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Needles and seed cones of Pinus premassoniana sp. nov., and associated pollen cone from the upper Miocene in East China
Review of Palaeobotany and Palynology 197 (2013) 78–89
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Research paper
Needles and seed cones of Pinus premassoniana sp. nov., and associated pollen cone from the upper Miocene in East China Su-Ting Ding a,b, Jing-Yu Wu a,b, Jun-Lin Chen a, Yi Yang a, De-Fei Yan a, Bai-Nian Sun a,b,⁎ a b
School of Earth Sciences & Key Laboratory of Mineral Resources in Western China (Gansu Province), Lanzhou University, Lanzhou 730000, China State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, CAS, Nanjing 210008, China
a r t i c l e
i n f o
Article history: Received 28 January 2013 Received in revised form 8 May 2013 Accepted 9 May 2013 Available online 26 May 2013 Keywords: Pinus premassoniana sp. nov. needles and seed cones associated pollen cone historical biogeography Miocene East China
a b s t r a c t A long shoot with needles and two immature seed cones, and some isolated organs of shoot, needles and seed cones from the late Miocene Shengxian Formation in Zhejiang Province, East China are described. A detailed comparison of the gross morphology and cuticle micromorphology of needles and seed cones with previously published Cenozoic fossils and related extant pine species reveals that the present fossils can be identified as a new species, Pinus premassoniana sp. nov., which has the closest affinity with extant Pinus massoniana. A pollen cone with copious bisaccate pollen grains from the same locality is identified as an indeterminate species of subgenus Pinus. The pollen grains show a verrucate external sculpture on the corpus and sacci and an alveolae internal structure in the sacci. The fossil needle cuticles and pollen cone of the genus Pinus from China are reported for the first time. The modern distribution indicates that P. premassoniana sp. nov. should also live under a warm and humid climate. Hitherto, there is no any reliable fossil record that has an affinity with the extant P. massoniana. The occurrence of the present fossils suggests that P. premassoniana has existed in East China since at least in the late Miocene. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Pinus L. with approximately 110 species is the largest genus of conifers widely distributed predominantly in the Northern Hemisphere (Richardson and Rundel, 1998; Farjon, 2005). The earliest accepted record of Pinus is Pinus belgica Alvin from the Lower Cretaceous of Belgium based on a permineralized cone (Alvin, 1960). Hitherto, over 50 species of isolated vegetative remains of Pinus have been described (e.g., Chaney, 1954; Mehringer and Ferguson, 1969; Miller, 1976, 1977; Robison, 1977; Stockey, 1983; Phipps et al., 1995; Saiki, 1996; McKown et al., 2002; Smith and Stockey, 2002; Erwin and Schorn, 2006; Xing et al., 2010; Klymiuk et al., 2011). However, fossil needles and cones preserved on the same long shoot are very rare. Hitherto, only one specimen as Pinus resurgens was described from the Miocene in Armissan, Southeastern France by de Saporta (1865). Most fossil species of Pinus are based on disassociated structures (e.g., cones, needles, winged seeds, and pollens). Therefore, the reconstruction of whole plants is questionable because of the lack of attachment or indisputable association evidence (Axelrod, 1986; Stockey and Ueda, 1986). The fossil record of pines from the Cretaceous to the Recent is typically considered good (Stults et al., 2010). In China, four cone species ⁎ Corresponding author at: School of Earth Sciences & Key Laboratory of Mineral Resources in Western China (Gansu Province), Lanzhou University, Lanzhou 730000, China. Tel./fax: +86 931 8915280. E-mail address: [email protected] (B.-N. Sun). 0034-6667/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.revpalbo.2013.05.004
from the upper Eocene–lower Oligocene of Hebei Province (Tao and Wang, 1983), the Miocene of Zhejiang Province (NIGMR, 1982), the Miocene of Yunnan Province (Xing et al., 2010) and the Pliocene of Yunnan (Tao and Kong, 1973), two needle species from the Eocene of Liaoning Province (WGCPC, 1978) and the Oligocene of Jilin Province (Guo and Zhang, 2002), and one seed species from the Oligocene of Hebei Province (WGCPC, 1978) were reported. In this study, we describe a long shoot with needles and seed cones, and some isolated organisms of shoots, needles and seed cones as a new species, Pinus premassoniana sp. nov., from the Miocene of Zhejiang. One pollen cone with in situ pollens as an indeterminate species of Pinus was also examined under the scanning electron microscopy (SEM). The fossil needle cuticles and pollen cone from China are reported for the first time. The comparison with selected extant and previously published fossil pines in this paper indicates that the gross morphological characters of needles and seed cones, as well as the needle cuticle micromorphological features, are important to the taxonomy of fossil Pinus. Moreover, the discovery of the present fossils provides us a rare opportunity to understand the Pinus evolution during the Miocene.
2. Material and methods The fossils described in this study were collected from the Shengxian Formation at Jiahu Village (29°09′N, 121°14′E), Tiantai County, Zhejiang Province, East China (Fig. 1). Based on lithostratigraphy and biostratigraphy researches (NIGMR, 1982; Li, 1984; BGMRZP, 1989; Liu et al.,
S.-T. Ding et al. / Review of Palaeobotany and Palynology 197 (2013) 78–89 100ºE
105ºE
115ºE
110ºE
120ºE
Yangtze River
30ºN
30ºN
Zhejiang
China 25ºN
25ºN
20ºN
20ºN
100ºE
105ºE
110ºE
115ºE
120ºE
Fig. 1. Map showing the fossil locality (black triangle) of Pinus premassoniana sp. nov. and the modern distribution of Pinus massoniana (broken line).
