Triatoma infestans (Klug) is the main vector of Chagas’ disease in the Southern Cone of Latin America between the latitudes 10° S and 46° S. The long-term effectiveness of the control campaigns is greatly dependent upon the vector population structure. Mitochondrial DNA (mtDNA) genes have been used in a number of T. infestans population genetic analyses. However, the maternally inherited markers as well as nuclear ribosomal DNA analyzed until the present exhibited low or limited levels of variation. Analyses based on microsatellite markers strongly supported the existence of some type of stratification in T. infestans populations and supported the hypothesis of vector population recovery from survivors of the insecticide-treated areas, highlighting the value of population genetic analyses in assessing the effectiveness of Chagas’ disease vector control programmes. Although phylogeographic studies have generally suggested a Bolivian Andean origin of T. infestans, they recovered two reciprocal monophyletic groups of T. infestans and Bolivian populations who were not basal as expected for an ancestral group. In addition, a non-Andean origin could not be excluded by mtDNA genealogies that included sylvatic bugs from Gran Chaco. On the other side, mitochondrial and microsatellite markers supported the hypothesis of two independent migration events of colonization and secondary contacts in southern South America. Since the phylogenetic analyses remain inconclusive, more sequences, not only from mitochondrial genes but also from nuclear genes, need to be examined. Trypanosoma cruzi (Chagas), a protozoan transmitted by hematophagous insects of the subfamily Triatominae (Hemiptera: Reduviidae). Transmission requires the contact of host skin or mucosa with the vector´s ejecta containing metacyclic forms of T. cruzi. The adults of both sexes and the five nymphal instars of the species belonging to Triatominae are bloodsuckers and at least one full blood meal is necessary for each molt. Therefore, the insects are potential vectors of T. cruzi from their earliest stages since they may acquire the infection from their first meal. At present, 147 species of Triatominae are formally recognized [Triatoma infestans (Klug) is one of the species most closely associated with man, widely distributed, and consequently of public health importance. It is the main transmitter of Chagas' disease in the Southern Cone of Latin America between the latitudes 10° S and 46° S. Across its distribution T. infestans is primarily restricted to domestic and peridomestic environments such as human dwellings, chicken coops and pig or goat corrals.Chagas' disease (American trypanosomiasis) is a public health problem with high socio-economic impact in Latin America, where about 7 to 8 million people are affected by the malady . The discognized . The veccognized . A generT. cruzi is available. The measures taken to reach the interruption of transmission of this parasitic and infectious disease rely on the vector control by insecticide treatment of infested dwellings; however, though important progress has been made by the Southern Cone Initiative, the long-term support for the surveillance phase is a new challenge for the region. The important accomplishments reached by the vector control programmes have been the elimination of T. infestans in Uruguay and Chile [T. cruzi transmission by T. infestans in Brazil. However, high levels of T. infestans reinfestation were observed in Argentina, Bolivia, and Paraguay after spraying [T. infestans by insecticide spraying. From 1962 to 2000, almost all the localities within the endemic area received insecticide treatment, but in most of them the entomological surveillance was not maintained after 20 years of the implemented programme [T. infestans provide new basis for understanding the evolutionary history, migration patterns, genetic structure and dynamics of vector populations and resolving questions on processes such as dispersal and recolonization of the species that directly affect the efficiency of control efforts. Particularly, the genetic analysis of vector populations may be useful in entomological surveillance of Chagas vectors control programmes, since it may give information about the source of the insects in reinfested areas. On the other side, it has been detected in some places that vector control failure is caused by resistance to pyrethroid insecticides [T. infestans produced contradictory results. Dujardin et al. [et al. [et al. [Currently, neither satisfactory treatment of the malady nor a preventive vaccine against nd Chile  and the spraying . In Argerogramme . Vector rogramme . The loncticides . In thisn et al. -16 and P [et al.  reported [et al. , 19 dete [et al. . Among tT. infestans population genetic analyses. Analyses of mitochondrial cytochrome B gene sequences revealed four haplotypes in nine geographical populations of T. infestans from Argentina, Bolivia and Brazil, and did not show variations within populations [T. infestans from five natural populations of Argentina, 10 of which presented variable nucleotide sites in the 16S sequence fragment and eight showed variable nucleotide sites only in that fragment [T. infestans was surveyed. DNA sequence comparisons yielded 18 haplotypes among 130 individuals from 16 localities that represented a large portion of the range of T. infestans in Argentina [ T. infestans populations; haplotype and nucleotide diversities varied from 0 to 0.84 and 0 to 0.29%, respectively. Genetic differentiation between populations was not significant in most of the comparisons with a few exceptions. However, these results were based on a genetic marker that presented low or no variation in the populations analyzed and should not be taken as conclusive evidence that T. infestans populations are genetically undifferentiated.Mitochondrial DNA (mtDNA) genes have been used in a number of ulations . Sequencfragment . A totalfragment -26, the rgentina . A totalet al. [On the other hand, Piccinali et al.  analyzedT. infestans from different localities of seven countries , using nuclear ribosomal DNA (ITS-1 and ITS-2) sequences, revealed very little genetic variation [The maternally inherited markers analyzed exhibited low or limited levels of variation , 27, 28.ariation . Among tariation , 31.et al. [T. infestans;, 30 of these loci were amplified and 10 for which different allele types could be resolved clearly were selected for genotyping. The high degree of intra-population variation detected in the microsatellite loci analyzed suggested that these markers provide a valuable molecular tool for genetic analysis of T. infestans populations [T. infestans from Argentina [T. infestans populations are subdivided. One of them was carried out comparing samples of T. infestans obtained within four houses of one of the localities analyzed , where there were no peridomestic environments such as chicken coops and pig or goat corrals, usually invaded by T. infestans [T. infestans populations was also strongly confirmed using the same markers with specimens collected from three peridomiciliary sites in Medanitos (Catamarca province), where specimens within human dwelling were not detected [T. infestans populations, three and two different genetic clusters were identified in Siete Arboles (Chaco province) and Medanitos (Catamarca province), respectively. One should expect mobile species not to show genetic differentiation over short distances as indicated by the results of the microgeographical analysis, unless geographical features and/or very strong selection pressure prevent gene flow. In this regard, T. infestans has been characterized as an insect with restricted dispersal over short distances and usually remains in the same house or in its immediate vicinity during its lifetime. Besides, a significant excess of homozygotes was observed in the samples from two of the four houses analyzed in Siete Árboles and two of the three houses analyzed in Medanitos, suggesting inbreeding and/or a high degree of subdivision in the population [García et al.  isolatedulations . These vrgentina . Departurgentina . Two micnfestans . The hetdetected . In agrepulation .et al. [T. infestans at microgeographical scale and the possibility for wild populations to actively recolonize insecticide treated villages. At base to the levels of genetic differentiation detected between populations of T. infestans in the Bolivian Andes, the authors inferred restricted genetic interchange between close but distinct sylvatic sites, which supported the hypothesis that wild T. infestans does not disperse by flying at high altitude. They also inferred restricted gene flow between domestic and sylvatic populations, which suggested limited short term role of wild insects in the process of recolonization of insecticide treated houses in the Andes. Pizarro et al. [et al. [T. infestans populations from an ecologically diverse but geographically small valley-mountain environment in the department of Chuquisaca in Southern Bolivia. There they found significant genetic differentiation at different hierarchical geographic level; between low altitude East and high altitude West, among the five communities analyzed, and among houses from one of them, where they observed significant differentiation between domestic and peridomestic populations. Five genetic clusters within the five communities were identified. One of them was a mixture of insects from three close localities and the other four clusters contained insects from primarily one locality. In agreement with the results obtained in other vector population genetic studies [T. infestans. Besides, the high degree of genetic structure at small geographic scale inferred by cluster analysis and assignment tests together to demographic data suggested that the domiciliary re-infestation is from residual population and from peri-domicile structures.Simultaneously, nine out of the 10 microsatellite loci previously isolated and characterized  were alset al.  to asseso et al.  also use [et al.  to analy studies , 35, 36,et al. [T. infestans from areas with different elapsed periods since the last insecticide treatment and from areas that never received treatment. T. infestans populations from insecticide-treated localities seemed to have retained a substantial proportion of genetic diversity. The levels of genetic diversity observed in the majority of the populations from insecticide treated areas were surprising considering that insecticide treatments produce severe population reductions (bottlenecks) and that random genetic drift should cause loss in genetic variation. The authors considered the possibility that the population crash during the bottleneck would have been overestimated. Allelic diversity decreases faster than heterozygosity, when a population experiences a reduction of its effective size, and generally develops a heterozygosity excess at selectively neutral loci [T. infestans populations. It was suggested that, since the populations of T. infestans are subdivided, a population bottleneck would result in several independent genetic drift effects that could randomly preserve different combinations of alleles in each subpopulation; if independent genetic drift events in the population were followed by a rapid population growth, high levels of genetic diversity could be preserved. The results obtained by Pérez de Rosas et al. [On the other side, Pérez de Rosas et al.  evaluateral loci . The resral loci , 35-37 ss et al.  supporteT. infestans would tend to accentuate the genetic differentiation through the process of genetic drift. Moreover, significant isolation by distance, with nearby sites apparently exchanging more genes than distant ones, was detected. However, some sites exhibited deviation from a pattern of isolation by distance, suggesting that allele frequencies in each of these sites drift independently of the geographic distances separating them, probably factors other than geographical distance could be involved in generating the structure of populations. The majority of T. infestans populations studied