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.     

Patterns and Processes of Genome-Wide Divergence Between North American and African Drosophila melanogaster

Genomic tools and analyses are now being widely used to understand genome-wide patterns and processes associated with speciation and adaptation. In this article, we apply a genomics approach to the model organism Drosophila melanogaster. This species originated in Africa and subsequently spread and adapted to temperate environments of Eurasia and the New World, leading some populations to evolve reproductive isolation, especially between cosmopolitan and Zimbabwean populations. We used tiling arrays to identify highly differentiated regions within and between North America (the United States and Caribbean) and Africa (Cameroon and Zimbabwe) across 63% of the D. melanogaster genome and then sequenced representative fragments to study their genetic divergence. Consistent with previous findings, our results showed that most differentiation was between populations living in Africa vs. outside of Africa (i.e., “out-of-Africa” divergence), with all other geographic differences being less substantial (e.g., between cosmopolitan and Zimbabwean races). The X chromosome was much more strongly differentiated than the autosomes between North American and African populations (i.e., greater X divergence). Overall differentiation was positively associated with recombination rates across chromosomes, with a sharp reduction in regions near centromeres. Fragments surrounding these high F_ST sites showed reduced haplotype diversity and increased frequency of rare and derived alleles in North American populations compared to African populations. Nevertheless, despite sharp deviation from neutrality in North American strains, a small set of bottleneck/expansion demographic models was consistent with patterns of variation at the majority of our high F_ST fragments. Although North American populations were more genetically variable compared to Europe, our simulation results were generally consistent with those previously based on European samples. These findings support the hypothesis that most differentiation between North America and Africa was likely driven by the sorting of African standing genetic variation into the New World via Europe. Finally, a few exceptional loci were identified, highlighting the need to use an appropriate demographic null model to identify possible cases of selective sweeps in species with complex demographic histories.THE study of genetic differentiation between populations and species has recently been empowered by the use of genomic techniques and analysis (e.g., Noor and Feder 2006; Stinchcombe and Hoekstra 2008). In the past decade, genetic studies of adaptation and speciation have taken advantage of emerging molecular techniques to scan the genomes of diverging populations for highly differentiated genetic regions (e.g., Wilding et al. 2001; Emelianov et al. 2003; Beaumont and Balding 2004; Campbell and Bernatchez 2004; Scotti-Saintagne et al. 2004; Achere et al. 2005; Turner et al. 2005; Vasemagi et al. 2005; Bonin et al. 2006, 2007; Murray and Hare 2006; Savolainen et al. 2006; Yatabe et al. 2007; Nosil et al. 2008, 2009; Turner et al. 2008a,b; Kulathinal et al. 2009). As a result, genome scans can identify candidate regions that may be associated with adaptive evolution between diverging populations and, more broadly, are able to describe genome-wide patterns and processes of population differentiation (Begun et al. 2007; Stinchcombe and Hoekstra 2008).Genome scans in well-studied genetic model species such as Drosophila melanogaster gain particular power because differentiated loci are mapped to a well-annotated genome. Moreover, the evolutionary history of D. melanogaster is rich with adaptive and demographic events with many parallels to human evolution. Most notable is the historical out-of-Africa migration and subsequent adaptation to temperate ecological environments of Europe, Asia, North America, and Australia. This has resulted in widespread genetic and phenotypic divergence between African and non-African populations (e.g., David and Capy 1988; Begun and Aquadro 1993; Capy et al. 1994; Colegrave et al. 2000; Rouault et al. 2001; Takahashi et al. 2001; Caracristi and Schlötterer 2003; Baudry et al. 2004; Pool and Aquadro 2006; Schmidt et al. 2008; Yukilevich and True 2008a,b). Further, certain populations in Africa and in the Caribbean vary in their degree of reproductive isolation from populations in more temperate regions (Wu et al. 1995; Hollocher et al. 1997; Yukilevich and True 2008a,b). In particular, the Zimbabwe and nearby populations of southern Africa are strongly sexually isolated from all other populations, designating them as a distinct behavioral race (Wu et al. 1995).D. melanogaster has received a great deal of attention from the population geneticists in studying patterns of sequence variation across African and non-African populations. Many snapshots have been taken of random microsatellite and SNP variants spread across X and autosomes, and these have generated several important conclusions. Polymorphism patterns in European populations are characterized by reduced levels of nucleotide and haplotype diversity, an excess of high frequency-derived polymorphisms, and elevated levels of linkage disequilibrium relative to African populations (e.g., Begun and Aquadro 1993; Andolfatto 2001; Glinka et al. 2003; Haddrill et al. 2005; Ometto et al. 2005; Thornton and Andolfatto 2006; Hutter et al. 2007; Singh et al. 2007). These results have been generally interpreted as compatible with population size reduction/bottlenecks followed by recent population expansions. On the other hand, African populations are generally assumed either to have been relatively constant in size over time or to have experienced population size expansions. They generally show higher levels of nucleotide and haplotype diversity, an excess of rare variants, and a deficit of high frequency-derived alleles (Glinka et al. 2003; Ometto et al. 2005; Pool and Aquadro 2006; Hutter et al. 2007; but see Haddrill et al. 2005 for evidence of bottlenecks in Africa).Previous work also shows that the ratio of X-linked to autosomal polymorphism deviates from neutral expectations in opposite directions in African and European populations with more variation on the X than expected in Africa and less variation on the X than expected in Europe (Andolfatto 2001; Kauer et al. 2002; Hutter et al. 2007; Singh et al. 2007). The deviation from neutrality in the ratio of X-autosome polymorphism may be explained by positive selection being more prevalent on the X in Europe and/or by a combination of bottlenecks and male-biased sex ratios in Europe and female-biased sex ratios in Africa (Charlesworth 2001; Hutter et al. 2007; Singh et al. 2007). The selective explanation stems from the argument that, under the hitchhiking selection model, X-linked loci are likely to be more affected by selective sweeps than autosomal loci (Maynard Smith and Haigh 1974; Charlesworth et al. 1987; Vicoso and Charlesworth 2006, 2009).The relative contribution of selective and demographic processes in shaping patterns of genomic variation and differentiation is highly debated (Wall et al. 2002; Glinka et al. 2003; Haddrill et al. 2005; Ometto et al. 2005; Schöfl and Schlötterer 2004; Thornton and Andolfatto 2006; Hutter et al. 2007; Singh et al. 2007; Shapiro et al. 2007; Stephan and Li 2007; Hahn 2008; Macpherson et al. 2008; Noor and Bennett 2009; Sella et al. 2009). This is especially the case in D. melanogaster because derived non-African populations have likely experienced a complex set of demographic events during their migration out of Africa (e.g., Thornton and Andolfatto 2006; Singh et al. 2007; Stephan and Li 2007), making population genetics signatures of demography and selection difficult to tease apart (e.g., Macpherson et al. 2008). Thus it is still unclear what role selection has played in shaping overall patterns of genomic variation and differentiation relative to demographic processes in this species.While there is a long tradition in studying arbitrarily or opportunistically chosen sequences in D. melanogaster, genomic scans that focus particularly on highly differentiated sites across the genome have received much less attention. Such sites are arguably the best candidates to resolve the debate on which processes have shaped genomic differentiation within species (e.g., Przeworski 2002). Recently, a genome-wide scan of cosmopolitan populations in the United States and in Australia was performed to investigate clinal genomic differentiation on the two continents (Turner et al. 2008a). Many single feature polymorphisms differentiating Northern and Southern Hemisphere populations were identified. Among the most differentiated loci in common between continents, 80% were differentiated in the same orientation relative to the Equator, implicating selection as the likely explanation (Turner et al. 2008a). Larger regions of genomic differentiation within and between African and non-African populations have also been discovered, some of them possibly being driven by divergent selection (e.g., Dopman and Hartl 2007; Emerson et al. 2008; Turner et al. 2008a, Aguade 2009). Despite this recent progress, we still know relatively little about large-scale patterns of genomic differentiation in this species, especially between African and non-African populations, and whether most of this differentiation is consistent with demographic processes alone or if it requires selective explanations.In this work, we explicitly focus on identifying differentiated sites across the genome between U.S., Caribbean, West African, and Zimbabwean populations. This allows us to address several fundamental questions related to genomic evolution in D. melanogaster, such as the following: (1) Do genome-wide patterns of differentiation reflect patterns of reproductive isolation? (2) Is genomic differentiation random across and within chromosomes or are some regions overrepresented? (3) What are the population genetics properties of differentiated sites and their surrounding sequences? (4) Can demographic historical processes alone explain most of the observed differentiation on a genome-wide level or is it necessary to involve selection in their explanation?In general, our findings revealed that most genomic differentiation within D. melanogaster shows an out-of-Africa genetic signature. These results are inconsistent with the notion that most genomic differentiation occurs between cosmopolitan and Zimbabwean reproductively isolated races. Further, we found that the X is more differentiated between North American and African populations and more strongly deviates from pure neutrality in North American populations relative to autosomes. Nevertheless, our article shows that much of this deviation from neutrality is broadly consistent with several demographic null models, with a few notable exceptions. Athough this does not exclude selection as a possible alternative mechanism for the observed patterns, it supports the idea that most differentiation in D. melanogaster was likely driven by the sorting of African standing genetic variation into the New World.     

