First Occurrence of Platycladus from the Upper Miocene of Southwest China and Its Phytogeographic Implications

Platycladus Spach is native to Central China, but its natural occurrences are very difficult to establish. According to molecular phylogenetic data, this genus might have originated since the Oligocene, but no fossil record has been reported. Here, we describe eight foliage branches from the upper Miocene in western Yunnan, Southwest China as a new species, P. yunnanensis sp. nov., which is characterized by foliage branches spread in flattened sprays, and leaves decussate, imbricate, scale-like and dimorphic. The leaves are amphistomatic, and the stomata are elliptical or oblong, haplocheilic, and monocyclic type. Based on a detailed comparison with the extant genera of Cupressaceae sensu lato, our fossils are classified into the genus Platycladus. The occurrence of P. yunnanensis sp. nov. indicates that this genus had a more southernly natural distribution in the late Miocene than at present. Molecular phylogeny and fossil records support a pre-Oligocene common ancestor for the genera Platycladus, Microbiota and Calocedrus. The separation of the three taxa was most likely caused by the arid belt across Central China during the Oligocene. In addition, the cooling down of the global temperature and the strengthening of Asian monsoon since the Miocene will further promote the migration of these genera.


Introduction
Platycladus Spach, one of the 30 genera in the Cupressaceae s.l., is a distinct genus of evergreen tree with one extant species, P. orientalis L., also known as Chinese arborvitae [1,2]. Platycladus orientalis currently occurs in Central China [2], and was introduced to North Korea and the Russian Far East due to its adaptation of a wide range of climate and soil conditions [1,3]. However, the natural distribution of Platycladus is difficult to distinguish owing to extensive cultivation and planting in the past [1,2].
The molecular phylogenetic data indicate that Platycladus and Microbiota Komarov form a clade that is closely related to Tetraclinis Masters and Calocedrus Kurz [4][5][6]. The divergence of the Platycladus-Microbiota clade is considered to be ca. 33 Ma (early Oligocene) [4,5]. The genus Tetraclinis has a wide historical distribution in western North America and Europe, but did not cross into Asia [7]. The floristic exchange of Calocedrus between eastern Asia and North America before the Oligocene via the Bering land bridge has been demonstrated [8,9]. In contrast, the earliest fossil record of Microbiota can only be traced back to the Pliocene in Russia [10], and to date no fossil record of Platycladus has been reported. In the present study, we describe a new species as Platycladus yunnanensis sp. nov. from the upper Miocene in West Yunnan Province, Southwest China, based on a detailed comparison of gross morphology and cuticular features with the extant Cupressaceae. As the first record of Platycladus, the occurrence of the present fossil species will provide us evidence to recognize the natural distribution and migration of this conifer in the past.

Geological setting
The fossil branches studied here were collected from the Miocene Nanlin Formation at Nongbie Village (24˚51'46"N, 98˚24'58"E; Fig. 1), Lianghe County, Yunnan Province, China. The Nanlin Formation unconformably underlies the Pliocene Mangbang Formation and consists mainly of conglomerates, sandstones, siltstones, mudstones and basaltic rocks [11] (Fig. 2). The Nanlin Formation has been assigned to the Miocene according to the plant fossil assemblage [11,12]. The basaltic rocks within the formation in Lianghe County were radiometrically dated at 7.20¡0.22 Ma and 6.77¡0.30 Ma using the K-Ar dating method [13]. Therefore, the fossiliferous layers studied here can be assigned to the late Miocene.

Fossil material and preparation
After photographing with a SONY NEX-7 (SEL30M35), the leaf fragments were sampled from the fossil bearing matrix with a scalpel and placed in water. The fragments were immersed in 10% HCl overnight, washed and then immersed in 40% HF for 48 h. After washing in distilled water, the fragments were macerated with 45% HNO 3 for 24 h. When the color of the fragments was changed from black to sandy beige, the samples were washed and then treated with 5% NH 4 OH for 5 min. The adaxial and abaxial cuticles were separated with a dissecting needle under a stereomicroscope. After staining with Safranin T, the cuticles were mounted on slides, embedded in glycerine jelly and sealed with nail polish, then photographed under a light microscope (Leica DM4000B). Unstained cuticles and unmacerated fragments were mounted on a stub and coated with gold, examined and photographed using a scanning electron microscope (JEOLJSM-6380LV).
All specimens and cuticle slides are housed in the Institute of Paleontology and Stratigraphy, Lanzhou University, China. Terminology on leaf morphology follows Fu et al. [1] and Farjon [2], while terms on foliar cuticle are adopted after Kvaček et al. [7] and Shi et al. [9,15,16].

Nomenclature
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Ethics Statement
All necessary permits were obtained for the described sampling sites in verbal or written form. Eight fossils of Platycladus were collected in Lianghe County, Yunnan Province, China. The field work is permitted by the local government. For the extant plant sampling sites, permits were obtained from the botanical garden offices in verbal. The extant plant materials did not involve endangered or protected species.

