Anatomically preserved fossil cornalean fruits from the Upper Cretaceous of Hokkaido: Eydeia hokkaidoensis gen. et sp. nov. 1

RESEARCH ARTICLE AMERICAN JOURNAL OF BOTANY Anatomically preserved fossil cornalean fruits from the Upper Cretaceous of Hokkaido: Eydeia hokkaidoensis gen. et sp. nov. 1 Ruth A. Stockey 2,4, Harufumi Nishida3, and Brian A. Atkinson 2 PREMISE OF THE STUDY: The basal asterid clade Cornales radiated during the Late Cretaceous. However, our understanding of early evolutionary patterns and relationships remain obscure. New data from five permineralized fruits in calcareous concretions from the Upper Cretaceous (Coniacian-Santonian) Haborogawa Formation, Hokkaido, Japan provide anatomical details that aid our knowledge of the group. METHODS: Specimens were studied from cellulose acetate peels, and three-dimensional reconstructions were rendered using AVIZO. KEY RESULTS: Fruits are drupaceous, roughly pyriform, 2.9 4.3 mm in diameter, with a fleshy mesocarp, transition, and a stony endocarp of four to five locules, with the septa forming a cross or star-like pattern in transverse section, distinct germination valves, and one apically attached anatropous seed per locule. Vascular tissue occurs in zones between the mesocarp and exocarp, in two rows within the septa, and prominent seed bundles can be traced throughout the fruit sections. Seeds have a single integumentary layer of radially flattened square to rectangular cells and copious cellular endosperm. A fully formed, straight, cellular dicotyledonous embryo, with closely appressed, spathulate cotyledons, is present within each seed. CONCLUSIONS: The unique combination of characters shown by these fruits is found in Cornaceae, Curtisiaceae, and Davidiaceae and allows us to describe a new taxon of Cornales, Eydeia hokkaidoensis gen. et sp. nov., with many similarities to extant Davidia involucrata. These fossils underscore the phylogenetic diversification of Cornales that was underway during the Late Cretaceous and support the hypothesis that a Davidia -like fruit morphology is plesiomorphic within Cornales. KEY WORDS Cornales; Cornus ; Cretaceous; Curtisia ; Davidia ; fossil fruits; Hokkaido; Hironoia The asterids comprise one of the most taxonomically and morphologically diverse clades of flowering plants ( Bremer, et al., 2004 ; Martínez-Millán, 2010 ; Friis et al., 2011 ). Early evolution of this clade is not well understood due to the lack of resolution in molecular phylogenies of the early-diverging lineages, Cornales and Ericales ( Xiang et al., 1998, 2011 ; Fan and Xiang, 2003 ; Schönenberger et al., 2005, 2010 ). This low resolution may be due to rapid initial diversifications of these orders. The earliest-diverging asterid order, Cornales, is currently recognized to contain 10 families (Cornaceae, Alangiaceae, Nyssaceae, Davidiaceae, Mastixiaceae, Curtisiaceae, Grubbiaceae, Hydrangeaceae, Loasaceae, and Hydrostachyaceae) and five clades in the most recent comprehensive treatment ( Xiang et al., 2011 ). 1 Manuscript received 2 April 2016; revision accepted 7 July 2016. 2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331 USA; and 3 Department of Biological Sciences, Chuo University, Tokyo, Japan 4 Author for correspondence (e-mail: stockeyr@science.oregonstate.edu) doi:10.3732/ajb.1600151 The Cornales are thought to have originated during the middle to Late Cretaceous according to the fossil record ( Knobloch and Mai, 1986 ; Takahashi et al., 2002 ; Friis et al., 2011 ; Atkinson, 2016 ) and molecular dating ( Schenk and Hufford, 2010 ; Xiang et al., 2011 ). Their rapid radiation during the Late Cretaceous, and lack of fossils of Cretaceous age, have limited our understanding of the evolutionary trends and relationships within the order. Fruits are by far the most taxonomically informative fossils in Cornales (Eyde, 1963, 1988 ; Atkinson, 2016 ). The oldest cornalean fossils are fruits from the Coniacian (Upper Cretaceous) of Japan ( Takahashi et al., 2002 ). Hironoia fusiformis Takahashi, Crane & Manchester (2002) was originally assigned to the nyssoids because of the presence of fibers in the endocarp. Other Late Cretaceous cornalean fruits include Suciacarpa starrii Atkinson (2016) described from permineralized fruits from the Campanian of western North America. In addition, Knobloch and Mai (1986) described several taxa, with elongate germination valves and a median infold, that they included in the mastixioids from the Maastrichtian (Upper Cretaceous) of central Europe. 1642 AMERICAN JOURNAL OF BOTANY 103 (9): 1642 1656, 2016; http://www.amjbot.org/ 2016 Botanical Society of America

SEPTEMBER 2016, VOLUME 103 STOCKEY ET AL. EYDEIA HOKKAIDOENSIS GEN. ET SP. NOV. 1643 In this paper, we describe a new genus and species of Santonian (Late Cretaceous) Cornales, Eydeia hokkaidoensis Stockey, Nishida & Atkinson based on permineralized fruits from four localities in the Tomamae area, Hokkaido, Japan. These are some of the most well-preserved Cretaceous cornalean fruits, including not only fleshy mesocarp (usually not preserved in fossil fruits), but seeds with embryos and cellular endosperm. These fruits are compared with those of other living and fossil Cornales and show closest similarities to fruits of Davidiaceae. Photoshop (Adobe, San Jose, California, USA). Three-dimensional reconstructions were rendered using AVIZO visualization software (TGS Software, San Diego, California, USA). Microscope slides are housed in the Paleobotanical