Ray Imaging of a Dichasium Cupule of Castanopsis from Eocene Baltic Amber

RESEARCH ARTICLE

Synchrotron X-ray imaging of a dichasium cupule of Castanopsis from Eocene Baltic amber

Eva-Maria Sadowski1,4 , Jörg U. Hammel2 , and Thomas Denk3

Manuscript received 30 May 2018; revision accepted 6 September PREMISE OF THE STUDY: The Eocene Baltic amber deposit represents the largest 2018. accumulation of fossil resin worldwide, and hundreds of thousands of entrapped 1 Department of Geobiology, University of Göttingen, arthropods have been recovered. Although Baltic amber preserves delicate plant Goldschmidtstraße 3, 37077 Göttingen, Germany structures in high fidelity, angiosperms of the “Baltic amber forest” remain poorly studied. 2 Institute of Materials Research, Helmholtz-Zentrum Geesthacht, We describe a pistillate partial inflorescence of Castanopsis (Fagaceae), expanding the Max-Planck-Str. 1, 21502 Geesthacht, Germany knowledge of Fagaceae diversity from Baltic amber. 3 Department of Palaeobiology, Swedish Museum of Natural History, Box 50007, 10405 Stockholm, Sweden METHODS: The amber specimen was investigated using light microscopy and 4 Author for correspondence (e-mail: eva-maria.sadowski@ synchrotron-radiation-based X-ray micro-computed tomography (SRμCT). geo.uni-goettingen.de) KEY RESULTS: The partial inflorescence is a cymule, consisting of an involucre of scales Citation: Sadowski, E.-M., J. U. Hammel, and T. Denk. 2018. Synchrotron X-ray imaging of a dichasium cupule of Castanopsis that surround all four pistillate flowers, indicating a dichasium cupule. Subtending bracts from Eocene Baltic amber. American Journal of Botany 105(12): are basally covered with peltate trichomes. Flowers possess an urecolate perianth of 2025–2036. six nearly free lobes, 12 staminodia hidden by the perianth, and a tri-locular ovary that doi:10.1002/ajb2.1202 is convex-triangular in cross section. The exceptional three-dimensional preservation suggests that the fossil belongs to the extant East Asian genus Castanopsis. The amber inclusion represents the first record of Castanopsis from Baltic amber and the first pistillate inflorescence of Fagaceae from Eurasia.

CONCLUSIONS: The partial female inflorescence reported here provides an important addition to acorns of Castanopsis described from middle Eocene strata of Europe. Furthermore, the intercontinental distribution of Castanopsis in the Eocene is confirmed. The amber fossil also broadens the picture of the Baltic amber source area, indicating oligotrophic, sandy, bog-like habitats. Finally, this study underscores the great benefit of SRμCT as a powerful tool to investigate plant inclusions from amber in a nondestructive way.

KEY WORDS Baltic amber forest; Castanopsis kaulii; DESY; dichasium cupule; evolution; Fagaceae; paleobotany; paleoecology; 3D reconstruction.

The Eocene Baltic amber is world famous for its insect inclusions, X-ray micro-computed tomography (SRμCT), has increasingly but plant inclusions are extremely rare (Weitschat and Wichard, been applied in the study of three-dimensionally preserved pale- 2010). Nevertheless, recent paleobotanical studies have demon- ontological objects (Friis et al., 2016). SRμCT has also been used strated that plant inclusions are preserved in high fidelity, leading for arthropod inclusions in amber to visualize and digitally dis- to novel insights into the evolution of specific plant taxa (Sadowski sect inclusions without destroying valuable specimens (Stebner et al., 2015, 2016a, b, 2017b) and the paleoecology of amber for- et al., 2016). For plant inclusions, X-ray imaging has been applied est ecosystems (Kaasalainen et al., 2017; Sadowski et al., 2017a). only in few studies of lauralean, fagalean, and coniferous inclu- Determining the exact botanical affinities of three-dimensional sions from Cretaceous amber of Myanmar, New Jersey, Spain, objects (flowers, seeds, and fruits) is challenging, because internal and France (Crepet et al., 2016; Moreau et al., 2017; Gandolfo structures may not be accessible using standard nondestructive et al., 2018; Kvaček et al., 2018). Here, we applied, for the first methods such as light microscopy. During recent years, a new time, SRμCT to a plant inclusion from Baltic amber, showing an powerful nondestructive method, synchrotron-radiation- based exceptional internal preservation of the specimen and allowing

its assignment to the genus Castanopsis in the Fagaceae (oak (Germany) and of the Herbarium of Alexander R. Schmidt (Jena). family). This finding not only adds to the fossil record of sub- Specimens examined: Castanopsis argentea, Java, Indonesia, family Castaneoideae, but also provides novel insight into the GOET019894; Lithocarpus fenestratus, East Bengal, GOET019902; diversity of Fagaceae from Baltic amber. Chrysolepis chrysophylla, Oregon, USA.

