Vipera aspis) shows a high level of color polymorphism. A blotched morph (cryptic) is common throughout its range , while a melanistic morph is frequently found in montane populations, presumably for thermoregulatory reasons. Besides, rare atypical uniformly colored individuals are known here and there. Nevertheless, we found in a restricted treeless area of the French Alps, a population containing a high proportion (>50%) of such specimens.The presence of intraspecific color polymorphism can have multiple impacts on the ecology of a species; as a consequence, particular color morphs may be strongly selected for in a given habitat type. For example, the asp viper (The aim of the study is to bring insight into the presence and function of this color morph by (i) studying the genetic structure of these populations using nine microsatellite markers, and testing for (ii) a potential local diversifying selection and (iii) differences in dispersal capacity between blotched and non-blotched vipers.Our genetic analyses support the occurrence of local diversifying selection for the non-blotched phenotype. In addition, we found significant color-biased dispersal, blotched individuals dispersing more than atypical individuals.We hypothesize that, in this population, the non-blotched phenotype possess an advantage over the typical one, a phenomenon possibly due to a better background matching ability in a more open habitat. In addition, color-biased dispersal might be partly associated with the observed local diversifying selection, as it can affect the genetic structure of populations, and hence the distribution of color morphs.The online version of this article (doi:10.1186/s12862-015-0367-4) contains supplementary material, which is available to authorized users. Simi[et al.,  found thFor these reasons, particular color morphs may be under strong selection in a given habitat type. In order to test such a selection, an effective method is to compare color variation with the genetic differentiation  of different populations to contrast the degree of adaptive variation and the degree of differentiation due to potential genetic drift -27. The Vipera aspis), which displays a high level of color variation in a large part of its distribution area . Blotched or zigzag morphs are common throughout V. aspis range; it is believed that these patterns have a cryptic function in European vipers, but also, once detected, reveal an aposematic signal to predators such as raptors [An appropriate model system to study this type of selection, and thus the evolution of color polymorphism, is the asp viper , whe raptors ). The adE) varied from 0.50 to 0.67 and observed heterozygosity (HO) varied from 0.52 to 0.65 , but no significant differences for AR , HO  and FST .We detected no evidence for null-alleles, scoring error due to stuttering or large allele dropout for the nine loci. In addition, we did not detect any significant linkage disequilibrium, or deviation from HWE within our 12 populations. For the nine microsatellite loci, the number of alleles per locus ranged from 3 to 32, with a total of 82 alleles across nine loci (Table ST) ranged from 0.00 to 0.24, with an overall FST value of 0.07 .The genetic differentiations between populations  from population 1 into 4. In addition, the evaluation of gene flow revealed that it was asymmetrical between most populations  between the mAIc of non-blotched , as well as within non-blotched individuals . However, sex biased dispersal was significant within blotched individuals, with females dispersing more than males .Considering individuals from all or strictly color polymorphic populations , we found significant differences  and hence were considered as extreme, meaning that the coloration differentiations between populations might evolve by selection and not by neutral processes. The Wilcoxon signed rank tests revealed that there are significant difference between PST calculated with FSTAT , and with heritabilities of 0.5  and 0.1 , but not when the heritability was set at 1 .The mean POur genetic analyses revealed a local diversifying selection and a color-biased dispersal . The combination of these results has several implications.Circaetus gallicus), which is a specific snake predator and can have a strong impact on viper populations [It is not known if non-blotched individuals are more frequently attacked by their predators (mainly raptors) than blotched individuals, in contrast to melanistic individuals ,30, but ulations .An alternative scenario explaining the observed results would be selection against the non-blotched phenotype outside the core region and no selection within it. The results obtained in this study are also in agreement with this hypothesis. Indeed, the mAIcs of blotched individuals were lower than those of non-blotched individuals, meaning than these latter may be poor dispersers, or that non-blotched dispersers may be unable to survive out of their restricted geographical range. Based on our investigation, it is currently impossible to untangle these two hypotheses. However, mark-recapture and telemetry studies should provide helpful information concerning potential differences in the dispersal capacities of the different color morphs. Moreover, studying the predation rate of color polymorphic decoys could highlight differences in detectability between color morphs.O) between monomorphic and polymorphic populations, (ii) the particularity of the habitat where non-blotched snakes are found and (iii) the observed differences in dispersal capacity between blotched and non-blotched individuals , a scenario involving a diversifying selection is more likely.Furthermore, we cannot exclude that the observed pattern might be due to a recent range expansion (coupled with founder effects) of asp vipers in the study area. Such event could result in a low genetic structure and in a non-random distribution of the different color morphs, as observed in this study. Nevertheless, given (i) the lack of differences in the genetic diversity (both for AR and HVipera seoanei), meadow viper  or Latifi’s viper , but itinoides: ,39; Colutrictor: ; Plethodinereus: ).et al. [Thamnophis elegans . Cox & Rabosky [Sonora semiannulata). In birds, Antoniazza et al. [Tyto alba). In addition, the analyses of contact zones between closely related gull species (genus Larus) showed that interspecific divergence in plumage melanism and orbital ring color, clearly exceeded neutral genetic differentiation [et al. [Ischnura elegans) populations in a given year, while two generations later (two years) population differentiation in morph frequencies fell behind neutral genetic differentiation. Consequently, it is consistent with a temporal heterogeneity in selection in these populations, meaning that selection might vary over time, where both spatial and temporal heterogeneities likely play an important role in promoting and maintaining polymorphism. In addition, in the Californian spider, Theridion californicum, characterized by at least eleven color morphs, genetic analyses of several populations revealed that such polymorphism is maintained through balancing selection, i.e. acting to maintain polymorphism across populations [The few studies focusing on color variation and the genetic structure of populations all showed that diversifying selection occurred. For example, Manier et al.  detected Rabosky  found tha et al.  found thntiation . Interes [et al.  showed tulations .V. aspis [Overall, these studies suggested that observed intraspecific color variations in both vertebrates and invertebrates are often the result of a local adaptation and are not due to a random genetic drift. Therefore, observed color polymorphism in these studies is not neutral from an evolutionary point of view. In this respect, recent studies highlighted that the presence of intraspecific color polymorphism might increase the adaptive potential of a species hence its long-term survival and capacity to deal with environmental variations (e.g. ). As a cV. aspis , melanisV. aspis . These rThe presence of important color polymorphism within a species may provide more opportunities to adapt and cope with different environmental pressures , leadingThe study site is located in the French Alps (Mont Blanc massif), between 1’100 and 2’100 m above sea level. We collected a total of 170 samples from blotched (N = 113) and non-blotched (N = 57) snakes between 2006 and 2010. Based on their location, we grouped the samples into 12 populations , both methods allowing an accuracy of about 10 m. We collected DNA samples for the genetic analyses from blood, ventral scales, tip of the tail (stored in 90% ethanol prior to DNA extraction), and/or buccal swab. In order to avoid duplicate samplings, we took dorsal and head pattern pictures  for each specimen. Color morphs of individuals were determined in the field. Several persons scored the phenotype of snakes simultaneously and none of the 170 individuals used in the present study had an ambiguous phenotype. Indeed, blotched (characterized by a zigzag-like pattern) and atypical non-blotched individuals  were sufficiently different to avoid misclassifications  according to the DNeasy® Blood & Tissue Handbook. In order to improve the quality of extractions, we modified a few steps: (i) overnight lysis was conducted for most samples, (ii) elution was performed twice using 100 μl of Buffer AE and finally (iii) incubation time was extended to 5 min so that a higher concentration of DNA was obtained. For buccal DNA sampling, the DNA extraction was also following the same protocol, except that swabs were placed into the DNeasy Mini spin column and centrifuged to remove the remaining liquid from them between steps 4 and 5 of the protocol provided by the manufacturer.V. aspis  and 0.25 – 0.5 mM of each primer  annealing temperature during 30 sec, 72°C for 45 sec, and a final extension of 72°C for 7 min  and two Vb-D17; ) were ammin (see  for moreIS), as well as pairwise subpopulation differentiation (FST), following Weir & Cockerham [IS) per locus and sample with a bootstrap procedure including 10,000 randomizations. We estimated expected (HE) and observed (HO) heterozygosities following the methods of Nei & Chesser [ST) as the dependent variable and the distance between sites as explanatory variable. We carried out permutation tests in order to detect significant differences in allelic richness, expected (HE) and observed (HO) heterozygosities and FST indices among the populations with a high versus a low amount of non-blotched individuals . We performed all summary statistics and tests using the software FSTAT Version 2.9.3.2 [p-value of 0.05 was adjusted using the Bonferroni correction [First, we tested for the presence of null-alleles, scoring error due to stuttering and large allele dropout with MICRO-CHECKER v 2.2.3 . We calcockerham . We comp Chesser  and alle Chesser  with gen 2.9.3.2 . The crirrection  due to mStructure version 2.3.4 [et al. [We used the software on 2.3.4 , a Bayeson 2.3.4 , to infe [et al. . This apBayesAss 3.0.3 [6 MCMC iterations, including a burn-in length of 3 x 105 iterations.In order to quantify unidirectional migration rates (m) between populations, we used the software ss 3.0.3 . This BaGenAlEx 6.5 [We tested for sex- and color-biased dispersal in our populations, using AlEx 6.5 . Then, wAlEx 6.5 ) betweenST, an analog of FST, which calculates the genetic differentiation at neutral genetic markers. Because QST calculation requires experimental estimates of additive genetic variances and since we did not estimate additive genetic variance for asp viper coloration, we will refer in this study to PST (phenotypic or pseudo-QST) rather than QST, as proposed by Saether et al. [ST was estimated with two different methods. First, we coded the color as a locus having only two alleles, blotched individuals being homozygotes coded 11 at this locus and non-blotched individuals being homozygotes 22. Then, we used FSTAT to calculate the pairwise PST between populations. Second, we estimated pairwise PST-values as a function of heritability (h2), within- A commonly used method to estimate population differentiation for a quantitative trait is a metric called Qr et al. . PST wasent}σb2; ,63):\docMS) with an ANOVA on color in JMP 10.0 . Because the heritability of the color pattern is not known, we used three different values of heritability . We estimated within-population variance MSb and MSw are the within- and between-population MS and n0 is a weighted average of the sample size for each population comparison estimated following Sokal & Rohlf [i the number of individuals in the ith population and a is the number of populations to be compared.We obtained the within- and between populations variances by extracting the mean squares . In addition, to check for significant difference between PST - and FST-values, we performed a Wilcoxon signed rank test [To test whether the coloration differentiations between populations evolved by neutral processes or selection, we compared Pv et al. , we consank test .Microsatellite genotypes and phenotypes: Dryad doi: 0.5061/dryad.87478.The samples have been taken with the authorizations of the local authorities .


