Liparis loeselii (L.) Rich.). We used amplified fragment length polymorphism (AFLP)-based assignment tests to quantify effective long-distance dispersal at two different regions in Northwest Europe. In addition, genomic divergence between fen orchid populations occupying two distinguishable habitats, wet dune slacks and alkaline fens, was investigated by a genome scan approach at different spatial scales  and based on 451 AFLP loci.Gene flow and adaptive divergence are key aspects of metapopulation dynamics and ecological speciation. Long-distance dispersal is hard to detect and few studies estimate dispersal in combination with adaptive divergence. The aim of this study was to investigate effective long-distance dispersal and adaptive divergence in the fen orchid  ‘outlier’ loci, potentially reflecting local adaptation to habitat-type, were identified with high statistical support. Of these, only one (0.22%) was a replicated outlier in multiple independent dune-fen population comparisons and thus possibly reflecting truly parallel divergence. Signals of adaptation in response to habitat type were most evident at the scale of individual populations.The findings of this study suggest that the homogenizing effect of effective long-distance seed dispersal may overwhelm divergent selection associated to habitat type in fen orchids in Northwest Europe. Gene flow in plants determines many key aspects of plant ecology including colonization and range expansion, and influences the potential responses to environmental changes. Effective long-distance seed dispersal,  can preserve genetic diversity at the local scale, which may in turn affect the efficiency of selection and local adaptation . QuantifMost orchid species are typically characterized by small, disjunct populations and are assumed to have a considerable dispersal potential because they produce a huge amount of dust-like, wind-dispersed seeds . Until net al. [ST-values) compared to most other plant families. Furthermore, isolation-by-distance was most frequently detected when the scale of the sampling exceeded 250 km, suggesting that below this scale, there is extensive seed dispersal. Phillips et al. [The family Orchidaceae is well known for its exceptional diversity, with approximately 26,000 species. A combination of strong genetic drift and natural selection has been proposed as the key to this immense species diversification ,8. A criet al.  found ths et al.  discusseLiparis loeselii (L.) Rich.) to study effective long distance gene flow and adaptive divergence. Fen orchid is a rare species declining throughout its distribution range that covers temperate parts of North-America and Europe. It occurs in early successional vegetation of coastal wet dune slacks and alkaline fens in plains and mountains [ovata Ridd. ex Godfery) occurring in dune slacks [et al. [Here, we chose the fen orchid (ountains . As suche slacks . Genetice slacks ). Howeve [et al. ) and comet al. [r = -0.22, p < 0.05), which may indicate a potential presence of size homoplasy or suboptimal concentrations in the PCR mix. The exclusion of fragments smaller than 200 bp for this primer combination (73 fragments) resulted in a non-significant correlation . To further reduce potential biases associated with the estimation of population parameters, we further reduced the number of fragments for this primer combination  from 17p < 0.0001), suggesting that less than 3% of all pairwise loci comparisons were not independent. This was further reduced to only 1.0% of significant pairwise loci comparisons (p < 0.0001) when repeating the logistic regressions on the subset of 266 loci.The AFLP band frequency distribution for the 451 polymorphic loci was asymmetric with relatively high occurrences at the low and high frequency ends of the distribution (results not shown). Pairwise logistic regressions between the 451 loci were significant for only 2.47% of all comparisons (s) (mean ± SD) calculated over all K subpopulations  was 91% (±5.5). This corresponds with a mean inbreeding coefficient of 0.83. The proportion polymorphic loci (PPL) at the 5% level, Nei’s gene diversity  and the rarity index  are given per population in Additional file j ranged from 0.13 to 0.35, with a mean of 0.20. Patterns in the unbiased Shannon diversity index are presented in Figure No ramets of the same genet were found among the samples. The estimated selfing rate  calculated for the estimated mean self-fertilisation rate of 91% was 0.09 (±0.1). The mean estimated value of ΦPT was 0.13 (p (rand > = data) = 0.001). Clustering at the population level is presented in the Neighbour-Joining (NJ) tree and in the PCoA in Figure p (rxy-rand > = rxy-data = 0.44)).There was a moderate genetic differentiation between the populations at the continental scale. The FThe simulations for the assignment procedure resulted in a fairly small increase in proportion of failures at increasing assignment stringency levels. Increasing the latter from a minimum log-likelihood difference (MLD) of 0 (i.e. no likelihood difference threshold between the most likely and the second most likely population) to MLD = 3  increased the average rate of failures  with 4.2% and 4.5% for the simulated data of Belgium & the Netherlands and Northwest France, respectively. Consequently, our AFLP-dataset proved to be adequately powerful. The average estimated rates of allocation success, of non-allocation and of allocations to the wrong population for different values of MLD and based on the simulated datasets (10 iterations × 1000 genotypes) are given in Additional file p = 0.001) when excluding locations with low sample size (n ≤ 5). Dispersal distances ranged from 1.95 to 152 km with a median geographical distance between the different combinations of source- destination populations of 50.6 km. When excluding locations with a low sample size (n ≤ 5), we obtained an estimate of the LDD rate between 9.3 and 17.7% and a mean dispersal distance of 74.8 km (range: 4.9 – 152). The main directions of LDD were northwest and northeast, each representing 33%  of all the different source – destination combinations. Re-allocation tests suggested seed dispersal between populations occupying different habitats . Another two individuals (2.2%) were allocated to a source population that was not sampled. This resulted in an estimate of the LDD rate between 15.2% and 28.2% for the sampled populations in Northwest France. The simulation analysis for the populations of Northwest France resulted in 71.5% correct allocations. This increased to a success rate of 99.9% (p = 0.05). LDD distances ranged from 1.64 to 220.7 km with a median geographical distance between the different combinations of source – destination population of 20 km. The main direction of LDD was southwest which represented 43% (14 out of 32) of the different source – destination combinations. Also at this regional scale, assignment tests suggested seed dispersal between populations occupying different habitats  different source – destination combinations. No putative immigrants from outside the sampled region were detected. This resulted in an estimate of LDD for the populations of Belgium and the Netherlands ranging from 16.5 to 30.5%. Simulation tests estimated an allocation success rate of 94.9%  putative immigrants from a population from Belgium/the Netherlands to a population located in Northwest France. Including the four sampled populations on the Dutch Wadden islands as putative source populations within the re-allocation tests resulted in a different assignment for three putative migrants (5%). For the re-allocation procedure of the samples from Northwest France, a higher number of putative source populations changed the assignment for seven out of 24 allocated individuals (29%) from a neighbouring French population to a population located in Belgium/the Netherlands. Removing loci that have a high probability of being homoplasious (73 loci) from the dataset increased the number of individuals that could not be allocated ) was identified as an outlier associated with habitat-type by both BAYESCAN and MCHEZA at the continental scale  Table ). No outp (α > 0.91)) in BAYESCAN. Of these, only one locus (0.2%) (ID 431) was a replicated outlier in three multiple pairwise population comparisons of which two were statistically independent, that is, comparisons that did not share a population. These comparisons included two different fen populations from the Netherlands  and three different dune-populations  were identified as outliers in at least one pairwise fen-dune comparison and not in the control fen-fen and dune-dune comparisons . The FST-values observed in this study confirm the rather low FST in orchids compared to other herbaceous families  seeds per capsule . Their ss et al. ). The ab. [et al. in a stut al.[ST . In cont.[ST [ST . The modon level . Under lon level . At inteon level ,20. Locaon level . Yearly on level   explain this nucleotide polymorphism by recombination events and random mutations. Comparable to this study, relative high population genetic diversity values  for the adult stage were also found in the predominantly autogamous food-deceptive orchid Neotinea maculata using AFLP loci [.The results of this study also indicate high admixture between populations of fen and dune slack habitats. Remarkable for a predominantly selfing species, we observed substantial genetic diversity within populations (mean PPL: 59%). A high level of AFLP polymorphism, the absence of identical genotypes, a similar asymmetric AFLP band frequency distribution and a relatively low level of linkage disequilibrium were also found for  selfing . MiyashiFLP loci .Previous studies have shown that AFLPs were efficient in assigning each individual to its population, especially at intermediate spatial scales and when population differentiation is weak ,26. The et al. [et al. [To date, few studies exist on the spatial scale of local adaptation at the genetic level [but see . Here, wet al. ). But, a [et al.  pointed There are several possible explanations for the lack of strong signals for adaptive divergence associated with habitat type in the fen orchid. One possible explanation is that the levels of genetic adaptive divergence across the genome are too low to be detected by a genome scan. If divergent adaptation occurs through moderate changes in allele frequencies at multiple sites, it is likely that none of these sites will exhibit substantial divergence between populations . Besideset al. [Founding effects from long-distance seed dispersal combined with local adaptation to regional variation, rather than drift-mediated selection, have been proposed by Phillips et al.  as key fLiparis loeselii (L.) Rich.) is a small, diploid (2n = 26) orchid that relies on regular disturbance for its survival [Fen orchid ) and the localities of existing populations in France, Belgium and the Netherlands are well known ,22,32. Tet al. [et al. [et al. [et al. [http://cran.r-project.org/web/packages/brainwaver/). Before performing further analysis, we excluded loci with frequencies below 5% and above 95% that may lead to spurious correlations and are therefore not considered reliable [Total DNA was extracted from silica dried leaf samples with the QuickPickTM