The results of the sequencing showed that all markers perfectly amplified their target loci for both samples. We also estimated the null allele frequency using INEst  software, which calculates the null allele frequency regardless of the effect of inbreeding , mean observed heterozygosity (HO), number of private alleles (PA), and fixation index (FIS). Allele richness (AR) and genetic differentiation among populations (FST) were determined by calculating the overall FIS according to the method of Weir and Cockerham (FST was tested using the log‐likelihood (G)‐based exact test in FSTAT. To test for departures from Hardy–Weinberg equilibrium (HWE) and linkage equilibrium, we conducted exact tests based on a Markov Chain method , using GENEPOP 4.0  from 1,000 iterations for K = 2 and K = 3, using random input orders.To infer the population structure of each species, we used a Bayesian clustering approach implemented in STRUCTURE 2.3, as calculated from microsatellite markers  , and haplotype diversity (Hd) . There were no fossil data for B. euphorbioides, so we used an uncorrelated lognormal relaxed clock for clock models and coalescence with constant size based on the substitution rates of chloroplast sequence for most angiosperm species  .For cpDNA analysis, intrapopulation genetic diversity levels were quantified using indices of nucleotide diversity per site , we used Mantel tests in GenAlEx 6.5 , and for the LGM from CHELSA. To reconstruct the historical distributions, we utilized three past climate models for LGM: the Community Climate System Model ; HE ranged from 0.184  to 0.557 (e‐JC) (mean of 0.401); AR ranged from 1.537 (e‐DN) to 3.676 (e‐HG) (mean of 2.497); PA ranged from 0 to 10 (mean of 4.375); FIS ranged from −0.021 (e‐SN) to 0.515 (e‐DN) (mean of 0.192). The Hardy–Weinberg exact test revealed significant deviation from HWE in four of the B. euphorbioides populations. For B. longiradiatum, HE was 0.328–0.556 and AR was 2.167–3.280; mean HE, AR, and FIS values were 0.474, 2.754, and 0.106, respectively.Genetic diversity parameters are shown in Table B. euphorbioides, rps16, trnSGCU–trnGUCC, and ndhA, produced 19 polymorphic sites. Among these, the trnS‐trnG (727–732 bp) region was the most variable, with nine polymorphisms, a polynucleotide repeat‐length of 5 bp, an indel, and a nucleotide substitution. The rps16 region (854–856 bp) had five polymorphic sites (three substitutions and three indels), and the ndhA region (825 bp) had four polymorphic sites . For B. euphorbioides, the combined cpDNA sequences provided a total of seven haplotypes (E1–E7), aligned with a consensus length of 2,415 bp  were identified based on 24 polymorphisms (six nucleotide substitutions and 18 indels) (Table The chloroplast non‐coding regions of bp Table . For B. s) Table .3.2B. longiradiatum, within‐population genetic diversity was relatively lower in B. euphorbioides: AR = 2.497, HE = 0.401, H = 1.12, and Hd = 0.067. In B. euphorbioides, genetic differentiation among populations  was higher than in B. longiradiatum . In B. euphorbioides, FIS was high, with a mean of 0.192 , than in the rear‐edge populations . Similarly, genetic diversity was higher for the pooled central populations  than cpDNA  analysis, whereas the opposite was true for the rear‐edge populations .At the regional level, for 3.3B. euphorbioides, the STRUCTURE analysis showed that the mean likelihood scores increased, with K values of 1–8, whereas ΔK decreased gradually, with K values of 2–7  of L(K) increased considerably from K = 4 .For 7 Figure . However4 Figure . Therefo4 Figure . At K = s Figure . In thiss Figure c, S2a, aB. euphorbioides, each population harbored only a single haplotype, except for e‐HG  .For G Figure . For B. S Figure . In the S Figure . LineageS Figure . FinallyS Figure . Based o) Figure . The Bay Figures b and S3b3.4B. euphorbioides , derived from haplotype E1, the ancestral haplotype and the only one present in the rear‐edge, also occurs in the central range. In other words, the rear‐edge certainly contributed to the higher genetic diversity of the central populations. Moreover, the distribution during the LIG inferred by ENM strongly supports the possibility that the current rear‐edge populations would have survived at that time.The inferred cryptic southern refugia must be verified by tracking the lineages of populations that occurred there. The populations we observed are living in an interglacial; the current climate is likely to be long lasting . In light of the fact that all central populations have private alleles, it is highly likely that the central region also represents interglacial refugia. To determine this, future studies should test leading edge samples collected around the northern end of the Baekdudaegan range.Our study is important and novel for several reasons. First, we clearly described the genetic characteristics of rear‐edge populations while inferring interglacial refugia in a phylogeographical framework. This particular case addresses fundamental aspects of species’ survival, along with the function of interglacial cryptic southern refugia, and the mechanisms whereby they maintain genetic diversity. Second, we show that the Baekdudaegan mountain system, an important refugium for many temperate plants during glacial periods, can also play this role in interglacial periods. Low genetic diversity in populations is typically expected to negatively impact species’ reproductive fitness and evolutionary potential and, ultimately, lead to extinction Dyke, . However4.3B. euphorbioides that conservation efforts should not be focused on a limited set of populations or habitats. All extant habitats, which represent interglacial refugia, have the evolutionary potential to guarantee the long‐term survival of the species. The low genetic diversity of the rear‐edge populations may in fact be beneficial to the species’ survival. Given that the B. euphorbioides populations we sampled here represent the species’ entire South Korean distribution, it should not be difficult to conserve all of these populations in situ. Thus, first, we recommend that all known populations be protected by law to prevent further damage by loss of individuals. An appropriate measure is to control the access roads by which people can enter  where the haplotypes are not shared among populations. Finally, the fact that pollen transfer could enable gene flow over several kilometers should be considered in restoration attempts.The authors have no conflict of interest to declare.Won‐Bum Cho: Conceptualization (supporting); Formal analysis (lead); Methodology (lead); Resources ; Software (lead); Visualization (lead); Writing‐original draft . Soonku So: Investigation ; Resources ; Validation (supporting); Visualization (supporting); Writing‐original draft (supporting). Eun‐Kyeong Han: Formal analysis (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing‐review & editing (supporting). Hyeon‐Ho Myeong: Investigation ; Methodology (supporting); Resources . Jong‐Soo Park: Methodology (supporting); Software ; Visualization (supporting); Writing‐original draft (supporting). Seung‐Hyun Hwang: Formal analysis (supporting); Investigation ; Resources (supporting). Joo‐Hwan Kim: Conceptualization (supporting); Supervision (supporting); Writing‐review & editing (supporting). Jung‐Hyun Lee: Formal analysis (supporting); Methodology (supporting); Project administration ; Supervision (lead); Visualization (supporting); Writing‐original draft ; Writing‐review & editing .Supplementary MaterialClick here for additional data file.


