Callisthene

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 : 228-233

Callisthene

Flower development— The flowers in both Callisthene species developed quickly and remained open for a single day and the petal usually dropped by the following morning. Microsporogenesis and development of male gametophyte occurred in about five days. Seven days before anthesis, the pollen was already formed and bicellular, but it was released only after the onset of anthesis. However, megasporogenesis only began the day before anthesis and even in freshly open flowers, the megaspore mother cells had yet to undergo meiosis. Only 72 hours after anthesis did tetrads of megaspores finally appear, and the female gametophyte did not mature until eight days after anthesis.

Microsporogenesis and gametogenesis— As observed in Salvertia and Vochysia , microsporangia development began very early in Callisthene species, while buds were still differentiating. The sporangia walls followed the Basic development pattern observed for the other two genera. In buds 2.0– 2.7 mm (ca. 10 days before anthesis), the anther was already tetralobed with a developed connective and vascular bundle ( Fig. 11A). At that stage, the anther wall already consisted of six layers: a uniseriate epidermis, a subepidermal layer which gives rise to the endothecium, two middle layers and a biseriate tapetum ( Fig. 11B and 11C).

During the development of the anther, the epidermal cells at the ventral side expanded and became vacuolated. As the microsporangia expanded, the epidermal cells became longer, except in the region between the sporangia of each theca, where they remained small and less vacuolated. In the mature anther, the epidermal cells covering the sporangia were relatively small ( Fig 11B and 11C).

The endothecium developed from the subepidermal layer ( Fig 11B). During sporogenesis, its cells increased radially and became vacuolated ( Fig. 11D and 11F). As the anther matured, some endothecium cells underwent periclinal divisions, which made this tissue biseriate in some places. In buds 5.9–6.5 mm onwards (ca. 5 dba), the endothecium cells had bar thickening linking the external and internal periclinal walls, similar to S. convallariodora ( Fig. 11H).

The middle layers cells below the endothecium underwent periclinal divisions and in buds 2.0– 2.7 mm (ca. 10 dba), this region was up to three layers thick ( Fig. 11B). With anther development, the middle layers near the tapetum were compressed and in buds 2.9–3.5 mm (ca. 8 dba), only one or two layers of this tissue remained ( Fig. 11C). In buds 8.0– 8.8 mm (ca. 3 dba), it was possible to see a middle layer in some points ( Fig. 11F).

The tapetum seems to be formed both by parenchymatic and sporogenous cells and this tissue completely surrounded the microspore mother cells (mmc). The tapetum was of glandular type and developed into a bi- or triseriate tissue. The tapetal cells near the mmcs were larger and originated from the sporogenous tissue ( Fig. 11D). Before meiosis, ca. 10 dba (buds 2.0– 2.7 mm), the tapetal cells showed a dense cytoplasm and were already binucleate ( Fig. 11B). After the development of microspores, the tapetum started to become disorganized ( Fig. 11D and 11E). In buds near anthesis, this tissue showed only remnants of cells that did not degenerate during pollen grain formation ( Fig 11F).

The intersporangial septum developed between each theca, and the epidermal cells in this region were smaller and less vacuolated.The subepidermal cells were also different and vacuolated ( Fig. 11G).At pre-anthesis, the subepidermal cells started to separate and disintegrate. After anthesis, when the endothecium cells loose water, dehiscence seemed to begin in this intersporangial septum ( Fig. 11H).

As observed in Vochysia and Salvertia , the sporogeneous cells underwent mitotic divisions before differentiating into microspore mother cells. The mmcs were already well differentiated in buds 2.0– 2.7 mm (10 dba), were polygonal, with dense cytoplasm and a large nucleus ( Fig. 11B). Meiosis began in buds ca. 3.0 mm (ca. 8 dba). In this phase, the mmcs were still in interphase, but a callose deposition could already be seen between the wall and the cytoplasm of those cells under Astra blue/Basic fuchsin staining ( Fig. 11C).

Meiosis was quick, and intermediate stages could not be followed in detail from sections, but in buds 3.6–3.9 mm (ca. 7 dba) we found a tetrahedral tetrad indicating that cytokinesis was simultaneous after meiosis II ( Fig. 11D). In buds 4.8–5.1 mm (ca. 6 dba), the unicellular microspores were free and already showed intine and exine walls ( Fig. 11F). Bicellular pollen grains (not shown) were observed from buds 8.0– 8.8 mm (ca. 3 dba) onwards, but pollen was released only during anthesis.

