Structure of staminate flowers , microsporogenesis , and microgametogenesis in Helosis cayennensis var . cayennensis ( Balanophoraceae )

González, A.M., Popoff, O.F. & Salgado Laurenti, C. 2013. Structure of staminate flowers, microsporogenesis, and microgametogenesis in Helosis cayennensis var. cayennensis (Balanophoraceae). Anales Jard. Bot. Madrid 70(2): 113-121. We analyzed the microgametogenesis and microsporogenesis of the male flowers of the holoparasitic Helosis cayennensis (Sw.) Spreng. var. cayennensis using optical and scanning electron microscopy. The unisexual flowers are embedded in a dense mass of uniseriate trichomes (filariae). Male flowers have a tubular 3-lobed perianth, with bilayered and non vascularized tepals. The androecium consists of three stamens with filaments and thecae connated into a synandrium. It has adnate a free central pistillode without megagametophyte. Staminal filaments, fused at their base to the perianth tube and distally free along a short section, have a single vascular bundle. The distal portion of the synandrium is formed by nine pollen sacs: six outer sacs are located laterally to each filament and three longer inner sacs. The anther wall consists of the epidermis, two parietal layers (that collapse at anther maturity), and an uninucleate secretory tapetum. There is no endothecium. During microsporogenesis, the stem cells produce tetrads of microspores by meiosis. The cytokinesis is simultaneous, forming tetrahedrally arranged tetrads. When pollen grains are in the tricellular state, the synandrium emerges from the mass of filariae, and anthers dehiscence occurs apically through longitudinal slits. In conclusion, despite the extreme reduction of flowers, the anatomic characteristics and gametophyte development of staminate flowers of H. cayennensis are perfectly normal and functional. They are thus highly similar to other genera of the holoparasitic subfamily Helosidoideae. Sterile parts of flowers and inflorescence maintain the same distinctive and aberrant features of the plant vegetative parts.


INTRODUCTION
Within Balanophoraceae, Helosis Rich. is a monotypic genus of the Helosiodoideae.This subfamily is characterized by an endogenous inflorescence with flowers embedded in a layer of filiform trichomes, and the connate staments of the male flowers forming a trimerous synandrium (Eberwein, 2000;Nickrent, 2002).
The only information of inflorescences and flowers of this genus are the morphological descriptions in taxonomic studies (Hansen, 1980b;Hansen & Engell, 1978;Martínez y Pérez & Rosas, 1995).Inflorescences are the only aerial portions of the plant to emerge endogenously from the rhizomes and are initially covered with a volva (Mauseth & al., 1992).The rest of the volva tissue remains at the base of the inflorescence and allows differentiation between H. cayennensis and H. mexicana (Liebm) B. Hansen varieties.The inflorescence is spadix-like, protogynous, 5-10 cm long, completely covered by peltate hexagonal scales.
Structure of staminate flowers, microsporogenesis, and microgametogenesis in Helosis cayennensis var.cayennensis (Balanophoraceae) Fagerlind (1938a, b) describes the distribution of flowers in the inflorescences of Helosis: the pistillate flowers are located in two rings around the axis of each scale; the staminate ones are on the periphery, under the corners of the scales.Staminate flowers of Helosis are characterized by a perianth with three apical lobes and an androecium with three fused stamens (Kuijt, 1969;Heide-Jørgensen, 2008).Erdtman (1966) and Hansen (1980a, b) describe pollen grains in H. cayennensis, H. mexicana and H. guyanensis as colpate and highlight the eurypalynous character.
No studies on the reproductive anatomy in the genus exist, except for a few details mentioned by Fagerlind (1938aFagerlind ( , 1945) ) and Umiker (1920).Therefore, the aim of this study is to provide a complete description of the morphology and anatomy of the inflorescence and staminate flowers of Helosis cayennensis var.cayennensis, including the development and structure of the male gametophyte, in order to complete the embryological studies underway in American species of the Balanophoraceae family.