Sturnus vulgaris) represent one of the most widespread and problematic avian invasive species in the world. Understanding their unique population history and current population dynamics can contribute to conservation efforts and clarify evolutionary processes over short timescales. European starlings were introduced to Central Park, New York in 1890, and from a founding group of about 100 birds, they have expanded across North America with a current population of approximately 200 million. There were also multiple introductions in Australia in the mid‐19th century and at least one introduction in South Africa in the late 19th century. Independent introductions on these three continents provide a robust system to investigate invasion genetics. In this study, we compare mitochondrial diversity in European starlings from North America, Australia, and South Africa, and a portion of the native range in the United Kingdom. Of the three invasive ranges, the North American population shows the highest haplotype diversity and evidence of both sudden demographic and spatial expansion. Comparatively, the Australian population shows the lowest haplotype diversity, but also shows evidence for sudden demographic and spatial expansion. South Africa is intermediate to the other invasive populations in genetic diversity but does not show evidence of demographic expansion. In previous studies, population genetic structure was found in Australia, but not in South Africa. Here we find no evidence of population structure in North America. Although all invasive populations share haplotypes with the native range, only one haplotype is shared between invasive populations. This suggests these three invasive populations represent independent subsamples of the native range. The structure of the haplotype network implies that the native‐range sampling does not comprehensively characterize the genetic diversity there. This study represents the most geographically widespread analysis of European starling population genetics to date.European starlings ( European starlings represent one of the most widespread and problematic invasive species in the world. Therefore, understanding their population history and current population dynamics can contribute to conservation efforts and clarify recent evolutionary processes.Here, we compare mitochondrial diversity in birds from North America, Australia, South Africa, and the native range in the United Kingdom. This study represents the most geographically widespread analysis of European starling population genetics to date. Based on previous research using nuclear markers, we predict low levels of population structure within North America. We predict that the mitochondrial diversity of the North American population will be lower than that of Australia, where multiple introductions were made Jenkins, , and the22.1N = 95) were obtained from starlings culled by the United States Department of Agriculture Animal and Plant Health Inspection Service (USDA APHIS) between 2014 and 2018 at 14 localities across the United States (Table N = 2) from National Museums Scotland, Edinburgh.North American tissue samples . Networks were constructed for the North American samples (1181 bp) and for the full dataset. To construct the latter, we trimmed the full dataset to 928 bp to accommodate the continental dataset with the shortest sequence length.Overlapping sequences were aligned using the software FST values), pairwise nucleotide diversity (π), haplotype diversity (h), Fu’s s,F and Tajima’s D in Arlequin v3.5.1.2  for each population was calculated using FSTAT v.2.9.4  included 20 haplotypes encompassing 53 polymorphic sites and did not indicate the presence of regional population structure Figure . When weFST values from pairwise comparisons between the native‐range and the three introduced populations ranged between 0.060 (North America) and 0.174  and were all statistically significant  and only significantly different for the Central versus Western US comparison  but not from the Australian  nor North American populations  ; although only 45 individuals were sampled, we identified 30 haplotypes in this population. Surprisingly, despite higher propagule pressure in Australia as compared to that of North America or South Africa, Australia harbored the lowest haplotype richness (R = 7.7). The North American population, which was intermediate in terms of propagule pressure, has retained the most genetic diversity (R = 14.7). Given the timescales involved, this is unlikely to be caused by novel mutations arising in North America  was found in the UK  albeit statistically significant difference between Central and Western samples but this may be due to the low sample size from the Central United States (N = 20). Overall, our findings are consistent with an earlier investigation of this population, which utilized allozyme data  and all significantly different from populations in the native range (FST ranged from 0.06 to 0.17). North America was most similar to the UK and Australia was least similar. These differences are likely caused by a combination of discrete introduction sources and founder effects. However, this could also be due to differences in timing of introductions; the Australian introduction occurred earlier than the others (mid‐19th century) so it is possible that these differences reflect shifts that occurred in the native range in the latter half of the 19th century.When we investigated genetic differentiation across continents, we found that invasive populations were genetically divergent  expansion model Figure . This maions Fu, . Given oions Fu, .It is also interesting to consider that differences in the environments of each of the three invasive ranges studies here may have influenced population expansion rates. The United Kingdom and surrounding parts of Europe (native range) are largely classified as temperate with a hot or warm summer  versus Australia (large), of similar or contrasting genetic backgrounds , North America versus South Africa,  and across different environments  versus Fiji ). Together with the recent development of genomic resources ; investigation (lead); writing–original draft (lead); writing–review and editing . Lee Ann Rollins: Formal analysis (supporting); investigation (supporting); methodology ; supervision (supporting); writing–review and editing . Julia M. Zichello: Conceptualization (lead); investigation ; supervision (lead); writing–review and editing .Supplementary MaterialClick here for additional data file.


