Symplasmic Isolation Contributes to Somatic Embryo Induction and Development in the Tree Fern Cyathea delgadii Sternb.

In this report, we describe studies on symplasmic communication and cellular rearrangement during direct somatic embryogenesis (SE) in the tree fern Cyathea delgadii. We analyzed changes in the symplasmic transport of low-molecular-weight fluorochromes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) and fluorescein (delivered to cells as fluorescein diacetate, FDA), within stipe explants and somatic embryos originating from single epidermal cells and developing during 16-d long culture. Induction of SE is preceded by a restriction in fluorochrome distribution between certain explant cells. Microscopic analysis showed a series of cellular changes like a decrease in vacuole size, increase in vacuole numbers, and increased density of cytoplasm and deposition of electron-dense material in cell walls that may be related with embryogenic transition. In somatic embryos, the limited symplasmic communication between cells was observed first in linear tri-cellular embryos. Further development of the fern embryo was associated with the formation of symplasmic domains corresponding to the four segments of the plant body. Using symplasmic tracers, we provided evidence that the changes in plasmodesmata permeability are corelated with somatic-to-embryogenic transition and somatic embryo development.

Weedy ferns (Polypodiopsida) in Argentina: diversity, distribution and impact on human activities and ecosystems.

Currently, in Argentina 368 species of true ferns (i.e. Polypodiopsida class) are distributed throughout the country, however, only four of them have been mentioned until now as weeds and ruderal species. The goal of this work was to generate an update of weedy ferns from Argentina, including morphology, distribution, and type of weed according to their impact on natural habitats and/or human activities. All Argentinian fern species were analyzed based on references, herbarium specimens, and field trips. As a result of our study 25 species were recorded from Argentina and classified as segetal, ecological, or aquatic weeds, and ruderal and/or toxic species. Current taxonomic identity, diagnostic characters, origins, habitats, geographical distribution, common names, and impact and potential risks were indicated by species. In addition, we provide a dichotomous key to species, presence of these species in southern South American countries, as well as and photographs in natural habitat. This work represents the first review on native and exotic ferns from Argentina that cause an impact on human activities or disturbe native habitats. The results provide information for the development of weed management tools and priority areas to implement them.

Simple and Divided Leaves in Ferns: Exploring the Genetic Basis for Leaf Morphology Differences in the Genus Elaphoglossum (Dryopteridaceae).

Despite the implications leaves have for life, their origin and development remain debated. Analyses across ferns and seed plants are fundamental to address the conservation or independent origins of megaphyllous leaf developmental mechanisms. Class I KNOX expression studies have been used to understand leaf development and, in ferns, have only been conducted in species with divided leaves. We performed expression analyses of the Class I KNOX and Histone H4 genes throughout the development of leaf primordia in two simple-leaved and one divided-leaved fern taxa. We found Class I KNOX are expressed (1) throughout young and early developing leaves of simple and divided-leaved ferns, (2) later into leaf development of divided-leaved species compared to simple-leaved species, and (3) at the leaf primordium apex and margins. H4 expression is similar in young leaf primordia of simple and divided leaves. Persistent Class I KNOX expression at the margins of divided leaf primordia compared with simple leaf primordia indicates that temporal and spatial patterns of Class I KNOX expression correlate with different fern leaf morphologies. However, our results also indicate that Class I KNOX expression alone is not sufficient to promote divided leaf development in ferns. Class I KNOX patterns of expression in fern leaves support the conservation of an independently recruited developmental mechanism for leaf dissection in megaphylls, the shoot-like nature of fern leaves compared with seed plant leaves, and the critical role marginal meristems play in fern leaf development.

Meristem development and activity in gametophytes of the model fern, Ceratopteris richardii.

Ceratopteris richardii is a model fern species widely used to analyze various developmental processes and their regulation in gametophytes. The form of mature C. richardii gametophytes depends on the activity of the marginal meristem, but knowledge on meristem formation and structure is limited. Therefore, we analyzed cellular events accompanying the development of gametophytes using cell lineage and proliferation analyses to explain the establishment and functioning of the marginal meristem. We show that: i) gametophytes are devoid of the apical initial cell or the apical cell-based meristem in the early developmental stages; ii) the cells that are predestined to form the marginal meristem divide according to a stable pattern; iii) only one transient initial cell is present in the marginal meristem, and the selection of a new functioning initial cell is related to a stable sequence of its divisions. Our results contribute to a better understanding of the developmental events underlying gametophyte growth and marginal meristem functioning in Ceratopteris. The principles, which were established in this study and enabled the identification of functioning initial cells, can be applied to analyze genetic and/or physiological mechanism(s) governing meristem maintenance in vascular plants, both in developmental and evolutionary contexts.