Collections, Department of Biological Sciences, Chuo University, Tokyo, Japan. RESULTS Systematics Order: Cornales Dumortier sensu Xiang et al. (2011). MATERIALS AND METHODS Five specimens of a small cornalean fruit have been identified in calcareous concretions from Hokkaido, Japan ( Table 1 ). Material comes from four different localities ( Fig. 1 ) in the Upper Cretaceous (Coniacian-Santonian) Haborogawa Formation ( Fig. 2 ), Yezo Group ( Table 1 ) in the Tomamae and Obira areas ( Tanaka, 1963 ; Moriya and Hirano, 2001 ; Okamoto et al., 2003 ; Takashima et al., 2004 ). The Haborogawa Formation overlies the Saku and Mikasa formations and is the synchronous shallow water facies of the Kashima Formation ( Takashima et al., 2004 ). The paleodepth is considered to be outer shelf ( Sliter and Baker, 1972 ; Takashima et al., 2004 ). Ages are determined based on ammonites and inoceramids embedded along with the fossil plants. Most specimens come from the Santonian (83.6 86.3 Ma) portion of the formation ( Table 1 ). Even though specimen 851051 comes from a locality where Coniacian-Santonian deposits crop out, this specimen was most likely collected from the Santonian portion of the section. Therefore, we assigned a minimum age to these fruits of 84 Ma. Permineralized plants have been known from Hokkaido since the original descriptions of Reiss (1907) and Stopes and Fujii (1911). Additional studies by Ogura (1930, 1932 ) brought more of this excellently preserved material to light. More recently, the permineralized floras were summarized by Nishida (1991). The fruits described in this paper, from the Tomamae area, are associated with conifer leaves of Pinus hokkaidoensis Stockey & Ueda (1986), Pinus haboroensis Stockey & Nishida (1986), and cone scales of Araucaria nipponensis Stockey, H. Nishida & M. Nishida (1994) as well as other conifer remains. The calcium carbonate concretions were prepared for study by cutting them into slabs on a water-cooled saw followed by the cellulose acetate peel technique ( Joy et al., 1956 ). The peels were mounted on microscope slides using the xylene-soluble mounting medium Eukitt (O. Kindler GmbH, Freiburg, Germany) or Entellan (EMD Millipore Corp., Darmstadt, Germany). Images were captured by a Better Light digital scanning camera (Better Light, Placerville, California, USA) using transmitted light, focused through a Leitz, Aristophot large-format camera using either Summar lenses or a Zeiss WL compound microscope, and processed using Adobe TABLE 1. Eydeia hokkaidoensis gen. et sp. nov., collection data. Specimen Locality Age Collector(s) 840208a Riverbed, Sankebetsu River Santonian a H. Nishida & M. Nishida 840208b Riverbed, Sankebetsu River Santonian a H. Nishida & M. Nishida 832489 Minorizawa, tributary of Sankebetsu River Santonian a H. Nishida & M. Nishida 851051 Takenokozawa, tributary of Nakafutamata River Coniacian-Santonian b R.A. Stockey, H. Nishida, & M. Nishida 960051 Gakkonosawa, tributary of Obirashibe River Santonian c T. Ohsawa Asakawa a Moriya and Hirano (2001) ; b Okamoto et al. (2003) ; c Tanaka (1963). Genus Eydeia Stockey, Nishida & Atkinson gen. nov. Generic diagnosis Fruit drupaceous, four- to five-loculate. Locules subtriangular to ellipsoidal in cross section. Valves elongate, composed of isodiametric. Septum with elongate and isodiametric. Mesocarp fleshy, parenchymatous. Endocarp vasculature in two rows within septa; vascular bundles small, without surrounding fibers. Seeds, one per locule, anatropous, apically attached, seed bundle prominent. Endosperm cellular, copious. Embryos with two, spathulate cotyledons. Species Eydeia hokkaidoensis Stockey, Nishida & Atkinson sp. nov. Specific diagnosis Fruits pyriform, without apical depression, stylar canal four- to five-armed. Septal ridges and ridges on valve present; septal ridges more pronounced. Central axis with elongate, large diameter in center. Transition, one to three layers, between endocarp and mesocarp. Mesocarp lacking, with outer zone of radially elongate parenchymatous cells, inner zone of smaller cells. At least one vascular bundle parallel to each germination valve ridge between mesocarp and endocarp. Locule lining of one to two rows of longitudinally elongate fibers, square to rectangular in cross section. Seed coat uniseriate with prominent cuticle. Endosperm with copious contents. Cotyledons of embryo flat, closely appressed, with three vascular bundles. Holotype hic designatus: 840208 C bot a (Chuo University) ( Figs. 3, 8 12, 14 16, 21 31, 33 35 ). Paratypes 840208 C bot b, 832489, 851051, 960051 (Chuo University). Locality Sankebetsugawa (Sankebetsu River bed), Tomamae area, Hokkaido, Japan. (44 20 N, 141 55 E). Stratigraphic position and age Haborogawa Formation, Santonian, Late Cretaceous. Etymology This genus is named in honor of the late Dr. Richard H. Eyde, for his numerous contributions to our understanding of the biogeography, morphology, anatomy, and evolution of Cornales that have served as the foundation for all further research in studies of fossil and living taxa. The specific epithet refers the island of Hokkaido where these specimens were found. Description Five small, drupaceous, fossil fruits of the same type have

