Hemiculter leucisculus  is a small, widespread, and native cyprinid fish with prominent habitat suitability and high invasive potential and is becoming the dominant species in freshwater ecosystems under intensified environmental disturbances. But how H. leucisculus acclimates to extremely heterogeneous environments remains unclear. In current study, the genetic structure of H. leucisculus was analyzed using Bayesian phylogenetic inference, haplotype network, and STRUCTURE base on cytb gene across 18 populations spanning 20 degrees of latitude and 18 degrees of longitude in China. The morphological diversification of body size and shape for H. leucisculus along the climate gradient was studied. The results showed that the 18 H. leucisculus populations were divided into 3 clusters: one cluster mainly from Huanghe River Basin, another cluster mainly from Yangzi River Basin, and H cluster containing Hainan and Beihai populations. The fish from southern populations were deeper bodied while individuals from northern populations were more slender. Inland individuals were more streamlined while coastal individuals were of deeper body. The partial Mantel test predicts that the potential mechanism underlining the intraspecies morphological diversification along climate gradients is primarily the divergent selection pressures among different environments, while genetic variation had less contribution to morphological differentiation. The formation of the Nanling Mountain Range could drive genetic differentiation between Beihai population and those from Yangzi River Basin. The present results highlight strong selective pressures of climate on widespread species and enrich morphological differentiation basis of acclimation for species with high habitat suitability and invasive potential.Sharpbelly  Hemiculter leucisculus has prominent habitat suitability and high invasive potential. This manuscript shows divergent selection pressures among different environments shape morphological diversification for sharpbelly. It predicts that divergent selective pressures are crucial in determining body shape in sharpbelly, while genetic variation has less contribution to morphological differentiation.Previous studies indicate  They were reported to be distributed across China, Vietnam, the Korea Peninsula, Mongolia, and the Fareast of Russia  from 18 locations . The data included mean annual temperatures, maximum temperatures of the warmest month, minimum temperatures of the coldest month, annual temperature differences (by subtracting the minimum monthly temperature from the maximum monthly temperature), and annual precipitation to provide site‐specific climatic information (Table http://earth.google.com/)  Table . Those v2.2cytb) was used as molecular marker  and 5′ CTCCGATCTCCGGA TTACAAGAC 3′ (reverse). The PCR amplification was performed in a final volume of 50 µl reaction mix with 25 µl 2 × Taq MasterMix , 4 µl primer mix, 8 µl template DNA, and RNase‐free water. Thermocycling condition was as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 58° for 30 s, 72°C for 30 s, and a final elongation step at 72°C for 10 min. The PCR products were isolated via gel electrophoresis and purified with the EasyPure Quick Gel Extraction Kit  and then sequenced by Tsingke Corporation .The total genomic DNA was extracted from pectoral fin tissue of specimens preserved in 95% ethanol using the EasyPure Genomic DNA Kit . The mitochondrial cytochrome b gene . Photographs were transformed to tps format using tpsUtil v. 1.75. Total 17 landmarks  to varying numbers of genetically distinct clusters (K). For each value of K = 1–10, ten independent iterations were run using the assumed admixture model with a burn‐in period of 250,000 generations, followed by a sampling phase of 750,000 Markov Chain Monte Carlo (MCMC) iterations. The uppermost level of population differentiation was detected using the web‐based tool STRUCTURE HARVESTER v. 0.6.94  and three sequences of H. bleekeri sampled at Changshu, Ankang, and Zhenjiang in Yangzi River Basin  were used as outgroups. Thirteen additional cytb sequences of H. leucisculus randomly picked from GenBank  of MrBayes v. 3.2.2  was conducted in Arlequin 3.5  was used in subsequent analysis. ANCOVA was performed with Log10CS as dependent variables and the two climate‐related PCs  as covariates. In addition, the interaction terms of both cPCs were treated as covariates. To evaluate the relative importance of each term, the effective size was estimated by calculating Wilk's partial eta squared (ηp2). To visualize significant interaction effects, the data were divided into inland (cPC2 ≥ median) and coastal populations (cPC2 < median) to depict variation along cPC1 . The data were also split based on median value of cPC1 to depict the variation along cPC2.Body size is represented by centroid size (CS), the square root of the summed squared distances from each landmark to the configuration centroid, and calculated using MorphoJ v. 1.06d. Logηp2 was calculated to estimate the effect sizes. All statistical analyses unless mentioned otherwise were performed using SPSS 20.0.To remove the effect of allometry, the residuals were held as morphology‐related PCs (mPCs) by regressing RWs against centroid size in MorphoJ v. 1.06d. To assess the extent of divergence along climatic gradients, multivariate analysis of covariance (MANCOVA) was employed with mPCs as dependent variables and the two cPCs and the interaction terms of both cPCs as covariates. To identify the sources of variation in case of significant model terms, post hoc ANCOVAs of the exact same structure were run as the final retained MANCOVA model. Wilk's H. leucisculus populations were calculated using MorphoJ v. 1.06d. The pairwise climate Mahalanobis distances between populations were based on cPC1 and cPC2 using StatMatch package in R  and Hainan (HN) populations. Seven populations containing Ankang (AK), Kaifeng (KF), Kunming (KM), Nantong (NT), Qiantangjiang (QT), Taihu (TH), and Tongling (TL) formed the second cluster. The remaining nine populations including Chongqing (CQ), Datong (DT), Luoyang (LY), Qujing (QJ), Qianxian (QX), Taigu (TG), Wuhan (WH), Xi'an (XA), and Zoucheng (ZC) composed the third cluster  to 0.96023 . There were varying degrees of genetic differentiation between populations, ranging from virtual panmixis , moderate genetic differentiation , higher genetic differentiation  to very high genetic differentiation .As shown in Table STF was 0.78731(Table p < .001), 23.86% of variation was accounted for among populations within groups, and 21.27% was accounted for within populations, suggesting that H. leucisculus populations were highly structured geographically.AMOVA showed the overall 3.2ηp2 magnitude, the interaction of cPC1 and cPC2 showed the strongest effect (ηp2 = 0.206), followed by cPC2 (ηp2 = 0.132) and cPC1 (ηp2 = 0.069). For the effects of interaction of cPC1 and cPC2, along cPC2, the fish in more southern sites  were getting bigger from coastal to inland  on body shape, followed by cPC1 (ηp2 = 0.205) and the interaction of cPC1 and cPC2 (ηp2 = 0.134). The post hoc ANCOVA data showed the main effects of 2 cPCs and interaction effects of them on every single mPCs , suggesting that the inland fish were longer and had more posteriorly positioned dorsal fins compared with coastal populations , indicating inland fish were slightly slender compared with coastal fish  suggests that inland fish tend to have marginally longer anal fin and larger eye than coastal fish . In northern populations (cPC1 < median), the inland fish develop more posteriorly positioned dorsal fins and shorter caudal peduncles compared with coastal fish . In spite of the significant correlation between pairwise morphological distances and FSTs estimated by Mantel test , the weighted evaluation result of partial Mantel test showed that the correlation was not significant  from Hainan sampling site in Changhuajiang River and Beihai sampling site in Nanliujiang River Basin. The STRUCTURE data also support the grouping of Beihai and Hainan populations, which reflects there exist potential barriers to gene flow between this cluster and the remaining populations. All the current data suggest the high genetic differentiation between the H cluster and the others. It could indicate ongoing speciation in Hemiculter genus in Nanliujiang River Basin and Hainan Island.In this study, both the BI tree and haplotype network based on Prochilodus lineatus, a freshwater fish widely distributed in the major rivers of South America, explained some geological and geographical events occurred millions of years ago , located at Nanliujiang River drainage, and populations from Yangtze River Basin, presumedly suggests the discrepant fish fauna between Nanliujiang River Basin and Yangzi River Basin. However, in both sides of Alps, the gene flow of bullheads Cotttus ferrugineus was unusually not impeded by this geographic barrier .The cluster H consists of HN population from Changhuajiang River Basin at Hainan Island and BH population from Beihai located in Nanliujiang River drainage. Although separated by Qiongzhou Strait, the two populations share fairly high genetic homogeneity. In addition, the NJ morphological tree also showed the two populations got together. According to Zhao and Huang , most ofH. leucisculus morphology and latitude  indicates inland fish are deeper bodied compared with coastal populations. The stagnant and/or low‐flow‐velocity habitats could account for the deeper bodied shape for fish from the populations of Kunming and Qujing, as fish from Kunming population resided in Dianchi Lake and Qujing population resided in a small pond.The correlation with cPC2 and morphology‐related PCs (mPC5 and mPC7) suggests that individuals from inland populations are more streamlined while fish of coastal populations are of deeper body. It is concurrent with fishes in the Pearl River, in which high‐elevation individuals were more narrow‐bodied ; data curation (lead); formal analysis ; investigation ; methodology (lead); project administration (lead); resources (lead); software (lead); validation (lead); visualization ; writing–original draft (lead); writing–review and editing . Long Zhu: Formal analysis ; investigation ; methodology ; resources . Kui Tang: Data curation ; investigation ; resources ; software ; validation . Mengyu Liu: Formal analysis ; investigation ; resources ; validation . Xue Xue: Data curation ; resources ; software . Gaoxue Wang: Conceptualization ; formal analysis ; investigation ; project administration ; resources ; supervision ; writing–original draft ; writing–review and editing . Zaizhao Wang: Conceptualization (lead); formal analysis (lead); funding acquisition (lead); investigation ; resources (lead); supervision (lead); writing–original draft ; writing–review and editing (lead).Table S1‐S6Click here for additional data file.


