Atrina pectinata, is one of the commercial bivalves in East Asia and thought to be recently affected by anthropogenic pressure (habitat destruction and/or fishing pressure). Information on its population genetic structure is crucial for the conservation of A. pectinata. Considering its long pelagic larval duration and iteroparity with high fecundity, the genetic structure for A. pectinata could be expected to be weak at a fine scale. However, the unusual oceanography in the coasts of China and Korea suggests potential for restricted dispersal of pelagic larvae and geographical differentiation. In addition, environmental changes associated with Pleistocene sea level fluctuations on the East China Sea continental shelf may also have strongly influenced historical population demography and genetic diversity of marine organisms. Here, partial sequences of the mitochondrial Cytochrome c oxidase subunit I (COI) gene and seven microsatellite loci were used to estimate population genetic structure and demographic history of seven samples from Northern China coast and one sample from North Korea coast. Despite high levels of genetic diversity within samples, there was no genetic differentiation among samples from Northern China coast and low but significant genetic differentiation between some of the Chinese samples and the North Korean sample. A late Pleistocene population expansion, probably after the Last Glacial Maximum, was also demonstrated for A. pectinata samples. No recent genetic bottleneck was detected in any of the eight samples. We concluded that both historical recolonization (through population range expansion and demographic expansion in the late Pleistocene) and current gene flow  were responsible for the weak level of genetic structure detected in A. pectinata.The pen shell,  Population connectivity plays a significant role on both evolutionary and ecological time-scales in both terrestrial and marine species Scapharca broughtoniiMactra veneriformisChlamys farreriMactra chinensisIn the coasts of China and Korea, the complex hydrology and bathymetry of Bohai Sea and Yellow Sea, including numerous bays, islands, and gyres, suggest the potential for restricted dispersal of pelagic larvae and geographical differentiation of marine bivalve in this region. Indeed, previous studies on Moreover, Pleistocene glaciations have played an important role in shaping patterns of genetic diversity Atrina pectinata, is common along the coasts of China, North Korea, South Korea and Japan. The fishery operates primarily along the coasts of Bohai Sea and Yellow Sea in China and along the eastern coast of Yellow Sea in North Korea A. pectinata is a broadcast spawner with external fertilization, and spawning times vary among populations, triggered by environmental cues such as temperature A. pectinata has a pelagic larval phase that lasts about 30 days The pen shell, A. pectinata is known to exhibit extensive morphological variations and five morphological forms of A. pectinata exist along the coast of China A. pectinata, which generally correspond to the five morphological forms observed. Four of the five cryptic species mainly distribute in the South China Sea. A. pectinata populations distributed in the Bohai Sea, Yellow Sea, and East China Sea correspond to a single cryptic species (Lineage I) found by Liu et al. A. pectinata has experienced severe population declines due to over-fishing, habitat loss, pollution, and other factors in the past decades A. pectinata along the coasts of Bohai Sea and Yellow Sea is still limited.As a lucrative commercial fishery, et al.A. pectinata along the west coast of South Korea using 13 microsatellite loci, and no significant genetic differentiation are detected. Considering its long pelagic larval phase and iteroparity with high fecundity, the genetic structure of A. pectinata could be weak at a fine scale. However, the unusual oceanographic characters around Bohai Sea and Yellow Sea, which may act as hydrographical and ecological barriers to larval dispersal, is likely to result in genetic differentiation among populations in this species.An A. pectinata were collected from eight geographic locations along coast of China and North Korea. Microsatellite markers and a portion of the mitochondrial COI gene were employed to address three key issues. First, we have estimated population genetic structure among populations. In particular, we attempted to test whether populations associated with the bays and gyres around Bohai Sea and Yellow Sea are genetically distinct, as previous studies suggested. Second, we have estimated the historical population demography of A. pectinata and discussed its response to climatic fluctuations during the Pleistocene, especially the LGM. Finally, we have tested whether the recent decrease in census population numbers has been accompanied by a genetic bottleneck in any population studied since such bottlenecks can compromise the future adaptive potential of populations.In the present study, samples of A. pectinata specimens was conducted in strict accordance with Animal Care Quality Assurance in China.Ethical approval was not required for this study because no endangered animals were involved. However, all handling of A. pectinata (n = 243) were collected from seven locations in China, and one in North Korea from Bohai Sea and Yellow Sea during 2008–2013 , and nucleotide diversity (π) of the COI sequences were calculated in DnaSP 5.10 OH) and expected (EH) heterozygosity were calculated by using the Excel Microsatellite Toolkit RA), inbreeding coefficient (ISF) were calculated with FSTAT2.9 FST values for microsatellite data and ΦST values for COI sequences with Arlequin 3.5 DA distance FST values and DA distance for microsatellite data were displayed in two dimensions via multidimensional scaling analysis using the SPSS16.0 software. To identify population structure, the software STRUCTURE 2.3 P or α <0.01). Potential source locality of the excluded individuals were identified based on probabilities larger than 0.1 The COI sequences were initially aligned using CLUSTAL X2 ΦST/(1–ΦST) and FST/(1–FST) for COI and microsatellites, respectively D test Fs test Isolation-by-distance (IBD) analyses were conducted for both COI and microsatellite data. Shoreline distances between sampled populations were estimated in km using Google Earth version 4.3 and plotted against genetic distance, pairwise To investigate whether any of the populations experienced recent genetic bottlenecks, Wilcoxon sign-rank test for heterozygosity excess was applied under three different models, namely, infinite alleles model (IAM), two-phase model (TPM) and stepwise mutation model (SMM), using the program Bottleneck 1.2.02 A. pectinata adults. An additional 45 sequences of two populations (ZS and FZ) in Southern China coasts were also retrieved from GenBank and incorporated in the analyses h) ranged from 0.653 to 0.947, and nucleotide diversity (π) ranged from 0.002 to 0.006 (h) and nucleotide diversity (π) of the PL population in Bohai Sea were generally lower than that of other populations. A total of 57 private haplotypes were identified, of which 32 were found in the seven populations in Northern China, five in HJ population in North Korea, and 20 in the two populations from Southern China coast (A 625-bp fragment of the COI gene was sequenced for 166 to 0.006 . Haplotyna coast . A dominna coast . The minRA), which is independent of sample size, was employed to compare among populations. The total allelic richness (RA) per locus ranged from 9.70 (KAP20) to 19.52 (KAP38). Average RA value was highest in LYG (15.86) and lowest in HJ (12.34). A total of 39 alleles were unique to a single population, ranging from two in LGD to eight in RZ. The observed and expected heterozygosities per locus varied from 0.679 (KAP20) to 0.930 (KAP11), and from 0.742 (KAP39) to 0.941 (KAP11), respectively. There was no significant difference in the average allelic richness, observed and expected heterozygosity among populations . No linkage disequilibrium was detected for 224 combinations in the 8 populations. Eight cases of locus-population combination out of 64 showed significant departure from Hardy-Weinberg equilibrium (HWE) after sequential Bonferroni correction . Departures were primarily related to excess of homozygosity at locus KAP32. No population deviated significantly from HWE by global tests across all loci. Analysis using MICRO-CHECKER suggested presence of null alleles at locus KAP32 in all populations. Thus, KAP32 was eliminated from subsequent analyses.Summary statistics for eight microsatellite loci in the eight populations from China and North Korea coast are showed in ΦST values based on COI data were low (–0.022∼0.081), and only two (both involving the HJ samples) out of 45 pair-wise ΦST values were significant after a Benjamini–Yekutieli correction, indicating high population connectivity  to 0.0233 (between PL and HJ) (P<0.05), and five of them were significant after a Benjamini–Yekutieli correction (P<0.01273). Based on microsatellite datasets, the lowest DA value was between PL and LYG (0.1390) and the highest DA value was between ZZD and HJ (0.2489) .AMOVA analyses were conducted for COI datasets with two groups: the Northern group (eight populations in coast of Northern China and North Korea) and the Southern group (ZS and FZ population). For microsatellite datasets, the AMOVA analyses were also conducted with two groups: the Northern China group (seven populations) and the North Korea group (HJ population). The AMOVA indicated no significant genetic differentiation at all levels  for COI datasets, while results for the nuclear microsatellite datasets  indicateectivity . Pairwis and HJ) . All of (0.2489) . Both thulations . The Strulations . The eigulations . An exclD and Fu’s Fs tests for the 10 populations, the northern group, the southern group, and the global group  are shown in Results of Tajima’s No significant heterozygotes excess (except HJ population under the IAM model) was detected under the infinite alleles model (IAM), two-phase model (TPM), and the stepwise mutation model (SMM) . These rA. pectinata, an important resource for the Chinese and Korea fishery. Patterns of genetic differentiation and population demography were estimated using both maternally-inherited COI sequences and microsatellites to reveal the components of the species population structure and population demography. We particularly focused on populations in the Yellow Sea and Bohai Sea, an area where A. pectinata was widely exploited and where the continental shelf were exposed during the Pleistocene ice ages and might shape the genetic variation. Indeed, we demonstrated that the COI sequences and microsatellites clarified the population demography and genetic structure of A. pectinata.Genetic data can make an essential contribution to the understanding of past and current processes that have shaped the evolution and genetic structure of marine species, as well as to conservation and management strategies A. pectinata along the west coast of South Korea, which show no genetic differentiation among samples probably due to the high levels of larval dispersal Scapharca broughtoniiMactra veneriformisChlamys farreriMactra chinensisChlamys farreriMactra chinensisA. pectinata might be responsible for the high gene flow among geographical samples. First, for species with a larval phase, pelagic larval duration (PLD) is assumed to influence the scale of connectivity A. pectinata (30 d) compared with Chlamys farreri (15 d) and Mactra chinensis (10 d) suggested that the larval dispersal potential of A. pectinata may be higher than that of Chlamys farreri and Mactra chinensis. Thus the larvae of A. pectinata might overcome barriers caused by the complex hydrology and bathymetry in Bohai Sea and Yellow Sea, which may result in high gene flow among geographical populations. Second, marine distribution patterns have been classified on a continuum from “oceanic” to “continental”, referring to requirement (or tolerance) for a suite of environmental conditions associated with primary productivity, freshwater influence and turbidity, all of which are present on continental margins and around high islands, and this has implications for the effectiveness of barriers to larvae dispersal A. pectinata could live in deeper ocean (to a depth of 100 m) compared to other species, so the larvae could be less impacted by ecological or hydrographical barriers