Pollen Morphology and Ultrastructure of Several Gnetum Species: an Electron Microscopic Study
Total Page：16
File Type：pdf, Size：1020Kb
Plant Syst Evol DOI 10.1007/s00606-015-1262-6 ORIGINAL ARTICLE Pollen morphology and ultrastructure of several Gnetum species: an electron microscopic study Maria Tekleva1 Received: 9 July 2015 / Accepted: 20 October 2015 Ó Springer-Verlag Wien 2015 Abstract Pollen grains of five Gnetum species have been Keywords Gnetales Á Gnetum pollen Á Granular studied in scanning and transmission electron microscopy: ultrastructure Á Microechinate sculpture G. africanum, G. funiculare, G. indicum, G. leptostachyum, and G. macrostachyum. The exine ultrastructure was described for the first time for G. funiculare, G. lep- Introduction tostachyum, and G. macrostachyum. The pollen grains are small, inaperturate, and microechinate. The sporoderm Pollen grains of Ephedra, Welwitschia and Gnetum have includes a rather thin tectum, granular infratectum, and been studied by means of light and electron microscopy lamellate endexine. The foot layer is indistinct or absent in (see review in Osborn 2000; Tekleva and Krassilov 2009). G. africanum, G. funiculare, and G. macrostachyum and Despite opinions on the absence of any connection between thin in G. indicum and G. leptostachyum. Gnetum africa- pollen of Gnetum and the other two genera of Gnetales in num differs from other studied species of the genus in early works (e.g., Wodehouse 1935; Gullva¨g 1966), it has having smaller supratectal microechini. They occur on become evident that the three genera share the same pattern considerably raised exine regions (islands) that are inter- of the sporoderm ultrastructure (e.g., see Osborn 2000). preted as an equivalent to the plicae in Ephedra and Wel- The only exception is G. africanum, the only studied witschia. In Asian species of Gnetum, a microechinus and African species of this genus, which was reported to have a area around it are interpreted as equivalent to the islands of baculate infratectum in contrast to all other studied mem- G. africanum and the plicae in Ephedra and Welwitschia. bers of Gnetales (Orel et al. 1986). The infratectum in G. africanum consists of few, widely The microechinate sculpture and spheroidal shape of spaced large granules in contrast to small densely packed Gnetum pollen are distinctly different from the polyplicate granules of other studied Gnetum species. A comparison of ellipsoidal pollen of Ephedra and Welwitschia. What could the published and original data on extant pollen of Gnetales have led to such a distinction and how it was acquired? and fossil ephedroid pollen shows a great similarity in the Why are pollen grains of Gnetum type almost absent in the sporoderm ultrastructure. Absence of Gnetum-like pollen fossil record? These and more questions are still pending in the fossil record may be due to their thin ectexine, when we think about this enigmatic group of seed plants, possible separation of the ect- and endexine or and accumulation of further data is needed to resolve at misinterpretation. least some of them. To answer these questions we describe and discuss pollen morphology and ultrastructure of five Gnetum spe- Handling Editor: Louis P. Ronse De Craene. cies. These species had been previously studied by the author but their description was not published and the data & Maria Tekleva (several photos) were only used for a comparison with [email protected] fossil gnetophytes in Tekleva et al. (2006) and Tekleva and 1 A.A. Borissiak Paleontological Institute, Russian Academy of Krassilov (2009). The sporoderm ultrastructure of G. Sciences, Moscow, Russia funiculare, G. leptostachyum, and G. macrostachyum is 123 M. Tekleva described for the first time. Additionally, our data on the Results pollen wall of modern Welwitschia and Ephedra, and fossil ephedroid pollen are used for comparison. Gnetum africanum Welw. (Figs. 1a, b, 2a–d) SEM Materials and methods The pollen diameter is about 12.2 lm. The sculpture is Extant pollen grains were obtained from the palynolog- microechinate. Microechini are very small, about or less ical collection of the Department of Higher Plants, M.V. than 0.2 lm high, rather widely spaced, blunt, with several Lomonosov Moscow State University (Welwitschia (5–20) microechini on considerably raised exine areas, the mirabilis Hook.f., Ephedra monosperma J.G.Gmel. ex latter looking like islands (Fig. 1a, b). C.A.Mey., and Gnetum indicum Merr.), from the her- baria of the Botanical Institute, St-Petersburg (Gnetum TEM africanum Welw. and G. funiculare Wight), and from the Biology Department’s Herbarium of the Chang-Mai The exine thickness is about 1.3 lm in island regions University, Thailand (G. leptostachyum Blume and G. (Fig. 2a, b) and about 0.45 lm between them (Fig. 2a, c). macrostachyum Hook.f.). For SEM, untreated pollen In islands, the ectexine consists of a homogeneous imper- grains were