Results
Family.   The lateral leaves are amphistomatic. The abaxial cuticle has two stomatal zones arranged on each side of the upper part (Fig. 4C). In the median nonstomatal zones, the epidermal cells are rectangular or with oblique end walls, usually elongate along the leaf long axis, 35-100 mm long and 15-30 mm wide, with a  stomata along the leaf long axis. The epidermal cells in the stomatal zones are irregular. The stomata are irregularly arranged, oriented longitudinally or orientation (Fig. 4G). The adaxial cuticle has fewer stomata. In the nonstomatal zones, the epidermal cells are rectangular or with oblique end walls, strongly elongate along the leaf long axis, strongly elongate, 48-110 mm long and 10-22 mm wide, with a length to width ratio up to 9. The stomatal zone is arranged on each side of the upper part along the leaf long axis, often containing two rows of stomata (Fig. 4D).
The facial leaves are amphistomatic. The abaxial cuticle is composed of a median nonstomatal zone and two broad stomatal zones on each side. In the nonstomatal zones, the epidermal cells are rectangular or with oblique end walls, more or less elongate, 30-80 mm long and 15-30 mm wide, with a length to width ratio up to 6. The anticlinal walls are straight or sometimes curved, the periclinal walls are smooth in the inner surface and rough on the outer surface. The stomatal zone is V-shaped, with 6-8 lines of stomata in the upper part, and decreases downward ( Fig. 4A; 5F). The stomata are irregularly arranged, mostly oriented longitudinally parallel to the long axis of the leaf, or oriented with a little deflexion (Fig. 4E, F). The epidermal cells in the stomatal zones are irregular. The adaxial cuticle has fewer stomata, with 2-3 lines of stomata on the upper part along the leaf long axis.
The lateral and facial leaves have similar stomatal complexes. The stomata complexes are elliptical or oblong, 30-48 mm long and 20-32 mm wide, haplocheilic, monocyclic (Fig. 5G-J). The guard cells are encircled by 5-7 subsidiary cells. The stomatal pits are usually open and shallow, elongate, elliptical in outline, 12-20 mm long and 4-9 mm wide (Fig. 5C, D). The guard cells are slightly sunken, usually forming a closed aperture, with periclinal walls inner cuticle surface almost smooth under the SEM. The subsidiary cells are usually two polar cells and the others lateral, quadrangular. The periclinal walls of subsidiary cells are usually smooth in the inner surface. The subsidiary cells are cambered outwards in the outer surface and form a distinct Florin ring around the stomatal pit (Fig. 5C, D). The Florin ring usually is elliptical in outline, 22-30 mm long and 14-25 mm wide, with a thickened, lobed rim 6-8 mm wide.

Affinities
The present fossil branches are spreading in flattened sprays, and possess decussate, imbricate and scale-like leaves. It is certain that the gross morphology of the present fossils are of the family Cupressaceae s.l. In the Cupressaceae (Table 1), the genera Cuninghamia R.Br., Taiwania Hayata, Athrotaxis D. Don, Sequoiadendron Buchholz, Sequoia Endl., Cryptomeria D. Don, Taxodium L. and Glyptostrobus Endl. have leaves that are helically inserted, differ from our fossils that have leaves arranged decussately [2]. The foliage branches of Cupressus L. and Widdringtonia Endl. often spread in a nonplanar arrangement [16]. The leaves of Callitris Vent., Actinostrobus Miq. and Fitzroya are arranged in whorls of 3 or 4. Juniperus differs from the present fossils in the absence of dimorphic leaves  (Table 1). However, most species of these genera are different from our fossils in leaf morphology ( Table 2). For example, Thujopsis dolabrata (Thunb. ex L. f.) Sieb. et Zucc. has obovateobdeltoid facial leaves with serrate margins [9], and its lateral leaves are dolabriform with a distinctly median groove of stomatal zone (Fig. 6A). Thuja sutchuenensis Franch. and T. koraiensis Nakai have broadly falcate lateral leaves ( Table 2), T. standishii (Gordon) Carrière possesses glands on the facial leaves (Fig. 6G), T. occidentalis L. and T. plicata Donn ex D. Don have ultimate branchlets often more numerous on the acroscopic side of lateral branchlets (Fig. 6C, F; [2]). Fokienia hodginsii (Dunn) A. Henry et H.H. Thomas possess a larger leaf sizes than our fossils [2], and the papillae around the stomata in F. hodginsii [18] are absent in the present fossils. The species within Chamaecyparis Spach possess rhombic to lanceolate facial leaves and broadly falcate to lanceolate lateral leaves, usually with acute apices (Fig. 6B, E). Xanthocyparis nootkatensis (D. Don) Farjon et Harder possesses ultimate branchlets often unilateral on the second highest order, and X. vietnamensis Farjon et Hiep has broadly falcate to lanceolate laterals with minutely serrate margins [2]. Papuacedrus papuana (F. Muell.) H.L. Li has much smaller facials than the laterals. Libocedrus bidwillii Hook. f. has acute apices in the facial and lateral leaves. Calocedrus species possess oblong to obtrullate facial leaves with serrate margins (Fig. 5N), and linearlanceolate laterals with acute apices (Fig. 6H). Therefore, we can conclude that all the species in Cupressoideae, except for Platycladus orientalis, are differ from the present fossils (Table 2). However, some minor differences can also be found between P. orientalis and our fossil branches, such as P. orientalis possessing smaller leaves (1.5-2 mm long and 1-1.5 mm wide) [2] than those in our fossils (2-3.2 mm long and 1.2-2.0 mm wide), and the stomata of our fossils being more or less elongate (Fig. 5G-J).
Zhang [12] reported some cupressaceous fossils as Calocedrus lantenoisii (Laurent) Tao from the Miocene Nanlin Formation, but without any figure and description. This fossil species has been widely reported from the Oligocene to the Miocene in Yunnan [12,19,20], and resembles the extant C. macrolepis Kurz in gross morphology [20]. Some fossil foliage shoots from the Oligocene in Guangxi, South China have been described as C. huashanensis [9]. The lateral leaves of C. huashanensis are falcate with acute to acuminate apices. In any case, the leaves with serrate and scariose margins in the extant and fossil Calocedrus [9] distinctly differ from those of our fossils. Fokienia shengxianensis He, Sun et Liu from the Miocene of Zhejiang, East China [18] has a leaf shape similar to that of our fossils. However, the leaves of F. shengxianensis are hypostomatic and the stomata are encircled by many papillae. Based on the comparisons above, all the extant species and previously fossil species of Cupressaceae are more or less different from our fossils, which supports their designation as a new fossil species of Platycladus.

