Patterns of cyto-nuclear linkage disequilibrium in Silene latifolia: genomic heterogeneity and temporal stability.

Non-random association of alleles in the nucleus and cytoplasmic organelles, or cyto-nuclear linkage disequilibrium (LD), is both an important component of a number of evolutionary processes and a statistical indicator of others. The evolutionary significance of cyto-nuclear LD will depend on both its magnitude and how stable those associations are through time. Here, we use a longitudinal population genetic data set to explore the magnitude and temporal dynamics of cyto-nuclear disequilibria through time. We genotyped 135 and 170 individuals from 16 and 17 patches of the plant species Silene latifolia in Southwestern VA, sampled in 1993 and 2008, respectively. Individuals were genotyped at 14 highly polymorphic microsatellite markers and a single-nucleotide polymorphism (SNP) in the mitochondrial gene, atp1. Normalized LD (D') between nuclear and cytoplasmic loci varied considerably depending on which nuclear locus was considered (ranging from 0.005-0.632). Four of the 14 cyto-nuclear associations showed a statistically significant shift over approximately seven generations. However, the overall magnitude of this disequilibrium was largely stable over time. The observed origin and stability of cyto-nuclear LD is most likely caused by the slow admixture between anciently diverged lineages within the species' newly invaded range, and the local spatial structure and metapopulation dynamics that are known to structure genetic variation in this system.

Detection of rare paternal chloroplast inheritance in controlled crosses of the endangered sunflower Helianthus verticillatus.

A variety of questions in population and evolutionary biology are studied using chloroplast DNA (cpDNA). The presumed maternal inheritance in angiosperms allows for certain assumptions and calculations to be made when studying plant hybridization, phylogeography, molecular systematics and seed dispersal. Further, the placement of transgenes in the chloroplast to lessen the probability of 'escape' to weedy relatives has been proposed since such genes would not move through pollen. In many studies, however, strict maternal inheritance is assumed but not tested directly, and some studies may have sample sizes too small to be able to detect rare paternal leakage. Here, we study the inheritance of cpDNA simple sequence repeats in 323 offspring derived from greenhouse crosses of the rare sunflower Helianthus verticillatus Small. We found evidence for rare chloroplast paternal leakage and heteroplasmy in 1.86% of the offspring. We address the question of whether one can extrapolate the mode of chloroplast transmission within a genus by comparing our results to the findings of another sunflower species study. The findings of occasional paternal transmission of the chloroplast genome are discussed in the framework of using these markers in studies of population and evolutionary biology both in Helianthus and other angiosperms.

High genetic diversity in a rare and endangered sunflower as compared to a common congener.

Determining the genetic structure of isolated or fragmented species is of critical importance when planning a suitable conservation strategy. In this study, we use nuclear and chloroplast SSRs (simple sequence repeats) to investigate the population genetics of an extremely rare sunflower, Helianthus verticillatus Small, which is known from only three locations in North America. We investigated levels of genetic diversity and population structure compared to a more common congener, Helianthus angustifolius L., using both nuclear and chloroplast SSRs. We also investigated its proposed hybrid origin from Helianthus grosseserratus Martens and H. angustifolius. Twenty-two nuclear SSRs originating from the cultivated sunflower (Helianthus annuus L.) expressed sequence tag (EST) database, and known to be transferable to H. verticillatus and its putative parental taxa, were used in this study thereby allowing for statistical control of locus-specific effects in population genetic analyses. Despite its rarity, H. verticillatus possessed significantly higher levels of genetic diversity than H. angustifolius at nuclear loci and equivalent levels of chloroplast diversity. Significant levels of population subdivision were observed in H. verticillatus but of a magnitude comparable to that of H. angustifolius. Inspection of multilocus genotypes also revealed that clonal spread is highly localized. Finally, we conclude that H. verticillatus is not of hybrid origin as it does not exhibit a mixture of parental alleles at nuclear loci, and it does not share a chloroplast DNA haplotype with either of its putative parents.

