By genotyping >580,000 SNPs in two ‘Afro-Bolivians’, and comparing these genomic profiles with data compiled from more than 57 African groups and other reference ancestral populations . Using tract length distribution of genomic segments attributable to distinct ancestries, we could date the time of admixture in about 400 years ago. This time coincides with the maximum importation of slaves to Bolivia to compensate the diminishing indigenous labor force needed for the development of the National Mint of Potosí.Overall, the data indicate that the genome of ‘Afro-Bolivians’ was shaped by a complex process of admixture occurring in America among individuals originating in different West-Central African populations; their genomic mosaics received additional contributions of Europeans and local Native Americans (e.g. Aymaras).The online version of this article (doi:10.1186/s12864-016-2520-x) contains supplementary material, which is available to authorized users. During the Transatlantic Slave Trade (TAST) from the fifteenth to the end of the nineteenth century, more than 12.5 million enslaved Africans boarded ships that were destined to the Americas. Most African slaves brought to the Americas came from West-Central Africa (~5.6 million) .During the last decade, geneticists have aimed to unravel the complex patterns of admixture occurring in America as a consequence of the European colonization and the TAST. In particular, the analysis of mitochondrial DNA (mtDNA) has been widely used in the literature –10. For Very little attention has so far been given to historically isolated populations whose history can also be traced back to the TAST, as is the case of the ‘Afro-Bolivian’ community of Tocaña, located in the Nor (North) and the Sud (South) Yungas valleys of Bolivia. The Yungas are located in the Department of La Paz, in the passageway that connects the Bolivian highlands and the tropics. During the colonial period, Spaniards initially used indigenous people as a workforce to exploit the region’s mineral wealth at the mines and Mint of Potosí, but soon they began to enslave Africans. Mortality rate of slaves was high in the region mainly due to the fact that they were forced to work in a region over 4,000 m above sea level and in very hard conditions . The firvia Río de la Plata, and at least between 1,500 and 3,000 Angolan enslaved people arrived at Río de la Plata every year during the first half of the seventeenth century [African enslaved people from West-Central Africa principally arrived in Upper Peru  century . It is a century . Later,  century  reportedAlthough ‘Afro-Bolivians’ were not included in the official National Census of 1996, it was estimated at that time that about 10,000 ‘Afro-Bolivians’ were mostly concentrated in the Yungas provinces, mainly in rural towns and villages such as Coroico, Irupana, Tocaña, etc. This small community adopted much of the technological and economic organization and cultural norms of the local indigenous Aymara . Only veSome inferences on their origins have been made based on linguistics . There a‘Afro-Bolivian’ groups have hitherto received very little attention within the scientific community that investigates the history of the TAST; probably because they constitute a relatively small community surrounded by a numerous Aymara-speaking population, and because they live in a geographically remote region. Similarly, genetics research on Bolivian populations has primarily focused on the Native American indigenous population excluding people of African ancestry. So far, mtDNA lineages in the departments of Beni, Chuquisaca, Cochabamba, La Paz, Pando, and Santa Cruz, which are distributed across three eco-geographically distinct regions , have been shown to be mainly of Native American ancestry –28. In cThe aim of the present study is to provide a first insight into the complex admixture processes experienced by individuals belonging to the geographically isolated ‘Afro-Bolivian’ community. In contrast to other ‘Afro-American’ communities that admixed in complex demographic circumstances , ‘Afro-Bolivians’ from the Yungas valleys constitute very isolated communities since their initial formation, and have remained surrounded by peoples of main Native American ancestry. These ‘Afro-Bolivians’ therefore constitute a sort of ‘genetic laboratory’ to gain new insight into the TAST.The genetic proximity of the two Tocaña profiles to different sub-Saharan African groups can be studied by examining the genetic distances in terms of Identity-by-State (IBS) values between these two Bolivians and each of the population sample sets in Africa. These analyses were carried out using separately the population sets from The 1000 Genomes Project (hereafter 1000G) and a large dataset of African populations (including those that contributed more slaves to the TAST). The exploratory analysis carried out with 1000G samples  African ancestry: 78.1 for #TOC1 and 82.1 % for #TOC2; (ii) European ancestry: 14.8 for #TOC1 and 2.6 % for #TOC2; and (iii) Native American ancestry: 7.1 for #TOC1 and 15.3 % for #TOC2.PCAdmix was performed in order to reveal the genome architecture of the two Afro-Bolivians analyzed Fig. . The anaK = 5 , this analysis . For an optimum 00 K SNPssis Fig.  confirmssis Fig. . FurtherTherefore, even though different reference population datasets and algorithms were used, percentages of admixtures obtained using PCAdmix and ADMIXTURE are broadly comparable for the three ancestral components. Both Tocaña have a similar proportion of African ancestry (79–81 %); the main difference between them is that #TOC2 has a larger proportion of Native American ancestry than #TOC1, which is balanced by a lower European ancestry in #TOC2.K = 12 . Some findings of this exploratory analysis are suggestive. First, it indicates that the two Bolivians have one predominant component (42–49 %) that coincides with the predominant component in West-Central Africa (56 on average); in particular, in the Yoruba , Hausa , Brong , and Mandenka ; Additional file Analysis of admixture was also carried out with the large set of African populations (>25 K SNPs) and the optimum Nigeria; 7, Hausa i) the fact that all the Native American groups used as reference populations are also admixed to different extents with Europeans and even with people of recent African ancestry [ii) the complex admixture nature of the two Tocaña individuals investigated. The analysis was carried out using the unmasked  and the masked  data of the Native American groups analyzed by Reich et al. [Additional analyses were carried out in order to further investigate the Native American component of ‘Afro-Bolivians’ as suggested by ADMIXTURE and PCAdmix. This analysis is somehow blurred by: . Also remarkable is the fact that the Native American component of the Tocaña does not share ancestry with the component that is more typical of the Llanos in Bolivia, represented here by the neighboring population of the Surui from the Mato Grosso do Sul, Brazil. The genomic patterns described are similar whether the unmasked , with TOCi being each of the two Tocaña individuals, and XNA the different Native American datasets used in the present study. D-statistics were highly significant (>98 K SNPs) for all the Native American datasets , which used four different datasets as surrogate populations to test for European admixture in the two Tocañas (XEU). D-statistics were again highly significant (>573 K SNPs) for the four different datasets used; Fig. We next computed D and D, with XEAF being the different East African datasets available. As shown in Fig. D-statistic was statistically significant (>148 K SNPs) for most of the population datasets from East Africa in both Bolivians (note that the threshold of significance was set to D-statistics < −2). This analysis was also very informative in showing that the Yoruba has a more important contribution to the Tocaña individuals than the East African populations, given that D-statistics showed more negative values when using East Africans as reference datasets; that is D. Also in good agreement with admixture proportions, D-statistics showed more negative values with #TOC2 than with #TOC1 in D indicating a larger West African ancestry of #TOC2. Finally, ADMIXTURE analysis indicated that, out of the East African population samples, the Bantu ethnic group of Luhya from Kenya seemed to show some genetic affinities with the African component of the two Tocaña  was used to date the time of admixture as recorded in the two ‘Afro-Bolivian’ genomes and using the track length distribution of genomic segments inherited from different ancestral groups , 35. TheThe results of the present study indicate that the two ‘Afro-Bolivian’ genomes are a mosaic of different ancestries. These genomes have a main sub-Saharan component (~80 %), with a minor contribution (~20 %) coming from Europe and Native America.The analyses suggest that West-Central Africa is the region that contributed the main proportion of African ancestry to ‘Afro-Bolivians’. The main signal comes from the Yoruba (Nigeria), but it seems also clear that there are other populations in West-Central Africa, mainly representing the Atlantic coast , that could also have contributed to the genome of the ‘Afro-Bolivians’ analyzed.D-statistics also indicated a seeming contribution of East African ancestry to the ‘Afro-Bolivian’ genomes. However, further analysis demonstrated that this signal could also be explained by computational artifacts provoked by the presence of European ancestry in the genome of the two Tocaña and in the East African populations.Stimulated by the recent findings of Heinz et al.  indicatiWhile the African ancestry of ‘Afro-Bolivians’ constitutes a complex mosaic of African ancestries, their Native American component is probably more homogeneous, even though the analysis is very complicated by the admixed nature of all reference Native American populations . The NatThe time of admixture, as reflected in the two ‘Afro-Bolivian’ genomes, could be derived from the tract length distribution of genomic segments inherited from distinct ancestries. The data indicate a main admixture event occurring at about 400 years ago between Africans, Europeans and Native Americans Fig. . InteresAmong the limitations of the present study is the fact that the reference populations in Africa, America and Europe, used in the present analyses may not