Indirect association studies routinely exploit linkage disequilibrium around genes, particularly in isolated populations where it is assumed to be higher. Here, we explore both the amount and the decay of linkage disequilibrium with physical distance along 211 gene regions, most of them related to complex diseases, across 39 HGDP-CEPH population samples, focusing particularly on the populations defined as isolates. Within each gene region and population we use r2 was found to be significantly different both between and within continental regions, a much higher proportion of r2 variance could be attributed to differences between continental regions . Similarly, while the proportion of SNP pairs with r2 > 0.8 was significantly different across continents for all distance classes, it was generally much more homogenous within continents, except in the case of Africa and the Americas. The only isolated populations with consistently higher LD in all distance classes with respect to their continent are the Kalash  and the Surui (America). Moreover, isolated populations showed only slightly higher proportions of SNP pairs with r2 > 0.8 per gene region than non-isolated populations in the same continent. Thus, the number of SNPs in isolated populations that need to be genotyped may be only slightly less than in non-isolates.Although the average rThe "isolated population" label by itself does not guarantee a greater genotyping efficiency in association studies, and properties other than increased linkage disequilibrium may make these populations interesting in genetic epidemiology. Linkage disequilibrium (LD) is the non-random association between allele frequencies at two loci. Recombination rate variation is the main determinant of LD ,2. It haLinkage disequilibrium implies correlation between loci, which means that information for untyped variants can be inferred from genotyped loci in LD with them. In recent years, LD has been exploited to the extent that it has become the cornerstone concept for research in genetic epidemiology of complex diseases, since it allows indirect association mapping, as implemented in the recent flurry of genomewide association studies . It is aIt has often been suggested that genetically isolated populations would offer increased statistical power to detect association because of the impact of their particular past demography on their genomic structure . LD in iHere we present data for 2 380 SNPs distributed across 211 gene regions in 39 worldwide populations representing human diversity (HGDP-CEPH Human Genome Cell Line Diversity Panel ). Gene r2 [2 larger than 0.8 in different physical distance classes and the overall per gene region. Our gene-centered approach provides an empirical view of the amount and pattern of LD decay with physical distance across worldwide human populations, including some considered as genetic isolates.Within each gene region and for each population we have quantified the extent of LD between all pairs of SNPs by means of r2  and dete2 between all possible SNP pairs within each gene region and with minor allele frequencies greater than 0.05 was computed for each population and continental region and plotted against physical distance . Results of the ANCOVA test show that the model and all of its components  are highly significant (p < 0.001). The amount of variation explained by the whole model was 14%; physical distance explains most of this (11%); while the continent and population within continent variables account for the remaining 2.8% and 0.5%, respectively. All mean r2 pairwise comparisons between continental regions were significant (after applying the Bonferroni correction for multiple comparisons), which means that significance could not be attributed to a single continent. When we compared average LD within continental regions, the proportion of pairwise population comparisons with significant differences (after applying the Bonferroni correction post-hoc) varies greatly among regions. In Europe only 6 out of 21 (28%) pairwise comparisons were significant and all of these six pairs contained the Orcadian population. In Central South Asia, the number of statistically significant comparisons was 15 out of 36 (41%) corresponding to the Brahui and Kalash populations. For the remaining continental groups, most comparisons (~80%) were significant: 11 out of 15 pairwise comparisons between Sub-Saharan African populations, 5 out of 6 in the Middle East – North Africa, 13 out of 15 in East Asia, and 8 out of 10 in the Americas. A similar pattern of significance was observed when comparing each continental and populational mean r2 value against the mean r2 across continents and across populations within each continent, respectively (data not shown).The mean re Figure . Populat2 greater than 0.8 was counted for each population and continental region and plotted against physical distance . In contrast, comparisons within continents are generally not significant, with a few exceptions. Sub-Saharan Africans  were significantly different (p < 0.05) in their proportion of SNP pairs with r2 > 0.8 for only two distance classes (0–10 and 250–300 kb), which were not significant after Bonferroni correction. The Middle East was significant in the first four distance classes (the first two after Bonferroni correction); this was also the case for seven distance classes in Central Asia (four after Bonferroni correction). European populations were heterogeneous for the 10–20, 30–40, and 300–400 kb distance classes, albeit none of these comparisons would be significant after Bonferroni correction. The number of heterogeneous distance classes in East Asia was five and two (60–70 and 70–80 kb) before and after Bonferroni correction, respectively. Finally, Americans were heterogeneous at all distance bins, and this effect cannot be attributed to any single population: dropping the apparently more differentiated population, the Surui, still results in 14 significant tests in 19 distance bins.The proportion of SNP pairs with r2 > 0.8 per gene region. In Sub-Saharan African populations, we compared the two Pygmy samples against the non-isolated Mandenka, Bantu, and Yoruba. In the Pygmy samples, the proportion of SNP pairs with r2 > 0.8 per gene region was only slightly lower than in non-Pygmies: 2.8% vs. 2.9% . In the Middle East and North Africa the only non-isolated population are the Palestinians; however, the proportion of SNP pairs with r2 > 0.8 per gene region was not significantly different between Palestinians and the three isolated populations . In the Central South Asian samples we compared the Kalash with all other Pakistani populations pooled together. We found significantly higher proportions of SNP pairs with r2 > 0.8 in the Kalash . In Europe, after dividing the populations in isolates  vs. non isolates  high-LD SNP pairs were 8.5% in isolates vs. 8.1% in non-isolates . In East Asia, we compared the Yakut against all other populations and found a slightly higher increase . Finally, in the Americas we compared the Surui vs. the rest and found the largest difference . In summary, we found that the proportion of SNP pairs with r2 > 0.8 was significantly higher in population isolates as compared to non-isolates in some but not all continental regions. Moreover, when analyzing the proportion of SNP pairs with r2 > 0.8 by physical distance classes between isolated and non-isolated populations within each continent, only the Kalash and the Surui displayed statistically significant larger proportions consistently for several distance bins after Bonferroni correction. It may be argued that removing SNPs with MAF < 0.05 populationwise would bias the results by dropping more SNPs in isolated populations; however, the proportion of polymorphic SNPs removed in African isolates (11.2%) was just slightly higher than in non-isolates (8.8%), as was also the case in East Asia (9.8% vs. 8.2%), while in Europe, the Middle East/North Africa, and Central South Asia, the difference was negligible .Several populations of the HGDP-CEPH panel are cultural, linguistic, or demographic isolates  to successively increasing amounts of LD in Middle East-North Africa, Central South Asia, Europe, East Asia, Oceania and America (with the highest amount of LD) [2 measure of LD between 21 685 SNPs pairs covering 211 autosomal gene regions. Moreover, we have specifically focused on those SNP pairs with high LD (so that each one can be used to tag the other) in a relevant subset of genes, as most of them may be implicated in common diseases. Note that our results may not be applied to the whole genome, but they are highly relevant to candidate-gene association studies. In this context, we have extended the previous observation that more tagSNPs are needed in the Yoruba than in Europeans or Asians [2 > 0.8 in Sub-Saharan Africans vs. 7.52% in Europeans; that is, 2.4 times as many SNP pairs showed high levels of information redundancy in Europeans than in Africans. We found a general pattern of greater LD differences between continents than within them, which would imply that genetic association studies should be more easily replicated within than between continents, as previously indicated in a set of dopamine and serotonin pathway genes [The amount of LD and the proportion of SNPs with rt of LD) ,12. Prevt of LD) ,12 or, ot of LD) ,28. A bat of LD) , outlinir Asians  to a widay genes . Similaray genes , with thay genes . Apparen2 > 0.8 per gene region when compared with the non-isolates in their respective continents. Conversely, if we take the proportion of SNP pairs with r2 < 0.8 as a rough indication of the minimum proportion of SNPs that are needed to capture the haplotype variation in a gene region, then the difference in the number of the SNPs that need to be typed in isolated populations compared to their non-isolate continental counterparts would be of 0.4% in the European isolates, 1.8% in the Yakut, 2.7% in the Kalash, and 11.3% in the Surui. Thus, genotyping costs may be slightly more economical in isolated than in outbred populations. However, association studies designed in the latter may have two practical advantatges: i) possibly larger sample sizes can be obtained in general populations, and ii) allele frequencies may be closer to those in reference HapMap populations, which allows more precise a priori statistical power calculations and prevents genotyping SNPs that can result monomorphic.A role has been suggested for genetically isolated populations in genetic epidemiology because of their predicted high levels of LD, which would facilitate the detection of genes involved in complex diseases by indirect association . In the θ = 4Neμ paramater were not significantly lower in isolated than in mainstream populations within each continent. Considering mutation rates (μ) as equal across populations, it follows that effective population sizes are not detectably lower in population isolates. A presumably reduced