1992), the geological age of the Shengxian Formation is regarded as the late Miocene (Ding et al., 2011). The carbonized needles were immersed in 10% HCl for 2 h, and then with 50% hydrofluoric acid overnight. The needle fragments were macerated into 30% HNO3 solution for ca. 48 h. When the black color changed to light brown, the specimens were washed and dripped with 5% NH4OH for ca. 5 min. After washing with distilled water several times, part of the cuticles was stained with 1% safranine solution. The cuticles were mounted on slides and embedded in glycerine jelly. The slides were observed and photographed using a Leica DM4000B light microscope. Another part of unstained cuticles was mounted on stubs and coated with gold under a sputter-coater, observed and photographed under a JEOLJSM 6380LV scanning electron microscope. For the pollen cone, several small fragments (microsporophylls) were removed from the middle part with the help of a dissecting needle and immersed in 10% HF solution in a plastic bottle for 12 h. After careful washing with distilled water, the fragments were divided into two parts. One part was placed on a concave glass slide, added with a drop of 65% HNO3 for several minutes until the materials became light brown and dripped with distilled water for several times to displace the nitric acid. Then, the specimen was removed to another glass slide with one drop of glycerol. In situ pollens were checked and photographed under the light microscope. Another part was mounted on a stub and coated with gold and examined under the SEM. All slides for LM and stubs for SEM are stored in the Institute of Palaeontology and Stratigraphy, Lanzhou University, Gansu Province, China. The extant specimens of Pinus massoniana for the morphological comparisons were collected in Tiantai County, Zhejiang Province. The terminology for morphological description of pine branches follows Farjon (2005) and Dörken et al. (2010), and that for seed cones follows Klaus (1980, 1989) and Mai (1986). Terms for cuticle micromorphology follow Yoshie and Sakai (1985), Kim et al. (1999) and Whang et al. (2001, 2004). Classification of the genus Pinus follows the recent scheme of Gernandt et al. (2005). 3. Results 3.1. Systematics Family: PINACEAE L. Genus: Pinus L. Subgenus: Pinus L. Section: Pinus sensu Gernandt, Geada López, Ortiz García & Liston. Subsection: Pinus sensu Gernandt, Geada López, Ortiz García & Liston. Species: Pinus premassoniana Su-Ting Ding et Bai-Nian Sun, sp. nov.
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Specific diagnosis: Winter bud ovoid-cylindric, apex acute, needles in fascicles of two, basal sheaths persistent. Needles amphistomatic, epidermal cells rectangular, end walls of epidermal cells vertical or oblique, anticlinal walls sinuous. Stomatal rows continuous, stomata oriented regularly along long axis of the needles, two guard cells surrounded by 6–8 subsidiary cells. Florin rings rise outwards and centripetally resulting in a volcanic shape (type C). Immature seed cones ovoid-conical, closed and symmetric. Mature seed cones symmetric, apex tapered, woody ovuliferous scales helically arranged. Apophyses rhombic to pyramidal in plane view, with a distinct transverse keel. Umbos dorsal and slightly sunken. An approximately erect mucro occurs on the upper umbo field, characteristic of the perexcentromucronate umbo type. Etymology: The specific epithet refers to the similarity with the extant species Pinus massoniana. Holotype: JH-417A (Plate I, 1) Isotype: JH-417B (Plate I, 2, 6) Paratypes: JH-083A (Plate I, 3), JH-083B (Plate I, 4), JH-790, JH-471, JH-566 (Plate I, 7, 8, 11–13), JH-041(Plate I, 9), JH-998 (Plate I, 10) Locality: Jiahu in Tiantai County of Zhejiang Province, China Stratigraphy: Shengxian Formation Age: late Miocene Repository: Institute of Palaeontology and Stratigraphy, Lanzhou University, Lanzhou, China. 3.2. Description Two long shoots are preserved (Plate I, 1–3). The winter bud is small, ovoid-cylindric, apex acute, 8 mm long and 3.3 mm wide (Plate I, 3, 4). The needles are held in a persistent basal sheath 4–8 mm long (Plate I, 3), in fascicles of two, preserved 6–10 cm long and 0.8–1 mm wide, margins serrate (Plate I, 1–5). Two immature seed cones borne at the apices of the long shoot, ovoid-conical, symmetric, closed, 1.2–1.4 cm long and 0.8–0.9 cm wide, have ca. 62 scales (Plate I, 1, 2, 6). Mature seed cones are isolated, conical, closed or open, symmetric, 5.2–7.4 cm long, and 2.8–3.5 cm wide, with a tapered apex (Plate I, 7–10). Each cone has 60 to 65 woody ovuliferous scales helically arranged around the axis. Cone scales expanded at each apophysis, up to ca. 2.5 cm long in the middle part of the cones (Plate I, 10, 11). In plane view, the apophyses are typically rhombic in the upper half of the cones, 1.0–1.4 cm wide and 0.7–1.0 cm high; however, the basal apophyses sometimes show a more pyramidal shape or nearly pentagonal, 0.6–0.9 cm wide and 0.7–0.9 cm high. Each apophysis possesses a distinct transverse keel. The umbos are dorsal, rhombic to elliptic in plane view, are relatively larger in the upper half of the cone (2–3 mm wide and 1.5–2.0 mm high) and smaller in the basal half (1.0–1.5 mm wide and 0.8–1.2 mm high). They are slightly sunken on the surface of the apophyses (Plate I, 11, 12). An approximate erect mucro occurs on the upper umbo field above the transverse keel, which are the characteristics of perexcentromucronate umbo type (Plate I, 11–13). Needles are amphistomatic; epidermal cells are rectangular on both adaxial and abaxial cuticle surfaces, 80–240 μm in length and 7–15 μm in width, extremely elongated in between, but much broader within the stomatal lines, with one to three interstomatal cells in the stomatal row (Plate II, 1–3); the end walls of the epidermal cells are vertical or oblique; the anticlinal walls sinuous, and the periclinal walls are granular in the internal surface (Plate II, 5). Stomatal rows are continuous, stomata oriented regularly along long axis of needles, have 3–5 lines per adaxial surface and 7–10 lines per abaxial surface. The stomatal complexes are monocyclic and elliptic, 40–50 μm in length and 28–36 μm in width; two guard cells surrounded by 6 to 8 subsidiary cells, and the lateral subsidiary cells are usually bigger than the polar cells (Plate II, 2, 4, 7). The external cuticle surface is slightly undulating, with the stomatal apparatus oriented parallel to the leaf axis. Each stoma is surrounded by an encircling ridge called a Florin ring (Buchholz and Gray, 1948). The Florin rings rise outwards and centripetally resulting
