Bathymodiolus platifrons  widely distributed in chemosynthesis‐based ecosystems in the Northwest Pacific. Three mitochondrial genes  and 6,398 genomewide single nucleotide polymorphisms (SNPs) were obtained from 110 individuals from four hydrothermal vents and two methane seeps. When using the three mitochondrial genes, nearly no genetic differentiation was detected for B. platifrons in the Northwest Pacific. Nevertheless, when using SNP datasets, all individuals in the South China Sea (SCS) and three individuals in Sagami Bay (SB) together formed one genetic cluster that was distinct from the remaining individuals. Such genetic divergence indicated a genetic barrier to gene flow between the SCS and the open Northwest Pacific, resulting in the co‐occurrence of two cryptic semi‐isolated lineages. When using 125 outlier SNPs identified focusing on individuals in the Okinawa Trough (OT) and SB, a minor genetic subdivision was detected between individuals in the southern OT (S‐OT) and those in the middle OT (M‐OT) and SB. This result indicated that, although under the influence of the Kuroshio Current and the North Pacific Intermediate Water, subtle geographic barriers may exist between the S‐OT and the M‐OT. Introgression analyses based on these outlier SNPs revealed that Hatoma Knoll in the S‐OT represents a possible contact zone for individuals in the OT‐SB region. Furthermore, migration dynamic analyses uncovered stronger gene flow from Dai‐yon Yonaguni Knoll in the S‐OT to the other local populations, compared to the reverse directions. Taken together, the present study offered novel perspectives on the genetic connectivity of B. platifrons mussels, revealing the potential interaction of ocean currents and geographic barriers with adaption and reproductive isolation in shaping their migration patterns and genetic differentiation in the Northwest Pacific.Studying population genetics of deep‐sea animals helps us understand their history of habitat colonization and population divergence. Here, we report a population genetic study of the deep‐sea mussel  BathymoB. platifrons  and a methane seep in the South China Sea (SCS)  Natsushima and Kaiyo of Japan Agency for Marine‐Earth Science and Technology (JAMSTEC), or by the manned deep‐submergence vehicle Jiaolong on‐board the Chinese R/V Xiangyanghong 9  and BP_atp6R (5′‐GGTTCTACCACCATCCTCG‐3′), the universal primer pair LCO1490 and HCO2198  were performed using a Mastercycler Gradient thermocycler  with the following program: 2 min initial denaturation at 94°C, 30 cycles of 30 s denaturation at 94°C, 30 s annealing at 50°C, 30 s extension at 72°C, and a final extension for 10 min at 72°C. The PCR products were separated using 1.0% agarose gel electrophoresis and purified using the Zymoclean™ Gel DNA Recovery Kit  following the manufacturer's protocol. Purified PCR products were bidirectionally sequenced on an ABI PRISM® 3730xl DNA Analyzer. Sequences obtained were visually checked and assembled into contigs using the DNASTAR Lasergene package . Each mitochondrial gene from all individuals was aligned using MUSCLE v.3.8.31 , and nucleotide diversity (π) of each mitochondrial gene were estimated using Arlequin v.3.5.2.2  model Kimura,  implemen Tajima,  and Fu's FS Fu, , implemeFST between local populations using Arlequin, with 10,000 permutations applied to test for significance. Furthermore, to investigate the population structure, TCS haplotype networks were reconstructed for each mitochondrial gene using POPART v.1.7 .Based on the recognition sites of the BsaXI enzyme, 2b‐RAD tags were extracted from the draft genome of Hobs) ≤0.5 among all individuals to avoid inclusion of paralogs  as assessed by the exact test implemented in Arlequin for each local population, with 100,000 dememorization steps followed by 1,000,000 steps in a Markov chain; (h) number of SNPs per locus ≤3.Detection of SNPs was carried out using the ref_map.pl pipeline implemented in Stacks v.1.41 , Hobs, π, and inbreeding coefficient (FIS), were calculate using Stacks. Two neutrality tests, including Tajima's D  and geographic distance (km), also with 10,000 permutations applied to test for significance. The approximate geographic distance between each pair of local populations was measured using the Latitude/Longitude Distance Calculator (http://jan.ucc.nau.edu/~cvm/latlongdist.html). An intermediate geographic point between the two sampling sites of IN and OH was separately generated to simplify the calculation.Values of pairwise 2.7K was set from 1 to 6, each with five replicates, using a burn‐in of 100,000 followed by 1,000,000 iterations. The optimal K was evaluated using STRUCTURE HARVESTER v.0.6.94  were defined according to the results of STRUCTURE and PCA based on the second outlier SNP dataset   for atp6  negative for local populations of B. platifrons  were detected to have a significantly (p < 0.05) positive value of Tajima's D in each local population (Supporting Information Table p < 0.05) negative value of Fu and Li's D* in the local populations of JR, DK, HK, and OH; nevertheless, no polymorphic loci were detected to have a value of Fu and Li's D* with statistical significance in the local population of IR or IN  detected only between the local population of JR and all the others in the OT‐SB region (FST range: 0.0139 to 0.0206) after Bonferroni correction  of B. platifrons in the Northwest Pacific  revealed the occurrence of two genetic groups  were identified  were identified  detected in all pairwise evaluations between the local population of JR and those in the OT‐SB region (FST range: 0.1953 to 0.2785) after Bonferroni correction  detected in all pairwise estimations between local populations in the OT‐SB region after Bonferroni correction  as well as those calculated between pairs of the three local populations in the M‐OT and SB (FST range: 0.0417 to 0.0778) were smaller  were larger  of B. platifrons in the Northwest Pacific  revealed two genetic groups  of B. platifrons in the OT‐SB region  uncovered two genetic groups , eight individuals (53.3%) in the local population of HK were identified to have mixed ancestry as either migrants or hybrid descendants  ranged from 0.0373 to 0.7033  revealed extensive gene flow between local populations of n Figure . Althoug4B. platifrons in the Northwest Pacific. By using the three concatenated mitochondrial genes, no significant genetic differentiation was detected between any pairs of the six local populations. Meanwhile, the haplotype network constructed based on each mitochondrial gene also revealed no obvious genetic structure. These results were consistent with previous studies based on mitochondrial markers, indicating a high dispersal capability and a lack of population differentiation of B. platifrons in the Northwest Pacific  in the Northwest Pacific or trapping a preexisting genetic barrier. Similar situations have also been reported from other Bathymodiolus mussels, such as those from the mid‐Atlantic Ridge , as well as the Pacific Deep Water through the Luzon Strait  toward the SCS, the M‐OT, and SB than that in the reverse directions. Such migration patterns indicated that the local populations in the S‐OT tended to be the source populations of B. platifrons in the Northwest Pacific, which was in agreement with the lower values of Hobs and π as well as higher values of FIS exhibited by the local populations in the S‐OT compared to those of the other local populations. However, this deduction should be treated with caution, since additional populations of B. platifrons elsewhere in the Northwest Pacific likely remain unsampled.The result of migration dynamic analyses revealed that gene flow of 5B. platifrons in the Northwest Pacific were identified in this study, which may have been formed due to the barrier effect of the Luzon Strait or the contact zone been trapped by it. Among them, one lineage is mainly distributed in the semi‐enclosed marginal SCS, while the other is mainly distributed across the OT‐SB region. In addition, a fine‐scale population structure was detected for B. platifrons in the OT‐SB region, which might be due to the existence of subtle geographic barriers between the S‐OT and the M‐OT. The occurrence of three individuals with a high genetic affinity to those in JR in the OH site of SB remains puzzling and warrants further investigation to test whether this is related to differences in habitat preferences . The mixed genetic backgrounds detected in the local population of HK in the S‐OT indicated that this area may represent a contact zone for larvae from different locations in the OT‐SB region. Moreover, the local populations in the S‐OT  may serve as a potential source of B. platifrons in the Northwest Pacific.By using genomewide SNPs rather than mitochondrial genes, two cryptic semi‐isolated lineages of B. platifrons. Together with several recent studies  database of NCBI under the accession number of SRP149310.The mitochondrial gene sequences were deposited in GenBank of National Center for Biotechnology Information (NCBI) under the accession numbers of JWQ, PYQ, TX, and JS conceived this project. JWQ, JS, HKW, CC, MN, and DF collected the samples. TX extracted DNA, conducted 2b‐RAD libraries under the guidance of JL, SW, and ZB, performed bioinformatics analyses, and drafted the manuscript. RJ helped interpret the roles of ocean currents in population connectivity among mussel populations. All authors contributed to improvement of the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Vicugna vicugna) is the most representative wild ungulate of the high Andes of South America with two recognized morphological subspecies, V. v. mensalis in the north and V. v. vicugna in the south of its distribution. Current vicuña population size  is the result of population recovery programs established in response to 500 years of overexploitation. Despite the vicuña’s ecosystemic, economic and social importance, studies about their genetic variation and history are limited and geographically restricted. Here, we present a comprehensive assessment of the genetic diversity of vicuña based on samples collected throughout its distribution range corresponding to eleven localities in Peru and five in Chile representing V. v. mensalis, plus four localities each in Argentina and Chile representing V. v. vicugna. Analysis of mitochondrial DNA and microsatellite markers show contrasting results regarding differentiation between the two vicuña types with mitochondrial haplotypes supporting subspecies differentiation, albeit with only a few mutational steps separating the two subspecies. In contrast, microsatellite markers show that vicuña genetic variation is best explained as an isolation by distance pattern where populations on opposite ends of the distribution present different allelic compositions, but the intermediate populations present a variety of alleles shared by both extreme forms. Demographic characterization of the species evidenced a simultaneous and strong reduction in the effective population size in all localities supporting the existence of a unique, large ancestral population  as recently as the mid-Holocene. Furthermore, the genetic variation observed across all localities is better explained by a model of gene flow interconnecting them rather than only by genetic drift. Consequently, we propose space “continuous” Management Units for vicuña as populations exhibit differentiation by distance and spatial autocorrelation linked to sex biased dispersal instead of population fragmentation or geographical barriers across the distribution.The vicuña ( Vicugna vicugna) is the most representative wild ungulate of the Andean high plateau in South America  and the southern vicuña (V. v. vicugna). The subspecies mensalis, inhabits the ‘moist puna,’ is smaller and darker than the southern vicuña and is distinguished primarily by the long growth of hair on the chest  range-wide phylogeographic structure linked with vicuña evolutionary history; (2) patterns of molecular genetic structure among vicuñas; and (3) links between patterns of genetic variation with phylogeographic history and barriers to gene flow. We further utilize this evidence to (4) describe and contrast the evolutionary history and patterns of gene flow among these populations in order to propose effective MUs for the species at broad scale.Samples were collected throughout the current distributional range of the vicuña  followinV. v. mensalis; as well as four Argentine and four Chilean localities currently designated as V. v. vicugna (n = 4), muscle (n = 2), blood (n = 333), and feces (n = 37). All samples were stored at -80°C in the Laboratorio de Genómica y Biodiversidad, Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad del Bío-Bío, Chillán, Chile or at CONOPA in Lima, Peru. Total genomic DNA was extracted from blood using the Wizard Genomic DNA Purification Kit . DNA from skin and muscle samples was purified using proteinase K digestion and a standard phenol–chloroform protocol . Thermocycling conditions were: 95°C for 10 min, followed by 30–35 cycles of 94°C for 45 s, 62°C for 45 s, 72°C for 45 s, then 72°C for 5 min. PCR products were purified using the GeneClean Turbo for PCR Kit (Bio101) following the manufacturer’s instructions. Products were sequenced in forward and reverse directions using BigDye chemistry on an ABI Prism 3100 semiautomated DNA analyzer, and consensus sequences were generated and aligned using Geneious v.9.1.5 . The final alignment was trimmed to 328 bp beginning at the 5′ left domain of the d-loop.Three hundred and eighty-five base pairs of the left domain of the mitochondrial control region (mtDNA-CR) was amplified using the camelid and vicuña-specific