A Korean synthetic pig breed, Woori-Heukdon , was developed by crossing parental breeds (Korean native pig [KNP] and Korean Duroc [DUC]) with their crossbred populations (F1 and F2). This study in genome-wide assessed a total of 2,074 pigs which include the crossbred and the parental populations using the Illumina PorcineSNP60 BeadChip. After quality control of the initial datasets, we performed population structure, genetic diversity, and runs of homozygosity (ROH) analyses. Population structure analyses showed that crossbred populations were genetically influenced by the parental breeds according to their generation stage in the crossbreeding scheme. Moreover, principal component analysis showed the dispersed cluster of WRH, which might reflect introducing a new breeding group into the previous one. Expected heterozygosity values, which were used to assess genetic diversity, were .365, .349, .336, .330, and .211 for WRH, F2, F1, DUC, and KNP, respectively. The inbreeding coefficient based on ROH was the highest in KNP (.409), followed by WRH (.186), DUC (.178), F2 (.107), and F1 (.035). Moreover, the frequency of short ROH decreased according to the crossing stage (from F1 to WRH). Alternatively, the frequency of medium and long ROH increased, which indicated recent inbreeding in F2 and WRH. Furthermore, gene annotation of the ROH islands in WRH that might be inherited from their parental breeds revealed several interesting candidate genes that may be associated with adaptation, meat quality, production, and reproduction traits in pigs. In the swine industry, crossbreeding has been widely used to exploit the phenomenon of heterosis, or hybrid vigor. The main benefit of the heterosis is increased performance of the resulting crossbred offspring over the average performance of its purebred parent pigs in traits of interest . AlthougIn recent years, the South Korea swine industry has been partly changing from emphasis on efficiently producing more pork, mostly by focusing on growth and reproduction, to addressing taste-oriented consumption. There has been consistent consumer demand in Korea for diversified and great-tasting pork, despite its higher price than the typical market pork that is mostly derived from the LYD system . In respRuns of homozygosity (ROH) are contiguous homozygous stretches that are inherited from each parent. Although ROHs are known to arise from several genetic factors, including genetic drift and population bottlenecks, it has been used as an indicator of selection signatures throughout the genome. In fact, ROHs have been widely used to quantify autozygosity in pigs ; becauseCurrently, there is still a lack of studies to elucidate genomic characteristics of the composite breeds that were generated by using the indigenous pigs. The main objective of this study were: 1) to estimate various parameters to clarify genomic population structure of the crossbreds with DUC and KNP populations; and 2) to detect and investigate ROH that could be an indicative of selection signature in WRH population.Sus scrofa (Sscrofa10.2 to Sscrofa11.1) according to manifest file of Illumina PorcineSNP60 v2 using PLINK v1.9  were used in this study, including DUC, KNP, and their crossbred F1 (DUC × KNP), F2 (F1 × DUC), and F3  populations. The dataset includes previously published genotype data  that conINK v1.9 .The quality control (QC) process for the genotype dataset was conducted separately for SNPs and animals using PLINK v1.9. We applied three different QC procedures for the initial raw SNP dataset to further analyze genetic diversity, population structure, and ROH. All three genotype subsets used the following process: 1) SNPs in sex chromosomes or unmapped in Sscrofa11.1; 2) SNPs with a call rate less than 90%. Individuals with a call rate less than 90% were removed. The ROH analysis was conducted using a subset of data without additional QC process. To assess genetic diversity, we also removed SNPs with a minor allele frequency (MAF) less than .05. Furthermore, SNP filtering based on pairwise linkage disequilibrium (LD) was conducted to minimize the reduction of informativeness of the dataset  using thO), expected heterozygosity (HE), and individual inbreeding coefficient (FHOM) using PLINK v1.9. The inbreeding coefficient was calculated based on homozygous genotypes as follows:To assess genetic diversity within populations, we calculated observed heterozygosity . The PCA plot was also considered a QC process because it reveals potential misclassification.To explore the pattern of genetic differentiation of samples, we conducted principal component analysis (PCA) using PLINK v1.9, and the first two principal components (PCs) were visualized using R v4.1.0  provided by p-value >.999) were used to form ROH islands (ROH = 41%), the threshold was set to 80% because SNPs with p-values higher than .999 were not found in this population, as described by ROH islands, which were defined as regions where SNPs in ROHs had  islands . FurtherUsing discovered ROHs, we calculated the inbreeding coefficient based on ROH using the following calculation proposed by https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/003/025/GCF_000003025.6_Sscrofa11.1/GCF_000003025.6_Sscrofa11.1_genomic.gff.gz, accessed on 15 June 2021). Furthermore, we also used pig quantitative trait locus information for ROH islands to annotate potential traits associated with selected regions .Gene annotation for ROH islands was conducted according to NCBI Sus scrofa Release 106 (The SNP coordinates for each chromosome (SSC) were updated from Sscrofa10.2 to Sscrofa11.1 according to manifest file provided from Illumina . After iThe top three PCs  explained approximately 39.3, 15.8, and 5.7% of total variation, respectively. As shown in O value (.477 ± .297), followed by WRH (.407 ± .164), F2 (.380 ± .163), DUC (.331 ± .160), and KNP (.223 ± .216). The average HE value was highest in WRH (.365 ± .133) and lowest in KNP (.211 ± .202). The inbreeding coefficient values (FHOM) were shown to have negative values in all five populations, exhibiting the highest value in DUC (−.002 ± .053), followed by WRH (−.024 ± .083), KNP (−.055 ± .104), F2 (−.115 ± .048), and F1 (−.417 ± .019).Genetic diversity estimates using a subset of 43,809 SNPs for 2,040 individuals are summarized in The mean number and size of discovered ROH per pig within each population are described in ROH), which was defined by ROH value was observed in KNP (0.409) followed by WRH (0.186), DUC (0.178), F2 (0.107), and F1 (0.035), which is the same order as mean ROH length. The correlation between FHOM and FROH for each population was high in most populations, including DUC (0.84), KNP (0.83), F2 (0.86), and WRH (0.88); however, F1 had a correlation of 0.10   . Therefohan FHOM .p-values higher than a threshold for each population, were observed for parental (DUC and KNP) and crossbred  populations. We identified a total of 365 ROH islands of 1-Mb bins throughout the autosomal regions , we conducted PCA and ADMIXTURE analyses . The mosanalyses . For theanalyses . In partanalyses  and cattanalyses . The ADManalyses . The genanalyses . At K = analyses . This stHowever, WRH was shown to have a somewhat dispersed cluster in PCA . This coROH value (.409)  .In ROH analyses, we found a clear difference in ROH patterns between parental breeds and their crossbred populations (F1 and F2). Among parental breeds, DUC had 24.4% short and 12.7% long ROH segments . AdditioThe discovery of ROH islands revealed numerous homozygous regions over five pig populations used in this study . FurtherCRTAM) gene. This gene was reported to have an association with adaptive immune response in cattle  of a ROH island, we found the Heat Shock Protein Family A Member 8 (HSPA8) gene, which is also known as Hsp70. HSPA8 is known to be associated with pork tenderness because this gene was down-regulated in tender samples  harboring the Cytotoxic And Regulatory T Cell Molecule (n cattle . As KNP  samples .ADAMTSL3) gene at 52–53 Mb on SSC7. ADAMTSL3 is a member of the ADAMTS superfamily of proteins, and this gene was previously reported as a candidate gene for body length in Large White pigs (CPEB1) gene at 52–53 Mb on SSC7. The CPEB1 is an RNA-binding protein that regulates mRNA translation by controlling the poly(A) tail length  between WRH and founder breeds, suggesting inheritance of homozygous region that might be potential signatures of selection.This study has shown genomic characteristics of crossbred pig populations derived from Korean Duroc and Korean native pigs as founder breeds. Population structure analysis showed genetic influence of founder breeds to crossbred populations. We also observed that WRH had two distinct subgroups due to newly introduced breeding group. For crossbred populations, the genetic diversity was gradually increased according to their crossbreeding stage . In ROH analyses, short ROHs were decreased, while medium and long ROHs were increased from F1 to WRH, suggesting that recent inbreeding is ongoing in WRH. In this study, there is a partial limitation to conclude on F1 due to its small samples size (N = 11). Furthermore, we identified shared ROH islands which contain candidate genes (


