Centrostephanus sylviae, which inhabits only the Juan Fernández Archipelago and the Desventuradas islands, separated by more than 800 km. For 92 individuals collected from Robinson Crusoe and Selkirk Islands (Juan Fernández Archipelago) and San Ambrosio Island (Desventuradas Islands), 7,067 single nucleotide polymorphisms (SNPs) were obtained. The results did not show a spatial genetic structure for C. sylviae; relative high migration rates were revealed between the islands. An analysis of the water circulation pattern in the area described a predominant northward water flow with periods of inverted flow, suggesting that larvae could move in both directions. Overall, this evidence suggests that C. sylviae comprises a single large population composed of individuals separated by more than 800 km.Most benthic marine invertebrates with sedentary benthic adult phases have planktonic larvae that permit connectivity between geographically isolated populations. Planktonic larval duration and oceanographic processes are vital to connecting populations of species inhabiting remote and distant islands. In the present study, we analyzed the population genetic structure of the sea urchin  Knowledge about the genetic structure of populations and gene flow between them is a central issue in population ecology. This information allows us to determine the importance of each population in a metapopulation context and the symmetry of reciprocal migration; key information for species management and conservation plans .Gene flow among populations of benthic marine organisms occurs mainly by the inter-population movement of larvae . The lifGenetic population variability has been used to study the genetic structure and gene flow among geographically isolated populations. Specifically, the use of thousands of single nucleotide polymorphisms (SNPs) obtained via next-generation sequencing represents a cost-effective alternative to obtain genetic data in non-model organisms .Jasus frontalis, which has a larval development of one year [The fragmented nature of the island systems, the span of the larval period and the type and scale of predominant oceanographic processes are all relevant in gene flow of the species inhabiting these islands; larval displacement is fundamental in determining the geographic extension of these populations , 14. Twoone year . Geneticone year , 18, sugCentrostephanus sylviae Fell 1975 [C. sylviae is unknown, a closely related species, C. rodgersii, has a larval development of four months [C. rodgersii between Australia and New Zealand [The sea urchin ell 1975  found hir months . It has  Zealand .Centrostephanus sylviae in the Juan Fernández Archipelago and the Desventuradas Islands. Using thousands of SNPs, population genetic structure and the degree of connectivity between populations (migrants between islands) of this species were described. Furthermore, a high-resolution reanalysis model analyzed the climatological currents between the islands to complement the genomic analysis. Considering the previous evidence for C. rodgersii in Australia and New Zealand, it was expected that the sea urchin C. sylviae would not show genetic differences between populations in the Juan Fernandez Archipelago and the Desventuradas Islands.The objective of this study was to estimate the degree of connectivity and population differentiation of the sea urchin Centrostephanus sylviae were collected from three oceanic islands . DNA was digested with the restriction enzymes Sbfl and Pstl following Kilian et al. (2012) [A small piece of muscle collected from each Aristotle’s lantern was placed in 96-well PCR plates with 80 μL of 99% ethanol for genetic analysis. DNA extraction and massive sequencing were performed at Dart Diversity Arrays Technology Pty Ltd., Canberra, Australia (. (2012) . FragmenDart Diversity’s bioinformatics service performed demultiplexing and removal of the DNA barcodes. More information about the detection of SNPs is described in Kilian et al. (2012). Raw SNP data from Dart were filtered using the dartR library  implemenST and heterozygosity implemented in the Fsthet library [To avoid bias in estimating the differences between populations, the loci identified as under selection were eliminated. Four different approaches were used for this: i) the method based on likelihood implemented in the outflank function of the dartR library, ii) the method based on principal components, implemented in the PCAdapt library , also im library  of the R library .IS) were estimated using GENETIX v 4.05 [Expected heterozygosity (Hexp.), observed heterozygosity (Hobs.), and the inbreeding coefficient (FX v 4.05 . AllelicX v 4.05 .C. sylviae. Three methods were used for this: i) A principal coordinates analysis (PCoA) was used to observe the distribution of individuals in a multivariate space qualitatively, using the dartR library implemented in the software R; ii) the paired FST index was assessed with the gl.fst.pop function implemented in the dartR library; iii) the most probable number of genetic clusters (K) was estimated using the Bayesian approach implemented in the Structure software [Population genetic structure was estimated using the SNP database for the three sampling sites of software . The admsoftware . The prosoftware .C. sylviae between the islands were estimated using the divMigrate function of the diveRsity library [Finally, the direction and magnitude of the gene flow of  library  implemenhttp://marine.copernicus.eu/) were used to understand circulation patterns between the Juan Fernandez Archipelago and the Desventuradas Islands. Briefly, CGLOPHY030 is a daily global reanalysis model [GLOBAGLOBAL_REANALYSIS_PHY_001_030 (CGLOPHY030) and CMEMSGLOBAL_ANALYSIS_FORECAST_PHY_001_024 (CGLOPHY024) products distributed by CMEMS .In the 92 individuals analyzed, 85,933 raw SNPs were obtained. After applying filters and removing outlier loci, 7,067 SNPs and 89 individuals were retained: 28 from Robinson, 31 from Selkirk, and 30 from San Ambrosio. The allelic richness and heterozygosity of these filtered data showed similar values among the three islands studied .ST were not detected between the pairs of sites compared  in the surface depth (mean 0–100 m) and the deep strata (100–1000 m), with brief periods of inverted flows (southward). Monthly longitudinal velocity time series showed eastward and westward flow connecting Selkirk and Robinson islands , with a Centrostephanus sylviae inhabiting the Desventuradas Islands and the Juan Fernández Archipelago are a single panmictic genetic population with strong gene flow between these two systems, even though they are separated by more than 800 km. Since adult echinoids have low mobility, migration and gene flow occur mainly via planktonic larvae.As expected, our results suggest that the sea urchin C. rodgersii shows weak genetic differentiation and no isolation over thousands of kilometers between eastern Australia and northern New Zealand [Strongylocentrotus droebachiensis inhabiting Atlantic Canada [Paracentrotus lividus in the Mediterranean [Tripneustes gratilla in the Philippines [S. franciscanus in California [Previous evidence showed high dispersal of larvae in sea urchins, which allows their populations to cover large geographic areas. For example,  Zealand . Other sc Canada , Paracenerranean , Tripneulippines , and S. lifornia , among oCancer pagurus have a planktonic larval duration of 3 months [Metacarcinus edwardsii, which has a planktonic development of 60 days  [Other benthic invertebrate taxa  with similar planktonic larval duration have large geographic populations, suggesting high dispersal of the larvae. For example, 3 months  and did 3 months . Moreove3 months  did not a, 1989) . In geneJasus frontalis. In this study, the individuals sampled in the Juan Fernández Archipelago and Desventuradas Islands systems showed a single panmictic population and high gene flow between islands, revealed by COI gene variability. This evidence is consistent with the pattern detected in the current study. Furthermore, Porobic et al. (2012) [J. frontalis populations between the Juan Fernández Archipelago and the Desventuradas Islands. The authors detected an asymmetric connectivity pattern between these islands, consisting of significantly greater larvae dispersal from Juan Fernández to the Desventuradas Islands. Our analysis using Nemo corroborates the analysis performed by Porobic et al. (2012) [C. sylviae did not show an asymmetric gene flow pattern as expected based on the biophysical model, suggesting that other factors could be involved in promoting the high gene flow observed in the current study. Therefore, it is necessary to continue studying dispersal patterns in this area to understand how the larval behavior, circulation (both mean and turbulent flow) and the biogeochemical environment relate to species connectivity between these two island systems, since it is known that the crustacean   describe. (2012)  used a b. (2012)  that use. (2012) . The veln  and sean (e.g.,  have larn (e.g., , so thatC. sylvae larvae could move from these islands separated by more than 800 km and continue to connect these geographic groups present on the different islands studied. This evidence is crucial to understand how species are connected in island systems that have marine protected areas. In 2015, Chile created the Nazca-Desventuradas Marine Park of approximately 300,000 km2 [Finally, ,000 km2  which in


Philaenus spumarius  , presently the most important vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al., 1987 in Europe. We found genome-wide divergence between P. spumarius and a very closely related species, P. tesselatus Melichar, 1899, at RAD sequencing markers. The two species may be identified by the morphology of male genitalia but are not differentiated at mitochondrial COI, making DNA barcoding with this gene ineffective. This highlights the importance of using integrative approaches in taxonomy. We detected admixture between P. tesselatus from Morocco and P. spumarius from the Iberian Peninsula, suggesting gene-flow between them. Within P. spumarius, we found a pattern of isolation-by-distance in European populations, likely acting alongside other factors restricting gene flow. Varying levels of co-occurrence of different lineages, showing heterogeneous levels of admixture, suggest other isolation mechanisms. The transatlantic populations of North America and Azores were genetically closer to the British population analyzed here, suggesting an origin from North-Western Europe, as already detected with mitochondrial DNA. Nevertheless, these may have been produced through different colonization events. We detected SNPs with signatures of positive selection associated with environmental variables, especially related to extremes and range variation in temperature and precipitation. The population genomics approach provided new insights into the patterns of divergence, gene flow and adaptation in these spittlebugs and led to several hypotheses that require further local investigation.Understanding patterns of population differentiation and gene flow in insect vectors of plant diseases is crucial for the implementation of management programs of disease. We investigated morphological and genome-wide variation across the distribution range of the spittlebug  Speciation involves the evolution of reproductive isolation and the buildup of genetic differentiation through selection and drift, but gene flow can counteract such divergence by homogenizing allelic variation and also by allowing recombination to oppose or break associations between loci underlying isolating traits . HoweverPhilaenus spumarius  , the meadow spittlebug, is a xylem-feeding vector of Xylella fastidiosa Wells et al., 1987, a plant pathogenic bacterium of South American origin that is emergent in Europe . It is native to the southern United States and it became established in late 1990’s in California, being a costly invasive species to agriculture. Population genetic structure studies based on DNA fingerprinting and mitochondrial DNA on this species have revealed highly differentiated geographic groups in the natural range and indicated that the likely sources of the California insects were in Texas  from their liquid excretion. Humidity and temperature are particularly limiting in the earlier nymphal stages  and cytochrome b (cytB), as well as on nuclear internal transcribed spacer 2 (ITS2) and elongation factor 1-alpha  DNA sequence analysis have questioned the species status of P. tesselatus, since individuals showing P. tesselatus-like male genitalia have the same or very similar sequences to P. spumarius  characterise the patterns of genome-wide differentiation of P. spumarius populations across the distribution range of the species; and (iii) detect local adaptation by finding genomic regions under selection and associated with environmental variation. The information on gene flow between populations and on the environmental factors associated with local adaptation, as well as on the most appropriate diagnostic methods for the identification of the closely related P. spumarius and P. tesselatus, will be important for future risk assessment of X. fastidiosa spread in Europe.In this study, we applied restriction site-associated DNA sequencing (RAD-seq), a reduced-representation sequencing approach that simultaneously discovers and genotypes thousands of single nucleotide polymorphisms for a large number of individuals . We had Philaenus spumarius were collected in 2010 and 2011 from eight populations (P. tesselatus was sampled in Morocco (MOR), from three main localities: near Azrou, near Rabat and near Ceuta. In total, 170 specimens were sequenced, including 