Heteroplasmy and Patterns of Cytonuclear Linkage Disequilibrium in Wild Carrot.

Organellar genomes are considered to be strictly uniparentally-inherited. Uniparental inheritance allows for cytonuclear coevolution and the development of highly coordinated cytonuclear interactions. Yet, instances of biparental inheritance have been documented across eukaryotes. Biparental inheritance in otherwise uniparentally-inherited organelles is termed leakage (maternal or paternal) and allows for the presence of multiple variants of the same organellar genome within an individual, called heteroplasmy. It is unclear what, if any, evolutionary consequences are placed on nuclear and/or organellar genomes due to heteroplasmy. One way of accessing cytonuclear interactions and potential coevolution is through calculating cytonuclear linkage disequilibrium (cnLD), or the non-random association of alleles between nuclear and organellar genomes. Patterns of cnLD can indicate positive or negative cytonuclear selection, coevolution between the nuclear and organellar genomes, non-traditional organellar inheritance, or instances of ancestral heteroplasmy. In plants, cytonuclear interactions have been shown to play a role in cytoplasmic male sterility which occurs in gynodioecious species and is associated with leakage. We used the gynodioecious species, Daucus carota L. spp. carota, or wild carrot, to investigate cnLD. We genotyped a total of 265 individuals from two regions of the USA at 15 nuclear microsatellites, the mitochondrial genes cox1 and atp9, and an intergenic region between trnS and trnG (StoG) in the plastid genome to calculate nuclear-nuclear LD (nucLD), cnLD, and organellar LD (i.e., within the mtDNA and between mtDNA and ptDNA) within the two regions. We were further able to identify cox1 and StoG heteroplasmy and calculate some of the same LD measures within heteroplasmic and homoplasmic (non-heteroplasmic) datasets. We used a Z-transformation test to demonstrate that heteroplasmic individuals display significantly higher levels of cnLD within both regions. In spite of this, within and between organellar LD is low to moderate. Given these patterns of LD in two regions of the USA in which gene flow has been shown to occur between crop and wild carrot, we suggest that heteroplasmy is an evolutionary mechanism which permits the maintenance of cnLD while also acting to disrupt organellar LD.

Pervasive Mitochondrial Sequence Heteroplasmy in Natural Populations of Wild Carrot, Daucus carota spp. carota L.

Exceptions to the generally accepted rules that plant mitochondrial genomes are strictly maternally inherited and that within-individual sequence diversity in those genomes, i.e., heteroplasmy, should be minimal are becoming increasingly apparent especially with regard to sequence-level heteroplasmy. These findings raise questions about the potential significance of such heteroplasmy for plant mitochondrial genome evolution. Still studies quantifying the amount and consequences of sequence heteroplasmy in natural populations are rare. In this study, we report pervasive sequence heteroplasmy in natural populations of wild carrot, a close relative of the cultivated crop. In order to assay directly for this heteroplasmy, we implemented a quantitative PCR assay that can detect and quantify intra-individual SNP variation in two mitochondrial genes (Cox1 and Atp9). We found heteroplasmy in > 60% of all wild carrot populations surveyed and in > 30% of the 140 component individuals that were genotyped. Heteroplasmy ranged from a very small proportion of the total genotype (e.g., 0.995:0.005) to near even mixtures (e.g., 0.590:0.410) in some individuals. These results have important implications for the role of intra-genomic recombination in the generation of plant mitochondrial genome genotypic novelty. The consequences of such recombination are evident in the results of this study through analysis of the degree of linkage disequilibrium (LD) between the SNP sites at the two genes studied.

Population genetics of Braun's Rockcress (Boechera perstellata, Brassicaceae), an endangered plant with a disjunct distribution.

Boechera perstellata is an endangered plant found only in middle Tennessee and north central Kentucky. After sampling 4 Tennessee and 3 Kentucky populations, genetic variability and population structure were examined for this species using isozymes, chloroplast DNA, and microsatellites (averaging 35, 29, and 27 individuals per population per class of marker, respectively). The only genetic variability detected for 23 isozymes was a fixed difference between Tennessee and Kentucky populations at 1 locus. Fixed differences between populations of the 2 states were also observed for 3 chloroplast markers. Polymorphism at 19 nuclear microsatellites was 74% at the species level and averaged 21% at the population level. However, observed heterozygosity was extremely low in all populations, ranging from 0.000 to 0.005. High F IS values (0.93) suggest that Boechera perstellata is a primarily selfing species. Tennessee populations have more genetic diversity than Kentucky populations of B. perstellata. Microsatellite markers revealed substantial genetic divergence between the states and genetic differences among populations within each state. Analysis of molecular variance indicates that most variability in this species occurs between the 2 states (49%) and among populations within each state (42%), with relatively little variation found within populations (9%). These data indicate that there is very little gene flow among populations of B. perstellata and that it is important to protect as many populations as possible in order to conserve the genetic diversity of this rare species.

