Springer-VerlagTokyo102650918-94401618-086030669031Journal
of Plant Research J
Plant Res16410.1007/s10265-004-0164-4
J Plant Res (2004) 117:323–328 Digital Object Identifier (DOI) 10.1007/s10265-004-0164-4
© The Botanical Society of Japan and Springer-Verlag Tokyo 2004
SHORT COMMUNICATION Harufumi Nishida • Kathleen B. Pigg • Kensuke Kudo • John F. Rigby
Zooidogamy in the Late Permian genus Glossopteris
Published online: July 16, 2004
Abstract We describe details of anatomically preserved fossil glossopterid ovules from the Late Permian of Queensland, Australia, that contain several pollen tubes at various stages of releasing flagellated sperm. Each sperm is approximately 12.7 mm long and 13.9 mm wide, with a conspicuous spiral structure comprised of a series of dots that resemble the position of basal bodies of flagella aligned along the multilayered structure (MLS). This configuration is similar to the helically arranged flagella in the sperm of cycads, Ginkgo, and many pteridophytes. However, the motile gametes of Glossopteris are considerably smaller than those of Ginkgo and cycads, and more similar in size, number of basal bodies, and number of gyres in their helix to pteridophyte forms. Glossopteris thus shares the intermediate stage of motile male gamete formation and apparently that of haustorial pollen tubes with cycads and Ginkgo. Key words Glossopteris · Gondwana · Gymnosperm · Seed plant · Sperm · Zooidogamy
Introduction The Glossopteridales are a group of extinct gymnosperms that dominated the southern hemisphere (Gondwana) during the Permian. They are typically reconstructed as trees bearing entire, tongue-shaped Glossopteris leaves with reticulate venation on long-shoot, short-shoot systems in tight helices, with aerenchymatous Vertebraria rooting structures, H. Nishida (*) Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-Ku, Tokyo 112-8551, Japan Tel. +81-3-38171886; Fax +81-3-38171880 e-mail:
[email protected] K.B. Pigg Arizona State University, Tempe, AZ, USA K. Kudo Sankyo Labo Service Corporation, Tokyo, Japan J.F. Rigby Queensland University of Technology, Brisbane, Australia
and pycnoxylic Araucarioxylon-type wood (Pant and Singh 1974; Pant 1977; Surange and Chandra 1978; Pigg and Trivett 1994). Separate micro- and megasporangiate fertile structures in this group are subtended by vegetative leaves. However, the exact structure and homologies of these organs have been an issue of considerable controversy, particularly because in the past some authors suggested that they might demonstrate an ancestral relationship with the angiosperms (Melville 1983; Retallack and Dilcher 1988). Studies of anatomically preserved plant remains from several sources in Antarctica and Australia have provided new insights into glossopterid structure, diversity, and potential affinities (Gould and Delevoryas 1977; Pigg and Taylor 1989; Taylor and Taylor 1992; Lindström et al. 1997; Pigg and McLoughlin 1997; McManus et al. 2002). The recent discovery from Homevale, Bowen Basin, Queensland, Australia, of fossil pollen tubes releasing motile sperm of Glossopteris added new evidence of zooidogamy among seed plants (Nishida et al. 2003). Zooidogamy is otherwise known only in living Ginkgo (Hirase 1896) and cycads (Ikeno 1896; Webber 1897; Caldwell 1907; Chamberlain 1909), and is strongly suggested in some Palaeozoic seed ferns (Benson 1908; Stewart 1951). In this paper, we supply additional details of the above fossils, and further discuss the implications of this find on the evolution of seed plant reproduction.
Materials and methods The plant material occurs as silicified peat in nonmarine sediments stratigraphically correlated with the Late Permian Blackwater Group (Gould and Delevoryas 1977; Pigg and McLoughlin 1997). The rock matrix contains a variety of plant organs including those of Glossopteris, noeggerathialeans, and ferns. Although the plant tissues suffer partial destruction to varying degrees, certain structures are superbly preserved. Silicified rocks containing plant fragments were slabbed by a diamond-blade saw. Serial sections were prepared by the modified acetate peel technique using
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full-strength, commercial grade (46%) hydrofluoric acid (Basinger and Rothwell 1977). Specimens were mounted on microscope slides with Canada balsam, and were observed and photographed using an Olympus BX 50 light microscope with an attached Olympus E-20 digital camera. To obtain the highest resolution, a 100 ¥ objective lens was used with microscopic slides oil-immersed on both upper and lower surfaces. Electronic images were processed in a color plate using Adobe Photoshop 4.0.1 J. Some digital images at higher magnification were color reversed to enhance structural detail.
