Plant Syst Evol (2012) 298:1635–1641 DOI 10.1007/s00606-012-0665-x

ORIGINAL ARTICLE

Structure of the stigma and style in Colletia and Discaria (: Colletieae)

M. M. Gotelli • B. G. Galati • D. Medan

Received: 20 April 2012 / Accepted: 31 May 2012 / Published online: 10 July 2012 Ó Springer-Verlag 2012

Abstract The ultrastructure of the stigmas and styles of spines, and presence of serial meristems in the leaf axils Colletia paradoxa, Colletia spinosissima, and Discaria (Miers 1860; Suessenguth 1953; Aagesen et al. 2005). americana was studied by transmission electron micros- The reproductive biology of some of the tribe, copy before and during anthesis. The purpose of the study among them Colletia paradoxa, Colletia spinosissima, and was to contribute a full description of possible interactions Discaria americana, is already known (Medan 1991; between the male gametophyte and the sporophytic tissues D’Ambrogio and Medan 1993; Medan and Basilio 2001). of the stigma and the style. Ultrastructure changes in the Flowers of both species of Colletia are homogamous, fra- stigmatic papillae and epithelial and sub-epithelial cells of grant, and entomophilous. Xenogamy and self-incompati- the stylar channels in relation to pollen tube growth are bility are observed in these species. There is strict separation discussed and compared between species. New pollen tube of the flowering periods of the species (D’Ambrogio and pathways are described for hollow styles. Medan 1993; Medan and Basilio 2001). D. americana is also self-incompatible and pollinated by insects, but the Keywords Stigma Á Style Á Colletia Á Discaria Á flowers are clearly protandrous (Medan 1991). Pollen tube Previous reports describe the stigma of some taxa in the tribe Colletieae as pertaining to the wet type, with con- spicuous secretion during the period of receptivity of the Introduction stigma, and the style as hollow, having three central chan- nels (Medan 1985, 1991; D’Ambrogio and Medan 1993; The tribe Colletieae is a monophyletic group that com- Medan and Basilio 2001). However, there are no studies of prises six genera that differ in flower and fruit traits changes in anatomy and ultrastructure occurring in the (Medan and Aagesen 1995; Aagesen 1999; Richardson stigma and the style before and during anthesis in Colletieae et al. 2000a, b; Medan and Basilio 2001). Distribution of in relation to pollen tube growth. the tribe is mainly associated with the Andes in South Studies on the ultrastructure of styles and stigmas are America, usually bellow 308 South (Medan and Basilio scarce, particularly for the hollow type of style, because 2001). Colletieae also includes a of Gondwanic within Angiosperms this type is less common. A single distribution, Discaria Hook. (Medan 1985; Arroyo et al. pattern of pollen tube growth (through the secretion) has 1995; Medan and Basilio 2001). The traditional diagnostic been described for hollow styles (Clarke et al. 1977; Pan- characters of the tribe are decussate leaves, abundance of dey 1997; Cresti et al. 1992). However, for solid and semi- solid styles different alternative routes have been observed (Wilms 1980). M. M. Gotelli (&) Á B. G. Galati Á D. Medan The purpose of this research was to contribute to general Grupo de Biologı´a Reproductiva en Plantas Superiores, knowledge of the family Rhamnaceae with a detailed Ca´tedra de Bota´nica Agrı´cola, Facultad de Agronomı´a, description of stigma and style structure in three species of Universidad de Buenos Aires, Av. San Martı´n 4453, C1417DSE, Buenos Aires, Argentina the tribe Colletieae, C. paradoxa, C. spinosissima, and e-mail: [email protected] D. americana, and to compare the species with regard to 123 1636 M. M. Gotelli et al. the pollen tube pathway through the stigma and style. We present in the papillae of D. americana (Fig. 1e, f). In this hypothesize there may be patterns of pollen tube growth in species the cell wall has three layers of different electron- hollow styles different from those described so far. density (Fig. 1c–f). During this stage secretion is con- spicuous in the three species (Fig. 1e, f). After anthesis papillae are mostly degraded, but some secretion remains. Materials and methods The style is hollow with three secretory central chan- nels, one for each carpel, in the three species (Fig.2a–c). Samples of C. paradoxa, C. spinosissima, and D. americana The style and stigmatic secretion react positively for lipids, were collected from individuals cultivated in the Lucien insoluble polysaccharids, and pectins (Fig. 2b–d). Hauman Botanical Garden of the Facultad de Agronomı´a, In young styles, epithelial and sub-epithelial cells of the Universidad de Buenos Aires. Reference specimens were stylar channels of C. paradoxa have abundant rough deposited in the Herbarium Gaspar Xuarez (BAA). endoplasmic reticulum, with cisterns parallel to the exter- For transmission electron microscopy, stigmas and nal tangential wall, and vacuoles and many mitochondria styles of flowers in pre-anthesis and anthesis were pre-fixed (Fig. 3a, b). Subepithelial cells have abundant pectic sub- overnight in 2.5 % glutaraldehyde in phosphate buffer stances in intercellular spaces (Fig. 3b). In C. spinosissima,