Ducetia japonica is one of the most widespread katydids in China, but little is known about its genetic structure and phylogeographic distribution. We combined the five‐prime region of cytochrome c oxidase subunit I (COI‐5P), 11 newly developed microsatellite loci coupled with an ecological niche model (ENM) to examine the genetic diversity and population structure of D. japonica in China and beyond to Laos and Singapore. Both Bayesian inference (BI) and haplotype network methods revealed six mitochondrial COI‐5P lineages. The distribution of COI‐5P haplotypes may not demonstrate significant phylogeographic structure . The STRUCTURE analysis based on microsatellite data also revealed six genetic clusters, but discordant with those obtained from COI‐5P haplotypes. For both COI‐5P and microsatellite data, Mantel tests revealed a significant positive correlation between geographic and genetic distances in mainland China. Bayesian skyline plot (BSP) analyses indicated that the population size of D. japonica's three major mitochondrial COI‐5P lineages were seemingly not affected by last glacial maximum . The ecological niche models showed that the current distribution of D. japonica was similar to the species’ distribution during the LGM period and only slightly extended in northern China. Further phylogeographic studies based on more extensive sampling are needed to identify specific locations of glacial refugia in northern China.Habitat fragmentation can lower migration rates and genetic connectivity among remaining populations of native species.  We investigated genetic diversity and population structure of D. japonica in China using the DNA barcoding fragment (COI‐5P) and 11 newly developed microsatellite loci. Three major lineages were nearly at a stable population size during LGM, and it does not appear that any lineages were adversely affected by climatic changes. The popTo date, phylogeographic analyses have been conducted in many insects  have been used for phylogeographic studies , West (India), and South   determine the phylogeographic structure and possible influential factors; (b) study the demographic history and gene flow among populations; and (c) identify the locations of putative refugia.While phylogeographic structure of many vertebrates has been examined in China  according to the manufacturer's protocol. PCR was performed using Premix Taq™ . Mitochondrial COI‐5P was amplified and sequenced using the universal primers LCO1490/HCO2198 , and 9 μl Hi‐DiTM formamide (Thermo Fisher Scientific). Microsatellite alleles were genotyped by size using GeneMapper 4.0 (ABI) and checked manually to reduce scoring error.We developed microsatellite loci specifically for 2.32.3.1A), observed heterozygosity (HO), expected heterozygosity (HE), and fixation index (FIS) for each locus and population. Polymorphism information content (PIC) was calculated in CERVUS v3.0. Genotypic linkage disequilibrium (LD) among all pairs of loci across all populations and Hardy–Weinberg equilibrium (HWE) tests for per locus and over all loci in each population were performed using GENEPOP 4.7 , haplotype diversity (h), and nucleotide diversity (π), were calculated using DnaSP 5.10  were all implemented in Arlequin 3.5  range from 0 to 1, where “0” indicates complete panmixia and “1” suggests a lack of migration  using SAMOVA 2.0   and population differentiation (2.3.2Kuwayamaea brachyptera (accession number HQ609356) was used as out‐group. Stationarity was considered to be reached when the average standard deviation of split frequencies was below 0.01. The initial 25% of trees were discarded as burn‐in, and the remaining trees were used to construct the Bayesian majority‐rule consensus tree. Haplotype networks were derived from PopArt 1.7 using MJ algorithm  significantly higher than population differentiation (GST). As PERMUT software needed at least three individuals per population, we excluded the populations with sample size less than 5 individuals.Mitochondrial COI‐5P sequences were edited with Seqman and EditSeq  and aligned with MUSCLE Edgar,  as impleSD = 0.004. The BEAST analysis was run 100 million generations with HKY + G + I model, strict molecular clock, random starting tree, the Yule process, and sampling every 10,000 steps. The first 10% of the trees were discarded as burn‐in. The maximum clade credibility tree was produced in TreeAnnotator v1.10.4.Divergence times were estimated using COI‐5P haplotype dataset with BEAST v1.10.4   were used to evaluate the population size dynamics of 2.3.4K method. The 20 replicates for the chosen K‐value were merged using CLUMPP 1.1.2  and the shortest geographic distance for both mitochondrial COI‐5P and microsatellite data. The Mantel tests were limited to the populations in mainland China, and excluded HNC (pops. 64–68), TWC (pops. 69–71), XZC (pop. 79), Laos (pop. 80), and Singapore (pop. 81), to avoid the influence of geographic barriers, such as Taiwan Strait, Qiongzhou Strait, and Tibetan Plateau. Geographic distances were calculated based on the latitude and longitude of sampling sites.To test for isolation by distance, Mantel tests were implemented with 1,000 permutations in Arlequin 3.5 to estimate correlation between genetic differentiation  at 2.5 arc‐min resolution. In addition to our sampling sites, 41 species’ distribution records of D. japonica were obtained from the literature  with low‐explanatory power was removed based on its relative contribution to the Maxent model. Finally, 8 environmental variables were used to predict distribution areas of D. japonica in China: annual mean temperature (bio1), mean diurnal range (bio2), isothermality (bio3), temperature seasonality (bio4), precipitation of wettest month (bio13), precipitation of driest month (bio14), precipitation seasonality (bio15), and precipitation of coldest quarter (bio19). Model validation was performed using the same settings. The accuracy of each model prediction was evaluated using AUC scores, where range from 0.5 (randomness) to 1 (exact match), and above 0.7 and 0.9 indicates good and excellent predictive power, respectively  displayed the widest geographic distribution, which was seen among samples originating in 23 populations from 4 groups. Meanwhile, H3 has more descendant haplotypes which also suggest it is most likely to be the ancestral haplotype  was seen in 8 populations from NEC and NC groups. The third most frequent haplotype, H12, was shared by 50 samples and occurred in 3 populations from NC and CC regions.We obtained 796 mitochondrial COI‐5P sequences that were each 658 bp long. None of COI‐5P sequences exhibited characteristics of nuclear pseudogenes (frame shifts or premature stop codons). We obtained 159 COI‐5P haplotypes (herein designated as H1‐H159) based on 138 polymorphic sites. Among 159 COI‐5P haplotypes, 12 haplotypes  were observed in more than one group, 22 haplotypes  were observed in different populations within groups, and 125 haplotypes were private to a single population  Table . HaplotyBI analysis revealed six mitochondrial COI‐5P lineages Figure . The COID. japonica COI‐5P haplotypes. The primary divergence within D. japonica commenced 1.61 Mya (HPD: 0.9–2.8 Mya) when two haplotypes (H117 and H119) from DDH population split from the rest. Lineage I split from the other populations around 0.96 Mya (HPD: 0.55–1.65 Mya). The split between lineage II and lineage III was dated to 0.76 Mya (HPD: 0.42–1.29 Mya)  Figure .D. japonica mitochondrial COI‐5P haplotype (Hd) and nucleotide diversities (π) were 0.931 and 0.01391, respectively. The haplotype (Hd) and nucleotide diversities (π) of 4 populations  were zero for lack of variable sites, and the low diversity may be irrelevant to sample size . For the remaining populations, Hd ranged from 0.069 (WMAS) to 0.955 (CYA) and π ranged from 0.0001 (WMAS) to 0.0233 (DDH).The overall The neutrality tests indicated few signs of demographic events in most of the populations, except for SDYT and ZQLF, which showed significant negative values in both tests Table . Four pop < .001) and lineage II (p < .01) was significantly negative, whereas lineage III was negative but not significant. Fu's Fs test was significantly negative values (p < .001) for all lineages. Significantly negative values of Fu's Fs and Tajima's D tests support an excessive number of rare haplotypes as a result of a recent population expansion. The unimodal mismatch distribution pattern suggested that three lineages underwent recent expansions  to 67 alleles (IAK5J) (Table E) ranged from 7.516 (JM9VE) to 27.742 (IAK5J). Shannon's information index (I) values across loci ranged from 2.111 (IIM2H) to 3.615 (IAK5J). Observed heterozygosity (HO) ranged from 0.449 (IB7C3) to 0.712 (H87V5), and expected heterozygosity (HE) and unbiased expected heterozygosity (uHe) values ranged from 0.732 (IIM2H) to 0.964 (IAK5J) and 0.733 (IIM2H) to 0.965 (IAK5J), respectively. Fixation index (FIS) ranged from 0.253 (H87V5) to 0.494 (IB7C3). These ranges suggest differential mutation rates among loci.Eleven microsatellite loci were genotyped for 610 A and NE across all loci were highest in DJH (15.364 and 9.445) and lowest in the GXME population (4.909 and 3.453). The highest and lowest Shannon's information index (I) was presented in DJH (2.369) and GXME (1.24), respectively. The mean HO, HE, and uHE across all loci were highest in JXJG (0.818), DJH (0.873), DDH (0.9), and lowest in XYS (0.384), QWZS (0.58), and QWZS (0.619), respectively.Mean NK = 2–15 , and K = 6 (ΔK = 3.408) . STRUCTURE tends to detect the uppermost level of hierarchical structure, which may explain why K = 2 was frequently reported, despite our suspicions of additional underlying structure  were assigned to two inferred clusters (Q > 0.80). Most individuals of SC, SWC, and XZC regions were inferred to be a mixture of two genetic components. Cluster I comprised samples of 3 populations in Jilin and LNDD population in Liaoning Province. Cluster II comprised samples of the remaining 2 populations in Liaoning, and BJHD population in Beijing and 2 populations in Hebei Province. Cluster III comprised samples of YLB population in Henan, SDPL and SDZY population in Shandong, JSQS population in Shanxi, HMH  and HJS population in Shanghai, and CDJY population in Sichuan Provinces. Cluster IV comprised samples of SDYT and SDJN population in Shandong, JSLF population in Shanxi, and SSYL population in Shaanxi Provinces. Cluster V comprised samples of WMAS population in Anhui, all populations in Zhejiang, CYA and CEMS populations in Sichuan Provinces, and XZMT population in Xizang. Cluster VI comprised samples of WGNJ population in Anhui, HMH  population in Shanghai, and all populations in Hunan, Jiangxi, Fujian, Guangxi, Yunnan and Hainan Provinces.The STRUCTURE analysis based on microsatellite data indicated that the mean estimated logarithm of probability of the data, L (K) value increased gradually from 5 Figure . Plots o) Figure . At K = ) Figure , the ind3.3HS = 0.548 ± 0.043) was significantly lower than total gene diversity (HT = 0.961 ± 0.014). Although NST (0.448 ± 0.051) > GST (0.430 ± 0.042), but not significant (p >.05). Therefore, COI‐5P