Evidence for paternal transmission and heteroplasmy in the mitochondrial genome of Silene vulgaris, a gynodioecious plant.

Gynodioecy refers to the co-occurrence of females and hermaphrodites in the same population. In many gynodioecious plants, sex is determined by an epistatic interaction between mitochondrial and nuclear genes, resulting in intragenomic evolutionary conflict, should the mitochondrial genome be maternally inherited. While maternal inheritance of the mitochondrial genome is common in angiosperms, few gynodioecious species have been studied. Here, the inheritance of the mitochondrial genes atpA and coxI was studied in 318 Silene vulgaris individuals distributed among 23 crosses. While maternal inheritance was indicated in 96% of the individuals studied, one or more individuals from each of four sib groups displayed a genotype that was identical to the father, or that did not match either parent. Given evidence that inheritance is not strictly maternal, it was hypothesized that some individuals could carry a mixture of maternally and paternally derived copies of the mitochondrial genome, a condition known as heteroplasmy. Since heteroplasmy might be difficult to detect should multiple versions of the mitochondrial genome co-occur in highly unequal copy number, a method was devised to amplify low-copy number forms of atpA differentially. Evidence for heteroplasmy was found in 23 of the 99 individuals studied, including cases in which the otherwise cryptic form of atpA matched the paternal genotype. The distribution of shared nucleotide sequence polymorphism among atpA haplotypes and the results of a population survey of the joint distribution of atpA and coxI haplotypes across individuals supports the hypothesis that heteroplasmy facilitates formation of novel mitochondrial genotypes by recombination.

The hierarchical spatial distribution of chloroplast DNA polymorphism across the introduced range of Silene vulgaris.

Silene vulgaris was introduced into North America sometime prior to 1800. In order to document the population structure that has developed since that time, collections were made from 56 local populations distributed among 9 geographical regions in eastern North America. Individual plants were characterized for chloroplast DNA (cpDNA) haplotype by restriction fragment size analysis of four noncoding regions of cpDNA amplified by polymerase chain reaction. A total of 19 cpDNA haplotypes were detected using this method. The overall gene diversity of 0.85 is quite similar to the diversity detected in these same regions of cpDNA in a previously published sample of S. vulgaris taken from across much of Europe. The spatial distribution of the North American cpDNA diversity was quantified by hierarchical F-statistics that partitioned the genetic variance into variation among local populations within regions, and variation among regions. The average FST among populations within regions was 0.66 and the FST among regions was 0.09. The among-region variation was due to both differences among regions in the frequency of two most common haplotypes, and to the presence of a number of region-specific haplotypes. In order to test for isolation by distance at the regional level, FST values were calculated for all possible pairs of regions, and regressed against the geographical distance between those regions. There was no evidence for isolation by distance. It is suggested that the local population structure is generated by recent extinction/colonization dynamics, and that the among-region structure reflects demographic events associated with range expansion following introduction to North America.

Associations among cytoplasmic molecular markers, gender, and components of fitness in Silene vulgaris, a gynodioecious plant.

