Contarinia vincetoxici, which is a monophagous parasite on the perennial herb White swallowwort (Vincetoxicum hirundinaria). This insect not only has a narrow feeding niche but also limited dispersal ability and an exceptionally long dormancy. Gall midge larvae (n = 329) were collected from 16 sites along its distribution range in Denmark, Sweden, and Finland. Using microsatellite loci and knowledge of the species and the regions' history, we investigated the role of landscape change, host plant distribution, insect population dynamics, and life history in shaping the population genetic structure of the insect. We devoted particular interest to the role of the insect's presumed poor dispersal capacity in combination with its exceptionally extended diapause. We found significant levels of local inbreeding , low‐level within‐population heterozygosity  with private alleles in all populations except two. We also found significant (p < .001) regional isolation‐by‐distance patterns, suggesting regularly recurring mainly short‐distance dispersal. According to approximate Bayesian computations, C. vincetoxici appears to have colonized the study area via wind‐aided flights from remote areas approximately 4600–700 years before present when the land has gradually risen above the sea level. Extremely long dormancy periods have allowed the species to “disperse in time”, thereby aiding population persistence despite generally low census population sizes.Species that exhibit very peculiar ecological traits combined with limited dispersal ability pose a challenge to our understanding of ecological and evolutionary mechanisms. This is especially true when they have managed to spread over long distances, overcome physical barriers, and colonize large areas. Climate and landscape changes, trophic web relations, as well as life history all interact to shape migration routes and present‐day species distributions and their population genetic structures. Here we analyzed the post‐glacial colonization of northern Europe by the gall midge  We analyzed the post‐glacial colonization of northern Europe by an insect with a narrow feeding niche, limited dispersal ability and an exceptionally long dormancy. We found substantial levels of local inbreeding and regional isolation‐by‐distance patterns. The species colonized the study area via wind‐aided flights from remote areas approximately 4600–700 years before present when the land has gradually risen above the sea level. The estContarinia vincetoxici  with an unusual life history. This monophagous gall midge is a tiny insect whose larva lives in flower galls of the perennial herb White Swallowwort, Vincetoxicum hirundinaria , which are routinely and effectively employed for such a purpose. Multilocus genotypes mirror the diversity of founders, dispersal pathways, and divergence times of populations  which were sampled in 1991. This sampling covers the current distribution of the host plant and gall midge populations in Fennoscandia. When galls were few at a site, they were all collected. When they were more numerous, collecting efforts were spread across the entire host plant patch.A total of 329 galls were collected from 16 sites ranging from SW Finland to SE and S Sweden and E Denmark  according to manufacturer instructions. Microsatellite sequences of m et al. . A totalm et al. . The fraTo test for genotypic linkage disequilibria among loci, we used contingency tables and an unbiased estimate of the exact probability obtained by Markov Chain Monte Carlo simulations and 10,000 permutations using Arlequin 3.5  and basic Jombart,  and “hieJombart, , respectJombart,  and consJombart, . To test2.4K (range 1–16), we conducted 20 independent runs with uniform priors using a burn‐in of 100,000 iterations followed by 100,000 Markov chain Monte Carlo iterations. The number of genetic clusters K in the data set was inferred in two steps. Firstly we used the ΔK method  implemented in the R package “CorrSieve” 1.6‐9 . Outputs of the Structure analysis were visualized with the Clumpak program , (2) standardized heterozygosity based on the mean expected heterozygosity (Hs_exp), (3) internal relatedness (IR), and (4) homozygosity by locus (HL) calculated by the R function GENHET , indicating a long‐term effect of mutations in a large ancestral population (scenarios 2–6). Lastly, we added a competing hypothesis where the population with the highest variation resulted from the admixture of different independently introduced lineages from an unknown source that gradually colonized the territory from different directions (scenarios 7–10). For the second and third assumptions, there are several alternative scenarios that differ in the times of divergence or admixture of different clusters. From these hypotheses, we selected a set of 10 hypothetical or possible evolutionary scenarios  was set to 0.22. We generated a reference table containing 10 million datasets, each scenario being given a uniform prior probability. Posterior probabilities of each scenario were estimated using local logistic regression on 1% of simulated datasets closest to the observed dataset after applying a logit (default) transformation of demographic and historical parameter values. Three populations  with the smallest sample size and low genotyping success were not used in this ABC analysis.To estimate divergence times and population history of gall midge populations, we used a coalescent‐based Approximate Bayesian Computation (ABC) algorithm in the program DIYABC 2.1.0  using 15 microsatellite loci. In total, 3.2% of data was missing. However, this was particularly due to the three populations with the smallest sample size  where on average 5 out of 15 loci were not amplified. Significant linkage disequilibria were found for almost all loci combinations (Table p < .001) from HWE were revealed in half of the populations at 1–6 loci  but showed very low within‐population observed (HO = 0.17–0.51) and expected heterozygosity (HE = 0.20–0.61) per population . The exceptionally high number of private alleles  was found in the population Res on Öland Island , FIT = 0.51  and FIS = 0.27 (ind/pop). Contrary to our prediction, the IBD analysis resulted in a continuous cline of genetic differentiation  with no sign of pattern breakdown , we therefore assumed two genetic clusters around the lake Mälaren in central Sweden (clusters III and IV) and one cluster of populations from southern Finland . This analysis suggested the highest heterozygosity in cluster II , outside the study area (or independent introductions of the species to Fennoscandia), was = 768 (516–1960) for the cluster I populations (t4) and 566 (210–952) for the cluster III populations (t3). Simultaneous admixture of cluster I and III populations and divergence between cluster III and IV (t2) was estimated around 133 (103–244) generations ago. Finally, population cluster V diverged from cluster IV 121 (43–246) generations before present (t1). This is in accordance with our prediction that older populations have higher genetic variability than younger (I and III vs. IV and V). However, the highest level of variation was found in the population that ABC analysis suggested as an admixture of two lineages  pattern across the whole study area. Despite the insect being tiny . Furthermore, being part of the natural life cycle, flight is timed to occur when host plants are suitable for oviposition, thus facilitating colonization. Other means of long‐distance dispersal are much less plausible in the gall midge. Zoochory is unlikely as the host plant is poisonous . Revealed molecular dating also rejects the possibility that dispersal occurred through the last land bridge which connected south tip of the peninsula with the mainland yet 10.3 ka BP before formation of the Littorina/Baltic Sea . Two pieces of evidence suggest that this may have occurred through aerial dispersal from the island of Gotland. First, there are no records of the gall midge along the eastern Baltic, and secondly, there are large populations of the host plant and numerous observations of the gall midge on this island . Unfortunately, we have no genetic data from Gotland and we cannot rule out the possibility of wind transport from even more distant areas. Based on the abundant occurrence of the host plant, the high genetic diversity of the gall‐midge on the island of Öland, and the fact that most of the southern regions have been above sea level for a long time, we assume that the area of cluster II was colonized soon after the first introduction from the cluster I, until it mixed with cluster III 800 BP.Our data indicate that long‐distance dispersal should have occurred in this species. This is interesting from an evolutionary perspective as it is probably the way in which  Björck, . Another4.3The prolonged larval diapause (of variable length) is an important aspect of the gall midge's life cycle, with considerable implications for its occurrence and population genetics. As our data show, the limited dispersal rate of the gall midge is not a hindrance to high occupancy in some sites. It appears that the high survival through long dormancy periods acts as a strong buffer against local extinction even in very small gall midge populations  of two different introductions to Fennoscandia, which coincided with subsequent colonization events towards north. In addition, extreme inbreeding was manifested in all local populations by elevated levels of homozygosity and a random pattern of linkage among loci. The fact that linkage included most combinations of loci across all data suggests that—rather than being a technical issue—the phenomenon decided by the species life history. The inbreeding pattern appears to be a result of high incidence of mating between closely related individuals rather than a consequence of geographic isolation of certain sites. On the other hand, lower level of inbreeding and high genetic variation, indicating a large effective population size of the gall‐midge found in the Resmo population on the island of Öland, may be associated with the one of the largest populations of White swallowwort in the region.Another aspect of the “low colonization—high survival” syndrome is the usually small size of host plant patches. This means that most local gall midge populations are also small, and small populations are generally subjected to high extinction risks. However, only a small fraction of a local population emerges from diapause to reproduce each year thus being exposed to extinction hazards ; data curation (lead); investigation (lead); writing – original draft ; writing – review and editing . Anna Cassel‐Lundhagen: Conceptualization ; investigation (supporting); writing – original draft ; writing – review and editing . Ane T. Laugen: Conceptualization ; writing – review and editing . Peter Kaňuch: Formal analysis (lead); visualization (lead); writing – original draft ; writing – review and editing .None declared.Appendix S1Click here for additional data file.


