Aspects of nonrandom turnover involved in the concerted evolution of intergenic spacers within the ribosomal DNA of Drosophila melanogaster

Polymerase chain reaction (PCR)-amplified, sequenced, and digitally typed intergenic spacers (IGSs) of the ribosomal (r)DNA in D. melanogaster reveal unexpected features of the mechanisms of turnover involved with the concerted evolution of the gene family. Characterization of the structure of three isolated IGS length variants reveals breakage hot spots within the 330-base-pair (bp) subrepeat array found in the spacers. Internal mapping of variant repeats within the 240-bp subrepeat array using a novel digital DNA typing procedure (minisatellite variant repeat [MVR]-PCR) shows an unexpected pattern of clustering of variant repeats. Each 240-bp subrepeat array consists of essentially two halves with the repeats in each half identified by specific mutations. This bipartite structure, observed in a cloned IGS unit, in the majority of genomic DNA of laboratory and wild flies and in PCR-amplified products, has been widely homogenized yet is not predicted by a model of unequal crossing over with randomly placed recombination breakpoints. Furthermore, wild populations contain large numbers of length variants in contrast to uniformly shared length variants in laboratory stocks. High numbers of length variants coupled to the observation of a homogenized bipartite structure of the 240-bp subrepeat array suggest that the unit of turnover and homogenization is smaller than the IGS and might involve gene conversion. The use of PCR for the structural analysis of members of the rDNA gene family coupled to digital DNA typing provides powerful new inroads into the mechanisms of DNA turnover affecting the course of molecular evolution in this family. Correspondence to: G. A. Dover     

CHROMOSOMAL POLYMORPHISM IN ISOFEMALE LINES AND CAGE POPULATIONS OF DROSOPHILA MELANOGASTER

Drosophila melanogaster populations in nature usually carry inversion polymorphisms. When they were transferred to and maintained in the laboratory as large cage populations, frequencies of polymorphic inversions were drastically decreased and finally eliminated. This “cage effect” was observed irrespective of the geographical origin of the population or the initial frequency of each inversion. The decrease and elimination of inversions in the cage was not overcome by changing conditions such as medium, temperature, or the number of isofemale lines (40-600) introduced. On the other hand, in the sets of isofemale lines derived from the same geographical origins as the cage populations, each of which was maintained as a small vial population, the inversion frequencies, though decreased from the initial frequencies, were kept at significantly high levels. The cage populations initiated with one or two isofemale lines also maintained the inversion polymorphisms that were as high as vial populations.     

Clinal and seasonal variations in initial retention capacity of virginDrosophila melanogaster females as a strategy for fitness

Summary The study of the first laboratory generation of isofemale lines ofDrosophila melanogaster, founded by wild flies collected in populations of various geographical origins, has shown that virgin females differ in their capacity to control egg deposition. This capacity to delay the ovulation process when no sexual partner is available is genetically determined. The present study shows that the frequency distribution of the various phenotypes varies progressively along a latitudinal cline from Afrotropical to European regions, or from neotropical to nearctic areas, while a few oriental populations appear off the cline. This variation in initial retention capacity, added to a similar variation in number of ovarioles conditioning a potential increase in fecundity for mated flies, induces variation in reactivity to insemination which may be an adaptation to a variable environment. Moreover, similar study of F1 virgin females from isofemale lines founded by wild flies collected in Spring, Summer and Autumn in two French populations over ten successive years revealed that seasonal genetic variations occur in this capacity to postpone egg-laying in case of non-insemination. The frequency of the different phenotypes follows cyclical seasonal variations. Long retention phenotypes are more frequent in Spring and Autumn samples, whereas short retention phenotypes, closer to the ancestral African type, are more common in Summer generations. This balancing selection temporarily modifies the genetic equilibrium of the population, enhancing its competitive ability. Temperature appears to be the primordial selective factor both in geographical and in seasonal variations.     

