Zoidogamy in Fossil Gymnosperms: the Centenary of a Concept, with Special Reference to Prepollen of Late Paleozoic Conifers RUUD J

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11713-11717, October 1996 Evolution

Zoidogamy in fossil gymnosperms: The centenary of a concept, with special reference to prepollen of late Paleozoic conifers RUUD J. POORT*, HENK VISSCHER*, AND DAVID L. DILCHERt *Laboratory of Palaeobotany and Palynology, Utrecht University, Budapestlaan 4, 3584 CD, Utrecht, The Netherlands; and tPaleobotany Laboratory, Florida Museum of Natural History, University of Florida, Gainesville, FL 32611 Contributed by David L. Dilcher, August 2, 1996

ABSTRACT This year is the centenary of the surprising because it serves as a haustorial organ to digest the tissues of discovery in 1896 of zoidogamy in extant cycadophytes and the nucellus and provide nutrients for the developing motile Ginkgo. But by coincidence, also in the same year, the concept antherozoids. ofprepollen was introduced. The morphology ofprepollen was Similar to those in extant cycadophytes and Ginkgo, the considered justification for the probable production of motile common presence of pollen chambers in ovules of late Paleo- antherozoids in extinct gymnosperms. In this paper, the zoic gymnosperms already had been well documented (5). history of the prepollen concept is briefly outlined. It is Following Renault's observations, the study of the fossilized emphasized that, in addition to well-known examples in content of pollen chambers received considerable attention. pteridosperms and cordaitaleans, a prepollen condition also Supposedly multicellular structures in pollen grains and a occurred among late Paleozoic conifers. variety of small bodies were interpreted as fossil evidence for the production of antherozoids (6-8). Indirectly, but more Toward the end of the 19th century, it was generally assumed convincingly, zoidogamy was suggested on the basis of the that seed plants, both extant and extinct, were uniformly morphology of the pollen wall. Following studies by Kidston characterized by siphonogamy. This fertilization mechanism (9), it gradually was realized that some pteridosperms pro- involves formation of a pollen tube, a tubular outgrowth of the duced pollen with the overall morphological organization of microgametophyte through which immotile gamete cells or many isospores or microspores of pteridophytes. A common nuclei are delivered to the archegonia. The mechanism is feature is the presence of a proximal aperture. In modern fundamentally different from zoidogamy, the fertilization by heterosporous pteridophyte spores, this aperture splits open, means of motile antherozoids, which can be observed in permitting the protrusion of the antheridia-bearing gameto- spore-bearing plants. phyte. This is true also in heterosporous lycopsids, which As early as 1887, the possibility of zoidogamy in extinct release internally produced antherozoids through a proximal gymnosperms was mentioned by the French palaeobotanist aperture (10). Hence, functional interpretation of similar Renault (1), but this view attracted little attention. Then in apertures in fossil gymnospermous pollen suggested zoid- 1896, a century ago this year, the assumption of uniform ogamy (11) siphonogamy in gymnosperms was definitively shown to be a false concept. By remarkable coincidence, the presence of Prepollen zoidogamy was independently discovered in both recent and in fossil plants in the same year. At present, fossil gymnospermous pollen characterized by proximal apertures and absence of distal specializations indic- Zoidogamy in Gymnosperms ative of the production of a pollen tube is known as prepollen. The prepollen concept is intimately linked with zoidogamy. In 1896, the Japanese botanists Hirase and Ikeno published This year, we are at the centenary of the concept. In 1896, their classic notes on the discovery of the production of Renault coined the termprepollinies for large pteridospermous antherozoids in Ginkgo biloba (2) and Cycas