Ceratitis capitata, is a cosmopolitan agricultural pest of worldwide economic importance and a model for the development of the Sterile Insect Technique (SIT) for fruit flies of the Tephritidae family (Diptera). SIT relies on the effective mating of laboratory-reared strains and natural populations, and therefore requires an efficient mass-rearing system that will allow for the production of high-quality males. Adaptation of wild flies to an artificial laboratory environment can be accompanied by negative effects on several life history traits through changes in their genetic diversity and symbiotic communities. Such changes may lead to reduced biological quality and mating competitiveness in respect to the wild populations. Profiling wild populations can help understand, and maybe reverse, deleterious effects accompanying laboratory domestication thus providing insects that can efficiently and effectively support SIT application.The Mediterranean fruit fly, In the present study, we analyzed both the genetic structure and gut symbiotic communities of natural medfly populations of worldwide distribution, including Europe, Africa, Australia, and the Americas. The genetic structure of 408 individuals from 15 distinct populations was analyzed with a set of commonly used microsatellite markers. The symbiotic communities of a subset of 265 individuals from 11 populations were analyzed using the 16S rRNA gene-based amplicon sequencing of single individuals (adults). Genetic differentiation was detected among geographically distant populations while adults originated from neighboring areas were genetically closer. Alpha and beta diversity of bacterial communities pointed to an overall reduced symbiotic diversity and the influence of the geographic location on the bacterial profile.Our analysis revealed differences both in the genetic profile and the structure of gut symbiotic communities of medfly natural populations. The genetic analysis expanded our knowledge to populations not analyzed before and our results were in accordance with the existing scenarios regarding this species expansion and colonization pathways. At the same time, the bacterial communities from different natural medfly populations have been characterized, thus broadening our knowledge on the microbiota of the species across its range. Genetic and symbiotic differences between natural and laboratory populations must be considered when designing AW-IPM approaches with a SIT component, since they may impact mating compatibility and mating competitiveness of the laboratory-reared males. In parallel, enrichment from wild populations and/or symbiotic supplementation could increase rearing productivity, biological quality, and mating competitiveness of SIT-important laboratory strains.The online version contains supplementary material available at 10.1186/s12863-020-00946-z. Ceratitis capitata (Diptera: Tephritidae) is a cosmopolitan species that affects numerous fruit crops worldwide [The Mediterranean fruit fly, orldwide . Femalesorldwide . Due to In the last decades, emphasis has been put on control strategies that have the least possible negative impact on environment. Among them, the sterile insect technique (SIT), as part of the area-wide integrated pest management (AW-IPM) is considered as a species-specific, environmentally friendly control method . SIT is The presence of genetically differentiated populations can lead to reduced mating compatibility between laboratory strains and SIT-targeted populations . GeneticBeing a SIT model for tephritids, medfly has been a target of extensive research in areas related to improvement of both rearing efficiency and biological quality of released males. Lots of resources have been invested in identifying the importance of gut microbiota . Studieswild’ character of the mass-reared colony. Refreshing the mass-reared strains periodically with wild material is a strategy that has been suggested to mitigate genetic issues occurring during mass-rearing [To ensure the efficacy of SIT applications, the biological quality and the continuous improvement of the mass-produced insects are of major importance. Adapting to the artificial environment poses considerable selection pressures on insects that may significantly alter both the genomic and the symbiotic profile. As a result, insects that will differ significantly from their wild counterparts, will be produced. Thus, it is required to develop a strategy that will allow maintaining the genetic and symbiotic diversity and preserve the ‘-rearing –31. Amon-rearing , 32. Up -rearing –31. The A total of 408 individuals belonging to fifteen different natural populations were analyzed  to 2.63 (Ccmic32) effective alleles per locus  and nine (Ccmic32)  and 1.105 (samples from Brazil and Honduras) .Klebsiella, Providencia, Tatumella, Citrobacter, Morganella, Enterobacter, Rahnella, and Pantoea (in order of reduced relative abundance)  and the same applied for Alphaproteobacteria   , followed by Enterobacteriaceae , followed by different Enterobacteriaceae genera, such as Klebsiella (~ 18%) and Morganella (~ 5%). In most of the remaining samples, Klebsiella was the prevailing genus, followed by Providencia , except for medflies from Honduras where Firmicutes had a higher relative abundance than Proteobacteria  . This analysis pointed to symbiotic communities of reduced diversity, with differential relative abundance and not differential OTUs presence, being the driving force of the bacterial profile structuring.As being a global pest, the invasion route and pathways of medfly have been well- studied in recent years , 35, 36.Our analysis included 15 medfly populations from Europe, Middle East, Australia, and the Americas . Sampling was expanded to regions previously not included in the population genetic studies of the species, covering Central America  and South America (with samples from Argentina and Bolivia) and more recent collections from previously studied regions. Our results are in accordance with the main proposed scenarios since a) samples from the Mediterranean basin cluster together, b) new samples from Central America are closely related to Mediterranean samples, c) the two samples from Australia are genetically very similar, which is consistent with a single invasion event (or of a single origin) and subsequent spread in different locations, and d) South America populations may form a discrete cluster, however not very different from the other medfly populations.Klebsiella oxytoca, has been identified as a major symbiont of natural populations [Pseudomonas communities have also been identified by different studies [Enterobacter is also a key member of medfly gut microbiota community, especially in laboratory populations, and has also been shown to positively influence rearing and biological quality of males produced for release purposes when supplementing larval and adult diet [Acinetobacter sp., Providencia sp., Morganella sp., and others [Few studies have been performed in the medfly to characterize microbiota communities from natural populations, aiming mainly to support SIT applications. Most of them suggest that medfly symbiotic communities constitute mainly of Gammaproteobacteria, and more precisely from rather few Enterobacteriaceae genera. Among them, ulations  and has ulations , 23. Min studies , 27, 28.ult diet –24. Micrult diet , 22, 26.d others , 22, 26 Klebsiella, Providencia, Enterobacter, Morganella, Citrobacter, and Pantoea, as being the major components of medfly microbiota communities [Rahnella in the populations from Australia_1 (1%), Australia_2 (8.3%), Nicaragua (3.2%) and Spain (3%). This genus has been rarely identified in fruit flies, with one study showing its presence in Anastrepha species [Rahnella has been found to be associated with Dendroctonus pondorosae, and it has been found to metabolize monoterpenes and diterpenes acids [Pseudomonas communities have also been described [Our analysis of the symbiotic profile of medfly expanded our knowledge on the microbiota structuring in natural populations of Europe, Australia, Middle East, and Central plus South America. Our data indicated that few Enterobacteriaceae genera (twelve) shaped symbiotic communities of the species, although many more are present in minor relative abundances in the different populations. This is in accordance with previous findings suggesting genera such as munities , 28, 38. species . Howeveres acids . Persistescribed , 27 and Previous studies also indicate that geographic origin can explain the differential presence of bacteria genera among different studies . HoweverExiguobacterium is abundant in Honduras and absent or in extremely low abundance in the rest geographically neighbouring populations. Although not previously found in medfly, Exiguobacterium has been isolated from Zeugodacus cucurbitae during an effort to identify putative attractants but it has not been further utilized [Albeit the Honduras population is genetically clustered with the rest Central American populations, their symbiotic profiles present major discrepancies. The genus utilized . In our All pairwise comparisons among the different samples showed statistically significant differentiation of the symbiotic profiles. Therefore, geographic origin is an important factor and this has been shown for medfly and fruit flies in general , 28, 42.Recent studies also point to the possible impact of methodological approaches (such as samples’ preservation) on the results. It has been shown in medfly that sample preservation in 70% ethanol influences analysis of gut microbiota . All theSIT, as well as other methodologies proposing population suppression through inducing sterility in natural populations using laboratory males, rely on the cost-efficient mass production and release of males that will be of high biological quality and competitiveness in the field. Cayol and colleagues studied wild and laboratory medfly populations and showed that mating compatibility was not negatively affected thus supporting the notion that the species has not yet evolved specific mating behaviours worldwide . HoweverPantoea, Enterobacter, and Klebsiella genera showing encouraging results [Performance in the field is an extremely important factor for the successful SIT application and can be affected both by inherent properties of the strains and the process of rearing. Rearing is a crucial step for SIT, and the initial fly material used in the rearing process as well as the genomic changes during the colonization process are important factors regarding the biological quality of the released individuals. Several studies have reported that life history traits can be affected during the laboratory adaptation process including reduction in developmental time, lifespan, dispersal ability and stress resistance, as well as early fertility and increased fecundity , 45. Any results , 19–24.  results , 48. HowSIT depends on the cost-efficient production and high performance of laboratory-reared males in the field. Genetic structure and symbiotic profile can influence mating compatibility of laboratory and natural populations thus affecting SIT performance. The present study expanded our knowledge on the genetic structure of medfly natural populations by including under-sampled regions and revisiting previously analyzed populations. Our findings are in accordance with the prevailing scenarios for the invasion events and pathways of this cosmopolitan species and did not find any indication for the presence of extremely sub-structured populations in the dataset analyzed. The symbiotic analysis shed light on the structure of the medfly natural populations’ microbiota, since eleven new samples were analyzed collected from 9 different countries. Our analysis pointed to the presence of a symbiotic community of reduced diversity, since 12 genera (mainly Enterobacteriaceae) are the key components of all symbiotic communities. However, the presence of a well differentiated microbiota community in medflies from Honduras calls for additional analysis, since important questions regarding whether it is a universal profile in the area or how that might affect insect’s behavior remain open. The genetic and the symbiotic profile of pest species in areas targeted for SIT must be studied and compared with the profiles of laboratory strains reared for release purposes. That way, either probiotic supplements or enrichment from the wild protocols can be applied to boost the efficiency of SIT.The genetic structure of fifteen medfly populations was analyzed with a set of eight microsatellite markers. Symbiotic microbiota communities were analyzed for eleven of them with high throughput Illumina sequencing of the 16S rRNA gene.Adult females and males were sampled and stored in 96–100% ethanol or propylene glycol at − 20 °C until DNA extraction. Samples’ details are presented in Additional file Medflymic30, Medflymic43, Medflymic74, Medflymic78 [Ccmic6, Ccmic9, Ccmic14, and Ccmic32 [https://www.geneious.com) loaded with the Geneious microsatellite plugin (https://www.geneious.com/plugins/microsatellite-plugin/) was used to visualize the traces, fit the Genescan 500 LIZ ladder, call peaks, predict bins, and create the amplicon size scoring matrix.The following microsatellite markers were used in the present analysis: flymic78 , Ccmic6, Ccmic32 . All forK = 1 to 10 and performed 20 repetitions for each potential K. STRUCTURE HARVESTER [K-value by calculating ln(K) and delta K, based on the modification described by Evanno et al. [Genetic variability parameters, including the effective number of alleles (Ne), the observed (Ho), and expected (He) heterozygosities under Hardy- Weinberg equilibrium, were measured using GenAlEx version 6.51 . AnalysiARVESTER  was usedo et al. ).DNAs were tested for quality and quantity using a Q5000 micro-volume UV-Vis spectrophotometer  and were stored in Eppendorf tubes at − 20 °C until PCR amplification and amplicon sequencing analysis were carried out.