Evaluating the role of frequency-dependent selection in controlling the expansion of clonal aggregations in the tropical forest's understory: Insights from a decade-long experiment.

Sexual reproduction, despite its associated costs and risks, is prevalent among many organisms, presumably to generate and maintain genetic diversity. This diversity is vital for adapting to environmental changes and combating natural enemies. Paradoxically, several clonal species also exhibit high genetic diversity. One theory for the maintenance of this genetic diversity is frequency-dependent selection, which favors rare genotypes over common ones, limiting the extent and dominance of a single clone, thereby preserving genetic diversity. Empirical evidence for this theory under natural conditions is sparse. Twelve years ago, we established in the forest fourteen genetically diverse plots where all plants had a unique genotype (rare genotypes) and paired with them clonal plots where all plants had the same genotype (common genotypes) to test whether common genotypes have a disadvantage and frequency-dependent selection is in action. Clones were created from cuttings from Piper cordulatum, a naturally clonally reproducing understory plant. We aimed to test if common genotypes are disadvantaged and if frequency-dependent selection is effective. Over the experiment's first ten years, herbivory, pathogen attacks, and plant size remained similar across both genotype categories. Intriguingly, clones exhibited superior survival during the initial five years. Survival rates equalize for rare and common genotypes by the decade's end. By year twelve, survival remained similar for rare and common genotypes. However, modeled survival projections based on the twelve-year-long trend suggest that common genotypes might experience increased mortality in the long run, consistent with the hypothesis of negative frequency-dependent selection. Moreover, plants in clonal plots exhibited lower fitness in terms of infructescence production at the plot level by the tenth year. Our findings suggest that having low genetic diversity in the neighborhood does not increase disease or herbivory susceptibility or reduce short-term survival. The impact of negative frequency-dependent selection is not immediate. However, it could eventually restrict the survival and reproduction of Piper clones in a tropical forest's understory, curbing the dominance of any single genotype and potentially enhancing population-wide genetic diversity.

Methods
Twelve years ago, we established fourteen experimental plots within a forest setting to investigate frequency-dependent selection among Piper cordulatum, a naturally clonally reproducing understory plant. Each plot was designated under one of two treatments: "All Clones," where all plants shared the same genotype, and "No Clones," where every plant possessed a unique genotype. This experimental design aimed to test the hypothesis that common genotypes, represented by the "All Clones" treatment, are at a disadvantage due to frequency-dependent selection, which might help preserve genetic diversity by limiting the dominance of any single clonal genotype.

Data collection was carried out systematically over the course of the experiment. Initial measurements were taken at 0, 6, 12, and 18 months post-transplantation. Subsequent censuses were conducted at extended intervals—specifically at 30, 42, 60 (5 years), and 120 months (10 years). In the final year, we harvested the above-ground parts of one focal plant per plot to measure the total leaf area, the extent of herbivory damage, and above-ground biomass (AGB).

During each census, we documented the number of plants that were alive or dead. We also assessed the extent of damage from herbivores and pathogens, calculating the percentage of total plant damage.

Furthermore, to determine whether plants with rare genotypes ("No Clones" treatment) exhibited higher reproductive success compared to those with common genotypes ("All Clones" treatment), we counted the total number of infructescences produced per plant and recorded the number of surviving plants in each plot during the reproductive season of the tenth year.