Niveoscincus ocellatus. 204 individuals from 29 populations across the distributional range of N. ocellatus were surveyed for variation at two mitochondrial genes , and two nuclear genes . Phylogenetic relationships were reconstructed using a range of methods , and the demographic histories of populations were assessed .The aim of this research was to identify the effects of Pleistocene climate change on the distribution of fauna in Tasmania, and contrast this with biotic responses in other temperate regions in the Northern and Southern Hemisphere that experienced glacial activity during this epoch. This was achieved by examining the phylogeographic patterns in a widely distributed Tasmanian endemic reptile, There was a high degree of mitochondrial haplotype diversity (96 unique haplotypes) and phylogeographic structure, where spatially distinct groups were associated with Tasmania’s Northeast and a large area covering Southeast and Central Tasmania. Phylogeographic structure was also present within each major group, but the degree varied regionally, being highest in the Northeast. Only the Southeastern group had a signature of demographic expansion, occurring during the Pleistocene but post-dating the Last Glacial Maximum. In contrast, nuclear DNA had low levels of variation and a lack of phylogeographic structure, and further loci should be surveyed to corroborate the mitochondrial inferences.N. ocellatus indicate Pleistocene range and demographic expansion in N. ocellatus, particularly in the Southeast and Central areas of Tasmania. Expansion in Central and Southeastern areas appears to have been more recent in both demographic and spatial contexts, than in Northeast Tasmania, which is consistent with inferences for other taxa of greater stability and persistence in Northeast Tasmania during the Last Glacial Maximum. These phylogeographic patterns indicate contrasting demographic histories of populations in close proximity to areas directly affected by glaciers in the Southern Hemisphere during the LGM.The phylogeographic patterns of The online version of this article (doi:10.1186/s12862-015-0397-y) contains supplementary material, which is available to authorized users. The glacial-interglacial oscillations of the Pleistocene have strongly influenced the distribution and evolution of many species . InitialRecently, an increasing number of phylogeographic studies have questioned the simplicity and universality of this classical southern Europe refugia model, describing ‘cryptic’ northern refugia –8 and ‘r2) during the early (~1.8 Myr) and middle Pleistocene (>130 kyr) )130 kyr) –30  , 28, 31 Plant studies indicate a wide range of phylogeographic patterns in Tasmania, but those of several Eucalypt species  have ledNiveoscincus ocellatus). This species is an excellent model for investigating the impacts of Pleistocene glacial cycles in Tasmania for two reasons: it has a wide geographic distribution across Tasmania, and it has clear distribution restrictions associated with climate and habitat type. Niveoscincus ocellatus ranges from sea level to high elevations, including previously glaciated regions  andN. ocellatus sampled, with 183 (12.9 %) of 1420 characters variable and 147 (10.4 %) parsimony-informative. Tree topologies were very similar between maximum parsimony and Bayesian tree-building methods, only differing in relationships at shallow phylogenetic levels, while the composition of major clades were consistent across analyses; therefore only the Bayesian inference tree is presented , and when testing for three populations recovered groups similar to that inferred by the phylogeny : Northeastern, Southeastern (but with inclusion of both Lake McKenzie and Dove Lake), and Northwestern (Mt. Oakleigh only). Given no appreciable increase in ΦCT with increasing number of populations in SAMOVA, three regional groups were defined for further population genetic analyses. The Northeastern and Northwestern (Mt Oakleigh and Dove Lake) clades each corresponded to respective groups, but the Lake Mackenzie clade (three haplotypes) was combined into the same group as the Southeastern clade  as two of the Lake Mackenzie haplotypes clustered monophyletically among Southeastern haplotypes, and this locality is geographically confluent with Southeastern clade localities. The same three groups are readily distinguished by large numbers (>19) of mutations in the TCS network  for RPS8, and 13 out of 656 (1.98 %) for β-globin. Subsequently, β-globin was successfully sequenced for 166 individuals in total . A total of 55 unique alleles were identified, with 39 (5.8 %) of 670 characters variable, and 30 (4.5 %) parsimony informative.18 CT had plateaued already at two groups, reflecting a western group  and the remainder. There was significant but weak population genetic structuring in the nuclear data when populations were grouped according to the inferred mtDNA groups .The Southeastern group was the only group with significant mtDNA Tajima’s ion Fig. . The samExtended Bayesian Skyline Plots conducted on multilocus data also indicated a stronger signal of recent population growth in the Southeast than the Northeast during the last 1.5 Myr, with the Northeast experiencing moderate growth and subsequent decline, and consequently no net change across the plot Fig. .Fig. 5TeN. ocellatus across its range revealed strong phylogeographic structure at multiple spatial scales. There was broad-scale structure based on mtDNA, with distinct groups corresponding to Tasmania’s Northwest, Northeast and Southeast . Additionally, phylogeographic structuring was evident within Northeast and Southeast groups, although the degree of structuring as well as the demographic history of each region differed. The Northeastern group had the highest levels of internal structure and coalescent analyses did not provide evidence for any substantial net demographic expansion; the populations appear to have persisted in isolation at or near their sampling sites during Pleistocene glaciations without substantial change in population size. This contrasts with the lower spatial structuring and evidence of greater demographic and spatial expansion in the Southeastern group. While populations within the Northwestern group also appeared to have some level of genetic structure, low sampling currently makes it difficult to reliably infer the biogeographic history of populations from this region. The nuclear β-globin locus provided corroborating evidence for the three groups suggested by mtDNA, but only within a population genetic (AMOVA)-rather than phylogeographic—framework. Estimated dates for the divergence of lineages from different regions and demographic expansion were firmly in the Pleistocene, but pre-date the LGM. The observed phylogeographic patterns suggest that Pleistocene climate—but not the LGM specifically-has been highly influential in shaping the distribution of N. ocellatus across Tasmania.Extensive genetic sampling of N. ocellatus populations are likely to reflect aspects of Tasmania’s climatic history. During the Pleistocene, high altitude areas including Ben Lomond, Mt Wellington, Mt Field, and the Tasmanian Central Plateau experienced repeated glacial and periglacial activity [N. ocellatus today [In reptiles, physiological processes essential for survival and reproduction are tightly linked to thermal opportunities , 49. Theactivity . While tus today .N. ocellatus between the highly structured Northeastern group and the comparatively less structured Southeastern group likely reflects historic differences in persistence of N. ocellatus populations at or near the sampling sites during the colder, more biologically limiting, glacial periods. The observed diversity of haplotypes and the individually monophyletic status of most sites within the Northeastern group provide support for the climatic suitability of northeast Tasmania for populations of temperate species during or immediately following Pleistocene glaciations, and not just the LGM. Additional support is provided by phylogeographic studies in two plant species, Eucalyptus regnans [Nothofagus cunninghamii [N. ocellatus at Ben Lomond is consistent with recolonisation from multiple, low elevation sources.The contrast of phylogeographic patterns in  regnans  and Nothinghamii , as wellinghamii . In contThe monophyletic population structure found in some populations in the Southeastern group including Coles Bay, Fortescue Bay and the combined Russell River and Margate population, indicates that in some of these lowland regions, populations may have either persisted in isolation for longer periods than other Southeast populations, or reflect founder effects from peripheral range expansion, the latter suggested by relaxed random walk analysis Fig. . RegardlEucalyptus globulus [N. ocellatus relaxed random walk analysis suggested persistence or early establishment in Northern Central Tasmania, and Northeast Tasmania immediately thereafter. This bears more similarity to temperate European species that persisted during glacials in ‘cryptic high latitude refugia’, including amphibians and reptiles [N. ocellatus.The patterns of glacial response inferred for other Tasmanian taxa bear some similarities with classical patterns in the Northern Hemisphere, where post-glacial recolonisation has often been rapid, and from a single or few source populations at lower latitudes and altitudes, leading to decreases in local genetic diversity . For exaglobulus . Howeverreptiles –56. Furtreptiles , 57, 58)cf. [N. ocellatus. The larger effective size of the nDNA markers confers slower lineage sorting relative to mtDNA, which is the favoured explanation for the phylogeographic discordance of these markers , as welsee also , 61).N. ocellatus do not correspond with any large-scale barriers in the current landscape or match the distribution of any previously reported biogeographic regions [N. metallicus [N. ocellatus, and are also non-saxicoline, and therefore it might not necessarily be expected that they share phylogeographic histories with N. ocellatus.The location of phylogeographic breaks between the Northwestern, Northeastern and Southeastern clades in  regions –64. Howetallicus , where stallicus –68, and tallicus ; it is iNiveoscincus ocellatus is a habitat specialist with low dispersal capacity [capacity , 70, 71,capacity , 73. If N. ocellatus given that it currently occupies a wide range of locations not presently connected by rocky habitats. Waters et al. [While poor connectivity between populations can explain why dispersal might be rare, it cannot be seen to prevent dispersal completely in s et al.  highlighs et al.  for an es et al. . There is et al. , 75, 76,N. ocellatus can also explain the maintenance of structure observed within clades such as those in the Northeast, where unique haplotypes are almost always associated with just one population. In addition, because competitive exclusion is density-dependent, it can only explain the maintenance of phylogeographic breaks in demographically stable areas. However, if climatic conditions change and there is an increase in the density of invaders, or an absence of residents in newly available suitable habitat, genes are more likely to move throughout a landscape, and result in a lack of phylogeographic structuring.Density-dependent effects can maintain barriers to gene flow at a variety of spatial scales . This meN. ocellatus became established and persisted at or near most contemporary sampling locations in Northeast Tasmania, and some low elevation sites in Southeast Tasmania, throughout the LGM and earlier Pleistocene glaciations. Other regions of central and Southeast Tasmania were uninhabited during the Pleistocene glaciations and were not recolonised from a single refugial population. Our findings contribute to the emerging picture of complexity in phylogeographic histories and of responses to Pleistocene glaciations across species and regions globally. Future studies should compare phylogeographic patterns in similar taxa  to verify the inferences of this study, and also examine the fine-scale spatial distribution of genetic variation between areas identified as Northeast and Southeast clades, to better understand the origin and maintenance of these groups .Niveoscincus greeni and N. pretiosus were employed as outgroups.Tissue samples were collected from 204 specimens representing 29 localities Fig. . Field sc. 547 bp) and ND4 (c. 873 bp), and two nuclear genes, β-globin  and RPS8 , were amplified and sequenced, as they exhibited useful levels of intraspecific variability in previous population studies of squamates . For ND2, ND4 and RPS8 amplification was carried out with an initial denaturation at 95 °C for 3 min, followed by 35 cycles of 95 °C denaturation for 25 s, 55 °C annealing for 30 s, and 72 °C extension for 90 s, with a final extension at 72 °C for 5 min after cycling. Amplification for β-globin followed the same protocol but with annealing at 60 °C.Total genomic DNA was extracted from tail or liver tissue samples following the Qiagen DNeasy Blood and Tissue Extraction Kit . A portion of two mitochondrial genes, ND2 . Th. Thc. 54s . Sequence data for ND2 and ND4 were concatenated, providing 1420 bp per individual. TCS v1.21  was usedhttp://popart.otago.ac.nz/index.shtml).Maximum parsimony and Bayesian tree-building methods were used for mtDNA data. Maximum parsimony trees were constructed in PAUP* v4.0  using eqhttp://cmpg.unibe.ch/software/samova2/). Population genetic statistics were then calculated for these groups using Arlequin 3.1 [ST), among populations within groups (ΦSC), and among groups (ΦCT), and P-values were estimated from 1000 permutations of haplotypes. Tajima’s D [Fs [Populations were assigned to a regional group based on patterns in the mitochondrial DNA phylogeny and Spatial Analysis of Molecular Variance . SAMOVAquin 3.1 . Haplotyjima’s D  and Fu’s’s D [Fs  were use’s D [Fs .http://beast.bio.ed.ac.uk/tutorials to improve mixing. Replicate runs were tested for convergence visually using Tracer v1.5 [Changes in effective population size were reconstructed using the multilocus Extended Bayesian Skyline Plot (EBSP) . Substitcer v1.5  and to eA relaxed random-walk analysis was also employed to reconstruct the spatiotemporal pattern of movements. This approach has similarities to relaxed molecular clock analyses in that it relaxes the constraint of constant movement rates through time . A log-nhttp://datadryad.org/review?doi=doi:10.5061/dryad.ff32k.The data set(s) supporting the results of this article are available in the Dryad repository, Additional file 1: Table S1.Table S2. Museum specimens. Table S3. Sampling locations of haplotypes illustrated in Fig. Table S4 Sampling locations of haplotypes indicated in Figure S3. Figure S1. Maximum parsimony tree for mtDNA variation. Figure S2. Maximum clade credibility tree from BEAST analysis. Figure S3. Minimum spanning network for mtDNA variation.Sampling details of specimens.


