Recognizing the Species of Thuja (Cupressaceae) Based on Their Cone and Foliage Morphology

Home , Thuja

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 species), 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). Leaf apex rounded with micro-teeth. Stomata rare on the abaxial surfaces of facial leaves but numerous on the adaxial surface, especially proximally (Figs. 3A, B). Subsidiary cells with low anticlinal walls, straight and continuous, margin jagged, periclinal wall with complicated ornamentation, the base of inner surface of papilla sometimes thickened (Figs. 5B, 9B, 11B). The inner surface of epidermal cells with low anticlinal walls, straight and continuous, margin smooth, periclinal wall with coarse particles (Fig. 11A).

T. occidentalis

Seed cones narrowly ovate (Fig. 2B), 8–12 mm×4–6 mm. Bract-scale complexes (3–) 4 (–5) pairs, the second whorl narrowly obovate, apex obtuse, base acute, umbo apex acute (Fig. 2G). Facial leaves with obvious glands, lateral leaves with inturned apex, appressed to the facial leaves, no overlapping between the adjacent lateral leaves (Fig. 2L). Leaf apex obtuse without micro-teeth. almost no stomata on the abaxial surfaces of facial leaves, 2 triangular shaped stomatal bands on the proximal part of adaxial surface (Figs. 3C, D). Subsidiary cells with low anticlinal walls, straight and continuous, margin denticulate (jagged), periclinal wall rough, the base of inner surface of papilla without thickening (Figs. 5D, 9D, 11D). The inner surface of epidermal cells with low anticlinal walls, straight and continuous, margin with coarse particles, periclinal wall with coarse particles (Fig. 11C).

T. plicata

Seed cones ovate (Fig. 2C), 10–16 mm×6–8 mm. Bract-scale complexes 4–6 (–7) pairs, the second whorl rhombic, apex obtuse, base cuneate, umbo apex acuminate (Fig. 2H).

104 • Phytotaxa 219 (2) © 2015 Magnolia Press Sun ET AL. Figure 2. Characters of cones, bract-scale complexes and tip branches of Thuja. A, F, K, T. koraiensis; B, G, L, T. occidentalis; C, H, M, T. plicata; D, I, N, T. standishii; E, J, O, T. sutchuenensis.—Arrows pointing to the umbo; Scale bars = 2 mm.

Facial leaves with obvious glands, lateral leaves with straight apex, overlapping between the adjacent lateral leaves (Fig. 2M). Leaf apex acuminate with no micro-teeth. Stomata rare on the abaxial surface of facial leaves, 2 triangular shaped stomatal bands on the proximal part of adaxial surface (Figs. 3E, F). Subsidiary cells with low anticlinal walls, straight and continuous, margin denticulate (jagged), periclinal wall smooth, the base of inner surface of papilla without thickening (Figs. 5F, 9F, 11F). The inner surface of epidermal cells with low anticlinal walls, straight and continuous, margin smooth, periclinal wall with coarse particles (Fig. 11E).

T. standishii

Seed cones broadly ovate (Fig. 2D), 7–12 (–14) mm×6–7 mm. Bract-scale complexes 4–5 (–6) pairs, the second whorl rhombic, apex obtuse, base obtuse, umbo apex acute (Fig. 2I).

Recognizing the species of Thuja Phytotaxa 219 (2) © 2015 Magnolia Press • 105 Figure 3. The distribution of stomata on the abaxial and adaxial surfaces of facial leaves and the apex of facial leaves. A–B, T. koraiensis; C–D, T. occidentalis; E–F, T. plicata.—DFL: abaxial facial leaves; VFL: adaxial facial leaves; Scale bars: A, C, E = 0.5 mm; B, D, F = 0.1 mm.

Facial leaves with obvious glands, lateral leaves with inturned apex, overlapping between the adjacent lateral leaves (Fig. 2N). Leaf apex obtuse or acute, without micro-teeth. almost no stomata on the abaxial surface of facial leaves, 2 triangular shaped stomatal bands on the proximal part of adaxial surface (Figs. 4A, B). Subsidiary cells with high anticlinal walls, straight and continuous, margin serrate, periclinal wall smooth, the base of inner surface of papilla without thickening (Figs. 6B, 10B, 12B).