effective population size is indeed the condition that would increase LD in isolated populations. The levels of isolation required to decrease Ne significantly and subsequently increase LD appear to have been rare in the human demographic history, at least in the populations sampled for the CEPH-HGDP panel. Examples of isolated populations with significantly increased LD are the Kuusamo Finns [It follows, then, that being labelled a population isolate by genetic, linguistic or cultural evidence is not sufficient to harbor increased LD to a point that would justify a significant reprieve in the genotyping burden for genetic association studies. This result agrees with a separate analysis  in the Cmo Finns  and the mo Finns ; in the mo Finns , althougmo Finns ; the Surmo Finns .2 > 0.8 threshold. We have shown that, under these conditions, the SNPs that would be needed to be typed are just slightly less in isolates. It is increasingly being recognized, and we provide empirical results to that effect, that the value of isolated populations in genetic epidemiology lies not in their higher LD brought about by a presumably reduced Ne, but due to other characteristics such as large and accessible families, deep genealogical records or a low environmental variance.The present study was designed to mimic the conditions under which most genomewide association studies are performed, namely: i) focus on gene regions; ii) common SNPs, usually defined through a MAF threshold in a reference population, and iii) use of tagSNPs, often defined with a r2 greater than 0.8. These results led us to conclude that the "isolated population" label by itself does not guarantee a greater genotyping efficiency in association studies, and that properties other than increased LD may make these populations interesting in genetic epidemiology.We have explored both the levels and decay of LD with physical distance along 211 gene regions mostly related to complex disease across 39 worldwide human populations. When focusing on the populations considered to be isolates, the main result of our gene-centered approach is that these isolates do not usually show increased levels of LD as measured by the proportion of SNPs with ra priori minor allele frequency (MAF) over 0.10, which were compiled from HapMap and dbSNP databases. Additionally, coding SNPs and other functional SNPs identified using PupaSNP Finder [We analyzed a total of 2 380 SNPs covering 211 gene regions with a mean of 11 SNPs per gene region, and a mean distance of 10.79 kb between consecutive SNPs  recommended by Rosenberg . The 51 , where part of the analyses such as control for replicate samples and basic analysis such as allele frequencies, expected heterozygosity and Hardy-Weinberg equilibrium Chi-square tests were performed. Haplotypes were estimated using fastPHASE [2 [Genotype data was collected and stored in a database within the SNPator  web enviastPHASE  for eachPHASE [2  for all 2 between all possible SNP pairs within each gene region was computed for each population and continental region. In order to achieve approximate normality, the r2 variable was square-root and Box-Cox transformed (λ = 0.34) using the car  package implemented in the R programme. A nested model of covariance analysis (ANCOVA) was applied using population as a factor nested in the continent variable. Distance class (as defined above) was treated as a covariable in order to control for the effect of physical distance on LD (as measured by r2). The experimental design can be written as the following linear model:The mean ryijk is the transformed r2 value, μ is the overall grand mean of the r2 transformed value; βXij is the effect explained by the physical distance class; Ti is the effect of the ith continent ; Rj(i) is the effect of the jth population  within continent i; and εijk is the residual error associated with the corresponding transformed r2 value of the ijkth element. Significance of mean r2 comparisons between continental regions and between populations within each region were conservatively evaluated for the whole model and using the Bonferroni correction for multiple comparisons.where for a couple of SNPs (within a given gene), 2 greater than 0.8 was counted for each population and continental region. Significance of differences in the proportion of SNP pairs with r2 > 0.8 across continental regions and among populations within each geographical region for all physical distance classes was evaluated through χ2 tests; p-values were corrected for multiple testing with the conservative Bonferroni correction. The mean r2 and proportion of SNP pairs with r2 above 0.8 are available in Additional file The proportion of SNP pairs with rEB supervised the SNP genotyping analysis, contributed to the analysis and interpretation of data, and drafted the manuscript. HL contributed to part of the statistical analysis and to the interpretation of data. CMS contributed to part of the statistical analysis. AME, AFA, MG, and AR selected the gene regions under study, carried out the SNP genotyping analysis and carried out descriptive statistical analyses on the data. FCas, AN and DC participated in the design and coordination of the study and helped to draft the manuscript. JG contributed to the design of the study and to part of the statistical analysis. FCal participated in the design of the study, supervised the statistical analysis, contributed to the interpretation of data and helped to draft the manuscript. JB conceived the study, contributed to the interpretation of data, and helped to draft the manuscript. All authors read and approved the final manuscript.Populational decay of linkage disequilibrium. For each continental region, mean r2 between all possible SNP pairs within a gene region and with MAF greater than 0.05 is plotted by distance class and population. The X-axis is not to scale.Click here for fileCharacteristics of population isolates. External evidence for the cultural, linguistic, demographic, or genetic distinctiveness of the populations considered as isolates in some analyses. Abbreviations: mitochondrial DNA (mtDNA), Y (Y chromosome), Haemoglobin (Hb), Single Nucletide Polymorphisms (SNPs), Restriction Fragment Length Polymorphisms (RFLP), Short Tandem Repeats (STRs).Click here for fileList of gene regions used in this study. SNP pairs refers to all possible SNP pairs within each gene region; mean SNP pairs is the average number of SNP pairs actually used per population after dropping those with MAF < 0.05. Abbreviations: CAN, cancer-related genes; GLY, genes involved in glycosylation; IMM, genes related to pathogen recognition and/or immune response; PSY, genes involved in neurotransmission or neurodevelopment; and others, genes belonging to other diverse functional categories.Click here for fileLinkage disequilibrium parameters for each population and distance class. Mean r2 and proportion of SNP pairs with r2 > 0.8 for each population and distance class. Abbreviations: N, number of SNP pairs; 2n, maximum sample size (chromosomes).Click here for file


Liocichla steerii), fit the general vicariant paradigm for a montane organism.The subtropical island of Taiwan is an area of high endemism and a complex topographic environment. Phylogeographic studies indicate that vicariance caused by Taiwan's mountains has subdivided many taxa into genetic phylogroups. We used mitochondrial DNA sequences and nuclear microsatellites to test whether the evolutionary history of an endemic montane bird, Steere's Liocichla (We found that while mountains appear to channel gene flow they are not a significant barrier for Steere's Liocichla. Recent demographic expansion was evident, and genetic diversity was relatively high across the island, suggesting expansion from multiple areas rather than a few isolated refugia. Ecological niche modeling corroborated the molecular results and suggested that populations of Steere's Liocichla are connected by climatically suitable habitat and that there was less suitable habitat during the Last Glacial Maximum.FST indices and an admixture analysis. Though both of these results are often used to suggest conservation action, we affirm that statistically significant results are not necessarily biologically meaningful and we urge caution when interpreting highly polymorphic data such as microsatellites.Genetic and ecological niche modeling data corroborate a single history--Steere's Liocichla was at lower density during the Last Glacial Maximum and has subsequently expanded in population density. We suggest that such a range-wide density expansion might be an overlooked cause for the genetic patterns of demographic expansion that are regularly reported. We find significant differences among some populations in  Past climatic cycles have had a profound impact on the levels and distribution of genetic diversity . DespiteCandidiopotamon rathbunae) [Fejervarya limnocharis) [Gekko hokouensis) [Apodemus semotus) [Rana sauteri) [Mustela sibirica) [The subtropical island of Taiwan is an area of high endemism as well as topologic, ecologic, and climatic diversity. Taiwan first emerged from the sea following the collision of the Eurasian and Philippine Sea plates approximately 5 mya ,5. Despithbunae) , a frog ocharis) , and a gouensis)  exhibit semotus) , a ranidsauteri) , and Sibibirica) . The obsibirica) .Pycnonotus sinensis and P. taivanus, [Phasianus colchicus, [Liocichla steerii) is a montane, resident bird species endemic to Taiwan. It inhabits forest edge and secondary growth from approximately 1000 m to 3000 m. Its dispersal ability was inferred to be low [The east-west pattern of genetic division has been found in lowland birds . We also considered whether any populations of Steere's Liocichla might be of special management concern for conservation efforts.