20–22 individuals from each sampling site except Morocco, from where only 7 specimens were included , since they were measured in different units. Three specimens were left out of the analysis due to missing values. The analysis was performed in R version 3.4.1  was used to evaluate if morphological characters of the aedeagus could separate on 3.4.1  using thon 3.4.1 .DNA was extracted from the head and thorax of each specimen using the DNeasy Blood & Tissue kit (Qiagen) following the manufacturer’s instructions and including an RNase A treatment step. Wings and abdomen were not used for DNA extraction to avoid extracting DNA of endosymbionts and parasites. The obtained DNA was assessed for the presence of a high molecular weight band on the agarose gel after electrophoresis, and it was quantified in Qubit 2.0 (Invitrogen), using Qubit dsDNA HS Assay kit.c oxidase subunit I (COI) by polymerase chain reaction (PCR). Primers used were: C1-J-2195 (5′–TTGATTTTTTGGTCATCCAGAAGT–3′) and TL2-N-3014 (5′–TCCAATGCACTAATCTGCCATATTA–3′)  and approximately 30 ng of DNA. PCR conditions were: an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 50 °C for 35 s and extension at 72 °C for 2 min, with a final extension period at 72 °C for 10 min.A subset of 48 specimens from the nine areas were sequenced for mitochondrial DNA  from whiATTA–3′) . PCR wasChromatograms were verified and edited using SEQUENCHER v. 4.0.5 (Gene Codes Corporation), they were aligned using CLUSTAL W on BIOEDIT v. 7.0.9  and subsSbfI (New England Biolabs). Six libraries were prepared, with 28 to 31 individually barcoded samples multiplexed. The libraries were sequenced on three lanes of an Illumina HiSeq 2000 in paired-end mode (2 × 100 bp) at Genepool (Ashworth Laboratories) (http://genepool.bio.ed.ac.uk/). The individuals from each population were distributed over the different libraries and lanes to avoid library or lane-specific biases.RAD libraries were prepared using a protocol by process_radtags from STACKS version 1.45 (ustacks. The minimum depth of coverage to build a stack (-m) was set to 10, the maximum number of nucleotide differences allowed between stacks to form a locus (-M) was set to 2. Then the STACKS module cstacks was used to build a catalog by merging stacks (loci) from multiple individuals, using the default options. The module sstacks was used to match loci from an individual against the catalog. Stacks with very high coverage were removed since they may represent highly repetitive regions and that may include non-orthologous sequences.The sequence reads from each run were examined by ion 1.45 , to remoion 1.45 . The tripopulations module was used to create a Variant Call Format (VCF) file with the bi-allelic genotypes of each individual for each variable nucleotide position. The minimum number of populations a locus must be present in to process a locus (-p) was set to the number of populations analyzed , the minimum percentage of individuals in a population required to process a locus for that population (-r) was set to 0.5 (50%). Only one SNP per locus was kept, using the option –write_random_snp. Other parameters  were tested and the differences were assessed by general patterns in the Principal Component Analysis.Finally, the The VCF file was then filtered using VCFTOOLS (v 0.1.14) , excludihttps://github.com/CoBiG2/RAD_Tools/blob/master/geste2baypass.py as of commit b99636e.The filtered VCF file with the SNP genotypes was converted into the file formats needed for the different analysis programs using PGDSPIDER 2.0.4.0 . For conIS in each population. Pairwise differentiation between populations (FST) were calculated in ARLEQUIN 3.5.1.3, and the significance of FST was obtained from permutation tests with 10,000 repetitions. Mantel tests between FST/(1-FST) matrices and the natural logarithm of the geographical distance (http://ape-package.ird.fr/) in R version 3.4.0, using 9,999 permutations.VCFTOOLS was used to calculate summary statistics of coverage and percentage of missing data. GENETIX v. 4.05.2 was used to obtain expected and observed heterozygosity, as well as Fdistance  were perPrincipal Components Analysis (PCA) was used as an exploratory tool of the population structure . ComputaP. tesselatus individuals from Morocco, which consisted of 8 populations and 127 individuals of P. spumarius. In order to compare morphological variation and genetic variation in P. spumarius, we used a dataset of the 32 individuals for which we had data for both morphometry and RAD-seq. We performed PCA for both types of data, as above, and we calculated Spearman correlations between the Principal Component scores obtained from both PCAs using R.The complete dataset consisted of 9 populations and 133 individuals. We also analyzed a dataset excluding P. spumarius RAD-seq dataset (without Morocco), two approaches were taken: one that detects outlier loci departing from expectation under neutral demographic models  and the data was extracted for each location using DIVA-GIS 7.5.0 (http://www.diva-gis.org). Associations between SNP allele frequency differences and the environmental variables were assessed with BAYPASS v. 2.1 (https://gitlab.com/StuntsPT/pyRona/blob/master/pyRona/R/Baypass_workflow.R) as of pyRONA v0.3.7   sequenced for mtDNA either showed the most common haplotype of the “W” haplogroup (H29) or a haplotype differing by only one substitution (H28 and H37) . All happrocess_radtags step in STACKS retained 647,870,180 reads. This corresponds to an average of 3,811,001 ± 3,524,799 (standard deviation) reads per individual. Thirty-seven individuals with lower numbers of reads  or large amounts of missing data (> 60%) were excluded from the analysis   . In all on pairs . There wulations . Moroccaulations .ST value obtained between Morocco and Portugal (FST =0.34), than between Morocco and the other populations   ranged from 0.0373 (in Morocco) to 0.0808 (in Greece) and mean observed heterozygosity (HO) from 0.0258 (in Morocco) to 0.0537 (UK). HO values were generally lower than expected under Hardy-Weinberg equilibrium (HWE) in all populations (average FIS of 0.346)  . For MorThe dataset without Moroccan samples consisted of 127 individuals and 2,083 SNPs. For this dataset, PCA revealed two distinct clusters along PC1, generally separating Greece and Turkey from the remaining populations . The latThe majority of specimens for which COI sequence was available, had a correspondence between the mtDNA and the genomic cluster. However, there were some specimens showing a mismatch consisting of a mtDNA haplotype belonging in a different genomic cluster ; Fig. S3N = 32), we computed Principal Components Analysis , while all the remaining were low and non-significant .For samples for which both morphometric and RAD-seq data was available  , one of When excluding these candidate SNPs  from the “full” dataset, creating a “neutral” dataset, the main pattern of structuring was maintained, differing only in admixture proportions at higher values of K (4 and 5) PCA in . When ane-value of 1E–30) with the draft genome of P. spumarius and seven with the transcriptome  suggests some level of recent or ongoing gene-flow between ntinuum” . Both tantinuum” . Structury, 2000 , but in ry, 2000 . Morphols status . A more P. spumarius showed some correlation with genetic variation, although the nature of this association was not fully clear. Clinal latitudinal variation, as well as elevation variation, in the shape of male genitalia had already been described in European populations of P. spumarius  compared to other pairwise comparisons in this study, and also a large number of COI haplotypes belonging to the “North-Eastern” haplogroup . This supports a likely origin of the North American P. spumarius from Northern Europe, perhaps with multiple colonization events and with large effective population sizes. In S. Miguel Island (Azores), only two COI haplotypes, differing by one substitution, have been found so far  than these two were from one another (mean FST =0.042 between UK and USA), showing the lowest genetic diversity (expected heterozygosity) of all P. spumarius populations here analyzed. These results suggest a likely origin of the Azores colonization from Northern Europe, and that this colonization is likely to have involved a bottleneck event leading to reduced genetic diversity. The low number of color morphs found in S. Miguel  between populations than expected from the neutral background, and also by finding loci that have allelic variation correlated with environmental variation. When discarding such candidate loci for selection from our dataset, the population structure patterns remained very similar to the neutral dataset, which means that these 9% of loci are not affecting the genome-wide neutral pattern of population structure. Despite the usefulness of RAD sequencing for detection of selection in natural populations , epistatic interactions among loci or genotype-by-environment interactions (P. spumarius is particularly known for its balanced polymorphism for dorsal color forms and the assessment of population variation in the color-associated loci (Population genomics approaches provide genome-wide information that is expected to reflect a baseline of neutral processes and, at the same time, allow detection of loci with signatures of selection, deviating from this baseline . We focuulations , this anulations . The larractions . Whole-gractions . The facted loci  remains P. spumarius transcriptome may indicate their location was mostly in non-coding regions, while the low number of hits with the partial genome denotes its incompleteness (P. spumarius which will provide important tools to further explore the molecular basis of adaptation in this species.In the environmental association analysis, the associated variables were primarily longitude and those related to the extreme values and range variation in temperature and precipitation. This analysis is tentative, since we have a low number of populations from a wide geographic range. The low number of hits of the candidate loci with the leteness . A more leteness , and newP. spumarius and related species of the genus Philaenus is relevant to the management of the eventual progression of the plant pathogenic bacterium X. fastidiosa, since their dispersal patterns might aid or constrain disease transmission. Also, the ecological characteristics of different taxa or local populations may be different, for example in host plant preference, ease of acquisition of X. fastidiosa or transmission efficiency. Understanding the specific ecology of the vectors has been shown to be crucial in the management of X. fastidiosa diseases in America (X. fastidiosa in Europe and other Mediterranean countries. The risk of X. fastidiosa transmission and disease progression is generally expected to be related to long-range human-assisted movements of infected plants and with shorter-range natural dispersal by vectors (P. spumarius have shown that it does not constitute a panmictic population and geographical distance is not the only factor restricting gene flow. Other factors have to be taken into account, including habitat fragmentation, barriers to gene exchange such as endosymbionts or behavioural differences, rapid climate changes that may cause major shifts in distribution ranges, as well as unpredictable adaptive responses (P. spumarius may be able to migrate occasionally, or consistently but without reproductive outcome, and spread the bacterium. More ecological studies on the abundance and distribution of this insect vector through the seasons and across years are needed to understand the dispersal patterns across geographical regions and the potential for disease spread. It will be important to understand the dispersal patterns from South Italy, a potential source of contamination by X. fastidiosa and particularly its subspecies pauca, which includes the strain associated with OQDS. This is especially true as previous work detected haplotypes from distinct haplogroups in Italy. For example, P. spumarius among the Mediterranean peninsulas.Understanding species divergence and the population genetic structure of  America . Integra vectors . Geneticesponses . Even wiP. spumarius and P. tesselatus, as well as of the population structure and adaptation in P. spumarius. We found genome-wide divergence between these two species, despite the lack of mitochondrial DNA differentiation between them. The population genomics approach taken here showed admixture but also co-occurrence of non-admixed individuals in contact zones of diverging mitochondrial lineages of P. spumarius. The potential role of Wolbachia in shaping these patterns of divergence and introgression should be further explored.In this study, morphological and genomic analysis allowed a more detailed view of the divergence between X. fastidiosa progression.The findings on species divergence and population structure described here point to the need for elucidating the dispersal and ecological requirements of the different taxa and local populations of these vectors for a better management of 10.7717/peerj.11425/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11425/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11425/supp-3Supplemental Information 3Each row shows aedeagus measurements for one individual. TotLen - total length of aedeagus; MidMeanLen - mean length of middle appendages; LowMeanLen - mean length of lower appendages; UpMeanLen - mean length of upper appendages; UpMeanCurv - mean curvature of upper appendages.Click here for additional data file.10.7717/peerj.11425/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.11425/supp-5Supplemental Information 5Click here for additional data file.