T. sutchuenensis

Seed cones broadly ovate or elliptic (Fig. 2E), 5–8 mm×3–4 mm. Bract-scale complexes 4–5 pairs, the second whorl obovate, apex rounded, base cuneate, umbo apex obtuse (Fig. 2J). Facial leaves without glands, lateral leaves with inturned apex, no overlapping between the adjacent lateral leaves (Fig. 2O). Leaf apex obtuse with micro-teeth. Stomata on both the abaxial and adaxial surfaces of facial leaves (Figs. 4C, D).

Figure 4. The distribution of stomata on the abaxial and adaxial surfaces of facial leaves and the apex of facial leaves. A–B, T. standishii; C–D, T. sutchuenensis.—DFL: abaxial facial leaves; VFL: adaxial facial leaves; Scale bars: A, C = 0.5 mm; B, D = 0.1 mm.

Subsidiary cells with low anticlinal walls, straight and continuous, margin denticulate (jagged), periclinal wall rough, the base of inner surface of papilla without thickening (Figs. 6D, 10D, 12D). The inner surface of epidermal cells with low anticlinal walls, straight and continuous, margin smooth, periclinal wall rough (Fig. 12C). The characters of cone morphology and foliage epidermal microstructure of the five living species of Thuja are listed in Table 2, and keys based on the characters of cone (Key 1) and foliage (Key 2) are provided.

Recognizing the species of Thuja Phytotaxa 219 (2) © 2015 Magnolia Press • 107 Figure 5. The arrangement of stomata and subsidiary cells on the abaxial surface of facial leaves. A–B, T. koraiensis; C–D, T. occidentalis; E–F, T. plicata.—SC: subsidiary cell; FL: Florin ring; Scale bars: A, C, E = 50 μm; B, D, F = 10 μm.

108 • Phytotaxa 219 (2) © 2015 Magnolia Press Sun ET AL. Table 2. Cone morphology and foliage epidermal microstructure characters of the five living species of Thjua Characters T. koraiensis T. occidentalis T. plicata T. standishii T. sutchuenensis Seed cone shape narrowly ovate narrowly ovate ovate broadly ovate ovate or elliptic *Seed cone length (mm) 7–11 8–12 10–16 (–18) 7–12 (–14) 5–8 *Seed cone width (mm) 6–9 4–6 6–8 6–7 3–4 * Bract-scale complexes 8–12 (6–) 8 (–10) 8–12 (–14) 8–10 (–12) 8–10 number Bract-scale complexes elliptic narrowly obovate rhombic rhombic broadly obovate shape Widest position of the middle distally middle middle distally Bract-scale complexes Bract-scale complexes 2.1 2.1 2.1 1.6 1.3 length (mm) Bract-scale complexes 1.0 1.1 1.3 1.6 1.1 width (mm) Bract-scale complexes 2:1 2:1 1.6:1 1:1 1.2:1 length/width Bract-scale complexes obtuse obtuse obtuse obtuse rounded apex Bract-scale complexes acute acute cuneate obtuse cuneate base Umbo apex obtuse acute acuminate acute obtuse Facial leaves apex obtuse, micro teeth obtuse, no tooth acuminate, no acute, no tooth obtuse, serrate tooth Lateral leaves apex inturned inturned straight inturned inturned Overlaping between the no no yes yes no adjacent lateral leaves Distribution of stomata on rare abaxially, but almost none abaxially, rare abaxially, 2 almost none present on the facial leaves many adaxially 2 triangular shaped triangular shaped abaxially, 2 abaxial and stomatal bands stomatal bands triangular shaped adaxial surfaces adaxially adaxially stomatal bands adaxially Anticlinal wall of low low low high low subsidiary cell Margin of subsidiary cell jagged jagged jagged Serrate jagged Ornamentation on inner smooth coarse particles smooth smooth coarse particles surface of subsidiary cell Inner surface of papilla base sometimes base never thickened base never base never base never thickened thickened thickened thickened Ornamentation on coarse particles coarse particles smooth smooth coarse particles periclinal wall of epidermal cell

Ornamentation on inner smooth coarse particles coarse particles smooth coarse particles surface of epidermal cell Note: * indicating those data cited from Farjon (2005)

