Experimental mito-nuclear introgression lines (MNILs) were established by backcrossing isofemale lines of D. subobscura originating from the same populations. MNILs were subjected to a series of life-history experiments designed to test the fitness of the bearers of different combinations of two main mtDNA haplotypes on their own nuclear background, as well as on the background of the opposite haplotype. By having 11 replicas of the four mito-nuclear combinations, we could test not only the adaptive significance of the differences between the two main haplotypes but also the influence of additional variation present within each of the 11 combinations on fitness. Testing the fitness of individuals of both sexes enabled us to examine if sex-specific selection has a role in maintaining the frequencies of the two mtDNA haplotypes in nature. Conducting the fitness assays on two different temperatures enabled us to test whether different temperatures favor specific mtDNA haplotypes or mito-nuclear genotypes and consequently promote stable sympatric mtDNA variation. The results show weak signature of genotype-by-environment interactions, and no sex-specific selection regarding differences between the two main haplotypes. However, individual models across different life-history components showed these two mechanisms at play in promoting mtDNA variability present in specific mito-nuclear crosses. Our models show that mito-nuclear interactions are, in fact, more important as units of selection.

Mitochondrial DNA variability of Drosophila subobscura Collin from Southeastern Serbia was studied with respect to Restriction Site Analysis (RSA) of complete mitochondrial genome and the nucleotide sequence of Cytochrome b (Cyt b) gene. The aim was to shed more light on the evolutionary forces that shape mtDNA variation of this species. Samples were collected from two sites in the foothills of the Balkan Mountains. No genetic differentiation was found between groups and most of the variation was observed within them. Restriction analysis revealed two main haplotypes and several rare ones. The sequencing of Cyt b gene showed larger number of haplotypes, among which, one is being the most common. The majority of singletons differed from the most frequent haplotype by one nucleotide change. Although some of the observed nucleotide differences may affect their host's fitness, the observed pattern of variation is consistent with the seasonal fluctuations in population size.

To explore local adaptation in wild populations at a fine spatial scale we characterized the genetic variability of eight closely located populations of Drosophila subobscura and its associations with microhabitat environmental conditions. Three different genetic markers were assessed: chromosomal inversions, a SNP of mitochondrial ND5 gene and nuclear microsatellites. Population genetic analyses of chromosomal variability revealed significant genetic differentiation between these populations. Gene arrangement frequencies on the E chromosome contributed most to these differences. We also investigated role of mitonuclear epistasis in mitochondrial genome differentiation and revealed weak linkage disequilibrium (LD) exclusively between O3+4 inversion arrangement and mitochondrial DNA haplotype I in two populations. In addition, the trend in the LD between O-ST chromosomal arrangement and haplotype II was general in the total sample. Microsatellite analysis revealed an absence of stochastic processes, like census reduction, upon population differentiation. Only a small amount of the genetic variation is related to geographic distance, while most (97%) is attributable to other factors and in some degree to microhabitat variables (temperature, humidity). The analysis of these factors revealed they effect inversion arrangement frequencies, especially E1+2+9, E-ST and O-ST. Even though this model organism is known for its high mobility and mostly large effective population size, the results presented here reveal that local adaptations can occur even at a small spatial scale. We propose that locally adapted alleles within chromosomal inversions, as well as joint selective pressures acting on mitochondria! and nuclear genomes, are responsible for the observed adaptation to microhabitat conditions.

