Gadus macrocephalus), a marine fish in the North Pacific, has already experienced major shifts in biomass and distribution linked to climate change. We estimated the magnitude and direction of connectivity between peripheral populations of Pacific cod at the southern edge of the species’ range, by conducting restriction site‐associated DNA (RAD) sequencing and individual assignment on fish collected around the Korean Peninsula during the spawning season. Three populations on the western, eastern, and southern Korean coasts were highly differentiated (STF = 0.025–0.042) and relatively small . Ten putative dispersers and estimates of contemporary migration rates revealed asymmetrical, west‐to‐east movement around the Korean Peninsula, at a higher rate than predicted by indirect estimates of connectivity (STF). Allele frequencies at 87 RAD loci were decisively correlated with strong marine temperature gradients between the warmer southern coast and the cooler waters of the eastern and western coasts. Despite relatively small sample sizes, our data suggest asymmetrical dispersal and gene flow, potentially involving adaptive alleles, between peripheral populations inhabiting markedly different thermal regimes. Our study emphasizes the conservation value of peripheral populations in high gene flow marine fish species.Small and isolated peripheral populations, which are often remnants of glacial refugia, offer an opportunity to determine the magnitude and direction of fine‐scale connectivity in high gene flow marine species. When located at the equatorial edge of a species’ range, these populations may also harbor genetic diversity related to survival and reproduction at higher temperatures, a critical resource for marine species facing warming ocean temperatures. Pacific cod  between highly differentiated peripheral populations around the Korean Peninsula. A total of 87 RAD loci had allele frequencies decisively correlated with strong marine temperature gradients between the warmer southern population and the cooler eastern and western populations. Our data suggest asymmetrical dispersal and gene flow, potentially involving adaptive alleles, between peripheral populations of a marine fish inhabiting markedly different thermal regimes.When located at the equatorial edge of a species’ range, small and isolated peripheral populations may harbor genetic diversity related to survival and reproduction at higher temperatures—a critical resource for adaptation to warming oceans. We use restriction site‐associated DNA (RAD) sequencing and individual assignment to reveal west‐to‐east movement of Pacific cod ( The interconnected marginal seas of the northwest Pacific are characterized by a complex glacial history  at 7 known spawning sites around the Korean Peninsula Figure , and calde novo construction of a reference database of RAD loci, SNP discovery, and genotyping, was completed using a combination of the Stacks v1.44 pipeline  were also removed. We used Genepop v4.2 , then quantified with Quant‐iT PicoGreen dsDNA Reagent (Invitrogen). RAD libraries were prepared according to Baird et al.  and EtteRousset, , 2014 to2.2F‐statistics , ISF, and observed heterozygosity (OH) per collection and pairwise STF between all collections  was varied from 1 to 9, and the optimum number was determined using the mean log‐likelihood  conducted with the R package Jombart,  was usedJombart, . The numln(chr)  and loci decisively correlated with temperature gradients were aligned to the Atlantic cod genome, 3ISF over loci remained close to zero for every collection, with a global locus ISF of −0.009, STF of 0.022, and ITF of 0.013.The final dataset consisted of 5,804 RAD loci and 243 individuals Table . Most in3.1STF between collections on different coasts was high (up to STF = 0.042) and significant, with no significant genetic differentiation between collections within coastal regions or between temporal replicates  of origin, using a reduced marker set. Overall assignment success to population of origin reached 100% using only 100 markers, and almost 90% with only 10 markers Figures .3.3All pairwise eastward migration rates per generation were almost an order of magnitude higher than pairwise westward migration rates . Removal of putative immigrants resulted in a 3.4% (Geoje 2014–2015) to a 461% (Pohang) increase in effective population size  to eN = 1,777 at Boryeong when the lowest minor allele frequency used was 0.05 , OutFLANK, and/or Bayenv2 aligned to sequences in the Atlantic cod genome with a mapping quality >10. Fifty‐eight of these (including 25 candidate outlier loci and 45 temperature‐associated loci) aligned within annotated protein‐coding regions of the Atlantic cod genome, 42 of which coded for proteins of known function Tables .4STF ≈ 0.02) made it possible to observe 10 individuals representing unidirectional, west to east dispersal; disequilibrium‐based estimates of contemporary population connectivity reflected asymmetrical migration eastward around the Korean Peninsula. The potential adaptive significance of this dispersal was indicated by 147 putatively adaptive loci, 87 of which were correlated with temperature and 58 of which could be annotated using the Atlantic cod genome. Our results support the use of peripheral populations in studies of population connectivity in high gene flow species, and the importance of such populations at the southern range edge as a potential source of adaptive genetic diversity.We analyzed over 5,800 RAD loci in 243 individuals across four spawning seasons to quantify the magnitude and direction of connectivity between peripheral populations of Pacific cod around the Korean Peninsula. We first identified three genetically distinct populations along the three coasts of the Korean Peninsula, consistent with findings from microsatellite DNA reported by Gwak and Nakayama . The rem4.1Our RAD sequencing results add to previous microsatellite studies  the Okinawa Trough, formed as sea levels fell in the Yellow Sea and East China Sea, and (2) a semi‐isolated inshore basin in the current East Sea  their consistency across different samples, (ii) the fisheries catch statistics showing a depletion of the stock 1950–2005 , another peripheral population in the eastern north Pacific, which is listed as a Species of Concern by the National Oceanic and Atmospheric Administration  t5 Figure , (iv) th5 Figure . Our NeHigh genetic differentiation and relatively small population sizes, as reported in this study, along with low genetic diversity  around the Korean Peninsula. This eastward dispersal is in accordance with the asymmetrical migration rates estimated with BA3‐SNPs; migration eastward around the Peninsula over the last two generations was on average five‐fold greater than westward migration. Although the number of dispersers we observed was relatively small, the magnitude of asymmetry presents a strong case for highly directional west‐to‐east connectivity. Gwak and Nakayama  found siUnidirectional dispersal from peripheral populations into core populations has been reported among northeastern Pacific cod . Although the assumptions of this approach are somewhat unrealistic  than between samples from the eastern Korean coast and sites in the north/northwestern Sea of Okhotsk ; formal analysis ; investigation (lead); methodology ; software (lead); validation ; visualization (lead); writing – original draft (lead); writing – review and editing . Thomas E. Helser: Conceptualization ; funding acquisition (lead); project administration ; resources ; supervision ; writing – original draft (supporting); writing – review and editing . Sukyung Kang: Conceptualization ; data curation ; funding acquisition ; resources ; writing – original draft (supporting); writing – review and editing . Woo Gwak: Conceptualization ; data curation ; resources ; writing – original draft (supporting); writing – review and editing . Michael F. Canino: Conceptualization ; writing – original draft (supporting); writing – review and editing . Lorenz Hauser: Conceptualization ; formal analysis ; funding acquisition (lead); investigation (supporting); methodology ; project administration ; resources ; supervision ; validation ; writing – original draft (lead); writing – review and editing .Supplementary MaterialClick here for additional data file.


