Increase in Quantitative Variation After Exposure to Environmental Stresses and/or Introduction of a Major Mutation: G × E Interaction and Epistasis or Canalization?

Why does phenotypic variation increase upon exposure of the population to environmental stresses or introduction of a major mutation? It has usually been interpreted as evidence of canalization (or robustness) of the wild-type genotype; but an alternative population genetic theory has been suggested by J. Hermisson and G. Wagner: “the release of hidden genetic variation is a generic property of models with epistasis or genotype–environment interaction.” In this note we expand their model to include a pleiotropic fitness effect and a direct effect on residual variance of mutant alleles. We show that both the genetic and environmental variances increase after the genetic or environmental change, but these increases could be very limited if there is strong pleiotropic selection. On the basis of more realistic selection models, our analysis lends further support to the genetic theory of Hermisson and Wagner as an interpretation of hidden variance.A common experimental observation in quantitative genetics is a higher phenotypic variance for quantitative traits in populations that carry a major mutation or are exposed to environmental stresses (e.g., heat shock) (Scharloo 1991; for a recent review see Gibson and Dworkin 2004). Part of the added variance must be genetic because the population responds to artificial selection. The lower variability of the wild type than that of the mutants has been interpreted as evidence for robustness or canalization (Waddington 1957): that is, under the new condition the magnitudes of gene effects across all trait loci increase relative to the original condition. The importance of canalization has been recognized for a long time and has been the subject of renewed interest recently (see de Visser et al. 2003 and Hansen 2006 for reviews).An alternative population genetic theory has been proposed by Hermisson and Wagner (2004), who suggest that the increase in genetic variance V_G after the change in environmental conditions or genetic background is a generic property of the population, with no need to introduce canalization (Waddington 1957). The theory appears simple. Under mutation–selection balance (MSB), the mutant alleles are at a selective disadvantage and there is a negative correlation between frequencies and effects of mutations: mutant alleles of small effects on the trait segregate at intermediate frequencies. After the change in genetic or environmental background, gene effects consequently change due to G × E interaction or epistasis, which reduces the negative correlation because genes that were previously of small effects and at intermediate frequencies may now have large effects. That is, the frequencies of alleles are determined by the previous MSB, while their new effects are at least partly determined by the new conditions. The genetic variance will therefore increase.Hermisson and Wagner (2004) found that the predicted increase in genetic variance can be substantial; however, the predicted increase is highly sensitive to the population size and can increase without bound with increasing population size (see their Figure 2 and Equation 16). Genetic variance would enlarge with the population size within a small population (Lynch and Hill 1986; Weber and Diggins 1990), but becomes insensitive to the population size within large populations (Falconer and Mackay 1996, Chap. 20). Hence the unbounded increase under the novel environmental condition appears to us as a downside of their theory, even though the predicted increase can be reduced if the changed environmental condition is not novel but there is previous adaptation to it (see their Figure 3).Open in a separate windowFigure 2.—Influence of the pleiotropic effect (s_p) on the increase of genetic variance Δ_G in units of the interaction parameter ξ for a “typical” situation with strength of stabilizing selection ω² = 0.1μ², mutation rate λ = 0.1 per haploid genome per generation, and population size N_e = 10⁶. The allelic pleiotropic effect on fitness and its variance effect on the trait independently follow gamma distributions with shape parameters β_s and β_v, respectively. The mean of a² across loci is E(v) = E(a²) = 10⁻⁴μ².Open in a separate windowOpen in a separate windowFigure 3.—Influence of shapes of distributions of mutational effects on (a) the variances at mutation–selection balance and (b) their increases after the genetic or environmental change. The squares represent the genetic variance and its increase and the triangles the environmental variance and its increase. The mutation rate is λ= 0.1 per haploid genome per generation, the population size is N_e = 10⁹, and the strength of real stabilizing selection is ω² = 0.1μ². Allelic effects on trait value (a), fitness (s), and residual variance (b) are assumed to be independently distributed such that v = a² follows a gamma () distribution with mean 10⁻⁴μ², s follows gamma (β_s) with mean s_p = 0.05, and b follows gamma (β_b) with mean 10⁻⁴μ².The basic model that Hermisson and Wagner (2004) employed is that the quantitative trait is under real stabilizing selection and mutant alleles have effects on the focal trait only by changing its so-called locus genetic variance. At the mutation–real stabilizing selection balance, some mutants can segregate at intermediate frequencies because of their small effects and therefore weak selection; and there are more such mutants the more strongly leptokurtic is the distribution of effects at individual loci. The unbounded increase of Hermisson and Wagner (2004) results from such a gene-frequency distribution; but it has been shown (see Barton and Turelli 1989; Falconer and Mackay 1996; Lynch and Walsh 1998) that solely stabilizing selection, whether modeled with a Gaussian (Kimura 1965) or a house of-cards approximation (Turelli 1984) or even the generalized form of Hermisson and Wagner (2004) (i.e., their Equation 14), cannot provide a satisfactory explanation for the high levels of genetic variance observed in natural populations under realistic values of mutation and selection parameters.A common observation is that one trait is controlled by many genes and one gene can influence many traits; i.e., pleiotropy is ubiquitous (Barton and Turelli 1989; Barton and Keightley 2002; Mackay 2004; Ostrowski et al. 2005). Recent detailed studies suggest that pleiotropy calculated as the number of phenotypic traits affected varies considerably among quantitative trait loci (QTL) (Cooper et al. 2007; Albert et al. 2008; Kenney-Hunt et al. 2008; Wagner et al. 2008). Such pleiotropic effects must influence the magnitude of the variance. Though some genes have little effect on the focal trait, they almost certainly affect other traits and therefore are not neutral. The inclusion of pleiotropic effects on fitness strengthens the overall selection on mutant alleles and, assuming such pleiotropic effects are mainly deleterious, maintains them at low frequencies. The genetic variance for a trait is therefore likely to be maintained at lower levels than that under only real stabilizing selection on the trait alone (Tanaka 1996). Although the gene-frequency distribution is much more extreme under this joint model, the relevant rate of mutation is genomewide and hence is much larger than that where mutation affects only the focal trait as is assumed in the real stabilizing selection model (Turelli 1984; Falconer and Mackay 1996). Taking into account empirical knowledge of mutation parameters, a combination of both pleiotropic and real stabilizing selection appears to be a plausible mechanism for the maintenance of quantitative genetic variance (Zhang et al. 2004). If pleiotropic selection is much stronger than real stabilizing selection, the association between frequency and effect of mutant alleles is weaker than that for a real stabilizing selection model. Further, if overall selection is stronger than recurrent mutation, the frequency distribution of mutant alleles will be extreme. Under those situations, the increase of genetic variance after the genetic or environmental change will be kept at lower levels than that of Hermisson and Wagner (2004), and hence the unbounded increase could be avoided.Further, Hermisson and Wagner (2004) assume that the environmental variance is not under genetic control (i.e., the variance of phenotypic value given genotypic value is the same for all genotypes) and therefore is not subject to change. This assumption conflicts with the increasingly accumulating empirical data that indicate otherwise (Zhang and Hill 2005; Mulder et al. 2007 for reviews). Direct experimental evidence is available that mutation can directly affect environmental variance, V_E (Whitlock and Fowler 1999; Mackay and Lyman 2005), and Baer (2008) provides what is perhaps the first clear demonstration that mutations increase environmental variances, on the basis of data for body size and productivity of Caenorhabditis elegans, and finds that the magnitudes of the increases are of the same order as those in the genetic variance.As real stabilizing selection on phenotype favors genotypes possessing low V_E (Gavrilets and Hastings 1994; Zhang and Hill 2005), a mutant that contributes little to V_E is more favored by stabilizing selection than one that contributes a lot. With all else being the same, mutants with small effect on V_E thus segregate at relatively high frequencies at MSB. That is, there is a negative correlation between the effect on V_E and the frequency of mutant genes. After the genetic or environmental change, some mutants that were previously of small effects on V_E have large effects due to G × E interaction or epistasis while their frequencies remain roughly the same as in the previous MSB. This certainly increases environmental variance.In this note, we first assume that mutant alleles can affect only the mean value of a focal quantitative trait and otherwise affect fitness through their pleiotropic effects (Zhang et al. 2004) and try to answer the following questions: How will the conclusion of Hermisson and Wagner (2004) be affected by taking into account the pleiotropic effect of mutants? Can the “unbounded increase” be avoided? We then further assume that mutant alleles can also directly affect the environmental variance of the focal trait (Zhang and Hill 2008) and investigate how both V_G and V_E change following the genetic or environmental change in the population.     

New insights into vegetation dynamics and settlement history in Hümmling,north-western Germany,with particular reference to the Neolithic

Palynological investigations on two well-dated peat profiles provide insights into Neolithic vegetation and settlement history from Hümmling in north-western Germany. The site selections allow comparisons between local and regional vegetation changes and are used to estimate the extent of Neolithic influence on the vegetation. The interpretation of the fossil spectra relied on radiocarbon dating, evaluation of pollen indicator taxa, non-pollen palynomorphs and multivariate techniques. During the late Mesolithic the vegetation was dominated by mixed oak forests while openings in forest cover were detected, with a decline in elm reflected in the regional pollen record around 4250 cal. b.c. The presence of humans is shown by settlement indicators that are first recorded at ca. 3800 cal. b.c. Vegetation changes were small between 4300 and 3600 cal. b.c. This suggests that regional vegetation was relatively resilient to small-scale disturbances. Possible indications of grazing were recorded in the spectra of the local pollen profile but there is no clear-cut evidence for Neolithic activity. Between 3520 and 2260 cal. b.c. decreases in forest cover were inferred from both profiles and increases in settlement indicators reflect farming activity. These changes coincide with the emergence in the area of the Funnel Beaker Culture and the subsequent Single Grave Culture. Both profiles suggest that settlement probably ceased between ca. 3230 and 3050 cal. b.c. This lull or cessation in activity was probably regional in character. After 2260 cal. b.c. human impact on the vegetation decreases and woodlands regenerate. The longevity of the regeneration phase—ca. 690 years—was probably connected with the low resilient capability of the vegetation on the poor soils.     