Purging of inbreeding depression and fitness decline in bottlenecked populations of Drosophila melanogaster

Drastic reductions in population size, or bottlenecks, are thought to significantly erode genetic variability and reduce fitness. However, it has been suggested that a population can be purged of the genetic load responsible for reduced fitness when subjected to bottlenecks. To investigate this phenomenon, we put a number of Drosophila melanogaster isofemale lines known to differ in inbreeding depression through four ‘founder‐flush’ bottleneck cycles with flush sizes of 5 or 100 pairs and assayed for relative fitness (single‐pair productivity) after each cycle. Following the founder‐flush phase, the isofemale lines, with a large flush size and a history of inbreeding depression, recovered most of the fitness lost from early inbreeding, consistent with purging. The same isofemale lines, with a small flush size, did not regain fitness, consistent with the greater effect of genetic drift in these isofemale lines. On the other hand, the isofemale lines that did not show initial inbreeding depression declined in fitness after repeated bottlenecks, independent of the flush size. These results suggest that the nature of genetic variation in fitness may greatly influence the way in which populations respond to bottlenecks and that stochastic processes play an important role. Consequently, an attempt intentionally to purge a population of detrimental variation through inbreeding appears to be a risky strategy, particularly in the genetic management of endangered species.     

Environmental control of ovarian dormancy in natural populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Drosophila melanogaster from Australia, Europe and North America enter an adult ovarian dormancy in response to short days and low temperatures. The independent effects of temperature and day length in the determination of dormancy have been examined only in one long-established laboratory line (Canton-S). In all other studies of natural or laboratory populations, dormancy has been assessed at either a single short day or a single moderately low temperature. Herein, we determine the relative roles of temperature, photoperiod, and their interaction in the control of ovarian dormancy in D. melanogaster from two natural populations representing latitudinal extremes in eastern North America (Florida at 27°N and Maine at 44°N). In both natural populations, temperature is the main determinant of dormancy, alone explaining 67% of the total variation among replicate isofemale lines, whereas photoperiod has no significant effect. We conclude that ovarian dormancy in D. melanogaster is a temperature-initiated syndrome of winter-tolerant traits that represents an adaptive phenotypic plasticity in temperate seasonal environments.     

Ribosomal DNA variation within and among individuals ofLisianthius (Gentianaceae) populations

Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of theLisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site differences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18S and 5.8S rRNA genes, a region inLisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.     

Genetics and the environment in interspecific competition: a study using the sibling species Drosophila melanogaster and Drosophila simulans

The outcome of interspecific competition of two closely related species may depend upon genetic variation in the two species and the environment in which the experiment is carried out. Interspecific competition in the two sibling species, Drosophila melanogaster and D. simulans, is usually investigated using longterm laboratory stocks that often have mutant markers that distinguish them. To examine competition in flies that genetically more closely resemble flies in nature, we utilized freshly caught wildtype isofemale lines of the two species collected at the same site in San Carlos, Mexico. Under ordinary laboratory conditions, D. melanogaster always won in competition. However, in hotter and drier conditions, D. simulans competed much more effectively. In these environmental conditions, there were genetic differences in competitive ability among lines with the outcome of competition primarily dependent upon the line of D. melanogaster used but in some cases also influenced by the line of D. simulans used. Differences in the measures of productivity and developmental time did not explain the differences in competitive ability among lines. This suggests that the outcome of competition was not due to differences in major fitness components among the isofemale lines but to some other attribute(s) that influenced competitive ability. When lines of flies were combined, the outcome of competition was generally consistent with competitive outcomes between pairs of lines. In several cases, the combination of lines performed better than the best of the constituent lines, suggesting that competitive ability was combined heterotically and that the total amount of genetic variation was important in the outcome of interspecific competition.     

Latitudinal Variation in Starvation Resistance is Explained by Lipid Content in Natural Populations of Drosophila melanogaster

One of the most common environmental stressors is a shortage or suboptimal quality of food, thus all animals deal with periods of starvation. In the present study we examine variation in starvation resistance, longevity and body lipid content and the correlations between traits along an environmental gradient using isofemale lines recently derived from natural populations of Drosophila melanogaster from South America. The use of isofemale lines and controlled rearing laboratory conditions allows us to investigate within and among population components of genetic variation and the potential associations among starvation resistance, longevity and body lipid content. All these traits were analyzed separately in females and males, improving our understanding of sexual dimorphism. Our results revealed significant differences among populations in starvation resistance and longevity. Actually, the opposing latitudinal cline detected for starvation resistance suggests that natural selection played an essential role in shaping the pattern of geographic variation in this trait. Moreover, we also detected a positive relationship between starvation resistance and body lipid content in both sexes, providing evidence for a physiological and/or evolutionary association between these traits. Conversely, starvation resistance was not correlated with longevity indicating that these traits might be enabled to evolve independently. Finally, our study reveals that there is abundant within population genetic variation for all traits that may be maintained by sex-specific effects.     

