Mitochondrial DNA size variation within individual crickets.

The mitochondrial DNA's of two closely related cricket species (genus Gryllus) share a size polymorphism as evidenced by analysis of restriction fragment patterns. Moreover, 12 of 100 field-collected crickets are heteroplasmic, that is these individuals have more than one size class of mitochondrial DNA. No heteroplasmy for restriction site variation is observed. Intraindividual variation in cricket mitochondrial DNA provides a useful marker for studying the transmission genetics of mitochondrial DNA. Available data on patterns of variation in mothers and offspring suggest that random segregation of mitochondrial DNA variants does not occur rapidly in cricket germ-cell lineages.

A cryptic lineage within the pupfish Cyprinodon dearborni suggests multiple colonizations of South America.

The coastal South American species Cyprinodon dearborni contains two lineages distinct at both mitochondrial and nuclear loci. One appears to be a long-term South American endemic, whereas the other is a more recent colonizer related to the widespread Cyprinodon variegatus.

Heteroplasmy of short tandem repeats in mitochondrial DNA of Atlantic cod, Gadus morhua.

The mitochondrial DNA of the Atlantic cod (Gadus morhua) contains a tandem array of 40-bp repeats in the D-loop region of the molecule. Variation among molecules in the copy number of these repeats results in mtDNA length variation and heteroplasmy (the presence of more than one form of mtDNA in an individual). In a sample of fish collected from different localities around Iceland and off George's Bank, each individual was heteroplasmic for two or more mtDNAs ranging in repeat copy number from two (common) to six (rare). An earlier report on mtDNA heteroplasmy in sturgeon (Acipenser transmontanus) presented a competitive displacement model for length mutations in mtDNAs containing tandem arrays and the cod data deviate from this model. Depending on the nature of putative secondary structures and the location of D-loop strand termination, additional mechanisms of length mutation may be needed to explain the range of mtDNA length variants maintained in these populations. The balance between genetic drift and mutation in maintaining this length polymorphism is estimated through a hierarchical analysis of diversity of mtDNA length variation in the Iceland samples. Eighty percent of the diversity lies within individuals, 8% among individuals and 12% among localities. An estimate of theta = 2N(eo) mu greater than 1 indicates that this system is characterized by a high mutation rate and is governed primarily by deterministic dynamics. The sequences of repeat arrays from fish collected in Norway, Iceland and George's Bank show no nucleotide variation suggesting that there is very little substructuring to the North Atlantic cod population.

Contrasting patterns of nonneutral evolution in proteins encoded in nuclear and mitochondrial genomes.

We report that patterns of nonneutral DNA sequence evolution among published nuclear and mitochondrially encoded protein-coding loci differ significantly in animals. Whereas an apparent excess of amino acid polymorphism is seen in most (25/31) mitochondrial genes, this pattern is seen in fewer than half (15/36) of the nuclear data sets. This differentiation is even greater among data sets with significant departures from neutrality (14/15 vs. 1/6). Using forward simulations, we examined patterns of nonneutral evolution using parameters chosen to mimic the differences between mitochondrial and nuclear genetics (we varied recombination rate, population size, mutation rate, selective dominance, and intensity of germ line bottleneck). Patterns of evolution were correlated only with effective population size and strength of selection, and no single genetic factor explains the empirical contrast in patterns. We further report that in Arabidopsis thaliana, a highly self-fertilizing plant with effectively low recombination, five of six published nuclear data sets also exhibit an excess of amino acid polymorphism. We suggest that the contrast between nuclear and mitochondrial nonneutrality in animals stems from differences in rates of recombination in conjunction with a distribution of selective effects. If the majority of mutations segregating in populations are deleterious, high linkage may hinder the spread of the occasional beneficial mutation.

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.

Molecular population genetics of mtDNA size variation in crickets.