MATERIAL AND METHODS
Helosis inflorescences were collected on the Apipé Grande Island, Ituzaingó department, province of Corrientes, Argentina.Voucher specimens are deposited in the Herbarium of the Botanical Institute of the Northeast, Corrientes (CTES;Gonzalez & Popoff No. 239, 18/12/2008).
For analysis of flowers with a scanning electron microscope (SEM), the fixed in FAA material was dehydrated in acetone ascending series, dried by critical point in CO2 and metalized with gold-palladium.SEM analyses were performed at the Electron Microscopy Service of the Universidad Nacional del Nordeste, Corrientes, using a microscope Jeol LV 5800-20 Kv.
Pollen grains were processed by the acetolysis technique (Erdtman, 1966).For LM analyses, grains were mounted on gelatin-glycerin (Johansen, 1940).Measurements of polar axis (P), equatorial diameter (E), area, colpus length, and thickness of exines were taken for 25 pollen grains.We calculated the minimum, maximum, and average of each parameter studied.Permanent preparations were deposited in the Herbarium Palinoteca CTES (PAL No. 2907).For SEM analyses temporary samples were mounted in a solution of distilled water and absolute alcohol (1:1) on aluminum foil, dried at room tem-A.M. González & al. perature and coated with gold.The terminology used for pollen morphology is taken from Punt & al. (1994Punt & al. ( , 2007)).

RESULTS
Helosis has oval spadix-like inflorescences, lacking the spathe typical of a true spadix (Fig. 1 A-G).They are located on a fragile peduncle.Young inflorescences are covered with tightly arranged scales that are peltate, capitate, and hexagonal in front view (Fig. 1 A).The scales become black and fall at anthesis, exposing the inflorescence surface densely covered by a cushion of pink trichomes or filariae (Fig. 1 B, C).The pistillate flowers are the first to develop, in acropetal mode, exposing their stigmas over the layer of filariae (Fig. 1  C).When the stigmas fall off, the filariae change from pink to light brown, and staminate flowers appear (Fig. 1 D).
Each staminate flower consists of a 3-lobed deep pink tubular perianth and a synandrium formed by three stamens (Fig. 1 E, F, H).The inflorescences are constantly visited by ants (Fig. 1 F, arrows), but no secretion was observed.After male flowers fall off, the whole inflorescence acquires a brownish color and the inflorescence axis bends (Fig. 1 G).

Anatomy of inflorescences
The inflorescence develops endogenously, the primordium is covered by a volva formed by two to four layers of tanniniferous parenchyma (Fig. 2 A), and this tissue is an extension of the outer layers of the rhizome.As the inflorescence elongates, this tissue brakes apart and the rests remain at the inflorescence base.
The peduncle lacks true epidermis; its surface is formed by a variable number of layers of parenchyma with thin walls and without intercellular spaces.The cytoplasm is tanniniferous and remarkably dense.The outermost layers of the protective tissue become centripetally disorganized; cells lose contact and detach (Fig. 2 B).Cuticle, stomata or trichomes were not observed.
The peduncle central region has numerous vascular bundles dispersed in a matrix of parenchyma, but there is no cortex or medullae (Fig. 2 B).Parenchymatic cells are tanniniferous with conspicuous nuclei and abundant simple starch grains with stellate fissures (Fig. 2 C).The vascular system is composed by numerous collateral vascular bundles, disposed disorderly (Fig. 2 B, D).The xylem is formed by short vessels with simple perforation plates; the walls have ingrowths toward the lumen cell (Fig. 2 E).The phloem has sieve tubes with composed sieve plates and abundant parenchymatic cells with conspicuous nuclei (Fig. 2 F).
The peltate scales that cover the young inflorescence lack an epidermis (Fig. 2 G, H).These scales consist of compact tanniniferous parenchyma with thin walls and poseess an unbranched vascular bundle.As the scales mature, the external tissues collapse centripetally.
The inflorescence surface lacks an epidermis and is totally covered by filariae (Figs. 1 B, C, 2 I, J).Filariae are multicellular trichomes 1.8-2.1 mm long, formed by 3-4 rows each consisting of 35-50 cells.These cells have a conspicuous nucleus and small starch granules.At the trichome the proximal cells are elongated and have tanniniferous cytoplasm.Distal cells are vacuolated, rounded and give the moniliform appearance to the trichome apex (Fig. 2 I).Trichome cell walls are cellulosic and thin, the contact walls between rows of cells are undulated (Fig. 2 K, L).The trichome apical portion has a detached and finely striated cuticle (Fig. 2 J-M).
The matrix of the inflorescence axis has similar anatomical features as that of the peduncle: a tanniniferous parenchyma and vascular bundles that innervate both scales and flowers.In a mature spadix cross-section, more than one hundred vascular bundles were found.