to larvae dispersal, and be transported by the coastal current in longer distance than in other species.The most common hypotheses explaining the genetic connectivity and differentiation among different geographical populations of marine organisms are that: (i) the dispersal ability A. pectinata was supported by the star-like shape of minimum spanning network, the relatively clear unimodal shape of the mismatch distribution, the negative and significant Tajima’s D and Fu’s Fs statistics, and the result of Bayesian skyline plots. However, if the migration rate between demes is high, population range expansion can lead to basically the same molecular signature as population demographic expansion A. pectinata. Because mutation rate may be “time dependent” A. pectinata at a more recent time after LGM, which was consistent with the range expansion on the East China Sea Shelf following the LGM. The late Pleistocene period (the past 1 million years) was punctuated by a series of large glacial-interglacial changes. During glacial periods, with a sea level drop of 130–150 m, the Bohai Sea and Yellow Sea were completely exposed at this time and the East China Sea was reduced to an elongated trough with an area less than a third of its present size A. pectinata might mix together due to the geographical range contraction during the glacial period, and then expand population range when favorable conditions emerged during interglacial periods. The periodic merging of populations could enhance historical gene flow among populations. Hence both population range expansion and demographic expansion might have influenced the pattern of genetic diversity for A. pectinata. In this case, the large scale connectivity of A. pectinata could reflect the recency of this expansion and insufficient time for population divergence. Similar conclusions have been reached on some other marine invertebrates When analyzing patterns of genetic differentiation, it is important to disentangle current gene flow from demographic processes that occurred in the population history Placopecten magellanicusPerna viridisFurthermore, the low but significant genetic differentiations detected between some Chinese samples and the North Korean sample might be the result of chaotic genetic patchiness. Such chaotic genetic patchiness could be caused by any of several factors including genetic drift before recruitment and natural selection at early life stage A. pectinata in Bohai Sea and Yellow Sea was probably result from both the historical recolonization (through population range expansion and demographic expansion in the late Pleistocene) and current gene flow . Furthermore, the lack of an isolation-by-distance supported the view that the lack of significant genetic population structure was caused by high contemporary gene flow. All of the eight samples were at mutation-drift equilibrium under different mutation models indicated that current gene flow played a more important role than historical recolonization in generating current genetic structure.Our results suggested that the strong connectivity and weak level of genetic structure of A. pectinata in coast of China and North Korea, there is no significant genetic differentiations among samples in Northern China and low but significant genetic differentiations between some of the Chinese samples and the North Korean sample, which might arise from both historical recolonization and current gene flow. Therefore, A. pectinata distributed in Northern China coast could be managed as a single unit. In addition, our results supported the conclusions of Bird et al.The results of our study revealed that, despite high level of genetic diversity within local populations of A file contained the genotypes at 8 microsatellite loci of all individuals in the study is available from the Dryad Digital Repository: doi:10.5061/dryad.52hn0.Figure S1Population structure of eight A. pectinata populations prepared using STRUCTURE program.(TIF)Click here for additional data file.Table S1Haplotype frequencies of COI gene in ten A. pectinata populations.(DOCX)Click here for additional data file.Table S2Matrix of pairwise ΦST values between ten populations based on COI datasets.(DOCX)Click here for additional data file.


Aedes japonicus japonicus, originally restricted to temperate East Asia, is now widespread in North America and more recently has become established in Europe. To ascertain the putative number of separate introductions to Europe and examine patterns of expansion we analyzed the genetic makeup of Ae. j. japonicus populations from five cemeteries in North Rhine-Westphalia and Rhineland-Palatinate, two western German federal states, as well as of specimens from populations in Belgium, Switzerland, and Austria/Slovenia. To do so, we genotyped individual specimens at seven pre-existing polymorphic microsatellite loci and sequenced part of the nad4 mitochondrial locus. We found evidence of two different genotypic signatures associated with different nad4 mitochondrial haplotypes, indicating at least two genetically differentiated populations of Ae. j. japonicus in Europe (i.e. two distinct genotypes). Belgian, Swiss, and Austrian/Slovenian populations all share the same genotypic signature although they have become differentiated since isolation. Contrary to expectations, the German Ae. j. japonicus are not closely related to those in Belgium which are geographically nearest but are also highly inbred. German populations have a unique genotype but also evidence of mixing between the two genotypes. Also unexpectedly, the populations closest to the center of the German infestation had the highest levels of admixture indicating that separate introductions did not expand and merge but instead their expansion was driven by punctuated human-mediated transport. Critically, the resulting admixed populations have higher genetic diversity and appear invasive as indicated by their increased abundance and recent spread across western Germany.The mosquito  AedesBiological invasions of potential disease vectors such as mosquitoes (Diptera: Culicidae) inflict more direct threat to human health than those of many other species because they can radically alter the frequency of local or exotic pathogen transmission to humans and wildlife Aedes japonicus japonicus Theobald, 1901, also known as the Asian bush mosquito. Although intercepted a few times in the early nineties in New Zealand’s ports Ae. japonicus, one of four, is originally restricted to climates with cold snowy winters in Japan and Korea A very recent expansion is that of Ochlerotatus japonicus and, more recently, Hulecoeteomyia japonicaAedes japonicus following the guidelines of Edman and colleagues Of note, the current generic name of this species is controversial as it is often called Ae. j. japonicus occurred in 2000 when larvae in water in tires at a used tire import platform in Normandy, France, were eliminated before adult emergence Ae. j. japonicus has never been caught outside these two locations, the populations were considered to be not expanding. Nonetheless, in 2012 a control campaign was started aiming at eliminating the species, although to our knowledge it is unclear if it succeeded. In 2008, larvae of Ae. j. japonicus were also found in northern Switzerland and in several locations near the border on the German side Ae. j. japonicus was also found distributed across a 50 km area around the Austrian-Slovenian border Ae. j. japonicus infestations have been recognized and the species is now prevalent in the northern part of the federal state of Rhineland-Palatinate and the southern part of the state of North Rhine-Westphalia up to the city of Cologne In Europe, the first detection of Ae. j. japonicus is a potential vector of several human encephalitis viruses as well as of dengue, chikungunya and Rift Valley fever viruses Aedes albopictus, Ae. j. japonicus is adapted to colder climate, which explains why the latter occurs in northern Europe while the former remains restricted to southern Europe and so far is only found occasionally in Southwest Germany Because Highly polymorphic DNA regions such as those associated with simple sequence repeats  are powerful tools for studying populations of introduced species both by revealing putative origins as well as changes in allelic frequencies through space and time Ae. j. japonicus in North Rhine-Westphalia and Rhineland-Palatinate in western Germany. Specifically, we aimed to answer the following questions: Did the West German mosquitoes originate from nearby existing populations in Belgium, or from populations farther away in Switzerland or Austria/Slovenia, or did other introduction events take place? If multiple introductions occurred are they remaining separate or are they mixing? How are German Ae. j. japonicus populations expanding?The objective of the present study was to examine the patterns of expansion of Ae. j. japonicus are not efficiently attracted to standard traps used in mosquito surveillance Aedes and Culex species Because adult male or female Ae. j. japonicus had been sent to us at ZALF in the framework of the online project “Mückenatlas” (http://www.mueckenatlas.de), we started a monitoring program focusing on cemeteries in the states of North Rhine-Westphalia and Rhineland-Palatinate (western Germany). In every cemetery sampled, Ae. j. japonicus larvae were collected from at least seven water containers to avoid over-sampling across siblings which would potentially bias the genetic analyses. Larvae were similarly sampled in Switzerland, while in Belgium they were collected from several used tires. Specimens from Austria/Slovenia were obtained from various containers scattered on the side of roads. The mosquitoes were processed as described in Kampen and colleagues In August 2012, after several specimens of Aedes j. japonicus CO1 sequences in GenBank. Specimens from Belgium, Switzerland and Austria/Slovenia were killed as larvae, stored in alcohol (70%) and identified using the dichotomous key from Schaffner Larvae from German sites were brought to the laboratory and reared to adults, then killed by exposing them to −20°C for a few minutes, and identified morphologically to species using the key developed by Schaffner and colleagues We examined individuals from five cemeteries in western Germany, one tire-recycling platform in Belgium, five locations in Switzerland and eight locations in Austria/Slovenia , Table 1nad4) region of the mosquito mitochondrial DNA .We amplified seven microsatellite loci currently available for I), mean number of alleles (Na), observed heterozygosity (Ho), and unbiased expected heterozygosity (uHe) for each population in GenAlEx 6.5 We examined departures from Hardy-Weinberg and obtained Shannon’s information index (K) using the method of Evanno et al. FST values and Nei’s index of divergence (both biased and unbiased), that formed the distance matrices analyzed with a principal coordinate analysis and were used in Mantel tests to examine the relationship between genetic and geographic distances. Pairwise FST values were also calculated with FSTAT 1.2 in order to check for significance using Fisher exact tests To uncover genetic discontinuities among specimens, we examined the relationships between individual multi-locus signatures using a Bayesian approach in STRUCTURE nad4 haplotypes matches to some extent the distribution of the two genotypes. Specifically, haplotype H5 occurs exclusively and is highly abundant in populations with a predominant genotype 2 signature . Instead, the two populations, Linz and Bonn-Hoholz, closer to the geographic “middle” of the five German sites, have more signs of admixture with genotype 1 and are therefore more similar to Swiss specimens than to the Bad Hönningen population, which is further south and therefore geographically closer to Switzerland . The population with a genotype 2 signature with the least admixed specimens and a low genetic diversity is Bad Hönningen, which of the five locations studied is the farthest from Bonn  are from German populations. These results mirror the results of the principal coordinate analysis on populations although it is hard to separate German populations, which is not surprising since they all have some degree of admixture between two introductions. Of note the green squares correspond to specimens from Bad Hönningen, which have the lowest genetic diversity (lowest levels of admixture).(JPG)Click here for additional data file.