Key 1. Key to species of Thuja based on cone characters

1. Bract-scale complexes obovate, widest distally...... 2 Bract-scale complexes elliptic or rhombic, widest in the middle...... 3 2. Bract-scale complexes narrowly obovate, apex obtuse, base acute...... T. occidentalis Bract-scale complexes broadly obovate, apex rounded, base cuneate...... T. sutchuenensis 3. Bract-scale complexes rhombic, length/width about 1:1...... T. standishii Bract-scale complexes elliptic, length/width more than 1.5:1...... 4

Recognizing the species of Thuja Phytotaxa 219 (2) © 2015 Magnolia Press • 109 4. Bract-scale complexes’ base acute, umbo apex obtuse...... T. koraiensis Bract-scale complexes’ base cuneate, umbo apex acuminate...... T. plicata

Figure 6. The arrangement of stomata and subsidiary cells on the abaxial surface of facial leaves A–B, T. standishii; C–D, T. sutchuenensis.—SC: subsidiary cell; FL: Florin ring; Scale bars: A, C = 50 μm; B, D = 10 μm.

Key 2. Key to species of Thuja based on foliage

1. No overlapping between adjacent lateral leaves...... 2 overlapping between adjacent lateral leaves...... 4 2. Stomata on both abaxial and adaxial surfaces of facial leaves...... T. sutchuenensis Stomata rare on abaxial surface of facial leaves but numerous on adaxial surface...... 3 3. Periclinal wall of subsidiary cells smooth...... T. koraiensis

110 • Phytotaxa 219 (2) © 2015 Magnolia Press Sun ET AL. Periclinal wall of subsidiary cells rough...... T. occidentalis 4. anticlinal wall of subsidiary cells low, margin jagged, periclinal wall of epidermal cells with coarse particles...... T. plicata anticlinal wall of subsidiary cells high, margin serrate, periclinal wall of epidermal cells smooth...... T. standishii

Figure 7. Characters of the stomatal area and non-stomatal area on the abaxial surface of facial leaves. A–B, T. koraiensis; C–D, T. occidentalis; E–F, T. plicata.—Scale bars = 50 μm.

Recognizing the species of Thuja Phytotaxa 219 (2) © 2015 Magnolia Press • 111 Figure 8. Characters of the stomatal area and non-stomatal area on the abaxial surface of facial leaves. A–B: T. standishii; C–D: T. sutchuenensis.—Scale bars: A, C = 50 μm; B, D = 100 μm.

Discussion

The seed cones of Thuja have 3–6 pairs of decussate and coriaceous bract-scale complexes with a small umbo near the apex of the bract. All the extant species have cyclocytic stomata, with 5–6 subsidiary cells and a Florin ring on the outer surface. The outer surface of the epidermal cells around the stomata is generally papillate. The anticlinal walls of both subsidiary cells and epidermal cells are straight. These highly consistent characters can be considered to reflect the close affinities among the species and imply a monophyletic origin, which supports the conclusion reached by earlier molecular research (Peng & Wang 2008).

Interspecific distinguishing characteristics

The five species of Thuja can be distinguished clearly with seed cone characters (Key 1, Table 2). They can be divided into two groups based on the shapes of the bracts and the position of the widest part of the bracts, i.e., T. occidentalis and T. sutchuenensis forming group 1 with obovate bracts with the widest part in the distal half vs. T. standishii, T. koraiensis and T. plicata forming group 2 with elliptic or rhombic bracts with the widest part in the middle. In group 1, T. occidentalis can be clearly distinguished as its bracts are narrowly obovate with an obtuse apex, and acute base, while T. sutchuenensis has broadly obovate bracts with rounded apex and cuneate base. In group 2, the bracts are either rhombic with a length/width of almost 1:1 as in T. standishii, or elliptic with a length/width larger than 1.5:1 (T.

112 • Phytotaxa 219 (2) © 2015 Magnolia Press Sun ET AL. koraiensis, T. plicata). T. koraiensis and T. plicata can be distinguished by the shape of the bract-scale complexes’ base and umbo apex (acute and obtuse vs cuneate and acuminate).

Figure 9. Characters of the outer surface and inner surface of stomata on the abaxial surface of facial leaves. A–B, T. koraiensis; C–D, T. occidentalis; E–F, T. plicata.—FL: Florin ring; Pa: papilla; Scale bars = 20 μm.