1644 A M E R I C A N J O U R N A L O F B OTA N Y FIGURES 1 AND 2 1. Collecting localities in the Santonian of the Tomamae area, Hokkaido, Japan (modified from Takashima et al., 2004). 1 = Sankebetsugawa, 2 = Minorizawa, 3 = Takenokozawa, 4 = Gakkonosawa. 2. Stratigraphic chart showing age and formation of specimen origins (asterisk) (modified from Takashima et al., 2004). been identified in four separate concretions (Figs. 3 7), from four localities in the Haborogawa Formation (Figs. 1, 2). Three of the specimens are four-loculed (Figs. 3, 6, 7), while two are five-loculed (Figs. 4, 5). Each locule has a germination valve (Figs. 3 7). Fruits are roughly pyriform in shape, 2.9 4.3 mm in diameter; however, none of these are complete, as parts have been lost in the saw cut. All of the specimens were completely peeled, and the two best preserved were reconstructed using AVIZO. Specimen 840208 C bot a (the holotype) is the basal two thirds to three quarters of a pyriform fruit (Figs. 30, 31), while paratype specimen 851051 (Fig. 7) represents the apical third of a fruit (Fig. 32). Preservation is somewhat variable with one specimen (Fig. 6) showing a dark coloration due to the presence of numerous fungi. Fruit wall Fruits have a fleshy mesocarp, composed of large par- enchymatous cells (Figs. 3, 5, 7, 8), that are abraded away to varying degrees among the specimens available. Exocarp or epidermis is missing on all the fruits (Figs. 3, 7, 11). The inner cells of the mesocarp (60 76 μm) are smaller than those toward the outside (150 270 μm) that are radially elongated (Figs. 3, 5, 8, 21). It is obvious that a portion of the mesocarp has been lost, either by abrasion and/ or bacterial degradation since a zone of pyrite occurs outside of the

S E P T E M B E R 2016, V O LU M E 103 S TO C K E Y E T A L. E Y D E I A H O K K A I D O E N S I S G E N. E T S P. N O V. 1645 FIGURES 3 7 Eydeia hokkaidoensis gen. et sp. nov. fruits. 3. Cross section of four-loculed fruit showing fleshy mesocarp, stony endocarp, four germination valves, and mature seeds with embryos. Holotype 840208 C bot #22a. 29. 4. Oblique cross section of five-loculed fruit endocarp. Paratype 832489 E2 top #34. 29. 5. Cross section of five-loculed fruit with small amount of mesocarp. Paratype 960051 #1. 26. 6. Cross section of four-loculed fruit with one preserved seed. Darker color due to fungal infection. Paratype 840208 C bot #191b. 23. 7. Cross section of four-loculed fruit with patches of mesocarp showing four seeds. Paratype 851051 #9. 22.

1646 A M E R I C A N J O U R N A L O F B OTA N Y FIGURES 8 11 Eydeia hokkaidoensis gen. et sp. nov. fruits. 8. Cross section of one locule showing parenchymatous mesocarp, germination valve, septum, and single seed with endosperm and embryo. Note separation of tissues for valve dehiscence. Holotype 840208 C bot #22a. 45. 9. Cross section of septum toward fruit periphery with germination valves on each side. Note distinct sclerenchyma cells of locule lining. Holotype 840208 C bot #22a. 74. 10. Cross section of fruit axis (septum) showing elongated with large lumens, near center; four vascular bundles, one to each seed. Holotype 840208 C bot #22a. 79. 11. Transverse section of one locule containing seed with endosperm and embryo with two cotyledons. Note transition between endocarp and mesocarp. Holotype 840208 C bot #22a. 107.

S E P T E M B E R 2016, V O LU M E 103 S TO C K E Y E T A L. E Y D E I A H O K K A I D O E N S I S G E N. E T S P. N O V. 1647 FIGURES 12 20 Eydeia hokkaidoensis gen. et sp. nov. fruits. 12. Transverse section of fruit near base showing dark ring of vascular tissue and several diverging traces into mesocarp. Holotype 840208 C bot #184. 43. 13. Slightly oblique transverse section of fruit near apex showing lobing of endocarp (septal ridge, at right), four-lobed stylar canal, and vascular tissue (dark bands). 851051 #45. 33. 14. Transverse section of seed vascular bundle. Holotype 840208 C bot #22a. 110. 15. Transverse section of seed vascular bundle. Holotype 840208 C bot #22a. 110. 16. Transverse section of seed vascular bundle. Note seed with endosperm tissue at top. Holotype 840208 C bot #4. 110. 17. Longitudinal section of fruit showing apical seed attachment. 832489 E2 top #0. 116. 18. Paradermal section of seed integument showing square to rectangular cell outlines. 851051 #30. 166. 19. Longitudinal section of fruit showing seed vasculature. 832489 E2 top #82. 97. 20. Longitudinal section of seed vasculature showing helical to scalariform tracheary elements. 832489 E2 top #82. 500.

1648 A M E R I C A N J O U R N A L O F B OTA N Y FIGURES 21 23 Eydeia hokkaidoensis gen. et sp. nov. fruit, transverse section showing vascular system in four-loculed fruit. All Holotype 840208 C bot #94a. 40. 21. Fruit with some endocarp bundles. 33. 22. Interpretive diagram of fruit tissues. Red = mesocarp; flesh colored = germination valve; dark blue = septum; green = endosperm; light blue = embryo; yellow = vascular tissue. 19.4. 23. Close-up of vascular bundles in septum. Note flat nature of cotyledons in cross section. 40.

S E P T E M B E R 2016, V O LU M E 103 S TO C K E Y E T A L. E Y D E I A H O K K A I D O E N S I S G E N. E T S P. N O V. 1649 FIGURES 24 27 Eydeia hokkaidoensis gen. et sp. nov. fruit. All Holotype 840208. 24. Transverse section of seed showing hypocotyls of embryo and cellular endosperm best preserved in outer layers. Note distinct cells of locule lining. C bot #11a. 104. 25. Transverse section of cellular embryo in region of hypocotyl. C bot #11a. 211. 26. Transverse section of embryo near cotyledonary node. C bot #51a. 161. 27. Transverse section of embryo showing two flat cotyledons with at least three vascular bundles (top). C bot #52a. 105.