Macridiscus multifarius with a planktonic larval stage of about 10 days is an ecologically and economically important species in the coastal regions of China. In this study, 3 mt-DNA markers  were used to investigate the population structure and demography of wild M. multifarius populations in 3 coastal localities of the East China Sea (ZS and ZP populations) and Beibu Gulf in the South China Sea (BH population). Sequences of 685 bp in COI, 350 bp in 12S rRNA, and 496 bp in ND1 were determined. High level and significant FST values were obtained among the different localities on the basis of either COI  or 12S rRNA  gene, indicating a high degree of genetic differentiation among the populations. FST values were significant but weak for the ND1 gene because it is highly conservative. The median-joining network suggested an obvious genetic differentiation between ZS and BH populations, and the finding is consistent with the results of our demographic analyses using the unweighted pair group method with arithmetic mean. Our study unraveled the extant population genetic structure of M. multifarius and explained the strong population structure of a species with a short planktonic larval stage species; this information could be useful for fishery management measures, including artificial breeding and conservation.The clam  Haliotis rubra, with a 6-day pelagic larval phase, has a dispersal distance of less than 15 km, that Ensis directus, with a 16-day pelagic larval phase, can disperse to about 111 km, and that Perna perna, with a 15 to 20-day pelagic larval phase, can disperse to about 235 km. Shanks et al. observed that propagules that spent in the water column dispersed further [The life history of most marine organisms includes a planktonic stage during which larvae disperse to distances that range from several meters to hundreds of kilometers from their location of release . In gene further  showed t further .Macridiscus multifarius L. F. Kong, Matsukuma & Lutaenko, 2012 [M. multifarius is found along the coast, and it has a shell length of 30–40 mm. It has a brief pelagic phase of about 10 days at 24–29°C and 20–25 ppt of salinity. Spawning of M. multifarius occurs in summer and peaks in July and August [M. multifarius. This clam is an important and commercially exploited species; however, M. multifarius populations have declined largely because of over-exploitation and habitat destruction [M. multifarius specimens collected from Nanji Island, Zhejiang Province, was less than 15 mm [M. multifarius populations has caused the price to increase, for example, in Zhoushan City, Zhejiang Province, the price has increased from ¥20 to more than ¥100 per kilogram in a few years [M. multifarius populations.The clam ko, 2012 , also cako, 2012 . In Chind August . The adutruction . A previan 15 mm . The decew years . TherefoM. multifarius juveniles into natural coastal areas were initiated by the Zhejiang Marine Aquaculture Research Institute in 2011  and 2012 . M. multifarius is also bred artificially; however, very little is known about the population dynamics. Management and conservation efforts could be improved by examining how environmental factors influence population connectivity and patterns of population genetic structure [Enhancement practices that involve the release of tructure . Populattructure , 10.M. multifarius populations in China. The objective of this study was to test the influence of planktonic larval stage duration on the genetic structure of M. multifarius. Our approach could add in the design of spatial fishery management and conservation strategies for species that inhabit the East China Sea and South China Sea.Genetic markers are useful tools for measuring genetic variation and gene flow among populations . In the M. multifarius were collected from 3 coastal localities in the East China Sea  and Beibu Gulf . These sites do not belong to a national park or a protected sea area or a relevant regulatory body concerned with wildlife protection or a private owner. We confirmed that the field studies did not involve endangered or protected species. Geographic locations and sample sizes of all the examined populations are provided in All experiments and animal sampling were reviewed and approved by the State Oceanic Administration of China and the Ethics Committee of Zhejiang Ocean University and performed according to national laws and regulations. Wild adult specimens of M. multifarius by using the salt-extraction procedure [Total genomic DNA was extracted from the adductor muscle of rocedure  with sliFor all markers, PCR was performed using the Taq Master Mix . The PCR primer sequences have been listed in http://www.ncbi.nlm.nih.gov/BLAST) to identify M. multifarius sequences. Then, the sequences were aligned using Clustal W [h), and nucleotide diversity (p), were calculated with DnaSP 4.0 [For all sequence analyses , genetic similarities were evaluated using BLAST  between the populations and testing their significance with 1000 permutations.Phylogenetic trees were constructed using the unweighted pair group method with arithmetic mean (UPGMA)  based onh) and nucleotide diversity (π) based on COI, 12S rRNA, and ND1 genes for the 3 populations are listed in Sequences of the 685-bp COI gene were determined in 61 specimens, and 33 polymorphic sites and 29 haplotypes were detected. Twenty-five haplotypes were found in only one population, 2 (H1 and H18) were found to be shared by two populations, and 2 (H11 and H13) were found to be shared by all three populations . A 350-bp > 0.05; S results for the 3 genes were not significant  and BH (p < 0.05) populations; this implies that M. multifarius may undergo population selection or expansion events.Not all of Tajima’s D results for the 3 genes were significant ; Nm was 0.627, indicating very low gene flow between these 2 localities. The other 2 pairwise Nm values (ZS and ZP populations and ZP and BH populations) were > 1, indicating an extensive genes flow among the populations. The same pattern was found using 16S gene data: pairwise FST ranged from 0.200 to 0.742 and pairwise Nm ranged from 0.174 to 2.012. The maximum FST value was also found between ZS and BH populations, while the Nm value was < 1. For ND1 gene data, the genetic structure was weak but significant and no differentiation was detected among the populations (FST < 0.05).Significant p < 0.001), except for ND1 gene results.AMOVA for COI, 12S rRNA, and ND1 genes on the basis of haplotype frequencies revealed that 75.56%, 41.98%, and 101.1%, respectively, of the genetic variation occurred within the populations, whereas 24.44%, 58.02%, and -1.1%, respectively, of the genetic variation occurred among the populations . AMOVA rM. multifarius among the haplotypes was determined using COI, 12S rRNA, and ND1 genes of Venerupis philippinarum (NC 003354.1), Paphia undulata (NC 016891.1), and Paphia amabilis (NC 016889.1) as the out-groups  with V. philippinarum and then clustered with the branch comprising P. undulata and P. amabilis.The phylogenetic relationship of t-groups . The 3 UM. multifarius populations by using 3 mtDNA markers. This result is consistent with the finding that species with a short planktonic larval stage have a short dispersal distance, especially those with low mobility as adults. Thus, marine bivalves with a short planktonic larval stage could have a strong population genetic structure.In this study, significant genetic structure was observed in M. multifarius populations was identified on the basis of all 3 mtDNA markers. Results of the COI gene showed high levels of haplotype diversity (h = 0.822–0.889) and low levels of nucleotide diversity (π = 0.004–0.006). This pattern is consistent with that observed in previous studies on other species from the East China Sea or South China Sea, for example, Mytilus coruscus (h = 0.818–0.972 and π = 0.00405–0.00747) [Chlamys nobilis (h = 0.641–0.966 and π = 0.00412–0.01306) [h = 0.257–0.678 and π = 0.001–0.002) and ND1 (h = 0.199–0.391 and π = 0.001–0.003) genes, a lower level of genetic variation and fewer haplotypes were obtained (7 haplotypes for 12S rRNA and 5 haplotypes for ND1), largely because they are in a low-variation region of the mitochondrial genome. In our study, the highest level of haplotype diversity was observed in the BH population. This result may be because the bio-resource of BH is more abundant in the southern tropical regions.Genetic diversity within and between populations provide a potential genetic resource for future adaptation, and can be critical for the fitness of a population . It is m0.00747)  and the 0.01306) . For 12SFST to assess gene flow, and a higher FST value indicates a lower level of gene flow (Nm) and higher genetic differentiation among populations [FST reflects the level of inbreeding within populations [FST values below 0.05 indicate negligible genetic differentiation, whereas values greater than 0.25 indicate high genetic differentiation within the analyzed population [FST values were obtained among different localities of M. multifarius populations on the basis of both COI  and 12S rRNA  genes; this, indicated a high degree of genetic differentiation among the M. multifarius populations. In addition, the highest FST values for both COI and 12S rRNA genes were between ZS and BH populations, which endorsed a signal of isolation due to distance. Populations may be divided by major geographic barriers such as land barriers and oceanographic patterns. Our result also showed a higher level of variation when compared with those of other bivalve studies, for instance, Li et al. [M. coruscus in the East China Sea by using the COI gene (FST = -0.03612–0.09774), and it showed no significant phylogeographic structure. The planktonic larval stage of M. coruscus was 35 days [FST value using the COI gene among populations of the mussel Mytilus galloprovincialis along the coast of Eastern and Southern China was between -0.0469 and 0.0509 [Coelomactra antiquata  [FST value between populations from the Bohai Sea and East China Sea was 0.95899, which showed a highly significant genetic differentiation between populations. These results endorsed a highly discussed hypothesis that marine species with an intensive planktonic larval stage would exhibit a high level of genetic differentiation. Nevertheless, with respect to the ND1 data, FST values were significant but weak (FST < 0.05), and high Nm values were detected among the populations; this may be because the ND1 gene is primitive and high conservative in this species.Researchers often use ulations . FST reulations  or the eulations . The prepulation . High ani et al.  characte 35 days , and it d 0.0509 . In contResults of the median-joining network suggested an obvious genetic differentiation between ZS and BH populations with respect to the COI and 12S rRNA genes, while results for the ND1 gene showed several ambiguous connections among the 3 populations. Similarly, demographic analyses using UPGMA for all haplotypes show 2 similar branches: one branch contains most (COI) or all (12S rRNA and ND1) haplotypes from the BH population, and the other branch contains most haplotypes from the BH population (COI) or haplotypes from all 3 populations. A much higher degree of genetic differentiation was observed between BH population and the other 2 populations, mostly because BH is located in the Beibu Gulf, South China Sea, and the Leizhou Peninsula  limits tM. multifarius on the basis of COI, 12S rRNA, and ND1 gene data; in addition, the BH population was isolated from the other populations. M. multifarius has a short planktonic larval stage, but it has a high population genetic structure that may facilitate fishery management, captive breeding, and restocking programs for aquaculture and conservation.In conclusion, our results showed that differentiations occurred among 3 populations of


