Zosterops borbonicus) a species that shows a striking, geographically structured plumage polymorphism on the topographically and ecologically complex island of Réunion, yet is monotypic on the relatively uniform neighbouring island of Mauritius.Oceanic islands provide unique scenarios for studying the roles of geography and ecology in driving population divergence and speciation. Assessing the relative importance of selective and neutral factors in driving population divergence is central to understanding how such divergence may lead to speciation in small oceanic islands, where opportunities for gene flow and population mixing are potentially high. Here we report a case of genetic and morphological structure in the Mascarene grey white-eye (Analysis of 276 AFLP loci in 197 individuals revealed prolonged independent evolution of Réunion and Mauritius populations, which is congruent with previous mtDNA assessments. Furthermore, populations on Réunion showed significant differentiation into three main genetic groups separating lowland from highland areas despite the small geographic distances involved. Genetic differentiation along the altitudinal gradient is consistent with morphometric analysis of fitness-related traits. Birds in the highlands were larger, yet had relatively smaller beaks than in the lowlands, suggesting the role of selection in shaping morphology and restricting gene flow along the gradient. No genetic differentiation between plumage morphs was detected in neutral markers, suggesting that plumage differences are of recent origin.in situ along the gradient cannot be ruled out at present. The lack of congruence between genetic groups and plumage morphs suggests that the latter are of recent origin and likely due to social or sexual selection acting on few loci. The presence of sharp and stable contact zones between plumage morphs suggests that they could be on independent evolutionary trajectories, yet whether or not they represent incipient species will require further research to directly assess the degree of reproductive isolation among them.Our results suggest a dual role of vicariance and natural selection in differentiating populations of a passerine bird in an oceanic island at very small spatial scales. We propose a combination of past microallopatry driven by volcanic activity and selection-constrained dispersal along steep ecological gradients to explain the striking levels of population structure found within the island, although the possibility that genetic differences evolved  Island archipelagoes have played a crucial role in the diversification of biotas, and oceanic islands have long been recognized as natural laboratories for the study of evolutionary processes -4. GeogrZosterops b. borbonicus) and Mauritius (Z. b. mauritianus) in the Mascarene Islands, some 800 km off the coast of Madagascar in the southern Indian Ocean [2), yet the species is composed of four distinct plumage morphs on the topographically and ecologically complex island of Réunion . The colour morphs on Réunion were first described as different species or as individual, age or sex variants [Here we examine patterns of genetic and morphological differentiation in the Mascarene grey white-eye endemic to Réunion . The divergence of these two clades  along with Z. mouroniensis and Z. semiflavus  from their closest continental relatives occurs deep within the Zosteropidae phylogeny and substantially pre-dates the divergence of all other western Indian Ocean Zosterops from their closest continental relative. Therefore, while multiple hypotheses for the direction of inter-island Zosterops colonization in the Mascarenes are plausible, based on mtDNA data and the sampling of all known living and extinct western Indian Ocean island forms, the Réunion population of Z. borbonicus can only result from a single colonization of the island and long-term divergence from the Mauritian population. This leaves open the possibility of an intra-island evolutionary radiation on Reunion.Analyses of mitochondrial DNA sequences revealed a deep phylogenetic break between reciprocally monophyletic populations of Réunion and Mauritius , and an Z. borbonicus morphs within Réunion, we carried out a survey of genome-wide genetic variation using amplified fragment length polymorphism (AFLP) to investigate inter-island divergence as well as the relationship between any genetic structure of populations within Réunion and the geographic distribution of colour morphs, geographic barriers, and ecological gradients. We also analyzed variation in fitness-related morphological traits to assess the role of selection in driving phenotypic divergence.While initial divergence could have resulted from drift or divergent selection in geographically separate areas within the island, three lines of evidence suggest the role of assortative mating in maintaining Réunion morphs separated: (1) The birds are good flyers and the geographic barriers separating the morphs are very narrow (0.3-5 km) relative to the birds' dispersal ability; (2) although intermediates occur, most sympatric grey and brown individuals in the highlands are easily assigned to either morph despite forming highly-social, mixed foraging groups; and (3) putative hybrids can be easily identified by a clear mixing of brown and grey colour, and are rare in nature . Given tA principal components analysis (PCA) revealed the separation of individuals along PC1 into two main clusters corresponding mainly to lowland (< 1000 m) and highland (>1000 m) populations Fig. . The PC12 = 0.512, F1,230 = 240.931, P < 0.0001), wing length  and tail length , yet explained less of the variance in bill length , bill depth , and bill width .Wing, tail and tarsus length means per site showed a strong positive correlation with elevation, whereas bill measurements showed a weak, inverse pattern Fig. . AccordiThe four pairs of selective primers used to generate AFLP markers yielded a total of 276 scorable loci, out of which 63 (23%) were polymorphic Table .Differentiation between the two islands accounted for almost one third of the variance in genetic variation, with structuring among populations within islands and within-population variation explaining 10% and 63% of the variance, respectively Table . A plot TRUCTURE 2.2 corroborated the marked separation of Mauritius with respect to Réunion. An optimal K of 4 revealed a Mauritian cluster and three additional clusters within Réunion . Within Réunion, differentiation between populations was remarkably high, with an average pairwise ΦPT of 0.148, and highest values were obtained in highland-lowland comparisons. A Mantel test of the correlation between a matrix of geographic distances , suggesting that factors other than mere isolation-by-distance are responsible for the observed genetic structure.gh Table , with an SD = 0.0. Within ST Fvalues per locus between Le Maido  and St. Leu (509 m) on the east side of the island. A distribution of ST Fvalues shows that differentiation is not due to just a few loci but to the contribution of many different ones, with 13 out of 44 loci (30%) showing ST Fvalues > 0.1  microallopatric isolation of ancestral populations into at least three disjunct demographic units, which has resulted in divergence in neutral genomic markers; (2) morphological differentiation along altitudinal gradients through local adaptation; (3) the appearance of distinct plumage morphs which now exist (or co-exist) in different parts of the island; and (4) the maintenance of these patterns in place over time, through a combination of restricted dispersal and pre and/or post-zygotic mechanisms of reproductive isolation. The combined effect of these different processes has likely given rise to the patterns of divergence and structure observed on Réunion, which are remarkable given the small geographic scale of the island and thus the opportunity for high levels of homogenizing gene flow in a highly vagile organism.The current pattern of geographic variation of Z. borbonicus populations were probably restricted to isolated patches. The presence of small areas (10 to 100 km2) with original basaltic shields at ground level in areas scattered around Réunion  suggests that these areas have remained free of lava for hundreds of thousands of years, and thus could have harboured forested areas capable of sustaining bird populations. Since the time of the last eruption of the "Piton des Neiges", as recent as 12,000 years ago, forested areas would have expanded throughout the island, bringing differentiated Z. borbonicus populations into contact. Given the very limited rates of dispersal inferred from our genetic and morphological results, and the apparent reluctance to cross barren areas such as river beds, large lava flows must have represented formidable barriers to dispersal among patches across the island.Although the establishment of isolated allopatric populations of birds in a small island is generally unlikely, the inferred pattern of microallopatry responsible for the genetic structure found in neutral markers could be tied to the active eruptive history of Réunion's volcanoes, the "Piton des Neiges" on the northwest, and the "Piton de la Fournaise" in the southeast. Whereas the island of Mauritius is about 8 million years old , the emeThe lack of congruence between the pattern of neutral structure found and the distribution of color morphs suggests that the latter evolved at a later stage and could indeed be of recent origin, as changes in plumage pigmentation can be fast in birds -35.Z. borbonicus, the altitudinal increase of size-related traits like wing, tail and tarsus length, combined with an opposite trend for diet-related traits like beak dimensions, suggests the role of natural selection in driving morphological divergence along steep altitudinal gradients, as has been documented in other similar avian systems [Zosterops species [Z. borbonicus , poses the question of whether genetic divergence represents in situ differentiation through isolation by adaptation  will be necessary to investigate this question, the morphological results suggest that selection may be an important factor in restricting gene flow up and down the slope, and thus may be important in at least maintaining this marked genetic break despite the short geographic distances involved.Phenotypic evolution on island populations can be fast under strong natural selection ,16,36-38 systems -41. Rapi species ,43, yet  species . Therefoolation: ,45, or rZ. borbonicus in the Mascarenes, where upon colonization, and despite the species' good flying capacity, dispersal rate has been dramatically reduced. The evolution of "behavioral flightlessness" has been documented in other members of the genus [Z. borbonicus [Our results are in line with previous work documenting the high diversification capacity of white-eyes (Zosteropidae) ,46. Membhe genus ,48,49, ahe genus ,46. Inderbonicus , which irbonicus ,52.Z. borbonicus is ongoing and represents a case of incipient speciation, or instead the process has been truncated by widespread gene flow upon secondary contact among morphs, remains to be addressed. Previous work on