Nucella lapillus L., a sedentary gastropod with direct development, is a good example: dispersal was traditionally assumed to be limited until studies with microsatellites disputed this idea. To shed some light on this controversy, the genetic structure of dogwhelk populations in northwest Spain was investigated with highly polymorphic AFLP markers giving special attention to the influence of hydrodynamic stress. In agreement with the expectations for a poor disperser, our results show a significant genetic structure at regional (<200 km) and areal scales (<15 km). However, the spatial genetic structure varied with wave-exposure in the present case study: IBD was evident under sheltered conditions but absent from the exposed area where genetic differentiation was stronger. Our results provide evidence that differences in wave-exposure can exert a detectable influence on the genetic structure of coastal organisms, even in species without a planktonic larva.Dispersal has received growing attention in marine ecology, particularly since evidence obtained with up-to-date techniques challenged the traditional view. The dogwhelk  The quantification of dispersal ability and connectivity between populations is a major challenge in marine ecology with implications for the conservation and management of coastal environments Estimating dispersal is challenging. Direct measures are only possible in few species Nucella lapillus illustrates the uncertainties surrounding the assessment of dispersal in marine organisms. This snail is the most common neogastropod in Eastern Atlantic intertidal rocky shores ranging from south Portugal to northern Russia, and it even reaches the northern west Atlantic coast of North America N. lapillus in many areas throughout its European range  to obtain indirect inferences about its dispersal ability. Sampling was designed to investigate the influence of wave exposure on genetic structure. We hypothesized that different hydrodynamic conditions could modify the dispersal capacity of the organisms with an impact on the resulting genetic structure. With this aim we sampled two different areas belonging to contrasting hydrodynamic environments.Here, we investigated the diversity and genetic structure of populations of AFLPs were chosen as molecular markers because of high reproducibility and potential to comprehensively scan the genotype i.e. a drowned river valley)   were collected in Galicia (NW Spain): five from sites scattered along a exposed stretch of coast ; and five from locations in a sheltered stretch, all of them within a single ria  . ImportaN. lapillusOnly subadults, identified by their shell traits We carried out a multistage study to select AFLP primer combinations that produced polymorphic, reproducible, and easily scorable patterns. In a first stage, 24 combinations were tested on six individuals from two sites . The 11 combinations that produced the most interpretable banding profiles were then re-tested on 24 individuals from six sites . The six combinations yielding the highest levels of polymorphism were thus retained and tested again on new, independent DNA extractions of the same 24 individuals to assess reproducibility . After incubation, the product was added to 6 µl of a ligation solution containing 0.43 µM EcoRI-adapters (5′-CTCGTAGACTGCGTACC-3′ and 5′-AATTGGTACGCAGTCTAC-3′), 4.3 µM Tru1I-adapters (5′-GACGATGAGTCCTGAG-3′ and 5′-TACTCAGGACTCAT-3′), 0.52 units of T4 ligase (Fermentas) and 10X ligation buffer. Ligation products were diluted 10-fold in Milli-Q H2O (Millipore Co.) and 10 µl were used for a preselective amplification with 0.3 µM EcoRI-primer with a T selective nucleotide (5′-GACTGCGTACCAATTC+T-3′), 0.3 µM Tru1I-primer with a C selective nucleotide (5′-GATGAGTCCTGAGTAA+C-3′), 2.5 mM MgCl2, PCR buffer, 0.04 µgµl−1 BSA, 0.2 µM dNTPs and 0.04 units of AmpliTaq polymerase (Applied Biosystems). Ten µl from the 10-fold diluted product of the preamplification were finally used for selective amplifications with 0.6 µM of each EcoRI and Tru1I primers, both designed with three extra selective nucleotides added to the 3′ end , 0.8 µM dNTPs, 2.5 mM MgCl2, 0.04 µgµl−1 BSA, PCR buffer, and 0.4 units of AmpliTaq Gold polymerase (Applied Biosystems). Preamplification was performed immediately after ligation whereas the products of the other reactions were kept overnight at −20°C. PCR reactions were performed in a Hybaid thermocycler model PxE. The 5′ end of the selective E-primers was labeled with FAM or NED fluorochromes.AFLP reactions were performed following the protocol of Vos et al. Negative DNA extraction controls were regularly included to screen for cross contamination. Furthermore, DNA extractions of 10% of individuals were duplicated on different dates and run blindly along the other extracts to avoid bias scoring during reproducibility tests PCR fragments were separated on a 3130×l Genetic Analyzer (Applied Biosystems). Fingerprint patterns were processed with the software GeneMarker v1.70 (Softgenetics) using the options suggested for AFLP and following common recommendations for these markers AFLP-SURV v1.0 Allele frequencies were estimated using the Bayesian method of Zhivotovsky FST). FST values were calculated for the whole data set as well as for each area (exposed and sheltered coast) following Lynch and Milligan (unweighted pair-group mean average) cluster analysis and for Principal Coordinates Analysis (PCoA) with the help of MVSP v3.12d. (Kovach Computing Services) and GeneAlEx v6.1 ΦPT, an analogue of FST, using the squared Euclidean distance matrix between AFLP phenotypes and allows a hierarchical analysis of the genetic structures . ΦPT is a band-based approach that does not depend so critically on specific assumptions that could underestimate genetic variability Allele frequencies were also used to estimate genetic differentiation between samples . Both distances were used unprocessed and log-transformed in all possible combinations. Mantel test was performed with the online application Isolation by Distance Web ServiceThe occurrence of isolation by distance (IBD) was explored with the Mantel test of correlation between the matrices of pairwise K) ranging 1–10. The ad hoc summary statistic ΔK of Evanno et al. K with the uppermost hierarchical level of population structure in our data. Anticipating that a superstructure might hide other structures at smaller spatial scales, STRUCTURE was run for the whole data set as well as for the populations within each area separately In an alternative approach, the genetic structure was further investigated with the Bayesian model-based clustering algorithms implemented in STRUCTURE v2.3.3 P<0.01, test T’). Sites CH2 and LF showed one private band each, although with no evident link to any other parameter.The six primer combinations produced a total of 230 AFLP loci, ranging from 29 to 52 per combination , 99 43%. LikewisFST (0.172) and ΦPT (0.254) statistics revealed the presence of highly significant genetic differences among sites (P<0.0001 and P<0.001 respectively)  than differences between populations within the same area . Pairwise ΦPT differentiation was always highly significant (P<0.01 after Bonferroni correction for multiple testing) and ranged from 0.021 for the comparison CH-CH2 (two sheltered sites separated 400 m) to 0.506 for SM-CH2 . Similarly, pairwise FST differentiation ranged from 0.014 for CH-CH2 to 0.405 for SM-S (Both ctively) ; as expeal AMOVA  confirmefor SM-S .The hierarchical structure was clearly illustrated both by the UPGMA cluster analysis see  of Nei’sN. lapillus might follow a model of isolation by distance. Mantel test confirmed this hypothesis . The correlation was still evident when the test was restricted to sheltered coast sites  but disappeared when only samples from exposed coast were considered in the analysis   .Using the complete data set, STRUCTURE detected two clusters that matched the two studied areas . The cluNucella lapillus possesses attributes typical of a low dispersal organism. The absence of a planktonic larva together with a sedentary adult stage constrained to rocky intertidal enclaves are traits linked to limited dispersion FST, IBD) found a significant genetic differentiation among sites, indicating that N. lapillus does not form the single panmictic population expected in organisms with long-distance dispersal. Instead, genetic differentiation was strong between areas (sites separated by 50–120 km) as well as between sites within a single area (distances <15 km). Even sheltered sites separated as little as 400 m along an uninterrupted rocky intertidal suitable for N. lapillus showed significant genetic differences. Our FST estimates are comparable to those obtained for other gastropods with direct development The dogwhelk N. lapillus) typically were <<1 km whereas the average distances in organisms without planktonic phase but with secondary dispersal mechanisms  would reach some 10 Km. Therefore, the mean dispersal distances estimated for N. lapillus fall well within the typical range found in species with very low dispersal capacity. Our results are also in agreement with a pioneer allozyme work conducted on N. lapillusST estimates (0.3326) and a three level structure that provided further support to the low dispersal of the dogwhelks.The slopes of the isolation by distance lines can be grossly translated into estimates of mean dispersal distances using the theoretical model proposed by Kinlan and Gaines N. lapillus, albeit statistically non-significant, are compatible with recolonization by a very low number of colonist (between 3 and 35) provided that census sizes were rapidly recovered after a short founding effect. Similarly, Piñeira et al. Littorina saxatilis, another marine gastropod with direct development, that had recovered from a massive oil spill.The low gene flow and restricted dispersal inferred with AFLP and allozyme data contrasts with the more recent proposal that dogwhelks must have larger dispersal ability and move longer distances than previously thought N. lapillus. The netted dogwhelk Nassarius reticulatus (L.) also showed a stronger genetic structure between exposed populations than between protected ones Our genetic markers reveal that, in addition to being genetically differentiated, our exposed and sheltered areas differed in the intensity and spatial pattern of their genetic structure. Exposed populations displayed stronger genetic differences among them but these differences did not conform to any spatial pattern. In comparison, populations inside the ria were genetically closer to each other but their differences fitted the expectations of an IBD pattern. Accordingly and after a hierarchical approach, STRUCTURE was still not able to resolve among sheltered IBD-patterned sites N. lapillus and N. reticulatus. Specifically, the populations from inside the ria are genetically indistinguishable in N. reticulatus while they retain significant levels of genetic differentiation in N. lapillus. Since both gastropods were studied in the same geographical context (NW Spain), it seems reasonable to presume that these differences in the intensity of the genetic structure in sheltered sites are possibly linked to the contrasting dispersal ability of each species. Thus, while veligers of N. reticulatus spend 1–2 months in the plankton and its adults are very mobile N. lapillus is a snail with direct development and a reputation for very sedentary adults  are genetically closer to open-coast populations than those from the upper reaches . STRUCTURE showed that the admixture proportion is higher in the individuals sampled in site M (closer to the entrance of the ria) which genetic composition assigned to the cluster dominating in exposed populations. The origins of the gradual increase of a distinct genetic composition towards the mouth of the ria are uncertain. Two hypotheses can be invoked to explain the admixture proportion found in individuals in the mouth of the ria. One explanation is that a fraction of the genome could have been inherited from a migrant ancestor. Alternatively, the occurrence of dogwhelks from two genetic clusters within the same location has formerly been related to the existence of individuals belonging to two different shell morphotypes (exposed and sheltered) eN) instead of migration rates (m) N. lapillus in this study vs. N. reticulatus in Barreiro et al. The situation is very different in exposed populations. Their unordered arrangement of genetic differences resembles the structure found in other coastal organisms studied at small-to-moderate spatial scales eN rather than m as proposed by Selkoe et al. eN over time in N. lapillus. However, some long-term monitoring surveys suggest that dogwhelks may experience appreciable changes in local abundance that, more importantly, may not be synchronous between sites eN, we would anticipate variations in the spatial genetic structure over time. Future work comparing population structure across age groups to test this prediction seems warranted.Most efforts to explain chaotic genetic patchiness have focused on recruitment from a larval pool, sometimes from distant sources, since this pattern is mostly detected in organisms with planktonic larvae. However, dogwhelks are direct-developers and our results suggest that dispersal is restricted, at least under some conditions. If limited dispersal were also applicable to wave-exposed populations, they should be mostly maintained by self-recruitment. Under this hypothesis, we would predict a spatially stable pattern of genetic differences over time and/or across age groups N. lapillus as a poor disperser and seem consistent with earlier allozyme studies as well as with predictions derived from its life history features In summary, using a sizeable number of reproducible AFLP markers, we found significant interpopulation differentiation at both regional (<200 km) and areal (<15 km) scales. Genetic differences followed an IBD pattern at regional scale as well as among sheltered populations within a ria, indicative of restrictions to gene flow. Thus, our results support the conventional view of Figure S1Nucella lapillus. UPGMA analysis based on Nei’s genetic distances between sites.(TIF)Click here for additional data file.Table S1Primer sequences used for the AFLP selective amplification and number of loci generated.(DOCX)Click here for additional data file.


