Vital & Nakamura. Androecium development of appendiculatum Mart. (): a taxonomic approach

Scientific Electronic Archives Issue ID: Sci. Elec. Arch. Vol. 11 (5) October 2018 Article link http://www.seasinop.com.br/revista/index.php?journal=SEA&page=article& op=view&path%5B%5D=739&path%5B%5D=pdf Included in DOAJ, AGRIS, Latindex, Journal TOCs, CORE, Discoursio Open Science, Science Gate, GFAR, CIARDRING, Academic Journals Database and NTHRYS Technologies, Portal de Periódicos CAPES.

Androecium development of Oxypetalum appendiculatum Mart. (Apocynaceae): a taxonomic approach

F. A. Z. Vital1 & A. T. Nakamura2

1 Universidade Federal de Lavras 2 Universidade Federal de Uberlândia

Author for correspondence: [email protected] ______Abstract: Circa 80% of the neotropical species of (Apocynaceae) belong to the “MOOG Clade”, whose interrelations are poorly established. This study describes the floral morphology, anther development, microsporogenesis, microgametogenesis, and pollinarium and pollen grain morphology of Oxypetalum appendiculatum to foster future taxonomic work. In addition to typical morphological features of Asclepiadoideae, as a bifid vinaceous appendix and a translator with a laterally expanded retinaculum, flowers present a clear division of stamens into three groups, with respect to size and arrangement in the flower, which has not yet been reported in the literature. Stamens present connate, subsessile filaments, bisporangiate anthers with six parietal layers, and dicotyledonous development. Endothecium is unthickened; secretory tapetum is biseriate on the adaxial side and multiseriate on the abaxial surface. Successive microsporogenesis produces linear tetrads. Single pollen grains, united in pollinia, are tricellular and inaperturate. Features as bisporangiate anthers and successive microsporogenesis delimit the subfamily. Unthickened endothecium may indicate a derived position of O. appendiculatum in the “MOOG Clade”. Keywords: Anther. Endothecium. Microsporogenesis. “MOOG Clade”. Subsessile Filament. ______

Introduction Four lineages of Asclepiadoideae grow in the With circa 355 genera and 3700 to 5100 New World: (1) Marsdenia (Marsdenieae); (2) species, Apocynaceae () occur on all the subgen. Mellichampia (A. Gray ex S. continents except Antarctica (RAPINI, 2012). Their Wats.) Sundell (Cynanchinae); (3) Asclepias specimens are easily recognized by the presence of (Asclepiadinae); and (4) a clade named “MOOG”, latex and a style head originating from the fusion of comprising subtribes Metastelmatinae Endl. ex the two carpels at style apex (RAPINI, 2012; Meissn., Orthosiinae Liede & Rapini, Oxypetalinae K. ENDRESS & BRUYNS, 2000). Asclepiadoideae, a Schum., and Gonolobinae (G. Don) Liede (LIEDE- derived subfamily, presents a series of androecium SCHUMANN ET AL., , 2005; RAPINI ET AL., , 2003). and gynoecium features, associated with a Third largest subtribe in the “MOOG Clade”, mechanism that increases pollination efficiency, which Oxypetalinae underwent generic rearrangements have evolved in close relation to the fusion of floral marked by the inclusion of various small genera into organs (ENDRESS, 2016). Among these innovations Oxypetalum and to avoid a paraphyletic is the presence of pollinia, which significantly reduces classification of this group (RAPINI ET AL., 2011; pollen loss during pollination (HARDER & ROUTLEY, LIEDE-SCHUMANN ET AL., 2014). With circa 130 2006; HARDER & JOHNSON, 2008). In addition, the neotropical species (SILVA et al., 2008), Oxypetalum precise location of the floral whorls allowed a fusion R. Br. is the largest genus of Oxypetalinae. Forty-four resulting in a gynostegium, which both prevents self- species, i.e. 47.3% of the 93 species known in Brazil, pollination and creates specific sites that guide grow in the state of Minas Gerais (SILVA et al., 2008; pollinators during pollinarium removal (HARDER & VIEIRA & SHEPHERD, 2002). AIZEN, 2010; VIEIRA & SHEPHERD, 2002).

