Adaptive significance of intrapopulational mitochondrial DNA variation in Drosophila subobscura and D. obscura

Mitohondrijska DNK (mtDNK) kodira gene uključene u fundamentalne biološke procese ćelijske respiracije i proizvodnje energije. Mitohondrijski genom je haploidan, nasleđuje se uglavnom materinski, gotovo nikad se ne rekombinuje i odlikuje ga relativno mala efektivna veličina populacije. Zbog navedenih odlika mtDNK, uslovi koji predviđaju postojanje i održavanje adaptivne varijabilnosti mtDNK unutar populacija su restriktivni. Analiziran je adaptivni značaj simpatričke mtDNK varijabilnosti, prisustvo mito-jedarnih interakcija, kao i selekcioni mehanizmi koji je održavaju: polno specifična selekcija, kao i selekcija u varijabilnim sredinskim uslovima. Korišćene su dve široko zastupljene palearktičke vrste roda Drosophila (iz Obscura grupe vrsta), D. subobscura i D. obscura. Prva vrsta je već ustaljeni model u ovom vidu evoluciono genetičkih istraživanja, i odlikuje se prisustvom dva učestala haplotipa u svim populacijama. Druga vrsta je kao model razvijena u ovoj disertaciji. Određena je njena genetička varijabilnost i izučena populaciona istorija kod nekoliko evropskih populacija. Izabrane su linije reprezentativnih haplotipova obe vrste i povratnim ukrštanjima su konstruisane eksperimentalne linije sa svim kombinacijama simpatričkih mtDNK haplotipova i jedarnih pozadina (nuDNK). Na dve eksperimentalne postavke koje su podrazumevale razlike u temperaturi i svetlosnom režimu analizirane su komponente životne istorije svih konstruisanih linija: otpornost na desikaciju, dužina razvića, preadultna vijabilnost, kao i procenat mužjaka (odnos polova). Rezultati su pokazali postojanje značajne genetičke varijabilnosti kako unutar tako i između populacija D. obscura. Kod ove vrste je uočena geografska struktuiranost i smanjenje genetičkog diverziteta u pravcu od zapada ka istoku. Pokazan je adaptivni značaj simpatričke mtDNK varijabilnosti obe vrste. Kod D. subobscura je on bio prisutan na nivou dva glavna haplotipa jedino za vijabilitet, a kod ostalih komponenti na nivou pretpostavljenih dodatnih nukleotidnih razlika karakterističnih za pojedinačne linije. Pokazano je da su mito-jedarni genotipovi primarne jedinice na koje deluje prirodna selekcija, a efekat ovih interakcija na fitnes je bio izraženiji kod D. obscura. Pečat polno specifične selekcije nije zabeležen kod D. subobscura, dok je kod D. obscura efekat ovog vida balansne selekcije bio umeren. Naposletku, eksperimenti su pokazali umeren efekat interakcija sredine i mito-jedarnog genotipa na fitnes kod D. subobscura, dok su kod D. obscura pokazali jači uticaj ovog vida balansne selekcije. Uz to kod D. obscura primećen je i efekat interakcija samog mitohondrijskog haplotipa i sredinskih faktora na fitnes jedinki. Posmatrajući obe vrste, analizirana sredinska varijabilnost se pokazala važnijom u održavanju mtDNK varijabilnosti u poređenju sa polno specifičnom selekcijom. Upotreba dve vrste sa različitim obrascima mtDNK varijabilnosti omogućila je uvid u adaptivne razlike na barem dva različita stupnja funkcionalne povezanosti mitohondrijskog i jedarnog genoma. Mali je broj eksperimentalnih radova koji ispituju mehanizme održavanja simpatričke varijabilnosti mtDNK, a rezultati ove studije umnogome doprinose njihovom boljem razumevanju.Mitochondrial DNA (mtDNA) encodes genes involved in fundamental biological processes of cellular respiration and energy production. The mitochondrial genome is haploid, usually maternally inherited, rarely undergoes recombination, and is characterised by a relatively small effective population size. Due to these characteristics of mtDNA, conditions predicting the existence and maintenance of adaptive mtDNA variation within populations are restrictive. The adaptive significance of sympatric mtDNA variability, the presence of mito-nuclear interactions, and selective mechanisms maintaining it were analysed, including sex-specific selection and selection in variable environmental conditions. Two widely distributed Palearctic species from the Drosophila genus (Obscura group species), D. subobscura and D. obscura, were used. The first species, a well-established model in evolutionary genetic research, features two common haplotypes in all populations. The second species was developed as a model in this dissertation. Genetic variability and population history of several European populations were determined as part of this thesis. Lines of representative haplotypes of both species were selected, and experimental lines with all combinations of sympatric mtDNA haplotypes and nuclear backgrounds (nuDNA) were constructed through backcrossing. All constructed lines of both species were subjected to life history experiments: desiccation resistance, developmental time, preadult viability, and sex ratio. Life history assays were conducted with two experimental setups involving differences in temperature and light regime. The results showed significant genetic variability both within and between populations of D. obscura. In this species, geographic structuring and a reduction in genetic diversity from west to east was observed. The adaptive significance of sympatric mtDNA variation was demonstrated in both species. In D. subobscura, this significance was not present at the level of two main haplotypes except in viability experiment, and in other components it was assumed to involve additional, line specific nucleotide differences. Mito-nuclear genotypes were identified as primary units affected by natural selection, with a more pronounced effect of these interactions on fitness observed in D. obscura. Regarding sex-specific selection, its impact was not observed in D. subobscura, while in D. obscura, the effect of this type of balancing selection was moderate. Finally, experiments showed a moderate effect of interactions between the environment and mito-nuclear genotype on fitness in D. subobscura, while in D. obscura showed a stronger influence of this type of balancing selection. Additionally, in D. obscura, an effect of interactions between the mitochondrial haplotype itself and environmental factors on the fitness of individuals was observed. Considering both species, environmental variability proved more important in maintaining mtDNA variation compared to sex-specific selection. The use of two species with different patterns of mtDNA variability provided insight into adaptive differences at least two different levels of functional connectivity between the mitochondrial and nuclear genomes. Few experimental studies have examined the mechanisms sustaining sympatric mtDNA variability, and the results of this study significantly contribute to a better understanding of these mechanisms

