Phytophthora infestans isolates collected from Fujian, China and to determine the role of sexual reproduction in the dynamics. Although the pathogen populations in this region were dominated by self-fertile genotypes, sexual reproduction only occurred occasionally and its contributions to the population genetic structure of P. infestans and epidemics of late blight in the region were limited. Only 49 genotypes were detected among the 534 isolates assayed and the pathogen populations displayed significant heterozygosity excess. Hierarchical analysis revealed that 21.42% of genetic variation was attributed to the difference among sampling years while only 4.45% was attributed to the difference among locations, suggesting temporal factors play a more important role in the population genetic dynamics of P. infestans than spatial factors in this region. We propose that clonal reproduction, combined with founder effects and long distance dispersal of sporangia, is responsible for the observed pattern of spatiotemporal dynamics in P. infestans.The mating system plays an important role in the spatiotemporal dynamics of pathogen populations through both its direct and indirect impact on the generation and distribution of genetic variation. Here, we used a combination of microsatellite and phenotypic markers to investigate the spatiotemporal distribution of genetic variation in  The mating system is one of the main evolutionary factors driving the spatial and temporal dynamics of genetic variation in pathogen populations. In nature, pathogens may vary in mating systems spatially and temporally and the ability to engage in various mating systems may be a ‘bet-hedging’ strategy that enables pathogens to adapt better to a range of biotic and abiotic environmentsMating systems affect the amount and distribution of genetic variation within pathogen populations. Clonal or inbreeding populations are expected to exhibit low genetic variation and a significant degree of non-random association among unlinked loci. In contrast, outcrossing populations are expected to display high genetic variation and random associations among neutral loci due to the re-assortment of unlinked genes during meiosis468P. infestans, the cause of late blight disease, is a notorious plant pathogen responsible for the Irish potato famine in the 1840s, and is still the most devastating disease of potato and tomato worldwide1013The oomycete P. infestans is considered to be a heterothallic oomycete in which the occurrence of sexual reproduction requires the presence of two opposite mating types (A1 and A2)P. infestans outside of Mexico reproduced asexually and was dominated by a single clonal lineage of A1 mating type termed HERB-1 and then US-1P. infestans globally17181920P. infestans populations due to the continuous rearrangement of existing alleles or generation of new alleles through intragenic recombination. Regular sexual reproduction in heterothallism can also homogenize the frequencies of A1 and A2 mating types attributed to balancing selectionP. infestans still reproduces primarily in a clonal manner and sexual reproduction is rare252627Despite the existence of self-fertile genotypes17181920P. infestans has high evolutionary potential in nature31P. infestans genotype called 13_A2 was first detected in the Netherlands in 2004 at a very low frequency333413_A2Although exhibiting low genetic variation overall, temporal analysis of its population dynamics indicates that Solanum crops and its production primarily relies on seed tubers imported from other parts of the country. This combination of landscape structure and production system may have important impacts on the population genetic dynamics and evolutionary trajectory of P. infestans. To understand the population genetic structure of P. infestans in Fujian and infer potential evolutionary mechanisms responsible for the structure, we performed a three-year molecular and phenotypic surveys in the regions. Therefore. The main objectives of this study are: i) to estimate genetic and phenotypic variation of P. infestans; ii) to investigate spatiotemporal population genetic dynamics of P. infestans; and iii) to determine whether sexual reproduction and genetic drift are responsible for the observed spatiotemporal genotypic diversity in this population of P. infestans.Fujian is a mountainous province located in Southeastern China and is classed as a winter potato production zone. Potato in this region always rotates with rice or other non-P. infestans were collected at random from 8 commercial fields located in Fujian Province in the early stage of epidemics during 2010–2012 (P. infestans isolates amplified with eight SSR markers (He) was similar across all populations, ranging from 0.37 in LH to 0.46 in ZZ  formed oospores with the A2 reference strain and were considered to be A1 mating type. The remaining 337 isolates (63.11%) formed oospores by themselves as well as with the A1 and A2 reference strains and were considered as self-fertile. No A2 mating type was detected in any of the populations from Fujian Province . Multilost and 2nd components accounted for 51.60% and 36.71% of total genetic variability, respectively  and log (Nm) among all 8 geographic populations was 0.11, which was not significant  performed on the eight populations indicated that 21.42% and 4.45% of the genetic variation was attributed to variations among years and among locations within year, respectively . At the P. infestans27P. infestans populations sampled before 2007Zymoseptoria tritici (Mycosphaerella graminicola), Zhan and colleagues assayed the pathogenicity of nearly 200 isolates sampled from four continents and found a consistent pattern of higher aggressiveness in one mating type over the otherSince its first report outside Mexico, the possible center of origin of P. infestans play an important role in the determination of its mating typesP. infestans. The self-fertile isolates detected in Gansu province, China can also produce oospores when they were inoculated onto potato leaves18We found that more than two-third of the isolates in the current study were self-fertile. It has been reported that environmental factors such as the types of media used to culture P. infestans populations from Fujian would have a moderate to high genetic variation, as reported in North and West Europe29P. infestans populations, but its contributions to the population genetic structure of the pathogen and epidemics of late light in the region is limited.Self-fertile isolates can not only produce oospores by themselves but also with the A1 and A2 isolates. It is thus expected that P. infestans than spatial factors in Fujian. This pattern of spatiotemporal population dynamics in P. infestans was also evident by comparing the population genetic structure of the pathogen from the same location but different years. XPI and XPII were sampled from two nearby fields planted with the same potato cultivar at one year interval but high genetic differentiation in SSR frequency, no identical genotypes, and different mating types were found in the two populations.Hierarchical analysis revealed that more than 20% of genetic variation was attributed to the difference in sampling years, while only ~5% was attributed to the difference in locations, suggesting temporal factors make more important contributions to the population genetic dynamics of P. infestans may account for the observed pattern of spatiotemporal population structure. Agriculture is characterized by a recurrent expansion (growing season) and contraction (harvest season) of both host and pathogen populations8P. infestans in Fujian is expected to be small and its genetic drift caused by the seasonal population contraction is expected to be severe in this region. Fujian is located in a subtropical temperature zone with long and hot summers. Its air temperature in July and August can reach 40 °C or higher and last for several hours in a day. At the night time, the air temperature in this season usually is not less than 25 °C. Such environmental condition further escalates genetic drift in P. infestans, causing rapid changes in population genetic structure over time. However, due to the potential for long distance movement of P. infestans sporangia42Genetic drift in terms of founder effect combined with long dispersal ability of P. infestans. Most potato seed tubers in Fujian are imported from other parts of China. The seed tubers are usually purchased from different producers and their quality is regulated poorly, bringing different sets of pathogen populations from different parts of the country each year. Natural populations usually display a negative correlation between the extent of gene flow and geographic distance, defined as isolation by distanceP. infestans in Fujian.Founder effects generated by infected seed tubers may also contribute to the observed pattern of population genetic dynamics of P. infestans in this region mainly reproduces asexually and the contribution of sexual reproduction to the population genetic structure of the pathogen is limited. This epidemic mode of reproduction that combines many cycles of asexual propagation with fewer cycles of sexual reproduction allows P. infestans to preserve favorable allele combinations that are well adapted to existing hosts and environments while retaining its ability to generate novel allele combinations when hosts and environments are changing, breaking the effect of Muller’s ratchetP. infestans can be regulated through good agricultural practices, strict quarantine procedures and public awareness of biosafety and biosecurity. To effectively control late blight, it is important to curb human-mediated gene flow by using clean seed and plant materials in addition to the dynamic use of resistance genes and chemicalsThough dominated by self-fertile genotypes, our analyses indicate that P. infestans were collected at random from 8 commercial fields located in Fujian province in the early stage of epidemics during 2010–2012 . The lyophilized mycelia were ground to powders with a mixer mill . Total DNA was extracted using Plant gDNA Miniprep Kit  according to the manufacturer’s instructions. The genomic DNA was suspended in 200 μL ultrapure water and stored at −20 °C until use.Mycelia (~100 mg) were obtained by culturing P. infestans isolates was amplified with eight SSR markers . Pi4B and PiG11 markers were developed by Knapova and GisiGenomic DNA from each of the P. infestans genomic DNA (~20 ng), 12.5 μL of 2× PCR Buffer Mix , 1.0 μM each of forward and reverse primers in a 2720 thermal cycler  with the following conditions: initiated with a cycle of 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 25 s at 56 °C (for PiG11), 57 °C (for pi56), 58 °C  or 61 °C (for Pi89) and 60 s at 72 °C, and finished with an elongation cycle of 5 min at 72 °C. PCR products from the eight markers were mixed into two groups with PiG11, Pi56, Pi02 and Pi33 as one group and Pi89, Pi4B, Pi04 and pi16 as another based on fluorescent dyes used to label the primers and expected sizes of amplifications. Mixed PCR products, were loaded into 96-well plates and sent to Ruiboxingke Biotechn. Co. LTD. (Beijing) to determine fragment sizes using an ABI3730XL automated DNA sequencer  in which a DNA size ladder was included in each of the samples. Alleles were assigned using GeneMapper software version 3.7 (Applied Biosystems).PCR amplifications were performed in a 25 μL volume containing 1 μL of P. infestans isolate was paired with an A1 tester (US970001), an A2 (US940480) or grown alone on rye B agar plates. The two mating type testers were kindly provided by Prof. Qinghe Chen in Fujian Academy of Agricultural Sciences, Fuzhou, China. After inoculation at 18 °C in the dark for 12–15 days when the colonies from testers and tested isolates were fused, a piece of tissue was picked off from the intersected edge and observed microscopically to determine the production of oospores. The isolates forming oospores with A2 tester were designated as A1 mating type. Similarly, the isolates forming oospores when paired with the A1 tester were designated as A2 mating type. The isolates forming oospores with both testers and alone were designated as self-fertile. Mating type determination was replicated twice for each isolate.Mating type was determined as described previouslyP. infestans isolates showed a maximum of two alleles per locus as expected for a diploid organism. However, for several cases, three alleles were detected in one locus. Loci with three alleles were modified to resemble diploid loci by assigning specific allele sizes and a binary representation of the presence or absence of specific alleles as described previously49Na) were calculated by POPGEN v3.2.In analyzing the variation in microsatellite loci, most FST) and gene flow (Nm) were estimated using GENALEX 6.5. The physical distance between pair of populations was estimated using the geographic coordinates of locations from which the populations were sampled and isolation by distance was evaluated by estimating the association between the extent of gene flow and logarithm of physical distance (km) in the populationsA. All population genetic parameters were estimated with clonal uncorrected data. Principal component analysis (PCA) was performed with GENALEX 6.5 to produce map depicting the distribution of genetic diversity across geographic space. Genetic distances between isolates was calculated using GENALEX6.5 and the phylogenetic trees of the 534 isolates and 49 genotypes were reconstructed by a neighbor joining approach using MEGA5 and MST goldsoftware programme as described by Salipante and HallPairwise population differentiation .


