Male fertility advantage within and between seasons in the perennial androdioecious plant Phillyrea angustifolia.

BACKGROUND AND AIMS: Androdioecy, the co-occurrence of males and hermaphrodites, is a rare reproductive system. Males can be maintained if they benefit from a higher male fitness than hermaphrodites, referred to as male advantage. Male advantage can emerge from increased fertility owing to resource reallocation. However, empirical studies usually compare sexual phenotypes over a single flowering season, thus ignoring potential cumulative effects over successive seasons in perennials. In this study, we quantify various components of male fertility advantage, both within and between seasons, in the long-lived perennial shrub Phillyrea angustifolia (Oleaceae). Although, owing to a peculiar diallelic self-incompatibility system and female sterility mutation strictly associated with a breakdown of incompatibility, males do not need fertility advantage to persist in this species, this advantage remains an important determinant of their equilibrium frequency. METHODS: A survey of >1000 full-sib plants allowed us to compare males and hermaphrodites for several components of male fertility. Individuals were characterized for proxies of pollen production and vegetative growth. By analysing maternal progeny, we compared the siring success of males and hermaphrodites. Finally, using a multistate capture-recapture model we assessed, for each sexual morph, how the intensity of flowering in one year impacts next-year growth and reproduction. KEY RESULTS: Males benefitted from a greater vegetative growth and flowering intensity. Within one season, males sired twice as many seeds as equidistant, compatible hermaphroditic competitors. In addition, males more often maintained intense flowering over successive years. Finally, investment in male reproductive function appeared to differ between the two incompatibility groups of hermaphrodites. CONCLUSION: Males, by sparing the cost of female reproduction, have a higher flowering frequency and vegetative growth, both of which contribute to male advantage over an individual lifetime. This suggests that studies analysing sexual phenotypes during only single reproductive periods are likely to provide inadequate estimates of male advantage in perennials.

A one-locus model of androdioecy with two homomorphic self-incompatibility groups: expected vs. observed male frequencies.

Androdioecy, the occurrence of males and hermaphrodites in a single population, is a rare breeding system because the conditions for maintenance of males are restrictive. In the androdioecious shrub Phillyrea angustifolia, high male frequencies are observed in some populations. The species has a sporophytic self-incompatibility (SI) system with two self-incompatibility groups, which ensures that two groups of hermaphrodites can each mate only with the other group, whereas males can fertilize hermaphrodites of both groups. Here, we analyse a population genetic model to investigate the dynamics of such an androdioecious species, assuming that self-incompatibility and sex phenotypes are determined by a single locus. Our model confirms a previous prediction that a slight reproductive advantage of males relative to hermaphrodites allows the maintenance of males at high equilibrium frequencies. The model predicts different equilibria between hermaphrodites of the two SI groups and males, depending on the male advantage, the initial composition of the population and the population size, whose effect is studied through stochastic simulations. Although the model can generate high male frequencies, observed frequencies are considerably higher than the model predicts. We finally discuss how this model may help explain the large male frequency variation observed in other androdioecious species of Oleaceae: some species show only androdioecious populations, as P. angustifolia, whereas others show populations either completely hermaphrodite or androdioecious.

Structural and content diversity of mitochondrial genome in beet: a comparative genomic analysis.

Despite their monophyletic origin, mitochondrial (mt) genomes of plants and animals have developed contrasted evolutionary paths over time. Animal mt genomes are generally small, compact, and exhibit high mutation rates, whereas plant mt genomes exhibit low mutation rates, little compactness, larger sizes, and highly rearranged structures. We present the (nearly) whole sequences of five new mt genomes in the Beta genus: four from Beta vulgaris and one from B. macrocarpa, a sister species belonging to the same Beta section. We pooled our results with two previously sequenced genomes of B. vulgaris and studied genome diversity at the species level with an emphasis on cytoplasmic male-sterilizing (CMS) genomes. We showed that, contrary to what was previously assumed, all three CMS genomes belong to a single sterile lineage. In addition, the CMSs seem to have undergone an acceleration of the rates of substitution and rearrangement. This study suggests that male sterility emergence might have been favored by faster rates of evolution, unless CMS itself caused faster evolution.