Diabrotica virgifera virgifera LeConte in southern Europe during the introduction (1996–2001) and establishment/spread (2002–2011) phases of its invasion. The Diabrotica microsatellite core-set was used to perform traditional population genetics analyses. Our results indicated that during the introduction phase genetic diversity and population genetic structure were lower overall as compared to the establishment/spread phase. Unusually high genetic differentiation was found between the Italy and southern Europe comparisons, including high differentiation between Italian populations separated by a short distance during the establishment/spread phase. STRUCTURE analysis revealed two genetic clusters during the introduction phase and two genetic clusters during the establishment/spread phase. However, bottlenecked populations were only detected during the invasion phase. A small but significant isolation by distance effect was noted in both phases. Serbia was the geographic source of WCR to Croatia and Hungary in the introduction phase, while the United States of America was the possible source of WCR to Italy in 2001. These introductory populations were the subsequent source of individuals sampled during the establishment/spread phase. Repeated introductions and admixture events in southern Europe may have resulted in genetically diverse WCR populations that have attained 83% of all known alleles worldwide.This study describes the genetics of the western corn rootworm (WCR),  Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) began its invasion of Europe ca. 1980, but the beetle was not officially recorded until 1992 from a maize field near Belgrade, Serbia ) and the natural logarithm (ln) of geographic distance in kilometres (km). These calculations were made for spatial comparisons among populations sampled during the introduction  and establishment/spread phases in the same region. An additional isolation by distance analysis was calculated without the Italian populations  in both phases to determine whether this pattern would hold if distant European populations were excluded from the analyses. Also, after identification of geographic source population of southern European populations, isolation by distance analysis was calculated without the identified source population to evaluate the role of source populations in the geographic distance analyses.Testing for isolation by distance was undertaken in FSTAT v2.9.3  using a Of all known alleles worldwide  ,18,21,22A ranged from two  to 11 (DVV-D8) with an overall mean of 4.2 alleles per locus  to 7.00 (Serbia) (O and HE) over all loci and populations were very similar for all European populations. The average HO per population ranged from 0.48 to 0.69, while the average HE ranged from 0.46 to 0.58. FIS estimates per locus and per population ranged from -0.87 (Serbia) to 0.65   , with Se  .IS values, indicative of an excess of heterozygotes , Italy  and Serbia (DVV-T2) were associated with negative Fozygotes . ConversAR was highly variable and ranged from 2.00  to 7.79  (O and HE) over all loci and populations were similar in all southern European populations, and comparable to the introduction phase. The average HO per population ranged from 0.57 to 0.63, while average HE ranged from 0.53 to 0.68. FIS estimates per locus and per population ranged from -0.162 to 0.103  to 13 (DVV-D8) with an overall mean of 5.3 alleles per locus . Overall, Italy) . Heterozto 0.103 .IS values. Conversely, significant deviations from HWE that were present in: Croatia for DVV-D4 and DVV-D2; Hungary for DVV-D2; Serbia for DVV-D2 and DVV-T2; and Italy for DVV-D2, DVV-D4, DVV-D8 and Dba07, were all associated with positive FIS values.Significant deviations from HWE were found in: Croatia for DVV-D2, Dba05 and DVV-D8; Hungary for DVV-T2; Serbia for DVV-T2 and Dba05; and Italy for DVV-T2 and Dba05. These deviations were also all associated with negative FO per population ranged from 0.52 to 0.60, while average HE ranged from 0.53 to 0.59. FIS estimates per locus and per population ranged from -0.87 (Serbia) to 0.60 (Serbia). Significant deviations from HWE were noted at only three loci from the 2011 USA population, and were associated with negative FIS values  which if they yielded low to no genetic differentiation within country/year and we subsequently treated country as a single putative population .We conducted preliminary data analyses   were found (Pairwise comparisons of F < 0.05) , Table 3re found , Table 3ST [ST = -0.007–0.22, P < 0.05) . Alternatively, significant yet moderate levels of differentiation  were found between southern European populations and Italian populations. Also, large and highly significant levels of differentiation  were found among Italian populations sampled in 2009 (Pairwise comparisons of FST  indicate < 0.05) , Table 3 in 2009 , Table 3K = 2, K, which was used to detect the actual number of clusters, confirmed that two genetic clusters (ΔK = 2) existed among the first population(s) introduced into southern Europe that arrived from the USA  existed among the inter-continental population comparison made (For populations collected during the establishment phase in Europe (2009–2011) and the US populations (sampled for comparative purposes) Bayesian genetic clustering detected two distinct genetic clusters K = 2, . The firson made .P < 0.05). In addition, Hungary and Serbia both experienced bottleneck events during the introduction phase, as indicated by a significant shifted mode non-normal distribution of alleles (P < 0.05). Croatia and Italy had a normal L shaped distribution of alleles, hence no indication of a bottleneck event.During the introduction phase Hungary and Serbia had a significant heterozygosity excess as indicated by the Wilcoxon sign-rank test .ST and highest Li→s values suggested that Serbia was the geographic source of individuals to Croatia and Hungary  (P < 0.001) (P < 0.001)  linearized F< 0.001) . When th< 0.001) . Serbia < 0.001) . After r< 0.001) .P < 0.001) (P < 0.001)  . Again w< 0.001) .In this study we widened the scope of our genetic survey of WCR to include countries neighbouring Croatia, where some of the first WCR introductions into Europe occurred. Our results are critical to further understanding the population dynamics of the WCR during the major phases of its European invasion over a 15 year period.During the introduction phase the number of alleles was low 19–27; 45%) in southern Europe compared to possible native populations from North America (Iowa or Illinois) that we also genotyped. Within a relatively short period of time and coinciding with what we have deemed to be the establishment/spread phase of the invasion, the number of alleles present in southern Europe approximately doubled. Of all known WCR alleles ,21,22 845% in souOur analyses revealed previously undiscovered alleles during the introduction and establishment/spread phases. That is, in the introduction phase two unique alleles were found while during the establishment/spread phase nine previously unrecorded alleles were found. The large number of unique alleles that we found possibly reflects multiple and on-going invasions into southern Europe from different geographic locations both from within Europe and the USA.Founder events during range expansions, agricultural management using crop rotation, and selection due to insecticide applications are known to affect how genetic diversity in insect species is partitioned . And inST estimates of populations sampled in Croatia, Serbia, and Hungary showed an absence of genetic structure. This indicates that these populations are genetically similar and exist as a single panmictic population over a large geographic area. These results were comparable to those found previously in studies examining the genetic structure of the WCR in central and south eastern Europe (CSE) [ST = 0.22) found between geographically close (<50km) Italian pairwise population comparisons during the establishment/spread phase (Venezia district vs. Pordenone district), suggesting possible multiple introductions (from the USA) and admixture events. In the well-known population genetics study by Ciosi et al. [ST estimates for populations in close proximity, in addition to the STRUCTURE results indicate a separate introduction from the USA, which may have resulted in the failure of the eradication program conducted in 2002 in the Venezia district [Microsatellite based Fpe (CSE) ,4,18. Coi et al.  the authi et al.  identifidistrict . Kim anddistrict  made it district .Genetic differentiation overall was lower in the introduction phase than in the establishment/spread phase. Intercontinental comparisons were highly differentiated. When comparing populations from the introduction phase with the same populations sampled during the establishment/spread phase moderate to high genetic differentiation was found for all comparisons. We first observed this in Lemic et al.  where thST estimates and at that point in time there was no evidence of genetic differentiation. However, during the establishment/spread phase we analysed three different regions close to the locations first invaded. There we found high genetic differentiation between pairwise population comparisons at relatively short distances of up to only 50 km. According to the STRUCTURE analyses conducted in numerous publications [K = 1) was found in southern Europe , distinct from the putative native source genetic cluster. During the establishment/spread phase also only one genetic cluster (K = 1) existed in this area, however, a single population from Italy (Venezia district) clustered with the USA populations. As already discussed this may indicate a second introduction into Italy from the USA in ca. 2001–2 and is contrary to the findings of Ciosi et al. [COI) gene. Nevertheless, this region is notable for its unusually high FST values in its pairwise comparisons with all other populations surveyed in this study.Italian populations sampled during the introduction phase revealed low Fications ,48,49 ini et al.  and Bermi et al.  who bothAnthomous grandis grandis Boheman). We only found that bottlenecks occurred in southern Europe during the introduction phase (1990–2001) and did not occur at all during the establishment phase (2002–2009) despite ongoing crop rotation. Contrary to the findings of Bermond et al. [The control methods practised in southern Europe and elsewhere in Europe focus on crop rotation –53. Cropon phase 90–2001 aA stronger isolation by distance pattern was found during the introduction phase compared to the establishment phase and both were significant. The strength of the isolation by distance pattern depended on whether Italian populations were included. Regardless, of the inclusion of Italy significant patterns do emerge, and suggest that populations may be fragmented, and we assume that this is occurring because of improved control practises. However, the impact it is having on overall genetic diversity is minimal, probably for the reasons discussed directly above related to the finding of the bottleneck results. When Serbia was removed from analyses no isolation by distance occurred, suggesting unrestricted gene flow during the first few years after introduction which initially created the southern European WCR population that then acted as the source for later CSE invasions ,19.Our results suggest Serbia as the initial source of WCR to its neighbouring countries. With the exception of the population from Venezia district , which was possibly a result of an introduction from the USA. Also, introductory populations were the subsequent source of individuals sampled during the establishment/spread phase. These results highlight that introductions of WCR to Europe from the USA are ongoing and must be factored into monitoring procedures, current management and control strategies. Genetic monitoring is an invaluable tool to support on ground pest management practises as it can inform managers and scientists alike as to the efficacy of practises by demonstrating how practises  impact upon the genetics of the populations being controlled.Empirical data and anecdotal evidence suggests that current control practises are working ,50 in soS1 Fig(TIF)Click here for additional data file.S2 FigK = 2 (A); K = 3 (B); K = 4 (C); K = 5 (D); K = 6 (E).Additional plots are presented for (TIF)Click here for additional data file.S1 TableDiabrotica microsatellite core-set [n: number of individuals.Alleles in parentheses are unique to the USA. The six loci comprise the core-set . Alleles(DOCX)Click here for additional data file.S2 Tablen = 975). Location names underlined were sampled during the introduction phase and again during the establishment/spread phase. Non-abbreviated location names are listed in Underlined values were significant after Bonferroni correction for multiple comparisons ((DOCX)Click here for additional data file.


