ISSN 1346-7565 Acta Phytotax. Geobot. 72 (1): 1–21 (2021) doi: 10.18942/apg.202009
A Multicarpellary Apocarpous Gynoecium from the Late Cretaceous (Coniacian–Santonian) of the Upper Yezo Group of Obira, Hokkaido, Japan: Obirafructus kokubunii gen. & sp. nov.
1,* 2 3 Yui Kajita , Mayumi Hanari Suzuki and Harufumi Nishida
1Iriomote station, Tropical Biosphere Research Center, University of the Ryukyus, 870, Uehara, Taketomi, Okinawa 907–1541, Japan. *[email protected] (author for correspondence); 2Tama, Tokyo 206–0003, Japan; 3Faculty of Science and Engineering, Chuo University, 1–13–27 Kasuga, Bunkyo, Tokyo 112–8551, Japan
Obirafructus kokubunii gen. & sp. nov. (family Incertae Sedis; order Saxifragales) is proposed based on a permineralized reproductive axis bearing at least 42 spirally arranged follicles. No bracts, perianth, stamens, nor their vestiges are present on the axis or the follicle stalk. It is therefore part of single flower and not an inflorescence. The axis is 57 mm long, woody, and contains scalariform perforations on the vessel walls. The flower is inferred to be unisexual, as in Cercidiphyllaceae (Saxifragales). The lower part, which may have borne male organs, is missing. The follicles consist of a conduplicate carpel with marginal placentas alternately bearing 90–100 seeds in two rows. The follicle has dorsal and ventral ridges and the ventral suture dehisces at maturity. The carpel probably has an apical style and stigma at anthesis. The ovules are bitegmic, anatropous. A nucellar cap plugs the micropyle. The seeds are slightly winged, which may represent hydrochory and/or anemochory. Based on these features, Obirafructus kokubunii probably inhabited a fluvial plain. The follicle clusters of Joffrea and Jenkinsella (fossil Cercidiphyllaceae) apparently resemble those of O. kokubunii; but they represent inflorescences. Obirafructus kokubunii adds a new morphotype to the past diversity of basal Saxifragales.
Key words: anatomy, Cercidiphyllaceae, eudicot, follicle, fossil, inflorescence,Jenkinsella , Joffrea, mor- phology, Saxifragales
The fossil described here was found in a cal- ing many follicles and is reminiscent of the mor- careous concretion that was collected along the phologies of some extant and comparable fossil Obirashibe River in Obira, Hokkaido, Japan, families of Saxifragales, sensu Angiosperm Phy- where Late Cretaceous shallow to offshore ma- logeny Group IV system (2016). Among the Saxi- rine sediments of the Haborogawa Formation of fragales, the fossil shows morphological similar- the Yezo Group occur. Nishida (2006) first tenta- ity to Cercidiphyllaceae, both living and fossil tively reported the specimen to be a ‘Cercidiphyl- forms (Swamy & Bailey 1949, Spongberg 1979, lum-like hamamelid’. The fossil-bearing strata Crane 1984, Crane & Stockey 1985, Feng et al. was exposed along the main stream of the Obi- 2000, Golovneva & Alekseev 2017). The Cercidi- rashibe River. The age has been estimated to cor- phyllaceae include only two extant species that respond to the Coniacian to Santonian period are restricted to Japan, China, and Korea. Their based on the associated marine fauna (Takashima fossil records extend back to the early-middle et al. 2004). An ammonite, Gaudryceras sp. (Fig. Aptian (Golovneva & Alekseev 2017). In this 1A), in the same concretion often occurs in the study, we describe the specimen as a new fossil Haborogawa Formation and does not contradict genus of Saxifragales, and particularly similar to the age estimation. Cercidiphyllaceae, without designating a particu- The Cercidiphyllum-like hamamelid fossil is lar family due to scarce information regarding a permineralized reproductive woody axis bear- Cretaceous plants. 2 Acta Phytotax. Geobot. Vol. 72
The fossil exhibits detailed anatomical infor- nearly mature before dehiscence. The fixed mate- mation reminiscent of the morphological and rials were dehydrated in an ethyl and tertiary-bu- functional diversity of basal Saxifragales, or tyl alcohol series and embedded in paraffin. Sec- more widely, of early Eudicots during their early tions 10 µm thick were cut with a RM2125RTF diversification in the Cretaceous. We compared rotary microtome (Leica Microsystems, Wetzlar, the seeds, follicles, and follicle clusters of the fos- Germany) and stained with Hematoxylin (Sig- sil with specimens of Cercidiphyllaceae to show ma), Safranin-O (Merck), and Fastgreen (Sigma). morphological diversity of the seemingly similar Sections of both the fossil and extant C. japoni- dispersal organs. For comparison, the follicle cum were mounted in Canada balsam and exam- anatomy of living Cercidiphyllaceae (i.e., Cerci- ined under a BX–50 light microscope (Olympus diphyllum japonicum Sieb. & Zucc. ex Hoffm. & Optical Co. Ltd., Tokyo, Japan). Images were Schult.) was also evaluated in this study. captured with a D–20 digital camera system (Pix- era Corporation, California, USA). The numerical geological ages used in this pa- Material and Methods per adhere to the International Chronostrati- graphic Chart (Cohen et al. 2013: revised version The fossil material was embedded in a calcar- May 2019). eous sandy siltstone concretion Kokubun NSM PP-9374 (original sample number: Kokubun HKP0001), which was collected in the middle Results reaches of the Obirashibe River, Obira town, Hokkaido, Japan. The concretion was cut into Systematic Description several pieces with a Mini-lab-cutter MC110 dia- mond slab saw (Maruto Instrument Co., Ltd., To- Phylum Magnoliophyta kyo, Japan) to obtain both transverse and longitu- Order Saxifragales dinal planes of the embedded reproductive shoot. Family incertae sedis For anatomical observations, the cutting planes were peeled sequentially using a cellulose-ace- Obirafructus Y. Kajita & H. Nishida gen. nov. tate peeling technique (Joy et al. 1956) to obtain Permineralized angiosperm reproductive shoot, consist- serial peels and to make microscope slides of the ing of a woody axis bearing a number of spirally arranged selected peels. Non-destructive images of the follicles. Axis in fruiting