The gene pool of the indigenous population of Siberia is a unique system for studying population and evolutionary genetic processes, analyzing genetic diversity, and reconstructing the genetic history of populations. High ethnic diversity is a feature of Siberia, as one of the regions of the peripheral settlement of modern human. The vast expanses of this region and the small number of aboriginal populations contributed to the formation of significant territorial and genetic subdivision. About 40 indigenous peoples are settled on the territory of the Siberian historical and ethnographic province. Within the framework of this work, a large-scale population study of the gene pool of the indigenous peoples of Siberia was carried out for the first time at the level of high-density biochips. This makes it possible to fill in a significant gap in the genogeographic picture of the Eurasian population. For this, DNA fragments were analyzed, which had been inherited without recombination by each pair of individuals from their recent common ancestor, that is, segments (blocks) identical by descent (IBD). The distribution of IBD blocks in the populations of Siberia is in good agreement with the geographical proximity of the populations and their linguistic affiliation. Among the Siberian populations, the Chukchi, Koryaks, and Nivkhs form a separate cluster from the main Siberian group, with the Chukchi and Koryaks being more closely related. Separate subclusters of Evenks and Yakuts, Kets and Chulyms are formed within the Siberian cluster. Analysis of SNPs that fell into more IBD segments of the analyzed populations made it possible to compile a list of 5358 genes. According to the calculation results, biological processes enriched with these genes are associated with the detection of a chemical stimulus involved in the sensory perception of smell. Enriched for the genes found, molecular pathways are associated with the metabolism of linoleic, arachidonic, tyrosic acids and by olfactory transduction. At the same time, an analysis of the literature data showed that some of the selected genes, which were found in a larger number of IBD blocks in several populations at once, can play a role in genetic adaptation to environmental factors. Genetic and demographic processes in populations, populationfluctuations, cross-breeding events, migrations and naturalselection affect the structure of genetic diversity in the genomesof individuals and populations as a whole. In particular,genetic and demographic processes lead to the formation oflinkage blocks of common origin .A segment having identical nucleotide sequences is IBD in twoor more individuals if they have inherited it from a commonancestor without recombination, that is, in these individualsthe segment has a common origin. The expected length of anIBD block depends on the number of generations that havepassed since the segment appeared in the last common ancestor.IBD segments can be used to reveal the demographic historyof populations, including bottleneck effects and gene flows inpopulations . Recent studies have showndifferences in IBD distribution between African, Asian, andEuropean populations, as well as IBD segments shared withancient genomes such as those of Neanderthals and Denisovans.Close relatives have rather long DNA fragments identicalwith each other and, accordingly, in most chromosomesthere are blocks of considerable length identical by descent(> 66.7 cM), as a result of which the expected length of thetotal IBD is about 1700 cM. Cousins and second cousins areexpected to have multiple regions (more than 2.5 expectedsegments) due to the presence of recent ancestors determiningtheir relationship. For first cousins, each IBD is expected tohave a total length greater than 62 cM and for second cousins,25 cM. Distant cousins that are fourth cousins or more distantare very likely to carry one or more regions from their nearestcommon ancestor. Such couples include the vast majorityof people in a particular population and are usually referredto as “unrelated” because the proportion and number of IBDacross the genome is expected to be relatively small betweenthem .IBD segments can also help in the detection of naturalselection signals in the human genome. Searching for regionswith an excess of IBD segments allows the identification ofgenomic regions in the human genome that are under veryrecent and strong selection, since selection generally increasesthe number of IBD segments among individuals in a population.Regarding the populations of the indigenous ethnic groupsof Siberia, it has been suggested that large-scale dispersal andmixing of populations probably may explain the unusuallyhigh proportion of IBD between populations .The purpose of this study was to analyze the structure ofthe gene pool of populations of the indigenous population ofSiberia, based on the identification of linkage blocks identicalby descent, and their intra- and interpopulation distribution.Genome-wide genotyping data were generated using InfiniumMulti-Ethnic Global-8 microarrays (Illumina) with over 1.7 millionmarkers. Samples with more than 5 % missing positions,as well as SNPs with more than 10 % missing genotypes,were excluded from the analysis. The data were preliminarilyfiltered by the minimum rare allele frequency . As a result, 886,889 autosomal SNPswere included in the final data setPopulations of the indigenous peoples of Siberia (N = 477)are represented by Altaians , Buryats , Kalmyks (N = 29), Kets(N = 15), Koryaks (N = 20), Chukchi (N = 25). The Koryakmaterial was collected in the Koryak AutonomousDistrict ofthe Kamchatka Region. A populationsample of the Chukchiwhose blood samples were collected in the villages of Lorino,Sireniki, Yanarykot and Novoe Chaplinoof the ChukotkaAutonomous Okrug belongs to the coastal group, Nivkhs(N = 13), Tatars , Tuvans (N = 28), Udeges(N = 15), Khantami , Khakases (T – Sagais of the Tashtyp districtN = 29 and S – Kachins of the Shirinsky district N = 26), Chulyms(N = 22), Evenks  N = 25 and Y – Yakut Evenks(N = 28) and Yakuts (N = 26).The material was deposited in the bioresource collection“Biobank of the Population of Northern Eurasia”. The characteristicsof the studied populations are presented in Table 1.Phasing of genotypes was carried out using Beagle 4.1software . The RefinedIBD algorithm, refined-ibd.16May19.ad5.jar version , was used to analyze genomeblocks identical by descent. To compare the populations, thesums of the average lengths of the IBD segments between pairsof individuals were obtained for the following length ranges:1.5–1.999 cM, 2–3.999, 4–7.999, 8–15.999 and >16 cM . A heat map with adendrogram based on the logarithm of the sum of the average IBD segment lengths between pairs of individuals was builtusing the heatmap.2 package in the R software environmentWe also identified SNPs that fell into a larger number ofIBD segments of the analyzed populations (the frequencyof SNPs in IBD was higher than the 99th quantile of thefrequency distribution), determined the belonging of theseSNPs to genes, and assessed the biological significance ofthe resulting list of these genes. For this analysis, we used theWebGestalt web resource (WEB-based Gene SeT AnaLysisToolkit); in particular, the analysis of KEGG paths and geneontologies (Gene Ontology) was conducted using the ORA method.For a more detailed analysis of the genetic relationship ofthe Siberian populations and to find out to what extent theirgenetic structure can be explained by recent local migrations,we isolated and analyzed DNA fragments that were inheritedwithout recombination by each pair of individuals from theirrecent common ancestor, that is, segments (blocks) identicalby descent (IBD).The populations inhabiting the territory of Siberia arecharacterized by a unique genetic and demographic history,which is reflected, among other things, in the distribution ofIBD blocks both within the populations and between them.We calculated the sums of the average lengths of the IBD segmentsbetween pairs of individuals and, based on their results,built a heat map with a dendrogram based on the logarithm ofthe sum of the average lengths of the IBD segments .The number of common segments among representatives ofdifferent populations is consistent with the geography of theirresidence, since the peoples living nearby can be influencedby common genetic and demographic processes. Analysis ofthe heat map demonstrates the clustering of the populationsof the Siberian group, linking peoples by place of origin.Among the Siberian populations, the Chukchi, Koryaks andNivkhs form a separate cluster from the main group of Siberianpopulations, with the Chukchi and Koryaks being moreclosely related. Separate subclusters of Evenks and Yakuts,Kets and Chulyms, Tuvans and Altaians are formed withinthe Siberian cluster.With the gradation of the IBD segments with differentmean length, the trend generally remains, but some differencesappear that more accurately characterize the recentadmixture between the peoples. For longer IBDs, the clustersare more in line with the current geographic location ofthe populations, reflecting the recent exchange of commonregions. In three length ranges with IBD sizes , populations are better divided into closelygeographically located pairs: Koryak-Chukchi, Yakut-Evenki The Kets almost equally share IBD blocks with the Chulyms(18.7–27.2–7.7 cM) and Khanty (23.4–24.4–4.8 cM forKhanty (K) and 25.9–30.1–7.9 cM for the Khanty (R)), whileamong the Khanty, a greater value of common IBD blocks isobserved in the Russian Khanty, which corresponds to theircloser geographical location compared to the Khanty of theBeloyarsky district.For the Khakas, who are more distant from the Kets, thevalues of the total IBD blocks are almost two to three timeslower than for the Khanty (9.8–10.4–2.5 cM for the Khakas (S)and 9.1–10.6–3.8 cM for Khakas (T)). Despite the fact that theEvenks, Tuvans and Yakuts are even more remote, for the Tuvans(12.3–12.8–2.1 cM) and the Yakuts (12.8–12.5–2.1 cM),there are similar values of total IBD blocks, for the Trans-Baikal Evenks (16.4–16.9–3.0 cM), it is worth noting thatthe values of the IBD blocks are greater than with the YakutEvenks (14.7–14.0–2.6 cM).With few exceptions, common IBD segments betweenSiberian populations are better explained by the geographicproximity of the populations rather than by their linguisticaffiliation. For example, the Yakut Evenks living in the territoryof Yakutia have more IBDs in common with the Yakuts(252.7 cM) than with the Transbaikal Evenks (102.5 cM). Atthe same time, the sum of the average lengths of IBD segmentsbetween pairs of individuals between two populations of theEvenks is comparable to the sum between the TransbaikalianEvenks and Yakuts (95.2 cM).In terms of intrapopulation