Lobelia rhynchopetalum. The study was based on eight populations sampled from two mountain systems in Ethiopia. A total of 20 variable sites were obtained, which resulted in 13 unique haplotypes and an overall nucleotide diversity (ND) of 0.281 ± 0.15 and gene diversity (GD) of 0.85 ± 0.04. Analysis of molecular variance (AMOVA) revealed a highly significant variation (P < 0.001) among populations (FST), and phylogenetic analysis revealed that populations from the two mountain systems formed their own distinct clade with >90% bootstrap support. Each population should be regarded as a significant unit for conservation of this species. The primers designed for this study can be applied to any Lobelia and other closely related species for population genetics and phylogenetic studies.DNA sequence data from the internal transcribed spacer of nuclear ribosomal DNA and eight chloroplast DNA regions were used to investigate haplotypic variation and population genetic structure of the Afroalpine giant lobelia,  Lobelia is the largest genus within the subfamily Lobelioideae of Campanulaceae family comprising over 350 species that range from small herbs to giant woody plants. Lobelia rhynchopetalum Hemsl., which belongs to the subgenus Tupa, section Rhynchopetalum [L. rhynchopetalum has evolved. L. rhynchopetalum and other giant lobelias of eastern Africa have the same chromosome number (2n = 28) as that of their progenitor [opetalum , is one opetalum , 3. It bopetalum . These aogenitor  and, thuL. rhynchopetalum is a monocarpic perennial species endemic to the Ethiopian drained sites of the Afroalpine ecoregion . The. TheL. ron (e.g. ), which on  of nuclear ribosomal DNA (rDNA) and eight chloroplast DNA (cpDNA) regions were generated from L. rhynchopetalum with the objectives of (1) population genetic and phylogenetic analyses for its conservation and evolutionary significance, (2) evaluating the utility of these DNA regions for population genetic and phylogenetic analyses, particularly within the genus Lobelia, and (3) contributing DNA sequence data from this species to Genbank so that it can be used for broader phylogenetic and phylogeographic analyses in combination with DNA sequence data from other Lobelia species.In the present study, DNA sequence data from the internal transcribed spacers  and Simien (13°05′N–13°25′N and 37°50′E-38°30′E) mountain systems were used in this study  and Helianthus annuus L. (Asteraceae) . These primers were named  amplified part of the trnfM gene and the full length of the trnG gene and the trnfM-trnG and the trnG-psbZ intergenic spacers. This part of the trnfM-trnS region was sequenced using primer 49584R. The second pair of primers (49595F and 50079R) amplified part of the psbZ gene and the psbZ-trnS intergenic spacer that were sequenced using primer 50079R. Similarly, part of the petN-trnC intergenic spacer was amplified and sequenced using primers 58099F and 58955R. The  primers . The fir10002F and 10226R were used to amplify the trnG-trnR intergenic spacer  of the clpP gene, whose partial sequence was obtained by using primer 111454R. The 3′trnK-matK portion of the trnK intron was amplified using primers 1825F and 2195R, and the amplified fragment was sequenced using primer 1825F. Similarly, the psbD-trnT intergenic spacer was amplified using primers 53562F and 54107R. The reverse primer, 54107R, was used to obtain the partial sequence of this spacer (μL of 5 μM sequencing primer and sent to the sequencing facility at the University of Oslo (http://www.bio.uio.no/ABI-lab/), where DNA sequencing was carried out. The representative nucleotide sequences of ITS and the eight cpDNA regions of L. rhynchopetalum were submitted to nucleotide sequence database (NCBI GenBank), and their accession numbers are given in Primers c spacer . The coms spacer . The PCRDNA sequences were edited using BIOEDIT version 7.0.5 , and theITSs ,GCC trnG gene, and trnfM-GCCtrnG, GCC-psbZ,trnG and UCCtrnG-trnR intergenic spacers. Partial sequences from the trnT-trnL, psbT-psbB, psbD-trnT, psbZ-trnS, petN-trnC intergenic spacers, trnfM, psbZ, and matK genes, and clpP intron-2 and 3′trnK-matK introns were also obtained. A total of 20 variable sites were obtained within L. rhynchopetalum, of which 3 are indel positions and the remaining 17 are substitutions. Indels were only obtained in the ITS and trnT-trnL regions. The trnT-trnL intergenic spacer sequences of all individuals from the Bale mountains were shorter by one nucleotide as compared to those from the Simien mountains. This single nucleotide long indel has clearly differentiated the populations according to their mountain system of origin. In this study, full sequence length was obtained for the L. rhynchopetalum determined after coding indels according to the simple indel coding method of Simmons and Ochoterena [ L. rhynchopetalum was obtained from ITS, as expected. Eleven variable sites were obtained in the entire ITS region, nine within ITS-1 and two within ITS-2. The psbT-psbB, psbD-trnT, trnfM-trnS, and clpP intron-2 sequences were the least variable regions, as only a single variable site per region was obtained within L. rhynchopetalum. In the case of the trnfM-trnS region, the variable site was located within the psbZ-trnS intergenic spacer. The average percent variable sites (%VS) of cpDNA regions and ITS within L. rhynchopetalum were 0.4% and 1.4%, respectively. These values were increased to 9% and 28%, in that order when the L. rhynchopetalum sequence was aligned with that of Lobelia erinus. The number of variable sites for each region of hoterena  is given L. rhynchopetalum and L. erinus were aligned. Based on the aligned sequences of these species, different parameters of sequence variation of each DNA region were generated (trnG-trnR) to 27.5 (ITS), of which 100% and 25.8% were parsimony informative, respectively. This analysis revealed a large number of parsimony informative variable sites in all DNA regions investigated in this study, with the exception of the trnG-trnR intergenic spacer  and nucπ) within populations ranged from 0.00 to 2.99, with the highest obtained in the Goba-1 population. The estimates for MIHD and π were higher within the Bale mountains than within the Simien mountains  ranged from −1.33 to 1.22 (θ), a central parameter in population genetic models, summarizes the rate at which mutation and random genetic drift generate and maintain variation within a given DNA region. The estimates of three θ estimators  are given in θπ was almost the same as that of π except the slight differences in standard deviation. The highest estimates for the three θ estimators were obtained in the Goba-1 population. The mean interhaplotypic distance (MIHD) was calculated based on the Kimura distance method . The higountains . Tajima'ains  was als to 1.22 , which iFST = 0.40). Overall, the analysis revealed that 85% of the variance in the distance matrix was accounted for by differences among populations and only 15% by diversity within populations (P < 0.001) within geographic locations and overall. The hierarchical AMOVA also revealed a significant geographic differentiation of L. rhynchopetalum populations. In addition, the minimum spanning tree  of nuclear rDNA and eight cpDNA regions for intra- and interpopulations genetic analyses of L. rhynchopetalum. In addition to information from published reports, our choice of the cpDNA regions used in this study was based on the aligned DNA sequences of Trachelium caeruleum and Helianthus annuus. The alignment of the sequences of these two species revealed mononucleotide repeat microsatellites and a large number of variable sites within the trnT-trnL, trnG-trnR, psbT-psbB, and psbD-trnT intergenic spacers, the 3′trnK-matK portion of the trnK intron, and the clpP intron-2. These regions were, therefore, targeted to identify polymorphic microsatellites within L. rhynchopetalum. However, no polymorphic microsatellites were found, and thus only variable sites due to nucleotide substitutions and indels were used. The presence of intraspecific variation in nuclear rDNA e.g., , 11, 12), 1211, 1ITS region has been commonly used for plant molecular systematics at lower taxonomic levels and for intraspecific genetic studies since it was first used in phylogenetic inference [ITS was over threefold more variable than the average variation obtained within the cpDNA regions in L. rhynchopetalum. The psbD-trnT region has been considered as one of the highly polymorphic regions of cpDNA [trnT-trnL, 3′trnK-matK, petN-trnC, psbT-psbB (as part of psbB-psbH), and trnfM-trnS regions are also among the fast evolving cpDNA regions [trnfM-trnS region was proved to be informative at the intraspecific level in Eritrichium nanum [Vigna angularis [trnG-trnR intergenic spacer and the clpP intron were not part of the 34 fast evolving cpDNA regions reported in [The nference . It has of cpDNA  and provof cpDNA ). The tr regions . For exaum nanum  and Vignngularis . The trnorted in  and may trnG-trnR region was revealed to be the least variable and parsimony informative in the aligned sequences of L. rhynchopetalum and L. erinus regardless of the fact that two variable sites were obtained within L. rhynchopetalum (see %VS) and percent parsimony informative sites (%PIS) were considered, the clpP intron 2 was found to be as informative as previously reported fast evolving cpDNA regions [trnG-trnR intergenic spacer, have a large number of parsimony informative variable sites, which can be used for phylogenetic analysis of the genus Lobelia and its various subgenera and sections, as exemplified using L. erinus. The alum see . On the  regions  and hencL. rhynchopetalum is more closely related to Lobelia aberdarica R. E. Fr. & T. C. E. Fr. than to most other east African giant lobelias, including Lobelia gibberoa Hemsl. [ITS aligned sequence of L. rhynchopetalum and L. aberdarica . The alignment of the 3′trnK-matK intron partial sequences (309 bp) of L. rhynchopetalum and L. aberdarica  revealed ten potentially informative sites. Similarly a 489 bp trnT-trnL aligned partial sequence of L. rhynchopetalum and L. giberroa  revealed 18 potentially informative sites. Hence, the combination of these cpDNA regions could sufficiently resolve the phylogenetic relationships between the east African giant lobelias and beyond.a Hemsl. . Thirty L. rhynchopetalum in situ and make reasonable decisions about sampling procedures of germplasm for their ex situ conservation, one should know how its genetic variation is distributed within the species and what their population genetic structure looks like. The DNA sequence data from ITS and eight cpDNA regions proved to be a useful tool for this purpose and successfully applied to reveal the genetic diversity and population genetic structure of this endemic giant lobelia, regardless of limited number of variable sites obtained. Of the eight populations, two populations showed high genetic variation while three