American Journal of Botany 101(12): 2136–2147, 2014.

HUGHMILLERITES VANCOUVERENSIS SP. NOV. AND THE CRETACEOUS DIVERSIFICATION OF CUPRESSACEAE1 BRIAN A. ATKINSON2,4, GAR W. ROTHWELL2,3, AND RUTH A. STOCKEY2 2Department

of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, Oregon 97331 USA; and of Environmental and Plant Biology, 315 Porter Hall, Ohio University, Athens, Ohio 45701 USA

3Department

• Premise of the study: Two ovulate conifer cones, one of which is attached terminally to a short leafy shoot, reveal the presence of a new species of Hughmillerites in the Early Cretaceous Apple Bay flora of Vancouver Island, British Columbia, Canada. This ancient conifer expands the diversity of Cupressaceae in the Mesozoic and reveals details about the evolution of Subfamily: Cunninghamioideae. • Methods: Specimens were studied from anatomical sections prepared using the cellulose acetate peel technique. • Key results: Vegetative shoots have helically arranged leaves that are Cunninghamia-like. Seed cones have many helically arranged bract/scale complexes in which the bract is larger than the ovuliferous scale. Each ovuliferous scale has three free tips that separate from the bract immediately distal to an inverted seed. Several ovuliferous scales show interseminal ridges between seeds. • Conclusions: This study documents a new extinct species of cunninghamioid conifers, Hughmillerites vancouverensis, expanding the record of the genus from the Late Jurassic to the Early Cretaceous. This new extinct species emphasizes the important role that conifers from subfamily Cunninghamioideae played in the initial evolutionary radiation of Cupressaceae. In light of recent findings in conifer regulatory genetics, we use H. vancouverensis to hypothesize that variations of expression in certain gene homologues played an important role in the evolution of the cupressaceous ovuliferous scale. Key words: conifer; Cretaceous; Cunninghamioideae; Cupressaceae; fossil; ovuliferous scale homologies.

Many members of basal Cupressaceae represent relics of clades that were much more diverse during the latter half of the Mesozoic (Stewart and Rothwell, 1993; Stockey et al., 2005; Atkinson et al., 2014; Shi et al., 2014). The fossil record of this family dates from the Early Jurassic (Escapa et al., 2008), which is roughly concordant with molecular divergence estimates of Cupressaceae (i.e., early mid Jurassic, Yang et al., 2012; Triassic, Mao et al., 2012). Throughout evolutionary history seed cones of Cupressaceae have undergone greater morphological change than those of any other conifer family (Farjon, 2005), and those changes have been the subject of many morphological and paleobotanical studies (Jagel, 2001; Jagel and Stützel, 2001; Farjon and Ortiz García, 2003; Schulz and Stützel, 2007; Escapa et al., 2010; Rothwell et al., 2011). Due to the long and inherently complex history of Cupressaceae, the evolutionary patterns of this family remain incompletely understood. Furthermore, studies of the genetic mechanisms responsible for morphological changes in conifer seed cones have only recently been initiated (Carlsbecker

1 Manuscript received 23 August 2014; revision accepted 15 October 2014. The authors thank Joe Morin, Gerald Cranham, Mike Trask, Thor Henrich, Sharon Hubbard, Graham Beard, and Pat Trask from the Vancouver Island Paleontological Society, the British Columbia Paleontological Alliance, and the Courtenay Museum for help with field collecting. This work was supported in part by grants from the National Science Foundation (NSF grant DGE-1314109 to B.A.A. and NSF grant EF-0629819 to G.W.R. and R.A.S.) and the Natural Sciences and Engineering Research Council of Canada (NSERC grant A-6908 to R.A.S.). 4 Author for correspondence (e-mail: [email protected])

doi:10.3732/ajb.1400369

et al., 2013). However, recent descriptions of several new Jurassic and Cretaceous fossil species (Escapa et al., 2010; Rothwell et al., 2011; Atkinson et al., 2014; Shi et al., 2014) and our growing knowledge of conifer regulatory genetics provide an opportunity to better clarify patterns of evolution in the Cupressaceae. In this paper, we describe an Early Cretaceous cupressaceous conifer as Hughmillerites vancouverensis sp. nov., providing an opportunity to expand our knowledge of the diversity of basal Cupressaceae as well as to improve our understanding of cupressaceous ovuliferous scale evolution. Coupled with recent data on the development of conifer cones, this study emphasizes the importance of coordinating paleobotany and regulatory genetics for deciphering the evolution of Cupressaceae. MATERIALS AND METHODS Hughmillerites vancouverensis is represented by two ovulate cones, one of which terminates a short segment of vegetative shoot. These specimens are preserved by cellular permineralization in calcium carbonate concretions derived from a carbonate-cemented graywacke matrix at the Apple Bay locality on northern Vancouver Island, British Columbia, Canada (Stockey et al., 2006). The locality is located on the beach along Quatsino Sound (50°36′21″N, 127°39′25″W; UTM 9U WG 951068) on northern Vancouver Island, British Columbia, Canada. Oxygen isotope analysis has dated the sediments to ca. 136 Ma (D. R. Gröcke, Durham University, UK, personal communication), which is in the Valanginian Stage of the Early Cretaceous (Cohen et al., 2013). The well-known cellulose acetate peel technique (Joy et al., 1956) was used to produce serial sections of the fossil specimens. Slides were prepared with Eukitt (O. Kindler GmbH, Freiburg, Germany) xylene-soluble mounting medium. Images were captured using a BetterLight digital scanning camera (Better Light, Placerville, California, USA) and processed using Adobe Photoshop 7.0 (San Jose, California, USA) and Pixelmator 2.0 (Pixelmator Team, Vilnius, Lithuania). The specimens are preserved in concretion numbers F-55439 and