revoluta (3). In the and cordaitalean pollen, characterized by a supposed multi- same year, Renault published the text of his monumental work cellular microgametophyte and assumed to be indicative of on Carboniferous and Permian plant fossils from France (4). zoidogamy (4). He considered this pollen to be intermediate By studying pollen grains present in pollen chambers of between pteridophyte spores and cycadophyte pollen, al- pteridospermous and cordaitalean ovules, Renault interpreted though he was unaware of the discoveries of Hirase and Ikeno. the supposedly internal cellular structure of the pollen as a The termprepollinies was used infrequently until it was revived multicellular microgametophyte. He hypothesized that these and anglicized by Schopf (12, 13). cells could have produced motile antherozoids rather than In the 1970s, the prepollen concept was elaborated by developing pollen tubes. Chaloner (14), and further comments were contributed by The discoveries of Hirase and Ikeno surprised the interna- Jonker (15, 16). More importantly, basic morphological and tional botanical community and had an immediate and tre- ultrastructural information became available, confirming the mendous impact on the comparative analysis of reproductive presence offunctional proximal sutures in prepollen, including biology in gymnosperms. Fertilization by means of motile the huge (up to 0.5-mm-long) pollen types of medullosalean antherozoids soon became universally recognized as the fun- pteridosperms for which Renault had used the term prepolli- damental reproductive strategy of all extant cycadophyte nies (17). As a result, prepollen is currently defined as "the genera, as well for Ginkgo. Zoidogamy provided a functional microspores of certain extinct seed plants characterized by explanation for the presence of the pollen chamber, long since proximal apertures and presumed proximal germination, known in the ovules of these taxa. The function of the pollen rather than the distal, equatorial or other typical apertures of tube in these taxa had to be regarded as exclusively nutritive, seed plant pollen grains" (18). Prepollen thus represents pollen that had not yet developed the capacity to produce The publication costs of this article were defrayed in part by page charge pollen tubes (Fig. la). payment. This article must therefore be hereby marked "advertisement" in In the definition of prepollen, emphasis is given to the accordance with 18 U.S.C. §1734 solely to indicate this fact. functional interpretation ofthe proximal aperture in the pollen

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a b zoids are released by wall decay, in the fossil pollen zoidogamy is supported morphologically by the presence of a preformed suture in the proximal wall (refs. 23 and 24; Fig. lb). c.... A more difficult problem to solve is the functional inter- **; pretation of fossil pollen characterized by the presence of a leptoma but without any indication of a proximal aperture. X...... Such pollen grains are likely to have produced pollen tubes, but it remains impossible to decide whether they represent zoidogamous or siphonogamous plants. Even the unique discovery of a fossil pollen grain with a well-preserved branched pollen tube (25) remains inconclusive. This particular grain is regu- larly cited to be indicative for siphonogamy in the late Paleo- zoic. However, other criteria, such as the presence of a pollen chamber in the corresponding ovules, strongly suggests zoidogamy. It should be realized that the origin of pollen tubes and siphonogamy are separate evolutionary steps (24, 26). An additional problem in the interpretation of apertures in fossil gymnosperm pollen. is the possibility of the presence of distinctive apertures (proximal, distal, or equatorial) that are FIG. 1. Scheme of zoidogamous (a and b) and siphonogamous (c) not related to pollen tube formation or the release of anthero- fertilization strategies of conifer pollen; outer pollen wall omitted. (a) zoids, but to harmomegathy, i.e,. the process by which