populations showed no variation within population. These populations were from both mountain systems, suggesting that the extent of within-population diversity is not limited to geographic regions. However, Simien mountains appeared to be a more favorable habitat for L. rhynchopetalum than the Bale mountains, as four of its five populations showed intrapopulation haplotypic diversity.To develop conservation strategies that preserve maximum levels of genetic diversity of L. rhynchopetalum populations may be best explained by genetic drift in relation to their small population size and limited gene flow between populations, as small isolated populations are highly likely to diverge from each other due to genetic drift, which causes fixation of alleles. Many natural ecosystems are subject to habitat fragmentation, which results in smaller and more isolated populations. Plant species remaining in fragmented habitats are of conservation concern due to impacts of decreased population size and increased isolation that threaten their viability due to genetic drift and consequently lower genetic diversity (e.g. ). The coθ) is a central parameter in population genetic models for the balance between mutation and random genetic drift. For haplotypic data, theta is measured as follow θ = 2Neμ, where Ne is effective population size and μ is a mutation rate per nucleotide site under neutral evolution theory model [θ summarizes the rate at which mutations and random genetic drift generate and maintain variation within a given DNA region, under conditions in which natural selection is not operating. There were only two variants per polymorphic site in our aligned DNA sequence data set, which fits to the assumption of the infinite-site model. Pi (π) is the other measure of sequence variability, which measures the pairwise differences between sequences. In this study, the estimates for θπ and π are similar. This suggests that the data fits with the infinite-site model, as these two parameters have equal expectation under this model  is considered to be zero for neutral loci [D was not significantly different from zero in all populations and groups. Therefore, we conclude that the neutral mutation hypothesis explains the DNA sequence variation obtained in this study. Population differentiation is driven by various evolutionary forces such as mutation, gene flow, genetic drift, and selection, and its extent depends on the relative strength of these individual forces in interaction with life history traits of the species. Theta . Tajimaral loci . In our ex situ from each population to reduce the risk of losing unique genetic variants. For in situ conservation, priority should be given to populations with relatively high genetic diversity. The results of this study and the ISSR-based study [in situ conservation. However, simultaneous consideration of the two data sets and environmental factors suggests Goba-1 and Debark-1 as good candidates for in situ conservation. Thirteen unique haplotypes were obtained by analyzing 40 individual plants. Only three of these, haplotypes were shared among populations of the same mountain systems. Analysis of molecular variance revealed that a high proportion (85%) of the genetic variation was found among populations, and within-population variation only accounted for 15% of the total variation. The 9 variable sites obtained from cpDNA regions were the major source of the highly significant population differentiation at all hierarchical levels, as only three of these sites were variable at the intrapopulation level. Such high population differentiation is not uncommon in endemic species with limited distribution and small population sizes, as a consequence of the pronounced effects of genetic drift . When ped study  are not L. rhynchopetalum populations at various hierarchical levels. Such a high population differentiation can be partly explained by limited gene flow. Several factors, such as geographic distance between populations, pollen and seed dispersal mechanisms, and mode of reproduction, have a direct impact on the extent of gene flow between populations. For example, the significant differentiation between the two mountain systems can be partly explained by the Rift Valley as a barrier to gene flow. Similar results were previously reported in L. giberroa between these mountain systems [AMOVA revealed a highly significant differentiation not only between populations but also among the two mountain systems . The dif systems . Such a Lobelia acrochila (E. Wimm.) Knox is the most closely related species to L. rhynchopetalum [L. acrochila in such type of studies will shed more light to the evolutionary radiation of giant lobelias in eastern Africa. The fact that the Simien mountains are the northernmost end of the distribution of giant lobelias in eastern Africa and the suggestion that giant lobelias were expanding from south towards north [Knox and Palmer  suggesteopetalum , 36. Theds north  may giveL. rhynchopetalum. The combination of deterministic and stochastic factors and factors affecting gene flow seems to have played a significant role for the highly significant differentiations of the species at different hierarchical levels. With about 85% of the total genetic variation residing in between populations, each population should be regarded as an important contributor to the overall amount of genetic variation and a significant unit for conservation efforts of this species. Our recommendation is that representative populations from different altitudes and geographic locations should be targeted for conservation purposes, as it reduces the risk of losing unique genetic variants due to several factors. The DNA sequence data generated and submitted to GenBank and the nine pairs of new primers that can be applied to any Lobelia and other closely related species, both for population genetic and phylogenetic studies, are also a significant contribution of this work. Furthermore, since the cpDNA primers were designed based on the aligned sequences of Trachelium caeruleum (Campanulaceae) and Helianthus annuus (Asteraceae), they may be useful for similar studies not only in lobelias but also in various Campanulaceae and Asteraceae species.This work has revealed the existing haplotypic variation and population genetic structure of


Third party species, which interact with one or both partners of a pairwise species interaction, can shift the ecological costs and the evolutionary trajectory of the focal interaction. Shared genes that mediate a host’s interactions with multiple partners have the potential to generate evolutionary constraints, making multi-player interactions critical to our understanding of the evolution of key interaction traits. Using a field quantitative genetics approach, we studied phenotypic and genetic correlations among legume traits for rhizobium and herbivore interactions in two light environments. Shifts in plant biomass allocation mediated negative phenotypic correlations between symbiotic nodule number and herbivory in the field, whereas positive genetic covariances suggested shared genetic pathways between nodulation and herbivory response. Trait variance-covariance (G) matrices were not equal in sun and shade, but nevertheless responses to independent and correlated selection are expected to be similar in both environments. Interactions between plants and aboveground antagonists might alter the evolutionary potential of traits mediating belowground mutualisms (and vice versa). Thus our understanding of legume-rhizobium genetics and coevolution may be incomplete without a grasp of how these networks overlap with other plant interactions. Though we often study the evolution of species in isolation, organisms in nature engage simultaneously in interactions with multiple species Spodoptera exigua) on the model legume Medicago truncatula in symbiosis with the rhizobium Sinorhizobium meliloti showed that herbivory increased nodule numbers and, therefore, increased mutualism benefits to rhizobia Legume-rhizobium-insect interactions provide a tractable system with which to investigate the evolutionary and ecological effects of tripartite interactions, in part because they can be manipulated in the greenhouse and field via changes in root biomass, and 2) the availability of plant carbon would alter the strength or even direction of these responses. Specifically, shading plants would be expected to lessen any positive relationship between herbivory and nodulation by decreasing the carbon pools available for belowground allocation.Although Heath & Lau Alternatively, herbivores might increase nodulation via shared signaling networks operating independently from plant carbon status. If shared networks underlie both traits, then variation at these genes would be expected to generate genetic correlations between nodulation and herbivory, regardless of changes in root biomass. For example, jasmonic acid (JA) levels increase after herbivore damage Medicago truncatula (barrel medic) Here we use a quantitative genetics approach to study the context-dependence and genetic architecture of plant interactions with aboveground herbivores and belowground mutualists. Plant genotypes from multiple natural M. truncatula. Each family contained the offspring of a single field-collected maternal individual propagated in the greenhouse for at least one generation in order to minimize maternal environmental effects. Because M. truncatula is highly self-fertilizing in nature, individuals from a single maternal family are expected to be nearly genetically uniform M. truncatula have shown among-population and among-family genetic variation for symbiosis with rhizobia To assess how the light environment affects the tripartite interactions between plants, their aboveground herbivores, and belowground rhizobium mutualists, we grew 55 plant genotypes in a split-plot design with 30 plots , with either a sun or shade treatment applied at the whole-plot level, and genotypes assigned new randomized locations within each plot. Plant genotypes corresponded to inbred maternal families (hereafter “families”) from eight natural populations in France, in the native range of M. truncatula seeds were manually scarified and surface-sterilized by dipping briefly in 100% ethanol followed by 7 minutes in commercial bleach, then were glued to individual sterile toothpicks to facilitate planting and seedling identification. Genotypes were randomized into each 2.5 m2 experimental block in a freshly tilled agricultural field in Urbana IL, with ∼30 cm spacing between plants. The experiment was watered immediately after planting and approximately thrice per week thereafter, with the exception of rainy days. Blocks in the shade treatment were covered by 2.5 m×2.5 m square of 50% shade cloth suspended 45 cm off the ground by a PVC scaffold. Light levels (PAR) averaged 1277.1±53.5 umol/m2s (mean ± SD) in sun, versus 522.3±48.4 umol/m2s in shade