stage elongate, eustelic; vessels concretions were also obtained using a ScanX- with scalariform perforation plates. Follicle elongate fusi- form to falcate, tapering distally, dehiscing along ventral mate-A130S145 X-ray CT scanner (Comscantec- longitudinal suture; lateral surface transversely striate; no Co., Ltd., Yokohama, Japan) to confirm the placenta marginal. Dorsal midrib of follicle and each side anatomical reconstruction and to search for other of ventral suture ridged, 3-veined, 1 dorsal and 2 ventral. notable structures in the concretion. Whole piec- Follicle wall 3-layered, with pericarp, vascularized meso- es of the sectioned concretion and the microscop- carp, and fibrous sclerenchyma endocarp. Seeds numer- ous, flat, in 2 rows along ventral suture, one above the ic slides were deposited in the Tsukuba Research other. Seeds bitegmic, anatropous, winged; wing devel- Department of the National Museum of Nature oped on upper side of main seed body. Seed coats 2, inner and Science in Tsukuba, Ibaraki, Japan. and outer; inner seed coat thin; outer seed coat constitut- For anatomical comparisons, follicles of ex- ing wing and lower fibrous layer. Nucellar wall thickened tant Cercidiphyllum japonicum were collected below endostome, forming a nucellar cap. from the Koishikawa Botanical Garden of the Graduate School of Science of the University of Obirafructus kokubunii Y. Kajita & H. Nishida Tokyo, Japan, and fixed with FAA (formalde- sp. nov. — Figs. 1B–D, 2–10, 11D & 12C, D hyde: 70% ethyl alcohol: acetic acid = 1 : 18 : 1). Permineralized angiosperm reproductive shoot consist- The follicles were determined to be immature or ing of a woody axis bearing more than 42 conduplicate February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 3
Fig. 1. X-ray CT images of calcareous concretion containing a part of reproductive shoot of Obirafructus kokubunii gen. & sp. nov., specimen Kokubun NSM PP-9374. (A) Ammonite, Gaudryceras sp., embedded in same nodule. Diameter of ammonite shell is about 1 cm. (B) Cross section of main axis, two follicles, and two follicle bases (arrowheads). (C) Radial longitudinal section showing main axis bearing stiped follicles containing numerous seeds. (D) Tangential longitudinal section of a follicle showing two rows of seeds. Note wing tissue on upper side of each seed. f, follicle; fs, follicle stalk; mx, main axis; s, seed; w, seed wing. B–D, Scale bar = 1 mm. follicles in 5 : 8 phyllotaxy. Axis in fruiting stage, more constituting wing and lower fibrous layer, attached to pla- than 57 mm long, 2–5 mm in diam., eustelic; vessels with centa on winged side, vascularized by a single bundle on scalariform perforation plates. Follicles elongate fusiform upper middle portion of wing to chalazal end of nucellus. to falcate, 30–40 mm long, 3 mm in diam., short-stiped, Nucellar wall membranous, thickened below endostome abruptly tapering distally, dehiscing along longitudinal to form a nucellar cap. ventral suture; lateral surface transversely striate; cross Typus. Kokubun NSM PP-9374 (fruits in concretion section nearly round with semicircular dorsal ridge and block and microscope slides prepared from it) deposited ventral ridge splits at middle along longitudinal suture. in the Paleobotanical Collection of the National Museum 3-veined, 1 dorsal and 2 on each side of ventral suture. of Nature and Science in Tsukuba, Ibaraki, Japan. Follicle wall 3-layered; pericarp thin, consisting of an epidermis with actinocytic stomata and hypodermis; me- Locality. Japan, Hokkaido, Obira, main stream socarp thick, parenchymatous, vascularized; endocarp of the Obirashibe River, approximately 44º04′53″N composed of fibrous sclerenchyma cells; placenta mar- 141º57′05″E. ginal. Seeds nearly 100, flat, tightly packed in each folli- cle, in 2 rows, one above the other, bitegmic, anatropous, winged on upper side, semicircular to oval, ca. 2 mm Type strata. Upper Cretaceous (Coniacian to long, 1 mm wide along horizontal plane, ca. 0.6 mm thick Santonian) Haborogawa Formation, Yezo Group. at middle; wing fleshy parenchymatous inside, on upper side of main seed body, as thick as nucellar portion, anvil- Etymology. The generic name comes from the shaped, with narrow marginal extensions horizontally town, Obira, where the fossil site is located. The around seed; nucellar portion semicircular to crescent- specific epithet honors Mr. Hakuji Kokubun, who shaped in horizontal plane, rounded columnar in tangen- tial plane at widest middle portion. Seed coat composed found the specimen. of inner and outer layers; inner seed coat mostly 1 cell thick, 3 cells thick around endostome; outer seed coat Morphological description. The specimen was 4 Acta Phytotax. Geobot. Vol. 72 embedded in a calcareous sandy siltstone concre- ably collateral, although the phloem is not well tion about 25 cm in diameter and broken along preserved. The stele is surrounded by thin cortex lateral and upper surfaces where the specimen observable as a black degraded layer (Fig. 3A). had been exposed (Fig. 2). A dark, woody axis The xylem contains thin secondary wood consist- with five elongate follicles and some isolated fol- ing of vessels with spiral, scalariform and annu- licle sections (Fig. 2A) is present on the lateral lar thickenings (Fig. 5C, E) and fibers with simple surface. More than 11 follicles on the main axis at pits (Fig. 5D). Scalariform perforation plates one end of the concretion were sectioned either were observed in some vessels (Fig. 5F). transversely or obliquely (Fig. 2A). The follicles The follicles were elongate fusiform to fal- radiated upward from the main axis with short cate, 30–40 mm long and up to 3 mm in diameter, internodes at angles of ca. 15° (Figs. 2A & 11D). with a basal short stalk 3–4 mm long, and tapered Although the lower part of the shoot was missing, distally (Figs. 2A, 4A & 6A, B, D). The follicle we estimated the entire follicle cluster of the re- stalks lacked nodal structures for other floral or- productive shoot to be about 75 mm long and 40 gans or bracts, branching vascular