Zosterops lateralis in Australia has shown that selection can act upon colonization and then remain static for thousands of years [Our results shed light on some of the evolutionary mechanisms underlying an extreme case of small-scale intra-island diversification in a passerine bird. The presence of three genetic clusters on Réunion island as revealed by neutral AFLP markers suggests the role of geography in differentiating populations at some time in the past despite the island's small size. In turn, morphometric data suggest the role of ecology-driven selection in differentiating populations and restricting gene flow across habitat types and geographic barriers. However, the fact that patterns of neutral genetic divergence are not geographically congruent with the distribution of plumage morphs, suggests that in addition to genetic drift and natural selection, social or sexual selection may have played a role in driving recent plumage divergence. Future research will be necessary to determine the degree of reproductive isolation between morphs, and thus the extent to which morphs represent incipient species. Thus, whether diversification in Réunion's of years . HoweverBirds were captured using standard mist nets at 11 sampling localities, eight on Réunion and three on Mauritius  on the correlation matrix of ln-transformed morphological variables. Based on results from the multivariate analysis, we analysed variation in morphological traits along the altitudinal gradient by means of regression analysis, with elevation as the predictor variable. Variables were transformed prior to analyses to meet model assumptions. Assumptions such as normality of the residuals and homogeneity of variances were met.Since there is no sexual dimorphism in plumage and morphology , both maet al. [et al. [EcoRI and MseI (Tru9) and fragments were ligated to oligonucleotide adapters with T4 DNA ligase. A random sub-sample of all restriction fragments was obtained through a pre-selective amplification using primers E-t and M-c, followed by four selective amplifications using primer pairs E-tct/M-cga, E-tcta/M-ctt, E-tag/M-cag, and E-tgc/M-cat  profiles were generated using a protocol modified slightly from Vos et al. , as deta [et al. . Brieflyat Table , with ean et al. , was 2.1e), and per-locus ST Fvalues based on allele frequencies where calculated using the method by Lynch and Milligan [FLP-SURV v. 1.0 [ST Fvalues using the ST Fanalogue ΦPT was calculated with GENALEX 6.0 [PT was calculated as VAP/(VAP + VWP) where VAP is the variance among populations and VWP is the variance within populations. Probability values of pairwise ΦPT were based on 9999 permutations.We estimated allelic frequencies using Zhivotovsky's  BayesianMilligan  as impleV v. 1.0 . A matriALEX 6.0 . ΦPT wasENALEX 6.0. We also examined patterns of population structure using the Bayesian assignment probability test in the program STRUCTURE 2.2 [et al. [To assess genetic structure among samples we conducted a principal coordinate analysis  on a geTURE 2.2 . This prTURE 2.2  and the  [et al. .ST F/(1-STF) values among localities and a matrix of the natural log of Euclidean geographic distances between localities. We used a Mantel test with 10,000 permutations of Spearman's rank correlation coefficient with a one-tailed exact test, as implemented in the program ISOLDE in the package GENEPOP[To examine the pattern of isolation by distance we tested the correlation between a matrix of pairwise e GENEPOP.ST Fagainst heterozygosity under the assumption of Hardy-Weinberg equilibrium to identify significant outlier loci with the program DFDIST[ST Fvalues based on 50,000 simulated loci with a mean ST Fequivalent to the "neutral" mean ST Fof the empirical distribution, which was obtained by trimming the 30% highest and lowest ST Fvalues [To assess the potential role of selection in driving genetic differentiation among populations, we examined plots of am DFDIST. SignifiT values .BM, CT and PH designed the study; BM, CT and BW carried out the field work; BM took the morphologic measurements in the field and run the morphometric analyses with input from PH; BM generated the molecular data and conducted all genetic analyses; BM and CT wrote the manuscript with contributions from all authors, who read and approved the final manuscript.


Here, we analysed the genetic structure of Baltic ciscoes , examining 271 individuals from 8 lakes in northern Germany using 1244 polymorphic AFLP loci. Six lakes had only one population of C. albula while the remaining two lakes had C. albula as well as a sympatric species (C. lucinensis or C. fontanae).Teleost fishes of the Coregonidae are good model systems for studying postglacial evolution, adaptive radiation and ecological speciation. Of particular interest is whether the repeated occurrence of sympatric species pairs results from θB = 0.22). Lower differentiation between allopatric (θB = 0.028) than sympatric (0.063-0.083) populations contradicts the hypothesis of a sympatric origin of taxa, and there was little evidence for stocking or ongoing hybridization. Genome scans found only three loci that appeared to be under selection in both sympatric population pairs, suggesting a low probability of similar mechanisms of ecological segregation. However, removal of all non-neutral loci decreased the genetic distance between sympatric pairs, suggesting recent adaptive divergence at a few loci. Sympatric pairs in the two lakes were genetically distinct from the six other C. albula populations, suggesting introgression from another lineage may have influenced these two lakes. This was supported by an analysis of isolation-by-distance, where the drift-gene flow equilibrium observed among allopatric populations was disrupted when the sympatric pairs were included.AFLP demonstrated a significant population structure (Bayesian Coregonus. Relative differences within and among lakes raises the possibility that multiple lineages may be present in northern Germany, thus understanding the postglacial evolution and speciation in the C. albula complex requires a large-scale phylogenetic analysis of several potential founder lineages.While the population genetic data alone can not unambiguously uncover the mode of speciation, our data indicate that multiple lineages may be responsible for the complex patterns typically observed in  Gasterosteus aculeatus L.) ; howeveiewed by ,11 have iewed by ,13. Furtiewed by -16. In aiewed by , whereasiewed by . SubsequCoregonus clupeaformis (Mitchill) and C. lavaretus (L.) complexes) and the North America ciscoes (C. artedi Lesueur complex) are comparatively well studied  which is only slightly modified by more recent stocking [C. albula populations in Lakes Stechlin and Breiter Luzin, as indicated by a low frequency of hybridization between these populations and C. albula from Lake Tollense. Secondary contact with another Coregonus lineage and subsequent introgression may have further shaped the genetic signature of the sympatric populations. Introgression is more likely for the autumn-spawning C. albula because the majority of all other European populations of the C. albula complex are autumn-spawners [TRUCTURE analyses, may have resulted in the spreading of introgressed genetic material into both populations. Introgression after secondary contact may explain why both C. albula and the spring-spawning C. fontanae and C. lucinensis of Lakes Stechlin and Breiter Luzin were genetically distinct to the allopatric C. albula in the other German lakes.Taken together, our results suggest the following evolutionary and phylogeographical hypothesis: All lakes in northern Germany including Lakes Stechlin and Breiter Luzin were invaded from a single refuge after the last glaciation about 12,000 years ago. This founder lineage of stocking . In two stocking . Subsequstocking ,32. Recestocking  may havespawners . Ongoinget al. [C. albula populations, with one group occurring in the east, and one in central and west Poland. These authors speculated that these may have evolved by colonization from different refugia or by introgression from C. sardinella [C. sardinella has also been suspected for the Lake Breiter Luzin populations [C. sardinella and C. albula populations in Russia. Accordingly, we cannot exclude that populations exist in East Europe which are part of a separate lineage involved into the secondary contact with the German sympatric populations, thus explaining the genetic similarity between Lakes Arend, Stechlin and Breiter Luzin. We would like to emphasize that this complex scenario is hypothetical at the moment. We cannot exclude that the sympatric populations had an allopatric divergence or originate from two different refugia [C. albula complex from several populations in Poland and Russia to be included in the analysis, as well as additional sympatric populations existing in Russia and Scandinavia [C. clupeaformis [C. albula complex [The source of the secondary contact could be from populations living farther east. This is suggested by the similarity between Lakes Stechlin and Breiter Luzin and the population from Lake Arend, which has a historical origin in Poland. Brzuzan et al.  demonstrrdinella . Interesulations . An alloulations  found lo refugia , followe refugia . Based ondinavia . Comparaeaformis  and sticeaformis  in North complex ,26 underCoregonus can be quantified and accounted for, thus the group remains an excellent model for the study of speciation in sympatry. Despite having used more than 1200 marker loci in a study of 10 populations, our data failed to provide conclusive evidence for whether spring-spawning Coregonus stem from parallel sympatric speciation. Parallel trends in niche differentiation and phenotypic differentiation are evident in both lakes with sympatric populations, but neutral genetic differentiation suggests allopatric speciation and introgression on secondary contact. It is clear that population genetic criteria alone cannot resolve the mechanisms of speciation, and that our analysis benefited from a large spatial scale of sampling. Examination of relative differences within and among lakes has raised the possibility that multiple lineages may be present in northern Germany. As a result, phylogenetic analysis and sampling of additional lakes from potential source populations from a broad geographic range is required for an unequivocal identification of the evolutionary mechanisms and phylogeography which have led to the ecological and genetic diversity found in the C. albula complex.The effects of hybridization and stocking on the commercially important C. albula occurs sympatrically with a spring-spawner; C. fontanae in Lake Stechlin and C. lucinensis in Lake Breiter Luzin. The remaining six populations of C. albula occur in allopatry in northern Germany, at variable geographic distances to Lakes Stechlin and Breiter Luzin  occur elsewhere in allopatry.Ten populations of Baltic ciscoes were sampled from eight lakes in Germany from 2000 to 2004 Fig. . In two zin Fig. , Table 1θ between the repeated samplings were not significantly different from zero (see results). All fish were caught with gill nets or trawls and stored on ice immediately after removal from the nets. Subsequently, clips from the pectoral fin were stored in pure ethanol.In total, 271 individuals were analysed, with the number of analysed specimens per population varying between 12 and 38. To verify that all procedures and analyses produced consistent results, the Lake Stechlin populations were sampled twice, in 2000/2001 and 2007. These samples were later combined because the pairwise DYE) und the utilization of two different fluorescent dyes permitted multiplexing, where two EcoRI primers were combined with one MseI primer (Table We examined population genetic diversity using amplified fragment length polymorphism (AFLP) in order to generate the large number of characters needed for genomic scans of non-model organisms ,60. We der Table .