David M. Rand

Ecological genetics of a mosaic hybrid zone: mitochondrial, nuclear, and reproductive differentiation of crickets by soil type

We investigated the effects that habitat variation has on the structure and dynamics of a hybrid zone between two closely related crickets in Connecticut. A collecting protocol was developed in which crickets were sampled from characteristic habitats on either side of the hybrid zone and from two distinct habitat types within the zone. Presumptive pure Gryllus pennsylvanicus were sampled from fields in northwestern Connecticut and represent “inland” populations. “Pure” Gryllus firmus were sampled from beaches along the coast and represent the “coastal” populations. Crickets from within the hybrid zone were sampled from two different soil types: the “loam” populations from loamy soils and the “sand” populations from sandy soils. Moreover, an attempt was made to identify closely adjacent sand and loam localities to determine the scale of habitat variation and its possible effects on hybrid‐zone structure. In general, there was little variation in morphological traits or in allozyme and mtDNA genotype frequencies among localities from within each of the four habitat types. Between each of the closely situated sand and loam localities within the hybrid zone, however, there were very significant differences in each of these sets of markers. In addition, crickets from hybrid‐zone populations were tested for reproductive isolation. The asymmetric outcome of hybrid crosses that exists across the zone (Harrison, 1983) also exists on a finer ecological scale within the zone. Thus, this hybrid zone is a mosaic of strikingly differentiated populations. The dynamics of hybrid zones with mosaic structures are discussed in contrast to the traditional clinal models. The data are also discussed in light of the semipermeable nature of species boundaries. The extent to which a species boundary is permeable varies not only from one genetic marker to the next, but also with the ecological and geographic context of species interaction.

Variable fitness effects of Wolbachia infection in Drosophila melanogaster

Maternally inherited Wolbachia bacteria are extremely widespread among insects and their presence is usually associated with parasitic modifications of host fitness. Wolbachia pipientis infects Drosophila melanogaster populations from all continents, but their persistence in this species occurs despite any strong parasitic effects. Here, we have investigated the symbiosis between Wolbachia and D. melanogaster and found that Wolbachia infection can have significant survival and fecundity effects. Relative to uninfected flies, infected females from three fly strains showed enhanced survival or fecundity associated with Wolbachia infection, one strain showed both and one strain responded positively to Wolbachia removal. We found no difference in egg hatch rates (cytoplasmic incompatibility) for crosses between infected males and uninfected females, although there were fecundity differences. Females from this cross consistently produced fewer eggs than infected females and these fecundity differences could promote the spread of infection just like cytoplasmic incompatibility. More surprising, we found that infected females often had the greatest fecundity when mated to uninfected males. This could also promote the spread of Wolbachia infection, though here the fitness benefits would also help to spread infection when Wolbachia are rare. We suggest that variable fitness effects, in both sexes, and which interact strongly with the genetic background of the host, could increase cytoplasmic drive rates in some genotypes and help explain the widespread persistence of Wolbachia bacteria in D. melanogaster populations. These interactions may further explain why many D. melanogaster populations are polymorphic for Wolbachia infection. We discuss our results in the context of host–symbiont co-evolution.

CYTONUCLEAR COADAPTATION IN DROSOPHILA: DISRUPTION OF CYTOCHROME C OXIDASE ACTIVITY IN BACKCROSS GENOTYPES

Abstract The cytochrome c oxidase enzyme (COX) is comprised of 10 nuclear‐encoded subunits and three mito‐chondrial‐encoded subunits in close physical association in the inner mitochondrial membrane. COX passes electrons from cytochrome c to molecular oxygen and pumps protons into the inner mitochondrial space for ATP production. Selection on nuclear‐mitochondrial interactions within species should lead to coadaptation of the proteins comprising this important enzyme. Under this model, there should be relatively little disruption of COX activity when mitochondrial genomes are crossed among strains within species. A more pronounced disruption of activity is expected when the mitochondrial genome is expressed in the nuclear background of a different species. We test these hypotheses in Drosophila using hybridization and backcrossing among lines of D. simulans and D. mauritiana. Disrupted cytonuclear genotypes were constructed using backcrosses between two lines of D. simulans (siI and si II) that introduced each divergent mitochondrial DNA (mtDNA) into each nuclear background due to maternal inheritance of mtDNA. Similar crosses were used to introduce eachD. simulans mtDNA into the D. mauritiana maI nuclear background. Reconstituted cytonuclear control genotypes were constructed by backcrossing the initial F1 females to males of the maternal genotype. COX enzyme activities were compared among these disrupted and reconstituted backcross genotypes within and between species. The disruption effect on COX activity was restricted to males of interspecific genotypes. These data support the coadaptation hypothesis and are consistent with predictions that the evolution of modifiers of male mitochondrial dysfunction is hindered by the maternal inheritance of mtDNA. New sequence data for nuclear encoded subunits of COX identified amino acids that may play a role in the disruption effect.