bundles or ax- mm wide (Fig. 2A). The shoot apex was embed- illary organs (Fig. 6D, E). One longitudinal ridge ded in the concretion (Fig. 2A). An eroded follicle along the adaxial surface of the follicle dehisces on the lateral broken side shows internal seeds in in the middle to form a ventral slit that extends a vertical row (Fig. 2B). The lateral surface of the from the base to the extreme tip of the follicle follicle is transversely striate. We used an X-ray (Figs. 4, 6A, B & 7B). The follicle trace derives CT scanner to examine the inside of the concre- directly from the main vascular cylinder as a tion, which was not used for making microscopic woody vascular bundle, which eventually sepa- sections, to confirm that there were only the three rates into a dorsal strand (ds, Fig. 3) and two ven- main structures, the single main axis, the folli- tral strands (vs, Fig. 3). The three traces are com- cles, and the seeds in the follicle (Figs. 1B–D & parable with the three main bundles supplying a 11D). typical conduplicate carpel. Each ventral strand The preserved main axis, about 57 mm long, 5 immediately divides radially into two bundles mm × 2 mm in maximum diameter at its base, (vs1 & vs2, Fig. 3), resulting in five traces at the and 2 mm × 1 mm distally, was slightly crushed distal end of the follicle stalk. vs1 continues dis- laterally. The axis is woody and eustelic (Fig. tally to the ventral ridge whereas vs2 further di- 3A). In one cross section, 32 follicles and the base vides to expand in the lateral wall of the follicle of isolated follicle were counted around the main (Figs. 3B & 7B, C). The ds enters the dorsal ridge axis (Fig. 4). The follicle phyllotaxy is spiral with of the follicle (Fig. 7A). No other traces, except eight clockwise and five counterclockwise paras- those entering the follicles, were observed, pro- tichies (Fig. 4B). Based on the reconstructed viding evidence that no other floral structures phyllotaxy, we estimated the minimum number originated from the shoot. The follicle consists of of attached follicles to be 42. Judging from the a single ovary containing numerous seeds (Figs. seed morphology and stage of carpel dehiscence, 2B & 6D). The ovary shows marginal placenta- we estimated that each follicle was at approxi- tion. The seeds are alternately attached on the mately the same stage of development (Fig. 4A). placentas located on the adaxial surfaces of the Although the number of vascular bundles was ventral ridges (Figs. 6B, C, 7B & 8B). We ob- difficult to count, the follicle parastichies (Fig. served two rows of seeds in cross and tangential 4B) that we observed suggest that there were sections in one ovary (Figs. 4, 6B & 8D), although originally five primary bundles in the vascular the radial longitudinal sections showed seeds cylinder of the main axis. The pith consisted of aligned in one row (Figs. 2B & 6C, D). Style and fibrous periphery and inner parenchymatous tis- stigmatic tissues were not identifiable at most dis- sues that were heavily crushed near the center tal tip of the follicle (Fig. 6A). The follicle wall or (Figs. 3 & 5A, B). The vascular bundles are prob- pericarp was divisible into three regions, exo- February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 5
Fig. 2. Calcareous concretion Kokubun NSM PP-9374 containing a reproductive shoot Obirafructus kokubunii gen. & sp. nov. (A) Entire specimen before sectioning. (B) Upper part of specimen showing a longitudinally broken surface of a follicle containing seeds. f, follicle; fs, follicle stalk; mx, main axis; s, seed. Scale bar = 10 mm (A), 1 mm (B).
Fig. 3. Obirafructus kokubunii gen. & sp. nov. (A) Cross section of female reproductive shoot showing structure of main axis (mx) and departing follicle bases. Scale bar = 1 mm. Slide Kokubun NSM PP-9374 Bbot#9. (B) Schematic drawing of lower portion of follicle showing vasculature. co, degraded black layer of cortex; ds, dorsal strand of a follicle; fs, follicle stalk; lv, lateral vascular bundles; p, pith of main axis; vs, ventral strand of a follicle distally dividing into vs1 and vs2. 6 Acta Phytotax. Geobot. Vol. 72
Fig. 4. Obirafructus kokubunii gen. & sp. nov. Cross section of female reproductive shoot. Slide Kokubun NSM PP-9374 Btop#99. (A) General structure. (B) Showing parastichies with eight clockwise (blue) and five counterclockwise (orange) lines. Follicles are subsequently numbered from proximal to distal. f, follicle; fs, follicle stalk; mx, main axis; vl, ventral slit. Scale bar = 5 mm. February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 7
Fig. 5. Obirafructus kokubunii gen. & sp. nov. Anatomical features of main axis. (A) Cross section of pith with axis center to right direction. Peripheral and central cells of pith are smaller and crushed respectively. Slide Kokubun NSM PP-9374 Bbot#9. (B) Longitudinal section of pith with axis center to right direction, showing fibrous periphery (to left) and inner parenchyma. Slide Kokubun NSM PP-9374 #77. (C–F) Xylem longitudinal sections. Slide Kokubun NSM PP-9374 B1#66. (C) Scalariform (sf) and spiral (sp) vessel thickenings. (D) Simple pits (pi) on fiber walls. (E) Scalariform (sf) and annular (an) vessel thickenings. (F) Scalariform perforation plates (arrowheads). Scale bar = 100 µm. 8 Acta Phytotax. Geobot. Vol. 72 carp, mesocarp and endocarp (Fig. 7). The exo- mal cells surrounding the entire seed (Fig. 9). The carp consists of the outer epidermis and a hypo- fibers were one to several cells thick, elongated in dermis 3–5 cells thick (Fig. 7). The hypodermis the direction of the funiculus and often filled with corresponds to the dark layers immediately be- brown substances (Figs. 8B, 9 & 10B, C). The fi- low the epidermis (Fig. 7). The epidermal cells brous layer extended to fill the margin of the lat- were light brown and polygonal in shape in the eral horn-like expansion of the wing (Fig. 9B). periclinal section with occasional actinocytic sto- The middle layer was thick, parenchymatous and mata (Fig. 7D). The hypodermal cells were flat- only developed in the upper winged portion of the tened in cross section, thick-walled, and probably seed (Figs. 8B, D, 9 & 10A). The parenchyma was contained a dark tannin-like substance (Figs. 4–15 cells thick and thickest in the middle of the 7A–C & 8A). The mesocarp was composed of pa- wing (Fig. 9A, B). Some parenchyma cells had renchyma (2–10 cells thick) and a nest of vascular pitted walls that we describe as idioblastic (Fig. bundles in the middle of the tissue (Fig. 7A–C). 