Salvertia convallariodora

Carmo-Oliveira, Renata, Custódio, Luciana Nascimento, Morretes, Berta Lange De & Oliveira, Paulo Eugênio, 2020, Early embryology of Vochysiaceae and some insights into its phylogeny and intrafamilial taxonomy, Phytotaxa 443 (3), pp. 211-257 : 217-222

Salvertia convallariodora

Flower development — Flowers took 25-30 days to develop and microsporogenesis and gametogenesis preceded megasporogenesis and gametogenesis by almost ten days. Microspores were already formed ca. 18 days before anthesis (dba), while a clear megaspore mother cell was only observed 11 dba. The detailed development is described below.

Microsporogenesis and gametogenesis— The anther of Salvertia convallariodora is tetrasporangiate, with all sporangia turned to the ventral side of the anther and to the style surface (introrse) ( Fig. 3A). In floral buds ca. 5.0 mm (ca. 25 days before anthesis-dba),the sporangia were covered by a uniseriate protodermis ( Fig. 3B). The development followed a Basic pattern (sensu Davis 1966), where two middle layers and the tapetum originated from divisions of the secondary parietal layers. In buds ca. 8.0 mm (ca. 23 dba), there were already at least four layers resulting from the parietal cells. At this phase, parietal and sporogenous cells divisions started to form the tapetum. Shortly after (8.0–9.0 mm and ca. 22 dba), the sporangium wall was already formed by a subepidermal layer, which originates the endothecium, two middle layers, a layer of tapetal cells of parietal origin, and layers of tapetal cells of sporogenic origin ( Fig. 3C). The middle layers and tapetal layers were concentric surrounding the sporangium. In buds ca. 9.1– 10.0 mm (some 21 dba), the anther wall was formed by the uniseriate epidermis, a pro-endothecium layer, a middle stratum with two (up to three) layers of cells and the tapetum with three to four layers ( Fig. 3D). With the growth of floral buds, epidermal cells became larger and vacuolated, especially in the dorsal surface. From buds ca.12.0 mm on (ca. 19 dba), epidermis cells between the sporangia increased in size and became ovate, covering the intersporangial septa ( Figs. 3E, 3F, 4A and 4C).

The subepidermal pro-endothecium layer was formed by long and vacuolated cells ( Fig. 3D). From buds 16.0– 17.2 mm (ca. 17 dba), those endothecium cells grew radially and later, in buds ca. 26.5–27.5 mm (ca. 6 dba), they formed bar lignified thickening that went from the internal to the external periclinal cell walls ( Fig. 4H), often forking before reaching the external cell wall. Some pro-endothecium cells sometimes divided resulting in a biseriate tissue in some points ( Fig. 4G).

The middle layer cells formed a three (sometimes four) seriate parietal strata between the endothecium and the tapetum. These parietal layers were compressed by the growth of the sporangia and collapsed during the pre-anthesis stages ( Figs. 4F)

The tapetum developed as a secretory, pluriseriate tissue originating both from parietal and sporogenic cells early in sporogenesis. Tapetal cells were binucleate and with dense cytoplasm. After microsporogenesis ( Fig. 3F), the tapetum started to disintegrate and often only a few cells remained before anther dehiscence.

During anther development, the intersporangial parenchyma increased in number and size of cells to form a septum between the two sporangia in each theca, following the increase of the endothecium and epidermal cells ( Fig. 4C). Longitudinal dehiscence of the anther occurred 1–2 days before anthesis along the epidermal cells near the intersporangial septum ( Fig. 4E and 4F).

The sporogenous cells underwent some mitotic divisions before giving rise to the microspore mother cells (mmc), which could be observed in buds 9.1–10.0 mm (ca. 22 dba) ( Fig. 3D). Shortly after, in buds 12.0–13.0 mm (ca. 19 dba), the mmcs were mature and ready for meiosis ( Fig. 3E). The cytokinesis was simultaneous, leading to tetrahedral tetrads of microspores ( Fig. 3F, 3G and 3H). The tetrads in buds 13.0 to 15.0 mm (ca. 18 dba) were enclosed in callose (blue in the double stained sections). Each microspore in the tetrad already showed three nuclei, a larger one in the centre and another two in the periphery of the microspore ( Fig. 3H). This indicates that microgametogenesis followed quickly after meiosis and tri-cellular pollen grains were formed inside the callose envelope. Shortly after (some 17 dba), the tri-cellular pollen grains were completely formed and free ( Fig. 4A and 4B).