belonged to areas with different elapsed periods since the last insecticide treatment and only three were from areas that never received treatment. It was considered that local differentiation by genetic drift during the reduction of population size by insecticide treatment could create significant structuring independent of distance. Pérez de Rosas et al. [Spatial genetic structure was also detectable at macrogeographical level , 39. This et al.  suggesteT. infestans populations from a region with the same insecticide treatment, Pérez de Rosas et al. [T. infestans populations using the same 10 microsatellite markers previously isolated [T. infestans are highly structured. Spatial genetic structure was detectable at macrogeographical and microgeographical levels from this region with the same insecticide treatment. Significant isolation by distance was also detected and seven different genetic clusters were identified. The six populations analyzed corresponded to six distinct clusters; one of these populations showed higher heterogeneity with approximately 30% of individuals corresponding to the seventh cluster identified. In this study, comparisons of the levels of genetic variability between two temporal samples from one of the localities analyzed were also carried out to assess the impact of the insecticide treatment. A second sample from Medanitos, collected 16 months after the capture of the first sample and subsequent to insecticide treatment, was also analyzed. The genetic diversity of the population was not significantly affected after insecticide use since different genetic parameters  remained stable; however, loss of low frequency alleles and no previously found alleles were detected. The authors stated that one explanation for the absence of diversity loss after insecticide treatment may be the size of the remnant population. The effective population size (eN) estimated for the population 16 months after insecticide treatment was substantially lower than the eN estimated before that treatment. Nevertheless, the population had only been studied for two generations after insecticide treatment and the remnant population size probably stayed high enough to maintain its genetic diversity at the pre-treatment level.In order to study the genetic structure of s et al.  examinedisolated  from sixet al. [T. infestans, of which nine were considered suitable for population genetics studies. These loci plus a 10th locus isolated later were used to analyze 352 insects collected in 21 houses of 11 rural communities from two neighboring rural areas in Santiago del Estero province (Argentina) that differed in their history of vector control [T. infestans populations from the core area, which were subjected to recurrent vector control actions, were highly structured and presented eight genetic clusters. Contrary to this, bug populations from peripheral area with sporadic vector control appeared to be less structured and less differentiated than those from the core area. Concordantly with the work mentioned above [T. infestans. Mitochondrial cytochrome B and/or COI gene sequences confirmed the identification of 20 of these sylvatic bugs as T. infestans. These mtDNA fragments and the microsatellite markers indicated the occurrence of unrestricted gene flow between sylvatic and domestic or peridomestic T. infestans populations from that area [T. infestans in the Gran Chaco may be an additional problem for vector control.On the other side, Marcet et al.  identifi control . This sthat area . The autet al. [T. infestans. With this purpose, a total of 314 insects from 22 domestic and peridomestic sites from one locality of Catamarca province (Argentina) were typed for 10 polymorphic microsatellite loci previously isolated [Recently, Pérez de Rosas et al.  proposedisolated . SignifiT. infestans was from Andean highland in Bolivia, where after it was introduced into human dwellings (~ 4500 years ago) the species was dispersed from this center throughout Latin American. As part of its dispersion associated with human migration it is estimated that, the vector would have invaded southern Brazil arriving from Argentina and Paraguay, and from this region it would have dispersed to the northeast of Brazil. According to this hypothesis, its current distribution may be interpreted as the result of the dispersal of the species from the site of origin in the Cochabamba valley of Bolivia, coupled with recent and rapid changes in the spread of T. infestans related to the activities of man, particularly between the 19th and 20th centuries [Based on arqueological findings and historical reconstructions, it has been suggested that the origin of enturies .T. infestans populations [et al. [Mitochondrial DNA (mtDNA) genes have been recognized particularly useful for phylogeographic studies in many species of insects, which involve migration pattern inferences . Initialulations . Two clu [et al.  were in et al. [T. infestans populations, named Andean and non-Andean. These authors detected differences in heterochromatin that have been considered the main cause of the variation in the DNA content between both groups, with approximately 30 % more heterochromatin in the Andean insects. On the basis of the observation that Andean samples came from geographic regions generally above 1800 m, whereas non-Andean populations were mainly from localities below 500 m, these authors suggested that heterochromatin and DNA variation reflect adaptative genomic changes that contribute to the ability of T. infestans to survive and reproduce in environments with different altitudes; populations with large genome would be better adapted to highlands (Andean regions), whereas populations with smaller genomes would do better in lowlands (non-Andean regions). Subsequently, Panzera et al.  identifiT. infestans in Bolivia highlands [T. infestans. However, Quisberth et al. [et al. [T. infestans allopatric group.Phylogenetic inferences carried out, using nuclear rDNA (ITS-1 and ITS-2) sequences, supported the previously suggested origin of ighlands  and two ighlands . In thish et al.  observed [et al.  found a  [et al. , 21, 27. [et al. , 29, theT. infestans, they recovered two reciprocal monophyletic groups of T. infestans [T. infestans not only in the Bolivian Andean valleys challenges this hypothesis. This finding prompted the hypothesis that the most ancient populations would be those of the dry subtropical Chaco forest in southeastern Bolivia, Paraguay and the north of Argentina [T. infestans. However, a non-Andean origin could not be excluded; first because only a single population in Gran Chaco was analyzed and second because higher genetic variability was detected in this population than in all the Bolivian Andean valley populations studied. Since in older populations more time would allow the accumulation of a higher level of genetic variation, the non-Andean region could be the center of origin and dispersal [et al. [T. infestans populations from Argentina [T. infestans. Although phylogeographic studies have generally suggested a Bolivian Andean origin of nfestans , 28, 29,rgentina . To thisispersal . In conc [et al.  using miT. infestans, either exhibited limited levels of variation or have not been very useful for phylogeographic inferences of the Chagas’ disease vector in Argentina [T. infestans observed at the 16S locus [On the other side, the maternally inherited markers analyzed until the present in rgentina , 28, 51.6S locus  as well 6S locus , 28, 51 6S locus , 39, 41.T. infestans populations covering almost the entire species range in Argentina [T. infestans was analyzed. Pérez de Rosas et al. [et al. [et al. [et al. [T. infestans that would have arrived to Argentina through the Andes. The authors also suggested that the presence in this area of populations that share some degree of ancestry with eastern populations would imply process of secondary contact between populations from the different dispersal lines. On the other side, it was considered that probably most of the other populations analyzed may have derived from the dispersal line of T. infestans in non-Andean lowlands associated to human migratory movements. These populations, which presented lower degree of genetic differentiation, showed higher heterogeneity (mix of different clusters) sharing different percentages of ancestry. It was suggested that the human internal migratory movement linked to regional economies, presumably associated with passive dispersal, would allow to maintain a higher genetic exchange between these populations of T. infestans.A multilocus approach with 10 microsatellite loci was performed to infer the phylogeography and migration patterns from different rgentina . Microsas et al.  identifi [et al.  and Barg [et al. , Pérez d [et al.  suggesteet al. [T. infestans using coalescence simulations and phylogeographic structure of this species in Chile. They also supported the hypothesis of two independent migration events of T. infestans in southern South America including a dual-origin of T. infestans in Chile.In summary, the last study favored the hypothesis of two independent migration events of colonization in Argentina and secondary contacts. Based on the analysis of a fragment of the mitochondrial COI gene, Torres-Pérez et al.  assessedT. infestans. The origin of reinfestations after insecticide treatment has remained controversial. The research supports the hypothesis of vector population recovery from survivors of the insecticide-treated areas, highlighting the value of population genetic analyses in assessing the effectiveness of Chagas’ disease vector control programmes. Moreover, the inferences made from these analyses in T. infestans populations demonstrated to be important, providing a complementary approach to help improve vector control strategies. On the other side, it is necessary to take into account the possible effect of the eradication campaign on the genetic structure of the vector populations since insecticide treatment would tend to accentuate the genetic differentiation among populations through the process of genetic drift. Further analyses should be undertaken to clarify if this effect may have epidemiological implications. The amount of genetic variation and differentiation through a species range is influenced by several factors, current and historical, which are often difficult to disentangle. It is possible that historical events, such as population bottlenecks, local extinctions and founder event, have probably played a major role in shaping their population genetic structures. Further analyses should be undertaken, since it is of considerable public health interest to know for example if genetic differentiation may be or could become associated with differences in the capacity of populations to act as vectors. More sequences, not only from mitochondrial genes but also from nuclear genes, need to be examined. In this respect, Fernández et al. [T. infestans. Based on their results it was inferred that the amplified regions should be useful for genetic analysis of T. infestans populations.Modern molecular approaches, employing DNA technology, afford vast new possibilities for studies of genetic population structure and evolution of z et al.  analyzed


