Harmful self-pollination drives gynodioecy in European chestnut, a self-incompatible tree.

PREMISE: Gynodioecy is a rare sexual system in which two genders (sensu Lloyd, 1980), cosexuals and females, coexist. To survive, female plants must compensate for their lack of siring capacity and male attractiveness. In European chestnut (Castanea sativa), an outcrossing tree, self-pollination reduces fruit set in cosexual individuals because of late-acting self-incompatibility and early inbreeding depression. Could this negative sexual interaction explain the presence of females in this species? METHODS: We studied gender variation in wild populations of European chestnut. In addition, we compared fruit set (the proportion of flowers giving fruits) and other key female fitness components as well as reproductive allocation between genders. We then performed emasculation experiments in cosexual trees, by removing nectar-producing fertile male inflorescences. We also removed sterile but nectar-producing male inflorescences from female trees, as a control. RESULTS: We found a highly variable proportion of male-sterile individuals in the wild in European chestnut. In the experimental plot, trees from each gender had similar size, flower density, and burr set, but different fruit set. Removing nectar-producing male inflorescences from branches or entire trees increased fruit set in cosexual but not in female trees. CONCLUSIONS: These results show that self-pollination impairs fruit set in cosexual trees. Female trees avoid these problems as they do not produce pollen but continue to attract pollinators thanks to their rewarding male-sterile inflorescences, resulting in a much higher fruit set than in cosexuals. This demonstrates that even outcrossed plants can benefit from the cessation of self-pollination, to the point that unisexuality can evolve.

Sexual interference revealed by joint study of male and female pollination success in chestnut.

Most seed plants produce both pollen and ovules. In principle, pollen export could interfere with pollen import through self-pollination, resulting in ovule usurpation and reduced fruit set. Evidence for such interference exists under experimental settings but its importance under natural conditions is unknown. To test for sexual interference in nature, it is necessary to study together mating system, through paternity analyses, and fruit set, the proportion of flowers giving seeds or fruits. We developed a new model combining both processes, using chestnut (Castanea) as case study. We carried out a paternity analysis in an intensively studied plot of 273 trees belonging to three interfertile chestnut species and including a range of individuals with more or less functional stamens, resulting in a large data set of 1924 mating events. We then measured fruit set on 216 of these trees. Fruit set of male-fertile trees was much lower than that of male-sterile trees. Our process-based model shows that pollen is not limiting in the study site and hence cannot account for reduced fruit set. It also indicates that self-pollination is high (74%) but selfing rate is low (4%). Self-pollen is less competitive than cross-pollen, reducing sexual interference, but not sufficiently, as many ovules end up being self-fertilized, 95% of which abort before fruit formation, resulting in the loss of 46% of the fruit crop. These results suggest that the main cause of reduced reproductive potential in chestnut is sexual interference by self-pollen, raising questions on its evolutionary origins.

Adaptive function of duodichogamy: Why do chestnut trees have two pollen emission phases?

PREMISE: Intersexual mating facilitation in flowering plants has been largely underexplored. Duodichogamy is a rare flowering system in which individual plants flower in the sequence male-female-male. We studied the adaptive advantages of this flowering system using chestnuts (Castanea spp., Fagaceae) as models. These insect-pollinated trees produce many unisexual male catkins responsible for a first staminate phase and a few bisexual catkins responsible for a second staminate phase. We hypothesized that duodichogamy increases female mating success by facilitating pollen deposition on stigmas of the rewardless female flowers through their proximity with attractive male flowers responsible for the minor staminate phase. METHODS: We monitored insect visits to 11 chestnut trees during the entire flowering period and explored reproductive traits of all known duodichogamous species using published evidence. RESULTS: In chestnuts, insects visited trees more frequently during the first staminate phase but visited female flowers more frequently during the second staminate phase. All 21 animal-pollinated duodichogamous species identified are mass-flowering woody plants at high risk of self-pollination. In 20 of 21 cases, gynoecia (female flower parts) are located close to androecia (male flower parts), typically those responsible for the second minor staminate phase, whereas androecia are often distant from gynoecia. CONCLUSIONS: Our results suggest that duodichogamy increases female mating success by facilitating pollen deposition on stigmas by means of the attractiveness of the associated male flowers while effectively limiting self-pollination.