® v4.0 (Applied Biosystems). Optimization yielded the following settings: bin width 1 bp, maximal peak width of 1.5 bp, scoring to presence (1) or absence (0) of fragments between 100 and 400 bp. Normalization to the sum of signal versus a fixed cut off value did not cause any differences, thus we used peak detection levels of 50 relative fluorescent units. Correct fit of the size standard was checked manually for all electropherograms. Larger fragments were excluded from the analyses to avoid errors from signal-strength drop off resulting from PCR or capillary loading bias. Only electrograms were evaluated which met the following criteria: smallest detected fragments < 50 bp and largest > 450, at least 5 fragments > 400 bp, no visible drop outs  in the entire spectrum, a minimum of 100 fragments in each primer combination and reproducible standards. We used 2 samples as standards in each PCR and subsequent analyses. They were considered reproducible only if they yielded > 95% fragment reproducibility to previous runs. Analyses of individual samples were repeated from restriction/ligation on until meeting the above described criteria. A second complete AFLP analysis of a random 10% of the samples yielded 96.08% reproducibility.Fragments were separated on an ABI Avant 3100 capillary sequencer with an internal size standard . Signal processing and binning were carried out using GeneMapperFLPDAT[URV[hs, the mean within-population expected heterozygosity Hs, and their 95% credibility values, were calculated using a Bayesian approach with 250,000 generations after a burn-in of 50,000 generations implemented in HICKORY 1.1 [IS). Because this value can be unreliable [Preparation of input data files for analyses was conducted by AFLPDAT. Data onLPDAT[URV. The expKORY 1.1 . To accoreliable , we repoθ) [RLEQUIN 3.2 [θ was calculated for all population pairs and tested for significant deviation from zero by a numerical resampling procedure in ARLEQUIN. All analyses used 50,175 permutations. An overall estimate of genetic differentiation using a Bayesian approach (θB) [ICKORY as described above . A modified Nei's genetic distance [HYLIP 3.6 [URV to calculate a majority-rule consensus tree in PHYLIP 3.6 [The genetic structure within and among all 10 populations was inferred from an analysis of molecular variance (AMOVA) and a global estimate of among-population differentiation (θ) , both caQUIN 3.2 . Pairwisach (θB)  was alsodistance  was calcYLIP 3.6 . SubsequYLIP 3.6 .10 direct geographical distances (km) and pairwise genetic distance (θ/1-θ) [n = 45 pairwise comparisons, PC-ORD 5.01 [10 = 0). Stepwise Mantel tests were subsequently separately performed by first removing the two spring-spawning populations C. lucinensis and C. fontanae  and then removing the two C. albula populations from Lakes Stechlin and Breiter Luzin . These tests aimed to determine whether IBD was disrupted by inclusion of the sympatric populations.An isolation-by-distance model was calculated by correlating the matrices of log (θ/1-θ)  between ORD 5.01 ). The geθB using HICKORY in order to obtain 95% credibility intervals for differentiation among the four populations that occur as sympatric pairs . Furthermore, STRUCTURE 2.2 [We calculated pairwise TURE 2.2 ,55 was uFST and heterozygosity per polymorphic locus were calculated for the two population pairs and compared with simulated null distributions (at p = 0.95 and p = 0.99) using DFDIST[FST were plotted against heterozygosity to determine those loci for which the neutral model could be rejected [p = 0.99, the probability that loci putatively under selection are shared in both lakes just by chance is 0.01 × 0.01 × 1264 = 0.1264. Accordingly, any locus with p > 0.995 or p < 0.005 found in both lakes would be a true outlier. At p = 0.95, up to three shared loci (0.05 × 0.05 × 1264 = 3.16) could be false positives.To evaluate those AFLP loci that may be subject to selection, a genome scan was conducted by assuming that genetic differentiation between sympatric populations is higher for those loci that are under divergent selection ,72. FST ng DFDIST,74. The rejected ,74. Thesp = 0.95 , and again calculated pairwise θB using HICKORY in order to obtain 95% credibility intervals for differentiation among the four populations that occur as sympatric pairs . Furthermore, a NJ tree was calculated based on Nei's distance between all 10 populations, based on the reduced matrix of supposedly neutral AFLP loci.Subsequently, we removed all loci putatively under positive selection in either Lakes Stechlin or Breiter Luzin at TM conceived of the study, carried out most of the statistical analyses and drafted the manuscript. KP carried out most of the molecular genetics studies. CE carried out statistical analyses. MTM carried out some statistical analyses and drafted the manuscript. BN established the molecular genetics protocol and carried out some molecular genetics studies. JF conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.θ between 10 populations of the Coregonus albula complexOverview on pairwise . Matrix of pairwise θ, as calculated by ARLEQUIN. Allopatric populations consist exclusively of C. albula. Sympatric populations are named by lake origin and the lake-specific species names.Click here for fileCoregonus populationsIsolation-by-distance model of . Scatter plot between log10 direct geographical distances (km) and genetic distance (θ/1-θ) for the 10 populations of the C. albula complex in 8 lakes of north Germany (a), the eight C. albula populations in eight lakes at either allopatry or sympatry (b), and the six allopatric C. albula populations (c).Click here for file


ND3 and ATP6 genes characterized by a clear association with temperature, which appear to be plausible targets of natural selection producing the association with climate. The same mutations have been previously shown to be associated with variation in mitochondrial pH and calcium dynamics. Our results indicate that natural selection mediated by climate has contributed to shape the current distribution of mtDNA sequences in humans.There is an ongoing discussion in the literature on whether human mitochondrial DNA (mtDNA) evolves neutrally. There have been previous claims for natural selection on human mtDNA based on an excess of non-synonymous mutations and higher evolutionary persistence of specific mitochondrial mutations in Arctic populations. However, these findings were not supported by the reanalysis of larger datasets. Using a geographical framework, we perform the first direct test of the relative extent to which climate and past demography have shaped the current spatial distribution of mtDNA sequences worldwide. We show that populations living in colder environments have lower mitochondrial diversity and that the genetic differentiation between pairs of populations correlates with difference in temperature. These associations were unique to mtDNA; we could not find a similar pattern in any other genetic marker. We were able to identify two correlated non-synonymous point mutations in the  Interestingly, it has since been shown that dN/dS ratios are largely inadequate when testing for natural selection within populations  remains by far the most widely used genetic marker in studies of human populations. One assumption behind inferences on past human demographic history is the selective neutrality of the genetic markers employed. There have been claims for natural selection affecting mtDNA, with temperature being highlighted as a possible selective force in a variety of taxa  includinulations . Here weThe most likely origin of anatomically modern humans lies in sub-Saharan Africa, where the most ancient remains (dated to approximately 200 000 years) have been found (2.(a)lustalX v. 2.0.9 and double-checked visually. The alignment covered the entire HVS-I region . The poly-C tract located between positions 16 184 and 16 193 proved difficult to align unambiguously and had not been successfully amplified in a substantial fraction of available sequences. Thus, this section was removed from all analyses.We downloaded over 5000 hyper-variable segment I (HVS-I) sequences from the HVRBase++ online resource . The genetic diversity for single nucleotide polymorphisms (SNPs) treated as phased haplotypes were obtained from table S1 in the electronic supplementary material in All autosomal (783), X- and Y-chromosome microsatellites  were downloaded from the HGDP-CEPH human genome diversity panel database v. 2.0 ((b)We assigned geographical coordinates to every population based on the information in the original articles. As very few authors provide sample coordinates, we had to rely on the names of the localities given or in some cases on the maps provided in the papers. Minimum temperature was obtained from WORLDCLIM ((c)rlequin v. 3.1 (rlequin (α was set to 1.12 for the whole genomes as estimated using Modeltest 3.6 (stat v. 2.9.3 (All genetic diversities and genetic distances for mtDNA used in the paper were computed as average pairwise difference in An v. 3.1 . The protest 3.6  and to 0test 3.6 . Summaryv. 