pollen Prepollen condition; release of motile antherozoids through proximal grains change in shape to accommodate variations in the aperture; no outgrowth of pollen tube; relatively large size (late Paleozoic). (b) Pollen grains with proximal release of motile anthero- volume of the cytoplasm caused by changing hydration. zoids; distal outgrowth of a haustorial pollen tube with an exclusively As far as prepollen is concerned, a wealth of morphological nutritive function (late Paleozoic and early Mesozoic). (c) Pollen and ultrastructural data is now available for a variety of pollen grains with distal outgrowth of a pollen tube functioning as a carrier types, found in situ in polleniferous organs of lyginopteridalean for immotile gamete cells or nuclei to migrate to the archegonium and medullosalean pteridosperms (17, 27-42). Although not (Mesozoic to Recent). always explicitly cited as prepollen, these studies firmly con- firm a prepollen condition in the Lyginopteridales and Medul- wall rather than to multicellular structures. At present, there losales. Pollen of other pteridosperms is frequently character- is a strong and justified reluctance to accept early records of ized by only a distal aperture. This also applies to pollen of a multicellular microgametophytes in some (pre)pollen. Notably number of cordaitalean taxa. However, a prepollen condition in permineralized material, effects of fossilization are a likely is also evident in some cordaitalean pollen types, such as the explanation for pseudocellular structures (19, 20). The early form-genus Felixipollenites (28, 38, 42, 43). records of fossilized antherozoids need to be reconfirmed. Yet The distinctive morphological and ultrastructural characters there is a subsequent record that justifies the link between of pteridosperm and cordaitalean prepollen have now at- prepollen and zoidogamy. In pollen present in the pollen tracted wide attention. However, despite conclusive evidence, chamber of the medullosalean ovule Pachytesta and corre- it is not yet been generally appreciated that pollen of some late sponding to Renault's and Schopf's prepollen, two cells that Paleozoic conifers could qualify as prepollen as well. closely resemble the immature paired antherozoids of modern cycadophytes were detected (21). Prepollen in Late Paleozoic Conifers It is obvious that the fossil prepollen by definition contrasts with the two pollen categories that are functionally recognized Modern conifers are siphonogamous. Except for apparently in extant gymnosperms: (i) pollen with a distal aperture inaperturate forms in the Taxales, conifer pollen is well known (leptoma) for the outgrow of a pollen tube with an exclusively to be characterized morphologically by a distal leptoma (44). nutritive function (release of motile antherozoids is not by One can recognize a wide variety in the overall shape of the preformed apertures but by wall decay; zoidogamy of cycado- pollen, even within a single family. In a number of genera of phytes, Ginkgo), and (ii) pollen with a leptoma for the outgrow the Pinaceae and Podocarpaceae, for example, pollen has two of a pollen tube that also serves as a carrier for immotile lateral and distally inclined expansions of the outer wall gamete cells or nuclei (siphonogamy of all other gymnosperm (sexine). These expansions may be hollow (saccate) or some- taxa; Fig. lc). The combination of the exclusively fossil pre- times filled with spongy sexinous structures (protosaccate or pollen with the two extant pollen categories provides a 3-fold saccoid). In other families, such differentiations are lacking. subdivision that is frequently followed to illustrate, in a general Regardless of these variations, the pollen tube develops in sense, progressive change in the reproductive strategy of most modern conifer pollen (except Taxales) from the distal gymnosperms (22). leptoma. In all modern conifers, the pollen tube functions as There are some controversies regarding the functional in- a carrier or transport system for the gamete cell or nucleus