− 1 and 32.4 μL sterile deionized water. The temperature profile used for the PCR was: 95 °C for 3 min followed by 8 cycles of 95 °C for 30 s, 30 s at 55 °C, 1 min at 72 °C and a final extension step of 72 °C for 3 min. The resulting amplicons were purified using the NucleoMag NGS Clean-up and Size Selection kit  following the manufacturer’s instructions. Indexed amplicons from all samples examined were mixed in equimolar ratio (8 nM) and sequencing was performed by Macrogen using a 2x300bp pair-end kit on a MiSeq platform.For the amplicon sequencing analysis, the hypervariable V3-V4 region of the bacterial 16S rRNA gene was amplified using MiSeq universal primers 341F and 805R . The firAfter sequencing, bioinformatic analysis was performed using USEARCH version 11  and QuanQ_m’HQ_m statistics, and were performed with 9999 permutations within PRIMER version 6+ [Richness, Simpson, Shannon, and Evenness indices of alpha diversity, which reflect the diversity of individual samples were calculated based on the “diversity” function of the “vegan” R package and plotted using “ggplot” function of the “ggplot2” package. Pair-wise ANOVA was used to identify significant differences of alpha diversity indices between the different locations. Beta diversity was analyzed to evaluate the similarity of bacterial communities from different locations using Generalized UniFrac distance  and visursion 6+ . Cap anaAdditional file 1:Table S1. Samples analyzed.Additional file 2:Table S2. Microsatellite markers used and diversity indices.Additional file 3:Table S3. Deviations from HWE.Additional file 4:Figure S1. Analysis of Molecular Variance.Additional file 5:Table S4. Pairwise genetic distances matrix (Nei 1972).Additional file 6:Figure S2. Identification of ‘true’ number of populations using the modification of Evanno and colleagues (2005).Additional file 7:Figure S3. Relative abundances of medfly microbiota at A) Phylum level, B) Class level, and C) Genus level.Additional file 8:Figure S4. Alpha diversity of the different samples and pairwise comparisons A) Richness index, B) Shannon index.Additional file 9:Table S5. PERMANOVA pairwise values .Additional file 10:Figure S5. MDS plot of the microbial profiles of male and female medflies.Additional file 11:Figure S6.The different OTUs (putative species) assigned to Klebsiella genus and their relative abundance in the medfly natural populations.Additional file 12:Figure S7.The different OTUs (putative species) assigned to Morganella genus and their relative abundance in the medfly natural populations.Additional file 13:Figure S8.The different OTUs (putative species) assigned to Providencia genus and their relative abundance in the medfly natural populations.Additional file 14:Figure S9.The different OTUs (putative species) assigned to Enterobacter genus and their relative abundance in the medfly natural populations.


Helicoverpa zea (Boddie) captured during 2002, 2005, 2016, and 2018 from Landisville and Rock Springs in Pennsylvania, USA were genotyped using 85 single nucleotide polymorphism (SNP) markers. Samples (n = 702) genotyped were divided into 16 putative populations based on collection time and site. Fixation indices (F-statistics), analysis of molecular variance, and discriminant analysis of principal components were used to examine within and among population genetic variation. The observed and expected heterozygosity in putative populations ranged from 0.317–0.418 and 0.320–0.359, respectively. Broad range of FST (0.0–0.2742) and FIS (0.0–0.2330) values indicated different genotype frequencies between and within the populations, respectively. High genetic diversity within and low genetic differentiation between populations was found in 2002 and 2005. Interestingly, high genetic differentiation between populations from two collection sites observed in 2018 populations was not evident in within-site comparisons of putative populations collected on different dates during the season. The shift of H. zea population genetic makeup in 2018 may be influenced by multiple biotic and abiotic factors including tropical storms. Continued assessment of these peripheral populations of H. zea will be needed to assess the impacts of genetic changes on pest control and resistance management tactics.Migrant populations of  Insect species living in temperate climatic zones with broad geographic distributions and long-range migration capacity disperse seasonally throughout habitable geographic regions when favorable conditions arise. Migration allows species to exploit food sources, avoid competition, and find mates that immigrated from other populations to reduce inbreeding . AdmixtuHelicoverpa zea (Boddie) (Lepidoptera: Noctuidae), commonly known as bollworm, corn earworm, or tomato fruitworm, is one of the several noctuid moths in the “heliothine complex” of pests in the Americas. Helicoverpa zea is a major pest of more than 300 plants species including economically important crop species such corn, cotton, soybean, and tomatoes [H. zea vary depending on the crop and the estimates are mostly based on an average of cost of control and crop losses of heliothine pest species. In 2019, approximately 1.3 million hectares of cotton cultivations  was infested by heliothine pests and the total economic damage (loss plus cost of control) was estimated to be over $116 million [H. zea in southern United States in 2012 was approximately $49 per ha, but could be greater with higher pest pressure [H. zea causes damage to both field and sweet corn ears [H. zea on cotton, corn, and soybean alone could exceed $100 per ha. Therefore, H. zea can be considered one of the most important pests of agriculture in North America.The polyphagous moth tomatoes ,11,12. E million . The cos million . Soybeanpressure ,15,16. Iorn ears ,18,19. Iorn ears . CombineH. zea within North America is well documented [H. zea to disperse over long, medium, and short ranges provides this species with several advantages including avoiding competition for food resources by following crop planting schedules during the growing season, reproducing with genetically diverse mates to maintain population genetic diversity, and adaptations to changing environmental and climatic conditions. With widespread planting of transgenic crops expressing Bacillus thuringiensis toxins (Bt crops) across the continent [H. zea is constantly under pressure to overcome deleterious effects of these Bt proteins. With high genetic diversity and panmixis in southern US populations [H. zea could be rapidly evolving resistance under the intense selection pressure from widespread use of Bt toxins in its host plants [H. zea populations is necessary to evaluate the potential to spread genetic changes that may contribute to tolerance to Bt toxins across the distribution of this species, there is a paucity of data and resources available to conduct population genetic studies on this important pest species. Our laboratory previously developed microsatellite markers for this species using resources available at the time [H. zea populations contributing migrants to these sink populations. The analysis of peripheral (sink) populations suggested that centers of source populations contributed many founders to the sink populations [Long range migration of cumented ,23,24 ancumented ,26,27. Tontinent , H. zea ulations ,30, thist plants ,34,35,36t plants . Althougthe time  and condthe time . The popthe time  indicateulations . In geneulations  and studH. zea populations was necessary to identify potential changes to genetic structure of this species. Our goals in this study were to (1) develop a validated set of single nucleotide polymorphism (SNP) markers that will provide higher resolution population genetic parameter estimates than microsatellite markers and (2) compare genetic makeup of recently collected populations from LV and RS with those collected in 2002 and 2005 from the same sites to evaluate changes to genetic makeup of H. zea over time. This study uses a panel of SNP markers to evaluate genetic composition changes over time and space in H. zea from two peripheral populations collected 11 to 16 years apart. We postulate that comparison of samples collected from sink populations before and after widespread control failures in Bt crops could detect genetic shifts in source populations. In addition, this study will establish baseline sink genetic structure data for H. zea and the SNP markers validated here will facilitate future population genetic studies of this species.With recent reports of control failures on Bt crops in the southern and eastern USA, evaluation of H. zea were sampled near corn fields using Hartstack pheromone traps [Adult male ne traps  from Aug1 progeny insects from a cross between a laboratory colony maintained at US Department of Agriculture (USDA) Southern Insect Management Research Unit, Stoneville, MS and an insect collected on a wild host in Stoneville, MS were submitted to a Cornell University Genotype-by-Sequencing (GBS) service facility to identify SNPs using the protocol described in Elshire et al. [H. zea genome was sequenced on single lane of a flowcell on Illumina HiSeq 2000 instrument to obtain 100 nucleotide single end reads. Tassel (v3.0.147) [H. zea [Genomic DNA samples extracted from 95 Fe et al. . Briefly3.0.147)  was used3.0.147)  was used3.0.147) . ScaffolDNA extracts from field collected samples were quantified using a Qbit Flex fluorometer and Quanti-iT PicoGreen assay kit  for double stranded DNA and adjusted to 60 ng/µl prior to SNP genotyping with the Fluidigm EP1 system using integrated fluidic circuits (IFCs) that can analyze 96 samples with 96 SNP markers. DNA from one laboratory control and 95 insects from each collection was genotyped using the Fluidigm EP1 instrument. Genotype data files were imported to Fluidigm Genotyping Analysis v4.1.3 software and genotypes were exported as comma separated text files that were opened in Microsoft Excel 365 for further processing. Nucleotide symbols in genotype data were converted to the two-digit numerical format of 01, 02, 03, and 04 to represent A, C, G. and T, respectively, to produce input data for analysis programs that use numerical input.HE) and observed (HO) heterozygosity, within (FIS) and among (FST) population fixation indices, pairwise FST, and for analysis of molecular variance (AMOVA). Deviations from Hardy–Weinberg equilibrium (HWE) across all populations using an exact test [The number of insects collected in pheromone traps in LV and RS vary depending on flight times of local populations and influx of dispersing insects. Therefore, initial analysis was performed by dividing the samples into 16 partitions (putative populations) based on the collection year and time of the collection . Subsequact test , based ohttp://taylor0.biology.ucla.edu/structureHarvester/; accessed on 07 May 2020) [STRUCTURE 2.3.4  was useday 2020)  to identH. zea using prior population assignments as well as no prior assignments. The DAPC analysis with non-prior population assignment was carried out to evaluate number of putative clusters (K) between two and 40. The Bayesian