Lymantria dispar L. 1758 (Lepidoptera: Erebidae) is one of the most dangerous forest pests of the Holarctic region. Outbreaks of gypsy moth populations lead to significant defoliation of local forests. Within the vast territory of the West Siberian Plain, we noted the outbreak front movement in the north-east direction with a speed 100–200 km per year. The reason for the outbreak’s movement is still unclear because L. dispar females are characterised by limited flight ability, which is not enough to support that movement per se. Herein, we analysed the mtDNA divergence pattern among L. dispar populations collected from the vast territory of the West Siberian Plain to determine the boundaries of populations and reveal the effect of the outbreak’s front movement on mtDNA patterns of populations. The 590-bp region of the cytochrome oxidase subunit I gene of the mitochondrial genome was sequenced for 220 specimens that were collected from 18 localities along a transect line (~ 1400 km). Our results clearly show that the gypsy moth populations of the vast Siberian territory are not subdivided. This result can be explained by extensive genetic exchange among local populations. Taking into account that the flight ability of L. dispar females is rather limited, we suggest that spreading occurs through ballooning of early instar larvae. This hypothesis was confirmed by the coincidence of the outbreaks’ movement direction with that of the dominant winds, complemented by the observation of ballooned larvae far from a forest edge.Gypsy moth  The spatio-temporal distribution of animal populations is an important topic for population ecology especially when populations of animals are able to significantly fluctuate. Many insect species are often considered as the model taxonomic group for population ecology studies. They are one of the most numerous animals on the Earth and widely distributed, while also being the main consumers of green biomass in the biosphere . Many heLymantria dispar L. 1758 (Lepidoptera: Erebidae) is one of the most dangerous forest pests of the Holarctic region, infesting over 300 plant species [L. dispar population dynamics is the regularity of outbreak emergence. The population dynamics of L. dispar is generally cyclical with a period of seven to nine years and is characterised by high amplitudes of population fluctuations. Some long-term shifts in the outbreaks cyclicity were found [Gypsy moth  species , 5. Outb species , 6, 7. Are found . The trare found .L. dispar populations, the synchronous character of outbreaks has been shown [L. dispar shows an asynchronous character of outbreaks. In addition, we noted for some regions the cases of travelling waves with the north-east direction  to determine boundaries of populations of Western Siberia, and ii) to compare mtDNA patterns between populations being at different phases of population cycle. There has been extensive collection of mtDNA data from L. dispar populations [In the present work, we aimed to explain the outbreak movement phenomenon by the analysis of mtDNA divergence patterns of populations collected from the vast territory of the West Siberian Plain in 2015–2016. In particular, we investigated the mtDNA divergence patterns of ulations , 24, 25.Insects were collected at the pupae or adult stages in July 2015–2016 in the West Siberian plain , Table 1L. dispar population phases. i) The ratio of previous/current year egg masses. It is easy to do in West Siberia in comparison to other regions because here females lay egg masses on the base of the tree stem that will be covered by a snow layer); ii) severe defoliation which shows the peak phase. iii) the heavy weight of female pupae means the rising phase because heavy females of L. dispar have higher fecundity (features of aphages adults) while small size vice versa the decline phase. iv) The high amount of parasitized larvae/pupae  indicating the decline phase. The rising phase was characterized by the bigger number of new egg masses then old ones; females pupae were heavy (heavier than 1.5 g) and parasitism level was low. The peak was characterized by severe defoliation of birch stands with extremely high numbers of egg masses (10-40/tree). The decline phase was characterized by the fewer number of new egg masses than old ones; the size of female pupae/exuvium was small  and parasite abundance was high. The “troughs” phase was assigned when extremely low population density (< 0.005 egg mass/tree) was registered.We characterised each locality in terms of population cycle phase, and based on it, the transect line was subdivided into six areas, referred to as ‘populations’. Distance between nearby populations was 150–200 km. Following criteria were used for the characterisation of In the outbreaking populations, insects were collected by net  or by hand . For low population densities , adult gypsy moths were caught via pheromone or light lures. Adults were stored in 95% ethanol. No permits for a field collection are required for this study, since the national forests in Russia are freely accessible. No protected species have been sampled.To determine the spatial-temporal distributions of pest outbreaks, we considered time-series data of gypsy moth densities, measured as the hectares of defoliated forest per year in the Sverdlovsk, Chelyabinsk, Tyumen, Kurgan, Omsk, and Novosibirsk oblasts . Initialx (i), were replaced by x´ = x + 1, which permitted us to transform the data with zero values of defoliation areas correctly: if x = 0, then ln x = -∞ but ln x´ = ln (0 + 1) = 0.To reduce the dispersion of the studied time series and switch over to the logarithmic scale, all values of the defoliation areas, Further, with the analysed time series, it was necessary to remove high-frequency noise. The Hunn filter was applied for this purpose :L(x(i))Removing high-frequency noise allows filtering high-frequency components of the time series with frequencies higher than 0.25/ year. The carried out transformation of the time series allows to work correctly with the data in the program, Statistica 10.0.pxy(k), can be calculated for two stationary time series, x and y, with mean values, μx and μy, and standard deviations, σx and σy [k = 0, k = ± 1, k = ± 2 is the time delay (lag).To assess the coherence of time series in population dynamics, the cross-correlation function is used. The cross-correlation function, x and σy :Pxy(k)=k = 0 and the value pxy(0) → 1. The time series are coherent with a delay equal to the value of k if the maximum of the cross-correlation function falls on the value, k = ± 1, k = ± 2, etc. and the value of the cross-correlation function, pxy(k) → 0, for any values of k for non-conjugate time series27.The absence of a time delay indicates that the time series are synchronous such that For a more visible demonstration of outbreaks moving, we used the detailed data of the vicinities of the Novosibirsk oblast for generation of a movie see . The outCOI) gene was sequenced for 220 specimens. The first part of the mtDNA amplicons (46 samples) was produced with a primer set, LepF1/LepR1, according to the original protocol [L. dispar mitochondrion genome, LepF2 5′-TACCGCTTAAACTCAGCCAT-3′ and LepR2 5′-GAGGTAAAGTAAGCTCGTGT-3′, which allowed a more effective acquisition of amplicons. The LepF1/R1 primer set produced an amplicon of the 1511–2168 mtDNA region according to GenBank Accession No. FJ617240 and LepF2/R2 produced a 1457–2363 region. PCRs were carried out in a 30-μL volume with ‘BioMaster HS-Taq PCR (2x)’  with PCR cycling at 95°C for 5 min, 35 cycles at 95°C for 15 s, 55°C for 30 s, 72°C for 1 min, and final elongation at 72°C for 5 min. The amplicons were purified via the Zymoclean Gel DNA Recovery Kit  according to the manufacturer’s instructions and sequenced with an automatic capillary sequencer with PCR primers under the BigDye v. 3.1 (Applied Biosystems) protocol.Total DNA was extracted from a leg of each specimen by incubation of homogenate in digestion buffer (see ). The 59protocol . A seconCOI gene were deposited in GenBank under accession numbers MK041668—MK041887.Nucleotide sequences of the To determine the boundaries of L. dispar populations of West Siberia plain we used a mitochondrial marker, in particular, a part of cytochrome oxidase I subunit (COI) gene. A mitochondrial marker indicates maternal inheritance of a studied population, therefore migration activity of adult males between populations in the previous season could be neglected. Moreover, males of current year collection were collected in localities in a period when they emerged, so taking into account the design of the study adult males could not migrate in considered season. The main evolution factor that affects the genetic pattern in our case should be a genetic drift. Indeed, since the local populations regularly suffer dramatic changes in effective population size, the genetic drift should strongly influence mtDNA variation Therefore we assumed that mtDNA variation would be different in different populations. As to other evolution factors, mutations have extremely low rate and close or identical mitochondrial haplotypes are found in distantly related populations of modern L. dispar range; and positive selection of mtDNA has extremely low probability .The alignments of nucleotide sequences were generated by the MUSCLE  that wasL. dispar populations. Populations of Europe and West Siberia are distant and genetic exchange is unlikely. The taken European area was characterised by an uninterrupted range of L. dispar and by similar scale. Populations of European territory were quite well characterised in a relation of mtDNA variation of L. dispar (L. dispar [www.boldsystems.org)  for 25 years have demonstrated that there is a cyclic component in a temporal context  was 22 years. For the Novosibirsk and Omsk oblasts, cycles were twice as frequent  were detected. Nucleotide diversity (Pi) was 0.00156 and the average genetic distance among individuals was 0.0016. Sixteen haplotypes (h) were observed with a haplotype diversity (Hd) of 0.669. Three haplotypes  made up 93% of the total sample, and they were found in almost all localities  of outbreak distributions has a directed movement in the north-east direction  the traffic intensity and road net in Siberia is much less than in Europe while mtDNA diversity is higher in Europe; and ii) Egg masses laid on the transport can not successfully overwinter in Siberia. It is important to note that in Siberia egg masses of L. dispar diapause under snow layer, laying eggs near the ground . The same scale of ballooning distance was also indirectly shown for the winter moth, Operophtera brumata L [We suggest that the spread of in et al . Accordiin et al , the spreed 1 km , 50. Yetrumata L , when rerumata L .L. dispar outbreaks for certain regions of West Siberia occurred as a travelling wave in a north and north-east direction, as recorded over the past quarter of a century  in the following years. The following facts indicate the main role of dispersal by ballooning: i) the genetic similarity of mtDNA patterns of low mobility species in the vast territory; ii) the correspondence of the direction of outbreak movement with dominant winds; and iii) the observation of ballooned larvae far from a forest edge. We assume that ballooning of Lepidoptera is underestimated to date.We can conclude that the vast territory of West Siberia (over 1000 km from West to East) with similar climatic conditions  and the same landscape (plain of forest-step zone) is inhabited by an non-subdivided population of S1 VideoTransparent colour indicates no outbreaks in a particular district; yellow indicates that the area of the outbreak is less than 1000 ha within the district; orange—1000–10000 ha; red—more than 10000 ha.