represent the patterns of variability existing at the time of the TAST. In addition, although we compiled information on hundreds of individuals representing dozens of Native American and European populations, and more specially population samples in Africa, the size of the reference populations might still be small. In the future, an improved database could help corroborate the present findings and add more details on the demographic processes that led to the configuration of present-day ‘Afro-Bolivian’ genomic architecture .With the arrival of new genotyping and computational technologies it is now possible to map with much better resolution the complex genomic mosaic of ‘Afro-Americans’. The case of ‘Afro-Bolivians’ is particularly interesting owing to their strong isolation from other African and Native American communities. Disentangling history of ‘Afro-Americans’ is hampered by the fact that many enslaved African people took unusual journeys after their enslavement. For example, Hans Jonathan, born as a slave on the Danish Caribbean island of St. Croix, was forced later in his life to migrate with his master to Copenhagen, Denmark, but when slavery was abolished in Denmark he was sentenced to go back as a slave to St. Croix. Jonathan escaped from slavery and finally landed in Iceland .The present article shows that genomics is useful to reconstruct the individual ancestries of ‘Afro’ communities living in the Americas, accounting for the complex processes of admixture occurring since their arrival to the American continent. Most of the previous attempts were based on the analysis of uniparental markers , 37, 38.n = 9) that showed a large membership of African ancestry in the analysis of a small panel of AIMs. We obtained written informed consent for all the donors prior the research, which includes consent for publication individual data. Rights of participants were safeguarded during the research and their identity was protected. The study was approved by the ethical commission of the Universidade de Santiago de Compostela  which depends on Ethics Committee of Galicia (Spain) and by the Ethics Committee of Universidad Mayor, Real y Pontificia de San Francisco Xabier de Chuquisaca (Bolivia). The study conforms with all applicable Spanish normative, this is to say, Law for Biomedical Research (Law 14/2007–3 of July), Law 41/2002 of Autonomy of the Patient, Decree SAS/3470/2009 for observational studies and Law 15/1999 of Data Protection.A collection of 105 saliva samples recruited from the Yunga’s region of Bolivia was previously characterized for mtDNA and a panel of Ancestry Informative Markers or AIMs . A smallSamples were genotyped using the Axiom® Genome-Wide Human Origins 1 Arrays at the Centro Nacional de Genotipado (CEGEN) of Santiago de Compostela (Spain). After processing the samples, the Affymetrix Genotyping Console™ (GTC) 4.1.2 Software package was used to generate QC metrics and genotype calls following commercial recommendations. This procedure includes generation of sample dish QC (DQC) values. Samples with a DQC value lower than the default DQC threshold of 0.82 were dropped from the study. The “AxiomGT1_all” algorithm was used for genotyping this group of high quality samples using all SNPs on the array. Unfortunately, not all the samples initially selected for genotyping contained enough DNA  for genome-wide SNP genotyping. Finally, only the analysis of two of the samples succeeded, with a total of 580,144 SNPs genotyped. These two samples have been recruited in the locality of Tocaña, and are referred to here as #TOC1 and #TOC2 .We intersected the SNP data obtained from the two ‘Afro-Bolivians’ with genome population data downloaded and processed from different SNP repositories; Additional file Data from The 1000 Genomes Project (hereafter 1000G) provides the dataset with the largest SNP overlap with the Tocaña data. The data from 1000G was retrieved from the original repository as done in Pardo-Seco et al. ; these aThe lowest SNP overlap occurred when intersecting the Tocaña SNP set with a large compilation of population samples that includes 1,161 Africans (representing 57 different populations/ethnic groups); these analyses involved 25,192 SNPs (0.7 % of missing data).ad hoc analyses were carried out using selected sub-sets of populations with the aim to increase the number of SNPs (specific numbers are given in the main text where relevant). For instance, analyses of the Native American component observed in the two Tocaña individuals were carried out using the unmasked (0.2 missing data) and masked (7.8 % missing data) dataset from Reich et al. [Some h et al. ; these dP-value = 1.PLINK  was usedAdmixture components of the two Bolivian individuals were performed using the ADMIXTURE software , which uLocal ancestry assignment of ancestry-specific haplotypes across the genome was carried out using PCAdmix 1.0 . PCAdmixcmdscale (library stats) from R (http://www.r-project.org). PCAmask [In order to discriminate among clusters of genetic variation in the population sets analyzed, multidimensional scaling (MDS) was carried out on a matrix of pairwise individual IBS values. MDS was performed using the function  PCAmask  was addiD-statistics [vs. different population sets [D-statistic was computed using the weighted block jackknife procedure (block size of 5 MB) [We additionally computed the four-population test, implemented as atistics , in ordeion sets , 47–49. D-statistics, we built an outgroup that is symmetrically related to all modern human population groups, by creating an individual profile possessing the ancestral alleles at all sites. The use of this artificial outgroup allowed us to infer D-statistics, thus ensuring that there is no differential gene flow between the outgroup and the population sets used; this is particularly important in our population context given that the three main continental ancestries are present in our sample sets. The construction of this outgroup involved the following steps. First, an auxiliary ancestral sample VCF file was generated containing all the ancestral alleles as alternative alleles. Second, all the chromosome sites from 1000G VCF files where parsed looking for “AA” codes, the ancestral alleles where detected and the alternative alleles where substituted with the ancestral alleles. Only the first four VCF columns where kept, then the ancestral allele was added, and the rest of the columns where filled with single dots. The genotype column was filled with reference homozygous (0/0) in cases when only the reference was reported, and with alternative heterozygous (0/1) when an ancestral allele different from the reference was reported.For the computation of Tracts [Finally, timing of admixture was estimated by way of analyzing the tract length of chromosomal segments attributed to different ancestries, using the software Tracts ; see alsTracts .


Capra hircus) have evolved in a large variety of locally adapted populations in response to different human and environmental pressures. In the present era, many indigenous populations are threatened with extinction due to their substitution by cosmopolitan breeds, while they might represent highly valuable genomic resources. It is thus crucial to characterize the neutral and adaptive genetic diversity of indigenous populations. A fine characterization of whole genome variation in farm animals is now possible by using new sequencing technologies. We sequenced the complete genome at 12× coverage of 44 goats geographically representative of the three phenotypically distinct indigenous populations in Morocco. The study of mitochondrial genomes showed a high diversity exclusively restricted to the haplogroup A. The 44 nuclear genomes showed a very high diversity (24 million variants) associated with low linkage disequilibrium. The overall genetic diversity was weakly structured according to geography and phenotypes. When looking for signals of positive selection in each population we identified many candidate genes, several of which gave insights into the metabolic pathways or biological processes involved in the adaptation to local conditions . This study highlights the interest of WGS data to characterize livestock genomic diversity. It illustrates the valuable genetic richness present in indigenous populations that have to be sustainably managed and may represent valuable genetic resources for the long-term preservation of the species.Since the time of their domestication, goats ( Capra hircus) in particular are one of the more important livestock species, because of their high potential of adaptation to harsh environments. They had a worldwide population of about 1006 million in 2013 (http://faostat3.fao.org/browse/Q/QA/E) and, together with cattle and sheep, they represent the most important source of meat, milk, and skin.Livestock species play a major socio-economic role in the world since they provide many goods and services to human populations. Goats . Consequently, a part of the highly valuable genetic resources captured from the wilds and gradually accumulated over 98% of their common history with humans is now threatened . Direct anthropic selection was relatively modest and until recently it was difficult to distinguish well-defined breeds. However, several phenotypic groups displaying specific morphological and adaptive characteristics have been identified. They will be referred hereafter here as populations. The three major groups are: (i) the Black goats with three sub-populations that have been recently officially recognized , (ii) the Draa population, (iii) and the Northern population. Besides these three main populations/breeds, the major proportion of Moroccan goats presents intermediate phenotypes and non-recognized local populations. The Black population is characterized by its dark color, long hair, a low water turnover and thus good resistance to water stress ]. A total of 44 individuals unambiguously assigned to one of the three main indigenous populations  were sampled . Sheared DNA was used for Illumina® library preparation by a semi-automatized protocol. Briefly, end repair, A tailing and Illumina® compatible adaptors (BiooScientific) ligation were performed using the SPRIWorks Library Preparation System and SPRI TE instrument (Beckmann Coulter), according to the manufacturer protocol. A 300–600 bp size selection was applied in order to recover the most of