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in a volcanic shape (Plate II, 8–10), which are the characteristics of Florin ring type C (Yoshie and Sakai, 1985). 3.3. Comparisons The present fossils are characterized by some typical features such as needles in fascicles of two, and seed cone scales have dorsal umbos. Therefore, the present fossils can be assigned to the genus Pinus based on the combined morphological features of the needles and seed cones (Richardson and Rundel, 1998; Frankis, 2002). Three isolated mature seed cones and one long shoot with immature cones occurred in the same horizon. These seed cones have similar cone-scale complexes and numbers of scales per cone. The present fossil shoot and the isolated seed cones both have the closest affinity with extant Pinus massoniana based on a detailed morphological comparison. Although the isolated mature cones are slightly elongate as compared with the immature cones, this shape variability can also be found in the seed cones of extant P. massoniana (Plate III, 2–4). Therefore, we considered that the present long shoot and the isolated seed cones should represent the same species. 3.3.1. Comparisons with extant Pinus Most current classifications recognize two major lineages within the genus Pinus, i.e., subgenus Pinus (diploxylon or hard pines) and subgen. Strobus (haploxylon or soft pines) (e.g., Little and Critchfield, 1969; Krupkin et al., 1996; Price et al., 1998; Liston et al., 1999; Gernandt et al., 2005; Klymiuk et al., 2011). The present fossils possess the needles in fascicles of two, which differ from most species of subgen. Strobus, õexcept for Pinus krempfii. However, the needles of P. krempfii have lanceolate-falcate shape and the basal sheaths are deciduous (Farjon, 2005), clearly different from our slender fossil needles and persistent sheaths. Subgen. Pinus consists of two sections, viz. sect. Pinus and sect. Trifoliae (Gernandt et al., 2005). However, some cuticular micromorphological features distinguishes our fossils from those of sect. Trifoliae. For example, the polar subsidiary cells of sect. Trifoliae are mostly shared and larger than the lateral subsidiary cells (Whang et al., 2001; Whang et al., 2004), whereas 1–3 cells between the stomata in row (Table 1) and the polar subsidiary cells are smaller than the
lateral subsidiary cells in the present fossil needles (Plate II, 2, 4, 7). Sect. Pinus consists of subsect. Pinus and subsect. Pinaster. However, one Himalayan species of subsect. Pinaster, Pinus roxburghii, possesses needles in fascicles of three (Farjon, 2005), and Mediterranean species of subsect. Pinaster usually has rectangular stomatal apparatus (Álvarez et al., 2009a). As demonstrated above, the present fossil needles can be further assigned to subsect. Pinus. Table 1 provides a list of needle morphology and cuticular features of subsect. Pinus. Only four species, viz. Pinus thunbergii, Pinus mugo, Pinus sylvestris, and P. massoniana, possess epidermal cells with vertical and oblique end walls and sinuous anticlinal walls, which are similar to the present fossil needles. However, P. thunbergii has 8–10 subsidiary cells, and the cells between stomata in row are mostly shared, P. mugo has needles of 1.5–2.2 mm in width and circular stomatal apparatus. P. sylvestris possesses rectangular stomata with 4–6 subsidiary cells and the type D Florin ring, which distinctly differs from those of our fossils. Based on a detailed comparison (Table 1), our fossil needles are significantly similar to the extant needles of P. massoniana (Plate III, 1, 6, 7), both on morphology and cuticular features. The present seed cones are characterized by the cone-scale complexes helically arranged around the axis, ovuliferous scales expanded at the apex, and each apophysis possesses a transverse keel and a dorsal umbo. According to the aforementioned features, these fossil seed cones can be assigned to the genus Pinus (Miller, 1976) and has affinity with subgen. Pinus (Frankis, 2002). Klaus (1980, 1989) subdivided the subgen. Pinus into two groups: viz. centromucronate type (mucro located in the center of umbo) and excentromucronate type (mucro located above the umbo). The current study shows that the mucro of our fossil cones lies above the keel; thus, the umbos can be a diagnostic of the excentromucronate type. Furthermore, the mucro is approximately erect and eccentric near the upper edge of the umbo (Plate I, 11–13), which can be further assigned to perexcentromucronate type. Klaus (1980) listed six species with perexcentromuronate umbos in sect. Pinus based on the external morphology of seed cones. Xing et al. (2010) checked the umbo types of all species in subgen. Pinus from the Herbarium of Kunming Institute of Botany, the Chinese Academy of Sciences (KUN), and some published accounts, such as the monograph of Farjon (2005) and the Gymnosperm database (Earle, 2008).
Plate I. 1–13. Pinus premassoniana sp. nov. 1. 2. 3. 4. 5. 6. 7–10. 7, 8. 9. 10. 11. 12. 13. 14.
Long shoot with needles and two immature seed cones. Specimen no. JH-417A (holotype). Scale bar = 1 cm. Specimen no. JH-417B (isotype). Scale bar = 1 cm. Long shoot with needles and a winter bud. Notice the persistent basal sheath (arrow). Specimen no. JH-083A. Scale bar = 1 cm. Long shoot. Showing the needles in fascicles of two and a small ovoid-cylindric winter bud. Specimen no. JH-083B. Scale bar = 1 cm. Showing the serrate margin of needles. Specimen no. JH-417A (holotype). Scale bar = 1 mm. An immature seed cone of specimen no. JH-417B (isotype). Scale bar = 1 cm. Seed cones. Scale bar = 1 cm. Specimen no. JH-566. Specimen no. JH-041. Specimen no. JH-998. A cone scale of specimen no. JH-566. Scale bar = 1 cm. Close-up of an apophysis from the middle portion of 7, showing the transverse keel and dorsal umbo. Notice an approximately erect mucro occurs on the upper umbo field above the transverse keel. Scale bar = 5 mm. Close-up of an umbo from the apophysis of Plate I, 12. Notice the perexcentromucronate umbo (arrow). Scale bar = 1 mm. A pollen cone of Pinus. Specimen no. JH-558. Scale bar = 1 cm.