(GIF)Click here for additional data file.S1 FigThe numbers on the first panel indicate names of districts: 1- Kyshtovka, 2- Ust’ Tarsky, 3- Vengerovo, 4- Severny, 5- Tatarsk, 6- Chany, 7- Kuibyshev, 8- Chistoozerny, 9- Barabinsk, 10- Ubinsky, 11- Kupino, 12- Zdvinsk, 13- Kargat, 14- Bagan, 15- Karasuk, 16- Krasnozerskiy, 17- Dovol’noe, 18- Kochki, 19- Chulym, 20- Kochenevo, 21- Kolyvan’, 22- Ordynsk, 23- Novosibirsk, 24- Moshkovo, 25- Bolotnoe, 26- Suzun, 27- Iskitim, 28- Toguchin, 29- Cherepanovo 30- Maslyanino.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 TableThe value of function/t-value are above the main diagonal and the temporal delay  is below.(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Gadus morhua) have been impacted by cyclic Pleistocene glaciations, where drops in sea temperatures led to sequestering of water in ice sheets, emergence of continental shelves, and changes to ocean currents. High‐resolution, whole‐genome mitogenomic phylogeography can help to elucidate this history. We identified eight major haplogroups among 153 fish from 14 populations by Bayesian, parsimony, and distance methods, including one that extends the species coalescent back to ca. 330 kya. Fish from the Barents and Baltic Seas tend to occur in basal haplogroups versus more recent distribution of fish in the Northwest Atlantic. There was significant differentiation in the majority of trans‐Atlantic comparisons (ΦST = .029–.180), but little or none in pairwise comparisons within the Northwest Atlantic of individual populations (ΦST = .000–.060) or defined management stocks (ΦST = .000–.023). Monte Carlo randomization tests of population phylogeography showed significantly nonrandom trans‐Atlantic phylogeography versus absence of such structure within various partitions of trans‐Laurentian, Northern cod (NAFO 2J3KL) and other management stocks, and Flemish Cap populations. A landlocked meromictic fjord on Baffin Island comprised multiple identical or near‐identical mitogenomes in two major polyphyletic clades, and was significantly differentiated from all other populations (ΦST = .153–.340). The phylogeography supports a hypothesis of an eastern origin of genetic diversity ca. 200–250 kya, rapid expansion of a western superhaplogroup comprising four haplogroups ca. 150 kya, and recent postglacial founder populations.The historical phylogeography, biogeography, and ecology of Atlantic cod ( Genetic structure can be influenced by a number of factors including habitat loss, changes to environmental conditions, overexploitation, and the introduction of invasive species  have proven useful in clarifying phylogenetic relationships among species  are included here, with the addition of the complete control region sequence (see below).A total of 153 Atlantic cod from 14 sampling locations were examined Tables . SamplesSampling locations in the Northwest Atlantic are designated by NAFO numbered divisions and lettered subdivisions Table . Various2.22, 0.4 μM forward and reverse primer, and 40 extension cycles. Primer details and annealing temperatures are given in Table DNA was extracted from heart tissue with the Qiagen DNeasy Blood and Tissue Kit according to the manufacturer's protocol . The complete mitochondrial genome of 89 fish was amplified in a series of 23 or 24 standard PCR fragments ranging from 534 to 1,345 bp, with an average overlap of 169 bp (88–273 bp). Primers were originally designed for use with Atlantic cod ; and a final extension at 68°C for 7 min.Five of the samples  were also amplified in a series of overlapping long‐range PCR fragments ranging from 2,876 to 5,741 bp, with an average overlap of 406 bp (77–846 bp). The primer pairs used were g01F/g05R1, g05aF/g07bR, g07bF/g10R, g11F/g14R, and g14F/g20R  and then visualized under UV light on a 1% agarose gel with 0.3 μg/ml ethidium bromide. PCR products were purified with an Exo‐SAP clean‐up procedure; this consisted of incubation at 37°C for 15 min with 0.1 U exonuclease I (USB) and 0.1 U shrimp alkaline phosphatase (USB), followed by denaturation at 80°C for 15 min. Purified PCR products were sent to Genome Quebec  for Sanger sequencing. Standard PCR products were sequenced in one or both directions; for fragments with extensive overlap, or where sequences showed no ambiguities, a single sequencing primer (either forward or reverse) was used. Long‐range PCR products were sequenced with a series of forward and reverse internal primers.® CustomSeq® resequencing multispecies microarray , defined as the difference between the two highest signal intensities divided by the sum of all intensities. Empirically, calls made on both strands with dS/N ≥ 0.13 are considered “strong” and reliable, either in agreement with the reference sequence (invariant) or for a SNP variant, and calls with 0.10 ≤ dS/N < 0.13 are considered “weaker” but reliable if in agreement with the reference . Variable sites were double‐checked across all genomes (microarray and/or chromatogram data). Positions at which ambiguous calls could not be reliably resolved were excluded from analyses. Sequences were aligned to the Atlantic cod reference sequence  and nucleotide (π) diversity indices , thus identifying any underlying structure and/or genetic barriers. The SAMOVA was calculated for K = 2–13  for mtDNA genomes were calculated in Arlequin v3.5.1 .Population pairwise genetic distances  was used to root both analyses. Analyses rooted with Alaska pollock  as outgroup produce substantially the same result.Phylogenetic relationships among haplotypes were analyzed with both Bayesian and neighbor‐joining (NJ) networks. The Bayesian network was assembled in MrBayes v3.2  v6  as the outgroup . These were combined with the 32 known partial mitogenome sequences with the addition of the 921‐bp control region sequence to each (GenBank Accession Numbers KX432219–KX432250). Results are reported for the combined set of 153 complete mitogenomes . A total of 887 variable sites were identified: 372 were parsimony informative . Of 706 coding region SNPs, 531 (75.2%) were third‐position transitions and 34 (4.8%) were first position transversions for alternative Leu codons. There were no frameshift or truncation mutations. The codon positions and substitution type of 87 SNPs imply amino acid substitutions, including three threefold variants .Complete mitogenomic sequences were obtained for 121 Atlantic cod from 14 locations  and a 29‐bp repeat in the intergenic region between tRNA Thr and tRNA Pro . Individual Atlantic cod from the Northeast Atlantic and adjacent waters have previously been shown to have one to six copies of the 40‐bp tandem repeat , which is excluded from all population comparisons below. Nucleotide diversity (π) within populations ranged from 0.0016 to 0.0028 . The SAMOVA identified the presence of K = 2 groups . With K = 3, BLT was separated from the others , and at K = 4, NOR was separated . Pairwise ΦST values (Table p = .024). None of the other Northwest Atlantic comparisons were significant. The QAS sample comprised genomes in two unrelated haplogroups (see below); repetition of the analysis with these haplogroups separated as QAS1 and QAS2 identified them as significantly different from all other populations, including each other (p < .001). There was no significant correlation between genetic and geographic distance including trans‐Atlantic comparisons , nor among populations in the Northwest Atlantic taken separately .A total of 142 distinct mitogenome sequences were identified. Of these, 139 were unique to single individuals, and three were found in two, five, and seven fish from Lake Qasigialiminiq. Population sample sizes ranged from 7 to 18 : NOR, BLT, and QAS were found almost exclusively in haplogroups B, E, and G .The Bayesian analysis identified 63 branches with posterior probabilities > .95 Figure , an addiK = 2), −15,080 (K = 3), and −15,831 (K = 4); the probability of two clusters was unity. The distribution of populations between the two groups was not significantly heterogeneous . The statistical parsimony network , and the four‐population trans‐Atlantic partition corresponding to Carr and Marshall (p = .0027). Most Northwest Atlantic‐only models were not significantly shorter than random, either with QAS included or excluded . Alternatives that excluded either FLM alone or together with QAS remained highly significant (p < .0001 in either case). This indicates that the diphyletic composition of QAS did not unduly lengthen random trees, and that trans‐Atlantic populations were significantly differentiated.The Monte Carlo dispersal models, matrices, and results for eight partitions are given in Table 3.3−8 substitutions/site/year, the most recent common ancestor of all Atlantic cod examined dates to 333 kya (95% highest posterior density (HPD) = 215–435 kya), and the superhaplogroup containing the more recent expansions in the Northwest Atlantic (FGI) dates to 145 kya (HPD = 75–170 kya). The coalescence times of the eight major haplogroups range from 47 to 120 kya, all well before the last glacial maximum . Among the 36 pairwise ΦST population comparisons, 12 were significant, all of which involved NOR, BLT, and/or QAS. QAS was significantly different from all other populations, except BLT, and none of the among‐North Atlantic stock comparisons were significant  and microsatellites showed significant differences between