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.     

Estimating Selection Intensity on Synonymous Codon Usage in a Nonequilibrium Population

Codon usage bias is the nonrandom use of synonymous codons for the same amino acid. Most population genetic models of codon usage evolution assume that the population is at mutation–selection–drift equilibrium. Natural populations, however, frequently deviate from equilibrium, often because of recent demographic changes. Here, we construct a matrix model that includes the effects of a recent change in population size on estimates of selection on preferred vs. unpreferred codons. Our results suggest that patterns of synonymous polymorphisms affecting codon usage can be quite erratic after such a change; statistical methods that fail to take demographic effects into account can then give incorrect estimates of important parameters. We propose a new method that can accurately estimate both demographic and codon usage parameters. The method also provides a simple way of testing for the effects of covariates such as gene length and level of gene expression on the intensity of selection, which we apply to a large Drosophila melanogaster polymorphism data set. Our analyses of twofold degenerate codons reveal that (i) selection acts in favor of preferred codons, (ii) there is mutational bias in favor of unpreferred codons, (iii) shorter genes and genes with higher expression levels are under stronger selection, and (iv) there is little evidence for a recent change in population size in the Zimbabwe population of D. melanogaster.CODONS specifying the same amino acid are called synonymous codons. These are often used nonrandomly, with some codons appearing more frequently than others. This biased usage of synonymous codons has been found in many organisms such as Drosophila, yeast, and bacteria (Ikemura 1985; Duret and Mouchiroud 1999; Hershberg and Petrov 2008). Conventionally, synonymous codons for a given amino acid are divided into two classes: preferred and unpreferred codons (Ikemura 1985; Akashi 1994; Duret and Mouchiroud 1999). Several observations indicate that codon usage is affected by natural selection. First, in species with codon usage bias, preferred codons generally correspond to the most abundant tRNA species (Ikemura 1981). Second, highly expressed genes usually have higher codon usage bias than genes with low expression (Sharp and Li 1986; Duret and Mouchiroud 1999; Hey and Kliman 2002). Third, the synonymous substitution rate of a gene has been shown to be negatively correlated with its degree of codon usage bias (Sharp and Li 1986; Bierne and Eyre-Walker 2006). The most commonly cited explanations of the apparent fitness differences between preferred and unpreferred codons are selection for translation efficiency, translational accuracy, and mRNA stability (Ikemura 1985; Eyre-Walker and Bulmer 1993; Akashi 1994; Drummond et al. 2005). Recently, it has been proposed that exon splicing also affects codon usage bias (Warnecke and Hurst 2007).From a population genetics perspective, the extent of codon usage bias is ultimately a product of the joint effects of mutation, selection, genetic drift, recombination, and demographic history. The Li–Bulmer model of drift, selection, and reversible mutation between preferred and unpreferred codons at a site is the most widely used model (Li 1987; Bulmer 1991; McVean and Charlesworth 1999). Applications of this model generally assume that the population is at mutation–selection–drift equilibrium. However, empirical studies have suggested that changes in the strengths of various driving forces may not be unusual. For example, in Drosophila melanogaster, there is evidence that the population size (Li and Stephan 2006; Thornton and Andolfatto 2006; Keightley and Eyre-Walker 2007; Stephan and Li 2007), recombinational landscape (Takano-Shimizu 1999), and mutational process (Takano-Shimizu 2001; Kern and Begun 2005) may have changed significantly over the species'' evolutionary history.Such changes cause departures from equilibrium. Theoretical models show that it takes a very long time, proportional to the reciprocal of the mutation rate, for the population to approach a new equilibrium state (Tachida 2000; Comeron and Kreitman 2002). Before reaching equilibrium, the population often shows counterintuitive patterns of evolution (Eyre-Walker 1997; Takano-Shimizu 1999, 2001; Comeron and Kreitman 2002; Comeron and Guthrie 2005; Charlesworth and Eyre-Walker 2007). Despite these theoretical results, details of the patterns of polymorphism and substitution rates following a recent change in population size, and their effects on estimates of strength of selection, have not been determined.The above findings point to the importance of incorporating nonequilibrium factors into the study of codon usage bias. To this end, we extend the Li–Bulmer model to allow population size to vary over time, by representing the evolutionary process by a transition matrix. By analyzing this matrix model, we show that a recent change in population size can result in erratic patterns of codon usage and that methods failing to take into account these demographic effects can give false estimates of the intensity of selection.To solve these problems, we propose a new method, which does not require polarizing ancestral vs. derived states using outgroup data (cf. Cutter and Charlesworth 2006), but requires only knowledge of preferred vs. unpreferred states defined by patterns of codon usage. We use information on both polymorphic and fixed sites, which enables both mutational bias and the strength of selection to be estimated, in contrast to previous methods that use information on polymorphisms alone. Simulations indicate that this method can accurately estimate both demographic and codon usage parameters and can distinguish between selection and demography. We use the new method to analyze a large D. melanogaster polymorphism data set (Shapiro et al. 2007) and find evidence for natural selection on synonymous codons. We use our approach to show that genes with shorter coding sequences and higher levels of expression are under significantly stronger selection than longer genes with lower expression.     