Ateles) are a widespread Neotropical primate with a highly plastic socioecological strategy. However, the Central American species, Ateles geoffroyi, was recently re-listed as endangered due to the accelerated loss of forest across the subcontinent. There is inconsistent evidence that spider monkey populations could persist when actively protected, but their long-term viability in unprotected, human-dominated landscapes is not known. We analyzed noninvasive genetic samples from 185 individuals in 14 putative social groups on the Rivas Isthmus in southwestern Nicaragua. We found evidence of weak but significant genetic structure in the mitochondrial control region and in eight nuclear microsatellite loci plus negative spatial autocorrelation in Fst and kinship. The overall pattern suggests strong localized mating and at least historical female-biased dispersal, as is expected for this species. Heterozygosity was significantly lower than expected under random mating and lower than that found in other spider monkey populations, possibly reflecting a recent decline in genetic diversity and a threat from inbreeding. We conclude that despite a long history of human disturbance on this landscape, spider monkeys were until recently successful at maintaining gene flow. We consider the recent decline to be further indication of accelerated anthropogenic disturbance, but also of an opportunity to conserve native biodiversity. Spider monkeys are one of many wildlife species in Central America that is threatened by land cover change, and an apt example of how landscape-scale conservation planning could be used to ensure long-term persistence.Spider monkeys (Genus:  Ateles geoffroyi  and kinkajou (Potos flavus). The polymerase chain reaction (PCR) products were purified using ExoSap-It (USB) and sequenced using the standard BigDye Terminator 3.1  or stepped elongation cycle sequencing protocols .We extracted DNA from fecal samples using QIAmp DNA Stool Mini Kits with an extended proteinase K digestion step, to maximize DNA yield. We confirmed the source species of all mammalian DNA by analyzing two overlapping, 400–500 bp segments of the mitochondrial cytochrome-b region using recommended protocols  to ensure amplification. Annealing temperatures ranged from 68 to 51°C with starting and denaturation at 94°C and extension at 72°C. The profile was as follows: 94°C (5 min), 17 cycles [94°C (30 sec)], 68-51°C , 72°C (30 sec)], 23 cycles [94°C (30 sec)], 50°C (30 sec each), 72°C (30 sec)], and 70°C (5 min). Sequencing protocols follow those used for species identification above. The resulting sequences were manually edited, aligned, and checked against accessioned sequences in Geneious Pro 5. Samples with identical mtDNA haplotypes that also displayed identical multilocus nuclear microsatellite genotypes (described below) were considered to be duplicate samples of the same individual and were removed. We calculated diversity statistics and tests of neutrality and variance in DNASP .We used information from Collins and Dubach  and Ascu2, 0.2 mmol/L dNTP mix, 0.5 μg BSA, and 0.02 μmol/L of each primer. Some sample DNA was more successfully amplified by increasing the MgCl2/dNTP ratio. We used touchdown PCR around the optimal annealing temperatures to ensure amplification of all alleles: 95°C (5 min), 35 cycles [95°C (45 sec)], Ta (45 sec), 72°C (45 sec)], and 72°C (7 min). All alleles were analyzed on an ABI 3730 and were scored in GeneMapper 4.0 (Applied Biosystems).We screened 23 nuclear microsatellite primer pairs previously amplified in platyrrhine primate species and chose eight easily scored and polymorphic loci to avoid downstream genotyping errors  and human buccal swabs were genotyped for all loci as positive controls, to predict allele size, and to assess contamination. Alleles were discarded if they resembled those found in human DNA rather than the spider monkey control. Two independent observers scored alleles visually (without automated binning) and we replicated PCR reactions to confirm our results. Following replication trials, samples that were missing data for more than two of the eight loci were removed from the analysis. We checked for indications of null alleles, allelic dropout, and stuttering based on patterns of homozygosity and allele size using MicroChecker . Finally, differentiation among sampling sites was weak but significant .We amplified the control region sequence from 185 individuals across 15 study sites and found 39 haplotypes. Nine study sites contained at least 10 unique individuals, our cutoff for inclusion in population-level analyses . Hence tP < 0.05) were only so at three or fewer study sites. Amplification results for all loci and individuals are described in Supporting Information. The Leon02, LL1110, and SB38 primers were adjusted to avoid PCR errors and florescent dye quenching. We found that the universal M13 tail system . There was no consistent pattern of gametic disequilibrium; any pair of loci that were significantly related at . All loci except for AB12 deviated strongly from HWE and tested positive for heterozygote deficiency across the restricted and global datasets. Inbreeding coefficients (Fit and Fis) were >0 in all loci .Genetic diversity as measured by allelic richness and expected heterozygosity was high in almost all loci and lowest in the tetranucleotide loci, Ce121 and AB12 , which aP < 0.05). There were six dyads that represent potential parent-offspring or sibling pairs (k > 0.5) within sampling sites, or IV (3), GU (2), and GM (1). Hence, samples from these sites may contain more than one generation.Genetic diversity across the six study sites that included >10 individuals was moderate . As expected in populations with low heterozygosity, D and Gst provided similar results in all locus and population comparisons hence only D is reported.Pairwise Fst values between sampling sites were low, even between pairs separated by long spatial distances . Howeverb = −7.34E-07, P = 0.001). The smallest distance class  maintained the only mean kinship value above 0.005 . The largest distance classes (approximately 22–28 km) exhibited the lowest mean kinship and significantly lower observed kinship than expected, despite having dyads with high kinship values .When analyzing spatial autocorrelation in kinship across the global sample of 163 individuals, and without assigning subpopulations based on sampling site, the regression of kinship to spatial distance was negative and significant  are similar by descent relative to random expectation. This also suggests a limitation on dispersal distances and an increased risk from inbreeding and declines in genetic diversity due to drift. One dyad in the largest distance class exhibited a high estimated kinship, possibly demonstrating a sibling pair that is spatially distant. We cannot confirm if this represents a long-distance dispersal event or an artificial translocation event .Our suite of nuclear microsatellites indicate spatial genetic structuring due to geographic distance, local mating, and close-kin associations within social groups. IBD explained a high proportion of the variance in differentiation among sampling sites and this was corroborated when we looked more closely at fine-scale patterns of kinship across all sampled individuals. We specifically found spatial autocorrelation over small distances and a significant reduction in close kin relationships over distances greater than 25 km. These results confirm the effect of localized mating on gene flow, as individuals within sampling sites  social groups in relatively intact forests in Yasuní National Park, Ecuador. Their results showed markedly more genetic diversity than in our sample. For example, the Yasuní social groups exhibit approximately the same number of alleles within individual social groups as was found in our entire population and higher expected heterozygosity .We expected a clear indication of the effects of forest cover loss on genetic structure via differentiation; instead these data suggest intense localized mating that could lead to a decline in overall genetic diversity. The comparatively weaker pattern of genetic structure in the maternally-inherited mitochondrial marker reinforces the theory of female-biased gene flow. However, most nuclear loci and sampling sites also had significantly lower than expected heterozygosity, consistent with a general loss of diversity. The diversity found in these loci is also lower than that reported for other spider monkey populations. Di Fiore et al.  analyzedThere are multiple explanations for heterozygote deficiency due to locus and population-specific phenomena (Allendorf and Luikart Microcebus murinus) groups and concluded that the predefined groups encompassed more than one reproductive subdivision. However, in our sample, estimations of allele and private allele richness are comparable across the study sites when sample size is taken into account.An alternative explanation is that our pooled samples contain more than one deme. Fredsted et al.  found a This study suggests that there is a genetic consequence for forest-dependent spider monkey populations from the accelerated human pressures on forest systems in southwestern Nicaragua. Collins and Dubach  suggesteThe heightened risk of inbreeding due to localized mating within social groups of spider monkeys is of substantial conservation concern. We found evidence of reduced diversity in nuclear DNA across almost all loci and sampling sites. This strongly suggests that functional diversity is also at risk in these animals. In primates, inbreeding depression has been linked to a variety of congenital malformations in wild populations and explicitly responsible for disease susceptibility in captivity (Charpentier et al. Restoring and maintaining connectivity across landscapes could enable the species to adapt to ongoing change. If these populations are losing genetic diversity, then maintaining gene flow is even more important. Connectivity with other spider monkey populations, such as those that are southeast of our research area, would greatly enhance the future viability of the Rivas population. However, threats to spider monkeys are not limited to the current availability of genetic diversity, but the continued conversion of forest habitat and dispersal corridors as well as hunting and animal capture. Given the precarious status of this population and the long recovery period that will likely be needed to restore genetic diversity, this landscape will require the protection from further animal extraction, conservation of core areas for habitat, connections between cores for migration, in addition to support for gene migration from outside populations.