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales. Two distinct nearly full-length Rhizobiales genomes were identified in leaf-pocket-enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2 -fixation. 15 N2 incorporation was active in ferns with N. azollae but not in ferns without. N2 O was not detectably released from surface-sterilized ferns with the Rhizobiales. N2 -fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2 O.

Fern Stomatal Responses to ABA and CO2 Depend on Species and Growth Conditions.

Changing atmospheric CO2 levels, climate, and air humidity affect plant gas exchange that is controlled by stomata, small pores on plant leaves and stems formed by guard cells. Evolution has shaped the morphology and regulatory mechanisms governing stomatal movements to correspond to the needs of various land plant groups over the past 400 million years. Stomata close in response to the plant hormone abscisic acid (ABA), elevated CO2 concentration, and reduced air humidity. Whether the active regulatory mechanisms that control stomatal closure in response to these stimuli are present already in mosses, the oldest plant group with stomata, or were acquired more recently in angiosperms remains controversial. It has been suggested that the stomata of the basal vascular plants, such as ferns and lycophytes, close solely hydropassively. On the other hand, active stomatal closure in response to ABA and CO2 was found in several moss, lycophyte, and fern species. Here, we show that the stomata of two temperate fern species respond to ABA and CO2 and that an active mechanism of stomatal regulation in response to reduced air humidity is present in some ferns. Importantly, fern stomatal responses depend on growth conditions. The data indicate that the stomatal behavior of ferns is more complex than anticipated before, and active stomatal regulation is present in some ferns and has possibly been lost in others. Further analysis that takes into account fern species, life history, evolutionary age, and growth conditions is required to gain insight into the evolution of land plant stomatal responses.

Are rates of species diversification and body size evolution coupled in the ferns?

PREMISE OF THE STUDY: Understanding the relationship between phenotypic evolution and lineage diversification is a central goal of evolutionary biology. To extend our understanding of the role morphological evolution plays in the diversification of plants, we examined the relationship between leaf size evolution and lineage diversification across ferns. METHODS: We tested for an association between body size evolution and lineage diversification using a comparative phylogenetic approach that combined a time-calibrated phylogeny and leaf size data set for 2654 fern species. Rates of leaf size change and lineage diversification were estimated using BAMM, and rate correlations were performed for rates obtained for all families and individual species. Rates and patterns of rate-rate correlation were also analyzed separately for terrestrial and epiphytic taxa. KEY RESULTS: We find no significant correlation between rates of leaf area change and lineage diversification, nor was there a difference in this pattern when growth habit is considered. Our results are consistent with the findings of an earlier study that reported decoupled rates of body size evolution and diversification in the Polypodiaceae, but conflict with a recent study that reported a positive correlation between body size evolution and lineage diversification rates in the tree fern family Cyatheaceae. CONCLUSIONS: Our findings indicate that lineage diversification in ferns is largely decoupled from shifts in body size, in contrast to several other groups of organisms. Speciation in ferns appears to be primarily driven by hybridization and isolation along elevational gradients, rather than adaptive radiations featuring prominent morphological restructuring. The exceptional diversity of leaf morphologies in ferns appears to reflect a combination of ecophysiological constraints and adaptations that are not key innovations.

Alternative stable states explain unpredictable biological control of Salvinia molesta in Kakadu.

Suppression of the invasive plant Salvinia molesta by the salvinia weevil is an iconic example of successful biological control. However, in the billabongs (oxbow lakes) of Kakadu National Park, Australia, control is fitful and incomplete. By fitting a process-based nonlinear model to thirteen-year data sets from four billabongs, here we show that incomplete control can be explained by alternative stable states--one state in which salvinia is suppressed and the other in which salvinia escapes weevil control. The shifts between states are associated with annual flooding events. In some years, high water flow reduces weevil populations, allowing the shift from a controlled to an uncontrolled state; in other years, benign conditions for weevils promote the return shift to the controlled state. In most described ecological examples, transitions between alternative stable states are relatively rare, facilitated by slow-moving environmental changes, such as accumulated nutrient loading or climate change. The billabongs of Kakadu give a different manifestation of alternative stable states that generate complex and seemingly unpredictable dynamics. Because shifts between alternative stable states are stochastic, they present a potential management strategy to maximize effective biological control: when the domain of attraction to the state of salvinia control is approached, augmentation of the weevil population or reduction of the salvinia biomass may allow the lower state to trap the system.