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (K _m = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (K _m = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.     

Estimating the Parameters of Selection on Nonsynonymous Mutations in Drosophila pseudoobscura and D. miranda

We present the results of surveys of diversity in sets of >40 X-linked and autosomal loci in samples from natural populations of Drosophila miranda and D. pseudoobscura, together with their sequence divergence from D. affinis. Mean silent site diversity in D. miranda is approximately one-quarter of that in D. pseudoobscura; mean X-linked silent diversity is about three-quarters of that for the autosomes in both species. Estimates of the distribution of selection coefficients against heterozygous, deleterious nonsynonymous mutations from two different methods suggest a wide distribution, with coefficients of variation greater than one, and with the average segregating amino acid mutation being subject to only very weak selection. Only a small fraction of new amino acid mutations behave as effectively neutral, however. A large fraction of amino acid differences between D. pseudoobscura and D. affinis appear to have been fixed by positive natural selection, using three different methods of estimation; estimates between D. miranda and D. affinis are more equivocal. Sources of bias in the estimates, especially those arising from selection on synonymous mutations and from the choice of genes, are discussed and corrections for these applied. Overall, the results show that both purifying selection and positive selection on nonsynonymous mutations are pervasive.SURVEYS of DNA sequence diversity and divergence are shedding light on a number of questions in evolutionary genetics (for recent reviews, see Akey 2009; Sella et al. 2009). Two of the most important questions of this kind concern the distribution of selection coefficients against deleterious mutations affecting protein sequences and the proportion of amino acid sequence differences between related species that have been fixed by positive selection. Several different methods have been proposed for studying each of these questions, using different features of data on polymorphism and divergence at nonsynonymous and silent sites.For example, the parameters of the distribution of selection coefficients against deleterious amino acid mutations have been estimated by contrasting the numbers of nonsynonymous and silent within-species polymorphisms and fixed differences between species (Sawyer and Hartl 1992; Bustamante et al. 2002; Piganeau and Eyre-Walker 2003; Sawyer et al. 2007); by fitting the frequency spectra of nonsynonymous and silent variants to models of selection, mutation, and drift (Akashi 1999; Eyre-Walker et al. 2006; Keightley and Eyre-Walker 2007; Kryukov et al. 2007; Boyko et al. 2008; Eyre-Walker and Keightley 2009); or by comparing levels of nonsynonymous and silent diversities between species with different population sizes (Loewe and Charlesworth 2006; Loewe et al. 2006). The results of these different approaches generally agree in suggesting that there is a wide distribution of selection coefficients against nonsynonymous mutations and that the mean selection coefficient against heterozygous carriers of such mutations is very small. The results imply that a typical individual from a human population carries several hundred weakly deleterious mutations (Eyre-Walker et al. 2006; Kryukov et al. 2007; Boyko et al. 2008); for a typical Drosophila population, with its much higher level of variability, the number is probably an order of magnitude greater (Loewe et al. 2006; Keightley and Eyre-Walker 2007).The presence of this large load of slightly deleterious mutations in human and natural populations, most of which are held at low frequencies by natural selection, has many implications. From the point of view of understanding human genetic disease, it means that we have to face the likelihood that susceptibility to a disease can be influenced by variants at many loci, each with small effects (Kryukov et al. 2007). The pervasive presence of deleterious mutations throughout the genome contributes to inbreeding depression (Charlesworth and Willis 2009) and may mean that the effective population size is reduced by background selection effects, even in regions of the genome with normal levels of genetic recombination (Loewe and Charlesworth 2007). Their presence may contribute so strongly to Hill–Robertson effects (Hill and Robertson 1966; Felsenstein 1974) that they cause severely reduced levels of diversity and adaptation in low-recombination regions of the genome (Charlesworth et al. 2010) and create a selective advantage to maintaining nonzero levels of recombination (Keightley and Otto 2006; Charlesworth et al. 2010). In addition, having an estimate of the distribution of selection coefficients against deleterious nonsynonymous mutations allows their contribution to between-species divergence to be predicted, providing a way of estimating the fraction of fixed nonsynonymous differences caused by positive selection (Loewe et al. 2006; Boyko et al. 2008; Eyre-Walker and Keightley 2009).It is thus important to collect data that shed light on the properties of selection against nonsynonymous mutations in a wide range of systems and also to compare the results from different methods of estimation, since they are subject to different sources of difficulty and biases. In a previous study, we proposed the use of a comparison between two related species with different effective population sizes for this purpose (Loewe and Charlesworth 2006; Loewe et al. 2006), using Drosophila miranda and D. pseudoobscura as material. These are well suited for this type of study, as they are closely related, live together in similar habitats, and yet have very different levels of silent nucleotide diversity, indicating different effective population sizes (N_e). This study was hampered by our inability to compare the same set of loci across the two species and by the small number of loci that could be used. We here present the results of a much larger study of DNA variation at X-linked and autosomal loci for these two species, using D. affinis as a basis for estimating divergence. We compare the results, applying the method of Loewe et al. (2006) with that of Eyre-Walker and Keightley (2009) for estimating the distribution of deleterious selection coefficients and with McDonald–Kreitman test-based methods for estimating the proportion of nonsynonymous differences fixed by positive selection. While broadly confirming the conclusions from earlier studies, we note some possible sources of bias and describe methods for minimizing their effects.     