μl of blood was collected via brachial venipuncture. Blood samples were stored in lysis buffer [A total of 122 individuals was sampled from 13 populations located throughout the breeding range of the species  following the manufacturer's instructions. Sequencing was performed on an ABI 3700 automated sequencer using BigDye kit v. 3.0 according to recommended protocols (Applied Biosystems). All products were sequenced in both directions. Usually, the amplification primers were sufficient to obtain >1,000 base pairs (bp) of unambiguous sequence. However, whenever an ambiguous stretch of sequence was produced, internal sequencing primers .Whole genomic DNA was extracted using a standard phenol-chloroform protocol followed by ethanol precipitation. The mitochondrial NADH dehydrogenase subunit 2 (ND2) gene was amplified from a subset of 91 individuals representing all populations  were usGarrulax canorus). Forward primers were fluorescently labeled, and the PCR cycling protocol followed the above for all loci except GC-GATA 15, for which we used an annealing temperature of 50°C. Fragment analysis of the amplification product was conducted on a Beckman CEQ 8000 sequencer (Beckman Coulter).All individuals were also genotyped at seven microsatellite loci. Four loci were developed specifically for Steere's Liocichla  and thrS, a neutrality statistic that is particularly sensitive to population demographic expansion [μt [t is the number of generations, and μ is the mutation rate. We obtained a 95% confidence interval (CI) for τ with 1000 replicates of bootstrapping in ARLEQUIN. We tested overall genetic structure of populations with a hierarchical analysis of molecular variance , number of haplotypes (nh), and haplotype diversity (h) for each population using the program DnaSP v. 4.9 . For eacxpansion . Its sigxpansion . We usedxpansion , as implsion [μt , where τ  in ARLE  and expected (He) heterozygosities using GENEPOP 4.0.9 . To corrFST and RST values for all populations. RST is analogous to FST but incorporates a stepwise mutation model of evolution that was specifically designed for microsatellite data [ST is small [K) given the data. We performed the "clustering of groups of individuals" analysis, considering clusters to contain a minimum of three individuals. We estimated the number and composition of clusters considering upper limits of K at 2-10, 15, and 20. We ran 10 repetitions for each maximum K. Using the groups identified in the mixture analysis, we conducted an admixture analysis with 100 realizations from the posterior of the allele frequencies. We repeated the admixture five times to confirm consistent results. We also performed a "spatial clustering of groups" analysis that incorporates geographical information in the estimation of the genetic population structure [To assess genetic structure between populations, we computed pairwise ite data . Next, wite data ,37. BAPSis small . We begaRST) and geographic distance, we performed a Mantel test using GenAlEx 6 [Finally, to test for an association between genetic distance  to evaluate our molecular analyses of demographic history and population structure. Briefly, the general ENM approach applied here generates a map of the expected distribution of a species using data on the environmental conditions where it is known to occur and randomly selected background locations in the study area . We usedTo construct the model, we used 19 bioclimatic variables from the WorldClim dataset with 2.5-minute spatial resolution  and 286 To estimate the geographic distribution and extent of suitable climate for Steere's Liocichla at the LGM , we applied the contemporary ecological niche model to a set 19 bioclimatic variables generated from CCSM3 paleoclimatic model. In doing so, we used the fade-by-clamp option in Maxent version 3.2 to remove heavily clamped pixels from the final prediction. Failing to remove such pixels from the region might lead to erroneously extensive predictions of suitable climate when transferring a model to a different time period.Distribution-based niche modeling makes at least three important biological assumptions. First, it assumes that a species distribution is at equilibrium with the present-day climate conditions. Second, it assumes that the distribution of the species is not strongly influence by biotic factors (e.g. competitors or predators). Third, it assumes that the climatic tolerances of the species are conserved over the timescale for which its distribution is being modeled. If any of these conditions is violated, the extent of climatically suitable habitat could be underestimated.-8 [A total of 1026 bp was obtained from the mitochondrial ND2 gene. There were 48 polymorphic and 20 parsimony informative sites resulting in 36 unique haplotypes. Nuclear copies of mitochondrial genes are known in birds . However-8  and assuThe microsatellite dataset showed no statistically significant deviations from Hardy-Weinberg equilibrium or evidence of lineage disequilibrium. There were a total of 137 alleles, and the average number of alleles per loci was 19.6. The number of alleles per locus ranged from 9 in lsgata21 to 51 in lsgata17. Allelic richness ranged from 4.00 in Aowanda to 7.57 in Liyuan  and geographic distances (km) when straight-line geographic distances were used . However, when least-cost geographic distances were considered, the Mantel test resulted in a positive and significant correlation . This suggests that dispersal for these birds is, at least somewhat, limited by altitude.The mixture analysis in BAPS indicated the presence of two distinct groups of populations, with the Meifeng population the sole member of one group and all other populations comprising the other group. The admixture analysis assigned almost every individual to the correct group Fig. . In contThe predicted geographic distribution of Steere's Liocichla for the present-day and LGM are shown in Fig. Intriguingly, despite the many assumptions that are made when constructing distributionbased niche models, both the contemporary and LGM predictions are consistent with the findings of the molecular analyses. The model predicts that populations of Steere's Liocichla are connected by climatically suitable habitats Fig. . This reWhile we found comparatively high levels of genetic variation within populations of Steere's Liocichla, these results demonstrate a lack of genetic differentiation among populations. This is in contrast to the deep genetic divisions reported for other montane species in Taiwan. The haplotype network showed no obvious structure with respect to geography. The AMOVA indicated no genetic differentiation in either mtDNA or microsatellites between north and south groups. However, significant amounts of differentiation were partitioned among populations, implying that populations are not entirely panmictic and that dispersal among populations is likely constrained. This was true of both mtDNA and microsatellites, though the percent of variation among populations was higher for mtDNA. This is expected because mtDNA has, on average, a four times more rapid time to coalescence than nuclear loci ,48. MantNiviventer culturatus, [Sphenomorphus taiwanensis, [L. omeiensis; [A lack of genetic structure has been found in other mountain species in Taiwan . This meiensis; ) was esteiensis; ). This sS, which is particularly sensitive to departures from population equilibrium [We also find evidence for a recent demographic expansion in Steere's Liocichla. For example, Fu's Filibrium , was negFST between the Beidongyanshan population and all other populations. This is surprising because there is no reason to expect restricted gene flow to or from the Beidongyanshan population; it is centrally located, only two km from the Meifeng population, and there is adequate habitat and a continuous population of Steere's Liocichla between Beidongyanshan and Meifeng. However, caution must be taken when interpreting FST distances associated with highly polymorphic loci. Hedrick [FST distances. There is evidence that the Beidongyanshan population has recently undergone a population bottleneck and this would explain why its gene frequencies might appear to differ from the other populations.Taken separately, the results of some of our analyses could be used as evidence that some populations should be of special management concern for conservation efforts. For example, we found significant differences in microsatellite  Hedrick  has showO) relative to its expected heterozygosity (HE), as indicated by the smaller than zero, and nearly significant, FIS value  A large HO relative to HE is expected shortly after a population bottleneck because rare alleles are quickly lost, resulting in a decrease in the HE. At the same time, HO levels are relatively unaffected until a new mutation-drift equilibrium can be reached because HO is mostly dictated by common alleles. Such a bottleneck may have been caused by anthropogenic disturbance from the construction of an agricultural research station at Beidongyanshan, from typhoon related disturbance, or from stochastic fluctuations in local demography. A change in FST caused by a bottleneck is likely ephemeral, existing only until a new mutation-drift equilibrium is reached. Thus, individuals sampled from Beidongyanshan in the future may not show significant FST differentiation from other populations. We note that in such a situation, FST may reflect biologically relevant differences between populations, but this differentiation might not actually result from restricted gene flow and might be ephemeral and not of management concern. Clearly, caution should be taken before making conservation recommendations based on the results of FST alone.First, the Beidongyanshan population had the lowest amount of allelic richness. Second, it had a high observed heterozygosity . This was probably caused by an excess of private alleles in the Meifeng population. Possibly as the result of sampling error, Meifeng had an inordinate number of private alleles and all sampled Meifeng individuals had at least one private allele. This would explain why the admixture analysis was able to accurately assign Meifeng individuals to population. Some authors have suggested private alleles are important for conservation. For example, Funk et al.  defined FST indices and an admixture analysis. Though both of these results are often used to suggest conservation action, we affirm that statistically significant results are not necessarily biologically meaningful.We did not find the deep genetic divisions reported in other montane species in Taiwan. Instead, populations of Steere's Liocichla have been historically connected by gene flow, though inter-population migration appears largely confined to middle elevations. We found evidence of a demographic expansion that can be dated to the Last Glacial Maximum. Genetic diversity was high across the geographic range, indicating expansion was range-wide, rather than expansion from isolated refugia. Ecological niche modeling corroborated genetic data and suggested that habitat for Steere's Liocichla may have been of poorer quality during the Last Glacial Maximum. Both sources of data are consistent with a single historical scenario--Steere's Liocichla was at lower density during the Last Glacial Maximum and has subsequently expanded in population density. We suggest that such a range-wide density expansion might be an overlooked cause for the genetic patterns of demographic expansion that are regularly reported. We find significant differences among some populations in BM performed laboratory work, carried out analyses, and drafted the manuscript. HM conceived of the study, collected samples, and participated in the design and coordination of the study. YP collected samples, performed laboratory work, and carried out analyses. KK performed ecological niche modeling analyses and helped draft the manuscript. CY collected samples and participated in the coordination of the study. HY participated in the design and coordination of the study. All authors read and approved the final manuscript.