Anopheles sinensis is a principal vector for Plasmodium vivax malaria in most parts of China. Understanding of genetic structure and genetic differentiation of the mosquito should contribute to the vector control and malaria elimination in China.An. sinensis populations using a 729 bp fragment of mtDNA ND5 among 10 populations collected from seven provinces in China.The present study investigated the genetic structure of ND5 was polymorphic by single mutations within three groups of An. sinensis that were collected from 10 different geographic populations in China. Out of 140 specimens collected from 10 representative sites, 84 haplotypes and 71 variable positions were determined. The overall level of genetic differentiation of An. sinensis varied from low to moderate across China and with a FST range of 0.00065 – 0.341. Genealogy analysis clustered the populations of An. sinensis into three main clusters. Each cluster shared one main haplotype. Pairwise variations within populations were higher (68.68%) than among populations (31.32%) and with high fixation index (FST = 0.313). The results of the present study support population growth and expansion in the An. sinensis populations from China. Three clusters of An. sinensis populations were detected in this study with each displaying different proportion patterns over seven Chinese provinces. No correlation between genetic and geographic distance was detected in overall populations of An. sinensis .ND5 gene of mtDNA is highly polymorphic in An. sinensis and has moderate genetic variability in the populations of this mosquito in China. Demographic and spatial results support evidence of expansion in An. sinensis populations.The results indicate that the  Anopheles sinensis is a principal vector of malaria in many regions in China along with An. lesteri, An. minimus, and An. dirus[An. sinensis has also been identified as the major malaria vector in many parts of other Asian countries, such as in Indonesia, South Korea, Thailand and Japan [An. sinensis was infected with P. vivax. The infection rate of An. sinensis in Taiwan was 0.02 % during 1947-1949 [An. sinensis is the principal vector in recent years [An. dirus,2. An. snd Japan . In Chinnt years . Malariant years .Although great success has been achieved since the launch of National Malaria Control Programme in 1955, malaria remains a serious public health problem in China -9. FalciAn. sinensis belongs to Hyrcanus group that includes approximately 30 morphologically indistinguishable species. In China, 25 taxa, including synonyms, have been recorded [An. sinensis could be separated into different forms on the basis of the egg deck width. Baisas and Hu [An. lesteri. Ho et al. [An. lesteri in China, but they noticed some distinct bionomic characters found in China as compared to those of the Philippine form. Particularly, the Chinese form of An. lesteri has a strong preference for human blood [recorded . The sturecorded  and Yao recorded  observeds and Hu  describeo et al.  also repan blood .An. sinensis[ITS2) region [An. sinensis populations in China, which indicates that An. sinensis could be divided at least into two populations [An. lesteri from Korea and Japan, An. lesteri from China and An. lesteri from the Philippines were all the same species [et al. [Anopheles species that was morphologically identical to An. sinensis, and Li et al. [An. sinensis. Rueda [An. belenrae sp. Nov. and An. kleini sp. Nov, based on their same morphologically identity with An. pullus and An. sinensis, respectively. Morphological identification of sinensis complex is extremely difficult, so that some members of An. lesteri, An. pullus (including the form yatsushiroensis) and two species (at least) are mixed in the population of An. sinensis in Korea.Cytogenetic studies have revealed two karyotypic forms, A (XY1) and B (XY2), in  sinensis, which c) region . Both fo) region , but onl) region ,18. Analulations . Recent  species . Ree et  [et al.  reportedi et al.  also obss. Rueda  designatVarious levels of population subdivision of the anopheline mosquito species have been observed from nearly panmictic across a wide geographic range to highly divergent within a short distance. A high rate of recurrent gene flow and/or recency of population expansion has made the populations of some anopheline species hardly differentiated -26. HoweAn. sinensis populations in China and the population divergence was not correlated with geographic distance or barrier in the range [An. sinensis populations should benefit malaria control programs in China and enable more appropriate control strategies to be developed. Unfortunately, there is very limited information about An. sinensis population genetics.A recent microsatellite study revealed two genetic pools indicating the coexistence of two genetic units in the sampled sites . In thishe range . A betteND5 gene was previously used as a powerful tool in elucidating the level of genetic and phylogenetic divergence between closely related species [An. sinensis populations from China based on NADH of dehydrogenase subunit 5 (ND5) gene of mitochondrial DNA (mtDNA).Mitochondrial DNA remains one of the most powerful and reliable tools for detecting population structure and inferring population differences due to its high and rapid mutational rate compared with nuclear DNA . Interes species . Herein,An. sinensis mosquitoes used in this study were collected during the period of July 2010 to September 2012 by light traps from ten sites located in seven provinces in China  were designed and synthesized for subsequent amplification of ND5 DNA fragments from An. sinensis genomic DNA samples. The ND5 primers were designed based on highly conserved regions of all available mosquito ND5 sequences from GenBank databases. Primer 3 software program was used to adjust the annealing temperature of two primer pairs. The primer pairs were used to amplify ND5 DNA fragments from genomic DNA samples extracted from An. sinensis collected from different regions to assess the polymorphism of the ND5 gene.A forward and reverse primer pair (5’-TGTGAACTGCAGGACACAT-3’ and 5’-GTTCTA CGGGCCTATCACC-3’), corresponding to The PCR was conducted in a 50 μL reaction mixture containing 1 μL of a 1:200 diluted genomic DNA, 25 pmol primers, 5 μL 10X reaction buffer, 2.5 mM MgCl2, 200 μM of each dNTP, and 1 U Taq polymerase. The cycling condition was 5 minutes denaturation at 94°C, followed by 35 cycles of 15 seconds denaturation at 94°C, 15 seconds annealing at 50°C, and 1 minute extension at 72°C, ending with a final extension for 5 min at 72°C. Amplified PCR products were purified and then unidirectionally sequenced for both strands for each one of the two regions by Sangon Biotech .ND5 gene sequences from 140 An. sinensis individuals were aligned by using BioEdit program 5.0.9 [An. sinensis from different provinces and/or sites, were estimated using the program DnaSP 4.9 [The am 5.0.9 . The Hapam 5.0.9  among ponaSP 4.9  and the naSP 4.9  tests weFST[ST evaluated was based on 1023 random permutations. Demographic analysis and spatial parameters were assessed using the distribution of pair wise sequence differences (mismatch distribution) of Rogers and Harpending [D and Fs values of Tajima and Fu, respectively, was evaluated by comparison with randomly generated values based on the observed (S) with 10,000 repeats. The Mantel test [Population pairwise net genetic distances based on Slatkin’s linearised FST, and hieFST,45 were FST. The sigrpending . We usedrpending  and Fu’srpending . The sigtel test  was perfAn. sinensis populations collected from different sites in China.A TCS software program 2.1 ,51 was uWe obtained ethical approval from the Ethical Review Committee of Chinese Center for Disease Control and Prevention (No.201214). Permission was also obtained from the directors and related departments of provincial CDC in seven provinces in China.An. sinensis either by PCR based on species-specific ITS2 sequences. Alignment of 30 representative ITS2 sequences of rDNA (accession number KC769646), in conjunction with different sites of collection, confirmed that only sequences of An. sinensis were used.All specimens used in this study were confirmed as ND5), was successfully amplified. Analyses were carried out on 729 bp of mtDNA dehydrogenase gene subunit 5 (ND5) from 140 An. sinensis individuals collected from ten sites in seven Chinese Provinces. A total of 84 haplotypes of mtND5 were determined in An. sinensis individuals collected from China (GenBank accession numbers: KC565754 – KC565837), in which 71 polymorphic sites were detected within these populations. The partial nucleotide sequences of ND5 gene showed variable polymorphic sites within and among An. sinensis populations. The frequencies of haplotypes clearly divided these populations into three clusters. Among them, one cluster (n = 51) had low variable sites including ancestral populations, another cluster (n = 32) displayed moderate polymorphism (at least two main constant variable sites) with nucleotide substitutions at positions (55 and 325) respectively, and the third population (n = 57) showed many variable sites with one main constant site at position 529. No characteristics of heteroplasmy and insertion or deletion events were shown within the analysed mtDNA sequence.A 836-bp segment of mitochondrial DNA (mtDNA), corresponding to coding region of NADH dehydrogenase gene subunit 5 , suggesting the existence of excessive rare nucleotide polymorphisms, with possible effects of purifying, selection, or population expansion. For other populations, all statistics tests did not reveal significant differences, suggesting that the nucleotide substitutions of the ND5 gene are consistent with neutral evolution theory.Tajima’s D test of departures from the neutral expectations did not show significant deviation from neutrality. However, Fu and Li’s An. sinensis in China  in the three clusters or overall populations respectively.Based on the haplotype polymorphism data of the mtDNA, the results revealed the presence of three clustered populations of An. sinensis samples, revealed generally patterns, characteristic of a population that has undergone a large expansion. According to the mismatch distribution analysis, the hypothesis of spatial experienced expansion could not be rejected in the three clusters and overall samples. A good fit of the spatial expansion was also observed for all clustered samples based on Sum of Squared deviation (SSD) values that were not significant in all the cases  in the three clustered populations and pooled samples  and geographic distance .Neutrality tests of Tajima's es Table . Howeveres Table . Fu's FsAn. sinensis samples collected from different sites, there were apparent differences in relative abundance for given groups. Figure An. sinensis in different geographical locations in China. Hainan and Yunnan provinces showed high abundance of cluster III . In contrast, CII became the majority in Guangxi and Sichuan . Although CI was present in all sites, it was noticeably more abundant than the other two clusters