Evolution and Phylogeny of Gnetophytes: Evidence from the Anatomically Preserved Seed Cone Protoephedrites eamesii gen. et sp. nov. and the Seeds of Several Bennettitalean Species Author(s): Gar W. Rothwell and Ruth A. Stockey Source: International Journal of Plant Sciences, Vol. 174, No. 3, Special Issue: Conceptual Advances in Fossil Plant Biology Edited by Gar Rothwell and Ruth Stockey (March/April 2013), pp. 511-529 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/10.1086/668688 . Accessed: 22/04/2013 15:53

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

. JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected].

.

The University of Chicago Press is collaborating with JSTOR to digitize, preserve and extend access to International Journal of Plant Sciences.

http://www.jstor.org

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions Int. J. Plant Sci. 174(3):511–529. 2013. Ó 2013 by The University of Chicago. All rights reserved. 1058-5893/2013/17403-0020$15.00 DOI: 10.1086/668688

EVOLUTION AND PHYLOGENY OF GNETOPHYTES: EVIDENCE FROM THE ANATOMICALLY PRESERVED SEED CONE PROTOEPHEDRITES EAMESII GEN. ET SP. NOV. AND THE SEEDS OF SEVERAL BENNETTITALEAN SPECIES

Gar W. Rothwell1,*,y and Ruth A. Stockeyy,z

*Department of Environmental and Plant Biology, Ohio University, Athens, Ohio 45701, U.S.A.; yDepartment of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, Oregon 97331, U.S.A.; and zDepartment of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada

A fossil seed cone with characters that have been hypothesized as transitional to the origin of crown group gnetophytes has been discovered in Lower Cretaceous deposits on Vancouver Island, British Columbia, Canada. This cone, described as Protoephedrites eamesii gen. et sp. nov., provides the first anatomically preserved fossil evidence for the evolution of gnetophyte cones with seeds. The cone is a compound shoot system consisting of a primary axis with nodes that bear bracts and axillary fertile shoots in an opposite/ decussate arrangement. The secondary fertile shoot axis produces one or two pairs of diminutive bracteoles and a pair of erect ovules in an opposite/decussate pattern. In contrast to crown group gnetophytes, bracteoles subtend rather than surround and enclose the ovules. Ovules have a single multiseriate integument that is adnate to the nucellus in the basal region and free distally. The micropylar tube is short with a thick integument, but it displays distinctive cells of the inner integumentary epidermis that are characteristic of gnetophyte seeds. Each seed produces a large pollen chamber with a uniseriate wall. Paired erect ovules conform to the pleisomorphic morphology previously hypothesized for Ephedra seed cones, thus supporting the proposal that ancestral gnetophyte seeds are borne terminally on oppositely arranged sporophylls. The combination of gnetophyte synapomorphies and putative pleisomorphic characters displayed by Protoephedrites broadens the known range of morphologies for the most ancient gnetophytes. Detailed comparisons to several species of Bennettitales confirm that there are fundamental structural differences separating the seeds of Gnetales from those of Bennettitales, support the hypothesis that the outer seed envelope evolved within the gnetophyte clade, and suggest that Bennettitales are not as closely related to Gnetales as hypothesized by some authors.

Keywords: Bennettitales seeds, Cretaceous, evolution, fossil Gnetales, seed cone anatomy, seed plant phylogeny.

Online enhancement: video.

Introduction characters usually resolve gnetophytes as the sister group to flowering plants at the apex of the spermatophyte tree Gnetophytes are among the most systematically important (Laconte and Stevenson 1990; Doyle and Donoghue 1992; and persistently discussed clades of living seed plants, being Nixon et al. 1994; Rothwell and Serbet 1994; Doyle 1998; repeatedly implicated in both the phylogeny of conifers and Hilton and Bateman 2006; Friis et al. 2007; Rothwell et al. in the origin of flowering plants (Doyle 1998; Mathews 2009), the results of analyses based on nucleotide sequence 2009). Yet, they are also one of the most enigmatic clades of characters of living species yield several conflicting topologies spermatophytes (Burleigh and Mathews 2004, 2007a, 2007b; (Rydin et al. 2002; Mathews 2009). Results of analyses Friis et al. 2007; Rothwell et al. 2009). Because of a unique based on some nucleotide sequences and employing certain suite of distinctive morphological characters (Chamberlain model assumptions resolve gnetophytes at the basal branch 1935; Eames 1952; Rydin et al. 2006a, 2006b; Friis et al. of the extant spermatophyte tree, while analyses of other nu- 2011) and long-branch separation from other spermato- cleotide sequences and/or employing other model assump- phytes in many nucleotide sequence phylogenies (Graham tions resolve gnetophytes either as the sister group to conifers and Isles 2009), systematic relationships and evolutionary or- or as variously nested within the conifer clade (Rydin et al. igins of the clade remain obscure (Mathews 2009; Taylor 2002; Rai et el. 2003, 2008; Mathews 2009; Finet et al. et al. 2009). Whereas systematic analyses of morphological 2010; Rai and Graham 2010). The paleontological record of gnetophytes also remains 1 Author for correspondence; e-mail: [email protected]. equivocal with respect to the most ancient occurrence, ances- Manuscript received April 2012; revised manuscript received October 2012. tral morphological characters, systematic relationships, and

511

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions 512 INTERNATIONAL JOURNAL OF PLANT SCIENCES phylogeny of the clade (Crane 1996; Rothwell et al. 2009; Tay- In this study, we describe the first permineralized fossil lor et al. 2009). On the basis of primarily palynological records evidence for reproductive anatomy and morphology of (Mu¨ller 1984; Osborn et al. 1993) or pollen contained within gnetophyte cones from the Valanginian Stage of the Lower compressed fructifications that lack the structural synapomor- Cretaceous, ;136 Ma. Protoephedrites eamesii gen. et sp. phies of gnetophytes (Taylor et al. 2009), some authors suspect nov. conforms to the morphology of a hypothesized struc- that the clade originated as early as the Permian (Wang 2004). tural intermediate in a previously proposed transformational There are intriguing gnetophyte-like megafossils in deposits as series leading to the basal crown group gnetophyte genus old as the Triassic (Ash 1972). However, specimens that display Ephedra (Thoday and Berridge 1912; Eames 1952; Ickert- a wide array of widely accepted synapomorphies for gneto- Bond et al. 2009), thus documenting a putative ancestral phytes appear first in the Lower Cretaceous (Crane and morphology and anatomy of gnetophyte seed cones and con- Upchurch 1987; Martill et al. 1993; Taylor et al. 2009; Friis firming hypothesized homologies of the seed position and et al. 2011; Kunzmann et al. 2011) at about the same time as ‘‘outer integumentary envelope’’ that characterize crown the oldest unequivocal megafossil evidence for flowering plants group gnetophytes. The new genus provides additional evi- (Yang et al. 2005; Friis et al. 2011). dence that the outer envelope of gnetophyte seeds evolved Palynological and dispersed seed data reveal that gnetophytes within the gnetophyte clade and that the outer integument diversified along with the flowering plants in the Lower Creta- of gnetophyte and angiosperm seeds evolved by parallel ceous (Crane and Lidgard 1989, 1990; Dilcher et al. 2005; evolution. Friis et al. 2011, 2013) but apparently suffered declining diver- Description of this new cone also provides an opportunity sity later in the Cretaceous (Crane and Lidgard 1990) and Pa- to refigure and analyze the seeds of several bennettitalean leogene (Taylor et al. 2009). The vast majority of Cretaceous species to the seeds of gnetophytes for a critical comparison gnetophyte megafossils are preserved as compression/impressions to the ovulate structures of Gnetales. This comparison em- (Mohr et al. 2007; for a review, see Friis et al. 2011), many phasizes that the seeds and seed-bearing structures of Gne- of which are similar to crown group species of Ephedra tales and Bennettitales show far fewer similarities than L., Welwitschia Hooker, and Gnetum L. (Dilcher et al. 2005; differences and are clearly much more structurally divergent Taylor et al. 2009; Friis et al. 2011), but those fossils provide than recently has been hypothesized by other authors (Friis little evidence for the structural transformations that led to et al. 2007, 2009, 2011, 2013). As a result, it becomes clear the origin and early diversification of the gnetophyte clade. that ovulate reproductive structures of gnetophytes share Heretofore, the almost total absence of permineralized fossil more morphological characters with Paleozoic cordaites and gnetophyte specimens that can be coded for a large number of ancestral conifers s.s. than they do with the ovulate repro- systematically informative morphological and anatomical char- ductive structures of Bennettitales. acters also has hampered attempts to more clearly resolve the evolution and phylogeny of the clade (Friis et al. 2007; Rothwell et al. 2009). Recent promising discoveries of anatomical evi- Material and Methods dence for the structure of gnetophyte-like seeds and gnetophyte diversity during the Lower Cretaceous (Rydin et al. 2004, Protoephedrites eamesii is preserved by cellular perminer- 2006a, 2006b; Friis et al. 2007, 2009, 2011, 2013) have bol- alization in a calcium carbonate concretion (University of stered earlier palynological and compression/impression evidence Alberta Paleobotanical Collections P13,158D) derived from for a Cretaceous radiation of Gnetales (Crane and Lidgard a carbonate-cemented graywacke matrix at the Apple Bay lo- 1989), but the structure and systematic relationships of some of cality on northern Vancouver Island, British Columbia, Can- those seeds are controversial and remain uncertain (for a critical ada. The locality is on the beach along Quatsino Sound evaluation, see Rothwell et al. 2009). Anatomical evidence for (50°369210N, 127°399250W; UTM 9U WG 951068), where the cones and vegetative features of those plants has yet to be material is most accessible at low tide (Stockey and Wiebe described. 2008). Sediments were previously regarded as Lower Creta- Phylogenetic relationships of Gnetales have been obscured ceous (Valanginian-Barremian), Longarm Formation equivalent even further by the recent reintroduction of an old structural (Jeletzky 1976; Haggart and Tipper 1994), which corresponds hypothesis (Berridge 1911) and by renewed assertions that to Jeletzky’s Barremian variegated clastic unit (Sweet 2000). there are systematically important structural similarities be- More recently, an oxygen isotope analysis has narrowed the age tween the seeds of Gnetales and Bennettitales (Friis et al. to ;136 Ma in the Valanginian Stage of the Early Cretaceous 2007, 2009, 2011, 2013), some of which conflict with evi- (D. R. Gro¨cke, personal communication). dence that has been accumulating for more than 100 yr The specimen was encountered in oblique transverse sec- (Solms-Laubach 1891; Lignier 1894, 1911; Wieland 1906, tion while making serial sections of another plant. Two hun- 1911, 1916; Stopes 1918; Sharma 1970a, 1970b; Crepet dred and sixty-eight sections were made from near the base 1972, 1974; Crepet and Delevoryas 1972; Nishida 1994; and toward the apex of the specimen before it was reoriented Ohana et al. 1998; Rothwell and Stockey 2002, 2010; and sectioned for longitudinal views of the cone apex. Prepa- Stockey and Rothwell 2003; Rothwell et al. 2009). Those as- rations were made using the well-known cellulose acetate sertions add additional uncertainty about relationships be- peel technique with 5% hydrochloric acid (Joy et al. 1956). tween the ovulate structures of Gnetales and Bennettitales Slides were mounted in Eukitt (O. Kindler, Freiburg) xylene- and necessitate that careful comparisons between seeds of the soluble mounting medium. Images were captured using two clades be made to clarify what similarities and differ- a PowerPhase digital scanning camera (Phase One, Copenha- ences actually exist. gen) and processed using Adobe Photoshop 7.0 (Adobe, San