Leiolepis ocellata is a lizard species distributing in topographically diverse habitats in northern Thailand. To explore its evolutionary history, 113 samples of L. ocellata were collected from 11 localities covering its distributional range in northern Thailand, and sequenced for mtDNA fragments (Cyt b and ND2). Pairwise comparisons across sampling localities yielded significant genetic differentiation (FST and Jost's D) but no clear pattern of isolation by distance could be demonstrated based on the Mantel test. Phylogenetic and network analyses highlighted six haplogroups. Their divergence times were estimated to occur during the Pleistocene, much more recent than major orogenic events affecting northern Thailand. Instead, the results suggested that lineage divergences, of particularly eastern and western haplogroups of the region, coincided with the major rivers in the region , indicating vicariance in response to riverine barriers. Furthermore, ecological niche modeling suggested an expansion of suitable habitats of L. ocellata, when LGM‐liked conditions. This expansion potentially facilitated their dispersal among adjacent localities leading to lineage diversification and genetic admixture, after the riverine divergence. Leiolepis ocellata, a lizard species distributed in topographically diverse habitats in northern Thailand, revealed 6 haplogroups. Lineage divergences, of particularly eastern and western haplogroups of the region, coincided with the major rivers in the region , indicating vicariance in response to riverine barriers. GeograpThe Indo‐Burma region is an intriguing biological hotspot for phylogeographic studies due to great biodiversity and complex landscapes and climates that are consequences of multiple paleogeographic events Hall, . That paLeiolepis, known as butterfly lizards, are diurnal and widely distributed in the Indo‐Burma region  restricted to northern Thailand , throughout northern Thailand , and ecological modeling. The main goals of this study were to explore how paleogeographic events, including orogenic development and river systems, and paleoclimate might explain observed phylogeographic and population structure of L. ocellata populations in northern Thailand.In this study, we sampled and sequenced 22.1L. ocellata were sampled and identified to species based on morphological characteristics  to use as outgroups. The lizards were randomly captured by hand and traps. Tissues of tail tip (<1 cm in length) were clipped using sterile scissors. Collected tissues were preserved in 95% ethanol and stored at −20°C until further analyses.In this study, s Figure  were cols Figure . These sr et al. , specimer et al. . Additio2.2b (Cyt b) and ND2L1 (5′‐CCAAAGATGGGCTTGATTGT‐3′) and ND2H1 5′‐AAGTATCCGGGTTGCATTTG‐3′) for partial nicotinamide adenine dinucleotide dehydrogenase 2 (ND2). Polymerase chain reaction (PCR) was performed using AccuStart II GelTrack PCR SuperMix (Quanta BioSciences). Concentration of the PCR ingredients followed the suggested protocol from the manufacturer. The reactions were executed with the following steps: 95°C for 2 min followed by 32 cycles of 95°C for 35 s, annealing temperature for 35 s (60°C for Cyt b and 55°C for ND2), 72°C for 1 min using Eppendorf Mastercycler gradient thermocycler. Products were visualized with 1.5% agarose gel electrophoresis and purified using NucleoSpin Gel and PCR Clean‐up (Macherey‐Nagel). The PCR products were sent for sequence analyses with the Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit following the protocol from the manufacturer.Genomic DNA was extracted using NucleoSpin tissue kit (Macherey‐Nagel). We amplified two mitochondrial regions using combinations of our designed primers CytbL (5′‐CCAACCAAGACCTTTGATCTG‐3′) and CytbH (5′‐AAGTATCCGGGTTGCATTTG‐3′) for partial cytochrome 2.3L. ocellata, genetic diversity indices, including number of haplotypes (n), haplotype diversity (h), and nucleotide diversity (π), were calculated using DnaSP5  haplotypes under Maximum Likelihood (ML) and Bayesian Inference (BI) approaches with L. belliana collected from Kamphaeng Phet Province (KP), serving as an outgroup. Dataset of mtDNA sequences were partitioned allowing the evolutionary model to specify each partition separately. Kakusan4  at a resolution of 30 s , which contained 1,071 bp of Cyt b  and 882 bp of ND2 , and defined 42 haplotypes. Genetic diversity among sampling localities was shown in Table n) ranged from 2 to 10, haplotype diversity (h) ranged from 0.30 to 0.95, and nucleotide diversity (π) ranged from 0.01% to 0.73%, for all localities. The overall genetic diversity h and π were 0.97 and 1.08%, respectively.The final alignments of concatenated partial mitochondrial DNA from 113 FST and Jost's D among all sampling localities were used to indicate genetic differentiation. Pairwise FST was significant (p < .05) for most comparisons, except for localities MN‐WS, while Jost's D was significant (p < .05) for most comparisons, except for localities MN‐MW and MN‐WS .Pairwise WS Table . There w3.2L. ocellata in northern Thailand (100% posterior probability) when L. belliana from KP was treated as an outgroup. Within the L. ocellata group, six haplogroups, A‐F, were identified. Haplogroup A  consisted of all L. ocellata from DK and one sample from WS. Haplogroup B  was closely related to haplogroup A and consisted of individuals from the southernmost localities, MW and BT. Haplogroup C  contained all samples from KY and MC, in the west of the region, with many samples from CS and some samples from CM. Haplogroup D  consisted of three samples from BT. Haplogroup E  consisted of samples from five localities in the east and south of northern Thailand . Finally, haplogroup F  was distributed in central northern Thailand, and found in MP, CS, CM, and TK.As concatenated mtDNA trees based on both BI and ML approaches provided similar topology, only the BI tree was shown in Figure L. ocellata formed six recognizable groups (A‐F) identified in the trees. Each locality harbored private haplotypes and most haplotypes were unique to their geographic localities except for two haplotypes shared between MN and WS.The network analyses of concatenated mitochondrial genes provided similar results to those of the phylogenetic trees but revealed slightly different genealogy Figure . A total3.3b mtDNA sequences, the coalescence times of the L. ocellata haplogroups were all estimated to have occurred during the Pleistocene . Those ranges were slightly contracted during the Holocene. Low habitat