in Henan and Jiangxi . Shanghai showed relatively even distribution of the three populations . These results are comparable to previous findings on An. sinensis and its member group of Hyrcanus An. lesteri[An. baimaii in India [An. darlingi malaria vector in Central and South America [An. albimanus[An. gambiae[An. arabiensis and An. funestus[It has generally been accepted that the degree of the polymorphisms of  species . The pre. lesteri,52,53. Ain India , An. dar America -57, An. albimanus in Latin. gambiae, An. ara funestus,59,60.D, Fu and Li’s D and F did not detect deviation from neutrality theory in the ND5 gene within any population of An. sinensis. Only Fu and Li’s D and F tests were significant when all populations from different sites pooled together, and this perhaps is due to a high level of polymorphism within ND5 gene (71/140). These results support the neutral theory of mitochondrial gene evolution and suggested that there is a large effective population size of An. sinensis in these locations.Neutrality tests of Tajima’s FST = 0.011; Nm = 42.33). This could be due to bias of low sample size in Hainan (n = 9) but similar samples were generated from (HE-N and SC-N), that give significant results with YN. Moreover, the two sites of Hainan and Yunnan are somehow similar in their conditions for mosquito habitats. Although distantly apart, both sites belong to typical temperate tropic zones where there are great seasonal variations in abundance of mosquito populations that reach their peak during summer [An. sinensis samples over several seasons. The climatic condition could impose selection on An. sinensis populations. In addition, An. sinensis breeds in a wide range of habitats including natural and artificial [Generally, the results showed that there is low-to-moderate genetic differentiation and high gene flow within different populations except the YN population that displayed a significantly high level of genetic differentiation and restricted gene flow with all populations except Hainan population , cluster II includes relatively less samples 23% (n = 32) where 41% (n = 57) belong to cluster III. Cluster I was found in all populations collected across the study zone , cluster two was found in 8 populations out of 10 (except HA and YN) where cluster III occured in 9 populations (except SC-C). The three clustered populations were assigned by at least one common haplotype or nucleotide substitution and/or amino acid(s) replacement. The proportion of the three clustered populations varied from site to site in China. For example, the cluster three of An. sinensis population is abundant in Hainan and Yunnan, but moderately abundant in Shanghai and Henan and absent in Guangxi. These results are similar to those reported in a previous study [An. sinensis populations collected across China from 20 collection sites (one pool included six populations and the second pool included eight populations). In that study the author reported that there was coexistence between two gene pools and specimens were assigned to one gene pool when it is remaining greater than 80%. A similar finding was reported in South Korea that An. sinensis includes two groups associated with the presence of a mountain functioning as a genetic barrier [Interestingly, the results of haplotypes and genealogy revealed the presence of three clustered populations of us study  that ind barrier . Normall36% n = 5, clusterFST = 0.313; P < 0.0001). The level of genetic differentiation is relatively similar to that previously detected in An. sinensis populations in China using microsatellites [An. gambiae and An. arabiensis malaria vectors in Africa [An. sinensis is an importance factor for malaria control and that suggests further examining at a local level for microgeographic scales in lack of isolation by distance (IBD), for understanding population connectivity and vector dispersal across multiple spatial scales.An AMOVA analysis using the three clusters found that 31.32% of the variance was attributed to between populations and 68.68% to within populations. A similar phenomenon of variance pattern was observed in comparing three clusters. There is substantial significant genetic differentiation among populations of the three clusters  of An. sinensis collected from China.Polymorphic positions of Click here for file


Greylag goose populations are steadily increasing in north-western Europe. Although individuals breeding in the Netherlands have been considered mainly sedentary birds, those from Scandinavia or northern Germany fly towards their winter quarters, namely over France as far as Spain. This study aimed to determine the genetic structure of these birds, and to evaluate how goose populations mix. We used mitochondrial DNA and microsatellites from individuals distributed throughout the European Atlantic flyway, from breeding sites in Norway and the Netherlands to stopover and wintering sites in northern and south-western France. The mtDNA marker  showed 23 different haplotypes. The genetic distances amongst individuals sampled in Norway, northern France and the Netherlands were low (range 0.012–0.013). Individuals in south-western France showed a slightly higher genetic distance compared to all other sampling areas (ranges 0.018–0.022). The NJ tree does not show evidence of any single clades grouping together all individuals from the same geographic area. Besides, individuals from each site are found in different branches. Bayesian clustering procedures on 14 microsatellites  did not detect any geographically distinct cluster, and a high genetic admixture was recorded in all studied areas except for the individuals from the breeding sites in Norway, which were genetically very close. Estimation of migration rates through Bayesian inference confirms the scenario for the current mixing of goose populations. Anser anser) is widespread throughout the Palearctic. In Europe, the main breeding populations are located in central and northern countries, and the species rarely breeds in Mediterranean areas , strong site fidelity to wintering areas and pair formation at wintering quarters indicated a population structure defined by wintering rather than nest-site philopatry (Anas platyrhynchos), single nucleotide markers were used to investigate population structure on a continental scale throughout the northern hemisphere. This genetic analysis found a general panmixia, suggesting that mallards form a single large, interbreeding population (Aythya fuligula) shows high breeding site fidelity, but mtDNA and microsatellite markers revealed an extensive population admixture on the wintering ground (Aythya ferina), genetic differentiation was observed among Eurasian breeding populations, but no evidence of genetic structure was detected for pochards sampled on European wintering grounds  and the pink-footed goose (Anser brachyrhynchus), and investigated the phylogenetic relationship between seven Anser species  and two wintering zones (northern France and south-western France). This study investigates to what extent populations are genetically differentiated. Genetic structure could have been increased by the fragmentation of breeding geese in separated areas, or on the contrary, a limited genetic structure could have been developed by (i) the widespread practice of amateur breeding and selling of geese , (ii) thKnowledge of the genetic structure and diversity of greylag goose populations is a necessary scientific basis to manage this emblematic species  and decirubrirostris subspecies . After extraction, genomic DNA was stocked at −20 °C.We analyzed feather samples from 174 greylag geese  collectebspecies . During bspecies . Feathertggttatgcatattcgtgcataga ′3) and H466 (5′ tttcacgtgaggagtacgactaat ′3) primers (2 (25 mM), 0.3 µl of each primer (25 pmol/µl), 2.5 µl 10 × buffer, 0.4 µl Taq polymerase , ddH2O and genomic DNA (20–100 ng/µl). The selected cycling profile included a 4 min preliminary denaturation cycle at 94 °C followed by 32 denaturation, annealing and extension cycles  before a final extension of 7 min. Negative controls were included for amplification procedures to detect contaminations.Partial mitochondrial control region (CR1 D-Loop 288 bp) was amplified in 144 of the 174 individuals  using L1 primers . PCR ampThe PCR product was purified using the EXO-SAP procedure with Exonuclease I  and Shrimp Alkaline Phosphatase . The purification cycle consisted of 30 min at 37 °C, then 15 min at 80 °C to deactivate the enzymes followed by a 10 min cooling-down step at 4 °C. DNA concentration was determined after electrophoresis in 1.8% agarose gels (TBE 1%) stained with ethidium bromide and visualized in a UV-trans illuminator Gel Doc XR  using the Molecular Imager ChemiDoc XRS System and Quantity One software .Sequencing was carried out at Macrogen Laboratories  in an ABI 3730xl Analyzer (Applied Biosystems).FinchTV , and sequences were aligned with ClustalW algorithm in BioEdit 7.05 (DnaSP version 5 (h), nucleotide diversity (π) and the mean number of pairwise differences (k) in the sampled areas. Demographic and/or spatial population expansion events were investigated using the mismatch distribution implemented in DnaSP v. 5. MEGA 5.0 (Anser anser anser (GenBank AF159962) from Finland and another of Anser anser rubrirostris (GenBank AF159963) from Slimbridge Wetland Center, England, were included as reference sequences in tree construction. A sequence of the lesser white-fronted goose  and the bean goose  were used as outgroups.Raw electropherograms were checked visually using dit 7.05 . The hapdit 7.05 . DnaSP version 5  was usedMEGA 5.0  was usedMEGA 5.0 , clusterMEGA 5.0 . SupportMEGA 5.0  after 1,FigTree 1.3.1 (jModelTest (Maximum likelihood (ML) and maximum parsimony (MP) trees were obtained through the DNAML, CONSENSE, DNAPARS programmes in PHYLIP 3.67 . Bootstree 1.3.1 . The besee 1.3.1  in jModeodelTest . MaximumArlequin 3.3 .A total of 169 of the 174 samples  were gener anser . We usedO) and expected heterozygosity (HE) for each locus and population were calculated in GenAlex v. 6  of individual multilocus scores in tix 4.05  to descrGenepop 3.4  (Ln probability of the data) and using ΔK statistics , 14 haplotypes were shared by 2–47 individuals. According to the study areas, we found a total of nine private haplotypes, the majority of which occurred in the Landes (SW France) population (The mtDNA marker sequences (CR1 D-Loop 288 bp) showed 23 haplotypes defined by ten polymorphic sites and distributed in eight locations . Among tpulation . The divpulation . HaplotyThe genetic distances recorded in Norway, northern France and the Netherlands were low (range 0.012–0.013). A slightly higher genetic distance was observed in south-west France in comparison to all other sampling sites (ranges 0.018–0.022), while the two breeding sites in Norway were genetically very close .anser subspecies, while the remainder were either grouped with the sequence relating to the rubrirostris subspecies or differed clearly from both subspecies. Very similar topologies were obtained from trees generated with other tree-building methods .The NJ tree shows that clades are composed of a wide variety of different geese from different areas. Individuals from each site were present in different branches . BesidesThe haplotype median-joining network  was concWhilst 88.85% of the total genetic variance shown in hierarchical AMOVA was within populations, the remaining 11.15% occurred among populations. This