Prunus sibirica L.), an ecologically and economically important tree species with a high degree of tolerance to a variety of extreme environmental conditions, is widely distributed across the mountains of northeastern and northern China, eastern and southeastern regions of Mongolia, Eastern Siberia, and the Maritime Territory of Russia. However, few studies have examined the genetic diversity and population structure of this species. Using 31 nuclear microsatellites, we investigated the level of genetic diversity and population structure of Siberian apricot sampled from 22 populations across China. The number of alleles per locus ranged from 5 to 33, with an average of 19.323 alleles. The observed heterozygosity and expected heterozygosity ranged from 0.037 to 0.874 and 0.040 to 0.924 with average values of 0.639 and 0.774, respectively. A STRUCTURE-based analysis clustered all of the populations into four genetic clusters. Significant genetic differentiation was observed between all population pairs. A hierarchical analysis of molecular variance attributed about 94% of the variation to within populations. No significant difference was detected between the wild and semi-wild groups, indicating that recent cultivation practices have had little impact on the genetic diversity of Siberian apricot. The Mantel test showed that the genetic distance among the populations was not significantly correlated with geographic distance . Our study represents the most comprehensive investigation of the genetic diversity and population structure of Siberian apricot in China to date, and it provides valuable information for the collection of genetic resources for the breeding of Siberian apricot and related species.Siberian apricot ( Prunus sibirica L.), an ecologically and economically important tree species, is widely distributed across the mountainous areas of northern and northeastern China, eastern Siberian, and Mongolia Siberian apricot  and leaf roller (Adoxophyes honmai) have made the originally fragile natural environment even worse In recent decades, almond products have become increasingly popular on the domestic and international market. Consequently, many almond processing plants have been established around the major areas of production in China. However, Siberian apricot resources are declining due to backward management patterns and deterioration of the natural environment Increased knowledge of the genetic diversity and population structure of Siberian apricot in China will provide the basis for protecting, utilizing, and improving our resources. Therefore, an assessment of the extent and nature of the genetic variation in Siberian apricot is important for breeding and genetic resource conservation programs. Traditionally, genetic diversity has been assessed based on morphological characteristics, which are often influenced by the environmental conditions. With the advent of molecular markers, including restriction fragment length polymorphisms, amplified fragment length polymorphisms, simple sequence repeats (SSRs), and single nucleotide polymorphisms, much progress has been made in understanding the genetic diversity and population structure of various species In this study, 31 nuclear SSR loci developed previously for this species A total of 672 individuals of Siberian apricot representing 22 populations were collected throughout the areas of distribution in China . A totalIn China, Siberian apricot has been cultivated for decades in an experimental forest. Currently, the main method of propagation is to sow seeds collected from the immediate area or near the region without selection. Three such populations were collected and designated as semi-wild type. All other populations were from the wild . Young lTotal genomic DNA was extracted from dry leaves collected from all localities using a modified version of the cetyl trimethylammonium bromide method Prunus armeniaca L.) Prunus persica L.) 5′-TGTAAAACGACGGCCAGT-3′) during synthesis. Amplification was performed in a 10-µL reaction mixture containing 1 µL of DNA template (10 ng/µL), 5 µL of 2X Taq mix, 0.4 µL of the forward primer (1 µM), 1.6 µL of the reverse primer, 1.6 µL of M13 primer (1 µM) with a fluorescent label , and 1.4 µL of ddH2O. The reaction conditions were: 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by 8 cycles of 94°C for 30 s, 53°C for 40 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. The products were separated in an ABI 3730×L DNA Analyzer using GeneScan-500LIZ as an internal marker . The amplicon fragments were sized using Gene-Marker 1.75 software . All rare alleles and private alleles were re-amplified. For the alleles from the homozygous loci, the purified PCR products were sent to sequence. For the alleles from the heterozygous loci, the targeted fragments were separated, cloned and sequenced following the protocol by Chen et al Thirty-one microsatellite loci were employed to study the genetic diversity on wild Siberian apricot accessions including 23 recently developed in Siberian apricot IS, FIT, and FST).FLEXBIN was used for automated binning of the microsatellite raw data Clustering based on a Bayesian model was used to evaluate the genetic structures of the Siberian apricot populations with the software package STRUCTURE ST and RST to calculate the genetic differentiation of all population pairs. The values of FST and RST were calculated using FSTAT version 2.9.3 ST/(1−FST) estimates http://www.movable-type.co.uk/scripts/latlong- vincenty.html), were tested using the Mantel test implemented by Isolation By Distance Web Service version 3.23 (http://ibdws.sdsu.edu/~ibdws/) The observed genetic variation among and within the populations and genetic groups was characterized by an analysis of molecular variance (AMOVA) using ARLEQUIN version 3.5 ST value of 0.071 across all loci, indicating moderate genetic differentiation among the sites. The values of gene flow ranged from 1.869 to 8.224, with an average of 3.595. Most loci conformed to HWE and no population had a particularly large number of loci that deviated from HWE.A total of 31 microsatellite loci were used to genotype 672 individuals of Siberian apricot . The genThe genetic structure of the Siberian apricot samples was investigated by a Bayesian-based population assignment analysis using STRUCTURE At the same time, the second largest ΔK at K = 7 was much larger than the remaining values. In addition, two clear peaks were observed at K = 10 and 14 . When K The population genetic parameters used in this study are summarized in The value of Na for the wild genotypes was significantly higher than that for the semi-wild genotypes . The numThe Ho and He values in genetic cluster C3 were slightly larger than those in the other genetic clusters  whereas A comparison of private alleles between the wild (152) and semi-wild (11) populations showed a significant difference between them . When alST value (0.06008). Seven genetic clusters revealed the highest percentage of variation (3.48%) among them, and it produced the second largest FST value (0.06002). With the populations grouped according to their geographical origin, a lower percentage of variation (1.98%) could be explained by geographic factors. When the populations were grouped according to their origin, a negative percentage of variation was detected among the groups.Our AMOVA revealed that a low percentage of variation was divided among natural populations, different origins, geographical distribution, and genetic clusters, respectively . About 9ST and RST) calculated for the 22 populations showed genetic differentiation between each population . The lowest values of FST were observed between P1-P2, P1-P3, P1-P4, P1-P17, P2-P4, P4-P5, P4-P17, P5-P17, P11-P12 and P13-P14. Populations from different genetic clusters appeared to be more differentiated from each other, corresponding well to the classification of the genetic cluster. Of the RST values, 10 (8 of which were from paired populations coming from different clusters) were not significantly different from 0. The pairwise genetic differentiation values (FST) between the four genetic clusters showed a higher genetic differentiation between three population pairs   .ST values were observed for pairwise comparisons between P16 and other populations. The geographic distance matrix ranged from 35.8 to 1,526.5 km, based on the latitude and longitude values of all 19 wild populations. Rousset’s genetic distance values [FST/(1–FST)] The highest FA genetic barrier prediction analysis using Monmonier’s maximum difference algorithm identified three putative barriers when all populations were included . The firPrunus armenica L.; Ho = 0.615, He = 0.621) Amygdalus nana L.; Ho = 0.339, He = 0.219) is reportedly even lower Heterozygosity is an important measurement of gene diversity Cinnamomum insularimontanum Hyata (Lauraceae) from southern Korea, a heterozygote deficiency was explained as a process of partial selfing rather than the presence of null alleles or a temporal Wahlund effect. A deficiency of heterozygotes in the tropical species Sextonia rubra (Mez) van der Werff was explained as an effect of biparental breeding due to limited pollen dispersal among relatives The Ho value was lower than the He value at all 31 loci , indicatAn AMOVA revealed that genetic variation within populations accounted for about 94.4% of the total . OutcrosST instead of RST when calculating the genetic differentiation among grapevine populations. Compared with RST, FST was more consistent with our other analysis. Though all the genetic differentiation between pairwise populations was significant, the lowest values of FST still could be observed between P1-P2, P1-P3, P1-P4, P1-P17, P2-P3, P2-P4, P4-P5, P4-P17, P5-P17, P11-P12 and P13-P14. An UPGMA dendrogram based on Nei’s unbiased genetic distance showed these population pairs had the shortest genetic distance  and an infinite-allele model, respectively. R-statistics was developed to take into account the high homoplasy inherent in microsatellite markers distance . In addie region , except  and P14  is not s and P14  supportsST values). This provided a new means of identifying the true optimum number of subsets. The result of AMOVA showed that the maximum FST value (0.06008) when all populations were grouped into four genetic clusters , whitebellied rat (Niviventer confucianus), striped field mouse (Apodemus agrarius), and other rodents feed and store Siberian apricot seeds, which makes long-distance gene flow possible and improves the level of genetic diversity.In this study, STRUCTURE identified four main genetic groups (clusters) . All genAccording to the results of our structure analysis, most of the populations that were geographically close were generally clustered into the same cluster. An analysis based on the Mantel test  showed tP16 was separated by the first predicted barriers, regardless of whether the three semi-wild populations were included or not . HoweverOur studies show a relatively high level of genetic diversity among Siberian apricot populations in China. However, a significant deficiency in heterozygotes was detected at the locus and population levels, which may be the result of low-level inbreeding. Our structure analysis clustered all of the populations into four genetic clusters. There was no significant difference between the wild and semi-wild groups, indicating that recent cultivation practices have had little impact on the genetic diversity of Siberian apricot. Our study represents the most comprehensive investigation of the genetic diversity and population structure of Siberian apricot in China and will provide valuable information for the collection of genetic resources for the breeding of Siberian apricot and related species.Figure S1UPGMA dendrogram of Siberian apricot populations based on Nei’s unbiased genetic distance.(TIF)Click here for additional data file.Table S1Primer information for 31 microsatellite loci used to analyze 672 Siberian apricot samples.(DOC)Click here for additional data file.Table S2Mean ancestry values for the four genetic groups inferred by STRUCTURE.(XLS)Click here for additional data file.Table S3Pairwise estimates of FST values based on data from 31 SSR loci among the model-based clusters inferred by Structure. Significant values at the 1% nominal level are bolded.(DOC)Click here for additional data file.