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 513

Fig. 1 Holotype of Protoephedrites eamesii gen. et sp. nov. All figures from UAPC-ALTA P13,158. A, Oblique cross section near apex of cone showing decussate arrangement of bracts, two bundles (arrowheads) that vascularize most apical pair of bracts, and histology at this level. D bot no. 252 3 16. B, Oblique section of cone showing bracts and axillary secondary shoots at two nodes. D bot no. 100 3 16. Scale bar ¼ 1 mm. C, Oblique section of cone showing bracts and dwarf shoots at two nodes. D bot no. 50 3 16. Scale bar ¼ 1 mm. D, Oblique cross section of cone axis showing features at base of specimen. D bot no. 2 3 16. b ¼ bract; o ¼ ovule; s ¼ secondary fertile shoot.

Jose, CA). Three-dimensional reconstructions were rendered Rothwell (2003), and Rothwell et al. (2009). Specimens and using AVIZO 3.1.1 visualization software (TGS Software, microscope slides are housed within the collections indicated San Diego, CA). in the original descriptions of each species. Figures of bennettitalean seeds from cones of Williamsonia bockii Stockey et Rothwell, Williamsonia sp., Foxeoidea con- natum Rothwell et Stockey, and Cycadeoidea maccafferyi Homology and Terminology Rothwell et Stockey are from previously described specimens, Terminology that has been used to describe the fertile struc- the more complete descriptions and sources of which are pre- tures of gnetophytes differs dramatically among authors who sented by Rothwell and Stockey (2002, 2010), Stockey and have studied such structures over the years (for informative

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions Fig. 2 Holotype of Protoephedrites eamesii gen. et sp. nov. All figures from UAPC-ALTA P13,158. A, Longitudinal section at apex of cone showing tiny tip of axis (arrowhead) between apical pair of bracts. D side no. 57 3 46. B, Cross section of cone axis near base, showing features of pith and stele and divergence of bract trace (BT) and two bundles to axillary secondary axis (ST). D bot no. 3 3 77. C–F, Series showing axillary

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 515 discussions, see Hufford 1996; Yang 2004), reflecting differ- ing narrow wing in major plane. Integument of thin-walled ent schools of botanical training and differing hypotheses of cells, consisting of inner zone of smaller cells and outer zone homologies for structures of the group. Some authors have of larger cells, with distinct inner and outer epidermises; in- employed flowering plant–centered or specialized terminol- ner epidermal cells enlarging to form palisade near apex. Nu- ogy (Thoday and Berridge 1912; Gifford and Foster 1989; cellus adnate to integument in basal region, free distally, Endress 1996; Hufford 1996), while other authors have forming large pollen chamber with uniseriate wall. employed more general terminology that is typically used for Holotype hic designatus. Peels and slides of specimen in gymnospermous seed plants (Eames 1952). P13158D (UAPC-ALTA). We regard gnetophytes as having compound cones Locality. Apple Bay, northern Vancouver Island, British (¼inflorescences of some authors; i.e., consisting of two or- Columbia, Canada (50°369210N, 127°399250W; UTM 9U ders of branching) in which there is a cone axis (i.e., stem ho- WG 951068). mologue) that produces bracts (i.e., leaf homologues) with Stratigraphic position and age. Longarm Formation equi- axillary fertile or dwarf shoots (i.e., secondary shoots; ¼flowers valent, Valanginian, Early Cretaceous. or female strobili of some authors) that consist of a stem that Etymology. The genus Protoephedrites is proposed to re- bears vegetative (i.e., bracteoles) and fertile (i.e., sporophylls) flect a combination of plesiomorphic characters and similari- leaf homologues. In living species, the number of seeds is hy- ties to the living genus Ephedra. The specific epithet eamesii pothesized to have been reduced to one, and ovulate sporo- recognizes the important contributions made to our under- phylls are considered to have been evolutionarily displaced to standing of gnetophyte morphology and evolution by the late the position of the secondary shoot tip (Thoday and Berridge Arthur J. Eames of Cornell University. 1912; Eames 1952; fig. 5), and the structure of P. eamesii supports those interpretations. Terminology employed for the description of bennettitalean seeds is the same as in the study Description by Rothwell et al. (2009). Cone structure. Protoephedrites eamesii is a cylindrical cone 7.8 mm in maximum diameter that consists of a cone axis ;3 mm in diameter that bears six pairs of bracts with Results axillary fertile dwarf shoots in an opposite/decussate arrange- ment (fig. 1; video 1, available in the online edition of the In- Systematics ternational Journal of Plant Sciences). As numbered from the apex to the base, nodes 1 and 2 bear only bracts (figs. 1A, Class—Spermatopsida 2A), whereas at nodes 3–6, the bracts subtend a secondary Order—Gnetales fertile shoot (fig. 1B,1C; video 1; table 1). Secondary shoots consist of an axis that bears one or two pairs of diminutive Family—Protoephedraceae Rothwell et Stockey fam. nov. bracteoles that subtend erect terminal seeds in an opposite/ Familial diagnosis. Gnetophyte plants with compound seed decussate arrangement (fig. 2C–2F). At nodes 3 and 6, both cones constructed of primary axis with decussately arranged secondary shoots bear two seeds, whereas at nodes 4 and 5, bracts and axillary fertile shoots; fertile shoots occurring in only one of the two axillary shoots bears seeds. The other ax- axils of most bracts. Axis of secondary shoot with pairs of illary shoot at each of these nodes either is incompletely de- diminutive decussately arranged bracteoles and near apical veloped or not fully preserved. Distal to the most apical pair pair of sporophylls with erect seeds; bracteoles not enclosing of bracts (i.e., borne at node 1), the cone axis is represented seeds. by a tiny, conical projection between the bracts (fig. 2A,ar- Type genus. Protoephedrites Rothwell et Stockey gen. nov. rowhead) that appears to consist of mature parenchyma cells. Generic diagnosis. Cones producing axillary fertile shoots Therefore, the cone has determinate growth and is preserved with apical pair of short sporophylls; seeds nearly sessile, having at a stage after which additional apical growth would not 180° rotational symmetry, multiseriate integument, and short have occurred. micropylar canal with radiating cells of inner epidermis; integ- At the base of the cone, the axis has an irregular shape ument otherwise undifferentiated. Nucellus attached to integ- and an undulating outer margin (fig. 1D). Progressing dis- ument to near midregion, free more distally, having large tally, the axis becomes rectangular in the midregion (fig. 1B, pollen chamber with uniseriate wall. 1C) and nearly round toward the apex (fig. 1A). The cone Type species. Protoephedrites eamesii Rothwell et Stockey axis has a pith consisting of parenchyma cells with dark con- sp. nov. tents (fig. 2B). The stele forms four crescent-shaped xylem Specific diagnosis. Seed cones ;8.7 mm wide, with ;6 bundles separated by pith rays (fig. 2B). Four large, intercon- decussate pairs of bracts and axillary fertile shoots. Seeds nected lacunae occur at the outer margin of the xylem bun- 1.0–1.3. mm long, 0.8–1.2 mm wide in major plane of sym- dles in positions where one would expect phloem to occur metry, 0.5–0.6 mm thick in minor plane of symmetry, show- (figs. 1D,2B). Tracheids are small and round to polygonal in secondary shoot in axil of bract (B), progressing from base (F) toward apex (C). C, Most apical level showing two erect ovules in cross section. Arrowhead identifies position of bract trace. D bot no. 221e 3 40. D, Level at base of ovule (left) and subtending sporophyll (right), showing diverged upper pair of bracteoles (arrows). D bot no. 212 3 40. E, Level slightly lower than in D, showing ovule base and sporophyll (left) and upper pair of bracteoles (arrows). D bot no. 209 3 40. F, Lowest level showing secondary shoot base at level where bract margins remain attached to cone axis. Note bulge at left of secondary axis, probably representing tiny bracteole of basal pair. D bot no. 201 3 40.