1650 AMERICAN JOURNAL OF BOTANY radially elongated cells ( Figs. 3, 21 ). A distinct zone of transition (sensu Morozowska et al., 2013 ), one to three cells thick, can be identified in some sections between the mesocarp and endocarp ( Figs. 8, 11 ). These cells are smaller than those of the mesocarp, have cell walls that are thinner than those of the regular, and are found in a distinct zone bounding the endocarp and mesocarp (see Morozowska et al., 2013 ). Near the fruit apex in one specimen ( Figs. 13, 32 ), a four-lobed stylar canal such as those in Alangium (Eyde, 1968 ) and Cornus ( Wilkinson, 1944 ) can be seen in transverse section. At this level, vascular strands that would eventually end up in the style are seen traversing the fruit ( Fig. 13 ). This course of the vascular traces reinforces the reconstruction of the fruit apex as being more truncated than elongate ( Fig. 32 ). The endocarp consists of a distinct fruit axis and four or five radially arranged septa forming a cross-shaped or five-armed pattern in transverse section ( Figs. 3 7 ). Fruits have four or five germination valves ( Figs. 3 7 ) that extend the length of the fruit ( Figs. 28, 29, 31 ; Appendices S1 S3 [see Supplemental Data with the online version of this article]). The endocarp is ridged; with a major ridge formed by the protruding ends of each septum and a minor ridge in the center of each germination valve ( Figs. 3, 5, 28, 29 ). The separation zone for the germination valves is very pronounced ( Figs. 3, 5, 8, 9, 11 ), and cells in this zone often show dark contents. In two of the specimens ( Figs. 3, 6, 8 ), the fruit is cracked open at the germination valve. This valve opening has a slightly irregular course, and cells separate from each other at the middle lamella. Cells of the septum and central axis are variable in anatomy, those of the central axis being composed of elongate with thick walls and relatively large lumens ( Figs. 3, 10 ). Most of the septum is composed of very thick-walled with small lumens ( Figs. 8, 9, 11 ). Germination valves are composed of thick-walled like those in the septum ( Figs. 8, 9, 11 ). Locules are ellipsoidal to subtriangular shape in cross section ( Figs. 3, 5 7, 8, 11 ). The locule lining is composed of distinctive cells that are radially elongate in some sections ( Figs. 9, 11 ) and more fiber-like than the typical cells of the septum or germination valve (Figs. 8 11 ). There is a single layer of these fiber-like cells on the septum and two layers of fiber-like cells on the germination valve ( Figs. 8, 9, 11 ). This innermost endocarp layer of the septum, as well as the valve, could have come off at the time of fruit opening. Fruit vascular system The vascular system of the fruit is complex with a cylinder of vascular tissue near the base that has at least 10 major bundles that arise and extend into the mesocarp ( Figs. 12, 31 ). There is one vascular bundle that follows each germination valve ridge, running in between the endocarp and mesocarp ( Figs. 28, 29, 31 ). In addition to this vascular bundle, there is a prominent seed bundle that runs up the inside of the fruit locule between the septa toward the fruit apex ( Figs. 14 16, 19 ). This bundle recurves to vascularize the funiculus of the seed and is the best-preserved bundle in the fruits, since the vascular bundles in the mesocarp are often infected with and/or replaced by fungi. Tracheary elements of the seed bundle show scalariform secondary wall thickenings (Fig. 20 ). Numerous small vascular bundles are common within the septum itself ( Figs. 21 23 ). Tracheary elements are smaller in diameter, and cell walls often appear blacker than those of the surrounding that show a brown color and a lower lumen to wall-thickness ratio ( Fig. 21 ). These septal bundles branch, forming two rows in each septum but are concentrated nearer the central zone of the fruit ( Figs. 22, 23 ). These numerous small bundles are often also accompanied by fungi. No vascular tissue has been identified either in the central axis of the fruit ( Figs. 22, 23 ). Seeds There is one apically attached, anatropous seed per locule ( Figs. 3, 7, 8, 11, 21, 24, 28, 29 ). Seed coats are one cell thick with a prominent cuticle ( Figs. 10, 11, 24 ). Integumentary cells are square to rectangular in paradermal section, flattened radially, and in close association with the endosperm ( Figs. 10, 18, 24 ). Seeds are elongate and roughly conform to the shape of the locule with a convex side adjacent to the septum and a flattened side adjacent to the germination valve ( Figs. 3, 8, 11, 24, 28, 29, 33, 34 ). In the holotype where all seed tissues are preserved, the outline of the seed is relatively smooth. By contrast, in seeds where endosperm and embryo are not preserved, the seed outline is somewhat undulating, presumably due to shrinkage. Endosperm tissue is preserved best in the outer two to three layers in the holotype specimen ( Figs. 3, 24, 25 ); however, complete cellularization is present, but can be seen only under certain lighting conditions (i.e., using transmitted light microscopy when the iris diaphragm is completely closed). The innermost endosperm cells (those next to the embryo cavity) also are better preserved in some sections ( Fig. 25 ). Endosperm cells are rectangular in cross section ( Fig. 24 ) and contain large numbers of cellular inclusions ( Fig. 16 ) that might represent proteins, starch, and/or fungi. Embryos are preserved in the holotype ( Figs. 3, 8, 11, 24 28, 34, 35 ). These are fully formed cellular, straight, dicotyledonous embryos, with closely appressed, spathulate cotyledons ( Figs. 25 27, 34, 35 ). The hypocotyl is 0.3 mm in diameter and at least 1.4 mm long ( Figs. 25, 30, 31 ). Just above the cotyledonary node, cotyledons are narrow ( Fig. 26 ), but rapidly expand and become flattened and closely appressed distally ( Figs. 27, 34, 35 ). There are at least three vascular bundles in each cotyledon ( Fig. 27 ). DISCUSSION These drupaceous fruits with woody endocarps, germination valves, one anatropous, apically attached seed per carpel with a single membraneous integument, are clearly attributable to Cornales (Eyde, 1963, 1967, 1988 ; Takahashi et al., 2002 ). Specifically, this fruit morphology is seen in Cornaceae, Alangiaceae, Davidiaceae, Nyssaceae, Mastixiaceae, and Curtisiaceae (Eyde, 1963, 1968, 1988 ; Mai, 1993 ; Yembaturova et al., 2009 ) ( Table 2 ). These families differ in endocarp anatomy and vasculature (Eyde, 1963, 1967, 1988 ; vascular tissue, tan = germination valves showing line of dehiscence (gold/yellow septum appears through opening between two germination valves). 32. Apex of endocarp in four-loculed fruit. Paratype 851051. 33. Longitudinal view of seeds showing elongate shape. Holotype 840208. 34. Longitudinal view of seeds showing embryos (green) inside. Note rapid expansion of flat cotyledons. Holotype 840208. 35. Basal view of four embryos in region of hypocotyls showing the rapid expansion and flattened nature of cotyledons below. (Note: embryo in upper right shows taphonomic split in the hypocotyl.) Holotype 840208.