Banksia attenuata . The results revealed an association between population genetic differentiation and the morphological divergence of postfire resprouting types in B. attenuata. A microsatellite allele has been shown to be associated with epicormic populations. Approximate Bayesian Computation analysis revealed a likely evolutionary transition from epicormic to lignotuberous resprouting in B. attenuata. It is concluded that the postfire resprouting type in B. attenuata is likely determined by the fire's characteristics. The differentiated expression of postfire resprouting types in different environments is likely a consequence of local genetic adaptation. The capacity to shift the postfire resprouting type to adapt to diverse fire regimes is most likely the key factor explaining why B. attenuata is the most widespread member of the Banksia genus.Resprouting is a key functional trait that allows plants to survive diverse disturbances. The fitness benefits associated with resprouting include a rapid return to adult growth, early flowering, and setting seed. The resprouting responses observed following fire are varied, as are the ecological outcomes. Understanding the ecological divergence and evolutionary pathways of different resprouting types and how the environment and genetics interact to drive such morphological evolution represents an important, but under-studied, topic. In the present study, microsatellite markers and microevolutionary approaches were used to better understand: (1) whether genetic differentiation is related to morphological divergence among resprouting types and if so, whether there are any specific genetic variations associated with morphological divergence and (2) the evolutionary pathway of the transitions between two resprouting types in  Plants resprout from aboveground or subterranean buds following damage to or the death of their aboveground tissue. Resprouting is a key functional trait that confers persistence and enables plants to survive diverse disturbance regimes. As a consequence, the plant community is resilient to severe disturbances, for example, fire and drought , which includes distinct lignotuberous and epicormic populations with marked morphological divergences in a speciose southwest Australian (SWA) ecosystem . There is a marked demarcation between the two forms, with northern populations being lignotuberous and southern populations being epicormic (Taylor and Hopper B. attenuata are stored on the plant for 5 years or more (serotiny), and seed release is stimulated by fire (Enright and Lamont Banksia attenuata is pollinated by nectar-feeding birds (predominantly honeyeaters of the Meliphagidae family), small marsupials and, to a lesser extent, bees and wasps  and examined regarding the following phenotypes: the presence of a lignotuber; the number of stems/trunks emerging from the ground; the height of the tallest stem/trunk; and the perimeter of the largest stem/trunk at 50 cm above the ground . To confirm whether the revealed pattern of divergence in postfire resprouting type is consistent across the species' range, an extensive survey was conducted, and resprouting type (epicormic or lignotuberous) was recorded for additional 17 populations throughout the species' distribution , mean effective number of alleles per locus (AE) and observed (HO) and unbiased expected (HE) heterozygosities, were calculated for each population using GenoDive  and assigns individuals to the different clusters based on the allele frequencies at each locus. An admixture model was employed in the analysis. K values ranging from 2 to the total number of populations  were tested. The burn-in period and run length of the Monte Carlo Markov Chain (MCMC) were 1 × 105 and 1 × 108 iterations, respectively. For each K, five runs were conducted. The optimum number of clusters (K) present in the dataset was evaluated according to Evanno et al.  using the program FDIST2  of divergence. The time period of bottleneck was assumed to a uniform distribution of 250–750 years, equivalent to 1–3 generations given a generation time of 250 years for Banksia attenuate  was set at average of 25,000 years before present with a standard deviation of 5000 years in a normal distribution, given that the possible age of sand dunes where lignotuberous populations were situated was estimated at 19,000–31,000 years . A reference table with 6 × 106 simulated datasets (2 × 106 for each of the three scenarios) was generated. The scenario selected as being most likely to explain the genetic data was the one with the highest posterior probabilities in 1500 of the closest datasets, which was cross-validated via logistic regression computed from 15,000 of the closest datasets. The confidence of the choice of scenario was evaluated following Cornuet et al. , narrow stems  (Table B. attenuata) up to 10 m tall. The northern range is drier and hotter, and its plants show a shorter growth season compared to southern part  (Table F'ST of 0.03 being observed and only five (out of 28) pairs of populations being significantly differentiated (P < 0.05 with the Bonferroni correction). Significant genetic differentiation was revealed between the lignotuberous and epicormic populations, associated with a mean F'ST of 0.27 (range 0.13–0.38), and all F'ST values were significantly greater than zero (P < 0.05). Analysis of Jost's D revealed that the significant microsatellite genetic differentiation between the lignotuberous and epicormic populations was mainly determined by three loci. Locus D155, C112, and A112 showed much higher differentiation between the lignotuberous and epicormic populations than that of within each resprouting type  Table . A similK = 2, and the most likely number of clusters was K = 2. The pattern of the clustering of individuals corresponded to the growth forms, that is, generating a cluster of lignotuberous populations and a cluster of epicormic populations, except that three epicormic individuals were marginally clustered with the lignotuberous populations  in the analysis including all six lignotuberous and eight epicormic populations  through a short period  of bottleneck (with population size of 50).The hypotheses regarding the evolutionary transitions between the two growth forms (corresponding to the two postfire resprouting types) were tested via approximate Bayesian computation examining three scenarios Fig A. The diB. attenuata were exclusively lignotuberous and showed distinctive morphological characteristics such as multiple stems and a short stature, whereas the southern populations were epicormic and presented a higher stature and usually a single stem. Among 20 resprouting Banksia species recorded in SWA, ten have been observed to resprout from lignotubers and/or epicormic buds and the others through rhizomes or root suckers , yet the F'ST values for these two populations with respect to the lignotuberous and remaining epicormic populations are markedly different (0.28 vs. 0.03). It is therefore likely that the differentiated expression of postfire resprouting types in B. attenuata between epicormic and lignotuberous forms is the consequence of local genetic adaptations. Differentiation in neutral markers may have also been tied with such local genetic adaptation, likely as a consequence of long time independent adaptation and evolution of the two resprouting types in local environment.It is theoretically predicted that random genetic drift in small isolated populations can lead to phenotypic divergence and genetic differentiation; however, both epicormic and lignotuberous populations of Local adaptation to divergent local environments can lead to dramatic phenotypic differences between populations, which can be accomplished in the presence of ongoing gene flow. Genetic adaptation and phenotype plasticity are two ways in which organisms can adapt to local environments . Based on an analysis involving approximate Bayesian computation, there was a possible evolutionary transition of the postfire resprouting type from epicormic to lignotuberous during range expansion in B. attenuata. It is worth noting that the evolutionary transition of the postfire resprouting type was concluded based on the detection of historical population bottleneck in lignotuberous populations and on the logic that the population that did not change size is more likely to be ancestral and to have kept ancestral characters. However, the bottleneck could have been caused by other factors than shift of fire-adaptation strategy, if less likely.The mode of postfire regeneration is a highly labile trait among banksias , in consistent with age estimate  of sand dunes where the studied lignotuberous populations were situated (Krauss et al. B. attenuata is the most widespread species of the Banksia genus. Indeed, banksias that are able to resprout via multiple means are usually widespread (Lamont and Connell The analysis of the population history of B. attenuata, a significant shift in the genetic composition of the populations with distinctive resprouting syndromes is not evident. In fact, neither type exhibits unique alleles across all eleven examined microsatellite loci. Even at locus BA-C112, which is associated with the resprouting type, the two forms share the same array of alleles. Although no cross-pollination experiments were conducted between the two forms, no morphological/ecophysiological differences that could possibly have resulted in their reproductive isolation have been reported (George Despite the evolutionary transition from epicormic to lignotuberous resprouting in d George . This red George  and thatd George . Further