Plate II. Cuticular structures of fossil needles of Pinus premassoniana sp. nov. (see on page 82) 1–4. 1. 2. 3. 4. 5–10. 5. 6. 7. 8. 9, 10.
Under LM. Abaxial epidermis. Scale bar = 100 μm. Adaxial epidermis. Scale bar = 100 μm. Showing the serrate margin. Scale bar = 100 μm. Stomatal apparatus. Scale bar = 20 μm. Under SEM. Showing the sinuous anticlinal walls and granular periclinal walls of the epidermal cells. Scale bar = 20 μm. Abaxial epidermis. Scale bar = 100 μm. Stomatal apparatus. Scale bar = 20 μm. Outer surface of abaxial epidermis. Scale bar = 100 μm. Florin rings, type C. Florin ring overarches stomatal pore resulting in a volcanic shape. Scale bar = 20 μm.
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Plate I.
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Plate II (caption on page 80).
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Plate III. Extant Pinus massoniana. 1. 2, 3. 4. 5. 6. 7.
Long shoot with needles. Scale bar = 5 cm. Mature seed cones. Scale bar = 1 cm. An immature seed cone. Scale bar = 5 mm. Close-up of an apophysis from the middle portion of 2. Notice an approximately erect mucro occurs on the upper umbo field above the transverse keel. Scale bar = 5 mm. Showing the stomatal lines and the serrate margin. Scale bar = 100 μm. Stomatal apparatus. Scale bar = 20 μm.
They confirmed that the perexcentromucronate umbo is only present in sect. Pinus. Therefore, the information suggested that the present fossil seed cones belong to sect. Pinus of subgen. Pinus. A total of seven extant species in sect. Pinus, viz. Pinus brutia, Pinus halepensis, Pinus pinea, Pinus resinosa, Pinus kesiya, Pinus massoniana and Pinus yunnanensis, possess perexcentromucronate umbo type (Xing et al., 2010). Three Mediterranean pines, P. brutia, P. halepensis and P. pinea, are assignable to subsect. Pinaster (Gernandt et al., 2005). They possess rounded apophyses and raised umbos (Xing et al., 2010) that are distinctly different from the rhombic apophyses and sunken umbos of the present cones. The other four species of subsect. Pinus (Gernandt et al., 2005) have rhombic apophyses and sunken umbos, similar to those of our fossil cones. However, a mucro in the umbo of the Northeast America species P. resinosa is absent (Farjon, 2005; Erwin and Schorn, 2006). P. kesiya and P. yunnanensis are also distributed in Southeast Asia, but the apophyses of P. kesiya are vallate and the umbos are surrounded by a vallum, which cannot be seen in our specimens. The apophyses of our cones are transversely keeled and the umbos are slightly sunken, whereas those of P. yunnanensis are cross
keeled (Xing et al., 2010) and somewhat protruding (Fu et al., 1999; Erwin and Schorn, 2006). Based on the detailed features of cone size, apophyses and umbos, the present fossil seed cones can be assigned to subsect. Pinus and closely resembles P. massoniana (Plate III, 2–5), consistent with the morphological comparisons of fossil needles. 3.3.2. Comparisons with fossil Pinus The records of fossil needles are frequently reported from the Cretaceous to Recent (e.g., Jeffrey, 1908; Chaney, 1954; Axelrod, 1966; Robison, 1977; WGCPC, 1978; Stockey, 1984; Axelrod, 1986; Stockey and Nishida, 1986; Stockey and Ueda, 1986; Wolfe and Schorn, 1990; Saiki, 1996; Axelrod, 1998a, 1998b; Guo and Zhang, 2002; Booth et al., 2003; Stults et al., 2010). However, most species have needles in fascicles of three to five, distinctly different from our fossil species that have two needles per fascicle. One species, Pinus bifoliata, from the upper Cretaceous in Hokkaido, Japan (Stockey and Nishida, 1986) possesses the needles in fascicles of two or three, but the leaves are hypostomatic and the stomata are scattered. The needles of Pinus allisonii from the Eocene of British Columbia (Stockey, 1984) are
Elliptical Elliptical to rectangular Elliptical Elliptical Elliptical Elliptical Rectangular Rectangular Elliptical to rectangular Elliptical Rectangular Circular Circular Circular Elliptical – Elliptical Rectangular
6–8 6–10 6–8 8–12 8–10 6–10 Ca. 6 8–12 6–8 6–8 4–6 6–8 6–8 4–6 6–8 – 6–8 6–8
C – C C B–C B–C C C C D D A C – C – A B
Zhejiang, China W China SW China SE Asia Japan & Korea China China Japan SE China Asia Europe NE America Europe Europe China Cuba Europe SE Asia
1.3–2 mm wide, but the width of present needles is no more than 1 mm. Hitherto, only two fossil needles of Pinus were reported from China. Guo and Zhang (2002) described a specimen as Pinus palaeopentaphylla from the Oligocene in Jilin Province, but it has needles in fascicle of five. The Eocene specimen from Liaoning Province possesses needles in fascicle of two or three, but the needles are only 0.3–0.6 mm wide (WGCPC, 1978). Xing et al. (2010) gave a detailed review of all documented fossil seed cones of subsect. Pinus from the Cenozoic in Eurasia and America. Among the seventeen species, only Pinus prekesiya, Pinus baileyi and Pinus salinarum possess perexcentromucronate umbo type. However, P. baileyi (Erwin and Schorn, 2006) and P. salinarum (Mai, 1986) have protruding umbos, which can be distinguished from