Ainsliaea linearis (riparian) and A. apiculata (non-riparian); A. oblonga (riparian) and A. macroclinidioides (non-riparian); Asteraceae] using an isolation-with-migration (IM) model based on variation at 10 nuclear DNA loci.Understanding demographic histories, such as divergence time, patterns of gene flow, and population size changes, in ecologically diverging lineages provide implications for the process and maintenance of population differentiation by ecological adaptation. This study addressed the demographic histories in two independently derived lineages of flood-resistant riparian plants and their non-riparian relatives [ca. 25,000 years ago for A. linearis and A. apiculata and ca. 9000 years ago for A. oblonga and A. macroclinidioides, although the confidence intervals of the parameters had broad ranges. The likelihood ratio tests detected evidence of historical gene flow between both riparian/non-riparian species pairs. The riparian populations showed lower levels of genetic diversity and a significant reduction in effective population sizes compared to the non-riparian populations and their ancestral populations.The highest posterior probabilities of the divergence time parameters were estimated to be This study showed the recent origins of flood-resistant riparian plants, which are remarkable examples of plant ecological adaptation. The recent divergence and genetic signatures of historical gene flow among riparian/non-riparian species implied that they underwent morphological and ecological differentiation within short evolutionary timescales and have maintained their species boundaries in the face of gene flow. Comparative analyses of adaptive divergence in two sets of riparian/non-riparian lineages suggested that strong natural selection by flooding had frequently reduced the genetic diversity and size of riparian populations through genetic drift, possibly leading to fixation of adaptive traits in riparian populations. The two sets of riparian/non-riparian lineages showed contrasting patterns of gene flow and genetic differentiation, implying that each lineage showed different degrees of reproductive isolation and that they had experienced unique evolutionary and demographic histories in the process of adaptive divergence. Theoreet al.) or evenl.[et al.). Recentl.[et al.; Hey,[9et al.[et al.[et al.[The flood-resistant riparian plants referred to as rheophytes (van Steenis) are remet al.; Imaichiet al.; Kato,[et al.; Tsukayaet al.; Setogucet al.; Nomura l.[et al.), increal.[et al.). Secondl.[et al.; Kato,[l.[et al.; Mitsui l.[et al.). The nol.[et al.). Third,l.[et al.), and mol.[et al.). TherefAinsliaea (Asteraceae); two independent lineages of flood-resistant perennial species, Ainsliaea linearis Makino and A. oblonga Koidz., and their closely related species distributed in the Japanese archipelago  and A. apiculata are isolated reproductively, mainly by divergent natural selection arising from their contrasting riparian and inland habitats, despite ongoing hybridization , is diss Figure. Its ranbe et al.). A receui et al.). Despitl.[et al.). This iAinsliaea oblonga, is endemic to Okinawa Island, part of the Ryukyu–Taiwan island chain, which is located between southeast China and mainland Japan , they show clear segregation in flooded and forest floor habitats. In common garden experiments, the leaf shapes of the two species were stable, suggesting that the morphological characteristics are fixed genetically. In addition, viable, fertile hybrids with an intermediate leaf shape can be produced with artificial crossing . In natural populations, however, intermediate phenotypes have not been seen, suggesting that the divergence of A. oblonga and A. macroclinidioides is likely maintained by ecological factors, such as habitat segregation that could lead to pre- or post-mating isolation.Another riparian species, n Figure. A. oblo. It hasAinsliaea (Asteraceae) using the IM model based on the multilocus datasets of nuclear gene sequences. The specific hypotheses addressed are the following. First, the initial events splitting the riparian and non-riparian species might be recent, despite their distinct morphological and ecological differences. Second, with adaptive divergence between the riparian and non-riparian populations, they could have exchanged genes since the initial split. The patterns of gene flow  might have implications for population dynamics, the relative strength of reproductive isolation, and selective pressures between the ecologically divergent riparian/non-riparian lineages. Third, the riparian specialists could have less genetic variation and smaller effective population sizes than the non-riparian populations owing to genetic drift. Comparative analyses of the two riparian species with different origins might show common or different patterns of divergence, providing implications for the process and maintenance of adaptive divergence to highly disturbed flooded habitats in this group of taxa.We investigated the divergence time, significance and directions of gene flow, and effective population size differences among riparian  and non-riparian  species in A. linearis, 12 of A. apiculata, 4 of A. oblonga, and 10 populations of A. macroclinidioides. The length of the aligned sequences for each locus ranged from 255 to 707 bp  are compared in TableA. macroclinidioides, the southern populations were excluded from the analyses because they clustered as distinct lineages in the phylogenetic analyses (see the results below). The genetic divergence between riparian and inland species was fairly small, and most of the polymorphisms were shared between them. The fixed nucleotide differences between A. linearis and A. apiculata averaged 0.8 per locus, while no fixed difference was observed between A. oblonga and A. macroclinidioides. We found that the riparian species tended to have fewer polymorphic sites and recombination events, and lower nucleotide diversities than the inland sister species.The sequence polymorphisms and divergence between each monophyletic pair of riparian and non-riparian species . None of the 10 loci showed evidence of being under selection using all of the statistics for the species examined, and we used all loci for the subsequent analyses. Note that inclusion or exclusion of the A27 locus in the analyses did not significantly change the results.None of the loci showed a consistently significant deviation from neutral expectations for Tajima’s , we perA. linearis formed a cluster . When K = 4, A. linearis and A. apiculata comprised different clusters, while A. oblonga and A. macroclinidioides were not separated from each other. The genetic admixture in the STRUCTURE analysis could reflect recent introgression between the two species .A. macroclinidioides populations exhibited distinct genetic structuring with regard to the northern (populations Am1–7) and southern (populations Am8–10) regions of the Ryukyu–Taiwan island chain, suggesting that A. macroclinidioides is a non-monophyletic group. The southern populations showed close relationships with Chinese outgroup species. There was no evidence of within-species geographical structuring for the four species, with the exception of A. macroclinidioides. Therefore, the southern populations of A. macroclinidioides were excluded from the riparian/non-riparian comparisons.By contrast, the phylogenetic and STRUCTURE analyses showed that the The maximum-likelihood estimates (MLEs) and 90% highest posterior density (HPD) of the population genetic parameters estimated by IM are shown in Tableca. 25,000 years ago  for A. linearis and A. apiculata and ca. 9000 years ago  for A. oblonga and A. macroclinidioides . The gene flow between riparian A. linearis and non-riparian A. apiculata was estimated to be unidirectional; the gene flow from A. apiculata to A. linearis was significantly greater than zero , while there was no evidence of gene flow in the opposite direction  from A. apiculata to A. linearis and 0.051 (2N2m2) in the opposite direction.The strict isolation model in which no gene flow occurred during the divergence of the two sets of riparian and inland species was rejected by likelihood ratio tests for both pairs  than in the opposite direction  = 0.526 and that in the opposite direction (2N2m2) was 2.499, suggesting that the amount and direction of gene flow between the two sets of riparian/non-riparian lineages differed in each group.More gene flow was detected between riparian s Figure. Both dis Figure. The genA. linearis was 8,643 , nearly one-third that of A. apiculata . The population size difference between A. oblonga and A. macroclinidioides was more remarkable. The riparian A. oblonga had the smallest effective population , nearly one-tenth that of the northern lineage of A. macroclinidioides . The effective sizes of the ancestral populations tended to be larger than the descendent populations, although the posterior probabilities had wide, flat distributions. This significant reduction in the population size of riparian species is consistent with the relatively low genetic diversity compared with the non-riparian species.We found that the effective populations of riparian species were significantly smaller than those of non-riparian species Tables. The estAinsliaea might have occurred over short evolutionary timescales. Given the wide confidence intervals, however, there is uncertainty in the divergence time estimates. Such wide intervals were likely due to relatively small data set. The previous studies showed that the divergence time parameter tended not to reach to convergence for the recently diverged populations or taxa . The ril.[et al.). Based l.[et al.). In anien et al.; Aguirrel.[et al.; Peccoudl.