Schistosoma japonicum is a significant public health risk in parts of China and elsewhere in Southeast Asia. To gain an insight into the epidemiology of schistosomiasis japonica, a detailed investigation of S. japonicum genetic population structure is needed.S. japonicum sampled on a geographical scale covering most major endemic areas.Using three mitochondrial DNA fragments and ten microsatellite loci, we investigated the genetic diversity within and structure among twelve populations of Schistosoma japonicum lineages from Indonesia, the Philippines and Chinese Taiwan were clearly distinct from each other and from those in mainland China. Within mainland China, there was some evidence for genetic divergence between populations from the mountain and lake regions. However, the analysis inferred no clear sub-population structure in the lake region of mainland China. High genetic diversity was found among S. japonicum populations of mainland China and this was significantly higher than those from island regions.S. japonicum.High genetic diversity within and substantial differentiation among populations were demonstrated in The online version of this article (doi:10.1186/s13071-015-0757-x) contains supplementary material, which is available to authorized users. Schistosoma japonicum, S. mansoni and S. haematobium, can infect humans [S. mansoni is endemic in Africa, the Middle East and South America and S. haematobium is found in Africa and the Middle East, whereas S. japonicum occurs in China, Indonesia and the Philippines. At present, this parasite is endemic in China in the lake/marshland regions of Anhui, Hubei, Hunan and Jiangxi provinces and in the mountainous areas of Sichuan and Yunnan provinces [S. japonicum in China by more than 90% [S. japonicum population genetic structure is required.Schistosomiasis is one of the most serious of human parasitic diseases, infecting an estimated 200 million people in 76 tropical and subtropical countries . Three mrovinces . Althougthan 90% , recent than 90% . To gainS. japonicum populations. Earlier studies using isoenzymes [S. japonicum lineages. Later, differences among, but not within, Chinese S. japonicum populations were detected using mt DNA sequences (nad1) [et al. [S. japonicum populations. Subsequently, a set of high-resolution microsatellite markers was used to investigate the genetic structure of S. japonicum populations from eight locations across China, and a high level of polymorphism was detected both between and within populations [S. japonicum populations from the lake and mountainous regions were genetically distinct [S. japonicum between the lake and mountainous regions in China [S. japonicum either from mainland China, or a few locations in mainland China and other Asian countries [S. japonicum across its entire range.Several classes of molecular markers have been applied to investigate the genetic variability of oenzymes  and mitooenzymes  reporteds (nad1) . Similar [et al.  applied ulations . Moreovedistinct . Similarin China ,11. Prevountries ,13, but S. japonicum to investigate the genetic relationships among populations using different classes of genetic markers simultaneously on the same samples.Most phylogeographical studies have relied on the analysis of mitochondrial DNA variation, because of its rapid evolution and maternal transmission without intermolecular recombination . In contS. japonicum samples from eight locations in China mainland (covering the lake and mountainous regions) and Taiwan, Indonesia, Japan and the Philippines. We explored the genetic relationships among these populations based on the analysis of three mt genes  and ten microsatellite loci from the same individual worms.In this work, we analyzed All procedures involving animals were carried out based on the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International. The study protocol followed institutional ethical guidelines that were approved by the ethics committee at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention .S. japonicum were obtained from twelve different geographical locations  from each mainland Chinese location were transported to the laboratory of NIPD, China CDC, Shanghai. Cercariae were released from pooled infected snails from each site and used to infect laboratory-raised rabbits. Finally, 45 days later, the adult schistosomes were perfused from the mesenteric veins of the infected rabbits and washed in saline before being preserved in 95% (v/v) ethanol at 4°C. The S. japonicum isolate from Leyte, the Philippines, was taken into culture originally in 1969 by Dr. Scholice. The material sent to us, as lyophilized adult worms, was provided by Dr. John Bruce, Centre for Tropical Diseases, University of Lowell, USA. The worms from Japan (Kofu) were gifted by Dr. Hiroshi Yamasaki, National Institute of Infectious Diseases, Tokyo, Japan. The other geographical samples (lyophilized adult worms) from Indonesia  and Taiwan (Changhua) were provided by Dr. John Cross, Uniformed Services University of the Health Sciences, Bethesda, USA. These preserved samples were from populations that had been maintained in laboratory animals.Adult individuals of s Figure , includiIn total, 42 worms were subjected to mitochondrial DNA analysis and 486 individuals were used for the microsatellite analysis Table . MicrosaGenomic DNA was extracted from individual adult schistosomes using the DNeasy Blood & Tissue Kit and Animal Tissues (Spin-Column) protocol from QIAGEN , and stored at −20°C until use.nad1), 1275 bp of NADH dehydrogenase subunit 4 (nad4) and 1748 bp of 16S-12S mitochondrial ribosomal DNA (16S-12S rRNA). Three pairs of primers were designed based on the complete reference mitochondrial sequence for S. japonicum (ID in GenBank: AF215860) as follows:We amplified three fragments of the mitochondrial genome: 891 bp of the gene encoding NADH dehydrogenase subunit 1 (nad1-F (5′-TTGGAGTTTGTCAGGCTTTAGG),nad1-R (5′-TATATCAAACACCATCAAAAGGAAC),nad4-F (5′-TGCTGTTTTATGCTATGCTACGAAG),nad4-R (5′-TACAAACGGACGGACCAATAAAAC),16S-12S rRNA-F (5′-ATAATGTTGCGTCTAAGGTC)and 16S-12S rRNA-R (5′-TAAACACTACCCATCAAATC).Taq polymerase , 1.25 mM MgCl2, 2 μl 10× reaction buffer and 1 μl dNTPs . The PCR protocol used a hot-start activation at 94°C for 10 min, followed by 35 cycles of denaturation for 30 s at 94°C, 30 s annealing  and extension  at 72°C, followed by a final extension at 72°C for 10 min. PCR products were examined using agarose gel electrophoresis (1% w/v) to validate amplification efficiency. Amplification products were then sent to BGI  for sequencing using an ABI 3730 DNA Analyzer.The amplifications were performed in 20 μl reactions containing 0.5 μM of each primer, ~ 50 ng of genomic DNA from an individual worm, Has S. japonicum worm was genotyped at ten microsatellite loci , which were characterized previously [et al. [The DNA of each individual eviously . The PCR [et al. . The PCRnad1, nad4 and 16S-12S rRNA sequences; hereafter “combined mtDNAs”) were identified using Arlequin 3.11 [4 bootstrap replicates. Schistosoma mansoni (ID in GenBank: NC_002545.1) was used as an outgroup. A median-joining network was generated to infer the relationships among the haplotypes of S. japonicum using Network [vs island region and lake vs mountainous regions in mainland China) were made using unpaired Student’s t test. Hierarchical analysis of molecular variance (AMOVA) was applied to partition the genetic variances into among regions (i.e. mountain and lake regions), among populations within region and within populations, for samples from mainland China, in Arlequin.Unique haplotypes for the mitochondrial DNA sequences (concatenated uin 3.11 . All uniuin 3.11 . A phylouin 3.11 . A conse Network . NucleotHe) and observed heterozygosity (Ho) in GenAIEx. In this and following analyses, the individuals for which data were missing were excluded, as were populations with N < 6. Third, the inbreeding coefficient (FIS), which measures the extent of nonrandom mating, was computed in Genepop 3 [FIS indicate heterozygote deficiency, whereas, negative FIS values suggest heterozygote excess. Finally, deviations from Hardy-Weinberg equilibrium (HWE) were tested in Genepop, with 104 permutations.Multiple genotypes from a single snail are often very similar to one another and possibly derived from a single miracidium by mutations occurring during the proliferation of cercariae inside the snail . We therenepop 3 . PositivFST) in GenAIEx, and the significance of the FST value was tested by 104 permutations. AMOVA was applied to quantify intra- and inter-population and region variances in Arlequin. A Mantel test was used to test whether there was a relationship between pairwise geographical distance (km) and pairwise genetic distance  in GenAIEx.Genetic population differentiation was estimated using Wright’s F-statistics . All the sequences were submitted to GenBank under accession numbers KP793743- KP793784 for nad1, KP793785-KP793826 for nad4, KP793827-KP793868 for 16S rRNA and KP793869-KP793910 for 12S rRNA.We successfully amplified and sequenced 42 Among the 42 samples sequenced, 34 haplotypes were identified Table . No haplvs 0.0005; N = 12, t = 2.52, P = 0.03). However, we detected no significant differences in diversity between populations from the mountainous area and lake region of mainland China . We recognize that sample sizes are small and this might influence the results. The four individuals from Anhui Guichi shared the same haplotype. In a further seven populations , each individual had a unique haplotype (Table P < 0.001).The nucleotide diversity (π) ranged from 0 to 0.005 (mean = 0.0016). Interestingly, the nucleotide diversities of most (five of eight) populations from mainland China were significantly higher than those from Chinese Taiwan and each of the other island regions (mean for mainland China 0.0022 Ho) was consistently lower than the expected heterozygosity (He), and most of the inbreeding coefficient (FIS) values were positive, with the exception of Indonesia and Japan  based on the microsatellite data and geographical distance  according to hierarchical AMOVA, significant genetic differentiation was detected among the populations. Pairwise 51 Table , indicat.91% accoBased on the highest value of ΔK, the most likely number (K) of clusters was eight Figure A. Using S. japonicum populations in four Asian countries to assess the degree of genetic subdivision within the species.To our knowledge, this report includes the widest geographic coverage to date, surveying 12 S. japonicum lineages from the mountain and lake regions of mainland China and from Japan, that were distinct according to the mitochondrial DNA markers, could not be separated by the microsatellite analysis.The two classes of markers, mitochondrial DNA and microsatellites, yielded results those were not completely congruent. Based on the phylogenetic tree , the Taiwanese lineage obviously diverged from the others. Moreover, the lineages from the mountain regions of mainland China, Indonesia, Japan and the Philippines were distinct from each other and from populations in the lake region. However, no substructure was detected among the lineages from the Chinese lake region. In agreement with the phylogenetic analysis, the PCoA analysis, based on ten microsatellite loci, showed that individuals from the Philippines, Indonesia and Chinese Taiwan formed distinct clusters according to their geographical origins. This pattern was further supported by a Bayesian approach in STRUCTURE. The S. japonicum [S. japonicum was first described from Taiwan in 1914 [Although some earlier studies failed to detect any evidence of major genetic dissimilarities at the DNA level between Chinese and Philippine aponicum , our stuaponicum , has sho in 1914 , there h in 1914 . Here, wOncomelania hupensis, the only intermediate host species complex involved in the transmission of S. japonicum [O. hupensis: one with a smooth shell in the mountain region and another with a ribbed shell in the lake region [O. hupensis quadrasi occurs in the Philippines, while O. h. nosophora is present in Japan, O. h. lindoensis in the Indonesian islands, and O.h. formosana and O. h. chiui are present in Chinese Taiwan [Oncomelania hupensis from China is genetically different from O. h. quadrasi in the Philippines [O. quadrasi populations in the Philippines have a substructure associated with their geographic origin [O. hupensis that exist in different regions might