IBD analysis, in general, individualsfrom populations of the Far North and Far East share more IBDs with specimensfrom the same group than individuals from the South Siberianpopulations such as the Altaians and Tuvans. At the same time,in the Chukchi, Koryaks, and Nivkhs, short IBD fragments of1.5–4 cM (55–57–59 %) make the largest contribution, whichmay indicate a bottleneck in the past during migrations tothe north and northeast and/or a strong isolation from otherpopulations inhabiting the territory of Siberia. At the sametime, in the Chulyms, Khanty (R), inhabiting the central part ofSiberia and having the largest sum of average lengths of IBDsegments between pairs of individuals, the largest contributionis made by IBDs longer than 8 cM (47–51 %), which indicatesa strong recent inbreeding within the populationThe most genetically heterogeneous Siberian populations,which have minimal values for the genomic inbreeding coefficient(FROH) , also have minimalvalues for the sum of the average lengths of IBD segments.This is most pronounced in the populations of Kalmyks, AginBuryats, and Tomsk Tatars .In Buryat populations, there are no significant differencesin the average total length between pairs of individuals withother populations, but there is a significant difference in thedistribution of IBD within populations. Thus, the Buryats (K)have a significantly larger average total length between pairsof individuals within the population (335.4 cM), comparedwith the Buryats (A) (163.5 cM), largely due to medium andlong IBD. The Agin Buryats have a much higher proportion ofshort IBDs (16–30–28–19–7 %) than the Kurumkan Buryats(9–24–29–25–13 %). Despite the fact that for the Buryats,who have a large total population, with their numbers almostdoubling from 237 thousand in 1926 to 461 thousand in2010, this difference between populations can be explainedby a sharp increase in the population of the village of Aginskoyefrom 451 people in 1908 up to 4556 people in 1939and 15 thousand in 2010, and the stability of the population ofthe village of Kurumkan, with a population of 5617 people in1979 and 5465 in 2010, located in one of the remote regionsof Buryatia.A similar dynamics is observed in the populations of theChukchi (20–36–22–12–11 %), Koryaks (24–33–17–11–15 %), Nivkhs (21–38–21–13–7 %) and Kalmyks (29–39–22–7–3 %), followed by a sharper decrease in the proportionof long IBD. The Chukchi, Nivkhs and Koryaks, which havea small population (up to 13 thousand), are characterized bythe absence of sharp fluctuations in the number of populationsover the past hundred years with an increase of 29–9–6 %,respectively, which could also contribute to a reduction in thetotal long IBD segments subject to a small number of closelyrelated marriages within the populationA total of 189,314 SNPs were obtained for 20 populations,falling into the largest number of IBD segments of the analyzedpopulations .Of these, 88,530 SNPs are located in intergenic regions, therest are located in the region of 5358 genes. Table 2 shows thegenes that were shown in four or more populations.From the list of 5358 genes most frequently found inIBD blocks, 1694 were annotated using the KEGG databaseaccording to WebGestalt. As a result, analysis taking intoaccount the Benjamini–Hochberg correction (FDR = 0.05)revealed molecular KEGG pathways enriched in these genes:the linoleic acid pathway hsa00591 (FDR = 0.0051) including17 genes , the arachidonic acid pathwayhsa00590(FDR = 0.0240) including 27 genes , tyrosine metabolism pathwayhsa00350 (FDR = 0.0240) including 18 genes , olfactory transduction pathwayhsa04740 (FDR = 4.55E-08) including 159 genes.The metabolic conversion of polyunsaturated fatty acids(PUFAs) such as linoleic acid into biologically active longchain PUFAs  such as arachidonicacid is essential for proper metabolism. LC-PUFAs and theirmetabolites are important structural and signaling componentsfor numerous biological systems, including brain developmentand function, innate immunity, and energy homeostasis. There are also foodsources of preformed LC-PUFAs in eggs and some meatscontaining arachidonic acid . The patterns found in thedistribution of IBD containing genes involved in fatty acid metabolism may indicate recent directional selection associatedwith adaptation to dietary habits in cold climates orreflect the influence of a Western diet .Positive selection in genes that affect the level of LC-PUFAs,as well as the metabolic efficiency by which LC-PUFAs areformed in populations of the Pygmies on Flores Island , Greenland Inuit  andNative Americans ,is also thought to be associated with dietary habits in coldclimates, although the exact selection pressure is unknown. An example is the similarity ofgenotypes in the genes of the olfactory system, which mayplay a role in the formation or maintenance of social bondsbetween individuals within a population . For such genotypes, higher rates of positive selectionhave been found .The analysis of gene ontologies  showed nine statistically significant biologicalprocesses (taking into account the Benjamini–Hochbergcorrection (FDR = 0.05)) associated with the detectionof a chemical stimulus involved in the sensory perceptionof smell .An analysis of the literature also revealed that a number ofgenes that fall into IBD blocks can play a significant role inthe formation of oncology and influence the treatment. For example, the AAGAB gene is included in the IBD blocks infive populations. AAGAB consists of 10 exons encoding a315 amino acid (aa) protein, the AAGAB  protein. AAGAB is widely expressed andinteracts with the gamma-adaptin and alpha-adaptin adapterprotein complexes, AP1 and AP2. It is involved in membranetransport and plays a role in endocytosis and protein sorting.Heterozygous AAGAB mutations cause pitted palmoplantarkeratoderma type 1 (PPKP1), a skin disease characterizedby punctate hyperkeratosis of the palms and soles . AAGAB is also a promising biomarker for chemotherapyresponse and outcome during breast cancer treatment.However, the exact role of AAGAB in the development ofbreast cancer is currently unclear and potentially requiresfurther study .Another gene, GSE1, which falls into IBD blocks in fivepopulations, may function as an oncogene in breast, stomach,and prostate cancer, and may also be important in the treatmentof patients with prostate cancer .IQCH-AS1 encoding antisense RNA IQCH 1 (IQCH-AS1)correlates with survival and diagnosis of cancer patients, butits role in the development of thyroid cancer and doxorubicinchemosensitivity remains unclear .The role of SMAD3 in the regulation of genes important forcell development, such as differentiation, growth and death,implies that changing its activity or suppressing its activitycan lead to the formation or development of cancer.Also, some of the genes presented in Table 1 may play a rolein human adaptation to environmental factors. For example,the IQCH gene may play a regulatory role in spermatogenesis and is also associated with adult growth inMongols .The CHRNA4 gene encodes for the α4β2 subcomponentof nicotinic receptors in the human brain. Individuals withcertain CHRNA4 genotypes have been shown to be better attracking and identifying multiple objects in visual search tasks. Polymorphisms in the CHRNA4 genealso seem to contribute to personality development by affectingthe degree of developmental sensitivity to both normal andadverse environmental conditions .The COL20A1 gene encoding type XX alpha 1 collagen isnoted in a number of genes with non-synonymous changeswith a high frequency in modern humans compared to archaichominids, which probably contributed to the developmentof unique human traits and is an interesting object for study.Thus, as a result of our study, new information was obtainedon the structure and composition of the gene pools of theindigenous peoples of Siberia, their genetic relationships andgenetic and demographic processes based on an analysis of thedistribution of linkage blocks identical in origin. The resultsobtained demonstrate the clustering of Siberian populations,linking peoples by place of origin, demonstrating a commonorigin and a high degree of kinship. The populations inhabitingthe territory of Siberia are characterized by a unique geneticand demographic history, which is reflected in the distributionof IBD blocks both within the population and betweenthem. The analysis of IBD blocks significantly complementsthe study of the formation and interaction of ethnic groups,but does not provide unambiguous answers for populationsdeveloping under conditions of complex ethnogenesis. Withfew exceptions, the overall IBD in Siberia is better explainedby the geographical proximity of the populations rather thanby their linguistic affiliation.Analysis of SNPs that fell into more IBD segments of theanalyzed populations made it possible to compile a list of5358 genes. According to the results of calculations, biologicalprocesses enriched in these genes are associated with thedetection of a chemical stimulus involved in the sensoryperception of odor. 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Homo sapiens originated in Africa, considerable uncertainty surrounds specific models of divergence and migration across the continent1. Progress is hampered by a shortage of fossil and genomic data, as well as variability in previous estimates of divergence times1. Here we seek to discriminate among such models by considering linkage disequilibrium and diversity-based statistics, optimized for rapid, complex demographic inference2. We infer detailed demographic models for populations across Africa, including eastern and western representatives, and newly sequenced whole genomes from 44 Nama (Khoe-San) individuals from southern Africa. We infer a reticulated African population history in which present-day population structure dates back to Marine Isotope Stage 5. The earliest population divergence among contemporary populations occurred 120,000 to 135,000 years ago and was preceded by links between two or more weakly differentiated ancestral Homo populations connected by gene flow over hundreds of thousands of years. Such weakly structured stem models explain patterns of polymorphism that had previously been attributed to contributions from archaic hominins in Africa7. In contrast to models with archaic introgression, we predict that fossil remains from coexisting ancestral populations should be genetically and morphologically similar, and that only an inferred 1–4% of genetic differentiation among contemporary human populations can be attributed to genetic drift between stem populations. We show that model misspecification explains the variation in previous estimates of divergence times, and argue that studying a range of models is key to making robust inferences about