the sunken umbos of our fossil cones. P. prekesiya from the Miocene of Yunnan (Xing et al., 2010) possesses ca. 50 cone scales, whereas the present cones have 60–65 scales; a clear vallum that encircles the umbo in P. prekesiya cannot be observed in our specimens. The mucro positions of Pinus arnoldii (Miller, 1973; Stockey, 1984), Pinus driftwoodensis (Stockey, 1983), Pinus princetonensis (Stockey, 1984) and Pinus yunnanensis (Tao and Kong, 1973) are not demonstrated in the descriptions. Therefore, a diagnosis of their umbo types is limited. However, all the four species have protruding umbos. Moreover, a well-preserved seed cone, Pinus prototabulaeformis (belongs to subsect. Pinus), has been reported from the upper Eocene–lower Oligocene in Hebei Province, China (Tao and Wang, 1983), but the apophyses show excentromucronate umbos. One seed cone as Pinus speciosa was also reported from the Miocene in Eastern Zhejiang (NIGMR, 1982). However, the denticulatomucronate umbo type of this cone (Xing et al., 2010) distinctly differs from the perexcentromucronate umbo type in our fossil cones. These comparisons suggest that the present fossil needles and isolated seed cones both have the closest affinity with extant Pinus massoniana. However, some minor differences can also be found between Pinus massoniana and the present fossils. For example, P. massoniana has a persistent basal sheath of 15–20 mm in length (Farjon, 2005) but the sheaths are only 4–8 mm in our fossils. The apophyses of the present cones have a distinct transversely keel, whereas the keel of P. massoniana is very weak (Farjon, 2005; Xing et al., 2010). As demonstrated above, none of the previously mentioned fossils, as well as any extant species, has concordant needle and cone features similar to our fossil specimens. Therefore, we describe them as a new species, P. premassoniana sp. nov., which has the closest affinity with extant P. massoniana from Southern and Eastern China.
Sinuous Mainly straight Mainly straight Straight & sinuous Straight & sinuous Mainly straight Straight & sinuous Straight & sinuous Straight Straight & sinuous Straight & sinuous Straight Straight & sinuous Mainly straight Straight & sinuous – Mainly straight Mainly straight Vertical & oblique Vertical & tapering Vertical & oblique Vertical & tapering Vertical & oblique Vertical & tapering Vertical & tapering Vertical & tapering Vertical & tapering Mainly vertical Vertical & oblique Tapering & oblique Vertical & oblique Vertical & oblique Vertical & oblique – Vertical & oblique Vertical & oblique
1–3 Several? Several? 1–several Mostly shared Mostly shared Mostly shared Mostly shared 1–4 Mostly shared 1–3 Mostly shared 1–several? 1–3 1–several – 1–2 Several?
Anticlinal walls of epidermal cells End walls of epidermal cells
4. Associated pollen cone
or 3 or 3
or 3
The explanation of Florin ring types follows Yoshie and Sakai (1985).
Ca. 1 1–1.5 1–1.2 0.5–1 1–2 Slender 0.8–1 b1 b1 Ca. 1 1–2 Ca. 1.2 1.5–2.2 1.5–1.8 0.8–1 1.5 1–2 b1 6–10 6–15 7–20 15–25 7–12 6–15 5–8 10–15 10–15 6–12 4–7 12–18 3–8 5–6 12–20 15–30 10–18 15–20 or 3 or 3
2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 P. premassoniana P. densata P. yunnanensis P. kesiya P. thunbergii P. tabuliformis P. hwangshanensis P. luchuensis P. taiwanensis P. densiflora P. sylvestris P. resinosa P. mugo P. uncinata P. massoniana P. tropicalis P. nigra P. merkusii
Needle width (mm) Needle number per bundle Species
Needle length (cm)
4.1. Systematics
a
Cells between stomata in row
Stomatal apparatus
Number of subsidiary cells
Type of Florin ringa
Distribution
S.-T. Ding et al. / Review of Palaeobotany and Palynology 197 (2013) 78–89 Table 1 Comparison of the present fossil needles with the extant species of sect. Pinus (refer to Gui and Li, 1963; Campbell, 1972; Yoshie and Sakai, 1985; Lendzian et al., 1986; Kim et al., 1999; Ickert-Bond, 2000; Stružková, 2002; Sweeney, 2004; Whang et al., 2004; Álvarez et al., 2009a, 2009b).
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Family: PINACEAE L. Genus: Pinus L. Subgenus: Pinus L. Species: Pinus sp. Specimen no. JH-558 (Plate I, 14; Plates IV, V). Locality and stratigraphy: Jiahu in Tiantai County of Zhejiang Province, China; Miocene Shengxian Formation. 4.2. Description The pollen cone is typically found isolated, ellipsoidal, 1.5 cm long and 0.5 cm wide (Plate I, 14). Microsporophylls are parenchymatous and contain resin canals (Plate IV, 5). Each microsporophyll bears two abaxial pollen sacs (Plate IV, 2, 3), and each pollen sac contains copious bisaccate pollen grains tightly clumped together (Plate IV, 1, 2, 4, 6). Pollen grains are 50–70 μm in total length (saccus to saccus). The corpus is 31–45 μm in width, 36–52 μm in length, and 6–15 μm in height (equatorial view; Plate V, 1, 2, 4, 12). Sacci are 22–34 μm in width, 28–37 μm in length, and 16–30 μm in height (polar view; Plate V, 3, 5, 6). The corpus exhibits a verrucate or microverrucate
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Plate IV. Microsporophyll and pollen morphology of associated Pinus pollen cone. 1. 2–6. 2, 3. 4. 5. 6.
