Vochysia (Poir, 1808)

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 : 222-228

Vochysia

Flower development— A detailed study was carried out for Vochysia cinnamomea only, and key stages of the other species were analysed and compared. In Vochysia cinnamomea , as in S. convallariodora , microsporogenesis and gametogenesis were much faster than megasporogenesis and gametogenesis. While ca. 18 days before anthesis the microspores were already formed, the megaspore mother cell was still in interphase. The bicellular pollen grains were already formed 10 days before anthesis, while the embryo sac was still undergoing mitosis and was totally differentiated only 4dba. A detailed account of the early embryology of V. cinnamomea is described below and differences observed in the other species are highlighted.

Microsporogenesis and gametogenesis— Microsporangia development was fast in V. cinnamomea . For very small buds ca. 2.0–4.0 mm (more than 35 days before anthesis-dba), it was already possible to observe the tetralobed anther and a well-developed connective. The vascular bundle could be observed in the centre of the anther surrounded by connective parenchymatic cells ( Fig. 7A). At this phase, the anther showed a protodermis and groups of archesporial cells in the ventral side. During the following stages, periclinal divisions of these subepidermal cells gave rise to the primary parietal layer and the sporogenous tissue ( Fig. 7B). Shortly after, this primary parietal cell divided to form two secondary parietal layers so that in buds 3.0–4.0 mm (more than 35 dba), the anther wall already showed four layers of cells between the epidermis and the sporogenous tissue, which characterizes the Basic development type (sensu Davis 1966). During this process, anthers quickly increased their volume.

In buds ca. 5.0 mm and larger (ca. 35 dba), the epidermis cells increased radially and showed large vacuoles ( Fig. 7C). On the sides of the sporangia, the epidermal cells developed thicker walls, which differed from the rest of the epidermis. During development, the epidermal cells in between the thecae increased in size and accumulated a content that stained red or purplish in the double stained sections. The remaining epidermal cells stained blue in most sections after double staining (Astra blue/basic fuchsin). In buds ca. 13.0–16.0 mm (some 10 dba), epidermal cells on the sides of sporangia had much thicker external periclinal walls and with development this thickening reached the other walls.

In buds 5.5 mm (ca. 35 dba), subepidermal cells had already undergone periclinal divisions, which originated the endothecium ( Fig. 7C). In buds 5.6–5.8 mm (ca. 33 dba), the endothecium was already well defined with two (or three) layers of cells ( Fig 7D). In later stages, those cells increased in size and became vacuolated. In buds ca. 8.0 mm (ca. 25 dba), the endothecium layers had large and vacuolated cells at the sides of the sporangia, while in between the sporangia they were radially flattened ( Fig. 7G and 7H).

Different from Salvertia , the endothecium cells did not develop any thickening. In buds ca. 18.0 mm (some 4 dba), the endothecium was already developed but some subsequent cell divisions were observed ( Fig. 8E). The epidermal cells of the sporangia region were relatively small with thick walls, while those in between the sporangia were larger ( Fig. 8E). This difference was linked to the mechanical dehiscence of the anther. While losing water and degenerating, larger cells between the sporangia pulled and broke the epidermis of the sporangia, releasing pollen in pre-anthesis buds.

The middle layers were formed by parenchymatic cells surrounding the sporogeneous tissue. They were radially short and became vacuolated as the anther matured. Some of these cells underwent periclinal divisions and during development this middle stratum was formed by three layers of cells. Later, during bud development, these layers were compressed and collapsed, as seen in buds 15.0 to 16.0 mm (ca. 8 dba) ( Fig. 8C). In the anthers of buds near anthesis, these layers were totally collapsed ( Fig. 8E).

The tapetum was observed in buds ca. 5.5 mm (ca. 35 dba), as a group of cells between the sporogenous cells and parietal cells. In this phase, the tapetal cells were very similar to the sporogenous ones, only a bit smaller ( Fig. 7C). Cell divisions in the parietal layers and sporogeneous cells indicate that both tissues contributed to originate the tapetum, but in the portion facing the connective, the tapetum cells originate from the sporogenous tissue only. The tapetum developed as a biseriate glandular or secretory tissue, with cytoplasmically dense, binculeate cells disposed radially. Nuclei duplication occurred in buds ca. 6.0 mm (some 30 dba) when the microspore mother cells were mature and prior to meiosis. During pre-anthesis, it was still possible to find remnants of tapetal cells which were not consumed during pollen grain development.

