Ferns Without Sex Chromosomes: How a Pheromone Decides Male or Female Fate

Homosporous ferns carry no sex chromosomes, yet their spores still give rise to individuals that make sperm, eggs or both. A new review published in Discover Developmental Biology by Dinh Nhan Lai and Yun Zhou pulls together the recent molecular and cellular findings that explain how this happens, focusing on the model fern Ceratopteris richardii, whose free-living gametophytes have become one of the most informative systems for studying plant sex determination. The review, published on 10 November 2025, synthesizes work on the pheromone antheridiogen, on mutants that override its signals, and on the formation of the multicellular meristem that defines the hermaphroditic form.

The core of the system is deceptively simple. Ceratopteris spores are genetically identical, but their sex type is not written in their genomes. Spores that germinate in the absence of antheridiogen, or early enough in a population that the pheromone has not yet accumulated, develop as hermaphrodites. These individuals initiate a multicellular marginal meristem and form egg-producing archegonia next to it. Once this program is set, it is generally irreversible: the meristem remains indeterminate, keeps producing cells, and continues to induce archegonia. Hermaphrodites also produce a few sperm-bearing antheridia, typically at the edges of the prothallus, so they retain the option of self-fertilization, though most wild ferns are thought to reproduce by outcrossing.

Hermaphrodites then begin secreting antheridiogen into their surroundings. Late-germinating spores bathed in the pheromone develop instead as males: ameristic gametophytes that lack both a meristem and archegonia but gradually produce numerous antheridia. When these mature, they rupture and release motile, flagellated sperm that swim into the archegonia of hermaphrodites to fertilize eggs, producing diploid embryos and advancing the life cycle. Through this intergametophytic signaling, antheridiogen adjusts the sex ratio of the population and promotes outcrossing, a strategy the review notes has now been confirmed by field evidence showing that it regulates sex ratios under natural conditions across diverse fern taxa.

Chemically, antheridiogens from several ferns have been identified as gibberellin-related compounds. In the fern Lygodium japonicum, the antheridiogen shares the ent-gibberellane skeleton of gibberellin A4 but differs in lacking a hydroxyl group at carbon 3 and carrying a methyl-esterified carboxyl group at carbon 6. The structure of the Ceratopteris antheridiogen itself remains unknown. Even so, indirect evidence suggests its signaling overlaps with the gibberellin pathway: treatment with gibberellin biosynthetic inhibitors reduced the proportion of males in gametophyte populations, implying shared or overlapping biosynthesis steps. Abscisic acid acts in the opposite direction, inhibiting antheridiogen-induced antheridium formation and male development, and mutants insensitive to abscisic acid allow male differentiation to proceed even when the hormone is present. One of the corresponding genes, GAIA1, encodes a close homolog of the Arabidopsis SnRK2 signaling component OST1.

Forward genetic screens have yielded a collection of sex-type mutants that map out the decision logic. Hermaphroditic (her) mutants ignore antheridiogen and develop as hermaphrodites regardless. Transformer (tra) mutants lose both meristem and archegonium formation, becoming ameristic males even under conditions that normally favor hermaphrodite development. Feminization (fem) mutants fail to make antheridia and instead undergo meristic female development. More recently, her7 mutations were traced to a brassinosteroid receptor-like kinase homolog on chromosome 29; five independent alleles carry missense mutations or small amino acid deletions in this gene. The HER7 protein localizes to the plasma membrane and cytoplasm, consistent with a receptor kinase role, and transcriptomic comparisons between wild type and her7 gametophytes revealed widespread antheridiogen-responsive gene expression changes, many dependent on HER7, including genes involved in brassinosteroid and gibberellin biosynthesis and signaling.

Perhaps the most striking recent molecular story concerns the maintenance of hermaphrodite identity. Once specified, hermaphrodites become insensitive to antheridiogen, whether self-produced or released by neighbors. Without this insensitivity, male development would dominate entire populations. The review highlights the HAIRY MERISTEM family GRAS-domain transcriptional regulator CrHAM as a central player. CrHAM protein preferentially accumulates in the meristems of hermaphrodites but is excluded from antheridia in males. Knockdown of CrHAM increased the male ratio in gametophyte populations and impaired hermaphrodite development, producing smaller prothalli, reduced meristem size and shallower meristem notches, with archegonia absent or greatly reduced and antheridia forming ectopically around and even within the meristem.