Effect of balancing selection on spatial genetic structure within populations: theoretical investigations on the self-incompatibility locus and empirical studies in Arabidopsis halleri.

The effect of selection on patterns of genetic structure within and between populations may be studied by contrasting observed patterns at the genes targeted by selection with those of unlinked neutral marker loci. Local directional selection on target genes will produce stronger population genetic structure than at neutral loci, whereas the reverse is expected for balancing selection. However, theoretical predictions on the intensity of this signal under precise models of balancing selection are still lacking. Using negative frequency-dependent selection acting on self-incompatibility systems in plants as a model of balancing selection, we investigated the effect of such selection on patterns of spatial genetic structure within a continuous population. Using numerical simulations, we tested the effect of the type of self-incompatibility system, the number of alleles at the self-incompatibility locus and the dominance interactions among them, the extent of gene dispersal, and the immigration rate on spatial genetic structure at the selected locus and at unlinked neutral loci. We confirm that frequency-dependent selection is expected to reduce the extent of spatial genetic structure as compared to neutral loci, particularly in situations with low number of alleles at the self-incompatibility locus, high frequency of codominant interactions among alleles, restricted gene dispersal and restricted immigration from outside populations. Hence the signature of selection on spatial genetic structure is expected to vary across species and populations, and we show that empirical data from the literature as well as data reported here on three natural populations of the herb Arabidopsis halleri confirm these theoretical results.

Detection of gene-anchored amplification polymorphism (GAAP) in the vicinity of plant mitochondrial genes.

A simple, semi-automatable method was established for assessing polymorphism in plant mitochondrial genome. A set of 41 mitochondrial markers based on the published Arabidopsis thaliana sequence was developed in Brassicaceae using a gene-anchored amplification polymorphism (GAAP) strategy. PCR primers were selected based on conserved coding regions of mitochondrial genes and used to amplify the corresponding 5' and/or 3' non-coding flanking regions in order to maximise sequence variability between haplotypes. The variations in fragment size were analysed on a LiCor DNA sequencer, but the methodology is compatible with various sequencing systems using denaturing polyacrylamide gels. One advantage of the method is that GAAP products can be directly sequenced (without any cloning steps) through labelled M13 consensus sequences. Mitochondrial GAAP loci gave clear and simple patterns (one or two bands) that were easy to score and highly reproducible. Nearly all mitochondrial loci examined in A. thaliana were conserved within the Brassicaceae family, and half of the primers generated products when DNA from a distant species, Beta vulgaris (Chenopodiaceae), was used as template. The GAAP markers revealed low levels of polymorphism within species but exhibited a high level of polymorphism among genera and families. Our results showed some discrepancies with respect to the published mtDNA sequence of A. thaliana.

A model for the evolution of high frequencies of males in an androdioecious plant based on a cross-compatibility advantage of males.

Lloyd's (1975) and Charlesworth & Charlesworth's (1978) phenotypic selection models for the maintenance of androdioecy predict that males (female-sterile individuals) must have an advantage in fertility (K) of at least two in order to invade a hermaphroditic population, and that their equilibrium frequency (x(eq)=(K - 2)/2(K - 1)) is always less than 0.5. In this paper, we develop a model in which male fertility is frequency-dependent, a situation not investigated in the previous models, to explore the conditions under which a high frequency of males (i.e. more than 50%) could be maintained at equilibrium. We demonstrate that a gametophytic self-incompatibility (GSI) locus linked to a nuclear sex determination locus can favour rare alleles through male function, by causing frequency-dependent selection. Thus, the spread of a female-sterility allele in a hermaphroditic population may be induced. In contrast with the previous models, our model can explain male frequencies greater than 50% in a functionally androdioecious species, as long as there is (i) dominance of female-sterility at the sex locus, and (ii) a few alleles at the self-incompatibility locus, even if the advantage in fertility of male phenotype is lower than two.

