Phytotaxa 219 (2): 101–117 ISSN 1179-3155 (print edition) www.mapress.com/phytotaxa/ PHYTOTAXA Copyright © 2015 Magnolia Press Article ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.219.2.1 Recognizing the species of Thuja (Cupressaceae) based on their cone and foliage morphology BIN SUN1, 4, 5, YI-MING CUI1, 4, 5, HAI-FENG WANG1, 5, DAVID K. FERGUSON1, 2, QIAO-PING XIANG1, QING- WEN MA3, 6 & YU-FEI WANG1, 6 1State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 2Department of Palaeontology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria 3Beijing Museum of Natural History, Beijing 100050, China 4University of Chinese Academy of Sciences, Beijing 100039, China 5These authors contributed equally to this work. 6Authors for correspondence: Yu-Fei Wang, E-mail: [email protected], Tel: 86-10-62836934, Address: No.20 Nanxincun, Xiang- shan, Beijing 100093, China; and Qing-Wen Ma, E-mail: [email protected], Tel: 86-10-67020722, Address: 126, Tianqiao ST, Bei- jing,100050, China. Abstract Thuja, with 5 extant species, exhibiting a disjunctive distribution between East Asia (3 species) and North America (2 spe- cies), was investigated with respect to the morphological characters of foliage and cones by LM and SEM. Here we provide 2 keys to all 5 species of Thuja based on the cones and foliage respectively, which not only can be used for identifying extant Thuja at the species level, but also have a great potential for recognizing and/or linking the fossil species to living ones, and further tracing the evolutionary history of the genus. Keywords Cone, Foliage, East Asia-North America disjunction, Species identification, Thuja Introduction The genus Thuja L. (Cupressaceae), with a disjunctive distribution between East Asia and North America, consists of five extant species. Of these, two species occur in North America, i.e., T. occidentalis occurs in the Northeastern United States, the Southern Applachian Mountains of the United States, southeastern Canada, and in the Great Lakes region of both countries. While T. plicata occurs in the coastal Northwestern United States, coastal British Columbia in Canada, and in the interior Rocky Mountains in both countries (data from http://esp.cr.usgs.gov/data/little/ ). The other three species grow in East Asia, i.e., T. koraiensis in the Korean Peninsula and Changbai mountain area of China, T. standishii in Honshu and Shioku, Japan and T. sutchuenensis in the Daba Mountains, Chongqing, China (Farjon 2005; Fu & al. 1999). All the species of Thuja are monoecious evergreen trees or shrubs with spreading branches, those of the second and third orders being disposed in a plane. Leaves scale-like, decussate, only the leaves on the primary branches decurrent. The ultimate (sub) branchlets have dimorphic leaves, i.e., ovate-rhombic in the middle and boat-like laterally, with a ridge less than 4 mm on the abaxial surface and overlap with the facial leaves, no obvious white stomatal band on the abaxial surface. Pollen cones terminal, solitary, subglobose, with 6–10 microsporophylls, bearing 2–3 abaxial bracts. Seed cones terminal on short, straight branchlets, mature and dehiscent in the same year. Bract-scale complexes 8–10, spreading, decussate, only 2–3 larger pairs are fertile, bracts umbonate. The flattened seeds 1–2 per cone scale, tapering towards both ends with two marginal wings of equal size and shape, surrounding the seed but leaving a notch at both ends (Farjon 2005; Fu & al. 1999; Schulz & al. 2005). The evolutionary history of Thuja is still controversial. For instance, McIver and Basinger (1989) proposed that it originated in North America, based on fossil and extant seed cone morphology, and suggested that all extant species except T. sutchuenensis might have arisen from an ancestor similar to T. polaris found in the Paleocene sediments of Ellesmere Island, Canadian Arctic Archipelago in North America. According to this scenario it spread from North America into Asia. Accepted by Libing Zhang: 11 Jun. 2015; published: 8 Jul. 2015 101 Recent molecular research expresses two different views. Li & Xiang (2005) based on the analysis of nrDNA ITS sequence, inferred that one clade (T. standishii, T. sutchuenensis and T. occidentalis) might have spread from East Asia into western North America via the Bering land bridge at 21.2 ± 14.7 Ma, while another clade (T. koraiensis and T. plicata) spread westward from east Asia into East North America via the North Atlantic land bridge. On the other hand, Peng & Wang (2008), based on multiple genes, including cpDNA, nrDNA ITS, LEAFY and 4CL, proposed that Thuja originated in the high latitudinal regions of North America. Thuja has a long fossil history from the Paleocene sediments in the Northern Hemisphere. The earliest unambiguous Thuja fossil T. polaris was reported from the Paleocene of the Canadian Arctic (Stockey