**4. Embryology**

Despite the fact that a large number of pollen grains are produced, no pollen tubes or fertilization can be observed [31, 33, 34]. The endosperm and embryo are formed in the absence of double fertilization. The formation of the endosperm occurs in three stages. The fusion of the polar nuclei has not been seen, in contrast, endosperm formation starts with a series of free nuclear divisions that result in a coenocytic structure of up to 12 nuclei, with six being the most frequently recorded (**Figure 5A** and **B**). The second stage in endosperm formation involves nuclei fusion (**Figure 5C**). An interruption occurs in the coenocyte wall and the nucellar cells wall, and then the nuclei of the coenocyte and the nucellar cells are fused. This fusion allows the entrance of both nuclear and maternal cytoplasmic material inside the coenocyte. Once inside the coenocyte, all the fused nuclei become one giant nucleus, reaching 120 x 60 μm in size (**Figure 5C**). The third stage of endosperm formation is the sequence of karyokinesis, producing about 12 nuclei of equal size (50 to 70 μm diameter) (**Figure 5D**). Cytokinesis then takes place, which generates the endosperm cells (**Figure 5E** and **F**). This process only occurs in one embryo-sac as the second embryo-sac is reabsorbed.

The egg cell divides forming a four-cell globular embryo only when the endosperm cytokinesis is complete (**Figure 5E** and **F**). The mature embryo is undifferentiated, globular and it is composed of up to about 24–32 cells; it completely lacks any cotyledons or outline of a radicle (**Figure 5G–I**). No suspensor formation has been verified.

The hypothesis of the existence of parthenogenesis is proposed for the Argentine species of *Lophophytum*, justified by the formation of embryo and endosperm in the absence of fertilization and the beginning of endosperm development is autonomous.

#### **Figure 5.**

*A, E: scheme of endosperm development. A B: cygote (em) and endosperm cytokinesis. C: fruits. D-E: seeds (light and scanning microscope). Scales: B: 100μm; C: 200μm; D-E: 500 μm.*

### **5. Fruit and seed**

In *Lophophytum* the fruits are achenes, reaching an average size of 2.5 x 1.5 mm in *L. mirabile*, and 2 x 1.2 mm in *L. leandri* [31, 34]. As they are indehiscent, they constitute the unit of dissemination or diaspore. The epicarp is derived from the outer epidermis of the ovary, with cells completely occupied by tannins; those in the upper portion of the ovary are differentiated into sclereids (**Figure 5G**). In *L. mirabile* the sclereids occupy the apical and basal portion of the fruit, and in *L. leandri* they spread along its side walls reaching the upper third of the fruit. The

*Anatomy, Embryology and Life Cycle of* Lophophytum*, a Root-Holoparasitic Plant DOI: http://dx.doi.org/10.5772/intechopen.99981*

mesocarp is made up of parenchyma cells, also with tannins. The endocarp develops from the internal epidermis of the carpel which differentiates into brachysclereids (**Figure 5H**).

The ovule nucellus tissue is digested during endosperm formation, therefore the seed lacks a seed coat. The mature seed is only made up of the endosperm and undifferentiated embryo; its cells completely lack tannins that are omnipresent in the remaining fruit (**Figure 5H** and **I**). The seed is spheroidal with a small wedge towards the upper part of the ovary (**Figure 5I**).

The inflorescence of *Lophophytum* has between 50 and 65 secondary rachises with pistillate flowers, each bearing between 350 and 700 fruits, with each plant producing an average of 25,000 fruits. These may remain aggregated since they are on the secondary rachis and they do not fall until the inflorescence axis becomes decayed. However, in both species of *Lophophytum* the larvae of Oxycorynus consume the parenchymatous axis of the secondary rachis and thus the fruits are separated from the rachis, facilitating their dispersal [35].

It has been recorded that the rodent *Dacyprocta aguti* L. (Rodentia, Agoutidae) digs up the plants of *L. mirabile* to consume the tubers and inflorescence axes, especially the staminate flowers. The female portion, with fruits, remains disintegrated in the ground. In the NW of Argentina, inhabitants have mentioned that the rodent *Agouti paca* L. (Rodentia, Agoutidae) consumes the plants of *L. leandri* very assiduously. However plants gnawed by animals have never been observed in the populations of *L. leandri* under observation in Misiones [31].