Rates of ecological divergence and body size evolution are correlated with species diversification in scaly tree ferns.

Variation in species richness across regions and between different groups of organisms is a major feature of evolution. Several factors have been proposed to explain these differences, including heterogeneity in the rates of species diversification and the age of clades. It has been frequently assumed that rapid rates of diversification are coupled to high rates of ecological and morphological evolution, leading to a prediction that remains poorly explored for most species: the positive association between ecological niche divergence, morphological evolution and species diversification. We combined a time-calibrated phylogeny with distribution, ecological and body size data for scaly tree ferns (Cyatheaceae) to test whether rates of species diversification are predicted by the rates at which clades have evolved distinct ecological niches and body sizes. We found that rates of species diversification are positively correlated with rates of ecological and morphological evolution, with rapidly diversifying clades also showing rapidly evolving ecological niches and body sizes. Our results show that rapid diversification of scaly tree ferns is associated with the evolution of species with comparable morphologies that diversified into similar, yet distinct, environments. This suggests parallel evolutionary pathways opening in different tropical regions whenever ecological and geographical opportunities arise. Accordingly, rates of ecological niche and body size evolution are relevant to explain the current patterns of species richness in this 'ancient' fern lineage across the tropics.

From phytoaccumulation to post-harvest use of water fern for landfill management.

We examined the potential of Azolla japonica as a remediating plant for leachate channels and post-accumulation use as fertilizer for landfill slope. The harvested biomass of Azolla after one month grown in leachate was 254% that of the initial biomass and the predicted annual harvestable biomass of Azolla using a growth model was 32 times that of the initial biomass. Na, Fe, Mn, Mg, and P were accumulated in Azolla at very high concentrations. Such rapid increase of biomass and high accumulation rates suggest that this plant could be an excellent remediating plant. The post-harvest use of Azolla as compost was studied for the management and use of phytoaccumulating Azolla. Metal contents of Azolla compost were below permissible limits for co-composting material. Nitrogen, organic matter, P, and Mg content of the Azolla compost improved the soil condition of the landfill and enhanced ecophysiological responses of the plants. The application of Azolla compost can improve management of sanitary landfills, including the restoration of vegetation. Considering its ease of harvesting, high accumulation rates, harvestable biomass and suitability for composting, Azolla can provide a suitable solution for sustainable management of leachate channels and landfill slopes.

Higher clonal integration in the facultative epiphytic fern Selliguea griffithiana growing in the forest canopy compared with the forest understorey.

BACKGROUND AND AIMS: The advantage of clonal integration (resource sharing between connected ramets of clonal plants) varies and a higher degree of integration is expected in more stressful and/or more heterogeneous habitats. Clonal facultative epiphytes occur in both forest canopies (epiphytic habitats) and forest understories (terrestrial habitats). Because environmental conditions, especially water and nutrients, are more stressful and heterogeneous in the canopy than in the understorey, this study hypothesizes that clonal integration is more important for facultative epiphytes in epiphytic habitats than in terrestrial habitats. METHODS: In a field experiment, an examination was made of the effects of rhizome connection (connected vs. disconnected, i.e. with vs. without clonal integration) on survival and growth of single ramets, both young and old, of the facultative epiphytic rhizomatous fern Selliguea griffithiana (Polypodiaceae) in both epiphytic and terrestrial habitats. In another field experiment, the effects of rhizome connection on performance of ramets were tested in small (10 × 10 cm(2)) and large (20 × 20 cm(2)) plots in both epiphytic and terrestrial habitats. KEY RESULTS: Rhizome disconnection significantly decreased survival and growth of S. griffithiana in both experiments. The effects of rhizome disconnection on survival of single ramets and on ramet number and growth in plots were greater in epiphytic habitats than in terrestrial habitats. CONCLUSIONS: Clonal integration contributes greatly to performance of facultative epiphytic ferns, and the effects were more important in forest canopies than in forest understories. The results therefore support the hypothesis that natural selection favours genotypes with a higher degree of integration in more stressful and heterogeneous environments.