FST of 0.716), which exceeds the highest values reported in freshwater fish. All locations showed low levels of genetic diversity as evidenced by low heterozygosities and a mean of only 2 alleles per locus, with few or no rare alleles. Many loci showed a discontinuous allele distribution, with missing alleles across the allele size range, suggestive of a population contraction. Accordingly, bottleneck episodes were inferred for all samples with a reduction in population size of 3–4 orders of magnitude. The reduced level of genetic diversity observed in all populations implies a strong impact of genetic drift, and suggests that along with limited gene flow, genetic differentiation might have been exacerbated by recurrent mortalities likely caused by flash flood and debris flows. Due to its low evolutionary potential the species might fail to cope with an intensification and altered frequency of flash flood events predicted to occur with climate change.A changing global climate can threaten the diversity of species and ecosystems. We explore the consequences of catastrophic disturbances in determining the evolutionary and demographic histories of secluded marble trout populations in Slovenian streams subjected to weather extremes, in particular recurrent flash floods and debris flows causing massive mortalities. Using microsatellite data, a pattern of extreme genetic differentiation was found among populations (global  Climate change poses a serious threat to species persistence. Many species will experience selection in new directions and at new intensities, and the degree to which species respond adaptively to dynamic and uncertain futures will determine their survival over the coming decades and millenia Freshwater salmonids are commonly subject to substantial environmental variability in the form of changes in stream flow at different time scales Salmo marmoratus, an endemism of the Adriatic Sea and its tributaries, currently restricted to Northern Italy, former Yugoslavia  and Albania Our model system is the stream-dwelling salmonid marble trout th century, and with a presumable occurrence interval of approximately 50 years , mostly due to the loss of rare alleles , Lipovscek , Sevnica  and Studenc .A total of 18 out of 24 loci were polymorphic, showing from 2 to 8 alleles per locus . All loc alleles . HoweverHo , He  and AR .All loci were at HWE after Bonferroni correction. A neutrality test using LOSITAN suggested no loci to be subject to balancing selection or directional selection. No difference in values of genetic diversity was found between EST-derived microsatellites and genomic microsatellites, including FST = 0.716; p<0.001). All pairwise FST and genetic distances between samples from different locations were high  and statistically significant . Accordingly, comparison of allele frequencies between pre- and post-flood samples using an exact test showed no temporal differences at Lipovscek (p = 0.993), Sevnica (p = 0.450) and Zadlascica (p = 0.985), while significant differences were found at Studenc (p<0.001) caused by loci CA059136 and Str151.A highly significant extreme overall genetic differentiation was found among samples  but not among (pre- and post-flood) samples within locations   . We condall loci . When pl10(r) were substantially below zero in all eight samples, suggesting that the past population size was larger than the current population size (10(θ) were negative, pointing to small current effective population sizes. Using a conservative mutation rate of μ = 10−4, N1 varied across locations between 3 and 41 individuals, with a maximum upper-bound CI of N1 of 396 individuals (−4 and 10−3), N1 values <than 1 were obtained, while lower mutation rates (i.e. 5×10−5) yielded unrealistic N0 values. The time of the bottleneck varied between 31 and 268 generations across locations (or roughly 100–800 years assuming a generation time of 2.7–3 years), with a maximum upper-bound CI of 2300 generations (roughly 7000 years).The MSVAR procedure for assessing past demographic history strongly supported a decline in all marble trout populations. All sampled points of logion size . All samividuals . Using hBottleneck-detection tests  using thSalvelinus fontinalis, a global FST of 0.37 was found among 26 populations from La Maurice National Park in Canada located 3–42 km apart Oncorhynchus clarkii, Taylor et al.FST of 0.32 and pairwise FST values up to 0.45 in populations from British Columbia, while the recent study of Pritchard et al.FST of 0.41 for Rio Grande populations. In bull trout Salvelinus confluentus, Taylor and Costello FST = 0.33 among 20 North-West America coastal locations, while recent papers reported an overall FST of 0.15 and pairwise FST values up to 0.31 in Alberta FST values up to 0.66 between lake samples in a 50 km area off Montana Freshwater fish species show a greater average degree of genetic differentiation among locations than marine species, resulting from the isolation of fish populations among drainages FST = 0.716; pairwise FST between drainages = 0.649–0.779; 49% private alleles sampled) exceeds the highest values reported in salmonid populations found above waterfalls and/or in small streams like the ones in this study. Such extremely high FST is indicative of complete genetic isolation that has persisted over time. This is concordant with the previous study of Fumagalli et al.FST of 0.66 in the same area using a smaller number of microsatellites. At present, the four locations in our study are completely isolated from each other and from the main river drainage by means of impassable natural barriers  that restrict dispersal abilities. Baloux and Lugon-Moulin Collectively, the extreme level of genetic differentiation found among Slovenian marble trout populations . The different pattern suggested for Italian and Slovenian populations of marble trout, with signatures of a bottleneck only observed in the latter, might be due to the particular geography and the extreme effect of meteorological events in the region of Slovenia studied. Rainfall data have been acquired since 1961 . While the annual mean precipitation was 2,400 mm in the 1961–2004 period, showing little variation across years, monthly rainfall showed up to 500-fold variation. Morphological features of the streams and watersheds in the region  are largely responsible for the high water flow in the stream after heavy rainfalls, and might explain the massive mortalities caused by flood events.The MSVAR procedure clearly suggested a population contraction with a reduction in population size of 3–4 orders of magnitude to current effective densities of around 3–41 (CI: 1–396) individuals per location. Bottleneck signatures were observed in all samples. First, most polymorphic loci presented a biallelic pattern with few (or none) rare alleles. Second, a discontinuous allele range was found at many loci . For inset al.In contrast with the extreme differentiation found at microsatellite loci, one single mitochondrial DNA control region haplotype (MA1) has been reported for marble trout in Slovenia The analysis of pre- and post-flood samples in our study allowed us to explore the genetic consequences of population bottlenecks. The effects of bottlenecks are directly related to the increase of stochastic events associated with small population sizes, leading in most cases to a loss in genetic variability The moderate reduction of allelic diversity and heterozygosity might be attributable to the particular demographic history of those populations. In his recent review, Bouzat On the question of how do the Slovenian population still maintain some polymorphism despite experiencing flood events for hundreds of years, one possible explanation is that our sampling is not representative of the entire population, and other compartments of the population might keep some genetic variation. One hypothesis could be that young fish (age 0+ and 1+) might be less sensible to flows because usually they are not found in the main stream but in more protected small tributaries, repopulating the stream even if all adults have been removed. However, field observations suggest that eggs and young fish are more vulnerable to floods than adults and that there is no recruitment at all when floods occur after reproduction . Alternatively, an upstream part of the population could be preserved. This hypothesis is supported by tagging data  that show non-tagged individuals in post-flood samples, whereas all pre-flood samples had been tagged, which suggests that the newly-arrived non-tagged individuals came from a compartment of the population located upstream, flushed by the flood.et al.Our findings strongly suggest that the evolutionary and demographic history of marble trout populations living in secluded Slovenian streams has been shaped by massive mortalities caused by disturbance factors such as flash flood and debris flows. Overall, genetic analysis revealed an extremely high genetic differentiation among remnant pure populations, together with much low levels of genetic diversity in all populations. Effective population sizes estimated using the MSVAR procedure ranged from 3 to 41 (CI:1–396) individuals per population. The low genetic variability found does not seem to affect the viability of the populations so far, as they have persisted up to the present despite recurrent and unpredictable disturbances. To mitigate their ecological impact, fish populations might exhibit several adaptations that result from trade-off among growth, reproduction and survival. According to life-history theory, recurrent floods can have important evolutionary consequences by selecting for life-histories that are synchronized to either avoid or exploit the direct and indirect effects of extreme flows Collectively, while the Slovenian marble trout populations have coped successfully with recurrent yet unpredictable disturbance events over time, the low genetic variation found in the species makes it difficult to assess its evolutionary potential since a genetically depauperate population might fail to adapt to future environmental change. Moreover, all populations are closed, and there is no potential for spontaneous colonization of new habitats or re-colonization after local extinction. It has been proposed that an effective population size of 500 individuals is large enough to maintain genetic diversity for key history traits All animal work was approved by the Ministry of Agriculture, Forestry and Food of Republic of Slovenia and the Fisheries Research Institute of Slovenia. Original title of the Plan: RIBISKO - GOJITVENI NACRT za TOLMINSKI RIBISKI OKOLIS, razen Soce s pritoki od izvira do mosta v Cezsoco in Krnskega jezera, za obdobje 2006–2010. Sampling was supervised by the Tolmin Angling Association (Slovenia).Salmo marmoratus individuals were caught using electrofishing at four tributaries of the Soca (1.Zadlascica), Baca (2.Lipovscek) and Idrijca (3.Sevnica and 4.Studenc) rivers in Slovenia  solution pH 5.2 (diluted in 0.08 M NaAC) and 2 µl proteinase K solution (20 mg/ml) for at least 8 hours at 56°C. After incubation at 96°C for 10 min, samples were centrifuged at 13,000 rpm for 11 min, and the supernatant was stored at −20°C.et al.et al.Salmo salar. We selected those loci with the highest genetic variation and a positive cross-species amplification in brown trout Salmo trutta. Additionally, 9 genomic microsatellite loci isolated and characterized from other salmonids, seven from brown trout  using the QIAGEN Multiplex PCR Kit. PCR reactions consisted of 2 µl template DNA, 5 µl QIAGEN Multiplex