Cell divisions at the endothecium layers, middle layers and tapetum created a relatively thick sporangia wall ( Fig. 7D).

The sporogenous cells underwent several divisions before differentiating into microspore mother cells (mmcs). In buds ca. 5.0 mm (more than 35 dba), they were still similar to the surrounding tapetal cells, although larger, occupying the central portion of the sporangia ( Fig. 7A). Shortly after, in buds ca. 5.6–5.8 mm (ca. 33 dba), the mmcs were already clearly differentiated and in the interphase before meiosis ( Fig. 7D).

Before meiosis started, the mmcs became rounder. A callose wall was formed between the protoplast and the wall of mmcs. The division started in the most central mmc and, in buds ca. 6.7 mm (ca. 30 dba), we observed two nuclei resulting from meiosis I ( Fig. 7E). In buds 6.9–7.8 mm (ca. 27 dba), we observed the final stages of meiosis with four nuclei in a tetrahedral arrangement still enveloped by a common cytoplasm and callose wall ( Fig. 7F). Cytokinesis was simultaneous and, in buds ca. 10 mm (ca. 23 dba), we observed tetrads still surrounded by callose but with isolated microspores ( Fig. 7G and 7H).

In buds ca. 11.0 mm (some 16 dba), the uninucleate microspores were already free with well-defined walls ( Fig. 8A). Shortly after, in buds 15.0–16.0 mm (some 8 dba), the pollen grains were already bicellular with a generative cell with an evident cell wall inside a vacuolated vegetative cell ( Fig. 8D). About three days before anthesis (buds 17.1- 18.0 mm), the mature bicellular pollen grains presented many starch grains ( Fig. 8F). The pollen was released in buds ca. 19 mm long (ca. 2 dba) and deposited on the surface of the style. During the onset of anthesis, the anther, which already had no pollen, was ejected from the flower.

No marked differences in pollen development were observed in the other species of Vochysia studied here. Microsporogenesis and gametogenesis followed V. cinnamomea stages and bicellular pollen was released before anthesis and deposited on the style surface.

Megasporogenesis and gametogenesis— In Vochysia cinnamomea , the mature ovule is hemianatropous with a short raphe and epitropous. The ovule is crassinucellate, with the micropyle formed by both integuments.

At the beginning of ovule formation, epidermal cells elongated and developed a dense cytoplasm, which stained intensely under Astra blue/basic fuchsin. Those cells were continuous with the transmitting tissue and form the placental obturator. The ovule primordia appeared as protuberances on the placenta, below the placental obturator and along the central axis of the ovary. They were observed in buds ca. 2 mm (more than 35 dba) and were trizonate, with a dermal layer, a subdermal and the ovule meristem (not shown).

In buds 5.6–5.8 mm (ca. 33 dba), each primordium was still a protuberance formed by small cells with a dense cytoplasm and relatively large nuclei. In buds 6.0– 6.7 mm (ca. 30 dba), the primordium was already curved upwards due to unilateral growth of the funicle ( Fig. 9A).

The inner integument resulted from periclinal divisions of epidermal cells followed by further oblique divisions in buds 5.6–5.8 mm. Those divisions produce cells that form a ring around the primordium. The outer integument developed in a similar manner together with the funicle ( Fig. 9A). In buds 7.5 mm onwards (some 27 dba), the outer integument was already larger than the inner integument ( Fig. 9B), and the micropyle was formed by both integuments (e.g. Fig. 9C).

In buds ca. 10.0 mm (ca. 20 dba) onwards, the epidermal cells divided in different directions, giving rise to integuments with up to three layers of small cells ( Fig. 9C). At pre-anthesis, the outer integument of the ovule consisted of up to seven layers. In abaxial side (towards the placenta), the outer integument had up to 13 layers of cells in some points and up to nine layers near the micropyle. The inner integument in mature ovules (ca. 17.1–18.0 mm and 5 dba) had three layers of cells around the nucellus ( Fig. 10A), but could have up to four layers in some points, such as the region near the micropyle.