(pH 7.2) and then post-fixed in OsO4 at 2 °C in the same epithelial and subepithelial cells have a large vacuole each, buffer for 3 h. After dehydration in an ethanol series, the many mitochondria, and amyloplasts (Fig. 3c). In the material was embedded in Spurr’s resin. Sections 1 lm cytoplasm of stylar epithelial cells of D. americana dict- thick were stained with toluidine blue O and observed with yosomes, mitochondria and amyloplasts are present near a bright-field microscope. Ultrathin sections (750–900 nm) the contact zone with the subepithelial tissue (Fig. 3d). In were prepared with a Sorvall ultramicrotome and then the latter species, epithelial cells have wall ingrowths stained with uranyl acetate and lead citrate (O’Brien and composed of a fibrillar substance of moderate electron- McCully 1981). The sections were observed and photo- density (Fig. 3d). Vacuoles present in the cells have graphed with a Jeol-JEM 1200 EX II TEM at 85.0 kV. fibrillar content of moderate electron-density and there is For bright-field microscope studies, stigma and styles of abundant intercellular substance in contact with the sub- flowers at anthesis were fixed in FAA (formalin, alcohol, epithelial tissue (Fig. 3d). acetic acid), dehydrated in an ethanol series, transferred to At anthesis, the secretion accumulates between the cell xylene, and then embedded in paraffin wax. Longitudinal wall of epithelial cells and the cuticle, which breaks down and transverse sections 10 lm thick were cut by following and liberates the secretion. The cytoplasm of epithelial standard botanical microtechniques. Slides and fresh stig- cells is reduced. In C. paradoxa and C. spinosissima pollen mas were stained with Sudan Black B to locate lipids tubes grow through the cytoplasm of sub-epithelial cells, (Pearse 1961), by the periodic acid–Schiff (PAS) reaction through the middle lamella present between these cells and to locate insoluble polysaccharides (Jensen 1962), and with through the secretion in the channel of the hollow style ruthenium red to locate pectins (Jensen 1962). (Fig. 4a–d), whereas in D. americana pollen tubes grow through the intercellular substance between the epithelial and subepithelial cells. Many mitochondria are found in Results the cells that surround pollen tubes and amyloplasts are no longer present (Fig. 4e, f). No pollen tubes were The stigma is trilobed and has unicellular stigmatic papil- observed growing through the secretion of the channel in lae. Even before anthesis abundant secretion occurs on the D. americana. stigmas of Colletia spinosissima and C. paradoxa (Fig. 1a, b) and very little on those of D. americana (Fig. 1c). At this stage, in both species of Colletia, stigmatic papillae Discussion have many mitochondria and rough endoplasmic reticulum (Fig. 1a). Mitochondria are also observed in D. americana, Copious secretion occurs on the stigmas of the three spe- with dictyosomes (Fig. 1c, d). Electron-dense vesicles in cies studied: C. paradoxa, C. spinosissima, and D. ameri- exocitosis and substances with the same electron-density cana. Therefore, the stigmas in both Colletia and Discaria (secretion) are prominent between the plasmalemma and belong to Group III (receptive surface with low to medium the cell wall, and outside the cell (Fig. 1c). papillae) of wet stigmas (Heslop-Harrison and Shivanna During anthesis, papillae appear much more vacuolated, 1977), which confirms previous reports for the tribe and membrane systems seem to start degrading in both (Medan 1985, 1991; Medan and Arce 1999; Medan and species of Colletia. Free ribosomes, rough endoplasmic Basilio 2001, Medan and Montaldo 2005). In Colletia the reticulum, lipid bodies, mitochondria, and dictyosomes are secretion occurs in preanthetic flowers, which is in 123 Structure of the stigma and style in Colletieae 1637