Staminate flowers
Staminate flowers are embedded in the dense layer of filariae from which they emerge as pollen matures (Fig. 2 N).The three tepals are fused at the base; each tepal is bilayered with cellulosic cell walls, tanniniferous cytoplasm, and scarce starch grains.In the free apical portion of each tepal, the inner layer is differentiated into thick-walled, sclerosed cells; there are no stomata (Fig. 4 A).Each staminate flower has a conoidal inferior pistillode, formed by a compact parenchy- The three staminal filaments have a quasi-triangular section, they are welded together at their inner vertices (Fig. 3 B,  C) and are shortly free at its most distal part.The filaments consist of tanniniferous parenchyma and vascular bundles identical to those described for the axis of the inflorescence.Anthers are welded along their length.In transection the synandrium has nine pollen sacs.Six outer pollen sacs are arranged in pairs, located laterally to each filament, and the three inner pollen sacs alternate with the outer ones (Figs. 3  H, 4 D).In longitudinal view, the inner pollen sacs are longer than the outer ones (Figs. 2 N, 3 A).
The staminate flowers are vascularized by three bundles that derive directly from the inflorescence axis and innervate each staminal filament without branching (Fig. 3 A-H).

Microsporogenesis and microgametogenesis
Sporogenous tissue completely fills the volume of the anther locules (Fig. 4 D).Cells are characterized by large, con-   When the callose wall disappears, the microspores are separated and gradually change their shape from polygonal to rounded.A very dense cytoplasm remains; the nucleus occupies a parietal position and divides by mitosis (Fig. 4 N, O).Progressively, the wall cells become thickened, and the generative cell nucleus splits resulting in a pollen grain with a vegetative nucleus and two voluminous and lenticular generative nuclei (Fig. 4 O-P).The microgametogenesis is completed with the formation of tricellular pollen grains before the dehiscence of the anthers.

Anther wall
At premeiotic state, the anther wall consists of the epidermis, one or two parietal layers and the tapetum, and all cells are characterized by large nuclei and thin cellulosic walls (Fig. 4 E, F).No cuticle or stomata were observed in the epidermis.At the state of free microspores, the parietal layers collapse while tangentially crushed (Fig. 4 G, H).The tapetum is of secretory type and is formed by a layer of large uninucleate cells, with very dense cytoplasm; the internal tangential wall is distended, so that the cells occupy much of the locule volume (Fig. 4 E-J).Tapetal cells retain their conspicuous nuclei and cellular integrity during microsporogenesis, but degenerate rapidly after the first mitosis of pollen grains (Fig. 4 O).
During anther development, endothecium or mechanical layers are not formed.At the three-cellular state of pollen grains, the synandrium emerges from the mass of trichomes, due to elongation of the filaments (Fig. 2 N).Dehiscence occurs by formation of three apical longitudinal grooves that join at the apex and open towards the base of the synandrium (Fig. 5 A).Dehiscence is caused by lysis of the epidermis, the parietal layers, and the remains of the tissue that connects the paired outer pollen sacs (Figs. 4 P, 5 A).The layers that separate the inner and outer pollen sacs also collapse.