Hydromantes strinatii using allozyme and mitochondrial markers showed a strong geographical genetic structure. This was likely the outcome of different evolutionary mechanisms leaving their signature despite the effects of the genetic drift due to the low population size typical of this species. Two highly divergent clades were identified in the eastern and central-western part of the range, with further geographic sub-structure. Nuclear and mitochondrial markers substantially recovered the same population groups but were conflicting in reconstructing their relationships. This apparent incongruence highlighted the action of different mechanisms such as secondary contacts and incomplete lineage sorting in originating the observed genetic variation. The troglophilic habit of this species provided the opportunity to show the importance of caves as local refugia in maintaining the genetic diversity through the persistence of local populations. Accordingly, high nucleotide and haplotype diversity, strong geographic genetic structuring and lack of expansion were evidenced. This signature was found in the populations from the Ligurian and Maritime Alps, in agreement with the complex orography and paleoclimatic history of this Mediterranean hotspot.The study of the European plethodontid salamander  Cave species are considered particularly fragile and endangered due to their strict ecological requirements, making them poorly able to face climatic variation. Also, their strong association with underground retreats constrains their populations to low size and poor genetic diversity . HoweverHydromantes are perfect candidates for these kind of studies. Their biological and ecological features would suggest species fragility: Hydromantes are lungless thus rely on cutaneous respiration and need steady moist and fresh environments to avoid dehydration and respiratory insufficiency. Moreover these species lack an aquatic larval stage since the metamorphosis is completed within the egg, requiring moist, fresh and steady habitats where females can attend to their eggs until hatching occurs [Hydromantes with underground retreats, such as caves and fissure systems, that are able to warrant the environmental conditions they need during unfavourable climatic periods such as Mediterranean summer. At the same time, European Hydromantes are generally characterized by old lineages and high genetic variation suggesting persistent and stable populations despite their very small size [The European plethodontid salamanders genus g occurs –13. For all size ,14–16.H. strinatii. Among the eight species of Hydromantes [H. strinatii is particularly suited for this type of study since the central and western part of its range is in the Ligurian and Maritime Alps, between Italy and southern France. This area is characterized by a very complex orography, including karst areas, and by a peculiar paleoclimatic history. The ice sheet covering the Western Alps during Quaternary glaciations showed a patchy distribution in the coastal zones and many peripheral areas remained free from ice. These refugial micro-areas now host a strikingly high species richness and endemism concentration of plant species [With the aim of explaining the apparently contrasting features of European plethodontids in the light of historical climate changes and localized retreats, we analyzed the genetic structure and phylogeography of romantes ,18, H. s species –21 and c species –24.H. strinatii. This allowed for the disentangling of the different evolutionary processes leading to the extant pattern of diversification in H. strinatii. We verified whether persistence in localized refugia contributed to the present genetic diversity of the species leaving the expected signature of high haplotypic and nucleotidic diversity associated to strong geographic structuring and lack of expansion. Finally, we tried to assess whether the distinctive topography and paleoclimatic history of the Ligurian and Maritime Alps was relevant in determining patterns and processes acting on H. strinatii by comparing the pattern of genetic diversity observed in the populations from this central-western part of the range vs. those observed in the easternmost populations, living in a more homogeneous environment.In this work, we used partial sequences of the mitochondrial genes cytochrome-b (cytb) and NADH-dehydrogenase sub-unit 2 (ND2) and allozyme analysis to study the genetic structure and the phylogeography of H. strinatii we studied belong to the collection of the Zoological Museum “La Specola” of the University of Florence  and were kindly provided by prof. Benedetto Lanza. Some specimens from a few localities were sampled on purpose under the licenses required by the corresponding authorities.The specimens of The permits of collecting, mark, collect tissues and release specimens was given by the Italian Ministry of the Environment (protocols DPN2009/0026530 and 0042634PNM07/08/13) according to the law DPR 357/97. The permits broadly allowed the collection of biological tissues without mentioning any protocol to be followed. No approval of ethics committee was required by both the Italian law and the EU directives on the use of animals for scientific purposes in force during the study period (Legislative Decree 116/92 and EU Directive 609/86).Hydromantes skin. Tissue samples were immediately frozen in a portable liquid nitrogen storable tank and sampled individuals were then released. No individuals were brought to the laboratory or sacrificed.For tissue sampling salamanders were captured manually and a small piece of about 2–3 mm was cut from the tail tip after keeping the tail on an instant-ice pack for about one minute. No anaesthetics were used to avoid possible mortality due to the high sensitivity of H. strinatii were analysed from 30 sites distributed throughout the species range  protocol .Partial sequences of the mitochondrial genes cytochrome-b (cytb) and NADH dehydrogenase subunit 2 (ND2) were obtained using specifically designed primers: HST-1f (5’-TTTATTGATTTACCAACCCCATCT-3’) and DEB-1r (5’-CAGGGGTGAAGTTTTCTGGAT-3’) for cytb; HND1-f (5’-TCAAGCTTATCCACCGGAAC-3’) and MAT2-r (5’-TTGGTGGTAGTCCGCCTAAG-3’) for ND2. Amplification was performed by polymerase chain reaction (PCR) in a volume of 25 μl containing MgCl2 (2.5 mM), the reaction buffer , the four dNTPs (0.2 mM each), the two primers (0.2 μM each), the enzyme Taq polymerase  and 2 μl of DNA template. PCR reactions were carried out in a Hain Lifescience Q-Sat 24 thermal cycler using the following program: denaturation at 94°C for 5 minutes followed by 35 cycles at 94°C for 30 s, 53°C for 1 min, 72°C for 1 min and a final elongation step at 72°C for 10 min.www.macrogen.com).Purification and sequencing reactions were outsourced to Macrogen Inc. (Electropherograms were independently checked by eye by two of the Authors using Chromas v.1.6 (Technelysium Pty Ltd) and sequences were then aligned using the software ClustalX v.1.83 .H. supramontis and H. imperialis and were used as outgroups (GenBank accession n. KJ834013-14 and KJ834057-58)In order to reconstruct the phylogenetic relationships between mitochondrial lineages, we conducted maximum likelihood (ML) and Bayesian inference (BI) analyses for the combined dataset of cytb and ND2 genes, and applying a partition strategy by genes in both cases. Sequences of both cytb and ND2 genes were obtained from Sardinian species We computed ML analysis in RAxML GUI v.1.3  using a Hydromantes lineages to the Messinian salinity crisis regardless of the markers used in their studies [Hydromantes species (represented by H. strinatii) and Eastern Sardinian species  at 5.33 ma, which is the end of the Messinian salinity crisis (5.96–5.33 ma) [http://www.fluxus-engineering.com) under Greedy FHP criterion [The distance-based least squares (LS) method  implemen studies ,17. Ther5.33 ma) . The sta5.33 ma)  as impleriterion  and defariterion  under thriterion .To investigate population genetic structure, we first ran our data in SAMOVA v.1.0  testingOnce the best number of groups was selected according to both SAMOVA and BAPS, all the subsequent analyses were carried out on each detected population group.Haplotype (h) and nucleotide (π) diversity as well as the gene flow parameter Nm were estimated for each cluster using DNAsp v5.10 .st distance matrix and running 1000 permutations.A hierarchical analysis of molecular variance  was carTajima’s D , Fu’s Fsst distance matrix was performed against a geographic distance matrix (with distances expressed in km) using 10000 permutations. Computations were done for each single group and for the whole population set.To verify if the population genetic structure conformed to an isolation by distance model (IBD), a Mantel test was executed in Arlequin v.3.5 . A geneta), observed heterozygosity (Ho), expected heterozygosisty (He), percentage of polymorphic loci under 99% criterion (P99). The softwares Genodive 2.0 [E) and the allelic richness (AR), respectively.Allozyme allele frequencies, conformity to Hardy-Weinberg equilibrium and linkage-disequilibria were calculated using the software Genepop 4.2  and testdive 2.0  and FSTAdive 2.0  were usest as implemented in Genepop 4.2 [st values was tested using Fisher’s method followed by Bonferroni correction [The partitioning of genetic diversity was analyzed for variable loci using the Fixation index Fepop 4.2 . The sigrrection .A Principal Component Analysis was carried out based on a matrix of covariance of allele frequencies as implemented in Genodive 2.0 , and thest for different subpopulations due to the high levels of natural fragmentation of Hydromantes populations. Priors were set choosing sampling location information (LOCPRIOR) and using both the mean value of Fst and the associated standard deviation obtained by previous calculations. Length of the burn-in period was of 100000 replicates followed by 100000 Markov chains Monte Carlo (MCMC) replicates, and fifteen runs were conducted for each K ranging from 2 to 12. The Evanno algorithm [The identification of population groups was obtained using BAPS v.6.0 ,57, settlgorithm , as impllgorithm , was uselgorithm  under thlgorithm .An analysis of molecular variance was carried out assuming a stepwise model as implemented in Genodive 2.0  and partst distance matrix vs. a geographic distance matrix (km). The IBD hypothesis was tested for the whole population set, for the two main mitochondrial clades and for the K groups recovered by STRUCTURE.A Mantel test was performed in Arlequin v.3.5  contrastThe 687-bp fragment of cyt-b sequenced had 59 variable positions (5 singletons) 54 of which were parsimony informative, and provided 24 different haplotypes over the 194 specimens scored. The obtained sequences have been deposited in GenBank (Accession n: KJ834015-38). Nucleotide percentages were: T 33.9%, C 22.0%, A 30.4%, and G 13.7%. The estimated nucleotide diversity (π) had a mean value of 0.028.The 702 bp fragment from ND2 gene had 54 variable positions (1 singleton), 53 of which were parsimony informative. The 194 specimens scored provided 18 haplotypes, available in GenBank (Accession n: KJ834039-56). Nucleotide percentages were: T 31.2%, C 23.2%, A 35.6% and G 10.0%. The estimated nucleotide diversity (π) had a mean value of 0.025.The alignment of the concatenated sequences was of 1389 bp, and provided an overall number of 30 haplotypes, as listed in Each haplotype was recovered in a single or a few geographically very close localities .All the phylogenetic reconstructions agreed in showing two well supported reciprocally monophyletic groups, including the haplotypes recovered in the eastern and western part of the range, respectively: the eastern clade (Clade