Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is one of the most destructive pests of corn in North America and is currently invading Europe. The two major invasive outbreaks of rootworm in Europe have occurred, in North-West Italy and in Central and South-Eastern Europe. These two outbreaks originated from independent introductions from North America. Secondary contact probably occurred in North Italy between these two outbreaks, in 2008. We used 13 microsatellite markers to conduct a population genetics study, to demonstrate that this geographic contact resulted in a zone of admixture in the Italian region of Veneto. We show that i) genetic variation is greater in the contact zone than in the parental outbreaks; ii) several signs of admixture were detected in some Venetian samples, in a Bayesian analysis of the population structure and in an approximate Bayesian computation analysis of historical scenarios and, finally, iii) allelic frequency clines were observed at microsatellite loci. The contact between the invasive outbreaks in North-West Italy and Central and South-Eastern Europe resulted in a zone of admixture, with particular characteristics. The evolutionary implications of the existence of a zone of admixture in Northern Italy and their possible impact on the invasion success of the western corn rootworm are discussed.The western corn rootworm,  In the early stages of biological invasions, the genetic diversity of populations may be reduced by bottlenecks Admixture may thus have a positive impact on invasion, through the generation of novel genotypes Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)) has involved multiple introductions into several disconnected geographic areas from a single source population in the northern USA th century and the early 2000s, WCR was introduced into Europe repeatedly from the Northern USA FST of 0.25 between CSE Europe and NW Italy. The largest outbreak, in CSE Europe, currently covers 16 countries extending from Austria to Ukraine and Southern Poland to Northern Greece. There has also been a westward expansion into eastern parts of Italy. In parallel, the NW Italian invasive outbreak has progressed eastwards. Since 2008, the NW and NE Italian and CSE European outbreak populations have been in close geographic proximity or even in contact, as in the Italian region of Veneto in natura. The aim of this study was to identify, document and characterize the putative admixture in Northern Italy, following contact between the WCR outbreak populations of NW and NE Italy and that of CSE Europe.The invasion of Europe by the western corn rootworm . For the Northern USA sample, DNA was extracted from individuals with the BioRad Aqua Pure isolation kit  according to the manufacturer’s instructions, whereas, for other samples, DNA was extracted with the DNeasy tissue kit , as explained above.Additional samples , Table 1All WCR samples were stored in 90–96% ethanol until DNA extraction. Template material for the polymerase chain reaction (PCR) amplification of microsatellite loci was obtained with two different protocols. For the NE Italian sample, the ‘salting out’ protocol of Sunnucks and Hales We amplified 13 WCR microsatellite loci  and mean expected heterozygosity (He) A and He were calculated with GENECLASS version 2.0.h ISF with GENEPOP ver. 4.0.1 A values between population samples by estimating allelic richness (AR) based on the smallest sample size, by the rarefaction method AR and He between samples were assessed in a one-sided Wilcoxon signed rank test  with locus as a repetition unit. The significance of the differences between the contact zone (Veneto) and the parental outbreak areas  was then assessed by combining the probabilities obtained for each sample from Veneto by Fisher’s method FST and by assessing genic differentiation between pairs of samples Genetic variation within samples was evaluated by determining the mean number of alleles per locus (K), defined as lying between 1 and the total number of sampling sites. Each run consisted of a burn-in of 5×104 iterations, followed by 105 iterations. We used the admixture model of ancestry together with the correlated allele frequencies model K was estimated with Evanno’s ΔK statistic K and the variability of log-likelihood between different runs. The most likely run was then represented with DISTRUCT version 1.0  on microsatellite allelic frequencies. We defined a geographic axis passing through the various sampling sites and we then projected each sampled population orthogonally on this axis . The proet al.Q, calculated with STRUCTURE version 2.3.3 The admixture rate was estimated (i) as described by Choisy For each sampling site within the contact zone (i.e. for each of the six Venetian target samples), we conducted six independent ABC analyses comparing various historical scenarios  or European  source populations and sG,N the effective size of the ghost population , were drawn from a uniform distribution bounded by 1000 and 20000 ; FiN and FG,N the effective number of founders of the European populations, were drawn from a Uniform distribution; the bottleneck duration of population i, iBD, was drawn from a Uniformi , NE Italy, NW Italy and CSE Europe, respectively) was founded by individuals originating from its source population it (and Gt for the ghost population) generations before the present . As WCR is univoltine, it and Gt are also the number of years before the present. 1t and Gt, 2t, 3t, and 4t were drawn from between one and five generations (or years) before the date of the first observation . For each locus, the coefficient P of the geometric distribution of repeat units by which a new mutant allele differs from its ancestor was drawn from an exponential distribution with a mean of 0.22.The prior distributions of the historical, demographic and mutational parameters used in the ABC analysis were as follows: tion see  and wereodel GSM . A mean We evaluated the ability of ABC to select the true scenario correctly, by analyzing test datasets simulated from known competing scenarios. For each scenario, one hundred such datasets were simulated with parameter values drawn from the same probability distributions as the priors. The posterior probabilities of each competing scenario were estimated for each simulated test dataset, with the same ABC procedure as described above, and were used to calculate type I and II errors in the selection of scenarios. Type I error is the proportion of simulations in which the scenario considered is excluded but is actually the true one. Type II error is the proportion of simulations in which the scenario considered is selected but is not the true one. Small Type 2 errors provide good confidence in the results even if the Type 1 errors are large.The main body of the present study concerns spatial analyses of WCR genetic variation. When the samples were not collected within the same year as in our case, a procedure must guarantee that no temporal confounding effect occurs. Such a temporal effect was hence tested using samples collected within the same area at various dates . Locations showing a significant temporal effect were removed from the spatial analysis.p>0.08 for all pairwise comparisons considered). These locations were thus kept and the 2007 samples were arbitrarily chosen for subsequent spatial analyses. A significant temporal differentiation was found in Friuli . Friuli was thus not considered for the spatial analyses.No temporal differentiation was found between samples collected in the NW Italy outbreak in Piedmont , in Lombardia  and in the CSE Europe outbreak (Hungary (Tolna) and Serbia (Voïvodine) in 2007 and Slovenia (Prekmurje) 2010; The Friuli sample of Buttrio was sampled in 2003, i.e. before the contact between the NW, NE Italian and CSE European outbreaks in 2008. Hence, it properly describes the north eastern Italian outbreak before any contact between the various outbreaks. Buttrio was therefore used in the ABC historical scenario comparison.He) was low to moderate and varied from 0.29 for Buttrio in NE Italy to 0.54 for San Donà di Piave in Veneto  and a marginally significantly larger heterozygosity  in Veneto compared to parental samples. Most pairwise comparisons of samples (92%) showed significant genetic differentiation  alleles per locus over all samples. Within samples, the mean number of alleles was between 1.77 (SD = 0.73) for Buttrio in NE Italy and 3.77 (SD = 2.43) in Oleggio in NW Italy . Mean exn Veneto . FIS estobserved . Samplesntiation . Veneto ntiation .(K) was estimated at K = 2, whatever the model used. The analysis of coancestry coefficients (Q) (for the model with the use of sampling location as prior information)  (ii) individuals from the east belonged to another cluster  and that (iii) a large number of individuals from Scorzè, San Donà di Piave, Borso del Grappa and Piove di Sacco are admixed between both clusters (with Q between 0.20 and 0.80). One individual from Summaga, a population belonging to the eastern genetic cluster, was assigned to the western genetic cluster (Q = 0.97).The number of genetic clusters rmation)  indicatep<0.0033 for each of these 6 loci; see 2 = 113.62, df = 26, p<10−5). The admixture rates obtained by the two different methods . A significant effect of geographic distance on allelic frequencies was found for six of the 13 loci studied (FST/(1-FST) between each sample and Oleggio, the sample located in the extreme west of the studied area was significant  is observed in the contact zone, i.e. in Veneto, where values are intermediate.The effect of the geographic distance on the The ABC procedure was used to calculate the posterior probabilities of the evolutionary scenarios describing the origin of each population sampled in Veneto. According to the highest probabilities with non overlapping confidence intervals, four of the six Veneto samples probably originate from admixture events: samples from Scorzè and San Dona di Piave probably originate from admixture between the outbreak populations of NW Italy and CSE Europe ; samplesSome of the largest posterior probabilities were only moderate  and someSince the first observation of the CSE European outbreak of WCR near Belgrade in 1992, a monitoring network has followed the progression of this pest in Europe The current contact zone between the different European outbreak populations  is located in Veneto. This zone has been intensively monitored with pheromone traps since the first captures of WCR in the Venetian region in 1998, leading to the implementation of an eradication program. Since 2008, the geographic distribution of WCR has been continuous in Northern Italy Our analysis of the population genetic structure of WCR in Northern Italy and CSE Europe showed that the WCR populations sampled in Veneto resulted mostly from contact and admixture between the NW Italian and CSE European outbreak populations. The admixture analysis, the Bayesian analysis of population genetic structure and the ABC analysis of scenario choice all indicate that the Veneto region is a zone of admixture, containing admixed individuals. The genomes of individuals sampled in Veneto could be attributed to both the genetically different clusters of NW Italy and CSE Europe, with various levels of admixture. Moreover, the rate of admixture between the NW Italian and CSE European outbreaks varied evenly across a gradient from west to east.According to the ABC analysis, most samples from the western part of Veneto have a simple NW Italian origin, whereas all the other sample populations result from single or double admixture events between the three parental outbreak populations . Double admixture between individuals originating from distant populations  accounted for the origin of the most westerly sample from Veneto (Borso del Grappa). This implies