SEPTEMBER 2016, VOLUME 103 STOCKEY ET AL. EYDEIA HOKKAIDOENSIS GEN. ET SP. NOV. 1651 FIGURES 28 35 Eydeia hokkaidoensis gen. et sp. nov. fruits. AVIZO three-dimensional reconstructions. 28. Whole specimen reconstruction of Holotype 840208. Transparent white = mesocarp, red = vascular tissue, orange/brown = endocarp, opaque brown = endosperm, green = embryo. Note that overlaying transparent units may alter color of structures beneath them. 29. Reconstruction of endocarp with seeds. Holotype 840208. Tan = germination valves, gold/yellow = septum and central axis, brown = seeds, dark red = vascular tissue. Note: dark red in septum indicates numerous small strands of vascular tissue (compare with yellow strands in Fig. 23 ). 30. Longitudinal view of fruit with mesocarp seen in Fig. 28 showing constriction at the base. Transparent gray = mesocarp. Holotype 840208. 31. Longitudinal view of fruit in Fig. 29 with mesocarp removed. Holotype 840208. Dark red =

1652 AMERICAN JOURNAL OF BOTANY TABLE 2. Comparison of endocarp structure among similar extant cornalean families with drupaceous fruits. Taxon No. of locules Locule shape Valve length Septum histology Valve histology Endocarp vasculature Eydeia hokkaidoensis 4 5 Subtriangular to ellipsoidal Elongate Elongate and isodiametric Longitudinal rows in septa Alangiaceae 1 2 Ellipsoidal Elongate Septum periphery Cornaceae (1 ) 2 (3 4 ) Ellipsoidal Elongate Elongate and isodiametric Septum periphery Nyssaceae 1( 3) Ellipsoidal, W-shaped, Short Fibers Fibers Septum periphery C-shaped Mastixiaceae 1 C-shaped Elongate Fibers Varying, fibers, Septum periphery parenchyma Davidiaceae 6+ Subtriangular Elongate Varying, fibers Fibers Longitudinal rows in septa Curtisiaceae 4 Subtriangular Elongate Central in fruit axis Notes: Data are from Eyde, 1963, 1967, 1968, 1988 ; Hammel and Zamora, 1990 ; Manchester et al., 1999, 2010 ; Noll, 2013 ; (families sensu Xiang et al., 2011 ). Yembaturova et al., 2009 ). Fossil fruits included in these taxa generally have a higher number of locules, but their endocarp anatomy and vasculature are very similar to the living species within cornalean families ( Eyde and Barghoorn, 1963 ; Eyde, 1997 ; Manchester et al., 2010, 2015 ). While these cornalean families vary in locule number from one to many ( Table 2 ), fruits in the extant genera of Alangiaceae, Nyssaceae, and Mastixiaceae consistently have fewer locules than the four or five locules seen in the fossil fruits described here. Fruits of Nyssaceae (sensu Xiang et al., 2011 ) have one to three locules/fruit that are ellipsoidal to C-shaped to nearly W-shaped (in some taxa), have short germination valves, and the endocarp is composed mostly of fibers ( Table 2 ) ( Eyde, 1963 ; Hammel and Zamora, 1990 ; Noll, 2013 ). The fruits described here, however, have four to five ellipsoidal to subtriangular locules, elongate germination valves, and endocarp composed of several cell types ( Table 2 ). Vascularization in Nyssaceae endocarps is concentrated in the septum periphery ( Eyde, 1963, 1967 ), while that of Eydeia occurs as two parallel rows in the septa ( Table 2 ). Modern Mastixiaceae usually have only locule and one seed per fruit, with C-shaped locules, fibers in the endocarp septum, and vascularization at the septum periphery ( Table 2 ) ( Eyde, 1963, 1967 ; Tiffney and Haggard, 1996 ; Stockey et al., 1998 ). Thus, these differ greatly from those of Eydeia with vascularized septa. While the valve histology in Mastixiaceae shows varying cell types including as in Eydeia, modern fruits often possess fibers and parenchyma that are lacking in Eydeia (Table 2 ). Alangiaceae (sensu Xiang et al., 2011 ) have only one to two locules per fruit, lack the elongate seen in Eydeia fruits, and have vascularization at the septum periphery ( Table 2 ) ( Eyde, 1967, 1968 ). The locules are ellipsoidal, rather than subtriangular in cross section as in Eydeia, although germination valves are as elongate as in Eydeia fruits (Table 2 ) (Eyde, 1968 ). Thus, the three families that share the largest number of characters with Eydeia are Cornaceae, Davidiaceae, and Curtisiaceae. The family Cornaceae ( Cornus L.) contains the largest number of extant species, and some characters such as number of locules/ fruit are variable within the genus ( Table 2 ) ( Eyde, 1988 ; Murrell, 1993 ; Xiang et al., 2006 ). The genus Cornus is currently recognized to include four major clades: blue- or white-fruited dogwoods, cornelian cherries, big-bracted dogwoods, and dwarf dogwoods ( Eyde, 1987, 1988 ; Xiang et al., 2006, 2011 ; Xiang and Thomas, 2008 ). Two is the most common number of locules/fruit. Within extant Cornus, only Cornus oblonga Wallich, in the blue- or white-fruited dogwoods, consistently has higher numbers of locules ( Eyde, 1988 ). There are usually three locules per fruit in C. oblonga (Eyde, 1988 ; Manchester et al., 2010 ); however, rarely four locules occur ( Fig. 36 ; Table 3 ). Locules in C. oblonga are ellipsoidal rather than subtriangular as in the Hokkaido fossil fruits ( Table 3 ; Fig. 36 ). While C. oblonga does have exterior ridges on the valves as in the Hokkaido fruits, septal ridges are absent ( Table 3, Manchester et al., 2010 ). Valves are elongate in C. oblonga and the Hokkaido fruits, and valve and septum histology are relatively similar; however, there are no