The ability of natural populations to adapt to new environmental conditions is crucial for their survival and partly determined by the standing genetic variation in each population. Populations with higher genetic diversity are more likely to contain individuals that are better adapted to new circumstances than populations with lower genetic diversity. Here, we use both neutral and major histocompatibility complex (MHC) markers to test whether small and highly fragmented populations hold lower genetic diversity than large ones. We use black grouse as it is distributed across Europe and found in populations with varying degrees of isolation and size. We sampled 11 different populations; five continuous, three isolated, and three small and isolated. We tested patterns of genetic variation in these populations using three different types of genetic markers: nine microsatellites and 21 single nucleotide polymorphisms (SNPs) which both were found to be neutral, and two functional MHC genes that are presumably under selection. The small isolated populations displayed significantly lower neutral genetic diversity compared to continuous populations. A similar trend, but not as pronounced, was found for genotypes at MHC class II loci. Populations were less divergent at MHC genes compared to neutral markers. Measures of genetic diversity and population genetic structure were positively correlated among microsatellites and SNPs, but none of them were correlated to MHC when comparing all populations. Our results suggest that balancing selection at MHC loci does not counteract the power of genetic drift when populations get small and fragmented. Human-induced habitat loss and habitat fragmentation are arguably the greatest threats to the survival and persistence of natural populations . These pGiven that neutral genetic variation may often provide an incomplete picture of the evolutionary potential of populations e.g., , it has Tetrao tetrix) is an ecologically well-studied species. Within Europe, it is a flagship and umbrella species for the conservation of open woodlands, heather moors, and bogs. It has a wide distribution from the United Kingdom in the West, to the mountains bordering the People's Republic of China and North Korea in the East (the European range is displayed in The black grouse (The aim of this study was to investigate if (1) small isolated populations have lower microsatellite, SNP, and MHC genetic diversity than large and continuous populations. We additionally tested (2) if the genetic diversity and population structure were different for MHC than for SNPs and microsatellites and (3) explored correlations between SNP and microsatellite markers.We sampled 320 black grouse from 11 European locations (hereafter referred to as populations) that were grouped into different categories according to their status . The popOf the 320 individuals included in this analysis, we obtained microsatellite genotypes for 307 individuals, SNP genotypes for 229 individuals and scored allelic variation at two (or three) MHC class IIB loci for 164 individuals. Not all individuals where we obtained full microsatellite genotypes could be scored for MHC or SNPs, and in a few cases, we were able to amplify the SNPs but were not able to genotype the individual at the microsatellite loci see .2, and 1 µl of Eppendorf PCR buffer (Eppendorf), 0.2 mM of each nucleotide, 0.5 mM of each primer, and 0.5 units Taq polymerase . PCR profiles comprised 35 cycles of 30-sec denaturation at 94°C, 30-sec annealing at 54°C (for BG loci) or 60°C (for TUT loci), and 30-sec extension at 68°C. PCR fragments were resolved by electrophoresis on an ABI 377 automated sequencer . Allele sizes were determined by reference to two standard samples run simultaneously: (1) the ROX 350 Ladder (Applied Biosystems); (2) a black grouse individual previously genotyped at the same loci. To rule out contamination of samples with exogenous DNA or PCR products, tubes with water instead of sample/template were included in the DNA extraction and PCR amplification procedure as negative control.DNA samples were genotyped at nine microsatellites: TUT1, TUT3, TUT4, BG10, BG12, BG15, BG16, BG18, and BG19 . The suc2, 1× GC, 0.1 mM dNTP , 0.25 µM of each primer, and 0.375 U FastStart Taq Roche polymerase . The protocol was the same as used for a study on the closely related willow/red grouse (Lagopus lagopus). The names and localization of the genes on the chicken  chromosomes, the sequences of the primers and the PCR profiles are compiled in http://www.genotyping.se). Twenty-one of the selected 34 SNPs (of which a majority were synonymous) were successfully amplified at multiplex yielding one SNP for every 376 bp in the genes bcl2, BRIP, CAAX, EPN, GCM, KELCH, LEPR, MBL, MICRO, NGF, PKP4, PROOPIO, TRANS, and YTH.Twenty-four protein-coding genes of length ranging between 324 and 809 bps were amplified in 15 µl reactions in an Applied Biosystems Gene Amp PCR Systems 2700 thermal cycler. Individual mixes contained approximately 40 ng DNA template, 1× PCR buffer, 1.5–2.5 mM MgCl(BLB) primer pair RNAF1a (5’-GACAGCGAAGTGGGGAAATA-3’) and RNAR1a (5’-CGCTCCTCTGCACCGTGA-3’), we amplified BLB alleles from gDNA, for details see BLB loci in black grouse and that this species has two to three BLB loci , nucleotide diversity Pi, total number of different MHC alleles in a population divided by sample size (MHC/pop), and mean number of MHC alleles per individual (MHC/ind). MHC Pi and theta k was calculated in Arlequin 3.5.1.2 . In previous MHC studies, generally only alleles present in two independent PCR reactions are regarded as confirmed . The nuc 3.5.1.2 . APD mea 3.5.1.2 , and we ST is argued to be appropriate to measure for biallelic markers such as SNPs but the value of FST may be affected by highly variable markers. For this reason, we compared classical FST with Jost's D (DEST)   also when correcting for geographic pairwise distance . As for microsatellites, SNP pairwise DEST and FST were highly significantly correlated . MHC pairwise DEST and FST were also highly significantly correlated . Since the fixation index FST and the differentiation index DEST were strongly significantly correlated for all marker types, we decided to only report FST values in the result section .FD (DEST)  for all tstraps)  and for tstraps)   (ST/(1 – FST) and natural logarithm of distance in kilometre between populations in Mantel tests  in the Vegan package in R . All statistical tests of multiple comparisons were Bonferroni corrected .The correlation between pairwise estimates of F1 – FST)  for diff1 – FST)  in R (10E) varied considerable, between 0.56 and 0.81  in one continuous population (Fin_C), one isolated population (Pol_I), and three small isolated populations . We could not detect linkage disequilibrium between loci or indication of null alleles for any locus. Values of microsatellite expected heterozygosity  and CAAX-200 in one small isolated population (Aus_SI). Likewise, linkage disequilibrium was detected for the loci pair GCM-273 and GCM-317 and for the loci pair MBL-442 and MBL-610. The amount of Hand 0.31 ; Fig. 2bSD 1.13)  . The RSCSD 1.13) . We did SD 1.13) . To deteE) and allelic richness (AR)  . The two measures of microsatellite genetic diversity, HE and AR were significantly correlated    and S1a. n = 11) .E and percentage polymorphic SNP loci  . Interestingly, small isolated populations also had significantly lower SNP polymorphic loci than isolated populations . The SNP diversity measures SNP HE   and S1b.s SNP HE  and SNP s SNP HE  were sigs SNP HE .k, there was a trend for MHC genetic diversity to be lower in small isolated populations compared to continuous populations . Pairwise FST ranged from 0.0166 (SweJ_C–Lat_C) to 0.343 (Eng_I–Neth_SI) and all pairwise FST tests were significant . Pairwise FST ranged from 0.001 (Fin_C–Lat_C) to 0.559 (Eng_I–Neth_SI)  . Forty-f_SI) (ST . As expe_SI) . Pairwise FST ranged from zero for 15 population pairs (mainly pairs with SweJ_C and Ger_SI) to 0.136 (Eng_I–Pol_I)  . All paiST. For both microsatellites and SNPs, Eng_I and Neth_SI was the most divergent pair, although they only differ in geographic distance at 630 km  for MHC and microsatellites were not correlated (with or without correcting for distance). However, when removing the small isolated category from the dataset (keeping the isolated and continuous categories), MHC and microsatellite FST/(1 – FST) were significantly correlated  . Pairwise measurements between FST/(1 – FST) for MHC and SNP were not significantly correlated neither when including all categories nor removing small isolated populations.Pairwise measurements between FST/(1 – FST) matrixes to the distance matrix (data not shown). However, when removing both small isolated and isolated populations from the analysis, leaving only continuous populations (n pop = 5), there was a significant isolation by distance for SNPs , but not for MHC or microsatellites.No isolation by distance  was found for any of the three marker types when we compared the FST/(1 – FST) for microsatellites and SNPs  also after controlling for geographical distance . Correlations were also observed between microsatellites (both HE and AR) and SNPs (both HE and Polymorphic loci) but not significantly so after Bonferroni correction .Pairwise estimates of Fand SNPs  were sigSmall and highly fragmented populations often display lower neutral genetic diversity compared to larger populations . Here, wSmall isolated populations of European black grouse showed significantly lower genetic diversity than continuous populations for both microsatellites and SNP markers . This re2 of quantitative traits) and neutral (HE) genetic diversity. Arguably, it is difficult to estimate “ecological meaningful” genetic diversity by solely measuring the neutral genetic diversity. In a recent review of MHC diversity and viability in natural populations, each case study included at least some populations that had undergone a decrease in population size   . A majorr study) . With lor study)  and woulr study)  depends r study) . Frequenr study) .ST/(1 – FST) correcting for geographic distance, ST/(1 – FST) were significantly correlated with the corresponding metric from microsatellites. This is interesting as it may suggest that drift is affecting microsatellites differently from MHC in small isolated populations. Logically, there are three different possible outcomes comparing pairwise MHC FST with FST in neutral markers. MHC class II FST (or G’ST) can be lower . In simulations, GST  is lower for genes under balancing selection (including overdominance/heterozygote advantage) than for neutral genes  to be lower compared to both microsatellite FST (0.142) and SNP FST (0.232)  . We did ST values. Isolated populations are likely subjected to stochastic events and therefore drift affects these populations more strongly. On the contrary, our samples from the continuous populations in Scandinavia reflect the natural dispersal of the species and genetic differences may be prominent only over larger distances . For MHC, no isolation by distance probably reflects the action of balancing selection in the past due to widespread disease organisms. For the neutral markers, the absence of isolation by distance at least in the isolated categories may reflect the strong action of drift leading to strong genetic differences among populations, which is also highlighted by FST/ (1 – FST) were significantly correlated for all combinations of population categories and markers. In addition, genetic diversity measures obtained from microsatellites and SNP were positively correlated . Moreover, microsatellite and SNPs revealed mainly the same patterns of lower genetic diversity in smaller populations compared to larger  involved in maintaining MHC variation. Black grouse is similar in the MHC organization compared to chicken . It was This study is, to our knowledge, the first one in combining microsatellites, SNPs and MHC markers to gain a wider and deeper knowledge of genetic diversity in natural populations of varying degree of isolation and size. We included several populations in each population category to identify possible differences in neutral and MHC genetic diversity. We show that small isolated populations of black grouse display significant lower neutral genetic diversity than continuous populations. In addition, genetic variation within the MHC complex also seems to be lowered. The power of balancing selection acting on MHC locally may therefore not be enough to counteract genetic drift in populations that are both small and isolated. However, the pattern of lower population structure in MHC than microsatellites or SNPs, suggests that selection on MHC is still operating across European black grouse populations.MHC allele GenBank accessions: BLB11 = GQ181215, BLB13 = JF509706, BLB14 = HQ108380, BLB16 = HQ108382, BLB18 = HQ108383, BLB21 = HQ108384, BLB22 = HQ108385, BLB23 = JF509707.