Callosobruchus chinensis, a stored product pest, is difficult to control. In the study, the goal was to explore the causes of the demographic history, dispersal path and genetic variations underlying the spatial and temporal distribution of C. chinensis in China. The phylogeography of C. chinensis was analyzed by distribution modelling (SDM) under six periods and the least-cost path (LCP) based on combined mitochondrial DNA. Our data showed that the geographical isolation of the genetic lineages and the distribution range of C. chinensis were restricted by climate in different times. The landscape structure had influence on the genetic differentiation of C. chinensis. Although the migration ability of C. chinensis is limited, the development of transportation and trade is helping the insect spread, along with the beans of its host.Callosobruchus chinensis (Coleoptera Bruchidae), is a pest of different varieties of legumes. In this paper, a phylogeographical analysis of C. chinensis was conducted to provide knowledge for the prevention and control of C. chinensis. A total of 224 concatenated mitochondrial sequences were obtained from 273 individuals. Suitable habitat shifts were predicted by the distribution modelling (SDM). Phylogeny, genetic structure and population demographic history were analyzed using multiple software. Finally, the least-cost path (LCP) method was used to identify possible dispersal corridors and genetic connectivity. The SDM results suggested that the distribution of C. chinensis experienced expansion and contraction with changing climate. Spatial distribution of mtDNA haplotypes showed there was partial continuity among different geographical populations of C. chinensis, except for the Hohhot (Inner Mongolia) population. Bayesian skyline plots showed that the population had a recent expansion during 0.0125 Ma and 0.025 Ma. The expansion and divergent events were traced back to Quaternary glaciations. The LCP method confirmed that there were no clear dispersal routes. Our findings indicated that climatic cycles of the Pleistocene glaciations, unsuitable climate and geographic isolation played important roles in the genetic differentiation of C. chinensis. Human activities weaken the genetic differentiation between populations. With the change in climate, the suitable areas of C. chinensis will disperse greatly in the future. Phylogeography is the study of the evolutionary history of species, explaining the present and past distribution patterns of species ,2,3. CliGeographic isolation is essential for the genetic structure of populations. A population may be physically separated when its original habitat becomes divided by natural barriers . These barriers prevent gene flow and result in the genetic differentiation of isolated subpopulations ,8. Some Cytisus oromediterraneus at four periods , indicated a similar potential range during MH and the present periods [Climate conditions are closely related to the survival of insects. The Pleistocene glaciations have been recent climatic cycles, usually resulting in genetic variation, which further affect population demographics ,24,25. T periods .Callosobruchus chinensis is widely distributed in China, breeding exclusively on many kinds of beans, such as kidney beans, cowpeas, lentils, chickpeas, broad beans and mung beans [C. chinensis may be derived from remaining populations on-site, it may be transmitted by flying or bean transportation, and it may also come from bean processing equipment [C. chinensis [C. chinensis population is likely to be affected by geography, climate and human activity [ng beans . Due to ng beans ,40. C. cquipment . Previouhinensis . Therefoactivity ,44.C. chinensis. It is difficult to directly observe pests’ population structure and long-distance migrations. Molecular markers therefore serve as a very useful tool to study these parameters [C. chinensis habitat at six periods was modelled. Phylogenetic and phylogeographical analyses, as well as the least-cost path (LCP), were conducted to determine possible structure-causing factors. This study provides dates on the mitochondrial gene data of the C. chinensis population samples, and therefore its genetics, in the main planting areas of mung bean. The study also provides knowledge for appropriate strategies in predicting pest infestations.In this study, our main goal was to estimate the importance of geographic barriers, climatic factors, geographic distance, and human intervention to the genetic structure and population demographic history of rameters . In thisC. chinensis were collected from 22 mung bean planting sites in China from 2017 to 2019 . The mitochondrial fragments of cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII), cytochrome b (Cyt-b) and 12S ribosomal RNA (12S rRNA) were amplified using PCR Master Mix. PCR-specific primers  reading frames were checked in MEGA X, which revealed no evidence of putative nuclear pseudogenes [BioEdit 7.1.7  was appludogenes ,49 in thC. chinensis in China, including 22 field collection points and 58 data points found by consulting related published literature and the GBIF   [C. chinensis between two adjacent time periods  by SDMtoolbox 2.4 [This study used data on 80 occurrence points of acility) . Species factors  for currer 2021) : (1) Laser 2021) . The parlbox 2.4 .ST (PhiST), Tajima’s D and Fu’s Fs. The values of Tajima’s D and Fu’s Fs were significantly negative, which indicated recent demographic expansions for species. To test a hypothesis of isolation by distance (IBD), we used a Mantel test which was calculated with the matrix of genetic distance (ΦST/(1 − ΦST)) versus the matrix of geographical distance (ln km) in the GenAlEx 6.501 software [The number of segregating sites (S), number and distribution of haplotypes (Hap), haplotype diversity (Hd), nucleotide diversity (Pi), standard deviation of haplotype diversity; standard deviation of Pi, and mismatch distributions of pairwise nucleotide differences were analyzed using DnaSP 5.0 . Recent software .C. chinensis, using the concatenated mitochondrial genes. BAPS 6.0 [PopART 1.7  was usedBAPS 6.0  was usedBAPS 6.0  was applC. chinensis, we reconstructed an intraspecific phylogeny based on concatenated mitochondrial haplotypes, with a set make up of a constant population size coalescent model, a relaxed uncorrelated lognormal molecular clock and a GTR  substitution model in BEAST 2.6.3 [C. chinensis, but was similar to rates obtained based on fossil and biogeographic events in some Coleoptera for the combined mitochondrial genes [To investigate the lineage divergence of ST 2.6.3 . Since tST 2.6.3 ,61,62. TST 2.6.3 , which mal genes ,65. Six al genes  was usedal genes  was usedC. chinensis. This involved, first, creating a friction layer by inverting SDM, and second, using shared haplotype data of mitochondrial genes (COI + COII + Cyt-b + 12S) of the C. chinensis population samples to calculate the cost of the diffusion path between geographic locations of different population samples. Third, we set the classification values of the lowest cost values 0.05, 0.02 and 0.01, which were shown in different colors in the diffusion connectivity layer. Finally, we summarized and standardized all the reclassified paired corridor layers, and determined the scattered corridors of C. chinensis in a clear landscape.SDMtoolbox 2.2 in ArcGIS 10.4.1 was used to create the least-cost paths of C. chinensis was accurate and reliable , COI (845 bp), COII (600 bp) and Cyt-b (809 bp) gene fragments. A total of 56 haplotypes were identified based on the concatenated mitochondrial genes. The concatenated mitochondrial haplotype diversity (Hd) ranged from 0.378 to 1, only the values of HB2 and GZ were less than 0.5, and especially the value of GZ was much smaller than the others. The nucleotide diversities ranged from 0.00021 to 0.00084. The values of SCW, SYQ, SD2, HL, HB2, TJ and GZ were less than 0.0005; however, the values of SJT and JX were higher than other populations  and geographical distances between populations for concatenated mitochondrial (12S + COI + COII + Cytb) genes   .C. chinensis populations have experienced a recent expansion , east China , central South China , southwest China  and Shaanxi.The LCP analysis of  periods . Change pulation . In the C. chinensis from 22 geographic sites in China. These C. chinensis beetles were analyzed for their patterns based on concatenated mitochondrial sequences . Our results showed that the genetic diversity (Hd) was seemingly high among populations, which was consistent with other Coleopterans, i.e., Cosmopolites sordidus (Germar 1824), Sitophilus zeamais (Motsch.), Callosobruchusmaculatus  [C. chinensis population had experienced a bottleneck effect. The results of multiple analysis  indicated that C. chinensis then underwent expansion and genetic variation. At the same time, it also showed that C. chinensis had high environmental adaptability [Molecular markers have proven to be very useful in solving the genetic structure of beetle populations . In this., 1775) ,69,70. Ttability .Hyalessa maculaticollis , Camaena cicatricosa , Metrocoris sichuanensis  and Scythropus yasumatsui Kono et Morimoto, have been studied [In China, the influencing factors on the phylogeographic structure of many species, such as Müller, 174, Metro studied ,71,72. G studied .Leptinotarsa Decemlineata (Say), Carabus solieri, and Epipyropidae, is due to geographical barriers [C. chinensis, which suggested that climate conditions are not an effective biogeographic barrier for Shanxi populations. In this study, these results indicated that the Taihang Mountains and Loess Plateau should be natural barriers of Shanxi  to restrict gene exchange. Their function of geographical isolation has been found in other species, e.g., S. yasumatsui [Sitodiplosis mosellana (Géhin) (SM) [Previous phylgeographic studies indicated that mountains have acted as geographical barriers to drive speciation in geographically-isolated populations ,74,75,76barriers ,78,79. Ibarriers , which bsumatsui . Moreovein) (SM) .Stomoxys calcitrans (L.) and Stomoxys niger niger Macquart [C. chinensis, the BAPS and BARRIER analysis indicated that the NM population had high genetic divergence. The NM population samples was collected from Hohhot, a region characterized by long and cold winters. The isolation by distance (IBD) analysis showed no relationship between genetic differentiation and geographic distances among the 22 populations. This indicates that genetic isolation may have been caused by other factors than geographical distance between population samples. Indeed, climatic conditions of a species’ habitat are often barriers to dispersal. These results suggest that climate played an important role in preventing migration between populations, which were consistent with the prediction results of the habitat suitability of C. chinensis in China. Temperature is the primary variable affecting the distribution and population dynamics of species [C. chinensis  and a contraction. The expansions agree with historical events, corresponding to the warming climate under the LIG and MH periods. Pre-LGM expansion has also been found in other plants and animals [C. chinensis occurred during the MH period, which was also found in C. maculatus [C. chinensis. The theory says that the separation and expansion of populations with climate fluctuations lead to gene exchange via lineage admixture [C. chinensis may retreat to shelters in eastern and southern China. After climate warming, these surviving C. chinensis populations are likely to expand rapidly and trigger gene flow between