Enterocytozoon bieneusi is a widespread parasite with high genetic diversity among hosts. Its natural reservoir remains elusive and data on population structure are available only in isolates from primates. Here we describe a population genetic study of 101 E. bieneusi isolates from pigs using sequence analysis of the ribosomal internal transcribed spacer (ITS) and four mini- and microsatellite markers. The presence of strong linkage disequilibrium (LD) and limited genetic recombination indicated a clonal structure for the population. Bayesian inference of phylogeny, structural analysis, and principal coordinates analysis separated the overall population into three subpopulations (SP3 to SP5) with genetic segregation of the isolates at some geographic level. Comparative analysis showed the differentiation of SP3 to SP5 from the two known E. bieneusi subpopulations (SP1 and SP2) from primates. The placement of a human E. bieneusi isolate in pig subpopulation SP4 supported the zoonotic potential of some E. bieneusi isolates. Network analysis showed directed evolution of SP5 to SP3/SP4 and SP1 to SP2. The high LD and low number of inferred recombination events are consistent with the possibility of host adaptation in SP2, SP3, and SP4. In contrast, the reduced LD and high genetic diversity in SP1 and SP5 might be results of broad host range and adaptation to new host environment. The data provide evidence of the potential occurrence of host adaptation in some of E. bieneusi isolates that belong to the zoonotic ITS Group 1. E. bieneusi isolates from pigs in China. The measures of LD and recombination events supported the occurrence of clonal evolution among the isolates from four study areas of China. Three subpopulations (SP3 to SP5) with potentially varied host ranges were identified within the isolates, which were genetically differentiated from two existing primate subpopulations SP1 and SP2. Population genetic analysis indicated that the isolates in SP1 and SP5 with clonal structure might be responsible for the cross-species transmission and thus have zoonotic potential, while the isolates in SP2, SP3, and SP4 with epidemic structure or host-adapted traits might colonize specific hosts. The data revealed the presence of clonality, potential host adaptation, and population differentiation of E. bieneusi in different hosts.This study explored the genetic characteristics of the ITS and four mini- and microsatellite markers and assessed the population structure and substructures in 101  Enterocytozoon bieneusi and whether it undergoes mating and a meiotic cycle are still unclear. E. bieneusi is the most common human microsporidian species and can colonize a variety of other mammals and birds [E. bieneusi among hosts. E. bieneusi exhibits high genetic diversity among isolates from different hosts [E. bieneusi genotypes have been identified in humans, companion animals, livestock, horses, birds, and wildlife based on DNA sequence analysis of the ribosomal internal transcribed spacer (ITS) and the established naming convention [Microsporidia are obligate intracellular eukaryotic parasites that infect a wide range of animals and are closely related to fungi ,2. Genomnd birds . This ubnd birds . Genotypnt hosts ,12. Overnvention ,14. The nvention ,12,15. Hnvention –22.E. bieneusi in animals has been assessed in numerous studies and pigs were recognized as the most significant reservoir [E. bieneusi genome with a length of about 6 Mb [E. bieneusi in primates [E. bieneusi populations from Peru, India, and Nigeria and no apparent geographic segregation of the isolates was observed. Nevertheless, two genetically isolated subpopulations (SP1 and SP2) were identified within the overall population. Significant linkage disequilibrium (LD) and limited recombination in SP1 support a clonal population structure, while the rapid expansion of some specified multilocus genotypes (MLGs) in SP2 obscures the limited genetic exchange because of its epidemic population structure [E. bieneusi in non-human primates from China were similar to those in humans [E. bieneusi in primates need to be substantiated in other hosts.The public health potential of eservoir ,17,23. Hout 6 Mb . Severalout 6 Mb . Coupledprimates –28. In ttructure ,27. Althtructure ,27. PopuE. bieneusi isolates that belong to nine genotypes in zoonotic Group 1 at the subtype level at five genetic loci, to assess the population structure and substructures, and to compare them with similar data from E. bieneusi subpopulations SP1 and SP2 in primates to examine the occurrence and extent of host segregation in different E. bieneusi subpopulations.The objectives of this study were to characterize 101 pig This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. Prior to experiments, the protocol of the current study was reviewed and approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University (approved protocol number SRM-08). For specimen collection, we obtained permission from animal owners. No specific permits were required for the described field studies, and the locations where we sampled are not privately owned or protected in any way. The field studies did not involve endangered or protected species.E. bieneusi were obtained from pigs in cities Changchun, Daqing, Harbin, and Qiqihar in northeast China. All of them were previously genotyped by PCR and sequence analysis of the ITS locus [E. bieneusi isolates in humans from India, Peru, and Nigeria and 5 isolates in captive baboons from Kenya were included in this analysis  [Genomic DNAs extracted from 101 pig fecal specimens were used for PCR amplification of a minisatellite marker MS4 and three microsatellite markers  as described . The secftware/) .http://www.ub.edu/dnasp/) was used to determine E. bieneusi genotypes at each of the five markers and the MLGs with consideration of both single base nucleotide substitutions and short insertions and deletions (indels) polymorphisms [http://darwin.uvigo.es/rdp/rdp.html) [http://cmpg.unibe.ch/software/arlequin35/; based on both segregating sites and indels) [ISA) were calculated with LIAN 3.5 (http://guanine.evolbio.mpg.de/cgi-bin/lian/lian.cgi.pl/query) on five-loci haplotypes [The software DnaSP version 5.10.01 (orphisms . Intrageorphisms . Recombidp.html) . Tests f indels) ,33. The plotypes .E. bieneusi by analyzing the intragenic and intergenic LD, ISA, neutrality, and recombination events (Rms). Wright’s fixation index (FST) calculated using Arlequin and gene flow (Nm) calculated using DnaSP were applied to evaluate the degree of genetic differentiation between E. bieneusi populations [We assessed population structure of ulations ,33.http://mrbayes.sourceforge.net/) was used in clustering nucleotide sequences using Markov chain Monte Carlo (MCMC) methods [http://www.molecularevolution.org/software/phylogenetics/modeltest) [http://tree.bio.ed.ac.uk/software/figtree/). Pairwise distance matrices among nucleotide sequences of MLGs were calculated using eachgap calculating method with the dist.seqs command in the software MOTHUR version 1.24.1 (http://www.mothur.org/wiki/Download_mothur) [http://biology-assets.anu.edu.au/GenAlEx) [http://pritch.bsd.uchicago.edu/software.html) to assess the presence of distinct subpopulations [http://www.fluxus-engineering.com/sharenet_rn.htm) to estimate the genetic segregation and evolutionary trend of E. bieneusi isolates [A Bayesian analysis implemented in the software MrBayes version 3.2.1 ( methods . The gendeltest) . An MCMCdeltest) . The max_mothur) . A princGenAlEx) . A Bayesulations . We alsoisolates .E. bieneusi isolates identified 9, 15, 5, 15, and 11 genotypes at the loci ITS, MS1, MS3, MS4, and MS7, respectively. Single nucleotide polymorphisms (SNPs) were the only source of genetic diversity at the ITS locus, while genetic variation at the other four loci included the lengths of trinucleotide TAC and TAA repeats at MS1, dinucleotide TA repeats at MS3, tetranucleotide GGTA repeats at MS4, and TAA repeats at MS7 and the SNPs outside the tandem repeat regions. Some MS4 fragments also carried isostructural GG to AA substitutions in the first tetranucleotide repeat. Gene diversity (Hd) at individual loci was calculated using DnaSP and is shown in E. bieneusi isolates . Due to the difference in substitution rate between SNPs and indels, two methods were used to estimate genetic diversity. The finite population genetic variance estimates that consider both SNPs and indels allowed identification of a total of 44 MLGs with a Hd value of 0.95 and a nucleotide diversity (Pi) value of 0.0197  and L  to assess the population structure of E. bieneusi using allelic profile data [ISA values  were obtained for the overall and individual pig E. bieneusi populations. The value of VD was greater than that of L for each data set as well. Thus, the populations tested were all in strong LD. Calculation of ISA and comparison of VD and L were also applied for the analysis of MLGs to avoid the possibility that LD would result from a clonal expansion of one or more MLGs which might mask the underlying equilibrium. The analysis showed the overall pig E. bieneusi population retained LD  when the isolates with the same MLG were treated as one individual (Nm) using the FST method. Pairwise analysis between geographic populations yielded FST values ranging from 0.327 to 0.506 and Nm values ranging from 0.24 to 0.48   . NeverthMedian-joining networks were used to infer the relationships between MLGs . The anaE. bieneusi ITS genotypes from different hosts and geographical regions, the relationship between genotypes and phenotypic traits such as host specificity and zoonotic potential remains unclear [E. bieneusi in humans and non-human primates [ISA, neutrality, and Rms to determine the genetic structure and substructures in E. bieneusi populations from pigs using both sequence and allelic profile data from five genetic loci. The presence of strong LD and very limited recombination supported the existence of significant clonal structure in the overall and individual pig E. bieneusi populations from four cities, northeast China. In addition, as illustrated in E. bieneusi populations has led to the expansion of several dominant MLGs, which might play a role in enhancing their adaptation to specific hosts. Two recent studies described that two other microsporidian species known to infect honeybees (Nosema apis and Nosema ceranae) were also under selective pressure and experienced a population expansion [FST and Nm and the distribution of the pig E. bieneusi isolates revealed the existence of geographic segregation among the cities surveyed.Despite advances in defining  unclear ,11. Anal unclear . MLST anprimates –28. Herexpansion ,44. The E. bieneusi isolates at the ITS locus [E. bieneusi isolates used for MLST analysis in