American Journal of Botany 101(12): 2136–2147, 2014; http://www.amjbot.org/ © 2014 Botanical Society of America

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F-55440. Wafers, peels, and microscope slides of the specimens are housed in the Museum of Natural and Cultural History, Condon Collection, University of Oregon, Eugene, Oregon, USA.

SYSTEMATICS Order— Coniferales sensu Eckenwalder (2009). Family— Cupressaceae Bartlett. Genus— Hughmillerites Rothwell, Stockey, Mapes et Hilton (2011). Species—Hughmillerites vancouverensis—Atkinson, Rothwell, et Stockey sp. nov.; F-55439; Figs. 1–4, 6, 8–16, 20, and 22. Specific diagnosis— Cone-bearing vegetative shoot at least 2.25 mm diam.; pith parenchymatous; secondary xylem pycnoxylic; stem cortex parenchymatous with scattered sclereids. Leaves awl-shaped, up to 3.0 mm long, 1.3 mm wide, and 1.0 mm thick. Mesophyll parenchymatous with scattered thick-walled cells; vascular trace single, terete with single abaxial resin canal; transfusion tissue in horizontally elongated zone; hypodermis sclerotic, 3–4 cells thick. Seed cones 2.1 cm long, 2.0 cm wide, subspheroidal with numerous helically arranged bract/ scale complexes. Cone axis up to 7.5 mm diam.; pith parenchymatous; secondary xylem cylinder pycnoxylic, continuous. Cortex of cone axis parenchymatous with resin canals, isolated sclereids, and sclereid nests. Bract tips falcate. Bract trace cylindrical shaped at origin from cone axis. Ovuliferous scale trace diverging vertically from bract trace. Central abaxial resin canal of bract/scale complex branching from cortical system; additional resin canals arising de novo, branching infrequently. Seeds flattened, winged, ca. 2.4 mm long, ca. 1.0 thick, and ca. 2.0 mm wide; integument thin, and papillate. Holotype here designated—Leafy shoot with terminal seed cone in F-55439 A bot, B top, B bot, C top, C bot, D top in the Museum of Natural and Cultural History, Condon Collection, University of Oregon, Eugene, Oregon, USA. Paratype—Seed cone in F-55440 A bot and B top. Locality—Coastal exposures at Apple Bay on Vancouver Island, British Columbia, Canada (50°36′21″N,127°39′25″W; UTM 9U WG 951068). Stratigraphic position and age—Longarm Formation equivalent, Valanginian, Early Cretaceous. Etymology— The species is named for Vancouver Island, where the fossils were collected. RESULTS Description— General features—Hughmillerites vancouverensis is represented by two ovulate cones (Figs. 4–6), one of which is attached terminally to a leafy shoot fragment (Figs. 1, 2). Ovulate cones are subspheroidal, up to 2.1 cm long and 2.0 cm wide, and are composed of numerous helically arranged bract/scale complexes (Figs. 4–6). The attached leafy stem

Figs. 1–3. Hughmillerites vancouverensis sp. nov., holotype F-55439. 1. Cross section of vegetative shoot to which seed cone in Fig 4 is attached, showing general features of stem and helically arranged leaves. B top #83, ×3. 2. Enlargement of stem in Fig. 1 with ring of resin canals surrounding woody vascular cylinder. Note diverging leaves. B top #83, ×7. 3. Cross section of leaf attached to shoot in Fig. 1 showing sclerotic hypodermis, mesophyll, vascular bundle (arrow), band of transfusion tissue (T), and central abaxial resin canal (R). B top #68, ×104.

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Figs. 4–6. Hughmillerites vancouverensis sp. nov. 4. Oblique cross section of seed cone showing general features. Note similarity of bract tips to vegetative leaves. Holotype F-55439 C top #47, ×3. 5. Oblique section of seed cone near tip of axis, showing diverging bract/scale complexes, bract/scale complexes at several levels, and large winged seeds. F-55440 B top #47, ×8. 6. Oblique section of seed cone displaying continuous cylinder of wood, free scale tips (arrows), and diverging bract/scale complexes with elongated, apically directed bract tips (upper left). Note several seeds in positions of attachment. Holotype F-55439 B side #92, ×6.

