The fern Dryopteris affinis ssp. affinis reproduces by obligate apogamous apomixis: unreduced (diploid) spores are formed through altered meiosis (diplospory) and the sporophyte embryo arises directly from a somatic cell of the gametophyte without fertilization (apogamy). A transcriptomic study of its gametophyte (Ojosnegros et al., 2024) identified more than a dozen candidate apomixis-related proteins homologous to Arabidopsis reproductive regulators — ARGONAUTE 4, 9 and 10, BABY BOOM, FERTILIZATION-INDEPENDENT ENDOSPERM, MSI1, and others — together with a striking set of flowering-, seed- and fruit-development gene homologs in a non-flowering, non-seed plant. None of these candidates has been functionally tested. The open problem is a mechanistic account of apogamous apomixis in ferns: (1) whether the identified candidates are causally involved in apogamy and diplospory; (2) whether the flowering/seed/fruit-development homologs are silenced relics or have been co-opted into asexual reproduction or gametophyte development; and (3) the molecular pathway leading from an unreduced spore to an apogamous embryo. No functional characterization in any apomictic fern has been reported to date; adjacent work in the sexual model fern Ceratopteris supports deep conservation of individual reproductive regulators but does not answer these questions.

Background

Land plants alternate between two free-living or embedded generations: a diploid sporophyte, which forms spores by meiosis, and a haploid gametophyte, which forms gametes by mitosis. In seed plants the gametophyte is reduced and enclosed in ovules and pollen, whereas in ferns it is a small, free-living, photosynthetic plant (the prothallus) that bears the male (antheridia) and female (archegonia) gametangia. Apomixis is asexual reproduction through the generative cycle, bypassing meiosis and fertilization; in ferns it combines diplospory (formation of unreduced spores through altered meiosis, so the gametophyte inherits the sporophyte's ploidy) and apogamy (formation of a sporophyte embryo directly from a somatic cell of the gametophyte, without fertilization). Apomixis is estimated to occur in about 10% of fern species, a higher frequency than in any other land-plant group. The European wood fern Dryopteris affinis ssp. affinis is obligately apogamous: its unreduced spores germinate into diploid gametophytes that produce antheridia but no archegonia, so the sporophyte can arise only from a gametophytic cell. A transcriptomic analysis of this gametophyte (Ojosnegros et al., 2024; ref. 1 is the preprint version of this work) annotated roughly one hundred Arabidopsis thaliana homologs and highlighted candidate apomixis-related proteins: ARGONAUTE 4, 9 and 10 (AGO; components of small-RNA-guided gene silencing, in which 20–24 nucleotide RNAs direct the repression of complementary genes), BABY BOOM (BBM; an AP2/ERF-family transcription factor whose ectopic expression induces somatic embryogenesis in Brassica napus and Arabidopsis), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MSI1 (both components of Polycomb Repressive Complex 2, a chromatin-silencing complex whose Arabidopsis mutants can initiate embryo or endosperm development without fertilization), and the RNA helicases FAS4, SWA1 and MEE29, together with MADS-box transcription factors of the AGL6 and AGL62 type (AGL = AGAMOUS-LIKE; MADS-box genes encode a large family of transcription factors with central roles in flowering, seed and fruit development in angiosperms). The same study reported homologs of Arabidopsis flowering-, seed- and fruit-development genes (e.g., FPF1, GA20OX1, KNAT1, TFL1, UFO, VIN3, MAN6, WOX13) in this non-flowering, non-seed plant, echoing an earlier report of flowering-related gene expression in the fern Ceratopteris richardii (Cordle et al., 2012). Two prior results anchor the functional questions: overexpression of the AINTEGUMENTA homolog CrANT promotes apogamy in C. richardii, indicating genetic conservation between fern apogamy and angiosperm somatic embryogenesis (Bui et al., 2017), and genome-scale analyses of C. richardii inferred that seed-development genes were co-opted from genes controlling fern sporangium development (Marchant et al., 2022). However, no apomictic fern — including D. affinis — has a transformation or genome-editing protocol, so every claim about the candidates' functions in apogamy has so far rested on homology, expression, predicted protein-protein interactions, and phylogenetic distance alone.

Problem Statement

Provide a mechanistic account of apogamous apomixis in the fern Dryopteris affinis — the molecular basis of gametophytic development, including apomixis, whose genomic machinery remains to be deciphered — answering three jointly linked questions that together define one research objective. (1) Causal involvement: determine whether the identified candidate proteins (ARGONAUTE 4, 9 and 10, BABY BOOM, FERTILIZATION-INDEPENDENT ENDOSPERM, MSI1, and the further annotated candidates such as FAS4, SWA1, MEE29, AGL6 and AGL62) are causally involved in apogamy and/or in the formation of unreduced spores. (2) Functional status of flowering/seed/fruit-development gene homologs: establish whether these homologs in this non-flowering, non-seed plant are silenced relics without current function or have acquired roles in asexual reproduction or gametophyte development. (3) Pathway: decipher the molecular pathway that leads from an unreduced spore (diplospory) to an apogamous embryo, connecting the meiotic and embryogenesis-initiation modules across the sporophyte and gametophyte generations. Together, these questions are aspects of a single molecular-mechanistic account of apogamous apomixis in D. affinis. The fern-versus-angiosperm conservation context — genetic conservation between fern apogamy and angiosperm somatic embryogenesis (Bui et al., 2017) and the proposed common genomic pathways in organ development between ferns and seed plants — motivates this account evolutionarily but is not itself a required deliverable.

