Late Eocene white pines (Pinus subgenus Strobus) from southern China

Fossil records indicate that the genus Pinus L. split into two subgenera by the Late Cretaceous, although subgenus Strobus (D. Don) Lemmon is less well documented than subgenus Pinus L., especially in eastern Asia. In this paper, Pinus maomingensis sp. nov. is established based on a compressed seed cone from the upper Eocene of the Maoming Basin of southern China. This species is attributed to genus Pinus, subgenus Strobus, section Quinquefoliae Duhamel, subsection Strobus Loudon based on the combination of morphological characters obtained from the cone scales, specifically from the terminal umbo, rhombic apophysis, and cuticle structure. Associated fascicles of needle leaves with deciduous sheaths and bulbous bases are recognized as Pinus sp. and also represent Pinus subgenus Strobus. This new discovery from the Maoming Basin constitutes the first megafossil record of subgenus Strobus from southern China and implies that the members of this subgenus arrived in the southern region of China by the late Eocene. The extant species of subgenus Strobus are mainly distributed in northern temperate and tropical to subtropical mountainous regions. We propose that the Maoming Basin was adjacent to a mountainous region during the late Eocene.

from the Late Cretaceous Aachen Fm. of northeastern Belgium 16 . More recent records of subgenus Strobus, for example ovulate cones, occur in the mid-Eocene strata 10 , no megafossils are known from the Paleocene 7 .
The megafossil record of subgenus Strobus in China is very restricted. Only two Pliocene fossil wood species from Yunnan Province, Pinus armandii Franchet and P. cf. armandii, were reported by Yi et al. 17,18 . In this study, we describe two species of subgenus Strobus based on a fossil pine cone and needle remains collected from the Huangniuling Fm. of the Maoming Basin in southern China (21°42′ N, 110°53′ E) (Fig. 1).
The Maoming Basin is a small, upper Mesozoic to Cenozoic sedimentary basin oriented along a northwestern to southeastern axis in Guangdong Province. The Upper Cretaceous to Neogene deposits of the Maoming Basin are subdivided into eight formations; in ascending order they are: the Sanyajiang Fm., Tongguling Fm., Shangdong Fm., Youganwo Fm., Huangniuling Fm., Shangcun Fm., Laohuling Fm., and Gaopengling Fm 19,20 . The Huangniuling Fm., from which the megafossils were recovered, consists principally of fluvial grey, yellow to white sandstones, siltstones and conglomerates with beds and lenses of varicolored mudstones and claystones, ranging from whitish pink to grayish green to brownish gray. Based on a magnetostratigraphic study by Wang et al. 21 , buttressed by palynological data by Aleksandrova et al. 22 , the age of the Huangniuling Fm. is considered to be late Eocene. Therefore, the occurrences of fossil pine cone and needle remains from Huangniuling Fm. provide new information on the distribution of subgenus Strobus and imply that the members of this subgenus arrived in the southern region of China by the late Eocene.   22 .

Systematics
Repository. The Museum of Biology of Sun Yat-sen University, Guangzhou, Guangdong Province,
Diagnosis. Seed cone oblong-elliptical to cylindrical. Ovuliferous scales thin with longitudinal ridges on abaxial side, spirally arranged. Apophyses rhomboidal to broadly rhomboidal; no sealing band. Umbo terminal, slightly sunken, bearing a slightly swelling transverse ridge in a diamond-shaped area.
Description. The seed cone ( Fig. 2A,B) is variably oblong to elliptical or cylindrical. The visible portion of the seed cone is 16 cm in length. The cone width, generally consistent over the middle and upper part of the cone, is ca. 5 cm. The basal part of the cone is more or less cuneate (probably due to incomplete preservation) and ca. 1 cm in diameter. The ovuliferous scales are thin, oblong-obovate, helically arranged around the axis, about 5 cm long, 2.4 cm wide, and about 0.1 cm thick in the middle, with a slightly expanded apophysis (Fig. 2D). The isolated ovuliferous scale (Fig. 2E) is rhomboidal to obovate, sessile, ca. 2.6 cm long by 2 cm wide. Apophyses are rhomboidal to broadly rhombic. Longitudinal ridges and furrows are clearly visible on the abaxial side of the scales. The umbones ( Fig Cuticles obtained from fossil needles preserve the following characters: Epidermal cells are rectangular and longitudinally oriented (Fig. 4I). The anticlinal walls of epidermal cells are straight or sinuous and slightly thickened. Stomatal complexes are paracytic, elliptical to oval in shape (Fig. 4J), and arranged in longitudinal rows. Well-developed cuticular flanges are preserved between guard cells and subsidiary cells. The polar subsidiary cells are smaller than the lateral subsidiary cells.