Paternal leakage, heteroplasmy, and the evolution of plant mitochondrial genomes.

Plant mitochondrial genomes are usually transmitted to the progeny from the maternal parent. However, cases of paternal transmission are known and are perhaps more common than once thought. This review will consider recent evidence, both direct and indirect, of paternal transmission (leakage) of the mitochondrial genome of seed plants, especially in natural populations, and how this can result in offspring that carry a mixture of maternally and paternally derived copies of the genome; a type of heteroplasmy. It will further consider how this heteroplasmy facilitates recombination between genetically distinct partners; a process that can enhance mitochondrial genotypic diversity. This will then form the basis for a discussion of five evolutionary questions that arise from these observations. Questions include how plant mitochondrial genome evolution can be placed on a sexual to asexual continuum, whether cytoplasmic male sterility (CMS) facilitates the evolution of paternal leakage, whether paternal leakage is more likely in populations undergoing admixture, how leakage influences patterns of gene flow, and whether heteroplasmy occurs in natural populations at a frequency greater than predicted by crossing experiments. It is proposed that each of these questions offers fertile ground for future research on a diversity of plant species.

Intraspecific variation in mitochondrial genome sequence, structure, and gene content in Silene vulgaris, an angiosperm with pervasive cytoplasmic male sterility.

In angiosperms, mitochondrial-encoded genes can cause cytoplasmic male sterility (CMS), resulting in the coexistence of female and hermaphroditic individuals (gynodioecy). We compared four complete mitochondrial genomes from the gynodioecious species Silene vulgaris and found unprecedented amounts of intraspecific diversity for plant mitochondrial DNA (mtDNA). Remarkably, only about half of overall sequence content is shared between any pair of genomes. The four mtDNAs range in size from 361 to 429 kb and differ in gene complement, with rpl5 and rps13 being intact in some genomes but absent or pseudogenized in others. The genomes exhibit essentially no conservation of synteny and are highly repetitive, with evidence of reciprocal recombination occurring even across short repeats (< 250 bp). Some mitochondrial genes exhibit atypically high degrees of nucleotide polymorphism, while others are invariant. The genomes also contain a variable number of small autonomously mapping chromosomes, which have only recently been identified in angiosperm mtDNA. Southern blot analysis of one of these chromosomes indicated a complex in vivo structure consisting of both monomeric circles and multimeric forms. We conclude that S. vulgaris harbors an unusually large degree of variation in mtDNA sequence and structure and discuss the extent to which this variation might be related to CMS.

Mitochondrial gene diversity associated with the atp9 stop codon in natural populations of wild carrot (Daucus carota ssp. carota).

Mitochondrial genomes extracted from the wild populations of Daucus carota have been used as a genetic resource by breeders of cultivated carrot, yet little is known concerning the extent of their diversity in nature. Of special interest is an SNP in the putative stop codon of the mitochondrial gene atp9 that has been associated previously with male-sterile and male-fertile phenotypic variants. In this study, either the sequence or PCR/RFLP genotypes were obtained from the mitochondrial genes atp1, atp9, and cox1 found in D. carota individuals collected from 24 populations in the eastern United States. More than half of the 128 individuals surveyed had a CAA or AAA, rather than TAA, genotype at the position usually thought to function as an atp9 stop codon in this species. We also found no evidence for mitochondrial RNA editing (Cytosine to Uridine) of the CAA stop codon in either floral or leaf tissue. Evidence for intragenic recombination, as opposed to the more common intergenic recombination in plant mitochondrial genomes, in our data set is presented. Indel and SNP variants elsewhere in atp9, and in the other 2 genes surveyed, were nonrandomly associated with the 3 atp9 stop codon variants, though further analysis suggested that multilocus genotypic diversity had been enhanced by recombination. Overall the mitochondrial genetic diversity was only modestly structured among populations with an F(ST) of 0.34.

Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates.

Genome size and complexity vary tremendously among eukaryotic species and their organelles. Comparisons across deeply divergent eukaryotic lineages have suggested that variation in mutation rates may explain this diversity, with increased mutational burdens favoring reduced genome size and complexity. The discovery that mitochondrial mutation rates can differ by orders of magnitude among closely related angiosperm species presents a unique opportunity to test this hypothesis. We sequenced the mitochondrial genomes from two species in the angiosperm genus Silene with recent and dramatic accelerations in their mitochondrial mutation rates. Contrary to theoretical predictions, these genomes have experienced a massive proliferation of noncoding content. At 6.7 and 11.3 Mb, they are by far the largest known mitochondrial genomes, larger than most bacterial genomes and even some nuclear genomes. In contrast, two slowly evolving Silene mitochondrial genomes are smaller than average for angiosperms. Consequently, this genus captures approximately 98% of known variation in organelle genome size. The expanded genomes reveal several architectural changes, including the evolution of complex multichromosomal structures (with 59 and 128 circular-mapping chromosomes, ranging in size from 44 to 192 kb). They also exhibit a substantial reduction in recombination and gene conversion activity as measured by the relative frequency of alternative genome conformations and the level of sequence divergence between repeat copies. The evolution of mutation rate, genome size, and chromosome structure can therefore be extremely rapid and interrelated in ways not predicted by current evolutionary theories. Our results raise the hypothesis that changes in recombinational processes, including gene conversion, may be a central force driving the evolution of both mutation rate and genome structure.

Paternal leakage and heteroplasmy of mitochondrial genomes in Silene vulgaris: evidence from experimental crosses.

The inheritance of mitochondrial genetic (mtDNA) markers in the gynodioecious plant Silene vulgaris was studied using a series of controlled crosses between parents of known mtDNA genotype followed by quantitative PCR assays of offspring genotype. Overall, approximately 2.5% of offspring derived from crosses between individuals that were homoplasmic for different mtDNA marker genotypes showed evidence of paternal leakage. When the source population of the pollen donor was considered, however, population-specific rates of leakage varied significantly around this value, ranging from 10.3% to zero. When leakage did occur, the paternal contribution ranged from 0.5% in some offspring (i.e., biparental inheritance resulting in a low level of heteroplasmy) to 100% in others. Crosses between mothers known to be heteroplasmic for one of the markers and homoplasmic fathers showed that once heteroplasmy enters a maternal lineage it is retained by approximately 17% of offspring in the next generation, but lost from the others. The results are discussed with regard to previous studies of heteroplasmy in open-pollinated natural populations of S. vulgaris and with regard to the potential impact of mitochondrial paternal leakage and heteroplasmy on both the evolution of the mitochondrial genome and the evolution of gynodioecy.

Recent advances in the study of gynodioecy: the interface of theory and empiricism.

BACKGROUND: In this review we report on recent literature concerned with studies of gynodioecy, or the co-occurrence of female and hermaphrodite individuals in natural plant populations. Rather than review this literature in its entirety, our focus is on the interplay between theoretical and empirical approaches to the study of gynodioecy. SCOPE: Five areas of active inquiry are considered. These are the cost of restoration, the influence of population structure on spatial sex-ratio variation, the influence of inbreeding on sex expression, the signature of cyto-nuclear coevolution on the mitochondrial genome, and the consequences of mitochondrial paternal leakage. CONCLUSIONS: Recent advances in the study of gynodioecy have been made by considering both the ecology of female:hermaphrodite fitness differences and the genetics of sex expression. Indeed theory has guided empiricism and empiricism has guided theory. Future advances will require that some of the methods currently available only for model organisms be applied to a wider range of species.

Mitochondrial heteroplasmy and paternal leakage in natural populations of Silene vulgaris, a gynodioecious plant.