deep history.Despite broad agreement that  An analysis of models of human populations in Africa, using some newly sequenced genomes, finds that human origins in the continent can best be described by a weakly structured stem model. Archaeological sites from the Middle Stone Age, of which some have been associated with H. sapiens, are also widely distributed across Africa. It is unclear whether these fossils and archaeological sites represent populations that contributed to contemporary H. sapiens as population precedents or were local ‘dead ends’. Attempts to reconcile genetic and palaeoanthropological data include proposals13 for a pan-African origin of H. sapiens in which populations in many regions of the continent contributed to the formation of H. sapiens beginning at least 300 ka.Decades of study of human genome variation have suggested a predominantly tree-like model of recent population divergence from a single ancestral population in Africa. It has been difficult to reconcile this finding with the fossil and archaeological records of human occupation across the vast African continent. For example, fossils such as those from the sites of Jebel Irhoud in Morocco14 or population coalescence and fragmentation13, but these approaches are more challenging to interpret and fit to data. However, new population-genetic tools now allow for inference on the basis of tens to hundreds of genomes from multiple populations and for greater model complexity16. Inspired by evidence for Neanderthal admixture with humans in Eurasia, several studies have shown that introducing an archaic hominin ‘ghost’ population contributing to African populations in the period surrounding the out-of-Africa migration event substantially improves the description of genetic data relative to single-origin models, mostly in western Africa7, but also in southern6 and central African17 populations. This has driven speculation about the geographical range of the ghost population, possible links to specific fossils and the possibility of finding ancient DNA evidence17. However, these studies share two weaknesses. First, they contrast only a single-origin model with an archaic hominin admixture model, leaving out other plausible models1  a tree-like model of isolation with migration. Alternative theoretical scenarios have been proposed, such as stepping-stone modelsstry Fig.. AncientWe therefore aim to discriminate between a broader set of demographic models by studying the genomes of contemporary populations. We take as our starting point four models , the Gumuz (recent descendants of a hunter-gatherer group from Ethiopia21) and eastern African agriculturalists (Amhara and Oromo from Ethiopia20). The Amhara and Oromo populations, despite speaking distinct Afro-Asiatic languages, are highly genetically similar22 so we combined the two groups for a larger sample size . We further find that back-to-Africa gene flow at the beginning of the Holocene epoch primarily affected the ancestors of the Ethiopian agricultural populations26, comprising almost 65% of their genetic ancestry. We observe considerable gene flow from the Amhara and Oromo into the Nama, a signal that is probably a proxy for the movement of eastern African caprid (goat) and cattle pastoralists28, here estimated to constitute a 25% ancestry contribution 2 ka. Although this gene flow is not apparent from the ADMIXTURE plot   Fig.  during tm) in each generation estimated as m = 6.3 × 10−5–1.3 × 10−4. For comparison, this is similar to the inferred migration rates between connected contemporary populations over the past 50 ka  compared with the Nama and populations from eastern Africa , with migration allowed to occur until 5 ka. A sampled lineage from the Nama, Mende and Gumuz have probabilities of being in stem 2 at the time of stem 1 expansion (135 ka) of approximately 0.145, 0.20 and 0.130, respectively, although these probabilities change over time, precluding the notion of a fixed admixture proportion.Under the continuous-migration model, one of the two stems (stem 1) diverges into lineages leading to contemporary populations in western, southern and eastern Africa, and the other (stem 2) contributes variable ancestry to those populations. This migration from stem 2 is highest with the Mende , although the size of this bottleneck is poorly constrained . After a long period of exchange with stem 2, stem 1 then fractures into stem 1E and stem 1S at 478 ka. The timing of this divergence was also poorly constrained . These populations evolve independently until 119 ka (101−125 ka) when stem 1S and stem 2 combine to form the ancestors of the Nama, with proportions of 30% and 70%, respectively. Similarly, stem 1E and stem 2 combine in equal proportions (50% each) to form the ancestors of the western and eastern Africans . Finally, the Mende receive a large additional pulse of gene flow from stem 2, replacing 19% (18–21%) of their population 25 ka (22−26 ka). The later stem 2 contribution to the western African Mende resulted in better model fits . This may indicate that an ancestral stem 2 population occupied western or central Africa, broadly speaking. The differing proportions in the Nama and eastern Africans may also indicate a geographical separation of stem 1S in southern Africa and stem 1E in eastern Africa.By contrast, in the multiple-merger model, stem populations merge with varying proportions to form the different contemporary groups. We observe a sharp bottleneck in stem 1 down to To assess the robustness of the inferred models to analysis and reference population choices, Supplementary Information sections 17, conditional site frequency spectra (cSFS)7 and the reconstruction of gene genealogies16. However, none of these studies considered a weakly structured stem. We validated our inferred models with additional independent approaches. We find that the observed cSFS  is well described by the merger model . Specifically, it is the addition of migration between stems that results in a qualitative improvement of the agreement , and recent migration can lead to the misordering of divergence events  have recently been used to estimate divergence between pairs of populations, as measured by the rate of coalescence between two groups divided by the mean within population coalescence. Simulations of RCCR accuracy, however, focus on a clean split between populations, whereby groups diverge without subsequent gene flow. Published estimatesnts Fig. . We sugg25 have fitted tree-like demographic models to African populations using distributions of allele frequencies or related statistics, finding inconsistent divergence times, some of which are older than those we find here. In Supplementary Information section 32.Other studiesAny attempt to build detailed models of human history is subject to model misspecification. This is true of previous studies, which often assumed that data inconsistent with a single-origin model should be explained by archaic hominin admixture. It is also true of this study. Although it is difficult to fully explore the space of plausible models of early human population structure, we sought to capture uncertainty in the model by exploring multiple parameterizations of early history. The best-fit models presented here include reticulation and migration between early human populations, rather than archaic hominin admixture from long-isolated branches Fig. . Element13. Other African populations, such as those from Central Africa, other Khoe-San groups or pre-Holocene ancient DNA samples, could further test our proposed models.We cannot rule out the possibility that more-complex models involving additional stems, more-complex population structure, or hybrid models including both weak structure and archaic hominin admixture, may better explain the data. Because parameters related to the split time, migration rates and relative sizes of the early stems were variable across models, reflecting a degree of confounding among these parameters, we refrained from introducing additional branches associated with more parameters during that period. Rather than interpreting the two stems as representing well-defined and stable populations over hundreds of thousands of years, we interpret the weakly structured stem as consistent with a population fragmentation-and-coalescence modelOur inferred models paint a consistent picture of the Middle to Late Pleistocene as a critical period of change, assuming that estimates from the recombination clock accurately relate to geological chronologies , indicating that this gene flow probably occurred in western and/or central Africa Table . Such an7 and assuming (or inferring) deep divergences. These perspectives have oriented interpretations of both genomic  and fossil (such as the evolution of early H. sapiens40) data. Here we have shown that a weakly structured stem model better captures the apparently inconsistent patterns of polymorphisms.Evidence for archaic hominin admixture in Eurasia has bolstered the plausibility of archaic hominin admixture having also occurred in Africa. Previous work that sought to explain patterns of polymorphism inconsistent with a single-origin model therefore focused on archaic hominin admixture as an alternative model, by referring to additional (ghost) branches required to fit the data as archaicPreferring models of a weakly structured stem to archaic-admixture models has a range of implications. First, with a weakly structured stem, there is no need to posit that an archaic hominin population in Africa remained reproductively isolated from the ancestral human lineage for hundreds of thousands of years before the initiation of gene flow. Instead, there would simply have been continuous or recurrent contact between two or more groups present in Africa.41, or an increased frequency of archaic-hominin-related haplotypes such as at EPAS1 among Tibetan people42. Under previous African archaic-hominin admixture models, the estimated 8–10% introgression rate is much higher than Neanderthal gene flow and would have plausibly been fertile ground for considerable selection for or against archaic-hominin-derived haplotypes39. By contrast, adaptation under a weakly structured stem would have occurred continuously over much longer periods. Polymorphism patterns that are inconsistent with the single-stem model predictions have been used to infer putative archaic admixed segments39, negative selection against such segments39 and pervasive positive selection43. However, such approaches are subject to large numbers of false positives in the presence of population structure with migration41, and their interpretation should be re-examined in the light of a weakly structured stem model within Africa.Second, there is evidence for both deleterious and adaptive archaic-hominin-derived