Chloroplast DNA haplotype variation and population differentiation in Sorbus aucuparia L. (Rosaceae: Maloideae).

Intra-specific chloroplast DNA (cpDNA) variation was studied in Sorbus aucuparia L., an entomophilous, mid-or early successional tree producing fleshy fruits. Eight PCR-amplified fragments of the chloroplast genome were screened for restriction fragment length polymorphisms, using one or two 4 bp-cutter restriction endonucleases. cpDNA variation was investigated on two geographical scales: (1) among four regions in France and Belgium; and (2) within the Belgian region. A total of 150 individuals from six populations were analysed. Fourteen polymorphisms were detected in six of the cpDNA fragments. All polymorphisms probably resulted from insertions or deletions, and allowed the identification of 12 haplotypes. The level of genetic differentiation computed on the basis of haplotype frequencies was similar on the two geographical scales considered (G(STc) = 0.286 among regions, G(STc) = 0.259 among populations within the Belgian region). These values are much lower than those obtained in nine previously studied temperate tree species, which are all wind-pollinated, late-successional species producing dry fruits. These results might primarily be accounted for by the contrasting life history traits of S. aucuparia. In order to obtain insights into the relative contribution of pollen and seeds to gene flow, G(STc) was also compared with previously obtained G(ST) estimates based on allozyme data.

Self-Incompatibility and Male Fertilization Success in Phillyrea angustifolia (Oleaceae).

Androdioecy is a rare breeding system in which low male frequency is expected in populations because males require a strong increase in their fertility to be maintained by selection. Phillyrea angustifolia L. has previously been reported as possibly functionally androdioecious. However, 1&rcolon;1 sex ratios have been reported and suggest functional dioecy. In this article, we compared both pollen tube growth and siring success of male and hermaphrodite pollen in two single-donor pollination experiments. We verified at both pre- and postzygotic levels that hermaphrodites produce functional pollen. Self-incompatibility was also clearly established. However, pollen from hermaphrodites was less efficient than male pollen. The probability of a pollen tube growing through the style was higher for male than for hermaphrodite pollen donors, and males sired twice as many fruits as hermaphrodites. The twofold male advantage in relative fecundity was mainly because of lower pollen fertility of hermaphrodites and possible cross-incompatibility among hermaphrodites.

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.

Zinc tolerance and accumulation in metallicolous and nonmetallicolous populations of Arabidopsis halleri (Brassicaceae).

Zinc tolerance was investigated in five populations of Arabidopsis halleri (syn.: Cardaminopsis halleri) raised from seeds collected from contaminated and uncontaminated sites. Tolerance was measured by determining the concentration which inhibited root growth (EC100 ). A. halleri populations from contaminated and uncontaminated sites were found to be Zn-tolerant compared with the Zn-nontolerant species Arabidopsis thaliana and A. lyrata subsp. petraea. At very high Zn concentrations, populations of A. halleri from uncontaminated sites were slightly less Zn-tolerant than those from contaminated sites. These observations support the hypothesis that in A. halleri, Zn tolerance is largely a constitutive property. One population from an uncontaminated site and one population from a contaminated site were studied for Zn uptake. Zinc content was measured in shoots and roots using a colorimetric test under laboratory conditions. The results showed that whatever their origin, individuals from both populations are Zn accumulators compared with the nonaccumulator species A. thaliana. Moreover, the population from the uncontaminated area accumulated Zn in its shoots and roots more quickly than the population from the contaminated site. These results suggest that, in A. halleri, Zn accumulation to very high concentration is a constitutive property.

Zinc tolerance and hyperaccumulation are genetically independent characters.

The hyperaccumulation of metals by a rare class of plants is a fascinating and little understood phenomenon. No genetic analysis has been possible since no intraspecific variation is known for this character. Here, we report on crosses between the zinc-hyperaccumulating and -tolerant species Arabidopsis halleri and the non-hyperaccumulating, non-tolerant species Arabidopsis petraea. The F2 segregates for both characters and it appears that the two characters are genetically independent. The data for tolerance are consistent with a single major gene for this character (although the number of genes for hyperaccumulation cannot be determined), and is probably not very large.