regions. Amusingly, the haplotypes distribution and Barrie analysis showed that AH population samples had genetic differentiation with adjacent populations and a higher value of Hd, which are also the potential refuges for C. chinensis. Moreover, the values of Hd and Pi of the GZ population were the smallest of all the populations. The location of the GZ population was far from the two refuges. Therefore, it is speculated that the pattern of genetic diversity of the GZ population may be largely due to the founder effect. The haplotypes distribution was related to the suitable distribution of C. chinensis during the late Quaternary, which confirmed the profound influence of changing climate on the present pattern of this weevil. The potential distribution of C. chinensis throughout the late Quaternary supported the hypotheses that the origin and dispersal of C. chinensis derived from South China and then spread from south to north. Similar patterns of origin and spread have been reported in other pests such as Carposina sasakii Matsumura and Grapholita molesta  [It has been demonstrated that Quaternary climate change has had a huge influence on many species’ distributions and population structures ,15. Our  animals ,93,94. Aaculatus . In thisdmixture ,96. Durik, 1916) ,97.C. chinensis populations. However, C. chinensis is highly migratory during a series of production, processing and commodity circulation processes, such as seed cultivation, harvesting, storage and transportation [C. chinensis is increasingly influenced by anthropogenic intervention.Finally, human activities promote the long-distance dispersal of insects, and facilitate gene flow to weaken the genetic differentiation between populations ,99,100. ortation ,100,101.ortation . As anthC. chinensis is widely distributed in China, and is an important and harmful beetle. In the future, global temperatures will be higher than they are now, due to the greenhouse effect. In recent years, some works have increasingly forecasted species’ distribution patterns under a future climate, which showed there is a trend of gradual expansion of some insects to the north of China [C. chinensis will spread widely in China. The survival rate of C. chinensis may increase in winter, and the number of generations of C. chinensis may increase. The prevention and control of this beetle is imperative. Several methods of analysis  have been employed to predict high suitability areas  and a high genetic connectivity region (Shanxi-Henan-Hebei), which need to be monitored. Given the evidence from this study that there is potential gene flow between geographic populations due to human factors, it is important to ensure that there are no C. chinensis  before the bean seeds are transported and traded.of China ,72. The


It pinpoints positively selected genetic variants in sequencing data by prioritizing high frequency, population-specific and functional derived alleles.In population genomics, polymorphisms that are highly differentiated between geographically separated populations are often suggestive of Darwinian positive selection. Genomic scans have highlighted several such regions in African and non-African populations, but only a handful of these have functional data that clearly associates candidate variations driving the selection process. Fine-Mapping of Adaptive Variation FineMAV statistic. To graphically visualise the FineMAV scores, it outputs the statistics as bigWig files, which is a common file format supported by many genome browsers. It is available as a command-line and graphical user interface. The software was tested by replicating the FineMAV scores obtained using 1000 Genomes Project African, European, East and South Asian populations and subsequently applied to whole-genome sequencing datasets from Singapore and China to highlight population specific variants that can be subsequently modelled. The software tool is publicly available at https://github.com/fadilla-wahyudi/finemav.We developed a stand-alone software that implements the FineMAV scores, using low or high-coverage whole-genome sequencing datasets, that can be used to prioritize a list of population specific, highly differentiated candidate variants for in vitro or in vivo functional screens. The tool displays these scores on the human genome browsers for easy visualisation, annotation and comparison between different genomic regions in worldwide human populations.The software tool described here determines genome-wide The online version contains supplementary material available at 10.1186/s12859-021-04506-9. Human whole-genome sequencing projects have contributed to the advancement of population genomics, specifically the unbiased detection of positive selection in human populations. In comparison to genotyping, sequencing mitigates ascertainment bias and captures greater genomic variation  making iFineMAV) is a statistical method that prioritizes functional SNP candidates under selection and depends upon population differentiation [FineMAV was developed to overcome a challenge that existing positive selection statistical methods face in that they are unable to distinguish between neutral, hitchhiked variants and true positively selected variants [FineMAV does this by incorporating methods that detect regions showing signatures of positive selection  and subject these regions to functional annotation under the assumption that it is unlikely for a deleterious or functional variant to reach high frequency in a given randomly mating population unless it confers some sort of functional advantage [Fine-Mapping of Adaptation Variation  equation to describe the disparate spread of derived alleles across populations [DAF) equation is used to determine sites with high frequency of derived alleles [FineMAV uses the Combined Annotation-Dependent Depletion (CADD) method which takes into account multiple variant annotations and condenses it into a single score called the C score [FineMAV calculations on whole-genome sequencing data and can output bigWig files which can be used to graphically visualise the scores on genome browsers. We test the software using the 1000 Genomes Project phase 3 dataset [To measure population differentiation, ulations . The der alleles . To anno C score . The C s C score . The phr C score . For exa C score . Incorpo C score . In this dataset  and whol dataset –8.FineMAV software works with high-throughput, massively parallel, sequencing data and relies on the information that can be extracted from variant call format (VCF) files (version 4.2 and above)  Table . The PytFineMAV software requires the user to provide the input data from the VCF file in a tab-delimited file format  for each population is not annotated in the INFO column, we suggest using the BCFtools fill-tags plugin to determine the AF first, and then piping it to the BCFtools query command for extraction. If the ancestral allele and/or the CADD_PHRED are not available in the VCF file, the software allows the user to supplement this information using the Ensembl Variant Effect Predictor (VEP) . The outFineMAV scores using the 1000 Genomes Project low coverage whole-genome sequences from African, European, East and South Asian continental populations to replicate the published results [FineMAV scores for high-coverage whole-genome sequencing datasets generated by the Singapore Sequencing Indian Project (SSIP) [We tested our implementation code by generating genome-wide  results . Subsequt (SSIP) , the Sint (SSIP)  and a dat (SSIP) .FineMAV score was estimated for genome-wide single nucleotide polymorphisms (SNPs) as only the ancestral allele could be unambiguously determined only for this class of variations. The derived allele for each SNP was calculated by multiplying three metrics: DAP, DAF and the CADD_PHRED score [DAP was calculated for each variant using the following equations: DAP equation is used when the population sizes are equal. In this software, the DAP is calculated using the derived allele frequency instead of counts in order to take into account population sizes that might be different.The ED score . DAP wasulations . The afoThe penalty parameter k et al.  for varyentclass1pt{minimaFineMAV calculations are done by splitting the file(s) into smaller chunks to optimise the random access memory (RAM) usage  for all four continental populations [FineMAV outliers across all four continental populations with the published data, only 5/300 variants did not overlap with the published results and all five of these variants were missing, because they did not pass our data filtering criteria.We initially tested our implementation code by generating genome-wide ulations  and obtaulations . When coFineMAV scores for three high-coverage whole-genome sequencing datasets: the SSIP [https://krishna.gs.washington.edu/download/CADD/v1.4/GRCh37/whole_genome_SNVs.tsv.gz) [ftp://ftp.ensembl.org/pub/release75/fasta/ancestral_alleles/homo_sapiens_ancestor_GRCh37_e71.tar.bz2 [Next, we used the pipeline to calculate the SSIP , SSMP [7the SSIP . These d.tsv.gz)  The FAST.tar.bz2 , 16. TheFineMAV statistic was able to replicate known positively selected variants as well as pinpoint novel ones from the three populations (Table EDAR) and rs2293766  in zonadhesin (ZAN). These variants have also been highlighted previously in several genomic scans for selection in East Asian populations [EDAR have confirmed its pleiotropic effects. The non-synonymous mutation has been found to be associated with hair thickness [ZAN encodes an acrosomal protein in the sperm called zonadhesin. A study employing Zan knockout mice found that their sperms remained fertile and had increased adhesion to the jelly-like coating of the egg (zona pellucida) of other species like pig, cow and rabbit [ZAN is responsible for species-specific binding, it can be speculated that a truncation, as a result of the nonsense mutation in rs2293766, could have mediated interbreeding between archaic humans and modern humans in Asia [The ns Table . Exampleulations , 18 and hickness –21, shovhickness , ear morhickness , 26, inchickness . ZAN encd rabbit . As ZAN FineMAV penalizes allele sharing between populations and highlights high frequency population-specific mutations. Some of the highest-scoring SNPs observed in SSIP are located in chromosome 16 , a SNP that has been reported to be positively selected in South Asian populations and was missing in our dataset [PRSS53 encodes a serine protease and is expressed in hair follicles [PRSS53) was excluded from this selection scan because it was not polymorphic in all three datasets. Within the top 10 FineMAV candidates in SSIP, three SNPs are in linkage disequilibrium with rs201075024 (PRSS53): rs35675346  (FBXL19), rs58726213 (NC_000016.9:g.31044683A > G) (STX4) and rs3747481  (PRR14), with r2 values of 0.45, 0.26 and 0.16, respectively. This suggests that the three SNPs may be neutral and tagging the PRSS53 rs201075024 variant. Interestingly, according to the Genotype-Tissue Expression (GTEx) database (V8 release), these SNPs were reported as expression and splicing quantitative trait loci (eQTL/sQTL) for PRSS53 in various tissues [As seen in Fig.  16 Fig. , Table 3ollicles  and rs20ollicles  and has ollicles . The funTSR1) and rs2229300  in glioma-associated oncogene family zinc finger 1 (GLI1) in the Singaporean Malay population  in the pre-rRNA-processing protein TSR1 homolog (turation . It was turation . Howeverications . Accordiitutions . The misitutions .SLC22A24) in which the global allele frequency is 0.75 [th highest scoring allele in Singaporean Malays , Singaporean Indian (SSIP) and Singaporean Malay (SSMP) populations.