Several pollen grains tightly clumped together (under LM). Scale bar = 50 μm. Under SEM. Microsporophyll with two abaxial pollen sacs. Scale bar = 200 μm. A sac filled with mature pollens. Scale bar = 200 μm. Showing several resin canals. Scale bar = 20 μm. Showing copious bisaccate pollen grains tightly clumped together in the sac. Scale bar = 50 μm.
ornamentation (Plate V, 7), and is entirely covered by two adjoining sacci on the distal surface (Plate V, 1, 5) or has a relatively broad sulcus (Plate V, 6, 9). The sacci also exhibit a verrucate external sculpture
(Plate V, 8) similar to that of the corpus. In some cases, the sacci were degraded or broken, thereby exposing an alveolae internal structure (Plate V, 10, 11).
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Plate V. Associated Pinus pollen cones—pollen morphology. 1–3. 4–12. 4. 5, 6. 7. 8. 9. 10, 11. 12.
Pollen grains. Under LM. Scale bar = 10 μm. Under SEM. Lateral surface of a pollen grain. Scale bar = 10 μm. Distal surface of a pollen grain. Scale bar = 10 μm. Showing verrucate ornamentation of corpus in 4. Scale bar = 2 μm. Showing the verrucate ornamentation of sacci in 5. Scale bar = 2 μm. Showing a relatively broad sulcus at the distal pole of 6. Scale bar = 5 μm. Showing the alveolae internal structure of the sacci. Scale bar = 10 μm. Several pollen grains exhibit the lateral surface and distal surface. Scale bar = 10 μm.
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4.3. Comparisons and discussion Considering the fragile nature of pollen cones in general (Phipps et al., 1995), a well-preserved pollen cone with a large number of pollen grains may further support that an in situ preservation occurred in the upper Miocene of Jiahu, Zhejiang. Moreover, the needles and seed cones were well preserved in a same long shoot, which can also be indicative of a rapid preservation that occurred at this locality. Based on the fossil assemblage and paraecology studies, Miocene Jiahu locality is believed to have a shallow, nearshore lacustrine environment (Ding, 2011), with Pinus premassoniana sp. nov. occupying at a marginal position. The present pollen cone is characterized by ellipsoidal shape and parenchymatous microsporophylls with two abaxial pollen sacs, and each pollen sac contains bisaccate pollen grains, which has a clear affinity with the genus Pinus. The fossil pollen cone is consistently found in association with the fossil shoots and seed cones, and its size and shape are similar to those of extant Pinus massoniana. Therefore, it is seemly that the pollen cone should be related to the organic attachment of Pinus premassoniana. However, some distinct differences can be observed between the pollen grains of extant P. massoniana (Li et al., 2011) and the present cone. For example, the grains of P. massoniana have larger sizes (96–110 μm in total length) than the grains in our pollen cone (50–70 μm), and the grains of P. massoniana possess a distinct gemmate corpus, whereas the external corpus sculpture of the present grains is verrucate or microverrucate (Plate V, 7). Moreover, one seed cone species named Pinus speciosa (NIGMR, 1982) and another winged-seed species (unpublished, see Ding, 2011) have also been described from the Miocene Shengxian Formation. Therefore, we consider that the present pollen cone cannot be assigned definitively to the species P. premassoniana. In general, morphological characteristics of pollen cones have minimal taxonomic values in the classifications of extant pines (Mirov, 1967; Little and Critchfield, 1969). However, some investigations have focused on various aspects of the development, morphology and taxonomy of pine pollen grains (Dickinson and Bell, 1970; Rowley and Walles, 1987; Kurmann, 1989). For example, the pollen grains of subgen. Strobus have an outline in which the sacci are more or less continuous with the corpus (haploxylon type), whereas the sacci are distinct from the corpus (diploxylon type) in the grains of subgen. Pinus (Weir and Thurston, 1977; Phipps et al., 1995; Kapp et al., 2002). The present fossil grains tend to exhibit a diploxylon type (Plate V, 5), and hence, can be assigned to the subgen. Pinus. Some palynological characteristics, such as overall size, cappa sculpture and saccus shapes have been used to differentiate the pine pollens at the species level (e.g., Cain, 1940; Hansen and Cushing, 1973; Sivak and Caratini, 1973; Bagnell, 1975). However, these systematic utilities have limited value for fossil pollens because the preservational variability may greatly influence the observed morphology among the grains (Whitehead, 1964; Mack, 1971; Phipps et al., 1995). Therefore, assigning our Miocene pollen cone to any extant or sporae dispersae species based on the pollen morphology is difficult. 5. Historical biogeography In previous systematic studies (e.g., Wang and Szmidt, 1993; Wang et al., 1999; López et al., 2002; Gernandt et al., 2005; Eckert and Hall, 2006), Pinus massoniana is excluded from the group of Eastern Asia pines, which consists of Pinus hwangshanensis, Pinus yunnanensis, Pinus kesiya, Pinus taiwanensis, Pinus thunbergii and Pinus tabuliformis. The occurrence of ancestral taxon for the Eastern Asia pines was estimated to have occurred from the late Oligocene to the Miocene based on molecular clock data (Eckert and Hall, 2006). However, a number of fossil records can provide some direct evidences to understand their historical biogeography. For example, Pinus prekesiya from the late
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Miocene of central Yunnan represents the ancestral stock of P. kesiya and P. yunnanensis, which suggests that the divergence between these two East Asia species may have taken place during the Neogene and triggered by the continuous uplift of the Qing-Tibetan Plateau and global cooling trend (Xing et al., 2010). This result concurs with the dispersal patterns from molecular clock dates (Eckert and Hall, 2006). Another fossil cone, Pinus prototabulaeformis, from the upper Eocene–lower Oligocene in Hebei, China, has a closer affinity with extant P. tabuliformis (Tao and Wang, 1983). The presence of P. prototabulaeformis suggests that the ancestral eastern Asia taxon might have begun during the late Eocene. However, this period is earlier than that from the molecular clock data (Eckert and Hall, 2006), consistent with Eckert and Hall's expectation for the diversification rate during the Eocene. Pinus massoniana is endemic in China, distributed in the southern, central and eastern parts of the nation, and mostly occurs in plains and mountains from near sea level to 1500 (2000) m elevation (Cheng and Fu, 1978; Farjon, 2005). This species is also one of the predominant tree species in the forest system of Zhejiang Province (Zhang and Zhang, 1993). The distribution of extant P. massoniana indicates that P. premassoniana should also live under a warm and humid ecology environment. Molecular dates show that the differentiation of the node of P. massoniana from Pinus merkusii occurred during the Paleocene to the Eocene (Eckert and Hall, 2006). As there is no any reliable fossil that has an affinity with extant P. massoniana, a further discussion on its evolution is limited. However, the occurrence of the present fossils suggests that P. massoniana (or its ancestral taxon) may have occurred in Southeast China (its present major distribution area) since at least the late Miocene. Acknowledgments The authors thank Frédéric M. B. Jacques (Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences) for help with French literature, Liang Xiao and Xiangchuan Li (Chang'an University, China) for their assistance in collecting fossils. This research was conducted under the National Basic Research Program (No. 2012CB822003), the National Natural Science Foundation of China (No. 41172022, 41272026), China Postdoctoral Science Foundation (No. 2012 M521813, 2011 M500960), the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20120211110022, 20100211120009), the Fundamental Research Funds for the Central Universities (No. lzujbky–2012–127), and the Foundation of Key Laboratory of Western China's Environmental Systems (Ministry of Education) in Lanzhou University. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.revpalbo.2013.05. 004. These data include Google maps of the most important areas described in this article. References Álvarez, S.G., Juaristi, G.M., Gutiérrez, J.S., García-Amorena, I., 2009a. Taxonomic differences between Pinus sylvestris and P. uncinata revealed in the stomata and cuticle characters for use in the study of fossil material. Review of Palaeobotany and Palynology 155, 61–68. Álvarez, S.G., García-Amorena, I., Rubiales, J.M., Morla, C., 2009b. The value of leaf cuticle characteristics in the identification and classification of Iberian Mediterranean members of the genus Pinus. Botanical Journal of the Linnean Society 161 (4), 436–448. Alvin, K.L., 1960. Further conifers of the Pinaceae from the Wealdon Formation of Belgium. Mémoires de l'Institut Royal des Science Naturelles de Belgique 146, 1–39. Axelrod, D.I., 1966. The Eocene Copper Basin flora of northeastern Nevada. University of California Publications in Geological Sciences 59, 1–125. Axelrod, D.I., 1986. Cenozoic history of some western American pines. Annals of the Missouri Botanical Garden 73, 565–641. Axelrod, D.I., 1998a. The Eocene Thunder Mountain flora of central Idaho. University of California Publications in Geological Sciences 142, 1–61.
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Research paper
Needles and seed cones of Pinus premassoniana sp. nov., and associated pollen cone from the upper Miocene in East China Su-Ting Ding a,b, Jing-Yu Wu a,b, Jun-Lin Chen a, Yi Yang a, De-Fei Yan a, Bai-Nian Sun a,b,⁎ a b
School of Earth Sciences & Key Laboratory of Mineral Resources in Western China (Gansu Province), Lanzhou University, Lanzhou 730000, China State Key Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology, CAS, Nanjing 210008, China
a r t i c l e
i n f o
Article history: Received 28 January 2013 Received in revised form 8 May 2013 Accepted 9 May 2013 Available online 26 May 2013 Keywords: Pinus premassoniana sp. nov. needles and seed cones associated pollen cone historical biogeography Miocene East China
a b s t r a c t A long shoot with needles and two immature seed cones, and some isolated organs of shoot, needles and seed cones from the late Miocene Shengxian Formation in Zhejiang Province, East China are described. A detailed comparison of the gross morphology and cuticle micromorphology of needles and seed cones with previously published Cenozoic fossils and related extant pine species reveals that the present fossils can be identified as a new species, Pinus premassoniana sp. nov., which has the closest affinity with extant Pinus massoniana. A pollen cone with copious bisaccate pollen grains from the same locality is identified as an indeterminate species of subgenus Pinus. The pollen grains show a verrucate external sculpture on the corpus and sacci and an alveolae internal structure in the sacci. The fossil needle cuticles and pollen cone of the genus Pinus from China are reported for the first time. The modern distribution indicates that P. premassoniana sp. nov. should also live under a warm and humid climate. Hitherto, there is no any reliable fossil record that has an affinity with the extant P. massoniana. The occurrence of the present fossils suggests that P. premassoniana has existed in East China since at least in the late Miocene. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Pinus L. with approximately 110 species is the largest genus of conifers widely distributed predominantly in the Northern Hemisphere (Richardson and Rundel, 1998; Farjon, 2005). The earliest accepted record of Pinus is Pinus belgica Alvin from the Lower Cretaceous of Belgium based on a permineralized cone (Alvin, 1960). Hitherto, over 50 species of isolated vegetative remains of Pinus have been described (e.g., Chaney, 1954; Mehringer and Ferguson, 1969; Miller, 1976, 1977; Robison, 1977; Stockey, 1983; Phipps et al., 1995; Saiki, 1996; McKown et al., 2002; Smith and Stockey, 2002; Erwin and Schorn, 2006; Xing et al., 2010; Klymiuk et al., 2011). However, fossil needles and cones preserved on the same long shoot are very rare. Hitherto, only one specimen as Pinus resurgens was described from the Miocene in Armissan, Southeastern France by de Saporta (1865). Most fossil species of Pinus are based on disassociated structures (e.g., cones, needles, winged seeds, and pollens). Therefore, the reconstruction of whole plants is questionable because of the lack of attachment or indisputable association evidence (Axelrod, 1986; Stockey and Ueda, 1986). The fossil record of pines from the Cretaceous to the Recent is typically considered good (Stults et al., 2010). In China, four cone species ⁎ Corresponding author at: School of Earth Sciences & Key Laboratory of Mineral Resources in Western China (Gansu Province), Lanzhou University, Lanzhou 730000, China. Tel./fax: +86 931 8915280. E-mail address: [email protected] (B.-N. Sun). 0034-6667/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.revpalbo.2013.05.004
from the upper Eocene–lower Oligocene of Hebei Province (Tao and Wang, 1983), the Miocene of Zhejiang Province (NIGMR, 1982), the Miocene of Yunnan Province (Xing et al., 2010) and the Pliocene of Yunnan (Tao and Kong, 1973), two needle species from the Eocene of Liaoning Province (WGCPC, 1978) and the Oligocene of Jilin Province (Guo and Zhang, 2002), and one seed species from the Oligocene of Hebei Province (WGCPC, 1978) were reported. In this study, we describe a long shoot with needles and seed cones, and some isolated organisms of shoots, needles and seed cones as a new species, Pinus premassoniana sp. nov., from the Miocene of Zhejiang. One pollen cone with in situ pollens as an indeterminate species of Pinus was also examined under the scanning electron microscopy (SEM). The fossil needle cuticles and pollen cone from China are reported for the first time. The comparison with selected extant and previously published fossil pines in this paper indicates that the gross morphological characters of needles and seed cones, as well as the needle cuticle micromorphological features, are important to the taxonomy of fossil Pinus. Moreover, the discovery of the present fossils provides us a rare opportunity to understand the Pinus evolution during the Miocene.