indicates a small differentiation between the sampled areas.P = 0.044) and Finnmark (P = 0.042)  . Fu’s FS= 0.042)  was onlyn = 169 individuals; Among the 15 microsatellites previously isolated by oH ranging from 0.374 to 0.484 and eH from 0.433 to 0.549). Geese from the Landes wintering area exhibited the highest number of private alleles  in each population . The ploSignificant departures from HWE, due to heterozygote deficit and related to positive FIS values, were observed in all populations .p < 0.001), while only 2.1% was distributed among populations. Overall fixation index F ST from AMOVA was 0.02105, indicating a low differentiation between areas.AMOVA analyses showed that 97.9% of the total genetic variance in geese was significantly distributed within populations . Analysis under TPM only confirmed a recent bottleneck event for the Nord population (P < 0.05).Bottleneck events tested under IAM revealed a significant excess of heterozygotes (evidence of a recent bottleneck) in Nord, Landes and Oise populations , suggesting that there is no strong relationship between geographic and genetic distances.The Mantel test calculated on geographic and genetic distances yielded a non-significant correlation coefficient  and the hierarchical AMOVA showed genetic variance to mainly occur within populations. These findings could be explained by a small differentiation between the sampled areas and a general admixture of greylag goose populations in our western European study region. However, it should be taken into account that genetic divergences in geese are characteristically very small, with the lowest interspecific divergence reported here for avian species . The genbspecies . Birds fer group . Althouger group  preliminAnser anser, Interestingly, the ANS19 sequence was recently found in the white Roman goose in Taiwan. This race is widely bred for commercial purposes, and has been found to originate from the European species  methods respectively. As seen in our previous analyses, no geographic clustering was observed inside these Structure groups. Almost all individuals, with few exceptions, showed admixed genotype regardless of the number of groups considered in the analysis.In our study of microsatellites, individuals from different geographic localities were found to be combined in the Factorial Correspondence Analysis representation. Bayesian structure analysis resulted in a best combination of four or seven groups, according to ΔThe high mixing of genotypes and the lack of geographic structure among our studied populations could be interpreted in the light of the data obtained through ringing activity and the extensive neck-banding programme carried out in Scandinavia from 1984 to 2004 . Ring rerubrirostris subspecies, have been spreading in Europe over the last few decades; their natural flyway toward wintering areas crosses other European countries  but does not reach France.Our findings are somewhat unexpected if one assumes that the fragmentation of breeding populations into separate areas during the first part of the last century , should Anser cygnoides). Even if descendants of Anser anser can also be found there, they are usually farmed in Europe . In some cases geese were released as part of assisted restoring projects and are now indistinguishable from the wild individuals . Besidesn Europe . Althougn Europe  or genotn Europe . The conn Europe .Chen caerulescens,Branta leucopsis,Limosa limosa,Our findings on the greylag goose genetic admixture are similar to those reported in the snow goose (2) compared to 2–5 fold values obtained in staging areas during migratory and premigratory periods .Our DNA-based estimates of migration during the wintering period indicated a low rate of exchange between our sampled areas. In five of eight areas the vast majority of individuals (86–95%) did not switch among the different zones, and a moderate exchange (about 20%) was only observed from Charente Maritime to the Netherlands and from Oise to Gironde. These results seem to support the hypothesis that the French wintering birds arrive from various areas, including zones that are not sampled here , while the contribution of the Norwegian population represents only a portion of the whole assemblage . This loOur data show evidence of genetic bottlenecks in just three groups under IAM, all located along the same flyway , and in a single case (Nord) under TPM. The discrepancy between IAM and TPM could be related to the different ability of the mutation models to detect bottleneck events. Empirical data suggest that TPM is the most appropriate model for microsatellite loci  while IAAnser anser anser and A. a. rubrirostris, as well as the sylvestris forms from Iceland, Scotland and Norway), and help to build an effective international management strategy for this migratory species (Greylag goose populations are steadily increasing in north-western Europe . The lar species .10.7717/peerj.1161/supp-1Appendix S1Click here for additional data file.10.7717/peerj.1161/supp-2Appendix S2Click here for additional data file.10.7717/peerj.1161/supp-3Appendix S3Click here for additional data file.10.7717/peerj.1161/supp-4Appendix S4Click here for additional data file.


Plasmodium falciparum and continues to produce ever-changing landscapes of genetic variation.Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in P. falciparum isolates from Malawi and used population genetics approaches to investigate genetic diversity and population structure and identify loci under selection.We performed whole-genome sequencing of 69 −4), moderately high multiplicity of infection (2.7), and low linkage disequilibrium (500-bp) were observed in Chikhwawa District, Malawi, an area of high malaria transmission. Allele frequency–based tests provided evidence of recent population growth in Malawi and detected potential targets of host immunity and candidate vaccine antigens. Comparison of the sequence variation between isolates from Malawi and those from 5 geographically dispersed countries  detected population genetic differences between Africa and Asia, within Southeast Asia, and within Africa. Haplotype-based tests of selection to sequence data from all 6 populations identified signals of directional selection at known drug-resistance loci, including pfcrt, pfdhps, pfmdr1, and pfgch1.High genetic diversity (π = 2.4 × 10The sequence variations observed at drug-resistance loci reflect differences in each country's historical use of antimalarial drugs and may be useful in formulating local malaria treatment guidelines. Plasmodium falciparum malaria, which accounts for 40% of hospitalizations of children <5 years old, 34% of outpatient visits by children of all ages, and 40% of hospital deaths. Year-round malaria transmission occurs in almost every part of the country and peaks during the annual rainy season, from December to May [http://files.givewell.org/files/DWDA%202009/AMF/Malawi_MIS_2010_Final.pdf). On the other hand, scaled up malaria interventions  have benefitted pregnant women by reducing the prevalence of peripheral parasitemia (from 23.5% to 5.0%) and placental malaria (from 25.2% to 6.8%) [An estimated 3.3 billion people worldwide are at risk of malaria. The majority of cases 81%) and deaths (91%) occur in sub-Saharan Africa, where children <5 years old and pregnant women bear the greatest burden of disease [% and dear to May . Since 2P. falciparum. An improved understanding of the parasite's genetic diversity, population structure, and natural selection will have practical implications for developing methods of disease control. In particular, genetic variation enables the parasite to overcome host immunity, antimalarial drugs, and vaccines to establish persistent infections and increase transmission [P. falciparum isolates obtained from children in the Chikhwawa district of Malawi to investigate the genomic epidemiology of P. falciparum in this area and to explore the impact of host immunity and antimalarial drugs on the parasite population. We have used allele frequency–based approaches to infer the parasite's demographic history in Malawi and discover genetic loci likely to be under balancing selection [STF), identified signals of directional selection at known drug-resistance loci, including pfcrt, pfdhps, pfmdr1, and pfgch1. These findings highlight potential roles of genomics in guiding malaria control efforts, such as monitoring of key drug biomarkers and informing drug policy, and changes in parasite populations structure that correspond to and can predict changes in malaria epidemiology.An integrated approach to malaria control is essential, and the application of genomics is one area that can provide biological insights into important evolutionary forces in smission –7. In thelection . ResultsThe study was performed in southern Malawi's Chikhwawa District , a rural area of intense perennial malaria transmission  . It is aPermission to conduct the study was granted by ethics committees of the College of Medicine, Malawi and the Liverpool School of Tropical Medicine. Written informed consent was obtained from a parent or guardian of each child.P. falciparum DNA levels were quantified using PicoGreen analysis and quantitative real-time polymerase chain reaction (PCR), using the Applied Biosystems stepOne RT-PCR system [Between December 2010 and July 2011, 93 whole-blood samples were collected from children participating in a clinical trial at Chikhwawa District Hospital. Blood was depleted of leukocytes by CF11 column filtration , and genR system . SamplesR system , with a http://www.sanger.ac.uk/resources/software/smalt) with default parameters, and SNPs were called using SAMtools (http://samtools.sourceforge.net). This process identified 115 965 SNPs across the 93 samples, 24 of which were discarded because of very low coverage . For the remaining 69 samples , we retained 88 655 high-quality SNPs (76.4%) in their nuclear genomes that met the following criteria: (1) biallelic; (2) quality scores of >30 ; (3) not in genomic positions at the very extremes of the coverage distribution , which could reflect deletions or copy number variants, respectively [var, rifin, and stevor gene families, or regions of low uniqueness; and (5) no SNP positions with ≥3 problematic genotypes (missing or mixed). Uniqueness was calculated by a sliding 54-bp window of contiguous sequence across the 3D7 reference genome and detecting the presence of this motif elsewhere in the genome. Only SNPs that were in unique positions were retained. Genotypes at SNP positions were called using ratios of coverage. Heterozygous calls were converted to the majority genotype if the coverage ratio was 80:20 or greater [mt772, mt1692, mt4179, and mt4952) were extracted from the alignments and the genotypes called as described above. Public accession numbers for sequence data are contained in SRA studies .Detailed description of the analysis pipeline has been described elsewhere , 16. Briectively ; (4) not greater , and the greater ) and othDnaSP [D test [D and F metrics [D and F metrics correlated highly with those from the Tajima D test  and were not analyzed further. To detect signals of directional selection, the integrated haplotype score (iHS) [P values were computed from standardized values based on a 2-tailed conversion from a Gaussian distribution [http://www.rqtl.org). For comparisons between populations , we first applied the principal component analysis (PCA), based on a matrix of pair-wise identity