to terpretation of the morphology of fossil pollen grains that are migrate to the archegonium and the egg cell before fertiliza- characterized by the presence of both proximal and distal tion occurs. apertures. Many such pollen grains have been described as Also, late Paleozoic and Mesozoic conifers are usually palynological form-genera based upon dispersed pollen from regarded as siphonogamous. In his monograph on Paleozoic late Paleozoic and early Mesozoic sediments. Well-known conifers, Florin compared saccate or saccoid fossil conifer examples are pollen types corresponding to the form-genera pollen with presumed modern counterparts (45). Yet pollen Jugasporites, Lueckisporites, and Triadispora. Considering the with both proximal and distal apertures was known or sus- proximal aperture to be a vestigial feature, such pollen some- pected to be produced by fossil conifers. Such pollen (Juga- times has been regarded as indicative of siphonogamy. In sporites) was described, for example, from polleniferous cones contrast, by neglecting the leptoma, similar forms have been of the late Permian conifer Ullmannia (46, 47). In 1984, named prepollen. In fact, this distinctive fossil pollen may be uniform siphonogamy in extinct conifers was challenged. compared functionally with the pollen of extant cycadophytes Again by coincidence, a prepollen condition for coniferous and Ginkgo. In contrast to this extant pollen, where anthero- pollen was reported from two independent sources. Downloaded by guest on September 29, 2021 Evolution: Poort et al. Proc. Natl. Acad. Sci. USA 93 (1996) 11715

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FIG. 2. Prepollen of late Carboniferous/early Permian Walchiaceae (form-genus Potonieisporites, known from the genera Walchia, Ernestio- dendron, and Otovicia). (a) Proximal face of specimen showing aperture. (X435.) (b) Section subparallel to longest axis; thick outer wall (sexine) with alveolate infrastructure; no indications of the presence of an aperture at the distal (lower) side. (x 1600.) (c) Section subparallel to short axis; inner wall (nexine) strongly folded due to compression; thick outer wall (sexine) with no indications of a distal aperture. (x 1600.) A detailed study of the genus Ortiseia from the Upper deduced that corresponding ovules are likely to possess pollen/ Permian of North Italy, and a reevaluation of the genera archegonial chambers. Walchia and Ernestiodendron by Clement-Westerhof (48) ini- By studying permineralized ovuliferous cones described as tiated the development of a natural concept of the Walchi- Lebachia lockhardii from the Upper Carboniferous of Kansas, aceae (48-52), the most prominent conifer family of the late Mapes and Rothwell (53, 54) arrived at similar conclusions. Paleozoic Euramerican floral province. One of the novel The ovules are characterized by a pollen chamber. Uncom- elements in this reevaluation is the concept that walchiaceous pressed pollen grains preserved in pollen chambers have the pollen represents prepollen. Light-microscopic and scanning characteristics of prepollen. Scanning electron microscopy electron microscopic studies of in situ pollen from pollenifer- reveals the proximal aperture, whereas a light-microscopic ous organs of Ortiseia demonstrate the presence of a proximal section indicates absence of a distal leptoma. aperture. No evidence was found for the additional presence Further morphological and ultrastructural studies (55) have of a leptoma that could support outgrowth of a pollen tube. confirmed the prepollen condition of in situ and dispersed Moreover, on the basis of detailed cuticular analysis, it was walchiaceous pollen. Meanwhile, Lebachia lockhardii had been

