Populus euphratica) using chloroplast DNA (cpDNA) sequences and nuclear microsatellites, and estimated its historical distribution using Ecological Niche Modeling (ENM).The effects of historical geology and climatic events on the evolution of plants around the Qinghai-Tibetan Plateau region have been at the center of debate for years. To identify the influence of the uplift of the Tianshan Mountains and/or climatic oscillations on the evolution of plants in arid northwest China, we investigated the phylogeography of the Euphrates poplar  contains supplementary material, which is available to authorized users. In eastern Asia, the effect of geological dynamics, e.g., the uplift of the Qinghai-Tibetan Plateau (QTP) and Quaternary climatic oscillations, on species diversification has been at the center of debate for several years –4. A lotNorthwest China is located in central Asia and is arid or semi-arid, with a cold and dry continental climate. The progressive extension of the uplift of the QTP was associated with the orogeny of the Tianshan Mountains, which achieved a significant elevation during the Miocene , 7. The Aconitum nemoru lineages Aconitum Populus euphratica Olivier) and P. pruinosa Schrenk, are the only tree species that have established in the world’s largest shifting-sand desert, the Taklimakan Desert [P. euphratica is naturally distributed in western China and adjacent Middle-Eastern countries. More than 61% of P. euphratica forests occur in China, of which about 91.1% are located in Xinjiang province [P. pruinosa has a more restricted distribution than P. euphratica, occurring in alluvial oasis communities in northwest China, Kazakstan, Tajikistan, Turkmenistan, and Uzbekistan [P. euphratica and P. pruinosa are endangered as a consequence of intensive water use and damming in northwest China [Two closely related desert poplars, the Euphrates poplar  the pattern of genetic diversity of the two desert poplars in northwest China; (2) the divergence and gene flow between Euphrates poplar populations to the north and the south of the Tianshan Mountains; and (3) the historical population demography of Euphrates poplar in response to climatic oscillations.In this study, we used both chloroplast and nuclear markers to explore the phylogeographical pattern of these two desert poplars in northwest China, with a particular focus on the widespread Euphrates poplar, and used Ecological Niche Modeling (ENM) to estimate their potential historical distributions, and finally identify how the uplift of the Tianshan Mountains and Quaternary climatic oscillations influenced the current population structure of desert plantsP. euphratica individuals were collected from 33 natural populations ), which covers the whole range of this species in northwest China . In addition, DNA samples from three P. ilicifolia supplied by the Royal Botanic Gardens (http://www.kew.org/) were used for chloroplast DNA (cpDNA) sequence analysis. All collection of specimens used in our study complied with related institutional, national, and international guidelines.Leaf samples from 552 P. trichocarpa [P. euphratica [The samples were screened for variation at 17 nuclear microsatellite loci that were either supplied by the International Populus Genome Consortium from genome sequence of chocarpa  or develphratica ; see Addphratica  for fluophratica . MicrosatrnK region was amplified using the primers trnK-1682F (5′- GGGTTGCCCGGGACTCGAAC-3′) and trnK-4292R (5′- TGGGTTGCTAACTCAATGG-3′), which were improved from the Demesure et al. [P. trichocarpa chloroplast complete genome (GenBank accession numbers NC_009143) [The chloroplast e et al.  original_009143) . A PCR w_009143) . SequencP. euphratica and P. pruinosa [Clonal reproduction is common in pruinosa . To idenpruinosa , by calcG representing the number of multilocus genotypes (MLGs) or multilocus lineages (when taking into account possible somatic mutations or scoring errors) discriminated in the sample and N representing the number of sampled ramets.Clonal diversity was estimated as:HO and HE), were calculated for each microsatellite locus using the FSTAT 2.9.3 program [A), HO, HE, and the inbreeding coefficient (FIS) were calculated for each population across all microsatellite loci. We used a permutation procedure (1000 permutations) to test whether a particular estimate of the overall FIS was significantly different from 0. For P. euphratica, the regional genetic diversity  was also calculated using values of HO, HS (average of HE for subpopulations), and FIS. Allelic richness (AR) and private allelic richness (PA) for each of the three regions were calculated by standardization for 25 individuals, using the hierarchical sampling method as executed in hp_rare 1.0 [FST with FSTAT [After the identification of ramets belonging to the same genets, replicates were removed from the data set to perform a subsequent analysis. The number of alleles, allele frequencies, and observed and expected heterozygosity  and an admixed origin of populations. After an initial test, where we varied the burn-in and run length, the burn-in was set to 500,000 with 1000,000 additional cycles. For both data sets, 20 replicate runs were conducted for each value of K from one to ten. The final posterior probability of K, Pr(X|K), and ΔK, where the modal value of the distribution is located at the real K [We used a Bayesian model-based clustering method implemented in the program STRUCTURE version 2.3 –32 to fiz et al. , the proe real K , were boe real K .P. euphratica, five alternative scenarios for three population groups, northern and southern Xinjiang, and the QGM region, were tested using the Approximate Bayesian Computer procedure [To identify the possible population history of rocedure  as perforocedure . ScenariP. euphratica begins to reproduce at the age of 8–10 years and reaches flourishing period at the age of 15–30 years [One million simulations were run for each scenario. Prior parameter distributions for population sizes and time frames (measured in generations) were set as following: uniform  for both current and ancestral effective population sizes, uniform  for divergence times t1, uniform  for t2 (with t2 > t1), and uniform  for the admixture rate. 30 years ; we thusP. euphratica , we used the coalescent approach implemented in Migrate-n version 3.6 [θ = 4Neμ, where μ = mutation rate) and historical migration rates  between pairs of population groups [FST calculations. For each replicate, we used 10 short chains  and three long chains  with 100,000 trees discarded as an initial ‘burn-in’ and a static heating scheme at four temperatures . To avoid the confounding effects of the differences in sample size on the estimate of gene flow, the software randomly picked 40 individuals from each group.To estimate the gene flow pattern among regional population groups of sion 3.6 . This prn groups . AnalyseP. euphratica, we used the software BayesAss 3.0 [6, we ran the analyses for 107 iterations, with a sampling frequency of 103. Delta values for the migration (m), allele frequencies (a) and inbreeding (f) switching proposals were adjusted so that the accepted numbers of changes were 30–50% of the total number of iterations. The delta values for a, m, and f were 0.2, 0.1, and 0.2, respectively. We performed 20 runs (each with a different seed) and selected the one with the lowest deviance for further analyses [To estimate contemporary gene flow (m) between the above three regional population groups of sAss 3.0 , which canalyses .trnK were aligned using ClustalX ver. 1.81  with subsequent manual adjustments. A matrix of trnK sequences was constructed for the 286 trees that we examined, and different cpDNA sequences were identified as haplotypes. The sequences for those haplotypes have been deposited in GenBank under accession numbers KY002202–KY002230. Average chloroplast gene diversity within populations (HS) and total gene diversity (HT) were calculated using the program ARLEQUIN version 3.1 [P. euphratica, respectively. The measure of population differentiation, GST, was estimated as (HT-HS)/HT. Relationships between haplotypes were examined via a haplotype network, which was constructed using the computer program Network version 4.5.1.6 [P. alba and P. laurifolia as outgroups and the substitution rate of the chloroplast sequence estimated in P. balsamifera and P. trichocarpa , Global Biodiversity Information Facility (http://www.gbif.org/), and previously published papers [http://www.worldclim.org/version1) [Species distribution models for T 3.3.3e , to predd papers , 38, 48.ersion1) . For theersion1)  and the ersion1) . The LIGersion1) . For theersion1)  were useP. euphratica, the following five uncorrelated and biologically significant bioclimatic variables were selected as predictors: (1) mean diurnal range (mean of monthly maximum temperature - minimum temperature), (2) minimum temperature of the coldest month, (3) mean temperature of the wettest quarter, (4) precipitation of the driest quarter, and (5) precipitation of the warmest quarter. For P. pruinosa, (1) annual mean temperature, (2) temperature annual range (maximum temperature of warmest month - minimum temperature of coldest month), (3) precipitation of wettest month, (4) precipitation of driest month, and (5) precipitation of coldest quarter were used. After a model testing with 25% of the data, model validation was then performed 20 independent replicates using default settings. For each run, the area under the receiver operating characteristic curve was calculated as an indicator of the accuracy of model prediction. The ENM was first analyzed based on worldwide records of the Euphrates poplar, and then analyzed based on records in China only to specify the suitability of its estimation in China.For P. euphratica and P. pruinosa, respectively. Following our analyses based on these microsatellite loci, 54 distinct MLGs were detected from the 122 samples of P. pruinosa, and 203 MLGs were detected from 551 samples of P. euphratica, with the clonal diversity (R) ranging from 0.077–0.821 within populations of P. pruinosa, and 0–0.947 within populations of P. euphratica  from our sample of 673 individuals  was similar for P. euphratica (0.594) and P. pruinosa (0.565). The mean values of HO and HE respectively across populations were 0.538 and 0.558 for P. euphratica, and 0.484 and 0.560 for P. pruinosa. After using a sequential Bonferroni correction, only one P. euphratica population that coexisted with P. pruinosa individuals, ALe, had a fixation index (FIS) that significantly deviated from zero, suggesting that all populations except ALe are under a Hardy–Weinberg equilibrium. No FIS significantly deviated from zero for each population of P. pruinosa; but there was a significant deviation for the species as a whole, possibly reflecting the existence of a Wahlund effect . Differentiation among populations was low within both species, but was relatively higher in P. euphratica  than in P. pruinosa . For P. euphratica, the FST values among populations within northern Xinjiang (0.047) were higher than those within both southern Xinjiang (0.020) and the QGM region ) and osa Fig. . The P. A comparison of the posterior probabilities (PPs) of the five scenarios using local linear regression indicated that Scenario 5 was the most likely , followed by the QGM region to southern Xinjiang , resulting in a relatively higher gene flow from northern Xinjiang to southern Xinjiang via the QGM region than the reverse , followed by migration from northern Xinjiang to the QGM region (mnorth → QGM = 0.3044 ± 0.0124). Other pairs of gene flow were all lower than 0.0066  among pairs of the three groups ranged from 0.0042 to 0.3137 Fig. . The hig066 Fig. , also