Stem—The stem fragment has a central pith, 1.25 mm in diameter (Figs. 1, 2). Pith is composed of parenchyma cells 44–50 µm in diameter. The lumens of most pith cells are empty, but some ←

fragment is 2.0 cm long and ~2.25 mm in diameter (Figs. 1, 2), with helically arranged leaves (Figs. 1–3) up to 3.0 mm long, 1.3 mm wide, and 0.5–1.0 mm thick.

Figs. 7–12. Hughmillerites vancouverensis sp. nov. 7. Transverse section of bract/scale complex somewhat distal to level of seed attachment, displaying three adaxial ovuliferous scale tips. Note central scale tip attached to bract more apically positioned than lateral tips. Bract trace divided into row of bundles at this level. Note central and lateral abaxial resin canals (R), scattered fibers, and sclerotic hypodermis (bottom). F-55440 B top #46, ×67. 8. Longitudinal section of bract showing divergence of ovuliferous scale tip (at left, arrow), elongated apical tip turned upright at 90° (right). Holotype F-55439 B side #88, ×4. 9. Oblique longitudinal section of bract/scale complex showing adaxial free ovuliferous scale tip, and ovule oriented with micropyle (arrow) toward cone axis. Note sclerotestal differentiation at micropyle and adaxial resin canals (R) at level of scale tip divergences. Holotype F-55439 B side #88, ×25. 10. Cross section of bract tip with central abaxial resin canal (R) and sclerotic hypodermis. Holotype F-55439 C bot #87, ×25. 11. Cross section of bract tip at higher magnification showing histological features similar to vegetative leaves. Note position of vascular bundle (arrow). (R = resin canal; T = transfusion tissue). Holotype F-55439 C top #85, ×56. 12. Oblique section of bract/scale complex showing three ovules in position of attachment. F-55439 Holotype C top #86, ×28.

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contain dark contents (Fig. 2). The pith is surrounded by a continuous cylinder of wood, 0.25 mm thick and 2.25 mm in diameter (Fig. 2). Secondary xylem contains radial rows of tracheids, 12–16 µm in diameter, and uniseriate rays (Fig. 2). The stem cortex is approx. 0.75 mm thick and is mostly parenchymatous, with scattered sclereids and a conspicuous ring of resin canals (Figs. 1, 2), 0.13−0.18 mm in diameter. Surrounding the cortex is a sclerotic hypodermis up to four cell layers thick. There is little evidence of epidermis due to abrasion. Leaves—Leaves are awl-shaped, slightly falcate, triangular in cross section, with incurved acuminate apices and decurrent bases. As is characteristic of cunninghamioid conifers, leaves have a single terete vascular bundle and a single abaxial resin canal (Figs. 1, 3). Surrounding the vascular bundle, there is a laterally elongated distinct zone of small cells that we interpret to be transfusion tissue similar to that seen in extant Cunninghamia R. Brown (Fig. 3). However, no wall thickening patterns have been identified on these cells. Mesophyll is parenchymatous with scattered thick-walled cells (Fig. 3), and is surrounded by a sclerotic hypodermis of thick-walled cells, three to four cells thick and an incompletely preserved epidermis (Figs. 2, 3). Bract/scale complexes—Bract/scale complexes are up to 6 mm long and 3.5 mm wide. They diverge from the cone axis at about at 70°–90° (Fig. 6), bend distally (Figs. 6, 8), and taper rapidly to a long and narrow bract tip up to 5 mm long (Figs. 6, 8, 10). They are narrowly attached, expanding laterally to the midregion before bending distally and narrowing. Transverse sections of bract tips closely resemble leaves in shape and histology (cf., Figs. 3, 10, 11). Most of each bract/scale complex consists of bract. The ovuliferous scale is represented by three separate, free tips that are positioned approximately in the midregion on the adaxial surface of the complex (Figs. 6–9). Complexes have three adaxial ovules/seeds (Fig. 12), each of which is attached immediately proximal to a free scale tip (Fig. 9). Most ground tissue within bract/scale complexes is parenchymatous with scattered fibers (Fig. 7). There is a hypodermis of thick-walled, dark brown fibers near the abaxial surface of the complex (Figs. 7, 8). A similar hypodermis occurs on the adaxial surface (Figs. 8, 9, 12), but is not always present at the level of scale tip separation (Fig. 7). The hypodermis is two to three cell layers thick on the abaxial surface of the bract/scale complex, consisting of cells with dark internal contents (Figs. 7, 8). The adaxial hypodermis is less completely developed on some bract/scale complexes (Fig. 7) than others, and is made up of cells, which are lighter in color than those on the abaxial surface (Fig. 8). Vascular tissue diverges to the complex as a single ovalshaped bundle in cross section (Figs. 13, 17). Progressing dis-