EN), climate adaptive potential and conservation value and lower genetic differentiation than second-growth forest tree populations, employing a keystone and long-lived conifer, eastern white pine . Genetic diversity and population structure of old-growth and second-growth populations of eastern white pine (EWP) were examined using microsatellites of the nuclear and chloroplast genomes and single nucleotide polymorphisms (SNPs) in candidate nuclear genes putatively involved in adaptive responses to climate and underlying multilocus genetic architecture of local adaptation to climate in EWP. Old-growth and second-growth EWP populations had statistically similar genetic diversity, inbreeding coefficient and inter-population genetic differentiation based on nuclear microsatellites (nSSRs) and SNPs. However, old-growth populations had significantly higher chloroplast microsatellites (cpSSRs) haploid diversity than second-growth populations. Old-growth EWP populations had significantly higher coalescence-based historical long-term EN than second-growth EWP populations, but the linkage disequilibrium (LD)-based contemporary EN estimates were statistically similar between the old-growth and second-growth EWP populations. Analyses of population genetic structure and inter-population genetic relationships revealed some genetic constitution differences between the old-growth and second-growth EWP populations. Overall, our results suggest that old-growth and second-growth EWP populations have similar genetic resource conservation value. Because old-growth and second-growth EWP populations have similar levels of genetic diversity in genes putatively involved in adaptive responses to climate, old-growth, and second-growth populations may have similar adaptive potential under climate change. Our results could potentially be generalized across most of the boreal and temperate conifer forest trees. Our study contributes to address a long-standing issue, advances research field and knowledge about conservation and ecological and climate adaptation of forest trees.Whether old-growth (OG) forests have higher genetic diversity and effective population size, consequently higher conservation value and climate adaptive potential than second-growth (SG) forests, remain an unresolved issue. We have tested the hypothesis that old-growth forest tree populations have higher genetic diversity, effective population size ( Pinus pinaster (Whether old-growth (OG) forests have higher conservation value and climate adaptation potential and provide better ecosystem functioning, productivity, and resilience than second-growth (SG) forests, remains a long-standing unresolved issue. Biodiversity provides the foundation of ecosystem stability, functioning, and productivity, as well as provisions of ecosystem services e.g., . There ipinaster . Furtherpinaster . TherefoBecause forest trees are normally the keystone species of the forest ecosystems, their genetic diversity has special significance and genetic diversity has been identified as the foundation of forest sustainability , 2020. OIriartea deltoidea  as a test species. EWP is an ideal candidate for this study as outlined below in Materials and Methods. We have used microsatellite DNA markers of the nuclear and chloroplast genomes, which are presumed to be selectively neutral, and SNPs in candidate nuclear genes putatively involved in adaptive responses to climate and underlying local adaptation to climate in this species. We tested the hypothesis that OG populations have higher genetic diversity and effective population size and lower inter-population genetic differentiation than SG populations, thus potentially higher conservation value and adaptation potential.The objective of this study was to examine whether old-growth forest tree populations have higher genetic diversity and effective population size, thus higher conservation value and adaptive potential, than second-growth forest tree populations, employing eastern white pine  species and has a wide natural range in eastern North America . HistoriWe sampled 33 populations throughout the species’ range for several research objectives, including investigating the range-wide population genetic structure and post-glacial phylogeography , geneticFifty individual trees were randomly sampled from each population, and needles were collected from individual sampled trees for DNA extraction. A 30 m buffer was left between sampled trees to minimize the chance of sampling siblings. Needles were stored at −20°C in sealed plastic bags, with a 5 g silica desiccant pack, before DNA extraction. DNA from needle samples was extracted and prepared as described in All sampled EWP individuals were genotyped for 12 nuclear microsatellite markers and a subset of 20 individuals per population for three chloroplast microsatellite markers as described in A subset of 22 randomly selected individuals per population was genotyped for SNPs in candidate genes putatively involved in adaptive responses to climate as described in Although we obtained the microsatellite and SNP data from O and HE) were calculated for the nuclear microsatellite and SNP markers. Number of alleles per locus (A), Shannon’s Information Index (I), haploid diversity (H), and unbiased haploid diversity (uH) were calculated for the chloroplast microsatellite markers. The number of rare alleles (ARare) was determined for nuclear microsatellites at the 1%  and for SNPs at the 5%  criteria. We also estimated the effective number of alleles (AE) per locus and fixation index/inbreeding coefficient (F or FIS) for the nuclear microsatellites and SNPs. We used one way ANOVA to test the statistical significance of differences in genetic diversity parameters and fixation index between old-growth and second-growth population groups. We also tested differences in allelic diversity, heterozygosity, and FIS between old-growth and second-growth population groups using the permutation test with 1,000 permutations in FSTAT  for each population from the nuclear microsatellite data. The historical long-term EN was estimated based on the coalescent method using the Markov chain Monte Carlo coalescent genealogy sampler LAMARC  for each population was calculated as Θ/4*10−3. The contemporary NE (LD) and its parametric 95% CIs were estimated using the LD method in NEEstimator v2  over the loci using FSTAT separately for two marker sets. The significance of differences in FIS and FST between old-growth and second-growth population groups was determined by a permutation test using 1,000 permutations in FSTAT. Hierarchical AMOVA .Genetic structure of EWP populations was determined by using a Bayesian model-based genetics approach, STRUCTURE . Multipl options , a 100,0We calculated We observed 252 alleles at 12 nuclear microsatellite loci: 200 in the old-growth populations and 213 in the second-growth populations. Of these, 161 were common between the two population types. The OG populations had 39 and SG populations had 52 private alleles. Only two of the OG-specific alleles were present in all four OG populations and absent in the five SG populations. Twenty-three of the 39 private alleles in OG were rare, whereas 14 of the 52 private alleles in SG were rare. We observed 29 chloroplast microsatellite haplotypes: 21 in OG populations and 19 in SG populations; of these, 10 were found only in OG and eight in SG, and 11 were common between OG and SG populations. A total of 88 alleles were observed at 44 SNP loci, 84 in four OG, and 85 in five SG populations. Of these, 81 were common between the OG and SG populations. Three alleles were specific to OG and four to SG. However, there was only one allele that was present in SG populations and not in OG populations. No evidence of linkage disequilibrium was observed.E), and observed (HO) and expected (HE) heterozygosity from each of the nuclear microsatellites and SNPs were similar between the old-growth and second-growth populations (p > 0.05). Likewise, the number of alleles per locus for the chloroplast microsatellites was not significantly different between OG and SG (p > 0.05). However, Shannon’s Information Index (I), haploid diversity (H), and unbiased haploid diversity (uH) for the chloroplast microsatellites were statistically higher in OG than in SG populations . On average, the SG populations had significantly higher number of rare alleles for SNP markers than the OG populations (F or FIS from F-statistics showed reversed trends between the nuclear microsatellites and SNPs (F or FIS than the OG populations for nuclear microsatellites (p > 0.05) as demonstrated by ANOVA, permutation test, and bootstrap 95% CIs. In contrast, SG populations showed higher excess of heterozygotes than OG populations for SNP markers . Therefore, overall, the inbreeding coefficients were not significantly different between the OG and SG populations. No instances of significant bottlenecks were identified in any population from the BOTTLENECK test.The genetic diversity levels for the mean number of alleles (A) per locus, effective number of alleles per locus  ranged from 109 to 322, with an average of 192 over the nine populations  than the SG populations of EWP (EN (LD) at the critical value of 0.01 ranged from 136 to 655, with an average of 340 over eight populations (EN (LD) for the Ontario French River (ONFR) population was estimated to be infinite; thus, this population could not be included in calculating means and significance of differences between the OG and SG populations. In the LD EN method, infinite estimate is obtained when, by chance, the actual sampling error is larger than the expected, resulting in negative estimate of EN (EN estimate is interpreted as infinite (EN (LD) than SG populations at 0.01 critical value, but the differences were not statistically significant (p > 0.05). EN (LD) estimates at the critical value of 0.03 ranged from 69 to 222, with an average of 145 over the nine populations (EN (LD) than OG populations, but the differences were not statistically significant (p > 0.05).The estimated historical long-term ulations . Overalls of EWP . The conulations . NE (LD)te of NE . In suchinfinite . On averulations . On averFST estimates based on microsatellite and SNP markers are provided in F-statistics parameters for all, OG and SG populations based on nuclear microsatellites and SNPs are presented in FST estimates for all populations was 9.2% for nuclear microsatellites and 6.7% for SNPs. The permutation test and the bootstrap 95% CIs for FST (p = 0.49 for microsatellites and 0.91 for SNP markers). Hierarchical AMOVA results were consistent with the FST results for inter-population genetic differentiation  analysis  and NJ tWe have demonstrated that genetic diversity for nuclear microsatellites and SNPs in candidate genes putatively involved in adaptive responses to climate is similar between old-growth and second-growth EWP populations, but OG populations have higher chloroplast microsatellite diversity than SG populations. We have also shown that OG populations on average have higher historical coalescence effective population size than SG populations, but OG and SG eastern white population have statistically similar contemporary effective population size.NE–4NE generations  in two pristine old-growth EWP stands in northern Ontario significantly reduced genetic diversity  in the post-harvest residual gene pool . Also, stm, multilocus outcrossing rates >90%; Pinus monticola), 80% of the empty seeds could be attributed to inbreeding  created fragmentations. Thus, the chloroplast genetic diversity in the studied OG populations may represent the pre-harvest ancestral chloroplast genetic diversity. Although, the bottleneck test based on nuclear microsatellites did not detect any significant bottleneck in the studied EWP populations, but this test may not be sensitive enough to detect bottleneck events in the studied populations as discussed above. Harvesting may have caused bottleneck enough for lowering chloroplast genetic diversity in the SG populations, which may not have been buffered by a lack of recombination in the chloroplast genome. The differences in chloroplast genetic diversity between the OG and SG populations were unlikely due to differences in post-glacial migration and evolution because both the OG and SG populations were from the same central lineage group inferred to have originated from the same post-glacial migration route  or four (nuclear and chloroplast microsatellites) other second-growth EWP populations from Quebec belonging to the species’ same central  lineage –S5. Alsoulations . TherefoEN than the second-growth populations, but old-growth and second growth EWP populations have similar contemporary EN. This is a significant finding because EN, particularly contemporary EN, has long been recognized to play a significant role in conservation and management of genetic resources  and 40% (nuclear SNPs) higher among the SG populations than among the OG populations of EWP, the differences were not statistically significant. Therefore, our FST results from both nuclear microsatellites and SNPs (Since the old-growth populations represented the intact EWP forest and the second-growth populations were potentially affected by fragmentation caused by harvesting and/or fire, a higher genetic differentiation among SG populations than among OG populations was expected. Although the and SNPs  suggest and SNPs . Althougand SNPs . Howeverand SNPs , 3. Indeand SNPs , and nucand SNPs . The priand SNPs , and theEN have high genetic conservation value. We observed that the OG and SG populations of EWP have similar levels of genetic diversity in nuclear microsatellites and SNPs in climate-responsive genes, inter-population genetic differentiation, and contemporary EN. Therefore, we can infer that the studied extant OG and SG EWP populations have