It is currently thought that most angiosperms transmit their mitochondrial genomes maternally. Maternal transmission limits opportunities for genetic heterogeneity (heteroplasmy) of the mitochondrial genome within individuals. Recent studies of the gynodioecious species Silene vulgaris and Silene acaulis, however, document both direct and indirect evidence of mitochondrial heteroplasmy, suggesting that the mitochondrial genome is at times transmitted via paternal leakage. This heteroplasmy allows the generation of multi-locus recombinants, as documented in recent studies of both species. A prior study that employed quantitative PCR (q-PCR) on a limited sample provided direct evidence of heteroplasmy in the mitochondrial gene atp1 in S. vulgaris. Here, we apply the q-PCR methods to a much larger sample and extend them to incorporate the study of an additional atp1 haplotype along with two other haplotypes of the mitochondrial gene cox1 to evaluate the origin, extent, and transmission of mitochondrial genome heteroplasmy in S. vulgaris. We first calibrate our q-PCR methods experimentally and then use them to quantify heteroplasmy in 408 S. vulgaris individuals sampled from 22 natural populations located in Virginia, New York, and Tennessee. Sixty-one individuals exhibit heteroplasmy, including five that exhibited the joint heteroplasmy at both loci that is a prerequisite for effective recombination. The heteroplasmic individuals were distributed among 18 of the populations studied, demonstrating that heteroplasmy is a widespread phenomenon in this species. Further, we compare mother and offspring from 71 families to determine the rate of heteroplasmy gained and lost via paternal leakage and vegetative sorting across generations. Of 17 sibships exhibiting cox1 heteroplasmy and 14 sibships exhibiting atp1 heteroplasmy, more than half of the observations of heteroplasmy are generated via paternal leakage at the time of fertilization, with the rest being inherited from a heteroplasmic mother. Moreover, we show that the average paternal contribution during paternal leakage is about 12%. These findings are surprising, given that the current understanding of gynodioecy assumes that mitochondrial cytoplasmic male sterility elements are strictly maternally inherited. Knowledge of the dynamics of mitochondrial populations within individuals plays an important role in understanding the evolution of gynodioecy, and we discuss our findings within this context.

Do recent findings in plant mitochondrial molecular and population genetics have implications for the study of gynodioecy and cytonuclear conflict?

The coexistence of females and hermaphrodites in plant populations, or gynodioecy, is a puzzle recognized by Darwin. Correns identified cytoplasmic inheritance of one component of sex expression, now known as cytoplasmic male sterility (CMS). Lewis established cytonuclear inheritance of gynodioecy as an example of genetic conflict. Although biologists have since developed an understanding of the mechanisms allowing the joint maintenance of CMS and nuclear male fertility restorer genes, puzzles remain concerning the inheritance of sex expression and mechanisms governing the origination of CMS. Much of the theory of gynodioecy rests on the assumption of maternal inheritance of the mitochondrial genome. Here we review recent studies of the genetics of plant mitochondria, and their implications for the evolution and transmission of CMS. New studies of intragenomic recombination provide a plausible origin for the chimeric ORFs that characterize CMS. Moreover, evidence suggests that nonmaternal inheritance of mitochondria may be more common than once believed. These findings may have consequences for the maintenance of cytonuclear polymorphism, mitochondrial recombination, generation of gynomonoecious phenotypes, and interpretation of experimental crosses. Finally we point out that CMS can alter the nature of the cytonuclear conflict that may have originally selected for uniparental inheritance.

Recombination and linkage disequilibrium among mitochondrial genes in structured populations of the gynodioecious plant Silene vulgaris.

The impact of intergenic recombination on the population genetics of plant mitochondrial genomes is unknown. In an effort to study this in the gynodioecious plant Silene vulgaris three-locus PCR/RFLP genotypes (based on the mitochondrial genes atpA, cox1, and cob) were determined for 239 individuals collected from 20 North American populations. Seventeen three-locus PCR/RFLP genotypes were found. Recombination was indicated by observation of each of the four two-locus genotypes possible when the two most common alleles are considered for each of two loci. Based on these common alleles the absolute values of standardized linkage disequilibrium |D'| between pairs of loci range from 0.17 to 0.78. This indicates modest disequilibrium, rather than the maximum value expected in the absence of recombination |D'=1|, or the linkage equilibrium expected if recombination is pervasive (D'=0). Values of D' did not depend on which pair of loci contributed alleles to the analysis. The direction of D' obtained for the common atpA and cox1 alleles was comparable in sign and magnitude to that obtained by examining similar information obtained in a prior study of European samples. All three loci indicated a high degree of population structure (average FST=0.63), which would limit the within-population genetic diversity required for intergenic recombination to create novel genotypes, if most mating is local. Thus, population structure acts as a constraint on the approach to linkage equilibrium.