suitability occurring in the Holocene appeared in the north of the Thanon Thong Chai range and upper course of the Ping River. At the present time, the distribution ranges were further contracted and mostly restricted to the central parts of the region. While the unsuitable habitats along the Thanon Thong Chai range and Ping river further isolated western populations (KY and MC), other unsuitable habitats along the Yom river in the east partially isolated eastern populations .Maxent predicted the distributions of s Figure . The AUC44.1L. ocellata may be the first phylogeographic and population genetic study of any terrestrial fauna confined to northern Thailand.Exploring the phylogeography and population genetics of organisms provides valuable insights for both evolutionary processes and conservation of biodiversity Avise, . The effL. ocellata as indicated by its high haplotype diversity (h) and nucleotide diversity (π). Based on Cyt b, the values of these diversity indices of L. ocellata (h = 0.95 and π = 0.98) were comparable to those observed in L. reevesii  and lack of shared haplotypes among populations other than for the MN‐WS. This population structure was likely a consequence of low dispersal ability of lizards . Juveniles usually dispersed to adjacent areas after maturation and remained in a particular burrow unless they were disturbed. It was also noticed that some adults protected their burrows and adjacent areas against other individuals indicating that territorial behavior that may exist in Leiolepis. Such habits of L. ocellata together with other geographical factors might further attribute to its phylogeographic structure at a regional scale.Analyses from the concatenated mtDNA implied strong population genetic structure of 4.2L. ocellata in which six haplogroups could be identified. To further infer the potential factors related to this phylogeographic pattern, divergence times were estimated from Cyt b tree using substitution rates from other reptiles (Agamidae), following the study of L. reevesii , and eastern  sides of the highest mountain of Thailand, Thanon Thong Chai range. Furthermore, the Pleistocene divergence time was much later than the time of orogenic events in northern Thailand, which took place during the Tertiary . Moreover, dispersal of L. ocellata during the range expansions or minor changes in river course may have resulted in secondary contact between haplogroups confined to different banks of the rivers ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology ; Project administration (lead); Validation ; Visualization (lead); Writing‐original draft (lead); Writing‐review & editing . Nontivich Tandavanitj: Conceptualization ; Formal analysis (supporting); Methodology ; Supervision ; Validation ; Visualization ; Writing‐review & editing . Chalita Kongrit: Conceptualization ; Formal analysis (supporting); Methodology ; Visualization ; Writing‐review & editing . Kritsayam Kongsatree: Resources . Phinit Kongpraphan: Resources . Wuttipong Dongkumfu: Resources . Detanan Kumsuan: Resources . Jenjit Khudamrongsawat: Conceptualization ; Formal analysis (supporting); Funding acquisition (lead); Investigation (supporting); Methodology ; Supervision ; Validation ; Visualization ; Writing‐review & editing .Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Phyllanthus emblica L. is a well-known medicinal and edible plant species. Various medicinal compounds in the fruit make it an important medicinal and promising economic material. The plant is widely distributed in Southwestern and Southern China. However, due to massive deforestation and land reclamation as well as deterioration of its natural habitat in recent years, the wild resources of this species have been sharply reduced, and it is rare to see large-scale wild P. emblica forests so far. In order to effectively protect and rationally utilize this species, we investigated the genetic diversity, genetic structure, and population dynamics of 260 individuals from 10 populations of P. emblica sampled from the dry climate area in Yunnan and wet climate area in Guangxi using 20 polymorphic EST-SSR markers. We found high genetic diversity at the species level (He = 0.796) and within populations (He = 0.792), but low genetic differentiation among populations (FST = 0.084). In addition, most genetic variation existed within populations (92.44%) compared with variation among the populations (7.56%). Meanwhile, the NJ tree, STRUCTURE, and hierarchical analysis suggested that the sampled individuals were clustered into two distinct genetic groups. In contrast, the genetic diversity of the dry climate group  was higher than that of the wet climate group , which might be attributed to the combined effects of altitude, precipitation, and geographic distance. Interestingly, only altitude and precipitation had significant pure effects on the genetic diversity, and the former was slightly stronger. In addition, DIYABC analysis suggested the effective population size of P. emblica might have contracted in the beginning of the Last Glacial Maximum. These genetic features provided vital information for the conservation and sustainable development of genetic resources of P. emblica, and they also provided new insights and guidelines for ecological restoration and economic development in dry-hot valleys of Yunnan and karst areas in Guangxi. Phyllanthus emblica is a perennial deciduous tree or shrub distributed across tropical and subtropical areas. It contains many medicinal compounds such as vitamin C, flavonoids, emblicol, emblicanin A/B, and is used as a daily medication by many ethnic minorities such as Tibetan group in China; its various therapeutic effects such as hepatoprotective, antimicrobials have also been confirmed in modern medical research  What is the level of genetic diversity of P. emblica in both of these typical climate areas? (2) What is the relative importance of geographic and environmental distance as well as precipitation and altitude to the genetic diversity of P. emblica? (3) What is the population dynamics of the species? We hoped to provide a reference for the protection and rational utilization of the wild resources of P. emblica as well as theoretical guidance for the ecological restoration and economic development of mountainous areas in China.SSR markers have been widely used in genetic diversity studies because of their co-dominance, high polymorphism rate, good reproducibility, and rapid excavation at a low cost . Based oP. emblica samples were collected from 10 wild