Myripnois dioica across its distribution as a representative of northern China flora to reconstruct plant demographic history, examine local adaptation related to environmental disequilibrium, and investigate the factors related to effective population size change. Our results indicate M. dioica originated from the northern area and expanded to the southern area, with the Taihang Mountains serving as a physical barrier promoting population divergence. Genome‐wide evidence found strong correlation between genomic variation and environmental factors, specifically signatures associated with local adaptation to drought stress in heterogeneous environments. Multiple linear regression analyses revealed joint effects of population age, mean temperature of coldest quarter, and precipitation of wettest month on effective population size (Ne). Our current study uses M. dioica as a case for providing new insights into the evolutionary history and local adaptation of northern China flora and provides qualitative strategies for plant conservation.The flora of northern China forms the main part of the Sino‐Japanese floristic region and is located in a south–north vegetative transect in East Asia. Phylogeographic studies have demonstrated that an arid belt in this region has promoted divergence of plants in East Asia. However, little is known about how plants that are restricted to the arid belt of flora in northern China respond to climatic oscillation and environmental change. Here, we used genomic‐level data of  Myripnois dioica as a case for providing new insights into the evolutionary history and local adaptation of northern China flora and provides qualitative strategies for plant conservation which have been generally overshadowed.Our current study uses  Genetice) Wang, . Alternae) Wang, . StudiesPhysical barriers containing multiple mountains and drainage systems have acted on species differentiation in northern China, such as the Taihang Mountains promoting intraspecies divergence , Campanula barbata L. (Campanulaceae), and Carex sempervirens Vill. (Cyperaceae)  investigate demographic history of M. dioica; (b) explore local adaptation of M. dioica to dry environmental conditions in northern China; (c) address specific mechanism for potential historical and environmental factors related to changes in effective population size of M. dioica.Genome‐wide data derived from restriction enzyme‐associated DNA sequencing (RAD‐seq) have been effective and well‐suited for phylogeographic studies and investigating adaptive evolution  method  with paired‐end sequencing on an Illumina HiSeq 2000 platform. The raw data were deposited in the NCBI Short Read Archive (SRA) (Bioproject No. PRJNA607823).We sampled 77 individuals from 16 populations spanning the geographic range of 2.2All 77 individuals from 16 populations were used for de novo SNP calling Table . We then2.3π), observed heterozygosity (HO), expected heterozygosity (HE), and fixation index (FIS) for each population. We performed pairwise population FST values using an analysis of molecular variance (AMOVA) with 1,000 permutations as implemented in ARLEQUIN 3.0  for each population was estimated using the molecular co‐ancestry method implemented in NEESTIMATOR 2.01    layer. For evaluation, models were calibrated on 75% of the data and evaluated on the remaining 25% using area under the curve (AUC) and replicated 10 times.Effective population size . We first conducted a principal component analysis (PCA) to obtain the first two principal components of the 19 bioclimatic variables (Clim_PC1 and Clim_PC2), and these values were used as points in two dimensions to calculate a pairwise distance matrix  analyses using ARLEQUIN   among populations. We divided the 19 climate factors into two types associated with energy and moisture factors and used the subsequent principal component (PC) axes to describe climatic variation. We estimated the date of colonization for each population (hereafter named population age). Bayesian reconstructions were performed for 16 M. dioica populations using MrBayes v3.2.6 under GTR+I+G model based on four makers   Makino selected as an outgroup based on Funk et al.  was 0.0347 ranging from 0.0303 to 0.0384 in populations JL and JW, respectively . Meanwhile, average expected heterozygosity (HE) and observed heterozygosity (HO) across all populations were 0.1433 and 0.1915  to 0.339 (HH vs. HX) indicating uneven genetic differentiation  and northern group  were separated by the Taihang Mountains , while in northern populations the values ranged from 3.3 to 15.6 (populations JH and HY). DIYABC estimations of the divergence history of M. dioica indicated that the scenario 3 had the higher posterior probability  , followed by a bottleneck at c. 0.033 Ma (95% CI: 0.003–0.094 Ma). Group S diverged from the ancestral population at c. 2.37 Ma (95% CI: 2.25–4.11 Ma), followed by expansion at 1.97 Ma (95% CI: 1.41–2.94 Ma) and a bottleneck at 0.046 Ma (95% CI: 0.029–0.097 Ma). Using TreeMix to infer migration patterns among different populations, we did not find significant gene flow between populations (not shown). Species distribution modeling revealed the optimal habitat for M. dioica has changed minimally during the LGM  Figure . For dem Figures . Group NM Figure , and the3.4r = 0.33, p < 0.001) . The first two RDA axes were significant and explained 12.20% of the constrained variance . We independently employed climate factors and population age for predicting eN in M. dioica populations. Our results showed no significant correlation between eN and population age  and Oryza glaberrima Steud. (Poaceae)  and s Figure . Neverthca Table . First, M. dioica are distributed throughout northern China, it is particularly troubling that many specimen locations from previous records cannot be found, and only a very few individuals are left in some places during our filed investigations. In this case, M. dioica may be currently under threat and it is necessary to take measures in advance to prevent further disappearance. Myripnois dioica is usually distributed in disturbed, open habitats, occurring on slopes of limestone, and even along the roadside. Human activities have caused species diversity loss by habitat destruction, suggesting that M. dioica habitats are under constant threat with some populations already having been extirpated ; Formal analysis (lead); Investigation (lead); Methodology (lead); Software (lead); Writing‐original draft (lead). Jacob B. Landis: Formal analysis (supporting); Methodology (supporting); Software (supporting); Writing‐review & editing (supporting). Yanxia Sun: Methodology (supporting); Software (supporting). Xianhan Huang: Methodology (supporting); Software (supporting). Xu Zhang: Investigation (supporting); Resources (supporting). Qun Liu: Investigation (supporting); Resources (supporting). Huajie Zhang: Investigation (supporting); Resources (supporting). Hang Sun: Conceptualization ; Funding acquisition ; Supervision ; Writing‐review & editing . Hengchang Wang: Conceptualization ; Supervision ; Writing‐review & editing . Tao Deng: Conceptualization ; Funding acquisition ; Supervision ; Writing‐review & editing .Supplementary MaterialClick here for additional data file.