Recognizing the species of Thuja Phytotaxa 219 (2) © 2015 Magnolia Press • 113 Figure 10. Characters of the outer surface and inner surface of stomata on the abaxial surface of facial leaves. A–B, T. standishii; C–D, T. sutchuenensis.—FL: Florin ring; Pa: papilla; Scale bars = 20 μm.

The gross foliar morphology of Thuja provides few useful characters for species delimitation except for three items, i.e., facial leaf apex, lateral leaf apex and overlapping between the adjacent lateral leaves (Table 2). Here the newly added epidermal and stomata microstructures provide enough information to clearly distinguish between the five extant species (Key 2, Table 2). For instance, stomata distributed equally on the abaxial and adaxial surfaces of facial leaves distinguish T. sutchuenensis from all the other species with only a few stomata present on the abaxial surface of the facial leaves but many on the adaxial surface. The high anticlinal wall and serrate margin of the subsidiary cells are unique features of T. standishii. Among the remaining 3 species, T. koraiensis is the only one with a thickened base on the inner surface of the papillae. T. occidentalis and T. plicata can be distinguished by the ornamentations on the inner surface of the subsidiary and epidermal cells (e.g. coarse and coarse vs smooth and smooth).

Figure 11. Characters of the inner surface of epidermal cells in stomatal area and non-stomatal area on the abaxial surface of facial leaves. A–B, T. koraiensis; C–D, T. occidentalis; E–F, T. plicata.—IP: inner surface of papilla; Scale bars = 20 μm.

Recognizing the species of Thuja Phytotaxa 219 (2) © 2015 Magnolia Press • 115 Figure 12. Characters of the inner surface of epidermal cells in stomatal area and non-stomatal area on the abaxial surface of facial leaves. A–B, T. standishii; C–D, T. sutchuenensis.—IP: inner surface of papilla; Scale bars = 20 μm.

Interspecific relationships

Thuja has a circum-Pacific distribution with three species in East Asia and two species in North America. Disjunction could have occurred in the late Miocene as the climate in the Northern Hemisphere became cooler and the treeline moved south. The uplift of the mountains on the west coast of North America caused a rain shadow which was responsible for the disappearance of the forests in the Great Plains of central USA about 10 million years ago, and led to the disjunction of the American populations. According to this scenario the American species T. plicata and T. occidentalis should be more closely related to one another than to the Asiatic species. However, based on the analysis of the ITS sequence of Thuja, Li & Xiang (2005) suggested that T. standishii is closest to T. sutchuenensis, and form a clade with T. occidentalis, while the other clade combines T. koraiensis and T. plicata. Peng & Wang (2008), based on multiple genes, i.e., cpDNA, nrDNA ITS, LEAFY and 4CL, inferred that T. occidentalis first diverged ~60 Ma, and then, at 51.1 ± 3.96 Ma, it split into two species pairs. The differentiation of T. standishii and T. sutchuenensis occurred at 23.7±5.04Ma, followed by the divergence of T. koraiensis and T. plicata at 14.7 ± 6.06Ma, T. koraiensis captured

116 • Phytotaxa 219 (2) © 2015 Magnolia Press Sun ET AL. the chloroplast genes of T. occidentalis and cross-fertilized with a species of the T. standishii–T. sutchuenensis branch, the hybrid individuals with the reorganized chloroplast DNA migrated into western North America, which became today’s Thuja plicata. Both molecular studies suggested that the phylogenetic relationship between T. occidentalis (ENA) and T. plicata (WNA) is quite distant although they have different opinions on the interspecific relationship of the genus. our survey reveals that T. occidentalis and T. plicata have numerous morphological differences. For instance, bracts narrowly obovate vs rhombic, base acute vs cuneate, lateral leaf apices inturned vs straight, no overlapping between the adjacent lateral leaves vs overlapping, coarse particles on the inner surface of the subsidiary cells and periclinal wall of epidermal cells vs smooth in both cases. All the above differences imply that their phylogenetic relationship is not as close as their geographical distribution might suggest.

