Phytophthora colocasiae that causes taro leaf blight is one of the most devastating diseases of taro which is widely distributed in India. Inter and intra-specific genetic diversity among P. colocasiae isolates collected from same field was assessed using amplified fragment length polymorphism (AFLP) marker. Seven primer pairs produced 431 markers, of which 428 (99.2 %) were polymorphic. Considerable genetic variability was displayed by the isolates. The average value of the number of observed alleles, the number of effective alleles, mean Nei’s genetic diversity, and Shannon’s information index were 1.993, 1.385, 0.261, and 0.420, respectively. Analysis of molecular variance (AMOVA) showed that the majority (85 %) of the diversity were present within populations of P. colocasiae. Dendrogram based on AFLP molecular data using the unweighted pair group method with arithmetic mean (UPGMA) classified the P. colocasiae isolates into two major clusters irrespective of their geographical origin. Clustering was further supported by principle coordinate analysis. Cophenetic correlation coefficient between dendrogram and original similarity matrix was significant (r = 0.816). The results of this study displayed a high level of genetic variation among the isolates irrespective of the geographical origin. The possible mechanisms and implications of this genetic variation are discussed. Colocasia esculenta (L.) Schott) is a major root crop of the family Araceae with wide distribution in tropics. It is the fourteenth most consumed vegetable in the world  or the PCR-based randomly amplified polymorphic DNA (RAPD). AFLP has been successfully used for analysis of genetic diversity in P. infestans  to study how this genetic diversity is distributed among and within populations of P. colocasiae.The objectives of the present study were: (1) to analyze the genetic diversity of P. colocasiae used in this study were obtained from same field of taro (Colocasia esculenta) showing typical symptoms of leaf blight across different geographical origins of India . Hyphae emerging from a diseased tissue were transferred to 2 % water agar. After 3–4 days, single hypha was transferred to a Potato dextrose agar  plate under a dissecting microscope. Each isolate was stored at −20 °C in 50 % glycerol (long-term storage) and at 15 °C on potato dextrose agar (PDA) slants in the dark (short-term storage). The P. colocasiae isolates were grown on PDA Petri dishes at 25–28 °C in the dark for mycelium production.For isolation, leaf tissue segments of 1–2 cm from leaf blight infected area were excised. The segments were sterilized in 1 % sodium hypochlorite for 2 min, rinsed twice with sterile distilled water, and placed onto Phytophthora colocasiae isolates were grown in Potato dextrose broth medium . For DNA isolation, small blocks (1 cm) of actively growing cultures were used to inoculate Erlenmeyer flasks (250 ml) containing 100 ml of autoclaved potato dextrose broth. The cultures were placed on a rotary shaker  at 50 rpm and incubated at 28 ± 2 °C. After 5–10 days, depending on the growth of each isolate, mycelia were harvested by filtration through cheesecloth, blotted dry with sterile paper towels, and used immediately for DNA isolation. DNA was isolated using a Genomic DNA purification kit  according to manufacturer’s instructions. The integrity and quality of the DNA isolated were evaluated by electrophoresis on 0.8 % agarose gel using a 1 kb DNA ladder as a DNA size marker.2, 50 M Tris–HCL, pH 7.5, 10 mM DTT, 1 mM ATP, 25 μg/mL BSA), EcoRI ligation adapter (30 ng) 0.5 μl, Taq I ligation adapter (150 ng) 0.5 μl, T4 DNA ligase (5 U/μl) 0.5 and 3 μl double deionized water, to a final volume of 20 μl. The mixture was incubated at 25 °C overnight. Pre-amplification PCR was performed after diluting the ligated DNA tenfold with double deionized water. A total volume of 25 μl reaction mixture containing 3 μl of the digestion/ligation mixture, 1.0 μl Taq1 primer (200 ng), 1.0 μl EcoRI primer (200 ng), 2.5 μl 10× PCR Buffer , 0.5 μl dNTPs (2.5 mM), 0.5 μl Taq DNA polymerase (1 U/μl) and 17.5 μl double deionized water was prepared. PCR reactions were performed with the following cycling parameters: 5 min at 95 °C; 30 cycles of 30 s denaturing at 94 °C, 60 s annealing at 56 °C, and 60 s elongation at 72 °C, ending with 4 °C pause. After checking for the presence of a smear of fragment by 1.5 % agarose electrophoresis, the amplification product was diluted 20 times with double deionized water. After pre-screening 36 primer pairs, 7 selective primer pairs were chosen for this study  or absence (0) by visual observation. In order to ensure credibility, only reproducible and well-defined bands were scored. Polymorphic and monomorphic bands were determined for each AFLP primer pair, but only polymorphic bands were included in the analysis. Bands were assumed to be independent, and those of identical size were assumed to have identical sequences. A dendrogram was constructed using genetic similarity matrices to display relationships between isolates using the Nei and Li  accordinP. colocasiae of any two regions .The similarity matrix was also used to perform a hierarchical analysis of molecular variance (AMOVA) , mean number of alleles per locus (A), effective number of alleles (AE), observed heterozygosity (HO), and expected mean heterozygosity (HE) with respect to Hardy–Weinberg equilibrium (Hedrick Allelic frequencies of AFLP marker were used separately to estimate the percentage of polymorphic loci ( Hedrick  using th Hedrick . Loci werBarD) statistic (Agapow and Burt rBarD = 0) can be rejected when the observed rBarD < 0.001, and it can be assumed that the sampled isolates probably originated from a population with a clonal mode of reproduction  were polymorphic. The bands were distinct and easy to score. There were differences in the numbers of AFLP loci produced by each of the different primer pairs, which presumably reflect differences in sequence composition in the genome. The highest number of amplification products (95) was obtained with the primer pair E + AG/T + AA, while the lowest (51) with E + GA/T + GT pair; the average number of bands among total 7 primer pairs was 61.5. The number of polymorphic fragments detected by each primer varied from 51 to 95, with an average of 61.1. The highest number of polymorphic bands (95) was produced by the primer pair E + AG/T + AA, whereas the primer E + GA/T + GT generated the lowest number of polymorphic bands (51) , effective number of alleles (NE) and Nei’s gene diversity values varied among populations studied. The population distance tree based on Nei’s gene diversity indices is presented in Fig. Population genetic parameters of P. colocasiae populations in this study was distributed on a small spatial scale with 85 % of the genetic diversity distributed within populations and only 14 % among populations (Table GST) was 0.091 which supports the AMOVA analysis indicating only limited genetic diversity among populations and significant diversity within populations. The pairwise Φ statistics were 0.147 indicating populations are considerably differentiated. The estimate of gene flow (Nm) among populations was 4.97.Analysis of molecular variance (AMOVA) based on AFLP data shows that a high percentage of the total genetic diversity of rBarD for four regions viz. Assam, Kerala, Andhra Pradesh and Odisha were 0.0386 (P < 0.050), 0.0290 (P < 0.100), 0.0203 (P < 0.050), 0.0164 (P < 0.033), respectively. The result indicates that the P. colocasiae populations are a population with a recombination mode of reproduction.The observed P. colocasiae was analyzed by 7 AFLP primer combinations on the basis of Nei and Li distance (Nei and Li The genetic relationship among 25 isolates of r = 0.816) markers supporting a good degree of confidence in the association obtained for 25 isolates of P. colocasiae. The results of the PCA derived on the basis of AFLP data illustrated similar trend to cluster analysis. The first three principal coordinate components accounted for 11.91, 20.91 and 29.16 % variation, respectively  is at least possible in this oomycetes.The dendrogram reconstituted based on the genetic similarity coefficient summarizes the interrelationship among P. colocasiae. It is well known that sexual recombination increases genotype diversity in populations, since it creates novel recombinants. Sexually reproducing populations make management of disease more difficult due to the constant appearance of new genotypes that increase the variability of features like fungicide resistance, higher aggressiveness, and better fitness in the population. However, there are several lines of evidence that indicate the absence or rare occurrence of sexual reproduction in P. colocasiae as compatible mating types (A1 and A2) are seldom found in the same field (Lin and Ko P. colocasiae populations. Alternatively, other mechanisms, such as mutation, translocations, chromosomal deletions and duplications are common in Phytophthora species (Goodwin P. colocasiae populations. Mitotic gene conversion was observed to occur at remarkably high frequencies in Phytophthora sojae documenting the potential for rapid generation of variation (Chamnanpunt et al. P. infestans have previously been shown to differ from their parents in several characteristics such as aggressiveness, growth rate, colony morphology and virulence (Caten and Jinks What is less clear is the cause of the high level of genetic variation in populations of  Goodwin , which mP. colocasiae populations could respond rapidly to selection exerted by newly introduced host resistance genes or fungicides, underlining the importance of relying on integrated disease management. Disease management programmes should focus on local scale than on a regional level since it is likely that effective packages at one particular location may not prove so in other regions. Even though this study does not provide information on the effective population size and only limited information on gene flow, it still provides important evidence on the evolutionary potential of the Indian P. colocasiae populations. The small sample sizes in this study, however, restrict the relevance of the analysis and the credibility of results for more generalized conclusions. Further studies should therefore be carried out, using larger populations from more extended geographical regions to gain insights into the origin of diversity in this important plant pathogen.For breeding programs aimed at reducing the negative effects of fungal pathogens, the evolutionary potential of populations is of prime importance. Fungal populations are considered to have high evolutionary potential when they have a mixed reproductive system, moderate gene or genotype flow, and large effective population size (McDonald and Linde


Linguistic and genetic studies on Roma populations inhabited in Europe have unequivocally traced these populations to the Indian subcontinent. However, the exact parental population group and time of the out-of-India dispersal have remained disputed. In the absence of archaeological records and with only scanty historical documentation of the Roma, comparative linguistic studies were the first to identify their Indian origin. Recently, molecular studies on the basis of disease-causing mutations and haploid DNA markers (i.e. mtDNA and Y-chromosome) supported the linguistic view. The presence of Indian-specific Y-chromosome haplogroup H1a1a-M82 and mtDNA haplogroups M5a1, M18 and M35b among Roma has corroborated that their South Asian origins and later admixture with Near Eastern and European populations. However, previous studies have left unanswered questions about the exact parental population groups in South Asia. Here we present a detailed phylogeographical study of Y-chromosomal haplogroup H1a1a-M82 in a data set of more than 10,000 global samples to discern a more precise ancestral source of European Romani populations. The phylogeographical patterns and diversity estimates indicate an early origin of this haplogroup in the Indian subcontinent and its further expansion to other regions. Tellingly, the short tandem repeat (STR) based network of H1a1a-M82 lineages displayed the closest connection of Romani haplotypes with the traditional scheduled caste and scheduled tribe population groups of northwestern India. German Zigeuner, French tzigane and names in several other European languages derive from a designation for a Manichaean sect that practiced sorcery and soothsaying in the last centuries of the Byzantine Empire. On linguistic grounds, Grellmann The Roma in England are traditionally known as gypsies because it was thought that they came from Egypt and were therefore ‘Rroma (singular Rrom), whereby the double rr in Romani orthography represents a uvular ‘r’ [R] as opposed to an apical ‘r’ [r]. The autonym Rroma is held to be