2.9.3 .To test the effect of past demography (distance from sub-Saharan Africa) and climate (minimum temperature), we built a linear model with within-population genetic diversity as the response variable, and with distance and temperature as predictors. The previous analysis assumes independence between populations and may thus overestimate degrees of freedom. Thus, we replicated all analyses based on genetic diversity with Mantel and partial Mantel tests. The different matrices were computed as pairwise difference in genetic diversity, absolute difference in minimum temperature and absolute difference in the geographical distance from the hypothetical sub-Saharan origin. The results from the Mantel tests were qualitatively similar to the ones obtained with the linear models and are reported in table S4 in the electronic supplementary material.R2 = 0.006, F1,107 = 1.688, p = 0.197) or complete mitochondrial genomes . To test whether the association between mtDNA and climate might have arisen by chance, we compared the proportion of variance in mtDNA diversity explained by climate to the same measure estimate for each of the 783 autosomal STRs individually. All pairwise correlations were computed with Mantel tests and partial Mantel tests.We also tested whether there may be an effect of sample size. There was no significant correlation between the number of individuals typed per population and genetic diversity for HVS I sequences ((d)R2) adopted for other models.For the analysis of candidate targets for natural selection, we recovered all polymorphisms with a minor allele frequency of 10 per cent or higher and computed their frequency in all 51 populations for which we had complete genome information. The mutations comprised 32 point mutations and two InDels. We used the MITOMAP database . This figure is low compared with the 84.9 per cent reported for the 54 HGDP-CEPH populations typed at 783 microsatellites . Since sub-Saharan Africa comprises some of the warmest areas on earth, it might be expected that minimum temperature and distance from Africa are related. In fact, distance from Africa and minimum temperature for the sampled populations are not correlated . Even when we accounted for the effect of distance from Africa, the relationship between HVS-I diversity and minimum temperature remained significant . Thus, both distance from Africa and low temperatures decrease within-population mtDNA diversity at HVS-I.Distance from Africa along landmasses explains 18.3 per cent of the variance in within-population genetic diversity for the first hypervariable segment of mtDNA (HVS-I) from 109 populations distributed worldwide  between genetic and physical distance, and rM = 0.22  between genetics and temperature. Testing for an effect of temperature after accounting for geographical distance with a partial Mantel confirms the correlation with temperature . While the correlation with climate is moderate, this effect is expected to colour analyses of population differentiation at large geographical scales.Does climate also influence population differentiation as measured with HVS-I sequences? To test for this, we produced matrices for between-population pairwise genetic distances, geographical distances along landmasses and difference in minimum temperature. When we ran separate Mantel tests, we obtained correlations of R2 = 0.293, F1,49 = 20.45, p < 0.001; figure 2c), and climate explains a far higher amount of variance . We also confirm that genetic differentiation between populations correlates with geographical distances  and differences in minimum temperature .To confirm that these patterns were not specific to the HVS-I sequences, we repeated the analyses above on a smaller dataset of complete mitochondrial sequences from 51 populations with HVS-I and HVS-II regions removed , we detected no effect of climate . mtDNA is haploid and inherited through the female line only without any genetic recombination. As such, it can be more sensitive to population bottlenecks , giving further support to a direct link between climate and within-population mitochondrial diversity.A potential explanation for the association between climate and the distribution of mitochondrial sequence variation could stem from populations in colder environments being smaller and more isolated. If this is the case, a similar demographic effect should be picked up by other genetic markers. To tackle this issue, we first tested whether minimum temperature was also a predictor for the mean genetic diversity of autosomal microsatellites in the HGDP-CEPH panel. While microsatellite diversity was strongly influenced by distance from Africa , but no signal for climate . The absence of any association with temperature for both microsatellites and SNPs argues against a spurious effect mediated by differences in mutation rates between classes of genetic markers.As autosomal microsatellites are characterized by a far higher mutation rate than mtDNA sequences, the signature of stronger bottlenecks in colder regions may have been partly erased by subsequent mutations replenishing the genetic diversity of populations from cold areas. We thus replicated the analysis on the mean heterozygosity of haplotypes constructed from phased autosomal SNPs , but, importantly, there was no trace of a signal from climate .Owing to its maternal inheritance, mtDNA is only affected by the past demography of the female line. For example, an association between climate and mitochondrial genetic diversity could arise if the ratio of male over female immigration was correlated with temperature. The same pattern could be generated if sex-specific variances in reproductive success covaried with climate. To test for such an effect, we repeated our analysis on the genetic diversity of 36 X-chromosome microsatellites typed in the HGDP-CEPH panel. Females transmit an X chromosome to both their daughters and sons, whereas males pass on their single copy to their daughters only. Thus, X-chromosome markers are more strongly affected by the past demography of the female line. Apart from this difference, autosomal and X-chromosome microsatellites should be largely comparable as both undergo recombination and do not seem to be characterized by significantly different mutation patterns in humans  and no relationship with minimum temperature , mirroring the results for autosomal and X-chromosome diversity. Having markers that are either passed down the male line or predominantly the female line provides us with a powerful test for an association between climate and mating system or sex-biased dispersal, which would be highlighted by a link between climate and the ratio of diversities at the two uniparental markers. When we modelled the distribution of ratios of diversity at the X and Y chromosomes, we found no effect of minimum temperature . This result indicates that minimum temperature does not seem to induce any systematic variation in mating system or sex-specific migration that could have created a spurious association between climate and mtDNA sequence distribution.The Y chromosome is only inherited down the paternal line, and thus constitutes a male analogue of mtDNA. If the effect we find on mtDNA is a feature of its lack of recombination, uniparental inheritance or small effective population size (relative to the autosomes and X chromosome), we would therefore expect to find a similar pattern on the Y chromosome. To investigate this, we tested whether diversity in microsatellites on the Y chromosome showed similar patterns to the rest of the genome. We found a significant decline in diversity with increasing distance from Africa . This smooth increase illustrates that the correlation with climate is not driven by a few populations from extreme environments but acts over a broad range of temperatures including the entire temperate zone. Indeed, the increase in the correlation with geography even extends to the 10–15°C minimum temperature bracket, which comprises areas such as parts of East Africa or large swathes of Southeast Asia, which are all regions that are believed to have been inhabited for a long time and have maintained sizeable human populations.ATP6 gene , and 10398G/A in the ND3 gene . The allele frequencies at these two SNPs are tightly linked  of the variance for 8701G/A and 15.3 per cent  for 10398G/A. For both polymorphisms, the G nucleotide is fixed or nearly fixed in sub-Saharan populations. The Americas show a similar gradient to the one found in Eurasia: the A nucleotide is predominant in Arctic populations close to the Bering Strait (the entry point to the Americas) and becomes less frequent further south. This leads to a strong correlation between minimum temperature and the two SNPs even when Beringian and American populations are considered in isolation .In the next step, we searched for specific nucleotides in our whole genome mtDNA dataset that may be the direct targets of selection. We retrieved all polymorphisms with a minor allele frequency of 0.1 or higher. We detected 34 such sites, including 32 SNPs and two insertion/deletion variants. Five of the SNPs represented non-synonymous changes , confirming that our results are not simply due to the loss of some haplotypes during an out-of-Africa bottleneck. Europe has been argued to have been recolonized only relatively recently, following the last glacial maximum. Thus, we also removed all European populations to test whether very recent bottlenecks during this period might be responsible for the association with climate. Again, the link with climate remained significant after all European populations were excluded . As the association between the two SNPs and climate is robust to the elimination of all African or European populations, we can conclude that it is a global pattern.Because the G nucleotides at both SNPs are nearly fixed in Africa and largely absent from Europe, one may argue that the effect is solely driven by these two continents. However, even after removing all African populations, we still find a significant link with climate for both SNPs . 8701G/A also leads to an alanine/threonine substitution in ATPase6, the F0 subunit 6 of complex V (ATP synthase). The NADH-ubiquinone oxidoreductase contributes to the generation of the proton electrochemical gradient that is then used by the ATP synthase to drive ATP production.The 10398G/A polymorphism has been linked to longevity et al. , 2004. TGiven the pivotal role of the oxidative phosphorylation (OXPHOS) cycle in metabolic processes, there are a large number of potential evolutionary explanations behind the strong correlation we observed. One hypothesis that has previously received considerable attention is a possible tradeoff between ATP synthesis and thermogenesis . Thus, tND3 and ATP6 may represent previously unrecognized important determinants of human metabolism.As humans expanded out of Africa eventually to colonize the entire globe, they experienced new environments. For many populations, this also meant exposure to far colder climates. Smooth clines in allele frequency correlated with climatic variables have been observed for SNPs in autosomal genes involved in common metabolic disorders, such as type 2 diabetes, obesity and hypertension . The sel