A) included the haplotypes recovered from samples 1 to 21 and the western clade (Clade B) contained those from samples 22 to 30 . These tThe estimates of K2P distance between the seven lineages gave values comprised between 0.8% and 5.4%. The minimum divergence of 0.8% (p-distance = 0.8%) was recovered between the sub-clades A2 and A3. The values of maximum divergence comprised between 5.0% and 5.4%  were always found in pairwise comparisons between sub- clades A vs. B.ct index grew from 0.79 to 0.95 for K between 2 and 7 and then slightly moved between 0.96 and 0.99 for K between 7 and 25  and the highest values recorded for the French Maritime Alps clade (B3) with π = 0.00472 and h = 0.846.Inferred levels of gene flow were very low both between groups (average Nm = 0.02) and within groups (Nm = 0.04–1.93) with the lowest values recorded within sub-clades A4 and B3.The Mantel test results showed no correlation between genetic and geographic distances within the seven groups detected. However, a pattern of IBD came out when considering the entire species range . A significant correlation was also observed within clade A , while clade B exhibited no IBD .H. strinatii provided weak and sometimes contrasting signals coherently supporting a past expansion only for clades A1 and B1.The demographic test statistics and the analysis of the mismatch distribution computed for each of the seven clades showed only weak signs of past demographic expansion . Both TaThe chronogram with the obtained confidence intervals  showed tNadh-dh, Np, Sod-1, Sod-2, Adk, Lap, Fum, Mpi) while the remaining 25 had from two to four alleles per population .Eight loci of the 33 studied resulted as monomorphic  as indicated by both the highest value of ΔK  and by tA Principal Component Analysis (PCA) explained 61.0% of variation with two axes, with the first axis explaining 40.6% of variation (p = 0.009). PCA recovered the larger part of the clusters identified by STRUCTURE , the onlThe AMOVA carried out took into consideration the five clusters inferred from STRUCTURE analysis  and showst estimates were always highly significant except when comparing samples from the eastern part of the range (1–11) where only 21 comparisons out of 55 were significant  were recorded when comparing the samples from the “Finalese” (15–18) vs. all the others with particularly high values vs. the eastern samples (even above 0.80). To investigate the correspondence of population genetic structuring to a model of IBD, Fst and geographic distance values were compared by a Mantel test. The results obtained showed a positive correlation across the whole range of the species  as well as within the eastern part of the range , while IBD did not hold for the central-western populations.The pairwise Fnificant . The oveH. strinatii despite the small range size. Large genetic divergence over small geographic scales is quite common in plethodontids, especially if living in geographically old areas [H. strinatii is not homogeneous across its range suggesting different processes leading to the extant Eastern , and Central-Western  population groups. Different levels of genetic variation in the E vs. CW populations were concordantly revealed by both mt and nc markers with E populations characterized by a low level of genetic diversity. A single main haplotype is shared by the larger part of E samples within each of the two sub-clades living in this area  together with a few rare singleton differentiated haplotypes. Also, the lowest values for allozyme variability parameters are recorded from these populations irrespective of the sample size. The pattern of differentiation between E populations conforms to the IBD model, linking genetic and geographic distances along a NW-SE axis. Since small population size and low gene flow, which are usually invoked to explain low genetic variation [Hydromantes, they cannot account for the lower genetic variation of the E H. strinatii populations. Our data rather support the idea that this pattern is due to a recent expansion in the E part of the range: significance of neutrality tests and mismatch analysis, low genetic variability, the presence of a few, widespread haplotypes and the geographic pattern of genetic diversification following an “expansion route”, are all in agreement with a recent expansion event [The results obtained, irrespective of the marker used, showed substantial geographical structuring in ld areas . Howeverariation , are genon event –68.st values for pairwise comparisons are highly significant. The IBD model does not hold in this area: there is no relationship between genetic and geographic distance with highly differentiated sample pairs found in the range of a few kilometres. Both nc and mt markers identified 3 groups in the C part of the range, but in the W area, the two data sets showed some incongruence. The samples from the “Finalese” (15–18) formed a monophyletic group with the E samples according to mt data, while the same populations resulted as the most differentiated according to the allozymes  was constrained at 5.33 ma, which is the end of the Messinian salinity crisis.Maximum clade credibility tree obtained from the mitochondrial dataset. Red bars at nodes represent the 95% Highest Posterior Densities of node ages. The split node between (TIF)Click here for additional data file.S2 FigMethods following Evanno et al. , as impl(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 TableAllozyme allele frequencies per population at the 25 polymorphic loci out of the 33 scored.(DOCX)Click here for additional data file.S3 Tablest) values based on allozyme data. * p <0.05; ** p < 0.01; *** p < 0.001;—not significantPairwise fixation index (F(PDF)Click here for additional data file.S1 Filect vs. K values according to SAMOVA (mt data); (b) by plotting log marginal likelihood vs. K values according to BAPS .Plot of the number of groups (K) obtained: (a) by plotting F(XLS)Click here for additional data file.


Genetic diversity analysis of triticale populations is useful for breeding programs, as it helps to select appropriate genetic material for classifying the parental lines, heterotic groups and predicting hybrid performance. In our study 232 breeding forms were analyzed using diversity arrays technology markers. Principal coordinate analysis followed by model-based Bayesian analysis of population structure revealed the presence of weak data structuring with three groups of data. In the first group, 17 spring and 17 winter forms were clustered. The second and the third groups were represented by 101 and 26 winter forms, respectively. Polymorphic information content values, as well as Shannon’s Information Index, were higher for the first (0.319) and second (0.309) than for third (0.234) group. AMOVA analysis demonstrated a higher level of within variation (86 %) than among populations (14 %). This study provides the basic information on the presence of structure within a genetic pool of triticale breeding forms.The online version of this article (doi:10.1186/s40064-016-1997-8) contains supplementary material, which is available to authorized users. Triticum species (AABB or AABBDD) and Secale cereale L. (RR). It combines grain quality and productivity typical for wheat with vigor, hardiness and high lysine content specific for rye  is a synthetic cereal crop that originated from a cross between http://www.factfish.com/catalog/crop).The history of triticale started in 1875 with the first report describing a fertile hybrid between wheat and rye issued by the Scottish botanist Wilson Wilson . SubstanT. timopheevii cytoplasm . 1275, 1582 and 260 markers developed based on wheat, rye and triticale genomes, respectively were used. After the elimination of 1279 redundant and nine rare markers (identified in <5 % of cases), their respective numbers dropped to 1829 with 760, 912 and 157 in wheat, rye, and triticale sets. Among 232 genotypes, there were 22 spring and 49 winter forms having nearly identical counterparts that were substituted by their representatives. Finally, the study was conducted on a set of 161 diverse triticale forms (17 spring and 144 winters).Based on principal coordinate analysis (PCoA) the first two coordinates explained the only small fraction of genetic variance . Nevertheless, three groups of data represented by most spring together with some winter forms plus two putative clusters of winter forms could be recognized Fig. .Fig. 1PrPopulation structure analysis followed by Evanno method  and Pop2 (97.3 %)  values varied among three groups of triticale forms  and low genetic differentiation among populations Pop2 and populations Pop1 and Pop3 .AMOVA analysis of the model-based populations Pop1, Pop2, and Pop3 showed that 86 % of variance was due to within population whereas 14 % (p < 0.001) to among-population differences. Pairwise Arabidopsis thaliana (43 %)     . Unfortunately, knowledge covering genetic diversity, the population structure of gene pool and heterotic groups that constitute the base for lines, and hybrid breeding is limited; however, this could be easily achieved via marker analysis and may deliver background for the development of crosses in breeding programs.Triticale is one of the most successful cereals in Poland with new varieties entering the market each year  and consisted of 193 winters and 39 spring inbreed lines were used  followed by spectroscopic (NanoDrop ND-1000) measurement of its quantity. The DNA samples were subjected to 1.2 % agarose gel stained with ethidium bromide (0.1 μg/ml) in TBE buffer to verify DNA integrity.http://www.triticarte.com.au) using high-resolution triticale array (DArT) representing markers from wheat (wPt), rye (rPt), and triticale (tPt) according to the method described by Badea et al.  and Jaccard genetic distance in PAST software. The forms were assumed to be identical if the differences between them did not exceed 5 % and if their molecular profiles, except when missing markers, were identical. The profiles of such individuals were merged.Preliminary visualization of putative structure among triticale breeding forms was calculated by the data from all non-redundant individuals and non-redundant markers. For this purpose, PCoA Gower  based onThe population structure was evaluated using Bayesian analysis of the genetic structure (K) carried out with STRUCTURE 2.2.3 , the number of unique markers, Shannon’s Diversity Index (I)  was performed with GenAlex using 999 permutations test to estimate the reliability of the data.Analysis of molecular variance—AMOVA (Φ


