Sceloporus occidentalis) have a patchy distribution at the northern edge of their range around the Puget Sound, Washington, where they almost exclusively occur on imperiled coastal habitats. The entire region was covered by Pleistocene glaciation as recently as 16,000 years ago, suggesting that populations must have colonized these habitats relatively recently. We tested for population differentiation across this landscape using genome-wide SNPs and morphological data. A time-calibrated species tree supports the hypothesis of a post-glacial establishment and subsequent population expansion into the region. Despite a strong signal for fine-scale population genetic structure across the Puget Sound with as many as 8–10 distinct subpopulations supported by the SNP data, there is minimal evidence for morphological differentiation at this same spatiotemporal scale. Historical demographic analyses suggest that populations expanded and diverged across the region as the Cordilleran Ice Sheet receded. Population isolation, lack of dispersal corridors, and strict habitat requirements are the key drivers of population divergence in this system. These same factors may prove detrimental to the future persistence of populations as they cope with increasing shoreline development associated with urbanization.Population dynamics within species at the edge of their distributional range, including the formation of genetic structure during range expansion, are difficult to study when they have had limited time to evolve. Western Fence Lizards ( As a consequence of their smaller size, peripheral populations often have lower genetic diversity compared to core populations4. Furthermore, the physiological constraints experienced by peripheral lineages can limit dispersal and gene flow among and between populations, which can restrict them to smaller geographic areas. As a result, peripheral populations can be relatively homogeneous with low genetic diversity due to low levels of dispersal and gene flow. Diversification at microgeographic scales is often correlated with either environmental heterogeneity or minor phenotypic changes  that contribute to a reduction in gene flow among populations8. Diversification at fine spatial scales can also occur when a species occupies a non-optimal niche or reaches the edge of a geographic range causing populations to become geographically fragmented, resulting in population structure10.Population divergence occurs across a broad range of spatial and temporal scales. The expected pattern of genetic variation, however, depends partly on whether the populations are located at the periphery or core of the range11. Populations near the edges or at the extremes of their preferred habitats and environments run up against physiological limitations or stressors, such as thermal maxima or minima, that can cause allele frequencies to shift as populations become smaller and more fragmented13. The low genetic diversity of species already at their physiological limits further restricts adaptive potential in the face of abiotic stressors15. However, this central-peripheral hypothesis is not always supported in natural systems16, and there is even evidence of groups excelling at the periphery17. Founder events and novel abiotic pressures can work together to shape patterns of genetic variation and population structure observed within species at the edge of their range.Intraspecific diversity is also a product of ongoing and historic environmental changes, including climate driven habitat change which can alter the genetic composition of a populationSceloporus occidentalis) spans a large geographic area in the Western North America, extending from Baja California, Mexico into the Puget Sound region, Washington19. Across this relatively large distribution, at least five distinct genetic groups can be identified that are separated by major biogeographic barriers18. The Pacific Northwest (PNW) group comprises the northernmost populations, extending from Northern California into Oregon and Washington, and it shares a most recent common ancestor with populations from the Western Sierra Nevadas20. Throughout much of their central and southern range, populations of S. occidentalis are geographically continuous and successful in nearly all habitats, exploiting both urban and natural environments. Yet, populations in Washington have a fragmented distribution, occurring solely along the Columbia River near the Oregon border, in the Cascade Mountains in central Washington, and on shoreline habitats around the Puget Sound in Western Washington  that tend to hatch more quickly, presumably due to their smaller size22. Despite physiological comparisons of northern to central or southern populations, few studies have directly addressed the ecological or habitat requirements for the northern populations30. In addition to investigating the genomic and morphological variation of S. occidentalis around the Puget Sound, we also aim to provide new information regarding their microhabitat requirements in this region.In contrast to California populations, few studies have focused on the ecology and life history of S. occidentalis in the Puget Sound and use genome wide single nucleotide polymorphisms (SNPs) and morphological data to test whether population divergence is present in the region. We then describe patterns of genetic and morphological variation and use demographic models to understand how populations have evolved through time. Lastly, since S. occidentalis in the Puget Sound region is less abundant and more fragmented than other populations throughout the species distribution, we discuss the ecological requirements necessary for their success at the northernmost extent of their range. These new data provide strong support for multiple genetically distinct groups in the Puget Sound region, which has important implications for conservation and population management.We present the first comprehensive study of the distribution of The research presented herein was conducted in accordance with the ethical standards and guidelines outlined by the University of Washington’s Institutional Animal Care and Use Committee (IACUC protocol #4367-02). Samples were collected with permission from the Washington Department of Fish and Wildlife (WDFW permit 20-144).Sceloporus occidentalis individuals from 19 localities across the Puget Sound Region and four localities from surrounding regions in Eastern Washington, with most sampling being conducted in 2020 32. We double-digested each sample using the digestion enzymes SbfI and MspI in CutSmart Buffer (New England Biolabs) for 7 h at 37 We extracted genomic DNA from liver biopsies using salt-extraction33. We conducted a reference-based assembly of the RAD loci using a draft of the S. occidentalis genome from Yosemite National Park, California35  procedure to test for the optimal number of subpopulations . To visualize and compare results, we used the program CLUMPAK39 to produce structure barplots. We repeated these procedures for datasets with 0% (236 SNPs) and 50%  missing data.We conducted genetic clustering analyses to estimate population structure. Using the 50% missing dataset, we ran a principal component analysis (PCA) using the R package ‘adegenet’ to establish general patterns of genetic diversity, and a discriminant analysis of principal components (DAPC) to determine if a sample’s locality could be determined by genomic data40. We used the 50% missing data dataset to maximize the number of loci in the analysis. Because the Puget Sound population is not geographically continuous throughout the region, our sampling was often confined to small, restricted geographic areas. To avoid including samples from siblings and other close familial relationships, we calculated the inbreeding coefficient among individuals using the option --relatedness2 in VCFtools41. Lastly, we used the program MEGA version X42 to calculate nucleotide diversity  from the 50% missing data dataset. To maximize the number of segregating sites, we projected the data down to a smaller sample size (N = 70) using the program easySFS (https://github.com/isaacovercast/easySFS). We optimized four single-population demographic models using Python scripts50: (1) two-epoch model with instantaneous size change (two parameters): T = time in the past at which size change happened; (2) exponential growth model (two parameters): T; (3) bottlegrowth with instantaneous size change followed by exponential growth (three parameters): T, and T, and TB = duration of bottleneck. We performed four rounds of model optimization under each model with 50 replicates each and 25 maximum iterations. For each model, we used the parameters from the best-scoring replicate as starting values for the next round of optimization. After the final optimization, we used the replicate with the highest likelihood for each model to calculate AIC scores and perform model selection51. For the top-ranked model, we conducted 100 replicate simulations to assess the goodness-of-fit of the model to the data. We tested the top-ranked model by comparing the empirical log-likelihood value to the values obtained from 100 parametric bootstrap replicates, with the expectation that the empirical value will fall within the range of simulated values. Finally, we obtained confidence intervals for parameters using bootstrapping (100 replicates) by re-sampling the SNP data with replacement, and then optimizing model parameters for each replicate using the same procedure described above. We converted the unscaled population parameters to demographic terms as follows: the time parameter T used the equation 52. To calculate L, where Moments output and we used a generalized lizard 53.To investigate population demographics, we analyzed samples from the Puget Sound region as a single population. We performed demographic analyses using the program S. occidentalis, we collected morphometric and meristic data from adult individuals. We included most samples used in the genetic dataset, with additional samples from some localities , measured from the tip of the snout to the vent; tail length (TL), measured from vent to the end of the tail; head length (HL), measured from the parietal eye to the tip of the rostrum; head width (HW), measured at the widest part of the head; right and left longest toe length , measured from the base to the tip of the toe; right and left femur length , measured from the ventral midline to the distal part of the knee, and five meristic counts: left femoral pores (LFP); right femoral pores (RFP); left longest toe lamellae (LTL); right longest toe lamellae (RTL); medial scales (MS); and dorsal scales (DS).To determine whether there are any patterns of morphological divergence in Puget Sound U test under the null assumption that the morphological distribution between the two groups are the same. The difference between males and females was statistically insignificant (p = 0.17), thus we analysed all individuals together. We made natural log transformations of all morphometric data. To account for allometric growth, we used R to size-correct the morphometric data by regressing each morphometric trait against SVL for all individuals and using the residuals in subsequent analyses (PCA and LDA)57. We normalized but did not size correct the discrete meristic data. We analyzed the normalized meristic and size-corrected and log-transformed morphometric data both separately and combined.To determine whether subpopulations within the Puget Sound show any morphometric variation, we performed multivariate statistics using a principal component analysis (PCA) and a linear discriminant analysis (LDA). We determined whether male and female individuals were significantly different by conducting a Mann–Whitney Sceloporus occidentalis individuals in the Puget Sound region. We collected the following data using visual encounter surveys: lizard body temperature, air temperature, lighting conditions, and the microhabitat and substratum that the lizard was observed on. We collected lizard body temperature data using a cloacal thermometer and air temperature using various external thermometers. All data collected were combined with other ecological studies on the population30. We did not collect ecological data for the lizards used in the genetic study.From April 1999 through October 2018, ecological data was collected for 466 Sceloporus occidentalis is discontinuous throughout the Puget Sound region. The majority of sites sampled had subpopulations confined to small, geographically isolated stretches of habitat. All but two localities , Puget Sound north , Puget Sound south , Kitsap Peninsula west , and Kitsap Peninsula south (KITs). The PCA reveals at least four genetic groups within the Puget Sound region with most samples clustering with those from geographically proximate areas, except for the KITs group which spans the entire Puget Sound region Fig. . The resten Fig. . Despiteted Fig. . When a les Fig. . Intrigurom Fig. B. In mosrom Fig. . HoweverAlthough the genomic data can be used to pinpoint the specific location of origin for most samples, the genetic diversity around the Puget Sound is, as expected, relatively