Fig. 1 Stigma: a–d, pre-anthesis; e, f, anthesis. a Colletia spino- (cw), and little secretion (s). d Discaria americana detail showing sissima, papillae with many mitochondria (m) and rough-type many mitochondria (m) and a dictyosome (d). e Disacaria americana, endoplasmic reticulum (err), and cell wall (cw), cuticle (c), and papillae with lipid bodies (lb), mitochondria (m), and dictyosomes (d). secretion (s). b Colletia paradoxa, papillae (p) and secretion (s). f Detail of a papillae and secretion (s)inDisacaria americana. Scale c Discaria americana, dictyosome (d), mitochondria (m), cell wall bars: a, e, f 1 lm, b 5 lm; c 500 nm; d 500 nm concordance with previous reports of stigmas being wet secretion during the receptive period is imprecise. Wet and receptive at early stages of development (Medan 1991; stigmas may have secretions primarily rich in lipids, as in D’Ambrogio and Medan 1993; Medan and Basilio 2001). Solanaceae, or rich in carbohydrates, as in Liliaceae In C. spinosissima and C. paradoxa, the exudate covers the (Goldman et al. 1994). Lipidic components seem to be whole stigma and connects the three receptive areas essential for pollen tube penetration in the stigma and (Medan and Basilio 2001). ulterior growth through the style (Lush et al. 1998; Raghavan (1997) believes the correlation between the Wolters-Arts et al. 1998). The observation of globules with morphology of the stigma surface and the amount of moderate electron-density in the secretions of the three

123 1638 M. M. Gotelli et al.

Fig. 2 Discaria americana. a–c, style; d, stigma. a Transverse section of the style. b Detail of one of the three channels. c Secretion in the channel stained with periodic acid–Schiff. d Papillae and secretion. Scale bars: a 200 lm, b 50 lm, c, d 20 lm

species studied suggests the presence of lipidic components 1969; Pate and Gunning 1972). Localized wall expansions in the these species, which was confirmed by its positive were found in the epithelial cells of the stylar channel of reaction to Sudan black B. The secretion also contains Ornithogalum caudatum (Tilton and Horner 1980) and insoluble polysaccharides and pectins. Citrus limon (Ciampolini et al. 1981). The cell walls of the According to Fahn (2000) elimination of secretions from transmitting tissue of the style of Oxalis paludosa and the cytoplasm may occur by an active molecular or ionic O. hispidula have ingrowths surrounded by the plasma process (eccrine secretion) or by membrane-bound vesicles membrane (Rosenfeldt and Galati 2009). Similar observa- (granulocrine secretion). The presence of dictyosomes, tions for Petunia hybrida were made by Herrero and rough endoplasmic reticulum, and mitochondria in the Dickinson (1979). According to these authors, this char- stigmatic papillae and epithelial cells of the stylar channels acteristic associated with the plasmodesmata ensures great in Colletia spinosissima, C. paradoxa, and D. americana, efficiency in cellular exchange. suggests a granulocrine secretion. The secretion is released Different paths in the growth of pollen tubes in solid by the disruption of the cuticle. According to Heslop- styles have been observed, but only one path in hollow Harrison and Heslop-Harrison (1982) this is not peculiar to styles. In this last type, pollen tubes have always been stigmas. reported to grow through the secretion filling the stylar Epithelial cells of the stylar channel in D. americana channel (Johri 1984). In Gladiolus, the mucilaginous form wall ingrowths toward the interior of the cell, a secretion is accumulated between the epithelial cell wall common characteristic of transfer cells (Gunning and Pate and the cuticle that covers the channel. The pollen tube