primers LthrArtio (5′- GGT CCT GTA AGC CGA AAA AGG A-3′), H15998V (5′-CCA GCT TCA ATT GAT TTG ACT GCG-3′), Loop007V (5′-GTA CTA AAA GAG AAT TTT ATG TC-3′), H362 (5′-GGT TTC ACG CGG CAT GGT GAT T-3′) . AmplifiS) and haplotypes (nh), haplotype diversity (h) (k) were estimated using ARLEQUIN 3.5.1.2 (The number of segregating sites (sity (h) , nucleot 3.5.1.2 . A stati 3.5.1.2  with defFifteen autosomal dinucleotide microsatellite loci , and expected heterozygosity (HE). The inbreeding coefficient FIS was estimated following We identified fecal samples that came from the same individual by searching for matching microsatellite genotypes using the Excel Microsatellite Toolkit  and elimK clusters. We tested values of K between 1 and 23, running STRUCTURE five times for each value of K, and using Evanno’s ΔK method to determine the most suitable number of clusters . We followed r = 0  and to test if the observed and expected r-values were significantly different form each other. Lastly, we tested for the presence of isolation by distance (IBD) using Mantel tests between the matrix of geographic distances between localities and the matrix of Fst estimated for either the mtDNA or the microsatellite loci.We tested for spatial autocorrelation in the data at various distance classes using Genalex 6.5 . We usedV. v. mensalis and the other of the individuals with the highest probability of belonging to V. v. vicugna based on the STRUCTURE results for K = 2 and with a minimum Q threshold of 0.75 indicating the corresponding population ancestry. The second dataset consisted of one population composed of all individuals with a Q-value > 0.5 based on the STRUCTURE results for K = 2 and the other population made up of all the remaining individuals. The third dataset consisted of the data for each locality separately. Each analysis was run independently three times for 200,000 iterations of the Markov Chain Monte Carlo algorithm with the first 10% of the simulations discarded as burn-in.We tested the relative likelihood of a gene flow vs. a genetic drift model using the program 2MOD  Supplem. Three dThe mtDNA of the northern and southern regions (one group per subspecies) were used to estimate Tajima’s D and Fu’s Fs statistics  with 10,-2.5 and 10-4.5  was for various markers in different populations, with ten populations showing no deviation from HWE for any locus. The remaining population showed between one and a maximum of eight locus not in HWE, with an overall mean of 2 loci per locality not in HWE. Overall no linkage disequilibrium was found in the sampling localities, with the exception of 12% o the microsatellite pairwise comparisons in Lauca, and one microsatellite pairwise comparison in Lagunillas, another one in Ascotán, and two in Santa Catalina . Estimates of genetic diversity excluding these loci were not significantly different from those estimated with all loci, thus, we kept these markers for further analyses . We found consistently moderate to high levels of genetic diversity (mean expected heterozygosity ranged from 0.45 to 0.78) and high values for allelic richness (mean RA ranged from 2.67 to 7.53) (h = 0.794) among 376 partial sequences of the 5′ end of the control region. Among variable sites, 37 (71.15%) were parsimonious informative. Haplotype (h) and nucleotide diversity (π) ranged between 0–0.92 and 0–0.35   relativeto 7.53) , guanacoto 7.53) , guigna to 7.53) . In the d 0–0.35 . GenBankK = 2 based on the ΔK method , while 13 were of mixed origin, and 2 were assigned to the Northern cluster . Overall, most of the individuals of mixed or incongruent heritage in both clusters were sampled in the localities of AN, SS and SA, which correspond to the contact zone between the North and South regions phi-st (FST ranged from low (0.037) to high values (0.612), with the greatest divergence observed between the CC and PI localities. D pairwise divergence values were larger than the FST values with significant values ranging between 0.19 and 0.95 and the highest divergence observed between the AY and PI localities. Furthermore, we also found a significant negative correlation  in average heterozygosity and pairwise FST between sampling localities suggesting that the observed divergence between localities may be driven by genetic drift rather than the build up of unique mutations present at the different localities phi-st . Significalities .FST including and excluding hybrids were 0.0779 and 0.0998, respectively, and both were significant (p < 0.005). A division between the North and South regions was also observed with the haplotype network calculated with the 57 mtDNA Control Region haplotypes  and 25 were found only in V. v. vicugna (South), while four where shared between the two subspecies and thus found in both the North and South Regions . Additionally, haplotypes 27, 29, and 30 found only on V. v. mensalis grouped together with those of V. v. vicugna, while haplotypes 33 and 34 found only on V. v. vicugna clustered with to the V. v. mensalis haplotypes. The 57 haplotypes are connected with a maximum of 48 mutational steps, with most genetic variation localized regionally and the main link between the two geographic regions of the network diverging by five mutations. Consistent with the STRUCTURE results, the haplotypes shared between the two regions occurred in the sampling localities in the contact zone .When dividing the samples into two groups representing the two clusters identified by STRUCTURE reflecting the two subspecies, the plotypes . Among tp < 0.001) and at both hierarchical levels, implying dispersal is limited at the spatial scale, with greater resemblance between individuals at shorter distances and decreasing resemblance as distance increases. Although both males and females presented a spatial autocorrelation pattern indicative of isolation by distance, these were significantly different from each other  with females presenting almost twice as much similarity than males at the smaller distance class . None the less, as geographical distance increases the differences between r-values in both sexes almost disappears . Equivalent results were obtained for this analysis using the microsatellite data .We found significant spatial autocorrelation among individuals for the species as whole  and Tajima’s D  consistent with a pattern of population expansion, however, Tajima’s D was not significant. For the South region both tests were not significant  suggesting a stable population history. These results are consistent with the presence of a major haplotype in the northern region (Hap2), while in the southern region two distantly related haplotypes occur at moderate to high frequency (Hap31 and 35) with many low frequency haplotypes in between them. The BSP analysis for each subspecies clearly shows a higher effective population size for V. v. mensalis  no demographic change, (2) bottleneck and (3) population expansion. The three independent runs for each sampling locality presented Gelman and Rubin’s statistics lower than 1.2. In all cases MSVAR detected evidence for major effective population size decline at all localities, consistent with current or recent small census sizes . All locV. v. mensalis) or the southern  groups, respectively. This apparent differentiation is supported by the divergence analysis that results in a large and significant ΦCT of 0.4. Nevertheless, some haplotypes are shared between the two groups of vicuñas, particularly at localities in the contact zone between 18°S  and 21°S . The data presented here evidences gene flow between the two vicuña types as reflected by haplotype sharing between animals of each type  deriving from localities at or near the contact zone between the two types . This is further supported the observation of phenotypically mensalis individuals that carry vicugna haplotypes , 29 , and 30  and conversely, phenotypically vicugna individuals that carry mensalis haplotypes .Here, we present the most comprehensive analysis to date of genetic variation in vicuña across the species range. The molecular markers used present contrasting patterns regarding vicuña evolutionary history with conflicting evidence regarding the split of the species into two different taxonomic units. For instance, analysis of the mtDNA haplotypes largely supports a split into two vicuña groups, with each group dominated by haplotypes specific to the northern , and supports a contact zone between the two clusters where animals in that region present composite genotypes at intermediate allele frequencies to those observed in more northern or southern vicuñas  including the extant Lake Poopó and the Coipasa and Uyuni saltpans. However, following its disappearance, a narrow area between the mountains to the east of the Atacama Desert and the Uyuni saltpan opened enabling contact between the two vicuña groups, which otherwise would have been separated by the presence of this large lake in the Andean altiplano.STRUCTURE analysis of the microsatellite data also identifies the presence of two clusters in the dataset  where individuals from localities at one end of the range distribution are more likely to resemble each other than individuals on the opposite end of the range. In such a scenario, instead of two populations being present, the data represents a single population with a gradient of intermediate genotypes between two contrasting extremes, as observed in  vicugna .The change in genetic similarity between animals at increased distance was also observed with the spatial autocorrelation analyses. While splitting the dataset by sex resulted in both groups showing approximately the same isolation by distance pattern, females were more similar to each other at smaller distances  than males. This pattern is consistent with females behaving more philopatric than males, with the later leaving their family groups upon becoming yearlings and form non-territorial bachelor groups which frequently have to move because of conflicts with local males with established territories . Yet, bor-values across further distances. Northern vicuñas inhabit a geographic range with higher habitat productivity and wider dietary resource availability than southern vicuñas. Thus, while northern vicuñas can find food resources in relative proximity . However, what is remarkable, is not only that all extant populations seem to have passed through this bottleneck at approximately same time, they all had a very similar ancestral effective population size . It is likely that a single large vicuña population occupied a wide range across the Andes prior to a relatively recent bottleneck that dramatically reduced the effective population size to less than 1,000 . The main consequence of this event was fragmentation and isolation of previously well connected vicuña populations within their present distribution area (9°S to 29° S) and the small effective population size of pocket populations that survived. This hypothesis is supported by comparison of the model of evolution under genetic drift against a model that also included gene flow and which unambiguously showed that the latter better explains the extant genetic variation. While this analysis does not indicate modern connectivity between these localities, as has been previously suggested for vicuña , it suppThe average bottleneck estimate across the vicuña populations is ∼7,600 YBP, but the range of variation from ∼700–125,000 YBP  reflect V. vicugna maintained its genetic and taxonomic identity through time. Moreover, this identity remained despite the human population expansion in South America  and their specialization in hunting vicuña (and guanaco)  , as wellguanaco) , 2012. Mguanaco) .Establishing MUs is difficult because of differentiation by distance and the influence of genetics pools at sampled localities. MU determination depends on geographic areas with independent demographic dynamics between populations, whose individuals present a well-defined genetic structure and low migration rates . At the Two captive populations were included in this study Abra Pampa (Argentina) and Picotani (Peru). These populations present lower genetic variation as measured with both types of molecular markers than their immediate wild neighboring populations . The Abra Pampa Experimental Station has had captive vicuñas since 1933 see . AlthougVicugna vicugna to date. These results suggest that the two morphological variants of vicuña, i.e., the northern V. v. mensalis and the southern V. v. vicugna, were until recently closely interconnected with each other, or probably part of a single large population that passed through a strong bottleneck that left small isolated populations across a vast geographic range. Furthermore, extant vicuña genetic variation is better explained by a model of isolation by distance rather than by two discrete populations. However, given the extent of the vicuña geographic range and variation in the environments therein, it is likely that vicuña populations differ to some extent due to adaptation to local environmental variables. We propose the use of continuous MU for vicuña conservation and that this data serve as a baseline for genetic variation monitoring to avoid further loss of genetic diversity in captivity.Here, we present the most extensive genetic analysis of Samples were collected following guidelines of the American Society of Mammalogists . SpecifiJM developed the ideas and obtained funding for the project. JW, BG, and JM collected the samples. JV, JC, RR, NA, and AC conducted the DNA analyses. AC, AA, DG-U, VV, and PO-tW analyzed the data. JM, BG, DG-U, JW, and PO-tW wrote the manuscript. All authors read, commented on 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.