fragments. DNA fragments were amplified by 12 cycles PCR using Platinum Pfx Taq Polymerase Kit (Life® Technologies) and Illumina® adapter-specific primers. Libraries were purified with 0.8× AMPure XP beads (Beckmann Coulter). After library profile analysis by Agilent 2100 Bioanalyzer  and qPCR quantification, the libraries were sequenced using 100 base-length read chemistry in paired-end flow cell on the Illumina HiSeq2000 .http://picard.sourceforge.net), GATK RealignerTargetCreator, GATK IndelRealigner   the site is called by all three variant callers with variant phred-scaled quality >100, (ii) the site is biallelic (iii) the minor allele count is at least 3 while counting only samples with genotype phred-scaled quality >30. The training set was used to build a Gaussian model using the tool GATK VariantRecalibrator using the following variant annotations from UnifiedGenotyper: QD, HaplotypeScore, MQRankSum, ReadPosRankSum, FS, DP, InbreedingCoefficient. The Gaussian model was applied to the full data set, generating a VQSLOD (log odds ratio of being a true variant). Sites were filtered out if VQSLOD < cutoff value. The cutoff value was set for each population by the following: Minimum VQSLOD = {the median value of VQSLOD for training set variants} − 3The whole mitochondrial genome (mtDNA) was assembled from a subset of random 20,000,000 reads using the ORGASM tool . We then extracted the sequence of the HVI segment of the control region for each individual in order to compare with the haplogroup references discovered worldwide (see below).The number of polymorphic sites and the number of haplotypes were calculated from the whole mitochondrial sequences using DNAsp  was calculated considering only the biallelic SNPs with no missing genotype calls. From Ho, the inbreeding coefficients (F) were calculated for each individual using population allelic frequencies over all 44 individuals. The relatedness among individuals was assessed using the pairwise identity-by-state (IBS) distances calculated as the average proportion of alleles shared using Vcftools.Neutral nuclear genomic variations were characterized to evaluate the level of genetic diversity present in Morocco and within populations. The total number of variants and the number of variants within each population were calculated. Allele frequencies and the percentage of exclusive variants  were estimated at the population scale using the Perl module vcf-compare of Vcftools . It was estimated in 5 segments of 2 Mb on different chromosomes . LD was estimated either by using the whole set of reliable variants or after discarding rare variants with a minor allele frequency (MAF) less than 0.05. For both estimations, we calculated r2 values between all pairs of bi-allelic variants (SNPs and indels) on the same segment using Vcftools. Inter-SNP distances (kb) were binned into the following 7 classes: 0–0.2, 0.2–1, 1–2, 2–10, 10–30, 30–60, and 60–120 kb and observed pairwise LD was averaged for each inter-SNP distance class and used to draw LD decay. Due to the insufficient number of individuals per population we made these estimations for the whole set of individuals without considering each population individually.Pairwise linkage disequilibrium (LD) was assessed through the correlation coefficient  was used for LD pruning. (ii) An analysis with the clustering method sNMF  Finally, the Fst index was estimated according to Weir and Cockerham  a principal component analysis (PCA) was done using an ockerham  for eachr2 > 0.95) in the reference group.A genome scan approach was performed using the XP-CLR method .To equilibrate the number of individuals per population, only 14 Black goats were randomly sampled among the 22. They were included with the 14 Draa and the 8 Northern individuals. Each population was tested using a reference group including individuals from the two other populations. The 0.1% genomic regions with highest XP-CLR scores revealed by the analysis were identified and lists of genes partially or fully covered by these regions were then established. To ensure the coverage of short genes , two segments of 1500 bp each surrounding both sides of genes were also considered. NCBI databases were used to identify coordinates of the 20700 annotated autosomal genes on the CHIR v1.0 genome assembly  enrichment analyses were performed using the application GOrilla  of reads to the CHIR v1.0 assembly. However, the mapped reads properly paired constituted 90.3% (±0.1%) of reads in average. After the filtering processes, a total of 24,022,850 variants were found to be polymorphic in the total dataset among which 22,396,750 were SNPs and 1,626,100 were small indels. There were a total of 15,948,529 transitions and 6,540,478 transversions leading to a ts/tv ratio of 2.44. Due to differences in quality among individuals, the number of variants called per individual was at least 23,273,239 and 24,003,837 on average. As a consequence, a total of 23,059,968 variants showed no missing genotype over the 44 samples, among which 22,963,257 were biallelic.n = 22), 1,887,724 only in the Draa population (n = 14) and 1,305,561 only in the Northern population (n = 8)  was 17.2% on average, ranging from 12.1% to 18.4%. The average inbreeding coefficient (F) was globally rather low (0.05 ± 0.07) and values were evenly distributed among populations. Similar average values were obtained for the Northern and Black populations (respectively 0.04 ± 0.07 and 0.04 ± 0.05). The Draa goats were slightly more inbred (average F = 0.07 ± 0.09), particularly due to one individual showing F = 0.32.Considering the 44 goats together, the average nucleotide diversity (π) calculated from 22,963,257 biallelic variants without missing genotype calls was 0.180. The Draa and the Black populations displayed similar π values amounting to 0.180 and 0.181 respectively. Among the 8 individuals representing the Northern population, π was slightly higher, amounting to 0.189. The observed percentage of heterozygote genotypes per individual , the average 2r value was 0.40 for the first bin (0–0.2 kb) and decayed to less than 0.20 in 5.4 kb . The pairwise Fst values varied from 0.001 for the Black-Draa comparison to 0.004 for the Northern-Draa comparison. Between the Black and Northern populations the pairwise Fst was 0.003.Among the three populations, the level of genetic differentiation over the whole nuclear genome was extremely low  explained 5.8% of variance. The first PC tended to distinguish the Northern and Draa populations while the Black populations formed an in-between group. The second PC acted predominantly to distinguish individuals within the Draa and the Northern populations Figure .K = 1). However, this criterion is not straightforward and when increasing until K = 3 we observed a weak pattern of genetic structure  . This result shows that despite a low number of variable sites, the dense variability found in the control region  concentrated most of the phylogenetic information. Thus, the HVI segment of the control region seems to be a good surrogate of the whole mitochondrial polymorphism. This study confirmed previous results based on the HVI segment of the control region  in comparison with the percentage of mapped reads (about 99%) would illustrate a possible fragmentation of the genome assembly used, we identified many high confidence variants  over the whole nuclear genomes of the 44 Moroccan goats studied. This is much higher than was found in all previous studies detecting variants in large sample cohorts from whole genome sequencing. For example, the human 1000 Genomes Project . The extent of LD reported without rare variants (r2 < 0.20 after 5.4 kb on average) is clearly shorter compared to all previous studies on farm animals, where it largely exceeds 10 kb for r2 = 0.20 . We do believe that this value should be considered in genome wide association and genome scan studies. Indeed most of studies remove rare variants for genotyping quality issues. In our case, the quality filtering produced reliable rare variants (about 45%) that would give a more realistic estimation of LD. To our knowledge, very few studies included rare variants to estimate LD  gene that is reported to be involved in coat color differentiation in goats  and in the Northern population (chromosome 22) matching un-annotated genes on the CHIR v1.0 assembly. This is probably due to either the incomplete annotation of the caprine genome or the fact that the selected functional mutations within each of these regions are not located within or close to a protein-coding gene. The incomplete genome annotation prevented us from identifying several known selected genes among Moroccan goat populations. For example, the huntingtin  and may play a role in autophagy triggered by ER stress  and organ development (GO:0048513). These two categories were clustered together with the enriched neuron maturation term (GO:0042551)  that regulate lipid homeostasis  Table . Hence, In the Draa population, which is raised in oasis/desert areas and well adapted to high temperatures  (Table melanocortin-1 receptor gene (MC1R) and promoting the synthesis of phaeomelanin, a yellow–red pigment  Table  in the DP) Table  by antagTRAP1, which encodes a mitochondrial chaperone protein  was previously associated with the number of vertebras in pigs (Mikawa et al., Finally, several strong signals of selection pointed to genes or pathways for which possible functions remained ambiguous. For example in the Northern population, the strong signal of selection associated with Our study characterized whole genome variation in the main goat indigenous populations at a countrywide scale in an unprecedented way. The whole genome data and the wide geographic spread of animals allowed for a precise characterization of the distribution of genomic diversity in various populations. The position of Morocco has made it subject to various colonization waves for domestic animals. Additionally, previous and present management schemes have favored gene flow between goat populations. This created and maintained a very high level of total genetic diversity that is weakly structured according to geography and populations. A part of the overall diversity corresponded to potentially adaptive variation, as several genes appeared to be under selection. The different populations studied appeared to bear specific adaptations, even when submitted to similar conditions such as