sutrnK data matrix was 2528 bp in length. A total of 25 haplotypes were identified in the 38 P. euphratica and P. pruinosa populations, based on 18 nucleotide substitutions and six indels  occurred in P. pruinosa, and three were shared by the two species . Another haplotype that differed from all 25 haplotypes mentioned above (by at least nine substitutions) was identified for the three P. ilicifolia individuals.The aligned cpDNA Hd) ranged from 0.476–0.668 and 0–0.810 within the five P. pruinosa populations, and 33 P. euphratica populations, respectively  than it was for P. pruinosa (GST = 0.183). Among the three regions of P. euphratica, the population from southern Xinjiang had the lowest chloroplast diversity and population divergence . The network could be roughly classified into five clades. Clade 1 (H01–H06), Clade 2 (H07–H15), and Clade 3 (H16–H18) were composed of haplotypes that only occurred in P. euphratica, except H15, which was located in the center of the network and was shared by the two species and was widespread (50.5%). Clade 4 (H19–H20) was located in the center of the network and was also shared by the two species, and Clade 5 (H21–H25) was composed of haplotypes that only occurred in P. pruinosa. The haplotype distribution in P euphratica populations from northern and southern Xinjiang was clearly different. Haplotypes from Clade 1 and Clade 3 were mainly distributed in the northern Xinjiang populations, while haplotypes from Clade 2 were mainly found in southern Xinjiang populations and eastern populations. H15 that mainly occurred in southern Xinjiang and QGM regions was found in three northern Xinjiang populations: YWX, BEJa, and BEJb.All 25 of the cpDNA haplotypes from P. alba and P. laurifolia were used as outgroups, all haplotypes from P. euphratica, P. pruinosa, and P. ilicifolia formed a monophyletic group with a support probability of 1 . All other haplotypes could be roughly classified into four lineages, with a support probability of higher than 0.7. The four P. pruinosa private haplotypes (H22–H25), that belonged to Clade 5 of the haplotype network, formed a lineage that was divergent at around 3.1 Ma (95% CIs: 1.5–5.0 Ma). Haplotypes from northern Xinjiang formed two lineages, which corresponded well to Clade 1 and Clade 3 in the haplotype network, and were divergent at around 2.1 Ma (95% CIs: 1.0–3.5 Ma) and at around 1.5–2.0 Ma, respectively. All other haplotypes formed the fourth lineage.The phylogenetic tree showed that, when f 1 Fig. . Within P. euphratica predicted completely separate potential distributions in China and those regions west of China, e.g., central Asia, Europe, and northern Africa, in all models, except for a slight connection at the western edge of the QTP , Middle Miocene 17–15 Ma), early late Miocene (11–10 Ma), and late Miocene (7–5 Ma) –15 Ma, e, 56. How5–20 Ma, P. euphratica, with the occurrence of a single, common, and often frequent ancestral haplotype (H15) in all populations of southern Xinjiang suggesting a significant historical bottleneck. Finally, the DIYABC analysis suggested that current Euphrates poplar distributions in the QGM region were created by an admixture of the northern and southern Xinjiang gene pools 336 generations ago . Sandy desert and gobi (stony desert) expansion has caused the habitat fragmentation of desert plants populations . We suggplotype H5 in all ongarica .P. euphratica  based on MLGs was similar for P. euphratica (0.594) and P. pruinosa (0.565). The genetic diversity at a population level was also comparable between the two species (HO = 0.484 and HE = 0.560 for P. euphratica and HO = 0.538 and HE = 0.558 for P. pruinosa). These values were lower than those stated in a previous study of the two species [P. alba  and P. tremula  [P. nigra  [We found that the overall gene diversity  , and P.  = 0.73) . This suP. euphratica from the three regions, populations from northern Xinjiang province held the highest nuclear and chloroplast genetic diversity, while populations from southern Xinjiang held the lowest genetic diversity  using STRUCTURE software. Figure S5. Modelled climatically suitable areas for Euphrates poplar. Figure S6. Modelled climatically suitable areas for P. pruinosa Table S1. Description of Populus pruinosa and P. euphratica populations analysed. Table S2. Description and references of the 17 microsatellite loci analysed for this study. Table S3. Diversity and differentiation for the 17 microsatellite loci analysed in Populus euphratica and P. pruinosa. Table S4. Variable sites of the aligned sequences of chloroplast DNA fragments in 25 haplotypes of Populus euphratic and P. pruinosa in northwest China. (DOCX 2025 kb)


Aframomum corrorima) is a perennial and aromatic herb native and widely distributed in southwestern Ethiopia. It is known for its fine flavor as a spice in various Ethiopian traditional dishes. Few molecular studies have been performed on this species so far. In the present paper, the ISSR technique was employed to study the genetic diversity in populations of cultivated A. corrorima.Korarima . Analysis of molecular variance (AMOVA) showed that 27.47% of the variation is attributed to the variation among populations and 72.53% to the variation within populations. The Fst (0.28) value showed a significant (p < 0.0001) genetic differentiation among populations. This was supported by the high coefficient of gene differentiation (Gst = 0.32) and low estimated gene flow (Nm = 1.08). A neighbor-joining dendrogram showed that the thirteen cultivated populations were separated into three clusters, which was in good accordance with the results provided by the two dimensional and three dimensional coordinate analyses. However, the clusters did not reveal clear pattern of populations clustering according to their geographic origin. This could be due to human mediated transfer of genetic material among different localities.Seven ISSR primers produced a total of 86 clearly scorable DNA bands. High levels of genetic diversity were detected in cultivated A. corrorima from the southwestern part of Ethiopia was relatively high. This finding should be taken into account when conservation actions, management policies for the species and site identification for in situ and ex situ conservation strategies are developed. Mizan Teferi II population displayed the highest genetic diversity; this population should be considered as the key site in designing conservation strategies for this crop. In addition, Jimma I and Jimma II populations with lowest genetic diversity, should also be considered due to the putative risk of extinction that they face because of the low genetic diversity.The genetic diversity in populations of  Aframomum corrorima (Braun) P.C.M. Jansen] belongs to the family Zingiberaceae and it is native to Ethiopia. It is a perennial tropical aromatic herb, often of large size, bearing flowers either terminally on aerial leaf shoots or from ground level [Korarima [nd level . Korarimnd level .Aframomum corrorima is widely distributed in Ethiopia, Sudan , Uganda (western), Tanzania (Usambara Mountains) and Eritrea [ Eritrea . The use Eritrea . Korarim Eritrea . Because Eritrea  was threAframomum species, the seeds of korarima have less pungent, milder and sweeter flavor. There is a demand for korarima in the neighboring countries and in Saudi Arabia where it has long been highly prized as a spice while there is a potential to expand its use to other regions [Korarima is a spice and medicinal plant of economic importance and an indigenous and endangered species of Ethiopia . The see regions . This spEnset ventricosum. Expansion of settlements, both urban and rural, and cultivation are creating “Vegetation Islands” [The moist montane forested land of the southwestern part of Ethiopia is dominated by forests that harbor genetic resources of vast social, economic and environmental importance. Every household has its own forest land with big trees used to hang traditional bee hives, to provide shade for different herb plants, like korarima and coffee, and to supply fruits, firewood and timber. This cultural practice helps the farmers to conserve the natural forest . HoweverIslands” .A. corrorima from southwestern Ethiopia, by using inter simple sequence repeat (ISSR) markers, to gain knowledge regarding conservation issues and best use of genetic resources.Korarima is one of the species in which genetic erosion is a real threat since its natural habitat, the humid mountain forests of southwestern Ethiopia, is being decimated at an increasing rate . To miniA. corrorima were randomly chosen from each of the 13 cultivated populations. The distance between sampled plants within each population was at least 20 m, in order to increase the likelihood of sampling inter-individual variation. Two very young healthy leaves were collected from each plant and sealed within plastic bags containing Silica gel. The samples were taken to the laboratory and stored at room temperature until used for DNA extraction.One hundred twenty nine 129) individuals, which corresponded to 13 populations , dNTPs (0.3 mM), Taq buffer (1× Thermopol reaction buffer) , MgCl2 (1.5 mM), primer (0.3 μM) and Taq polymerase (1 U μl−1) and sterile double-distilled water added to 25 μl. The amplification program was 4 min pre-heating and initial denaturation at 94 °C, followed by 40 cycles of 15 s at 94 °C, 1 min at the specific annealing temperature for each primer, and 1 min and 30 s extension at 72 °C. The lid temperature was held at 105 °C. A final extension for 7 min at 72 °C followed. The PCR products were stored at 4 °C until loaded on gel for electrophoresis.The polymerase chain reaction (PCR) was conducted by using a Biometra 2000 T3 Thermo cycler . PCR amplification was carried out in a 25 μl reaction as follows: template DNA  was used to estimate the size of each PCR product. Electrophoresis was carried out for 2 h at constant voltage of 100 V. The gels were stained with ethidium bromide and observed on a UV light and photographed using Biodoc Analyse 2.0 gel documentation system .Aframomum corrorima is a diploid with chromosome number (2n = 48) [A. corrorima samples using selected primers. The resulting binary data matrix of the ISSR phenotypes was analyzed using POPGENE version 1.31 [ST) and the level of gene flow (Nm) from Gst according to [m = 0.5 (1 − Gst)/Gst). Nei’s unbiased genetic identity (I) [Since ISSR markers are dominant and 2n = 48) , it is aion 1.31 , to estiion 1.31 , Shannonrding to  where Nmtity (I)  and geneThe hierarchical analysis of molecular variance (AMOVA) was used to calculate variance components within and among populations. Partitioning the total variation to different hierarchical level was done using Arlequin (version 3.0b) software . The un-The pattern of variation among individual samples was further examined by principal coordinate analysis (PCoA) which was performed based on Jaccard’s coefficient of similarity . The calA. corrorima individuals and 79 (91.86%) were found to be polymorphic. The total number of bands per primer varied from 6 (UBC-853) to 18 (UBC-841), with an average of 12.29 fragments per primer. The highest values of Nei’s gene diversity (0.43) and Shannon’s information index (0.62) were exhibited by primer UBC-835. In contrast, primer UBC-853 showed the lowest values of Nei’s gene diversity and Shannon’s information index . The mean Nei’s gene diversity and Shannon’s information index values for all the primers were 0.34 and 0.51, respectively  ranged from 31.40% in the Jimma I population to 81.40% in the Mizan Teferi II population. Similarly, the highest values of Shannon’s diversity index and Nei’s gene diversity for cultivated korarima population was recorded for Mizan Teferi II . Although samples of Jimma I, Jimma II and Jimma III populations were collected from geographically close sites in Jimma, populations Jimma I  and Jimma II  exhibited the lowest values in all parameters of genetic diversity estimates (Table st), estimated according to [m) was estimated to be 1.08 individuals per generation between populations.The genetic differentiation among populations (Grding to  was 0.32Inter-population genetic distance ranged from 0.06 to 0.41 for the 13 populations Table . Among tST: 0.28) and found to be highly significant (p < 0.0001).Analysis of molecular variance (AMOVA) was carried out on the overall ISSR data of cultivated korarima populations Table , firstlyCT < 0.01; p > 0.05)   region, while 73.64% of the variation was within the two regions. The variation among the two regions was very low and non significant , showed three major clusters Fig. . ClusterThe dendrogram produced by UPGMA for all 13 populations is presented in Fig. A. corrorima into three clusters  showed the grouping of individuals using two and three dimensional coordinates. Two dimensional (2D) coordinate analyses partitioned the 129 individuals of A. corrorima populations using ISSR marker. The extent and pattern of genetic diversity among 129 cultivated individuals of korarima from 13 populations were estimated using seven di-nucleotide ISSR markers. Based on the observed results, the application of ISSR DNA finger-printing technique was efficient and successful for revealing the diversity among individuals and populations of A. corrorima, scoring a range from 5 to 18 polymorphic bands. From all the populations, 86 discernible ISSR fragments were generated among which 79 were polymorphic. The overall percentage of polymorphism (94.48%) revealed by this study suggests that genetic diversity of the studied individuals and populations is high. Long-lived perennials have higher gene diversity compare to plants that are annuals or short-lived perennials [A. corrorima might be due to long life span. The mean Nei’s genetic diversity (GD) and Shannon’s information index (I) values for all primers were 0.34 and 0.51 respectively, indicating an immense genetic diversity at species level. This result is in accordance with the result obtained in other perennial plants in the Zingiberaceae family like Curcuma alismatifolia from Thailand  [Nigella sativa L.)  [A. corrorima in southern Ethiopia by Eyob et al. [The present study reports genetic diversity parameters for cultivated Ethiopian rennials –27. The I: 0.43)  and in tI: 0.54) , teff (GI: 0.54)  and sorgI: 0.54)  using ISThe success of a crop-improvement program largely depends on the availability and knowledge of the genetic resources in a germplasm collection. Since areas of high genetic diversity mostly contribute more accessions than those of low diversity for further and future collection, breeding and conservation activities should be based in areas with high genetic diversity. Among the 13 populations from southwestern Ethiopia studied, the highest percentage of polymorphic bands (PPB), gene diversity (GD) and Shannon’s diversity index (I) respectively were recorded in Mizan Teferi II population    value also confirmed high genetic differentiation among populations. The higher genetic differentiation might be due to genetic drift resulted from small population size and limited gene flow. AMOVA analysis based on further grouping of the populations into regions and zones resulted in non-significant variation. The absence of genetic divergence between regions and among zones could be attributed to the widespread practice of exchanging seeds and planting material between relatives and migrants from one location to the other during trade exchange.The overall degree of genetic differentiation of 2) Table , is muchtyledons , 26. Bretyledons . In linem smaller than 2 provides a substantial opportunity for genetic population divergence [m) was estimated to be 1.08 individuals per generation between populations (Table st (0.32) value which shows high variation among populations.Gene flow through a movement of individuals on a geographic scale , in conjvergence . The levns Table , suggestIn this study, the inter-population genetic distance ranged from 0.06 to 0.41 Table . The maxst = 0.32) obtained in this study. No clear grouping of individuals from the same population is observed in the NJ dendrogram  Fig. . The firram Fig.  and 3D (ram Fig.  PCoA anaram Fig.  contribuA. corrorima populations from southwestern Ethiopia. The results obtained, validate once more that ISSR are useful markers in genetic diversity studies, due to the very high level of polymorphism detected by the primers. The findings should be taken into account when conservation actions, management policies for the species and site identification for in situ and ex situ conservation strategies are developed. Among the thirteen populations, all genetic diversity estimators indicate higher genetic diversity in Mizan Teferi II population. Therefore, this population should be considered as a key site in designing conservation strategies for this crop. Populations like Jimma I and Jimma II showed the lowest genetic diversity and should also be considered during conservation strategies due to the putative risk of extinction that they face because of low genetic diversity.In the present study, seven ISSR markers have been used to study the genetic variability of


Retropinna semoni) is a native fish with a broad distribution spanning inland and coastal drainages of south-eastern Australia. Previous studies have demonstrated variability in population genetic structure and movement behaviour  across the southern part of its geographic range. Some of this variability may be explained by the existence of multiple cryptic species. Here, we examined genetic structure of populations towards the northern extent of the species’ distribution, using ten microsatellite loci and sequences of the mitochondrial cyt b gene. We tested the hypothesis that genetic connectivity among rivers should be low due to a lack of dispersal via the marine environment, but high within rivers due to dispersal. We investigated populations corresponding with two putative cryptic species, SEQ-North (SEQ-N), and SEQ-South (SEQ-S) lineages occurring in south east Queensland drainages. These two groups formed monophyletic clades in the mtDNA gene tree and among river phylogeographic structure was also evident within each clade. In agreement with our hypothesis, highly significant overall FST values suggested that both groups exhibit very low dispersal among rivers . Microsatellite data indicated that connectivity among sites within rivers was also limited, suggesting dispersal may not homogenise populations at the within-river scale. Northern groups in the Australian smelt cryptic species complex exhibit comparatively higher among-river population structure and smaller geographic ranges than southern groups. These properties make northern Australian smelt populations potentially susceptible to future conservation threats, and we define eight genetically distinct management units along south east Queensland to guide future conservation management. The present findings at least can assist managers to plan for effective conservation and management of different fish species along coastal drainages of south east Queensland, Australia.Freshwater fishes often exhibit high genetic population structure due to the prevalence of dispersal barriers  whereas population structure in diadromous fishes tends to be weaker and driven by natal homing behaviour and/or isolation by distance. The Australian smelt (Retropinnidae:  Genetic structure in aquatic fauna is strongly influenced by the characteristics of the ambient environment. Freshwater species typically exhibit higher levels of genetic differentiation than those living in estuarine or marine habitats . These gRetropinna spp.) is a common fish species distributed throughout the rivers of south-eastern Australia  following R. semoni would display high population structure among rivers similar to southern populations; and (ii) that genetic structure within rivers would be low due to dispersal within rivers.In most studies to date, diadromous behaviour has been shown to facilitate genetic connectivity among river catchments and typically results in “isolation-by-distance” (IBD) patterns of population genetic structure . In AustA total of 389 individual samples were collected from 15 locations in south-east Queensland, Australia . SamplesR. semoni were amplified and genotyped using primers developed by taq polymerase . The following basic thermocycler settings for PCR were performed: initial denaturation at 94 °C for 4 min, followed by 35 cycles at 94 °C for 1 min, 57 °C for 30 s, 72 °C for 1 min and a final extension at 72 °C for 7 min. Fluorescently labelled PCR products were pooled and added to 10 µl of Hi-Di™ formamide with 0.1 µl of GeneScan™ 500 LIZ size standard. Fragment analysis was conducted on an ABI PRISM 3130 Genetic Analyzer  according to the manufacturer’s instructions. Data were scored using GENEMAPPER version 3.1 software .Genomic DNA was extracted from fin tissue using the DNeasy Blood and Tissue kits  following the manufacturer’s directions. Microsatellite markers developed for b region of the mtDNA genome was selected for sequence analysis. The primers HYPSLA and HYPSHD  site from which one additional sample was included for analysis. Samples from four additional sites not included in microsatellite analysis were also sequenced—two sites from the Mary River and two sites from the Brisbane River . In totad HYPSHD  were used HYPSHD .Microsatellite genotype frequencies were checked for the presence of null alleles, large allele dropout and stuttering artefacts using MICRO-CHECKER v2.2.3 . Tests fFIS) was estimated in FSTAT 2.9.3  in ARLEQUIN. Two hierarchical arrangements of the 15 populations were analysed where the highest level was either (a) two groups (species), SEQ-N  and SEQ-S  or (b) catchment division, grouped into eight rivers. These were: Mary , Noosa (NSD), Mooloolah , Brisbane , Logan , Coomera , Nerang  and Currumbin . Three hierarchical levels of variation were analysed for each arrangement: among rivers (FCT), among populations within rivers (FSC) and among all populations (FST).ARLEQUIN was used to evaluate the geographic structuring of genetic variation. K) from 1 to 16 were carried out to verify that the estimates of K were consistent across runs. The burn-in length was set at 250,000 iterations followed by a run phase of one million iterations. The generated results were imported into the software STRUCTURE HARVESTER ) based on microsatellite DNA loci was carried out using a Mantel test  in ARLEQUIN. Genetic distance was represented as m is the proportion of immigrants in a focal population i that arrive from a source population j (F0) immigrants from unsampled populations. Here we used the F0 immigrants.BAYESASS v1.3 was used to calculate contemporary migration rates over the past few generations, where lation j . Migratilation j . We alsolation j  to estimR. tasmanica: JN232589; and one representing R. semoni: JN232588 , but likely belongs to a southern lineage of R. semoni which are known to have a closer mtDNA relationship with R. tasmanica than to northern lineages (A neighbour-joining (NJ) tree analysis was performed using the HKY distance model in Geneious version 9.1.5 with 1,000 bootstrap replicates . In addiJN232588 . The R. lineages .After Bonferroni correction, 3 out of 15 populations exhibited deviations from HWE in only one or two loci . All locFIS values, indicating that most of the populations had slight heterozygote deficits.Population genetic diversity