cognate with oma, a collective term for the ancient aboriginal populations of the Indian subcontinent. Many The name by which Roma designate themselves is th century. A legacy of the migration is that some Roma refer to themselves as Sinti, an adjectival form derived from Sindh, the name of the Indus river. Morgenstierne The Roma route of migration has long been the object of linguistic study. Grierson CHRNE, i.e. cholinergic receptor, nicotinic, epsilon en route explicitly proposed by linguists in the 19th century The Indian ancestry of Roma has been studied using mtDNA, Y-chromosomal and autosomal studies Although the Indian origin of the European Roma populations is linguistically and genetically well-established, accurate identification of their South Asian source has remained a matter of debate. Some linguistic studies argue that the proto-Romani founder population must have been in northwestern India In contrast with the maternal lineages, where three South Asian-specific lineages were reported There are examples where a single Y-chromosomal hg is associated with the expansion of a population from a homeland, e.g. hg C3 with Genghis Khan's army To detect possible source populations of European Roma paternal lineages within, we genotyped first M82-H1a1a hg) in a large number of Indian populations to produce a high-resolution dataset of hg H1a1a-M82 and thereafter, 17 Y-chromosome short tandem repeats (STRs) . To reduHg H1a1a-M82 accounts for more than 12% of all male lineages in South Asia, with a highest incidence of 20% amongst South Indians and a lowest incidence of 0.2% among northeast Indian populations  and S3. Within India, language group-wise expansion times and variance were observed to be highest among Dravidian speakers , followed by Austroasiatic speakers , and lowest among Indo-European speakers . Just as with the expansion time of hg O2a-M95 among Austroasiatic speakers The geographical distribution of hg H1a1a-M82 is largely restricted to South Asia, and its significant occurrence among European Roma populations strongly links the Roma to the Indian subcontinent. The high frequency of H1a1a-M82 among all the Roma groups and their reduced genetic diversity relative to South Asian populations can most likely be attributed to their recent migration from India  and 2. IIn order to ascertain the closest population group among northwestern Indians, we redrew the network of Roma haplotypes exclusively within the northwestern Indian variation . It is hThis suggestion also enables a resolution of the controversy on the Punjab and the Gangetic plain. The In order to conduct an intra-group comparison of Y-chromosome variation, we estimated the genetic distances  from 15 loci Y-STR haplotypes and made a multidimensional scaling plot . The firRroma and oma and resolve the controversy about the Gangetic plain and the Punjab in favour of the northwestern portion of the diffuse widespread range of the In conclusion, the analysis of Y-chromosome hg H1a1a-M82 variation in 214 ethnic groups from India shows that northwest Indian populations are the closest to the hg H1a1a-M82 variants observed in the present-day European Roma populations. Although only entire genome analyses can provide the richest narrative of history and migration, the Y-chromosome hg H1a provides an exceptional record of Indian and European Roma-specific paternal heritage, including their exodus from northwestern India and subsequent recent expansion. Our phylogeographical study of hg H1a1a-M82 enables now to better understand the temporal and spatial parameters of this migration. This first genetic evidence of this nature allows us to develop a more detailed picture of the paternal genetic history of European Roma, revealing that the ancestors of present scheduled tribes and scheduled caste populations of northern India, traditionally referred to collectively as the Samples were collected with the informed written consent from 3498 unrelated healthy individuals belonging to 57 populations from all the four linguistic groups of India  and S3. Text S1Detailed .(DOC)Click here for additional data file.Figure S1Multidimensional scaling plot of the Rst distances of the population groups based on haplogroup H1a1a-M82 Y-STR data.(TIF)Click here for additional data file.Table S1Details of the Indian and Nepali samples included in the present study and haplogroup H1a1a-M82 frequencies.(DOC)Click here for additional data file.Table S2The 15 loci Y-STR profile of haplogroup H1a1a-M82 belonging to Indian, Afghani and European Roma population, used in the present analysis.(DOC)Click here for additional data file.Table S3Regionwise haplogroup frequency in India and Nepal.(DOC)Click here for additional data file.Table S4The frequency of haplogroup H1a1a-M82 among different world populations.(DOC)Click here for additional data file.Table S5Mean pairwise Fst between different studied groups for haplogroup H1a1a-M82.(DOC)Click here for additional data file.Table S6Modal H1a1a-M82 Y-STR haplotype of different population groups.(DOC)Click here for additional data file.Table S7Average mutational distances from Roma Modal haplotype.(DOC)Click here for additional data file.


Ensatina eschscholtzii in order to assess the relative contribution of genetic and ecological divergence for the development of RI.Reproductive isolation (RI) is widely accepted as an important "check point" in the diversification process, since it defines irreversible evolutionary trajectories. Much less consensus exists about the processes that might drive RI. Here, we employ a formal quantitative analysis of genetic interactions at several stages of divergence within the ring species complex By augmenting previous genetic datasets and adding new ecological data, we quantify levels of genetic and ecological divergence between populations and test how they correlate with a restriction of genetic admixture upon secondary contact. Our results indicate that the isolated effect of ecological divergence between parental populations does not result in reproductively isolated taxa, even when genetic transitions between parental taxa are narrow. Instead, processes associated with overall genetic divergence are the best predictors of reproductive isolation, and when parental taxa diverge in nuclear markers we observe a complete cessation of hybridization, even to sympatric occurrence of distinct evolutionary lineages. Although every parental population has diverged in mitochondrial DNA, its degree of divergence does not predict the extent of RI.Ensatina, the evolutionary outcomes of ecological divergence differ from those of genetic divergence. While evident properties of taxa may emerge via ecological divergence, such as adaptation to local environment, RI is likely to be a byproduct of processes that contribute to overall genetic divergence, such as time in geographic isolation, rather than being a direct outcome of local adaptation.These results show that in  In a Darwinian sense, species formation is an outcome of a continuum of gradual evolution, from ecological races and biotypes, to hybridizing taxa and, ultimately, to "good" biological species that no longer cross . The lonAlternatively, if selection is strong, reinforcement of reproductive barriers may lead to full RI (e.g. green-eyed tree-frog ) or, if Reproductive isolating mechanisms are generally understood to accumulate gradually as a function of molecular genetic distance, a surrogate for time since divergence . SubstitGambusia fishes )terflies ,54).per se does not result in reproductively isolated taxa. The patterns of clinal genetic variation or weak genotypic clustering commonly found between ecologically divergent taxa  in nature probably result from processes similar to those in Ensatina. The fact that we consider species formation to be "incomplete" results only from our assumption that species will only endure in time in the presence of RI, rather than being genetically isolated in most of their genomes, or particular genomic regions controlling for ecologically relevant traits. Hybrid zones between ecological taxa  where transition in ecological traits remains abrupt despite the lack of complete RI or absolute restrictions to gene flow, constitute the evidence that RI is not necessary, or even advantageous, for ecological differentiation. While it is irrefutable that natural selection is an important force in species diversification, that process may well be uncorrelated with the development of RI.The genic view of species postulates that "species are groups that are differentially adapted and, upon contact, are not able to share genes controlling these adaptive characters, by direct exchanges or through intermediate hybrid populations. These groups may or may not be differentiated elsewhere in the genome" . Our resSince the time of the modern synthesis, we have made major progress in understanding how RI may arise in various geographic contexts . Whereas RI is the ultimate test as to whether two populations should be considered species, this process only reflects a single axis of the whole process of species formation. This limitation has been both technological and conceptual. However, recent technological advances now allow us to operate not only at the level of the organism, but also on the genes responsible for the traits we attribute to species, and how they interact with the local environment. Extending this same approach to other response variables that are now more tractable, such as restriction of gene flow in selected versus neutral loci, and comparison across several hybrid zones with different levels of divergence, will soon enable us to break from assumptions of what a "good" species should be, and directly illuminate the multidimensional nature of species formation.To determine the best predictors for the development of reproductive isolation we followed a three-step approach. First, we distinguish the range of pure parental populations (i.e. not affected by ongoing gene flow) from areas of secondary contact (i.e. where genetic introgression occurs). Second, we focus on the ranges of pure parental populations to infer the amount of genetic and ecological divergence (predictor variables) prior to secondary contact and without the confounding effect of genetic and ecological intermediates. Third, we focus on individuals collected at the center of secondary contacts to determine the frequency of genetic admixture (response variable) and statistically infer relationships between predictors and the response variable.Ensatina, ecologically and morphologically distinct groups are subdivided into several genetically distinct units, with varying degree of genetic divergence [Because we want to predict how reproductive isolation (RI) develops along the whole continuum of species formation, our operational units need to be at any diagnosable stage of divergence along the gradient from genetically distinct populations, ecomorphological groups (subspecies), and reproductively isolated taxa. In vergence . TherefoEnsatina complex, as in other organisms with low dispersal, genetic variability has a fractal spatial structure [klauberi, croceater, 4 within platensis, 8 within oregonensis, picta, 2 coastal plus a third inland population of xanthoptica, and eschscholtzii. The individual multilocus genotypes were assigned to groups maximizing Hardy-Weinberg (HW) and linkage equilibria at a uniform level of diagnosability . For purposes of this study, localities with a mean assignment to a single cluster higher than 0.8 pp were considered "parental localities", while those with assignments lower than 0.8 pp were considered "admixed localities". Polygons encompassing parental localities represent the range of genetically pure "parental populations" (N = 20), whereas intervening polygons represent areas of "secondary contact"  support the ring species hypothesis, in particular the pattern of clade origin in northern coastal California, and southwards colonization around the Central Valley [Ensatina has been subjected to the dynamic geologic history of California. The formation of island isolates together with the movement of tectonic plates resulted in periods of geographic isolation having varying lengths between populations around the ring [In a ring species, the accumulation of genetic divergence depends both on the bidirectional colonization around the main geographic barrier, and on restrictions to gene flow caused by periods of geographic isolation. At the level of this analysis, we cannot always differentiate between the effect of time and space, but both factors are known to affect the diversification process in l Valley ,33. Morethe ring . As a cothe ring ,22,24. Wthe ring . Here, wb (cyt b) from 344 salamanders sampled in 212 unique sites within the range of the 20 pure parental populations. We used previously published data from 263 individuals [FJ151653 - FJ152002; see Additional file Ensatina and intra-population variability. Sequences were visually aligned in SEQUENCHER  and deposited in GenBank (accession numbers JN022615 - JN022695). We calculated corrected average pairwise differences between pure parental populations  using tns (PiXY .pp≥0.95) and calculating Nei's D [To estimate divergence across nuclear genes (nDNA), we used the 22 to 27 allozymic loci described above, which are selectively neutral based on Hardy-Weinberg equilibrium [ Nei's D  with GEN Nei's D .Ensatina colonized disparate habitats throughout California, from wet and cold climates characteristic of northern California, to arid habitats in the southern part of the range. These steep climatic gradients are likely to constitute important physiological challenges for ectothermal and lungless organisms such as Ensatina. Moreover, California is marked by distinct physiographic regions that are characterized by specific plant communities [Ensatina is contiguous across most vegetation types, changes in color pattern, and therefore subspecies limits, are spatially concordant with turnover of vegetation composition. We here account for the putative effect of ecological divergence by independently evaluating the effect of climate and vegetation, since they would be attributed to distinct ecological processes important to Ensatina and are generally recognized as major ecological factors that could drive RI.During the southwards expansion of the complex, munities . AlthougEnsatina around the main geographic barrier that defines the ring, the Central Valley. We gathered observation points of Ensatina in California by compiling information from field records at the Museum of Vertebrate Zoology (UC Berkeley) and surveying herpetological collections from most North American natural history museums using HerpNET on 5 May 2009. From a total of 12,366 observations, we accounted for sampling biases by reducing the number of points to spatially unique samples - defined according to the georeferences associated with the specific localities of the specimens. Samples were assigned to pure parental populations or contact zones in between, using polygons defined by the genetic data (above). We then queried the samples for 19 continuous bioclimatic variables . Ecological divergence was calculated based on 3,404 assigned spatially unique observations of Ensatina around the Central Valley of California. Because we did not find associations between these traits and RI, we did not consider possible confounding effects of interaction between climate and vegetation any further.We evaluated ecological divergence with respect to current climate and vegetation using the distribution of solution ) and 55 Ensatina in California using a principal component analysis (PCA) on the 19 bioclimatic variables, which includes records in areas occupied by parental populations and contact zones. Using the first two principal components (PC axes), we then calculated the area of climatic space occupied by each parental population. Using the subset of records assigned to each of the 20 pure parental populations, we calculated their respective climatic areas by using a kernel density function [We first described the entire climatic space occupied by function  . Here, the use of the word "hybrid" referring to a genetically admixed individual does not imply any a priori knowledge about the degree of RI between the respective parental taxa, but only that those taxa are genetically diagnosable at the level of our analysis.pp, and calculated the frequency of hybrids (F1 and backcrosses).We assessed the frequency of genetically admixed individuals based on their multilocus genotypes for the allozyme data described above, using the Bayesian assignment test implemented in the program NEW HYBRIDS . This mez and s options off, see manual), basing the assignment solely on the multilocus genotypes. We pseudo-replicated the Markov chain Monte Carlo from different starting points and ran the analysis long after reaching stability to assure convergence to the same result. Although the assignment test implemented in NEW HYBRIDS is mainly driven by HW and linkage equilibria, genetic differentiation between parental populations is known to affect the power of discriminating admixed individuals. Therefore, we did not compute frequency of admixture in secondary contacts between populations that diverged less that 0.1 (DN) in allozyme loci. Because we have more than one transect across some hybrid zones (contact zones across the ring), we calculated the frequency of hybrids by pooling individuals and computed means across the multiple transects.Because the program works under a two-population model, we performed individual runs for each contact, including the samples from localities within the secondary contact as our query sample, and from localities in the range of both parental populations as reference samples for the assignment. Because the limits of some contact zones are better defined as a genetic gradient rather than an abrupt change, we did not discriminate our reference samples as genetically pure . PC2 is responsible for 32.5% of the variation and reflects drier and warmer gradients . Colors and labels are in agreement with Figure Click here for fileCollecting localities for allozymic data, sample sizes, and geographic location. Underlined names represent localities at the center of the contact zones.Click here for fileCollecting localities for mtDNA data, geographic location and GenBank Accession numbers.Click here for file


Tectura paleacea  belongs to a seagrass obligate lineage that has shifted from the Caribbean in the late Miocene, across the Isthmus of Panama prior to the closing of the Panamanian seaway, and then northward to its modern Baja California – Oregon distribution. To address whether larval entrainment by seagrass beds contributes to population structuring, populations were sampled at six California/Oregon localities approximately 2 degrees latitude apart during two post-settlement periods in July 2002 and June 2003. Partial cytochrome oxidase b (Cytb) sequences were obtained from 20 individuals (10 per year) from each population in order to determine the levels of population subdivision/connectivity. From the 120 individuals sequenced, there were eighty-one unique haplotypes, with the greatest haplotype diversity occurring in southern populations. The only significant genetic break detected was consistent with a peri-Point Conception (PPC) biogeographic boundary while populations north and south of Point Conception were each panmictic. The data further indicate that populations found south of the PPC biogeographic boundary originated from northern populations. This pattern of population structure suggests that seagrass patches are not entraining the larvae of T. paleacea by altering flow regimes within their environment; a process hypothesized to produce extensive genetic subdivision on fine geographic scales. In contrast to the haplotype data, morphological patterns vary significantly over very fine geographic scales that are inconsistent with the observed patterns of genetic population structure, indicating that morphological variation in T. paleacea might be attributed to differential ecophenotypic expression in response to local habitat variability throughout its distribution. These results suggest that highly localized conservation efforts may not be as effective as large-scale conservation efforts in near shore marine environments.The seagrass limpet  Haliotis , found that genetic distance was independent of geographic distance over a 300 km sampling range, and suggested that limited larval dispersal is involved in generating genetic differentiation. Along the entire California coast, Moberg & Burton Strongylocentrotus) also have very heterogeneous genetic compositions on both large and small geographic scales suggesting that, “the larval pool is not well mixed geographically , despite long planktonic larval duration.”The effects of larval dispersal on marine invertebrate ecology and evolution has long been recognized From these and other studies, a picture of the role of larval dispersal in contributing to the genetic makeup of populations is emerging, and it is one that often significantly differs Tectura paleacea , a stenotopic species found only on its obligate seagrass host PhyllospadixT. paleacea is patchy on local and regional scales throughout the Northeastern Pacific  within each locality. These patches were subsequently mapped using permanent markers embedded into surrounding boulders and triangulation techniques. All size classes of T. paleacea and their associated Phyllospadix blades were collected during timed searches from each Phyllospadix patch. Specimens of T. paleacea and blades of Phyllospadix were stored in 95% EtOH. Relative abundance was estimated in terms of T. paleacea individuals collected per person per unit search time. One-way ANOVA was used to test whether relative abundances for each locality were significantly different .Populations of Tectura depicta collected at San Diego, CA by Kristina D. Louie (unpublished).A total of 763 individuals were collected and deposited in the University of California Museum of Paleontology see . MorpholT. paleacea adult individuals . Total genomic DNA was isolated using the “Animal Tissues” protocol included with the DNEasy Tissue Kit from QUIAGEN, Inc. Valencia CA. A 450 nucleotide fragment of cytochrome oxidase b mtDNA (Cytb) was amplified and sequenced using the following primers: cobF 5′ - GGW TAY GTW YTW CCW TGR GGW CAR AT - 3′; cobR 5′ - GCR TAW GCR AAW ARR AAR TAY CAY TCW GG - 3′ Small pieces of foot tissue were excised from a total of 120 T. depicta  was calculated as the sum of the covariance components FST , FSC (within group variation), and FCT (between group variation). Significance tests were based on 16,000 permutations.Patterns of haplotype diversity were examined on regional scales by grouping individuals according to phylogenetic affinities. To further test if this grouping accurately reflects population connectivity, each population was serially allocated to unique blocks  and AMOVA analyses arlequinv2.0. Haplotype and nucleotide diversity was estimated as a means to identify recent changes in effective population sizes. Tajima's π) estimated from the mean number of pairwise nucleotide differences (π) was selected due to its incorporation of allele frequencies, making this statistic more biologically meaningful as compared to others  Further population genetic data analyses were also carried out using T. paleacea individual collected from each locality during 2002 and again during 2003. A total of 50 individuals were measured from each locality except in the case of Cape Arago where only 20 individuals were collected in 2003 resulting in a total of 45 individuals being measured from this locality. In total, 295 individuals were measured for morphological analyses. Phyllospadix blade width was measured at the exact point from which each T. paleacea individual was removed from the blade. The relationship of these variables was examined across all localities using ANOVA and correlation statistics. Analyses were done with the statistical software package SPSS v11 .Length, width, and height measurements were taken from 25 T. paleacea spend their entire lives on Phyllospadix blades and stems. In turn, the width of these plant structures constrains T. paleacea shell width soon after individuals reach adult size therefore shell width is a reflection of ecophenotypic variation in the plant host and not in the associated limpet. The maximum width of 1.5–2.0 mm is reached when individuals are approximately 3.5 mm long The relationship between locality and morphology was examined by comparing length:height ratios (LHR) of individuals from each locality. This measure has been established as an appropriate indicator of ecophenotypic variation in marine plant limpets previously T. paleacea was examined relative to each associated Phyllospadix blade (or stem) width across all localities. (2) ANOVA of Phyllospadix blade width was compared across all localities. In order to test if Phyllospadix blade width at any given locality was significantly affecting shell LHR, (3) the degree of ovalness, measured as width:length ratios (WLR), was compared across all localities.The contribution of blade and stem constraints to the observed variation in LHR was tested using a series of three comparisons. (1) Shell width of Of the 120 individuals sequenced, there were eighty-one unique haplotypes present. Twenty individuals were examined from each population resulting in the following haplotype distributions across the six sampled populations: 8 unique haplotypes from Simpson Reef, Cape Arago, OR (SR); 11 from Trinidad Head, Trinidad, CA (TH); 12 form Horseshoe Cove, Bodega, CA (HC); 13 from Monterey