trans‐Pacific populations, and no distinction among geographically proximal locations . This includes the complete set of 153 genomes aggregated broadly as [NOR + BLT], FLM, North, and South of the Laurentian Channel (Ia). (The influence of QAS in Model Ia is discussed below.) It also includes partitions that separate NOR and BLT. Model IIa corresponds to the partition in Carr and Marshall (p > .4).In the Monte Carlo analysis, all five models that examined trans‐Atlantic partitions  alone consistently suggest an absence of genetic differentiation within regions and trans‐Atlantic differences in haplotype frequencies . Shared mitogenomic haplotypes are not seen in marine populations of Atlantic cod, and the presence of multiple distinct, private haplotypes within a single population is unprecedented in marine fish mitogenomic studies. The major haplogroups in Lake Qasigialiminiq are polyphyletic, and, by implication, of distinct temporal origins, although a single colonization event by a mixed population is also possible. The QAS fish assigned to haplogroup F is most closely related to an individual in NGB and is likely a more recent introduction from a lineage that is otherwise restricted to the Northwest Atlantic.p = .0065 vs. .0105). In the various partitions of the Northwest Atlantic in Model III, the two variants that include QAS separately are highly structured (IIIb and e: p < .001), whereas variants that group it with NAFO 2H or exclude it altogether are not  or shared predominantly with BLT cod (E). This suggests a closer phylogeographic affinity with the east than with the west; this is supported by recent findings that the Greenland cod populations were colonized from Iceland  separated from the mainland by a narrow strait in the Barents Sea. Based on a 970 bp mtDNA cytochrome b sequence, Zelenina, Makeenko, Volkov, and Mugue , and that although Flemish Cap fish occur predominantly in haplogroup F, they are also present in every other haplogroup except the two smallest (D and H). Likewise, the NGB data do not support the Grand Banks as a possible refugium.During the last glacial maximum, suitable habitat occurred in the Northeast Atlantic, in the western Atlantic south of the ice sheets, and may have occurred along the continental shelves of Atlantic Canada including the Flemish Cap, the Grand Banks, and in the Davis Strait between Baffin Island and western Greenland ; KX432219–KX432250 (control regions). Primer sequences: uploaded as online supporting information (Table DNA sequences: GenBank accession numbers on Table .None declared.SMC and HDM conceived the ideas; HDM and LAL collected the samples; LAL and HDM collected the data; LAL and SMC analyzed the data; SMC and LAL funded the project, and LAL led the writing with contributions from all authors. Click here for additional data file.


Ancient DNA genome-wide analyses of Neolithic individuals from central and southern Europe indicate an overall population turnover pattern in which migrating farmers from Anatolia and the Near East largely replaced autochthonous Mesolithic hunter-gatherers. However, the genetic history of the Neolithic transition in areas lying north of the European Neolithic core region involved different levels of admixture with hunter-gatherers. Here we analyse genome-wide data of 17 individuals spanning from the Middle Neolithic to the Early Bronze Age (4300-1900 BCE) in order to assess the Neolithic transition in north-central Poland, and the local impacts of hunter-farmer contacts and Late Neolithic steppe migrations. We evaluate the influence of these on local populations and assess if and how they change through time, reporting evidence of recurrent hunter-farmer admixture over three millennia, and the co-existence of unadmixed hunter-gatherers as late as 4300 BCE. During the Late Neolithic we report the appearance of steppe ancestry, but on a lesser scale than previously described for other central European regions, with evidence of stronger affinities to hunter-gatherers than to steppe pastoralists. These results help understand the Neolithic palaeogenomics of another central European area, Kuyavia, and highlight the complexity of population interactions during those times. Recent ancient DNA (aDNA) genome-wide studies have particularly addressed the extent of genetic admixture between Neolithic farmers and hunter-gatherers (HG) and found evidence of regional variability in the level and nature of the admixture in Iberia20, south-east16, central15, north-eastern14, and northwestern Europe8. In regions such as the Baltic, however, genomic data has shown that hunter-gatherer populations adopted farming without a significant genetic input from farming populations14. In light of these results, north-central Europe is particularly important to understand the genetic history of prehistoric Europeans. Here, the line of maximum north and north-eastern reach of settlement of Neolithic farmers of Linear Pottery culture (LBK) and post-Linear Pottery cultures (post-LBK) ran through the North European Plain. These farmers represented the first stage of neolithization of this part of Europe and they were allochthonous populations whose ancestry can be traced back to Anatolian early farmers, with only minor admixture of European hunter-gatherers obtained in south-eastern Europe and the Carpathian Basin16. In the territories like north-central Poland these farmers came into contact with well established hunter-gatherers communities of the Baltic region, which in turn occupied habitats not attractive for the agricultural lifestyle of LBK and post-LBK cultures. As a consequence, a borderland between the Neolithic farmer and HG worlds was constituted where farmers and HGs coexisted with each other for over one thousand years (during the fifth millennium BCE)23.The knowledge about the genetic history of prehistoric Europeans has increased substantially during the past three years following a series of studies that have provided new insights about the ancestry and affinities of Neolithic, Chalcolithic and Bronze Age populations from various parts of Europe26. During the 5th millennium BCE, following the dissolution of the LBK, Kuyavia continues to be populated by post-Linear Pottery Neolithic cultural units of local expression such as the Stroke Band Pottery and later the Middle Neolithic Brześć Kujawski Group of the Lengyel culture (BKG)27. This cultural tradition is also known as the ‘Danubian Neolithic’ for its clear cultural ties with Carpathian Basin cultures on the one hand, and contrast to subsequent indigenous cultures on the other. After this phase, Kuyavia went through cultural transformations dominated by pan-European Middle and Late Neolithic cultural units like Funnel Beaker culture (TRB), Globular Amphora culture (GAC) and Corded Ware culture (CWC).The Kuyavia region is one of the most representative areas of north-central Poland from the point of view of these processes when considering the archaeological record. Farming was introduced to this region with the arrival of LBK farmers ca. 5400 BCE28, appears, and becomes the main culture associated with the Neolithic expansion across Scandinavia and Atlantic Europe. This culture became responsible both for the final neolithization of territories like Poland, and for a further spread of agriculture to northern Europe. It has been suggested that the TRB communities were formed by indigenous northern European Mesolithic people who adopted farming locally rather than by incoming exogenous Danubian farmers from central Europe29. This model is supported by archaeological data which points to cultural, social, economic, and ideological transformations29. However, nuclear aDNA studies show that Scandinavian TRB farmers are genetically similar to today’s southern Europeans (particularly Sardinians), like other Early Neolithic farmers from central Europe30, albeit with the appearance of hunter-gatherer mitochondrial DNA haplogroups34. The results raise interesting questions about how and when did this admixture occur. Was it a short-term phenomenon that occurred at the end of LBK and post-LBK cultural tradition and reflected a disruption of the current cultural system and a massive transition of local HGs to agriculture; or rather was it a long term process lasting several hundred years within post-LBK cultural units like Rössen, Lengyel, or the Brześć Kujawski group in Poland? However, these specific questions cannot be directly addressed based on the current data as they require a much larger number of individuals from the various Central and Northern European post-LBK Middle Neolithic cultures.Around 4000 BCE, the Trichterbecherkultur, or Funnel Beaker culture29. The ancestry of the GAC had been disputed but recent data shows an absence of steppe ancestry in this culture15. The Corded Ware Culture, which have had a major input from migrating steppe pastoralists9, appears in the same region by the early part of the 3rd millennium BCE.By around 3100 BCE, the TRB culture was replaced by the Globular Amphora Culture across most of central and eastern Europe. This involved an economic transition marked by an increase in elements of pastoralism as well as farmingThe Kuyavia region is particularly useful for a population history analysis of the Polish lowlands for a number of reasons: (1) it was one of its most intensively settled regions during the entire Neolithic period and all major cultural units characteristic of central Europe are represented within it; (2) it has one of the best archaeological records of the Neolithic settlement in Poland, including well preserved human skeletons; and (3) it was situated on the north and north-east edge of the ‘farmers world’ during the Early and Middle Neolithic being particularly exposed to contacts with HGs.Here we therefore investigate the population affinities, changes in genetic structure, and admixture, among Neolithic, Eneolithic, and Bronze Age populations from the Kuyavia region, north-central Poland. We report the results of a genomic time series of 17 individuals, spanning from the Middle Neolithic to the Early Bronze Age (4300-1900 BCE)  appear on ADMIXTURE as mostly composed of the same genetic component present among Anatolian and LBK Early Neolithic farmers, and with a small HG component , with X as N22 (Poland_BKG_N22) or N42 (Poland_BKG_N42), and Y as one of the published Early/Middle Neolithic and Mesolithic hunter-gatherer populations present in the merged dataset ; Poland_BKG_N22, Loschbour_WHG/Hungary_WHG), and detected that N22 forms a clade with WHG from Luxembourg and