Sterile parts of the inflorescence
Of the 17 Balanophoraceae genera six are grouped in the Helosiodoideae subfamily: Helosis, Corynaea, Ditepalanthus, Exorhopala, Scybalium, and Rhopalocnemis (Eberwein, 2000;Nickrent, 2002;Takhtajan, 2009).The presence of peltate scales falling from the inflorescence before anthesis is a characteristic of the genera in this subfamily.The scales of Helosis have a simple anatomical structure, as they consist of a compact parenchyma with abundant tannins and starch and are vascularized by a single vascular bundle.Engell (1979) found an identical structure in the scales of Corynaea crassa.A peculiarity observed in Helosis, is that its scales lack a true epidermis, and the scale surface is instead formed by several cell layers, which gradually collapse forming a protective covering.Presence of such a protective coating instead of the true epidermis was also observed on the peduncle of the inflorescence.
Sterile portions of Helosis inflorescences display anatomical features observed in the vegetative body of several holoparasites: replacement of the epidermis by a multilayered protective tissue, almost universal presence of tanniniferous cells and development of ingrowts in tracheary elements.Such peculiar features had already been described for the vegetative body of Helosis by Hsiao & al. (1993) and Shu-Chuan & al. (1993), as well as in other holoparasites, including Lophophyton leandrii and L. mirabile (González & Mauseth, 2010;Sato & González, 2013) and Ombrophytum subterraneum (Mauseth & Montenegro, 1992).
The filariae or paraphyses that cover the inflorescence of Helosiodoideae subfamily and surround the flowers are considered nectar-secreting structures by Heide-Jørgensen (2008).In Rhopalocnemis, these structures are indeed interpreted as nectariferous by van Steenis (1931), due to the secretion and scent they produce.Engell (1979) also came to similar conclusions about the filariae in Corynaea crassa after studying their anatomy using inflorescences fixed in a liquid preservative.The filariae of Helosis have an identical anatomy to C. crassa, in which the distended cuticle in the apical portion would indicate their secretory nature.Although Helosis living inflorescences had no secretion or scent (detectable by humans), they were covered with ants, suggesting that the absence of nectar could be explained by the fact that the ants remove the nectar as it is secreted.

Flower
Balanophoraceae is one of the eudicots families characterized by trimerous flowers (Endress, 1996).The presence of an irregularly lobed perianth is restricted to male flowers of subfamily Helosiodoideae (Harms, 1935).According to Fagerlind (1938a), the tepals of Rhopalocnemis and C. crassa are fused, but in the latter species perianth segments are free (as observed by Engell, 1979, too).In H. cayennensis (this study) and Exorhopala ruficeps (Eberwein & Weber, 2004), tepals are fused for a short part at the base.The presence of an inner epidermis formed by sclereids in H. cayennensis is consistent with the description of the perianth of C. crassa.
The synandrium is a characteristic of subfamily Helosiodoideae (Umiker, 1920;Kuijt, 1969;Engell, 1979;Eberwein & Weber, 2004).Studies on the flowers vascular supply in the subfamily show that the androecium always consists of three stamens but there are discrepancies in the number of pollen sacs among genera or even within a same species.In E. ruficeps, Eberwein & Weber (2004) described trimerous staminate flowers with anthers fused into a globose synandrium consisting of 10 to 16 pollen sacs.These authors consider that the inner pollen sacs do not correspond to any particular stamen, and, therefore, transferred E. ruficeps to Helosis ruficeps.Engell (1979)'s description of staminate flowers of C. crassa agrees with that of Helosis staminate flowers studied here but C. crassa flowers have only three to six pollen sacs.Helosis flowers have three stamens, each with its own vascular bundle; nevertheless they have nine pollen sacs, in which the three inner ones alternate with the outer ones, making it difficult -as in H. ruficeps-to assign with certainty which pollen sac pertains to which stamen.
Presence of a synandrum and stamens reduced to an undefined or variable number of pollen sacs is frequently observed in families with flowers with a high level of reduction, as in Araceae (Grayum, 1990) and Myristicaceae (De Wilde, 2000;Sauquet, 2003).This reduction is a character common in par-  Huaxing & Gilbert, 2003), or in Balanophora, Langsdorffia, Thonningia and Rhopalocnemis genera (Balanophoraceae, Kuijt, 1969;Eberwein & al., 2009).In Apodanthaceae, the anthers form a tube that surrounds the sterile gynoecium, in which neither thecae nor stamens can be recognized, pollen sacs are arranged in pairs, and the dehiscence is produced by a transverse aperture through the two rings of the pollen sacs (Blarer & al., 2004).