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions

Table 1 Characters of Protoephedrites eamesii gen. et sp. nov. as Compared with Potential Gnetophyte Outgroups and Crown Group Gnetophyte Genera Secondary Leaf Outer Integument shoot more arrangement Elongated envelope with Nucellus Large This content downloadedfrom 128.193.162.72 onMon,22Apr 201315:53:03PM Cone No. nodes Bract or less enclosed on secondary Seed micropylar surrounding differentiated attached to pollen Species sizea on axis arrangement by bract shootb Seed borne orientation tube integument sclerotesta integument chamber Cordaixylon dumusum Large Numerous Specialized Absent Helical At tip of long Erect Absent Absent Present At chalaza Present four rankedc sporophyll only

All usesubject toJSTORTerms andConditions Barthelia furcata Large Numerous Helical Absent Helical At tip of long Erect Absent Absent Present ? ? sporophyll Thucydia Helical mahoningensis Large Numerous Helical Absent At tip of long Inverted Absent Absent Present ? ? sporophyll Protoephedrites eamesii Small 6 Opposite/decussate Present Opposite/decussate At tip of short Erect Absent Absent Absent In basal Present sporophyll region, free distally Ephedra spp. Small 4–6 Opposite/decussate Present Opposite/decussate Terminal on Erect Present Present Absent In basal Present secondary axis region, free distally Welwitschia spp. Large Numerous Opposite/decussate Present Opposite/decussate Terminal on Erect Present Present Absent In basal Present secondary axis region, free distally Gnetum spp. Large Numerous Opposite/decussated Present Opposite/decussate Terminal on Erect Present Present Absent In basal Present secondary axis region, free distally a Large: greater than 3 cm; small: ;1 cm or less. b Vegetative leaf homologues on secondary axis of cordaites and walchian conifers are termed vegetative or sterile scales. Those on gnetophytes are termed bracteoles, but we interpret all to be homologous in terms of organography. c Rothwell 1977. d Fused into collar. ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 517

The inner cortex of the axis is constructed of closely spaced parenchyma cells with clear lumens (figs. 1, 2B). Scat- tered cells with thicker dark walls occur throughout the cor- tex (fig. 2B) and may represent sclereids. There is also an outer cortex constructed of cells that consistently show dark, thicker walls than the inner cortical cells (figs. 1, 2B). To- ward the periphery of the cortex, some cells are radially aligned (fig. 1B [right], 1D [left]), suggesting that some sec- ondary cortical development has occurred. The outermost cell layer of the cone axis forms an epidermis of cells with uniform arrangement and consistent size (fig. 1B, bottom). Otherwise, epidermal cells are little differentiated from cells of the outer cortex. Bracts are scale-like with rounded apices (fig. 1A,1B), 3 mm long, 3 mm wide, and 0.5 mm thick at the base. They di- verge from the cone axis at an acute angle and then curve distally (fig. 1C, right) to more or less enclose an axillary fer- tile shoot (figs. 1B,1C,2C–2F; video 1). In a distally pro- gressing series of cross sections (fig. 2C–2F), each bract first separates from the cone axis at the center (fig. 2F) and then progressively toward the margins (fig. 2C–2E), forming a shallow pocket in which the fertile shoot axis diverges (figs. 1B,2F). Section views of the bracts reveal that mesophyll consists of closely spaced parenchyma cells surrounded by a uniseriate epidermis of small cells (fig. 2A,2C–2F). Some of the meso- phyll cells have amber contents, whereas others have empty lumens (fig. 2C–2F). There is a prominent layer of rectangu- lar cells adjacent to the adaxial epidermis (fig. 2C–2E), but the rest of the mesophyll cells are randomly arranged (fig. 2C–2F). In longitudinal views, the parenchyma cells are arranged in irregular axial rows (fig. 2A). There is a small, inconspicuous trace at the center of the bract (fig. 2C, arrow- head) that can be identified by a few tracheids to the abaxial side of which is a lacuna that probably represents the posi- tion of phloem (fig. 2C, arrowhead). Axillary fertile shoots diverge from the cone axis at about a45° angle within a small pocket formed by separation of the bract center at a lower level than the bract margins (figs. 1B,2C–2F; video 1). Overall, axillary shoots are ;3mm long. In cross sections, each is oval at the base and displays two bulges on opposite sides and in the plane that parallels Fig. 3 Holotype of Protoephedrites eamesii gen. et sp. nov. All the periphery of the axis (i.e., lateral plane). Progressing dis- figures from UAPC-ALTA P13,158. A, Tracheids with uniseriate tally, the basal bulges disappear, and two small, scale-like bordered pits and with biseriate pitting (bottom right). D bot no. bracteoles diverge in a plane that is at right angles to the 166 3 500. B, Tracheids with scalariform pitting and bordered pits. D basal bulges (fig. 2D,2E, arrows). At still more distal levels, bot no. 166 3 410. the shoot produces two short stalks that each represent a spo- rophyll with an erect terminal ovule (figs. 1C,2C–2E) and that are oriented in the same plane of section as the basal cross section, ranging 5–10 mm (mean, 6.8 mm) in diameter. bulges (fig. 2C). Therefore, each axillary dwarf shoot appears Tracheids are densely packed and either randomly or radially to have three nodes of paired organs arranged in an opposite/ arranged (fig. 2B). They appear to be radially arranged in the decussate taxis. best-preserved xylem (fig. 2B, bundle at top right), indicating Ovules. The erect ovules are oval with a narrow wing in that there is at least some secondary xylem production. No cross sections, displaying 180° rotational symmetry (figs. 1C wood rays are preserved, and there is no indication of cell [left], 2C,4A). Individual ovules measure 1.0–1.3 mm long, size gradation, such that each cauline bundle probably con- 0.75–1.3 mm wide, and ;0.5 mm thick in the midregion. sists primarily of metaxylem and secondary xylem tracheids. They are roughly ovoid in longitudinal sections (fig. 4E). In longitudinal sections, the tracheids display wall thicken- Each narrows distally to a diminutive micropylar tube (fig. ings on all faces that range from scalariform bars (fig. 3A, 1C, right) that extends through an integument that is several 3B) to uniseriate and biseriate pits (fig. 3A, bottom right). cell layers thick (fig. 4B). The nucellus is adnate to the integ-

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions Fig. 4 Holotype of Protoephedrites eamesii gen. et sp. nov. showing features of ovules. All figures from UAPC-ALTA P13,158. A, Cross section at level where nucellus, represented by cuticle, is free from integument, showing symmetry and histology of integument. Inner epidermis torn away from rest of integument except at top. D bot no. 145 3 112. B, Cross section near apex showing features of multicellular integument surrounding nearly closed micropylar canal. Note radially elongated cells of inner integumentary epidermis. D bot no. 155 3 163. C, Longitudinal section at level of pollen chamber, with uniseriate pollen chamber wall and internal contents. D bot no. 168 3 160. D, Oblique longitudinal section at level of pollen chamber showing features of integument. D bot no. 217 3 192. E, Oblique longitudinal section showing nucellus attached to integument in basal region and free distally. Note cellular basal region of nucellus and pollen chamber with contents more apically. Inner epidermis torn away from other integumentary cells at sides. D bot no. 164 3 165.

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 519

The nucellus consists of thin-walled parenchyma cells sur- rounded by an outer cuticle in the basal region, and it forms a large pollen chamber distally (fig. 4C–4E). There is no evi- dence of a megaspore, a basal hollow, or megagametophyte tissue, suggesting that ovules are relatively immature. The pollen chamber of some ovules forms a uniseriate wall (fig. 4C,4D). In other ovules, cells of the pollen chamber wall are incompletely preserved, and the outer cuticle is quite promi- nent (fig. 4A,4E). There is an amber, noncellular substance within the pollen chamber of most ovules (fig. 4A,4C–4E). By comparison to ovules of living and other fossil gymno- sperms, this substance can be identified as the residue of cells that have broken down to form the pollen chamber. These data support the interpretation that the ovules are immature, possibly preserved at about the stage of pollination (Rothwell 1971).

Structure of Bennettitalean Ovules and Seeds Seeds of numerous permineralized bennettitalean species have been described from exquisitely well-preserved speci- mens, and those studies have demonstrated that the seeds of Bennettitales have a common basic structure that differs from their gnetalean counterparts in many important features (for a detailed comparison of reproductive structures, see Roth- well et al. 2009). In earlier studies, we have described and figured the features of the ovulate fructifications for species of Cycadeoidea (¼Bennettites; Rothwell and Stockey 2002; Rothwell et al. 2009), Williamsonia (Stockey and Rothwell 2003; Rothwell et al. 2009), and Foxeoidea (Rothwell and Stockey 2010), so the descriptions that follow are limited to features of the seed integument and nucellus that have been Video 1 Still photograph from a video (available in the online interpreted differently by other authors (Berridge 1911; Tho- edition of the International Journal of Plant Sciences) showing day 1911, 1921; Friis et al. 2007, 2009, 2011, 2013). holotype of Protoephedrites eamesii gen. et sp. nov. AVIZO The outer limit of the single seed integument in Williamso- reconstructions of cone based on all longitudinal sections, showing nia bockii, Williamsonia sp., and Cycadeoidea maccafferyi morphology of cone axis (a), bracts (b), and seeds (s). Node numbers lies directly adjacent to interseminal scales (figs. 5, 6A–6C, are indicated within parentheses next to bract identifiers (b). 7). In section views, interseminal scales are most easily distin- Animation of this reconstruction rotates around axis. guished from the seed integument by a prominent epidermis and frequently also by a small cellular discontinuity that helps establish the outer limits of the seeds (figs. 5A [right ar- ument in the basal region (fig. 4E), becoming free below the row], 6A,6C,7B–7D). Because no interseminal scales are level of a large pollen chamber (fig. 4A,4C–4E). produced by Foxeoidea connatum, all seeds are directly adja- The ovule integument consists of an inner epidermis distal cent to other seeds (fig. 6D,6E,6G). We address the struc- to the level of separation from the nucellus (fig. 4A,4C–4E), ture of Williamsonia sp. and W. bockii first, because they a zone of thin-walled parenchyma several cell layers thick have the simplest and most easily understood integumentary (fig. 4), an outer zone of cells with darker walls and amber structure. This is followed by the description of F. connatum, cell contents (fig. 4A,4E), and an outer epidermis (fig. 4A). which lacks interseminal scales and has seeds that are fused In the midregion of the ovules, the thin-walled cells often are to each other except at the apex. Cycadeoidea is described pulled away from the inner epidermis (fig. 4A,4E), but frag- last, because it has the most complex integument and is most ments of broken cell walls in the areas of separation (fig. 4A, easily confused unless seed structure of the other bennettita- 4E [left]) and cellular continuity at more distal levels (fig. lean genera is clearly understood. 4E, top) reveal that the discontinuity is taphonomic in origin. Williamsonia sp. The cone of Williamsonia sp. (Roth- Immediately above the level of separation from the nucellus, well et al. 2009) is preserved at about the developmental the inner integumentary epidermis is quite prominent, con- stage where meiosis occurs to form the megaspore and mega- sisting of cells that are relatively isodiametric in cross section gametophyte. Therefore, some ovules have a parenchymatous (fig. 4A). Toward the apex of the ovule, in the region of the nucellus throughout (i.e., premeiosis; fig. 5A), while others pollen chamber, the inner epidermal cells expand in the ra- show a central hollow in the nucellus that represents the dial plane (fig. 4D), forming a distinctive palisade that also megaspore or early precellular stage of the megagametophyte lines the micropylar canal (fig. 4B). (fig. 5D). The nucellus consists of thin-walled parenchyma