Sensitivity of three tree ferns during their first phase of life to the variation of solar radiation and water availability in a Mexican cloud forest.

PREMISE OF THE STUDY: Regeneration niche differentiation promotes species coexistence and diversity; however, the ecological implications for the initial life phases of the majority of pteridophytes are unknown. We analyzed the sensitivity of gametophytes and juvenile sporophytes of the tree ferns Alsophila firma, Cyathea divergens, and Lophosoria quadripinnata to variation in light and water availability. METHODS: We evaluated gametophyte desiccation tolerance using saturated salt solutions and gametophyte solar radiation tolerance by direct exposure. We also transplanted juvenile sporophytes in environments with 7% and 23% canopy openness and two watering levels. KEY RESULTS: The response of photosynthetic efficiency and water content suggest that the gametophytes of the three species require high relative humidity, tolerate direct solar radiation for up to 30 min and that the response is not species-dependent. Sporophyte size and gas exchange were greater in the more open site, but decreased watering had a lesser effect on these variables in the more closed site. Relative growth rate correlated with the net assimilation rate and leaf weight ratio. Juvenile sporophytes of A. firma were more shade tolerant, while those of C. divergens and L. quadripinnata acclimatized to both environments. CONCLUSIONS: Specialization to humid habitats in the tree fern gametophyte restricts the species to humid forests, while differences in the plasticity of the sporophyte facilitate coexistence of the species.

Developmental morphology of the typical cordate gametophyte of a homosporous leptosporangiate fern, Lygodium japonicum (Lygodiaceae), focusing on the initial cell behavior of two distinct meristems.

PREMISE OF THE STUDY: Understanding the origin and early evolution of vascular plants requires thorough consideration of the gametophyte generation of ferns and lycophytes. Unfortunately, information about this generation is quite limited. To reveal the origin and evolution of varied gametophyte shapes, we used comparative morphological studies of meristem behavior of gametophytes of Lygodium japonicum, which exhibit the typical cordate shape. METHODS: Microscopic images of epi-illuminated growing gametophytes cultured from spores were captured periodically using a metallurgical microscope equipped with a digital camera to analyze the cell lineage in the meristem. KEY RESULTS: Gametophytes form from two meristems: the apical-cell-based meristem and the multicellular meristem. The triangular apical cell produces six to eight derivatives from two lateral facets, then disappears. Subsequently, the multicellular meristem, with a row of several rectangular cells, forms in the notch. These rectangular cells divide asynchronously in the periclinal and anticlinal walls to produce cells to both lateral sides and downward. Usually two, and sometimes three, cells located at the center of the meristem divide at a slower pace in the periclinal and anticlinal planes than others at the periphery. The cells at the periphery are pushed away and become involved in the wing base. CONCLUSIONS: The triangular apical cell behaves as a permanent initial cell. In the multicellular meristem, however, two or three central cells behave as initial cells that are transient and regulated in a position-dependent manner. The organization and behavior of both meristems are shared with the ribbon-shaped gametophytes of Colysis.

Effects of the naturally-occurring contaminant microcystins on the Azolla filiculoides-Anabaena azollae symbiosis.

Harmful algal blooms (HABs) contaminate aquatic ecosystems and are responsible for animal poisoning worldwide. We conducted a toxicity test with the aquatic fern and the biofertilizer, Azolla filiculoides. The sporophytes were exposed to three concentrations (0.01, 0.1 and 1µgmL(-1)) of a microcystin (MC) cyanobacterial crude extract and purified MC-LR. The growth of A. filiculoides decreased only at 1µgmL(-1) crude extract concentration while with MC-LR it decreased at all the tested concentrations, indicating that the presence of other compounds in the crude extract altered toxicity and stimulated the fern growth at lower concentrations (0.01 and 0.1µgmL(-1)). Both phycoerythrocyanin and allophycocyanin levels decreased in all the concentrations of crude extract and MC-LR. The phycocyanin had a marked increase at 0.1µgmL(-1) crude extract concentration and a marked decrease at 1µgmL(-1) MC-LR concentration. These changes in the phycobiliprotein content indicate a shift in the antenna pigments of the cyanobionts of A. filiculoides. The changes in two oxidative stress enzymes, glutathione reductase for the crude extract assay and glutathione peroxidase for MC-LR assay, points towards the induction of stress defense responses. The low bioconcentration factor in both crude extract and MC-LR treatments can suggest the low uptake of microcystins, and indicates that the aquatic fern can be used as a biofertilizer and as animal feed but is not suitable for MC phytoremediation.

