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.

Genetic diversity assessment of Helichrysum arenarium (Asteraceae) for the genetic restoration of declining populations.

Helichrysum arenarium (L.) Moench (Asteraceae) is a self-compatible, insect-pollinated herb occurring in sand grasslands, and is declining and endangered in many parts of its European distribution range. A recovery plan of H. arenarium has been conducted in southern Belgium, involving plant translocations. We developed multiplex genotyping protocol for nine microsatellite markers previously published for Helichrysum italicum and two newly developed microsatellite markers for H. arenarium. Eleven polymorphic loci were associated (pooled) in two multiplex panels, to assess the genetic status of the only small remaining population in Belgium and of three large German populations used as seed source for propagating transplants. The small Belgian population was characterized by high clonality, with only two, however heterozygous, genets detected. The three large German populations showed high genetic diversity (H e ranging from 0.635 to 0.670) and no significant inbreeding coefficient values, despite expectations of geitonogamous selfing. Management practices (grazing livestock) increasing seed dispersal distances, inbreeding depression at early stages of development, and mechanisms preventing or delaying selfing might be hypothesized to explain the observed patterns. The two Belgian genotypes remained within genetic variation range of German populations so that the high genetic differentiation between Belgian and German populations (F ST values ranging from 0.186 to 0.206) likely resulted from genetic drift effects and small sample size. Transplants obtained from seeds sampled from the three large source populations from Germany constitute a highly diverse, noninbred gene pool, and are thus of high genetic quality for plant translocations.

Paternal leakage of plastids rescues inter-lineage hybrids in Silene nutans.

BACKGROUND AND AIMS: Organelle genomes are usually maternally inherited in angiosperms. However, biparental inheritance has been observed, especially in hybrids resulting from crosses between divergent genetic lineages. When it concerns the plastid genome, this exceptional mode of inheritance might rescue inter-lineage hybrids suffering from plastid-nuclear incompatibilities. Genetically differentiated lineages of Silene nutans exhibit strong postzygotic isolation owing to plastid-nuclear incompatibilities, highlighted by inter-lineage hybrid chlorosis and mortality. Surviving hybrids can exhibit variegated leaves, which might indicate paternal leakage of the plastid genome. We tested whether the surviving hybrids inherited the paternal plastid genome and survived thanks to paternal leakage. METHODS: We characterized the leaf phenotype (fully green, variegated or white) of 504 surviving inter-lineage hybrids obtained from a reciprocal cross experiment among populations of four genetic lineages (W1, W2, W3 and E1) of S. nutans from Western Europe and genotyped 560 leaf samples (both green and white leaves for variegated hybrids) using six lineage-specific plastid single nucleotide polymorphisms. KEY RESULTS: A high proportion of the surviving hybrids (≤98 %) inherited the paternal plastid genome, indicating paternal leakage. The level of paternal leakage depended on cross type and cross direction. The E1 and W2 lineages as maternal lineages led to the highest hybrid mortality and to the highest paternal leakage from W1 and W3 lineages in the few surviving hybrids. This was consistent with E1 and W2 lineages, which contained the most divergent plastid genomes. When W3 was the mother, more hybrids survived, and no paternal leakage was detected. CONCLUSIONS: By providing a plastid genome potentially more compatible with the hybrid nuclear background, paternal leakage has the potential to rescue inter-lineage hybrids from plastid-nuclear incompatibilities. This phenomenon might slow down the speciation process, provided hybrid survival and reproduction can occur in the wild.

The decoupled evolution of the organellar genomes of Silene nutans leads to distinct roles in the speciation process.

There is growing evidence that cytonuclear incompatibilities (i.e. disruption of cytonuclear coadaptation) might contribute to the speciation process. In a former study, we described the possible involvement of plastid-nuclear incompatibilities in the reproductive isolation between four lineages of Silene nutans (Caryophyllaceae). Because organellar genomes are usually cotransmitted, we assessed whether the mitochondrial genome could also be involved in the speciation process, knowing that the gynodioecious breeding system of S. nutans is expected to impact the evolutionary dynamics of this genome. Using hybrid capture and high-throughput DNA sequencing, we analyzed diversity patterns in the genic content of the organellar genomes in the four S. nutans lineages. Contrary to the plastid genome, which exhibited a large number of fixed substitutions between lineages, extensive sharing of polymorphisms between lineages was found in the mitochondrial genome. In addition, numerous recombination-like events were detected in the mitochondrial genome, loosening the linkage disequilibrium between the organellar genomes and leading to decoupled evolution. These results suggest that gynodioecy shaped mitochondrial diversity through balancing selection, maintaining ancestral polymorphism and, thus, limiting the involvement of the mitochondrial genome in evolution of hybrid inviability between S. nutans lineages.