Preparation, microscopy, and imaging MATERIALS AND METHODS For an optimal visualization of the inclusion, the amber specimen Age and origin of Baltic amber was carefully ground manually with wet silicon carbide papers (grit sizes 25.8–5.0 μm; Struers, Sarasota, Florida, USA), creating The amber specimen (GZG.BST.21967) investigated here derives an even and smooth facet orientated parallel to the inclusion. To from a collection that was probably established in Königsberg (to- remove scratches, the specimen was finally polished using a leather day Kaliningrad, Russia). The biggest deposit of Baltic amber is cloth and a toothpaste suspension. The amber specimen and extant located near Kaliningrad, where the Jantarny mine yields several pistillate inflorescences of the Castaneoideae were examined un- hundred tons of amber each year (Kosmowska-Ceranowicz, 1997). der a Carl Zeiss AxioScope A1 compound microscope (Carl Zeiss, The “Blue Earth” is the main source layer for Baltic amber, and pol- Oberkochen, Germany), using incident and transmitted light si- len and dinoflagellates suggest a late Eocene age (Priabonian 34–38 multaneously (Figs. 1 and 3D). Samples of extant pistillate inflores- Ma; Kosmowska-Ceranowicz et al., 1997). This age estimate is fur- cences were further studied under a Carl Zeiss Stereo Discovery V8 ther supported by the known stratigraphic range of a great num- dissecting microscope (Carl Zeiss, Oberkochen, Germany) with in- ber of conifer taxa recently described from Baltic amber (Sadowski cident light only (Fig. 3A–C, E–I). Images were taken with a Canon et al., 2017a). The frequently suggested Lutetian age of the Blue EOS 5D digital camera (Canon, Tokyo, Japan). The software pack- Earth (Ritzkowski, 1997) is problematic, since it is based on a dating age HeliconFocus 6.0 (Helicon Soft, Kharov, Ukraine) was used to method of glauconites that can potentially be biased toward older create stacked photomicrographic composites of up to 125 individ- ages due to contamination and/or reworked glauconites (Clauer ual images, which allow visualization of the three-dimensionality of et al., 2005; Grimaldi and Ross, 2017). Small amounts of Baltic the specimens. amber extend to older and younger sediments (Lower Blue Earth, Lutetian and Lower Gestreifter Sand, upper Oligocene), resulting in X-ray tomography of the fossil and image reconstruction an approximate age range of 23–48 Ma for all strata bearing Baltic amber (Standke, 1998, 2008). However, whether the Oligocene am- Imaging of the fossil using SRμCT (Fig. 2) was done at the beam- ber is in fact redeposited Eocene amber has recently been discussed line P05 of the storage ring PETRA III (Deutsches Elektronen- (Standke, 2008). Synchrotron—DESY, Hamburg, Germany) operated by Helmholtz-Zentrum Geesthacht (Haibel et al., 2010; Greving Specimen description and identification et al., 2014; Wilde et al., 2016). The amber piece was mounted on a beamline standard sample-stub with beeswax and imaged using The morphology of the amber fossil was compared with mor- an attenuation contrast setup (Greving et al., 2014). The photon en- phological descriptions of extant Fagaceae (for references and de- ergy applied was 15 keV. A total of 1200 radiographic projections tailed information, see Table 1) and with herbarium material of were recorded at equal steps between 0 and π. The tomographic extant Castaneoideae (Herbarium Göttingen, GOET; Herbarium reconstruction algorithm “back-projection of filtered projections” Alexander R. Schmidt, Jena, Germany). Larson-Johnson (2016) (Huesman et al., 1977) was used after binning the raw data two used 89 morphological characters in her total evidence approach times to yield 32-bit floating point image stacks with isotropic voxel to Fagales. Of these, about 15 could be used if the present fossil was size of 2.62 μm in the reconstructed image volume. Visualization included. However, this data set was used only in combination with was done using Avizo software version 9.1.1 (Thermo Scientific, molecular data. Unfortunately, there is currently no molecular phy- Waltham, Massachusetts, USA; the video files are available in logeny available for Castanopsis. Using morphological characters Appendixes S1–S3; see Supplemental Data with this article). alone, and in particular of a single organ that may have evolved in parallel in unrelated taxa, may produce erroneous phylogenetic sig- nals. Therefore, we decided not to undertake a phylogenetic analysis RESULTS but instead argue for the generic placement of the fossil on the basis of shared sets of characters with modern Castanopsis. Order—Fagales