Discussion
Classification of the species with terminally-positioned umbones. Over 40 taxonomic treatments have been proposed for the genus Pinus 4,23,24 . The early classificatory systems are based mainly on morphology 25,26 , whereas more recent studies use molecular phylogenetic approaches 4,6,23,27 . The recent system of Pinus by Gernandt et al. 6 based on rbcL + matK gene sequences and morphological characters is relatively widely accepted. This classification includes two subgenera (Pinus and Strobus), four sections (Pinus, Trifoliae Duhamel, Quinquefoliae, Parrya Mayr) and 12 subsections. The strictly North American section Parrya is restricted to the subsections Cembroides Engelmann, Nelsoniae Van Der  phological characters: (i), thin ovuliferous scales helically arranged around the axis; (ii), the apex of the ovuliferous scale is slightly inflated with an apophysis and terminal umbo. Consequently, this fossil cone can be assigned to the genus Pinus, according to distinguishing features summarized by Miller 9 . The umbo position and absence of a sealing band on the lower side of apophysis exclude a close affinity to the subgenus Pinus 28 , and judging from its distinct terminal umbo, this cone can be easily classified into the subgenus Strobus. The isolated ovuliferous fossil scale is considered to be conspecific with the cone due to the morphological similarities of the apophysis and umbo. Wing development and the size of seeds also are important in Pinus classification 5 . However, seeds discovered in the middle and upper part of the present cone provide only limited evidence for classification, principally because it is difficult to distinguish whether these seeds are winged or not.
Comparisons with fossil cone taxa. Pinus existed at the onset of the Cretaceous and is well documented for the Cretaceous and Cenozoic 29 . Relatively few Cretaceous fossils have affinity with the subgenus Strobus and some specimens originally attributed to this subgenus were reassigned to the subgenus Pinus or other genera. Pinus magothensis Penny, from the Magothy Fm. of Delaware, U.S.A. is 9-10 cm long by 3-4 cm wide with thin flattened scales, each subtending two winged seeds; and with a thin apophysis and an inconspicuous terminal umbo 13 . There is no detailed description for the apophysis and umbo of P. magothensis, therefore it is difficult to compare it with P. maomingensis. Pinus magothensis was considered to be the most important evidence for the subgenus Strobus during the Cretaceous. However, Miller 29 thought the characters were insufficient to show conclusive affinity with this subgenus due to the lack of details involving internal structure. As a result, the cone was reassigned to Pityostrobus by Miller and Malinky 14 . Willyard et al. 15 also accepted this reassignment.
The fossil record of pines continues into the Paleogene and Neogene where fossil seed cones are more abundant and are overwhelmingly assigned to the subgenus Pinus 10,14 . The diversity of cone types, as well as the megafossil record of external impressions, implies that a number of species in various subsections were already in existence during the Eocene 10 . Those cones, such as Pinus lindgrenii Knowlton 30 , P. balfouroides Axelrod 31 and P. sanjuanensis Axelrod 10 , appear assignable to the subsections of section Parrya and are excluded for comparison here. We focus on those fossil cones that exhibit affinity to the section Quinquefoliae, subsection Strobus sensu Gernandt et al. 6 .
The middle Eocene cone Pinus delmarensis Axelrod 10 , from the Del Mar Fm. near San Diego, California (U.S.A.) is larger (estimated as 26 cm long) than P. maomingensis, but is the most similar fossil species to our specimens. The similarities involve overall shape and the size of the ovuliferous scale and terminally positioned umbo. However, the lack of detailed published information on the apophysis and umbo of P. delmarensis precludes an informative comparison. Axelrod 10 observed that P. delmarensis was very similar to the extant species P. lambertiana Douglas. However, the extant species P. lambertiana is readily distinguished from P. maomingensis (see the comparison with extant species below), indicating that additional detailed comparisons with P. delmarensis are unnecessary.