this study all belong to phylogenetic Group 1 with zoonotic potential [E. bieneusi isolates used for comparative analysis are also Group 1 members [E. bieneusi isolates in these subpopulations. As observed in Pigs are considered a potential reservoir for human microsporidiosis based on genotypic features of TS locus ,17,23. T members ,27. ThesE. bieneusi isolates in the former three subpopulations. The high diversity of E. bieneusi isolates in SP1 and SP5 may enable responses to environmental challenges and adaptations to new hosts [E. bieneusi infections and have zoonotic potential. This is supported by the broad host range of E. bieneusi ITS genotypes D, IV, and EbpC in the two subpopulations.The stronger LD and higher occurrence of specific MLGs observed in SP2 to SP4 than SP1 and SP5 suggest the presence of higher clonality of ew hosts . Thus, ME. bieneusi isolates belonging to the ITS genotypes CS-4, Henan-I, Henan-IV, PigEBITS3, and EbpC. The former four genotypes were shown to infect a very limited number of host species as shown in E. bieneusi isolate with ITS genotype EbpC in SP4 supported the zoonotic nature of some E. bieneusi isolates.Reduced gene flow between primate SP1 and SP2 and between pig SP5 and SP3 might promote the emergence of advantageous haplotypes in SP2 and SP3 and allow these haplotypes to remain intact despite the possibility of recombination . These pE. bieneusi to cope with host variations and environmental challenges. The haplotype network suggests that clusters SP3/SP4 may have been derived from SP5 and that SP2 may have arisen from SP1. Thus, SP2 to SP4 with host-specific features might serve as recurrent transitions to asexuality from otherwise SP1 and SP5 with sexual potential and some of the isolates in SP2 to SP4 might have outcompeted or partially displaced their relatives in SP1 and SP5 under certain ecological conditions. This process would limit host range and result in the emergence of highly successful E. bieneusi genotypes. It has been suggested that some asexual microsporidian populations might originate independently several times from their sexual ancestors [E. bieneusi might have a sexual phase in its life cycle, sex could be lost or cryptic, and the parasite could switch to obligate asexuality when the population structure becomes epidemic.Population genetic analysis is an indirect but powerful way to assess reproductive modes that are often difficult to uncover in some microorganisms . Sexual ncestors ,49. ThesE. bieneusi in pigs. Combined with the MLST data from primate E. bieneusi isolates, five distinct subpopulations have been defined. Among them, the very strong LD and low genetic recombination are indicative of the epidemic or host-adapted characteristics of SP2 to SP4, whereas the weakened LD and higher genetic diversity in SP1 and SP5 may represent higher potential for cross-species transmission of E. bieneusi infections. These data demonstrate the existence of genetic structure within E. bieneusi ITS Group 1 and the evolutionary potential for adaptation to host species in some of the subpopulations. Nevertheless, additional MLST data from other hosts including humans in China are needed for in-depth assessment of the potential for zoonotic transmission, host adaptation, and population differentiation of E. bieneusi isolates in different hosts.In conclusion, we have shown an overall clonal population structure of S1 TableRecombination events assessed using the methods GENECONV, MaxChi, and SiScan.(DOC)Click here for additional data file.S2 TableP values for intergenic linkage disequilibrium among genetic loci based on allelic profile data from pig Enterocytozoon bieneusi populations.Pairwise (DOC)Click here for additional data file.S3 TableEnterocytozoon bieneusi subpopulations.Proportion of the specimens from each city that belong to each of the three pig-derived (DOC)Click here for additional data file.S4 TableEnterocytozoon bieneusi referred in this study.Host range and geographical distribution of ITS genotypes of (DOC)Click here for additional data file.


Martes foina) populations.Population genetic diversity and structure are determined by past and current evolutionary processes, among which spatially limited dispersal, genetic drift, and shifts in species distribution boundaries have major effects. In most wildlife species, environmental modifications by humans often lead to contraction of species’ ranges and/or limit their dispersal by acting as environmental barriers. However, in species well adapted to anthropogenic habitat or open landscapes, human induced environmental changes may facilitate dispersal and range expansions. In this study, we analysed whether isolation by distance and deforestation, among other environmental features, promotes or restricts dispersal and expansion in stone marten  located inside a large forest complex. Genetic differentiation between this site and all others was 20% higher than between other sites separated by similar distances. The genetic uniqueness index of NE1 was also twofold higher than in other sites. Past demographic history analyses showed recent expansion of this species in north-eastern Poland. A decrease in genetic diversity from south to north, and MIGRAINE analyses indicated the direction of expansion of stone marten.Our results showed that two processes, changes in species distribution boundaries and limited dispersal associated with landscape barriers, affect genetic diversity and structure in stone marten. Analysis of local barriers that reduced dispersal and large scale analyses of genetic structure and demographic history highlight the importance of isolation by distance and forest cover for the past colonization of central Europe by stone marten. This confirmed the hypothesis that human-landscape changes (deforestation) accelerated stone marten expansion, to which climate warming probably has also been contributing over the last few decades.The online version of this article (10.1186/s12898-017-0156-6) contains supplementary material, which is available to authorized users. The genetic structure of a species is driven by past and ongoing processes within its populations –5. TheseA number of genetic signatures may be used to detect demographic changes, particularly range expansions or contractions . ColonizWithin a species range, variations in genetic diversity and its structure are often related to population connectivity by gene flow , 14. AniDispersal restricted by anthropogenic habitat changes depends on species adapting to new human-modified habitats. For some species, human-induced changes may enhance dispersal and connectivity and may also accelerate colonization of new areas , 9. SpecMartes foina) is a species that has adapted to anthropogenic habitats. It began to colonize Europe alongside Neolithic human societies and is now widespread across large parts of Europe from Portugal to Eastern Russia [The stone marten  explore the genetic diversity and structure of the stone marten in Poland, (2) investigate its demographic history, in light of the hypothesis about the recent expansion of stone marten populations in north-eastern Europe and (3) analyze the impact on gene flow of potential environmental barriers. We predicted that: (1) stone marten colonized north-eastern Poland in the last decades when the climate warmed and human-induced habitat changed, (2) there are no barriers for dispersal of habitat-opportunistic stone marten in a mosaic of agriculture and natural habitats, but (3) large primeval forest complexes are a barrier for dispersal between populations of this species, and thus affect its pattern of genetic variability.2 piece of skin or muscle, were placed in ethanol and kept frozen at − 20 °C until DNA extraction. The locations of the samples were accurate to 0.5 km.Tissue samples of 298 individuals were collected in Poland between 1994 and 2015 from eight sites in five regions: north-east (NE), central-east (CE) south-east (SE), south-west (SW) and north-west (NW) Poland. DNA samples were obtained from carcasses of martens killed by cars and from hunters or trapped individuals. We only trapped individuals in NE1 and NE2 sites, where we acquired permission from private land owners to conduct studies on their properties. All marten capture and handling procedures were approved by the Ministry of Environment and the Local Ethics Committee for Animal Experiments at the University of Białystok . Tissue samples, a 1 cmWe extracted DNA from tissue samples using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. Twenty-two microsatellite loci developed for martens were used to genotype all individuals: Ma8, Lut615, Mlut27, Mp0059, Mf3.2, Mf4.10, Mf3.7, Mf6.5, Mvi57, Mvi072, Ma1, Ma2, Gg454, Mel1, Mer041, Mar43, Mar15, Mf4.17, Mf8.8, Mf8.10, Mar08, Mf1.3 –40. MicrA), mean number of private alleles, observed heterozygosity (HO), unbiased expected heterozygosity (uHE) and inbreeding coefficients (FIS) using FSTAT 2.9.3 [Ar) according to the smallest sample size (N = 12) using FSTAT.We tested for deviation from Hardy–Weinberg equilibrium and linkage disequilibrium between pairs of loci within each sample site with GENEPOP 4.4  using deAT 2.9.3  and GENAAr and HE for two periods: 1994–2007 and 2008–2015 for NE1, because only from site NE1 did we obtain a minimum of ten individuals in each period. We tested differences among Ar and HE using Friedman’s test [To test the potential influences of different sampling periods on genetic diversity, we compared n’s test .K statistic [Genetic structure was explored using individual-based Bayesian clustering analyses with the program STRUCTURE v. 2.3.4  , TESS 2.tatistic .