Discussion
The genus Platycladus only contains one extant species, P. orientalis, native to S Gansu, Hebei, Shaanxi and Shanxi of China, and introduced or status uncertain in Korea and the Russian Far East [1,2]. It is therefore very difficult to establish its original natural range [1,2]. Wilson [21] argued that the species occurs naturally  [2] is certain that the occurrences in Yunnan and Sichuan are not indigenous, and there had been no evidence in the fossil record to indicate a historical natural distribution more southernly than at present. In the present study, the occurrence of P. yunnanensis sp. nov. in western Yunnan suggests that this genus might have a more southernly natural distribution during the late Miocene, which is beyond the bounds of the modern natural distribution of this genus (Fig. 7) if Farjon's opinion is correct. The climate of the natural range of Platycladus orientalis is characterized by relatively dry and moderately cold winter conditions [2]. However, P. orientalis is introduced extensively in China due to its toleration of a wide range of climate and soil conditions [3,22]. The present fossil leaves have open stomatal pits and shallowly sunken guard cells, usually reflecting a humid climate [9]. Therefore, the late Miocene P. yunnanensis sp. nov. should also grow under a humid subtropical climate in West Yunnan. Such a climate inference is supported by previous quantitative analyses of several late Miocene floras in southwestern China [23][24][25].
On the basis of molecular phylogenetic studies within the Cupressaceae s.l., two Asian species, Platycladus orientalis and Microbiota decussate, form a clade [4,5]. Mao et al. [4] suggested that the age of the node of Platycladus-Microbiota is ca. 33 Ma (Early Oligocene). However, the earliest fossils of Platycladus and Microbiota [10] only date back to the late Miocene and Pliocene, respectively. Phylogenetic data indicate that the clade Platycladus-Microbiota is closely related to the genera Tetraclinis and Calocedrus [4,5]. The earliest fossils of Tetraclinis [7] and Calocedrus [9] are documented from the Oligocene. However, Kvaček et al. [7] indicated that the genus Tetraclinis had migrated between western North America and Europe during the Oligocene or Miocene through the North Atlantic land bridge, but did not cross Asia. Brunsfeld et al. [26] indicated that the clade Platycladus-Microbiota is close to Calocedrus, but Tetraclinis is the sister taxon to Thuja and Thujopsis based on the rbcL sequences.
The extant eastern Asian Calocedrus usually occurs in mixed evergreen coniferbroadleaved forests in the subtropical or tropical montane areas [27]. However, extant Platycladus and Microbiota are found in a climate of relatively dry and very cold winters [2]. The molecular phylogenetic studies indicate that the divergence between Platycladus and Microbiota, as well as the species among Calocedrus both happened during the Oligocene [4,8]. If the molecular phylogenetic evidence proved to be correct, the separation between the clade Platycladus-Microbiota and the genus Calocedrus was most likely a result of the broad arid belt across Central China during the Oligocene [28][29][30]. In addition, the the cooling down of the global temperature [31] and the strengthening of Asian monsoon [32][33][34][35][36][37] since the middle Miocene further promoted the migration of these genera. The deep split between eastern Asian and North American Calocedrus before the Oligocene via the Bering land bridge has been demonstrated [8,9]. However, due to the inadequate paleobotanical data, the differentiation of Platycladus and Microbiota is not well defined.