Family Repository of specimens —Fagaceae Dumort. Specimen GZG.BST.21967 (former collection no. Oe 186) is origi- Genus—Castanopsis (D.Don) Spach nally derived from the amber collection of Joachim Oehlke, which was later incorporated into the Hoffeins Amber Collection. The Species—Castanopsis kaulii sp. nov. (Figs. 1 and 2) specimen is now housed in the Geoscientific Collections of the University of Göttingen. Samples of extant pistillate inflorescences Diagnosis—Cymule: four pistillate flowers, forming a dichasium of Castanopsis argentea (Blume) A.DC., Lithocarpus fenestratus cupule; surrounded by five large bracts, followed by numer- (Roxb.) Rehder, and Chrysolepis chrysophylla (Douglas ex Hook.) ous bracteoles. Bracts: widely trullate to obovate in shape, apex Hjelmq. were taken from specimens of the Herbarium Göttingen acute-obtuse, margin remotely serrate with teeth of different December 2018, Volume 105 • Sadowski et al.—Castanopsis from Eocene Baltic amber • 2027 like fused developed developed Structures scales (in form of (in form remnants) surrounded by by surrounded a cup- of involucre scales numerous separating walls between flowers between Interseminal Absent to present present Absent to Each flower Each flower Well- Absent Absent Stigma punctiform punctiform punctiform punctiform punctiform punctiform Minute, Minute, Minute, Minute, Minute, Minute, 3 3 6–9 Style (rarely 4) (rarely 4–6) 4–5) (2 or) 3 3 (rarely 3 (rarely 3 (rarely 3 (rarely Ovary triangular in section cross triangular in section cross 4–6 loculed) incompletely incompletely 6–9- loculed Inferior, Inferior, 3- loculed; Inferior, Inferior, 3- loculed; Inferior, 3 (rarely 3 (rarely Inferior, Inferior Inferior Inferior, Inferior, 6 Staminodia perianth perianth than perianth or elongated than perianth perianth 12, shorter than 12, shorter than 6–12, shorter Slightly longer 6–8, shorter than walled” walled” walled” walled” walled” walled” walled” walled” Trichomes peltate at peltate base of bracts; solitary, unicellular, acute simple, trichomes inside of perianth peltate; solitary, solitary, peltate; acute simple, trichomes inside and outside of perianth peltate; solitary, solitary, peltate; unicellular, acute simple, trichomes inside and outside of perianth peltate peltate perianth with pale trichomes “Thin- “Thin- “Thin- “Thick- Bracts tomentose, Bracts tomentose, ovaryPubescent subtended by 5 by subtended by bracts, followed higher- numerous imbricate order bracteoles subtended by by subtended primary bract, by followed higher- numerous imbricate order bracteoles per cymule, 1–7 by followed bracteoles order bracts form order a whorl of scales flowers around Subtending bracts Subtending All flowers All flowers All flowers All flowers One primary bract Numerous Two acute bracteoles acute Two higher Numerous Perianth two nearly whorls, acute; ovate free, thick relatively 6 lobes in two nearlywhorls, acute; ovate free, rather thick, fleshy, soft and relatively flexible suburceolate, suburceolate, campanulate, or cyathiform, more less deeply lobed; lobes thick, stiff, carnose or thin sepals in two whorls, short lobes rounded 6–8- toothed Urecolate; 6 lobes in Urecolate; Urceolate, cyathiform; Urceolate, Urceolate, Urceolate, Urceolate, 6 distinct Urceolate, 6- lobed Urceolate, Pistillate flowers Pistillate cymule; involucre cymule; involucre of imbricate scales enclosing all of flowers cymule cupule); (dichasium pseudo- pedicel side flowers present; meet at abaxial side of primary flower cymule; dichasium cupule; undeveloped pseudo- pedicel; meet side flowers abaxially of primary flower flowers per flowers cymule, with its each flower cupule (flower own cupule), cupules or less fused more cymule; flower cupule dichasium cupule 4 flowers per 4 flowers Solitary or 3–5(–7) per Solitary or 3–5–7 (1–)3 or more per (1–)3 or more Solitary or in pairs 1–3 per cymule; Morphological comparison of pistillate inflorescences of the Castaneoideae. Sources of data: Camus, 1929, 1952–54a, b; Brett, 1963; Soepadmo, 1970; Jones, 1986; Kubitzki, 1987, 1988; 1970; Jones, Kaul, 1993; 1963; Soepadmo, 1929, 1952–54a, b; Brett, Camus, of data: Sources Castaneoideae. of the inflorescences Morphological of pistillate comparison

Castanopsis kaulii Castanopsis Genus Castanopsis Lithocarpus Chrysolepis Notholithocarpus Castanea TABLE 1. Nixon, 1997; Huang et al., 1999. 1997; Huang et al., Nixon, 2028 • American Journal of Botany December 2018, Volume 105 • Sadowski et al.—Castanopsis from Eocene Baltic amber • 2029