that individuals from the eastern outbreak populations  migrated to the western part of Veneto, suggesting that long-distance dispersal occurs. Long-distance dispersal is also suggested by the detection of an individual genetically assigned to the western genetic cluster in Summaga, a population from the eastern genetic cluster. Recent studies have suggested that long-distance dispersal is common in WCR and that invasive populations of WCR are expanding through stratified dispersal The occurrence of a cline in the Veneto region in 2009, even with such a large width (about 100 km), is consistent with the short period of time between secondary contact (2008) and sampling (2009). The WCR has a considerable capacity for dispersal, as shown by the rapid rate of expansion of the CSE Europe outbreak population , may thus provide information about the balance between dispersal and selection Harmonia axyridisMelanoides tuberculataSilene vulgarisWe found that genetic variation was greater in the zone of admixture than in the parental outbreak areas. Moreover, the genetic variation within the zone of admixture is approaching that of the Northern USA source population and thus displays substantial restoration of the genetic variation lost during the introduction and establishment of the invasive outbreaks. Indeed, the mean allelic richness and the mean heterozygosity of the zone of admixture represent 70% and 77% of that of the Northern USA source population, respectively, whereas the NW Italian, the NE Italian and CSE Europe outbreaks display a mean of 59, 41 and 63%, of the allelic richness and 64, 47 and 76% of the heterozygosity found in the Northern USA, respectively , (ii) and (iii) represent the “simple origin” scenarios, scenarios (iv), (v) and (vi) are “single admixture” scenarios and (vii), (viii) and (ix) are “double admixture” scenarios. Historical and demographic parameters were identical for all introduction models. Time 0 is the present and represents the year of the study (2010). The Veneto population was founded 1t generations before the present, had an effective number of founders VenetoNF with the population remaining at this size for 1BD generations (bottleneck duration) and then reached a larger stable effective population size VenetoNS. The putative source populations, the Central South-Eastern European (CSE Europe) and North-West (NW) Italian outbreak populations, diverged from the USA population 4t and 3t generations ago with an effective numbers of founders CSENF and NWItalyNF, bottleneck durations 4BD and 3BD and stable effective population sizes CSENS and NW ItalyNS, respectively. The North-East (NE) Italian population was founded 2t generations ago from the CSE European population, with an effective number of founders NEItalyNF, a bottleneck duration 2BD and an effective population size NEItalyNS. When admixture occurs, the admixture rates ar and 1-ar are the genetic contribution of each of the source populations to the origin of the Veneto population. An unsampled population called the “Ghost population” was included into the analysis to allow “double admixture” scenarios (scenario (vii), (viii) and (ix)) between NE and NW Italy and CSE outbreak populations. This “Ghost population” is the result of a single admixture between two of the three putative source populations (the populations involved depend on the scenario), Gt generations ago (Gt>1t and Gt<2t), with an effective number of founders GhostPopulationNF, a bottleneck duration GBD and an effective population size GhostPopulationNS. The rates of admixture corresponding to the “Ghost population” are Gar and 1-Gar. For all models, populations were assumed to be isolated from each other, with no exchange of migrants.(TIF)Click here for additional data file.Figure S2Percentage of the mean expected heterozygosity (He) and allelic richness (AR) of the Northern USA source population  for each sample from the three putative parental outbreak areas  and for samples from Veneto. Dark gray bars indicate the mean expected heterozygosity (He) of the various samples, whereas light gray bars correspond to their allelic richness (AR).(TIF)Click here for additional data file.Table S1Mean pairwise STF comparisons between Northern Italian  and Central and South-Eastern European (CSE Europe) samples of western corn rootworm (WCR).(DOC)Click here for additional data file.


At present the Croatian Turopolje pig population comprises about 157 breeding animals. In Austria, 324 Turopolje pigs originating from six Croatian founder animals are registered. Multiple bottlenecks have occurred in this population, one major one rather recently and several more older and moderate ones. In addition, it has been subdivided into three subpopulations, one in Austria and two in Croatia, with restricted gene flow. These specificities explain the delicate situation of this endangered Croatian lard-type pig breed.In order to identify candidate breeding animals or gene pools for future conservation breeding programs, we studied the genetic diversity and population structure of this breed using microsatellite data from 197 individuals belonging to five different breeds.The genetic diversity of the Turopolje pig is dramatically low with observed heterozygosities values ranging from 0.38 to 0.57. Split into three populations since 1994, two genetic clusters could be identified: one highly conserved Croatian gene pool in Turopoljski Lug and the"Posavina" gene pool mainly present in the Austrian population. The second Croatian subpopulation in Lonjsko Polje in the Posavina region shows a constant gene flow from the Turopoljski Lug animals.One practical conclusion is that it is necessary to develop a "Posavina" boar line to preserve the "Posavina" gene pool and constitute a corresponding population in Croatia. Animals of the highly inbred herd in Turopoljski Lug should not be crossed with animals of other populations since they represent a specific phenotype-genotype combination. However to increase the genetic diversity of this herd, a program to optimize its sex ratio should be carried out, as was done in the Austrian population where the level of heterozygosity has remained moderate despite its heavy bottleneck in 1994. Many studies on the diversity of various pig breeds have been reported in the literature in recent years. As early as the 1990's, several research groups have worked on harmonizing data from these various genetic studies in multiple pig populations from Europe and China PiGMaP -4). For . For 4])Larson et al. ,7 have sth century. At present, this subpopulation comprises 90 breeding animals and over the last 60 years, it has not undergone any immigration of pigs from other origins [In this study, our aim was to illustrate the diversity and genetic relationships of local pig breeds originating from Croatia, Serbia, Hungary and Bosnia-Herzegovina. The Croatian Turopolje breed has undergone a complicated recent breed history. In 1991, the last Turopolje pigs of the Posavina region in Croatia were collected to establish a nucleus herd for further conservation programs. However, in the autumn of the same year the Yugoslavian wars started and put a stop to this project initiated by the SAVE Foundation and EURONATURE. Six animals were rescued and placed in the Zagreb zoo. Four of these animals, together with two boars imported from the Posavina region in 2001, constituted the founder population for the Turopolje breed in Austria, which comprises at present 324 herdbook animals. A recently conducted pedigree analysis showed the high levels of inbreeding  and the poor variability of the Austrian gene pool ,11. AfteBecause in the Austrian population the level of inbreeding is very high, our aim was to genetically characterize sub-populations and individuals from the Croatian pig breed in Turopoljski Lug, which could be used for future breeding programs in Austria. We studied the genetic diversity within the three Turopolje populations and their genetic relationships. In order to take into consideration, the influence that the instable demographic and political situation during and after the Croatian war may have had on the breeding of Turopolje pigs in the Save delta, we included animals from other local pig breeds localized next to this area i.e. the Black Slavonian or Fajferica, a local lard-type breed of the eastern part of Croatia, the Bosnian mountain pig, a local mixed population on the Bosnian side of the Save river and Austrian and Serbian Mangalica pigs, which originate from the border region of Serbia and Croatia Figure .Hair samples were collected on field trips in Croatia, Serbia and Bosnia Herzegovina in 2009 and 2010. Samples of extracted DNA for parentage controls were also used, which included all unrelated Austrian pig individuals and part of the Croatian individuals sampled in 2003 and five Turopolje pigs sampled in 1998. A total of 177 pigs were sampled i.e. 93 Turopolje pigs (29 from the herd in Turopoljski Lug (TL), 39 from the herd of the Lonjsko Polje national park (LP) and 25 from the Turopolje population of Austria (AUT), 41 Black Slavonian pigs, 23 Mangalica pigs from the Austrian (AUT) herdbook , 11 Serbian Black Mangalica pigs from the national Zasavica park (SER), and nine pigs from a valley in Bosnia and Herzegovina, Republika Srpska. Since the small-sized Bosnian samples may cause distortion in the phylogenetic studies, they were excluded but the Bosnian mountain pigs were retained for the analysis of gene diversity and population structure. Samples of 20 unrelated Austrian Pietrain pigs were also included, as outgroup individuals since this breed is quite distant from the lard-type breeds of the Balkan countries both in terms of production traits mean values and genetic units. Finally, the DNA from 197 animals from eight populations and five breeds was used. The experimental data in this paper were obtained according to the legislative regulations regarding experimentals research on animals in Austria, Croatia, Serbia and Bosnia i Herzegovina.All animals were genotyped for 25 microsatellite markers belonging to the panel recommended by the ISAG-FAO Advisory Committee for genetic distance studies . Twelve IS was calculated according to Weir and Cockerham [Allele frequency, number of alleles, observed heterozygosity (HO) and gene diversity (HE) were calculated across different loci and populations using the GENETIX 4.05.2 software package . Mean nuockerham  using GEockerham . To accoockerham  was applockerham .ST [k distance [Molecular genetic relationships among populations were derived using Wright's FST  and the ST . Individdistance . Among tdistance ,23.k matrix was performed using the SAS® software [All genetic distances were calculated using the software package POPULATIONS 1.2.28 . The stasoftware .6 iterations following a burn-in period of 105 iterations, applying an admixture model, correlated frequencies model and the parameter of individual admixture alpha set to the same values for all clusters and with a uniform prior. The resulting individual genotype membership coefficients were displayed using DISTRUCT [Population subdivisions and genetic differentiation can be tested by several clustering procedures. However, the algorithm of Pritchard et al. , implemeDISTRUCT .S0227 and SW951 to 0.83 for loci SW939 and S0097. A significant deviation from HWE was observed for loci SW951, S0387 and SW2410 and although the existence of null alleles for these three loci was not demonstrated, they were excluded from further analyses The results are based on 19 microsatellite loci from 181 animals; sample size (N), observed heterozygosity (HO), gene diversity (HE), mean number of alleles (MNA), the rarefacted mean number of alleles on a sample with 22 genes (RMNA), Wright's FIS coefficients, with a 95% confidence interval ranging