contribute to the genetic divergences apparent among populations of its parasite, S. japonicum [Genetic divergence observed among regional Chinese and Asian populations in this study may relate to the distribution and morphs of aponicum . In maine region ,33. A sie Taiwan . Oncomellippines , and thec origin . These daponicum .FST values ranged from 0.01 to 0.51, indicating varied levels of pairwise population-genetic differentiation. In fact, strong population genetic differentiation has been detected between the mountain and lake regions of China on a large geographical scale [In the present study, al scale ,10. Thisal scale , our ManS. japonicum, based on microsatellites [S. japonicum populations, according to both mitochondrial DNA and microsatellite markers. We also observed marked deviations from HWE in most populations, in agreement with findings of a previous study on S. japonicum [S. mansoni [Corresponding well with the previous studies indicating high genetic diversity of tellites  and mitotellites , in thisaponicum  and simi mansoni . This siS. japonicum populations were demonstrated using both mitochondrial DNA and microsatellites. Future research should aim to generate a comprehensive link between the population nucleotide diversity/structure differences and corresponding phenotypic variation, such as host preferences and differences in pathology, as well as possible differences in the response to anti-schistosome vaccines and immunodiagnostics. Overall, the present findings provide fundamental biological and evolutionary information on S. japonicum, with significant implications for the control and elimination of this parasite in Asia.In conclusion, high genetic diversity within and substantial genetic differentiation among


Crop diversity managed by smallholder farmers in traditional agrosystems is the outcome of historical and current processes interacting at various spatial scales, and influenced by factors such as farming practices and environmental pressures. Only recently have studies started to consider the complexity of these processes instead of simply describing diversity for breeding purposes. A first step in that aim is to add multiple references to the collection of genetic data, including the farmers’ varietal taxonomy and practices and the historical background of the crop.dicoccum). By contrast, the genetic diversity assessed using 14 microsatellite markers was relatively limited. When compared to the genetic diversity found in a large collection of tetraploid wheat, it corresponded to free-threshing tetraploid wheat. Within Morocco, the two studied regions differed for both genetic diversity and variety names. Within regions, neither geography nor variety names nor even breeding status constituted strong barriers to gene exchange despite a few significant patterns.On the basis of interview data collected in a previous study, we sampled 166 populations of durum wheat varieties in two traditional Moroccan agrosystems, in the Pre-Rif and Atlas Mountains regions. Using a common garden experiment, we detected a high phenotypic variability on traits indicative of taxonomical position and breeding status, namely spike shape and plant height. Populations often combined modern (short) with traditional-like  statures, and classical durum squared spike shape (5 flowers/spikelet) with flat spike shape (3 flowers/ spikelet) representative of primitive domesticated tetraploid wheat  contains supplementary material, which is available to authorized users. With the Green Revolution, most of breeding effort has been devoted to the development of elite cultivars adapted to favorable lands where high input agriculture is predominantly practiced . The attStudies of landraces often focus on describing their genetic diversity in order to identify valuable and diversified genetic resources; the genetic information is generally only coupled with the geographical origin of the accessions ,14. To iDisentangling these mechanisms requires as first step integrating additional information to the morphologic and genetic characterization of landraces, in order to consider them in their environmental and social context . For insTriticum turgidum ssp. durum) is a selfing tetraploid cereal. The outcrossing rate is generally considered lower than 5% by breeders, and has been estimated between 1 and 3% in Ethiopian landraces [Triticum turgidum ssp. dicoccoides) in the mountains of Fertile Crescent around the 8th millennium BC. Firstly it led to a non-brittle hulled subspecies (T. t. ssp. dicoccum) which was one of the first cereals domesticated [. Then, human selection of mutations at multiple loci in particular at Tg – tenacious glume – and Q loci, [T. t. ssp. polonicum, T. t. ssp. carthlicum, T. t. ssp. turgidum, T. t. ssp. durum). Hulled and free-threshing forms (from 7th and 3rd millenium BP respectively) played a crucial role in the development of Mediterranean civilizations. They were widely spread out with the early agricultural movements leading to agriculture systems based on tetraploid wheat. Nowadays, elite durum varieties and landraces grown in different environments are of major importance for grain production in the Mediterranean basin [Durum wheat  and an important source of staple food. Most of the cultivated varieties in the Great Plains result from breeding programs developed by the Moroccan National Institute of Agricultural Research (INRA) or by international research centres . These modern varieties are single-genotype based, early flowering spring (i.e. they do not require low temperature to induce flowering and/or they are insensitive to day length) with a short stature due to dwarfing genes, [maroccanum [dicoccum [In Morocco, tetraploid wheat represents a major crop .A few studies have described the genetic diversity of Moroccan landraces in terms of allele number and specific alleles ,47. KeheTriticum turgidum and products of modern breeding)? (ii) How is the Moroccan genetic and morphologic diversity structured relative to two factors: the geographic location at small and large scales, and the farmers’ taxonomy . These two factors are potentially important determinants of gene flow through seed exchange or intercrossing. (iii) What are the consequences of the introduction of modern varieties? Do the traditional and modern genetic pools remain distinct and how can the diversity of traditional varieties be affected on the long term?In the present work, we enriched these interviews with genetic and morphologic data to address three questions: (i) How can we locate the Moroccan cultivated populations in the historical background of tetraploid wheat domestication? Do we find the different evolutionary steps described above  and including wild and domesticated subspecies. We considered three kinds of markers: The spike shape is an indicator of the taxonomical position, the stature of the plants is an indicator of breeding status, and a set of microsatellite markers gives us access to the neutral genetic diversity. The results are presented with respect with these three kinds of markers, and considered together to give insights into our three axes of investigation. For the interpretation, we also made use of the qualitative information gathered by the interviews of Chentoufi et al. [To address these questions, we used two data sets. One, denoted as the Moroccan sample, is made of a sample of Moroccan farmers’ population, identified by their varietal name and their farmer of origin ; the othi et al. .We sampled 166 durum wheat populations in two Moroccan regions (Moroccan sample). These populations belonged to 30 different varieties that were classified as modern or traditional. Each region was subdivided into zones including geographically close villages  per population and of two Moroccan modern varieties used as checks.Triticum aestivum, BW), and three corresponding to a shape indicative of different subspecies of the tetraploid species Triticum turgidum . Even if this description is referring to different subspecies of tetraploid wheat, it cannot be considered sufficient to confidently assign a plant to a subspecies. Variability for spike type was observed among and within populations. Based on within-populations frequencies of spike types, different categories of pure and mixed populations were defined (see Methods for details).Using a combination of spike shape and flow cytometric analysis, each progeny was assigned to one of four spike types: one corresponding to hexaploid bread wheat . The other zones were dominated by the classical DW type , corresponding to the usual stature of traditional varieties, and short (S), indicative of modern breeding Figure . As for The tall type T was the most frequent within each region and each zone, except in zone ZN2 in the Pre-Rif Table , was chavs traditional) and plant stature  was not strict , a population containing a majority of short individuals .Triticum turgidum (INRA collection).One seed per Moroccan population  and Moroccan check (2 varieties) was genotyped with 14 microsatellite loci, as well as 495 samples from a large collection of wild and domesticated subspecies of He = 0.327) to Xgwm285 . When computed only over this set, the diversity of ssp. durum decreased to He = 0.496 and Rs = 4.53.Among the subspecies of es Table . Within He = 0.48, Rs = 4.69 for the Pre-Rif and He = 0.39, Rs = 3.45 for the Atlas Mountains, P > 0.01). Most alleles identified in Morocco were included in the allele pool of the INRA collection: only 4 private alleles were detected among the 91 identified. These four alleles were present in the Pre-Rif samples at loci Xgpw7577, Xgwm312, Xgwm495 and Xgwm537, with a frequency ranging from 0.3% to 1.86%.Overall, the Moroccan sample exhibited the lowest levels of genetic diversity. The level of genetic diversity was not significantly different between the two sampled regions  was tricky since there was no clear and repeatable value of K for which the Bayesian Information Criteria (BIC) value was lowest. Practically, we chose K = 10, a value from which the BIC decreased or increased only by negligible amounts in many runs of the analysis  as well as the repeatability of the obtained clustering based on 15 additional runs with K = 10.The choice of the optimal number of clusters , a cluster only made of ssp. dicoccum individuals (C10), and a cluster only made of ssp. durum (C9). Three more clusters corresponded to a relative homogeneous composition regarding the taxonomical position and/or to a relatively high average membership probability, although to a lower extent: a cluster almost only made of ssp. dicoccum individuals (C7), a cluster almost only made of ssp. durum (C3), and a more atypical cluster, including accessions of ssp. polonicum and ssp. turgidum and well distinguished from the others (C8). Finally, four clusters  were a mixture of two to four different subspecies, and occupied the central part of the graph. This group of four clusters will be denoted as the “central pack” in the following.Among the 10 clusters identified in the