similar value for genetic resource conservation of the species. Since old-growth stands are considered to have the highest species biodiversity, conservation, and ecosystem functioning value (Genetic diversity provides the basis for conservation of genetic resources of a species and its populations. Effective population size is an important parameter of high genetic conservation relevance see . Therefong value , and manng value , old-groEastern white pine has experienced multiple episodes of post-glacial range expansion and retraction , encountEN, but their contemporary EN is similar to that of second-growth populations. Overall, it could be inferred that the studied OG and SG EWP populations have similar genetic conservation value and adaptive potential under climate change. Nevertheless, there are some genetic constitution differences between the OG and SG EWP populations, which may have been caused by high grading harvesting in the parental stands of the SG populations. As the studied SG populations are most likely one generation apart from the OG populations, significant genetic erosion has not yet occurred in these populations. However, genetic diversity may be reduced in SG populations and differentiation between OG and SG populations may be increased if the extant EWP SG populations are not harvested and managed in a genetically sustainable manner. Because extant OG EWP populations are rare, which may be harvested in near future, and most of the EWP forests are SG, it is essential that SG forests are sustainably managed for conservation of their genetic resources, and measures should be taken so that no genetic erosion happens in future generations.Old-growth and second-growth populations of EWP have similar levels of genetic diversity in nuclear microsatellites and SNPs located in candidate genes putatively involved in adaptive responses to climate, and statistically similar levels of inter-population genetic differentiation. However, OG populations have higher chloroplast microsatellites haploid genetic diversity than SG populations. Old-growth EWP populations have higher coalescence-based historical Our study is based on a limited number of genetic markers, although including both putatively neutral microsatellites and adaptively selective SNPs in climate-responsive candidate genes, genome-wide genetic diversity may provide much more comprehensive and more precise inferences. Also, an ideal sampling design should include adjacent OG and SG stands or preharvest OG and postharvest SG populations from the same stand. However, such design was not possible because appropriate OG and SG EWP stands in the same neighborhood could not be found as discussed in Material and Methods. And a study before and after harvest in the same OG populations will require controlled experiments and decades of waiting for SG populations to attain an appropriate sampling age.via pollen and seed dispersal, paternal transmission of the chloroplast genome, and long generation life cycle. Therefore, the results from our study could possibly be generalized for most of the boreal and northern temperate conifer and potentially to other forest trees. Boreal and temperate forest biomes are the largest terrestrial biomes on earth. Forests cover about one-third of the earth’s land surface, about 33% of which is covered by the boreal zone and about 25% by the temperate zone. Thus, boreal and temperate forest biomes cover about 20% of the planet’s terrestrial surface. As such, they have immense environmental, ecological, economic, and social important. Conifers, such as Pinus and Picea are the dominant components of the boreal forest, which represents the huge carbon sink. Thus, both the boreal and northern temperate conifer forest trees are hugely important for addressing the climate change challenges. Their conservation and sustainable management are important for the sustainability, stability, and functioning of boreal and temperate ecosystems, provision of ecosystem and environmental services, which are vital for the sustenance of human and other life. Also, many of the boreal and temperate forest trees are anticipated to extend their rages northward under climate change. Their potential to adapt to new climate conditions will depend upon their adaptive and evolutionary potential, which could be reflected by genetic diversity in genes involved in adaptive responses to climate. Old-growth forests, due to their existence for long time and surviving through various temporal variations in climate, are expected to have higher adaptive potential under changing climate conditions. However, most of the old-growth forests have been replaced by second-growth forests, and whether the adaptive potential of these forests is lower than primary old-growth forests is a question of broad implications. Our study addresses this very basic and applied question. Thus, overall, our study should have very wide implications for the conservation and ecological adaptation of forest trees, especially under climate change and the advancement of research in these areas.While our study used EWP as a test species, the results have broad implications and significance. EWP is a good representative of most of the temperate and boreal conifer forest trees because of its similar biological traits. Like EWP, most of the boreal and northern temperate conifer forest trees have predominantly outcrossing mating system, severe inbreeding depression, selection against inbreds at an early developmental stage, extensive and long-distance gene flow https://bmcevolbiol.biomedcentral.com/track/pdf/10.1186/s12862-016-0624-1 and https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0158691&type=printable.Publicly available datasets, produced earlier by the authors of this article, were analyzed in this study. This data can be found at: OR conceived, supervised, and directed the study, obtained the funding, reanalyzed the data, and rewrote and revised the manuscript. JZ conducted the field sampling and all lab work, produced the genotype data, performed initial data analysis, and prepared the initial draft of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Varroa destructor, a novel parasite of Apis mellifera originating from Asia, was investigated using population genetics to determine how the genetic structure of the parasite changed in distinct European populations of its new host. To do so, mites infesting two categories of hosts in four European regions were compared: (a) adapted hosts surviving through means of natural selection, thereby expected to impose strong selective pressure on the mites, and (b) treated host populations, surviving mite infestations because acaricides are applied, therefore characterized by a relaxed selection imposed by the host on the mites. Significant genetic divergence was found across regions, partially reflecting the invasion pattern of V. destructor throughout Europe and indicating local adaptation of the mite to the host populations. Additionally, varying degrees of genotypic changes were found between mites from adapted and treated colonies. Altogether, these results indicate that V. destructor managed to overcome the genetic bottlenecks following its introduction in Europe and that host‐mediated selection fostered changes in the genetic structure of this mite at diverse geographic scales. These findings highlight the potential of parasites to adapt to their local host populations and confirm that adaptations developed within coevolutionary dynamics are a major determinant of population genetic changes.Comparative studies of genetic diversity and population structure can shed light on the ecological and evolutionary factors governing host–parasite interactions. Even though invasive parasites are considered of major biological importance, little is known about their adaptative potential when infesting the new hosts. Here, the genetic diversification of  Varroa destructor across sympatric adapted and nonadapted populations in four regions of Europe. Our results reveal that host‐mediated selection fostered changes in the genetic structure of this mite at diverse geographic scales. These findings highlight the potential of parasites to adapt to their local host populations and confirm that adaptations developed within coevolutionary dynamics are a major determinant of population genetic changes.We investigated the genetic structure of the honey bee ectoparasite  PCRs were conducted as detailed in Beaurepaire et al.  and observed heterozygosity (HO), were compared using Fstat v. 2.9.3  was suitable to accurately represent the genetic diversity of the mite groups, rarefaction analyses were conducted using the ADZE software  between mite groups treated with different acaricides was compared using a Student t test. Finally, to investigate the relationships between the genetic distance obtained with Dest and the spatial distance separating the mite groups  in each mite group was computed using the R package 3p‐values > .05). The AMOVA confirmed our a priori grouping, indicating that sampling regions and host groups  were significantly (p <.01) structuring V. destructor populations  and number of alleles (NA = 2–4) were found across the seven markers over all samples , while mites from Sweden were more markedly differentiated from mites of these two regions . Surprisingly, the genetic divergence levels between samples from these three locations and mites from Norway were thrice as high  and nonsignificant  were found when these two groups were compared with the mites infesting the French adapted population, despite the fact that one of the treated host group was in the same apiary as the adapted honey bees. Also, the lowest level of divergence in the Netherlands occurred between the two adapted groups , in spite of the ~100 km separating them. In parallel, the pairwise allelic comparisons revealed that mites from the adapted host population in France and from Tiengemeten in the Netherlands were not significantly genetically different  did not differ significantly from that obtained when mite groups treated with oxalic acid were compared with each other .The comparison of genetic diversity across mite groups infesting the different host populations revealed that the mean number of alleles and the observed heterozygosity levels did not differ significantly between these groups  varied between groups, but only represented a minor proportion of the sampled mites  showed 139 distinct mite genotypes over all groups Table . A totalp <.05, Figure Overall, contrasting patterns of MLG diversity could be observed when comparing mites from adapted and treated populations across the different locations Figure . Notably4V. destructor is shaped by their interactions with their local host populations. These findings reveal that sympatric and allopatric mite populations are experiencing significant levels of genetic divergence that are probably caused by both unique host population traits and environmental differences across locations.Our results suggest that the genetic structure of European R2 = 0.14, p =.001) was found between the genetic and geographic distance separating the mite groups, indicating that geographic isolation can explain a small part of the genetic differences found between distant mite populations. Additionally, the pattern of genetic differentiation documented here appears to be further explained by the invasion history of the parasite in Europe. Historical reports of this invasion showed that V. destructor arrived in Europe through two routes. First, the mite is believed to have been introduced in 1971–72 in Eastern European countries and to slowly spread to the Northern regions of the continent, including Sweden  were found across the honey bee populations, including many rare MLGs private to specific regions and populations. Notably, the distribution of MLGs significantly differed across the populations studied, and strong and significant differences were also found when performing pairwise allelic differentiation analyses between regions. Altogether, these results suggest that the mites are adapting to their local host populations, despite the relatively recent genetic bottlenecks caused by both the host shift and introduction of the mite in Europe .In addition to the diversification of V. destructor being no exception  were not significantly genetically different, while the mite infesting susceptible colonies in these two regions were. This may suggest parallel evolution similar to their honey bee hosts ; Formal analysis (lead); Visualization (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Tjeerd Blacquière: Conceptualization ; Data curation (supporting); Methodology ; Validation (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Bjorn Dahle: Conceptualization ; Data curation (supporting); Methodology ; Validation (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Vincent Dietemann: Conceptualization (supporting); Investigation (supporting); Methodology (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Yves Le Conte: Conceptualization ; Investigation (supporting); Methodology ; Validation (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Locke Barbara : Conceptualization ; Data curation (supporting); Methodology ; Validation (supporting); Writing‐original draft (supporting); Writing‐review & editing (supporting). Peter Neumann: Conceptualization ; Funding acquisition (lead); Methodology ; Project administration ; Resources (lead); Writing‐original draft (supporting); Writing‐review & editing (supporting). Alexis Beaurepaire: Conceptualization (lead); Data curation ; Formal analysis (supporting); Methodology (lead); Project administration (supporting); Supervision (lead); Validation (lead); Writing‐original draft (lead); Writing‐review & editing (lead).Supplementary MaterialClick here for additional data file.