alleles in contemporary genomes in the form of a depletion of Neanderthal ancestry in regulatory regions46 47, Ishango in the Democratic Republic of Congo (20–25 ka)48 and Nazlet Khater in Egypt (35–40 ka)49, have morphological features that may reflect recent gene flow from archaic hominins48, and have been used in support of previously inferred archaic admixture scenarios25. The weakly structured stem model is not incompatible with archaic admixture having occurred in the ancestry of these fossils, but would imply, by contrast, that such individuals are unlikely to have contributed much ancestry to contemporary humans. The fossil record of Africa is sparse during the earlier time period of the stems (≳200 ka). Of the fossils that date to this period, some are fairly similar overall in morphology to contemporary humans , whereas others are similar in some morphological features to contemporary humans ; others are different enough in morphology to have been assigned to species other than H. sapiens . If, as our model predicts, the genetic differences between the stems were similar to those among contemporary human populations, the most morphologically divergent fossils are unlikely to represent branches that contributed appreciably to contemporary human ancestries.Third, multiple studies have shown a correspondence between phenotypic differentiation, usually assessed by measurements of the cranium, and genetic differentiation among human populations and between humans and Neanderthals22, as well as the 1000 Genomes Project (1KGP) populations18. These included the Amhara, Bakiga, Gumuz, Nama (newly generated), Oromo, Somali and Zulu populations from the ADRP and ESN, GWD, LWK, MSL, YRI, CEU, GBR, CHB and PJL from 1KGP (these groups are defined elsewhere18). After filtering for relatedness and retaining Nama individuals with more than 70% estimated Khoe-San ancestry, we focused on data from 289 individuals, including 44 Nama. These were merged with the high-coverage Neanderthal genome from Vindija Cave23. We kept variants from regions that fell within the 1KGP strict-callability mask, overlapped with at least 100 continuously called base pairs in the Neanderthal genome and were annotated as intergenic. ADMIXTURE and principal component analyses were done on a subset of variants filtered to remove variants in high linkage disequilibrium (r2 threshold of 0.1). Additional details on the data and sequencing are available in Supplementary Information section We generated a sequencing dataset by combining existing and newly recruited populations who are now part of the African Diversity Reference Panel (ADRP)2. Unbiased linkage disequilibrium statistics were computed from all variants in retained intergenic regions24, for pairs of variants separated by recombination distances r = 5 × 10−6–5 × 10−3 Morgans. These were assigned to 16 recombination distance bins, and average statistics were computed within each bin (Supplementary Information section 18.We used multi-population linkage disequilibrium and pairwise diversity statistics to fit parameterized demographic models to the dataModel parameters include population sizes and size changes, split times, continuous migration rates and admixture times and proportions. The simplest model we tested was a bifurcating tree-like structure, allowing for subsequent migrations and recent known admixture events. To include ancestral population structure, we tested models that included multiple stem groups, each of which were allowed their own population size and could be connected by continuous migration. We tested multiple scenarios of early population structure, including long-lasting continuous migration between stem populations and scenarios of periods of isolation with subsequent merger events (Supplementary Information section 16 to reconstruct genome-wide gene genealogies from the focal populations in the merged ADRP and 1KGP datasets (Supplementary Information section 56 to simulate genomic data for equal numbers of samples for each population in our inferred models. We then applied Relate to these simulated datasets using the same mutation and recombination rates and generation time.We used RelateFurther information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-06055-y.Supplementary InformationThis file contains further discussion, methods and data, Supplementary Tables S1–S8, Supplementary Figs. S1–S40, and Supplementary References.Reporting SummaryPeer Review FileSupplementary DataThis zipped file contains inferred demographic models in Demes format for all models presented in the main text and all alternative models used in validation of our main results that are discussed in the Supplementary Information.


Salmo trutta complex) populations, we analyzed both mitochondrial DNA sequences and genotypes at 10 microsatellite loci of fish caught in the Black Sea and from nine river catchments in Georgia, flowing into either the Black or Caspian seas. The results show that: (1) there is substantial genetic differentiation among Ponto‐Caspian trout populations, both among the populations of different nominal species and within those of the same species; (2) the genetic distance between conspecific populations from the Black and Caspian Sea basins exceeds that among the populations within the same basin. Moreover, within drainages, genetic distance correlates with the geographic distance; (3) the Black Sea itself is not a barrier to gene flow among the watersheds draining into the Black Sea; (4) some populations in the headwaters of the rivers draining into the Black Sea Basin fall out of this pattern and likely form a separate, non‐anadromous (resident) taxon, previously described from northeastern Turkey as Salmo rizeensis. This hypothesis is supported by mitochondrial DNA phylogeny. The presence of both anadromous and resident populations in a single river basin calls for a substantial re‐thinking of speciation patterns and taxonomy of Eurasian brown trout.To assess the genetic structure of Ponto‐Caspian brown trout ( Salmo rizeensis from NE Turkey and distinct from Salmo labrax dominated in the Black Sea basin.The analysis of 238 samples of brown trout collected from 19 Georgian locations was studied by means of mitochondrial DNA sequencing and population genetic analysis based on the study of 10 microsatellite loci, showed intensive gene flow between drainages of the rivers flowing into the Black Sea, but not between the Black and Caspian Sea basins. The study identified a riverine form of brown trout, matrilineally monophyletic with  Subsequently, Black and Caspian Sea salmonids were recognized as anadromous forms of brown trout, originating from the drainages of these basins , distinct from related fish from the Mediterranean, Persian Gulf, and Atlantic drainages. This interpretation was later supported by the genomic data of Segherloo et al.  some genetic factors of anadromy have been identified  haplotypes were analyzed in tandem with 10 microsatellite markers. We addressed four working hypotheses: (1) the populations from the Black and Caspian Sea drainages are isolated. This hypothesis is supported based on prior mitochondrial DNA analysis . Permits were obtained from the Ministry of Environmental Protection and Agriculture of Georgia  and from the Agency of Protected Areas . We collected tissue samples of 238 individual fish from 13 locations in the Black Sea basin, and six locations from the Caspian Sea basin. The locations from the Black Sea basin were small rivers (1) Chvana and (2) Akavreta (riv. Chorokhi (Coruh) drainage); (3) river Chakvistskali; (4) river Kintrishi; (5) coastal area of the Black Sea; rivers (6) Shareula, (7) Krikhula, and (8) Sakraula (drainage of the riv. Rioni); (9) Tsentskali ; rivers (10) Magana, (11) Khuberi, (12) Nenskra, and (13) Dolra (drainage of the riv. Enguri). The locations from the Caspian Sea basin were (14) riv. Snostskali (drainage of the river Tergi (Terek)); (15) Khiso's Alazani (drainage of the river Sulak); (16) river Aragvi, (17) river Ilto, (18) river Iori, and (19) river Ktsia (drainage of the river Kura (Mtkvari)). From each location, 4–21 samples (12.5 on average) were collected , and the unique sequences were deposited to GenBank (accessions # OP849668–OP849680). For phylogenetic analyses, an 841‐bp‐long region of cytochrome b was used. The sequences were obtained from 83 individuals collected in Georgia, and aligned with the 44 published homologous sequences of brown trout from western Eurasia, including those from the Ponto‐Caspian basin . After 3 min denaturation at 95°C, samples were sequenced on ABI 3031xl genetic analyzer. For analysis, we used GeneMapper vers. 5.0 (ThermoFisher). We used the software MICRO‐CHECKER ver. 2.2.3  using the software BEAST ver. 2.6.3 , to visualize clustering of the populations in PCA space. The software used was SPSS 23.0 for Windows  was set from 1 to 19 with five independent runs for each. The most probable K was chosen using the online tool Structure Harvester  between geographic populations of the Black Sea versus Caspian Sea basins and (2) among individual populations within each of the two sea basins, Caspian and Black, separately. In addition, we calculated the pairwise RST value  were evaluated using Arlequin ver. 3.5  and the sum of squared differences (RST‐like), for validating the concurrence of the estimates based on these two metrics.Finally, population genetic structure was evaluated by analysis of molecular variance (AMOVA) using ARLEQUIN ver. 3.5.2.2  and six groups .2.5Rxy and the significance level p between two distance matrices: geographic riverine distances among the studied locations and genetic distances calculated as FST  and RST . Since populations from the of the Black and the Caspian basins appear to be effectively isolated, even if they are geographically close  in contrast with the previous study, which did not discover the presence of haplotypes clustering with nominal S. rizeensis in Georgia, the current study identified a resident population from the upper region of the Rioni, which clustered with high statistical support with S. rizeensis from the Chorokhi headwaters in Turkey  and that from Sevan Lake in Armenia  occupy a separate position, distinct from the haplotypes of Caspian Sea origin, Kura and Sulak River drainages, and from northern Iran samples, which share the same cytochrome b haplotype. The haplotypes from the Black Sea drainage are distributed among three most common haplogroups, shared among the specimens from the Black Sea and the main rivers  flowing into it from the east, and nine rare haplotypes different from the “main” ones with one, two, or three mutated positions. The most deviated cluster of haplotypes from the Black Sea drainage is that marking nominal S. rizeensis