During integument development, the nucellar cells also underwent mitosis and contributed to ovule expansion. Up to buds ca. 6.0– 6.7 mm (ca. 30 dba), the nucellus was homogeneous with small, undifferentiated cells with large nuclei. In buds ca. 7.5 mm (ca. 27dba), it was already possible to observe a larger sporogenous cell with a prominent nucleus ( Fig. 9B). In buds ca. 10 mm (ca. 20dba), the parietal cells divided to form more layers and the sporogenous cell was evident ( Fig. 9C). From this phase on, the ovules grew quickly and in buds ca. 11 mm (some 15 dba) they were already long and bent upwards, with integuments fully developed. Nucellar parietal cells divided continuously during ovule development and formed many layers, which separated the megaspore mother cell from the nucellar epidermis ( Fig. 9E). In these buds, it was possible to see that some nucellar epidermal cells also divided but they did not do so continuously, so that in the mature ovule the nucellar tissue around the megagametophyte was mostly of parietal origin. In the chalazal region around the end of the vascular bundle, there was a group of small cells with thin walls and minute vacuoles, which clearly stained and constitute the hypostase ( Fig. 9E). This tissue is more clearly differentiated in buds ca. 13.0 mm (ca. 10 dba) and the cells keep the same features until anthesis. Some of these cells may be destroyed in the final stages of embryo sac development.

In buds ca. 11.0–13.0 mm (ca. 15 dba), the megaspore mother cell (MMC) was separated from the cellular epidermis by many layers of cells ( Fig. 9E). In this phase, the nucellar parietal cells started to accumulate starch. Later, during the development of the embryo sac, these nucellar cells were consumed or collapsed, except those at the micropylar portion. At pre-anthesis, the nucellar tissue at the micropylar end still presented a lot of starch grains that were easily observed in polarized light or iodine (Lugol’s) stain ( Fig. 10G and 10H)

In buds ca. 7.5 mm (ca. 27 dba), the sporogenous cell was recognized by its size and prominent nucleus ( Fig. 9B). This cell was separated from the epidermis by a parietal cell that originated from the archesporial cell. Afterwards ( Fig. 9C, 9D and 9E), the sporogenous cell was more distinct.

During ovule development, the sporogenous cell was pushed to the chalazal region by the continuous divisions of the parietal cells. A megaspore mother cell (MMC) was observed in buds ca. 12.0 mm (ca. 15 dba) near the chalazal end ( Fig. 9E).

In buds ca. 15 mm (ca. 12 dba), it was possible to observe the chalazal megaspore and the remains of the other megaspores, which formed a linear tetrad after meiosis ( Fig. 9F and 9G). In buds ca. 16.0–17.0 mm (ca. 5 dba), the embryo sac was already in the tetranucleate stage and, from buds ca. 18.0 mm on (ca. 3 dba), it was totally differentiated with two synergids, eggcell, a binucleate central cell, and three antipodals, characterizing a Polygonum - type embryo sac ( Fig. 10A, 10B and 10C). In buds 17.1–18.0 mm (ca. 3 dba), antipodals were still present and polar nuclei were in the middle of the central cell ( Fig. 10A and 10B). However, during pre-anthesis, the antipodals degenerated and the polar nuclei, not yet fused, were observed a bit closer to the egg apparatus ( Fig. 10C).

In buds ca. 17.0 mm (ca. 3 dba), one of the ovules in each locule had stopped developing and the inner integument cells were collapsed. Later, the chalazal cells also collapsed and the embryo sac never went beyond the tetranucleate phase of development. In buds ca. 19.0 mm (ca. 2 dba), the inner integument was totally collapsed and from this phase on, only one ovule per locule continued to develop.

The other Vochysia species had similar megasporogenesis and gametogenesis stages. A single embryo sac developed using reserves from the starch rich nucellus ( Fig. 10D). The same ovule abortion occurred in all species of Vochysia studied here (e.g. Fig. 10E) and a single ovule per locule reached maturity. In the mature ovule, a single egg apparatus, with two synergids and the egg cell, was observed at anthesis ( Fig. 10F and 10G).