Identification of the Major Sex-Determining Region of Turbot (Scophthalmus maximus)

Sex determination in fish is a labile character in evolutionary terms. The sex-determining (SD) master gene can differ even between closely related fish species. This group is an interesting model for studying the evolution of the SD region and the gonadal differentiation pathway. The turbot (Scophthalmus maximus) is a flatfish of great commercial value, where a strong sexual dimorphism exists for growth rate. Following a QTL and marker association approach in five families and a natural population, we identified the main SD region of turbot at the proximal end of linkage group (LG) 5, close to the SmaUSC-E30 marker. The refined map of this region suggested that this marker would be 2.6 cM and 1.4 Mb from the putative SD gene. This region appeared mostly undifferentiated between males and females, and no relevant recombination frequency differences were detected between sexes. Comparative genomics of LG5 marker sequences against five model species showed no similarity of this chromosome to the sex chromosomes of medaka, stickleback, and fugu, but suggested a similarity to a sex-associated QTL from Oreochromis spp. The segregation analysis of the closest markers to the SD region demonstrated a ZW/ZZ model of sex determination in turbot. A small proportion of families did not fit perfectly with this model, which suggests that other minor genetic and/or environmental factors are involved in sex determination in this species.SEX ratio is a central demographic parameter directly related to the reproductive potential of individuals and populations (Penman and Piferrer 2008). The phenotypic sex depends on the processes of both sex determination and sex differentiation. Exogenous factors, such as temperature, hormones, or social behavior, can modify the gonad development pathway in fish (Baroiller and D''Cotta 2001; Piferrer and Guiguen 2008). Both genetic (GSD) and environmental sex determination has been reported in this group (Devlin and Nagahama 2002; Penman and Piferrer 2008), although primary sex determination is genetic in most species (Valenzuela et al. 2003). Among GSD, single, multiple, or polygenic sex-determining (SD) gene systems have been documented (Kallman 1984; Matsuda et al. 2002; Lee et al. 2004; Vandeputte et al. 2007).Sex determination in fish can evolve very rapidly (Woram et al. 2003; Peichel et al. 2004; Ross et al. 2009). Different sex determination mechanisms have been reported between congeneric species and even between populations of the same species (Almeida-Toledo and Foresti 2001; Lee et al. 2004; Mank et al. 2006). The evolution of sex chromosomes involves the suppression of recombination between homologous chromosomes probably to maintain sex-related coadapted gene blocks (Charlesworth et al. 2005; Tripathi et al. 2009). The sex determination pathway appears to be less conserved than other developmental processes (Penman and Piferrer 2008). However, differences are more related to the top of the hierarchy in the developmental pathway, while downstream genes are more conserved (Wilkins 1995; Marín and Baker 1998). As a consequence, the SD master gene in fish can vary among related species (Kondo et al. 2003; Tanaka et al. 2007; Alfaqih et al. 2009). In this sense, fish represent an attractive model for studying the evolution of SD mechanisms and sex chromosomes (Peichel et al. 2004; Kikuchi et al. 2007).A low proportion of fish species have demonstrated sex-associated chromosome heteromorphisms (Almeida-Toledo and Foresti 2001; Devlin and Nagahama 2002; Penman and Piferrer 2008). This is congruent with the rapid evolution of the SD region in fish, and thus in most species the male and female version of this chromosome region appears largely undifferentiated. In spite of this, indirect clues related to progenies of sex/chromosome-manipulated individuals or to segregation of morphologic/molecular sex-associated markers indicate that mechanisms of sex determination in fish are similar to other vertebrates (Penman and Piferrer 2008). With the arrival of genomics, large amounts of different genetic markers and genomic information are available for scanning genomes to look for their association with sex determination. Quantitative trait loci (QTL) (Cnaani et al. 2004; Peichel et al. 2004) or marker association (Felip et al. 2005; Chen et al. 2007) approaches have been used to identify the SD regions in some fish species. Also, microarrays constructed from gonadal ESTs have been applied to detect differentially expressed genes in the process of gonadal differentiation (Baron et al. 2005). Further, the increased genomic resources in model and aquaculture species have allowed the development of both comparative genomics (Woram et al. 2003; Kikuchi et al. 2007; Tripathi et al. 2009) and candidate gene (Shirak et al. 2006; Alfaqih et al. 2009) strategies to identify and characterize the SD region in fish. This has permitted the identification of the SD region in eight fish, including both model and aquaculture species (reviewed in Penman and Piferrer 2008).The turbot is a highly appreciated European aquaculture species, whose harvest is expected to increase from the current 9000 tons to >15,000 tons in 2012 (S. Cabaleiro, personal communication). Females of this species reach commercial size 4–6 months before males do, explaining the interest of the industry in obtaining all-female populations. Although some differences between families can be observed in the production process at farms, sex ratio is usually balanced at ∼1:1. Neither mitotic nor meiotic chromosomes have shown sex-associated heteromorphisms in turbot (Bouza et al. 1994; Cuñado et al. 2001). The proportion of sexes observed in triploid and especially gynogenetic progenies moved Cal et al. (2006a,b) to suggest an XX/XY mechanism in turbot with some additional, either environmental or genetic, factor involved. However, Haffray et al. (2009) have recently claimed a ZZ/ZW mechanism on the basis of the analysis of a large number of progenies from steroid-treated parents. These authors also suggested some (albeit low) influence of temperature in distorting sex proportions after the larval period. Finally, hybridizations between brill (Scophthalmus rhombus) and turbot render monosex progenies, depending on the direction of the cross performed, which suggests different SD mechanisms in these congeneric species (Purdom and ThaCker 1980).In this study, we used the turbot genetic map (Bouza et al. 2007, 2008; Martínez et al. 2008) to look for sex-associated QTL in this species. The identification of a major QTL in a specific linkage group (LG) in the five families analyzed prompted us to refine the genetic map at this LG and to perform a comparative genomics approach against model fish species for a precise location and characterization of the putative SD region. Also, sex-associated QTL markers were screened in a large natural population to provide additional support to our findings and to obtain population parameters at sex-related markers that could aid in interpreting the evolution of this genomic region.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Multiple Sclerosis—Current Etiological Concepts