Genomic Consequences of Background Effects on scalloped Mutant Expressivity in the Wing of Drosophila melanogaster

We have evaluated the extent to which SNPs identified by genomewide surveys as showing unusually high levels of population differentiation in humans have experienced recent positive selection, starting from a set of 32 nonsynonymous SNPs in 27 genes highlighted by the HapMap1 project. These SNPs were genotyped again in the HapMap samples and in the Human Genome Diversity Project–Centre d''Etude du Polymorphisme Humain (HGDP–CEPH) panel of 52 populations representing worldwide diversity; extended haplotype homozygosity was investigated around all of them, and full resequence data were examined for 9 genes (5 from public sources and 4 from new data sets). For 7 of the genes, genotyping errors were responsible for an artifactual signal of high population differentiation and for 2, the population differentiation did not exceed our significance threshold. For the 18 genes with confirmed high population differentiation, 3 showed evidence of positive selection as measured by unusually extended haplotypes within a population, and 7 more did in between-population analyses. The 9 genes with resequence data included 7 with high population differentiation, and 5 showed evidence of positive selection on the haplotype carrying the nonsynonymous SNP from skewed allele frequency spectra; in addition, 2 showed evidence of positive selection on unrelated haplotypes. Thus, in humans, high population differentiation is (apart from technical artifacts) an effective way of enriching for recently selected genes, but is not an infallible pointer to recent positive selection supported by other lines of evidence.IN the last 50,000–100,000 years (KY), humans have expanded from being a rare species confined to parts of Africa and the Levant to their current numbers of >6 billion with a worldwide distribution (Jobling et al. 2004). Paleontological and archaeological evidence suggests that key aspects of modern human behavior developed ∼100–50 KYA in Africa (Henshilwood et al. 2002) and behaviorally modern humans then expanded out of Africa ∼60–40 KYA (Mellars 2006). The physical and biological environments encountered outside Africa would have been very different from those inside and included climatic deterioration reaching a glacial maximum ∼20 KYA and subsequent amelioration that permitted the development of agricultural and pastoral lifestyles in multiple independent centers after ∼10 KYA. Neolithic lifestyles would have led to further changes including higher population densities, close contact with animals, and novel foods, in turn leading to new diseases (Jobling et al. 2004). It is likely that genetic adaptations accompanied many of these events.Adaptation, or positive natural selection, leaves an imprint on the pattern of genetic variation found in a population near the site of selection. This pattern can be identified by comparing the DNA variants in multiple individuals from the same and different populations and searching for signals such as unusually extended haplotypes (extended haplotype homozygosity, EHH) (Voight et al. 2006; Sabeti et al. 2007; Tang et al. 2007), high levels of population differentiation (International Hapmap Consortium 2005; Barreiro et al. 2008; Myles et al. 2008), or skewed allele frequency spectra (Carlson et al. 2005). These signals become detectable at different times after the start of selection and are all transient, being gradually eroded by both molecular processes such as mutation, recombination, or further selection and population processes such as migration or demographic fluctuations, with the survival order extended haplotypes < population differentiation < allele frequency spectra (Sabeti et al. 2006). The absolute timescales of survival are not well understood, but extended haplotype tests typically detect selection within the last 10 KY (Sabeti et al. 2006) while unusual allele frequency spectra may detect much older selection. For example, it has been suggested that the signal associated with the FOXP2 gene (Enard et al. 2002) may predate the modern human–Neanderthal split ∼300–400 KYA (Krause et al. 2007), although such an interpretation has been questioned (Coop et al. 2008). However, despite significant uncertainties and limitations, population-genetic analyses are well placed to provide insights into many of the important events within the timescale of recent human evolution.In principle, it should be possible to survey the genome for sites of selection and then interpret this catalog in the light of archaeological, climatic, and other records. Progress toward such a goal has, however, been limited: many factors can confound the detection of selection and only genotype data from previously ascertained SNPs, rather than full resequence data, have thus far been available throughout the whole genome. In practice, the strategy used has therefore been to search the genome for signals that can be detected in available genotype data, such as extended haplotypes or population differentiation, and evaluate the significance of the regions identified by comparing them with empirical distributions of the same statistic, models that incorporate information about the demography, or biological expectations (McVean and Spencer 2006). However, it remains unclear how effective this strategy is: What false positive and false negative rates are associated with its applications? Further evaluation is desirable.The International HapMap Project has carried out the highest-resolution study so far of genetic variation in a set of human populations. In an article published in 2005, genotypes of >1 million SNPs were reported from 270 individuals with ancestry from Africa (Yoruba in Ibadan, Nigeria: YRI), Europe (Utah residents with ancestry from northern and western Europe: CEU), China (Han Chinese in Beijing, China: CHB), and Japan (Japanese in Tokyo, Japan: JPT) (International HapMap Consortium 2005). This article highlighted 32 SNPs from 27 genes that showed particular evolutionary interest because of a combination of two factors: they were nonsynonymous, that is, they changed an amino acid within a protein-coding gene and thus were likely to alter biological function, and they also exhibited a high level of population differentiation equal to or exceeding that of rs2814778, a SNP that is associated with strong biological evidence for population-specific selection. This SNP underlies the FY*0 (Duffy blood group negative) phenotype; FY*0 homozygotes do not express the Duffy blood group antigen on red blood cells and are consequently highly resistant to infection by the malarial parasite, Plasmodium vivax. The *0 allele is nearly fixed in Africa and rare outside, and it is widely believed that this is due to selection for resistance to vivax malaria.However, a number of studies have emphasized that large differences in allele frequency between populations can arise without positive selection: for example, a highly differentiated SNP in the Neuregulin I gene was not accompanied by unusual patterns in adjacent SNPs (Gardner et al. 2007), and large frequency differences can be quite common in empirical data sets, particularly in comparisons between Africa or America and the rest of the world, where population bottlenecks and “allele surfing” may have occurred during the exit from and entrance to these continents, respectively (Hofer et al. 2009). We wished to measure the extent to which the high population differentiation observed at the 27 HapMap genes might have resulted from positive selection and the extent to which it reflected other origins such as demographic factors, chance, or errors. We therefore retyped the same SNPs in the HapMap samples and in a large additional set of human populations and applied alternative tests for selection, either based on long-range haplotypes or based on full resequence data. For the latter, sequence data for 5 of the genes were available from public sources, and four new data sets were generated for this project. We found that, while genotyping errors led to some artifactual high differentiation signals, population differentiation was a useful but by no means infallible guide to recent selection detected by other methods.     

Paternal Leakage and Heteroplasmy of Mitochondrial Genomes in Silene vulgaris: Evidence From Experimental Crosses