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Vital & Nakamura. Androecium development of Oxypetalum appendiculatum Mart. (Apocynaceae): a taxonomic approach

Since they are used to define subfamilies captured with a photomicroscope or a trinocular photo (CIVEYREL et al., 1998; ENDRESS & BRUYNS, stereomicroscope. 2000; VERHOEVEN & VENTER, 1998) and tribes For scanning electron microscope (VERHOEVEN & VENTER, 1998; ENDRESS et al., examination, pollinaria of flowers at anthesis were 2014), androecium features are taxonomically removed, fixed in Karnovsky’s solution with 0.05 M important in Apocynaceae. Though still little explored, sodium cacodylate buffer (pH 7.2) (KARNOVSKY, pollinarium features, mainly with regard to sporangium 1965). Samples were washed in 0.05M sodium formation and organization, should also be quite cacodylate buffer and post-fixed in osmium tetroxide useful for that matter. 1%, between 1-4 hours, at room temperature. They The androecium of Oxypetalum was studied were dehydrated in acetone solution, submitted to and made remarkable contributions to the CO2 critical drying point using a BAL-TEC, CPD-030, embryological studies of Apocynaceae addressing fixed on a metallic support with silver cement, and mainly microsporogenesis and microgametogenesis coated with gold (10 nm) using a BAL-TEC SCD-050 (CORRY, 1882; FYRE & BLODGETT, 1905; device. Prepared material was observed and GUIGNARD, 1917; MEYER, 1938; RAU, 1940). electromicrographed with a LEO-EVO 40 XVP Based on such studies, Maheshwari (1970) scanning electron microscope. reported the plasticity of mother cell division in microspores and characterized the family as Results heterogeneous since different species of a given Anther Wall genus can present simultaneous or successive Oxypetalum appendiculatum Mart. presents cytokinesis. Therefore, embryology studies can be an five bithecal, bisporangiate anthers (Fig. 1 - except k) important tool to elucidate kinship relationships and in convex position on the gynostegium (Fig. 1a). foster the family . Anthers can be classified into three groups, according This work thus analyzes the floral morphology to their position in the flower bud: (1) two big inferior of Oxypetalum appendiculatum, to describe anthers; (2) two medium-sized intermediate anthers; androecium development, anatomical aspects of and (3) a small superior anther (Fig. 1a). anther development, microsporogenesis, and Anther wall development conforms to the microgametogenesis, in addition to pollinarium and dicotyledonous type. Although anthers present pollen grain morphology. Such information can help different sizes, their wall development, understand how the reproductive structures of microsporogenesis, and microgametogenesis follow Apocynaceae have developed and provide resources the same pattern. Yet anthers and microsporangia of for the family systematics. the same anther do not develop concomitantly (Fig. 1a – 3c). Methods Locule formation begins in the two basal Botanical materials of Oxypetalum anthers, where the contiguous locules develop first to appendiculatum were collected in a rocky outcrop form a pollinarium. Then, the adjacent locules of basal (campo rupestre) within the boundaries of the town of and median anthers form simultaneously. Finally, the Lavras, Minas Gerais. Herborized specimens were adjacent locules of the intermediate anthers and of the deposited at the ESAL Herbarium, Federal University superior anther complete the development. of Lavras (voucher number ESAL 27.511). Longitudinal sections show that locules are located on Buds and flowers at pre-anthesis, anthesis, the inferior half of anthers (Fig. 3d) and that their and post-anthesis were collected to analyze height varies according to anther size (Fig. 1a). histologically anther walls, microsporogenesis, and Anatomically, the anther primordium of O. microgametogenesis. Material was fixed in appendiculatum presents uniseriate protoderm with Karnovsky’s solution for 24 hours (Karnovsky 1965), cuboid cells containing evident nuclei. The ground gradually dehydrated in an ethanol series (10%, 20%, meristem of anthers divides into two parts: one with 30%, 40%, 50%, and 60%) and conserved in ethanol cells presenting a dense aspect, which will form a 70%. microsporangium, the other, with bigger cells showing Samples were dehydrated in a growing a hyaline aspect, will constitute an anther (Fig. 1b). ethanol series, infiltrated, and embedded in At the second stage of bud development, a hydroxyethyl methacrylate (Leica®) to make up radially elongated primary parietal layer appears in the permanent slides. Circa 5 µm cross and longitudinal locule region (Fig. 1c). It undergoes a periclinal sections were cut using a rotary microtome, stained division (Fig. 2d) to originate the inner and outer with Toluidine Blue O (pH 4.7) (O’BRIEN et al., 1964), primary parietal layers (Fig. 1d - e). The layer and mounted in Acrilex 500 ® colorless glass varnish subjacent to the primary parietal layer also elongates (PAIVA et al., 2006). All the slides were analyzed with radially and undergoes a periclinal division to originate optical and stereo microscopes and images were the sporogenous cells, externally, and more meristematic cells, internally (Fig. 1c - d). 131