Asymmetric character displacement in mixed oak stands.

Ecological character displacement (ECD) refers to a pattern of increased divergence at sites where species ranges overlap caused by competition for resources. Although ECD is believed to be common, there are few in-depth studies that clearly establish its existence, especially in plants. Thus, we have compared leaf traits in allopatric and sympatric populations of two East Asian deciduous oaks: Quercus dentata and Quercus aliena. In contrast to previous studies, we define sympatry and allopatry at a local scale, thereby comparing populations that can or cannot directly interact. Using genetic markers, we found greater genetic divergence between the two oak species growing in mixed stands and inferred that long-term gene flow has predominantly occurred asymmetrically from the cold-tolerant species (Q. dentata) to the warm-demanding later colonizing species (Q. aliena). Analysis of leaf traits revealed greater divergence in mixed than in pure oak stands. This was mostly due to the later colonizing species being characterized by more resource-conservative traits in the presence of the other species. Controlling for relevant environmental differences did not alter these conclusions. These results suggest that asymmetric trait divergence can take place where species coexist, possibly due to the imbalance in demographic history of species resulting in asymmetric inter-specific selection pressures.

Sixty Years from the First Disease Description, a Novel Badnavirus Associated with Chestnut Mosaic Disease.

Although chestnut mosaic disease (ChMD) was described several decades ago, its etiology is still not clear. Using classical approaches and high-throughput sequencing (HTS) techniques, we identified a novel Badnavirus that is a strong etiological candidate for ChMD. Two disease sources from Italy and France were submitted to HTS-based viral indexing. Total RNAs were extracted, ribodepleted, and sequenced on an Illumina NextSeq500 (2 × 150 nt or 2 × 75 nt). In each source, we identified a single contig of ≈7.2 kb that corresponds to a complete circular viral genome and shares homologies with various badnaviruses. The genomes of the two isolates have an average nucleotide identity of 90.5%, with a typical badnaviral genome organization comprising three open reading frames. Phylogenetic analyses and sequence comparisons showed that this virus is a novel species; we propose the name Chestnut mosaic virus (ChMV). Using a newly developed molecular detection test, we systematically detected the virus in symptomatic graft-inoculated indicator plants (chestnut and American oak) as well in chestnut trees presenting typical ChMD symptoms in the field (100 and 87% in France and Italy surveys, respectively). Datamining of publicly available chestnut sequence read archive transcriptomic data allowed the reconstruction of two additional complete ChMV genomes from two Castanea mollissima sources from the United States as well as ChMV detection in C. dentata from the United States. Preliminary epidemiological studies performed in France and central eastern Italy showed that ChMV has a high incidence in some commercial orchards and low within-orchard genetic diversity.

The oak syngameon: more than the sum of its parts.

One of Anthropocene's most daunting challenges for conservation biology is habitat extinction, caused by rapid global change. Tree diversity has persisted through previous episodes of rapid change, even global extinctions. Given the pace of current change, our management of extant diversity needs to facilitate and even enhance the natural ability of trees to adapt and diversify. Numerous processes contribute to this evolutionary flexibility, including introgression, a widespread yet under-studied process. Reproductive networks, in which species remain distinct despite interspecific gene flow, are called syngameons, a concept largely inspired from work focusing on Quercus. Delineating and analyzing such species groups, empirically and theoretically, will provide insights into the nonadditive effects on evolution of numerous partially interfertile species exchanging genetic material episodically under changing environmental conditions. To conserve tree diversity, crossing experiments designed with an empirical and theoretical understanding of the constituent syngameon should be set up to assist diversification and adaptation in the Anthropocene. Our increasingly detailed knowledge of the oak genome and of oak interspecific and intraspecific phenotypic variation will improve our ability to sustain the diversity of this tree through an unpredictable and unprecedented future.

