Apis mellifera in China, eastern honey bees (Apis cerana) were the only species used for beekeeping in this region. In the mountains of Changbai, populations of A. cerana are considered a distinct ecotype of the species which formed through the distinct selective pressures in this area over time.Exploration of adaptive evolution of organisms in response to environmental change can help to understand the evolutionary history of species and the underlying mechanisms of adaptation to local environments, thus guiding future conservation programmes. Before the introduction of A. cerana in Changbai Mountain. A total of 3,859,573 high-quality SNP loci were yielded via the whole-genome resequencing of 130 individuals in 5 geographic regions.We performed a measure of 300 wing specimens of eastern honey bees and obtained the geometric morphological variation in the wing of A. cerana in Changbai Mountain. Genetic differentiation at the subspecies level exists between populations in Changbai Mountain and remaining geographic regions, and a significant reduction in the effective population size and an excessive degree of inbreeding may be responsible for a substantial loss of population genetic diversity. Candidate genes potentially associated with cold environmental adaptations in populations under natural selection were identified, which may represent local adaptations in populations. Our study provided insights into the evolutionary history and adaptation of A. cerana in Changbai Mountain, as well as its conservation.Corresponding geometric morphology and population genomics confirmed the particularity of the The online version contains supplementary material available at 10.1186/s12864-022-08298-x. Apis cerana is native to Asia with a long history of managed beekeeping, the populations have also experienced severe declines in recent decades [A. cerana are considered a distinct ecotype of the species [A. cerana bees is bound to affect the survival of upstream predators in the future. In terms of economic value, A. cerana in Changbai Mountain bears excellent traits such as rapid reproduction, strong maintenance of colonies, collection capability, resistance to adversity, and cold tolerance [A. mellifera, habitat loss and fragmentation, overexploitation of biological resources, intensive agricultural development, pathogenic microbial infection, overuse of pesticides, global warming, and atmospheric pollution, have caused a massive decline in the population size and distribution density of A. cerana in Changbai Mountain [A. cerana in Changbai Mountain. Collectively, the diversity of A. cerana in Changbai Mountain should be further clarified.Nowadays, the number of pollinators has been declining considerably; in particular, the invertebrate pollinators represented by honeybees and butterflies are on the verge of extinction , 2. Apis decades , 4.In No species . This ecolerance . Thus, iMountain , 8; its Mountain , and theMountain –12. WithApis cerana cerana were incubated at constant lower temperature until emergence, resulting in the increase of wing veins of adults, which may be the wing veins that disappeared in the process of evolution [A. cerana in Changbai Mountain. In addition, there are differences in adaptation to various climates between eastern honey bees and western honey bees. It is generally believed that the adaptation of western honey bees to temperate climate conditions is a key step in their evolution [A. cerana populations inhabiting the mountains of Changbai may have a more effective social defense system than A. mellifera through the combination of complex molecular and behavioral mechanisms [A. cerana in Changbai Mountain has developed distinct traits in response to mountain conditions, particularly strong resistance to the cold  [A. cerana in Changbai Mountain is lacking.Shape and veins of insect wings contain a wealth of ecological and behavioural information –15. Geomvolution . Thus, cvolution . Native volution –24, therchanisms . A. cera− 40 °C) . While p− 40 °C) , 26, a s. cerana samples of each ecotype were downloaded from the literature [A. cerana in Changbai Mountain, and helped in identifying candidate genes related to their adaptation to the local cold environment. This paper offers an in-depth investigation of the genetic basis and uniqueness of the environmental adaptation of A. cerana in Changbai Mountain, thus rendering directions for the scientific and effective conservation of the population.In this study, we collected samples from 64 sampling sites in the Changbai Mountain, northern, southern, Qinghai–Tibetan, and northwestern regions. Moreover, resequencing data from 66 Aterature . A comprA. cerana samples from the published literature . After quality control, a total of 3.86 million SNPs were identified, of which 253,656 were in exonic regions and 52,161 were in non-homologous regions , and a total of 2,237,786 Indels were detected, of which 15,588 were in exonic regions . Population genetic structure was first evaluated using ADMIXTURE [K = 2–5, most Jilin individuals and all Heilongjiang individuals in Changbai Mountain were from the same ancestor, and all Liaoning individuals were of mixed lineage and had different ancestral origins. In particular, when K = 4 and K = 5, the origin of the Liaoning individuals was more complex, with individual samples containing a mixture of 3 or more ancestral subgroups, and few Jilin samples showed different degrees of admixture. According to the cross-validation errors, when K = 4, the CV error value (0.22113) was the lowest, which was the best K value. All regions were divided into 4 genetic components, the individuals from Tibetan (XZ01-XZ08) and Hainan (HI01-HI10) formed an ancestral cluster  and Fst values of ain Fig. A. The hiain Fig. B. Pairedntiation , 30, 31.on Table . Fst forA. cerana, and A. cerana in Changbai Mountain had the lowest He, highest Ho, and highest F , observed heterozygosity (Ho), inbreeding coefficient (F), and linkage disequilibrium (LD) for each geographic regions of  F Table , indicatcay Fig. . PresumaA. cerana in various regions, including Changbai Mountain populations, coincided with the cyclical variation in historical global temperature, which peaked during the most recent major interglacial period of the Pleistocene, approximately 13,000–11,000 years ago, when the global climate was warm, followed by a sharp decline, consistent with the findings of Chen et al. [A. cerana peaked at MIS5. The difference lied in the fact that the A. cerana population in Changbai Mountain exhibited a more moderate expansion and contraction of the effective population and the population size was at a lower level than A. cerana populations in other regions .We performed a selective sweep analysis between Changbai Mountain individuals and Hainan individuals . Genomic regions with the top 5% values for the Fst and θπ ratios (log2(θπ ratio)) in each pairwise comparison were considered to be potentially selective-sweep regions Fig. A. Genes In this work, we paid considerable attention to the genes related to cold adaptation in the Changbai Mountain region. According to the decreasing order of Fst values, we focused on the genes involved in the top 20 bins. Among these, four structural constituent of cuticle protein and four lipid storage droplets surface-binding protein were included . Based on previous studies on cold tolerance of honeybees, we found many of these genes involved in the GO terms and KEGG pathways to be associated with cold adaptation in insects , 35. In The pathway enrichment of the selected genes combined with the functional annotation of the genes themselves resulted in the identification of several highly enriched functional categories, including structural constituent of cuticle (4), zinc ion binding (4), serine/threonine-protein kinase (5), lipid storage droplets surface-binding protein (4), and G-protein-coupled receptor 1 family (4) . Through the selective sweep analysis of population evolution, we attempted to determine the candidate genes that may be related to local adaptation. Because the Tibetan and Changbai Mountain populations belong to the cold-tolerant honeybee ecotype, the same method was used to perform the selective sweep analysis between the Tibetan and Hainan individuals , JL06 (belongs to JL-JL), JL07 (belongs to JL-TH), and JL13 (belongs to JL-BS) in Jilin Province; LN01 and LN09 in Liaoning Province; and HL03 in Heilongjiang Province . The results of multiple tests by the LSD method showed that the individuals in the northwestern region, Qinghai–Tibetan region, and Changbai Mountain had larger forewings, and no notable differences were observed between the individuals. Individuals from Changbai Mountain differed significantly from the individuals in the northern and southern regions in terms of forewing size (P < 0.05) . A. cerana from the Qinghai–Tibetan region, northwestern regions, and Changbai Mountain had larger hindwings, and non-significant differences were observed between these individuals, whereas the individuals from Changbai Mountain differed notably (P < 0.05) from the northern and southern region individuals , whereas no significant correlation with longitude was observed (P > 0.05) .The wing size is expressed in terms of the centriod size (CS) of the wing. One-way ANOVA revealed a significant difference in CS values of forewings of 05) Fig. A. Hindwials Fig. B. StepwiA. cerana in Changbai Mountain, with no significant differentiation observed between the individuals from Jilin and Heilongjiang, and significant differentiation observed between Liaoning individuals.The first two principal components of forewings and hindwings occupied 77.74% and 83.29% of the total variance, respectively, both of which were able to illustrate main differences in forewings and hindwings among the geographical regions. Results of forewing PCA showed that individuals from Jilin province and Heilongjiang province were concentrated in the positive half-axis of PC1 and PC2, whereas an individual from Liaoning province (LN09) was in the negative half-axis of PC1 and PC2 . Results of hindwing PCA showed that individuals from Jilin province and Heilongjiang province were concentrated in the positive half-axis of PC1, whereas an individual from Liaoning province (LN01) was in the negative half-axis of PC1 . Taken together, a certain degree of wing morphological differentiation was observed within Apis cerana in Changbai Mountain, and a low level of genetic diversity tends to increase inbreeding. The previous analysis of genetic diversity of A. cerana in Changbai Mountain, which were based on SSR and mtDNA markers, also reflected the current situation of a single genetic structure and low genetic diversity [A. cerana in Changbai Mountain.A certain number of wild bee colonies are present in Changbai Mountain, mostly in the hollow caves of trees, rock caves in steep mountain cliffs, rock crevices, underground caves, and other hidden places. Eastern honey bees have the germplasm characteristic of flight escape, and a certain number of wild colonies are obtained from various reared colonies and are constantly replenished. In recent decades, human capture and the outbreak of sacbrood virus have greatly changed the population dynamics of eastern honey bees in Changbai Mountain , 36. Theiversity –39. It hA. cerana was affected by cyclical climate changes during the Quaternary ice age [A. cerana in Changbai Mountain. According to the Quaternary glacial remains around the Tianchi volcanic cone, the basic geomorphic features of the Tianchi caldera were formed in the MIS4 and were maintained to the present day after basic stereotypes [A. cerana.The results of population history analysis show that biogeography and climate change have seriously affected the Changbai Mountain population, and glaciation and local volcanic events have likely brought about the bottleneck effect of population history. Available evidence demonstrated that the  ice age , and tha ice age , 42, inereotypes , which iA. cerana. The effective size of the A. cerana in Changbai Mountain increased significantly for once from the last glacial maximum of 15,000 years ago to the Holocene, indicating that global warming was beneficial to the Changbai Mountain population. The period of maximum extent of continental icesheet was 18,000 years ago, and since then, glaciers began to retreat. The Holocene warm period in the northeast was the best period for vegetation development, and the enhanced summer monsoon precipitation due to warming led to ecological recovery [A. cerana in Changbai Mountain during 5000 years ago.The population decline during the MIS4, which lasted until the last glacial maximum of approximately 15,000 years ago, is presumed to be mainly due to the paleoglaciation, grand development of the ice sheet, low snowline, and small dust flow, indicative of a cold and wet climate , and therecovery . Global recovery , the ecorecovery , 49, whirecovery , 51, caurecovery –54. In arecovery , and a srecovery . TherefoA. cerana populations in each geographical region increased with latitude and altitude. Body size in insects may be related to cold resistance, highland populations of A. cerana are larger, darker and have longer body hair [A. cerana. Larger wings imply larger insects who adapt to cold conditions at high altitudes and long overwintering periods through an increase in body size [We explored the selected genes in the evolution of the Changbai Mountain population from multiple perspectives by conducting the selective sweep analysis. The KEGG pathways involved in the selected genes with a high FST value are all related to the metabolism of lipid energy substances, and energy metabolism is crucial for the cold resistance of honeybees , 35. Theody hair . The supody size , 61. Theody size , have a ody size , and havody size , 65.A. cerana in Changbai Mountain were morphologically similar to the Korean and Japanese populations, and may be classified as the same taxon [A. cerana being significantly different from that of the other two honeybees, and suggested that the Jilin population may be a novel subspecies. Gene flow blocking is responsible for population differentiation, prompting differentiation into independent populations that produce genetic traits adapted to the local environment [A. cerana in Changbai Mountain bears varying degrees of genetic differentiation within its population. The genetic differences were negligible among the local individuals of Jilin province, which exhibited a low genetic differentiation compared with Heilongjiang individuals, whereas a significant and major genetic differentiation was noted between the Jilin and Liaoning individuals. It is also proved by the results of wing geometric morphology.The subspecies-level genetic differences among the individuals from Changbai Mountain and Northern, Northwestern, Southern, and Qinghai-Tibetan regions were observed , 31, 66,me taxon , 68. Worme taxon . Zhou etme taxon  confirmeironment . A recenironment . On the In summary, our research shows that eastern honey bees from Changbai Mountain have genetic and morphological singularity. However, populations in the core area of Changbai Mountain are under threat from the outer reserves, it may lead to irreversible loss of local adaptations in native populations, genetic homogenisation, and pose a risk of genetic contamination , 72. TheA. cerana in Changbai Mountain showed a gradual change with altitude and latitude. A certain degree of wing morphological differentiation was observed within A. cerana in Changbai Mountain. Genetic differences were observed between Changbai Mountain and other geographic regions at subspecies level, which has a lower effective population size and genetic diversity, and a higher degree of inbreeding. Selected genomic regions associated with adaptation of populations to cold environments were identified, and these genes may represent local adaptations of population, which serve as excellent candidates for further research. Geometric morphological and colony genomic evidence confirmed the special evolutionary status of A. cerana in Changbai Mountain, which will contribute to future conservation programmes.The body and wing sizes of A. cerana samples were collected from 64 sampling sites in 5 geographical regions of China, including northern, northwestern, Qinghai–Tibetan, southern regions, and Changbai Mountain. All the samples were taken from germplasm reserves or original rearing colonies, with priority given to samples from wild hives (e.g. tree cave hives) and traditional-type hives (e.g. tree barrel hives). The samples were transported in anhydrous ethanol and stored in an ultra-low temperature refrigerator at − 80 °C.One workerbee was randomly selected from each colony in the sampling site for whole-genome resequencing. First, genomic DNA was extracted and quantified precisely, and DNA samples with a total volume of ≥ 1.5 μg were used for library construction. The inspected DNA samples were randomly fragmented into 350-bp-long fragments through Covaris, followed by high-quality library construction using the TruSeq Library Construction Kit, the library was prepared through various steps such as end repair, polyA tailing, sequencing junction, purification, and PCR amplification. The constructed library was sequenced by Illumina. Qubit3.0 was then used for preliminary quantification, and the insert size of the library was measured by Agilent 2100 after diluting the library to 1 ng/μL. Then, the effective concentration of the library was accurately quantified by Q-PCR. Finally, according to the effective concentration of library and data output requirements, the Illumina platform was used for PE150 sequencing.Effective high-quality sequencing data were mapped to the reference genome  by usingA total of 130 samples were collected for variant detection. SNP detection was performed using SAMtools , and higBased on the results of the population structure analysis, the SNP data obtained from the Jilin and Heilongjiang samples were selected to perform genetic diversity index calculation, LD analysis, population history analysis, and selection elimination analysis. GCTA64 software was used for PCA, and the first three principal components were plotted. The distance matrix was calculated using Treebest-1.9.2 software, and based on this data, a phylogenetic tree was constructed using the neighbour-joining method. The bootstrap values of the tree were obtained after up to 1000 calculations.VCFtools was used to calculate π, Fst, He, Ho, and F. For calculating π, the parameter was set to “–window-pi 40,000 –window-pi-step 20,000”; and for calculating Fst, the parameter was set to “–fst-window-size 40,000 –fst-window-step 20,000”.2), and data with r2 greater than 0.1 were used as the decay value of the LD.LD analysis was performed using Haploview software. The SNP spacing on the genome was fitted to the linkage disequilibrium coefficient , Hainan (HI01-HI10), and Tibet (XZ01-XZ08) were selected for selection sweep analysis . We compared the Changbai Mountain individuals with the Hainan individuals and the Tibet individuals with the Hainan individuals. We calculated the genome-wide distribution of Fst values and θπ ratios for each sliding window (in 20-kb windows with 10-kb step size). The Fst values were Z-transformed, and the θπ ratios were log2-transformed. The putative selection targets were extracted based on being in the top 5% of log-odds ratios for both Fst and θπ. The GO enrichment analysis result was compared with the Pfam database to obtain GO terms, and enrichment analysis was performed using goseq. KOBAS was adopted for KEGG enrichment, the species used was A. mellifera, and the BlastX parameter was set to -evalue 1e-5 -m8.The 10 worker bees were randomly selected from 30 sampling site, and 300 honeybee samples were collected, which basically covered all climatic regions in China . The right forewings and hindwings of the worker bees were removed with forceps, and the wings were fixed by pressing them in the middle by using two slides to make specimens. The specimens were then photographed using a German Leica LEICA-M165FC fluorescence microscopy imaging system. During image acquisition, all specimens were photographed under the same standard and scale.The forewings and hindwings of the samples were digitally marked using TPS Util and TPS Dig software, respectively , 78, witOne-way ANOVA and LSD multiple tests were performed using SPSS software , and theAdditional file 1. Additional file 2.Additional file 3.