Enhydra lutris, populations have suffered at least one historic population bottleneck stemming from the fur trade extirpations of the eighteenth and nineteenth centuries. We examined genetic variation, gene flow, and population structure at five microsatellite loci in samples from five pre-fur trade populations throughout the sea otter's historical range: California, Oregon, Washington, Alaska, and Russia. We then compared those values to genetic diversity and population structure found within five modern sea otter populations throughout their current range: California, Prince William Sound, Amchitka Island, Southeast Alaska and Washington. We found twice the genetic diversity in the pre-fur trade populations when compared to modern sea otters, a level of diversity that was similar to levels that are found in other mammal populations that have not experienced population bottlenecks. Even with the significant loss in genetic diversity modern sea otters have retained historical structure. There was greater gene flow before extirpation than that found among modern sea otter populations but the difference was not statistically significant. The most dramatic effect of pre fur trade population extirpation was the loss of genetic diversity. For long term conservation of these populations increasing gene flow and the maintenance of remnant genetic diversity should be encouraged.All existing sea otter,  Enhydra lutris, is a lutrine carnivore in the mustelid family, and the only member of the genus Enhydra. The species is represented by three subspecies defined by skull morphometrics: the Russian, E.l. lutris; the Northern, E.l. kenyoni; and the Southern, E.l. nereisEnhydritheriumThe sea otter, Sea otters are sexually dimorphic with males typically 34% heavier and 8% longer than females Sea otter populations have suffered from historical periods of population fragmentation due to extirpations associated with significant human hunting for their luxurious pelts. Sea otters once ranged throughout coastal regions of the north Pacific rim from the islands of northern Japan to central Baja California, Mexico In an effort to re-establish sea otter populations throughout their former range, management authorities made several translocations from the 1950s through the 1970s Knowledge of the geographic location of surviving sea otter populations during periods of population bottlenecks and of their persistence is limited, although some post-bottleneck survey data are available for estimating population growth rates Given this history of population extirpation and fragmentation, it is evident that all extant sea otter populations incurred population bottlenecks of varying severity and duration. The impact of these bottlenecks on genetic variation and genetic relationships (genetic population structure) within surviving sea otter populations remains unclear, inspiring several studies th and 19th centuries A survey of genetic variation in several extant sea otter populations has revealed levels of variation in nuclear microsatellites that are also relatively low in both remnant and translocated populations, and comparable to variation in several other mammalian species that have experienced severe population bottlenecks To test the hypothesis that all modern sea otter populations have potentially altered inter-population genetic relationships due to fur trade extirpation and population fragmentation we obtained genotypes for five microsatellite loci from DNA extracted from bones of sea otters throughout the range that lived prior and up to the fur trade. We then compared genetic diversity and population structure from these extinct populations to similar data previously reported for extant sea otters E. l. kenyoni  and E. l. nereis (CA) Five microsatellite loci .All of the 98 OLDCA sea otter remains were obtained from the Diablo Canyon archaeological site (CA-SLO-2) on the central coast of California in San Luis Obispo County. The site was excavated in the 1960s and was originally reported by Greenwood in 1972 The 40 OLDOR samples were from five archaeological sites in Oregon: near Little Whale Cove (35-LNC-43) on the northern Oregon coast The 34 OLDWA genetic samples were obtained from archaeological bone samples from the Makah Indian village site of Ozette, near Neah Bay, WA. Excavated materials are currently curated by the Makah Cultural and Resource Center in Neah Bay. Although the Ozette village appears to have been occupied for approximately 2000 years OLDAK comprised samples from several different sites. The majority of the 56 OLDAK samples (42) were obtained from skulls taken during the fur trade and curated at the Smithsonian Institution in Washington DC. They range from 100–200 YBP and were taken from various locations throughout the Aleutian Islands to Prince William Sound. They are ancestral to the current Alaskan sea otter population. Eight samples were from the Chaluka excavation on Umnak Island, ca 3500–4000 YBP The 39 OLDRU sea otter samples were obtained from the Kapsyul excavation site on Urup Island in the Kuril Islands of Russia, approximately 100–800 YBP In all bone sampling from pre-fur trade individuals, caution was used to prevent multiple sampling from the same individual and to prevent sample contamination. To minimize the chances of obtaining more than one sample per individual, three precautions were taken: (1) samples were taken from a wide array of sites; (2) a narrow set range of skeletal elements  was utilized; and (3) after amplification, samples were compared for identical genotypes and, if found, one was removed. Control of potential contamination of the ancestral bone samples followed aspects of protocols described previously 2), and 4 µl DNA/RNA free dH2O to make up final volume. The amplification profile was as follows: one cycle of 94°C (240 s), 35 cycles of 94°C (30 s)+53–57°C (30 s)+72°C (30 s) and one cycle of 72°C (300 s). PCR products were stored at 4°C or −20°C until analysis on an ABI 310 single-capillary system or 3100 sixteen-capillary system in Genescan mode. Allele scoring for each locus was performed using Genotyper Software, version 2.0 or Genescan Software, version 3.0.Microsatellites for bone samples were amplified and screened using a GeneAmp PCR 9600 thermal-cycler  in 10 µl containing 1 µl of 100–250 ng/µl purified DNA template, 0.5 µM/µl forward and reverse primer, 4 µl PCR Mastermix 2× , heterozygosity, linkage disequilibrium and genotyping failures such as null alleles and other errors. Sequential Bonferroni adjustments were used to determine significance levels for all simultaneous tests making the significance p value 0.01 General descriptive statistics of the loci was determined using GENEPOP 4.0.10 To determine the relative stability of the genetic diversity measured over time BOTTLENECK software was used To examine the population structure between geographic regions, we calculated the genetic distance between each region using Nei's standard distance Finally we re-ran GENEPOP 4.0.10 and POPULATIONS 1.2.30 software on geneotypes assigned to populations based on STRUCTURE analysis results to determine potential significant differences in genetic structure between modern and pre-exploitation sea otters. Post STRUCTURE population grouping names were based on the most numerous geographic population of origin. For example a STRUCTURE assigned AK/CA grouping would have primarily AK individuals followed by CA individuals.Departures from Hardy Weinberg expectations were statistically significant for one locus throughout the pre-fur trade populations due to an excess of homozygotes within individuals in all populations, Bonferroni corrected alpha is 0.01 . This deHE) was 0.766  for OLDAK (p = 0.011) and OLDCA (p = 0.016) due to all loci exhibiting heterozygosity deficiency. In contrast, no modern populations were significant for non-expected heterozygote deficiency based on mutation-drift equilibrium under the IAM model.FST values were statistically significant between most population comparisons (based on geographic sampling location) regardless of the era .Microsatellite K and WA . The FSCalculated Nei's genetic distances were significantly higher within pre-fur trade sea otters when compared to values found within modern otters most likely due to the higher number of alleles found within the former  and WA/AK/RU and AK/CA/RU  and for modern .For population structure based on STRUCTURE assignments, microsatellite ulations . In pre-Fst values were not significantly different when the remnant groups were compared using sampled or structure assigned populations .Finally the STF and genetic distances values STF estimates and genetic distances found among pre fur trade otters are likely inflated due to the effect of null alleles and should be regarded as conservative when interpreting population structure and gene flow.The greatest impact of the fur trade of the 18th and 19th centuries on sea otter genetics is the dramatic loss of genetic diversity. Modern populations have lost almost half their genetic heterozygosity and over 66% of the number of alleles within microsatellite loci. The numbers of alleles found within pre-fur trade samples was significantly larger than that found within extant populations. In addition the number of alleles found in the pre fur trade populations are likely an underestimate of the true number due to the null alleles found in ancient samples. Their presence underestimates genetic diversity, which generally results in increased STF estimates among pre-fur trade and modern sea otters were comparable and not statistically significantly different. Populations that are directly comparable between ancestral and modern groups are the OLDCA and OLDAK populations and the current remnant populations CA and the remnant groups from Alaska (PWS and AM). For example the STF estimate between OLDCA and OLDAK was 0.225 while the STF estimate between CA and PWS was 0.294 and between CA and AM was 0.194  The modern translocated groups that are founded by two populations are the populations with the highest growth rates and the largest, healthiest otters. Perhaps one way to assist the threatened populations with the lowest diversity such as CA would be to boost their genetic diversity by encouraging historical gene flow through future translocations. The future health of sea otter populations is not certain under current conditions but the maintenance and enhancement of the remaining genetic diversity is crucial and should be a high priority in any management plan.


