Abstract Self-incompatibility can limit the availability of compatible mates in small and isolated populations, eventually reducing average seed set to the point that the long-term persistence of the populations can be impaired. This phenomenon, named the S-Allee effect, is caused by the loss of alleles (S-alleles) at the self-incompatibility locus (S-locus) due to the intense genetic drift experienced by small populations. Quantifying the diversity of S-alleles is therefore of direct interest for biological conservation, but efficient genotyping methods have been lacking so far because of technical challenges associated with the typically extreme levels of polymorphism and complex genomic structure of the S-locus. We used two alternative approaches to genotype the S-locus using NGS sequencing technologies in four natural populations of the endangered Brassica insularis in Corsica. First, we used an NGS amplicon-sequencing approach using generalist primers for each of the two classes of Brassica S-alleles. Second, we obtained whole genome shotgun short-read resequencing data and analyzed them with a recently developed bioinformatic pipeline dedicated to hypervariable loci, which we successfully validated on a public dataset comprising 119 cultivated accessions of B. oleracea . By combining the two approaches in natural populations of B. insularis we identified 31 distinct S-alleles and obtained fully resolved S-locus genotypes for 319 out of 326 sampled individuals. The number of S-alleles varied from four in the smallest population to 18 in the largest one. As a result, the smallest population exhibited very low proportions of compatible individuals, potentially threatening its persistence. We conclude that introducing individuals carrying S-alleles currently absent from the population could help rescue fertility.

Abstract Female choice is a central process of sexual selection that has shaped numerous phenotypes in animals. It may operate broadly across sexually reproducing organisms, including plants, not only because sex differences in sexual selection arise from anisogamy, wich refers to the unequal investment in gametes between sexes, but also because it can emerge from simple variation in female reproductive morphology. Here, we provide the first empirical test of cryptic female choice in an angiosperm by examining whether pollen and pistil traits jointly influence paternal fertilization success. Using experimental pollen competition, paternity analyses, and trait measurements, we show that pistil traits can bias paternity toward pollen donors with specific pollen traits. These results demonstrate that cryptic female choice operates in plants, paralleling mechanisms described in animals.

Cryptic female choice - female-mediated bias in fertilization after mating - is well established in animals and can also occur in plants when multiple pollens compete on the same pistil. However, whether interactions between pollen and pistil tissues after pollen deposition contribute to this process remains unknown. Here, we experimentally test whether such interactions mediate cryptic female choice in the angiosperm Brassica rapa. We quantified fertilization success of pollen donors competing on the same pistil using paternity analyses, and in parallel, made semi-in vivo assays to measure pollen tubes trajectories emerging from the excised styles and growing toward unfertilized ovules for each donor-recipient pair. We show that pollen tube growth towards ovules predicts higher fertilization success under pollen competition. Thus, we document a previously unobserved mechanism of cryptic female choice based on physical interactions between male and female components of reproduction. In addition, different recipient plants favour different pollen donors, consistent with non-directional female choice. Plants with longer styles bias paternities more strongly towards the most successful pollen donor. Overall, our study demonstrates that interactions between pollen tubes and pistil tissues after pollen germination enable plants to bias paternity toward particular donors.

Sexual dimorphism in plants may emerge as a result of sex-specific selection on traits enhancing access to nutritive resources and/or to sexual partners. Here we investigated sex-specific differences in selection of sexually dimorphic traits and in the spatial distribution of effective fecundity (our fitness proxy) in a highly dimorphic dioecious wind-pollinated shrub, Leucadendron rubrum. In particular, we tested for the effect of density on male and female effective fecundity. We used spatial and genotypic data of parent and offspring cohorts to jointly estimate individual male and female effective fecundity on the one hand and pollen and seed dispersal kernels on the other hand. This methodology was adapted to the case of dioecious species. Explicitly modelling dispersal avoids the confounding effects of heterogeneous spatial distribution of mates and sampled seedlings on the estimation of effective fecundity. We also estimated selection gradients on plant traits while modelling sex-specific spatial autocorrelation in fecundity. Males exhibited spatial autocorrelation in effective fecundity at a smaller scale than females. A higher local density of plants was associated with lower effective fecundity in males but was not related to female effective fecundity. These results suggest sex-specific sensitivities to environmental heterogeneity in L. rubrum. Despite these sexual differences, we found directional selection for wider canopies and smaller leaves in both sexes, and no sexually antagonistic selection on strongly dimorphic traits in L. rubrum. Many empirical studies in animals similarly failed to detect sexually antagonistic selection in species expressing strong sexual dimorphism, and we discuss reasons explaining this common pattern.