[et al.), but thA. linearis and A. apiculata, which coexist on Yakushima Island, comprised distinct genetic clusters in the phylogenetic analysis, suggesting that they have accumulated genetic differentiation since their split. By contrast, the riparian species A. oblonga and its sister species A. macroclinidioides, which coexist on Okinawa, had no fixed nucleotide differences, and they were not clustered separately. A previous study demonstrated the origin of the flood-resistant riparian species Solenogyne mikadoi, an endemic species in the Ryukyu Islands . It shoA. apiculata to riparian A. linearis. One possible explanation of the unidirectional gene flow from riparian to non-riparian species is that the genes of riparian species are more difficult to move to non-riparian species inhabiting forest floor habitats. The nucleotide diversity of the riparian species was low, suggesting that their genomic components have undergone selection for specific types that have greater fitness in riparian habitats, but lower fitness in forest floor habitats. Although the statistical test suggested the existence of gene flow, the population migration rates (2Nm) between A. linearis and A. apiculata were below 1.0 for both directions, suggesting that the riparian/non-riparian species have been isolated genetically and are well-differentiated . The li (Wright).A. oblonga and A. macroclinidioides, was estimated to have exchanged genes at a relatively high rate since their divergence. The population migration rate from riparian A. oblonga to non-riparian A. macroclinidioides was 2.499 and that in the opposite direction was 0.526, suggesting that there was sufficient gene flow to prevent genetic differentiation between the two species. In the STRUCTURE analysis, the genetic admixture and absence of population clustering in the two species also indicate relatively recent gene flow . The grtsumoto,), leadin,[et al.). The coAinsliaea was estimated to be nearly zero (m1 and m2 = 0.000), indicating that these regionally subdivided populations have long been strictly isolated. The estimated date of their divergence, ca. 0.8 mya, is congruent with the geological history that the Ryukyu–Taiwan island arc had separated from the nearby continent . Overall, the contrasting patterns of divergence  highlight the relative importance of adaptation to new ecological habitats for phenotypic evolution and speciation in this group of taxa.The gene flow between the northern and southern lineages of . NotablA. oblonga, which is thought to have speciated quite recently, was the lowest of the four species studied. Presently, the riparian species A. linearis occurs only on Yakushima Island and forms abundant metapopulations in the river systems . Given that the plants in riparian flooded habitats suffer severe selective pressures, the invasion of flooded habitats is likely achieved by small numbers of founders, leading to reduced genetic diversity and effective population sizes through genetic drift. The IM model assumes the absence of a population structure in the ancestral population and the sudden split, thus the model can be viewed as a simple case of divergence. In general, the effective population sizes of the descendant populations are well estimated, but the estimates of NA are reported to be sensitive to model misspecification . Theui et al.). A. obleworski,). Thus, This study demonstrated the recent origin of flood-resistant riparian plants, which are remarkable examples of plant ecological adaptation to abiotic environments. The population demographic analyses showed evidence of historical gene exchange between riparian/non-riparian species with parapatric distribution. The two independently evolved riparian species showed reduced genetic diversity and effective population sizes, implying that strong natural selection by flooding frequently reduced diversity of riparian populations. The small sizes of effective populations of riparian species could have promoted the fixation of adaptive trait divergence in highly disturbed flooded habitats. Our results presented a potential case of rapid population differentiation and speciation by ecological adaptation in plants, and adaptive divergence between riparian and non-riparian species has been achieved in the face of gene flow.A. linearis on Yakushima Island and four populations of A. oblonga on Okinawa Island were collected  and GenBank database. CHS region was amplified using primers developed by Alvarez et al.[et al.[We determined the sequences of 10 nuclear loci. Three regions, GA2ox1, GTF, CPPS1, were amplified using primers originally developed based on the sequences of Asteraceae and a related family, which were obtained from the Compositae Genome Project Database . Haplotl.[et al.). To ensS), average number of pairwise nucleotide differences per site  within the 10 loci using the four-gamete test . This test is based on a prediction that follows from the neutral theory of molecular evolution, which states that loci exhibiting high rates of divergence between species will also exhibit high levels of variation within species.For each species, we calculated the number of segrega-ting sites , and θ tterson,) for eac Kaplan,). To eva was estFu & Li,) were es,[et al.). In addl.[et al.) across et al.[et al.[P distance as the metric, and ambiguous states were ignored in the analysis.Previous studies have indicated that the four species comprise monophyletic relationships in nuclear ribosomal and chloroplast DNA data . To estet al.) implemeet al.) becausel.[et al.). Only tet al.[5 iterations, following 3.0 × 105 iterations as the burn-in period. The number of clusters (K) was set from 1 to 20, and all runs were replicated 20 times to test the stability of the results. The most likely number of clusters was estimated according to the model value (ΔK) based on the second-order rate of change with respect to K of the likelihood function following the procedure described by Evanno et al.[In addition, a Bayesian clustering analysis using STRUCTURE ver. 3.1  was conno et al..t), bidirectional migration rates (m1 and m2), and the effective population sizes of the ancestral (θA) and descendent populations (θ1 and θ2). After several preliminary runs to optimize prior boundaries for the six parameters, we conducted runs for 3–5 × 106 MCMC steps following a 300,000 burn-in period from which genealogies were saved every 100 steps. To ensure proper mixing, the run was performed using 20 independent chains and 10 swap attempts per step under Metropolis coupled with a geometric heating scheme of parameters g1 = 0.05 and g2 = 0.1. To check the convergence, plotted trend lines and the effective sample size (ESS) values were examined for each run. In addition, three independent runs for each data set were examined for consistency. An infinite site model of mutation was applied to all loci. The analyses were performed using the largest non-recombining block for each locus . We comμ). Mutation rates of nuclear genes for the studied samples were unknown. Thus, we calibrated the mutation rates of the GA2ox1 and CHS genes by comparing the sequences of perennial species in various taxa of Asteraceae because the studied species are perennials, although they can bloom and produce seeds within the year of germination . A previous phylogenetic study by Kim et al.[c. 33–38 million years ago (mya). We calculated the average nucleotide divergence (K) in the sequences between the tribes Cardueae and Mutisieae and tribes LALV and Astroid using species from various taxonomic groups. The mutation rate per site per year (μ) was then calculated by μ = K/2 T, in which T = 35.4 mya was used for each of the GA2ox1 and CHS regions. The geometric mean of the mutation rates for GA2ox (6.79 × 10–9 substitutions/site/year) and CHS (8.26 × 10–9 substitutions/site/year) was estimated to be 7.49 × 10–9 substitutions/site/year, and this value was used to rescale the IMa parameter estimates.Demographic parameters were scaled to the neutral mutation rate (im et al. estimateThe authors declare that they have no competing interests.YM designed the study, collected and assembled plant samples, carried out the molecular analyses and drafted the manuscript. HS participated in the overall design of the study, collected some of the samples, and contributed to drafting the manuscript. Both authors have read and approved the final manuscript.Table S1. Haplotype compositions. The numbers represent the haplotypes of individuals at each locus.Click here for fileTable S2. Results of neutrality tests for each of the 10 loci and each species. For Ainsliaea macroclinidioides, the southern populations were excluded in the analyses.Click here for fileTable S3. Sampling localities. Sampling sites are shown in Figure Click here for file


Sheep is important in the socio-economic lives of people around the world. It is estimated that more than half of our once common livestock breeds are now endangered. Since genetic characterization of Nigerian sheep is still lacking, we analyzed ten morphological traits on 402 animals and 15 microsatellite DNA markers in 384 animals of the 4 Nigerian sheep breeds to better understand genetic diversity for breeding management and germplasm conservation.FST, FIT and FIS statistics across all loci were 0.088, 0.394 and 0.336 respectively. Yankasa and Balami are the most closely related breeds (DA = 0.184) while WAD and Balami are the farthest apart breeds (DA = 0.665), which is coincident with distance based on morphological analysis and population structure assessed by STRUCTURE.Morphological traits of Uda and Balami were significantly (P < 0.05) higher than Yankasa, which were both higher than West African Dwarf (WAD) sheep. Stepwise discriminant analysis showed tail length, rump height, chest girth, ear length and chest depth as the most discriminating variables for classification. Mahalanobis distances show the least differentiation between Uda and Balami and the largest between WAD and Balami sheep. While 93.3% of WAD sheep were correctly assigned to their source genetic group, 63.9% of Yankasa, 61.2% of Balami and 45.2% of Uda were classified correctly by nearest neighbour discriminant analysis. The overall high Polymorphism Information Content (PIC) of all microsatellite markers ranged from 0.751 to 0.927 supporting their use in genetic characterization. Expected heterozygosity was high for all loci (0.783 to 0.93). Mean heterozygote deficiency across all populations (0.171 to 0.534) possibly indicate significant inbreeding (P < 0.05). Mean values for These results suggest that within-breed genetic variation in Nigerian sheep is higher than between-breeds and may be a valuable tool for genetic improvement and conservation. The higher genetic variability in Yankasa suggests the presence of unique ancestral alleles reflecting the presence of certain functional genes which may result in better adaptability in more agro-ecological zones of Nigeria. These genetic characteristics are potentially useful in planning improvement and conservation strategies in Nigerian indigenous sheep. The population of sheep in Nigeria is currently estimated at 33.9 million making up 3.1% of the world’s