Results A total of five pollen tubes were found in the pollen chambers of several of the numerous ovules attached on the adaxial surface of a single Dictyopteridium-type glossopterid reproductive organ (Gould and Delevoryas 1977). The ovulate structure is incurved and laminar, around 2.0 mm high, 5.5 mm wide and thickened centrally into a midrib (Fig. 1, 1, 2). This particular specimen was found isolated in the matrix, however, similar ovulate structures, each bearing around a dozen ovules, were borne within a bud subtended by vegetative leaves assignable to Glossopteris homevalensis Pigg et McLoughlin (Pigg and McLoughlin 1997). We can thus demonstrate that these were the reproductive structures that belonged to the G. homevalensis plant. A more complete description of the ovulate reproductive structures and ovules will be presented in a later report. Ovules are borne on the adaxial surface of the structure, sometimes on a small stalk and are ovoid, 1.2–1.3 mm long ¥ 0.8–0.9 mm wide and 0.6–0.7 mm thick (Fig. 1, 1, 2). They have relatively simple pollen chambers that occasionally contain well-developed pollen tubes (Fig. 1, 3). Each ovule has a megagametophyte with a single archegonium containing an egg cell (Fig. 1, 4). The entire body of the archegonium is occupied by the egg, and is 180 mm long and 89 mm wide. A possible egg nucleus, 113 mm long and 73 mm wide and appearing darker than the surrounding material, fills most of the egg (Fig. 1, 5). The presence of a welldeveloped archegonium suggests that the ovule was developmentally mature and ready for fertilization. The archegonium has two neck cells, which are usually better preserved than are neighboring megagametophyte cells, and have cytoplasm similar in color to that of the egg cell (Fig. 1, 5– 7). Based on our observations, it is unlikely that the neck cells further divided before fertilization to produce four neck cells as in Ginkgo (Lee 1955) or more as in Encephalartos villosa (Sedgwick 1924). In some pollen chambers, remains of germinated Protohaploxypinus pollen grains were found (Fig. 2, 8–11). Pollen of this type is characterized by a conspicuous reticulate pattern on its sacci and prominent striae on the corpus, and was previously recognized from Homevale (Gould and Delevoryas 1977). Glossopterid pollen with similar morphology is also reported from South Africa (Zavada 1991) and Antarctica (Lindström et al. 1997).
In the pollen chamber of certain ovules that are also identified as Glossopteris homevalensis, pollen tubes at earlier developmental stages were found (Fig. 2, 9, 10). Five pollen tubes of varying developmental stages found within the pollen chamber of a single ovule were studied in serial section (Fig. 2, 12–15). The micropylar end of the pollen tube (which was the distal end of the original pollen grain) forms a slightly elongate haustorial structure (Fig. 2, 16). The haustorium appears to branch at its attachment to the nucellus (Fig. 2, 16, arrow), but whether it branched highly as in Ginkgo (Friedman 1987) or did not branch as in most cycads (Choi and Friedman 1991) still remains uncertain. Of the five pollen tubes preserved, two have a single immature spermatogenous cell (Fig. 2, 11–15; pollen tubes #2, 5), while two contain a pair of young sperm (Fig. 2, 12– 15, 17; pollen tubes #1, 3) and the fifth pollen tube (pollen tube #4) is ruptured, releasing mature sperm. This combination of stages in pollen-tube development found together within a single ovule suggests a gradate maturation of sperm. There is no clear evidence of additional prothallial, sterile, or tube cells, possibly because of poor preservation. Only a small dark structure similar in color to that of the single spermatogenous cell or young sperm cells, found at the bottom of pollen tube #2, can be compared with one or both of the prothallial or sterile cells (Fig. 2, 12, arrow). A pair of released sperm, one with its entire shape preserved intact and the other only partly remaining have been recognized (Fig. 2, 14, 18; Nishida et al. 2003). The complete sperm is about 12.7 mm long and 13.9 mm wide and shows a spiral structure that is strikingly similar to the flagellate band, or multilayered structure (MLS), typical of both charophycean algae (Cooper 2000) and land plants with motile gametes including bryophytes, pteridophytes (Bierhorst 1971; Gifford and Foster 1989), Ginkgo (Li et al. 1989), and cycads (Norstog 1986). The MLS is comprised of a series of basal bodies to which each flagellum is typically attached. In our material, the basal bodies or basal plate to which they are attached are represented by a single line of small, white dots occurring at more or less regular intervals (Fig. 2, 19). Colorreversed and enhanced images more clearly demonstrate the basal body alignment (Fig. 2, 20). The basal bodies have diameters of less than 0.3 mm, which is very close to the diameter of those common in flagella and cilia of eukaryotes (0.2 mm; Cooper 2000). Within one complete mature sperm, 37 basal bodies were counted, based on optical sectioning of the specimens (Fig. 2, 21). Nishida et al. (2003) reconstructed the MLS as a linear structure nearly 45 mm long, bearing more than 50 basal bodies at a mean interval of 0.57 mm within two counterclockwise gyres. Flagella themselves were not identified.