haplotype distribution may not demonstrate significant phylogeographic structure.In this study, the maximum K2P distance of COI‐5P sequences is 1.41%. The mean gene diversity  were delimited. Group I included all populations of NEC, NC, and TWC, and 12 populations  of CC. Group II only included XZMT population from XZC alone. Group III included 23 populations  of CC and 4 populations  of SWC. Group IV included all populations of SC, HNC, Laos, Singapore, and GZGY population of CC, and 3 populations  of SWC.The SAMOVA based on mitochondrial COI‐5P dataset showed that Fp < .001).Results of the AMOVA test on mitochondrial COI‐5P and microsatellite data in different populations and regional groups are shown in Table p < .05, 10,100 replicates) for the majority of pairwise comparisons. Pairwise FST between populations based on mitochondrial COI‐5P (Kimura‐2P) and microsatellite data  ranged from 0 to 1 and from 0.013 to 0.311, respectively  within NEC regions, and the FST values with the remaining 2 populations (LNDD and LNSY) were higher based on microsatellite data. Based on mitochondrial COI‐5P, lower FST occurs only among JLTH, JLJL, and LNFC, and the FST values exceeded 0.8 between them and JLLJ. Furthermore, lower FST often was observed between geographically adjacent populations, for example, HBBD and HBSP , XYS and XGZ , HMH and HJS , and CYA and CDJY . Our results indicated a low or moderate gene flow, as observed by the high and significant FST values. The Singapore lineage is much more divergent from the others; FST ranged from 0.429 to 0.997 for mitochondrial COI‐5P, and FST ranged from 0.117 to 0.364 for microsatellite data.In both mitochondrial COI‐5P and microsatellite data, population differentiation was significant (ly Table . There wFST between the two nearest populations (GZSY and GZKS) was 0.23881 for microsatellites and 0.932 for COI‐5P. Mantel tests revealed a significant correlation between genetic differentiation and geographic distance for both mitochondrial COI‐5P  and microsatellites .The geographic distance among populations ranged from 2.6 km (GZSY vs. GZKS) to 3,781.94 km (JLJL vs. DDH) in mainland China. Pairwise 3.4D. japonica is mainly located in the east of Heihe–Tengchong population density line, especially Northern and Central China. The distribution predictions under current climatic conditions were largely congruent with the actual distribution of D. japonica. The predicted current distribution of D. japonica was similar to that of the LGM period and only slightly extended in Northern China  were unique to a population, which also implies high level genetic differentiation among D. japonica populations. Several singletons stemmed from haplotype H2, H3, H28, H63, and H83 separated by a single substitution. This star‐like topology is often associated with recent demographic expansions . The high nucleotide diversity (π = 0.023) of DDH population can be explained by the existence of H117 and H119. High Hd and low π were observed in most populations, which probably resulted from rapid population growth of ancestral populations  and Pakistan to Solomon Islands   and lineage III (0.05 Mya) expansion may have occurred earlier than the LGM. Three major lineages were nearly at a stable population size during LGM, and it does not appear that any lineages were adversely affected by climatic changes. High levels of lineage diversity and signals of greater demographic stability suggest a lower impact of glaciations  were significantly correlated with meteorological factors and its host plants  and larger population sample sizes could potentially reveal more fine‐scaled patterns. In summary, our results indicated that D. japonica in mainland China remained almost stable over long periods of time. Although our present study provides some insight for the D. japonica genetic patterns, the study for D. japonica does not stop here. Further phylogeographic studies based on more extensive sampling would be needed to identify specific locations of glacial refugia.Given that the fossil record for The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential competing interests.ZZJ: Conception of the ideas. ZYX, GB, ML, and WWJ: Conduction of the fieldwork and collection of the data. ZZJ, ZYX, and GB: Analysis of the data. ZZJ and ZYX: Preparation of the manuscript.Fig S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.


The population prehistory of Xinjiang has been a hot topic among geneticists, linguists, and archaeologists. Current ancient DNA studies in Xinjiang exclusively suggest an admixture model for the populations in Xinjiang since the early Bronze Age. However, almost all of these studies focused on the northern and eastern parts of Xinjiang; the prehistoric demographic processes that occurred in western Xinjiang have been seldomly reported. By analyzing complete mitochondrial sequences from the Xiabandi (XBD) cemetery , the up-to-date earliest cemetery excavated in western Xinjiang, we show that all the XBD mitochondrial sequences fall within two different West Eurasian mitochondrial DNA (mtDNA) pools, indicating that the migrants into western Xinjiang from west Eurasians were a consequence of the early expansion of the middle and late Bronze Age steppe pastoralists (Steppe_MLBA), admixed with the indigenous populations from Central Asia. Our study provides genetic links for an early existence of the Indo-Iranian language in southwestern Xinjiang and suggests that the existence of Andronovo culture in western Xinjiang involved not only the dispersal of ideas but also population movement. One of Located at the intersection of the ancient “Silk Road,” Xinjiang has played an important role in bridging the exchanges of cultures, goods, languages, and population movements . A recenHowever, all those ancient DNA studies of Xinjiang were confined to the northern and eastern parts of this region. Considering the large geographic range and diverse ecosystems of Xinjiang, such studies in western Xinjiang are in great need to gain a more comprehensive understanding of the prehistoric demography of Xinjiang populations. In recent decades, a number of cultural remains and archaeological sites in western Xinjiang, showing the traits that are characteristic of the middle and late Bronze Age Eurasian Steppe (Steppe_MLBA) cultures  , were inIn this study, we collected 15 ancient samples from the XBD cemetery, the earliest archaeological site excavated in western Xinjiang to the best of our knowledge. We then enriched and sequenced the complete mitochondrial genomes of the XBD individuals through designed target probes. By comparing the mitochondrial DNA (mtDNA) of the XBD individuals with that of ancient and extant Eurasians, we explored the early population movement in western Xinjiang.The XBD cemetery is located in the westernmost region of Xinjiang, adjacent to the eastern edge of the Pamir Plateau . This reTM kit . Sequencing was carried out on an Illumina HiSeq 2000 platform at Novogene Inc. .DNA was extracted from teeth powder (∼50 mg) with the method previously described . The lib-l 2000). The duplicate reads were removed by the DeDup v0.12.1  based on the frequencies of sub-haplogroups was performed with the R libraries “factoextra”, “FactoMineR”, and “ggplot2”.The coalescence time of each lineage was estimated using the ρ statistic-based method and the maximum likelihood (ML) method implemented in PAML software v4.9g  with theStrict contamination precautions for ancient DNA were taken, and wet lab works were carried out in a dedicated clean room facility specially designed for ancient DNA studies at Jilin University. All samples showed a short fragment length (55–90 base pairs) and postmortem chemical modifications at 3′ and 5′ ends that are expected for ancient DNA . The conWe obtained complete mitochondrial sequences to an average coverage of 187- to 4,130-fold across all 15 individuals sequenced in this study. A total of 14 different mitochondrial haplogroups belonging to five macro-haplogroups, such as U, H, T, R, and I, were observed . All theWe used 540 present-day mitochondrial sequences obtained from PhyloTree database  who wereThe prehistory of Xinjiang is of considerable interest given its special geographic location in connecting the East and the West Eurasians. Multiple genetic studies showed that since the Bronze Age, the populations in Xinjiang had exhibited high genetic diversity and extensive admixture with various populations . The admThe discovery of the Tocharian manuscript from the northern rim of the Tarim Basin and the Indo-Iranian manuscripts from the southern edge provides direct evidence for the dispersal of Indo-European languages into the region . It is nTaken together, the systematic mtDNA analysis on ancient samples from the westernmost part of Xinjiang provides us a unique opportunity to investigate the population origin of Xinjiang with a broader geography. We find that the 15 XBD individuals fall within the range of the ancient western Eurasian variation, and the formation of the ancestry legacy of XBD is related to the expansion of the middle and late Bronze Age steppe herders who might speak Indo-Iranian languages and admixed with the indigenous populations in the West or Central Asia during their expansion. Additionally, integrating the archaeological and genetic evidences in this study, the existence of the Andronovo culture in western Xinjiang involved not only the dispersal of ideas but also population movement. We recognize that such study on samples from a broader region and time sequences is required to obtain a more comprehensive understanding of the population prehistory of Xinjiang.HRA001154 (http://bigd.big.ac.cn/gsa-human).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: The BIG Data Center Genome Sequence Archive (GSA) under accession number YC, LJ, and SG conceived and supervised the study. CN, YZ, YX, and CL performed the laboratory work. YW and DW provided archaeological materials and associated information. CN, H-XZ, FZ, and SW analyzed the data. CN, YC, FZ, SG, and H-XZ wrote the manuscript with the input from all co-authors.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.