Hungary to the exclusion of the BKG individuals from the same archaeological context (−0.420 < Z < −0.136) ; Poland_BKG_N42, Loschbour_WHG/Hungary_WHG), −2.687 < Z < −2.321]. These results are closer to the significance threshold (Z < 3), which is expected given the admixture of N42 with farmer populations, as shown in other analyses ] and f4-statistics tests [f4] applied to X chromosome SNPs. Both analysis produced similar results showing that on the X chromosome N42 is symmetrically related to Anatolian Neolithic farmers and to Loschbour_WHG , but with a slightly higher HG component  with X as Early and Middle Neolithic populations from northern Europe. Results indicate that the Polish TRB were as symmetrically related to the Scandinavian TRB as to other Middle Neolithic central European populations from Poland, Germany, and Hungary (0.287 < Z < 2.025) , with X as the groups in the time series  (Z = −2.302) by the end of the Middle Neolithic, the TRB individuals, dated to ca. 3400 BCE, shared as much genetic drift with hunter-gatherers as with Early Neolithic farmers , suggests those differences are not statistically significant  Z = −2.02 by theers Fig. . This soers Fig. . The Pol36. While the CWC show evidence of steppe ancestry, as seen by the appearance of a Caucasus Hunter-Gatherer and Iranian Neolithic component in ADMIXTURE, the same analysis and qpAdm  that show different patterns of affinities with hunter-gatherers and steppe populations  is genetically significantly closer to WHG than to steppe individuals (Z = −4.898), a result which is in contrast with those for CWC from Germany (Z = 2.336), Estonia (Z = 0.555), and Latvia (Z = 1.553) , the Polish CWC are statistically more similar to WHG than to other central/northern European CWC groups , but symmetrically related to WHG and Corded_Ware_Latvia (Z = −2.859) due to their higher HG ancestry   2, however in that case the resurgence of HG ancestry was found within the Funnel Beaker (TRB) complex dated to 4000-3000 BCE, while our data shows that admixture had occurred earlier and involved farmers representing post-Linear Pottery culture units. Consequently, the turning point in terms of hunter-farmer admixture was not between Danubian and “indigenous” Neolithic north-central European TRB and GAC cultures , but rather within the Danubian Neolithic tradition - between LBK and post-LBK cultural units. This may be in accordance with the demographic decline between these phases of the Danubian Neolithic seen not only in the Kuyavia region27, but in Central Europe in general39; it is possible that the close contacts with HG were a key for the success of post-LBK societies, which more effectively and permanently colonized the Polish lowlands.The analysis of a human time series from north-central Poland that spans from the Middle Neolithic to the Early Bronze Age (4300-1900 BCE) provides insights into the affinities and admixture patterns, and adaptive processes of farming populations in the Kuyavia region. A resurgence of WHG genetic component, with its most significant impact around the end of the Middle Neolithic period, has been identified in other studies2. The presence of two outliers from the middle and late phases of the BKG in Kuyavia associated with typical Neolithic burial contexts  of each petrous bone was identified, isolated, and then ground to a fine powder42, and took place in dedicated ancient DNA labs in Dublin and Lodz, in adherence with strict anti-contamination protocols.A first subset of 28 samples was analysed in University College Dublin with the purpose of screening for overall DNA quality and complexity from this geographical area and archaeological sites. A second set of 10 new samples was prepared in the University of Lodz. All extractions followed the same protocol, extracting approximately 50 mg of bone powder per sample43 as outlined in1, where blunt end repair was performed using NEBNext End-Repair (New England Biolabs Inc.) and Bst was inactivated by heat (20 minutes at 80 °C). For the creation of full UDG-treated libraries, we followed44.We divided the DNA extracts of all samples into two portions of 12.5 uL. One portion was converted into libraries without any type of uracil-DNA glycosylase (UDG) treatment in order to identify the molecular damage patterns typical of ancient DNA molecules. The other portion was used for UDG-treated libraries once the screening stage was completed. For the non UDG-treated libraries, we used a modified version of Libraries were quantified using a DNA-1000 chip on an Agilent 2100 Bioanalyser platform, and a Qubit 2.0 Fluorometer (Thermo Fischer Scientific), prior to pooling.The samples allocated for screening at UCD were sequenced at the UCD Conway Institute of Biomolecular and Biomedical Research, on an Illumina MiSeq platform, using 65 base pair (bp) and single-end sequencing. All final, UDG-treated, and the remaining 10 samples for screening, were sequenced on an Illumina NextSeq platform at the University of Lodz, using 100 bp single-end sequencing.45 was used to trim adapter sequences with minimum overlap set to 1 (−O 1) and minimum length to 17 bp (−m 17). Reads were aligned to the hg19 build of the human genome using the Burrows-Wheeler Aligner46 with disabled seed (−l 1000) and with a minimum QC quality filter of 30. Duplicated sequences were removed using samtools v0.1.19-96b5f2294a47. General sequencing information is presented in Supplementary Table An ancient DNA bioinformatics pipeline was applied for processing of raw data. Cutadapt v1.548 to verify deamination patterns and frequencies and analysed length distributions on the samples from the screening steps. The 17 deep sequenced samples showed characteristic deamination patterns, with frequencies of substitution ranging from 0.13 to 0.34 on the 5′ end (C > T), and from 0.08 to 0.30 on the 3′ end (G > A)  with the Genome Analyzer Tool Kit’s (GATK) Pileup tool for the 594,924 autosomal positions present in the Affymetrix’s Human Origins dataset, described in54. We included only a subset of 646 European, West Asian, and Near Eastern samples comprising 46 modern populations from the broader Human Origins dataset. All ancient samples from the dataset described on the previous section were projected onto these first two principal components by using the options lsqproject:YES and shrinkmode:YES.Principal component analysis (PCA) was performed using the smartpca software (v.13050) from Eigensoft 5.0.155. As suggested by the authors, the dataset was pruned for linkage disequilibrium with the option–indep-pairwise 200 25 0.4 using PLINK. This left us with 305255 SNPs for the analysis. Each K value from the range 2 to 14  was repeated 5 times with random seeds. Cross validation errors  on each individual  had X as the cultures/individuals, Y as the first population in the dataset to test against, and Z as the second with Yoruba as outgroup. The parameter f4mode: YES was used for these tests.We applied qpAdm from ADMIXTOOLS56. This package allows to investigate if a target population or individual can be modelled as a result of admixture between two or more test populations. We followed the approach used in53, using the O9 outgroup list as a base, and when testing for the significance of lower rank models including the dropped population in the outgroup list to test for robustness of the results. Models were only accepted if both tests, with and without the dropped population as an outgroup, produced p > 0.05. In a few cases when the highest model was the only statistically significant, we added an extra outgroup to further test robustness (Supplementary Table We estimated admixture coefficients and modelled all ancient Polish group using 57 (Supplementary Table Relatedness analysis was performed on the four Corded Ware samples, that either archaeologically or during the analysis showed possible familial relationships (Supplementary Fig. Supplementary InformationSupplementary Tables


Quercus chenii, a deciduous oak species representative of the East China flora. Based on the nuclear microsatellite variation at 14 loci, we identified multiple genetic boundaries that were well associated with persistent landscape barriers of East China. Redundancy analysis indicated that both geography and climate explained similar amounts of intraspecific variation. Ecological differences along altitudinal gradients may have driven the divergence between highlands and lowlands. However, range expansions during the Last Interglacial as inferred from approximate Bayesian computation (ABC) may have increased the genetic diversity and eliminated the differentiation of lowland populations via admixture. Chloroplast (cp) DNA analysis of four intergenic spacers  identified a total of 18 haplotypes, 15 of which were private to a single population, probably a result of long-term isolation among multiple montane habitats. A time-calibrated phylogeny suggested that palaeoclimatic changes of the Miocene underlay the lineage divergence of three major clades. In combination with ecological niche modeling (ENM), we concluded that mountainous areas with higher climatic stability are more likely to be glacial refugia that preserved higher phylogenetic diversity, while plains and basins may have acted as dispersal corridors for the post-glacial south-to-north migration. Our findings provide compelling evidence that both topography and climate have shaped the pattern of genetic variation of Q. chenii. Mountains as barriers facilitated differentiation through both geographic and environmental isolation, whereas lowlands as corridors increased the population connectivity especially when the species experienced range expansions.Major topographic features facilitate intraspecific divergence through geographic isolation. This process may be enhanced by environmental isolation along climatic gradients, but also may be reduced by range shifts under rapid climatic changes. In this study, we examined how topography and climate have interacted over time and space to influence the genetic structure and evolutionary history of  Tectonically driven uplift and climatically driven erosion create high-elevation mountains and deep river valleys upon the earth . Interacin situ diversification among isolated habitats  to distinguish between geographic and climatic effects on genetic divergence, and to test whether environmental isolation has also contributed to the local patterns of intraspecific variation. Third, we used approximate Bayesian computation (ABC) to explore the past demographic history, and used least-cost path calculations