Genetic Divergence under Uniform Selection. II. Different Responses to Selection for Knockdown Resistance to Ethanol among DROSOPHILA MELANOGASTER Populations and Their Replicate Lines

We have tested the hypothesis that genetic differences among conspecific populations may result in diverse responses to selection, using natural populations of Drosophila melanogaster. Selection for ethanol tolerance in a tube measuring knockdown resistance was imposed on five West Coast populations. In 24 generations the selected lines increased their mean knockdown times, on average, by a factor of 2.7. An initially weak latitudinal cline was steepened by selection. The two southernmost populations showed the same increases in the selected character, but differed consistently in their correlated responses in characters related to ethanol tolerance. This result indicates that the populations responded to selection by different genetic changes. Selection decreased female body weight and increased resistance to acetone, suggesting components of the response unrelated to ethanol metabolism. The Adhs allele was favored by selection in all populations at the onset, but increased in frequency only in the selected lines of the southernmost population. There was a correlation between latitude and Adh frequency changes, suggesting that fitnesses of the Adh alleles were dependent on the genetic background. Genetic background also had a large effect on the loss of fitness due to selection. Genetic drift between replicate lines caused more variation in selection response than initial genetic differences between populations. This result demonstrates the importance of genetic drift in divergence among natural populations undergoing uniform selection, since the effective population sizes approached those of small natural populations. Drift caused greater divergence between selected replicates than control replicates. Implications of this result for the genetic model of selection response are discussed.     

Selection in Finite Populations with Multiple Alleles. III. Genetic Divergence with Centripetal Selection and Mutation

Natural selection for an intermediate level of gene or enzyme activity has been shown to lead to a high frequency of heterotic polymorphisms in populations subject to mutation and random genetic drift. The model assumes a symmetrical spectrum of mutational variation, with the majority of variants having only minor effects on the probability of survival. Each mutational event produces a variant which is novel to the population. Allelic effects are assumed to be additive on the scale of enzyme activity, heterosis arising whenever a heterozygote has a mean level of activity closer to optimal than that of other genotypes in the population.-A new measure of genetic divergence between populations is proposed, which is readily interpreted genetically, and increases approximately linearly with time under centripetal selection, drift and mutation. The parameter is closely related to the rate of accumulation of mutational changes in a cistron over an evolutionary time span.-A survey of published data concerning polymorphic loci in man and Drosophila suggests than an alternative model, based on the superiority of hybrid molecules, is not of general importance. Thirteen loci giving rise to hybrid zones on electrophoresis have a mean heterozygote frequency of 0.22 +/-.06, compared with a value of 0.23 +/-.04 for 16 loci classified as producing no hybrid enzyme.     

Evolution of Fitness in Experimental Populations of Vesicular Stomatitis Virus

The evolution of fitness in experimental clonal populations of vesicular stomatitis virus (VSV) has been compared under different genetic (fitness of initial clone) and demographic (population dynamics) regimes. In spite of the high genetic heterogeneity among replicates within experiments, there is a clear effect of population dynamics on the evolution of fitness. Those populations that went through strong periodic bottlenecks showed a decreased fitness in competition experiments with wild type. Conversely, mutant populations that were transferred under the dynamics of continuous population expansions increased their fitness when compared with the same wild type. The magnitude of the observed effect depended on the fitness of the original viral clone. Thus, high fitness clones showed a larger reduction in fitness than low fitness clones under dynamics with included periodic bottleneck. In contrast, the gain in fitness was larger the lower the initial fitness of the viral clone. The quantitative genetic analysis of the trait ``fitness' in the resulting populations shows that genetic variation for the trait is positively correlated with the magnitude of the change in the same trait. The results are interpreted in terms of the operation of MULLER's ratchet and genetic drift as opposed to the appearance of beneficial mutations.     