Endotherms, ectotherms, and mitochondrial genome-size variation

The patterns of mitochondrial genomesize 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 endothermic 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.

Ecological genetics in the North Atlantic: environmental gradients and adaptation at specific loci.

The North Atlantic intertidal community provides a rich set of organismal and environmental material for the study of ecological genetics. Clearly defined environmental gradients exist at multiple spatial scales: there are broad latitudinal trends in temperature, meso-scale changes in salinity along estuaries, and smaller scale gradients in desiccation and temperature spanning the intertidal range. The geology and geography of the American and European coasts provide natural replication of these gradients, allowing for population genetic analyses of parallel adaptation to environmental stress and heterogeneity. Statistical methods have been developed that provide genomic neutrality tests of population differentiation and aid in the process of candidate gene identification. In this paper, we review studies of marine organisms that illustrate associations between an environmental gradient and specific genetic markers. Such highly differentiated markers become candidate genes for adaptation to the environmental factors in question, but the functional significance of genetic variants must be comprehensively evaluated. We present a set of predictions about locus-specific selection across latitudinal, estuarine, and intertidal gradients that are likely to exist in the North Atlantic. We further present new data and analyses that support and contradict these simple selection models. Some taxa show pronounced clinal variation at certain loci against a background of mild clinal variation at many loci. These cases illustrate the procedures necessary for distinguishing selection driven by internal genomic vs. external environmental factors. We suggest that the North Atlantic intertidal community provides a model system for identifying genes that matter in ecology due to the clarity of the environmental stresses and an extensive experimental literature on ecological function. While these organisms are typically poor genetic and genomic models, advances in comparative genomics have provided access to molecular tools that can now be applied to taxa with well-defined ecologies. As many of the organisms we discuss have tight physiological limits driven by climatic factors, this synthesis of molecular population genetics with marine ecology could provide a sensitive means of assessing evolutionary responses to climate change.

Mutation and selection at silent and replacement sites in the evolution of animal mitochondrial DNA

Two patterns are presented that illustrate the interaction of mutation and selection in the evolution of animal mtDNA: 1) variation among taxa in the ratio of polymorphism to divergence (rpd) at silent and replacement sites in protein-coding genes, and 2) strand-differences in polymorphism and divergence at ‘silent’ sites that suggest a mutation-selection balance in the evolution of codon usage. Cytochrome b data from GenBank show that about half of the species pairs tested have a significant excess of amino acid polymorphism, relative to divergence. The remaining half of species pairs do not depart from neutrality, but generally do show an excess of amino acid polymorphism. Sequences from Drosophila pseudoobscura displaying a signature of an expanding population show a slight, but non-significant, deficiency of amino acid polymorphism suggestive of recently intensified selection on mildly deleterious mutations. Genes whose reading frames lie on the major coding strand of Drosophila mtDNA show a preponderance of T → C substitutions, while genes encoded on the minor strand experience more A → G than T → C substitutions between species at both silent and replacement sites. However, silent mutations at third codon positions are introduced into the population in proportions opposite to those observed as fixed differences between species (e.g., an excess of T → C polymorphisms are found at the ND5 gene on the minor coding strand). The high A+T content of insect mtDNAs imposes strong codon usage bias favoring A-ending and T-ending codons resulting in a distinct mutation-selection balance for genes encoded on opposites strands. Thus, at both replacement and silent sites, mutations that appear to be constrained in terms of divergence between species are in excess within species. The data suggest that mildly deleterious mutations are common in mitochondrial genes. A test of this, and a competing, hypothesis is proposed that requires additional sequence surveys of polymorphism and divergence. An important challenge is to tease apart the impact of mutation and selection on levels of polymorphism versus divergence in a genome that does not generally recombine.

An Incompatibility between a Mitochondrial tRNA and Its Nuclear-Encoded tRNA Synthetase Compromises Development and Fitness in Drosophila