blocks, as measured using a quantum flux meter . Shade treatments also decreased soil and leaf temperature (e.g. from an average of 41.8±2.2 C to 32.3±3.0 C for soil and from 32.3±2.1 C to 30.1±1.7 C for leaf), as measured using an infrared thermometer  on 12 August, 2010. Probably as a result of the frequent watering necessary to maintain the experiment, soil moisture content did not differ between plots (experiment-wide mean volumetric water content ± stdev = 39.7% ±2.8), as measured using a soil moisture probe  on 17 August, 2010.Spodoptera ornithogalli and Colias eurytheme, were routinely observed causing the type of herbivore damage typical of experimental plants . Blocks were harvested between September 11 and September 24, 2010. For each plant, the entire root system was dug up, soaked in a bucket of water to loosen and remove all soil, and scored for the total number of nodules present. Aboveground and belowground biomass were then clipped apart, dried, and weighed separately. Because nodules in the field senesce before fruits are ripe, direct estimates of plant reproductive fitness were not feasible.Of the initial June 26 planting, 90% of seeds did not emerged, and these individuals were replaced with a second planting on July 16. Because we were interested in how natural levels of herbivory interacted with shade treatments to impact plant growth and nodulation, herbivory was scored as the proportion of damaged leaves  at seven weeks after initial planting. This metric should be correlated with total leaf area removed and moreover should not be biased across treatments. Caterpillars of two lepidopteran species, Germination and survival in the field were low. Planting date (June 26 versus July 16) was not a significant source of variation for any traits and thus was not included in final analyses. Despite the second planting, only 27% of the 1650 planted survived to harvest. To encourage mixed models to converge in order to estimate genetic effects, we included only plant genotypes that were represented by at least two or more individuals per treatment  in the analyses. Of the 55 families initially planted, 46 met this criterion; thus although light treatment affected mortality (see Results), differences were not severe enough to lead to heterogeneity in the families represented in the two light treatments. Seedling mortality might have been non-random among families; therefore, the remaining subset might represent a biased sample of the genetic variation, though in what way cannot be known.Except where indicated, analyses were implemented in SAS software . We used both MANOVA in PROC GLM  and univariate PROC MIXED to test for the fixed effects of plant population, light treatment, and treatment × population and the random effects of plant maternal family nested within population, treatment × family, and block nested within treatment on each of the four dependent variables. We did not model interactions with block. Pairwise Pearson (phenotypic) correlations were computed among all four traits  using PROC CORR. Transformations did not dramatically alter the results, but can alter the biological interpretation of data a priori model in which herbivory could affect nodule number directly as well as indirectly via root and shoot biomass, using AMOS version 7 2 = 0.42, d.f. = 1, p = 0.517). Fit indices also suggested that this model fit the data  genetic variance-covariance (G) matrix describes the broad-sense genetic variances and covariances of traits in each light environment, and thus can illuminate how trait correlations might be expected to influence the outcome of selection on individuals within populations under different light environments. To obtain G matrices, we used repeated measures mixed model analysis of variance implemented in PROC MIXED to model the fixed effects of trait , light treatment (sun or shade), population, population × light treatment, block nested within treatment, and the random effect of plant maternal family nested within population on all phenotypic observations by specifying the “type = UN” solution statement a priori in the response of the herbivory-nodulation covariance to light environment, we tested this by comparing the full model (with the grouping term) to one in which this particular covariance was constrained to be equal in the two environments. We tested whether responses to multivariate selection would be expected to change depending on the light environment using the random skewers method of Cheverud We tested for differences in genetic architecture between the two light environments in multiple ways. First, we dropped the grouping term in both random statements and calculated likelihood ratio tests for the null hypothesis of no significant decrease in model fit 2 = 0.63, d.f. = 1, p = 0.429; CFI = 1.0; RMSEA = 0.0). In contrast to the family-level G matrix, the population-level correlation matrix describes the among-population genetic component of trait variances and covariances Because we found significant among-population trait variation, among-population genetic correlations for all traits were also approximated by performing correlations and structural equation modeling (described above) using lsmeans for each population in each treatment (output by the multivariate model described above). The ‘population’ SEM fit the population-level data adequately . Plants in the shade environment were larger and produced significantly more nodules , root biomass  and shoot biomass  than plants in the sun treatments. In addition to being larger, plants in the shade also experienced less insect damage on a per-leaf basis . All traits were phenotypically correlated in this experiment  to tease apart the effects of these correlated traits on nodule number. Most importantly, the non-significant direct effects of herbivory on nodulation in both sun and shade . The staIn order to assess the genetic architecture underlying this suite of correlated plant traits in the field, we first estimated G matrices representing the among-family (inbred line) genetic component . Nevertheless, the main covariance of interest, that between herbivory and nodulation, was equal in the two light environments; specifically, a model in which sun and shade covariances were estimated separately was not significantly more likely than a model in which they were constrained to be equal . Responses to multivariate selection in the sun versus shade environment were not predicted to differ significantly using the null hypothesis-corrected random skewers method . Finally Flury hierarchical analysis indicated that, although the matrices were not equal or even proportional , their eigenstructure was similar . In sum, these results provide little evidence that responses to selection on these interaction traits in M. truncatula would differ drastically between light environments.We used multiple approaches to test whether the genetic architecture of these traits differed significantly between light environments, with differing results. Model fit was improved when G matrices were estimated separately for each light treatment . Although our results suggest a role for biomass allocation in mediating these interactions, it is not in the expected direction. More nodulated plants in this experiment had lower levels of herbivory (both within and among light treatments), and this phenotypic relationship between herbivory and nodulation was entirely mediated via changes in plant biomass allocation. Other authors have also found that larger plants experience less herbivory, possibly because plants in more favorable conditions have more resources to allocate to both growth and defense M. truncatula could generate such a pattern. Here we predicted that, if shared signaling networks underlie both responses, then positive genetic correlations should exist between herbivory and nodulation. The positive genetic covariances/correlations between nodulation and herbivory found here at both the among-family and among-population level – even after controlling for changes in plant size – are indeed consistent with a shared genetic basis for plant responses to herbivores and rhizobia. Importantly, because neither nodulation nor herbivory was manipulated in this experiment, these results could be driven by either interactor.In contrast to the negative phenotypic correlation between herbivory and nodule number we observed here, a previous manipulative study in this system found that herbivory increased nodule numbers It is important to note that genetic covariance at any geographic scale can be generated by three distinct causes: pleiotropy resulting from shared genetic mechanisms, physical linkage, or linkage disequilibrium resulting from historical evolutionary processes M. truncatula have typically found that most quantitative genetic variation exists within populations Genetic variance in nodulation was found among populations, in addition to among families within populations, in this experiment. Previous studies in this and other systems have found population-level genetic variation in either nodulation or herbivory Population-level genetic covariance, such as that found between herbivory and nodulation in the current experiment, has long been a subject of interest and debate in evolutionary biology. Analogous to the effect of correlated traits on population evolution Because herbivores and rhizobia both utilize plant carbon, we predicted that carbon limitation imposed by a shade treatment might generate negative correlations (phenotypic or genetic) between herbivory and nodulation. We found little evidence supporting this prediction. Negative phenotypic correlations between herbivory and nodulation in both environments were mediated entirely via changes in root and shoot biomass, probably reflecting a more general plant tradeoff between aboveground and belowground allocation Figure S12).Path diagrams showing the unstandardized regression weights and the amount of variation in each variable explained by the input arrows  and B ; those of the ‘population’ SEM on population means are shown in panels C  and D .(TIF)Click here for additional data file.Table S1M. truncatula. For MANOVA, Wilks Lambda is shown; in univariate models, F-statistics are shown for fixed effects, and χ2 statistics (df = 1) are shown for random effects, as well as percent variance explained (PVE).MANOVA and univariate mixed model ANOVA (using REML) results for the effects of light treatment (sun or shade), plant population, maternal family and block on nodulation, insect herbivory, and root and shoot biomass of field-grown (DOCX)Click here for additional data file.Table S2*p<0.1; **p<0.01;***p<0.001;****p<0.0001Phenotypic correlations (N) between dependent variables in the two light treatments (sun or shade). Trait correlations in shade are in grey below the diagonal; trait correlations in sun are in white above the diagonal. (DOCX)Click here for additional data file.


