Widespread coexistence of self-compatible and self-incompatible phenotypes in a diallelic self-incompatibility system in Ligustrum vulgare (Oleaceae).

The breakdown of self-incompatibility (SI) in angiosperms is one of the most commonly observed evolutionary transitions. While multiple examples of SI breakdown have been documented in natural populations, there is strikingly little evidence of stable within-population polymorphism with both inbreeding (self-compatible) and outcrossing (self-incompatible) individuals. This absence of breeding system polymorphism corroborates theoretical expectations that predict that in/outbreeding polymorphism is possible only under very restricted conditions. However, theory also predicts that a diallelic sporophytic SI system should facilitate the maintenance of such polymorphism. We tested this prediction by studying the breeding system of Ligustrum vulgare L., an insect-pollinated hermaphroditic species of the Oleaceae family. Using stigma tests with controlled pollination and paternity assignment of open-pollinated progenies, we confirmed the existence of two self-incompatibility groups in this species. We also demonstrated the occurrence of self-compatible individuals in different populations of Western Europe arising from a mutation affecting the functioning of the pollen component of SI. Our results show that the observed low frequency of self-compatible individuals in natural populations is compatible with theoretical predictions only if inbreeding depression is very high.

Polygamy or subdioecy? The impact of diallelic self-incompatibility on the sexual system in Fraxinus excelsior (Oleaceae).

How flowering plants have recurrently evolved from hermaphroditism to separate sexes (dioecy) is a central question in evolutionary biology. Here, we investigate whether diallelic self-incompatibility (DSI) is associated with sexual specialization in the polygamous common ash (Fraxinus excelsior), which would ultimately facilitate the evolution towards dioecy. Using interspecific crosses, we provide evidence of strong relationships between the DSI system and sexual phenotype. The reproductive system in F. excelsior that was previously viewed as polygamy (co-occurrence of unisexuals and hermaphrodites with varying degrees of allocation to the male and female functions) and thus appears to actually behave as a subdioecious system. Hermaphrodites and females belong to one SI group and functionally reproduce as females, whereas males and male-biased hermaphrodites belong to the other SI group and are functionally males. Our results offer an alternative mechanism for the evolution of sexual specialization in flowering plants.

Evolutionary dynamics of quantitative variation in an adaptive trait at the regional scale: The case of zinc hyperaccumulation in Arabidopsis halleri.

Metal hyperaccumulation in plants is an ecological trait whose biological significance remains debated, in particular because the selective pressures that govern its evolutionary dynamics are complex. One of the possible causes of quantitative variation in hyperaccumulation may be local adaptation to metalliferous soils. Here, we explored the population genetic structure of Arabidopsis halleri at fourteen metalliferous and nonmetalliferous sampling sites in southern Poland. The results were integrated with a quantitative assessment of variation in zinc hyperaccumulation to trace local adaptation. We identified a clear hierarchical structure with two distinct genetic groups at the upper level of clustering. Interestingly, these groups corresponded to different geographic subregions, rather than to ecological types (i.e., metallicolous vs. nonmetallicolous). Also, approximate Bayesian computation analyses suggested that the current distribution of A. halleri in southern Poland could be relictual as a result of habitat fragmentation caused by climatic shifts during the Holocene, rather than due to recent colonization of industrially polluted sites. In addition, we find evidence that some nonmetallicolous lowland populations may have actually derived from metallicolous populations. Meanwhile, the distribution of quantitative variation in zinc hyperaccumulation did separate metallicolous and nonmetallicolous accessions, indicating more recent adaptive evolution and diversifying selection between metalliferous and nonmetalliferous habitats. This suggests that zinc hyperaccumulation evolves both ways-towards higher levels at nonmetalliferous sites and lower levels at metalliferous sites. Our results open a new perspective on possible evolutionary relationships between A. halleri edaphic types that may inspire future genetic studies of quantitative variation in metal hyperaccumulation.

Evidence for the long-term maintenance of a rare self-incompatibility system in Oleaceae.

