Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

Sex-specific effects of sympatric mitonuclear variation on fitness in Drosophila subobscura.

BACKGROUND: 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. RESULTS: We found functional differences between these sympatric mtDNA haplotypes, but these effects were contingent upon the nuclear genome with which they were co-expressed. Further, we demonstrate a significant mitonuclear genetic effect on adult sex ratio, as well as a sex × mtDNA × nuDNA interaction for adult longevity. CONCLUSIONS: The observed effects suggest that sex specific mitonuclear selection contributes to the maintenance of mtDNA polymorphism and to mitonuclear linkage disequilibrium in this model system.

Population History Shapes Responses to Different Temperature Regimes in Drosophila subobscura.

Drosophila subobscura is considered a good model species for investigation of a population's ability to adapt and cope with climate changes. Decade long research has shown that inversion frequencies change in response to environmental factors indicating their role in adaptation to novel environments. The mechanisms behind organisms' responses to temperature are complex, involving changes in physiology, behavior, gene expression and regulation. On the other hand, a population's ability to respond to suboptimal conditions depends on standing genetic variation and population history. In order to elucidate the role of local adaptation in population response to the changing temperature, we investigated the response to temperature in D. subobscura individuals originating from two different altitudes by combining traditional cytogenetic techniques with assessing the levels of Hsp70 protein expression. Inversion polymorphism was assessed in the flies sampled from natural populations and in flies reared in laboratory conditions at three different temperatures after five and sixteen generations and Hsp70 protein expression profile in 12th generation flies at the basal level and after heat shock induction. Our results indicate that local adaptation and population history influence population response to the changing temperature.

Absence of linkage disequilibria between chromosomal arrangements and mtDNA haplotypes in natural populations of Drosophila subobscura from the Balkan Peninsula.

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.

Vitamin D receptor, CYP27B1 and CYP24A1 genes polymorphisms association with oral cancer risk and survival.

BACKGROUND: Genetic polymorphisms of vitamin D receptor gene (VDR) and genes involved in vitamin D metabolism pathway, CYP27B1 and CYP24B1, may affect individual susceptibility to oral squamous cell carcinoma. The purpose of this study was to evaluate the associations between VDR, CYP27B1 and CYP24A1 gene polymorphisms with oral cancer risk and survival. METHODS: Study cohort consisted of 110 patients with oral cancer and 122 healthy controls. The genotypes of the analysed genes were determined by PCR-RFLP or real-time PCR method. RESULTS: The significant decrease of oral cancer risk was observed in individuals with heterozygote genotype of CYP24A1 gene (rs2296241) (odds ratio 0.281, P = 0.000) in comparison with wild type. Patients with VDR FokI ff wild type genotype had significantly worse overall survival (P = 0.012, log rank) compared with heterozygous and mutated genotype combined. A stratified analysis by the lymph node involvement and tumour stage showed that ff is associated with poor survival in groups with and without lymph node involvement (P = 0.025, P = 0.040, respectively) and in stage III tumours (P = 0.026). Multivariate Cox's regression analysis revealed that VDR FokI could be considered an independent prognostic factor. CONCLUSION: Our findings indicate that CYP24A1 gene polymorphism might have an influence on the susceptibility to oral cancer. VDR FokI polymorphism was associated with worse survival and could be considered as an independent prognostic marker.

Sex ratio and relatedness in the Griffon vulture (Gyps fulvus) population of Serbia.