to infer the potential migration routes, to test whether lowlands have increased the genetic connectivity among populations especially when the species experienced range expansions. Finally, we reconstructed a fossil-calibrated phylogeny to determine the divergence times of major intraspecific lineages, and to detect how palaeoclimatic changes since the Neogene have influenced the evolutionary history of Q. chenii.Here, we combine information from two sets of molecular markers, i.e., bi-parentally inherited nuclear microsatellites (nSSRs) and maternally inherited chloroplast (cp) DNA sequences, and use an integrative approach including landscape genetic, phylogeographic, fossil-calibrated phylogenetic, and ecological niche model (ENM) analyses to clarify the associations between topography, climate, and the genetic variation of Q. chenii in China  and two species from genus Castanea (C. mollissima and C. henryi) were used as outgroups. Leaf tissues were quickly dried with silica gel and stored at room temperature in the laboratory. Spatially explicit information was recorded for each population using a handheld GPS unit . Voucher specimens of all individuals of Q. chenii and outgroups are deposited in the Herbarium of Nanjing Forestry University (HNFU) -trnG(UCC), and trnS(GCU)-trnT(GGU), were sequenced for six to ten individuals per population following protocols in Total genomic DNA was extracted from 30 mg leaf tissue of each individual using a Plant Genomic DNA Kit . Four chloroplast intergenic spacers, All the 419 samples were genotyped at 14 nSSR loci, including quru-GA-0M07, quru-GA-1H14, quru-GA-1M17, and quru-GA-2G07 , ssrQrZAn), inbreeding (f), and genotyping failures (b) simultaneously. The statistical significance was tested by comparing deviance information criterion (DIC) values between the full model and the null allele-ignored model (n is fixed at 0). Linkage disequilibrium (LD) for all locus pairs and deviation from Hardy-Weinberg equilibrium (HWE) in each population were tested by randomization in FSTAT 2.9.3.2  and genetic diversity within populations (HS) were estimated for each population, and compared between highland and lowland groups  tested by 10,000 permutations in ARLEQUIN 3.5  from 1 to 11 with 500,000 iterations as burn-in followed by 500,000 Markov Chain Monte Carlo (MCMC) iterations. Both the Ln Pr(X|K) selection method  and genetic admixture index (AD) with the geographical locations (latitude and longitude) and climatic conditions of each population using a general linear model (GLM). Genetic admixture index was calculated as the standard deviation (SD) of probabilities of population membership (Q) to each of the three genetic clusters (K = 3) inferred by STRUCTURE, and normalized to a range of 0 to 1 following the method of r | < 0.70; http://www.worldclim.org/) at 30″ resolution for the present. Elevation was also used as a climatic variable because it captures vertical variation of microclimatic features  were used as geographic variables to quantify the spatial pattern in a rectangular form  in the forward stepwise selection for partial RDA models including all the geographic variables (PCNM1–PCNM10) or climatic variables  and partial RDA using R package ‘vegan’ . RDA prolar form . PCNM waosphere’ . An equaosphere’ . Based osphere’ Radj2 in hS; total genetic diversity, hT) with PERMUT 2.0 (Hd) with DNASP 5.10. Genetic differentiation between highland and lowland populations was evaluated by AMOVA using ARLEQUIN 3.5. The phylogeographic structure was assessed by comparing the difference between two measures of population differentiation (GST and NST) with 10,000 permutations in PERMUT 2.0. Mismatch distribution analysis (under the demographic expansion model), Tajima’s D and Fu’s FS test were performed through ARLEQUIN 3.5 to detect the demographic expansion for the species.Sequences of the four cpDNA fragments were proofread and aligned in BIOEDIT 7.2.5 , and theRMUT 2.0 , and forQuercus and Castanea. Second, we used the result from the most recent phylogenetic study of East Asian oaks (Quercus (including section Cerris and section Quercus) to 35.89 Ma . A combination of uncorrelated lognormal relaxed clock and Bayes-skyline coalescent prior was selected for node age estimation through Bayes factor (BF) calculations based on marginal likelihoods obtained by stepping-stone sampling and path sampling. For the preferred combination, two independent MCMC runs were performed, each with 100 million generations and sampled every 10,000 generations. Tree and log files were combined through LOGCOMBINER 1.8.4, and then passed to TRACER 1.7.1 (Phylogenetic relationships and divergence times among cpDNA haplotype lineages were estimated using Bayesian inference (BI) as implemented in BEAST 1.8.4 . We applian oaks  as a secER 1.7.1  for asseQ. chenii. Species occurrence records were mainly collected from the fieldwork, the literature, and the database of the Global Biodiversity Information Facility , the Chinese Virtual Herbarium  and the Plant Photo Bank of China . We filtered the data by removing duplicate records and retaining only one record among all locations falling within the same 2.5′ × 2.5′ grid. Finally, a total of 54 occurrence points were obtained for Q. chenii. The environmental layers of the eight bioclimatic variables (http://www.worldclim.org/) with a resolution of 30″ for the present, the Mid Holocene (∼6 ka), the Last Glacial Maximum , and the Last Interglacial  under the Community Climate System Model 4 (CCSM4). All layers were clipped to the same spatial range . The optimal settings of feature types (linear and quadratic) and regularization multiplier  were selected through the R package ‘ENMeval’ . No consistent genotypic disequilibrium was found between any locus pairs across all populations, so all loci were used for further analyses. Significant deviation from HWE was only detected in one of the 18 populations  ranged from 3.849 to 5.901, and genetic diversity within populations (HS) varied from 0.585 to 0.713 . The highest pairwise FST values were observed for JZ-ZN, corresponding to two montane habitats located at the northwest (the Dabie Mountains) and southeast (the Tianmu Mountains). A higher level of genetic differentiation was detected among highland populations  than among lowland populations  , with only 0.43% of the variation partitioned among groups, and the most (94.33%) partitioned within populations  in each population was detected with increasing elevation  , and two were dominated by cluster II (QII: 0.93–0.94)  than in highland populations (mean of AD = 0.134)  within both high and low elevation regions  and genetic admixture index (AD) were not associated with longitude or latitude , but correlated with elevation and climatic variables. The model that best explained genetic diversity contained elevation , with mean diurnal temperature range (bio2), temperature seasonality (bio4), and mean temperature of driest quarter (bio9) as covariates. The model that best explained AD included only elevation (P < 0.01) . Other covariates, including mean diurnal temperature range (bio2) and mean temperature of wettest quarter (bio8), showed marginally significant (0.05 < P < 0.10) or insignificant (P > 0.10) positive correlations with genetic diversity , and both partial RDA models corresponding to geography  and climate  were significant. The full model explained 12.91% of the total variance. The percentages of genetic variance attributed to geography and climate alone were 6.22% and 6.10%, respectively. Only 0.59% of the overall genetic variance was explained by the collinearity between geographic and climatic variables . Among those, three showed the highest scores on the first three axes, i.e., elevation on the RDA1, temperature seasonality (bio4) on the RDA2, and PCNM9 on the RDA3 -trnG(UCC), trnS(GCU)-trnT(GGU), and concatenated cpDNA were 718, 639, 607, 902, and 2,866 bp, respectively (hS = 0.126) compared with the total genetic diversity (hT = 0.917) at the species level. Intraspecific differentiation at cpDNA markers was significant , with 88.65% of the total genetic variation partitioned among populations, and 11.35% partitioned within populations . The genetic divergence between highlands and lowlands was also not significant . Although the mismatch distribution for all haplotypes was bimodalP = 0.222) and Harpending’s Raggedness index  did not reject the sudden expansion model. Following D test  and Fu’s FS test  also did not show evidence of extensive demographic expansion.The lengths of consensus sequences after alignment of ectively Figure 6Both median-joining network and phylogenetic analysis grouped all the 18 haplotypes into three clades 7. CladeQ. chenii. Historical distributions for the LIG, LGM, and Mid Holocene are shown in The mean AUC value (± SD) on the test dataset was 0.921 ± 0.031, indicating a good fit of ENM to the observed occurrence data of Q. chenii indicated that a higher level of genetic differentiation occurred among highland populations than among lowland populations  in China , although the same region may also have been a refugium for the latter, implying a probable scenario of interspecific introgression due to secondary contact or long-term co-occurrence in a shared refugium.Our analyses suggested that an area surrounded by multiple geographic barriers, i.e., the Tianmu Mountains, the Huangshan Mountains, the Baiji Mountains, and the Huaiyu Mountains, may have acted as a refugium for istocene , and proistocene . Thus, tna e.g., . Our infQ. chenii was strongly influenced by both topography and climate. Palaeoclimatic changes of the Miocene may have driven the lineage divergence of chloroplast haplotypes. Persistent landscape barriers in East China may have facilitated population differentiation through both long-term geographic isolation and environmental isolation along altitudinal and other climatic gradients. By contrast, post-glacial range shifts along plains and basins may have increased the genetic connectivity among lowland populations via admixture.Our study illustrates that the pattern of genetic variation of YL and YF conceived and designed this research. YL and XZ collected samples and performed experiments. YL analyzed the data. YL led writing with substantial contributions from YF. All authors read and approved the final manuscript.This research was financially supported by the National Natural Science Foundation of China , the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYLX15_0922).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.