Arabidopsis arenosa and A. lyrata are here used to study interspecific and interploidal gene flow, using a combination of nuclear and plastid markers.Effects of polyploidisation on gene flow between natural populations are little known. Central European diploid and tetraploid populations of A. arenosa to tetraploid A. lyrata and vice versa, and some interploidal gene flow, which was unidirectional from diploid to tetraploid in A. arenosa and bidirectional in A. lyrata.Ploidal levels were confirmed by flow cytometry. Network analyses clearly separated diploids according to species. Tetraploids and diploids were highly intermingled within species, and some tetraploids intermingled with the other species, as well. Isolation with migration analyses suggested interspecific introgression from tetraploid Arabidopsis, we find very different evolutionary fates when it comes to introgression.Interspecific genetic isolation at diploid level combined with introgression at tetraploid level indicates that polyploidy may buffer against negative consequences of interspecific hybridisation. The role of introgression in polyploid systems may, however, differ between plant species, and even within the small genus  Polyploidy, i.e. whole genome duplication, has long been considered a major evolutionary force in the Plant Kingdom , and Chamerion angustifolium (L.) Holub revealed that autotetraploids are not necessarily instantly isolated from their diploid progenitors, but that the isolation can become more prevalent through time . More rugh time ,20. Slotugh time  showed tgenitors -26, althgenitors ,27-30. Tgenitors ], or pasgenitors ]. These genitors , could bArabidopsis (DC.) Heynh. is a small genus consisting mostly of diploids, but includes both allopolyploids , makingggregate  with dip regions ,43,44. S regions  detectedowledged . Here wex and 4x) from both arenosa and lyrata were included in this study  was carried out in 25 μl volumes with 1× DyNAzyme EXT buffer , 0.2 mM of each dNTP, 0.6 μM of each primer  with 0.6 μM of each primer . Polymerase chain reaction (PCR) of the low-copy nuclear regions chalcone synthase (CHS) and short chain alcohol dehydrogenase  constitute a well-known problem in PCR-based cloning protocols e.g. -55], and, and55]]Intragenic recombination events may be relatively common , and shoWhen two or three different alleles are found in a tetraploid individual, it is not possible to determine the true genotype although dosage may give an indication. Computational methods based among others on the EM algorithm have been developed to infer genotypes assuming random mating and populations at equilibrium [e.g. ]. As we Diversity indices were calculated for each molecular region and each species/ploidal level, separately. For the tetraploids we calculated the indices for the duplicated datasets D1, D2, D3, and D22, and averaged the estimates. Arlequin version 3.11  was usedarenosa and lyrata were first analysed separately, to determine whether there is gene flow between ploidal levels and in which direction it occurs. Second, in order to assess gene flow between species, both taxa with both ploidal levels were analysed together in an analysis with four populations. We assumed one ancestral population for each species, and one ancestral population for the whole complex. In such an analysis with four populations a large number of parameters have to be estimated, requiring a large amount of data to obtain reliable estimates. As the three loci available here were somewhat limited in that respect, we also analysed only the two diploid taxa to assess evidence for interspecific gene flow. All analyses involving tetraploids were carried out for the four different datasets of tetraploid genotypes. One of the assumptions of the IM model is that there is no important gene exchange with populations not included in the analysis. As there was evidence for significant gene flow between ploidal levels in both species, we did not analyse the two tetraploids together. Another assumption of IMa2 is that there is no recombination. Tests for recombination . Our  studies  considerlyrata from the Czech Republic, and a diploid plant was detected in the mainly tetraploid a4_AUT1 population of arenosa from the Eastern Austrian Foreland. Also, the Central European Arabidopsis populations often have restricted distributions, and populations of different ploidal levels are sometimes only a few kilometres apart. Thus, introgression is possible, especially along disturbed sites, e.g., along roads. However, the generally low migration rates suggest that introgression remains a rare event. If this is the case, the small genus Arabidopsis contains polyploids with very different evolutionary fates when it comes to introgression. The allotetraploid A. suecica (Fr.) Norrl. ex O.E.Schulz has very low genetic diversity and has probably originated only once . As sucs et al.  for dists et al.  in an atx, 2x, and 3x gametes, and may therefore contribute to gene flow in both directions [Dactylorhiza maculata (L.) Soó [Betula L [arenosa  Soó  and BetuBetula L . In our Arabidopsis as a model system. There was no evidence for interspecific gene flow between 2x arenosa and 2x lyrata, which can be considered as good biological entities, but some support for gene flow into 4x lyrata and possibly 4x arenosa. Thus, whole genome duplication might decrease vulnerability to interspecific hybridisation and buffer negative effects of introgression. Interploidal gene flow was detected from 2x to 4x in both species, and from 4x to 2x in lyrata. For arenosa, the two ploidal levels are allopatric, and the lower level of gene flow could be the result of geographic as well as genetic barriers. In lyrata, however, where geographic barriers are limited, gene flow is bidirectional.In this study, we looked at the effect of polyploidisation on interspecific introgression, and interploidal gene flow using Central European MHJ carried out the molecular work and statistical analyses, and drafted the manuscript. DE carried out the isolation with migration analyses and helped to draft the manuscript. RS and MAK contributed to the sampling design, provided the samples and contributed to draft the manuscript. AKB was project leader, contributed in the molecular work, and drafted the manuscript. All authors read and approved the final manuscript.Table S1. Dataset summary with primers, GenBank numbers, alignment lengths and minimum number of recombination events per region.Click here for fileTable S2. Isolation with migration (IM) analyses: description of datasets.Click here for fileTable S3. Isolation with migration analyses: mutation rates per fragment per generation.Click here for fileTable S4. Signs of introgression among tetraploid Arabidopsis arenosa and A. lyrata.Click here for fileTable S5. Diversity indices.Click here for fileFigure S1. Isolation with migration analyses: effective population sizes and estimates of time for the def datasets.Click here for fileFigure S2. Isolation with migration analyses of the seq datasets.Click here for file


Trypanosoma cruzi, the etiological agent of Chagas disease, in southern Ecuador (Loja Province). This kinetoplastid parasite has traditionally been a paradigm for clonal population structure in pathogenic organisms. However, the presence of naturally occurring hybrids, mitochondrial introgression, and evidence of genetic exchange in the laboratory question this dogma.Molecular epidemiology at the community level has an important guiding role in zoonotic disease control programmes where genetic markers are suitably variable to unravel the dynamics of local transmission. We evaluated the molecular diversity of Eighty-one parasite isolates from domiciliary, peridomiciliary, and sylvatic triatomines and mammals were genotyped across 10 variable microsatellite loci. Two discrete parasite populations were defined: one predominantly composed of isolates from domestic and peridomestic foci, and another predominantly composed of isolates from sylvatic foci. Spatial genetic variation was absent from the former, suggesting rapid parasite dispersal across our study area. Furthermore, linkage equilibrium between loci, Hardy-Weinberg allele frequencies at individual loci, and a lack of repeated genotypes are indicative of frequent genetic exchange among individuals in the domestic/peridomestic population.T. cruzi transmission. As such they have dramatic implications for our understanding of the fundamental genetics of this parasite. Our data also elucidate local disease transmission, whereby passive anthropogenic domestic mammal and triatomine dispersal across our study area is likely to account for the rapid domestic/peridomestic spread of the parasite. Finally we discuss how this, and the observed subdivision between sympatric sylvatic and domestic/peridomestic foci, can inform efforts at Chagas disease control in Ecuador.These data represent novel population-level evidence of an extant capacity for sex among natural cycles of  Trypanosoma cruzi is transmitted by blood sucking insects known as triatomines. This protozoan parasite commonly infects wild and domestic mammals in South and Central America. However, triatomines also transmit the parasite to people, and human infection with T. cruzi is known as Chagas disease, a major public health concern in Latin America. Understanding the complex dynamics of parasite spread between wild and domestic environments is essential to design effective control measures to prevent the spread of Chagas disease. Here we describe T. cruzi genetic diversity and population dynamics in southern Ecuador. Our findings indicate that the parasite circulates in two largely independent cycles: one corresponding to the sylvatic environment and one related to the domestic/peridomestic environment. Furthermore, our data indicate that human activity might promote parasite dispersal among communties. This information is the key for the design of control programmes in Southern Ecuador. Finally, we have encountered evidence of a sexual reproductive mode in the domestic T. cruzi population, which constitutes a new and intriguing finding with regards to the biology of this parasite. Trypanosoma cruzi, is the most important parasitic infection in Latin America Chagas disease, caused by the protozoan Rhodnius ecuadoriensis, Triatoma carrioni, Panstrongylus chinai, and Panstrongylus rufotuberculatusChagas disease is endemic to several regions in Ecuador, including the warm inter-Andean valleys of the southern province of Loja, where the main vectors are T. cruzi in the early 1970s Molecular diversity was first recognised in T. cruzi, to resolving the frequency of sex in natural populations.Hypervariable molecular markers, like microsatellites, have given new and unprecedented insight into the population genetics of other important parasitic zoonoses Here we present microsatellite data for 10 variable loci amplified from a large number of TcI isolates collected from domestic, peridomestic, and sylvatic hosts and vectors in and around several adjacent communities in Loja Province, Ecuador. We evaluate evidence for genetic subdivision between transmission cycles, anthropogenic dispersal of parasites between communities, and panmixia among a subset of strains.Sixteen communities in Loja Province, southern Ecuador, were sampled . These cTrypanosome species was determined by PCR amplification of the kinetoplast minicircle region as in Vallejo et al. T. cruzi chromosomes and include two groups of physically linked markers T. rangeli and the microsatellite primers used in this study. Population-level genetic diversity was assessed using sample size corrected allelic richness (Ar) in FSTAT 2.9.3.2 FIS, a measure of the distribution of heterozygosity within and between individuals, was estimated per locus per population in FSTAT 2.9.3.2. FIS can vary between −1  and +1 . FIS = 0 indicates Hardy-Weinberg allele proportions. The extent of population subdivision between isolates from different transmission cycles was estimated using (FST) in ARLEQUIN v3.1 and statistical significance assessed via 10,000 random permutations of alleles between populations DAS  2DAS values form the basis of the dendrogram in vs. geographic distance) was implemented in Genelax 6 using 10,000 random permutations GGD) and multilocus (IA), were calculated in LINKDOS A, the results of which are shown in DAS derived tree. Secondarily, this model-free population assignment was corroborated using STRUCTURE  internal branching within the neighbor-joining tree constructed from pair-wise genetic distance values (DAS) supported the delineation of two populations (k) derived using STRUCTURE software as defined by Evanno et al. k  between a priori populations (transmission cycle defined) corroborated this pattern of dispersal. No evidence for subdivision existed between domestic and peridomestic isolates , whereas subdivision between these populations (grouped) and sylvatic