Background: Once a widespread species across the region of Southeast Europe, the Griffon vulture is now confined to small and isolated populations across the Balkan Peninsula. The population from Serbia represents its biggest and most viable population that can serve as an important reservoir of genetic diversity from which the birds can be used for the region's reintroduction programmes. The available genetic data for this valuable population are scarce and as a protected species that belongs to the highly endangered vulture group, it needs to be well described so that it can be properly managed and used as a restocking population. Considering the serious recent bottleneck event that the Griffon vulture population from Serbia experienced we estimated the overall relatedness among the birds from this population. Sex ratio, another important parameter that shows the vitality and strength of the population was evaluated as well. Methods: During the annual monitoring that was performed in the period from 2013-2021, we collected blood samples from individual birds that were marked in the nests. In total, 169 samples were collected and each was used for molecular sexing while 58 presumably unrelated birds from different nests were used for inbreeding and relatedness analyses. The relatedness was estimated using both biparentally (10 microsatellite loci) and uniparentally (Cytb and D-loop I of mitochondrial DNA) inherited markers. Results: The level of inbreeding was relatively high and on average it was 8.3% while the mean number of relatives for each bird was close to three. The sex ratio was close to 1:1 and for the analysed period of 9 years, it didn't demonstrate a statistically significant deviation from the expected ratio of 1:1, suggesting that this is a stable and healthy population. Our data suggest that, even though a relatively high level of inbreeding can be detected among the individual birds, the Griffon vulture population from Serbia can be used as a source population for restocking and reintroduction programmes in the region. These data combined with previously observed genetic differentiation between the populations from the Iberian and Balkan Peninsulas suggest that the introduction of foreign birds should be avoided and that local birds should be used instead.

Stress Resistance Traits under Different Thermal Conditions in Drosophila subobscura from Two Altitudes.

Global warming and climate change are affecting many insect species in numerous ways. These species can develop diverse mechanisms as a response to variable environmental conditions. The rise in mean and extreme temperatures due to global warming and the importance of the population's ability to adapt to temperature stress will further increase. In this study, we investigated thermal stress response, which is considered to be one of the crucial elements of population fitness and survival in fast-changing environments. The dynamics and variation of thermal stress resistance traits in D. subobscura flies originating from two natural populations sampled from different altitudes were analysed. Three different temperature regimes (25 °C, 19 °C, and 16 °C) were used for the F1 progeny from both localities to establish six experimental groups and investigate stress resistance traits: desiccation resistance, heat knock-down resistance, starvation resistance, and chill-coma recovery time. We detected that laboratory thermal conditions and population origin may have an effect on the analysed traits, and that sex also significantly influences stress resistance. Individuals from the lower altitude reared at higher temperatures show inferior resistance to thermal shock.

Temperature-Specific and Sex-Specific Fitness Effects of Sympatric Mitochondrial and Mito-Nuclear Variation in Drosophila obscura.

The adaptive significance of sympatric mitochondrial (mtDNA) variation and the role of selective mechanisms that maintain it are debated to this day. Isofemale lines of Drosophila obscura collected from four populations were backcrossed within populations to construct experimental lines, with all combinations of mtDNA Cyt b haplotypes and nuclear genetic backgrounds (nuDNA). Individuals of both sexes from these lines were then subjected to four fitness assays (desiccation resistance, developmental time, egg-to-adult viability and sex ratio) on two experimental temperatures to examine the role of temperature fluctuations and sex-specific selection, as well as the part that interactions between the two genomes play in shaping mtDNA variation. The results varied across populations and fitness components. In the majority of comparisons, they show that sympatric mitochondrial variants affect fitness. However, their effect should be examined in light of interactions with nuDNA, as mito-nuclear genotype was even more influential on fitness across all components. We found both sex-specific and temperature-specific differences in mitochondrial and mito-nuclear genotype ranks in all fitness components. The effect of temperature-specific selection was found to be more prominent, especially in desiccation resistance. From the results of different components tested, we can also infer that temperature-specific mito-nuclear interactions rather than sex-specific selection on mito-nuclear genotypes have a more substantial role in preserving mtDNA variation in this model species.

Genetic Diversity Analysis of Mitochondrial Cytb Gene, Phylogeny and Phylogeography of Protected Griffon Vulture (Gyps fulvus) from Serbia.