123 Structure of the stigma and style in Colletieae 1639

Fig. 3 Pre-anthesis, style. a Colletia paradoxa, epithelial cells with spinosissima, with amyloplasts (a) and mitochondria (m) d Epithelial rough-type endoplasmic reticulum (err) and mitochondria (m); cells of Discaria americana, with dictyosomes (d), amyloplasts (s), secretion in the hollow style. b Colletia paradoxa, connective tissue cell wall invaginations (arrows), and intercellular substance (is)in of the style with plasmodesms (arrow head), mitochondria (m), and contract with the sub-epithelial tissue. Scale bars a–d 1 lm rough-type endoplasmic reticulum (err). c Epithelial cells of Colletia penetrates the cuticle and grows through the secretion Ultrastructural differences between the epithelium cells (Clarke et al. 1977; Pandey 1997). In Cyclamen, Polygala of the channel in the studied species can be correlated with vayredae, and Lilium longiflorum pollen tubes develop the different pollen tube paths. The presence of epithelial inside the stylar channel in the same way as in other species and subepithelial cells with cytoplasm rich in organelles and with hollow styles (Lord and Kohorn 1986; Cresti et al. a copious secretion in the lumen of the channel in the two 1992; Reinhardt et al. 2007; Castro et al. 2008). In this species of Colletia could favor growth of the pollen tubes study, we observed three different paths for the pollen tube both through the cytoplasm of these cells and the channel in both species of Colletia: through the secretion in the secretion. Conversely, very vacuolated epithelial cells, with channel, through the middle lamella between epithelial and characteristics of transfer cells, abundant amyloplast, and subepithelial cells of the stylar channel, and through the limited channel secretion in D. americana may lead pollen cytoplasm of subepithelial cells. Unlike in Colletia,in tubes to grow through the thickened middle lamella D. americana pollen tubes grow only through the middle between the epithelial and subepithelial cells, which is lamella located between epithelial and subepithelial chan- probably the most favorable medium. The presence of nel cells. Considering previous reports and given that the starch in the epithelial cells of this last species is notable. style of the three studied species is hollow and provided According to Rosenfeldt and Galati (2000) starch is com- with a well differentiated epithelium, the pollen tube monly present in the peripheral parenchyma of the trans- pathways observed here were unexpected. Growth of pol- mitting tissue and is consumed by the pollen tube during len tubes through the middle lamella or inside the cellular growth and not by the cells that contain it. Similar events lumen has, so far, previously been observed only in the occur in D. americana, in which the starch is present in the transmission tissue of solid and semi-solid styles (Wilms epithelial cells, marking a probable reason for pollen tube 1980, Weber and Frosch 1995, Hristova et al. 2005, Gotelli growth through the middle layer and not through the cyto- et al. 2010). plasm of these cells as occurs in both species of Colletia.