Inheritance of chloroplast DNA is not strictly maternal in Silene vulgaris (Caryophyllaceae): evidence from experimental crosses and natural populations.

Chloroplast DNA (cpDNA) is maternally inherited in the majority, but not all, of angiosperm species. The mode of inheritance of cpDNA is a critical determinant of its molecular evolution and of its population genetic structure. Here, we present the results of investigations of the inheritance of cpDNA in Silene vulgaris, a plant used in a variety of studies in which cpDNA is an important component. PCR/RFLP markers were used to compare mother and offspring cpDNA genotypes sampled from two natural populations, and mother, father, and offspring genotypes obtained from controlled greenhouse crosses. Ten of 215 offspring cpDNA genotypes studied in the controlled crosses and three of 156 offspring from natural populations did not match that of the mother, demonstrating rare nonmaternal inheritance. That the chloroplast genome is occasionally transmitted through pollen is discussed in the context of using S. vulgaris cpDNA as a marker in studies of seed dispersal and when considering the joint evolution of the chloroplast and mitochondrial genomes.

Variable populations within variable populations: quantifying mitochondrial heteroplasmy in natural populations of the gynodioecious plant Silene vulgaris.

Populations of mitochondria reside within individuals. Among angiosperms, these populations are rarely considered as genetically variable entities and typically are not found to be heteroplasmic in nature, leading to the widespread assumption that plant mitochondrial populations are homoplasmic. However, empirical studies of mitochondrial variation in angiosperms are relatively uncommon due to a paucity of sequence variation. Recent greenhouse studies of Silene vulgaris suggested that heteroplasmy might occur in this species at a level that it is biologically relevant. Here, we use established qualitative methods and a novel quantitative PCR method to study the intraindividual population genetics of mitochondria across two generations in natural populations of S. vulgaris. We show incidences of heteroplasmy for mitochondrial atpA and patterns of inheritance that are suggestive of more widespread heteroplasmy at both atpA and cox1. Further, our results demonstrate that quantitative levels of mitochondrial variation within individuals are high, constituting 26% of the total in one population. These findings are most consistent with a biparental model of mitochondrial inheritance. However, selection within individuals may be instrumental in the maintenance of variation because S. vulgaris is gynodioecious. Male sterility is, in part, regulated by the mitochondrial genome, and strong selection pressures appear to influence the frequency of females in these populations.

EST databases as a source for molecular markers: lessons from Helianthus.

Expressed sequence tag (EST) databases represent a potentially valuable resource for the development of molecular markers for use in evolutionary studies. Because EST-derived markers come from transcribed regions of the genome, they are likely to be conserved across a broader taxonomic range than are other sorts of markers. This paper describes a case study in which the publicly available cultivated sunflower (Helianthus annuus) EST database was used to develop simple sequence repeat (SSR) markers for use in the genetic analysis of a rare sunflower species, Helianthus verticillatus, as well as the more widespread Helianthus angustifolius. EST-derived SSRs were found to be more than 3 times as transferable across species as compared with anonymous SSRs (73% vs. 21%, respectively). Moreover, EST-SSRs whose primers were located within protein-coding sequence were more readily transferable than those derived from untranslated regions, and the former loci were no less variable than the latter. The utility of existing EST databases as a means for facilitating population genetic analyses in plants was further explored by cross-referencing publicly available EST resources against available lists of rare or invasive flowering plant taxa. This survey revealed that more than one-third of all plant-derived EST collections of sufficient size could conceivably serve as a source of EST-SSRs for the analysis of rare, endangered, or invasive plant species worldwide.

Relative effects of nocturnal vs diurnal pollinators and distance on gene flow in small Silene alba populations.

Silene alba exists in natural metapopulations throughout its range and is visited by a suite of both diurnal and nocturnal pollinators. Pollen-mediated gene flow may help reduce genetic isolation of subpopulations. Here, we compared the relative effects of nocturnal vs diurnal pollinators on pollen-mediated gene flow in subpopulations separated by two distance treatments. We established populations consisting of genetically marked individuals in an old field in Tennessee (USA). Electrophoretic examination of seedlings produced by plants exposed to nocturnal, diurnal and control pollinator treatments and separated by either 20 or 80 m allowed us to directly measure pollen-mediated gene flow. Gene flow was more common between populations separated by only 20 m. Nocturnal pollinators were responsible for most gene flow between populations, regardless of distance. Diurnal pollinators played only a small role in pollen-mediated gene flow. The results suggest that nocturnal pollinators are better than diurnal pollinators at moving pollen between small S. alba subpopulations. However, their effectiveness declines as the distance between subpopulations increases, making them relatively ineffective at moving genes between isolated subpopulations.