information criterion (BIC) was then used to evaluate the relevance of different K values to population structure. Assignment values for the selected number of clusters were then generated for each individual using DAPC [H. zea partitions corresponding to the date and location of collection were assigned to the prior population set. All statistical analysis results are reported as mean ± standard deviation (s.d.).Discriminant analysis of principal components (DAPC)  implemening DAPC . DAPC fiH. zea genome, respectively, and 234,623 reads (26.2%) could not be aligned. Since we used adult insects to obtain genomic DNA for the GBS library, microbial contaminants present in the tissue pieces used  may have contributed to the sequence tags that could not be mapped to the genome. Mean coverage depth per individual was 77.8 ± 9.9 and the depth per site was 61.9 ± 64.2. Mean missingness for individuals and sites were 0.28 ± 0.10 and 0.28 ± 0.30, respectively. Genotypes with sequencing depth between 3 and 127 were selected for further analysis and 6,891 biallelic SNPs with more than 20% missing data were filtered out. After Tassel pipeline analysis, a total of 15,669, 7477 and 2664 VCF, unfiltered hapmap, and filtered hapmap SNPs, respectively were detected within GBS reads from 95 H. zea. Compared to other species of insects, the low number of SNPs most likely resulted from high repetitive DNA content from the EcoR I/Pst I double digest that was excluded from GBS library construction and low levels of genetic polymorphism in the parents used in generating F1 individuals used in the study. In addition, filtering for loci with multiple alleles and missing data may have further reduced the number of useable SNPs identified in this study. Nevertheless, the number of SNPs recovered was sufficient to develop assays for population genetic studies. A total of 384 polymorphic SNPs were manually selected to assign one SNP per scaffold in the largest scaffolds that had a computationally validated SNP for developing assays. Primers developed for the SNP loci were used to verify amplification of H. zea genomic DNA using real time polymerase chain reaction (PCR). After PCR verification, 96 loci were selected to develop Fluidigm SNP assays. The SNPs were developed for the unpublished version 6 of the H. zea genome, assembly version 5 was published [Sequencing of the GBS partial genomic library yielded a total of 252,303,111 reads that had 178,205,561 good barcoded reads. Filtering resulted in 894,576 tags, of which 601,201 (67.2%) and 58,752 (6.6%) were aligned to unique and multiple positions of the After a preliminary analysis of data using ARLEQUIN, loci and samples with more than 10% missing data in one or more populations and non-informative monomorphic loci across all populations were excluded from further analysis to retain a total of 85 SNP loci. Initial data set and the final genotype data used in this analysis are given in Observed and expected heterozygosities were calculated by pooling all 702 and samples ranged from 0.0014 (locus Hz6-105) to 0.871 (Hz6-406) and 0.0014 (Hz6-105) to 0.500 (Hz6-954), respectively. Mean observed heterozygosity in putative populations ranged from 0.3168 ± 0.2334) in 05LV-Aug24 to 0.4181 ± 0.2586 in 18LV-Aug06 and mean expected heterozygosity ranged from 0.3203 ± 0.1570 in 05LV-Aug19 to 0.3596 ± 0.1636 in 18LV-Aug06 . Relativp ≤ 0.01). However, deviations from HWE at these loci were inconsistent in exact tests performed on individual populations  and 05LV-Sep03 (0.1467) significantly deviated from zero at p ≤ 0.05 . Comparison of LV population pairs from 2005 resulted in non-significant FST for the pair 05LV-Aug19 and 05LV-Aug24, but significantly deviated from zero (p ≤ 0.01) when compared with 05LV-Sep03. FST estimates for 05LV populations paired with LV populations from 2016 and 2018 (16LV- and 18LV-) were significantly high ranging from 0.1325 to 0.2365 . In contrast, FST estimate for 02RS-Sep03 and 05LV-Sep03 was non-significant and that for 16LV-Sep02 and 16RS-Sep01 was 0.0996. When analysis was performed by pooling all samples collected within a year from each site into 6 populations, FST for 02RS and 05LV pair was essentially zero, but FST values for all other pairs ranged from 0.0996 between 2016 LV and RS populations to 0.2280 between LV populations collected in 2016 and 2018  compared to the estimate for the LV and RS populations from 2016  genomes indicate that LD due to physical proximity may not exist beyond a few thousand base pairs [H. zea used in this study is not sufficient to identify haplotype blocks or genomewide associations (GWAS), LD observed here may indicate unrecognized sub-structuring in populations analyzed in this study.Linkage disequilibrium (LD) was detected between several pairs of the SNP loci used in the final genetic analyses, but the number of linked loci varied across 16 putative populations . No linkomosomes  and physse pairs , haplotyse pairs ,60,61. Ase pairs ,64,65,66Simulations with STRUCTURE resulted in ΔK peak reaching a value 623 at K value of 3, indicating three population clusters A. Depictp-value of 0.4626 ± 0.1682 and tests for differentiation between all pairs of samples resulted in p-values greater than 0.2383, indicating no significant substructure in putative populations. Global AMOVA performed with ARLEQUIN as a weighted average over loci from the 16 putative populations indicated that high genetic diversity in individuals within the total population contributed to 96.89% of the total variation with an FIT of 0.031 (p = 0.00001) and non-significant inbreeding coefficient (FIS) of −0.058 . AMOVA performed by partitioning the genotypes into groups based on collection time, location or cluster assignments resulted from DAPC analysis also indicated that greatest molecular variance occurred in individuals within the total population . Little differentiation observed between putative populations collected in September of 2016 at both LV and RS indicated there was some gene flow between two sites. Insects collected at both sites during the 2018 growing season showed no genetic differentiation among putative populations collected on different dates within each site. When populations collected in different years within the same site were compared, FST values for 05LV/16LV, 05LV/18LV, and 16LV/18LV pairs were 0.1710, 0.1392, and 0.2280, respectively. Similarly, comparison of RS population between years yielded FST estimates of 0.1237, 0.1068, and 0.1950 for 02RS/16RS, 02RS/18RS, and 16RS/18RS pairs, respectively. Comparisons of populations from two sites collected within the same year yielded FST estimates of 0.0996 and 0.1852 for 16LV/16RS and 18LV/18RS population pairs, respectively. Overall, within a site, populations that paired more recent times (2016 versus 2018) yield higher FST estimates than pairs that used 02 or 05 years. Similarly, across sites, populations with the most recent times (2018) yield higher FST estimates. Relatively high FST estimates between populations from LV and RS in 2016 and 2018 and lower FST values for other comparisons outlined above may point to a change in genetic composition of H. zea populations in recent years. Analyses conducted by partitioning the populations by collection date indicates that there is a temporal shift in genetic composition within each site during each year, with insects collected in late August and September of 2016 and 2018 exhibiting higher differentiation than those from 2002 and 2005. It is possible that mountainous terrain may act as a physical barrier to restrict intraspecific gene flow that might result in significant genetic differentiation within and between years. However, determining the actual forces that influenced recent genetic differentiation between LV and RS populations could not be done without a detailed study of intrinsic and extrinsic factors, but one can only speculate many possible reasons including change in crop landscape and pest management practices as well as insect dispersal patterns influenced by different airflow patterns due to changing climatic conditions.In the present study, we used 85 SNP loci to estimate genetic parameters of insects collected in 2002, 2005, 2016, and 2018 from two peripheral habitat sites in Pennsylvania. Non-significant Helicoverpa species may contribute to low genetic differentiation among populations across large geographical regions [Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in Australia and India [H. zea. A previous microsatellite-based population genetics study of H. zea using insects collected in 2002 and 2005 from LV and RS concluded asynchronous influx of migrants from core populations, contributing to low genetic differentiation and high genetic diversity [FST estimates due to differences in genotype frequencies between populations.Long-range dispersal in heliothine species is well documented ,72,73,74 regions ,76,77,78nd India ,80,81,82iversity . This deiversity ,30,68. ASpodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), has been modeled using coupled bio-physical processes, and validated with SNPs that function as markers of natal origins [Helicoverpa zea may be using similar nocturnal wind currents during migration, albeit from an overwintering range that is much wider and in closer proximity to our study sites. The high genetic differentiation between 2018 LV and RS populations compared to other years may reflect the variation in aerobiological processes [https://www.ncdc.noaa.gov/monitoring-content/sotc/tropical-cyclones/2018/annual/tws_atl_latest.gif; accessed on 6 July 2020). It is possible that high winds from these storms could have forced source populations with different genotype frequencies from coastal areas toward LV to alter the genetic composition of that population. However, it is not possible to accurately predict the causes of high FST estimates between LV and RS populations without developing prediction models that combine weather data with extensive genetic and ecological datasets.The migration of another noctuid, the fall armyworm,  origins ,84. The  origins ,84,85. Hrocesses  resultinrocesses  indicateH. zea populations collected at RS in 2002 and at LV in 2005, but populations collected in 2018 from LV and RS exhibited high genetic differentiation. The populations within each site had no genetic differentiation. DAPC and STRUCTURE analyses indicate some sub-structuring in H. zea populations, in contrast to previous studies that did not show population structure in H. zea. Although H. zea are capable of dispersal over a large geographic range which contribute to genetic homogenization and panmixia [H. zea populations and there may be a multitude of underlying factors that may have contributed to this change, including environmental, climatic, and genetic factors. Yearly variations in gene flow rates in H. armigera influenced by various factors have been reported [H. zea populations may be undergoing such variations in geneflow. These changes are likely transient within a year, and by comparisons within 2002 and 2016, significance in local variation among these sites is not consistent. Inconsistency could be influenced by effective population sizes that in turn impact the effects of random genetic drift at each site and gene flow between LV and RS. If high genetic differentiation observed between populations collected in 2018 is not a temporary phenomenon, H. zea management practices, crop biotechnology, and insect resistance management models may be impacted.In summary, this study generated a set of SNP markers suitable for population genetic studies and identified high genetic diversity and low genetic differentiation in panmixia ,39, genereported ,82 and HH. zea populations from two sites about 150 km apart in Pennsylvania, USA collected in 2002, 2005, 2016, and 2018 indicated a temporal variation in genetic composition of insects collected in 2016 and 2018. Population genetic parameter estimates suggested a trend toward population differentiation, although it may be a transient or temporary phenomenon. This shift may be a result of several factors including annual changes in migratory patterns, climatic conditions, wind patterns, host plant availability, pest management practices, and limited gene flow between sites. Further evaluation of populations of this species is needed to determine if this shift is temporary and the impact on insecticide and Bt resistance management tactics.Population genetic analyses of