Anthemis cotula) is a globally invasive, troublesome annual weed but knowledge of its genetic diversity, population structure in invaded regions and invasion patterns remains unstudied. Therefore, germplasm from 19 A. cotula populations (sites) from three geographically distinct invaded regions: the Walla Walla Basin (located in southern Washington) and the Palouse (located in both northern Idaho and eastern Washington), Pacific Northwest, USA and Kashmir Valley, India were grown in the greenhouse for DNA extraction and sequencing. A total of 18 829 single-nucleotide polymorphisms were called and filtered for each of 89 samples. Pairwise FST, Nei’s genetic distance, heterozygosity, Wright’s inbreeding coefficient (F) and self-fertilization rates were estimated for populations within and among the three regions with a total of 19 populations comprised of 89 individuals. Overall measurements of genetic variation were low but significant among regions, populations and individuals. Despite the weak genetic structure, two main genetic clusters were evident, one comprised of populations from Palouse and Kashmir Valley, the other comprised of populations from the Walla Walla Basin. Significant selfing was observed in populations from the Walla Walla Basin and Palouse but not from Kashmir Valley, indicating that Mayweed chamomile in the Pacific Northwest, USA could persist with low pollinator or pollen donor densities. Although FST values between the regions indicate Palouse populations are more closely related to Kashmir Valley than to Walla Walla Basin populations, based on Migrate-n analysis, panmixis was the most likely model, suggesting an unrestricted gene flow among all three regions. Our study indicated that Kashmir Valley populations either originated from or shared the origin with the Palouse populations, suggesting human-mediated migration of A. cotula between regions.Mayweed chamomile ( Due to global trade of plant materials and human movement, alien species have increasingly invaded new places, displaced native species, negatively affected biodiversity and altered ecosystem structure and function (Anthemis cotula) is an annual, bushy, ill-scented weed that originated in the Mediterranean region and eventually became a global invader  that producers are beginning to adopt in response to the changes in climate  What is the genetic diversity and population structure of these A. cotula populations? and (ii) Is there gene flow among A. cotula populations across regions? Based on its biology and ecology, possible repeated intrapopulation crossing events, and likely migration patterns, we expected genetic diversity to be low and population structure to be weak in A. cotula populations.To address these gaps, we collected seeds from the 19 A. cotula populations (i.e. sampling sites or farms) were collected from three regions: the Palouse  and the Walla Walla/Tucannon Basin  in the Pacific Northwest, USA; and Kashmir Valley, India   filled with a commercial greenhouse soil mix  under a 15-h photoperiod of sunlight and supplemental artificial light (photosynthetic photon flux = 595 µmol m−2 s−1) with an average temperature of 22.9 ± 0.26 (mean ± SE) °C and ambient humidity of 43.7 ± 9.5 % (mean ± SE). Twenty seeds from each of the 19 populations were planted into individual pots for a total of 95 pots, which were distributed on the greenhouse bench in a randomized complete block design. Pots were regularly watered as needed and not fertilized  and stored in vials at −80 °C until further processing.Thirty days after seeding, when the seedlings were at the 3- to 5-leaf stage, leaf samples were collected from five randomly selected individuals of each population  and ground using a TissueLyser . DNA was extracted from prepared tissue using a BioSprint 96 DNA Plant Kit (Qiagen) according to manufacturer’s instructions. Genotyping by sequencing libraries were prepared from A. cotula samples by ‘LGC Genomics’ following High-throughput automatic plant DNA extraction was performed with After sequencing, the library groups were de-multiplexed with Illumina bcl2fastq software  allowingk-th nearest neighbour imputation using the ‘impute’ package .Contigs for single-nucleotide polymorphism (SNP) calling were created using Cluster Database at High Identity with Tolerance for expressed sequence tags (CD-HIT-est)  to groupP-values for whether or not the variants could be considered adaptive . The qvalue package  below 0.1 were considered adaptive. Single-nucleotide polymorphism with a corrected P-value above 0.1 was considered neutral.To test for an association between geographic and genetic distance, and to perform analysis of molecular variance (AMOVA) with adaptive variants, the PCADAPT R package    and discA Linux command line tool ‘fineRADstructure’  of Nei’sA. cotula in the Palouse region of the Pacific Northwest. For the spatial analysis, the Walla Walla Basin and Kashmir Valley regions, which had few populations, were removed leaving the 13 Palouse samples that were most of the A. cotula populations in our study. Pairwise FST values calculated from the neutral SNPs were used for the spatial analysis and the physical distance matrix for the Mantel test was calculated using the ‘pointDistance’ function  and expected heterozygosity (HE) were calculated using the ‘gl.basic.stats’ function in the ‘dartR’ library in R . Self-fertilization rates were estimated for each of 19 populations, three regions and for all regions combined using robust multilocus estimate of selfing (RMES)  of the hot chains used to estimate likelihood approximations migrate were: T = 1, T = 1.5, T = 3, T = 1.0 × 106. We estimated the potential migration pathways with the migration rate  between regions. The estimated average effective genetic diversity  was calculated and the Bezier curve ln (likelihood) approximation from Migrate-n was used to select the most likely model, among our four models.Migrate-n version 3.7.2  was used package  was usedA. cotula populations had two clusters, while the BIC identified a single cluster to be optimal. Although LEA also identified one cluster to be optimal, with K = 2, its clustering matched the one found by DAPC–AIC (K = 2). The two clusters were samples from the Walla Walla Basin versus all other samples (K = 2 and K = 3. Most of the grouping in ‘fineRADstructure’ corresponded to sampling location (i.e. population). The grouping structure in the fineRADstructure plot  was 2.2 %. In region-wise estimation significant selfing was detected in the populations from the Walla Walla Basin and Palouse but not in Kashmir Valley populations based on the permutation test  based on permutation testing with AMOVA (seeP = 0.15). Considering Palouse populations, 7.76 % of the genetic variation was explained by populations and population significantly contributed to overall genetic variation based on the AMOVA permutation test. The full hierarchical AMOVA performed with the adaptive SNPs yielded roughly similar results except that the percent explained (23 %) by region drastically increased [see].Overall measurements of genetic variation were low with the hierarchical AMOVA attributing 1.2 % of the genetic variation to sampling region, 7.0 % of the genetic variation to population within region and 27.8 % between samples within population . VariatiFST was calculated between populations and between regions to develop an understanding of the relative levels of genetic differences between samples from different populations or regions. The pairwise FST values between populations ranged from 0.018 between Tensed and Thornton populations to 0.147 between Dayton2 and Parker Farm populations, although all FST values were determined to be highly significant through bootstrapping   where nearby sites were dissimilar to each other (Pairwise rapping) . The ovech other , this is[see]. The average effective genetic diversity  estimate (θ) for panmixis model was 0.076. Although the panmixis model was found to be the most likely, the model with three regions  with Walla Walla Basin and Palouse sourcing migration to Kashmir Valley (model 2) was used to generate estimates of genetic diversity for each population (i.e. region in this case). Using this approach, the path with the highest estimated migration rate  was Palouse to Kashmir Valley and the lowest from Walla Walla Basin to the Palouse  suggests they are derived from one generalist population that can adapt locally to geographically and climatically diverse locales  and pairwise ronments . The low locally .A. cotula populations , there was no isolation-by-distance signal suggesting that differentiation through either selection, drift or a combination of both has occurred rapidly, overwhelming gene flow among populations. Rapid dissemination is likely at least facilitated by human transports. Compared to other globally invasive asters . Moderate inbreeding and significant selfing (2.5 and 2.1 %) in A. cotula populations from Palouse and Walla Walla Basins suggest there are prevalent local isolation and potential genetic bottlenecks. Additionally, relatively low rates of selfing in Kashmir Valley indicate that A. cotula populations are adaptively outcrossing with the capacity for self-fertilization when isolated or when pollinators are limited, potentially contributing to its invasiveness, as it does for other species  wheat to India , potentially contaminated with A. cotula, have been frequently shipped from the Pacific Northwest to India  climates, whereas other parts of India are mostly tropical. Other possibilities include multiple introductions or an admixed introduction from the USA, and our results closely mirrored the results of Ambrosia artemisiifolia.Although Migrate-n analysis supports panmixis among the populations of to India , mostly to India . Both Wato India . Second,ears ago . Additioears ago . Chickpeears ago . One posA. cotula populations, our study was limited by not having samples from the native range of A. cotula. While we have included A. cotula populations from its two key invading ranges , we were unable to obtain seeds from the native range (Mediterranean region) which would have allowed us to compare the invasive genotypes with those of native ones. Previous studies have shown that invasive species that have both selfing and outcrossing strategies in their native ranges often have higher levels of self-pollination in the invasive range , archaeophytes (i.e. anciently introduced) and native populations.Despite revealing interesting genetic information among and within ve range . While wA. cotula have some capacity for self-fertilization, they could invade and establish even with a limited number of propagules; thus, management efforts should aim to prevent invasion to new fields at local scale and to new regions at global scale. In a previous study, individuals (within a population) of A. cotula explained a higher proportion of variation than the populations for phenotypic traits  markets.At the global scale, A. cotula, improved sanitary practices will be important for limiting the spread of herbicide resistance genes and other problematic genotypes. Established measures include limiting machinery movement, improving cleaning of equipment, ensuring use of certified seed and using new technology to clean grain of seeds during harvest   among A. cotula populations. FST values are below the diagonal, and P-values are above the diagonal.A. cotula populations.A. cotula populations and among individual samples.plab049_suppl_Supplementary_MaterialsClick here for additional data file.