Biochemical and growth performance of the aquatic macrophyte Azolla filiculoides to sub-chronic exposure to cylindrospermopsin.

Physiological and biochemical effects of cylindrospermopsin (CYN), a cyanobacterial toxin that inhibits protein synthesis and released during a harmful cyanobacterial bloom, has been overlooked in plants. Therefore, at the present research, the toxic effects (physiological and biochemical) of a crude extract containing CYN were assessed in the aquatic fern Azolla filiculoides exposed to three concentrations (0.05, 0.5 and 5 µg CYN mL(-1)). At 5 µg CYN mL(-1), fern growth rate has showed a drastic decrease (0.001 g g(-1) day(-1)) corresponding to a 99.8% inhibition, but at the concentrations of 0.05 and 0.5 µg CYN mL(-1) the growth rate was similar to the control plants. Growth rate also indicated a IC50 of 2.9 µg CYN mL(-1). Those data point to the presence of other compounds in the crude extract may stimulate the fern growth and/or the fern is tolerant to CYN. Chlorophyll (a and b), carotenoids and protein content as well as the activities of glutathione reductase (GR) and glutathione-S-transferase (GST) has increased at 5 µg CYN mL(-1) which may indicate that photosynthesis and protein synthesis are not affected by CYN and the probable activation of defense and detoxifying mechanisms to overcome the effects induced by the presence of CYN. Low uptake of cylindrospermopsin (1.314 µg CYN g(-1) FW) and low bioconcentration factor (0.401) point towards to a safe use of A. filiculoides as biofertilizer and as food source, but also indicate that the fern is not suitable for CYN phytoremediation.

[Investigation on resource and quality assessment of Cibotii Rhizoma].

The study is aimed to understand the resource and the current situation of the use of Cibotii Rhizoma and provide the basis for protecting and utilization. The method of literature survey, field survey and quality assessment were applied in the study. The results showed that all the Cibotii Rhizoma came from wild resource and was mainly founded in Fujian, Guangxi, Guizhou, Yunnan, Guangdong, Hunan, Jiangxi, Sichuan, Chongqing, Zhejiang, etc. It contains over 5 000 000 kg in the area which total is about 7 000 hm2. The annual output is over 850 000 kg. At present, there is no cultivated resources. Based on the investigation and market sampling analysis from various regions, the results showed that the quality of the collected crude drugs conformed with the regulations of the Chinese pharmacopoeia. However the qualification rate of decoction pieces of Cibotii Rhizoma in market was only 56.4%. At present, the resource of Cibotii Rhizoma could meet the needs of medinal uses. It is important to protect the wild resource which is less and less because of the environmental factors. It also need to make a standard of processing method to ensure the safety, and solve quality problem of the decoction pieces.

Sector analysis and predictive modelling reveal iterative shoot-like development in fern fronds.

Plants colonized the terrestrial environment over 450 million years ago. Since then, shoot architecture has evolved in response to changing environmental conditions. Our current understanding of the innovations that altered shoot morphology is underpinned by developmental studies in a number of plant groups. However, the least is known about mechanisms that operate in ferns--a key group for understanding the evolution of plant development. Using a novel combination of sector analysis, conditional probability modelling methods and histology, we show that shoots, fronds ('leaves') and pinnae ('leaflets') of the fern Nephrolepis exaltata all develop from single apical initial cells. Shoot initials cleave on three faces to produce a pool of cells from which individual frond apical initials are sequentially specified. Frond initials then cleave in two planes to produce a series of lateral merophyte initials that each contributes a unit of three pinnae to half of the mediolateral frond axis. Notably, this iterative pattern in both shoots and fronds is similar to the developmental process that operates in shoots of other plant groups. Pinnae initials first cleave in two planes to generate lateral marginal initials. The apical and marginal initials then divide in three planes to coordinately generate the determinate pinna. These findings impact both on our understanding of fundamental plant developmental processes and on our perspective of how shoot systems evolved.