FIGURE 1. Cymule of Castanopsis kaulii from Baltic amber, GZG.BST.21967. (A) Lateral side of the cymule, showing two of four flowers from the side, surrounded by bracts. (B) Proximal view, showing the rectangular attachment scar (arrowhead) of the cymule. (C) Distal view, exhibiting four pistillate flowers. (D) Two pistillate flowers (distal view), each with three styles that are exposed at the top, exhibiting their circular-shaped cross section. (E) Singular flower with three entire, linear styles. (F) Singular style with punctiform stigma at the apex (arrowhead). (G) Simple, acute trichomes (arrowhead) emerging from the inside of an individual flower. (H–J) Peltate trichomes with a flat discoidal and multicellular cap. Bars = 1 mm (A–C), 200 μm (D, F), 500 μm (E), 100 μm (G–J). sizes. Bracteoles: numerous, narrow, margins irregular dentate. central trichome tuft in each flower (Fig. 2A). Base of cymule and Flowers: obovate; six tepals arranged in two whorls, glabrous, of outer bracts covered with peltate trichomes, shortly stalked, bear- nearly free, elliptic acute; gynoecium inferior, ovaries tri-locular, ing a flat discoidal cap roundish in outline (Fig. 1H); cap composed triangular in cross section, ovaries of side flowers meeting abax- of randomly oriented rectangular cells radiating from the center ially of primary flower; three styles per flower, linear, exceeding (Fig. 1I, J; trichome type 13 [= “thin-walled” peltate] sensu Jones, the perianth, bending outward, stigma punctiform; 12 stami- 1986). nodes, covered by perianth, anthers dorsifixed. Trichomes: two types; (1) solitary, curled or straight, forming a central trichome Remarks—The amber fossil shares relevant features with extant tuft in each flower; (2) peltate, shortly stalked, located on outer Fagaceae, including sessile pistillate flowers arranged in clusters surface of cymule. Interseminal scales: located at the base of flow- and surrounded by a cupule, six-lobed perianth, inferior ovary, ers, shorter than perianth, non-fused, non-vascularized, irregu- 6–12 staminodes, and terete styles, each with a punctiform stigma larly arranged. (Soepadmo, 1972; Kubitzki, 1993). Among Fagaceae, the amber specimen can readily be differentiated from Trigonobalanoideae, Holotype—GZG.BST.21967 (former coll. no. Oe 186) Castanea Mill., Notholithocarpus P.S.Manos, C.H.Cannon et S.H.Oh and Quercus L. by the following characteristics: the ar- Repository—Hoffeins Amber Collection, Geoscientific Collections rangement of flowers in dichasia (always solitary in Quercus and of the University of Göttingen, Germany Notholithocarpus; Camus, 1952–54a, b; Forman, 1964), the punctiform, inconspicuous stigma (stigma elongated, broad, capitate or Locality—Samland, Kaliningrad rosulate in Quercus; broadly capitate, discoid and notched in trig- onobalanoids; Camus, 1936–38; Forman, 1964; Lozano-C. et al., Stratigraphy—Blue Earth layer, late Eocene 1979), the glabrous, cylindric style (pilose or tomentose in trigono- balanoids; Forman, 1964; Lozano-C. et al., 1979; flattened, grooved Etymology—The species in named after Robert B. Kaul (Nebraska) in some Quercus; Camus, 1936–38), and the number of styles (6–9 in honour of his work on reproductive morphology and evolution in Castanea; Camus, 1929). of extant Fagaceae. Three (or more) flowers per cymule (Fig. 3A, E), bracts and bracteoles surrounding the cymule (Fig. 3B, E, G), three styles Description—One detached cymule (Fig. 1A), 2.2 × 2 × 1.5 mm, per flower and punctiform, inconspicuous stigmas (Fig. 3C, D, F, with four pistillate flowers (Figs. 1C and 2C), proximal side with I) are typical of Castanopsis, Lithocarpus Blume and Chrysolepis rectangular attachment scar (Fig. 1B). Cymule surrounded by five Hjelmq. (Table 1; Soepadmo, 1972). These genera also possess pel- large bracts (Fig. 2C), bracts 560–1400 μm wide × 1100–1200 μm tate trichomes (Camus, 1929, 1952–54a; Jones, 1986; Kvaček and long, widely trullate to obovate, apex acute to obtuse, bract mar- Walther, 1988). However, pistillate flowers of Chrysolepis differ from gin irregular, remotely serrate with teeth of different sizes; outer the amber specimen by fewer elongated staminodes that exceed the bracts followed by numerous narrower bracteoles/scales (Fig. 2C), perianth (Fig. 3H, I), the shallowly lobed perianth (Fig. 3I), and the margins of bracteoles irregular dentate. Main vascular cylinder well-developed separating walls between the cupules (Camus, 1929; of cymule branching into four traces, each leading to one flower Hjelmqvist, 1948). The peltate trichomes of the amber specimen (Fig. 2A). Each flower 357–500 μm wide × 893–1071 μm long correspond to the “thin-walled” peltate trichomes of Lithocarpus (styles excluded), urecolate. Perianth glabrous (Fig. 1D), six tepals and Castanopsis (Jones, 1986), as they are multicellular and round- originating above the inferior ovary, nearly free, elliptic, acute in ish in shape. shape, arranged in two whorls. At least five imbricate interseminal, Castanopsis and Lithocarpus are mainly distinguished by their non-vascularized scales are irregularly arranged around the flowers cupules: Castanopsis possesses a dichasium cupule that encloses (Fig. 2A–C, E). The immature inferior gynoecium forms a narrow all flowers of a cymule, whereas Lithocarpus has a flower cupule pseudo-pedicel (Fig. 2A); ovaries convex triangular in cross section in which each flower of a cymule is surrounded by its own cupule (Fig. 2C, E), tri-locular (Fig. 2F), in cross section ovaries of side (Kaul, 1988). Modern interpretations of Fagaceae cupular evolu- flowers meeting on the abaxial side of the primary flower (Fig. 2C); tion suggest that the dichasium cupule is a synapomorphy of all each flower with three long, linear styles that bend outward slightly, Fagaceae, whereas the flower cupule is derived from a reduced di- exceeding the perianth (Fig. 1E), 120–160 μm wide × 860–980 μm chasium cupule (Oh and Manos, 2008). long; stigma inconspicuous, terminal pore at the apex of each style In the flower cupule of Lithocarpus, cupular scales are present at (punctiform; Fig. 1F); 12 staminodes inside the perianth (Fig. 2D), anthesis, forming an involucral cup around each flower (Soepadmo, consisting of filaments and anthers (Fig. 2B), anthers dorsifixed, st- 1970; Abbe, 1974). Later, “the cupule is surrounded by an uninter- aminodes shorter than perianth (Fig. 2B). Space between styles, sta- rupted spiral of bracts or by annular rings of lamellae” (Forman, mens and inner perianth filled with numerous solitary unicellular 1966: 400). Although the amber fossil possesses interseminal scales trichomes, curled or straight, simple, acute apex, singular (Fig. 1G; (Fig. 2A, B, E), they do not form a cup-like structure that completely trichome type 1 = solitary unicellular sensu Jones, 1986), forming a envelopes each flower, but are irregularly distributed between the 2030 • American Journal of Botany December 2018, Volume 105 • Sadowski et al.—Castanopsis from Eocene Baltic amber • 2031