Spodoptera frugiperda, is a polyphagous pest capable of feeding over 80 plant species and was indigenous to the Western Hemisphere. Within a span of 4 years, FAW has established itself throughout most of the regions in Africa and Asia causing significant losses in maize production. Owing to its revamped distribution range, it would be prudent to analyze the ensuing genetic changes and study the emerging phylogeographic patterns across the world. In this regard, we would like to provide a current snapshot of genetic diversity of FAW in India 2 years after the initial introduction and compare it with the worldwide diversity in order to trace the origins and evolutionary trajectories of FAW in India. We have investigated around 190 FAW samples from different regions in India for strain identity and polymorphism analysis on the basis of partial mitochondrial cytochrome oxidase I (COI) gene sequences. Apart from the ancestral rice and corn strain haplotype, our study demonstrates the presence of 14 more haplotypes unique to India at a haplotype diversity of 0.356. We were also able to record inter-strain hybrid haplotypes of rice and corn strains in India. Regional heterogeneity within Indian populations seems to be quite low representative of extensive migration of FAW within India. Distribution analysis of pairwise differences and rejection of neutrality tests suggest that the FAW population in India might be undergoing expansion. Our data is consistent with the findings suggesting a recent and common origin for invasive FAW populations in Asia and Africa, and does not indicate multiple introductions to India. This study reports the highest genetic diversity for Indian FAW populations to date and will be useful to track the subsequent evolution of FAW in India. The findings would have important ramifications for FAW behavior and composition throughout the world.Fall Armyworm (FAW),  Spodoptera frugiperda (J.E. Smith), is a polyphagous pest native to Western Hemisphere and is capable of feeding over 80 plant species. In 2016, it was first reported outside America in the African subcontinent and became notorious for its rapid expansion throughout the subcontinent and significant economic losses in maize production in a short span of time3. In 2018, reports of its presence in the Indian subcontinent were abounded and it was again found to be majorly associated with maize crops across different regions in the country6. Within a span of 2 years, FAW has established itself throughout most of the regions in India and has been found to attack maize, sorghum, sugarcane, and millets11. Additionally, FAW invasion has been reported from 19 Asian countries including Myanmar, China, Vietnam, Japan, Korea and in February, 2020, it was detected in Queensland, Australia as well (https://gd.eppo.int/taxon/LAPHFR/distribution). The rapid dispersal of FAW to these widespread regions and varied landscapes is alarming and poses a major threat to the food security efforts of different countries.Fall Armyworm (FAW), Spodopterafrugiperda is known to exist as two genetically distinct but morphologically identical sister strains, designated as ‘Rice strain’ (R strain) and ‘Corn strain’ (C strain) which differ in their host preferences. R-strain is most consistently found in millet and grass species associated with pasture habitats while the C-strain prefers corn, cotton and sorghum13. Apart from the variable host distribution, the strains exhibit restricted mating potential and changes in pheromone composition as documented by several studies15. Owing to similar morphological phenotypes, the two strains of FAW are mainly classified on the basis of polymorphisms in mitochondrial gene cytochrome c oxidase subunit 1 (COI) and a nuclear gene triosephosphate isomerase (tpi)17. The correspondence between the two markers is highly significant for the identification of FAW strains in the Western Hemisphere. However, when the invasive populations from Africa, India and southeast Asia were examined for strain identity by using the two markers, the results were found to be highly discordant. In general, Tpi gene was found to accurately predict the host association in invasive populations19. The discordance between the two markers was reflective of the hybrid nature of the invasive populations which was subsequently corroborated with whole-genome studies21. Barring the detection of two maternal COI lineages and a limited number of Tpi haplotypes, genetic variability in the invasive populations was found to be very low. In addition, detection of a unique African Tpi ‘rice’ haplotype in the populations from India and south east Asia further strengthened the hypothesis that the invasive populations shared a recent and a common origin19. Though the notion was challenged by whole mitochondrial sequencing analysis, but there was consensus on highly reduced genetic diversity in invasive populations indicative of a small number of founders22.Several studies have focussed on genetic analysis to understand the origins of FAW at the newly invaded regions and to ascertain its crop preferences and insecticide resistance potential. 22. The Indian population exhibited high homogeneity with East and South African populations with the majority showing mitochondrial rice strain COI and nuclear corn strain Tpi marker18. Mitochondrial diversity of the analyzed specimens was found to be very low when compared to the native population21. Thereafter, the dissemination of FAW in disperse regions, potential subsequent introductions in India and adaptive changes could possibly lead to deviations from the original structure. We were interested in finding whether the Indian FAW populations still conformed to the findings of the previous studies or were they beginning to show digression. This study was designed to capture the diversity of FAW populations in India in the last 2 years, to understand how FAW has adapted to the Indian landscape and to compare the same across the newly expanded range of FAW across the world.Most of the studies analyzing the genetic diversity of FAW in India were primarily undertaken in 2018, when FAW was first detected in India and had spread to limited regions within the countryS. frugiperda was found to be a serious pest in maize, sorghum and was occasionally found on sugarcane and cotton in different parts of the country. 92 FAW specimens were collected from 39 different regions in India and Nepal during the year 2018–20 and were subjected to molecular identification at Division of Genomic Resources, ICAR-NBAIR, Bengaluru, India. The generated COI sequences showed 100% similarity with S. frugiperda and were deposited in NCBI GenBank database. The sequences and the specimen details were submitted to the BOLD database and DNA barcodes were generated. A list of all COI sequences generated at ICAR-NBAIR is provided in Supplementary File ; and one to the Rice Strain category. The relative distribution of COIB haplotypes, CSh4 and CSh2, has been used as an indicator to study the descent of FAW populations in America. FAW populations collected from Florida and the East coast of America majorly belong to CSh4 haplotype while those from Texas and most other parts of America show a predominance of CSh2 haplotypeTpi gene were studied, which identified four haplotypes in Indian populations (n = 66), i.e. TpiCa1a homozygous (n = 39), TpiCa2a homozygous (n = 6), TpiCa2b homozygous (n = 4) and TpiRa1a homozygous (n = 4). Besides this, thirteen specimens demonstrated the heterozygous corn-strain haplotypes  as observed by their overlapping DNA sequence chromatographs  in the dataset at a nucleotide diversity of 0.00313. The occurrence of non-synonymous mutations (60%) was higher than the synonymous ones. We also identified 16 different haplotypes from Indian COIA sequences at a haplotype diversity of 0.356, out of which 13 haplotypes belonged to the rice strain while three belonged to the corn strain. As per our knowledge, this study catalogs the highest genetic diversity for FAW reported from India to date. The majority of the analyzed COI sequences belonged to a single rice haplotype  which was distributed throughout the country. This was followed by the predominant ‘C’ strain haplotype  and another ‘R’ strain haplotype from India . The remaining haplotypes from India were found as singletons ; 2. Africa, 3. India and 4. Asia-II . Along with the previously defined 183 specimens from India, we analyzed 163 COI sequences from America, 150 from Africa and 76 from rest of the Asian countries for polymorphisms. For a comparative study, analysis from all sequences was performed using the same 460 bp region of COI gene which was used for the analysis of Indian populations. In the process, we observed 27, 3, 5 haplotypes, respectively from America, Africa and East-Asia. The predominant ‘R’ and ‘C’ COI haplotypes from America  represent the principal haplotypes in all the invaded regions and are presumed to represent the ancestral haplotypes introduced to these regions. Other than the ancestral haplotypes, Africa, India and Asia-II were represented by 2, 14 and 3 haplotypes specific to these regions, respectively. Neutrality test statistics for both African and rest of Asian populations suggested that the populations are still evolving rather neutrally in these places . The results are shown in Table Similar results were corroborated with DAPC analysis and the resultant membership table which indicated two clear discriminant clusters between America and India Fig. .Figure 6Spodoptera frugiperda provides an excellent model system in this regard since it is believed to have expanded its distribution range very recently and has been well documented and characterized for its diversity in its native as well as the extended range. Most of the studies analysing the diversity of Indian FAW populations were performed around 2 years ago from a few locations where it was first detected in India22. Thus we thought it was imperative to understand the diversity of the Indian FAW population over the course of the last 2 years after the expansive migration of FAW across India.An ideal design for any population genomics study would involve diligent tracing of the evolutionary course of a population over geography or time. Tpi genes, we did not find much variation between our dataset and the previous study from India by Nagoshi et al.18. The structure and composition of the FAW strains for COI locus was well preserved over this period. We found a disagreement of nearly 90% between COI and Tpi gene markers for the prediction of host association as has been ubiquitously reported for the populations from most