10A). We observed a single vascular bundle in the The mesocarp was thickest at the ventral and dor- wing parenchyma below the outer fibrous cells sal ridges of the follicle and contained a columnar (Fig. 9A). In the seed, the vascular bundle ran mass of thick-walled sclerenchymatous fibers that through the central part of the seed wing and developed along the external surface of the ven- reached the chalazal base (Figs. 9A & 12C, D). tral and the dorsal bundles, respectively (Figs. The innermost layer of the outer seed coat was 7A, B & 8A, B). The endocarp was a single-cell one-cell thick and consisted of small, rounded po- layer of sclerenchymatous, transversely elongat- lygonal cells filled with dark brown material ed fibers (Figs. 7A, C & 8C), but did not develop (Figs. 8B, 9C, D & 10A–C). The inner seed coat in the placental region (Figs. 6C & 7B). was thin and composed of a single layer of flat We counted at least 60 seeds in one of the cells with dark brown material, except at the mi- largest follicles, although it was broken and in- cropylar end where it was three cells thick and complete. From the estimated follicle length and formed the endostome ring (Figs. 8B, 9A, C, D & seed thickness we calculated that there were ~ 10C–E). We observed papillate protrusions on 90–100 seeds in each follicle. The embryos and cells toward the inside of the endostome (Fig. other tissues, except for some organic vestiges, 10D). The anticlinal walls of the tegmic cells had were not preserved in the nucellus. We regarded minute sinuations (Fig. 10E). The main nucellar the seeds to be mature because the follicles have body of the seed was located below the wing started to dehisce (Figs. 4 & 6B). The seeds are (Figs. 6C, 9A & 12C, D). The nucellus had a trans- flat, semicircular to oval, about 2 mm long, 1 mm lucent membranous wall (Fig. 9A, D) that thick- wide, and 0.6 mm thick (Figs. 2B, 4A, 6B–D & ened inside the endostome to form a nucellar cap, 9A). The upper half of the seeds (distal direction which was a small mound-like mass of cells plug- in the follicle) consists of a fleshy layer of paren- ging the endostome (Fig. 10C). chyma that formed an anvil-shaped wing (Figs. 6C, 8D & 9A, B). It is possible that the wing rep- Follicle Wall of Cercidiphyllum japonicum resented a horn-shaped lateral extension in tan- We also examined the follicles of Cercidi- gential view (Figs. 8D & 9A, B). The seeds were phyllum japonicum as the representative of an ex- attached to the placenta by a short funiculus that tant species with follicles like those of Obirafruc- continued to the seed wing (Figs. 6C & 12C, D). tus. Obvious dorsal and ventral ridges are not The ovules were bitegmic and anatropous; the mi- formed in C. japonicum. Three major vascular cropyle formed on the placental side (Figs. 6C & bundles supply the follicle; one dorsal and one on 8B). The outer seed coat consisted of three layers, each side of the ventral suture line (Fig. 13A, B). outer, middle and innermost (Figs. 8B, 9 & 10A– Longitudinal fibrous bundles were externally as- C). The outer layer consisted of the outer epider- sociated with each vascular bundle (Fig. 13 A, B). mis with sparse obtuse trichomes and fibrous der- Nests of vascular bundles were in the lateral walls February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 9
Fig. 6. Obirafructus kokubunii gen. & sp. nov. Sections of follicle. (A) Tangential longitudinal sequential sections through ventral suture at follicle top. Upper slide Kokubun NSM PP-9374 Ctop#1. Lower slide Kokubun NSM PP-9374 Ctop#8. No typical stigmatic features are recognized. (B) Cross section showing pairs of winged seeds. Slide Kokubun NSM PP-9374 Btop#3. (C) Radial longitudinal section through placenta (pl). Arrowhead indicates ventral strand of follicle. Slide Kokubun NSM PP-9374 B1#67. (D) Radial longitudinal section of shoot showing main axis and attached follicles. Slide Kokubun NSM PP-9374 B1#67. (E) Longitudinal section showing main axis and attached follicle stalk. Slide Kokubun NSM PP-9374 B1#77. dr, dorsal ridge of follicle; fs, follicle stalk; mx, main axis; nu, nucellus; vr, ventral ridge of follicle; w, seed wing. Scale bar = 1 mm. 10 Acta Phytotax. Geobot. Vol. 72
Fig. 7. Obirafructus kokubunii gen. & sp. nov. Follicle structures. A, and B: Cross sections. C: Longitudinal section. D: Periclinal section. (A) Dorsal ridge. Slide Kokubun NSM PP-9374 Cbot#19. (B) Ventral ridge that dehisced at its middle. Slide Kokubun NSM PP-9374 Cbot#19. (C) Lateral wall showing three pericarp layers. Note lateral vascular bundles (lv) in parenchymatous mesocarp. Slide Kokubun NSM PP-9374 Btop#5. (D) Outer epidermis with stomata. Slide Kokubun NSM PP-9374 B1#77. ds, dorsal vascular strand; en, fibrous endocarp; ep, epidermis; fb, fibers; gc, guard cells; hy, hypodermis; pl, placenta; sd, seed coat; st, seed trace; vs, ventral vascular strand. Scale bar = 100 µm. February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 11