populations  which were distributed in two main climate areas: arid areas (Yuanmou County and Binchuan County in Yunnan) and humid areas (Mashan County in Guangxi)  , 2. The Guangxi)  showed sGuangxi) . The fivP. emblica using the PCR conditions described above. Allele sizes for the tagged PCR products were obtained using an ABI 3730 sequencer with a GeneScan 500 LIZ Size Standard and GeneMapper 4.1 .Genomic DNA was isolated with the modified CTAB method . All samNa), the effective number of alleles (Ne), Shannon’s diversity index (I), the observed heterozygosity (Ho), the expected heterozygosity (He), inbreeding coefficient (FIS), outcrossing rate t [t = (1 − FIS)/(1 + FIS)] (Nm), percentage of polymorphic loci (PPL), and genetic differentiation index (FST). Polymorphic information (PIC) was calculated using PowerMarker version 3.25 (POPGENE version 1.31  was used + FIS)] , gene flion 3.25 .2) were obtained from WorldClim version 2  and geographical distance or environmental variables (Euclidean distance) with 9999 permutations. To further explore the relative importance of altitude and precipitation to genetic diversity, we also conducted a similar correlation analysis with them as environmental distance (Euclidean distance), respectively.P. emblica, several approaches were used in this study. Firstly, the Bayesian clustering analysis of the population structure was carried out using STRUCTURE version 2.3.4 (5 and 1 × 105 Markov chain Monte Carlo (MCMC) replications. A total of 20 independent runs were performed for each K value ranging from 1 to 20. The most likely number of clusters was evaluated using both ΔK and the log-likelihood value with STRUCTURE HARVESTER version 0.6.94  was performed using POPULATIONS version 1.2.30  at 95% confidence interval (CI). In order to gain insight into the population dynamic history of P. emblica, the ABC framework in DIYABC v. 2.0  was carried out to estimate the partitioning of genetic variation between and within the populations using ARLEQUIN version 3.0 . The effC v. 2.0  was usedNa), expected heterozygosity (He), and Shannon’s diversity index (I) ranged from 18 to 40, 0.485 to 0.921, and 1.324 to 2.851, respectively, with the means of 28.1, 0.796, and 2.232, respectively  in all 10 populations were 100%; however, the genetic diversity of each population was different. The YMB population had the highest genetic diversity (He = 0.819), whereas MSE had lowest genetic diversity (He = 0.604) (He = 0.792) was at a high level; however, the average genetic diversity of the five populations of P. emblica from dry climate areas of Yunnan (He = 0.786) was higher than that of the five populations from wet climate areas of Guangxi (He = 0.673) (He = 0.796) of P. emblica was also at a high level  . Moreove= 0.673) . In addigh level .The PCA results show that PC1 (97.81%) not only divided the seven precipitation-related bioclimatic variables and altitude into two clusters, but also made a clear distinction between the populations in dry and wet climatic areas . The altNa) and Shannon’s diversity index (I), while geographic distance explains more variation of expected heterozygosity (He) (I) was significant (p = 0.036 < 0.05).According to the Mantel tests, both environmental distance and geographic distance had significant effects on three genetic diversity indices . Compareity (He) . In addiNa) and Shannon’s diversity index (I), while precipitation was only significantly related to Shannon’s diversity index (I) and its effect was weaker than that of altitude  was 0.084 (FST < 0.15). The populations YMB, BCA, BCB, BCC, and MSD had significant deviations from the Hardy-Weinberg equilibrium (p < 0.05 or p < 0.01) (FIS) of these five populations were positive; correspondingly, their outcrossing rates (t) were < 1  . The inbwere < 1 , indicatK reached the maximum value (ΔK = 577.307) at K = 2 (K) has the highest value and least variance at K = 4 ; 4.66% of the genetic variations were attributed to the differences among the groups, 90.53% were attributed to the differences within populations, and 4.81% were attributed to the differences among populations within groups  rather than to the variation among populations (7.56%) . At the n groups .Pcrit) are listed in Pcrit = 0.02 and Pcrit = 0.01, effective population size of BCC was the largest, whereas MSA had the smallest effective population size. The DIYABC results showed that the optimal model was Scenario 7  individuals , and the mean genetic diversity of the 10 populations investigated in the present study (He = 0.792) was similar to that reported for this species in Thailand (He = 0.619) (He = 0.753) (He = 0.22 or 0.23). Compared to congeneric herbs, such as P. amarus  and P. fraternus  (P. emblica showed higher genetic variation at the species level (0.796) and within populations (0.792), but lower genetic differentiation among populations (FST = 0.084) in the present study. Outcrossing species usually have higher genetic diversity than inbreeding species ; however, for the other eight populations, the inbreeding coefficients (FIS) were positive, and the outcrossing rates (t) were lower than 1, indicating insufficient heterozygotes and the possibility of inbreeding. According to our long-term field investigations, this could be attributed to human interference in the habitats of these eight populations, resulting in insufficient heterozygotes and a high inbreeding coefficient. This might also be attributed to hybridization (inbreeding) between adjacent individuals with the same genotype. The richness of plant genetic diversity is related to the complexity of its habitat and ecological factors such as precipitation, temperature, and soil   and Sri = 0.753) , but sig = 0.26) , P. embl species . A previcrossing . In the and soil , which ical Asia .Nm) of these populations of P. emblica was >1, indicating that gene flow was high enough to resist the genetic differentiation among populations caused by genetic drift, thus achieving a homogenizing effect  was higher than that in the wet climate group , and both of these values were higher than the genetic diversity of Camellia nitidissima at the population level . Moreover, the AMOVA suggested that genetic variation within the populations of the dry climate group (96.24%) was also higher than that within the populations of the wet climate group (93.36%). The Mantel tests confirmed that the genetic diversity of this species was affected by altitude, precipitation and geographic distance. Interestingly, the partial Mantel tests showed that