Mitochondrial DNA haplotype frequencies in natural and experimental populations of Drosophila subobscura.

The evolution of Drosophila subobscura mitochondrial DNA has been studied in experimental populations, founded with flies from a natural population from Esporles (Majorca, Balearic Islands, Spain). This population, like other European ones, is characterized by the presence of two very common (>96%) mitochondrial haplotypes (called I and II) and rare and endemic haplotypes that appear at very low frequencies. There is no statistical evidence of positive Darwinian selection acting on the mitochondrial DNA variants according to Tajima''s neutrality test. Two experimental populations, with one replicate each, were established with flies having a heterogeneous nuclear genetic background, which was representative of the composition of the natural population. Both populations were started with the two most frequent mitochondrial haplotypes, but at different initial frequencies. After 13 to 16 generations, haplotype II reached fixation in three cages and its frequency was 0.89 by generation 25 in the fourth cage. Random drift can be rejected as the force responsible for the observed changes in haplotype frequencies. There is not only statistical evidence of a linear trend favoring a mtDNA (haploid) fitness effect, but also of a significant nonlinear deviation that could be due to a nuclear component.     

Conditional Hitchhiking of Mitochondrial DNA: Frequency Shifts of Drosophila Melanogaster Mtdna Variants Depend on Nuclear Genetic Background

Tests were performed of the selective neutrality of mitochondrial DNA (mtDNA) variants from geographic populations of Drosophila melanogaster in Argentina (ARG) and Central Africa (CAF). The two populations were completely reproductively compatible. The two distinct mtDNA haplotypes from the two populations were competed in replicate experimental populations on three nuclear genetic backgrounds: homozygous ARG, homozygous CAF, or hybrid ARG/CAF. Mitochondrial haplotype frequencies did not change significantly on either of the two homozygous nuclear backgrounds, and there was no change after experimental perturbation of haplotype frequencies. On the hybrid background, the ARG haplotype frequency increased significantly for the first two generations in all replicate populations but then did not change in subsequent generations. After perturbation, the ARG haplotype frequency increased in only one of four replicates. There is no evidence for selective differences among mtDNA variants in homozygous nuclear contexts or for nuclear-mitochondrial coadaptation. While some ``fitness' difference among mtDNA variants is required to account for the observed frequency shifts, it appears that in these hybrid populations, mtDNA is hitchhiking on fitness variation among hybrid segregating nuclear genes. These results have implications for the use of mtDNA in the study of hybrid zones and gene flow.     

Frequencies of Mitochondrial DNA Haplotypes in Laboratory Cage Populations of the Mosquito, Aedes Albopictus

A laboratory cage experiment was undertaken to study changes over time in the frequencies of two mitochondrial DNA (mtDNA) haplotypes in the mosquito, Aedes albopictus, under two conditions: bidirectionally compatible matings and unidirectionally incompatible matings. Frequencies were monitored for 10 generations in three replicate cages for each of the two conditions above. In cages with bidirectionally compatible strains, changes in haplotype frequencies were nondirectional and neither haplotype increased in frequency. Statistical analysis of relative proportions of the two haplotypes in each generation indicated that the magnitude of the observed fluctuations could be expected under an assumption of random genetic drift alone. In cages with unidirectionally incompatible matings, mtDNA of females that lay inviable eggs upon mating with males of another strain, decreased significantly in the F1 generation and was completely replaced in the F2 generation.     

Divergent evolution of life span associated with mitochondrial DNA evolution

Mitochondria play a key role in ageing. The pursuit of genes that regulate variation in life span and ageing have shown that several nuclear‐encoded mitochondrial genes are important. However, the role of mitochondrial encoded genes (mtDNA) is more controversial and our appreciation of the role of mtDNA for the evolution of life span is limited. We use replicated lines of seed beetles that have been artificially selected for long or short life for >190 generations, now showing dramatic phenotypic differences, to test for a possible role of mtDNA in the divergent evolution of ageing and life span. We show that these divergent selection regimes led to the evolution of significantly different mtDNA haplotype frequencies. Selection for a long life and late reproduction generated positive selection for one specific haplotype, which was fixed in most such lines. In contrast, selection for reproduction early in life led to both positive selection as well as negative frequency‐dependent selection on two different haplotypes, which were both present in all such lines. Our findings suggest that the evolution of life span was in part mediated by mtDNA, providing support for the emerging general tenet that adaptive evolution of life‐history syndromes may involve mtDNA.     