The current Brazilian population is the product of centuries of admixture between intercontinental founding groups. Although previous results have revealed a heterogeneous distribution of mitochondrial lineages in the Northeast region, the most targeted by foreign settlers during the sixteenth century, little is known about the paternal ancestry of this particular population. Considering historical records have documented a series of territorial invasions in the Northeast by various European populations, we aimed to characterize the male lineages found in Brazilian individuals in order to discover to what extent these migrations have influenced the present-day gene pool. Our approach consisted of employing four hierarchical multiplex assays for the investigation of 45 unique event polymorphisms in the non-recombining portion of the Y-chromosome of 280 unrelated men from several Northeast Brazilian states.FST genetic distances show similarities between Northeast Brazil and several other European populations, indicating multiple origins of settlers. Regarding Native American ancestry, our findings confirm a strong sexual bias against such haplogroups, which represented only 2.5% of individuals, highly contrasting previous results for maternal lineages. Furthermore, we document the presence of several Middle Eastern and African haplogroups, supporting a complex historical formation of this population and highlighting its uniqueness among other Brazilian regions.Primary multiplex results allowed the identification of six major haplogroups, four of which were screened for downstream SNPs and enabled the observation of 19 additional lineages. Results reveal a majority of Western European haplogroups, among which R1b-S116* was the most common (63.9%), corroborating historical records of colonizations by Iberian populations. Nonetheless, We performed a comprehensive analysis of the major Y-chromosome lineages that form the most dynamic migratory region from the Brazilian colonial period. This evidence suggests that the ongoing entry of European, Middle Eastern, and African males in the Brazilian Northeast, since at least 500 years, was significantly responsible for the present-day genetic architecture of this population. Present-day Brazilians are the result of centuries of admixture between three main ethnic groups, namely Native Americans, Europeans and Africans . As the Besides Portuguese settlers, other European groups also invaded or populated the country, such as Spanish, Dutch, French, English, Italian, German, Middle-Eastern and Japanese populations. Most of these settlements initially took place in the Northeastern and Southern regions. In fact, the Northeast was the most targeted area for migratory events during the colonial period and received the greatest number of European and African individuals , 3–5. ThUniparental markers are useful for disentangling the complex processes that shaped the current population . Indeed,Given its non-recombining nature and low mutation rates, single nucleotide polymorphisms (SNPs) located in the Y-chromosome are a useful tool for investigating historical events and serve as a valuable counterpart for our mtDNA data –17. The In this paper, a sample of 280 unrelated male individuals from several Northeastern states is analysed in a hierarchical assay for determining Y-chromosomal ancestral lineages Fig. . Four suN ≥ 15), showing that there is a homogenous distribution of Y-chromosome haplogroups in the investigated locations .Data obtained from the Major South America Multiplex is shown in Table Considering the globally widespread presence of certain Y-chromosome haplogroups, we performed the investigation of 33 downstream SNPs through multiple Multiplex reactions in order to provide higher phylogenetic resolution of the paternal ancestry composition of this population Table , Fig. 3.To address the lack of data regarding European lineages in many states from Northeastern Brazil, we carried out the sub-typing of eight R1b-M269 downstream SNPs in 147 samples with the R-M207 derived allele –21.R1b-S116* was the most common haplogroup in this subset of samples, corresponding to 63.9% of individuals, while R1b-M529 showed a frequency of 14.3% as the second most frequent. Haplogroup R1b-L23* was observed in 6.1% of samples, R1b-M167 and R1b-U152 shared a frequency of 5.4%, R1b-U106 was assigned to 4.1% of samples, and lastly, R1b-M153 was the least frequent, accounting for 0.7% of the subjects.FST genetic distances based on R1b-M269* sub-type frequencies. A multidimensional scaling plot demonstrates the distances between the Brazilian population investigated here and previous data obtained from potential European colonizer populations , followed by G-M201 (28%), J2-M172 24%), and J1-M267 (10%). Additionally, Multiplex 1 was performed on five samples assigned to haplogroup KLT-M9, all of which belonged to rare sub-haplogroup T-M70, typically attributed to Shepardic populations  than in Rio Grande do Norte and Maranhão . This haRegarding the remaining M269 derived lineages found in frequencies ranging from 2.8–0.4% in our sample, R1b-U106 has been reported as most frequent in Northwestern Europe, R1b-M153 and M167 were reported in Iberian populations and their descendants, mostly in the Basques , 35–38, Indications of multiple colonizer sources are also observed through the presence of haplogroups G-M201, J-M267, and J-M172, common in the Middle East and Near Eastern regions , 39. TheSuch discussion may be further extended to the presence of other Middle Eastern and North African haplogroups E-M78 and E-M81 in our findings, both present in 2.9% of individuals. One should consider that similar frequencies are found in Iberian populations, meaning this contribution could be from a European colonizer source . HoweverWith regards to E haplogroups in our findings, the presence of M2 derived markers, which is restricted to Sub-Saharan Africa, seems to be a product of the transatlantic slave trade, responsible for the arrival of West African individuals mostly to the Northeast region of Brazil during the seventeenth century. The contribution of haplogroups E-M2*, E1b1a-M191, and E1b1a-M154 show that the ancestral background of these Brazilian men is derived from a signature of the Bantu expansion, which is consistent with prior studies , 41. IntFinally, the least frequent haplogroups in our sample, accounting for a total of 1.4%, are derived from the Q-M242* polymorphism, which is confined to the American continent and Amerindian populations –45. SuchIn conclusion, our data brings biological evidence to historical records stating the importance of intercontinental arrivals to Northeast Brazil since the colonial period. Through the analyses of 45 Y-chromosome SNPs, we demonstrated that Iberian ancestry is represented in the majority of individuals. Still, the presence of other non-western European lineages is a strong indicator of the continued presence of multiple historically relevant occupations. Furthermore, the frequency of Middle Eastern haplogroups may suggest more recent immigrations, while common African Bantu lineages probably reflect the transatlantic slave trade. Overall, these results reveal the complex structure of the ancestral male genetic background of the Brazilian Northeast, and contribute to the knowledge of South American demographic history.We tested a total of 280 unrelated male samples from the Northeastern region of Brazil, distributed in eight states as follows: i) 82 from Piauí, ii) 46 from Ceará; iii) 118 from Rio Grande do Norte; iv) 15 from Paraíba; v) nine from Pernambuco; vi) two from Alagoas; vii) two from Sergipe; viii) and six from Bahia, as shown in Fig. In total, 45 SNPs were analysed in this work Fig. . SNP typFST) were computed using the Arlequin software v.3.5.2.2  analysis and haplogroup frequency distribution analysis (Pearson’s Chi-squared test) were performed on R software v.3.5.3 (Haplogroup frequencies were determined by direct counting. Population genetics parameters such as comparisons, diversity values and population pairwise genetic distances (.3.5.2.2 . FST val v.3.5.3 . Haplogr v.3.5.3 , 22. For v.3.5.3  into ourAll data generated or analysed during this study are included in the supplementary information files.Additional file 1. Genotypic and SNP data. All genotypes found for the samples analysed in this study as well as chosen SNP information can be found in Additional file 1.