Ecological and evolutionary factors, together with abiotic conditions, affect biogeo-graphic patterns of genetic entities. The spatial and temporal variability of chromosomal inversions of Drosophila subobscura suggests that this species can serve as a good model for studying the effects of environmental change on the genetic structure of natural populations. A comprehensive meta-analysis of the association of environmental and climatic variables with inversion diversity patterns was performed on 20 D. subobscura populations from the central part of the Balkan Peninsula. Environmental data consisted of 3 sets of variables related to temperature and precipitation, extracted from 2 climatic databases, averaged over a 3 month period, and using biological instead of calendar dates of sampling. Arrangement frequency patterns are likely driven by a synergistic effect of factors related to temperature and precipitation. The frequencies of standard chromosomal arrangements tend to co-vary positively with precipitation, whereas parameters related to temperature appear to favor higher frequencies of the inverted and more complex chromosomal arrangements. A complex relationship among local environmental variables is evident from the results and reflects the probable effect of an altitudinal shift; the altitudinal gradient of inversions is different from their latitudinal gradient. The pattern of inversions is generally not associated with environmental variables, and a particular inversion cannot be a predictive genetic marker of global climate change. Populations in different habitats are subjected to habitat-specific selection regimes, while demographic factors and population history also affect the genetic variability pattern observed.

Growing anthropogenic influence on every aspect of environment arise important issues regarding the ability of populations and species to adapt to variant pressures. Lead is one of the most present contaminants in the environment with detrimental influence on organisms and populations. In combination with genomic stress, lead may act synergistically, leading to reduction in adaptive values. We sampled two Drosophila subobscura populations, from ecologically different habitats and established differences in genetic backgrounds and population histories. In order to establish different levels of genome heterozygosity, series of intra-line, intra-population and between population crosses were made. The progeny was reared on a standard Drosophila medium and a medium with 200μg/mL of lead acetate and right wing of approximately 4000 individuals was used for geometric morphometric analysis of wing size. Results showed that lead significantly reduces wing size and that magnitude of this reduction is dependent on genetic background, indicating synergistic effect of genomic and environmental stress. There is also an indication of strong female origin influence on the outcome of hybridization when source of environmental stress is lead. Our results showed that the genetic structure of populations is of great importance for population fitness in anthropogenic induced stressful conditions. Further studies of synergistic effect of genetic and environmental stress are needed, as well as studies of its outcome in natural populations. [Projekat Ministarstva nauke Republike Srbije, br. 173012]

Over the past century, man-made electromagnetic fields (EMF) have changed the natural environment, representing one of the most common and rapidly increasing environmental factors as technology advances. Numerous studies of biological effects of exposure to EMF have been performed on Drosophila during the last 30 years, showing contrasting results. In an attempt to determine whether EMF exposures might affect the development of Drosophila, the present study examines phenotypic variations through direction and range of changes in several fitness components and wing length and width, of Drosophila subobscura isofemale lines, affected by 50 Hz EMF. Embryonal and early post-embryonal stages were exposed to homogeneous sinusoidal 0.25 mT EMF of 50 Hz. The EMF exposure caused a significantly prolonged developmental time and significantly increased the wing size with the opposite direction of directional asymmetry compared to the control group. These results shows that even such a weak EMF has the potential to modulate, through developmental pathways, some of the fitness-related traits, wing size, and wing asymmetry in D. subobscura even after one generation of exposure, showing that extremely low frequency (ELF) EMFs have relevant consequences on development, adult morphology, and fitness.

As a response to the long-term presence of heavy metals in the environment, populations can evolve resistance. Its maintenance may have detrimental effect on population's fitness, causing a fitness cost. Lead is one of the widely distributed elements in the environment exhibiting high toxicity on organisms. By analyzing developmental stages viability and developmental time, we evaluated fitness cost in Drosophila subobscura flies adapted to low lead concentration and control flies derived from the same wild population, as well as their hybrids. Significant changes in specific developmental stages viability were detected in both lines, as well as their hybrids, suggesting complex response to low lead concentration. The results show that a long-term exposure to low lead concentration may have a significant impact on a population's survival, especially in a changing environment conditions.

