Pollen limitation of female reproductive success at fine spatial scale in a gynodioecious and wind-pollinated species, Beta vulgaris ssp. maritima

In sexually polymorphic plants, the spatial distribution of sexes is usually not random. Local variation in phenotype frequencies is expected to affect individual fitness of the different phenotypes. For gynodioecious species, with co-occurrence of hermaphrodites and females, if sexual phenotypes are structured in space and pollen flow is spatially restricted, local pollen availability should vary among patches. Female fitness may thus be low when hermaphrodites are locally rare. To test this hypothesis, we analysed how the reproductive output of females varied among patches within two natural study sites of the gynodioecious wind-pollinated Beta vulgaris ssp. maritima. Plants growing in female-biased areas and experiencing pollen limitation were found to have low fruit and seed sets but did not reallocate resources towards better offspring. Our results show that fine-scale processes influence individual fitness and the evolution of sex ratio in sexually polymorphic plants.     

Effects of fine‐scale genetic structure on male mating success in gynodioecious Beta vulgaris ssp. maritima

Plant mating systems are known to influence population genetic structure because pollen and seed dispersal are often spatially restricted. However, the reciprocal outcomes of population structure on the dynamics of polymorphic mating systems have received little attention. In gynodioecious sea beet (Beta vulgaris ssp. maritima), three sexual types co‐occur: females carrying a cytoplasmic male sterility (CMS) gene, hermaphrodites carrying a non‐CMS cytoplasm and restored hermaphrodites that carry CMS genes and nuclear restorer alleles. This study investigated the effects of fine‐scale genetic structure on male reproductive success of the two hermaphroditic forms. Our study population was strongly structured and characterized by contrasting local sex‐ratios. Pollen flow was constrained over short distances and depended on local plant density. Interestingly, restored hermaphrodites sired significantly more seedlings than non‐CMS hermaphrodites, despite the previous observation that the former produce pollen of lower quality than the latter. This result was explained by the higher frequency of females in the local vicinity of restored (CMS) hermaphrodites as compared to non‐CMS hermaphrodites. Population structure thus strongly influences individual fitness and may locally counteract the expected effects of selection, suggesting that understanding fine scale population processes is central to predicting the evolution of gender polymorphism in angiosperms.     

Disentangling the causes of heterogeneity in male fecundity in gynodioecious Beta vulgaris ssp. maritima

Variation among individuals in reproductive success is advocated as a major process driving evolution of sexual polymorphisms in plants, such as gynodioecy where females and hermaphrodites coexist. In gynodioecious Beta vulgaris ssp. maritima, sex determination involves cytoplasmic male sterility (CMS) genes and nuclear restorers of male fertility. Both restored CMS and non-CMS hermaphrodites co-occur. Genotype-specific differences in male fitness are theoretically expected to explain the maintenance of cytonuclear polymorphism. Using genotypic information on seedlings and flowering plants within two metapopulations, we investigated whether male fecundity was influenced by ecological, phenotypic and genetic factors, while taking into account the shape and scale of pollen dispersal. Along with spatially restricted pollen flow, we showed that male fecundity was affected by flowering synchrony, investment in reproduction, pollen production and cytoplasmic identity of potential fathers. Siring success of non-CMS hermaphrodites was higher than that of restored CMS hermaphrodites. However, the magnitude of the difference in fecundity depended on the likelihood of carrying restorer alleles for non-CMS hermaphrodites. Our results suggest the occurrence of a cost of silent restorers, a condition supported by scarce empirical evidence, but theoretically required to maintain a stable sexual polymorphism in gynodioecious species.     

Gynodioecy in structured populations: understanding fine-scale sex ratio variation in Beta vulgaris ssp. maritima

Natural selection, random processes and gene flow are known to generate sex ratio variations among sexually polymorphic plant populations. In gynodioecious species, in which hermaphrodites and females coexist, the relative effect of these processes on the maintenance of sex polymorphism is still up for debate. The aim of this study was to document sex ratio and cytonuclear genetic variation at a very local scale in wind-pollinated gynodioecious Beta vulgaris ssp. maritima and attempt to elucidate which processes explained the observed variation. The study sites were characterized by geographically distinct patches of individuals and appeared to be dynamic entities, with recurrent establishment of distinct haplotypes through independent founder events. Along with substantial variation in sex ratio and unexpectedly low gene flow within study sites, our results showed a high genetic differentiation among a mosaic of genetically distinct demes, with isolation by distance or abrupt genetic discontinuities taking place within a few tens of metres. Overall, random founder events with restricted gene flow could be primary determinants of sex structure, by promoting the clumping of sex-determining genes. Such high levels of sex structure provide a landscape for differential selection acting on sex-determining genes, which could modify the conditions of maintenance of gynodioecy in structured populations.     