indices are shown in FST values between the 15 populations were significant and ranged from −0.018 to 0.404. The SEQ-N populations were more diverged from one another than the populations in the SEQ-S group. The lowest pairwise FST value  was observed between populations NRD and NRU. The highest genetic divergence  was observed between populations NSD and MLU. Out of 105 comparisons, only two comparisons were non-significant (P > 0.05) and each of these pairs was from within the same river (Logan-Albert and Nerang). However, one site of each of these pairs contains a small number of samples which probably reduces the power to detect the significant FST values. Generally FST comparisons revealed much less divergence among populations within the same river than between populations from different rivers (Most of the pairwise t rivers .K = 8 clusters (Average log probability of data Ln[P(DK)] ± SD = − 15125.4 ± 0.584618)   indicatiTRUCTURE . ΔK alsoulations  and neglulations .FCT = 0.05), but also among populations within groups (FSC = 0.19)  higher than the FSC value (among sites within rivers) in both groups  ( = 0.19) . There wh groups  and 3Bii2 =0.3687, p = 0.001; BRD, BRU, LAD, LAU, CMD, CMU, NRD, NRU, CRD, and CRU)  (There was a significant correlation between genetic differentiation and stream distance among populations in the SEQ-S group (Rand CRU) , but notand MLU) .F0 migrants using the “detection of first generation migrants” option in GENECLASS2 , there were highly significant FST values, which indicated that populations were not panmictic. The results of the significant genetic differentiation among rivers were also consistent with the results of population structure revealed from the STRUCTURE analysis, suggesting restricted gene flow and limited dispersal among populations in both groups. Limited dispersal was supported by our first-generation migrant detection analysis in Geneclass2, which demonstrated that less than 10% of individuals in each population were immigrants.Based on previous studies of Australian smelt , we had Maccullochella mariensis (Elusor macrurus (Cherax dispar (Neoceratodus fosteri (The sample from Tinana Creek (MRD site), was differentiated from the rest of the populations in the SEQ-N group . This miariensis , Mary Rimacrurus , freshwax dispar  and Aust fosteri .R. semoni populations in the SEQ-S group than the SEQ-N group suggests that more current or recent gene flow occurs within and among the catchments in SEQ-S group than for the SEQ-N group. This could be because the catchments in the SEQ-S group have been connected more recently and/or more often than those in the SEQ-N group. This could be because during extreme flood events, SEQ-S catchments might be connected by freshwater plumes flowing into Southern Moreton Bay. Another plausible reason might be that these cryptic species (SEQ-N and SEQ-S) have different microhabitat preferences, with SEQ-N being less tolerant of brackish conditions . In this model, equilibrium between genetic drift and gene flow may be reached in species where the life time dispersal distance is less than the range. Here, a relationship between stream distance and genetic differentiation should be evident . In thisectively . Using ob divergence of 4% between SEQ-N and SEQ-S samples is close to the 3.6% divergence observed for the full mitochondrial molecule reported by R. semoni in southern Queensland (KX421785 corresponds to SEQ-N and KX421784 corresponds to SEQ-S). The level of cyt b divergence between the SEQ groups and southern lineages of R. semoni is very large (15–17%) and adds to previous studies that have highlighted the likely existence of a cryptic species complex within the taxon currently referred to as R. semoni  along the south-east Queensland drainages were detected in  dataset  demonstr10.7717/peerj.4654/supp-1Table S1Departures from Hardy-Weinberg equilibrium (following Bonferroni correction) are indicated by an asterisk (∗).Click here for additional data file.10.7717/peerj.4654/supp-2Table S2No means no evidence for null alleles. See Click here for additional data file.10.7717/peerj.4654/supp-3Table S3K). The mean value of K, the ad hoc statistic of Evanno, Regnaut & Goudet (2005) was used to summerize the second-order rate of change in LnP(D). The bold value of K = 8 represents the most likely number of genetic groups indicated by both test statistics. NA = not applicable given that K cannot be calculated for these value of K.The mean and standard deviations (SD) of the log-likelihood values (LnP[D]) for different hypothesised numbers of genetic populations (Click here for additional data file.10.7717/peerj.4654/supp-4Table S4Click here for additional data file.10.7717/peerj.4654/supp-5Figure S1K means number of clusters and ΔK means the corresponding ΔK statistics calculated according to Click here for additional data file.10.7717/peerj.4654/supp-6Data S1Click here for additional data file.


Aphelandra aurantiaca. Also, we assessed its present-day genetic structure under different past demographic scenarios.Theory predicts that habitat fragmentation, by reducing population size and increasing isolation among remnant populations, can alter their genetic diversity and structure. A cascade of effects is expected: genetic drift and inbreeding after a population bottleneck, changes in biotic interactions that may affect, as in the case of plants, pollen dynamics, mating system, reproductive success. The detection of the effects of contemporary habitat fragmentation on the genetic structure of populations are conditioned by the magnitude of change, given the few number of generations since the onset of fragmentation, especially for long-lived organisms. However, the present-day genetic structure of populations may bear the signature of past demography events. Here, we examine the effects of rainforest fragmentation on the genetic diversity, population structure, mating system (outcrossing rate), indirect gene flow and contemporary pollen dynamics in the understory herb A. aurantiaca were sampled in large (4), medium (3), and small (5) forest fragments in the lowland tropical rainforest at Los Tuxtlas region. Variation at 11 microsatellite loci was assessed in 28–30 reproductive plants per population. In two medium- and two large-size fragments we estimated the density of reproductive plants, and the mating system by analyzing the progeny of different mother plants per population.Twelve populations of A. aurantiaca possess high genetic variation (He = 0.61), weak genetic structure (Rst = 0.037), and slight inbreeding in small fragments. Effective population sizes (Ne) were large, but slightly lower in small fragments. Migrants derive mostly from large and medium size fragments. Gene dispersal is highly restricted but long distance gene dispersal events were detected. Aphelandra aurantiaca shows a mixed mating system (tm = 0.81) and the outcrossing rate have not been affected by habitat fragmentation. A strong pollen pool structure was detected due to few effective pollen donors (Nep) and low distance pollen movement, pointing that most plants received pollen from close neighbors. Past demographic fluctuations may have affected the present population genetic structure as Bayesian coalescent analysis revealed the signature of past population expansion, possibly during warmer conditions after the last glacial maximum.Despite prevailing habitat fragmentation, populations of A. aurantiaca despite dozens of generations since the onset of fragmentation in the region of Los Tuxtlas. Instead, past population expansion is compatible with the lack of observed genetic structure. The predicted negative effects of rainforest fragmentation on genetic diversity and population structure of A. aurantiaca seem to have been buffered owing to its large effective populations and long-distance dispersal events. In particular, its mixed-mating system, mostly of outcrossing, suggests high efficiency of pollinators promoting connectivity and reducing inbreeding. However, some results point that the effects of fragmentation are underway, as two small fragments showed higher membership probabilities to their population of origin, suggesting genetic isolation. Our findings underscore the importance of fragment size to maintain genetic connectivity across the landscape.Habitat fragmentation has not increased genetic differentiation or reduced genetic diversity of  Tropical rainforests sustain much of global biodiversity, including most endemic plant species of the world . UnfortuTheorized genetic consequences of habitat fragmentation have focused on effects brought about by reductions in population size and increasing spatial isolation between remnant populations . These cThe impact of rainforest fragmentation on the genetic structure of plant populations is highly variable, depending on life history, life-span, and mating system . At the ca. 45% of vascular plant diversity and are the richest plant communities in lowland tropical rainforests  in a highly fragmented tropical rainforest in southern Mexico. Also, using contemporary genetic data, we infer the demographic history to understand the current distribution of genetic diversity. Aphelandra aurantiaca is an important species of tropical rainforest understory, whose population dynamics is affected by the presence of sunflecks or forest light-gaps  reduced genetic variation and effective population sizes, (2) higher population differentiation as a consequence of genetic isolation, (3) higher inbreeding, (4) lower out-crossing rate, and (5) higher differentiation among pollen pools, (6) reduced number of pollen donor parents, and (7) decreased effective pollination neighborhood due to a decrease in plant abundance. Complementary, we assessed the present day genetic structure of A. aurantiaca under different past demographic scenarios.We assessed the potential effects of habitat fragmentation on the genetic structure of Aphelandra aurantiaca (Scheidw.) Lindl. is an understory herb of neotropical rainforests from southern Mexico to Bolivia. It is a self-compatible species that bears inflorescences with yellow floral buds that turn red when flowers open and produce nectar. In the rainforest of Los Tuxtlas in Mexico, this species is pollinated by the hummingbird Phaethornis longirostris  altough it is also visited by butterflies and bumble-bees , three medium (n = 97) and four large fragments (n = 138)  .A. aurantiaca to determine its genetic structure  and expected and observed heterozygosity (He and Ho) using Arlequin v.3.5.1.3 (P(1)) was calculated by rarefaction with HP−RARE (FIS) was estimated using GENETIX v.4.05  using a linkage disequilibrium method in NeEstimator V2 (Ne–4Ne generations) using the program Bottleneck 1.2.02 (Heq) (We estimated effective population sizes (mator V2 . We emplk 1.2.02 . Populat02 (Heq) .Ne, one with a decline, and one with an expansion. We ran one million simulations for each scenario. The parameter settings and priors are shown in Ne was set at 10–10,000 before present, assuming generation time of one year for the species , implemented in DIYABC. We estimated the posterior probabilities using a logistic regression approach on the first 1% simulations closest to the observed dataset. To check the confidence of model choice we estimated type I and type II error rates by simulating 500 pseudo-observed data sets  framework to infer past demographic history, as implemented in the software DIYABC 2.0 . ABC cho species . We usedata sets .Rst and a hierarchical analysis of molecular variance (AMOVA) using Arlequin v.3.5.1.3 program  was inferred with the Evanno ΔK method (K)   to visuaNetStruct 1.2 program  infers gadegenet package , a multivariate analysis implemented in the  package  