A rare homomorphic diallelic self-incompatibility (DSI) system discovered in Phillyrea angustifolia (family Oleaceae, subtribe Oleinae) can promote the transition from hermaphroditism to androdioecy. If widespread and stable in Oleaceae, DSI may explain the exceptionally high rate of androdioecious species reported in this plant family. Here, we set out to determine whether DSI occurs in another Oleaceae lineage. We tested for DSI in subtribe Fraxininae, a lineage that diverged from subtribe Oleinae c. 40 million yr ago. We explored the compatibility relationships in Fraxinus ornus using 81 hermaphrodites and 25 males from one natural stand and two naturalized populations using intra- and interspecific stigma tests performed on F. ornus and P. angustifolia testers. We uncovered a DSI system with hermaphrodites belonging to one of two self-incompatibility (SI) groups and males compatible with both groups, making for a truly androdioecious reproductive system. The two human-founded populations contained only one of the two SI groups. Our results provide evidence for the evolutionary persistence of DSI. We discuss how its stability over time may have affected transitions to other sexual systems, such as dioecy.

CATION EXCHANGER1 Cosegregates with Cadmium Tolerance in the Metal Hyperaccumulator Arabidopsis halleri and Plays a Role in Limiting Oxidative Stress in Arabidopsis Spp.

Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium(2+)/hydrogen(+) antiporter, cation/hydrogen(+) exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants.

Evidence of various mechanisms of Cd sequestration in the hyperaccumulator Arabidopsis halleri, the non-accumulator Arabidopsis lyrata, and their progenies by combined synchrotron-based techniques.

Arabidopsis halleri is a model plant for Zn and Cd hyperaccumulation. The objective of this study was to determine the relationship between the chemical forms of Cd, its distribution in leaves, and Cd accumulation and tolerance. An interspecific cross was carried out between A. halleri and the non-tolerant and non-hyperaccumulating relative A. lyrata providing progenies segregating for Cd tolerance and accumulation. Cd speciation and distribution were investigated using X-ray absorption spectroscopy and microfocused X-ray fluorescence. In A. lyrata and non-tolerant progenies, Cd was coordinated by S atoms only or with a small contribution of O groups. Interestingly, the proportion of O ligands increased in A. halleri and tolerant progenies, and they were predominant in most of them, while S ligands were still present. Therefore, the binding of Cd with O ligands was associated with Cd tolerance. In A. halleri, Cd was mainly located in the xylem, phloem, and mesophyll tissue, suggesting a reallocation process for Cd within the plant. The distribution of the metal at the cell level was further discussed. In A. lyrata, the vascular bundles were also Cd enriched, but the epidermis was richer in Cd as compared with the mesophyll. Cd was identified in trichomes of both species. This work demonstrated that both Cd speciation and localization were related to the tolerance character of the plant.

The joint evolution and maintenance of self-incompatibility with gynodioecy or androdioecy.

Mating systems show two kinds of frequent transitions: from hermaphroditism to dioecy, gynodioecy or androdioecy, or from self-incompatibility (SI) to self-compatibility (SC). While models have mostly investigated these two kinds of transitions as independent, empirical observations suggest that, to some extent, they can evolve jointly. Here, we study the joint evolution and maintenance of SI and androdioecy or SI and gynodioecy by the means of phenotypic models. Our models focus on three parameters: the unisexuals׳ advantage relative to that of the hermaphrodites due to resource reallocation, inbreeding depression and the selfing rate. We assume no pollen limitation or discounting. We show that SI helps the maintenance of androdioecy, but favors the loss of gynodioecy, and also that androdioecy facilitates the maintenance of SI, whereas gynodioecy does not affect it. We finally investigate how gynodioecy and androdioecy may affect the diversification of SI groups, especially considering an evolutionary pathway through SC intermediates. We show that while androdioecy prevents the increase of the number of SI groups, under certain conditions of inbreeding depression and selfing rates, gynodioecy allows it.

The homomorphic self-incompatibility system in Oleaceae is controlled by a hemizygous genomic region expressing a gibberellin pathway gene.