Emergence of gynodioecy in wild beet (Beta vulgaris ssp. maritima L.): a genealogical approach using chloroplastic nucleotide sequences

Gynodioecy is a breeding system where both hermaphroditic and female individuals coexist within plant populations. This dimorphism is the result of a genomic interaction between maternally inherited cytoplasmic male sterility (CMS) genes and bi-parentally inherited nuclear male fertility restorers. As opposed to other gynodioecious species, where every cytoplasm seems to be associated with male sterility, wild beet Beta vulgaris ssp. maritima exhibits a minority of sterilizing cytoplasms among numerous non-sterilizing ones. Many studies on population genetics have explored the molecular diversity of different CMS cytoplasms, but questions remain concerning their evolutionary dynamics. In this paper we report one of the first investigations on phylogenetic relationships between CMS and non-CMS lineages. We investigated the phylogenetic relationships between 35 individuals exhibiting different mitochondrial haplotypes. Relying on the high linkage disequilibrium between chloroplastic and mitochondrial genomes, we chose to analyse the nucleotide sequence diversity of three chloroplastic fragments (trnK intron, trnD-trnT and trnL-trnF intergenic spacers). Nucleotide diversity appeared to be low, suggesting a recent bottleneck during the evolutionary history of B. vulgaris ssp. maritima. Statistical parsimony analyses revealed a star-like genealogy and showed that sterilizing haplotypes all belong to different lineages derived from an ancestral non-sterilizing cytoplasm. These results suggest a rapid evolution of male sterility in this taxon. The emergence of gynodioecy in wild beet is confronted with theoretical expectations, describing either gynodioecy dynamics as the maintenance of CMS factors through balancing selection or as a constant turnover of new CMSs.     

Different CMS sources found in Beta vulgaris ssp maritima: mitochondrial variability in wild populations revealed by a rapid screening procedure

Summary Mitochondrial DNA (mtDNA) variation in natural Beta maritima populations has been characterized by way of Southern blot hybridizations of total DNA using non-radioactive probes and chemiluminescent detection. It was found that the previously described N (normal) mitochondrial type could be subdivided into three subtypes. A new mitochondrial genotype (type R) was distinguished in addition to the previously described type S. Both are male-sterile cytoplasms and can produce a. segregation of sexual phenotypes in their progenies depending on the nuclear background. The populations contained at least two to four different mitochondrial genotypes.     

Crop-weed interactions in the Beta vulgaris complex at a local scale: allelic diversity and gene flow within sugar beet fields

Crop-wild hybrids and weed beets are the main source of agronomic concern for sugar beet production all over Europe. In order to understand the dynamics of crop-wild interactions and the evolution of weediness in Beta vulgaris, we investigated genetic features of bolting individuals occurring at a local scale, i.e. within two sugar beet fields of the French northern area of sugar beet production. By analysing ploidy level, mitochondrial DNA and microsatellite polymorphism, the genetic diversity and the genetic relationships among three different classes of individuals (variety, in-row and out-row weed-beets) from a given field were examined. Such genetic analyses provide a unique opportunity to obtain evidence for the weeds origin and the evolutionary hypotheses previously stated. All the individuals shared in common the Svulg mitochondrial haplotype, and thus a common maternal origin. Conversely, the large genetic diversity at microsatellite loci highlighted the large diversity of the pollinator plants (cultivated and wild plants) during the-seed production process, as well as during the further evolution of weed beets in the sugar production area. Received: 23 April 2001 / Accepted: 15 June 2001     

Reproductive capacities in the sexual forms of the gynodioecious species Thymus vulgaris L.

The reproductive capacities of the females (male-steriles or mS) of Thymus vulgaris L. have been investigated, in terms of size and weight of plants, seed production, germination ability and pollination. The data, collected during nine years under controlled conditions and in natural situations are discussed in comparison with the results known in the literature. It is suggested that the large diversity of characteristics promoting sexual polymorphism could justify the high and variable rates of mS met in natural conditions in T. vulgaris.     