Fig. 8. Obirafructus kokubunii gen. & sp. nov. Follicle and seed structures. A, and B: Radial longitudinal sections of follicle. C, and D: Tangential sections of follicle. (A) Dorsal ridge tissues. Slide Kokubun NSM PP-9374 Bbot#9. (B) Placental side of seed (arrowhead shows endostome). Slide Kokubun NSM PP-9374 B1#68. (C) Paradermal view of fibrous endocarp (en). Note simple-pitted cell walls. Slide Kokubun NSM PP-9374 Bbot#9. (D) Two rows of seeds in follicle. Slide Kokubun NSM PP-9374 Btop#3. fb, fibers; hy, hypodermis; is, inner seed coat; lw, lateral wall of follicle; os, outer seed coat; pa, parenchyma; w, seed wing. Scale bar = 200 µm. of the follicle. The pericarp of Cercidiphyllum ja- Discussion ponicum can be divided into three layers (Fig. 13). The exocarp consists only of the outer epi- Obirafructus kokubunii gen. nov. & sp. nov. is dermis (Fig. 13). The mesocarp, in which the vas- characterized by a woody axis, which helically cular bundles run, is parenchymatous and usually bears numerous conduplicate follicles containing eight cells thick (Fig. 13). We observed three col- abundant seeds in two rows along the marginal umns of vertically oriented fibers in the mesocarp placenta. The absence of any vestiges of bracts, of young carpel sections, one external to the dor- perianth or stamens on the axis or in the axil of sal vascular bundle and two external to the ven- each follicle stalk suggests that the specimen of tral vascular bundles (Fig. 13A, B). Nests of fibers O. kokubunii represents a single flower rather occurred dorsally in the mature mesocarp where than an inflorescence (Figs. 6D, E & 11D). This the fibers are somewhat directed either vertically view is also supported by the internode anatomy, or obliquely (Fig. 13C). The mature endocarp was given that the follicle traces are derived directly about 12 cells thick at its thickest and was com- from the main woody axis without associated posed of horizontally-elongated sclerenchyma- traces of possible axillary organs (Fig. 3). tous fibers (Fig. 13C). The endocarp was not Although the morphotype, an elongated floral formed in the placental regions (Fig. 13B, C). axis with many follicles, is not known in extant 12 Acta Phytotax. Geobot. Vol. 72
Fig. 9. Obirafructus kokubunii gen. & sp. nov. Seed structures. (A) Oblique cross section showing endostome (es) and inner seed coat (is). Single seed vascular bundle (sv) runs through upper central portion of wing (w) and nearly reaches chalazal base. Note thin and translucent nucellar wall (nw). Slide Kokubun NSM PP-9374 Btop#93. (B) Cross section at marginal portion of seed wing. Note epidermis with obtuse trichomes (ot) on fibrous dermal cells. Slide Kokubun NSM PP-9374 Btop#9. (C) Cross section of seed coats of two vertically aligned seeds. Only outer epidermis and middle layer of outer seed coat are shown for lower seed. Slide Kokubun NSM PP-9374 B#30. (D) Horizontal longitudinal section of seed coat near chalazal end. Slide Kokubun NSM PP-9374 Bbot#9. fb, fibers; ie, innermost layer of outer seed coat; pa, parenchyma. Scale bar = 100 µm.
Saxifragales, we inferred that Obirafructus 1986, Stevens 2001 onwards, Soltis et al. 2018). kokubunii belongs to that order and the most Apical styles and stigmas occur in living Cercidi- closely related living relative is Cercidiphyllace- phyllaceae, and the stigmas are characterized by ae, based on the following. Plants having vessels unicellular papillae (Swamy & Bailey 1949, with scalariform perforations, conduplicate and Spongberg 1979). In fossil Cercidiphyllaceae so ventricidal carpels, and marginal placenta, as in far reported, the stigmatic regions are not pre- O. kokubunii, are only known in saxifragalean served (Crane 1984, Crane & Stockey 1985, Feng families (Stevens 2001 onwards, Golovneva & et al. 2000, Golovneva & Alekseev 2017). Al- Alekseev 2017, Soltis et al. 2018). Among the though the style and the stigmas were not ob- Saxifragales, follicles (i.e., apocarpous gynoecia) served in O. kokubunii, it is possible that they occur in Cercidiphyllaceae and Paeoniaceae. In originally formed but shrunk during follicle de- particular, the former resembles O. kokubunii velopment. The stigma of Cercidiphyllum japoni- with regard to the formation of numerous com- cum actually shrivels after anthesis and falls pressed, squarish, winged seeds (Mohana Rao sometime before the follicle dehisces (Spongberg February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 13
Fig. 10. Obirafructus kokubunii gen. & sp. nov. Seed structures. (A) Cross section of winged portion of outer seed coat showing pitted idioblasts in parenchyma layer (pa) and innermost layer (ie). Slide Kokubun NSM PP-9374 Cbot#19. (B) Oblique periclinal section of lower portion of outer seed coat, showing fibrous outer layer (fb) and innermost layer. Slide Kokubun NSM PP-9374 Bbot#9. (C) Radial longitudinal section through entire micropyle, showing endostome (es), inner seed coat (is) and nucellar cap (nc). Slide Kokubun NSM PP-9374 B1#68. (D) Cross section of endostome. Note papillate protrusions of cells in micropylar opening. Slide Kokubun NSM PP-9374 Btop#93. (E) Paradermal view of inner seed coat. Slide Kokubun NSM PP-9374 Btop#5. Scale bar = 50 µm. 14 Acta Phytotax. Geobot. Vol. 72
1979). Stockey 1985, 1986, Mohana Rao 1986, Feng et The Saxifragales are estimated to have ap- al. 2000, Remizowa et al. 2009, Golovneva & peared about 120 million years ago, during the Alekseev 2017). We compared the follicle clus- Aptian age (Magallón et al. 2015), and fossil Cer- ters among species belonging to different genera cidiphyllaceae have been reported from the Low- of Cercidiphyllaceae, Cercidiphyllum japonicum, er Cretaceous- (at the earliest from the lower- Joffrea speirsii, and Jenkinsella jiayinense and middle Albian) to Cenozoic-era strata, mainly O. kokubunii. The complexity of the follicle clus- based on impression/compression specimens, ters in Eocercidianthus (not discussed here), was with the exception of some pyritized materials the same as in C. japonicum (Krassilov 2010). from the Eocene London Clay (Crane 1984, The female reproductive shoot of C. japonicum Crane & Stockey 1985, Feng et al. 2000, Golov- superficially appears as a multicarpellate flower, neva & Alekseev 2017). The oldest possible an- although it is a short inflorescence composed of cestral ally of extant Cercidiphyllum has been de- 2–6(–13) monocarpous flowers (Fig. 11A, Swamy scribed as ‘Eocercidiphyllites plants’ from the & Bailey 1949, Tucker & Grimes 1999). In C. ja- Turonian of the Negev Desert in Israel (Krassilov ponicum, each carpel is subtended by a bract, et al. 2005, Krassilov 2010). This morphotype as- which may be a tepal (Yan