those related to a warm/desert context. This would demonstrate the potential of different indigenous livestock populations to constitute complementary reservoirs of possibly adaptive diversity that would be highly valuable in the context of global environmental changes. However, these populations are threatened due to their substitution by more productive cosmopolitan breeds that should not have the potential to become locally adapted to harsh environments. It is thus extremely important to promote the sustainable management of these genetic resources with emphasis on both overall neutral and adaptive diversity. This study has also identified several genes as potentially under selection and further studies are needed to depict the underlying mechanisms.The accession numbers of the 44 samples in the BioSamples archive, the accession numbers of the sequencing data and aligned bam files in the ENA archive are reported in the Table PT, FP, SJ, PF designed the study. PT and FP supervised the study. BB, MB, MI, MC, AB, AC sampled individuals. AA, SE produced whole genome sequences. BB, FJA, IS, FB, EC, SS, KL, MI, LC analyzed the data and interpreted the results. BB, FJA, FP, KL, SJ, IS, AA wrote the Manuscript. All authors revised and accepted the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trypanosoma cruzi, a genetically heterogeneous, multi‐host zoonosis, provides an ideal system to examine the sylvatic diversification of parasitic protozoa. In Bolivia, T. cruzi I, the oldest and most widespread genetic lineage, is pervasive across a range of ecological clines. High‐resolution nuclear (26 loci) and mitochondrial (10 loci) genotyping of 199 contemporaneous sylvatic TcI clones was undertaken to provide insights into the biogeographical basis of T. cruzi evolution. Three distinct sylvatic parasite transmission cycles were identified: one highland population among terrestrial rodent and triatomine species, composed of genetically homogenous strains , and two highly diverse, parasite assemblages circulating among predominantly arboreal mammals and vectors in the lowlands . Very limited gene flow between neighbouring terrestrial highland and arboreal lowland areas  but strong connectivity between ecologically similar but geographically disparate terrestrial highland ecotopes  strongly supports ecological host fitting as the predominant mechanism of parasite diversification. Dissimilar heterozygosity estimates  and mitochondrial introgression among lowland strains may indicate fundamental differences in mating strategies between populations. Finally, accelerated parasite dissemination between densely populated, highland areas, compared to uninhabited lowland foci, likely reflects passive, long‐range anthroponotic dispersal. The impact of humans on the risk of epizootic Chagas disease transmission in Bolivia is discussed.An improved understanding of how a parasite species exploits its genetic repertoire to colonize novel hosts and environmental niches is crucial to establish the epidemiological risk associated with emergent pathogenic genotypes.  Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae), the aetiological agent of Chagas disease, a pervasive zoonosis that is eclectic with respect to host species and tissues it can inhabit, provides a model system to examine the genetic diversification of parasitic protozoa.Host–parasite relationships are assumed to be ecologically specialized, tightly co‐evolved systems driven by either mutual modification (synchronous cospeciation) or exaptation into novel environmental niches, often accompanied by host switching    Fig. . Study sTriatoma infestans and terrestrial rodents (Akodon boliviensis and Phyllotis osilae). Triatomine sampling (T. infestans and T. guasayana) was also undertaken in neighbouring Toro Toro, an area of similar ecology to Cotopachi, situated at ~2700 m. North of Cotopachi, sylvatic Rhodnius robustus were collected from Chapare, a dense temperate montane forest (Yungas) in the westernmost foothills of the Andes.The Cotopachi site in Cochabamba department is located in a densely populated area of open dry Andean puna , ~20 km southwest of Cochabamba city at an elevation of ~2600 m. Here, TcI parasites were sampled from wild T. infestans in Tupiza, a region of high‐altitude (~3200 m) montane grasslands in Potosí department.South of Cochabamba, TcI parasites were isolated from wild Rhodnius pictipes) and mammals  in areas of savannah grassland, interspersed with large stands of evergreen palm trees, on the boundary between Llanos de Moxos and the moist forests of northwestern Santa Cruz. The study sites in North Beni  were remote, largely uninhabited, open savannah grasslands with occasional lone standing trees, bordered by riverine forests. Here TcI parasites were isolated from R. robustus, P. opossum and D. marsupialis. Both study sites in North and East Beni were situated at low‐lying altitudes , and parasite sampling was undertaken using similar methods described for other departments.Sampling was undertaken in two regions of Beni department, a sparsely populated province in eastern lowland Bolivia. Ecologically, Beni is a patchwork of two principal vegetation types. The majority of the department is covered by lush savannah grassland (Llanos de Moxos). Along riverine alluvial plains and to the northern and western borders of the area, this ecotope is supplanted by dense Amazonian moist forests. To the east, Beni borders another moist forest (Madeira‐Tapajós), which extends into Brazil and Santa Cruz department. In East Beni , TcI parasites were isolated from triatomines . Biological clones were obtained from primary cultures by plate cloning according to Yeo et al. (m HEPES buffer (pH 7.2), 30 mm haemin, 10% (v/v) heat‐inactivated foetal calf serum, 2 mm sodium glutamate, 2 mm sodium pyruvate and 25 μg/mL gentamycin . Genomic DNA was extracted using the Gentra PureGene Tissue Kit , according to the manufacturer's protocol. Clone genotypes were confirmed as TcI using a triple‐marker assay , Tupiza (n = 15), Toro Toro (n = 43), North Beni (n = 26) and East Beni (n = 87).A panel of 199 biological clones derived from 68 o et al.  to minimet al. , an alternate set of primers was designed to improve amplification efficiency: ND4 forward (5′‐TTYTTCCCAATATGTATBGTMAG‐3′) and ND4 reverse (5′‐TGTATTAYCGAYCAATTYGC‐3′), and reactions were performed using the same conditions as previously   between microsatellite genotypes calculated in microsat v1.5d  tree based on pairwise distances . A  Jombart , was uset et al. .10.5061/dryad.b8465). Population‐level genetic diversity was evaluated using sample‐size‐corrected allelic richness (Ar) in fstat 2.9.3.2  and +1 . FIS = 0 indicates Hardy–Weinberg allele frequencies. Sample‐size‐corrected private (population‐specific) allele frequency per locus (PA/L) was calculated in hp‐rare . Population‐level heterozygosity indices were also calculated in arlequin v3.11 and associated significance levels for P‐values derived after performing a sequential Bonferroni correction to minimize the likelihood of type I errors (Rice IA), was calculated in multilocus 1.3b . Additional mitochondrial multilocus sequence typing (mtMLST) data from 24 previously published TcI strains were included in selected analyses, as indicated   was calculated using dnasp v5.10.1 . In total, 10 122 alleles were identified, corresponding to 178 unique multilocus genotypes (MLGs). Multiple (≥3) alleles were observed at 0.83% of loci. Levels of intrastrain genetic diversity were high; multiclonality, that is the presence of multiple, different genetic clones, was observed in 65/68 strains. Identical intraclonal genotypes were sampled in five isolates . Clones were initially categorized into five populations based on geographical origin, consisting of three high‐altitude  and two low‐altitude groups (North and East Beni). All populations demonstrated uniformly high numbers of unique MLGs and low frequencies of repeated MLGs (Table DAS). Ten genetic clusters were defined among the 199 clones submitted to DAPC, once three principal components (PCs) were retained and analysed . A full list of isolate assignments to DAPC populations is included in Table S1 (Supporting information), and a multidimensional scaling plot of the DAPC results is shown in Fig. K = 10 , which was instead assigned to cluster 2 in the lowlands 1 group. Within the highlands group, isolates from different sampling areas and sources (hosts and vectors) were distributed across clusters 8 and 10, while cluster 1 comprised only a subset of clones from Triatoma infestans found in Tupiza and Toro Toro. The lowlands 1 group encompassed all strains from North Beni (only cluster 2) and approximately half of the isolates from Rhodnius spp. and Didelphis marsupialis in East Beni . Lastly, the lowlands 2 group contained all remaining East Beni clones, including those isolated from Rhodnius spp., D. marsupialis, Philander opossum and Sciureus spp.Patterns of isolate clustering were evaluated using two different methodologies: nonparametric population assignment (DAPC) and a NJ analysis based on pairwise genetic distances , one composed of all North Beni clones and the same portion of East Beni clones (DAS lowlands 1), the other containing the remaining East Beni strains (DAS lowlands 2). As previously, CV‐05 cl1 from Chapare clustered as an outlier among North and East Beni isolates. Comparison of branch lengths in Fig. DAS = 0.151 and 0.431 for highlands and lowlands, respectively). Within this clade, there was strong evidence for the existence of local geographical clusters in Tupiza (100% BS) and Toro Toro (73% BS), which clustered basally to the remaining highland strains.A NJ tree based on the same microsatellite data was constructed and further corroborated the DAPC strain assignments. A clear division between highland and lowland populations was observed, with isolates segregating into two well‐supported clades (64% BS) Fig. . SimilarDAS‐supported strain assignments (Table Ar = 1.92–2.22) and numbers of private alleles per locus (PA/L = 0.19–0.42), compared to the lowlands  , whereas both lowland populations demonstrated more pronounced deviations from H–W allele frequencies  .Population genetic indices were calculated using both a priori geographical and DAPC/ts Table . OverallFST) between a priori populations support a genetic demarcation between highland and lowland areas  or