Avicennia species, A. germinans and A. schaueriana, from the Western Hemisphere using nuclear and chloroplast DNA markers.Mangrove plants grow in the intertidal zone in tropical and subtropical regions worldwide. The global latitudinal distribution of the mangrove is mainly influenced by climatic and oceanographic features. Because of current climate changes, poleward range expansions have been reported for the major biogeographic regions of mangrove forests in the Western and Eastern Hemispheres. There is evidence that mangrove forests also responded similarly after the last glaciation by expanding their ranges. In this context, the use of genetic tools is an informative approach for understanding how historical processes and factors impact the distribution of mangrove species. We investigated the phylogeographic patterns of two Avicennia bicolor, A. germinans and A. schaueriana are independent lineages, hybridization between A. schaueriana and A. germinans is a relevant evolutionary process. Our findings also reinforce the role of long-distance dispersal in widespread mangrove species such as A. germinans, for which we observed signs of transatlantic dispersal, a process that has, most likely, contributed to the breadth of the distribution of A. germinans. However, along the southern coast of South America, A. schaueriana is the only representative of the genus. The distribution patterns of A. germinans and A. schaueriana are explained by their different responses to past climate changes and by the unequal historical effectiveness of relative gene flow by propagules and pollen.Our results indicate that, although A. bicolor, A. germinans and A. schaueriana are three evolutionary lineages that present historical and ongoing hybridization on the American continent. We also inferred a new evidence of transatlantic dispersal for A. germinans, which may have contributed to its widespread distribution. Despite the generally wider distribution of A. germinans, only A. schaueriana is found in southern South America, which may be explained by the different demographic histories of these two species and the larger proportion of gene flow produced by propagules rather than pollen in A. schaueriana. These results highlight that these species responded in different ways to past events, indicating that such differences may also occur in the currently changing world.We observed that The online version of this article (doi:10.1186/s12862-015-0343-z) contains supplementary material, which is available to authorized users. Mangrove forests are unique tree communities that occupy narrow elevation ranges within the intertidal zones of tropical and subtropical regions. Compared with tropical and subtropical terrestrial plant communities, the few species that occupy these forests are characterized by physiological and ecological traits that make them highly adapted to the coastal environment . The latRhizophora mangle L. (Rhizophoraceae) has expanded its distribution southward along the Brazilian coast since the last glacial maximum (LGM) [Avicennia germinans L. (Acanthaceae) populations have expanded their ranges northward since the LGM [Avicennia species of the Western Hemisphere: Avicennia germinans and A. schaueriana Moldenke. The former is a widespread species found throughout most of the ACEP region, whereas the latter is restricted to the Atlantic coast of South America and the southern Caribbean [In the ACEP region, for example, um (LGM) . Further the LGM ,19. For  the LGM ,20, rein the LGM . To expaaribbean ,22   Vierh., we also included previously analyzed sequences [To evaluate the distribution of the genetic variation, we sequenced two intergenic spacers of the chloroplastidial genome and one region of the nuclear ribosomal internal transcribed spacer (ITS). The mer sets , and themer sets . The seqlleagues . The seqequences ,23. For equences ,23, we ctrnD-trnT and trnH-trnK spacers were concatenated and will henceforth be jointly referred to as DTHK. To understand the genealogical relationships among the ACEP region samples, we applied the median joining method [http://popart.otago.ac.nz/index.shtml [We assembled and manually verified the chromatograms using CLC Genomics Workbench 4.9 software (CLC Bio). When we detected evidence of heterozygotes, three new amplifications and sequencing reactions were conducted as follows: only consistent double peaks were considered to be an indicator of a heterozygous site. The alignment and phasing of the entire dataset were performed using MUSCLE  and PHASg method  implemenex.shtml ), using ST values, considering the haplotype frequency using Arlequin 3.5 [ST matrix was then dimensionally represented using multidimensional scaling (MDS) in R software [ST, Nei’s coefficient of multiple alleles gene differentiation analog to Wright’s FST [p/ms = {(1/GSTbipar − 1)(1 + FIS) − 2 (1/GSTmat − 1)}/(1/GSTmat − 1)) [ST of each marker (GSTbipar for ITS and GSTmat for DTHK) and the previous average values of FIS estimated for A. germinans (0.174) and A. schaueriana (0.242) using microsatellites [IS, is constant through time and also across the species populations.We next determined the haplotype frequencies of each sample and calculated the haplotype diversity (h), nucleotide diversity (π), and estimates of group pairwise Φquin 3.5 . For fursoftware . The gloht’s FST ,36, wereht’s FST , consideat − 1)) , given ttellites . By doina priori hypotheses regarding A. germinans and A. schaueriana based on several factors: a) the geographic influences of the American continent, b) the effects of contemporary near-surface marine currents on the genetic diversity of ACEP mangrove species [a posteriori groups regarding the genealogical analysis and the geographic distribution of haplotypes. The criteria for determining the best hypothesized arrangement were a significant departure from a random distribution and the maximum variance among groups (ΦCT). We used PERMUT software [ST, which considers both haplotype frequencies and their divergence, and GST, which only considers haplotype frequencies, based on 10,000 random permutations.To better understand the phylogeographic patterns of the observed genetic variation, and because the previously analyzed sequences were obtained from a few samples from each location ,23, we a species ,25,26,40 species , which isoftware  to test CT criterion and the sample arrangements previously inferred using other genetic markers, such as microsatellites [S [S [We evaluated the demographic fluctuations using several summary statistics and considered the groups that best met the maximum significant Φtellites ,18,23,26tellites  and Fu’slites [S  using Arlites [S  and D* alites [S  computedlites [S . Assuminlites [S . We thenes [S [S  for the es [S [S , D* and es [S [S  for the es [S [S . These aes [S [S . Regardies [S [S  are signes [S [S . SignifiA. germinans and A. schaueriana from the Brazilian Institute of the Environment and Natural Renewable Resources - IBAMA (currently Chico Mendes Institute for Biodiversity Conservation - ICMBio). We confirm that A. germinans and A. schaueriana are not endangered or protected species.We obtained two licenses (Nos. 17159 and 17130) to collect the leaves and propagules of Avicennia species on the scale of the entire Western Hemisphere, we obtained samples of A. germinans and A. schaueriana from northeastern and southern South America and studied them together with previously evaluated samples using cpDNA and nDNA markers [h) and nucleotide (π) diversities . This difference suggests that the gene flow of A. germinans through its propagules was similar to the gene flow by pollen; however, in A. schaueriana, the movement of genes by seeds was one to two times higher than that by pollen.Population differentiation analyses indicated that there was intraspecific genetic divergence between the evaluated samples of T values  of 0.568ST values indicated that there was substantial genetic structure, which is more readily observed when one considers the pairwise ΦST values for each species, which were mostly significant, except for the cpDNA marker and the A. schaueriana samples  from the ACEP region [A. germinans and A. schaueriana indicated that a posteriori hypotheses provided better explanations for the observed molecular variation. The a priori models considered different combinations of the expected effects of the AMMC, the American continent and/or prominent surface marine currents on the genetic diversity. The hypothesized groupings of Ag1, Ag4, As1, As8 and As9 were based on previous studies that used molecular markers to evaluate the genetic variation [a posteriori models because the ΦCT values were not significantly different from random distributions, and due to the highly negative values of among-group variance, they failed to reasonably explain the genetic diversity  when the former was analyzed  and the a priori scenarios, namely models Ag1 and As1, based on previous studies [a priori and a posteriori hypotheses. When A. germinans model Ag1 was considered, we found signs of expansion in the AgNPc and AgNAtl groupings, whereas AgSPc showed indications of background selection. When each population was considered, only AgALC, AgPNB and AgTMD did not present signs of demographic changes  difference between the mean values of GST  and NST . In contrast, for A. schaueriana , the differences between these indexes were not significant (P > 0.05): for ITS and DTHK markers, the mean values of GST were 0.384 and 0.442, respectively, whereas the mean values for NST were 0.302 and 0.400, respectively.Regarding the PERMUT analysis, we observed that for Avicennia species from the ACEP region may be considered three different evolutionary lineages independent from the lineage composed of A. alba and A. marina, which are IWP species  [A. schaueriana and A. germinans from several locations within the zone of sympatry for these species, indicating that this biological process may be more common than previously believed. We favor hybridization/introgression rather than an ancestral polymorphism as the most likely mechanism generating this haplotype sharing due to the positions in the phylogenetic networks and the relatively high frequencies of the shared haplotypes. The branches where these haplotypes occurred, however, are not more related to the IWP species, as would be expected for this biological process  . Herein,s Figure , and twoAvicennia than previously believed, adding a relevant report to the large list of examples of hybridization in mangrove species. Based on the morphological and molecular data, interspecific gene flow has been described for the genera Rhizophora, Bruguiera (Rhizophoraceae), Sonneratia (Lythraceae), Lumnitzera (Combretaceae) and Avicennia [These observations suggest that introgressive hybridization is a more widespread process on both coasts of the American continent for vicennia ,23,52-57A. bicolor, A. germinans and A. schaueriana presented clear genetic differentiation despite the evidence for introgression previously discussed here and elsewhere [A. germinans and A. schaueriana was not obvious.At the species level, lsewhere . ConversA. germinans clearly clustered into different groups. The samples from the west coast of Central America were mostly phylogenetically separate from the remaining haplotypes, which can be readily visualized in ITS and DTHK haplotype networks  [The Pacific and Atlantic samples of s Figure . Howevers Figure A, there s Figure B and beclvaceae) , whose plvaceae) ,23,25,40A. germinans [A. germinans transatlantic seed dispersal.Individuals from both sides of the Atlantic Ocean shared ITS and DTHK haplotypes, and those haplotypes that were different were phylogenetically closely related Figures ; this oberminans . Despiteerminans ,60, we aA. germinans [R. mangle [Hibiscus L., a sea-dispersed plant, [This finding supports and extends the role of transatlantic dispersal as a relevant evolutionary process for the mangrove species erminans  and R. m. mangle , whose p. mangle . Moreoved plant, ,62 and ed plant, . This mod plant, .A. germinans propagules in salt and fresh water and their high buoyancy  [A. germinans and A. schaueriana [A. germinans and R. mangle [. The observation of both long-distance dispersal and limited dispersal was also observed for mangrove species from the Eastern Hemisphere, e.g., Rhizophora [Ceriops (Rhizophoraceae) [Kandelia (Rhizophoraceae) [Interestingly, despite the high longevity of  rotten) , LDD is  rotten) . We haveaueriana . Moreove. mangle . The obsizophora ,66, Cerioraceae) -69 and Koraceae) -72 speciA. germinans samples, there was also generally substantial and significant genetic differentiation, as measured by global GST, its comparison with NST and the pairwise ΦST  for the DTHK marker may be explained by the remarkable genetic diversity of this marker that was observed in AsPAR compared to other A. schaueriana samples . During America ,76. The After extinction events occurred due to Quaternary environmental changes, populations would have become more isolated . This diA. schaueriana and model Ag1 and considering each sample separately for A. germinans) presented signs of recent demographic change. For both species, in aggregate, we found signs of population growth for different evolutionary scenarios across the samples , there were consistent indications that recent demographic expansion occurred along the entire A. schaueriana distribution regardless of the model that was assumed  and lowAvicennia species can also provide valuable information about the responses of these plants to current global climate change. For example, despite their close phylogenetic relationship, A. germinans and A. schaueriana have responded differently since the last glaciation, and it is, thus, likely that their distinct ecological features may also influence their future in the face of the currently changing world. Considering this information about the past, our current endeavor is to understand potential impacts of the current climate changes on the neutral genetic variation of A. germinans and A. schaueriana.In addition to these retrospective conclusions, the novel details that our findings revealed about the evolutionary history of the ACEP region