Corythucha ciliata Say, 1832) is one of the most abundant and widespread pests on plane trees (Platanus spp.) across the globe. The native range of the species is in North America, but it has been introduced to Europe (1964), South America (1985), Asia (1995), Australia (2006), and Africa (2014). To understand the genetic background behind this successful colonisation, we analysed a fragment (1356 bp) of the mitochondrial DNA. The 327 individuals revealed 17 haplotypes forming two separated groups. One group includes North American and Japanese individuals, while the other group comprises North American, European, and Asian individuals. We found a much higher genetic diversity in the native area  than in the invaded areas . The longer DNA fragment provided detailed information about the genetic structure of the species both in its native range and in the invaded areas, but the shorter DNA fragment could not provide a clear link between the genetic variation and the geographic origin.The sycamore lace bug  is of North American origin, but after its introduction to Europe (1964), South America (1985), Asia (1995), Australia (2006), and Africa (2014), it became an abundant and widespread pest on plane (Platanus spp.) trees. We analysed a 1356 bp long fragment of the mtDNA (COI gene) of 327 sycamore lace bug individuals from 38 geographic locations from Europe, Asia, and North America. Seventeen haplotypes (17 HTs) were detected. C. ciliata populations from North America exhibited higher haplotype diversity (12 HTs) than populations from Europe (6 HTs), Asia (4 HTs), or Japan (2 HTs). The haplotypes formed two haplogroups separated by at least seven mutation steps. One of these mutation steps includes HTs from North America and Japan. Another includes HTs from North America, Europe, and Asia. Haplotypes from Asia Minor, the Caucasus, and Central Asia are linked to haplotypes from Europe, while haplotypes found in Japan are linked to haplotypes found in North America only. The incorporation of published data from the GenBank into our dataset  did not show any structure according to the geographic origin of the individuals.The sycamore lace bug ( Understanding the success of an invasive insect species requires knowing the source and pathway(s) of invasion and the spatial distribution of intraspecific diversity ,2. WithoCorythucha ciliata Say, 1832) is in North America, where its main hosts are Platanus spp.  Vuill., 1912), nematodes  and generalist predatory insects  proved to be possible biological control agents [Various methods to control SLB have been investigated. Yoon et al. ) examinex of SLB . Entomopl agents ,67,68.C. ciliata in the last decade. One single individual was analysed with microsatellites markers from China [Pseudacysta perseae, Heidemann 1908) using a genome skimming approach. Yang et al. [C. ciliata in the GenBank [C. ciliata were published in 2020 [A few genetic studies have been conducted on om China . The genom China ,62,70 asom China ,72. Kochom China  publisheg et al. ,74 analy GenBank . Some pr in 2020 , but thi in 2020  analysedAdelphocoris fasciaticollis Reuter, 1903 (Hemiptera: Miridae) [Merodon sp., Diptera: Syrphidae) [Anopheles darlingi Root, 1926 (Diptera: Culicidae) [Scarabaeus (Coleoptera: Scarabaeidae) [Pyllonorycter platani  [Several studies used a fragment that was longer (>1100 bp) than the barcode fragment of COI. Some examples of this method include the plant bug Miridae) , hoverflrphidae) , Anophellicidae) , Scarababaeidae) , and Pylariidae) .Corythucha ciliata both in its native and invaded area, (ii) to explore the species’ phylogeographic pattern across three continents , (iii) to revisit the possible introduction events of the species, and (iv) to reanalyse our data with the already published datasets.Our aims were (i) to reveal the genetic structure of C. ciliata from Europe, Central Asia, Japan and North America and one Hungarian population of Corythucha arcuata   . All samA 1356 bp long region of the COI gene was amplified for 327 individuals by using Pat (5′-TCC AAT GCA CTA ATC TGC CAT ATT A-3′), and LCO1490-J-1514 (5′-GGT CAA ATC ATA AAG ATA TTG G-3′) primers ,84. PCR Sequences were generated at the Eurofin’s Laboratory . All sequences are available at NCBI GenBank (accession numbers OM033605-621).Corythucha arcuata  sequence (OM033622) was used as an outgroup. Genetic distances were estimated using the Kimura 2-parameter and computations were completed in MEGA 5.02 [Three hundred and twenty-seven individuals were used for mitochondrial DNA (COI) analyses . SequencEGA 5.02 .We applied jModeltest 2.1.2 ,89 to sePopulation structure: Patterns of molecular diversity based on the mtDNA sequences between and within populations were assessed by estimating nucleotide diversity (π) , transitGenetic distances between groups  and within groups were estimated using the Kimura 2-parameter and computations were completed in MEGA 5.02 .Demographical expansion: Population dynamics analyses were performed on different geographical scales: overall dataset, between natural and invaded area, within natural and within invaded area, between continents, and within continents, with special emphasis on European populations. Arlequin 3.5.1.2 with 10,000 permutations  was usedPhylogeographical analysis: Spatial analysis of molecular variance (SAMOVA) was performed using SAMOVA v1.0 . The proQGIS 2.18.11  was usedSeventeen haplotypes were detected on the 1356 bp long fragment of the COI gene from 327 individuals from 38 localities . The varC. arcuata and C. ciliata 8.49–8.93%.The haplotypes formed two haplogroups (A and B), which were separated by at least seven mutation steps . The 14 The genetic distance between populations was 0.00–0.65%; within populations, 0.00–0.52%; and the overall mean distance (TOTAL DATASET) was 0.20%. Overall, haplotype diversity (h) was 0.73, and nucleotide diversity (π) was 0.20% .Altogether 12 haplotypes were detected among the sequences of the 40 specimens collected in North America (five sampling locations). Ten of these haplotypes  were unique. HT4 was the most common (32.50%) and was found in all populations except Orlando . This haNeutrality test results showed that neither Tajima’s D nor Fu’s Fs values were significant. Mismatch distributions showed multimodal (SSD = 0.041) waves, associated with a constant population size ,110.Average sequence divergence between invaded and natural groups (0.46%) was higher than at the intrapopulation level . The genetic distance within the natural group was nearly three times higher (0.35%) than within the invaded group (0.12%).Fu’s Fs and Tajima’s D values were not significant. Mismatch distribution (SSD = 0.020) shows a multimodal shape, which suggests a constant population size ,110.Five haplotypes were detected among the sequences of the 250 specimens collected in Europe (29 locations). Two of these were common (HT1 44.40% and HT5 31.20%) while HT9 (23.20%), HT15 (0.80%), and HT17 (0.40%) were unique for Europe. HT9 was common in the populations from Central Europe and the Balkan Peninsula. Europe is represented in haplogroup B only . HaplotyMismatch distributions show a slightly unimodal (SSD = 0.015) shape for the European dataset. Unimodal distributions are generally associated with a sudden/recent population expansion or bottleneck ,110.Four haplotypes were observed among the 37 specimens collected in Asia (four locations) with one being (HT3) unique to the continent. The population from Japan differs from the other Asian populations unambiguously because HT3 and HT4 were only observed there, while no other Asian or European haplotype was detected there. HT1 (24.32%) and HT5 (51.35%), common in Europe, were found from Asia Minor, the Caucasus and Central Asia, and are included in Haplogroup B. Haplotypes of the Japanese population are present in both haplogroups. Diversity indices are slightly lower than the North American values .The results of the neutrality tests  did not provide significant values.Intrapopulation divergence was two times higher in the Asian (0.22%) group than in the European (0.10%). The Asian group was better differentiated (due to the population from Japan) than the European group.The FCT values reached a plateau at K = 4 (FCT = 0.702) by SAMOVA, but the four groups did not consist with the geographic distribution.Results