PCR Master Mix, 0.2 µl 10 µM forward and reverse primers, and water up to 10 µl. PCR conditions were as follows: 3 min at 95°C, 35 cycles of 30 sec at 94°C, 90 sec at 57°C and 1 min at 72°C, and final elongation for 5 min at 60°C. PCR products were visualized in 1.8% agarose gels and screened for microsatellite polymorphism using an ABI 3130 AVANT automatic capillary sequencer (Applied Biosystems). Alleles were sized according to a Liz500 (50–500 bp) marker.Ho) and expected (He) heterozygosities per locus using GENETIX version 4.05 Within-sample genetic diversity statistics were assessed by observed , describing the expected distribution of Wright's inbreeding coefficient FST vs. He under an island model of migration with neutral markers. This distribution is used to identify outlier loci that show excessive high or low FST compared to neutral expectations. Such outlier loci are candidates for being subject to selection. We used the 0.99 criterion in order to minimize false positives as suggested by the authors.Neutrality of the markers was tested using the software LOSITAN F-Statistics FST values were calculated. Genetic differentiation was also partitioned among locations  and among samples within locations (pre- and post-flood samples). Significance tests were assessed with 10,000 permutation tests. In all cases, significance levels were corrected for multiple comparisons using Bonferroni CE) and graphically represented by Multidimensional Scaling (MDS) analysis using STATISTICA version 7.0 (StatSoft). Isolation-by-Distance (IBD) was tested using a Mantel test implemented in GENETIX, by correlating linearized genetic distance (FST/(1−FST)) vs. geographic distance .Population structure was explored using non-hierarchical and hierarchical et al.5 iterations followed by 106 additional Markov Chain Monte Carlo (MCMC) iterations were performed. A minimum of 5 runs were performed for each K to check repeatability of results.A model-based clustering algorithm as implemented in the software STRUCTURE r = N0/N1 , the time since the population started to expand or decline tf = ta/N0 (where ta is the number of generations since the beginning of the expansion/decline) and the genetic parameter θ = 2N0μ. A mutation rate of μ = 10−4 was used 10(r), log10(tf) and log10(θ) and starting values of 1 for θ at each locus, r and tf. The analysis was performed for an exponential model of population change. We used 50,000 thinned updates and a thinning interval of 50,000 steps, with an initial 10% discarded as burn-in. Convergence was assessed using Tracer v1.4 Historical demographic changes were inferred using the Bayesian coalescent-based approach implemented in MSVAR He) at polymorphic loci. Thus, in a recently bottlenecked population, the observed gene diversity is higher than the expected equilibrium gene diversity (Heq) which is computed from the observed number of alleles (k), under the assumption of a constant-size (equilibrium) population et al.crit estimated after 10,000 simulations and assuming the population to be at equilibrium. In all simulations, three different values of θ were used . A range of mutation models were examined and conservative values were used for ps (frequency of one-step mutations) and Δg (average size of non one-step mutations), ps = 0.90 and Δg = 3.5, respectively.Finally, we tested for a recent genetic bottleneck episode with two different approaches. First, we used the software BOTTLENECK Table S1Allele frequencies at all loci. Samples labelled as in (DOC)Click here for additional data file.


Genetic studies of populations from the Indian subcontinent are of great interest because of India's large population size, complex demographic history, and unique social structure. Despite recent large-scale efforts in discovering human genetic variation, India's vast reservoir of genetic diversity remains largely unexplored.To analyze an unbiased sample of genetic diversity in India and to investigate human migration history in Eurasia, we resequenced one 100-kb ENCODE region in 92 samples collected from three castes and one tribal group from the state of Andhra Pradesh in south India. Analyses of the four Indian populations, along with eight HapMap populations (692 samples), showed that 30% of all SNPs in the south Indian populations are not seen in HapMap populations. Several Indian populations, such as the Yadava, Mala/Madiga, and Irula, have nucleotide diversity levels as high as those of HapMap African populations. Using unbiased allele-frequency spectra, we investigated the expansion of human populations into Eurasia. The divergence time estimates among the major population groups suggest that Eurasian populations in this study diverged from Africans during the same time frame (approximately 90 to 110 thousand years ago). The divergence among different Eurasian populations occurred more than 40,000 years after their divergence with Africans.Our results show that Indian populations harbor large amounts of genetic variation that have not been surveyed adequately by public SNP discovery efforts. Our data also support a delayed expansion hypothesis in which an ancestral Eurasian founding population remained isolated long after the out-of-Africa diaspora, before expanding throughout Eurasia. The Indian subcontinent is currently populated by more than one billion people who belong to thousands of linguistic and ethnic groups ,2. GenetThe high diversity and the deep mitochondrial lineages in India support the hypothesis that Eurasia was initially populated by two major out-of-Africa migration routes ,15-17. Pad hoc fashion [Recently, insights into population structure were gained from analyses of data from high-density SNP arrays ,19-26. A fashion . A lack To overcome the limitations and biases associated with SNP microarrays, we used the PCR-Sanger sequencing method to resequence a 100-kb ENCODE region in 92 Indian samples from four population groups  from the south Indian state Andhra Pradesh and combined our results with eight HapMap populations that are resequenced for the same region . By examWe sequenced one 100-kb ENCODE region-ENr123  in four different Andhra Pradesh ethnic groups representing three castes, Brahmin, Yadava, and Mala/Madiga, and one tribal group Irula Figure . We chosTo generate a comparable dataset, we applied the same SNP calling criteria on 722 HapMap individuals who were sequenced using the same protocol in the ENCODE3 project . We thenAmong the 1,484 total SNPs, 234 (15.8%) are specific to Indian populations , Watterson's θ estimator, nucleotide diversity (π), and observed SNP heterozygosity (H) for each population and continental group  as 'continental groups'. For continental groups, 152 unrelated individuals were randomly selected from HapMap African, European, and East Asian samples, respectively . At the population level, 24 individuals were randomly selected from each HapMap population, and all individuals from south Indian populations were included in the analyses. After sample size normalization, we measured genetic diversity using various summary statistics, including the number of segregating sites . In contrast, Brahmins and HapMap GIH have lower diversity levels, comparable to HapMap European and East Asian populations . However, when the Indian populations were included, the correlation became non-significant . This lack of correlation is due to large variation in H among the Indian populations (60.02 π 10-5 in Brahmins to 95.12 π 10-5 in the Irula). This result demonstrates great variation in diversity among groups within India.Several studies have shown that heterozygosity decreases with increasing distance from eastern Africa, presumably due to multiple bottlenecks that human populations experienced during the migration ,30. AmonADMIXTURE analysis at the individual level  on the genetic distances between populations using the normalized dataset. When all populations are included in the analysis, the first principal component PC1) accounts for 93% of the total variance and separates African and non-African populations , and the largest ST Fvalue is between HapMap GIH and the tribal Irula (10.4%).Next, we examined the divergence between Indian and non-Indian populations using pairwise ue Table . At the ue Table . Among Iue Table , the sma∂a∂i (Diffusion Approximation for Demographic Inference) [The complete sequence data allow us to obtain an accurate derived-allele frequency (DAF) spectrum. At both the continental and population levels, the DAF spectra in our dataset are characterized by a high proportion of low-frequency SNPs, as expected for sequencing data  .∂a∂i can simultaneously infer population parameters in models involving three populations, we first estimated the parameters associated with the out-of-Africa event using the African continental group and two continental Eurasian groups. We started from a simplified three-population divergence model based on the out-of-Africa model described in ∂a∂i [Af Tbefore any population divergence, and the population size changes from the ancestral population size A Nto Af Nin Africa. At time B Tthe Eurasian ancestral population with a population size of B Ndiverges from the African population, while the African population size Af Nremains constant until the present. The two Eurasian groups split from the ancestral population B Nat time 1-2T, with initial population sizes of 1_0 Nand 2_0N, respectively. Both populations experience exponential population size changes from the time of divergence to reach the current population sizes 1 Nand 2N.Because  in ∂a∂i  and asse-8 per base pair per generation , the ancestral population size is estimated to be between 13,000 and 14,000 for all models  are comparable to the size of the Eurasian ancestral population . At the time of the Eurasian population divergence, the population sizes of the two Eurasian continental groups in each model (1_0 Nand 2_0N) are consistently smaller than the African and the Eurasian ancestral population sizes, with one exception for the estimated European population size  in the Africa-East Asia-Europe model. These results suggest that the Eurasian population experienced population bottlenecks at the time of their divergence. Among Eurasians, East Asians have the smallest effective population size at the time of divergence  for each parameter, are shown in Table ∂a∂i at the same time, we fixed the parameters of the out-of-Africa epoch  in the model based on the parameters estimated from the three-population model with the highest likelihood (Africa-East Asia-European), as described in ∂a∂i [1_0N, 11,410; CI, 4,568 to 28,665) is comparable to the Eurasian ancestral population size , consistent with the high diversity we observed in these Indian samples.To further examine the population history among Eurasian populations, we constructed a four-population model containing all four continental groups Figure . Because in ∂a∂i . A modelWhen individual populations are analyzed, the patterns are largely consistent with the results from continental groups  in the Irula, Mala/Madiga, and Yadava samples are higher than those of the HapMap African populations. The genetic diversity difference among Indian populations has been observed previously in mitochondria [By sampling and resequencing 92 south Indian individuals we found 137 novel SNPs in the 100-kb region. These new SNPs represent approximately 30% of the total SNPs in these individuals. This result is consistent with several previous studies that showed that genetic variants in Indian populations, especially the less common variants, are