Are fern stomatal responses to different stimuli coordinated? Testing responses to light, vapor pressure deficit, and CO2 for diverse species grown under contrasting irradiances.

The stomatal behavior of ferns provides an excellent system for disentangling responses to different environmental signals, which balance carbon gain against water loss. Here, we measured responses of stomatal conductance (gs ) to irradiance, CO2 , and vapor pressure deficit (VPD) for 13 phylogenetically diverse species native to open and shaded habitats, grown under high- and low-irradiance treatments. We tested two main hypotheses: that plants adapted and grown in high-irradiance environments would have greater responsiveness to all stimuli given higher flux rates; and that species' responsiveness to different factors would be correlated because of the relative simplicity of fern stomatal control. We found that species with higher light-saturated gs had larger responses, and that plants grown under high irradiance were more responsive to all stimuli. Open habitat species showed greater responsiveness to irradiance and CO2 , but lower responsiveness to VPD; a case of plasticity and adaptation tending in different directions. Responses of gs to irradiance and VPD were positively correlated across species, but CO2 responses were independent and highly variable. The novel finding of correlations among stomatal responses to different stimuli suggests coordination of hydraulic and photosynthetic signaling networks modulating fern stomatal responses, which show distinct optimization at growth and evolutionary time-scales.

Azolla domestication towards a biobased economy?

Due to its phenomenal growth requiring neither nitrogen fertilizer nor arable land and its biomass composition, the mosquito fern Azolla is a candidate crop to yield food, fuels and chemicals sustainably. To advance Azolla domestication, we research its dissemination, storage and transcriptome. Methods for dissemination, cross-fertilization and cryopreservation of the symbiosis Azolla filiculoides-Nostoc azollae are tested based on the fern spores. To study molecular processes in Azolla including spore induction, a database of 37 649 unigenes from RNAseq of microsporocarps, megasporocarps and sporophytes was assembled, then validated. Spores obtained year-round germinated in vitro within 26 d. In vitro fertilization rates reached 25%. Cryopreservation permitted storage for at least 7 months. The unigene database entirely covered central metabolism and to a large degree covered cellular processes and regulatory networks. Analysis of genes engaged in transition to sexual reproduction revealed a FLOWERING LOCUS T-like protein in ferns with special features induced in sporulating Azolla fronds. Although domestication of a fern-cyanobacteria symbiosis may seem a daunting task, we conclude that the time is ripe and that results generated will serve to more widely access biochemicals in fern biomass for a biobased economy.

Multiflagellated sperm cells of Ceratopteris richardii are bathed in arabinogalactan proteins throughout development.

UNLABELLED: • PREMISE OF THE STUDY: Sperm cell differentiation in ferns involves the origin of an elaborate locomotory apparatus, including 70+ flagella, and the structural modification of every cellular component. Because arabinogalactan proteins (AGPs) are implicated in molecular signaling and in regulation of plant development, we speculated that these glycoproteins would be present during spermiogenesis in ferns.• METHODS: Using ß-glucosyl Yariv reagents that specifically bind to and inhibit AGPs and immunogold localizations with monoclonal antibodies JIM13, JIM8, and LM6, we examined the specific expression patterns of AGPs and inhibited their function during sperm cell development in the model fern Ceratopteris richardii.• KEY RESULTS: Developing sperm cells stained intensely with Yariv phenylglycosides, demonstrating the presence of AGPs. JIM13-AGP epitopes were widespread throughout development in the expanding extraprotoplasmic matrix (EPM) in which flagella elongate, cytoplasm is eliminated, and spherical spermatids become coiled. JIM8 and LM6 epitopes localized to the plasmalemma on growing flagella and on the rapidly changing sperm cell body. Spermatids treated with ß-glucosyl lacked an EPM and formed fewer, randomly arranged flagella.• CONCLUSIONS: We demonstrated that AGPs are abundant in the EPM and along the plasmalemma and that the three AGP epitopes have specific expression patterns during development. Coupled with inhibition studies, these results identify AGPs as critical to the formation of an extraprotoplasmic matrix and the consequent origin and development of flagella in an orderly and precise fashion around the cell. We speculate that AGPs may play additional roles as signaling molecules involved in cell shaping, cytoskeletal development, vesicle trafficking, and cytoplasmic elimination.