The inheritance of mitochondrial genetic (mtDNA) markers in the gynodioecious plant Silene vulgaris was studied using a series of controlled crosses between parents of known mtDNA genotype followed by quantitative PCR assays of offspring genotype. Overall, ∼2.5% of offspring derived from crosses between individuals that were homoplasmic for different mtDNA marker genotypes showed evidence of paternal leakage. When the source population of the pollen donor was considered, however, population-specific rates of leakage varied significantly around this value, ranging from 10.3% to zero. When leakage did occur, the paternal contribution ranged from 0.5% in some offspring (i.e., biparental inheritance resulting in a low level of heteroplasmy) to 100% in others. Crosses between mothers known to be heteroplasmic for one of the markers and homoplasmic fathers showed that once heteroplasmy enters a maternal lineage it is retained by ∼17% of offspring in the next generation, but lost from the others. The results are discussed with regard to previous studies of heteroplasmy in open-pollinated natural populations of S. vulgaris and with regard to the potential impact of mitochondrial paternal leakage and heteroplasmy on both the evolution of the mitochondrial genome and the evolution of gynodioecy.MATERNAL inheritance of the mitochondrial genome seems to be the usual case in angiosperms, with only occasional reports of paternal leakage (Birky 2001). The mode of inheritance has several interesting consequences for the evolution of the plant mitochondrial genome and plant mating systems. One is that maternal inheritance contributes to homoplasmy, or within-individual genetic homogeneity, in that it precludes the mixing of mitochondrial genomes of differing origin at the time of fertilization. Homoplasmy is further enforced by repeated sampling events associated with the transmission of a finite number of mitochondria from mother to daughter cells during mitotic or meiotic events (Birky 2001). This within-individual genetic drift is sometimes known as vegetative sorting (McCauley and Olson 2008). Paternal leakage would allow the possibility of mitochondrial heteroplasmy (within-individual cytoplasmic genetic diversity) when it leads to some degree of biparental inheritance. With homoplasmy the mitochondrial genome evolves as an effectively asexual lineage. While intra- or intermolecular recombination associated with repeat sequences often found in noncoding regions of plant mitochondrial genomes can result in structural rearrangements (Mackenzie and McIntosh 1999), there is limited opportunity for such events to generate novel genotypic combinations. Heteroplasmy enhances the possibility that recombination can occur between divergent genomes and generate novel genotypes.A second consequence of the mode of inheritance concerns the evolution of gynodioecy or the co-occurrence of female and hermaphrodite individuals. This phenomenon is often ascribed to the interaction between mitochondrial genes that confer cytoplasmic male sterility (CMS) and nuclear genes, known as restorers, that counteract the effects of CMS and restore male function (Frank 1989), a topic that continues to be the object of much study by plant evolutionary biologists (McCauley and Bailey 2009). The evolutionary dynamics of these interactions are usually evaluated on the basis of the assumption of pure maternal inheritance of mitochondrial genes. This maximizes the potential for genetic conflict between a CMS gene and its restorers, since a difference in the mode of inheritance between the mitochondrial and nuclear genomes results in a difference in their respective currency of fitness. With paternal leakage, pollen production is no longer unimportant for the fitness of the mitochondrial genes carried by a hermaphrodite (Wade and McCauley 2005).Recently there has been increased appreciation of the potential role of paternal leakage and heteroplasmy in the evolution of the mitochondrial genomes of a broad array of eukaryotes (Kmiec et al. 2006; White et al. 2008). This includes studies of the plant genus Silene, which have provided evidence of at least occasional paternal transmission of mitochondria in several species, as well as mitochondrial heteroplasmy. Observations supporting the possibility of mitochondrial paternal leakage and heteroplasmy in the genus Silene are especially intriguing given the occurrence of gynodioecy in this genus. Evidence of paternal leakage comes primarily from two types of observation. First are observations of mitochondrial genotypes that most likely result from intra- or intergenic recombination (see studies by Städler and Delph 2002 for S. acaulis and McCauley et al. 2005; Houliston and Olson 2006; and McCauley and Ellis 2008 for S. vulgaris). Second, direct evidence of heteroplasmy in S. vulgaris comes from studies that utilize real time quantitative PCR (q-PCR) to quantify the within-individual diversity of mitochondrial marker genes (Welch et al. 2006; Pearl et al. 2009). The likelihood that heteroplasmy is due to paternal leakage in S. vulgaris was inferred from observations by Pearl et al. (2009) of heteroplasmic offspring of open-pollinated homoplasmic mothers. A second observation by Pearl et al. (2009) bears on the inheritance of heteroplasmy. Heteroplasmic mothers were more likely than homoplasmic mothers to produce heteroplasmic offspring, but this heteroplasmy was also lost between generations in many cases, in keeping with the theory of vegetative sorting.One interesting result from Welch et al. (2006) and Pearl et al. (2009) is that incidents of heteroplasmy and apparent leakage do not seem to be evenly distributed among the natural populations from which samples were taken. Most of the heteroplasmic individuals documented by Welch et al. (2006) were from just one of the three populations studied. Similarly, while the apparent leakage rate observed by Pearl et al. (2009) was ∼8% when all 14 study populations are considered together, if the rate is calculated on a population-by-population basis, it exceeds 10% in 3 of them and is zero in 3 others (see their Supplementary Table 2). Population-to-population variation in the rate of leakage might suggest that variable environmental conditions influence leakage or that any genetic variation that influences the traits that determine mode of inheritance is geographically structured.Much of the current evidence for mitochondrial paternal leakage in Silene is indirect in that it is derived from observations of apparent recombinant genotypes or of heteroplasmy. While this evidence is compelling, alternate explanations, such as mutational hotspots within the genes under study, are at least possible. Even the evidence of leakage presented by Pearl et al. (2009) was based on mother–offspring comparisons of individuals collected from natural populations, in which the pollen donor was unknown. Though some evidence for paternal leakage and heteroplasmy reported in McCauley et al. (2005) comes from controlled crosses of S. vulgaris, those crosses were few in number and any incidents of heteroplasmy were based on qualitative observations rather than the q-PCR method used more recently. Thus, it would be valuable to conduct a large number of controlled crosses between S. vulgaris individuals of known mitochondrial genotype to assay directly the rate and magnitude of paternal leakage and any resulting heteroplasmy and also to assay the degree to which heteroplasmy is transmitted between generations. Taken together, this information would allow one to begin to ask, not only about the origins of mitochondrial heteroplasmy in Silene, but also about the degree to which the frequency of mitochondrial heteroplasmy in natural populations results from gains through paternal leakage vs. loss from vegetative sorting. Furthermore, since the among-population heterogeneity in levels of heteroplasmy and leakage summarized above could be due to either real differences between populations in factors promoting these phenomena or simply ascertainment bias associated with differences between populations in the level of polymorphism of the q-PCR markers, it would be valuable to test for a population effect in an experimental setting.Here we present comparisons of parent and offspring mitochondrial genotypes obtained by q-PCR following three types of controlled crosses in which either (1) the two parents are homoplasmic for different alleles of a marker gene, (2) both parents are homoplasmic for the same allele, or (3) the maternal parent is heteroplasmic. In the first cross type any contribution of the pollen donor to the offspring mitochondrial genotype would be detectable. This quantifies the likelihood of leakage. Knowing the natural population from which the pollen donor and pollen recipient trace their respective ancestry allows investigation of the possibility of a population effect without the confounding effects of varying levels of marker polymorphism present in field studies. In the second cross type, any observed mother–offspring difference would most likely be due to error of some sort (or the unlikely possibility of mutation at the SNP that defines the marker). Thus, these crosses act as a control by estimating the experimental error rate. The third type of cross measures the frequency with which heteroplasmy is transmitted maternally to offspring or is lost. Taken together this study represents what is, to our knowledge, the first attempt to combine experimental crosses and q-PCR methodology to examine mitochondrial genome inheritance and heteroplasmy in a plant species; important information given that it is not yet clear how widespread mitochondrial leakage and heteroplasmy are in the genus Silene, in other gynodioecious species, or in other species of plants in general.     

ObStruct: A Method to Objectively Analyse Factors Driving Population Structure Using Bayesian Ancestry Profiles

Bayesian inference methods are extensively used to detect the presence of population structure given genetic data. The primary output of software implementing these methods are ancestry profiles of sampled individuals. While these profiles robustly partition the data into subgroups, currently there is no objective method to determine whether the fixed factor of interest (e.g. geographic origin) correlates with inferred subgroups or not, and if so, which populations are driving this correlation. We present ObStruct, a novel tool to objectively analyse the nature of structure revealed in Bayesian ancestry profiles using established statistical methods. ObStruct evaluates the extent of structural similarity between sampled and inferred populations, tests the significance of population differentiation, provides information on the contribution of sampled and inferred populations to the observed structure and crucially determines whether the predetermined factor of interest correlates with inferred population structure. Analyses of simulated and experimental data highlight ObStruct''s ability to objectively assess the nature of structure in populations. We show the method is capable of capturing an increase in the level of structure with increasing time since divergence between simulated populations. Further, we applied the method to a highly structured dataset of 1,484 humans from seven continents and a less structured dataset of 179 Saccharomyces cerevisiae from three regions in New Zealand. Our results show that ObStruct provides an objective metric to classify the degree, drivers and significance of inferred structure, as well as providing novel insights into the relationships between sampled populations, and adds a final step to the pipeline for population structure analyses.     

Effects of acriflavine on the mitochondria and kinetoplast of Crithidia fasciculata. Correlation of fine structure changes with decreased mitochondrial enzyme activity

The effects of acriflavine on the fine structure and function of the mitochondria and the kinetoplast in Crithidia fasciculata have been investigated. A mitochondrial fraction was prepared by differential centrifugation of cells broken by grinding with neutral alumina. Isolated mitochondria or intact cells revealed by spectrophotometric measurements the presence of cytochromes a + a ₃, b, c ₅₅₅ and o. After cells were grown in acriflavine for 3–4 days, the fine structure of the mitochondria and their cytochrome content were affected. Cells grown in 5.0 µM acriflavine had a threefold decrease in cytochrome a + a ₃ and decreased respiratory activity. The mitochondrial preparation from these cells had a fivefold decrease in cytochrome a + a ₃ and a less but significant decrease of other cytochromes present. There was also a decrease in the mitochondrial enzyme activities of NADH, succinic and L-α-glycerophosphate oxidases, and succinic and L-α-glycerophosphate dehydrogenases. Dyskinetoplastic cells could be demonstrated after growth in 1.0 µM acriflavine. At 5 µM, 80–90% of the cells were dyskinetoplastic. The kinetoplastic DNA was condensed, nonfibrillar, and did not incorporate thymidine-³H. The mitochondria in these cells had few cristae and were shorter and more swollen than the controls. Acriflavine may induce the fine structure effects we have observed and may affect the formation of the mitochondria in C. fasciculata.     

Two d-2-Hydroxy-acid Dehydrogenases in Arabidopsis thaliana with Catalytic Capacities to Participate in the Last Reactions of the Methylglyoxal and ��-Oxidation Pathways