References

  1. Ojosnegros S, Alvarez JM, Gagliardini V, Quintanilla LG, Grossniklaus U, Fernández H (2024) Transcriptomic analyses in the gametophyte of Dryopteris affinis: apomixis and more. bioRxiv preprint, doi:10.1101/2024.03.15.585305, https://doi.org/10.1101/2024.03.15.585305 (preprint version of ref. 2)
  2. Ojosnegros S, Alvarez JM, Gagliardini V, Quintanilla LG, Grossniklaus U, Fernández H (2024) Transcriptomic analyses in the gametophytes of the apomictic fern Dryopteris affinis. Planta 260:111, doi:10.1007/s00425-024-04540-w, https://doi.org/10.1007/s00425-024-04540-w (published version of ref. 1)
  3. Fernández H, Ojosnegros S, Álvarez JM, Vázquez F, Goya N, Quintanilla LG, Flexas J, Peña A, Grossniklaus U (2026) Comprehensive in silico characterization of proteins associated with light responses, transport, and stress-related processes in the gametophyte of the fern Dryopteris affinis ssp. affinis. BMC Genomics 27:508, doi:10.1186/s12864-026-12935-0, https://doi.org/10.1186/s12864-026-12935-0
  4. Bui LT, Pandzic D, Youngstrom CE, Wallace S, Irish EE, Szövényi P, Cheng C-L (2017) A fern AINTEGUMENTA gene mirrors BABY BOOM in promoting apogamy in Ceratopteris richardii. The Plant Journal 90(1):122-132, doi:10.1111/tpj.13479, https://doi.org/10.1111/tpj.13479
  5. Cordle AR, Irish EE, Cheng C-L (2012) Gene expression associated with apogamy commitment in Ceratopteris richardii. Sexual Plant Reproduction 25:293-304, doi:10.1007/s00497-012-0198-z, https://doi.org/10.1007/s00497-012-0198-z
  6. Marchant DB, Chen G, Cai S, Chen F, Schafran P, Jenkins J, Shu S, Plott C, Webber J, Lovell JT, He G, Sandor L, Williams M, Rajasekar S, Healey A, Barry K, Zhang Y, Sessa E, Dhakal RR, Wolf PG, Harkess A, Li F-W, Rössner C, Becker A, Gramzow L, Xue D, Wu Y, Tong T, Wang Y, Dai F, Hua S, Wang H, Xu S, Xu F, Duan H, Theißen G, McKain M, Li Z, McKibben MTW, Barker MS, Schmitz RJ, Stevenson DW, Zumajo-Cardona C, Ambrose BA, Leebens-Mack JH, Grimwood J, Schmutz J, Soltis PS, Soltis DE, Chen Z-H (2022) Dynamic genome evolution in a model fern. Nature Plants 8:1038-1051, doi:10.1038/s41477-022-01226-7, https://doi.org/10.1038/s41477-022-01226-7
  7. McConnell H, Lanclos JR, Willis K, Gjording N, Stockmann G, Lind C, Maloof JN, Plackett AR, Di Stilio VS (2026) LEAFY demonstrates functions in reproductive development of the gametophyte but not the sporophyte of the fern Ceratopteris richardii. Development 153:dev204808, doi:10.1242/dev.204808, https://doi.org/10.1242/dev.204808
  8. Plackett ARG, Catoni M (2025) Gene networks are conserved across reproductive development between the fern Ceratopteris richardii and the flowering plant Arabidopsis thaliana. bioRxiv preprint, doi:10.1101/2025.03.18.643782, https://doi.org/10.1101/2025.03.18.643782
  9. Ekrt L, Férová A, Koutecký P, Vejvodová K, Hori K, Hornych O (2024) An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns. American Journal of Botany 111(5):e16332, doi:10.1002/ajb2.16332, https://doi.org/10.1002/ajb2.16332
  10. Hörandl E (2024) Apomixis and the paradox of sex in plants. Annals of Botany 134(1):1-18, doi:10.1093/aob/mcae044, https://doi.org/10.1093/aob/mcae044
  11. Kawakami SM, Kawakami S (2024) Unreduced spore formation in a spontaneous chimeric pinnule in an artificially produced haploid Anisocampium niponicum (Athyriaceae, Polypodiales). Journal of Plant Research 137(2):161-165, doi:10.1007/s10265-023-01511-2, https://doi.org/10.1007/s10265-023-01511-2
  12. Woudenberg S, Dipp Alvarez M, Rienstra J, Levitsky V, Mironova V, Scarpella E, Kuhn A, Weijers D (2024) Analysis of auxin responses in the fern Ceratopteris richardii identifies the developmental phase as a major determinant for response properties. Development 151:dev203026, doi:10.1242/dev.203026, https://doi.org/10.1242/dev.203026
  13. Schulz R, Theißen G (2025) Ceratopteris richardii U6 promoters and plants expressing Cas9 endonuclease as tools for efficient genome editing in a fern. bioRxiv preprint, doi:10.1101/2025.01.12.632625, https://doi.org/10.1101/2025.01.12.632625