from -0.20 to -0.04, were estimated for the Serbian Mangalica and Turopolje TL breeds, suggesting an excess of heterozygotes due to non-random mating. In all other populations FIS values were not significantly different from zero. Overall, low average diversity values  were observed in all three Turopolje populations  illustrating the risk status of this breed.Negative Fp = 0.013, two sided test p = 0.027).Applying the Wilcoxon sign-rank test  for geneST values was observed between the Turopolje TL and Turopolje AUT populations (FST = 0.26). Lower differentiation levels ranging from FST = 0.05 to FST = 0.09, were observed between the Black Slavonian and Mangalica breeds (AUT and SER), Bosnian mountain, and the Austrian Turopolje pig populations and between the Turopolje TL Turopolje LP populations , although its FIS value is significantly lower than zero, suggesting that there are more heterozygous animals than expected. This contradiction may be explained by the breeding history of this population. The nucleus herd is managed by a farmers' collective and at present, although the population is split into two herds, the breeders are using only one or in the past two boars for reproduction. This mating strategy results in genetic and phenotypic uniformity and high levels of inbreeding within the boar lines. The merging of such boar lines is comparable to cross-breeding, which is documented here by the negative FIS value. Situations in the Austrian Turopolje herd with high heterozygosity values reaching 0.48 and in the nucleus herd in Turopoljski Lug differ completely. This is due to of the fact that a narrow sex ratio  has been maintained during the last 17 years.The genetic diversity within the different Balkan pig breeds studied is low compared to that of conventional breeds playing a key role in global meat production. Numerous studies have reported observed heterozygosity values for different populations of the Landrace, Large White, Pietrain and Duroc breeds with values ranging from 0.67 to 0.50 ,3-5,23. The Turopolje herd of the national park Lonjsko Polje, genealogically a metapopulation of Turopolje TL and Turopolje AUT, shows the highest levels of genetic diversity (HO = 0.57), which is the consequence of using Turopoljski Lug animals since 1994.ST value of 0.24, which is comparable to the results reported by SanCristobal et al. [ST = 0.21) and Fabuel et al. [ST = 0.14 - 0.15). The Turopolje island population in Turopoljski Lug is highly differentiated compared to all the other studied breeds (mean FST = 0.21). The highest FST value is obtained within the Turopolje breed surprisingly with a coefficient of 0.26 between Turopolje TL and Turopolje AUT. The Black Slavonian is moderately differentiated  and overlaps with the Austrian Turopolje and the Mangalica breeds, a result also observed in the individual-based phylogenetic tree  and polymorphic information content of the 22 markers typed in this study; markers with a * were excluded from further analyses due to deviations from the Hardy-Weinberg equilibrium.Click here for fileST estimates among eight pig populationsPopulation differentiation based on F. Values are inferred from 19 microsatellite markers; on the diagonal FIS values are shown in bold.Click here for filePrincipal component analysis. Description: Plot of the first three principal component axes based on individual genetic distance DK matrices; ellipsoids contain 75% of the animals; 1 red = Mangalica AUT, 2 orange = Mangalica SER, 3 green = Black Slavonian, 4 blue = Pietrain, 5 black = Turopolje AUT, 6 black = Turopolje Lonjsko Polje, 7 black = Turopolje Turopoljski Lug.Click here for fileDistribution of ln(X|K) from ten iterations ranging from K = 2 to K = 10. Values of log likelihood of the multilocus genotypic data, ln(X|K), as a function of the number of clusters, K (ten runs); the largest values of ln(X|K) are presented with black dots.Click here for file


A recent discovery of Iron Age burials (Pazyryk culture) in the Altai Mountains of Mongolia may shed light on the mode and tempo of the generation of the current genetic east-west population admixture in Central Asia. Studies on ancient mitochondrial DNA of this region suggest that the Altai Mountains played the role of a geographical barrier between West and East Eurasian lineages until the beginning of the Iron Age. After the 7th century BC, coinciding with Scythian expansion across the Eurasian steppes, a gradual influx of East Eurasian sequences in Western steppes is detected. However, the underlying events behind the genetic admixture in Altai during the Iron Age are still unresolved: 1) whether it was a result of migratory events , or 2) whether it was a result of a local demographic expansion in a ‘contact zone’ between European and East Asian people. In the present work, we analyzed the mitochondrial DNA lineages in human remains from Bronze and Iron Age burials of Mongolian Altai. Here we present support to the hypothesis that the gene pool of Iron Age inhabitants of Mongolian Altai was similar to that of western Iron Age Altaians (Russia and Kazakhstan). Thus, this people not only shared the same culture (Pazyryk), but also shared the same genetic east-west population admixture. In turn, Pazyryks appear to have a similar gene pool that current Altaians. Our results further show that Iron Age Altaians displayed mitochondrial lineages already present around Altai region before the Iron Age. This would provide support for a demographic expansion of local people of Altai instead of westward or eastward migratory events, as the demographic event behind the high population genetic admixture and diversity in Central Asia. Historically, Central Asia has been a crossroad between West and East Eurasian people leading to the current high population genetic admixture and diversity. The origin of this diversity may go back as early as the Iron Age, more than two thousand years ago, with the dispersal of mounted pastoral nomads across the Eurasian steppes Archaeological work conducted by a Spanish–French–Mongolian team in the Mongolian Altai during the period 2005–2007 discovered burial sites belonging to the Pazyryk culture. This was the first time that this culture was found in Mongolia The Pazyryks have traditionally been associated with the Eastern Scythians. Scythians, whose history is well-known from the ancient texts of Herodotus (484–425 BC), was the name that the Greeks gave to a number of separate Indo-European-speaking nomadic groups living in the region encompassing the Pontic-Caspian steppe (in Eastern Europe) and the steppe of Central Asia. The Scythian culture (7th-2nd century BC) flourished in this region from local Indo-European peoples that emerged at the Pontic-Caspian steppe in about 2000BC and expanded eastward until they reached the Altai Mountains. Advances in technology that favoured mounted nomadic pastoralism were the triggers for the expansion of Scythian culture across the Western Eurasian steppe. The end of Scythian period might be related with the westward migrations of nomadic Turkomongolian tribes coming from East Asia since the 3rd century BC, which marked the end of Indo-European domination of the steppe Archaeological findings, almost entirely provided by burial site discoveries, documented that the Scythians had European morphological features These molecular data raise two likely hypotheses for the origin of the genetic diversity and admixture among the Iron Age inhabitants of the Altai: 1) people holding west Eurasian lineages arrived at Altai Mountains with the eastward migration of Scythians and, once settled, they began to establish relationships with the neighbouring communities from East Asia holding East Eurasian lineages; 2) this was the result of the admixture between the native people inhabiting either sides of the Altai Mountains (people with West Eurasian lineages in Western Altai and East Eurasian lineages in the Eastern Altai), as a result of a demographic expansion during the Scythian period. Hence, the second hypothesis would provide support to the cultural transmission against the demic diffusion during the Scythian period.The skeletal remains unearthed by our team from Bronze and Iron Age tumuli in the Mongolian Altai offer a unique opportunity to get further insights into the ancient Altaians. Here, we aim to shed light on the origin of the current east-west population admixture in Central Asia, specifically in the Altai region, by analyzing the mitochondrial DNA lineages of these skeletons. Owing to the clear geographical structuring of the East and West Eurasian lineages in Central Asia population, mtDNA is particularly suited for the study of admixture in this region Skeletal remains from 19 individuals of Bronze and Iron Age For DNA extraction, 0.1 g of powder was extracted from teeth pulp cavities; when bones were used, 0.5 g of powder was collected from the internal compact tissue. After DNA treatment and extraction  was amplified and sequenced, and coding region informative polymorphisms for haplogroup assignment were analysed by PCR-RFLPs. The PCR reactions were carried out in a final volume of 50 µl and Taq polymerase (Bioline®) was used. Each PCR reaction consisted of an initial denaturation step (5 min at 94°C) followed by 39 cycles of PCR  and a final extension step of 5 min at 72°C, or of 10 min if the amplified segment was to be cloned. Amplified fragments were then visualized with Ethidium Bromide staining in a 3% agarose gel For all samples, the fragment containing the majority of HVRI mutations was cloned using the Topo TA Cloning® kit  following the manufacturer’s instructions. The colonies were harvested and subjected to PCR with M13 universal primers; for each sample 10 inserts of the right size were subsequently sequenced.For coding region analysis 10 coding region segments, determining the 10 Eurasian haplogroups, were analysed by PCR-RFLPs. Restriction sites and the primers used to amplify each specific fragment of the coding region are shown in For genetic sex determination, X and Y Amelogenin loci and the SRY gene (sex-determining region Y gene) were analyzed using primers and conditions described respectively by Beraud-Colomb et al. Independent replication for four teeth and one bone were performed at the Institut de Biologia Evolutiva (CSIC-UPF) using the methodology previously described by Lalueza-Fox et al. Sequence raw data was analysed with Sequence Scanner v1.0 (Applied BioSystems) program and sequences were subsequently aligned with BioEdit software version 7.0.0 Haplogroups were clustered according to their geographic origin following West Eurasian haplogroups: R0: R0a’b, HV; N1; JT; UK; W and X.East Eurasian haplogroups: M: C, D, G, Z, M9, M10, M11, M13; A; B; F and N9a.South Asian haplogroups: M*, U2a-c, U9, R*, R1–R2, R5–R6, N1d.For comparative purposes, mtDNA sequences between positions 16051 and 16400, from ancient For each population, the number of different haplotypes (K), the number of polymorphic sites (S) Slatkin’s linearized FST pairwise genetic distance matrices between population (MDS) was used to represent genetic distances in a two-dimensional space using SPSS ver. 17.0 (SPSS Inc.).www.fluxus-engineering.com). Positions of HVRI were weighted according to their site-specific mutation rate following the weight scheme proposed by Phylogenetic networks Spatial frequency distribution maps of East Eurasian lineages in Pre-Iron Age and Iron Age periods were obtained using Surfer version 8.05 (Golden Software).Ancient DNA was retrieved and replicable unambiguous results were obtained for the 19 individuals analysed. The genetic sex determination was possible in fourteen of the nineteen individuals . With exThe cloning process was applied to the most informative HVRI regions of all individuals. The results further verify