MC1R) is responsible for normal pigment variation in humans and is highly polymorphic with numerous population-specific alleles. Some MC1R variants have been associated with skin cancer risk.The melanocortin 1 receptor gene  than in other genes for all subjects. A large degree of population differentiation, determined by FST, was also present, particularly between Asia and all other populations, due to the p.R163Q (c.488 G>A) polymorphism. The least amount of differentiation was between the United States, Northern Europe, and Southern Europe. Tajima's D statistic suggested the presence of positive selection in individuals from Europe.Allele frequency data were compiled on 55 single nucleotide polymorphisms from seven geographically distinct human populations . MC1R.This study further quantifies the degree of population-specific genetic variation and suggests that positive selection may be present in European populations in  MC1R, MIM#155555), a member of the G protein-coupled receptors superfamily, is the major contributor to normal pigment variation in humans. It is a small, highly polymorphic gene consisting of one exon with 951 coding nucleotides on chromosome 16q24.3.Differences in human skin pigmentation have been attributed to genetic variation in several different genes -3. AmongMC1R variants and red hair, light skin, poor tanning ability and heavy freckling [MC1R SNPs in hair, eye, and skin pigmentation[MC1R variants are also associated with increased risk of malignant melanoma in a variety of populations [MC1R polymorphisms in melanoma risk appears to extend beyond its effect on pigmentation in most of these investigations, and to be linked to melanomas harboring mutations in the BRAF oncogene[Numerous studies have demonstrated associations between specific reckling -9. A recmentation. The funmentation-13. Seveulations -22 The e oncogene.MC1R, or selection for functionally relevant variants that led to lighter skin pigmentation occurred[Several hypotheses have been generated in an effort to understand the evolutionary history of skin pigmentation in humans. It has been suggested that as humans migrated out of Africa to climates with more limited exposure to sunlight, relaxation of functional constraints in pigmentation genes, including  occurred. This co occurred-27. It h occurred. In addi occurred.MC1R, in the form of single nucleotide polymorphisms (SNPs), is significantly different across populations from different geographic regions [MC1R is an exception to this observation. It has been shown to be more polymorphic in individuals of European descent than in those from Africa [MC1R from populations around the world, further quantified the large differences in the distribution of variants across populations, with a prominent difference between light and dark-pigmented individuals [MC1R genetic variation by characterizing nucleotide diversity, population specific differentiation (FST), and to study measures of selection.Genetic variation of  regions ,30. In m regions ,32. MC1Rm Africa ,30. A coividuals . The goaMC1R in these analyses. AFs are shown in Table Allele frequency (AF) data was compiled from a total of 2306 individuals who were grouped into seven populations, based on geographic location Table . The actMC1R, as measured by π, was 10.1 × 10-4 for all populations ; it ranged from a low of 3.6 × 10-4 in subjects from India to a high of 11.1 × 10-4 from US subjects (Table -4). θ, the population mutation parameter, was quite variable across populations, ranging from 6 × 10-4 in India to 47.3 × 10-4 in Southern Europe. In Southern European subjects, θ was the highest, 43.6 × 10-4, in Italy.In order to correct for the variable population sizes, which could contribute to the absolute number of SNPs identified, π and θ, measures of nucleotide diversity, were calculated. The overall nucleotide diversity for all SNPs in MC1R genetic variation . As was seen for all SNPs, θ for NS SNPs was the highest in Italy (36.6 × 10-4).Nucleotide diversity was also calculated for NS SNPs only to further understand the contribution of these SNPs to on Table . π in NSMC1R has a higher degree of nucleotide diversity in comparison to other groups of genes  than in other sets of genes .In addition to a high degree of inter-population variability, es Table . StudiesST statistic, a pairwise measure of population differentiation, has been used extensively to compare the degrees of heterozygosity across populations[MC1R FST was calculated for each of the described populations  to 0.232 (Southern Europe). Papua New Guinea had relatively modest degrees of population differentiation when compared to all populations except Asia, where it was very large. The least amount of population differentiation was found in comparisons between the United States, Northern Europe, and Southern Europe.ST values for all of these comparisons were between 0 and 0.03, suggestive of little differentiation (data not shown).We also performed analyses on the subpopulations that comprised the Northern Europe  and the Southern Europe  groups. FMC1R locus, is an area of active investigation. Previous work suggested that the high degree of variation in MC1R is not due to selection but rather to a relaxation of functional constraint outside of Africa [Several studies have identified signals of positive selection in pigmentation genes in subjects from East Asia, and Europe but not from those in Africa,38,39. Wf Africa . In ordeS test of neutrality developed by Fu (1997) and based on θ is a powerful method to further evaluate the polymorphic patterns under population growth and genetic hitchhiking. These values are shown in Table S was statistically significant in the grouped Southern European population  but only in the subpopulation from Spain . The Northern European population group also had a statistically significant FS value . The FS values were not statistically significant in the other populations. Fu and Li's D values were comparable in scope to Tajima's D in this study but p-values were not obtainable due to software limitations (data not shown).The FMC1R is a small, highly variant gene. This study evaluated the nucleotide diversity, population-specific differentiation, and tested for positive selection of MC1R based on a compilation of previously published data. We used allele frequencies from studies that reported sequencing the entire gene and used the 951 coding bp of MC1R as the reference sequence. The genotype data used for these analyses was derived from the sample size and reported AFs, as a result, we were unable to assess the haplotype structure of MC1R. Also, we could not assess the flanking sequences of MC1R because the exact regions sequenced were not reported in the published data.MC1R than in other groups of genes. It should be noted that these differences may be somewhat skewed because our study evaluated only one, small gene, MC1R, while the other studies evaluated between 7 and 4950 different genes [MC1R . Overal history . This al history .ST statistic was calculated to further assess the degree of population-specific differentiation in MC1R. Numerous population-specific SNPs and an overall high degree of population differentiation, as measured by FST between populations, are present in MC1R, particularly in Asians. Minimal differentiation was noted between Southern Europe, Northern Europe, and the United States. These individuals were identified in previous studies as Caucasian (i.e. of European descent), and likely share some degree of common ancestry. Although frequency of specific variants differ across populations of European descent , the sun/Ireland,27,30. Tpulations.MC1R for the presence of selection. These data, which are based on the very large sample size described herein, suggest that positive selection may be present in the Southern European population as a whole, as well as in its three subgroups, Greece, Italy, and Spain, based on the presence of negative Tajima D values and statistically significant p-values. The data also suggest the presence of some degree of positive selection in the Northern European population; but only the subgroup from the Netherlands had statistically significant p-values. It should be noted that Tajima's D statistic assumes that all nucleotides are equally mutable, subject to the same population dynamics, and can be misleading if a significant population bottleneck occurred. We also used Fu's FS test to address the presence of positive selection versus neutral evolution of MC1R. This was statistically significant only in the Southern and Northern European groups and the subpopulation from Spain and further suggests the presence of positive selection in the European populations. However, our data are also limited because we were only able to study the 951 bp of coding sequence in MC1R and were not able to assess the larger genomic region.Tajima's D statistic tests whether or not a gene or genomic region is evolving randomly  or if the region is under selection . It is based on the spectrum of AFs at different sites, as well as on population size. Tajima's D statistic was used to test MC1R gene for evidence of positive selection with conflicting results. Some studies suggested that purifying selection is present in Africa and that relaxation of functional constraint in non-African populations, instead of positive selection, is present[et al [DCT gene among individuals of Chinese ancestry. In their study, MC1R interpretations were limited because of the different SNPs genotyped between the Perlegen and HapMap data sets studied. In a study of 118 putative skin pigmentation genes, data were consistent with positive selection in subjects from Europe  and in Asians [MC1R could not be evaluated in that study due to ascertainment criteria. It was also suggested that at least weak, recent positive selection may be present in MC1R, based on the AF variability between CEPH Utah and East Asian HapMap samples[MC1R may be under positive selection in some populations, although additional studies are needed to further evaluate this finding.Several studies have evaluated genetic adaptation of the s present,27,40. Oand DRD2). Unfortup samples. Our datMC1R genetic variation, illustrates the complexity of this variation across numerous populations, and suggests that positive selection plays a role in European populations. Understanding of population-specific genetic variation in MC1R and the role it plays not just in skin pigmentation, but sun sensitivity and melanoma risk, has the potential to impact clinical care and public health.This study further quantifies the degree of MC1R SNPs from populations around the world were compiled as described [MC1R in distinct populations. From the studies included in Gerstenblith et al [MC1R variants in individuals from Spain was also included [AF data of escribed , from twth et al , we restth et al ,27,40-47th et al ,49, thatth et al , that weth et al , or to eth et al . In addiincluded . PopulatMC1R sequence (NM_002386) was used as the template. Since data files were created from AFs, we were unable to assess the haplotype structure of MC1R. Analyses were performed that were not dependent on haplotype structure. Nucleotide diversity (π), the average number of nucleotide substitutions per site between two sequences, was calculated with the Jukes and Cantor correction [ST[S test[MC1R values [Genotype data files were created from AFs for each population Table  and wererrection . Theta (rrection . Geneticction [ST. ArlequiST[S test under a ST[S test,56. OtheR values -36.MC1R, analyzed the MC1R frequency in each population, summarized all data for the population genetics analyses, and participated in data analysis and interpretation. AMG participated in the study design and oversaw statistical analyses and data interpretation; LM participated in data analysis and interpretation. MCF and KP participated in the study design and data interpretation. MTL was responsible for the conception and design of the study, and oversaw data analysis and interpretation. All authors contributed to the manuscript's writing and review.SAS participated in the study design, performed all the population genetics analyses, data interpretation, and wrote the manuscript. MRG reviewed all manuscripts related to