Lead is one of the most present contaminants in the environment, and different species respond differently to this type of pollution. If combined with genomic stress, lead may act synergistically, causing significant decrease of fitness components. We used two genetically diverse Drosophila subobscura populations (regarding both putatively adaptive inversion and microsatellite loci polymorphisms) originating from two ecologically distinct habitats. To establish different levels of genome heterozygosity, series of intraline, intrapopulation and interpopulation crosses were made. The progeny were reared on a standard medium and a medium with 200 μg/mL of lead acetate. Development time was significantly extended to all groups reared on lead. The progeny of intraline crosses showed significantly extended development time compared to all other groups. The obtained results suggest that genome heterozygosity reduces the effect of lead pollution.

Local adaptation to environmental stress at different levels of genetic polymorphism in various plants and animals has been documented through evolution of heavy metal tolerance. We used samples of Drosophila subobscura populations from two differently polluted environments to analyze the change of chromosomal inversion polymorphism as genetic marker during laboratory exposure to lead. Exposure to environmental contamination can affect the genetic content within a particular inversion and produce targets for selection in populations from different environments. The aims were to discover whether the inversion polymorphism is shaped by the local natural environments, and if lead as a selection pressure would cause adaptive divergence of two populations during the multigenerational laboratory experiment. The results showed that populations retain signatures from past contamination events, and that heavy metal pollution can cause adaptive changes in population. Differences in inversion polymorphism between the two populations increased over generations under lead contamination in the laboratory. The inversion polymorphism of population originating from the more polluted natural environment was more stable during the experiment, both under conditions with and without lead. Therefore, results showed that inversion polymorphism as a genetic marker reflects a strong signature of adaptation to the local environment, and that historical demographic events and selection are important for both prediction of evolutionary potential and long-term viability of natural populations.

A number of recent studies have shown that the pattern of mitochondrial DNA variation and evolution is at odds with a neutral equilibrium model. Theory has suggested that selection on mitonuclear genotypes can act to maintain stable mitonuclear polymorphism within populations. However, this effect largely relies upon selection being either sex-specific or frequency dependent. Here, we use mitonuclear introgression lines to assess differences in a series of key life-history traits (egg-to-adult developmental time, viability, offspring sex-ratio, adult longevity and resistance to desiccation) in Drosophila subobscura fruit flies carrying one of three different sympatric mtDNA haplotypes.

Contamination represents environmental stress that can affect genetic variability of populations, thus influencing the evolutionary processes. In this study, we evaluate the relationship between heavy metal contamination (Pb) and phenotypic variation, assessed by coefficients of variation (CV) of life-history traits. To investigate the consequences of population origin on variation of life history traits in Drosophila subobscura in response to different laboratory conditions we compared populations from relatively polluted and unpolluted environments. Prior to experiment, flies from natural populations were reared for two generations in standard Drosophila laboratory conditions. Afterwards, all flies were cultured on three different media: one medium without lead as the control, and the other two with different concentrations of lead. Coefficients of variation (CV) of life- history traits (fecundity, egg-to-adult viability and developmental time) were analyzed on flies sampled in generations F2, F5 and F8 from these three groups. In later generations samples from both polluted and unpolluted environments showed the increased fecundity variation on media with lead. This increase is expressed more in population from unpolluted environment. On contrary, population from unpolluted environment had increased variation of developmental time in earlier, F2 generation, compared to the population from polluted environment. Our results showed that the response to heavy metal contamination depends on the evolutionary history of the populations regarding habitat pollution.

Differences in heavy metal tolerance among separate populations of the same species have often been interpreted as local adaptation. Persistence of differences after removing the stressor indicates that mechanisms responsible for the increased tolerance were genetically determined. Drosophila subobscura Collin (Diptera: Drosophilidae) populations were sampled from two localities with different history of heavy metal pollution, and reared for eight generations in the laboratory on a standard medium and on media with different concentrations of lead (Pb). To determine whether flies from different natural populations exposed to the Pb-contaminated media in the laboratory show population specific variability in fitness components over generations, experimental groups with different concentrations of lead were assayed in three generations (F2 , F5 , and F8 ) for fecundity, developmental time, and egg-to-adult viability. On the contaminated medium, fecundity was reduced in later generations and viability was increased, irrespective of the environmental origin of populations. For both populations, developmental time showed a tendency of slowing down on media with lead. Faster development was observed in later generations. Preadaptation to contamination, meaning higher fecundity, higher viability, and faster egg to adult development in all studied generations, was found in D. subobscura originating from the locality with a higher level of heavy metal pollution.