Coix lachryma-jobi L.) is an important crop used as food and herbal medicine in Asian countries. A drug made of Job’s tears seed oil has been clinically applied to treat multiple cancers. In this study, the genetic diversity of Job’s tears accessions and the fatty acid composition, triglyceride composition, and anti-proliferative effect of Job’s tears seed oil were analyzed using morphological characteristics and ISSR markers, GC-MS, HPLC-ELSD, and the MTT method. ISSR analysis demonstrated low genetic diversity of Job’s tears at the species level  and the accession level , and strong genetic differentiation (GST = 0.6702) among all accessions. It also clustered the 11 accessions into three cultivated clades corresponding with geographical locations and two evidently divergent wild clades. The grouping patterns based on morphological characteristics and chemical profiles were in accordance with those clustered by ISSR analysis. Significant differences in morphological characteristics, fatty acid composition, triglyceride composition, and inhibition rates of seed oil were detected among different accessions, which showed a highly significant positive correlation with genetic variation. These results suggest that the seed morphological characteristics, fatty acid composition, and triglyceride composition may be mainly attributed to genetic factors. The proportion of palmitic acid and linoleic acid to oleic acid displayed a highly significant positive correlation with the inhibition rates of Job’s tears seed oil for T24 cells, and thus can be an important indicator for quality control for Job’s tears.Job’s tears ( Coix lachryma-jobi L.), which belongs to the Coix genus, the Andropogoneae tribe, and the Gramineae family [Job’s tears , AFLP (amplification fragment length polymorphism) and ISSR (inter-simple sequence repeat) are useful tools to evaluate the genetic variation of plants. Among these markers, ISSR is attractive because it requires no previous DNA sequence information and is highly variable, reproducible, and cost effective ,37. ISSRThe objectives of this study were to (i) analyze the genetic diversity of 11 Job’s tears accessions from the main distribution regions in China based on morphological characteristics and ISSR markers; (ii) detect the fatty acid and triglyceride compositions of seed oil from different accessions using GC-MS and HPLC-ELSD; (iii) assess the anti-proliferative effect of seed oils from different accessions on T24 cells using the MTT method; and (iv) clarify the relationship between genetic variation and active component composition of Job’s tears seeds, and further impact on anti-proliferative effect. Understanding the genetic diversity and indicators of quality could provide valuable information for the utilization of, breeding of, and conservation strategy establishment for Job’s tears.Eleven Job’s tears accessions were included in this study , Table 1Coix species and varieties [The shape, size, color and texture of seed are important in the classification of arieties ,43. FourEleven quantitative morphological characteristics were measured on the same 15 seeds of each accession. Seed weight, weight of seed kernel and weight of the hull and the testa were measured with a scale sensitive to 0.0001g. Seed thickness, seed length, seed width, thickness of seed kernel, length of seed kernel and width of seed kernel were measured by a digimatic calipers . The length-width ratio of seed and the corresponding seed kernel were calculated in Excel.The Job’s tears seeds germinated in a light-proof environment until the seedlings reached a height of 3–4 cm. Total genomic DNA was extracted from the seedlings using a modified CTAB (cetyltrimethyl ammonium bromide) method , and the2, 0.2 mM each of dNTPs, 0.2 μM of primers, 0.5 U of Taq DNA polymerase (TaKaRa), and approximately 20 ng of template DNA. The amplifications were performed in a Eppendorf Mastercycler Gradient PCR machine  with the following program: an initial denaturation at 94°C for 4 min; 40 cycles of denaturation at 94°C for 45 s, annealing at 50–55°C (depending on different primers) for 45 s, elongation at 72°C for 1.5 min; and a final extension at 72°C for 10 min.One hundred ISSR primers screened in this study were synthesized by Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. (China) according to the primer set published by the University of British Columbia . PCRs were performed in 20 μL reaction mixtures containing 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 2 mM MgClThe amplification products were electrophoresed in a horizontal gel apparatus  using 1.5% agarose gel in 1× TAE buffer (pH 8.0) at 100 V for 45 min. The gels were stained with 0.8 μg/mL ethidium bromide for approximately 30 min, and then photographed under UV light using the UVP-GDS8000 Gel Documentation System .Acetonitrile and methylene dichloride were chromatographically pure and purchased from Tedia Company, Inc. .The Job’s tears seeds were dehulled and ground into a powder. One gram of powder was accurately weighed and soaked in 25 mL of mobile phase  for 2 h at 25°C; then, it was ultrasonically extracted for 30 min. The extract was diluted to 25 mL and filtered through a Millipore filter (0.45 μm). The filtrate was used for HPLC-ELSD (High Performance Liquid Chromatography- Evaporative Light Scattering Detector) analysis.TM C18 column  was maintained at 30°C for analysis. Acetonitrile and methylene dichloride (67:33) were used as the mobile phase with a flow rate of 0.6 mL/min. A drift tube temperature of 45°C and a nebulizer gas (N2) flow rate of 1.5 L/min were set as the detection conditions for ELSD. An injection volume of 10 μL was used for quantitative analysis.HPLC-ELSD analyses were carried out on an Agilent 1200 HPLC system  equipped with an evaporative light scattering detector (ELSD) 3300 . An XtimateAcetone, sulfuric acid, methanol, ether, sodium sulfate, and n-hexane were analytically pure and purchased from Hangzhou Gaojing Fine Chemical Co., Ltd .Five grams of dehulled Job’s tears seed powder was extracted by reflux with 50 mL acetone for 1 h. This was done twice. The extracts were combined and evaporated by rotary vacuum at 60°C. Then, the extract was added to a 10 mL solvent of sulfuric acid and methanol (1:9), extracted by reflux at 60°C for 2 h, extracted with ether, washed to a neutral pH with distilled water, dehydrated with sodium sulfate, evaporated to remove the ether, and dissolved with n-hexane to a volume of 5 mL.GC-MS analyses were carried out on the Agilent 7890A-5975C  with electron impact ionization (70 eV). A DB-5 fused silica capillary column  was used. The column temperature was maintained at 60°C for 1 min; then, it was programmed to increase from 60°C to 200°C at a rate of 15°C/min, maintained at 200°C for 5 min, raised to 280°C at a rate of 8°C/min, and finally held at 280°C for 1 min. The carrier gas used was helium, with a flow rate of 1 mL/min and a split ratio of 50:1. Mass range was 50–550 m/z and the injector temperatures were set at 220°C. The components were identified by comparison of mass spectra with those stored in the available database (NIST version 2008).Ethanol, sorbitane monooleate (Span 80), and polyoxyethylene-80-sorbitan monooleate (Tween 80) were analytically pure and purchased from Sinopharm Chemical Regent Co., Ltd. . MTT and dimethyl sulfoxide (DMSO) were purchased from Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. (China). RPMI-1640 medium , new-born calf serum, penicillin, and streptomycin were purchased from Hyclone . The human bladder carcinoma T24 cells were purchased from ATCC .To obtain the seed oil of Job’s tears, 30 g dehulled seed powder was ultrasonically extracted with 300 mL of 100% ethanol for 1 h, filtered to remove the residue, and evaporated by rotary vacuum. Then, 100 μL oil, 10 μL emulsifier of Span 80 and Tween 80 (2:3), and 890 μL cell medium were mixed and vortexed to obtain a 10% ethanol extract emulsion.2 atmosphere.The human bladder carcinoma T24 cells were cultured in RPMI-1640 cell medium supplemented with 10% new-born calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and a 5% CO3 cells/well) were inoculated into a 96-well polystyrene plate and incubated for 18 h. The test group, emulsifier group, positive group, and control group were treated with 0.5% or 0.33% ethanol extract emulsion, 0.5‰ or 0.33‰ emulsifier, 100 μg/mL MMC (mitomycin C), and pure medium, respectively. After incubation for 24 h, 20 μL MTT (5 mg/mL) was added to each well and incubated for 4 h before removing the medium, and 150 μL DMSO was then added to each well to dissolve the precipitate. The absorbance was measured by a Multifunctional Microplate Reader SpectraMax M5  at a wavelength of 490 nm, and the inhibition rate was calculated according to the following formula:The MTT assay procedure used was as follows: T24 cells  of all 15 morphological characteristics were performed with SPSS 19.0 . The Euclidean distances between Job’s tears accessions based on morphological characteristics were calculated using the between-groups linkage method in SPSS 19.0.h), Shannon's Information index (I), Neiʼs coefficient of accession differentiation (GST), and gene flow among all accessions (Nm) were calculated using PopGene 1.32 [The discernible and reproducible DNA bands ranging from 250 to 2000 bp were scored 1 for presence and 0 for absence to construct the binary data matrix for statistical analysis. The parameters of genetic diversity as percentage of polymorphic fragments (PPF), Neiʼs expected heterozygosity , seed thickness (0.687 cm), seed length (1.023 cm), seed width (0.809 cm), weight of seed kernel (0.091 g) and width of seed kernel (0.596 cm), and the minimum values of these 6 characteristics were recorded in AHBZ  . AHBZ reIn the PCA scatter plot constructed by the 15 morphological characteristics, the Job’s tears accessions can be divided into 3 groups, which corresponded to the southern and central, northern, and wild groups, respectively . The souAmong the three divided groups, the accessions in the wild group possessed the biggest, heaviest, and roundest seeds. The accessions in the northern group possessed the smallest, lightest, and narrowest seeds. The southern and central group contained accessions with seeds of moderate weight and size. The weight and size of seed kernels were not significantly different among the three groups, whereas the shape of