low. The maximum genome-wide pairwise distance between any two genetic clusters is 0.089%, which is for the comparison between samples from opposite ends of the Puget Sound (Camano Island vs. Anderson Island). The minimum pairwise distance between any two genetic clusters is 0.030%, which is for the comparison between samples from Joemma and Chambers Creek , and we ranked the models by their AIC scores  to understand the population history of es Table . The topes Table ; Fig. S7es Table . The twoNone of the morphological traits evaluated here can be used to clearly distinguish subpopulations from one another Table . We presS. occidentalis in the Puget Sound region. The distribution of the Puget Sound population is primarily restricted to south-facing beaches and occasionally deforested areas that receive high sun exposure. Of the lizards recorded, 78.5% (n = 465) of individuals were in open, sunlit area (no shading or light filtering). On coastlines, we found individuals on driftwood a majority of the time , with most being on shorelines that meet forested hills. Additionally, despite prior records stating that the populations hibernate from late September through mid May27, we observed individuals basking from April through October. These activity windows are presumably dependent on annual climate patterns. Lastly, we observed body temperatures ranging from 19.8–37.8 °C (n = 465), with a mean of 34.1 °C and median of 34.8 °C, consistent with body temperatures documented for the species59.Using data collected from 466 lizards observed between 1999–2020, we provide updated information on the ecology and natural history of Sceloporus occidentalis at the northern edge of the species range. The genomic data demonstrate that despite spanning a microgeographic scale and lacking any pronounced ecological differences, the Puget Sound region comprises multiple genetically identifiable subpopulations. Although genetic diversity is relatively low, in many cases there is sufficient genomic differentiation to identify the specific location from which a lizard originated.We investigated the geographic distribution, genetic and morphological variation, and phylogenetic and demographic history of 60. Considering the low genetic diversity observed in the group, the relatively high Despite low genetic diversity  and 0%  missing data datasets , Canada, with two from the citizen-science platform iNaturalist and one from a scientific study64. Older records also indicate the presence of S. occidentalis in B.C., but with no specific locality information67. We expect that these records represent either accidentally or purposefully translocated individuals, yet it remains unclear whether they have or will be able to establish sustainable populations. If more individuals are detected from the region, genomic testing could prove useful in determining their geographic origin.By solely focusing on S. occidentalis populations22, which may provide an explanation for their fragmented distribution in the Puget Sound region. In addition to females dedicating more resources to rearing offspring at higher latitudes22, the northern populations also have substantially reduced physiological growth rates. In a laboratory-based experiment, hatchlings from Deschutes County, Oregon exposed to 34 °C versus 27 °C environments for 12 h had a growth rate 1.4× greater. Given the same conditions but only 6 h of exposure to the specified temperature, the growth rate increased to 1.7× that of the colder temperature22. Additionally, Oregon populations showed fewer hours of activity per day than those in California23. The opportunity for Puget Sound subpopulations to have extra sunlight provided by inhabiting south facing beaches or full sun areas may be critical for their success. Further, the cool temperatures of the Puget Sound region , may necessitate exposure to additional hours of warmth for survival. In our field surveys, we only detected two localities , and removes the cover of riparian vegetation71. Although no studies have directly examined impacts on lizard populations, armoring can have strong detrimental effects on species assemblages and abundance73. Fortunately, armor removal can effectively restore these key elements of shoreline habitats75.Extensive shoreline development and urbanization could limit future population expansion of S. occidentalis occurred one mile south of Lincoln Park, Seattle. We were unable to detect S. occidentalis in Lincoln Park. However, the locality from which the specimens were found is now developed with little to no natural habitat remaining, so it is unlikely that the population has persisted. As coastal development in the Puget Sound region continues, more isolated groups may, or already have, become threatened. As such, expanded studies on the ecology and population demographics of the species in the region will prove useful for potential reintroductions. Our study provides important genetic information for guiding the selection of source populations to be used for reintroductions by demonstrating that unique alleles are present in many subpopulations across the Puget Sound region. One approach could be to translocate gravid females from a locality that shares the distinct alleles associated with the extirpated group. Another approach could utilize translocated individuals from multiple subpopulations to increase the genetic diversity of the reintroduced group, considering the low genetic diversity within any given subpopulations. Lastly, and especially pertinent for locally extirpated groups with an unknown genetic heritage , reintroduction efforts could utilize individuals from Chuckanut, considering that the assemblage at this locality is known to be human introduced.Urban development may have already led to extirpation of local lizard assemblages, thus population reintroductions may become necessary. Historical museum records indicate that S. occidentalis population would be expected to continue a northward expansion, but the adaptive potential of the group may not be great enough to keep up with the rampant rate of anthropogenic change.Although a warming climate may lead to natural expansion of the Puget Sound population, occupying the northernmost extent of the distributional range exposes them to threats that the remainder of the species does not face. We provide evidence for a population that has undergone a relatively recent and expansive growth, which could indicate continued expansion as the climate becomes more favorable. However, multiple compounding factors limit this success. The pattern of colonization limited to highly specific stretches of south-facing coastal habitats with high sun exposure, including islands and peninsulas, has likely limited gene flow and accelerated genetic drift producing the genetic patterns described herein. Further, geographic discontinuity coupled with expanded urban development may impede the group’s ability to take advantage of the rapidly warming climate. The northernmost Supplementary Information.


Cymodocea nodosa along a latitudinal gradient extending across the Atlantic-Mediterranean boundary . Our main goal was to search for potential outlier loci that could underlie adaptive differentiation of populations across the latitudinal distribution of the species. We hypothesized that such polymorphisms could be related to variation in photoperiod-temperature regime occurring across latitudes. The three populations were clearly differentiated and exhibited diverse levels of clonality and genetic diversity. Cymodocea nodosa from the Mediterranean displayed the highest genotypic richness, while the Portuguese population had the highest clonality values. Gran Canaria exhibited the lowest genetic diversity (as observed heterozygosity). Nine SNPs were reliably identified as outliers across the three sites by two different methods , and three SNPs could be associated to specific protein-coding genes by screening available C. nodosa transcriptomes. Two SNPs-carrying contigs encoded for transcription factors, while the other one encoded for an enzyme specifically involved in the regulation of flowering time, namely Lysine-specific histone demethylase 1 homolog 2. When analyzing biological processes enriched within the whole dataset of outlier SNPs identified by at least one method, “regulation of transcription” and “signalling” were among the most represented. Our results highlight the fundamental importance signal integration and gene-regulatory networks, as well as epigenetic regulation via DNA (de)methylation, could have for enabling adaptation of seagrass populations along environmental gradients.Plant populations distributed along broad latitudinal gradients often show patterns of clinal variation in genotype and phenotype. Differences in photoperiod and temperature cues across latitudes influence major phenological events, such as timing of flowering or seed dormancy. Here, we used an array of 4,941 SNPs derived from 2b-RAD genotyping to characterize population differentiation and levels of genetic and genotypic diversity of three populations of the seagrass  Environmental heterogeneity in space and time can impose a strong selective pressure driving adaptive divergence of populations . QuantifArabidopsis thaliana accessions collected throughout the European range of the species  across geographical clines is directly related to aspects of phenotypic and genetic divergence among populations in land plants . For ins species . In part species .Zostera marina) and the general unfeasibility to perform molecular genetic studies across multiple generations. The reproduction of seagrasses under controlled conditions is indeed still challenging, and life cycles are often too long, which prevent identifying heritable variations . Such studies have aided in resolving main geographic differentiation and structure of seagrass populations e.g., . IndicesCymodocea nodosa (Ucria) Ascherson is a marine dioecious angiosperm present throughout the Mediterranean Sea and the adjoining Atlantic coasts and the dominant species structuring subtidal seagrass ecosystems along North West Africa and South West Europe  and SST], and evolutionary contexts   in response to heatwaves, in terms of diversion of carbon reserves and biomass allocation (t Europe . Recent t Europe . Large iversity) . Populatversity) . The higversity) . An impolocation . HoweverRestriction-site-associated DNA sequencing (RAD-Seq) techniques represent a family of cost-effective techniques compared to e.g., whole-genome sequencing that can be employed in non-model species without a reference genome and guarantee high-resolution population genomics data for demographic analyses . In addiC. nodosa distributed along the Atlantic-Mediterranean transition region , thus encompassing most of the latitudinal distribution range of the species . 2b-RAD libraries were prepared following a modification of the protocol described by https://github.com/z0on/2bRAD_denovo. Briefly, genomic DNA (∼100 ng) was digested using the type 2b restriction enzyme BcgI to produce uniform fragments of 36-bp, to which adaptors were ligated on the cohesive ends. The fragments were then amplified with barcoded adaptors and purification of the target bands was carried out by 2% agarose gel-electrophoresis. Subsequently, gel fragments were cleaned using a MinElute Gel Extraction Cleaning Kit (Qiagen) and pooled equimolarly into a single pool. Four individuals from each locality were used as technical replicates . These technical replicates were used in the analysis step to assess the overall accuracy of genotyping, set quality filtering criteria, and quantify error rates between samples. In total, 70 C. nodosa samples  were successfully sequenced on two lanes of an Illumina NovaSeq platform, generating 50-bp paired-end reads, at the Science for Life Laboratory (SciLifeLab) Genomics, SNP&SEQ Technology Platform in Uppsala University, Sweden. Only reads 1 (“forward reads”) were used for this analysis.Genomic DNA was extracted using the NucleoSpin® 96 Plant II kit (Macherey-Nagel) following the manufacturer’s instructions. DNA quality and quantity were checked through 1% agarose gel electrophoresis, and the QubitC. nodosa, the analysis followed a modified de novo pipeline available at https://github.com/z0on/2bRAD_denovo. Reads were first ‘demultiplexed’ based on barcodes and then adaptors were removed, then a quality filtering was performed using the Fastx-toolkit , Naples . As no genome sequence is currently available for -toolkit . Only re-toolkit  allowing-toolkit .SNP-calling was performed using The Genome Analysis Toolkit (GATK) version 3.8 . A firsthttps://github.com/z0on/2bRAD_denovo). Technical replicates were discarded from further analyses after examination for concordance in genotype estimates. Individual genetic variation was explored by a Principal Component Analysis (PCA) using the R package adegenet v. 2.1.3  as well as FIS across all loci for each population were calculated using the R package diveRsity 1.9.90  were recalculated keeping only distinct MLLs per population.Our final dataset was thinned in order to keep one SNP per RAD fragment with maximal allele