An animal model for acute multiple sclerosis (ms) is experimental allergic encephalomyelitis (eae). eae is produced by intradermal injection of a protein component of central nervous system (cns) myelin. Ultrastructural studies of eae and of a peripheral nerve analog, experimental allergic neuritis (ean), have revealed an orderly sequence of cellular events leading to the destruction and removal of myelin with sparing of axons (primary demyelination). Acute ms has not been studied electron microscopically, but the ultrastructural similarities between ean and a case of acute Landry-Guillain-Barré syndrome, a primary demyelinating disease of the peripheral nervous system, suggest that a similar sequence of events might be found in acute ms. While the pathological findings support a cellmediated or delayed hypersensitivity response, there is also evidence for the pathogenetic role of circulating antibodies. Among such evidence is included the finding that sera from animals with eae and humans with acute ms rapidly produce a reversible block of complex (polysynaptic) electrical activity when applied to cns tissue cultures, which suggests a possible mechanism for transient symptoms in ms. Epidemiological and other studies link ms with a viral cause, although no direct evidence that ms is caused by a virus exists. Viral and immunological mechanisms are not mutually exclusive in considering pathogenetic possibilities for ms, for it can be postulated that a viral infection of the central nervous system acts as a triggering agent for a series of immune responses, including production of a bioelectric blocking antibody and demyelination mediated by sensitized cells, the combination of which ultimately produces the total clinical picture of ms.     

Mating-System Variation,Demographic History and Patterns of Nucleotide Diversity in the Tristylous Plant Eichhornia paniculata

Inbreeding in highly selfing populations reduces effective size and, combined with demographic conditions associated with selfing, this can erode genetic diversity and increase population differentiation. Here we investigate the role that variation in mating patterns and demographic history play in shaping the distribution of nucleotide variation within and among populations of the annual neotropical colonizing plant Eichhornia paniculata, a species with wide variation in selfing rates. We sequenced 10 EST-derived nuclear loci in 225 individuals from 25 populations sampled from much of the geographic range and used coalescent simulations to investigate demographic history. Highly selfing populations exhibited moderate reductions in diversity but there was no significant difference in variation between outcrossing and mixed mating populations. Population size interacted strongly with mating system and explained more of the variation in diversity within populations. Bayesian structure analysis revealed strong regional clustering and selfing populations were highly differentiated on the basis of an analysis of F_st. There was no evidence for a significant loss of within-locus linkage disequilibrium within populations, but regional samples revealed greater breakdown in Brazil than in selfing populations from the Caribbean. Coalescent simulations indicate a moderate bottleneck associated with colonization of the Caribbean from Brazil ∼125,000 years before the present. Our results suggest that the recent multiple origins of selfing in E. paniculata from diverse outcrossing populations result in higher diversity than expected under long-term equilibrium.THE rate of self-fertilization in hermaphrodite organisms is expected to affect a number of important features of population genetic structure and diversity. Most directly, homozygosity increases as a function of the selfing rate and thus reduces the effective population size (N_e), up to twofold with complete selfing (Pollak 1987; Charlesworth et al. 1993; Nordborg 2000). Further, because of increased homozygosity, crossing over rarely occurs between heterozygous sites, thus increasing linkage disequilibrium (LD). Higher LD causes stronger hitchhiking effects such as selective sweeps, background selection, and Hill–Robertson interference, all of which are expected to further reduce the amount of neutral genetic variation within populations (reviewed in Charlesworth and Wright 2001).Population genetic processes resulting from inbreeding may be further augmented by demographic and life-history characteristics associated with the selfing habit. In particular, selfing populations can be founded by single individuals, resulting in striking reductions in diversity as a result of genetic bottlenecks and reproductive isolation. The capacity for uniparental reproduction gives many selfers prolific colonizing ability and the capacity to establish after long-distance dispersal, especially in comparison with obligate outcrossers (Baker 1955; Pannell and Barrett 1998). The colonization–extinction dynamics typical of many selfing species and limited pollen-mediated gene flow also increase differentiation among populations, resulting in considerable population subdivision (Hamrick and Godt 1990, 1996; Schoen and Brown 1991). Although the total amounts of among-population variation may be less affected by these processes (Pannell and Charlesworth 1999; Ingvarsson 2002), the demographic and life-history characteristics of many selfing species are likely to result in nonequilibrium conditions occurring in selfing populations.In many taxa where selfing has evolved it may be of relatively recent origin (Schoen et al. 1997; Takebayashi and Morrell 2001; Foxe et al. 2009; Guo et al. 2009). Where selfing has recently established, demographic forces associated with colonization may be as important as the mating system per se in structuring patterns of diversity. For example, if selfing originates through the establishment of a small number of founders, we would expect a sharp reduction in diversity relative to the outcrossing progenitor and a strong signature of a genetic bottleneck. In contrast, if selfing has evolved recently through the spread of genetic modifiers of small effect, newly established populations may retain significant amounts of ancestral polymorphism from their outcrossing progenitors. In this latter case populations may retain considerably more variation than expected under long-term equilibrium predictions.Molecular evidence for reduced nucleotide diversity and greater differentiation among populations of selfing taxa compared to populations of related outcrossing taxa has been reported from Leavenworthia (Liu et al. 1998, 1999), Arabidopsis (Savolainen et al. 2000; Wright et al. 2002), Solanum (Baudry et al. 2001), Mimulus (Sweigart and Willis 2003), Amsinckia (Perusse and Schoen 2004), and Caenorhabditis (Graustein et al. 2002; Cutter et al. 2006; Cutter 2008). In each case the reduction in diversity was more severe than the twofold reduction predicted for selfing populations at equilibrium. This indicates that factors in addition to the mating system are reducing diversity, but it has been difficult to uncouple the relative importance of genetic hitchhiking from the ecology and demographic history of selfing taxa. This challenge parallels similar difficulties in efforts to distinguish selective from demographic explanations in population genetic studies of Drosophila (Haddrill et al. 2005; Ometto et al. 2005; Thornton and Andolfatto 2006; Jensen et al. 2008). However, in many plant populations, especially those with annual life histories and small structured populations, demographic processes may play a more prominent role in causing reduced diversity than increased hitchhiking associated with selfing.Molecular population genetic studies of selfing in plants have generally focused on either small samples from a large number of populations (e.g., Sweigart and Willis 2003; Nordborg et al. 2005) or relatively large within-population samples from a small number of populations (e.g., Baudry et al. 2001). Ideally, a deeper sampling both within and among populations combined with independent ecological and historical information is required to improve understanding of the interplay of demographic and selective factors. Here we address these issues by examining patterns of nucleotide diversity within a large sample of populations of Eichhornia paniculata (Pontederiaceae), an annual species for which there is considerable ecological and demographic information (reviewed in Barrett and Husband 1997).E. paniculata occurs primarily in northeastern (N.E.) Brazil and the Caribbean islands of Cuba and Jamaica. Various lines of evidence suggest that Brazil is the original source region for Caribbean populations (reviewed in Barrett et al. 2009). Populations of E. paniculata exhibit striking mating-system diversity, ranging from predominantly outcrossing to those that are highly selfing (outcrossing rate, t = 0.002–0.96; n = 54 populations) (Barrett and Husband 1990; Barrett et al. 1992). Variation in mating system is associated with the evolutionary breakdown of the species'' tristylous genetic polymorphism and the spread and fixation of selfing variants capable of autonomous self-pollination (Barrett et al. 1989). Populations of E. paniculata are characterized by three morph structures: trimorphic with long-, mid-, and short-styled morphs (hereafter L-, M-, and S-morphs); dimorphic, with two floral morphs, most commonly the L- and M-morphs; and monomorphic, primarily composed of selfing variants of the M-morph. The morph structure and presence of selfing variants within populations explain ∼60% of the variation in outcrossing rates among populations (Barrett and Husband 1990). Trimorphic populations are largely outcrossing, dimorphic populations display mixed mating, and monomorphic populations are highly selfing. Patterns of allozyme variation indicate a reduction in diversity with increased selfing rates and greater among-population differentiation (Glover and Barrett 1987; Barrett and Husband 1990; Husband and Barrett 1993). Finally, studies of the inheritance of mating-system modifiers (Fenster and Barrett 1994; Vallejo-Marín and Barrett 2009) in combination with allozyme (Husband and Barrett 1993) and molecular evidence (Barrett et al. 2009) indicate that the transition from outcrossing to selfing in E. paniculata has occurred on multiple occasions.The goal of our study was to investigate the relation between mating-system variation and neutral molecular diversity for a large sample of E. paniculata populations encompassing most of the geographical range. This was accomplished by collecting multilocus nucleotide sequence data from 225 individuals sampled from 25 populations including trimorphic, dimorphic, and monomorphic populations. Because it has been previously demonstrated that this sequence of morph structures is strongly associated with increasing rates of self-fertilization (see Barrett and Husband 1990), we predicted a decrease in neutral diversity and increases in F_st and linkage disequilibrium from floral trimorphism to monomorphism. This extensive population-level sampling across a wide range of selfing rates allowed us to investigate the relative importance of mating system, geography, and current population size in structuring genetic variation. We also applied the approaches of Bayesian clustering (Pritchard et al. 2000; Falush et al. 2003; Gao et al. 2007) and divergence population genetics (Wakeley and Hey 1997; Hey and Nielsen 2004; Becquet and Przeworski 2007) to investigate the demographic history of E. paniculata and to provide a framework for understanding island colonization and the transition from outcrossing to selfing.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 μm canavanine was effective in root tips whereas only 50 μm was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO₃ resulted in a loss of NRA. NO₃⁻ protected nitrate reductase from this inactivation in both root tip and mature root sections.     