It has been suggested that the dynamics of chloroplast DNA (cpDNA) or mitochondrial DNA (mtDNA) genetic markers used in studies of plant populations could be influenced by natural selection acting elsewhere in the genome. This could be particularly true in gynodioecious plants if cpDNA or mtDNA genetic markers are in gametic disequilibrium with genes responsible for sex expression. In order to investigate this possibility, a natural population of the gynodioecious plant Silene vulgaris was used to study associations among mtDNA haplotype, cpDNA haplotype, sex and some components of fitness through seed. Individuals were sampled for mtDNA and cpDNA haplotype as determined using restriction fragment length polymorphism (RFLP) methods, sex (female or hermaphrodite), fruit number, fruit set, seeds/fruit and seed germination. The sex of surviving germinating seeds was also noted. All individuals in the population fell into one of two cytoplasmic categories, designated haplotypes f and g by a unique electrophoretic signature in both the mtDNA and cpDNA. The subset of the population carrying haplotype g included a significantly higher proportion of females when compared with the sex ratio of the subset carrying the f haplotype. Haplotype g had a significantly higher fitness when measured by fruit number, fruit set and seeds/fruit, whereas haplotype f had significantly higher fitness when measured by seed germination. Offspring of individuals carrying haplotype g included a significantly greater proportion of females when compared with offspring of individuals carrying the f haplotype. Other studies of gynodioecious plants have shown that females generally have higher fitness through seed than hermaphrodites, but in this study not all fitness differences between haplotypes could be predicted from differences in haplotype-specific sex ratio alone. Rather, some differences in haplotype-specific fitness were due to differences in fitness between individuals of the same sex, but carrying different haplotypes. The results are discussed with regard to the potential for hitchhiking selection to influence the dynamics of the noncoding regions used to designate the cpDNA and mtDNA haplotypes.

Genetic and demographic dynamics of small populations of Silene latifolia.

Small local populations of Silene alba, a short-lived herbaceous plant, were sampled in 1994 and again in 1999. Sampling included estimates of population size and genetic diversity, as measured at six polymorphic allozyme loci. When averaged across populations, there was very little change between samples (about three generations) in population size, measures of within-population genetic diversity such as number of alleles or expected heterozygosity, or in the apportionment of genetic diversity within and among populations as measured by F(st). However, individual populations changed considerably, both in terms of numbers of individuals and genetic composition. Some populations doubled in size between samples, while others shrank by more than 75%. Similarly, expected heterozygosity and allele number increased by more than two-fold in individual populations and decreased by more than three-fold in others. When population-specific change in number and change in measures of genetic diversity were considered together, significant positive correlations were found between the demographic and genetic variables. It is speculated that some populations were released from the demographic consequences of inbreeding depression by gene flow.

A quantitative genetic analysis of nuclear-cytoplasmic male sterility in structured populations of Silene vulgaris.

Gynodioecy, the coexistence of functionally female and hermaphroditic morphs within plant populations, often has a complicated genetic basis involving several cytoplasmic male-sterility factors and nuclear restorers. This complexity has made it difficult to study the genetics and evolution of gynodioecy in natural populations. We use a quantitative genetic analysis of crosses within and among populations of Silene vulgaris to partition genetic variance for sex expression into nuclear and cytoplasmic components. We also use mitochondrial markers to determine whether cytoplasmic effects on sex expression can be traced to mitochondrial variance. Cytoplasmic variation and epistatic interactions between nuclear and cytoplasmic loci accounted for a significant portion of the variation in sex expression among the crosses. Source population also accounted for a significant portion of the sex ratio variation. Crosses among populations greatly enhanced the dam (cytoplasmic) effect, indicating that most among-population variance was at cytoplasmic loci. This is supported by the large among-population variance in the frequency of mitochondrial haplotypes, which also accounted for a significant portion of the sex ratio variance in our data. We discuss the similarities between the population structure we observed at loci that influence sex expression and previous work on putatively neutral loci, as well as the implications this has for what mechanisms may create and maintain population structure at loci that are influenced by natural selection.

Population-specific gender-biased hybridization between Dryopteris intermedia and D. carthusiana: evidence from chloroplast DNA.