Invoking adaptation to decipher the genetic legacy of past climate change.

Persistence of natural populations during periods of climate change is likely to depend on migration (range shifts) or adaptation. These responses were traditionally considered discrete processes and conceptually divided into the realms of ecology and evolution. In a milestone paper, Davis and Shaw (2001) Science 292:673 argued that the interplay of adaptation and migration was central to biotic responses to Quaternary climate, but since then there has been no synthesis of efforts made to set up this research program. Here we review some of the salient findings from molecular genetic studies assessing ecological and evolutionary responses to Quaternary climate change. These studies have revolutionized our understanding of population processes associated with past species migration. However, knowledge remains limited about the role of natural selection for local adaptation of populations to Quaternary environmental fluctuations and associated range shifts, and for the footprints this might have left on extant populations. Next-generation sequencing technologies, high-resolution paleoclimate analyses, and advances in population genetic theory offer an unprecedented opportunity to test hypotheses about adaptation through time. Recent population genomics studies have greatly improved our understanding of the role of contemporary adaptation to local environments in shaping spatial patterns of genetic diversity across modern-day landscapes. Advances in this burgeoning field provide important conceptual and methodological bases to decipher the historical role of natural selection and assess adaptation to past environmental variation. We suggest that a process called "temporal conditional neutrality" has taken place: some alleles favored in glacial environments become selectively neutral in modern-day conditions, whereas some alleles that had been neutral during glacial periods become under selection in modern environments. Building on this view, we present a new integrative framework for addressing the interplay of demographic and adaptive evolutionary responses to Quaternary climate dynamics, the research agenda initially envisioned by Davis and Shaw (2001) Science 292:673.

Cryptic no more: soil macrofossils uncover Pleistocene forest microrefugia within a periglacial desert.

Despite their critical importance for understanding the local effects of global climate change on biodiversity, glacial microrefugia are not well studied because they are difficult to detect by using classical palaeoecological or population genetics approaches. We used soil macrofossil charcoal analysis to uncover the presence of cryptic glacial refugia for European beech (Fagus sylvatica) and other tree species in the Landes de Gascogne (southwestern France). Using botanical identification and direct radiocarbon dating (140 (14) C-dates) of macrofossil charcoal extracted from mineral soils, we reconstructed the glacial and postglacial history of all extant beech stands in the region (n = 11). Soil charcoal macrofossils were found in all sites, allowing the identification of up to at least 14 distinct fire events per site. There was direct evidence of the presence of beech during the last glacial period at three sites. Beech was detected during Heinrich stadial-1, one of the coldest and driest intervals of the last glacial period in Western Europe. Together with previous results on the genetic structure of the species in the region, these findings suggest that beech persisted in situ in several microrefugia through full glacial and interglacial periods up to the present day.

Distinct male reproductive strategies in two closely related oak species.

Reproductive strategies of closely related species distributed along successional gradients should differ as a consequence of the trade-off between competition and colonization abilities. We compared male reproductive strategies of Quercus robur and Q. petraea, two partly interfertile European oak species with different successional status. In the studied even-aged stand, trees of the late-successional species (Q. petraea) grew faster and suffered less from intertree competition than trees of the early-successional species (Q. robur). A large-scale paternity study and a spatially explicit individual-based mating model were used to estimate parameters of pollen production and dispersal as well as sexual barriers between species. Male fecundity was found to be dependent both on a tree's circumference and on its environment, particularly so for Q. petraea. Pollen dispersal was greater and more isotropic in Q. robur than in Q. petraea. Premating barriers to hybridization were strong in both species, but more so in Q. petraea than in Q. robur. Hence, predictions based on the competition-colonization trade-off are well supported, whereas the sexual barriers themselves seem to be shaped by colonization dynamics.

Efficient mitigation of founder effects during the establishment of a leading-edge oak population.