elongate in the center of the septum as in the Hokkaido fruits ( Table 3 ). By far, the most useful character in distinguishing our fruits from those of C. oblonga and Cornus as a whole, is the position of the vascular tissue, which is at the septal periphery ( Fig. 36 ) and does not occur as two rows of separate bundles in the septa as in Eydeia (Table 3 ). Within the fossil record of Cornaceae, fruits with three to six locules have been reported ( Manchester, 1994 ; Manchester et al., 2010 ). Cornelian cherries ( Cornus subg. Cornus ) have the oldest fossil record within Cornus dating back to the Paleocene of North America ( Manchester et al., 2010 ). Most fossil cornelian cherries are represented by endocarps in which the locule number is higher than that observed in living species within the subgenus Cornus, which normally have one to two locules per fruit. Cornus multilocularis (Gardner) Eyde from the Eocene London Clay flora has endocarps with three to six subtriangular locules, overlapping in number with the fossil fruits described here ( Table 3 ). Similar to those seen in Eydeia, the septa of C. multilocularis contain elongated (figs. 5R, 5S, 5T of Manchester et al., 2010 ). However, C. multilocularis, like all cornelian cherries, has endocarps with large secretory cavities ( Manchester et al., 2010 ), which are absent in the endocarps of Eydeia. While the endocarp vasculature of C. multilocularis is confined to the septal periphery ( Manchester et al., 2010 ), Eydeia fruits have two rows of vascular bundles within the septa. Cornus clarnensis Manchester (1994) from the Eocene Clarno Nut Beds, placed in the big-bracted dogwoods, has three locules per endocarp, unlike the four to five locules seen in endocarps of Eydeia ( Table 3 ). Vascular tissue in C. clarnensis is confined to the periphery of the septa, and the endocarp is composed of isodiametric ( Manchester, 1994 ), while in Eydeia two rows of vascular tissue occur in the septa and the endocarp contains both isodiametric and elongate ( Table 3 ). The genus Curtisia Aiton (Curtisiaceae) has fruits that show a large number of similarities to those of Eydeia (Table 3 ). The four locules per fruit, subtriangular locules, elongate germination valves,

SEPTEMBER 2016, VOLUME 103 STOCKEY ET AL. EYDEIA HOKKAIDOENSIS GEN. ET SP. NOV. 1653 FIGURE 36 Transverse section of Cornus oblonga fruit. R. H. Eyde collection, Smithsonian, 382, slide 4, row 6, section #2. 50. isodiametric in the septa, and germination valves are all similar to those in the Hokkaido fruits ( Table 3 ) ( Manchester et al., 2007 ; Yembaturova et al., 2009 ). In addition, Curtisia has been reported to have elongated parenchymatous cells in the mesocarp and zone of transition between the endocarp and mesocarp (Table 3 ) (figs. 2A, 2D, 2K of Yembaturova et al., 2009 ). Curtisia, however, lacks any septal ridges or external ridges on the valves and elongate in the septum ( Table 3 ). The most striking difference between Eydeia fruits and those of Curtisia is the presence of a central vascular strand in the fruit axis of Curtisia (Table 3 ) (Eyde, 1988 ; Manchester et al., 2007 ; Yembaturova et al., 2009 ). Fossils of Curtisia quadrilocularis (Reid & Chandler) Manchester, Xiang & Xiang (2007) from the Paleocene/Eocene London Clay Flora are represented by pyritized endocarps that show all of the characters seen in those of extant C. dentata (Burm.F.) C.A.Sm. ( Table 3 ). The family Davidiaceae today is represented only by Davidia involucrata Baillon, the dove tree, from central China ( Eyde, 1963 ; Manchester, 2002 ). Extant Davidia fruits have six or more locules, while Eydeia fruits have four or five locules ( Table 3 ). Davidia involucrata fruits have subtriangular locules in cross section, septal ridges, exterior valve ridges, and elongate germination valves similar to those in Eydeia ( Table 3 ). Endocarp vasculature, occurring in two longitudinal rows in the septa, is the most striking similarity of Eydeia fruits to those of Davidia (Table 3 ) (Eyde, 1963, 1967 ; Atkinson, 2016 : fig. 7C). However, the endocarp histology and presence of in the mesocarp, lack of transition between endocarp and mesocarp, and a lack of radially elongate cells in the mesocarp of Davidia enable us to distinguish Eydeia from this genus ( Table 3 ). In addition, fruits of extant Davidia have very pronounced ridges on the valves, as well as additional minor ridges on the valves and septa ( fig. 6C of Manchester, 2002 ), that are lacking in Eydeia. The presence of fibers in the endocarp septa and valves in extant Davidia is also a noticeable difference from what is seen in Eydeia (Table 3 ). Fossil fruits described as Davidia antiqua (Newb.) Manchester (2002) from the Paleocene of North America show subtriangular locules and have septal ridges, elongate germination valves and vasculature like that seen in D. involucrata and Eydeia (Table 3 ). However, there are six to eight locules per fruit and the endocarp histology is fibrous like that observed in D. involucrata (Table 3 ) (Manchester, 2002 ). Davidia antiqua fruits, however, lack prominent ridges on the germination valves ( Table 3 ) ( Manchester, 2002 ). Since the mesocarp is not anatomically preserved in these fossil fruits, only endocarp characters can be used in identification. The ridges on the germination valves in Eydeia