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tally, the bundle expands laterally and divides into a row of several bundles (Figs. 7, 18). In the midregion of the bract/ scale complex a small bundle divides vertically (Fig. 18, at arrow) and extends toward the adaxial surface proximal to the position of seed attachment then terminates. Distally within the bract, the number of vascular bundles in the row diminishes (Fig. 7) until there is a single bundle in the tip (Figs. 10, 11, at arrow). Near the bract tip, the solitary vascular bundle occurs within a band of what appears to be transfusion tissue like that seen in vegetative leaves (Figs. 10, 11). The central resin canal of the bract/scale complex divides from a cortical resin canal in the cone axis. This resin canal is located abaxial to the vascular bundle (Fig. 17). Progressing distally, in the bract/scale complex, a small number of additional abaxial resin canals typically arise de novo (Figs. 7 [at right], 12). At about the level of seed attachment, three additional resin canals arise de novo adaxial to the vascular bundle (Fig. 9) with one terminating near the base of each ovuliferous scale tip. Cone axis—The cone axis is ca. 7.5 mm in diameter consisting of a parenchymatous pith, a cylinder of vascular tissue, and a largely parenchymatous cortex with a ring of resin canals (Fig. 13). As in the stem, the pith is ~3.0 mm in diameter, with a small percentage of cells containing dark contents (Fig. 13). Pith cells are 44–50 µm in diameter. The cylinder of wood (Fig. 13) is 1.0 mm thick, consisting of radially aligned rows of tracheids that are 14.0–19.5 µm in diameter, and uniseriate vascular rays. Pith rays are also uniseriate except above the divergence of bract/scale complex traces, where they are multiseriate. Protoxylem tracheids have helical secondary wall thickenings and secondary xylem tracheids show uniseriate circular bordered pits (Fig. 14). One specimen shows a zone of radially aligned thin-walled cells at the periphery of the secondary xylem that represents vascular cambium and a thin layer of secondary phloem (Fig. 16). The cortex is largely parenchymatous, but it contains both isolated sclereids and nests of thick-walled sclereids (Fig. 15). Within the cortex, there are also resin canals with epithelial linings of thin-walled cells (Figs. 13, 15). Epidermis is not preserved. Seeds—The three ovules/seeds that are borne on each scale proximal to the free scale tips occur within a shallow concavity on the adaxial surface of the complex (Figs. 9, 12). Interseminal ridges are typically absent, but they appear to occur on a small number of bract/scale complexes (Fig. 12). Seeds preserved within the cones are of two distinctly different size ranges and stages of maturation. Less mature ovules are up to 1.0 mm long, 0.5 mm wide, and 0.5 mm thick. In cross sections, ovules display 180° rotational symmetry, with narrow, lateral wings (Figs. 6, 12, 19). They are inverted with the micropyle facing the cone axis (Figs. 9, 20). More mature seeds

←

Figs. 13–18. Hughmillerites vancouverensis sp. nov. 13. Cross section of cone axis showing pith, continuous cylinder of wood, and cortex with ring of resin canals (R). Holotype F-55439 C top #86, ×4. 14. Radial longitudinal section of xylem showing secondary tracheids with uni-seriate circular bordered pits (left) and protoxylem tracheids with helical wall thickenings (right). Holotype F-55439 B side #174, ×1399. 15. Cortex of cone axis showing resin canals with epithelial lining (R), cortical parenchyma, isolated sclereids, and sclerotic nest. Holotype F-55439 C top #86, ×133. 16. Histology of cone axis showing secondary xylem (X), vascular cambium (VC), secondary phloem (P), and primary cortex (C) in Fig 12. Holotype F-55439 C top #86, ×350. 17. Transverse section of bract/scale complex near base, showing single vascular bundle above central resin canal (R). F-55440 B top #112, ×34. 18. Transverse section of bract/scale complex near attachment of seeds, showing horizontal divisions of vascular bundle and vertically diverging ovuliferous scale trace (arrow). Note large abaxial resin canal (R). F-55440 B top #112, ×96.

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(Fig. 5) are up to 2.5 mm long, 2.0 mm wide, and 1.0 mm thick. The integument is thin and papillate (Figs. 19–21), consisting of endotesta, a sclerotesta, and a thin sarcotesta (Fig. 21). The nucellus appears to be attached to the chalazal end only (Fig. 20). In most specimens, it consists of a zone of cells that surround a hollow center, but in a small number of specimens the nucellus contains a megaspore membrane (Fig. 19) and a megagametophyte with golden-colored cell walls (Figs. 20, 22). One specimen shows a cavity within the cellular megagametophyte, which probably represents the position of an embryo (Figs. 20, 22).