Nucleotide sequence analysis of a region of cricket (Gryllus firmus) mtDNA showing discrete length variation revealed tandemly repeated sequences 220 base pairs (bp) in length. The repeats consist of 206 bp sequences bounded by the dyad symmetric sequence 5'GGGGGCATGCCCCC3'. The sequence data showed that mtDNA size variation in this species is due to variation in the number of copies of tandem repeats. Southern blot analysis was used to document the frequency of crickets heteroplasmic for two or more different-sized mtDNAs. In New England populations of G. firmus and a close relative Gryllus pennsylvanicus approximately 60% of the former and 45% of the latter were heteroplasmic. From densitometry of autoradiographs the frequencies of mtDNA size classes were determined for the population samples and are shown to very different in the two species. However, in populations where hybridization between the two species has occurred, the frequencies of size classes and cytoplasmic genotypes in each species' distinct mtDNA lineage were shifted in a manner suggesting nuclear-cytoplasmic interactions. The data were applied to reported diversity indices and hierarchical statistics. The hierarchical statistics indicated that the greatest proportion of variation for mtDNA size was due to variation among individuals in their cytoplasmic genotypes (heteroplasmic or homoplasmic state). The diversity indices were used to estimate a per-generation mutation rate for size variants of 10(-4). The data are discussed in light of the relationship between genetic drift and mutation in maintaining variation for mtDNA size.

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.

Mitochondrial DNA transmission genetics in crickets.

This paper presents the results of a single generation study of the transmission genetics of mitochondrial DNA in the field cricket Gryllus firmus. In this species, individuals heteroplasmic for at least two different-sized mitochondrial genomes can be collected easily from natural populations. The frequencies of mtDNA size variants in heteroplasmic females and samples of their offspring were estimated by densitometry of autoradiographs. The variance in mitochondrial genotype frequencies among the offspring of heteroplasmic females indicates that, through genetic drift, fixation would take several hundred animal generations. Differences between the observations and data on mtDNA transmission in yeast and cows are discussed in light of the differences in organelle sampling regime and early developmental events in these species. Our data also show shifts in genotype frequencies in the transmission from mother to offspring that suggest a bias in favor of smaller genomes. The nature of mtDNA size variation in natural populations of crickets is discussed in reference to a mutation-selection balance.

Neutral and non-neutral evolution of Drosophila mitochondrial DNA.

To test hypotheses of neutral evolution of mitochondrial DNA (mtDNA), nucleotide sequences were determined for 1515 base pairs of the NADH dehydrogenase subunit 5 (ND5) gene in the mitochondrial DNA of 29 lines of Drosophila melanogaster and 9 lines of its sibling species Drosophila simulans. In contrast to the patterns for nuclear genes, where D. melanogaster generally exhibits much less nucleotide polymorphism, the number of segregating sites was slightly higher in a global sample of nine ND5 sequences in D. melanogaster (s = 8) than in the nine lines of D. simulans (s = 6). When compared to variation at nuclear loci, the mtDNA variation in D. melanogaster does not depart from neutral expectations. The ND5 sequences in D. simulans, however, show fewer than half the number of variable sites expected under neutrality when compared to sequences from the period locus. While this reduction in variation is not significant at the 5% level, HKA tests with published restriction data for mtDNA in D. simulans do show a significant reduction of variation suggesting a selective sweep of variation in the mtDNA in this species. Tests of neutral evolution based on the ratios of synonymous and replacement polymorphism and divergence are generally consistent with neutral expectations, although a significant excess of amino acid polymorphism within both species is localized in one region of the protein. The rate of mtDNA evolution has been faster in D. melanogaster than in D. simulans and the population structure of mtDNA is distinct in these species. The data reveal how different rates of mtDNA evolution between species and different histories of neutral and adaptive evolution within species can compromise historical inferences in population and evolutionary biology.

Sexually antagonistic cytonuclear fitness interactions in Drosophila melanogaster.