Target enrichment of long open reading frames and ultraconserved elements to link microevolution and macroevolution in non-model organisms.

Despite the increasing accessibility of high-throughput sequencing, obtaining high-quality genomic data on non-model organisms without proximate well-assembled and annotated genomes remains challenging. Here, we describe a workflow that takes advantage of distant genomic resources and ingroup transcriptomes to select and jointly enrich long open reading frames (ORFs) and ultraconserved elements (UCEs) from genomic samples for integrative studies of microevolutionary and macroevolutionary dynamics. This workflow is applied to samples of the African unionid bivalve tribe Coelaturini (Parreysiinae) at basin and continent-wide scales. Our results indicate that ORFs are efficiently captured without prior identification of intron-exon boundaries. The enrichment of UCEs was less successful, but nevertheless produced substantial data sets. Exploratory continent-wide phylogenetic analyses with ORF supercontigs (>515,000 parsimony informative sites) resulted in a fully resolved phylogeny, the backbone of which was also retrieved with UCEs (>11,000 informative sites). Variant calling on ORFs and UCEs of Coelaturini from the Malawi Basin produced ~2000 SNPs per population pair. Estimates of nucleotide diversity and population differentiation were similar for ORFs and UCEs. They were low compared to previous estimates in molluscs, but comparable to those in recently diversifying Malawi cichlids and other taxa at an early stage of speciation. Skimming off-target sequence data from the same enriched libraries of Coelaturini from the Malawi Basin, we reconstructed the maternally-inherited mitogenome, which displays the gene order inferred for the most recent common ancestor of Unionidae. Overall, our workflow and results provide exciting perspectives for integrative genomic studies of microevolutionary and macroevolutionary dynamics in non-model organisms.

On the function of flower number: disentangling fertility from pollinator-mediated selection.

In animal-pollinated angiosperms, the 'male-function' hypothesis claims that male reproductive success (RS) should benefit from large floral displays, through pollinator attraction, while female RS is expected to be mainly limited by resource availability. As appealing as this theory might be, studies comparing selection strength on flower number in both sexes rarely document the expected asymmetry. This discrepancy could arise because flower number impacts both pollinator attraction and overall gamete number. In this study, we artificially manipulate floral displays to disentangle the fertility versus pollinator attraction components of selection, both in terms of mating and RS. In females, flower number was under strong fertility selection, as predicted in the absence of pollen limitation. By contrast, in males, flower number was mainly under sexual selection, which in turn increased male RS. However, these selection patterns were not different in males with artificially increased floral displays. This suggests that sexual selection acting on flower number in males does not occur because flower number increases pollinator attraction, but rather because more pollen is available to disperse on more mates. Our study illustrates the power of disentangling various components of selection with potentially sex-specific effects for understanding the evolution of sexual dimorphism.

Reproductive isolation among lineages of Silene nutans (Caryophyllaceae): A potential involvement of plastid-nuclear incompatibilities.

Early stages of speciation in plants might involve genetic incompatibilities between plastid and nuclear genomes, leading to inter-lineage hybrid breakdown due to the disruption between co-adapted plastid and nuclear genes encoding subunits of the same plastid protein complexes. We tested this hypothesis in Silene nutans, a gynodioecious Caryophyllaceae, where four distinct genetic lineages exhibited strong reproductive isolation among each other, resulting in chlorotic or variegated hybrids. By sequencing the whole gene content of the four plastomes through gene capture, and a large part of the nuclear genes encoding plastid subunits from RNAseq data, we searched for non-synonymous substitutions fixed in each lineage on both genomes. Lineages of S. nutans exhibited a high level of dN/dS ratios for plastid and nuclear genes encoding most plastid complexes, with a strong pattern of coevolution for genes encoding the subunits of ribosome and cytochrome b6/f that could explain the chlorosis of hybrids. Overall, relaxation of selection due to past bottlenecks and positive selection have driven the diversity pattern observed in S. nutans plastid complexes, leading to plastid-nuclear incompatibilities. We discuss the possible role of gynodioecy in the evolutionary dynamics of the plastomes through linked selection.