The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae d-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of d- and l-lactate, d-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for d-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for l-lactate and glycolate, respectively, and a K_m value for d-lactate of ∼160 μm. Knock-out mutants showed impaired growth in the presence of d-lactate or methylglyoxal. Collectively, the data indicated that the protein is a d-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific d-2-hydroxyglutarate (d-2HG) conversion in the presence of an organic cofactor with a K_m value of ∼580 μm. Thus, the enzyme was characterized as a d-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total d-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in β-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of d-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.l- and d-lactate dehydrogenases belong to evolutionarily unrelated enzyme families (1). l-Lactate is oxidized by l-lactate:NAD oxidoreductase (EC 1.1.1.27), which catalyzes the reaction l-lactate + NAD → pyruvate + NADH, and by l-lactate cytochrome c oxidoreductase (l-lactate cytochrome c oxidoreductase, EC 1.1.2.3), which catalyzes the reaction l-lactate + 2 cytochrome c (oxidized) → pyruvate + 2 cytochrome c (reduced). Both groups are found in eubacteria, archebacteria, and eukaryotes. All known plant sequences belong to the EC 1.1.1.27 group (1). On the other hand, d-lactate is oxidized by d-lactate:NAD oxidoreductase (d-lactate:NAD oxidoreductase, EC 1.1.1.28), which catalyzes the reaction d-lactate + NAD → pyruvate + NADH, and by d-lactate cytochrome c oxidoreductase (d-lactate cytochrome c oxidoreductase, EC 1.1.2.4), which catalyzes the reaction d-lactate + 2 cytochrome c (oxidized) → pyruvate + 2 cytochrome c (reduced).Although l-lactate dehydrogenase belongs to the most intensely studied enzyme families (2, 3), our knowledge about the structure, kinetics, and biological function of d-LDH³ is limited. d-LDHs have mainly been identified in prokaryotes and fungi where they play an important role in anaerobic energy metabolism (4–10). In Saccharomyces cerevisiae and Kluyveromyces lactis, a mitochondrial flavoprotein d-lactate ferricytochrome c oxidoreductase (d-lactate cytochrome c oxidoreductase), catalyzing the oxidation of d-lactate to pyruvate, is required for the utilization of d-lactate (8, 11). In S. cerevisiae it was suggested that d-LDH is involved in the metabolism of methylglyoxal (MG) (12).In eukaryotic cells, d-lactate results from the glyoxalase system (13, 14). This system is the main MG catabolic pathway, comprising the enzymes glyoxalase I (lactoylglutathione lyase, EC 4.4.1.5) and glyoxalase II (hydroxyacylglutathione hydrolase, EC 3.1.2.6). MG (CH₃-CO-CHO; see structure in Fig. 4) is a cytotoxic compound formed primarily as a by-product of glycolysis through nonenzymatic phosphate elimination from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (15), and its production in various plants is enhanced under stress conditions such as salt, drought, cold, and heavy metal stress (16, 17). Moreover, the overexpression of glyoxalase I or II was shown to confer resistance to salt stress in tobacco and rice (17, 18). It is assumed that the role of the MG pathway, from MG synthase to d-lactate cytochrome c oxidoreductase in the extant metabolism, is to detoxify MG, whereas in the early state of metabolic development it might function as an anaplerotic route for the tricarboxylic acid cycle (15).Open in a separate windowFIGURE 4.Scheme showing the involvement of AtD-LDH in the methylglyoxal pathway and of AtD-2HGDH in the respiration of substrates from proteolysis and/or lipid degradation. d-Lactate resulting from the glyoxalase system is converted to pyruvate by AtD-LDH. The electrons originated may be transferred to the respiratory chain through cytochrome c in the intermembrane space. d-2-HG produced in the peroxisomes (as shown in supplemental Fig. S3) is transported to the mitochondria and converted to 2-ketoglutarate by AtD-2HGDH. Electrons are donated to the electron transport chain through the ETF/ETFQO system. Dotted files represent possible transport processes. 2-KG, 2-ketoglutarate. CIII, complex III. CIV, complex IV. e⁻, electron. ETF, electron transfer protein. ETFQO, ETF-ubiquinone oxidoreductase. GSH, glutathione. Pyr, pyruvate. TCA cycle, tricarboxylic acid cycle; UQ, ubiquinone.Glyoxalase I catalyzes the formation of S-d-lactoylglutathione from the hemithioacetal formed nonenzymatically from MG and glutathione, although glyoxalase II catalyzes the hydrolysis of S-d-lactoylglutathione to regenerate glutathione and liberate d-lactate. Glyoxalase I and II activities are present in all tissues of eukaryotic organisms. Glyoxalase I is found in the cytosol, whereas glyoxalase II localizes to the cytosol and mitochondria (13, 19, 20). Although glyoxalase I and II were extensively characterized, there are only few reports on the characterization of d-LDH. Recently, Atlante et al. (13) showed that externally added d-lactate caused oxygen consumption by mitochondria and that this metabolite was oxidized by a mitochondrial flavoprotein in Helianthus tuberosus.The complete sequence of Arabidopsis thaliana opened the way to search for genes encoding d-LDHs. Based on similarity with the d-LDH from S. cerevisiae (DLD1), an A. thaliana ortholog was identified. In this study, the isolation and structural and biochemical characterization of the recombinant mature d-LDH from A. thaliana (AtD-LDH) and its paralog, which was found to be a d-2-hydroxyglutarate dehydrogenase (AtD-2HGDH), is described. Whereas AtD-LDH has a narrow substrate specificity and the preferred substrates are d-lactate and d-2-hydroxybutyrate, AtD-2HGDH showed activity exclusively with d-2-hydroxyglutarate. Based on gene coexpression analysis and analysis of corresponding knock-out mutants, the participation of these previously unrecognized mitochondrial activities in plant metabolism is discussed.     

Age Structure,Changing Demography and Effective Population Size in Atlantic Salmon (Salmo salar)

Effective population size (N_e) is a central evolutionary concept, but its genetic estimation can be significantly complicated by age structure. Here we investigate N_e in Atlantic salmon (Salmo salar) populations that have undergone changes in demography and population dynamics, applying four different genetic estimators. For this purpose we use genetic data (14 microsatellite markers) from archived scale samples collected between 1951 and 2004. Through life table simulations we assess the genetic consequences of life history variation on N_e. Although variation in reproductive contribution by mature parr affects age structure, we find that its effect on N_e estimation may be relatively minor. A comparison of estimator models suggests that even low iteroparity may upwardly bias N_e estimates when ignored (semelparity assumed) and should thus empirically be accounted for. Our results indicate that N_e may have changed over time in relatively small populations, but otherwise remained stable. Our ability to detect changes in N_e in larger populations was, however, likely hindered by sampling limitations. An evaluation of N_e estimates in a demographic context suggests that life history diversity, density-dependent factors, and metapopulation dynamics may all affect the genetic stability of these populations.THE effective size of a population (N_e) is an evolutionary parameter that can be informative on the strength of stochastic evolutionary processes, the relevance of which relative to deterministic forces has been debated for decades (e.g., Lande 1988). Stochastic forces include environmental, demographic, and genetic components, the latter two of which are thought to be more prominent at reduced population size, with potentially detrimental consequences for average individual fitness and population persistence (Newman and Pilson 1997; Saccheri et al. 1998; Frankham 2005). The quantification of N_e in conservation programs is thus frequently advocated (e.g., Luikart and Cornuet 1998; Schwartz et al. 2007), although gene flow deserves equal consideration given its countering effects on genetic stochasticity (Frankham et al. 2003; Palstra and Ruzzante 2008).Effective population size is determined mainly by the lifetime reproductive success of individuals in a population (Wright 1938; Felsenstein 1971). Variance in reproductive success, sex ratio, and population size fluctuations can reduce N_e below census population size (Frankham 1995). Given the difficulty in directly estimating N_e through quantification of these demographic factors (reviewed by Caballero 1994), efforts have been directed at inferring N_e indirectly through measurement of its genetic consequences (see Leberg 2005, Wang 2005, and Palstra and Ruzzante 2008 for reviews). Studies employing this approach have quantified historical levels of genetic diversity and genetic threats to population persistence (e.g., Nielsen et al. 1999b; Miller and Waits 2003; Johnson et al. 2004). N_e has been extensively studied in (commercially important) fish species, due to the common availability of collections of archived samples that facilitate genetic estimation using the temporal method (e.g., Hauser et al. 2002; Shrimpton and Heath 2003; Gomez-Uchida and Banks 2006; Saillant and Gold 2006).Most models relating N_e to a population''s genetic behavior make simplifying assumptions regarding population dynamics. Chiefly among these is the assumption of discrete generations, frequently violated in practice given that most natural populations are age structured with overlapping generations. Here, theoretical predictions still apply, provided that population size and age structure are constant (Felsenstein 1971; Hill 1972). Ignored age structure can introduce bias into temporal genetic methods for the estimation of N_e, especially for samples separated by time spans that are short relative to generation interval (Jorde and Ryman 1995; Waples and Yokota 2007; Palstra and Ruzzante 2008). Moreover, estimation methods that do account for age structure (e.g., Jorde and Ryman 1995) still assume this structure to be constant. Population dynamics will, however, likely be altered as population size changes, thus making precise quantifications of the genetic consequences of acute population declines difficult (Nunney 1993; Engen et al. 2005; Waples and Yokota 2007). This problem may be particularly relevant when declines are driven by anthropogenic impacts, such as selective harvesting regimes, that can affect age structure and N_e simultaneously (Ryman et al. 1981; Allendorf et al. 2008). Demographic changes thus have broad conservation implications, as they can affect a population''s sensitivity to environmental stochasticity and years of poor recruitment (Warner and Chesson 1985; Ellner and Hairston 1994; Gaggiotti and Vetter 1999). Consequently, although there is an urgent need to elucidate the genetic consequences of population declines, relatively little is understood about the behavior of N_e when population dynamics change (but see Engen et al. 2005, 2007).Here we focus on age structure and N_e in Atlantic salmon (Salmo salar) river populations in Newfoundland and Labrador. The freshwater habitat in this part of the species'' distribution range is relatively pristine (Parrish et al. 1998), yet Atlantic salmon in this area have experienced demographic declines, associated with a commercial marine fishery, characterized by high exploitation rates (40–80% of anadromous runs; Dempson et al. 2001). A fishery moratorium was declared in 1992, with rivers displaying differential recovery patterns since then (Dempson et al. 2004b), suggesting a geographically variable impact of deterministic and stochastic factors, possibly including genetics. An evaluation of those genetic consequences thus requires accounting for potential changes in population dynamics as well as in life history. Life history in Atlantic salmon can be highly versatile (Fleming 1996; Hutchings and Jones 1998; Fleming and Reynolds 2004), as exemplified by the high variation in age-at-maturity displayed among and within populations (Hutchings and Jones 1998), partly reflecting high phenotypic plasticity (Hutchings 2004). This diversity is particularly evident in the reproductive biology of males, which can mature as parr during juvenile freshwater stages (Jones and King 1952; Fleming and Reynolds 2004) and/or at various ages as anadromous individuals, when returning to spawn in freshwater from ocean migration. Variability in life history strategies is further augmented by iteroparity, which can be viewed as a bet-hedging strategy to deal with environmental uncertainty (e.g., Orzack and Tuljapurkar 1989; Fleming and Reynolds 2004). Life history diversity and plasticity may allow salmonid fish populations to alter and optimize their life history under changing demography and population dynamics, potentially acting to stabilize N_e. Reduced variance in individual reproductive success at low breeder abundance (genetic compensation) will achieve similar effects and might be a realistic aspect of salmonid breeding systems (Ardren and Kapuscinski 2003; Fraser et al. 2007b). Little is currently known about the relationships between life history plasticity, demographic change and N_e, partly due to scarcity of the multivariate data required for these analyses.Our objective in this article is twofold. First, we use demographic data for rivers in Newfoundland to quantify how life history variation influences age structure in Atlantic salmon and hence N_e and its empirical estimation from genetic data. We find that variation in reproductive contribution by mature parr has a much smaller effect on the estimation of N_e than is often assumed. Second, we use temporal genetic data to estimate N_e and quantify the genetic consequences of demographic changes. We attempt to account for potential sources of bias, associated with (changes in) age structure and life history, by using four different analytical models to estimate N_e: a single-sample estimator using the linkage disequilibrium method (Hill 1981), the temporal model assuming discrete generations (Nei and Tajima 1981; Waples 1989), and two temporal models for species with overlapping generations (Waples 1990a,b; Jorde and Ryman 1995) that differ principally in assumptions regarding iteroparity. A comparison of results from these different estimators suggests that iteroparity may often warrant analytical consideration, even when it is presumably low. Although sometimes limited by statistical power, a quantification and comparison of temporal changes in N_e among river populations suggests a more prominent impact of demographic changes on N_e in relatively small river populations.     