Plant DNA kit , except for the samples collected in 2003 for which the CTAB-based method  was usedet al. , with re [et al. ) using t [et al.  with Raw [et al.  with the [et al. , the cor [et al.  to corre [et al.  and braireliable .IS = s/(2-s)). Genetic diversity was investigated by computing the proportion of polymorphic loci (PPL) at the 5% level and Nei’s gene diversity  using standardized sample sizes (i.e. with a maximum of eight samples per population). Frequency down-weighted marker values  were calculated with AFLPDAT [As the fen orchid can reproduce vegetatively, we first checked whether the dataset contained similar ramets of the same genet with AFLPDAT  by setti AFLPDAT .et al. [Spatial patterns of genetic diversity were inferred based on the Shannon index using a geographical ‘sliding window’ approach as in Arrigo et al. . The anaST using AFLP-SURV1.0 with 100 permutations and assuming the selfing rate estimated from the data by INSTRUCT. In addition, pairwise ΦPT were estimated using the AMOVA analysis of GenAlEx 6.5 [http://tree.bio.ed.ac.uk/software/figtree/) was used to display the midpoint rooted dendrogram.As a measure of population differentiation we calculated FAlEx 6.5 . The sigAlEx 6.5  on whichAlEx 6.5  to obtaiPT and pairwise geographic distances was performed as implemented in GenAlEx 6.5 (999 permutations).Furthermore, a Bayesian clustering approach implemented in INSTRUCT  was run Population likelihood assignment tests for individuals based on their multi-locus genotype were used to estimate contemporary, effective seed dispersal events. We followed the procedure of Duchesne & Bernatchez , developTaking into account impacts of founding events , the number of different source (seed population) – destination (sampled population) combinations where source and destination are not identical, was considered as the lower bound of the estimated seed migration rate. The proportion of putative immigrants among the total of unambiguously assigned individuals was considered as the upper bound of the estimated migration rate. Seed dispersal distance and direction were inferred for each putative immigrant.Assignment tests were performed within two independent sites in which we are quite certain to have allocated all the fen orchid populations: i) Northwest France and ii) Belgium and the Netherlands with the populations on the Dutch Wadden Sea Islands excluded because of missing data for one large population of the Dutch Wadden Sea Islands Figure . This alST by comparing observed FST-values with values expected under neutrality. Outlier locus detection was performed based on 451 polymorphic loci by two commonly employed approaches implemented by the programs MCHEZA [In comparison with expectations for neutral evolution, marker loci with excess differentiation are considered to indicate candidate regions under divergent selection. Outlier loci display unusually high values of Fs MCHEZA  and BAYEs MCHEZA . A detaiAlthough DFDIST was shown less reliable than BAYESCAN with AFLP data , we expeWe defined a locus as a significant outlier if it was detected by both MCHEZA and BAYESCAN in at least one of the overall between-habitat comparisons of the pooled population samples  or if it was detected by BAYESCAN in at least one population pairwise comparison in which the pair of populations were located on different habitats. The significant outlier was not retained if it also occurred as an outlier in comparisons between populations of the same habitat type (control comparisons). We defined replicated outliers as outliers associated with different habitats in at least two different, statistically independent pairwise population comparisons thus providing evidence for truly ‘parallel’ divergence.The data set supporting the results of this article is available in the Dryad repositoryHigh levels of effective long-distance dispersal may blur ecotypic divergence in a rare terrestrial orchid.Dryad. doi: 10.5061/dryad.sb68v http://doi.org/10.5061/dryad.sb68v.Vanden Broeck, A., Van Landuyt, W., Cox, K., De Bruyn, L., Gyselings, R., Oostermeijer, G., Valentin, B., Bozic, G., Dolinar, B., Illyés, Z., Mergeay, J. (2014): AFLP data from: The authors declare that they have no competing interests.AV, WV, RG, LD, GO and JM designed the research, AV, WV, RG, GO, BV, GB, BD and IZ generated the data, AV and WV performed the research, AV, KC, RG analyzed the data, AV wrote the paper and all authors commented on multiple drafts of the manuscript. All authors read and approved the final manuscript.Description of the 38 sampling locations of Liparis loeselii.Click here for fileNumber of DNA fragments generated by 4 AFLP primer-enzyme combinations used in Liparis loeselii.Click here for fileEffect of different values of the minimal log likelihood difference (MLD) on the estimated rates of allocation success, of allocations to the wrong population and of non-allocations calculated with the simulation procedure of AFLPOP 1.1 and based on 1000 simulated genotypes and 10 iterations.Click here for fileRe-allocation results of Liparis loeselii individuals performed with AFLPOP 1.1 for a minimal log likelihood difference (MLD) = 1.Click here for filePosterior probabilities for the model including selection of putative outlier loci detected with BAYESCAN 2.01 in pairwise population comparisons of Liparis loeselii.Click here for fileDetailed description on the search of putative outlier loci.Click here for file


Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum, which plays a crucial role in the transmission of schistosomiasis. The endemic area of S. japonicum is strictly consistent with the geographical distribution of O. hupensis.O. hupensis from four ecological landscapes were selected for analysis of genetic diversity by screening eight microsatellite DNA polymorphic loci.A total of 24 populations of Ho) and expected (He) heterozygosities varied from 0.331 to 0.57 and from 0.888 to 0.974, respectively. The mean of polymorphism information content (PIC) for all populations was 0.940, appearing polymorphic for all loci. For the fixation index of F-Statistics, Fit and Fst were 54.95 and 37.62 %, respectively. Variation of O. hupensis chiefly exists among individuals, accounting for 60.58 % of the total variation determined by Analysis of Molecular Variation (AMOVA). Variation among individuals within populations, among populations within groups and among groups only accounted for 26.60, 8.04 and 4.78 %, respectively. This distribution of variation suggests that genetic differences principally originate from within-populations rather than among-populations. Moreover, UPGMA cluster analysis showed that the populations spreading within middle and lower reaches of the Yangtze River  clustered together first, then gathered with the populations in the high mountains , coastal hills (FJFQ and FJFZ) and Karst landform (GXBS and GXYZ) successively.The number of alleles per locus ranged from 29 to 70 with an average of 45.625 and that of effective alleles were 18.5 to 45.8 with an average of 27.4. The observed (Oncomelania hupensis in mainland China, which is critical for the epidemiological investigation and surveillance of S. japonicum.This study provides novel insight into the theoretical source of genetic differentiation of  Schistosoma japonicum, remains one of the most prevalent parasitic diseases and effects severe socio-economic and public health losses in China  were determined using Arlequin . The corO. hupensis sampled, 396 specimens were scanned at the genetic level across eight polymorphic loci of microsatellite DNA. The lengths of amplified fragments for a total of 6,196 microsatellite DNA loci were obtained.From the 24 populations of O. hupensis showed that the number of alleles per locus ranged from 29 to 70 with an average of 45.625, and that of effective alleles were 18.5 to 45.8 with an average of 27.4. The GXYZ and HNHS populations had the minimum and maximum average Na values, respectively. The average He within populations ranged from 0.888 to 0.974, and the average Ho ranged from 0.331 to 0.57. The populations with the highest and lowest Ho values were HNHS and GXYZ, respectively. The average PIC for all populations of O. hupensis was 0.940 .No significant linkage disequilibrium was found between all pairs of the eight loci examined (P < 0.01), which indicated the independent behaviour of all loci. Analysis with Genepop software showed the possible occurrence of null alleles, which may lead to deviations from HWE and result in exaggerated levels of genetic differentiation [Significant deviation from Hardy-Weinberg equilibrium (HWE) was observed: 47 out of 192 (24.48 %) possible single exact locus tests  between geographic distance and genetic distance among populations is positive  and genetic distribution of all populations accorded with the Isolation-by-distance Model  showed that O. hupensis populations based genetic distance showed that the populations spread in the landscape of middle and lower reaches of Yangtze River  clustered together first and then gathered with the populations of high mountains , coastal hills (FJFQ and FJFZ) and Karst land form (GXBS and GXYZ) successively  from all populations was higher than (0.764) obtained from previous result [The abundance of the number of heterozygotes and the amount of genetic information in a population is directly proportional to the IC value , 39. Ress result . This siFst for all loci was 0.376, which means that 37.6 % of genetic variation was among populations and 72.4 % was among individuals within populations. The analysis of AMOVA displayed that genetic variation among individuals (60.58 %) were far higher than that within populations (26.60 %), while among populations and among groups are (8.04 %) and (4.78 %) respectively. This implies that, genetic diversity is strongly derived from among-individuals rather than among-populations. However, the average Fst (0.376) and genetic variation among populations (8.04 %) were higher than values obtained from the previous results (0.048 and 4.8 %) respectively, revealing genetic variation among populations increased along with geographical distance [Furthermore, this study reveals that the average distance . The ManO. hupensis spreading along the river within the large population, or gene drifting for surged water flow in the lakes and marshes landscape [The phylogenetic tree constructed by UPGMA also showed that populations in neighboring geographical locations generally cluster together, which was consistent with the Mantel test results. The cluster sequence of geographical populations showed us that the population from the karst landscape of Guangxi autonomous region maybe the most original one, then the population from the littoral hill part of the Fujian province, the population from the mountainous region of the Sichuan and Yunnan provinces and the population from the region of swamps and lakes in the middle and lower reaches of the Yangtze River, respectively. Regarding as the largest population spread throughout the middle and lower reaches of the Yangtze River , the popandscape . Then thandscape  and DNA andscape , 41, 42.O. hupensis, an important snail intermediate host of S. japonicum in China mainly originates from among-individuals rather than among-populations. It also reveals that the populations within subspecies have closer consanguinity than between subspecies in the mass, nevertheless, genetic variations exist within subspecies. These findings further provide important information on genetic structure of O. hupensis and strengthen our knowledge about diffusion trend and tracking to the source of Oncomelania in mainland China. Ultimately, these findings will help us develop more effective guidelines for controlling the spread and distribution of Oncomelania and consequently prevent the transmission of Schistosomiasis in China. Our data offers a better understanding of the genetic differentiation of Oncomelania hupensis, enhancing our ability to effective and efficient surveillance of Schistosomiasis.This study has shown that the genetic diversity of