FIGURE 2. SRμCT sections of Castanopsis kaulii; f1–f4 = singular flowers, sta = staminodes, an = anther, fi = filament, tr = trichome (simple, acute), p = perianth, s = style, o = ovary, l1–l3 = locules, isc = interseminal scales, br1–br5 = bracts, bl = bracteole, v = vascular cylinder, v1–v4 = tracts forcing from vascular cylinder. (A) Longitudinal section through the cymule. (B) Longitudinal section through f1, magnified; note the dorsifixed anther. (C) Cross section through the base of the cymule. (D) Cross section through f1, showing 12 anthers (an1–an12) and three styles (s1–s3). (E) Cross section through the base of f4 and f3, with interseminal scales (isc). (F) Cross section through the ovule of f1 exhibiting three locules (l1–l3). Bars = 500 μm (A), 100 μm (B–D), 50 μm (E, F). flowers without a specific ring- or spiral-like pattern (Fig. 2E), as we of Castanopsis from Europe are compressed fruits from middle would expect for a flower of Lithocarpus. Furthermore, cupules of Eocene lignite deposits of Germany (Geiseltal flora, MP13, 44.3 each flower in Lithocarpus are fused (Camus, 1952–54a; Soepadmo, Ma; Mai, 1976). The trigonous shape of these fruits, as well as 1970) and vascularized (Kaul, 1987), while the interseminal scales their quadrant-like cross section, indicate that more than one of the amber fossil are singular, non-fused, and lack vascular traces nut was enclosed by a cupule, as it occurs in extant Castanopsis (Fig. 2A–C, E). Hence, we conclude that they do not represent (Mai, 1976). Fossil fruits of Castanopsis are most abundant from cupular scales. the upper Oligocene to lower Miocene of numerous fossil lo- Besides the flower/cupular morphology, Lithocarpus is also calities of Central Europe (e.g., Germany [Zülpich, Frielendorf, distinct from the amber fossil in the elongated staminodes, which Kleinsaubernitz], Poland [Turow, Osieczów], Hungary [Noszvaj/ overtop the perianth (Fig. 3F; Camus, 1952–54a; Soepadmo, 1970), Eger], Austria [Leoben]; for a complete list of fossil localities, see although pistillate cymules of androgynous inflorescences in cer- Mai, 1989: table 1). Castanopsis disappeared from Europe at the tain Lithocarpus species may possess underdeveloped staminodes end of the Pliocene (Mai, 1989). (Camus, 1952–54a). A number of leaf impressions and wood fossils further suggest Several Castanopsis species exhibit basal structures separating that the genus was present in East Asia during the Paleogene. From the flowers and fruits of a cupule (Hjelmqvist, 1948), which is simi- late Eocene (Priabonian) strata of European Russia, Vikulin (2011) lar to the interseminal scales of the amber fossil. They further share reported Castanopsis timensis (Palib.) Iljinskaja and Castanopsis sp. the triangular outline of the ovary in cross section (Hjelmqvist, Although these leaf imprints resemble modern Castanopsis species, 1948) and the arrangement of flowers, with side flowers meeting the generic affinity cannot unambiguously be established without at the abaxial side of the primary flower (Fig. 2C; Hjelmqvist, 1948; diagnostic leaf epidermal characteristics. From upper Oligocene de- Soepadmo, 1970). In summary, the combination of the following posits of South China, Huang et al. (2018) described wood remains features justifies the assignment of the amber fossil to Castanopsis: as Castanopsis nanningensis Huang et al. and C. guangxiensis Huang flowers are arranged in a cymule; flowers are encircled by numerous et al., which they compared to modern East Asian Castanopsis spe- bracts and bracteoles; side flowers meet at abaxial side of the pri- cies. Thus, there is no unambiguous fossil record of Castanopsis mary flower; three cylindric styles per flower; triangular outline of from the Eocene of East Asia. the ovary in cross section; punctiform stigma; staminodes hidden Dispersed pollen of castaneoids (corresponding to the par- by the perianth; presence of “thin-walled” peltate trichomes; and aphyletic subfamily Castaneoideae Oerst. that includes the ex- lack of separating walls or fused cup-like cupules surrounding each tant genera Castanea, Castanopsis, Chrysolepis, Lithocarpus, and flower. Notholithocarpus) generally does not give further insight into the fossil history of particular genera. This is due to the ornamentation of the pollen surface (smooth striation, microreticulate, rod-like DISCUSSION fused), which is virtually identical among all castaneoids and, thus, noninformative at the generic level (Crepet and Daghlian, 1980; Fossil records of Castanopsis Praglowski, 1984). One exception is pollen similar to Castanopsis cuspidata (section Pseudopasania Camus) from western Greenland Permineralized fruits of Castanopsis crepetii Manchester from late (Aamaruutissaa Member of the Hareøen Formation, Hareøen; mid- Ypresian (early Eocene) strata of North America (Manchester, 1994) dle Eocene, late Lutetian-early Bartonian; Grímsson et al., 2015). represent the oldest member of this genus. As in extant Castanopsis, These dispersed pollen grains show distinct ornamentation not re- the endocarp of fruits in C. crepetii is fused with the cupule, and ported for other genera in Castaneoideae and share diagnostic fea- cupules completely enclose a single nut. Furthermore, the endo- tures with extant Castanopsis (C. cuspidata (Thunb.) Schottky; see carp (nutshell) anatomy of C. crepetii is identical to that in modern discussion in Grímsson et al., 2015). Castanopsis (Manchester, 1994). Up to now, the only known fossil of a Castanopsis-like pistillate In Europe, fossils of wood, fruits, and leaves of Castanopsis inflorescence is from early to middle Eocene deposits of Tennessee derive from middle Eocene to middle Miocene strata (Mai, 1989). (United States) and has been described as Castanopsoidea colum- However, leaf impression fossils identified as Castanopsis are biana Crepet et Nixon (Crepet and Nixon, 1989). Due to the lack doubtful, because identical leaf morphologies evolved in various of diagnostic characteristics of Castanopsoidea, affinities to “either extant and extinct members of Fagaceae (Kvaček and Walther, Castanopsis or Chrysolepis or their common ancestor” were dis- 1988, 1989, 2012; Mai, 1989). Also, some pre-Oligocene fos- cussed (Crepet and Nixon, 1989: 847). Castanopsoidea is dissimilar sil fruits with presumed affinities to Castanopsis from Central to the Baltic amber inclusion in that the styles of Castanopsoidea Europe (Mai, 1989) are questionable and would need to be reex- columbiana are twice as long as those in Castanopsis kaulii (2 mm amined. Among those, fruit-like structures from the Paleocene of in Castanopsoidea, Crepet and Nixon, 1989; 860–980 μm in France belong to the extinct betulaceous genus Palaeocarpinus Castanopsis kaulii). In contrast to Castanopsoidea, the exceptional Crane (Pigg et al., 2003). The oldest unambiguous fossil records three-dimensional preservation of Castanopsis kaulii provides the 2032 • American Journal of Botany December 2018, Volume 105 • Sadowski et al.—Castanopsis from Eocene Baltic amber • 2033