The Iberian Peninsula is considered a reservoir of genetic diversity and the source for the recolonization of continental Europe by several mammal species, after the last glaciation period. Here, we intended to characterize the genetic patterns and origins of one of those species, the roe deer, through the analysis of different genetic markers, as there is a major knowledge gap about the species’ populations at the western edge of roe deer distribution in the Iberian Peninsula. We found that western Iberia is a diversity hotspot for roe deer, with shared gene pools with other European and Iberian regions, but also with unique genetic elements, particularly the case of the relict population of Peneda Gerês National Park. Due to the distinct genetic diversity that was observed in western Iberian populations, we highlight the importance of these populations as sources of resilience against global changes. Our results provide useful information for the management and conservation of this species in the Iberian Peninsula. We advise transboundary management between Portugal and Spain as a rule, as well as careful evaluation of reintroduction actions, that should take in account the genetic data, in order to maintain the genetic heritage of roe deer in Europe.Capreolus capreolus) is native and widespread in Europe and its phylogeography has been clarified in the last decades. Southern peninsulas are considered as reservoirs of genetic diversity and the source for the recolonization of Europe after the last glacial maximum. Even though roe deer populations have been genetically characterized, there is a major knowledge gap about the populations at the western edge of its distribution. To fill this caveat, and based on mitochondrial and nuclear DNA data, we aim to: (i) characterize the genetic diversity and structure of roe deer in western Iberia; (ii) clarify the origins and phylogeographical affinities of these populations, namely the relict population from Peneda Gerês National Park  and the likely allochthonous populations from central and south (CS) Portugal; (iii) discuss the implications of our findings for the management and conservation of the roe deer. Three major genetic clusters were inferred based on nuclear genotypes and were structured in a similar way as the three major mtDNA clades present in Iberia. Patterns inferred with nuclear markers confirmed PNPG as a relict population. Roe deer from CS Portugal share haplotypes with Central Europe rather than with other western Iberian populations, confirming its mainly allochthonous origin. Our results highlight western Iberia as a diversity hotspot for roe deer. We highlight the role of intraspecific genetic diversity as a source of resilience against ongoing global changes; the need for transboundary management and the importance of genetic data to inform management and conservation. When considered, repopulation or translocation measures should follow the IUCN Law of Reintroductions and meticulously conducted in order to preserve the genetic heritage of the species.The roe deer ( The Mediterranean Basin is one of the very few World’s biodiversity hotspots located outside the tropics . While tCapreolus capreolus) is one of the most abundant and widespread wild ungulates in Europe. This species is native to the European continent and its distribution ranges from Scandinavian to the Balkans and Middle East, and to the Iberian Peninsula, at its southwestern distribution limit [Cervus elaphus [Sus scrofa [The roe deer  side of Iberia. Randi et al.  highligho et al. , should eninsula . Given teninsula ,8, and teninsula  and wildeninsula ), it is Taking this into account, several questions remain unanswered about the origins of the western Iberia roe deer populations. Is the population from the north-western Portugal (PNPG) indeed a distinct relict population, as suggested by Randi et al. (2004)? Does this population distribution range extend to other areas in north-western Iberia? Or are the populations in northern Portugal the result of natural colonization from populations in north-western Spain ? On the ® cards stored at room temperature for posterior usage. A total of 116 samples were collected and submitted to DNA isolation.Roe deer samples were collected from different regions in the northern, central and southern Portugal, and from northern and north-western Spain . Spanish® kit from Genomed®, following the manufacturer’s recommendations. Samples were also genotyped for 9 microsatellites with autosomal origin: CSSM66, INRA006, BM6506, BM1818, BM757, NVHRT48, NVHRT21, RT1, NVHRT16 [® Multiplex PCR Kit™ with a total volume of 20ul of reaction mix and 2ul of extracted DNA, following manufacturers conditions, in three multiplex sets . Success of PCR multiplex amplification was verified in an agarose gel. Successfully amplified products were enzymatically purified and electrophoresis of PCR products  was conducted in an automatic sequencer ABIPRISM® 3730-XL DNA Analyzer (Applied Biosystems™). Electrophoretograms were analyzed using Peak Scanner™ v1.0 (Applied Biosystems™) and allele calling was performed manually. Because DNA from antlers is often present in low quantity and quality, it was not possible to retrieve enough data from all samples. Thus, we only considered genotypes for which information for at least eight of the nine microsatellite markers was available.DNA was isolated from blood and tissue samples using the salt extraction method . DNA ext®Taq DNA Polymerase (following the manufacturer’s conditions) and an annealing temperature of 60 °C. PCR products were visualized in agarose gels, purified and sequences in an automatic sequencer ABIPRISM® 3730-XL DNA Analyzer da Applied Biosystems™. Mitochondrial DNA sequences were aligned using CLUSTALW algorithm on MEGA 5 [A fragment of d-loop region  was amplified using the primers L-Pro e H-16493 . PCR rean MEGA 5 , with sua posteriori, based on genetic diversity and structure results: CS Portugal, PNPG, Trás-os-Montes, NW Iberia and Valsemana) were estimated following Slatkin [divMigrate of the diveRsity R package [The inference of genetic structure and individual probability of assignment of each individual to each genetic cluster was performed, without any a priori information, on STRUCTURE v2.3.4 ,31. Resu Slatkin  and Nei  Slatkin . Samples Slatkin . Calcula Slatkin . In orde Slatkin . We esti package . Signifi package .Mitochondrial D-loop sequences generated in this study were used to infer mitochondrial haplotypes present in NW Iberia. Number of haplotypes was inferred using DnaSP . Mean geq = 89%) with individuals sampled in the putative relict population of Peneda Gerês National Park; a second cluster (CSP) was strongly associated with individuals from the putative allochthonous populations south of Douro river (q = 88% both in Alentejo and Beiras); the third cluster (NWI) was mostly associated with individuals from north-western Spain  and, at a lesser extent, northeastern Portugal (q = 50% in Trás-os-Montes). Mean genetic distances, estimated from microsatellite data  or other studies  where found to be shared with other European regions. On the other hand, while haplotypes from West clade were also found elsewhere in Europe, all haplotypes assigned to this clade, that were sampled in Iberia—including H8 —were exclusively sampled in this peninsula. No haplotypes from East clade  were sampled in Iberia, based on available information.The Iberian roe deer population is highly diverse and genetically structured, with elements characteristic of three of the main European clades: West, Central, and Celtic-Iberian this study ,9,15,46.C. capreolus garganta, [The structure and diversity patterns inferred from nuclear DNA highlight the distinctiveness of the roe deer populations in Peneda Gerês, thus corroborating the hypothesis advanced by Randi et al.  that thiarganta, ), despitarganta, . Lorenziarganta,  analysedarganta, ,50, it iLikewise, both the structure and diversity patterns inferred from nuclear and mitochondrial DNA identify the populations south of Douro river  also as distinct from other western Iberian populations. Contrarily to populations in southern Spain, which are dominated by West clade haplotypes , CS PortOther populations show a mostly admixed origin, such as the case of Trás-os-Montes and NW Iberia . The existence of shared haplotypes between regions from northern Portugal and north-western Spain corroborates the hypothesis of natural colonization of individuals from transboundary population . PopulatThe Celtic-Iberian haplotypes were the most common in the fenced population of Valsemana and also in Asturias, in line with what was observed by Royo et al. , but curStructure and diversity patterns inferred with nuclear markers were consistent with the patterns observed in mtDNA. Concordant structure patterns inferred from mtDNA and microsatellite data were previously reported by Biosa and collaborators , for cenGenetic differentiation is observed both at a continental and a regional scale, and non-intuitive barriers to gene flow occur. We believe this pattern is also patent in the mitochondrial signature of roe deer in the Iberian Peninsula, as the majority of the haplotypes from Iberian Peninsula are exclusive from this region. Similarly, unique haplotypes are found in other geographic regions, including Italy and Balkans, the last being related with glaciation periods ,55,56. OIberia is a genetic diversity hotspot for roe deer ,8,15, beC.c garganta, C.c. canus and C.c. decorus are valid subspecies is not debated here. However, the matching between genetic and morphological variation may be an important clue to ecologically relevant differences and adaptations to different climatic regions, terrains or land-uses. As rewilding initiatives are proliferating in Europe, genetic diversity patterns can be used as a relevant proxy for choosing suitable populations for translocations, within a conservation context. Captive-reproduced stocks of ungulates are common in some regions of Europe e.g., [As pointed by Royo et al. , geneticpe e.g., , but chope e.g., ,61. Accope e.g.,  have intpe e.g.,  is that


Octopus vulgaris Cuvier, 1797, occur in the northeastern Atlantic (NEA) throughout Macaronesia, comprising the Azores, Madeira and Canaries, and also the Cabo Verde archipelago. This octopus species, found from the intertidal to shallow continental-shelf waters, is largely sedentary, and the subject of intense, frequently unregulated fishing effort. We infer connectivity among insular populations of this octopus. Mitochondrial control region and COX1 sequence datasets reveal two highly divergent haplogroups (α and β) at similar frequencies, with opposing clinal distributions along the sampled latitudinal range. Haplogroups have different demographic and phylogeographic patterns, with origins related to the two last glacial maxima. FST values suggest a significant differentiation for most pairwise comparisons, including insular and continental samples, from the Galicia and Morocco coasts, with the exception of pairwise comparisons for samples from Madeira and the Canaries populations. Results indicate the existence of genetically differentiated octopus populations throughout the NEA. This emphasizes the importance of regulations by autonomous regional governments of the Azores, Madeira and the Canaries, for appropriate management of insular octopus stocks.Exploited, understudied populations of the common octopus,  Oceanic islands are a source of diverse biogeographic paradigms, with those related to genetic isolation, population differentiation and speciation of terrestrial fauna and flora being particularly common . In the Octopus vulgaris Cuvier, 1797, represents a species complex, with O. vulgaris sensu stricto found in the NEA and Mediterranean Sea. Several allopatric, cryptic Atlantic putative species have been referred to as Type I, from Caribbean Sea and the Gulf of Mexico, Type 2 from the western South Atlantic along the Brazilian coast, and Type 3, mainly along the coast of South Africa [O. insularis [O. vulgaris (s.s.) are synonymous [The common octopus, nsularis , has recnonymous .Octopus vulgaris (s.s.) occurs mainly in intertidal and shallow subtidal rocky or sandy habitat. During the day this species frequents dens, but at night it becomes more active, displaying relatively short-distance movements within a home range. Inshore reproductive migrations have been reported [O. cyanea Gray, 1849 [reported . Semelpareported  and prodreported . Planktoreported . In one reported , age ranreported , Canariereported , northwereported , Senegalreported , and thereported . MaturatO. vulgaris are highly sensitive to environmental factors, particularly during the larval stage, but they are also affected by fishing activity [The population dynamics of activity . Diverseactivity . Neverthactivity .O. vulgaris populations in the NEA has been rarely studied. There have been no studies on island population connectivity for this species [The genetic structure of  species . A micro species  along th species .O. vulgaris populations within the Mediterranean Sea. Significant population genetic structure was detected along the eastern Mediterranean coast, with haplotypes highly divergent from those found in Atlantic waters, with a correlation between genetic and geographic distances [Genetic studies have focused on istances . Signifiistances . Recent istances . On the istances .O. vulgaris (s.s.). The population structure of this species, which, in post-paralarval stages has limited dispersal and unusual reproductive behavior, that is subjected to intense, frequently unregulated, fishery effort along the Macaronesian coast, is analyzed. Patterns inferred for this species are related to biotic and abiotic process affecting marine invertebrates in general, to describe a phylogeographic scenario for the NEA oceanic insular environment. As a result, genetic patterns are relevant to delineation of units used in fisheries management.The taxonomic status of octopuses captured in the NEA is investigated to determine their relationship with O. vulgaris were obtained from six locations in the North Atlantic archipelagos of Azores, Madeira, Canaries and Cabo Verde, and two continental localities in Galicia  and the Atlantic Moroccan coast .Samples comprising 296 specimens  of an coast , Table 1O. vulgaris was amplified using de novo designed primers OvulCR3F (5’-GAAAATCTTTCGTGCAAATTACACCACA-3’) and OvulCR4R (5’-TGTTAATGGTCAGGGTCTAAATTCAACTAAAT-3’), located in flanking tRNA-Glu and COX3 genes, respectively. The partial COX1 gene, homologous to the standard barcoding COX1 fragment, was amplified to obtain a complementary dataset using the new primers OvulCOX11F (5’-TGAATATTYTCAACAAATCAYAAAGAYATTGG-3’) and OvulCOX12R (5’-GGGTGACCAAARAATCAAAATARRTGTTG-3´). Amplification was performed in a representative sample of specimens (N = 42) harboring distinct hypervariable control region haplotypes which collapse in a reduced number of more conserved COX1 haplotypes.Two mitochondrial loci, a hypervariable and non-coding control region, and a 5’ partial segment of the COX1 gene, were analyzed. The complete mitochondrial control region of 2, 200 μM of dNTP, 0.5 μM of each primer, 0.15 units of GoTaq Flexi DNA Polymerase (Promega), and 10–50 ng of total DNA. PCR amplification protocol consisted of 95°C for 3 min, followed by 35 cycles including 95°C for 40 s, 60°C for 40 s, 72°C for 40s, then 72°C for 7 min, performed using a GeneAmp 9700 thermal cycler (Applied Biosystems).Reactions were performed in a volume of 15 μL containing GoTaq Flexi Buffer , 1.5–3.5 mM MgClPCR products were treated with Exo-SAP-It  to digest primers and deactivate unused dNTPs, and sequenced in both senses using a BigDye 3.1 sequencing kit . Extension products were purified with Montage SEQ 96 (Millipore) and separated and detected in a 3730xl Genetic Analyzer (Applied Biosystems). After chromatogram revision and trimming with Sequence Scanner (Applied Biosystems), sequences were aligned using Clustal X  implemenh) and nucleotide (π) diversity estimations, FST estimates for population differentiation, and diverse population parameters, including Tajima’s D and Fu’s FS, were obtained with Arlequin v.3.5 [K; K max = 5) was estimated after five multiple independent runs with 50,000 Markov Chain Monte Carlo (MCMC) iterations, with both correlated and uncorrelated allele frequency models. The hypothesis of an isolation-by distance scenario was tested with IBD and the Mantel test [The basic haplotype (in v.3.5 . The hapin v.3.5 . Geneticin v.3.5 . The numtel test .Octopus species clades within the Atlantic and Pacific oceans [N = 289) for Octopus species using MEGA v.7 [6 MCMC chain length. Demographic dynamics were investigated under the distribution of pairwise differences in mismatch distributions [The model of DNA sequence evolution to best fit the data was selected using jModeltest v2.1 . Moleculc oceans . GeminatMEGA v.7 , and ii)MEGA v.7 . The tMRMEGA v.7 , 41; theMEGA v.7  selectedibutions  and the ibutions .O. vulgaris samples were from four insular localities , and two off the continent . These samples provide an appropriate mtDNA sequence dataset to appraise Macaronesian phylogeographic scenarios, and to evaluate taxonomic homogeneity within O. vulgaris (s.s.) [NEA s (s.s.) .Control region and COX1 sequences were submitted to GenBank and are available with accession numbers MN704980–MN705275, and MN705276–MN705317, respectively.h) of 0.8783, and a nucleotide diversity (π) of 0.02039. At the population-level, diversity estimates varied, with h ranging 0.653 to 0. 8771, and π 0.002 to 0.02, depending on the presence or absence of highly divergent haplotypes within a population . As expected for this segment, the base proportion was highly skewed towards AT (82.35%), with polymorphic sites accounting for 10.5% of sequence length. Three specimens, from Azores (N = 2) and Canaries (N = 1), had a 63 bp duplication starting at position 144, which was removed from the final alignment. A total of 64 haplotypes were detected, with a haplotype diversity , whereas the lowest values were obtained from the Azores and Canaries samples . Samples from Galicia and Cabo Verde had the lowest nucleotide diversity (π) values, π = 0.0018 and 0.0049, respectively, and the haplotype set was relatively homogeneous.The only negative Tajima’s The additional alignment of COX1 641 bp length sequences, elaborated from a subsample of 42 individuals which showed different haplotypes at the CR sequence, consisted in only 13 COX1 haplotypes with a nucleotide diversity (π) of 0.0063.h = 0.797, π = 0.0058), while haplogroup β  exhibited a typical star-conformation.The haplotype network built on control region sequences reveals two highly divergent haplogroups (α and β) with a near equal frequency , with 35 and 29 haplotypes, respectively, differentiated by at least 20 mutations . Within-The haplogroup structure is also observed, but a lower resolution, from COX 1 sequences data set acquired from this and others studies. A total of 41 Atlantic-Mediterranean haplotypes were mined from GenBank and BOLD repositories, resulting in a consensus alignment containing 58 variable positions. The COX1-based network revealed the two most common and ubiquitous haplotypes, reported from the North Atlantic, including the Mediterranean Sea, to be identical or closely related to those we report . In contHaplogroup frequency differs at different sampling sites. Haplogroups also have opposing clinal distributions. In the extreme southern (Cabo Verde) and northern  sampling locations, only one haplogroup, α or β, respectively, was detected .P < 0.05, 10,100 replicates) for the majority of pairwise comparisons. Exceptions involve comparisons between the Madeira, Canaries (P = 0.637), and Morocco (P = 0.053) samples. However, the Morocco sample had a low, but significant FST value relative to the Canaries sample. Excluding these estimates, the mean FST value, FST = 0.1447, was high. The highest differentiation values were estimated from comparisons involving the Cabo Verde sample  under the uncorrelated allele frequency model. The correlated allele frequency model produces four populations (k = 4), but with reduced support. The three estimated groups include the Galicia sample (cluster 1), the Morocco, Madeira, Canaries and Azores samples (cluster 2), and the Cabo Verde sample (cluster 3). For the less-supported correlated model, cluster 4 includes only the Azores sample. The probability of cluster membership exceeded 0.6 for all samples  .A pattern consistent with an isolation-by-distance scenario was observed, resulting in a significant regression between the linearized  0.0016) . A ManteD was negative for both haplogroups, it was only significant for the high value for haplogroup β . Both Fu values were large, negative, and significant (P < 0.001).Haplogroup-specific mismatch distributions (MD) had different profiles. The haplogroup α MD was multi-modal, with a distribution that better fitted a spatial than sudden expansion model. In contrast, the haplogroup β MD was unimodal, with a distribution that was similar to both sudden and spatial models . The τ vA Bayesian coalescent analysis suggests haplogroup-specific demographic patterns. Haplogroup α had a skyline plot, resulting from a stable population from 50 Kybp (thousand years before present) to 10 Kybp, after which the population continued to grow in size. This increment is near parallel to that observed for haplogroup β, also near 10 Kybp. The estimated effective population size is slightly higher for the older and genetically diverse haplogroup α .Octopus species on both sides of the Panama Isthmus, based on all available COX1 data, suggests that O. insularis, O. maya , O hubbsorum , and O. mimus  have diverged the least, and are involved in a putative geminate relationship (O. mimus and O. hubbsorum) with 99% bootstrap support . In a basal position is O. vulgaris, while O. bimaculoides  holds a divergent position (p-distance between the geminate Atlantic pair (O. insularis and O. maya) and Pacific clade (O. mimus/hubbsorum) was 0.06. We suggest approximate mutation rates of 0.0086 and 0.012 mutations per site per million years (My) using estimated dates of Panama Isthmus closure of 3.5 Mya [Sepia officinalis Linnaeus, 1758 [Dosidicus gigas d’Orbigny, 1835 [The reconstructed phylogenetic relationships of tionship . When phs et al. , and 2),position . The mea 3.5 Mya , and 2.5 3.5 Mya , respect 3.5 Mya , and witus, 1758  and Humbny, 1835 .Octopus control region mutation rate was unavailable for coalescence dating of haplogroups, so we indirectly approximated this from COX1 calculations. Relationships between COX1 and CR mutation rates were obtained by estimations from respective homologous data sets. The ratio between each gene-specific ϑ  equals the ratio of mutation rates between COX1- and CR-linked loci, as N is identical for the same sample [COX1, and ϑ CR, respectively. The mutation rate for the CR (μ CR) was 3.53 times that of COX1(μ COX1). Based on this COX1 calibration, our μ CR estimate was 5.44 x 10−8 per site per year.A direct, specific and reliable estimate of e sample . Thus, ϑt = τ / (2 μCR) with a generation time of 0.57 years [Dating of demographic or spatial expansions was obtained by 57 years –16, 18. Haplogroup coalescence BSL dating agrees with expansion estimates from mismatch distributions. Haplogroup α was traced to about 50–60 Kya, whereas the coalescence of haplogroup β occurred more recently (8–11 Kya), after the last glacial maxima (LGM). It is during the recent period that both haplogroups experienced drastic increases in population size.h (0.8783) and particularly π values (0.0204), are indicative of mixing of historically isolated lineages, herein referred to as haplogroups α and β. When calculated separately, the h and π value of each haplogroup decreases significantly . A global sample of the giant squid, Architeuthis dux  (N = 43) has both lower control region haplotype (h = 0.613) and nucleotide (π = 0.0017) diversity [h = 0.786, π = 0.0018) and Pico  are similar to values for A. dux, indicative of exceptionally low global genetic diversity [O. vulgaris (0.004) in the Atlantic [h and π are consistent with general patterns for animal COX1 diversity  [Overall diversity values, iversity . These giversity . For COXAtlantic . Populat 0.0036) , but hig 0.0036) .p-distance, 1.6%, is unremarkable , lower than the estimate between eastern and western Atlantic O. vulgaris (2.6%) of Sales et al. [Enteroctopus dofleini Wülker, 1910, 2.9%, suggests cryptic lineage speciation [Pareledone [Octopus haplogroups reported herein likely represent intraspecific lineages.The mean COX1 divergence between haplogroups (1.0%) is an overestimate because it is calculated on sequence data from selected specimens harboring different CR haplotypes. The highest COX1 s et al. , and sims et al. . In conteciation . Considereledone , the OctD and Fu’s FS estimated values are not always informative with regard to population size dynamics. Our values are affected by the mixing of divergent haplogroups within most populations, except for those from