Sugar transport across the peritubular face of renal cells of the flounder

The transport of some sugars at the antiluminal face of renal cells was studied using teased tubules of flounder (Pseudopleuronectes americanus). The analytical procedure allowed the determination of both free and total (free plus phosphorylated) tissue sugars. The inulin space of the preparation was 0.333 ± 0.017 kg/kg wet wt (7 animals, 33 analyses). The nonmetabolizable α-methyl-D-glucoside entered the cells by a carrier-mediated (phloridzin-sensitive), ouabain-insensitive process. The steady-state tissue/medium ratio was systematically below that for diffusion equilibrium. D-Glucose was a poor inhibitor of α-methyl-glucoside transport, D-galactose was ineffective. The phloridzin-sensitive transport processes of 2-deoxy-D-glucose,D-galactose,and 2-deoxy-D-galactose were associated with considerable phosphorylation. Kinetic evidence suggested that these sugars were transported in free form and subsequently were phosphorylated. 2-Deoxy-D-glucose accumulated in the cells against a slight concentration gradient. This transport was greatly inhibited by D-glucose, whereas α-methyl-glucoside and also D-galactose and its 2-deoxy-derivative were ineffective. D-Galactose and 2-deoxy-D-galactose mutually competed for transport; D-glucose, 2-deoxy-D-glucose, and α-methyl-D-glucoside were ineffective. Studies using various sugars as inhibitors suggest the presence of three carrier-mediated pathways of sugar transport at the antiluminal cell face of the flounder renal tubule: the pathway of α-methyl-D-glucoside (not shared by D-glucose); the pathway commonly shared by 2-deoxy-D-glucose and D-glucose; the pathway shared by D-galactose and 2-deoxy-D-galactose.     

Anecdotal,Historical and Critical Commentaries on Genetics: In the Beginning Was the Worm …

The replicative life span (RLS) of Saccharomyces cerevisiae has been established as a model for the genetic regulation of longevity despite the inherent difficulty of the RLS assay, which requires separation of mother and daughter cells by micromanipulation after every division. Here we present the mother enrichment program (MEP), an inducible genetic system in which mother cells maintain a normal RLS—a median of 36 generations in the diploid MEP strain—while the proliferative potential of daughter cells is eliminated. Thus, the viability of a population over time becomes a function of RLS, and it displays features of a survival curve such as changes in hazard rate with age. We show that viability of mother cells in liquid culture is regulated by SIR2 and FOB1, two opposing regulators of RLS in yeast. We demonstrate that viability curves of these short- and long-lived strains can be easily distinguished from wild type, using a colony formation assay. This provides a simplified screening method for identifying genetic or environmental factors that regulate RLS. Additionally, the MEP can provide a cohort of cells at any stage of their life span for the analysis of age-associated phenotypes. These capabilities effectively remove the hurdles presented by RLS analysis that have hindered S. cerevisiae aging studies since their inception 50 years ago.THE budding yeast Saccharomyces cerevisiae is a popular model system for studying fundamental processes of cellular aging (reviewed in Steinkraus et al. 2008). Analyses over the past 50 years have led to the idea that budding yeast can be used to study three types of cellular aging. Replicative aging describes the division potential of individual cells and relies on the asymmetric cell divisions of budding yeast that yield distinct mother and daughter cells. Replicative life span (RLS) is defined as the number of times an individual cell divides before it undergoes senescence (Mortimer and Johnston 1959). Chronological aging describes the capacity of cells in stationary phase (analogous to G₀ in higher eukaryotes) to maintain viability over time, which is assayed by their ability to reenter the cell cycle when nutrients are reintroduced (Longo et al. 1996). Finally, budding yeast have been used to study clonal senescence, which is analogous to the Hayflick limit imposed on mammalian tissue culture cells and characterized by a finite number of times a population of cells can divide. Although wild-type yeast populations do not senesce, this phenomenon has been observed in mutant strains such as those lacking telomerase components (Lundblad and Szostak 1989; Singer and Gottschling 1994).While genetic screens have been applied to examine clonal and chronological aging (Lundblad and Szostak 1989; Powers et al. 2006; Murakami et al. 2008), they have been limited in their application to studying replicative aging (Kaeberlein and Kennedy 2005; Kaeberlein et al. 2005b). This limitation arises from the arduous nature of isolating replicatively aged yeast cells. The current “gold standard” for isolating aged mother cells is by micromanipulation, where daughter cells are counted and removed after every division (Park et al. 2002). Although micromanipulation is currently the only method capable of accurately measuring RLS in yeast, it is severely constrained by the small number of cells that can be analyzed. Thus, genetic analysis of the regulation of RLS has been limited to a candidate gene approach (reviewed in Steinkraus et al. 2008).True genetic analysis of RLS will require large populations of aged cells. However, there are two confounding issues that make isolation of aged individuals difficult. First, single-cell pedigree analysis has shown that age-associated phenotypes, such as replicative life span potential, segregate asymmetrically between mother and daughter cells, rendering age-associated phenotypes nonheritable (Egilmez and Jazwinski 1989; Kennedy et al. 1994). Thus, daughter cells are generally “reset” to a young state with every generation. Second, when age is measured in terms of cell divisions, an unfractionated population is predominately young. The fraction of the population at an age of n cell divisions is ∼1/2ⁿ. Individual cells that reach the median RLS, which is ∼26 generations for haploid cells of the S288C strain background (Kaeberlein et al. 2005a), represent an insignificant fraction of the total population. In fact, it is unlikely that any cell reaches such an advanced age because nutrient depletion will limit the division potential of the population (Dickinson and Schweizer 1999).As an alternative to micromanipulation, methods were developed to isolate aged cells from liquid cultures (Smeal et al. 1996; Sinclair and Guarente 1997; Chen and Contreras 2007). However, due to the exponential growth of progeny cells, these populations are technically limited to 7–12 generations before nutrient depletion interferes with replicative aging. While sequential rounds of growth and purification are possible, the inability to continuously follow an undisturbed cohort of cells prevents the measurement of RLS by these methods. Instead, purification methods are primarily used for the examination of molecular changes associated with aging cells. Unfortunately, low yields and loss of viability due to purification methods diminish their utility for analyzing phenotypes that affect cells of advanced age. As an alternative to purification from natural populations, a strategy to genetically regulate the replicative capacity of daughter cells and avoid the limits imposed by exponential growth has been described (Jarolim et al. 2004). While this system effectively prevents division of daughter cells, it unintentionally decreases the median RLS of mother cells to four cell divisions, thus restricting its usefulness.Here we describe the development of a novel genetic selection against newborn daughter cells, the “mother enrichment program” (MEP), which restricts the replicative capacity of daughter cells while allowing mother cells to achieve a normal RLS. We demonstrate that upon induction of the selection, the viability of MEP strains growing in liquid culture is determined by the RLS of the initial population of mother cells. MEP cultures therefore allow the comparison of RLS between strains without the need for micromanipulation. Additionally, because MEP cultures are not subject to nutrient limitation, single-step affinity purification of aged cells can be achieved at any point during their life span. Together, these capabilities substantially resolve the technical hurdles that have made replicative aging studies in S. cerevisiae exceptionally challenging.     

Assembly of the Mitochondrial Membrane System: Nuclear Suppression of a Cytochrome b Mutation in Yeast Mitochondrial DNA

In a previous study, a mitochondrial mutant expressing a specific enzymatic deficiency in co-enzyme QH₂-cytochrome c reductase was described (Tzagoloff, Foury and Akai 1976). Analysis of the mitochondrially translated proteins revealed the absence in the mutant of the mitochondrial product corresponding to cytochrome b and the presence of a new low molecular weight product. The premature chain-termination mutant was used to obtain suppressor mutants with wild-type properties. One such revertant strain was analyzed genetically and biochemically. The revertant was determined to have a second mutation in a nuclear gene that is capable of partially suppressing the original mitochondrial cytochrome b mutation. Genetic data indicate that the nuclear mutation is recessive and is probably in a gene coding for a protein involved in the mitochondrial translation machinery.     