Direct and indirect estimates of the selfing rate in small and large individuals of the bumblebee pollinated Cynoglossum officinale L (Boraginaceae).

We measured the relationship between selfing rates and flower number in an experimental population of bumblebee pollinated Cynoglossum officinale, with plants differing in flower number. Results were compared with the prediction of a model based on pollen dynamics and pollinator behaviour. The selfing rate, as measured by multilocus oligonucleotide DNA fingerprinting, increased with flower number and ranged from 0% to 70%. Flowers on large plants received an equal number of visits from bumblebees as flowers on small plants. On large plants more flowers in a row were visited, inducing geitonogamy. The overall relationship between selfing rate and number of flowers can be explained by pollen dynamics and pollinator behaviour without invoking postpollination processes such as differential pollen tube growth and abortion.

Ligand binding was acquired during evolution of nuclear receptors.

The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organisms. The analysis of the NR gene duplication pattern during the evolution of metazoans shows that the present NR diversity arose from two waves of gene duplications. Strikingly, our results suggest that the ancestral NR was an orphan receptor that acquired ligand-binding ability during subsequent evolution.

The spatial genetic structure of cytoplasmic (cpDNA) and nuclear (allozyme) markers within and among populations of the gynodioecious Thymus vulgaris (Labiatae) in southern France.

Recent advances in molecular biology have allowed the development of techniques to contrast spatial differentiation in nuclear and cytoplasmic genes and thus provide important data on relative levels of gene flow by pollen and seed in higher plants. In this paper, we compare the spatial structure of nuclear (allozymes) and cytoplasmic (cpDNA) genes among populations of the gynodioecious Thymus vulgaris in southern France. Based on a combination of three restriction enzymes (CfoI, EcoRV, and PstI), eight chlorotypes (combination of three restriction enzyme patterns revealed by Southern hybridization of Beta vulgaris cpDNA) were identified in the 13 studied populations. One chlorotype was particularly abundant and was detected in nearly all populations. Only one chlorotype was specific to a single population. Up to four different chlorotypes were observed in some populations. An FST of 0.238 (P < 0.002) for cpDNA haplotypes indicates spatial structure of cytoplasmic genes among the studied populations. Similar patterns were found within a single young population (CAB) structured in patches and surrounded by a continuous cover of T. vulgaris where the FST is 0.546 (P < 0.002). No significant correlation between sex and chlorotype nor between cpDNA diversity and female frequency was detected. Allozyme markers showed markedly less spatial structure (FST = 0.021 among populations and 0.019 in the CAB population, P < 0.001). This difference between cpDNA and nuclear allozyme markers suggests that pollen dispersal is more important than seed dispersal both among and within populations.

Genotypic diversity revealed by allozymes and oligonucleotide DNA fingerprinting in French populations of the aquatic macrophyte Sparganium erectum.

The vast majority of perennial plants reproduce sexually and vegetatively at the same time. This may lead to important variation among clonal plant populations in their amount of genotypic diversity. In order to verify this assumption, we compare the clonal diversity of 10 natural populations of the aquatic clonal macrophyte Sparganium erectum in France. Diversity was quantified by DNA fingerprinting and allozyme electrophoresis for a sample of 10 shoots per population. Two DNA probes (CA)8 and (TAA)6TA, were selected among 10 synthetic oligonucleotide probes containing simple repeat motifs. Both allozymes and DNA fingerprints revealed different amounts of diversity among populations. Five populations consist of a single genotype, whereas two populations were genetically highly diverse. In four of the monomorphic populations, absence of fingerprints diversity was combined with uniformly heterozygous allozyme loci, suggesting that each population was composed of a single clone. In the highly diverse populations, the level of clonal diversity combined with the allele segregation of the two allozyme loci Lap and Est suggests frequent seedling recruitment. The origin of new genotypes remains unclear but the absence of widespread genotypes together with the discrete distribution of Sparganium erectum populations implies that new genotypes are locally produced through sexual reproduction.

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.