Bay, Monterey, CA (MB); 19 from Coal Oil Point Reserve, Santa Barbara, CA (CR); and 18 from Bird Rock, La Jolla, CA (BR). The haplotype relationships determined using a neighbor-joining algorithm are shown in T. paleacea populations changed significantly across the sampled range. When plotted against localities by latitude, abundance data appear to have a normal distribution with the highest abundances at the center of the range of T. paleacea  region and the two populations sampled south of the PPC region see , with 32π) are significantly smaller in northern population as compared to populations found south of the PPC region  between four groups of populations (p<<0.00) between four groups  of populations that did not correspond to the same four population groupings  found for blade width alone (see A positive correlation between blade width and shell width was observed . ANOVA oulations . Averagelone see . The lonp<<0.00) indicating that overall shell shape is characterized by elongated shell length and low apex height. The opposite was true for TH and SR populations, which had significantly smaller LHR (p<<0.00) indicating that shell shape is shorter in length with a relatively high apex. The three populations found between the most northern and southern extremes  were statistically indistinguishable form each other (p = 0.206), and were characterized by an intermediate shell shape.ANOVA and Tukey post hoc multiple comparisons of length:height ratios (LHR) across all localities indicated a statistically significant break between three groups  of populations . BR had T. paleacea-like marine plant limpet in the subtropical Pacific comes from a Pleistocene deposit in the Sea of Cortez (Lindberg unpublished). As seagrass limpets expanded their range into the temperate North Pacific during the late Pliocene and early Pleistocene, populations in the subtropical Pacific and Caribbean went extinct T. paleacea and T. depicta, the sister taxon of T. paleaceaT. depicta is restricted from northern Baja to central California reflecting ancestral distributions.A diverse patellogastropod fauna found in Tertiary fossil deposits from the Dominican Republic include species that were closely associated with marine plants T. paleacea exists somewhere between Santa Barbara and Monterey . While this region encompasses the well-known biogeographic boundary of Point Conception T. depicta, and concordant patterns seen in a variety of taxa that share a similar coastal distribution across the Northeastern Pacific AMOVA results indicate that a barrier to gene flow between northern and southern populations of T. paleacea populations may have gone extinct entirely south of Point Conception during the late Pliocene and early Pleistocene. If this was the case then these dispersal events represent range expansion into areas where T. paleacea had previously gone extinct. This raises the possibility that southern populations are being maintained as sink populations deriving from northern larval source populations. This scenario would have significant impact on conservation strategies being implemented in rocky intertidal environments along the California-Oregon coast.From a phylogenetic perspective, haplotype relationships predicted from NJ and ML analyses further support the presence of a partial barrier to gene flow in the PPC region. As expected, due to the low levels of genetic variation present between our sampled populations, the tree resulting from ML analysis has less resolution than the NJ tree and neither have significant support values . NonetheIt has been generally accepted that seagrass canopies alter the hydrodynamic conditions of the space they occupy T. paleacea with Phyllospadix would suggest increased levels of population structure due to decreased flow environments and subsequently increased larval retention within Phyllospadix beds. While we did not directly measure the level of larval dispersal ability associated with any given seagrass bed, our data indicate that the presence of any larval retention due to attenuated flow in seagrass beds is not enough to act as a population structuring force for populations of T. paleacea distributed along the California-Oregon coast. The lack of collection site-specific population structure indicates high levels of gene flow between sites. Given the lack of dispersal ability by adults, the possibility of this gene flow being a result of adult dispersal as opposed to larval dispersal is unlikely. While adult dispersal potential may exist via individuals hitchhiking on drifting clumps of seagrass, this has yet to be documented.Given these findings from previous studies, the obligate association of T. paleacea. This result is consistent with Avise's The data presented here lend support to the hypothesis that a biogeographic boundary within the PPC region is acting as the primary mechanism driving genetic population structuring in T. paleacea, the boundaries between these clusters do not correspond to the PPC region, the biogeographic boundary associated with the phylogeographic break between northern and southern populations  occur along the coast of North America. Individuals of T. paleacea feed exclusively on the leaf epidermis of P. scouleriP. torreyiPhyllospadixP. serrulatus from P. scouleri and P. torreyi, few data support the separate species designation of P. scouleri and P. torreyiP. scouleri and P. torreyi. These data, combined with data that indicate latitudinal variability in P. scouleri and P. torreyiT. paleacea populations living on these seagrasses.Morphological variation within Phyllospadix beds provide a partial defense against many larger benthic predators due to the inaccessibility of thin floating blades where T. paleacea is found. However, smaller predators, such as the sea star Leptasterias hexactis, are commonly found in these beds T. paleacea individuals show no escape response as a result of contact with L. hexactis individuals. Instead, they suggest that T. paleacea is chemically camouflaged. If T. paleacea uses chemical camouflage as its primary defense against predators, it is unlikely that predation intensity could explain the region-specific variability seen in shell morphology. If we are to identify the mechanisms regulating ecophenotypic plasticity within T. paleacea, detailed studies examining the ecological role of T. paleacea in seagrass habitats and its ecophenotypic plasticity potential are needed.T. paleacea that is concordant with the biogeographic barrier at the PPC region in California. Historical data suggest that extant populations originated in southern subtropical regions and have made their way across this barrier only since the late Pliocene or early Pleistocene. Although larval dispersal was not directly measured, the pattern of population structuring observed indicates that seagrass communities, which are known to alter flow regimes within their environment, do not affect larval dispersal ability enough to impact on the connectivity of T. paleacea populations. Furthermore, morphological patterns are not concordant with the molecular patterns observed. These findings support the role of local habitat variability in maintaining differential ecophenotypic expression across the distribution of T. paleacea, suggesting that highly localized conservation efforts may not be as effective as large-scale conservation efforts in near shore marine environments with diverse life history components.This study identifies population structure of Figure S1The resulting strict consensus tree topology using ML analysis. ML analysis resulted in two trees with the likelihood score 1542.74406 and few nodes with significant bootstrap support. Bootstrap values  are indicated above each branch. Southern clades are highlighted in red while northern clades are in black.(TIF)Click here for additional data file.Table S1(DOC)Click here for additional data file.


Vigna unguiculata ssp. unguiculata var. spontanea) populations from West Africa, using 21 isozyme loci, 9 of them showing polymorphism.Cowpea is a highly inbred crop. It is part of a crop-weed complex, whose origin and dynamics is unknown, which is distributed across the African continent. This study examined outcrossing rates and genetic structures in 35 wild cowpea (Outcrossing rates ranged from 1% to 9.5% (mean 3.4%), which classifies the wild cowpea breeding system as primarily selfing, though rare outcrossing events were detected in each population studied. Furthermore, the analyses of both the genetic structure of populations and the relationships between the wild and domesticated groups suggest possibilities of gene flow that are corroborated by field observations.As expected in a predominantly inbred breeding system, wild cowpea shows high levels of genetic differentiation and low levels of genetic diversity within populations. Gene flow from domesticated to wild cowpea does occur, although the lack of strong genetic swamping and modified seed morphology in the wild populations suggest that these introgressions should be rare. Agricultural systems present spectacular and well studied examples of evolutionary changes . Indeed,Vigna unguiculata (L.) Walp., plays an important role in the livelihood of millions of poor people in the developping countries of the tropical region where it is used as food, animal feed or as a cash crop , varied according to the ecological context of the populations. In rather undisturbed habitats the observed inbreeding tended to be higher than the inbreeding equilibrium while this trend was reversed in disturbed habitats (field and roadside). Field and roadside are subjected to frequent disturbances and are characterized notably by ground transfers causing possible rearrangements in the soil seed bank. Moreover, outcrossing rates may vary among years . Standard errors of the mating system parameters were estimated based on 500 bootstraps considering the maternal family array as the resampling unit.First, the outcrossing rates  program , an exte program  based onF was then compared to the expected inbreeding coefficient at equilibrium Fe = (1-t)/(1 + t) [F and Fe are equal [F and Fe are expected to reflect the amount by which a population deviates from inbreeding equilibrium. FeF was tested for difference from zero using a paired non-parametric Wilcoxon signed rank test. Moreover, any differences in (Fe – F) values according to the degree of disturbance in the habitat of collection, i.e., natural vs. disturbed , were checked using a Mann – Withney U test./(1 + t) . If popure equal . Discreptmts to zero using a Wilcoxon signed rank test. A tmts > 0 value means that biparental inbreeding does exist beside selfing of flowers. To test the heterogeneity of tm among the populations analysed, a Chi-square test was carried out, as suggested by Godt and Hamrick [Chi-square distributed, with n – 1 degrees of freedom where n is the number of populations. At the population level, diversity parameters were estimated using the Popgene software version 1.3 [P), observed heterozygosity (Ho), and the expected heterozygosity under Hardy-Weinberg equilibrium (He). Fixation indices were estimated with Fstat, version 1.2 [FIS) were computed to assess the deviation from Hardy-Weinberg equilibrium following Weir and Cockerham [Lastly, in order to verify the contribution of biparental inbreeding versus autogamy to observed inbreeding, we compared  Hamrick . The tession 1.3 . These ision 1.2 . Inbreedockerham  and wereGenetic structure among populations was studied by analyzing both genetic differentiation and spatial genetic autocorrelation in the wild and domesticated populations.First, an analysis of molecular variance (AMOVA)  was carrr[r values were tested with 999 permutations. In the case of domesticated cowpea, represented by 7 populations only, spatial autocorrelation was assessed with a Mantel’s test (999 permutations) between Nei’s distances and geographic distances.Second, spatial autocorrelation of the wild populations was analyzed using the GenAlEx 6.1 software program . Geneticr. r valueThe possibility of genetic exchange between the wild and domesticated compartment were studied as follows.A Principal Coordinates Analysis (PCO)  and domesticated (D) populations was implemented in the R software [rW-D) for Wild - Domesticated populations pairs was computed for several distance classes (100 km up to 500 km) and tested with 999 permutations.Based on the algorithm published in Smouse and Peakall , the comsoftware  in orderRSP, JBT, KT, RG, JTO and ABS contributed to plant material collection. EBK performed lab analyses. EBK, RSP and PC conceived of the study, analyzed data, interpreted results and wrote the paper. GMM and PG reviewed the paper prior to submission and provided valuable comments on the interpretation and presentation of results. RSP secured funding. All authors read and approved the final manuscript.