from the Chorokhi drainage, and closely related to it, haplotypes from Shareula  with the riverine distance from the Black Sea coast. Genotype frequencies in none of the studied locations deviated significantly from Hardy–Weinberg equilibrium expectations.There was no significant evidence for the presence of null alleles and large allele dropout for any population/locus. The number of alleles per locus within populations varied from 6 (for locus BG935388) to 34 (for locus SSsp2201). The mean allelic richness was the lowest (2.8) for the population from Chvana  separation of the individuals from the Black and Caspian Sea basins along the first PCA axis, with the highest loadings on loci Ssa408Uos and BG935388; (2) the second axis, with the highest loading on the locus Ssa410Uos, separated genotypes of fish from the drainage of Tergi (location 14), Sulak (15) and Kura (16–19); and (3) the second axis separated genotypes from Shareula (location 6), Chvana (location 1), Akavreta (location 2), and Dolra (location 13) from the genotypes from other locations of the Black Sea basin . When the populations from Shareula and Chvana, which showed the highest genetic differentiation from the other populations of the Black Sea basin, were excluded from the Black Sea cluster, the pairwise FST and RST values dropped to 0.189 and 0.239, and when the populations of nominal S. ciscaucasicus were excluded from the Caspian Sea cluster, the pairwise FST and RST values dropped to 0.145 and 0.140. In most cases, the differences among the local populations are significant after stepwise Bonferroni correction is applied  between this population and the other populations from the Rioni Basin was 0.300, which substantially and significantly exceeded that for most of the other populations. Remarkably, fish from Shareula had a haplotype clustering with that of the nominal resident S. rizeensis. There was also another population (Chvana) quite distant from the other populations of the Black Sea drainage (average FST = 0.347).The fish collected in the Black Sea were closest genetically to the populations from the small rivers entering the sea  explained by the differences among the groups; this was lower than the variance between populations of the same group, especially in case of FST‐based calculation  clustered with mitochondrial haplogroup of S. rizeensis. In this case the component of the between‐basin variation FCT increased from 0.077 to 0.083 (FST) and from 0.209 to 0.252 (RST).AMOVA analysis results are shown in Table on Table . We repe3.3FST values for the populations from the Black Sea drainage: . For the Caspian Sea drainage, correlation was higher . Correlation of RST values with geographic distance was lower  than the correlation of FST with geography for the Black Sea drainage, but higher  for the Caspian Sea drainage. Significant correlation between the geography and genetics for those locations that are not in the same river drainage means that the Black Sea is not an impenetrable barrier to migration among populations from different rivers. Moreover, the genetic distance between the fish found in the Black Sea or that from the locations of different rivers close to the sea was commonly smaller than the genetic distances among distant populations from within the same river basin. In other words, isolation by distance appears to be a stronger driver of genetic differentiation than water salinity/transit through the Black Sea.There was significant positive correlation between the ln‐transformed geographic (riverine) distances and pairwise 4Addressing the first hypothesis formulated in the introduction of this paper, we suggest that the populations of brown trout from the Black and the Caspian Sea basins are genetically isolated from one another. Previously, this hypothesis was confirmed based on mitochondrial DNA analysis , from the south , S. ischchan from Lake Sevan, and Salmo oxianus from the Aral Sea drainage. Our current results, based on the mitochondrial sequences, do not contradict these findings in the synonymization of S. ciscaucasicus and S. caspius. However, microsatellite profiles of the fish from the drainages of the rivers Tergi and Sulak are distinct related to the fish from a larger Kura drainage; besides, these forms are phenotypically more distinct than some species recognized by Segherloo et al.—see Ninua et al. , is usually anadromous and rarely produces resident forms; however, resident forms of S. salar are landlocked and isolated from the anadromous fish, most likely, since early Holocene  from the Pacific Ocean; in this species, multiple genes are identified that influence life history; anadromy can be present or absent within the same polymorphic populations, depending upon unique constellations of alleles aggregating in individuals  is ancestral form related to the residential rainbow trout, and transition to the resident freshwater life history is secondary rather than primary in this group.The genetic basis of anadromy is better studied in rainbow trout  and resident S. rizeensis, marked with a distinct mitochondrial haplogroup. Ninua et al. (S. labrax) than reciprocally monophyletic brown trout haplogroups from the Black and the Caspian/Aral Sea drainages. These observations and the analysis of recombinant alleles reported in this paper support the maintenance of genetic differences between resident and anadromous trout.Much less is known about the variation in life history and reproductive strategies of brown trout from the Ponto‐Caspian drainages, including wide‐spread Sabaneev  considerSabaneev . Turan eSabaneev  was the a et al.  showed tFST of each of these populations to the Black Sea population is 0.23–0.26, whereas the distance of the other 10 river populations to the Black Sea population varies between 0.01 and 0.18, depending on the distance from the sea coast. These distances are comparable with the genetic distance between S. labrax and S. caspius. One of these populations (Shareula) is marked with a mitochondrial haplotype that clusters with S. rizeensis , showing high genetic distances, both from each other and from other trout populations from the Black Sea drainage. The pairwise S. rizeensis by Turan et al. (S. trutta) and the differences between rainbow trout and steelhead (O. mykiss), the presence of a similar pattern in S. labrax is not surprising. Haplotype divergence between S. rizeensis and “S. coruhensis” described in the paper of Turan et al.  are effectively isolated from the rest of the populations from the Black Sea basin, and are probably genetically determined residential forms similar to that described as n et al. . Considen et al.  suggestsn et al. . AnotherS. labrax coexists with resident form, sharing a mitochondrial haplogroup with S. rizeensis from northeastern Turkey. A better understanding of the genetic background and palaeoecological factors that triggered the divergence between the resident and anadromous trout in this area can be achieved with further genomic studies of the group.In conclusion, our study shows that brown trout from the studied area is comprised of several distinct genetic clusters, coinciding with both separation of the sea basins and the differentiation of life histories. Fish from the Caspian Sea basin shows higher genetic differentiation among the river catchments, whereas in the Black Sea basin anadromous Levan Ninua: Conceptualization ; data curation (lead); formal analysis ; investigation (lead); methodology ; project administration (supporting); visualization ; writing – original draft ; writing – review and editing . David Tarkhnishvili: Conceptualization ; formal analysis ; methodology ; supervision (supporting); visualization ; writing – original draft ; writing – review and editing . Cort Lewis Anderson: Conceptualization ; methodology ; project administration (lead); supervision (lead); writing – review and editing (supporting).None declared.


Quadrivisio lutzi inhabitscoastal lagoons, highly unstable environments subject to sudden inflow of marinewater. Our aim was to evaluate how the genetic composition varies in thesepopulations. Brazilian populations were compared by 16S rRNAand COI gene sequences. The genetic structure of four Rio deJaneiro amphipod populations was evaluated during the period of 2011-2019 byCOI. Rio de Janeiro population was compared with Alagoasand São Paulo populations, which was genetically distinct, at species level. The genetic structure in Rio de Janeiro showed the Imboassicasubpopulation as the most divergent , followed by Lagamar population . The geographic distance and urbanization around these lagoonsexplain the degree of genetic isolation of these amphipod subpopulations.Paulista and Carapebus populations were not structured. Temporal variation inhaplotype number and frequency were evident in both populations that wereevaluated (Carapebus and Imboassica). Changes in salinity and water volumevariation at these lagoons may be responsible for the observed changes ingenetic composition, which may be the results of genetic drift effects overtemporally fluctuating size subpopulations, without loss of geneticdiversity.The freshwater/brackish amphipod  Complex coastal lagoon systems are observed along the Brazilian coast . At the Quadrivisio lutzi , and 16S rRNA (16S) mitochondrialgenes were performed by polymerase chain reaction (PCR) using universal primersand primers designed specifically for Q. lutzi . PCR reactions were submitted in aMastercycler gradient thermocycler  to thefollowing cycles: 1 cycle at 94 ºC for 4 min; 36 cycles for 1 min at each of thefollowing temperatures: 94 ºC, 48 ºC -59 ºC (COI) and 52 ºC-57ºC (16S) and 72 ºC; and one final cycle at 72 ºC for 10 min.All PCR products were purified and sequenced by Macrogen Inc., Korea, using theautomated Sanger dideoxide method.Amplifications of the cytochrome Q. lutzi . PCR reaCOI sequences was done by aligning withDaphnia pulex (Accession No. NC000844) and Parhyalehawaensis (Accession No. NC039402) COI gene, usingthe 16S, OQ361834-OQ361842;COI, OQ401341-OQ401385).Sequences were edited with ChromasPro  and Gene16S and COI genes, using MEGA 11 software.Trees were constructed based on maximum likelihood (ML) and Bayesian inference(BI) at the 16S, COI and concatenated datasets, using evolutionary models determined by jModelTest 2.1 , Maeridae; Quadrimaerapacifica (Accession No. AB432980), Maeridae; and Gammaruspulex (Accession No. AJ269626), Gammaridae. ForCOI: E. nkjaf ; Melita nitida ,Melitidae; and G. pulex (Accession No. MN400977).COI trees were performed only for Alagoas and Rio deJaneiro populations, because it was not possible to obtain more amphipods fromUbatuba, São Paulo, although sampling efforts were made. A heuristic search ofthe ML tree was performed using Garli 2 software  was