In flowering plants, outcrossing is commonly ensured by self-incompatibility (SI) systems. These can be homomorphic (typically with many different allelic specificities) or can accompany flower heteromorphism (mostly with just two specificities and corresponding floral types). The SI system of the Oleaceae family is unusual, with the long-term maintenance of only two specificities but often without flower morphology differences. To elucidate the genomic architecture and molecular basis of this SI system, we obtained chromosome-scale genome assemblies of Phillyrea angustifolia individuals and related them to a genetic map. The S-locus region proved to have a segregating 543-kb indel unique to one specificity, suggesting a hemizygous region, as observed in all distylous systems so far studied at the genomic level. Only one of the predicted genes in this indel region is found in the olive tree, Olea europaea, genome, also within a segregating indel. We describe complete association between the presence/absence of this gene and the SI types determined for individuals of seven distantly related Oleaceae species. This gene is predicted to be involved in catabolism of the gibberellic acid (GA) hormone, and experimental manipulation of GA levels in developing buds modified the male and female SI responses of the two specificities in different ways. Our results provide a unique example of a homomorphic SI system, where a single conserved gibberellin-related gene in a hemizygous indel underlies the long-term maintenance of two groups of reproductive compatibility.

Plant Defensin type 1 (PDF1): protein promiscuity and expression variation within the Arabidopsis genus shed light on zinc tolerance acquisition in Arabidopsis halleri.

Plant defensins are recognized for their antifungal properties. However, a few type 1 defensins (PDF1s) were identified for their cellular zinc (Zn) tolerance properties after a study of the metal extremophile Arabidopsis halleri. In order to investigate whether different paralogues would display specialized functions, the A. halleri PDF1 family was characterized at the functional and genomic levels. Eleven PDF1s were isolated from A. halleri. Their ability to provide Zn tolerance in yeast cells, their activity against Fusarium oxysporum f. sp. melonii, and their level of expression in planta were compared with those of the seven A. thaliana PDF1s. The genomic organization of the PDF1 family was comparatively analysed within the Arabidopsis genus. AhPDF1s and AtPDF1s were able to confer Zn tolerance and AhPDF1s also displayed antifungal activity. PDF1 transcripts were constitutively more abundant in A. halleri than in A. thaliana. Within the Arabidopsis genus, the PDF1 family is evolutionarily dynamic, in terms of gain and loss of gene copy. Arabidopsis halleri PDF1s display no superior abilities to provide Zn tolerance. A constitutive increase in AhPDF1 transcript accumulation is proposed to be an evolutionary innovation co-opting the promiscuous PDF1 protein for its contribution to Zn tolerance in A. halleri.

The five AhMTP1 zinc transporters undergo different evolutionary fates towards adaptive evolution to zinc tolerance in Arabidopsis halleri.

Gene duplication is a major mechanism facilitating adaptation to changing environments. From recent genomic analyses, the acquisition of zinc hypertolerance and hyperaccumulation characters discriminating Arabidopsis halleri from its zinc sensitive/non-accumulator closest relatives Arabidopsis lyrata and Arabidopsis thaliana was proposed to rely on duplication of genes controlling zinc transport or zinc tolerance. Metal Tolerance Protein 1 (MTP1) is one of these genes. It encodes a Zn(2+)/H(+) antiporter involved in cytoplasmic zinc detoxification and thus in zinc tolerance. MTP1 was proposed to be triplicated in A. halleri, while it is present in single copy in A. thaliana and A. lyrata. Two of the three AhMTP1 paralogues were shown to co-segregate with zinc tolerance in a BC1 progeny from a cross between A. halleri and A. lyrata. In this work, the MTP1 family was characterized at both the genomic and functional levels in A. halleri. Five MTP1 paralogues were found to be present in A. halleri, AhMTP1-A1, -A2, -B, -C, and -D. Interestingly, one of the two newly identified AhMTP1 paralogues was not fixed at least in one A. halleri population. All MTP1s were expressed, but transcript accumulation of the paralogues co-segregating with zinc tolerance in the A. halleri X A. lyrata BC1 progeny was markedly higher than that of the other paralogues. All MTP1s displayed the ability to functionally complement a Saccharomyces cerevisiae zinc hypersensitive mutant. However, the paralogue showing the least complementation of the yeast mutant phenotype was one of the paralogues co-segregating with zinc tolerance. From our results, the hypothesis that pentaplication of MTP1 could be a major basis of the zinc tolerance character in A. halleri is strongly counter-balanced by the fact that members of the MTP1 family are likely to experience different evolutionary fates, some of which not concurring to increase zinc tolerance.