Phenotypic Consequences of Aneuploidy in Arabidopsis thaliana

Aneuploid cells are characterized by incomplete chromosome sets. The resulting imbalance in gene dosage has phenotypic consequences that are specific to each karyotype. Even in the case of Down syndrome, the most viable and studied form of human aneuploidy, the mechanisms underlying the connected phenotypes remain mostly unclear. Because of their tolerance to aneuploidy, plants provide a powerful system for a genome-wide investigation of aneuploid syndromes, an approach that is not feasible in animal systems. Indeed, in many plant species, populations of aneuploid individuals can be easily obtained from triploid individuals. We phenotyped a population of Arabidopsis thaliana aneuploid individuals containing 25 different karyotypes. Even in this highly heterogeneous population, we demonstrate that certain traits are strongly associated with the dosage of specific chromosome types and that chromosomal effects can be additive. Further, we identified subtle developmental phenotypes expressed in the diploid progeny of aneuploid parent(s) but not in euploid controls from diploid lineages. These results indicate long-term phenotypic consequences of aneuploidy that can persist after chromosomal balance has been restored. We verified the diploid nature of these individuals by whole-genome sequencing and discuss the possibility that trans-generational phenotypic effects stem from epigenetic modifications passed from aneuploid parents to their diploid progeny.THE genome of aneuploid individuals contains incomplete chromosome sets. The balance between chromosome types, and the genes they encode, is compromised, resulting in altered expression of many genes, including genes with dosage-sensitive effects on phenotypes. In humans, only a few types of aneuploid karyotypes are viable (Hassold and Hunt 2001), highlighting the deleterious effect of chromosome imbalance. The most commonly known viable form of aneuploidy in humans is Down syndrome, which results from a trisomy of chromosome 21 in an otherwise diploid background. Down syndrome patients exhibit many specific phenotypes, sometimes visible only in a subset of patients (Antonarakis et al. 2004). For phenotypes found in all Down syndrome patients, the penetrance of each phenotype varies between patients (Antonarakis et al. 2004). Despite the increasing amount of information available about the human genome and the availability of a mouse model for Down syndrome (O''Doherty et al. 2005), the genes responsible for most of the phenotypes associated with Down syndrome are still unknown (Patterson 2007; Korbel et al. 2009; Patterson 2009). Recently, detailed phenotypic analyses of as many as 30 aneuploid patients have allowed the identification of susceptibility regions for several specific phenotypes (Patterson 2007, 2009; Korbel et al. 2009; Lyle et al. 2009), but the specific genes remain to be identified. Understanding the physiology of aneuploidy is not only relevant to those individuals with aneuploid genomes but also to understanding cancer since most cancerous cells are aneuploid (Matzke et al. 2003; Pihan and Doxsey 2003; Storchova and Pellman 2004; Holland and Cleveland 2009; Williams and Amon 2009) or the consequences of copy number variation and dosage sensitivity (Dear 2009; Henrichsen et al. 2009).Plants are more tolerant of aneuploidy than animals (Matzke et al. 2003) for reasons that remain unclear. Since the discovery of the Datura trisomic “chromosome mutants” by Blakeslee (1921, 1922), viable trisomics of each chromosome type have been described in numerous species. Trisomics exhibit phenotypes specific to the identity of the triplicated chromosome (Blakeslee 1922; Khush 1973; Koornneef and Van der Veen 1983; Singh 2003). More complex aneuploids, i.e., individuals carrying more than one additional chromosome, can be viable as well and have been observed in many plants species, especially among the progeny of triploid individuals (McClintock 1929; Levan 1942; Johnsson 1945; Khush 1973). Some species appear to be more tolerant of complex aneuploidies than others, suggesting a genetic basis for aneuploidy tolerance (Satina and Blakeslee 1938; Khush 1973; Ramsey and Schemske 2002; Henry et al. 2009). Aneuploid individuals frequently appear spontaneously within polyploid plant populations, presumably due to a failure to equally partition the multiple chromosome sets at meiosis (Randolph 1935; Doyle 1986). These aneuploids exhibit few or subtle phenotypic abnormalities and can often compete with their euploid progenitors (Ramsey and Schemske 1998). Plants therefore provide an excellent opportunity for a genome-wide investigation of aneuploid syndromes: sample size is not limited, phenotypes can be described and assessed in detail, and plant aneuploid populations provide a complex mixture of viable karyotypes.In this article, we report our investigation of the relationship between phenotype and karyotype in populations of aneuploid Arabidopsis thaliana plants. All simple trisomics of A. thaliana have been previously isolated and phenotypically characterized (Steinitz-Sears 1962; Lee-Chen and Steinitz-Sears 1967; Steinitz-Sears and Lee-Chen 1970; Koornneef and Van der Veen 1983), demonstrating that they are tolerated in A. thaliana. We previously reported that aneuploid swarms—populations of aneuploid individuals of varying aneuploid karyotypes—could be obtained from the progeny of triploid A. thaliana individuals (Henry et al. 2005, 2009). Using a combination of a quantitative PCR-based method and flow cytometry, we were able to derive the full aneuploid karyotype of each of these individuals (Henry et al. 2006). We further crossed triploid A. thaliana to diploid or tetraploid individuals and demonstrated that at least 44 of the 60 possible aneuploid karyotypes that could result from these crosses (aneuploid individuals carrying between 11 and 19 chromosomes) were viable and successfully produced adult plants. Taken together, these populations and methods make it possible to explore the basis of aneuploid syndromes in A. thaliana. In this study, we were able to phenotypically characterize at least one individual from 25 different aneuploid karyotypes falling between diploidy and tetraploidy. We demonstrated that specific phenotypes are affected by the dosage of specific chromosome types. The effect of the dosage of specific chromosome types on traits was additive and could be used to predict the observed phenotype. The availability of multiple generations of aneuploid and euploid individuals allowed us to investigate potential long-term effects of aneuploidy as well as parent-of-origin effects on aneuploid phenotypes.     