evaluatedusing  network  was perf network  for amph network , showingArlequin . The genArlequin  and Fu  and to São Paulo populations. Alagoas and São Paulo were the most divergent  populations. The high divergence observedbetween Rio de Janeiro and Alagoas population was confirmed withCOI gene sequences (d = 0.1472).The degrees of divergence among the Brazilian populations of equences  which shFST and diversity parameters for the four studied populations areshown in d = 0.0009 ± 0.0002.Imboassica showed a significantly high FST from all other populations (0.163-0.238), showing that Rio deJaneiro population is structured. Paulista amphipod population did not showsignificant differences from Lagamar and Carapebus; and Lagamar showedsignificant, but low level of structuring with Carapebus . Other lagoons from which a few amphipodswere collected in previous sampling events, but in which they were no longerfound , were not included in theanalysis. Pairwise genetic distance, arapebus .A total of 22 haplotypes with a total of 24 segregating sites of which 10 wereparsimony informative, were found at Rio de Janeiro population. The mostfrequent haplotype (H1) was represented at all subpopulations . Each suFST, showed that Imboassica amphipod subpopulation increased itsdivergence from Lagamar and Carapebus-Paulista subpopulations from 2016 to 2018;and Carapebus diverged significantly from Lagamar and Paulista in 2019, while nosuch differences were show previous years  and Carapebus subpopulation of the year 2015 showed deviationfrom neutrality.Diversity parameters (haplotype and nucleotype diversity) also changed duringtime in Carapebus and Imboassica subpopulations, increasing in 2018/2019compared to 2015/2016 . In moreQuadrivisio. The levels of divergence found betweenthem are higher than those found at 16S locus for conspecificcrustacean populations  also support themultispecific status of the genus Quadrivisio.COI gene has great potential to complement traditionaltaxonomy in the identification of crustacean species  with high levels of divergence, a strong indication of thepresence of more than one species in Brazil for the genusto 25.7% . The hig species . In accoQuadrivisio  in the years of 2018/2019 compared to previous years.The genetic changes were evident on the increased diversity of haplotypes andthe change of the most common haplotype at each subpopulation. Deviation fromneutrality indicates population expansion at Carapebus in 2015‚ which predictlarge population size. Population growth determine the increase of amphipodswith rare haplotypes, therefore retaining diversity , when la2) than Carapebus lagoon . During 2014 and most of 2015, survivingand newborn amphipods had to live in areas close to the sea under a salinityrange of 8-13 ppt, on which they are able to osmoconform (unpublished data). Atthe end of 2015, salinity dropped, and amphipods needed to activate thehyper-regulation system, which would have demanded time and energy, causingprobably population size reduction in the amphipod at Carapebus lagoon.In both lagoons, the change of the most frequent haplotype may have happened by adrastic temporarily reduction in population size, followed by a sweepstakeschance event that led to the increase in frequency of new dominant haplotypesmainly due to the effect of genetic drift. At Imboassica, certainly the suddenlagoon volume reduction and salinity increase may explain the severe reductionin population size. However, for Carapebus subpopulation, may not be the case.The environmental instability caused by the sudden intrusion of seawater inCarapebus and Imboassica lagoons has driven changes in fish assemblage . EuryhalWe do not understand exactly how and when different mechanisms of osmoregulationare activated in the new born amphipods or in adult amphipods, which remainedmost of their life in a specific level of salinity. Therefore, we cannot ruleout completely the possibility that selection may have taken place whenpopulation size was still elevated, acting against amphipods not well adapted toa specific new salinity regimen.Q.lutzi by genetic drift acting over a fluctuating population size,which causes changes in haplotype frequencies, without diversity loss.The instability in coastal lagoons due to strong water volume and salinityvariations has driven changes in the genetic composition of The high diversity and endemism observed in coastal lagoons  and the


Sphagneticola trilobata (L.) Pruski is a prevalent and widely distributed invasive plant in South China. To investigate the molecular mechanisms underlying its rapid adaptation, we employed DNA methylation-sensitive amplified polymorphism (MSAP) and simple sequence repeat (SSR) analysis to study 60 S. trilobata individuals collected from Fuzhou (FZ), Haikou (HK), Jinghong (JH) and Guangzhou (GZ).In this study, we computed the Shannon diversity index (I) of SSR and MSAP as 0.354 and 0.303, respectively. The UPGMA phylogenetic tree and PCoA analyses showed that MSAP had a better discriminatory power to distinguish populations from different regions. Notably, the GZ population was found to be the most distinct from the other three populations. Moreover, Mantel analysis revealed a significantly higher correlation between epigenetic distance and geographic distance as compared to genetic distance and geographic distance. Consequently, the correlation between epigenetic distance and geographic distance observed to be markedly stronger than that between genetic distance and geographical distance on Mantel analysis.S. trilobata populations in various regions displayed a high of complementary genetic and epigenetic diversity, which was a key feature contributing to their rapid invasion. Interestingly, the correlation between epigenetics and geographical distance was significantly stronger than that observed for genetics and geographical distance. These findings indicated that the epigenetic mechanism of S. trilobar exhibited high plasticity, leading to significant differences in methylation pattern across different populations.The The online version contains supplementary material available at 10.1186/s12870-023-04277-w. Flaveria bidentis was introduced from Tianjin and quickly spread throughout in northern China. Its ability for sexual reproduction and multiple introductions have led to the maintained of high levels of genetic diversity, which in turn has facilitated local adaptation and invasion [With the globalization of the economy and trade, the rapid expansion in the scope and frequency of human activities has led to the ubiquity of invasive plants, resulting in significant economic losses and ecological damage on a global scale. Genetic and evolutionary processes are believed to be key determinants in the establishment and spread of invasive species. Invasive species must adapt to their new environment in order to survive and reproduce , and geninvasion . On the Sphagneticola trilobata (L.) Pruski , belongs to the Asteraceae, a perennial herb native to tropical South America [S. trilobata was introduced to China as a ground cover plant, and S. trilobata quickly fled to the wild after settlement, and then developed and spread rapidly in southern China [S. trilobata has the ability to inhibit the growth and development of surrounding species, which poses a significant threat to local biodiversity and causes the imbalance of local ecological diversity. This is due to its rapid coverage of occupied open spaces through allelopathic therapy and asexual reproduction [S. trilobata can invade other remote areas through water flow or human activities as its stem can directly develop into a complete plant [ America . This pl America . It was rn China .Previousoduction , 9. Morete plant , 11. CurS. trilobata leaves has been shown to inhibit the germination of seeds and the growth of seedlings from both competitors and its own seeds, resulting in a high number of seed sterilizations and few seedlings in wild populations [S. trilobata primarily encroaches on new habitats through asexual clonal reproduction. In general, clonal reproduction typically leads to reduced gene flow between individuals within a species and a low level of genetic diversity, which is not conducive to adapting to changing environmental conditions. However, this was contradictory to the fact that S. trilobata was highly adaptable to diverse environments. Some researches have shown that epigenetics can provide clonal plants with more adaptive strategies. Invasive species tend to have higher phenotypic plasticity than non-invasive or native species, and even though invasive species populations in invasive areas were able to evolve greater plasticity than native populations [Alternanthera philoxeroides, which predominated in clonal reproduction, responded rapidly to environmental stresses mainly through epigenetic variation [S. trilobata's environmental adaptations has been poorly reported. There was still unclearly about how S. trilobata itself adapted quickly in a short term. Therefore, it was hypothesized that S. trilobata might adapt quickly to the stresses of different environments in a similar manner.The extract from ulations . Consequulations . For insariation . Thus faEpigenetics provides clonal plants with more adaptive strategies. It is one of the best examples of how cloning can replace the slow mechanism of natural selection. Epigenetics depends to varying degrees on asexual reproduction and preserves or reverses gene ability to buffer plants against current challenges and future rapid environmental changes . In addiDNA methylation, RNA interference, and histone modification are widely recognized as essential components of epigenetic regulation. Among them, cytosine methylation stands out as the most important epigenetic regulation strategy and plays a central role in the epigenetic control of gene expression . FurtherSince DNA methylation-sensitive amplified polymorphism (MSAP) does not require sequencing of a reference genome, it can be directly used to assess the methylation status of randomly distributed genomic cytosines. This method has the advantage of being easy to operate and inexpensive, and is currently the most widely used method for detecting cytosine methylation, which often occurs at the CG site in the promoter region of the DNA sequence . MSAP isS. trilobata have mainly focused on examining changes in phenotype or physiological indices in response to different environmental factors. However, there are few explorations based on genetics and epigenetics [S. trilobata, this study explored the genetic and epigenetic structures and differences among populations of S. trilobata under different habitats by using molecular markers SSR and MSAP.Previous studies on the invasion mechanism of genetics . For betSphagneticola trilobata (L.) Pruski is a common invasive plant widely distributed in South China. It has been recorded in Guangdong, Guangxi, Fujian, Hainan and Yunnan province. It can adpapt to highly heterogeneous environments, such as parks, scenic spots, riversides, roadsides, fields, etc. S. trilobata, triangular-shaped leaves with toothed edges that are 5–10cm long and 2–5cm wide. The leaves are arranged alternately on the stem and are slightly hairy on both surfaces. The plant produces yellow daisy-like flowers with a diameter of approximately 2–3 cm, which bloom throughout the year.such as its triangular-shaped leaves and hairy surfaces. The plant's growth habit and morphology also vary depending on its environment, with plants growing in full sunlight typically being more compact and bushy than those growing in shade [in shade .S. trilobata distribution records in China were obtained from literature reports and previous data collected by the laboratory. Maxent software and ArcGIS were utilized to predict contemporary suitable areas, and samples were gathered from highly suitable regions. As this species was introduced in the 1970s and has undergone a certain period of evolution, reference data from contemporary climate were chosen as the environmental factor data. The environmental data were collected from China Meteorological Administration.s website (http://www.cma.gov.cn/). In this study, a total of 120 samples were collected solely from highly suitable areas in four different regions: Guangzhou, Guangdong Province (GZ), Haikou, Hainan Province (HK), Fuzhou, Fujian Province (FZ), and Jinghong, Yunnan Province (JH), respectively. Thirty samples were collected from each region (Table S. trilobata in the area); 2) Ten samples were randomly selected from each quadrat and ensure each plant is more than 50 m apart; 3) Harvest the mature leaves after the third pair of leaves on the plant; 4) A total of 120 samples were collected from four populations, and the mature leaves were dried and numbered using silica gel from each region. DNA was extracted from each sample and stored at − 20 °C. Total genomic DNA extraction was extracted using the plant genome DNA extraction kit (Beijing Tiangen).The current 2O. The PCR conditions were set as follows: 95 ℃, 3 min; 34 cycles of ; and a final extension at 72 ℃ for 5 min, and finally stored at -20 °C. In the end, PCR products were separated by 6% non-denaturing polyacrylamide gel electrophoresis [Based on the transcriptome data, six pairs of SSR primers were selected for capillary electrophoresis. The volume of Polymerase Chain Reaction (PCR) system was 10 μL, consisting of 1 μL of DNA template, 5 μL of 2 × Taq PCR Master Mix, 0.5 μL of upstream primers (the 5’ end of the upstream primer was labeled with phosphorescent dye during fluorescence PCR), 0.5 μL of downstream primers and 3 μL of ddHphoresis .Using EcoRI and two restriction sites 5′-CCGG-3′, the restriction endonucleases MspI and HpaII with different sensitivity to methylation were cut at 37 °C for 12 h. The resulting DNA digested products were connected and amplified by PCR. After 2% agarose gel detection, 8 pairs of primers were selected for PCR selective amplification. The selective PCR products were detected with 6% polypropylene gel, and then the color was observed by silver staining . There were four cytosine methylation patterns in DNA  and Python (3.10.5) were utilized for data analysis and figure drawing.The SSR bands of The numbers of observed alleles (Na) in SSR ranged from 1.656 to 1.750, while the number of effective alleles (Ne) range from 1.319 to 1.530. The average values of Na and Ne were 1.703 and 1.406, respectively. In MSAP, Na ranges from 1.603 to 2.000, while the Ne ranges from 1.148 to 1.344, with the average values of 1.798 and 1.275, respectively. The average values of gene diversity (H) and the Shannon diversity index (I) of both SSR and MSAP were 0.236, 0.184 and 0.354, 0.303, respectively, with little variance . This suggested that the differentiation among populations was mainly attributed to epigenetics. Genetic and epigenetic gene flow (Nm) were 0.652 and 0.276, respectively, which were less than 1, indicating that gene flow among S. trilobata populations in different regions was limited by geographical distance.AMOVA analysis Table  revealedThe total methylated rates in the four populations Fig.  were 23.S. trilobata were 0.472 and 0.440, respectively. In Alternanthera philoxeroides, the Shannon Diversity Indexes (I) of genetic diversity and epigenetic diversity were 0.002 and 0.046, respectively [S. trilobata has relatively higher genetic and epigenetic diversity. The Shannon Diversity Index of genetic and epigenetic diversity in HK was low. The reason could be HK population was located in Hainan Province, which was an independent island, resulting in less frequent gene exchange between HK population with the other populations. As the earliest region in China to introduce S. trilobata, GZ has the longest time for the formation of its escape zone and natural community, which may be the reason have led to its highest genetic diversity. During the process of clonal reproduction, plants evolve complex adaptation with defense mechanisms through potential epigenetic traits due to the stress they feel from the environment, and are genetically mediated to produce phenotypic changes called phenotypic plasticity. Studies had shown that invasive species tend to had higher phenotypic plasticity than non-invasive or native species, even that populations of invasive species in invasive regions were able to evolve greater plasticity than native populations [In this study, the Shannon Diversity Indexes (I) of genetic diversity and epigenetic diversity in ectively .These reulations . The FZ S. trilobata, heredity and epigenetics were mutually supportive.After analyzing the UPGMA phylogenetic tree and PCoA results, it was found that the MSAP had a pronounced effect than the SSR clustering. The epigenetic molecular markers seemed to be able to better distinguish regions, which indicated that epigenetics was more malleable during adaptation to different habitats. It was even more obvious in the PCoA result plot of MSAP that the GZ population was clearly separated from the rest of the region, which was a very interesting phenomenon. As the GZ region was the first to be introduced and cultivated in China, and its population has undergone a long-term stage of adaptive evolution, whereas other regions were introduced and cultivated at a later time. This observation also supports the notion that genetic and epigenetic diversity indices of species increase proportionally with the stage of adaptive evolution.This result was consistent with the findings of Shi et al., indicating that during clonal reproduction, plants evolved complex adaptations with defense mechanisms through underlying epigenetic traits due to environmental pressures, and improved adaptive ability through gene mediation . MoreoveHelleborus foetidus were related to environment and epigenetic distance [Linum catharticum from both dry and wet grasslands [S. trilobata just showed a significantly weakly correlated, where the habitats of our four regions were not in extreme opposition. Besides, Ne, H, I and altitude were significantly positively correlated in genetics, as well as PIC, Na, Ne, H, and I in epigenetics were all positively correlated with the highest temperature  and fully methylated rate (%) were both the highest in HK population, whereas those in GZ population were the lowest. The hemi-methylated rate (%) was the highest in FZ population. The non-methylated rate was the highest in GZ population and the lowest in HK population. Overall, the methylation level of GZ population was significantly lower than that of other populations, indicating that there were some differences in methylation patterns between different populations. This could be attributed to variations in local temperature and habitats at the sampling sites. In cloned plants, epigenetic modifications can created new modules over time and accumulate them for adaptations [The methylation rate in plants typically falls within the range of 20% to 40% . In this compact , resultiptations , 38. Theptations , 40.Alternanthera philoxeroides in the same environment, which can be maintained until 10 generations before gradually disappearing, indicating that epigenetic variation was likely caused by environmental induction and spontaneous mutations [Arundo donax, which showed that epigenetic diversity was higher than genomic diversity, suggesting its invasion under different environmental conditions was attributed to higher epigenetic variability [For example, Shi et al. (2019) discovered a substantial amount of epigenetic differences within the clone population of utations . Similariability .S. trilobata indicated that the methylation patterns of cloned plants produced significant differences in invasion regions. These results suggest that epigenetic mechanisms partially compensate for the limited gene exchange between populations due to clonal reproduction. This indicates that epigenetics plays a supportive role in the process of adaptation and evolution, and exhibits greater plasticity in aiding clonal plants to adapt to invasion.The differences in methylation patterns in different regions of S. trilobata populations in different regions had high genetic and epigenetic diversity, which have played a mutually reinforcing role in facilitating its rapid invasion. The underlying drivers for these changes are attributed to diverse environmental pressures, which have been one of the primary factors responsible for S. trilobata's successful invasion. Notably, the correlation between epigenetics and geographic distance was significantly higher than that observed for genetic and geographic distance. By virtue of the significant difference in methylation patterns among different populations, the epigenetic mechanism of S. trilobata have endowed it with greater plasticity, thereby enabling it to adapt rapidly to changing environments. Overall, this study lays a foundation for a deeper understanding of how cloned plants undergo rapid adaptation.As can be concluded from this study, Additional file 1: Table S1. Components of enzyme digestion system for MSAP (20 μl). Table S2. Formula of adapters for MSAP. Table S3. System of ligation for MSAP (20 μl). Table S4. Reaction system of pre-amplification (20 μl). Table S5. Reaction system of selection PCR amplification. Table S6. Sequences of adapters and primers for MSAP. Table S7. Reaction system of SSR amplification (10μl). Figure S1. Sampling sites distribution of Sphagneticola trilobata. Figure S2. Correlation between epigenetic (a) and genetic diversity (b) of Sphagneticola trilobata and environmental factors.