ST statistics, and the three-population test were used to investigate the underlying genetic structure and ancestry of the different ethno-linguistic groups. Our analyses revealed a genetic component for Sudanese Nilo-Saharan speaking groups (Darfurians and part of Nuba populations) related to Nilotes of South Sudan, but not to other Sudanese populations or other sub-Saharan populations. Populations inhabiting the North of the region showed close genetic affinities with North Africa, with a component that could be remnant of North Africans before the migrations of Arabs from Arabia. In addition, we found very low genetic distances between populations in genes important for anti-malarial and anti-bacterial host defence, suggesting similar selective pressures on these genes and stressing the importance of considering functional pathways to understand the evolutionary history of populations.East Africa is a strategic region to study human genetic diversity due to the presence of ethnically, linguistically, and geographically diverse populations. Here, we provide new insight into the genetic history of populations living in the Sudanese region of East Africa by analysing nine ethnic groups belonging to three African linguistic families: Niger-Kordofanian, Nilo-Saharan and Afro-Asiatic. A total of 500 individuals were genotyped for 200,000 single-nucleotide polymorphisms. Principal component analysis, clustering analysis using ADMIXTURE, F East Africa is a strategic region to study human genetic diversity due to the presence of ethnically, linguistically, and geographically diverse populations. North African and sub-Saharan African populations in East Africa are connected by the Nile River. It has been proposed that the Nile River Valley acted during history as a genetic corridor allowing gene flow between North and South in Eastern Africa1East Africa complexity can be seen in the fact that all families of continental African languages are represented in the region. Continental African languages have been classified into four major language families: Afro-Asiatic, Nilo-Saharan, Niger-Kordofanian (or Niger-Congo), and Khoisan. Afro-Asiatic, spoken predominantly by northern and eastern African pastoralists and agro-pastoralists, covering North Africa, includes the Semitic, Cushitic, and ancient Egyptian (Coptic) languages. Nilo-Saharan, spoken predominantly by eastern and central African pastoralists, includes in its main Chari-Nile branch the Central Sudanic and Eastern Sudanic  languages. Niger-Kordofanian, spoken predominantly by agriculturalist populations across western, eastern, central, and southern Africa, includes the Bantu languages3et al.6In an extensive and detailed study, Tishkoff Here, we focus on the region of Sudan and South Sudan with some other external related populations ; we refer as Sudanese Region to the ensemble see . The gen156ST to identify putative signals of adaptive selection in these populations, with a focus on immunological adaptation in anti-malarial, anti-bacterial and anti-fungal defence genes.The first aim of this study was to provide new insights into the genetic history of East African populations by analysing six Sudanese ethnic groups belonging to the main African linguistic families spoken in the region , in addition to ethno-linguistic neighbouring groups . We assessed the genetic diversity and relationships between these different ethno-linguistic groups to clarify the genetic history of East Africa. The second aim of the study was to use genetic distance estimated as FWe applied a principal component analysis (PCA) to investigate the population structure of the new populations genotyped in this study from the Sudanese region . PC1  with previously studied populations of special interest for this analysis: QatarTo test whether these particular sets of Immunochip SNPs (140 K and 14 K data sets) can recover population structure, we extracted 1000 Genome data from world-wide populations and observed that the genetic structure between them is maintained across the different data sets of SNPs used . In addiST statistic, a measure of global population differentiation, confirmed the PCA clustering  was found both in the pair Arabs and Nubians, located at the Nile River Valley, and in the pair Beja and Ethiopians, located at the coast. Among North-East populations, Nubians had the highest FST values when compared with Beja and Ethiopians (average FST of 0.006 and 0.007 respectively). South-West populations showed higher population differentiation among themselves than North-East populations. When comparing North-East populations with South-West populations, all comparisons have a high FST (between 0.044 and 0.054). Copts, with a strong individual heterogeneity, are more similar to Arabs (FST = 0.019) than to any other East African population. Copts and South-West populations are the most distant populations (FST > 0.1). Fulani had on average lower FST values when compared to South-West  than to North-East populations . These values show a complex situation beyond the simple North African versus Sub-Saharan Africa main differentiation.Pairwise Fustering . Populatoutliers . The lowST) between populations are correlated. We found a significant positive correlation between genetic and geographic distance .To test the hypothesis that geographically close populations are genetically similar, we performed a Mantel test to determine to which extent geographic and genetic distances , whereas the South-West cluster  share an ancestry component (light blue) with sub–Saharan samples. The North-East cluster  shows both components, although the main component (~70%) is that detected in North Africa and Middle East , supporting a common origin with Egypt (or other North African/Middle Eastern populations). They are known to be the most ancient population of Egypt and at k = 4 , they shk = 5 show their own component  than North African (<40%) or sub–Saharan (<15%) and at omponent . They ha3 statistics)−5): those of the North-East cluster  and Fulani. Populations from the North-East cluster: Beja, Ethiopians, Arabs and Nubians  between populations using the genetic variation in genes involved in defence against different agents. We selected among the genes genotyped in the Immunochip those associated with resistance/susceptibility to malariaST of those genes was compared to the mean FST of a set of randomly selected SNPs from genic regions with the same sample size and similar MAF, using a permutation test (10.000 permutations). All pairwise comparisons showed that the mean FST score of malaria-related genes was significantly lower than the mean FST score of the sampling distribution  and Afro-Asiatic Ethiopians . Y-chrom1et al.k = 6, which is different from the Sudanese component  related to Nilotes of South Sudan, but not to other Sudanese populations or sub-Saharan populations . This anomponent  and do nThe presence of the core of Nilo-Saharan languages in the confluence of the two Nile rivers suggests that the Sudanese region is the place of origin of the Nilo-Saharan linguistic family despite their fragmented distribution, as shown by the location of the Nubian language21Population displacement, whether it is followed with cultural or genetic exchange with local populations, would explain why not every Nilo-Saharan speaking group has this genetic component (as is the case of Nubians) and not every population that has it is mainly formed by Nilo-Saharan speakers (as is the case of Niger-Kordofanian speaking Nuba).k = 4  language and occupy a large area in Africa’s Sahel. Their origin is still controversial, as mitochondrial DNA indicates a West African and traces of North African origina-priori hypothesis was that selective pressures on host defence genes induced by similar infections would determine lower genetic distances between populations, as compared with a genome-wide distribution: a de facto convergent evolution in host defence. Similar signals of convergent evolution in the TLR1/2/6/10 cluster were recently reported between European and Rroma populations living in the same geographic areaThe second objective of our study was to analyse how infectious pressures affected the genetic variation of East African populations. The In this work, we analyse genotyping data for almost 140,000 SNPs in nine East African populations from Sudan, South Sudan and Ethiopia. Our main results add new and interesting features to the North East African genetic complexity, with new populations that define a genetic component in southern Nilo-Saharan speakers that cannot be related to a North-African or other sub-Saharan components. These populations should be included in further population genetics and epidemiological studies to have a representative sample of the genetic diversity of the region of East Africa. Moreover, a functional analysis shows similar genetics signals related to genes involved in antimalarial and antibacterial immune response. These findings suggest convergent evolution of the immune system of various ethnic groups in East Africa due to the major common selective pressures attributable to parasitic and bacterial infections acting on these populations.Saliva samples were collected from 500 individuals belonging to nine east African populations based on self-reported ethnicity. The samples used in the present research were collected and studied with ethical approval and informed consent. All experimental protocols were approved by the IRB of University of Medical Sciences and Technology in Khartoum and that of Universitat Pompeu Fabra  in Barcelona and were carried out in accordance with the approved guidelines. The population samples from the Sudan belonged to: the Afro-Asiatic ; the Nilo-Saharan ; and the Niger-Kordofanian linguistic families . In addition to these populations, we also collected samples from neighbouring populations: Nilo-Saharan speaking Nilotes (n=50) from South Sudan, and Afro-Asiatic speaking Ethiopians (n=40) currently living in Khartoum (Sudan). Samples from Niger-Kordofanian speaking Fulani (n=40), a nomadic group that usually traverse Africa’s Sahel, were also analysed. These samples were genotyped on the Immunochip (Illumina Infinium single-nucleotide polymorphism microarray), a custom-made, high-density genotyping array containing 195,806 single-nucleotide polymorphisms (SNPs) and 718 small insertion-deletionshttp://d-maps.com/carte.php?num_car=737&lang=en and http://d-maps.com/carte.php?num_car=740&lang=en, using Adobe Photoshop 7.0.1. software . Data are publicly available at the web page of JB (http://biologiaevolutiva.org/jbertranpetit/wp-content/uploads/2015/02/SudanImmunochip.zip) or under request.Nine samples were identified as being second or third-degree relatives by identity by descent (IBD) matrix (IBD > 0.185) and were removed from the analyses. SNPs with a call rate below 99% and on sexual chromosomes were also removed. After these steps, a total of 143,602 SNPs and 447 samples remained: 27 Copts, 36 Beja, 39 Ethiopians, 112 Arabs, 79 Nubians, 35 Darfurians, 37 Nuba (18 Nilotic and 19 Niger-Kordofanian speakers), 49 Nilotes, and 33 Fulani. This data set is further referred to as the “140 K” data set.For comparative studies, a Middle Eastern population (Qatar)To study the genetic relationships among East African ethno-linguistic groups, we used principal components analysis (PCA) as implemented in the Eigensoft packageST valuesST between populations. Mantel test was calculated using the R ADE4 packagePopulation differentiation was estimated using classical pairwise Fk = 2 through 10) were tested successively and the optimal value of k was estimated by ten-fold cross-validation. Clustering results were visualized with DistructPopulation admixture was analysed using ADMIXTUREthree-population test implemented in the ADMIXTOOLS software package3 , where a negative value of the f3 statistic implies that population X (target population) was the result of an admixture event between the two ancestral populations of A and B (source populations). We tried every combination of source populations for each of our nine target populations and estimated the mixing coefficient (α) with Yoruba as the outgroup population. It is the proportion of the admixture of the target population given by the source population A, while 1 − α is the proportion given by the source population B. For each comparison we kept the results with a significantly negative value of the f3 statistic after multiple testing correction .To formally test whether admixture happened within the studied populations, and to measure its extend, we used the To take advantage of the particular design of the array used, groups of functionally related genes were analysed to look for particular signals in a given population. Genes related with resistance/susceptibility to malariaST statistics. For each SNP we computed the FST between each pair of groups or populations using the BioPerl module PopGenST index is correlated with heterozygosity3435ST score of those genes was compared to the sampling distribution of the average FST value of a subset of randomly selected genomic SNPs with the same sample size and similar MAF values than those of the functional categories. P values were calculated using a permutation test (10.000 permutations).For every pair of groups  we detected unusual allele frequency differentiation among them by FHow to cite this article: Dobon, B. et al. The genetics of East African populations: a Nilo-Saharan component in the African genetic landscape. Sci. Rep.5, 9996; doi: 10.1038/srep09996 (2015).Supplementary Figures 1-6


Schistosoma japonicum remains a major challenge to human and animal health. Earlier microsatellite-based studies reported possible definitive-host-specific private alleles within S. japonicum, opening the possibility that different definitive hosts might harbour different parasite strains. Previous investigations have also detected near-identical multilocus genotypes in populations of adult worms - possibly the result of mutations occurring during the asexual (intramolluscan) phase of clonal expansion. Research has also revealed extensive deviations from Hardy-Weinberg Proportions (HWP) and conflicting results among studies. The present study was performed to examine some of the potential effects of infrapopulation structure on microsatellite-based studies of the transmission ecology of S. japonicum. Potential sources of bias considered included organotropic distribution of worms, non-random mating and corrections for clonal expansion.Stool samples from naturally infected hosts were used to infect snails in the laboratory and thereby expose mice. 274 individual worms were typed at seven microsatellite loci. Removal of individuals bearing duplicate MLGs  had an impact on both HWP and organotropic genetic differentiation. The study found no evidence that heterozygote deficiencies were caused by a Wahlund effect. Female-male pairings appeared to be random and there was no evidence for mate choice by heterozygosity. There was some indication that excess heterozygosity, induced by clonal expansion, can offset heterozygote deficiencies caused by small population size or populations fragmented by parasite control efforts.S. japonicum in China, where control efforts have greatly fragmented parasite populations.The view is supported that miracidia are preferable to adult worms in investigations into host-specific parasite lineages. Where adults must be used, extreme care should be taken with regard to sampling if infrapopulations of small animals are compared with those of larger animals; this is because of organotropic patterns in genetic variation and the tendency to sample from different organs in differently sized hosts. As corrections for clones may accentuate signals of population subdivision, corrections should only be made if tests for clonal expansion prove positive. Finally, evidence for heterozygote deficiency caused by small sample size, calls for carefully designed random and comprehensive sampling strategies for The online version of this article (doi:10.1186/s13071-016-1454-0) contains supplementary material, which is available to authorized users. Schistosoma (Trematoda: Digenea). At least 261 million people required preventive treatment for schistosomiasis in 2013 and the disease can cause serious and debilitating illness ; hSchistosoma spp. can differ )n differ . Similarn differ . These pearchers  noted thearchers  if male earchers –12, rathS. japonicum. Significant heterozygote deficiencies, across all or most loci sampled, have been reported for miracidia . This suggests that in the present study, and perhaps in earlier studies, inbreeding or non-random mating may be responsible for the observed deficiencies in heterozygotes, rather than population structure or age structure . Inbreeding or non-random mating may be a feature of S. japonicum in China as control measures drive down prevalence and populations become fragmented and isolated.Unlike most earlier studies involving microsatellite variation in ost loci , 13, 15 ost loci . The difaponicum , 16. At .e. FST) . Consequtson’s r ) the comtson’s r . A plot S. japonicum populations  is offset by the excess induced by clonal reproduction at the intramolluscan stage. Should clonal expansion be having an effect on heterozygosity, one would expect to observe an increase in allelic diversity with concomitant decrease in genotypic diversity [t-test, t = 2.6241, P = 0.0475); this supports the view that clonality and/or the inclusion of niMLGs has an offsetting effect on a naturally occurring deficiency of heterozygotes in S. japonicum populations.Clonal reproduction, or rapid population expansion from a small number of individuals with many offspring, as might occur during cercariogenesis at the intramolluscan stage of the life-cycle, is expected  to generiversity . Indeed,ST value between them (0.026) and the difference in extent of genetic variation  heterozygote excess at only two loci (and slight excess at two more), the females showed excesses at all loci, with four out of the seven being moderate. Although, according to earlier studies [Schistosoma) spp. but further work to map the loci to the sex chromosomes would be required to support this.In addition to clonality, an excess of heterozygotes can result from a small effective population size. As already mentioned this scenario might be expected to arise in the face of persistent disease control in China. A common explanation for this is that excesses occur when allele frequencies differ between the sexes in small populations . Indeed, studies , the mic studies . The patS. japonicum must be based. Small sample size alone can effect an “excess” heterozygosity proportional to 1/2 N (where N is sample size) [S. japonicum, where samples tend to come from accessible villages, or areas where transmission is known, and sampling is costly and therefore limited. Even where many stool samples are taken or many infected snails collected, these may represent the reproductive output of a small number of adults – a community; this leads to a situation where the small number genotypes, all of which are quite discrete, leads to a slight heterozygote excess in the progeny. Larger samples would lead to mixing of several such communities, thus effecting a counterbalancing “Wahlund effect”. Indeed, a recent study has sought to develop a correction method for such “family structure”, as this was found to lead to heterozygote excess in studies of Schistosoma mansoni based on miracidia [A simpler, and perhaps equally valid, explanation for the excess heterozgosity observed in earlier studies is the small sample sizes upon which all studies of natural populations of le size) . In addiiracidia . Unlike iracidia . DiffereST values were low between sub-populations, except for the value of 0.0254 between Liver and Mesentery. The lack of differentiation between Hpv and Liver might be attributable to their close proximity and difficulties in determining the precise origin of worms in that anatomical region; however, there was a similar lack of differentiation between Hpv and Mesentery. The differentiation between the Liver and Mesentery sub-populations may be explained by the fact that immature and/or unpaired males tend to predominate there. It could be that differing MLGs between the sexes lie at the root of the difference. The G’ST values for the males and females taken separately lend support to this possibility; these being 0.027 and −0.052, respectively . The mating studies indicated that unpaired males were more heterozygous than paired males, although this was not signifincant, it could add to heterozygote excess in samples from small rodents, where the liver and Hpv are often preferentially sampled. Figure S. japonicum.G’In addition, there was some evidence for variation between infrapopulations of different laboratory host individuals. The chi-squared test comparing infrapopulations across mice was significant and DAPC indicated that the infrapopulations of two the mice may be genetically distinct populations. In view of this it also appears necessary to use laboratory animals that are as homogeneous as possible and to check