Once a widespread and common species across the region of southeast Europe, the Griffon vulture is now confined to small and isolated populations across the Balkan Peninsula. The population from Serbia with 290 couples represents its biggest and most viable population that can serve as an important reservoir of genetic diversity from which the birds can be used for the region's reintroduction or recolonization programs. To estimate the level of genetic diversity, the mitochondrial Cytb gene from 58 unrelated birds sampled during the marking in the nests was sequenced and compared to the homologous Griffon vulture sequences available in publicly accessible online databases. Phylogeographic analysis based on Cytb sequences showed that the most frequent haplotype is found in all Griffon vulture populations and that each population possesses private haplotypes. Our data suggest that the Griffon vulture population from Serbia should be used as a source population for restocking and reintroduction programs in the region. The observed genetic differentiation between the populations from the Iberian and Balkan Peninsulas suggest that the introduction of foreign birds from remote populations should be avoided and that birds from indigenous or neighboring populations, if available, should be used instead.

Heterozygosity maintains developmental stability of sternopleural bristles in Drosophila subobscura interpopulation hybrids.

Interpopulation hybridization can lead to outbreeding depression within affected populations due to breakdown of coadapted gene complexes or heterosis in hybrid populations. One of the principal methods commonly used to estimate the level of developmental instability (DI) is fluctuating asymmetry (FA). We used three genetically differentiated Drosophila subobscura populations according to inversion polymorphism analysis and measured the variability of sternopleural bristle number and change in FA across generations P, F1, and F2 between intra- and interpopulation hybrids of D. subobscura. The mean variability of sternopleural bristle number in intra- and interpopulation hybrids of D. subobscura across generations cannot determine whether the changes at the level of developmental homeostasis are due exclusively to genomic coadaptation or to heterozygosity. Phenotypic variance (V(p)) and FA of sternopleural bristle number was higher in interpopulation than in intrapopulation hybrids across generations. F1 hybrids were more developmentally stable compared to each parental population in both intra- and interpopulation hybrids. The most probable mechanism providing developmental homeostasis is heterozygote or hybrid superiority, also called overdominace. However, V(p) was higher and FA lower in the F2 generation when compared to F1, due mainly to crossing-over in the formation of F2.

Life History Traits in Two Drosophila Species Differently Affected by Microbiota Diversity under Lead Exposure.

Life history traits determine the persistence and reproduction of each species. Factors that can affect life history traits are numerous and can be of different origin. We investigated the influence of population origin and heavy metal exposure on microbiota diversity and two life history traits, egg-to-adult viability and developmental time, in Drosophila melanogaster and Drosophila subobscura, grown in the laboratory on a lead (II) acetate-saturated substrate. We used 24 samples, 8 larval and 16 adult samples (two species × two substrates × two populations × two sexes). The composition of microbiota was determined by sequencing (NGS) of the V3-V4 variable regions of the 16S rRNA gene. The population origin showed a significant influence on life history traits, though each trait in the two species was affected differentially. Reduced viability in D. melanogaster could be a cost of fast development, decrease in Lactobacillus abundance and the presence of Wolbachia. The heavy metal exposure in D. subobscura caused shifts in developmental time but maintained the egg-to-adult viability at a similar level. Microbiota diversity indicated that the Komagataeibacter could be a valuable member of D. subobscura microbiota in overcoming the environmental stress. Research on the impact of microbiota on the adaptive response to heavy metals and consequently the potential tradeoffs among different life history traits is of great importance in evolutionary research.

Highly contiguous assemblies of 101 drosophilid genomes.

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

The discovery, distribution, and diversity of DNA viruses associated with Drosophila melanogaster in Europe.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

Genetic diversity of the Griffon vulture population in Serbia and its importance for conservation efforts in the Balkans.