Development of highly polymorphic microsatellite markers for Campanula glomerata L. (Campanulaceae).

BACKGROUND: Campanula glomerata L. (Campanulaceae) is a self-incompatible, insect-pollinated herb occurring in calcareous grasslands, and is declining and (critically) endangered in many parts of its European distribution range. It often exists as small and isolated populations. A recovery plan of C. glomerata has been implemented in southern Belgium, involving plant translocations. METHODS AND RESULTS: We developed microsatellite markers using an enriched genomic library and characterized 16 loci in 111 individuals from eight populations. These 16 loci were highly polymorphic, with 11 to 31 alleles per locus for a total of 329 alleles, and expected heterozygosity (He) ranging from 0.470 to 0.938. CONCLUSIONS: These highly polymorphic loci constitute a promising tool for detailed genetic analyses: assigning individuals to distinct multilocus genotypes will allow quantifying pollen dispersal, clonal propagation and sexual recruitment and identifying admixed seed progeny and their pollen donors. Evaluating the genetic status of existing populations and a genetic monitoring of the translocated populations will contribute to optimize success in restoring viable and evolutionary resilient populations.

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.

Congruent population genetic structures and divergence histories in anther-smut fungi and their host plants Silene italica and the Silene nutans species complex.

Study of the congruence of population genetic structure between hosts and pathogens gives important insights into their shared phylogeographical and coevolutionary histories. We studied the population genetic structure of castrating anther-smut fungi (genus Microbotryum) and of their host plants, the Silene nutans species complex, and the morphologically and genetically closely related Silene italica, which can be found in sympatry. Phylogeographical population genetic structure related to persistence in separate glacial refugia has been recently revealed in the S. nutans plant species complex across Western Europe, identifying several distinct lineages. We genotyped 171 associated plant-pathogen pairs of anther-smut fungi and their host plant individuals using microsatellite markers and plant chloroplastic single nucleotide polymorphisms. We found clear differentiation between fungal populations parasitizing S. nutans and S. italica plants. The population genetic structure of fungal strains parasitizing the S. nutans plant species complex mirrored the host plant genetic structure, suggesting that the pathogen was isolated in glacial refugia together with its host and/or that it has specialized on the plant genetic lineages. Using random forest approximate Bayesian computation (ABC-RF), we found that the divergence history of the fungal lineages on S. nutans was congruent with that previously inferred for the host plant and probably occurred with ancient but no recent gene flow. Genome sequences confirmed the genetic structure and the absence of recent gene flow between fungal genetic lineages. Our analyses of individual host-pathogen pairs contribute to a better understanding of co-evolutionary histories between hosts and pathogens in natural ecosystems, in which such studies remain scarce.

Effects of contemporary shifts of range margins on patterns of genetic structure and mating system in two coastal plant species.

Species' geographical ranges are often restricted due to niche limitation resulting in geographical isolation and reduced population size at range margins. Under the "abundant center" paradigm, static marginal populations are thus expected to show higher genetic differentiation and lower genetic diversity than core populations. Low mate availability may also drive shifts toward higher propensity for selfing in geographically marginal populations. However, these predictions remain to be validated for contemporary range shifts occurring under current environmental change. This study is devoted to bridging this gap and assesses the spatial patterns of genetic structure and mating system across the geographical range of two coastal plant species characterized by contrasting contemporary range dynamics: the receding myrmecochorous Dune pansy (Viola tricolor subsp. curtisii) and the widespread expanding hydrochorous Rock samphire (Crithmum maritimum). Both species exhibited high propensity for selfing, with indications of inbreeding depression acting at early life stages. In Dune pansy, a biogeographical break was observed between core and marginal populations, with trailing-edge populations showing higher levels of genetic differentiation, reduced genetic diversity, and higher levels of selfing estimated through progeny arrays. In contrast, genetic structuring was weak in Rock samphire and no clear spatial trends were observed in genetic diversity nor in mating system, likely the result of efficient long-distance seed dispersal by sea-surface currents. Our study highlights that key species differences in life-history traits related to dispersal and/or mate limitation modify the expectations of genetic diversity loss and mating system shift in contemporary range-expanding populations, as compared with historical core populations.