patterns of MLGs for such host-induced effects.ST). Nevertheless, removal of clones/niMLGs did improve the success of assignment of worms to sub-populations by DAPC, and sub-populations appeared more cohesive and distinct in PCAs for datasets with clones removed. There was no evidence that the removal of clones inflated FST between populations by random loss of MLGs from a sub-sample of the data; however, it is still possible that the reduction in sample size effected departures from HWP. In view of the impact of correcting for clones, it is recommended that studies likely to be affected by this phenomenon should report their results for both corrected and original datasets, as which dataset is best may depend on the aims of the particular study; for example, the relative contribution of clones to a population may be of interest. In addition, compliance with HWP is only critical if subsequent analyses assume HWP.The results have shown that the presence of clones arising from the amplification of parasite numbers during cercariogenesis at the intramolluscan stage, and mutations generating niMLGs at this stage, has a significant impact on heterozygosity, with more loci likely to deviate from HWP. Comparisons of allelic and genotypic diversity between the raw and clone corrected datasets revealed a significant signature of clonal reproduction. Indeed, the effects of clonal expansion appear to be able to offset any deficiency in heterozygotes present in a clone corrected dataset. The presence of clones/niMLGs appeared to have less potential impact on measures of genetic divergence among subpopulations (e.g. FST 0.024). The effect might be more pronounced if more slowly maturing males represent a distinct strain. The implications of this are that sex-based differences in allele frequencies together with the predominance of males (often immature) in the liver, could lead to biased sampling of worms where infrapopulations of small animals (e.g. rodents) are compared with those of larger animals (e.g. bovines), because the livers and hepatic-portal-veins of small animals are more easily sampled than the mesenteric blood vessels, and the mesentery tends to be the preferred source of worms when sampling larger animals. The present authors have also found that the livers are generally sold as food, with the mesentery being a cheaper and more available source of worms. Consequently, the recommendations of this study are that extreme care is taken to avoid such sampling bias when comparing adult worms between definitive host groups and that results be examined both with and without removal of duplicate MLGs (attributed to clones). Corrections for clones should only be made if tests for the effects of clonal expansion prove positive , and the full dataset used wherever possible. The findings support the case for sampling at the miracidial stage, when studying parasite divergence among host-groups, so as to avoid problems of clonal expansion and niMLGs, possible selection though immune responses of laboratory hosts, as well as sampling bias in collecting worms from hosts. The detection of heterozygote deficiencies probably due to a fragmented and isolated population structure effected by prolonged snail control measures, also calls for a need to employ random and comprehensive sampling strategies for S. japonicum in China. Although, the use of miracidia is preferable in the study of host-group specific parasite lineages, it is not suitable for study of transmission ecology and the intramolluscan processes. Additional work is required to develop methods for the study of the entire transmission process for S. japonicum.The study found no evidence that heterozygote deficiencies, found in the clone corrected dataset and typical of earlier studies, were caused by a Wahlund effect. The results did, however, include patterns of heterozygosity differences between the sexes that were consistent with some loci being in pseudoautosomal regions of the sex chromosomes. Mating appeared to be random, with no evidence for assortative or disassortative mating, nor any preference for more heterozygous mates. There was little distinction between the mesentery and hepatic-portal-vein sub-populations; however, the sub-population of the liver formed a relatively discrete sub-population. It is possible that this distinction was, at least in part, due to the predominance of males in the hepatic sub-population, as the males and females showed some genetic differentiation (G’S. japonicum can only be constructed from the combined results of several population genetic studies covering all of these variables.The present study was limited to a single field sample and to a particular set of microsatellites. The findings may vary with different microsatellite loci or additional samples from the same or other areas, provinces or habitats, and also with other definitive host types as the source of eggs. In addition, the deviations from HWP reflect mating and union of gametes in the previous generation only. Consequently, further work is required to investigate the structure of infrapopulations across generations, regions, habitats and host-groups, and with different microsatellite loci. A true picture of the transmission ecology of


Trogia venenata was the leading culprit for SUDs. At present, relatively little is known about the genetics and natural history of this mushroom. In this study, we analyzed the sequence variation at four DNA fragments among 232 fruiting bodies of T. venenata collected from seven locations. Our ITS sequence analyses confirmed that all the isolates belonged to the same species. The widespread presence of sequence heterozygosity within many strains at each of three protein-coding genes suggested that the fruiting bodies were diploid, dikaryotic or heterokaryotic. Within individual geographic populations, we found significant deviations of genotype frequencies from Hardy-Weinberg expectations, with the overall observed heterozygosity lower than that expected under random mating, consistent with prevalent inbreeding within local populations. The geographic populations were overall genetically differentiated. Interestingly, while a positive correlation was found between population genetic distance and geographic distance, there was little correlation between genetic distance and barium concentration difference for the geographic populations. Our results suggest frequent inbreeding, geographic structuring, and limited gene flow among geographic populations of T. venenata from southwestern China.During the past 40 years, more than 400 Sudden Unexplained Deaths (SUDs) have occurred in Yunnan, southwestern China. Epidemiological and toxicological analyses suggested that a newly discovered mushroom called  Since 1978, more than 400 apparently healthy persons have died suddenly without any obvious causes in Yunnan, southwestern China . Those STrogia and named the LWM Trogia venenata. At present, this species is only found in the mountains in central and western parts of Yunnan Province.Morphologically, the fruiting body of LWM is white when fresh and looks like a ginkgo leaf. Its fruiting body is usually about 5cm long and 3cm wide, typically found as clusters on rotten wood. Fruiting bodies of LWM are fragile and easy to break. Based on its morphological features and sequences at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene, Yang et al.  classifiAgaricus species, Lepiota cristata, etc) [T. venenata from Yunnan will also show frequent gene flow and limited geographic differentiation.Previous studies have shown that saprophytic fungi are generally opportunistic in their ecological distributions and are found wherever substrates (e.g. dead organic matter such as rotten wood) and growth conditions permit . Indeed,ta, etc) . Such geta, etc) . Here, wCryptococcus gattii [C. gattii [L. cristata, MGGA identified long distance dispersal and recombination [Multilocus sequence typing (MLST)  and mults gattii . In addi. gattii , 16. In bination .T. venenata from seven sites in Yunnan province representing the broad geographic distribution range of SUDs. ITS sequences were first obtained from representative samples at each of the seven sites to confirm species identity. These samples were then analyzed for sequence variation at the following three protein-coding genes: the second largest subunit of RNA polymerase II gene (rpb2), the translation elongation factor 1-α gene (tef1-α), and the β tubulin gene (β-tub). The sequences were analyzed to identify the patterns of genetic variation within and among populations. In addition, we also analyzed the putative relationships between genetic differentiation and the concentration of the heavy metal barium related to the seven sampled sites in T. venenata. A high concentration of barium in this mushroom was originally suspected as a major contributor to SUDs [T. venenata was unlikely a contributor to SUDs [In this study, we collected 232 fruiting body samples of  to SUDs . DespiteBased on the patterns of SUDs from earlier epidemiological surveys, we chose seven sites for this study. These sites cover the known geographic distribution of SUDs and are located in five counties: Tengchong, Dayao, Xiangyun, Binchuan and Heqing in Yunnan Province. All seven sites are located in difficult-to-access and remote mountainous regions. The fruiting bodies of our samples were obtained from the surface of tree stumps, fallen tree trunks, and rotten wood. Of these seven sites, five were from forests close to five villages that had reported cases of SUDs while two were from communities that had never reported SUDs . The sevThe genomic DNA of each of the 232 samples was individually extracted from dried fruiting bodies using the CTAB method slightly modified for fungi . To confITS) regions of the nuclear ribosomal RNA gene cluster, rpb2, tef1-α and β-tub. PCR amplifications of these four fragments were conducted using the following primer pairs: ITS4 (5' TCCTCCGCTTATTGATATGC 3') and ITS5 (5' GGAAGTAAAAGTCGTAACAAGG 3') for the ITS region; RPB2-577f (5' TCAGTCCAGAGGTTTCGGTTGT 3') and RPB2-577r (5' GACCGTGTCGTAGCAAGA 3') for the rpb2 gene; TEF-630f (5' CTTGTTCTAGTGGAGCGAGGAT 3') and TEF-630r (5' TTGGCAGGGTCGTTCTTC 3') for the tef1-α gene; TUB-s (5' ATCAC(A/T)CACTCICTIGGTGGTGG 3') and TUB-r (5' CATGAAGAA(A/G)TGIAGACGIGGG 3') for the β-tub gene. The ITS primers were optimized from White et al. [Psathyrella gracilis [ITS); KT971373 ~ KT971604 (rpb2); KT971605 ~ KT971836 (tef1-α); KT971837 ~ KT972068 (β-tub).In this study, the following four nuclear DNA fragments were analyzed: the internal