We advocate the advantage of an evolutionary approach to conservation biology that considers evolutionary history at various levels of biological organization. We review work on three separate plant taxa, spanning from one to multiple decades, illustrating extremes in metapopulation functioning. We show how the rare endemics Centaurea corymbosa (Clape Massif, France) and Brassica insularis in Corsica (France) may be caught in an evolutionary trap: disruption of metapopulation functioning due to lack of colonization of new sites may have counterselected traits such as dispersal ability or self-compatibility, making these species particularly vulnerable to any disturbance. The third case study concerns the evolution of life history strategies in the highly diverse genus Leucadendron of the South African fynbos. There, fire disturbance and the recolonization phase after fires are so integral to the functioning of populations that recruitment of new individuals is conditioned by fire. We show how past adaptation to different fire regimes and climatic constraints make species with different life history syndromes more or less vulnerable to global changes. These different case studies suggest that management strategies should promote evolutionary potential and evolutionary processes to better protect extant biodiversity and biodiversification.

Two figures showing the results of the randomization tests.

BACKGROUND AND AIMS:The pseudometallophyte Noccaea caerulescens is an excellent model to study evolutionary processes, as it grows both on normal and on heavy-metal-rich, toxic soils. The evolution and demography of populations are critically impacted by mating system and, yet, information about the N. caerulescens mating system is limited.METHODS:Mean selfing rates were assessed using microsatellite loci and a robust estimation method (RMES) in five metallicolous and five non-metallicolous populations of N. caerulescens in Southern France, and this measure was replicated for two successive reproductive seasons. As a part of the study, the patterns of gene flow among populations were analysed. The mating system was then characterized at a fine spatial scale in three populations using the MLTR method on progeny arrays.KEY RESULTS:The results confirm that N. caerulescens has a mixed mating system, with selfing rates ranging from 0·2 to 0·5. Selfing rates did not vary much among populations within ecotypes, but were lower in the metallicolous than in the non-metallicolous ecotype, in both seasons. Effective population size was also lower in non-metallicolous populations. Biparental inbreeding was null to moderate. Differentiation among populations was generally high, but neither ecotype nor isolation by distance explained it.CONCLUSIONS:The consequences of higher selfing rates on adaptation are expected to be weak to moderate in non-metallicolous populations and they are expected to suffer less from inbreeding depression, compared to metallicolous populations.

A table showing the results of the generalized linear model.

We advocate the advantage of an evolutionary approach to conservation biology that considers evolutionary history at various levels of biological organization. We review work on three separate plant taxa, spanning from one to multiple decades, illustrating extremes in metapopulation functioning. We show how the rare endemics Centaurea corymbosa (Clape Massif, France) and Brassica insularis in Corsica (France) may be caught in an evolutionary trap: disruption of metapopulation functioning due to lack of colonization of new sites may have counterselected traits such as dispersal ability or self‐compatibility, making these species particularly vulnerable to any disturbance. The third case study concerns the evolution of life history strategies in the highly diverse genus Leucadendron of the South African fynbos. There, fire disturbance and the recolonization phase after fires are so integral to the functioning of populations that recruitment of new individuals is conditioned by fire. We show how past adaptation to different fire regimes and climatic constraints make species with different life history syndromes more or less vulnerable to global changes. These different case studies suggest that management strategies should promote evolutionary potential and evolutionary processes to better protect extant biodiversity and biodiversification.