The Elucidation of the Structure of Thermotoga maritima Peptidoglycan Reveals Two Novel Types of Cross-link

Thermotoga maritima is a Gram-negative, hyperthermophilic bacterium whose peptidoglycan contains comparable amounts of l- and d-lysine. We have determined the fine structure of this cell-wall polymer. The muropeptides resulting from the digestion of peptidoglycan by mutanolysin were separated by high-performance liquid chromatography and identified by amino acid analysis after acid hydrolysis, dinitrophenylation, enzymatic determination of the configuration of the chiral amino acids, and mass spectrometry. The high-performance liquid chromatography profile contained four main peaks, two monomers, and two dimers, plus a few minor peaks corresponding to anhydro forms. The first monomer was the d-lysine-containing disaccharide-tripeptide in which the d-Glu-d-Lys bond had the unusual γ→ϵ arrangement (GlcNAc-MurNAc-l-Ala-γ-d-Glu-ϵ-d-Lys). The second monomer was the conventional disaccharide-tetrapeptide (GlcNAc-MurNAc-l-Ala-γ-d-Glu-l-Lys-d-Ala). The first dimer contained a disaccharide-l-Ala as the acyl donor cross-linked to the α-amine of d-Lys in a tripeptide acceptor stem with the sequence of the first monomer. In the second dimer, donor and acceptor stems with the sequences of the second and first monomers, respectively, were connected by a d-Ala⁴-α-d-Lys³ cross-link. The cross-linking index was 10 with an average chain length of 30 disaccharide units. The structure of the peptidoglycan of T. maritima revealed for the first time the key role of d-Lys in peptidoglycan synthesis, both as a surrogate of l-Lys or meso-diaminopimelic acid at the third position of peptide stems and in the formation of novel cross-links of the l-Ala¹(α→α)d-Lys³ and d-Ala⁴(α→α)d-Lys³ types.Peptidoglycan (or murein) is a giant macromolecule whose main function is the protection of the cytoplasmic membrane against the internal osmotic pressure. It is composed of alternating residues of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)² cross-linked by short peptides (1). The composition of the peptide stem in nascent peptidoglycan is l-Ala¹-γ-d-Glu²-X³-d-Ala⁴-d-Ala⁵, where X is most often meso-diaminopimelic acid (meso-A₂pm) or l-lysine in Gram-negative and Gram-positive species, respectively (2, 3). In the mature macromolecule, the last d-Ala residue is removed. Cross-linking of the glycan chains generally occurs between the carboxyl group of d-Ala at position 4 of a donor peptide stem and the side-chain amino group of the diamino acid at position 3 of an acceptor peptide stem (4→3 cross-links). Cross-linking is either direct or through a short peptide bridge such as pentaglycine in Staphylococcus aureus (2, 3). The enzymes for the formation of the 4→3 cross-links are active-site serine dd- transpeptidases that belong to the penicillin-binding protein (PBP) family and are the essential targets of β-lactam antibiotics in pathogenic bacteria (4). Catalysis involves the cleavage of the d-Ala⁴-d-Ala⁵ bond of a donor peptide stem and the formation of an amide bond between the carboxyl of d-Ala⁴ and the side chain amine at the third position of an acceptor stem. Transpeptidases of the ld specificity are active-site cysteine enzymes that were shown to act as surrogates of the PBPs in mutants of Enterococcus faecium resistant to β-lactam antibiotics (5). They cleave the X³-d-Ala⁴ bond of a donor stem peptide to form 3→3 cross-links. This alternate mode of cross-linking is usually marginal, although it has recently been shown to predominate in non-replicative “dormant” forms of Mycobacterium tuberculosis (6).Thermotoga maritima is a Gram-negative, extremely thermophilic bacterium isolated from geothermally heated sea floors by Huber et al. (7). A morphological characteristic is the presence of an outer sheath-like envelope called “toga.” Although the organism has received considerable attention for its biotechnological potential, studies about its peptidoglycan are scarce (8–11), and in particular the fine structure of the macromolecule is still unknown. In their initial work, Huber et al. (7) showed that the composition of its peptidoglycan was unusual for a Gram-negative species, because it contained both isomers of lysine and no A₂pm. Recently, we purified and studied the properties of T. maritima MurE (12); this enzyme is responsible for the addition of the amino acid residue at position 3 of the peptide stem (13, 14). We demonstrated that T. maritima MurE added in vitro l- and d-Lys to UDP-MurNAc-l-Ala-d-Glu. Although l-Lys was added in the usual way, yielding the conventional nucleotide UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys containing a d-Glu(γ→α)l-Lys amide bond, the d-isomer was added in an “upside-down” manner, yielding the novel nucleotide UDP-MurNAc-l-Ala-d-Glu(γ→ϵ)d-Lys. We also showed that the d-Lys-containing nucleotide was not a substrate for T. maritima MurF, the subsequent enzyme in the biosynthetic pathway, whereas this ligase catalyzed the addition of dipeptide d-Ala-d-Ala to the l-Lys-containing tripeptide, yielding the conventional UDP-MurNAc-pentapeptide (12).However, both the l-Lys-containing UDP-MurNAc-pentapeptide and d-Lys-containing UDP-MurNAc-tripeptide were used as substrates by T. maritima MraY with comparable efficiencies in vitro (12). This observation implies that the unusual d-Lys-containing peptide stems are likely to be translocated to the periplasmic face of the cytoplasmic membrane and to participate in peptidoglycan polymerization. Therefore, we have determined here the fine structure of T. maritima peptidoglycan and we have shown that l-Lys- and d-Lys-containing peptide stems are both present in the polymer, the latter being involved in the formation of two novel types of peptidoglycan cross-link.     

Alternative Splicing Modulates Ubx Protein Function in Drosophila melanogaster

Polyploidy is an important aspect of the evolution of flowering plants. The potential of gene copies to diverge and evolve new functions is influenced by meiotic behavior of chromosomes leading to segregation as a single locus or duplicated loci. Switchgrass (Panicum virgatum) linkage maps were constructed using a full-sib population of 238 plants and SSR and STS markers to access the degree of preferential pairing and the structure of the tetraploid genome and as a step toward identification of loci underlying biomass feedstock quality and yield. The male and female framework map lengths were 1645 and 1376 cM with 97% of the genome estimated to be within 10 cM of a mapped marker in both maps. Each map coalesced into 18 linkage groups arranged into nine homeologous pairs. Comparative analysis of each homology group to the diploid sorghum genome identified clear syntenic relationships and collinear tracts. The number of markers with PCR amplicons that mapped across subgenomes was significantly fewer than expected, suggesting substantial subgenome divergence, while both the ratio of coupling to repulsion phase linkages and pattern of marker segregation indicated complete or near complete disomic inheritance. The proportion of transmission ratio distorted markers was relatively low, but the male map was more extensively affected by distorted transmission ratios and multilocus interactions, associated with spurious linkages.POLYPLOIDY is common among plants (Masterson 1994; Levin 2002) and is an important aspect of plant evolution. Widespread paleopolyploidy in flowering plant lineages suggests that ancient polyploidization events have contributed to the radiation of angiosperms (Soltis et al. 2009; Van de Peer et al. 2009a). Whole genome duplications are thought to be the sources of evolutionary novelty (Osborn et al. 2003; Freeling and Thomas 2006; Chen 2007; Hegarty and Hiscock 2008; Flagel and Wendel 2009; Leitch and Leitch 2008). Other attributes of polyploids considered to promote evolutionary success include increased vigor, masking of recessive alleles, and reproductive barriers arising from loss of one of the duplicate genes (Soltis and Soltis 2000; Comai 2005; Otto 2007; Van de Peer et al. 2009b). Among crop species, polyploidy likely contributed to trait improvement under artificial selection (Paterson 2005; Udall and Wendell 2006; Dubcovsky and Dvorak 2007; Hovav et al. 2008).Disomic inheritance in polyploids, in contrast to polysomic inheritance, presents opportunities for duplicated genes to diverge and evolve new functions. The relative age of whole genome duplications and the extent of homology between subgenomes greatly influence chromosomal pairing at meiosis (Soltis and Soltis 1995; Wolfe 2001; Ramsey and Schemske 2002). Polysomic inheritance resulting from random chromosome pairing is associated with doubling of a single set of chromosomes. Disomic inheritance resulting from preferential pairing is often associated with polyploidy arising from combinations of divergent genomes. The evolutionary process of diploidization leads to a shift from random to preferential pairing that is not well understood but is genetically defined in systems such as Ph1 of wheat (Triticum aestivum) and PrBn of Brassica napus (Riley and Chapman 1958; Vega and Feldman 1998; Jenczewski et al. 2003). The degree of preferential pairing also affects allelic diversity and the ability to detect linkage. Accurate information about chromosome pairing and whole or partial genome duplications is thus important for both evolutionary studies and in linkage analysis.Such information is extremely limited in the C4 panicoid species Panicum virgatum (switchgrass), which is now viewed as a promising energy crop in the United States and Europe (Lewandowski et al. 2003; McLaughlin and Kszos 2005) and is planted extensively for forage and soil conservation (Vogel and Jung 2001). Little is known about either its genome structure or inheritance. Much current bioenergy feedstock development is focused on tetraploid cytotypes (2n = 4x = 36) due to their higher yield potentials, and an initial segregation study indicated a high degree of preferential pairing in a single F1 mapping population (Missaoui et al. 2005). A once-dominant component of the tallgrass prairie in North America, switchgrass is largely self-incompatible (Martinez-Reyna and Vogel 2002) with predominantly tetraploid or octoploid cytotypes (Hultquist et al. 1997; Lu et al. 1998). Limited gene flow appears possible between different cytotypes suggested by DNA content variation within collection sites and seed lots (Nielsen 1944; Hultquist et al. 1997; Narasimhamoorthy et al. 2008). True diploids appear to be rare (Nielsen 1944; Young et al. 2010). Multivalents in meiosis have not been observed in tetraploids or F1 hybrids between upland and lowland tetraploids, although rare univalents occurred (Barnett and Carver 1967; Martinez-Reyna et al. 2001). However, polysomic inheritance may occur with random bivalent pairing (Howard and Swaminathan 1953).Sustainable production of switchgrass for bioenergy to meet the goal of reducing greenhouse gas emissions will require advances in feedstock production that include improvements in yield (Carroll and Somerville 2009). Switchgrass has extensive genetic diversity and potential for genetic improvements, but each cycle of phenotypic selection can take several years (McLaughlin and Kszos 2005; Parrish and Fike 2005; Bouton 2007). Detailed understanding of genome structure to enable efficient marker-assisted selection (MAS) can speed this process considerably. Complete linkage maps are therefore required to both understand chromosome pairing and allow MAS.We report the construction of the first complete linkage maps of two switchgrass genotypes. The linkage maps provide genetic evidence for disomic inheritance in lowland, tetraploid switchgrass. Gene-derived markers enabled a comparative analysis to sorghum, revealing syntenic relationships between the diploid sorghum genome and the tetraploid switchgrass subgenomes. Transmission ratio distortion and multilocus interactions were analyzed in detail to document their potential influence on map accuracy and map-based studies in switchgrass.     