Nuclear and chloroplast DNA phylogeography reveals vicariance among European populations of the model species for the study of metal tolerance, Arabidopsis halleri (Brassicaceae).

Arabidopsis halleri is a pseudometallophyte involved in numerous molecular studies of the adaptation to anthropogenic metal stress. In order to test the representativeness of genetic accessions commonly used in these studies, we investigated the A. halleri population genetic structure in Europe. Microsatellite and nucleotide polymorphisms from the nuclear and chloroplast genomes, respectively, were used to genotype 65 populations scattered over Europe. The large-scale population structure was characterized by a significant phylogeographic signal between two major genetic units. The localization of the phylogeographic break was assumed to result from vicariance between large populations isolated in southern and central Europe, on either side of ice sheets covering the Alps during the Quaternary ice ages. Genetic isolation was shown to be maintained in western Europe by the high summits of the Alps, whereas admixture was detected in the Carpathians. Considering the phylogeographic literature, our results suggest a distinct phylogeographic pattern for European species occurring in both mountain and lowland habitats. Considering the evolution of metal adaptation in A. halleri, it appears that recent adaptations to anthropogenic metal stress that have occurred within either phylogeographic unit should be regarded as independent events that potentially have involved the evolution of a variety of genetic mechanisms.

Nuclear microsatellite loci for Arabidopsis halleri (Brassicaceae), a model species to study plant adaptation to heavy metals.

PREMISE OF THE STUDY: Arabidopsis halleri is a model species to study the adaptation of plants to soils contaminated by zinc, cadmium, and lead. To provide a neutral genetic background with which adaptive genetic markers could be compared, we developed highly polymorphic neutral microsatellite markers. METHODS AND RESULTS: Using a microsatellite-enriched library method, we identified 120 microsatellite loci for quantitative trait locus (QTL) mapping analysis, of which eight primer pairs were developed in a single multiplex for population genetic studies. Analyses were performed on 508 individuals from 26 populations. All loci were polymorphic with six to 23 alleles per locus. Genetic diversity varied between 0.56 and 0.76. CONCLUSIONS: Our results demonstrated the value of these eight microsatellite markers to investigate neutral population genetic structure in A. halleri. To increase the resolution of population genetic analyses, we suggest adding them to the 11 markers previously developed independently.

Diallelic self-incompatibility is the main determinant of fertilization patterns in olive orchards.

Self-incompatibility (SI) in flowering plants potentially represents a major obstacle for sexual reproduction, especially when the number of S-alleles is low. The situation is extreme in the commercially important olive tree, where in vitro pollination assays suggested the existence of a diallelic SI (DSI) system involving only two groups (G1 and G2). Varieties belonging to the same SI group cannot fertilize each other, such that successful fruit production is predicted to require pollination between varieties of different groups. To test this prediction, we explored the extent to which the DSI system determines fertilization patterns under field conditions. One hundred and seventeen olive cultivars were first genotyped using 10 highly polymorphic dinucleotide Simple Sequence Repeat (SSR) markers to ascertain varietal identity. Cultivars were then phenotyped through controlled pollination tests to assign each of them to one of the two SI groups. We then collected and genotyped 1440 open pollinated embryos from five different orchards constituted of seven local cultivars with known group of incompatibility groups. Embryos genotype information were used: (i) to assign embryos to the most likely pollen donor genotype in the neighbourhood using paternity analysis, and (ii) to compare the composition of the pollen cloud genetic among recipient trees in the five sites. The paternity analysis showed that the DSI system is the main determinant of fertilization success under field open pollination conditions: G1 cultivars sired seeds exclusively on G2 cultivars, and reciprocally. No self-fertilization events were observed. Our results demonstrate that DSI is a potent force determining pollination success among varieties within olive orchards used for production. They have the potential to improve management practices by guiding the selection of compatible varieties to avoid planting orchards containing sets of varieties with strongly unbalanced SI groups, as these would lead to suboptimal olive production.