that the data of the sequences obtained represent the consensus in each individual as shown in The majority of the retrieved sequences (58%) fit into East Eurasian lineages; namely to haplogroups A, C, D and G. On the other hand, 42% of the individuals belong to West Eurasian mtDNA haplogroups  . ConsideEvidence of a perfect admixture between East Eurasian and West Eurasian lineages is also observed in other Iron Age populations from central Asia . On the The number of mtDNA haplotypes, Nei gene diversity Network analysis using ancient Asia populations  and 4 diSeven of the sixteen different haplotypes found in this study (43.75%) are shared with other ancient populations used for comparison. Concerning these haplotypes, it is evidenced that K and T1 West Eurasian lineages detected in our Pazyryk sample were also found in Bronze Age samples from Siberia and Kazakhstan .For non-shared haplotypes, additional haplotype comparisons were performed including present-day data of Eurasian populations  representation of the Slatkin genetic distance between pairs of ancient and modern populations ST values for pairs of populations reveal that the Pazyryk populations seem to be genetically homogeneous. Notwithstanding, according to With exception of the Pazyryk samples from Mongolia previously reported by Central Asians exhibit high frequencies of East Eurasian mtDNA lineages, which are otherwise virtually absent in populations from the Indo-Gangetic region and westwards, along with a high prevalence of lineages of Western Eurasian origin It is widely accepted that animal domestication , particularly of the horse, gave the inhabitants of the Central Asian steppes the opportunity to expand geographically in different directions The high rate of success of the genetic analyses performed in the present study, evidenced by the amplification of both nuclear and mitochondrial DNA, suggests that the DNA of the samples is well preserved. In accordance with other authors Concerning Bronze Age samples from the Mongolian Altai mountains analyzed in the present study, 100% of the mtDNA lineages  belong to East Eurasian haplogroups, an opposite profile to that detected in the West side of the Altai Half of the shared haplotypes between ancient populations from Central Asia and South Siberia represent lineages present in both pre-Iron Age and Iron Age populations and all of these lineages have a west Eurasian origin. Moreover, considering both shared and non-shared haplotypes, it seems that the number of West Eurasian lineages does not increase in the Iron Age. These results allow us to hypothesise that the substrate of mtDNA lineages is already present in pre-Iron Age populations of the central Asia and that in the Iron Age (Scythian period) a population expansion lead to the admixture of pre-existing lineages. Thus, the admixture profile observed in the region during the Iron Age would not derive from a migratory movement from west to east, as has been hypothesised, but would represent a local population expansion in different directions. This population expansion, however, would be probably be a consequence of the introduction of new technology by the adoption of a new culture, supporting the idea of cultural transmission against the demic diffusion during Scythian period.The Pazyryk groups analysed so far appear to be genetically homogeneous and they did not present significant genetic differences to current Altaians. These results suggest that roots of the current genetic diversity and admixture of the Altai region in Central Asia could be traced back to the Iron Age.Table S1Description of primers used and their references.(DOCX)Click here for additional data file.Table S2Ancient and current populations used in comparative analysis. References corresponds to reference number in the manuscript.(DOC)Click here for additional data file.Table S3Alignment of clone sequences.(PDF)Click here for additional data file.


Although two geographically adjacent orange-spotted grouper populations in China showed negligible genetic divergence, significant population differentiation was observed in wild grouper populations distributed in a wide geographical area from China, through Malaysia to Indonesia. However, the Mantel test rejected the isolation-by-distance model of genetic structure, which indicated the genetic differentiation among the populations could result from the co-effects of various factors, such as historical dispersal, local environment, ocean currents, river flows and island blocks. Our results demonstrated that microsatellite markers could be suitable not only for genetic monitoring cultured stocks but also for revealing the population structuring of wild orange-spotted grouper populations. Meanwhile, our study provided important information for breeding programs, management of cultured stocks and conservation of wild populations of the orange-spotted grouper.In the present study, we employed microsatellite DNA markers to analyze the genetic diversity and differentiation between and within cultured stocks and wild populations of the orange-spotted grouper originating from the South China Sea and Southeast Asia. Compared to wild populations, genetic changes including reduced genetic diversity and significant differentiation have taken place in cultured grouper stocks, as shown by allele richness and heterozygosity studies, pairwise F Epinephelus coioides), an economically high valued marine food fish species belongs to the subfamily Epinephelinae  [The orange-spotted grouper (ranidae) , which iranidae) . Howeverranidae) . Over thranidae) . Accordiranidae) . At presranidae) .Scophthalmus maximus), Atlantic salmon , Sea bream (Pagrus major), Japanese flounder , Arctic charr , common carp (Cyprinus carpio) using DNA fingerprinting techniques such as microsatellite and mitochondrial markers [However, aquaculture practices are likely to reduce the genetic diversity by founder effects, inbreeding and random genetic drift in hatchery-reared stocks, which may cause loss of disease resistance and reduce environmental adaptability, thus limit the genetic potential for selective breeding –9. For t markers –15. Unfo markers ,17, therMarine fishes are considered to be genetically less differentiated compared to freshwater fishes due to high gene flow in open marine environment . The disWe have recently isolated polymorphic microsatellite markers and shown that these markers have the potential to characterize the genetic diversity and analyze the population structure of the orange-spotted grouper . In the Our samples were mainly obtained from the South China Sea, from which almost all the cultured and wild orange-spotted grouper in China mainland presently originated, due to its ideal subtropical climate. Sampling locations are shown in Caudal fin or muscle tissue samples of the orange-spotted grouper were clipped and immediately preserved in 90% ethanol until DNA extraction was performed. Genomic DNA was isolated according to the phenol-chloroform extraction protocol by Hoelzel and Green .2, 1XPCR buffer, 1U Taq DNA polymerase (Fermentas), and 20ng template genomic DNA. PCR program was as follows: 5 min at 94 °C followed by 30 cycles of 30 s at 94 °C, 30 s at the annealing temperature , Allelic richness (Ar), observed heterozygosity (Ho) and expected heterozygosity (He) using the program FSTAT 2.9.3 . Since a5 iterations after a 105 burn-in length with K ranging from 1 to 7. The most likely K value was inferred by calculating ΔK using the method of Evanno et al. [Inter-population genetic differentiation was estimated with pairwise Fst values and significance tests of pairwise Fst was computed by a permutation with 10,000 replicates using ARLEQUIN 3.11 . Furthero et al. .e) at each locus varied from 0.460 (Mbo061) to 0.916 (D496) with a mean value of 0.716.Microchecker analysis showed no evidence for null alleles and allele stuttering. Examination of the Hardy-Weinberg equilibrium (HWE) showed that two loci (CA-6 and Ec_157) ,21 exhibFor the Chinese samples, a statistically significant (P < 0.05) reduction of allele richness (Ar) was observed in cultured stocks of orange-spotted grouper  compared to the wild populations  . The meaPairwise Fst analysis showed significant genetic structure among all the seven samples except two comparisons where the differentiation was not statistically significant . The genGadus morhua) [Seriola lalandi) [Lates calcarifer) [This is the first study of genetic diversity and differentiation of the orange-spotted grouper between cultured stocks and wild populations using microsatellite markers. In this study, we found that the number of alleles per locus varied between 4.286 and 17.143, and the expected heterozygosity varied from 0.460 to 0.916 . The pol morhua) , sea bre morhua) , king filalandi)  and Asiacarifer) , suggestcarifer) .Several studies have shown that aquaculture practices reduce genetic variability in hatchery-reared stocks of various fish species –15. As hWith respect to genetic variation changes of the cultured stocks relative to wild populations, we should not neglect the stock enhancement programs for some grouper species in China. In recent years, the government-sponsored hatcheries of the orange-spotted grouper have released a number of hatchery-reared fingerlings into the South China Sea . HoweverEpinephelus marginatus) and Hawaiian grouper (Epinephelus quernus) was comparable [Genetic analysis involving allele richness and heterozygosity showed that genetic diversity among the orange-spotted grouper, dusky grouper  [Ctenopharyngodon idella) [Pairwise Fst, Structure and AMOVA analysis revealed significant differentiation between the cultured stocks and wild populations , Table 4o salar) , grass c idella) ,55 and A idella) . The gen idella) . The ran idella) . The orast = 0.011) was observed between two wild populations collected in different locations (Taiwan and Guangdong) of the South China Sea [st and Structure analysis  [Epinephelus adscensionis) [Regarding the differentiation among wild populations, the insignificant differentiation between two Chinese wild populations (HNI and DYB) indicated that these two samples should be considered as one population . This rehina Sea . Howeveranalysis . Signifitivelis) , dusky gtivelis) , Rock grnsionis) . This dinsionis) , it is bThe detection of differentiation between and within cultured stocks and wild populations in our study provided much valuable information for the breeding program of this species. The significant genetic differentiation among these wild populations might be used to produce hybrid vigour, although further experiments are required. At the same time, our results supplemented the previous population genetics studies, which showed important implications for the conservation of this species.To our knowledge, this study was the first genetic monitoring research for cultured stocks and wild populations of the orange-spotted grouper. Our work demonstrated that genetic changes, including reduced genetic diversity and significant differentiation, have taken place in cultured grouper stocks compared to the wild populations due to founder effects and random genetic drifts during aquaculture practices. This result provided important information for ongoing breeding programs and stock enhancement programs. We also found considerable population differentiation among the orange-spotted grouper populations distributed in a wide geographical area from China, through Malaysia to Indonesia, although negligible genetic differentiation was observed between Chinese populations. This could result from the co-effects of various factors, but not merely a matter of isolation-by-distance. The genetic differences among geographical populations could provide more choice for the selective breeding work of the orange-spotted grouper.