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Fig. 4 Anthesis, style. a–d, Colletia paradoxa; e, f, Discaria through the secretion (s). c Pollen tube growing through the cell americana. a Pollen tube (pt) growing through the middle lamella cytoplasm of an epithelial cell. d Detail of c. e Pollen tube growing between epithelial and sub-epithelial cells. Large amount of secretion through the middle lamella of epithelial and sub-epithelial cells. (S) and remains of the cuticle (arrowhead). b Pollen tube (pt) growing f Detail of e. Scale bars a–f 1 lm

Conclusion References

This research has resulted in an original contribution to Aagesen L (1999) Phylogeny of the tribe Colletieae, Rhamnaceae. knowledge of the anatomy and ultrastructure of the style Bot J Linn Soc 131:1–43 Aagesen L, Medan D, Kellermann J, Hilger HH (2005) Phylogeny of and stigma in the family Rhamnaceae. Two new pollen the tribe Colletieae (Rhamnaceae)—a sensitivity analysis of the tube pathways are described for hollow styles: through the plastid region trnL-trnF combined with morphology. Syst middle lamella and through the cell cytoplasm, in addition Evol 250:197–214 to the already known path through the channel secretion. Arroyo TM, Caviares L, Pen˜aloza A, Riveros M, Faggi AM (1995) Relaciones fitogeogra´ficas y patrones regionales de riqueza de