between the two lowland populations (FST = 0.087 and P < 0.0001 for North and East Beni). However, elevated FST values between closest highland and lowland study sites  indicate very limited gene flow, suggesting a powerful role for altitude and/or ecotope in structuring parasite populations. Interestingly, the extent of genetic subdivision between the most geographically distant highland populations  and adjacent areas of Beni (distance = ~155 km) was equivalent .Estimates of subdivision , among populations within groups  and among individuals within populations. Strikingly, 23% of total genetic variation was attributed to differences between highlands and lowlands, while 4.5% and 7% were present at the population and the individuals within population levels, respectively.Finally, a hierarchical L = −4845.23, Bayesian tree L = −4848.13, P = 0.12). A second ML tree was assembled using 24 additional outgroup sequences representing known TcI mitochondrial diversity, including a small population of domestic Bolivian isolates  were sequenced and concatenated into a 3684‐bp alignment. Twenty‐four unique haplotypes were identified from a total of 48 variable sites (~1.3% sequence diversity). ML and Bayesian phylogenies constructed from concatenated data were not significantly different  separated highland and lowland populations into two major clades, each with strongly supported internal structuring. The highland group was largely homogeneous, with a number of geographically dispersed strains sharing identical mitochondrial haplotypes. The mitochondrial topology also confirmed the existence of Tupiza‐ and Toro Toro‐specific populations (98/1.0), in agreement with the nuclear tree.Bol/Chile), while genetically distinct from sylvatic strains circulating in the same area (63/1.0), were grouped within the main highlands clade. As previously, lowland strains were subdivided into two well‐supported clades (74/1.0) with higher overall levels of genetic diversity, compared to highland isolates . No evidence of recombination between highland and lowland strains was observed, even in Chapare, a zone of ecological transition. Across the more ‘gentle’ ecological cline of East–North Beni, several instances of genetic hybridization were apparent. Three clones from the mixed East–North Beni group (DAS lowlands 1) and three isolates from the East Beni‐specific population (DAS lowlands 2) received unambiguously different phylogenetic positions in the maxicircle topology and are likely the progeny of multiple, independent mitochondrial introgression events . However, the strength of the effect was significantly larger among lowland isolates . Furthermore, when focusing on highland clones from approximately the same spatial scale as their lowlands counterparts [i.e. omitting the local subpopulation of Tupiza isolates identified in the DAS tree (n = 6)], little evidence for spatial structuring remained . Concordant with estimates of FST between populations, the differing extent of spatial genetic structuring suggests accelerated parasite dispersal among geographically disparate highland areas by comparison with adjacent lowland foci  among highland and lowland isolates, Mantel's tests were conducted using alternate nuclear and mitochondrial data sets. Nuclear IBD was detected within both highland and lowland populations  or lowland  populations using mitochondrial sequence data, potentially the result of lower population genetic resolution at these loci, but also consistent with the occurrence of mitochondrial introgression among lowland isolates.Interestingly, no IBD was detected in either highland . Deep internal nuclear branching patterns in both lowland groups were indicative of stable, undisturbed, long‐term genetic diversification, with correspondingly high levels of diversity. Mitochondrial introgression occurring among genetically distinct strains in Beni supports prolonged historical interactions between these two populations. Consistent with high intrahost and vector clonal diversity, these data support intense, local transmission and/or low rate of genotypic extinction . Dissimilar heterozygosity estimates between highlands (excess) and lowlands (deficit) suggest a recent hybrid origin for some highland strains or fundamental differences in mating systems between these two populations , a single clone (CV‐05 cl1) isolated from Rhodnius robustus in the Andean foothills was more closely related to lowland Beni strains on the basis of both nuclear and mitochondrial markers, suggesting the existence of an additional, under‐sampled transmission cycle and potential hybridization zone in Chapare, northern Cochabamba.While minimal parasite interaction was observed between neighbouring terrestrial and arboreal transmission cycles (high Rhodnius vectors (R. robustus and Rhodnius pictipes). In general, sylvatic Rhodnius species are promiscuous feeders, which can actively migrate at night to colonize domestic environments  . Previous T. cruzi studies that favoured constrained, extant co‐evolutionary scenarios were probably limited by sampling bias , strongly suggest ecological host fitting is the predominant mechanism of sylvatic T. cruzi diversification , we were unable to detect hybridization across the highland–lowland cline. However, mitochondrial introgression was observed among a subset of lowland strains between East and North Beni. Evidence of intra‐TcI genetic exchange in a primary Amazonian forest , and this level was comparable to that observed between neighbouring lowland areas (~155 km). Terrestrial clones also displayed limited genetic IBD, a lack of private alleles and excess heterozygosity, all potentially attributable to a recent population bottleneck and/or founder event followed by clonal propagation.Rhodnius vectors have a far greater capacity for auto‐dissemination than T. infestans and smaller rodents , microsatellite primers , microsatellite genotypes , concatenated maxicircle sequence alignment, mitochondrial ML phylogeny and microsatellite NJ phylogeny are all available from Dryad: doi: Appendix S1 DatasetClick here for additional data file.Table S1 Panel of Bolivian T. cruzi TcI biological clones assembled for analysis.Click here for additional data file.Table S2 Panel of microsatellite loci and primers employed in this study.Click here for additional data file.


The main goal of the current work was to infer the demographic history of seven Spanish goat breeds  based on genome-wide diversity data generated with the Illumina Goat SNP50 BeadChip . Five additional populations from Europe (Saanen and Carpathian) and Africa  were also included in this analysis (N = 80) for comparative purposes.Our results show that the genetic background of Spanish goats traces back mainly to European breeds although signs of North African admixture were detected in two Andalusian breeds . In general, observed and expected heterozygosities were quite similar across the seven Spanish goat breeds under analysis irrespective of their population size and conservation status. For the Mallorquina and Blanca de Rasquera breeds, which have suffered strong population declines during the past decades, we observed increased frequencies of large-sized (ROH), a finding that is consistent with recent inbreeding. In contrast, a substantial part of the genome of the Palmera goat breed comprised short ROH, which suggests a strong and ancient founder effect.Admixture with African goats, genetic drift and inbreeding have had different effects across the seven Spanish goat breeds analysed in the current work. This has generated distinct patterns of genome-wide diversity that provide new clues about the demographic history of these populations.The online version of this article (doi:10.1186/s12711-016-0229-6) contains supplementary material, which is available to authorized users. Since their domestication in the Fertile Crescent, goats are bred by humans as a source of milk, meat and fiber. Breeding has led to substantial changes in the patterns of genomic diversity of caprine breeds . IntensiAccording to the classification criteria established by the Spanish Ministry of Environment, Rural and Marine Affairs , in SpaiSampling was performed by trained veterinarians in the context of sanitation campaigns and parentage controls that were not directly related with our research project. For this reason, it was not necessary to obtain permission from the Universitat Autònoma de Barcelona Committee of Ethics in Animal Experimentation. In all instances, veterinarians followed standard procedures and relevant national guidelines to ensure appropriate animal careBlood samples were collected by jugular venipuncture in Vacutainer EDTA-containing tubes. We sampled 176 individuals from seven Spanish goat breeds . In addition, we performed a moderate linkage disequilibrium (LD) pruning step with the command—indep 50 5 2 of the PLINK software, as recommended in the PLINK manual [Following the instructions of the manufacturer , we genoK manual  and in aK manual . This isK manual . After tK manual  was usedo) and expected (He) heterozygosities for each population, whereas pairwise FST values were obtained with Arlequin 3.5 [PLINK  was usedquin 3.5 . Populatquin 3.5 , which cPLINK  was alsoo values of 0.38, 0.38 and 0.42, respectively. Only the Palmera breed had a lower level of genetic diversity, with Ho and He values less than 0.29. The MDS in Fig. Descriptive statistics of genetic diversity and differentiation are in Additional file The results of the admixture analysis agreed well with the findings outlined above . These results are also similar to those from a genome-wide survey of Italian goats [In 2005, Pereira et al.  charactean goats  and a woe = 0.39) and Malagueña  were quite similar to those found for much smaller endangered populations, such as Bermeya  and Blanca de Rasquera . This unexpected observation could be due to a technical bias i.e. that only the SNPs with intermediate frequencies, in a representative sample of goat breeds, were selected to form part of the Goat SNP50 BeadChip [The levels of genetic diversity that we found in breeds with large and expanding population sizes, such as Murciano-Granadina  than in Spanish 6.68 Mb) breeds  of the ROH present in Spanish goat breeds. We also demonstrate that these ROH features reflect demographic history much better than simple diversity statistics (e.g. observed and expected heterozygosities).