the pure effects of altitude and precipitation only on Shannon’s diversity index (I) was significant, which implied that this genetic diversity index was more sensitive to the environmental response to some extent. Just as it is widely used in microbial and forest landscape researches (I) is also a key indicator for genetic diversity assessment of woody plants. This study supported the conclusion that altitude significantly affects the distribution of genetic variation in populations . Recent studies have proposed that genetic diversity basically exhibited a high-low-high pattern with increasing altitude . Given that intermediate populations between the two climate zones were not sampled and compared, in future researches, further work should be carried out to study the populations distributed in more climatic gradients, altitude gradients and distance gradients to reveal the general effects of altitude, precipitation and geographical distance on genetic diversity.According to the niche breadth variation hypothesis, molecular genetic variation is related to environmental variation, and high level of genetic variation is an adaptive strategy of plants for responding to heterogeneous environments . The cursearches , Shannonulations . In thisulations . On the altitude . Howeveraltitude . Althougaltitude . The genaltitude . TherefoF = 0.095). The genetic differentiation among the populations in the wet climate group (FST = 0.071) was higher than that among the populations in the dry climate group (FST = 0.042), and it might be the result of combined effects of isolation by distance (IBD) and environmental selection. The effective population size of the dry climate group was larger than that of the wet climate group, and the effective population sizes of BCC was the largest (83.5), whereas MSA had the smallest effective population size (10.1). However, DIYABC analysis suggested the effective population size of P. emblica might have contracted in the beginning of the Last Glacial Maximum. The above results supported that genetic differentiation was associated with decline in the effective population size   , implyinulations . It is wrn China . Based oP. emblica. However, this famous plant species which has important medicinal, commercial, and ecological values is being consumed at a very high and alarming rate, with the increase in human population leading to the increase in the demand for such plant materials. In Yunnan and Guangxi, the natural P. emblica forests are commonly being cut down for fodder, fuel, and wood. Because of the negative influence of climate change and human activities, the native vegetation types in the dry-hot P. emblica habitat have declined and are almost non-existent. The existing habitat types mainly include semi-savannah vegetation similar to the valley savannah vegetation in Africa (P. emblica in the humid climate area of Guangxi is karst landform, where plants live in harsh and extremely fragile conditions with serious soil erosion and high ecological sensitivity. Over-exploitation is a serious threat to the survival of this species, and currently, it is difficult to find well-preserved wild P. emblica forests in China. At the same time, the harsh and fragile habitats of P. emblica are also facing various threats, including climate change and human activities, and it is difficult for the ecosystem to recover naturally after these disturbances. Therefore, it is crucial to protect and rationally utilize the wild resources of P. emblica as well as to put efforts into the ecological restoration of its habitats.China has abundant resources of n Africa , and in P. emblica is a species with excellent tolerance to drought and barren soil because of its well-developed roots, and is often used as a pioneer tree species for forestation. Meanwhile, its high genetic diversity provides more possibilities to breed high-quality varieties of this species to be used as an important medicinal economic plant. The genetic resource conservation of local P. emblica populations should be considered mainly for the ecological restoration of dry-hot valleys and karst areas. In addition to in situ conservation of the populations with high genetic diversity, ex situ germplasm collection for different purposes, such as breeding and conservation, is also significant for achieving our goals. Compared to the five populations from the Guangxi area, the five populations from the dry-hot valley of Yunnan had higher genetic diversity, particularly the YMA and YMB populations, and attention should be payed to this difference. Furthermore, given that the genetic variation mainly existed within populations, more individuals should be selected and propagated within populations used for ecological restoration in Yunnan dry-hot valley area and Guangxi karst area. In the present study, P. emblica samples were divided into two gene clusters, which should be taken into account when collecting germplasm resources and carrying out ecological restoration. Finally, environmental protection should be advocated in order to increase local farmers’ awareness of the protection of this valuable species, and it should be forbidden to cut down P. emblica trees in its ecological restoration areas.P. emblica in two different typical climate areas of China by polymorphic EST-SSR markers, P. emblica had more genetic diversity at the species level and within populations, but less genetic variation among populations. However, the genetic diversity of P. emblica in the dry climate area of Yunnan was higher than that in the wet climate area of Guangxi, and both altitude and precipitation had significant pure effects on this result. In addition, we found that P. emblica populations had experienced a bottleneck event in the beginning of the Last Glacial Maximum. The findings of this study will provide new ideas and guidance for the protection and rational development and utilization of P. emblica resources, and they will also provide a scientific basis for the ecological restoration and economic development in Yunnan dry-hot valley areas and Guangxi karst areas.According to our investigations of the genetic diversity and genetic structure of The original contributions presented in the study are included in the article/HM conceptualized the study. XL, YM, and ZL contributed to the investigation. XL and YW were in charge of data curation. XL and HM wrote the original draft, and HM reviewed. All authors agree to be accountable for the final manuscript.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.