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions 520 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 5 Ovules of immature Williamsonia sp. All figures from UAPC-ALTA P14,653. A, Midlongitudinal section of apical half of ovule at about stage of meiosis, showing solid nucellar apex (n) surrounded by single multiseriate integument. Note nucellar tip consists of solid tissue of longitudinally elongated, randomly arranged cells. Note also that nucellus (n) terminates below apex of micropylar canal and that inner epidermal cells are radially elongated distal to nucellus (top), where they partially close micropylar canal. Arrows identify positions where interseminal scales (i) abut integument. Horizontal lines identify extent of single integument and are placed at three positions that roughly correspond to levels at which cross sections on the same plate and of the same letter were made. D1 side no. 40 3 120. B, Cross section of seed apex at about level of line BinA surrounded by interseminal scales (i). Line identifies extent of single integument. Note outer epidermis of round cells and inner epidermis of radially elongated cells that occlude micropylar canal. D top no. 15 3 190. C, Cross section of micropylar region at about level of line C in A surrounded by interseminal scales (i), where randomly arranged parenchyma cells of solid nucellar apex (arrow at n) fill micropylar canal. Line identifies extent of single integument. D top no. 10 3 180. D, Cross section below micropylar region at about level of line D in A surrounded by interseminal scales (i), where randomly arranged parenchyma cells of nucellus (n) are interrupted at center by space that represents noncellular stage of megagametophyte. Line identifies extent of single integument. D top no. 20 3 140. cells that are axially elongated (fig. 5A), randomly arranged, growth. The parenchymatous nucellar tissue extends into and tightly packed in cross sections (fig. 5B–6D), with the the micropyle of the integument as a solid cellular plug (fig. appearance of parenchyma that is the product of primary 5A, top).

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 521

The integument is four to six cell layers thick in the apical seeds, the nucellus of F. connatum extends distally as a solid region (fig. 5, horizontal lines). At the apex, it consists of cellular plug of parenchymatous tissue that fills the micropy- a distinct outer epidermis of round cells without internal con- lar canal (fig. 6G) except at the most apical levels (fig. 6F). tents (fig. 5B), two to four layers of isodiametric thick-walled The plug consists of uninterrupted tissue made up of thin- sclereids (fig. 5A,5B), and an inner epidermis of radially walled cells that are isodiametric and randomly arranged in elongated cells that partly occlude the micropylar canal (fig. cross sections (fig. 6G) and axially elongated in longitudinal 5A,5B). At successively more proximal levels, the sclereids sections (fig. 6E). It is clear that there is no channel through become progressively thinner walled and more axially elon- the apical plug, nor that such a putative channel has been gated, and the cells appear to be parenchymatous (fig. 5). In closed by cellular proliferations (fig. 6E,6G). the midregion, there is a layer of cells immediately within the The integument of F. connatum consists of several layers of outer epidermis with darker and thicker appearing walls than irregularly sized and shaped cells (fig. 6E–6G), some of the other cells of the integument (fig. 5D). At the level of the which are sclereids (fig. 6G) while others are thin walled (fig. apex of the nucellar plug, cells of the inner epidermis abruptly 6E–6G). As also is characteristic of other bennettitalean change shape, being isodiametric in more basal cross sections seeds, there is a distinct inner epidermis of cells that are radi- (fig. 5A,5C,5D) and axially elongated in longitudinal views ally expanded distal to the apex of the nucellar plug (fig. 6E, (fig. 5A). 6F), although not as prominently as in many other species Williamsonia bockii. Ovules or seeds of W. bockii are (cf. figs. 5B,6B,6F). In the region of the nucellar plug, cells preserved at more mature stages than those of Williamsonia of inner epidermis are axially elongated fig. 6E) and isodia- sp., typically having a nucellus that is thin and discontinuous metric in cross sections (fig. 6G). Seeds of the type specimen in the seed body (fig. 6A) and forming a solid parenchyma- typically have integumentary tissue that has been taphonomi- tous plug in the micropylar canal (fig. 6A,6C). Some speci- cally disrupted in the basal region but that forms a continu- mens show cellular megagametophytes, and others also have ous cellular zone distally (fig. 6E,6F). This demonstrates what has been interpreted to be an immature embryo that the integument of this species originally consisted of (Stockey and Rothwell, 2003). In this species, the nucellar a single continuous zone of tissue (lines in fig. 6E–6G). plug displays an epidermis of round cells that lack internal Cycadeoidea maccafferyi. The preserved cone surface contents, and the interior consists of closely spaced, ran- of C. maccafferyi specimens shows round micropyles of seeds domly arranged cells, many of which have dark contents (fig. (fig. 7A, arrowheads), each of which is surrounded by inter- 6A,6C). As with the specimens of Williamsonia sp., cells of seminal scales (fig. 7A). Putative bracts and microsporangiate the nucellar plug in W. bockii show no evidence of a central sporophylls that probably subtended and enclosed the ovu- canal or of cellular proliferations that could have occluded late zone are not preserved on this specimen. The most apical such a canal (figs. 5A,5C,6A,6C). levels of the micropylar tube in seeds of this species typically In longitudinal sections of W. bockii, the tip of the micro- have been lost as a result of abrasion, and no seeds were cut pyle is often abraded (fig. 6A, top). The integument displays in midlongitudinal section. However, oblique sections (fig. an outer epidermis of radially elongated, peg-like cells and is 7B, arrow at n) and cross sections (fig. 7C, arrow at n) of the usually separated from the surrounding interseminal scales micropylar region reveal a nucellar plug that is constructed by a narrow space (fig. 6A). Within the outer epidermis, of an uninterrupted zone of thin-walled parenchyma (fig. 7B,ar- there are two to six layers of axially elongated cells (fig. 6A) row at n) and that is comparable to the nucellar plugs in other of variable sizes (fig. 6B,6C). In cross sections distal to the bennettitalean seeds (cf. figs. 5A,5C,6A,6C,6E,6G,7B). nucellar plug, there is a prominent internal epidermis of radi- As is characteristic of species of Cycadeoidea (Wieland ally elongated cells similar to those of Williamsonia sp., but 1911), the integument is more complex than in other bennet- an inner epidermis is difficult to identify at more proximal titalean genera. The inner margin of the integument does not levels (fig. 6A,6C). We emphasize that in W. bockii, similar show as distinct an epidermis as the other genera (fig. 7B). to all of the bennettitalean seeds studied, the integument is This may be the result, in part, of the absence of sections dis- clearly a single structure with cellular continuity throughout tal to the nucellar plug, where such cells have been observed (horizontal lines in figs. 5, 6A–6C,6E–6G,7B–7D). Only in in other species of Cycadeoidea (e.g., Cycadeoidea wielandii; species of Cycadeoidea spp. are there large tubular cells at fig. 3C of Wieland 1911). The innermost zone of the integu- the seed periphery that are separated from the otherwise un- ment in C. maccafferyi is constructed of several layers of rel- interrupted integument (fig. 7), and that discontinuity has been atively thin-walled, radially elongated cells (fig. 7B,7C)to convincingly documented by Stopes (1918) to be developmental/ the inside of several layers of thick-walled sclereids (fig. 7B– taphonomic in origin. 7D). As is also characteristic of other Cycadeoidea species Foxeoidea connatum. The absence of interseminal scales (Wieland 1911), prominent ribs constructed of thin-walled from F. connatum (fig. 6D) is unique among bennettitalean cells radiate from the sclerotic zone of the integument (fig. cones. However, other features of the fructification allow for 7C), immediately below the micropylar tube in C. maccaf- confident assignment of the species to the Bennettitales feryi (fig. 7C). Those ribs diminish proximally (fig. 7D) and (Rothwell and Stockey 2010). Seeds of F. connatum lie di- are nearly absent at the chalaza of the seeds. rectly adjacent to each other (fig. 6D) and are fused to each To the outside of the integumentary tissues already de- other except in the apical region (fig. 6D,6E). This is partic- scribed are elongated tubular cells (fig. 7B) that occupy the ularly important for demonstrating that there is no additional space between the rest of the integument and the epidermises structure surrounding or enclosing the seed integument in of the surrounding interseminal scales (fig. 7B–7D). In cross Bennettitales. As is also characteristic of other bennettitalean sections, the tubular cells appear as prominent round (fig.