Natural selection is expected to cause convergence of life histories among taxa as well as correlated evolution of different life-history traits. Here, we quantify the extent of convergence of five key life-history traits (adult fire survival, seed storage, degree of sexual dimorphism, pollination mode, and seed-dispersal mode) and test hypotheses about their correlated evolution in the genus Leucadendron (Proteaceae) from the fire-prone South African fynbos. We reconstructed a new molecular phylogeny of this highly diverse genus that involves more taxa and molecular markers than previously. This reconstruction identifies new clades that were not detected by previous molecular study and morphological classifications. Using this new phylogeny and robust methods that account for phylogenetic uncertainty, we show that the five life-history traits studied were labile during the evolutionary history of the genus. This diversity allowed us to tackle major questions about the correlated evolution of life-history strategies. We found that species with longer seed-dispersal distances tended to evolve lower pollen-dispersal distance, that insect-pollinated species evolved decreased sexual dimorphism, and that species with a persistent soil seed-bank evolved toward reduced fire-survival ability of adults.

Despite the recent advances in generating molecular data, reconstructing species-level phylogenies for non-models groups remains a challenge. The use of a number of independent genes is required to resolve phylogenetic relationships, especially for groups displaying low polymorphism. In such cases, low-copy nuclear exons and non-coding regions, such as 3' untranslated regions (3'-UTRs) or introns, constitute a potentially interesting source of nuclear DNA variation. Here, we present a methodology meant to identify new nuclear orthologous markers using both public-nucleotide databases and transcriptomic data generated for the group of interest by using next generation sequencing technology. To identify PCR primers for a non-model group, the genus Leucadendron (Proteaceae), we adopted a framework aimed at minimizing the probability of paralogy and maximizing polymorphism. We anchored when possible the right-hand primer into the 3'-UTR and the left-hand primer into the coding region. Seven new nuclear markers emerged from this search strategy, three of those included 3'-UTRs. We further compared the phylogenetic potential between our new markers and the ribosomal internal transcribed spacer region (ITS). The sequenced 3'-UTRs yielded higher polymorphism rates than the ITS region did. We did not find strong incongruences with the phylogenetic signal contained in the ITS region and the seven new designed markers but they strongly improved the phylogeny of the genus Leucadendron. Overall, this methodology is efficient in isolating orthologous loci and is valid for any non-model group given the availability of transcriptomic data. (C) 2013 Elsevier Inc. All rights reserved.

Summary Serotiny, the retention of mature seeds in closed fruits within the canopy for over a year, is a common trait in fire‐prone environments. When competition with adult plants prevents seedling establishment between fire events and in the absence of post‐release soil seed dormancy, strong serotiny, i.e. the retention of all seeds until the next fire, appears as the best strategy. Despite the low levels of inter‐fire seed recruitment for several species in both Australian and South African fire‐prone environments, considerable variation in the duration of fruit retention is nevertheless observed among species. Our aim is to predict optimal age‐specific reproductive schedules in a perennial, serotinous species, when cone maintenance is costly. We focus on species where adults are killed by fire, without a soil seed‐bank. We explicitly consider a trade‐off between growth (which determines plant survival), seed production and seed maintenance. In our model recruitment relies upon fire events. We use dynamic programming to determine, for given fire regimes, the optimal pattern of resource allocation. We further study the effect of changes in fire regime on the viability of populations adapted to some historical fire regime. We find that, whenever maximal plant survival probability is low, the optimal strategy consists in reducing resource allocation to seed maintenance while increasing resource allocation to annual seed production. This illustrates a trade‐off between current and future reproduction. A low rather than a strong level of serotiny should evolve whenever the variance of fire intervals is large and the mean fire interval is low. Low levels of serotiny could constitute a bet‐hedging strategy with decreasing predictability of the arrival of fire. Once adapted to some historical fire regime, serotinous populations are highly sensitive to a change in mean fire frequency and to an increase in the variance of fire intervals. Populations adapted to a historically high level of variance in fire return are more robust to changes in fire regime. Synthesis: Life‐history trade‐offs and low predictability of fire intervals may favour low rather than strong levels of serotiny even when recruitment essentially occurs just after fire events.