## **6. Taxonomic value of floral characteristics**

A vegetative body lacking stems and leaves makes it necessary to look for other characters of taxonomic value, such as those related to the floral parts [31]. Several morphological characteristics of the staminate and pistillate flowers allow easy distinction of material from the two Argentine species of *Lophophytum* (**Table 1**).

In flowers of *L. leandri* the perianth pieces have been described by Burkart [25] as reduced ovaries. In the present study it is confirmed that the fleshy excrescences accompanying the stamens do not show any female reproductive structures that could be considered as reduced ovaries, nor any remnants of them. Hansen


#### **Table 1.**

*Differential morpho-anatomical characteristics of pistillate flowers (PF) and staminate flowers (SF).*

[17, 26] described them as parts of the perianth and used them in the taxonomic delimitation of the species.

In the core Eudicot, the absence of perianth parts is not common, except in wind-pollinated plants and the Balanophoraceae [16, 40]. In the *Lophophytum* species studied, the protective function of flowers is carried out by woody bracts covering the inflorescence*. L. leandri* shows an additional second protective line, represented by the clavate bracts, which accompany each pistillate flower.

## **7. The evolutionary trend in the gynoecium and embryo sac of the Balanophoraceae**

The analysis of the anatomy and development of pistillate flowers and the study of the functional architecture of the ovules, carpels and embryo-sac provide embryological data of great importance to complement the phylogenetic studies of the family Balanophoraceae, and even of the order Santalales.

The presence of four vascular bundles in the ovary, two ovules, and two styles and stigmas, suggests the occurrence of two carpels in *Lophophytum*. The bi-carpellated ovary is a widespread condition in the Balanophoraceae s.l., except in *Balanophora* [41], and *Dactylanthus* [42] that have a single carpel and one style.

The reduction of ovules, fusion of the ovules with the carpels, and the number of loculi are variable characteristics in the family (**Table 2**). The complete fusion between the ovules and the carpels determines the absence of a locular cavity in *Balanophora* [41] and Helosis [46–49]. In *Corynaea* [45] and *Rhopalocnemis* [44] they have a single locule due to the absence of postgenital fusion. In *Lophophytum*, the two ovarian cavities are determined by the postgenital fusion of the tip of the placenta with the apex of the ovary.

The ovules of *Lophophytum* are the only ones in the whole family that are still distinguishable from the placenta, although they are ategmic. In *Corynaea*, *Dactylanthus* and *Rhopalocnemis* [42, 44, 49] the term placental-nucellar complex (PNC) has been used instead of ovules, as the boundary between the nucellus and the placenta is blurred. The most extreme reduction occurs in *Helosis* and *Balanophora*, where there are no recognizable ovules; the ovary, placenta and nucellus are completely fused into a compact mass where the embryo sacs develop, there is no ovarian cavity [41, 44, 50]. All genera in the family, except *Balanophora* (with 1 MMC), have two MMCs, but only one ES completes its development and forms an embryo.


**Table 2.**

*Morpho-embryological features known for pistillate flowers in species of Balanophoraceae s.l.*

*Anatomy, Embryology and Life Cycle of* Lophophytum*, a Root-Holoparasitic Plant DOI: http://dx.doi.org/10.5772/intechopen.99981*

**Figure 6.**

*Hypothetical line of sequences of congenital fusion and reductions within the gynoecium of the Balanophoraceae at the embryo-sac development stage. Without scales bars.*

Among the genera of the family, monosporic, bisporic and tetrasporic ES have been described, with a bisporic ES with Allium-like development being the predominant type (**Table 2**). A new type of embryo sac (bisporic four-celled embryo sac, provided with a typical egg apparatus and a uni-nucleated central cell) has been described for Helosis [49].

In the **Table 2**, a hypothetical line of successive steps that includes several major modifications, such as: a gradual reduction in the integuments; gradual loss of identity of the ovule and placenta, both structures that are still recognized in *Lophophytum*, while in the other genera of the Balanophoraceae it is not possible to discern discrete ovules, presenting a PNC, a consequence of this reduction is the loss of the chalaza, funicle and absence of vascularization; and progressive fusing of the placenta/ovules/carpels, with the consequent reduction of the ovarian cavity, until its complete disappearance, which is found in *Helosis*, *Balanophora* and in some *Loranthaceae* (**Figure 6**).

*Lophophytum* [31] and *Balanophora* [50] studies show the rotation of the ES growing within the nucellus, with the egg- apparatus oriented towards the apex of the ovary, in the region that is more favorable to pollen tube access.