Tropheus moorii across a wide sandy bay is linked to observed differences in mate preferences between the two lineages. This hypothesis predicts bi-directional nuclear gene flow and was rejected by congruent patterns of introgression in mtDNA, AFLP and microsatellite markers, with admixture confined to the populations west of the bay. This pattern can be explained on the basis of habitat changes in the course of lake level fluctuations, which first facilitated the development of a symmetric admixture zone including the area corresponding to the present sand bay and then shaped asymmetry by causing local extinctions and cessation of gene flow when this area became once more inhabitable. This conforms with previous assumptions that habitat dynamics are a primary determinant of population-level evolution in Tropheus. In this respect, Tropheus may be representative of species whose preferred habitat is subject to frequent re-structuring.Introgression patterns between divergent lineages are often characterized by asymmetry in the direction and among-marker variation in the extent of gene flow, and therefore inform on the mechanisms involved in differentiation and speciation. In the present study, we test the hypothesis that unidirectional introgression between two phenotypically and genetically distinct lineages of the littoral, rock-dwelling cichlid fish  Patterns of introgression between diverging lineages inform about mechanisms involved in differentiation and speciation. For instance, asymmetries in the direction of gene flow and among loci can elucidate various pre- and postzygotic processes that determine the evolutionary trajectories of the involved lineages. In animals, potential sources of asymmetric introgression include asymmetries in mate choice, driven, for example, by lineage-specific mating preferences and dominance relationships , geograpTropheus moorii, mitochondrial and phenotypic variation suggested unidirectional introgression between two genetically distinct color morphs across a major habitat barrier , which argues against cytonuclear incompatibilities and sex-specific  hybrid mortality that could otherwise explain the lack of introgressed mitochondrial haplotypes in the populations east of Mbete Bay. Lineage-specific dispersal behavior, i.e., a higher dispersal propensity of yellow-blotch than of bluish Tropheus, is rejected by similar and high levels of genetic differentiation among bluish-morph populations west of the bay  with a strict molecular clock . The various datasets required different run lengths, but all analyses were run until post-burn-in ESS for all parameters exceeded 200. The median and corresponding 95% highest posterior density (HPD) intervals were visualized with Tracer 1.6 (available from http://beast.bio.ed.ac.uk/tracer).Population size trajectories through time were inferred by means of Bayesian skyline plots BSPs;  in BEASTP values . Nuclear and mtDNA markers consistently reflected a cline in admixture between bluish and yellow-blotch Tropheus genomes in the populations west of Mbete Bay and an abrupt drop in admixture proportions in the populations east of the bay  was distinct and significant, except for weak or no differentiation across all three marker types between Chaitika and Nakaku as well as between Tanganyika Lodge and Kasakalawe Lodge; in both cases, sampling locations are connected by continuous rocky habitat. Additionally, mitochondrial differentiation estimated by ϕST was non-significant in three comparisons among adjacent populations  and narrowly missed significance based on FST estimates in three other population pairs .Both mitochondrial and nuclear markers displayed a high level of genetic diversity within and differentiation among populations  to a divergence time parameter of 0.489 (mean of four IMa2 runs). Higher divergence time parameter estimates (mean estimates of 0.905–1.016) were obtained between the “bluish lineage” haplotype groups from the different admixed locations west of Mbete Bay , as opposed to “pure bluish populations” (Nakaku and Chaitika) and “pure yellow-blotch populations” . Although Mbete Bay represents a divide between genetic and phenotypic clusters for several cichlid species  contains supplementary material, which is available to authorized users.Suppl material 3Suppl material 2Suppl material 1


Cycas taiwaniana complex consists of six endangered species that are endemic to South China. In this study, we investigated the relationship between phylogeographic history and the genetic structure of the C. taiwaniana complex. To estimate the phylogeographic history of the complex, we assessed the genetic structure and divergence time, and performed phylogenetic and demographic historical analyses. Two chloroplast DNA intergenic regions (cpDNA), two single-copy nuclear genes (SCNGs), and six microsatellite loci (SSR) were sequenced for 18 populations. The SCNG data indicated a high genetic diversity within populations, a low genetic diversity among populations, and significant genetic differentiation among populations. Significant phylogeographical structure was detected. Structure and phylogenetic analyses both revealed that the 18 populations of the C. taiwaniana complex have two main lineages, which were estimated to diverge in the Middle Pleistocene. We propose that Cycas fairylakea was incorporated into Cycas szechuanensis and that the other populations, which are mainly located on Hainan Island, merged into one lineage. Bayesian skyline plot analyses revealed that the C. taiwaniana complex experienced a recent decline, suggesting that the complex probably experienced a bottleneck event. We infer that the genetic structure of the C. taiwaniana complex has been affected by Pleistocene climate shifts, sea-level oscillations, and human activities. In addition to providing new insights into the evolutionary legacy of the genus, the genetic characterizations will be useful for the conservation of Cycas species.Historical geology, climatic oscillations, and seed dispersal capabilities are thought to influence the population dynamics and genetics of plants, especially for distribution-restricted and threatened species. Investigating the genetic resources within and among taxa is a prerequisite for conservation management. The  Geological events and climate fluctuations during the Pleistocene have profoundly shaped the distribution and speciation of organisms . Most trCycas L. is the sole genus of the Cycadaceae, and its 117 species are mainly distributed in tropical and subtropical regions of Southeast and East Asia, East Africa, Oceania, and Madagascar  and gymnosperms (naked seeds) . Therefodagascar . Cycas id genera . South Ca  to investigate the patterns of genetic variation of the C. taiwaniana complex; 2) to examine the historical population demography relative to climate fluctuations; and 3) to reconstruct phylogenetic relationships and estimate divergence time among populations. The genetic data can contribute to the management of the endangered species and can provide insights into the genetic structure and history of the C. taiwaniana complex.In this study, we used two chloroplast intergenic spacers , two single-copy nuclear genes (SCNGs), and six microsatellite markers  to explore how the Quaternary climate oscillations may have influenced the current population structure of the C. taiwaniana complex were obtained from 18 populations in Hainan, Guangdong, and Fujian provinces of South China  method  method .trnS-trnG and atpB-rbcL) and two unpublished single-copy nuclear genes (EX929503 and FJ393265) were sequenced for complete analysis  . For SCNGs, the PCR reactions contained 5 µl of DNA template, 0.8 µl of each primer (forward and reverse), 10 µl of 2× Taq Master Mix (Takara), and 3.4 µl of double-distilled water. The PCR amplification of cpDNA began with an initial denaturation of 4 min at 95°C; followed by 35 cycles of 94°C for 45 s, annealing at 53–55°C for 60 s, and 72°C for 90 s; and a final extension at 72°C for 10 min. For SCNGs, the procedure began with an initial denaturation of 4 min at 95°C; followed by 34 cycles of 95°C for 30 s, 55–58°C for 60 s, and 72°C for 60 s; and a final extension at 72°C for 10 min. The product sizes were determined on 1.5% agarose gels in TBE buffer stained with ethidium bromide, and the products were then purified with the Gel Extraction Mini Kit. The PCR products were sequenced using an ABI 3730XL automated sequencer with the BigDye(r) Terminator v3.1 Cycle sequencing kit (Applied Biosystems). The sequences in this study have been deposited in GenBank .Two chloroplast intergenic spacers (O genes)  using TBCycas  and haplotype diversity (Hd) were calculated with DnaSP, version 5.0 (HT), average within-population diversity (HS), and two parameters of genetic differentiation (GST and NST) were estimated using Permut version 2.0  with Arlequin, version 3.0  and raggedness index as well as their significance values were calculated using Arlequin, version 3.0. Finally, Bayesian skyline plot (BSP) analyses were performed with BEAST, version 2.3.0  dataset. Prior to combining the sequences, the congruence was examined using the partition–homogeneity test (7 generations.Bayesian inference (BI) as implemented in MrBayes, version 3.2.6  was usedity test . The besity test . FurtherCycas was unavailable, we applied the evolutionary rates for seed plants of 1.01 × 10−9 substitutions per site per year for synonymous sites for cpDNA and 6.1 × 10−9 (5.1–7.1 × 10−9) substitutions per site per year for synonymous sites for SCNGs . Divergence times among populations were estimated using BEAST, version 2.3.0 . A Yule or SCNGs . The MCMsion 1.7  with ESSNA), effective number of alleles (AE), expected heterozygosity (HE), observed heterozygosity (HO), fixation index (F), and percentage of polymorphic loci (PPB). Gene flow (Nm) between pairwise populations was estimated based on Nm = (1 − FST)/4 FST (ST) was calculated with Arlequin, version 3.0 (FST/(1 − FST). The geographic distance between sampling locations was determined using the R package /4 FST , and thesion 3.0 . Exact tsion 3.0 . AMOVA wsion 3.0  accordinsion 3.0  was perfsion 3.0  accordin package .C. taiwaniana complex was assessed in several ways. First, the individual-based principal coordinate analysis (PCoA) was performed with GenALEx, version 6.5 (K) from K = 1 to 18 were run 20 times. Each run contained a burn-in of 105 iterations and 105 replications of MCMC. The most likely number of K was estimated in Structure Harvest, version online, version 0.6.9  implemented with Wilcoxon test method was selected in bottleneck analysis. We also tested for a bottleneck by using a mode shift model (We performed bottleneck effect test of 18 populations in the ft model  in BOTTLft model .trnS-trnG and 717 bp for atpB-rbcL) with a rate of homogeneity . A total of 17 polymorphic sites and nine haplotypes (H1–H7) across 177 individuals were identified (C. changjiangensis had the highest haplotype diversity (Hd = 0.64), and CHA2 had the highest nucleotide diversity (Pi = 0.0011) (HS = 0.095) was much lower than the total diversity (HT = 0.772) . For FJ, 29 haplotypes with 32 polymorphism loci were identified . The overall estimated genetic diversity was larger for EX  than for FJ . The total genetic diversity in both EX and FJ was high. NST was significantly larger than GST, indicating a phylogeographic structure of haplotype distribution  and expected heterozygosity (HE) across all populations were 0.620 and 0.565, respectively (F) were negative in populations HAI2, TAI1, FAIRY1, FAIRY2, FAIRY3, FAIRY4, FAIRY5, CHA3, and SZE1, suggesting that these nine populations were deviated from HWE , supporting an overall pattern of IBD in the C. taiwaniana complex  indicated that the populations were grouped into three