Crossocalyx hellerianus, a strictly epixylic dioicous liverwort . Studied populations were located in Fennoscandia and Central Europe, with localities differing in availability of substrate and the population connectivity, and their populations consequently different in size, density, and prevailing reproductive mode. A set of nine polymorphic microsatellites was successfully developed and used. Identical individuals were only found within populations. Especially in large populations, the majority of the individuals were genetically unique. Resampled number of genotypes, mean number of observed alleles per locus after rarefaction, and Nei’s gene diversity in large populations reached high values and ranged between 4.41–4.97, 3.13–4.45, and 0.94–0.99, respectively. On the contrary, the values in small populations were lower and ranged between 1.00–4.42, 1.00–2.73, and 0.00–0.95, respectively. As expected, large populations were found to be more genetically diverse than small populations but relatively big diversity of genotypes was also found in small populations. This indicated that even small populations are important sources of genetic variation in bryophytes and processes causing loss of genetic variation might be compensated by other sources of variability, of which somatic mutations might play an important role. The presence of SGS was discovered in all populations. Large populations possessed less SGS, with individuals showing a pronounced decrease in kinship over 50 cm of distance. Apparent SGS of small populations even at distances up to 16 meters suggests the aggregation of similar genotypes, caused predominantly by the deposition of asexually formed gemmae. Although no strong kinship was detectable at the distances over 16 meters in both small and large populations, identical genotypes were occasionally detected at longer distances (20–80 m), suggesting effective dispersal of asexual propagules.Patterns of genetic variation and spatial genetic structure (SGS) were investigated in  Ptilidium pulcherrimum, which showed that less than 1% of produced spores were deposited on substrate suitable for establishment [The structure of genetic diversity on fine scales within populations and on larger scales among populations may bring valuable insights into the reproductive systems of studied organisms including the assessment of reproductive effort, rates of sexual and vegetative reproduction, dispersal capacity of diaspores and levels of gene flow among populations. Bryophytes are generally considered to possess high dispersal capacity of their sexually originating spores , 2, whiclishment . On the lishment –16. Genelishment .Barbilophozia attenuata Mart. (Loeske), which is a species closely related to our object of study, possessing a similar reproduction mode.Recent studies of population genetic variability and spatial genetic structure using DNA fingerprinting methods have shown a remarkable variability of results, showing the uniqueness of parameters of individual reproduction systems in different taxa. One of the most interesting findings is that the level of genetic differentiation among bryophytes reproducing mostly or exclusively vegetatively was in several cases surprisingly high –21. The Crossocalyx hellerianus (Nees ex Lindenb.) Meyl., a minute, circumboreally distributed dioicous epixylic liverwort . Boyllaceae ), whereayllaceae , 31. On yllaceae , inhabityllaceae . With reyllaceae  and theryllaceae , and Endyllaceae  accordinThe study populations, located in Scandinavia and Central Europe, differ in size, density, prevailing reproductive mode, and population connectivity. Thus, these populations represent a suitable study system for investigations on patterns of genetic variation with regard to the above mentioned population characteristics. The studied liverwort moreover produces sexual and vegetative diaspores of potentially very similar dispersal capacities with respect to their size, which facilitates the inference on dispersal efficiency. We hypothesized that the population size or density and prevailing reproductive mode would be mirrored in the population genetic diversity and fine-scale spatial genetic structure. Microsatellite markers, which have been developed for his study, further allowed for the assessment of gene flow levels among populations and rates between sexual and asexual reproduction.Crossocalyx hellerianus. The Czech forests represent small extant fragments of herb-rich and acidophilous montane mixed old-growth forests with the tree composition and herb vegetation approaching the natural one, dominated mostly by beech with spruce admixtures. The amount of suitable decaying wood is only high in the Boubínský prales National Nature Reserve among the Czech forests. Consequently, C. hellerianus is relatively common only in this reserve, while the other Czech localities support only very small populations of the liverwort  and in the Czech Republic  were investigated for possible occurrence.In populations, where C. hellerianus at a minimum distance of 20 cm; the maximum distance depended on the patchy distribution of species on each sampled log  species in the Czech Republic according to IUCN criteria [All necessary permits were obtained for field studies to collect species material. Metsähallitus issued the permission for entry into Finish localities and Nature Conservation Agency of the Czech Republic issued the entry permission into Czech localities. No special permission is required for sampling of criteria , which hA SSR-enriched genomic library was constructed using a biotin-streptavidin capture method . Screeni2, 0.2 mM dNTPs, 0.3 μM primers, 0.25 U Taq polymerase  in the manufacturer’s reaction buffer, and sterile water to make up a final volume of 5 μL. Amplifications were performed with an initial denaturation of 3 min at 94°C, followed by 45 cycles of 1 min denaturation at 94°C, 30 s at primer-specific annealing temperature . The probability that individuals shared the same multilocus genotypes (MLG) were derived from sexual reproduction involving recombination (Psex) calculated in the GenClone 2.0. Samples with missing data were excluded from all above mentioned computations.Nei’s gene diversity (Ĥ) was calculated using Arlequin v3.5 . Number  program . With resoftware  was usedsex assessment, linkage disequilibrium analysis was performed to assess whether marker distributions resulted from sexual or asexual reproduction. Multilocus linkage disequilibrium was tested using the index of association modified to remove the effect of number of loci analyzed (rd [In addition to Pyzed (rd ) and calST-like method, using the sum of squared size differences. The RST-like method was preferred because a preliminary allele permutation test performed in SPAGeDi 1.4 software [ST, which is likely to provide a biased estimate of gene flow [ST-like method showed that variation among populations within regions was slightly higher than variation between the two geographic regions (CZ vs. FI). In addition, the pairwise RST values for all populations were computed. The significance of AMOVA components and of pairwise RST values was tested using 10,000 permutations.Hierarchical structure of genetic variation was examined using analysis of molecular variance (AMOVA) in Arlequin v 3.5  with calsoftware  was signene flow . The folij) based on Nason’s kinship coefficient [ij per distance class was tested using 1,000 random permutations of individuals.To reveal the fine-scale spatial genetic structure (SGS), a spatial autocorrelation analysis was conducted in SPAGeDi 1.4 software . Distancfficient  were calThe spatial extent of clonal dispersal was quantified using distance classes and population assignment defined as above. The percentage of clones within each of the distance classes was calculated using pairwise comparisons which included identical genotypes and they were plotted against the upper boundaries of classes. In addition, the maximum distance among samples of the same genotype was recorded for each population.Crossocalyx hellerianus were developed (Nine polymorphic microsatellite markers from the liverwort eveloped . All geneveloped . The fineveloped . Identicg), mean number of observed alleles per locus (Na) after rarefaction, and Nei’s gene diversity (Ĥ) varied from 1.00 to 4.97, 1.00 to 4.45, and 0.233 to 0.995, respectively . On the contrary, the pairwise RST values between FI populations except the most remote population N did not exceed the value of 0.1. Even in case of population N, the pairwise comparisons with the remaining FI populations  revealed generally lower RST values than those observed among CZ populations separated by even shorter geographic distances . The pairwise RST values among geographically close populations  were higher among CZ populations  was relatively high for some of the putative clones from small CZ populations, but negligible for majority of individuals from large CZ and FI populations  tend to have bigger pool of genotypes/alleles. In large populations, the majority of the individuals were genetically unique, whereas small populations showed higher ratio between N/Ng. The reduced variation in smaller populations may result from processes such as bottleneck, genetic drift or inbreeding [C. hellerianus support the earlier views that even small populations are important sources of genetic variation in bryophytes [Abies alba dominated forest towards broad-leaved forest dominated by Fagus sylvatica with only a minor percentage of spruce . Interebies 15% ). The poT values .Fully identical individuals were only found within populations. A large diversity of multilocus genotypes within populations appears to be common in both liverworts , 20, 23 Crossocalyx hellerianus, as the frequency of these events is massively outweighed by the gemmae production. The study [C. hellerianus colony exceeded the spore production nearly five times, while the ability to germinate in both types of propagules was similar [C. hellerianus is also indicated by high values of linkage disequilibrium . Significant and rather high linkage disequilibrium was also found in the large FI population N, although the percentage of patches with perianths (25.9%) was comparable with other large FI populations. Nevertheless, the slightly lower genetic variation as inferred from Ng, Na and Ĥ values was congruent with linkage disequilibrium. This pattern could be