Within-Range Translocations and Their Consequences in European Larch

In contrast to biological invasions, translocations of individuals within a species range are understudied, due to difficulties in systematically detecting them. This results in limited knowledge about the corresponding processes and uncertainties regarding the status of extant populations. European larch, a forest tree whose fragmented native distribution is restricted to the Alps and to other Central European mountains, has been massively planted for at least 300 years. Here we focus on the genetic characterization of translocations having taken place within its native range. Microsatellite variation at 13 nuclear loci and sequence data of two mitochondrial DNA fragments were analyzed on the basis of a comprehensive range-wide population sample. Two complementary methods (Geneclass and Structure) were used to infer translocation events based on nuclear data whereas mitochondrial data were used for validation of these inferences. Using Geneclass, we found translocation events in a majority of populations. Additional cases of translocation and many instances of admixture were identified using Structure, thanks to the clear-cut ancestral genetic structure detected in this species. In particular, a strong divide between Alpine and Central European populations, also apparent at mitochondrial markers, helped uncover details on translocation events and related processes. Translocations and associated admixture events were found to be heterogeneously distributed across the species range, with a particularly high frequency in Central Europe. Furthermore, translocations frequently involved multiple geographic sources, some of which were over-represented. Our study illustrates the importance of range-wide investigations for tracing translocations back to their origins and for revealing some of their consequences. It provides some first clues for developing suitable conservation and management strategies.     

Healing of Euchromatic Chromosome Breaks by Efficient de novo Telomere Addition in Drosophila melanogaster

Many arthropod species are infected with maternally inherited endosymbionts that induce a shift in the sex ratio of their hosts by feminizing or killing males (cytoplasmic sex-ratio distorters, or SRDs). These endosymbionts can have profound impacts on evolutionary processes of their hosts. Here, I derive analytical expressions for the coalescent effective size N_e of populations that are infected with SRDs. Irrespective of the type of SRD, N_e for mitochondrial genes is given by the number of infected females. For nuclear genes, the effective population size generally decreases with increasing prevalence of the SRD and can be considerably lower than the actual size of the population. For example, with male-killing bacteria that have near perfect maternal transmission, N_e is reduced by a factor that is given to a good approximation by the proportion of uninfected individuals in the population. The formulae derived here also yield the effective size of populations infected with mutualistic endosymbionts or maternally inherited bacteria that induce cytoplasmic incompatibility, although in these cases, the reduction in N_e is expected to be less severe than for cytoplasmic SRDs.SIMPLE null models are essential in science. In population genetics, this role is filled by the Wright–Fisher model and its retrospective counterpart, the Kingman coalescent. Both of these models have proven to be immensely useful in spite of the fact that natural populations usually violate the assumptions made in these models. The reason for this is that often, the Wright–Fisher model can be rescaled so that it behaves in many important respects like a more complex population model. This rescaling is achieved through the concept of the effective population size, N_e. Roughly speaking, a complex population model is said to have a certain N_e if the haploid Wright–Fisher model with population size N_e experiences the same amount of random genetic drift as the complex model. Reflecting the different ways in which drift can be measured, N_e can be defined in different ways, e.g., as the inbreeding, the variance, or the coalescent effective population size. Different definitions often produce the same value for N_e, but may also yield drastically different numbers (Kimura and Crow 1963).The coalescent effective population size is defined through the factor by which time needs to be rescaled in a complex population model to produce the standard coalescent with time scale given by the population size N (Nordborg and Krone 2002). It has been argued that this is the most useful definition for N_e because “the coalescent essentially embodies all of the information that can be found in sampled genetic data” (Sjödin et al. 2005). More recently, Wakeley and Sargsyan (2009) have proposed two extensions of the coalescent effective population size in which they advocate including a mutation parameter in the definition and also allowing for a nonlinear relationship between N_e and N.One frequently encountered feature in natural populations that complicates population genetics is infection with maternally inherited endosymbionts. In particular, many arthropod species harbor a great number of phylogenetically diverse microorganisms that influence their hosts'' biology in different ways (Bourtzis and Miller 2003; Bourtzis and Miller 2006; Bourtzis and Miller 2009). Because of their maternal transmission, many of these microorganisms—for example, the bacteria Wolbachia pipientis and Cardinium hertigii—have evolved intricate manipulations of their hosts'' reproductive system that allows them to spread in a host population through exploitation of male hosts (reproductive parasitism, reviewed in Engelstädter and Hurst 2009a). Most manipulations involve a shift in the sex ratio of their hosts (both primary and at the population level), and the inducing endosymbionts are consequently referred to as cytoplasmic sex-ratio distorters (SRDs). In some species, genetic males develop into females if they are infected (“feminization”: Martin et al. 1973; Rigaud 1997; Bouchon et al. 1998; Hiroki et al. 2002). More commonly, infected males are killed by the endosymbionts early in their development (male killing: reviewed in Hurst et al. 2003). The adaptive advantage of this strategy is seen in an early fitness boost in the surviving females in a brood, for example, through reduced sibling competition or cannibalism of the dead brothers (Hurst 1991; Hurst and Majerus 1993; Jaenike et al. 2003). Some examples for species infected with male-killing or feminizing SRDs are given in Huigens and Stouthamer 2003).

TABLE 1

Empirical examples for cytoplasmic SRDs with parameter estimates

Holocene anthropogenic landscapes in the Balkans: the palaeobotanical evidence from southwestern Bulgaria

Palaeoecological reconstructions from the region of southwestern Bulgaria were used for inferring the human impact on the vegetation and landscape during the last 8 millennia. They are based on data from pollen analyses of lakes and peat-bogs, plant macrofossils, archaeobotanical finds and radiocarbon dating. During the early Holocene, after 7900?cal. b.p. (5950?cal. b.c.) the climate changed to cooler summers, milder winters and higher precipitation resulting in the formation of a coniferous belt dominated by Pinus sp. and Abies alba. These favorable environmental pre-conditions had a positive influence on the Neolithisation of the Balkans after the 8200?cal. b.p. (6250?cal. b.c.) cold event, which caused drought in the Eastern Mediterranean. Direct evidence from wood charcoal records from the Neolithic settlement layers in the study area shows a slight modification of the surrounding woodlands and an increase of the light-demanding components, probably expressed through larger forest border zones and thinning out of the wood stands. The increase in the number of settlements in the valleys of southwestern Bulgaria intensified the human activity visible in the palaeobotanical record from 6950?cal. b.p. (5000?cal. b.c.) onwards. Between ca. 5700–5100?cal. b.p. (3800–3200?cal. b.c.) signs of anthropogenic influence on the vegetation are virtually absent. The intensity of human impact increased notably after 3200?cal. b.p. (1400–1250?cal. b.c., approx. Late Bronze Age), documented by a rise of pollen anthropogenic indicators. The final transformations in the natural forest cover after 2750?cal. b.p. (800?cal. b.c. onset of the Iron Age) marked the reduction of the coniferous forests dominated by Abies alba and Pinus sp. and the expansion of Fagus sylvatica and Picea abies. These vegetation changes are contemporaneous with increase of the palaeofire activities and the next peak of anthropogenic indicators. The changes in the landscape during the Roman period and the medieval period reflect regional environmental features and were forced by the diversification of anthropogenic activity.     

Rational design and characterization of D-Phe-Pro-D-Arg-derived direct thrombin inhibitors

The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH₂. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH₂, competitively inhibits α-thrombin''s cleavage of the S2238 chromogenic substrate with a K_i of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′.     