ψ) set at 0.6 [ψ at 0.4 and 0.0. We considered ten replicate runs of 20,000 burn-in iterations followed by 30,000 iterations. The number of clusters was set to range from K = 2 to K = 10. The preferred K was selected by comparing the individual assignment results and the deviance information criterion (DIC) [K values, and the most likely value of K was selected by visually assessing the point at which DIC first reached the plateau of the DIC curve.Secondly, TESS was run using a Convolution Gaussian prior for spatial admixture (BYM) model with the spatial interaction parameter (t at 0.6 . This paon (DIC) . Mean DINext, DAPC was used to identify genetic clusters by sequential clustering and model selection. This method provides a description of the genetic structuring using coefficients of alleles in linear combinations that give the largest between-group and smallest within-group variances in these loadings. In contrast to analyses in STRUCTURE and TESS, DAPC cluster detection within the genetic data does not consider any assumptions about HW proportions or linkage equilibrium . The mosFST [DEST [FST dependency for the amount of within-site variation, using FSTAT and GENALEX, respectively . Differentiation between sample sites based on FST was represented by a dendrogram using the program MEGA v.6 [FST values observed between a site and all other sites [The level of genetic differentiation was estimated by FST , and JosT [DEST , which cMEGA v.6 . Geneticer sites . To deteFST/(1 − FST) and the logarithm of geographical Euclidian distance, measured as a straight-line between the central point of each site, using the Isolation by Distance Web Service (http://ibdws.sdsu.edu/~ibdws/) [We tested the presence of isolation by distance (IBD) across the study area using a Mantel test . The Man~ibdws/) , for allDassign) was chosen to extract parameter estimates. Posterior densities were visually inspected, and the mode (point estimate) and 95% highest posterior density interval were computed on those densities.Current rates of migration between populations were estimated using a Bayesian MCMC method implemented in BIMR 1.0, which is effective at estimating migration rate when genetic structure is weak . Twenty In order to test for recent population contractions or expansions, we used the program BOTTLENECK v.1.2.02 . We testhttp://kimura.univ-montp2.fr/~rousset/Migraine.htm) under single population models with a single continuous past variation in population size (OnePopVarSize) and two past variations in population size (OnePopFounderFlush) [Nµ, 2Nancµ, and Dg/2N, as well as two extra composite parameters, namely Nratio and Dg*µ, where µ is the mutation rate per generation per locus, Dg is the time of the demographic change in generations, N = Ncur is the current population size and Nanc is the ancestral population size, expressed as the number of genes. Second, we fixed pGSM, the parameter of the geometric distribution of mutation step size under the GSM, at 0.3 based on the results obtained in the previous analysis and inferred the remaining four parameters of the OnePopFounderFlush model: 2Nµ, 2Nfounderµ, 2Nancµ and Dg/2N, as well as four extra parameters: Nancratio, NcurNfounderratio and NfounderNanc ratio, where Nfounder is the founder population size, and Dg*µ. In the OnePopFounderFlush demographic model, NfounderNanc ratio = Nfounder/Nanc allows the quantification and testing of a first discrete change in population size  while NcurNfounder ratio = Ncur/Nfounder equivalently characterizes the second continuous change in population size, typically an expansion following the founder event. Extra parameters were used to better characterize the timing, strength and direction of the demographic events, e.g. Nratio = Nanc ratio = Ncur/Nanc quantifies the strength of the change between current and ancestral population sizes—it is < 1 for a contraction, and > 1 for an expansion. Past changes in population size are thus significant when the Nratio’s value 1 lies outside theirs 95% confidence intervals (CI) [Nµ, 2Nancµ, 2Nfounderµ, Dg/2N and Dg*µ) into biological ones , we used a mutation rate of 5 × 10−4 per locus per generation for all microsatellite loci, a classical average value derived from many different species [Populations demographic history was further explored using MIGRAINE 0.5 (erFlush) , 64. To erFlush) . All anaerFlush) , 63. Fir species . We firsTwenty-two loci were genotyped for the 298 individuals sampled from the eight study sites. Thirteen individuals with high relatedness to others (QGM > 0.7) and thirteen with missing genotypes were removed from this final dataset. Finally, 272 full multi-locus genotypes were obtained with 0.30% missing data. After sequential Bonferroni correction (p < 0.000027), deviation from HWE was detected in sites NE2 and CE2 for locus Ma1. Null alleles were found for one locus (Ma1) in four sites and for four loci in one or two sites only  per locus within each sample site ranged from 3.43 to 4.33, the allelic richness (Ar) from 2.98 to 3.80 and the number of private alleles from zero to 0.33 (Table HO) over all loci was 0.51 (SE = 0.02) and ranged from 0.47 to 0.57, while unbiased expected heterozygosity (uHE) was 0.53 (SE = 0.02) and ranged from 0.47 to 0.59 , and expected heterozygosity showed a decreasing trend from south to north . The mean number of alleles  values  were all obtained by comparing NE1 with the other populations; and (2) despite the fact that NE1 is located inside NE2, the FST value between NE1 and NE2 was higher than between NE2 and NE3. Similar results were obtained using DEST estimator , but when NE1 was removed from the analyses the IBD relationship significantly explained a significant proportion of the variation . The slope of regression of genetic and geographic distances from NE1 to other sites was similar to slope between distance matrices excluding NE1 . The mean differentiation between NE1 and the other sites was on average 20.2% higher than the regression line of genetic vs geographic distances of martens populations between the other sites  with other sites, including a neighboring site 20 km away. First, pairwise comparisons between NE1 and other sites gave the highest clusters –71.HE = 0.47) and observed heterozygosity (HO = 0.47) of NE1 are similar to results of isolated populations of other mammals, e.g. the edible dormouse Glis glis (HE < 0.42), Ethiopian wolf Canis simensis (HE = 0.38–0.54) and fisher Pekania pennanti (HE = 0.47–0.56) [Ar and uHE, there was no evidence of bottlenecks . However, analyses with MIGRAINE showed significant founder and expansion events in site NE1, suggesting that stone marten recently colonized this area and that the low local genetic diversity at this site may be related to the founder event.The distinct genetic structure of NE1, an isolated population inside a large forest complex, has probably been maintained by low gene flow from neighboring sites. In general, isolated populations with low gene flow are prone to losing alleles, reducing genetic diversity and viability as a result of genetic drift –74. Due 47–0.56) . DespiteMartes martes) has been confirmed by habitat selection analyses of radio-tracked martens [The large genetic differentiation in and low migration rate into NE1 confirmed that this stone marten population is separated from the other sites by a large forest complex, creating an isolated population. The avoidance of this large forest complex by stone marten in contrast to pine marten . This was confirmed by clustering methods implemented in STRUCTURE, TESS and DAPC. Greater structures in stone marten populations, with higher probabilities of assignment to groups have been observed elsewhere in Europe ; however the geographic areas considered in these studies were slightly larger than in our study [Comparison of the genetic diversity and genetic structure of stone marten from all other sites (except site NE1) suggested an absence of barriers and high dispersal rates between sites, causing low genetic structuring across large parts of Poland. However, genetic uniqueness of martens inhabiting sites increased with the increase proportion of forest cover around the sites. This analysis confirmed that forests may restrict gene flow between sites but that probably only large forest complexes reduce it to a level that affects genetic structure. A lack of distinct genetic structure and small divergence between all sites except NE1 was indicated by ur study –81. A weur study , 27. In ur study , which aAr and uHE decreased from south-west to north-east Poland; the highest genetic diversity was recorded in SE1 site and Ar or uHE decreased to the north, both north-west or north-east. The gradual loss of genetic variability is typical during colonization of new territories because of population bottlenecks and founder effects [The lack of evident genetic structure and large similarity between stone marten inhabiting distantly-located sites in Poland may also reflect demographic processes, notably the history of colonisation of Poland by this species. The signal of expansion detected by MIGRAINE indicates a recent demographic expansion of stone marten in NE Poland. The arrangement of Neighbor-joining tree branches indicated the directions of population expansions: the populations from SW Poland gave rise to the populations of central Poland and individuals from these populations then expanded into the north-eastern regions. Also, the DAPC genetic structure results suggested the direction of differentiation of populations, where group 1—consisting of the south-west sites—originated group 3 , which in turn initiated the existence of the most north-eastern group 2 and isolated group 4. The direction of migration, indicated by BIMR analysis, from all sites to NE2 may also reflect the direction of current expansion. Furthermore, genetic diversity usually decreased along the expansion axis . In Pola effects .The past demographic inferences obtained with MIGRAINE analyses suggested stone marten colonized NE Poland relatively recently (17–45 generations), which is confirmed by observations of stone marten presence in this region. Assuming a generation time of 2 years we can estimate the time of colonization to be around 34–90 years ago but this estimation should be treated with caution as it may be slightly biased by non-synchronous sampling. The proportion of stone to pine martens collected from hunters and road-killed animals in the zoological collection of the Mammal Research Institute, Polish Academy of Sciences has increased, over successive decades, since the 1980s  for the same loci (as used in this study) were higher when considering populations from southern Europe (Bulgaria) than when considering populations from south-western Europe (France, Spain and Portugal; Table The stone marten probably colonized Europe post-8000 YBP, which was associated with the spread of Neolithic societies , 86. In Our results showed that two processes, namely changes in species distribution boundaries and limited dispersal associated with landscape barriers within the species range, probably affect genetic diversity and structure in stone marten in Poland. In landscapes less modified by humans, with large forest complexes, dispersal is limited, and structure between stone marten populations increases. Analysis of local barriers that reduced dispersal and large scale analyses of genetic structure and diversity highlight the importance of anthropogenic landscapes for the past colonization of central Europe by stone marten. This confirmed the hypothesis that human-landscape changes accelerated stone marten expansion, to which climate warming has also probably been contributing over the last few decades. The ongoing colonization process in north-eastern Europe and Asia may be explained by these two changes in biotic and abiotic conditions.Additional file 1. Additional figures and tables.Additional file 2. Row experimental data.