explained by low portion of gametophytes arising from sexually produced spores or as a result of inbreeding. Mating may occur among haploid siblings originating from the same sporophyte as a result of non-existing mechanism to distinguish among differently related gametes [C. hellerianus. Especially small CZ populations showed high values of linkage disequilibrium, rather low number of genotypes and aggregation of similar genotypes, which is consistent with the assumption of low recombination efficiency. Therefore, genetic variation in small CZ populations has most likely been caused by somatic mutations, past genetic variation prior to population reduction, and/or establishment by multiple genotypes, although we cannot rule out the contribution of sexual reproduction with respect to the facts discussed above.Other explanations of remarkable genetic diversity in predominantly and/or seemingly asexual bryophytes may involve e.g. population establishment by multiple genotypes, or periodical occurrence of sexual reproduction generating novel recombinant genotypes. Recruitment of new genotypes from neighboring populations seems to be a rather improbable and rare event in the studied system, as no identical MLG were shared among populations, not even between the spatially closest populations Z and Y, distant only 4 km. Occasional and unobserved sexual reproduction, which might be a major source of variation in large populations with stable reproductive system even with only small number of reproducing individuals per generation , also prhe study  reported similar . Prevail gametes . InbreedST values was found in most of the studied populations. Genetic differentiation was rather low among the FI populations , whereas the values among the CZ populations mostly exceeded 0.1 . We suppose that suitable substrate, enabling step-by-step dispersal [Estimates of genetic differentiation among populations reflect the amount of gene flow between them . Isolatieded 0.1 . This imispersal , 62 suppispersal , 18. Genispersal , 49.Crossocalyx hellerianum [Direct observations of propagule dispersal in lerianum  showed tlerianum . Anyway,C. hellerianus, where the amount of decaying wood is generally low and essentially all available substrate is occupied. Random SGS cannot be achieved in the absence of sexual reproduction, evidenced by high values of linkage disequilibrium and absence of perianths in small CZ populations. Asexual reproduction by gemmae represents here the most important and efficient role in maintaining the populations. This is in agreement with previously postulated conclusions in vascular plant studies [High values of kinship coefficients observed in most of the small populations provided the evidence for aggregation of similar genotypes. This can be caused by the relatively low level of genetic diversity resulting from bottleneck and/or founder effect, prevailing asexual reproduction or breeding of related individuals, as discussed above. Spatial distribution reflects both substrate availability and the mode of reproduction. If suitable habitats are evenly distributed and spore production is frequent, allowing effective dispersal at the middle and long distances, randomness in distribution, reflected in the absence of SGS, can be achieved . This is studies , 64.C. hellerianus, large populations possessed less SGS than small populations, with their individuals showing marked decrease in kinship over 50 cm distances and appearing to be without any obvious kinship on distances exceeding 16 m. This result reflects higher population density and more frequent spore production observed in large populations, both allowing more efficient dispersal of different or novel MLGs on farther distances, which reduces the pattern of SGS. Anyway, even in large populations, the plants continue to produce gemmae massively, contributing to aggregation of genotypes and presence of SGS over short distances. Vegetative reproduction by gemmae obviously contributes to economic balance avoiding the costly production of sporophytes [Recent investigations of SGS in seed plants, reviewed in  showed trophytes .C. hellerianus and the small-scale pattern of SGS in a closely related liverwort species, Barbilophozia attenuata [B. attenuata, whereas small CZ populations of C. hellerianus differed in noticeably strong SGS. Whereas the kinship coefficients reached zero over 8–10 m in B. attenuata, they approached zero not earlier that at distance of 16 m and turned negative at distance of 500 m in C. hellerianus, reflecting the aggregation of genotypes over larger distances in the latter species. This might infer that B. attenuata produces sporophytes more often or the gemmae of C. hellerianus have better dispersal capacity. The latter explanation can be supported by the difference in propagule weight, because the smaller gemmae of C. hellerianus have about eight times smaller volume than the gemmae of B. attenuata.Comparison of SGS shape between studied populations of ttenuata  shows siC. hellerianus. We observed most of clones to be dispersed only within logs at short distances in large populations, whereas small CZ populations showed significant portion of clones dispersed at distances up to 10 m Click here for additional data file.S2 FigThe plot was generated using 1,000 random samples of 1 to 9 loci. Resampling of loci indicated that our set of nine loci had sufficient haplotypic resolution, as even the use of approximately 7 loci would reveal the majority of MLGs detected in this study.(TIF)Click here for additional data file.S3 FigPlots were generated for each population separately (A) small populations and (B) large populations, using 1,000 random samples of individuals to see if the relationship reached a plateau. Resampling of individuals indicated that increased sampling would yield higher number of MLGs in large populations (B), whereas in small populations the number of MLGs mostly tended to reach a plateau (A). The estimated number of MLGs was substantially lower in small populations (1–8 MLGs) than in large populations  at smaller sampling sizes (N ranging from 5 to 16), corresponding to the maximum sampling size in small populations. Therefore, sampling in small populations was probably rather comprehensive despite lower number of individuals in population, whereas in large populations the clonal diversity estimates could be underestimated.(TIF)Click here for additional data file.S4 Figsex) was plotted against the particular repeated multilocus genotypes (MLG) for populations (A) small CZ populations, (B) large CZ population and (C) large FI populations. If the probability is below significance threshold (Psex < 0.05), the respective individual is not likely to be the result of a distinct event of sexual reproduction. Thus we can conclude that individuals with identical genotypes, which occur more than once in the population and their Psex < 0.05, were probably established from asexual propagules .Probability of sexual reproduction (P(TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 FileAbbreviations of localities correspond to (XLSX)Click here for additional data file.S3 FileAbbreviations of localities correspond to (XLSX)Click here for additional data file.S1 TableF values is marked as *** P < 0.001; ** P < 0.01; * P < 0.05.Significance of (DOCX)Click here for additional data file.


GATM) plays a vital role in energy metabolism in muscle tissues and is associated with multiple clinically important phenotypes. However, the genetic diversity of the GATM gene remains poorly understood within and between human populations. Here we analyzed the 1,000 Genomes Project data through population genetics approaches and observed significant genetic diversity across the GATM gene among various continental human populations. We observed considerable variations in GATM allele frequencies and haplotype composition among different populations. Substantial genetic differences were observed between East Asian and European populations (FST = 0.56). In addition, the frequency of a distinct major GATM haplotype in these groups was congruent with population-wide diversity at this locus. Furthermore, we identified GATM as the top differentiated gene compared to the other statin drug response-associated genes. Composite multiple analyses identified signatures of positive selection at the GATM locus, which was estimated to have occurred around 850 generations ago in European populations. As GATM catalyzes the key step of creatine biosynthesis involved in energy metabolism, we speculate that the European prehistorical demographic transition from hunter-gatherer to farming cultures was the driving force of selection that fulfilled creatine-based metabolic requirement of the populations.The glycine amidinotransferase gene ( GATM) (chromosome 15q15.3) encodes a mitochondrial enzyme, L-arginine:glycineamidinotransferase  that catalyzes the first critical step of indigenous creatine biosynthesis by converting arginine and glycine to ornithine and guanidinoacetate (GAA). The GATM gene is 41,203 bp in size, and mutations in this gene cause hereditary Cr deficiency syndromes (OMIM 602360),which are further characterized by severe mental retardation, speech delay, epilepsy, autism, and hypotonia2GATM expression and creatine synthesis in the myocardium have been observed in heart failure patientsGATM locus have also been reported to be significantly associated with chronic kidney diseases relating renal function5GATM are significantly associated with statin-induced  myopathyThe phosphocreatine (PCr)/creatine (Cr) system acts as a rapidly available buffer for phosphate-bound energy storage and transmission in organs that demand high and fluctuating energy. These buffer system components  are highly abundant in skeletal muscles at a concentration range of 20-40 Mmmes OMIM 02360,whiGATM gene has been implicated in several chronic diseases and drug response-relevant phenotypes among different human populations, we screened for continental-wide diversity across this gene. Analysis of the 1,000 Genomes Project data (http://www.1000genomes.org) has indicated significant continental-wide genetic diversity among human populations at the GATM locus. Furthermore, statistical analysis has revealed that the GATM gene in European populations underwent positive selection.Human populations have encountered substantial environmental challenges as they colonized various parts of the world. Local adaptations to various environments have largely promoted genetic diversity, which is illustrated by their phenotypic differentiation. The availability of whole genome data from continental populations around the world has provided excellent opportunities to identify genetic diversity or selection signatures across various loci. Because the GATM gene in 1,092 unrelated individuals from four continental regions, including Europe, East Asia, Africa, and America, from the 1,000 Genomes Project phase I. The FST differentiation ratio  calculated for the GATM locus compared to all the other 54,740 genome-wide SNPs encompassing genes ranked GATM as the 33rd most highly differentiated gene (FST(CEU_JPT) = 0.56; whereas populations within these continental groups showed a high level of similarity  among the 14 