incompletely captured by HapMap populations ,29,36. Mchondria , and Y cchondria  studies.ST Festimates) and population structure, we inferred the divergence time and migration rate among continental groups using the program ∂a∂i. The estimates of continental ST Fvalues and PCA results show that the greatest population differentiation occurs between African and non-African groups, while the least amount of differentiation occurs between Europeans and Indian populations. This is consistent with the estimates of divergence time between continental groups based on the three-population models . The SNP calls from the remaining 92 samples that passed quality control were then combined with the SNP calls from eight HapMap non-admixed populations studied by ENCODE3, including individuals from the Centre d'Etude du Polymorphisme Humain collection in Utah, USA, with ancestry from Northern and Western Europe (CEU), Han Chinese in Beijing, China (CHB), Japanese in Tokyo, Japan (JPT), Yoruba in Ibadan, Nigeria (YRI), Chinese in Metropolitan Denver, CO, USA (CHD), Gujarati Indians in Houston, TX, USA (GIH), Luhya in Webuye, Kenya (LWK), and Toscani in Italy (TSI), to create a final dataset containing 722 individuals from 12 populations.After merging the HapMap and the south Indian data sets, 112 loci that are fixed in all 12 populations were removed from the dataset. Thirteen tri-allelic SNPs were also removed because most analyses in this study are designed for bi-allelic SNPs. For SNPs that are fixed in certain populations, genotypes were filled-in using the hg18 reference allele because the reference allele information was used in the SNP calling process .P-values from each test were obtained and transformed to Z-scores. Twelve Z-scores were combined to a single Z-score and transformed to a single P-value for each SNP. Bonferroni correction was used, and 48 SNPs that failed the test at the 0.01 level  were removed. The ancestral/derived allele states of each SNP were determined using the human/chimpanzee alignment obtained from the UCSC database . Minor-.panTro2  under PuFor the 137 SNPs that are specific to our samples , we performed a validation experiment using an independent platform (Roche 454). When the minor allele is present in more than five individuals at a given locus, five individuals with the heterozygous genotype were randomly selected for validation. Among the 137 SNPs, we successfully designed and assayed 119 SNPs in 211 individual experiments. For the validation pipeline, we used PCR to amplify regions around the variants using the same primers as those used in the initial variant detection pipeline. In order to make genotype calls on all experiments simultaneously and also to reduce the cost of Roche 454 sequencing, we pooled PCR reactions in ten different pools and each pool was sequenced using a quarter of a Roche Titanium 454 sequencing run. The analysis was done using the Atlas-SNP2 pipeline available at the BCM-HGSC . Reads fS, θ, π, H and Tajima's D), and the confidence intervals for θ and π were calculated using the Population Genetics and Evolution Toolbox [ST Festimates between populations were calculated by the method described by Weir and Cockerham [Sequence-analysis statistics  [∂a∂i implements a series of methods to infer population history based on sequence data. We compared three different three-population out-of-Africa models and three four-population out-of Africa models to test the effect of adding different parameters to the model  . Using aBp: base pair; CI: confidence interval; DAF: derived-allele frequency; kb, kilo-base; kya, thousand years ago; PC: principal component; PCA: principal components analysis; SNP: single nucleotide polymorphism; STR: short tandem repeat.The authors declare that they have no competing interests.JX, LJ, and FY conceived and designed the study. JX, WSW, YH, CH, and FY performed the analysis and wrote the manuscript. AS and DM generated sequencing data and performed the validation experiment. MB collected the Indian samples. RG, LJ, and FY participated in project coordination. All authors read and approved the final manuscript.Supplemental text, four supplemental tables, and nine supplemental figures.Click here for file


Madagascar is located at the crossroads of the Asian and African worlds and is therefore of particular interest for studies on human population migration. Within the large human diversity of the Great Island, we focused our study on a particular ethnic group, the Antemoro. Their culture presents an important Arab-Islamic influence, but the question of an Arab biological inheritance remains unresolved. We analyzed paternal (n=129) and maternal (n=135) lineages of this ethnic group. Although the majority of Antemoro genetic ancestry comes from sub-Saharan African and Southeast Asian gene pools, we observed in their paternal lineages two specific haplogroups (J1 and T1) linked to Middle Eastern origins. This inheritance was restricted to some Antemoro sub-groups. Statistical analyses tended to confirm significant Middle Eastern genetic contribution. This study gives a new perspective to the large human genetic diversity in Madagascar.  Many population movements took place across the western Indian Ocean, notably for colonial and commercial purposes, and these have been highlighted through linguistic, crop, cattle, archaeozoological and commensal archaeological data –4. Madagth century, and finally the Antemoro. This last migration may have taken place around the last quarter of the 15th century. The Antemoro would have been able to root their beliefs and traditions in the region as they shared cultural and social similarities with the first migrants in the area )ST values between Antemoro and other Malagasy people were computed on HVI (nt16065-16363) and represented by a MDS plot. Haplogroup frequencies were visualized on a PCA plot. Finally, data obtained from the HVI were compared with data available in the literature for Malagasy populations, southern, central and western Africa, eastern Africa, northern Africa, Middle East, south Asia, parts of southeast Asia, Oceania and Europe  and the Antalaotra  have population genetic diversities within the same order of magnitude as those found generally in populations from southern Madagascar  . In contST values  with one Antandroy and one Antanosy individual, respectively from the southern coast of the Great Island. Three Malagasy populations were not considered for statistical calculations due to the low number of individuals in the sample , and the Andriana was excluded as it is an inbred population ,30. NeveST = 0.041, p-value <0.05). Some Antemoro groups differ from other populations from southern Madagascar 65].[65].64][.[65].64]Analysis of maternal lineages revealed haplogroups usually found in Madagascar. The vast majority of HVI haplotypes were shared with at least one Malagasy population from the south and the Highlands. This result reflects the existence of many intra-population movements and extensive interbreeding in Madagascar. Genetic diversity was very homogeneous and suggests important recent arrivals; the ‘Polynesian motif’  was found among others, reflecting the migration of Austronesian Malays most especially in the Antalaotra group. The genetic diversity of the Antemoro most especially for the Anteony, was also very close to African populations  ,4, the fThis study has highlighted a Middle Eastern biological trace in Madagascar consistent with the Middle Eastern cultural tradition of the population involved. The results of the Antemoro gene pool analysis suggest a Middle Eastern origin to some of the Y chromosome variation associated specifically with haplogroups J1 and T1, but this does not exclude an origin of this variation from unsampled/not studied African or Southeastern Asian populations. However, there is no Middle Eastern origin for the mtDNA gene pool. Nevertheless, this link is based on very few haplogroups, and the ethnogenesis and origin of all Antemoro groups, as well as the genetic impact of Middle Eastern populations around the Indian Ocean fringe during the last millennium remain open to discussion. Although, genetic analyses do not prove or refute a single theory concerning the geographical origin of the Antemoro, this study has suggested a Middle Eastern biological trace in the southeast coast of Madagascar. A study of the genetic contribution of Malagasy people from the two previous migrations to the coast (Onjatsy and ZafiRaminia) would be necessary. The groups in the north of the island where Arab trading posts have been described could also be of particular interest. The Antemoro migration would have arrived in the north in Vohemar before moving along the Malagasy coast to the south. Moreover, more data from archipelagos like the Kilwa islands would allow us to possibly relate the Antemoro migrations to the history of these areas. Gathering information on population movement from Middle East/Southwest Asia around the Indian Ocean would also be necessary. Futures objectives for studies on Malagasy populations will be to work at a genome wide scale in order to infer more accurate proportions and dates of admixture. File S1Supplementary tables and figures. Figure S1, MDS plot of FST between Malagasy populations and the Comoros using NRY haplogroup frequencies . Figure S2, MDS plot of FST computed from Y haplogroup frequencies between the Antemoro and populations from various geographic regions . Figure S3, MDS plot computed from FST values between Malagasy populations, based on HVI data. Figure S4, PCA computed from mitochondrial haplogroups frequency in Malagasy and Comoros populations. Figure S5, MDS plots of FST computed from HVI haplotypes between the Antemoro and populations from various geographic regions. Table S1, Y-STR profiles in the Antemoro. Table S2, Database used for the analysis of Y haplogroup frequencies. Table S3, Database used for the analysis of the seven Y-STR markers. Table S4, J1 haplotype references used for the median-joining network. Table S5, T1 haplotype references used for the median-joining network. Table S6, HVI and HVII profiles in the Antemoro. Table S7, Database used for HVI analysis. Table S8, Table of population pairwise FST values based on Y haplogroup frequencies. Table S9, Shared haplotypes between the Ampanabaka, other Malagasy populations and the Comoros, using 17 Y-STR markers. Table S10, Shared haplotypes between the Anteony, other Malagasy populations and the Comoros, using 17 Y-STR markers. Table S11, Shared haplotypes between the Antalaotra, other Malagasy populations and the Comoros, using 17 Y-STR markers. Table S12, Shared haplotypes between the Ampanabaka and the database using seven YSTR markers. Table S13, Shared haplotypes between the Antalaotra and the database, using seven Y-STR markers. Table S14, Shared haplotypes between the Anteony and the database using seven Y-STR markers. Table S15, Population pairwise FST values based on HVI in our three Antemoro groups. Table S16, Shared HVI haplotypes between the Ampanabaka and the other Malagasy populations. Table S17, Shared HVI haplotypes between the Antalaotra and other Malagasy populations. Table S18, Shared HVI haplotypes between the Anteony and other Malagasy populations. Table S19, Shared HVI unique haplotypes between the Ampanabaka and populations from the database. Table S20, Shared HVI unique haplotypes between the Antalaotra and populations from the database. Table S21, Shared HVI unique haplotypes between the Anteony and populations from the database. (PDF)Click here for additional data file.