Discussion Glossopteris is similar to charophycean algae, bryophytes, and pteridophytes in having flagellated male gametes as do the zooidogamous seed plants, Ginkgo and cycads. Two
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Fig. 1. 1–7 Zooidogamy of Glossopteris homevalensis. Specimen H397018. 1, 2 Serial transverse sections of a Dictyopteridium-type ovulate organ, with different ovules sectioned in 1 and 2. Arrowhead indicates the ovule that contains pollen tube and sperm. Slides E2l Atop#1 and #31(E2 lat. slide 5 in Nishida et al. 2003), respectively. 3, 4 Longitudinal sections of the ovule indicated by arrow in 2. Note pollen tubes (numbered in red in 3; numbers corresponding to those in 11–17) and a large archegonium in megagametophyte (mg) in 4. Slides E2lAtop#31
and #39, respectively. Scale bar 1 mm. 5 Part of 4 enlarged, showing egg cell and two archegonial neck cells (arrows, same in 6). Scale bar 100 mm. 6, 7 Two neck cells in different ovules. 6 is an enlargement of 5 (longitudinal section), whereas 7 shows a transverse section. Note larger size and better preservation of neck cells compared with surrounding, less organized megagametophyte cells. Slides E2lAtop#39 and E1#62. Scale bar 10 mm
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previously described Palaeozoic seed fern ovules, a medullosan (Stewart 1951) and a lyginopterid (Benson 1908) also show evidence of motile gametes. Motile sperm of charophycean algae, bryophytes, and the lycopsids (except Isoetes and Phylloglosum) are biflagellate with a compact MLS. In contrast, Glossopteris, along with ferns, horsetails, Isoetes, Phylloglossum, cycads and Ginkgo are multiflagellate, and have an elongated helical MLS (Bold and Claire 1985; Renzagalia and Garbary 2001). The Glossopteris sperm is structurally similar, but much smaller than sperm of Ginkgo, cycads, and the putative medullosan, all of which are unusually large. Mature, released Ginkgo sperm is around 82 mm long and 49 mm wide (Hirase 1896), that of Cycas revoluta is 160 mm long and 70 mm wide (Ikeno 1898), and that of Zamia floridana may be up to 222–332 mm long and 222–306 mm wide (Webber 1897; Norstog 1986). In contrast, Glossopteris sperm is of comparable size to that of all other vascular plants, both flagellated and nonmotile. In general, throughout land plants, there is a correlation between the size of the MLS and the number of flagella. In the cycad Zamia, which has one of the largest plant sperm known, the MLS is a helical band 1,600 mm long, exhibiting six gyres, and bearing several tens of thousands of flagella arranged in 8–12 rows at tight intervals (0.1 mm) (Norstog 1986). Ginkgo’s MLS attains 300 mm in length, as calculated from previously published figures (Hori and Miyamura 1997), with 1,000 flagella on three gyres (Li et al. 1989), although the details of flagellar alignment are uncertain. Since the number of flagella generally correlates with that of the basal bodies, the shorter MLS with a single row of basal bodies suggested for Glossopteris is on the low end of those known for gymnosperms, but greater than those of pteridophytes. The MLS of the fern Pteridium, for example, exhibits two and a half gyres bearing ca. 40 flagella (Manton 1959), whereas larger sperm of the water fern Marsilea have 85–120 flagella (Mizukami and Gall 1966). In contrast to the taxa with motile male gametes, the conifers, gnetophytes, and angiosperms all have siphonogamous pollen tubes that carry nonmotile gametes directly to the female gamete.