Populus davidiana and Populus tremula are among the most geographically widespread and ecologically important tree species in Northern Hemisphere. Whole-genome resequencing data of 41 individuals of P. davidiana and P. tremula throughout Eurasia was conducted, finding that genetic differentiation was evident between the two species, the STF values between P. davidiana and P. tremula was 0.3625. The ancestors of the two aspen diverged into P. davidiana and P. tremula species approximately 3.60 million years ago (Mya), which was in accordance with the rapid uplift of Qinghai–Tibet Plateau (QTP) around the Miocene/Pliocene boundary. The two species experienced a considerable long-term bottleneck after divergence, with population expansion beginning approximately 20,000 years ago after the end of the last glacial maximum. Although the majority of regions of genomic differentiation between the two species can be explained by neutral evolutionary processes, some outlier regions have also been tested that are significantly influenced by natural selection. We found that the highly differentiated regions of the two species exhibited significant positive selection characteristics, and also identified long-term balancing selection in the poorly differentiated regions in both species. Our results provide strong support for a role of linked selection in generating the heterogeneous genomic landscape of differentiation between P. davidiana and P. tremula. These results provide the detailed and comprehensive genomic insights into genetic diversity, demography, genetic burden, and adaptation in P. davidiana and P. tremula.Forest trees can increase our understanding of how evolutionary processes drive the genomic landscape and understand speciation due to the majority of forest trees being distributed widely and able to adapt to different climates and environments.  Increasing our understanding of how evolutionary processes drive the genomic landscape of variation is fundamental to a better understanding of the genomic consequences of speciation. Understanding how and why genomes diverge during speciation has received considerable attention in evolutionary biology research. . GeneralA growing quantity of genome-wide data are becoming available with the development of high-throughput sequencing technology, and intense research activity has resulted in the discovery of substantial patterns of genetic variation and population divergence among multiple related species with considerably increased accuracy . A univePopulus davidiana Dode and Populus tremula L. are two of the most ecologically important and geographically widespread tree species of the Northern Hemisphere. Both are keystone species, display rapid growth, with high tolerance to environmental stresses and long-distance pollen and seed dispersal via wind  and are keystone taxa in boreal forest communities  was used to analyze 41 P. davidiana and P. tremula trees to explore population structure, estimate population divergence time points, identify the historical demographic processes and infer the overall patterns of genomic divergence. This study provides insights into the evolutionary history and genetic diversity of the two species, in addition to describing examples of the mechanisms by which the species can adapt to regions with variations in climate and also provides the important reference value for understanding the mechanism of the formation of the geographical distribution patterns of other plant populations in Eurasian.Both P. davidiana and 21 individual of P. tremula . Additionally, we further discarded site types that likely cause mapping bias based on three criteria: Where total read coverage was particularly low (<100) or extremely high  across all P. davidiana and P. tremula samples reads or sites were completely filtered out; reads or sites that included >20 mapping quality scores of zero within the whole sample were discarded. These quality control steps resulted in only high quality reads being kept.A total of 20 individual of  tremula . A paire Archive  at the Bmmomatic  to removmmomatic  was usedersion 3 . SAMtoolersion 3  was usedersion 3  to correAfter filtering, we implemented two complementary strategies for downstream analysis  is a claP. davidiana and P. tremula, and sites with less than 10% of their data missing were used, the number of coancestry clusters (K) ranging from 1 to 6. Principal component analysis (PCA) was performed on all SNPs using the smartpca program in PCAngsd software (http://www.popgen.dk/software/index.php/PCAngsd). A Tracy–Widom test was used to determine the significance level of eigenvectors. The phylogenetic tree was constructed using neighbor-joining (NJ) with TreeBest software (http://treesoft.sourceforge.net/treebest.shtml), with P. tremuloides Michx. used as an outgroup. We downloaded the data from the Short Read Archive (SRA) at NCBI and the accession numbers is SRP065065.we used the program NGSadmix, which is based on genotype likelihoods to directly estimate individual admixture proportions from next generation sequencing data  to inferP. davidiana and P. tremula species based on the site frequency spectrum. Allele frequencies in the 41 samples were calculated using the realSFS module in ngsTools software so as to construct the required two-dimensional joint site frequency spectrum (2D-SFS), which was estimated with 100,000 coalescent simulations in each model. Alternative models of historical events were fitted to the joint site frequency spectra data. All parameter estimates were global ML estimates from 50 independent fastsimcoal2.1 runs, with 100,000 simulations per likelihood estimation  and 40 cycles of the likelihood maximization algorithm. The best model was identified through the maximum value of likelihoods and Akaike’s information criterion (AIC); simulated datasets were compared with the observed site frequency spectra to evaluate the fit of the best demographic model   to infersoftware . We assuand size .STF. Both hp and STF were calculated by a sliding window method (100 kb windows sliding in 10 kb steps). Variants were filtered when the minor allele frequency was less than 5% and the missing genotypes frequency was more than 50%. For comparing groups (groups 1 and 2), the regions with maximum STF values (top 5%) and minimum θπ1/θπ2 (top 5%) were identified as selected regions for group 1, and vice versa.The polymorphism levels in each group were quantified by pairwise nucleotide diversity (θπ) and the genetic differentiation in two populations was quantified by pairwise P. davidiana and P. tremula over non-overlapping 10 Kbp windows for calculating Fay & Wu’s H (2) between SNPs with pairwise distances larger than 1 Kbp were calculated using VCFtools v0.1.12b  the relative node depth (RND), calculated by dividing the dxy of the P. davidiana and P. tremula species