Evolution of Young Sex Chromosomes in Two Dioecious Sister Plant Species with Distinct Sex Determination Systems.

In the last decade, progress has been made in methods to identify the sex determination system in plants. This gives the opportunity to study sex chromosomes that arose independently at different phylogenetic scales, and thus allows the discovery and the understanding of early stages of sex chromosome evolution. In the genus Silene, sex chromosomes have evolved independently in at least two clades from a nondioecious ancestor, the Melandrium and Otites sections. In the latter, sex chromosomes could be younger than in the section Melandrium, based on phylogenetic studies and as no heteromorphic sex chromosomes have been detected. This section might also exhibit lability in sex determination, because male heterogamy and female heterogamy have been suggested to occur.In this study, we investigated the sex determination system of two dioecious species in the section Otites (Silene otites and its close relative Silene pseudotites). Applying the new probabilistic method SEX-DETector on RNA-seq data from cross-controlled progenies, we inferred their most likely sex determination system and a list of putative autosomal and sex-linked contigs. We showed that the two phylogenetically close species differed in their sex determination system (XY versus ZW) with sex chromosomes that derived from two different pairs of autosomes. We built a genetic map of the sex chromosomes and showed that both pairs exhibited a large region with lack of recombination. However, the sex-limited chromosomes exhibited no strong degeneration. Finally, using the "ancestral" autosomal expression of sex-linked orthologs of nondioecious S. nutans, we found a slight signature of dosage compensation in the heterogametic females of S. otites.

Polymorphic nuclear markers for coastal plant species with dynamic geographic distributions, the rock samphire (Crithmum maritimum) and the vulnerable dune pansy (Viola tricolor subsp. curtisii).

Identifying spatial patterns of genetic differentiation across a species range is critical to set up conservation and restoration decision-making. This is especially timely, since global change triggers shifts in species' geographic distribution and in the geographical variation of mating system and patterns of genetic differentiation, with varying consequences at the trailing and leading edges of a species' distribution. Using 454 pyrosequencing, we developed nuclear microsatellite loci for two plant species showing a strictly coastal geographical distribution and contrasting range dynamics: the expanding rock samphire (Crithmum maritimum, 21 loci) and the highly endangered and receding dune pansy (Viola tricolor subsp. curtisii, 12 loci). Population genetic structure was then assessed by genotyping more than 100 individuals from four populations of each of the two target species. Rock samphire displayed high levels of genetic differentiation (FST = 0.38), and a genetic structure typical of a mostly selfing species (FIS ranging from 0.16 to 0.58). Populations of dune pansy showed a less pronounced level of population structuring (FST = 0.25) and a genotypic structure more suggestive of a mixed-mating system when excluding two loci with heterozygote excess. These results demonstrate that the genetic markers developed here are useful to assess the mating system of populations of these two species. They will be tools of choice to investigate phylogeographical patterns and variation in mating system over the geographical distribution ranges for two coastal plant species that are subject to dynamic evolution due to rapid contemporary global change.

When new human-modified habitats favour the expansion of an amphibian pioneer species: Evolutionary history of the natterjack toad (Bufo calamita) in a coal basin.

Human activities affect microevolutionary dynamics by inducing environmental changes. In particular, land cover conversion and loss of native habitats decrease genetic diversity and jeopardize the adaptive ability of populations. Nonetheless, new anthropogenic habitats can also promote the successful establishment of emblematic pioneer species. We investigated this issue by examining the population genetic features and evolutionary history of the natterjack toad (Bufo [Epidalea] calamita) in northern France, where populations can be found in native coastal habitats and coalfield habitats shaped by European industrial history, along with an additional set of European populations located outside this focal area. We predicted contrasting patterns of genetic structure, with newly settled coalfield populations departing from migration-drift equilibrium. As expected, coalfield populations showed a mosaic of genetically divergent populations with short-range patterns of gene flow, and native coastal populations indicated an equilibrium state with an isolation-by-distance pattern suggestive of postglacial range expansion. However, coalfield populations exhibited (i) high levels of genetic diversity, (ii) no evidence of local inbreeding or reduced effective population size and (iii) multiple maternal mitochondrial lineages, a genetic footprint depicting independent colonization events. Furthermore, approximate Bayesian computations suggested several evolutionary trajectories from ancient isolation in glacial refugia during the Pleistocene, with biogeographical signatures of recent expansion probably confounded by human-mediated mixing of different lineages. From an evolutionary and conservation perspective, this study highlights the ecological value of industrial areas, provided that ongoing regional gene flow is ensured within the existing lineage boundaries.