A New Family of Ty1-copia-Like Retrotransposons Originated in the Tomato Genome by a Recent Horizontal Transfer Event

The use of β-lactam antibiotics has led to the evolution and global spread of a variety of resistance mechanisms, including β-lactamases, a group of enzymes that degrade the β-lactam ring. The evolution of increased β-lactam resistance was studied by exposing independent lineages of Salmonella typhimurium to progressive increases in cephalosporin concentration. Each lineage carried a β-lactamase gene (bla_TEM-1) that provided very low resistance. In most lineages, the initial response to selection was an amplification of the bla_TEM-1 gene copy number. Amplification was followed in some lineages by mutations (envZ, cpxA, or nmpC) that reduced expression of the uptake functions, the OmpC, OmpD, and OmpF porins. The initial resistance provided by bla_TEM-1 amplification allowed the population to expand sufficiently to realize rare secondary point mutations. Mathematical modeling showed that amplification often is likely to be the initial response because events that duplicate or further amplify a gene are much more frequent than point mutations. These models show the importance of the population size to appearance of later point mutations. Transient gene amplification is likely to be a common initial mechanism and an intermediate in stable adaptive improvement. If later point mutations (allowed by amplification) provide sufficient adaptive improvement, the amplification may be lost.THE extensive use of β-lactam antibiotics has led to the evolution and spread of many chromosomal-, plasmid-, and transposon-borne resistance mechanisms (Livermore 1995; Weldhagen 2004). Prominent among these mechanisms is a class of enzymes, β-lactamases, that hydrolyze the β-lactam ring (Ambler 1980; Poole 2004). TEM-1 β-lactamase, encoded by the bla_TEM-1 gene, hydrolyzes both penicillins and early cephalosporins (Matagne et al. 1990). As bacteria developed resistance, stable extended-spectrum cephalosporins (ESCs) were introduced, leading to evolution of TEM sequence variants with improved ESC hydrolysis (Petrosino et al. 1998). Resistance to β-lactams can also result from mutations that reduce levels of outer membrane proteins involved in uptake, altered target proteins (penicillin-binding proteins) to reduce β-lactam binding, or increased expression of efflux pumps that export the antibiotics (Poole 2004; Martínez-Martínez 2008; Zapun et al. 2008).Resistance to β-lactam antibiotics is linearly correlated with the lactamase level over a large range (Nordström et al. 1972) and resistance to β-lactam antibiotics can be provided by increasing enzyme levels. An early illustration of this process is the finding that Escherichia coli can develop ampicillin resistance by amplifying its ampC gene (Edlund and Normark 1981). Similar amplification has been observed in both eubacteria and eukaryotes (Craven and Neidle 2007; Wong et al. 2007) in response to various selective pressures, including antibiotics (Andersson and Hughes 2009; Sandegren and Andersson 2009). In an unselected bacterial population, the frequency of cells with a duplication of any specific chromosomal region ranges between 10⁻² and 10⁻⁵ depending on the region (Anderson and Roth 1981), whereas a point mutation in that gene is expected to be carried by perhaps 1 cell in 10⁷–10⁸ (Hudson et al. 2002). Thus, the rate of duplication formation is ∼10⁻⁵/cell/division and further increases ∼0.01/cell/division (Pettersson et al. 2008) while the base substitution rate is ∼10⁻¹⁰/cell/division/base pair (Hudson et al. 2002). Thus, it is apparent that variants with an increased level of any enzyme activity are more likely to owe the increase to a gene copy number change than to a point mutation. Furthermore, because of the high intrinsic instability of tandem amplifications, haploid segregants are expected to take over the population when the selection pressure is released (Pettersson et al. 2008).To examine the importance of gene amplification in bacterial adaptation to cephalosporins, several independent Salmonella typhimurium lineages carrying the bla_TEM-1 gene were allowed to develop resistance to progressively increased concentrations of cephalothin (a first-generation cephalosporin) and cefaclor (a second-generation cephalosporin). As these lineages developed resistance to higher antibiotic levels, amplification of the bla_TEM-1 gene was the primary and most common resistance mechanism, which in some cases was followed by acquisition of rare point mutations that provided stable resistance.     

Rate of Adaptation in Large Sexual Populations

Adaptation often involves the acquisition of a large number of genomic changes that arise as mutations in single individuals. In asexual populations, combinations of mutations can fix only when they arise in the same lineage, but for populations in which genetic information is exchanged, beneficial mutations can arise in different individuals and be combined later. In large populations, when the product of the population size N and the total beneficial mutation rate U_b is large, many new beneficial alleles can be segregating in the population simultaneously. We calculate the rate of adaptation, v, in several models of such sexual populations and show that v is linear in NU_b only in sufficiently small populations. In large populations, v increases much more slowly as log NU_b. The prefactor of this logarithm, however, increases as the square of the recombination rate. This acceleration of adaptation by recombination implies a strong evolutionary advantage of sex.IN asexual populations, beneficial mutations arising on different genotypes compete against each other and in large populations most of the beneficial mutations are lost because they arise on mediocre genetic backgrounds or acquire further beneficial mutations less rapidly than their peers—the combined effects of clonal interference and multiple mutations (Gerrish and Lenski 1998; Desai and Fisher 2007). Exchange of genetic material between individuals allows the combination of beneficial variants that arose in different lineages and can thereby speed up the process of adaptation (Fisher 1930; Muller 1932). Indeed, most life forms engage in some form of recombination, e.g., lateral gene transfer or competence for picking up DNA in bacteria, facultative sexual reproduction in yeast and plants, or obligate sexual reproduction in most animals. Some benefits of recombination for the rate of adaptation have recently been demonstrated experimentally in Caenorhabditis reinhardtii (Colegrave 2002), Escherichia coli (Cooper 2007), and Saccharomyces cerevisiae (Goddard et al. 2005); for a review of older experiments, see Rice (2002).Yet the benefits of sex become less obvious when one considers its disadvantageous effects: recombination can separate well-adapted combinations of alleles and sexual reproduction is more costly than asexual reproduction due to resources spent for mating and, in some cases, the necessity of males. The latter—in animals often termed the twofold cost of sex—implies that sexual populations can be unstable to the invasion of asexual variants. As a result, the pros and cons of sex have been the subject of many decades of debate in the theoretical literature (Crow and Kimura 1965; Maynard Smith 1968; Felsenstein 1974; Barton 1995a; Barton and Charlesworth 1998), and several different potentially beneficial aspects of sex have been identified, including the pruning of detrimental mutations (Peck 1994; Rice 1998) and host–parasite coevolution or otherwise changing environments (Charlesworth 1993; Ladle et al. 1993; Bürger 1999; Waxman and Peck 1999; Gandon and Otto 2007; Callahan et al. 2009). In the opposite situation of relatively static populations, it has been proposed that recombination is favored in the presence of negative epistasis (Feldman et al. 1980; Kondrashov 1984, 1988)—a situation when the combined detrimental effect of two unfavorable alleles is greater than the sum of the individual effects. While this may sometimes be a significant effect, most populations, especially microbes, are likely to be under continuing selection and the benefits of sex for speeding up adaptation are likely to dominate.The Fisher–Muller hypothesis is that sex speeds up adaptation by combining beneficial variants. Moreover, it has been demonstrated by Hill and Robertson (1966) that linkage decreases the efficacy of selection. This detrimental effect of linkage, known as the “Hill–Robertson effect,” causes selection for higher recombination rates, which has been shown by analyzing recombination modifier alleles at a locus linked to two competing segregating loci (Otto and Barton 1997; Iles et al. 2003; Barton and Otto 2005; Roze and Barton 2006; Martin et al. 2006). Hitchhiking of the allele that increases the recombination rates with the sweeping linked loci results in effective selection for increased recombination.Experiments and simulation studies suggest that the Hill–Roberston effect is more pronounced and selection for recombination modifiers is stronger in large populations with many sweeping loci (Felsenstein 1974; Colegrave 2002; Iles et al. 2003). However, the quantitative understanding of the effect of recombination in large populations is limited. Rouzine and Coffin have studied the role of recombination in the context of evolution of drug resistance in human immunodeficiency virus, finding that recombination of standing variation speeds up adaptation by producing anomalously fit individuals at the high fitness edge of the distribution (Rouzine and Coffin 2005; Gheorghiu-Svirschevski et al. 2007). The effects of epistatic interactions between polymorphisms and recombination on the dynamics of selection have recently been analyzed by Neher and Shraiman (2009). Yet none of these works consider the effects of new beneficial mutations. In the absence of new mutations (and in the absence of heterozygous advantage that can maintain polymorphisms) the fitness soon saturates as most alleles become extinct and standing variation disappears. Thus the crucial point that must be addressed is the balance between selection and recombination of existing variation and the injection of additional variation by new mutations.Here, we study the dynamics of continual evolution via new mutations, selection, and recombination using several models of recombination. Our primary models most naturally apply when periods of asexual reproduction occur between matings, so that they approximate the life style of facultatively outcrossing species such as S. cerevisiae, some plants, and C. elegans, which reproduce asexually most of the time but undergo extensive recombination when outcrossing. The models enable us to study analytically the explicit dependence of the rate of adaptation and of the dynamics of the beneficial alleles on the important parameters such as the outcrossing rate and population size. In an independent study N. H. Barton and J. Coe (personal communication) calculate the rate of adaptation for obligate sexual organisms using several different multilocus models of recombination, including the free recombination model studied here. The relation of our work to theirs, as well as to that of Cohen et al. (2005, 2006) who have also studied the effects of recombination with multiple new mutations, is commented on in the discussion.When deleterious mutations can be neglected, the rate of adaptation is the product of the rate of production of favorable mutations NU_b (N being the population size and U_b the genomewide beneficial mutation rate), the magnitude of their effect, and their fixation probability. The fixation probability is dominated by the probability that the allele becomes established, i.e., that it rises to high enough numbers in the population that it is very unlikely to die out by further stochastic fluctuations. In a homogeneous population a single beneficial mutation with selective advantage s has a probability of establishment and eventual fixation of (in discrete generation models, P_e≈2s) (Moran 1959). In a heterogeneous population, however, a novel beneficial mutation can arise on different genetic backgrounds and its establishment probability will thus vary, being greater if it arises in a well-adapted individual. But even well-adapted genotypes soon fall behind due to sweeps of other beneficial mutations and combinations. To avoid extinction, descendants of the novel mutation thus have to move to fitter genetic backgrounds via recombination in outcrossing events (Rice 2002). As a result the establishment probability decreases as the rate of average fitness gain, v, in the population increases. But the rate of average fitness gain or, equivalently, the rate of adaptation itself depends on the establishment probability. These two quantities therefore have to be determined self-consistently.In this article we analyze several models via self-consistent calculations of the fixation probability of new mutations. For a given production rate of beneficial mutations NU_b, we find that interference between mutations is of minor importance if the recombination rate r exceeds . In this regime, the rate of adaption is v ≈ NU_bs² as found for sequential mutations or in the absence of linkage. At recombination rates below , however, v grows only logarithmically with log NU_b. We find this behavior in all our models and argue that it obtains more generally. The prefactor of the log NU_b increases with the square of the recombination rate, implying a strong benefit of recombination in large populations.