Qualea (Marcano-Berti, 1969)

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 : 233-242

Qualea

Flower development — The early embryology is detailed mainly for Q. parviflora , but significant differences from the other species are pointed out. As in the other studied genera, the microsporogenesis in Qualea parviflora precedes the megasporogenesis and only in buds ca. 5.5 mm (ca. 11 days before anthesis-dba), when the microspores were already free in the sporangia, the meiosis started in megaspore mother cells. Bi-cellular pollen grains were observed nine days before anthesis, but at the start of anthesis embryo sacs were still in the tetracellular stage and matured only ca. 72 hours after anthesis (3 daa).

Microsporogenesis and gametogenesis— The microsporangia differentiate very early, when the bud itself was differentiating. In buds less than 2.0 mm (more than 33 dba), the anther appears as a group of meristematic cells below the epidermis ( Fig. 15A and 15B). In the following stages, the anther was already bithecal and tetrasporangiate. The connective was ample as in the other species. In buds at ca. 2.2 mm (ca. 30 dba), it was possible to observe the separation of subepidermal layers, which led to sporangia formation ( Fig. 15C). In slightly larger buds, the archeosporial cells divided giving rise to the sporogenous tissue and parietal layers which surrounded and isolated the sporogeneous cells from the epidermis ( Fig. 15D). The subepidermal cells layer differentiated into the endothecium, the two layers below formed the middle layers and the most internal one formed part of the tapetum, in a typical Basic type sporangium wall (sensu Davis 1966).

Anticlinal divisions of the epidermal cells allowed this tissue to expand as the anther grew. In buds 3.0– 3.5 mm onwards (ca. 30 dba), epidermal cells grew in size and became vacuolated. As the anther matures those cells became flatter ( Fig. 15D, 15E, 15F and 15G). In the region between the sporangia of each theca, the epidermal cells remained smaller than the others ( Fig. 15H).

The subepidermal layer developed into the endothecium. In buds 3.8–4.2 mm onwards (ca. 26 dba), these cells became more flattened and vacuolated ( Fig. 15F). Later, they increased in volume and some of them divided forming a biseriate tissue in some places. After microspore differentiation (buds 6.5–6.85 mm, ca. 5 dba), the endothecium cells were vacuolated and showed bar thickening similar to those observed in Salvertia and Callisthene ( Fig. 16D, 16E, 16F). In the mature anther, these endothecium cells were larger in the proximal portion of the sporangia ( Fig. 16C and 16D).

The middle layer cells originated either from the subepidermal layers or from parenchyma cells where the anther was linked to the connective, surrounding the tapetum and sporogeneous tissues. Up to buds 3.8–4.2 mm (ca. 27 dba), the cells of the innermost middle layers underwent divisions, giving rise to three distinct layers ( Fig. 15D). At this stage, those cells were flattened and vacuolated as the endothecium. As the anthers developed the middle layers were compressed. In buds 5.5–6.0mm (ca. 10 dba), we can observe two middle layers below the endothecium, the innermost layer already collapsed ( Fig. 15H and 16A). Even during anther dehiscence, one of the middle layers could be still observed.

The tapetum at the most distal region of the sporangium originated from the innermost layer of parietal cells, below the middle layers. Outer sporogeneous cells and parenchymatic cells at the proximal side of the sporangium also participate in tapetum formation, so that this tissue continuously surrounded the sporogeneous cells. In buds 3.8–4.2 mm (ca. 25 dba), the tapetal cells underwent divisions and had two nuclei, while the microspore mother cells (mmcs) were in interphase before meiosis. The tapetum cells showed dense cytoplasm, characterizing the tissue as glandular or secretory. After mmcs meiosis, the tapetum started to disorganize and was mostly absorbed at pollen grain maturation stage ( Fig. 15G, 15H and 16A), however, even in a day before anthesis buds, it was possible to observe remnants of tapetal cells that were not totally consumed.

