1 of 12 Aquatic Conservation: Marine and Freshwater Ecosystems, 2025; 35:e70070 https://doi.org/10.1002/aqc.70070 Aquatic Conservation: Marine and Freshwater Ecosystems ARTICLE A Simple and Effective Method for the Cryopreservation of Quillwort Spores Sérgio L. Cavalheiro-Filho 1 | Naiara V. Campos 1 | Anderson R. Gripp 1 | Laís B. Jordão 1 | Allysson B. Cavalcante 2 | Francisco A. Esteves 1 | Rodrigo L. Martins 1 | Lísia M. S. Gestinari 1 1 Instituto de Biodiversidade e Sustentabilidade NUPEM, Universidade Federal do Rio de Janeiro, Macaé, Brazil | 2 Vale S.A., Carajás, Brazil Correspondence: Naiara V. Campos (naiaravcampos@gmail.com) Received: 9 September 2024 | Revised: 1 November 2024 | Accepted: 13 January 2025 Funding: This work was supported by Fundação Coordenação de Projetos, Pesquisas e Estudos Tecnológicos (Project Nº 23513 and 22016). Keywords: desiccation | endangered species | freshwater | germplasm | Isoëtes | mining | new techniques | spore viability | ultrafreezing ABSTRACT Quillworts are highly threatened worldwide and many Isoëtes species have been suffering population decreases. Isoëtes cangae is a lycophyte endemic from a single Amazon lake that has been challenging due to climate change and deforestation of the Amazon rainforest. The development of strategies for ex situ conservation of lycophyte germplasm is urgent to prevent biodi- versity losses. Spores of I. cangae were obtained from specimens collected in situ. Samples were submitted to three different moisture conditions: hydrated, air-dry desiccation and drying in silica gel. Desiccated samples were stored in an ultrafreezer (−80°C) or liquid nitrogen (−196°C) and the hydrated spores at room temperature. The spore viability was evaluated through spore germination (G) and sporeling emergence (SE) indices after 30, 90, 180 and 275 days of storage. The growth of emerged spo- rophytes was evaluated after 3 months. Desiccated–cryopreserved spores had high G% and SE%, even after 275 days of storage, and did not differ from hydrated spores. Hydrated spores had higher daily emergence and a longer time to maximum sporophyte emergence than desiccated spores, but not in all storage periods. Sporophytes that emerged from hydrated and desiccated spores had a similar development. Both desiccated methods and temperatures of storage maintained spore viability high. The method- ologies tested in this study are novel for the genus and represent a significant step towards the ex situ conservation of quillworts. 1 | Introduction Ferns and lycophytes are two distinct lineages of spore-bearing vascular plants comprising ca. 12,000 species (PPG I 2016; Sessa and Der 2016). Brazil harbours a substantial number of 1412 species of ferns and lycophytes (Flora e Funga do Brasil 2024). Although lycophytes represent only 10% of these taxa, the family Isoetaceae has been the target of recent worldwide studies and con- sequently, the number of taxa and endemic species both increased for the group (Pereira, Guimaraes, and Watanabe 2019; Brunton and Pereira 2020; Brunton et al. 2019; Brunton, Garrett, et al. 2021; Brunton, Ivanova, and Sokoloff 2021; Zheng et al. 2020; Fischer and Lobin 2022). The genus Isoëtes hosts 29 species, with a remark- able 22 endemic to the country (Prado et al. 2015; Pereira 2024). Isoëtes L., whose representatives are known as quillworts, constitutes a unique genus of extant plants within the Isoetaceae (Larsén and Rydin 2016). Two Brazilian species of Isoëtes are endemic to the ironstone outcrops of Serra dos Carajás (Pará state, Brazil): Isoëtes cangae J.B.S. Pereira, Salino & Stützel and Isoëtes serracarajensis J.B.S. Pereira, Salino & Stützel (Pereira et al. 2016). These species differ re- garding their area of occurrence, habitat, morphoanatomy, ecophysiology and reproductive strategies (Pereira et al. 2016; Caldeira et al. 2021; Cavalheiro-Filho et al. 2021; Zandonadi et al. 2021). I. cangae is a submerged species that occurs exclu- sively in the Amendoim Lake while I. serracarajensis has an amphibian habit and is found in upland swamps and ponds (Pereira et al. 2016). © 2025 John Wiley & Sons Ltd.