Numerous plant species are shifting their range polewards in response to ongoing climate change. Range shifts typically involve the repeated establishment and growth of leading-edge populations well ahead of the main species range. How these populations recover from founder events and associated diversity loss remains poorly understood. To help fill this gap, we exhaustively investigated a newly established population of holm oak (Quercus ilex) growing more than 30 km ahead of the nearest larger stands. Pedigree reconstructions showed that plants belong to two non-overlapping generations and that the whole population originates from only two founder trees. The four first-generation trees that have reached maturity showed disparate mating patterns despite being full-sibs. Long-distance pollen immigration was notable despite the strong isolation of the stand: 6 per cent gene flow events in acorns collected on the trees (n = 255), and as much as 27 per cent among their established offspring (n = 33). Our results show that isolated leading-edge populations of wind-pollinated forest trees can rapidly restore their genetic diversity through the interacting effects of efficient long-distance pollen flow and purging of inbred individuals during recruitment. They imply that range expansions of these species are primarily constrained by initial propagule arrival rather than by subsequent gene flow.

Fine-scale environmental control of hybridization in oaks.

Natural hybridization is attracting much interest in modern speciation and conservation biology studies, but the underlying mechanisms remain poorly understood. In particular, it is unclear why environmental changes often increase hybridization rates. To study this question, we surveyed mating events in a mixed oak stand and developed a spatially explicit individual-based hybridization model. This model, where hybridization is frequency-dependent, pollen is nonlimiting and which allows immigrant pollen to compete with local pollen, takes into account species-specific pollen dispersal and sexual barriers to hybridization. The consequences of pollen limitation on hybridization were studied using another simple model. The results indicate that environmental changes could increase hybridization rates through two distinct mechanisms. First, by disrupting the spatial organization of communities, they should decrease the proportion of conspecific pollen available for mating, thus increasing hybridization rates. Second, by decreasing the density of conspecifics, they should increase pollen limitation and thus hybridization rates, as a consequence of chance pollination predominating over deterministic pollen competition. Altogether, our results point to a need for considering hybridization events at the appropriate level of organization and provide new insights into why hybridization rates generally increase in disturbed environments.

Stronger spatial genetic structure in recolonized areas than in refugia in the European beech.

Extant rear-edge populations located in former glacial refugia remain understudied despite their high conservation value. These populations should have experienced strong genetic drift due to their small size and long isolation. Moreover, the prolonged action of isolation by distance in refugial areas should result in stronger regional spatial genetic structure (SGS) than in recolonized areas, but empirical tests of this prediction are scarce. To fill this gap, we first used a set of 16 microsatellite markers to investigate the genetic structure of European beech in France in 65 populations from three refugial areas and one control recolonized (nonrefugial) area. Then, using the same approach, we reanalysed published isozyme data from 375 populations distributed across the entire species range. We found stronger genetic differentiation among populations in refugia than in recolonized areas. However, contrary to expectations, regional SGS was lower within refugia than within recolonized areas. Published studies presenting similar analyses suggest that our results could have generality across different biogeographical settings and types of organisms. Strong and prolonged genetic drift in refugial areas could have erased the signature of range expansions that is still visible in recolonized areas. Our results therefore suggest that Pleistocene population isolation has played a key role in increasing the genetic complexity of extant rear-edge populations.

Direction and extent of organelle DNA introgression between two spruce species in the Qinghai-Tibetan Plateau.

A recent model has shown that, during range expansion of one species in a territory already occupied by a related species, introgression should take place preferentially from the resident species towards the invading species and genome components experiencing low rates of gene flow should introgress more readily than those experiencing high rates of gene flow. Here, we use molecular markers from two organelle genomes with contrasted rates of gene flow to test these predictions by examining genetic exchanges between two morphologically distinct spruce Picea species growing in the Qinghai-Tibetan Plateau. The haplotypes from both mitochondrial (mt) DNA and chloroplast (cp) DNA cluster into two distinct lineages that differentiate allopatric populations of the two species. By contrast, in sympatry, the species share the same haplotypes, suggesting interspecific genetic exchanges. As predicted by the neutral model, all sympatric populations of the expanding species had received their maternally inherited mtDNA from the resident species, whereas for paternally inherited cpDNA introgression is more limited and not strictly unidirectional. Our results underscore cryptic introgressions of organelle DNAs in plants and the importance of considering rates of gene flow and range shifts to predict direction and extent of interspecific genetic exchanges.