frequency  developed for . nodosa  and used. nodosa . Only a . nodosa , using 1. nodosa . Clonal  samples . ObserveEx 6.503 .ST and PCA, respectively, i.e., BayeScan v. 2.1 ] by using the blastn algorithm , in respect to Faro and Gran Canaria populations  (C. nodosa population from Ria Formosa lagoon (Faro), one MLL largely dominated over the other (C. nodosa population from Ebro Delta exhibited the highest genotypic richness with 11 MLGs over 12 samples (R = 0.91), while in Gran Canaria 6 distinct MLGs were identified (R = 0.45). In Faro, a single MLG was identified with microsatellite markers  were negative for all populations, with the lowest value observed in Faro (o) was highest in Faro, intermediate in Ebro and lowest in Gran Canaria  indicated above have been obtained following clones’ removal from the dataset. Genetic diversity and differentiation analyses repeated using SSR markers  was significant for each population pair comparison   both with SSRs and SNPs markers. Genotypic richness (R) has been found to be highly variable across C. nodosa populations, even at small geographical scales  has been historically considered monoclonal. IS that is influenced by clonality. All three populations exhibited strongly negative FIS values based on the SNP genotyping, but the most clonal population (Faro) exhibited the lowest FIS (-0.51). Negative FIS has been shown to be strongly linked to the degree of clonality in facultative sexually reproducing species driven by genetic drift, both in theoretical (IS has been shown to be not as sampling-sensitive as genotypic richness (R) and has been suggested as the measure of choice for assessing the importance of clonal reproduction in seagrasses  (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FLOWERING LOCUS T (FT) (Lysine-specific histone demethylase 1 homolog 2 (LDL2). In Arabidopsis, this gene is involved in H3K4 (de)methylation of target genes including FLC. FLC is down-regulated by LDL2 and ldl2 mutants display increased H3K4me3 levels at FLC compared to wild type  in Glycine (Gly). We cannot establish the real effect of this substitution at the protein level, but as it will modify the charge of the peptide, it could potentially result in a loss of interactions with other molecules or could induce differences in the regulation of other genes related to the flowering pattern , which is in an ecologically important life history trait. The timing of floral transition is critical to reproductive success in plants and varies across latitudes in response to changes in photoperiod and temperature patterns . In Arab C (FLC) . FLC inhS T (FT) . The regS T (FT) . Chr1_42ild type . LDL2 isild type  and contild type . A similild type . The mut pattern . It is w pattern , whereas pattern . In addis of FLC ; while operature . We cannPWWP domain protein 3, which is specifically involved in the regulation of FLC and flowering time. PWWP domain proteins (PDPs) function together with other components  to regulate the function of the PRC2 histone methyltransferase complex, thereby facilitating the maintenance of H3K27me3 on FLC (EARLY FLOWERING MYB PROTEIN (EFM). In Arabidopsis, JMJD5 has a histone demethylase activity (H3-K36 specific) and interacts with EFM to repress the floral pathway integrator FT, thus negatively regulating flowering  in C. nodosa populations from contrasting thermal environments, as well as a low level of gene body methylation (predicted by high CpGO/E ratio) in transcripts with a differential expression depending on plants’ origin  along latitudinal clines would allow a higher power for detecting signature of selection associated to local environmental settings in seagrasses.While we acknowledge that our study including only three


The population of Mexico has a considerable genetic substructure due to both its pre-Columbian diversity and due to genetic admixture from post-Columbian trans-oceanic migrations. The latter primarily originated in Europe and Africa, but also, to a lesser extent, in Asia. We analyze previously understudied genetic connections between Asia and Mexico to infer the timing and source of this genetic ancestry in Mexico. We identify the predominant origin within Southeast Asia—specifically western Indonesian and non-Negrito Filipino sources—and we date its arrival in Mexico to approximately 13 generations ago (1620 CE). This points to a genetic legacy from the seventeenth century Manila galleon trade between the colonial Spanish Philippines and the Pacific port of Acapulco. Indeed, within Mexico we observe the highest level of this trans-Pacific ancestry in Acapulco, located in the state of Guerrero. This colonial Spanish trade route from East Asia to Europe was centred on Mexico and appears in historical records, but its legacy has been largely ignored. Identities and stories were suppressed due to slavery, assimilation of the immigrants as ‘Indios’ and incomplete historical records. Here we characterize this understudied Mexican ancestry.This article is part of the theme issue ‘Celebrating 50 years since Lewontin's apportionment of human diversity’. BeforAlthough Native Americans, Spanish Europeans and sub-Saharan African individuals were the most numerous in Mexico during the colony (as reported by Spanish written records and a census from the seventeenth century ), other . 2 (a)K = 5 clusters distinguishes the broad continental components: sub-Saharan African, European, Native American, East Asian and Melanesian . This is consistent with the lowest cross validation (CV)-error criterion obtained at K = 5 , where the five continental references differentiate. Higher K values show substructure within continental clusters, mostly among Native American populations. The Austronesian language-speaking groups in Southeast Asia cluster together with the rest of the East Asian populations, so we use the latter as references for local ancestry inference of Southeast Asian and East Asian segments (see §3 for details). This shared clustering of East Asian ancestry with the Austronesian groups of Southeast Asia (K = 5 clusters) is supported by the lowest CV-error criterion when running ADMIXTURE .Genome-wide ancestry proportions were estimated with unsupervised maximum-likelihood clustering (ADMIXTURE)  includinK = 5. Sub-Saharan African ancestry reaches 33.4% in individuals from coastal states known for their Afro-Mexican presence [Cluster membership proportions for the admixed Mexican individuals consist mainly of Native American and European components, followed in decreasing order by sub-Saharan African, East Asian and Melanesian. The proportions differ within the Mexican subregions as reported in previous studies . For inspresence , namely In this study, we considered a fourth continental origin for the sources that contributed to admixture in Mexico: Asia . East Asc). Moreover, the comparison between East Asian ADMIXTURE proportions and IBD sharing with Southeast Asia shows that Guerrero exhibits an exceptional amount of both compared to the remainder of cosmopolitan Mexicans (d). These results led us to further characterize the subcontinental roots within Asia of these individuals.An independent line of evidence demonstrating Asian ancestry in Guerrero comes from the identification of genomic segments inherited from common ancestors  between Asian populations and Mexico c. MoreovMexicans d. These  (b)Phasing  followed by local ancestry inference were applied to the merged dataset (see §3 for details) using SHAPEIT2  and RFMiTo pinpoint the origin of the Asian component in Mexico, a MAAS-MDS was performed with a reference panel of East Asian, Southeast Asian and Oceanian populations. Only cosmopolitan Mexicans having more than 5% combined East Asian and Melanesian ancestry were included in this analysis, so as to ensure that sufficient sites remained in each individual after masking the other ancestries. This yielded one individual from Sonora, one from Oaxaca, one from Yucatan and 12 from Guerrero. The Sonora and Yucatan individuals clustered near Chinese reference populations, while the Oaxaca and Guerrero individuals clustered within maritime Southeast Asia, with the latter showing a heterogeneous distribution across several source populations on the first two principal components (PCs) . No cosm (c)In an admixed population, the length distribution of segments originating from each ancestral source is informative of the time and history of admixture events; for instance, longer segments reflect more recent events for which recombination has had less time to act. Modelling these segment length distributions under different demographic scenarios allows us to identify the more likely history that explains the observed distribution of ancestry tracts. For the Asian-derived segments identified in the Guerrero individuals with over 4% of the ancestry, we performed this fitting using the Tracts algorithm, testing three different models to contrast possible demographic dynamics with the likelihoods given the observed lengths (see §3 for details). We applied a Bayesian Information Criterion (BIC) to compare the fit of these models. The model with a single admixture pulse fit the data best, as compared to the models with continuous migration, i.e. multiple recurrent pulses of the same ancestry across generations .We found the maximum-likelihood parameters for this single pulse model to be consistent with an Asian admixture event 13 generations ago, or around 1620 CE using a sex-averaged estimate of 30 years per generation . This daThe East Asian ancestry in the individuals from Sonora and Yucatan, both distant states from Guerrero, has a very different source within Asia and appears to represent post-colonial migration events, perhaps the immigration of Chinese labourers from the Guangdong Province to northern Mexico  and the  (d)Southeast Asian ancestry was observed in Mexicans from Guerrero, particularly from the Pacific port of Acapulco. This profile suggests a genetic legacy of the Manila Galleon, which used Acapulco as its port of disembarkation in Mexico. Limited historical records indicate that the proximal source of the thousands of ‘Chinos’ who arrived in this city was the Philippines . Our genOverall, our results reveal an understudied origin for the historically neglected passage of large numbers of Asian immigrants into Mexican territory during the colonial period. This origin suggests that unraveling the full history of the slave trade within southeast Asia and across the Pacific to Mexico can be pursued through the genetic footprint of present-day admixed populations.. 3 (a)To study the ancestral components of admixed Mexicans, a total of 369 cosmopolitan individuals from ten sampling locations across Mexico were reanalyzed using previously generated and publicly available genotype data [ (b)https://www.coggenomics.org/plink2/), retaining only the intersection set of markers shared across all panels. In order to consider the genetic structure affinity between East Asian and Southeast Asian populations, an additional ADMIXTURE run was performed including three Filipino populations that are not Negrito, namely, Luzon, Visayas and Igorot individuals. This merged dataset included 118 562 SNPs before LD pruning, and 76 005 SNPs after LD pruning .The genetic clustering analyses were performed with ADMIXTURE v. 1.3.0 in unsupervised mode. The proportions of five well-differentiated, continental source populations were found for all samples via unsupervised clustering: sub-Saharan African, European, Native American, East Asian and Melanesian. Each continental signal was estimated with an equal number of reference samples when possible. In order to include the largest number of markers, the cosmopolitan Mexican dataset was merged with whole genome reference data from the 1000 genomes consortium dataset  and the  (c)To compute IBD sharing between Mexicans and Southeast Asian individuals, we used hap-IBD considering segments down to 1.5 cM . Default (d)The cosmopolitan Mexican and continental references were merged with Plink version 1.90 beta, considering only the intersecting set of markers. Each continental reference panel and admixed Mexican panel was phased separately with SHAPEIT2 and default parameters. Phased haplotypes were given to RFMix v. 1.5.4 . The repBecause we were investigating events that occurred during the historical period, long after the admixture of East Asian and Melanesian ancestries in prehistoric southeast Asian peoples, after running RFMix we relabelled segments from either of these sources simply as ‘Southeast Asian’ (in the process merging contiguous segments). We then masked all other segments  and performed all ancestry-specific analyses only on these ‘Southeast Asian’ ancestry segments. Details for each analysis and run are provided in  (e)To investigate the dates of Asian admixture in Guerrero, while ignoring the precise labelling of which non-Asian ancestries were counter-party to those admixture events, we applied a general ‘non-Asian’ label to all other ancestry segments called by RFMix above, namely, African, European, Native American. This allowed us to use a simple, robust model with few parameters to focus only on the question at hand, the dates of Asian and non-Asian pulses, without needing to simultaneously investigate subsidiary questions such as the identities of those non-Asian pulses. Using a maximum-likelihood approach, three models were tested using Tracts  to find  (f)For the MAAS-MDS analyses, local ancestry was inferred with RFMix using five continental references separately by array. Each array was merged separately with whole-genome sequencing data from 1000 genomes and HGDP for each of the five RFMix runs, allowing for a far higher density of SNPs than a full merge of all five arrays would have, and thus yielding more accurate local ancestry calls. The five continental references were identical to the panel used for genome-wide ancestry clustering. East Asian and Melanesian components were afterwards merged in analyses focusing on localizing the Asian ancestry origins. Local ancestry calls were performed with 803 636 SNPs for Array A, 518 409 SNPs for Array B, 561 339 SNPs for Array C and 542 878 SNPs for Array D .We applied a threshold of more than 5% combined East Asian and Melanesian in order for a sample to be considered in the MDS analysis. Samples from cosmopolitan Mexicans included seven populations from Array A and three populations from Array B . After aThe MAAS-MDS analysis considered the tracts from the combined East Asian and Melanesian ancestry merge, thus masking intercontinental components such as sub-Saharan African, European and Native American, especially in cosmopolitan Mexicans. The analysis was run with average pairwise distances as a dissimilarity measure . All ind