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions Fig. 6 Seeds of Williamsonia bockii (A–C) and seed cone and seeds of Foxeoidea connatum (D–G). A, Midlongitudinal section of somewhat abraded seed apex flanked by interseminal scales (i), showing solid cellular plug of nucellar tissue (arrow at n) filling micropylar canal of single integument. Pollen tubes (pt) are preserved at right of nucellus. Horizontal line B indicates level at which cross section in B was made. Horizontal

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 523

7C,7D), oval (fig. 7C,7D), or elongated (fig. 7B) objects pairs of opposite/decussately arranged bracteoles (whorled in that are situated between the solid cellular integumentary tis- some species), seeds with a prominent pollen chamber, and sues and the interseminal scales (fig. 7B–7D). Separation of a nucellus that is adnate to the integument basally and free those large tubular cells from the otherwise uninterrupted integ- distally (Chamberlain 1935; Gifford and Foster 1989). Like- ument (fig. 7B–7D) is characteristic of most species of Cycadeoi- wise, both living gnetophytes and Protoephedrites have a dis- dea (Lignier 1894; Wieland 1906; Stopes 1918; Rothwell and tinctive inner epidermis of the micropylar canal that consists Stockey 2002) and upon casual observation could be mistaken of radially elongated palisade (Rothwell et al. 2009; Friis for either a cupule or as remnants of another structure enclosing et al. 2011). These characters clearly place Protoephedrites the integument. However, Stopes (1918) has convincingly docu- within the Gnetales (Chamberlain 1935; Gifford and Foster mented that the separation has a developmental/taphonomic 1989), but other characters of Protoephedrites eamesii are origin in Bennettites albianus Stopes (¼Cycadeoidea albianus; novel for the clade (table 1). text-fig. 7 of Stopes 1918), which strongly implies that the tu- All living gnetophyte species (Chamberlain 1935; Gifford bular cells of other species of Cycadeoidea were previously and Foster 1989) as well as a variety of dispersed fossil seeds continuous with the otherwise uninterrupted single integument from Cretaceous deposits (for a comprehensive review, see of bennettitalean seeds as well. Friis et al. 2011) have an outer envelope (two envelopes in Gnetum spp.) surrounding and enclosing the seed integu- Discussion ment, which is widely, although not universally, regarded as having originated from fused subtending bracteoles (for a con- Among gymnospermous clades, Protoephedrites shares temporary discussion of interpretations, see Yang 2004). By con- monosporangiate compound seed cones with Cordaitales, trast, bracteoles of Protoephedrites are diminutive subtending Coniferales, and Gnetales (table 1). This contrasts with the structures that neither are united nor enclose the seed. There simple seed cones of bennettitaleans and most cycads and are also two seeds near the tip of the secondary shoot in Pro- with the ovulate sporophylls of many other gymnospermous toephedrites that occur in positions consistent with their be- groups (Taylor et al. 2009). Cordaiteans typically produce ing borne on short, near-apical sporophylls, whereas only bracts in a distinctive four-ranked pattern not found in other one seed is produced at the tip of the secondary axis by all gymnosperms (Rothwell 1977), but in one genus the bracts crown group gnetophyte species (Chamberlain 1935; Eames are helically arranged (i.e., Shanxioxylon Wang et al. 2003). 1952; Gifford and Foster 1989). The seed integument in spe- Bracts of ancestral conifers and many modern conifer seed cones also are arranged in a helical pattern (table 1), but other cies of Ephedra, Welwitschia, Gnetum, and a variety of dis- Cretaceous, Cenozoic, and Recent species have an opposite/ persed Cretaceous fossil seeds (Friis et al. 2011, 2013) is decussate bract arrangement (e.g., Metasequoia and many spe- extended to form a narrow micropylar tube, but that of Pro- cies of cupressoid Cupressaceae; Farjon 2005). Protoephedrites, toephedrites is not. However, Protoephedrites, crown group other fossil, and all living gnetophytes also produce bracts in an gnetophytes, and dispersed fossil gnetophyte seeds all display opposite/decussate (whorled in some species) pattern (Friis an inner integumentary epidermis that forms a distinctive et al. 2011), but unlike species of Cupressaceae, seeds are not specialized epidermis within what is otherwise a relatively borne in an adaxial or axillary position with respect to the undifferentiated integument (Rothwell et al. 2009). bracts. Rather, the seeds of Protoephedrites and gnetophytes It is difficult to compare Protoephedrites to previously de- are borne near or at the tip of the secondary shoot (table 1). scribed fossil gnetophyte cones because the latter are all pre- In cordaiteans and ancestral walchian conifers, vegetative served as compressions or impressions that display a largely scales and sporophylls are helically arranged on the second- disjunct character set as compared with the anatomical pres- ary axis, while those of Protoephedrites and gnetaleans are ervation of P. eamesii (Taylor et al. 2009; Friis et al. 2011). in an opposite/decussate (whorled in some species) arrange- The most important characters that Protoephedrites shares ment. Additional similarities of Protoephedrites and crown with the compressed fossils are compact cones and an opposite/ group gnetophytes are erect seeds subtended by one or more decussate bract arrangement.

line C indicates extent of single integument at level where cross section in C was made. Note peg-like cells at exterior of integument face epidermises of interseminal scales. VIPM 123 side no. 60 3 70. B, Cross section of seed apex distal to apex of nucellar plug surrounded by interseminal scales (i). Line indicates extent of single integument. Note radially expanded cells of inner epidermis of integument. VIPM 123 top no. 5 3 130. C, Cross section of seed apex surrounded by interseminal scales (i) at level where micropylar canal is filled with parenchyma cells that form solid nucellar plug within single integument. Line indicates extent of integument. Note that nucellar plug consists of randomly arranged cells with outer epidermis. VIPM 123 top no. 5 3 135. D, Oblique section of Foxeoidea cone consisting of central axis surrounded by only seeds. Micropylar region of several adjacent ovules shown in cross sections at top. P13,158 D bot no. 252 3 10. E, Midlongitudinal section of seed apex showing solid plug of nucellar tissue (arrow at n) filling micropylar canal below somewhat radially expanded inner epidermal cells at apex of micropylar canal. Lines F and G indicate extent of integument at levels from which cross sections in F and G were made. Note that single integument is torn below but consists of solid tissue of interdigitating cells above line F. P13,158 D bot no. 190 3 50. F, Cross section of seed apex distal to nucellar plug showing solid tissue of interdigitating cells and inner epidermis that make up single integument. Line indicates extent of integument. P13,158 D top no. 45 3 90. G, Cross section of seed apex at level of line G in E, showing randomly arranged cells of nucellar plug (arrow at n) filling micropylar canal. Line indicates extent of single integument that is torn like specimen in E and that consists of tightly packed cells with prominent inner epidermis. P13,158 top no. 45 3 85.

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions Fig. 7 Cone surface and seeds of Cycadeoidea maccafferyi. A, Surface of specimen showing interseminal scales surrounding and directly adjacent to seed micropyles (arrowheads). BO 120 surface 3 9. B, Cross section of seed surrounded by interseminal scales (i) below apex, where large tubular cells form radiating ribs and other tubular cells lie adjacent to solid tissue of single integument. Note micropyle filled with solid plug of nucellar tissue (arrow at n) at this level. Line identifies extent of integument. BO 211 side no. 100 3 26. C, Oblique section of seed micropylar region flanked by interseminal scales (i). Micropylar canal filled with solid cellular plug of nucellar tissue (arrow at n) at this level. Line identifies extent of single integument at this level. Note detached tubular cells at periphery of integument. 1884 A top no. 10 3 20. D, Cross section of seed and surrounding interseminal scales (i) at midlevel, showing narrow ribs and detached tubular cells at periphery of single integument. Line identifies extent of single integument at this level. 1884 A top no. 138 3 40.

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 525

Fig. 8 Transformational series of morphologies hypothesized by Eames (1952) for the evolution of Ephedra seed cones. Longitudinal sections showing cone axis, diverging bracts, and axillary secondary shoots with bracteoles and erect ovules terminal on sporophylls. Cross sections show bract subtending seeds surrounded by bracteoles, with the cone axis represented by small circle at top. Note that morphology of Protoephedrites eamesii conforms closely to the hypothetical ‘‘ancestral’’ structure at the beginning of the transformational series (A). In original fig. 2 of Eames (1952, p. 83), A–E were designated E–I. Original figure caption (fig. 2 of Eames 1952) reads, ‘‘Diagrams of fertile shoots of Ephedra showing progressive stages in evolutionary modification ... based on living species; E–I, ovulate, hypothetical, based on anatomy and comparisons with microsporangiate shoot. In the longitudinal diagrams the sporophylls are shown, for convenience, as though anterior-posterior, though actually lateral as shown in transverse diagrams.’’

Among the living genera of gnetophytes, Protoephedrites the inner micropylar epidermis and sarcotesta. Although the appears to share several characters with species of Ephedra imaging techniques employed by those authors to study the (table 1). The irregular shape of the axis below the basal gnetophyte-like dispersed seeds do not provide for the level node of P. eamesii (fig. 1D) suggests that the most proximal of resolution, clarity of histological detail, or spectrum of dif- preserved level represents the actual base of the cone. If cor- fering colors displayed by tissues of well-preserved specimens rect, then seed cones of P. eamesii were only a few centime- that are prepared as cellulose acetate peel sections, the de- ters long, with only about six pairs of bracts (i.e., nodes) and scriptions of those seeds appear to be detailed and accurate. axillary fertile shoots. In this respect, P. eamesii is similar to At the same time, such studies provide no evidence about the Ephedra, which has quite small seed cones with only a few structure of bennettitalean seeds. Friis et al. (2007, 2009, pairs (or whorls) of bracts and axillary structures (Stevenson 2011) do present photographs of one specimen of Cycadeoi- 1993). However, in contrast to seed cones of Ephedra, which dea morierei Lignier that is represented by a small number of typically produce fertile secondary shoots with seeds only in thin sections and seeds that have been dislodged from the axils of the most apical bracts (Cutler 1939; Yang 2004; cone, as well as interpretative diagrams to support their as- table 1), P. eamesii produces axillary fertile shoots through- sertion. However, their figures of the relevant structures are out the cone. In this latter character, Protoephedrites is more at too low a magnification to allow the reader to determine reminiscent of Welwitschia and Gnetum, which also produce whether their hypothesis is correct (figs. 129, 131–137 of seeds throughout the length of seed cones. In Gnetum, the Friis et al. 2009). bracts at each node are adnate, forming a collar of tissue, In their tacit questioning of the accuracy of descriptions and and the internodes are much longer than for Protoephedrites the more recent understanding of bennettitalean ovulate organs, and the other living gnetophyte genera. Friis et al. have resurrected an old alternative hypothesis of ben- nettitalean seed structure (Carruthers 1870; Berridge 1911; Thoday 1911) that asserts that there are close structural similar- Comparative Structure of Gnetalean and Bennettitalean ities between the seeds of Gnetales and Bennettitales (Friis et al. Seed Cones and Seeds 2007, 2009, 2011, 2013). That view contrasts dramatically Seeds of superb anatomical preservation have been de- with our own observations, descriptions, and understanding of scribed and illustrated for numerous species of Bennettitales, bennettitalean seed cones and seeds (Rothwell and Stockey and for many years, the structure of those seeds has been 2002; Stockey and Rothwell 2003; Rothwell et al. 2009), as generally regarded as well understood. More recently, Friis well as with data presented in the text and/or photographs and et al. (2007, 2009, 2011, 2013) have described a variety of diagrams of articles by many other authors on several spe- dispersed early Cretaceous seeds in which the integument is cies of Cycadeoidea (¼Bennettites of some authors; Wieland enclosed in an outer envelope, similar to that of Gnetales. 1906, 1911, 1916; Stopes 1918; Crepet 1972, 1974; Crepet Those authors apparently have not conducted detailed stud- and Delevoryas 1972), several species of Williamsonia (Sew- ies of bennettitalean seed anatomy. Nevertheless, they have ard 1913; Sahni 1932; Sharma 1970a, 1970b), Cycadeoidella concluded that gnetalean seeds and bennettitalean seeds japonica Ogura (Nishida 1994), Bennetticarpus yezoites share a common basic structure, including an outer envelope Ohana, Kimura et Chitaley (1998), and Williamsoniella coro- surrounding the seed integument and histological features of nata Thomas (Crane and Herendeen 2009).