Anopheles gambiae s.s. and Anopheles arabiensis are the two major and widespread malaria vectors in sub-Saharan Africa. Several studies have examined the ecology and population dynamics of these vectors. Ecological conditions along the Kilombero valley in Tanzania influence the distribution and population density of these two vector species. It remains unclear whether the ecological diversity within the Kilombero valley has affected the population structure of An. gambiae s.l. populations. The goal of this study was to characterise the genetic structure of sympatric An. gambiae s.s and An. arabiensis populations along the Kilombero valley.Genetic diversity is a key factor that enables adaptation and persistence of natural populations towards environmental conditions. It is influenced by the interaction of a natural population's dynamics and the environment it inhabits. Mosquitoes were collected from seven locations in Tanzania: six from the Kilombero valley and one outside the valley (~700 km away) as an out-group. To archive a genome-wide coverage, 13 microsatellite markers from chromosomes X, 2 and 3 were used.An. arabiensis populations was observed, as opposed to An. gambiae s.s., which was genetically undifferentiated across the 6,650 km2 of the Kilombero valley landscape. It appears that genetic differentiation is not attributed to physical barriers or distance, but possibly by ecological diversification within the Kilombero valley. Genetic divergence among An. arabiensis populations (FST = 0.066) was higher than that of the well-known M and S forms of An. gambiae s. s. in West and Central Africa (FST = 0.035), suggesting that these populations are maintained by some level of reproductive isolation.High levels of genetic differentiation among An. arabiensis genetic divergence. The impact of the observed An. arabiensis substructure to the prospects for new vector control approaches is discussed.It was hypothesized that ecological diversification across the valley may be a driving force for observed  The genetic structure of a population is shaped by interactions between the behaviour of individuals and their prevailing environment . These fFST values or other parameters. Using this information it may be possible to deduce which features/factors are responsible for restricting or promoting movements of genes between or within these populations [The method commonly used to study patterns of gene flow includes identifying genetic units within an ecological landscape and the features that help to shape the spatial distribution of these units . This inulations ,6. Althoulations ,7,8. Theulations . Bayesiaulations , animalsulations ,11 and pulations .Anopheles gambiae complex, include the two primary vectors (An. gambiae s.s. and Anopheles arabiensis) of human malaria in sub-Saharan Africa, that are responsible for an estimated 240 million cases and 280,000 deaths worldwide, with over 80% occurring in Africa [An. gambiae complex and major vectors of malaria [An. gambiae s. s. is associated with more humid climates than An. arabiensis, which has a greater tolerance for drier environments [An. gambiae s.s. are highly anthropophagic [An. arabiensis are more zoophagic, exophagic [Mosquitoes of the n Africa . These t malaria . Althougronments ,15. Addipophagic ,16, endoxophagic , and exoxophagic ,19. A stxophagic  are compxophagic .An. gambiae s. s and is thought to be influenced by environmental heterogeneity [An gambiae s.s. subpopulations, which exist in sympatry, have been revealed in West and Central Africa [An. arabiensis populations [An. arabiensis population differentiation [Population substructure is more pronounced in ogeneity . For exal Africa ,23. Studulations -26. Howentiation , except ntiation .Anopheles gambiae s.s. subpopulations have been studied intensively and are of great epidemiological importance as they have been suggested to undermine available malaria vector control efforts. For example the two molecular forms (S and M) of An. gambiae s.s. have been reported to respond differently to control interventions, as the S form has developed resistance to pyrethroids (insecticides used for impregnating bed-nets) while the M form remains largely susceptible [ceptible ,29. In aceptible ,18. Therceptible ,31. The ceptible ,32.An. gambiae s.s. and An. arabiensis within the Kilombero valley (6650 km2) located in southern Tanzania. Although there are remarkable reductions in transmission intensities, this valley experienced some of the most intense malaria transmission in the world [An. gambiae s. s. and An. arabiensis in it. In this study therefore, a Bayesian clustering analysis was used and 13 polymorphic microsatellite loci originally designed for use in An. gambiae s. s. [This study, characterised the population structure of he world ,34. Epidhe world ,35-37. Ihe world ,39 or whhe world ,38,39, bae s. s.  were empAn. gambiae s.s. and An. arabiensis, are widely distributed along the valley, which is oriented from the northeast to southwest (7°44'-9°26'S/35°33'-36°56'E), The valley lies between the densely forested escarpment of the Eastern Arc mountains and covers an area of 6650 km2 [>700 km west of the Kilombero Valley, as an out-group. In each village, five CDC light traps were placed in different randomly selected houses for three consecutive nights. Every morning, traps were retrieved and mosquitoes identified according to their morphological differences. Mosquitoes visually identified as belonging to the An. gambiae complex were individually stored on silica gel in Eppendorf tubes and taken for further molecular analysis to determine whether they were An. gambiae s.s. or An. arabiensis.The Kilombero Valley is located in south-eastern Tanzania Figure . The two6650 km2 . Estimat6650 km2 ,42. Mosq®. DNA extraction was performed using a Qiagen 96 Biosprint® work station in 96 well plates using a BioSprint96 DNA Blood Kit . Each individual of the An. gambiae complex was identified to species level using species-specific PCR primers [Prior to DNA extraction, individual mosquitoes were rehydrated in 100 μl of ultra pure water overnight. The following day, 10 μl of proteinase K and 90 μl of ATL buffer were added to each individual tube, and homogenized in a Qiagen Tissue Lyser primers .An. gambiae microsatellite loci [An. gambiae s.s., and only 13 gave good amplification in both An. gambiae s.s. and An. arabiensis. Consequently only these 13 were used in this study. These markers provide representative coverage of the entire genome as they are distributed relatively evenly throughout the genome as follows: X chromosome: AGXH25, AGXH100 and AGXH71; Chromosome 2: AG2H175, AG2H85, AG2H164, AG2H197 and AG2H675; and Chromosome 3: AG3H127, AG3H249, AG3H311, AG3H811 and AG3H9 [We screened PCR primers for 20 ite loci . These wnd AG3H9 .® 3130 Genemapper (Applied Biosystems) to identify alleles.Microsatellite loci were PCR-amplified for each mosquito sample. Each 25 μl PCR reaction consisted of 12.5 μl of Multiplex master mix, 2.5 μl primer mix and 9 μl of RNase free water. The primer mix was made to a final volume of 250 μl, consisting of 10 pmol of each primer. The forward primer in each reaction was labeled with a fluorescent marker  compatible with ABI PRISM  capillary electrophoresis. DNA amplification was completed in MJ Research PTC-200 thermal cyclers . A 5 min denaturation step at 95°C was followed by 29 cycles of 20 s at 95°C, 30 s at 55°C and 30 s at 72°C. A final incubation at 72°C was extended for 1 hour to alleviate problems associated with addition of non-template nucleotide (dA) to the PCR products. PCR products were mixed with a GeneScan  size standard and deionized formamide as directed by the manufacturer. Mixtures were run on an ABI 3130 Genetic analyzer. Output was analysed using ABI PRISMArlequin [Arlequin software was used to calculate pair-wise FST values [FST distance. The neighbour-joining algorithm implemented in neighbour, a part of the Phylip software package [FST distances. The cladogram was drawn using the drawtree programme, also provided in Phylip. As an estimate of gene flow, the number of migrants per population per generation (Nm) was calculated for FST according to the equation, Nm ≈ (1-FST)/4FST [structure software [K), chosen in advance. This model calculates the probabilities of each individual for each subgroup, within which Hardy-Weinberg (H-W) equilibrium and linkage equilibrium are met. These probabilities are used to infer the membership of each individual at their most probable subgroup, and these are referred to as membership coefficients which sum to 1. The probability distribution of an individual X in a putative population K (P(X/K)) was also plotted. Based on allele diversity, individuals with unique alleles are grouped together into assumed population (K) which is pre-determined. The K value with the maximum posterior probability (P(X/K)) is retained and assumed to be the most probable number of clusters in that putative population.Microsatellite allele and genotype frequencies were examined using Arlequin  developeArlequin . Each miArlequin . SignifiArlequin . The ArlT values , and 10, package  was usedST)/4FST . A Bayessoftware , individAn. gambiae s. l. mosquitoes were collected in this study from the seven localities. Of these, 113 (18.7%) were An. gambiae s. s. and 490 (81.2%) were An. arabiensis. A total of 386 (288 An. arabiensis and 98 An. gambiae s. s.) mosquitoes were screened for the 13 microsatellite loci.A total of 603 female An. arabiensis (28%) and 10 out of 39 tests in An. gambiae s.s. (25.6%) were significant (P < 0.00044). Further inspection in An. arabiensis populations revealed that nine out of 21 tests (42%) involving X-linked loci significantly deviated from H-W expectations and no test out of nine tests (0%) in An. gambiae s.s. significantly deviated from H-W expectations , . It has been established that linkage disequilibrium is predicted to approach zero for an ideal population, in the absence of forces such as genetic drift, population mixing, mutation and natural selection [An. arabiensis suggests the existence of population subdivision [The non-random association between polymorphism at different loci is measured by the degree of linkage disequilibrium. Linkage disequilibrium was determined  and 33.3, Figure . In An. election . It was,division .FST estimates as a measure of differentiation between the two species and among populations. Results are presented in Table 2. Anopheles arabiensis was genetically distant from An. gambiae s.s. (FST ranging from 0.21-0.27)  whereas the level of genetic divergence between An. arabiensis populations was substantially higher (FST ranging from 0.006 to 0.1). The product of effective population size Ne and the migration rates m was used to estimate the amount of gene flow within each species based on the observed FST values. Thus there is a low amount of gene flow between the two species . These values are in line with other reported studies [An. arabiensis populations indicate that there is a limited number of migrants between some populations  and populations from Mikeregembe, Mkamba and Ilonga forming the second cluster (Cluster II), , i.e. corresponding to the number of postulated Mendelian populations. As shown in Figure An. arabiensis population clusters from K = 4 and K = 5 showed a similar range of likelihood and the highest likelihood was observed at K = 3. Thus the most likely number of clusters (K) is three. This corresponds to three distinct genetic clusters: 1) The An. gambiae s. s. cluster 2) the An. arabiensis cluster I . and 3) the An. arabiensis cluster II Bayesian clustering analysis was applied for all populations within and outside the Kilombero Valley. This approach is advantageous in that it does not require a prior population classification, but instead estimates the shared population ancestry based on observed genotypes, under the assumption of the presence of a H-W equilibrium and linkage equilibrium within each cluster . Thus, bAn. gambiae s.s. and An. arabiensis in the Kilombero Valley, southern Tanzania. Both species occur in the Kilombero Valley with An. arabiensis more widespread. The An. gambiae s.s. populations sampled appear to represent a single genetic population, whereas An. arabiensis appear to occur as two discrete populations that are found in sympatry at some sites  the genome-wide signature of departures from H-W equilibrium and high linkage