Selection for Chaperone-Like Mediated Genetic Robustness at Low Mutation Rate: Impact of Drift,Epistasis and Complexity

Genetic robustness is defined as the constancy of a phenotype in the face of deleterious mutations. Overexpression of chaperones, to assist the folding of proteins carrying deleterious mutations, is so far one of the most accepted molecular mechanisms enhancing genetic robustness. Most theories on the evolution of robustness have focused on the implications of high mutation rate. Here we show that genetic drift, which is modulated by population size, organism complexity, and epistasis, can be a sufficient force to select for chaperone-mediated genetic robustness. Using an exact analytical solution, we also show that selection for costly genetic robustness leads to a paradox: the decrease of population fitness on long timescales and the long-term dependency on robustness mechanisms. We suggest that selection for genetic robustness could be universal and not restricted to high mutation rate organisms such as RNA viruses. The evolution of the endosymbiont Buchnera illustrates this selection mechanism and its paradox: the increased dependency on chaperones mediating genetic robustness. Our model explains why most chaperones might have become essential even in optimal growth conditions.MUTATIONAL (or genetic) robustness is defined as the constancy of a phenotype in the face of deleterious mutations (Sanjuan et al. 2007). Selection drives populations to adapt to their environment by the fixation of successive advantageous mutations. However, in approaching a fitness optimum—i.e., a genotype that is maximally adapted—they have to cope with an increasing proportion of deleterious mutations and, when at the optimum, they experience only neutral and deleterious mutations (Silander et al. 2007). Therefore any mechanism that would reduce the effect of deleterious mutations, i.e., increase mutational robustness, could be favored by natural selection when at, or near, an optimum of fitness. Indeed, the general observation that for a large range of organisms, mutations have little effect on fitness, suggests that selection for robustness is pervasive (Melton 1994; Winzeler et al. 1999). Three main mechanisms that are not mutually exclusive could explain how genetic robustness has arisen. First, in the “intrinsic hypothesis” (de Visser et al. 2003) robustness could simply be a by-product of some biologically relevant functions. Second, mutational robustness could be a by-product of the selection for nongenetic perturbations such as environment changes or intrinsic noise (Wagner 2005). Third, mutational robustness could be selected for because it is adaptive in itself. In the following we restrict our attention to this “adaptive hypothesis” (de Visser et al. 2003).Chaperone proteins, proteins that help other proteins to fold properly, have been shown to buffer the effect of deleterious mutations in diverse organisms (Rutherford 2003). In lineages that have accumulated deleterious mutations, the overexpression of the chaperone GroESL in Escherichia coli (Fares et al. 2002) or Salmonella typhymurium (Maisnier-Patin et al. 2005) resulted in an improved fitness. However, such robustness appears to come at a cost, as the buffering was visible only in carbon-rich media (Fares et al. 2002), and it is also known that GroESL-mediated refolding of proteins is ATP dependent. Chaperones can also buffer against environmental perturbations (such as heat shock); however, the observation that groESL evolved under positive selection and is overproduced in obligate intracellular endosymbionts (Moran 1996; Fares et al. 2004), for which environmental perturbations are assumed to be very weak, suggests that genetic robustness could be the direct target of selection.Selection for a modifier of genetic robustness, i.e., a gene modulating the effect of mutations, has been mainly studied in the context of high mutation rates, as the effect of the modifier allele affects the fitness of mutants (Wagner 2005). Under some theoretical frameworks, it has been suggested that the intensity of selection acting on a modifier of robustness would be of the order of the mutation rate (Gardner and Kalinka 2006). Therefore it has been presumed that selection for genetic robustness is relevant only in very large populations having a high mutation rate, such as RNA virus populations. In agreement with these ideas, artificial life experiments (Wilke and Adami 2001; Azevedo et al. 2006) and experimental data on viruses (Montville et al. 2005; Sanjuan et al. 2007) have shown that robustness varies between organisms and that it can be selected for under high mutation rates. It has also been shown by Krakauer and Plotkin (2002) that drift, i.e., stochastic effects due to the finite size of populations, can promote selection for robustness even when more robust alleles are costly, as suggested in the case of chaperone overexpression. However, again this effect was examined only under high mutation rates.When mutations are very rare, populations experience at the most the presence of a single mutant. In such conditions, the population fitness at equilibrium does not depend on the mutation rate but only on drift (Sella and Hirsh 2005; Tenaillon et al. 2007). Two factors modulate how drift affects fitness:

1. Epistasis, defined here as a local property of the adaptive landscape, describes how the selective effects of mutations depend on the genetic background in which they arise. Epistasis is negative (positive) if two mutations have a lower (higher) fitness when simultaneously present within a genome than expected if they did not interact. Negative epistasis increases selection against mutation-loaded individuals and therefore reduces the effect of drift on population fitness (Charlesworth 1990; Tenaillon et al. 2007).
2. Phenotypic complexity, defined as the number of independent mutable traits that contribute to fitness (Orr 2000; Tenaillon et al. 2007), also affects population fitness in finite populations: complex organisms are more sensitive to the action of drift (Hartl and Taubes 1998; Poon and Otto 2000; Tenaillon et al. 2007).