Characodon is the earliest diverging lineage of which three species have been described: C. lateralis, C. audax, and C. garmani, with the latter, considered extinct. Characodon lateralis and C. audax are classed as endangered, and have been the subject of taxonomic controversy since their description: previous studies have recognized a genetic differentiation in two groups separated by the El Salto waterfall, but morphological analyses contradict these genetic results. We perform a phylogeographic study using the mitochondrial cytb gene and d-loop region to elucidate the evolutionary history of C. lateralis and C. audax. The results with both markers show the presence of two highly differentiated haplogroups; one distributed north and the other distributed south of the waterfall, with genetic distances of 1.7 and 13.1% with cytb and d-loop respectively, and divergence calculated to have occurred 1.41 Mya. Significant genetic structure was found within each haplogroup and suggests the existence of at least four Evolutionary Significant Units (ESUs) within the examined populations. The possible processes identified as contributing to the formation of differentiated genetic groups are isolation, low population size, recurrent bottlenecks, and the strong sexual selection exhibited by the genus.The subfamily Goodeinae is a group of fishes endemic to the Mexican highlands. Most of the species are restricted to small and isolated streams or springs. Within this subfamily, the genus  Characodon is the earliest diverging lineage of the Goodeinae subfamily, sharing a common ancestor with the rest of Goodeinae around 15.5 Mya  of waterfall and C. lateralis south (below) of it  ). The ge15.5 Mya . The genateralis , C. garm garmani  and C. aC. audax , all of C. audax . The gener basin , whereaser basin . Characow) of it .Characodon lateralis and C. garmani were described in 1866 and 1898 respectively, based on a brief description of morphological characters with limited diagnostic value. This led to C. garmani being considered synonymous with C. lateralis until C. audax and C. lateralis are very similar in morphology, with limited diagnostic traits that differentiate both species , with further specimens from Cerro Gordo and Los Berros forming distinctive morphological clusters by themselves  and a portion of the control region (d-loop), based on extensive sampling of the 11 currently known populations of Characodon, and seven of the ten Evolutionary Significant Units (ESUs) described in Knowledge of the genetic variation within and between populations is important for both, taxonomy and conservation. We conduct a phylogeographic study using the mitochondrial cytochrome b gene . We obtain pectoral fin clips and preserved tissues in absolute ethanol, frozen at −75 °C, and deposited in the tissue collection of the Laboratorio de Biologia Acuatica of the Universidad Michoacana de San Nicolas de Hidalgo. We returned all specimens to the water following tissue extraction. We extracted DNA by digesting tissues with Buffer ATL (QIAGEN) and Proteinase K and purifiying using the BioSprint DNA Blood Kit (QIAGEN) according to the manufacturer’s instructions.cytb) and a portion of the control region (d-loop), via polymerase chain reaction (PCR) using samples from 59 and 56 individuals respectively. We used the primers HA and LA , and 1 µl (ca. 10-100 ng) of DNA template. The PCR procedure is described in cytb: MW208628–MW208647 and, d-loop: MW208648–MW208661)  see .http://popart.otago.ac.nz/index.shtml).To evaluate the geographic correspondence of haplotype distribution, we conducted a phylogenetic network estimation for each gene using the median-joining algorithm, using the default parameters as implemented in PopArt v1.7. (cytb and d-loop) using the web-based program ALTER   were grouped and the southern populations (SP)  were grouped. In the second hypothesis (H2) we considered C. audax, the rest of the northern populations (RNP), and all SP  as independent groups, based upon previous genetic studies .We collapsed to haplotypes the DNA sequences of each of the two markers  con studies . In the cytb partition using the mutation rate in teleosts of 0.76–2.2%/million years  model on branch length and calibrated the on years . We esti species . Given t species  and esti species  and summ species .p- distances for both genes considering two different arrangements according to the results obtained in the species tree analyses: H1) the two recovered haplogroups of Characodon in the haplotype network, in which all northern populations (NP) were grouped and all southern populations (SP) were grouped, and H3) C. audax as a differentiated group (population from El Toboso within NP), with the rest of the NP as another group, and SP samples grouped as: La Constancia + Los Berros as a single group and Amado Nervo as an independent group.To quantify the genetic differences between assumed genetic groups or populations, we calculated the uncorrected Characodon, we conduct an analysis of molecular variance (AMOVA) using 10,000 permutations in Arlequin v3.5.1.3  for each gene, under the same scenarios used in genetic distances and AMOVA analyses. We applied a Bonferroni correction , nucleotide diversity (π) and haplotype diversity (h) for each gene in Arlequin v3.5.1 , polymorphic sites (n v3.5.1  under thcytb and d-loop). We implemented all species delimitation analyses under three hypotheses, in the same manner as the time-calibrated species-tree. We implemented the A10 analysis (species delimitation using a fixed guide tree) (θs) the inverse-gamma prior IG , with mean 0.002/(3-1) = 0.001 (τ0) the inverse-gamma prior IG , with a mean 0.002, while the other divergence time parameters were specified by the uniform Dirichlet distribution   using th = 0.001 . We assiribution : Eq. 2. cytb mitochondrial gene (1081 base pairs (bp)) and 56 sequences of the d-loop (384 bp) distributed in 11 localities across the distribution range of Characodon in the Mezquital basin  was highly supported, with Pp = 1.0 for all relationships   .cytb and d-loop respectively. When genetic distances were calculated for four groups: (1) C. audax, (2) the RNP as an independent group, (3) La Constancia + Los Berros, and 4) Amado Nervo, the values for cytb range from 0.3% between C. audax and RNP to 1.8% between C. audax and La Constancia + Los Berros and C. audax and Amado Nervo. The genetic distances for d-loop range from 0.5% between C. audax and RNP, to 13.3% between RNP and Amado Nervo, and between C. audax and Amado Nervo (The uncorrected genetic distances between the two haplogroups (NP and SP) were 1.7% and 13.1% for do Nervo .cytb, the AMOVA arrangements showed ΦST values of 0.92 for four groups and 0.94 for two groups, with ΦSC values of 0.22 for the four groups and 0.39 for two groups, and ΦCT values for both arrangements of 0.90, with all fixation indices showing significant results (p  < 0.05). For d-loop, the ΦST values were 0.96 and 0.97 for four and two groups, respectively. The ΦSC values were 0.28 and 0.43 for four and two groups, and the ΦCTvalue was 0.95 for both arrangements, with all fixation indices showing significant results (p <  0.05) (For <  0.05) .ST values for cytb and d-loop for NP and SP showed high and significant genetic differentiation (>0.90) (The pairwise Φ (>0.90) . When th (>0.90) .cytb and 0.955 for d-loop), while for the NP the haplotype diversity was 0.760 for cytb and 0.659 for d-loop   .C. audax, rest of Northern populations, Amado Nervo, and La Constancia + Los Berros) , and hypothesis H3 of four putative species ( Berros) .ca. 1.41 Mya  shows significant genetic differentiation , genetic drift due to recurrent bottlenecks induced by the drying events, and the strong sexual selection that occurs in the species, as has been suggested before to explain morphological differences in Characodon populations (FST) has been found in the more dimorphic species, implying lower gene flow between populations, as could be happening in Characodon  showen in the present study corroborates previous studies using mitochondrial . C. lateralis, collected in the upper stretches of the Rio Mezquital, correspond to the same species that Günther used in his description in 1866. The collector of the type material, Dr. Berthold Seemann, was appointed as a naturalist on the voyage of exploration of the American west coast and Pacific on the HMS Herald, 1847–1851. As such, he visited Durango during December 1848 and February 1849. His detailed account is given in his Narrative of the voyage of H.M.S. Herald  (p genetic distances were much lower (0.3% for cytb and 0.5% for d-loop) than the divergences found between recognized species within Goodeidae  exhibits the most divergent body shape, differing significantly from all other investigated populations of the NP, with the variation mainly related to body height, proportions of the caudal peduncle, head shape, and the length of the anal and dorsal fin bases.The results presented herein for the northern populations shows onzalez)  and 3, btb gene; . In the  another . Whereasp genetic distances (cytb >0.4% and d-loop >1.4%) that are higher than the differences between C. audax and other populations within NP. However, due to the low sample size from Amado Nervo and the use of exclusively mitochondrial markers, these results should be viewed with caution. We recommend that further research should be carried out on the Amado Nervo population utilising other molecular markers and SNPs to clarified the genetic structure and taxonomy of the genus. Our results also support the morphological study of In the case of the SP, the genetic structure results show significant differences among La Constancia, Los Berros, and Amado Nervo (SP group)  and 3, iC. audax, the genetic result presented herein and the contradictory results in morphological evidence prevent any conclusion about its taxonomic status. We consider C. audax to be a valid species pending a more extensive and integrative taxonomic study. According to our results, if C. audax is confirmed to be endemic to El Toboso, the rest of the Northern populations would have to be considered as C. lateralis, whereas the SP emerges as an undescribed taxon. All the above highlight the necessity for an extensive integrative taxonomic study to elucidate the taxonomic status of the genetically and morphologically differentiated groups.According to the results presented herein, we suggest the existence of two well-differentiated and putative species, one north and the other south of the El Salto Waterfall. In the case of Characodon, with h >0.7 for cytb, and h >0.4 for d-loop (N = 3). The high genetic diversity contrasts with the decline in population size and the low population estimation for species living in springs arising from desert areas, indicating that the populations seem to maintain diversity despite their depauperate nature  and the other distributed to the south (SP) of the El Salto waterfall. We suggest that this waterfall acts as a biogeographic barrier that isolated the two groups at ca. 1.41 Mya. We also found genetic differentiation within each group. In NP the species C. audax, endemic to the El Toboso watercourse, do not share haplotypes with the rest of the populations and show significant genetic differentiation, although the genetic divergences are low. Within the SP, the three studied populations show genetic differentiation, concordant with morphological differentiation observed in previous studies (C. lateralis is unknown, and recent morphological studies were unable to distinguish differences between populations. This highlights the necessity for an extensive integrative taxonomic study. Regardless of taxonomic uncertainty, the conservation of extant populations of Characodon is urgently needed.We recognized two well-differentiated and highly divergent genetic and geographic groups within the genus  studies . The tax10.7717/peerj.11492/supp-1Supplemental Information 1PCR procedures for each genes is shown in the first table, also the primers used for each genes, in the second table are the specimen information and, also de genbank access numberClick here for additional data file.