Fascioloides magna from all enzootic North American regions were revealed in close relation with geographical distribution of its obligate definitive cervid hosts for the first time.Population structure and genetic interrelationships of giant liver fluke c oxidase subunit I  and nicotinamide dehydrogenase subunit I  were applied as a tool. The concatenated data set of both cox1 and nad1 sequences (789 bp) contained 222 sequences that resulted in 50 haplotypes. Genetic data were analysed using Bayesian Inference (BI), Maximum Likelihood (ML) and Analysis of Molecular Variance (AMOVA).Variable fragments of the mitochondrial cytochrome F. magna, which separated the parasite into western and eastern populations. Western populations included samples from Rocky Mountain trench (Alberta) and northern Pacific coast (British Columbia and Oregon), whereas, the eastern populations were represented by individuals from the Great Lakes region (Minnesota), Gulf coast, lower Mississippi, and southern Atlantic seaboard region  and northern Quebec and Labrador. Haplotype network and results of AMOVA analysis confirmed explicit genetic separation of western and eastern populations of the parasite that suggests long term historical isolation of F. magna populations.Phylogenetic analysis revealed two major clades of F. magna adults towards any definitive host species; the detected haplotypes of giant liver fluke are shared amongst several host species in adjacent populations.The genetic makeup of the parasite’s populations correlates with data on historical distribution of its hosts. Based on the mitochondrial data there are no signs of host specificity of  Spatial distribution of parasites is closely coupled with distribution of their hosts; parasites require suitable hosts for nutrients and other resources, and thus they cannot occur in territories where susceptible hosts are absent. Therefore, the maximum geographical range of a parasite must equal the combined ranges of its hosts. The hosts also represent the only mean of dispersal for the majority of parasitic taxa and in the case of parasites with multi-host lifecycles, the parasite’s dispersal is driven by its most mobile host. Furthermore, geographical distribution and relationships of the parasites of terrestrial hosts may be affected by historical contractions and expansions of their hosts’ distribution, including recent bottlenecks caused by humans , 2. On tInfluence of hosts and parasites on their respective biodiversity is of particular interest, since each partner in a host-parasite association potentially exerts a selective pressure on the other . The relFascioloides magna, represents a very interesting parasitic model characterized by a wide spectrum of intermediate and definitive hosts, large spatial distribution, potential to colonize new territories and adapt to new host species. It parasitizes a wide range of definitive ruminant hosts, especially cervids. The fluke has a strong predilection to liver parenchyma where it is localized in thin-walled fibrous pseudocysts. It is generally accepted that F. magna is of North American origin and may have co-evolved with ancestral cervids, Odocoileus spp. [F. magna is enzootic in five major areas across the United States and Canada: (1) the northern Pacific coast, (2) the Rocky Mountain trench, (3) northern Quebec and Labrador, (4) the Great Lakes region, and (5) Gulf coast, lower Mississippi, and southern Atlantic seaboard.Giant liver fluke, eus spp. . CurrentCervus elaphus canadensis, white-tailed deer Odocoileus virginianus, caribou Rangifer tarandus, black-tailed deer Odocoileus hemionus columbianus and mule deer Odocoileus hemionus hemionus) and domestic ruminants  are known to be susceptible to F. magna infection but only white-tailed deer, caribou, and wapiti contribute significantly to maintaining its population [F. magna were finely tuned due to low number of flukes within individual deer. On the other hand, potential for translocation of liver flukes in wapiti is higher due to increased F. magna egg production and subsequent release to the environment [Concerning the host spectrum of giant liver fluke in North America, a variety of free-living  northern Italy, (2) Czech Republic and south-western Poland, and (3) Danube floodplain forests . In Eurbe River . In Eurond Radix  and defiama dama , 12.F. magna and the subsequent course of colonisation and migratory routes of this alien parasite in Europe recently were unravelled [cox1 + nad1 datasets revealed two genetically separated clades of European F. magna. The Italian population represented one phylogenetic clade while the second one included populations from the Czech focus and the Danube floodplain forests. Results clearly indicated that F. magna was introduced from North America to Europe at least twice; first to Italy and independently to the Czech Republic. As comparative samples, several F. magna specimens from North America were included in the above-mentioned work. The overall analysis of North American and European giant liver fluke populations clearly confirmed a western North American origin of the Italian population; these flukes clustered with specimens from Alberta (Canada) and Oregon (USA). On the other hand, representatives of the Czech Republic and Danube floodplain forests displayed close genetic relationships with parasites from the south-eastern USA. These data showed for the first time relatively high genetic molecular diversity of North American F. magna individuals [F. magna from a more extensive dataset including all five enzootic North American regions using previously applied mitochondrial cox1 and nad1 molecular markers [The origin of European populations of ravelled . Phylogeividuals . However markers , 14 in oF. magna from 37 livers originating from all five North American enzootic regions were included in the analysis  and caribou (Rangifer tarandus), while specimens from the Rocky Mountain trench (RMT) originated from wapiti. In the northern Pacific coast (NPC), F. magna samples were isolated from Roosevelt elk (Cervus elaphus roosevelti) and black-tailed deer. In Minnesota, belonging to the Great Lakes region (GLR), all flukes came from white-tailed deer. Finally, the Gulf coast, lower Mississippi, and southern Atlantic seaboard (SAS) enzootic region was represented by F. magna from wapiti and white-tailed deer; in particular flukes were obtained from US states of Mississippi (MS), Louisiana (LA), Georgia (GA), Florida (FL), and South Carolina (SC).Flukes from the enzootic region of northern Quebec and Labrador (NQL) were collected from muskox  and nicotinamide dehydrogenase subunit I , the previously designed primers and PCR conditions were applied [cox1 and nad1 datasets was performed in SeaView 4.2 [https://inkscape.org).For amplification and sequencing of partial mitochondrial cytochrome  applied , 14. The applied . Sequenc applied , 17. ConView 4.2 . Distribhttp://tree.bio.ed.ac.uk/software/tracer). Model of molecular evolution for the BI analysis was selected in PartitionFinder [cox1 and nad1 sequences of the closest available relative, Fasciola hepatica (GenBank Accession No. NC002546). The topology used to position a root for the trees from the ML and BI analyses was obtained in PhyML by a ML analysis of translated amino acid sequences under the LG model. Amino acid sequence analysis was used due to a relatively deep level of genetic variation between the two genera, which created a very long branch when analysed in the DNA mode.Phylogenetic reconstruction of the relationships between mtDNA haplotypes was performed using concatenated data with Maximum Likelihood algorithms (ML) in PhyML 3.0  and BayeonFinder  allowingF. magna (see Table Hd), nucleotide diversity (Pi) and neutrality tests  for population sets used in the second AMOVA analysis were calculated for concatenated data in DNASP 5.10.1 [Genealogical information contained in the two mtDNA genes was visualized using haplotype network in the software TCS 1.21 . To explP 5.10.1 . Significox1 mtDNA fragment  and 405 bp nad1 fragment (134 aa + stop codon) revealed 32 cox1 (CO1-Ha) and 28 nad1 (ND1-Ha) mitochondrial haplotypes  and four nad1  haplotypes were detected exclusively in the European populations of the parasite [The analysis of 384 bp cox1 (transitions (ts)/transversions (tv) ratio; 40/3), 36 substitutions were not responsible for change in amino acid sequence while seven mutations underwent the non-synonymous substitutions. As for nad1, 16 substitutions out of 23  were silent whereas seven mutations changed the amino acid sequence. The concatenated dataset of both cox1 and nad1 sequences (789 bp) contained 222 sequences that resulted in 50 haplotypes  were characterized by single cervid hosts; AB/wapiti, BC/Roosevelt elk, and OR/black-tailed deer Table . HoweverF. magna from geographically distant regions – SAS and NQL. Even more diverse was lineage no. 5, which included representatives of all eastern populations: SAS, NQL and GLR. Additionally, besides heterogeneity in geography, the three heterologous lineages  were characterized by the most diverse spectrum of definitive hosts – wapiti, caribou, muskox and white-tailed deer  of eastern enzootic regions was polyphyletic. Some of the respective sublineages within clade A mirror the geography/host pattern. The most homogenous internal lineages were no. 2 , nos. 4 and 6 , and no. 7  .Table 5F. magna populations from all enzootic regions in North America (NA); the Rocky Mountain trench (RMT), the northern Pacific coast (NPC), northern Quebec and Labrador (NQL), the Great Lakes region (GLR), and the Gulf coast, lower Mississippi, and southern Atlantic seaboard (SAS). The most straightforward finding was explicit genetic separation of western (NPC and RMT) and eastern  populations of the parasite.Current data provide a complete picture of the genetic structure of Parasites in general rely almost entirely on their hosts for dispersal ranging from small to large scales . The geoF. magna, in particular wapiti, white-tailed deer, and caribou. According to Pybus [F. magna are separated in detached pockets across North America and the parasite may have co-evolved with the ancestral Odocoileus spp. Giant liver fluke has originally been widespread in white-tailed deer in major wetland habitats throughout NA where wapiti and caribou sympatric with white-tailed deer encountered F. magna in overlapping contaminated regions.The genetic structure of NA giant liver fluke populations needs to be related to the historical and current distribution of obligate definitive NA cervid hosts of to Pybus , currentF. magna corresponds very probably to historical distribution, extirpation, re-introduction, and current occurrence of two obligate definitive hosts of giant liver fluke; white-tailed deer in eastern and wapiti in the western part of NA. Odocoileus spp. is native to North America and its populations were well established in the south-eastern USA during the Pleistocene epoch [th century unrestricted hunting, deforestation and extensive agricultural development led to dramatic declines in the white-tailed deer populations in the south-eastern USA [th and in 20th century increased the white-tailed deer populations, especially in the south-east where re-establishing deer populations was considered essential [The distinction of two mitochondrial lineages of ne epoch . In the tern USA . Implemessential .th century, wapiti were restocked from the Yellowstone National Park and expanded in number and geographic distribution within the Banff National Park (BNP). A significant exchange