Acknowledgements

This research was supported by the China National Key Basic Research Program (2014CB954201), and the National Natural Science Foundation of China (Nos. 30990241; 41072022, 41210001, 31370254, 31300186). Prof. David K. Ferguson was supported by the Chinese Academy of Sciences Visiting Professorship for Senior International Scientists at the Institute of Botany (Nos. 90004F1005).

References

Akhmetiev, M.A. (1973) Sikhote-alin Miocene flora (Botchi River). Nauka Press, Moscow. [In Russian] Bennike, O. (1990) The Kap København Formation: stratigraphy and palaeobotany of a Plio-Pleistocene sequence in Peary Land, North Greenland, Vol. 23. Commission for Scientific Research, Greenland. Brown, R.W. (1939) Fossil plants from the Colgate Member of the Fox Hills Sandstone and adjacent strata. US Geological Survey Professional Papers (U.S.) 189: 239–275. Brown, R.W. (1962) Paleocene flora of the Rocky Mountains and Great Plains. Geological Survey Professional Papers (U.S.) 375: 1–119. Dilcher, D.L. (1974) Approaches to the identification of Angiosperm leaf remains. Botanical Review 40: 1–157. Farjon, A. (2005) A monograph of Cupressaceae and Sciadopitys. Royal Botanic Gardens, Kew, 643 pp. Fu, L.G., Yu, Y.F. & Farjon, A. (1999) Cupressaceae. In: Wu, Z.Y. & Raven, P.H. (Eds.) Flora of China. Vol. 4. (Cycadaceae through Fagaceae). Beijing, Science Press, pp. 62–77. Heer, O. (1870) Die miocene Flora und Fauna Spitzbergens. Kungliga Svenska Vetenskaps Akademiens Handlingar 8: 1–98. Heer, O. (Ed.) (1882) Die fossile Flora Grönlands, Theil 1. In: Oswald, H., Cramer, K.E., Nordenskiöld, A.E. & Schröter, C. (1883) Flora fossilis arctica. Vol. 6. 61 pp. Huzioka, K. & Uemura, K. (1973) The late Miocene Miyata flora of Akita Prefecture, northeast Honshu, Japan. Bulletin of the National Science Museum, Tokyo 78: 661–738. Knowlton, F.H. (1905) Fossil plants of the Judith River beds. US Geological Survey Bulletin 257: 129–155. LePage, B.A. (2003) A new species of Thuja (Cupressaceae) from the Late Cretaceous of Alaska: Implications of being evergreen in a polar environment. American Journal of Botany 90: 167–174. Li, J.H. & Xiang, Q.P. (2005) Phylogeny and biogeography of Thuja L. (Cupressaceae), an East Asian and North American disjunct genus. Journal of Integrative Plant Biology 47: 651–659. McIver, E.E. & Basinger, J.F. (1989) The morphology and relationships of Thuja polaris sp. nov. (Cupressaceae) from the early Tertiary, Ellesmere Island, Arctic Canada. Canadian Journal of Botany 67: 1903–1915. Newberry, J.S. (1868) Notes on the later extinct floras of North America, with descriptions of some new species of fossil plants from the Cretaceous and Tertiary strata. Annals of the Museum of Natural History of New York 9: 1–76. Peng, D. & Wang, X.Q. (2008) Reticulate evolution in Thuja inferred from multiple gene sequences: Implications for the study of biogeographical disjunction between East Asia and North America. Molecular Phylogenetics and Evolution 47: 1190–1202. Schulz, C., Knopf, P. & Stuetzel, T.H. (2005) Identification key to the cypress family (Cupressaceae). Feddes Repertorium 116: 96–146. Schweitzer, H.J. (1974) Die „tertiaren“ Koniferen Spitzbergens. Palaeontographica Abteilung B Palaeophytology 149: 1–89. Stockey, R.A., Kvaček, J., Hill, R.S., Rothwell, G.W. & Kvaček, Z. (2005) The fossil record of Cupressaceae s. lat. chapter 6. In: Farjon, A. (2005) A monograph of Cupressaceae and Sciadopitys. Royal Botanic Gardens, Kew, 63 pp. Sveshnikova, I.N. (1967) Late Cretaceous Coniferae of the USSR. I. Fossil Coniferae of the Viliuyian Depression. Palaeobotanica 6: 178–204.