Competition between Mitochondrial Haplotypes in Distinct Nuclear Genetic Environments: Drosophila Pseudoobscura Vs. D. Persimilis

A test for coadaptation of nuclear and mitochondrial genomes was performed using the sibling species, Drosophila pseudoobscura and D. persimilis. Two lines of flies with ``disrupted' cytonuclear genotypes were constructed by repeated backcrossing of males from one species to females carrying mitochondrial DNA (mtDNA) from the other species. Each ``disrupted' strain was competed in population cages with the original stock of each species from which the recurrent males were obtained during the backcrossing. As such, the two species' mitochondrial types were competed reciprocally in the nuclear genetic environments of each species. The trajectories of mtDNA haplotypes were followed in discrete-generation population cages using a PCR-four-cutter approach. A significant increase in the frequency of D. pseudoobscura mtDNA was observed in each of four replicate cages with a D. pseudoobscura nuclear background. In the D. persimilis nuclear background, one cage actually showed an increase in frequency of D. pseudoobscura mtDNA, although together the four replicate cages show little change in frequency. These results were repeated after frequency perturbations and reinitiation of each cage. An analysis of fitness components revealed that fertility selection greatly outweighed viability selection in these cytonuclear competition experiments. The asymmetry of the fitnesses of the mtDNA haplotypes on the two genetic backgrounds is consistent in direction with the previously reported asymmetry of female fertility in backcrosses between these two species. While our experiments do not allow us to identify mtDNA as the sole source of fitness variation, at a minimum the data indicate a fitness association between nuclear fertility factors and the D. pseudoobscura mtDNA on its own genetic background.     

Multiple hybridization events between Drosophila simulans and Drosophila mauritiana are supported by mtDNA introgression

The study of speciation has advanced considerably in the last decades because of the increased application of molecular tools. In particular, the quantification of gene flow between recently diverged species could be addressed. Drosophila simulans and Drosophila mauritiana diverged, probably allopatrically, from a common ancestor approximately 250,000 years ago. However, these species share one mitochondrial DNA (mtDNA) haplotype indicative of a recent episode of introgression. To study the extent of gene flow between these species, we took advantage of a large sample of D. mauritiana and employed a range of different markers, i.e. nuclear and mitochondrial sequences, and microsatellites. This allowed us to detect two new mtDNA haplotypes (MAU3 and MAU4). These haplotypes diverged quite recently from haplotypes of the siII group present in cosmopolitan populations of D. simulans. The mean divergence time of the most diverged haplotype (MAU4) is approximately 127,000 years, which is more than 100,000 years before the assumed speciation time. Interestingly, we also found some evidence for gene flow at the nuclear level because an excess of putatively neutral loci shows significantly reduced differentiation between D. simulans and D. mauritiana. Our results suggest that these species are exchanging genes more frequently than previously thought.     

Positive relationships between genetic diversity and abundance in fishes

Molecular markers, such as mitochondrial DNA and microsatellite loci, are widely studied to assess population genetics and phylogeography; however, the selective neutrality of these markers is increasingly being questioned. Given the importance of molecular markers in fisheries science and conservation, we evaluated the neutrality of both mtDNA and microsatellite loci through their associations with population size. We surveyed mtDNA and microsatellite data from the primary literature and determined whether genetic diversity increased with abundance across a total of 105 marine and freshwater fishes, with both global fisheries catch data and body size as proxies for abundance (with an additional 57 species for which only body size data were assessed). We found that microsatellite data generally yielded higher associations with abundance than mtDNA data, and within mtDNA analyses, number of haplotypes and haplotype diversity were more strongly associated with abundance than nucleotide diversity, particularly for freshwater fishes. We compared genetic diversity between freshwater and marine fishes and found that marine fishes had higher values of all measures of genetic diversity than freshwater fishes. Results for both mtDNA and microsatellites generally conformed to neutral expectations, although weaker relationships were often found between mtDNA nucleotide diversity and ‘abundance’ compared to any other genetic statistic. We speculate that this is because of historical events unrelated to natural selection, although a role for selection cannot be ruled out.     