We analysed the spatial distribution of genetic diversity to infer gene flow for Baillonella toxisperma Pierre (Moabi), a threatened entomophilous pollinated and animal‐dispersed Central African tree, with typically low density (5–7 adults trees/km2). Fifteen nuclear and three universal chloroplast microsatellites markers were used to type 247 individuals localized in three contiguous areas with differing past logging intensity. These three areas were within a natural forest block of approximately 2886 km2 in Gabon. Expected heterozygosity and chloroplast diversity were Henuc = 0.570 and Hcp = 0.761, respectively. FIS was only significant in one area (FIS = 0.076, P < 0.01) and could be attributed to selfing. For nuclear loci, Bayesian clustering did not detect discrete gene pools within and between the three areas and global differentiation (FSTnuc = 0.007, P > 0.05) was not significant, suggesting that they are one population. At the level of the whole forest, both nuclear and chloroplast markers revealed a weak correlation between genetic relatedness and spatial distance between individuals: Spnuc = 0.003 and Spcp = 0.015, respectively. The extent of gene flow (σ) was partitioned into global gene flow (σg) from 6.6 to 9.9 km, seed dispersal (σs) from 4.0 to 6.3 km and pollen dispersal (σp) from 9.8 to 10.8 km. These uncommonly high dispersal distances indicate that low‐density canopy trees in African rainforests could be connected by extensive gene flow, although, given the current threats facing many seed disperser species in Central Africa, this may no longer be the case.

Long-term demographic surveys, needed to obtain accurate information on population dynamics and efficiently manage rare species, are still very scarce. Matrix population models are useful tools to identify key demographic transitions and thus help setting up conservation actions. Furthermore, the combination of ecological, demographic and genetic data is likely to improve the identification of the threats acting upon populations and help conservation decisions. In this paper we illustrate the power of this approach on Brassica insularis, a Mediterranean endemic plant species, rare and endangered in Corsica (France). In four populations of this species, a long-term demographic survey (2000–2009), genetic analyses (in 2000 and 2009) and survey of ecological variables (climatic variables, competition and herbivory) were performed. By using both deterministic and stochastic matrix model analyses, we assessed the viability of each population and tested for both spatial and temporal variations in demographic vital rates. Populations exhibited differing demographic behaviours and environmental stochasticity occurred in populations. Significant correlations between climatic variables and vital rates were detected. Stochastic simulations suggested that three out of the four populations studied might present a high risk of extinction on the short-term and should actively be managed, or at least surveyed. It could be, however, that two of these populations are experiencing density-dependent regulation, rather than being declining. Microsatellite diversity was slightly reduced in a single population and similar in the three others, consistently with expectations based on population census size and geographic area, as well as with diversity at the S-locus observed in 2000. The combination of all data led to specific recommendations for managing each population. We discuss the implications for conservation of such a general approach.