Assessing genetic biodiversity and population structure of minor breeds through the information provided by neutral molecular markers, allows determination of their extinction risk and to design strategies for their management and conservation. Analysis of microsatellite loci is known to be highly informative in the reconstruction of the historical processes underlying the evolution and differentiation of animal populations. Guadarrama goat is a threatened Spanish breed which actual census (2008) consists of 3057 females and 203 males distributed in 22 populations more or less isolated. The aim of this work is to study the genetic status of this breed through the analysis of molecular data from 10 microsatellites typed in historic and actual live animals.ST of 0.074, GST of 0.081 and RST of 0.085. Percentages of variation among and within populations were 7.5 and 92.5, respectively. Bayesian clustering analyses pointed out a population subdivision in 16 clusters, however, no correlation between geographical distances and genetic differences was found. Management factors such as the limited exchange of animals between farmers (estimated gene flow Nm = 3.08) mostly due to sanitary and social constraints could be the major causes affecting Guadarrama goat population subdivision.The mean expected heterozygosity across loci within populations ranged from 0.62 to 0.77. Genetic differentiation measures were moderate, with a mean FGenetic diversity measures revealed a good status of biodiversity in the Guadarrama goat breed. Since diseases are the first cause affecting the census in this breed, population subdivision would be an advantage for its conservation. However, to maintain private alleles present at low frequencies in such small populations minimizing the inbreeding rate, it would necessitate some mating designs of animals carrying such alleles among populations. The systematic use of molecular markers will facilitate the comprehensive management of these populations, which in combination with the actual breeding program to increase milk yield, will constitute a good strategy to preserve the breed. Before the intensification and industrialisation process of the last decades, European livestock farming was generally extensive and closely linked to the use of farmland. This is still the case for small ruminants and especially for goats, where not only local breeds do not benefit from modern breeding techniques but they also are about to disappear. Thus, the decline of local breeds and their production systems are raising concern about the importance of European agro-ecosystems and cultural landscapes maintenance. The goat is among the earliest species to be domesticated . Goats aHowever, in contrast to high productive foreign goat breeds, most local breeds are not subject to breeding programs to improve production traits, which would increase their genetic ability for productivity and consequently their profitability. Due to the extensive conditions of animal management, existing breeding strategies applied to local breeds are constrained by poor pedigree recording. In this way, the lack of pedigree records can lead to both a limited genetic progress for the selected trait and a suboptimal inbreeding control. The use of highly variable molecular genetic markers, such as microsatellites, is one of the most powerful means for studying genetic diversity and pedigree reconstruction because of their high degree of polymorphism, random distribution across the genome and neutrality with respect to selection -5.Guadarrama goat, a rustic breed which has been exploited in mountain areas in the centre of Spain since XVIII century, constitutes a good illustrative example. Guadarrama goat is a threatened breed whose actual population consists of 3057 females and 203 males distributed in 22 herds, which correspond to an effective population size of 763 individuals considering only the unequal number of males and females [This breed is mainly used for milk production, although it is also exploited in a local meat industry. Since 1998 Guadarrama breeders association has established a breeding program to increase milk yield. There are a high degree of disconnectedness among the herds of this breed and scarce pedigree information due to the low spread of the artificial insemination. Therefore, genetic evaluations of animals for milk yield are only comparable at the intra-herd level. For this reason, a pilot project for pedigree reconstruction based on molecular markers information (10 microsatellite) has been established in 2003 as an alternative to pedigree recording.The aim of this work is to evaluate the genetic status of the Guadarrama goat breed making use of the molecular information generated in the breeding program analyzing both, their genetic diversity and its population structure or subdivision using clustering methods.Fresh blood samples were obtained between years 2004-2007 from 6635 goats pertaining to 20 different herds, which constitutes the whole animals of the Guadarrama breed. Figure ® 310 Genetic Analyzer (Applied Biosystems).Ten microsatellite markers were studied: ILSTS005 , CSSM31 F-statistics FIS, FST, and FIT [ST ≈ 1/(1+4Nm) assuming genetic markers neutrality and an island model. Heterozygosities, mean number of alleles across populations, FIS within populations and Gst were calculated with GENETIX 4.03 software [ST values for pairwise comparisons of the 20 Guadarrama goat herds and their significance level for genetic differentiation and Rst were tested with FSTAT. Significance levels were set using the sequential Bonferroni correction . GENCLASS 2.0 package [ST values and the geographic distances between populations. The population structure was analyzed by cluster techniques with the software STRUCTURE 2.1 [ST mean values for each cluster were also estimated. BAPS was run setting the maximum number of cluster at 20. Results were based on 50 simulations from the posterior allele frequencies. Finally, locus by locus AMOVA analysis considering groups and populations as sources of variation was assessed by ARLEQUIN 3.1 software package [The expected heterozygosity corrected for sampling bias , the obs and FIT  jackknif and FIT . Gene fl and FIT  FST ≈ 1/software . Further package  under a  package  was used package  was perfTURE 2.1  and BAPSTURE 2.1 ,27. Due TURE 2.1 , STRUCTU package .A total of 170 alleles were detected at the 10 microsatellite loci assessed in the 6335 goats genotyped. Table F-statistics and gene flow (Nm) for each locus across the 20 herds of Guadarrama goat breed. Mean values of FIS and FST across loci were 0.023 and 0.074, respectively.Table Results of the Fisher's exact test for Hardy-Weinberg (HW) equilibrium across loci and populations, considering the heterozygote deficit as the alternative hypothesis, are shown in Table IS estimates within the 20 Guadarrama goats populations, are shown in Table IS, was positive in most populations when averaged across loci, raging from -0.015 (population 10) to 0.066 (population 18). Average value of FIS across loci and populations was 0.022.The mean number of alleles across loci, the mean observed and expected heterozygosities and the FST values of pair-wise comparisons among the 20 herds (matrix not shown) of Guadarrama goats, showed an overall genetic differentiation FST of 0.074 (SD 0.011) and pair-wise FST values ranging from 0.027 (pop13 vs. pop19) to 0.165 (pop12 vs. pop20). Significant (α = 0.05) genetic differentiation was found after sequential Bonferroni correction  in 92 out of 190 population pairs.FST analysis. The marker MCM53 showed two private alleles, which were present only in population 15. The marker BM8125 had only one private allele found in population 1. On the other hand, populations 4, 12, 14 and 17 did not show private alleles at any microsatellite analyzed.Results from GENECLASS assignment test revealed that about 77% of the animals were assigned to the population they were collected from. The higher percentages (91.2% to 97.2%) of individuals assigned to its original population occurred in populations 7, 10, 15, 18 and 20 while the lower correspond to populations 4 (68.2%) and 19 (69.0%). Assignment of individuals was consistent with the extent of genetic divergence reported in the FST values and the geographical distances .The Mantel test including the 20 populations of Guadarrama goats and the relative distances among them Figure , depicteFigure Estimated α values averaged 0.04 for K varying from 2 to 19, indicating that most individuals were essentially from one population or another. However, for K values ranging from 20 to 23, α values varied from 1.50 to 2.50 indicating that most individuals were admixed.Using BAPS package the highest likelihood was also obtained with K = 16 .ST in each of the 16 inferred clusters, for the high estimate of L(K)  among the 15 replicates ran for K = 16. Clusters 2, 7, 11 and 12, had moderate to high proportions of members from two of the original populations. Clusters 11 and 12 were essentially a mixture of animals from populations 5 and 14 and populations 7 and 19, respectively. Cluster 5 seems to be the most heterogeneous group, containing moderate proportions of animals from populations 1, 9, 2 and 6.Table ST) and of the allele size (Rst) among groups, among populations within groups and within populations were estimated. In both cases, the highest percentage of variation (92-93%) corresponded to the within population component. Components among groups and among populations within groups showed low and similar magnitudes (3-4%).Table In order to maintain genetic diversity, breeding strategies that increase effective population size minimizing genetic drift effect should be implemented. Microsatellite markers in combination with recent statistical methodologies represent a useful tool for the conservation and management of endangered breeds.A breeding program focused on improving Guadarrama goats milk yield has been carried out in Spain since 1998. In the present work, the actual situation concerning genetic diversity and population structure of this breed has been evaluated using the molecular information derived from 10 microsatellites loci and the use of clustering methods.The total number of alleles per locus in the present study ranged from 9 to 36. This fact suggested that all markers used were appropriated to analyze genetic diversity in this breed. A more appropriate measure of genetic variation within a population was gene diversity (average expected heterozygosity). Gene diversity estimated in this breed was 0.70, which was in the range (0.3 to 0.8) to be useful for measuring genetic variation . This vaST = 0.074) in the Guadarrama goat population were detected. Similar FST value was found in a large analysis [Although only a seven percent of the total genetic variability could be attributed to differences among subpopulations, evidences of a moderate genetic subdivision   and usinF-statistics results, subdivision processes more than inbreeding (average FIS across loci was 0.022 ± 0.017), could be the cause of the observed genetic differences between populations. Furthermore, populations analyzed were not in HW equilibrium, as it is revealed by the smaller observed than expected heterozygosity. The heterozygote deficiency is probably reflecting a subdivided population structure (Wahlund effect) rather than selection against heterozygotes.Genetic differences were not correlated with geographic distances among populations (Mantel test) therefore management factors such as the limited exchange of animals between farmers mostly due to sanitary, social and cultural reasons could constitute the major causes affecting Guadarrama goat population subdivision. In this breed tuberculosis and paratuberculosis are the main causes of mortality and culling. These kinds of diseases have high prevalence in the affected herds. Thus, subdivision would be an advantage preserving the breed from the dissemination of such diseases. Reproductive isolation, consequence of the local use and management of the breed, reduces the effective population size and contributes to the genetic subdivision. Considering Wright' In this work we have demonstrated that Guadarrama goat genetic diversity is still conserved. Management factors such as the limited exchange of animals between farmers (estimated gene flow Nm = 3.08) could be the major causes affecting Guadarrama goat population subdivision. Since diseases are the first cause affecting Guadarrama goat census, population subdivision would be an advantage for the conservation of the breed. In such cases, additional constraints, such as the minimum levels of contribution of each population should be included in the conservation strategy . To mainMSN has developed the design of the work, the statistical analyses and wrote the manuscript. JHC has contributed in markers selection and genotyping design, and in manuscript revision. MM has made the animal genotyping and has contributed in the paper discussion. AMC has collaborated in manuscript writing, and results interpretation. FJC has contributed in animal genotyping. CC has collaborated in some technical aspects. JJJ has contributed in the statistical analyses. PDT is the breeder association secretary and has developed the sample collection.All authors have been read and approved the final manuscript.