et al., 2005). The genus was found mostly as foliage (e.g., Brown 1939, 1962; Heer 1870, 1882; Knowlton 1905; Newberry 1868; Schweitzer 1974; Sveshnikova 1967) and rarely as branches with cones (e.g., LePage 2003; Schweitzer 1974; Mclver & Basinger 1989; Akhmetiev 1973; Huzioka & Uemura 1973; Bennike 1990). Due to the fragmental preservation of fossils, the precise species identification is difficult, which impedes the evaluation of the hypotheses regarding its origin, and further tracing its evolutionary history. Therefore, a precise species identification system for fossils based on foliage and seed cones is urgently needed. Here we provide a new species identification system based on detailed morphological surveys of the seed cone and foliage of all 5 extant species of Thuja under Light Microscope and Scanning Electron Microscope. Material and Methods The herbarium sheets from the Peking herbarium (PE), examined at the Institute of Botany, Chinese Academy of Sciences are listed in Table 1. Foliage and seed cones taken from the herbarium sheets were soaked in 10% HCl for 2 hours. After washing in distilled water, they were dried and flattened, and then photographed using a Nikon D300 digital camera and Nikon SMZ1000 stereo microscope. The scale leaves from the middle of the branch were soaked in ℃ a 1:1 mixture of 30% H2O2 and 99% CHCOOH in a 60 water bath for 8 hours, then rinsed with water three times, soaked in 10% KOH for 1 min and again rinsed with water three times. The scale leaves were then placed inner surface up, the mesophyll gently brushed away, and the remaining cuticle dyed with Safranine. A Leica DM2500 optical microscope was used for observation and photography. The leaf epidermis samples were fixed to a labelled stub, coated with gold using a Hitachi E-1010 sputter coater, and scanned with a Hitachi S-4800 SEM. The terminology follows that of Farjon (2005) for gross morphology and Dilcher (1974) for cuticle analysis. Specific terms used in foliage are illustrated in Fig. 1. Results Generic description Seed cones terminal on short branch, ovate or elliptic. Bract-scale complexes 3–6 (–7) pairs, coriaceous and decussate. A small umbo is present near the top of the bract (Fig. 2A–J). Branches spreading, alternate, branches of second and third orders disposed in a plane. Leaves scale-like, decussate, dimorphic with flattened facial leaves, and folded lateral leaves (Fig. 2K–O). Stomata densely distributed on the adaxial surface of the facial leaves (Figs. 3, 4). Stomata cyclocytic, randomly oriented, with 5–6 subsidiary cells and a Florin ring on the outer surface, stomatal aperture steep-sided (Figs. 5–8). Subsidiary cells with straight and continuous anticlinal walls (Figs. 7–10). Outer surface of epidermal cells in stomatal zone generally papillate (Figs. 5, 6, 9–12), papillae orbicular or elliptic, solitary or in groups of 2–3. Long axis of epidermal cells on abaxial surface of facial leaves usually radially arranged with the leaf’s tip acting as the center of radiation. Epidermal cells generally polygonal close to the lower edge and oblong in the central area (Figs.11, 12). 102 • Phytotaxa 219 (2) © 2015 Magnolia Press SUN ET AL. FIGURE 1. Diagram of foliage and epidermis. A, Terms of tip branches and leaves; B, Longitudinal view (of a cut along the dashed line in a), showing the relationship between adaxial surfaces and abaxial surfaces of facial leaves; C, Relationship between the surfaces of facial leaves and lateral leaves. RECOGNIZING THE SPECIES OF THUJA Phytotaxa 219 (2) © 2015 Magnolia Press • 103 TABLE 1. Herbarium sampling data. Name Collector Collection number Bar code Collection site Herbarium T. koraiensis Xiaoquan Wang et al. T004 00206885 Changbai Mountain County, PE China Shene Liu 9444 02906001y0009 Jilin, China PE Jiaju Qian 3392 00019487 Changbai Mountain County, PE China T. occidentalis V. Bates, I. Sandra Elsik 124 00019472 Rimouski County, Québec, PE Canada Liguo Fu & Ronghou 18 00019456 Nanjing, Jiangsu, China PE Zhang W. Hess & M. T. Hall 6863 00019337 Pennsylvania, U.S.A. PE T. plicata W. Hess, K. Winter 7052 Skamania, Washington State, PE 01816322 U.S.A. J. Riser 1 01869379 Idaho, Benewah Co., U.S.A. PE R. Halse 1676 00206321 Hood River, Oregon, U.S.A. PE T. standishii C. Maximowicz s.n. 00019528 Honshu, Japan PE Liguo Fu & Ronghou 102 00019526 Jiujiang, Jiangxi, China PE Zhang F. G. Meyer 18014 00047267 Yunoko, Nikko, Japan PE T. sutchuenensis Zhenyu Li et al. 11304 00206042 Chengkou, Sichuan, China PE Specific descriptions: T. koraiensis Seed cones narrowly ovate (Fig. 2A), 7–11 mm×6–9 mm, bract-scale complexes 4–6 pairs, the second whorl elliptic, apex obtuse, base acute, umbo apex obtuse (Fig. 2F). Facial leaves with obvious glands, lateral leaves with inturned apex, appressed to the facial leaves, no overlapping between the adjacent lateral leaves (Fig. 2K).