The Levant is a region in the Near East with an impressive record of continuous human existence and major cultural developments since the Paleolithic period. Genetic and archeological studies present solid evidence placing the Middle East and the Arabian Peninsula as the first stepping-stone outside Africa. There is, however, little understanding of demographic changes in the Middle East, particularly the Levant, after the first Out-of-Africa expansion and how the Levantine peoples relate genetically to each other and to their neighbors. In this study we analyze more than 500,000 genome-wide SNPs in 1,341 new samples from the Levant and compare them to samples from 48 populations worldwide. Our results show recent genetic stratifications in the Levant are driven by the religious affiliations of the populations within the region. Cultural changes within the last two millennia appear to have facilitated/maintained admixture between culturally similar populations from the Levant, Arabian Peninsula, and Africa. The same cultural changes seem to have resulted in genetic isolation of other groups by limiting admixture with culturally different neighboring populations. Consequently, Levant populations today fall into two main groups: one sharing more genetic characteristics with modern-day Europeans and Central Asians, and the other with closer genetic affinities to other Middle Easterners and Africans. Finally, we identify a putative Levantine ancestral component that diverged from other Middle Easterners ∼23,700–15,500 years ago during the last glacial period, and diverged from Europeans ∼15,900–9,100 years ago between the last glacial warming and the start of the Neolithic. Population stratification caused by nonrandom mating between groups of the same species is often due to geographical distances leading to physical separation followed by genetic drift of allele frequencies in each group. In humans, population structures are also often driven by geographical barriers or distances; however, humans might also be structured by abstract factors such as culture, a consequence of their reasoning and self-awareness. Religion in particular, is one of the unusual conceptual factors that can drive human population structures. This study explores the Levant, a region flanked by the Middle East and Europe, where individual and population relationships are still strongly influenced by religion. We show that religious affiliation had a strong impact on the genomes of the Levantines. In particular, conversion of the region's populations to Islam appears to have introduced major rearrangements in populations' relations through admixture with culturally similar but geographically remote populations, leading to genetic similarities between remarkably distant populations like Jordanians, Moroccans, and Yemenis. Conversely, other populations, like Christians and Druze, became genetically isolated in the new cultural environment. We reconstructed the genetic structure of the Levantines and found that a pre-Islamic expansion Levant was more genetically similar to Europeans than to Middle Easterners. It includes Lebanon, Syria, Jordan, Israel, Palestine, and often Cyprus and historical Armenia. The region has been central to human cultural development, embracing the earliest civilizations, agricultural communities, and the rise of the first urban cities. The genetic diversity based on uniparental markers (i.e. Y-chromosome and mtDNA) of the Levantine populations shows a strong correlation with geography In this study we analyze newly-generated genome-wide data from Lebanon in addition to individuals from 48 published global populations 2 . Christianity in Lebanon dates back to the first century CE, whereas Islam was brought to the Levant through the Islamic expansions in 635 CE. In 986 CE, the Druze faith developed as a movement within Islam, and from 1030 AD, a person could only be Druze if born Druze. This correlation of genetic structure within Lebanon with cultural traits was previously described by Haber et al. K = 2, which showed the lowest cross-validation error  plot based on the identity-by-state (IBS) matrix shows strong stratification in Lebanon by religion, with separate clusters for Christians, Muslims, and Druze, irrespective of their geographic origin . The reson error , ChristiIn order to assess the population structure of Levantine populations more generally, an MDS  and a no2<0.4) since LD can bias cluster analysis. However, identification of haplotypes shared between groups is a valuable tool to infer population history events The previous analyses are based on linkage disequilibrium (LD) pruned data  error, however the CV effectiveness in predicting the “truth” K can be challenged when considering closely related populations K value ADMIXTURE requires the assignment of a specific population number (K). We chose to assign a K = 10  since itK = 10  shows thK = 10 an ancestral component (light green) with a geographically restricted distribution representing ∼50% of the individual component in Ethiopians, Yemenis, Saudis, and Bedouins, decreasing towards the Levant, with higher frequency (∼25%) in Syrians, Jordanians, and Palestinians, compared with other Levantines (4%–20%). The geographical distribution pattern of this component (ST = F0.035) than to the Arabian Peninsula/East Africa component (light green) (STF = 0.046). Our estimates show that the Levantine and the Arabian Peninsula/East African components diverged ∼23,700-15,500 y.a., while the Levantine and European components diverged ∼15,900-9,100 y.a. We note here that our divergence time estimates are based on the assumption that “effective population sizes” have not significantly changed overtime. We make this assumption, and obtain divergence times from genetic data which appear to coincide well with archeology.ADMIXTURE identifies at omponent  correlatomponent . We founThe estimated time of divergence between the Levantine component and other Middle Easterners overlaps with evidence from archeological findings of a major cultural development in the Levant during the early Epipaleolithic period  From the first ventures out of Africa, to admixture with archaic humans, to the earliest Neolithic transition, the developments in the Levant have marked the history of modern humans. However, the Levant had been underrepresented in genome-wide studies and little is known about its population structure. In this study, we show a multilayered history of the Levantines with multiple components that might be traced to different historical population events. We propose that the Levant and Middle Eastern modal components diverged after the LGM during the early Epipaleolithic period, which was characterized by behavioral variability and innovations accompanied by major life-style and technological changes in the Levant Finally, we show that although population movements and expansions during the Epipaleolithic marked the emergence of a Levantine component and made the Levantines genetically similar, recent cultural developments, such as the founding and spread of major world religions, have had a strong impact on population stratifications in the Levant.Populations like the Levantines, where geography is not the only major correlate of genetic variation, are unusual. In addition to their importance in understanding human evolution and history, these unusual stratifications can be hard to control in association studies for mapping complex disease susceptibilities and therefore require particular attention.bhusers.upf.edu/~mhaber/PLOS/Samples were collected from 1,341 Lebanese subjects with informed consent approved by the IRB of the Lebanese American University. Genotyping was performed on Illumina 610K or 660K bead arrays. PLINK N x N matrix of the genome-wide IBS pairwise distances was constructed using PLINK on pruned SNPs. The MDS was performed in the R environment The smartpca, part of the EIGENSOFT 3.0 package PCA was performed using Samples  were phased with SHAPEIT ROLLOFF analysis The clustering algorithm ADMIXTURE STsF were calculated using ADMIXTURE at K = 10. In order to map our STF estimates into population divergence dates some information about population demography is needed. Li et al. STF and the effective population size Ne  Contour maps showing the distribution of the ancestral components were generated using Surfer 8 (Golden Software) implementing the Kriging method.Figure S1Lebanon religion groups structure inferred by ADMIXTURE analysis of >240K autosomal SNPs. A) Each horizontal line represents ancestry probabilities of an individual in the 2–4 constructed ancestral populations. B) Cross-validation plot for the Lebanese dataset.(TIF)Click here for additional data file.Figure S2Principle component analysis of >240K SNPs showing the top two components. A) Plot shows global diversity using 50 populations. B) Magnification of West Asia region showing the Levantine populations in their regional and religion context.(TIF)Click here for additional data file.Figure S3Stratified random sampling of 75 Lebanese samples. A) 25 samples from each of the three main religion groups in Lebanon were randomly chosen from the 1,341 samples illustrated in (TIF)Click here for additional data file.Figure S4Raw coancestry matrix shows relationships between the Levantines and the world populations. A) Intensity of the colors reflects the number of haplotype chunks donated to the Levantines. The vertical line is a visual aid to reflect the Levantine split observed in the tree. Horizontal lines distinguish the major geographic regions. B) coancestry matrix with an alternative color scale.(TIF)Click here for additional data file.Figure S5Principle component analysis generated with fineSTRUCTURE using ChromoPainter's coancestry matrix showing the top two components. A) Plot shows global diversity using 50 populations. B) Magnification of West Asia region showing the Levantine populations in their regional and religion context.(TIF)Click here for additional data file.Figure S6World population structure inferred by ADMIXTURE analysis of >240K autosomal SNPs. A) Each horizontal line represents ancestry probabilities of an individual in 2–10 constructed ancestral populations. Levantine population names are shown in blue. B) Cross-validation plot for the world dataset.(TIF)Click here for additional data file.Table S1Location of the 75 Lebanese samples used in the comparative analyzes.(XLS)Click here for additional data file.Table S2Populations selected for this study.(XLS)Click here for additional data file.Table S3Ancestry probabilities of individuals considering 10 ancestral populations. Highlighted cells indicate individuals have >60% of one component. Standard errors were estimated using 200 bootstrap replicates.(XLS)Click here for additional data file.Text S1Description of the ROLLOFF analysis.(PDF)Click here for additional data file.