of AMOVA demonstrated that the largest variation (44.64%) occurs among natural and invaded groups , while 3Our data  were supplemented by the results of Yang et al.  190 ind. Yang etThe haplotype SLB2 was detected in 43.52% of the individuals across the entire invaded area, but it could not be confirmed in the native area. The other common haplotype was SLB5 (37.33%), which was present across all continents. In our samples from Europe , Asia Minor (SLB5), Caucasus (SLB2), and Central Asia,  we detected only two haplotypes with various patterns. There were only two, albeit different, haplotypes from Japan . Yang et al.  detectedNone of the neutrality tests  provided significant results.The FCT values reached a plateau at K = 8 (FCT = 0.652) by SAMOVA, but the set of the eight groups did not consist with the geographic distribution.p < 0.01; FSC = 0.376, p < 0.01), indicating that there may be factors limiting the gene flow among regions.AMOVA analysis detected the largest variation (57.47%) among natural and invaded groups , while oAltogether five haplotypes were observed in the invaded area, where SLB2 and SLB5 were the two most common haplotypes (47.17% and 38.16%). SLB2-SLB4 were detected only from the invaded area.In the invaded area we detected moderate haplotype diversity with low nucleotide diversity indices . Haplotype diversities were moderate and nucleotide diversities were low in both the Far East  samples and in the European samples .We observed 0.51% average sequence divergence between invaded and natural populations. Within-group divergence of the invaded area was approximately half (0.21%) of the natural range of within-group divergence (0.40%). Sequence divergence was 0.30% within the Asian group and 0.08% within the European group, while the sequence divergence between Asia and Europe was 0.25%.The neutrality tests  usually were not significant in the most of investigated groups  except the invaded group, where the mismatch distribution (SSD = 0.019) had an unimodal shape. This is a common finding in populations that experienced recent population expansion or bottleneck ,110.The question of genetic diversity and the term of the invasion is interesting because approximately 60 years have passed since the invasion began in Europe. Regardless, we revealed only two haplotypes  in Europe. Only 20 years have passed since the invasion began in the Far East, yet we revealed five haplotypes  there.Nine haplotypes were detected in the native range where SLB1 (40%), SLB5 (27.5%), and SLB8 (15%) were the most common and SLB6–12 were unique. From North America, we observed high haplotype diversity with low nucleotide diversity  and revealed average sequence divergence (0.40%) that was nearly two times higher than in the invaded area (0.21%). Fu’s Fs and Tajima’s D values were not significant. Mismatch distribution (SSD = 0.035) shows multimodal shape, which is usually associated with constant population size ,110.Corythucha ciliata populations. Interspecific divergence of the COI gene in the plant bugs (Miridae) was reported as 6.30% [C. ciliata belongs. The interspecific divergence values between C. arcuata and C. ciliata varies 8.49–8.93% in our study.We detected moderate haplotype diversity (17 HTs) on the long (1356 bp) fragment of the COI gene in the as 6.30% . Park etas 6.30%  detectedApolygus species (Miridae) 0.40% [Scolopocelis). In our study, we also detected 44 times higher interspecific divergence. COI sequences of C. ciliata showed higher genetic differentiation than avocado lace bug (P. perseae), where altogether nine haplotypes from 469 individuals with 16 polymorphic sites were found [Intraspecific distances for other Heteropteran species were reported 0–7.72% (mean distance 0.74%) , and fore) 0.40% . In our e) 0.40%  revealedre found . The hapre found . While wre found . Furtherre found  as well.re found . Severalre found  report tThe genetic structure  togetherSeveral papers have analysed the applicability of COI fragments of different lengths and locations on the mtDNA. Roe & Sperling  suggest Corythucha ciliata. New populations need to be included in future analyses, particularly in Asia.We detected significantly higher values in most of the diversity indices  values on the longer fragment than on the barcode fragment. Our study could not reveal all possible links and connections in the invaded areas of


Listronotus oregonensis, a major pest of several apiaceous crops in North America. To undertake genetic studies on this species, we assembled the first complete genome sequence for L. oregonensis. Then, we used both haplotype discrimination with mitochondrial DNA (mtDNA) and a genotyping‐by‐sequencing (GBS) approach to characterize the genetic population structure. A total of 220 individuals were sampled from 17 localities in the provinces of Québec, Ontario, Nova Scotia (Canada), and the state of Ohio (USA). Our results showed significant genetic differences between distant populations across North America, indicating that geographic distance represents an important factor of differentiation for the carrot weevil. Furthermore, the GBS analysis revealed more different clusters than COI analysis between Québec and Nova Scotia populations, suggesting a recent differentiation in the latter province. In contrast, we found no clear evidence of population structure associated with the four cultivated apiaceous plants tested  using populations from Québec. This first characterization of the genetic structure of the carrot weevil contributes to a better understanding of the gene flow of the species and helps to adapt local pest management measures to better control this agricultural pest.Population genetic studies of insect pests enhance our ability to anticipate problems in agroecosystems, such as pest outbreaks, insecticide resistance, or expansions of the host range. This study focuses on geographic distance and host plant selection as potential determinants of genetic differentiation of the carrot weevil  In addition, a whole‐genome assembly was produced and released as the first draft genome for this species. More specifically, these genomic resources for the carrot weevil allowed us to examine the role of geographic distance and host plant selection in structuring populations.The main objective of this study was to investigate the genetic structure of 22.1D. carota L. var. sativus), celery (A. graveolens L. var. dulce), parsley (P. crispum L.), and celeriac (A. graveolens L. var. rapaceum), using carrot‐bait traps  species of host plant . For each individual, the six legs were removed from the body and frozen at −80°C for at least one hour to facilitate tissue grinding. Legs were then crushed in extraction buffer directly into the 1.5‐ml Eppendorf tube using a pestle (Ultident Scientific). Samples were incubated overnight at 56°C (16 h) in the lysing buffer, and all subsequent steps followed the recommendations from the extraction kit. DNA was eluted in 100 μl of distilled water and stored at −20°C. Concentration and purity of extracted DNA were analyzed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and a Qubit fluorometer (Invitrogen). DNA amount per sample was normalized to between 100 and 500 ng/μl for mtDNA (COI) sequencing and to 2 ng/μl for the GBS approach.2.3Fifty weevils from the rearing colony, aged between 2.5 and 3.5 months, were used to extract gDNA using the method described above. The concentration was assessed on the Qubit fluorometer (Invitrogen) system using the Qubit dsDNA HS Kit. Prior to library preparation, extraction quality was confirmed on the Agilent 2100 Bioanalyzer using the Agilent DNA 7500 Kit. Libraries were then prepared for both long‐read and short‐read sequencing. The sequencing libraries for the Oxford Nanopore MinION were prepared using the Ligation Sequencing Kit (SQK‐LSK110) following the manufacturer’s recommendations. The resulting library was sequenced on R9.4.1 MinION flow cells (Oxford Nanopore Technologies) for 72 h or to pore exhaustion. A total of five libraries, from the same DNA extraction, were generated and sequenced. Short‐read sequencing library preparation was carried out at McGill and Genome Québec Innovation Center  and sequenced (paired‐end 2 × 150 bp) on a single lane of NovaSeq 6000 (Illumina).2.4Sequences from the five nanopore runs were