total . IncreasSheep biodiversity have been described using morphological measurements ,6 or chaD-loop region in Nigerian sheep [The Nigerian sheep is still genetically unimproved, and the pressure of modern genetic improvement has increased the need to better understand natural genetic variation in Nigerian sheep breeds, as well as formulate germplasm conservation policies. The only genetic diversity study of genetic variation among Nigerian sheep breeds using microsatellite markers covered a limited geographical area . Therefoan sheep . The inf2 values ≥0.01  according to Weir and Cockerham (1984), GST and Shannon index values for all 15 microsatellite markers analyzed in Nigerian sheep breeds are shown in Table Polymorphism Information Content (PIC) and F statistics  was observed for BM8125 while the lowest value of 0.022 was seen in DYMS1. Inbreeding values within and across breeds can also be attributed to selection. FST and GST are indices of population subdivision. Global FST, GST and GST’ over all loci were all significantly different (p < 0.001). The population differences examined by global analysis of FST (coefficient of multilocus genetic differentiation fixation index) for each of 15 microsatellite loci across the four sheep breeds revealed that most of the total genetic variation corresponds to differences among individuals within breeds (91.2%) and 8.8% result from differences among breeds. Values of GST ranged from 0.033 for OarFCB304 to 0.211 for OarHH47 with a mean of 0.120  results revealed that the greatest variation 60.716%) is within individuals, 30.545% among individuals within populations and 8.739 among populations which are consistent with the Fts Table . Figure 6% is witPopulation structure assessed by STRUCTURE software and barplots generated by DISTRUCT are presented in Figure Phenotypic characterization has been shown to be an accessible and easy-to-use tool in conservation and breeding programs . This coLama guanicoe guanicoe in Argentina. The higher values recorded for intensively managed animals might be as a result of better nutrition and management practices as they tend to gain more attention than those reared semi-intensively. Riva et al. [The larger values reported for the conformation traits of males are in consonance with earlier reports on sheep , goats  and catta et al.  reportedThe three canonical functions (linear combinations of the continuous variables that summarize variation between the four sheep breeds) obtained could be used as criterion for establishing phenotypic standards for Nigerian sheep. The closeness between Uda and Balami sheep compared to their WAD and Yankasa counterparts might be as a result of near biometric convergence, which may function as a guide to genetic and evolutionary relationships between the two breeds. The longer distance between WAD and other breeds, especially Balami and Uda revealed that phenotypic differences are maintained in part by the reduction of gene flow among populations separated by large distances as well as physical-ecological barriers. The WAD sheep predominate in the trypano-endemic humid zones of Southern Nigeria. The intermediate morphology of Yankasa sheep might be maintained by natural selection on body size of individuals inhabiting intermediate or a range of different environments . Selective advantage might therefore favor Yankasa sheep from the biogeographical context, although more heterotic gains might be attained from crosses involving WAD and Uda or Balami. This is because populations are dynamic units which adapt physiologically and genetically to their environments and sensitive to, and within limits, responsive to any change in their environmental conditions. An appreciable percentage of animals were classified into their distinct breeds. However, some level of intermingling was observed between Uda and Balami, which could partly be attributed to indiscriminate crossbreeding due to geographical proximity.et al.[The high number of alleles observed in Yankasa must have contributed to its adaptability to more agro-ecological zones in Nigeria compared to other sheep breeds that may confer selective advantages . Geneticet al. reportedet al..Different indices used in this study demonstrate the existence of genetic diversity among Nigerian sheep breeds. Variations in allelic richness were observed across all loci. Mean values among the various loci ranged from 3.66 in OarHH47 to 14.73 in OarJMP29, with mean values of 8.63. Dalvit et al.  found siST) and Nei’s coefficient of gene variation (GST) [ST analysis in this study reveal that the proportion of gene variation among the breeds is still low.Differences among populations are commonly quantified by the use of one of several statistics, including Wright’s inbreeding coefficient (Fon (GST) . The raton (GST) . These von (GST)  observedon (GST)  and Euroon (GST) . The obson (GST) . The resThe Shannon index of Nigerian sheep breed in this study revealed low species richness and evenness since all the indices were below 3.5, the mark set for high species evenness and richness . This miFST, FIT and FIS over all loci considered in this study supports the suitability of some microsatellite markers over the others in the study of genetic diversity in Nigerian sheep breeds. Selection based on estimated breeding values rather than phenotypic merits can lead to the extensive use of a small number of elite individuals in purebred livestock populations, and could potentially increase inbreeding [IS) was observed to be 33.5% whereas the total population (FIT) exhibited 39.5% deficit with significant values (p < 0.05). Some investigations have reported relatively low variability in local non-selected breeds, as is the case of certain Nigerian [Differences in the values of global breeding . It has breeding . On the Nigerian  and ChinNigerian .FST results. Finally, the trend in genetic relationship between these Nigerian sheep breeds is the same for delta mu square, kinship coefficient and proportion of shared alleles. Indeed, our recent molecular characterization of Nigerian sheep using mtDNA sequences of the D-loop found 96 haplotypes, but only 5 haplotypes are common to all the breeds [The relative higher genetic identity is probably due to the continuous crossing between populations at least in the recent past . The AMOe breeds , showingThe closest genetic distance between Yankasa and Balami at 0.184 and the farthest distance between Balami and WAD (0.665) may be reflective of their geographical locations in Nigeria since Yankasa and Balami are more of northern than southern breeds. This is further confirmed by the greater genetic distance between WAD and Balami observed in this study. These results are supported by Adebambo et al.  among thResults from the STRUCTURE analysis revealed that varying the number of presumed ancestral populations (K) produces clusters that are consistent with the observed morphological categorization. The first level of clustering (K = 2) reflects the presence of two clusters in the four breeds examined and further evaluation revealed a third strong cluster. This result suggests that the four breeds originated from three ancestral populations which diverged as a result of several years of adaptation and domestication. Further evaluation of the clusters revealed the presence of sub clusters and admixtures which are indicative of substantial gene flow between these breeds. WAD is the only breed with a minimal case of admixture. This may be due to geographical delineation of the breed and the breeding practices of the owners of these animals predominantly in the southern part of the country. Yankasa possess alleles which are shared by the other three breeds and this is consistent with the results discussed above.The mitochondrial data showed a different relationship in the neighbor-joining tree for the four Nigerian sheep breeds . In thatMorphological and genetic diversity of Nigerian sheep breeds is eroding gradually. These results suggest that within-breed genetic variation observed in Nigeria sheep is more than between-breed and this variation could be a valuable tool for genetic improvement and conservation. The higher genetic variability in Yankasa may mean the presence of unique alleles reflecting the presence of certain functional genes which may possibly be related to better adaptability of Yankasa in more agro-ecological zones of Nigeria. The higher level of heterozygosity in this study provides the basis for further improvement through selection of primarily Yankasa as well as the other breeds.2 . Nigeria is bounded by Niger, Benin and Cameroon Republics on the North, West and East, respectively  with a total area of 923,768 km2 and Taq DNA polymerase  using annealing temperatures shown in Table DNA was extracted from 50 μl of whole blood using the ZymoBead™ Genomic DNA Kit  according to the manufacturer’s recommendations and DNA yield and quality were assessed using a Nanodrop ND-100 Spectrophotometer . The DNA was amplified by PCR in a MyCycler™ Thermal Cycler  using 15 microsatellite markers selected from the FAO recommended list  describeDNA fragment analysis of microsatellite markers was carried out using the Applied BioSystems 3730xl DNA Analyzer  at the Cornell University Genomics Core Facility. GeneMapper Software version 3.0  (which cMeans, standard deviations, standard errors and coefficients of variation were computed for all the traits measured using the GLM procedure of SAS statistical package . SourcesA model-based Bayesian clustering analysis was used to infer population structure and the level of admixture in the sheep breeds implemented in STRUCTURE v2.3 . The STRBL: Body length; BW: Body weight; CD: Chest depth; CG: Chest girth; EL: Ear length; FCL: Fore cannon bone length; HW: Height at withers; RH: Rump height; RW: Rump width; TL: Tail length.The authors declare that they have no competing interests.IGI and BOA conceived the study; BOA, IGI, OAA, MOO, CONI designed the study; IGI and MO obtained funding for the study; BOA, MAA, AY, MW, TMS, OOA, BMI, SAA, GOO and JOE collected phenotypic data and contributed to morphological data analysis; BOA, AY, and OOA carried out morphological data analysis; BOA, MAA and MD carried out the molecular lab analysis; BOA, MAA, SOP, MD, KK and IGI carried out the molecular data analysis and interpretation, BOA, MAA, IGI, MO, AY and SOP wrote the manuscript. All authors read and approved the manuscript.