Ambystoma laterale-jeffersonianum complex from 10 sites in the northern part of their distribution.Unisexuals of the blue-spotted salamander complex are thought to reproduce by kleptogenesis. Genome exchanges associated with this sperm-dependent mode of reproduction are expected to result in a higher genetic variation and multiple ploidy levels compared to clonality. However, the existence of some populations exclusively formed of genetically identical individuals suggests that factors could prevent genome exchanges. This study aimed at assessing the prevalence of genome exchange among unisexuals of the A. jeffersonianum haplome. One of these groups exclusively reproduced clonally, even when found in sympatry with lineages presenting signature of genome exchange. Genome exchange was site-dependent for another group. Genome exchange was detected at all sites for the three remaining groups.A total of 235 individuals, including 207 unisexuals, were genotyped using microsatellite loci and AFLP. Unisexual individuals could be sorted in five genetically distinct groups, likely derived from the same paternal Prevalence of genome exchange appears to be associated with ecological conditions such as availability of effective sperm donors. Intrinsic genomic factors may also affect this process, since different lineages in sympatry present highly variable rate of genome exchange. The coexistence of clonal and genetically diversified lineages opens the door to further research on alternatives to genetic variation.The online version of this article (10.1186/s12862-018-1200-7) contains supplementary material, which is available to authorized users. Unisexuality in vertebrates is often considered an ephemeral anomaly from the usual course of evolution. Interspecific hybridization is the most common trigger of deviation from sexual reproduction, the standard mode of reproduction among vertebrates . Most unPoecilia formosa complex [Rana esculenta) [Ambystoma laterale-jeffersonianum complex [Three modes of reproduction are known in sperm-dependent unisexuals. The most common is gynogenesis, in which the male genome is not incorporated into the unreduced genome of the eggs, making this reproduction mode totally clonal. Known unisexuals reproducing by gynogenesis include hybrids from the  complex , the Poe complex  and the  complex . In the culenta) . In klepculenta) . Genome  complex , 9, but Ambystoma laterale-jeffersonianum complex is lower than that found in sexual species, but higher than that found in clonal organisms [A. laterale, haplome designated “L”), unisexual individuals of this complex may include the haplome of either the Jefferson salamander , the small-mouth salamander , the tiger salamander  or the streamside salamander .The genetic diversity found in unisexuals of the rganisms , 10–13. rganisms , 15. Howrganisms , 16–18. rganisms –22, but Ambystoma unisexuals incorporate genetic material from the different sexual species, but the gene flow remains unidirectional. The exact mechanism of genome replacement remains unknown, and since it is difficult to assess whether haplome permutation occurs in the same reproduction event (genome replacement) or through several generations, some authors prefer to use the term genome exchange [Kleptogenesis has then been proposed to explain the relatively high genomic diversity and the presence of five different species despite the single origin of all unisexuals . Since texchange .Different factors have been recognized as affecting the prevalence of genome exchange. A relatively high amount of genetic diversity was found within clutches, both in the field  and in tAmbystoma laterale-jeffersonianum complex to determine factors affecting male genome inclusion in natural conditions. To address this objective, a sampling was performed in 10 sites from Quebec (Canada), where a previous survey revealed an unusually high genetic homogeneity among individuals from the same location [The objective of the present study was to assess the prevalence of genome exchange in the northern part of the distribution of unisexuals of the location .A. jeffersonianum haplome. This haplome was not expected to have been exchanged recently since Ambystoma laterale is the only sexual species of this complex present in the region of study [A. laterale haplome(s) of unisexuals and sympatric sexual species A. laterale. Genome exchanges were expected to result in high genetic diversity for a given group within the site, in addition to a genetic similarity with sympatric A. laterale host individuals.To address this objective, individuals were first partitioned in different evolutionary groups according to their of study . Genome Sampling was conducted over two consecutive years during the reproduction period in the southern Quebec region. A total of 10 sampling sites were chosen according to the presence of Ambystoma unisexuals reported in previous surveys  to confirm genetic differentiation among groups. Finally, the origin of mtDNA was assessed for all d genome .Ambystoma laterale: LL) and unisexuals , as well as for determination of ploidy levels [Microsatellite loci were used for discrimination between the contributing sexual individuals .We used eight loci amplifying the J-genome and four loci amplifying the L-genome , 33. An PCR amplification was performed in a 12.5 μL reaction with approximately 30–40 ng of DNA, 1.25 μL of 10× Taq reaction buffer, 0.4 mM of dNTP mix, 0.6 mM of each primer and 0.2 U of Taq DNA polymerase. PCR consisted of an initial denaturation of 30 s at 92 °C, followed by 45 cycles of 92 °C for 30 s, 57 °C for 30 s and 68 °C for 45 s, with a final extension of 5 min at 68 °C. PCR products were run on denaturing 6% polyacrylamide (19:1 acrylamide:bis-acrylamide) gel. Silver nitrate staining was used to visualize results .A subsample of 71 individuals was selected to confirm genetic differentiation among groups inferred with microsatellites. AFLP analyses were performed according to a protocol modified from Xiong et al. . The MspA. laterale nuclear genome and unisexual mtDNA.The presence of unisexual mtDNA in unisexuals was confirmed using Noël et al.’s protocol . Individpolysat for R v. 3.2 is appropriate for such datasets [polysat analysis, but they were considered for other analyses. The segregation of the homologous loci allowed the use of the function meandist2, which extrapolates the unknown alleles of the partial heterozygotes by weighting each possibility (ex: A or B) according to allele frequency in the population dataset.The genetic analyses of the unisexuals of the Ambystoma complex represent a challenge because haplome(s) from different species are present in the same individual, and individuals present different ploidy levels. In addition, dominance can occur in partial heterozygosities for polyploid individuals. For instance, triploid individuals AAB and ABB will both be genotyped AB at this locus, leaving an undetermined allele. Some functions of the package datasets . When thBruvo2. This distance corresponds to the add-loss model proposed by Bruvo et al. [true. Neighbour-joining trees were built from the distance matrix with the nj function of the ape package in R. The trees were visualised with FigTree v 1.4.0 [bruvo.boot of the package proppr [To assess genetic distance between unisexual individuals and partition them in genetic groups according to their microsatellite multiloci genotype, we used the distance measure o et al. , behaves v 1.4.0 . To assee proppr  was usedmantel in R. A Mantel correlogram was also computed between the two matrixes using the mantel.correlog function with 20 classes.To assess spatial segregation of genotypes, we tested for the correlation between the abundance of genotypes among sites and geographic distance; a Mantel test was performed on Hellinger distance matrix for the abundance of genotypes with the function polysat-Bruvo2 distance was used to quantify the genetic variation within groups with respect to the multiploidy constraint. The total dispersion in each genetic group was computed from the microsatellite distance matrix using the function betadisper of the package vegan in R [aov and HSD.test. Dispersion intra-sites and inter-sites were also obtained and compared by the same method, with modification of the groups’ attributions.Dispersion from the centroids of the groups considering the gan in R , which rA. laterale individuals were found was weighted to account for the different number of individuals with the function allelic.richness of the package hierfstat in R [For a comparison between unisexuals and sexual host individuals, allelic frequencies of pure individuals LL were obtained and compared to the LL part of the genome of LLJ triploid unisexuals. Allelic frequencies were computed for the pure individuals LL with Genepop on the web v. 4.2 . The fretat in R .A. jeffersonianum species, the J-haplome is restricted to a clonal mode of transmission. This can be used as a reference to assess diversity originating from mutations. In the absence of genome exchange, a similar diversity was expected between the J-specific and the L-specific loci while genome exchanges only increase diversity for the L-specific loci. The function allelic.richness of the package hierfstat in R was used to weight the number of alleles for each genetic group per site. The number of alleles was weighted according to the number of haplomes for each specific set of loci. As an example, a group of three individuals LLJ and one individual LJ would be n = 7 and n = 4 for L- and J-specific loci, respectively. This was performed for the set of eight loci for the J-haplome  and the set of 4 loci for the L-haplome . The number of alternative alleles was calculated by removing the minimal number of alleles of the group from the weighted sum to control for the ploidy level. For each group’s weighted ploidy level, the group’s sum of all haplomes of each specific set of loci (L or J) was divided by the total number of individuals of the group. As an example for the L-haplome, a group of three individuals LLJ and one individual LJ would have a basic weighted ploidy level of (2 + 2 + 2 + 1)/4 = 1.75 L-alleles and (1 + 1 + 1 + 1)/4 = 1 J-alleles under strictly clonal reproduction without mutation. The mean number of alternative alleles was compared between the J-specific and the L-specific loci for each genetic group per site, since the J-haplome can be used as a reference for a scenario of clonal reproduction in the study area.We first assessed genome exchange by comparing the mean number of alternative alleles between the J-specific and the L-specific loci for each genetic group per site. In the absence of the neighbor. Bootstraps were used to assess the relative support of the different groups: 1000 replicates were generated with the executor seqboot of Phylip [consens with the extended method of majority rule and compared to the real tree.The simple-match coefficient was used to calculate the distance matrix from the presence-absence AFLP matrix. We first assessed the consistency between AFLP and microsatellite data sets using a Mantel test performed between the distance matrix for AFLP bands and the distance matrix of the corresponding individuals for microsatellites. We also validated the different clusters obtained from microsatellites by performing neighbour-joining on the AFLP dataset. The distance matrices were