Gadus morhua L.) across the species distribution.Marine fishes have been shown to display low levels of genetic structuring and associated high levels of gene flow, suggesting shallow evolutionary trajectories and, possibly, limited or lacking adaptive divergence among local populations. We investigated variation in 98 gene-associated single nucleotide polymorphisms (SNPs) for evidence of selection in local populations of Atlantic cod (per se.Our global genome scan analysis identified eight outlier gene loci with very high statistical support, likely to be subject to directional selection in local demes, or closely linked to loci under selection. Likewise, on a regional south/north transect of central and eastern Atlantic populations, seven loci displayed strongly elevated levels of genetic differentiation. Selection patterns among populations appeared to be relatively widespread and complex, i.e. outlier loci were generally not only associated with one of a few divergent local populations. Even on a limited geographical scale between the proximate North Sea and Baltic Sea populations four loci displayed evidence of adaptive evolution. Temporal genome scan analysis applied to DNA from archived otoliths from a Faeroese population demonstrated stability of the intra-population variation over 24 years. An exploratory landscape genetic analysis was used to elucidate potential effects of the most likely environmental factors responsible for the signatures of local adaptation. We found that genetic variation at several of the outlier loci was better correlated with temperature and/or salinity conditions at spawning grounds at spawning time than with geographic distance These findings illustrate that adaptive population divergence may indeed be prevalent despite seemingly high levels of gene flow, as found in most marine fishes. Thus, results have important implications for our understanding of the interplay of evolutionary forces in general, and for the conservation of marine biodiversity under rapidly increasing evolutionary pressure from climate and fisheries induced changes in local environments. When genotype by environment interactions take place, populations can evolve traits that confer a Darwinian fitness advantage in their local habitat. The process and the resulting patterns are termed "local adaptation" . For adaIt is notoriously difficult to separate genetic from environmental effects when traits of presumed adaptive value are measured directly in the wild . FurtherGadus morhua L.) has been the subject of numerous population genetic studies and thus represents one of the best studied marine fish species within the field. Population genetic studies have demonstrated low, albeit significant genetic differentiation on various hierarchical levels, ranging from clear transatlantic differences [Hb I) most likely under environmental selection. Recently, Andersen et al. [Pantophysin (Pan I) locus in cod [et al. [et al. [The Atlantic cod .In this study, we tested the hypothesis that populations of Atlantic cod are adapted to local environmental conditions and therefore display genomic signatures of divergent selection. We applied genome scan methods to identify outlier loci potentially associated with adaptive population divergence on global, regional and local scales as well as an explorative landscape genetic analysis focusing on environmental factors suspected to be involved in local adaptation.ST values, see additional file et al [First, we scanned for global outlier loci by including a selection of major population groups from the western Atlantic (Canada), Greenland, Iceland, northeast Arctic cod, Norwegian coastal cod, North Sea, English Channel and the Baltic Sea , Salmo salar) populations [Drosophila). Furthermore, overall genetic differentiation of "neutral" loci for the EST derived loci and our own candidate loci was almost identical . With an alpha level of 0.05 we would expect five significant outliers, which in fact is not so far from the present results. However, the statistical support for individual outliers identified here is generally much stronger. We should expect only one outlier by chance with a Bayes factors above 2 (log10), but the computed values are almost exclusively above 2 (log10), which is generally interpreted as "decisive". Four loci have log10 values as high as 5, which was the value ascribed to posterior probabilities of 1 (Bayes factor is infinity). Accordingly, we are still convinced that the majority of these outliers are affected by directional selection and not just outliers by chance. We think that our evidence for selection is as strong as possibly achievable given the statistical method and the number of loci at hand.Hsp90, have been shown to be involved in various stress responses in fish including temperature, salinity and pollution [Aromatase plays a regulatory role in sex determination, gametogenesis, central nervous system development and sex behaviour [rhodopsin is a pigment gene involved in the formation of photoreceptor cells and the perception of light [As evidenced above, the identified outlier genes originate both from randomly chosen gene associated SNPs and from specifically targeted SNPs in candidate genes of known functions. Interestingly, a relatively high proportion of candidate genes showed evidence of being subject to selection compared to the randomly chosen SNPs (app. 30% versus app. 6%). The identified outlier genes code for proteins with highly divergent functions  using the proteinase K/chelex method . OverallST values were estimated by Weir and Cockerham's θ [ST between samples used in the global analyses were estimated for each of the loci identified as outliers in BAYESCAN. In addition, pairwise multi-locus FST were estimated for all loci, excluding the outlier loci  to identify loci under selection from locus specific Bayes factors. A Bayes factor of infinity, corresponding to a posterior probability of 1, was assigned a log10 value of 5. In order to alleviate potential problems arising from allele frequency correlations between samples ,59, we rhttp://ingrid.ldgo.columbia.edu). Salinity data were collected from NOAAs Global Ocean Data Assimilation System  and from the oceanographic database managed by the International Council for the Exploration of the Sea . Environmental data were integrated in a Geographic Information System (ArcGIS 9 from ESRI) in order to extract data covering a latitude/longitude grid of 0.5° *0.5° to 1° *1° around each sample position. Environmental variables used in SAM were means of a sampling period from 1990 to 2007. The null hypothesis of no association between an allele and an environmental variable was rejected if the examined variable explained the observed allelic distribution better than a model with a constant only. We changed the critical P value from 0.05 to 0.01 and 0.001 for the implemented Wald test to demonstrate robustness of significant associations. Since only two alleles were present at each locus, leaving only one independent variable, we present the results as associations between locus and environmental variable.A spatial analysis, using the program SAM , was perEN conceived and designed the study and drafted the manuscript. JH-H analysed the majority of the data, participated in the design of the study as well as drafting and commenting on the manuscript. NAP participated in the design of the study, data analysis and commented on previous drafts of the manuscript. VL participated in the design of the study and commented on manuscript drafts. TM, TJ, CM, G-LT and RO provided data and commented on manuscript drafts. GC participated in the design of the study and in drafting and commenting of the manuscript. All authors have read and approved the final version of the manuscript.STPairwise F. Pairwise Fst for eight samples used in the global analysis , otherwise Homo sapiens (b) is used as reference.ces from  or own sogy.org, ). Where Click here for fileNovel SNPs analysed in this study. New SNPs analysed in this study with PCR primers to amplify a fragment containing the SNP.Click here for file


Four economically important Australian sheep flocks, three pure breeds  and a crossbred population (Merino × Border Leicester), along with an inbred Australian Merino museum flock were analysed.The success of genome-wide scans depends on the strength and magnitude of linkage disequilibrium (LD) present within the populations under investigation. High density SNP arrays are currently in development for the sheep genome, however little is known about the behaviour of LD in this livestock species. This study examined the behaviour of LD within five sheep populations using two LD metrics, D' and x2') for markers spaced up to 20 cM exceeded the non-syntenic average within the White Faced Suffolk, Poll Dorset and Macarthur Merino. LD decayed faster within the Merino and Merino × Border Leicester, with LD below or consistent with observed background levels. Using marker-marker LD as a guide to the behaviour of marker-QTL LD, estimates of minimum marker spacing were made. For a 95% probability of detecting QTL, a microsatellite marker would be required every 0.1 – 2.5 centimorgans, depending on the population used.Short range LD (0 – 5 cM) was observed in all five populations, however the persistence with increasing distance and magnitude of LD varied considerably between populations. Average LD (x2') between populations. The strength and magnitude of LD was found to differ markedly between breeds and aligned closely with both observed levels of genetic diversity and expectations based on breed history. This confirmed that breed specific information is likely to be important for genome wide selection and during the design of successful genome scans where tens of thousands of markers will be required.Sheep populations were selected which were inbred (Macarthur Merino), highly heterogeneous (Merino) or intermediate between these two extremes. This facilitated analysis and comparison of LD (x Mapping genes of interest within animal genomes has been a lengthy and expensive task. In the past, the technique of choice has been within family linkage analysis, requiring the construction of large multigenerational pedigrees. A faster and more economical way to narrow the genetic interval surrounding a gene of interest is through whole genome scans and linkage disequilibrium (LD) mapping. The power of LD mapping lies in its ability to exploit historical recombination within populations of unrelated animals to track the sequence variations which contribute to phenotypic variation. Linkage disequilibrium refers to the ability of an allele from one marker to predict the allelic status at a second marker. The extent of LD serves to inform the number of markers required for a whole genome scan. A population with extensive LD will require a lower marker density as large tracts of the genome will be redundant to those surrounding it. Conversely if LD persists over short distances many more markers will be required to obtain the same power to detect association. Recombination events, population dynamics including drift and admixture as well as breed selection bottlenecks all serve to influence the extent of LD. With this in mind, it is important to quantify the extent of LD within different breeds as this is likely to have an impact on the success of gene mapping experiments.The potential application of LD has prompted investigation into its magnitude and persistence within a number of livestock species including cattle -4, pig ,6 and shAN = 8.13), gene diversity (HE = 0.70) and allelic richness (AR = 8.13). The MER also appeared the most distinct as measured by private allelic richness (pAR = 0.58). The closed population of Macarthur Merinos (EMAI) contained the lowest amount of diversity, with estimates of AN (3.03) AR (3.13) and pAR (0.09) less than half that of the next lowest population . Information describing the chromosomal location and the polymorphism observed at each marker is contained within Additional file FST (Table FST = 0.035), indicating of the five groups analysed, these two are the most closely related. The next lowest FST was observed between the MER and MxB (FST = 0.043). This is likely a reflection of the common Merino contribution to both populations. The highest FST values