Helichrysum italicum (Roth) G. Don; Asteraceae) is a perennial plant species native to the Mediterranean region, known for many properties with wide application mainly in perfume and cosmetic industry. A total of 18 wild H. italicum populations systematically sampled along the eastern Adriatic environmental gradient were studied using AFLP markers to determine genetic diversity and structure and to identify loci potentially responsible for adaptive divergence. Results showed higher levels of intrapopulation diversity than interpopulation diversity. Genetic differentiation among populations was significant but low, indicating extensive gene flow between populations. Bayesian analysis of population structure revealed the existence of two genetic clusters. Combining the results of FST - outlier analysis (Mcheza and BayeScan) and genome-environment association analysis  four AFLP loci strongly associated with the bioclimatic variables Bio03 Isothermality, Bio08 Mean temperature of the wettest quarter, Bio15 Precipitation seasonality, and Bio17 Precipitation of driest quarter were found to be the main variables driving potential adaptive genetic variation in H. italicum along the eastern Adriatic environmental gradient. Redundancy analysis revealed that the partitioning of genetic variation was mainly associated with the adaptation to temperature oscillations. The results of the research may contribute to a clearer understanding of the importance of local adaptations for the genetic differentiation of Mediterranean plants and allow the planning of appropriate conservation strategies. However, considering that the identified outlier loci may be linked to genes under selection rather than being the target of natural selection, future studies must aim at their additional analysis.Immortelle ( It occupies only 2% of the world's land area and is the habitat for great number of species2. Heterogeneous environments and numerous past events are responsible for significant plant diversity and a large number of endemic species in this area2. The Mediterranean basin is the meeting point of three continents; a place where humans began to exert their influence on biota and environment very early3. Today, human activity continues to pose a threat to the biodiversity in the Mediterranean, mainly because population density is highest along the coastal region, where numerous refugia have been discovered4. Refugia are known to act as climate-stable zones that are crucial for the long-term survival of species and genetic diversity. Populations that survived in glacial refugia show high rarity index values  and high genetic diversity due to long-term isolation5. Populations that emerged after the Last Glacial Maximum show lower genetic variation6 and lower rarity as a result of consecutive founder events during postglacial colonization8. It is very important to preserve these areas of numerus refugia, which are vital for the evolutionary processes of Mediterranean plant species4.The Mediterranean basin area is one of the largest biodiversity hotspots in the world10, and the impact of each factor on the evolutionary processes of a species must be considered for appropriate conservation planning.Climate change, overexploitation of natural populations, degradation, and fragmentation of natural habitats are the direct drivers affecting biodiversity12. From a theoretical perspective, the interaction between gene flow and natural selection has been well studied13, but there are still too few empirical studies to clarify the establishment of local adaptation and maintenance at the molecular level in natural populations. For non-model perennial and outcrossing species, genomic and evolutionary research is more difficult to conduct, and the genetic background of adaptive traits remains poorly understood15. Plant species that have wide geographic distribution and thereby are exposed to a range of environmental conditions, are often locally adapted, in part because of higher gene flow16. Discovering the mechanisms of adaptability of plant species is important for predicting their survival due to climate change or to what extent certain plant species will develop or activate adaptive mechanisms to the new conditions18.Local adaptation is an important tool of species through which natural populations are phenotypically and genotypically separated to better adapt to new habitat conditions and survive21. Genotyping of random loci throughout the genome allows the detection of outlier loci characterized by a higher degree of differentiation between populations than would be expected for neutral loci25. Identification of genetic regions under selection is commonly performed using two methods: outlier loci detection and genome-environment association analyzes. Both approaches assume that only a small number of loci are under selection26. Detection of outlier loci is a population-level analysis based on estimates of population genetic differentiation (FST), whereas genome-environment analysis examines correlations between population allele frequencies and environmental variables29. Using these methods, local adaptation along environmental gradients was studied in several plant species: Eruca sativa Mill.30; Eucalyptus camaldulensis Dehnh.31; Abies alba Mill.32; Liriodendron chinense (Hemsl.) Sarg.21; Geropogon hybridus (L.) Sch.Bip.33; Diplotaxis harra (Forssk.) Boiss.34, Populus tremula L.35, and Festuca pallescens (St.-Yves) Parodi36.Genetic signatures of natural selection are revealed by genome scanningH. italicum, a typical Mediterranean representative of thermophilic species. The species is naturally distributed along the eastern Adriatic coast and on the islands along the northwest-southeast environmental gradient. It grows on calcareous and well-drained soils: from sea level to 2200 m a.s.l.37. It is an outcrossing, thermophilic perennial and entomophilous plant species. As an anemochorous species, it is easily dispersed by wind, which contributes significantly to the spread of the species. It belongs to the genus Helichrysum Mill. and the family Asteraceae. The genus Helichrysum has more than 500 species worldwide38, with the species H. italicum distributed in the Mediterranean region. Herrando-Moraira et al.39 revised the classification of the entire H. italicum complex and proposed a new division of the H. italicum subspecies: (1) H. italicum subsp. italicum , (2) H. italicum ssp. microphyllum (endemic to Crete), (3) H. italicum ssp. siculum (endemic to Sicily), and (4) H. italicum ssp. tyrrhenicum . Numerous studies on the medicinal properties and biological activity of H. italicum were conducted, and are summarized in Guinoiseau et al.40, Maksimovic et al.41, and Ninčević et al.42. There have been several studies on the genetic diversity of Helichrysum spp.46 but none of them focused on the eastern Adriatic natural populations. Owing to many beneficial properties it is used in many commercial products, which increased the demand for H. italicum. This has led to overexploitation of natural populations, especially on the eastern Adriatic coast, where the collection of H. italicum contributes significantly to the local economy . In general, the use of medicinal and aromatic plants (MAP) has a long tradition on the eastern Adriatic coast. Commercial gathering is an important source of main or additional income in many regions48. Wild collection is still the main way to supply the market with medicinal and aromatic plants, as production is still insufficient. The negative impacts of wild collection are manifested in the loss of biodiversity, inconsistent plant material and lower prices. Increasing demand and resulting overexploitation, destructive harvesting techniques along with habitat loss and degradation49 put great pressure on many medicinal plants, leading to a decrease in genetic diversity and a reduction in the species potential to respond to environmental changes50. Therefore, it is of great importance to assess genetic biodiversity for efficient conservation of plant genetic resources and their use in plant breeding programs48.To study local adaptation along the eastern Adriatic environmental gradient characterized by increasing temperatures and precipitation, we chose H. italicum populations systematically sampled along the eastern Adriatic environmental gradient to elucidate genetic diversity within and among populations, identify candidate loci subjected to selection, and investigate their relationship with environmental conditions. Possible effects of past climatic variability and overexploitation on the genetic diversity and structure of natural H. italicum populations are considered, as well as the importance of local adaptations to climatic conditions as important promoters of genetic differentiation.In this research, an AFLP genome scan was performed on a total of 18 natural H. italicum was analyzed using AFLP markers. Four combinations of primers resulted in 693 polymorphic AFLP markers. The overall error rate for all combinations of selective PCR primers was 4.87%.Diversity within and between 18 populations of HE) was calculated within each of the analyzed populations based on gene frequencies assuming equilibrium according to Hardy–Weinberg (Table HE = 0.134), and the highest in the population P04 Rab (HE = 0.153). The total gene diversity (HT) was 0.147, and an average gene diversity within populations (HW) was 0.142.The Shannon information index ranged from 0.330 (P16 Živogošće) to 0.383 (P04 Rab) with an average value of 0.355 (Table FST) at the level of all populations was 0.036. The highest value of FST was calculated between populations P04 Rab and P13 Hvar (FST = 0.069), and the lowest between populations P09 Kistanje and P10 Unešić (FST = 0.002).The Wright coefficient of genetic differentiation (P(φ) < 0.001. A matrix of φST values was calculated between all pairs of populations by individual analyzes , P06 Pag (Miškovići), and P07 Obrovac) and the populations of Dalmatia  supported by bootstrap value of 96% ] and the rate of change between successive values of K (ΔK) for different numbers of ancestral populations (K = 1–11) were obtained. The highest value of ΔK was found at K = 2 (ΔK 386.84) followed by K = 3 (ΔK = 101.29). For K values greater than three, the average [ln P(X|K)] values decreased, while the standard deviations between the different runs for each K increased considerably and, therefore, the ΔK values were notably lower (from 6.50 to 0.08)  Fig. .K = 2 based on the run with the highest [ln P(X|K)], and populations were assigned to a particular cluster (A and B). The results were congruent with those obtained using the distance-based method. At K = 2 populations mainly from North Adriatic and Kvarner Bay  Pag, P06 (Miškovići) Pag, and P07 Obrovac) were assigned to the cluster A, while populations from central and south of investigated area   were assigned to the cluster B.The proportions of membership of each individual in each of the gene pools were calculated for ΔK at K = 2 could be an artifact resulting from significantly low likelihoods for K = 151, we used the BAPS program to verify data obtained from STRUCTURE. The results showed agreement with those from STRUCTURE for K = 2. The best partitions obtained log marginal likelihoods of −89,691.83 at P = 1 (without using geographic coordinates as informative priors) and −90,102.87 at P = 1 . Both analyzes, with or without spatially informative priors classified the populations identically into two clusters as revealed by the STRUCTURE analysis  values and the matrix of natural logarithms of geographic distances (in km) between the analyzed populations was r = 0.510 and was highly significant (PMantel < 0.0001). The coefficient of determination was R2 = 0.260, revealing that 26% of the genetic differentiation between the analyzed populations can be explained by their spatial distance  was found in 26 cases, and in five cases a strong negative correlation (r < −0.70), out of 171 pairs examined, respectively (Table Nineteen bioclimatic variables accounted for in this study were highly correlated. A strong positive correlation (10(PO) threshold used to determine the significance of loci that showed atypical values at a false-positive probability level of 0.01 . No locus was under balancing selection . With a 99% confidence level using the Mcheza program, a total of 21 outlier loci (4.71%) under possible selection were detected, of which ten (2.24%) were under positive selection  and 11 (2.47%) under balancing selection  and explained 20.17% (adjusted R2 = 0.2017) of the inherent genetic variation, indicating an important role of these environmental variables in shaping the distribution of AFLP genotypes. The first two RDA axes were significant and explained 19.82% and 8.10% of the variation, respectively. Variable Bio18 Precipitation of warmest quarter, significantly associated with RDA axis 1, contributed to the partitioning among northern, central and southern H. italicum populations. Both variables Bio03 Isothermality and Bio04 Temperature seasonality were associated with RDA axis 2 differentiating central H. italicum populations from the rest  and two precipitation-related  were selected for linear model redundancy analysis (RDA). The optimal RDA model included three variables: Bio18, Bio04 and Bio03. The model was highly significant  Sch. Bip.)55 