FIGURE 3. Pistillate inflorescences of extant Castanopsis argentea (A–D), Lithocarpus fenestratus (E, F), and Chrysolepis chrysophylla (G–I). (A) Overview of a pistillate inflorescence of Castanopsis argentea (GOET019894). (B) Three-flowered cymule; note the large subtending bract. (C) Singular flower, detached from the cymule. (D) Style; arrowhead indicates punctiform stigma. (E) Overview of pistillate inflorescence of L. fenestratus (GOET019902). (F) Three-flowered cymule, showing staminodes overarching the perianth (arrowhead). (G) Overview of pistillate cymule at the base (arrowhead) of a staminate inflorescence of Chrysolepis chrysophylla. (H, I) Cymule (H) and singular flower (I), note the elongated staminodes (arrowhead). Bars = 1 mm (A, B, E, G, H), 500 μm (C, F, I), 100 μm (D). same set of informative characters that can be observed in extant well-preserved plant inclusions such as the Castanopsis cymule Castanopsis and allows its definite generic assignment. described in the present study provide important additions to the Numerous inclusions of fagaceous affinities have previously Eocene flora of the Baltic amber. been reported from Baltic amber, but they mostly represent stellate Extant Castanopsis species are dominant constituents in East trichomes and staminate inflorescences with affinities to Quercus Asian broad-leaved evergreen forests (Aoki et al., 2014). The re- and Castanea (Conwentz, 1886; Kirchheimer, 1937). Czeczott cent distribution extends from Northeast China, Korea, Taiwan, (1961) hypothesized that Fagus succinea Goepp. et Menge, an in- and Japan to the Himalayas; however, most species are found in clusion of a trigonous fruit from Baltic amber (Goeppert, 1853; Southeast Asia (Malay Peninsula, Indonesia, Thailand, New Guinea, Conwentz, 1886), might be affiliated toCastanopsis . However, the Philippines; Soepadmo, 1972; Kaul, 1988). Extant Castanopsis particular inclusion was later assigned to Trigonobalanus succinea mainly occurs in Quercus-Castanea forests of mid-montane or (Goepp. et Menge) Forman (Trigonobalanoideae), since the trigo- near-coast evergreen lowland areas, associated with further ev- nous shape as well as the discoid, slightly concave stigmas are in- ergreen species in the families Fagaceae (Quercus, Lithocarpus, dicative for trigonobalanoid taxa (Forman, 1964; Mai, 1967). Thus, Trigonobalanus Forman), Lauraceae (e.g., Actinodaphne Nees, Castanopsis kaulii represents the first unambiguous fossil record of Cinnamomum Schaeff.), Magnoliaceae (e.g., Magnolia L., Manglietia this genus from Baltic amber, as well as the first report of a pistillate Blume), Hamamelidaceae (Exbucklandia R.W.Brown), Altingiaceae inflorescence fossil of Fagaceae from Eurasia. (Altingia Noronha), and Ericaceae (Rhododendron L.). Further habitats of extant Castanopsis encompass mixed mesophytic for- Paleoecological implications of Castanopsis kaulii for the “Baltic est with deciduous and evergreen tree species and rarely extremely amber forest” wet habitats, such as swamps and mires (C. foxworthyi Schottky, C. fulva Gamble, C. borneensis King). In Laos, Taiwan, Yunnan, The Baltic amber source area (also called “Baltic amber forest”) and Japan, Castanopsis also inhabits coniferous forests, associated was composed of coastal lowland swamps, raised bog habitats, ri- with Pinaceae (Abies Mill., Keteleeria Carrière, Pinus, Pseudotsuga parian forests and mixed mesophytic conifer-angiosperm forests, Carrière, Tsuga (Endl.) Carrière) and cupressaceous taxa (spe- intermingled by open areas, such as meadows (Sadowski et al., cies in the genera Chamaecyparis Spach, Cryptomeria D.Don, 2017a). These habitat types are characterized by high conifer di- Cunninghamia R.Br. ex A.Rich., Taiwania Hayata; Soepadmo, 1972; versity, including Cupressaceae (e.g., Quasisequoia couttsiae (Heer) Mai, 1989, and references therein). L.Kunzmann, and Calocedrus Kurz), Pinaceae (e.g., Nothotsuga In Oligocene-Miocene European floras, Castanopsis was mainly protogaea L.Kunzmann et Mai, Pseudolarix Gordon, Pinus L.), associated with Pinus subgen. Pinus, Symplocos Jacq., Quercus sect. Sciadopityaceae (Sciadopitys cf. tertiaria Menzel emend. Weyland, Lobatae, Laurophyllum Goepp. (Lauraceae) and Myrica L., forming Kilpper et Berendt) and Geinitziaceae (Cupressospermum saxoni- Castanopsis-Lauraceae forests (also called “Castanopsietum oligo- cum Mai; Sadowski et al., 2016a, 2017a). miocenicum taphocoenosis”; Mai, 1989, 1995). These forests further The “Baltic amber forest” also contained numerous angio- comprised several conifer species, such as Quasisequoia Sriniv. et sperm taxa, such as graminids (Rhynchospora Vahl, Cyperaceae; E.M.Friis, Sciadopitys (Thunb.) Siebold et Zucc. and Taxodium Rich., Poaceae; Sadowski et al., 2016b), carnivorous plants (Roridulaceae; as well as Mastixiaceae, Magnoliaceae, and deciduous plants (e.g., Sadowski et al., 2015), and dwarf mistletoes (Arceuthobium Carya Nutt., Acer L., Juglans L.; Mai, 1989). The floristic composition M.Bieb., Viscaceae; Sadowski et al., 2017b). However, the knowl- of the Castanopsis taphocoenosis mostly resembles mixed meso- edge of the Baltic amber flora, especially of its angiosperms, is phytic forests of extant East Asia but also comprised North American far from being complete, since the most comprehensive accounts flora elements (e.g.,Sequoia Endl.). In contrast to the majority of ex- on plants from Baltic amber (e.g., Goeppert and Berendt, 1845; tant Castanopsis species, Paleogene and Neogene Castanopsis mainly Goeppert and Menge, 1883; Conwentz, 1886, 1890; Caspary and occurred on acidic and oligotrophic soils in peat-like habitats, river Klebs, 1907) have not been fully revised. Fagaceae inclusions, es- sands, and dunes (Mai, 1976, 1989; Gee et al., 2003). pecially staminate inflorescences and stellate trichomes, are by far The ecological characteristics of extant and extinct Castanopsis the most abundant plant inclusions in Baltic amber (Weitschat are consistent with the current reconstruction of the “Baltic am- and Wichard, 1998). According to Caspary (1881) and Conwentz ber forest” (Kaasalainen et al., 2017; Sadowski et al., 2017a) where (1886), Baltic amber Fagaceae are very diverse, comprising about raised bog habitats, riparian forests, and riversides provided fa- 14 species in the genera Quercus (10 spp.) and Castanea (4 spp.). vorable conditions for Castanopsis. As in the Castanopsietum However, revisions by Kirchheimer (1937), Czeczott (1961), and oligo-miocenicum taphocoenosis, plant taxa with affinities to Iljinskaja (1982) accepted only four to six fagaceous species from East Asian and North American floras are present in Baltic am- Baltic amber (three to four Quercus spp. and one Castanea sp.). ber, such as Pinus, Sciadopitys, and Taxodium (Sadowski et al., Kirchheimer (1937) furthermore pointed out that generic as- 2016a, 2017a) which were likely associated with Castanopsis kau- signments of the staminate inclusions to Castanea, Castanopsis, lii. Besides conifers, other Fagaceae probably co-occurred with C. Pasania (= Lithocarpus), and Quercus are difficult, because di- kaulii in the “Baltic amber forest,” similar to the Oligocene-Miocene agnostic characters were mostly not preserved. Therefore, Castanopsis-Lauraceae forests (Mai, 1989). 2034 • American Journal of Botany