used to build the neighbour-joining tree, using the executor f Phylip . Distancn = 207, 88%) compared to A. laterale (LL), the unique contributing sexual species captured. The capture of A. laterale (LL) was restricted to three sites: . The unisexuals were dominated by triploids , but also included diploid  and a few tetraploid  unisexuals  mutation steps from the direct neighbours for a given allele  and 8 of them explained the association to a given group . In addition, the five genetic groups were supported by high bootstrap values for microsatellite data of the J-genome. These groups were also fully consistent with those based on multilocus genotypes and the divergent alleles of AmaD42, AjeD378, AjeD13 and AjeD23 loci. The relationships among groups slightly differed among the datasets due to the inconsistent position of group D  and the correlation between AFLP and LJ-microsatellites distance matrices was high .The individuals of these five genetic groups were also spatially structured Fig. . Each sip = 0.083); it became significant only when the sites from the Montreal region were considered . The D group was the only group detected outside the Montreal region. Moreover, this group was also characterized by complete allopatry between diploid (sites E01 and E02) and triploid (site E03) unisexuals  to group D-3n   and E01 (n = 36) presented a single genotype per site for loci AjeD23, AjeD37 and AjeD422. No more than three genomotypes in low abundance and differing by a single mutation step, were found for loci AjeD94, AjeD283 and AjeD346.Several lines of evidence indicate that groups A and D-2n displayed a typical pattern of clonal reproduction. First, the two groups have very low variation within a given site. This variation was likely due to mutations, since the different microsatellite multilocus genotypes of a given location are separated by a single mutation step. Despite the low diversity over all individuals, groups A and D-2n displayed the major portion of their genetic diversity among sites suggesting the accumulation of mutations in isolated populations. Moreover, none of these groups displayed the highly divergent alleles found in sexual species populations, except for the tetraploid individual, for which the supplementary L-haplome seemed to come from a sexual l et al.  were excA. maculatum [A. laterale individuals were found at those sites, despite an extensive sampling effort in the E01 population .Ambystoma maculatum is not part of the complex, but its sperm can trigger the development of unisexuals’ eggs in the laboratory [A. maculatum haplome in the genome of an unisexual offspring seems unviable [A. maculatum. They proposed that A. maculatum males can discriminate between their conspecific and LJJ unisexuals, preventing them to access to A. maculatum sperm and reproduce [boratory . Howeverunviable . In the unviable  to concleproduce . In abseeproduce .A. maculatum do not discriminate diploid unisexuals as much as they do with LJJ unisexuals, allowing this diploid lineage to persist by gynogenesis. Reproduction by gynogenesis with A. maculatum is for now the most plausible explanation for the exclusively clonal and diploid populations found in E01 and E02 sites, since any attempt for genome exchange or ploidy elevation would lead to unviability.The context is however different for E01 and E02 populations. Since distant E03 populations displayed the same genetic profile than E01 and E02 populations, one can expect they persist since post glacial expansion. Although this is speculative, it is possible that male A. laterale populations. Since none of the unisexuals of group A displayed the highly divergent alleles, although the frequency of those alleles was high in the sympatric A. laterale populations, there was no evidence of genetic exchange with the sexual species. Indeed, all individuals from group A were highly similar, regardless of their sites of origin (M02 and M04 to M07). Individuals from group A were also detected in sympatry with those from groups B and E in the M02 population. Since individuals from groups B and E of this site seem to have acquired those highly divergent alleles by genome exchange, the lack of exchanges between the group A and the sympatric populations of A. laterale cannot be related to environmental conditions, as is proposed for diploid unisexuals from group D.However, ecological reasons can hardly be invoked for the clonal reproduction of individuals from the A group , since they coexist with sympatric sexual A. laterale, because A. jeffersonianum is absent and A. maculatum is either absent or very rare in this region  were ecologically very different, relative to the other sites . It seeGenome replacement is a mechanism suggested by several authors who have studied the similarity between unisexuals and local sexual species , 18, 26.It is not possible to distinguish the resulting effect of genome replacement that occurs throughout a single reproductive event from subsequent ploidy elevation and reduction processes performed in distinct generations. Although these two hypotheses are not mutually exclusive, a certain prevalence of genome replacement seems necessary to explain the genetic variability observed in this study: the very low abundance of diploid unisexuals and tetraploid individuals in most populations makes the subsequent ploidy elevation and reduction hypothesis unlikely to explain the genetic diversity found within some of the main genetic groups.A. laterale populations than in A. jeffersonianum populations [A. laterale individuals. Even if both hypotheses are not mutually exclusive, the hypothesis of gene flow from A. laterale host individuals to the unisexuals is more parsimonious to explain the different patterns observed between the groups than a hypothesis of recombination alone.Recombination is also a mechanism that can reorganize genetic variation among the complex. It has been observed that recombination occurs between homologous chromosomes, and more rarely between homeologs , 17. It ulations . RecombiAmbystoma laterale-jeffersonianum complex was proposed by Bogart and Bi [A. jeffersonianum is absent from the sampled area.Another mechanism that can explain the nuclear diversity of the t and Bi . The occA major criticism of the genome replacement theory is the absence of LL or LLL individuals with the unisexual mitochondrial haplotype . ResearcAmbystoma complex revealed high variability in the prevalence of genome exchange, as well as different factors preventing the occurrence of this process. Although occasional, this capacity to acquire genetic material from sexual species can provide higher genetic variation and an evolutionary advantage to kleptogens. It is therefore striking that strictly clonal unisexual lineages can coexist with genetically diversified kleptogenetic lineages, unless they have an advantage of their own.Lineages located in the northern part of the distribution of unisexuals of the Additional file 1:Table S1. Description of the environmental variables collected. Table S2. Primers and ranges of the loci used in the study. Sequence of the primers, specific range of alleles when known and reference are given. Table S3. Consensus score of the J-alleles for the five main genetic groups. Table S4. Allelic frequency for the 8 AFLP loci explaining the association to each group. Figure S1. Distribution of the sampling sites according to environmental conditions. (DOCX 241 kb)Additional file 2:Sheet 1: Unisexuals’ Microsatellite scores. Individuals are also associated to their main genetic group , to a specific genotype  and to its biotype (ex: LLJ). Sheet 2: AFLP scores. The presence/absence of the different bands are labelled according to the combination used (GCNN) and the size of the band. Sheet 3: Polysat Matrix. Microsatellite scores in polysat format. Sheet 4: Polysat POP. Two matrices for population assignment in polysat: according to the sampling site or according to the main genetic group of each individual. Sheet 5: Matrices for weighting J. Matrices used to weight J-alleles and J-genotype. Sheet 6: Matrices for weighting LJ. Matrices used to weight LJ-alleles and LJ-genotype. Individual lines have been multiplied to account for ploidy variation. Sheet 7: LL alleles. Microsatellite scores of the LL individuals found, in table and in population format. Sheet 8: Statistics on m-sat scores. Allelic means, standard deviation and other parameters for each loci, including differences between alleles and the mean and its nearest neighbours, per alleles, per locus. (XLSX 181 kb)Microsatellite and AFLP scores of sampled individuals.


Geography of Genetic Variants (GGV) browser (http://popgen.uchicago.edu/ggv/) provides maps of allele frequencies in populations distributed across the globe.One of the key characteristics of any genetic variant is its geographic distribution. The geographic distribution can shed light on where an allele first arose, what populations it has spread to, and in turn on how migration, genetic drift, and natural selection have acted. The geographic distribution of a genetic variant can also be of great utility for medical/clinical geneticists and collectively many genetic variants can reveal population structure. Here we develop an interactive visualization tool for rapidly displaying the geographic distribution of genetic variants. Through a REST API and dynamic front-end, the http://popgen.uchicago.edu/ggv/) which employs an API to access frequency data (http://popgen.uchicago.edu/freq_api/). Python and javascript source code for the website and the API are available at: http://github.com/NovembreLab/ggv/ and http://github.com/NovembreLab/ggv-api/.GGV is implemented as a website (Bioinformatics online. Genetics researchers often face the problem that they have identified one or many genetic variants of interest using an approach such as a genome-wide association study and then would like to know the geographic distribution of the variant. For example, the researcher may hope to address: (i) implications for genomic medicine . A simpContemporary population genetics researchers are also faced with the challenge of large, high-dimensional datasets. For example, it is not uncommon for a researcher in human genetics to have a dataset comprised of thousands of individuals measured at hundreds of thousands or even millions of single nucleotide variants (SNVs). One common approach to visualizing such high-dimensional data is to compress the SNV dimensions down to a small number of latent factors, using a method such as principal components analysis  uses the scalable vector graphics and mapping utilities of D3.js  Rare variants. Many alleles are rare (e.g. (2) Uncertainty in frequency data. We use varying transparency in a population’s pie chart: estimated frequencies with higher levels of sampling error (e.g. those from samples with n < 30) are made more transparent, and hence less visible, on the map ((3) Overlapping populations. We use force-directed layouts of the populations such that no two points are overlapping each other, and yet the points will be pulled towards their true origins  Unc the map . (3) Ove origins . Also, bSTF is relatively low globally  and the National Institute of General Medical Sciences under training grant award number T32GM007197 (JHM).Conflict of Interest: none declared.Supplementary DataClick here for additional data file.