Schistosoma mansoni. Chemotherapy is the main measure for controlling schistosomiasis, and the current drug of choice for treatment is praziquantel (PZQ). Although PZQ is efficient and safe, its repetitive large-scale use in endemic areas may lead to the selection of resistant strains. Isolates less susceptible to PZQ have been found in the field and selected for in the laboratory. The impact of selecting strains with a decreased susceptibility phenotype on disease dynamics and parasite population genetics is not fully understood. This study addresses the impact of PZQ pressure on the genetics of a laboratory population by analyzing frequency variations of polymorphic genetic markers.Schistosomiasis has a considerable impact on public health in many tropical and subtropical areas. In the new world, schistosomiasis is caused by the digenetic trematode S. mansoni population with decreased susceptibility to PZQ. In our experiments we also observed that female worms were more susceptible to PZQ than male worms.Infected mice were treated with increasing PZQ doses until the highest dose of 3×300 mg/Kg was reached. The effect of PZQ treatment on the parasite population was assessed using five polymorphic microsatellite markers. Parasitological and genetic data were compared with those of the untreated control. After six parasite generations submitted to treatment, it was possible to obtain a S. mansoni and increased endogamy. The understanding of how S. mansoni populations respond to successive drug pressure has important implications on the appearance and maintenance of a PZQ resistance phenotype in endemic regions.The selective pressure exerted by PZQ led to decreased genetic variability in  Schistosomiasis causes severe health problems and economic losses in transmission areas in the Americas, Africa, and Southeast Asia. In regions where treatment is available, such as Brazil, chemotherapy has led to decreased morbidity of the disease. However, parasite resistance to drugs is of general concern. Drug resistance is known in some species of helminths, and there are reports of decreased susceptibility in schistosomes, although such reports are not widespread. The impact of consecutive drug treatment on parasite populations at transmission sites can be evaluated by the genetic makeup of the population. For example, the decrease in the genetic diversity of these populations upon chemotherapy can be interpreted as the selection of less sensitive parasites. In this study, we used a laboratory mouse model of schistosomiasis that was treated with increasing amounts of praziquantel (the drug of choice for human treatment) until we reached the highest dose of 3×300 mg PZQ/kg. Our results clearly indicate that the parasite population showed a decrease in genetic diversity after measuring allelic and genetic diversity. This observation confirms that drug selection occurred in this population. The work highlights the need for genetic monitoring of populations under drug pressure, resistance mechanisms, and the dynamics of parasite populations upon treatment. S. haematobium, while others are trapped in the host tissues where they produce a granulomatuous immune-inflammatory process that represents the main cause of pathology Schistosomiasis is a disease that affects approximately 206 million people in 76 countries. On a global scale, 779 million people are at risk of contracting the disease Schistosoma mansoni+2 influxes suggests that the sites of action are Ca2+ ion channels in the membrane of tegument and muscle cells Schistosoma that have been demonstrated include the following: reduction in the level of glutathione (GSH) Praziquantel (PZQ) is the drug used for treatment of human schistosomiasis and is inexpensive and effective against all human schistosome and some cestode species S. mansoni strains regarding susceptibility to schistosomicidal drugs was demonstrated in different endemic regions, such as Senegal, Egypt and Brazil S. mansoni in endemic regions may emerge by different mechanisms. The main causes may be high parasitic loads, fast reinfection, the presence of immature worms, inefficient human immune responses, the use of low quality drugs, differences in evaluation, and cure criteria or actual drug resistance The availability of only one drug to treat schistosomiasis is a concern, and too little investment has been made toward the development of alternatives mansoni less susceptible to PZQ was published by Fallon and Doenhoff S. mansoni populations are being investigated with the use of highly polymorphic microsatellite markers The first study that showed it is possible to induce S. Schistosoma populations at endemic sites perform under drug pressure are necessary for determining the most efficient treatment strategy for infected populations and for the design and modeling of control programs S. mansoni population will experience a decrease in genetic diversity and susceptibility to the drug.Understanding how drug pressure affects the genetic structure of a parasite population in a controlled setting raised questions relevant to the endemic setting related to transmission, chemotherapy effects, and epidemiology of the disease In vivo studies were conducted in compliance with the guidelines of the Collegiate of Animal Experimentation (COBEA) and approved by the Commission on the Ethical Use of Animals (CEUA-FIOCRUZ) protocol number L-018/09 approved on Jan. 23, 2009.S. mansoni (LE strain) life cycle was maintained using Biomphalaria glabrata snails as intermediate hosts and Swiss mice as definitive hosts according to Pellegrino and Katz S. mansoni (LE strain) by a subcutaneous route.The To induce decreased susceptibility, increasing doses of an aqueous suspension of PZQ were administered to infected mice by oral gavage. The protocol used for parasite selection was based on Fallon and Doenhoff For DNA extraction of individual parasites, worm pairs were separated and digested in extraction buffer  and 20 µg/ml of proteinase K (Sigma) and incubated for 12 hours at 37°C, followed by incubation at 95°C for 5 min. Nucleic acids were precipitated with cold absolute ethanol and resuspended in 30 µl of TE buffer and rehydrated for 12 hours at 4°C.loci for each population. Allelic and genotypic data were identified for a total of 250 worms. PCR reactions and scoring of the polymorphic microsatellites were conducted as previously described We analyzed five polymorphic microsatellite Genetic data was analyzed with the Fstat software version 2.9.3.2 PZQ selection resulted in a striking increase in the number of recovered parasites after treatment with this drug, compared with the number recovered after equivalent treatment of non-selected parasites. Thus, after seven parasite generations of selection using doses of PZQ that increased from 200 mg/kg to 900 mg/kg, we observed a greater than three-fold increase in the degree to which the proportion of worm load in the Non-selected group had been reduced by drug treatment (47.3%), compared with the level of reduction in the Selected group 14.58% - . These fin vitro males displayed higher sensitivity to PZQ. These experiments differ from ours in that we assessed PZQ sensitivity in vivo. It has been suggested that the environment in which the parasite is located affects the parasite physiology and drug sensitivity Female adult worms were shown to be more susceptible than males to PZQ exposure. We observed that after exposure to 3×300 mg/kg of PZQ, both male and female worms showed a significant decrease in susceptibility. S. mansoni. The number of alleles found in the Non-selected population decreased in the Selected population from 98 to 42. The number of genotypes found in the Non-selected population and the Selected population decreased from 265 to 112. The allele and genotype number for each locus decreased after successive treatments. The Selected population (S) showed a lower total number of alleles per genotype locus than that of Non-selected parasites  and Inbreeding Coefficient (Fis). Our results show that there is a significant genetic differentiation between the first and seventh generations of the S population (Rst = 0.240). In contrast, without PZQ pressure, we did not observe a significant differentiation in the Non-selected populations (Rst = 0.080). Genetic diversity data reveal that decreased susceptibility selection contributes to the decrease of genetic variability of parasites selected with PZQ. Next, we measured the level of inbreeding between the Selected and Non-selected populations. According to Wright is value of 0.309 indicates a high level of inbreeding in the Selected population. The Fis index shows that there is a strong increase in endogamy in the Selected population (0.134 to 0.309). Our results with both indexes suggest that the Selected S. mansoni population is less genetically diverse as a result of PZQ pressure. These results agree with a report by Norton and colleagues S. mansoni. We did not observe significant differences in the genetic variability of male and female worms (data not shown) nor in populations passaged in parallel but not submitted to drug pressure.The degrees of population structuring and diversity among Selected and Non-selected populations were evaluated by genetic diversity analysis . We speculate that in the field, one possible reason for resistance or decreased susceptibility not being widespread is perhaps its biological cost. The creation of a genetic map constructed using microsatellites, combined with the future development of a SNP map, means that genetic tools will be invaluable in both investigating the genetic profile of populations and identifying markers or genes that convey resistance.


Signal transducer and activator of transcription 3 (STAT3) is a potent regulator of gliomagenesis through its induction of angiogenesis, host immunosuppression, and tumor invasion. Gain of function mutations result in constitutive activation of STAT3 in glioma cells, making STAT3 an attractive target for inhibition in cancer therapy. Nevertheless, some studies show that STAT3 also participates in terminal differentiation and apoptosis of various cell lines and in glioma with phosphatase and tensin homolog (PTEN)-deficient genetic backgrounds. In light of these findings, the utility of STAT3 as a prognostic indicator and as a target of drug therapies will be contingent on a more nuanced understanding of its pro- and anti-tumorigenic effects. Glioblastoma multiforme (GBM) is the most common primary malignancy of the central nervous system (CNS), with an incidence of 3.19 per 100,000 and a five-year survival rate of less than 5% . GBM’s mAnother potential target of GBM molecular therapy is signal transducer and activator of transcription (STAT) 3, a member of the STAT family of proteins that mediates cytokine signaling and nuclear transcription. STAT3 is an attractive focus for therapeutic intervention since it represents a point of convergence for multiple oncogenic signaling cascades. STAT3 has emerged as a key initiator and master regulator of mesenchymal transformation in malignant gliomas . Constitin vitro versus in vivo model, and therapeutic modality underscore our incomplete understanding of the role of STAT3 activation in GBM. In fact, some studies have suggested that STAT3 can act, paradoxically, as a tumor suppressor by participating in terminal differentiation and apoptosis. The utility of STAT3 as a prognostic indicator and therapeutic target is, therefore, contingent on further clarification of its pro- and anti-tumorigenic effects. Here, we review the role of STAT3 activation in gliomagenesis, and summarize the most recent laboratory and translational strategies for targeted STAT3 inhibition. Several preclinical studies have demonstrated