Galicia and Cabo Verde, where values are negative (as would be expected for a recent expansion event). Otherwise D and FST values were positive, indicative of bottleneck events. However, these positive values result from increased nucleotide diversity relative to the number of segregating sites, and in a deficiency of alleles, related to the partitioning of sample sequences into divergent haplogroups. Considering each haplogroup separately, D and FST values are negative (more so for β), as expected from an expansion population process.Different Tajima’s O. vulgaris, the COX1 and control region data from our and mined data, suggests two main divergent haplogroups exist throughout the eastern Atlantic. While population structure is complex, haplogroup β has a distribution focused in northern waters, while haplogroup α is primarily southern; both occur in the Mediterranean Sea. The southernmost-detected private haplotypes occur in haplogroup α from Cabo Verde and South Africa samples [The total mitochondrial evidence for  samples .O. vulgaris ‘Type I’ as proposed by Norman et al. [The near identical (single mutation) COX1 haplotypes in South Africa , Cabo Ven et al.  for the Environmental adaptive patterns in haplogroup distributions have been described for invertebrates . HoweverThe deep open ocean surrounding insular habitats presents barriers to dispersal-driven population differentiation in octopuses . We descO. vulgaris, when estimated from microsatellite loci [Samples from Madeira and the Canaries belong to a single population unit. The distance between them (about 450 km) exceeds the 200 km considered sufficient for promoting genetic differentiation in Iberian coastal populations of ite loci . HoweverPatella candei , is highly divergent from conspecific Macaronesian populations [Lasaea clams [Chthamalus stellatus  at the Azores also differ from those of other Macaronesian islands [Megabalanus azoricus , is, in contrast, genetically similar to the Madeira and Canaries populations [The Macaronesia core diverged slightly from the continental Moroccan coast and northern Azores archipelago. Isolation of the Azores population from other Macaronesia archipelagos is a likely outcome of the unidirectional flow of the Azores Current , explainulations ; populatea clams  and the  islands . The Azoulations .The strong genetic divergence of the Cabo Verde sample is consistent with a recurrent pattern observed for other marine invertebrate populations –5, 61. TO. vulgaris, and its relationship with geographic distance among sample locations, as reported for continental populations [The isolation-by-distance model accounts for observed divergence in ulations , 25. TheResults suggest a biogeographically congruent population genetic structure in the northeastern Atlantic, with three well-differentiated groups, including Macaronesia, northern continental Iberia, and southern Cabo Verde. Within the Macaronesia group, low, but significant levels of differentiation were present in the Macaronesian core (Madeira and Canaries archipelagos), and the more marginal northern Azores archipelago and continental coast of Morocco.O. vulgaris [High degrees of genetic differentiation are consistent with short life cycles, phylopatric behavior, progeny care, sedentary lives, and direct development and low dispersal capability of benthic octopus paralarvae . These fvulgaris ; the femO. vulgaris. Haplogroup α was traced back to around 50–60 Kya, whereas coalescence of haplogroup β occurred more recently, around 8–11 Kya. Both estimates are framed in the context of subtropical East Atlantic Macaronesia paleoclimatology, based on local opal maxima records for glacial terminations [The matched dating of demographic or spatial expansions, and coalescence time estimates for the divergent haplogroups α and β, reinforce the role of major, recent climate events in shaping the evolutionary history of northeastern Atlantic inations  and globinations . Thus, tinations . The curinations , 67. ThuThe “Sea-Level Sensitive” (SLS) dynamic model  for mariO. vulgaris is sensitive to SST, with temperature limits of 7–33°C [Throughout the northeastern Atlantic, it has been identified potential LGM marine glacial refugia, including the Macaronesia archipelagos, Iberian Peninsula, and Mediterranean Sea . The souf 7–33°C . Thus, mThe recent LGM-related haplogroup β is more frequent in northern latitudes, and absent from the southernmost Cabo Verde. This haplotype has experienced a recent expansion in more acceptable climatic conditions. At the most northern site  it is the only detected haplogroup; its low diversity is consistent with an expected outcome of the evolutionary legacy of the ice ages . HaplogrM. azoricus most recent haplogroup β sampled in Azores, Madeira, and Canaries [Pollicipes pollicipes  [O. vulgaris haplogroup α. All roughly date to the interval following the MIS6 glacial maxima, including the MIS5 interglacial. The recent expansion of O. vulgaris haplogroup β after the LGM seems to be a common pattern among North Atlantic marine taxa [Studies dating mitochondrial haplogroups in Macaronesian marine invertebrates have demonstrated synchronized coalescence with comparable glacial/interglacial recent Pleistocene episodes. The Azorean barnacle Canaries , and hapn, 1789) , resembline taxa .Macaronesian populations occur through the latitudinal midrange of the Atlantic study area, and consistently exhibit a mix of clinally distributed haplogroups α and β. The low level of gene flow among archipelagos and along the continental coast preserves haplogroup ratios. This pattern differs from others resulting from simultaneous vicariant events following glacial events, with secondary contact . We repoh) diversities manifest a weak latitudinal (north-south) cline, with highest diversity values at the Cabo Verde population, mainly because of the high proportion of private alleles, indicative of the role of this area for sustaining a refugial population. However, nucleotide (π) diversities have higher values within mid-latitudinal populations, reflecting the contribution of divergent haplotypes to α and β haplogroups. The haplotype network constructed from a limited sample focused on the Macaronesia area replicates a network pattern observed from a complete set of available COX1 data from the O. vulgaris species sampled worldwide  main stock in western Africa , but its status and fishery catch remain unknown.Fisheries statistics  for hipelago . Our res


Canis lupaster), a recently recognized canid species, to understand if demographic trends of this generalist and opportunistic carnivore reflect the increase in food availability that emerged after the arrival of the Neolithic economy in North Africa. We screened nuclear and mitochondrial DNA in samples collected throughout Algeria and Tunisia, and implemented coalescent approaches to estimate the variation of effective population sizes from present to ancestral time. We have found consistent evidence supporting the hypothesis that the African wolf population experienced a meaningful expansion concurring with a period of rapid population expansion of domesticates linked to the advent of agricultural practices.The diffusion of Neolithic technology together with the Holocene Climatic Optimum fostered the spread of human settlements and pastoral activities in North Africa, resulting in profound and enduring consequences for the dynamics of species, communities and landscapes. Here, we investigate the demographic history of the African wolf ( The beginning of this impact dates back to approximately 12 000 years BP in the eastern Mediterranean, from where it expanded westwards during the following millennia . In NortCanis lupaster), recently recognized as a distinct species of canid [The African wolf . Recent phylogenetic studies conducted on the species identified two main populations in Northwestern and in Eastern Africa [Little is still known about basic biological and ecological aspects of the African wolf because of its previous misidentification as a golden jackal Sampling was carried out in Algeria across different ecosystems between 2014 and 2016. DNA extraction followed specific protocols for different sample types .(b)® (Affymetrix) and sequenced using DLH primer using the Big-Dye Terminator v. 3.1 Cycle Sequencing protocol (Applied Biosystems). Electropherograms were checked and aligned using GENEIOUS v. 7.1.5 (https://www.geneious.com).Mitochondrial (mtDNA) control region was amplified using primers DLH and ThrH . Amplifi(c)A set of 47 microsatellite loci was amplified in five multiplex reactions for tissue samples following  and 25]25] (elec(d)n = 22), and then together with 46 sequences from Algeria and Tunisia retrieved from previous works [p-distance model. Phylogeographic relationships among the different mtDNA haplotypes were estimated using the median-joining (MJ) network algorithm [Mitochondrial diversity was assessed using sequences generated in this study , and for the subset of 13 microsatellites in Algeria (n = 18 + 2 genotypes from non-invasive samples) and Tunisia (n = 27), the latter previously generated in our laboratory [adegenet package [The 47 microsatellite dataset was evaluated for deviations from Hardy–Weinberg (HW) equilibrium using GENALEX v. 6.5 , and locboratory . Diversiboratory . Populatboratory  (for det package .Isolation by distance (IBD) was evaluated through Mantel tests implemented in GENALEX v. 6.5 for both molecular markers. The same software was used to test population structure between the two sampling areas (Algeria and Tunisia) through an analysis of molecular variance.(e)Demographic history of the African wolf was inferred using mitochondrial and microsatellite loci separately, compiling data from Algeria and Tunisia in a single dataset.p-value < 0.05) of Tajima's D and Fu's Fs were taken as evidencing a scenario of demographic expansion. Past population dynamic trend was inferred using Extended Bayesian Skyline Plot (EBSP) implemented in BEAST v. 2.3.2 [For mtDNA, we estimated mismatch distributions and Harpending's raggedness statistics , and tesv. 2.3.2 . We usedv. 2.3.2  and prevv. 2.3.2 , and HKIv. 2.3.2   from present to ancestral time with a coalescent approach using the method VarEff  implemen3.(a)π) diversities . After merging our dataset with 46 available sequences from Algeria and Tunisia and trimming the fragment to 223 bp, we observed 26 haplotypes with 21 segregating sites . Ten haplotypes were found for the first time in our study. Two haplotypes (H6 and H13) were shared among Algeria and Tunisia  and nucleotide , but a positive correlation was observed for the microsatellite dataset  suggesting some degree of IBD, which is expected in a panmictic population with a very wide range and limited dispersal . This is also partially evidenced in the PCA .We found no correlation between genetic and geographical distances for mtDNA ((b)r = 0.022, p-value = 0.06). Both tests of neutrality showed negative values, indicating deviations from the expectations under the mutation–drift equilibrium. Tajima's D indicated negative but non-significant deviations , while the Fu's Fs value was negative and significant , supporting population expansion. The EBSP supported a population increase to the present, as indicated by the slope of the median effective female population size and the posterior density favouring one population change at ca 50 kya , and the observed raggedness value was not significantly different from that expected in expanding populations  and a second one in the Holocene (approx. 6720–3840 years BP), as detected in mtDNA and microsatellites, respectively. This should be further investigated using additional molecular tools.Discrepancies in inferring demographic histories using different molecular markers are not uncommon ,49,50, aFavourable climate change during the Late Pleistocene ,56 triggThe Holocene African wolf expansion detected using microsatellites is remarkably concurrent with African cattle demographic history with pronounced population expansion approximately 5000 years BP . The incAlthough signatures of human-driven demographic processes are well reported in the literature, there is little evidence of favourable historical coexistence of post-Neolithic humans and wild mammals in Africa. Here, we provide evidence of population expansion of an opportunistic species during the Neolithic human revolution, which is possibly related to the increase of human-related resources, such as food availability. Surprisingly, this idea has been little explored.