Theoretical and empirical studies have shown that selection cannot maintain a joint nuclear-cytoplasmic polymorphism within a population except under restrictive conditions of frequency-dependent or sex-specific selection. These conclusions are based on fitness interactions between a diploid autosomal locus and a haploid cytoplasmic locus. We develop a model of joint transmission of X chromosomes and cytoplasms and through simulation show that nuclear-cytoplasmic polymorphisms can be maintained by selection on X-cytoplasm interactions. We test aspects of the model with a "diallel" experiment analyzing fitness interactions between pairwise combinations of X chromosomes and cytoplasms from wild strains of Drosophila melanogaster. Contrary to earlier autosomal studies, significant fitness interactions between X chromosomes and cytoplasms are detected among strains from within populations. The experiment further demonstrates significant sex-by-genotype interactions for mtDNA haplotype, cytoplasms, and X chromosomes. These interactions are sexually antagonistic--i.e., the "good" cytoplasms in females are "bad" in males--analogous to crossing reaction norms. The presence or absence of Wolbachia did not alter the significance of the fitness effects involving X chromosomes and cytoplasms but tended to reduce the significance of mtDNA fitness effects. The negative fitness correlations between the sexes demonstrated in our empirical study are consistent with the conditions that maintain cytoplasmic polymorphism in simulations. Our results suggest that fitness interactions with the sex chromosomes may account for some proportion of cytoplasmic variation in natural populations. Sexually antagonistic selection or reciprocally matched fitness effects of nuclear-cytoplasmic genotypes may be important components of cytonuclear fitness variation and have implications for mitochondrial disease phenotypes that differ between the sexes.

A statistical test of a neutral model using the dynamics of cytonuclear disequilibria.

In this paper we use cytonuclear disequilibria to test the neutrality of mtDNA markers. The data considered here involve sample frequencies of cytonuclear genotypes subject to both statistical sampling variation as well as genetic sampling variation. First, we obtain the dynamics of the sample cytonuclear disequilibria assuming random drift alone as the source of genetic sampling variation. Next, we develop a test statistic using cytonuclear disequilibria via the theory of generalized least squares to test the random drift model. The null distribution of the test statistic is shown to be approximately chi-squared using an asymptotic argument as well as computer simulation. Power of the test statistic is investigated under an alternative model with drift and selection. The method is illustrated using data from cage experiments utilizing different cytonuclear genotypes of Drosophila melanogaster. A program for implementing the neutrality test is available upon request.

Neutrality tests of conservative-radical amino acid changes in nuclear- and mitochondrially-encoded proteins.

The neutralist-selectionist debate should not be viewed as a dichotomy but as a continuum. While the strictly neutral model suggests a neutralist-selectionist dichotomy, the nearly neutral model is a continuous model spanning strict neutrality through weak selection (Ns approximately 1) to deterministic selection (Ns>3). We illustrate these points with polymorphism and divergence data from a sample of 73 genes (31 mitochondrial, 36 nuclear genes from Drosophila, and six Arabidopsis data sets). In an earlier study we used the McDonald-Kreitman (MK) test to show that amino acid replacement polymorphism in animal mitochondrial genes and Arabidopsis genes show a consistent trend toward negative selection, whereas nuclear genes from Drosophila span a range from negative selection, through neutrality, to positive selection. Here we analyze a subset of these genes (13 Drosophila nuclear, ten mitochondrial, and six Arabidopsis nuclear) for polymorphism and divergence of conservative and radical amino acid replacements (a protein-based conservative-radical MK, or pMK, test). The distinct patterns of selection between the different genomes is not apparent with the pMK test. Different definitions of conservative and radical (based on amino acid polarity, volume or charge) give inconsistent results across genes. We suggest that segregating fitness difference between silent and replacement mutations are more visible to selection than are segregating fitness differences between conservative and radical amino acid mutations. New data on the variation among genes with different opportunities for positive and negative selection are as important to the continuum view of the neutralist-selectionist debate as is the distribution of selection coefficients within individual genes.

Adaptive maintenance of genetic polymorphism in an intertidal barnacle: habitat- and life-stage-specific survivorship of Mpi genotypes.