et al. 2007). In addi- semblage consists of Eocercidiphyllites Krassilov tion, rudiments of a floral apex and a second car- (leaves and shoots), Eocercidianthus Krassilov pel of single flower were observed in the inflores- (fascicle with follicles), and Eocercidispermum cence (van Heel 1986, Endress 1993). The inflo- Krassilov (seeds) (Krassilov et al. 2005). There is rescence of Jo. speirsii consists of flowers com- considerable variation in morphology of inflores- prised of either a single or a pair of carpels; a cences/infructescences among fossils of Cercidi- bract occurs between the carpel stalk or pedicel phyllaceae, as described in the next paragraph, and the peduncle (Fig. 11B, Crane & Stockey which has resulted in nomenclatural problems 1985, 1986). In Je. jiayinense the flowers always when classifying fossil taxa. As a result, the no- comprise a pair of carpels with a joint scar be- menclature has been revised by synonymizing tween the pedicel and the peduncle, although most names under the genus Jenkinsella Reid & bracts are not present (Fig. 11C, Feng et al. 2000). Chandler (Golovneva & Alekseev 2017). Detailed In C. japonicum, the adaxial surface of each car- taxonomic comparisons and stratigraphic and pa- pel faces away from the main inflorescence axis leophytogeographic distributions of Jenkinsella (Fig. 11A, Swamy & Bailey 1949), whereas in Je. jiayinense (G. P. Feng, C. S. Li, Zhilin, Y. F. Wang jiayinense, the carpels first face each other and & Gabrielyan) Golovneva & P. Alekseev and oth- the carpel stalk later twists and the ventral slit er fossil Cercidiphyllaceae can also be consulted turns to face the outside of infructescence (Fig. in Golovneva & Alekeev (2017). Joffrea speirsii 11C, Feng et al. 2000). If O. kokubunii was de- Crane & Stockey from the Paleocene Paskapoo rived from cercidiphylloids with a compound in- Formation in North America is an exceptional ex- florescence, the elongated flower O. kokubunii ample of a wholly reconstructed fossil of Cercidi- should be phylogenetically derived from such an- phyllaceae (Crane & Stockey 1985). cestral forms through complete reduction of the We assume that the flowers of Obirafructus bracts and other associated floral organs. How- kokubunii flower were pistillate and produced ever, O. kokubunii is older than the estimated di- multiple follicles (Fig. 11D), although the possi- vergence time of cercidiphylloids (Magallón et bility that stamens or a perianth was attached to al. 2015). It is also reasonable to consider that O. the missing basal part of the main axis cannot be kokubunii represents a floral morphotype earlier denied. Living and fossil Cercidiphyllaceae are than the establishment of cercidiphylloid inflo- dioecious and their single follicle or follicle pairs rescences during the early Cretaceous diversifi- corresponds to a pistillate flower (Fig. 11A–C, cation of the saxifragalean stem groups. Saxi- Swamy & Bailey 1949, Spongberg 1979, Crane & fragales are not supported by any morphological February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 15 B A vl vl
br fs jo br pe pd pe C D
vl
vl fs pd pe jo
Fig. 11. Comparison of reproductive shoots. (A) Cercidiphyllum japonicum (modified from Fig. 19 in Crane & Stockey 1986). (B) Joffrea speirsii (modified from Fig. 20 in Crane & Stockey 1986). C( ) Jenkinsella jiayinensis (modified from Fig. 29 of “Nyssidium jiayinense” in Feng et al. 2000). (D) Obirafructus kokubunii gen. & sp. nov. br, bract; fs, follicle stalk; jo, joint of follicle stalk and peduncle; pe, peduncle and branch of peduncle; pd, pedicel vl, ventral slit. synapomorphies (Hermsen et al. 2006). The has been the focus of most research since Sun et present morphological variation in the order is al. (1998, 2002) proposed that it was the oldest derived from diversifications in the early Creta- angiosperm, from the late Jurassic, and that its ceous period (Magallón et al. 2015) and possible reproductive structure represented the most subsequent extinctions. primitive floral state. However, later researchers The elongated flowers and radially arranged revised the age of Archaefructus, stating that it follicles of Obirafructus kokubunii are reminis- corresponded to the Barremian–Aptian age (~125 cent of Archaefructus Sun, Dilcher, Zheng & Ma) and that the elongated ‘flower’ was a bisexu- Zhou, Archaeanthus Dilcher & Crane, and other al inflorescence (Friis et al. 2003, Zhou et al. fossil magnoliids and ranunculids. Archaefructus Fig.2003, Ji et al. 2004). Additionally, Archaefructus11 16 Acta Phytotax. Geobot. Vol. 72
Fig. 12. Comparison of seed structures. A, and B: Cercidiphyllum japonicum (modified from Swamy & Bailey 1949, Krassilov 2010). C, and D: Obirafructus kokubunii gen. & sp. nov. A, and C: Vertical longitudinal sections. B, and D: Sections at broken lines in (A & C), respectively. is, inner seed coat; nu, nucellus; os, outer seed coat; sb, seed base attached to placenta; sv, seed vascular bundle; w, wing. differs from Obirafructus kokubunii in that it is a thus is regarded as representing a stem group of smaller, herbaceous plant, although fossil speci- extant Magnoliaceae (Friis et al. 2011). In the mens were compressed and the anatomical struc- Magnoliaceae, abaxial dehiscence is common, ture could not be observed (Friis et al. 2003, Zhou but the carpels of Archaeanthus dehisce adaxial- et al. 2003, Ji & al. 2004). Archaefructus eoflora ly (Dilcher & Crane 1984, Romanov & Dilcher Ji, Li, Bowe, Liu & Taylor is reported to demon- 2013). The carpel structure of Archaeanthus is strate laminar placentation (Ji et al. 2004), which apparently similar to that of O. kokubunii; how- is distinct from the marginal placentation of Obi- ever, Archaeanthus has hairy stigmatic crests rafructus. Archaeanthus from the late Albian to along the ventral ridges (Dilcher & Crane 1984, the early Cenomanian age of North America ex- Romanov & Dilcher 2013). Such a stigmatic re- hibits bisexual, multicarpellary, apocarpous flow- gion was not present in O. kokubunii, which rep- ers that bear 100–130 loosely spaced condupli- resents a significant difference from Archaean- cate follicles (Dilcher & Crane 1984). Archaean- thus. February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 17