Lutzomyia umbratilis is an important Leishmania guyanensis vector in South America. Previous studies have suggested differences in the vector competence between L. umbratilis populations situated on opposite banks of the Amazonas and Negro Rivers in the central Amazonian Brazil region, likely indicating a species complex. However, few studies have been performed on these populations and the taxonomic status of L. umbratilis remains unclear.L. umbratilis samples from the central Amazonian region in Brazil by analyzing mtDNA using 1181 bp of the COI gene to assess whether the populations on opposite banks of these rivers consist of incipient or distinct species. The genetic diversity was fairly high and the results revealed two distinct clades ( = lineages) with 1% sequence divergence. Clade I consisted of four samples from the left bank of the Amazonas and Negro Rivers, whereas clade II comprised two samples from the right bank of Negro River. No haplotypes were shared between samples of two clades. Samples within clades exhibited low to moderate genetic differentiation (FST = −0.0390–0.1841), whereas samples between clades exhibited very high differentiation (FST = 0.7100–0.8497) and fixed differences. These lineages have diverged approximately 0.22 Mya in the middle Pleistocene. Demographic expansion was detected for the lineages I and II approximately 30,448 and 15,859 years ago, respectively, in the late Pleistocene.Phylogeographic structure was estimated for six L. umbratilis. These findings suggest that the Amazonas and Negro Rivers may be acting as effective barriers, thus preventing gene flow between populations on opposite sides. Such findings have important implications for epidemiological studies, especially those related to vector competence and anthropophily, and for vector control strategies. In addition, L. umbratilis represents an interesting example in speciation studies.The two genetic lineages may represent an advanced speciation stage suggestive of incipient or distinct species within  In the last three decades, molecular genetic markers have been widely used for elucidating the population structure and evolutionary genetics in insect vectors, and these studies have contributed to differentiate members of cryptic species complexes, primarily in malaria Leishmania guyanensis Floch, 1954 that causes American Cutaneous Leishmaniasis (ACL), Lutzomyia umbratilis Ward & Fraiha, 1977 (Diptera: Psychodidae), occurs in northern South America, including Bolivia, Brazil, Colombia, French Guyana, Peru, Suriname and Venezuela L. umbratilis has been registered in the Amazonian region L. umbratilis populations are more susceptible to evolving into differentiated populations, incipient species and, ultimately, reproductively isolated species.The main vector of Lutzomyia umbratilis has been implicated in Le. guyanensis transmission in Brazil Le. guyanensis transmission in the states of Pará L. umbratilis is recognized as the most important vector, the number of human cases of ACL was considered the highest in Brazil, comprising ∼57.4% of the autochthonous cases in the region Leishmania braziliensis Vianna, 1911 was identified in L. umbratilisLeishmania seems to vary in the central Amazonian Brazil region Lutzomyia umbratilis populations naturally infected with Le. guyanensis have been observed east of the Negro River and north of the Amazonas River; however, at south of the Amazonas River fluvial system have not been reported evidences of natural infections by Leishmania for this species Le. guyanensis transmission cycle, where L. umbratilis populations display distinct degrees of vector competence between the opposite sides. However, only a few studies have tested the role of this barrier in L. umbratilis genetic subdivision.L. umbratilis populations from Manaus and Manacapuru  from Amazonas, Brazil. The results of this study revealed significant differences in the life cycle, fecundity, fertility, emergence degree and adult longevity between populations, which are indicative of intrinsic biological differences L. umbratilis populations of this fluvial system showed significant differences in the bristle lengths of 4th instar larvae and in the number and size of the spines of armature of the female genital atrium L. umbratilis remains unclear.A biological analysis, under laboratory conditions, was conducted with two COI) is one of the largest protein-coding genes, which functions in electron transport and ATP synthesis and exhibits the most conserved amino acid sequence Genes encoding mitochondrial DNA (mtDNA) have been widely employed in population studies, molecular taxonomy and phylogenetic relationships of many organisms L. umbratilis populations on opposite sides of the Negro River and on the left bank of Amazonas River in the central Amazonian Brazil region by analyzing an 1181 bp fragment of the COI gene from mtDNA to assess whether L. umbratilis populations on both sides comprise incipient or distinct species.We analyzed the phylogeographic structure of L. umbratilis specimens were sequenced from six locations from the central Amazonian Brazil region using an 1181 bp fragment of the COI gene (One hundred and eleven COI gene . NucleotL. umbratilis. The two samples of clade II (H29 to H52) contained five fixed mutations . Phylogenetic relationships demonstrated by Bayesian Inference (BI) analysis also revealed two monophyletic clades, but with moderate support  were strongly supported (1.00) on an isolated cluster. A similar topology tree was visualized by Maximum-Likelihood (ML) analysis (data not shown).L. umbratilis genetic diversity. Within clade I, the Rio Preto da Eva sample exhibited the lowest haplotype and nucleotide diversities, whereas Manaus and Cachoeira Porteira samples had the highest haplotype diversity. Within clade II, two samples showed slightly high haplotype diversity, as did clades I and II. The nucleotide diversity was slightly low in all analyses.The intra-population genetic diversity measures for each sample, combined data and for two clades are shown in D test P<0.05) and for clades I and II (P<0.001), reflecting significant deviations from the neutral model likely due an excess of rare haplotypes consistent with recent population expansion or positive selection (Fs P<0.0001), and for BR-174 Highway and Manacapuru (P<0.05), rejecting the mutation-drift equilibrium and strongly favoring the interpretation of recent population expansion or selective sweeps. Mismatch distribution was unimodal for all samples, except Rio Preto da Eva (data no shown), and for clades I and II (r) calculated for all samples and clades did not reject the null hypothesis of sudden demographic expansion. The sum of squared deviations (SSD) goodness-of-fit test was also not significant   was moderately high between Rio Preto da Eva and Manaus; it likely reflects the small Manaus sample size (n = 4). Similarly, the FST value within clade II between two samples was low (0.0548). Unlikely, the samples between clades I and II yielded very high FST values and were highly significant after the Bonferroni correction (0.7100 to 0.8497). This result was also observed between clades I and II (0.7776).Within clade I,  0.1841) . MoreovexyD), the number of net nucleotide substitutions per site between populations (aD), the number of shared polymorphisms (sS) and the number of fixed differences (fS). Similarly, FST, the highest xyD and aD values were detected between clades I and II. Furthermore, few or no shared polymorphisms or fixed differences were found between samples of two clades, particularly in comparisons between Manacapuru and three BR-174 Highway, Rio Preto da Eva and Manaus samples . Interestingly, Manacapuru versus Cachoeira Porteira and Novo Airão versus Cachoeira Porteira, which are geographically further apart, harbored more shared polymorphisms and less fixed differences . Although the FST value (0.0569) was statistically significant between Cachoeira Porteira and Rio Preto da Eva (both from clade I), the xyD and aD values were low, and no fixed differences were found between them. As described above, the genetic divergence observed between the populations does not fit the isolation-by-distance model. Analyzing all samples together, the correlation between genetic and geographic distances was negative (r = −0.063116) and not significant (P = 0.366), perhaps because geographically closer populations are more differentiated, which likely can be attributed to the rivers acting as barriers.K) between clades I and II were 1.0% and 9.99, respectively, whereas between L. umbratilis and the closely related species L. anduzei were ∼5.8% and 12.78, respectively. Given an evolutionary rate of 2.3% per million years estimated for the COI gene in insects Sequence divergence and the average number of nucleotide differences  was detected between two clades. These results combined with the lack of shared haplotypes and presence of fixed differences between populations of these clades suggest that the Amazonas and Negro Rivers may be acting as effective barriers, thus preventing gene flow between L. umbratilis populations from opposite sides.In this study, the phylogenetic analyses revealed the presence of two distinct et al.The genetic differentiation detected in this study supports the biological and morphological differences initially observed by Justianiano FST values detected between samples of two clades were similar to those described by Soto et al.Lutzomyia longipalpis Lutz & Neiva, 1912 from Central and South America using polymorphisms of the ND4 gene from mtDNA (FST = 0.52 to 0.933), over a much larger geographic area than in the present study. Another study analyzed nine populations of the L. longipalpis complex from northeastern region and state of Minas Gerais, Brazil, using Ctyb gene sequences from mtDNA and a larger geographic area FST = 0.184) than that observed here. Comparing these findings, our very high FST values over short geographic distances appear rare in sandflies species, and according to Wright Anopheles cruzii complex L. umbratilis, although this hypothesis was moderately supported in the phylogenetic analyses.The L. umbratilis samples within each clade (samples from the same side of the rivers) was low, even when populations were separated by geographic distances ranging 353.67 to 394.02 km apart  of this study and among four clades of L. longipalpis complex (9.47–10.91%) using COI gene L. umbratilis populations from opposite banks was ∼0.22 Mya during the middle Pleistocene. Although this comparison involves different genetic markers, this divergence time suggests that L. umbratilis diversification from the central Amazonian region may be more recent than that estimated for the L. longipalpis complex based on two genes  that between two closely related species L. umbratilis is also consistent with population expansion , one from municipality of Autazes , and two from Amajari  were sequenced as shown in the The sequences were edited by using the BIOEDIT K values, number of polymorphic sites, transition and transversion rates) and Tajima's DFs tests D, was implemented to test strict neutrality, whereas the neutrality test, Fu's Fs, was estimated to test population size stability. The latter test is more powerful for detecting population expansion and genetic hitchhiking. The mismatch distribution (an observed distribution of pair-wise nucleotide differences among haplotypes) was computed for each sample, combined data and for clades I and II in ARLEQUIN, 3.1 r) SSD) between observed and expected mismatch distributions were calculated as a test statistic to validate the estimated sudden expansion model. Genetic differentiation, on the basis of FST pairwise between samples and between clades, was inferred in ARLEQUIN, 3.1 xyD) aD), the number of shared polymorphisms between population pairs (sS) and number of fixed differences between population pairs (fS) were calculated in DNASP, 4.0 The intra-population genetic diversity measures  and L. anduzei  are deposited in GenBank under the accession numbers JQ839256 to JQ839261.The correlation between straight-line geographic distances and Table S1Variable sites of haplotypes of Lutzomyia umbratilis and Lutzomyia anduzei. H, haplotypes; H1 to H52, haplotypes of Lutzomyia umbratilis; H53 to H58, haplotypes of Lutzomyia anduzei. The dots indicate identical nucleotides with the H1. The frequency of each haplotype is provided in (DOC)Click here for additional data file.