As has been shown for many kinds of organisms, barriers to interspecific hybridization may differ in strength between reciprocal crosses, resulting in a bias in the probability that one or the other species may be the maternal or paternal parent of hybrids. The fern Dryopteris Xtriploidea, the "backcross" hybrid between the diploid D. intermedia and the tetraploid D. carthusiana, occurs in large numbers in nature, providing an opportunity to investigate whether such a bias exists. Differences in the chloroplast genome distinguishing the two parental species were discovered in the sequence of the trnL region following amplification by polymerase chain reaction (PCR), including a Mse I restriction site. This allowed rapid identification of the donor of the chloroplast genome, and therefore the maternal parent of each hybrid, assuming chloroplast DNA to be maternally inherited in Dryopteris. Analysis was carried out on 127 hybrids, shown to be of independent origin using allozymes, occurring at three localities in Virginia and West Virginia. When samples from all localities were pooled, 91 possessed the D. carthusiana trnL genotype and 36 possessed the D. intermedia genotype, a ratio that is significantly different (P < 0.001) from the null hypothesis of no gender bias. The strength of the bias differed significantly among the three sites, however, with bias at the West Virginia site much stronger (5.6:1 carthusiana:intermedia; P < 0.001) than at either Virginia site (1.55:1 and 1.43:1 carthusiana:intermedia, respectively; P > 0.05 in both cases). The cause of the strong bias in the West Virginia sample is unknown, as is the cause of the population differences. Causes of bias could include differences between the parental species related to their ploidy difference, including sizes of gametes and/or gametangia, sperm motility, breeding system (D. intermedia is outcrossing while D. carthusiana is selfing), or the nature and strength of interspecific isolating mechanisms.

Linkage disequilibrium and phylogenetic congruence between chloroplast and mitochondrial haplotypes in Silene vulgaris.

Both the chloroplast and mitochondrial genomes are used extensively in studies of plant population genetics and systematics. In the majority of angiosperms, the chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) are each primarily transmitted maternally, but rare biparental transmission is possible. The extent to which the cpDNA and mtDNA are in linkage disequilibrium is argued to be dependent on the fidelity of co-transmission and the population structure. This study reports complete linkage disequilibrium between cpDNA and mtDNA haplotypes in 86 individuals from 17 populations of Silene vulgaris, a gynodioecious plant species. Phylogenetic analysis of cpDNA and mtDNA haplotypes within 14 individuals supports a hypothesis that the evolutionary histories of the chloroplasts and mitochondria are congruent within S. vulgaris, as might be expected if this association persists for long periods. This provides the first documentation of the evolutionary consequences of long-term associations between chloroplast and mitochondrial genomes within a species. Factors that contribute to the phylogenetic and linkage associations, as well as the potential for intergenomic hitchhiking resulting from selection on genes in one organellar genome are discussed.

Indirect measures of gene flow and migration: FST not equal to 1/(4Nm + 1).

The difficulty of directly measuring gene flow has lead to the common use of indirect measures extrapolated from genetic frequency data. These measures are variants of FST, a standardized measure of the genetic variance among populations, and are used to solve for Nm, the number of migrants successfully entering a population per generation. Unfortunately, the mathematical model underlying this translation makes many biologically unrealistic assumptions; real populations are very likely to violate these assumptions, such that there is often limited quantitative information to be gained about dispersal from using gene frequency data. While studies of genetic structure per se are often worthwhile, and FST is an excellent measure of the extent of this population structure, it is rare that FST can be translated into an accurate estimate of Nm.

Local population structure and sex ratio: evolution in gynodioecious plants.

Although the influence of population structure on evolution has been explored previously in a variety of theoretical studies, there are few examples of specific traits whose fitness is likely to be modified by the local structure. Here we focus on a specific trait, sex expression in gynodioecious plants, and derive a model in which the fitness of females and hermaphrodites is a function of the local sex ratio. By using the concept d genes. As a consequence, when local demes vary in sex ratio, a polymorphism for a cytoplasmic male sterility (CMS) allele can be maintained in the absence of nuclear alleles that restore male function. When of subjective frequencies, it is shown that among-deme variance in the local sex ratio reduces the average fitness of females when pollen availability limits fertility. In contrast, sex ratio variance increases the fitness of hermaphrodites from the perspective of maternally inherited genes and lessens the negative impact of pollen limitation on hermaphrodite fitness when it is measured from the perspective of biparentally inheriterestorer alleles are introduced into the model, polymorphism cannot be maintained simultaneously at both the cytoplasmic and nuclear loci. In that case, the CMS allele spreads to fixation, and the equilibrium frequency of females is an inverse function of the equilibrium frequency of the restorer allele, which increases with increased structure. The results exemplify how population structure can greatly alter the fitness and evolution of a frequency-dependent trait.