by the dxy between the P. davidiana population and P. tremuloides. For all population genetic parameters, Wilcoxon ranked-sum tests were used to examine the significance of differences between outlier regions and the remainder of the genome. (4) dxy, which was calculated based on the posterior probability of the sample allele frequency at each locus and was then averaged over each 10 Kbp window.To assess the occurrence of selection in outlier windows displaying either exceptionally high or low differentiation, multiple population genetic parameters of the two unions of outer regions were compared with the remaining portion of the genome by a variety of additional population genetic statistics in both species. Firstly, we used ANGSD to estimate sample allele frequency probabilities between populations of the & Wu’s H , Fu and & Wu’s H  and θπ. v0.1.12b . And we v0.1.12b  to calcuv0.1.12b  to calcu. davidiana and 21 P. tremula whole-genome resequenced data were generated for downstream analysis. The genomes of the two aspen and P. trichocarpa are highly conserved  was 2. Further population sub-structuring was observed in P. davidiana and P. tremula. Eighteen different models were formulated to simulate the past population histories of P. davidiana and P. tremula that differed in terms of: (A) AsymmetricMigration without population expansion; (B) NoMigration without population expansion; (C) AsymmetricMigration with population expansion; (D) complex model, including a bottleneck in N; (E) complex model, including a bottleneck in S  (bootstrap range [BR]: 3.58–3.65 Mya). The current effective population sizes (Ne) of P. tremula (Ne-P. tremula) and P. davidiana (Ne-davidianaP. ) are 905,400  and 1,893,583 , respectively. The effective population sizes of the two species are all significantly higher than their common ancestor . The migration rate (m) is also clear among the two species, the lowest generation migration rate (m) between P. davidiana and P. tremula (4.43 × 10−8 and 2.52 × 10−7), not an accident due to the large geographical distance between the two populations.A coalescent simulation-based method was employed to infer demographic histories of Ne) over historical time was also evaluated in the P. davidiana and P. tremula populations. Higher resolution of recent population size changes is expected when more haplotypes are used , randomly distributed throughout the genome, these outlier windows possibly affected by natural selection.Comparing the empirical distribution of inter-specific ve sweep  and deteP. davidiana population was the center of adaptability and diversification. Throughout the genome, we observed that the genetic diversity parameters π, θW, and HE of the P. davidiana population were highest, and the P. tremula population had the lowest genetic diversity  also varied markedly; specifically, the average distance over which LD decayed to half of its maximum value. The STF is a relative measure of differentiation and is thus sensitive to any processes that alter intra-species genetic variation, we quantified and compared inter-specific genetic differentiation between two unions of outlier windows and the rest of the genome using several additional approaches  . The divergence time frame of P. tremula and P. davidiana was also in accordance with the rapid uplift of Qinghai–Tibet Plateau (QTP) around the Miocene/Pliocene boundary   could have fragmented the distribution of the ancestral population of P. davidiana and P. tremula and driven the speciation divergence. Geographical isolation had impeded gene flow between the populations . This demographic is consistent with many other forest trees in Eurasia . Moreovetrongest .STF value would be expected to be high in those regions with a low recombination rate if natural selection was the principal evolutionary factor for genetic differentiation of the populations of the two species (STF) and absolute divergence (dxy) will be higher, depending on intraspecific genetic diversity in areas with lower rates of recombination (STF and recombination rate (ρ) in both P. davidiana and P. tremula populations. As a consequence, our results highlight that linked selection and ρ were important factors of genomic differentiation between P. davidiana and P. tremula populations  in both late FST . The morlate FST . A more late FST . Therefo process . HoweverP. davidiana and P. tremula populations was significantly high. In comparison to purifying and positive selection, long-term balancing selection favors the maintenance of advantageous polymorphisms for many generations, which instead result in genomic regions with elevated genetic diversity and reduced STF , which was in accordance with the rapid uplift of Qinghai–Tibet Plateau (QTP) around the Miocene/Pliocene boundary. The two species experienced a considerable long-term bottleneck after divergence, with population expansion beginning approximately 20,000 years ago after the end of the last glacial maximum. Coalescent simulations suggest that stochastic genetic drift and historical demographic processes can largely explain the genome-wide patterns of differentiation between species. However, there is an excess of regions displaying extreme inter-specific genetic differentiation in the observed data compared with demographic simulations. We infer that heterogeneous genomic divergence is strongly driven by linked selection and variation in recombination rate in the two species. Instead of being clustered into a few large genomic “islands” as is expected under a model of speciation with gene flow, regions of pronounced differentiation are characterized by multiple signatures of positive selection in both species, and are distributed throughout the genome at many small, independent locations. Regions displaying exceptionally low differentiation are likely candidate targets of long-term balancing selection. Our research highlights that more information need to be integrated into future work when interpreting genomic variation during speciation. These include strict neutral theory, demographic fluctuations, genetic drift, geographical isolation, gene flow, sources of adaptation, positive selection (advantageous mutations) and purifying selection (deleterious mutations).Here we provide insights into the speciation and recent evolutionary histories of two closely related forest tree species, http://bigd.big.ac.cn/gsa, CRA001674 and CRA001683.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: ZH performed the experiments and wrote the study. AL designed the research.Financial support for this research was provided by the Fundamental Research Funds of China West Normal University (Grant No. 19E044) and the Open Fund of State Key Laboratory of Tree Genetics and Breeding (Chinese Academy of Forestry) (Grant No. TGB2019002).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.