Evaluating ND1 and Cytb mitochondrial genes as markers for diversity analysis of protected White-tailed eagle species from Serbia

White-tailed eagle is the biggest bird of prey in Central and Southeast Europe. In Serbia it inhabits the Vojvodina province and the valleys of Danube, Sava, Tisa and Tamiš. Anthropogenic pressure on its habitats in Europe caused a decline in its numbers, but due to the strict laws protecting both species and its habitats, birds’ numbers are now steady and increasing. In Serbia, as a strictly protected species it is a subject of different conservation programs. The available genetic data for this population are scarce and it is necessary to assess its genetic diversity to improve the existing conservation efforts. ND1 and Cytb mitochondrial genes can be used to estimate the populations’ adaptation to different environmental conditions and their variability can potentially be used to evaluate differentiation between populations. To assess the genetic diversity of White-tailed eagle in Serbia we used mitochondrial ND1 and Cytb nucleotide sequences from 40 unrelated birds collected in nests. ND1 and Cytb nucleotide sequences variability was evaluated using standard parameters of genetic diversity (PGD). Acquired values were compared with the available data for the variability of the D-loop region which showed that combined ND1/Cytb nucleotide sequences PGD provide comparable results. Using publicly available sequences we reconstructed haplotype networks for ND1, Cytb, ND1/Cytb and D-loop which further showed the applicability of ND1/ Cytb in population genetics analyses. Phylogeny reconstructed using combined ND1/Cytb sequences identified two branches in Serbian white-tailed eagles. Although the majority of substitutions were nonsynonymous, no selective pressure was detected. Our data suggest that combined ND1/Cytb sequence variability provides sufficient information to be used for population comparison, population differentiation analyses and phylogeny reconstruction, but also gives a tool to potentially identify adaptations to different environmental conditions.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Orthorhombic Crystal Structure of Grossular Garnet (Suva Česma, Western Serbia): Evidence from the Rietveld Refinement