Genetic Diversity and Fitness of Fusarium graminearum Populations from Rice in Korea

Fusarium graminearum is an important fungal pathogen of cereal crops and produces mycotoxins, such as the trichothecenes nivalenol and deoxynivalenol. This species may be subdivided into a series of genetic lineages or phylogenetic species. We identified strains of F. graminearum from the Republic of Korea to lineage, tested their ability to produce nivalenol and deoxynivalenol, and determined the genetic composition and structure of the populations from which they were recovered. Based on amplified fragment length polymorphism (AFLP), PCR genotyping, and chemical analyses of trichothecenes, all 249 isolates from southern provinces belonged to lineage 6, with 241 having the nivalenol genotype and 8 having the deoxynivalenol genotype. In the eastern Korea province, we recovered 84 lineage 6 isolates with the nivalenol genotype and 23 lineage 7 isolates with the deoxynivalenol genotype. Among 333 lineage 6 isolates, 36% of the AFLP bands were polymorphic, and there were 270 multilocus haplotypes. Genetic identity among populations was high (>0.972), and genotype diversity was low (30 to 58%). To test the adaptation of lineage 6 to rice, conidial mixtures of strains from lineages 3, 6, and 7 were inoculated onto rice plants and then recovered from the rice grains produced. Strains representing lineages 6 and 7 were recovered from inoculated spikelets at similar frequencies that were much higher than those for the strain representing lineage 3. Abundant perithecia were produced on rice straw, and 247 single-ascospore isolates were recovered from 247 perithecia. Perithecia representing lineage 6 (87%) were the most common, followed by those representing lineage 7 (13%), with perithecia representing lineage 3 not detected. These results suggest that F. graminearum lineage 6 may have a host preference for rice and that it may be more fit in a rice agroecosystem than are the other lineages present in Korea.Fusarium graminearum (teleomorph: Gibberella zeae) causes head blight of small grains, including rice, wheat, and barley (23). The fungus was first reported on rice in Italy by Cattaneo (4) as Botryosphaeria saubinetii Niessland. This rice disease has since been recorded in other countries, including Brazil, China, India, Japan, Nepal, and Uganda (11, 31). The disease usually does not cause heavy damage, but under conditions that favor disease development, e.g., high humidity, it may be severe. Chung et al. (7) found that an isolate from wheat could infect rice and other plants and also could cause a postemergence blight in rice. Wheat isolates of F. graminearum can cause significant disease on rice, but under greenhouse conditions no trichothecenes were detected in the infected rice florets (14). In addition, Nepalese rice contained no detectable contamination with trichothecenes even though F. graminearum occurs in Nepal (11).The fungus can produce the 8-ketotrichothecene mycotoxins nivalenol (NIV) and deoxynivalenol (DON). Most of the biosynthetic genes for the synthesis of 8-ketotrichothecenes are tightly linked in the TRI gene cluster (9). TRI7 and TRI13 are required for acetylation and oxygenation of the oxygen at C-4 to produce NIV and 4-acetyl nivalenol (4-ANIV), respectively, from DON. PCR-based methods to identify polymorphisms in both genes were developed as simple, reliable diagnostic tools for differentiating strains with DON and NIV chemotypes (20, 21). There are regional differences in the distribution of the two chemotypes. Maize and wheat in North America and Europe commonly are contaminated with DON (9), while strains with NIV chemotypes are commonly recovered from cereal crops in Asia (15, 17). In the Republic of Korea, strains with the DON chemotype often cause maize ear rot, while strains with the NIV chemotype commonly are recovered from barley (17, 35). A severe epidemic of Fusarium head blight on wheat and barley occurred in 1963 in southern Korea (5, 6). Humans and farm animals consuming moldy cereals exhibited typical signs of trichothecene intoxication involving vomiting, dizziness, nausea, abnormal pain, and diarrhea (9). The natural occurrence of NIV and DON has been reported in barley and maize in Korea (17, 35, 41), but there have been few surveys of Fusarium mycotoxins in Korean rice.O''Donnell et al. (30) divided F. graminearum into seven phylogenetic lineages based on the genealogical concordance of six genes. The phylogenetic separation has been used to raise these seven and four additional lineages to species status (36). The geographic location often influences the lineage present, e.g., lineage 7 is the most common in the United States, and lineage 6 dominates in China. Lineage and trichothecene chemotype are not correlated (45), and the lineages are morphologically cryptic. Members of all lineages are cross-fertile with strains belonging to lineage 7 and in some cases with strains of other lineages (1, 2, 19, 25), a pattern that suggests that the members of all of the lineages belong to a single biological species.Studies of F. graminearum populations have been made in different geographic regions, e.g., China (12), Europe (42), the United States (48, 49), and Argentina (34). Populations of F. graminearum have high levels of genotypic diversity, which suggests that recombination occurs regularly in F. graminearum populations. Most studies have focused on populations from wheat, barley, and corn, and there is little information on F. graminearum populations from rice.Severe epidemics of Fusarium head blight of rice occurred in August 2001 after heavy rainfall during the rice flowering period in southern Korea. Lesions on or discoloration of the glumes were common, with infected grains first appearing to be white and later yellow, salmon, or carmine. Sometimes the entire seed was colonized. Infected grains were lightweight, shrunken, and brittle. Our objectives in the present study were (i) to determine the frequency at which F. graminearum occurs in plants with rice head blight; (ii) to determine the number and relative frequency of the F. graminearum lineages present; and (iii) to evaluate the strains for their sexual fertility, genetic relatedness, virulence, and toxin-producing abilities. Our working hypotheses were that sexually fertile strains from lineage 6 would dominate in the population and that these strains would be the most aggressive toward rice. We expected most of the lineage 6 strains to produce NIV and for there to be a high level of genetic variation, as assessed by neutral (amplified fragment length polymorphism [AFLP]) markers. We evaluate here F. graminearum population diversity in Korea and provide new information on the pathogenic capabilities of strains belonging to several of the known lineages of this very widespread fungal species.     