ST = 0.01) and no correlation between genetic and geographic distance (r2 = − 0.02 p = 0.56), indicating high gene flow and no population structure. There is some evidence that kochia populations are genetically depauperate compared to other weed species, but genetic diversity did not differ between glyphosate susceptible and resistant populations or individuals. The inbreeding coefficients suggest there are 23% fewer heterozygotes in these populations than expected with random mating, and no variation was found within the chloroplast. These results indicate that any alleles for herbicide resistance can be expected to spread quickly through kochia populations, but there is little evidence this spread will reduce the species’ genetic variation or limit the species’ ability to respond to further selection pressure.Kochia, a major weed species, has evolved resistance to four herbicide modes of action. Herbicide resistance appears to spread quickly, which could result in diminished standing genetic variation, reducing the ability of populations to adapt further. Here we used double digest restriction enzyme associated sequencing to determine the level of gene flow among kochia populations and whether selection for glyphosate resistance reduces genetic variation. Canadian Prairie populations show little to no genetic differentiation (F At the individual level, this reduction can be detected in persistent genomic signatures of selective sweeps such as those associated with human evolution (reviewed by3), animal e.g.5 and crop domestication e.g.7. The evolution of herbicide resistance (HR) in agricultural weeds results from strong selection pressure. The spread of these resistance alleles through populations provides a growing number of study systems for understanding the consequences of selective sweeps at the individual and population level10. For practical reasons, weed managers are interested in understanding whether or not the standing variation for other herbicide modes of action is likely to be lost from populations following this selection. This could delay the evolution of additional HR within the population resulting from the time expected for new mutations conferring this resistance to arise in the population14. However, even when selection pressure is strong, loss in genetic variation depend on multiple factors, including the genetic basis of the resistance, mating system, population size, spatial or temporal variation in selection pressure, and gene flow9.A key area of interest in evolutionary biology is understanding the consequences of selection for genetic diversity and the future ability of populations to adapt. One potential consequence of strong selection is a reduction in the raw material, the genetic variation, available for selectionBassia scoparia (L.) A.J. Scott syn. Kochia scoparia (L.) Schrad.) is native to Europe and Asia and introduced to Canada, the United States, Africa, and South America. It is an annual noted for early germination, tolerance of arid and saline conditions, and a tumble weed habit. Kochia is wind pollinated and produces a large amount of pollen. However, it is also self-compatible, and as a result, the species is likely predominately outcrossing, but with high levels of variablity15. It has North American herbarium collections dating from the 1880s (reviewed by15). Kochia was found to be the fastest spreading alien species in the western USA from 1880 to 198016. It causes significant yield losses (30–60%) in crops such as winter wheat and sugar beet15, a problem exacerbated by the evolution of multiple herbicide resistance17.Kochia  inhibitors20, synthetic auxins21 and glyphosate24. Individuals with multiple herbicide resistance to all four modes of action have been detected in Kansas17, while individuals combining ALS, glyphosate (GR) and synthetic auxin resistance were documented in Alberta, Canada in 201725. Kochia populations with ALS inhibitor resistance were first detected in Canada in 198826 and the point mutations conferring this resistance have become nearly ubiquitous throughout the Prairie provinces in less than 20 years27. Glyphosate resistance was first detected in Kansas in 200722 and was widespread in the USA’s Great Plains and confirmed in all three Canadian Prairie provinces by 201330. This suggests that glyphosate resistance arose de novo in Texas after 33 years of glyphosate use31 and 11 years of intensified glyphosate use following the introduction of glyphosate resistant crops32. This resistance appears to have then spread through populations, rather than being a common variant in the species’ standing variation or emerging repeatedly de novo across the range. The glyphosate resistance (GR) mechanism described for kochia is increased copy number and expression of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme, which is inhibited by glyphosate in susceptible plants33. EPSPS copy number correlates with glyphosate resistance, with four or more copies resulting in resistance24. Inheritance of increased copy number follows a single locus Mendelian pattern, as the gene copies are in a tandem array on a single chromosome34. At this time, no other mechanisms of glyphosate resistance for kochia have been described.The species has evolved resistance to four herbicide modes of action37. Here we used double digested restriction enzyme associated markers and 26 populations from the Canadian Prairies to understand the current genetic variation in kochia populations and how selection for glyphosate resistance may have changed this variation. Specifically, we investigated three questions: (1) what is the level of gene flow and inbreeding among the kochia populations? (2) what is the current level of genetic diversity in these populations? and (3) do populations where high EPSPS copy number (EPSPSCN) has been introduced or individuals with high EPSPSCN show evidence of reduced diversity?While the spread of herbicide resistance genes, via pollen and seed, depends on the interconnectivity of populations, the selection of novel herbicide resistance from standing variation in a population depends on variation being available. In addition to selection for multiple HRs, often which have resulted from changes at a single locus , kochia’s history as an introduced species may limit available variation as only a subset of the variation available within a species is expected to be introduced to a new region29, none of the 12 individuals sampled had increased EPSPSCN  and 362 (95%) of individuals had sufficient coverage in the de novo and reference based pipelines, respectively. However, the sibling sets were excluded in the majority of analyses, resulting in the inclusion of 89 high EPSPS individuals and 206 low EPSPS individuals (Table 39). In total, 3440 loci were identified by both pipelines. Population analyses were run on all four data sets, but statistical results were very similar  was very low at 0.01, and pair-wise FST values ranged from 0 to 0.07 . AMOVAs indicated the majority of molecular variation (79.8%) was attributed to individuals, with less than 1% of the variation explained by differences among populations and no variation explained by population status (resistant or susceptible)  averaged 2.73 × 10–3 (range 1.02–4.58 × 10–3), and the average proportion of loci showing variation within individuals (HL) was 13% (range 5% to 23%).Overall population differentiation (Fe) Table , nor dide) Table . The anae) Table . The oveThe unweighted pair group method with arithmetic mean dendrograms included groupings mixed by province, population and EPSPS status Fig. . While sm) was calculated as 16.4 for comparison to40.The number of migrants among the populations (Nn) nor the proportion of variable loci (HL) differed by population type or individual EPSPS status , which may indicate a lack of variation.Thirty-six de novo consensus loci aligned to the chloroplast, but all were fixed across individuals. This may indicate that too few regions were used to detect variation, that markers fell in invariant regions, or that there is little variation in the chloroplast. Previous attempts in our laboratory to find variability in ST 0.01) is lower than the moderate level41 reported for 13 North Dakota and Minnesota populations based on 45 microsatellites (GST = 0.0940). While we note that calculating the number of migrants from FST has been criticized as underlying assumptions are likely to be violated43, our estimate would be six times higher than that of40, which suggests a higher level of connection between these populations. The level of genetic differentiation observed here for kochia is also lower than the average genetic differentiation reported for outcrossing wind-pollinated species  based on allozymes44. Other weedy species with similarly low FST include the wind-pollinated grasses Apera spica-venti L.  and Alopecurus myosuroides Huds. (FST = 0.023 from AFLPs46), as well as the outcrossing and self-incompatible Rosa rugosa Thub. (FST = 0.045 from microsatellites47). Populations of GR plants with similar genetic differentiation include the outcrossing and self-incompatible Lolium perenne ssp. multiflorium (Lam.) Husnot (FST = 0.006–0.088 from microsatellites48); and some population pairs of the obligate outcrossing species Amaranthus palmeri S. Watson in the USA (e.g. FST = 0.052 for R-S pairs from Arizona from SNPs49).Here, we determined that these 26 kochia populations, sampled from across the Canadian Prairie provinces, showed high levels of gene flow. This was indicated by: 1) the very low levels of genetic differentiation (Table  the veryIS) of 0.23, indicating a 23% higher level of homozygosity than expected by random mating. As material used here was grown from openly pollinated seed, this could indicate that inbreeding is occurring in many populations. Given the low level of genetic differentiation between populations, these high inbreeding coefficients are unusual; plants are generally expected to have either low or high values for both FST and FIS, as self-pollination increases divergence while outcrossing reduces divergence. For example, for two species with low FST values mentioned above, R. rugosa and A. palmeri, the FIS values were estimated at 0.04347 and 0.01649, respectively. In contrast, central European populations of Amaranthus retroflexus L. had inbreeding coefficients similar to the higher values found here at 0.382, but FST was 0.27 indicating strong population differentiation . However, a similar relationship to that observed here was reported for L. perenne ssp. multiflorium, which had FIS estimates ranging from 0.396 to 0.517 despite low values for genetic differentiation48. The authors suggested that this could result from genetic bottlenecks caused by glyphosate selection, but noted that FIS values did not correlate with the frequency of GR plants. Similar processes may be contributing to the high FIS values estimated in this study, since both high and low ESPSCN populations have been subject to selection pressure.We observed an overall inbreeding coefficient (FE) in these populations averaged 0.28, lower than the previous report for kochia of 0.35 (Nei’s gene diversity51 or h in40). While this may represent a reduction in genetic diversity, these populations are further north along kochia’s invasion path and may have had lower initial genetic diversity. Alternatively, this difference may be the result of using different genetic markers. Kochia’s genetic diversity is higher than the average of 0.16 for outcrossing wind-pollinated species from allozyme studies44. However, the percentage of variable loci (variable vs. variable and fixed loci) was 53%, similar to the 51% of variable allozyme loci in outcrossing species52. It is lower than genetic diversity of 0.678–0.824 estimated for GR populations of L. perenne ssp. multiflora48, but slightly higher than estimates for 42 GR populations of the highly self-pollinating Conyza canadensis (L.) Cronq. 