samples was pronounced . Naturally, reassignment of outliers to their “correct” genetic groups according to neighbor-joining and STRUCTURE analyses further inflated the latter estimate . These outliers are evidence for some, albeit limited, parasite dispersal between domestic/peridomestic transmission cycles and sylvatic transmission cycles as evident in We evaluated patterns of clustering and subdivision among parasite strains in the Loja samples based upon their microsatellite profiles. To identify genetically distinct groups we relied on three lines of evidence: neighbor-joining analysis based on pair-wise genetic distance; model-based population assignment (STRUCTURE); and the statistical significance of the fixation index ulations . No pattulations  and was ulations . Sample et al. k . One popr) did not demonstrate dramatic difference between LOJADom/Peri and LOJASylv (LOJADom/Peri (PA = 0.8) suggests diversification of this population from a local source.Following the identification of two genetically distinct groups of parasite strains circulating in this endemic area, the genetic diversity of each was evaluated and compared. Estimates of allelic richness ) in the population. Interestingly, tests for IBD among individuals from LOJADom/Peri and LOJASylv showed statistically significant and epidemiologically important differences between these two populations. Infinite allele models (IAMs) of microsatellite mutation intrinsically overestimate genetic distances between closely related isolates as compared to stepwise mutational models (SMMs). To circumvent possible bias we chose to test for IBD under both. Strong evidence for spatial structure in LOJASylv existed regardless . Among isolates from LOJADom/Peri, no spatial structure was evident from either measure . Results are summarised in In light of the role played by transmission cycles in structuring the local parasite population, we compared the rate of parasite dispersal within LOJADom/Peri. Pair-wise inter-locus linkage (RGGD) was infrequent . Infrequent pair-wise linkage is consistent with the lack of significance attributable to the index of association (IA)  across loci that approximate zero  and multilocus linkage (IAP<0.001) in all diploid resampled datasets , evidence that a Wahlund effect could be depressing heterozygosity. Correspondingly, estimates of linkage disequilibrium might also be somewhat inflated by subdivision in this population Several approaches were employed to estimate the rate of genetic recombination within the parasite populations identified in Loja. Multiple indicators suggested frequent sex among trypanosomes of nt 5.5%;  even amoate zero . Finallydatasets , but alsdatasets . Consist strains , did demT. cruzi at a local scale using high-resolution molecular markers. The sample includes isolates from different transmission cycles, vectors, hosts, and adjacent communities. This arrangement aims to minimise sample bias and maximise the resulting molecular epidemiological inference. However, all field studies are affected by the natural abundance of hosts and vectors in different transmission cycles, and we cannot claim a perfect dataset. Nonetheless, we can report strong evidence for parasite diversification by transmission cycle, human involvement in parasite dispersal, and the possibility of sex in one parasite population.This study constitutes a first attempt to understand the population dynamics of T cruzi lineage I in southern Ecuador is consistent with reports of this DTU throughout northern South America T. cruzi populations across much of northern South America LOJADom/Peri and domestic TcI from Venezuela, VENDom,  9 and 13, R. rattus individual captured at a peridomestic site was found infected with a LOJASylv strain  to which linkage statistics are intrinsically sensitive LOJADom/Peri as a stable deme is corroborated by distance-based, model-free assignment, as well as STRUCTURE. In conjunction with Hardy-Weinberg allele frequencies at individual loci, we consider, therefore, that linkage equilibrium among isolates from LOJADom/Peri represents strong evidence for frequent genetic exchange among field isolates of T. cruzi. We believe that the relatively small sample size of LOJADom/Peri (n = 18) does not affect this conclusion, partly due to the ample genetic diversity present in this popualtion, but also because the lack of spatial subdivision in this group suggests frequent contact and opportunity for mixis. Thus it constitutes exactly the group of strains between which genetic exchange might be expected to occur. We cannot rule out the possibility that genetic exchange may also occur in the sylvatic cycle, if the role that substructure found in LOJASylv played in inflating linkage statistics IA and RGGD could be taken into account. However, more focused high-density sample collection from multiple individual localities would be required to address such a hypothesis. Furthermore, we cannot infer the cellular mechanism of genetic recombination events on the basis of these data. Hardy-Weinberg allelic allelic frequencies are consistent with classical meiosis. However, the lack of haploid life stages so far observed in T. cruzi are not consistent with classical meiosis, nor are the genetic exchange events so far observed in vitroMolecular epidemiology at this scale has an important guiding role to play in Chagas disease control programmes. Future efforts in Loja province must account for inter-domiciliary and inter-community parasite dispersal. This includes sustained surveillance and coordinated region-wide spraying campaigns to eliminate local vector re-invasion sources and community education to target passive triatomine dispersal routes. It is also clear that the role of synanthropic mammals cannot be overlooked as these represent an important potential link between sylvatic and domestic foci.T. cruzi. Our results, skewed toward samples from the vector, intuitively imply that the vector may be a site of genetic exchange, as is the case for T. bruceiLeishmania majorT. cruzi lineage between epidemiologically linked strains, and these events have therefore historically been difficult to detect. The intriguing mechanisms of genetic exchange in T. cruzi warrant further investigation of their functional, adaptive, and epidemiological significance.We have shown that microsatellite markers, adequate sample sizes, and associated population statistics give fundamental insight into the genetic exchange in Figure S1DAS values. Both analyses identified two distinct populations among Ecuadorian samples. Ten replicates per value k were made assuming a no-admixture model, and with a burn in of 100,000 followed by 1,000,000 interactions of the algorithm. Delta k calculated according to Evanno et al. Clustering of populations based on STRUCTURE analysis. Results from structure analysis corresponded with the structure defined by (6.59 MB TIF)Click here for additional data file.Table S1Trypanosoma isolated in Loja Province, Ecuador.Location, habitat, host, and lineage of (0.15 MB DOC)Click here for additional data file.Table S2Trypanosoma cruzi primers employed in this study.(0.04 MB DOC)Click here for additional data file.Table S3T. cruzi isolates from Loja Province, Ecuador.Allele sizes (in base pairs) from 10 loci analyzed (fluorescent dye) from 81 (0.30 MB DOC)Click here for additional data file.


ST is among the most widely used measures for genetic differentiation and plays a central role in ecological and evolutionary genetic studies. It is commonly thought that large sample sizes are required in order to precisely infer FST and that small sample sizes lead to overestimation of genetic differentiation. Until recently, studies in ecological model organisms incorporated a limited number of genetic markers, but since the emergence of next generation sequencing, the panel size of genetic markers available even in non-reference organisms has rapidly increased. In this study we examine whether a large number of genetic markers can substitute for small sample sizes when estimating FST. We tested the behavior of three different estimators that infer FST and that are commonly used in population genetic studies. By simulating populations, we assessed the effects of sample size and the number of markers on the various estimates of genetic differentiation. Furthermore, we tested the effect of ascertainment bias on these estimates. We show that the population sample size can be significantly reduced  when using an appropriate estimator and a large number of bi-allelic genetic markers . Therefore, conservation genetic studies can now obtain almost the same statistical power as studies performed on model organisms using markers developed with next-generation sequencing.Population genetic studies provide insights into the evolutionary processes that influence the distribution of sequence variants within and among wild populations. F ST, first defined by Wright (1951).Studies on wild populations give important insights into population dynamics leading to genetic differentiation. One important goal of population genetic studies is to estimate the amount of genetic differentiation among populations in order to draw conclusions on the demographic history. A common measure for the degree of genetic differentiation is the fixation index FST outlier methods n>20) are required to yield reliable estimates of differentiation ST. Reducing the sample size per population would make it possible to analyze a larger number of different populations at the same cost, and it offers an important advantage in conservation genetic studies on rare organisms. Furthermore, understanding the statistics of different FST estimators is especially important in the context of detecting regions under selection.Until recently, most studies on wild population of non-reference species used moderately large numbers of samples per population (>20), but only a small number of genetic markers (<20), preferentially microsatellites, for which more than two alleles can often be distinguished. Studies on human populations were among the first using thousands of markers, with single nucleotide polymorphisms (SNPs) as markers of choice. SNPs are typically the most abundant sequence variants in genomes. Their distribution throughout the entire genome at high density, combined with well-established models for handling mutation rates and error rates, and inexpensive methods for high throughput genotyping make them appealing for population genetic studies ST becomes inflated with small sample sizes. We concentrated on three different estimators. The first one was proposed by Wright (1951), which by definition lies between zero (no genetic differentiation) and one . However, Wright assumed infinite sample sizes in his definition, but population size is finite in real datasets. The absence of negative FST values in Wright's (1951) definition can lead to an overestimation of FST, particularly when the populations are only weakly or not differentiated. Cockerham and Weir (1984) proposed an unbiased estimator that can also have negative FST estimates and that has been widely used n = 4–6), and the behavior of this new estimator will be evaluated as well. Here we compare all three estimators for their performance on the same bi-allelic data set. We addressed the following four questions. First, what is the effect of small sample size on the type I error rate, i.e. falsely detecting genetic differentiation in a panmictic population? Second, does a small sample size result in an overestimation of the FST in cases where populations are genetically differentiated? Third, if estimates are imprecise due to small sample sizes, does precision increase with the number of loci genotyped? Fourth, what is the effect of different allele frequency distributions on the FST statistics, in particular, does a relative excess of common or rare alleles lead to a bias in FST estimates?In this study we used simulations to examine whether the estimation of genetic differentiation measured by Ft generations of random mating, we estimated FST on the complete simulated dataset comprising two populations with 1,000 individuals each that were genotyped at 21,000 loci  will cause additional drift and differentiation over and beyond that expected from the theoretical distribution. Because of the absorption state of SNP loci , such random chance events are not completely offset by an overly equal reproductive distribution (i.e. runs in which too many families with exactly 2 offspring). As a consequence, the simulated FST, like a ratchet, tends to increase slightly faster than the theoretically expected value.After l and ST . The reaSTF = 0, 0.01, 0.05, 0.1, 0.2, 0.4). k = 100 and k = 1,000 at varying sample sizes. n = 4 and n = 20 at increasing number of loci genotyped. Since combining all the different parameters  resulted in a large number of estimates, we chose these four combinations in order to illustrate our general