When population genetics serves genomics: putting adaptation back in a spatial and historical context.

Recent advances in molecular biology have opened new perspectives for the study of plant adaptation, especially at the intraspecific level. Nowadays, scientists employing -omic results in multiple scientific fields can be optimistic of their chances of revealing mechanisms involved in adaptive population divergence. However, the investment required by integrative studies greatly reduces the number of experiments that can be performed. In this context, a comprehensive choice of accessions under study is crucial. We maintain this choice could be appreciably enlightened by population genetics because it helps putting adaptive population divergence in a spatial and historical context. As an example, we highlight the usefulness of knowledge about population genetic structure in the integrative study of metal tolerance in Arabidopsis halleri.

Quantitative trait loci analysis of mineral element concentrations in an Arabidopsis halleri x Arabidopsis lyrata petraea F2 progeny grown on cadmium-contaminated soil.

SUMMARY: This study describes the quantitative trait locus (QTL) analysis of cadmium (Cd), zinc (Zn), iron (Fe), potassium (K), magnesium (Mg) and calcium (Ca) accumulation in the pseudometallophyte Arabidopsis halleri under conditions of Cd excess using an interspecific A. halleri x Arabidopsis lyrata F(2) population. *Our data provide evidence for the implication of one major QTL in Cd hyperaccumulation in A. halleri, and suggests that Cd tolerance and accumulation are not independent in A. halleri. Moreover, the major loci responsible for Zn hyperaccumulation in the absence of Cd appear to be the same when Cd is present at high concentrations. *More than twofold higher Fe concentrations were measured in A. halleri shoots than in A. lyrata, suggesting a different regulation of Fe accumulation in the hyperaccumulator. *With the exception of Ca, the accumulation of Cd was significantly correlated with the accumulation of all elements measured in the F(2) progeny, suggesting pleiotropic gene action. However, QTL analysis identified pleiotropic QTLs only for Cd, Zn and Fe. Mg accumulation was negatively correlated with Cd accumulation, as well as with dry shoot biomass, suggesting that it might indicate cellular damage.

Variability of zinc tolerance among and within populations of the pseudometallophyte species Arabidopsis halleri and possible role of directional selection.

We estimated the level of quantitative polymorphism for zinc (Zn) tolerance in neighboring metallicolous and nonmetallicolous populations of Arabidopsis halleri and tested the hypothesis that divergent selection has shaped this polymorphism. A short-term hydroponic test was used to capture the quantitative polymorphism present between edaphic types, among and within populations. We measured six morphological and physiological traits on shoots and roots to estimate the response of A. halleri to Zn. In order to assess the adaptive value of Zn tolerance polymorphism, we compared differentiation of quantitative traits with that of molecular markers. Zinc tolerance of metallicolous populations was, on average, higher than that of nonmetallicolous populations according to the morphological and physiological traits measured. Phenotypic variability within edaphic types was very high and mainly explained by polymorphism among individuals within populations. Genetic differentiation for photosystem II yield of leaves (a measure of photosynthetic efficiency) was greater than the differentiation for microsatellite and thus, probably shaped by divergent selection. Overall, these results suggest that, in the sampled populations, Zn tolerance has been increased in metallicolous populations through selection on standing genetic variation within local nonmetallicolous ancestral populations.

Genetic architecture of zinc hyperaccumulation in Arabidopsis halleri: the essential role of QTL x environment interactions.