DISCUSSION Basal cupressaceous conifers usually have vegetative shoots with helically arranged, awl-shaped or sometimes scale-like leaves and terminal seed cones with numerous helically arranged bract/scale complexes (Farjon, 2005; Schulz and Stützel, 2007). Bract/scale complexes are composed mostly of bract; the bract and scale are largely or completely fused, with seeds attached to the adaxial surface. In contrast, the cupressoid Cupressaceae generally have oppositely arranged scale-like leaves at maturity, and seed cones have a small number of oppositely arranged diverging or highly fused bract/scale complexes (Gadek et al., 2000; Farjon, 2005). In the cupressoids, the ovuliferous scale is typically diminutive and often is initiated late in development (Jagel, 2001) such that the seeds appear to be borne in the axil of the bract (Takaso and Tomlinson, 1989b; Tomlinson et al., 1993; Farjon, 2005). Among the cupressoids, some cone scale complexes are fused to each other during development (e.g., Juniperus spp.), but many split apart at maturity (e.g., Callitris spp.). Unlike the cupressoid Cupressaceae, Hughmillerites vancouverensis possesses vegetative shoots with helically arranged awl-shaped leaves, terminal seed cones with many helically arranged bract/scale complexes, three free scale tips, and seeds borne on the adaxial surfaces of scales. Due to this combination of characters, it is appropriate to assign H. vancouverensis to the basal Cupressaceae. The most basal members of extant Cupressaceae (i.e., Cunninghamia, Taiwania Hayata, and Athrotaxis D. Don) have bract/scale complexes with small adaxial ovuliferous scales that may or may not have a free tip at maturity (Stewart and Rothwell, 1993; Jagel, 2001; Jagel and Stützel, 2001; Schulz and Stützel, 2007). Other basal cupressaceous genera such as Taxodium Rich., Cryptomeria D. Don, and Glyptostrobus Endl. (Subfamily: Taxodioideae sensu Gadek et al., 2000) produce mature seed cones in which the scale is comparable in size to the bract, and the seeds are erect (Takaso and Tomlinson, 1989a, 1990; Schulz and Stützel, 2007). Bract/scale complexes of the Sequoioideae (sensu Gadek et al., 2000), Sequoiadendron Buchholz, Sequoia Endl., and Metasequoia Miki, have peltate complexes and do not display a conspicuous ovuliferous scale (Takaso and Tomlinson, 1992; Jagel, 2001; Schulz and Stützel, 2007). Among living Cupressaceae, H. vancouverensis is most similar to the basal members of Cupressaceae (sensu Gadek et al., 2000; Mao et al., 2012), Cunninghamia, Taiwania, and Athrotaxis, in having terminal seed cones with numerous separate helically arranged bract/scale complexes (Jagel, 2001; Schulz

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and Stützel, 2007). Most of the complex is composed of bract tissue and the ovuliferous scale is diminutive. Athrotaxis cupressoides D. Don and A. latifolia Hook. differ from H. vancouverensis by having leaves that are appressed to the stem (Farjon, 2005). Unlike H. vancouverensis, Athrotaxis has an ovuliferous scale that lacks a thin free tip; however, there is a conspicuous swelling slightly distal to the attachment of seeds, and each scale has 3–6 seeds per scale (Jagel, 2001) (Table 1). The monotypic genus Taiwania differs from Hughmillerites vancouverensis in lacking a free ovuliferous scale tip at maturity (Liu and Su, 1983; Farjon and Ortiz García, 2003), while H. vancouverensis has three free scale tips. Taiwania has 1–2 seeds per scale (Farjon and Ortiz García, 2003; Schulz and Stützel, 2007), while H. vancouverensis consistently has three (Table 1). Resin canals within the bracts of Taiwania branch, whereas in H. vancouverensis, the lateral resin canals arise de novo. The extant cupressaceous conifers most similar to H. vancouverensis are among the species of Cunninghamia. Cunninghamia is the only extant genus within the subfamily Cunninghamioideae (Gadek et al., 2000) and is represented by the two living species C. lanceolata (Lambert) Hooker and C. konishii Hayata. Two permineralized Late Cretaceous species of Cunninghamia, C. taylorii Serbet, Bomfleur et Rothwell (2013) and C. hornbyensis Brink, Stockey, Beard et Wehr (2009) have been described from central Alberta, Canada and Hornby Island, British Columbia, Canada, respectively. It is evident that Cunninghamia was well established by the Late Cretaceous. In addition, there are several reports of Cenozoic Cunninghamia that have been described based on compression and impression fossils from North America, Europe, and Asia (e.g., Miki, 1941; Lakhanpal, 1958; Szafer, 1958; Tanai and Onoe, 1961; Matsuo, 1967, 1970; Givulescu, 1972; Kimura and Horiuchi, 1978; Meng et al., 1988; Walther, 1989; Dillhoff et al., 2005, 2013; Dolezych and Schneider, 2007; Du et al., 2012; Yabe and Yamakawa, 2012). Hughmillerites can be distinguished from living Cunninghamia by several characters. The bract/scale complexes of Cunninghamia have an ovuliferous scale that is a single structure with a trilobed free tip (Farjon and Ortiz García, 2003; Serbet et al., 2013; Shi et al., 2014), while in Hughmillerites vancouverensis the scale is represented by three free scale lobes that separate individually from the fused bract/scale complex. Also, interseminal ridges like those that occur between the seeds in H. vancouverensis are absent from species of Cunninghamia. Resin canals within bract/scale complexes of Cunninghamia branch and form a continuous system, while the lateral bract canals of H. vancouverenis are typically unbranched and new resin canals within the bract/scale complex originate de novo (Table 1). Although H. vancouverensis differs from Cunninghamia by several characters, it can be assigned to the Cunninghamioideae by having helically arranged, awl-shaped leaves with acuminate tips and seed cones that have more than two inverted seeds/ ovules per scale (Page, 1990; Gadek et al., 2000). Several previously described permineralized Mesozoic and Cenozoic conifers are assignable to the Cunninghamioideae (Stopes and Fujii, 1910; Miller, 1975; Miller and Crabtree, 1989; Nishida et al., 1992; Saiki and Kimura, 1993; Brink et al., 2009; Rothwell et al., 2011; Atkinson et al., 2014). One of these conifers is Mikasastrobus hokkaidoensis Saiki et Kimura from the Upper Cretaceous of Japan. Unlike Hughmillerites vancouverensis, the bract/