the seed kernel varied from oblate in the wild group to oblong in the northern group , Fig 2.In total, 103 reproducible fragments, of which 91 were polymorphic (84.47%), were produced in amplification for 218 samples from 11 Job’s tears accessions by 10 selected ISSR primers . The numh), Shannon's Information index (I) and percentage of polymorphic fragments (PPF) of all accessions were 0.2051, 0.3261 and 84.47%, respectively. The genetic diversity was different among all accessions. As seen in h = 0.0335, I = 0.0492, PPF = 8.74%) was detected in wild accessions compared to the cultivated accessions . The wild accession ZJLD showed the lowest genetic diversity , while the cultivated accession ZJTS showed the highest genetic diversity . The genetic diversity level of ZJJY  was the lowest among the cultivated accessions, and almost equal to the genetic diversity  of wild accessions.The Neiʼs expected heterozygosity  was found among all accessions. The analysis of molecular variance (AMOVA) also showed that 73.42% of total genetic variability occurred among all accessions, indicating significant genetic differentiation of Job’s tears. Most of the genetic diversity of clade III (GST = 0.6447) and clades IV and V (GST = 0.7442) was detected among accessions. Relatively low GST values of 0.4313 and 0.3770 in clade I and clade II, respectively, indicated that most genetic diversity was detected within accessions. Compared with the low gene flow (Nm) of 0.2460 in all accessions, the gene flow of subdivided cultivated clades was relatively higher, ranging from 0.2756 in clade III to 0.8262 in clade II; the gene flow of clade IV and V (0.1718) was even lower  in all accessions, but significant correlation between genetic and geographic distance matrices  was detected in the cultivated accessions when the wild accessions were excluded.GC-MS analysis detected four fatty acids in Job’s tears seed oil from different accessions. They showed a general order of abundance of oleic acid > linoleic acid > palmitic acid > stearic acid , with thIn the PCA scatter plot constructed by the fatty acid compositions of seed oil, the Job’s tears accessions can be divided into four groups: the wild group (SCCD and ZJLD), the northern group , the central group , and the southern group  . The groHPLC-ELSD analysis showed that the content of nine triglycerides in seed oil were significantly different in the 11 Job’s tears accessions . 1,2-LinAccording to the PCA scatter plot constructed by the triglyceride compositions of seed oil, the Job’s tears accessions can be divided into four groups: the wild group (SCCD and ZJLD), the northern group , the central group , and the southern group  . The groThe inhibition rates of Job’s tears seed oil were significantly different among accessions, varying from -1.80% (SCCD) to 101.59% (ZJTS) at a concentration of 0.33% and from 5.84% (SCCD) to 102.42% (ZJTS) at a concentration of 0.5% . There wCompared with the control group, the seed oil of almost all cultivated accessions significantly inhibited the proliferation of T24 cells in a concentration-dependent manner, while the seed oil of wild accessions (SCCD and ZJLD) showed no repressive effect at either concentration. The oil of the ZJTS accession showed the most potent repressive effect against T24 cells, with an inhibition rate of 101.59% at a concentration of 0.33% . The GXXA significant correlation can be seen between inhibition rates and content of single fatty acids. The inhibition rates showed a significant or extremely significant positive correlation with palmitic acid content  and linoleic acid content , and a significant or extremely significant negative correlation with oleic acid content  . The inhThe inhibition rates at both concentrations demonstrated a significant positive correlation with 1,2-linolein-3-olein, 1,2-linolein-3-palmitin and 1,3-palmitin-2-linolein content, and a significant negative correlation with 1,2-olein-3-linolein and triolein content . The inhThe seed oil of cultivated Job’s tears accessions significantly inhibited the proliferation of T24 cells. Triglycerides were confirmed as the main active components of Job’s tears seed oil . In thisSeveral studies have indicated that palmitic and linoleic acid can inhibit the growth of cancer cells, and that oleic acid has the opposite effect. Linoleic acid has been verified to suppress the growth of human colorectal cancer cells ,50, humaThe fatty acid and triglyceride compositions were significantly different among the 11 Job’s tears accessions, and the chemical profiles showed an extremely significant positive correlation with genetic variation. Typically, the yield and variability of secondary metabolites can be influenced by physiological variations, environmental conditions, geographic variation, genetic factors, and evolution . HoweverIn this study, the 11 Job’s tears accessions were divided into two to three cultivated groups corresponding with geographical locations, and one to two evidently divergent wild groups by seed morphological characteristics, ISSR data, or chemical profiles. The inhibition rates for T24 cells were also different among the divided groups. ISSR markers are stable in all tissues throughout the lives of the plants, and are independent of environmental, pleiotropic, and epistatic effects . The ISSh = 0.21, I = 0.33) and accession level  was lower than that of the Gramineae species Miscanthus sinensis  and Elymus sibiricus , as detected by ISSR markers [The genetic diversity of Job’s tears at the species level  . The souST value. Typically, Nm > 1 is considered to be sufficient to prevent accession differentiation [m = 0.2460) of all Job’s tears accessions is not sufficient to counteract the genetic differentiation caused by genetic drift, and has resulted in great genetic differentiation (GST = 0.6702) among all accessions. The Job’s tears resources of China can be divided into three ecotypes  according to morphological, biological, and agronomic traits [ST = 0.6447) can be attributed to low gene flow (Nm = 0.2756), while the low genetic differentiation among northern accessions of clade I (GST = 0.4313) and central accessions of clade II (GST = 0.3770) can be attributed to relatively higher gene flow . The geographic pattern of cultivated accessions indicated that gene flow was restricted by geographic distance. Job’s tears is an anemophilous plant, but the distance between southern, central, and northern accession regions is too great for the pollen to cover. In addition, the seeds are too heavy to be carried to distant regions by wind, and they fall only around their mother plants, which makes seed dispersal even more difficult than pollen dispersal without artificial spread. The genetic structure of Job’s tears is affected by its breeding system, gene flow, life form, and geographic range.The genetic structure of a species is affected by multiple evolutionary factors including the breeding system, gene flow , life form, and geographic range . Job’s tntiation . The lowc traits , which sThe low genetic diversity of Job’s tears accessions was identified in this study and supported by genetic diversity estimates for Chinese and Korean accessions –30. TherS1 Table(XLSX)Click here for additional data file.


Comparative analyses of the control of mammalian microbiomes by host genetic architecture reveal striking conserved features that have implications for the evolution of host–microbiome interactions.http://www.genomebiology.com/2015/16/1/191See related Research article:  Genome Biology, is the first to use genome-wide analyses in a human study population to attribute variation in microbiome composition to genetic variation at specific loci in the host genome.Genetic and environmental factors converge in many different ways and bring about remarkable diversity in complex traits. Our growing understanding of the role of the microbiome in complex diseases naturally leads us to examine the concept of the microbiome as a complex trait, and hence to measure genetic versus environmental control of microbiome composition. Quantitative genetic analyses in mouse models, which allow many environmental factors to be measured or controlled, have established that genetic variation at a number of loci affects compositional features of the gut microbiome, supporting our view of the microbiome as a complex, polygenic trait –3. A stuAccurate dissection of genetic and environmental contributions to the control of complex traits in human study populations is a holy grail of complex trait research. This goal is elusive because of the confounding effects of environmental factors that cannot be controlled or estimated. Indeed, initial studies of microbiome composition in a large cohort of obese and lean twin pairs that relied on relatively crude statistical analyses showed only a trend toward greater differences between the microbiomes of dizygotic twin pairs than between those of monozygotic pairs  . AnalysiBlekhman et al.  have devThe methodologies used by Blekhman and colleagues are an elegant display of the power of bioinformatics and quantitative genomics. Equally exciting is the opportunity for these techniques to be used in comparative studies to reveal a broad understanding of mammalian genetic architectures that affect the compositional landscape of the microbiome. One of the first observations from such comparative studies is that the genetic architecture revealed by any single study is highly context-dependent and only a small number of associations are replicated across studies. The context-dependency stems from the relatively strong contributions of environmental and stochastic factors that affect microbiome composition. Despite the context-dependency, four general features of genetic architecture that influence microbiome composition are shared between humans and mice, and these features merit discussion because they shed light on the very nature of host–microbiome interactions and their evolutionary relationships.This feature was consistently observed in mouse studies of the gut microbiota, and data from the human populations reported by Blekhman et al.  extend tIn general, the effect sizes of the associations account for less than 10 % of the phenotypic variation, with the largest effect size reported for any microbiome QTL being 19 %. This general feature teaches us that the cumulative effect of genetic variation on a number of taxa, rather than large effects from a small number of loci on a few taxa, is the main driver of microbiome composition. This feature is likely to explain why factors such as diet can overcome genetic influence , 9. FromIn each of the studies, multiple instances are observed in which QTL peaks for different taxa of the microbiome overlap, implying that variation at a single genomic region influences several different microorganisms, some of which are phylogenetically diverse. In several instances, genes that are involved in innate immunity underlie these pleiotropic QTL peaks, providing a mechanistic basis for how such pleiotropy could be mediated. Blekhman et al.  extend tPerhaps the most intriguing conserved feature of the architecture is the consistent finding of overlapping QTLs that are associated with both the control of the microbiome and predisposition to complex diseases or physiological traits related to complex disease. Fig. Clearly, there is much to learn about host–microbiome interactions and their relationships with human and animal disease. For now, humans have 'weighed-in’ among the models in which genome–environment interactions and the microbiome are being studied, and these studies will provide tremendous insights into the mechanistic details of diseases as well as their distributions across populations.