Geographic variation in the structure of oak hybrid zones provides insights into the dynamics of speciation.

Studying geographic variation in the rate of hybridization between closely related species could provide a useful window on the evolution of reproductive isolation. Reinforcement theory predicts greater prezygotic isolation in areas of prolonged contact between recently diverged species than in areas of recent contact, which implies that old contact zones would be dominated by parental phenotypes with few hybrids (bimodal hybrid zones), whereas recent contact zones would be characterized by hybrid swarms (unimodal hybrid zones). Here, we investigate how the hybrid zones of two closely related Chinese oaks, Quercus mongolica and Q. liaotungensis, are structured geographically using both nuclear and chloroplast markers. We found that populations of Q. liaotungensis located around the Changbai Mountains in Northeast China, an inferred glacial refugium, were introgressed by genes from Q. mongolica, suggesting historical contact between the two species in this region. However, these introgressed populations form sharp bimodal hybrid zones with Q. mongolica. In contrast, populations of Q. liaotungensis located in North China, which show no sign of ancient introgression with Q. mongolica, form unimodal hybrid zones with Q. mongolica. These results are consistent with the hypothesis that selection against hybrids has had sufficient time to reinforce the reproductive barriers between Q. liaotungensis and Q. mongolica in Northeast China but not in North China.

Sex-biased dispersal promotes adaptive parental effects.

BACKGROUND: In heterogeneous environments, sex-biased dispersal could lead to environmental adaptive parental effects, with offspring selected to perform in the same way as the parent dispersing least, because this parent is more likely to be locally adapted. We investigate this hypothesis by simulating varying levels of sex-biased dispersal in a patchy environment. The relative advantage of a strategy involving pure maternal (or paternal) inheritance is then compared with a strategy involving classical biparental inheritance in plants and in animals. RESULTS: We find that the advantage of the uniparental strategy over the biparental strategy is maximal when dispersal is more strongly sex-biased and when dispersal distances of the least mobile sex are much lower than the size of the environmental patches. In plants, only maternal effects can be selected for, in contrast to animals where the evolution of either paternal or maternal effects can be favoured. Moreover, the conditions for environmental adaptive maternal effects to be selected for are more easily fulfilled in plants than in animals. CONCLUSIONS: The study suggests that sex-biased dispersal can help predict the direction and magnitude of environmental adaptive parental effects. However, this depends on the scale of dispersal relative to that of the environment and on the existence of appropriate mechanisms of transmission of environmentally induced traits.

The 'New Wave' in plant demographic inference: more loci and more individuals.

Plant population genetic surveys are starting to take full advantage of technological advances in genotyping methods and of methodological advances in demographic inference. In this issue of Molecular Ecology, Keller et al. (2010) illustrate this trend with a particularly convincing study of rangewide genetic variation in a North American poplar, using both SNP and sequence data. They first investigate population genetic structure by estimating the most likely number of genetic clusters using a more formal approach than most other studies to date. They proceed by estimating gene flow among the inferred populations and by testing predictions on the distribution of low frequency alleles derived from recent work on range expansions.

Origin of spatial genetic structure in an expanding oak population.