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions 526 INTERNATIONAL JOURNAL OF PLANT SCIENCES

We previously have described and illustrated ovulate struc- ner epidermal cells of the micropylar canal do not become tures from the cones of several bennettitalean taxa (Rothwell transversely divided during development (figs. 5A,5B,6B,6E, and Stockey 2002, 2010; Stockey and Rothwell 2003; Roth- 6F) as do comparable cells of Gnetum (Thoday 1921). well et al. 2009) and have found them to contrast markedly There is also an intriguing similarity between the exterior with comparable structures of Gnetales in several important shape of the inner pair of bracteoles covering the integu- characters. In general, Gnetales bear their seeds in unisporan- ment of Gnetum and the outer surface of the integument giate cones of compound construction with parts that show of Cycadeoidea, particularly immediately below the elon- opposite/decussate (or whorled) arrangement, whereas ben- gated micropylar tube (cf. fig. 1 of Berridge 1911 with nettitalean cones are both monosporangiate and bisporan- figs. 5, 6 of Wieland 1911). However, closer examination giate, are of simple construction, and bear their parts in reveals that shape to be produced by nonhomologous a helical arrangement. The seeds of Gnetales are borne on structures in Gnetum and the seeds of the Bennettitales. secondary shoots and are either subtended by or enclosed by Whereas in Gnetum, in which that shape is formed by fused pairs (or whorls) of bracteoles (Wieland 1911; Stopes a vascularized structure that covers the integument and is 1918; Rothwell and Stockey 2002; Rothwell et al. 2009). In not attached to the integument above the chalaza, in Cyca- contrast, bennettitalean sporophylls are borne directly on the deoidea the similar shape is produced by the unvascularized receptacle of the simple cone, and the erect, terminal seeds are integument, a single structure adjacent to the nucellus that is not subtended by (or enclosed by) one (namely, Ephedra, Wel- attached to the nucellus into the midregion of the seed and witschia, and several species of dispersed Cretaceous seeds; that shows cellular continuity from the apex to the tip of the Friis et al. 2011, 2013) or two (namely, Gnetum spp.; Thoday micropylar tube. Also, the micropylar tube of bennettitalean 1921) vascularized envelopes that are derived from bracteoles seeds shows no evidence of the cellular proliferations that that subtend the seed on a secondary shoot (table 1 of Roth- close the micropyle of Gnetum after pollination. well et al. 2009). The latter structure of the seed cone and Careful examination of the nucellus in the apical regions of seeds is characteristic of Gnetales but not of Bennettitales. bennettitalean and gnetophyte seeds reveals additional impor- As stressed by Berridge (1911), Thoday (1911, 1921), and tant structural and biological differences between the two Friis et al. (2007, 2009, 2011, 2013), there are intriguing his- clades. The nucellus of bennettitalean seeds consists of a single tological similarities between epidermal cells of the micropyle structure of continuous cellular construction from the inside to (i.e., integument) in bennettitalean species and the seeds of the outside that surrounds the megaspore, megagametophyte, Gnetum. In both clades, the inner epidermis of the integu- and/or embryo and that extends into the base of the micropylar ment consists of isodiametric cells in cross sections at some canal as a solid plug of tissue. We wish to stress that in all ana- levels (cf. figs. 5C,5D,6E,6G with figs. 1, 7 of plate 1 of tomically preserved bennettitalean seeds for which histological Thoday 1921) and of radially elongated epidermal cells that preservation is adequate for detailed examination, solid plug of partly occlude the micropylar canal at other levels (cf. figs. tissue consists of cells that are isodiametric, tightly packed, lon- 5B,6B,6E with figs. 2–5, 9 of plate 1 of Thoday 1921). gitudinally elongated, and randomly arranged in cross sections However, the radially elongated cells occur at the apex of the (figs. 5A,5C,6A,6C,6E,6G,7B; fig. 63-(1) and plate 28, fig. 2 micropylar tube in the bennettitalean species, whereas simi- of Wieland 1906; text-fig. 3C andplate11,figs.18,21,25of larly appearing cells occur at one (fig. 1 of Berridge 1911) or Seward 1913; text-fig. 11, plate 21, fig. 18, and plate 22, fig. 3 two levels (text-fig. 1 of Thoday 1921) lower down the mi- of Stopes 1918; fig. 1E,1F, plate 1, fig. I and plate 2, fig. J of cropylar canal in Gnetum. Conversely, the more isodiametric Sharma 1970a; fig. 2 of Sharma 1970b; fig. 10 of Crepet inner epidermal cells occur at the most distal levels of the mi- 1972; plate 64, fig. 30 and plate 67, fig. 42 of Crepet 1974; cropylar canal (and possibly also at a lower level) in Gnetum figs. 14–16 of Nishida 1994; plate 2, figs. 4, 5 and plate 3, (fig. 1 of Berridge 1911; text-fig. 1 and figs. 1, 7 of Thoday fig. 1 of Ohana et al. 1998; fig. 3 [bottom arrow] of Crane 1921), whereas such cells consistently occur as a single zone and Herendeen 2009). Although the cross section of the mi- in the region of the nucellar plug that terminates below the cropylar tube of Gnetum that has been closed by cellular radially elongated epidermal cells in bennettitalean seeds proliferations (fig. 3 of Berridge 1911) does look superficially (figs. 5A–5C,6A–6C,6E–6G). Bennettitalean seeds have no like the nucellar plug of bennettitalean seeds (figs. 5C,6G), pollen chamber, while gnetalean seeds have a pollen chamber the inner epidermis of the integument (figs. 5C,5D,7G) and/ at the apex of the nucellus (fig. 1 of Berridge 1911; fig. 85 of or the outer epidermis of the nucellus (fig. 6C) clearly sepa- Pearson 1929), and that pollen chamber is not exerted into rate the nucellus from the integumentary tissues in bennetti- the micropylar canal, as is the nucellar plug of Bennettitalean talean seeds. Moreover, there often is a space between the seeds (figs. 5A,5C,6A,6C,6E,6G,7B; for a detailed dis- nucellar plug and the integument (fig. 6C,6G) in bennettita- cussion, see Rothwell et al. 2009). lean seeds. In Gnetum seeds cut at comparable levels (fig. 3 Radial elongation of the inner epidermal cells is apparently of Berridge 1911), no such epidermis or separation is present associated with postpollination closure of the micropylar ca- because the cells are all of integumentary origin. nal in Gnetum (fig. 1 of Berridge 1911) and also with trans- Structure of the integument and nucellus in the apical re- verse divisions of the radially elongated cells during maturation gion of bennettitalean seeds has been confused further by in- in that genus (Thoday 1921). By contrast, the inner epidermal complete preservation in some specimens and by differing cells at the distal levels of bennettitalean seeds are already radi- terminology used by different authors for the apical plug allyelongatedinthemostimmature ovules available for study (i.e., nucellus) in well-preserved specimens (e.g., ‘‘nucellar (fig. 5A,5B; fig. 5 of Crepet 1972; fig. 10 of Crepet and Dele- beak’’ of Wieland 1906 and Sharma 1970b; ‘‘nucellus’’ of voryas 1972) as well as in more mature seeds. Moreover, the in- Seward 1913 and Nishida 1994; ‘‘nucellar mass’’ of Stopes

