Pteris multifida is a typical species of Pteridaceae (PPGI, 2016). It has fertile leaves with sporangia in the intramarginal position (Supplementary Figure 1). To observe the sporangium development and cell lineages, we established a technique of co-staining and confocal imaging in Pteris multifida. Sporangia were fixed in ClearSee to improve the transparency for deep imaging (Supplementary Figure 1E; Kurihara et al., 2015). Calcofluor white (CW) stains polysaccharides of the cell wall (Ursache et al., 2018). Basic fuchsin (BF) is a commonly used red fluorescent dye for lignin, a product of the phenylpropanoid pathway (Yokoyama et al., 2007; Ursache et al., 2018). Recently, we showed that the sporopollenin wall of vascular plants also contains derivatives of the phenylpropanoid pathway (Xue et al., 2020), and BF could stain microspore sporopollenin wall. To observe both the cell lineages of sporangia and sporopollenin wall, co-staining of BF and CW was performed (Supplementary Figure 1E). The CW signals colored green clearly show the cell wall of sporangial layers (Figure 1A). The weak CW signals indicate newly formed cells in the sporangium (Figure 1A). The BF signals colored red are mainly detected in the cytoplasm of all the sporangial layers (Figure 1B), indicating that the phenylpropanoid derives might be synthesized in these cells. BF staining shows the cytokinesis of the sporangial cell, which is consistent with the CW staining showing newly formed cell walls. Therefore, the co-staining can observe the different cell layers and cell divisions in sporangium (Figure 1C). We performed a Z-stack of a series of 2-dimensional (2D) images to re-constitute the 3-dimensional (3D) structure of the sporangium (Figures 1D, and Supplementary Figure 2). The transverse light section of the sporangium is called the X-Z planes (Figures 1E,F). The periclinal light section of the sporangium is called the X-Y planes (Figure 1G). The anticlinal light section of the sporangium is called the Y-Z planes (Figure 1H). The morphology of the sporangium is observed in these light sections for a comprehensive analysis of the 3D organ structure, cell division, and developmental process (Supplementary Figure 2).

To observe the sporangium development in Pteris, this co-staining method was used in this study. The sporangium originates from a single superficial primordial cell of the receptacle, which is called the sporangial initial cell (Figure 2A; Bower, 1891; Wilson, 1999). The outer growth of the sporangial initial cell establishes the proximal-distal axes (Y-axis in this study) of the sporangium. The formation of an oblique cell wall indicates that this sporangial initial cell undergoes an asymmetric division (Figure 2B). Thereafter, both the basal and apical cells undergo further cell expansion (Figure 2C). The second round of asymmetric cell division occurs both in the apical and basal cells. The basal cell divides into two daughter cells, occupying about 2/3 of the base of the sporangium (Figure 2C). In the apical cell, an anticlinal oblique cell division was observed (Figure 2C). This cell division produces a daughter cell and a new apical cell. The daughter cell occupies 1/3 of the base of the sporangium (Figure 2C). The medio-lateral (Z-axis in this study) and adaxial-abaxial axis (X-axis in this study) are established after these cell divisions. At this stage, the sporangium consists of four cells: a new apical cell and three basal cells (Figure 2C).

The three basal cells perform several rounds of cell division to form the three-rowed sporangial stalk (Figures 2D,E,F,H). However, only the upper layer of the sporangial stalk will undergo further cell division, and all of them are horizontal divisions (Figure 2F). The apical cell performs three oblique divisions in different directions, forming three epidermal cells and another apical cell (Figures 2E,F). Finally, this apical cell produces a cap cell and an enclosed tetrahedral mother initial cell through a transverse cell division (Figures 2G,H). The three epidermal cells and the cap cell surrounding the tetrahedral mother initial cell further develop into the sporangial epidermis (Figures 2G,H). The sporangium now consists of a stalk and a capsule (Figure 2H). The capsule is constituted of four epidermis cells and an enclosed mother initial cell (Figure 2H).

After the establishment of the capsule, the epidermis cell layer only performs anticlinal cell division (Figure 3). The tetrahedral mother initial cell performs four rounds of oblique cell divisions to produce the primary tapetum layer and an enclosed sporogenous cell (Figures 3B–D). The primary tapetum cells undergo anticlinal cell division to increase the cell numbers before the sporogenous cell mitoses (Figure 3E). The sporogenous cell undergoes multiple symmetric mitoses to produce the spore mother cells (Figures 4A–D). Usually, each sporangium contains about 64 spores. This indicates that the sporogenous cell undergoes 4 mitoses to produce 16 spore mother cells with each of them further divided into 4 spores. The primary tapetal layer produces the outer tapetal layer and inner tapetal layer through a periclinal cell division (Figures 4A–C). The morphology of the two layers of tapetal cells is quite similar at this stage (Figure 4C). CW staining shows both sporogenous cells and tapetum cells contain cellulose walls which is similar to the regular plant cell walls during the mitotic stage (Figures 4A–C). However, the cell walls of the spore mother cells and inner tapetum cells are gradually degraded after their last mitosis (Figures 4D,E).