1. In a context of increasing human impact on ecosystems and species distributions, population restoration (introductions, reintroductions, reinforcements) is an essential management tool, especially for plant species with limited colonization ability. However, detailed demographic surveys following restoration and comparisons of demographic rates between restored and natural populations, although essential for identifying the key factors of restoration success, are lacking.2. We compared the demography over 10 years of six natural and two experimentally introduced populations of the narrowly endemic, cliff-dwelling, self-incompatible plant species Centaurea corymbosa. We analysed the fate of two cohorts of individuals that emerged simultaneously from seed introduction and natural germination. We then built a matrix model of population dynamics (using 6 years of data) and compared the demographic rates and asymptotic growth rate between the natural and introduced populations.3. Overall, survival rates were higher in the introduced than in the natural populations, either due to better habitat conditions at the cliff scale or to better conditions in microsites selected for seed introduction compared to those reached by chance following natural seed dispersal.4. In contrast, introduced populations exhibited lower fecundity than natural populations, probably due to the introduction protocol which led, in combination with self-incompatibility, to severely reduced mate availability.5. Despite clear differences in population dynamics between introduced and natural populations, no significant difference in the asymptotic growth rates could be detected, because the higher survival compensated for the lower fecundity in introduced populations.6. Synthesis and applications. Creating new populations of C corymbosa in suitable unoccupied sites seems straightforward, provided that the introduction protocol allows sufficiently high fecundity. This key parameter for restoration success can be optimized by sowing seeds from several sources at high density and in several consecutive years, which should increase mate availability for self-incompatible flowering individuals. We suggest that population introduction might be successful for many (endemic) plant species whose geographical range is mainly limited by low colonization ability, especially in Mediterranean landscapes. We show that the simultaneous monitoring of restored and natural populations enables identification of the key parameters to be targeted for management optimization of restored populations.

Self-incompatibility systems prevent self-fertilization in angiosperms. Although numerous S alleles are usually maintained by negative frequency-dependent selection, the number of S alleles can be low in small populations, which limits mate availability and reduces fecundity in endangered populations of self-incompatible plants. Despite the increasing evidence of the negative effect of self-incompatibility in small populations, the direct link between the number and the distribution of S alleles and their reproductive consequences has been rarely reported. Brassica insularis is a rare self-incompatible species with medium to very small populations. Results of a previous study showed that the smallest population has very few S alleles. We investigated whether reduced mate availability affects reproduction in this species. We compared the pollination success and the fruit set in 4 populations differing in population size and number of S alleles. Our results suggest that reproduction may be negatively affected by the low S-allele diversity in the smallest population. Nevertheless, other populations also had reduced fruit set that could not be attributed to self-incompatibility alone.

Various methods have been proposed to estimate inbreeding depression and to assess its consequences for natural populations. As an alternative to controlled crosses, the use of molecular markers has allowed direct investigation of inbreeding depression in natural populations, but usually suffers from low statistical power. Here, we investigated the effect of inbreeding depression on survival in two populations of the rare species Brassica insularis, using both controlled crosses and a marker-based approach. We compare the respective merits of the two approaches for studying inbreeding depression. We also use information from the molecular markers to dissect in detail patterns of inbreeding depression in this species. A posteriori, we find that combining the approaches was not necessary to obtain simple point estimates of inbreeding depression. However, using molecular markers may give insight into the genetic basis of inbreeding depression, such as the occurrence of epistatic interactions among deleterious alleles or purging.

The existence of genetic variability for dispersal is a crucial issue for organisms facing increased habitat fragmentation and climate change. We study the genetic basis and evolutionary potential for diaspore traits related to dispersal in Centaurea corymbosa. Using diaspores collected in natural conditions in four of the six extant populations of this narrow-endemic plant species and diaspores produced in a common garden experiment, we study the variation for pappus and achene sizes, and diaspore mass. Using a sample of achenes from the common garden experiment, we find that the best predictor of terminal velocity is a linear combination of pappus length, achene width, and achene weight. We find significant differences among populations for all traits in both conditions, as well as significant differences among families within population. Although the differences among populations for some traits are not exactly the same in controlled conditions compared to natural conditions, the ranking of populations according to their mean trait values is consistent in both conditions. Our study is therefore one of the first to show a correlation between phenotypic differentiation for dispersal traits in natural conditions vs. controlled conditions. We also show evidence of genetic variation for traits commonly thought to be involved in dispersal ability, suggesting the potential for evolutionary changes following environmental change and management actions.