Foeniculum vulgare populations from the region of Istria have been subjected to a genetic and chemical study. Headspace-gas chromatography analysis of volatile secondary metabolites and PCR-RFLP analysis of the ribosomal DNA internal transcribed spacer region has been chosen to analyze the chemical and genetic traits of single plants, respectively. Large intrapopulation and interpopulation differences have been observed in both chemical profiles and restriction patterns of PCR products. The data from chemical and genetic analyses were pooled and used to assess allele frequencies of three putative genetic loci on individual populations. The pooled allele frequencies were used to determine interpopulation distances for phenogram reconstruction. The combined use of chemical and genetic datasets for genetic variation analysis proved to be a more comprehensive approach for such a study, compared to the use of single datasets, even using such relatively simple analytical tools.Wild  Foeniculum vulgare Mill.) has been used for centuries in the Mediterranean basin for both culinary and medicinal purposes. Besides its high content of essential oils [Fennel  as a well-proven technique in this research field ,9,32,33, samples ,35, as i samples ,23 would samples . Thus, w samples  in combi samples  in theirDue to the outbreeding nature of fennel , we assutrans-anethole, estragole, and fenchone content) are also in agreement with the analytical results published by Marotti et al. [trans-anethole and estragole share a common biosynthetic pathway [trans-anethole or only estragole, and a whole range of ratios between them. Fenchone was present in the majority of plants analyzed, but its absence was not strictly chemotype-dependent. Most of the nine plant populations studied exhibited a high heterogeneity, but mean population values have shown significant differences between populations. The average erc and arc values are displayed in The same sets of fennel samples were used for both chemical and genetic analyses, and were collected during the flowering season. We avoided the use of samples at the ripe stage, because higher levels of secondary metabolites could inhibit PCR of the extracted DNA . Althougi et al. . Moreove pathway . TherefoFoeniculum vulgare Mill. var. vulgare) [Foeniculum vulgare Mill. var. dulce), a cultivated variety containing prevalently trans-anethole [erc from 1.0 to 0.44), which is not a characteristic of sweet fennel. There were only two cases of plants with low erc and consequently high arc lacking fenchone, one from each population (Flengi and Plomin).The vast majority (over 90%) of all fennel plants assayed contained various amounts of fenchone, a terpene found in the bitter fennel variety (vulgare) , which ivulgare) . Thus, oanethole . HoweverThe sporadic absence of fenchone in fennel plants should be attributed to fenchone biosynthesis as a dominant trait. The non-relatedness between the absence of fenchone and the variability in phenylpropanoid contents observed in such cases indicates a significant number of allelic variants in corresponding genes.trans-anethole and estragole biosynthesis. Although environmental conditions could influence to some extent the phenylpropanoid biosynthesis [On the other hand, a wide distribution in phenylpropanoid composition observed by us  and otheynthesis ,41, a wiynthesis  in obtaiynthesis . Consequerc and arc as good indicators for allele frequencies of the corresponding genes.The wide distribution in the profile of phenylpropanoids, and their high content in fennel plants , also sufp allele frequency in a population. The choice for 15% of fenchone content in the essential oil, as a criterion between putative fp monoallelic and biallelic plants, was made on the basis of the histogram shown in fa homozygotes , a larger one of putative fp homozygotes  and the largest one of putative heterozygotes . Although such an approach for genetic assessment could be more arguable as in the case of phenylpropanoid composition, it is supported by the distribution of those three groups and allowed us to evaluate this trait in a codominant manner as well.On the contrary, besides its presence at the ripe stage, fenchone timewise accumulation pattern was found to be rather hard to predict. As a consequence, the arbitrary approach described in Despite the observed intra-population heterogeneities, the pooled allele frequency data revealed differences between populations . Becauseerc frequency. Apparently, the chemical criteria alone could not resolve the relationships between these populations.The relationships based on chemical data are depicted in MspI endonuclease was selected for large-scale PCR-RFLP analyses because it provided the highest degree of polymorphism among the samples. Two distinct groups of banding patterns occurred by using MspI  lacks a MspI restriction site; thus no restriction products are generated. This form was present in a varying, but considerable amount, in all fennel samples. The second allelic form (itsB), carrying one MspI restriction site (generating 529 bp and 153 bp restriction products), was also present in all samples, whereas the third allelic form (itsC), carrying another MspI restriction site , was present in about 36% of all samples. The itsC frequencies were highly variable among populations . They differ in erc and fp frequencies, but there are similarities in the ITS locus. There is a complete lack of the itsC allele in two of them (Flengi and Padna), but their common denominator is a slight, but apparent drift in the itsB frequency going from north to south as well. The first outgroup (Plomin) is located on the eastern Istrian coast, facing the Kvarner Gulf. This population has a rather low itsC frequency, similar to Vodnjan population, while the fp and erc frequencies are similar to those from Ližnjan and Flengi populations, respectively.The clustering pattern in the dendrogram of combined data  differs erc and the highest fp frequencies among all, whereas its itsB and itsC frequencies were more typical of the northwestern populations. It also displayed the highest chemical homogeneity. The Rabac population is probably a recently established offspring from a cultivated bitter fennel plantation, without the time needed to become sufficiently cross-pollinated from neighboring populations and consequently diversified. Excluding the Rabac population, a generalized statement about two allele frequency drifts could be made; the itsC is decreasing from north to south and from the coast to the inland area, whereas erc is decreasing from north to south and from the western to the eastern coast.The outermost outgroup (Rabac), located also on the eastern coast, differed substantially from all other populations. The most evident differences were the lowest To corroborate the observed allelic drifts in Istrian fennel populations, more of the latter should be examined, possibly along with topological, and meteorological data at the time of flowering, with the aim to obtain an objective explanation of such an allelic distribution. Anyway, the employed approach of combining chemical and genetic analysis for plant population studies looks promising, with little effort needed for method development. Consequently, it could be a valuable, high-throughput tool for population data gathering. Another option using this approach, which should be also considered in future work, is a proper sample pooling technique, for a further reduction of experimental efforts.One hundred and fifty-five fennel samples were collected during the flowering period in the Istria region (Slovenia and Croatia) from wild growing populations, on nine different locations: Ankaran, Buje, Flengi, Ližnjan, Padna, Plomin, Rabac, Rovinj, and Vodnjan . Ten to Template DNA was extracted from leaf samples of individual plants according to a previously published protocol . The exterc) was calculated as the ratio between estragole peak area divided by the sum of peak areas from both fennel phenylpropanoids, namely trans-anethole and estragole. In the same way, the trans-anethole relative content (arc) was also calculated. The obtained percentages were used as a measure of allele frequencies in the putative phenylpropanoid biosynthesis gene locus. Allele frequencies per population were calculated as the arithmetic averages obtained from single plant data. Gas chromatograms were also scored for the presence or absence of fenchone peak. The following criteria were used for fenchone scoring; score 2 : fenchone peak area > 15%; score 1 : fenchone peak area < 15%; score 0 : fenchone peak area = 0%. The sum of all scores was divided by the number of haploid genomes, in a given population, and used so as to calculate the fenchone presence allele frequency (fp) or complementally, fenchone absence allele frequency (fa).Leaf samples from individual plants were subjected to the analysis of essential oil components by using headspace-gas chromatography as described in our previous paper . The estPCR amplification of the nrDNA ITS region was performed by using a TGradient thermal cycler . The primers used  were ITSAluI, HaeIII, MseI, MspI, RsaI, SmaI, TaqI, and Tsp509I . For each endonuclease restriction reaction, 10 μL of the PCR product was incubated, for 16 h with 3 U of the corresponding enzyme, 1× appropriate buffer and 1× BSA, in a final volume of 15 μL, at the optimal enzyme incubating temperature. MspI was finally selected for large-scale PCR-RFLP analyses. In this case, the restriction reactions were performed, using the abovementioned conditions, at 37 °C. Examples of PCR-RFLP products using MspI are shown in During preliminary tests, PCR products were subjected to endonuclease restriction by using several enzymes: itsA, itsB, and itsC, respectively) of the ITS locus in a sample. Population allele frequencies were calculated as arithmetic averages of single samples from a given population and used for data analysis.Ten microliters of each PCR-RFLP product were loaded onto and separated in 1.5% agarose gels, containing 0.5 μg/mL of ethidium bromide, at 105 V for 1.75 h. Samples were bracketed (every 8 samples) with a 100 bp DNA size marker  as an aid in sizing the PCR-RFLP products. Gel visualization and image acquisition was performed under UV light. A Coolpix 8400  CCD camera was used to acquire gel images at the following settings: shutter speed 0.5 s, aperture F3.0, ISO setting 100, JPEG file format. The images were electronically filtered through a red channel filter and further used for analysis. For densitometric image analysis, Sorbfil TLC Videodensitometer software  was used. Densitometry was performed on 684 bp, 529 bp, and 344 bp electrophoretic bands of PCR-RFLP products  and the The allele frequencies calculated from genetic and chemical datasets  were useOur findings indicate a high degree of genetic polymorphism and chemical variability both between and within fennel populations. The obtained gas chromatography data for phenylpropanoids and fenchone were employed for allele frequency estimation for their respective loci, in a similar manner as PCR-RFLP data. Population analyses were conducted on both chemical and genetic data separately, as well as in combination. The separate use of genetic and chemical data did not reveal an unambiguous relationship between both datasets, but the combined use of genetic and chemical information, due to their complementarity, proved to be useful in obtaining a more detailed and comprehensive insight into relationships between fennel populations in study, rather than the use of single data sets alone.