A spore mother cell undergoes meiosis to produce a tetrad. During this process, the spore mother cell wall is gradually degraded, and some materials are accumulated outside to form a tetrad wall (Supplementary Figure 3). This cell wall transition is quite similar to Arabidopsis (Xue et al., 2021). After the cell wall degradation, the spore mother cells turn to round and separate from each other (Figure 5A–C). The outer tapetum becomes a very thin cell layer while the inner tapetum layer is enlarged which adheres closely to the spore mother cells (Figures 5A,B, and Supplementary Figure 4). This result indicates a functional differentiation of the inner and outer tapetum layers. After meiosis, the microspores are enclosed in the tetrad wall (Figure 5D). CW stains Arabidopsis tetrad wall green (Supplementary Figure 3). However, the tetrad wall of Pteris shows no CW signals (Figures 5A,B). This indicates that the composition of the tetrad wall of Pteris is different from the callose in angiosperms. The cell wall of the outer tapetum is also degraded at this stage (Figure 5D). During sporangium development, the epidermis cells undergo anticlinal division to increase the volume of the locule (Figures 5A–C). At the meiosis stage, some epidermis cells are thinner than others, suggesting that these cells are differentiated into the stomium cell (Figure 5D).

At the later tetrad stage, the haploid spores are covered by a sporopollenin wall (Figures 6A,B). In seed plants, sporopollenin material is from the tapetum. However, in the Pteris spore, sporopollenin is gradually accumulated after the degradation of the tapetum (Figures 6A–D and Supplementary Figure 5). Thus, epidermis or spores may also provide materials for sporopollenin formation. The annulus is a special structure on the sporangium, consisting of a row of cells that are unevenly thickened on the epidermis of the sporangium. It plays a role in dehiscence and spore dispersal (Noblin et al., 2012). The BF staining shows strong signals in the thickened annulus, indicating the lignification occurs in fern sporangium (Figure 6D and Supplementary Figure 6).

Sporangia at different development stages were collected from Pteris multifida Poir. grown in the campus of Shanghai Normal University (Shanghai, China) between April 1, 2020, and October 29, 2020. Each fertile leaf has a large number of sporangia at different development stages. For each development stage, at least 3 sporangia were imaged. The voucher specimens (NO.20200401001, NO.20200701001, and NO.20201029001) are deposited in the spore function laboratory herbarium of Shanghai Normal University.

Calcofluor white (CAS: 4404-43-7, Product no. F3543, Sigma) stains the primary cell wall and callose. However, the primary cell wall of the tapetum and the spore mother cells degrade during meiosis and become extremely difficult to discern (Wilson, 1958b). Basic Fuchsin (CAS: 58969-01-0, Product no. B0904, Sigma) stains the phenylpropanoid phenolics. We showed the expression of these pathway genes in tapetum (Xue et al., 2020), and this dye also stains the sporopollenin wall (Jia et al., 2021). Furthermore, these two dyes show non-overlapped emission wavelengths. Thus, we used these dyes for the staining. Toluidine Blue (CAS: 3209-30-1, Product no. 89640, Sigma).

ClearSee solutions were prepared by mixing xylitol powder, sodium deoxycholate, and urea in water as previously described (Kurihara et al., 2015). Sporangia were fixed in ClearSee solution, placed under vacuum for 15 min, and then were treated for two weeks, washed three times in pure water, followed by staining in a mixture of Calcofluor white (0.01M, Sigma)) and Basic Fuchsin (0.001M, Sigma) under 4 degrees in the dark for two days. The sporangia were washed with pure water, and then immediately observed by confocal scanning laser microscopy (objectives were 60x, and lasers were 405nm and 561nm). Sporangia at different developmental stages were fixed overnight in FAA [ethanol 50% (v/v), acetic acid 5.0% (v/v) and formaldehyde3.7% (v/v)], dehydrated in a graded ethanol series (50, 70, 80, 90, 95, and 100% twice), transferred to acetone (100% twice) and embedded in Spurr’s epoxy resin. Sections (1μm) were cut on a Leica UC7. Sections after staining with toluidine blue were taken photographs using an Olympus DX51 digital camera.

For Calcofluor white, we used 405 nm for excitation and the emission spectra were recorded in the range of 418-468 nm. For basic fuchsin, we used 561nm excitation and emission spectra were recorded in the range of 600-650 nm. All fluorescence images were obtained with an Olympus FV3000 laser scanning microscope. The 3D reconstruction was performed by Olympus FV31S-SW.