disequilibrium between loci, (ii) higher FST estimates between An. arabiensis than between An. gambiae s.s. populations, (iii) higher levels of gene flow between An. gambiae s.s. than An. arabiensis populations and (iv) Bayesian probability distribution at K = 3. This clearly indicates that An. arabiensis gene pool across the Kilombero Valley is not homogeneous. In contrast, An. gambiae s. s. showed low levels of genetic differentiation with high level of gene flow across populations, indicative of a homogeneous gene pool.In this study, 13 microsatellite loci were used to describe the genetic structure of populations of An. arabiensis populations may be a consequence of several factors that need further investigations. Cytogenetic evidence suggests that An. arabiensis is an ancient species from which other members of the complex descended and that this species inhabited East Africa more than 6,000 yrs ago [An. arabiensis populations inhabiting Mkamba and Ilonga, as opposed to An. arabiensis populations outside and along the rest of the Kilombero Valley, such as Kaliua, Ukindu and Malinyi. It is important to note that Mikeregembe is a fishing camp [An. arabiensis existing in Mkamba and Ilonga may have been introduced in Mikeregembe by fishermen from Mkamba (20 kms away) who regularly visit the camp for fishing activities. These mountains also create unequal climatic conditions across the Kilombero Valley which correlate very well with the abundance of the two malaria vector species [An. gambiae s.s is commonly found in close proximity with forested Eastern Arc mountain areas, where the climate is stable and annual rainfall is higher than in the rest of the valley , (ii) An. arabiensis is commonly found in riverine areas where the climate is less stable and annual rainfall is lower  [An. arabiensis populations inhabiting sites along the Eastern Arc mountains are subjected to a higher degree of environmental heterogeneity typical to these mountains, promoting genetic variation and the emergence of new adaptive genotypes [Population substructure observed in Ilonga, Mikeregembe and Mkamba, among  yrs ago , indicat yrs ago . Consequ yrs ago  estimateing camp  thus it  species ,54. For 0 mm/yr) . Therefoenotypes .FST), the level of genetic divergence between An. gambiae s. s. populations is lower (mean FST = 0.006) than that between An. arabiensis populations (mean FST = 0.066). The degree of divergence between An. arabiensis populations (mean FST = 0.066) is even greater than the reported divergence between the well-known M and S forms of An. gambiae s.s. in West Africa (mean FST = 0.035), [An. arabiensis subpopulations are maintained by some degree of reproductive isolation. This hypothesis can also be supported by the absence of hybrids in sympatric An. arabiensis populations , . From thFST estimate reported in this study is higher than the reported FST estimate for An. arabiensis from west, south and east Africa (0.035-0.038) where extensive An. arabiensis population differentiation was observed [FST estimate observed in this study (0.066 - 0.100) is in line with the largest reported An. arabiensis FST estimate (0.080-0.215). This is a comparison between mainland and island An. arabiensis populations [An. arabiensis genetic differentiation was neither a result of any obvious physical barrier nor geographic distance, consistent with reports from other studies [An. arabiensis [An. arabiensis was recorded for markers located on chromosome X. 42% of tests involving markers on chromosome X deviated from Hardy-Weinberg equilibrium, in contrast to 0% in An. gambiae s.s. This does not only suggest the population to be structured, but also supports the hypothesis developed by Stump and colleagues that sex-linked differentiation is characterised by some degree of reproductive isolation mechanisms controlled by genes located in genomic regions within X chromosome [The observed ,27. The ulations , where g studies ,27. It cabiensis . In thisromosome .An. arabiensis subgroups existing within the Kilombero Valley may be of great significance to malaria epidemiology. Anopheles arabiensis mosquitoes are reported to be phenotypicaly diverse. For example, An. arabiensis is more zoophilic and exophilic [An. arabiensis are able to tolerate higher temperatures and lower humidities than An. gambiae s.s. [An. arabiensis and accelerate disease transmission. Therefore, if the observed population substructure is ignored, degrees of medically important phenotypes in An. arabiensis species, such as vectorial capacity, host preference and insecticide resistance, could be obfuscated. Thus, further investigations are needed to thoroughly understand the epidemiological significance of the observed population substructures.The observed genetic xophilic , and higxophilic . Also Aniae s.s. . This phAn. arabiensis in East Africa has a complex genetic structure with distinct populations occurring in sympatry, apparently maintained by some degree of reproductive isolation. It was hypothesized that ecological differences rather than a physical barrier or geographical distance are responsible for the observed An. arabiensis population substructure. Further investigation is needed not only to clearly establish the specific mechanisms underlying genetic differentiation in An. arabiensis populations, but also to identify the number of discrete malaria vector populations sustaining transmission along the Kilombero Valley, important for guiding the implementation of available and future control strategies. Proposed new genetic control strategies such as sterile insect technique (SIT) and genetically modified mosquitoes (GMM), require a homogeneous population structure for success. The observed level of genetic substructure may pose a challenge to the successful implementation of these approaches [An. arabiensis populations appear to be maintained by some degree of reproductive isolation, multiple genetic modifications of An. arabiensis may be needed if genetic control is to be successful. Furthermore, the observed An. arabiensis population substructure may have different malaria transmission intensity, as populations with distinct genetic composition may have different vectorial capacities [This study provides evidence that proaches ,63. Sincpacities . FurtherAn. gambiae complex, suggesting the possibility of population expansion [An. gambiae and An. arabiensis along the Kilombero Valley.The study showed that there may still be another level of genetic subdivision within the xpansion , with poThe authors declare that they have no competing interests.KRN, BGJK, GCL and HMF designed this study. KRN carried out the laboratory and SFS work. KRN and YL analysed and interpreted the data. KRN, HMF, GCL and BGJK drafted the manuscript. All authors read and approved the manuscript.P < 0.00044) of deviations from Hardy - Weinberg expectations for 13 microsatellite loci in populations of An. arabiensis and An. gambiae s. s. collected from within the Kilombero/Ulanga Valleys, TanzaniaP-values indicating statistical significance  between An. . The total numbers of mosquitoes screened in each location are in brackets. Underlined values indicate inter-species comparison between An. gambiae and An. arabiensis. The non-underlined values indicate intra-species comparisons, i.e. within An. gambiae and An. arabiensis. Values in italics are statistically significant. Significance levels at ¥P < 0.05, $P < 0.01 and *P < 0.001. 'Ag' = An. gambiae s. s. and 'Aa' = An. arabiensis.Click here for fileAn. gambiae s. l. populations within and outside Kilombero Valley Estimated number of migrants between . 'Ag' stands for An. gambiae s. s. and 'Aa' stands for An. arabiensis.Click here for file


North African populations are distinct from sub-Saharan Africans based on cultural, linguistic, and phenotypic attributes; however, the time and the extent of genetic divergence between populations north and south of the Sahara remain poorly understood. Here, we interrogate the multilayered history of North Africa by characterizing the effect of hypothesized migrations from the Near East, Europe, and sub-Saharan Africa on current genetic diversity. We present dense, genome-wide SNP genotyping array data  from seven North African populations, spanning from Egypt to Morocco, and one Spanish population. We identify a gradient of likely autochthonous Maghrebi ancestry that increases from east to west across northern Africa; this ancestry is likely derived from “back-to-Africa” gene flow more than 12,000 years ago (ya), prior to the Holocene. The indigenous North African ancestry is more frequent in populations with historical Berber ethnicity. In most North African populations we also see substantial shared ancestry with the Near East, and to a lesser extent sub-Saharan Africa and Europe. To estimate the time of migration from sub-Saharan populations into North Africa, we implement a maximum likelihood dating method based on the distribution of migrant tracts. In order to first identify migrant tracts, we assign local ancestry to haplotypes using a novel, principal component-based analysis of three ancestral populations. We estimate that a migration of western African origin into Morocco began about 40 generations ago ; a migration of individuals with Nilotic ancestry into Egypt occurred about 25 generations ago (approximately 750 ya). Our genomic data reveal an extraordinarily complex history of migrations, involving at least five ancestral populations, into North Africa. Proposed migrations between North Africa and neighboring regions have included Paleolithic gene flow from the Near East, an Arabic migration across the whole of North Africa 1,400 years ago (ya), and trans-Saharan transport of slaves from sub-Saharan Africa. Historical records, archaeology, and mitochondrial and Y-chromosome DNA have been marshaled in support of one theory or another, but there is little consensus regarding the overall genetic background of North African populations or their origin and expansion. We characterize the patterns of genetic variation in North Africa using ∼730,000 single nucleotide polymorphisms from across the genome for seven populations. We observe two distinct, opposite gradients of ancestry: an east-to-west increase in likely autochthonous North African ancestry and an east-to-west decrease in likely Near Eastern Arabic ancestry. The indigenous North African ancestry may have been more common in Berber populations and appears most closely related to populations outside of Africa, but divergence between Maghrebi peoples and Near Eastern/Europeans likely precedes the Holocene . We also find significant signatures of sub-Saharan African ancestry that vary substantially among populations. These sub-Saharan ancestries appear to be a recent introduction into North African populations, dating to about 1,200 years ago in southern Morocco and about 750 years ago into Egypt, possibly reflecting the patterns of the trans-Saharan slave trade that occurred during this period. The census size of Mediterranean North Africa exceeds 160 million people Prior genetic studies, largely from uniparentally inherited markers, have not resolved the location origin of North African populations or the timing of human dispersal(s) into North Africa. Analyses based on the frequencies of a small number of autosomal genetic polymorphisms and uniparental markers have shown that the genetic landscape follow an east-west pattern with little to no difference between Berber- and Arab-speaking populations Less controversial is the observation that many North African populations now speak Arabic and that this language shift occurred primarily after the Arabic conquest 1,400 ya. This Arabic shift is well documented, but it remains unknown how deeply recent migrations  from the Arabian and Iberian Peninsulas shaped the genetic diversity of current North African populations. In addition, sub-Saharan influence has been detected in North African samples by all types of genetic markers analyzed, although it is unknown how recent this gene flow might have been Genomic models of admixture in human populations have largely been confined to cases of two-way admixture such as African-American ADMIXTUREADMIXTURE, we explored k = 2 through 10 ancestral populations to investigate how assumptions regarding k impact our inference of population structure in North Africa. Log likelihoods for successively increasing levels of k continue