Oryza sativa) worldwide. Knowledge on genetic diversity and structure of weedy rice populations will facilitate the design of effective methods to control this weed by tracing its origins and dispersal patterns in a given region. To generate such knowledge, we studied genetic diversity and structure of 21 weedy rice populations from Sri Lanka based on 23 selected microsatellite (SSR) loci. Results indicated an exceptionally high level of within-population genetic diversity (eH = 0.62) and limited among-population differentiation (stF = 0.17) for this predominantly self-pollinating weed. UPGMA analysis showed a loose genetic affinity of the weedy rice populations in relation to their geographical locations, and no obvious genetic structure among populations across the country. This phenomenon was associated with the considerable amount of gene flow between populations. Limited admixture from STRUCTURE analyses suggested a very low level of hybridization (pollen-mediated gene flow) between populations. The abundant within-population genetic diversity coupled with limited population genetic structure and differentiation is likely caused by the considerable seed-mediated gene flow of weedy rice along with the long-distance exchange of farmer-saved rice seeds between weedy-rice contaminated regions in Sri Lanka. In addition to other effective weed management strategies, promoting the application of certified rice seeds with no weedy rice contamination should be the immediate action to significantly reduce the proliferation and infestation of this weed in rice ecosystems in countries with similar rice farming styles as in Sri Lanka.Increased infestation of weedy rice—a noxious agricultural pest has caused significant reduction of grain yield of cultivated rice ( Weeds continue to pose great challenges for crop productivity around the world, despite of decades of intensive efforts and practices for weed control and eradication Oryza sativa f. spontanea: Poaceae) is a noxious weed infesting rice fields worldwide. This weed is the same biological species of cultivated rice (O. sativa), with annual and predominantly inbreeding characteristics Weedy rice  markers were used to study weedy rice populations collected from a broad range of rice cultivation regions across Sri Lanka. The primary objective of this study is to address the following questions: (1) Does abundant within-population genetic diversity exist in Sri Lankan weedy rice? (2) Is there any spatial genetic structure or differentiation among weedy rice populations across the country? (3) Does long-distance gene flow occur among weedy rice populations, which is associated with genetic diversity of Sri Lankan weedy rice? The answer of these questions will provide a useful guideline for designing effective strategies to control and manage weedy rice in Sri Lanka and in other countries that have similar or the same rice farming styles and seed management as in Sri Lanka.Oryza sativa f. spontanea) populations with a total of 449 individuals sampled in the “Yala” (May∼August) season from the Dry and Wet zones of Sri Lankan major rice planting regions in 2009 were included in this study (O. sativa) varieties (each with three plants) and 15 wild rice (O. nivara) accessions (each with one plant) provided by the National Rice Research and Development Institute of Sri Lanka were used as references in analyses. The rice varieties were At308, At362, Bg300, Bg352, Bg358, Bg379-2, Bg379-25, Suduheenati, and Suwandal. All weedy rice seed samples were deposited in the Department of Agricultural Biology, Faculty of Agriculture, University of Ruhuna, Sri Lanka.Twenty-one weedy rice  with a light/dark cycle of 16/8 h in May, 2012. The total genomic DNA was extracted from fresh leaf tissues of a single plant at about 10-day seedling stage following a modified cetyltrimethyl ammonium bromide (CTAB) protocol http://archive.gramene.org/markers/). The forward primers of the 23 SSR primer pairs were fluorescently labeled with three different colors  for the fluorescence detection of amplified DNA products  loci  were sel2+), 0.2 mM each of dNTPs , 0.2 µM each of SSR primers, 50 ng of genomic DNA template, and 0.4 U of Taq polymerase . The reaction procedure started with denaturation at 94°C for 4 min, followed by 28∼30 cycles of 30 s at 94°C, 30 s at 55°C, and 40 s at 72°C, and ended with 7 min extension at 72°C.The polymerase chain reactions (PCR) were carried out on a 2720 Thermal Cycler  with a 10 µl volume containing 1×buffer (with MgTM -500 LIZ) for denaturing at 94°C for 5 min and then cooled at 4°C. The mixture was separated on ABI 3130xl capillary electrophoresis genotyper .According to the size and fluorophore color of the DNA fragments, the amplified PCR products from 4∼5 SSR primer pairs were added to the mixture of 9 µL Hi-Di Formamide (to denature the dsDNA) and internal lane size standard  rice sample, given the co-dominant nature of SSR markers. The amplified products were coded either as homozygous genotypes having two equally sized fragments or heterozygous genotypes having two different sized fragments, based on their molecular weight (bp) of the internal lane size standard (GeneScan-500LIZ). All the allelic scores were performed using the software GeneMarker ver. 2.4.2 .aN); (2) number of effective alleles per locus (eN); (3) observed heterozygosity (oH); (4) Nei's unbiased expected heterozygosity (eH) F). In addition, the analysis of molecular variance (AMOVA) was carried out to dissect the distribution of genetic diversity within and among weedy rice populations, and between the Dry and Wet zones stF by FSTAT software (http://www2.unil.ch/popgen/softwares/fstat.htm) and subsequently transformed in F′st, a standardized measure of genetic differentiation stF (max). The standardized values of genetic differentiation (stF′), were subsequently obtained by dividing each stF by the correspondent stF (max).Genetic diversity was estimated for the 21 weedy rice populations with 449 individuals based on the 23 SSR loci. The calculated genetic parameters included: (1) number of observed allele per locus  was used to determine genetic divergence and relationships between weedy populations, in comparison with wild and cultivated rice groups, based on their similarity matrix. The cluster analysis was conducted using the software NTSYS ver. 2.02 To examine whether there existed an isolation-by-distance pattern, the Mantel test was conducted to examine the correlation between genetic distance of weedy rice populations and spatial distance of their geographical localities. The analysis was performed using the software GenAlEx ver. 6.5 mN) N drawn from a large pool of populations exchange a fraction m of migrants each generation, and that the mutation rate u is negligible as compared to the migration rate mM value using the software Arlequin ver. 3.5 eH) of the weedy rice population and the average number of migrants of the weedy rice population with all other paired populations was analyzed using the linear regression model installed in the SPSS software ver. 19.0.0 (http://www.spss.com/).To estimate the level of gene flow, the pair-wise migrants per generation (ad hoc statistic ΔK http://taylor0.biology.ucla.edu/structureHarvester/). In addition, genetic structure of weedy rice populations was superimposed to their geographical locations using the frequencies of STRUCTURE file by Microsoft Excel  and within-population genetic diversity (eH = 0.37-0.69), although great diversity was detected among different populations (stF 0.17) was detected among weedy rice populations  per locus. However, the number of effective alleles (eN) was much lower than the number of observed alleles (aN), with an average of 3.4 alleles per locus. This suggested a large number of rare alleles in Sri Lankan weedy rice samples. Obviously, each of the analyzed weedy rice populations presented a high level of genetic diversity as measured by eH, although varied considerably among weedy rice populations (eH = 0.69), even though a relatively less number of samples  were included for analysis. The lowest genetic diversity was also detected in a northern population , although 30 individuals were included to represent the population. There was no considerable difference in genetic diversity between populations from Dry zone and Wet zone (oH) that was in good correlation the high level of fixation index (0.87∼0.99), indicating the self-pollination mating system and an extremely low hybridization rate of weedy rice. However, all the above results suggested the significantly high level of genetic diversity within weedy rice populations from Sri Lanka.Results indicated a total number of 272 alleles from the 23 selected SSR markers, all of which were polymorphic in the examined weedy rice samples, with an average of ∼11.8 alleles  accessions were included in the cluster analysis as references. The results of cluster analysis showed a clear two-group pattern, in which all the weedy rice populations included in the analysis were clustered as one group. This group was evidently separated from the cultivated- and wild-rice groups at the similarity coefficient value of 0.42. However, all the Sri Lankan weedy rice populations showed a relatively close but variable genetic relationship at the similarly coefficient values of ∼0.72–0.98. The cultivated and wild rice sub-groups were somehow distantly related, showing separation at the similarity coefficient value of 0.52.To illustrate genetic relationships of the 21 Sri Lankan weedy rice populations across the country, a UPGMA dendrogram  was cons2r = 0.011, P<0.01) between the genetic-distance and spatial-distance matrices of the weedy rice populations, supporting the finding that there was neither obvious genetic structure nor pattern of isolation-by-distance (IBD) among the weedy rice populations across the country.Noticeably, the clustering of weedy rice populations did not follow the rule of “isolating-by-distance”. In other words, many weedy rice populations from the same geographical locations were not grouped together. This trend could be evidently reflected by some populations that were genetically distinct from other populations with a close spatial distance collected from the same geographical regions , 2. In cmN) between each pair of all populations was calculated using cultivated rice as a reference. Pair-wise migration data (mN) per generation, although significant variation was detected with a range between0.14-122. In addition, gene flow between weedy rice populations and cultivated rice was also detected with an average number of migrants per generation to be 0.17, ranging between 0.08-0.34. Correlation analysis indicated a significant positive correlation  between the expected heterozygosity (eH) and the average number of migrants per generation of a particular weedy rice population with other populations  of cultivated rice was also found in most of the weedy rice populations . The genIn addition, when genetic structure of each of the weedy rice populations was plotted to the geographical locations, the proportion of genetic