were observed for every pair-wise combination of populations which included the EMAI animals. A cluster based method was used to estimate the minimum number of sub-populations (K) required to explain the total sum of genetic variation observed [K = 4) differentiated the MER, MxB and EMAI as distinct populations, however the fourth cluster contains both the WFS and PD. The undifferentiated genetic unit containing both the WFS and PD is in keeping with the low FST reported for these breeds and is also consistent with breed history, as the White Faced Suffolk was founded in part by the Poll Dorset. Cluster analysis also illustrated subpopulation diversity. Figure The level of relatedness between ovine populations was investigated by calculation of pair-wise ST Table . The smaobserved . Figure 2', a standardised chi-square statistic suitable for use with multi-allelic markers [2' derived from chromosome 18 marker pairs were plotted as a function of increasing genetic distance  exceeded the average derived from non-syntenic markers (orange line) for closely spaced markers in each of the five populations tested. For example, average LD for markers separated by less than 5 cM in WFS (x2' = 0.167 ± 0.076) was well above the average observed using non-syntenic markers in the same population  [Linkage disequilibrium was estimated for all marker pairs using the metric x markers . The val x2' = 0.7 ± 0.076The proportion of microsatellite pairs in significant LD was determined as a function of genetic distance Table . As expebj) used to quantify this curve for each population. The value for bj is inversely proportional to the extent of LD, meaning high values of bj indicate a low persistence of disequilibrium with distance [b = 9.015) followed by the MxB (4.875), PD (1.066), WFS (0.802) and EMAI (0.239).To examine the decline in LD, decay with distance was modelled and plotted  and the only other investigation of LD in sheep which employed D' [bj) was calculated for each population. Despite the marked variation in population heterozygosity and genetic variability shown in the diversity and structure analyses  using a probabilistic relationship (see equation 4). This used the proportion of marker pairs which display LD in a given range (LDR) to estimate the probability of detecting marker-QTL LD (PR). A genome scan performed using unrelated animals and markers spaced at 2 cM intervals is predicted to identify 99% of QTL within the Macarthur Merino population , a total of 35,000 markers would be required at 0.1 cM intervals across the genome. This minimum marker number is reduced 5 – 8 fold when the other commercial sheep populations are considered  and its decay with distance was measured within five sheep populations across a single chromosome OAR18). Studies which use multi-allelic markers to measure LD in livestock species have mainly calculated D' 8. Studie,5, howev2' ,16. Compce Table  and manyOnly one previous investigation reported on the level of LD found within sheep populations . These a2' metric, was found to differ markedly between breeds. Table bj, the coefficient of LD decay, for the five populations. LD decayed fastest within the commercial Merino (b = 9.02). Conversely, LD persisted over the largest distance and decayed slowest within the Macarthur Merino population (b = 0.24). This neatly fits both the known breed history for each population and the objective measures of genetic diversity  based on markers spread across the genome . The majThe study consisted of 460 Australian commercial sheep from four populations; White Faced Suffolk , Poll Dorset , Merino  and Merino × Border Leicester . Animals were selected from between 3 and 11 properties across Australia to ensure the recruitment of as many unrelated individuals as possible. The MER is a wool breed, the PD and WFS meat breeds and the MxB a terminal composite which has been selected for both wool and meat production. A fifth population was also included in the study. The Elizabeth Macarthur Agricultural Institute Merinos  are maintained as descendants of the original nineteenth century Macarthur Merinos and are a single, closed flock. DNA from the WFS, PD, MER and MxB was prepared from whole blood using QIAamp DNA mini kits  following the manufacture's instructions, whilst DNA from each EMAI animal was extracted using standard phenol/chloroform methods.hh47 from MSP1. MSP2 was used to estimate levels of non-syntenic LD. The forward primer of each marker pair was fluorescently labelled and after multiplex PCR was performed, the products were separated using an ABI 3130 × l Genetic Analyser . GeneMapper v3.7 software  was used for allele sizing and binning. The name, genomic location, observed allelic size range and polymorphism associated with each marker is presented in Addition file 1.Two panels of microsatellites were used. Microsatellite panel 1 (MSP1) consisted of nineteen markers selected to span 113 cM of ovine chromosme (OAR) 18. Marker locations (in cM) were taken from the CompLDB integrated map . The aveHE), average number of alleles per locus (AN), allelic richness (AR) and private allelic richness (pAR) were performed using the complete data set (MSP1 and MSP2) in HP-RARE v1.0 [ was used to evaluate population relatedness using pair-wise estimates of FST. The presence of population substructure was investigated using MSP2 data and an admixture ancestry model-based clustering method as implemented in STRUCTURE v2.2 [K = 1 – 5) were performed using 50,000 Markov chain steps after a burn-in period of 20,000 steps.Four indices of genetic diversity were used to compare the amount of diversity within each ovine population. Calculations of gene diversity (ARE v1.0 . FSTAT 2URE v2.2 . Three r2' (formula 1), has recently been proposed as the measure of choice for use with multi-allelic markers such as microsatellites [ij [Two measures were considered. The first metric, xtellites . The sectellites  as a mulites [ij . D' was ites [ij .where,andDij = P(AiBj) - P(Ai)P(Bj) where P(Ai) is the frequency of allele i at marker A, P(Bj) is the frequency of allele j at marker B. N is the population size and n is the number of alleles at the marker with the smaller number of alleles.2' and D' require two-marker haplotype frequency estimation. This was performed using the Expectation-Maximisation (EM) algorithm and 20 initial conditions for each of 5000 permutation tests. The maximum likelihood estimate of haplotype frequencies was then used to estimate D' and x2'. The EM algorithm, D' and associated p-value calculations were implemented in PyPOP release 0.6.0 [2' was performed with R statistical software [Both xse 0.6.0  whilst tsoftware . LD derisoftware . This prsoftware ij is the LD between microsatellite pair i of population j, separated by genetic distance (in cM) dij, and where bj expresses LD decay with distance for population j, and eij equates to the model residual. Parameter bj was calculated using the nls function set in R.where LDR) which had x2' values exceeding a defined threshold (T) was termed LDR. The number of markers in this range was denoted MR and the probability of finding QTL with LD > T with at least one marker in the given range is (PR). The relationship between each is given in formula 4 as: [Calculations regarding genome wide association studies were made using formula 4. The proportion of marker pairs within a given cM distance range (la 4 as: R) 0 – 5 cM; number of markers (MR) = 5). The value of LDR was determined empirically where T was set to either x2' > 0.2 or the 5% critical threshold for significance. T > 0.2 represents the threshold estimate of detecting QTL between SNP taken from [2 and x2' are comparable. The second question examined the number of markers (MR) required to obtain a 95% probability of detecting QTL given the observed magnitude of LD in each population . The number of markers was converted into the total required for a genome scan assuming a genome size of 3,500 cM.Two separate questions were addressed  as a function of genetic distance within five sheep populations.Click here for fileMean D' as a Function of Increasing Genetic Distance.Click here for fileThe Proportion of Marker Pairs in Significant LD Using D'.Click here for file


Locusta migratoria belongs to a monotypic genus, and is an infamous pest insect with exceptional migratory ability – with dispersal documented over a thousand kilometers. Its distributional area is greater than that of any other locust or grasshopper, occurring in practically all the temperate and tropical regions of the eastern hemisphere. Consequently, minimal population substructuring is expected. However, in marked contrast to its high dispersal ability, three geographical subspecies have been distinguished in China, with more than nine being biologically and morphologically identified in the world. Such subspecies status has been under considerable debate.Highly migratory species are usually expected to have minimal population substructure because strong gene flow has the effect of homogenizing genetic variation over geographical populations, counteracting random drift, selection and mutation. The migratory locust L. m. tibetensis; (2) the South China group, containing locusts from the Hainan islands; this corresponds to the Southeast Asia oriental tropical subspecies L. m. manilensis; (3) the North China group, including locusts from the Northwest and Northern China (the Asiatic subspecies L. m. migratoria), Central China and Eastern China regions. Therefore, the traditional concept on Locusta subspecies status established from Uvarov in 1930s needs to be revised. The three groups of locusts probably have separate evolutionary histories that were most likely linked to Quaternary glaciations events, and derived from different ancestral refugial populations following postglacial expansions.By multilocus microsatellite genotyping analysis, we provide ample genetic evidence for strong population substructure in this highly migratory insect that conforms to geography. More importantly, our genetic data identified an unexpected cryptic subdivision and demonstrated a strong affiliation of the East China locusts to those in Northwest/Northern China. The migratory locusts in China formed three distinct groups, viz. (1) the Tibetan group, comprising locusts from Tibet and nearby West China high mountain regions; this is congruent with the previously recognized Tibetan subspecies, The migratory locust populations in China have differentiated into three genetically distinct groups despite high dispersal capability. While this clarified long-standing suspicions on the subspecific diversification of this species in China, it also revealed that the locusts in the vast area of East China are not the oriental subspecies but the Asiatic subspecies, an unexpected substructuring pattern. The distribution pattern of the three locust groups in China may be primarily defined by adaptive differentiation coupled to Quaternary glaciations events. Our results are of general significance both for locust research and for phylogeographical study of flora and fauna in China, illustrating the potential importance of phylogeographical