clades. However, there were some admixtures when the K value was 3. Low genetic composition was detected among C. lingshuigensis, C. hainanensis, C. taiwaniana, and C. changjiangensis. The UPGMA and structure analyses were supported by the PCoA  had a significant excess of heterozygosity and deviated from mutation-drift equilibrium (P = 0.1). The phylogenetic tree constructed from the concatenated cpDNA–scnDNA dataset was partly unresolved with C. pectinata as the outgroup (C. taiwaniana complex were obtained with high support: one clade comprised C. fairylakea and C. szechuanensis (clade A), and the second clade (clade B) comprised C. hainaniana, C. lingshuigensis, C. changjiangensis, and C. taiwaniana  of 170 plant species  was much larger than the average within-population diversity (HS = 0.095), suggesting that the level of genetic differentiation among populations is high (GST = 0.977 and NST = 0.952). In addition, NST was significantly greater than GST, which indicates the presence of a distinct phylogeographical structure in the C. taiwaniana complex. The FST values obtained by cpDNA were also much greater than the average value of 0.67 previously estimated from other maternally inherited markers  is well supported with PP = 1. The other clade B  located in Hainan Island and Fujian Province is supported by PP = 0.97. However, the relationship within C. hainanensis, C. changjiangensis, C. lingshuigensis, and C. taiwaniana is not very clear. The clade including species from Hainan Island is geographically isolated from species from South China, indicating that limited genetic exchange may have promoted the divergence of the two clades. Hainan Island was separated from Mainland China by the Qiongzhou Strait, which formed approximately 2.5–25 million years ago  in research . The divresearch . The recresearch . A compiciation” .Cycas species had different population dynamics to respond to glacial and interglacial influences. Cycas debaoensis . It is possible that refugia, temperature changes, and recent human activities caused the contractions in the C. taiwaniana complex.Although the Pleistocene ice events were evident in Europe , the clibaoensis , Cycas sicipinna , Cycas mipinnata , and Cycnanensis  have expungensis  have expC. taiwaniana complex than many other Cycas species in China. However, severe habitat loss and illegal exploitation for trading and ornament have severely threatened the existing populations of C. taiwaniana complex. C. hainanensis, C. changjiangensis, C. lingshuigensis, and C. taiwaniana are regarded as endangered (EN), while C. szechuanensis and C. fairylakea are critically endangered (CR) species . Therefore, we proposed to protect all the existing wild populations of C. taiwaniana complex and their habitats in situ. Some species and populations with unique haplotypes, such as C. hainanensis, C. changjiangensis, C. lingshuigensis, C. szechuanensis, C. fairylakea, and populations LING1, HAI2, FAIRY1, FAIRY5, and CHA1-4 should be given the highest priority protection. We also recommend the ex situ conservation for those critically endangered species (C. szechuanensis and C. fairylakea) and important populations  by collecting seeds or seedlings from wild  and the National Natural Science Foundation of China (grant no. 31070304).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.


We found that Mas-VBIA has similar genetic diversity to present-day Asian populations and higher diversity than that of contemporary Europeans. Our studies revealed close genetic links between the Mas-VBIA and two other Iron Age populations from the Jutland peninsula and from Kowalewko, located in western Poland. We disclosed the genetic connection between the Mas-VBIA and ancient Pontic-Caspian steppe groups. Similar connections were absent in the chronologically earlier Kowalewko and Jutland peninsula populations. The collected results seem to be consistent with the historical narrative that assumed that the Goths originated in southern Scandinavia; then, at least part of the Goth population moved south through the territory of contemporary Poland towards the Black Sea region, where they mixed with local populations and formed the Chernyakhov culture. Finally, a fraction of the Chernyakhov population returned to the southeast region of present-day Poland and established the archaeological formation called the “Masłomęcz group”.For years, the issues related to the origin of the Goths and their early migrations in the Iron Age have been a matter of hot debate among archaeologists. Unfortunately, the lack of new independent data has precluded the evaluation of the existing hypothesis. To overcome this problem, we initiated systematic studies of the populations inhabiting the contemporary territory of Poland during the Iron Age. Here, we present an analysis of mitochondrial DNA isolated from 27 individuals  excavated from an Iron Age cemetery (dated to the 2 As a result, an increasingly precise map of the genetic history of a human population is generated. Despite the progress in our understanding of the demographic processes that took place in Europe since its first peopling, the map still has a numerous blank spaces4. They are especially frequent in the case of the Early Bronze Age (EBA) and later periods, when more complex demographic and cultural events occurred11. Importantly, not all geographical regions have been sufficiently sampled12. The majority of data are confined to Central-West Europe, whereas Eastern Europe is scarcely represented1.During the last decade, genetics and genomics have become new driving forces that have stimulated the rapid development of studies on the human past. Archaeogenomics has quickly evolved from analyses of single individuals to studies involving dozens of subjects13. The works of Chylenski et al. and Lorkiewicz et al.15 suggest that the matrilineal genetic makeup of people living in the Vistula River Basin during the Early Neolithic (EN) and the Middle Neolithic (MN) better resembled the Funnel Beaker culture populations becherkultur), which were a mixture of Mesolithic Hunter Gatherers and Neolithic farmers, than the Linear Pottery culture-associated populations , which were mostly composed of Neolithic farmers17. The studies of autosomal DNA of the Globular Amphora culture (GAC) population inhabiting the Vistula River Basin during the Late Neolithic (LN) showed no presence of Yamnaya steppe herder ancestry (YAM)18. At the same time, the Corded Ware culture (CWC) groups that later succeeded GAC have as much as 75% of their genetic makeup attributed to YAM . During that time, cremation of the dead became a common custom; thus, our knowledge on these peoples is based almost exclusively on archaeological artifacts. They indicate that during the BA, several local cultures developed in this region20. All of them were, to a large extent, related to each other, and consequently, they are usually considered as one, called the Lusatian culture, existing from the EBA to the early Iron Age (IA). At the beginning of the IA (approximately 600 B.C.), an alternative to Lusatian culture, called the Pomeranian culture (PC), was developed. The PC dominated the region and spread between the Oder and Bug Rivers until the end of the 3rd century B.C. , the lowland of contemporary central Poland was occupied by the Przeworsk culture  was connected with further cultural and demographic transformations in the region that is present-day Poland20. A new grouping called the Wielbark culture was established, most likely under the influence of Goths and Gepids. To learn more about these processes, we have commenced systematic studies of the populations inhabiting the territory corresponding to present-day Poland in the first centuries AD. Our initial analyses were focused on a large group of individuals living in the region between Oder and Vistula Rivers in the IA 21. Biological material was collected from a biritual cemetery located in central Wielkopolska  in Kowalewko. Former archeological and anthropological studies showed that approximately 500 people were buried there between 1 and 200 A.D. Our recently published report revealed a high genetic diversity of the Kow-OVIA8. Interestingly, women and men from Kowalewko had a significantly different genetic history. In general, the collected data showed how the interactions between newcomers (most likely Goths) and autochthonous communities derived from other cultural traditions could shape the new, local genetic substructure.The turn of the epochs  and is believed to represent the next stage of the Goth migrations. The genetic makeup of individuals living at that time in this region has not yet been determined. The biological material was collected from one of the richest IA cemeteries located in east Poland near Masłomęcz village. The cemetery is attributed to the IA Wielbark culture, i.e., to the same culture as the recently characterized site at Kowalewko. This relation in terms of culture and time with geographical separation provided a unique opportunity to obtain the imprint of the demographic processes underlying the genetic variability of the population living during the IA in Central-East Europe. Our analysis involved 27 individuals. We assigned mitochondrial DNA (mtDNA) haplogroups and determined complete mtDNA genome sequences for all of them. We found a high intrapopulation genetic diversity of the studied group. It showed close matrilineal relationship with two other IA populations living, respectively, at the Jutland peninsula and between the Oder and Vistula Rivers. In contrast to our earlier findings concerning Kowalewko (contemporary Western Poland), we found no evidence of distinct genetic history between females and males from Masłomęcz. This observation suggests that both populations were formed or functioned in different ways. Thus, the data presented here provide new insights into the processes that led to the formation of region-specific genetic substructures within the populations inhabiting the Vistula River Basin in the IA.This paper is a continuation of our previous work and concentrates on the human population that lived between the Vistula and Bug Rivers (contemporary eastern Poland) in the IA. The studied group is younger than the Kow-OVIA (2Masłomęcz), geographical context (between Vistula and Bug rivers) and time (the IA)], included 27 individuals, whose burial sites previously had been thoroughly characterized archaeologically and anthropologically . The studied group, called the Mas-VBIA [abbreviation describing the exact location  based on two fragments of the HVS (hypervariable sequence) region of mitochondrial genome (regions: HVS-I between nucleotide position (np) 16033 and 16365 and HVS-II between np 73 and 340)  Fig.  and nucl40) Fig. . The HD To exclude the possibility that the presence of two potentially maternally related individuals in the relatively small tested group will affect the results of our consecutive analyzes, we removed the two individuals from the tested group - one from each pair with identical mtDNA sequences.mtDNA haplogroup H was the most common in the Mas-VBIA 32%); T2 and U5a were the second most common (16% each). To place the Mas-VBIA in the Central European space-time, we compared mtDNA haplogroup frequencies within the set of populations that lived in this region from the Mesolithic until the present , for details, see Materials and methods, Tables %; T2 andTo look wider on the Mas-VBIA ancestry and its relationship with other contemporary populations, we assembled selected ancient populations in the European Population Transect dataset (EPT)   to one group because of their high genetic homogeneity and analyzed 41 different combinations of populations’ groupings . In the first step, we determined an optimal division of the populations from CEPT, excluding