in R 3.1 package . Based o package . We follgstudio   were estimated for paired populations using the program BayesAss v 3.0 . Becauseeration) . To checeration) . The runL_home using L_home, is the likelihood of the individual genotype within the population where it was sampled. GENECLASS2 assumes Hardy–Weinberg equilibrium and species, sexual reproduction. The probability of each individual to be encountered in a given population was estimated using 100,000 simulated individuals with a threshold value of 0.01.To detect possible first-generation migrants and their source population we used GENECLASS2 . We used2) within four fragments, two medium size fragments and two populations from large fragments. We did not include small fragments due to the lack of enough samples of maternal families (mother and progeny). Plant density was characterized in each population by counting the number of flowering individuals. In each plot, we collected leaf tissue and mature infructescences of maternal plants (n = 33). We germinated seeds and collected the leaf tissue from the emergent seedlings for DNA extraction. The number of maternal families varied between populations and each family between 2 and 11 individual plants (t) and pollen pool structure (ΦFT) we used six highly polymorphic unlinked microsatellite loci  (We established a 50 × 20 m plots (200 ml plants . To estind 1808)  (Suárez-nd 1808)  and analF), multilocus outcrossing rate (tm), single-locus outcrossing rate (ts), and biparental inbreeding due to mating among relatives (tm − ts) using MLTR  sampled by different maternal families using the TwoGener method  and selfing (s), we used the formulaWe estimated the differentiation of pollen pools , the effective number of pollen donors d is the density of reproductive plants (e plants .p < 0.05) were observed in all populations, likely due to heterozygotes deficit rather than null alleles. Across populations, 10 loci displayed a significant heterozygotes deficit and one (1810) displayed a significant excess. Exact test of linkage disequilibrium indicated significant deviations at six out of 55 possible primer pair’s comparisons. Deviations of Hardy–Weinberg equilibrium could be explained by age structure caused by overlapping generations and patchy plant distribution that can create Wahlund-like effects.We did not detect evidence of null alleles. Significant deviations from Hardy–Weinberg equilibrium  and similar among fragments. High genetic diversity values are expected for long-lived perennial plants , ranging −0.066–0.29 for populations, and statistically similar between fragments of different size (range: 0.05 to 0.114), but slightly lower for large fragments . Geographic distances between populations are given in Overall, levels of mean population genetic diversity of ragments . Mantel’Ne) are relatively large in all fragment sizes (Ne = 205.7 (CI [123–480.6])) is not significantly higher than in small fragments (Ne = 119.2 (CI [86.4–176.7])). However, effective population size tends to decrease with reduction of fragment size. The lowest estimated Ne corresponds to one small fragment (19.2 at fragment 3 SM) while the highest value (infinite Ne estimate) corresponds to a large fragment (9 Lrg).Effective population sizes (nt sizes . AverageHO) was lower than expected (Heq), suggesting an absence of recent genetic bottlenecks in fragments of all sizes . All fragments sizes exhibited significant allele deficiency (Under the Stepwise Mutation Model (SMM) and Two-Phase Mutation Model (TPM), the bottleneck test failed to detect a recent genetic bottleneck. The proportion of heterozygotes observed . The PCA representation exhibited a good recovery of the posterior predictive distribution and the observed data , with most genetic variance (96.2%) within populations, while only 3.8% of the variance was among populations within fragment size classes.Populations showed a lack of genetic differentiation despite isolation by fragmentation. The hierarchical AMOVA analysis indicated weak genetic differentiation among all fragments (K = 2 (LnP = −11100.43) and K = 3 (LnP = −10965.43) . For K = 3, cluster I is also composed by almost all populations , while cluster II and III included two main populations   . For K =ctively) .p < 0.05). The network indicates that these communities are dispersed over the landscape without a clear pattern . Conditional genetic distances (cGD) are shown in The Population Graph consisted of 12 populations connected by 24 edges that exhibited a significant conditional covariance. The topology showed that all populations formed a single interconnected network, indicating gene dispersal . No IBD p < 0.01)  occurred from large to both medium and small fragments . However < 0.01) . These r < 0.01) .A. aurantiaca at Los Tuxtlas is variable among populations. Densities ranged from 0.42 to 3.18 individuals/m2; similar values have been reported by 2. The density of reproductive individuals in our study ranged from 0.18 to 1.6 individuals/m2.Plant density of Aphelandra aurantiaca has a mixed mating system (tm = 0.81), altough mating system estimators of each population are not related to fragment size (FT), were high and significantly higher than zero for all populations , with a relatively low number of effective pollen donors (Nep), and short pollen distance movement in this species (Nep) was relatively low  (Fis(s.d.)] were slightly lower for adult maternal plants [0.03(0.15)] than for seedlings [0.05(0.04)]. Furthermore, we detected biparental  inbreeding in all populations (tm − ts ranged from 0.02 to 0.24), that did not differ between populations. The effective pollination neighborhood ranged from less than 0.6 to 14.4 m2, whereas pollen distance movement ranged from half a meter up to 1.1 m  . Averageto 1.1 m . We suggA. aurantiaca despite dozens of generations since the onset of fragmentation in the region of Los Tuxtlas. Regardless of fragment size, populations possess private alleles and high genetic diversity (He = 0.61), similar to those of long-lived perennial herbs (Graptophyllum reticularum (He = 0.31), Graptophyllum ilicifolium (He = 0.43) and Ruellia nudiflora (I = 0.26) (Ne) showed that most populations are effectively large, suggesting that habitat fragmentation has not as yet reduced Ne enough to detect an impact on genetic diversity.Habitat fragmentation has not produced genetic differentiation or immediate reductions in genetic diversity of al herbs , but hig = 0.26) . EffectiRst = 0.037) is supported by a number of analyses, suggesting that A. aurantiaca populations have remained genetically connected. Most genetic variation is contained among individuals within populations (96%) rather than between populations (3%) in fragments of different size. A similar result was obtained for other understory tropical herbs in the same region  ) followed by subsequent vegetation expansion events . In A. aars ago) , and witars ago) . FurtherPhaethornis longirostris, and their consequences on reproductive output of A. aurantiaca.The highest rates of migrants and first-generation migrants derived from the largest and medium fragments, underscore the importance of relatively large forest patches to prevent genetic isolation. Moreover, pollinators may use a series of different fragment sizes to forage, helping to maintain connectivity across the landscape . SpecifiA. aurantiaca, analyses do not allow us to determine which process is the most important contributor to gene flow. Natural gene flow often follows a leptokurtic distribution, implying that most genes move over short distances and only a small fraction move over long distances. Pollen dispersal kernels are often short, resulting in self-pollination or gene exchange among closely related individuals  and seed dispersal . The description of A. aurantiaca as a selfing species with a mixed mating system agrees with values found in other hummingbird-pollinated plants in the Neotropics , suggesting that the potential pollen donors contribute little to Nep. The moderate biparental inbreeding in A. aurantiaca could be explained by the limited seed dispersal and mating among close relatives, which may be due to hummingbirds’ moving among relatively close plants . High plant density may contribute also to increase inbreeding and shorten pollen dispersal distance in A. aurantiaca. However, assessing whether high plant density reduces pollen dispersal makes necessary an extensive sampling of populations with different densities. We suggest caution when interpreting results of TwoGener given that it assumes uniform individual distribution.Forest fragmentation does not seem to affect the contemporary pollen dynamics of  Tuxtlas . The polA. aurantiaca is the result of different factors acting simultaneously. Despite extensive forest fragmentation of Los Tuxtlas rainforest, A. aurantiaca maintains high genetic diversity and low genetic differentiation between populations, suggesting effective gene flow. Habitat fragmentation has not affected the outcrossing rate and pollen dynamics within populations. Demographic history and life history characteristics are important to explain the current pattern of low population structure of A. aurantiaca, rather than recent fragmentation effects. We propose that past demographic dynamics, large effective populations, long distance gene dispersal events, and life history characteristics of this species, such as mixed mating system, overlapping generations, and ability to re-sprout after forest disturbance , ameliorate the effects of fragmentation. In addition, higher gene flow originated from medium and large fragments favour genetic connectivity and confirm their importance as genetic reservoirs and gene sources. Conservation efforts must be directed to preserve these fragments. However, small fragments should not be overlooked, as they may act as stepping stones to increase/maintain connectivity among fragmented populations, especially for species whose gene flow is aided by animals. Our findings should contribute significantly to the development of effective conservation strategies for A. aurantiaca and species with similar mating systems and pollinators.Current genetic structure of 10.7717/peerj.2764/supp-1Table S1Click here for additional data file.10.7717/peerj.2764/supp-2Table S2Click here for additional data file.10.7717/peerj.2764/supp-3Table S3Click here for additional data file.10.7717/peerj.2764/supp-4Table S4P < 0.05 indicates significance of Wilcoxon’s signed –rank test.TPM: two-phase model of mutation; SMM: step-wise mutation model. *Click here for additional data file.10.7717/peerj.2764/supp-5Table S5Bold numbers indicate membership probabilities for individuals in their home population.Click here for additional data file.10.7717/peerj.2764/supp-6Table S6home), where L is the ratio of the likelihood computed from the population where the individual was sampled (Lhome). Fifteen migrants were identified across the different fragment sizes. Average membership probability to resident population was estimated in DAPC.Footnote: Resident fragment refers to the fragment size an individual was found at, fragment source indicates the most likely origin. L was estimated as L= -LOG , using Fast Greedy and geographic coordinates algorithm implemented in NetStruct. Each node represents an individual, with colors representing the three community assigned. (B) SAD Analysis for the network, show the distributions of the SA values for each community.(A) Community detection on Click here for additional data file.10.7717/peerj.2764/supp-10Figure S4Click here for additional data file.10.7717/peerj.2764/supp-11Data S1Click here for additional data file.10.7717/peerj.2764/supp-12Data S2Click here for additional data file.