by state values, followed by the cross-population long-range LD method, XP-EHH [STF [P values for the XP-EHH estimates were calculated using a Gaussian approximation. A significance threshold of P < .0006 was established for both iHS and XP-EHH, using a simulation approach. We used the ranked STF statistics to identify the informative polymorphism driving the clustering in the PCA. LD was measured using the 2r metric [For the high-quality SNPs n = 88 655) in the Malawian population, we estimated the genetic diversity by calculating the average pair-wise nucleotide diversity (π). We used DnaSP  software8 655 in  metrics  to identre (iHS)  was usedribution . The MOIribution . We estiribution . Second,ribution  were est, XP-EHH  and popuEHH [FST . P value2 metric , calculaP = .035, by the Wilcoxon signed rank test on differences). Regions with elevated diversity may encode an antigenic or polymorphic locus that may be useful for vaccine approaches. Modest positive correlations were observed between recombination rate and diversity .We identified 88 655 high-quality biallelic SNPs  in robu2r values (>0.5) that extended beyond 1 kb (Supplementary Table 1). We found no evidence of low recombination rates in these regions, and none of the genes were in the lowest 10th percentile of the recombination rates distribution for either genetic cross. One particular gene, piesp2 , showed a pattern of LD extending to 1 kb, suggesting that this region is under recent positive selection.To examine the potential effects of recombination, we looked at levels of LD in the Malawian population, which decayed rapidly within a few 100 bp and reached a baseline level within 500 bp. The decay in LD was more rapid in Malawian parasites than in Asian parasites, as previously reported  that extend to several genes, suggesting that drug selection has produced chromosomal segments of selective sweeps. We did not detect selection signals at pfcrt, but there was evidence of selection within 3 kb of the gene. This may be expected with regression back to the pfcrt wild-type alleles after withdrawal of chloroquine (CQ) from Malawi >15 years ago. Signals were also detected within 10 kb of pfubp1, a homologue of a Plasmodium chabaudi gene linked to artemisinin resistance. In Kenya, pfubp1 alleles were recently found to be under directional selection and associated with reduced in vitro susceptibility to artemisinin [msp6 and msp3.8, pfama1, trap, and msp7 on chromosomes 10, 11, 13, and 14, respectively. These genes are expressed predominantly in merozoites and are thought to be primarily under balancing selection, consistent with other observations in Asian and African populations [msp3.8 and members of the clag gene family [PF3D7_1343700 but none of the so-called K13-propeller mutations  associated with artemisinin resistance in Cambodia [Computation of iHS for the Malawian SNPs identified 14 chromosomal loci likely to be under positive directional selection , reCambodia .Table 1Supplementary Figure 1), with coding, nonsynonymous, synonymous, and intergenic sites more skewed than expected under neutrality. This observation indicates recent population expansion, as has been demonstrated in other African populations [D test to evaluate each polymorphic gene across the genome identified regions likely to be under balancing selection. There was a strong positive correlation between π and Tajima D values , providing potential evidence of increasing diversity with balancing selection. For the 2073 genes (51.1%) with at least 5 SNPs computed for Tajima D, the majority of values (96.5%) were negative. To negate any confounding effects of population expansion and difficulties in establishing significance levels, we report 19 genes with Tajima D values of >1  involved in merozoite invasion of erythrocytes and previously reported to be under immune selection [Interrogation of the allele frequency spectrum of different classes of nucleotide sites showed an excess of rare alleles , only 8% were polymorphic in Malawi. The comparison of Malawian samples to samples from each of the 5 populations, using the cross population long-range haplotype method (XP-EHH), identified regions potentially under positive directional selection at or near known drug-resistance loci  on chromosome 12 is unique to Malawi in this study. The pfgch1 gene is the first gene in the folate biosynthesis pathway, and adaptive selection could have resulted from SP pressure [pfgch1 locus during SP selection [pfgch1 [trap gene are thought to reflect genetic adaptation to divergent host ligands [The observed selective sweep surrounding the GTP cyclohydrolase gene  were used to confirm that the Malawian samples were of African origin. Haplotypes were formed using 4 established continent-specific polymorphisms  [mt  was consistent with the known multiple copies of the organelle in a P. falciparum cell [STF metric to measure genomic divergence across the 6 populations. At a stringent genome-wide cutoff , we identified the most divergent loci between Malawian samples and the other 5 populations. The frequency of alleles encoding known drug targets and their divergence between populations is shown in Supplementary Table 2 and Table The populations of ressures , 18. Pri mt4952) . Two haprum cell . There wpfcrt-K76T alleles after CQ withdrawal differs between Malawi (0%) and Kenya (31%), a disparity that has been observed previously [pfcrt-K76T alleles differs between Mali (62%) and Burkina Faso (36%), where CQ is still used. Third, pfcrt-K76T alleles have reached fixation in Cambodia and Thailand; indeed, CQ remains the first-line treatment for Plasmodium vivax malaria in these 2 countries and thus may continue to select for the resistant genotype in P. falciparum [pfdhps-K540E alleles have reached fixation in Malawi, despite the drug policy change from SP to ACT in 2007. The persistence of these alleles may be maintained by the use of SP as the first-line drug for IPTp and the use of trimethoprim/sulfamethoxazole, an antifolate antibiotic used widely for various indications. Although STF values may reflect differences in allele frequency due to differential selective pressure, they may also simply reflect random genetic drift. Further analysis at the gene level provided evidence of a high correlation between maximum STF and XP-EHH , suggesting that some signals of directional selection were detected using the population differentiation approach. However, it is possible that extreme XP-EHH could reflect demographic characteristics rather than selection pressure.The interpopulation differences at drug resistance loci likely reflect local historical parasite adaptation to drug pressure, leading to fixation or near fixation of the implicated resistance alleles, as evidenced by the following observations. First, reductions in the prevalence of eviously , 45. Theeviously . Second,lciparum . Fourth,P. falciparum population structure in Africa [D values in the African  populations indicate a historical population expansion in Africa. Second, balancing selection acts on a similar set of genes (including those predominantly expressed in merozoites and involved in erythrocyte invasion) in Guinean and Malawian samples, suggesting that it acts on similar antigenic targets irrespective of differing population demographic characteristics. For example, msp3.8 has the highest Tajima D value in both Malawi and Guinea. Third, signatures of directional selection at drug resistance loci appear to be population specific as expected, reflecting historical differences in antimalarial drug use. For example, there are strong signatures of directional selection around pfcrt in Guinea (where CQ was used until 2006) but not in Malawi (where CQ was withdrawn in 1998). In contrast, there is evidence of strong selection in pfdhps in Malawi but only weak selection in Guinea, where SP was never introduced as first-line treatment for malaria [Finally, a comparison of our data with those from a recent analysis from West Africa (Gambia and Guinea) highlights several key points about the n Africa . First,  malaria .P. falciparum whole-genome sequences can be used to understand malaria biology and impact disease control. Estimates of parasite genetic diversity, LD, and MOI will improve our understanding of various aspects of malaria epidemiology, including infection dynamics, transmission levels, pathogenesis mechanisms, and drug efficacy. For example, the high nucleotide diversity, short-range LD, and MOI found in Malawian samples reflect the high transmission history of the district in which they were collected. Additionally, we have provided insights into the parasite population structure within Malawi and placed it in the context of other populations, facilitating the recognition of potentially imported cases into the country and targeting of effective malaria control measures. Our whole-genome analytical approach has detected evolutionary genetic signatures and genes under selective pressure due to drug resistance or virulence. For example, we observed differences at drug resistance loci between geographically distinct populations due to differing histories of antimalarial drug use. However, we also observed evidence of selection pressure in genes with unknown functions, which can be investigated in future experiments.Our study demonstrates how the large and growing number of The Journal of Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.Supplementary materials are available at


Anopheles sinensis is one of the most abundant vectors of malaria and other diseases in Asia. Vector control through the use of insecticides is the front line control method of vector-borne diseases. Pyrethroids are the most commonly used insecticides due to their low toxicity to vertebrates and low repellency. However, the extensive use of insecticides has imposed strong selection pressure on mosquito populations for resistance. High levels of resistance to pyrethroid insecticides and various mutations and haplotypes in the para sodium channel gene that confers knockdown resistance (kdr) have been detected in An. sinensis. Despite the importance of kdr mutations in pyrethroid resistance, the evolutionary origin of the kdr mutations is unknown. This study aims to examine the evolutionary genetics of kdr mutations in relation to spatial population genetic structure of An. sinensis.Anopheles sinensis were collected from various geographic locations in China. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of the para-type sodium channel gene were conducted to analyse kdr allele frequency distribution, kdr codon upstream and downstream intron polymorphism, population genetic diversity and kdr codon evolution. The mitochondrial cytochrome c oxidase COI and COII genes were amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion and gene flow patterns.Adults or larvae of kdr codon L1014 of para-type sodium channel gene. A patchy distribution of kdr mutation allele frequencies from southern to central China was found. Near fixation of kdr mutation was detected in populations from central China, but no kdr mutations were found in populations from southwestern China. More than eight independent mutation events were detected in the three kdr alleles, and at least one of them evolved multiple times subsequent to their first divergence. Based on sequence analysis of the mitochondrial COI and COII genes, significant and large genetic differentiation was detected between populations from southwestern China and central China. The patchy distribution of kdr mutation frequencies is likely a consequence of geographic isolation in the mosquito populations and the long-term insecticide selection.Three non-synonymous mutations  were detected at the kdr insecticide-resistant alleles in An. sinensis from southern and central China. Local selection related to intense and prolonged use of insecticide for agricultural purposes, as well as frequent migrations among populations are likely the explanations for the patchy distribution of kdr mutations in China. On the contrary, the lack of kdr mutations in Yunnan and Sichuan is likely a consequence of genetic isolation and absence of strong selection pressure. The present study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.Our results indicate multiple origins of the The online version of this article (doi:10.1186/s13071-016-1513-6) contains supplementary material, which is available to authorized users. The development of insecticide resistance is regarded as an empirical model of an adaptive trait  whose orpara sodium channel gene (knockdown resistance kdr) and the detoxification of insecticides in mosquitoes involving detoxification enzymes  that metabolise the insecticide before it reaches its target (metabolic resistance) [para sodium channel gene have been shown to be associated with the use of pyrethroids, the most commonly used class of insecticide for malaria control worldwide [para sodium channel gene is highly conserved across insect species, indicating convergent evolution [kdr mutations: either the mutations arose once and then spread, or have evolved independently in different populations across a broad landscape. For instance, in Anopheles gambiae, the main African malaria vector, non-synonymous mutations at position L1014 of the para sodium channel gene occurred at least 4–5 times independently [para sodium channel gene were derived from single mutational steps from the common ancestor whereas others were the result of two mutational steps [kdr mutations were also found in Musca domestica and in the aphid Myzus persicae [Bemisia tabaci, where the L925I and T929V mutations appeared to have originated once and then spread through migration and global trade [Resistance to insecticides in mosquitoes involves multiple mechanisms, including the target-site insensitivity caused by mutations in the istance) –19. Mutaorldwide . The spevolution . There aendently . Some ofal steps . Multiplpersicae , 22. An al trade .An. sinensis, the most abundant and important malaria vector species in southeast Asia [para sodium channel gene are known, including the L1014F [kdr allele variants in An. sinensis across China and Southeast Asia. For example, the complexity of landscape could impose barriers to gene flow between the mosquito populations. Selection based upon the intensity and duration of pyrethroid use could also determine allelic distribution of kdr. On the other hand, genetic variation based on neutral markers reflects population structure resulting from demographic factors. In this case, the cytochrome c oxidase genes of the mitochondrial genome are used as the neutral reference. Based on the unique characteristics of maternal inheritance, no recombination, high variability relative to nuclear DNA, and low effective population size, the mitochondrial DNA (mtDNA) has been a marker of choice for studies of genetic diversity and population structure.In ast Asia –32, foure L1014F , 33–37, e L1014F , 35, L10e L1014F , 36, 37 kdr mutations. The specific objectives were to (i) determine the kdr allele distribution in multiple sites over a large geographic region; (ii) elucidate the evolutionary origin(s) of kdr mutations; and (iii) examine the role of landscape and demographic history of An. sinensis on the evolution of kdr mutations using mitochondrial DNA sequence data.The central goal of this study was to understand how local selection and migration have shaped the distribution of Anopheles sinensis mosquitoes were collected from 15 localities in China, including five sites in central China, seven sites in south China and three sites in southwest China  and 2708R (5'-AAA AAT GTT GAG GGA ARA ATG TTA-3') for COI (~900 bp) and primers COIIF (5'-TCT AAT ATG GCA GAT TAG TGC A-3') and COIIR (5'-ACT TGC TTT CAG TCA TCT AAT G-3') for COII (~800 bp). Reaction mix consisted of 10.5 μl 2X SYBR® Green PCR Master Mix , 1 μl of template DNA (5–20 ng), 0.5 μl of 10 μM each primer, and 8.5 μl water. Conditions for polymerase chain reaction (PCR) were 95 °C for 3 min, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, followed by an additional extension step at 72 °C for 6 min. Products of PCR were visualised on 1 % agarose gel and then purified and sequenced from both ends with the ABI Big Dye Terminator Cycle Sequencing Kit.An. sinensis sodium channel gene sequence (GenBank accession: DQ334052) to amplify a 1285 bp fragment that encompasses the kdr target codon position L1014, an upstream intron (intron-1) and a downstream intron (intron-2). Both primers are located in the coding region. Two internal primers, 566 F (5'-CAC TGC TGT AAA ACC CTG TGT-3') and 855R (5'-CTG TTT GCT AGG CAG TTT GC-3') were used for sequencing. We amplified the 1285 bp fragment from individual specimen and a total of 273 specimens were examined. Reaction mix consisted of 10.5 μl 2X SYBR® Green PCR Master Mix , 1 μl of template DNA (5–20 ng), 0.5 μl of 10 μM each primer, and 8.5 μl water. PCR cycles involved an initial denaturing step at 95 °C for 3 min, 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, followed by an additional extension step at 72 °C for 6 min. Amplifications were performed in a Bio-Rad MyCycler Thermal Cycler. Products of PCR were visualised on 1 % agarose gel and then purified and sequenced from both ends with ABI Big Dye Terminator Cycle Sequencing Kit.We designed a pair of primers  based on the kdr allele distribution in An. sinensis populations from southern China to central China was shaped by geographic isolation and/or by insecticide selection. We asked two questions in particular, i.e., (i) What is the kdr allele frequency distribution pattern and the phylogenetic relationship of kdr haplotypes? and (ii) What is the spatial genetic structure of An. sinensis mosquito populations in China? We used the para sodium channel gene sequence data to address the first question used mtDNA sequence data to address the second.This study addressed the central question as to whether the patchy para sodium channel gene encompassing the kdr codon L1014, intron-1 and intron-2 were aligned in Bioedit version 7.0 [D [D* and F* [Sequenced 1285 bp fragments of the sion 7.0 . DNA polsion 7.0 . The twon 7.0 [D , Fu and * and F*  and F te* and F* . Partiti* and F* . A Bayes* and F*  was usedA statistical parsimony network was constructed using the TCS v1.21 software  to inferFCT), among populations within groups (FSC) and among populations among groups (FST). Pairwise FST and ФST among all populations were calculated. Significance values were adjusted for multiple comparisons using the false discovery rate (FDR) approach [FST/(1- FST) with 10,000 randomizations [COI and COII gene sequences were aligned using Bioedit version 7.0 . The numapproach . Pairwisapproach  to inferapproach  using thapproach . Euclidiizations .FCT values after the 100 independent simulated annealing processes was retained as the best grouping of populations.To examine the proportion of total genetic variance between groups of populations and identify possible genetic barriers between them, the spatial genetic structure of haplotypes (combined COI and COII sequences) was analysed using the Spatial Analysis of Molecular Variance (SAMOVA 2.0 software) . This soWe performed the genetic landscape shape (GLS) interpolation analysis based on the pairwise genetic and geographical distance matrices using the Alleles-In-Space (AIS) software . AIS is D, Fu’s Fs and Ramos-Onsins and Rozas’s R2 statistic in DnaSP. Significantly negative D, Fs or small R2 values indicated recent population expansion, whereas positive or high values indicated population decline. Population expansion parameter τ  was estimated using the moment method, assuming the infinite sites model [r) over 1000 simulated samples of pairwise nucleotide differences. All P-values were corrected for multiple testing using the false discovery rate (FDR) approach [Demographic history of the 15 populations was elucidated based on the neutrality indices Tajima’s es model . Deviatiapproach .Gene flow and migration rates between all pairwise populations were estimated using the Bayesian coalescence-based approach implemented in LAMARC version 2.1.10 . Felsenspara-type sodium channel gene generated a 1285 bp PCR fragment, including a 187 bp exon encompassing the kdr target codon position L1014, its upstream intron 905 bp and exon 74 bp, and its downstream intron 64 bp and exon 55 bp. The PCR amplification of the COI and COII genes of the mitochondrial genome resulted in 900 bp and 774 bp fragments, respectively. In order to elucidate the kdr allele frequency distribution pattern and the phylogenetic relationships of kdr haplotypes, the para sodium channel gene sequence data were analysed for kdr allele frequency distribution, kdr codon upstream and downstream intron polymorphism, population genetic diversity and kdr codon evolution. Because mtDNA is haploid and maternally inherited, isolated populations will drift to different haplotype frequencies faster and achieve approximately twice the level of differentiation comparing to nuclear markers. So, the mtDNA sequence data were analysed for population variations, genetic differentiation, spatial population structure, population expansion and gene flow patterns.The PCR amplification of kdr codon L1014. Overall, the distribution of kdr mutant allele frequencies varied by geographic location. Populations in central China  reached a near fixation for kdr at codon L1014 mutations, whereas the southern populations  exhibited a low frequency of kdr mutations . In central China, L1014F was the predominant mutation and L1014C was also common in three provinces  in central China  were detected at the kdr target codon position L1014, an upstream intron (intron-1), and a downstream intron (intron-2). Among the 344 total samples, 273 samples were sequenced, and a total of 71 polymorphic sites were observed. Of these sites, 69 were bi-allelic and two (at the second and third nucleotides of the kdr codon L1014) had three variants  and nucleotide diversity (Pi) were 0.82 and 0.07, respectively. Genetic diversity varied significantly among geographical regions  = 46.3, P < 0.0001) and nucleotide diversity  = 47.3, P < 0.0001), whereas in the south and southwest populations, high haplotype (0.92 ± 0.005 and 0.85 ± 0.13) and nucleotide diversity (0.07 ± 0.005 and 0.06 ± 0.03) were detected.The overall haplotype diversity  account for the observed pattern. Although the AMOVA indicated that most of the variation (~70 %) occurred among populations among regions , the percentage of variation found among populations within the same region (3.2 %) as well as among the three regions (27.9 %) was also highly significant . Both the FST and ФST values (based only on haplotype frequencies) were significant for 61 and 55 out of the 105 pairwise comparisons, respectively, after Bonferroni correction for multiple comparisons  for constructing a TCS genealogical network. The network revealed at least eight