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 527

1918; ‘‘nucellar stalk’’ of Sahni 1932 and Ohana et al. 1998; pollen and seed cones (fig. 2 of Eames 1952) that illustrates ‘‘plug’’ of Sharma 1970b; ‘‘nucellar plug’’ of Stockey and this hypothesis. The structure of P. eamesii is so similar to Rothwell 2003, Rothwell et al. 2009, and Rothwell and the hypothesized ancestral morphology in this transfor- Stockey 2013). Additional confusion has arisen as a result of mational series that the seed cone portion of that figure some authors having identified what they interpret to be is reproduced as figure 8. Protoephedrites eamesii differs a pollen chamber in bennettitalean seeds (Lignier 1894; Wie- from that ancestral hypothesized morphology (fig. 8A) land 1906; Stopes 1918; Ohana et al. 1998). However, none only by having sporophylls and subtending bracteoles that of those identifications are based on structures that compare are shorter than hypothesized by Eames (1952; cf. video favorably with the pollen chambers eroded into the apex of 1, fig. 8). In both Protoephedrites and the hypothesized the nucellus in most other gymnosperms (Rothwell 1971; ancestral morphology of Eames (fig. 8A), each secondary Serbet and Rothwell 1995). Rather, the pollen chamber is shoot bears two erect seeds on near apical sporophylls identified below the apical plug of nucellar tissue in those and are subtended by a pair of bracteoles that do not en- bennettitalean taxa, in the position that has been called an close the seeds. Successive hypothesized morphologies archegonial chamber in cycads (Chamberlain 1935). No pol- lead to the loss of one of the seeds (fig. 8B), shortening of len (comparable to that of other gymnosperms) has ever been the sporophyll (as is already present in P. eamesii), enclo- discovered in this region of anatomically preserved bennetti- sure of the seed within the subtending bracts to produce talean seeds, and there is no opening through the apex of the the outer envelope (fig. 8C), and displacement of the spo- nucellus that would allow for pollen to reach that area of the rophyll to the apex of the secondary shoot (fig. 8D,8E). ovules or seeds. Therefore, a pollen chamber like that of gne- tophytes is not present in bennettitalean seeds. As a result of the studies and analyses of bennettitalean seed structure de- Summary and Conclusions tailed in this discussion, it is clear that the reconstructions of bennettitalean seeds presented by Thoday (1921, text-fig. 5) An anatomically preserved specimen has been discovered and Friis et al. (2009 [fig. 129], 2011 [fig. 5.25]) are not ac- in Early Cretaceous sediments of western Canada that pro- curate with respect to the structure of the nucellus, the struc- vides the first fossil evidence for internal seed cone anatomy ture of the integument, and/or the putative presence of an and organ homologies of stem group Gnetales. Protoephe- additional envelope of tissue enclosing the integument. drites eamesii gen. et sp. nov. demonstrates that at least some ancestral gnetophyte seed cones bore bracts with axillary fer- tile shoots in the opposite/decussate arrangement that charac- Plesiomorphic Characters of Gnetalean Seed Cones terizes Gnetales as a whole. This supports the hypothesis that By comparison to both potential gnetophyte outgroups such morphology represents a synapomorphy for the gneto- (i.e., cordaiteans and walchian conifers) and the crown group phyte clade. The occurrence of two oppositely positioned genera of Gnetales (i.e., Ephedra, Welwitschia, and Gnetum; seeds near the apex of the fertile dwarf shoot confirms the table 1), Protoephedrites displays several characters that can previously hypothesized ancestral position of the gnetophyte be hypothesized as being ancestral within gnetophytes. These seed at the tip of a sporophyll. Likewise, the presence of a pair putatively ancestral characters are (1) monosporangiate com- of small bracteoles subtending the naked seed of P. eamesii pound cones (2) with opposite/decussate bract arrangement, supports the hypothesis that the outer envelope of crown (3) secondary fertile shoots more or less enclosed by subtend- group gnetophyte seeds is derived from such a pair of bracte- ing bract, (4) erect seeds (5) borne apically on (6) opposite oles (fig. 8). In addition, the absence from Protoephedrites of sporophylls near the secondary shoot apex with (7) subtend- bracteoles that fully surround and enclose the integument ing bracteoles unfused and (8) not enclosing the seed, and (9) strongly suggests that the outer envelope of crown group gne- a short micropylar tube. This combination suggests that tophyte seeds evolved within the gnetophyte clade (fig. 8). some characters typically considered to be synapomorphies Detailed study of anatomically preserved ovules and seeds for gnetophytes (i.e., seed enclosed by an outer envelope and produced by several species of Bennettitales (i.e., Williamsonia narrow elongated micropylar tube) may actually be derived sp., Williamsonia bockii, Foxeoidea connatum,andCycadeoi- within the gnetalean clade and not characteristic of stem dea maccafferyi) and careful comparisons to previously de- group gnetophytes (table 1). If this is correct, then features of scribed bennettitalean ovulate structures reveal that all have Protoephedrites may aid in identifying other fossil stem a simple integument that is not surrounded by an additional group Gnetales that could help clarify the pattern of charac- vascularized envelope and that the nucellus extends into the ter transitions leading to the stem group genera. micropylar tube as a solid plug of tissue. Contrary to an old In this regard, a previously hypothesized transformational hypothesis (Berridge 1911; Thoday 1911) and more recent as- series leading to the presence of a single seed at the apex of sertions by other authors (Friis et al. 2007, 2009, 2011, the secondary shoot and with an outer envelope formed from 2013), such seeds are not structurally equivalent to those of subtending bracteoles (i.e., Thoday and Berridge 1912; Gnetales and do not provide compelling data for a close sys- Eames 1952) is most intriguing. According to this hypothesis, tematic relationship between Gnetales and Bennettitales. the structure of modern Ephedra seed cones is derived from Despite the basal position of Ephedra among living gneto- an ancestral morphology in which there were two oppositely phyte genera in the results of virtually all systematic analyses positioned seeds at the tips of near-apical sporophylls and of the clade, comparisons of P. eamesii and Ephedra spp. to with subtending bracteoles that did not enclose the seed. the seed cones of potential gnetophyte outgroups and to Eames (1952) developed a transformational series for both other crown group Gnetales imply that small cone size, rela-

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions 528 INTERNATIONAL JOURNAL OF PLANT SCIENCES tively simple cone structure, and the occurrence of seeds only Acknowledgments in the axils of the most apical bracts may represent specializa- tions within the clade. The recognition that Protoephedrites This study is dedicated to Dr. Thomas N. Taylor of the represents an extinct representative of Gnetales provides the University of Kansas, in grateful appreciation of the un- first internal anatomical evidence for a stem group representa- flinching support that he has offered each of us as a tireless tive of the gnetalean clade. It also helps in the development of mentor and for being a steadfast friend throughout the a search image for additional fossil representatives of the clade, course of our professional careers. We thank Ashley A. Kly- which will provide vital data for confirming structural homolo- miuk, University of Kansas, for assistance with developing gies of gnetophyte organs, for understanding the sequence of three-dimensional images of the cone. This study was sup- morphological transformations that led to crown group gneto- ported in part by the National Science Foundation (grant phytes, and that ultimately will be needed for determining both EF-0629819) and the National Science and Engineering Re- the sister group relationships and the clade age of Gnetales. search Council of Canada (grant A-6908 to R. A. Stockey).

Literature Cited

Ash SR 1972 Late Triassic plants from the Chinle Formation in organ complexes in Gnetales. Int J Plant Sci 157(suppl):S113– northeastern Arizona. Palaeontology 15:598–618. S125. Berridge DM 1911 On some points of resemblance between gneta- Farjon A 2005 A monograph of Cupressaceae and Sciadopitys. Royal lean and bennettitalean seeds. New Phytol 10:140–144. Botanic Gardens, Kew. Burleigh JG, S Mathews 2004 Phylogenetic signal in nucleotide data Finet CR, E Timme, CF Delwiche, F Marle´taz 2010 Multigene from seed plants: implications for resolving the seed plant tree of phylogeny of the green lineage reveals the origin and diversification life. Am J Bot 91:1599–1613. of land plants. Curr Biol 20:2217–2222. ——— 2007a Assessing among-locus variation in the inference of Friis EM, PR Crane, KR Pedersen 2009 Early Cretaceous mesofossils seed plant phylogeny. Int J Plant Sci 168:111–124. from Portugal and eastern North America related to the Bennettitales- ——— 2007b Assessing systematic error in the inference of seed Erdtmanithecales-Gnetales group. Am J Bot 96:252–283. plant phylogeny. Int J Plant Sci 168:125–135. ——— 2011 Early flowers and angiosperm evolution. Cambridge Carruthers W 1870 On fossil cycadean stems from the secondary University Press, Cambridge. rocks of Britain. Trans Linn Soc Lond 26:675–708. Friis EM, PR Crane, KR Pedersen, S Bengston, PCJ Donoghue, GW Chamberlain CJ 1935 Gymnosperms. University of Chicago Press, Chicago. Grimm, M Stampanoni 2007 Phase contrast enhanced synchro- Crane PR 1996 The fossil history of Gnetales. Int J Plant Sci 157(suppl): tron-radiation X-ray analysis of the Cretaceous seeds links Gnetales S50–S57. to extinct Bennettitales. Nature 450:549–552. Crane PR, PS Herendeen 2009 Bennettitales from the Grisethorpe Friis EM, KR Pedersen, PR Crane 2013 New diversity among Bed (Middle Jurassic) at Cayton Bay, Yorkshire, UK. Am J Bot 96: chlamydospermous seeds from the Early Cretaceous of Portugal 284–295. and North America. Int J Plant Sci 174:530–558. Crane PR, S Lidgard 1989 Angiosperm diversification and palaeo- Gifford E, AS Foster 1989 Comparative morphology of vascular latitudinal gradients in Cretaceous floristic diversity. Science 246: plants. 3rd ed. WH Freeman, San Francisco. Graham SW, WJD Iles 2009 Different gymnosperm outgroups have 675–678. (mostly) congruent signal regarding the root of flowering plant ——— 1990 Angiosperm radiation and patterns of Cretaceous phylogeny. Am J Bot 96:216–227. palynological diversity. Pages 377–407 in PD Taylor, GP Larwood, Haggart JW, HW Tipper 1994 New results in Jura-Cretaceous eds. Major evolutionary radiations. Oxford University Press, stratigraphy, northern Vancouver Island, British Columbia. Geol Oxford. Surv Can Curr Res 1994E:59–66. Crane PR, G Upchurch 1987 Drewria potomacensis gen. et sp. nov., Hilton J, RM Bateman 2006 Pteridosperms are the backbone of seed- an early Cretaceous member of Gnetales from the Potomac Group plant phylogeny. J Torrey Bot Soc 133:119–168. of Virginia. Am J Bot 74:1722–1736. Hufford, L 1996 The morphology and evolution of male reproduc- Crepet WL 1972 Investigations of North American Cycadeoids: tive structures in Gnetales. Int J Plant Sci 157(suppl):S95–S112. pollination mechanisms in Cycadeoidea. Am J Bot 59:1048–1056. Ickert-Bond SM, C Rydin, SS Renner 2009 A fossil-calibrated ——— 1974 Investigations of North American cycadeoids: the relaxed clock for Ephedra indicates an Oligocene age for the reproductive biology of Cycadeoidea. Palaeontographica 148B: divergence of Asian and New World clades and Miocene dispersal 144–169. into South America. J Syst Evol 47:444–456. Crepet WL, T Delevoryas 1972 Investigations of North American Jeletzky JA 1976 Mesozoic and Tertiary rocks of Quatsino Sound, cycadeoids: early ovule ontogeny. Am J Bot 59:209–215. Vancouver Island, British Columbia. Geol Surv Can Bull 242:1–243. Cutler HC 1939 Monograph of the North American species of the Joy KW, AJ Willis, WS Lacey 1956 A rapid cellulose peel technique genus Ephedra. Ann Mo Bot Gard 26:373–427. in palaeobotany. Ann Bot, NS, 20:635–637. Dilcher DL, ME Bernardes-De-Oliveira, D Pons, TA Lott 2005 Wel- Kunzmann A, BAR Mohr, V Wilde, MEC Bernardes-de-Oliveira 2011 A witschiaceae from the Lower Cretaceous of northeastern Brazil. Am putative gnetalean gymnosperm Cariria orbiculiconiformis gen. nov. et J Bot 92:1294–1320. spec. nov. from the Early Cretaceous of northern Gondwana. Rev Doyle JA 1998 Molecules, morphology, fossils, and the relationship Palaeobot Palynol 165:75–95. of angiosperms and Gnetales. Mol Phylogenet Evol 9:448–462. Laconte H, DW Stevenson 1990 Cladistics of the spermatophytes. Doyle JA, MJ Donoghue 1992 Seed plant phylogeny reanalyzed. Brittonia 42:197–211. Brittonia 44:89–106. Lignier O 1894 Ve´ge´tative fossils de Normandie: structure et Eames AJ 1952 Relationships of the Ephedrales. Phytomorphology affinities du Bennettites morieri (Sap. & Mar.). Mem Soc Linn 2:79–100. Normandie Caen 18:5–78. Endress PK 1996 Structure and function of female and bisexual ——— 1911 Le Bennettites morieri (Sap. Et Mar.) Lignier se