Ips typographus, is a major pest, capable of killing spruce forests during large population outbreaks. Recorded dispersal distances of individual beetles are typically within hundreds of meters or a few kilometers. However, the connectivity between populations at larger distances and longer time spans and how this is affected by the habitat is less studied, despite its importance for understanding at which distances local outbreaks may spread. Previous population genetic studies in I. typographus typically used low resolution markers. Here, we use genome‐wide data to assess population structure and connectivity of I. typographus in Sweden. We used 152 individuals from 19 population samples, distributed over 830 km from Strömsund (63° 46′ 8″ N) in the north to Nyteboda (56° 8′ 50″ N) in the south, to capture processes at a large regional scale, and a transect sampling design adjacent to a recent outbreak to capture processes at a smaller scale (76 km). Using restriction site‐associated DNA sequencing (RADseq) markers capturing 1409–1997 SNPs throughout the genome, we document a weak genetic structure over the large scale, potentially indicative of high connectivity with extensive gene flow. No differentiation was detected at the smaller scale. We find indications of isolation‐by‐distance both for relative (FST) and absolute divergence (Dxy). The two northernmost populations are most differentiated from the remaining populations, and diverge in parallel to the southern populations for a set of outlier loci. In conclusion, the population structure of I. typographus in Sweden is weak, suggesting a high capacity to disperse and establish outbreak populations in new territories.The Eurasian spruce bark beetle,  We investigated the genetic structure of the Eurasian spruce bark beetles over 800 km in Sweden using Restriction site associated DNA yielding thousands of markers across the genome. Our study show that population structure over this scale is weak, but we find indications of isolation by distance and that the Northern populations tended to be differentiated from the Southern ones. The stations were distributed throughout Sweden, and samples were gathered during the peak activity period in the spring of 2019 . DNA was extracted using the Qiagen blood and tissue kit, following the protocol suggested for insect DNA. The whole insect was homogenized with the TissueLyser for 4 min at 30 Hz. The final DNA elution was done in 60 μl buffer EB. We monitored DNA concentration with a NanoDrop, and performed Qubit analyses on a subset of the samples  as nanodrop quantification may inflate the estimated concentration. To further assess DNA quality, all extractions were run on agarose gels to estimate fragment size. Samples with concentrations below ~13 ng/μl or smeared profiles on the gel, indicative of decomposed DNA, were omitted from further analyses.The DNA extraction protocol, the RADtag library protocol, and summaries of the RADtag library preparation have been deposited in a public repository (https://www.scilifelab.se/facilities/ngi‐stockholm/http://ugc.igp.uu.se/our‐services/ngs‐technologies/).For the two transects, two single‐digest RADtag libraries were created from 160 samples per library. All samples from each transect were pooled in the same library to avoid any cross‐library batch effects. Of the 320 samples, 13 constituted individual duplicates originating from the same extract, and were split into two different tubes and treated as separate samples before library preparation to enable us to assess the repeatability of the method. From each sample, 500 ng DNA was used for restriction digest with the restriction enzyme SbfI from New England Biolabs. The DNA was sheared to allow for identification and removal of PCR duplicates. Individual barcoding was performed with 40 P1 inline barcodes of 8 bp differing by at least three sequence positions, combined with four P2 index barcodes of 8 bp differing by at least six sequence positions. Both libraries were amplified with a 16 cycle PCR. We performed size selection using SPRI beads throughout the library preparation, with a final size selection following the PCR step. The bulk of the final library consisted of fragments in the size range of 380–730 bp. The RADtag libraries were sequenced on separate lanes on an SP flow cell as paired‐end 150 bp reads on an Illumina NovaSeq 6000 platform by National Genomics Infrastructure – Stockholm, Sweden . The RADseq data were demultiplexed by the NGI sequencing facility to sublibraries based on the index barcodes on the P2 adapter. These libraries were then demultiplexed with the bioinformatics pipeline Stacks 2.53 . The reads were aligned to a concatenated reference genome consisting of the nuclear genome assembled by Powell et al.  as implemented in Plink 1.90b4.9 , and Euclidean distances were calculated from the first four principal components using Plink 1.90b4.9 . Weighted FST was transformed to FST/(1 − FST) following Rousset . The error rate after filtering was estimated to 1.25% using the seven duplicate pairs . After removing duplicates and down sampling of the dataset, the regional dataset contained 1997 non‐pruned and 1409 pruned SNPs from 152 individuals and 19 sampling locations. The final average sample coverage for the regional non‐pruned dataset was 49× , with the third principal component driving the trend , and weak support between the northern and southern regions , with the second principal component driving the trend . The linear discriminant analysis of sampling locations has an accuracy of 0.059 in assigning an individual to its original sampling location. The assignment to southern and northern regions was higher (accuracy of 0.89), but this was an artifact of the individuals assigning correctly to the southern region with much higher number of individuals , absolute divergence Dxy did not increase significantly with distance , and pairwise genetic differentiation of Euclidean distances was borderline significant  were differentiated between the northern populations that were most strongly differentiated from all other populations and the entire group. For We also examined if there were specific genomic regions showing indications of being under selection, and if outlier regions under stronger selection also were more differentiated. Across the entire dataset, we found elevated nucleotide diversity for 15 coherent outlier regions from 52 windows above the 95% quantile, whereof three regions from 11 windows were above the 99% quantile Figure . The midFST outliers above the 99%. The genes found in this region, include e.g. calpain‐A‐like isoform X1, involved in the organization of the actin‐related cytoskeleton during embryogenesis in Drosophila, PREDICTED: roquin‐1 gene and PREDICTED uncharacterized protein LOC109546387  also has high connectivity, but a mountain range structures the population into two subpopulations ; formal analysis (lead); methodology (supporting); visualization (lead); writing – original draft . Shruti Choudhary: Methodology ; writing – review and editing . Kajsa Svensson: Data curation ; investigation ; methodology . Martin N. Andersson: Resources ; writing – review and editing . Carsten Kirkeby: Funding acquisition ; project administration ; writing – review and editing . Daniel Powell: Formal analysis ; resources . Fredrik Schlyter: Resources ; writing – review and editing . Anna Maria Jönsson: Funding acquisition ; supervision (supporting); writing – review and editing . Mikkel Brydegaard: Funding acquisition ; investigation ; methodology ; project administration ; writing – review and editing . Bengt Hansson: Funding acquisition ; investigation ; methodology ; project administration ; supervision (supporting); writing – review and editing . Anna Runemark: Conceptualization (lead); data curation (lead); formal analysis ; funding acquisition ; investigation (lead); methodology (lead); project administration ; resources ; supervision (lead); visualization (supporting); writing – original draft (supporting).We declare no conflicts of interest.Figure S1Click here for additional data file.Table S1Click here for additional data file.