Leaf samples were collected from a total of 205 trees at six sites of naturally growing olive populations in Israel. The genetic analysis included a multi-locus lineage (MLL) analysis, Rousset’s genetic distances, Fst values, private alleles, other diversity values and a Structure analysis. The analyses also included scions and suckers of old cultivated olive trees, for which the dominance of one clone in scions (MLL1) and a second in suckers (MLL7) had been shown earlier.Naturally growing populations of olive trees are found in the Mediterranean garrigue and maquis in Israel. Here, we used the Simple Sequence Repeat (SSR) genetic marker technique to investigate whether these represent wild var. The majority of trees from a Judean Mts. population and from one population from the Galilee showed close genetic similarity to scions of old cultivated trees. Different from that, site-specific and a high number of single occurrence MLLs were found in four olive populations from the Galilee and Carmel which also were genetically more distant from old cultivated trees, had relatively high genetic diversity values and higher numbers of private alleles. Whereas in two of these populations MLL7 (and partly MLL1) were found in low frequency, the two other populations did not contain these MLLs and were very similar in their genetic structure to suckers of old cultivated olive trees that originated from sexual reproduction.sylvestris. The similarity in genetic structure of these two populations with the suckers of old cultivated trees implies that wild trees were used as rootstocks. Alternatively, trees at these two sites may be remnants of old cultivated trees in which the scion-derived trunk died and was replaced by suckers. However, considering landscape and topographic environment at the two sites this second interpretation is less likely.The genetic distinctness from old cultivated olive trees, particularly of one population from Galilee and one from Carmel, suggests that trees at these sites might represent wild var. The online version of this article (doi:10.1186/s12870-016-0947-5) contains supplementary material, which is available to authorized users. Prunus dulcis and P. orientalis) [Vitis vinifera subsp. vinifera and V. vinifera subsp. sylvestris) [Malus domestica and M. sylvestris) [Olea europaea [The domestication of crop species started 13,000 to 10,000 years before present by gradual selection of desirable traits and of adaptations to agricultural environments [entalis) , grapes vestris)  and applvestris) . In addivestris) , as showeuropaea , 8. BothOlea europaea subsp. europaea var. europaea originated from wild var. sylvestris (Mill) Lehr by artificial selection from wild populations [It is generally accepted that the cultivated olive ulations . Recentlulations . Howeverulations .sylvestris, often called ‘oleaster’, resembles cultivated olives except for its shrubby growth and smaller leaves and fruits [sylvestris is often questionable [sylvestris and could show that current conditions are suitable for its presence in the southwest Levant, i.e., modern Israel.Wild var. d fruits . These ctionable . Howevertionable  could idsylvestris in the Mediterranean area. Supporting this hypothesis, other studies using DNA [sylvestris requires caution in view of the possibility of gene flow between cultivated and wild populations.Studies employing different molecular marker techniques to investigate the relationship between cultivated and wild olives and to map the distribution of wild olives in the Mediterranean area have been conducted before, e.g. –22. In ssing DNA  and allosing DNA  variatiosing DNA . The stusing DNA  as well sing DNA , 26 thussylvestris in the region perhaps is not likely and needs to be studied. Several studies included samples of naturally growing olive trees from the southeastern Mediterranean to infer the distribution and genetic diversity among population of ‘oleaster’ around the Mediterranean [sylvestris in the west Mediterranean [sylvestris potentially could have enormous importance in breeding programs aiming at the introduction of wild alleles conferring valuable traits that were lost during the domestication process [In Israel, naturally growing populations of olive trees can be found in the Mediterranean maquis and garrigues of the Carmel and western Galilee mountain ranges. Considering that it is likely that olives have been cultivated continuously in the area for at least 6,000 years , 27–30, erranean , 19, 25.erranean  but ques process . On this process , as recesylvestris, feral (var. europaea) or perhaps as abandoned groves, we used SSR markers for the analysis of six naturally growing olive populations sampled at close to far distances from extant cultivated groves. In a previous study we already used a multi-locus lineage (MLL) analysis with the same SSR markers to infer cultivar identity in the same region [sylvestris), if such exist, will be genetically different from cultivated olives and feral populations. On this background, a population of naturally growing olives from outside the hypothetical natural distribution range of wild olives in the region [To determine the identity of naturally growing olive populations in Israel as wild var. e region . We coule region  was inclThe genetic analysis included 205 naturally growing olive trees sampled in six populations in Israel Table ; Fig. 1.In comparison, samples of suckers (collected at the base of tree trunks) and scions, from presumably grafted trees, belonged to 141 and 18 MLLs, respectively  in Israel was here investigated by using SSR variation in naturally growing populations. Based on a Bayesian analysis of SSR markers, it has recently been suggested that 38 trees sampled outside cultivated groves in Israel are presumably feral [oleaster’ trees that were collected within  and outside  the putative distribution range of var. sylvestris, it did not include the most common cultivars in the southeast Mediterranean region, which renders the conclusions rather hypothetical. Here, a genetic comparison between grafted old olive trees (i.e. 288 scions and 281 suckers) and naturally growing olive trees from different populations in Israel provided more comprehensive information on their identity as feral, cultivated or genuinely wild, and furthermore allowed us to obtain evidence for the possible source of rootstocks of grafted old trees.The existence of wild olive trees , two trees were assigned to site specific MLLs, and MLL7 was present in one individual , where wild var. al Table . In addial Table , and theal Table . Thus, aal Table , and hasThe remaining four populations  are very different in their genetic composition from BGR and ZUR. Trees from IDM  and the three Carmel populations  were found to be similar in terms of relatively high genetic diversity values Table , and gensylvestris). Similarly, a recent genetic survey of scions and rootstocks of old olive trees in the Iberian Peninsula suggested that old olive trees were grafted on wild growing trees [First, IDM and NOR should be considered wild populations. This interpretation would confirm previous reports that indicated that supposedly wild populations from Carmel and Galilee genetically resemble wild olive populations from Turkey and Syria , 21, 26.ng trees . Based ong trees  additionSecond, the interpretation of the similarity in genetic structure between IDM and NOR on the one hand and suckers of old cultivated olive trees on the other hand can be reversed: such interpretation would imply that the naturally growing trees at IDM and NOR are remnants of old cultivated trees in which the scion-derived trunk died and was replaced by suckers. If this interpretation of IDM and NOR as essentially feral should be correct, populations BOR and OFR may represent an intermediate stage in the transition of orchards into naturally growing populations of feral origin. However, as naturally growing trees at NOR and IDM grow in conditions that are unsuitable for agriculture  growing in natural surroundings and resembling the shrubby phenotype of southeastern Mediterranean var. sylvestris; identification of plants was based on the Analytical Flora of Israel [sylvestris) is not included in the Red List of the Israeli Flora [Naturally growing olive trees can be found sparsely in Israel in natural habitats surrounding cultivated groves and residential areas. Surveys were conducted in the Carmel and Galilee to locate populations of at least 20 olive trees , and grow in patches as small shrubs , the sampled population grows remotely from contemporary olive groves  following the manufacturer’s protocol. Simple Sequence Repeat (SSR) markers used in olive trees –48 had pAnalysis of multi-locus genotypes (MLGs) and grouping of MLGs into multi-locus lineages (MLLs) was done using Genotype 1.2 . To estiV.2.3.4 [Structure V.2.3.4  was usedV.2.3.4 , 10 indeV.2.3.4 .


Xenostrobus securis) recent invasion in Europe (two-year old), while in older introductions such epigenetic signature of invasion was progressively reduced. Decreased methylation was interpreted as a rapid way of increasing phenotypic plasticity that would help invasive populations to thrive. This epigenetic signature of early invasion was stronger than the expected environmental signature of environmental stress in younger populations sampled from ports, otherwise detected in a much older population (>90 year old) of the also invasive tubeworm Ficopomatus enigmaticus established in similar locations. Higher epigenetic than genetic diversity found in X. securis was confirmed from F. enigmaticus samples. As reported for introduced plants and vertebrates, epigenetic variation could compensate for relatively lower genetic variation caused by founder effects. These phenomena were compared with epigenetic mechanisms involved in metastasis, as parallel processes of community  and organism (cancer) invasions.Epigenetics, as a DNA signature that affects gene expression and enables rapid reaction of an organism to environmental changes, is likely involved in the process of biological invasions. DNA methylation is an epigenetic mechanism common to plants and animals for regulating gene expression. In this study we show, for the first time in any marine species, significant reduction of global methylation levels during the expansive phase of a pygmy mussel ( This is a natural response to changes in abiotic factors15675Due to its importance in adaptation mechanisms, epigenetics must be involved in the process of biological invasions facilitating the establishment of exotic organisms in recipient ecosystems11131411To date, the available data about invasion epigenetics refer predominantly to terrestrial species. The epigenetics of marine invasions is largely understudied yet. Biological invasions follow a sequential process of arrival, establishment, expansion, and eventually accommodation within the recipient ecosystem1920Xenostrobus securis. This species is invading the coastal waters of Japan231825Here we present a proof-of-concept study aimed at challenging our hypothesis of epigenetic signature in invasive populations, based on the New Zealand pygmy mussel Ficopomatus enigmaticus from markedly different environments. This is a worldwide well-established marine invader2728To check if the results may be generalizable we have also analyzed a few individuals from comparatively older introduced populations of Australian tubeworm X. securis and F. enigmaticus specimens analyzed in this study were all adults, collected in winter time . The sampling sites represented different environments , published national and regional reports and research papers. The date of the first record of the species was verified with the published literature resources and on-line databases  were identified de visu and preserved in ethanol for further genetic and epigenetic analysis. Total DNA was extracted from a small piece of foot muscle with the E.Z.N.A Mollusc DNA kit , following manufacturer´s instructions. Five individuals of F. enigmaticus were sampled from each site and preserved in ethanol as well. DNA was extracted from the whole body employing a method based on silica gel columns , following manufacturer’s instructions.The two species here studied have a short planktonic larval stage, are tolerant to wide salinity, temperature and pollution ranges and can disperse through shipping pathway27® 2.0, and normalized to 100 ng/μ for subsequent analysis.The tubes with DNA samples were stored at 4 °C for immediate analysis and aliquots were frozen