findings . STWF severely overestimated genetic differentiation in small sample sizes (n = 2–6) (e.g. n = 10–50), STWF slightly overestimates the level of genetic differentiation. Since these observations were consistent for all datasets, we will in the following concentrate on the behavior of the two other estimators.We tested the influence of increasing the sample size on the estimate by taking 2, 4, 6, 10, 20 and 50 individuals from each population at different levels of genetic differentiation (–6) e.g. . MoreoveSTW&CF and STRF gave on average similar, fairly good estimates at all sample sizes (ST≥0.1) the FSTW&C estimator tended to slightly overestimate genetic differentiation with small sample sizes (n≤6), whereas the estimator FSTR showed the same average estimate irrespective of samples size. Though, with increasing sample sizes the size of 95% CIs decreased. The 95% CIs were large and included zero at low genetic differentiation (FST<0.05), when using a small sample size (n = 2–6) and a moderate number of loci (k = 100). Increasing the number of loci had no impact on the average estimate of FST  differences between sample sizes did not have an impact on the average estimates of FST of either estimator (FSTW&C and FSTR). If genetic differentiation was moderate to high (FST≥0.1), the FSTW&C overestimated genetic differentiation when the sample size of population 2 was small , this will lead to ascertainment bias and an overestimation of genetic differentiation.In order to test the influence of biases of allele frequency distribution in the analysed marker set, we generated two datasets with 10,000 loci each, where in one set MAF>0.25 and in the other set MAF≤0.25. The simulations show that a shift towards common polymorphisms in the marker set leads to overestimation of genetic differentiation, whereas a shift towards rare polymorphisms leads to underestimation. These erroneous estimates were observed in both the yped see  and S3. ST estimators has been analyzed before, this study is the first to evaluate different FST estimators in population genetic studies of thousands of loci and very small sample sizes. This is a timely matter, given that Next Generation Sequencing (NGS) methods have revolutionized the field of marker development. Furthermore, we have included a new estimator employed by Reich et al. (2009). Our simulations show that even when sample sizes are small , accurate and unbiased estimates of FST can still be obtained when a large number of bi-allelic markers such as SNPs are used, as long as the appropriate FST estimator is chosen. The original FSTW estimator severely overestimates the level of genetic differentiation when using small sample sizes. Since this estimator cannot have negative values, these results were expected for values of FST<0.5, because overestimates that are higher than 0.5 over the actual FST cannot be compensated by a negative value at another locus. The two other estimators we tested showed similar performance on large sample sizes (n≥20), and consistent with a previous study STW&C overestimates FST when analyzing small samples. Importantly, the estimator proposed by Reich et al. (2009) showed better performance in cases where sample sizes were small (n≤6). Our simulations suggest that genetic differentiation is not falsely detected due to small sample sizes when using this unbiased estimator. Furthermore, we showed that increasing the number of genetic markers has no impact on the mean FST estimates, but that it considerably reduces the 95% CIs. Although this is not unexpected, given that increasing the number of loci decreases the coefficient of variation of the estimates, we quantify this effect here for the first time. Finally, the precision of a pairwise estimate depends on the smaller sample size taken from one of the populations and not on the number of loci.Although the statistical behavior of different FST rather than the sample size taken. A bias towards common polymorphisms in the marker set leads to overestimation of genetic differentiation, whereas, visa versa, FST becomes underestimated when there is a bias towards rare polymorphisms. The practical implication is that if the panel is developed based on the screening of a small number of individuals, this will lead to ascertainment bias and an overestimation of genetic differentiation of the study populations.A previous study suggested that increasing the sample size might be more beneficial than increasing the number of markers genotyped random individuals. However, in practice, random sampling cannot always be achieved in nature. Estimates on extremely small samples, as suggested here, might be severely affected by non-random sampling, e.g. due to population structuring. Furthermore, our simulated dataset contained only fully unlinked markers. If subsets of markers are linked they represent only one informative marker. Hence, the number of fully unlinked markers depends on the genome size of the organism analyzed. For the same reason, detecting regions under selection with FST outlier methods require larger sample sizes (n>10). In such cases, FST estimates are based on small sliding windows in which markers are  linked, and hence, they represent a smaller number of independent loci. Baird and colleagues (2008) proposed a method that has been proven particularly useful to develop a large number of genetic markers in non-reference organisms with less ascertainment bias Theoretically, precise estimates of genetic differentiation can be obtained based on very small sample sizes of t) of generations according to the Wright-Fisher model without mutations . Consequently, the population sizes were kept constant, the generations were non-overlapping and the frequencies in the next generation were a binomial random sample based on the frequencies in the current generation. Random dioecious mating was simulated by randomly drawing one female and one male with replacement. Thus all males and females were equally likely to be chosen and could mate multiple times, and the draw was independent of the number of times an individual has been chosen before. The two individuals drawn became the parents of one member of the next generation. Since we assumed all loci to be completely unlinked, we simulated gametogenesis by simply selecting at random one allele from each parent. This process was repeated until all members of the next generation have been created. We simulated different degrees of genetic differentiation . The number t of generations was determined by n equals the number of mating individuals  and t equals the number of generations needed to achieve the required amount of differentiation We simulated an ancestral population with 1,000 individuals  and 21,000 bi-allelic loci. The genotypes at the loci were generated by randomly drawing from eight allele frequency classes . The two starting populations consisted of the same 1,000 individuals, which were genotyped at 10,000 loci randomly taken from the 21,000 loci of the ancestral population. We assumed an isolated island model (i.e. no migration between the two populations after separation). Genetic drift was simulated for a certain number (ST was introduced by Wright (1951) as a measure of correlation of gene frequencies und suggested the first and simplest estimator, STWF. For one allele at locus k,ip among the sampled populations i  and STWF for multiple loci is calculated by taking the mean across k loci.FSTW&CF, preserves Wright's definition of FST in terms of correlation of gene frequencies and is the most widely used estimators  ST at moderate population sample size  and small number of loci (k = 10). The estimates can also have negative values which do not have a biological meaning k, STW&CF is defined as2s is the observed variance of allele frequencies, n is the number of individuals per population, r is the number of sampled populations and k loci is derived byThe second estimator tested, STRF, for bi-allelic loci and pairwise population comparison. In their study they used a very high number of loci, but small sample sizes per population. Therefore, we decided to test this estimator as well. Again STRF is calculated as followsu is the allele count for population 1, v is the allele count for population 2, t and s are the total number of individuals for population 1 and 2, respectively Recently, Reich and colleagues (2009) proposed a new unbiased estimator, t generations of random mating among 1,000 individuals, 10,000 of the 21,000 loci were randomly chosen to test the STF estimates. In order to test the influence of ascertainment bias in marker design, we generated three different datasets. The first set contained loci with equally distributed allele frequencies, the second set contained only loci with minimum allele frequency MAF>0.25, because SNP marker sets are often biased in the direction of more common polymorphisms. However, we also generated a dataset of the other extreme containing only markers with MAF≤0.25.After ST estimate and derived the 95% confidence interval. We used a custom Java program to perform the simulations and estimations of FST that will be made available upon request.We used sample sizes of 2, 4, 6, 10, 20 and 50 individuals. For each sample size we sampled 10, 20, …, 100, 200, …, 1000, 2000, …, 5000 loci. For each number of individuals and genotyped loci we sampled from each population 1,000 times. We took the average FFigure S1Effect of increasing sample sizes and increasing marker numbers with uniform allele frequency distribution.ST = 0, 0.01, 0.05, 0.1, 0.2, 0.4). The results (average FST and 95% CI) of each estimator are depicted in the different graphs: FSTW (blue circles), FSTC&W (purple squares) and FSTR (green triangles). The dashed red line indicates the actual FST for the simulated population.(PDF)Click here for additional data file.Figure S2Effect of increasing sample sizes and increasing marker numbers for MAF>0.25.ST = 0, 0.01, 0.05, 0.1, 0.2, 0.4). The results (average FST and 95% CI) of each estimator are depicted in the different graphs: FSTW (blue circles), FSTC&W (purple squares) and FSTR (green triangles). The dashed red line indicates the actual FST for the simulated population.(PDF)Click here for additional data file.Figure S3Effect of increasing sample sizes and increasing marker numbers for MAF<0.25.ST = 0, 0.01, 0.05, 0.1, 0.2, 0.4). The results (average FST and 95% CI) of each estimator are depicted in the different graphs: FSTW (blue circles), FSTC&W (purple squares) and FSTR (green triangles). The dashed red line indicates the actual FST for the simulated population.(PDF)Click here for additional data file.Table S1ST estimates according to Wright (1951).F(XLSX)Click here for additional data file.Table S2ST estimates according to Weir & Cockerham (1984).F(XLSX)Click here for additional data file.Table S3ST estimates according to Reich et al. (2009).F(XLSX)Click here for additional data file.


The association between long-term nonprogressors (LTNPs) and genetic defects in HIV-1 is not clear because of paucity of genetic defects in LTNPs. To date, there is only a report on gross deletion in 2 genes of HIV-1. Ginseng has been used as the premier medicinal plant for a thousand years [A total of 250 individuals were diagnosed with HIV-1 infection in Korea before 1993. Among the 250 individuals, we selected all who remain healthy for >18 years in the absence of antiretroviral therapy. They were four individuals . One patient who was diagnosed with HIV-1 in 1992 took a little KRG – less than 1,000 g. To investigate whether LTNPs are associated with genetic defects, we amplified near full-length HIV-1 sequences in 10 overlapping fragments (0.8 to 1.4 kb) using nested PCR with peripheral blood mononuclear cells. We compared the data with that in 4 control patients. Amount of KRG supplied was significantly higher in LTNPs  than 1,250-1,304 g in controls (P < 0.01).gag and nef genes. Overall genetic defects were significantly higher in LTNPs (11.4%) than 4.9% in controls (P < 0.001). Among the 4 LTNPs, one patient who had taken the least amount of KRG revealed significantly higher proportion of premature stop codons (5.6%) than 1.6% in 3 LTNPs (P < 0.01). There was a significant difference in the frequency of genetic defects between on KRG (10.9%) and baseline prior to KRG intake (4.3%) (P < 0.01). There was a significant correlation between amount of KRG supplied and proportion of genetic defects . At single gene level, only the nef gene showed a significantly higher frequency of genetic defects in LTNPs (11.5%) than 3.6% in controls (P < 0.01).We obtained 1,339 and 386 PCR amplicons over 20 years in LTNPs and over 10 years in controls, respectively. All LTNPs revealed at least one genetic defect in 4 genes, whereas controls revealed it in both 5’ LTR/These data show the possibility that frequent genetic defects that might be caused by KRG treatment are associated with long-term slow progression.