In the northern acorn barnacle, Semibalanus balanoides, genotype frequencies of three genetic markers were tracked over time in four types of intertidal habitats. These habitats were selected to represent natural variation in several environmental parameters, specifically the degree of physical stress experienced by barnacles. Frequencies for one allozyme locus (Gpi) and a presumably neutral mtDNA marker were homogeneous among habitats in each temporal sample. Similarly, no temporal stratification in genotype frequencies was evident across the five sampling intervals: from planktonic larvae sampled in March to juveniles collected at the end of June. In contrast to the Gpi and mtDNA loci, Mpi genotypes significantly changed in frequency in two habitats in the high intertidal zone. On exposed substrate, the Mpi-FF homozygote increased in frequency, whereas the alternative homozygote, Mpi-SS, significantly decreased in frequency. Barnacles that were protected from environmental stress at high intertidal heights by the Ascophyllum nodosum algal canopy demonstrated the opposite pattern. In both habitats, the change in frequency of the heterozygote was intermediate to that of the homozygous genotypes. Furthermore, these patterns of genotype-by-environment association reflected a pulse of genotype-specific mortality that occurred over a two-week interval subsequent to metamorphosis from the larval to the adult form. These data indicate that each Mpi homozygote is the highest fitness genotype in some portion of the intertidal environment. Using the Levene (1953) model to evaluate the spatial variation in genotypic fitness, the stable maintenance of the Mpi polymorphism is predicted under certain subsets of conditions. Environmental heterogeneity in the intertidal zone translates to spatial variation in selection pressures, which may result in the active maintenance of the Mpi polymorphism in this species.

Gene flow versus local adaptation in the northern acorn barnacle, Semibalanus balanoides: insights from mitochondrial DNA variation.

In reciprocal transplant experiments, Bertness and Gaines (1993) found that Semibalanus balanoides juveniles that had settled in an upper Narragansett Bay estuary survived better in that estuary that did juveniles from coastal localities. The observed pattern of survivorship led to the claim that local adaptation may result from a combination of limited gene flow between and strong selection within these habitats. Here we test the hypothesis that limited gene flow has led to habitat-specific population differentiation using sequence and restriction fragment length polymorphism analyses of the mitochondrial DNA D-loop region of S. balanoides. Samples were analyzed from replicated coastal and estuary localities in both Narragansett Bay, Rhode Island, and Damariscotta River, Maine. The patterns of F(ST) indicate that gene flow between coast and estuary is extensive (Nm > 100) and is not lower in the estuary with lower flushing rates (Narragansett Bay). Given the high estimate of genetic exchange, adaptations for unpredictable environments seem more likely than local adaptation in this species because loci that respond to selection in one generation are essentially homogenized by the next seasons' settlement. Nevertheless, these estimates of neutral gene flow can help identify the strength of selection necessary for local adaptation to accumulate in Semibalanus.

Environmental heterogeneity and balancing selection in the acorn barnacle Semibalanus balanoides.

The northern acorn barnacle Semibalans banlanoides occupies several intertidal microhabitats which vary greatly in their degree of physical stress. This environmental heterogeneity creates distinct selection regimes which can maintain genetic variation in natural populations. Despite considerable attention placed on the link between spatial variation in fitness and balancing selection at specific loci, experimental manipulations and fitness estimates for molecular polymorphisms have rarely been conducted in the wild. The aim of this transplant experiment was to manipulate the level of physical stress experienced by a cohort of barnacles in the field and then investigate the spatial variation in fitness for genotypes at three loci: two candidate allozymes and the mitochondrial DNA control region. The viability of mannose-6-phosphate isomerase (Mpi) genotypes was dependent on the level of physical stress experienced in the various treatments; alternative homozygotes were favoured in alternative high stress-low stress environments. In contrast, the fitness of genotypes at other loci was equivalent among treatments and unaffected by the manipulation. Evaluated in the light of balancing selection models, these data indicate that the presence of multiple environmental niches is sufficient to promote a stable Mpi polymorphism in barnacle populations and that allelic variation at this locus reflects the process of adaptation to the heterogeneous intertidal landscape.

Concerted evolution and RAPping in mitochondrial VNTRs and the molecular geography of cricket populations.