Megasporogenesis and gametogenesis— In Salvertia convallariodora , the mature ovules are hemianatropous/ epitropous, with the micropyle turned to the apex of the ovary. They are crassinucellate and bitegumented, with micropyle formed by both integuments. The ovule primordia appeared as protuberances on the placenta in buds ca. 8.0 mm (ca. 24 dba). At this stage, epidermal cells in the upper region of the placenta divided actively and had a dense cytoplasm. These cells were contiguous to transmitting tissue and would form the placental obturator. In buds ca. 9.5 mm (ca. 22 dba), periclinal cell divisions in the ovule primordium indicated the formation of the inner and outer integuments. The outer integument originated near the base of the inner integument ( Fig 5A). For buds 11.0–12.0 mm on (ca. 21 dba), the ovule started to curve upwards by the unilateral proliferation of funicle cells. The curvature increased up to 90 o, turning the micropyle to the ovary apex. The integuments developed through periclinal, anticlinal and even oblique divisions of epidermal and subepidermal cells. These divisions formed two rings of cells around the primordium. The outer integument ring was asymmetrical and followed the funicle and ovule bending upwards. The inner integument developed a bit earlier (e.g. Fig. 5B) but later on, in buds 16.0– 17.2 mm (ca. 17 dba), the outer integument already covered the inner integument ( Fig. 5C).

The outer integument cells underwent divisions and formed a multilayered tissue of small cells, up to 10 or 12 layers in the mature ovule at anthesis. The number of cell layers was even higher, up to 18, in the base near the funicle and placenta. The inner integument had ca. eight layers of cells in mature ovules.

The nucellus was formed by undifferentiated cells during the first stages of integument development. However, in buds 13.0–15.0 mm (ca. 19 dba), a larger sporogenous cell with a prominent nucleus was observed. In this phase, this cell was separated from the nucellar epidermis by two parietal cell layers derived from the primary parietal cells ( Fig. 5B).

For buds 13.0–15.0 mm on (ca. 19 dba), the ovules grew quickly and, in buds ca.17.0 mm (ca. 15 dba), they were curved upwards and the outer tegument completely covered the inner tegument ( Fig. 5C). The megaspore mother cell (MMC) was isolated from the nucellar epidermis by several parietal cell layers resulting in a crassinucellate ovule ( Fig. 5D). A hypostase of relatively small, thin-walled and vacuolated cells could be seen at the chalazal end, above the funicle vascular bundle ( Fig. 5C).

A megaspore mother cell in meiotic prophase ( Fig. 5E) was observed in buds 22.0–23.0 mm (ca. 10 dba). This cell was isolated from the micropyle by many parietal layers of cells. In buds ca. 22.0 mm (ca. 10 dba), the MMC was clearly differentiated and isolated. Starch grains in nucellar and parietal cells were observed in this phase. Even during anthesis, there was a multilayered nucellar tissue around the embryo sac.

We found a linear tetrad of megaspores ( Fig. 5F) in buds ca. 24.0 mm (ca. 9 days before anthesis). The chalazal megaspore enlarged quickly and originated a single embryo sac. In buds 25.0–26.0 mm (ca. 7 dba), we observed a binucleate embryo sac ( Fig. 5G) and in buds ca. 29.0 mm (ca. 4 dba), this embryo sac was already in the tetranucleate state ( Fig. 5H). In each locule of the ovary, only one of the ovules developed to maturity. The other ovule stopped developing during the growth of the embryo sac. The inner tegument cells collapsed, followed by the outer integument ( Fig. 6A and 6B).

In buds ca. 34.0 mm (pre-anthesis), the embryo sac was already completely organized. In the pre-anthesis stage, the long Polygonum - type embryo sac showed the two synergids and the egg-cell at the micropylar end, a central cell with free polar nuclei, and three persistent antipodals at the chalazal end (details in Fig. 6C, 6D and 6E). During development of the embryo sac, most nucellar cells were either destroyed or compressed, and during anthesis, only a few layers separated the gametophyte from the micropyle ( Fig. 6C and 6E).