of wapiti between BNP and Kootenay National Park through Vermilion Pass provided a natural dispersal mechanism for F. magna from British Columbia into Alberta  throughout the core of Canada and USA, with very limited ecological conditions suitable for maintaining the giant liver fluke. Consequently, western and eastern populations of ulations . The effulations , 33 as wled deer  and cariled deer . Howeverpes Fig.  point toF. magna individuals from geographically distant eastern populations of the parasite , where overlapping distribution of white-tailed deer and caribou may have played an important role. Caribou were present in North America as early as the glacial periods of the middle Pleistocene in Beringia [th century, peripheral populations of caribou within the United States were extirpated and populations that occurred from Minnesota to Maine, in New York, Wisconsin and Michigan disappeared [F. magna in overlapping contaminated wetland habitats, thus facilitating its movement into new areas and establishing new parasite populations. The present fluke population in caribou in the northeast (NQL) is most probably a residual population that survived the caribou extirpations in more southern regions. In addition, giant liver fluke further spilled over into local muskox populations that were overlapping with infected caribou in NQL region.Phylogenetic reconstruction of the relationships between concatenated mtDNA haplotypes using Maximum Likelihood and Bayesian Inference, as well as haplotype network reflecting genealogical information congruently revealed genetic admixture of Beringia . In the appeared , 38. Whiappeared  and overappeared , 41. ConO. virginianus from south Florida showed genetic differentiation attributable to pleistocene climatic oscillations [In contrast to the general pattern of mixing in the eastern populations, some populations seem to retain genetic distinctiveness. For example, all but one haplotype from Florida and Georgia created a separate cluster in the haplotype network Fig. . Despitellations . Thus, cCervus elaphus in North America [C. e. roosevelti) along the Pacific coast of British Columbia and US states Washington/Oregon (NPC enzootic region) is immediately adjacent to the Rocky Mountain elk (C. e. canadensis) population in the RMT enzootic region. The Coastal Mountains to the west (where the Roosevelt elk originated) are separated from the Rocky Mountains to the east (origin of Rocky Mountain elk) only by a broad lowland area (the Interior Plateau) [F. magna populations. The current shared genetic makeup of the western populations of F. magna could be explained by admixing of the fluke population in association with distribution of different wapiti subspecies in this region.Pertinent to the genetic relatedness of western populations of giant liver fluke (NPC and RMT) confirmed by statistical testing  and phylogenetic analyses (Maximum Likelihood and Bayesian Inference), the distribution of six populations (subspecies) of  America  needs toPlateau) , which pF. magna adults towards any definitive host species; the detected haplotypes of giant liver fluke are shared amongst several host species in adjacent populations. Similar genetic patterns of geographic isolation with shared hosts broadly displayed e.g., winter ticks Dermacentor albipictus on cervids in North America [Based on the mitochondrial data, it can be concluded that there are no signs of host specificity of  America , suggestF. magna reflect historical distribution, past extirpation, subsequent re-introduction, and current occurrence of the obligate definitive cervid hosts of giant liver fluke. The present study provides missing pieces of the puzzle and completes the comprehensive dataset on mitochondrial structure and population diversity of all (NA and European) F. magna populations. Comparison of original results with published data [F. magna, a high level of molecular diversity in mitochondrial haplotypes evident between and within respective enzootic regions was anticipated. While the total number of concatenated haplotypes in NA populations ranged from three (RMT) and eight (each in NQL and NPC) to 26 (SAS) (present study) only two haplotypes were determined in Danube floodplain forests and four in each of Italy and the Czech Republic [Genetically diverse NA populations of hed data  revealedRepublic . As expeF. magna from Italy, Alberta, and Oregon, thus confirming the western NA origin of fascioloidosis in the first European focus – Italy [F. magna samples [F. magna populations should be assessed by multilocus population genetic markers, such as microsatellites, polymorphic and codominant markers which were recently designed specifically for giant liver fluke [The only haplotype common for parasites from both continents was haplotype 1, assessed in  – Italy . After c samples  two addier fluke  and can


The genetic provenance of domesticated plants and the routes along which they were disseminated in prehistory have been a long-standing source of debate. Much of this debate has focused on identifying centers of origins for individual crops. However, many important crops show clear genetic signatures of multiple domestications, inconsistent with geographically circumscribed centers of origin. To better understand the genetic contributions of wild populations to domesticated barley, we compare single nucleotide polymorphism frequencies from 803 barley landraces to 277 accessions from wild populations.We find that the genetic contribution of individual wild populations differs across the genome. Despite extensive human movement and admixture of barley landraces since domestication, individual landrace genomes indicate a pattern of shared ancestry with geographically proximate wild barley populations. This results in landraces with a mosaic of ancestry from multiple source populations rather than discrete centers of origin. We rule out recent introgression, suggesting that these contributions are ancient. The over-representation in landraces of genomic segments from local wild populations suggests that wild populations contributed locally adaptive variation to primitive varieties.This study increases our understanding of the evolutionary process associated with the transition from wild to domesticated barley. Our findings indicate that cultivated barley is comprised of multiple source populations with unequal contributions traceable across the genome. We detect putative adaptive variants and identify the wild progenitor conferring those variants.The online version of this article (doi:10.1186/s13059-015-0712-3) contains supplementary material, which is available to authorized users. The domestication of plants and animals around 10,500 YBP initiated the development of complex human societies and provided the raw material on which modern agriculture still depends . Barley Extensive archeological remains at human Neolithic sites capture the timing and phenotypic transition from wild to cultivated barley across the Near East , 12, 13 Multiple lines of evidence, presented here, indicate that barley landraces have mosaic ancestry, reflecting the contribution of all major geographic portions of the range of the wild progenitor species. A broad contribution of wild progenitor populations to the landraces is consistent with archeological evidence for a gradual transition to cultivation . This isK = 2) while Asian landraces correspond to the Eastern partition . In summary, western wild barley populations appear to contribute most directly to the genetic constitution of African and European landraces, while eastern wild barley populations made a greater contribution to Asian landraces.To investigate the contribution of wild to domesticated barley we first examined the extent of population structure among landraces using genotyping data from 6,152 SNPs in 803 landrace accessions collected in Europe, Asia, and North Africa , the largest genome-wide proportion of ancestry derives from the Southern Levant wild population (57 %)  between geographic distance and genetic contribution of the Central Asian wild population to the Asian landraces . A similar pattern is observed in all other comparisons between the proportion of ancestry and distance from each wild population. This correlation is consistent with isolation by distance, with r equal to -0.28 and -0.27 for comparisons to Northern Levant and Syrian Desert wild populations, respectively. There is very limited correlation with distance (0.04) for Northern Mesopotamia and Southern Levant populations. Within each landrace population, individual samples have distinct genetic compositions, with some accessions carrying higher proximate wild ancestries than the average in their population. For example, the Northern Mesopotamia wild population contributed 12 % of the genomic segments in Asian landraces  to any of the wild populations  Core Collection from the USDA. These 803 individuals include all landraces collected in Europe, Asia, and North Africa constituting the range of dissemination of cultivated barley in human pre-history Fig. .We also make use of the 284 wild barley accessions from the Wild Barley Diversity Collection (WBDC)  analyzedA collection of 2,446 landraces and cultivated accessions from the NSGC were genotyped with 7,864 SNPs using the Illumina Infinium SNP genotyping platform (hereafter referred to as the 9K). The 9K chip contains 5,010 SNPs discovered in a panel of 10 barley varieties, composed primarily of European two row cultivars. In addition, a set of 2,832 SNPs used for the existing BOPA (Barley Oligo Pooled Assay 1 and 2) on the Illumina Golden Gate genotyping platform  was inclWe used automated genotype calling implemented in the software ALCHEMY . ALCHEMYet al. [SNP quality control procedures consisted of the removal of SNPs that were monomorphic, had more than 10 % missingness, or had more than 10 % heterozygosity [see reference et al. . We retaThe 284 accessions from the WBDC were genotyped with 3,072 SNPs , a subseet al. [We used the consensus genetic map described in  which iset al.  with theet al. . This mahttp://www.r-project.org/). Experimentally phased haplotypes are used in two analyses where they are critical to inference, that is, the estimation of admixture proportions and assessment of identity by state between wild and landrace accessions.We infer the phase of heterozygous sites (approximately 0.1 % of sites) using PHASE v.2.1.1 , 45 for r2 [Linkage disequilibrium (LD) as measured by r2  is calcur2  was usedK) ranging from 1 to 7  and the collection of landraces   from each wild population per iteration, without replacement. As in the landrace assignment, we used windows of 75 SNPs. An average of 16.4 % of genomic segments per individual could not be assigned with confidence (probability of assignment <0.95) to any population of origin. Window sizes smaller than 75 SNPs resulted in > 80 % of the genome being unassigned (data not shown). In summary, among genomic segments that are assigned with high confidence, 69 % are correctly assigned to the population of origin. A notable exception to assignment of genomic segments of wild individuals back to population of origin occurred in the Northern Levant population, where proportional assignment to the Northern Levant wild population averaged 43 %, with 31 % of segments assigning to the geographically proximate Southern Levant  analysis between the wild and landrace barley lines is used to test for shared genomic segments between populations, consistent with recent introgression. The IBS analysis used PLINK v.1.90  with winhttps://github.com/AnaPoets/BarleyLandraces. The raw genotyping data for the 2,446 accessions in the NSGC are available in Figshare, http://figshare.com/articles/Raw_Genotyping_Data_Barley_landraces_are_characterized_by_geographically_heterogeneous_genomic_origins/1468432.All code used for analysis and for figures can be found in the GitHub repository,


