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.

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.