Lineages of Silene nutans developed rapid, strong, asymmetric postzygotic reproductive isolation in allopatry.

Reproductive isolation can rise either as a consequence of genomic divergence in allopatry or as a byproduct of divergent selection in parapatry. To determine whether reproductive isolation in gynodioecious Silene nutans results from allopatric divergence or from ecological adaptation following secondary contact, we investigated the pattern of postzygotic reproductive isolation and hybridization in natural populations using two phylogeographic lineages, western (W1) and eastern (E1). Experimental crosses between the lineages identified strong, asymmetric postzygotic isolation between the W1 and the E1 lineages, independent of geographic overlap. The proportion of ovules fertilized, seeds aborted, and seeds germinated revealed relatively little effect on the fitness of hybrids. In contrast, hybrid mortality was high and asymmetric: while half of the hybrid seedlings with western lineage mothers died, nearly all hybrid seedlings with E1 mothers died. This asymmetric mortality mirrored the proportion of chlorotic seedlings, and is congruent with cytonuclear incompatibility. We found no evidence of hybridization between the lineages in regions of co-occurrence using nuclear and plastid markers. Together, our results are consistent with the hypothesis that strong postzygotic reproductive isolation involving cytonuclear incompatibilities arose in allopatry. We argue that the dynamics of cytonuclear gynodioecy could facilitate the evolution of reproductive isolation.

Demographic history of the trace metal hyperaccumulator Noccaea caerulescens (J. Presl and C. Presl) F. K. Mey. in Western Europe.

Noccaea caerulescens (Brassicaceae) is a major pseudometallophyte model for the investigation of the genetics and evolution of metal hyperaccumulation in plants. We studied the population genetics and demographic history of this species to advance the understanding of among-population differences in metal hyperaccumulation and tolerance abilities. Sampling of seven to 30 plants was carried out in 62 sites in Western Europe. Genotyping was carried out using a combination of new chloroplast and nuclear neutral markers. A strong genetic structure was detected, allowing the definition of three genetic subunits. Subunits showed a good geographic coherence. Accordingly, distant metallicolous populations generally belonged to distinct subunits. Approximate Bayesian computation analysis of demographic scenarios among subunits further supported a primary isolation of populations from the southern Massif Central prior to last glacial maximum, whereas northern populations may have derived during postglacial recolonization events. Estimated divergence times among subunits were rather recent in comparison with the species history, but certainly before the establishment of anthropogenic metalliferous sites. Our results suggest that the large-scale genetic structure of N. caerulescens populations pre-existed to the local adaptation to metalliferous sites. The population structure of quantitative variation for metal-related adaptive traits must have established independently in isolated gene pools. However, features of the most divergent genetic unit (e.g. extreme levels of Cd accumulation observed in previous studies) question the putative relationships between adaptive evolution of metal-related traits and subunits isolation. Finally, admixture signals among distant metallicolous populations suggest a putative role of human activities in facilitating long-distance genetic exchanges.

Development of Nuclear Microsatellite Loci and Mitochondrial Single Nucleotide Polymorphisms for the Natterjack Toad, Bufo (Epidalea) calamita (Bufonidae), Using Next Generation Sequencing and Competitive Allele Specific PCR (KASPar).

Amphibians are undergoing a major decline worldwide and the steady increase in the number of threatened species in this particular taxa highlights the need for conservation genetics studies using high-quality molecular markers. The natterjack toad, Bufo (Epidalea) calamita, is a vulnerable pioneering species confined to specialized habitats in Western Europe. To provide efficient and cost-effective genetic resources for conservation biologists, we developed and characterized 22 new nuclear microsatellite markers using next-generation sequencing. We also used sequence data acquired from Sanger sequencing to develop the first mitochondrial markers for KASPar assay genotyping. Genetic polymorphism was then analyzed for 95 toads sampled from 5 populations in France. For polymorphic microsatellite loci, number of alleles and expected heterozygosity ranged from 2 to 14 and from 0.035 to 0.720, respectively. No significant departures from panmixia were observed (mean multilocus F IS = -0.015) and population differentiation was substantial (mean multilocus F ST = 0.222, P < 0.001). From a set of 18 mitochondrial SNPs located in the 16S and D-loop region, we further developed a fast and cost-effective SNP genotyping method based on competitive allele-specific PCR amplification (KASPar). The combination of allelic states for these mitochondrial DNA SNP markers yielded 10 different haplotypes, ranging from 2 to 5 within populations. Populations were highly differentiated (G ST = 0.407, P < 0.001). These new genetic resources will facilitate future parentage, population genetics and phylogeographical studies and will be useful for both evolutionary and conservation concerns, especially for the set-up of management strategies and the definition of distinct evolutionary significant units.