the HGCN and CEM. The division was considered to be optimal if the intragroup variability (Fsc) was minimal and intergroup variability (Fct) was maximal. Considering earlier observations made by other authorsNext, to determine to what extent particular populations contributed to the genetic structure of the contemporary Central European population, we performed an analogous analysis, but the studied group was extended by the CEM (CEPT with excluded HGCN). Once again, the populations of the EN/MN  were treated as one group, and we analyzed 91 combinations of the other fossil populations and the CEM  was influenced by four major cultures: BBC, CWC, UC and YAM Fig. . However8. In this report, we focused on the maternal genetic history of the Mas-VBIA group that lived in the eastern part of contemporary Poland (the region located between the Vistula and Bug Rivers) during the 2nd to 4th century A.D. We found that the intrapopulation diversity of the Mas-VBIA (π = 0.016263) was beyond expectations. It was as high, as is currently observed for Asian populations (π between 0.013 and 0.017), and exceeded the values typical for non-isolated European populations (π between 0.006 and 0.01), as well as the intrapopulation diversity previously determined for its western counterpart – the Kow-OVIA (π = 0.0079). However, it should be noted here that due to the postmortem DNA damage, the diversity estimates could be inflated. Accordingly, one can hypothesize that people living between the Vistula and Bug Rivers in the IA formed genetically diversified, non-isolated populations. The haplogroup frequencies observed for the Mas-VBIA were similar to those reported for the Kow-OVIA. Haplogroup H frequency was intermediate between CWC and BBC and lower than in CEM. Interestingly, the frequencies of the mtDNA haplogroups U5a (characteristic for Eastern Hunter Gatherer groups) and U5b (characteristic for Western Hunter Gatherer groups) were a significantly different in the Mas-VBIA and other IA populations studied so far (the JIA and Kow-OVIA). The Mas-VBIA was characterized by the absence of mtDNA haplogroup U5b and the high frequency of mtDNA haplogroup U5a (14.8%), whereas in the JIA and Kow-OVIA, mtDNA haplogroup U5b was more prevalent than U5a. As noticed previously8, the domination of U5b over U5a in Central Europe was observed only during the IA. Therefore, either the process that led to the temporal increase of U5b mtDNA haplogroup frequency did not affect regions east of Vistula, or its effects were subsequently removed by another demographic event.To extend our knowledge on the issues described above, we initiated broad genetic studies of the populations inhabiting the territory of present-day Poland in the IA. In our earlier paper, we characterized the maternal genetic makeup of the individuals living in the west part of contemporary Polandet al.32 showed that there are clear genetic links, even at the mtDNA level, between the central European CWC and BBC populations and the YAM. However, if close genetic connections between the Mas-VBIA and YAM were a result of the LN migratory events, the same should be observed in the remaining IA populations of the region. Therefore, we propose that the presence of the Pontic-Caspian steppe genetic component in the Mas-VBIA is a result of the migratory event or events that occurred in the IA period. Considering the high prevalence of the mtDNA haplogroup U5a in the Mas-VBIA, one can hypothesize that the direction of this movement was from east to west.All high dimensional analyses of genetic diversity  within the Mas-VBIA and other ancient and extant populations were consistent and showed that the Mas-VBIA was closely connected with the Kow-OVIA and had a similar genetic structure to that of Central European LN/EBA populations. Within the group of three IA Central European populations, the Mas-VBIA was the closest related with the Kow-OVIA, and the distance between the JIA and Mas-VBIA was shorter than that between the JIA and Kow-OVIA. Furthermore, the Mas-VBIA had close genetic connections with the YAM, located in the eastern parts of Europe. We did not observe these connections in either the Kow-OVIA or the JIA. Earlier, Haak 14. Thus, here we provide an additional line of evidence that Danubian Neolithic populations were a common genetic background of all Central-North and Central-East populations in Europe.Another interesting observation was the genetic proximity between the BEC and virtually all subsequent populations . This proximity was most evident when shared mtDNA haplotypes were analyzed. We found that the haplotypes shared between all LN and IA populations were nearly always present in the BEC. The BEC is part of a TRB cultural horizon that spread from North to Central Europe during the MN in the form of the sequence of Danubian populations33, and the second, with the spread of the local Oksywie culture that appeared at the Baltic seashore at approximately the 2nd century B.C34. In addition, there are at least two hypotheses concerning the origin of Goths. One of them assumes that the homeland of the Goths was located in the southernmost part of the Germanic territories other than in Scandinavia35. Considering the results obtained for both the Kow-OVIA and Mas-VBIA, one can assume that they are, to a large extent, consistent with the postulated chronology of early migrations of Goths and their settlement in Central-East Europe. The high genetic diversity of the Mas-VBIA strongly corresponds with the suggested role of Masłomęcz at that time22. The archaeological findings indicate that in the 2nd and 3rd centuries A.D., it was one of the major cultural and political Goth centers. In addition, the genetic relationships reported here between the Mas-VBIA and both earlier characterized IA populations (the Kow-OVIA and JIA) support the opinion that southern Scandinavia was the homeland of the Goths. According to some authors, the process of their migration through the contemporary territory of Poland, which was connected with the spread of the Wielbark culture, can be divided into the following 6 stages, named with the letters A-F . Then, they spread west along the Baltic seashore (stage B). In stage C, the Goths moved south and ousted the Przeworsk culture from the northwest part of contemporary Poland, including Kowalewko (1st and 2nd centuries A.D.). The Goth’s settlements established in this area could have a rather temporal character suggested by the fact that the genetic structure of the Kow-OVIA population was biased by sex. Males from the Kow-OVIA were closely related with the JIA and females with Neolithic farmers8 . During the next stage (D), the Goths spread east of the Vistula, and then they migrated along the Vistula and Bug Rivers towards the Black See and established the Chernyakhov culture by mixing with Pontic–Caspian steppe populations (stage E). Finally, a part of the Chernyakhov culture population moved back and established a large settlement near Masłomęcz called the “Masłomęcz group”   Fig. . Such a 36. Genomic libraries were prepared following the protocol of Meyer37, with omission of the initial sonication step due to natural fragmentation of aDNA. In order to maximize the chance of amplification of each unique DNA template molecule, six separate PCR reactions were set up for each library. PCR amplifications were performed in 25 µl, with 3 µl of the DNA library template, 12.5 µl of 1x AmpliTaq Gold® 360 Master Mix (Life Technologies), 0.5 µl of indexing primer (10 µM) and 0.5 µl of PCR primer IS4 (10 µM)38. The PCR profile was as follows: initial denaturation , 12–16 cycles of 94 °C (30 s), 60 °C (30 s), 72 °C (45 s) and final extension . PCR reactions for the same library were pooled and purified with AMPure® XP beads (Agencourt-Beckman Coulter)38. The quality and size distribution of the libraries were verified with a High Sensitivity DNA kit and 2100 Bioanalyzer system (Agilent), while the DNA concentration was determined with a Qubit fluorimeter and Qubit dsDNA HS Assay Kit (ThermoFisher Scientific), according to the manufacturer’s protocols.For the purposes of this study, 27 samples (teeth) were obtained from 27 individuals from the Masłomęcz archeological site  and TruSeq SBS Kits v5-GA (Illumina) was applied. Seven to eight libraries were pooled per lane. On average, 6.3 mln 75 bp-long reads were collected per library. Libraries with small human DNA content below 5% were enriched in mtDNA or whole human genome sequences using MYbaits  and an in-solution hybridization capture procedure, according to the recommendations of the manufacturer. mtDNA enrichment was performed with MYTObaits (Arbor Biosciences), a set of 80-bp long tiling probes designed based on rCRS (Cambridge Reference Sequence). Two rounds of enrichment were applied, each followed by 14 amplification cycles. Enriched libraries were sequenced using GAIIx (Illumina) again. On average, 6 mln 75 bp-long reads were collected per mtDNA-enriched library and human genome content was in the range of 55–74%  were filtered with AdapterRemoval45 and estimated whether the human DNA damage parameters for each sample were typical for aDNA: (i) λ, the fraction of nucleotides positioned in single-stranded DNA overhangs context, (ii) δs, C → T deamination probability in the single-stranded overhangs context, and (iii) δd, C → T deamination probability in the double-stranded DNA context  16033–16365, and HVS-II np 73–340) (Supplementary Table Haplotype diversity (HD) was analyzed with Arlequin ver. 3.5.147 with respect to Phylotree build 17 (http://www.phylotree.org/)48. Only samples with a haplogroup score ≥0.8 and average coverage ≥3 were used in downstream analyses. To assess changes in temporal mtDNA haplogroup frequency from the Mesolithic to the present day, we performed Ward clustering with Euclidean distance on 23 haplogroups . We used a set of 27 samples from this study and 14 ancient populations from Central/North Europe  on haplogroup frequencies of the Mas-VBIA and published ancient mtDNA data of populations from across Europe and West Asia. We called this group EPT (European Population Transect) , with public data from 73 extant populations (Supplementary Table http://R-project.org).Cluster significance was tested by performing 10,000 permutations with the pvclust package in R ver. 3.3.0. A PCA was conducted with prcomp of the vegan package in R ver. 3.3.0 . Additionally, for newly reported samples from this study, no missing nucleotides were allowed in the selected HVS-I range, and at least 3x coverage was expected for 95% of the nucleotides. To examine genetic affinities on the sequence level, we calculated genetic distances (Fst)50 values were calculated in Arlequin ver. 3.5.1 for both datasets separately, with the associated substitution model and gamma values selected with jModel test 0.1 AIC and BIC51. P-values were calculated by performing 10,000 permutations. Genetic distances were visualized on an MDS plot with the metaMDS function from the vegan package in R ver. 3.3.0.Pairwise and Slatkin’s Fst51 (see Supplementary Information).To examine whether genetic affinities between particular populations from CEPT were a result of a shared genetic structure, we have conducted an Analysis of Molecular Variance (AMOVA) in 41 combinations of CEPT populations (excluding HGCN and CEM) (Supplementary Table 29 in CEPT (Supplementary Table 31 in LN/EBA and IA populations (Supplementary Table To further test genetic affinities and account for the temporal succession of archaeological cultures in Central Europe, we conducted an analysis of shared ancestral haplotypes as described inSupplementary TablesSupplementary information