Oryza glumaepatula in Costa Rica. We also evaluated the likelihood of gene flow between wild and commercial rice species because the latter is commonly sympatric with wild rice populations. Introgression may change wild species by incorporating alleles from domesticated species, increasing the risk of losing original variation. Specimens from all known O. glumaepatula populations in Costa Rica were analyzed with 444 AFLP markers to characterize genetic diversity and structure. We also compared genetic diversity estimates between O. glumaepatula specimens and O. sativa commercial rice. Our results showed that O. glumaepatula populations in Costa Rica have moderately high levels of genetic diversity, comparable to those found in South American populations. Despite the restricted distribution of this species in Costa Rica, populations are fairly large, reducing the effects of drift on genetic diversity. We found a dismissible but significant structure (θ = 0.02 ± 0.001) among populations. A Bayesian structure analysis suggested that some individuals share a significant proportion of their genomes with O. sativa. These results suggest that gene flow from cultivated O. sativa populations may have occurred in the recent past. These results expose an important biohazard: recurrent hybridization may reduce the genetic diversity of this wild rice species. Introgression may transfer commercial traits into O. glumaepatula, which in turn could alter genetic diversity and increase the likelihood of local extinction. These results have important implications for in situ conservation strategies of the only wild populations of O. glumaepatula in Costa Rica.Wild crop relatives are an important source of genetic diversity for crop improvement. Diversity estimates are generally lacking for many wild crop relatives. The objective of the present study was to analyze how genetic diversity is distributed within and among populations of the wild rice species  Oryza sativa) as their major staple food and it represents 19% of the world’s per capita energy consumption (http://www.sustainablerice.org/). Wild rice species are valuable resources for breeding programs. There are 21 identified wild rice species; six of them are diploid and capable of hybridizing with commercial O. sativa (Oryza glumaepatula is the only diploid (AgpAgp) native rice species in America.Crop wild or weedy relatives (CWR) are an important source of genetic diversity for modern agriculture . Geneticsumption . One-fif. sativa . These w. sativa . Oryza gO. glumaepatula are generally small and fragmented due to specific habitat requirements . Additionally, land use changes have negatively impacted habitat availability  crops are able to move via gene flow into wild populations  however, previous reports  in a seasonal wetland in the Guanacaste Province  restricted to a small area of less than 1 Ha. The second site is located in the Medio Queso wetland (MQ) in northeastern Costa Rica . Along riverbanks, farms with livestock and maize, bean and rice plantations are common. O. glumaepatula is commonly found in MQ, with large patches of individuals on both banks of the river and in multiple sites along the MQ wetland. Along the river unconnected patches of >100 specimens are commonly found. Separation between populations may increase during the rainy season when river volume increases . Therefore, we were only able to collect 15 specimens  from Universidad de Costa Rica and represent two varieties “CR1821” and “CR5272,” which were released by the Ministry of Agriculture in the early 1970s and have been used for more than 30 years. The Comisión Institucional de Biodiversidad issued permit VI-3150-2010 to Griselda Arrieta-Espinoza authorizing field collections.In GU, we found less than a 100 pecimens . In MQ, pecimens . A boat ®  kit protocol. DNA quantity and quality were determined by gel electrophoresis and via Nanodrop quantification.Total DNA was extracted from 50 to 150 mg of dried leaf material or from 100 to 250 mg of fresh tissue. Flag leaves were used for DNA extraction as it is customary for mature plants since the flag leaf is the last to senesce  The flagIndividual genotypes were determined using AFLP following  with modO. glumaepatula and aDNA control from the Applied B iosystemskit; water was used as negative control. The selective amplification of the samples was carried out using these primer combinations twice in independent experiments and in different thermal cyclers (Hybaid and GeneAmp 9700) to assure reproducibility. Based on this data, eight combinations  were chosen for this study.Selective amplification standardization: 32 of the 64 available combinations were randomly used to determine which primers generated the most polymorphic peaks in samples of AFLPs band analysis: amplified fragments were sized on an ABI 3100 sequencer and band scoring was conducted using GeneMarker v.9.1 software. Only AFLP peaks between 150 and 500 bp were considered. The same individual was scored at least three times, to eliminate false positives. We only retained bands that had a consistent intensity (intensity higher than 500), were reproducible and, were separated by at least 2 bp from each other. Bands were scored manually as 1 (present) or 0 (absent) for each fragment within individuals. Two different analysts scored the electropherograms and the consensus between both was recorded as the multilocus genotype for each individual. We combined data from all markers (primer and selective nucleotide combination) for subsequent diversity analyses.FIS = 0.7 as estimated by To allow comparison with previous studies, band frequencies and the Shannon diversity index were estimated using GenAlEx 6.5 . Bayesiaf = 0), a model with inbreeding (f ≠ 0) but no genetic structure among populations (θII = 0). A model where Hickory chooses a random f-value from the posterior distribution , while estimating other parameters was also tested. This last model (f-free model) may circumvent bias introduced in f estimates from small samples and dominant markers  were sufficient for stable Q and α estimates. Multiple runs of Structure were conducted to test for the most likely number of possible clusters (K). We changed the number of clusters from K = 1 to K = 6 and twenty replications were conducted for each K value. The most likely number of clusters was inferred using StructureHarvester . A total of 444 AFLP bands were scored in all individuals. Genetic diversity estimates for O. glumaepatula are shown in r < 0.05, p > 0.05). Populations located on opposite sides of the Medio Queso River had comparable levels of genetic diversity. GU was the smallest and most isolated, but isolation and population size did not significantly affect genetic diversity. Both Shannon Indexes and Bayesian estimates of heterozygosity were comparable across sites  across O. glumaepatula populations (including GU), suggesting a significant heterozygote deficit. Hickory estimated a low but significant genetic structure among sites (θ = 0.023 ± 0.003). If GU is excluded from the model, genetic structure estimates do not differ significantly. When O. sativa individuals are included in the analysis, as expected, there is an increase in genetic structure (θ = 0.08 ± 0.005), suggesting differences in allele frequencies among wild and cultivated species. Similar results were shown by the neighbor-joining tree  and GU were clustered in an intermediate clade close to O. sativa individuals.According to Hickory the full model fit to the data better (DIC = 5248.19) than the model without inbreeding (DIC = 5252.19) or the model without structure (DIC = 5643.89), suggesting a significant departure from Hardy–Weinberg equilibrium (HWE). Average inbreeding was ing tree . CommercO. glumaepatula populations in MQ and grouped individuals into a single cluster . When Guanacaste was included in the analysis, Structure suggested K = 2 as the most likely number of clusters  were assigned to one cluster, whereas O. glumaepatula individuals were assigned to a second cluster. Structure indicates that wild rice individuals at S3 and GU appear to share a significant proportion of their genomes with O. sativa  and are separated by large distances between each other and from other populations in Central America or the Caribbean  among sub-populations separated by less than 15 km within the same river. Migration between sub-populations may occur at the beginning of the rainy season (May), when seeds and culms are likely carried along the river. In a review by O. glumaepatula in Costa Rica shows that it is only likely to occur in the MQ wetland . Our analyses showed that O. glumeapatula individuals appear to be admixed with cultivated O. sativa  organisms into non-GM cultivars and their wild/weedy relatives  this site is currently endangered by the construction of a road along the northern border of Costa Rica, which could imperil the largest O. glumaepatula population in Costa Rica and a likely stepping-stone between Costa Rican and Nicaraguan wild rice populations.In Costa Rica, gene flow between cultivated and wild rice species may pose an important biohazard because it allows commercial traits to be transferred into the only populations of elatives . Biosafe10.7717/peerj.1875/supp-1Supplemental Information 1Oryza glumaepatula individuals in Costa Rica and 19 Oryza sativa individuals. The data file includes a header row. First column indicates individual ID numbers. Second column indicates the location of analysed individuals. Successive columns hold AFLP data, with ones (1) indicating a band presence and (0) indicating the absence of a scorable band.AFLP data set for Click here for additional data file.