at −20 °C for long-time preservation. Isolated DNA was quantified using a fluorometric method with QubitX. securis samples using the universal primers designed by Geller et al.F. enigmaticus due to the absence of COI gene references in publically available databases  at the moment of this study , using the primers and protocol described in Distel et al.et al.DNA barcoding was performed for each individual to verify the identity of the species and for the further reference. The mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified from PCR products were examined on 2% agarose gel stained with SimplySafeTM . Positive amplicons (evidenced by clear single band of the expected size) were sequenced by Macrogen Inc. (The Netherlands) with ABI3730xl DNA sequencer (Applied Biosystems).www.ncbi.nlm.nih.gov/).Obtained DNA sequences were edited with BioEdit v7.2.5et al.EcoRI/HpaII or EcoRI/MspI. Both enzymes (MspI and HpaII) recognize and cleave CCGG target sequences, but cleaving by HpaII is blocked when the inner or outer C is methylated at both strands; while cleaving in MspI is blocked when the outer cytosines are fully or hemi-methylated; cleaving in both enzymes is blocked when both cytosines are methylated –and/or when nucleotide polymorphism occurs in the restriction target thus the sequence is not recognized by the enzymesThe methylation-sensitive amplified polymorphism approach or MSAP was used to detect polymorphism in DNA methylation patterns. The protocol for global methylation analysis was conducted following Díaz-Freije EcoRI-AAG, HpaII-TCC and EcoRI-AAG, HpaII-TAC. HpaII primers were end-labeled using 6-FAM reporter moleculeF. enigmaticus and five X. securis samples (XAv1–5) from the Bay of Biscay were analyzed again with the same protocols.The resulting DNA fragments were ligated with linkers and PCR amplified using two primer combination: EcoRI-HpaII and EcoRI-MspI primer combinations, yielding a new score matrix according to the methylation state. The type of epigenetic variation detected with MSAP loci was categorized following Salmon et al.MSAP individual and population profiles were analyzed using the R package msap v.3.2.2.Type I = restriction site no methylation: both enzymes cut at the restriction site,-Type II = methylation of internal C: HpaII does not cut and MspI does cut,-Type III = methylation of external C or hemimethylation: HpaII does cut and MspI does not, and-Type IV = hypermethylation or mutation in restriction site: neither enzymes cut.-et al.The study developed by Fulnecek and KovarikEvery locus was classified as either Methylation-susceptible loci (MSL) or Non-methylated loci (NML), depending on whether the observed proportion of methylated states across all samples exceeded a user-defined error rate-based threshold . Only those fragments showing polymorphism, with at least two occurrences of each state, were used for subsequent analysisThe following analyses were performed in MSAP using the R package msap v.3.2.2.ST values , and principal coordinates analyses (PCoA) followed by analysis of molecular variance (AMOVA)44The epigenetic (MSL) and genetic (NML) differentiation among populations and between pairs of populations was assessed by means of ɸThe mean proportion of methylated loci was compared among populations using classic ANOVA. Analysis of residuals was done and normality was checked with Shapiro-Wilk test; if it was significant or not interpretable Welch F test was performed instead of ANOVA. In that case clear outlier data were removed for pairwise analyses. Medians were compared among populations with Kruskal-Wallis test. Post-hoc pairwise comparisons were made with Tukey’s honest significance tests for means, and Mann-Whitney for medians. Software PASTEcoRI-HpaII and EcoRI-MspI profiles as standard AFLPs using the option meth implemented in the R package msap. This is a second measure of genetic variation that scores all the loci, not only NML. Consistent results for the two measures would reinforce the conclusions about genetic differences between populations. For confirmation ɸST values were also obtained and AMOVA performed for population pairs using GenAlEx software44Finally, genetic differences between pairs of populations were also assessed by comparing X. securis and 15 F. enigmaticus adults, from three different introduced populations for each species, were barcoded for species confirmation and analysed for AFLP and MS-AFLP variation. The most frequent haplotypes of COI and 18 S rRNA gene sequences obtained from the analyzed X. securis and F. enigmaticus individuals (648 and 656 nucleotides respectively), are available in NCBI GenBank database with the accession numbers KX129960-KX129962 and KX129957-KX129959. Comparison of the acquired sequences with existing references in nucleotide databases confirmed unambiguously the species identity of the individuals analyzed.In total 82 X. securis samples . The genetic differentiation based on all the AFLP loci provided higher statistical significance for pairwise ɸST, as expected from the higher number of loci examined and distant populations of likely multiple originX. securis populations (data not shown). Significant differences occurred between F. enigmaticus FMed and FNZ samples , but not between FAtl and FMed , neither between FCant and FNZ , probably due to small sample sizes.Detected AFLP variation was considerable in the two species. In total 380 AFLP loci were found in the X. securis from different locations is summarized in There was considerable variation in MSL between individuals within each population. The proportional occurrence of each type of methylation for For the individual number of methylated loci in the analyzed mussels, Shapiro-Wilk test  was not statistically significant, meaning the distribution did not deviate significantly from normality. The mean proportion of methylated MSL loci exhibited strong statistical significance among groups  and the same occurred for medians . From a total variance of 42.323, 14.47% was due to variation among populations. For genetic variation (NML) the populations were also globally significantly different , but the variance due to among-population variation was lower, 10.29% of the total variance . Epigenetic diversity (at MSL) was indeed significantly higher than genetic diversity (NML), Shannon’s index being I = 0.5754 (SD = 0.1189) and I = 0.2510 (SD = 0.1483) respectively, W = 31834 (P < 0.0001).On the other hand, global epigenetic differences (i.e. in MSL) were detected among X. securis dataset could be visualized from the PCoA (X. securis from the Mediterranean lagoon (XMed) and Atlantic international port (XAtl) samples overlapped partially, with the new expansive population from the Bay of Biscay port (XCant) clearly differentiated. For NML . Despite not significantly difference for epigenetic variation, these two populations did differ significantly for their genetic variation  and 83 NML (99% polymorphic). As in the case of X. securis, more fully methylated –or mutation in restriction site- (Type IV) than hemimethylated (Type III), internally methylated (Type II) and unmethylated loci (Type I) were detected  than for NML (genetic). The average Shannon’s diversity indices were I = 0.5869 (SD: 0.1055) and I = 0.2843 (SD: 0.0873) for MSL and NML respectively, significantly different according to Wilcoxon signed rank test . According to lower genetic than epigenetic diversity, the PCoA for this species . Accordingly, significant difference did not occur between any pair of samples . The pairwise differences at MSL between the more recently introduced Napier international port population (FNZ) and the other two samples were significantly different .The three population samples analyzed did not exhibit significant genetic differences among them based on the 83 NML loci examined  were similarly methylated. These populations (XAtl and XMed), with a significant differentiation in genetic variation and their very different stress level, were expected to be significantly different for global methylation4X. securis is likely under expansion in the northwest Iberia sampling regionOur study is the first reporting changes in methylation patterns of invertebrates likely associated with the process of biological invasions. In invasive F. enigmaticus analysis, the results pointed to the same direction. For this species the older population, sampled from a small fishing port, seemed to be more methylated than populations from differentially disturbed settings . Although for quantitative tests (proportion of methylated loci) statistical significance of p < 0.05 was not reached, highly significant differences were found for ɸST analysis between the more recent population established in the New Zealand port and the two older ones  even adult individuals would exhibit a global signature of decreased methylation - compared to those from earlier established populations.Taken together, the results of this study would support our departure hypothesis of relaxed methylation control in early invasions. All the individuals here analyzed were adults, thus ontogenetic methylation changes can be reasonably excluded as a reason for global methylation differences. The phenomenon of reduced methylation could be a function of the invasion phase (expanding populations versus the accommodated ones), and would have a role in invasion processes. At organism level, in vertebrates DNA methylation switches-off gene expression during the development but can also arise when ageing, perturbing the animal organism and causing disease482751X. securis analyzed here were reported from the respective regions at least one decade ago53F. enigmaticus, especially the older introduced population in Bay of Biscay –although not the more recent introduction in New Zealand, could be an example. Epigenetic variation may thus be linked to the ‘age’ of the introduced population1314The clear understanding of epigenetic contribution to the species invasiveness is difficult from observational studies. There is poor knowledge on the current status of the local invasive populations  and the details of the invasion history . Yet, in the present case there is no doubt XCant population is very young and in early expansive phase1825X. securis and F. enigmaticus. Not a minor discovery, we provided here the first evidence of methylation in the two studied species. To date, regulation of gene expression through DNA methylation has been described for other molluscs55F. enigmaticus from the three analyzed populations did not differ much. It differed more among X. securis samples. Still, genetic diversity was clearly and significantly lower than epigenetic diversity in both species. Epigenetic differences with similar genetic variation were reported in a few studies of introduced populations of plants and vertebrates1314Another interesting finding in our study was significantly higher epigenetic than genetic diversity in both highly invasive species, This proof-of-concept study could open new perspectives for better understanding marine biological invasions, their ecology and intrinsic mechanisms. The epigenome analysis of donor and introduced populations along with the population status assessment  is highly desirable. Discovering the key genes involved in adaptation mechanisms of marine invaders, as well as their epigenetics would provide useful information for prioritizing risks associated with particular invasive species and undertaking adequate response measures.How to cite this article: Ardura, A. et al. Epigenetic signatures of invasive status in populations of marine invertebrates. Sci. Rep.7, 42193; doi: 10.1038/srep42193 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Taenia saginata is a tapeworm found in cattle worldwide. Analysis of genetic diversity in different geographical populations of T. saginata not only helps to understand the origin, transmission and spread of this organism, but also to evaluate the selection pressures acting on T. saginata and how it is responding to them. However, there are few reports of the genetic variability of T. saginata populations in different regions of the world, including Lao PDR and Thailand. We report the genetic diversity of T. saginata populations in Lao PDR and northeastern Thailand together with sequences of T. saginata from other countries deposited in GenBank.cox1 sequence analysis revealed that 15 and 8 haplotypes were identified in 30 and 21 T. saginata isolates from Lao PDR and northeastern Thailand, respectively. Fifty-three haplotypes were