Vital & Nakamura. Androecium development of Oxypetalum appendiculatum Mart. (Apocynaceae): a taxonomic approach

Figure 1. Longitudinal sections of Oxypetalum appendiculatum buds and flowers. A: General aspect of androecium. Left to right, two big inferior anthers, two medium-sized intermediate anthers and a small superior anther. B: Anther primordium showing meristematic tissue. C: Anther primordium showing protoderm, primary parietal layers (WHITE ARROW) and sporogenous cells during mitosis (*). D: Division of primary parietal layers (BLACK ARROW). E: Anther primordium with inner and outer primary parietal layers (*). F: Anther with six parietal layers: epidermis, unthickened endothecium (BLACK ARROWHEAD), two middle parenchymatous layers (WHITE ARROWHEAD), and two strata of tapetum. G: Anther at pre- anthesis, and immature anther wings. H: multiseriate tapetum (*). I: Mature anther with wall lignify and pollinium already isolated in locule. To note stomium break. J: Anther opening. K: Enconter of two adjacent anther wings forming guide rail. AS = anthers wings; CMM = microspore mother cells; FV = vascular bundle; GI = gynoecium; MI = microspore; PC = procambium; PO = pollinium; PR = protoderm; TA = tapetum; TM = meristematic tissue. SCALE BARS: 200 µm (I); 125 µm (A, G); 100 µm (B, J, K); 50 µm (C, D, E, F, H).

The outer primary parietal layer divides to undergo mitotic division and differentiates directly into originate the outer secondary parietal layer, which the tapetum. The meristematic cells of the inner divides again to form the endothecium and the medial region of the anther locule (anther abaxial surface) layer. The inner secondary parietal layer does not divides periclinally and anticlinally to form a tissue 132