Microsporogenesis and gametogenesis were not followed in detail for C. fasciculata , but pollen production and release were similar in timing and stages of those observed in C. major .

Megasporogenesis and gametogenesis— In Callisthene major , ovules were crassinucellate and bitegumented with micropyle formed by both integuments. The ovule primordia appeared as protuberances on the axial placenta. The placental obturator was also formed from a similar protuberance above the ovule primordia ( Fig. 12A). In mature ovaries at pre-anthesis, this tissue appeared as part of the placenta and was continuous with transmitting tissue along the style.

In buds ca. 4.8 mm (ca. 6 dba), the archesporial cells had already underwent mitosis, giving rise to parietal cells and sporogenous tissue. In this same phase, it was already possible to see the concomitant development of the outer and inner integuments ( Fig. 12A and 12B). The parietal cells divided continuously during ovule development. The nucellar epidermal cells also suffered some divisions ( Fig. 12B) but only formed three or four layers of cells at some points. The integuments surrounded the nucellus from early stages ( Fig. 12C). In the mature ovule, the inner integument had three or four layers and only two layers in the micropyle. The outer integument was formed by up to seven layers of cells in the abaxial side (towards the placenta). The layers were often the result of epidermal cell divisions and growth, and they continued to grow even after anthesis. The integument cell growth up to 48 hours after anthesis made the micropyle half as long as the entire ovule.

As mentioned before, the megasporogenesis in Callisthene species started much later relative to the microsporogenesis than in Salvertia and Vochysia . Sporogenous tissue was not differentiated until buds were 4.8–5.1 mm (ca. 6 dba). Sporogenous cells apparently arose from multiple archesporial cells, and at this stage were larger and with a prominent nucleus when compared to the surrounding parietal cells ( Fig. 12B). They were kept at the chalazal end of the ovule due to successive divisions of the parietal cells. The day before anthesis, the ovule was totally differentiated and had several elongated megaspore mother cells (MMCs) (at least eight in each ovule) ready to initiate meiosis. They were located in the chalazal region and isolated from the micropyle by at least five layers of parietal cells ( Fig. 12C and 12D). The MMCs remained in interphase for up to three days after anthesis while the ovule continued to grow in size and parietal cell divisions kept them further to the chalazal region. While the MMCs were still immature, the pollination and pollen tube growth occurred. The pollen tube reached the ovary about 24 hours after pollination and were observed growing in the placental obturator ( Fig. 12F).

Only 72 hours after anthesis (3 days after anthesis-daa) was it possible to find dividing cells and linear meiotic tetrads with a chalazal megaspore already larger than the others ( Fig. 12G and 12H respectively). The remaining megaspores of each tetrad degenerated. The tetrads were far apart from the micropyle, isolated by many layers of parietal cells from the nucellar epidermis ( Fig. 13A).

Only 96 hours after anthesis (4 daa), the chalazal megaspore of each tetrad started mitotic divisions to originate the embryo sac ( Fig. 13A, 13B and 13C). Multiple embryo sacs developed concomitantly. Five days after anthesis, we observed several bi- or tetranucleate embryo sacs developing in each ovule of the pistil, and all ovules had multiple embryo sacs ( Fig. 13D). From this stage on, the embryo sacs elongated quickly towards the micropyle, growing through the parietal layers ( Fig. 13E). Tetranucleate embryo sacs showed two pairs of nuclei in each end separated by a huge central vacuole.

After the third mitotic division, the embryo sacs differentiated into Polygonum - type gametophytes, which matured seven or eight days after anthesis. Up to four mature embryo sacs could be observed in a single ovule of Callisthene major , but this number was even higher in C. fasciculata . Eight days after anthesis, the polar nuclei were already fused and near the egg apparatus in the mature embryo sacs ( Fig. 13F). At this stage, the embryo sacs elongated up to the micropyle (or even past the micropyle in C. fasciculata ) near the pollen tubes, which were still growing in the placental obturator, and fertilization ensued ( Fig. 14A). At this stage, it was often possible to observe the egg apparatus in the micropylar end and persisting antipodals at the chalazal end of the long embryo sacs. Using Herr solution and DIC microscopy, it was possible to remove the integuments and show the various competing embryo sacs in Callisthene fasciculata ( Fig. 14B and 14C). Several embryo sacs were effectively fertilized, and some embryos developed concomitantly in each ovule ( Fig. 14D), however, usually only a single embryo reached maturity (transient polyembryony).