The Underlying Structure of Adaptation under Strong Selection in 12 Experimental Yeast Populations

The aims of this study were to determine (i) whether adaptation under strong selection occurred through mutations in a narrow target of one or a few nucleotide sites or a broad target of numerous sites and (ii) whether the programs of adaptation previously observed from three experimental populations were unique or shared among populations that underwent parallel evolution. We used archived population samples from a previous study, representing 500 generations of experimental evolution in 12 populations under strong selection, 6 populations in a high-salt environment and 6 populations in a low-glucose environment. Each set of six populations included four with sexual reproduction and two with exclusively asexual reproduction. Populations were sampled as resequenced genomes of 115 individuals and as bulk samples from which frequencies of mutant alleles were estimated. In a high-salt environment, a broad target of 11 mutations within the proton exporter, PMA1, was observed among the six populations, in addition to expansions of the ENA gene cluster. This pattern was shared among populations that underwent parallel evolution. In a low-glucose environment, two programs of adaptation were observed. The originally observed pattern of mutation in MDS3/MKT1 in population M8 was a narrow target of a single nucleotide, unique to this population. Among the other five populations, the three mutations were shared in a broad target, sensing/signaling genes RAS1 and RAS2. RAS1/RAS2 mutations were not observed in the high-salt populations; PMA1 mutations were observed only in a high-salt environment.     

Genetic Changes Accompanying Increased Fitness in Evolving Populations of Escherichia Coli

Two populations of Escherichia coli, each initiated with a single clone containing a derivative of the plasmid pBR322, were maintained for long periods in glucose-limited continuous culture. In both populations, after an extensive number of generations had elapsed, clones were isolated in which the transposon Tn3 from the plasmid had integrated into the bacterial chromosome. In both cases examined, the transpositions were shown to increase relative fitness approximately 6-7%, in the environment in which the populations were maintained. The loci of integration were mapped to approximately 13.2 min (population 1) and approximately 32.8 min (population 2).     