Vital & Nakamura. Androecium development of Oxypetalum appendiculatum Mart. (Apocynaceae): a taxonomic approach whose cells present thin walls, dense content, and a occur concomitantly in all the anthers or in the conspicuous nucleus (Fig. 1e). microsporangia of one anther (Fig. 2c). Later, the medial layer and the tapetum divide The archesporial cells begin differentiating periclinally to forms two layers each (Fig. 1f). when four cells below the primary parietal layer Therefore, at this stage, the anther presents six stretch and divide periclinally to form, internally, the parietal layers: epidermis, unthickened endothecium, meristematic cells that will constitute the multiseriate two middle parenchymatous layers, and two strata of tapetum (Fig. 1). The four sporogenous cells develop tapetum. Anther wall formation follows the into four cell columns that undergo transversal dicotyledonous type. mitoses to fill the lower half of the anther locule with It is worth noting that, on the anther abaxial microspore mother cells arranged in an inclined surface, the cells adjacent to the locule, which began manner. They become more evident than the other differentiating at the previous stage, form a tapetum microsporangium cells (Fig. 2d) and undergo a radial (Fig. 1f) that is biseriate on the locule adaxial face and mitosis to form eight microspore mother cells, also multiseriate on its abaxial surface, which is oriented called microsporocytes (Fig. 2c-e-f). towards the vascular bundle. The former presents Each microspore mother cell undergoes a cuboid cells with well-defined walls and a conspicuous meiosis to form a microspore dyad and, immediately nucleus located in the central region (Fig. 1f), while after, a microspore tetrad with walls developing after the later has radially elongated to cuboid cells. The each division (Fig. 2g), characterizing a successive tapetum of O. appendiculatum is secretory (Fig. 1f-g). microsporogenesis. These microspore tetrads are At pre-anthesis, the uniseriate epidermis linear (Fig. 2h). At the end of this stage, cytokinesis presents cuboid to slightly convex cells, whose size and callose deposition, which separates the varies (Fig. 1g-h). The lateral extremities of anther microspores, take place (Fig. 2h) and sporoderm develop a protruding border, the “anther wing”, which deposition has already begun. gives it its characteristic triangular shape (Fig. 1g). In Microgametogenesis then begins and cells the distal region of wing, although epidermis undergo asymmetric mitosis to originate bicellular elongates radially, it remains single-layered with thin- pollen grains (Fig. 2i). The vegetative cell with its walled cells and is covered with a thin cuticle (Fig. 1g). central spherical nucleus is located in the central Epidermis, endothecium, and middle layers become region, while the generative cell is found around it radially thinner and are compressed in the stomium (Fig. 2i). The former, which occupies most of the region. Some tapetum cells can become binucleate young gametophyte, undergoes a division that (Fig. 1g). originates two spermatic cells (Fig. 3k). Mature pollen At the stage corresponding to the maximum grain is tricellular and inaperturate (Fig. 2j, k, l, m). degree of flower maturity, when anthesis occurs, Sporoderm is smooth and does not present any exine epidermis, endothecium, and middle layers are ornamentation (Fig. 2l-m). obliterated in the stomium region, which turns towards In this species, pollen grains are arranged the inner part and contacts the gynostegium (Fig. 1i). individually in compartments (Fig. 2j-m), but Tapetum has already begun to degenerate (Fig. 1i-j). agglutinated in pollinia (Fig. 2k, l, m; Fig. 3 a-d), and Anther wings grow longer and anther parenchyma, enclosed in an amorphous (Fig. 2l-m) and hyaline which shows secondary wall deposition, lignifies. On (Fig. 2k) layer. the adaxial face of anther, in the region between thecae, the epidermis and part of the septum also Pollinarium lignify (Fig. 1j). The rest of the epidermis, the vascular The secretory epidermis of the style head bundle, and the region surrounding the locule, with secretes the translator, which comprises a circa six cell layer, that includes part of the septum retinaculum and caudicles (Fig. 4a). This fluid separating the thecae, do not lignify (Fig. 1i-j). The secretion deposits in the slits created by the junction distal part of anther wings shows the formation of the of adjacent anther wings (Fig. 4a, f). When anthesis guide rails (Fig. 1k). occurs, this fluid contacts a pollinium nestled within an anther locule to form a pollinarium, that is, a set Microsporogenesis and Microgametogenesis composed by a pollinium (reproductive portion) and a Microspore development begins in the anther translator (sterile portion) (Fig. 4a-d). primordium when the protoderm involves the The caudicles, segments of the translator that meristematic tissue into a differentiation that forms are in direct contact with the pollinia, presents two anther microsporangia. Both are separated by the expansions at apex (Fig. 4a). They show visible vacuolated cells constituting the septum, clearly damages when a pollinarium is removed from a showing the sporangium initial cells, which are flower, revealing that the translator is firmly attached isodiametric and present dense cytoplasm (Fig. 2a). to the outer epidermis of anther (Fig 4b, c). Microsporogenesis and microgametogenesis do not

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Vital & Nakamura. Androecium development of Oxypetalum appendiculatum Mart. (Apocynaceae): a taxonomic approach

Figure 2. Oxypetalum appendiculatum microsporogenesis and microgametogenesis Anther cross sections, except F, K and L, that are longitudinal sections. I-J: Scanning electron microscope. A: Anther primordium showing protoderm and meristematic tissue. B: sporogenous cells during mitosis (BLACK ARROW). C: Overview of buds showing anthers in diferent height. D: Differentiated sporogenous cells. E, F: Microspores. G: Dyad (*); anther wall after division (BLACK ARROWHEAD). H: Late tetrads (*). I: Bicellular pollen grains. J: Mature pollen grains arranged individually in compartments. K: Pollinarium showing tricellular pollen grain; WHITE ARROW indicate two spermatic cells. L: Overview of pollinium showing each pollen grain at compartments. M: Inaperturate pollen grain. SCALE BARS: 200 µm (C); 100 µm (F); 50 µm (A, B, D, E, H, I, K); 25 µm (G); 20 µm (L); 10 µm (M).