Trachidermus fasciatus) is a small catadromous fish, and has been listed as a second-class state protected aquatic animal since 1988 in China. To elucidate the underlying mechanism of population genetic structuring and genetic adaptations to local environments, RAD tags were sequenced for 202 individuals in nine populations across the range of T. fasciatus in China. The pairwise FST values over 9,271 filtered SNPs were significant except that between Dongying and Weifang. All the genetic clustering analysis revealed significant population structure with high support for eight distinct genetic clusters. Both the minor allele frequency spectra and Ne estimations suggested extremely small Ne in some populations , which might result from recent population bottleneck. The strong genetic structure can be partly attributed to genetic drift and habitat fragmentation, likely due to the anthropogenic activities. Annotations of candidate adaptive loci suggested that genes involved in metabolism, development, and osmoregulation were critical for adaptation to spatially heterogenous environment of local populations. In the context of anthropogenic activities and environmental change, results of the present population genomic work provided important contributions to the understanding of genetic differentiation and adaptation to changing environments.Understanding the patterns of genetic diversity and adaptation across species’ range is crucial to assess its long-term persistence and determine appropriate conservation measures. The impacts of human activities on the genetic diversity and genetic adaptation to heterogeneous environments remain poorly understood in the marine realm. The roughskin sculpin ( Ne). Ne is a key parameter to understand evolutionary processes and the viability of the overexploited and endangered populations as it determines the rate of genetic drift and inbreeding , belonging to the family Cottidae, is a small carnivorous catadromous fish dwelling on the bottom of water  from catadromous to noncatadromous. Considering its great biological, ecological, and economic importance in China, T. fasciatus had been included in the List of the Wildlife under Special State Protection as a second-class state protected aquatic animal by Chinese government in 1988.The roughskin sculpin  of T. fasciatus, likely due to the lack of power of genome-wide genotyping methods. Moreover, previous studies restricted to neutral markers can provide only limited insight to the mechanisms of local adaptation. Across its widely distributed range for T. fasciatus, the temperature, salinity, the levels of pollution, and pathogen conditions are largely different  is one of the rapid and cost-effective methods for genome-wide single nucleotide polymorphism (SNP) discovery and genotyping of nonmodel species without reference genomes .A total of 202 individuals of 013–2017  online. Raw reads from Illumina runs were demultiplexed into separated files according to individuals’ indexes. Reads with adaptors were removed using Cutadapt , and furRAD contigs were assembled using an optimized method as implemented in RADassembler . This pihttps://github.com/lyl8086/VCF_filter; Last accessed June 5, 2019): 1) only biallelic SNPs were retained; 2) SNP quality ≥ 30; 3) minimum coverage depth for an individual ≥ 6; 4) minimum genotype quality (GQ) for an individual ≥ 15; 5) minimum individual coverage for a population ≥ 12, that is at least 12 individuals were required to be genotyped for a SNP in each population; 6) total missing rate for all individuals of each SNP ≤ 0.1; 7) maximum observed heterozygosity (HO) for each population ≤ 0.5; 8) minor allele frequency (MAF) ≥ 0.05, but SNPs with MAF ≥ 0.2 in at least one population were retained. The final VCF file was converted into other formats necessary for subsequent analyses using PGDSpider 2.0.1.0 , observed (HO) and expected heterozygosity (HE) and FIS, were estimated using “populations” in Stacks. Pairwise genetic differentiation (FST) between populations and their significance were evaluated using Arlequin 3.5.2.2 (FCT) and differentiation among populations within groups (FSC), three groups were defined considering both the geographic origin of populations  and geographic distance among them  was tested for values ranging from 1 to 10 with ten replications for each tested K value. In order to accurately estimate the true number of subpopulations, four supervised estimators  of K implemented in StructureSelector  method implemented in the R package Adegenet 2.1.0  for each population was calculated with NeEstimator v2  were applied. Furthermore, in order to provide an illustration of the extent to which populations exhibit an L-shaped distribution, MAF spectrum for each population were plotted in R 3.5.0 (Contemporary effective population size (mator v2 . This pr R 3.5.0 . MAF spe R 3.5.0 . Deviati R 3.5.0 . In ordeFST-based method implemented in BayeScan 2.1 (FST outliers. BayeScan identifies outlier loci with a Bayesian model that decomposing FST coefficients into a population-specific component (beta) shared by all loci, and a locus-specific component  shared by all the populations using a logistic regression. BayeScan was performed using default parameters, and outlier loci were identified using a posterior odds of 10 with a 5% FDR. Secondly, a genotype–environment association method as implemented in Bayenv 2.0  was used. Because minor alleles may also be useful for identifying selection in naturally occurring organisms  were used for downstream structure analysis.RAD sequencing of 202 HO and HE average over the filtered 13,681 loci varied across the nine sampling locations  were similar to HE, and FIS were not high across all the sites. Qingdao showed the lowest HO, HE, and Pi, indicating the lowest genetic diversity among the nine populations. Rongcheng, Wendeng, Qingdao, and Fuyang displayed generally low level of percentage of polymorphic loci (46.69–58.48%), suggesting that lots of SNPs were monomorphic with one allele fixed, likely resulting from small populations after bottlenecks.Estimates of FST values over 9,271 SNPs between populations were significant (P < 0.0014 after B-Y FDR correction), except that between Dongying and Weifang. FST ranged from –0.0038 between the two closest populations (Dongying and Weifang) to 0.1916 between Qingdao and Fuyang . The hierarchical AMOVA .Most of the pairwise d Fuyang . The FSTal AMOVA  showed sK = 8 generated by Clumpak followed the patterns as indicated by the pairwise FST values: Dongying and Weifang formed into one group, and the other seven populations came out as distinct groups respectively  in comparison with other populations.The analysis using Admixture and StructureSelector indicated that there were highest supports for eight genetic groups  to infinite  of stickleback genome. These genes were found to be involved in diverse functions, and were putatively involved in local adaptive processes of T. fasciatus. Using Blast2Go, 74 loci were annotated and assigned to 138 GO terms. Most of the candidate loci containing SNPs putatively under selection could be categorized as having functions associated with catalytic activity, binding and hydrolase activity functions; cellular metabolic processes, biological regulation, development and response to simulation; or transport, signal transduction, cell communication and biosynthetic process , MU2 , MU3 (Qinhuangdao), MU4 (Dongying & Weifang), MU5 (Rongcheng), MU6 (Wendeng), MU7 (Qingdao), and MU8 (Fuyang). The Delta K method could identify high-level genetic structure, which indicated the highest support for three major groups  between six clusters using outlier loci were listed in the Structure results based on neutral loci were similar to that on all loci (data not shown). Similarly, there was also high support for eight genetic clusters in nine populations of s K = 3, a, the sis K = 3, . Therebys K = 6, c, corresT. fasciatus populations by integrating data of putatively neutral and adaptive genetic variation at the genomic level. This is the first use of genome-wide SNP markers to assess a broad-scale population differentiation and signatures of selection in the T. fasciatus. Our study contributes to the growing body of literature identifying significant population structure and local adaptation in marine systems and has important implications for the conservation management of T. fasciatus and other endangered marine species.The present population genomic study delineated fine population genetic characteristics of the nine FST values were higher than those based on microsatellites. A good understanding of how ecological variants of fishes can impact their population structure will provide more comprehensive implications for conservation management and decision-making.The results provided a higher resolution of population structure compared that identified in our previous study based on microsatellite data . In partT. fasciatus depends on different habitats including oceans, estuaries and rivers , and the census populations size were sharply reduced  has been sharply reduced since recent decades, and the drying-up of the river course has occurred frequently. Specially, the Rongcheng population is mainly distributed in Swan Lake. Swan Lake is a semienclosed coastal lagoon, the major environment perturbations might result in bottlenecks and small Ne values, which will further strengthen genetic drift effects.In the presence of limited gene flow between fragmented populations, random genetic drift is expected to increase genetic differentiation among populations . Current species . Theorete > 1000 . The smaWith the advantages of identification of genome-wide polymorphic markers, RAD sequencing can facilitate insight into studies of the ecological, evolutionary and conservation genomics , especiaT. fasciatus to the local environment. One locus of interest is located in the Glycerol-3-phosphate dehydrogenase (GPDH) gene, which encodes enzyme that catalyzes the reversible redox conversion of dihydroxyacetone phosphate to sn-glycerol 3-phosphate. GPDH is a major link between carbohydrate metabolism and lipid metabolism, and is crucial in electron transport chain of the mitochondria. Indeed, T. fasciatus expends a vast amount of energy during the catadromous and reproduction processes. Previous physiological studies indicated that the digestive system of T. fasciatus was degenerative since its migration from freshwater to saltwater  signaling pathway  might have been lived completely in saltwater, which has been confirmed by otolith microchemistry analysis , which was involved in MHC protein binding, antigen processing and presentation of exogenous peptide antigen. TAP1 functions as the membrane transporter associated with antigen processing that are involved in transport of peptides that bind to MHC molecules , six AUs (based on adaptive loci), and three ESUs . CUs with lower genetic diversity and Ne require high conservation priority, such as MU7 (Qingdao) & MU8 (Fuyang) or AU5 (Qingdao) & AU6 (Fuyang), which show high level of isolation with other populations. In particular, MU6 (Wendeng) deserves special attention given the lowest Ne, which is related to low fitness risk. However, the census population size of MU6 might be relative large, which could be much larger than that of MU8  for the breeding and growth of T. fasciatus also suggested its sensitive to environmental changes. Currently it is difficult for T. fasciatus to survive, migrate, breed, and completed the entire life cycle  might show higher fitness  than recruited in clusters found elsewhere.Another government-promoted conservation action on ancement . A largeT. fasciatus populations, fine population genetic structure and genomic signatures of adaptations across the species’ range were detected. Genetic clustering analysis revealed significant population structure with high support for eight distinct clusters among nine populations. The strong genetic structure could be attributed to habitat fragmentation and genetic drift due to anthropogenic activities. A substantial number of genetic variants appeared to be under differential selective pressure across the range of T. fasciatus. Annotations of candidate adaptive loci suggested that the genes involved in diverse functions like metabolism, development and osmoregulation are critical for the adaptation to environmental heterogeneity across the species’ range.By analysis of genome-wide data of nine Genome Biology and Evolution online.Supplementary_Material_evz118Click here for additional data file.