populations mentioned in the 1,000 Genomes Project, and their derived allele frequencies were relatively different from those of the East Asian and European population groups (Statins are anti-hypercholesterolemia drugs prescribed across the globe for lowering lipoprotein (LDL) concentrations. Several ed genes . We alson groups .GATM gene, we applied a modified composite of multiple signals (CMS)lnRsbDAFFSTGATM locus . The elevated nucleotide diversity of the GATM gene compared to that observed in the entire genome revealed significant continental-wide population differences at the GATM locus, especially between East Asians and Europeans. Significant genetic diversity in haplotype diversity and LD patterns was also observed among these populations. The combined CMS results revealed positive selection across this locus in European populations. According to the ancient demography of Europeans, an important prehistorical event involving European communities was the transition from hunter-gatherer to farming culturesGATM gene might have undergone selection during the European transition from hunter-gatherer towards early farming culture. Although our estimated selection time analysis assumed more ancient GATM selection, which was incongruent to the timing proposed for European demographic shift towards agricultural society, it was difficult to accurately perform selection time estimation from thecurrent data.Modern human populations experienced a series of migrations with founder effect and subsequent population expansionGATM locus based on its haplotype composition among different populations. The European predominant haplotype  was distinct from that of East Asians’ . These considerable population variations in haplotype composition rendered it difficult to accurately predict the existing haplotype from tagged SNPsGATM gene has been associated with several important biomedical and drug relevant phenotypes567We observed substantial genetic divergence at the GATM-deficient mice exhibit decreased fat deposition as well as reduced cholesterol levels21222425GATM locus might be contributory genetic factors in this scenario. The minor allele of the GATM cis-eQTL  has been reported in association with reduced GATM expression in Europeans-Americans populationFST between CEU and JPT for rs9806699 eQTL was 0.41, with an empirical P value = 3.6e−3. The predominance of this allele in East Asian populations may contribute to the low incidence of statin-induced myopathy in East Asians compared to Europeans, as revealed in epidemiological studies2122i.e., rs906699 hasGATM locus of Europeans. This selection event has resulted in an alteration in the requirement for creatine biosynthesis for energy metabolism during the prehistorical transition from foraging toward farming culture among Europeans. We also conducted an in-depth characterization of the genetic variation and haplotype structure involving the GATM gene among various human populations. We assumed that the significant genetic diversity at this gene locus might account for the epidemiological differences in the predisposition of creatine-associated biomedical consequences and relevant drug responses. In addition, this information provides useful resources for the design and development of epidemiological and/or anthropological studies involving the GATM gene.In conclusion, combined CMS statistical analysis of whole-genome sequence data from the 1,000 Genomes Project has determined that ancient fixed selection occurred in the http://www.1000genomes.org). These individuals belonged to 14 populations from sub-Saharan Africa, East Asia, Europe, and the Americas. The Sub-Saharan Africans included Yoruba in Ibadan (YRI) in Nigeria; Luhya in Webuye (LWK) Kenya, and African ancestry people from Southwest United States (ASW). The European groups included residents from Northern and Western European ancestry (CEU), Toscani in Italy (TSI), British in England and Scotland (GBR), Finnish in Finland (FIN), and Iberians in Spain (IBS). The East Asians included Han Chinese in Beijing (CHB) China, Southern Han Chinese (CHS) in China, and Japanese in Tokyo (JPT), Japan. The American groups comprised Mexican ancestry individuals in Los Angeles, California (MXL); Colombians in Medellin, Colombia (CLM); and Puerto Ricans in Puerto Rico (PUR). The genetic variant datasets files (vcf format) released by the 1,000 Genomes project phase I were processed to acquire only the SNP genotype, while the rest of variants including INDELs and SVs were discarded. Total 36,820,992 SNPs from each sample of all fourteen population groups were selected for downstream analysis.The genomic data of a total of 1,092 unrelated individuals from the 1,000 Genomes Project were directly downloaded from the website . The haplotype frequencies were then separately calculated for each population.In above equation lnRsb was implemented in the CMS instead of XP-EHH, and iHS and ΔiHH were not integrated as they both reduced the power in cases where sweeps had reached a high frequency, fixation, or high recombination rateDAF analysis was performed according to Grossman et al. and the mean values of the CEU vs. CHB/JPT and CEU vs. YRI comparisons were used to calculate the CMS score. In the case of lnRsb, the more significant population in these comparisons was integrated into the CMS. Unlike the study conducted by Grossman et al.To identify the signals underlying positive selection, the combined CMS methodp(si) is the empirical p-value of the ith test. We assumed that 1% of the genome was under positive selection (π = 0.01).where; lnRsb score within the GATM gene) was selected as core SNP and an estimated 181 kb around this core SNP was assumed to have undergone natural selection , with an EHH value >0.25 and at a genetic distance of 0.055 cM. Simulation analysis was then performed to estimate the selection time of the above selected region in Europeans using the msms software−8 and the effective population size of Europeans as 20,000, and generated 85*2 haplotypes in each simulation. To estimate the selection coefficient and selection time, we set the selection coefficient as 0.01, 0.05, 0.07, 0.1, 0.5, and 1 and performed 10,000 simulations for each selection time, ranging from 310 to 1,500 generations, with each step comprising 10 generations. Next, we defined the mean values of the numbers of segregating sites as St for generation t and the numbers of distinct haplotypes as Ht for generation t. S0 is the observed number of segregating sites, and H0 is the observed number of distinct haplotypes. Genetic distance was calculated as follows:The SNP rs1153857 LD analysis using phase I data from the 1000 Genomes Project was calculated for the CEU, CHB, and YRI populations using the Haploview software .


Commelina communis with 12 microsatellite loci. Eight metallicolous and six non-metallicolous populations of C. communis were sampled from cupriferous sites and surrounding non-contaminated areas in China. Neither significant reduction in genetic diversity nor apparent founder and bottleneck effects were observed in metallicolous populations of C. communis. Based on Bayesian and Neighbor-Joining clustering analyses and a principal coordinates analysis, all sampled populations were found to be mainly separated into three genetic groups, corresponding well to their geographical locations rather than edaphic origins. Moreover, a significant and strong correlation between population genetic divergence and geographical distance were detected by Mantel test  and multiple matrix regression with randomization . However, the effect of copper concentration on genetic patterns of C. communis was not significant . Our study clearly demonstrated that the extreme edaphic conditions in metalliferous areas had limited effects on the genetic variability in C. communis. Geographic distance played a more important role in affecting the genetic structure of C. communis than soil composition did. In C. communis, the geographically disjunctive populations on metalliferous soils had multiple origins and evolved independently from nearby non-metallicolous populations.Pseudometallophytes are excellent models to study microevolution and local adaptation to soil pollution, as they can grow both on metalliferous and contrasting non-metalliferous soils. Although, there has been accumulating evidence for the effects of edaphic conditions and geographical isolation on the genetic structure of pesudometallophytes, it is still a difficult problem in evolutionary biology to assess their relative importance. In this study, we investigated the spatial patterns of genetic variability, population differentiation and genetic groups in pseudometallophyte  Environmental stress experienced by living organisms plays an important role in local adaptation and evolution of new species . Soils cGenetic variation at intra- and inter-population level have been considered as the preconditions of plant local adaptation or evolution . The extGenerally, geography plays an important role in population genetic divergence and speciation. The greater physical distance among populations will lead to the stronger internal genetic differentiation . Heavy mCommelina communis L. (Commelinaceae), commonly known as dayflower, is distributed extensively throughout China except for a few provinces, such as Qinghai, Hainan, Xinjiang, and Tibet  test the relative importance of geographic distance and soil composition on shaping genetic differentiation of C. communis populations; (3) explore whether C. communis populations established in metalliferous and non-metalliferous soils had a single or multiple independent origins.To accurately evaluate the effects of edaphic conditions and geographical factors on the microevolutionary processes of pesudometallophytes, multiple replicates of M and NM populations separated by large geographic distances should be analyzed. In this study, 14 us study . Twelve communis . The speC. communis . Sampled M populations were discontinuously distributed in three geographical regions . Six NM populations were also sampled from nearby non-metalliferous soils. In each population, leaves of C. communis were collected from twenty individuals separated by at least 5 m and immediately dried in silica gel for genomic DNA extraction. In addition, 10 rhizospheric soil samples were randomly collected from 5 to 15 cm in depth at each sampling site.Eight natural copper mines located in different geographic areas of China were selected as sampling sites for M populations of 3 (3:1) for at least 12 h, then digested at 120°C for 2 h. For the extractable Cu concentrations, soil samples (three replicates per site) were extracted with diethylenetetraminepenta acid (DTPA) solution.Sampled soils were dried and sieved through a 2-mm mesh. The concentrations of total and extractable Cu in soils were analyzed using atomic absorption spectrometry . For the total Cu concentrations, prepared soils (three replicates per site) were cold-digested with HCl and HNOC. communis using the DNA extraction Kit . The quality of extracted DNA was measured by NanoDrop spectrophotometer .The genomic DNA was extracted from dried leaves of Taq PCR MasterMix , 0.6 μM of each primer. Three fluorescent compounds  were used to