transcribed spacer  using a BLAST search [Raw ITS sequences using both the forward and reverse primers were assembled for each isolate and edited using SeqMan software (DNA STAR package) . These sT search . Here, bT search  to inferT search . Only unITS within the ribosomal rRNA gene cluster within fungi, the heterozygous sites observed within a diploid/dikaryotic individual for the ITS gene fragment could be due to variations among the copies within an individual ribosomal RNA gene cluster and not between homologous chromosomes [rpb2, tef1-α and β-tub. These three genes have been widely used for fungal phylogenetic and population genetic studies and they are commonly considered single-copy genes within a haploid nucleus in fungi.Due to the multiple copy nature of omosomes . Thus, oT. venenata are not known. However, in the majority of basidiomycete fungi, their sexual fruiting bodies are developed from dikaryotic or heterokaryotic mycelia. Thus, the genotype information from the nuclear genome of fruiting bodies is typically considered equivalent to a diploid. Evidence for diploidy in wild mushrooms can be found in the sequencing chromatographs. Specifically, the presence of heterozygous nucleotide sites in a sequencing chromatograph for a single-copy gene in a mushroom is commonly considered as evidence that the mushroom developed from a diploid, dikaryotic or heterokaryotic mycelia. The analyses of diploid genotypes in population genetics differ from those of haploid organisms. For example, in haploid organisms, the allele information at a single gene fragment can be directly observed from the DNA sequences. In contrast, in diploid organisms, the allele information can’t be directly observed from the polymorphic nucleotide sites due to uncertainty in the relationships among polymorphic nucleotide sites within each sequenced gene fragment.At present, the mating system and reproductive life cycle of FST values [Thus, to analyze the three nuclear gene sequence information fully, we first inferred the putative haplotype sequences for each individual mushroom at each of the three nuclear loci respectively using the Bayesian method implemented in the program PHASE 2.1 . The infT values  and deteT values . The locT values .http://clumpak.tau.ac.il and our STRUCTURE output files were uploaded as a compressed file (.zip) to the site directly to conduct the analysis.Aside from the above population genetic analyses, we also estimated the number of genetic clusters using the program STRUCTURE version 2.3.3 . The STRThe modes of reproduction for fungi in nature are commonly inferred based on the allelic relationships in natural populations. In diploid/dikaryotic/heterokaryotic organisms, there are two types of allelic relationships. The first is between alleles from the same locus, the commonly known Hardy-Weinberg equilibrium. The null hypothesis of the HWE test is that the observed genotype frequencies at each locus are not significantly different from those expected under the null model of random association. Here in this study, tests of HWE at each locus for each geographic population were conducted using the program GenAlEx version 6.5. For loci that showed significant deviations from HWE, we further compared whether the observed heterozygosity was significantly higher or lower than those expected under random mating. In this test, Ho is the observed heterozygosity, He is the expected heterozygosity and the Fixation Index F  exhibits values ranging from -1 to +1. Values close to zero are as expected under random mating, while substantial positive values indicate inbreeding. Negative values indicate excess of heterozygosity, due to negative assortative mating or significant heterozygote advantage.T. venenata. In our tests, because the geographic samples are genetically differentiated (see below in A). Because IA can be influenced by the number of polymorphic loci , we also standardized the IA value by the number of loci to generate the rBarD value for comparisons among populations. The null hypothesis for IA is that there is random association  among alleles (or diploid genotypes in our case) at different loci, a P-value of <0.05 would indicate that the null hypothesis should be rejected. Phylogenetic incompatibility is another indicator of recombination at the population level. The observed value was compared to that of 500 randomizations in which individuals were randomized across populations.The second test is linkage equilibrium, assessing the associations among alleles at different loci. In diploid organisms, the test is also called genotypic equilibrium where the observed allelic relationships within a locus are fixed and only associations among the diploid genotypes at different loci are tested. Fixing the diploid genotype at each locus in this test is to eliminate the effects of potential deviations of HWE on the genotypic equilibrium calculations. The program MULTILOCUS version 1.3b  was usedFST values. The quantitative relationship between genetic difference and geographic distance were assessed using the Mantel test [DNA sequence divergence among populations was estimated using pairwise tel test . The geoThe GenBank accession numbers for the sequences generated in this study are KT967978 to KT968080 for the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster; KT971373 to KT971604 for the second largest subunit of the nuclear RNA polymerase B gene (rpb2); KT971605 to KT971836 for the translation elongation factor 1-α gene (tef1-α); and KT971837 to KT972068 for the β tubulin gene (β-tub).T. venenata . T. venenata and its closely related species Trogia infundibuliformis. Here, we used the ITS sequence of Clitocybula oculus as the outgroup. Only bootstrap values greater than 90 are shown for the NJ tree. Our results showed that one ITS sequence type was found in five geographic populations  while most others were geographically more limited  trees containing all the representative haplotype sequences for the three gene fragments in each population, with one tree for each of the three gene fragments, were obtained using PAUP*4.0b10 –S3 Figs.rpb2 gene fragment, we found 19 variable sites. PHASE analyses identified a total of 45 haplotypes among these 232 strains. None of the 45 haplotypes at the rpb2 locus were shared by all seven geographic populations. The most frequent haplotype was HAP24 and it was found in four local populations . The second most frequent haplotype was HAP30 and most of the strains in the XY population shared this haplotype. Six of the 45 inferred haplotypes were found only once each. The phylogenetic relationships among the haplotypes are presented in Among the 497 aligned nucleotide sites for the tef1-α gene fragment, the 563 aligned nucleotides contained seven variable sites and a total of eight haplotypes in the T. venenata populations. Among them, HAP2 was the most frequent, representing about 70% (324/464) of all the haplotypes at this locus and it was distributed in six of the seven local populations except for JT. However, more than one third of HAP2 was found in the SY population. Another frequent haplotype was HAP4, most abundantly distributed in the JT population but also found in four other local populations. Five of the eight haplotypes  were only found in one local population each, with HAP3 found only in one fruiting body in the LXJ population .The haplotype information obtained above was used to analyze the population structure of Our analyses identified abundant genetic variations within each of the seven local populations. The overall genotypic diversity for the whole sample of 232 individuals was 0.83, suggesting that over 83% of the time, two randomly drawn fruiting bodies from the total population will have genotypes different in at least one of the assayed loci. However, a range of genotypic diversity was found among the seven local populations, from a low of 0.15 in Xiangyun to a high of 0.98 in Jizushan . The resFST values between pairs of local populations was found  was found between Jizushan and Lixiji, separated by about 67 kilometers, while the highest (0.615) was found between Xiangyun and Jietou, separated by about 221 kilometers. Overall, the Mantel test results showed a significant positive correlation between genetic difference and geographical distance among these seven populations (P = 0.018)  .The results of AMOVA also suggested significant genetic variations within populations . The anaThe number of clusters K = 4 was inferred because the standard deviation of posterior probability was the lowest for that K . Our anarpb2 locus, with the observed heterozygosities lower than those expected under the hypothesis of random mating. For the tef1-α locus, two populations (JT and XY) were monomorphic, one population (SY) showed slight excess of observed heterozygosity while four  had excess of homozygotes. For the β-tub locus, four local populations  showed excess homozygosity while three  had slight excess of heterozygosity.Based on the genotypes and allele frequencies for the seven populations, we assessed whether the local populations were in HWE, as would be expected if the local populations were randomly mating. Our overall results rejected the null hypothesis of HWE for each of the seven local populations . We thenT. venenata. For the total samples, the observed rBarD value was 0.049 (P = 0.962), and for the clone-corrected sample, it was 0.046 (P = 0.974)  as an important component of their reproduction that generates significant HW disequilibrium within loci and linkage disequilibrium among loci.In addition to HWE tests, the SNPs were also analyzed for their associations with each other using the MULTILOCUS software. SNPs located within the same DNA fragment were considered genetically linked. In this analysis, each variable nucleotide site was treated as a locus and different nucleotides at the same site were viewed as different alleles. The results of our analyses were consistent with recombination in natural populations of = 0.974) . These r= 0.974) . However= 0.974) , consistT. venenata populations from Yunnan province is unknown. Here, we evaluated the relationships between these two variables to examine whether there is any potential correlation between them for the seven local populations. Our Mantel test showed a slightly negative correlation between levels of barium concentration and their genetic distances  that we sequenced were polymorphic in our samples. Among these four fragments, the ITS fragment from direct PCR and sequencing showed a large number of heterozygous sites in chromatograms in 129 of the 232 isolates (55.6%), rendering their chromatograms unreadable for significant portions of the gene fragment. The unreadable chromatographs were not due to base substitutions at one or a few single nucleotide sites, because base substitution polymorphisms are almost universally readable as was shown in our sequences of the three protein-coding genes. Rather, they were due to prevalent insertions and deletions, as shown by the alignments of the 118 ITS sequences (103 from direct PCR sequencing and 15 from cloning the PCR product first followed by sequencing). The high failure rate (55%) in direct sequencing of PCR products of the ITS suggested that this locus is not a good barcode for species identification of T. venenata using PCR amplification followed by traditional Sanger sequencing method. However, the frequent within-strain ITS sequence heterogeneity should not be a problem for barcode identification if next-generation sequencing methods such as Roche 454 and Illumina are used. Given that the dominant approach for ITS sequence-based specimen identification of fungi is Sanger sequencing, other markers e.g. the three protein-coding genes analyzed here, should be explored as alternative barcode markers. Specifically, the patterns of intra- and inter- species sequence variation should be analyzed at these loci for the taxa closely related to T. venenata to help define the barcode gap among the closely related species in this genus.All four gene fragments  while the remaining six geographical populations each had genotypic diversities ranging from 0.92 for the JT population in Tengchong to 0.98 for the JZSH population in Binchuan  were mainly based on haplotype and genotype frequencies at the analyzed loci. However, STRUCTURE analyses were mainly based on the patterns of allelic and haplotype associations within and among individuals. The different results in the two approaches can be shown by the relationships between two local populations, Jietou and Qushi, from Tengchong. These two populations were located very close to each other. FST analysis showed that they were statistically significantly differentiated due to differences in allele frequencies. However, STRUCTURE analyses grouped them into the same genetic cluster due to their shared alleles and haplotype associations at the three analyzed loci.Our STRUCTURE results were overall consistent with the rpb2, tef1-α and β-tub) had haplotypes that are broadly distributed among several local populations. For example, at the rpb2 locus, HAP24 was shared among four local populations  while HAP30 was found in three local populations. At tef1-α, HAP2 was found in six of the seven local populations while HAP4 was distributed among five geographic populations. Similarly, HAP1 of the β-tub locus was distributed in four local populations .Despite the observed genetic differentiation, our analyses based on data from the three gene fragments (haplotype inferences and AMOVA test) also showed evidence for gene flow. Specifically, the haplotype comparisons identified that each of the three loci  is the more likely explanation. In addition, the excess heterozygotes observed at the few locus-population combinations were likely due to selection against homozygotes. For example, in the saprophytic button mushroom Agaricus bisporus, homozygote disadvantage has been demonstrated, consistent with inbreeding depression [At present, there is no information about the mating system and reproductive behavior of pression .R-amino-4S-hydroxy-5-hexynoic acid and 2R-amino-5-hexynoic acid, are likely the main contributors to the toxic effects of T. venenata in humans [T. venenata could not explain all the SUDs cases. Other factors such as human host genotypes and environmental factors were also likely involved. Even with the two toxins, we know almost nothing about the genes and molecular processes involved in their synthesis and their detailed toxicity mechanisms.We observed no association between genetic distance and barium concentration difference. Our result further suggested that barium is unlikely an important factor in the biology and toxicity of these mushrooms. The current evidence suggests that two unusual amino acids, 2n humans , 55. Hown humans , the twoCandida albicans [Cryptococcus gattii [Aspergillus terreus [T. venenata contain different concentrations of these two toxic amino acids. And, if the toxin concentrations were different, could they explain the differences in SUD mortality rates among the communities? Because these two amino acids are presented at very low concentrations in fruiting bodies of T. venenata [Previous studies have shown that genetically divergent populations of human fungal pathogens such as albicans , Cryptocs gattii , 57, and terreus  can diffvenenata , large aT. venenata from southwest China is a diploid, dikaryotic or heterokaryotic species. Among the four sequenced gene fragments, the ITS fragment had a large number insertions and deletions, making the ITS sequence chromatographs from direct PCR and sequencing reactions of many fruiting bodies unreadable. There was abundant sequence variation within and among isolates at the three protein-coding genes and the geographic populations were overall genetically differentiated. Our analyses suggest prevalent inbreeding and low levels of gene flow among the local populations. The lack of statistically significant correlation between population genetic distance and barium concentration difference among the seven geographic populations of T. venenata suggest that the genetic differentiation observed at the three loci among local populations is unlikely a significant contributor to the toxic effects of T. venenata in humans.In conclusion, the newly discovered poisonous mushroom S1 FigA total of 45 unique rpb2 haplotypes were found in our samples. For each rpb2 haplotype (HAP), the first number represents the haplotype assignment; the characters after represent the county/community from where the strains were sampled; the last number represents the total number of strains belonging to the specific haplotype in that local population. Only representative sequences of unique haplotypes from each location are shown.(TIF)Click here for additional data file.S2 FigA total of 8 unique tef1-α haplotypes were found in our samples. For each tef1-α haplotype (HAP), the first number represents the haplotype assignment; the characters after represent the county/community from where the strains were sampled; the last number represents the total number of strains belonging to the specific haplotype in that local population. Only representative sequences of unique haplotypes from each location are shown.(TIF)Click here for additional data file.S3 FigA total of 41 unique β-tub haplotypes were found in our samples. For each β-tub haplotype (HAP), the first number represents the haplotype assignment; the characters after represent the county/community from where the strains were sampled; the last number represents the total number of strains belonging to the specific haplotype in that local population. Only representative sequences of unique haplotypes from each location are shown.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Genome sequencing of the 5,300-year-old mummy of the Tyrolean Iceman, found in 1991 on a glacier near the border of Italy and Austria, has yielded new insights into his origin and relationship to modern European populations. A key finding of that study was an apparent recent common ancestry with individuals from Sardinia, based largely on the Y chromosome haplogroup and common autosomal SNP variation. Here, we compiled and analyzed genomic datasets from both modern and ancient Europeans, including genome sequence data from over 400 Sardinians and two ancient Thracians from Bulgaria, to investigate this result in greater detail and determine its implications for the genetic structure of Neolithic Europe. Using whole-genome sequencing data, we confirm that the Iceman is, indeed, most closely related to Sardinians. Furthermore, we show that this relationship extends to other individuals from cultural contexts associated with the spread of agriculture during the Neolithic transition, in contrast to individuals from a hunter-gatherer context. We hypothesize that this genetic affinity of ancient samples from different parts of Europe with Sardinians represents a common genetic component that was geographically widespread across Europe during the Neolithic, likely related to migrations and population expansions associated with the spread of agriculture. The analysis of the genome of the Tyrolean Iceman, a 5,300 year old mummy from Central Europe, revealed a surprising recent common ancestry with modern Sardinians for this ancient genome. However, this study was limited both by the availability of data from Sardinians and by a lack of genomic data from other ancient European samples. Here, we use genomic data from modern Sardinians and from ancient European individuals from different geographic regions and cultural contexts, to demonstrate that this ancestry component is shared among individuals associated with the onset of agriculture in Europe. Our results thus suggest that the Iceman's Sardinian ancestry actually reflects a more widespread genetic component related to the migration of people during the Neolithic transition in Central Europe. Subsequent analyses revealed that the remains were from a ∼45-year-old male, with a Most studies of the genetic structure of prehistoric European populations to date have described patterns of variation in the mitochondrial DNA (mtDNA), primarily due to the limitations of sequencing the nuclear genome from ancient samples with low endogenous DNA contents. Nevertheless, the limitation of only investigating the maternal lineage has been offset by the capacity to investigate larger sample sizes, both spatially and temporally distributed, which has yielded considerable insight into the genetic history of Europeans brana1) from the Iberian Peninsula ajv70) from Sweden gok4) from the same study as the Swedish hunter-gatherer P192-1, K8) We compiled several sets of single nucleotide polymorphism (SNP) genotype data for our analysis. For each dataset, data from the ancient genomes was merged into a reference set of modern populations, using only SNPs present in the modern data. Three complementary reference datasets were used: SNP array genotype data from the Human Genome Diversity Panel (HGDP); high coverage whole-genome sequencing data from Complete Genomics (CG); and low coverage whole-genome sequencing data from the 1000 Genomes project and Sardinians (1000G/Sardinia). For the 1000G/Sardinia dataset only population-level allele frequency data was used, to circumvent lower genotype accuracy in the low-coverage experimental design. Although the CG dataset does not include individuals from Sardinia, we included it to investigate the relationship of the ancient individuals with Eurasians using high-coverage whole-genome data. The ancient genomes we used comprised both previously published and newly generated data. In addition to the Iceman genome, we included partial genomic DNA data from five individuals distributed across Europe . We broak ranging from k = 2 to k = 8   , indicatTreeMixWe then investigated the relationship between the Iceman and all pairs of European populations in the 1000G/Sardinia dataset as described above. We find that all significant tests fall into one of two categories, involving either the Finns (FIN) or Sardinians . In all To gain further insight into the demographic history of the Iceman, we used a coalescent-based method k, ancestry proportions are similar among the different ancient samples, and comparable to other European populations. We note that at k = 3, populations from Southern Europe show a small proportion of ancestry related to Middle Eastern populations, which is absent from the two hunter-gatherer (HG) individuals, but present in the farmer (F) individuals at a comparable level  from the F group , which was discovered geographically close to contemporary Basque populations, also shows the highest proportion of Basque ancestry. For the Thracian individuals from Bulgaria, no clear pattern emerges. While P192-1 still shows the highest proportion of Sardinian ancestry, K8 more resembles the HG individuals, with a high fraction of Russian ancestry. Because those two individuals also had the lowest number of SNPs, we wanted to investigate the accuracy of cluster assignments from the modern individuals. We inferred ancestry proportions for each modern individual for all SNP subsets corresponding to the ancient samples, and calculated the root mean squared error (RMSE) per population and k value compared to the full dataset. We find that the accuracy is very high for the Iceman (RMSE< = 0.01) and remains high for gok4 and the HG samples , where all but one comparisons are non-significant (Z< = 3) , whereas ajv70 splits after the FIN but before other Northern Europeans (95% bootstrap) . Finally, although K8 clusters with Northern European populations, its position in the tree is not resolved (3% Bootstrap). We note however that despite the reduced number of SNPs for K8, the relationships among the modern populations are consistent with the full dataset and generally well supported (bootstrap >90%), except within the Southern European group (minimum bootstrap 53%). It is therefore possible that the inconclusive pattern for K8 either reflects a possible higher level of modern DNA contamination  , whereas(Z< = 3) , again lotstrap) . In the tion see  or a morTreeMix analysis of the merged dataset with the Iceman described above, allowing up to five mixture events. The first four inferred edges are highly significant (p<10−10 for m = 1 to m = 4), the last one however to a much lesser extent (p = 0.009). In addition, both the amount of variance explained and the residuals do not change substantially after m = 4, indicating possible issues with overfitting the model at m = 5. We therefore report estimates of the tree with four inferred mixture events in the remainder of this section. The first inferred edge corresponds to sub-Saharan African admixture in Southern European populations (edge weight w = 0.027), which is consistent with previous estimates of 1%–3% sub-Saharan African ancestry in those populations via North African gene flow w = 0.075) to present-day Japanese (JPT), consistent with evidence of circumpolar gene flow m = 3 migrations, an edge is added between the ancestral Europeans and the TSI, with a surprisingly high proportion (w = 0.35) (w = 0.03). The LWK have previously been reported as showing a signal of gene flow of possible Neolithic Middle Eastern or European origin To further investigate the history of the populations in the 1000G/Sardinia dataset, we investigated patterns of admixture. We first turned to the  = 0.35) , which i3f statistics 3f for all populations in the 1000G/Sardinia dataset as targets, using two sets of combinations of source populations. In the first set, we use all pairs of remaining modern populations as potential sources, whereas in the second analysis, we always use the ancient samples as one of the source populations. We find a large number of significant tests (Z≤−3), which we group based on interpretation , which remains significant if we replace the non-Africans with either HG or F ancient samples (max Z = −3.4). The TSI show the signature of African admixture as described when using CEU, GBR or Sardinia as European source populations (max Z = −4.4), whereas for the IBS only YRI and GBR as source populations gives a significant result (Z = −3.4). We also confirm the Asian-related admixture in the FIN when using the other Northern European populations as one source (max Z = −5.8). The remainder of the significant results within Europe follows the pattern of a south-to-north gradient of genetic similarity, with Sardinians and Finns at either ends of the gradient (Sardinia – IBS/TSI – CEU/GBR – FIN), consistent with a model of isolation by distance as previously described 3f tests using Sardinians as one source population and TSI/IBS as the target population are significant irrespective of which Northern European population is used as the other source  are significant. A complementary explanation suggested by recent results would be a scenario of increasing levels of admixture between arriving farmers and local HG populations during the Neolithic transition 3f test. This is indeed what we observe in the majority of the cases. For example, tests using either TSI or IBS as target population remain largely significant if we replace Sardinians with the Iceman as one of the source populations. In addition, in tests with CEU or GBR as target populations, the HG samples ajv70 and brana1 can replace the FIN, whereas the Iceman and P192-1 from the farmer groups can replace the southern source populations , Z = −25.4), tests using the HG samples only reach significance with the TSI as the second source population. Taken together, these results suggest that the observed patterns can at least in part be explained by different levels of HG ancestry proportion in Northern versus Southern Europe, as previously suggested We proceeded to investigate these signals of admixture in a less parameterized way, using retation , S5. A lr source , whereasulations . HoweverThe HGDP dataset also contains genotype data for Neanderthal, Denisova, and Primate samples in addition to modern humans. We therefore used this dataset to perform PCA as previously described The discovery of a genetic affinity between the Iceman and modern-day Sardinians showcased both the power and some of the remaining limitations of the availability of nuclear genomic data from historical human samples gok4 and the Thracian P192-1 closely resemble the Iceman in their relationship with Sardinians, making it unlikely that all three individuals were recent migrants from Sardinia. Furthermore, P192-1 is an Iron Age individual from well after the arrival of the first farmers in Southeastern Europe , perhaps indicating genetic continuity with the early farmers in this region. The only non-HG individual not following this pattern is K8 from Bulgaria. Interestingly, this individual was excavated from an aristocratic inhumation burial containing rich grave goods, indicating a high social standing, as opposed to the other individual, who was found in a pit The results of the analyses including additional ancient genomes provide mounting evidence that the Iceman's genetic affinity with Sardinians reflects an ancestry component that was widespread in Europe during the Neolithic. Despite their different geographic origins, both the Swedish farmer et al. argue for a substantial contribution of Mesolithic settlers, but also find evidence for a later contribution of people from the mainland during the Bronze and early Copper Ages, which is compatible with the age of the farmer individuals analyzed here Sardinians have long been recognized as forming a distinct outlier within contemporary European genetic diversity e.g. , often ik = 3), they generally resemble Southern European populations rather than Middle Eastern ones. A possible explanation for this observation could be that there was an initial wave of migration from the Middle East into Southeastern Europe during the early Neolithic transition, followed by a subsequent expansion of a derived Southern European population with some fraction of Middle Eastern ancestry towards Central and Northern/Western Europe  is significantly negative , indicating that the Iceman is more closely related to the Swedish farmer. However, the test D shows an even stronger signal in the same direction , which would suggest that the Iceman is also more closely related to the Iberian hunter-gatherer than to Sardinians. Given the observed discontinuity in the relationship of hunter-gatherers and farmers with modern Europeans, we believe that this pattern is at least in part due to unaccounted biases as described above. We therefore did not analyze the relationship of more than one ancient sample combined with modern samples using D-tests throughout the study. The aforementioned issues also highlight the need for improved statistical methods that will allow the analysis of ancient and modern samples in joint fashion, while at the same time accounting for differences in data quality and biases due to DNA damage and differences in sequencing technology. The incorporation of these methods with additional genomic and archaeological data from key spatial and temporal points will be crucial in the quest to disentangle the complex population history of the European continent.Finally, an important caveat with analyses based on allele sharing such as the D-test is their sensitivity to confounding factors such as correlated errors in a pair of samples used in the test The two individuals from Bulgaria used in this study originate from two separate excavations, both associated with Iron Age Thracian culture. The first individual (P192-1) was excavated from a pit sanctuary near Svilengrad, Bulgaria, dated to 800–500 BCE. The other individual (K8) was found in the Yakimova Mogila Tumulus in southeastern Bulgaria, dated