The maintenance of mitochondrial genetic variation by negative frequency‐dependent selection

Mitochondrial genes generally show high levels of standing genetic variation, which is puzzling given the accumulating evidence for phenotypic effects of mitochondrial genetic variation. Negative frequency‐dependent selection, where the relative fitness of a genotype is inversely related to its frequency in a population, provides a potent and potentially general process that can maintain mitochondrial polymorphism. We assessed the change in mitochondrial haplotype frequencies over 10 generations of experimental evolution in 180 seed beetle populations in the laboratory, where haplotypes competed for propagation to subsequent generations. We found that haplotypes consistently increased in frequency when they were initially rare and decreased in frequency when initially common. Our results have important implications for the use of mtDNA haplotype frequency data to infer population level processes and they revive the general hypothesis that negative frequency‐dependent selection, presumably caused by habitat heterogeneity, may commonly promote polymorphism in ecologically relevant life history genes.     

Cytoplasmic Incompatibility and Mating Preference in Colombian Drosophila Pseudoobscura

MACRAE and ANDERSON observed a large frequency change of mitochondrial DNA (mtDNA) haplotypes in a population initiated with two allopatric strains of Drosophila pseudoobscura, BogER from Colombia and AH162 from California. They concluded that mtDNA haplotypes in D. pseudoobscura are not always selectively neutral. NIGRO and PROUT suggested, however, that a maternally transmitted incompatibility system, similar to the one they observed in two strains of D. simulans from Italy, could account for the observed mtDNA frequency changes. SINGH and HALE postulated that a mating preference between the strains BogER and AH162 in MACRAE and ANDERSON's experiment, in the form of negative assortative mating, could also account for the mtDNA frequency changes. We report two experiments designed to test the hypotheses: that a maternally transmitted cytoplasmic incompatibility system exists between D. pseudoobscura strains BogER and AH162; and, that BogER females mate preferentially with AH162 males. Our results do not support either hypothesis.     

Mitochondrial DNA effects on fitness in Drosophila subobscura

We tested different fitness components on a series of conspecific mtDNA haplotypes, detected by RFLPs in Drosophila subobscura. Additionally, haplotype VIII, endemic to the Canary Islands, was tested upon its own native nuclear DNA background and upon that of the rest of mtDNAs tested herein. We found that both nuclear and mitochondrial DNA can have a significant effect upon their hosts' fitness, and that negative selection is one of the mechanisms that can intervene in this species' mtDNA haplotype pattern. We discuss the importance of this mechanism in relation to genetic drift, in the form of periodic population bottlenecks, and how the latter can enhance the former. We also detected a significant positive effect of haplotype VIII upon fitness that could explain in part the dominance of this endemic haplotype on some of the Canary Islands, and a mitochondrial heterosis involving this haplotype when on a foreign nuclear DNA background.     

Population dynamics of the 2 major mitochondrial DNA haplotypes in experimental populations of Drosophila subobscura.

The evolution of Drosophila subobscura mitochondrial DNA has been studied in experimental populations, founded with flies from a natural population from Calvià (Majorca, Balearic Islands, Spain). This population, like others founded in Europe, is characterized by the presence of 2 very common (>95%) mitochondrial haplotypes (named I and II) and rare and endemic haplotypes that appear at very low frequencies. Four experimental populations were established with flies having a heterogeneous nuclear genetic background, which was representative of the composition of the natural population. The populations were started with haplotypes I and II at an initial frequency of 50% each. After 33 generations, the 2 haplotypes coexisted. Random drift could be rejected as the only force responsible for the observed changes in haplotype frequencies. A slight but significant linear trend favouring a mtDNA (haploid) fitness effect has been detected, with a nonlinear deviation that could be due to a nuclear component. An analysis of chromosomal arrangements was made before the foundations of the cages and at generation 23. Our results indicated that the hypothesis that the maintenance of the frequencies of haplotypes I and II in natural populations could be due to their association with chromosomal arrangements remains controversial.     

Within‐population genetic effects of mtDNA on metabolic rate in Drosophila subobscura

A growing body of research supports the view that within‐species sequence variation in the mitochondrial genome (mtDNA) is functional, in the sense that it has important phenotypic effects. However, most of this empirical foundation is based on comparisons across populations, and few studies have addressed the functional significance of mtDNA polymorphism within populations. Here, using mitonuclear introgression lines, we assess differences in whole‐organism metabolic rate of adult Drosophila subobscura fruit flies carrying either of three different sympatric mtDNA haplotypes. We document sizeable, up to 20%, differences in metabolic rate across these mtDNA haplotypes. Further, these mtDNA effects are to some extent sex specific. We found no significant nuclear or mitonuclear genetic effects on metabolic rate, consistent with a low degree of linkage disequilibrium between mitochondrial and nuclear genes within populations. The fact that mtDNA haplotype variation within a natural population affects metabolic rate, which is a key physiological trait with important effects on life‐history traits, adds weight to the emergent view that mtDNA haplotype variation is under natural selection and it revitalizes the question as to what processes act to maintain functional mtDNA polymorphism within populations.     