components of different weedy rice populations showed a pattern of somewhat graduate change from south to north in Sri Lanka, i.e., red in northern populations and green in southern populations. This indicated south-to-north genetic differentiation of weedy rice populations. However, no such differentiation was obviously observed between western and eastern populations or between the Wet and Dry zones .et al., rich genetic diversity of weedy rice can significantly enhance its competition ability over cultivated rice Understanding genetic diversity and the possible mechanisms contributing to the formation and maintenance of such diversity within and among weedy rice populations is essential for designing strategies for the effective control of this weed stF value. Previous studies from China, USA, and Italy AMOVA revealed much greater genetic diversity within the Sri Lankan weedy rice populations (83%) than that among populations (15%) and between the two zones (2%). These results suggest that genetic diversity of Sri Lankan weedy rice mainly exists within populations, with limited genetic differentiation among populations, which gains supports from the relatively low The diversity pattern of Sri Lankan weedy rice, having abundant within population genetic diversity and limited genetic differentiation, suggests considerable level of gene flow, which is an important driving force to homogenize differences among populations Usually, the formation of a strong genetic structure of plant populations at a given spatial region is associated with limited gene flow vice versa. For example, the Ma-1 population from the Matara District was genetically more closely related to populations (Am-2 and Am-4) from another distant District than populations from its own District. In contrast, some spatially close populations  showed a distant genetic relationship. All these results suggested a loose genetic structure of Sri Lankan weedy rice populations, most likely caused by substantial gene flow. Data from the analysis of pair-wise migration among the 21 weedy rice populations confirmed our hypothesis of a high level of gene flow, up to 122 migrants per generation. Even between populations  with a great spatial distance, the detected gene flow can be as high as ∼1.3 migrants per generation , indicating the importance of gene flow to create and accumulate within-population genetic diversity in Sri Lankan weedy rice populations.As a relatively strict self-pollination plant, weedy rice has extremely low frequency of pollen-mediated gene flow Results based on the STRUCTURE analysis also strongly support our hypothesis of exceed gene flow among different rice planting regions, which leads to the limited spatial genetic structure of Sri Lankan weedy rice populations. The profile of STRUCTURE analysis indicates the considerable sharing of genetic components among different rice planting regions. For example, the northern weedy rice populations, Po-2 (from Polonnaruwa), Pu-1 and Pu-2 , and Am-4 (from Ampara), shared considerable amount of genetic components with the southern populations from Matara District, obviously suggesting the exchange of genetic materials among populations across these districts. STRUCTURE analysis did not suggest an obvious admixture of different genetic components in weedy rice populations. These results, together with the extremely low observed heterozygosity and high fixation index, clearly indicated the lack of hybridization, which is usually caused by pollen-mediated gene flow, between weedy rice populations, although the possibility of crop-to-weed pollen-mediated gene flow within the same rice fields cannot be excluded To ensure the sustainable rice production, effective weedy rice control and management is critical in rice ecosystems where the infestation of weedy rice becomes inevitable. Weedy rice seeds can be accidentally harvested alone with the harvested cultivar seeds and spread to other rice fields through cultivar-seed distribution when these seeds are used for planting in the next seasons Our findings of extensive seed-mediated gene flow in this study not only provide reasonable explanations for the increased genetic diversity within populations and reduced genetic differentiation among populations in Sri Lankan weedy rice, but also have important implications for weedy rice control in this country . As indicated, it is common that Sri Lankan rice farmers save their own rice seeds for planting in the next generation. The exchange of the saved rice seeds between different villages and districts is the common practice for seed distribution in Sri Lanka. Given the situation of weedy rice infestation in Sri Lanka and our findings from this study, we consider that the current rice seed distribution pattern is largely responsible for the rapid spread of weedy rice across the entire country, causing serious infestation. Therefore, we propose to break the long-distance movement and spread of weedy rice by only using certified clean rice seeds provided either by the government agencies or licensed seed production companies. This measure will be a key step in the weedy rice control program in combination with other effective methods. Without this step, the continual introduction of weedy rice through contaminated rice seeds will make the control of this weed very difficult, even though other measures are effective. Therefore, clean seed sources without the contamination of weedy rice can significantly minimize the spread and infestation of this weed.Currently in Sri Lanka, 90% of rice seed supplies are provided by farmers' self-saved seeds, and the distribution of rice seeds is essentially on a farmer-to-farmer basis. In the entire country, only ∼10% of the certified seeds are provided and distributed by government-based seed production agencies, e.g. Department of Agriculture (4%), and private seed production companies (6%) Table S1Pair-wise genetic differentiation among the collected weedy rice populations and cultivated rice. The genetic differentiation was estimated by stF values calculated based on the FSTAT software (DOCX)Click here for additional data file.Table S2Pair-wise standardized genetic differentiation among the collected weedy rice populations and cultivated rice. The genetic differentiation was estimated by the stF′ values obtained based on the division of the original stF values by the stF-estimator  (DOCX)Click here for additional data file.


Native populations of Atlantic salmon in Poland, from the southern Baltic region, became extinct in the 1980s. Attempts to restitute salmon populations in Poland have been based on a Latvian salmon population from the Daugava river. Releases of hatchery reared smolts started in 1986, but to date, only one population with confirmed natural reproduction has been observed in the Slupia river. Our aim was to investigate the genetic differentiation of salmon populations in the southern Baltic using a 7K SNP (single nucleotide polymorphism) array in order to assess the impact of salmon restitution in Poland.One hundred and forty salmon samples were collected from: the Polish Slupia river including wild salmon and individuals from two hatcheries, the Swedish Morrum river and the Lithuanian Neman river. All samples were genotyped using an Atlantic salmon 7K SNP array. A set of 3218 diagnostic SNPs was used for genetic analyses.ST over polymorphic loci was high (0.177). A strong genetic differentiation was observed between the Lithuanian and Swedish populations (FST = 0.28).Genetic structure analyses indicated that the individuals from the investigated populations were clustered into three groups i.e. one clade that included individuals from both hatcheries and the wild population from the Polish Slupia river, which was clearly separated from the other clades. An assignment test showed that there were no stray fish from the Morrum or Neman rivers in the sample analyzed from the Slupia river. Global FWild juvenile salmon specimens that were sampled from the Slupia river were the progeny of fish released from hatcheries and, most likely, were not progeny of stray fish from Sweden or Lithuania. Strong genetic differences were observed between the salmon populations from the three studied locations. Our recommendation is that future stocking activities that aim at restituting salmon populations in Poland include stocking material from the Lithuanian Neman river because of its closer geographic proximity.The online version of this article (doi:10.1186/s12711-015-0121-9) contains supplementary material, which is available to authorized users. Salmo salar, L) has considerable economic, social and environmental importance since it contributes to global and local economies through aquaculture, wild stock fisheries and recreational sport [Atlantic salmon  by the end of the 1980s . Year 19Salmon samples from 28 individuals at each location were collected in 2011 from five locations in the southern Baltic: in Poland, wild parr from the Slupia river (PS) and parr from the two hatcheries Gabriel-Żelkówko (PHG) and Aquamar-Miastko (PHA); in Sweden, wild smolts from the Morrum river (SM); and in Lithuania, wild adults from the Neris (LN) river that is a tributary of the Neman river  less than 0.01 and they were removed. Finally, 3218 polymorphic SNPs remained for the analyses.Ho and He) were calculated using the Arlequin 3.5.1.2 software [ST estimator of Weir and Cockerham [ST was estimated in two datasets both for all SNPs and only outlier SNPs. The first dataset comprised the full geographic distribution i.e. the Latvian-Polish PL pooled population , the Lithuanian LN and Swedish SM populations, and the second dataset contained only the Polish PS, PHA and PHG populations.The number of polymorphic SNPs and their observed and expected heterozygosity  [The hierarchical island model with 100 000 simulations implemented in Arlequin was used to detect outlier SNPs. SNPs that had Felection . Signifie (BEST)  that supe (BEST) .The five salmon samples used in this work represent wild and hatchery populations. One hundred and forty individuals were genotyped using 5568 SNPs, of which 3218 were diagnostic markers. The number of polymorphic SNPs for each population varied and ranged from 2461 for the LN population to 3030 for the SM population. For the PS wild population and the PHA and PHG populations, the numbers of polymorphic SNPs were similar i.e. 2645, 2654 and 2620, respectively  population while for SM, it was null. Before Bonferroni correction, deviations from Hardy-Weinberg expectations ranged from 23 for the PHG to 88 for the PS populations .The average population specific FIS estimated for each population was also non-significant (P < 0.05)  was equal to 0.226 which indicates a high level of differentiation. For comparison, a test was performed for a scenario that comprised only the wild populations and overall FST was even higher and reached 0.242. Overall FST was lowest (0.016) for the scenario that comprised only the Polish populations.AMOVA was conducted for three scenarios: “Countries”, “Wild populations”, and “Polish populations” Table . The larST analysis of the various geographic locations showed that PL vs. SM (0.21) and SM vs. LN (0.27) were genetically differentiated, while PL vs. LN and PL vs. SM were not. When only the Polish populations were analyzed, PS vs. PHG and PS vs. PHA had nearly equal FST values at 0.02. The SM population was the most genetically differentiated while the three polish PS, PHG and PHA populations had very similar, but lower, levels of within-population differentiation than SM. Finally, the population that was the least genetically differentiated was LN.All pairwise comparisons of genetic differentiation between populations were significant (P < 0.05) Tables  and 4. PST values were outside the 99% quantile were identified as potential candidates for divergent selection . Pairwise FST values increased significantly for all comparisons between populations in both analyses except for PS vs. PHA and PS vs. PHG for which FST values decreased non significantly (P = 0.073) from 0.019 to 0.014 for PS vs. PHA and from 0.022 to 0.015 for PS vs. PHG  tree was constructed and the branches were supported by high bootstrap values Figure . The NJ In this study, a 7K SNP microarray was used to analyze the genetic relationships between hatchery and wild salmon populations from the southern Baltic. The Polish wild population from the Slupia river (PS) and the two hatchery Polish populations PHA and PHG formed one clade. However, the PS individuals that were caught were born in the Slupia river where is located a recovering and naturally reproducing salmon population. Bayesian analysis and assignment tests showed that wild specimens sampled from the Slupia river were not the progeny of fish straying from Sweden or Lithuania but were the progeny of fish released from the hatcheries that were established by using imported stocking material from the Daugava river. Salmon eggs have been imported several times i.e. in 1985, 1987 and each year between 1994 and 1999 . PreviouIS estimates for all Polish populations revealed that the genetic diversity of the Polish salmon does not seem to be affected by inbreeding. Therefore, the Polish hatchery Aquamar (PHA) stock consisting of a large number of specimens  may be sufficient to preserve a hatchery strain against unfavorable factors such as inbreeding effects, genetic drift and loss of genetic diversity [IS values need to be interpreted with caution since negative selection processes or domestication occurring in hatchery stocks cannot be excluded. In spite of the observed small, but significant, differences between the wild PS population and PHA and PHG hatchery stocks, the wild population was closely related to the stocking material.Fiversity ,37,38. NThe three Polish populations, PS, PHA and PHG separated well from the Lithuanian LN and Swedish SM populations. The results obtained from the genetic structure analysis suggested that the most significant subdivision is the geographic subdivision. The Polish populations irrespective of the site of sampling had the same ancestral population, which was confirmed by the results of NJ reconstruction and PCoA.The stocking material used in the two sampled hatcheries in Poland came from Latvia (Daugava river) as eyed eggs and were imported each year between 1994 and 1999. Initially, the stocking material was reared and released at the smolts stage and later, an own-hatchery stock, located in Aquamar, was created. Currently, all stocking material in the Slupia basin is based on releases of smolts that are marked by clipping the small adipose fin near the tail. According to the results of studies on scales and tagging experiments, the rate of potential straying of Baltic salmon is relatively low (on average 4%) and the risk of contamination for neighboring native populations is near zero .Based on microsatellite analyses , salmon However, in this study, the lowest genetic variability was observed for the LN population, which may be the result of a large reduction in effective population size in the past. Therefore, the genetic quality of this new potential stocking material from the Neman river needs to be analyzed to assess the genetic consequences on the recently established salmon population in the Słupia river in Poland prior to any releases.The LN population is characterized by the smallest number of polymorphic SNPs and smallest mean number of alleles. In contrast, among all analyzed populations, diversity measures were greatest for the SM population. The current annual wild production of smolts is about 50 000 per year in the Neman and 60 000 in the Morrum rivers . Both saSalmo trutta m. trutta) and Atlantic salmon, underwent a bottleneck event [Previous research showed that Salmonidae, including sea trout  differed from southern Sweden and Lithuanian populations. Wild smolts from the restituted population in the Slupia river originated from fish released from hatchery stocks. No stray fish or progeny of fish straying from Sweden or Lithuania were found in the restituted population of the Slupia river. Our recommendation is that future stocking activities that aim at restituting salmon populations in Poland, include stocking material from the Neman river because of its closer geographic proximity.


Investigating the molecular evolution of human genome has paved the way to understand genetic adaptation of humans to the environmental changes and corresponding complex diseases. In this review, we discussed the historical origin of genetic diversity among human populations, the evolutionary driving forces that can affect genetic diversity among populations, and the effects of human movement into new environments and gene flow on population genetic diversity. Furthermore, we presented the role of natural selection on genetic diversity and complex diseases. Then we reviewed the disadvantageous consequences of historical selection events in modern time and their relation to the development of complex diseases. In addition, we discussed the effect of consanguinity on the incidence of complex diseases in human populations. Finally, we presented the latest information about the role of ancient genes acquired from interbreeding with ancient hominids in the development of complex diseases.  Geneticists have made significant progress in understanding the genetics behind many human diseases. These accomplishments include monogenic disease such as Huntington's disease. On the other hand, the discovery of genetic determinants for complex diseases such as diabetes, Crohn's disease, ischemic heart disease, stroke and some types of cancer , schizophrenia, and bipolar disorder is still poorly understood , 2. HoweThere are several factors that determine the amount of inter- and intragenetic diversity in human populations, which in turn is reflected in different phenotypes, including healthy and diseased phenotypes. These include mutation rates and recombination events that create and reorganize genetic diversity on the molecular level. Moreover, other factors are capable of changing the population size such as migration rates in or out of the population and birth and death rates. In addition, cultural behavior of human populations, such as selective or directed marriages or consanguinity, is also capable of effecting allelic frequencies within populations –14.Generally, genetic, historical, and archeological evidences supported the Out-of-Africa hypothesis, which emphasized the elevated diversity of the original African population –18. On tIt is well known that the main driving forces of evolution in any population are mutation, natural selection, genetic drift, and gene flow. The ability of these driving forces to perform their role is dependent on the amount of genetic diversity within and among populations. Genetic diversity among populations rises from mutations in genetic material, reshuffling of genes through sexual reproduction, and migration of individuals among populations (gene flow) . The effThe migration of human populations to new and different geographical habitats with different environmental challenges such as new climate, food varieties, and exotic pathogens acted as selective pressure on human populations that lead to adaptive changes in population genetic makeup to cope with these new challenges in order to achieve the golden goal of survival . This seGenetic differentiation among human populations is significantly influenced by geographical isolation due to the accumulation of local allele frequency differences . It was F-statistics (FST) (FST = 5 × 10−4) followed by Asia (FST = 9 × 10−3), Africa (FST = 1.7 × 10−2), and America (FST = 2.6 × 10−2). Generally, the genetic differentiation among two European populations separated by a thousand km is at least one order of magnitude lesser than in African, American, or Asiatic populations [It was found that when comparing two nearby populations, Europe was found to be the continent with the smallest genetic differentiation, in relation to geographic distances measured using ulations . OCTN1 gene has increased in frequency due to recent positive selection and that disease-causing variants in linkage disequilibrium with 503F have hitchhiked to relatively high frequency, thus forming the inflammatory bowel disease 5 (IBD5) risk haplotype. Moreover, association results and expression data support IRF1, which is nearby of 503F hitchhiking variants, as a strong candidate for Crohn's disease causation [ GPX1 locus (3p21) and recent selective sweep in the vicinity of the locus were observed in Asian populations [ GPX1 locus is a selenoprotein gene characterized by the integration of selenium into the primary sequence as the amino acid selenocysteine. Selenoproteins have antioxidant properties, and thus interindividual differences in selenoprotein expression or activity could encompass an effect on risk for a range of complex diseases, cancers, neurodegenerative disorders, and diabetes complications [ GPX1 gene can illustrate the role of selenoprotein genetic variants in the etiology of various human complex diseases [Negative selection, also called purifying selection, is the most well-known form of natural selection . Negativausation . This maausation –46. On tulations . GPX1 loications –51. Infodiseases –55. An adiseases , 57. Baldiseases . Based odiseases .All mentioned above forms of selection create characteristic molecular fingerprint also called selection signature. These selection signatures could be in the form of differences in rate of nucleotide diversity, allele frequency spectra, haplotype diversity, or genetic differentiation within or among population genomes . As mentp value = 0.0022 and 0.0054, respectively, for systolic blood pressure (SBP) and diastolic blood pressure (DBP).Natural selection signatures have been detected on many complex diseases . Among t SH2B3 gene that is associated with blood pressure showed significant differentiation between European and non-European populations with FST p value = 0.0042 and branch length p value = 0.0088. It was found that the allele (T) was rare in African and Asian populations with q = 0.03 and 0.01, respectively, while