Potential preadaptation to anthropogenic pollution: evidence from a common quantitative trait locus for zinc and cadmium tolerance in metallicolous and nonmetallicolous accessions of Arabidopsis halleri.

As a drastic environmental change, metal pollution may promote the rapid evolution of genetic adaptations contributing to metal tolerance. In Arabidopsis halleri, genetic bases of zinc (Zn) and cadmium (Cd) tolerance have been uncovered only in a metallicolous accession, although tolerance is species-wide. The genetic determinants of Zn and Cd tolerance in a nonmetallicolous accession were thus investigated for the first time. The genetic architecture of tolerance was investigated in a nonmetallicolous population (SK2) by using first backcross progeny obtained from crosses between SK2 and Arabidopsis lyrata petraea, a nonmetallophyte species. Only one significant and common quantitative trait locus (QTL) region was identified explaining 22.6% and 31.2% of the phenotypic variation for Zn and Cd tolerance, respectively. This QTL co-localized with HEAVY METAL ATPASE 4 (AhHMA4), which was previously validated as a determinant of Zn and Cd tolerance in a metallicolous accession. Triplication and high expression of HMA4 were confirmed in SK2. In contrast, gene duplication and high expression of METAL TOLERANT PROTEIN 1A (MTP1A), which was previously associated with Zn tolerance in a metallicolous accession, were not observed in SK2. Overall, the results support the role of HMA4 in tolerance capacities of A. halleri that may have pre-existed in nonmetallicolous populations before colonization of metal-polluted habitats. Preadaptation to metal-contaminated sites is thus discussed.

Characterization and multiplexing of 21 microsatellite markers for the herb Noccaea caerulescens (Brassicaceae).

PREMISE OF THE STUDY: Multiplexed microsatellite markers were developed for population genetic studies in the pseudometallophyte Noccaea caerulescens (Brassicaceae), a model species to investigate metal tolerance and hyperaccumulation in higher plants. METHODS AND RESULTS: Microsatellite loci were isolated through pyrosequencing of an enriched DNA library. Three multiplexes combining four previously published and 17 newly designed markers were developed. The new markers were screened in metallicolous and nonmetallicolous populations from southern France. The total number of alleles per locus ranged from five to 18. The observed heterozygosity per locus and per population ranged from 0 to 0.83, and expected heterozygosity ranged from 0 to 0.89. CONCLUSIONS: The investigated loci showed reasonable to high levels of polymorphism at the regional scale. The multiplex set should be helpful in investigating genetic diversity, population structure, and demographic history in N. caerulescens at various spatial scales.

Selfish male-determining element favors the transition from hermaphroditism to androdioecy.

According to the current, widely accepted paradigm, the evolutionary transition from hermaphroditism toward separate sexes occurs in two successive steps: an initial, intermediate step in which unisexual individuals, male or female, sterility mutants coexist with hermaphrodites and a final step that definitively establishes dioecy. Two nonexclusive processes can drive this transition: inbreeding avoidance and reallocation of resources from one sexual function to the other. Here, we report results of controlled crosses between males and hermaphrodites in Phillyrea angustifolia, an androdioecious species with two mutually intercompatible, but intraincompatible groups of hermaphrodites. We observed different segregation patterns that can be explained by: (1) epistatic interactions between two unlinked diallelic loci, determining sex and mating compatibility, and (2) a mutation with pleiotropic effects: female sterility, full compatibility of males with both hermaphrodite incompatibility groups, and complete male-biased sex-ratio distortion in one of the two groups. Modeling shows that these mechanisms can explain the high frequency of males in populations of P. angustifolia and can promote the maintenance of androdioecy without requiring inbreeding depression or resource reallocation. We thus argue that segregation distortion establishes the right conditions for the evolution of cryptic dioecy and potentially initiates the evolution toward separate sexes.