IT) of 0.073 ± 0.02, was partly due to the homozygote excess within breeds (FIS = 0.026 ± 0.02) and to a larger extent due to genetic differentiation among breeds (FST = 0.048 ± 0.01). The genetic distance, STRUCTURE and Principal Component Analyses concluded that the Siri cattle are most distinct among the investigated cattle populations. Furthermore the analysis of genetic structure indicated that the most probable number of clusters is four. All analysis showed that a significant amount of genetic variation is maintained in local cattle populations of which Shahabadi and Purnea are distinct from the recognized breeds of the area and needs recognition as breeds.India has 34 recognized breeds of cattle in addition to many more not characterized and accredited so far. It is imperative to characterize all the cattle germplasm of the country so as to have better breeding and conservation options. Thus, present study was planned for assessing genetic diversity and relationship between three local cattle populations  and two established cattle breeds (Bachaur and Siri) of eastern India by using 21 FAO and ISAG recommended microsatellite markers. A total of 243 unrelated DNA samples of five cattle populations were collected from respective habitats. A total of 304 microsatellite alleles were identified with number of alleles at one locus ranging from 5 to 29. The average observed heterozygosity lie within the narrow range of 0.681 ± 0.04 in Purnea to 0.721 ± 0.03 in Siri. Mean estimates of observed and expected heterozygosity over all loci and breeds were 0.704 ± 0.02 and 0.720 ± 0.01, respectively. In the overall population, the homozygote excess (F A large and divergent range of agro-ecological zones in India has helped to develop number of cattle populations. Cattle are the largest livestock species in India and constitute 37.5% of its total livestock population  and recognized cattle breeds of eastern India (Bachaur and Siri). Twenty one microsatellites  were amplified in five multiplex PCR. We intend to evaluate not only the current diversity but also to know their relationship for the conservation of the genetic diversity in the context of biodiversity management programs.et al.et al.et al.Shahabadi, Purnea and Gangatiri are non-descript cattle from Eastern India Figure . Gangatiet al.et al.Bachaur and Siri are recognized cattle breeds of Eastern India. Bachaur, a famous draught purpose cattle breed is concentrated in Bachaur Pargana  of Bihar state. The habitat of Bachaur has shrunken with time to northern part of Bihar bordering Nepal. The animals are white to grey in colour. The cows are poor milker, producing an average of 2.2 kg of milk per day and are managed under extensive management  and 1 min at 72°C and a final extension of 5 min at 72°C. Amplified fragments were separated by capillary electrophoresis using an ABI PRISM 310 automatic sequencer . Fluorescently labeled fragments were detected and sized using GeneMapper .Samples of the populations included in this study represented animals of the original autochthonous phenotype. Blood samples from 243 individuals were collected  from different villages of habitat Figure  while avo), number of private alleles, effective number of alleles (Ne), observed (Ho) and expected heterozygosity (He) and Hardy-Weinberg equilibrium (HWE). Wright’s statistics FIS (f), FST (θ) and FIT (F) and Nei’s  confirmed the remarkable level of diversity in the studied populations. Among populations, observed heterozygosity ranged from 0.681 ± 0.04 to 0.721 ± 0.028 with the lowest value found in Purnea cattle and the highest in Siri cattle. Most of the indigenous breeds including Kherigarh-0.57  . Since blood samples were collected from different villages, presence of a hidden substructure cannot be ruled out. In fact, animals of the same population but belonging to different villages could derive from genetically different founders. A significant homozygote excess was also observed in other studies on indigenous cattle breeds such as Sahiwal FIS = 0.32  amounted to 7.3% (P < 0.001). An overall significant deficit of heterozygotes (FIS) of 2.6% occurred in the analyzed loci because of inbreeding within populations. All loci except six contributed to heterozygote deficit within populations. The studied populations showed a low but significant genetic differentiation among five populations (FST = 0.048). All loci contributed to the differentiation with the highest values found for ETH25 (19.6%). Genetic differentiation of similar magnitude has been reported among cattle breeds of Orissa and hill cattle of Kumaun (0.044) from India  as the closest populations while the most differentiated were Purnea and Siri (0.044) (Table m = 32.809) populations and least between Purnea and Siri (Nm = 5.408). Overall Nm value also indicated high rate of genetic flow between the populations (10.230 ± 1.922) (Table Pair-wise genetic differentiations quantified by F4) Table . Similar4) Table . In accoet al.Among methods not assuming predefined structure, tree-based methods use genetic distance between individuals and tree construction algorithm such as UPGMA or Neighbour-joining to group them in clusters. Similarly, multivariate analyses can help in defining clusters of individuals. However these graphical methods are loosely connected to statistical procedures allowing the identification of homogeneous clusters of individuals. An alternative approach to delineate clusters of individuals on the basis of their genotypes at multiple loci was also performed using a Bayesian approach employed in software STRUCTURE. It works by grouping individuals into clusters (K) such that Hardy-Weinberg equilibrium is maximized within clusters. Likely value of K which best captured the variation present in the data was four based on modal value of K versus ΔK distribution Figure  followinIt is vital to report that lesser known cattle populations too have high genetic diversity. Unfortunately, animals of these populations are continuously decreasing due to change in agricultural practices as mechanization is fast replacing the traditional practices involving these animals, procedures performed by breeders to increase efficiency (crossing with available exotic or crossbred semen) and apathy of government agencies since these populations are not recognized as distinct breeds. The present study established the uniqueness of two such populations; Purnea and Shahabadi from the recognized cattle breeds of Eastern India. Conservation of genetic variation in these populations should be considered by breeders, in the interest of long term future of the populations in their native tract. To begin with, breed societies need to be created, that will be responsible for registration of these cattle populations as breeds, complete maintenance and improvement of the breed to make it economically sustainable in the transforming agricultural scenario of the country.