label the forward primers for the automated sequencers analysis  and expected (He) heterozygosity, the number of alleles (Na) and the Polymorphism Information Content (PIC) were calculated with PowerMarker v3.23 software  and the number of ‘private’ alleles (Npr) for each population was estimated by FSTAT 2.9.3.2 . To determine whether there was a significant association between the geographic location and genetic diversity in C. communis populations, multiple regression analyses were performed in the SPSS with latitude and longitude as covariates and M-NM as fixed factor. Complementarily, we performed paired t-test to compare levels of genetic diversity between nearby pairs of populations established in metalliferous and non-metalliferous soils .The average levels of Cu concentration and genetic variation between M and NM populations were compared with independent- C. communis, a Bayesian model-based clustering, regardless of an individual’s geographic origin, was performed in STRUCTURE to infer the number of potential genetic clusters (K) (K was estimated from the posterior probability of the log-likelihood distribution Pr (X|K) (K) (To identify the patterns of genetic structure in ters (K) . The cluPr (X|K)  and its X|K) (K) .C. communis, an analysis of molecular variance (AMOVA) was performed with Arlequin 3.5.1 ; (2) a arrangement based on edaphic types with Cu-contaminated sites  vs. non-contaminated sites . In each analysis, 1, 000 permutations were used to test the significance levels of variance components. To test the correlation between the genetic (Pairwise FST) and geographic distances among populations of C. communis, the Mantel test was conducted in Arlequin 3.5.1 with 10, 000 permutations  tree was constructed with Cavalli-Sforza’s and Edwards chord distance (Dc) based on microsatellite allele frequencies . Compared to non- metalliferous areas, the average Cu concentrations was almost 50 times higher at the metalliferous sites (Table 1). In addition, large variances in Cu concentrations among the metalliferous sites were also detected. The highest values were reached up to 3400 ± 320 mg kg-1 for the total Cu and 535 ± 89 for the extractable Cu mg kg-1 at population GZ, whilst the lowest concentrations of total and extractable Cu were 1393 ± 183 and 193 ± 42 mg kg-1, respectively at population TKY.An overview of geographical locations and soil characteristics for the 14 P > 0.0001). A total of 135 different alleles were found across all the 14 populations of C. communis. The number of alleles among polymorphic loci ranged from four to 28, and the average value per locus was 11.25 (Supplementary Table Ho) and expected (He) heterozygosity ranged from 0.552 to 0.905 and 0.567 to 0.913, with a mean value of 0.764 and 0.771, respectively .Over the 12 SSR loci, no evidence of null alleles or scoring error was detected using MICROCHECKER. Deviations from linkage equilibrium were not significant after Bonferroni corrections (Commelina communis showed high levels of genetic diversity (Table 2). The mean number of alleles  for M and NM populations of C. communis ranged from 1.833 (TKY) to 6.500 (DY) and 2.500 (LFZ) to 5.167 (GF), respectively. No significant associations between genetic diversity and population latitude (P = 1.0) and longitude (P = 0.9) were detected by multiple regression analyses. The parameters of average gene diversity indices were relatively high in most populations from metalliferous soils, with the average values of 3.558 for Ar, 0.440 for Ho and 0.555 for He (Table 2). There were no significant differences between M and NM populations in average Ar , oH , and eH . Paired t-test considering nearby populations established in metalliferous and non-metalliferous soils did not show either significant difference in Ar , oH , or eH . Private alleles were detected in both M and NM populations of C. communis and no significant differences were observed in average Npr between M and NM populations . However, the total number of private alleles within M populations (24) was almost fourfold that within NM populations (7). The populations with most private alleles were DY (7) and JKL (5) (Table 2).FST value was 0.325 . Except three pairs of populations , all the values of pairwise FST were significantly different from 0 , indicating that geography is an important factors affecting the genetic structure of C. communis. Moreover, the results of MMRR revealed that the genetic distance had a significant correlation with geographic distance . However, the effect of environmental factor (copper concentration) on genetic patterns of C. communis was not significant .The overall pairwise K values. A clear peak in the value of ΔK was detected at K = 3 . In general, three gene pools were evident. The north China populations were predominantly composed of a single gene pool, whereas the other two gene pools were mostly detected in the east-central China populations and the central China populations . In addition, a general picture of admixture genotypes also emerged in our analysis and the ancestry of several genotypes in the 14 populations could be traced to more than one gene pool . Several genotypes form one population shared alleles with other populations, indicating that these populations likely maintained frequent gene flow . The PCoA results supported the findings from STRUCUTRE, demonstrating that genetic clustering of populations was in concordance with their geographic origins . However, there was an overlap of population LFZ from central China with population JKL from east-central China, indicating that intensive gene flow might occur between these two populations . The relationships between populations in the NJ tree showed that populations sampled from the metalliferous and non-metalliferous sites were not clearly separated, but divided into three distinct clusters, broadly corresponding to three geographical areas . All the populations located in east-central China formed one cluster (cluster I). The populations from central China and north China comprised another two major clusters (cluster II and cluster III). C. communis populations with close geographic distances had relatively low genetic divergence .The number of subpopulations (K) was identified based on maximum likelihood and ΔC. communis , although variation among subpopulations were significant in both groupings . Grouping of the individual samples according to geographical distribution allowed 10.47% of the total variation (P < 0.001) to be accounted for by differences among the three geographic origins (Table 3A). Edaphic separations, however, showed little contribution to the total genetic variation .The results of AMOVA showed that most of the genetic variability occurred within populations of C. communis populations , indicating that all populations sampled from metalliferous areas did not experience apparent founder and bottleneck effects during their recent colonization.Bottleneck analysis under the TPM showed that there were no significant heterozygosity excesses in the 14 C. communis. The populations of C. communis from metalliferous soils had not undergone any apparent founder and bottleneck events during their colonization history. All sampled C. communis populations mainly clustered into three groups, corresponding well to their geographical locations rather than edaphic origins. Geographically distant M populations showed closer genetic similarity to their nearby NM populations than to each other. Both Mantel and MMRR analyses suggested that geographic distance played a more important role in determining the population structure of C. communis than soil composition did. Moreover, all Cu-contaminated populations branched into separated genetic groups, supporting the hypothesis of multiple and independent origins of M populations in C. communis.Our data showed that severe soil conditions at metalliferous areas did not significantly affect the levels of genetic variability in Biscutella laevigata, Arabidopsis halleri, Silene paradoxa and Sedum alfredii, the levels of genetic diversity in M populations were significantly reduced due to strong founder and bottleneck effects (C. communis (Table 2). For example, the DY population originating from Cu-contaminated sites had the highest number of private alleles as well as the highest value of genetic diversity indices (Table 2). Moreover, in contrast to other studies (Table 4). These results suggested that the selective pressure caused by Cu contamination might be not perceivably stronger for C. communis populations on metalliferous soils, or its effect on genetic variability had been balanced by other factors.Heavy metal tolerance is a fundamental condition for pseudometallophytes to survive on metalliferous soils. The evolutionary scenario assumes that metal-tolerant genotypes initially have low frequencies in natural populations ; therefo effects . However studies , our larC. communis could be significantly determined by the interplay and balance between local genetic drift and gene flow. The flowers of C. communis lack nectaries, and generalist pollinators, such as flies and bees, have been reported as their main pollinators . Several populations shared alleles with other populations in spite of their far geographic distance. For example, individuals from population ZH located in north-China had inferred ancestry from east-central and central China. Similarly, mixed inferred ancestry was also found in population LFZ . The population LFZ from central China had a close genetic relationship with east-central China populations . There was an overlap of population LFZ from non-metalliferous soils with metalliferous population JKL from east-central China , indicating that intensive gene flow might occur. Moreover, the AMOVA results showed that geography only accounted for 10% of the total genetic variation in our study (Table 3). Thus, all these results suggested that gene flow might play an important role in maintaining high genetic diversity in populations of C. communis. In addition, frequent nuclear DNA mutations in the M populations also could increase the population genetic diversity and reverse the initial founder and bottleneck effects , suggesting that genomic mutations may enhance the levels of genetic diversity in M populations of C. communis. However, we have to keep in mind that our study was based on only a small set of randomly chosen microsatellites. Because SSRs are neutral markers  and may also be in disequilibrium with Cu selected gene, such mutations could be just replenishing genetic diversity in neutral regions of the genome and the necessary mutations for coping with Cu might be still at low frequency in M populations. Therefore, the overall genomic effect of the bottleneck might be small and quickly erased by gene flow from nearby NM populations or from other M populations. It also should be pointed out that C. communis is an out-crossing species, which can produce a lot of seeds every year. Each plant produces 5–80 bisexual flowers and each fruit has 2 elliptic, or 1 elliptic and 2 semi-elliptic, or 4 semi-elliptic seeds , while that derived from the group of contaminated vs. non-contaminated populations was not significant . Both Mantel and MMRR analyses also indicated