to 450–400 BCE. DNA was extracted from teeth using clean room procedures to prevent contamination, following an established protocol http://picard.sourceforge.net) as well as low quality alignments (MQ<30) and bases . Due to the higher coverage of the Iceman genome, we performed diploid SNP genotype calling using the Bayesian algorithm implemented in ‘bcftools’ of the SAMtools suite. For all other ancient samples, haploid genotypes were obtained by randomly sampling reads if positions were covered by multiple reads. For all analyses involving only one ancient sample, we excluded DNA damage sites (C>T/G>A transitions) where the ancient sample shows the damage allele (T/A). For analyses including multiple ancient samples, all transitions were excluded.The ancient genome data used for this study come from a number of different sources, which differ both in sample preparation as well as sequencing technology. In order to minimize biases due to these differences as much as possible, we applied very stringent quality control filters during the processing of the different datasets, using the same processing pipeline for each sample when possible. For all samples, pileup files from the aligned reads (reference genome build hg18) were obtained using the ‘mpileup’ command of SAMtools Genotype calling was performed using BAM files containing the already aligned reads Phred-scaled variant quality score >40Read depth ≥6 or ≤30Distance from gaps ≥5 base pairsNot within repeat regions Sequencing reads were obtained from the European Nucleotide Archive (ENA), for two samples, Ajv70 (accession ERS084011) and Gok4 (ERS084013). Reads were aligned to the reference genome using bwa The aligned and processed sequencing reads from https://github.com/jstjohn/SeqPrep), the sequencing reads were aligned to the reference genome using bwa with seeding disabled. Pre-and post-capture BAM files from the same sample were then merged and processed as described above. Details on analyses of ancient DNA damage patterns can be found in After merging of overlapping paired end reads  in BAM format were obtained from et al.For all population genetic analyses, data from the ancient genomes was merged with three different reference datasets of present-day human populations, using only autosomal data. We followed the approach of Skoglund ftp://ftp.cephb.fr/hgdp_supp10/. This dataset was designed for population genetic studies by including 13 different SNP panels with well-defined ascertainment schemes. We used the combined data for all panels  as the master dataset for merging with the ancient genomes, after removing SNPs with >10% missing data. For all subsequent analyses, the relevant subset of SNPs corresponding to the desired ascertainment scheme was then extracted from the master dataset. Individuals used for SNP discovery in the ascertainment panels were excluded from all analyses.Data for 934 unrelated samples from the HGDP from 52 populations, genotyped on the Affymetrix Axiom Human Origins SNP array was obtained from Whole-genome sequence data of 46 samples from 10 populations sequenced to high coverage by Complete Genomics ftp://ftp.1000genomes.ebi.ac.uk). Population allele frequencies were calculated for each SNP, excluding related individuals, for a final dataset of 841 individuals. Both datasets were merged, excluding variants failing QC filters in any dataset or with mismatching alternative alleles. To further minimize potential biases from the merging of the two datasets, we further removed all variants not passing the more stringent “strict” accessibility mask from the 1000 Genomes project. The remaining variants were converted to hg18 coordinates using the liftover tool from the UCSC genome browser (http://genome.ucsc.edu/) for the final merge with the ancient genomes.The data for this combined dataset were obtained from low-coverage whole-genome sequencing. Therefore, we restricted the analysis to population-level by using only allele frequencies and not individual genotypes. We obtained allele frequency data from 452 unrelated Sardinian individuals sequenced to low coverage as part of the SardiNIA project k = 2 through k = 8 ancestral clusters by running ADMIXTURE with default settings. In order to maximize the accuracy for the initial clustering, we used all SNPs where a genotype was observed for any ancient sample prior to removal of damage SNPs. For each value of K, ten replicate runs were performed, and the run with the greatest likelihood was selected for further analysis. The cluster membership proportions for each ancient sample were then obtained by maximizing the log-likelihood function  on the genotype matrix.In addition, we also performed principal component analysis (PCA), separately for each ancient genome together with the 263 contemporary individuals, using only non-missing SNPs of the respective ancient sample. The initial PCA was performed using the modern samples only, followed by projection of the ancient samples onto the inferred principal components. All analyses were carried out in R using singular value decomposition , we quantified the amount of derived allele sharing between the ancient and modern genomes. For each ancient genome, we extracted all variants showing the derived allele and calculated the proportion of variants with matching alleles for each of the modern genomes. The sharing rate was normalized by the rate for the individual with the lowest sharing rate with the Iceman , for easier comparison among different ancient genomes.TreeMixFor population-level datasets (1000G/Sardinia), we used Additionally, we used formal tests of admixture as implemented in the three- and four-population tests et al.et al.Divergence times of the Iceman with contemporary genomes was estimated using a previously described coalescent method Figure S1k = 2 to k = 8 ancestral clusters on the HGDP individuals, and the corresponding cluster proportions inferred for the ancient samples.ADMIXTURE results for HGDP. Panels show the results for ADMIXTURE runs for (PDF)Click here for additional data file.Figure S2PCA results for HGDP. Panels show the results for PCA on the HGDP individuals for subsets of SNPs with data in the respective ancient sample. Each point represents an individual, with plot symbol and color indicating population of origin. The position of the ancient samples was inferred by projecting onto the PC space calculated using the modern samples only.(PDF)Click here for additional data file.Figure S3Derived allele sharing with CG genomes. Normalized derived allele sharing rate of the Iceman with the full set of whole genomes from Complete Genomics. Each circle represents the rate of sharing with a particular genome, grouped by population of origin (circle color). Positions on the y-axis have added jitter for ease of visualization. Population names with suffix correspond to the respective ancestry tracts  of individuals of populations with known recent admixture .(PDF)Click here for additional data file.Figure S4D-tests of Iceman with European populations from CG. Each diamond indicates the value of the D-statistic for a single D-test involving the Iceman and a pair of modern European populations, indicated on the y-axis. A significant deviation from the x = 0 line indicates a closer relationship of the ancient sample to one of the populations in the tested pair. Significance at Z = 3 is indicated with black diamonds, and the line shows the corresponding standard error of the D-statistic. Populations with EUR suffix correspond to the European ancestry tracts of individuals of populations with known European admixture .(PDF)Click here for additional data file.Figure S5D-tests of ancient samples with European populations from 1000G/Sardinia. In each panel, diamonds indicate the value of the D-statistic for a single D-test involving the respective ancient samples and a pair of modern European populations. Significance at Z = 3 is indicated with filled diamonds, and the line shows the corresponding standard error of the D-statistic. Plot colors indicate different pairs of geographic regions within Europe.(PDF)Click here for additional data file.Figure S6m = 0 to m = 5 mixture events.Results of TreeMix analysis of the Iceman with 1000G/Sardinia. Shown are maximum-likelihood trees and the matrices of pairwise residuals for all models allowing from (PDF)Click here for additional data file.Figure S7k.RMSE for HGDP cluster proportions. Panels represent the results for ancestral cluster membership accuracy for each subset of SNPs corresponding to the ancient samples. Accuracy is measured as root mean square error (RMSE) of cluster membership proportions for each population of origin and value of (PDF)Click here for additional data file.Figure S8Results of TreeMix analysis of the ancient samples with 1000G/Sardinia. Shown are maximum-likelihood trees and the matrices of pairwise residuals for all ancient samples, without mixture edges. Bootstrap support for the position of the ancient sample is indicated by the numbers next to the branches.(PDF)Click here for additional data file.Figure S9PCA with archaic hominins and Iceman in HGDP. Plot symbols indicate the position of an individual, projected onto PC space inferred using Chimp, Neanderthal and Denisova. Individuals are colored according to continental region of origin.(PDF)Click here for additional data file.Figure S10D-tests of archaic hominins with CG populations and Iceman. In each panel, diamonds indicate the value of the D-statistic for a single D-test involving the respective archaic hominin sample and pairs of Iceman/CG population on the y-axis. A significant shift to the left (D<0) indicates a closer relationship of the ancient sample to the Iceman compared to the modern population. Significance at Z = 3 is indicated with filled diamonds, and the line shows the corresponding standard error of the D-statistic. Plot colors indicate geographic origin of the CG population.(PDF)Click here for additional data file.Table S1D-Tests of Iceman – 1000G/Sardinia with outgroup.(XLSX)Click here for additional data file.Table S2Divergence time estimates of the Iceman with Complete Genomics genomes.(XLSX)Click here for additional data file.Table S3Divergence time estimates of the Iceman with 1000G/Sardinia.(XLSX)Click here for additional data file.Table S43f statistic with modern populations.Signals of admixture using the (XLSX)Click here for additional data file.Table S53f statistic with ancient samples.Signals of admixture using the (XLSX)Click here for additional data file.Table S6TreeMix parameters.(XLSX)Click here for additional data file.