The grossular garnet from rodingite-type rock from the Suva Česma area in western Serbia is characterized with its weak anisotropic nature. Because its anisotropy could indicate a non-cubic lower symmetry, SEM-EDS and Rietveld powder refinement methods were used. The SEM-EDS results have shown that the garnet has a (Ca3.00Mn0.01)3.01(Al1.82Fe0.15Ti0.02)1.99(Si2.97Al0.03)3.00O12 chemical composition (i.e., Grs91Adr08), which can be more specifically explained as ferric iron containing grossular. The next step further used Rietveld powder refinements of the various crystal structures in the Ia‾3d, R‾3c, R‾3, I41/a, Fddd, C2/c, and I‾1 space groups as well as a single mixture, which was followed by a comparative analysis of the R-values, site occupancy factors, and bond lengths and angles. The synthesis of these results showed both that the studied grossular garnet is not cubic and that it crystallized in the disordered Fddd space group with the final RB = 5.29% and RF = 1.75%. It was presumed that the grossular formed at temperatures between 150 and ~600 °C

Nucleotide diversity of Cyt b gene in drosophila subobscura Collin

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

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

Adaptive significance of intrapopulational mitochondrial DNA variation in Drosophila subobscura and D. obscura

Mitohondrijska DNK (mtDNK) kodira gene uključene u fundamentalne biološke procese ćelijske respiracije i proizvodnje energije. Mitohondrijski genom je haploidan, nasleđuje se uglavnom materinski, gotovo nikad se ne rekombinuje i odlikuje ga relativno mala efektivna veličina populacije. Zbog navedenih odlika mtDNK, uslovi koji predviđaju postojanje i održavanje adaptivne varijabilnosti mtDNK unutar populacija su restriktivni. Analiziran je adaptivni značaj simpatričke mtDNK varijabilnosti, prisustvo mito-jedarnih interakcija, kao i selekcioni mehanizmi koji je održavaju: polno specifična selekcija, kao i selekcija u varijabilnim sredinskim uslovima. Korišćene su dve široko zastupljene palearktičke vrste roda Drosophila (iz Obscura grupe vrsta), D. subobscura i D. obscura. Prva vrsta je već ustaljeni model u ovom vidu evoluciono genetičkih istraživanja, i odlikuje se prisustvom dva učestala haplotipa u svim populacijama. Druga vrsta je kao model razvijena u ovoj disertaciji. Određena je njena genetička varijabilnost i izučena populaciona istorija kod nekoliko evropskih populacija. Izabrane su linije reprezentativnih haplotipova obe vrste i povratnim ukrštanjima su konstruisane eksperimentalne linije sa svim kombinacijama simpatričkih mtDNK haplotipova i jedarnih pozadina (nuDNK). Na dve eksperimentalne postavke koje su podrazumevale razlike u temperaturi i svetlosnom režimu analizirane su komponente životne istorije svih konstruisanih linija: otpornost na desikaciju, dužina razvića, preadultna vijabilnost, kao i procenat mužjaka (odnos polova). Rezultati su pokazali postojanje značajne genetičke varijabilnosti kako unutar tako i između populacija D. obscura. Kod ove vrste je uočena geografska struktuiranost i smanjenje genetičkog diverziteta u pravcu od zapada ka istoku. Pokazan je adaptivni značaj simpatričke mtDNK varijabilnosti obe vrste. Kod D. subobscura je on bio prisutan na nivou dva glavna haplotipa jedino za vijabilitet, a kod ostalih komponenti na nivou pretpostavljenih dodatnih nukleotidnih razlika karakterističnih za pojedinačne linije. Pokazano je da su mito-jedarni genotipovi primarne jedinice na koje deluje prirodna selekcija, a efekat ovih interakcija na fitnes je bio izraženiji kod D. obscura. Pečat polno specifične selekcije nije zabeležen kod D. subobscura, dok je kod D. obscura efekat ovog vida balansne selekcije bio umeren. Naposletku, eksperimenti su pokazali umeren efekat interakcija sredine i mito-jedarnog genotipa na fitnes kod D. subobscura, dok su kod D. obscura pokazali jači uticaj ovog vida balansne selekcije. Uz to kod D. obscura primećen je i efekat interakcija samog mitohondrijskog haplotipa i sredinskih faktora na fitnes jedinki. Posmatrajući obe vrste, analizirana sredinska varijabilnost se pokazala važnijom u održavanju mtDNK varijabilnosti u poređenju sa polno specifičnom selekcijom. Upotreba dve vrste sa različitim obrascima mtDNK varijabilnosti omogućila je uvid u adaptivne razlike na barem dva različita stupnja funkcionalne povezanosti mitohondrijskog i jedarnog genoma. Mali je