that the geographic distance rather than soil composition had a strong influence on genetic differentiation among populations of C. communis . In addition, M and NM populations were not clearly separated, but divided into three distinct clusters, broadly corresponding well with their geographical locations rather than edaphic types. Geographically adjacent C. communis populations showed relatively low genetic divergence, compared to populations far apart  and their extractable fractions (1196 ± 688) at metalliferous sites in Table 1), which may increase diverging selection and reduce gene flow between their study M and NM populations of C. communis. Moreover, ecogeographic distance has long been regarded as the most important factors to affect the gene flow in plants . The population structure of C. communis was analyzed at a relatively small geographic scale in the previous research, concentrated and mainly limited in eastern China  exert more influence than geographic distance on the genetic structure of pseudometallophytes . In contn plants . Althougrn China . The releography .C. communis, the geographically disjunctive M populations in our study clearly distributed into three distinct genetic clusters rather than one . All populations of C. communis from Cu-contaminated soils have closer genetic relationships with geographically adjacent NM populations than other distant conspecific M populations . Thus, our data indicated that the widely geographic distribution of M populations in C. communis today more likely evolved independently from nearby NM populations rather than from one single tolerant ancestral population (Because the metalliferous sites are often fragmented, the hypothesis of multiple and independent origins of M populations has been proposed and validated in some pseudometallophytes . For C. pulation .C. communis. The populations of C. communis from metal contaminated sites have not experienced apparent historical founder and bottleneck events during their recent colonization. The high levels of genetic diversity in the M populations of C. communis may be resulted from extensive gene flow between neighboring NM populations or geographically close M populations, high frequency of genetic mutations, and/or a high number of tolerant plants in the initially established populations. The patterns of genetic structure in C. communis were primarily influenced by geographic distance rather than soil composition. Thus, we speculate that geography plays a more important role than edaphic condition in shaping the genetic structure of C. communis. In addition, the widely distributed M populations of C. communis may have evolved independently from multiple origins.This study clearly demonstrated that the extreme edaphic conditions in metalliferous areas had limited effects on the genetic variability in JL and WZ designed the study. HX and YS collected the data. JL, LT, YG, YL analyzed the data. JL and WZ drafted the manuscript. LS, RY, and XW critically revised the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fagus hayatae is an important relict tree species in subtropical China, whose biogeographical patterns may reflect floral responses to climate change in this region during the Quaternary. Previous studies have revealed phylogeography for three of the four Fagus species in China, but study on F. hayatae, the most sparsely distributed of these species, is still lacking. Here, molecular methods based on eight simple sequence repeat (SSR) loci of nuclear DNA (nDNA) and three chloroplast DNA (cpDNA) sequences were applied for analyses of genetic diversity and structure in 375 samples from 14 F. hayatae populations across its whole range. Both nDNA and cpDNA indicated a high level of genetic diversity in this species. Significant fixation indexes and departures from the Hardy–Weinberg equilibrium, with a genetic differentiation parameter of Rst of 0.233, were detected in nDNA SSR loci among populations, especially those on Taiwan Island, indicating strong geographic partitioning. The populations were classified into two clusters, without a prominent signal of isolation‐by‐distance. For the 15 haplotypes detected in the cpDNA sequence fragments, there was a high genetic differentiation parameter (Gst = 0.712) among populations. A high Gst of 0.829 was also detected outside but not within the Sichuan Basin. Consistent with other Fagus species in China, no recent population expansion was detected from tests of neutrality and mismatch distribution analysis. Overall, genetic isolation with limited gene flow was prominent for this species and significant phylogeographic structures existed across its range except for those inside the Sichuan Basin. Our study suggested long‐term geographic isolation in F. hayatae with limited population admixture and the existence of multiple refugia in the mountainous regions of the Sichuan Basin and southeast China during the Quaternary. These results may provide useful information critical for the conservation of F. hayatae and other Chinese beech species.The beech species  Fagus L. had a widespread distribution across the continents of the Northern Hemisphere during the Tertiary  using chloroplast and nuclear genome sequences, but the bias against analyses of the chloroplast genome weakened the phylogeographical analyses, and the demographic history of this species remains elusive.nd Hsieh , and witnd Hsieh . Researco et al. , who deto et al.  analyzedF.  hayatae in China and attempted to address the following three questions: (1) What is the spatial genetic structure in F. hayatae populations? (2) Where were the potential glacial refugia for F. hayatae? (3) What can be implied about the demographic history of F. hayatae? The results of this study should provide critical insights for understanding the distribution and history of Chinese beeches in the subtropical region, as well as the relationships among different Fagus species.In this study, we applied microsatellite markers of nuclear DNA (nDNA) and chloroplast DNA (cpDNA) sequence fragments to examine phylogeography of F. hayatae populations were implemented during July–September 2012, covering the whole distribution range of the species. A total of 375 individuals in 14 populations were sampled  or 58°C (sfc0378 and sfc1143) or 60°C  and 2 min at 72°C, with a final 10 min at 72°C. Amplification products were checked by capillary electrophoresis in an ABI 3730xl sequenator . Allele sizing was performed with GeneScan™ 500 LIZ® size standard (ABI) and assigned with Allelogram v2.2 .A preliminary screening of microsatellite markers, or simple sequence repeat (SSR) markers, was performed using 40 SSR primers with 8% nondenaturing polyacrylamide gel electrophoresis for separation, as described by Tanaka et al. , Asuka eA)  (Nei Rst)  Nei  over allely) Nei  over allely) Nei  were carely) Nei , the nulely) Nei . Among pK) with a burn‐in period of 105 steps followed by 106 replicates. The optimum was detected by calculating the parameter ΔK  approach over all loci was performed for individual clustering under a model assuming admixture and noncorrelated allele frequencies using Structure v2.1  were selected after a screening of 20 primers described by Taberlet et al. , 1 μL each of forward and reverse primers (10 μmol/L), 1 μL of template DNA (about 100 ng), and 7 μL of ddH2O. Amplifications were carried out using the same PCR cycler as those for SSR amplification in section For amplification and sequencing of cpDNA, three polymorphic primers  and aligned using Clustal X v1.81 . A statistical parsimony network of haplotypes was generated using TCS v1.21 with a median‐joining algorithm using a maximum‐parsimony (MP) approach  with parameters sampled every 1000 steps. Based on 1000 replicates, the bootstrap support values of the tree nodes resulting from maximum‐likelihood (ML) approaches with Akaike information criterion and Bayesian information criterion (BIC) procedures, with the best substitution models TPM3uf and F81, respectively, inferred by jModelTest 2  and average number of pairwise differences between haplotypes (π) (Nei and Li Gst) making use of haplotype frequencies was also used (Pons and Petit r) could be estimated with FI over all loci among populations, Rst and Gst Nst, a measure of genetic differentiation taking into account the distance between haplotypes, was estimated and compared with Gst using the program PermutandCpSSR v2.0  .The values of K value . Populations S1, S2, C1, E1, E3, Z1, and Z2 were clustered into one group and the others into another group, but there was mismatching between populations and genetic groups for some individuals . Haplotype H1 was fixed in the populations sampled in the Sichuan Basin, and the haplotypes fixed in the populations in Taiwan Island were more diverse. Additionally, a major specific haplotype was fixed with high frequency in most populations  indicated not only many interior nodes but also many missing intermediate haplotypes  were lower than those outside ; and values among all 14 populations  were higher than the mean   but little for those inside. The relative rate of pollen over seed flows (r) was 10.6 among the 14 populations.The results showed significantly high genetic differentiation among all 14 populations with Nst values  than Gst values  (P < 0.001). However, there was no significant difference between Nst (0.009) and Gst (0.005) among populations in the Sichuan Basin (P > 0.1).Phylogeographic structures were detected among all 14 populations, and populations outside the Sichuan Basin, with correspondingly significantly higher D and Fu's Fs tests of neutrality were not significant among all 14 populations and for populations outside the Sichuan Basin, while there were conflicting results between the two tests among populations within the Sichuan Basin (Table S3). However, all the mismatch distributions were roughly multimodal , suggesting no signals of population expansion in the analysis.The results of Tajima's A) was 21.75 among the 14 F. hayatae populations  must also result from isolation and fragmentation by mountains in subtropical China. It is noteworthy that F. hayatae had Rst = 0.233, comparable to other Fagus species distributed in China . Two clusters each with mixed populations in different locations and many mismatched individuals also indicated that genetic distance was not correlated with geographic distance Fig. . It was F. hayatae (Gst = 0.712), as well as of other Fagus species  suggested that these populations may have had a single origin and long existed within an enclosed environment, that is the Sichuan Basin.The results of tests between F. hayatae and other Fagus species and that population T1 and populations in the Sichuan Basin were more ancient elements of F. hayatae. Zhang et al.  plant species assembled in the mountains of subtropical China. Ying  and geographical distance (Dist).Figure S2. Relationship of ΔK with the number of clusters (K).Figure S3. Mismatch distributions for populations (a) inside and (b) outside the Sichuan Basin, and (c) over the 14 populations, with black lines representing the distributions expected for expanding populations and red lines representing the observed mismatch distance.Click here for additional data file.