Silene uniflora is predominantly a coastal species, but it has also colonized isolated, disused mines with phytotoxic, zinc-contaminated soils. To test whether rapid, parallel adaptation to anthropogenic pollution has taken place, we used reduced representation sequencing (ddRAD) to reconstruct the evolutionary history of geographically proximate mine and coastal population pairs and found largely independent colonization of mines from different coastal sites. Furthermore, our results show that parallel evolution of zinc tolerance has occurred without gene flow spreading adaptive alleles between mine populations. In genomic regions where signatures of selection were detected across multiple mine-coast pairs, we identified genes with functions linked to physiological differences between the putative ecotypes, although genetic differentiation at specific loci is only partially shared between mine populations. Our results are consistent with a complex, polygenic genetic architecture underpinning rapid adaptation. This shows that even under a scenario of strong selection and rapid adaptation, evolutionary responses to human activities  may be idiosyncratic at the genetic level and, therefore, difficult to predict from genomic data.The impact of human-mediated environmental change on the evolutionary trajectories of wild organisms is poorly understood. In particular, capacity of species to adapt rapidly (in hundreds of generations or less), reproducibly and predictably to extreme environmental change is unclear.  Modification of the natural environment by humans has significant implications for biodiversity . Rapid hMimulus guttatus . In Great Britain and Ireland, metal mining activities had largely ceased by the early 20th century, but the legacy of spoil heaps and soils contaminated with heavy metals forms a patchwork of highly localized and drastically altered environments across the landscape . However, only the Irish mine population was significantly more copper tolerant than the respective local coastal population —as has constitutive tolerance in non-mine specialists .Although our experiments do not provide a direct measure of fitness in the wild, given the association between zinc tolerance, levels of zinc contamination in soil, vegetative growth, and flower production in uniflora ; 1978, ouniflora . Althouguniflora . Therefon per population; WWA-M = 25, WWA-C = 28, SWA-M = 28, SWA-C = 27, ENG-M = 26, ENG-C = 27, IRE-M = 28, IRE-C = 27) were genotyped at 74,064 SNPs. On average “local” mine and coastal populations were 20.8 km apart . Genetic differentiation between populations was high  than between coastal populations (mean FST = 0.25). Mine populations were also substantially differentiated from their local coastal population (mean FST = 0.36), suggestive of very limited gene flow between differentially adapted populations at the local level. In support of this, analysis of molecular variance , whereas f4 for the coastal population quartet . Comparisons of quartets with three mine populations and one coastal population .To investigate the genetic basis of mine-coast differentiation and degree of molecular convergence in adaptation, we conducted pairwise t genome  online. t genome A and SNPt genome B was sigt genome . In totat genome A. There t genome B, with fFundulus heteroclitus; Mimulus guttatus; Fundulus grandis; It is currently unclear whether the adaptive variation that underpins tolerance and colonization of the mine habitat has arisen through new independent mutations in each population  have direct links to these physiological differences between tolerant and nontolerant Silene: 1) PSD2 encodes a form of phosphatidylserine decarboxylase which is located in the tonoplast  WRKYtrations . Quercetinc ions ; and 3) for NaCl . Furthere shoots .RWP1 influencing the parallel evolution of zinc-tolerant populations. Of the three genes, only the scaffold containing RWP1 had consistently lower genetic diversity in the mine populations , whereas for WRKY23 and PSD2 diversity was only lower in the mines from West Wales and Ireland  was taken as confirmation of zinc tolerance  and only the best scoring hit from each predicted amino acid sequence was retained.DNA was extracted from silica dried leaf tissue using Qiagen DNeasy Plant tissue kits. DNA quality was assessed using agarose gel electrophoresis and DNA was quantified using a Promega Quantus fluorometer with Quantifluor dsDNA kits. For draft genome assembly, four NEBnext Ultra II libraries were prepared for SUTF1P and each was sequenced using illumina MiSeq v3 600 bp PE cartridges. Adapter and quality trimming were performed using cutadapt v2.1  and Trimsearched  against EcoRI-HF and MspI. For this study, size selection was conducted with a pippin prep (468–546 bp) and one pool of 230 uniquely barcoded individuals was sequenced on five lanes of an illumina HiSeq 2500  at the Earlham Institute. Raw reads were demultiplexed, trimmed to 90 bp and low-quality reads were discarded, resulting in an average of 4.76 M reads per sample (s.d. 2.01 M). Reads were mapped to the draft genome using bowtie v2.3.4  was calculated using Arlequin v3.5.2.2 . To track genetic relationships among populations, we simulated a single chromosome that was 50,000 bp long with a uniform mutation rate of 7.5 × 10−9—based on estimates for S. latifolia  and each of the four mine populations. We ran 100 independent replicates for each of the three colonization scenarios described above.Population genetic structure across t v2.1.3  in R andt v2.1.3 . AMOVA wt v2.1.3  To assesv3.5.2.2 , pairwis v0.1.16  and averatifolia —and a re 446 cM;  and genovulgaris . In the f4 statistics ,); ,); and ,)]. If ,) is the correct tree, the allele frequency difference between A and B will not be correlated with the frequency difference between C and D, that is, the correlation in frequency differences (f4) would not deviate from zero  descriptions and associated references. Systematic searches were performed using gene names with and without the terms “stress” and “heavy metal” using Google Scholar.To investigate the level of parallel evolution at the molecular level, we calculated Weir and Cockerham’s R v1.4.0  and signR v1.4.0 . To inveR v1.4.0  using thMolecular Biology and Evolution online.msab141_Supplementary_DataClick here for additional data file.