Castanopsis sclerophylla (Lindl. & Paxton) Schotty populations and decoupled the effects of habitat loss and fragmentation per se. We selected seven nuclear and six cpDNA microsatellite loci and genotyped 460 individuals from mainland and island populations, which were located in the impoundment created in 1959. Number of alleles per locus of populations in larger habitats was significantly higher than that in smaller habitats. There was a significant relationship between the number of alleles per locus and habitat size. Based on this relationship, the predicted genetic diversity of an imaginary population of size equaling the total area of the islands was lower than that of the global population on the islands. Re-sampling demonstrated that low genetic diversity of populations in small habitats was caused by unevenness in sample size. Fisher's α index was similar among habitat types. These results indicate that the decreased nuclear and chloroplast genetic diversity of populations in smaller habitats was mainly caused by habitat loss. For nuclear and chloroplast microsatellite loci, values of FST were 0.066 and 0.893, respectively, and the calculated pollen/seed dispersal ratio was 162.2. When separated into pre-and post-fragmentation cohorts, pollen/seed ratios were 121.2 and 189.5, respectively. Our results suggest that habitat loss explains the early decrease in genetic diversity, while fragmentation per se may play a major role in inbreeding and differentiation among fragmented populations and later loss of genetic diversity.Generally, effect of fragmentation per se on biodiversity has not been separated from the effect of habitat loss. In this paper, using nDNA and cpDNA SSRs, we studied genetic diversity of  Habitat fragmentation is a landscape-scale process involving both habitat loss and the breaking apart of habitats, i.e. narrow sense fragmentation or fragmentation per se A commonly adopted method of identifying the genetic consequences of habitat fragmentation is to study the relationship between genetic diversity and habitat size or population size  populations in island habitats of different sizes and adjacent mainland habitats in the Qiandao Lake region. The focal species C. sclerophylla is a dominant species of evergreen broad-leaved forests, and is the most common evergreen broadleaved tree in the Qiandao Lake region C. sclerophylla is wind-pollinated and gravity-dispersed, but rodents may play a role in the secondary dispersal of seeds. For such a species, formation of land-bridge islands may have a minor impact on the extensive pollen flow, but the water surrounding the islands is a critical barrier for seed dispersal. Available studies show that, in general, seed dispersal is much more limited than pollen dispersal In this study, we examined genetic diversity of C. sclerophylla populations on islands varying in size and on adjacent mainland. Because losses of individuals and associated genetic diversity happen simultaneously with the loss of habitats that they occur, whereas genetic diversity of fragmented populations is lost slowly over subsequent generations In the present study, we used polymorphic microsatellite loci of both nuclear and chloroplast genomes to detect genetic composition of The study involved a common tree species, which is not endangered or protected species. The study location Qiandao Lake region is neither privately-owned nor protected area. Therefore, no specific permissions were required for the study.Castanopsis sclerophylla and Cyclobalnopsis glauca (Thunb.) Oersted ( = Quercus glauca Thunb) Pinus massoniana Lamb. Pure P. massoniana forests are estimated to dominate 60% of the land cover in this region The study sites were located in Qiandao Lake National Forest Park (QLFP) in Zhejiang Province, China . The mea2 when the water-level is 108.0 m a.s.l., the altitude of the dam, and several hundred of smaller islands in this impoundment. Previous surveys indicated C. sclerophylla occurred mainly on islands and adjacent mainland in the southeastern sub-region of QLFP C. sclerophylla was found on 13 islands, including Laoshan Island , the largest one. The peak altitudes of the islands range from 105.4 m to 408.5 m. Leaves of C. sclerophylla were collected from 11 of the 13 island populations, except two extremely small populations of less than 5 individuals, and 3 mainland populations in QLFP , and 3) strongly fragmented habitats, including islands <10 hm2  (There are 1078 islands larger than 2500 m in QLFP . Table 1 and BN) .C. sclerophylla populations, the samples were classified into two cohorts: pre-fragmentation and post-fragmentation, based on the age of the individuals. The classification was based on the relationship between basal diameter and age constructed by Zhang Estimated or true population sizes of the sampled populations ranged from 6 to more than 4000 . In largC. cuspidata var. sieboldiiTM thermocycler . A denaturation period of 4 min at 94°C was followed by 36 cycles of 40 s at 94°C, 30 s at 55°C and 40 s at 72°C, and then 10 min at 72°C for final extension. The PCR products were separated in 6% polyacrylamide denaturing gels. After electrophoresis, bands were revealed using the following modified silver-staining procedure.Total DNA was extracted with a modified CTAB procedure C. sclerophylla. Six primer pairs produced clear and distinguishable bands, and among them three were polymorphic. The six pairs of primers were applied to all samples.Primers for chloroplast microsatellite loci developed for species of the Fagaceae NA) and allelic richness (AR) HO) and expected (HE) heterozygosities were calculated using TFPGA FIS) was estimated as the average for each population, and also for pre-and post-fragmented cohorts using FSTAT stats′ in R environment The nuclear microsatellite data were used to test for linkage disequilibrium for each locus pair across all populations in FSTAT version 2.9.3.2 FST was estimated using bootstrap analysis in TFPGA using loci as replicates to determine if FST estimates were significantly different from a null hypothesis of panmixia Genetic differentiation among populations was estimated for both nuclear DNA and cpDNA data set (θc) using TFPGA C. sclerophylla populations When a population has gone through a severe and recent genetic bottleneck, it will show a transient excess of heterozygotes r) of the amount of dispersal by pollen , where S is number of alleles, n is number of individuals and α is the Fisher's α.We assessed the relationship between sample size and number of alleles detected in pre-or post-fragmentation cohorts of each habitat type to account for potential biases resulting from unequal sample sizes. We checked allele accumulation curves or the number of alleles for a certain number of sampled individuals in pre-or post-fragmentation cohorts of each habitat type using SPECACCUM using the software package ‘et al. We followed the general logic of Yaacobi vice versa.We also examined the relative roles of habitat loss and fragmentation per se on genetic diversity using Quinn and Harrison's E ranged from 0.013 (Ccu90) to 0.823 (Ccu33). At the population level, mean number of alleles per locus ranged from 3.00 in strongly fragmented populations SH and TD to 4.71 in the mainland population XS. There was a significant difference in mean number of alleles per locus (NA) among different habitat types . The post hoc Tukey HSD test indicated values of NA in slight-and moderate-fragmented habitats were significantly higher than that of strongly-fragmented habitats. There was no significant difference in HO and HE among habitat types.Across the 460 individuals scored, seven polymorphic loci produced a total of 39 alleles. The number of alleles per locus ranged from 2 (Ccu90) to 10 (Ccu93), and HUsing the program BOTTLENECK, no population was found to have experienced a recent bottleneck under the models of TPM or SMM. No significant relationship was found between distance to the nearest occupied habitat and parameters of genetic diversity of islands.x), and the following relationship was determined: y = 3.410+0.139 ln(x) . Using this equation, we may obtain the predicted value for the number of alleles per locus of an imaginary population of size equaling the total area of the islands. The predicted value (4.37) is smaller than the value of the global population of all the islands (4.57)  was significantly related to habitat size (39 ln(x)  (adjustes (4.57) , indicats (4.57) , also inF = 5.23, P = 0.014)  . Post-fr= 0.014) . A re-sa= 0.014) . SimilarThree of the six cpDNA microsatellite loci were polymorphic with a total of 10 alleles. Combining information from those alleles allowed the identification of five haplotypes among the 460 individuals. Haplotype II was the most frequent overall while haplotypes IV and V were found in only one individual, in populations XS and LS, respectively. Eight populations contained only one haplotype, while population XS had three haplotypes. The mean Nei's gene diversity of the populations was 0.010, and the Nei's gene diversity of the global population was 0.102. In the studied region, chloroplast haplotypes exhibited a distribution gradient , indicatR2 = 0.1524, P = 0.092). Based on this relationship, the predicted number of haplotypes of an imaginary population of a size equaling the total area of the islands was 1.91, much smaller than the observed value (3 haplotypes) in the island populations.The number of haplotypes was marginally significantly related to habitat size (adjusted C. sclerophylla in the study area (r = 162.2). Post-fragmentation cohorts had a higher FIS than pre-fragmentation cohorts (0.380 v.s. 0.348), and a higher FST by cpDNA SSRs (0.915 v.s. 0.878). However, nuclear FST values were nearly the same in pre-and post-fragmentation cohorts (0.073 v.s. 0.072). The calculated values of pollen/seed dispersal ratios were 121.2 and 189.5 in the pre-and post-fragmentation cohorts, respectively.The estimatedC. sclerophylla populations in the studied region.Habitat destruction leads to habitat loss and fragmentation per se, resulting in a decrease in genetic diversity. Fifty years after dam construction, we found a positive relationship between habitat size and number of alleles per locus using nuclear microsatellites or number of cpDNA haplotypes, a pattern indicating smaller habitats harbor populations of lower genetic diversity. This pattern was frequently observed in the fragmented populations C. sclerophylla populations. Habitat loss directly decreases the area of suitable habitats, resulting in a decrease in population size or even leading to local extirpation of some species. Genetic diversity may also decline during this process. The positive relationship