the anti-tumor effects of STAT3 knockdown using small interfering RNA (siRNA), micro-RNA (miRNA), or small molecule inhibitors ,26,27,28STAT proteins are a family of cytoplasmic transcription factors that are activated by tyrosine kinases and mediate cellular response to inflammatory and proliferative signals ,30. ThesThese molecules play a significant role in regulating the STAT3 signaling cascade, and under normal physiologic conditions, represent important checkpoints in the activation and deactivation of cell proliferation . HoweverPersistent STAT3 activation—secondary to intrinsic hyperactivity, aberrant upstream signaling, or defective negative regulation—can lead to abnormal survival and tumorigenesis . ConstitExtracellular cytokine dysregulation can also result in abnormal autocrine or paracrine stimulation of cell surface receptors involved in downstream STAT3 signaling. This mechanism has been described in multiple myeloma, where IL-6 overexpression led to increased JAK/STAT3 activity , as wellet al. demonstrated similar rates of STAT3 activation in anaplastic astrocytomas (AA) and GBMs  [et al. expanded on these findings by describing a positive correlation between glioma grade and extent of STAT3 activation. Constitutive activation was detected in 60% of primary high grade/malignant gliomas, secondary to JAK2, EGFR and/or EGFRvIII kinase hyperactivity. In comparison, only 27%, 29%, and 57% of Grade I, II, and III gliomas, respectively, were found to have similar activation [et al. reported STAT3 activation in only 9% of AA and 9% of GBM samples, and found no correlation with tumor grade [The presence of STAT3 activation in GBMs varies considerably, with detection rates of most retrospective, immunohistochemistry-based studies ranging from 9%–83% . These dtivation . These ctivation ,59. In cor grade .et al. observed constitutive activation of STAT3 in 90% of human GBM tumors and GBM cell lines. The majority of STAT3 activity in U251 cells was catalyzed by gp130-associated JAKs secondary to IL-6, a cytokine secreted by GBM cells both in vivo and in vitro [The mechanisms of STAT3 activation in GBMs are similar to those found in other cancer cell lines. Rahaman in vitro . Severalin vitro ,63,64,65de novo GBMs in older patients [STAT3 gain-of-function mutations have not been reported, but amplifying mutations of STAT3 activators have been identified in several studies. In particular, missense mutations of EGFR genes may lead to constitutive downstream activation, and have been implicated in patients ,67,68. Bpatients .et al. demonstrated a negative correlation between transcription inhibitor PIAS3 and phospho-STAT3 expression, with subsequent PIAS3 overexpression leading to decreased STAT3 transcriptional activity and cell proliferation [et al. observed a greater frequency of PTPRD expression loss in GBM versus lower grade gliomas. Decreased PTPRD expression was also predictive of poorer prognosis in GBM patients [Contrary to amplifying mutations, decreased expression of STAT3 repressors can cause constitutive activation of STAT3. Brantley feration . Proteinferation ,71. FurtOnce activated, STAT3 drives several pro-oncogenic mechanisms to promote cell proliferation and survival, immune suppression, invasion, and angiogenesis.in vitro and in vivo studies of STAT3 inhibitors suggest that STAT3 downregulation can induce cell cycle arrest and apoptosis, often in a dose-dependent manner [et al. observed that even transient STAT3 inhibition results in irreversible growth arrest and loss of self-renewal capacities in GBM stem cells [Under normal physiologic conditions, STAT3 activity is tightly regulated via ligand-dependent receptor and non-receptor tyrosine phosphorylation . Coordint manner ,78,79. Ft manner . Sherry em cells . STAT3 tem cells . Togetheet al. demonstrated decreased STAT3 activation and migratory behaviors across five different GBM cell lines in vitro [Infiltration and migration are characteristic of glioma tumor cell migration preferentially occurs along white matter tracts, accounting for the characteristic “butterfly lesions” frequently observed crossing the corpus callosum, perineuronal satellitosis, and perivascular or subpial spread . STAT3 iin vitro . Though in vitro ,84,85. Iin vitro . Furtheret al. noted that VEGF expression in several cancer cell lines correlated with constitutive STAT3 activity, and that STAT3 inhibition led to reduced Src-induced VEGF expression [in vitro overexpression of the viral chemokine receptor US28 has been shown to activate several downstream transcription factors such as hypoxia inducible factor-1 and STAT3, resulting in VEGF promoter activation [VEGF-mediated angiogenesis is critical for the survival of most tumors, providing the requisite nutrients for accelerated growth and progression . Constitpression . Resultstivation ,91.et al. found that vascular remodeling and the creation of a more hypoxic tumor microenvironment paradoxically resulted in a more invasive, glycolysis-dependent tumor phenotype [et al. demonstrated that anti-VEGF therapy in glioma patients results in markedly elevated levels of STAT3 expression, and that STAT3 inhibitors could enhance the efficacy of antiangiogenic treatment strategies [VEGF is often overexpressed in GBM ,92,93, arategies . FurtherTumor-mediated immune suppression at both the local and systemic levels has been well described in GBM patients . T cell et al. demonstrated that siRNA-mediated STAT3 suppression triggered the release of soluble factors such as interferon (IFN) gamma-inducible protein 10 (IP-10), Rantes, IL-8, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-β; furthermore, exposure to these paracrine signaling molecules induced DC maturation and activation [STAT3 has been established as an important negative regulator of the host’s antitumor immune response. STAT3-associated immunosuppressive mediators generally fall into two categories: (1) soluble or membrane-bound proteins produced by glioma cells and (2) immune cell populations recruited by the tumor. Secreted factors include IL-10 (an inhibitor of Th1 activity), VEGF, and TGF-beta, which inhibit T cell, B cell, natural killer (NK) cell, and monocyte functions ,95,96. Vtivation .et al. demonstrated that direct interaction with glioma cells led to STAT3 activation in tumor infiltrating macrophages. In return, MG/macrophage-derived factors were shown to activate STAT3 signals in tumor cells, perpetuating glioma pathogenesis and progression [Microglia (MG) make up almost one third of the non-tumor cells found in GBM tissue sections and are responsible for secreting MMP-9, EGF, and VEGF. In the homeostatic state, MG act as resident antigen presenting cells with proinflammatory, anti-neoplastic functions. However, relatively recent evidence suggests that exposure to glioma-derived chemokines induce constitutive STAT3 activation in MG, with consequent suppression of antitumor mechanisms or even tolerance to tumor antigens ,100,101.gression .STAT3 also recruits and promotes the proliferation of T regulatory cells (Tregs), which suppress effector lymphocyte activity within the tumor microenvironment ,104. TreWhile STAT3 activation is clearly implicated in GBM survival through invasion, differentiation, angiogenesis, and immunosuppression, recent studies in GBM and other tumors have suggested a role for STAT3 in tumor suppression. Most studies illustrate STAT3 involvement in growth inhibitory signaling, terminal cell differentiation, and apoptosis. For example, the IL-6 growth-inhibitory and terminal differentiation signal has been correlated with STAT3 transcriptional activity in prostate cancer ,109. SimIn stratified squamous epithelium, constitutively active STAT3 is responsible for terminal differentiation through enhanced activation of keratin 13. Inhibition of STAT3 via overexpression of the tumor suppressor PTEN promotes proliferation and tumorigenesis . Moreoveet al. have shown that in PTEN-deficient GBM, endogenous STAT3 inhibition prevents STAT3-mediated transcriptional IL-8 repression, resulting in increased tumor proliferation and invasiveness [The role of the JAK-STAT pathway is well established in astrocyte differentiation ,123,124.siveness . Furtheret al. report that STAT3 expression in colorectal carcinoma is correlated with higher-grade tumors and poorer survival [versus tumor suppressor function on the tumor genetic background, potentially confounding the results.Given STAT3’s established role in GBM tumorigenesis and tumor suppression, it is a reasonable candidate as a prognostic marker. Jin survival . Converssurvival . Such diet al. report that JAK/STAT activation correlates with higher-grade gliomas and is an independent prognostic indicator of decreased survival [In GBM, survivin positivity has been suggested as a strong prognostic indicator of significantly poorer survival and higher malignant grade, owing to its anti-apoptotic activity ,128. Regsurvival ,129. RecA number of approaches have been developed to target STAT3 or its downstream effects and thereby inhibit glioma growth. Specific antitumor therapies against STAT3 are summarized in in vivo glioma inhibition in a dose-dependent manner and is partially associated with G1 arrest in GSCs [Potential drugs that directly inhibit STAT3 activation include the naturally occurring triterpenoid oleanolic acid, which also suppresses the M2 polarization of tumor-associated macrophages by suppressing IL-10 secretion . Small m in GSCs . STAT3 k in GSCs . Oligode in GSCs . Further in GSCs .Other drug targets include STAT3 associated genes that inhibit tumor cell migration or invasion . AG490 iSTAT3 inhibition is also useful synergistically with other treatment modalities. Resveratrol, a grape polyphenol, has been shown to enhance glioma radiosensitivity by inhibiting STAT3 signaling, rendering future promise for more effective radiotherapy . ResistaWhile STAT3 is clearly an integral pathway to tumor growth and invasion, lifting the “brakes” on immune function through STAT3 checkpoint blockade combined with tumor-specific vaccine therapy may show promise for more robust anti-tumor responses . Such chin vitro cell line experiments, which may limit its relevance for clinical GBM pathogenesis. Further research on the pro- and anti-tumorigenic effects of its activation is needed to maximize the possibilities of STAT3 as a prognostic indicator and a target for molecular therapy.Recent studies continue to define the role of STAT3 as a constitutively active element in gliomagenesis. Activation of STAT3 has been implicated in tumor activation through the production of anti-apoptotic and glioma stem-cell maintenance factors, pro-invasive enzymes, and angiogenic elements such as VEGF. Additionally, STAT3 helps to orchestrate immune evasion of GBM through downstream increases in Tregs and decreases in activated circulating lymphocytes. On the other hand, studies have also shown that STAT3 may paradoxically facilitate tumor suppression by contributing to cell differentiation, growth inhibitory signaling, and apoptosis. It is important to note that much of the evidence supporting STAT3’s role as a potential tumor suppressor has been obtained through