Weedy dandelions have a worldwide distribution and thrive in urban environments despite a lack of sexual reproduction throughout most of its range. North American dandelions, introduced from Eurasia, are believed to be primarily, if not exclusively, apomictic triploids. In some European populations, apomicts co‐occur with diploid sexual individuals and hybridizations can create genetically unique apomicts, which may subsequently disperse and establish new populations globally. Using six nuclear microsatellite markers and a cpDNA intergenic spacer, we investigate the impact of this unusual natural history on population structure and diversity in three urban Boston area dandelion populations. Our results show high levels of genetic diversity within populations, spatial population structure, and seasonal genotypic differentiation in flowering times. We find evidence that sexual reproduction and recombination, presumably in Europe, and extensive gene flow drive these patterns of diversity and create the appearance of panmixia despite the lack of evidence for local sexual reproduction. This study investigates population genetics, clonal diversity, and genotypic differences in flowering time of weedy dandelions in an urban area. We find population structure among sites and that specific genotypes are flowering during certain seasons. Populations exhibit high genetic diversity, are asexual, and mimic panmixia as a result of mass dispersal from European source populations. Wiggers, is a highly successful, cosmopolitan, short‐lived perennial species that thrives in disturbed environments. Dandelion likely originated 30 million years ago in the western Himalayas, and its recent history and center of diversity is in western Asia and Europe  to characterize clonal diversity in three populations using six polymorphic microsatellite markers and the trnH‐psbA intergenic spacer region of chloroplast DNA, (2) to assess spatial structure and flowering time patterns as an indicator of life history diversity, and (3) to test for linkage disequilibrium, the signature of clonal reproduction, and panmixia to reveal the relative importance of sexual reproduction, dispersal and somatic mutation in the establishment of local population structure.22.1Sampling for this study was performed over two years on a monthly basis April through November in 2017 and 2018; plants flowering well beyond this period are found but flowering is sporadic and their numbers are low. We collected 568 dandelion samples from three stable, undisturbed  sites covering approximately a 20 km range from north to the south of Boston, MA: Prospect Hill Monument, Somerville, MA; Cedar Grove Cemetery, Boston, MA; and 12 Moon Farm in Milton, MA, respectively. Leaf tissue was collected only from individuals in flower, separated at least four meters apart, and placed in separate Ziploc bags. Samples were kept fresh or stored in −20°C until the time of extraction. Leaf tissue was typically not stored in the freezer for longer than one month before DNA extraction.2.2Approximately 100 mg of leaf tissue was used for DNA extraction using the DNeasy Plant Mini Kit  following manufacturer's protocol with slight modifications. Frozen leaf tissue was measured out and manually crushed using sterilized ceramic mortar and pestles. Lysation buffer (200 µl) was added directly onto the leaf tissue to aid in manual crushing. The elution buffer was heated at 65°C for 10 min prior to the final DNA elution step to increase DNA yield. Whole genomic DNA was quantified and checked for purity on a NanoDrop 2000C. DNA dilutions on samples were performed to a level of 0.5–0.7 ng. PCR was performed using the diluted DNA as the template. PCR for the trnH‐psbA intergenic spacer region was performed according to primers and PCR specifications from Hamilton . PCRs fo2.3pegas version 0.11 , summer (July–August), and fall (September–November) were assigned based on the month of collection. The R package Jombart,  was usedpoppr version 2.8.0  was performed in the R package Hedrick,  were caln = 551 for the three sites set as “populations” as well as the three seasons set as the “populations.” Two additional analyses were performed: (1) with the cpDNA haplotype groupings set as “populations” for n = 91 samples in order to visualize nuclear–cytoplasmic recombinant groupings established at each population site and (2) with the clone‐corrected dataset where all multilocus genotypes were represented once and any samples with missing data removed for n = 270. For all STRUCTURE analyses, admixture model and correlation between alleles were assumed. For the total dataset of n = 551, an initial simulation was performed for K = 2 through K = 20 for burn‐in period of 200,000 with an additional 200,000 Markov Chain Monte Carlo (MCMC) replicates over five iterations in order to implement post hoc tests of optimal K clusters. For both the haplotype subset of n = 91 and clone‐corrected subset of n = 270, an initial simulation of K = 2 through K = 15 for burn‐in period of 200,000 with an additional 200,000 MCMC replicates over five iterations was performed. These initial simulations were run through Structure Harvester  making these markers very useful for our study (Table st between the three populations showed the least differentiation between PH and CG (Gst = 0.018) and more differentiation between MF and PH (Gst = 0.049) and MF and CG (Gst = 0.049). Pairwise Gst calculated between the seasons showed the least differentiation between spring and summer genotypes (Gst = 0.004); followed by Summer and Fall (Gst = 0.010); the most differentiation between spring and fall genotypes (Gst = 0.046).The six microsatellite markers showed substantial variability with 14–39 alleles, high levels of genetic diversity (Hexp = 0.77–0.94), and evenness  chosen, suggesting substantial seed flow of clones into sites. Some multilocus genotypes (clones) are found in all three populations but many are specific to populations, and the frequency of the genetic groups varied among sites  as well as among seasons within populations (p < .001).Bayesian STRUCTURE analyses and AMOVA all detected widespread diversity and population structure among the three sites. While STRUCTURE analyses may not be the most appropriate tool for clonal populations , to assess the fit of our dandelion data to these models  as well as for each of the three populations separately. There were 256 different genotypes (56%) among the 456 samples, 202 being detected once only and 54 genotypes occurring more than once; the most frequent genotype was detected 32 times with 21 individuals in Moon Farm, 8 in Cedar Grove, and 3 in Prospect Hill. The proportion of different genotypes among the samples was similar for each site; 90 in 156 samples for Prospect Hill, 116 in 184 for Cedar Grove, and 69 in 116 for Moon Farm , while the second and third largest clones with 28 and 26 samples, respectively, both flowered primarily in the spring of both years (86% and 69% respectively). The fourth largest clone with 17 individuals flowered about equally in fall and spring of 2017, but was not detected in any population in 2018.Many of the 256 genotypes share common alleles in some loci but have different alleles at other loci, suggesting recombination as the source of genetic diversity. Indeed, in the entire dataset, there is only one pair of different clones that differs by a single allele at a single locus. All other clones differ from each other by multiple alleles at multiple loci, a pattern of variation not expected by local somatic mutation.Further, a careful analysis of the STRUCTURE output showed that regardless of the assumed number of genetic groups, approximately 10% of the individuals in the entire population are the result of admixture . For the clone‐corrected analyses, 22%–32% of the multilocus genotypes are defined as having mixed ancestry. This reflects the role of sexual reproduction and recombination in the history of these individuals, though the timing, in Europe or in North America, of this is unknown. We addressed this timing issue, by examining the level of mixed ancestry as determined from the STRUCTURE analysis, with the prevalence of the various clones. From the clone‐corrected data in which each multilocus genotype is represented once in the analysis, none of the 15 largest clones represented by 5–32 individuals/clone in the full population have mixed ancestry at K = 12. These large clones are defined as >90% pure in the analysis. However, 32% of the genotypes are identified as having mixed ancestry (<80% of their genome coming from a single source) possibly suggesting that recombinants are more recent arrivals or evolved locally.4Taraxacum species, closely related sexual members coexist in certain populations mainly in some areas of Europe and gene flow between apomictic and sexual types is possible, leading to newly formed and genetically unique apomicts  and showed a less distinct signature of panmixia than the other two populations. These sampling strategies allowed us to assess the full array of genotypes in the area and the cumulative effects of population‐level processes.We detected three major cpDNA haplotype lineages, consistent with King  in her NTaraxacum in North America have high diversity and high numbers of different genotypes, the source of that variation is not as clear. Rogstad et al.  seemed surprising given the proximity of these sites, the prevalence of dandelions throughout the region, and the enormous level of gene flow via seed needed to generate the levels of diversity seen. The genetic structure in flowering times Figure  suggestsWhile the prevailing view is that all genetic diversity is generated in Europe and dispersed to North American, we cannot discount the possibility of some cryptic local sexual recombination and its possible contribution to the genotypic diversity found in these populations. One interesting finding was that the 15 largest, most frequently occurring clones with 5–32 sampled individuals per genet, were never recombinant or of mixed ancestry based on STRUCTURE assignment analyses, suggesting that frequency may be correlated with time since establishment. This may indicate that recombinants are being generated in, and continuously dispersed from, Europe. Alternatively, this pattern might be achieved by dispersal and establishment of older clones from Europe in the past, followed by local cryptic recombination. Regardless, the propensity for dandelions to thrive in disturbed habitats created by humans makes this study system ripe for exploring urbanization, global climate change and local adaptation or selection of lineages. Further investigations of environmental conditions within each of the sites may reveal the factors that drive the establishment of population structure.The authors report no competing interests.Lisa Mazumder: Conceptualization ; Data curation (lead); Formal analysis (lead); Investigation ; Methodology ; Validation ; Visualization (lead); Writing‐original draft (lead); Writing‐review & editing . Rick Kesseli: Conceptualization ; Data curation (supporting); Formal analysis (supporting); Funding acquisition (lead); Investigation ; Methodology ; Project administration (lead); Resources (lead); Supervision (lead); Validation ; Visualization (supporting); Writing‐original draft (supporting); Writing‐review & editing .5® (accessions MW451112‐MW451204). Microsatellite data as well as additional figures not pictured herein are available at 10.6084/m9.figshare.14544672.Chloroplast DNA sequences of the trnH‐psbA intergenic spacer are available in GenBank