it has a high minor allele frequency of q = 0.47 in the European population [p value of 0.008 and branch length analyses p value of 0.0072. Most of the observed genetic differentiation in SLE associated SNPs allele frequencies differences was driven by differences between African and European populations with FST AFR-EUR p value of 0.0028 or the Eurasia split in the branch length analysis p value of 0.001. For instance, a risk SNP (rs6705628) identified in Asian samples had a low allele frequency in Europeans q of 0.01 but high allele frequency in Africans q of 0.36 and Asians q of 0.19 [p value of 0.0354, which was likely attributed to the Eurasia split from Africa. At the individual T2D SNP level, the rs8042680 in PRC1 gene showed the most significant selection signal. This SNP has a high derived protective allele frequency in European but is rare in African and absent in Asian populations [p value of 0.034. The individual CHD SNP showing the most significant selection signal was rs599839 in PSRC1 gene, which was also significantly associated with low-density lipoprotein (LDL) [At the individual SNP level, a nonsynonymous SNP (rs3184504) inpulation . Moreove of 0.19 , 64. In ulations , 64. An in (LDL) , 65, 66.p value of 0.017 or total branch length p value of 0.01. Majority of the observed differentiation was mapped to the African lineage in the maximum likelihood (ML) branch length analysis p value (AFR) of 0.0002. The most two significant SNPs (rs1465618 and rs103294) are located in THADA gene and near LILRA3 gene, respectively. Moreover, multiple SNPs  in the THADA gene have been reported to be associated with a variety of complex traits or diseases such as cleft palate [p value of 0.0006. In addition, the significant colorectal cancer SNP rs4925386 in LAMA5 gene has higher derived allele frequency in Africans, but relatively low frequencies in Asians and Europeans.Furthermore, several genetic differentiation analyses of GWA studies of SNPs associated with different types of cancers, such as breast, prostate, and colorectal cancers were performed. The most significant collective evidence of global population differentiations was observed in the 34 SNPs associated with prostate cancer with a global FST t palate , polycyst palate , platelet palate , type 2 t palate , 75. Thit palate , 76, 77.BRCA1) [ UGT2B4 gene, associated with increased risk of breast cancer in Nigerians and African Americans, shows molecular signatures of recent positive selection or balancing selection [ PPP2R5E gene, which is involved in the negative regulation of cell growth and division. PPP2R5E gene encodes a regulatory subunit of the tumor suppressing protein phosphatase 2A and resides in a naturally selected genomic region in the Caucasian population of the HapMap [Even though  Peto et BRCA1) , 80. NotBRCA1) –86. On tBRCA1) , 88. Morelection . Furthere HapMap . This obIt was suggested that prehistoric selection events that may favored some genetic variants in ancient lifestyles, such as hunter-gatherers lifestyle, are not advantageous any longer. On the contrary, these positively selected genetic variants have become disadvantageous in modern societies with modern lifestyles. Many complex diseases, such as diabetes, obesity, hypertension, inflammatory or autoimmune diseases, allergies, and cancers, may have been by-products of these disadvantageous prehistoric selection events that are not fit with modern and more sedentary lifestyles. An excellent example is the thrifty gene hypothesis and the evolution and increased incidence of diabetes in modern populations. The thrifty gene hypothesis was first suggested by Neel, who suggested that diabetes predisposition genotypes in modern times were advantageous genotypes historically . These pAdditional examples of detrimental health costs of historical natural selection leading to nowadays complex diseases are inflammatory and autoimmune diseases, such as type 1 diabetes, inflammatory bowel disease, Crohn's disease, celiac disease, and rheumatoid arthritis. This can be justified by the hygiene hypothesis especially in North European populations . The “hyp = 0.008) [As we mentioned above, cultural behavior of human populations, such as directed marriages or consanguinity, is capable of effecting allelic frequencies and genetic diversity within populations. Complex diseases can be affected by consanguinity when they are controlled by multiple rare genes and transmitted in an autosomal recessive manner . Unfortu= 0.008) . In anot= 0.008) . The con= 0.008) . He sugg= 0.008) , 117. ThNeanderthals, ancient hominids, and modern humans have coexisted for thousands of years and interbred outside of Africa especially in Europe and Asia . This le∗0702, found in Neanderthals, is common in modern Europeans and Asians but is rarely seen in Africans [Moreover, recent studies showed that the increased rates of type 2 diabetes in Europeans and Asians compared to Africans are due to interbreeding with ancient Neanderthals. It was found that many genes associated with complex diseases such as systemic lupus erythematosus, primary biliary cirrhosis, Crohn's disease, and diabetes mellitus type 2 have been introgressed from Neanderthals into non-African modern humans . Though Africans .Population genetics and molecular evolution studies have paved the way to gain better understanding of genetic adaptation of human in order to cope with environmental and lifestyle changes. Understanding the effect of evolutionary driving forces on human complex traits, such as natural selection, facilitated our ability to understand the relationship between genetic diversity, adaptive phenotypes, and complex disease. Huge amount of population genetics data for different human populations is available and waiting to be investigated deeply integrating both population genetics and molecular evolution contexts. Molecular signatures of genetic variations such as single nucleotide polymorphism, copy number variation, and genomic structural variations should be investigated and linked with human adaptation, the changing environment, and complex diseases. In addition large scale investigations about changes in lifestyles and the development of complex diseases are needed, especially in the Arabian Gulf area where drastic lifestyle changes accrued after the petroleum discovery. Integrating information about population genetics, molecular evolution, environmental changes, epidemiology, and social and cultural studies is an immediate need. These multidisciplinary efforts can elucidate the relationship between molecular evolution concept and complex diseases and improve our understanding of the evolutionary mechanisms in disease susceptibility, resistance, or progression, in turn facilitating disease prevention, diagnosis, and treatment.


Leptographium longiclavatum and Grosmannia clavigera are fungal symbionts of MPB that aid the beetle to colonize and kill their pine hosts. We investigated the genetic structure and demographic expansion of L. longiclavatum in populations established within the historic distribution range and in the newly colonized regions. We identified three genetic clusters/populations that coincide with independent geographic locations. The genetic profiles of the recently established populations in northern British Columbia (BC) and Alberta suggest that they originated from central and southern BC. Approximate Bayesian Computation supports the scenario that this recent expansion represents an admixture of individuals originating from BC and the Rocky Mountains. Highly significant correlations were found among genetic distance matrices of L. longiclavatum, G. clavigera, and MPB. This highlights the concordance of demographic processes in these interacting organisms sharing a highly specialized niche and supports the hypothesis of long-term multipartite beetle-fungus co-evolutionary history and mutualistic relationships.Over 18 million ha of forests have been destroyed in the past decade in Canada by the mountain pine beetle (MPB) and its fungal symbionts. Understanding their population dynamics is critical to improving modeling of beetle epidemics and providing potential clues to predict population expansion.  Mutualistic symbioses are strong evolutionary drivers, enabling specialization and diversification of host and microbial symbionts Dendroctonus are serious forest pests in North America. The mountain pine beetle  forms complex multipartite symbioses with many microbes including bacteria, single cell yeasts, and filamentous fungi (both Basidiomycota and Ascomycota) that include many tree pathogens Pinus contorta Douglas ex Loudon) and other conifer species 2, which could generate a feedback loop of global warming which results in climatic conditions more conducive to further MPB population expansion The bark beetle-microbe system represents a complex community of symbionts, which provides an opportunity to study the impact of multipartite symbiont associations on population structure, ecosystem health, and pest management http://www.for.gov.bc.ca.hfp/mountain_pine_beetle/facts.htm) P. banksiana Lamb.) and has spread further east, attacking lodgepole × jack pine hybrids MPB is a native pest in North America and it attacks many pine species in western North America When the MPB attacks pine trees, female adults bore through the bark and deposit microbes Grosmmania clavigera (Rob.-Jeffr. & R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf., Leptographium longiclavatum S.W. Lee, J.J. Kim & C. Breuil and Ophiostoma montium (Rumbold) Arx are the major ophiostomatoid fungi that appear to be exclusively associated with the MPB. Although these fungi share the same beetle host and colonize the same host trees, they have different pathogenic/virulence profiles and possibly roles. G. clavigera has been identified as a primary and aggressive invader of sapwood after beetle infestation and it is commonly isolated from the MPB mycangia, a special beetle adaptation for spore transport Grosmannia clavigera can kill mature or young lodgepole pine in the absence of the MPB when inoculated at a density similar to that of a beetle mass attack Leptographium longiclavatum also can cause necrotic tissue around inoculations point both on the phloem and the sapwood O. montium has been considered to be a weak pathogen L. longiclavatum in the MPB-fungal interactions has received little attention; it has only been recently described as having previously been misidentified due to its similarity to G. clavigeraL. longiclavatum and G. clavigera are closely related L. longiclavatum has been relatively more abundant in the recently introduced MPB outbreak locations (northern BC and AB) when compared with the Rocky Mountains locations L. longiclavatum is more adaptable/competitive in the cold boreal climate G. clavigera by L. longiclavatum with increasing latitude. It could be that niche competition is regulated by cold tolerance in these similar species The role of Grosmannia clavigera populations formed four major genetic clusters  that resembled the observed north-south population differentiation