The use of chloroplast DNA polymorphism in studies of gene flow in plants.

In many species of plants, the dispersal of genes is mediated by the movement of both seeds and pollen. The relative contributions of seed and pollen movement to total gene flow can be difficult to estimate. Chloroplast DNA (cpDNA) may prove useful for resolving this problem. Over the past several years, studies of numerous species of plants have shown that intraspecific variation in cpDNA is often sufficiently abundant to serve as a marker for studies of gene flow. Recent theoretical models have shown that estimates of population structure based on cpDNA polymorphism should be especially sensitive to the impact of seed movement on gene flow, because cpDNA is often maternally inherited.

Contrasting the distribution of chloroplast DNA and allozyme polymorphism among local populations of Silene alba: implications for studies of gene flow in plants.

The distribution of chloroplast DNA (cp-DNA) length variants was analyzed within and among 10 local populations of Silene alba, a dioecious angiosperm. The populations displayed considerable allele frequency variation, resulting in an estimate of Wright's Fst of 0.67 over a 25 x 25 km portion of the species' range. By contrast, a concurrent analysis of the genetic structure of these same populations based on seven polymorphic allozyme loci yielded an estimate of Fst of 0.13. The two Fst estimates are significantly different from one another when their respective confidence limits are estimated by jackknifing. The results of a breeding study were consistent with maternal inheritance of the cpDNA variants. With maternal inheritance the genetic structure of the cpDNA should reflect seed movement, whereas the genetic structure of the nuclear-encoded allozyme loci should reflect the movement of both seeds and pollen. Comparison of the two markedly different Fst estimates in the context of recent models of the population genetics of organelles suggests that the movement of both seeds and pollen contributes significantly to gene flow.

Genetic consequences of local population extinction and recolonization.

Theoretical results have shown that a pattern of local extinction and recolonization can have significant consequences for the genetic structure of subdivided populations; consequences that are relevant to issues in both evolutionary and conservation biology. The nature of those consequences depends largely on the mode of colony formation. Extinction and recolonization can either increase or decrease the genetic differentiation of local populations and can lead to a loss of the genetic diversity stored in an array of populations. Recent ecological studies of two insect species have revealed population structures resembling, in part, that considered in the models. They serve to illustrate the potential complexity of the processes of extinction and recolonizatiion in nature.

The survival of mutants at very low frequencies in tribolium populations.

Forty population cages, each with 499 adult T. castaneum of the wild-type UPF strain, received a bb female newly mated with UPF males. Half of the immigrants had a Chicago Black genetic background, the other half a UPF background. These conditions simulate, respectively, the fate of a rare, genetically differing immigrant or the fate of a mutation in populations of considerable size. Adults were censused for 11 discrete generations. The semi-dominant autosomal black gene survived in 26 out of 40 cultures by the end of the experiment, demonstrating its selective advantage at these very low frequencies. The gene increased from an initial frequency of 0.002 to 0.055 (at generation 11) in at least one replicate. Although frequency-dependent fitness has been shown for black at higher frequencies, no such dependence could be demonstrated at the low frequencies of this study. The cultures simulating mutations (immigrants with native backgrounds) had a higher average gene frequency, different distribution of gene frequencies across replicates, and a lower extinction rate of black than did the cultures with alien background immigrants. The observations only partially fitted expectation based on a branching process model. The data show a tendency for the persistence of a few heterozygotes in cultures and for a deficiency of cultures that lost the mutant or those with many heterozygotes. The increase in frequency of black cannot be attributed to increased reproductive success of heterozygotes. The advantage of heterozygotes appears due to delayed developmental period as a result of tactile stimulation and probable differential cannibalism among pupae.