The conduplicate follicular morphotype is O. kokubunii and living Cercidiphyllum japoni- shared among other late Early to Late Cretaceous cum based on observations and previous studies, fossil forms of earlier magnoliids and ranuncu- given that the known anatomical details of fossil lids (Krassilov 2010). Protomonimia H. Nishida Cercidiphyllaceae are limited and depend on the & Nishida, Hidakanthus Nishida, Ohsawa, H. state of preservation of the fossil. Follicles of O. Nishida, Yoshida & Kanie, and Keraocarpon kokubunii and C. japonicum show a typical con- Ohana, T. Kimura & Chitaley are included in the duplicate structure with three major vascular former, while Hyrcantha Krassilov & Vachra- bundles in their three-layered walls, but differ in meev and Ternaricarpites Krassilov & Volynetz follicle wall (pericarp) proportions and histologi- are included in the latter (Krassilov et al. 1983, cal features. Nishida & Nishida 1988, Nishida et al. 1996, The pericarp in Obirafructus kokubunii and Ohana et al. 1999, Dilcher et al. 2007, Krassilov Cercidiphyllum japonicum is divisible into exo- & Volynetz 2008). In the former forms, which are carp, mesocarp, and endocarp (Figs. 7 & 13, Mo- permineralized and possible eumagnoliid struc- hana Rao 1986). The exocarp of O. kokubunii had tures from the Turonian to Santonian of Hokkai- hypodermal cells containing tannin-like sub- do, Japan, the carpels developed adaxial hairy stances, while C. japonicum lacks a hypodermis stigmatic crests rather than the terminal stigmatic but produces tannin-containing cells in the outer area, although the presence of stigmatic hairs was layer of the mesocarp (Figs. 7A–C, 8A & 13, Mo- only confirmed in Protomonimia (Nishida & hana Rao 1986). The mesocarp of both O. kokubu- Nishida 1988, Nishida et al. 1996, Ohana et al. nii and C. japonicum resemble each other in their 1999). Obirafructus kokubunii has a solitary fol- parenchymatous nature, thickness, presence of licle with a short stalk on a woody axis and de- vascular bundles, and occurrence of a vertical fi- hisces adaxially. Hircantha, however, produces ber column along the major three vascular bun- basally united follicles on an herbaceous axis dles in the follicle (Figs. 7 & 13, Mohana Rao (Krassilov et al. 1983, Dilcher et al., 2007) and 1986). In contrast to the thick endocarp of C. ja- Ternaricarpites has dorsicidal follicles with an ponicum, the endocarp of O. kokubunii is thin abaxial dehiscent crest (Krassilov & Volynetz and consists of a single layer of fibrous cells (Fig. 2008). These morphotypes can be interpreted as 7A, C). The endocarp of C. japonicum is also being derived during earlier diversification of composed of fibrous cells, although it is much pre- to early Eudicots before the appearance of thicker (ca. eight cells thick) than that of O. the core Eudicots, including Saxifragales. There- kokubunii (Figs. 7A, C & 13, Mohana Rao 1986). fore, we hypothesize that O. kokubunii represents In young follicle walls of C. japonicum, the endo- a new, permineralized fossil genus of Saxifrag- carp is thin, but thickens during a later develop- ales represented by a single carpellate floral axis mental stage via periclinal cell division (Mohana without assigning it to a particular family. We Rao 1986). The transverse orientation of the fi- propose that O. kokubunii represents a new mor- brous cells of the mesocarp in both genera prob- photype that will aid in reconstructing the past ably helps to protect the follicle from being diversity of the Saxifragales, and more widely, crushed and further increases mechanical tension Eudicots in general. when the follicles dry and dehisce. The winged seeds or samaras with a one-sid- Comparison with Cercidiphyllum japonicum and ed anatropous chalazal wing are widely seen in Paleoecology of Obirafructus taxa of Cercidiphyllaceae (e.g., Cercidiphyllum The general morphology of follicles and seeds japonicum, Jenkinsella jiayinense, and Joffrea in Obirafructus kokubunii and the Cercidiphylla- speirsii; Stockey & Crane 1983, Crane & Stockey ceae show many similarities, but there are also 1985, Feng et al. 2000, Krassilov 2010, Golovne- anatomical and histological differences between va & Alekseev 2017). The seeds of Obirafructus them. We compared the anatomical structures of kokubunii and C. japonicum were derived from 18 Acta Phytotax. Geobot. Vol. 72
Fig. 13. Cercidiphyllum japonicum. Follicle cross sections. (A) Dorsal portion of immature follicle. Note absence of hypodermis. (B) Ventral portion of follicle in A. (C) Follicle at a later developmental stage showing pericarp structures. Note thick endocarp. Ventral slit (vl) to right. ds, dorsal strand; en, fibrous endocarp; ep, outer epidermis; fb, fibers; me, mesocarp; pl, placenta; s, seed; vs, ventral strand. Scale bar = 100 µm. February 2021 Kajita & al. — Obirafructus kokubunii gen. & sp. nov. 19 bitegmic, anatropous ovules and the micropyle derived from ancestral forms with equally devel- was formed by the outer and inner seed coats oped peripheral wings. The mid-Albian seeds of (Figs. 8B, 10C & 12A, C, Mohana Rao 1986, En- Eocercidispermum from Israel are interpreted as dress & Igersheim 1999). The outer seed coat on representing such an ancestral Cercidiphyllum the funicular side was indistinguishable from the morphotype (Krassilov 2010). funicular tissue in the two species (Figs. 6C, 8B Crane & Stockey (1985) recognized a vascu- & 12A, C, Endress & Igersheim 1999). The ob- lar ‘hair-pin loop’ in the seed wing as a common tuse trichomes of the outer epidermis and the fi- feature of Cercidiphyllum-like plants, and Obi- brous cell layers in the outer seed coat observed rafructus kokubunii shows similar seed vascula- in Obirafructus kokubunii (Figs. 8B & 9) were ture (Fig. 12C). The vascular bundle in C. japoni- not seen in C. japonicum (Mohana Rao 1986, En- cum, however, forks once at the hair-pin, leaving