Oncorhynchus tshawytscha) is the time at which adults migrate from the ocean to breed in freshwater. We investigated whether allelic variation at the circadian rhythm genes, OtsClock1a and OtsClock1b, underlies genetic control of migration timing among 42 populations in North America. We identified eight length variants of the functionally important polyglutamine repeat motif (PolyQ) of OtsClock1b while OtsClock1a PolyQ was highly conserved. We found evidence of a latitudinal cline in average allele length and frequency of the two most common OtsClock1b alleles. The shorter 335 bp allele increases in frequency with decreasing latitude while the longer 359 bp allele increases in frequency at higher latitudes. Comparison to 13 microsatellite loci showed that 335 and 359 bp deviate significantly from neutral expectations. Furthermore, a hierarchical gene diversity analysis based on OtsClock1b PolyQ variation revealed that run timing explains 40.9 per cent of the overall genetic variance among populations. By contrast, an analysis based on 13 microsatellite loci showed that run timing explains only 13.2 per cent of the overall genetic variance. Our findings suggest that length polymorphisms in OtsClock1b PolyQ may be maintained by selection and reflect an adaptation to ecological factors correlated with latitude, such as the seasonally changing day length.A critical seasonal event for anadromous Chinook salmon ( Arabidopsis, Oryza, timeless, has been shown to affect the incident of diapause, a seasonal event in Drosophila (Many organisms use day length (photoperiod) as an environmental cue to regulate seasonal changes in behaviour, growth, development, reproduction, dormancy and migration . In planOncorhynchus tshawytscha) is the time at which adults migrate from the ocean to their natal rivers to breed. Intra-annual variability in both water flow and temperature limits access to discrete spawning habitats within these rivers, and consequently, locally adapted migratory populations have evolved low inter-annual variability in their seasonal return time. Thus, the timing of this event is primarily an adaptation to long-term average conditions rather than a proximate response to current conditions. Photoperiod is a stable, long-term environmental cue that fishes could use to coordinate their population-specific migratory runs with these seasonally varying conditions  as potential candidate loci for migration time in Chinook salmon  of the Pacific Salmon Commission . Average allele length is defined as the sum of all allele lengths of individuals in a given population divided by the total number of individuals in that population. To determine if regression coefficients for the candidate gene alleles differed significantly from neutral expectations, we compared these values to a distribution of regression coefficients  for 441 alleles (13 microsatellite loci) from the CTC microsatellite baseline dataset.Calculations of allelic frequencies were performed using p v. 3.3 . We calc v. 4.02 . AssociaFst, allele frequency, average allele length) to geographical distance . Mantel tests were performed with 10 000 permutations using the software program Passage  is primarily characterized by the insertion and deletions consisting of both glutamine (Q) and proline (P) repeats  revealed a significant association between frequency and latitude albeit in the opposite direction. The short 335 bp allele increases in frequency at lower latitudes  while the long 359 bp allele increases in frequency at higher latitudes  .We found a significant association between average allele length of t=0.013) a,b.To test for departure from neutral expectations, we calculated the regression coefficient of allele frequency on latitude for each of the 441 microsatellite alleles and compared these values to those for the two candidate alleles. The regression coefficients for 335 bp (−0.016) and 359 bp (0.012) were exceeded by less than 1 per cent of the microsatellites.OtsClock1b alleles further, we performed partial Mantel tests of association and compared allele frequency (335 and 359 bp), average allele length and pairwise Fst estimates to latitude and longitude. We found a significant positive association between all three measures of genetic distance  and latitude  and short PolyQ alleles (335 and 338 bp) in southern populations.We discovered an analogous pattern of geographical distribution for the two less common length variants (338 and 383 bp). The short 338 bp allele is only present in 12 populations located south of 48° latitude while the long 383 bp allele is present in 9 populations located north of 48° latitude. One exception to this distinct geographical separation occurs in the Umpqua River, where two individuals have a 383 bp allele. In summary, the overall trend in Fst estimate among the 42 populations based on OtsClock1b was 0.142 while the overall Fst estimate based on microsatellite loci was 0.073. We found no significant association between pairwise Fst estimates based on OtsClock1b allele frequency and pairwise Fst estimates based on microsatellite allele frequencies .The overall OtsClock1b PolyQ variation showed that genetic variance related to run timing was 0.056, indicating that run timing explained 40.9 per cent of the overall genetic differences among populations. Genetic variance related to differences among populations was 0.065 (47.4%) while the remaining 0.016 (11.7%) resulted from differences among regions. By contrast, a hierarchical gene diversity analysis of 13 microsatellite loci revealed that genetic variance related to run timing was 0.010, indicating that run timing only explained 13.2 per cent of the overall genetic differences among populations. Genetic variance related to differences among populations was 0.057 (75.0%) while the remaining 0.0096 (11.8%) resulted from differences among regions.A hierarchical gene diversity analysis of Clock PolyQ domain in natural populations  while the shorter variant was only present in southern populations (less than 48°). We hypothesize that the observed clinal variation in OtsClock1b PolyQ alleles could reflect an adaptation to photoperiodic parameters correlated with latitude as the seasonal variation in day length is more pronounced in Alaska compared to California. A recent study by Clock PolyQ domain , which the authors propose may be indicative of local adaptation to latitudinal gradients in the seasonal rate of change of photoperiod.Clock PolyQ domain modifies the amplitude of the pacemaker which, in turn, can have significant effects on the entrainment behaviour of organisms to light and other resetting stimuli. For Chinook salmon, the duration of the daily photoperiod throughout the breeding season is steadily increased as one moves north. Natural length variation in the OtsClock1b PolyQ may also modify the circadian pacemaker amplitude such that selection for the conservation of pacemaker amplitude during the breeding season may produce the observed latitudinal cline in OtsClock1b allele frequency.OtsClock1b PolyQ alleles as has been demonstrated for the threonine–glycine-encoding (Thr–Gly) repeat region of the Drosophila circadian rhythm gene, PERIOD 20 and (Thr–Gly)17, with the longer allelic variant predominating in the northern Europe and the shorter variant predominating in the southern region 20 variants are not sensitive to changes in temperature and show, on average, a period slightly shorter than 24 hours. Therefore, the two major (Thr–Gly) variants appear to be adapted to the thermal environments in which they predominate, (Thr–Gly)17 in southern and (Thr–Gly)20 in northern Europe.Other ecological factors correlated with latitude may also contribute to the clinal variation in OtsClock1b PolyQ variants as has been documented for the Drosophila Period gene. However, the relationship between the Clock gene and temperature has yet to be explored in any model organism.Temperature is a critical environmental variable particularly during the freshwater stages of Chinook salmon life history. Seasonal fluctuations in temperature can create thermal barriers to salmon migrating to freshwater spawning habitats . ConsequThe primary aim of this study was to investigate whether there was any association between variation in allele frequency and migration timing among Chinook salmon populations along the west coast of North America. Considering the clinal variation in run time diversity, one would predict increasing gene diversity in the southern populations. We, however, found no evidence for this trend. In fact, California's Trinity autumn and Trinity spring populations were essentially fixed for the 335 bp allele .Fst values based on OtsClock1b were not significantly associated with those based on microsatellites indicating that they do not reflect similar patterns of population structure and/or history. Thus, while we cannot rule out the possibility that the spatial distribution of OtsClock1b allelic variants is influenced by historical separations, our findings provide evidence for clinal variation probably maintained by selection.Pairwise OtsClock1b PolyQ variants revealed that almost half of the overall genetic variance is related to run timing (40.9%) with the remaining variation attributed to differences among regions and populations . These results contrast sharply with those obtained for assumed neutral microsatellite loci, which indicate that run timing explains only 13.2 per cent of the overall genetic variance. Our microsatellite findings are in accord with those reported by The hierarchical gene diversity analysis of eight OtsClock1b allele frequencies . Therefore, the large percentage of genetic variance attributed to run timing is likely a reflection of the strong latitudinal cline in allele frequency as primarily summer runs inhabit the northern range of this species where average allele length is large. To test this, we excluded the summer run populations and reanalysed data from 20 autumn and spring run populations in Oregon, Washington and California and found that the overall genetic variance related to run timing decreased to 20 per cent while the differences among populations increased to 79 per cent. Thus, other environmental factors or life-history traits correlated with latitude probably confound the analysis.We discovered several examples where temporally divergent runs within a river have similar Clock genes in Chinook salmon show distinct patterns of length variation in the functionally significant PolyQ domain. In contrast to the highly conserved PolyQ of OtsClock1a, we identified eight length variants of the OtsClock1b PolyQ domain. Based on the OtsClock1b PolyQ variation, we found evidence for a latitudinal cline in average allele length and frequency as well as a large component of genetic variance explained by run timing. These results contrast sharply with those obtained for presumed selectively neutral microsatellite markers suggesting that the observed variation may be maintained by selection and reflect an adaptation to ecological factors correlated with latitude such as photoperiod.Duplicated