As in extant Southeast Asian evergreen-broadleaved for- and A. R. Schmidt (Göttingen) kindly provided access to herbarium ests where Castanopsis co-occurs with Trigonobalanus, T. suc- specimens. T.D. acknowledges funding from the Swedish Research cinea formed part of the “Baltic amber forest” (Conwentz, 1886; Council (VR). We are grateful to two anonymous reviewers for Forman, 1964; Mai, 1967). It is noteworthy that the extinct trigo- constructive suggestions. nobalanoid taxa Trigonobalanopsis Z.Kvaček et H.Walther and Eotrigonobalanus Z.Kvaček et H.Walther (not directly related with extant Trigonobalanus s.l.) appear to have been part of taphocoe- DATA ACCESSIBILITY noses that were antagonistic to the Castanopsis-Lauraceae forests, as these particular trigonobalanoid taxa of the European Paleogene The SRμCT videos and volume rendering of Castanopsis kau- generally avoided oligotrophic habitats (Mai, 1967, and references lii are available at FigShare (Appendix S1: video showing the 3D therein; Kvaček and Walther, 1988; Mai, 1989). volume rendering of Castanopsis kaulii from Baltic amber, https:// In the early Eocene of North America (Nut Bed Flora; doi.org/10.6084/m9.figshare.7041821; Appendix S2: SRμCT video Manchester, 1994) and in the middle Eocene flora of the Geiseltal of Castanopsis kaulii reconstruction in cross section, https://doi. (Germany; Mai, 1976), Castanopsis was part of paratropical forests. org/10.6084/m9.figshare.7041815; Appendix S3: SRμCT video of However, both floras are older and more warmth-loving than the Castanopsis kaulii reconstruction in longitudinal section, https:// “Baltic amber forest.” This is also reflected in the exceptionally high doi.org/10.6084/m9.figshare.7041863). number of conifers from the younger Baltic amber, which indicate a warm-temperate climate for the latter (Sadowski et al., 2017a). SUPPORTING INFORMATION