of the newly invaded regions across the world19. Tpi gene loci provided a better estimate for host preference in the specimens investigated in our study.During our preliminary investigation of strain defining loci from COI and 29. It is divided into 15 different agro-climatic zones on the basis of soil type, climate, water availability etc. (http://mowr.gov.in/agro-climatic-zones), which is partly responsible for the observed diversity in India. Previous studies on the intra-species diversity of certain insect pests in India have suggested geographical sub-structuring within different regions of India32. We looked at the distribution of the different haplotypes in India but most of the novel haplotypes in India at present are too infrequent and dispersed to attribute them to specific ecozones of India. Moreover, looking at the rampant distribution of the predominant rice and corn haplotypes throughout India, the migration of FAW within India does not anyhow seem restricted. However, future studies would help to shed more light on regional associations or microcosms of different haplotypes in India and their adaptive advantage over other haplotypes in the Indian landscape.When polymorphisms in mitochondrial COIA gene were studied in Indian populations, we found several novel haplotypes predominantly belonging to the COIA ‘R’ strain, which were unique to India. Several population genetic test statistics suggested population expansion in India while indicating that the populations in Africa and other East Asian countries were still evolving neutrally. We tried to investigate the reasons behind the same. First, we analyzed the distribution of different haplotypes in India to understand the impact of regional influences in shaping the genetic diversity in India. India is considered as one of the most biodiverse countries of the world and contains four major biodiversity hotspots in a dense cluster22. On that basis, we think it would be prudent to analyze the diversity in Indian populations with multiple gene loci to conclusively trace the geographic origins or monitor the outflows from India. However, based on our analysis from a large dataset of FAW COI sequences, we found that the mtCOI gene was useful in sub-structuring the populations from different geographical groups but did not indicate introduction of multiple COI haplotypes to India.As an alternate hypothesis, we tried to investigate whether multiple introductions of FAW could contribute to the higher diversity seen in India. In this regard, shared polymorphism analysis or network studies from partial COIA sequences did not provide any evidence for multiple introductions to India. Most of the novel haplotypes seen in India belonged to the COIA ‘R’ strain category and were not found in America or any other part of the world. In fact, India, Africa and other regions of Asia shared common ancestral haplotypes and similar polymorphisms with each other, signifying a common source for all these populations. However, a recent study involving whole-genome sequencing of FAW populations from New World and invaded regions has proposed multiple origins for FAW in the invaded countriesConversely, on a different note, it is important to state that within our primary data generated from 92 FAW specimens collected from 39 different regions of India, we did not observe similar diversity levels as those found in other FAW sequences reported from India. We could only find the two ancestral COIA ‘rice’ and ‘corn’ haplotypes which are reported from invasive populations of Africa and East Asia. The observed haplotype diversity was much in line with that found in Africa , 0.5 μL DreamTaq DNA polymerase (5 U/μL), 5 μL DNA (50 ng/μL), and 35 μL sterile water. Thermo-cycling parameters used for the study consisted of an initial denaturation of 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at specific temperatures for 1 min, extension at 72 °C for 1 min. The amplified products were analyzed on 1.5% agarose gel electrophoresis. Each specimen PCR sample was bi-directionally sequenced and checked for homology, insertions and deletions, stop codons, and frame shifts by using NCBI-BLAST and ORF finder. The COIA generated sequences showed 100% similarity as S. frugiperda and were then deposited in NCBI GenBank database and accession number was retrieved for all the populations. Further the sequences and the specimen details were submitted to the BOLD database and DNA barcodes were generated.92 FAW specimens were collected from 39 different regions in India and Nepal during the years 2018–2020. The larvae from each collection were stored in absolute ethanol at − 80 °C until DNA extraction. Genomic DNA was isolated from single larva by using QIAGEN DNeasy blood and tissue kit, Germany, following the manufacturer’s protocols. The remaining larvae were kept as voucher specimens at − 80 °C. DNA thus obtained was subjected to PCR amplification using a Bio-Rad C1000 Thermal Cycler. Each PCR reaction of 50 μL consisted of 5 μL 10X DreamTaq master mix, 2 μL of 10 mM dNTP mix, 1 μL (20 pmol/μL) each of gene-specific forward and reverse primer  was performed using the R package poppr v2.8.643. The hierarchy for this analysis was based on four predetermined geographical regions America, Africa, India and Asia-II. The percentage of observed variance within and between groups was calculated initially among all the regions and then the same analysis was performed between different combinations of two groups . The significance of observed variance between groups was tested by a randomization test with 1000 permutations. This test was performed using the accompanying R package ade4 v1.7.15 and the function randtest44.Population structure was studied from the alignment file using AMOVA46. This method was applied to the aligned sequence file to find the extent of discrimination between different predefined groups. The balance between discrimination power and overfitting was maintained by selecting the optimal number of principal components for analysis using the a-score optimization test from the function optim.a.score. We have retained eight principal components as the optimum number based on the a-score optimization test. As there are four geographical groups, we retained three discriminant functions. Predefined groups were then randomized and the analysis was repeated with the new randomized data to confirm that discrimination between clusters does not occur by chance. DAPC analysis was also used to study the level of admixture in the predefined groups. Samples with less than 50% posterior membership probability were considered admixed while the samples with more than 50% posterior membership probability were allotted with their respective group.A second method discriminant analysis of principal components, was used to further study the population structure. This analysis was performed using the R package adegnet v2.1.3Supplementary Information.


Haliotis discus samples from three different locations around the Korean peninsula to assess population structure, utilizing Genotyping-by-Sequencing (GBS) method. Using PstI enzyme for genome reduction, we demonstrated the resultant library represented the whole genome region with even spacing, and as a result 16,603 single nucleotide variants (SNVs) were produced. Genetic diversity and population structure were investigated using several methods, and a strong genetic heterogeneity was observed in the Korean abalone populations. Additionally, by comparison of the variant sets among population groups, we were able to discover 26 Korean abalone population-specific SNVs, potentially associated with phenotype differences. This is the first study demonstrating the feasibility of GBS for population genetic study on H. discus. Our results will provide valuable data for the genetic conservation and management of wild abalone populations in Korea and help future GBS studies on the marine mollusks.Continuous monitoring of the present genetic status is essential to preserve the genetic resource of wild populations. In this study, we sequenced regional Pacific abalone  Haliotis discus is known as the most popular and highly valued species for commercial fisheries resources owing to the quality of meat and traditional consumer preferences [H. discus species has a wide geographic distribution in coastal waters of East Asia, and two subspecies exist: H. discus hannai inhabiting throughout Korea and northeastern Japan, and H. discus discus is distributed in the southern coastal areas of Korea and southwestern Japan [Abalones are a type of marine gastropods belonging to the family of reef-dwelling snails, Haliotidae. There are about 70 different species distributed across tropical and temperate coastal areas, such as Australia, the United States, or East Asia. Abalones have been a valuable food source for humans worldwide, and they are also an important research resource for ecological and evolutionary studies due to their diverse phenotypic appearances and global distribution . Among aferences , 3. The rn Japan .Esox lucius (Pike), restriction site-associated DNA sequencing (RAD-seq) not only resolved population genetic structure with better resolution than microsatellites but also were able to identify candidate loci under selection [For appropriate conservation of wild animals, continuous monitoring of the population structure and genetic status is essential because the maintenance of genetic diversity is important for whole species protection by providing disease- or stress-resistance genes to the gene pool . Therefoelection , and in election . Thus, ielection . Of the election , the othelection .Ruditapes philippinarum (manila clam), the genomic best linear unbiased prediction (GBLUP) study of Perna canaliculus (greenshell mussel), and the population studies of Haliotis fulgens and Haliotis laevigata along the Western Australian coast [H. discus.The recent advances in sequencing technology have led to enable sampling of the genome more densely and efficient production of more accurate and sufficient genetic information , 20. Onean coast –26. Yet,H. discus hannai) collected from each of three seas  in Korea and two outgroup populations, one Japan population (H. discus discus) and one red abalone population (H. rufescens). Using the variant information, we evaluated the genetic diversity and the population structure of H. discus populations inhabiting around the Korean peninsula and elucidated the impacts of geographical features and ocean currents in the gene flow among marine mollusks. Furthermore, by comparison of variant sets against red abalones, morphologically very different