Laodelphax striatellus. In this study, we characterized the alimentary canal microbiota of L. striatellus collected from seven regions in China by sequencing 16S rDNA. The insects harbored a rich diversity of microbes, mainly consisted of bacteria from phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Tenericutes. The composition and abundance of microbiota were more similar as the geographic distance decreased between the populations and clustered by geographic location into three groups: temperate, subtropical, and tropical populations. Although the abundance and species of microbes differed among the populations, the various major microbes for each population performed similar functions based on a clusters of orthologous group analysis. Greater diversity in ecological factors in different regions might lead to higher microbial diversity, thus enabling L. striatellus to adapt or tolerate various extreme environments to avoid the cost of long-distance migration. Moreover, the abundance of various metabolic functions in the Kaifeng populations might contribute to higher fecundity in L. striatellus.Almost all insects harbor commensal bacteria in the alimentary canal lumen or within cells and often play a pivotal role in their host’s development, evolution, and environmental adaptation. However, little is known about the alimentary canal microbiota and their functions in sap-sucking insect pests of crops, which can damage plants by removing plant sap and by transmitting various plant viruses, especially in the small brown planthopper,  Acetobacter pomorum in the gut can fully restore the developmental delay of malnourished Drosophila larvae by activating the insulin signaling pathway, which controls developmental rate, body size, energy metabolism, and intestinal stem cell activity  is one of the most destructive pests of a wide range of crops, especially rice, wheat, and maize, and is widely distributed in Asian, European, and American countries . The region V3 + V4 in 16S rDNA was amplified with specific primers 5′-ACTCCTACGG GAGGCAGCA-3′ and 5′-GGACTACHVGGGTWTCTAAT-3′. Then the PCR products were purified using VAHTS DNA clean beads  to construct libraries by Biomarker Technologies Company . Finally, the libraries were paired-end (PE) sequenced on the Illumina HiSeq 2500 platform at Biomarker Technologies Company.DNA was extracted from approximately 30 alimentary canals (per replication) of R2 means the higher interpretation degree of group variance, and the larger group variance means that the P-value is less than 0.05, indicating the high reliability of the test. The sample Alpha diversity index was evaluated using Mothur (version v.1.30) software. Shannon indices were used to measure species diversity. The higher Shannon index value indicates the higher species diversity of the sample.According to the overlap relationship between the PE reads, the PE-tagged sequence data from the HiSeq sequencing were merged into one tag by FLASH software to produce the raw data . For obtQIIME software was used for each OTU taxonomic level to assign the sequence with the highest abundance as the representative sequence. Multiple sequence alignment of all replications and the Python language tool were used to construct the phylogenetic tree. Each branch in the tree represents one genus, and the evolutionary distance between two genera was taken as the sum of the branch lengths from each of the two terminal nodes to their common ancestor. On the basis of the abundance of microbial species in all samples, the SparCC method was used to infer positive or negative correlations among the different microbes in all replications at the genus level .L. striatellus from different regions, we created high-quality Venn diagrams using the microbial effective sequences tags in the R statistical environment  effect size (LEfSe) was used to find biomarkers that differed significantly among the different groups . The mett-test was used to test for differences in abundance by functional category on different populations, with P-value threshold of 0.05. We then calculated the abundance of different genera by function and tested for significant differences among the seven populations using SPSS version 22 .Gene functions for microbial communities in the different populations were inferred from clusters of orthologous groups (COG) information using PICRUSt software . First, L. striatellus were collected from different regions for rearing separately on rice seedlings in a clean growth chamber at 25°C and 60% relative humidity with 16-h light/8-h dark to establish separate homogeneous populations. For different populations, one female and one male were fed separately into a growth chamber. Ten pairs of insects were reared separately to determine the number of developmental days per instar and count the number of eggs for each pair.More than 20 pairs of adult T-test was used to compare the sets of data.After a pair of adults laid eggs, we measured the development time of offspring at each age and calculated the time when the offspring changed age. When the offspring had matured adults, we counted their number to assess the fertility of different populations. Males and females were also counted from each pair for calculating ratio of females to males. More than two sets of data were statistically analyzed with one-way ANOVA by SPSS statistics 22. The L. striatellus from the seven populations were binned to 115 OTUs after filtering out spliced reads, chloroplast, and mitochondrial sequences  analysis showed that the differences between the groups were much greater than within the groups, indicating that microbial diversity differed greatly among the seven regions (Kosakonia and Undibacterium) and one specific to Kaifeng (Candidatus_Endoecteinascidia)  . This reulations .Acinetobacter, Arsenophonus, Glutamicibacter, and Staphylococcus was significantly higher in the Kaifeng population , subtropical , and tropical (Sanya) , which showed higher diverse than other groups, were clustered separately  . Populat (Sanya) . This obparately . The UPGd branch .L. striatellus using a heat map analysis. The results showed that the major microbial richness varied among the different regions, and the microbes clustered into three clades. The genera from Sanya, which were of high abundance, mainly grouped with clades I’ those from Beijing, Baoding, and Kaifeng mainly grouped with clades II and III; and those from Huai’an, Changsha, and Kunming grouped in clades III  were specific to the tropical region, three genera  to the temperate, and none to the subtropical  found for the Kaifeng population  from 10 regions in China differed in diversity in response to environmental heterogeneity  from six locations across Europe was also determined by the environment and may be relevant to host ecology  consists of wheat and gramineous weeds (Bromus japonicus). However, insects from Kaifeng  obtain nutrients from wheat, rice, and gramineous weeds   , subtropical populations  and tropical population (Sanya). Since the pathways . The annusgalli) , and theequalis) . HoweverAcrosternum hilare; Hemiptera: Pentatomidae) and harlequin cabbage bug  are significantly influenced by temperature  was also associated with a cold, wet climate  and negatively influences the insecticide resistance of the host brown planthopper   . Thus, wL. striatellus varied among the different populations, the main microbial functions in the seven populations were similar in abundance. The relative abundance of Rickettsia was higher in Kunming and Sanya populations. However, functional analysis showed that these two genera had the same functional types and similar functional abundance, which had little effect on the main microbial functions. In particular, the abundance of many metabolic functions in the Kaifeng insects was significantly higher than that in the other populations. Based on our observation in recent years, the population density of L. striatellus in Kaifeng, up to about 2,000/m2 in May and June, was 10 times greater than in the other regions , subtropical populations , and tropical population (Sanya), which might be due to factors such as latitude, temperature, and humidity; i.e., greater diversity in the ecological factors in different regions might result in microbial diversity in the alimentary canal. Although the abundance and species of microbes differed among the populations, the various major microbes for each population performed similar functions based on a COG analysis. Perhaps the microbiota could assist L. striatellus in adapting or tolerating various extreme environments to avoid the cost of long-distance migration. Moreover, the abundance of various metabolic functions in the Kaifeng populations might contribute to the higher fecundity and ratio of females to males of L. striatellus, which in turn may be associated with the outbreaks of viral diseases in this region in recent years. Our results imply that the insect’s alimentary canal microbiota is not only a factor in determining insect optimal survival in diverse environments but also an important driving force for its population expansion and outbreaks.Clearly, the plant sap-sucking insect PRJNA486401 and PRJNA559703).The nucleotide sequences used in this study were deposited in the National Center for Biotechnology Information (SRA accession: XW designed the experiments and reviewed the manuscript. XW and WL analyzed the data and wrote the manuscript. WL, XZ, NW, and YR processed the samples, and isolated and sequenced DNA. WL, XZ, NW, and YR collected the samples and edited the manuscript. All authors read and approved the final manuscript.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.