pattern of MPB  Do populations conform to isolation-by-distance patterns, and if so, at what scale? (3) Do the fungal symbionts and the MPB have congruent population structures? By analyzing the genetic structure of L. longiclavatum and comparing it with G. clavigera and their beetle vectors, we can infer the ecological and evolutionary processes shaping their population structures such as historical isolation and drift, offering insights into demographic expansion patterns and improving management strategies for beetle epidemics.In this investigation, we describe the population genetics structure of Most of our sampling was conducted on crown land, where permits are not required for sampling trees and tree pests for scientific purposes. Permits were also obtained when required to cover both the collection and transport of fungal materials. Parks Canada provided permits for the mountain parks Yoho. Alberta Tourism, Parks, and Recreation provided permits for collection in the Kakwa, Cypress Hills and Canmore. We also received permission from Tembec to collect in their forests around Sparwood. We sampled from dead or dying trees in the middle of the largest forest pest outbreak in Canada's history and no specific permission were required for these activities. We did not work on endangered species.G. clavigera cluster “North” , 5 from the cluster “Rocky” , and 1 from cluster “SBC” (MP in southern BC) G. clavigera populations We sampled a total of 241 fungal isolates from BC and AB, Canada. Fungi were isolated directly from MPB mycangia /exoskeleton and beetle galleries within the infected lodgepole pines from 17 locations , S1 betwG. clavigeraL. longiclavatum isolates (data not shown). Ten polymorphic markers were selected for the current investigation , 1 µL of AmpliTaq DNA Polymerase  and 20 ng of template DNA. Reaction mixture for 14 µl contained 0.71× PCR buffer, 25 mM each dNTP, 0.71 mM MgCL2, 0.71 µM DMSO, 0.71 µM primer, 0.35 µM of each primer , 0.35 U of AmpliTaq DNA Polymerase , and 5 ng of template DNA. The condition for PCR amplifications was described in L. longiclavatum (SL-KW 1436) has been used as the reference isolate to ensure genotyping consistency.Genotyping was performed on the LI-COR 4300 DNA analyzer on denaturing polyacrylamide gels with molecular size standards 50–350 bp (IRD-700/800 dye) (LI-COR) and analyzed using the LI-COR SAGA software version 2. The holotype of ST. Heterozygosities, gene diversity, number of alleles and private alleles were estimated across all loci for each population using GenAlEx6 A matrix of 218 isolates × 10 microsatellite loci was generated. The data was clone-corrected by removing all the repeated genotypes (multilocus haplotypes) from a single geographic population . PolymorL. longiclavatum by distance-based methods including Principal Coordinates Analysis (PCoA) implemented in GenAlEx6 and AMOVA in Arlequin v.3.11 We analyzed the spatial genetic structure of L. longiclavatum populations was also quantified using FSTST was calculated and evaluated using a randomization test with 1,000 iterations using Arlequin v. 3.11 AMOVA estimates the hierarchical partitioning of genetic variation among populations and regions MAT) locus evolution and sexual reproduction in Grosmannia species demonstrated the presence of two MAT alleles in L. longiclavatum populations at almost equal frequencies A) was computed in the populations using Multilocus 1.3b A allows the calculation of linkage disequilibrium within populations. When there is no linkage disequilibrium because of frequent recombination event, the value of IA would approach zero A increases Investigation of the mating-type . These analytical tools utilize Bayesian clustering algorithms that can help construct population expansion models as well as evolutionary and ecological processes among populations The population structure and genetic clustering of K) K distinct populations that minimize Hardy-Weinberg disequilibrium between loci within groups by including prior information, for example, the geographical location of populations K) was defined from 1 to 12, and each run was conducted with the admixture and correlated allele frequency model, with 100,000 MCMC generations burn-in period followed by 900,000 MCMC generations. Three to five independent runs were performed, and the delta log likelihood value for each K was determined to ensure convergence and consistency among runs. The spatial sampling location of each population was included as input prior. We used Structure Harvester <http://taylor0.biology.ucla.edu/structureHarvester> to estimate the optimal value of K using the ΔK method (rate changes in the log likelihood) as in STRUCTURE 2.3.3 is a model-based clustering method to analyze the association of individual isolates from different geographical locations, to infer clusters and to test for population admixture K) present in the data by incorporating geographical coordinates of individuals as prior information to detect discontinuities in allele frequencies. We used an admixture model (CAR) with a burn-in of 10,000 iterations period followed by 60,000 iterations from which estimates were obtained. We performed five independent runs on each K (K = 2–10) with spatial interaction influence ψ at 0.6 . The optimal value of K was determined by the lowest value of the deviance information criterion (DIC). Data from TESS were also visualized graphically using the software DISTRUCT K.TESS 2.3 K) from 2 to 17, with five replicates for each K. The output of BAPS was used to perform an admixture analysis to determine individual coefficients of ancestry with regard to the inferred number of clusters. In the admixture analysis, 100 iterations were used to estimate the admixture coefficient for individuals. We also used 100 reference individuals from each cluster, and we repeated the admixture analysis 50 times per each individual.BAPS v. 5.2 uses a stochastic optimization procedure rather than the MCMC algorithms used in STRUCTURE and TESS Isolation-by-distance (IBD) was tested by calculating the correlation between genetic and geographical pair-wise distance matrices in GENEPOP (with ISODE) In order to investigate the origin/source of the fungal populations, in particular in newly expanded populations, GENECLASS 2.0 L. longiclavatum populations. We analyzed the 17 populations individually and we also pooled the 17 populations into major genetic clusters identified using the distance and Bayesian clustering methods described above. The Sign and Wilcoxon significance tests were used to determine whether loci displayed a significant excess of H > HEQ (genetic diversity expected under mutation drift equilibrium) or deficit in gene diversity under a mutation drift equilibrium for loci evolving under the infinite allele model (IAM), stepwise mutation model (SMM) and two-phase mutation models (TPM) (70% SMM and 30% IAM) Since MPB was only recently introduced in northern AB and in many parts of northern BC, we used BOTTLENECK v. 1.2 L. longiclavatum by approximate Bayesian computation (ABC) http://www1.montpellier.inra.fr/CBGP/diyabc/) using the pooled population data set of major genetic clusters (from 17 sampling populations) inferred from the Bayesian clustering methods and distance methods described above.Based on the historical knowledge of the presence of MPB in northern BC and AB, as well as the results obtained with Bayesian clustering methods, we compared the posterior probabilities of three competing scenarios that could have given rise to the epidemic populations of ST values and maximum likelihood estimate of admixture proportion. Then the posterior probability of each scenario was estimated using logistic regression on the 0.1% of the simulated data set closest to the observed data set.We considered three competing scenarios that differed in the order of population divergence, as well as the origin of admixed individuals in epidemic populations . The ABCWe compared the scenarios by calculating their relative posterior probabilities by polychotomous logistic regression L. longicavatum, G. clavigera and mountain pine beetles (MPB), we compared the significance of congruence among genetic distance matrices with CADM (congruence among distance matrices) L. longiclavatum, G. clavigeraTo identify the congruent patterns among the population genetic structures of e) for all the samples and each population separately was high, with values ranging from 0.276  to 0.465 , and overall allelic diversity ranging from 1.7  to 3.1   . In geneops, BC) . The numops, BC) , we idenThe number of alleles per locus varied between 2 and 13, and gene diversity per locus ranged from 0.149 to 0.746 . The proA) that deviated significantly from 0  was greater than 3  (p<0.001)  performed on the 17 populations indicated that 13.42 and 86.58% of the genetic variation was attributed to variations among and within populations, respectively (p<0.001) . To furtp<0.001) .ST was common across geographic groupings, suggesting strong genetic differentiation (ST) between populations generally were non-significant within the three geographic groupings, supporting their genetic similarities. Fairview and Yoho  and Kakwa and Fox Creek  were the most and least differentiated populations, respectively (Significant population pair-wise Fntiation . With thectively  indicatiectively .ST/1-FST) and geographic (Km) distances was observed for the entire data set , as well as the data set containing all populations in BC and the Rocky ; however, there was no strong evidence of isolation by distance within each of three genetic clusters; ‘BC’ , “Rocky”  and “North”   .Results from GENECLASS produced an overall low correct assignment rate to home (24%) . Populations belonged to one of three assignment classes: 1) generally high , 2) low (8 populations ranged from 45.5 to 12.5%), and 3) extremely low (6 populations below 6% to zero) .The overall rate of individual assignment to each of the three inferred clusters increased substantially to a minimum assignment of 95% in “BC”, but remained fairly low in “North” ranging from 5 to 28% in 5 populations, indicating a low level of differentiation among populations within the same genetic cluster . Out of The possibility of population bottlenecks/founder events, or population expansion was tested on the 17 populations with the program BOTTLENECK. The Sign and Wilcoxon's tests were used to detect significant excess or deficits in gene diversity L. longiclavatum. We used the ABC method to compare the alternative evolutionary scenarios of the demographic histories with our data. The DIYABC analysis provided the strongest support for scenario 3  C. Moreau and Ips typographus L.) The observed population structure (e.g. divergence between BC and Rocky populations) may have several possible explanations. Post-glacial expansion of the MPB followed by population differentiation along re-colonization path has been proposed L. longiclavatum populations were possibly established following glacial retreat and remained isolated until the current MPB outbreak.Given the dispersive nature of MPB and the distance