A majority of studies reporting human genetic variants were performed in populations of European ancestry whereas other global populations, and particularly many ethnolinguistic groups in other continents, are heavily underrepresented in these studies. To investigate the extent of this disproportionate representation of global populations concerning variants of significance to thrombosis and hemostasis, 845 single nucleotide polymorphisms (SNPs) in and around 34 genes associated with thrombosis and hemostasis and included in the commercial Axiom Precision Medicine Research Array (PMRA) were evaluated, using gene frequencies in 3 African  and 14 non-African  populations. Among the populations studied, Europeans were observed to be the best represented population by the hemostatic SNPs included in the PMRA. The European population also presented the largest number of common pharmacogenetic and pathogenic hemostatic variants reported in the ClinVar database. The number of such variants decreased the farther the genetic distance a population was from Europeans, with Yoruba and East Asians presenting the least number of clinically significant hemostatic SNPs in ClinVar while also being the two genetically most distinct populations from Europeans among the populations compared. Current study shows the lopsided representation of global populations as regards to hemostatic genetic variants listed in different commercial SNP arrays, such as the PMRA, and reported in genetic databases while also underlining the importance of inclusion of non-European ethnolinguistic populations in genomics studies designed to discover variants of significance to bleeding and thrombotic disorders.The online version contains supplementary material available at 10.1186/s12920-022-01220-0. Hemostasis is a complex and vital physiological process that involves activation of clotting factors as well as blood platelets, clot retraction, fibrinolysis and vascular repair . HundredA large number of common and rare genetic variants in hemostasis were identified in the past thanks to modern high-throughput genotyping and sequencing approaches that deciphered the human genome. Some of these variants are associated with altered phenotype and, hence, are accessible from public disease database repositories such as ClinVar  and OnliFourteen populations were included in the analyses: African , African Americans in Southwest USA (ASW), ad-mixed American , East Asian , European , South Asian . Except for Somalis (n = 95), genotype data were accessible from the 1000 Genomes Project (n = 4973) . For thehttps://www.ncbi.nlm.nih.gov/clinvar/) [The analysis of the Affymetrix Axiom Array was performed according to the protocol described in the AxiomTM 2.0 Assay Manual Workflow User Guide by Thermo Fisher Scientific Inc. , as we previously described . Genotyplinvar/) . Varianthttp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/). In the final analysis, we only included the SNPs that were represented in both the Affymetrix genotype array and the phase-determined population data obtained from the 1000 genomes project phase 3.Haplotype phasing was estimated with the Shapeit v2 method . DuplicaPrincipal component analysis (PCA) was performed with the R software version 3.6.1  using faUnder the predefined inclusion criteria, 845 hemostatic SNPs in 34 genes were found to be eligible for downstream analyses  are identical or whether at least one of these populations deviates from other populations. Post-hoc analyses with Bonferroni corrections were then performed to investigate pairwise relationships in gene frequency among the 6 populations. This generated 6 pairs of populations that displayed statistically significant differences in allele frequencies: EAS‒EUR (P < 0.05), YRI‒EUR (P < 0.05), SOM‒YRI (P < 0.05), EAS‒AMR (P < 0.001), EAS‒YRI (P < 0.001), and SAS‒YRI (P < 0.001). No other pairs showed statistically significant differences in gene frequencies. The EAS and YRI populations showed the lowest correlation in gene frequencies between any pair of population compared, with Pearson's correlation coefficient (r) corresponding to 0.72 , indicating a significant European admixture in the latter population.To investigate whether there were differences in gene frequencies between the populations, allele frequencies for the 845 SNPs were statistically examined over 6 populations  using Kruskal–Wallis test . The Krur values then decreased as the genetic distance from Europeans increased in the following order: AMR (r = 0.98) > EUR and SAS (r = 0.97) > SOM (r = 0.96) > EAS (r = 0.92) > YRI (r = 0.89).To investigate dispersion of the 845 SNPs over populations, Pearson correlations between allele frequencies of each population and the expected mean allele frequencies for the 6 populations was examined (graphs not shown). The test showed that allele frequencies in Europeans and in populations with genetic proximity to Europeans had the best correlations with the expected mean allele frequencies. The Pearson In Fig. SERPINE1 whose pathogenicity status is disputed, have alleles that are prevalent in non-African populations but not in the 2 African populations investigated . In Fig. VKORC1) – is shown with regard to 8 different SNPs in 10 different populations: SOM, ASW, CEU, CHB, GIH, JPT, LWK, MXL, TSI, YRI. The VKORC1 gene is shown as an illustrative example partly because of its pharmacogenetic importance (warfarin resistance/sensitivity) and partly because of the high proportion of selected SNPs that given the allele frequencies could be expected to be informative as regards ethnicity. Surprisingly, a mere 8 different multi-SNP haplotypes of VKORC1 represent the whole spectrum. Those with a frequency less than one percent were excluded  and examined SNPs in a region of 1 Mbps encompassing the PROC gene—one of the 34 genes studied. As expected, the resulting haplotype chart  = 14.6, P = 0.012. Post hoc analysis with Wilcoxon signed-rank tests was conducted with a Bonferroni correction applied, which showed a significant difference between EUR and EAS (P < 0.05) and between YRI and EAS (P = 0.008).To examine the representation of populations with respect to the clinically relevant variants, we combined the drug response pharmacogenetic variants involved in thrombosis therapy and common pathogenic SNPs that alter function of hemostasis genes (n = 17), which we then investigated with Friedman’s two way analysis of variance by ranks. Friedman’s mean rank was largest in Europe and decreased with increasing genetic distance from Europe: EUR (4.4) > AMR (3.9) > SAS (3.8) > SOM (3.6) > YRI and EAS (2.7 each). This suggested that EUR was the best-represented population by the DNA variants investigated and that YRI and EAS were the least represented. There was a statistically significant difference in allele frequencies of the clinically relevant SNPs between the 6 populations, chi squared  and in CYP2C9  genes. These eight variants are known to be associated with warfarin dose requirement [Nine pathogenic hemostatic SNPs and eight drug response DNA variants with at least 1% AAF were identified in the populations studied Fig. . In the uirement . One of uirement –21 and, rs118203905 (FV R306T), also called Factor V Cambridge and associated with APC resistance [rs41276738 (VWF R854Q) linked to type 2 N von Willebrand disease [rs73015965 (PLG K38E) and rs148685782 (FGG A108G) associated with type I plasminogen deficiency [rs121918478 , is a single nucleotide variant in factor II gene creating an amino acid substitution at position 461 of the protein (FII R461W), which causes hereditary factor II deficiency disease [rs137852380  in F8 gene (FVIII G89D) [VKORC1 loss of enzyme activity [The EUR population appeared to have the most pathogenic SNPs reaching at 1% AAF Fig. , right. sistance , was com disease , reachedficiency  and redu disease . The othII G89D) . Finally3905 FV R6T, also https://www.ebi.ac.uk/gwas). At the time of writing , 78% of all individuals and 49% of all studies in the NHGRI-EBI Catalog involve European populations  pathogenic and drug response hemostatic variants identified on ClinVar database when SNPs included in the Axiom PMRA were employed. Yet, Friedman’s two way analysis of variance by ranks obviously showed statistically significant differences between the populations with respect to these clinically relevant variants. Interestingly, the pattern of Friedman’s mean ranks mirrored nearly perfectly the genetic shape shown in the PCA  in Italy rather than to Luhya in Webuye, Kenya, despite the latter country being a neighbor to Somalia. Yet, Horn of African populations and other ethnolinguistic groups in Africa are not represented in large genome ventures such the 1000 genomes.Another limitation is the merging of non-African continental and subcontinental populations. Obviously, it might be expected that there is some variation in allele frequencies within a particular continental or subcontinental population. The variation in gene frequencies, however, is much greater within African populations as we observed in this study between the Somali and other African populations when analyzed the CFTR gene variants and flanking polymorphic SNPs on individuals from PGD families. Heterozygous calls in each individual were phased using massive parallel sequencing genomic data for reference populations. For founder mutation carriers, the haplotyping approach facilitated near perfect phasing accuracy. Their results demonstrate the feasibility of replacing classical haplotype phasing with population-based phasing, thus eliminating the need for recruiting additional family members in the PGD procedure.Finally, we would like to discuss the use of haplotype information in the field of medical genetics. First, the importance and usefulness of haplotyping is well established through traditional linkage analysis in families with inherited disease. Second, there are also other circumstances where genetic phasing plays a role. For example, hematopoietic stem cell transplants using haplotype matched recipients perform better clinically than those using allele matched but haplotype mis-matched patients . To mentIn precision medicine, by definition, it is expected that many diagnostic tests for selection of appropriate therapies will be based on the patient's genetic characteristics. Skewed representation of global populations in genetic databases affects the effectiveness of such endeavors, especially when modelled on interfering anti-sense oligonucleotides. For example, one could speculate of miRNAs as potential cancer biomarkers and therapeutic targets. However, the breakthrough of circulating miRNA-based cancer therapy has yet to come.VKORC1 rs61742245 polymorphism to control warfarin resistance may be considered as a way to improve the safety and efficacy of warfarin use but, unfortunately, there is limited evidence to support routine testing due to the even greater impact of diet, possible lack of compliance, and environmental factors on the warfarin levels monitored in blood. However, as a proof of principle, our VKORC1 example shows the power of a population-based phasing approach when applied to GWAS data. The basis for such analysis is linkage disequilibrium of polymorphisms in the vicinity of disease-causing founder mutations, including pharmaco-genetically important variants.In this study, our results demonstrate the importance of considering ethnicity when prescribing warfarin for the prophylaxis and treatment of venous thrombosis and pulmonary embolism. Warfarin has a very narrow therapeutic range. If the dose is too high, the patient may be at risk of serious bleeding and if the dose is too low there is an increased risk of thrombosis. Pharmacogenetic testing of the VKORC1) based on only 8 SNPs are highly correlated across populations and could be used not only for population studies but also for disease gene mapping.In conclusion, this study highlights the importance of diversity in genomics research pertaining to DNA variants of clinical importance to thrombosis and hemostasis. By including more African, Asian and indigenous ethnic groups in genomics studies, more clinically relevant hemostatic variants of significance to thrombosis and bleeding disorders would presumably be discovered and reported to the different clinical genetic databases as well as be included in different commercial SNP arrays. Haplotype-based methods offer a powerful approach to disease gene mapping, based on the association between causal mutations and the ancestral haplotypes on which they arose . We confAbdimajid Osman is Associate Professor of Medical Genetics and senior Clinical Scientist at Linköping University Hospital. Jon Jonasson is Associate Professor of Clinical Genetics and senior consultant Clinical Geneticist at Linköping University Hospital.Additional file 1: Table S1. A list of 845 SNPs in genes associated with hemostasis. ClinVar annotations are shown on the column farthest to the right.Additional file 2: Fig. S1. Principal component analysis of 849,267 high-quality markers that were included in the Axiom Precision Medicine Research Array. The Somali data were projected onto PC1 and PC2 data from PGG population (https://www.pggpopulation.org/) as we previously described [escribed .Additional file 3: Table S3. Pearson correlations for 845 haemostatic gene variants with at least 1% MAF in at least 1 population.Additional file 4: Fig. S2. Population multiplot for 845 hemostatic gene variants. Allele frequencies are shown on the axis.Additional file 5: Table S2. Multi-SNP haplotype phasing for hemostatic genes constructed with the Shapeit v2 method. Each tab in the Excel file shows haplotypes for one hemostatic gene. Additional file 6: Fig. S3. Population haplotypes in a region of 1 cM (1 Mbp) encompassing the PROC gene. Rows and columns represent haplotypes and SNPs, respectively. Haplotypes of the same color are identical.