Association between chloroplast DNA and mitochondrial DNA haplotypes in Prunus spinosa L. (Rosaceae) populations across Europe

Chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) were studied in 24 populations of Prunus spinosa sampled across Europe. The cpDNA and mtDNA fragments were amplified using universal primers and subsequently digested with restriction enzymes to obtain the polymorphisms. Combinations of all the polymorphisms resulted in 33 cpDNA haplotypes and two mtDNA haplotypes. Strict association between the cpDNA haplotypes and the mtDNA haplotypes was detected in most cases, indicating conjoint inheritance of the two genomes. The most frequent and abundant cpDNA haplotype (C20; frequency, 51 %) is always associated with the more frequent and abundant mtDNA haplotype (M1; frequency, 84 %). All but two of the cpDNA haplotypes associated with the less frequent mtDNA haplotype (M2) are private haplotypes. These private haplotypes are phylogenetically related but geographically unrelated. They form a separate cluster on the minimum-length spanning tree.     

Mitochondrial inheritance and the detection of non-parental mitochondrial DNA haplotypes in crosses of Agaricus bisporus homokaryons

This study evaluates mtDNA transmission in Agaricus bisporus, as well as the occurrence of non-parental haplotypes in heterokaryons produced by controlled crosses. Sixteen crosses were performed with blended liquid cultures, using different combinations of 13 homokaryotic strains. For each cross, different mtDNA haplotypes were present in each homokaryon. Heterokaryons generated from these crosses were subject to genetic analysis with RFLP markers to identify (i). karyotic status, (ii). mtDNA haplotype, and (iii). the occurrence of non-parental mtDNA haplotypes. These analyses generally supported the occurrence of uniparental mitochondrial (mt) inheritance in A. bisporus, with one mtDNA haplotype usually favoured in the new heterokaryon. The preponderance of one mtDNA haplotype in a new heterokaryon did not necessarily show a correlation with a greater mycelial growth rate for the parent homokaryon possessing that haplotype. Mixed mtDNA haplotypes and non-parental haplotypes were also identified in the heterokaryons from some crosses. Evidence for the occurrence of two mtDNA haplotypes in one heterokaryotic mycelium was observed in 8 of 16 crosses, suggesting the maintenance of true heteroplasmons after three successive subculturing steps. Non-parental mtDNA haplotypes were seen in heterokaryons produced from 7 of 16 crosses. The mating protocol described can be utilized to generate novel mtDNA haplotypes for strain improvement and the development of strain-specific markers. Mechanisms of mt selection and inheritance are discussed.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Dynamics of mitochondrial polymorphism in a natural population of Drosophila littoralis

During seven years, we observed stable mtDNA polymorphism in a local population of Drosophila littoralis. Using RFLP, a number of mitochondrial haplotypes were revealed, two of which were the core and in condition of stable equilibrium. To explain the absence of fixation of one haplotype, we checked a hypothesis that the D. littoralis population had a complex structure, being subdivided into several partially isolated races existing on the same territory. Analysis of highly hypervariable nuclear sequence of retrotransposons Tv1 showed positive correlation of the mitochondrial haplotype with a particular allelic form of Tv1. This supports the proposal that the D. littoralis natural population forms the population system consisting of genetically differentiated races.     

An ancient mitochondrial polymorphism in Adalis bipunctata linked to a sex-ratio-distorting bacterium

Sex-ratio-distorting microbes are common parasites of arthropods. Although the reasons they have invaded and spread though populations are well understood, their subsequent dynamics within those populations are virtually unknown. We have found that different strains of a male-killing Rickettsia bacterium infecting the beetle Adalia bipunctata are associated with distinct mitochondrial haplotypes, which is expected as both the mitochondria and the bacteria are maternally transmitted. These mitochondrial haplotypes shared a common ancestor >2 million years ago, and their overall diversity is significantly greater than expected under neutrality from comparisons with a nuclear gene. Furthermore, a variety of statistical tests show strong deviations from neutrality in mitochondrial but not in nuclear genes. We therefore conclude that natural selection is probably maintaining a polymorphism of different Rickettsia strains in this species. Despite the age of the different mitochondrial haplotypes, there is very little genetic diversity within them. Furthermore, there is considerable variation in mitochondrial haplotype and bacterial strain frequency between populations, despite it being thought that this species has fairly low levels of population structure. We conclude that the fitness of these male killers may be negatively frequency dependent or different strains may be favored in different populations. These hypotheses await experimental confirmation.     