CONCLUSIONS Additional Supporting Information may be found online in the supporting information for this article. For the first time, we applied synchrotron-radiation-based X-ray micro-computed tomography (SRμCT) to a minute plant inclu- APPENDIX S1. Video showing the 3D volume rendering of sion from Baltic amber revealing the exquisitely preserved internal Castanopsis kaulii from Baltic amber. structures of a pistillate partial inflorescence. Indeed, the exceptional three-dimensional preservation of the fossil suggests a close APPENDIX S2. SRμCT video of Castanopsis kaulii reconstruction relationship with extant Castanopsis (Fagaceae), allowing the de- in cross section. scription of a new fossil species, Castanopsis kaulii sp. nov. This fossil represents the first record of this genus from Baltic amber and APPENDIX S3. SRμCT video of Castanopsis kaulii reconstruction the first pistillate inflorescence of the Fagaceae from Eurasia. in longitudinal section. The occurrence of C. kaulii in Baltic amber further broadens our knowledge about the Fagaceae of the Baltic amber flora, which were LITERATURE CITED more diverse than recently suggested. The floristic composition of the “Baltic amber forest” with a prominent conifer contribution Abbe, E. C. 1974. Flowers and inflorescences of the “Amentiferae”. Botanical is comparable to Castanopsis-Lauraceae forests of the Oligocene- Review 40: 159–261. Miocene of Central Europe. Along with numerous conifer species, Aoki, K., S. Ueno, T. Kamijo, H. Setoguchi, N. Murakami, M. Kato, and Y. C. kaulii likely grew on oligotrophic, acidic, and sandy soils, such as Tsumura. 2014. Genetic differentiation and genetic diversity of Castanopsis in mires and bog-like habitats, within the Baltic amber source area. (Fagaceae), the dominant tree species in Japanese broadleaved evergreen Castanopsis kaulii also supports affinities of the Baltic amber flora forests, revealed by analysis of EST-associated microsatellites. PLoS ONE to the East Asian region, as previously suggested on the basis of co- 9: e87429. nifer inclusions from Baltic amber. Brett, D. W. 1963. The inflorescence ofFagus and Castanea, and the evolution of Finally, a review of the fossil record of Castanopsis shows that the cupules of the Fagaceae. New Phytologist 63: 96–118. while unambiguous fossil records are known from Eocene depos- Camus, A. 1929. Les chataigniers—Monographie des genres Castanea et Castanopsis. Encyclopédie Économique de Sylviculture III. Paul Lechevalier, its of western North America and Europe, the Eocene fossil record Paris, France. from East Asia is ambiguous. This suggests an initial range exten- Camus, A. 1936–38. Les chênes—Monographie du genre Quercus, Tome I, sion of Castanopsis across the North Atlantic land bridge (Tiffney Genre Quercus sous-genre Cyclobalanopsis, sous-genre Euquercus (Sections and Manchester, 2001). Cerris et Mesobalanus). Encyclopédie Économique de Sylviculture VI. Paul Lechevalier, Paris, France. Camus, A. 1952–54a. Les chênes—Monographie du genre Quercus, Tome III ACKNOWLEDGEMENTS (1er partie), Genre Quercus sous-genre Euquercus (Sections Protobalanus et Erythrobalanus) et Monographie du genre Lithocarpus. Encyclopédie SRμCT scanning was made possible by funding from the Deutsches Économique de Sylviculture VIII. Paul Lechevalier, Paris, France. Elektronen-Synchrotron (DESY; proposal no. I-20150309). We Camus, A. 1952–54b. Les chênes—Monographie du genre Quercus, Tome III (2me partie), Genre Quercus sous-genre Euquercus (Sections Protobalanus thank the DESY (beamline PETRA III/IBL P05, operated by the et Erythrobalanus) et Monographie du genre Lithocarpus. Encyclopédie Helmholtz-Zentrum Geesthacht, Hamburg, Germany), F. Stebner Économique de Sylviculture VIII. Paul Lechevalier, Paris, France. (Bonn), and P. T. Rühr (Köln) for operating X-ray imaging analy- Caspary, R. 1881. Ueber neue fossile Pflanzen der blauen Erde, d.h. des Bernsteins, sis. Many thanks to C. and H. W. Hoffeins (Hamburg), E. M. Friis des Schwarzharzes und des Braunharzes. In Schriften der physikalisch-ökon- and V. Belivanova (Stockholm), S. S. Renner (Munich), and L. J. omischen Gesellschaft zu Königsberg, vol. 22, Sitzung am 27. Mai 1881, 22– Seyfullah (Vienna) for their support. M. Appelhans (Göttingen) 23. Commission, Königsberg. December 2018, Volume 105 • Sadowski et al.—Castanopsis from Eocene Baltic amber • 2035

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