Int. J. Plant Sci. 168(7):985–997. 2007. Ó 2007 by The University of Chicago. All rights reserved. 1058-5893/2007/16807-0002$15.00 POLLEN ONTOGENY IN EPHEDRA AMERICANA (GNETALES) 1, 2, Allison S. Doores,* Jeffrey M. Osborn, * and Gamal El-Ghazaly y *Division of Science, Truman State University, Kirksville, Missouri 63501-4221, U.S.A.; and yPalynological Laboratory, Swedish Museum of Natural History, SE-104 05 Stockholm, Sweden Several investigations have focused on mature pollen of Ephedra; however, little is known about pollen ontogeny. This article is the first to describe the complete pollen developmental sequence in the genus. Com- bined light, scanning electron, and transmission electron microscopy were used to document all major devel- opmental stages in Ephedra americana. Microspore mother cells are characterized by an unevenly thickened callose envelope. Both tetrahedral and tetragonal tetrads occur, but the majority of tetrads exhibit tetrahedral geometry. Pollen wall development is initiated in regions that will become characteristic plicae. The majority of exine deposition, including formation of the tectum, infratectum, foot layer, and endexine, occurs throughout the tetrad stage, although endexine deposition continues into the free microspore stage. The intine forms during the late free microspore stage. Two types of infratectal elements occur. Granular infratectal elements are the predominant type and develop throughout ontogeny, whereas columellar elements form in the early tetrad stage but subsequently become indiscernible. Mature grains are elliptic, have a series of longitudinal plicae, and are inaperturate, yet the exine is very thin in the furrow regions between plicae. Pollen grains with straight and undulated furrows co-occur in the same pollen sac, with the straight morphology dominating. Because several key characters are revealed only during an investigation of the full ontogenetic sequence, this study emphasizes the importance of integrating pollen developmental characters into phylogenetic analyses. Keywords: pollen ontogeny, pollen development, Ephedra americana, Gnetales, Coniferales, basal angiosperms, anthophyte. Introduction (Samigullin et al. 1999; Bowe et al. 2000; Donoghue and Doyle 2000). Because these molecular data have also been Ephedra is a member of the gymnospermous order Gne- disputed by other molecular studies (e.g., Rydin et al. 2002), tales, a rare clade also including Welwitschia and Gnetum. developmental and paleobotanical investigations have become The Gnetales are generally regarded as monophyletic, with increasingly important in reconciling the phylogenetic differ- Ephedra basal to the derived Gnetum and Welwitschia (Arber ences between morphological and molecular data. and Parkin 1908; Doyle 1996; Friedman 1996; Price 1996; Despite the phylogenetic importance of the group, limited Bowe et al. 2000; Ickert-Bond et al. 2003; Rydin et al. 2004). information is available about the geologic history of Gnetales. Although the generic relationships within Gnetales are gener- The majority of data about gnetalean evolution through geo- ally well recognized, their phylogenetic position among seed logic time come from palynological evidence. The record of plants has received considerable attention in recent years. dispersed, ‘‘polyplicate’’ pollen resembling that of Ephedra Many morphologically based phylogenetic studies have placed and Welwitschia, at the light-microscope level, extends from Gnetales as the closest living relatives to angiosperms (e.g., the Lower Permian to recent sediments (see Osborn et al. 1993 Arber and Parkin 1908; Crane 1985; Doyle and Donoghue and references therein; Crane 1996; Osborn 2000; Dilcher 1992), and along with the fossil groups Bennettitales and et al. 2005). Polyplicate pollen is also produced by several Pentoxylales, Gnetales and angiosperms have comprised the angiosperm groups (e.g., Arales, Laurales, and Zingiberales); ‘‘anthophyte clade’’ (e.g., Crane 1985; Doyle and Donoghue however, the pollen walls of these polyplicate pollen types are 1992; Osborn 2000). A series of molecular studies, however, structurally and chemically different from those of Gnetales have questioned this phylogenetic interpretation and have in- (Osborn et al. 1993; Hesse et al. 2000). A limited number of dicated that Gnetales are more closely allied with other gym- Gnetalean megafossils have been described; examples include nosperms, specifically, that they are nested within conifers Masculostrobus clathratus (Ash 1972), Drewria potomacensis (Crane and Upchurch 1987), Piroconites kuesperti (van 1 Author for correspondence; current address: School of Science, Konijnenburg-van Cittert 1992), Craytonia cotyledon (Rydin The College of New Jersey, Ewing, New Jersey 