Dioscorea humilis (Dioscoreaceae), a dioecious geophyte extending 530 km from the Valparaíso to the Bío-Bío Regions, as a case study to disentangle the spatio-temporal evolution of populations in conjunction with latitudinal environmental changes since the Last Inter-Glacial (LIG) to the present. We used nuclear microsatellite loci, chloroplast (cpDNA) sequences and environmental niche modelling (ENM) to construct current and past scenarios from bioclimatic and geographical variables and to infer the evolutionary history of the taxa. We found strong genetic differentiation at nuclear microsatellite loci between the two subspecies of D. humilis, probably predating the LIG. Bayesian analyses of population structure revealed strong genetic differentiation of the widespread D. humilis subsp. humilis into northern and southern population groups, separated by the Maipo river. ENM revealed that the ecological niche differentiation of both groups have been maintained up to present times although their respective geographical distributions apparently fluctuated in concert with the climatic oscillations of the Last Glacial Maximum (LGM) and the Holocene. Genetic data revealed signatures of eastern and western postglacial expansion of the northern populations from the central Chilean depression, whereas the southern ones experienced a rapid southward expansion after the LGM. This study describes the complex evolutionary histories of lowland Mediterranean Chilean plants mediated by the summed effects of spatial isolation caused by riverine geographical barriers and the climatic changes of the Quaternary.The effects of Pleistocene glaciations and geographical barriers on the phylogeographic patterns of lowland plant species in Mediterranean-climate areas of Central Chile are poorly understood. We used  The diversification of this small group followed the retreat of the marine transgressions of the middle Miocene (15-11 Ma) which covered central Chile, providing new lands available for plant colonization from the late Miocene onwards Dioscorea humilis is a dioecious, diploid (2n = 14), dwarf geophyte with a widespread distribution spanning five central Chilean regions (530 km), from its northernmost limit in Valparaíso to its southernmost limit in Bío-Bío Dioscorea humilis occurs in the lowland depression between the coastal mountain range and the Andes. It includes two subspecies, the widespread D. humilis subsp. humilis and the narrow parapatric Maule coastal endemic D. humilis subsp. polyanthes (F. Phil.) Viruel, Segarra-Moragues & Villar The Dioscorea humilis has a sprawling habit with shoots creeping among rock crevices. Flowers are tiny and inconspicuous; those of males are produced in pauciflorate racemes, and those of females are generally solitary. The pollination mechanisms are unknown, but flower morphology suggests the implication of a small-sized insect. The wingless seeds are produced in capsules which are sustained by spirally curled peduncles that attach capsules close to the ground or inside rock crevices, suggesting extremely short-distance seed dispersal D. humilis. Additionally, Environmental Niche Modelling (ENM) was estimated on the current range extension of the infraspecific genetic groups and projected to two past scenarios, the Last Glacial Maximum (LGM) and Last Inter-Glacial (LIG). Phylogeographical patterns obtained from molecular markers, together with the estimated past variation in range extension, were investigated to elucidate the effect of Pleistocene glaciations and geological and hydrological barriers in the evolutionary history of lowland central Chilean species with limited dispersal abilities like D. humilis.We used nuclear microsatellite markers and cpDNA sequences to document the current patterns of population genetic diversity and structure in Necessary permits for fieldwork and sampling were obtained from the Corporación Nacional Forestal (CONAF-Chile).D. humilis were collected throughout its entire distribution range. Fifteen populations (Dhh01-Dhh15) corresponded to D. humilis subsp. humilis and two populations (Dhp01-Dhp02) to D. humilis subsp. polyanthes (D. humilis subsp. humilis (Dhh01-Dhh08) were located North of the Maipo river basin, growing in semi-arid Mediterranean-type climate areas, whereas the other seven populations (Dhh09-Dhh15) were located South of it (D. humilis subsp. polyanthes (Dhp01-Dhp02), were growing in humid Mediterranean-type climate areas Fresh leaves from a total 558 individuals from 17 populations of lyanthes , Fig. 1.th of it , Fig. 1.trnT-L and trnL-F Two plastid regions, A*), observed heterozygosity (HO), genetic diversity within populations (HS), inbreeding coefficient (FIS) and population differentiation (FST) using FSTAT v. 2.9.3.2 FST) was calculated with ARLEQUIN 3.11 FST values (i.e. FST/(1−FST); Allele frequencies and genetic diversity indices were calculated in all populations using GENETIX 4.05 DA genetic distances Pairwise Population genetic structure was investigated by means of Analysis of Molecular Variance (AMOVA) which was performed in ARLEQUIN 3.11 to partition in different population groups according to the taxonomical or geographical membership. The significance of the analyses was tested with 1000 replicates.K genetic clusters from one to 19, with ten replicates for each K. The analyses were performed with a burn-in period and a run length of the Monte Carlo Markov Chain (MCMC) of 7×105 and 7×106 iterations, respectively. The most likely number of genetic clusters (K) was determined according to Evanno et al.Bayesian clustering was also used to infer population genetic structure using STRUCTURE 2.1 S), the number of haplotypes (h), the haplotype diversity index (Hd) and the average number of pairwise nucleotide differences between DNA sequences (θπ) Haplotype polymorphism was estimated within populations and within genetic and geographical groups through the analysis of the number of segregating sites  and Last Interglacial (LIG) conditions. A set of 19 bioclimatic variables (Table S1 in www.worldclim.org) plus the altitude were used, and GIS layers with 30 sec resolution were clipped to the extent of central Chilean regions using DIVA-GIS D. humilis  was conducted to evaluate the potential distribution of the geographical groups of DA population genetic distances FSTD. humilis, and the correlation between the 20 environmental variables and latitude, using the Mantel test with 1000 random permutations.Additionally, we assessed pairwise correlations between all 20 environmental variables studied and pairwise D. humilis data fit these requirements since no significant increase in the area under the curve (AUC) values was observed when using all variables compared to those from the reduced set of variables  to build the model and test data (25%) to test the accuracy of the model. Fifteen subsample replicates were performed in each run using the default options and 1000 iterations. Model accuracy was assessed with the AUC value of the receiver-operating characteristic curve (ROC) D. humilis ssp. humilis. The low number of known populations of D. humilis subsp. polyanthes (Dhp01-02) precluded a confident ENM analysis of this taxon.ENM were conducted for the two northern (Dhh01-Dhh08) and southern (Dhh09-Dhh15) population groups of D. humilis subsp. humilis have remained stable during at least since LIG.ENMs were projected to LGM (c. 21 ka BP), with 2.5 arc-minutes resolution D. humilis using PAST 2.17c A complementary ENM approach was done through a Principal Component Analysis (PCA), which was constructed with the raw data obtained from the 19 climatic variables and the altitude for each population of D. humilis  and 9 (11.39%) were exclusive to D. humilis subsp. humilis and D. humilis subsp. polyanthes, respectively  to 0.598 (Dhp01), and unbiased expected heterozygosities from 0.357 (Dhh12) to 0.521 (Dhh10) . Five ofe excess .D. humilis subsp. humilis and D. humilis subsp. polyanthes, except for observed heterozygosity (Ho). Surprisingly, the more restricted endemic D. humilis subsp. polyanthes showed significantly higher (p = 0.033) average Ho (D. humilis subsp. humilis failed to find significant differences at any of the tested indices (No significant differences were detected for the tested genetic diversity indices between rage Ho . Similar indices .p<0.05) levels of population differentiation were observed among populations (results not shown). Higher average FST values were found between populations of both subspecies (average FST = 0.295) than among populations within subspecies . Similarly, a higher average differentiation was observed between northern and southern populations groups of D. humilis subsp. humilis (average FST = 0.198), than among populations within northern populations (Dhh01-Dhh08 FST = 0.069) and southern populations (average Dhh09-Dhh15 FST = 0.109) of D. humilis subsp. humilis with differentiation between the groups not being statistically significant  or to cluster 2  to differences among populations within groups (D. humilis subsp. humilis populations into four genetic clusters did not increase the variance among groups (11.81%) but lowered the proportion of variance among populations within groups (5.34%).Non-hierarchical AMOVA attributed 19.03% of the total variation to among populations of  humilis . In hiern groups . The groD. humilis subsp. polyanthes separated at a large distance from populations of D. humilis subsp. humilis  and D. humilis subsp. humilis  populations  and southern  geographical groups of D. humilis subp. humilis . Three haplotypes  were restricted to some western populations of northern D. humilis subsp. humilis, whereas haplotypes V, VI and VII were restricted to some southern populations of D. humilis subsp. humilis , than in D. humilis subsp. polyanthes  (D. humilis subsp. humilis (Dhh01-Dhh08) showed higher haplotype diversity  compared to the southern group (Dhh09-Dhh15) of populations  (Haplotype diversity was higher in = 0.500) , as expe= 0.534) .TCS estimated a 95% maximum connection of 17 steps incorporating all eight haplotypes into the network and inferred three missing haplotypes . The hapDA and FST genetic distances  and all but three temperature-derived variables  were significantly correlated to genetic distances . At the subspecies level, the variables with the largest contributions to the ENM of D. humilis subsp. humilis were altitude and the climatic variables bio8 (mean temperature of wettest quarter), bio9 (mean temperature of driest quarter) and bio15  , reproductive (winged seeds) and distribution range , characteristics that, contrary to D. humilis should predispose them to higher levels of genetic diversity  and 2  on opposite shores of the Yangtze river D. humilis, such as the snake Philodryas chamissonis Wiegmann D. humilis subsp. humilis  and cooling LGM, . The pre the LGM , precede the LGM .D. humilis subsp. humilis Click here for additional data file.Appendix S2Microsatellite allele frequencies, Bayesian estimation of genetic clusters and plastid haplotype frequencies in populations of Dioscorea humilis.(DOC)Click here for additional data file.Appendix S3Comparison of microsatellite genetic diversity in yam species.(DOC)Click here for additional data file.