Even though Cyprus was an important crossing point for the westward spread of olive, and one of the primary regions of domestication, its genetic recourses remain uncharted at a great extent. Throughout the centuries, a number of ancient olive trees remain in the same orchards, contributing to Cypriot oleiculture and society. In an attempt to explore this monumental genetic pool, a survey was conducted to identify centennial olive trees in rural provinces of Cyprus. Microsatellites were employed in order to study their genetic composition (including rootstocks when feasible) and to establish possible associations among genotypes. High numbers of specific alleles, suggestive of the distinctiveness of this germplasm, were detected, and both grafting and rootstock propagation was verified. Moreover, it was determined by Bayesian structural and network reticulate analysis that centennial olives can be divided in two discrete genetic clusters having intermediate admixed accessions. Furthermore, it was determined that all contemporary Cypriot cultivars, that were included in the present study, were highly affiliated exclusively to one genetic group, a strong evidence of selection among elite clones. The information acquired from the current study reveals the genetic rareness of this material and its contribution to the current olive germplasm. Olea europaea L.) is the most symbolic species in the Mediterranean Basin due to its ecological, economical and cultural importance [O. europaea var. sylvestris), wild forms (Olea oleaster Hoff. Link) and domesticated forms classified as O. europaea var. europaea [O. europaea, the genotypes spread by cutting or seed migration caused great confusion on cultivar identity and nomenclature [The olive tree , while the remaining were located after interviews with the owners and locals. The countryside was divided in four prefectures  and after a systematic examination, 52 centennial olive trees were recognized and sampled ; Fig 1. In order to detect potential genetic relationships among the contemporary and ancient Cypriot olive germplasm, certified varieties by the Cypriot Ministry of Agriculture, Rural Development and Environment were included in the study. In total, 12 Cypriot cultivars from the Gregoriou  collectiTaq , 1X reaction buffer, 320 μM dNTPS and 0,4 μM of each primer. Unambiguous loci where also evaluated when needed with the Type-it Microsatellite PCR Kit . PCR amplification was performed using a Bio-Rad PTC-200 thermocycler  with the following temperature profile: initial denaturation step for 5 min at 95°C followed by 34 cycles of 45 s at 95°C, 45 s at 50–55°C, 45 s at 72°C and a final elongation step for 10 min at 72°C. SSR primers were selected based on their efficacy and robustness previously described [®  software. Seven olive accessions  were analyzed in replicates  to test the reproducibility of allele identification, and constantly included in each run in order to detect possible allele size shifts.Total DNA was extracted from olive leaves using the NucleoSpin Plant II Kit . DNA concentration was determined spectrometrically and its quality was established by agarose gel electrophoresis. After an initial screening, 14 carboxyfluorescein (FAM) labeled SSR primer pairs were chosen because of their consistency in amplification and polymorphism . Amplifiescribed –19. SSR Ho), the expected heterozygosity (He), the polymorphic information content (PIC) and the probability of null alleles (F Null) using the Cervus software [Allele sizes were converted into a data matrix table and a Maximum Likelihood analysis was conducted using the SH-aLRT algorithm and the W-IQ-TREE tool . The levsoftware . Primerssoftware . The sigsoftware , under dsoftware  in ordersoftware .A total of 52 centennial olive trees along with 20 cultivars were analyzed using 11 out of 14 SSR loci. The SSR profiles led to the identification of 43 different genotypes among the 72 accessions of the present study. Furthermore, the microsatellite profiles from the canopy and from the root suckers differed in four out of six olive trees, which is an indisputable indication of grafting. Replicate PCR amplifications and fragment analyses revealed the reproducibility of the detected SSR loci, including rare alleles (data not shown). The analysis revealed a total of 109 alleles, ranging from two to 14 alleles for the UDO99-011 and UDO99-043/GAPU 103A loci, respectively, and an average of 7.79 alleles per locus. The GAPU 103A locus showed the highest number of specific alleles (seven). Allelic frequencies ranged from 0.007  to 0.96, presenting polymorphism for each locus. In addition, 27 specific alleles were detected among the centennial olives ; four wePIC) was calculated. The highest PIC value was 0.730, while the lowest was 0.077 for loci GAPU 59 and UDO99-011, respectively, with an average of 0.499. The likelihood of null alleles incidence ranged from -0.272 to 0.683. For the 11 out of the 14 loci analyzed, the observed heterozygosity was significantly higher than the expected values (Hardy-Weinberg equilibrium). Finally, a considerable heterozygosity deficiency was detected for UDO99-011, UDO99-031 and UDO99-039 loci. These results suggest a selection of elite clones and a declination from random mating (panmixia).Analysis of molecular variance revealed that a high proportion (92%) of the total genetic diversity was allocated within the populations. The highest variability was recorded for the ancient Cypriot genotypes , followed by modern Cypriot and Greek cultivars . Fst values also suggested the presence of significant divergence between populations .The majority of the Cypriot olive genotypes (58 out of 63) were organized in two distinctive clusters , while all Greek taxa and a few Cypriot olive trees were organized in a third (III) diverse group . Thirty-Four out of six centennial olive trees that were tested were found to be grafted , indicating that the majority of the olive trees of the present study are grafted. Still, several modern cultivars shared the same genetic composition with centennial olives, revealing an active participation of the ancient Cypriot olive germplasm to the current via vegetative/clonal propagation. For instance, cv 'Klirou 2' and cv Analiontas 1' were identical to the accession of 'Poli Chrysochous'; furthermore, cv 'Klirou 1' was indistinguishable from the 'Aglisides' and 'Potamiou' genotypes, while cv 'Lefkara 1', cv 'Flasou' and cv 'Evrihou 3' were integrated in the second biggest genetic group containing eight entries . FinallyIn order to additionally resolve the genetic relationships among the olive entries, a reticulate analysis was employed and a network was constructed by coalescent simulations . Based oThe pattern of genetic admixture and germplasm structure was additionally portrayed using a Bayesian-based approach and the visual outline of substructures among the olives is presented in Out of 43 different genotypes identified, only a few centennial trees had an identical genetic profile to contemporary cultivars, while the rest were different . This isIdentical genotypes were revealed mainly from entries that were collected in close geographical proximity (same village), with the exception of the Kato Moni village. On the contrary, Hosseini-Mazinani et al.  identifiGrafting has been described as the transition to domestication and this technique was developed almost four millennia ago . The SSRO. europaea var. sylvestris was common in the Levant [The high genetic variability of rootstocks raises questions about their origin. For an explanation, several possibilities can be proposed: primarily, as suggested by Diez et al.  for the e Levant . Alternae Levant . Conseque Levant The majority of the centennial olive trees and the local multiclonal varieties of 'Ladolia' were clustered in two discrete groups in all the analyses carried out. The grouping highlights the close intra population relations, possible due to common ancestry and the diversification among clusters . StructuThis similarity could either highlight their common genetic background since Cyprus is referred as a possible passage for the dispersal of olives westward of the Levant ,29, or iExtensive genetic differences were detected among the Cypriot cultivars. Morphological descriptors , as wellSince there is little resemblance of contemporary Cypriot cultivars to the second cluster of ancient genotypes, it can be concluded that the former germplasm has suffered a reduction of genetic variability. It is known that the domestication process involves a selective bottleneck and a shift in the direction of fixing alleles associated to desirable agronomic characters . Hence, The study of Cypriot centennial olive trees was fruitful since the collection of this unique and diverse material can elucidate the agricultural practises and the domestication procedures that led to the establishment of the current olive germplasm. The extensive genetic diversity observed, points out that these genotypes are probably not clones of the ancient Cypriot cultivar 'Ladolia', but have a diverse origin. Cypriot scions were divided in two discrete clusters and modern cultivars were directly affiliated to one, while rootstocks were mainly affiliated to Greek genotypes. Based on the data of the current study it is safe to formulate the conclusion that oleiculture in Cyprus has a complex history. It is possible that hybridization between local or/and foreign material, followed by selection of elite genotypes has repeatedly occurred in different eras with the goal of ameliorating the existing genotypes. Rootstocks of centennial olives retain their ‘hidden’ rich genetic diversity and further focused studies can provide clues for the spread of olive germplasm around the Mediterranean basin. Hence, the collection and maintenance of centennial olives is a critical first step towards breeding and evolutionary unravelling of the oleiculture history through different eras.S1 Table(XLSX)Click here for additional data file.


Liquidambar formosana) is a relatively fast-growing ecological pioneer species. It is widely used for multiple purposes. To assess the genetic diversity and genetic differentiation of the species, genic SSR markers were mined from transcriptome data for subsequent analysis of the genetic diversity and population structure of natural populations. A total of 10645 potential genic SSR loci were identified in 80482 unigenes. The average frequency was one SSR per 5.12 kb, and the dinucleotide unit was the most abundant motif. A total of 67 alleles were found, with a mean of 6.091 alleles per locus and a mean polymorphism information content of 0.390. Moreover, the species exhibited a relatively moderate level of genetic diversity (He = 0.399), with the highest was found in population XY (He = 0.469). At the regional level, the southwestern region displayed the highest genetic diversity (He = 0.435) and the largest number of private alleles (n = 5), which indicated that the Southwestern region may be the diversity hot spot of L. formosana. The AMOVA results showed that variation within populations (94.02%) was significantly higher than among populations (5.98%), which was in agreement with the coefficient of genetic differentiation (Fst = 0.076). According to the UPGMA analysis and principal coordinate analysis and confirmed by the assignment test, 25 populations could be divided into three groups, and there were different degrees of introgression among populations. No correlation was found between genetic distance and geographic distance (P > 0.05). These results provided further evidence that geographic isolation was not the primary factor leading to the moderate genetic differentiation of L. formosana. As most of the genetic diversity of L. formosana exists among individuals within a population, individual plant selection would be an effective way to use natural variation in genetic improvement programs. This would be helpful to not only protect the genetic resources but also attain effective management and exploit genetic resources.Chinese sweetgum ( Liquidambar formosana Hance) belongs to the genus Liquidambar of family Altingiaceae  database. A short read archive under accession numbers PCR amplifications were performed in 25 μL reaction volumes as follows: 12.5 μL of 2 × Taq MasterMix , approximately 50 ng of DNA, 5 pmol reverse primer and 5 pmol forward primer with the 5′ end labeled with fluorescent dyes  and sterile double-distilled water added to 25 μL. The amplifications were performed with a touchdown profile, with the first denaturation at 94°C for 5 min, followed by 10 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 63°C and then extension for 45 s at 72°C, with a 1°C decrease in the annealing temperature each cycle; the following 20 cycles included denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s, with a final extension at 72°C for 7 min. All the PCR reactions were performed in the same thermal cycler . The PCR products were separated by a ABI 3730XL capillary electrophoresis analyzer  with a GeneScan-500LIZ size standard. Fragments were genotyped for their presence/absence at each locus, and the allele sizes were scored using GeneMaker 2.2.0 software  and visually checked twice to reduce genotyping errors. The genotyping error was computed by the blind replication of approximately 13.5% of the amplifications . The repeated genotypes were compared to the previous ones, and the number of allelic differences was counted.Na), the effective number of alleles (Ne), Shannon's information index (I), expected heterozygosity (He), observed heterozygosity (Ho), and Wright's F statistics parameters  were computed using POPGENE32 software  and geographic distance (km) to analyze the isolation by distance among populations. The significance was evaluated by performing 10,000 randomization  carried a single SSR locus and 1454 (16.06%) contained more SSR loci.A total of 80482 unigenes comprising approximately 56.25 Mb in L. formosana transcriptome data were calculated (Table n = 6 (23.42%), containing the most dinucleotide repeats.The frequencies of different types of genic SSR motifs in ed Table . ApproxiOf the 72 genic SSR primer pairs designed, 45 pairs successfully produced the expected product by amplification, with a success rate of 62.5%, but only 14 primer pairs showed abundant polymorphisms. The characteristics of the polymorphism primers were presented in Table L. formosana.MICRO-CHECKER analysis showed that several loci exhibited null alleles in some populations. At LF 15, LF 26, LF 72, null alleles were detected in the 17, 20, and 19 populations, respectively Table . So, theNa) per locus ranged from 3 (LF 40) to 10 (LF 69), with a mean of 6.0909, whereas the effective number of alleles (Ne) per locus varied from 1.2709 (LF 40) to 2.8047 (LF 17), with an average value of 1.9266. The Shannon's Information Index (I) had an average of 0.8178 and ranged from 0.3823 (LF 40) to 1.2657 (LF 19). Observed heterozygosity (Ho) and expected heterozygosity (He) varied from 0.2012 (LF 40) to 0.6411 (LF 17), and 0.2133 (LF 40) to 0.6439 (LF 17), with an average of 0.4090 and 0.4322, respectively. He was higher than Ho at 10 loci, with the exception being LF 25, in accordance with the mean inbreeding coefficient at the total population (Fit = 0.056). The value of the polymorphism information content (PIC) ranged from 0.192 (LF 40) to 0.580 (LF 17 and LF 19) with a mean of 0.3904 (Table PIC showed that highly polymorphic loci (PIC > 0.5) comprised 36.4% of all loci, whereas moderately polymorphic (0.25 < PIC < 0.5), and lowly polymorphic (PIC < 0.25) loci occupied 36.4% and 27.2%, respectively.A total of 67 alleles were generated by the 11 SSR loci in all samples. The number of alleles , whereas the lowest was detected in population BWL (He = 0.302). In population SZHN, He (0.341) was higher than Ho (0.287), which implied that there was a deficit of heterozygosity in the population, possibly due to the existence of inbreeding. We assessed the level of genetic variation among four different regions, and these results showed that the highest genetic diversity  and the largest number of private alleles (n = 5) was found in the Southwestern populations, whereas the lowest genetic diversity  and a relatively low number of private alleles (n = 1) were discovered in the Central populations.The genetic diversity at the population and region level is presented in Table Fis) per locus varied from −0.1915 (LF 25) to 0.0476 (LF 69) with a mean of −0.0213, and these results indicated an excess of heterozygosity. However, a deficiency of heterozygosity was discovered at six loci: LF 3, LF 29, LF 32, LF 40, LF 49, and LF 69. Furthermore, the inbreeding coefficient at total populations (Fit) ranged from −0.1354 (LF 25) to 0.1818 (LF 69) with an average of 0.056. Moreover, the genetic differentiation (Fst) ranged from 0.0461 (LF 40) to 0.1409 (LF 69) with a mean of 0.0757, which indicated moderate differentiation among populations (Table The inbreeding coefficient within populations (K = 16.13) was at the value K = 3 . These results provided further evidence that geographic isolation was not the primary factor leading to moderate genetic differentiation of L. formosana.The Mantel test revealed that no correlation was found between genetic distance and geographic distance in L. formosana was one SSR locus per 5.28 kb, which is lower than that of Larix gmelinii (one SSR locus per 2.87 kb)   may be helpful for targeting genic SSRs of plant materials at different taxonomic levels.The mean frequency of SSR loci detected in He = 0.399) detected in our study was only relatively moderate. The genetic diversity was lower than the average value (He = 0.650) of outcrossing plants using SSR markers  and the National Forest Genetic Resource Platform (2005DKA21003).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.


