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Abstract PremiseAntheridiogen systems are a set of pheromonal mechanisms that control sex expression in fern gametophytes. However, antheridiogen has rarely been studied outside of the laboratory, and little is known about its function in natural settings. Combining predictions based on field and laboratory study, we tested whether the sexual structure of gametophytic colonies of a tree fern were attributable to antheridiogen. MethodsGametophytic colonies of the antheridiogen‐producing tree fernCyathea multiflorawere collected at La Selva Biological Station in Costa Rica in January 2019. The sex of each gametophyte was determined, mapped, and spatial statistic approaches were used to examine the distribution of sex in each colony. ResultsIn all gametophytic colonies, males were most common, representing 62–68% of individuals. No hermaphroditic gametophytes were identified in any colony. A quadrat‐based method showed female gametophytes were not clustered in each colony, while male gametophytes were clustered. In two of the colonies, theK(r) test statistic for males was greater than expected compared to random simulations of sex expression, indicating male sex expression was spatially associated with females. ConclusionsThis study provides the first documentation of spatial sex expression in natural settings of gametophytes of an antheridiogen‐producing tree fern species. The profound impact of antheridiogen on gametophytic sex expression in field settings suggests this system is intimately tied to mating system, fitness, and genetic diversity inCyathea multiflora.