they can travel, the potential for isolated/differentiated vector-symbiont populations should be low L. longiclavatum populations could be attributed to the frequent short-distance and long-distance aerial dispersal , as well as the ‘rainout’ mechanism of MPB invasion and infestation in the past decade L. longiclavatum populations.The “North” cluster represents recently established populations at the margin or beyond the historic MPB range. The relatively weak genetic differentiation among L. longiclavatum in northern and southern BC have retained high genetic variability. This is consistent with the multiple MPB epicentres hypothesis and the long-term persistence of beetle populations across BC L. longiclavatum in northern and central BC is comparable. If colonization/expansion had only originated from a southern population following the end of the glacial period, we would have observed a decreasing diversity from south to north with increasing latitude G. clavigera and MPB L. longiclavatum in central BC or the presence of glacial refugia The substructured populations of L. longiclavatum populations were consistent with MPB expansion from BC into Alberta through the Pine Pass and Yellowhead Pass  Despite the genetic differentiation among three clusters, the populations were not completely isolated. Gene flow and admixed populations were observed. The individual assignments revealed that individuals in the newly established populations in the “North” cluster (northern BC and Alberta) most probably originated from the “BC” cluster (west of the Rocky Mountains). Gene flow estimations among G. clavigera and MPB also revealed a likely western source for the newly established eastern populations L. longiclavatum populations suggested that the populations introduced into northern BC and Alberta have gone through drift, the founder effect and local adaptation despite multiple, constant MPB introductions.The most probable scenario from our ABC analyses also supports that the newly established population in northern Alberta had been from admixture of populations from BC and the Rocky Mountains. Previous analysis of G. clavigeraL. longiclavatum populations from Valemount and the locations along the Rocky Mountains. More importantly, Kakwa a population sampled slightly north of Valemount, displays a ‘star’ pattern, with high genetic relatedness to eight other populations and could play a similar role. The low barriers to migration existing along the Rocky Mountain Trench may serve as sources for the continued spread of beetles carrying L. longiclavatum to healthy trees north and east of the Rockies due to its proximity to the mountain passes In this study, the population from Valemount, which is situated on the western slopes of the Rocky Mountains , is geneL. longiclavatum is the source of genetic variation L. longiclavatum has never been observed and it has been presumed to reproduce mainly via asexual spores called conidia, while the sexual cycle of G. clavigera has been observed sporadically and may be more prevalent L. longiclavatum population Fungi can be sexual, asexual or exhibit a mixture of both types of reproduction. The type of reproduction and mating system has been known to affect fungal population characteristics L. longiclavatum and G. clavigera also inferred long-term evolutionary history and co-existence of MPB with these fungal associates as different MPB populations can carry different symbiont fungal species. Pathogen dissemination has been influenced by the range expansions of the vector in many other studies Concordant signals in the demographic expansion and transmission pattern between The correlated population structures among the fungal and MPB symbionts may reflect local adaptation and co-evolutionary processes L. longiclavatum populations in the latest regions affected by epidemics and any that can be isolated from the infected jack pines L. longiclavatum and G. clavigera with the MPB might be different, future research should also investigate and characterize the abilities of these fungi under different environmental conditions including temperature and elevations to determine if they have various adaptations, e.g. different cold tolerance The expansion of the MPB's range to higher latitudes and elevations is further than ever observed before Figure S1Graphical representation of three scenarios modelled in DIYABC for demographic expansion patterns of three groups of population (clustering the independent geographic locations) with the posterior probabilities. Scenario 1 indicated the divergence of “BC” population from the ancestral “Rocky” population. The “North/Epidemic” population emerged from the “BC” population. Scenario 2 is similar to scenario 1, but suggesting the “North” population emerged from the “Rocky” population which diverged from “BC”. In scenario 3, the “North” population was an admixture of “BC” and “Rocky” populations.(TIF)Click here for additional data file.Figure S2Population structure based on individual assignment inferred from STRUCTURE (K = 2–4) and BAPS (K = 6). Each individual is represented by a line partitioned into K segments that represent the individual's estimated membership fractions in K clusters.(TIF)Click here for additional data file.Figure S3Population structure of Leptographium longiclavatum populations using TESS for K = 4. Each individual is represented by a thin vertical line, which is partitioned into K segments that represent its estimated population group membership fractions. Black lines separate individuals from geographical site locations (labeled as in (TIF)Click here for additional data file.Figure S4Splits network showing relationships of the analyzed populations. Distances between the populations were estimated by the Nei's distance over 10 microsatellie loc.(TIF)Click here for additional data file.Figure S5ST/1- FST.Plot of isolation-by-distance for the entire  population, and each of the three genetic “BC”, “North”, and “Rocky” clusters, Y  =  ln (km), X  =  F(TIF)Click here for additional data file.Table S1Microsatellite profiles for 241 strains (strains in grey were not included in the clone corrected data set).(XLSX)Click here for additional data file.Table S2Prior distributions and the demographic and historical parameters estimated in DIY ABC analyses. .(XLSX)Click here for additional data file.Table S3Admixture analysis for 218 individuals of L. longiclavatum from 17 locations: average membership coefficients in three clusters inferred from STRUCTURE.(XLSX)Click here for additional data file.Table S4ST calculated with Arlequin (assessed after 100 permutations).Pairwise F * p<0.05; ** p<0.01.(DOC)Click here for additional data file.


Xanthomonas axonopodis pv. manihotis (Xam), the main bacterial pathogen of cassava. Recently, diversity and population dynamics of Xam in the Colombian Caribbean coast were estimated using AFLPs, where populations were found to be dynamic, diverse and with haplotypes unstable across time. Aiming to examine the current state of pathogen populations located in the Colombian Eastern Plains, we also used AFLP markers and we evaluated the usefulness of Variable Number Tandem Repeats (VNTRs) as new molecular markers for the study of Xam populations.Molecular typing of pathogen populations is an important tool for the development of effective strategies for disease control. Diverse molecular markers have been used to characterize populations of Xam populations from the Colombian Eastern Plains. These two typing strategies clearly separated strains from the Colombian Eastern Plains into distinct populations probably because of geographical distance. Although the majority of analyses were congruent between typing markers, fewer VNTRs were needed to detect a higher number of genetic populations of the pathogen as well as a higher genetic flow among sampled locations than those detected by AFLPs.The population analyses showed that AFLP and VNTR provide a detailed and congruent description of Xam isolates in order to obtain a more detailed overview of the pathogen to improve the strategies for disease control.This study shows the advantages of VNTRs over AFLPs in the surveillance of pathogen populations and suggests the implementation of VNTRs in studies that involve large numbers of  In order to generate effective mechanisms for the control of plant diseases, it is crucial to gain insights into the diversity and population dynamics of plant pathogens,2. PathoXanthomonas axonopodis pv. manihotis (Xam) is the causal agent of cassava bacterial blight disease (CBB), the most important bacterial disease of cassava. The most common symptoms of CBB are angular leaf spots, stem exudates, cankers, blight, wilt and dieback[Xam is an example of a pathogen that presents diverse degrees of variability in different geographical zones and interesting population processes, including genetic flow and instability of populations in different geographical regions[Xam populations have been characterized in different countries in South America and Africa, starting in the 1980s. These studies showed that the South American populations were more diverse than those from Africa[Xam populations from Colombia were classified as highly diverse and showed significant levels of genetic flow between them, in spite of their distant geographical origins in the country[Xam populations were mainly studied in three regions of Colombia: the Caribbean region, the Eastern Plains and the province of Cauca[Xam populations from the Caribbean and Eastern Plains were dynamic and presented a higher genetic diversity when compared with populations from Cauca[Xam. Additionally, we identified that Caribbean populations change rapidly over time, since it was already possible to establish a temporal differentiation compared to the populations characterized by Restrepo and collaborators in the 1990s[ dieback,7. Xam i regions. Xam popm Africa-14. Parthe 1990s. Despitehe 1990s. HoweverXam population studies. These include Restriction Fragment Length polymorphisms (RFLPs), Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Amplified Fragment Length Polymorphisms (AFLPs)[Mycobacterium tuberculosis[Bacillus anthracis[Staphylococcus aureus[Xylella fastidiosa[Xanthomonas citri pv. citri[Ralstonia solanacearum[Xanthomonas oryzae pv. oryzicola[Xam when cost is a limiting factor and when the access to especialized laboratory equipment is restricted.A number of different molecular markers have been implemented for  (AFLPs),14,16. N (AFLPs),10,16. T (AFLPs),18. Rece (AFLPs),19. One  (AFLPs),21. VNTRerculosis,23, Bacianthracis and Stapus aureus. More reastidiosa, Xanthompv. citri, Ralstonanacearum, and theryzicola. VNTRs hryzicola,31. An aryzicola,19. All Xam populations in the Eastern Plains of Colombia using two types of neutral molecular markers. The Eastern Plains is the second most important region for cassava cultivation in Colombia. In contrast to the Caribbean cassava fields, Eastern Plains fields are considerably small and their growers are not commercially allied for trading of their produce. In this study, we isolated strains from