dress & Igersheim 1999). However, in Jo. speir- a tiny bundle on the opposite side (Fig. 12A, sii, a finely striate seed surface was observed and Krassilov 2010). If this branching is a rudimen- may possibly show the impressions of the fibrous tary ovule-supplying bundle, it may support the layer in the outer seed coat (Crane & Stockey idea that the primitive character state of the an- 1985). The inner seed coat of O. kokubunii had giosperm ovule in the early stage of carpel/ovule 1–3 cell layers that were composed of dark brown evolution was multiovulate, which has been de- cells (Figs. 9C, D & 10), while the inner seed coat duced from Evo-Devo studies (Yamada et al. of C. japonicum was a three-cell layer of paren- 2016, Nishida et al. 2018). In the seed vasculature chyma (Endress & Igersheim 1999, Mohana Rao of O. kokubunii, such branching was not ob- 1986). served. A nucellar cap was seen in Obirafructus We here consider the habit and habitat of Obi- kokubunii (Figs. 8B & 10C), but not observed in rafructus kokubunii based on the anatomical fea- Cercidiphyllum japonicum (Endress & Igersheim tures of the specimen. The follicles were hypoth- 1999). However, several families in Saxifragales esized to have matured because they have started form a nucellar cap (Stevens 2001 onwards). In C. to dehisce (Figs. 4 & 6B). The large number of japonicum, the ovule is epitropous (the micropyle seeds and the small size of the seed-wing indicate is located above the raphe), and the chalazal side non-zoochoric dispersal as common among both of the seeds expands conspicuously to form a fossil and living Cercidiphyllaceae. The samaras wing in a longitudinal downward manner to the in Cercidiphyllaceae are clearly adapted for ane- follicle base (Fig. 12A, B). In O. kokubunii, the mophily, and the follicles twisting to face the ovule was apotropous (the micropyle was located ventral opening on the outside of the infructes- under the raphe), and the wing of the seed was cence represent a characteristic of efficient seed formed by lateral extension of the anvil-shaped dispersal. The narrower, thicker, and anvil- parenchymatous tissue above the main seed body shaped wing tissue of O. kokubunii (Figs. 6C, 8D (Figs. 8D & 9A, B), with limited expansion on the & 9A, B) appears to be suitable for both ane- charazal side (Fig. 6B). We cannot conclude that mophily and hydrochory. Floating with the main the difference in ovule position with regard to the seed body downward might be an adaptive char- raphe between O. kokubunii and C. japonicum is acteristic that promotes safe seed germination, taxonomically significant because the ovule posi- given that the seed is covered and protected by tion may sometimes vary within the same ovary the wing tissue on sedimentary soil surfaces or (Decraene et al. 2000). It is interesting to note the desiccating shallow water at fluvial margins. that Krassilov (2010) reported anomalies in the The seed coat with sclerenchymatous fibers in O. shape of the wing of C. japonicum, including a kokubunii might increase seed durability. The nu- symmetrical samara developing a bilateral wing. cellar cap plugging the micropyle (Fig. 10C) may Such anomalies suggest that the widespread non- have protected the nucellus and embryo from ei- symmetrical seeds in the Cercidiphyllaceae are ther desiccation or water immersion. We there- 20 Acta Phytotax. Geobot. Vol. 72 fore suspect that O. kokubunii grew on a fluvial Dilcher, D. L. & P. R. Crane. 1984. Archaeanthus: an ear- plain and dispersed its seeds via both water and ly angiosperm from the Cenomanian of the Western Interior of North America. Ann. Missouri Bot. Gard. wind. With regard to the study on vessel perfora- 71: 351–83. tion by Lens et al. (2016), plants with scalariform Dilcher, D. L., G. Sun, Q. Ji, & H. Li. 2007. An early in- perforations tend to grow in environments with- fructescence Hyrcantha decussata (comb. nov.) from out drought stress. For example, scalariform per- the Yixian Formation in northeastern China. Proc. forations occur in Cercidiphyllum japonicum, Natl. Acad. Sci. U.S.A. 104: 9370–9374. which prefers margins of water or wet environ- Endress, P. K. 1993. Cercidiphyllaceae. In: Kubitzki, K., J. G. Rohwer & V. Bittrich (eds.), The Families and ments (Swamy & Bailey 1949, Ishida & Ohtani Genera of Vascular Plants, Flowering Plants, Dicoty- 1974). Although the wood anatomy of O. kokubu- ledons: Magnoliid, Hamamelid and Caryophyllid nii is unknown, the scalariform perforations in families, vol. 2, pp. 250–252. Springer-Verlag, Berlin. the main axis support similar habitat preferences. Endress, P. K. & A. Igersheim. 1999. Gynoecium diver- sity and systematics of the basal eudicots. Bot. J. Linn. Soc. 130: 305–393. We are grateful to Mr. Hakuji Kokubun for donating the Feng, G. P., C. S. Li, S. G. Zhilin, Y. F. Wang & I. G. Ga- specimen. Thanks are due to Mr. Yusuke Takebe, Chuo brielyan. 2000. Nyssidium jiayinense sp. nov. (Cerci- University, who prepared X-ray CT images. Special diphyllaceae) of the Early Tertiary from north–east thanks to Prof. Toshihiro Yamada, Osaka City University, China. Bot. J. Linn. Soc. 134: 471–484. for identifying the ammonoids in the concretion. Materi- Friis, E. M., J. A. Doyle, P. K. Endress & Q. Leng. 2003. als of Cercidiphyllum japonicum were provided by Koi- Archaefructus–angiosperm precursor or specialized shikawa Botanical Garden, Graduate School of Science, early angiosperm? Trends Plant Sci. 8: 369–373. the University of Tokyo. This study was partly supported Golovneva, L. B. & P. I. Alekseev. 2017. Taxonomy and by Chuo University Personal Research Grant to HN in morphological diversity of infructescences Jenkin- 2015 and 2017. We are thankful for the financial support sella co-occurred with Trochodendroides leaves in from the Tropical Biosphere Research Center, University the Cretaceous and Paleogene. Palaeobotany 8: 92– of the Ryukyus. 121. Hermsen, E. J., K. C. Nixon & W. L. Crepet. 2006. 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Received December 11, 2019; accepted May 7, 2020