to increase substantially as k increases , 80% of the ancestry in North African individuals is assigned to a cluster defined by its maximum frequency in Near Eastern and European populations  and decreases into Egypt and westward across North Africa .A gradient also appears in the higher analysis . Assumink = 6 through 8, all North African populations except for Tunisians have sub-Saharan ancestry, present in most individuals, though this ancestry varies between 1%–55%. Interestingly, eastern populations (i.e. Libya and Egypt) share ancestry assigned to both the Bantu-speaking Luhya and the Nilotic-speaking Maasai, whereas western populations share ancestry mainly with the Luhya. Of note is that the South Moroccan and western Saharan populations contain considerable variation across individuals in the amount of sub-Saharan ancestry ; as all populations derive the majority of their ancestry from an Out-of-Africa ancestral population, and the OOA bottleneck is the primary signature in OOA populations, we believe this assumption is valid. Estimates for population eN were taken from Li et al. We estimate F35–0.063 . In orde “t” see . Since tst estimates using cluster-based allele frequencies from ADMIXTURE among the Maghrebi, European and Near Eastern ancestries, when we considered higher order k = 5∶8 ancestral clusters. As indicated in k estimates and assumptions regarding eN, as Near Eastern populations have a greater estimated eN than European. Although these divergence time estimates may not be precise, as they do not adequately model ancient migration, they do suggest that the population divergence between the ancestral Maghrebi population and neighboring Mediterranean populations occurred at least 12,000 ya and indeed more likely predated even the Last Glacial Maximum.We then attempted to obtain more accurate estimates of divergence time by controlling for recent migration. We calculated a second set of Fst and 2nd cousin genetic equivalent pairs were present in this sample, even though donors declared themselves to be unrelated during the sampling process. Analysis of long runs of homozygosity (ROH) indicate that the Tunisian population averaged almost twice as much of their genome is in ROH than other North African populations, 230 Kb versus 120 Kb respectively  using the GERMLINE software ectively . The patOur cluster-based analysis identified five distinct ancestries in North Africa that we refer to as: Maghrebi, European, Near Eastern, eastern and western sub-Saharan Africa. In order to test whether sub-Saharan African ancestry was an ancient or recent migration signature, we considered the length of sub-Saharan haplotypes. First, to assign local, ancestry-specific haplotypes across a genome, we implemented a new principal component-based admixture deconvolution approach (PCADMIX) for three ancestral populations see  [20],  occurs 50–60 Kya, geographically dividing North African and Near Eastern populations; b) North Africans experience a separate bottleneck; c) gene flow maintains similarity between the two geographically distinct populations; d) the gene flow then ceases or slows roughly between 12–40 Kya in order to allow sufficiently distinct allele frequency distributions to form. In contrast, we find it more parsimonious to describe model where: a) an OOA migration occurs [concurrent with a bottleneck]; b) OOA populations and North Africans diverge between 12–40 Kya when a migration back-to-Africa occurs. These models should be further tested with genomic sequence data, which have better power to detect magnitude and timing of bottlenecks, and to estimate the true joint allele frequency spectrum.A scenario where North African Maghrebi ancestry is the result of More recently, the substantial, east-to-west decline of Near Eastern ancestry  could reIn summary, although paleoanthropological evidence has established the ancient presence of anatomically modern humans in northern Africa prior to 60,000 ya k = 2  in total were tested successively. Log likelihoods for each k clusters are available in st based on allele frequencies was calculated in ADMIXTURE for each identified cluster at k = 8. Given the high heterogeneity in Qatari population, we present individuals with the lowest sub-Saharan, European and North African ancestries and higher Near Eastern ancestry, based on ADMIXTURE. Multidimensional scaling (MDS) was applied to the pairwise IBS Matrix of 279,528 SNPs using PLINK 1.07 software k = 5–8) were obtained using st estimate, are unlikely to be younger if the range of eN sizes is realistic.An unsupervised clustering algorithm, In previous work, imputation accuracy was tested in a sample of Algerian Mozabites and other populations from the Human Genome Diversity Project (HGDP-CEPH) We estimate the amount of DNA shared identically by descent (IBD) using the GERMLINE software phased genotype data  to determine exact posterior probabilities along each chromosome. PCADMIX relies on Principal Components Analysis (PCA) to quantify the information that each SNP contributes to distinguishing the ancestry of a genomic region. PCADMIX is publicly available at sites.google.com/site/PCADMIX. We use Singular Value Decomposition in R to perform PCA on the phased genotypes of the ancestral representatives. We project admixed individuals on the basis of principal components, and compute the observed ancestry “score” for a haplotype i in the jth window as the weighted average jgijL, where ijg is a column vector of the haplotype's alleles (coded as 0 or 1) in window j, standardized by the mean and standard deviation of that SNP's frequency in the ancestral populations. jL is a matrix for which the entry in the kth row, lth column is the loading of SNP l in the window on principal component k. We use a forward-backward algorithm to identify the probability of ancestry at each window, conditional on the ancestry scores. For the forward-backward algorithm in our HMM, we used a haploid version of the transition and emission probabilities in the Viterbi algorithm of Bryc et al.p, the probability of recombination between windows, and qj, the frequency of the target population's chromosomes in the admixing ancestral pool.Local ancestry was assigned with a new PCA-based method, PCADMIX. This method uses 2<0.8 in order to remove highly linked alleles from different populations, which can lead to spurious ancestry transitions. Second, chromosomes for each individual in a population are artificially strung together to create two haploid genomes for the individual; this step increases the amount of information used for PCA, and it is of special relevance given that Europeans, Near Easterners and North African are differentiated with an Fst of only ∼0.05. Then, PCA on a number k≤3 of ancestral populations is performed and the admixed population is projected into the determined k≤3 PCA space. PC loadings are used as weights in a weighted average of the allele values in a window of 40 SNPs. These window scores are then used as observed values in a HMM to assign posterior probabilities to the ancestry in each window (where chromosome were considered separately). Information on using PCADMIX in Egyptians is available in First, ancestral populations are thinned for SNPs with rWe tabulated the length and number of genomic tracts (i.e. phased haplotypes) assigned to particular population ancestries for the South Moroccan and Egyptian population samples . We used a posterior probability threshold of 0.8, optimized for concordance with ADMIXTURE ancestry proportions . The maxWe modify Pool and Nielsen Figure S1ADMIXTURE results for k = 10 ancestral clusters in our North African populations, Spanish Basque, Near Eastern Qatari, western Africans, HapMap3 Kenyan Luhya and Maasai and Italian Tuscans. B) Log likelihoods for each of the k clusters tested.A) (TIF)Click here for additional data file.Figure S2k = 10). North African populations are all indicated in turquoise.We used multidimensional scaling (MDS) to discriminate clusters of genetic variation within Africa and neighboring regions. MDS was applied to the pairwise, individual identity-by-state (IBS) matrix of 279,500 SNPs using PLINK 1.07 software (TIF)Click here for additional data file.Figure S3Long runs of homozygosity compared across North African populations and neighbors. –homozyg –homozyg-window-kb 5000 –homozyg-window-het 1 –homozyg-window-missing 1 –homozyg-snp 25 –homozyg-kb 500 –homozyg-gap 100.(TIF)Click here for additional data file.Figure S4k = 3 ancestral populations. The proportion of Population A's ancestry in an admixed individual is estimated by: a given haplotype's (black square) distance from the line connecting the means of PCA1 and PC2 for the two other populations, as a proportion of the haplotype's distance from all edges. B) Simulated ancestry assignment with and without LD filtering. The black arrow indicates a region of simulated European ancestry that is incorrectly classified  as African when no linkage disequilbrium (LD) filtering is used, and whose ancestry is left undecided when LD filtering is implemented (r2<0.8).Implementation of PCADMIX. A) A principal components analysis is first run for (TIF)Click here for additional data file.Figure S5ADMIXTURE and PCADMIX ancestry estimations in (A) South Moroccans and (B) Egyptians. In both cases PCADMIX was required to assign ancestry with a posterior probability of 0.95. The 0.95 threshold substantially reduces the proportion of the genome assigned by PCADMIX. In South Moroccans, the reduction in assigned ancestry occurs primarily in the European and to a lesser extent in the Berber component. For the Egyptians, the reduction in assigned ancestry is dramatically reduce Near Eastern (or Arabic) ancestry.Comparison of (TIF)Click here for additional data file.Figure S6A) PCADMIX applied to a South Moroccan individual using Saharawi, Basques and Luhyan as ancestral populations. Segments are assigned to ancestries with a posterior probability higher than 0.8. B) PCADMIX applied to the same South Moroccan individual as in A) using Tunisian, Basque and Luhya as the ancestral populations. Segments are assigned to ancestries with a posterior probability higher than 0.8.(TIF)Click here for additional data file.Figure S7We capture admixture proportions by independently running LAMP (TIF)Click here for additional data file.Figure S8n = 38). Initially we assigned ancestry for k = 3 ancestral populations  using a 0.8 posterior probability threshold, shown in . Then we assumed a different set of 3 ancestral populations  shown in . In the third step, we assumed the Sub-Saharan ancestry, assigned in A, represented truly divergent sub-Saharan haplotypes given the high Fst between this ancestry and all others. E) We layered these haplotypes on top of [C]  deconvoluted chromosomes.Shown is the admixture deconvolution for chromosome 1 using PCADMIX for 19 Egyptian individuals ((TIF)Click here for additional data file.Figure S9A) We present the average assigned ancestry (>0.8 posterior probability) across chromosome 1 for each of 4 ancestries assigned in the Egyptians: Maghrebi (Saharawi), European (Basque), Near Eastern (Qatari), Sub-Saharan (Maasai).(TIF)Click here for additional data file.Figure S10A) Distribution of the number and length in centimorgans of migrant Sub-Saharan (Luhya) tracts distributed by length found in the South Moroccan population. B) Distribution of the number and length in centimorgans of migrant Sub-Saharan (Maasai) tracts distributed by length found in the Egyptian population. Red bar indicates the minimum threshold cutoff employed in the migration parameter analysis. Please note the different scales along the X-axis.(TIF)Click here for additional data file.Table S1Name, sample size and country of origin for populations newly genotyped in the present study as well as for populations published previously. References are included in the table.(DOC)Click here for additional data file.Table S2st after removed putative admixture events.Additional estimates of F(DOC)Click here for additional data file.Table S3ADMIXTURE.Significance of the comparisons of ancestry assignment using PCADMIX and (DOC)Click here for additional data file.Text S1Assigning local ancestry with PCADMIX.(DOC)Click here for additional data file.Text S2Concordance between ADMIXTURE and PCADMIX.(DOC)Click here for additional data file.Text S3Chromosome 1 Ancestry Deviations.(DOC)Click here for additional data file.