Bolinopsis infundibulum is distributed throughout cooler northern latitudes and B. vitrea warmer. Here, we examined the global population structure for species of Bolinopsis with genetic and morphological data. We found distinct evolutionary patterns within the genus, where B. infundibulum had a broad distribution from northern Pacific to Atlantic waters despite many physical barriers, while other species were geographically segregated despite few barriers. Divergent patterns of speciation within the genus suggest that oceanic currents, sea-level, and geological changes over time can act as either barriers or aids to dispersal in the pelagic environment. Further, we used population genomic data to examine evolution in the open ocean of a distinct lineage of Bolinopsis ctenophores from the North Eastern Pacific. Genetic information and morphological observations validated this as a separate species, Bolinopsis microptera, which was previously described but has recently been called B. infundibulum. We found that populations of B. microptera from California were in cytonuclear discordance, which indicates a secondary contact zone for previously isolated populations. Discordance at this scale is rare, especially in a continuous setting.The study of evolution and speciation in non-model systems provides us with an opportunity to expand our understanding of biodiversity in nature. Connectivity studies generally focus on species with obvious boundaries to gene flow, but in open-ocean environments, such boundaries are difficult to identify. Due to the lack of obvious boundaries, speciation and population subdivision in the pelagic environment remain largely unexplained. Comb jellies (Phylum Ctenophora) are mostly planktonic gelatinous invertebrates, many of which are considered to have freely interbreeding distributions worldwide. It is thought that the lobate ctenophore  The midwater zone of the world ocean is an enormous region of seemingly limitless connectivity inhabited by mostly holoplanktonic species, which spend their entire lives drifting or swimming in the water column . SpeciesLobatolampea, comb jellies are holoplanktonic. Although most species of ctenophores are thought to be weak swimmers, they are expected to disperse long distances during their lifespan, propelled mostly by ocean currents , some of which have been synonymized. Bolinopsis infundibulum  is a large species thought to have a global distribution that encompasses most of the North Pacific, Arctic, and N. Atlantic oceans. Bolinopsis vitrea  includes the well-studied coastal species, is genus . Bolinophrodites . There aitrea L.  is thougitrea L. . In 1912onal use .Mnemiopsis leidyi  with newly designed primers (B. infundibulum sensu strictu (s.s.) truly had a broad distribution that spanned the Arctic from the Bering Sea to the Northern Atlantic. In contrast, we found differentiation among all the other lineages we sequenced, even where no barriers were evident. The present work confirms that the name Bolinopsis microptera is appropriately applied to Bolinopsis from Washington State to California, and we formally reverse the proposed synonymy . We included eight more images of B. infundibulum s.s. photographed by Erland Svensen in Norway. We included seven of our own images of B. microptera each from Friday Harbor, WA and San Luis Obispo, CA, and 19 images of B. microptera from the Monterey Bay area, CA. We also included 11 images of B. mikado from Tokyo Bay, Japan, 15 images of B. aff. vitrea from Hawaii, nine images of B. vitrea s.s. from Florida (photographed by Richard Collins) and two of our own from the Bahamas. We read the landmarks with the geomorph package v.4.0.0 , B. mikado, and Mnemiopsis leidyi. Results were plotted in the Tidyverse  and Friday Harbor Labs (WA) were maintained at the Monterey Bay Aquarium (MBA). Five individuals of F2 CA and three individuals of wild-caught WA were placed in a wide diffusion tube . Samples of larvae were taken at 17- and 24-days post-hatching for DNA sequencing.In order to test reproductive incompatibility and potential hybridization between eastern Pacific ion tube ; Table 1COI, nuclear H3, MDH, LDH1, LDH2, and PK (SI ST), inbreeding (GIS), individual population assignment (parentage), and a hybrid index for wild eastern Pacific populations and experimental progeny based on allelic data. The hybrid index is an estimated likelihood that the progeny had a higher affinity to one parent population. Since it is haploid, the COI fragment was excluded from GenoDive analyses addressing inbreeding and HWE. To compare progeny with parents, we summarized gene diversity statistics for the COI sequences with the program DnaSP v.6.12.03x64  of the progeny from the captive spawning experiment (WA×CA) for the following loci: mitochondrial nd PK SI . To test12.03x64 .B. infundibulum s.s. as an outgroup (O)  is based on jackknife resampling, where statistically significant D > 0 indicates gene flow between populations P2 and P3, statistically significant D < 0 indicates gene flow between populations P1 and P3 and D = 0 indicates ancestral polymorphisms. Tests were performed on phased sequence fragments of LDH2 and PK with roup (O) .B. microptera from the eastern Pacific to explore the evolution and connectivity of a species . The F3 animals in the culture were collected in December 2018. Statistical programs further detailed in An F3 cultured individual of B. microptera and use them in separate population genetic analyses from the resulting fastsq file by aligning them to the M. leidyi mitogenome with BWA MEM v.0.7.17. We then assembled using the Geneious v.11.1.5 assembler with standard parameters. The assembly was polished using Pilon v.1.22 with an Illumina WGS library constructed with the same DNA. The assembly was then trimmed to only include one full copy of the mitogenome.We isolated mitochondrial reads from PacBio sequencing to extract whole mitochondrial genomes from nuclear genomic data, determine gene order for Mnemiopsis mitochondrial genome. A minimum spanning network of mitochondrial genomes was estimated with PopArt v.1.7.Shotgun and ezRAD sequence data were quality checked with fastQC and trimmed with Trimmomatic v.0.35. We filtered mitochondrial data and PhiX sequencing control from nuclear loci with BWA MEM v.0.7.17, seqtk, and SAMTools v.1.7-1. Separately, nuclear or mitochondrial reads were then mapped to the PacBio nuclear or mitochondrial assemblies with BWA MEM and reformatted with seqtk and SAMtools. We aligned whole mitochondrial genomes within Geneious Prime and compared them to the p value < 1e-4 to test whether the minor allele frequency (MAF) was significantly different from zero. We tested each population for deviations from Hardy-Weinberg equilibrium (HWE) separately with the same quality filters as previous analyses . We calculated proportions of sites that deviated significantly from HWE within R and these sites were excluded from statistical analyses.To understand population connectivity and metapopulation dynamics, we ran separate analyses of mitochondrial and nuclear genomes within the program ANGSD v0.935. We removed low-quality sites for all analyses by estimating and plotting the average read depth and quality of sequencing read scores for nuclear genomes of all individuals. Visualizations enabled us to limit low quality reads, paralogs, and repetitive regions of the genome. Potentially paralogous regions were excluded by discarding reads that had multiple hits to the reference assembly and by limiting read depths from 10 to 100x coverage based on the mean read-depth distribution. SNP site mapping quality was also increased by filtering for excessive mismatches (C = 50); we removed sites with missing data, excluded spurious and improperly paired reads and computed per-base alignment qualities (baq = 1) to resolve false variants. In order to select for polymorphic sites, we set the ST), population structure (admixture), and inbreeding. Population genetic statistics were estimated in two ways. First, the PacBio B. microptera sequence was designated as ancestral, and results were “folded”. Second, reads were mapped to M. leidyi as the ancestor and results were “unfolded.” From those analyses we calculated summary statistics with realSFS (We used ANGSD to estimate genotype likelihoods (GL) and the MAF from counts of alleles  to calcu realSFS . NucleotM. leidyi (NC016117) as the outgroup to estimate Tajima’s D, Fay and Wu’s H, and the overall DHEW statistic.To test for selection and population contraction or expansion on the mitochondrial genomes, we used DH with 50,000 coalescent simulations with alpha = 0.05 for each population and ST) where unweighted is the mean of the per-site ratios and the weighted is the ratio between the sum of α and the sum of β . We used the Rscript estAveError.R (included with the ANGSD package) to estimate Z-scores and set the limit for significance at |alpha| = 3.0.To differentiate between introgression versus ancestral polymorphisms among eastern Pacific populations of To infer whether populations were self-fertilizing or randomly mating, we used ANGSD to calculate heterozygosity and the variances of each population mean with 10 bootstrap replicates within a probabilistic framework estimated from genotype probabilities. We estimated inbreeding coefficients for each individual using the program ngsF v1.2.0. The ngsF analysis used 20 initial searches from random starting points to avoid convergence on local maxima. The run with the highest maximum likelihood was used as a starting value for the final run.Bolinopsis and Mnemiopsis leidyi included in Sanger, ezRAD, and shotgun sequencing efforts , and B. mikado (Japan)  distance  supported that B. microptera from the eastern Pacific Ocean were distinct from B. infundibulum s.s. The nuclear fragment for H3 contained shared ancestral polymorphisms between the three lineages, while attempts to amplify LDH1 and MDH were unsuccessful for B. infundibulum s.s.  . We provfragment . There wdistance , which wdistance . K2P disdistance . Distinc America  , NorCal , and SoCal . The highest levels of mitochondrial diversity were found in California populations, in part because they included both the California and the Washington mitochondrial lineages . In both species, the eight meridional canals are fed near their aboral ends by four interradial canals arising from the infundibulum, each of which bifurcates once into eight paired adradial canals. In B. infundibulum s.s., all eight adradial canals connect directly to the extreme aboral end of the meridional canals (B. microptera (B. infundibulum s.s.), the four branches that feed the subtentacular meridional canals (stmc) meet the stmc somewhere between the aboral tips of the comb rows to the level of about the 30th comb plate above the aboral ends of the comb rows. The position of this connection point varies between animals—likely associated with overall size—but it is consistent within each animal. At the aboral end of each comb row, below the junction with the adradial canal, each stmc then narrows, curves upward towards the statocyst, and ends blindly. The blind extension of the meridional likely sustains the ciliary groove that is adjacent to it, in the same way that the meridional canals provide sustenance for the comb rows. In one specimen from California, the left canal projection was very slightly wider than the right  was different from the M. leidyi mitogenome gene order (COX1-COX3-ND3-COX2-12S-16S-ND4-ND2-ND4L-ND5-ND1-CYTB-ND6) despite being a closely related species. The B. microptera mitogenome also contains one unidentified reading frame (URF) that appears to encode a 55-amino acid peptide. All 12 B. microptera mitogenome ORFs used the TAA termination codon.The B. microptera from the eastern Pacific Ocean. Network topologies and within/among population divergence of whole mitochondrial genomes and the COI fragment showed the same pattern, where the WA mitochondrial lineage was divergent (K2P∼2%) but sympatric with CA populations  and showed different patterns of population subdivision than mitochondrial data. The WA mitochondrial lineage was also present in CA populations but the WA nuclear lineage was restricted to Washington. Admixture plots of nuclear versus mitochondrial data clearly illustrated cytonuclear discordance in California populations where the WA mitochondrial lineage was admixed throughout California but