FOXQ1, MAST2, and CDH4, were found to play a role in hair follicle development and human cancer, signal transduction, and tumor repression, respectively. These showed strong indications of natural selection in Philippine Negritos, a group of aboriginal hunter-gatherers living in the Philippines. MTTP, which has associations with metabolic syndrome, body mass index, and insulin regulation, showed a strong signature of selection in Southeast Asians, including Indonesians. Functional annotation analysis revealed that genes and genetic variants underlying natural selections were generally enriched in the functional category of alternative splicing. Specifically, many genes showing significant difference with respect to allele frequency between northern and southern Asian populations were found to be associated with human height and growth and various immune pathways. In summary, this study contributes to the overall understanding of human local adaptation in Asia and has identified both known and novel signatures of natural selection in the human genome.Genetic studies of human local adaptation have been facilitated greatly by recent advances in high-throughput genotyping and sequencing technologies. However, few studies have investigated local adaptation in Asian populations on a genome-wide scale and with a high geographic resolution. In this study, taking advantage of the dense population coverage in Southeast Asia, which is the part of the world least studied in term of natural selection, we depicted genome-wide landscapes of local adaptations in 63 Asian populations representing the majority of linguistic and ethnic groups in Asia. Using genome-wide data analysis, we discovered many genes showing signs of local adaptation or natural selection. Notable examples, such as  HBB and G6PD, whose mutants and deficiency alleles were found to confer resistance to malaria respectively, LCT, whose variant allows lactose tolerance to persist throughout adulthood, SLC24A5, which contributes to skin pigmentation diversity, and EDAR, one of whose polymorphisms may cause variation in hair morphology High-throughput DNA genotyping and sequencing technologies and novel statistical approaches advanced genome-wide detection of molecular signature of natural selection in the human genome NAT2 gene supporting acetylation in human adaptation to farming in Central Asia, positive selection on NRG-ERBB4 pathway in Middle East, natural selection on an ABCC11 SNP determining earwax type in East Asians, and EPAS1 and EGLN1, which are likely to be responsible for high-altitude adaptations in Tibetans However, most of the previous genome-wide studies of natural selection have concentrated on population samples collected from international collaborative efforts such as the Human Genome Diversity Panel (HGDP), the International HapMap Project, and the 1000 Genome Project We therefore attempted to provide the first comprehensive landscape of local adaptations in Southeast Asia using genome-wide analysis of 63 populations obtained from the HUGO Pan-Asian SNP consortium, where 54,794 autosomal single-nucleotide polymorphisms (SNPs) in 1513 individuals were genotyped using Affymetrix GeneChip Human Mapping 50K Xba Array Taking advantages of the dense population samples, we drew a general yet comprehensive picture of human local adaptation, particularly in Southeast Asia. First, we characterized an overview of putative local adaptation signals using between-population comparisons, which provided a guide for further, finer-scale examinations. However, since the marker density was not sufficiently high in our data, we mainly focused on allele-frequency-based analysis rather than haplotype-based analysis. We classified populations into several sub-groups according to the genetic structures revealed in a previous study After removing some populations showing evident admixture as reported in a previous study ST, of genome-wide SNP markers in each pair. As the total sample size of each group was not always similar, some groups might have much larger or smaller sample size than others. We randomly sampled the same number of individuals from each group so that the sample size of groups under comparison is comparable. The most commonly used means of identifying putative signals of selection has been the outlier-based method ST values within genes. In order to control the bias resulting from the number of genotyped SNPs or the size of genes, here we employed a sliding window strategy  ST-based approach also appeared on the list produced by the XP-CLR test  was best illustrated by our functional analysis . The OMIPLCG2 with the SNP in the gene reaching FST of 0.55. These findings indicated that signaling pathways have been under natural selection and might have played an important role in Negritos adapting to local environment in Malaysia.Another group of Negritos in Malaysia showed a great variation in keratinocyte differentiation and epidermal cell differentiation compared with Indonesian population (Benjamini FDR corrected p<0.01). There were a series of KEGG signaling pathways showing enrichment of divergence between the Negritos and Indians , for exaIn summary, giving clues to biological basis of regional natural selection, our functional analyses provided a great deal of insight into human local adaptation based on allele frequency differentiation between populations. A similar procedure of functional analysis was also performed upon signals identified by XP-CLR analysis .ST value among that of all the SNP markers within it. We chose the top ten candidates from each group pair, as shown in In order to better understand selective pressures upon Asian populations that might not have previously been evaluated, we ranked candidate genes according to the highest FMLKL, PPP1CC, DAPP1, and ABCA12.Considering the many group comparisons and candidate genes, we only highlighted the top candidate gene from each pair, as shown in FOXQ1 showing the strongest signal of positive selection in comparisons with most other Asian groups was involved in the development and morphogenesis of the hair follicle. In Malaysian Negrito population, the outstanding candidate WNT4 had something to do with gamete generation and specificity, indicating the influence of selection on human reproduction. Additional candidate genes specific to some groups were also identified  on chromosome 20. The whole genomic picture of selective signals in these comparing pairs confirmed the most significant signature of local adaption in Philippine Negritos  and southern populations . MTTP protein catalyzes the transport of lipoproteins in the process of lipid metabolism and its variants are associated with plasma cholesterol levels and body mass index DAPP1 protein, an adaptor protein, regulates antigen receptor signaling downstream of phosphatidylinositol 3-kinase and is closely related to human immune system ST scores upon the whole genome loci in the comparisons between Southeast Asian populations and northern populations  showed remarkable evidence of selection signatures  and southern populations (Indian)  . This inIn this study, we performed the first comprehensive genome-wide scan for natural selection in 63 Asian populations and identified a number of putative selection signals. These may further the understanding of human local adaptation in Asia. One of the great advantages of our data is the fact that it was produced by sampling populations of a high geographic resolution and covering a large number of Asian populations, particularly Southeast Asian populations.To draw a general picture of genome-wide signs of local adaptation in Asian populations, we investigated significant SNPs underlying population differentiation with respect to allele frequency. Using the sliding-window-based approach, we adopted a 1% genome-wide cutoff with respect to population differentiation across windows and indicated top 200 genes as putative selection signals in each group pair. Our results showed that Negritos and Indians had the most significant divergence regarding the allele frequency. Further, functional analysis of candidate genes suggested that natural selection among Asian populations is likely to induce mutations that play roles in alternative splicing. Selection divergence from northern Asian populations and southern Asian populations aimed at genes related to cadherin and a plenty of immune and related disease pathways. Another intriguing enrichment was about biological pathways of human height and growth, making Japanese and Korean populations different from Philippine Negritos. Moreover, Philippine Negritos and Malaysian Negritos demonstrated great genetic divergence and carried genes with polymorphisms whose allele frequency differed from other Asian populations.Despite the fact that we did have observed some enrichment of genes, a part of categories, mainly for cell adhesion-related terms, do harbor many genes from protocadherin gamma gene cluster that are physically closely located in a region of 100 kb . AlthougST approach and better understand the selective signals, we turned to the results of XP-CLR analysis, and highlighted notable examples of candidate genes showing large numbers of extreme SNPs achieving peak FST and XP-CLR scores  and southern populations , which was supported by FST-based analysis. We believe that these genes merit further study because they are all important for human development or immunity, especially PIK3R3, since it is involved in the insulin receptor signaling pathway, which has been suggested as an adaptive pathway among African Pygmies To confirm the reliability of the candidate signals identified by FR scores . GeneralHLA region varies dramatically across broad geographical populations, and it has been found to be a predictor of northern versus southern ancestry in Europe and other parts of the world HLA showed notably high FST and XP-CLR scores in the comparisons of certain Asian populations, especially between Indians and SouthernChinese&Thai1, and between Han Chinese populations and southeastern populations  (IFNG and LEPR reflected the difference between northern and southern Asian populations. As people who reside at higher latitudes (north) are generally taller than those living at lower latitudes (south), these two genes may be in association with the stature of Asian individuals.In this study, our results provided a great deal of evidence for local adaptation in Southeast Asian populations that burden historical and geographical admixture, and in Negritos that have received increasing amounts of attention from the research community. Previous studies have identified some significant and comprehensive local adaptation signatures in African pygmies that are similar to Negritos on stature but differ substantially in genetic makeup. Most of the signatures lie in the immunity-related genes because of the different microbial environments in different habitats. For example, the e&Thai2) . Our fune&Thai2) . EspeciaTYRP1 expression in response to differential UV radiation based on latitude DDB1, which protects the skin from solar UV exposure, has different alleles fixed in continents from different latitudes Considering the differences between northern and southern populations, the most remarkable and comprehensive clue may be related to skin pigmentation. It has been proved, from Africa to eastern Europe and eastern Asia, that microRNA regulation acts as a rheostat to optimize However, this study has two principal limitations. The first is the low SNP density relative to the high-density data available in public resources and therefore may not fully represent the entire genome. But these SNP markers randomly distribute across the genome, thus providing a background of genome-wide information Despite its limitations, our study can be regarded overall as a useful guide for further evaluation of local adaptation in Asian populations. The strongest signs of local adaptation in Asian populations may have been novel candidates, suggesting the advantage of data, such as the data evaluated here, which cover wide geographic areas within Asia. This may be particularly true of candidates discovered among the Negritos. A more comprehensive collection of greater numbers of samples across a wider geographic range will be necessary for further studies of positive selection, especially in the populations whose members exist across special geographic regions, such as the Negritos and Tibetans. More importantly, high-resolution data such as genome-wide SNP data and next generation sequencing data will facilitate investigation of the genetic basis of local adaptation in a more effective and detailed way than has previously been possible. The development of novel and sophisticated methods of deciphering signs of natural selection will assist researchers and improve the biological understanding of human adaptive evolution.The HUGO Pan-Asian SNP consortium constructed a database (PanSNPdb) containing data of human genetic diversity among Asians by sampling 1,719 unrelated individuals among 71 populations from China, India, Indonesia, Japan, Malaysia, the Philippines, Singapore, South Korea, Taiwan, and Thailand ST following Weir and Hill ST value of the top 3 SNPs in each window as the value of it and defined 1% tail of the distribution as the threshold. Those windows above were considered regions underlying local adaptation. After that, for each individual window, we sorted SNPs based on their FST and selected the top n SNPs (n = int(N/i), N represents the total number of SNPs in a certain window. If N<20, i = 5; if N> = 20, i = 10), which were thought to be candidate SNPs. Finally we mapped these candidate SNPs onto genes and picked out the top 200 genes as candidate genes for local adaptation.To detect signs of local adaptation in Asian populations, we used an allele-frequency-based approach. For each pair of groups, we calculated unbiased estimates of FST analysis, we applied another allele-frequency-based method: the cross-population composite likelihood ratio test (XP-CLR) To confirm selection signals identified by Fhttp://david.abcc.ncifcrf.gov/) to perform functional analysis upon candidate genes acquiring significant SNPs in each pair of group comparisons We implemented DAVID Bioinformatics Database Click here for additional data file.Figure S2Signatures of local adaptation in Southeast Asian populations associated with MTTP and DAPP1.(PDF)Click here for additional data file.Table S1Characterization of datasets and group division.(PDF)Click here for additional data file.Table S2Selection candidates detected by XP-CLR approach.(XLSX)Click here for additional data file.Table S3Candidate genes overlapping with XP-CLR genes.(XLS)Click here for additional data file.Table S4Functional enrichment analysis.(XLSX)Click here for additional data file.Table S5Top 10 candidate genes in 36 comparing pairs.(XLS)Click here for additional data file.Table S6ST approach.Candidate genes underlying local adaptation identified by F(XLS)Click here for additional data file.Table S7Outstanding candidate genes demonstrating strong signs of local adaptation in Asia.(XLS)Click here for additional data file.Table S8Candidate genes overlapping with 14 literature genes.(XLS)Click here for additional data file.Text S1Results of XP-CLR analysis.(PDF)Click here for additional data file.