cassava fields located at the provinces of Meta and Casanare, located at the Eastern Plains of Colombia, from 2011 to 2012. The collected isolates were typed using both AFLPs and VNTRs markers. This study highlights the usefulness of VNTR markers for characterizing populations of Xam. This study provides an updated distribution of distinct populations of Xam in the Eastern Plains of Colombia.The aim of this study was to evaluate the diversity of current Cassava crops in the Meta and Casanare provinces of Colombia were sampled from 2011 to 2012  and then incubated at 28°C for 48 h. White, viscous bacterial colonies, typical of Xam were found in high populations in all plates coming from symptomatic tissue. These were confirmed as Xam using primers directed to the C-terminus of the gene coding for PthB, now called TALE1Xam[Xam strains and it has been implemented for Xam identification[Xam strains, which represented the genetic diversity of the pathogen in the 1990s in the Colombian Eastern Plains, were used as reference strains. Reference strains were kindly provided by Dr. Valérie Verdier from IRD .For isolation of the bacterium, a 1 cm-diameter leaf disk with infected and healthy tissue was obtained from each sample. The disk was disinfected with 1% hypochlorite and washed in sterile water three times. The tissue was ground in 200 μl of 10 mM MgClALE1Xam (Additiofication. A singlXam isolates were grown overnight in 5 ml of liquid Phi (Φ) medium  at 220 rpm and 28°C. Total DNA was obtained using the PureLink™ genomic DNA mini kit according to the manufacturer instructions . The DNA quality was checked in 0.8% agarose gel electrophoresis, and it was quantified using a NanoDrop spectophotometer ND1000 .EcoRI and MseI to generate the AFLPs[MseI + 0/EcoRI + 0. The thermal profile used was: 20 cycles at 94°C for 30 sec; 56°C for 60 sec; 72°C for 60 sec. A 1:25 dilution of the PCR product was used as template for the selective amplification with four primer combinations  , which wXam. Loci wi et al.,.2, 3 mM PCR primers, 1.3 mM dNTPs and 1 unit of Taq DNA polymerase . Thermal profile was conducted as follows: 3 min at 95°C, 35 cycles consisting of 20 sec at 95°C, 30 sec at 52–58°C, and 60 sec at 72°C, with a final extension step at 72°C for 10 min. When a bacterial colony was used as the direct source of the template, an additional step of 95°C for 10 min at the beginning of the thermal profile was added. Amplified products were separated on 1% agarose gels and then sequenced using the primers listed in the Additional fileVNTR loci were amplified either from genomic DNA or from a fresh bacterial colony. Each PCR reaction contained 10-50 ng of genomic DNA, 2.5 mM MgClSequences were aligned using MUSCLE and thenPT) with 1000 permutations using GenAlEx 6.5[ST values[Molecular Variance Analysis (AMOVA) was conducted to determine genetic differentiation among sampled provinces estimating the genetic differentiation among population value .Isolates were then classified into haplotypes, which were established with an infinite allele model and a threshold of 0 using GenoDive 2.0b20. The cloXam isolates were obtained from four fields located in Orocué in the province of Casanare. Samples collected in Orocué came from small plots where cassava is cultivated for self-consumption of smallholder farmers, in contrast to the fields visited in the other locations.A total of 101 isolates were collected at four locations in the Eastern Plains of Colombia. From these, 47 isolates were collected in La Libertad (Meta) from an experimental field that contained 96 representative cassava accessions from the Eastern Plains. The experimental field was visited with permission of the International Center for Tropical Agriculture (CIAT). In contrast, other sampled locations presented one or a maximum of two cassava varieties per field. Commercial field crops at Granada and Fuente de Oro (Meta) presented a comparatively low number of samples with typical CBB symptoms. Only three isolates were obtained from Granada and one isolate was obtained from Fuente de Oro. In addition, 50 EcoRI + T/MseI + T, EcoRI + T/MseI + A and EcoRI + C/MseI + A were used. Primer combination EcoRI + G/MseI + A did not produce polymorphic bands among the evaluated isolates. AFLP selective amplifications were run twice for each isolate. Band patterns were consistent between replicates.One-hundred and one isolates and ten reference strains were characterized by both AFLP and VNTR markers. The characterization with AFLPs was performed with four combinations of selective primer pairs. AFLP band patterns obtained with selective amplifications were clear to read after detection with silver staining. A total of 57 polymorphic bands were generated when primer combinations Xam isolates were also characterized using five VNTR loci. PCR amplicons of VNTRs were strong and highly reproducible. Sequencing of VNTR loci showed that the number of alleles per locus ranged from 7 to 17 . On the other hand, five genetic clusters were estimated when isolates were characterized using VNTRs , however this correlation was not as evident as the one estimated using the AFLP markers. FST values from the populations estimated using both techniques were compared. FST values of the five populations obtained for the VNTR analysis were lower than the FST values from the populations generated with the AFLP analysis, indicating that VNTRs detected a higher genetic flow between populations.We then evaluated if there were distinguishable genetic clusters of the pathogen in the Eastern Plains region. When isolates were assigned to estimate genetic clusters using AFLP markers, they were grouped in two well-differentiated genetic clusters Figure A. Each g Figure B. In theAn analysis of haplotype assignment was conducted to determine the number and distribution of haplotypes among sampled locations. A haplotype was defined with a 100% similarity threshold for both AFLP and VNTR loci. Both approaches generated a highly similar number of haplotypes for each sampled location and for reference strains Table . In addiXam isolates, evidencing a potential temporal differentiation. This was observed with both types of markers  and Orocué (Casanare). In contrast, Fuente de Oro and Granada were the source of only a few samples for this study. The difference in the number of isolates was due to great differences in disease incidence among locations. In contrast to La Libertad and Orocué, cassava fields in Granada and Fuente de Oro are constantly rotated by growers or substituted by other types of crops and this could have contributed to a reduction in the incidence of CBB in these locations.Xam populations. This is supported by several observations. First, both techniques were able to genetically differentiate the populations of Xam between sampled locations. Second, global clustering patterns were constant in both types of markers. For instance, clustering in distance trees and haplotype networks was clearly defined by the geographical origin of isolates, although AFLPs displayed a better geographical clustering  was larger than that for AFLPs (k = 2). This finding suggests that VNTRs were able to detect a more detailed structuring of  Figure . These gn 500 km,14,15.If we compare the current populations from the Caribbean and the Eastern Plains, it is evident that the pathogen is more diverse in the Caribbean. A total of 57 AFLP haplotypes were detected among 160 isolates from the Caribbean region, when using 80% similarity as a threshold.. In the Xam populations. In contrast, the geographical differentiation detected at the Eastern Plains populations could also be associated with the fact that growers in Orocué are indigenous people who do not move over large geographical distances. This phenomenon could reduce the exchange of propagative material infected with Xam, hence enhancing genetic differentiation between Eastern Plain locations.In contrast to the locations at the Eastern Plains, most of the Caribbean populations did not display a geographically-dependent genetic differentiation. These dXam populations. Remarkably, only 5 VNTR loci offered a very similar panorama of the pathogen populations to that obtained by 57 AFLP loci. This finding is relevant for further studies on the population dynamics of Xam, because VNTR markers provide a faster and less expensive characterization of bacterial isolates, as has been reported for several pathogenic microorganisms[Xam survey studies we recommend the use of VNTRs. The rising number of sequenced genomes available nowadays, provides an additional advantage to identify new VNTR loci, hence improving the characterization of several pathogens[Xam strains have been released[Xam strains using the information obtained from the 65 draft genome sequences. Our goal is to obtain a small sets of VNTRs with a high discriminatory power, aiming to implement them in studies that involve a large number of isolates to provide a more accurate description of evolving processes taking place in Xam populations.In this study, we were able to assess the usefulness of VNTRs for the study of rganisms,24,25,49rganisms. For futathogens,21,50,51released, providiXam using VNTRs as molecular markers. Here we demonstrated that a small number of VNTR loci could offer a similar panorama of the status of the pathogen to that offered by AFLPs markers. Because VNTRs represent a fast and simple tool to type Xam populations, their implementation will allow a constant and adequate surveillance of the pathogen, which could provide information to improve the efficiency of strategies for disease control, such as the deployment of resistant varieties.This study represents the first attempt to type populations of http://doi.org/10.5061/dryad.t173v.The data sets supporting the results of this article are available in the Dryad Digital Repository:DNA sequences are available in Genbank database: .This study did not involve any human material, or human data. No experimental procedure was performed in vertebrate or invertebrate animals for the development of this research.The authors declare that they have no competing interests.CT was involved in the conception and design of the study, sampling, bacterial isolation, molecular characterization using AFLPs and VNTRs, data analyses and who wrote the manuscript. NAR performed DNA extraction, the evaluation of 3 VNTR loci, VNTR data analyses and drafting of the manuscript. LP contributed in the evaluation 3 VNTR loci, VNTR data analyses and drafting of the manuscript. CM carried the sampling and data acquisition. AT participated in the data acquisition and revised the content of the manuscript. SR was involved in the conception and design of the study, drafting and revising the manuscript. RK was involved in the conception and design of the study and the design of the VNTR strategy. AB participated in the conception and design of the project, funding acquisition, editing and revisiting of manuscript. All authors read and approved the final manuscript.Primers used for the AFLP amplification and VNTR amplification and sequencing.Click here for file