with one exception the WA nuclear lineage was restricted to Washington .B. microptera and were the most similar within California, regardless of whether the animal had a WA mitotype. We detected one individual that was a putative WA/CA hybrid from the SoCal population  and distance matrices for multidimensional scaling (MDS) of mitochondrial versus nuclear genomes . PCA plolations. . MDS corlations. .Sanger-sequenced data also showed the same pattern of discordance where the WA mitochondrial lineage was found throughout California but the WA nuclear lineage was not .TD = 0.711) indicating a lack of rare alleles and possible population contraction, while for the CA populations it was slightly negative  indicating an abundance of rare alleles and possible recent expansion. These results also were opposite of what we found for mitochondrial genomes (Levels of nucleotide (π) and genetic diversity (Watterson’s θ) were similar in the nuclear genomes of all three populations , but con genomes . Per-ind genomes .We analyzed gene flow and demographic histories among the three Eastern Pacific populations. Pairwise FST values estimated using both allele frequencies from Sanger and WGS nuclear data showed significant differentiation between WA and CA populations, but not within CA . DirectiM. leidyi as an outgroup. These tests showed significant introgression between NorCal and SoCal and also between WA and SoCal. We performed PCA with the same data to examine population structure and found that the first PC explained 36% and the second only 1.5% of variation between populations. While the WA population was the most distinct, SoCal was slightly more similar, surprisingly, to WA than to NorCal (We used ABBA-BABA tests (D-statistics) to differentiate between incomplete lineage sorting  and introgression (ongoing gene flow) between the populations for 14,396 sites in the nuclear genomes, with o NorCal .Bolinopsis at the Monterey Bay Aquarium, resulting in many thousands of progeny (WA×CA). We collected two different batches of ∼100 progeny 1 week apart for sequencing analyses. All of the ∼200 progeny sequenced for COI had an identical mitotype (data not shown). We used a number of tests on the progeny and putative parents to determine whether the progeny represented “hybrids”: (1) whether multiple parents contributed to crosses, (2) whether there was asymmetrical reproduction, and (3) whether populations were randomly mating. Because all progeny sequences were identical for the COI fragment, it initially appeared either that one individual self-fertilized or one ‘mother’ spawned. However, sequences of the four nuclear Sanger loci were variable among the progeny and revealed several heterozygous positions for all markers, indicating that at least two parents contributed to genetic diversity (p ≤ 0.001), but the SoCal and WA×CA populations did not. The hybrid index results showed all but one of the progeny had a higher affinity to the NorCal population. We used ABBA-BABA tests to distinguish gene flow (introgression) from ancestral polymorphism in the two polymorphic markers for which an outgroup was sequenced (LDH2 and PK). ABBA-BABA tests were not significant for LDH2 but showed that for the PK fragment, the highest probability of gene flow was between CA and the progeny  was described from the shores of St. Matthew (“Matthaei”) Island in the Bering Sea by Mertens and identified in the Gulf of Georgia (Washington Territory) by A. Agassiz . A few years later, Alexander Agassiz re-examined his drawings and descriptions of Washington specimens from Rosario Strait (Washington State) about 20 miles from and contiguous with both Friday Harbor and the Gulf of Georgia . He concluded that Bolinopsis ctenophores from the western coast of North America were distinct from Mertens’ arctic B. septentrionalis because they were more elongated, had shorter lateral lobes, and had more complicated “windings” of the canals in the lobes . The new species was designated as Bolina microptera  that was in discordance.Hybrid zones occur when distinct populations meet, mate, and produce progeny. Hybridization between distinct populations or species can occur for a number of reasons, whether they are exogenous (due to the environment) or endogenous (due to genetic differentiation) . Two hypound, WA . Patternound, WA  detectedAs the presence of the WA genotype in CA was rare, it is plausible that the Washington mitotype invaded the California populations via adaptive introgression, or that cytonuclear incompatibilities existed between California mitotypes and Washington genotypes. We examined a number of hypotheses that could explain the patterns we saw.Bolinopsis ctenophores could accelerate these processes. Our data indicated the presence of selection on mitochondria in the Washington population, which had the smallest effective population size. Tajima’s D values for the WA mitochondrial genomes also were negative; while rare alleles were abundant, this can be interpreted as evidence for a recent selective sweep or population expansion after a bottleneck. We also found that all of the populations deviated from Hardy-Weinberg expectations and were therefore not randomly mating. It is plausible that sea level changes over time historically isolated the Washington population in a neritic rather than oceanic environment, thereby contributing to patterns of selection, genetic drift, and local adaptation.Reproductive isolation can be driven by a few individuals that colonize and rapidly expand to fill a new area. Selection and drift in the new population cause a rapid shift to a new co-adapted combination of alleles . Local aBolinopsis ctenophores are abundant in the summer, and are present but rare in late fall and winter months (pers. obs.), but present in low numbers throughout the year. We did not observe spatial or temporal segregation between the two mitotypes collected in the Monterey Bay area, further indicating a well-mixed population. However, divergent reproductive histories  could be a remnant of local adaptation in northern populations despite evidence of connectivity in pelagic habitats.Another common mechanism of reproductive isolation is asynchronous spawning, which could perpetuate divergence between Washington and California populations despite a lack of obvious contemporary geographic boundaries. In our laboratory experiment, Washington and California adults spawned asynchronously. In the Monterey Bay area, r months , whereasNatural selection has greater influence on systems with larger population sizes and species with large populations may be particularly likely to exhibit selection-driven phylogeographic structure . The effBolinopsis, are self-fertile simultaneous hermaphrodites, begin adult reproduction relatively young, spawn continuously  must have had significant effects on the patterns of local retention, gene flow, and speciation of Bolinopsis. We found California represented a massive zone of cytonuclear discordance in B. microptera, a pattern seldom detected in open populations. Geography and historical sea-level changes may have contributed to isolation of the WA population of B. microptera. The dock at Friday Harbor Laboratories, where we collected Washington ctenophores, is in a protected bay in the Salish Sea, over 100 km from open Pacific coastal waters. Isolation, combined with accelerated rates of mitochondrial evolution, small effective population sizes, and selection, could have driven population differentiation on a relatively fast timescale. Additional collections from the waters offshore of Washington state would be informative and would help to ascertain whether discordance was due to inbreeding (selfing), reproductive isolation , complex local oceanic circulation patterns, or some combination of the above. Future studies of ctenophore population genetics can illuminate the evolution and biodiversity of this rarely studied group and help to understand worldwide population subdivision in what were thought to be mostly cosmopolitan species.Our results revealed cryptic drivers of genetic isolation and population subdivision, which stand in stark contrast to the cosmopolitan model traditionally applied to pelagic species.


Valeriana officinalis L.) is an important medicinal plant revealing sedative, hypotensive, anti-spasmodic and anxiolytic activity. The purpose of the study was to determine the intraspecific variability of the common valerian growing wild in Poland and the ‘Lubelski’ landrace, as to their developmental traits, chemical composition and selected genetic parameters. Both wild-growing populations (19) and the landrace (1) were evaluated under ex situ conditions. Observations of the underground organs parameters, both developmental and chemical (according to the European Pharmacopoeia) were carried out in the first year of the plant’s development, while the characteristics of the aboveground organs, followed by the sowing value of seeds —in the second year. The genetic analyses were performed using the NGS-DArT-seq method. Results indicate the presence of five different gene pools covering the regions of population’s origin, with a gene flow within and between them. A high level of developmental and chemical variabilities among the wild-growing populations was noticed, however without a clear relation to the region of the origin. The mass of underground organs ranged from 107.4 to 403.6 g FW × plant−1 with the content of sesquiterpenic acids at the level of 0.004–0.094%. Population no 18 was distinguished by the highest content of sesquiterpenic acids and the relatively high mass of underground organs, followed by the admixture of the gene pool, typical for the ‘Lubelski’ landrace. Unlike the ‘Lubelski’ landrace, the wild-growing populations were characterized by a high amount of an essential oils (3.90 to 10.04 mL/kg), which may be promising from the perspective of their potential use. In turn, the sowing value of the seeds obtained from the populations, expressed as the germinability, was rather low (25.25–62.25%).Common valerian ( Valeriana officinalis L.) (Caprifoliaceae family) is a perennial plant, native to Europe and Asia. It grows mainly on wet meadows, water banks, low peat bogs and in moist forests [Valerianae radix)—rhizomes with roots and stolons—are listed in the European Pharmacopoeia and classified by the European Medicines Agency as a traditional herbal drug [The common valerian , comparable to the ‘Lubelski’ landrace (375.0 g FW × plant−1), which is considered a high-yielding form [It was shown that the examined populations differed both in the developmental and chemical traits. Regarding the first year of the plants’ development, it was observed that the diameter of the rhizome was at a level of 1.7–6.0 cm (CV = 0.24). The mass of underground organs ranged from 107.4 to 403.6 g FW × plantV = 0.35 . Populating form . The investigated raw materials were subjected to a chemical analysis, to determine the total content of sesquiterpenic acids  and essential oils, which are listed in the EP monograph as quality markers of Valeriana radix . It was In our work, the content of the essential oils in the underground organs of the populations investigated, varied between 3.9 and 10.4 mL/kg CV = 0.27), while in the case of the ‘Lubelski’ landrace, it was slightly lower (6.3 mL/kg) . A simil, while iIn the second year of the plant’s development, the populations investigated were characterized with regards to the traits of the aboveground organs. The height of the plants ranged from 110 (population No. 2) to 143 cm (population No. 18) (CV = 0.07), while the number of flowering shoots per plant was from five (population No. 2 and 16) to 15 (population No. 1) (CV = 0.27). These features differentiated the populations at a relatively low degree, and corresponded well with the ‘Lubelski’ landrace  . AccordiIn Central Europe, the common valerian blooms from June to July, when its hermaphrodite flowers are frequently visited by pollinators, especially hoverflies of the Eristalis genus . The speThe results described above indicate a remarkable polymorphism, both morphologically and chemically, within the common valerian species. However, no clear relationship between the traits examined and the geographical location of the populations was recognized. The phenotypic plasticity observed may be associated with the ploidy level of the common valerian, corresponding with the ‘basic