are smaller than those of D. involucrata but are consistently present in unabraded specimens. Recently, fruits of Davidia sp. were briefly described by Manchester et al. (2015) from the Upper Cretaceous (Campanian) of Drumheller, Alberta, Canada. These have yet to be fully described, but a manuscript is currently in preparation (R. Serbet, University of Kansas, personal communication, 2016). Like some of the fruits described here, they have five locules and septal ridges on the endocarp ( Table 3 ); however, there are a number of fruits in the collection with aborted locules that are not seen in any specimens of Eydeia. Locule shapes, however, vary from subtriangular to pentagonal in cross section, and ridges are lacking on the valves ( Table 3 ). Mesocarp characters are missing, and the valve lengths have not been reported. There are some elongate in the center of the endocarp, but these are shorter than those seen in Eydeia (R. A. Stockey and B. A. Atkinson, personal observations; Table 3 ). Comparison to other fossil genera of Cornales Four additional extinct fossil genera of Cornales have been described from fruits from the Upper Cretaceous and Paleocene: Browneia Manchester & Hickey (2007), Amersinia Manchester, Crane & Golovneva (1999), Suciacarpa Atkinson (2016), and Hironoia Takahashi, Crane & Manchester (2002). Fruits described as Browneia serrata (Newberry) Manchester & Hickey from the Paleocene of North America are borne in infructescences similar to those of Camptotheca Decne. (Nyssaceae). Most of these fossil fruits are preserved as compression/impression fossils ( Manchester and Hickey, 2007 ), but partially permineralized specimens show an elongated germination valve like that seen in Eydeia; however, the endocarps are unilocular in Browneia ( Manchester and Hickey, 2007 ). Thus, this taxon can be eliminated from further consideration. Amersinia obtrullata Manchester, Crane & Golovneva (1999) infructescences have fruits that occur in globose to ellipsoidal heads. While these are known from several localities in North America and Asia, they are usually represented by compression/ impression fossils. However, permineralized material is known from the Almont Flora of North Dakota ( Manchester et al., 1999 ). We reexamined the thin sections of Amersinia obtrullata from Almont for comparative purposes and attempted to see more details of cells in the endocarp and mesocarp. Fruits of A. obtrullata are obtrullate, rather than pyriform as in the Hokkaido specimens. Amersinia fruits typically show three locules, but a few specimens with four locules have been observed as in the Eydeia ( Table 3 ) ( Manchester et al., 1999 : fig. 4.35). Amersinia fruits have septal ridges and isodiametric in the valves as in Eydeia, but lack ridges on the valves ( Table 3 ). In addition, A. obtrullata fruits have ellipsoidal locules in cross section, short valves, and fibers in the septa and center of the fruit ( Table 3 ). Radially elongate cells are lacking in the mesocarp of A. obtrullata ; however, appear to be present (authors personal observations). We could not determine

1654 AMERICAN JOURNAL OF BOTANY TABLE 3. Comparison of endocarp structure among similar fossil and extant cornalean species. Taxon Age Eydeia hokkaidoensis No. of locules Locule shape in cross section Santonian 4 5 Subtriangular to ellipsoidal Septal ridges Exterior ridge(s) on valve Valve length Septum histology Present Present Elongate Hironoia fusiformis Coniacian 3 (4) Subtriangular Present Present Elongate Elongate amd isodiametric Suciacarpa starrii Campanian 4 C-shaped Absent Absent Short Elongate and secretory cavities Histology of central axis Elongate Elongate and isodiametric Elongate Valve histology and secretory cavities Amersinia obtrullata Paleocene 3 ( 4 ) EllipsoidalPresent Absent Short Fibers Fibers Davidiaceae Davidia sp. Campanian5 Subtriangular to pentagonal Present Absent? Elongate Elongate Endocarp vasculature Longitudinal rows in septa Septum periphery, periphery of fruit axis Longitudinal row in septa Septum periphery, periphery of fruit axis? Longitudinal rows in septa D. antiqua Paleocene 6-8 Subtriangular Present Absent Elongate Fibers Fibers Fibers Longitudinal rows in septa D. involucrata Extant 6+ Subtriangular Present Present Elongate Varying, fibers Cornaceae Cornus oblonga Extant 3 (4) Ellipsoidal Absent Present Elongate C. clarnensis Eocene 3 Ellipsoidal Absent Absent Elongate C. multilocularis Eocene 3 6 Subtriangular Absent Absent Elongate Elongate Curtisiaceae Curtisia quadrilocularis Paleocene/ Eocene 4 Subtriangular Absent Absent Elongate Curtisia dentata Extant 4 Subtriangular Absent Absent Elongate Fibers Fibers Longitudinal rows in septa Elongate Vascular tissue and isodiametric Vascular tissue and isodiametric Transition Sclereids in mesocarp Radially elongated cells in mesocarp Present Absent Present?????? Absent? Present Absent?????? Absent Present Absent Septum periphery Present? Absent? Absent Septum periphery???? Septum periphery??? Central in fruit axis??? Central in fruit axis Present Present Present Notes: Data are from Chandler, 1962 ; Eyde, 1963 ; 1968 ; 1988 ; Manchester, 1994, 2002 ; Manchester et al., 1999, 2007, 2010, 2015 ; Takahashi et al., 2002 ; Yembaturova et al., 2009 ; R. Serbet, University of Kansas, personal communication, 2016; Atkinson, 2016.