from H. discus, Korean Pacific abalone population-specific variants were discovered, which may be implicated with phenotypically different traits between groups. The results of the present study will provide an assessment of the utility of GBS for genetic analysis of abalone species and contribute to the management and genetic conservation of the wild Pacific abalone populations.In this present study, we performed GBS in a total of 102 regional abalones consisting of three regional Pacific abalone populations (H. discus hannai-specific single nucleotide variants (SNVs), Goseong, Yeosu, Taean, and Red samples were used. No permissions were required for sample collection in this study because they were obtained during the season in which regional fishing communities allow harvesting.Five abalone populations (102 samples) were used in this study . Of thesPstI  at 37 C for 2 hours and heated to 65°C for 20 minutes to inactivate the enzyme. DNA libraries for genotyping-by-sequencing (GBS) were constructed according to the protocols as described previously [PstI was followed by ligation of adapters with specific barcode for each sample. The sets of 102 ligations were purified using QIAquick PCR Purification Kit (Qiagen). Ligation samples were pooled and 5μl were amplified in 50μl reaction by PCR using AccuPower Pfu PCR Premix  and 25 pmol of Illumina adaptors:Total genomic DNA was extracted from muscle tissue using the DNeasy Blood and Tissue Kit  following the manufacturer’s instruction. The amount of DNA was quantified using the standard procedure of Quant-iT PicoGreen dsDNA Assay Kit  with Synergy HTX Multi-Mode Reader  and normalized to 20 ng/μl. DNA (200ng) was digested with 8U of High-fidelity eviously , 28 with5’- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT—3’and5’- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT—3’.PCR cycles consisted of 98°C for 5 min followed by 18 cycles of 98°C for 10 s, 65°C for 5 s, and 72°C for 5 s, with a final extension step at 72°C for 5 min. The PCR product was also purified using QIAquick PCR Purification Kit (Qiagen) and then evaluated the distribution of fragment sizes with BioAnalyzer 2100 . The GBS library was sequenced in the Illumina NextSeq500  with the length of 150 bp single-end reads following the manufacturer instruction. Read count distribution of the GBS library is shown in H.discus hannai constructed in our previous study [P-values < 0.05.Before conducting variant calling analysis, we conducted de-multiplexing of GBS data using GBSXtoolkit . Reads ous study , using Bus study  with theus study  to creatus study  was usedus study  for minoFST values with a 50Kb sliding window for each pair of populations was calculated using VCFtools 4.0 [Principal component analysis (PCA), based on the SNV information of Korea and Japan samples (91 samples), was conducted using SNPRelate R package . For popools 4.0 . Lastly,ools 4.0  with 10,ools 4.0  since GeHaliotis discus hannai draft genome, constructed in our previous study, and its gene annotation to build database. Korean abalone-specific SNVs were then extracted based on the existence of non-synonymous (missense and nonsense) SNVs present only in Korean abalone populations  at orthologous positions compared to H. rufescens species, and in this step, only fixed SNVs were considered. Further, these sets were subjected to enrichment analysis to identify significant SNVs using SnpSift (version 4.2) CaseControl [SnpEff (version 4.2)  was usedeControl . CaseConeControl . For FiseControl . Korean PstI GBS library was sequenced using Illumina technology (NextSeq500), and it generated approximately 204.8 million reads from 102 samples which equated to a total of 17.43 Gbp of sequence data (H. discus hannai which was constructed in our previous study [PstI.The resultant nce data . On averH. discus samples (Korea and Japan). The average number of SNVs in each population varied from 3,407 ± 381.8 to 4,186 ± 348.8; the least number of SNV set was observed in the Yeosu abalone population, while the highest value was found in the Japan abalone population , with a relatively low value in Japan population (9.126 x 10−4). To investigate the relationships among populations, PCA was performed. Eigenvector 1 clearly separated samples into Korea and Japan group, and eigenvector 2 distinguished Goseong samples (Korea) from Yeosu and Taean samples (Korea) , followed by Goseong (0.1617) and Taean (0.1888). Among Korean individuals, comparison between Yeosu and Taean population had lower overall FST value compared to the Goseong to Yeosu and Goseong to Taean pairs (2 = 0.046) between the genetic and geographical distribution  on a per-site basis were estimated from the SNV data. Average nucleotide diversity (pi) of three Korean  (Korea) . To furt (Korea) . These f (Korea) . With K  (Korea) . Among tan pairs . Similarribution .H. discus hannai)-specific SNVs. Among them, 26 SNVs were found within coding regions, from which 23 genes were identified by BLAST search as genes encoding hypothetical protein LOTGIDRAFT_163461, hypothetical protein CGI_10016004, adenosine 3’-phospho 5’-phosphosulfate transporter 1-like isoform X2 exhibited multiple protein mutations (2H2-type), carbonic anhydrase , and chitin binding domain-containing protein (chitin binding domain), and some of them were previously reported to be associated with shell formation of abalones [Additionally, in order to search candidate SNVs accounting for phenotypic differences of Korean abalone populations against red abalones, we further extracted fixed non-synonymous SNVs that were significantly enriched, from 23,123 SNVs for 86 Korean abalone samples and 11 red abalone samples, based on SNV annotation and CaseControl analysis. We identified a total of 809 Korean abalone  [In contrast to terrestrial species, marine organisms generally show reduced intraspecific genetic differentiation among geographic regions –57. This hannai) –67. Also hannai) . The uni2 = 0.046), indicating that the geographical distribution contributed partly to the genetic differentiation among the populations  and Gadus macrocephalus (Pacific cod) have presented significant differentiation in the East sea areas as well [Donax deltoides  at the point of different currents converge [To assess population differentiation, we investigated patterns of the genetic structure of four regional Pacific abalone populations using 23,123 SNVs, collected from each of the three seas surrounding the Korean peninsula and from coastal waters of Japan. Our results suggested the existence of genetic heterogeneity among Korean groups which was strongly affected by the geographical features of the peninsula and major ocean currents congregating around it. First of all, regression analysis from Mantel test showed that the genetic distance among individuals slightly increased with geographical distance  = 0.001, Rulations . Also, F as well , 70, andconverge . Similarconverge  found diAt the Korean peninsula, the Kuroshio Current flowing northeastward splits into the Yellow Sea Warm Current and the East Korea Warm Current . The EasH. discus and H. rufescens. The shell length of H. rufescens can reach up to 31cm, making it the largest abalone species in the world, and the shell length of H. discus varies between 10cm to 15cm [H. rufescens’s shell is thicker and stronger, so that it is more resistant to shell-boring ectoparasite infection such as Polydora spp. than H. discus species [H. discus populations and H. rufescens population to search for candidate SNVs potentially associated with the phenotype differences. For this analysis, we used only fixed non-synonymous SNVs with moderate to high impacts on gene function, and we were able to discover 26 SNVs existing in coding regions which were significantly enriched in the case-control analysis. Some of these SNVs were located in shell formation related genes such as vWA domain-containing protein 7, Kunitz-like protease inhibitor, carbonic anhydrase alpha, and chitin binding domain-containing gene [There are differences in morphological and physiological characteristics between  to 15cm . Also, H species . Due to ing gene –54, 76, 3, shell matrix proteins (SMP), pigments, lipids, polysaccharides, and glycoproteins [Crassostrea gigas, Mytilus coruscus, Lottia gigantea, Pteriida fucata, etc. [2 to HCO3- in Nacrein [3 forms. It is usually found together with vWA in SMPs, and both are typically domains of collagen, the fundamental component of ECM [Haliotis asinina and also found from the mantle cells of diverse mollusks [P. fucata species [H. discus hannai-specific SNVs presented here are the first to be reported in this study. Moreover, since the present study had a limitation in a relatively small sample size of H. rufescens population, and thus they were hard to represent the genetic characteristics that covered species’ range, future studies including a higher number of samples are needed to further validate these preliminary findings. Also, most population-specific genetic differences are likely to be neutral, and it is above the capability of this study to determine how each SNV functionally affects the gene. Thus, we cautiously suggest these identified SNV sets as promising candidates to guide further investigation.The formation of the molluskan shell is regulated by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, mantle . This maproteins , 76. Theproteins , 77. vWAproteins . They arta, etc. , 52–54.  Nacrein . And chit of ECM , 48. Kun species . HoweverH. discus populations around the Korean peninsula and elucidated the impacts of the geographical features and the ocean currents in the gene flow among marine mollusk populations. Based on the patterns of population genetic structuring in the FST, PCA, and STRUCTURE, the ocean currents congregating around the Korean peninsula were key factors of genetic heterogeneity among groups as the directionalities of ocean currents worked as a directional bias in gene flow. Besides, by comparison of variant sets, we identified Korean Pacific abalone-specific variants, which might be associated with phenotypically different traits between groups. Our results will provide valuable data for the genetic conservation and management of wild abalone populations in Korea and help the future GBS studies on the marine mollusks.This study evaluated the genetic diversity and the population structure of S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(a) Mean L(K) (±SD) over seven runs from K = 1 to K = 6; (b) Delta K (ΔK); (c) Estimation following Evanno et al.(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Appendix(GZ)Click here for additional data file.