merged before being simultaneously basecalled, trimmed of adaptor sequences, and filtered based on quality scores using Guppy  with the flags qscore_filtering and min_qscore 7 set. The Illumina short reads were trimmed of adaptors and filtered based on a minimum Q score of 30 using Fastp  having different molecular evolutionary rates , 0.5 μl of dNTP (10 mM), 1.5 μl of 10 μM forward and reverse primers, 0.5 μl of MgCl2 (25 μM), and 2 μl of template DNA. PCRs were realized using a Mastercycler® thermo‐cycler (Eppendorf) with the following cycler conditions: 94°C during 3 min ; 5 cycles at 94°C for 30 s (denaturation), 45°C for 30 s , and 72°C for 30 s (extension); followed by 35 cycles at 94°C for 30 s (denaturation), 48°C for 30 s , and 72°C for 30 s (extension); and 72°C for 10 min . PCR product sizes were visualized by electrophoresis on a 1.5% agarose gel (BioShop). The amplified PCR fragments were sequenced with a 3730xl DNA Analyzer (Applied Biosystems) at the Genome Québec Innovation Center and McGill University . Then, forward and reverse sequences were assembled and edited to 685 bp using CLC Main Workbench V20.0 (Qiagen). Sequence alignment was performed on MEGA X, using MUSCLE with the default options  was amplified by PCR using the universal primers LCO1490 (5′‐GGT CAA CAA ATC ATA AAG ATA TTG G‐3′) and HCO2198 (5′‐TAA ACT TCA GGG TGA CCA AAA AAT CA‐3′)  and nucleotide (π) diversity were generated using ARLEQUIN v3.1  were calculated to compare each pair of populations based on haplotype frequency. Networks to depict relationships among haplotypes were also produced using Pop Art with a statistical parsimony network (TCS network)  using BWA , and the inbreeding coefficients (FIS). FST values were calculated using the fstats function to compare each pair of populations for each variable site  with the function dudi.pca from the ade4 package  and the geographic distance using the Mantel test with 1000 permutations was performed using the adegenet package.GBS reads were processed using the software Stacks v.2.54  under 0.05 and a posterior probability  above 0.91, which correspond to a strong relationship on the Jeffreys scale . Then, a locus‐specific loading plot was created using a threshold of 0.001 to identify the SNPs that most contribute to separating individuals on the first two components. Additional PCAs were produced using only the outlier SNPs. Then, the position of the outlier SNPs was retrieved from the genome assembly and compared for both methods. For the SNPs located in predicted genes, their putative function was determined by comparing the coding sequence with the National Center for Biotechnology Information (NCBI) protein database by means of BLASTX  and 1367.4 ± 0.7 Mb in females (N = 6). This draft consisted of 41,689 contigs with a L50 of 5172 contigs and an average G + C content of 30.95% , under the project ‟Carrot weevil population geneticsˮ (CAWE001‐20–CAWE206‐20). Corresponding GenBank accession numbers are MW471412–MW471617.We obtained 220 sequences of 685 bp of the partial COI from 17 populations of S) were observed, with a haplotype diversity (H) ranging between 0.562 and 0.964 (Table π) was low for all populations and ranged from 0.002 to 0.003. A total of 23 haplotypes were identified (Hap1–Hap23) across carrot weevil populations  compared with other geographic regions (N = 20). The AMOVA detected a significant population structure where most of the total genetic variability in carrot weevils was among geographic regions (45.55%) and within populations  revealed several negative values , QC‐DE , and QC‐LL .Based on the network analysis, Ontario and Ohio were the most genetically distant from Québec and Nova Scotia populations, Nova Scotia being genetically closer to Québec than to other populations Figure . PopulatFST values in COI were low and nonsignificant in the majority of the Québec samples, indicating low genetic differentiation, except for QC‐DE (FST = [0.000–0.232]) , followed by Nova Scotia (FST = [0.180–0.370]) and Ontario (FST = [0.121–0.295]). The rearing colony population was found to be similar to the field populations in Québec, as also shown by the network analysis  , with most of the genetic variations (98.80%) being observed within sampling locations . Paired‐end reads were aligned to the carrot weevil reference genome, which led to the identification of 7393 SNPs. Observed heterozygosity varied little across the populations (Ho = 0.150–0.169), and the lowest value was observed in the rearing colony (QC‐AAFC) with a Ho of 0.150 (Table FIS) was relatively high and ranged between 0.052 and 0.126.We sequenced 157 individuals from 17 populations of 50 Table . Nucleotr² = 0.6989, p = 0.01; Figure A principal component analysis of the data obtained by GBS revealed a clear partition between the Nova Scotia populations and all the other tested populations Figure . AlthougThe membership probabilities of the DAPC analysis for each individual revealed a strong admixture among the populations from Québec and to a lesser degree, with those from Ontario Figure . The twoFST values obtained with GBS indicated a low genetic differentiation among Québec populations (FST ranging between 0.000 and 0.013), with the exception of QC‐AAFC , while Nova Scotia (FST ranging between 0.127 and 0.179) and Ohio (FST ranging between 0.076 and 0.168) populations were significantly more differentiated. A linear regression of the FST values obtained with GBS and COI revealed a significant correlation between both genotyping approaches  and contained a conserved domain encoding a DDE superfamily endonuclease (pfam03184).The Bayesian method for outlier detection (BayeScan) identified 15 SNPs putatively under selection Figure , while t4Our study revealed significant genetic differences between distant populations of the carrot weevil across North America, indicating that geographic distance represents a key factor of differentiation. In contrast, no clear evidence of sympatric speciation or genetic differentiation associated with the host plant was observed in the populations of Québec collected from four different cultivated apiaceous plants .Analyses of both mitochondrial and nuclear DNA allowed the characterization of the genetic structure of carrot weevil populations at two timescales. Mitochondrial DNA goes much further back in the evolutionary history of a species compared with nuclear DNA that reveals more recent changes Avise, . For exaAnthonomus grandis Boheman) is not an efficient flyer  are isolated by distance, while at the local scale (~50 km), there is a continuous genetic flow among individuals.Both analyses (mtDNA and GBS) showed great haplotype diversity and complex genetic structure among populations of carrot weevils, which is characteristic of native species , a pest of rosaceous, with geographic distance being a strong driver of population differentiation. For the fall webworm (Hyphantria cunea Drury), although geographic distance and host plant were correlated, geographic distance was the main factor contributing to genetic variations between populations  showed a genetic differentiation between individuals that feed on a native host plant, the northern water milfoil (Myriophyllum sibericum Komarov), and individuals that prefer a congeneric introduced species, the Eurasian water milfoil (Myriophyllum spicatum L.)  was previously feeding on the ancestral downy hawthorn (Crataegus mollis Torrey and Gray) but developed host shift for the domesticated apple tree , leading to sympatric host race formation , to maintain its populations in the agricultural environment, a pattern that may prevent host plant specialization. However, the impact of wild host plants on the carrot weevil population dynamics remains to be determined. Van Tol et al. (Otiorhynchus sulcatus Fabricius) to feed and reproduce on numerous species of less‐preferred host plants contributes to their establishment in different habitats.At the finer geographic scale, comparison of carrot weevil populations captured from different host plants did not reveal significant genetic differentiation. A slight clustering by hosts was detected by a discriminant analysis on the GBS data, but this result was only partially supported by additional analyses. Two genome scan approaches were compared to identify outlier SNPs linked to host plant