In recent years, many animal species have been shown to exhibit a variable number of tandem repeats (VNTR) in the control region of their mitochondrial DNA. To characterize the nucleotide polymorphism of a VNTR system, this region was sequenced in individuals from two species of crickets (genus Gryllus) collected at a mainland locality and an island locality within each species. The data reveal a clear pattern of concerted evolution: homogeneity among repeats within individuals and populations, but heterogeneity among the tandem arrays from divergent populations, and between species. The patterns of nucleotide polymorphisms within the arrays show several instances of partial homogenization where derived nucleotides have not swept through all the repeats in the array. These serve as examples of concerted evolution "caught in the act". Additional repeat associated polymorphisms (RAPs) shed light on the molecular basis of the insertion and deletion of repeat units and, with the concerted evolution of VNTRs, provide phylogeographic resolution of island and mainland populations within and between species. The polymorphisms within VNTRs also provide a simple system in which to examine the balance of mutation and drift and offer a unique view of the histories of genomic and organismal phenomena in the patterns and processes of molecular evolution.

A multi-locus assessment of connectivity and historical demography in the bluehead wrasse (Thalassoma bifasciatum).

The pelagic larval stage of most coral reef fishes might allow extensive dispersal or, alternatively, some level of local recruitment might be important. Molecular markers can be used to obtain indirect estimates of dispersal to evaluate these alternatives, yet the extent of gene flow among populations is known for only a small number of species. The use of such markers must take into account the properties of the markers and the demographic history of the population when making inferences about current gene flow. In the Caribbean bluehead wrasse, Thalassoma bifasciatum, previous studies have found both substantial levels of local recruitment, in studies interpreting otolith microchemistry and, conversely, a lack of genetic differentiation inferred from mitochondrial DNA (mtDNA) restriction-fragment length polymorphism (RFLP) data and allozymes. However, if subtle differentiation exists, larger sample sizes and highly variable markers may be required to discern it. Here we present results from mitochondrial control region sequence and microsatellite data that indicate a lack of genetic differentiation at both small and large spatial scales. However, historical processes, such as changes in population size, may have affected the current distribution of genetic variation.

Thermal habit, metabolic rate and the evolution of mitochondrial DNA.

The hallmarks of animal mitochondrial DNA (mtDNA) are a rapid rate of sequence evolution, a small genome carrying the same set of homologous genes, maternal inheritance and lack of recombination. Over the past few years, a variety of different observations has challenged these accepted notions of mitochondrial biology. Notable examples include evidence for variable rates of mtDNA sequence evolution among taxa, evidence for large and variable mitochondrial genome sizes in certain groups, and a growing number of cases in metazoans of 'paternal leakage' in the inheritance of mtDNA. Several recent studies have uncovered different lines of evidence suggesting that an organism's thermal habit, or metabolic rate, can influence the evolution of mtDNA.

Endotherms, ectotherms, and mitochondrial genome-size variation.

The patterns of mitochondrial genome-size variation were investigated in endothermic and ectothermic species to examine the role that thermal habit might play in the evolution of animal mitochondrial DNA (mtDNA). Data on mtDNA size (the modal, largest, and smallest mtDNA reported within a species), the percent variation in mtDNA size (the difference in size between the largest and smallest mtDNAs divided by the model genome size for a given species), and the frequency of heteroplasmic individuals (those carrying more than one mtDNA length variant) were tabulated from the literature. Endotherms showed significantly less variation in mtDNA size and tended to have smaller mtDNAs than ectotherms. Further comparisons between endothermic and ectothermic vertebrates revealed that the largest genome and the percent variation in genome size were significantly smaller in the former than the latter. There was no difference between endotherms and ectotherms in the frequency of heteroplasmy. These data are discussed in light of two hypotheses: (1) more intense directional and purifying selection for small genome size in the cytoplasms of species with higher metabolic rates and (2) reduced mutation pressures generating mtDNA size variants in endotherms relative to those in ectotherms. The general trends are consistent with the selection hypothesis but in certain species mtDNA size variation appears to be governed by mutational pressures. To test these competing hypotheses further, comparative studies are proposed where mitochondrial genome size is quantified in sister taxa and tissue types with very different metabolic rates.