Ipomoea hederacea is a self-compatible species whose geographic origin is contested, and previous work suggests that although there are signals of adaptation (significant leaf shape and flowering time clines), no population structure or neutral genetic differentiation of I. hederacea populations was detected. Here, we use DNA sequence data to characterize patterns of genetic variation to establish a more detailed understanding of the current and historical processes that may have generated the patterns of genetic variation in this species. We resequenced ca. 5000 bp across 7 genes for 192 individuals taken from 24 populations in North America. Our results indicate that North American I. hederacea populations are ubiquitously genetically depauperate, and patterns of nucleotide diversity are consistent with population expansion. Contrary to previous findings, we discovered significant population subdivision and isolation-by-distance, but genetic structure was spatially discontinuous, potentially implicating long-distance dispersal. We further found significant genetic differentiation at sequenced loci but nearly fourfold stronger differentiation at the leaf shape locus, strengthening evidence that the leaf shape locus is under divergent selection. We propose that North American I. hederacea has experienced a recent founder event, and/or population dynamics are best described by a metapopulation model , leading to low genetic diversity and a patchy genetic distribution.Disentangling the historical evolutionary processes that contribute to patterns of phenotypic and genetic variation is important for understanding contemporary patterns of both traits of interest and genetic diversity of a species.  For example, the advance and subsequent retreat of glaciers ~20,000 years ago has played a major role in shaping the geographical distributions and patterns of genetic variation of many plants and animals in North America  Is there evidence of hierarchical population structure and genetic differentiation at the nucleotide level? (2) How genetically diverse are I. hederacea populations, and are there geographical patterns associated with nucleotide diversity? (3) What are the potential historical processes that have generated the patterns that we observe now?In the current study we use Sanger sequencing to collect DNA sequence data for a large sample of individuals from many populations in Ipomoea hederacea (L.) Jaquin (Convolvulacaea) is an herbaceous weedy vine that has a NA range spanning from the southern United States  up through the Great Lakes region  was flash frozen in liquid N2, lysed with two 6-mm glass beads at a frequency of 30 Hz for 30 sec, and then genomic DNA was extracted and purified using a QIAGEN DNeasy Plant Mini kit; we used Tris-EDTA (pH 8.0) heated to 65° to elute DNA rather than the elution buffer provided. All DNA samples were quantitated using a Qubit and diluted to a final concentration of 10 μg/mL. We randomly selected seven nuclear genes from an unannotated transcriptome of I. hederacea . Primers were designed using a combination of Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and NetPrimer (http://www.premierbiosoft.com/netprimer/index.html) to amplify short fragments (~500−800 bp) from within these nuclear genes; although the sequences we use do not represent entire genes, for simplicity, we refer to them as nuclear loci throughout. A list of primer sequences can be found in Supporting Information, Table S1.A ~4-cmTaq buffer, 2.6 mM MgCl2, 0.25 mM dNTPs, 0.25 mM forward and reverse primers, 1U Fermentas Taq polymerase [Fisher Scientific], and 20 ng DNA template) on an Eppendorf Mastercycler using the following polymerase chain reaction (PCR) protocol; 2 min at 95°, then 45 sec at 95°, 30 sec at 55° (−0.2° each cycle), 1 min at 72°, for 30 cycles, then 30 sec at 95°, 30 sec at 49°, 1 min at 72°, for 10 cycles, with a final extension of 10 min at 72°. PCR products were verified on 1.5% ethidium bromide stained agarose gels and then purified by adding 2 U of Fermentas SAP and 4 U of Fermentas ExoI to the remaining PCR products, and incubating them for 30 min at 37°. Sequencing was carried out in 10-μL reaction volumes  using the following protocol; 1 min at 96°, then 10 sec at 96°, 5 sec at 50°, 4 min at 60°, for 45 cycles, then 4 min at 60°. Sequences were obtained using an Applied Biosystems 3730 DNA Analyzer by the Centre for the Analysis of Genome Evolution and Function (University of Toronto).All seven loci were amplified in 20-μL reaction volumes , and edited all chromatographs and verified polymorphic sites (both single-nucleotide polymorphisms [SNPs] and heterozygous sites) manually to ensure robust scoring. Each individual haplotype was reconstructed from unphased diploid sequences using PHASE . We deteI. hederacea. InStruct uses a Markov Chain Monte Carlo algorithm to probabilistically assign individuals to a cluster based on their genetic profiles, and searches for the optimal number of genetic clusters (K) to describe the data. We explored K = 1 to K = 20 clusters and determined the optimal number of clusters based on the deviance information criterion (We previously explored population structure using dominant amplified fragment-length polymorphisms (AFLP) markers , but we FST, and the linearized-FST transformation (LinFST = FST/(1 – FST); Rxy) for the three pairs of matrices , and used Mantel tests consisting of 9999 permutations to generate a null distribution of Rxy for each pair of matrices, for which the observed Rxy was then compared to for statistical significance.We examined whether our populations displayed IBD . We usedI. hederacea populations in NA. To do this, we calculated pairwise nucleotide differences . We first describe the levels of nucleotide diversity both within and across nces θπ;  and the s θπ; w;  for eachs θπ; w; . To tests θπ; w; , and thes θπ; w; ; locus nπ; w; FS  and Ramow; FS  in two ways: First, we regressed the within-population means of diversity against geographic location using both latitude and longitude as independent variables. Second, we split the populations into north and south groups (12 north and 12 south populations) depending on their geographic location with respect to the clinal boundary , we calculated pairwise LD (r2) between all SNPs using the ‘genetics’ .Clustering did not correspond to geographic location ; individRxy = 0.072, P = 0.10; FST , with individual populations exhibiting a range of diversity levels ; latitude and longitude reveal similar results, likely because they are significantly correlated for our populations . Our results show that leaf shape genotype was likewise not a good predictor of nucleotide diversity , in agreement with our results for Tajima’s D. We detected a significant deviation from neutrality in southern populations, but not in the north . Four loci showed significantly negative Tajima’s D, and one showed positive Tajimas’ D, while the remaining two did not deviate from neutral expectations (Table S2).We found that Tajima’s D did not deviate from neutrality for any individual population . Howeverhe north . Becausehe north . Heterozhe north ; howeverFIS = 0.86, P < 0.0001), yielding an elevated population-wide estimate of selfing . An analysis of molecular variation of all populations confirmed a lack of admixture; 72% of the molecular variation is partitioned among individuals, 16% partitioned among populations, and the remaining 12% within individuals.A global estimate of Wright’s inbreeding coefficient revealed a high level of inbreeding . Individual SNP sites , which are directly comparable with the leaf shape locus, exhibit a range of FST values, with about 50% showing significant genetic differentiation . After removing these, we found that the distribution of FST nearly mirrored that for all SNPs , so we base our interpretations on the whole set. Despite a strong overall signal of global genetic differentiation at SNPs, FST at the leaf shape locus was nearly four times greater  and was in the 96th percentile of the range of FST values for the SNPs. At the leaf shape locus, 40% of the variation was partitioned within populations, 57% among populations and the remaining 3% within individuals.A multilocus estimate of global genetic differentiation revealed significant population structure with ntiation . We deteI. hederacea, we assessed nucleotide diversity across ~5 kb of DNA sequence for a large representative NA sample. Our analyses revealed three major findings: First, we detected significant population genetic structure, but genetic clusters were patchily distributed and not associated with geography or leaf shape. Second, we found that levels of nucleotide diversity are overall low, and patterns are consistent with population expansion . Third, we found significant genetic differentiation at sequenced loci, but this was not enough to explain the much stronger differentiation at the leaf shape locus. Below we highlight two potential demographic models that are consistent with our sequence data—a founder event associated with colonization of NA, and metapopulation dynamics—and describe the challenge of detecting genetic evidence for each hypothesis. Finally, we address how our current results strengthen our previous interpretation of an adaptive leaf shape cline.To understand the historical processes that may have been responsible for patterns of phenotypic and genetic variation in I. hederacea may potentially indicate long-distance dispersal (LDD). For example, four of the populations we sampled are mostly assigned to their own genetic cluster . These four populations are all in closer geographic proximity to one of the other 20 populations than they are to one another, suggesting potential LDD events, in agreement with past suggestions of agriculturally mediated dispersal  were distributed in clumps in areas of recolonization after the last glacial retreat in Europe, which potentially suggests that I. hederacea populations are the result of random colonization events. However, cases of such irregular genetic subdivision are rare  compared to within individuals (12%). These results are additionally supported by the high average selfing rates of 92% and 93.7% (depending on how it is estimated) that we found. Our findings are in agreement with previously documented selfing rates using a combination of progeny arrays  using allozyme markers . Combined, these findings suggest a general lack of outcrossing, which may contribute to the discontinuous population structure of I. hederacea.Long-distance dispersal is expected to reduce overall IBD by facilitating gene flow and thus eroding the buildup of genetic differentiation. Our results only partially support the notion of reduced IBD under a model of rare LDD events; pairwise Nei’s D and cant IBD . In addiays 93%; , or F-ste.g., I. hederacea  It recently colonized NA and (2) NA populations are exhibiting metapopulation dynamics. The particularly low level of genetic variation, and lack of geographical patterns in diversity, may be explained by a recent and severe founder event because we would otherwise predict the buildup of neutral molecular variation beyond the observed levels and expect to detect reductions in diversity along the range expanding front , leading to a high-turnover rate. These contemporary forces may be causing I. hederacea populations to behave more akin to a single large metapopulation, with high extinction and colonization rates driving the genetic diversity and structure patterns we observe throughout its eastern NA range. Under this scenario, recent founder events associated with colonization are not a necessary requirement of the metapopulation hypothesis, because frequent and strong bottlenecks may reduce and maintain a minimal number of haplotypes.Alternatively, populations of I. hederacea’s range will be necessary. However, I. hederacea’s major center of occurrence is in the eastern United States, whereas its most closely related congener I. nil is found predominantly in Mexico and parts of South America (http://www.gbif.org). Although some analyses suggest taxonomic/phylogenetic uncertainty between these two species  and adaptation . Future work that investigates geographic patterns of genetic variation in the chloroplast genome of I. hederacea (chloroplast DNA is maternally inherited through seed only, I. hederacea.Our findings on patterns of neutral polymorphism and structure, combined with previous work on life-history and morphological traits (