history in shaping the divergence and distribution patterns of widespread species with strong dispersal ability. Patterns of population genetic differentiation of an organism are shaped by various factors, such as geographical barriers, ecological difference, and historical processes, as well as the dispersal ability of the species. Highly migratory species are usually expected to have minimal population substructure over their distributional ranges because strong gene flow can counteract the isolating effects of geographical distance and physical barriers, and even remove genetic differentiation due to local adaptation . HoweverLocusta migratoria, belonging to a monospecific genus, is one of the most important agricultural pests in the world, and outbreaks were recorded as early as in 13th century BC [Locusta migratoria migratorioides, L. m. tibetensis, and L. m. manilensis. There is considerable debate on the reliability of the subspecific status so identified [The migratory locust ntury BC . Its disntury BC , occurrintury BC . Such a ntury BC ,8. Consentury BC -12, threentified ,13,14, sentified . This isentified ,14.Additionally, phylogeographical study in China as a whole is still underpresented , and gennull) vary from 0.048 (BaM) to 0.217 (F) among population samples  and L. m. manilensis (CC & EC).Figure betensis ; (2) thenilensis ; (3) theST values between the locust populations. FST values between populations of different groups range between 0.023 – 0.149, and are significantly different from zero, indicating that the three groups are moderately to strongly differentiated  and not significantly different from zero for most pair-wise comparisons   of the microsatellite genotype data are shown in Figure K = 3) for the Chinese locust populations (the mean Dirichlet parameter Alpha (α) for degree of admixture is 0.041 at K = 3), corresponding to the three major groups identified in the aforementioned phylogenetic approach . At higher K values (4–8), the locusts in North China group keep splitting further, albeit apparently irregularly . Thus one possibility is that the observed population structure might reflect the effect of gene flow barrier between the island and the continent populations, with simply an artifact of insufficient sampling in South China. Several lines of evidence argue against this suggestion. First, the minimum distance between Hainan islands and the main continent is only 20 km , we believe that historical process, such as historical climatic fluctuations played a primary role. The impact of Pleistocene glaciations cycles on floral and faunal distributions is now well recognized, being a major force shaping population diverging patterns in many organisms [c. 1,000 km eastward into the lowlands, and the northern boundary of broadleaved evergreen/warm mixed forests was displaced southward by c. 1,000 km [Artemisia and Chenopodiaceae [Among the other processes likely involved in the divergence of the locust populations ; they demonstrated a closer genetic affinity of North China locusts to those in central Asia. The lowland shores and surrounding reed-beds and deltas of several important rivers in these areas could serve as favorable breeding areas of the locusts during glacial times.The most likely source of origin of locusts in North China is glacial refugia in the Black, Caspian and possibly Aral Seas basins regions, given that (1) recent climatic modelling studies have identified these regions as potential refugia areas along with the well known Iberian Peninsula, Italy, and the Balkans , (2) thegratoria ,10,27, agratoria  , considering recorded invasions of locusts from the Philippines to the Taiwan islands [ST>0.11 between SCJSJ and L/R), suggesting local isolation of geographical populations over a sufficiently long period of time. Pollen evidence suggests that the southern and eastern edges of the Tibetan plateau had favorable climatic conditions during the last glacial [There is not enough evidence to deduce how the South China group of locusts  , includiHow is the substructuring pattern of locust populations in China maintained given the strong dispersal ability of this insect? Distributional patterns of species are molded by a number of factors, including barriers to dispersal, physical and biological factors that make particular regions of habitat unsuitable for viability and/or reproduction . The actet al. [In summary, the migratory locust populations in China have differentiated into three distinct groups despite high dispersal capability, and the locusts in the vast area of East China are not the oriental subspecies but the Asiatic subspecies. It suggests that these groups of locusts have separate evolutionary histories most likely molded by Quaternary glaciations events, and derived from different ancestral refugial populations following postglacial expansions. The population substructuring patterns observed in the migratory locusts, as reported here and in Chapuis et al. , are of L. m. migratoria , the oriental migratory locust L. m. manilensis (in E. and S. Asia and the Pacific region), the Tibetan migratory locust L. m. tibetensis (in Tibet and nearby W. China), and the African subspecies L. m. migratorioides (in Africa south of the Sahara and off shore Atlantic islands). The last one was used as the outgroup. Solitary Locusta migratoria individuals were collected between 1998 and 2001, stored either in DMSO-salt solution or in absolute ethanol at 4°C (Table ® 400HD (ROX) as the internal size standard. Sizes of the amplified microsatellites were scored by GeneScan 3.7 and manually checked for every allele. A blank control was carried out along each set of DNA extractions and PCR amplifications to monitor any possible cross contamination. Samples that did not amplify at more than two loci were excluded from further analysis.Locust samples studied here includes four subspecies based on locality and morphology, viz. the Asiatic migratory locust Note that mitochondrial DNA of locusts is of little use for population genetic studies due to the presence of numerous pseudogenes in the nuclear genome , and nucHeterogeneity testing was carried out for the two sexes and multiple samples collected from the same areas before pooling them in analysis, and no genetic difference was observed . Basic pST or θ), measures of population subdivision, were calculated using FSTAT 2.9.3 [Wright's F-statistics (FAT 2.9.3  and ARLEL. m. tibetensis), locusts in Mengxin region in North China  the Asiatic subspecies (L. m. migratoria) and locust in East and South China the oriental subspecies (L. m. manilensis) [AMOVA  was performed using the program implemented in ARLEQUIN. AMOVA was used to examine which grouping of the Chinese locusts has the maximum among group variance, and whether the traditional taxonomic classification of the Chinese locust has a high among group variance. The traditional taxonomic classification is as follows Figure ; Table 1ilensis) ,10,12. TPHYLIP ver. 3.6  was used4–106) after a burn-in period of various lengths (104–106). We found that stationarity was reached with a burn-in period of 1 × 104 iterations, and data collection for ≥1 × 105 iterations produced highly consistent results. Independent runs with different K values each with several replicates were then performed using a burn-in period of 1 × 105 iterations and data collection for 1 × 106 iterations, with a model of correlated allele frequencies. A criterion recommended for selecting the appropriate K value is the estimated posterior probability of the data, P(K/X) . For complex datasets with many groups, this criterion is difficult to apply. We have observed that the Dirichlet parameter Alpha (α) for degree of admixture appears to be a more reliable indicator of the 'correct' K value. For the clustering pattern with the most appropriate population structure (at the simulated 'correct K'), admixture among populations (the inferred clusters) should be minimal, and therefore α should be minimal; for values smaller or larger than the 'correct' K, α should always be larger. Thus, the smallest K with the smallest α is most likely the real structure contained in the data. It is expected that departures of data from HWE may lead to overestimating K. While this could particularly be a problem for closely related populations, it should have little influence on divergent populations. Graphical display of the results of STRUCTURE was done with the program DISTRUCT by N. A. Rosenberg [A Bayesian clustering analysis implemented in the program STRUCTURE  was alsoosenberg .Principal component analysis (PCA) was performed with PCA-GEN , incorpoLNY carried out the molecular genetic studies and participated in data analysis. YJJ participated in some field work, genotyping data collection, analysis and coordination, and helped to revise the manuscript. ZSH participated in some field work and genotyping studies. GMH coordinated the study and the Royal Society UK-China joint project, and helped to revise the manuscript. DXZ conceived of and designed the study, carried out field work, participated in data analysis and coordination, and wrote the manuscript. All authors read and approved the final manuscript.Table S1. Pair-wise FST values of locust populations studies here (upper triangle: + statistically significant at 0.05 level; lower triangle: pair-wise FST; pop, population samples that are numbered as in Table Click here for file


Anopheles darlingi is the most important malaria vector in the Neotropics. An understanding of A. darlingi's population structure and contemporary gene flow patterns is necessary if vector populations are to be successfully controlled. We assessed population genetic structure and levels of differentiation based on 1,376 samples from 31 localities throughout the Peruvian and Brazilian Amazon and Central America using 5–8 microsatellite loci.RS = 7.62, mean HO = 0.742), and low levels for the Belize and Guatemalan populations . The Bayesian clustering analysis revealed five population clusters: northeastern Amazonian Brazil, southeastern and central Amazonian Brazil, western and central Amazonian Brazil, Peruvian Amazon, and the Central American populations. Within Central America there was low non-significant differentiation, except for between the populations separated by the Maya Mountains. Within Amazonia there was a moderate level of significant differentiation attributed to isolation by distance. Within Peru there was no significant population structure and low differentiation, and some evidence of a population expansion. The pairwise estimates of genetic differentiation between Central America and Amazonian populations were all very high and highly significant . Both the DA and FST distance-based trees illustrated the main division to be between Central America and Amazonia.We found high levels of polymorphism for all of the Amazonian populations (mean Ne among the populations may have contributed to the observed genetic differentiation. All of the data suggest that the primary division within A. darlingi corresponds to two white gene genotypes between Amazonia (genotype 1) and Central America, parts of