mounted on SEM stubs (covered with nail forate tectum and granular infratectum (Fig. 2d). The tec- varnish) and sputter-coated with gold–palladium (Cam- tum thickness is more or less uniform within the island, scan, Hitachi) or gold (Tescan). The pollen grains were about 0.29 lm, except for the central (top) part where the observed and photographed under SEMs: Camscan tectum has a conical process (a microechinus) and may (pollen of Welwitschia, Ephedra,andallGnetum spe- reach up to 0.46 lm thick (Fig. 2b). The tectum decreases cies, except for G. indicum), Hitachi (fossil ephedroid in thickness towards the valley regions (Fig. 2b, c). The pollen) with accelerating voltage 20 kV, and Tescan (G. infratectal granules are large, 0.2–0.6 lm in diameter, and indicum) with accelerating voltage 30 kV. For TEM, widely spaced; one to four granules were observed per individual pollen and fragments of sporangia (hydrated) island in each particular section (Fig. 2a, b, d). The foot were fixed with 1 % OsO4, dehydrated in an ethanol layer is indistinct or absent. The endexine is less electron series, stained with uranyl acetate, dehydrated in ace- dense than the ectexine, about 0.45 lm thick, uniform in tone, and embedded in epoxy resin according to Meyer- thickness around the pollen grain. It consists of five to eight Melikian et al. (2004). At least 10 pollen grains were anastomosing lamellae, which are clearly distinct in the studied under TEM for each species and 20 pollen grains outer part and closely appressed to each other in the inner under SEM for each species. Fossil pollen grains come part (Fig. 2a–d). The valley is represented by the endexine from the Furao Formation, borehole XHY2008, late and probably by a thin tectum (Fig. 2c). Maastrichtian, Amur (Heilongjiang) River area, Zeya- Bureya Basin (see Markevich et al. 2011 for detailed Gnetum funiculare Wight (Figs. 1c, d, 2e–g) information). They were picked from the residue and studied with light (LM), scanning (SEM) and transmis- SEM sion (TEM) electron microscopy. The pollen grains were sectioned with LKB-3 (pollen of Welwitschia, Ephedra, The pollen diameter is about 14 lm. The sculpture is and all Gnetum species, except for G. indicum)and microechinate. Microechini are about 0.5 lm high, regu- Leica UC6 (G. indicum, fossil ephedroid pollen) ultra- larly distributed, blunt, are located on small, distinct and microtomes. The ultrathin sections were post-stained considerably raised exine areas (islands); there are one or with lead citrate and examined under Jeol 100 B (all two microechini per each island (Fig. 1c, d). studied pollen grains) and Jeol 400 (fossil ephedroid pollen) TEMs with accelerating voltage 80 kV. TEM In this paper slightly or considerably raised exine areas with microechini in Gnetum pollen are called islands The exine thickness is about 1.3 lm in island regions and (Fig. 1b–e, g–i, arrowheads) and areas between them— about 0.8 lm between them (Fig. 2e). In islands, the ectex- valleys. These are not palynological terms but they make it ine consists of a homogeneous imperforate tectum and easy to describe the sporoderm ultrastructure of Gnetum granular infratectum (Fig. 2f, g). The tectum thickness is pollen. Otherwise, the pollen terminology follows Hesse more or less uniform within the island, about 0.09 lm, it et al. (2009). thins towards the margins (towards the valley, Fig. 2g). The 123 Gnetum pollen Fig. 1 Extant Gnetum pollen and fossil ephedroid pollen, SEM. a, from Furao Formation, Late Maastrichtian. Arrowheads border island b G. africanum. c, d G. funiculare. e, f G. indicum. g G. regions. Scale bar 3 lm for a–d, g–i;5lm for e, f;10lm for j leptostachyum. h, i G. macrostachyum. j Fossil ephedroid pollen infratectal granules are small, densely packed, scarcely dis- Gnetum indicum Merr. (Figs. 1e, f, 2h–j) cernable, about 0.02 lm in diameter. The foot layer is indistinct or absent. The endexine is about the same electron SEM density as the ectexine. It is uniform in thickness around the pollen grain, about 0.8 lm, and consists of six to eight The pollen diameter is 18 lm. The sculpture is anastomosing lamellae, which are clearly distinct in the outer microechinate. Microechini are about 0.5 lm high, regu- part and densely appressed to each other in the inner part of larly distributed, blunt, there are one or rarely two echini this layer (Fig. 2e, f). The valley is represented by the per each slightly raised area (island, Fig. 1e, f). endexine and a thin tectum (Fig. 2f, g). 123 M. Tekleva 123 Gnetum pollen b Fig. 2 Sporoderm structure of Gnetum africanum (a–d), G. funicu- packed, about 0.04 lm in diameter. The granules that lie lare (e–g), and G. indicum (h–j), TEM. a Section through a whole on the endexine are often smaller than the granules located pollen grain, arrow indicates one of the valley regions. b Island region, arrowheads point to the microechini, arrows indicate closer to the tectum.