Trypoxylon is a genus of solitary crabronid wasps whose populationgenetics is poorly known. The purpose of the present study was to investigate thegenetic variation and differentiation among five populations of Trypoxylonalbitarse, a species widely distributed throughout the Neotropics, withrecords from Panama to northern Argentina. Eight species-specific microsatellite lociwere used for genotyping 96 adult wasps  sampled at five sitesin Brazil. The analysis of allelic richness and private alleles indicated highgenetic diversity in the populations sampled. Pairwise comparisons using theFst and Dest indices revealed significant differentiation for all, but one pair ofpopulations. Fst, Dest, AMOVA and assignment test values pointed to inter-population differentiation.Additionally, the analysis of population structure using Bayesian and PCA methodscharacterized two alternative genetic groups. The Mantel test indicated nocorrelation between genetic and geographic distances. Despite evidence ofconsiderable dispersal capacity for T. albitarse, the data indicatelow to moderate population structuring in this species. Among aculeate members of the order Hymenoptera, the family Crabronidae is a group ofwasps that stands out for its worldwide distribution as well as for exhibiting a widevariety of nesting and foraging strategies. About 9,000 species have been described and mostTrypoxylon is the most diverse genus of the family Crabronidae, withabout 634 described species, which have a worldwide geographical distribution is widely distributed throughout the Neotropics, with records from Panama to northernArgentina, and is easily found on the walls of human constructions, such as buildingsand bridges, especially if located near forested areas , Viçosa (state of Minas Gerais), Lavras (state ofMinas Gerais), São Carlos (state of São Paulo) and Guarapuava (state of Paraná)(Specimens of  Paraná). The samTrypoxylon albitarse nests with mud tubes found on the walls ofhuman constructions were carefully opened with fine-tipped tweezers. When immatureindividuals in the pupal stage were found, the cocoons were placed in individual,duly labeled, plastic tubes, taken to the laboratory and kept at room temperatureuntil the emergence of the adult stage, when the sex was determined, followed bystorage of the material at −20 °C for subsequent DNA extraction.T. albitarse using protocols based on eitherphenol-chloroform  in afinal volume of 10 μL. Amplification consisted of an initial denaturation step at 94°C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing ofthe primers at the specific temperature for each locus indicated by Total DNA was extracted after the maceration of three pairs of legs or the mesosomaof adult specimens of Approximately 13% of the samples were retyped to verify the reproducibility of thedata and confirm the genotypes considered when weak or double peaks were found. Tominimize the occurrence of missing data, PCR was performed up to three times in casesof the absence of amplification for a given locus.The amplification products were analyzed after a run in the MegaBace-1000 automatedsequencer . Fragment size wasestablished by comparing the peaks in the set of samples to the ET550R size marker inthe MegaBace Fragment Profiler program, version 1.2.T. albitarse males develop fromunfertilized eggs and are haploid, whereas females develop from fertilized eggs andare diploid. Based on this feature, only females were used in the genetic analysis.To avoid biased results due to the familial structure of the nests, only one femaleper nest was considered in the final dataset. The number of wasps analyzed persampling site varied from 12 to 37 , which ranged from 1 to 5. In each run, 1,000,000 repetitions of burn-inwere conducted, followed by 5,000,000 MCMC repetitions. The estimated probabilitiesfor each run were used to estimate ΔK  was performed to identify the distribution ofgenetic variation in the geographic samples using the GenAlEx program . This prFis) (The inbreeding coefficient (Fis)  wasveriFis) . The sofThe Kingroup2 program  from the five sampling siteswere genotyped for nine microsatellite loci. As locus TALB12 wasmonomorphic in all samples, only eight loci were considered in the analysis. The lackof amplification for some loci, even after repetitions of the PCR, generated amissing data rate of nearly 3%.Fst and Dest indices for pairs of populations were obtained for all loci. The resultsindicated significant genetic differentiation between all pairs of populations, butone (Lavras vs. Guarapuava) . Overall Fst and Dest values  indicatedsignificant population differentiation. The distribution of genetic variation withinand among populations was determined using AMOVA (Estimates of differentiation using the rapuava) . The Peang AMOVA . The assK = 2) .The differentiation pattern obtained through Bayesian analysis identified twoalternative genetic clusters (K = 2) . A similK = 2) . Both anFis values over all loci were positive and significantly different from zero inthe five populations, ranging from 0.154 to 0.285 .The test carried out in the program BOTTLENECK resulted in significant values,indicating a possible bottleneck effect for all the populations studied. The Wilcoxonsign-rank test for heterozygosity excess, using the two phase mutation model,resulted in the values 0.187 (p < 0.01), 0.632 (p < 0.01), 0.204 (p <0.001), 0.187 (p < 0.01) and 0.101 (p < 0.01) for Ilhéus, Viçosa, Lavras, SãoCarlos and Guarapuava, respectively.r) were close to25% , low mean relatedness values between pairs of femalesof each population were observed .Although some pairwise estimates of relatedness . Comparatively, the smallest variationin He among populations (0.14 to 0.25) occurred at locus TALB09 andeven the highly polymorphic locus TALB07 exhibited a low level ofvariation (0.86 to 0.93). Thus, mean expected heterozygosity did not differsubstantially among populations (range: 0.54 to 0.63)  , which wdiffered , suggestT.albitarse population revealed that all loci, except TALB03,had at least a significant p-value (< 0.05). However following the Bonferronicorrection, most deviations were found not to be significant. Deviations fromHardy-Weinberg equilibrium are generally associated with inbreeding, populationstructuring, the effects of natural selection, preferential mating, and/or the presenceof null alleles  among populations could be partially attributed to the presumed philopatry ofTrypoxylon females. If an area is colonized by a small number offemales, this results in a founder effect. If the growth of local populations occurs bythe reproduction of the original nests, with the construction of satellite nests bydaughters of the founding females, local populations would consist of few familialgroups. Thus, despite the dispersal of males, some structuring would be possible due tothe behavior of daughters nesting close to their native site. This hypothesis is inaccordance with the possible recent demographic bottlenecks detected in all populationsof T. albitarse studied. However, kinship analysis showed low geneticrelatedness among females of the same population, suggesting that the dispersal abilityof males may be sufficient to generate a low r values among females ofthe same population. It is worthy of note that this result is not due to the lowresolution power of the genetic markers employed, as r values used toestimate the relatedness among individuals from the same nest were consistent with thoseexpected for a predominantly monogamous genetic system  and reportedthat specimens marked during nesting activity were observed up to 42 days after marking.Based on these findings, the author proposed that this species has good dispersalcapacity. Studies involving other species of the genus also suggest that these waspshave a high emigration rate (T. albitarse is not coherent withthe inter-population genetic differentiation observed in the present investigation andthe one by Little information is available on flight and dispersal capacity among species ofion rate . HoweverT. albitarse in a more limited geographical scale, aswell as phylogenetically related species, are needed to obtain more conclusive resultsabout the effects of gene flow and female philopatry on the genetic differentiationamong populations of Trypoxylon species.In conclusion, this study makes a significant contribution to knowledge on thepopulation genetics of cabronid wasps, which has been rarely studied. Given therelevance of these wasps to the phylogeny of bees and the present findings of moderateinter-population heterogeneity, further studies involving the genotyping of otherpopulations of