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Figs. 19–22. Hughmillerites vancouverensis sp. nov. 19. Cross section of large seed with lateral wings and well-developed integument. Note megaspore membrane (m) toward center and shrunken nucellus (n). F-55440 B top #37, ×42. 20. Oblique longitudinal section of large seed showing attachment of nucellus at chalaza, and cellular megagametophyte (mg). Note cavity toward center that may represent embryo (not preserved). Holotype F-55439 B side #119, ×34. 21. Cross section of mature seed showing histology of integument. Note papillate epidermis. F-55440 B top #30, ×380. 22. Oblique longitudinal section of large seed in Fig. 20 at high magnification showing megagametophyte (mg), nucellus (n), and embryo cavity. Holotype F-55439 B side #119, ×67.

scale complexes of Mikasastrobus have a single free ovuliferous scale tip (Saiki and Kimura, 1993). In addition, there are 4–5 seeds per scale in Mikasastrobus (Saiki and Kimura, 1993;

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Table 1), whereas H. vancouverenis has only three. Also, Mikasastrobus lacks interseminal ridges like those of H. vancouverenis. Parataiwania nihongii M. Nishida, Ohsawa et Nishida is a Late Cretaceous conifer with ovuliferous scales represented by a single free tip (Nishida et al., 1992), which distinguishes it from Hughmillerites vancouverensis (i.e., with three free scale tips). Other differences between P. nihongii and H. vancouverensis are seed number (P. nihongii has four seeds per scale; H. vancouverensis has three; Table 1) and interseminal ridges, which are absent in P. nihongii (Nishida et al., 1992) and present in H. vancouverensis. Moreover, there are three resin canals at the origin of the bract/scale complex of P. nihongii, whereas H. vancouverensis has a single resin canal at that level. The fossil genus Cunninghamiostrobus includes two species from the Late Cretaceous, C. hueberi Miller (1975) and C. yubariensis Stopes & Fujii, (1910; Ogura, 1930; Ohana and Kimura, 1995), and one species from the Oligocene, C. goedertii Miller and Crabtree (1989; Miller, 1990). Similar to Hughmillerites, each species of Cunninghamiostrobus has three seeds per scale (Table 1). However, Cunninghamiostrobus hueberi and C. goedertii have one free ovuliferous scale tip and C. yubariensis lacks a conspicuous ovuliferous scale tip (Stopes and Fujii, 1910; Ohana and Kimura, 1995), as opposed to the three free tips seen in H. vancouverensis. All three species of Cunninghamiostrobus have up to three resin canals at the origin of the bract/scale complex, while H. vancouverensis has only one. Also, the resin canals branch within the bract/scale complex in Cunninghamiostrobus, while additional resin canals typically arise de novo in H. vancouverensis. The two previously described Cretaceous species of Cunninghamia differ from Hughmillerites vancouverensis in several ways. Cunninghamia hornbyensis is an anatomically preserved Late Cretaceous conifer that was described from vegetative leafy shoots (Brink et al., 2009). Similar to Hughmillerites vancouverensis, the leaves of C. hornbyensis have a single vascular bundle, central resin canal, and a horizontally elongated zone of transfusion tissue within the mesophyll (Brink et al., 2009). However, C. hornbyensis leaves are much larger than the leaves of H. vancouverensis. The number of leaf resin canals in C. hornbyensis ranges from one to five, while H. vancouverensis consistently has one resin canal. Resin canals can also be found adaxially in the mesophyll of C. hornbyensis (Brink et al., 2009), while leaves of H. vancouverensis lack adaxial resin canals. Cunninghamia taylorii has ovulate cones with bract/scale complexes that consist of a large bract and a single free ovuliferous scale tip (Serbet et al., 2013), while the ovuliferous scales of Hughmillerites vancouverensis have three conspicuous, free scale tips. There is a branching resin canal system within the bracts of C. taylorii (Table 1), which differs from H. vancouverensis where additional resin canals arise de novo within the bract. Bract resin canals adaxial to vascular strands and interseminal ridges like those of H. vancouverensis are absent from C. taylorii. Hubbardiastrobus cunninghamioides Atkinson, Rothwell, et Stockey (2014) is a cunninghamioid collected from the same Early Cretaceous locality as Hughmillerites vancouverensis. These two conifers are similar in that they both have three free scale tips, three seeds per scale, and one resin canal at the origin of the bract/scale complex (Table 1). However, cone shape is cylindrical in Hubbardiastrobus, while in Hughmil-