The Griffon vulture was once a widespread species across the region of Southeast Europe, but it is now endangered and in some parts is completely extinct. In the Balkan Peninsula the largest Griffon vulture inland population inhabits the territory of Serbia. We present, for the first time, the genetic data of this valuable population that could be a source for future reintroduction programs planned in South-eastern Europe. To characterize the genetic structure of this population we used microsatellite markers from ten loci. Blood samples were collected from 57 chicks directly in the nests during the ongoing monitoring program. We performed a comparative analysis of the obtained data with the existing data from three native populations from French Pyrenees, Croatia, and Israel. We have assessed the genetic differentiation between different native populations and determined the existence of two genetic clusters that differentiate the populations from the Balkan and Iberian Peninsulas. Furthermore, we analysed whether the recent bottleneck events influenced the genetic structure of the populations studied, and we found that all native populations experienced a recent bottleneck event, and that the population of Israel was the least affected. Nevertheless, the parameters of genetic diversity suggest that all analysed populations have retained a similar level of genetic diversity and that the Griffon vulture population from Serbia exhibits the highest value for private alleles. The results of this study suggest that the Griffon vulture populations of the Balkan Peninsula are genetically differentiated from the populations of the Iberian Peninsula, which is an important information for future reintroduction strategies.

Effect of microhabitat variability on body size in Drosophila subobscura.

We assessed the variation in thorax size, wing size and wing loading in populations of Drosophila subobscura from two ecologically different habitats and within each habitat sampled during three periods of the day. The traits analyzed differed between laboratory reared samples and field collected samples. Differences were mainly caused by environmental factors and genotype x environmental interactions. While there were no significant differences between populations for particular periods of the day, within-population analysis for each sex showed specific differences. Results showed that wing loading was the least variable character in natural populations, also showing the lowest level of sexual dimorphism. The data are discussed from the aspect of the variability of gene arrangement frequencies over daytime periods obtained previously for the same samples. They are consistent with models of maintenance of genetic variability in multi-niche habitats, and are in favour of a type of reactive behaviour dependent on ecological niche qualities on a daily rhythm scale in D. subobscura.

The effect of lead on fitness components and developmental stability in Drosophila subobscura.

We analyzed the developmental time, egg-to-adult viability, and developmental stability (fluctuating wing size asymmetry) in Drosophila subobscura, maintained for six generations on different concentrations of lead. Development time is significantly affected by generation and lead concentration, but interaction of these factors is not a significant source of variability for this fitness component. Generation and the interaction generation x concentration of lead significantly affect egg-to-adult viability. Levene's test of heterogeneity of variance showed that variability of FA is not significant in any of the samples. Within both lead concentrations females showed significantly higher FA indices for the wing width than males. Within sexes, a significantly higher FA was found only in females for wing width FA between the control and the lower concentration of lead. The results show that if strong relationship between FA and the studied fitness components exists, it results in a stronger selection of unstable genotypes under lead as a stress factor and, consequently, FA needs to be used with caution as a biomarker in natural populations under environmental stress.

Mating behavior as an indicator of quality of Drosophila subobscura males?

According to current theoretical predictions, any deleterious mutations that reduce nonsexual fitness may have a negative influence on mating success. This means that sexual selection may remove deleterious mutations from the populations. Males of good genetic quality should be more successful in mating, compared to the males of lower genetic quality. As mating success is a condition dependent trait, large fractions of the genome may be a target of sexual selection and many behavioral traits are likely to be condition dependent. We manipulated the genetic quality of Drosophila subobscura males by inducing mutations with ionizing radiation and observed the effects of the obtained heterozygous mutations on male mating behavior: courtship occurrence, courtship latency, mating occurrence, latency to mating and duration of mating. We found possible effects of mutations. Females mated more frequently with male progeny of nonirradiated males and that these males courted females faster compared to the male progeny of irradiated males. Our findings indicate a possible important role of sexual selection in purging deleterious mutations.

Adaptive Role of Inversion Polymorphism of Drosophila subobscura in Lead Stressed Environment.

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.

Effect of lead pollution on fitness and its dependence on heterozygosity in Drosophila subobscura.

Lead is one of the most present contaminants in the environment, and different species respond differently to this type of polution. 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.