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Vital & Nakamura. Androecium development of Oxypetalum appendiculatum Mart. (Apocynaceae): a taxonomic approach

Figure 3. Oxypetalum appendiculatum pollinarium. A-C: Scanning electron microscope. Longitudinal sections (D) and cross section (E-F). A: Overview of pollinarium. B. Pollinaria showing the pollen grains at individually compartments. C: Damaged caused when a pollinarium is removed from anther (WHITE ARROW). D: Overview of pollinarium tied to anther. E: Translator, with retinaculum showing “clipping mechanism” and two caudicles in basal part. F: Projection on the gynostegium epidermis fixes the translator. AN = adjacent anther; AS = anther wings; C = caudicles; FI = filament; PO = pollinaria; RE = retinaculum; BLACK ARROW = denticle. SCALE BARS: 250 µm (F); 200 µm (A, D, E); 100 µm (B); 20 µm (C).

The translator presents a winged expansion at The pollinium, in turn, is involved in a film (Fig. retinaculum apex (Fig. 4a) that reaches the apical and 4 a-c). External observation clearly shows the adaxial regions. This projection on both sides creates depressions delimiting the chambers in which each two hooks facing opposite directions that are pollen grain nestles (Fig. 4a-b). When viewed through responsible for the fixation on the pollinating agent, an optical microscope, this film is crystalline (Fig. 3h). allowing the dispersal of the whole pollinarium (Fig. It involves each pollen grain in individual 4a, d, e). At retinaculum apex, the anther denticles are compartments and the whole pollinium. an active part of this “clipping mechanism” morphology (Fig. 4e). At the basal part of retinaculum, Discussion a projection on the gynostegium epidermis fixes the The bilocular anther of Oxypetalum translator and, consequently, the pollinarium (Fig. 4e, appendiculatum develops according to the f). dicotyledonous type. It is constituted by six parietal 135