and sage 56. Our results are comparable with studies on the above species, because the species have similar life traits. They are thermophilic, xerophytic, outcrossing perennials. Although these species generally have a wider range, their distribution pattern overlaps on the eastern Adriatic coast. The overall genetic diversity of H. italicum populations, indicated by the Shannon information index, showed intermediate values (0.355) compared to the populations of S. officinalis (0.387) and T. cinerariifolium (0.223). All three species are of great economic value, either as medicinal plants or, in the case of T. cinerariifolium, as a source of the potent natural insecticide pyrethrin. Overexploitation has been observed in all three species but may have most affected the genetic diversity of T. cinerariifolium, the species that has been extensively collected over long periods. In the case of H. italicum, collection of wild populations has increased more recently, in the last decade, and the impact on overall genetic diversity will become apparent in the future. Collection of immortelle from natural habitats is legally regulated in Croatia by the Nature Protection Act57 and the Ordinance on Collection of Native Wild Species58. Nevertheless, it is necessary to systematically monitor habitats, as harvesting regulations are often not respected. Results of the research showed that differentiation between populations of H. italicum was low, indicating extensive gene flow between populations59, and compared to S. officinalis and T. cinerariifolium it was the lowest. The AMOVA results showed greater intrapopulation genetic variation in H. italicum, which is typical for outcrossing plant species54. The greater genetic variation within populations compared to variation between populations was also observed in another outcrossing plant species from the family Asteraceae Xanthium italicum Moretti, naturally distributed on Corsica60. The similar partitioning of genetic diversity between and within populations was also found in the studies by Grdiša et al.55 and Jug-Dujaković et al.56.Recent studies have been conducted in the study area and have contributed significantly to the knowledge of species that are typical representatives of the Mediterranean climate, e.g., Dalmatian pyrethrum  and B, with populations from the central and southern part of the study area . These findings are congruent with the Fitch-Margoliash tree. The eastern Adriatic coast and the coastal Dinarides were heavily glaciated at various times during the Pleistocene63, which is well documented in the study of Quaternary sediments along the north of east Adriatic coast and the islands of Krk, Rab and Pag, as well as northern Dalmatia63. Much evidence suggests that glaciers were the barriers along the eastern Adriatic coast64 and may have been the reason for the separation of these two ancestral groups for a long time. We assume that the populations of H. italicum survived in the mini-refugia  during various unfavorable climatic events and reconnected when climatic conditions became favorable again. This theory is confirmed by the existence of four refugia for the eastern Adriatic coast and the coastal Dinarides out of a total of 52 recognized putative refugia in the Mediterranean65. Many authors also confirmed the existence of several Pleistocene microrefugia for this study area74.The results of the model-based methods showed clustering of H. italicum was found below the Kvarner Bay, near the Zrmanja river canyon (Obrovac), which is supported in a study by Marjanac and Marjanac64 that there is evidence of glaciation in the Obrovac area. Grdiša et al.55, also identified strong genetic differentiation of the north Adriatic genetic group for T. cinerariifolium with similar phylogeographic split at Kvarner Bay, near the Zrmanja canyon. In another study, Liber et al.75 found a distinct cluster at Kvarner Bay for S. officinalis, which also suggests the existence of refugia in the northern Adriatic. In the study by Rešetnik et al.72, a phylogeographic split is found between genetic clusters of S. officinalis located further south . Glasnović et al.73, in their studies of Edraianthus tenuifolius, also indicated the possible presence of these two separate "refugia within refugia" during the LGM. The results can also be compared with studies of Edraianthus tenuifolius68, Campanula pyramidalis76 and Cardamine maritime77, which showed a similar phylogeographic split, but in the area of the Neretva Valley . The rarity  in the northwestern H. italicum populations was higher than in the other parts of the study area, indicating long-term survival of the populations in the north5. The Shannon information index of H. italicum was also higher in the northern Adriatic populations. The high rarity of the northern Adriatic populations (Kvarner Bay) was also reported by Grdiša et al.55 for T. cinerariifolium and Liber et al.75 for S. officinalis. The lower DW values of H. italicum populations from the central and southern part of the study area might indicate later dispersal.The separation of populations of H. italicum populations, a total of 446 AFLP markers were used to identify loci thought to be under natural selection. A total of 21 loci that exhibited atypical values (FST) (4.71%) were detected using the Mcheza program, of which 10 (2.24%) were under positive selection and 11 (2.47%) were under balancing selection, while nine (2.02%) loci under positive selection were identified using the BayeScan program. Based on the results of both programs, Mcheza and BayeScan, seven AFLP loci were under positive selection in H. italicum. Less than 5% of the large number of markers commonly examined by this method are identified as outliers18. Nosil et al.25 reported that 5–10% of loci are outliers when summarizing data from available studies. In the study of the species Mikania micrantha Kunth78, 14 outlier loci (2.9%) were identified using the Dfdist and BayeScan programs, while in the analysis of the species Eruca sativa Mill.30 nine loci were identified, but only three of them were confirmed by another method (1.6%). In mountain populations of Sideritis scardica Giseb.79, a lower number of loci were also identified under selection by BayeScan (3.10%) compared to Mcheza (5.31%). Similar results were obtained by other authors who emphasize the more conservative approach of the Bayesian method, that works more efficiently as it detects a larger number of outlier loci with a lower presence of false positives82. Therefore, it is recommended to use more methods that incorporate different approaches and use different algorithms to increase the probability that the detected loci are truly adaptive.In 18 83. The correlation between allele frequency variation and climatic variables were assessed by using the Spatial Analysis Method (Samβada), which does not depend on genetic models and operates at the individual level84. Using all three methods , five (1.121%) loci under selection associated with bioclimatic traits were identified in H. italicum populations. Comparison with studies of other species confirms that only a small number of loci are potentially affected by natural selection. Müller et al.33 detected 11 (8.9%) outlier loci correlated to annual precipitation in the species Geropogon hybridus (L.) Sch. Bip. using the Mcheza, BayeScan and the spatial analysis method (SAM). Yang et al.21 used both frequency based (Dfdist and BayeScan) and correlation based (MLM) methods and showed that six outlier loci were strongly associated with at least one climate factor  in Liriodendron chinense (Hemsl.) Sarg. Authors Oberprieler et al.34 reported that using three methods  732 AFLP loci screened revealed only 1.6% (full dataset) and 0.4% (reduced data set) of all loci were found to be under selection in Diplotaxis harra (Forssk.) Boiss. In the study of Sideritis scardica Giseb.79, seven outlier loci were identified by Mcheza, BayeScan, and Samβada and associated with bioclimatic variables with precipitation identified as a significant environmental factor driving adaptive genetic variation. Temperature and precipitation were the most important environmental factors triggering adaptation in Rhododendron oldhamii85, Keteleeria davidiana var. formosana86, Salix species87, and alpine species91. Another method, LFMM has been used to test gene-environment associations while estimating the effects of hidden factors that represent background residuals of population structure. The advantage of the LFMM method is that it has a low rate of false positives and negatives92, and it also offers the best compromise between detection capabilities and error rates when dealing with complex hierarchical neutral genetic structure93. The LFMM has recently been used in several studies. For example94 genetic divergence in Pinus bungeana Zucc. ex Endl. was investigated and six environmental variables were identified that were related to the ecological habitat of the species and were correlated with the highest number of environmentally associated loci. In the study of Li et al.95, annual mean temperature, annual precipitation and slope were considered to be the most important environmental factors associated with adaptive genetic divergence in Cunninghamia konishii Hayata. Our results obtained by combining FST—outlier analysis (Mcheza and BayeScan) and genome-environment association analysis  showed that the most important environmental variables for adaptive genetic divergence in H. italicum populations on the eastern Adriatic coast were: Bio3 Isothermality, Bio08 Mean temperature of wettest quarter, Bio15 Precipitation seasonality, and Bio17 Precipitation of driest quarter. The observed natural populations of H. italicum inhabit sites under Mediterranean climate characterized by hot summers and cool winters with unevenly distributed precipitation prevailing in winter. Mediterranean plants cope with drought by developing different mechanisms that allows them to survive unfavorable or insufficient precipitation distribution96. In our study, the variable Bio15 Precipitation seasonality describes the differences between minimum and maximum precipitation values; therefore, our results suggest that H. italicum populations might be adapted to large fluctuations/amplitudes in the amount of precipitation. In Mediterranean climates, large rainfall amplitudes are common, and Mediterranean species strive to adapt and survive extremes. As Mediterranean region is highly vulnerable to climate change96 future projections for global climate change state that more uneven temporal distributions of precipitation are expected97. Climate models suggest that the hydrological cycle will intensify due to rising temperatures and increasing evaporation, leading to more storms and precipitation in particular areas, while drought will occur in areas far from storms98. According to Giorgi and Lionello99 and Matusik et al.100 the decrease of precipitation and increase of warming is expected in the future, together with increased inter-annual variability of both precipitation and temperature, which will cause greater frequency of extremely arid periods. Plant adaptation to seasonal water stress in Mediterranean climate is an important driver of genetic differentiation96.When adaptive loci are combined with climate variables, there is also the possibility of determining which climate factors are responsible for adaptive evolutionH. italicum also identified Bio03 Isothermality as important bioclimatic variable, together with Bio04 Temperature seasonality and Bio18 Precipitation of the warmest quarter as the main bioclimatic variables distinguishing northern, southern and central Adriatic populations. The variable Bio18 Precipitation of the warmest quarter contributed to the partitioning of the northern populations of H. italicum, as they are exposed to greater amounts of precipitation during the warmer months, leading to an adjustment of the populations in this direction. The two variables Bio03 Isothermality and Bio04 Temperature seasonality influenced the differentiation of the central H. italicum populations from the rest. Both variables are a measure of temperature heterogeneity, with Bio03 Isothermality quantifying how temperatures vary in relation to annual oscillations, while Bio04 temperature seasonality measures temperature changes throughout the year101. As mentioned above, the Mediterranean region is characterized by strong seasonality, both in temperature and precipitation, and the climate results in strong, opposing pressures on species102. Seasonality is an important component of climate that influences the availability of resources and thus the distribution of species in the environment at both temporal and spatial scales103. Central inland H. italicum populations cope with the wide range of daytime and nighttime temperatures, as well as cold winters and high summer temperatures, and are most likely adapted to temperature fluctuations, making them the most likely candidates for species persistence under ongoing climate change.The RDA approach used to study the contribution of bioclimatic variables to the genetic structure of natural populations of 89, but they are also poorly informative because they are dominant and biallelic, involving only reduced and anonymous part of the genome. The key weakness of genome scans is that they often