Pinus florissanti Lesquereux 32 was established based on an ovoid seed cone with large scales from the upper Eocene Florissant Fm., Colorado, subsequently supplemented with seeds and needles by MacGinitie 33 . MacGinitie 33 also reassigned P. sturgisi Cockerell 34 to this species. Pinus florissanti was considered to be related to extant P. ponderosa Douglas ex C. Lawson (subgenus Pinus) 32,33 , but Axelord 10 suggested that this fossil species was more closely allied to P. flexilis E. James (subgenus Strobus, section Quinquefoliae, subsection Strobus) because the characters of the cone and needles of P. florissanti were similar to those of the extant P. flexilis. Millar 7 also supported the close affinity of P. florissanti with subsection Strobus. The cone of P. florissanti is shorter but wider (11 cm long by 6 cm wide 33 ) than our specimens, and the cone scales are shorter and narrower (4.5 cm long by 1.5 cm wide 33 ). Furthermore, this fossil species differs from our specimens in having conical and rhomboidal umbo.
Pinus echinostrobus Saporta 35 from the upper Oligocene of Armissan in Aude, France, has an ovate, flat and slightly striated apophysis with a terminal umbo. The nearest living relative of this species was considered to be P. koraiensis Siebold et Zuccarini 36 which is distinguished from our specimen by having a reflexed apex with a basal-uncinate umbo. The cone of P. echinostrobus differs from our specimen in that the cone scales are shorter (1.0-1.4 cm long by 1.2-1.4 cm wide) and the apophysis is slightly curved and basally uncinate at the end of the umbo.
Pinus grossana Ludwig 37 was recovered from the lower Miocene of Rockenberg locality of Wetterau, Germany. This cone is similar in width (5.5 cm) but is much longer (23 cm) than the present specimen according to the descriptions of Mai 36 . The thin cone scale with stripes on the abaxial side and the rhomboidal and slightly convex apophysis with a slightly reflexed apex are both similar to our specimens, although P. grossana is much wider and its length is unknown. In addition, this species differs from P. maomingensis in its erect, conical umbo. Ludwig 37 thought that the nearest relative of this species was P. lambertiana. However, Mai 36 considered that the overall features of the cone and the structure and testa anatomy of its associated winged seed indicated a relationship with extant P. wallichiana Jackson. This extant species is different from P. maomingensis in having wedge-shaped cone scales and grooved apophyses with an obviously incurved apex and a blunt umbo.
Pinus letzii Kirchheimer 38 was described from the upper Miocene of the lower Rhenish Basin, Germany. The cones were 6-12 cm long by 2.5-3.5 cm wide and the cone scales were 2.5-3 cm long by 1-1.7 cm wide. The closest relative of P. letzii is thought to be P. dalatensis de Ferré 36,39 . The cone of P. letzii is shorter and narrower than that of P. maomingensis. Additionally, the flat apophyses of P. letzii are triangular or pentagonal in shape, with a triangular and terminal sunken umbo, whereas P. maomingensis bears rhombic to broadly rhombic apophyses with a diamond shaped and sunken umbo.
Pinus monticola var. fossilis 40 from the Pliocene of Siberia, Russia, differs from our present specimens in possessing more robust and thicker cone scales. This species was once thought to be similar to the extant American species P. monticola Douglas ex D. Don 41 , but Axelrod 10 assessed its affinities as more nearly allied to extant Asian species, notably P. armandii Franchet of central China. The extant species P. armandii differs from P. maomingensis in having a triangular or rhombic and thickened apophysis with an obtuse umbo. The Miocene cone P. itelmenorum Dorofeev 42 from the Mammoth Mountain flora of Aldan River, Russia, is similar to P. monticola var. fossilis. However, this relatively complete fossil cone bears more massive and broader, thicker scales. Pinus itelmenorum also shows a relationship with the extant species P. armandii and its relatives, and this fossil species can also be distinguished from our specimens.  Table 2 for detailed comparison. Pinus dalatensis and P. morrisonicola Hayata show less similarity with our specimens in having smaller cone scales with recurved or slightly recurved terminal umbones. Additionally, P. morrisonicola bears an obtuse apophysis apex. Pinus dabeshanensis Cheng and Law has a weakly developed umbo, but the apex of the apophysis is obtuse and the upper lateral side of the apophysis clearly is reflexed. The umbones of P. chiapensis (Martínez) Andresen, P. peuce Grisebrach, and P. kwangtungensis Chen are flat, straight, or slightly incurved, and this character is different from that of P. maomingensis. The species P. fenzeliana Handel-Mazzetti, P. parviflora Siebold et Zuccarini, and P. wangii Hu et Cheng bear sunken to slightly sunken umbones which are very similar to the present specimens. However, the apex of apophysis of P. parviflora is rounded and its lateral side is reflexed. The lateral side of the apophysis of P. wangii is slightly incurved.