Ipomoea pes-caprae (Convolvulaceae), a pantropical plant with sea-drifted seeds, is found globally in the littoral areas of tropical and subtropical regions. Unusual long-distance seed dispersal has been believed to be responsible for its extraordinarily wide distribution; however, the actual level of inter-population migration has never been studied. To clarify the level of migration among populations of I. pes-caprae across its range, we investigated nucleotide sequence variations by using seven low-copy nuclear markers and 272 samples collected from 34 populations that cover the range of the species. We applied coalescent-based approaches using Bayesian and maximum likelihood methods to assess migration rates, direction of migration, and genetic diversity among five regional populations. Our results showed a high number of migrants among the regional populations of I. pes-caprae subsp. brasiliensis, which suggests that migration among distant populations was maintained by long-distance seed dispersal across its global range. These results also provide strong evidence for recent trans-oceanic seed dispersal by ocean currents in all three oceanic regions. We also found migration crossing the American continents. Although this is an apparent land barrier for sea-dispersal, migration between populations of the East Pacific and West Atlantic regions was high, perhaps because of trans-isthmus migration via pollen dispersal. Therefore, the migration and gene flow among populations across the vast range of I. pes-caprae is maintained not only by seed dispersal by sea-drifted seeds, but also by pollen flow over the American continents. On the other hand, populations of subsp. pes-caprae that are restricted to only the northern part of the Indian Ocean region were highly differentiated from subsp. brasiliensis. Cryptic barriers that prevented migration by sea dispersal between the ranges of the two subspecies and/or historical differentiation that caused local adaptation to different environmental factors in each region could explain the genetic differentiation between the subspecies. For most land plants that have sessile life forms, seed dispersal represents the only way by which populations can exchange individuals and maintain migration and gene flow between distant populations over which pollen cannot move One of the most effective modes of long-distance dispersal of seeds is sea dispersal, which can disperse seeds >100 km Ipomoea pes-caprae (L.) R. Br. (Convolvulaceae), Canavalia rosea Sweet. (Fabaceae), Vigna marina (Burm.) Merr. - Vigna luteola (Jacq.) Benth. (Fabaceae), and Hibiscus tiliaceus L. - Hibiscus pernambucensis Arruda . All species in this group are believed to have developed mechanisms for long-distance seed dispersal by ocean currents. Ipomoea pes-caprae has among the widest ranges of these species.Pantropical plants with sea-drifted seeds Ipomoea pes-caprae (L.) R. Br., commonly known as beach morning glory, is a creeping vine found on beaches throughout the tropical and subtropical regions of the world Ipomoea pes-caprae subsp. pes-caprae and Ipomoea pes-caprae subsp. brasiliensis (L.) van Ooststr, which are morphologically different in the shape of the leaves and the dimensions of the calyx and corolla pes-caprae is only distributed in the northern part of the Indian Ocean (from the Arabian Peninsula to Indonesia) and subsp. brasiliensis is found in tropical and sub-tropical areas worldwide, except for the northern Indian Ocean I. pes-caprae has achieved and maintained this extraordinarily wide range around the globe was the first question for this study. The circumglobal range of the species distribution includes several possible land and ocean barriers Ipomoea pes-caprae subsp. brasiliensis uses a wide range of insect pollinators, gene flow by pollen is not likely between two neighboring but distant populations when they are separated by hundreds of kilometers of ocean . Sea dispersal by drifted seeds would be the only possible way to keep migration between the two populations, and, indeed, the species has seeds that can float on seawater for more than 90 days I. pes-caprae subsp. pes-caprae and subsp. brasiliensis was the second question. Taxonomists have considered that the morphological differences between the two are at the subspecies level How I. pes-caprae, including both subspecies, collected from 34 populations that cover much of the global range of the species.To answer these questions, we tested whether 1. Long distance trans-oceanic dispersal of seeds results in low genetic divergence among continents, 2. Known biogeographic barriers such as continents and boundaries in ocean currents correspond to patterns of genetic differentiation, and 3. Genetic differentiation within subspecies is lower than genetic differentiation between subspecies. We used nucleotide sequences of seven low-copy nuclear markers and performed a coalescent-based approach using Bayesian and maximum likelihood inference. This is a powerful method to demonstrate the amount and direction of migration among populations and is useful to determine the geographic structure of the species over its global range. We studied 272 samples of I. pes-caprae were collected across the range of this species .Leaf samples of 272 individuals from 34 populations of  species . Five ofTotal DNA was isolated from silica-gel leaf pellets using the cetyltrimethyl ammonium bromide (CTAB) extraction method as was described in a previous study Ipomoea and other species from the published literature as candidate loci for PCR amplification: Beta-amylase (Primers 3F and 4R) Ipomoea batata EST data in GenBank. We then obtained PCR primers from the literature, or designed primers from the EST sequence data in GenBank. We performed PCR amplification for the 20 loci using a sample of I. pes-caprae subsp. brasiliensis collected from the Philippines and selected seven loci that produced only a single band. All other loci that produced multiple bands or no bands were not considered further in this study. In the second step, we tested whether clear sequence data could be obtained for the seven candidate markers by the direct sequencing method even though we were using samples of subsp. pes-caprae and subsp. brasiliensis from around the world. We used 12 individuals from 6 populations that well covered the range of subsp. brasiliensis: West Atlantic (Panama-Colon), East Atlantic (Ghana), West Pacific (Philippine), East Pacific (Panama-Vera Cruz), Indian Ocean  and a population of subsp. pes-caprae (UAE). All the seven loci yielded clear sequence data for direct sequencing using an ABI 3500 automated sequencer . In the last step, to make sure that there was no variation within primer regions, we designed internal specific primers for all seven loci , chalcone synthase gene (Chs), triosephosphate isomerase gene (Tpi), heat shock protein 90 (Hsp-90), anthocyanidin synthase (ANS), acetolactate synthase (ALS), and 1 gene from the EST database of the NCBI (EST). For three of the seven loci , all sequenced regions were in coding regions, while for the other 4  sequenced regions included both coding and noncoding regions , according to the manufacturer's instructions. The cycle sequencing reactions were carried out using the ABI BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems), and the sequencing reaction products were purified by ethanol precipitation. The purified PCR products were sequenced directly on an ABI 3500 automated sequencer .In all of the experiments explained earlier, PCR was performed in reaction volumes of reaction mixtures (30 µL/reaction) containing c. 10–30 ng of template DNA, 0.12 µL (0.6 units) of ExTaq DNA polymerase , 3 µL of ExTaq PCR buffer [10 mmol/L Tris-HCL (pH 8.3), 50 mmol/L KCL, 1.5 mmol/L MgClWaxy and HSP-90. All the obtained sequences were visually edited and aligned by SEQSCAPE V2.5 software (Applied Biosystems). All new sequence data obtained in this study were deposited in GenBank (accession nos. KF296474-KF296517 and KF296525-KF296536). For further population genetic studies, we also used the sequence data for Waxy and HSP-90 (GenBank accession nos. KF296518-24 and KF296558-62) which had been published in our previous study We determined nucleotide sequences of population samples for five out of the seven markers, the exceptions being Ipomoea batata EST data from GenBank. To determine the phylogeographic relationship among populations, a statistical parsimony network was inferred for each locus using TCS1.06 Sequences were aligned using BioEdit v. 5.0.9.1 Recombination is believed to be common in nuclear loci and to influence the results of genealogy-based analyses I. pes-caprae was investigated using a model-based clustering algorithm implemented in STRUCTURE V. 2.3 The genetic structure of We also calculated population pairwise Fst I. pes-caprae, we applied a coalescent-based approach pes-caprae from the northern part of the Indian Ocean were included as a group into the analyses. According to the linear setting of the five regional groups, we employed the stepping-stone migration model with asymmetric rates pes-caprae, but the migration rate involving subsp. pes-caprae was estimated between the subspecies and all other samples of subsp. brasiliensis. Starting parameters for migrant values and θ were generated from a few trial runs. Bayesian MCMC coalescent modeling was used to provide parameter estimates based on full likelihood estimation and the decreased computation time of the approximation in comparison to maximum likelihood estimates. Bayesian parameters included an update frequency of 0.5, a Metropolis–Hastings sampling algorithm for both θ and M; uniform priors were placed on θ from 0 to 0.002 and M from 0 to 5000. Starting parameters for migrant values and θ were generated from the first few trial runs. An adaptive heating scheme with four chains and a swapping interval of one was applied. We used the Felsenstein 84 model of evolution, and set the transition to transversion ratio to two as default values in the program. Six independent MCMC runs of varying length and burn-in were conducted which produced similar results. Hence, we presented mean results of all runs. Using tracer v1.5 4) followed by five long chains (length 5.0×105) with a sample increment of 100 for both runs were conducted, and the first 15,000 generations were discarded as burn-in at the beginning of each chain. An adaptive heating scheme with four chains and a swapping interval of one was applied. Maximum likelihood estimates were verified with three replicate Markov Chain Monte Carlo (MCMC) simulation runs to ensure the convergence of similar values for θ.To assess migration rates, the direction of migration, and the genetic diversity among populations of I. pes-caprae. ANS, HSP-90, TPI, and Waxy included five, one, three, and one indel sites, respectively, and those indels were excluded from further analyses. The aligned length of the nucleotide sequences ranged from 425 to 785 bp with a total length of 4075 bp after excluding gaps and recombination sites (Chs was the most polymorphic (k = 8.721) and Waxy the least (k = 1.140) (brasiliensis (πt = 0.0068) than in subsp. pes-caprae (πt = 0.0027). The ratio of synonymous to non-synonymous polymorphism was compared within and between populations. We did not find any fixed non-synonymous mutations within a population. The diversity at noncoding and synonymous sites was higher than that at nonsynonymous sites (t = 0.0098) than the other comparisons between groups, which were Kt = 0.0054 between the East Atlantic and Indian Oceans, Kt = 0.0037 between the Indian and West Pacific, and Kt = 0.0021 between the East Pacific and West Atlantic  . The levus sites . The divAtlantic .pes-caprae had at least one unique haplotype separated from subspecies brasiliensis with two to 20 steps, but also at least one haplotype was shared between the two subspecies except for Chs. For subsp. brasiliensis, there was at least one haplotype in all of the loci shared between the populations of the Indian Ocean and West Pacific regions, between the West and East Atlantic regions, and between the Atlantic and East Pacific regions. In three cases  networks possessed one main haplotype shared among all populations of geographical regions, and in two cases (ALS and EST) haplotypes were evenly shared between the two subspecies. In the remaining four loci, there were at least two major close haplotypes that were shared in all geographical regions . In order to visualize the degree of haplotype endemicity and haplotype sharing among regional populations, we used pie charts in the haplotype network  are shown in  setting . In K>3, setting .The results of population pairwise Fst also suggested a similar pattern of population differentiation and relatively less differentiation among populations within regions .I. pes-caprae populations (WP = 0.00625), and lowest in the East Atlantic region (θEA = 0.00300). The most probable estimates of migration rates (M) ranged from 360.0 to 4993.7, with the highest migration observed from the West Pacific to the Indian Oceanic region (M = 4993.7). In contrast, estimates of migration rates between the two subspecies (M = 360.0 and M = 430.0) were lower. The migration between populations was asymmetrical, as is shown by the number of migrants (Nm) in the Coalescent-based Bayesian estimates by MIGRATE-N indicate asymmetric migration among the ulations . The BayI. pes-caprae subsp. brasiliensis throughout its global range  from the East Pacific The East Pacific Ocean and Africa are both strong barriers for many other sea-dispersal plants such as mangroves  and AEP , the gen and AEP  in the WIO to EA = 0.511) and much larger eastward migration (NmEA to IO = 2.403) (Another mismatch between genetic clustering and biogeographic boundaries shown in East Africa is more complicated. Two populations (6 and 7 in = 2.403) . We stilbrasiliensis. The Malay Peninsula region is known to shape distinct genetic structures in some mangrove plants between the Indian Ocean and West Pacific regions brasiliensis, the migration between the two regions was the highest among the population pairs studied Click here for additional data file.Figure S2Haplotype networks of five low copy nuclear genes of Ipomoea pes-caprae. Each haplotype is shown as a circle, the size of which is proportional to the number of individuals that have the haplotype. A small open circle connecting haplotypes represents a mutational step between the haplotypes.(TIF)Click here for additional data file.Figure S3STRUCTURE analysis of Ipomoea pes-caprae populations from K = 2 to K = 6. Vertical bars represent the membership coefficients (Q) of individual plants. The horizontal axes correspond to the regional grouping of populations. Left to right: subsp. pes-caprae, Indian Ocean, West Pacific, East Pacific, West Atlantic, and East Atlantic regions. Numbers at the upper side designate the population as are shown in (TIF)Click here for additional data file.Table S1Geographic location and sample size of Ipomoea pes-caprae populations in this study.(DOC)Click here for additional data file.Table S2List of primers used for sequencing of nuclear regions in this study.(DOC)Click here for additional data file.Table S3Summary of Genetic Divergences and Differentiations between each two regions.(PDF)Click here for additional data file.Table S4Population pairwise average Fst for Ipomoea pes-caprae calculated rom seven low-copy gene sequences. Population pairs within a rectangler of dotted lines show the ones within a geographic region employed in this study (names of regions are shown in gray letters).(PDF)Click here for additional data file.Table S5Maximum likelihood estimates (MLE), 95% confidence interval of θ and migration rates (M) and number of migrants (Nm) obtained from MIGRATE-N.(PDF)Click here for additional data file.


Lymantriadispar), one of the most recognized pests of forests and ornamental trees in the world. We assessed genetic diversity and structure in eight geographic populations of gypsy moths from China using five polymorphic Inter simple sequence repeat markers, which produced reproducible banding patterns. We observed 102 polymorphic loci across the 176 individuals sampled. Overall genetic diversity  was 0.2357, while the mean genetic diversity within geographic populations was 0.1845 ± 0.0150. The observed genetic distance among the eight populations ranged from 0.0432 to 0.1034. Clustering analysis , revealed strong concordance between the strength of genetic relationships among populations and their geographic proximity. Analysis of molecular variance demonstrated that 25.43% of the total variability  was attributable to variation among geographic populations. The results of our analyses investigating the degree of polymorphism, genetic diversity (Nei’s and Shannon) and genetic structure, suggest that individuals from Hebei may be better able to adapt to different environments and to disperse to new habitats. This study provides crucial genetic information needed to assess the distribution and population dynamics of this important pest species of global concern.This study provides the first genetic characterization of the gypsy moth from China ( Lymantriadispar Linnaeus (Lepidoptera: Lymantriidae), is a destructive defoliator with a broad host range, and one of the most recognized pests of forests and ornamental trees in the world. Indeed, it is the only forest insect to be included in the American Quarantine regulations [Lymantriadispardispar and the Asian gypsy moth (AGM), Lymantriadisparasiatica, based on preferred host plant usage and the females’ flight abilities. The EGM was originally found in Europe, but was accidentally introduced into North America from France in 1869 [o and 58o north, covering an area of more than 20 provinces or autonomous regions [The gypsy moth, ulations . The gyp in 1869 , where i in 1869  and to t in 1869 . AccordiGenetic variability is a crucial indicator of the capacity of a species or population capacity to adapt to the environment . SpeciesL. ddispar.  subspecies and the two Asian subspecies asiatica and japonica [Previous research using microsatellite DNA markers to assess genetic variability in four gypsy moth populations from Japan, eastern Russia, China, and North America revealed that genetic variation in the North American population was lower than that in the Asian populations . Amplifijaponica . In all In this study, we assessed the genetic diversity of the gypsy moth within China using Inter Simple Sequence Repeat (ISSR) markers. The ISSR technique entails the amplification of DNA fragments located between two closely and inversely oriented microsatellites . PrimersLdispar.  sourced from eight locations . Compared to traditional insect DNA extraction methods, such as SDS  and CTABAristaeomorpha foliacea ISSR work [Ldispar.  DNA; however, twenty-seven just produced single patterns. Ultimately, five ISSR primers capable of producing reproducible and unambiguous multiple , 0.75 U Taq DNA Polymerase  and ddH2O to the final volume. The amplification reaction was performed in a DNA Engine Thermal Cycler PTC-200 . The conditions were 94oC for 5 min, 35 cycles of 94oC for 45 s, annealing at their respective temperature for 45 s and 72oC for 90 s, and final extension at 72oC for 10 min. For each primer, replicates and negative controls were included to ensure the consistency of results.All PCR amplifications were carried out in a 20 µL volume, containing 10 ng of template DNA, 2 µL 10× PCR buffer , 1 µM of primer , 240 µM of each dNTP , 1.5 mM of MgClTo analyze the PCR product, 5 µL of product was added to 1 µL of 6× loading buffer , and this mixture was loaded in a 2% agarose gel  for electrophoresis with 0.5× TAE (Tris/Acetic/EDTA) electrophoresis buffer. After running at 100 V for 1 h, the gel was stained in ethidium bromide (EB) buffer for 3 min, rinsed with distilled water and photographed in a gel documentation machine . A DL2000 DNA marker  was run as a standard with each primer.The presence or absence of bands was scored as a 1 or a 0, thusour data was consisted by a presence/absence (1/0) matrix of 102 multiple bands from 176 individuals.Ldispar.  populations was obtained by Nei’s original measurements [The program PopGene32  was usedurements . And a dFST, with 95% confidence intervals (CI) obtained by bootstrapping 1000 replicates over all loci.To examine the partitioning of genetic variance within and among samples from different geographical regions, Analysis of molecular variance (AMOVA)  was carrOverall, 102 different bands, varying from 280 to 2,000 base pairs, were obtained by the five primers screened for 176 individuals across the eight geographic populations. All primers screened in this work were polymorphic, and the number of polymorphic bands per primer ranged from 16, for IT2, to 23, for UBC847. The primer which amplified the highest mean number of bands per population was IT1 (16.87 polymorphic bands); whereas that producing the lowest mean value was IT3 (9.25 polymorphic bands). Hebei was the geographic population with the highest number of bands (84 polymorphic bands); whilst Arxan, Inner Mongolia was the geographic population with the lowest mean number of bands  and Shannon’s information index (I = 0.3735) showed relatively high overall genetic diversity for gypsy moth from China. Among the different geographic populations, Hebei showed the highest genetic diversity , while Arxan, Inner Mongolia showed the lowest  .Ldispar.  ranged from 0.0432 to 0.1034, suggesting a large genetic base  was estimated as 1.466 via the formula: Nm = (1- FST)/2FST [AMOVA conducted on ISSR markers confirmed the presence of significant genetic differentiation among the eight geographic populations /2FST . This vaLymantriaxylina Swinhoe and Lymantriamonacha Linnaeus when monitoring with attractant the next years . To determine the genetic composition and structure of the gypsy moth from China, we conducted a large-scale analysis of genetic variability across eight different geographic populations, utilizing novel ISSR molecular markers [Ldispar.  in China.The AGM has received close attention as a potential pest species over the past few decades because, unlike EGM, their eggs could complete diapause under a lower chill requirement , their l markers . This inLdispar.  with AFLP which demands of preliminary pedigree analysis, independent inheritance and alletic assignment of AFLP markers [The efficiency of molecular markers is evaluated by the degree of polymorphism detected among the populations under investigation. All five primers, UBC818, U847, IT1, IT2 and IT3 used for analyses in this study were polymorphic. Unlike another previous genetic study of  markers , the ISSLdispar.  populations from different localities, revealed that the Hebei population (HB) had the highest percentage of polymorphic loci (82.35%), suggesting this population could be the longest-existence of the eight populations or could be the largest population. Individuals from Arxan, Inner Mongolia (MA) showed the least genetic diversity, probably because of the frequent eradication measures undertaken by the local government every year.Polymorphism of a population depends on the existence of genetic variants, represented by the number of alleles per locus and by their frequency distribution in the population . The resH = 0.2410, I = 0.3707), agreeing with the data for percentage of polymorphic loci. This suggests that individuals from Hebei possessing greater information content may be better able to adapt to different environments and, therefore, to disperse to new habitats.Our analyses revealed high genetic diversity in the gypsy moth from China (as measured by both the Nei’s and Shannon’s index), which may be explained by the great dispersal capabilities at adult stage of AGM. Hebei (HB) had the highest genetic diversity of all the geographic populations  lending further support to the finding of strong genetic structuring among the Chinese AGM. What is noteworthy is that there exists large degree of genetic separation among the five northern Chinese populations, which might be driven by the accumulation of genetic differences via adaptation of these populations to their differing environments.Cluster analyses utilizing UPGMA revealed stronger genetic relationships among geographically proximate locations, suggesting that more closely related populations share a common origin, or evolutionary history. Our results also revealed high overall genetic differentiation among populations  and genetic relationships among different populations all indicate that individuals from Hebei pose the greatest threat as potential pests, and should be closely monitored by the phytosanitary department. The genetic information on the Chinese AGM obtained in this study will assist in elucidating crucial information on the distribution and population dynamics of this important pest species of global concern.


Fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the Atlantic coast of North America. Several populations of this species have successfully adapted to environments contaminated with toxic aromatic hydrocarbon pollutants such as polychlorinated biphenyls (PCBs). Previous studies suggest that the mechanism of resistance to these and other “dioxin-like compounds” (DLCs) may involve reduced signaling through the aryl hydrocarbon receptor (AHR) pathway. Here we investigated gene diversity and evidence for positive selection at three AHR-related loci  in F. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern US, including extremely polluted and reference estuaries, with a focus on New Bedford Harbor , a PCB Superfund site, and nearby reference sites.The non-migratory killifish F. heteroclitus loci. Genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. However, for AHR2 the New Bedford Harbor population had significant FST values in comparison to the nearest reference populations. Tests for positive selection revealed ten nonsynonymous polymorphisms in AHR1 and four in AHR2. Four nonsynonymous SNPs in AHR1 and three in AHR2 showed large differences in base frequency between New Bedford Harbor and its reference site. Tests for isolation-by-distance revealed evidence for non-neutral change at the AHR2 locus.We identified 98 single nucleotide polymorphisms within three AHR-related loci among all populations, including synonymous and nonsynonymous substitutions. Haplotype distributions were spatially segregated and F-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other F. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. However, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in AHR1 and AHR2, and specific AHR2 SNPs and haplotypes that are associated with the PCB-resistant phenotype in the New Bedford Harbor population. The results suggest that AHRs, and especially AHR2, may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants.Together, these data suggest that  These “dioxin-like compounds” (DLCs) are capable of interfering with embryonic development and eliciting acute and chronic effects on reproduction, immune function, and other essential processes ,23Funduled sites .The molecular mechanism(s) underlying the DLC resistance are not known for any of these populations. However, the characteristics of the resistant phenotype provide important clues, which can be illustrated using the NBH population as an example. First, killifish embryos from NBH are less sensitive to the developmental toxicity of 3,3′,4,4′,5-pentachlorobiphenyl PCB-126) than embryos from a reference site . Second, than embTheoretical considerations suggest that adaptation to extreme pollution such as that found in NBH and other contaminated sites is more likely to result from major gene effects rather than polygenic adaptation ,28. KillF. heteroclitus AHR pathway, including two AHR paralogs , an AHR nuclear translocator (ARNT2), and AHR repressor (AHRR)  The twSecond, tree-based methods to test for positive selection were implemented in PAML v.4 . For thCT) but also calculated variation among all populations (FST) and among populations within each class (FSC). To reduce Type 1 errors, we assessed significant differences at p < 0.0001.Third, we used analysis of molecular variance (AMOVA) to determine differentiation among populations and between sets of populations classified as polluted or reference. All nucleotide variants in our analyses are from the coding region of each transcript and represent a combination of synonymous and nonsynonymous polymorphisms, which may display variable signals of population structure and be under different degrees of selection. Selection may result in significant differences in F-statistics for particular nucleotides if they are not evolving under neutral conditions. The locus-by-locus AMOVA feature of Arlequin  was usedWe compared the frequency of polymorphisms identified in at least one test for selection in fish collected from New Bedford Harbor and Scorton Creek, Massachusetts. For these positions, we constructed sequence logos (Weblogo v. 3 ) to dispST) were calculated between each population pair with Arlequin. Geographic and genetic distances were regressed with a web-implementation of Isolation By Distance v3.16 [ST/(1-FST) in place of FST. The relationships were unchanged and we only report results using FST for genetic distance.Statistical tests of isolation-by-distance were carried out for each gene among all populations and by studying populations from reference and polluted sites separately. Geographic distances between populations were determined by calculating a smoothened coastal distance between locations that ignored small inlets, when present. Pairwise genetic distances (multilocus Fce v3.16  with 100The data supporting the results of this article (unique haplotypes for each AHR locus) are available in the Dryad Digital Repository: http://dx.doi.org/10.5061/dryad.t2888.p-dioxin; PAHs: Polycyclic aromatic hydrocarbon; SH: Sandy Hook; SC: Scorton Creek; SNPs: Single nucleotide polymorphisms; TAD: Transactivation domain.TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; PCB-126: 3,3′,4,4′,5-pentachlorobiphenyl; ARNT: AHR nuclear translocator; AHRR: AHR repressor; AMOVA: Analysis of molecular variance; AHR: Aryl hydrocarbon receptor; BEB: Bayes Empirical Bayes; CYP1A: Cytochrome P450 1A; DLC: Dioxin-like compound; FP: Flax Pond; JB: Jamaica Bay; NBH: New Bedford Harbor; YC: Newark Yacht Club; ns-SNPs: Nonsynonymous SNPs; PC: Piles Creek; PCB: Polychlorinated biphenyl; PCDD: Polychlorinated dibenzo-The authors declare that they have no competing interests.AMR carried out the population genetic and statistical analyses and drafted the manuscript. SIK participated in the design of the study, sequencing and sequence analysis, carried out the haplotype analyses, and edited the manuscript. DGF participated in sequencing and sequence analysis and edited the manuscript. BRE participated in the design of the study, collection of fish, and sequencing. DN participated in the design of the study, led the collection of fish, and edited the manuscript. DC participated in the collection of fish. VMV participated in the data analysis involving geographic distances. MEH conceived of the study, participated in its design and coordination, helped to draft the manuscript, and edited the manuscript. All authors read and approved the final manuscript.Materials and Methods. In the top panel, circles refer to unique haplotypes, wedges are colored by sampled population, and numbers within wedges refer to the number of alleles with that haplotype in the population represented by that color. Numbers outside of the circles refer to the haplotype number as shown in the bottom panel. In the bottom panel, black wedges indicate the percentage of site-specific (SS) haplotypes at each site and colored wedges indicate haplotypes shared among populations.AHR1 haplotype frequencies and distributions. Haplotypes were reconstructed using PHASE and genealogical relationships among the haplotypes were estimated using TCS software, as described in Click here for fileMaterials and Methods. For additional description, see legend to Additional file AHR2 haplotype frequencies and distributions. Haplotypes were reconstructed using PHASE and genealogical relationships among the haplotypes were estimated using TCS software, as described in Click here for fileMaterials and Methods. For additional description, see legend to Additional file AHRR haplotype frequencies and distributions. Haplotypes were reconstructed using PHASE and genealogical relationships among the haplotypes were estimated using TCS software, as described in Click here for fileFundulus heteroclitus. The locus-by-locus AMOVA feature of Arlequin was used to determine F-statistics for each variable nucleotide. Nucleotide positions that were significantly different among populations within each class (FSC), among all populations (FST), and between the set of populations classified as polluted as compared to those classified as reference (FCT) were assessed using a significant cutoff of p < 0.0001. SS=synonymous site; NS=nonsynonymous site; nonsynonymous positions are also shaded. Nucleotide number (nt#) refers to the position with respect to the ATG translational start (A=1) unless otherwise specified.Locus-by-locus F-statistics for AHR-related loci from Click here for fileFundulus heteroclitus were collected for this study.Pairwise geographic distances (km) for locations where Click here for filePCR primers.Click here for file