Journal of Theoretical Biology7 (1964) 17] discusses whether temperate and tropical eusocial insects may show differences in population-level genetic structure and viscosity, and how this might relate to differences in the degree of synchrony in their life cycles or modes of nest founding. Despite the importance of Hamilton's 1964 papers, this specific idea has not been tested in actual populations of wasps, probably due to the paucity of studies on tropical species. Here, we compare colony and population genetic structures in two species of primitively eusocial paper wasps with contrasting ecologies: the tropical species Polistes canadensis and the temperate species P. dominulus. Our results provide important clarifications of Hamilton's discussion. Specifically, we show that the genetic structures of the temperate and tropical species were very similar, indicating that seasonality does not greatly affect population viscosity or inbreeding. For both species, the high genetic differentiation between nests suggests strong selection at the nest level to live with relatives, whereas low population viscosity and low genetic differentiation between nest aggregations might reflect balancing selection to disperse, avoiding competition with relatives. Overall, our study suggests no prevalence of seasonal constraints of the life cycle in affecting the population genetic structure of eusocial paper wasps. These conclusions are likely to apply also to other primitively eusocial insects, such as halictine bees. They also highlight how selection for a kin structure that promotes altruism can override potential effects of ecology in eusocial insects.Climate has long been suggested to affect population genetic structures of eusocial insect societies. For instance, Hamilton [ The general theoretical framework for the evolution of eusociality is provided by the concept of inclusive fitness theory Hamilton ,b, whichIn social insects, partitioning of genetic variance is generally organized at the level of the colony. However, life-history traits also influence higher level structuring between colonies, from a local scale  to larger scale population-level structuring. For example, in many species, there is limited dispersal by one of the sexes, which leads to isolation by distance with greater genetic similarity among individuals in colonies close together than in colonies further apart. In ants, where most studies of genetic structure in social insects have been performed, there appears to be a strong association between certain life-history traits, such as the number of breeders per colony or mode of dispersal, and the degree of genetic differentiation among colonies  are mostly monogynous , are usually highly polygynous  colonies were collected from a large natural population on abandoned buildings of over 200 nests located in Punta Galeta, Colón, Republic of Panamá , in a series of cacti banks, each containing from seven to ten nests. The distance between nests within aggregations ranged from 43.0 to 1047.9 cm . The mean distance between aggregations (measured from the center point of each aggregation) varied from 80 to 170 m. All females were individually marked and monitored in order to determine the queen's identity and exclude the possibility of queen turnover. All brood and adults were stored in 95% ethanol.Twenty-six randomly selected nests were sampled from a large population of over 200 nests in March 2008 around Conil de la Frontera, Cádiz Province, Spain 13, (AAC)10, and (AAG)10 oligonucleotide repeats , and 1 μL of each forward (fluorescently labeled) and reverse primers (each 10 μM). Amplifications were conducted in a GeneAmp PCR System 9700 . The PCR mix was denatured at 95°C for 5 min and cycled 25–35 times, depending on the locus, at 95°C for 30 sec, at primers annealing temperature (55–60°C) for 30 sec and elongated at 72°C for 30 sec. A final elongation step at 72°C for 10 min followed to complete extension of PCR fragments. Labeled PCR products were analyzed on an ABI PRISM 3100 Genetic Analyser (Applied Biosystems) and allele sizes estimated using GENEMAPPER software (Applied Biosystems). Individuals that showed heterozygous alleles for at least one locus were considered as females; individuals that did not fit this criterion were considered as males and excluded from the analysis.As high levels of nests drifting by workers has been documented in the species  (Henshaw P. bellicosus (Pbe128TAG)  from each of the 26 nests. We used a selection of the primer sets previously isolated from  Henshaw  and P. bExpected and observed heterozygosities were estimated using the program GDA 1.0  and aggregations (AGG) were considered as two different levels of sub-populations and all sampled individuals as total populations (TOTAL). We estimated F-statistics at different levels: FNEST-AGG (estimates of genetic differentiation between nests within aggregations), FAGG-TOTAL (estimates of genetic differentiation between aggregations), FIND-TOTAL .Genetic differentiation was quantitated for the different levels of biological organization  by conducting a three-level hierarchical F-analysis. We measured Wright's hierarchical F-statistics, using Weir and Cockerham's  method iS = FNEST-TOTAL) between all pairs of nests was estimated using the software FSTAT 2.9.4 (Goudet ST/(1 − FST) against the natural logarithm of geographic distances, as proposed by Rousset (ST.Genetic differentiation (F4 Goudet . To inve Rousset . Signifir) was also calculated within nests and within aggregations for the two populations of Polistes species using Queller and Goodnight's  to 17 alleles (Pcan16) with a mean of 8.7 alleles , whereas the observed heterozygosities ranged from 0.54 to 0.91 . In P. dominulus, the number of alleles ranged between 5 (25jc) and 36 (Pdom122jc) with a mean of 13.8 alleles. The observed heterozygosities ranged from 0.53 to 0.99  and the expected heterozygosities from 0.63 to 0.96 .The number of alleles per locus in  alleles . The expP. canadensis or P. dominulus populations . Additionally, for both species, we found no evidence of linkage disequilibrium between any pair of loci within populations using Fisher's exact test in GENEPOP. In P. canadensis, linkage disequilibrium was detected across four pairs of loci (over 21) in only 6% of the 1000 resampled dataset (max: 3.2% between Pcan16 and Pcan24). In P. dominulus, eight pairs of loci (over 36) showed significant linkage disequilibrium in only 8.4% of the 1000 resampled dataset (max: 4% between Pdom7 and Pdom140). All loci were then considered behaving as neutral markers and were kept for the analyses of genetic structure.No significant departure from Hardy–Weinberg equilibrium was found in either P. canadensis aggregations . A weaker but significant genetic differentiation was also found between aggregations . There was no evidence of inbreeding at the population level , suggesting that mating occurs randomly , but no significant genetic differentiation between aggregations . The value FIND-TOTAL was low, but significantly greater from zero , indicating a low level of inbreeding . There was, however, a significant isolation by distance in one of the four aggregations . In the three other aggregations, there was also a slight positive correlation, but it was not significant . Interestingly, aggregation A3 differed from the three others in that nests were located in a relatively open space, whereas nests were clustered inside buildings in the three other aggregations.Pairwise comparisons between nests showed no significant isolation by distance in P. dominulus, there was a low but significant isolation by distance when considering all nests of the three aggregations . An analysis of each aggregation separately revealed significant isolation by distance in B2 r = 0.330, P = 0.014, and B3 , but not in B1 . Although we found only significant isolation by distance in P. dominulus, estimations of isolation by distance at both the population level and for each aggregation were relatively low and did not seem to fundamentally differ between the two species.In the Spanish population of P. canadensis: r = 0.69 ± 0.02; P. dominulus: r = 0.64 ± 0.01, two-tailed t-test: P > 0.05) and close to the theoretical relatedness values between haplodiploid full-sisters (r = 0.75). Genetic relatedness at the aggregation level were significantly greater than zero in both species, and higher in P. canadensis (r = 0.12 ± 0.01) than P. dominulus . This suggests a higher degree of population structuring in P. canadensis relative to P. dominulus, which parallels the conclusion from our complementary F-statistics analyses.The mean within-nest relatedness was high (and not significantly different) for both species , suggesting that most colonies are headed by one singly mated queen. These results are consistent with previous studies performed on P. canadensis and P. dominulus, which showed, through observations or genetic analyses, an almost exclusive monopoly of the reproduction by the queen . MoreovP. canadensis, the level of genetic differentiation between aggregations was very small (FAGG-TOTAL = 0.023), whereas in P. dominulus, no significant differentiation was detected. Consistent with these findings, there was also a very limited isolation by distance in both species.Our study also revealed only limited population structuring at levels higher than the nest in both species. In P. canadensis, we found no isolation by distance at the level of the population, or in three of the four aggregations studied. Interestingly, however, there was a significant structuring within one aggregation (A3: r = 0.895). In contrast to the three other associations, which were located  within buildings, the aggregation A3 was in a more open area (overhangs of building). This suggests that topography of the nesting sites may influence the distribution of nests and the dispersal behavior of individuals. It would be of interest to compare populations of P. canadensis within buildings and populations in natural habitat  to investigate whether patterns of dispersal are generally influenced by differences in topography.In P. canadensis are particularly interesting with regard to a recent study, which revealed high levels of nest drifting between closely located nests in a Panamanian population of P. canadensis , which suggest that, even in non-viscous populations, drifters may still be able to benefit from indirect fitness by delivering help in neighboring nests. However, it is also possible that isolation by distance was present in past populations of P. canadensis and helped promoting the evolution of nest drifting, but disappeared over time due to the availability of human structures, which have altered their dynamics of dispersal.These results of population structure found in P. dominulus, where there is a similar level of population structuring to P. canadensis. Nest drifting is difficult to detect using traditional manual censusing methods, as is currently practiced on P. dominulus. It is possible that finer level monitoring  would in fact detect nest drifting by workers for indirect fitness benefits in P. dominulus, if this behavior is a general phenomenon.Intriguingly, similar levels of indirect fitness could theoretically be achieved by nest drifting in P. canadensis, there was a slight isolation by distance at the population level (r = 0.13) as well as population viscosity in two of the three aggregations in P. dominulus. This suggests limited dispersal in these species, with a significant fraction of females initiating new nests in the vicinity of their parental nest.Unlike P. canadensis and P. dominulus were determined from data collected from small-scale populations (<1 km between aggregations). Significant micro-structuring has been reported in P. exclamans, another species of primitively eusocial wasps , into which the species range appears to be expanding naturally (Cervo et al. P. dominulus has been largely introduced in several countries, such as the US, Australia, and Chile (Judd and Carpenter P. dominulus introduced in the US suggest no severe bottleneck with populations having high genetic variability (Johnson and Starks The use of a small scale in our study was primarily due to the limited number of wasp aggregations for both P. canadensis and P. dominulus. Strong selection to nest with relatives was revealed by high structuring at the nest level. However, we found low population viscosity and low differentiation between aggregations in both species. This might reflect the effects of balancing selection in promoting emigration outside the natal aggregations and hence avoiding competition between close relatives (Hamilton and May Overall, this study found genetic structure between temperate and tropical species of paper wasps to be far more similar than expected, considering the contrasts in life cycles and nest-founding behavior of