Comparisons with extant cone taxa. Almost all members of subsection
After detailed comparisons with the extant species, the results show that, although the cones of Pinus fenzeliana are somewhat smaller than the cone of P. maomingensis, it is the most similar extant species given its cone's general shape, possession of both a broadly rhombic apophysis with slightly reflexed upper lateral side and a sunken umbo. Nevertheless, there is a subtle difference in the umbonal area. The middle part of the umbonal area of P. maomingensis is sunken and slightly folded, and the transversely stretched ridge is situated near the center of the umbonal area, and extends to the slightly reflexed apex. This character seems more similar to the cone scale apex of P. sibirica Du Tour of the subsection Cembrae sensu Price et al. 4 , but a key difference is that P. sibirica has a basal-uncinate umbo. Although we find two slight protrusions on the center umbonal area of our specimens, there is insufficient evidence to conclude that the fossil cone is a member of the subsection Cembrae sensu Price et al. 4 . Pinus maomingensis illustrates the close relationship between the subsections Strobus and Cembrae of Price et al. 4 which were combined into one subsection (subsection Strobus) by Gernandt et al. 6 .
The cone morphology and epidermal structure of the cone scales of Pinus maomingensis was compared with modern species and was found to have similarities both to P. armandii and P. fenzeliana. Pinus armandii is similar to our specimens in the general size and shape of its cone and cone scales. However, epidermal structures of the fossil cone are more similar to those of P. fenzeliana. Both of these species  possess three kinds of epidermal cells: longitudinally elongate cells, irregular polygonal cells, and irregularly shaped cells. They also share longitudinally elongate cells and irregularly polygonal cells over the ribs and furrows. However, P. fenzeliana has longer cells over the ribs. The trichome bases of P. fenzeliana have one ring of epidermal cells, and the shapes of these cells are different from those of the fossil cone. Pinus armandii has shorter epidermal cells over the ribs, and the trichomes have two rings of epidermal cells surrounding their bases. However, they are distinguished from P. maomingensis by smaller numbers and the trapezoidal shape of epidermal cells. Therefore, based on the detailed comparisons, we conclude that our specimens represent a new species of subgenus Strobus, section Quinquefoliae, subsection Strobus. We formally describe it as Pinus maomingensis sp. nov., in light of the following defining characters: (i) the shape and size of the cone; (ii) the shape and size of the cone scales; (iii) the shape, size and umbo of apophyses; (iv) epidermal structures.
The fossil needles. Abundant fossil needles of pinaceous affinity were collected from the Huangniuling Fm. of the Maoming Basin. A majority of the specimens have a bulbous base and deciduous sheaths. These characters also are diagnostic of needle fascicles of the subgenus Strobus [3][4][5] . Because scales of the fascicle sheaths abscise as the needles elongate in most species of this subgenus 4 , we speculate that our specimens were preserved in different growth stages, as we found a pair of sheath scales arising from surrounding bud scales that had not yet abscised. The bracts that subtend the fascicles are non-decurrent in subgenus Strobus 4,5,28 ; this feature is clearly displayed in several fossil specimens. Moreover, characters of well-preserved fossil fascicles are very similar to subgenus Strobus needle fascicles described by Stults et al. 43 from a Pliocene deposit in the coastal plain of the Gulf of Mexico. Both of these taxa have short, bulbous fascicle bases. Both species have deciduous sheaths. Consequently, we believe that the fossil needle fascicles associated with the same sediments as P. maomingensis also belong to subgenus Strobus.