identified from 98 sequences. Phylogenetic tree and haplotype network analyses revealed that global isolates of T. saginata were genetically divided into five groups . Taenia saginata isolates from Lao PDR and northeastern Thailand belonged to either Group A or B. Taenia saginata from western Thailand clustered in groups C1, C2 and D, and populations from the northeast and western Thailand were found to be genetically distinct. Taenia saginata isolates in Lao PDR and Thailand were also found to be genetically diverse but the degree of genetic differentiation was low.Mitochondrial Taenia saginata populations from Lao PDR and northeastern Thailand are genetically distinct from the population in western Thailand and it is proposed that T. saginata has been dispersed by different transmission routes in Southeast Asia.The online version of this article (doi:10.1186/s13071-017-2079-7) contains supplementary material, which is available to authorized users. Taenia saginata, Taenia solium or Taenia asiatica [Taenia saginata, known as the beef tapeworm, has a global distribution which includes Thailand [T. saginata have an active discharge of gravid proglottids and occasionally present with epigastric pain, nausea, weight loss and poor appetite [T. saginata can cause economic losses in terms of food production [Taeniosis is a parasitic zoonosis caused by infection with asiatica , 2. TaenThailand , 4 and LThailand , 6. The oduction –10 and toduction .T. saginata is very limited. Mitochondrial DNA has been used as a genetic marker for detection of intraspecific variation or cryptic species [c oxidase subunit 1 gene (cox1) is a commonly used marker for studying geographical populations of T. saginata [T. saginata isolates from northern and northeastern regions of Thailand based on the partial cox1 sequences (924 bp). Although the distribution range of T. saginata is known for Lao PDR, which borders Thailand [T. saginata has been reported to date.Information on the global genetic diversity of  species , and cytsaginata , 13, 14.Thailand , 6, no ecox1 sequences  of 51 T. saginata isolates from Lao PDR and northeastern Thailand. We also included 47 sequences already deposited in GenBank, to better understand the genetic diversity and character of T. saginata populations in these countries.In this study, we performed molecular-phylogenetic tree and haplotype network analyses based on the complete n = 30) and northeastern Thailand (n = 21). In Lao PDR, specimens were obtained from patients with taeniosis in Xebungfai District, Khammouane Province, central Lao PDR  during a parasitological survey conducted by the Lao Ministry of Health in June 2015 [Opisthorchis viverrini) in Lao PDR [The 51 taeniid samples examined in this study were collected in Lao PDR  was amplified by PCR using the primers Taenia trnW/F (5′-GTT ATG TTA GAC TAG ATG TTT TCA-3′ for the Taenia transfer RNA-Try gene) and Taenia rrnl/R  [Taq  was used as a DNA polymerase. PCR amplification consisted of an initial denaturation step of 98 °C for 30 s, followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 90 s, with a final cycle of 72 °C for 5 min. Amplicons were cleaned using ExoSAP-IT  and used as templates for direct DNA sequencing. Samples for DNA sequencing were prepared using a BigDye Terminator Cycle Sequencing Ready Reaction kit  by primer walking and run on a 3730 × l DNA Analyzer .Genomic DNA samples were extracted from individual taeniid proglottids using a DNeasy Blood & Tissue kits  according to the manufacturer’s instructions. The complete it gene) . TaKaRa T. saginata cox1 sequences were aligned using ClustalW [www.fluxus-engineering.com).The ClustalW  and a phClustalW  and suppClustalW . A netwoT. saginata cox1 sequences from this study and GenBank were calculated: genetic variability at polymorphic sites between populations (S), haplotype numbers (h), haplotype diversity (Hd), nucleotide diversity (π), population mutation rates based on the number of segregation sites (θω) and mean number of pairwise differences (θπ) [Fs tests [The following measures for population genetics based on complete ces (θπ) –25 were ces (θπ) . Statistces (θπ)  and Fu’sFs tests .T. saginata based on the diagnostic nucleotide (= adenine) at position 723 of cox1 [T. saginata sequences deposited in GenBank. The complete sequence analysis revealed that 15 and 8 haplotypes were detected in 30 and 21 T. saginata isolates from Lao PDR and northeastern Thailand, respectively. Fifty-three haplotypes were identified in 98 sequences, which included 47 sequences from GenBank. The haplotypes, haplotype frequencies, and accession numbers for the 98 T. saginata cox1 sequences are presented in Table All of the 51 taeniid isolates collected in Lao PDR and northeastern Thailand were identified as  of cox1 , althougT. saginata could be genetically divided into five groups: A, B, D and two subgroups (C1 and C2)  from western Thailand, Japan, Korea, Indonesia, Djibouti and Ethiopia, as well as other haplotypes from China , Mongolia (H35), Ecuador (H27), Ethiopia , and unspecified African countries (H28 and H42). Haplotype H29 was predominant in subgroup C2 and was composed of isolates from western Thailand, Cambodia, China and Belgium, and other haplotypes were from China , western Thailand (H32 and H33), Indonesia (H34), Nepal (H40), Ecuador (H36) and Brazil (H37). Subgroups C1 and C2 were closely linked and formed firework-type structures around the two predominant haplotypes H24 and H29, respectively. Interestingly, Group D was composed of a few haplotypes  from Kanchanburi Province, western Thailand and Bangkok, and T. saginata from northeastern and western Thailand were genetically distinct populations.In addition to phylogenetic tree analysis, the median-joining network also revealed that her Fig. . Group AT. saginata populations from Lao PDR and northeastern Thailand, several population genetics indices based on the complete cox1 sequences are presented in Table T. saginata haplotypes identified from Lao PDR and northeastern Thailand was 15 and 8, respectively, and the number of segregation sites was 20 (1.23%) in Lao PDR and 10 (0.67%) in northeastern Thailand. The overall number of haplotypes was 53 out of 98 sequences and 57 sites (3.79%) were segregated. The haplotype diversity (Hd) was as high as 0.926 ± 0.038 and 0.819 ± 0.064 in T. saginata populations from Lao PDR and northeastern Thailand, respectively. The global Hd (0.968 ± 0.020) was also high in T. saginata including the western Thai population (0.868 ± 0.076). In contrast, the nucleotide diversity (π) was as low as 0.00252 ± 0.0003 and 0.0015 ± 0.00034 in T. saginata from Lao PDR and northeastern Thailand, respectively. Overall the nucleotide diversity of haplotypes of T. saginata worldwide was as low as 0.00381 ± 0.00021, including the western Thai population (0.0236 ± 0.00041). θπ was less than θω in T. saginata populations from Lao PDR and northeastern Thailand. Neutrality in T. saginata populations from Lao PDR and northeastern Thailand was not significant by Tajima’s D test  and Fu’s Fs test . However, a pairwise Fu’s Fs test showed a significant value  in the Lao population. In haplotypes overall, Tajima’s D value  and a Fu’s Fs value  were significant.To estimate the genetic diversity in T. saginata infection in people in central Lao PDR and northeastern Thailand. We explored genetic diversity based on the complete cox1 sequences of T. saginata populations from these regions and also included T. saginata data available from GenBank. To our knowledge, this is the first molecular identification and genetic diversity analysis of T. saginata from Lao PDR.In this study, we present molecular evidence of T. saginata could be genetically divided into five groups and that T. saginata populations from Lao PDR and northeastern Thailand belonged to Group A and Group B. Subgroup C1 included the western Thai population, and Group D was also composed of western Thai isolates, but was differentiated from subgroup C2. Taenia saginata populations from northeastern and western Thailand are geographically and genetically isolated and, to date, there is no evidence that they are sympatrically distributed in Thailand. It has been suggested that haplotype H11 may be an ancestral haplotype and that other haplotypes in the Lao population have diverged from it  and Ecuadorian haplotypes (H36), and many of the global T. saginata isolates are unclear in Subgroups C1 and C2. Further study using other molecular markers is necessary to explain the complex phylogenetic relationships that exist between these T. saginata populations.Phylogenetic tree and haplotype network analysis both revealed that h and π values in T. saginata from northeastern Thailand were lower than those from the Lao population. θπ was less than θω in T. saginata populations from Lao PDR and northeastern Thailand. While the results of Tajima’s D test were not significant in the Lao and northeastern Thai populations, Fu’s Fs test showed that the Lao population is genetically differentiated . This discrepancy between the two statistical tests is considered to explain why more haplotypes were detected compared with the number of segregated sites, and indicated that the genetic diversity in the Lao population was high. However, compared with other global T. saginata isolates, the degree of genetic differentiation in the Lao and northeastern Thai populations was low. This also supports the finding that Groups A and B are linked to each other, in particular, haplotype H6 in Group B was linked to haplotype H30 from China in Subgroup C1. Group B formed a firework-like structure, which was differentiated by the small base substitution rate around predominant haplotypes H3 and H11. Taenia saginata populations are dispersed in different geographic localities where genetic variation is influenced by the genetic bottleneck effect that arises from decreases in the population size. However, because the sample-size that was examined in this study was small, further study using more samples from around the world is necessary to more accurately clarify the effect of such genetic events in geographically different populations.The T. saginata isolates from northern and northeastern Thailand based on partial cox1 sequences (924 bp). In their study, haplotypes A and B were predominant, and haplotype A was found in ten countries, including Thailand. These authors proposed that haplotype A shared the same ancestor as T. saginata populations from around the world. However, the findings of our network analysis differ from those in their report because the complete cox1 sequence of their haplotype B is not available, and the haplotype in our study corresponding to their haplotype B has not been identified. Nonetheless, their haplotype A is identical to haplotype H29 in our study, but it was only found in Thailand, Belgium, China and Cambodia as shown in Fig. cox1 sequences because we analyzed the complete sequence , whereas only 924 bp was analyzed previously [Anantaphruti et al.  identifiuences 92 bp. In tT. saginata populations are distributed in Lao PDR and Thailand is probably closely associated with the artificial movement of cattle as an intermediate host. The artificial movement of cattle may have played an important role in the dispersal of liver fluke Fasciola species in Asia [T. saginata populations in Lao PDR and Thailand, the following scenario is possible: T. saginata originating in southern provinces of China might have been transmitted, via northwestern Thailand and Myanmar, to the western regions of Thailand (western route), and via northern Lao PDR and Vietnam to northeastern regions of Thailand (eastern route). Network analysis using more T. saginata specimens from different Asian countries, including China, is necessary to verify this scenario and to provide a more detailed estimate of the genetic diversity of T. saginata. The sample size in population genetics analysis can modulate the neutrality [The fact that genetically different  in Asia , 31. To utrality  and the While taeniosis has a low impact on human health, it is an important zoonosis that can cause serious economic losses in cattle –11. ConsTaenia saginata from Lao PDR and northeastern Thailand showed high haplotype diversity, but the degree of genetic differentiation was low. Lao and northeastern Thai populations were also found to be genetically distinct form the T. saginata population in western Thailand, suggesting that T. saginata is spread by different transmission routes in Southeast Asia.