differentiation, but only one outlier was detected and shared by the two methods. In addition, the frequency of these alleles varied greatly among individuals collected from a given host and, while the frequency of an allele differed slightly between populations, its presence was not mandatory to develop on a given. Interestingly, the outlier SNP detected by the two approaches was located in a gene with a conserved domain generally associated with transposable elements, which are known to contribute to genome evolution and adaptation in insects     species such as the red palm weevil, From an applied perspective, these results help to refine current integrated pest management strategies to control carrot weevil populations. The genotype characterization of the pest populations at the local scale represents an opportunity to find markers of interest that are associated with specific biological functions or biochemical processes. For example, insecticide resistance threatens the success of pest control programs, with carrot weevils from commercial fields in Ontario having developed resistance to the foliar insecticide phosmet (Telfer et al., The authors have no conflict of interest to disclose.Supplementary MaterialClick here for additional data file.


Globodera pallida is among the most significant plant-parasitic nematodes worldwide, causing major damage to potato production. Since it was discovered in Idaho in 2006, eradication efforts have aimed to contain and eradicate G. pallida through phytosanitary action and soil fumigation. In this study, we investigated genome-wide patterns of G. pallida genetic variation across Idaho fields to evaluate whether the infestation resulted from a single or multiple introduction(s) and to investigate potential evolutionary responses since the time of infestation. A total of 53 G. pallida samples  were collected and analyzed, representing five different fields in Idaho, a greenhouse population, and a field in Scotland that was used for external comparison. According to genome-wide allele frequency and fixation index (stF) analyses, most of the genetic variation was shared among the G. pallida populations in Idaho fields pre-fumigation, indicating that the infestation likely resulted from a single introduction. Temporal patterns of genome-wide polymorphisms involving (1) pre-fumigation field samples collected in 2007 and 2014 and (2) pre- and post-fumigation samples revealed nucleotide variants  with significantly differentiated allele frequencies indicating genetic differentiation. This study provides insights into the genetic origins and adaptive potential of G. pallida invading new environments. Globodera pallida and Globodera rostochiensis [Globodera pallida was first discovered in Idaho in April 2006 [G. pallida in Idaho caused several countries to ban all fresh U.S. potato imports, though these markets have since been reopened [G. pallida in Idaho, containment and eradication of infested fields has been ongoing [As major agricultural pathogens, plant-parasitic nematodes cause numerous diseases in plants and are responsible for more than US$100 billion in annual agriculture loss worldwide . Among tchiensis . Althougchiensis ,5. Potatchiensis . Both spril 2006 . While ireopened . Since tGlobodera pallida initially evolved in South America, then was introduced into Europe, and from there spread to North America [G. pallida in Idaho resulted from a single introduction [G. pallida populations in Idaho stemmed from a single introduction or resulted from many. Knowledge on this is important from a regulatory perspective because population-level genetic variation is a key determinant of the strength of evolutionary adaptability; several introductions could lead to high within-species genetic diversity, which would increase the likelihood and pace of adaptation to the nematodes’ new environment. America ,10. A spoduction . HoweverGlobodera pallida is a sedentary endo-parasite that undergoes sexual reproduction. Second-stage juveniles invade plant roots and establish feeding sites. As development continues, fertilized females enlarge to form cysts containing 200 to 600 eggs. Eggs require hatching factors, which are specific chemical cues released from host roots that induce hatching. In the absence of the host, eggs can remain dormant in the soil for many years [G. pallida [G. pallida. Since G. pallida was discovered in Idaho in 2006, eradication efforts have aimed to contain and eradicate it through phytosanitary action and soil fumigation [ny years . Plantin pallida . Crop roStudies of the genetic structure of populations provides important insights into the origins of invasive populations and their potential for adaptation to local and changing environmental conditions. Populations are generally thought to adaptively evolve through one of two main routes: wait for the appearance of a rare novel mutation which will sweep through the population if advantageous, or alternatively, through standing  genetic variation . A thirdF-statistic/fixation (stF) index (differentiation outlier method). The second strategy tests the association between allele frequencies and environmental variables . There are two main strategies aimed at identifying regions of the genome that may be responsible for local adaptation, particularly for non-model organisms ,19. The stF is a widely used pairwise measure of population-level genetic differentiation and structure [stF values range from zero to one; a zero value implies that the two populations under investigation are interbreeding freely and completely share genetic diversity, while a value of one implies complete isolation and an absence of shared genetic diversity. Local adaptation leads to strong genetic differentiation between populations, but only at specific genetic loci. Thus, loci with very high stF values compared to the rest of the genome are commonly identified to be under local adaptation and are referred to as outliers [tructure . It is foutliers . Geneticoutliers . Here, rG. pallida field populations to: (1) evaluate the genetic variation of G. pallida across fields to determine if the infestation was a result of a single or multiple introduction(s), and (2) explore temporal patterns of genome-wide polymorphisms to evaluate the adaptive potential of G. pallida populations in Idaho since the time of infestation.In this study, a whole genome sequencing approach coupled with analysis strategies mentioned above were applied to Idaho G. pallida populations from Idaho fields, the whole genomes from 53 cyst samples were sequenced; a population maintained in the greenhouse and a population from Scotland were also analyzed for comparative purposes. The yield of Illumina reads passing quality control measures varied from ~25 to 50 million reads for 52/53 samples  in some of the post-fumigation samples  were considered in subsequent analyses.To compare patterns of genome-wide genetic variation among  samples . One sam samples . This obG. pallida populations. Each survey  to find the optimum minimum coverage threshold per survey. The total number of SNPs per survey decreased with increasing minimum coverage threshold . The genome-wide SNP-specific stF values calculated through pairwise population comparisons with BIN258 ranged from 0.0 to 0.175 . Accordingly, we identified these twelve SNPs as significantly differentiated SNPs in BIN258 field population compared to the four field populations detected in 2007 . The twelve SNP outliers  were located on two scaffolds of G. pallida genome: HG821129 and HG821413. The annotation results revealed genes (coding regions) associated with one of the SNP outliers. The single SNP outlier detected on scaffold HG821413 was associated with CLAVATA3/ESR (CLE-1) gene and identified as a non-synonymous mutation (amino acid replacement in the polypeptide). SNP outliers in the HG821129 scaffold  were placed in an intergenic region (non-coding) (When a field where infestation was more recently detected in 2014 (BIN258) was included in the analysis with the four 2007 fields , an average to 0.175 a. For th-coding) . G. pallida population maintained in a greenhouse at the University of Idaho, established from cysts collected from the BIN25 field population, was also included in the analysis for comparative purposes (survey III). stF analysis comparing