Spatial genetic structure (SGS) results from the interplay of several demographical processes that are difficult to tease apart. In this study, we explore the specific effects of seed and pollen dispersal and of early postdispersal mortality on the SGS of a seedling cohort (N = 786) recruiting within and around an expanding pedunculate oak (Quercus robur) stand. Using data on dispersal (derived from parentage analysis) and mortality (monitored in the field through two growing seasons), we decompose the overall SGS of the cohort into its components by contrasting the SGS of dispersed (i.e. growing away from their mother tree) vs. nondispersed (i.e. growing beneath their mother tree) and initial vs. surviving seedlings. Patterns differ strongly between nondispersed and dispersed seedlings. Nondispersed seedlings are largely responsible for the positive kinship values observed at short distances in the studied population, whereas dispersed seedlings determine the overall SGS at distances beyond c. 30 m. The paternal alleles of nondispersed seedlings show weak yet significantly positive kinships up to c. 15 m, indicating some limitations in pollen flow that should further promote pedigree structures at short distances. Seedling mortality does not alter SGS, except for a slight increase in the nondispersed group. Field data reveal that mortality in this group is negatively density-dependent, probably because of small-scale variation in light conditions. Finally, we observe a remarkable similarity between the SGS of the dispersed seedlings and that of the adults, which probably reflects dispersal processes during the initial expansion of the population. Overall, this study demonstrates that incorporating individual-level complementary information into analyses can greatly improve the detail and confidence of ecological inferences drawn from SGS.

Plant traits correlated with generation time directly affect inbreeding depression and mating system and indirectly genetic structure.

BACKGROUND: Understanding the mechanisms that control species genetic structure has always been a major objective in evolutionary studies. The association between genetic structure and species attributes has received special attention. As species attributes are highly taxonomically constrained, phylogenetically controlled methods are necessary to infer causal relationships. In plants, a previous study controlling for phylogenetic signal has demonstrated that Wright's FST, a measure of genetic differentiation among populations, is best predicted by the mating system (outcrossing, mixed-mating or selfing) and that plant traits such as perenniality and growth form have only an indirect influence on FST via their association with the mating system. The objective of this study is to further outline the determinants of plant genetic structure by distinguishing the effects of mating system on gene flow and on genetic drift. The association of biparental inbreeding and inbreeding depression with population genetic structure, mating system and plant traits are also investigated. RESULTS: Based on data from 263 plant species for which estimates of FST, inbreeding (FIS) and outcrossing rate (tm) are available, we confirm that mating system is the main influencing factor of FST. Moreover, using an alternative measure of FST unaffected by the impact of inbreeding on effective population size, we show that the influence of tm on FST is due to its impact on gene flow (reduced pollen flow under selfing) and on genetic drift (higher drift under selfing due to inbreeding). Plant traits, in particular perenniality, influence FST mostly via their effect on the mating system but also via their association with the magnitude of selection against inbred individuals: the mean inbreeding depression increases from short-lived herbaceous to long-lived herbaceous and then to woody species. The influence of perenniality on mating system does not seem to be related to differences in stature, as proposed earlier, but rather to differences in generation time. CONCLUSION: Plant traits correlated with generation time affect both inbreeding depression and mating system. These in turn modify genetic drift and gene flow and ultimately genetic structure.

More introgression with less gene flow: chloroplast vs. mitochondrial DNA in the Picea asperata complex in China, and comparison with other Conifers.

Recent work has suggested that rates of introgression should be inversely related to levels of gene flow because introgressed populations cannot be 'rescued' by intraspecific gene flow if it is too low. Mitochondrial and chloroplast DNA (mtDNA and cpDNA) experience very different levels of gene flow in conifers due to their contrasted maternal and paternal modes of transmission, hence the prediction that mtDNA should introgress more readily than cpDNA in this group. Here, we use sequence data from both mtDNA and cpDNA to test this hypothesis in a group of closely related spruces species, the Picea asperata complex from China. Nine mitochondrial and nine chloroplast haplotypes were recovered from 459 individuals in 46 natural populations belonging to five species of the Picea asperata complex. Low variation was found in the two mtDNA introns along with a high level of differentiation among populations (G(ST) = 0.90). In contrast, we detected higher variation and lower differentiation among populations at cpDNA markers (G(ST) = 0.56), a trend shared by most conifer species studied so far. We found that cpDNA variation, although far from being fully diagnostic, is more species-specific than mtDNA variation: four groups of populations were identified using cpDNA markers, all of them related to species or groups of species, whereas for mtDNA, geographical variation prevails over species differentiation. The literature suggests that mtDNA haplotypes are often shared among related conifer species, whereas cpDNA haplotypes are more species-specific. Hence, increased intraspecific gene flow appears to decrease differentiation within species but not among species.