detect false positives due to deviations from Hardy–Weinberg equilibrium and population structure model assumption104. Demographic events such as bottleneck, allele surfing during population expansion, secondary contact, and isolation by distance can mimic selection105, making it difficult to differ selection from demography106 and consequently to draw inferences about selection. Sampling strategy helps reduce occurrence of false positives by including abundant number of sampled individuals, more than 10 per site for allele frequency-based methods 108. Another way to reduce number of false positives and have more confidence in the outliers found is to use different approaches simultaneously and set strict thresholds, as was performed in this research. Four (0.89%) loci detected by all four methods  are expected since only small number of them are affected by natural selection. These loci are possibly linked to genes under selection due to the the ‘hitchhiking effect’, but their location and function is unknown due to a lack of prior knowledge about the genome structure of the species under study. Therefore, this research is the first step in finding evidence for adaptive divergence of H. italicum and additional analyzes involving the discovery of the location and function of detected loci are needed. Adaptation to precipitation and temperature oscillations in H. italicum populations appears to be the most important trait to be further investigated using genotype–phenotype association studies.The development of numerous genome scanning and spatial statistical methods has facilitated analyzes and our knowledge of adaptability in non-model species, but there are some limitations to these methods. AFLPs are useful marker systems with high reproducibility and ability for discovering polymorphism without previous information of the genomeH. italicum on eastern Adriatic coast. Extended sampling in the Mediterranean region should also be included in future analyses as the results obtained and conclusion derived in this study may not be applicable to the entire distribution range of the species. On the eastern Adriatic coast, in addition to longer-term problems such as degradation, habitat loss and climate change, increased demand for H. italicum has led to overharvesting, which could also lead to a long-term decline in genetic diversity. Evaluation of genetic diversity allows the identification of populations that have lower genetic diversity and are more vulnerable so conservation measures can be focused on them. Conservation of entire habitats through monitoring in the wild (in situ) can help legislators respond in a timely manner by adopting regulatory measures regarding the amounts of plant material allowed to be collected. Ex situ conservation should include seed banks with special attention to covering the most genetic diversity of the species109. Another possible solution is to promote the cultivation of H. italicum based on knowledge gained from molecular analysis and the adaptive potential of the species, which should be incorporated into breeding programs.The analyzes performed in this study and those proposed for future investigations could be the basis for future conservation strategies of H. italicum populations was collected in July 2014 and 2015. In addition, the seed samples of each individual were collected and stored in the Collection of Medicinal and Aromatic Plants at the University of Zagreb, Faculty of Agriculture under the Accession numbers MAP02672-MAP02689 . The collection of plant and seed specimens was carried out within the National Programme for Conservation and Sustainable Use of Plant Genetic Resources for Food and Agriculture in the Republic of Croatia and therefore in accordance with relevant institutional, national, and international guidelines and legislation. Voucher specimens were identified by Tonka Ninčević and Marija Jug -Dujaković and are deposited in the ZAGR Virtual Herbarium, Zagreb, Croatia . Sampling sites are listed in Table H. italicum along the environmental gradient from northwest to southeast on the eastern Adriatic coast. Populations P01 to P06 were sampled in the northern part of the study area, populations P07–P10 in the central part and populations P11 to P18 in the southern part of the study area. The average distance between sampling sites was 145.83 km, with minimum and maximum distance of 7.93 km and 415.94 km, respectively. The climatic data on precipitation and temperature conditions of each sampling site were taken from the WorldClim database at a spatial resolution of approximately 1 km2110 (available at: www.worldclim.org). Generally, the average annual temperature increases from the northern to the southern parts of the study area, while the amount of precipitation decreases. Precipitation deficit becomes greater and lasts longer from the northern to southern part of the eastern Adriatic coast111.Leaf tissue from twenty-five individuals from 18 wild DNA samples were extracted from 23 to 25 individuals per each of the 18 populations. Total genomic DNA was isolated from 25 mg of silica gel dried leaf tissue using a DNeasy Plant Mini Kit (Qiagen®). DNA concentrations were measured using a P300 NanoPhotometer (Implen®).112 with minor modifications proposed in Carović-Stanko et al.113. Four combinations of selective primers were chosen for amplification: FAM-EcoRI-ACA + MseI-CAC, NED-EcoRI-AGA + MseI-CAC, VIC-EcoRI-ACG + MseI-CGA and PET-EcoRI-AGC + MseI-CGA. The amplified fragments were separated by capillary electrophoresis in an ABI3130xl Genetic Analyzer .The AFLP analysis was done as proposed in Vos et al.114. Genetic diversity within the populations was estimated by calculatating the percentage of polymorphic bands (%P), the number of private alleles (Npr) and the Shannon's information index (I)115. The Shannon’s information index was calculated as I =  − Σ (pi log2pi), where pi is the phenotypic frequency115. The frequency down-weighted marker values (DW) were calculated according to Schönswetter and Tribsch5 using AFLPdat116 in R117.AFLP fragments were scored using the GeneMapper v. 4.0 software (Applied Biosystems) and fragments from 100 to 500 bp were analyzed. Error rates were estimated using the scanAFLP v. 1.2118) was used to partition the total AFLP diversity between and within H. italicum populations. Analysis was performed using the program Arlequin v. 3.5.2.2119 and the significance of the φST values was calculated based on 10,000 permutations. Pairwise population comparisons examined with AMOVA yielded a matrix of φST values corresponding to the proportion of total variance shared between two populations that can be interpreted as the distance average between any two populations120.Analysis of molecular variance  due to the outcrossing nature of H. italicum. The calculated allele frequencies were used to analyze genetic diversity within and between populations as described in Lynch and Milligan123. Total gene diversity (HT), average gene diversity within populations (HW), average gene diversity between populations that exceeds that observed within populations (HB), and Wright’s FST were used to describe the genetic structure of populations.Based on the frequency of amplified fragments of each AFLP marker in each analyzed population, allelic frequencies were calculated using the Bayesian method with a non-uniform prior distribution of allele frequencies according to ZhivotovskyDNEI72) was calculated between all populations pairs of using AFLP-Surv v. 1.0122. An unrooted Fitch–Margoliash tree based on pairwise Nei’s standard genetic distances between populations was created using the FITCH program v. 3.6b (PHYLIP124). The bootstrap method was used to create 1,000 distance matrices using the AFLP-Surv, and the bootstrap values were calculated using the FITCH and CONSENSE programs within PHYLIP software package.Standard Nei genetic distance  was set from 1 to 11 and 30 runs per K were performed on the Isabella computer cluster at the University of Zagreb, University Computing Centre (SRCE). An admixture model with correlated allele frequencies was applied with a burn-in period of 200,000 steps and 1,000,000 MCMC replicates after burn-in. The detection of the most likely number of clusters was performed as described by Evanno et al.128, and implemented in STRUCTURE HARVESTER v. 0.6.94129. Results from independent runs were clustered and averaged using Clumpak130 to obtain the Q-value matrix.The genetic structure of 127 to verify data obtained from STRUCTURE. Mixture analysis was performed both without the geographic coordinates as an informative prior  and with this prior  set to 20 with each run replicated 10 times. The best K value was assessed using the log marginal likelihood values of the best partitions and the distribution of posterior probabilities for different K values. To detect admixture between clusters the results of the mixture analysis were used as input to the population admixture analysis132 with default settings.Another Bayesian model-based analysis was performed using BAPS133 was performed by the Mantel's test based on 10,000 permutations using the program NTSys-pc v.2.10 s134 between the matrix of FST / (1−FST) values and the matrix of natural logarithms of geographic distances (in km) between the analyzed populations.Isolation by distanceH. italicum sampling site are shown in Table 135 and implemented in Mcheza136, (ii) a Bayesian approach as implemented in BayeScan v. 2.0124, and (iii) a spatial analysis method27 implemented in Samβada v. v0.5.184, (iv) a latent factor mixed model (LFMM)92 implemented in R package lfmm137, and (v) a linear model redundancy analysis (RDA)138 implemented in R package vegan v. 2.5–7139. Analyzes were restricted to loci with dominant allele frequencies between 5 and 95% across the whole dataset to avoid a bias in global FST estimates140.Values of 19 environmental variables (11 temperature-related and eight precipitation-related) representing the annual trends, seasonal variations, and extremes in temperature and precipitation, for each 18 FST values than the majority of neutral loci in a sample. In contrast, loci under balancing selection are expected to exhibit significantly lower FST values. The neutral distribution of FST values was determined by 1,000,000 iterations using the 'Neutral mean FST' and 'Force mean FST options. Outlier loci were selected with a confidence interval (CI) of 99% and a false discovery rate (FDR) of 0.1136. BayeScan evaluates individual loci within a hierarchical Bayesian model that decomposes genetic variation into population- and locus-specific effects. For each locus, two models are defined that include or exclude the effect of natural selection. The posterior probabilities of these two models are then estimated using a reversible-jump MCMC approach. We used 20 pilot runs with 5,000 iterations to fit the proposal distribution to acceptance rates between 0.25 and 0.45. Analyzes were performed with a burn-in of 50,000 iterations, a sample size of 10,000, and a thinning interval of 50, resulting in a total number of 550,000 iterations. The logarithm of Posterior Odds [log10(PO)] greater than 1.5 was considered as a 'very strong' evidence for selection141. The false discovery rate142 (FDR) was set to 0.01 to fit the corresponding log10(PO) significance threshold.The methodology used in Mcheza is based on the assumption that loci under directional selection have significantly higher P < 0.01). A model was considered significant only if both tests rejected the corresponding null hypothesis84.In Samβada, the spatial analysis method was used to calculate multiple univariate logistic regressions to test the probability of the presence of an allelic variant given the values of environmental variables of the sampling sites. Significance was assessed with both the log-likelihood G ratio and Wald test using Bonferroni correction (143) implemented in the R package lfmm137. In LFMMs, associations between genetic variation and environmental variables are tested, while estimating the effects of hidden factors representing background residual levels of population structure. Thus, in LFMMs, environmental variables are introduced as fixed effects, while population structure is modeled by latent factors. The number of latent factors (K) was set to two based on the results of STRUCTURE and BAPS analyses. Regularized least squares were calculated using a ridge penalty (lfmm_ridge) and estimated the genomic inflation factor (GIF) was estimated based on median z-scores (lfmm_test). To account for multiple testing, p values were converted to q values using the R package qvalue144. Significant associations were selected based on a false discovery rate (FDR) of 5% (q < 0.05).Gene-environment associations have also been identified using latent factor mixed models  were excluded before analysis. Hellinger-transformed allele frequencies145 were calculated in vegan based on the AFLP allele frequencies estimated using AFLP-Surv. The optimal model was determined using the ordiR2step function in vegan using a forward selection procedure with 10,000 permutations. To test the significance of the RDA model and constrained-axis, the vegan function anova.cca was run with 10,000 permutations.Linear model redundancy analysis (RDA), implemented in the R package vegan v. 2.5–7Supplementary Information.