During anther development, the parenchymatic cells in between the sporangia of each theca grew and became vacuolated. The subepidermal cells at this region were connected to endothecium and grew in a similar way, although they were a bit smaller. Later proliferation of these cells originated the intersporangial septum. At the pre-anthesis stage these cells dissociated and the tissue disintegrated ( Fig. 16C). Dehydration of the endothecium led to the dehiscence of the anther, which occurred at the onset of anthesis ( Fig. 16D, 16E and 16F).

In buds 2.2–2.8 mm (ca. 31 dba), the sporogeneous tissue was already differentiated, with larger cells with relatively large nuclei ( Fig. 15C). During the following stages, the sporogeneous cells underwent some mitotic divisions and originated many mmcs. In buds ca. 4.2 mm (ca. 27 dba), mmcs were polygonal, with thin cell walls, dense cytoplasm and a large nucleus, and seemed mature for meiosis ( Fig. 15D). Callose deposition occurred as described for the other genera, to a certain extent isolating each mmcs. The meiotic process was fast and slightly larger buds (4.8–5.4 mm, ca. 17 dba) already showed tetrahedral tetrads, from simultaneous cytokinesis ( Fig. 15F and 15G). In buds 5.5–6.0 mm (ca. 11 dba), we observed unicellular microspores with a central nucleus ( Fig. 15H) and soon after (buds 6.5–6.9 mm, ca. 5 dba), we observed bicellular pollen grains with a vegetative and a generative cell. In this phase the pollen wall with exine and intine was already defined and could be observed under double staining ( Fig. 16A and 16B). The vegetative cell contained many starch grains at this stage.

Pollen development was similar between the species of Qualea . Despite size differences of the anthers and details of pollen structure, there was no marked difference among the species in timing and pollen release process.

Megasporogenesis and gametogenesis— In Qualea species, the ovules are hemianatropous/epitropous, crassinucellate and bitegumented, with the micropyle formed by both integuments. The number of ovules varied from species to species, but at least three pairs started to develop in each locule. The basal pair started to develop first, followed by the remaining pairs. They appeared as protuberances in the central axis of the ovary, which resulted from fusion of the three carpels. In buds ca. 6.0 mm (ca. 10 dba), it was possible to observe ovules in different stages of development from the base to the tip of the ovary ( Fig. 17A).

The development of the ovule in Qualea parviflora was similar to the other genera, with a unilateral growth at the ovule base which bent the micropyle to the ovary apex. The outer and inner integument had simultaneous dermal origin. They developed initially as rings that circled the nucellus ( Fig. 17B). During the integument formation, the epidermal cells and parietal cells, initially derived from the archesporial cells, underwent periclinal and anticlinal mitotic divisions and contributed to ovule expansion ( Fig. 17C). Division of epidermal cells contributed to nucellus formation. Parietal cells also divided continuously throughout ovule development and form a thick, multilayered, parietal stratum. In buds ca. 6.0 mm (ca. 10 dba), in the basal ovules, the megaspore mother cells were separated from the nucellar epidermis by several layers of parietal cells ( Fig. 17D). Later, during anthesis, the embryo sac was separated by ca. 20 layers of cells. The presence of starch grains in this tissue was observed from buds ca. 6.5 mm (ca. 7 dba) up to 18 days after anthesis, at the beginning of embryo development.

The mature ovule at pre-anthesis showed the outer integument formed mostly by three or four layers of cells. The inner integument showed two or three layers. In the basal ovules, the integuments grew long enough to reach the placental obturator at the ovary apex. Thus, the micropyle was very long, especially due to the external integument.

The vascular bundles reached the chalazal end of the ovules where a group of small cells with dense cytoplasm and prominent nucleus formed the hypostase. Although less conspicuous, the hypostase cells remained apparently active at least until anthesis. In Qualea parviflora and Q. multiflora , open flowers only had the basal pair of ovules mature. The upper pairs had differentiated integuments but no sporogeneous tissue. They never fully developed, and fruits had only two seeds per locule. About three pairs can mature in Q. cordata var. dichotoma and up to 12 pairs of ovules per locule may mature in Q. grandiflora .