types’ described within the species ,38,39,40p <0.0001) and weakly correlated with the annual precipitation . There was no significant correlation between the genetic distance and the average annual temperature and the altitude of the collection sites. The agglomerative hierarchical clustering (AHC), based on the Ward method, indicated the presence of four main clusters composed of six, eleven, two and one accessions, respectively  to 0.104 (the ‘Lubelski’ landrace–population No. 6). The Mantel test showed that the genetic distance was moderately positively correlated with the geographical distance , based on the genetic distance, revealed that the first three PCs together accounted for 58.8% of the total variation. The first, second and third PCs accounted for 38.6%, 13.5% and 6.7%, respectively. A two-dimensional PC plot (PC1 vs. PC2) of the common valerian accessions is shown in A structural analysis of the populations using the Bayesian model was carried out using the STRUCTURE software. For each accession, the proportion of its genome derived from the different clusters was estimated and the accessions were assigned to a cluster when 70% or more of their inferred genome belonged to the cluster, whereas the accessions with a lower percentage were considered to be admixed. The second-order likelihood delta K calculated, following Evanno et al. , indicatV. officinalis. The admixture of 55% was described in populations with di- and tetraploid specimens [The structure analysis of the investigated wild-growing populations revealed the presence of different gene pools in the studied geographical regions. The most distinctive were populations No. 2, 3, 4, 5, 6 and 7, inhabiting the Bieszczady Mountains. This is most likely due to the presence of natural barriers in the topography. However, they do not provide full isolation and there is gene flow both within and between the geographical regions. Moreover, on the basis of the obtained picture of the population structure, which indicates a high genetic similarity of the populations from the Noteć River Valley, the Nida Basin and the Kielce Upland, it can be concluded that the common valerian occurs in the form of a metapopulation with free gene flow in a significant area of Poland. Such an assumption is supported by the widespread occurrence of the common valerian throughout the entire country, but its verification will be possible only after examining a larger number of wild-growing populations representing all geographical regions of Poland. Interestingly, there is a genetic distinctness of the ‘Lubelski’ landrace, from the wild-growing populations. The background of this is probably a different ploidy level, as it was mentioned before. In the wild-growing populations from the Middle Mazovian Lowland, a significant admixture of the gene pool, typical for the cultivated form, was observed. It can therefore be assumed that we are dealing with the uncontrolled spread of the cultivated form beyond the plantation area. Native species are not being surveyed in this context. However, the ploidy analysis did not reveal the presence of tetraploid individuals in these two populations. Therefore, we are probably faced with hybrids between the diploid and tetraploid forms, which have stabilized at the level of 2n = 14. On the basis of the results obtained, it is impossible to say whether the hybridization took place in the Central Mazovian Lowland as a result of crossing the cultivated form with the wild plants or whether there was a hybridization of the two naturally occurring forms in the Lublin region, from where the cultivated form was selected. The hybrids among the forms with different ploidy levels have been proven to exist within pecimens ,40,47,48V. officinalis is a species related to fresh habitats and margins of water courses and, as can be seen, water availability is, in addition to the spatial isolation, a factor determining the genetic makeup of the species in the region. Disturbance in the water conditions of the habitat, as a result of climatic changes, may lead to a significant reduction of the occurrence of this species in Poland.Due to the presence of the gene flow between populations, the value of the correlation between the genetic and geographical distances was only at a moderate level. There is a report in which it is demonstrated that there is a significant link between the geographical distance and the relative size of the genome . It seemIn agricultural practice, the underground organs of the common valerian are usually harvested in the first autumn of the plant’s vegetation. During this period, the plants are at the vegetative stage and form a basal rosette of pinnatisect leaves with rhizomes and numerous roots. In the following year, the plants form flowering shoots and seeds. In our work, observations were carried out in the first and second years of the plant’s development, covering the terms of both the raw and reproductive materials.The objects of the study were 19 wild-growing populations and ‘Lubelski’ landrace (20 accessions) of the common valerian introduced to ex situ conditions. The seeds of wild-growing populations were collected in 2015 from natural sites covering five geographical regions of Poland, i.e., the Noteć River Valley, the Central Mazovian Lowland, the Kielce Upland, the Nida Basin and the Bieszczady Mountains . The locIn February 2017, the seeds of the wild-growing populations and the ‘Lubelski’ landrace were sown (by a single seed technique) into multi-pots filled with a peat substrate, in a greenhouse. In May, the well rooted seedlings (30 per accession) were planted in 60 × 40 cm spacing in a field located at the Experimental Station of the Vegetable and Medicinal Plant Department, WULS-SGGW .The seed specimens were deposited in the National Centre for Plant Genetic Resources (Polish Gene Bank), under the accession numbers given in In the first year of the plants’ development, at the beginning of plants’ dormancy (October 2017), the underground organs were collected from 10 randomly chosen plants, per accession. The following observations and measurements were carried out: the diameter of rhizomes (cm) and mass of the roots and rhizomes (g FW per plant). The raw materials were purified, dried at 35 °C, ground and subjected to the chemical analysis .. In the second year of the plants’ development, when they were in full bloom (June 2018), observations on the parameters of the aboveground organs were determined, namely: the plant height (cm), the number of flowering shoots per plant, plant lushness, the anthocyanin colouring of the shoots, the colour of the petals, the teething of the leaf margin and the width of the leaf sections. These traits were assessed using a 3-step-scale, which was previously elaborated. At the seed ripening stage (August 2018), the seeds were collected, purified and evaluated as to their sowing value . The obsValerianae radix monograph 07/2015:0453 [The analyses  were carried out, according to the European Pharmacopoeia, 015:0453 . The anaThe following analyses were carried out according to the International Seed Testing Association (ISTA) protocols : 1000 seThe test was determined by the calculation of an average of eight replications containing 100 seeds, multiplied by 10.2×s; temp. 21 °C for 24 h. Four replications were used with 100 seeds each. The seeds were spread out evenly in sterile Petri dishes (8 cm) filled with filter paper. A number of germinated seeds in each dish was counted every day. The germination rate, expressed as an average time (days) needed for the single seed germination, was carried out. At the end of the test (21 days after sowing the seeds) the results were recorded as a germination percentage (%). The tests were determined in climatic chambers , in the controlled conditions: PPFD = 150 µE/mDNA was extracted from young healthy leaves of 9–10 plants from wild-growing populations and 14 plants from ‘Lubelski’ landrace. The tissue was dried in active silica gel and ground into a fine powder in a bead mill . The Genomic Mini AX Plant kit  was used for the extraction. The DNA quality was assessed in 2% agarose gel, and the quantity and purity were assessed using a spectrophotometer . Genotyping was performed using the DArTseq method in a commercial laboratory  which developed this technology. DArTseq™ is a combination of the DArT complexity reduction methods and sequencing on Illumina NGS platforms ,51,52. Tip is the relative frequency of the ith allele of the SNP loci. The genetic distance was measured using the unbiased Nei coefficient [pix and piy are the frequencies of the ith allele in populations x and y and n is the sample size. In order to determine the genetic structure, the agglomerative hierarchical clustering (AHC), according to Ward’s method, the principal coordinate analysis (PCoA) and the model-based Bayesian clustering algorithms were performed. To determine the number of clusters (K), the admixture and the correlated allele frequency model was used. Ten independent runs were set for each K value within the range of 1–10 with a burn-in period of 10,000 and 50,000 Markov chain Monte Carlo replications after the burn-in without prior information about the origin of the sample. The most probable value of K was determined using the method described by Evanno et al. [3 permutations). The analyses were performed using Microsoft Excel 2016, XLSTAT Ecology , GenAlEx 6.501 [The SNP markers were scored as binary data, indicating the presence (1) or absence (0) of a marker in the genomic representation of each sample, as described by Cruz et al. , and thefficient , accordio et al. . The anno et al. . A correEx 6.501 , STRUCTUEx 6.501 , CLUMPAKEx 6.501  and QGISp < 0.05.The data were subjected to a statistical analysis using the Statistica 12 software . The mean values were compared using the one-way analysis of variance (ANOVA), by Tukey’s test, then expressed as mean with the standard deviation (±SD). The differences between the individual means were considered to be significant at The results indicate a high level of variability among the wild-growing common valerian populations, in respect to the developmental and chemical traits, but not strictly related to their geographical localization. In turn, the molecular analysis showed such an association, where five gene pools covering the regions of the population’s origin were observed, with the obvious distinctiveness of those located in the Bieszczady Mountains, as the most isolated. The gene flow was noticed within and between all examined regions, which indicates the affinity of the common valerian widespread in Poland. In the study, the wild-growing populations, recognized earlier as diploids, were compared to the ‘Lubelski’ landrace (tetraploid), a high-yielding model form, rich in sesquiterpenic acids. Among the populations, the one from the Mazowian Lowland (No. 18) and Bieszczady (No. 5), appeared to be closely related to the landrace, which was reflected in their highest mass of underground organs, followed by the highest content of sesquiterpenes. In the case of population No. 18, such a similarity was visibly associated with the huge admixture of the gene pool, typical for the ‘Lubelski’ landrace. Taking into account the results of the molecular analysis and the ploidy levels, it may be suspected that population No. 18 is a hybrid between the di- and tetraploid forms. Irrespective of the genetic relationship, the wild-growing populations were characterised by a high amount of essential oils in the underground organs, which may be promising from the point of view of practice. However, the sowing value of the seeds obtained from the examined populations, expressed as, the germinability and germination rate, was rather low. The present work confirms the thesis that the wild-growing populations of the common valerian may constitute a valuable source of genes for future cultivating programs. It also indicates the need of further screening of natural resources of the species, to find pure and/or hybrid polyploid accessions, usually characterized by a higher level of heterozygosity, in comparison with the diploids. This is especially important when considering the inbreeding tendencies within the wild-growing populations, followed by a degradation of their natural habitats associated with the recently observed climate changes, which altogether make the common valerian a threatened species.