Litsea cubeba L., also named as Makauy, is a traditional herb and has been used as cooking condiment or tea brewing to treat diseases for aborigines. The present study was undertaken to explore the chemical compositions of the fruit essential oil of L. cubeba (LCEO) and the immunomodulatory effect of LCEO on dendritic cells and mice. The LCEO was analyzed using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) with direct injection (DI/GC) or headspace-solid phase microextraction (HS-SPME/GC). In total, 56 components were identified, of which 48 were detected by DI/GC and 49 were detected by HS-SPME/GC. The principal compounds were citral . An immunosuppressive activity of LCEO was investigated with bone marrow-derived dendritic cells (DCs) which have a critical role to trigger the adaptive immunity. Additionally, the inhibitory effect of LCEO on immune response was elucidated by performing the contact hypersensitivity (CHS) responses in mice. Our results clearly showed that LCEO decreases the production of TNF-α and cytokine IL-12 in a dose-dependent manner in lipopolysaccharide (LPS)-stimulated DCs. CHS response and the infiltrative T cells were inhibited in the tested ears of the mice co-treated with LCEO. We demonstrate, for the first time, that the LCEO mainly containing citral exhibits an immunosuppressive effect on DCs and mice, indicating that LCEO can potentially be applied in the treatment of CHS, inflammatory diseases, and autoimmune diseases. In the immune system, various immune cells are highly communicative with each other by various cytokines and are in charge of the defense against foreign pathogen infection and maintaining health. Inflammation is a major component of our immune response. Although inflammation is a natural defense, the persistence of the process for abnormally long periods can be harmful and has been recognized as a major risk factor for various human diseases, including cardiovascular disease, metabolic disorder, neurological disease, and cancer ,2. Thus,Litsea cubeba L. belongs to the family Lauraceae, which is widely distributed in Japan, Taiwan, Southern China, and Southeastern Asia [L. cubeba are spicy condiments, frequently used in the aboriginal cuisine of Taiwan [L. cubeba on DCs and its relative immune response in vivo. In this study, L. cubeba essential oil (LCEO) was extracted from the fresh fruits. The inhibitory effect of the LCEO on dendritic cell activation was examined. In addition, the contact hypersensitive response was conducted to examine the in vivo immunosuppressive effects in mice. ern Asia . All parern Asia . Fruits f Taiwan . The essf Taiwan . Its phaf Taiwan ,13, antif Taiwan , anticanf Taiwan ,15, antif Taiwan , and insf Taiwan ,16. HoweL. cubeba berries of Thai origin and the yield was ca. 3.0% [L. cubeba from Taiwan, and the yields were 13.9% ± 0.09% and 4.0% ± 0.03%, v/w, respectively [L. cubeba from Taiwan, and the yields were 4.5% ± 0.2% [L. cubeba contains 3%–5% of essential oils which are rich in citral [L. cubeba by steam distillation was 3.7% ± 0.4%. The yield is similar to that reported in these published studies.Punyarajun and Nandhasri 1981) extracted essential oils from unripe ca. 3.0% . Ho et aectively . Liu and81 extracn citral . In the As shown in The headspace-solid phase microextraction (HS-SPME) method has been reported to be an excellent tool for the analysis of herbs because it is simple, fast, and does not leave any residues . In thisComparative analysis of these compounds identified by these two methods, showed that 56 compounds were detected in total, of which, 48 were identified by DI/GC and 49 by HS-SPME/GC. As shown in L. cubeba essential oils (LCEO) on dendritic cells (DCs), the viability of mouse bone marrow-derived DCs treated with the different concentrations of LCEO was examined. The result showed no significant effect of 5 × 104-, 1 × 105-, 2 × 105- and 4 × 105-fold diluted LCEO on DCs, although 5 × 104-fold diluted LCEO exhibited a little cytotoxic effect  cells in the tested ears was counted. The results showed that infiltrative CD3+ cells were significantly reduced by the presence of LCEO  were homogenized for 2 min with 1200 mL of deionized water. The homogenate was put into a 5 L round-bottom flask and steam-distilled for 4 h to extract the essential oils. The oil was dried over anhydrous sodium sulfate. The prepared samples were immediately stored in brown flasks at −20 °C (freezer) prior to analyses by gas chromatography (GC) and bioassays.Fresh fruits of (1) Direct injection analytic method (DI): 1 μL of essential oil was injected into the gas chromatograph injection unit. All experiments in the present study were performed in triplicate.(2) Headspace-solid phase microextraction (HS-SPME) analysis: A 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane fiber  was exposed to each sample (1 mL) as placed in a 22 mL vial  for 20 min at 25 °C; the fiber was then injected into the gas chromatograph injection unit.(3) Analysis of GC: quantitative analyses of the volatile compounds were performed using an Agilent 7890A GC  equipped with a DB-1  fused-silica capillary column with a flame ionization detector. The oven temperature was held at 40 °C for 1 min and then raised to 150 °C at 2 °C/min and held for 1 min, and then increased from 150 to 200 °C at 10 °C/min and held for 3 min. Injector and detector temperatures were maintained at 250 °C and 300 °C, respectively. The nitrogen gas flow rate was 1 mL/min. Kovats indices were calculated for the separated components relative to a C5-C25 n-alkanes mixture . The met(4) Analysis of GC-MS: qualitative analyses of volatile compounds were identified using an Agilent 7890B GC  equipped with a DB-1  fused-silica capillary column coupled to an Agilent model 5977 N MSD mass spectrometer (MS) (Agilent model 5977 N MSD mass spectrometer). The GC conditions in the GC-MS analysis were the same as in the GC analysis. The injector temperature was maintained at 250 °C. The helium gas flow rate was 1 mL/min. The electron energy was 70 eV at 230 °C. The constituents were identified by matching their spectra with those recorded in an MS library (Wiley 7n). The constituents were confirmed by comparing the Kovats indices or GC retention time data with data published in the literature or those of authentic standards. The method used was modified as previously described [C57BL/6 mice, which were purchased from Taiwan, were used in this study. All animals were housed in a specific pathogen-free facility in the Division of Laboratory Animals, China Medical University. All mice were maintained and handled according to standard protocols and the protocols was approved  by the Institutional Animal Care and Use Committee, China Medical University. The bones of mice were collected and bone marrow-derived dendritic cells (DCs) were prepared as previously described .4-, 105-, 2 × 105-, and 4 × 105-fold dilution in final) at 37 °C in 5% CO2/air for 24 h. The cells were then harvested and the viability measured according to manufacturer’s instruction.The cytotoxicity of LCEO was examined by Cell Counting Kit-8 (CCK-8) assay . The LCEO was diluted into 50-fold diluted stock with dimethyl sulfoxide. The DCs were treated with LCEO at different concentrations (5 × 10Escherichia coli 055:B5 (Sigma) or LPS + LCEO  for 6 h for TNF-α and 24 h for IL-12. The production of TNF-α and IL-12p70 was measured using the ELISA kit . Cytokine production was measured by enzyme-linked immuno sorbent assay (ELISA) as described previously ,7,8. The2,4-Dinitro-1-fluorobenzene -stimulated hypersensitivity was conducted as previously described ,24. BrieU-test was used. In order to assess the significance of the differences in the numbers of CD3+ T cells, the Student’s t-test with a two-tailed distribution and two-sample equal variance was used. Values of ** p < 0.01 and *** p < 0.001 were considered highly significant. A value of * p < 0.05 was considered significant.In order to assess the significance of the differences in the levels of the cytokines and the increase of thickness of ear, the Mann–Whitney A total of 56 components were identified in LCEO. Forty-eight were detected by DI/GC, and 49 were detected by HS-SPME/GC. The principal compounds were neral and geranial . LCEO inhibits DC functioning. Thus, LCEO may be useful in the treatment of inflammatory diseases.