broj eksperimentalnih radova koji ispituju mehanizme održavanja simpatričke varijabilnosti mtDNK, a rezultati ove studije umnogome doprinose njihovom boljem razumevanju.Mitochondrial DNA (mtDNA) encodes genes involved in fundamental biological processes of cellular respiration and energy production. The mitochondrial genome is haploid, usually maternally inherited, rarely undergoes recombination, and is characterised by a relatively small effective population size. Due to these characteristics of mtDNA, conditions predicting the existence and maintenance of adaptive mtDNA variation within populations are restrictive. The adaptive significance of sympatric mtDNA variability, the presence of mito-nuclear interactions, and selective mechanisms maintaining it were analysed, including sex-specific selection and selection in variable environmental conditions. Two widely distributed Palearctic species from the Drosophila genus (Obscura group species), D. subobscura and D. obscura, were used. The first species, a well-established model in evolutionary genetic research, features two common haplotypes in all populations. The second species was developed as a model in this dissertation. Genetic variability and population history of several European populations were determined as part of this thesis. Lines of representative haplotypes of both species were selected, and experimental lines with all combinations of sympatric mtDNA haplotypes and nuclear backgrounds (nuDNA) were constructed through backcrossing. All constructed lines of both species were subjected to life history experiments: desiccation resistance, developmental time, preadult viability, and sex ratio. Life history assays were conducted with two experimental setups involving differences in temperature and light regime. The results showed significant genetic variability both within and between populations of D. obscura. In this species, geographic structuring and a reduction in genetic diversity from west to east was observed. The adaptive significance of sympatric mtDNA variation was demonstrated in both species. In D. subobscura, this significance was not present at the level of two main haplotypes except in viability experiment, and in other components it was assumed to involve additional, line specific nucleotide differences. Mito-nuclear genotypes were identified as primary units affected by natural selection, with a more pronounced effect of these interactions on fitness observed in D. obscura. Regarding sex-specific selection, its impact was not observed in D. subobscura, while in D. obscura, the effect of this type of balancing selection was moderate. Finally, experiments showed a moderate effect of interactions between the environment and mito-nuclear genotype on fitness in D. subobscura, while in D. obscura showed a stronger influence of this type of balancing selection. Additionally, in D. obscura, an effect of interactions between the mitochondrial haplotype itself and environmental factors on the fitness of individuals was observed. Considering both species, environmental variability proved more important in maintaining mtDNA variation compared to sex-specific selection. The use of two species with different patterns of mtDNA variability provided insight into adaptive differences at least two different levels of functional connectivity between the mitochondrial and nuclear genomes. Few experimental studies have examined the mechanisms sustaining sympatric mtDNA variability, and the results of this study significantly contribute to a better understanding of these mechanisms

Корунд из дистенско-силиманитског шкриљца Боболош-Брза Паланка

Korund iz distensko silimanitskog škriljca lokalnosti Bobološ je detaljnije mineraloško-kristalografski ispitan pomoću nekoliko metoda. Dobijeno je učešće korunda od oko 15 %, čime je ova stena postala značajna kao sirovina za aluminijske opeke. Takođe je ispitan plavi korund safirskog tipa koji se ovde javlja pri čemu je dobijena sledeća jedinična ćelija: a0=4,7600 /8/ A, c0=12,98 /1/ A i V0=254,6 /4/ A3

Stress Resistance Traits under Different Thermal Conditions in <i>Drosophila subobscura</i> 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

Population History Shapes Responses to Different Temperature Regimes in <i>Drosophila subobscura</i>

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

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