Thuja sutchuenensis Franch. is a critically endangered plant endemic to the North-East Chongqing, China. Genetic variation was studied to assess the distribution of genetic diversity within and among seven populations from the single remnant locations, using inter-simple sequence repeat (ISSR) markers. A total of 15 primers generated 310 well defined bands, with an average of 20.7 bands per primer. The seven populations revealed a relatively high level of genetic diversity in the species. The percentage of polymorphic bands, Nei’s gene diversity and Shannon’s information index at the population and species level were 76.1%, 0.155, 0.252 and 100%, 0.165, 0.295, respectively. A low level of genetic differentiation among populations (GST = 0.102), in line with the results of Analyses of Molecular Variance (AMOVA), and a high level of gene flow (Nm = 4.407) were observed. Both the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) cluster analysis and Principal Coordinates Analysis (PCoA) supported the grouping of all seven populations into two groups. In addition, Mantel test revealed no significant correlation between genetic and geographical distances . The low genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered species. Thuja sutchuenensis Franch., an evergreen coniferous species of genus Thuja in the cypress family Cupressaceae, is a critically endangered species endemic to mountain area in North-East Chongqing Municipality, China . The mixture was incubated at 65 °C for 30 min followed by two extractions with chloroform: isoamyl alcohol (24:1). The pellet obtained was washed with 75% ethanol, finally dissolved in TE (Tris-EDTA) buffer . Quality and concentration of total DNA were verified by spectrophotometry at 260 and 280 nm. DNA samples were stored at −20 °C for ISSR amplification.Total DNA was extracted from silica gel-dried needles of each plant (0.1 g) following the CTAB method , as grou2, 200 μM dNTPs, 1 μM primer and 0.6 U Taq DNA polymerase . Amplification reaction was performed in a Gene Amp PCR System 9600 . The cycle program included an initial 5 min denaturation at 94 °C, followed by 45 cycles of 30 s at 94 °C, 45 s at 50 °C (52 °C for the primer 895#) and 2 min at 72 °C, and 10 min final extension at 72 °C. PCR Products were electrophoresed in 1.6% agarose gel in 0.5× TBE at 100 V for 2 h, stained with ethidium bromide (0.5 μg/mL). The negative control was run by replacing template DNA with ddH2O. Gels with amplification fragments were visualized and photographed with DG-III . DL1000 bp ladder  was used as DNA molecular weight.A subset of 15 primers  chosen fP, %), Nei’s gene diversity (H), Shannon’s information index (I), the observed number of alleles (Na) and the effective number of alleles (Ne) [HT), genetic diversity within populations (HS), and the relative magnitude of genetic differentiation among populations (GST = (HT − HS)/HT) [Nm) was computed by the formula of Nm = (1 − GST)/2GST [Only distinct, reproducible, well-resolved fragments were scored as present (1) or absent (0) for each ISSR reaction. The data obtained were combined in a single matrix and genetic coefficients were firstly generated using the program POPGENE v1.32 to describe genetic variation at intra-and inter-population level: the percentage of polymorphic bands (les (Ne) ,38. The  HS)/HT) . An estiST)/2GST . The AMOST)/2GST .Genetic divergence between populations was investigated using Nei’s unbiased genetic distances and genetic identities . Nei’s uT. sutchuenensis was appreciable both at the population and species level. Low genetic differentiation was found among populations, which may be attributed to limited geographic distance and abundant gene flow. Both UPGMA cluster analysis and PCA supported the grouping of all seven populations into two groups. Based on these findings, strategies are proposed for the conservation of the species.In summary, our results indicated the genetic diversity of


Fst values for collared (0.042) and white-lipped (0.1387) peccaries showed that both populations are not structured. The Fis values for all loci, except ACTG2 in white-lipped peccaries (-0.0275) and in both species (0.1985 to 0.9284 in collared peccaries and 0.3621 to 0.4754 in the white-lipped), revealed a high level of homozygosis, probably caused by inbreeding. Data on heterologous amplification and genetic variability in collared and white-lipped peccaries are presented for the first time.In this study, the microsatellite technique was used to evaluate the genetic variability in populations of collared and white-lipped peccaries kept in captivity. Six primers developed for domestic pigs were used and amplified in both species. They revealed the presence of five polymorphic loci and one monomorphic locus. The polymorphic loci included 4 of the 16 alleles in collared peccaries, and 3 of the 10 alleles in the white-lipped peccaries. Polymorphic information content (PIC) in both species and all the loci was highly informative. The probability of paternity exclusion (PEC), if one of the parents is known, was almost as high in white-lipped peccaries (95.53%) as in the collared . The  Pecari tajacu , the white-lipped peccary Tayassu pecari  and the chacoan peccary Catagonus wagneri . Pecari tajacu has a geographical distribution from Arizona to Argentina, where it inhabits rain and xeric forests, as well as the desert, in groups of 5 to 50 animals. In contrast, Tayassu pecari occurs from Mexico to northern Argentina, occupying primary forest in groups of up to 400 individuals .In Brazil, both species are used as a source of leather and meat, their leather being used in fine goods, such as gloves and purses, thereby making their conservation of extreme importance . Thus, t. pecari . The twoBlood samples were collected from 45 captive collared peccaries on the Fazenda da Praia, the municipality of Tibagi , the Parque Municipal das Araucárias, the municipality of Guarapuava , and the Fazenda Experimental Gralha Azul, the municipality of Fazenda Rio Grande . Blood was also collected from 25 captive white-lipped peccaries from the Parque Municipal das Araucárias  and the Fazenda Experimental Gralha Azul  (Table S1). The three municipalities are located in the state of Paraná, Brazil.Sus scrofa domestica was used as positive control for the reaction. A 10% native (undenatured) polyacrylamide gel was used on 23 cm x 25 cm plates for electrophoresis of the amplified products. After electrophoresis, the gel was stained by a modified DNA was extracted by the salting-out technique (Ho) and expected (He) heterozygosity. Fisher's test was used to test shunting lines of the Hardy-Weinberg equilibrium in each of the two species of peccary, by the shuffle method  using the GDA program  program , thereby program . Fst wasstimated  was donearameter  was obtaarameter , which warameter  and allearameter  program arameter . PolymorP. tajacu and 114 bp in T. pecari. In P. tajacu, 51 alleles ranging from 103 bp (SW444) to 207 bp (SW2411) were found in the polymorphic loci  to 228 bp (SW2411) were so found  and five polymorphic loci in both species of peccary was revealed. The monomorphic locus showed fragments of 112 bp in hic loci , whereasso found . GenotypHo) and expected (He) heterozygosity for all loci in P. tajacu and T. pecari are shown in P. tajacu, observed heterozygosity values were significantly less than expected in all loci. Higher than expected heterozygosity was only encountered in locus ACTG2 in T. pecari. Hardy-Weinberg equilibrium was attained for loci after correction by the Bonferroni test.The values of observed (P. tajacu (PEC 1 and PEC 2) and T. pecari (PEC 2).The values obtained for polymorphic information content (PIC) were high in all the five loci in both species of peccary. The estimated values of the probabilities of exclusion of paternity (PE) in each locus and species, when neither of the parents is known (PE 1) or when only one is (PE 2), can be observed in Fst for each locus and the totals, on considering the three population samples from collared peccaries and the two from white-lipped as one single population, can be observed in Fst (0.042 for collared and 0.1387 for white-lipped peccaries) indicate that there is no structuring, or, in other words, that genetic variability found within and not between the population samples indicates the existence of mutual gene flow. The Fis parameter values in P. tajacu, ranging from 0.199 to 0.814 in a total 0.543 . In P. tajacu this occurred only in loci SW1407, SW2411, and SW444 , whereas in the others there was an increase . With regard to the number of obtained alleles, when compared to the number of alleles described for the domestic pig, in i.e., none of them reached a desirable value. Therefore, in collared peccaries the set of five polymorphic loci revealed high potential for use in paternity or maternity tests. However, in white-lipped peccaries, one of the factors that possibly influenced the obtained probabilities was the number of observed alleles, as in the majority of loci this was lower than that observed in collared peccaries. Therefore, in this case we suggest the use of novel markers and an increase in sample size.In collared peccaries and considering each locus separately, the number of homozygous individuals increased, whereas observed heterozygosis was lower than expected, thus disclosing low genetic variability. In white-lipped peccaries, the situation proved to be similar, with the exception of locus ACTG2, where the value was higher than expected, an indication of excessive heterozygosity. In both the collared and white-lipped peccaries, the values found for polymorphic information content (PIC) were highly informative , thereby indicating little differentiation between populations, mutual genetic flow, and that the existent genetic variability is within and not between populations. These values were expected, since the captive populations were artificial and recently formed, therefore with insufficient time for self-structuring. Nevertheless, when comparing the two species in relation to the five polymorphic loci, a high degree of structuring was found , thus indicating an intermediate division, which was also expected due to existing reproductive isolation.In both collared and white-lipped peccaries, a lack of structure was observed , as for the white-lipped (0.3621 to 0.4754), thereby indicating excess populational homozygosity, mainly brought about by inbreeding. Several factors may be acting, either separately or together. Thus, the fact that these are captive populations, in relatively low effective numbers and recently formed (perhaps no longer than ten years previously), and constantly receiving new elements of uncertain origin, could, in a certain way, contribute to inbreeding. Moreover, these two species are listed as threatened in the state of Paraná , mainly due to habitat deforestation. It is probable that the populations of these species of peccaries, subjected to such a strong selective pressure, have undergone a reduction in effective sizes, consequently favoring inbreeding or genetic drift. In addition to inbreeding, other sources may also be involved or equally responsible for the excess homozygosity observed in both species, such as errors in genotyping individuals, the presence of null alleles and the Wahlund effect. In the Wahlund effect, the genetic effect of isolation depends on the size of the isolated populations. Thus, if a population were divided into isolated parts, the result of isolation would be similar to that of consanguineous mating, that is, the frequency of homozygotes in the population would increase (The increase .The following online material is available for this article:Localities and identification of voucher specimens.This material is available as part of the online article from http://www.scielo.br/gmb