Cone shape

Size (cm) Present Present Absent Absent Absent Absent Absent Absent Absent Absent

3 3 3 3 3 4 3 3 3 3–6

0

1

1

1

1

0

1

3

3

3

Present

Present

Present

Present

Absent

Present

Present

Absent

Absent

Absent

1

1

3

1

3

3

3

1

1

1

Cone axis

Cone axis

Cone axis

Cone axis

Cone axis

Cone axis

Cone axis

Cone axis

Cone axis

De novo

3

1

3

1

1

3

3

1

1

1

Present

Present

Present

Present

Absent

Present

Present

Absent

Absent

Absent

Present

Absent

Present

Absent

Absent

Present

Present

Absent

Present

Present

Source notes: 1Rothwell et al., 2011; 2Atkinson et al., 2014; 3Miller, 1975; 4Ohana and Kimura, 1995; 5Nishida et al., 1992; 6Serbet et al., 2013; 7Miller, 1990; 8Farjon, 2005; 9Radais, 1894. a Presence or absence of adaxial resin canals in transverse section at a level where seeds are attached. Boldface indicates similarity to Hughmillerites vancouverensis.

Late Jurassic Ovoid 3.0-4.3 x 2.2-3.6 Except Hughmillerites at tip juddii1 Hughmillerites Early Subspheroidal 2.1 x 2.0 Except vancouverensis Cretaceous at tip Early Cylindrical 1.3-1.5 x 0.4 Except Hubbardiastrobus at tip cunninghamioides2 Cretaceous Ovoid 3.0-3.5 x 2.5 Except Cunninghamiostrobus Cretaceous at tip hueberi3 Cylindrical 6.5 x 2.5 Throughout Cunninghamiostrobus Cretaceous yubariensis4 Cretaceous Ellipsoidal 2.2 x 1.6 Except Parataiwania at tip nihongii5 Late Ovoid 3 x 2.3 Except Cunninghamia Cretaceous at tip taylorii6 Ovoid 4.0 x 3.0-4.5 Except Cunninghamiostrobus Oligocene at tip goedertii7 Extant Ovoid 2.5-4.0 x 2.5-3.5 Except Cunninghamia at tip lanceolata1, 6, 8 Extant Ovoid 1.5-3.0 x 1.5-3.0 Throughout Athrotaxis selaginoides9

Age

First No. of No. of No. of division of resin canals resin canals Resin canals No. of free tipsof bract/scale at origin to Bract/scale with bract Resin canal adaxial Bract/scale seeds/ Interseminal ovuliferous complex bract/scale resin canal trace at branching to vascular fusion scale ridge scale trace vertical complex origin divergence in bract strandsa

Seed cone characters of basal Cupressaceae (modified from Atkinson et al., 2014).

Character Taxon

TABLE 1.