Fig. 2. 8–21 Zooidogamy of Glossopteris homevalensis. Specimen H397018. 8–11 Pollen residues in pollen chamber (at arrows), and a young pollen tube (blue arrowhead in 9). 10 is an enlargement of 9. Note micropyle (white arrowhead), and a residue with reticulate pattern on saccus (blue arrow) in 8. 2 Pollen tube in 11 has one large spermatogenous cell. i Integument, n nucellus. Slides E2lAtop#32, E2lat#3, and E2lAtop#2, respectively. Scale bar 50 mm. 12–15 Serial sections of a pollen chamber of the ovule indicated by arrow in 2, showing five pollen tubes each numbered in red. Arrow in 12 shows possible residues of either or both prothallial and/or sterile cells. Two arrows in 14 indicate released sperm enlarged in 18–20. Slides E2lAtop#34, 33, 31, and 30. Scale bar 100 mm. 16 1 Pollen tube enlarged, showing its haustorial nature. Arrow shows possible branching. Slide E2lAtop#36. Scale bar 50 mm. 17 3 Pollen tube enlarged, showing two young sperm. E2lAtop#31. Scale bar 50 mm. 18–20 Released sperm in 14 enlarged. Scale bars 10 mm. 18 Pair of sperm in the same focal plane. Arrowhead points toward bottom side of each sperm. 19 Pair of sperm each optimized at best focal plane, showing basal body alignment by arrows. 20 Negative color image of 19. Red arrowheads point to some basal bodies of an entire sperm. 21 Suggested reconstruction of an entire sperm with spiral MLS. Dark dots show observed basal body positions. Pale dots show possible positions. Red arrowheads correspond to those in 20
Siphonogamy has also been documented for the Palaeozoic seed fern Callistophyton and the Bennettitales (Rothwell 1972; Stewart and Rothwell 1993; Stockey and Rothwell 2003). Three functional modes have been hypothesized in the evolution of pollen (Doyle and Donoghue 1986; Doyle 1988; Friedman 1993; Rothwell and Serbet 1994). The first, “prepollen”, is initiated from a relatively unspecialized microspore in certain Palaeozoic seed ferns and cordaite lineages (Stewart and Rothwell 1993). In this mode, an endosporic microgametophyte that is not elongated into a pollen tube releases motile sperm from a proximal germination suture rather than the distal suture characteristic of all other seed plants. The second mode is represented by Ginkgo, cycads and Glossopteris, where elongated, sometimes branched, haustorial pollen tubes germinate from a distal aperture, then release motile sperm proximally by rupture of the pollen tube wall. The third is demonstrated by plants with siphonogamy, including conifers, gnetophytes, and angiosperms. Here an elongate, sometimes branched, pollen tube germinates distally and serves as a carrier of nonmotile gametes rather than as an haustorium. These three modes suggest that a progressive change occurred, probably independently, in the reproductive biology of several seed plant lineages. The present findings confirm certain features of Glossopteris in addition to the fertilization mechanism, which can be important in understanding the relationships of Glossopteris to other seed plants. Whereas the reduction to a single archegonium per megagametophyte in G. homevalensis ovules could be considered a derived state, the presence of an haustorial pollen tube with motile male gametes has traditionally been considered a character of reproductively more “primitive” seed plants. Changes from a zooidogamous to a siphonogamous pollen tube occurred probably more than once among seed ferns and conifers (Friedman and Gifford 1997), and it remains unknown whether similar convergence had occurred in the descendants, (if any), of Glossopteris. Acknowledgments We thank Mr. Hiromichi Yano for preparing the microscopic slides. The field collection at Homevale was supported by Grants for Overseas Survey from the Ministry of Education, Culture, Sports, Science and Technology No. 04041034 to Prof. Masahiro Kato, University of Tokyo, and No. 08041135 to Dr. Motomi Ito, University of Tokyo, to whom we are deeply grateful. The work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology No. 07640933 to H.N., and NSF grant BSR-9006625 and an Arizona State University Faculty Grant-in-Aid to K.B.P.
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