Monochamus galloprovincialis) using a landscape genetics approach. To account for the unknown scale of gene flow and the multiple local confounding effects of evolutionary history and landscape changes on inference, we developed a way of resampling study areas on multiple scales and in multiple locations (sliding windows) in a single large-scale sampling design. Landscape analyses were conducted in 3*104 study areas ranging in scale from 220 to 1,000 km and spread over 132 locations on the Iberian Peninsula. The resampling approach made it possible to identify the features affecting the gene flow of this species but also showed high variability in inference among the scales and the locations tested, independent of the variation in environmental features. This method provides an opportunity to explore the effects of environmental features on organism gene flow on the whole and reach conclusions about general landscape effects on their dispersal, while limiting the sampling effort to a reasonable level.The use of multiple sampling areas in landscape genetic analysis has been recognized as a useful way of generalizing the patterns of environmental effects on organism gene flow. It reduces the variability in inference which can be substantially affected by the scale of the study area and its geographic location. However, empirical landscape genetic studies rarely consider multiple sampling areas due to the sampling effort required. In this study, we explored the effects of environmental features on the gene flow of a flying long-horned beetle ( Landscape genetics examines the relationship between landscape and environmental features and genetic structure . It enabTo integrate these factors and deal with the variability of the results in landscape genetic analysis, several authors have highlighted the importance of two aspects: matching the study design to the process being investigated . We first characterized the broad-scale genetic structure of the beetle across the study area, and we used the ”isolation-by-resistance” (IBR) framework to model beetle dispersal as a function of three relevant landscape features , called IBR hypothesis . For each of these IBR hypothesis, we then carried out a landscape genetic analysis based in 30,576 resampled areas of extents ranging from 220 to 1,000 km and distributed in 132 sampling locations on the Iberian Peninsula. Lastly, we searched for the main landscape features affecting gene flow in M. galloprovincialis and we analyzed how the scale and location of the study area impacted inference.In order to provide a more complete and comprehensive picture of the general effects of the landscape on the dispersal of organisms, there is a need to integrate various scale and landscape level replications of sampling areas in more empirical landscape genetic studies. However, such exploration often remains limited due to the substantial sampling efforts required. Resampling methods  are an i2) with altitudes ranging from sea level up to 2,444 m. M. galloprovincialis specimens were trapped between 2011 and 2013 at 137 sites spread over the Iberian Peninsula. We used multifunnel traps baited with a volatile attractant  installed during the summer to catch flying adults. The traps used had a radius of attraction of 100 m  was estimated for each deme, each inferred cluster and for the whole dataset using GENEPOP 4.2 (n > 19). Loci exceeding a rate of 7% of null alleles across populations were discarded from further analysis. Allelic richness was computed for each deme using rarefaction . Specimens were genotyped at 12 microsatellite loci , with α = 1, 2, …, n, be a set of n values of sample scores on a PCA axis where uα is the vector of spatial coordinates of the αth observation. In geostatistics, spatial dependence is described in terms of dissimilarity between observations expressed as a function of the separating distance (N(h) is the number of data pairs for a given lag vector h, z(uα) and z(uα + h) the score values of all sample locations separated by a vector h. The more alike the observations at points separated by h are, the smaller h is called a variogram and represents the average rate of change of z with distance. Its shape describes the pattern of spatial variation in terms of general form, scales and magnitude , we considered three environmental features to be potential drivers of dispersal .We then selected the environmental features considered to be the most likely to influence the dispersal of M. galloprovincialis and is thought to affect its foraging dispersal. As the dispersal behavior of this beetle in reaction to pine density is not known, we modeled this parameter according to two alternative scenarios. (1) High pine densities are positively correlated with beetle dispersal . In this scenario, a dense pine cover represents a corridor for dispersal due to the large amount of resources available. Conversely, a low pine density would represent a barrier. (2) High pine densities are negatively correlated with beetle dispersal . For this second scenario, it was assumed that a dense pine cover provides sufficient resources for local populations, which would therefore not need to disperse. This scenario assumes increased dispersal in low pine cover areas. To model resistance based on pine density, we considered the sum of densities of all pine species encountered in a grid cell, because in the Iberian Peninsula, M. galloprovincialis shows no preference for pine species. As shown in previous studies, this beetle will live in the dead wood of any of the pine species considered in this study , the pine density from http://www.efi.int/; original resolution: 1 × 1 km) and elevation from ARCGIS 9.3 . Null distances were not encountered at a grain size of 10 × 10 km, as none of the sampling sites fell within neighboring sites in the same grain. As temperature, elevation and pine density are continuous parameters, we did not assign particular resistances to particular values, but directly used the values . Pairwise resistance distances were estimated based on random walk probabilities (R2), where R2 is the r-squared value of regression between variables. VIF values >10 are usually considered evidence for collinearity between environmental features (Resistance distances were computed using the gdistance package . Raster eriments . Temperabilities  and compbilities . We consfeatures . We did p < 0.05) and IBD should be non-significant with IBR partialled out  and resistance distances  and the geographic distances  using partial Mantel tests . Partialgenetics  and has genetics . Howevergenetics , and aregenetics . To overgenetics . This megenetics .The above Partial Mantels test and commonality analysis were conducted in areas of various spatial scales and in various locations in order to estimate the effects of scale and location on the inference of the landscape genetic analysis for the four IBR hypothesis . First, nested sampling areas (sliding windows) were generated over the full extent of the Iberian Peninsula. These areas were constructed as circles of diameters ranging from 220 to 1,000 km (steps of 20 km) and centered on each sampling location , two loci exhibited substantial null allele frequencies (>7%) and were therefore not considered for further analysis (Mon 01 and Mon 27). A significant heterozygote deficit was detected at four loci . Corresponding null allele frequencies were low (<7%), so these loci were kept. After the removal of incomplete genotypes (n = 58) and biased loci, we obtained a total of 992 individuals genotyped at ten loci. The average number of alleles per locus was 10.2 (range: 6–24). The number of alleles per deme (using rarefaction) ranged from 1.32 to 1.64 and Fis estimates from −0.27 to 0.38  between the three populations of large size (n > 19) were moderate .Individuals formed an optimum number of two clusters under STRUCTURE analysis . The cluThe data points of the variogram were grouped into 26 distance classes ranging from 0 to 1,252 km, with a distance interval of 50 km. The variogram revealed that the first axis of the PCA corresponded to a highly spatially structured pattern . The semAnalyses were conducted on both the whole dataset  and within the East Iberian cluster . Within each dataset, the number of alleles observed across all individuals and loci was 116 and 102, respectively.Over the whole study area (whole dataset), we generated a total of 30,576 sampling areas. The mean number of individuals within sampling areas varied from 89.18 (SD: 42.42) on the smallest scale (220 km) to 644.58 (SD: 158.07) on the largest scale  or inflection of the curves  of the number of areas with supported IBR hypotheses observed on scales ranging from 400 to 600 km corresponded to the increase in dissimilarity in the variogram . Support was scarcely detected on the smallest spatial scale (220–400 km) and was generally more often detected with increasing scale. A larger study area had a larger sampling leading to better detection of positive correlations via partial Mantel tests . Beyond ariogram . Thus, s mammals  and inse mammals , this stMonochamus species, substantial flying distances have been measured . Most variation in the distribution of support for IBR was observed on small and intermediate scales (220–600 km), the smallest scale being larger than areas at which landscape genetic analysis are usually conducted . On thesfeatures . This obenturies , resultis tested .M. galloprovincialis. This effect of differentiated genetic clusters on inference was also observed in the strength of the correlations: we observed a decrease in the mean Mantel r for large-scale study areas  which always overlapped the two genetic clusters. It is difficult to predict exactly to what extent genetic divergence derived from evolutionary history can contribute, or not, to the detection of an effect of environmental features on gene flow, as this structure can also derive from old and stable barriers to dispersal. While the vast majority of species exhibit differentiated genetic linages derived from evolutionary history , another oligophagous pine–associated insect .Click here for additional data file.10.7717/peerj.4135/supp-2Figure S2Mean: black; ±SD: greyClick here for additional data file.10.7717/peerj.4135/supp-3Figure S3Click here for additional data file.10.7717/peerj.4135/supp-4Table S1Fis, Fis estimate of deme, computed without Mon01 and Mon 27.Long. and Lat. refer to the geographic coordinates of the sampling sites; N, is the number of individuals of demes; A, mean allelic richness; AR, corrected allelic richness, accounting for variation in deme size; Click here for additional data file.10.7717/peerj.4135/supp-5Table S2®), 0.75 µL Dream Taq Green Buffer , 1 µM Betaine, 0.24 µL dNTP (10 µM) and deionized H2O. PCR amplifications were run on a Veriti® 96 well fast thermal cycler (Applied Biosystems®) using the following settings: a first denaturation step at 95 °C for 10 min; 40 cycles of denaturation (30 s at 95 °C), hybridization (30 s at 55 °C) and elongation (1 min at 72 °C), and a final elongation step at 72 °C for 10 min. One µL of PCR products was denatured in a mix of 10 µL of formamide and 0.3 µL of 600 Liz marker before being run on an ABI PRISM 3500 sequencer (Life Technologies®). Genotypes were read using GENEMAPPER V 4.1 software (Applied Biosystems®).Multiplexed PCR were performed in a 10 µL reaction volume using 25 ng of genomic DNA, 0.4 U of DreamTaq DNA Polymerase (Thermo ScientificClick here for additional data file.10.7717/peerj.4135/supp-6Table S3Click here for additional data file.