The effective population size (Ne) represents the number of individuals that can contribute genes equally to the next generation and is usually smaller then the actual size of a population. The aims of this study were: (i) to assess the Ne for two Drosophila subobscura populations sampled from geographically close, but ecologically and topologically distinct habitats, (ii) to compare the results obtained from two independent approaches for estimating Ne [an ecological (capture-mark-release-recapture) and a molecular (microsatellite data, linkage disequilibrium and heterozygote excess)], and (iii) to obtain a long-term Ne estimation using a variety of mutational models for D. subobscura populations. The ecological method showed that the beech wood population (B) had a significantly larger Ne compared to the oak wood population (O). Observed sex ratio was in favor of females in both populations studied. The microsatellite analysis of populations showed that short-term effective population size in beech wood was larger when compared to population from oak wood, which is in concordance with results obtained by the ecological method. Long term Ne of both natural populations is infinite according to a variety of mutational models. Our results confirmed concordance between ecological and molecular methods in Ne estimation, but also suggested that ecological approach showed less robustness. Molecular approach provides a promising opportunity for more effective monitoring of Ne in Drosophila subobscura. However, the biology, demography and history of the populations may affect different estimators differently so we suggest that ecological and molecular approach should be combined in Ne estimation.

The aim of the current study was to analyze the direction and range of changes in wing size and shape in both sexes of Drosophila subobscura (Collin, 1936) flies that originated from two natural populations with different evolutionary history (sampled from ecologically distinct habitats) maintained during seven generations in laboratory conditions on different lead (Pb) concentrations. The results showed significant wing size variability differences across seven generations of rearing on lead for both populations. Wing size is negatively correlated with lead level, in contrast with wing shape in which significant variation was observed in just one of the populations. According to our results, wing size seems to be more affected by lead pollution in both tested populations compared with wing shape. Our data suggest that presence of lead in higher concentration over extended period of time may reduce the stability of wing morphology and consequently reduce the fitness of exposed individuals. Therefore, specific stress that persists over multiple generations could increase the probabilities for extinction of populations composed of sensitive individuals.

Background: The Balkan Peninsula was one of three main refugia for many European species during the last glaciation. During that period, glacial-interglacial climate oscillations probably shaped the habitat characteristics and influenced the population genetic structures of the Balkan species we see today, including Drosophila subobscura.Hypothesis: Some central Balkan gorges were refugia for D. subobscura. We can detect the effects of these refugia by identifying specific patterns of genetic variability using different genetic markers. We expect conspecific populations to be geographically structured and to differ in particular parameters of population genetic structure.Methods: We analyse the genetic structure of six D. subobscura populations that occupy geographically and ecologically different habitats from the central part of the Balkans using inversion polymorphism, microsatellites, and mtDNA markers.Results: The variability generally fits the European population distribution, with specific gene pool structure in some local populations. Populations from gorges are distinctive in particular rare gene arrangements, mtDNA haplotype diversity providing negative Tajima D-values, whereas the analysed microsatellite loci did not show significant departures from neutrality.Conclusions: The degree and pattern of the differences leaves open the question of the refugial existence of D. subobscura in the central Balkans. The inversion polymorphism data show that some endemic arrangements are present with high frequency. But the molecular markers do not fully support the refugial hypothesis. The adaptive divergence we see among the populations indicates that local adaptations at the molecular level can occur despite high gene flow and large effective population sizes.