Adaptive diversity in genetic control of egg-laying behavior in Drosophila melanogaster

Summary

In the absence of insemination or of a suitable oviposition site, Drosophila melanogaster females react with egg retention patterns which differ as to the position in the genital tract at which the mechanism operates (ovarioles for initial retention; uterus for oviposition blocking) and to physiological basis. A study of the first laboratory generation of isofemale lines, founded by using three different samples of wild flies collected the same year from a natural population, showed a significant genetic variability in initial egg retention by virgin females and in the capacity for oviposition blocking by mated flies reacting to an unfavorable oviposition site. Comparison of isofemale lines showed that these two adaptive behavior patterns also differ in their genetic determination. They are controlled, and therefore can react, independently to selection pressures resulting from seasonal variation in the natural environment.     

Micro-spatial population differentiation in activity of glycerol-3-phosphate oxidase (GPO) from mitochondria of Drosophila melanogaster

Replicate mass-bred laboratory populations of D. melanogaster were derived from females collected in the Tahbilk winery cellar and from females collected outside but from within two kilometres of the cellar. When mitochondrial extracts from larvae were assayed for specific activity of glycerol-3-phosphate oxidase the cellar populations had levels only 50% of those from the outside area, confirming an earlier report of such a difference among isofemale lines derived from these same areas. This micro-spatial differentiation occurred when larvae were raised on a medium supplemented with both sucrose (5% w/v) and ethanol (4% v/v), known to effect high GPO activity, but was not detected when the larvae were raised on unsupplemented medium.A heritable basis for larval GPO activity variation was confirmed in a set of 32 isogenic second chromosome substitution lines and measured in a subset of 4 of these lines about 25 generations later. A reciprocal cross using two isogenic substitution lines with the highest and lowest activities suggested the difference was attributable to genes acting additively and that there were no maternal or paternal effects. The detection of a collection site difference in GPO enzyme activity in the isogenic lines suggests that polymorphic variation on the second chromosome is responsible for the differentiation at the winery.Variation in adult GPO activity did not show a dependence on the winery location from where the isogenic lines were derived nor was there an effect of line. Adult GPO activity was significantly higher than that detected in larval tissues and did not show a dependence on the sugar/ethanol level in the growth medium.     

Coevolution of Drosophila melanogaster mtDNA and Wolbachia Genotypes

Maternally inherited microorganisms can influence the mtDNA pattern of variation in hosts. This influence is driven by selection among symbionts and can cause the frequency of mitochondrial variants in the population to eventually increase or decrease. Wolbachia infection is common and widespread in Drosophila melanogaster populations. We compared genetic variability of D. melanogaster mitotypes with Wolbachia genotypes among isofemale lines associated with different geographic locations and time intervals to study coevolution of the mtDNA and Wolbachia. Phylogenetic analysis of D. melanogaster mtDNA revealed two clades diverged in Africa, each associated with one of the two Wolbachia genotype groups. No evidence of horizontal transmission of Wolbachia between maternal lineages has been found. All the mtDNA variants that occur in infected isofemale lines are found in uninfected isofemale lines and vice versa, which is indicative of a recent loss of infection from some maternal fly lineages and confirms a significant role of Wolbachia in the D. melanogaster mtDNA pattern of variation. Finally, we present a comparative analysis of biogeographic distribution of D. melanogaster mitotypes all over the world.     

Characterization of subrepeat regions within rDNA intergenic spacers of the edible basidiomycete Lentinula edodes

For 16 commercial cultivars of Lentinula edodes, DNA fragments for the nuclear rDNA intergenic spacers IGS1 and IGS2 were amplified and analyzed. IGS1 contained a subrepeat region, named SR1, and IGS2 contained a pair of direct repeats and a subrepeat region, named SR2. Three and five types of subrepeats were found in SR1 and SR2, respectively. Heterogeneity in the lengths of IGS1 and IGS2 arose mainly from the number of different kinds of subrepeats within SR1 and SR2. The DNA fingerprints from the PCR products targeting SR1 and SR2 were specific for each of the 16 cultivars, and had enough variation for discrimination among the cultivars. This result suggests that the DNA fingerprints targeting SR1 and SR2 are useful for investigations of L. edodes cultivars.     