samples obtained from field and greenhouse G. pallida populations had a low average stF of 0.009. All genome-wide SNP-specific stF values calculated through pairwise population comparisons with the greenhouse population were less than 0.05 . A han 0.05 b. For thG. pallida population obtained from Scotland was used as an outside point of reference. When the Scotland population (Luff) was included in the analysis with the 2007 Idaho field populations , an average stF of 0.05 was obtained (survey IV). This was the largest average stF obtained compared to analyses including BIN258  or greenhouse . In the analysis where stF was calculated for every SNP, the between population pairwise comparisons involving the Scotland G. pallida population had a genome-wide stF distribution that reached up to 0.3 . The SNP-specific allele frequency analysis supported this observation with statistical evidence. From a total of 8296 SNPs, 1855 SNPs had p-values that exceeded the genome-wide significance threshold , indicating significant genetic differentiation between Idaho and Scotland G. pallida populations. These 1855 SNP sites were located on 257 scaffolds of G. pallida genome.In order to provide an external context for our analyses, which were limited to Idaho field samples, a p to 0.3 c. The reG. pallida field populations: BIN25, BIN54, BIN258 . All the results presented here were obtained utilizing four biological replicates per field and from two time points . Genome-wide SNP-specific stF values calculated for pre- and post-fumigation sample comparisons provided evidence for significant genetic change in some regions in the genome over time. In order to provide an analytical context for pre–post comparisons, we used stF values resulting from pre–pre comparisons and post–post comparisons as references from each field. All genome-wide SNP-specific stF values resulted from comparison between BIN25 pre-fumigation samples (25-0 vs. 25-0) were less than 0.17. This included all possible pairwise comparisons between the four replicates of BIN25-0 (pre-fumigation). Similarly, comparisons between all BIN25 post-fumigation samples (25-1 vs. 25-1) had genome-wide SNP-specific stF values less than 0.12. However, the comparisons between BIN25 pre- and post-fumigation samples (25-0 vs. 25-1) had SNP-specific stF values that reached up to 0.35, indicating high genetic differentiation between pre- and post-fumigation samples obtained from this field based on our threshold stF of 0.25 , we found 73 sites (out of 956) with p-values that exceeded the standard genome-wide significance threshold (p < 5 × 10−8). These 73 SNP outliers  were located on twelve scaffolds in the G. pallida genome resource  had genome-wide SNP-specific stF values less than 0.02. But comparisons between BIN54 pre- and post-fumigation samples (54-0 vs. 54-1) had SNP-specific stF values as high as 0.24, which was closer to our threshold stF value of 0.25 . These 22 SNP outliers were located on twelve scaffolds in the G. pallida genome (The comparisons between BIN54 pre-fumigation samples (54-0 vs. 54-0) had genome-wide SNP-specific  of 0.25 . When SNa genome b. Annotaa genome . stF values resulting from comparison between BIN258 pre-fumigation samples (258-0 vs. 258-0) were less than 0.03. Similarly, comparisons between BIN258 post-fumigation samples (258-1 vs. 258-1) had genome-wide SNP-specific stF values less than 0.07. The comparisons between BIN258 pre- and post-fumigation samples (258-0 vs. 258-1) had SNP-specific stF values that reached up to 0.16 . These twelve SNP outliers were located on four scaffolds in the G. pallida genome . This is important from a regulatory perspective since several introductions could lead to high within-species genetic diversity, which would increase the likelihood and pace of adaptation to the nematodes’ new environment. Our assessment of population genetic structure using the stF index revealed patterns of genetic interrelatedness among G. pallida populations obtained from the Idaho fields. First considering the four fields, BIN25, BIN26, BIN54, and BON64, all 16 cyst samples from these four fields were collected in 2007 before fumigation began; therefore, any genetic effect that might occur from fumigation can be disregarded. The average stF for these four Idaho fields was estimated to be 0.009, which is very low. Wright suggested the following qualitative guidelines for the interpretation of stF: the range 0.0 to 0.05 indicates little genetic differentiation, the range 0.05 to 0.15 indicates moderate genetic differentiation, the range 0.15 to 0.25 indicates great genetic differentiation, and values above 0.25 indicate very great genetic differentiation [stF of 0.009 obtained for the four Idaho fields indicates close genetic relationships among the nematodes in these fields. Understanding the genetic structure of parasite populations and the process of genome evolution and adaptation is essential for crafting effective control strategies . Accordintiation . Based ostF guidelines are general recommendations; stF is a relative rather than an absolute measure of genetic differentiation, and the value depends on the choice of populations or heterozygosity within populations. For example, stF values can be as low as 0.006 within one species, while that of another species can be as high as 0.714 [stF between human populations being 0.15 (15%) [Baylisascaris schroederi had stF values ranging from 0.01 to 0.02 among different populations [G. pallida field populations in Idaho with a G. pallida population obtained from Scotland and a population maintained in a greenhouse at the University of Idaho. When the Scotland population was added to the analysis with the Idaho fields , the average stF notably increased to 0.05. This is a 10-fold increase in relative terms and moderate genetic differentiation according to Wright’s general guidelines [G. pallida populations obtained from the same area in France had stF values less than 0.1, while populations obtained from different European countries had stF values that showed a 10-fold increase and reached up to 0.45 [G. rostochiensis had stF values ranging from 0.02 to 0.40 among different populations [stF remained the same (0.009). This low stF value supports the finding that the G. pallida populations in Idaho fields and greenhouse are genetically similar in both relative and Wright’s terms. In fact, the greenhouse population was originally collected from the infested field BIN25 and maintained on susceptible potato (Solanum tuberosum) “Désirée” or “Russet Burbank”. The relative stF values confirmed close genetic relationships among the nematodes in Idaho fields. However, Wright’s as 0.714 . For clo15 (15%) . The aniulations . Therefoidelines  between ulations . When thstF results. We found no statistically significant differences in SNP-specific allele frequencies among pre-fumigation G. pallida populations obtained from these four different Idaho fields. Further, we found no statistically significant differences in allele frequencies among Idaho field and greenhouse populations. However, analysis between Idaho and Scottish populations revealed 1855 SNP sites with significantly differentiated allele frequencies (p < 5 x 10−8), indicating greater genetic differentiation between the Idaho and Scotland populations. Moreover, we obtained consistent results for all biological replicates for all above analyses according to the CMH statistical test. Based on these results, we demonstrated that most of the genetic variation was shared among G. pallida populations from Idaho fields, indicating that the infestation likely resulted from a single introduction. It is common for plant-parasitic nematodes to gain entry to new areas through agricultural commodities . After establishment and integration are achieved, mainly through local nematode reproduction, nematodes easily spread to nearby fields [G. pallida seems to have spread from field to field in Idaho depending on the introduction. Our results are consistent with a recent spatial pattern analysis of the G. pallida infestation in Idaho, which revealed that nearby infested fields contributed to the infestation of new fields through a contagion effect scenario [Our analysis on SNP-specific allele frequencies performed to detect any genomic regions with strong genetic differentiation among