The Genetic Structure of Natural Populations of DROSOPHILA MELANOGASTER. Xviii. Clinal and Uniform Genetic Variation over Populations

It has been reported in the previous papers of this series that in the eastern United States and Japan there is a north-to-south cline of additive genetic variance of viability and that the amount of the additive genetic variance in the northern population can be explained by mutation-selection balance. To determine whether or not the difference in the genetic variation in northern and southern populations can be explained by the differences in mutation rate and/or effective population size, numerical calculations were made using population genetic parameters. In addition, the average heterozygosities of the northern and southern populations at ten of 19 polymorphic structural loci surveyed were estimated in relation to the cline of additive genetic variance of viability, and the following findings were obtained. (1) The changes in mutation rate and population size cannot simultaneously explain the difference in additive genetic variance and inbreeding decline between the northern and southern populations. Thus, the operation of some kind of balancing selection, most likely diversifying selection, was suggested to explain the observed excess of additive genetic variance. (2) Estimates of the average heterozygosities of the southern population were not significantly different from those of the northern population. Thus, it was strongly suggested that the excess of additive genetic variance in the southern population cannot be caused by structural loci, but by factors outside the structural loci, and that protein polymorphisms are selectively neutral or nearly neutral.     

Temperature-Related Divergence in Experimental Populations of DROSOPHILA MELANOGASTER. I. Genetic and Developmental Basis of Wing Size and Shape Variation

The effects of environmental temperature on wing size and shape of Drosophila melanogaster were analyzed in populations derived from an Oregon laboratory strain kept at three temperatures (18°, 25°, 28°) for 4 yr. Temperature-directed selection was identified for both wing size and shape. The length of the four longitudinal veins, used as a test for wing size variations in the different populations, appears to be affected by both genetic and maternal influences. Vein expression appears to be dependent upon developmental pattern of the wing: veins belonging to the same compartment are coordinated in their expression and relative position, whereas veins belonging to different compartments are not. Both wing and cell areas show genetic divergence, particularly in the posterior compartment. Cell number seems to compensate for cell size variations. Such compensation is carried out both at the level of single organisms and at the level of population as a whole. The two compartments behave as individual units of selection.     

Effective Size of Populations under Selection

Equations to approximate the effective size (N(e)) of populations under continued selection are obtained that include the possibility of partial full-sib mating and other systems such as assortative mating. The general equation for the case of equal number of sexes and constant number of breeding individuals (N) is N(e) = 4N/[2(1 - α(I)) + (S(k)(2) + 4Q(2)C(2)) (1 + α(I) + 2α(O))], where S(k)(2) is the variance of family size due to sampling without selection, C(2) is the variance of selective advantages among families (the squared coefficient of variation of the expected number of offspring per family), α(I) is the deviation from Hardy-Weinberg proportions, α(O) is the correlation between genes of male and female parents, and Q(2) is the term accounting for the cumulative effect of selection on an inherited trait. This is obtained as Q = 2/[2 - G(1 + r)], where G is the remaining proportion of genetic variance in selected individuals and r is the correlation of the expected selective values of male and female parents. The method is also extended to the general case of different numbers of male and female parents. The predictive value of the formulae is tested under a model of truncation selection with the infinitesimal model of gene effects, where C(2) and G are a function of the selection intensity, the heritability and the intraclass correlation of sibs. Under random mating r = α(I) = -1/(N - 1) and α(O) = 0. Under partial full-sib mating with an average proportion β of full-sib matings per generation, r & β and α(O) & α(I) & β/ (4 - 3β). The prediction equation is compared to other approximations based on the long-term contributions of ancestors to descendants. Finally, based on the approach followed, a system of mating (compensatory mating) is proposed to reduce rates of inbreeding without loss of response in selection programs in which selected individuals from the largest families are mated to those from the smallest families.     