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions ROTHWELL & STOCKEY—EARLY CRETACEOUS GNETOPHYTE SEED CONE 529

reproduisait probablement par Parthe´noge´ne`. Bull Soc Bot Fr 58: Ephedra: Cretaceous fossils and extant molecules. Proc Natl Acad 224–227. Sci USA 101:15571–15576. Martill DM, PM Brito, S Wenz, PR Wilby 1993 Fossils of the Rydin C, SQ Wu, EM Friis 2006b Liaoxia (Gnetales): ephedroids Santana and Crato Formations, Brazil. Field Guides to Fossils, no. from the Early Cretaceous Yixian Formation in China. Plant Syst 5. Palaeontological Association, London. Evol 262:239–263. Mathews S 2009 Phylogenetic relationships among seed plants: Sahni B 1932 A petrified Williamsonia (W. sewardiana, sp. nov.) persistent questions and the limits of molecular data. Am J Bot from the Rajimahal Hills, India. Palaeontol Indica, NS, 20:1–19. 96:228–236. Serbet R, GW Rothwell 1995 Functional morphology and homolo- Mohr BAR, BEC Bernardes-de-Oliveira, RF Loveridge. 2007 The gies of gymnospermous ovules: Evidence from a new species of macrophyte flora of the Crato Formation. Pages 537–565 in DM Stephanospermum (Medullosales). Can J Bot 73:650–661. Martill, G Bechly, RF Loveridge, eds. The Crato fossil beds of Seward AC 1913 A petrified Williamsonia from Scotland. Philos Brasil: window into an ancient world. Cambridge University Press, Trans R Soc B 203:101–136. Press. Sharma BD 1970a On the structure of a Williamsonia cf. W. scotica Mu¨ ller J 1984 Significance of fossil pollen for angiosperm history. from the Middle Jurassic rocks of the Rajmahal Hills, India. Ann Ann Mo Bot Gard 71:419–443. Bot, NS, 34:289–296. Nishida H 1994 Morphology and evolution of Cycadeodales. J Plant Sharma BD 1970b On the structure of seeds of Williamsonia Res 107:479–492. collected from the Middle Jurassic rocks of Amarjola in the Nixon K, WL Crepet, DW Stevenson, EM Friis 1994 A reevaluation Rajmahal Hills, India. Ann Bot, NS, 34:1071–1078. of seed plant phylogeny. Ann Mo Bot Gard 81:484–533. Solms-Laubach H 1891 On the fructification of Bennettites gibso- Ohana T, T Kimura, S Chitaley 1998 Bennetticarpus yezoites sp. nov. nianus, Carr. Ann Bot 5:419–454. (Bennettitales) from the Upper Cretaceous of Hokkaido, Japan. Stevenson DW 1993 Ephedraceae. Pages 428–434 in Flora of North Paleontol Res 2:108–119. America Editorial Committee, ed. Flora of North America. Vol 2. Osborn J, TN Taylor, MR de Lima 1993 The ultrastructure of fossil Pteridophytes and Gymnosperms. Oxford University Press, New ephedroid pollen with gnetalean affinities from the Lower York. Cretaceous of Brazil. Rev Paleobot Palynol 77:171–184. Stockey RA, GW Rothwell 2003 Anatomically preserved Williamso- Pearson HHW 1929 Gnetales. Cambridge University Press, Cam- nia (Williamsoniaceae): evidence for Bennettitalean reproduction in bridge. the Late Cretaceous of western North America. Int J Plant Sci 164: Rai HS, S Graham 2010 Utility of large, multigene plastid data set in 251–262. inferring higher-order relationships in ferns and relatives (Mon- Stockey RA, NJP Wiebe 2008 Lower Cretaceous conifers from Apple ilophytes). Botany 97:1444–1456. Bay, Vancouver Island: Picea-like leaves, Midoriphyllum piceoides Rai HS, HE O’Brien, PA Reeves, SW Graham 2003 Inference of gen. et sp. nov. (Pinaceae). Botany 86:649–657. higher-order relationships in the cycads from a large chloroplast Stopes MC 1918 New Bennettitalean cones from the British Creta- data set. Mol Phylogenet Evol 29:350–359. ceous. Philos Trans R Soc B 208:389–440. Rai HS, PA Reeves, R Peakall, RG Olmstead, SW Graham 2008 In- Sweet A 2000 Applied research report on two samples of Cretaceous ference of higher-order conifer relationships from a multi-locus age from Vancouver Island, British Columbia, as requested by J. plastid data set. Botany 86:658–669. Haggart (GSC Pacific, Vancouver). Geological Survey of Canada, Rothwell GW 1971 Ontogeny of the Paleozoic ovule Callosperma- Paleontological Report ARS-2002-02. rion pusillum. Am J Bot 58:706–715. Taylor TN, EL Taylor, M Krings 2009 The biology and evolution of ——— 1977 The primary vasculature of Cordaianthus concinnus. fossil plants. 2nd ed. Elsevier, Amsterdam. Am J Bot 64:1235–1241. Thoday MG 1911 The female inflorescence and ovules of Gnetum Rothwell GW, WL Crepet, RA Stockey 2009 Is the anthophyte africanum,withnotesonGnetum scandens. Ann Bot 25:1109–1135. hypothesis alive and well? new evidence from the reproductive ——— 1921 Anatomy of the ovule and seed of Gnetum gnemon structures of Bennettitales. Am J Bot 96:296–322. with notes on Gnetum funiculare. Ann Bot 85:37–53. Rothwell GW, R Serbet 1994 Lignophyte phylogeny and the Thoday MG, EM Berridge 1912 The anatomy and morphology of evolution of spermatophytes: a numerical cladistic analysis. Syst the inflorescences and flowers of Ephedra. Ann Bot 26:953–985. Bot 19:443–482. Wang S-J, J Hilton, T Baolin, J Galtier 2003 Cordaitalean seed plants Rothwell GW, RA Stockey 2002 Anatomically preserved Cycadeoi- from the Early Permian of north China. I. Delimitation and dea (Cycadeoidaceae), with a reevaluation of the systematic reconstruction of the Shanxioxylon sinense plant. Int J Plant Sci characters for the seed cones of Bennettitales. Am J Bot 89:1447– 164:89–112. 1458. Wang X-Q 2004 New Permian gnetalean cone as fossil evidence for ——— 2010 Independent evolution of seed enclosure in Bennetti- supporting current molecular phylogeny. Ann Bot 94:281–288. tales: evidence from the anatomically preserved cone Foxeoidea Wieland GR 1906 American fossil cycads. Carnegie Institution of connatum gen. et sp. nov. Pages 51–66 in CT Gee, ed. Plants in Washington Publication 34. Washington, DC. Mesozoic time: morphological innovations, phylogeny, ecosystems. ——— 1911 A study of some American fossil cycads. V. Further Indiana University Press, Bloomington. notes on seed structures. Am J Sci 32:133–155. Rydin C, M Ka¨llersjo¨ , EM Friis 2002 Seed plant relationships and ——— 1916 American fossil cycads. Vol 2. Taxonomy. Carnegie the systematic position of Gnetales based on nuclear and Institution of Washington Publication 34. Washington, DC. chloroplast DNA: conflicting data, rooting problems, and the Yang Y 2004 Ontogeny of triovulate cones of Ephedra intermedia monophyly of conifers. Int J Plant Sci 163:197–214. and origin of the outer envelope of ovules of Ephedraceae. Am J Bot Rydin C, KR Pedersen, PR Crane, EM Friis 2006a Former diversity 91:361–368. of Ephedra (Gnetales): evidence from Early Cretaceous seeds from Yang Y, B-Y Geng, DL Dilcher 2005 Morphology and affinities of an Portugal and North America. Ann Bot 98:123–140. Early Cretaceous Ephedra (Ephedraceae) from China. Am J Bot 92: Rydin C, KR Pedersen, EM Friis 2004 On the evolutionary history of 231–241.

This content downloaded from 128.193.162.72 on Mon, 22 Apr 2013 15:53:03 PM All use subject to JSTOR Terms and Conditions