The association between inversion In(3R)Payne and clinally varying traits in Drosophila melanogaster

In Drosophila melanogaster, inversion In(3R)Payne increases in frequency towards low latitudes and has been putatively associated with variation in size and thermal resistance, traits that also vary clinally. To assess the association between size and inversion, we obtained isofemale lines of inverted and standard karyotype of In(3R)Payne from the ends of the Australian D. melanogaster east coast cline. In the northern population, there was a significant association between In(3R)Payne and body size, with standard lines from this population being relatively larger than inverted lines. In contrast, the inversion had no influence on development time or cold resistance. We strengthened our findings further in a separate study with flies from populations from the middle of the cline as well as from the cline ends. These flies were scored for wing size and the presence of In(3R)Payne using a molecular marker. In females, the inversion accounted for around 30% of the size difference between cline ends, while in males the equivalent figure was 60%. Adaptive shifts in size but not in the other traits are therefore likely to have involved genes closely associated with In(3R)Payne. Because the size difference between karyotypes was similar in different populations, there was no evidence for coadaptation within populations.     

Sexual dimorphism of body size and sternopleural bristle number: a comparison of geographic populations of an invasive cosmopolitan drosophilid

Zaprionus indianus is a cosmopolitan drosophilid, of Afrotropical origin, which has recently colonized South America. The sexual dimorphism (SD) of body size is low, males being almost as big as females. We investigated 10 natural populations, 5 from America and 5 from Africa, using the isofemale line technique. Three traits were measured on each fly: wing and thorax length and sternopleural bristle number. Two indices of SD were compared, and found to be highly correlated (r > 0.99). For the sake of simplicity, only the female/male (F/M) ratio was further considered. A significant genetic variability of SD was found in all cases, although with a low heritability (intra-class correlation of 0.13), about half the value found for the traits themselves. For size SD, we did not find any variation among continents or any latitudinal trend, and average values were 1.02 for wing length and 1.01 for thorax length. Bristle number SD was much greater (1.07). Among mass laboratory strains, SD was genetically much more variable than in recently collected populations, a likely consequence of laboratory drift. Altogether, SD, although genetically variable and prone to laboratory drift, is independent of size variations and presumably submitted to a stabilizing selection in nature.     

Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer

DNA sequences from the intergenic spacer (IGS) region of the ribosomal operon were amplified by the polymerase chain reaction (PCR) technique using two primers derived from 16S and 23S rRNA conserved sequences. The PCR products, cleaved by 4 base cutting restriction enzymes, were used to differentiate Nitrobacter strains. This method offered a convenient alternative to serological testing for characterization of Nitrobacter isolates and enabled a large number of strains to be genotypically characterized easily and rapidly. This method was successfully used to characterize natural populations of Nitrobacter from various soils and a lake. A diversity was demonstrated in various soils, and in a lake both in freshwater and in sediments. Strains closely related to both WL and LL were found in these eco-systems. It seems that the diversity of Nitrobacter populations was not associated with global environments but may be related to the presence of locally coexisting niches.Non-commun abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism - IGS intergenic spacer     

NATURAL VARIATION IN THE EXPRESSION OF THE HEAT-SHOCK PROTEIN HSP70 IN A POPULATION OF DROSOPHILA MELANOGASTER AND ITS CORRELATION WITH TOLERANCE OF ECOLOGICALLY RELEVANT THERMAL STRESS

Although Hsp70, the principal inducible heat-shock protein of Drosophila melanogaster, has received intense scrutiny in laboratory strains, its variation within natural populations and the consequences of such variation for thermotolerance are unknown. We have characterized variation in first-instar larvae of 20 isofemale lines isolated from a single natural population of D. melanogaster, in which larvae are prone to thermal stress in nature. Hsp70 expression varied more than twofold among lines after induction by exposure to 36°C for one hour, with an estimated proportion of the variation due to genetic differences of 0.24 ± 0.08. Thermotolerance with and without a Hsp70-inducing pretreatment, survival at 25°C, and developmental time also varied significantly. As expected, expression of Hsp70 correlated positively with larval thermotolerance. By contrast, lines in which larval survival was high in the absence of heat stress showed lower than average Hsp70 expression and lower than average inducible thermotolerance. This conditional performance suggests an evolutionary trade-off between thermotolerance and the ability to produce higher concentrations of Hsp70, and survival in a benign environment.     

Ancestral population reconstitution from isofemale lines as a tool for experimental evolution

Experimental evolution is a powerful tool to study adaptation under controlled conditions. Laboratory natural selection experiments mimic adaptation in the wild with better‐adapted genotypes having more offspring. Because the selected traits are frequently not known, adaptation is typically measured as fitness increase by comparing evolved populations against an unselected reference population maintained in a laboratory environment. With adaptation to the laboratory conditions and genetic drift, however, it is not clear to what extent such comparisons provide unbiased estimates of adaptation. Alternatively, ancestral variation could be preserved in isofemale lines that can be combined to reconstitute the ancestral population. Here, we assess the impact of selection on alleles segregating in newly established Drosophila isofemale lines. We reconstituted two populations from isofemale lines and compared them to two original ancestral populations (AP) founded from the same lines shortly after collection. No significant allele frequency changes could be detected between both AP and simulations showed that drift had a low impact compared to Pool‐Seq‐associated sampling effects. We conclude that laboratory selection on segregating variation in isofemale lines is too weak to have detectable effects, which validates ancestral population reconstitution from isofemale lines as an unbiased approach for measuring adaptation in evolved populations.     