Background. The roundworm Ascaris lumbricoides infects 0.8 billion people worldwide, and Ascaris suum infects innumerable pigs across the globe. The extent of natural cross-transmission of Ascaris between pig and human hosts in different geographical settings is unknown, warranting investigation.Methods. Adult Ascaris organisms were obtained from humans and pigs in Europe, Africa, Asia, and Latin America. Barcodes were assigned to 536 parasites on the basis of sequence analysis of the mitochondrial cytochrome c oxidase I gene. Genotyping of 410 worms was also conducted using a panel of microsatellite markers. Phylogenetic, population genetic, and Bayesian assignment methods were used for analysis.Results. There was marked genetic segregation between worms originating from human hosts and those originating from pig hosts. However, human Ascaris infections in Europe were of pig origin, and there was evidence of cross-transmission between humans and pigs in Africa. Significant genetic differentiation exists between parasite populations from different countries, villages, and hosts.Conclusions. In conducting an analysis of variation within Ascaris populations from pig and human hosts across the globe, we demonstrate that cross-transmission takes place in developing and developed countries, contingent upon epidemiological potential and local phylogeography. Our results provide novel insights into the transmission dynamics and speciation of Ascaris worms from humans and pigs that are of importance for control programs. Ascaris lumbricoides, with around 760 million cases worldwide [Ascaris suum infects innumerable pigs across the globe and is especially common in organic and extensive farming systems [Ascariasis is caused by infection with the giant roundworm orldwide . Althougorldwide –4. It waorldwide , 5. The A. lumbricoides and A. suum worms are morphologically indistinguishable, there has been much debate as to whether they represent the same or different species [A. suum can infect humans and that A. lumbricoides can infect pigs, with host preference in the efficiency of infection establishment [Ascaris infections in developed countries are predominantly of pig origin [Ascaris from the 2 host species [Because adult  species , 9. In a species . Experimlishment , 11. Altg origin –4, 12, wg origin –16. Howeg origin . Hybrids species .Ascaris on a macroscale and microscale. Overall, the data support structuring of Ascaris populations between countries [Ascaris from Africa has not been extensive. Given that humans arose on this continent [Ascaris parasite here.Molecular markers have been used to investigate geographical differences in the population structure of ountries , 18–21. ountries , 20, 22,ontinent , it is pAscaris worms acquired from human and pig hosts in areas where A. lumbricoides is considered endemic or nonendemic. The level of Ascaris cross-transmission between pig and human hosts was investigated, as was the geographical structuring of parasite populations. The results provide novel insights into the evolutionary origins of Ascaris in humans and pigs.In this study, we used mitochondrial and microsatellite markers to characterize a large collection of adult The host and sampling location of the worms are summarized in Table Genomic DNA was extracted from worm gonads or muscle tissue , 19, usiAscaris cox1 gene was amplified using primers As-Co1F and As-Co1R [A 450-bp fragment of the  As-Co1R , and the As-Co1R . Eight m As-Co1R .cox1 sequences were assembled and manually edited using Sequencher v4.8 . A 383-bp consensus sequence was obtained for each sample and aligned using MacClade v4.08 . Collapse v1.2 was used to identify samples with identical haplotypes. BLAST was used to search for exact sequence matches in GenBank and novel haplotypes were submitted to GenBank (accession numbers KF719094–151). A minimum-spanning parsimony network was constructed in TCS v2.1. jModeltest v2.1.3 was used to determine the best-fitting nucleotide substitution model [p-distances between clusters.on model , using aon model  obtainedA), observed heterozygosity (HO), and expected heterozygosity (HE). Numbers of private alleles (NP) and allelic richness (RA) were calculated using FSTAT v2.9.3.2 [ST (a measure of genetic differentiation) between populations were generated, and permutation tests of genetic differentiation (101 000 permutations) were conducted. Genetic distances between parasite populations  [Ascaris populations from different host types, countries, villages, or individuals (in which the number of worms per village/host was ≥6).Microsatellite allele sizes were determined using PeakScanner v1.0 (Applied Biosystems). PowerMarker v3.25  was usedv2.9.3.2 . To takev2.9.3.2 . Pairwisstances)  were estK from 1 to 14 were tested, and 20 independent runs were performed for each K. To determine the most likely true value of K, the mean Ln P(D) value (an estimate of posterior probability) was determined for each K and plotted against K. If there was no obvious peak in the plot, ΔK was calculated and plotted against K [K. To examine whether further substructuring existed, samples were assigned to one of 2 groups on the basis of a Q value of >0.5 in the STRUCTURE output. The simulation was run again for each group separately, testing values of K from 1 to 10, using a burn-in of 100 000 and a run length of 1 000 000 to improve stability between runs. The presence of cross-transmission and hybrids between pig and human worms was investigated using STRUCTURE, NewHybrids [Population structure was also inferred from the microsatellite data set using Bayesian analysis in STRUCTURE v2.3.4 , 35. An gainst K . This anwHybrids , and BAPwHybrids , as descwHybrids , 18.cox1 sequences from 319 Ascaris worms collected from humans and 217 worms collected from pigs . A maximum likelihood tree was constructed on the basis of these haplotypes and included additional, recently published Ascaris cox1 sequences [Supplementary Figure 1). The tree revealed the same 3 groupings of Ascaris sequences with strong bootstrap support for separation between the 3 groups. Mean p-distances were 2.8% between clusters A and B, 5.4% between clusters A and C, and 4.2% between clusters B and C. None of the sequences from other ascarids fell into these groups. Whereas clusters A and B contained sequences from Ascaris collected from both host species and all 4 continents, cluster C contained only sequences from Europe and Africa.In a minimum spanning parsimony network of all haplotypes, 3 main groups could be observed Figure . Clusterequences  ; this was more apparent when Ascaris from humans in the United Kingdom were classified as pig Ascaris (P = .041). There was no difference in observed or expected heterozygosity between human and pig Ascaris .Microsatellite data were analyzed from 246 human and 164 pig worms . Populations were then subdivided on the basis of village or individual host, and genetic distances were determined . The same major division between worms from pig and human hosts was observed, with further clustering by location. Ascaris from humans in Uganda divided into 2 main clusters, one containing worms from Habutobere and Musezero and the other containing worms from other villages. The worms from Bangladesh grouped with those from Zanzibar. Ascaris worms from humans in the United Kingdom appeared most similar to worms from pigs in Tanzania and Denmark. Ascaris populations from different pig hosts in the United Kingdom were genetically differentiated.Genetic flow between on Table . There ws Figure A. Populad Figure B. The saK  was most likely 2. A bar plot from a representative STRUCTURE run is shown in Figure 2C. It was apparent that the 2 clusters corresponded to host type, apart from worms from humans in Europe, which mainly clustered with worms from pigs. To investigate further substructuring in the data set, worms were designated to one of 2 groups on the basis of the STRUCTURE output, and the simulation was run again for each group separately. Additional structure was evident, with a likely K of 4 for each group. The 4 clusters for human worms loosely corresponded to location .To investigate whether there were any cases of cross-transmission and hybridization between worms from human hosts and worms of pig origin, simulations using BAPS, NewHybrids, and STRUCTURE were conducted Table . HybridsAscaris from different locations across the globe using mitochondrial and microsatellite markers including better sampling of African isolates. We found strong genetic differentiation between Ascaris originating from human and pig hosts. However, there was also evidence of cross-transmission of Ascaris between pigs and humans, with practically all European human Ascaris infections originating from pigs and sporadic zoonotic and anthroponotic transmission in areas of endemicity.Here, we describe the first large-scale molecular analysis of human and pig cox1 haplotypes identified add to the growing database of Ascaris cox1 sequences from human and pig hosts [–C, identified previously [p-distances between cluster C and clusters B and A were surprisingly high but still lower than the 10% that is normally seen for cox1 sequences between different helminth species [cox1 haplotypes from worms of human or pig origin. Nevertheless, there were constellations of Ascaris haplotypes originating predominantly from 1 host species, suggesting that there are local barriers to the exchange of genetic material between pig and human Ascaris and adaptations to particular host species. Interestingly, haplotypes from human Ascaris in the United Kingdom and Denmark cluster practically exclusively with pig worms, indicating a zoonotic origin for these infections.The ig hosts , 39, 40.eviously , 40. The species . In addi species . The extAscaris in developing countries. These results are in accordance with findings from a number of published studies [Ascaris from humans in Europe clustered with pig worms rather than human worms from other areas, providing further evidence that Ascaris infection is a zoonosis in developed countries [cox1 haplotype (H64). These Ascaris parasites came from an organic abattoir in Bedfordshire, but the location of the farm(s) where the pigs were reared is not known. It is possible that a human Ascaris transmission cycle exists in pigs, an extremely surprising result which warrants further investigation. No examples of hybridization between pig and human worms were found in any location. In contrast, 7.8% of worms in China (n = 258) and 4% in Guatemala (n = 24) were identified as hybrids using 23 microsatellite markers, indicating the potential for exchange of genetic material between pig and human Ascaris populations [Ascaris worms in different locations. Alternatively, the 8 markers we used may not provide sufficient resolution to identify hybrids.Using different approaches for analysis of microsatellite data, we observed genetic differentiation between human and pig  studies –18, 43. ountries –4, 12. Uountries , 1 of 14ountries . The difulations , 18. TheAscaris populations from different countries. Additional structuring of parasite populations at the village or individual host level was also apparent, particularly in Uganda where the largest sample of worms originated. This may be related to the well-documented overdispersed distribution and clustering of Ascaris infections at household level [As anticipated , 18, 20,ld level , 45. Genld level . The 4 cAscaris in Africa. Since the evolution of early hominids is thought to have taken place here [Ascaris. The oldest ascarid eggs discovered so far were found in France [Ascaris originated. It is clear that, ascariasis has been intimately intertwined with humans for thousands of years, as recently demonstrated by infections discovered within the body of King Richard III [Because of greater sampling, our study provides a better picture of the genetic diversity of ace here , it is pn France . Howeverhard III .Ascaris in human and pig hosts is contested. It is unclear whether A. lumbricoides and A. suum are separate species derived from a common ancestor, multiple host colonization events took place after geographical subdivision of host populations [A. lumbricoides and A. suum are really a single species with regional variants [A. lumbricoides and A. suum are 2 separate species, since there are high levels of genetic differentiation between pigs and human worms in sympatric areas. Although hybrids between pig-derived and human-derived worms have been identified, these are rare and likely to have reduced fitness, otherwise population structuring between the hosts would not be observed [Ascaris populations infecting pigs or humans. However, the mitochondrial data and the fact that no single diagnostic marker has been identified to distinguish between A. lumbricoides and A. suum favor an alternative model of multiple host switches over time, with subsequent merging of worm populations in the 2 hosts [The older evolutionary origin of ulations , or A. lvariants . This divariants , we suggobserved . Regardiobserved . This co 2 hosts . This ex 2 hosts .Ascaris-endemic communities with anthelmintic drugs, and there are real prospects for local elimination of disease by treatment scale-up [The current mainstay of control programs against ascariasis in humans is preventive chemotherapy, involving periodic treatment of preschool and school-aged children in scale-up . In areaAscaris from humans and pigs. Although A. lumbricoides and A. suum appear to be 2 separate species over most of their range, the process of speciation is not so far advanced that they are entirely host specific or unable to exchange genetic information, and cross-transmission between pig and human hosts takes place across the globe. The level of cross-transmission is likely to depend on local farming and hygiene practices. However, it is possible that we underestimated the zoonotic potential of A. suum, as we only analyzed adult worms . It is probable that A. suum commonly undergoes visceral larvae migration in humans, but because of host preferences most larvae are expelled on return to the intestine. Zoonotic and anthroponotic transmission could lead to greater morbidity because hosts are less well-adapted to the parasite. In addition, potential exchange of genetic information between A. lumbricoides and A. suum could allow the spread of drug-resistance and virulence genes between parasite populations in different hosts, although it is also possible that if humans are treated but pigs are not, the untreated parasite population will act as refugia, diluting the alleles linked to resistance.In conclusion, our results provide novel insights into the transmission dynamics and speciation of Supplementary materials are available at The Journal of Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.