53). Unfortunately, few other studies using approaches such as ddRADseq to identify SNPs report the number of loci that were identified, but that were invariant. Our estimate is higher than that for two species of bee-pollinated perennial Rhododendron in Japan, where 23% (144 loci of 675) of loci were variable; this was the only other study we located that reported this information54. Similarly, kochia’s nucleotide diversity  was 0.0036, which is lower than estimates of 0.0047 from AFLPS for A. myosuroides46, but higher than the nucleotide diversity estimated overall for core and invasive populations of Mercurialis annua L. . With increasing numbers of GBS studies examining genetic diversity, we anticipate that sufficient context to evaluate the potential for a particular weed to adapt from standing variation will soon be available14.Currently, it is difficult to evaluate kochia’s level of genetic diversity compared to other species, as it is unclear what levels should be expected in weedy or outcrossing plants. This challenge results from the variety of molecular markers used over the last 50 years and the variety of information presented by SNP studies. Genetic diversity  did not differ between high EPSPSCN populations or individuals and their low EPSPSCN counterparts. The bootstrapped confidence intervals of the inbreeding coefficient for low and high EPSPSCN individuals did not overlap , but random permutation tests indicated no statistical significance (p = 0.057). As a result, if kochia populations are depauperate or rich in comparison to expectations, this is the case whether or not the individuals or populations have high EPSPSCN, indicating that we have no evidence that selection for EPSPSCN has altered genetic diversity in these populations. This study sampled at an early stage in the spread of high EPSPSCN individuals across the Prairie Provinces (i.e. prior to fixation); sampling kochia populations after fixation of GR or after the spread of additional HR genes would provide additional insights.Whether kochia populations are more or less diverse than expected, there were no differences associated with high EPSPSCN within population or individuals. Similarly, population genetics parameters  and auxinic-resistant kochia (0 to 18%) in a 5-year period25. With the refinement of our understanding of kochia’s genome and the generation of a chromosome level assembly for the species, it will become possible to use this data to look at the signatures of selection near the EPSPS gene and determine whether this event is associated with a hard or soft sweep and better explore the origin of GR resistance in this species and its consequences58.The high gene flow among populations suggests that kochia on the Canadian Prairies, and perhaps beyond, could be considered a single population. Estimates from tracking tumbling kochia suggest that approximately 90% of seeds are dispersed over the first kilometer60. Based on this study, we expect the spread of GR will result in little change in kochia’s capacity to evolve additional herbicide resistance from standing variation. Swift, comprehensive, and ongoing action would be needed to curtail the spread of herbicide resistance genes from points of evolution in kochia populations. The species will need to be managed as a whole, as there are no smaller, individually controllable units require coordination and cooperation among producers and levels of government36. Future work expanding the geographic coverage of sampling, and investigating the genetic variation of these populations as GR and auxinic resistance spread, would provide further insights.A potential consequence of this high gene flow is that evolving locally adapted ecotypes would require extremely strong selection in kochia. However, the evolution of locally adapted ecotypes has been considered a key feature of successful invasions30. In these surveys, populations were considered resistant if they had individuals not controlled by glyphosate at 900 g ae/ha in greenhouse screens30. We extracted DNA from twelve individuals from four, seven and two pairs from Alberta, Saskatchewan and Manitoba, respectively. We also used six groups of reciprocally related progeny, sibling plants resulting from reciprocal controlled crosses between high and low EPSPSCN individuals from within populations in Alberta and Saskatchewan61. In total, this included 312 individuals from 26 populations and 72 progeny. The maps of the locations of these populations were made in QGIS Desktop 2.18.1562 with layers available and downloaded from Natural Earth .Plant material was from bulk-collected seed from population pairs where high EPSPSCN individuals had been detected (resistant) and from where they had not (susceptible) in relatively close geographic proximity. These populations were sampled and identified during surveys to determine the extent of GR kochia in Alberta , Saskatchewan (2013) and Manitoba (2013) Fig. 28–30. InSeed were germinated and grown in the greenhouse at Agriculture and Agri-Food Canada’s Ottawa Research and Development Centre. Young leaves were collected and DNA extracted using FastDNA kit . All material from the greenhouses and residual debris following seed cleaning were autoclaved before disposal.33. Specifically, two replicates were averaged to determine the EPSPS:ALS ratio. We measured the DNA concentrations of samples using a NanoDrop ND-1000 and 8000 Spectrophotometers , corrected their concentration to 5 ng/μL and conducted Quantitative PCR (qPCR) using an Eppendorf Mastercycler ep cycler. The specific primers for EPSPS: 5′ GGCCAAAAGGGCAATCGTGGAG 3′ and 5′CATTGCCGTTCCCGCGTT TCC 3′63, and ALS ALS890F: 5′AGCCTGTGTTGTATGTGGGA 3′ and ALS999R: 5′ AGCGCCCAAACCCATTAAAG 3′61 were used and produced products of 102 and 110 bp, respectively. BioRad strip wells containing 10 μL ABI Power Sybr Green MM (2X) , 0.5 μL of the appropriate forward and reverse primer (5 μM), 10 ng of gDNA, and 7 μL of dH2O were used for the qPCR reactions33. Cycle parameters were initial denaturing at 95 °C for 15 min, followed by 95 °C for 30 s and annealing and extension at 60 °C for 60 s, for a total of 40 cycles. The ALS reference gene was used to standardize the EPSPSCN using the equation R = 2−ΔCTsample−ΔCTcalibrator to produce the estimate of the ratio between EPSPS and ALS33.Quantitative PCR was used to determine relative EPSPSCN compared to ALS following the method described inDouble digested restriction enzyme associated marker library preparation and sequencing were completed at the University of Georgia using a 3DRAD based protocol. The enzymes used to generate the markers were HindIII (A|AGCTT) and NdeI (C|ATATG), and the project was designed to result in 300 million paired end reads for each of four plates.64, and the population map was created in QGIS 2.18.2562 with data from Natural Earth .Distances between the populations were calculated from their GPS coordinates using the Geographic Distance Matrix Generator v1.2.338 and custom R (3.4.3 “Kite-Eating Tree”) scripts65. STACKS (process_radtags) was used to demultiplex and filter data. In total, 1.6 billion reads were received and 1 billion were retained for an average of 1.5 million reads per individual. Stacks parameters were determined using a subset of samples as recommended66 and both the de novo and reference pipelines were followed. The parameters used were (M = n =) 5, (m =) 3, the minimum minor allele frequency allowed was 0.05, the maximum observed heterozygosity allowed was 0.7, and a random SNP was used from each locus. For the reference based pipeline, tags were aligned to kochia’s draft genome67 using Bowtie 2 version 2.1.039. Average coverage was 20.2 × for the reference pipeline and 15.8 × for the de novo pipeline. Individuals with less than 10 × coverage were excluded. Additionally, we removed loci with more than four alleles identified within full sibling groups, loci with 3 or fewer individuals represented in a population, and individuals with fewer than 60% of the loci. To identify loci associated with the chloroplast, consensus loci sequences were aligned to an assembly of kochia’s chloroplast67.Data were analyzed using STACKS v1.4465. Observed heterozygosity (HO), within-population gene diversity (HS), overall gene diversity (HT), bootstrapped estimates of the inbreeding coefficient (FIS) with confidence intervals, and levels of genetic differentiation among populations (FST) were all calculated by hierfstat68. Mantel tests were conducted with ade469. Bootstrapped values for FST, associated p-values and Nei’s genetic distance (DST) were calculated with StAMPP70. BayeScan71 (version 2.1), used with default parameters except an increased prior of 300, produced estimates of FST with upper and lower limits for each population. The R packages boa72 and coda73 were used to assess BayeScan results and model convergence. Allelic richness estimates were generated by PopGenReports74. Unweighted pair group method with arithmetic mean trees were calculated using poppr75, while AMOVAs were calculated with 1000 permutations by poppr.manova with the “ade4” method69. The proportion of shared alleles among groups and k-means clustering were estimated (testing k = 1 to 40) with adegenet76. The program fineRADStructure was used to further investigate clustering77. A custom R script processed STACKS’ haplotype files containing all SNPs for each loci in order to calculate the proportion of polymorphic nucleotide sites (Pn) and the percentage of heterozygous loci (HL). The function oneway_test from coin78 estimated the p-value for comparing low and high EPSPSCN individuals or populations using 100,000 permutations. The proportion of shared alleles was calculated by adegenet76. Following40, the number of migrants were estimated with Nm = 0.25((1 − FST)/FST)79.Population genetics parameters were calculated in R80, gdata81, pegas82, phytools83, reshape84, Hmisc85 and vcfR86 were used for data handling and manipulation, while ggplot87 and colorspace88 were used for plotting.The packages apeSupplementary Legends.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Tables.