Androecium anatomy and pollen morphology
Analyses of the floral vascularization suggest an extreme reduction of Helosis flowers.Indeed only the stamens possess vascular supply (which originates directly from the inflorescence axis), whereas, the perianth and the pistillode lack any vascularization.
Anther walls in Helosis possess an epidermis, parietal layers, and a secretory tapetum.Umiker (1920) describes Helosis anthers having a plasmodial tapetum and tetrahedral tetrads formed by simultaneous cytokinesis.He postulates that Helosis is an apomictic plant and observed no endothecium or other indication of anther dehiscence.The endothecium is indeed absent also in other parasitic plants, as Ditepalanthus (Fagerlind, 1938b), Balanophora (Fagerlind, 1945), and Pilostyles (Rutherford, 1970).However, our observations indicate that staminate flowers emerge from the mass of filariae by the growth of staminal filaments, so that the pollen sacs are exposed to atmosphere; the synandrium dehiscence occurs through apical openings without mechanical layers; pollen is released to tricellular state.These observations are consistent with those of Lotsy (1901) for Rhopalocnemis.Engell (1979) describes the same anatomy in anthers of C. crassa, he mentions that some male flowers do not emerge from the mass of filariae, and probably do not release pollen (but these could have been young flowers, as it occurs in Helosis).
Maybe one of the earliest works on apical dehiscence is that of Harris (1905), who classified them into seven types and already mentions Balanophoraceae alongside the Araceae in type 1 or "Araceous Type", often characterized by anthers united in one synandrium.Dehiscence through apical openings without endothecium is a common feature in flowers with stamens attached at synandrium (Maheshwari, 1950;Eames, 1961;Esau, 1965;Davis, 1966).However, later studies showed that this correlation cannot be generalized and even is wrong in monocots with poricidal dehiscence (Gerenday & French, 1988) or in families like Begoniaceae (Tebbitt & Maciver, 1999).
The staminate flowers of Helosis have a rudimentary gynoecium; totally inferior.In H. ruficeps, Eberwein & Weber (2004) described staminate flowers without rudimentary gynoecium, however it is evident in the photographs of the inflorescence.Of the related genera, their presence was described in C. crassa (Engell, 1979).
Lophophytum is another genus of Balanophoraceae where the anatomy and development of the male gametophyte was studied (Sato & González, 2013), but belongs to the subfamily Lophophytoideae (Takhtajan, 2009) in which the inflorescences lack filariae.In Lophophytum the stamens are free and have a longitudinal dehiscence with normal stomium and endothecium.
Poricidal dehiscence in Helosis flowers may represent an adaptation to the presence of the dense mass of filariae that could hinder pollen release.Pollen is in fact released when the anthers emerge and are exposed above the dense layer of filariae.
In conclusion, despite the extreme reduction of flowers, the anatomic characteristics and gametophyte development of staminate flowers of Helosis cayennensis are perfectly normal and functional.They are thus highly similar to other genera of the holoparasitic subfamily Helosidoideae.Sterile parts of flowers and inflorescence maintain the same distinctive and aberrant features of the plant vegetative parts.
spicuous nuclei and one or two nucleoli, and the cytoplasm is very dense (Fig.4 E).The shape of these cells change from polygonal to rounded, and cells separate from each other and from the tapetum to become microspore mother cells (MMC; Fig. 4 G).Meiosis is initiated simultaneously; MMCs are surrounded by a callose wall (Fig. 4 H).Meiosis takes place in the usual way, thin chromosomes appear during prophase I; chromosomes acquire their maximum condensation and are located in the equatorial plane in the metaphase I; they move to the poles, and the CMM reach anaphase I forming a dyad of cells enclosed in a callose wall (Fig. 4 H-K).Meiosis II occurs rapidly and cytokinesis forms tetrads simultaneously, the four microspore are surrounded by callose walls (Fig. 4 L, arrow).The tetrads are tetrahedral (Fig. 4 M).