Vital & Nakamura. Androecium development of Oxypetalum appendiculatum Mart. (Apocynaceae): a taxonomic approach layers: epidermis, endothecium, two middle layers, lignification, whereas the other subfamilies present and a biseriate secretory tapetum, resembling the lignification occurring in different anther regions, as description of Oxypetalum banskii Roem. et Schult. apex or base or even along its whole extension subsp. banskii (VALENTE, 1977). Bilocular anthers (ENDRESS & BRUYNS, 2000). Valente & Costa are inherent and exclusive to the individuals of (2005) showed the presence of endothecial bars of Asclepiadoideae. Since the other subfamilies of thickening subjacent to the locules in the anthers of Apocynaceae present tetralocular anthers (ENDRESS Marsdenia loniceroides E. Fournier. & BRUYNS, 2000), except Trachelospermum fragrans Phylogenetically, M. loniceroides is inserted in (), which is also bisporangiate (Sud tribe Marsdenieae, a tribe with endothecial thickening 1984), they indicate a synapomorphic character state. that is not part of the “MOOG Clade”, while O. When they analyzed the pattern of anther appendiculatum, inserted in tribe Asclepiadeae, within development of Rauvolfia serpentina (L.) Benth. ex the “MOOG Clade” (LIEDE-SCHUMANN et al., 2005), Kurz, Ghimire, Ghimire & Heo (2011) concluded that does not present endothecial thickening. This leads to this species also presents six parietal layers, conclude that endothecial thickening can be a secretory tapetum, and anther development of the character inherent only to ancestral tribes of this dicotyledonous type. Thus, the anthers of clade. It is suggested that the absence of endothecial Rauvolfioideae Kostel (Rauvolfia L.) and thickening in O. appendiculatum is justified by the fact Asclepiadoiedae R. Br. ex Burnett (Oxypetalum R. that the function of assisting anther opening is Br.) are anatomically similar, although these perfectly performed by the lignified tissues of anthers. subfamilies are located at opposite extremes of the In addition, the secondary thickening of the anther evolutionary derivation of Apocynaceae. walls, which imprisons the pollinator, helps the In addition, the anthers of Vallaris foetida (L.) pollination process (KOTOVSKI, 2013). Raf., another species of Rauvolfioideae with The secretory tapetum, which is biseriate on dicotyledonous development, present 10 to 14 parietal the adaxial face and multiseriate on the abaxial face layers (MAHESHWARI, 1970). Therefore, the anther of the studied species, plays many functions, among development of the dicotyledonous type can be which microspore nutrition and pollen grain considered a constant in family Apocynaceae, development, production of locular fluid, callase, although the number of parietal layers may vary polysaccharides, exine precursors, viscin ligaments, among members. The number of parietal layers may orbicules, proteins, enzymes, and “pollenkitt”/tryphine represent a constant character among species of a (DEMARCO, 2015; PACINI, 2010). A secretory given genus, as observed in Oxypetalum, but it seems tapetum with voluminous anthers and many to have evolved more than once, independently. microspores/pollen grains is a feature inherent to the Manning (1996), who also addressed parietal species of Apocynaceae (DEMARCO, 2015; PACINI layers, analyzed 125 families of angiosperms and et al., 1985). verified that their endothecium walls present a Biseriate tapetum has been reported in characteristic thickening. In family Apocynaceae, the species of subfamily Asclepiadoideae as Cynanchum endothecium is constituted by elongated cells whose callialata, daemia, and Tylophora indicates walls present lignified secondary thickening in the (MAHESWARI, 1964). Nonetheless, these layers do form of trabeculae, a character that is not constant in not multiply on the anther abaxial surface as in O. all the subfamilies (KOTOVSKI, 2013; SIMÕES et al., appendiculatum. The multiseriate tapetum of O. 2006). Kotovski (2013) used the pattern of appendiculatum might be an autapomorphy, but more endothecium lignification of Allamanda L. as a studies are needed to prove such assertion. Demarco taxonomic tool, justifying that the homogenous (2015), who analyzed the secretory structures of lignification of endothecium is a feature that maintains Asclepiadeae (Asclepiadoideae), concluded that the this genus in Rauvolfioideae. glandular tapetum cells secrete a lipid substance that However, O. appendiculatum shows no both covers the whole pollinium and is an exine evidence of thickening, since its endothecium exhibits precursor. little apparent parenchymatous cells in the anther. The hyaline film that covers the pollinia of O. Anther lignification, nevertheless, is massive, as a appendiculatum, which, according to Demarco (2015), whole, and the distal ends of wings exhibit a guide is composed of sporopollenin secreted by the rail. Simões et al., (2006) reported that the formation tapetum, both covers the pollinia and creates of guide rails directing the pollinator’s mouth compartments isolating pollen grains one from apparatus and legs is also found in the endothecial another (VERHOEVEN & VENTER, 2001). Matelea thickening of tribe Mesechiteae Miers. denticulata (Vahl) Fontella and E.A. Schwarz present (Apocynoideae). a hyaline film similar to the one found in the species of Rauvolfioideae (except genera of Oxypetalum (SCHILL & JAKEL, 1978). In these Tabernaemontaneae G. Don) and Periplocoideae R. species, such presence may be explained by the Br. ex Endl do not show specialized anther proximity of subtribes Gonolobinae (Matelea) and 136