Cytoplasmic male sterility in hybrids of sterile wild beet (Beta vulgaris ssp. maritima) and O-type fertile sugar beet (Beta vulgaris L.): molecular analysis of mitochondrial and nuclear genomes

The organization of the mitochondrial genome of B3, B4 and B5generations of hybrids created by backcrossing sterile wild beet Betamaritima with a fertile O-type sugar beet line was studied usingrestriction fragment length polymorphism (RFLP) analysis. Random amplifiedpolymorphic DNA (RAPD) analysis was used to study restoration of the fertile(O-type) sugar beet genotype in hybrids after multiple backcrossings.Restriction of mtDNAs from the cytoplasm of B. maritimaandhybrids revealed BamHI, EcoRI andXhoI restriction patterns different from those for sterileand fertile sugar beet lines. The most conspicuous feature of our accession ofsterile wild beet mtDNA was the absence of the 10.7-kbEcoRI fragment detected in the cytoplasm of S-type sterileB. maritima and sugar beet. The hybridization of digestedmtDNAs with coxII, atpA andatp6 homologous probes revealed alterations within thesegene loci that distinguished wild beet and hybrids from sugar beets.Characteristic hybridization profiles for the wild beet and B3, B4 and B5hybrids were observed for all probes regardless of the restrictase used todigest mtDNA. Notable changes in atpA andatp6 genes resulted when probes that comprised the5flanking sequences of these genes and a small part of the coding sequences wereused. RFLP analysis of the sterile B. maritimamitochondrial genome further supported the unique character of this source ofwild beet sterility. The genotypic differences between hybrids and parentalaccessions were determined by scoring PCR-RAPD reaction products for nineselected primers. The diversity of the B. maritimagenotyperesulted in a lower genetic similarity index in comparison with hybrids,sterileand fertile lines of sugar beet. The dendrogram obtained after cluster analysisdistinguished hybrids as a group that differed from wild beet and themaintainersugar beet line used for backcrossing. These results may indicate incompleterestoration of the fertile sugar beet genotype in hybrids.     

Identification and mapping of RAPD and RFLP markers linked to a fertility restorer gene for a new source of cytoplasmic male sterility in Beta vulgaris ssp. maritima

 The present study shows that the recently described mitochondrial H haplotype is associated with cytoplasmic male-sterility (CMS). This new source of CMS appears to be different from the mitotype E-associated CMS most frequently found in natural populations. A mitotype H progeny with a sexual phenotype segregation was used to identify a gene restoring male fertility (R1H ). Using bulk segregant analysis (BSA), nine RAPD markers linked to this restorer locus were detected and mapped. The comparison with other Beta genetic maps shows that the closest RAPD marker, distant from R1H by 5.2 cM, belongs to the same linkage group as the monogermy locus. In order to determine the position of R1H more precisely, four RFLP loci within this linkage group were mapped in the segregating progeny. It thus became possible to construct a linkage map of the region containing the RFLP, RAPD and R1H loci. The closest RFLP marker was located 1.7 cM away from R1H. However, a nuclear gene restoring the ‘Owen’ CMS which is currently used in sugar beet breeding is reportedly linked to the monogermy locus, raising the question of a possible identity between the new CMS system and the ‘Owen’ CMS. Received: 15 September 1997 / Accepted: 1 December 1997     

The linkage disequilibrium between chloroplast DNA and mitochondrial DNA haplotypes in Beta vulgaris ssp. maritima (L.): the usefulness of both genomes for population genetic studies

The structure and evolution of the plant mitochondrial genome may allow recurrent appearance of the same mitochondrial variants in different populations. Whether the same mitochondrial variant is distributed by migration or appears recurrently by mutation (creating homoplasy) in different populations is an important question with regard to the use of these markers for population genetic analyses. The genetic association observed between chloroplasts and mitochondria (i.e. two maternally inherited cytoplasmic genomes) may indicate whether or not homoplasy occurs in the mitochondrial genome. Four-hundred and fourteen individuals sampled in wild populations of beets from France and Spain were screened for their mitochondrial and chloroplast polymorphisms. Mitochondrial DNA (mtDNA) polymorphism was investigated with restriction fragment length polymorphism (RFLP) and chloroplast DNA (cpDNA) polymorphism was investigated with polymerase chain reaction PCR-RFLP, using universal primers for the amplification. Twenty and 13 variants for mtDNA and cpDNA were observed, respectively. Most exhibited a widespread geographical distribution. As a very strong linkage disequilibrium was estimated between mtDNA and cpDNA haplotypes, a high rate of recurrent mutation was excluded for the mitochondrial genome of beets. Identical mitochondrial variants found in populations of different regions probably occurred as a result of migration. We concluded from this study that mtDNA is a tool as valuable as cpDNA when a maternal marker is needed for population genetics analyses in beet on a large regional scale.     