a b c d e FIG. 3. Pollination model for late Paleozoic prepollen-bearing conifers (after refs. 51 and 52). (a) Portion of an ovuliferous cone with airflow pattern indicated by curved arrow. (b) Capture of prepollen by a pollination droplet at the top of the micropyle. (c) Enlargement of (b), showing prepollen absorbed by pollination droplet. (d) Retraction of droplet into micropyle. (e) Prepollen inside pollen/archegonial chamber where release of motile antherozoids takes place through the proximal aperture of the prepollen grains. Downloaded by guest on September 29, 2021 11716 Evolution: Poort et al. 11716 Evolution: Poort et al. ~~~~~Proc. Nati. Acad. Sci. USA 93 (1996) applied to typify the Emporiaceae (54). The presence of 1. Renault, B. (1887) Mgm. Soc. Hist. Nat. Sa6ne-et-Loire 1887, prepollen has now become part of the diagnosis of two late 155-160. Paleozoic conifer families. The coniferous prepollen types 2. Hirase, 5. (1896) Bot. Mag. Tokyo 10, 325-326. so far to the late Paleo- 3. Ikeno, 5. (1896) Bot. Mag. Tokyo 10, 367-368. recognized correspond (or resemble) 4. Renault, B. (1896) Bassin HouilleretPermien dAutun etd'Epinac. zoic form-genera Nuskoisporites and Potonieisporites (Fig. 2). Flore Fossile (Imprimerie Nationale, Paris). In the Lower Permian of Argentina, dispersed pollen (Can- 5. Brongniart, A. (1881) Recherches surles Graines Fossiles Silicifiges nanoropollis) that has been associated with the Ferugliocla- (Imprimerie Nationale, Paris). daceae (56) might show a prepollen condition, but further 6. Oliver, F. W. (1904) Trans. Linn. Soc. London 6, 361-400. studies are required. On the other hand, the presumed pres- 7. Oliver, F. W. (1909) Ann. Bot. 23, 73-116. ence of prepollen in the late Permian Majonicaceae (57) has 8. Benson, M. (1908) Bot. Gaz. 45, 409-412. to be In situ to the 9. Kidston, R. (1906) Philos. Trans. R. Soc. London B 196,413-445. rejected. pollen corresponds form-genus 10. Slagg, R. A. (1932) Am. J. Bot. 19, 106-127. Lueckisporites and incudes forms with both proximal and distal 11. Wodehouse, R. P. (1936) Bot. Rev. 2, 67-88. apertures. Similarly, the coniferous families Ullmanniaceae 12. Schopf, J. M. (1938) State Geol. Surv. Ill. Rep. Invest. 50, 1-74. (late Permian) and Voltziaceae (Triassic) produced pollen 13. Schopf, J. M. (1948) 1. Paleontol. 22, 681-724. (Jugasporites and Triadispora) that apparently had developed 14. Chaloner, W. G. (1970) Geosci. Man 1, 47-56. the capacity to form distal haustorial pollen tubes (46, 47, 58). 15. Jonker, F. P. (1974) Adv. Pollen-Spore Res. 1, 50-61. In contrast to the siphonogamy in extant conifers, these 16. Jonker, F. P. (1977) Adv. Pollen-Spore Res. 2, 6-13. 17. Millay, M. A., Eggert, D. A. & Dennis, R. L. (1978) Micropal- families were still characterized by zoidogamy. Consequently, eontology 24, 303-315. conifers represent the only group of gymnosperms in which the 18. Punt, W., Blackmore, S., Nilsson, S. & Le Thomas, A. (1994) three functional steps in pollen evolution can be recognized L. P. P. Contrib. Ser. 1, 1-71. (Fig. 1). 19. Potoni6, R. (1970) Beih. Geol. Jahrb. 87, 1-222. Coniferous prepollen is characterized by a single, monosac- 20. Taylor, T. N. (1973) Rev. Palaeobot. Palynol. 16, 157-165. cate or monosaccoid, expansion of the sexine. Despite the 21. Stewart, W. N. (1951) Am. Midl. Nat. 46, 717-742. size of the to 300 the 22. Meyen, S. V. (1984) Bot. Rev. 50, 1-111. relatively large prepollen grains (up atm), 23. Traverse, A. (1988) Paleopalynology (Unwin Hyman, Boston). overall shape of this differentiation is likely to indicate wind 24. Friedman, W. E. (1993) Trends Ecol. Evol. 8, 15-21. pollination (45, 52, 55). Both in the Walchiaceae and the 25. Rothwell, G. W. (1972) Science 175, 772-774. Emporiaceae, the ovules are inverted, with a downward- 26. Rothwell, G. W. & Serbet, R. (1994) Syst. Bot. 19, 443-482. projecting micropyle. This would suggest a pollination drop 27. Eggert, D. A. & Taylor, T. N. (1971) Bot. Gaz. 132, 30-37. mechanism for facilitating the entry of prepollen in the pollen 28. Millay, M. A. & Taylor, T. N. (1974) Palaeontographica B 147, chamber (51-54). The presence of such a mechanism is 75-99. an observed cluster of at the of the 29. Stidd, B. M., Leisman, G. A. & Phillips, T. L. (1977) Am. J. Bot. supported by prepollen tip 57, 827-836. nucellar beak of an ovule of the walchiaceous genus Otovicia 30. Taylor, T. N. (1978) Can. J. Bot. 56, 3105-3118. (51, 52). Noting the occurrence of prepollen in the genus, the 31. Eggert, D. A. & Rothwell, G. W. (1979)Am. J. Bot. 66, 851-866. reconstructed pollination mechanism for Otovicia may serve as 32. Millay, M. A. & Taylor, T. N. (1979) Bot. Rev. 45, 301-375. a model of the pollination biology of Walchiaceae and Em- 33. Taylor, T. N. & Millay, M. A. (1981) Rev. Palaeobot. Palynol. 