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Oxypetalinae (Oxypetalum) in the “MOOG Clade” structure: (1) Rauvolfioideae present colporate or (DEMARCO, 2015; LIEDE-SCHUMANN et al., 2005). porate pollen; (2) Apocynoideae and Periplocoideae The pollinia of Periplocoideae and have porate pollen; (3) Secamonoideae and Secamonoideae are not coated with a hyaline film Asclepiadoideae (Oxypetalum), which present true (Verhoeven & Venter 2001). Tribe Fockeeae pollinia, produce inaperturate pollen (ENDRESS & (Asclepiadoideae) presents a reduced pollinial wall BRUYNS, 2000). This feature ratifies the presence of quite similar to that of species of subfamily O. appendiculatum in Asclepiadoideae. Secamonoideae, guaranteeing its maintenance as the Subsessile stamen and absence of most basal among Asclepiadoideae (POTGIETER & endothecial thickening characterize genus ALBERT, 2001). Oxypetalum R.Br. The position of O. appendiculatum Our results indicate successive within the group of species derived from this genus is microsporogenesis in O. appendiculatum. Species consolidated by the fact that anther filaments do not Calotropis procera (Aiton) Dryand. and specimens of form a tube. Anther arrangement in three groups, Asclepias L., pertaining to tribe Asclepiadeae, present divided according to size and position, has never been a microsporogenesis pattern similar to that of O. reported in the literature. On the other hand, the lack appendiculatum, indicating that such development of endothecial thickening is a character shared only by pattern is shared by the tribe members (CORRY, species of tribe Asclepiadeae. 1882; DICKO-ZAFIMAHOVA, 1980; ENDRESS & Bilocular anther, biseriate tapetum, BRUYNS, 2000). Successive microsporogenesis can successive microsporogenesis, linear tetrads, and even be considered a synapomorphy in inaperturate pollen grains are reported as inherent to Secamonoideae and Asclepiadoideae, whose species the species of subfamily Asclepiadoideae, where O. present true pollinia (ENDRESS & BRUYNS, 2000). appendiculatum is inserted. The linear tetrads produced by this type of Such features as anther wall development of microsporogenesis, as observed in the studied the dicotyledonous type, secretory tapetum, presence species, are also common in species that present of pollinia, and tricellular pollen grains ratify the pollen grains agglutinated in pollinia (DEMARCO, taxonomic features of family Apocynaceae. 2015; DICKO-ZAFIMAHOVA, 1980). Oxypetalum appendiculatum presents five References pollinaria. Each consists of a pair of hanging pollinia, CIVEYREL, L.; ROWE, N. Phylogenetic relationships two caudicles, and a central corpuscle, named of Secamonoideae based on the plastid gene matK, retinaculum, with winged lateral expansion. Pollinia morphology, and biomechanics. Annals of the orientation in relation to the translator was used to Missouri Botanical Garden. v. 88, p. 583-602, 2001. classify Asclepiadoideae into three tribes: (1) Marsdenieae, with erect pollinia; (2) Gonolobeae, with COCUCCI, A.A.; MARINO, S.; BARANZELLI, M.; horizontal pollinia; and (3) Asclepiadeae, with hanging WIEMER, A.P.; SERSIC, A. The buck in the pollinia (SWARUPANANDAN et al., 1996). Because milkweed: evidence of male-male interference among of its hanging pollinia, O. appendiculatum is inserted pollinaria on pollinators. New Phytologist. v. 203, p. in tribe Asclepiadeae. 280-286, 2014. Size, shape, pollinia orientation, and caudicle and retinaculum positions are mainly used to delimit CORRY, T.H. On the mode of development of the species (SINHA & MONDAL, 2011; SCHUMANN, pollinium in Asclepias cornuti, Decaisne. Transactions 1895; SILVA et al., 2008; FOURNIER, 1885). In O. of the Linnean Society of London. v. 2, n. 75-84, 1882. appendiculatum, the winged expansion at retinaculum apex is a character that helps identifying the species DEMARCO, D. Micromorphology and histochemistry (SILVA et al., 2008). Both the retinaculum and the of the laticifers from vegetative organs of caudicles present a shiny surface and corneous asclepiadoideae species (Apocynaceae). Acta consistency, but retinaculum distinguishes from Biológica Colombiana. v. 20, p. 57-65, 2015. caudicles through its reddish brown chestnut color and translucent caudicles characterize genus DICKO-ZAFIMAHOVA, L. Etude ontogenique de la Oxypetalum (SILVA et al., 2008). pollinie de Calotropis procera (Asclepiadaceae): Finally, the tricellular pollen grains of O. apport de la microscopie photonique. Grana. v. 19, p. appendiculatum are a character shared by most 85-98, 1980. species of Apocynaceae. Nevertheless, bicellular pollen grains have already been reported in ENDRESS, P.K. Development and evolution of pusillus L. and Rauvolfia serpentina (L.) extreme synorganization in angiosperm flowers and Benth ex Kurz (LAMBA, 1976). With regard to pollen diversity: a comparison of Apocynaceae and aperture in family Apocynaceae, the three Orchidaceae. Annals of Botany. v. 117, p. 749–767, development stages are reflected in the pollen 2016. 137

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