Abundance and length polymorphism of microsatellite repeats in Beta vulgaris L.

Simple sequence repeats (SSRs) are known to exhibit high degrees of variability even among closely related individuals. Their usage as nuclear genetic markers requires their conversion into sequence-tagged sites (STSs). In this paper we present the development of simple sequences as STSs for Beta vulgaris. This species comprises wild, cultivated, and weedy forms; the latter are thought to originate from accidental hybridisation between the other two. Two partial genomic libraries were screened with simple sequence motifs (AT, CA, CT, ATT, GTG, and CA, CT, respectively). Clones of 22 CA, nine CT, eight ATT, and one GTG sequence were obtained. AT micro satellites were present in compound motifs, not recognised by the probe. Sequence comparisons revealed that 20 CA clones containing short motifs (<16 bp) were variants of a previously described approximately 320-bp satellite DNA (Schmidt et al. 1991), and hence did not correspond to unique loci. Polymorphism of one (ATT)₁₅ and three (CT)_n, with n=15, 17 and 26, was detected by PCR on a sample of 64 plants from the different forms of B. vulgaris. 13 (ATT), 13 (CT), nine (CT) alleles and one (CT) allele were detected. One of the ATT alleles was much larger than the others (>800 bp). Genetic variability was high among wild beets, lower among cultivated beets, and intermediate among weed beets. One allele of each locus was found at high frequencies in cultivated beets and, to a lower extent, in weed beets. The combination of three polymorphic loci allowed the individual identification of 17/17 wild and 15/15 weed beets, and 21/32, mostly homozygous, cultivated beets.     

The Activation of Cinnamate by an Enzyme from Leaves of Spinach Beet (Beta vulgaris L. ssp. vulgaris)

The preparation of an extract from leaves of spinach beet (Betavulgaris L. ssp. vulgaris), which catalysed, the activationof cinnamate, is described. The reaction required CoA and wasmost rapid under nitrogen in the presence of thiol reagents.An enzyme which activated acetate was also present but was distinguishableby its indifference to thiol reagents and air during the reactionand its greater stability during storage under air. Extractscatalysing cinnamate activation could be prepared only fromrapidly growing leaves at the height of the growth season.     

Spatial structure of nuclear factors involved in sex determination in the gynodioecious Thymus vulgaris L.

Theoretical models have shown that the maintenance of high frequencies of females in gynodioecious species can be explained by the spatial structure of cytoplasmic and nuclear genes involved in sex determination. Whereas spatial structure of cytoplasmic factors which cause male-sterility has been studied in several taxa, that of nuclear factors that restore male-fertility (restoration factors) has received little attention. In this paper, we estimate spatial variation in the frequency of restoration factors associated with different cytoplasmic male-sterilities in the gynodioecious Thymus vulgaris. Clonal replicates of five female plants bearing at least four different male-sterilities and originating from five different populations (i.e., five cytogenotypes), were reciprocally transplanted into the original populations. Following open pollination at each site, seeds were harvested, germinated and grown to flowering. The frequency of hermaphrodites in each progeny was used to estimate the frequency of restoration factors in each population. For all cytogenotypes, there was marked variation in the rate of restoration among populations, indicating that spatial structure in nuclear restoration factors may influence the variation in female frequency in T. vulgaris. For three out of the five cytogenotypes, higher restoration rates were obtained for females introduced into their original population, which agrees with the theoretical prediction, under some hypotheses, that restoration factors are selected for in populations that contain the associated male-sterility (Gouyon and Couvet, 1985). The very low restoration rate in the progeny of one female clone in three populations indicates that stochastic events, such as founder effects, could lead to a local absence of restoration factors and thus the high female frequency (up to 90%) observed in some populations. Differences among cytogenotypes suggest that some male-sterilities are associated with rarer restoration factors and are thus more likely to cause patches of females to develop in young populations.     