Functional conservatism among Drosophila simulans flies experiencing different thermal regimes and mitochondrial DNA introgression

Drosophila simulans possesses three different mitochondrial haplotypes (siI, II and III) that are nonrandomly geographically subdivided with a 3% interhaplogroup variation. The aim of this study was to determine whether perturbation of mitochondrial metabolism and ROS management by temperature variation and mtDNA introgression would influence the development of aerobic capacity and the intensity of oxidative stress in D. simulans at different ages. Environmental temperature divergences during development had few impacts on metabolic capacities. Our data suggested strong functional conservatism of mitochondrial haplotypes between the D. simulans lines studied. This conservatism was expressed by the low divergences in either mitochondrial or ROS buffering enzyme activities, or even markers of ROS damage even after disruption of coevolved genomes. Disruption of coevolved mitochondrial and nuclear genomes through mtDNA introgression induced no clear divergence on metabolic phenotype at any state of development. Reduction of cytochrome c oxidase activity that was observed after introgression of one mitochondrial haplotype will require further investigation to delineate whether it is associated with any modification of mito-nuclear interactions.     

Extensive paternal mtDNA leakage in natural populations of Drosophila melanogaster

Strict maternal inheritance is considered a hallmark of animal mtDNA. Although recent reports suggest that paternal leakage occurs in a broad range of species, it is still considered an exceptionally rare event. To evaluate the impact of paternal leakage on the evolution of mtDNA, it is essential to reliably estimate the frequency of paternal leakage in natural populations. Using allele‐specific real‐time quantitative PCR (RT‐qPCR), we show that heteroplasmy is common in natural populations with at least 14% of the individuals carrying multiple mitochondrial haplotypes. However, the average frequency of the minor mtDNA haplotype is low (0.8%), which suggests that this pervasive heteroplasmy has not been noticed before due to a lack of power in sequencing surveys. Based on the distribution of mtDNA haplotypes in the offspring of heteroplasmic mothers, we found no evidence for strong selection against one of the haplotypes. We estimated that the rate of paternal leakage is 6% and that at least 100 generations are required for complete sorting of mtDNA haplotypes. Despite the high proportion of heteroplasmic individuals in natural populations, we found no evidence for recombination between mtDNA molecules, suggesting that either recombination is rare or recombinant haplotypes are counter‐selected. Our results indicate that evolutionary studies using mtDNA as a marker might be biased by paternal leakage in this species.     

Empirical evaluation of cytonuclear models incorporating genetic drift and tests for neutrality of mtDNA variants: data from experimental Gambusia hybrid zones

Statistical tests of genetic drift and of the neutrality of mtDNA are presented using empirical time‐series data on multi‐generational changes in cytonuclear disequilibria within replicated experimental hybrid populations of two species of live‐bearing Poeciliid fishes (Gambusia holbrooki and G.affinis) which were monitored over a period of two years (three generations). Cytonuclear disequilibria D and D (which measure departures from random associations of cytoplasmic and nuclear genotypes) over the three generations of the experiment were non‐zero for all replicate populations. For each of five nuclear loci, the observed measures of D and D were highly concordant between replicates during each generation. Significant departures from expectations were observed after one and two generations. A statistical measure of goodness of fit of observed changes in cytonuclear disequilibria (and implicitly of the neutrality of the mtDNA markers) was calculated for each nuclear locus. When the results for the replicates were combined into an overall test of neutrality, the fit to the random union of zygotes (RUZ) model was rejected for four of the five nuclear loci (P < 0.05). A simple genetic drift model does not explain the temporal changes in composite cytonuclear genotypic frequencies. Frequencies of parental G. holbrooki mitochondrial alleles and nuclear genotypes exceeded expected values during most time periods, implying some selective advantage of offspring produced by G. holbrooki females. Expansion of cytonuclear models to explicitly address questions of genetic drift and neutrality have general relevance to studies of natural populations. This revised version was published online in July 2006 with corrections to the Cover Date.