Memory and Fitness Optimization of Bacteria under Fluctuating Environments

Bacteria prudently regulate their metabolic phenotypes by sensing the availability of specific nutrients, expressing the required genes for their metabolism, and repressing them after specific metabolites are depleted. It is unclear, however, how genetic networks maintain and transmit phenotypic states between generations under rapidly fluctuating environments. By subjecting bacteria to fluctuating carbon sources (glucose and lactose) using microfluidics, we discover two types of non-genetic memory in Escherichia coli and analyze their benefits. First, phenotypic memory conferred by transmission of stable intracellular lac proteins dramatically reduces lag phases under cyclical fluctuations with intermediate timescales (1–10 generations). Second, response memory, a hysteretic behavior in which gene expression persists after removal of its external inducer, enhances adaptation when environments fluctuate over short timescales (<1 generation). Using a mathematical model we analyze the benefits of memory across environmental fluctuation timescales. We show that memory mechanisms provide an important class of survival strategies in biology that improve long-term fitness under fluctuating environments. These results can be used to understand how organisms adapt to fluctuating levels of nutrients, antibiotics, and other environmental stresses.     

Genetic Divergence Among Marine and Lagoon Atherina boyeri Populations in Greece Using mtDNA Analysis

Genetic differentiation and phylogenetic relationships among 15 Atherina boyeri populations from several marine and lagoon or lake sites in Greece were investigated using mtDNA analysis. PCR-RFLP analysis of 12s, 16s rRNA genes and D-loop revealed 23 haplotypes. All the lake or lagoon populations, as well as the Kymi and Kalymnos populations that originated from sites with lagoonlike environmental conditions, showed haplotypes 1-6, clearly distinguishable from the marine populations, which exhibited types 7-23. The genetic divergence values estimated between the lagoon and the marine populations ranged from 5.55 to 10.45%. The high genetic differentiation observed between these two types of populations is also highlighted by the dendrograms obtained using UPGMA and maximum parsimony methods.     

Adaptive Divergence in Experimental Populations of Pseudomonas fluorescens. IV. Genetic Constraints Guide Evolutionary Trajectories in a Parallel Adaptive Radiation