Colombia and Venezuela (genotype 2), and are in agreement with previously published mitochondrial COI gene sequences interpreted as incipient species. Overall, it appears that two main factors have contributed to the genetic differentiation between the population clusters: physical distance between the populations and the differences in effective population sizes among the subpopulations.We detected a large amount of population structure in Amazonia, with three population clusters within Brazil and one including the Peru populations. The considerable differences in  Anopheles (Nyssorhynchus) darlingi is the most efficient malaria vector in the Neotropics, and is responsible for transmission of Plasmodium falciparum, P. vivax and P. malariae .Many of the anopheline species responsible for malaria transmission are members of species complexes composed of closely related cryptic species . Most no species , as welliae s.s. ,34,35. Tiae s.s. . The recA. darlingi in the Brazilian Amazon  and Peruvian Amazon, within Central America in Guatemala and Belize, and between the incipient species described as genotypes 1 and 2 . Eight microsatellite loci .All individuals collected from Peru and Brazil were classified as A. darlingi from eastern Amazonian Brazil [ADC107) did not amplify in any of the individuals collected from Peru and three loci  did not amplify in any of the individuals from Belize or Guatemala. Seven loci were genotyped from 350 individuals in Peru, five loci from 276 individuals from Central America , and all eight loci from 57 individuals from BV and 58 from PLT, Brazil (Table N = 254) [N = 381) [A. darlingi. The levels of polymorphism were high in Amazonian Brazil and Peru, and low in Central America, based on the 5 shared loci: in Peru, the number of alleles per locus ranged from 5–11 and the observed heterozygosity from 0.44 – 0.86 ; in Brazil, the number of alleles per locus ranged from 6–22 and the observed heterozygosity from 0.74 – 0.93  [RS = 4.3, mean HO = 0.457)   and from= 0.836) ,38; and,FIS, within some samples from Peru (6 of 49 tests), Brazil , and Central America (4 of 30 tests) indicated heterozygote deficits . The majority of the genetic diversity in A. darlingi is accounted for by within-population differences among individuals.An AMOVA using the five population clusters detected with the Bayesian analysis as the groupings, found that 18.9% of the variance was explained at the among groups level, and 77.9% at the within populations level. In an AMOVA using the two genotypes, Amazonia and Central America, as the groupings, 20.1% of the total variance was explained at the between groups level and 70.1% within populations. All of the global FST = 0.047, 33.3% P < 0.05), except for between GOL and all other subpopulations there was a moderate amount of significant differentiation (FST range of 0.1063–0.1489) , and within the four Amazonian population clusters the mean FST ranged from -0.002 to 0.082 (70.6% P < 0.05) . The mean pairwise estimates of genetic differentiation between Central America and the four population clusters in Amazonia were high (FST = 0.2161 – 0.3625) (Table FST = 0.0751 – 0.1813), particularly between Peru and WCA Brazil, Peru and NEA Brazil, and NEA and WCA Brazil (Table P < 0.05) after correction for multiple tests. Estimates of gene flow revealed little or no recurrent gene flow between genotypes 1 and 2, and reduced gene flow among the population clusters in Amazonia  there was mostly low non-significant differentiation . Loci ADC137 and ADC01 (only amplified in the Amazonian populations) did not show significant differentiation among the Amazonian population clusters. The highest level of differentiation among all of the Amazonian population clusters was at locus ADC02.The significant differentiation between genotypes 1 (the 4 population clusters in Amazonian Peru and Brazil) and 2  was genomewide, as shown by independent analyses for each of the five loci revealing significant differentiation between them (data not shown). The differentiation between the genotypes varied in magnitude, with the highest level of differentiation observed at locus R2 = 0.5972, P = 0.001; Amazonia: R2 = 0.3088, P = 0.0001) , there was a significant positive association between distance and FST. The results suggest that the genetic differentiation observed between A. darlingi populations is primarily due to restricted gene flow by geographic distance. Although, between Peru populations and those in NEA Brazil and SEAC Brazil the average distance is 2451 km and 2270 km and the mean pairwise FST is 0.1484 and 0.0959, respectively; and, the average distance between Peru and Central American populations is 2923 km and the mean pairwise FST is 0.3625, which demonstrates that the large genetic differentiation is not accompanied by correspondingly large difference in geographic distance.Tests of isolation by distance were performed separately for all of the populations together, for each population cluster, and for all of Amazonia. For all of the populations together (4–5028 km) and all of Amazonia (4–2878 km), there was a significant positive correlation between geographic distance and genetic differentiation based on the Mantel test . Under the linkage disequilibrium, the highest overall effective population size estimates for the five population clusters were observed in Central America  and NEA Brazil , based on the 5 common loci. The overall Ne estimate for Peru was high based on 5 loci , and more moderate based on all 7 amplified loci , which demonstrates a discrepancy in the calculation based on 5 vs. 7 loci. The Ne estimates for BV and PLT, Brazil  and including only the 5 loci amplified from all populations, we were able to compare the results of these two treatment methods. The differentiation and mean heterozygosity  [A. gambiae populations in west Africa separated by > 200 km (FST = 0.034–0.167) [FST = 0.104) [A. funestus populations from west, central, and eastern Africa (FST = 0.110) [A. darlingi [e.g., rivers or mountains).Substantial population structure was found in Amazonia, which was undetected with more conservative nuclear markers and isozymes ,10,24,71ntiation ,73. The = 0.114) , among A4–0.167)  and thos= 0.104) , as welldarlingi , althougdarlingi  and betwdarlingi . The maiWithin the WCA Brazil population cluster there was little genetic structure and differentiation, and the isolation-by-distance model explained nearly all of the differentiation observed . Within A. darlingi in the Peruvian Amazon that detected high homogeneity among populations (within 60 km) irrespective of different habitat types [Ne among the Peru subpopulations (93.6 – 8) that may contribute to the small significant differentiation observed among many of the localities. Anopheles darlingi appears to be panmictic in this region of Peru. There is some evidence of a population expansion in MAZ, NAU, and SAE. The expansion in NAU and SAE is reflected in a very large Ne in these localities. Prior to 1991, A. darlingi had not been reported around Iquitos, the major Peruvian Amazon city [A. darlingi into the Peruvian Amazon possibly from PLT, Brazil, where there is the most genetic similarity, in the early 1990's and the resultant increase in malaria [Within Peru there was no significant population structure and low differentiation among the seven subpopulations, in agreement with an earlier RAPD-PCR analysis of at types . We detezon city ,44. This malaria ,46.RS = 4.3, mean HO = 0.457) as compared to within Amazonia , and there was no evidence of isolation-by-distance. Low haplotype and nucleotide diversity was also observed within Central America with mtDNA COI sequences as compared to within South America [A. darlingi populations from only a few individuals from the Colombian population is also consistent with the data [FST range of 0.1063–0.1489, all P values < 0.05), GOL is separated from the other subpopulations by the Guatemalan Highlands and the Maya Mountains  and Central America (genotype 2) [white, ribosomal ITS, and mitochondrial COI sequence data together established a deep divergence between genotypes 1 and 2 [A. dirus, A. scanloni, and A. baimaii  in Thailand (mean FST = 0.263) [A. gambiae M and S forms  . Earlier 1 and 2 ,28, inte 1 and 2 . In the  1 and 2 . The ind 1 and 2 ,68. The = 0.263) , A. gamb 0.1–0.3 ; mean FS = 0.203 ), and be.12–0.27 ; mean FS = 0.349 ) using m = 0.349 ,28, thatFST values do not translate into meaningful rates of gene flow [FST, and the differentiation will be underestimated as compared to neutral equilibrium values. Therefore, the low level of differentiation measured within Peru and WCA Brazil may be an underestimation as well as an overestimation of gene flow; and, the differentiation and gene flow between the genotypes and population clusters may be underestimating the current degree of isolation. Despite possible departures from MDE, our large sample sizes and number of populations add statistical power to our study.With the detection of a recent population expansion or the departure from MDE in many of the populations in Amazonia and two populations in Central America, the ene flow . In the A. darlingi between Amazonia (genotype 1) and Central America, parts of Colombia and Venezuela (genotype 2). It appears that two main factors have contributed to the genetic differentiation between the population clusters: physical distance between the populations and the differences in effective population sizes among the subpopulations. Knowledge of A. darlingi's population genetic structure is essential to an understanding of malaria epidemiology and for the success of potential genetic control strategies (release of transgenic mosquitoes refractory to Plasmodium infection) that will rely on the ability to target all populations and will require a thorough understanding of the forces that produce and maintain the population structure, especially gene flow [A. darlingi's population structure, and specifically these genotypes, into account.Overall, there was a large amount of population structure in Amazonia, and a primary division within ene flow . ControlA. darlingi specimens, carried out the analyses, and drafted the manuscript. JHV did most of the PCR and genotyping at the New York State Department of Health Wadsworth Center Genotyping Core facility, assisted with the structure analysis, and gave general microsatellite analysis advice. SPY managed all of the A. darlingi collections in Peru. MMP together with JEC provided samples from PLT, BV and those from eastern Amazonian Brazil [A. darlingi from Guatemala. NLA provided all of the A. darlingi samples from Belize. JEC participated in the study design, coordinated collaborations and A. darlingi collections, and helped revise the manuscript. All authors read and approved the final manuscript.LM designed the study, extracted and prepared the n Brazil . VMS pron Brazil . NP provA. darlingi in Central America, Peru and Brazil. The data provided represent the number of alleles, heterozygosity and inbreeding coeffient values of each microsatellite locus at each locality.Summary of microsatellite variation at 5–8 loci for Click here for file