The number of needles per fascicle is almost constant within many species of pines, and frequently has been used as a species-specific character in many taxa 2,4 . Among the Haploxylon pines, the number of needles per fascicle always is five in section Quinquefoliae, but ranges from one to five in section Parrya. Fossil needle fascicles collected from the Huangniuling Fm. of the Maoming Basin mostly consist of five needles, although several specimens are preserved as three or four needles, possibly due to the preservation conditions of fossils or needles that fell singularly from mature fascicles with deciduous sheaths 44 . The margins of fossil needles are finely serrate. The needles of the extant species in the same section Quinquefoliae are finely serrate in eastern Asia and entire in North America 10 . Species of section Parrya that grow in China also possess finely serrate needles.
The anatomical characters of needles, such as the number of vascular bundles, the number and position of resin canals, and cuticular structure also provide important characters for Pinus classification [45][46][47][48] . Although a large number of fossil needles were collected from the Huangniuling Fm. in the Maoming Basin, most are preserved as impressions, and only a few specimens are preserved replete with cuticle fragments which show features of epidermal cells and stomatal complexes. Since cuticle characters of fossil needles were incomplete and lack anatomical structure in cross section, it is unreasonable to refer them to a certain species. These fossils are recognized as Pinus sp.
Biogeographic implications. The fossil record indicates that genus Pinus split into two subgenera by the Late Cretaceous. The earliest definitive representative of the subgenus Strobus was discovered in the Late Cretaceous (Santonian) Aachen Fm. of northeast Belgium 16 . During the Paleogene, the subgenus Strobus was common in Eurasia and North America 10,36,49 but very rare in China. Pinus maomingensis sp. nov. and Pinus sp., collected from the Huangniuling Fm. in the Maoming Basin of Guangdong Province, assuredly belongs to subgenus Strobus. The discovery of these fossils indicates that white pines were distributed in southern China by at least the late Eocene.
Palynological assemblages from the Huangniuling Fm. of the Maoming Basin of Guangdong suggest that the late Eocene was warm and humid 22 . In addition, many fossil plants collected in the Maoming Basin, such as Podocarpaceae, Arecaceae, Dipterocarpaceae, Annonaceae, Juglandaceae, Euphorbiaceae, Myrtaceae, Fagaceae, Altingiaceae, and Lauraceae 22 , provide evidence for tropical-subtropical components in the Eocene Maoming flora. The subgenus Strobus mainly inhabits the north and tropical-subtropical mountainous regions of China and grows well in habitats characterized by temperate and moist climate 5,50-52 . Presently, however, white pines do not occur in or adjacent to the Maoming Basin. Currently, only two species-Pinus fenzeliana and P. kwangtungensis-are naturally distributed in mid to high altitude areas of southern China. These two species grow at an altitude of ca. 1000 m in the northern part of Guangdong Province (Lechang and Ruyuan Mountains) and the Wuzhi Mountains of Hainan Island 5 . Because of these distributional patterns, we propose that the Maoming Basin was adjacent to a mountainous region during the late Eocene.

Methods
A seed cone, one isolated ovuliferous scale and large numbers of needles were collected from the Huangniuling Fm. of the Maoming Basin, southern China. This cone and the isolated ovuliferous scale are preserved as compressions. Cuticular fragments were obtained from the distal part of the ovuliferous scales of the compressed cone (MMJ2-1-005b). Most needles are impressions, and only a few needle fragments preserved, albeit poorly, epidermal remains. Leaf cuticular fragments were obtained Scientific RepoRts | 5:16390 | DOI: 10.1038/srep16390 from specimen MMJ3-002a. Specimens were photographed using a Canon EOS 500D digital camera. Photomicrography of the seeds was done with using a Leica S8ap0 and Image-Pro software. The terminology for morphological description of subgenus Strobus follows Klaus 53 , Fu et al. 5

and Earle 3 .
Fossil cuticle pieces were removed from the specimens and immersed in 30% HNO 3 solution overnight, and then washed in distilled water 2 or 3 times. The samples then were treated with very weak (ca. 1-2%) ammonia for 4 hours, and then washed with distilled water. Cuticles were mounted on glass slides and observed and photographed using a Nikon microscope in transmitted light and processed with Adobe Photoshop CS5 (Adobe Inc., San Jose, California, USA). All the fossil specimens and slides are stored at the Museum of Biology of Sun Yat-sen University, in Guangzhou, China.