AbstractChironomus “annularius” sensu Diptera, Chironomidae) from three populations of the Caucasus region . We found 17 banding sequences in the Caucasian populations. We observed inversion polymorphism in almost all chromosome arms except for arm G. The genetic distances between all the studied populations of Ch. “annularius” were calculated using Nei criteria (1972). In spite of relative geographic proximity, the genetic distances between populations of the Caucasus are quite large, and they do not form a single cluster of Caucasian populations. The population of the South Caucasus goes to the European cluster, the population of the Central Caucasus goes to the Asian cluster and the population of Eastern Ciscaucasia does not belong to any of the outlined clusters. Principal component analysis (PCA) shows a similar picture. Two of the Caucasian populations do not follow Hardy-Weinberg expectation, there being a marked deficiency of heterozygotes in arms A, B and C, arguably, due to negative selection of heterozygotes or founder effect. All the obtained data are indicative of the complex genetic structure of Caucasian populations of Ch. “annularius” and total complexity microevolution processes occurring in the Caucasus region.The study presents data on the karyotype characteristics and features of chromosomal polymorphism of  Chironomusannularius from the 18th century (Ch.annularius name is necessary. The most complete descriptions of Ch.annularius morphology and karyotype were presented by Ch. “annularius” sensu http://www.faunaeur.org) the species is common in Western  and Eastern Europe . Also, according to There are a great number of publications that mention the name of  century . AccordiCh. “annularius” sensu Ch. “annularius” populations using an arbitrary system of chromosome mapping or without any mapping  described the karyotype of  mapping . The karulations , 2012.Ch. “annularius” from one Central Caucasian population.Ch. “annularius” from three Caucasian populations. In addition, it was also very important to compare the chromosomal polymorphism characteristics of Ch. “annularius” from the Caucasus with earlier studies.The aim of the work was to present the description of karyotype characteristics and chromosomal polymorphism of Chironomus in the karyological study. We provide the collection sites and abbreviations of earlier studied populations . The slides of the chromosomes were prepared using the ethanol-orcein technique  of all the studied populations using original and previous data of We performed a principal component analysis   Fig. . The nucCh. “annularius” banding sequences pool. In the studied populations, 15 of those sequences are present, and two banding sequences have been found for the first time, providing 17 banding sequences in the Caucasian populations  and only in the heterozygous state (annA1.5 – 0.139) Tables .Arm B has three banding sequences: annB1, annB2, and annB4  and only in the heterozygous state (annD1.4 – 0.028) Tables , 4.Arm E has two banding sequences: annE1 and annE2 , Siberian, Kazakhstan and Nearctic (USA) populations are available due to PageBreakArm A. Most earlier studied populations , Kazakhstan population and one Siberian population (Pond in Berdsk). The arm is monomorphic in populations of Europe, South Caucasus and rest of the Siberian populations, where only genotypic combinations annC1.1 was present. In addition, the arm is monomorphic in both Nearctic populations where the other genotypic combination annC3.3 was found . The populations of the South Caucasus and Eastern Ciscaucasia are generally close to Asian populations (Siberia and Kazakhstan) by all the parameters of chromosomal polymorphism. The population of Central Caucasus is close to the European population by the average number of heterozygous inversions per larvae, number of banding sequences per population and number of genotypic combinations per population. The percentage of heterozygous larvae in population of Central Caucasus is lowest (72%) among all the Holarctic populations (81-90%) . The homozygotes annA1.1 were observed 2.22 times more frequently than it was expected and heterozygotes annA1.2 should be occurred 2.29 times more frequently than they were observed. One can observe an even more complex picture in the population of Eastern Ciscaucasia where the frequencies of genotypic combinations do not follow Hardy-Weinberg expectation across three arms: arm A , arm B , and arm C . In the arm A the heterozygotes annA1.2 should be occurred 1.78 times more frequently than they were observed, homozygotes annA2.2 were observed 7.17 times more frequently than it was expected, also expected combinations annA2.3 and annA3.3 were not found at all. In arm B the homozygotes annB2.2 and annB4.4 were observed 1.5/2.1 times more frequently than it was expected and heterozygotes annB2.4 should be occurred 3.77 times more frequently than they were observed. Finally, in arm C the homozygotes annC1.1 and annC2.2 were observed 1.5/1.2 times more frequently than it was expected and heterozygotes annC1.2 should be occurred 1.5 times more frequently than they were observed.Among Caucasian populations, the frequencies of genotypic combinations in all arms of PageBreakCh. “annularius” in the South Caucasus for the first time. Earlier , within the radius of ca 100 km. Most of them were drilled in the 50–60s of the 20th century for the aims of animal husbandry. Considering this, we can expect a lot of new records of this species from habitats situated beside those wells. The puddle that served as collection site is quite small  and thus the total size of the population is not so big. Possibly this population is relatively young and just over 50–60 years old. It can be presumed that initially a very small number of individuals from some nearby habitats established this population and the influx of new migrants is not so large. It is quite possible that most part of the larvae here could be relatives and so the inbreeding could occur quite often. Possibly, there was not enough time for the population to come to the equilibrium. Perhaps we see the founder effect that can also explain the observed picture with Hardy-Weinberg expectation.Ch. “annularius” and total complexity of microevolution processes occurring in the Caucasus region. In spite of geographic proximity, one Caucasian population is separated from other populations of the Caucasus at the level of subspecies.All the obtained data are indicative of the complex genetic structure of Caucasian populations of


In this work, we compare the genetic differentiation and structure of populations of two closely related Centaurium species that display a large variation in floral morphology across two geographic regions in Europe. The populations of both species show opposite patterns of genetic differentiation and structure, and the patterns are reversed between the two regions. For both species, we find a strong link between these patterns and the differences in floral morphology observed in the sampled populations. Centaurium species that display large variation in floral morphology across two distinct geographic regions in Europe (mainland Europe and the UK). Our results showed that genetic differences between regions and populations within regions were similar for both species, but that patterns of genetic structure largely depended on the observed variation in floral morphology. Populations of Centaurium erythraea showed higher genetic differentiation and clear isolation by distance (IBD) in mainland Europe, but limited IBD in the UK. Opposite patterns were found in Centaurium littorale, with higher genetic differentiation and significant IBD in populations sampled in the UK and lower genetic differentiation in Continental populations with no pattern of IBD. Overall, these results indicate that variation in floral morphology has a profound impact on structuring of genetic diversity, with populations displaying low levels of herkogamy showing the strongest patterns of genetic structuring and the reverse pattern in populations showing high levels of herkogamy.The genetic diversity and structure of plant populations are determined by the interaction of three distinct processes: gene flow, genetic drift and natural selection. These processes are to some extent dependent on the mating system of plants, which in turn is largely determined by floral morphology and the level of herkogamy in particular. In this study, we used molecular markers to investigate the impact of floral morphology on genetic differentiation and structure in two closely related  The genetic diversity and structure of plant populations are determined by the interaction of gene flow, genetic drift and natural selection, processes that are influenced by the geographic distribution of plant populations and population demography ; 2012 wiDifferences in environmental pressures have been shown to influence plant mating systems, a major determinant of population genetic structure . OutcrosSelfing rates are strongly determined by flower characteristics such as flowering time and floral morphology . One of Changes in flower morphology and evolution to higher rates of selfing during range expansion as a mechanism for reproductive assurance might further reduce the genetic variation in plant populations . ParticuCentaurium species that show large variation in floral morphology across two distinct regions in Europe (UK and mainland Europe)  display a stronger population genetic structure, but less pronounced patterns of IBD in comparison with more herkogamous populations .In this study, we investigated variation in genetic structure of two closely related Centaurium erythraea and C. littorale are both biennial, short-lived herbs that belong to the Gentianaceae. Although both species have a wide distribution in Europe and sometimes can be found in sympatry, they usually grow in different environments , but also some bees  and small flies   . When suanthesis .C. erythraea populations and low levels of herkogamy in UK populations, while opposite patterns have been described for C. littorale  to a fine powder.DNA was extracted using Isogen’s QuickPick DNA extraction kit . Amplified fragment length polymorphism (AFLP) was used to genotype all individuals from each population using the standard protocol of Fis) was set to 0.5 to account for an expected deviation from Hardy–Weinberg genotypic proportions (The dominant (binary) data obtained from AFLP with the four different primer–enzyme combinations were further analysed. The number of polymorphic loci and expected heterozygosity for each population were determined using AFLP-SURV using a Bayesian method with non-uniform prior distribution, which is the default for non-haploid organisms to obtain the most accurate results . To incoportions . DiffereK-value  ranging from 1 to the total number of sampled populations . The optimal K-value was then determined by calculating ΔK as suggested by Based on the AFLP data, population structure was estimated using an admixture model  in the software package STRUCTURE  . A princPT) and the geographic distance separating these populations was evaluated with the Excel package GenAlEx  and the log-transformed geographic distance matrices for all populations of both species separately, split by region (UK or Continent).To assess the relative importance of genetic drift and gene flow between populations on genetic differentiation, the relationship between pairwise population genetic distance (φ GenAlEx . φPT is PT) were calculated using the AFLP data with the excel package ‘GenAlEx’  or region (P = 0.549 and P = 0.347), respectively.Four different primer–enzyme combinations resulted in a total number of 1935 scored loci of which 246 were polymorphic with allele frequencies varying between 1 and 99 % and mean error rates between 0.016 and 0.04 %  and 142 . No signK values for K = 2 for C. littorale and K = 3 for C. erythraea[see]. Differences