Discoglossus montalentii. In contrast with expectations based on its insular, restricted and continuous distribution, we found evidence of 3 phylogroups, whose rather ancient divergence (Early-Middle Pleistocene) was likely primed by climatic changes that occurred during the ‘middle Pleistocene revolution’. Furthermore, their differentiation explained most (68%) of the overall genetic diversity that was observed. These results and the growing evidence from intra-island phylogeographies, suggest that island populations frequently may not conform to some long-standing assumptions, including long-term stability, range-wide panmixia and the correlation of effective population size to the island size. As a consequence, both for theoretical and for applied purposes, the extensive use of these assumptions in the study of island populations warrants a careful re-examination.Island populations have been extensively used as model systems in ecology, biogeography, conservation and evolutionary biology, owing to the several simplifying assumptions that they allow. Nevertheless, recent findings from intra-island phylogeographic studies are casting doubts on the generality of some of these underlying assumptions. We investigated the phylogeography, historical demography, and population genetic structure of the Corsican endemic frog,  Islands are among the most important biodiversity hotspots on earth The island of Corsica is one of the more than 5000 islands in the Mediterranean basin Discoglossus montalentii, is a species endemic to the mountain regions of the island of Corsica, specifically in the central portion of the island. Once attributed to the Tyrrhenian Painted frog D. sardus, populations of D. montalentii were then attributed to a separate species based on studies of their allozyme variation Discoglossus  de Corse. Frogs were captured with nets at their breeding sites. We collected tissue samples from toe-tips after anaesthetizing frogs by submerging them in a 0.1% solution of MS222 (3-aminobenzoic acid ethyl ester). No individuals were brought to the laboratory or sacrificed. After the completion of the sampling procedure, all the individuals were immediately released at the collection site. Tissue samples were stored in 96% ethanol until further analyses.D. montalentii were collected from 6 populations located on the main mountaintop of the island of Corsica, spanning the whole species range  were amplified by polymerase chain reactions (PCR), then purified and sequenced by Macrogen Inc. (www.macrogen.com). The primers used to amplify the cytb fragment were 494dismod (AACATYTCRGCGCTATGAAA) and cytdis (ATCGATTTAGAAGYTTRTTTTC); whereas primers 12SZ-L (AAAGGTTTGGTCCTAGCCTT) 12SF-H (CTTGGCTCGTAGTTGCCTGGCG) were used to amplify the 12s fragment 2 (2.5 mM), the reaction buffer , the 4 dNTPs (0.2 mM each), the 2 primers (0.2 µM each), the enzyme Taq polymerase , and 2 µL of DNA template. PCR cycling conditions were: one step at 94°C for 4 min followed by 35 cycles at 94°C for 1 min, 52°C (cytb) or 50°C (12s) for 1 min, 72°C for 1 min, and a single final step at 72°C for 10 min. All sequences obtained were deposited in GenBank  to check the electropherograms by eyes and we produced a sequence alignment with clustalxdnasp 5 mega 5.1. Since no differences were detected by a partition-homogeneity test paup* 4.0b10 We used the program network 4.6.1.0 (www.fluxus-engineering.com), using the default option for all parameters in the analysis.The phylogenetic relationships among haplotypes were investigated using the median-joining algorithm for phylogenetic network estimation samovaK), ranging from 1 to 6. The optimal K was assessed as the one for which FCT (i.e. the genetic variance due to between groups divergence) was the highest and statistically significant. To verify its consistency, we ran the analysis 5 times for each K value with 1000 independent annealing processes. The partition of the total genetic variation into its hierarchical components was evaluated through the analysis of molecular variance . A tree topology was obtained using the Neighbor-Joining algorithm (NJ) as implemented in mega, using the Tamura-Nei distance (which is the best approximation of the HKY distance available in mega) and 1000 bootstrap re-samplings to evaluate nodal support. The LS procedure in DAMBE was run using the NJ tree topology, the ‘softbound’ option and the ‘MLCompositeTN93’ genetic distance, as suggested by Xia & Yang et al.D. montalentii and all other species of the genus Discoglossus. Finally, a bootstrap re-sampling procedure (with 1000 pseudoreplicates) was used to assess the standard deviation of the time estimates.The time to the most recent common ancestor (TMRCA) of the main haplogroups was estimated using the distance-based least squares (LS) method recently described by Xia & Yang cytb gene and a fragment of 448 bp of the 12s gene. The combined dataset  included 39 variable positions (29 parsimony informative) defining 16 haplotypes, whose geographic distribution among populations is shown in cytb gene fragment, no indels, stop codons, or non-sense codons were found. For the whole mtDNA dataset, haplotype diversity was 0.867 (±0.028 s.d.), while nucleotide diversity was 0.0073 (±0.0037 s.d.).For all 72 individuals analysed, we obtained a fragment of 935 bp of the The phylogenetic network among haplotypes is shown in samova analysis indicated 3 groups of populations (K = 3) as the best option of population grouping. Indeed, 68% of the total variation was explained at the among groups level with K = 3, while 51%, 64% and 57% was explained with K = 2, K = 4 and K = 5, respectively. One group comprised all of the populations carrying haplotypes from clade S , a second group included populations 2 and 3, carrying haplotypes of the clade W at higher frequencies, and the third group comprised just population 1, where clade E was the most represented. The amova analysis , 0.91% to the among-population within-group level (FSC = 0.03), and 31.55% to the within-population level (FST = 0.68).The analysis  was perfR2 statistic, and by the large negative and significant value of the FS statistic.The mismatch distribution and the values of the demographic test statistics computed separately for the 3 clades, are shown in The chronogram showing the TMRCA of the main groups of haplotypes is shown in D.montalentii, as presented here, supports this revised view and exemplifies some of its major implications well . Each group was found to be geographically restricted to a portion of the species range: clade W in the north-western part, clade E in the north-eastern part, and clade S in the southern part of the island. Furthermore, the TMCRA analysis traced the split-time among these groups back to approximately the transition from Early to Middle Pleistocene.c.1.2-0.5 Ma D. montalentii was forced to move from mountain regions to lower valleys, where more suitable humidity and temperature conditions persisted The Early to Middle Pleistocene transition , or alternatively, they may have occurred earlier in the history of these populations and their signatures were subsequentely erased.Historical demographic analyses indicated a recent demographic expansion for the southern clade S, but not for clades E and W. This parallels what has been recently found for D. montalentii, as we have inferred here, shows several similarities with what has been previously observed in other Corsican endemic species, particularly with respect to the spatial distribution of phylogeographic breaks, as well as a more structured pattern of variation in northern than in southern Corsica The Pleistocene evolutionary history of D. montalentii, as the whole set of data now available for this species could justify its elevation from ‘Near Threatened’ to ‘Vulnerable’ , since no data on population structure were available until now. The pattern emerging from our study does not fit with this prediction. Indeed, populations appeared to be fragmented into 3 main groups of ancient origin (Early-Middle Pleistocene). Moreover, most of the genetic diversity found was allocated among groups (68%), rather than within them. Although these results deserve to be explored further based on a panel of nuclear loci (e.g. to assess the impact of secondary contacts on the levels of population genetic diversity), the 3 groups identified here define 3 distinct evolutionary significant units (ESUs) that would warrant management for conservation see also .The data presented here add to the recently growing literature on genetic diversity of island populations in showing that microevolutionary processes acting within islands can be extremely relevant in explaining the current patterns of genetic diversity of island populations. Far from discounting the role of other processes see e.g. , these rTable S1Uncorrected pairwise divergence among the 16 composite haplotypes found in D. montalentii, and their genbank accession numbers. Uncorrected pairwise divergence (p-distance) among the 16 composite haplotypes found in D. montalentii. Divergences for the 12S (448 bp) and CytB (935 bp) fragments are given below and above the diagonal respectively. The Genbank accession number of each haplotype is given below the distance matrix for both the 12S and the CytB fragments.(DOCX)Click here for additional data file.