32, poriaceae (Fig. 3). 27-62. 34. Taylor, T. N. (1982) Rev. Palaeobot. Palynol. 37, 29-53. Conclusions 35. Rothwell, G. W. & Mickle, J. E. (1982) Rev. Palaeobot. Palynol. 36, 361-374. 36. Mickle, J. E. & Leary, R. L. (1984) Rev. Palaeobot. Palynol. 43, In conclusion, we emphasize that during the late Paleozoic, 343-357. zoidogamy was the rule rather than the exception among all 37. Rothwell, G. W. & Eggert, D. A. (1986) Trans. R. Soc. Edinburgh major gymnosperm groups, including the conifers. Prepollen- Earth Sci. 77, 47-79. bearing plants gradually became extinct during the Permian. 38. Taylor, T. N. & Taylor, B. L. (1987) Soc. Bot. Fr. 134 (Actual. The most recent known genus with prepollen, the conifer Bot., 1987, 2), 121-140. Ortiseia, did not survive the ecological crisis at the Permian- 39. Taylor, T. N. (1988) in Origin and Evolution of Gymnosperms, ed. Triassic transition as a Beck, C. B. (Columbia Univ. Press, New York), pp. 177-217. (55, 59). However, possibly parallel 40. Meyer-Berthaud, M. (1989) Palaeontographica B 211, 87-112. trend in a variety of late Paleozoic gymnosperms (24, 26), the 41. Stewart, W. N. & Rothwell, G. W. (1993) Paleobotany and the capacity to form haustorial pollen tubes had already developed Evolution of Plants (Cambridge Univ. Press, Cambridge, U.K.). and zoidogamous taxa could continue to dominate gymno- 42. Taylor, T. N. & Taylor, B. L. (1993) The Biology and Evolution of sperm plant life during a considerable part of the Mesozoic. It Fossil Plants (Prentice-Hall,- Englewood Cliffs, NJ). is not yet possible to estimate accurately when siphonogamy 43. Millay, M. A. & Taylor, T. N. (1976) Rev. Palaeobot. Palynol. 21, originated, or when and why this reproductive strategy started 65-92. 44. Kurmann, M. H. & Zavada, M. 5. (1994) in Ultrastructure ofFossil its rise to dominance over zoidogamy. Yet we have to be Spores and Pollen, eds. Kurmann M. H. & Doyle, J. A. (R. Bot. grateful that there are still a few "living fossils" that have Gardens, Kew, U.K.), pp. 123-137. survived the progressive elimination of zoidogamous plants. 45. Florin, R. (1939-1945) Palaeontographica B 85, 1-729. Fossil pollen may help to hypothesize on the waxing and 46. Potoni6, R. & Schweitzer, H. J. (1960) Palaontol. Z. 34, 27-39. waning of zoidogamy, but we first needed the pollen of Cycas 47. Grebe, H. & Schweitzer, H. J. (1962) Fortschr. Geol. Rheinl. and Ginkgo to reveal, a century ago, that the very concept of Westfalen f2, 201-224. is not a but a 48. Clement-Westerhof, J. A. (1984) Rev. Palaeobot. Palynol. 41, zoidogamy among gymnosperms hypothesis 51-166. reality. 49. Visscher, H., Kerp, J. H. F. & Clement-Westerhof, J. A. (1986) Acta Bot. Neerl. 35, 87-99. We thank Hans Kerp, Han Van Konijnenburg-Van Cittert, William 50. Clement-Westerhof, J. A. (1988) in Origin and Evolution of DiMichele, Gar Rothwell, and William Stern for helpful comments. Gymnosperms, ed. Beck, C. B. (Columbia Univ. Press, New This work was supported by The Netherlands Organization for Sci- York), pp. 298-337. entific Research and The Netherlands Life Science Foundation. This 51. Kerp, JI H. F, Poort, R. T Swinkels, H. A. JI M & Ve-rwer, R is Netherlands Research School of Sedimentary Geology publication (1989) Cour. Forschungsinst. Senckenberg 109, 137-151. no. 960901 and University of Florida Contributions to Paleobiology 52. Kerp, J. H. F., Poort, R. J., Swinkels, H. A. J. M. & Verwer, R. no. 477. (1990) Rev. Paleobot. Palynol. 62, 205-248. Downloaded by guest on September 29, 2021 Evolution: Poort et al. Proc. Natl. Acad. Sci. USA 93 (1996) 11717

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