Phytophthora infestans (Mont.) de Bary, the causal agent of potato late blight, is responsible for tremendous crop losses worldwide. Countries in the northern part of the Andes dedicate a large proportion of the highlands to the production of potato, and more recently, solanaceous fruits such as cape gooseberry  and tree tomato (Solanum betaceum), all of which are hosts of this oomycete. In the Andean region, P. infestans populations have been well characterized in Ecuador and Peru, but are poorly understood in Colombia and Venezuela. To understand the P. infestans population structure in the Northern part of the Andes, four nuclear regions  and one mitochondrial (Cox1) region were analyzed in isolates of P. infestans sampled from different hosts in Colombia and Venezuela.P. infestans isolates from crops within several regions of Colombia and Venezuela, revealing the presence of clonal populations of the pathogen in this region. We detected low frequency heterozygotes, and their distribution patterns might be a consequence of a high migration rate among populations with poor effective gene flow. Consistent genetic differentiation exists among isolates from different regions.Low genetic diversity was found within this sample of P. infestans is a clonal population with some within-clone variation. P. infestans populations in Venezuela reflect historic isolation that is being reinforced by a recent self-sufficiency of potato seeds. In summary, the P. infestans population is mainly shaped by migration and probably by the appearance of variants of key effectors such as Avr3a.The results here suggest that in the Northern Andean region  Phytophthora infestans (Mont.) de Bary  for Ras,  for β-tubulin, [GenBank: GU258058 and GU258059] for Cox1 and [GenBank:GU258052-GU258057] for Avr3a. Additionally, sequences for ITS were deposited for all the strains employed under accession numbers .Haplotype reconstruction was conducted using DNAsp v4.90.1  implemenβ-tubulin, Ras and Cox1, from P. infestans isolates collected worldwide were retrieved from GenBank and included in the analyses , nucleotide substitution rate (θ) and haplotypic diversity (HD) were calculated for each data set from the Northern Andean region, including Colombia and Venezuela (NA) and for the rest of the world, corresponding to available sequences from other geographical origin (R) using DnaSP v.4.90.1 . For theTo test the hypothesis of genetic structure  among these populations, a permutation test for Hudson's statistics  was perf-tubulin, Ras, Avr3a and Cox1 with DnaSP v.4.90.1  as an outgroup. Details of these tests are explained elsewhere [A pairwise comparison of the rates of nonsynonymous nucleotide substitutions per nonsynonymous site (dN) and synonymous nucleotide substitutions per synonymous site (dS) were estimated for i (1986)  implemeni (1986) . CODEML lsewhere .β-tubulin. The haplotype most frequently found (H1) was present in Colombia and Venezuela. Venezuelan P. infestans showed only two haplotypes, H1 and H8. The Central Andes showed four haplotypes, one of them restricted to this zone (H10). In the Eastern Andes three haplotypes were found for β-tubulin, and H11 was only found in this region. The Southwestern Andes showed the highest number of different haplotypes for this gene (9), five of them  restricted to this area. For Ras, haplotype the same nomenclature was used as the one in Gomez-Alpizar et al., 2007 [Ras region showed two haplotypes (H2 and H13) with only one haplotype (H2) found in the Southwestern, Central and Eastern Andes. Venezuelan isolates were heterozygotic (H2/H13) and the H13 haplotype was exclusive to this country. Cox1 showed two haplotypes (H1 and H5), and H1 was present in all regions. All isolates from the Central Andes belonged to only this haplotype, which was also the most common one in the Southwestern and Eastern Andes. H5 was the most common haplotype in Venezuela , two haplotypes for Cox1 (H1 and H5), and just one haplotype for Ras (H2) and Avr3a (H1).Nucleotide diversity (π) and substitution rate were low in all genes analyzed in the R and W datasets Table . Again, x1 Table . The Norβ-tubulin haplotypes showed a dominant haplotype, H1, present in Central America and South America . For Cox1, two haplotypes (H1 and H5) were widely distributed among regions and connected directly in the network. The three remaining haplotypes were derived from H5; two of them were restricted to Central America (Mexico) and one, H4, to South America (Ecuador)  .The estimated gene networks for the W dataset Figure  showed aa Figure . H7 was ) Figure . For the) Figure . H2 was Ras and Cox1, between the Eastern Andes and Venezuela and between the Central Andes and Venezuela . Colombia is situated in a critical geographical position, acting as a bridge between Central and South America. According to FAO statistics, this country has a commercial history in trading potatoes with several countries in Europe, Asia, North and South America, and the Caribbean [This is the first time that a detailed population genetic analysis of aribbean . This suRas, only 2 out of 13 haplotypes found globally could be observed in the region. The A2 mating type was discovered in Ecuador and Colombia [The low genetic diversity found on global and regional scales in the Northern Andean region (Colombia and Venezuela), in both mitochondrial and nuclear regions, is consistent with previous reports in other countries of South America . Even foColombia ,26, and Colombia ,16,48, oβ-tubulin and Avr3a, more than two steps separated four and two haplotypes, respectively. The more divergent haplotypes were found in samples from S. betaceum and corresponded to the mitochondrial haplotype Ia. Recently it has been suggested that a new species, P. andina, with an Ia mitochondrial haplotype, could be associated with S. betaceum [P. infestans sensu lato. The presence of this new species in the population of the southwestern Andes could explain the different pattern observed in Avr3a and β-tubulin.The gene networks for the different regions showed, as a general pattern, that the relationships between the haplotypes in each population of the Northern Andean region are consistent with variation within a clonal population and that haplotypes are discriminated by one or two steps. However, in the Southern Andes, at least for betaceum ,23,49. HP. infestans is mainly explained by the variability present within each population that is generated by the existence of different low-frequency alleles, particularly for β-tubulin and Avr3a in the Southern Andes. Nevertheless, the variation detected in this study was enough to show some genetic differentiation. At the regional level, the P. infestans population in Venezuela appeared to be isolated from the Central and Eastern Andes populations. Indeed, at the mitochondrial level Venezuelan isolates belonged largely (11 out of 12) to the Ia haplotype, and just one corresponded to IIa, the most common haplotype in the North Andean region. This and the presence of a particular haplotypic composition for the nuclear genes may suggest that the Venezuelan population probably has been structured by different population events. Reasons for the apparent genetic isolation of the Venezuelan isolates of P. infestans have been discussed elsewhere [The genetic variation of lsewhere . The estβ-tubulin. This could be the result of a long history of self-sufficiency in terms of potato seed tuber supply in the eastern Andes as well as avoiding movement of the pathogen on plant tissue. Second, results obtained with the mitochondrial gene Cox1 and nuclear genes suggested that historically the Southwestern and the Eastern P. infestans populations have been isolated, but recent gene flow could be taking place.Additionally, two significant patterns were observed. First, subdivision was found between the Eastern Andes with the rest of Colombia for Avr3a may have resulted in a different pattern of sequence polymorphisms in the sampled population in comparison to the other nuclear regions analyzed. However, low genetic diversity was found for this gene. No genetic subdivision could be detected in the Northern Andean region, in contrast with what was found for β-tubulin and Ras. At the amino acid level interesting patterns emerged. Previous studies reported only three polymorphic positions in a sampling of isolates from S. tuberosum from different locations worldwide. Two alleles were characterized based on those amino acid positions, C19 K80 I103 and S19 E80 M103, with avirulent and virulent phenotypes, respectively [S. tuberosum R3-expressing plants remains to be determined in order to define their potential effect on host resistance at the subspecific level.The selection imposed on a gene with a known function in host recognition as ectively . Here weectively . Accordiectively  have shoectively . It has ectively , while tectively . This scAvr3a gene may be related to the plant species from which the isolates were collected. Armstrong et al. [S. tuberosum from different locations in the world contained only two alleles. In this study we found both previously reported haplotypes AVR3aEM (H1) in S. tuberosum as well as in other Solanum species, and AVR3aKI (H3) restricted to S. muricatum. The other four reported variants were not present in S. tuberosum. Because of this, it is required to establish the correlation of each of these variants and their host range. Additionally, the taxonomic status of the P. infestans-related species P. andina should be clarified in order to understand the real sympatry of the two species and their respective contribution of alleles to the population. Furthermore, a more intensive sampling is needed in order to correlate polymorphisms with the host. Finally, the wider picture of the population structure in this region suggests that the low steps in the networks, together with the low genetic diversity of this pathogen and the evidence of the presence of selection in the effector gene, might be consistent with a scenario of random dispersion of new mutations by drift that in some cases might be fixed by selection. We need further research in other populations and more data from effector genes and neutral loci to really understand the Phytophthora infestans diversification process in South America.Polymorphisms of the g et al.  reportedP. infestans population genetic analysis suggests that a clonal polymorphic population inhabits the region with population subdivision between Colombia and Venezuela as a result of potato seed self-sufficiency. Given the observed variation at an avirulence locus, the study of this type of loci could be useful to understand the epidemiological behavior of this pathogen on different hosts and in response to other ecological variables. Avirulence genes are thought to coevolve in an arms race with corresponding resistance genes in the host plant. Thus avirulence genes or other effectors could be excellent candidates for understanding the evolutionary forces responsible for the diversity observed, particularly for clonally reproducing populations of this pathogen. As was said before, comparisons of other populations and neutral vs. avirulence loci are needed to understand the population dynamics of Phytopthora infestans.The Northern Andean MC acquired data, carried out and analyzed the molecular genetic studies, and drafted the manuscript. AG carried out the molecular genetic studies and drafted the manuscript. RS participated in acquisition of data and drafted the manuscript. CS participated in the molecular genetic analyses, and drafted and critically revised the manuscript. AR, AG, AV, MM, GF and LEL participated in acquisition of data and in revising the manuscript. NJG, AB, CS, and SR conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Phytophthora infestans isolates analyzed in this study.Click here for fileGeographical distribution of the sampling sites.Click here for fileRas, Cox1, β-tubulin and ITSSequences retrieved from the GenBank public database employed in this study for .Click here for fileGene networks showing relationships among haplotypes for each gene in each North Andean region.Click here for fileGene networks showing relationships among haplotypes for Colombia and Venezuela.Click here for fileMismatch distribution for all analyzed regions.Click here for file