reference run Table B, three durum C3 and C9 included almost exclusively varieties registered from the 50ies, whereas all landraces were included in the central pack (data not shown).Interestingly, the two homogeneous clusters of ssp. Overall, the 15 additional runs gave similar qualitative results. The 6 clusters not belonging to the central pack were easily recognizable in the new clusterings, even if some were subdivided or merged in a few runs. A central pack was always present but with different subdivisions and a different number of clusters among runs. We thus decided to group some clusters into “metagroups” and evaluated the repeatability of these metagroups as the average proportion of individuals of the metagroup of the reference run, that was assigned to a similar metagroup in the new runs, or that clustered altogether to another metagroup in case of merging. We obtained a high repeatability score  assigned to the C9 cluster containing the most recent varieties Figure C. The Moum Table . The unsHe and Rs: we detected 5 significant differences out of 42 tests. In the Pre-Rif Region, only ZN4 had a lower allelic richness compared to the three other zones. In the Atlas Mountains, ZS3 (at the highest elevation) and ZS5 exhibited the lowest level of genetic diversity, whereas ZS2 was the most genetically diverse zone. As expected for a selfing species, genotypes were highly homozygous. A few accessions were heterozygote at one locus at least: they represented 13% of our sample in both regions  and between zones within regions . Most of the genetic variance was observed within zones (70.7%) whereas the remaining variance was almost equally partitioned among zones within regions (14.7%) and among regions (14.6%). One hundred and twelve different genotypes were identified in the Moroccan data set. Only three were shared between the two regions: two were identified in modern-like populations and encompassed respectively 2 and 4 different varietal names (different between regions); the third was present in ten Atlas traditional populations (belonging to 4 different varieties) and in one population described as Krifla beda, a traditional variety of the Pre-Rif. We applied DAPC to the Moroccan sample: individuals from different regions were overall assigned to different clusters. Only 9 individuals were assigned to clusters made of individuals from another region  was performed with zones nested within regions. It detected significant differentiation both between regions  and of the spike-type class of the population (DC-type for cluster B vs DW-type for cluster A) . The two others reflected a combination of the zone of origin (ZS3 and ZS6 for cluster B es Table .Oryza sativa) and African rice (Oryza glaberrima) in different African countries i.e. occurrence of varieties described as ‘hybrid’, [We identified in the Moroccan sample individuals and whole populations as bread wheat, even if these populations had been explicitly designated by farmers as durum wheat. In some countries, farmers consciously grow mixtures of durum and bread wheat ,45 but shybrid’, . Interesdicoccum clusters of the collection  cultivation in Morocco, but it was documented during the first part of the twentieth century [Plants with ancestral spike shape of the dicoccum type were observed within Moroccan landrace populations but they were free-threshing at maturity. Moreover, they did not cluster with the specific ssp.  century . Here, t century , even th century . Such meTriticum turgidum INRA collection. Moroccan genetic diversity was relatively reduced and similar to that of the group of modern varieties from the INRA collection registered since the 1950ies. Such result is however not surprising considering the restricted geographical range of our sampling. Oliveira et al. [carthlicum which is supposed to result from hybridization with bread wheat, [As a whole, the high morphological diversity in the Moroccan tetraploid wheat sample did not correspond to a high and/or original genetic diversity relative to the a et al.  also idea et al. . Moreoved wheat, , the fred wheat,  and wered wheat, .al. [Chentoufi et al.  inventoral. . Such hial. . Howeveral. . This isWithin regions, although variety names were rather localized , only a Besides this, we can point out some mechanisms potentially involved in these patterns (and lacks of pattern) and open tracks of future research to decipher them.First, the large proportion of mixed populations (on the base of spike type and stature) suggests the occurrence of intermixing and genetic exchanges between varieties with different morphological composition . These exchanges might have different sources: unconscious mixing in threshing areas used by different farmers and for different varieties, unreliability of seed exchange networks (e.g. markets providing seed lots that do not correspond to the declared names) , limitedSecond, the taxonomy of varieties and how farmers experience this taxonomy is questioned by our results. Indeed, our morphological and genetic data suggest that names of some varieties are interchangeable Table , for insvs the four other zones). Assessing the relative effect of local adaptation and gene flow would help understanding this pattern. For instance, local adaptation may arise from different selective pressures along the altitudinal gradient in the Ziz valley [Finally, we have shown that the two well-differentiated groups of varieties of the Atlas Mountains partially overlapped with the two geographic groups of zones  and barley (selfing) ,59. In Met al. [Our study, together with the interviews reported in Chentoufi et al. , provideFollowing these different tracks requires to concentrate investigations at a small scale, potentially different for each question and to integrate contributions from different disciplines : consideClimatic, economic and political contexts have been changing rapidly in traditional agrosystems during the last few decades following for instance the development of communication routes and agricultural policies, as described for Ethiopia in Samberg et al. . UnderstChentoufi et al.  surveyedBased on these results, we followed two different sampling strategies in the two regions. In the Pre-Rif region, we adopted a sampling strategy allowing the collection of as many as possible of the wheat varieties. In total, 65 durum wheat populations were sampled. These populations corresponded to 18 different varieties: 17 identified during the initial survey of Chentoufi et al.  and one For each of the 166 wheat populations, 30 spikes from 30 randomly chosen plants were harvested in the farmer’s field.During the 2011/2012 growing season, 20 seeds from each individual spike were sown in an ear-row design (1 m length and 0.3 m between rows) at the experimental station of the IAV Hassan 2 . Two Moroccan durum wheat modern varieties  were used as checks to account for potential environmental variation at the field station: 74 rows of Karim and 39 of Marzak were sown across the experiment (20 seeds per row).In the following, we will denote as a progeny the maternal family corresponding to the seeds from a given spike.i.e. flat ear with only three flowers per spikelet (denoted as DC); this corresponds to the shape expected for plants carrying the q allele of the Q gene [T. turgidum ssp. carthlicum or of bread wheat T. aestivum, species status was checked by flow cytometric analysis for all progenies of the populations where it was observed protocol adapted from [Q allele-carrying plants [We recorded two key morphological traits that are informative on durum wheat history, namely spike shape and plant height. We classified progenies into four categories of spike: (i) dicoccum-like spikes, e Q gene  but noteted from . Hexaplog plants ). These Average plant height was recorded for each progeny by averaging 3 measures taken across the row Figure . For theFor each population composed of thirty collected spikes, we randomly picked one spike. One seed per spike and one for each Moroccan check were sown for DNA extraction.Genomic DNA was extracted from 100 mg of fresh leaf tissue of three weeks old seedlings. The extraction was performed following a protocol adapted from Tai and Tanksley (1991). DNA quality was checked on agarose gel and the DNA concentration was estimated using spectro-fluorometry. Five samples of the Pre-Rif belonged to progenies identified as hexaploid wheat  and were excluded from the genetic analysis. Thus, 163 Moroccan individuals were included in the final data set: 161 populations (Moroccan sample) and two checks.Triticum turgidum conserved at INRA (INRA collection). It included 53 accessions of the wild ancestor T. turgidum ssp. dicoccoides, 94 accessions of ssp. dicoccum, 34 of ssp. carthlicum, 29 of ssp. polonicum, 33 of ssp. turgidum and 252 of ssp. durum subsets of this sample have been analyzed in [durum accessions allowed distinguishing among them 69 landraces from Lebanon, Jordan, Syria and Turkey and 161 varieties of durum wheat registered after 1950 (122 from France and 39 from other countries).In addition, we analyzed a set of 495 DNA samples of genotypes from a large collection representing different subspecies of lyzed in ,63. Avaihttp://wheat.pw.usda.gov/GG2/index.shtml, [2, 0.2 mM of each dNTP, 0.5 units of Taq DNA polymerase and 1-4 pM of each primer. The forward primer was labeled with one of the three fluorophores . PCR was carried out as follows: after 5 min at 94°, 35 cycles were performed with 30 s at 94°C, 30 s at either 55 or 60°C (depending on the locus), and 1 min at 72°C, followed by final extension step of 30 min at 72°C. Amplified products were detected on an ABI 3130xl Genetic Analyser  and analyzed using the GENEMAPPER V3.7 software (Applied Biosystems).Fourteen nuclear SSR markers used in previous assessments of durum wheat diversity and mapping to a single, genome specific, locus sequences available at x.shtml, ,61 were Frequencies of spike types and of plant statures among progenies were computed for each population and each zone.T. aestivum) individuals. This class included pure populations of bread wheat and mixed populations with a majority of bread wheat progenies associated with T. turgidum, (ii) DW and (iii) DC, corresponding to pure populations of respectively DW and DC spike type, (iv) DW_DC, corresponding to mixed populations combining DC and DW spike types, and (v) DC-DW-CA, corresponding to mixed populations including these three spike types. Note that the CA type was always rare within populations.Then, frequencies within populations were used to assign each population to a class. First, for each of the two morphological traits, we denoted as pure populations the populations where all sampled progenies were of the same type (i.e. spike type or stature) with the allowance of one outlier, and as mixed populations the ones combined different types, with at least two progenies of each. For spike types, we defined five classes: (i) BW, corresponding to populations containing bread wheat .a), mean allelic richness Rs, [T. turgidum, whole