08628, U.S.A.; e-mail et al. 2003), and Welwitschiella austroamericana (Dilcher [email protected]. et al. 2005). Fertile reproductive shoots and seeds containing 2 Deceased. characteristics of both Gnetales and conifers have also been as- Manuscript received November 2006; revised manuscript received March signed to the morphospecies Ephedra archaeorhytidosperma 2007. (Wang 2004). Recently, early Cretaceous seeds and in situ 985 986 INTERNATIONAL JOURNAL OF PLANT SCIENCES pollen grains have been assigned to the genera Ephedra and examined/imaged using a JEOL JEM-100SX transmission Ephedrispermum (Rydin et al. 2006). electron microscope at 80 kV. Ephedra is the largest of the three extant genera of Gne- Additional specimens were dehydrated in a graded ethanol tales. The genus comprises ca. 50 species, which occur pri- series, critical-point dried, and macerated using a syringe nee- marily in xeric habitats in both the Old and New World. dle. These specimens were mounted onto aluminum stubs, Pollen morphology has been used as a source of important sputter-coated with a layer of gold, and examined/imaged taxonomic characters. Previous studies on mature pollen of using a JEOL JSM-6400 scanning electron microscope at Ephedra show that the pollen grains are elliptic to elongate 20 kV. and range from 20 to 80 mm in length and from 16 to 50 mm in width. Grains are characterized by a series of longitudinal ribs, or plicae, that typically have a psilate ornamentation. Results Plicae number ranges from four to 19 (Steeves and Barghoorn 1959). The polyplicate pollen of Ephedra is typically consid- Pollen cones in Ephedra americana are compound; the ered inaperturate. However, the exine is considerably thinner cone axis bears several pairs of bracts, which separate the mi- within the plicae furrows (El-Ghazaly and Rowley 1997; crosporangiate structures. Each microsporangiate shoot has Osborn 2000), and the entire exine is completely discarded an axis bearing a pair of bracteoles that enclose a microspo- before pollen tube germination (El-Ghazaly et al. 1998). Species rophyll with several pollen sacs. of Ephedra are principally distinguished from one another Pollen sacs within the same cone generally show consistent palynologically by the number of plicae, plica height and de- ontogenetic timing of the pollen grains; however, a limited gree of slope, and furrow morphology. The furrow may be amount of variation occurs among pollen sacs. Pollen grains either straight or undulated/branched. Pollen with both types within a single pollen sac show consistent and synchronous of furrow morphologies has also been shown to occur within development. Pollen ontogeny is presented below as it occurs a single species and within a single microsporangium (El-Ghazaly in four major stages, the microspore mother cell, tetrad, free and Rowley 1997; Ickert-Bond et al. 2003). microspore, and mature pollen grain stages. Once the first Although several studies have focused on the morphology tectal elements are laid down, each developing plica is char- of mature pollen in Ephedra, no published studies have ex- acterized by crest and margin regions, with a furrow region amined the ultrastructural aspects of pollen ontogeny in the between adjacent plicae. These characters are illustrated in genus. Investigations of pollen development can potentially figure 1 as they appear in the nearly mature pollen wall, to generate many important characters for assessing phyloge- serve as structural landmarks for comparison with ontoge- netic relationships that cannot be observed when only mature netic data. grains are studied (Blackmore and Barnes 1991; Kreunen and Osborn 1999; Taylor and Osborn 2006). Microspore Mother Cell Stage The primary objective of this article is to comprehensively Microspore mother cells are large and typically occur document pollen wall development in Ephedra americana clumped together near the tapetum. Each microspore mother Hum. & Bonpl. ex Willd. Key ontogenetic characters are also cell is surrounded by an unevenly thickened layer of callose compared with those of both saccate and nonsaccate pollen of ranging from 2.2 to 5.7 mm in thickness (fig. 2A–2C). The conifers, as well as with basal-angiosperm pollen, in an effort thinner areas of callose, however, are often one-tenth the to help clarify the phylogenetic position of Gnetales. width of the thick areas, with an average thickness of 0.33 mm and a maximum of only 0.67 mm (fig. 2C). The callose is divided into two distinct layers: a thick inner layer that is Material and Methods more electron dense and a thin outer layer that is less electron dense (fig. 2D). The outer layer is evenly thickened and is also Sixty-five pollen cones of Ephedra americana were col- lected from the greenhouse of