Paternal leakage sustains the cytoplasmic polymorphism underlying gynodioecy but remains invasible by nuclear restorers.

Cytoplasmic male sterility (CMS) in plants often results in gynodioecious populations, composed of hermaphrodites and male-sterile females. All models of gynodioecy assume maternal inheritance of the cytoplasmic alleles and postulate a variety of negatively frequency-dependent mechanisms to maintain the cytoplasmic polymorphisms observed in many natural populations. However, in some plant species, mitochondria are transmitted at least occasionally by pollen, a process called paternal leakage. We show that even a small amount of paternal leakage is sufficient to sustain a permanent, stable cytoplasmic polymorphism. Because only hermaphrodites provide pollen in gynodioecious species, the effects of paternal leakage are biased and occur more often from the non-CMS male-fertile haplotype to the CMS male-sterile haplotype. We also show that a nuclear restorer disrupts the polymorphic cytoplasmic equilibrium, leading to fixation of both the CMS allele and the restorer. Although a dominant nuclear restorer fixes, it fixes much more slowly than in the standard CMS models. Although a stable cytonuclear polymorphism is possible with "matching alleles" nuclear restoration, oscillations to low frequencies present a risk of loss by drift. Paternal leakage enhances the stability of joint cytonuclear polymorphism by reducing the chance that a CMS allele is lost by drift.

Genetics and adaptation in structured populations: sex ratio evolution in Silene vulgaris.

Theoretical models suggest that population structure can interact with frequency dependent selection to affect fitness in such a way that adaptation is dependent not only on the genotype of an individual and the genotypes with which it co-occurs within populations (demes), but also the distribution of genotypes among populations. A canonical example is the evolution of altruistic behavior, where the costs and benefits of cooperation depend on the local frequency of other altruists, and can vary from one population to another. Here we review research on sex ratio evolution that we have conducted over the past several years on the gynodioecious herb Silene vulgaris in which we combine studies of negative frequency dependent fitness on female phenotypes with studies of the population structure of cytoplasmic genes affecting sex expression. This is presented as a contrast to a hypothetical example of selection on similar genotypes and phenotypes, but in the absence of population structure. Sex ratio evolution in Silene vulgaris provides one of the clearest examples of how selection occurs at multiple levels and how population structure, per se, can influence adaptive evolution.

Offspring sex ratio under inbreeding and outbreeding in a gynodioecious plant.

Silene vulgaris is a gynodioecious plant native to Eurasia and now found throughout much of North America. Using hermaphrodite plants from three geographic regions (Stamford, NY; Broadway,VA; and Giles Co., VA) and four local populations within each region, we employed a hierarchical crossing design to explore the geographic structure of sex determining genes. Sex determination in this species is cytonuclear involving multiple cytoplasmic male sterility and nuclear restorer loci. Due to dominance effects within nuclear restorer loci, self-fertilization of hermaphrodites heterozygous at restorer loci should produce some homozygous recessive female offspring. Female offspring may also result from outcrossing among related individuals. At greater geographic and genetic distances, mismatches between cytoplasmic and nuclear sex determining genes should also produce high frequencies of female offspring if coevolution between cytoplasmic and nuclear sex determining alleles occurs independently among widely separated populations. We found evidence of dominance effects among nuclear restorer loci but no evidence of nuclear-cytoplasmic mismatches at the regional level. Of 63 maternal lines, 55 produced at least one female offspring when self-fertilized. Outcrossing within populations produced significantly fewer female offspring than self-fertilization. Outcrossing among regions produced the lowest proportion of female offspring, significantly fewer than outcrossing among populations within regions. Regions responded differently to among-region outcrossing with pollen donors from the two Virginia regions producing far fewer female offspring with New York dams than crosses among New York populations. These results indicate that nuclear restoration is complex, involving multiple loci with epistatic interactions and that most hermaphrodites in nature are heterozygous at one or more restorer locus. Further, regional differences in restorer frequencies indicate significant genetic structure for sex determining genes at large geographic scales, perhaps reflecting invasion history.