To study the genetic relationship of Kazakhs from East Kazakhstan to other Eurasian populations by examining paternal and maternal DNA lineages.Whole blood samples were collected in 2010 from 160 unrelated healthy Kazakhs residing in East Kazakhstan. Genomic DNA was extracted with Wizard® genomic DNA Purification Kit. Nucleotide sequence of hypervariable segment I of mitochondrial DNA (mtDNA) was determined and analyzed. Seventeen Y-short tandem repeat (STR) loci were studied in 67 samples with the AmpFiSTR Y-filer PCR Amplification Kit. In addition, mtDNA data for 2701 individuals and Y-STR data for 677 individuals were retrieved from the literature for comparison.There was a high degree of genetic differentiation on the level of mitochondrial DNA. The majority of maternal lineages belonged to haplogroups common in Central Asia. In contrast, Y-STR data showed very low genetic diversity, with the relative frequency of the predominant haplotype of 0.612.The results revealed different migration patterns in the population sample, showing there had been more migration among women. mtDNA genetic diversity in this population was equivalent to that in other Central Asian populations. Genetic evidence suggests the existence of a single paternal founder lineage in the population of East Kazakhstan, which is consistent with verbal genealogical data of the local tribes. In terms of population genetics, Central Asia is one of the least studied regions in the world. The studies conducted in the region, based on scarce genetic data, indicate that the Central Asia population is a mix of Eastern and Western populations ,2. Kazakshezhire,” states that the Naiman people living in Tarbagatay region are descendants of one ancestor, named Toktar-kozha, who came from the territory of modern Uzbekistan and was a Sart by origin. Based on the data from “shezhire,” we formed a hypothesis of uniform paternal descent of the Naiman tribe. The aim of this study was to better understand the origins and differentiation of the Kazakh ethnic group and to investigate the genetic relationship between this population and other Eurasian populations.The current study focused on the Kazakh population of the East Kazakhstan Province, because recently there have been many reports on the neighboring populations of Xinjiang Uyghur Autonomous Region and Altai regions. East Kazakhstan is populated by the Naiman tribe. Their genealogical narrative, “A total of 160 blood samples were collected from healthy adult individuals, 67 men and 93 women, during an expedition to Tarbagatay region, East Kazakhstan in 2010. Prior to the expedition, ethical approval was received from the Ethics Committee of the National Center for Biotechnology of the Republic of Kazakhstan . The Ethics Committee approved the informed consent form and questionnaire form designed specifically for the study.The ethnic origin of sampled individuals was ascertained up to three generations. Blood was taken with the informed consent signed by all donors. In addition, all participants completed the questionnaire that included information on the geographic origin, nationality, maternal and paternal pedigree, and health issues. Related individuals were not included into sampling. In addition, mtDNA haplogroup data for 2701 individuals and Y-short tandem repeat (STR) haplotype data for 677 individuals of different ethnic backgrounds were retrieved from the literature to establish the genetic relationship of Kazakhs from East Kazakhstan with other populations  and blood collecting needles (Terumo®). Blood samples were stored in portable refrigerators until DNA was extracted. DNA extraction and purification was performed with Wizard® genomic DNA Purification Kit , according to the manufacturer’s instructions.Polymerase chain reaction (PCR) amplification. Mitochondrial DNA polymorphisms were typed for 160 individuals by using conventional PCR, with primers specific to hypervariable segment I (HVS-I) (2(Lytech). The PCR protocol was: 94°C for 10 minutes, followed by 35 cycles of denaturation at 94°C for one minute, annealing at 55°C for one minute, extension at 72°C for one minute, and a final extension step at 72°C for 7 minutes. PCR products were initially checked in 1.5% agarose gel and sequenced on ABI 3730xl DNA analyzer . (HVS-I) . PCR reaSequence alignment and haplogroup analysis. The obtained sequences were compared with the revised Cambridge Reference Sequence  to find polymorphisms by using SeqScape v 2.6 software . Identified HVS-I polymorphisms were used for assignment of mtDNA haplogroups. We also included published data on other Eurasian populations for comparative analysis  according to the manufacturers’ instructions. It includes 17 Y-specific STR loci  that were typed in three multiplexes on an ABI3100 Genetic Analyzer (Applied Biosystems/Hitachi). A quality control check was performed by successful completing of the quality assessment exercise in 2011. After certification, the data were deposited to the Y-Chromosome Haplotype Reference Database (accession number YA003700).Data analyses. Fragment sizes were determined using the GeneScan 3.1.2 software (Applied Biosystems) and allele designations were performed using the Genotyper 2.5.2 software (Applied Biosystems). We also included published data on Altaian Kazakhs, Kalmyks, Kazakhs, Kyrgyzs, Mongolians, Uighurs, and Uzbeks . The statistical significance  was estimated by permutation analysis, using 10100 permutations. The STATISTICA package  was used for multidimensional scaling (MDS) analysis. Y predictor by Vadim Urasin, v.1.5.0 was used for Y-STR haplogroup prediction (http://predictor.ydna.ru/).The study of mtDNA haplogroup variation in the population of East Kazakhstan region revealed that the majority of maternal lineages were distributed among haplogroups that were common in Central Asian region .Comparison of 33 Eurasian populations based on genetic distances was performed and the results are presented as a multi-dimensional scaling plot . The popIn fact, all of them had equal haplogroups A, B, C, D, F, G, H, and M, suggesting that these lineages were in the common maternal gene pool from which these different lineages had emerged. However, there were some notable differences between them. For example, Kazakhs from Xinjiang had higher frequencies of West Eurasian lineages H, T, U, and Z than Mongolians, Altaian Kazakhs, or Kazakhs from East Kazakhstan. Kazakhs from East Kazakhstan had greater diversity of haplogroups present in their mtDNA gene pool than the groups from South Siberia, Xinjiang, or Mongolia. Since recent data showed a common ancestry of indigenous Altaians with Native Americans , we compAnalysis of 17 fast evolving Y-STRs provided additional details that helped to elucidate the paternal diversity in the Kazakh population. A total of 26 different haplotypes were identified in 67 individuals. Twenty-four haplotypes were nonrecurring. Paternal genetic variation within the population was rather low. Relative frequency of haplotype 15-12-29-23-10-13-12-13,18-10-12-15-19-15-17-19-12  was 0.612 . The hapRst values showed that population of East Kazakhstan remained distinctive even with a reduced number of Y-STRs. Based on the Slatkin’s Rst matrix, the populations genetically closest to the Tarbagatay population were Kazakhs from Kara-Kalpakia (Kazakhs2), southeastern Altaian Kazakhs (Altaian Kazakhs2), Uighurs from South East Kazakhstan (Uighurs1), and Uzbeks from Kara-Kalpakia (Uzbeks) . Our popThis study found a high degree of genetic differentiation on the level of mitochondrial DNA, but very low genetic diversity of Y-STR data.Genetically close subpopulations of Kazakhs from Altai, Kazakhstan, and Xinjiang showed a similar mtDNA composition consisting of mainly East Eurasian haplogroups. Affinities among these populations may result from their common origin or a recent admixture resulting from geographic proximity. Genetic distances between populations can be related to geographic distances, according to a model of isolation by distance . ComparaThe paternal lineages present in the Tarbagatay population were different from the populations of Kazakhs that have been studied before, with an exception of southeastern Altaian Kazakhs, who had 19 haplotypes (21.35% out of the sample size) identical with the most common haplotype observed in East Kazakhstan. This could be explained by historical data indicating that Kazakhs began migrating to the Altai region in the 19th century. According to Oktyabrskaya , there wshezhire,” which states that the Naiman tribe has a common single ancestor from Uzbekistan. These verbal genealogical data are consistent with the results of the MDS plot. An alternative explanation for our results is provided by historian Sultanov . Not a single 17 Y-STR haplotype was found to be common between South and East Kazakhstan region. This implies different paternal origin of the tribes and genetic substructuring among Kazakhs.In addition, we genotyped 99 male individuals from South Kazakhstan region populated by a different tribe (accession number YA003729, The comparison with the central “star cluster” profile, described by Zerjal et al , showed The studied subpopulation represents a genetically isolated group with a single paternal founder lineage different from the “star cluster” lineage. The presented results reveal different migration patterns in East Kazakhstan region, showing more migration among women. Partially, this paternal genetic uniformity could be explained by local traditions, such as exogamy, that were strictly followed in the past and played a crucial role in conservation of the unique genetic properties. The population of Madjar from Torgay area had been following same traditions in the past aiming to avoid inbreeding . NonetheOur finding on active migration of maternal DNA requires additional explanation and further support from complementary sources, especially since the studied nomadic tribe is scarcely described in historical sources. Apart from additional genotyping of the Kazakh population, further cultural and historical studies are needed to gain more knowledge on the cultural processes that have greatly influenced genetic variability of this and other populations.