PHENOTYPIC PLASTICITY AND REACTION NORMS IN TEMPERATE AND TROPICAL POPULATIONS OF DROSOPHILA MELANOGASTER: OVARIAN SIZE AND DEVELOPMENTAL TEMPERATURE

The plasticity of ovariole number relative to developmental temperature was studied in three populations of Drosophila melanogaster at both ends of the cline: a temperate French population and two equatorial Congolese. Ovary size was much greater in the French flies, in agreement with an already known latitudinal cline. Among isofemale lines, significant differences in genetic variability were observed between populations with a maximum variability at intermediate temperatures. Parameters of phenotypic variability (CV and FA) were not statistically different among lines or populations, but a significant increase at low temperature was demonstrated for both. The shapes of the response curves (i.e., the norm of reaction) were analyzed by adjusting the data to a quadratic equation. The parameters of the equation were highly variable among lines. On the other hand, the temperature for maximum value of ovarioles (TMV) was much less variable and exhibited only a slightly significant difference between temperate and tropical flies (22.2°C vs. 22.7°C). During its geographic extension toward colder places, D. melanogaster underwent a large, presumably adaptative, increase in ovariole number but very little change in the norm of reaction of that trait.     

Increase in the degree of inversion polymorphism in Drosophila bipectinata populations transfereed to laboratory conditions

Thirteen laboratory populations (six mass cultures and seven isofemale lines) of Drosophila bipectinata were established from files collected from six different geographic localities (four in the north and two in the south) in India. These mass cultures and isofemale lines were maintained in food bottles in the laboratory for varying number of generations by transferring about 50 files (females and males in equal number) in each generation. After several generations, all the laboratory populations were analysed chromosomally to determine the frequency of different inversions. The results indicate that all the laboratory populations remained chromosomally polymorphic due to the persistence of inversions which were originally present ion the populations. The quantitative data on the frequency of inverted gene orders and the level of inversions heterozygosity in laboratory populations are compared with those in corresponding natural populations. This comparison clearly shows that there is a considerable increase in the frequency of inverted gene orders and the level of inversion heterozygosity in laboratory populations when compared with corresponding natural populations. This is most likely to be due to selective advantage of inversion heterozygotes under stringent competition in laboratory populations. Thus heterotic buffering is associated with chromosome inversion of Drosophila bipectinata.     

The endosymbiont <Emphasis Type="Italic">Wolbachia</Emphasis> in Eurasian populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The endosymbiotic α-proteobacteria Wolbachia is widely spread among arthropods and Filariidae nematodes. This bacterium is transmitted vertically via a transovarian route. Wolbachia is a cause of several reproductive abnormalities in the host species. We analyzed the isofemale lines created using flies collected from Drosophila melanogaster natural populations for infection with the endosymbiont Wolbachia. Wolbachia were genotyped according to five variable markers: the presence of insertion sequence IS5 in two loci, the copy number of two minisatellite repeats, and an inversion. Overall, 665 isofemale lines isolated from the populations of D. melanogaster from Ukraine, Belarus, Moldova, Caucasus, Central Asia, Ural, Udmurtia, Altai, West and East Siberia, and Far East in 1974 through 2005 were used in the work. The samples from Ukrainian, Altaian, and Middle Asian populations were largest. The infection rate of D. melanogaster populations from Middle Asia, Altaian, and Eastern Europe (Ukraine, Moldavia, and Belarus) with Wolbachia amounted to 64, 56, and 39%, respectively. The D. melanogaster population from the Caucasus displayed heterogeneity in the genotypes of this cytoplasmic infection. The Wolbachia genotype wMel, detected in all the populations studied, was the most abundant. The genotype wMelCS2 was always present in the populations from Middle Asia and Altai and was among the rare variants in the D. melanogaster populations from the Eastern Europe. Single instances of the Wolbachia genotype wMelCS occurred in a few flies from the Central Asian and Altai populations, but was not found this genotype in the other regions.