Modern cattle originate from populations of the wild extinct aurochs through a few domestication events which occurred about 8,000 years ago. Newly domesticated populations subsequently spread worldwide following breeder migration routes. The resulting complex historical origins associated with both natural and artificial selection have led to the differentiation of numerous different cattle breeds displaying a broad phenotypic variety over a short period of time.This study gives a detailed assessment of cattle genetic diversity based on 1,121 individuals sampled in 47 populations from different parts of the world  genotyped for 44,706 autosomal SNPs. The analyzed data set consisted of new genotypes for 296 individuals representing 14 French cattle breeds which were combined to those available from three previously published studies. After characterizing SNP polymorphism in the different populations, we performed a detailed analysis of genetic structure at both the individual and population levels. We further searched for spatial patterns of genetic diversity among 23 European populations, most of them being of French origin, under the recently developed spatial Principal Component analysis framework.Overall, such high throughput genotyping data confirmed a clear partitioning of the cattle genetic diversity into distinct breeds. In addition, patterns of differentiation among the three main groups of populations—the African taurine, the European taurine and zebus—may provide some additional support for three distinct domestication centres. Finally, among the European cattle breeds investigated, spatial patterns of genetic diversity were found in good agreement with the two main migration routes towards France, initially postulated based on archeological evidence. Bos taurus) and humped zebuines (Bos indicus) i.e. between the Mediterranean sea and Iran) from the wild extinct aurochs Bos primigenius primigenius and a second separate one which lead to zebus in the Indus valley (including Rajasthan and present day Pakistan) from the wild extinct aurochs B. p. namadicusB. p. opisthonomousB. p. primigenius), which had been present until the Middle Age, and domestic stocks might have occurred to a substantial extent As part of the Neolithic agricultural revolution, the domestication of cattle, which occurred about 8,000 years ago, changed the social and economical life of most human populations e.g.The recent advent of high-throughput and cost effective genotyping techniques makes it possible to provide a detailed genome wide assessment of the genetic structure and relationships among cattle populations. This might in turn allow to refine previous pioneering works usually performed on a small number of genetic markers e.g.. We prese.g. European and African taurines or zebus). Interestingly, populations of hybrid origin displayed generally higher levels of polymorphism than their population of origin as exemplified for i) the two synthetic breeds, BMA and SGT, which result from crosses between European taurines and zebus We first performed a joint analysis based on SNP data generated for all 1,121 individuals representing 47 different populations  genotyped for 44,706 SNPs from this study and three previously published studies see  to provie.g.2r dropped quickly toward its asymptotic value when physical distances were above 200 kb. In our data set, SNP genome coverage was homogeneous with a very small proportion of inter-marker distances less than 20 kb  based on all available SNP information allowing to refine and extend previous reports Besides, the neighbor-joining (NJ) tree based on Allele Sharing Distances (ASD) unambiguously separated individuals according to their population of origin  as confifrappee.g. K = 10) while the infusion of zebus in SGT and BMA was of South American origin consistent with recent historical data We finally performed a model-based unsupervised hierarchical clustering of the individuals using the program Finally, individuals belonging to some breeds such as HOL (and HO2), ANG (and RGU), HFD, BSW (and BRU) tended to be clearly assigned to a single cluster when K = 10. This latter trend was further confirmed for most populations  when increasing the number of inferred clusters toward the number of populations (K = 47).STF across populations was found equal to 0.190 with an average ISF almost null (−0.007) leading to an ITF of 0.185. Note that within all populations, ISF were also found close to zero  although moderately negative values (<−0.1) were observed for ND2 STF computed for each pair of populations  among the two analyses showed that genetic variability between populations explained 32.8% of the whole genetic variability .We thus decided to extent the PCA described above by performing a so-called between-class PCA e.g., classesWe finally constructed a neighbor-joining (NJ) tree based on Reynolds genetic distances relating the 47 cattle populations and including American Bison (OBB) as a rooting outgroup . Given tOverall, these different analyses support a global partitioning of world-wide cattle diversity into EUT, WAT and ZEB. Although not excluding the possibility of strong founder effects, this might suggest three distinct domestication events Within EUR, grouping of the breeds on the NJ tree  was striOverall, among European cattle, both NJ tree and PCA results suggested strong spatial patterns of genetic diversity. Hence, as pioneered by Cavalli-Sforza and collaborators for the reconstruction of the early history of human populations PCA does not explicitly incorporate geographical information because the optimization criterion relies on the maximization of the genetic variance. Thus, PCA may fail to detect spatial structuring if this is not associated with the most pronounced genetic differentiation. Recently, after the works of I1PCAI = 0.73 and 1sPCAI = 0.87). Hence the first axis in both PCA and sPCA unambiguously captures global spatial patterns while separating populations according to a North/South gradient (2PCAI = 0.14 and 3PCAI = 0.06) while the second and third sPCA axes displayed a Moran's I above 0.5 (2sPCAI = 0.63 and 3sPCAI = 0.80). This suggested that PCA might fail to identify relevant spatial patterns on these additional axes making it difficult to interpret the underlying variance in terms of geography. We thus focused subsequently on the first three sPCA axes (22sPCAI = −0.63) displayed a relatively high negative spatial autocorrelation suggesting a strong local spatial pattern. This axis actually separated JE2 and GNS (data not shown) which are closely geographically related (Channel Islands) but most probably because of complete isolation of JER since the 18th century As detailed in gradient . NeverthPCA axes . Note thThe coordinates of each breed on the first three sPCA axes were synthesized on th century had been extensively reported in MAN. The grouping of the NOR with the Channel Islands and Northwestern France breeds (ii) used to be more controversial The grouping of French breeds is mostly in agreement with previous classification based on historical data, morphological characters (mostly craniometric and morphometric data), geographical proximity e.g. Southern Europe, Northern Africa, India or Middle East) may provide additional useful insights to draw a more precise picture of the genetic history of cattle. As exemplified in the present study, such extension is straightforward because of the easy to share nature of SNP data and the widespread use and cost effectiveness of the Bovine SNP50 genotyping assay.More generally, including data for populations from other parts of the world . Based on available pedigree information, every attempt was made to ensure that samples were typical of the breeds and to limit relationships. In addition, genotyping data were also included for 33 other breeds and 4 american bisons (Bison bison) For the purpose of this study 296 individuals belonging to 14 different French cattle breeds were genotyped on the Illumina BovineSNP50 chip assay http://cran.r-project.org/web/packages/qvalue/index.html). A total of 585 SNPs exhibiting q-value<0.05 in at least one breed were then discarded from further analysis. Thus, 44,706 SNPs were finally considered for the study leading to an average marker density of 1 SNP every 56.9 kb over the genome , sPCA aims at finding independent synthetic variables that maximize the product of the genetic variance and spatial autocorrelation measured by Moran's IX'(L+L')X where L synthesizes spatial structure among populations via a neighboring graph (in our study a Gabriel neighboring graph was chosen) connecting the populations on the geographical map IsPCA was carried out on a between-breed level using the R packages ijx where ijx represents the proportion of alleles alike in state averaged over all genotyped SNPs. A neighbor-joining tree ASD were computed for each pair of individuals using all available SNP information by a simple counting algorithm: for a given pair of individuals i and j, ASD was defined as 1-frappe. The program was allowed to run for 10,000 iterations, with pre-specified numbers of clusters varying from K = 2 to K = 47 (number of distinct populations). Convergence of the algorithm was empirically assessed by considering estimated cluster membership and data likelihood. Graphical displays of the results were done with the program DistructUnsupervised hierarchical clustering of individuals based on SNP genotyping data was performed using the maximum likelihood method described in F-statistics ITF, STF and ISF were estimated respectively in the form of F, θ and fISF was derived from the average of these two quantities over all the SNPs. In order to evaluate the reliability of the ISF estimates we computed the mean and standard deviation over 10,000 samples of 5,000 randomly chosen SNPs.The global Figure S1Decay of average pairwise r2 with inter-marker distance for the different populations.