between both habitat area or population size and genetic diversity observed in many studies confirmed the negative impact of habitat loss It is not surprising that habitat loss leads to reduced genetic diversity of both nuclear and chloroplast genomes of Fagus sylvatica L. In the present study, C. sclerophylla populations have been fragmented for only five decades, i.e., less than two generations. Also, C. sclerophylla may resprout, which permits persistence in demography and prolongs generation time That fragmentation per se had not played a major role in genetic diversity in the fragmented landscape seems to contrast with most previous observations et al.et al. Anthyllis vulneraria, a rosette-forming legume. Our study shows a large population contains lower genetic diversity than several small ones, indicating that fragmentation per se may have a positive impact on genetic diversity in C. sclerophylla. It is well known a heterozygous landscape may maintain high genetic diversity caused by differential selection Another plausible reason is that fragmentation per se has weak or no negative or even positive impacts on genetic diversity, as observed in species diversity C. sclerophylla populations seems to be more tolerant to fragmentation, which has also been observed in other plant species, such as oaks in France Pinus elliottii var. densaet al. C. sclerophylla, low cpDNA diversity within populations may have limited the effects of fragmentation per se. When seed dispersal is limited, most populations had only one haplotype. Although some other populations had two or three haplotypes, the frequency of the dominant haplotype was lower than 0.9 in only two populations (C. sclerophylla has a strong ability to resprout. When disturbed, C. sclerophylla regenerates mainly by resprouting Compared to nuclear DNA diversity, chloroplast DNA genetic diversity in ulations . Also, CC. sclerophylla is wind-pollinated and gravity-dispersed, it has a high pollen-to-seed dispersal ratio. This ratio is among the highest for plants at a range-wide scale, and much higher than 17, the median ratio of 93 taxa C. sclerophylla was small because of the limited range in the present study, as scale plays a critical role in estimating the ratios 2, pollen-to-seed dispersal ratio was 1.15–2.16 in the congener C. fargesii Franch. Since C. sclerophylla, while fragmentation per se has no negative effect, but does have a weak positive impact. Our results suggest habitat loss may explain the early decrease in genetic diversity, while fragmentation per se may play a major role in differentiation among fragmented populations and a later decrease in genetic diversity.In conclusion, by decoupling the effects of habitat loss and fragmentation per se, we found, in recently fragmented habitats, habitat loss may play a major role in the decrease of nuclear and chloroplast genetic diversity of


Ancestry-informative markers (AIMs) show high allele frequency divergence between different ancestral or geographically distant populations. These genetic markers are especially useful in inferring the likely ancestral origin of an individual or estimating the apportionment of ancestry components in admixed individuals or populations. The study of AIMs is of great interest in clinical genetics research, particularly to detect and correct for population substructure effects in case-control association studies, but also in population and forensic genetics studies.This work presents a set of 46 ancestry-informative insertion deletion polymorphisms selected to efficiently measure population admixture proportions of four different origins . All markers are analyzed in short fragments (under 230 basepairs) through a single PCR followed by capillary electrophoresis (CE) allowing a very simple one tube PCR-to-CE approach.HGDP-CEPH diversity panel samples from the four groups, together with Oceanians, were genotyped to evaluate the efficiency of the assay in clustering populations from different continental origins and to establish reference databases. In addition, other populations from diverse geographic origins were tested using the HGDP-CEPH samples as reference data. The results revealed that the AIM-INDEL set developed is highly efficient at inferring the ancestry of individuals and provides good estimates of ancestry proportions at the population level.In conclusion, we have optimized the multiplexed genotyping of 46 AIM-INDELs in a simple and informative assay, enabling a more straightforward alternative to the commonly available AIM-SNP typing methods dependent on complex, multi-step protocols or implementation of large-scale genotyping technologies. Initial studies of human genetic variation focused on Short Tandem Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs) These investigations have identified a large number of candidate gene variants showing strong association with specific conditions or phenotypes and subsequent replication studies and meta-analysis have strengthened or weakened these initial findings. One of the major problems in case-control association studies is the presence of undetected population structure that can lead to finding false positive associations when an excess of ancestry differentiated markers stratifies the case and the control groups. Alternatively false negative results may occur if real associations are missed if weak while greater allele frequency differentiation exists between study and control groups due to differences in ancestry AIMs show high allele frequency divergence between different ancestral or geographically distant populations and are especially useful in inferring the likely ancestral origin of an individual or estimating the apportionment of ancestry components in admixed individuals or populations. Ancestry information can then be used to perform genetic matching or correct substructure effects in case-control association studies. In the population genetics field AIMs are used mainly to estimate ancestry proportions in admixed populations and assess the structure of those populations. Furthermore, AIMs are of great interest in forensic genetics, with the potential to provide an intelligence tool in criminal investigations. In the absence of any other investigative leads, AIM genotypes obtained from evidential material could indicate the likely ancestry of the donor, and therefore help direct the course of investigations In recent years several studies have been published reporting AIM sets varying greatly in the type of polymorphism, the number of loci involved and the genotyping strategies, ranging from simple PCR followed by capillary electrophoresis (e.g. INDEL sets) to more laborious and resource-intensive technologies (e.g. SNP typing by SNaPshot and TaqMan assays). The reported AIM sets have also focused attention on different population group comparisons, depending on the ancestral contributors to the admixed populations under study, or otherwise comprise more generic panels aimed at efficient population differentiation at the continental level. The great majority of AIM panels described to date use SNPs and only a minority apply STRs In this study we followed an approach that brings together highly informative short binary INDELs that combine the desirable characteristics of the other genetic markers most commonly used In this study a set of 46 AIM-INDELs was selected to efficiently measure population admixture proportions of four different origins . We have optimized the multiplexed genotyping of the 46 AIMs in a simple and informative assay, enabling a more straightforward alternative to AIM-SNP typing methods dependent on multi-step protocols or implementation of genotyping technologies that are expensive, complex and platform-dependent. In addition, we established reference databases using the HGDP-CEPH diversity panel samples The current study was approved by the Institute of Molecular Pathology and Immunology of the University of Porto institutional review board. Besides the HGDP-CEPH diversity panel human cell line samples, all other samples involved in the study are long-lasting anonymized DNA extracts previously obtained with informed written consent from healthy individuals for research purposes.A total of 1002 DNA samples were used in this study comprising: i) reference samples from the HGDP-CEPH diversity panel standardized subset H952 http://www.marshfieldclinic.org/mgs/; An initial pool of candidate INDELs was assembled by collecting previously available population data on this type of polymorphism included in the Marshfield Diallelic Insertion/Deletion Polymorphisms database website (PCR amplification of the 46 AIM-INDELs used the QIAGEN Multiplex PCR kit (Qiagen) at 1× Qiagen multiplex PCR master mix, 0.1 µM of all primers (sequence details in ST genetic distances and linkage disequilibrium tests were assessed using Arlequin v3.5.1.2 http://taylor0.biology.ucla.edu/structureHarvester/). In a second phase, when using reference samples as training sets to test for “unknown” individuals or populations, STRUCTURE analyses were carried out using the same parameters as before or selecting the “Use Population Information” option. In these cases, allele frequencies were updated using only the reference individuals with POPFLAG = 1 data (option under the Advanced tab). Here, 3 independent runs were performed only for the appropriate number of clusters, as evaluated by the initial analysis. Unless otherwise indicated, results are presented for the default settings considering the “Admixture Model” and correlated allele frequencies. CLUMPP v1.1.2 distruct v1.1 Estimation of allele frequencies, exact tests of Hardy-Weinberg equilibrium (HWE), FPrincipal component analysis (PCA) was performed as an additional and independent approach to estimate the number of populations present in the data set. We used R 2.11.1 http://mathgene.usc.es/snipper/; The efficiency of the 46 AIM-INDEL set for assigning individuals to population groups was further evaluated by one-out cross-validation based on a flexible single profile analysis system very similar to STRUCTURE, calculating likelihood ratio values obtained with a Bayesian classification algorithm implemented in the “Snipper app suite” website  through a single PCR followed by capillary electrophoresis . The worAfter optimization of the method we created a database including HGDP-CEPH diversity panel genetic data, commonly used by the research community as reference populations for the four groups AFR, EUR, EAS, NAM and also from Oceania (complete database included in File S1).ST for each marker. With few exceptions, the vast majority of the INDELs show high allele frequency differentials and genetic distances between at least two