123 Structure of the stigma and style in Colletieae 1641

especies en la flora del bosque lluviosos templado de Sudame´- Medan D, Aagesen L (1995) Comparative flower and fruit structure in rica. In: Armesto J, Villagra´n C, Arroyo MC (eds) Ecologı´ade the Colletieae (Rhamnaceae). Bot Jahrb Syst 117:531–564 los bosques nativos de Chile. Editorial Universitaria, Santiago de Medan D, Arce M (1999) Reproductive biology of the Andean- Chile, pp 71–99 disjunct genus Retanilla (Rhamnaceae). Plant Syst Evol 218: Castro S, Silva S, Stanescu I, Silveira P, Navarro L, Santos C (2008) 281–291 Pistil anatomy and pollen tube development in Polygala Medan D, Basilio AM (2001) Reproductive biology of Colletia vayredae Costa (Polygalaceae). Plant Biol 11:405–416 spinosissima (Rhamnaceae) in Argentina. Plant Syst Evol Ciampolini F, Cresti M, Sarfatti G, Tiezzi A (1981) Ultrastructure of 229:79–89 the stylar canal cells of Citrus limon (Rutaceae). Plant Syst Evol Medan D, Montaldo NH (2005) Ornithophily in the Rhamnaceae: the 138:263–274 pollination of the Chilean endemic Colletia ulicina. Flora 200: Clarke AE, Considine JA, Ward R, Knox RB (1977) Mechanism of 339–344 Pollination in Gladiolus: roles of the stigma and pollen-tube Miers J (1860) On the tribe Colletieae, with some observations on the guide. Ann Bot 41:15–20 structure on the seed in the family of the Rhamnaceae. Ann Mag Cresti M, Blackmore S, Van Went JL (1992) Atlas of sexual Nat Hist. 5: 76–96, 200–216, 267–273, 370–381, 482–492; 6: reproduction in flowering . Springer, Berlin 5–14. [Reprinted with illustrations in: Miers J (1851–1861) D’Ambrogio A, Medan D (1993) Comportamiento reproductivo de Contributions to Botany. I. Willams, Norgate, Edinburgh, Colletia paradoxa (Rhamnaceae). Darwiniana 32:1–14 London p 230–304, plates 33–42.] Fahn A (2000) Structure and function of secretory cells. Adv Bot Res O’Brien TP, McCully ME (1981) The study of plant structure. 31:37–75 Principles and selected methods. Termarcarphi, Melbourne Goldman MHS, Goldberg RB, Mariani C (1994) Female sterile Pandey AK (1997) Introduction to the embryology of Angiosperms. tobacco plants are produced by stigma specific cell ablation. CBS Publishers and Distributors, Daryaganj EMBO J 13:2976–2984 Pate JS, Gunning BES (1972) Transfer cells. Annu Rev Plant Physiol Gotelli M, Galati B, Medan D (2010) Structure of the stigma and style 23:173–196 in sunflower (Helianthus annuus L.). Biocell 34:133–138 Pearse AGE (1961) Histochemistry, theoretical and applied, 2nd edn. Gunning BES, Pate JS (1969) Transfer cells: plant cells with wall Little Brown, Boston ingrowths, specialized in relation to short distance transport of Raghavan V (1997) Molecular embryology of flowering plants. solutes—their occurrence, structure and development. Protopl- University Press, Cambridge, pp 33–35 asma 68:107–133 Reinhardt S, Ewald A, Hellwig F (2007) The Anatomy of the stigma Herrero M, Dickinson HG (1979) Pollen–pistil incompatibility in and style from Cyclamen persicum (Mill.) cv. ‘‘pure white’’ and Petunia hybrida—changes in the pistil following compatible and its relation to pollination success. Plant Biol 9:158–162 incompatible intraspecific crosses. J Cell Sci 36:1–18 Richardson JE, Fay MF, Cronk QCB, Chase MW (2000a) A revision Heslop-Harrison J, Heslop-Harrison Y (1982) The specialized cuticles of the tribal classification of Rhamnaceae. Kew Bull 55:311–340 of the receptive surfaces of angiosperm stigmas. In: Cutler DF, Richardson JE, Fay MF, Cronk QCB, Bowman D, Chase MW Alvin KL, Price CE (eds) The plant cuticle, Linnean Society (2000b) A phylogenetic analysis of Rhamnaceae using rbcL. and Symposium No. 10. Academic Press, London, pp 99–120 trnL-F plastid DNA sequences. Am J Bot 87:1309–1324 Heslop-Harrison Y, Shivanna KR (1977) The receptive surface of the Rosenfeldt S, Galati BG (2000) Stigma and style morphology in angiosperm stigma. Ann Bot 41:1233–1258 Ceiba insignis (Bombacaceae). Phytomorphol 50:69–74 Hristova K, Lam M, Feild T, Sage TL (2005) Transmitting tissue Rosenfeldt S, Galati BG (2009) The structure of the stigma and the ECM distribution and composition, and pollen germinability in style of Oxalis spp. (Oxalidaceae). J Torrey Bot Soc 136:33–45 Sarcandra glabra and Chloranthus japonicas (Chloranthaceae). Suessenguth K (1953) Rhamnaceae. In: Engler A, Prantl K (eds) Die Ann Bot 96:779–791 Natu¨rlichen Pflanzenfamilien, vol 20d, 2nd edn. Duncker & Jensen WA (1962) Botanical histochemistry. Freeman, San Francisco Humblot, Berlin, pp 7–173 Johri BM (1984) Embryology of Angiosperms. Springer, Berlin Tilton VR, Horner HT Jr (1980) Stigma, style, and obturator of Lord EM, Kohorn LU (1986) Gynoecial development, pollination, Ornithogalum caudatum (Liliaceae) and their function in repro- and the path of pollen tube growth in the tepary bean, Phaseolus ductive process. Am J Bot 67:1113–1131 acutifolius. Am J Bot 73:70–78 Weber M, Frosch A (1995) The development of the transmitting tract Lush WM, Grieser F, Wolters-Arts M (1998) Directional guidance of in the pistil of Hacquetia epipactis (Apiaceae). Int J Plant Sci Nicotiana alata pollen tubes in vitro and on the stigma. Plant 156:615–621 Physiol 118:733–741 Wilms HJ (1980) Development and composition of the spinach ovule. Medan D (1985) Fruit morphogenesis and seed dispersal in Colletiae Acta Bot Neerl 29:243–260 (Rhamnaceae) I. The genus Discaria. Bot Jahrb Syst 105: Wolters-Arts M, Lush WM, Mariani C (1998) Lipids are required for 205–262 directional pollen-tube growth. Nature 392:818–821 Medan D (1991) Reproductive phenology, pollination biology, and gynoecium development in Discaria americana (Rhamnaceae). N Z J Bot 29:31–42

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