in the way populations were clustered could be distinguished between the two geographic regions. In C. littorale sampled in mainland Europe  clustered with UK populations rather than with the other Continental populations  were genetically more distant to the other C. littorale populations  from each other in mainland Europe than in the UK  [see]. The average among-population genetic distance between Continental C. littorale populations was 0.198 and 11 out of 28 comparisons had a φPT < 0.15 [see]. In the UK, on the other hand, C. littorale populations were significantly more differentiated from each other with an average pairwise genetic distance of 0.304, although two populations  were remarkably similar to each other (φPT = 0.073). For both species, among-population variation was highest when comparing populations of both regions (average φPT = 0.348 for C. erythraea and φPT = 0.333).Pairwise population genetic distances varied considerably, between 0.082 and 0.564 for C. littorale and C. erythraea differed significantly between both regions, among populations within regions and within populations among all populations (The AMOVA indicated that ulations . For botPT) were significant for C. erythraea populations both in mainland Europe and in the UK  higher levels of herkogamy than C. littorale populations . In contrast, in UK populations of C. littorale, populations had significantly higher levels of herkogamy (P = 0.01) than populations of C. erythraea  (C. erythraea populations in the European mainland were higher (P < 0.001) than those between C. littorale populations . Genetic distances between UK populations of C. erythraea are lower than those between C. littorale populations , but the difference was not significant (P = 0.104)  . Similar= 0.104) .Centaurium species that were sampled in It is generally expected that the geographic distribution and mating system of plant species can shape their population genetic diversity and structure via processes such as gene flow, genetic drift and natural selection  of C. erythraea indicate that adjacent populations are genetically more similar to each other and that dispersal limitation rather than genetic drift is the main determinant of genetic structure , while the sampled populations in the UK were more geographically distant to each other. Reduced levels of herkogamy were found in the same UK populations  (G.0982.13).H.J. and R.B. designed the study, D.S. collected the samples, H.J. and A.M. analysed the data, A.M. wrote the first draft of the paper, all authors read and approved the final draft.None declared.The following additional information is available in the online version of this article—Figure S1. Labelled distribution map of Centaurium erythraea (green) and Centaurium littorale (blue) including study area and sampled populations. Continental populations are denoted by circles while triangles represent populations in the UK.Table S1. Used primer–enzyme combinations for amplified fragment length polymorphism (AFLP) barcoding and the initial number of loci, number of polymorphic loci and mean error rate for each of these combinations.Table S2. Determining the optimal K-value for Centaurium erythraea and Centaurium littorale populations by calculating ΔK with Structure Harvester.Table S3. Pairwise φPT values between Centaurium erythraea populations.Table S4. Pairwise φPT values between Centaurium littorale populations.Supporting MaterialClick here for additional data file.


Grapholita molesta, in its native range of China.Refugial populations in Quaternary glaciations are critical to understanding the evolutionary history and climatic interactions of many extant species. Compared with the well-studied areas of Europe and Northern America, refugia of species in eastern Asia remain largely unknown. Here, we investigated the phylogeographic history of a globally important insect pest, the oriental fruit moth Genetic structure analyses unveiled three distinct groups and a set of populations with admixture. Approximate Bayesian Computation (ABC) analyses support range expansion of this moth from southwest groups of Yunnan and Sichuan to northern and eastern China. A set of admixed populations was found around these two ancestral groups. This pattern of genetic structure points to two refugia located in the Yunnan region and Sichuan Basin. The split of the two refugia was dated to 329.2 thousand years ago in the penultimate glacial period. One of the lineages was exclusively found around the Sichuan Basin, indicating the formation of endemic populations in this refugium. Ecological niche model analysis suggested a shrinking distribution from the LIG period to the MID period in the Sichuan lineage but a wide and stable distribution in the other lineage.Our results for the first time suggest that Yunnan and Sichuan jointly served as two large-scale refugia in eastern Asia in Quaternary glaciations, helping to maintain genetic diversity overall.The online version of this article (10.1186/s12862-018-1269-z) contains supplementary material, which is available to authorized users. Quaternary climatic oscillations (2.6 million years ago–present) have profoundly impacted the current distribution and genetic diversity of extant species , 2. SpecIdentification of refugia has always been challenging , 9. RespRecent developments around hypothesis-based approaches, such as Approximate Bayesian Computation (ABC) , and theThe impacts of Quaternary climate on the distribution and genetic diversity of extant species have been well studied in Europe and North America , 2, 17. Unlike Europe and North America, the climate was warmer in eastern Asia with less area covered by ice sheets in the LGM , 19. PatGrapholita molesta  (Lepidoptera: Tortricidae) is a major pest of stone and pome fruit, especially species of Rosaceae [The oriental fruit moth (OFM), Rosaceae , inflictRosaceae . Larvae Rosaceae , 40. OurRosaceae . A weak Rosaceae . In smalRosaceae . These sRosaceae , 42. In Rosaceae , 44. StrRosaceae . The higIn southwestern China near Yunnan province, a highly diverged mitochondrial lineage was identified in populations from the Sichuan Basin, indicating this area is a putative refugium during glaciations for OFM . Thus, bIn this study, we explored genetic diversity and population structure within and around the original area of OFM in southwestern China. We chose 12 microsatellite loci developed from genome-scale analysis in and two mitochondrial genes in the current study because both sets of genetic markers can reveal genetic differentiation at small and large scales , 41, 44.FST as low as 0.0025 for all populations and 100% probability of detecting an FST as low as 0.005, indicating that the markers genotyped in our study had strong statistical power to investigate population differentiation in OFM , another was formed by populations from Sichuan and two surrounding populations (SC), and the third was formed by seven populations from the northern and eastern region (NE) Fig. .Fig. 1PhBAPS analysis using microsatellites identified a similar genetic pattern. Different clusters from those in the core populations of Yunnan and Sichuan were identified in surrounding populations Fig. . BAPS anSTRUCTURE analysis indicated the optimal number of clusters was two Fig. . IncreasIn DAPC analysis, an inverted V-shaped pattern for the distribution of populations was evident. Populations from Yunnan and Sichuan located at two tips. Populations from NE and AM connected the two groups at the top position Fig. , suggestr = 0.311, p = 0.002), however, when based on mitochondrial genes, no IBD was present . Incongruent between microsatellites and mitochondrial genes in IBD was confirmed by no significant correlation between pairwise FST values calculated from them. Outliers of IBD plots based on mitochondrial genes were found in population pairs involving Sichuan and admixture populations. When we excluded those populations, IBD was present in an analysis based on mitochondrial genes  and significant correlation was found between pairwise FST values calculated from two sets of markers .Finally, we tested isolation by distance and correlation between two sets of genetic markers. Based on microsatellites, there was significant IBD in populations of OFM  for replicate runs of current potential distribution was 0.859 and the standard deviation was 0.01. The modeled current potential distribution of OFM was basically the same to its actual distribution Fig. . OFM is When occurrence sites of Sichuan lineage were used in analysis, the present and past potential distributions of the OFM are shown in Fig. P < 0.01; Additional file P < 0.01; Additional file P = 0.088; Additional file We evaluated the niche overlap between Sichuan lineage Fig.  and the In this study, we investigated the population genetic structure and demographic history of OFM in its purported native range mainly around Yunnan and Sichuan. By dense sampling and using phylogeographical and ecological niche modelling approaches, we were able to identify putative glacial refugia of this species.Both nuclear and mitochondrial markers unveiled strong genetic structure in populations of OFM, as previously found for this species in China . Three dBased on the identified population genetic structure, there are two possible scenarios around OFM refugia. One of these has refugia in locations with highly admixed populations. This pattern has been found in other species , reflecting the fact that populations in refugia areas represent sinks to which other populations disperse. However, the three populations of OFM with the highest levels of admixed clusters are located in higher latitude and altitude regions compared to Yunnan and Sichuan, making them unlikely to represent sinks. Further analysis using DAPC revealed that these admixed populations are at intermediate positions between the Yunnan and Sichuan groups.In species with a single refugium, a star-like haplotype network and decreased genetic diversity from the refugium to newly colonized areas are expected , neitherWe validated refugia by niche modelling the distribution of OFM under palaeoclimates. Species distribution models reconstruct past distribution of species by projecting the relationship between empirical observations and environmental data to earlier time periods using palaeoclimatic data , 47. ThiAs far as we are known, this is the first identification of Yunnan and Sichuan Basin jointly as refugia in East Asia. Both regions located in the eastern Qinghai-Tibet Plateau, which is the highest and largest plateau in the world. Since QTP is known to have been affected by large climatic changes in the Quaternary , regionsBased on mitochondrial genes, we trace the divergence time of OFM between Yunnan and Sichuan to 329.2 kya, which is coincident with the first stage of Penultimate Glacial Maximum (PGM) (333–316 kya)  as estimOur study showed endemic populations of OFM in Sichuan Basin. This indicated the strong influence of climate oscillations on the adaptation of the OFM before PGM, as evidenced by the presence of endemic species in these areas , 51. NicThis study revealed strong genetic structure around the suspected native range of OFM. Two refugial regions of OFM were identified in Yunnan and Sichuan Basin, likely shaped in the first stage of the PGM. Analyses based on both microsatellites and mitochondrial genes showed haplotypes restricted to Sichuan, suggesting an endemic population in this region. The findings suggest a strong impact of the Quaternary climate on the genetic structure of OFM. Further study using genome-wide SNP could help to investigate the structure of populations in this region in more detail and reveal the adaptive mechanism of OFM to local climate oscillations in the Quaternary.Larvae of OFM were sampled from