SUMMARY: This study sought to determine the main genomic regions that control zinc (Zn) hyperaccumulation in Arabidopsis halleri and to examine genotype x environment effects on phenotypic variance. To do so, quantitative trait loci (QTLs) were mapped using an interspecific A. halleri x Arabidopsis lyrata petraea F(2) population. *The F(2) progeny as well as representatives of the parental populations were cultivated on soils at two different Zn concentrations. A linkage map was constructed using 70 markers. *In both low and high pollution treatments, zinc hyperaccumulation showed high broad-sense heritability (81.9 and 74.7%, respectively). Five significant QTLs were detected: two QTLs specific to the low pollution treatment (chromosomes 1 and 4), and three QTLs identified at both treatments (chromosomes 3, 6 and 7). These QTLs explained 50.1 and 36.5% of the phenotypic variance in low and high pollution treatments, respectively. Two QTLs identified at both treatments (chromosomes 3 and 6) showed significant QTL x environment interactions. *The QTL on chromosome 3 largely colocalized with a major QTL previously identified for Zn and cadmium (Cd) tolerance. This suggests that Zn tolerance and hyperaccumulation share, at least partially, a common genetic basis and may have simultaneously evolved on heavy metal-contaminated soils.

Using Arabidopsis to explore zinc tolerance and hyperaccumulation.

Identifying the particular gene or genes underlying a specific adaptation is a major challenge in modern biology. Currently, the study of naturally occurring variation in Arabidopsis thaliana provides a bridge between functional genetics and evolutionary analyses. Nevertheless, the use of A. thaliana to study adaptation is limited to those traits that have undergone selection. Therefore, to understand fully the genetics of adaptation, the vast arsenal of genetic resources developed in A. thaliana must be extended to other species that display traits absent in this model species. Here, we discuss how A. thaliana resources can significantly enhance the study of heavy-metal tolerance and hyperaccumulation in the wild species Arabidopsis halleri.

Zinc distribution and speciation in Arabidopsis halleri x Arabidopsis lyrata progenies presenting various zinc accumulation capacities.

The purpose of this study was to investigate the relationship between the chemical form and localization of zinc (Zn) in plant leaves and their Zn accumulation capacity. An interspecific cross between Arabidopsis halleri sp. halleri and Arabidopsis lyrata sp. petrea segregating for Zn accumulation was used. Zinc (Zn) speciation and Zn distribution in the leaves of the parent plants and of selected F(1) and F(2) progenies were investigated by spectroscopic and microscopic techniques and chemical analyses. A correlation was observed between the proportion of Zn being in octahedral coordination complexed to organic acids and free in solution (Zn-OAs + Zn(aq)) and Zn content in the leaves. This pool varied between 40% and 80% of total leaf Zn depending on the plant studied. Elemental mapping of the leaves revealed different Zn partitioning between the veins and the leaf tissue. The vein : tissue fluorescence ratio was negatively correlated with Zn accumulation. The higher proportion of Zn-OAs + Zn(aq) and the depletion of the veins in the stronger accumulators are attributed to a higher xylem unloading and vacuolar sequestration in the leaf cells. Elemental distributions in the trichomes were also investigated, and results support the role of carboxyl and/or hydroxyl groups as major Zn ligands in these cells.

Genomic pattern of adaptive divergence in Arabidopsis halleri, a model species for tolerance to heavy metal.

Pollution by heavy metals is one of the strongest environmental constraints in human-altered environments that only a handful of species can cope with. Identifying the genes conferring to those species the ability to grow in polluted areas is a first step towards a global understanding of the evolutionary processes involved and will eventually improve phytoremediation practices. We used a genome-scan approach to detect loci under divergent selection among four populations of Arabidopsis halleri growing on either polluted or nonpolluted habitats. Based on a high density of amplified fragment length polymorphism (AFLP) markers (820 AFLP markers, i.e. approximately 1 marker per 0.3Mb), evidence for selection was found for some markers in every sampled population. Four loci departed from neutrality in both metallicolous populations and thus constitute high-quality candidates for general adaptation to pollution. Interestingly, some candidates differed between the two metallicolous populations, suggesting the possibility that different loci may be involved in adaptation in the different metallicolous populations.