populations further supported the y fields ,28. Therscenario .G. pallida was more recently detected in 2014 (BIN258). The cysts collected from BIN258 prior to fumigation were used for this analysis (similar to the four fields mentioned above). These five fields  had a low average stF of 0.013, indicating very little genetic differentiation between them, similar to the results of the previous analysis. This suggests that BIN258 likely stemmed from the original infestation. However, SNP-specific allele frequencies analysis revealed twelve SNP sites with strong genetic differentiation in BIN258 compared to other Idaho fields (p < 5 × 10−8). Of the twelve outlier SNP sites observed in the analysis involving BIN258, eleven were found to be on non-coding regions of the genome. One outlier was associated with a CLE gene that is expressed in nematode parasitic stages, suggesting a role for their encoded proteins in plant parasitism [G. pallida detection in Idaho. During this time period, the field had not undergone methyl bromide fumigation but was planted with potato. Thus, approximately seven generations are estimated to have occurred between 2007 and the sample collection at BIN258, assuming one generation per year/season based on the observation that cyst nematodes are monocyclic [G. pallida and the model nematode Caenorhabditis elegans suggest that rapid and strong adaptation can happen in fewer than ten generations [Next, we compared fields where infestation was originally detected (in 2007) to a field where rasitism . This SNnocyclic . The deterations ,32. FurtG. pallida to soil fumigants. For this, 24 G. pallida samples were collected from three of the fields at two different time-points: (1) pre-fumigation and (2) the year immediately following fumigation (post-fumigation). This approach allowed us to look at potential genetic changes immediately surrounding fumigation treatments. If this genetic change is significant, it’s likely due to the survival of treatment resistant nematodes in post-fumigation populations, indicating potential for adaptation. Our analyses revealed higher genome-wide stF values between pre- and post-fumigation sample comparisons for the three fields evaluated , as compared to values between pre-fumigation comparisons and post-fumigation comparisons. This method provided the benefit of having a relative stF value to detect any genetic differentiation due to treatments, rather than having to rely on general stF guidelines. In certain regions of the genome, the SNP-specific stF values of pre–post comparisons relative to pre–pre and post–post comparisons, had a 10-fold difference and provided evidence for significant genetic change post-fumigation. Strong genetic differentiation like this due to contrasting habitats (non-fumigated vs fumigated) can be indicative of local adaptation that helps an organism to survive in a particular environment. To confirm local adaptation, one has to eliminate alternative explanations such as genetic drift. The stF, determined by genetic drift under selective neutrality, will affect all loci across the genome in a similar and predictable fashion [stF values for a selected loci [stF results. Our next goal was to investigate patterns of genome-wide polymorphisms to evaluate the adaptive potential of  fashion , and thited loci , and thip < 5 × 10−8), 107 outlier SNP sites were found between pre- and post- fumigation samples across all three fields. This further supports the existence of genomic regions with strong genetic differentiation among pre- and post-fumigation populations. Strong outlier “signals” like these are taken to be indicative of local adaptation relative to background “noise” caused by migration, drift, or any other demographic changes that affect the whole genome equally [G. pallida provides important implications for its eradication, i.e., the eradication strategies could now focus on eliminating the source of adaptive genetic variation.To further support this observation, genome-wide SNP-specific allele frequencies were calculated and compared between pre- and post-fumigation samples. When statistical testing was applied to detect any statistically significant changes in SNP allele frequencies under more conservative thresholds  from fields in southern Idaho according to guideline protocols and using an agreed upon sampling protocol outlined by USDA-APHIS ). The sampling method used by USDA-APHIS involved collecting 22.42 kg of soil samples per hectare, with cysts extracted from soil using the Fenwick flotation method [G. pallida was confirmed by morphological and molecular methods [G. pallida cysts  were processed. This included cyst samples from five different G. pallida infested fields in Idaho that were located within a 10 km radius in Bingham (BIN) and Bonneville (BON) counties  in Idaho and a European population from Scotland (Luffness)  .Samples consisting of 50 cysts  were used for DNA extraction; fewer cysts  were used from the Scotland population due to limited availability. For DNA extraction, cysts of pooled samples were crushed for 2 min with a motorized micro pestle and processed with QIAampDNA Micro kit  following the manufacturer’s protocol. Total genomic DNA was sheared for 50 s using a Diagenode Bioruptor Pico  to obtain peak library fragment sizes of ~500 bp, and genomic libraries were prepared using the NEBNext_Ultra II DNA Library Prep Kit for Illumina  following the manufacturer’s instructions. Whole genome sequencing (pool-seq) was performed using Illumina HiSeq 3000 for 2 × 150 bp reads (paired-end) at the Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR. Raw reads are available from NCBI’s  Sequence Read Archive (SRA) under BioProject number PRJNA679610. http://jgi.doe.gov/data-and-tools/bbtools (accessed on 18 March 2021)). Filtered reads were then mapped to the G. pallida reference genome  using BWA mem  [G. pallida reference genome, and all the reads that mapped ambiguously were removed using SAMtools (version 1.3) [After sequencing, raw reads were trimmed to remove adaptors and filtered for quality using bbduk-BBtools  (2-r1039) . In ordeion 1.3) . For theG. pallida populations, respectively (stF was estimated (for surveys I to IV) using the formula:tH = total heterozygosity and sH = average heterozygosity within each population.SNP calling and allele frequency computation (at every base in the reference genome) were performed for each replicate of every population using SAMtools (mpileup) . Then, pectively . First, Heterozygosity was calculated using major and minor allele frequencies of the populations being analyzed. The polymorphic sites where coverage was between 30X and 200X with a minimum minor allele frequency of 2% across all populations were considered here, and all sites failing to meet these criteria were discarded . This fistF values were calculated for all possible pairwise population comparisons. The pairwise stF values were calculated using POPOOLATION2 software package [n-fold minimum coverage thresholds (10X–70X) were applied for each survey in order to determine the optimal minimum coverage for each particular survey  and involved a two-step procedure . First,  package  with a mr survey . The calntiation . The FstG. pallida populations. To identify highly differentiated SNPs, the Bonferroni-corrected significance threshold and more stringent standard genome-wide significance threshold of 5 × 10−8 [Second, genome-wide SNP-specific allele frequencies were calculated and compared between populations . Here, SNPs were identified in each survey using identical filtering criteria, i.e., SNP sites where coverage was between 40X and 200X were considered, with a minimum minor allele frequency of 2% across all populations. All sites failing to meet these criteria were discarded. Since four independent measurements of the allele frequencies were obtained per population , Cochran–Mantel–Haenszel test statistics (CMH test)  were imp5 × 10−8  were useG. pallida reference genome was used to convert the synchronized file of scaffolds into a gene-based file [The scaffolds with SNP outliers  were further analyzed to identify coding regions which corresponded to significantly differentiated sites. The information in WormBase ParaSite database (Version:WBPS14 (WS271)) on annotated exons (coding regions/genes) of the sed file . Once th