independent mutation events from seven distinct intron haplotypes, giving rise to nine kdr haplotypes  between them. Haplotypes H04-L1014F (kdr L1014F codon TTT) and H04-L1014F (kdr L1014F codon TTC) are results of single mutational steps from the common ancestor H04-L1014, but with connection (T-5-C) between these kdr haplotypes. The remaining two kdr haplotypes (H05-L1014F and H02-L1014F) are also derived from single mutational steps from the distinct ancestors with a mixed geographical distribution from south to north.Among the 65 polymorphic sites detected within intron-1 and intron-2, six ) showed to be highly polymorphic , and a total of 18 haplotypes were identified based on these six polymorphic sites . Among the 15 populations, An. sinensis from southern China exhibited higher haplotype and nucleotide diversity in COI and COII than those in central China . Population differentiation was significant between the southwest and south/central China regions . Both the FST and ФST values (based only on haplotype frequencies) were significant for 30 and 26 out of the 105 pairwise comparisons, respectively, after Bonferroni correction for multiple comparisons , suggesting that the genetic structure observed in the An. sinensis populations could be partially explained by distance isolation  showed nonsignificant D- and F-values, indicating an excess of rare nucleotide site variants compared to what would be expected under a neutral model of evolution. The observed smooth and unimodal mismatch distribution indicates sudden population expansion in 13 out of the 15 populations, and this was consistent with the mismatch distribution derived under the model of sudden expansion  and populations from south/central China. The AMOVA analysis indicated that the percentage of genetic variation between these two regions (~28 %) is much higher than that among populations within each of the regions (~3 %). Such regional genetic differentiation was also manifested by the observation of more private alleles in each of the regions than within individual populations of a region. Our data show that Fs statistic) did not provide evidence of selection at the kdr introns. This finding concurs with a relatively low rate of insecticide use in agriculture and indoor spraying in Yunnan as compared to central China. Apart from limited migrations, the absence of strong selective pressure may in part explain the lack of insecticide-resistance alleles in Yunnan. Lower genetic variation was observed in the introns of the kdr locus in populations from central China when compared to the other populations. These populations showed a high frequency of kdr mutation (L1014F), suggesting that low variation at the adjacent introns could be a consequence of a recent selective sweep associated with intense insecticide use following the selection and/or fixation of the resistance genotypes. A selective sweep occurs when an allele rapidly increases its frequency due to positive or directional selection. Through genetic hitchhiking, the frequency of linked alleles in the flanking regions of the locus under selection can also increase, thus reducing genetic variation [For the two Yunnan populations, neutrality tests  and a downstream intron (intron-2) of the kdr codon suggests at least eight independent origins of kdr alleles in An. sinensis populations from south to central China. Multiple origins of kdr alleles were also reported in the Afrotropical mosquito vector An. gambiae [kdr haplotypes H01-1014 F and H01-L1014C from the same ancestor were the most widespread in An. sinensis populations from central China and evolved multiple times subsequent to their first divergence. This phenomenon could be explained by the selection pressure of insecticides experienced in these populations, which favors kdr resistant alleles. Therefore, the frequency of haplotype (H01-L1014) carrying the wild-type allele is much lower than that of haplotype (H01-L1014F) harbouring kdr mutations. However, the kdr haplotype L1014S from two distinct ancestors was detected only in the Guangdong population, suggesting a recent origin of this mutation that is unique to the populations in southern China.Information on whether resistance-associated mutations evolved independently us study  of kdr gkdr insecticide-resistant alleles in An. sinensis from southern and central China. Local selection related to intense and prolonged use of insecticide for agricultural purposes, as well as frequent migrations among populations are likely the explanations for the patchy distributions of kdr mutations in China. On the contrary, the lack of kdr mutations in Yunnan and Sichuan is likely a consequence of genetic isolation and absence of strong selection pressure. The present study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.Our results indicate multiple origins of the


The genetic relationship between original Atlantic salmon populations that are now extinct in the southern Baltic Sea and the present-day populations has long been controversial. To investigate and clarify this issue, we successfully genotyped individuals of the historical populations from the Oder and Vistula Rivers using DNA extracted from dried scales with the Atlantic salmon single nucleotide polymorphism array.FST of 0.2515 for all pairs of loci, which indicates a high level of genetic differentiation among the groups analyzed in this study. Pairwise FST values were significant for all comparisons and the highest values were found between present-day reintroduced Slupia River salmon and extinct Vistula River Atlantic salmon. Bayesian analysis of genetic structure revealed the existence of substructures in the extirpated Polish populations and three main clades among studied stocks.Our results showed a global The historical salmon population from the Oder River was genetically closer to present-day salmon from the Neman River than to the historical salmon from the Vistula River. Vistula salmon clearly separated from all other analyzed salmon stocks. It is likely that the origins of the Atlantic salmon population from the Morrum River and the Polish historical native populations are different.The online version of this article (doi:10.1186/s12711-016-0208-y) contains supplementary material, which is available to authorized users. Salmo salar L. ranges from northern Portugal to the Pechora River in northwest Russia and encompasses Iceland, the British Isles and the Baltic Sea [Currently, the European distribution of the Atlantic salmon ltic Sea . Baltic ltic Sea . The natHistorically, the largest population of salmon ascended the Vistula River and migrated to the tributaries of the upper Vistula River . In the Salmo salar) have, to date, resulted in three hypotheses. The first one argues that Baltic salmon may have derived from a refugial population that survived in eastern pre-glacial lakes and colonized the Baltic basin before a marine strait connected the Baltic Sea to the North Sea [Ongoing discussions about the post-glacial colonization routes that were followed by the Baltic salmon  array as in , our aimR and private allelic richness PAR ranged from 1.116 to 1.230 and 0.010 to 0.050, respectively. We detected 17 SNPs that deviated from Hardy–Weinberg equilibrium (P ≤ 0.05) after Bonferroni correction for multiple comparisons  to 1608 (Morrum) and that the mean number of alleles ranged from 1.571 (Neman) to 1.786 (Morrum). Observed heterozygosity per locus/population ranged from 0.235 (Vistula) to 0.339 (Neman) and expected heterozygosity from 0.241 (Vistula) to 0.32 (Slupia and Neman) , whereas pairwise FST values were significant before and after Bonferroni correction (P ≤ 0.05) for all tests, with higher values between present-day Slupia (SSP) salmon and the extinct Vistula (SVex) salmon (Table FST was lowest for the Oder River (SDex) pair. Estimation of global FST by AMOVA for all pairs of loci was equal to 0.2515, which indicates a high level of genetic divergence among the analyzed populations. Overall FIS reached −0.0065.Population-specific on Table . By contThe STRUCTURE software was used to examine the relationships among the salmon populations. The maximum value of ∆K was found for K = 3 and the mean log likelihood against K showed a clear plateau at K = 3. Bayesian analysis revealed the existence of substructures in the extirpated populations SDex and SVex Fig. . These sDuring the last decade, many new fish passes have been constructed on Pomeranian rivers in the southern Baltic area (e.g. seven in the Slupia River basin) and a previous fish pass on the Włocławek dam in the Vistula River was rebuilt in 2015. These systems allow the passage of Atlantic salmons and have increased their migration but the recovery program that still continues is only moderately successful . In the Our findings demonstrate that preserved fish scales can be a very valuable source of genomic DNA in population genetic studies. However, analyses based on markers from non-coding regions such as microsatellites often lead to a large amount of allele dropout , whereasIn our study, the lowest level of heterozygosity, most departures from HWE and the lowest allelic richness were found for the salmon individuals that were sampled from the Vistula River. These results can be explained by a reduction in population size that was inferred from a drop in commercial and research catches prior to complete extinction in the 1960 s .Säisä et al.  suggesteOur data showed that the historical salmon population from the Oder River was genetically closer to salmon from the Neman River than from the Vistula River. Salmon from the Vistula River were clearly separated from all other analyzed stocks. Although a mitochondrial DNA study reported that salmon populations from the Neman River clustered with populations from the eastern Baltic Sea including the Daugava River , our datResults from the STRUCTURE analysis provided evidence of population substructures in the samples from the Vistula and Oder Rivers, which originate from the presence of migrant fish since among the samples from both of these extinct populations, potential migrant fish from the Neman River were detected (PCoA and assignment test). This may be explained by the presence of strayers from the Neman River that could have been caught in Polish rivers or the effect of stocking activities. In our analysis, the salmon population from the Morrum River and the Polish present-day line introduced from the Daugava River stock each formed a cluster, which indicates that the post-glacial origin of the Atlantic salmon from southern Sweden and the origins of the Polish populations from the Vistula and Oder Rivers differ. Since the formation of the southern Baltic rivers occurred much earlier than that of rivers from the Baltic Main Basin, this may explain the differences among Polish stocks and populations from the south Scandinavian Peninsula.Native Atlantic salmon populations from the southern Baltic Sea originated from diverse sources after the retreat of the glacial ice sheet. Although the polish historical salmon populations from the Oder and Vistula Rivers clustered together, they were separated by a significant genetic distance. The population from the Oder River was more closely related to the present-day Neman River stock than that from the Vistula River and genetic substructure was revealed in these historical populations. The Polish native populations had a different origin and clustered separately from the Morrum and Daugava River salmon.