The outcome of hematopoietic stem cell transplantation (HSCT) is shaped by both clinical and genetic factors that determine its success. Genetic factors including human leukocyte antigen (HLA) and non-HLA genetic variants are believed to influence the risk of potentially fatal complications after the transplant. Moreover, ethnicity has been proposed as a factor modifying the risk of graft-versus-host disease. The populations of Latin America are a complex array of different admixture processes with varying degrees of ancestral population proportions that came in different migration waves. This complexity makes the study of genetic risks in this region complicated unless the extent of this variation is thoroughly characterized. In this study we compared the HLA-A and HLA-B allele group profiles for 31 Latin American populations and 61 ancestral populations from Iberia, Italy, Sub-Saharan Africa, and America. Results from population genetics comparisons show a wide variation in the HLA profiles from the Latin American populations that correlate with different admixture proportions. Populations in Latin America seem to be organized in at least three groups with (1) strong Amerindian admixture, (2) strong Caucasian component, and (3) a Caucasian-African gradient. These results imply that genetic risk assessment for HSCT in Latin America has to be adapted for different population subgroups rather than as a pan-Hispanic/Latino analysis. Hematopoietic stem cell transplantation (HSCT) is a curative therapy used for the treatment of malignant and nonmalignant hematologic diseases, congenital immune deficiencies, solid tumors, and metabolic diseases . Its outGraft-versus-host disease (GVHD) is a major complication affecting the success of the transplant and the survival of the patients. Despite the fact that most transplants are performed with high levels of compatibility in terms of HLA, a significant proportion of these transplants is affected by GVHD. Apart from clinical factors , a genetLatin America is a region where the most dramatic human migrations have taken place, from the early northeastern Asian bands of hunter-gatherers that conquered the last continent humanity had expanded to , throughIn this study, we used population genetics tools to compare the HLA profiles of 31 LAP and 61 ancestral populations in order to characterise their diversity and classify them according to their genetic makeup.A selection of 92 populations from Latin America, Iberia, Italy, and sub-Saharan Africa with available DNA-based typing data for HLA-A and HLA-B allele groups was made and their details are shown in The remaining 61 populations are native population samples from the three ancestral regions that have contributed majorly to the Latin American gene pool: Amerindians (22 populations), Caucasians from Europe , and SSA (20 populations). In the Caucasian population group, a sample of Italians was selected to complement the Iberian populations in view of the important immigration from this country into some LAP. In total, the population array included 384,446 chromosomes. HLA frequency data was extracted from journal articles and/or the Allele Frequencies database website . The appA database containing the frequencies for 47 HLA-A and HLA-B allele groups from the 92 populations that were selected was built. When the available data were at high resolution, the data were reduced to two-digit allele groups. The database was constructed on the Multi-Variate Statistical Package  and was independently checked for accuracy.The HLA-A and HLA-B profiles of the 92 populations were analysed by clustering analysis and Principal Coordinates Analysis (PCO), both based on Euclidean distances. The clustering analysis was performed dually and dendrograms were generated for both analyses. The clustering method was based on minimum variance of squared Euclidean distances with a randomized input order. The Eigenanalysis for the PCO was performed at an accuracy of 1E-7 and axes were extracted according to Kaiser's rule .Additionally, three ancestry-specific HLA allele groups were compared between population subgroups in order to illustrate the relative contribution of each ancestral population across LAP. A dendrogram based on squared Euclidean distances was generated by the comparison of 47 HLA-A and HLA-B allele group frequencies present in the 61 ancestral populations and the 31 LAP. The results for this analysis are shown in A closer look at the clusters shows that there is a correlation between the geographic location of the ancestral populations and the branching within the clusters. Within the Amerindian cluster, 4 groups form a South American lowland group, a South American Andean group, a Central American group, and a more distinct North American-Alaskan group. A similar correlation is seen within the SSA cluster: western Africans split from the southern, eastern and central African populations. Some of the LAP cluster with the Amerindians, such as the Peruvian mestizos from Arequipa, or with the SSA, as the Cuban Mulattos and the Afro-Brazilians from Paraná. However, 90% of the LAP cluster within a distinct group which includes the Iberians and Italians.The LAP-Iberian+Italian cluster splits further in distinct subgroups. Most of the Spanish populations, and minority populations from Spain, cluster in their own groups. Also, there is a broad group that clusters all of the remaining Brazilian and Cuban populations, and another one that clusters the Portuguese, Italian, and Argentinians from La Plata, the region of Cuyo, and Buenos Aires. Finally, the last cluster includes the admixed populations from Mexico, Colombia, Venezuela, Costa Rica, as well as the South American immigrants to Madrid and the Mexican and pan-Hispanic samples from the United States.A dual-clustering method was applied to the dataset in order to identify the groups of alleles that are most variable between the populations. The results from this analysis are shown in The results from the PCO are shown in When the LAP are included in the analysis , the resA few populations seem to cluster closely enough to the ancestral populations to be considered part of those clusters. This is the case of the Cuban Mulattos and the Afro-Brazilians from Paraná, the Cuban Whites, the Brazilian Caucasians from Paraná, and the Peruvians of Arequipa. In turn, it must be noted that a population classified as Amerindian, the Argentinian Toba from the city of Rosario, show significant Caucasian admixture and, consequently, lie closer to the admixed Mexicans than the Peruvians do.To further illustrate the differential admixture patters present in LAP based on their HLA profile, 3 allele groups which are present in one of the ancestral populations and absent or nearly absent in the other two  were selected in order to evaluate their frequency among the LAP groups . As seenFinally, The results obtained after comparing 47 HLA-A and HLA-B allele groups among the LAP and their ancestral populations show that there is widespread variation between the genetic profiles of these admixed or exported populations. In the cluster analysis it is clear that most LAP have substantial Caucasian components, with the exception of some populations such as the Peruvians from Arequipa or the Afro-Brazilians from Paraná. This is in agreement with the uneven process of population replacement and the collapse of many Native American groups that took place throughout the continent. However, PCO analysis showed that most LAP sit on a wide admixture arch that approaches the ancestral clusters. A few populations fall very close to or in the ancestral clusters, but most are scattered in intermediate regions. Interestingly, population samples that are likely to be a mixture of several LAP, such as those of the USA Hispanic immigrants and the Ibero-American expatriates in Madrid, sit in the center of the distribution. In fact, the heterogeneity of the Hispanic population in the US has been described using other markers , 69, shoThe stronger Caucasian component in some LAP can be attributed to recent European migration , such asOn the Caucasian-SSA axis of admixture, several Brazilian and Cuban populations can be found. It seems that for these populations, Amerindian admixture is very low or absent. This has been noted by others  and it iOur study is limited by both the availability of population data and the need to use HLA allele group data for comparison as opposed to high-resolution allele frequencies or haplotype frequencies. It is likely that an analysis of high-resolution frequencies would give finer results, but it would seriously diminish the number of populations that can be included in the analysis. However, the use of 47 allele groups from the most polymorphic genes in the human genome gives robustness to the analysis.The effect of ethnicity on complications after HSCT has been suspected for many years , 77 but It is likely that the evidence for differential outcome in different ethnic groups could be explained, at least in part, by differences in allele frequencies in genes that are relevant to the immune response and that show variable interethnic polymorphism, such as the cytokine genes . MoreoveLAP show widespread variation in their genetic profiles, and this complicates genetic association studies made in these populations. There is noticeable variation not only between regions and countries, but also between areas of the same country , 86. FurIt is also important that a better definition of individual ancestry in LAP is reached in view of the evident limitations of both self-reported  and rese