peach orchards on different trees to avoid the collection of siblings. In total 461 individuals were collected from 21 populations across China Table , Fig. 2.cox1 and nad5) with a total length of 1386 bp were sequenced according to the method of [For mitochondrial markers, two protein-coding genes , expected heterozygosity (HE) and the polymorphic information content (PIC) were analyzed using the macros in Microsatellite Tools [AR) of each locus and inbreeding coefficient (FIS) between individuals within each geographical population were detected using FSTAT version 2.9.3 [For microsatellite loci, allele frequencies, observed heterozygosity , total number of mutations (η), number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (Pi), Tajima’s D and average number of nucleotide differences (K) from both single and combined mitochondrial sequences were calculated in DnaSP version 5.10.01 [For mitochondrial genes, polymorphic sites, DNA polymorphism, linkage disequilibrium, the number of polymorphic sites  analyses, to explore the population genetic structure of OFM.A distance. POPTREE2 constructs phylogenetic trees from allele frequency data by using the neighbor-joining (NJ) method and the unweighted pair-group method with arithmetic mean (UPGMA) [First, to reveal the phylogenetic relationships among populations, POPTREE2  was used (UPGMA) .K) was set to 5, 10, 15 and 20 respectively to ensure convergence and consistency of the results because the initial K value may affect the initial assignment of simulation and reduce the possibility of finding only a local mode.Second, a Bayesian analysis of population genetic structure (BAPS) analysis was conducted using BAPS version 6.0  based onK from 1 to 10, and a burn-in of 500,000 iterations followed by 2000,000 Markov Chain Monte Carlo iterations to identify the number of clusters. All results were collected and submitted to the online software STRUCTURE HARVESTER version 0.6.94 [K using both the Ln Pr (X|K) and the Delta (K) methods.Third, the population genetic structure was analyzed by STRUCTURE version 2.3.4  based onn 0.6.94  to deterFourth, discriminant analysis of principal components (DAPC) implemented in the R package ADEGENET 1.4–2  was usedFST/(1-FST)) and geographic distance (ln km) using ade4 version 1.7–4 implemented in R  with 999 replicates. This method was also used to test the correlation between FST values calculated from microsatellites and mitochondrial genes. The values of FST were calculated in FREENA version 4.0 with ENA  model [Finally, IBD was tested in populations of OFM using a Mantel test correlating genetic distance (s) model  for micrs) model .We used SPLITSTREE version 4.14.5  to exploGrapholita dimorpha was used as an outgroup (GenBank Number: KJ671625) [Taking advantage of the mitochondrial molecular clock estimated in insects , we estiJ671625) .We also constructed a haplotype network based on statistical parsimony in the program TCS version 1.21  to reveaFirst, we investigated the population history by estimating the changes in the effective population size over time using a Bayesian skyline plot (BSP)  implemenSecond, we used an ABC method implemented in DIYABC version 2.1.0  to inferIn step 1, the relationships among the three major groups were compared by using the 32 assembled data sets. A total of nine possible scenarios were tested, regarding split and admixture events.In step 2, two competing scenarios regarding the ancestral population were tested based on the results of step 1, of which one population was derived from the admixture of the other two populations. Fifteen datasets that supported the most plausible scenario in step 1 were retained for analysis in this step.In step 3, the origins of four populations with admixture, i.e., GXNN, GDGZ, GSTS, QHHD, were tested individually by adding them to the best scenario of step 2. A hierarchical tournament approach was used in this step , in whicMethods for scenario comparisons and validation are described in . Based oThird, gene flow between Sichuan and Yunnan populations was estimated using the isolation-by-migration (IM) model in IMa2 , 84 baseFor niche modelling, 326 occurrences, throughout its entire distribution range in East Asia, were obtained from the published literature, websites (CABI) and collected samples in our laboratory. Occurrences were rarefied to 178 sites by excluding imprecise points and retaining one occurrence in a grid cell (30 min of a longitude/latitude degree). Seven bioclimatic variables  that mosBoth niche modelling and molecular data revealed that populations around Sichuan Basin were distinct from other regions (see results section). We obtained 49 occurrence sites located in the distribution areas of Sichuan lineage  and clusters from other regions. Figure S6. Scenarios in Step 1 of DIYABC analysis. Figure S7. Scenarios in Step 2 of DIYABC analysis. Figure S8. Scenarios in Step 3–1 of DIYABC analysis. Figure S9. Scenarios in Step 3–2 of DIYABC analysis. 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Nylanderia fulva, which was recently introduced into the United States from South America.Social insects are among the most serious invasive pests in the world, particularly successful at monopolizing environmental resources to outcompete native species and achieve ecological dominance. The invasive success of some social insects is enhanced by their unicolonial structure, under which the presence of numerous queens and the lack of aggression against non-nestmates allow high worker densities, colony growth, and survival while eliminating intra-specific competition. In this study, we investigated the population genetics, colony structure and levels of aggression in the tawny crazy ant, N. fulva is associated with a shift in colony structure. This species exhibits a multicolonial organization in its native range, with colonies clearly separated from one another, whereas it displays a unicolonial system with no clear boundaries among nests in its invasive range. We uncovered an absence of genetic differentiation among populations across the entire invasive range, and a lack of aggressive behaviors towards conspecifics from different nests, even ones separated by several hundreds of kilometers.We found that this species experienced a genetic bottleneck during its invasion lowering its genetic diversity by 60%. Our results show that the introduction of Overall, these results suggest that across its entire invasive range in the U.S.A., this species forms a single supercolony spreading more than 2000 km. In each invasive nest, we found several, up to hundreds, of reproductive queens, each being mated with a single male. The many reproductive queens per nests, together with the free movement of individuals between nests, leads to a relatedness coefficient among nestmate workers close to zero in introduced populations, calling into question the stability of this unicolonial system in which indirect fitness benefits to workers is apparently absent.The online version of this article (10.1186/s12862-018-1336-5) contains supplementary material, which is available to authorized users. Understanding the evolutionary factors affecting population structure and ecological assemblage is a central question in ecology. This question is especially complex in the context of biological invasions, as introductions usually prompt severe shifts in genetic structure and life history traits of invasive species, and profoundly disturb the ecological community of native species , 2. PopuSocial insects are among the most abundant and successful species at invading terrestrial environments. Despite the taxonomic diversity of invasive species, most of them share life history traits that may facilitate their introduction and dominance in new ecosystems . Among tUnicoloniality is a common trait in invasive ant species and several hypotheses have been proposed to account for its evolution in introduced populations , 10. A fSupercolonies may extend across large geographic distances, with populations ultimately consisting of a single, vast supercolony with no aggression towards colony-mates , 23–27. Nylanderia fulva, is native to South America from Brazil to Argentina along the border of Uruguay and Paraguay  coefficients between pairs of nests against the ln of their geographical distance (Slatkin 1993). The significance of the correlation was tested using Mantel tests implemented in GENEPOP ON THE WEB [FactoMineR R package [4 burn-in period and 1 × 105 iterations of the MCMC. The log-likelihood value and the ΔK method [For both native and introduced ranges, the hierarchical partitioning of the genetic diversity among localities, among nests within localities, and within nests was assessed using analysis of molecular variance (AMOVA) implemented in Arlequin . We asseng FSTAT . We inve THE WEB . We visuSlatkin 13. The si package . SimulatK method  implemenK method  were useK method . The ovefij is the frequency of the allele i at the locus j.We estimated the number of queens per nest, the number of matings per queen, genetic relatedness among nestmate workers, and possible production of queens through thelytokous parthenogenesis using samples from the introduced range. The presence of multiple queens per nest was assessed directly from field observations, and polygyny was confirmed genetically when all the worker genotypes from a nest could not be assigned unambiguously to a single queen. We estimated queen mating frequency by establishing artificial subcolonies containing a single queen and ~ 100 workers using Texas colonies. Care was taken to remove all brood to ensure that all the new workers produced in a subcolony were the offspring of the introduced queen. Each subcolony was kept under standard rearing conditions over a three month period or until the queen produced at least eight worker pupae. All mother queens and their newly produced pupae (X ± SD = 10.0 ± 2.6) were genotyped at all 13 microsatellite loci at the end of the experiment. The number of matings per queen was inferred by reconstructing parental genotypes from mother-offspring inferences using the maximum likelihood algorithm implemented in COLONY 1.2 program . As geneRelatedness coefficients (r) among nests were estimated using COANCESTRY v.1.0 , followiSolenopsis invicta), to control for the ability of N. fulva to be aggressive. Each combination was replicated three times, yielding a total of 36 encounter type assays between nestmates, 36 between non-nestmates from the same locality, 18 between non-nestmates from a different locality, and 12 against a fire ant. Aggression levels were compared using ANOVA tests between groups using R software [http://www.inkscape.org/).Within a week of collection, standardized aggression tests were conducted by placing two workers in a 5 cm petri dish arena for 5 min. Workers were not starved before the beginning of the experiment. The arena floor was covered with filter paper to prevent odor transfer between replicates, and the sides were coated with Fluon to prevent escapes. Interactions were scored on a 5-level scale: levels 1 (ants still or huddled together) and 2  were considered non-aggressive behaviors, whereas levels 3 (biting and quickly releasing), 4 (prolonged biting > 3 s) and 5  were considered as agonistic. Aggression tests were conducted with three nests per locality for four localities separated by at least 30 m in the introduced range in Texas Additional file 2:Table S1. List of sample names with information on localities and accession numbers. (PDF 38 kb)Additional file 3:Table S2. Primer sequences, PCR optimization and multiplexing for each of the markers used in our study. This also includes the methods used to estimate detection of null alleles and linkage disequilibrium for the microsatellite markers analyses. (PDF 48 kb)