Impatiens glandulifera) plants originating from native and non‐native regions of their distribution. When genetic relationships were explored by PCoA using SNP and SilicoDArT marker data, the first, second, and third principal coordinates explained altogether 37.4% and 31.0% of the variability, respectively. Samples from the UK, Canada, and Pakistan were grouped together, while Indian plants were clearly distinct based on SNP markers but relatively close to the UK–Canada–Pakistan group based on SilicoDArT markers. Constructed trees differentiated individuals into clusters resembling the PCoA patterns. The Bayesian BAPS analysis performed for the SNP data revealed that the individuals were distributed in seven clusters, representing samples from each of the four Finnish populations, India, Pakistan, and the combination of the UK and Canada. Similar clustering was visible in the UPGMA tree. The Indian cluster did not display any ancestral gene flow with the others, while the Pakistani cluster showed ancestral gene flow only with the combined UK and Canada cluster. Furthermore, the latter cluster displayed ancestral gene flow with the Finnish populations varying from 0% to 3.1%. The BAPS analyses conducted for the SilicoDArT data differ slightly: The individuals were distributed in nine clusters, and the Indian cluster exhibited ancestral gene flow with the mixed cluster including Canadian, Pakistani, and UK samples, and one Finnish sample. The AMOVA showed that 45% and 26% of variation was present among the I. glandulifera groups/populations and the rest within them based on SNP and SilicoDArT markers, respectively. The Bayesian BAPS analyses and the gene flow networks were the most informative tools for resolving relationships among native and introduced plants. It is notable that the small sample sizes for non‐Finnish plant materials may affect the accuracy of the gene flow and other estimates.We conducted genomic characterization based on SNP and SilicoDArT markers on the invasive Himalayan balsam ( Impatiens glandulifera) plants originating from the native and non‐native regions of their distribution. Considerable genetic differentiation and the presence of migration between distantly located populations indicate that genetic drift and human activities have facilitated differentiation and further spread of I. glandulifera.We conducted genomic characterization based on SNP and SilicoDArT markers on the invasive Himalayan balsam ( Their dispersal may occur either intentionally or accidentally, often being promoted by human activities, such as agriculture, horticulture, aquaculture, transportation, and recreation , is a tall, self‐pollinated annual plant, which has been introduced widely as a garden ornamental, first to England in 1839 from its native distribution area in Kashmir in the Himalayas  analyses generating large numbers of markers have become an increasingly frequent approach for molecular characterization and for studies on genetic variation and differentiation  show a closer genetic relationship to the plants from the native area of distribution  than those from other regions of introduction, and plants in the areas of introduction show considerable differentiation due to genetic drift and anthropogenic effects, for example, seed dispersal commercially and by home gardeners. Additionally, we hypothesized that the two marker types provide similar results of genetic relationships and differentiation patterns.In this study, we employed GBS using both SNP and SilicoDArT marker techniques to perform a comprehensive genome‐wide analysis of the genetic structure on 22.1I. glandulifera representing its native area of distribution included seven samples from India  and five samples from Pakistan (originating from populations PA‐3 and PA‐4), while samples representing introduced material included eight samples from Canada , six samples from the UK (originating from populations UK‐2 and UK‐3), and 68 samples from Finland , a total of 94 samples , following the DArT genotyping protocol of Kilian et al.  and monomorphic loci. Call rate and reproducibility were filtered for both types of markers to the threshold of 0.95. All SNP loci were filtered for deviations from HWE, but no loci were deleted upon filtering. The frequency distribution of polymorphism information content (PIC) values was computed for both marker types after filtering. Filtered data were used for subsequent diversity and genetic structuring analyses. Data filtering was performed using the R 4.0.2  and expected heterozygosities (HE), mean within‐population genetic diversity (HS), total genetic diversity (HT) and corrected HT (HTP), and the total genetic diversity among populations DST and corrected DST (DSTP)  and corrected FST (FSTP), as well as the inbreeding coefficient (FIS), were computed  Nei, . In addited Nei, .Principal coordinate analyses (PCoA) were applied to investigate genetic relationships among individuals from different groups/populations using R 4.0.2 , 0.115 (DSTP = 0.132), and 0.448 (FSTP=0.482) for SNPs, respectively, and 0.166 (HTP = 0.169), 0.024 (DSTP = 0.028), and 0.146 (FSTP = 0.163) for SilicoDArt markers, respectively. On the other hand, average within group/population diversities was equal, 0.141 and 0.142 for SNP and SilicoDArt markers, respectively. The observed (HO) and expected heterozygosities (HE) per population were meaningful only for the four Finnish population samples (FI‐1–FI‐4), for which HO values equaled 0.075, 0.174, 0.132, and 0.074 and HE values 0.071, 0.174, 0.132, and 0.111, respectively, based on SNP markers. Thus, only FI‐4 showed a considerably different (lower) value for HO in comparison with HE.The SNP markers showed a higher total genetic diversity and a higher genetic diversity and genetic differentiation among groups/populations compared with SilicoDArt markers, the Genetic relationships among individuals within groups/populations were first explored by PCoA. Based on SNP and SilicoDArT marker data, the first, second, and third principal coordinates  between SNP and SilicoDArT distance matrices, as determined by a Mantel test, which proved a good fit between SilicoDArT and SNP marker data sets.Unrooted trees were constructed based on pairwise genetic distances and cluster analysis, to show the relationships among the 84 genotypes Appendix  and eighAppendix  based onI. glandulifera genotypes were assessed also using the Bayesian BAPS analysis, which revealed that based on SNP markers the individuals were distributed in seven clusters  and the rest within them based on SNP and SilicoDArT markers, respectively, which indicates a high degree of differentiation among groups/populations, especially based on SNP marker data  compared with SilicoDArt markers  and expected heterozygosities (HE) per population were meaningful and calculated only for the four Finnish population samples (FI‐1–FI‐4), for which HO values equaled 0.075, 0.174, 0.132, and 0.074, and HE values 0.071, 0.174, 0.132, and 0.111, respectively. Thus, only FI‐4 showed a deficiency of heterozygotes compared with the expected heterozygosity. Previously, Nagy and Korpelainen , while individuals from the fourth Finnish population were far apart. Samples from the UK, Canada, and Pakistan grouped together, while Indian plants were clearly distinct based on SNP markers but relatively close to the UK–Canada–Pakistan group based on SilicoDArT markers. Overall, the relationship between the native Pakistani samples show a relatively close relationship with the UK and Canadian samples.Phylogenetic trees constructed to show the relationships among the genotypes differentiated the individuals into clusters resembling the patterns observed by PCoA. The four Finnish populations were distinct from the rest, but one of them showed the presence of two subgroups, similarly as by PCoA. Relationships among individuals were not fully consistent, partly depending on the marker type, despite a significant positive correlation and a good fit found between SNP and SilicoDArT distance matrices. The only Canadian sample showed a close relationship with most UK samples, and Indian and Pakistani samples were mostly distinct. The observed inconsistency may reflect an error in the clustering analysis due to small sample sizes.Studying the population structure is a key point for understanding patterns of gene flow and for inferring the history of populations. The Bayesian clustering approaches implemented, for example, in BAPS  show closer genetic relationship to the plants from the native area of distribution in the Himalayas than those from other regions of introduction. The Bayesian BAPS analysis and the following gene flow network were the most informative tools for resolving relationships among native and introduced plants.The genome profiling of I. glandulifera across Europe, thus confirming our second hypothesis. Multiple introductions, considerable genetic differentiation, and drift effects among I. glandulifera plants across geographic regions have been suggested also in microsatellite‐based studies by Hagenblad et al. ; data curation (lead); formal analysis ; funding acquisition (lead); investigation ; methodology ; project administration (lead); resources (lead); software (supporting); supervision (lead); validation ; visualization (supporting); writing—original draft (lead); and writing—review and editing (lead). Sakina Elshibli: formal analysis ; investigation ; methodology ; software (lead); validation ; visualization (lead); and writing—original draft (supporting).Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.Appendix S5Click here for additional data file.