Genetic variation for sexual dimorphism in flower size within and between populations of gynodioecious Thymus vulgaris

There has been very little empirical study of quantitative genetic variation in flower size in sexually dimorphic plant species, despite the frequent occurrence of flower size differences between sexual phenotypes. In this study we quantify the nature of quantitative flower size variation in females and hermaphrodites of gynodioecious Thymus vulgaris. In a field study, females had significantly smaller flowers than hermaphrodites, and the degree of flower size dimorphism varied significantly among populations. To quantify the genetic basis of flower size variation we sampled maternal progeny from 10 F₀ females in three populations (across the range of variation in flower size in the field), performed controlled crosses on F₁ offspring in the glasshouse and grew F₂ progeny to flowering in uniform field conditions. A significant population * sex interaction was again observed, hence the degree of sexual dimorphism shows genetic variation among populations. A significant family * sex interaction was also observed, indicating that the degree of sexual dimorphism shows genetic variation among families. Females showed significantly greater variation among populations and among families than hermaphrodites. Female flower size varied significantly depending on the degree of stamen abortion, with morphologically intermediate females having flowers more similar to hermaphrodites than to other females. The frequency of female types that differ in the degree of stamen abortion varied among populations and families and mean family female flower size increased as the proportion of intermediate female types increased across families. Variation in the degree of flower size dimorphism thus appears to be a result of variation in the degree of stamen abortion in females, the potential causes of which are discussed.     

Founder effects and sex ratio in the gynodioecious Thymus vulgaris L.

Thymus vulgaris is a gynodioecious species (in which females and hermaphrodites coexist) with a highly variable frequency of females among natural populations (5–95%) and a high average female frequency (60%). Sex determination involves both cytoplasmic genes responsible for male sterility, i.e. the female phenotype, and specific nuclear factors responsible for the restoration of male fertility, and thus a hermaphrodite phenotype. In this study, molecular markers of the mitochondrial genome have been used to quantify the cytoplasmic diversity in 11 clumps of individuals observed in four recently founded populations. The very low diversity within patches in conjunction with the strong diversity among patches strongly suggests that clumps of individuals are the result of single matrilinear families. In clumps that contain mainly females, all the analysed females showed the same cytoplasmic pattern. This pattern differed from that shown by neighbouring hermaphrodites, indicating that the determination of sex is locally cytoplasmic. A comparison of genetic diversity before and after fire in one population showed that disturbances may cause a reduction in genetic diversity and a concurrent induction of local cytoplasmic determination of sex. Such cytoplasmic determination of sex in colonizing populations, together with the greater seed set of females, may largely improve the colonizing ability of the species.     

The effects of local spatial structure on epidemiological invasions.

Predicting the likely success of invasions is vitally important in ecology and especially epidemiology. Whether an organism can successfully invade and persist in the short-term is highly dependent on the spatial correlations that develop in the early stages of invasion. By modelling the correlations between individuals, we are able to understand the role of spatial heterogeneity in invasion dynamics without the need for large-scale computer simulations. Here, a natural methodology is developed for modelling the behaviour of individuals in a fixed network. This formulation is applied to the spread of a disease through a structured network to determine invasion thresholds and some statistical properties of a single epidemic.     

Fine scale spatial structuring of sex and mitochondria in Silene vulgaris

Fine scale spatial structure (FSSS) of cytoplasmic genes in plants is thought to be generated via founder events and can be amplified when seeds germinate close to their mother. In gynodioecious species these processes are expected to generate FSSS in sex ratio because maternally inherited cytoplasmic male sterility genes partially influence sex expression. Here we document a striking example of FSSS in both mitochondrial genetic markers and sex in roadside populations of Silene vulgaris. We show that in one population FSSS of sexes influences relative fruit production of females compared to hermaphrodites. Furthermore, FSSS in sex ratio is expected to persist into future generations because offspring sex ratios from females are female-biased whereas offspring sex ratios from hermaphrodites are hermaphrodite-biased. Earlier studies indicated that pollen limitation is the most likely mechanism underlying negative frequency dependent fitness of females. Our results support the theoretical predictions that FSSS in sex ratio can reduce female fitness by decreasing the frequency at which females experience hermaphrodites. We argue that the influence of FSSS on female fitness is complementary to the influence of larger scale population structure on female fitness, and that population structure at both scales will act to decrease female frequencies in gynodioecious species. Better comprehension of the spatial structure of genders and genes controlling sex expression at a local scale is required for future progress toward understanding sex ratio evolution in gynodioecious plants.