groups (39 with δ≥0.4 and 44 with δ≥0.3). No significant departures from Hardy-Weinberg equilibrium were found in the studied populations and pairwise linkage disequilibrium exact tests did not detect significant associations within the marker set.Patterns of INDEL variability observed in the HGDP-CEPH samples from the population groups AFR, EUR, EAS and NAM are detailed in One interesting finding was the occurrence of an unexpected third allelic state  for MID360 and MID2264. Sequencing analysis confirmed our observations as a result of additional sequence length variants within the amplicon fragments. For MID360 the third allelic state observed is due to a T insertion associated with the short allele, 8 bases downstream of the targeted polymorphism . Conversely for MID2264, allele 3 corresponds to a T deletion occurring in the long allele background . Interestingly, the MID360 variant alleles were only found in AFR samples whereas the MID2264 variants seemed specific of EUR, further contributing to the differentiation of the two groups.Before implementing the HGDP-CEPH diversity panel reference database, a preliminary evaluation on the performance of a panel comprising 44 INDELs at the time (without MID94 and MID1734) had been performed using 48 samples with origin in each of the four groups under study  yet allow a clear spatial separation of four different groups . LikewisReference HGDP-CEPH diversity panel genetic data from the four population groups  was used to estimate ancestry proportions of individuals/populations from different geographic locations. We tested samples from Angola, Portugal, Taiwan and Brazil . The individual and global admixture estimates obtained with genetic data only (no prior population information) correspond well with expected patterns, knowing the origin of the subjects ; Table 2The AIM-INDEL panel was primarily designed as a tool for ascertaining ancestry from four major population groups. Nonetheless, as there is general interest in AIM panels able to distinguish populations at the broader continental level, we extended our study to HGDP-CEPH Oceanian samples and assessed the ability of the panel to differentiate populations with origin in all five continent regions. Following the same evaluation strategy as before, the assay proved to consistently recognize a fifth cluster corresponding to Oceanians and that K = 5 captures most of the structure in the dataset  show mosST, allele differentials ä, informativeness of assignment index InThe main objective of this study was to provide a simple tool for inferring ancestry and estimate admixture proportions from four different population origins that can be widely applied to genetic studies. We describe a new AIM assay comprising 46 INDELs that are simply analyzed in a multiplex PCR followed by CE detection. With this approach we were able to combine the ancestry informative power of biallelic markers (exemplified by AIM-SNP panels) with the simplified analysis based in fragment size separation (as in STR typing). The methodology of the assay is straightforward and can be readily and inexpensively implemented in any molecular genetics laboratory. In contrast, the majority of AIM sets published in recent years involve more complex genotyping protocols or are limited to specific platforms not available to all laboratories and therefore requiring additional resources ST values obtained with the 46 AIM-INDELs  and representative testing samples with different origins and admixture levels. From the genetic characterization of the reference ancestral samples we observed a high level of differentiation from the chosen INDELS, as expected from the selection criteria. Although some markers revealed lower differences than those expected from previous data, this is possibly due to the samples representing each group and allele frequency estimation strategies being different M-INDELs  are clean4 AIM-SNP set for equivalent population groups  may contribute to this reduced differentiation for East Asians. Another important aspect is the proximity of the East Asian and Native American gene pools. Considering the history of modern humans these groups have diverged over the shortest time, and furthermore, the original peopling of Americas from Beringia involved a significant bottleneck effect that is still reflected in Native American variability. Despite this slightly reduced level of differentiation in the AIM-INDELs selected, STRUCTURE, PCA and cross validation studies together support the capacity of the panel to properly distinguish both groups.The population assignment cross validation studies based on Bayesian likelihood ratios provided additional evidence of the utility of the assay, particularly for forensic applications where single profiles are often analyzed one at a time. Here the error rates in classifications considering either four or five population groups were low (1.26% and 1.54% respectively). The AIM-INDEL panel achieves very high accuracy for population assignment in the five broad continental regions, similar to results observed by Paschou et al. when using subsets of 50 SNPs ascertained by PCA and estimation of Ip value = 0.136), the three-group membership proportion estimates at K = 3 showed a noticeable increase in the Native American component to 29.5%  include significant East Asian communities, despite having joined these populations rather recently. When using Brazilian samples from such geographic areas, particularly as case and control samples for association studies, a preliminary four-group analysis is recommended to detect the presence of East Asian ancestry amongst individuals in the study. Otherwise there is considerable risk that the a priori rejection of this hypothesis based on three-group analyses could lead to an over-estimation of the Native American proportion in the global admixture estimates (data not shown) due to a strong bias caused by the presence of East Asian individuals in the population under study. Conversely, when “forcing” a four-group analysis in south-American tri-hybrid populations, it is possible that the fourth East Asian component can produce a spurious fraction of membership arising from the Native American component, due to the close relationship of the East Asian and Native American population groups. In summary, we advocate adopting an approach taking due regard for the particular population under study. Consideration of the known recent population history and demographics helps make appropriate adjustment for the different principal ancestral contributors. In the special case of south-American populations, we recommend a preliminary study taking advantage of the full potential of the AIM-INDEL assay to identify and possibly exclude East Asian study subjects, and subsequently perform a comprehensive three-group analysis. The AIM-INDEL assay can be efficiently used in three-group analyses AFR/EUR/NAM, similarly to AIM panels are regularly applied in population genetics studies to analyze admixed populations by estimating admixture proportions both at the individual and population level. Depending on the historical context of populations under study, there are different principal ancestral contributors to the formation of the current ancestry characteristics of the region. For example, Brazil and the majority of south-American countries underwent admixture between the pre-existent Native Americans, colonizing Europeans and later African influences resulting from the slave trade to create essentially tri-hybrid populations. In such cases, it is appropriate for genetic studies to perform three-group analyses of ancestry estimates. Our study analyzed ancestry proportions in Belém. We first considered the possibility of a fourth EAS minor ancestral contributor in initial analyses and K = 4 resulted in a low level but detectable fraction of membership of this cluster at 8.8% . Howeverto 29.5%  which isto 29.5% . NeverthIn conclusion, we have optimized the multiplexed genotyping of 46 AIM-INDELs in a simple and informative assay, enabling a more straightforward alternative to the commonly available AIM-SNP typing methods dependent on multi-step protocols and/or implementation of dedicated genotyping technologies. The AIM-INDEL assay produces accurate individual ancestry estimates of four different origins, which can be applied to the correction of false positive results due to population stratification between case and control samples in association studies. Most effectively it can be used as a simple and inexpensive tool for the initial screening of individuals prior to expensive GWA studies or to allow precise matching of ancestries amongst case and control samples. Finally, given the relatively high efficiency in population assignment of individuals from all five continental origins, the multiplex represents a tool of considerable potential in forensic applications.Figure S1Analysis of population samples from four different continental origins using a preliminary set of 44 AIM-INDELs (without MID94 and MID1734). A) ancestral membership proportions (based on STRUCTURE results from 3 independent runs treated in CLUMPP and plotted with distruct); B) estimated ln probability of the data (−lnP(D) obtained with STRUCTURE and plotted using Structure harvester); C) principal component analysis 3D plots; D) estimation on population assignment success Click here for additional data file.Figure S2Ancestral membership proportions in the Brazilian city of Belém using HGDP-CEPH diversity panel genetic data of three main ancestral contributors as training sets. A) bar plots based on STRUCTURE results from 3 independent runs treated in CLUMPP and plotted with distruct ; B) triangular plots based on STRUCTURE results from the run with highest −lnP(D) .(PDF)Click here for additional data file.Figure S3Analysis of HGDP-CEPH diversity panel samples from five continental origins using a set of 46 AIM-INDELs. A) ancestry membership proportions ; B) estimated ln probability of the data (−lnP(D) obtained with STRUCTURE and plotted using Structure harvester); C) principal component analysis 3D plots. D) estimation on population assignment success Click here for additional data file.Table S1PCR primer sequences used in the multiplex.(PDF)Click here for additional data file.Table S2Allele frequencies, δ and FST values for the 46 AIM-INDELs in HGDP-CEPH diversity panel population samples from Africa (AFR), Europe (EUR), East Asia (EAS) and Native America (NAM).(PDF)Click here for additional data file.File S1Genotypic data (STRUCTURE format) for the 46 AIM-INDELs in HGDP-CEPH diversity panel population samples from Africa, Europe, East Asia and Native America.(TXT)Click here for additional data file.