(0.05 MB PDF)Click here for additional data file.Figure S2http://genome.ucsc.edu/).Distribution of inter-SNP physical distances based on the Btau_4.0 bovine genome assembly ((0.00 MB PDF)Click here for additional data file.Figure S3Between breed PCA for the 47 different bovine populations. Populations are plotted according to their coordinates on the first two (A) and first and third (B) principal components on the eigenanalysis.(0.12 MB PDF)Click here for additional data file.Figure S4Neighbor-Joining tree relating the 47 cattle populations and American bisons (OBB) outgroup based on Reynolds genetic distances computed using allele frequencies at 27,527 SNPs polymorphic (MAF>0.01) in at least two zebus, two WAT and two EUR breeds. Reliability of the nodes (percentage over 100 Bootstrap samples) are indicated for each node.(0.05 MB PDF)Click here for additional data file.Figure S5Gabriel neighboring graph modeling the spatial structure of breeds projected on the geographic map.(0.03 MB PDF)Click here for additional data file.Figure S6sPCA results. Projection of the breed coordinates on the first (A), second (B) and third (C) sPCA principal components onto the geographical map. The area of the square is proportional to the absolute value of the score while the color of the square (black or white) corresponds to its sign (positive or negative). D) 3D representation of the breed coordinates on the first three sPCA principal components (breed names are colored according to the synthetic score obtained in (0.10 MB PDF)Click here for additional data file.Table S1Sample Description. The land of origin, country of sampling  and type of the populations  are indicated, together with the number of individuals sampled. For each breed, marker polymorphism is summarized through average heterozygosity computed across the 44,706 SNPs considered in this study and the proportion of SNPs with a MAF above 0.05.(0.03 MB XLS)Click here for additional data file.Table S2http://genome.ucsc.edu/).SNP bovine genome coverage based on the Btau_4.0 bovine genome assembly ((0.01 MB XLS)Click here for additional data file.Table S3IS.Within population F(0.01 MB XLS)Click here for additional data file.Table S4ST for each pair of populations.F(0.05 MB XLS)Click here for additional data file.Table S5I spatial autocorrelation associated to the corresponding principal components are given.Comparison of PCA and sPCA. For each analysis, eigenvalues, percentage of genetic variance explained and Moran's (0.01 MB XLS)Click here for additional data file.


Understanding of the magnitude and direction of the exchange of individuals among geographically separated subpopulations that comprise a metapopulation (connectivity) can lead to an improved ability to forecast how fast coral reef organisms are likely to recover from disturbance events that cause extensive mortality. Reef corals that brood their larvae internally and release mature larvae are believed to show little exchange of larvae over ecological times scales and are therefore expected to recover extremely slowly from large-scale perturbations.Seriatopora hystrix, are mostly self-seeded and some populations are highly isolated, a considerable amount of sexual larvae (up to ∼4%) has been exchanged among several reefs 10 s to 100 s km apart over the past few generations. Our results further indicate that S. hystrix is capable of producing asexual propagules with similar long-distance dispersal abilities , which may aid in recovery from environmental disturbances.Using analysis of ten DNA microsatellite loci, we show that although Great Barrier Reef (GBR) populations of the brooding coral, Patterns of connectivity in this and probably other GBR corals are complex and need to be resolved in greater detail through genetic characterisation of different cohorts and linkage of genetic data with fine-scale hydrodynamic models. Larval dispersal and reproductive population connectivity  of most marine populations is poorly understood 2, of which ∼21,000 km2 consists of coral reef) and comprises ∼2,900 separate reefs Seriatopora hystrix Dana 1846, is a widespread and common species on the GBR The Great Barrier Reef (GBR) is the world's largest reef system , which are significant for all but 3 comparisons , (3) Geographically distant sites are sometimes genetically similar , (4) Populations in the Ribbon Reefs tend to be more genetically similar than populations elsewhere.The AMOVA  indicates a high level of genetic structuring in this coral species , suggesting that most recruitment is highly localised. This is supported by the indices of pairwise genetic differentiation  OspSTF values obtained here and in previous work K = 2 is unrealistically small given the high population structure in this species based on Approximately 2.5% of the samples (25) were found to have multi-locus genotypes that occur more than once. Eleven of those occur within sites and one sample of each pair was removed before analysis as it was assumed that clones within a collection site were produced through fragmentation. In addition, 14 multi-locus genotypes are shared between some of the sites sampled . These aS. hystrix is very common and widespread on the GBR, and many potential source populations were therefore not sampled in this study. Hence, it is possible that some immigrants have been assigned to a population they have not originated from. Given that geographically close populations are generally genetically more similar than geographically distant ones , however, we are confident that the estimates of the spatial scales over which these larvae have dispersed are reasonably accurate. Some of the migrants are inferred to be sourced from nearby sites , while others appear to have been transported over distances of 10 s to 100 s of kilometres . However, there is no assignment of migrants between the extremes of the sampling range, providing further confidence that these results provide reasonably accurate estimates of recent dispersal distances.An exclusion test conducted in GeneClass v2.0 Significant Linkage Disequilibrium (LD) was found in 165 out of 1,035 comparisons (15.9%), and at the following sites (numbers of locus pairs in parentheses): Osprey Rf (5), Yonge Rf (1), Ribbon Rf 5_2003 (11), Lizard Island 1 (1), Lizard Island 2 (17), Emily Rf (30), Agincourt Rf (19), Tongue Rf 1 (1), Tongue Rf 2 (16), Flora Rf (5), Myrmidon Rf (4), Davies Rf 2 (25) and Big Broadhurst Rf (29). All the sites with large numbers of LD also show significant heterozygote deficits involving 3–9 loci . HoweverS. hystrix, supporting earlier findings based on allozyme analysis of the same species on the GBR S. hystrix populations occurs within 100 m of the natal colony, a finding also supported by our data.This study shows a high level of genetic structuring among most GBR populations of the coral STF values not significantly different from zero, consistent with an almost continuous north-south reef matrix along the Ribbon Reefs acting as a stepping stone for coral dispersal. There are, however, exceptions to this pattern. For example, the two sites sampled at Ribbon Rf 5 are very genetically divergent  of larval release compared to broad-cast spawning corals In addition to recent long-distance dispersal of asexually produced larvae, migration of sexually produced larvae can be inferred from the exclusion test results . S. hystS. hystrix at the two Sudbury reefs was unusually large and the colonies on this reef were also large . This suggests that a disproportionally large number of larvae are produced here and the likelihood of some larvae reaching other reefs and establishing themselves is relatively high.The combination of high levels of Linkage Disequilibrium (LD), heterozygote deficits at large number of loci, and high genetic diversity in some of the populations  is also S. hystrix is extremely sensitive to heat and light stress In conclusion, our study is the first in which a considerable level of recent migration over spatial scales of 10 s to 100 s of km has been shown in a brooding coral on the GBR. Seriatopora hystrix (Scleractinia: Pocilloporidae) were collected between March 2003 and February 2005, and their genotypes at 10 microsatellite loci were determined. The samples originated from 21 collection sites on the GBR and one site in the Coral Sea (Osprey Reef)  of the coral ey Reef) , spanninDNA was extracted following a slightly modified method used for the black tiger shrimp Chromatograms were imported into the MegaBACE Genetic Profiler Software Suite version 2  to determine the fragment sizes  present in the samples. All automatic scoring was checked manually, and samples that yielded ambiguous or no signal were re-amplified and rerun.STF values were calculated using an AMOVA approach STF values was based on 9,999 permutations. The probabilities of identity by sexual reproduction were calculated using the software package GENCLONE 2.0 ISF estimated from each population S. hystrix is known to show some level of self-fertilisation Genotypic Linkage Disequilibrium (LD) and deviations from Hardy-Weinberg Equilibrium (HWE) were assessed in GENEPOP (web version 3.4) by estimation of exact p-values by the Markov chain method The fully Bayesian model-based clustering method implemented in STRUCTURE v2.2 P or α≤0.01). Potential source reefs of the excluded individuals were identified based on probabilities >0.1.To identify first generation migrants, an exclusion test was conducted in GeneClass v2.0