SEPTEMBER 2016, VOLUME 103 STOCKEY ET AL. EYDEIA HOKKAIDOENSIS GEN. ET SP. NOV. 1655 the presence or absence of transition with certainty; however, they appear to be absent ( Table 3 ). Suciacarpa starrii Atkinson (2016) was recently described from the Campanian of Sucia Island and Little Sucia Island, Washington State. Only two fruits of this species are known. Like the Hokkaido fruits, they have been described as four-loculed, with elongate in the septum, isodiametric in the valve and longitudinal rows of vascular bundles in the septa ( Table 3 ) ( Atkinson, 2016). They differ greatly in the shape of the locules in cross section, which in Suciacarpa are more similar to taxa of the Mastixiaceae and Nyssaceae ( Tables 2, 3 ). Suciacarpa fruits lack septal ridges, external valve ridges, have short germination valves, and elongate in the septa, all contrasting to the conditions seen in Eydeia fruits ( Table 3 ). In addition, S. starrii fruits have numerous secretory cavities similar to those seen in cornelian cherries ( Cornus subg. Cornus ; Manchester et al., 2010 ) in both the valves and septum, making these very distinct from Eydeia. Details of the mesocarp are unknown for Suciacarpa (Atkinson, 2016 ). Hironoia fusiformis Takahashi, Crane & Manchester (2002) is the earliest accepted cornalean fossil from the Upper Cretaceous (Coniacian) of northeastern Japan (Honshu). The species is based on mesofossils from the Kamikitaba locality in northeastern Honshu ( Takahashi et al., 2002 ). These fruits commonly have three locules, but four locules have also been observed ( Table 3 ) ( Takahashi et al., 2002 ). Like Eydeia fruits, the locules are subtriangular in cross section, septal and valve ridges are present, and the valves are elongate with isodiametric ( Table 3 ). There are some differences in septal histology including the presence of what we interpret as elongate in the septa and isodiametric in the center of the fruit in Hironoia (Table 3 ). The vascular system in the endocarp, however, is distinctly different from that of Eydeia, with vascular bundles at the periphery of the septum as in Cornaceae, Alangiaceae, Nyssaceae, and Mastixiaceae ( Tables 2, 3 ) and additional bundles at the margins of the central axis rather than in the center as in Curtisiaceae ( Tables 2, 3 ). Details of the mesocarp have not been described for these fruits, except surficially with SEM, and the material is unavailable for study at the present time. Therefore, the presence of transition, in the mesocarp or tangentially elongate cells in the mesocarp could not be confirmed ( Table 3 ). Thus, these new fruits from Hokkaido, while showing distinctive cornalean characters, can be distinguished from those of both living and fossil taxa by a mosaic of characters, supporting our recognition of Eydeia hokkaidoensis as a new genus and species. Eydeia hokkaidoensis shows closest similarities to the Davidiaceae ( Davidia ) based on fruit vasculature; however, other characters of Davidia such as those of the mesocarp, and endocarp histology, contrast with those reported for Eydeia (Table 3 ). The excellent preservation seen in Eydeia fruits reveals details of the structure of embryos, endosperm, and the membraneous seed coat in cornalean fossils. Endosperm is cellular with numerous spherical inclusions that have the appearance of stored proteins as have been reported in Curtisia ( Yembaturova et al., 2009 ) or perhaps starch as in Cornus ( Chopra and Kaur, 1965 ). Embryos are straight with cylindrical hypocotyls and broad, flattened spathulate cotyledons, each with three major vascular bundles as seen in Davidia, Nyssa, Alangium, and Cornus (Harms, 1898 ; Takhtajan, 2000 ). Integuments are thin in most Cornales, and detailed descriptions are rare; however, see the structure of Curtisia in Yembaturova et al. (2009). The seed coat of Eydeia hokkaidoensis is only one cell thick, is in close contact with the endosperm, and appears to be covered with a cuticle. Details of these tissues in extant Cornales need to be reexamined in the future so that well-preserved fossils can be compared across the order. Eyde (1963, 1967 ) suggested that Davidia fruits showed the ancestral fruit type in the Nyssaceae-Mastixiaceae-Davidiaceae lineage in Cornales. There are several Davidia -like characters in Eydeia, including the fruit vascular pattern, which Eyde (1967) hypothesized to be the ancestral pattern for Cornales. The fossil record of Eydeia shows that this vascular pattern was present in fruits that were widespread in the Tomamae area of Hokkaido during the Santonian. As more cornalean fossils are recovered from Cretaceous sediments, we are coming to a better understanding of early fruit morphology within the order Cornales. The current study represents part of an ongoing investigation that will allow us to increase the fossil database and identify the characters that both extant and fossil fruits can provide for elucidating patterns of early cornalean evolution. ACKNOWLEDGEMENTS The authors thank Midori Matsumoto and Takeshi Ohsawa Asakawa, Chiba University for providing some of the material for study and the late Makoto Nishida, Chiba University and Toshihiro Yamada, Kanazawa University for help with field logistics and geologic data. Stan Yankowski, Smithsonian Institution, and Steven Manchester, Florida Museum of Natural History, University of Florida made the R. H. 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