Mitochondrial transcription, translation, and respiration require interactions between genes encoded in two distinct genomes, generating the potential for mutations in nuclear and mitochondrial genomes to interact epistatically and cause incompatibilities that decrease fitness. Mitochondrial-nuclear epistasis for fitness has been documented within and between populations and species of diverse taxa, but rarely has the genetic or mechanistic basis of these mitochondrial–nuclear interactions been elucidated, limiting our understanding of which genes harbor variants causing mitochondrial–nuclear disruption and of the pathways and processes that are impacted by mitochondrial–nuclear coevolution. Here we identify an amino acid polymorphism in the Drosophila melanogaster nuclear-encoded mitochondrial tyrosyl–tRNA synthetase that interacts epistatically with a polymorphism in the D. simulans mitochondrial-encoded tRNATyr to significantly delay development, compromise bristle formation, and decrease fecundity. The incompatible genotype specifically decreases the activities of oxidative phosphorylation complexes I, III, and IV that contain mitochondrial-encoded subunits. Combined with the identity of the interacting alleles, this pattern indicates that mitochondrial protein translation is affected by this interaction. Our findings suggest that interactions between mitochondrial tRNAs and their nuclear-encoded tRNA synthetases may be targets of compensatory molecular evolution. Human mitochondrial diseases are often genetically complex and variable in penetrance, and the mitochondrial–nuclear interaction we document provides a plausible mechanism to explain this complexity.

Nuclear–Mitochondrial Epistasis and Drosophila Aging: Introgression of Drosophila simulans mtDNA Modifies Longevity in D. melanogaster Nuclear Backgrounds

Under the mitochondrial theory of aging, physiological decline with age results from the accumulated cellular damage produced by reactive oxygen species generated during electron transport in the mitochondrion. A large body of literature has documented age-specific declines in mitochondrial function that are consistent with this theory, but relatively few studies have been able to distinguish cause from consequence in the association between mitochondrial function and aging. Since mitochondrial function is jointly encoded by mitochondrial (mtDNA) and nuclear genes, the mitochondrial genetics of aging should be controlled by variation in (1) mtDNA, (2) nuclear genes, or (3) nuclear–mtDNA interactions. The goal of this study was to assess the relative contributions of these factors in causing variation in Drosophila longevity. We compared strains of flies carrying mtDNAs with varying levels of divergence: two strains from Zimbabwe (<20 bp substitutions between mtDNAs), strains from Crete and the United States (∼20–40 bp substitutions between mtDNAs), and introgression strains of Drosophila melanogaster carrying mtDNA from Drosophila simulans in a D. melanogaster Oregon-R chromosomal background (>500 silent and 80 amino acid substitutions between these mtDNAs). Longevity was studied in reciprocal cross genotypes between pairs of these strains to test for cytoplasmic (mtDNA) factors affecting aging. The intrapopulation crosses between Zimbabwe strains show no difference in longevity between mtDNAs; the interpopulation crosses between Crete and the United States show subtle but significant differences in longevity; and the interspecific introgression lines showed very significant differences between mtDNAs. However, the genotypes carrying the D. simulans mtDNA were not consistently short-lived, as might be predicted from the disruption of nuclear–mitochondrial coadaptation. Rather, the interspecific mtDNA strains showed a wide range of variation that flanked the longevities seen between intraspecific mtDNAs, resulting in very significant nuclear × mtDNA epistatic interaction effects. These results suggest that even “defective” mtDNA haplotypes could extend longevity in different nuclear allelic backgrounds, which could account for the variable effects attributable to mtDNA haplogroups in human aging.

INTERTIDAL MICROHABITAT AND SELECTION AT MPI: INTERLOCUS CONTRASTS IN THE NORTHERN ACORN BARNACLE, SEMIBALANUS BALANOIDES

Barnacles were sampled from various microhabitats in the rocky intertidal at multiple sites in two years. At sites in which there were large differences among microhabitats in temperature profiles, Mpi genotype frequencies were consistently and significantly different. Genotype frequencies for another allozyme locus (Gpi) as well as a DNA marker shown to be neutral (the mtDNA control region) were statistically homogeneous among thermal microhabitats at all sites in both years. The data indicate that temperature and/or desiccation mediated selection is operating at Mpi or a linked locus and that Mpi genotypes experience differential mortality in the various habitat types. If the relative fitness of genotypes is dependent on habitat type, the Mpi polymorphism may be actively maintained by a Levene model of balancing selection (Levene 1953). Because barnacle larvae are produced in abundance each year and spend five to eight weeks dispersing in the water column, there is little opportunity for the accumulation of adaptive divergence over the environmental grain size relevant in intertidal habitats. The Mpi polymorphism may be an important component of a suite of traits involved in the adaptation of barnacles to heterogeneous environments. Due to the relatively high concentration of mannose in a variety of algal groups, the metabolism of mannose may substantially affect individual performance and fitness in this and other species that feed on algae and phytoplankton. Because the Mpi locus is one of the most strongly polymorphic in marine organisms, these findings may be relevant for a diversity of other such species.

ADAPTIVE MAINTENANCE OF GENETIC POLYMORPHISM IN AN INTERTIDAL BARNACLE: HABITAT- AND LIFE-STAGE-SPECIFIC SURVIVORSHIP OF MPI GENOTYPES

Abstract.— 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.