Fauna of Drosophilidae in Serbia has been studied for 40 years. This paper presents the results of faunistic research conducted on Mt. Goc in June 2011 in two forests (beech and oak), which are significantly different in the type of vegetation and microclimatic factors. In the oak forest eight species of Drosophila were registered, while in the beech forest only five species were recorded. A relatively small number of species recorded is, among other things, the result of extremely bad weather conditions during the collection attempts. An interesting non-drosophilid fly was also found in the sample - Opomyza florum Fabricius, 1794 (Diptera: Opomyzidae). Since there is very little data concerning the distribution of O. florum in Serbia, this paper is an invitation to entomologists to further study this species, including faunistical, ecological and behavioral aspects, keeping in mind its potential economic impact

The genetic structure of Drosophila subobscura population from the Lazar?s River Canyon (Serbia) was studied with respect to restriction site polymorphism of mitochondrial DNA and chromosomal inversion polymorphism. The aim was to shed more light on the role of cytonuclear interactions in shaping mitochondrial DNA variability in this species. Similar to other populations of D. subobscura two main haplotypes (I and II) were found, as well as less common ones that appeared at very low frequencies. The frequency distribution of haplotypes did not depart from neutrality. We did not find statistically significant linkage disequilibrium between the haplotypes belonging to haplogroups I and II and any of the chromosomal arrangements. However, when we compared the data in hereby analyzed population and two previously analyzed populations we observed that haplotype I is more frequent in populations where standard inversion arrangements are less frequent. Pattern of the observed mitochondrial variability could be influenced either directly by environmental variability or through environmentally specific cytonuclear coadaptation.

The genetic structure of Drosophila subobscura from the Balkan Peninsula was studied with respect to restriction site polymorphism of mitochondrial DNA in populations from the Derventa River Gorge and Sicevo Gorge (Serbia). To investigate the role of cytonuclear interactions in shaping mitochondrial DNA variability in natural populations of this species, the study was complemented with the analysis of linkage disequilibria between mitochondrial haplotypes and chromosomal inversion arrangements. Similar to other populations of D. subobscura, two main haplotypes (I and II) were found, as well as a series of less common ones. The frequencies of haplotypes I and II accounted for 25.8% and 71.0%, respectively, in the population from the Derventa River Gorge, and for 32.4% and 58.1%, respectively, in the population from Sicevo Gorge. One of the haplotypes harbored a large insertion (2.7 kb) in the A+T rich region. The frequency distribution of both haplotypes did not depart from neutrality. Contrary to prior studies, we did not detect any significant linkage disequilibrium between the two most frequent mtDNA haplotypes and any of the chromosomal arrangements in either of the populations. We conclude that linkage disequilibrium is not a general occurrence in natural populations of D. subobscura, and we discuss how transient coadaptations, ecologically specific selective pressures, and demographics could contribute to population-specific patterns of linkage disequilibrium.

Populations of Drosophila subobscura from the urban area of Belgrade and from the locality, Deliblato, which is not under strong anthropogenic influence, were studied with the aim to characterize and compare their genetic structure by examining chromosomal inversion polymorphism. Additional analysis and comparison of this type of polymorphism with several other populations from different habitats in the central Balkans, was done. The obtained results indicate higher heterozygosity in the population from Belgrade. Despite being ecologically marginal and under strong and complex influences, this population did not show a decline in the number of inversions and it is not highly differentiated compared to the referent populations.

Fluctuating asymmetry (FA), the increased variation of bilateral symmetry in a sample of individuals, can indicate disturbance in developmental stability caused by environmental and/or genomic stress. This developmental instability was analyzed in Drosophila subobscura maintained for seven generations on two different concentrations of lead in laboratory conditions. The FA4 index showed that the genotypes reared on the higher lead concentration were in developmental homeostasis, except for males in the F7 generation, for both wing size parameters. The results show that different degrees of lead pollution cause different responses to selection of the exposed population in laboratory conditions.