The functional data are consistent at the level of gene expression. Male-specific GAMYB genes were up-regulated in CrHAM knockdown gametophytes, and in antheridiogen treatment assays a considerable number of young knockdown hermaphrodites failed to maintain meristem activity and gradually converted into ameristic males, explaining the elevated male ratio. Transcriptomic analysis of differentially expressed genes pointed to roles in cell growth and prothallus expansion, and knockdown plants showed reduced size and cell number due to decreased division activity. A CLAVATA1 homolog, CrCLV1a, and an auxin biosynthesis gene, the YUCCA homolog CrYUC1, were downregulated in knockdown hermaphrodites; both are normally expressed at higher levels in hermaphrodites than in males, paralleling the meristem-specific roles of their counterparts in the liverwort Marchantia.

Spatial regulation adds another layer. A transcriptional reporter showed the CrHAM promoter active in nearly all cells of the hermaphrodite prothallus, yet the protein itself is enriched specifically at the meristem, raising the question of what controls this restriction. In Arabidopsis, type II HAM genes are negatively regulated by microRNA171, which is expressed in the epidermis and moves a limited distance into deeper layers, generating a gradient of HAM protein. The CrHAM coding sequence contains a miR171-binding site identical to that in the Arabidopsis HAM1-3 genes, and two Ceratopteris MIR171 genes, CrMIR171B and CrMIR171C, produce mature microRNAs nearly identical to their Arabidopsis counterparts. Overexpression of CrMIR171B reduced CrHAM expression and produced defects resembling CrHAM knockdown, including an increased male ratio and smaller prothalli with shallower meristem notches, evidence for a conserved yet lineage-specific miR171-HAM module in ferns.

The review also covers the remarkable plasticity of the male fate. When antheridiogen is removed or depleted, male identity cannot be maintained, and male gametophytes initiate de novo meristems and produce archegonia, converting into hermaphrodites. Recent work combining long-term, non-invasive time-lapse confocal imaging with computational analysis of a ubiquitous fluorescent nuclear marker reconstructed the full lineage trajectory of this conversion. It showed that the entire new meristem descends from a single non-antheridium cell, the meristem progenitor cell, whose lineage keeps dividing while other lineages become mitotically inactive. A mathematical model demonstrated that stochastic cell division combined with inhibitory signals from actively dividing cells is sufficient to establish the progenitor lineage, and treatment with the S-phase inhibitor aphidicolin disrupted conversion and shrank the resulting meristems, confirming that cell cycle re-entry is essential.

Division geometry matters as well. The orientation of the progenitor cell’s first division determines the proliferative behavior of its descendants: an anticlinal division yields two outer-layer lineages with similarly high activity, while a periclinal division produces one highly active outer daughter and one less active inner one. Once de novo meristems are established, outermost-layer cells divide significantly more often than inner cells, matching earlier observations in normal hermaphrodite meristems and recent findings in the epiphytic fern Phlebodium pseudoaureum, where the outermost layer likewise drives de novo meristem formation. The authors argue that these shared strategies, driven by positional signals from the outermost layer, are conserved across fern species from diverse taxa. Looking forward, they identify hormonal crosstalk among gibberellins, abscisic acid and brassinosteroids, the function and regulation of the Ceratopteris MIR171 family, and the integration of stable transformation, forward genetics, quantitative live imaging, deep learning-based image analysis, computational modeling and multi-omics approaches as the directions most likely to yield a comprehensive picture of the regulatory logic underlying fern sex determination and meristem activity.

Subject of Research: Sex determination and meristem development in fern gametophytes

Article Title: Sex-type specification and meristem development in fern gametophytes

Article References: Lai, D. N., & Zhou, Y. (2025). Sex-type specification and meristem development in fern gametophytes. Discover Developmental Biology, 236(1), Article 1. https://doi.org/10.1007/s00427-025-00728-6

Image Credits: AI Generated

DOI: 10.1007/s00427-025-00728-6

Keywords: fern gametophytes, sex determination, antheridiogen, Ceratopteris richardii, meristem, CrHAM, miR171, gibberellin, abscisic acid, brassinosteroid, cell lineage, hermaphrodite