The capacity for phenotypic evolution is dependent upon complex webs of functional interactions that connect genotype and phenotype. Wrinkly spreader (WS) genotypes arise repeatedly during the course of a model Pseudomonas adaptive radiation. Previous work showed that the evolution of WS variation was explained in part by spontaneous mutations in wspF, a component of the Wsp-signaling module, but also drew attention to the existence of unknown mutational causes. Here, we identify two new mutational pathways (Aws and Mws) that allow realization of the WS phenotype: in common with the Wsp module these pathways contain a di-guanylate cyclase-encoding gene subject to negative regulation. Together, mutations in the Wsp, Aws, and Mws regulatory modules account for the spectrum of WS phenotype-generating mutations found among a collection of 26 spontaneously arising WS genotypes obtained from independent adaptive radiations. Despite a large number of potential mutational pathways, the repeated discovery of mutations in a small number of loci (parallel evolution) prompted the construction of an ancestral genotype devoid of known (Wsp, Aws, and Mws) regulatory modules to see whether the types derived from this genotype could converge upon the WS phenotype via a novel route. Such types—with equivalent fitness effects—did emerge, although they took significantly longer to do so. Together our data provide an explanation for why WS evolution follows a limited number of mutational pathways and show how genetic architecture can bias the molecular variation presented to selection.UNDERSTANDING—and importantly, predicting—phenotypic evolution requires knowledge of the factors that affect the translation of mutation into phenotypic variation—the raw material of adaptive evolution. While much is known about mutation rate (e.g., Drake et al. 1998; Hudson et al. 2002), knowledge of the processes affecting the translation of DNA sequence variation into phenotypic variation is minimal.Advances in knowledge on at least two fronts suggest that progress in understanding the rules governing the generation of phenotypic variation is possible (Stern and Orgogozo 2009). The first stems from increased awareness of the genetic architecture underlying specific adaptive phenotypes and recognition of the fact that the capacity for evolutionary change is likely to be constrained by this architecture (Schlichting and Murren 2004; Hansen 2006). The second is the growing number of reports of parallel evolution (e.g., Pigeon et al. 1997; ffrench-Constant et al. 1998; Allender et al. 2003; Colosimo et al. 2004; Zhong et al. 2004; Boughman et al. 2005; Shindo et al. 2005; Kronforst et al. 2006; Woods et al. 2006; Zhang 2006; Bantinaki et al. 2007; McGregor et al. 2007; Ostrowski et al. 2008)—that is, the independent evolution of similar or identical features in two or more lineages—which suggests the possibility that evolution may follow a limited number of pathways (Schluter 1996). Indeed, giving substance to this idea are studies that show that mutations underlying parallel phenotypic evolution are nonrandomly distributed and typically clustered in homologous genes (Stern and Orgogozo 2008).While the nonrandom distribution of mutations during parallel genetic evolution may reflect constraints due to genetic architecture, some have argued that the primary cause is strong selection (e.g., Wichman et al. 1999; Woods et al. 2006). A means of disentangling the roles of population processes (selection) from genetic architecture is necessary for progress (Maynard Smith et al. 1985; Brakefield 2006); also necessary is insight into precisely how genetic architecture might bias the production of mutations presented to selection.Despite their relative simplicity, microbial populations offer opportunities to advance knowledge. The wrinkly spreader (WS) morphotype is one of many different niche specialist genotypes that emerge when experimental populations of Pseudomonas fluorescens are propagated in spatially structured microcosms (Rainey and Travisano 1998). Previous studies defined, via gene inactivation, the essential phenotypic and genetic traits that define a single WS genotype known as LSWS (Spiers et al. 2002, 2003) (Figure 1). LSWS differs from the ancestral SM genotype by a single nonsynonymous nucleotide change in wspF. Functionally (see Figure 2), WspF is a methyl esterase and negative regulator of the WspR di-guanylate cyclase (DGC) (Goymer et al. 2006) that is responsible for the biosynthesis of c-di-GMP (Malone et al. 2007), the allosteric activator of cellulose synthesis enzymes (Ross et al. 1987). The net effect of the wspF mutation is to promote physiological changes that lead to the formation of a microbial mat at the air–liquid interface of static broth microcosms (Rainey and Rainey 2003).Open in a separate windowFigure 1.—Outline of experimental strategy for elucidation of WS-generating mutations and their subsequent identity and distribution among a collection of independently evolved, spontaneously arising WS genotypes. The strategy involves, first, the genetic analysis of a specific WS genotype (e.g., LSWS) to identify the causal mutation, and second, a survey of DNA sequence variation at specific loci known to harbor causal mutations among a collection of spontaneously arising WS genotypes. For example, suppressor analysis of LSWS using a transposon to inactivate genes necessary for expression of the wrinkly morphology delivered a large number of candidate genes (top left) (Spiers et al. 2002). Genetic and functional analysis of these candidate genes (e.g., Goymer et al. 2006) led eventually to the identity of the spontaneous mutation (in wspF) responsible for the evolution of LSWS from the ancestral SM genotype (Bantinaki et al. 2007). Subsequent analysis of the wspF sequence among 26 independent WS genotypes (bottom) showed that 50% harbored spontaneous mutations (of different kinds; see Open in a separate windowFigure 2.—Network diagram of DGC-encoding pathways underpinning the evolution of the WS phenotype and their regulation. Overproduction of c-di-GMP results in overproduction of cellulose and other adhesive factors that determine the WS phenotype. The ancestral SBW25 genome contains 39 putative DGCs, each in principle capable of synthesizing the production of c-di-GMP, and yet WS genotypes arise most commonly as a consequence of mutations in just three DGC-containing pathways: Wsp, Aws, and Mws. In each instance, the causal mutations are most commonly in the negative regulatory component: wspF, awsX, and the phosphodiesterase domain of mwsR (see text).To determine whether spontaneous mutations in wspF are a common cause of the WS phenotype, the nucleotide sequence of this gene was obtained from a collection of 26 spontaneously arising WS genotypes (WS_A-Z) taken from 26 independent adaptive radiations, each founded by the same ancestral SM genotype (Figure 1): 13 contained mutations in wspF (Bantinaki et al. 2007). The existence of additional mutational pathways to WS provided the initial motivation for this study.

TABLE 1

Mutational causes of WS