The basal ovules showed multiple archesporial cells, which originated several sporogeneous cells. In buds ca. 5.5 mm (ca. 15 dba), the sporogenous cells were larger than the remaining nucellar cells, with dense cytoplasm and prominent nucleus ( Fig. 17C). In buds ca. 5.5–6.0 mm (ca. 10 dba), the megaspore mother cells (MMCs) occupied the centre of the nucellus and were already in the interphase before meiosis. They were isolated from the nucellar epidermis by many layers of parietal cells ( Fig. 17D). Even before meiosis, the MMCs were placed at the chalazal end by successive divisions of the parietal cells. In buds ca. 6.15 mm (ca. 7dba), we observed the first phase of meiosis ( Fig. 17E) and some resulting dyads ( Fig. 17F). In the same ovule, we observed both undivided MMCs and dyads. In other buds at the same stage, we observed linear tetrads already formed ( Fig. 17G). Several MMCs underwent meiosis simultaneously and it was possible to observe up to six tetrads in the same ovule. In other buds of similar size (ca. 7 dba), it was possible to observe more than one megaspore growing in each tetrad ( Fig. 17I), but usually only the chalazal megaspore continued the gametogenesis process. However, since several tetrads were developing, it was difficult to rule out the development of more than one embryo sac from each tetrad. In buds 6.5–6.9 mm onwards (ca. 5 dba), binucleate embryo sacs were growing among the nucellar cells ( Fig. 18A). It was possible to observe at least four embryo sacs in each ovule.

Callose deposition was observed at the beginning of megasporogenesis, around the MMCs. At the linear tetrad stage the callose surrounded all megaspores and, even at the tetranucleate embryo sac, it was possible to see some callose at the chalazal end ( Fig. 18B).

In buds 7.0– 7.5 mm (ca. 2 dba), the tetranucleate embryo sacs were growing towards the micropyle through the nucellar parietal tissue ( Fig. 18B), but the third mitotic division occurred only ca. 24 hours after anthesis in the fully expanded embryo sacs (e.g. Fig. 18C, 18D and 18E). Several embryo sacs were observed growing simultaneously in most ovules ( Fig. 18F). The mature gametophyte was of the Polygonum - type with persisting antipodals at the chalazal end, the egg apparatus at micropylar end, and a middle cell with two unfused polar nuclei, which were already near the egg apparatus ( Fig. 19A, B, C, and D). We observed pollen tubes growing at the placental obturator 96 hours after anthesis in Qualea parviflora ( Fig. 19E), but fertilization was clearly observed only six days after anthesis ( Fig. 19F). The nucellus was mostly consumed during embryo sac development, but some remnants were observed even 18 days after anthesis when globular embryos were already observed (Fig, 19G).

As mentioned before, the species studied varied in the number of mature ovules developed, from three pairs inside each locule in Q. parviflora , Q. multiflora ( Fig. 20A), in which only the basal one matured; to six pairs per locule in Q. cordata var dichotoma ( Fig. 20B), in which three pairs matured; and up to eight pairs per locule in Q. grandiflora ( Fig. 20C), in which at least six pairs may form seeds . However, we observed several embryo sacs in each ovule of all species (e.g. Fig. 20D and 20E). At anthesis, the embryo sacs were in the tetra or octanucleate stage (in Q. cordata var. dichotoma ) and were mature only from two (in Q. cordata var. dichotoma ) to six days after anthesis ( Q. parviflora ). Multiple fertilizations were observed in ovules of all species and multiple embryos were also common ( Fig. 20F). Although more than one embryo may be formed from different embryo sacs in an ovule, the mature seeds were strictly monoembryonic in all the studied species.