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lerites vancouverensis cones are subspheroidal. Hubbardiastrobus also lacks interseminal ridges (Atkinson et al., 2014) like those of Hughmillerites vancouverensis. Yet another difference between Hubbardiastrobus and Hughmillerites is the prescence of adaxial resin canals within the bract/scale complexes of Hughmillerites, which are absent from Hubbardiastrobus complexes. Monotypic Hughmillerites, i.e., H. juddii (Seward et Bancroft) Rothwell, Stockey, Mapes et Hilton (2011) was described from the Late Jurassic of Scotland. In agreement with Hughmillerites vancouverensis, H. juddii has three free ovuliferous scale tips, three seeds per scale, interseminal ridges, one resin canal at the divergence of the bract/scale complex, and adaxial resin canals in ovuliferous scale tissue (Table 1). Several features (Table 1) that distinguish the two species of Hughmillerites are (1) the cone shape of H. juddii is ovoid, while in H. vancouverensis it is subspheroidal, (2) seed cones of H. juddii are larger than those of H. vancouverensis, (3) bract tips of H. vancouverensis are much more elongated than those of H. juddii, and H. juddii has a single large abaxial resin canal within the bract (Rothwell et al., 2011), while H. vancouverensis has a relatively small central resin canal with occasional lateral resin canals that originate de novo. Furthermore, the bract/ scale complex central resin canal of H. juddii arises de novo at the base of the complex, while the central resin canal of H. vancouverensis branches from the axial resin canal system (Table 1). From these comparisons (Table 1), it is clear that Hughmillerites vancouverensis represents a new species of Hughmillerites. This newly described conifer increases the diversity of Cupressaceae during the Cretaceous due to its unique combination of characters (Table 1). Hughmillerites vancouverensis also extends the stratigraphic and geographic ranges of the genus from the Late Jurassic of eastern Scotland to the Early Cretaceous of western North America. Initial diversification and evolution of Cupressaceae—The Cunninghamioideae is the earliest-diverging group within the Cupressaceae (Gadek et al., 2000; Farjon, 2005; Shi et al., 2014). A recent phylogenetic analysis that included living and fossil Cupressaceae found that Cunninghamia and Cretaceous Cunninghamia-like fossils form a monophyletic group, thus expanding the Cunninghamioideae to include extinct species (Shi et al., 2014). Furthermore, this analysis supports the hypothesis that the Cunninghamioideae played a major role in the initial diversification of Cupressaceae. Recent paleontological discoveries have demonstrated that the bulk of diversity of the cunninghamioids occurred in the Cretaceous (Brink et al., 2009; Rothwell et al., 2011; Serbet et al., 2013; Atkinson et al., 2014; Shi et al., 2014), suggesting that Cupressaceae initially diversified during the Late Jurassic and Cretaceous and was composed largely of cunninghamioid species. The earliest cunninghamioids are known from the Jurassic and Early Cretaceous. Many of those plants (Hughmillerites juddii, Hughmillerites vancouverensis, and Hubbardiastrobus cunninghamioides) have ovuliferous scales with three free scale tips (Rothwell et al., 2011; Atkinson et al., 2014). This morphology is reminiscent of living Cunninghamia at the onset of ovuliferous scale development; however, as the scale matures, tips fuse with one another proximal to seed attachment and form a single three-lobed structure (Farjon and Ortiz García, 2003). Therefore, it is possible that other extinct cunninghamioids with one scale tip may have had three free tips early in

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development as well. In the extant cunninghamioids, the remaining portion of the ovuliferous scale is considered to be continuous with the bract from the base of the complex up toward the scale tips (Schulz and Stützel, 2007). In other words, most of the ovuliferous scale is confluent with the base of the bract. The occurrence of free scale tips is also seen in the pinaceous conifer mutant Picea abies var. acrocona. Instead of a unified scale that is typical of the wild-type P. abies, P. abies var. acrocona possesses a mutation in which the ovuliferous scale resembles a small shoot with 2–3 slightly fused tips that typically bear ovules (Carlsbecker et al., 2013). Morphological and genetic analysis of P. abies var. acrocona seed cones found that ovuliferous scales demonstrated normal gene expression except that they showed an absence of an AGAMOUS-LIKE6/SEPALLATA homologue, DAL14, normally expressed in the wild type (Carlsbecker et al., 2013). It is possible that the lack of this homologue may be linked to a suppression of determinate development of ovuliferous scales in P. abies var. acrocona seed cones, thus allowing for the development of multiple fertile scales at their apices (Carlsbecker et al., 2013; Ruelens and Geuten, 2013). Since all extant conifers probably share a common ancestor (Rai et al., 2008), it is logical to hypothesize that a DAL14 homologue could play an important role in the evolution/ reduction of the ovuliferous scale in Cupressaceae (and other conifer families) as well as in Pinaceae (Carlsbecker et al., 2013; Ruelens and Geuten, 2013). Rothwell et al. (2011) proposed a transformational series that depicts the ovuliferous scale evolution and reduction from Voltzialean ancestors to living Cupressaceae. Within this transformational series, cupressaceous conifers such as Hughmillerites and Hubbardiastrobus have an intermediate ovuliferous scale morphology. Conifers with reduced ovuliferous scales similar to Taiwania have ovuliferous scales that are highly reduced in which they lack free scale tips (Rothwell et al., 2011). It is possible that the progressive reduction of the ovuliferous scale observed throughout the evolution of Cupressaceae is a response to the increasing upregulation during determinate development caused by a DAL14 homologue, thus terminating growth early in development. Along with a great deal of confluence between the bract and ovuliferous scale, this heterochronic hypothesis explains the lack of conspicuous scales and the superficial simple cone appearance of many cupressaceous species. However, more genetic studies are needed to test this hypothesis and to determine more specific functions of genes in conifer reproductive organ development (Carlsbecker et al., 2013; Ruelens and Geuten, 2013). The description of Hughmillerites vancouverensis sp. nov. extends the occurrence of Hughmillerites from the Late Jurassic to the Early Cretaceous and also expands the diversity of Cretaceous cunninghamioids. The addition of this extinct species emphasizes the growing importance of Cunninghamioideae with respect to the initial diversification of Cupressaceae. The growing number of recent discoveries from developmental and genetic studies now enables us to hypothesize links between gene regulation and evolutionary changes in the morphology of cupressaceous seed cones. As a result, we are optimistic that the continued interplay between developmental genetics and paleontology can ultimately resolve not only the pattern of evolution within the Cupressaceae, but also the regulatory mechanisms that underlie those evolutionary changes.

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