Moroccan sample, regions within Morocco, and zones within regions. Differences in the amount of genetic diversity between groups were tested by Wilcoxon signed-rank tests, comparing values for the same loci in different groups, using R (R Development Core Team 2008). For these tests, we used the significance threshold P = 0.01.The mean number of alleles per locus  was visually examined, and the number of clusters was determined as the value of K above which BIC values decreased only subtly. In order to obtain reliable group membership probabilities, the number of Principal Components (PC) retained to compute the discriminant functions was determined using the function optim.a.score, which examines the trade-off between power of discrimination and over-fitting. Here we retained 11 to 13 PC depending on the runs. We then projected the Moroccan genotypes as supplementary individuals onto the discriminant functions and examined their assignment to the different clusters. A DAPC analysis was run on the Moroccan sample alone, using the same process as described for the INRA collection.We first ran DAPC on the INRA collection, including only the domesticated subspecies of Within Morocco, a hierarchical analysis of molecular variance was performed with zones nested within regions, using ARLEQUIN v 3.5 . The sigFST) was estimated using the method of Weir and Cockerham [FST estimates based on variety names: Namely, each individual was described by its variety name, which was considered as equivalent to a homozygous genotype at a single locus. These FST values were compared with the classical FST values based on microsatellite genotypes. It is a way to assess the discrepancy between the information provided by the genotype and by the name of the variety of origin of a sample.Genetic differentiation between zones within region (ockerham , with thockerham . To quanIn order to depict the genetic relationships between populations, DARwin version 5.0 was used to compute a genetic dissimilarity matrix using the simple matching index . The dis


Datura stramonium, we compared the degree of phenotypic differentiation (PST) of leaf resistance traits  against neutral genetic differentiation (FST) at microsatellite loci. Results showed that phenotypic differentiation in defensive traits among-population is not consistent with divergence promoted by genetic drift and restricted gene flow alone. Phenotypic differentiation in scopolamine concentration was significantly higher than FST across the range of trait heritability values. In contrast, genetic differentiation in trichome density was different from FST only when heritability was very low. On the other hand, differentiation in atropine concentration differed from the neutral expectation when heritability was less than or equal to 0.3. In addition, we did not find a significant correlation between pair-wise neutral genetic distances and distances of phenotypic resistance traits. Our findings reinforce previous evidence that divergent natural selection exerted by herbivores has promoted the among-population phenotypic differentiation of defensive traits in D. stramonium.Defensive traits exhibited by plants vary widely across populations. Heritable phenotypic differentiation is likely to be produced by genetic drift and spatially restricted gene flow between populations. However, spatially variable selection exerted by herbivores may also give rise to differences among populations. To explore to what extent these factors promote the among-population differentiation of plant resistance of 13 populations of  Datura stramonium is consistent with a scenario of varying selection or genetic drift and restricted gene flow.Most species consist of a series of populations that are often phenotypically differentiated . HeritabQST), it is necessary to contrast this hypothesis against a null model of differentiation at adaptively neutral loci , or high recombination between molecular neutral marker loci and quantitative trait loci  is an analogous index to QST  provides an optimal system for studying among-population differentiation in resistance traits. Because of its wide distribution , D. stramonium is exposed to different environmental conditions and to a wide diversity of herbivore species  vary widely across populations . Selecti species . Resistarichomes  and troprichomes , of whicrichomes . These srichomes  with negrichomes . Recent richomes . HoweverD. stramonium is accounted by neutral processes (genetic drift and restricted gene flow) or divergent natural selection. To do so, we compared the degree of phenotypic differentiation of resistance traits by means of PST estimated for the whole range of values of heritability, with the neutral expectation set by allelic divergence at microsatellite loci (FST). We expect that PST of each resistance character would be significantly higher than the index of population differentiation in neutral molecular markers (FST), since previous studies have detected contrasting selection exerted by herbivores on the three characters.Here, we assessed to what extent population differentiation in resistance leaf traits  of Datura stramonium L. (Solanaceae) is an annual herb commonly found in roadsides, cultivated areas and disturbed environments in Mexico, the United States, Canada, and Europe , which also feeds from other members of the Solanaceae family  as resistance traits against herbivory. These traits have shown heritable basis  from a sample of 20 leaves per plant by means of High Precision Liquid Chromatography (HPLC). Details of the extraction method and HPLC conditions can be found elsewhere  to explore if restricted gene flow and genetic drift (FST) alone can account for this differentiation or if there is a signal of differentiation promoted by divergent selection on resistance traits . To estimate PST values we fitted a linear model for each resistance trait, under the assumption that the distribution of resistance traits was normally distributed. The population term was considered as a random effect. To test the hypothesis that PST is higher than FST, a Monte Carlo test was carried out, approaching a sample of 10,000 deviates from both PST and FST by means of their estimated error. PST error was estimated from the likelihood errors of its components (variances among- and within-populations), while FST error was obtained by bootstrapping .In order to obtain trapping . The 10,FST and PST for all populations. Neutral marker variation can be used as a neutral expectation against which the phenotypic divergence of traits can be compared (PST) is the result of neutral processes rather than selection, differentiation among populations in these traits should correlate positively with differentiation in selectively neutral markers (FST) . Statistical analyses were performed using JMP® version 9.0.0 .We further evaluated the pair-wise Pearson’s correlation between compared . If resirs (FST) . We evalD. stramonium . After the subsequent univariate ANOVAs were applied, we found significant differences in trichome density , atropine , and scopolamine concentration . Mean leaf trichome density and mean atropine and scopolamine concentration per population are shown in A multivariate analysis of variance (MANOVA) detected significant multivariate differences in the studied resistance traits of 13 populations of FST was 0.228 (S.E. = 0.039), which is well above the minimum detectable value (FST = 0.01) that our sample and markers allowed with a statistical power of 0.94.Genetic differentiation as estimated by differences in allele frequency at microsatellite loci was moderate. PST) and neutral genetic marker divergence (FST) showed that PST values for scopolamine concentration were significantly higher than the FST in all values of h2 .We found no significant correlations between pair-wise aracters . Most coD. stramonium is not consistent with divergence promoted by genetic drift and restricted gene flow alone . Using this FST value, the indirect estimate of gene flow (Nm) is 0.846, suggesting restricted gene flow among populations of D. stramonium, and not sufficient to prevent differentiation by genetic drift  when it is not possible to obtain the amount of additive genetic variation   has been detected previously by D. stramonium to support our estimation of PST values.Although lations) , concluslations) . Thus isl Mexico . Phenotyl Mexico . Previouectively . Also, gectively  and geneectively  has beenPST index higher than FST means that divergent selection might be involved in population differentiation of resistance traits, at least for scopolamine, it is relevant to ask to what extent natural selection by herbivores is responsible for population differentiation in this character. In D. stramonium, several lines of evidence strongly suggest that differentiation in resistance is accounted by for herbivores. Differential and contrasting selection gradients on resistance to herbivores were detected between two populations of this species in a reciprocal transplant experiment  and Lema daturaphila (in two populations). In contrast, scopolamine was positively selected in one population where the specialist Lema daturaphila was the main herbivore, whereas trichome density was positively selected in two populations , and negatively selected in one population with the E. parvula (D. stramonium and data presented here suggests that population differentiation can be potentially adaptive.Because a periment . Likewis parvula . Thus, aPST and FST among population across the whole range of heritability, suggesting that differentiation at quantitative traits and neutral molecular loci is decoupled. Theoretically, if the pace of differentiation is dictated by genetic drift only, it is expected that differentiation indices will be perfectly and positively correlated (r = 1, PST = FST; PST > FST, any positive pair-wise correlation across populations, depicts a scenario where differentiation in quantitative traits exceeds the neutral expectation and suggests divergent selection (PST < FST, any positive pair-wise correlation across populations, portrays a scenario where differentiation at neutral molecular loci surpasses that of quantitative characters suggesting a strong effect of genetic drift; however at moderate values of FST stabilizing selection might be favoring the same phenotype across populations (PST > FST and are uncorrelated (dotted line in D. stramonium. Under this scenario there is opportunity for divergence driven by selection in resistance traits. Our results suggest that the higher PST than FST for scopolamine, together with spatial variation in resistance traits and the existence of a selection mosaic detected previously by Furthermore, we did not detect any significant correlation between the pair-wise election . On the ulations . When PS10.7717/peerj.1411/supp-1Supplemental Information 1Datura stramonium from 13 populations in Central Mexico.Data of trichome density, atropine and scopolamine concentration in plants of Click here for additional data file.