In this article, we attempt to further clarify the role of drift on the evolution of chaperone-like genetic robustness and to decouple the effect of drift from the effect of the mutation rate. We use Fisher''s geometric model of adaptation (Fisher 1930), to map phenotype to fitness under an assumption of a vanishing mutation rate and extract exact analytical solutions for the genetic properties of the population at mutation–selection–drift equilibrium (MSDE). We examine how these genetic properties change under various population sizes and epistasis parameters and in organisms ranging in phenotypic complexity.     

The Genetic Signature of Conditional Expression

Conditionally expressed genes have the property that every individual in a population carries and transmits the gene, but only a fraction, φ, expresses the gene and exposes it to natural selection. We show that a consequence of this pattern of inheritance and expression is a weakening of the strength of natural selection, allowing deleterious mutations to accumulate within and between species and inhibiting the spread of beneficial mutations. We extend previous theory to show that conditional expression in space and time have approximately equivalent effects on relaxing the strength of selection and that the effect holds in a spatially heterogeneous environment even with low migration rates among patches. We support our analytical approximations with computer simulations and delineate the parameter range under which the approximations fail. We model the effects of conditional expression on sequence polymorphism at mutation–selection–drift equilibrium, allowing for neutral sites, and show that sequence variation within and between species is inflated by conditional expression, with the effect being strongest in populations with large effective size. As φ decreases, more sites are recruited into neutrality, leading to pseudogenization and increased drift load. Mutation accumulation diminishes the degree of adaptation of conditionally expressed genes to rare environments, and the mutational cost of phenotypic plasticity, which we quantify as the plasticity load, is greater for more rarely expressed genes. Our theory connects gene-level relative polymorphism and divergence with the spatial and temporal frequency of environments inducing gene expression. Our theory suggests that null hypotheses for levels of standing genetic variation and sequence divergence must be corrected to account for the frequency of expression of the genes under study.IN genetically and ecologically subdivided populations, some individuals will experience a local environment very different from others, making it difficult to evolve a single adaptation adequate for all local conditions. Phenotypic plasticity allows organisms to respond adaptively to spatially and temporally varying environments by developing alternative phenotypes that enhance fitness under local conditions (Scheiner 1993; Via et al. 1995). Examples of alternative phenotypes, i.e., polyphenisms, include the defensive morphologies in Daphnia and algae induced by the presence of predators (e.g., Lively 1986; DeWitt 1998; Harvell 1998; Hazel et al. 2004); the winged and wingless morphs of bean beetles responding to resource variation (e.g., Abouheif and Wray 2002; Roff and Gelinas 2003; Lommen et al. 2005); and bacterial genes involved in traits such as quorum sensing, antibiotic production, biofilm formation, and virulence (Fuqua et al. 1996). The developmental basis of such alternative phenotypes often lies in the inducible expression of some genes in some individuals by environmental variables. That is, all individuals carry and transmit the conditionally expressed genes but only a fraction of individuals, φ, express them when environmental conditions are appropriate.The genes underlying plastic traits should experience relaxed selection due to conditional expression. Wade and co-workers have shown that genes hidden from natural selection in a fraction of individuals in the population by X-linked (Whitlock and Wade 1995; Linksvayer and Wade 2009) or sex-limited expression (Wade 1998; Demuth and Wade 2007) experience relaxed selective constraint. In Drosophila spp., sequence data for genes with maternally limited expression quantitatively support the theoretical predictions both for within-species polymorphism (Barker et al. 2005; Cruickshank and Wade 2008) and for between-species divergence (Barker Et Al 2005; Demuth and Wade 2007; Cruickshank and Wade 2008). Furthermore, male-specific genes in the facultatively sexual pea aphid have been shown to have elevated levels of sequence variation due to relaxed selection (Brisson and Nuzhdin 2008). Genes with spatially restricted expression in a heterogeneous environment should likewise experience relaxed selection. Adaptation to the most common environment in an ecologically subdivided population (Rosenzweig 1987; Holt and Gaines 1992; Holt 1996) allows deleterious mutations to accumulate in traits expressed in rare environments (Kawecki 1994; Whitlock 1996).Here we extend these results by quantifying the consequences of relaxed selection on conditionally expressed genes. Specifically, we show that, with weak selection, spatial and temporal fluctuations in selection intensity generate approximately equivalent effects on mean trait fitness, even with low rates of migration between habitats, resulting in a great simplification of analytical results. Our analytical approximations are supported with deterministic and stochastic simulations, and we note the conditions under which the approximations fail. We then derive general expressions for (1) the expected level of sequence polymorphism within populations under mutation, migration, drift, and purifying selection with conditional gene expression; (2) the rate of sequence divergence among populations, for dominant and recessive mutations; and (3) the reduction in mean population fitness due to accumulation of deleterious mutations at conditionally expressed loci. We find that the rate of accumulation of deleterious mutations for conditionally expressed genes is accelerated and the probability of fixation of beneficial mutations is reduced, causing a reduction in the fitness of conditional traits and an inflation in sequence variation within and between species. Our results suggest that evolutionary null hypotheses must be adjusted to account for the frequency of expression of genes under study, such that signatures of elevated within- or between-species sequence variation are not necessarily evidence of the action of diversifying natural selection. Furthermore, if conditional expression is due to spatial heterogeneity, we show that the level of genetic variation in a sample will often depend on whether or not genotypes were sampled from the selective habitat, the neutral habitat, or both. In the discussion we address the scope and limitations of our theory, as well as its implications for the maintenance of genetic variation, adaptive divergence between species, constraints on phenotypic plasticity, and evolutionary inference from sequence data.     

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.