2. Material and methods The fossils described in this study were collected from the Shengxian Formation at Jiahu Village (29°09′N, 121°14′E), Tiantai County, Zhejiang Province, East China (Fig. 1). Based on lithostratigraphy and biostratigraphy researches (NIGMR, 1982; Li, 1984; BGMRZP, 1989; Liu et al.,
S.-T. Ding et al. / Review of Palaeobotany and Palynology 197 (2013) 78–89 100ºE
105ºE
115ºE
110ºE
120ºE
Yangtze River
30ºN
30ºN
Zhejiang
China 25ºN
25ºN
20ºN
20ºN
100ºE
105ºE
110ºE
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120ºE
Fig. 1. Map showing the fossil locality (black triangle) of Pinus premassoniana sp. nov. and the modern distribution of Pinus massoniana (broken line).
1992), the geological age of the Shengxian Formation is regarded as the late Miocene (Ding et al., 2011). The carbonized needles were immersed in 10% HCl for 2 h, and then with 50% hydrofluoric acid overnight. The needle fragments were macerated into 30% HNO3 solution for ca. 48 h. When the black color changed to light brown, the specimens were washed and dripped with 5% NH4OH for ca. 5 min. After washing with distilled water several times, part of the cuticles was stained with 1% safranine solution. The cuticles were mounted on slides and embedded in glycerine jelly. The slides were observed and photographed using a Leica DM4000B light microscope. Another part of unstained cuticles was mounted on stubs and coated with gold under a sputter-coater, observed and photographed under a JEOLJSM 6380LV scanning electron microscope. For the pollen cone, several small fragments (microsporophylls) were removed from the middle part with the help of a dissecting needle and immersed in 10% HF solution in a plastic bottle for 12 h. After careful washing with distilled water, the fragments were divided into two parts. One part was placed on a concave glass slide, added with a drop of 65% HNO3 for several minutes until the materials became light brown and dripped with distilled water for several times to displace the nitric acid. Then, the specimen was removed to another glass slide with one drop of glycerol. In situ pollens were checked and photographed under the light microscope. Another part was mounted on a stub and coated with gold and examined under the SEM. All slides for LM and stubs for SEM are stored in the Institute of Palaeontology and Stratigraphy, Lanzhou University, Gansu Province, China. The extant specimens of Pinus massoniana for the morphological comparisons were collected in Tiantai County, Zhejiang Province. The terminology for morphological description of pine branches follows Farjon (2005) and Dörken et al. (2010), and that for seed cones follows Klaus (1980, 1989) and Mai (1986). Terms for cuticle micromorphology follow Yoshie and Sakai (1985), Kim et al. (1999) and Whang et al. (2001, 2004). Classification of the genus Pinus follows the recent scheme of Gernandt et al. (2005). 3. Results 3.1. Systematics Family: PINACEAE L. Genus: Pinus L. Subgenus: Pinus L. Section: Pinus sensu Gernandt, Geada López, Ortiz García & Liston. Subsection: Pinus sensu Gernandt, Geada López, Ortiz García & Liston. Species: Pinus premassoniana Su-Ting Ding et Bai-Nian Sun, sp. nov.
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Specific diagnosis: Winter bud ovoid-cylindric, apex acute, needles in fascicles of two, basal sheaths persistent. Needles amphistomatic, epidermal cells rectangular, end walls of epidermal cells vertical or oblique, anticlinal walls sinuous. Stomatal rows continuous, stomata oriented regularly along long axis of the needles, two guard cells surrounded by 6–8 subsidiary cells. Florin rings rise outwards and centripetally resulting in a volcanic shape (type C). Immature seed cones ovoid-conical, closed and symmetric. Mature seed cones symmetric, apex tapered, woody ovuliferous scales helically arranged. Apophyses rhombic to pyramidal in plane view, with a distinct transverse keel. Umbos dorsal and slightly sunken. An approximately erect mucro occurs on the upper umbo field, characteristic of the perexcentromucronate umbo type. Etymology: The specific epithet refers to the similarity with the extant species Pinus massoniana. Holotype: JH-417A (Plate I, 1) Isotype: JH-417B (Plate I, 2, 6) Paratypes: JH-083A (Plate I, 3), JH-083B (Plate I, 4), JH-790, JH-471, JH-566 (Plate I, 7, 8, 11–13), JH-041(Plate I, 9), JH-998 (Plate I, 10) Locality: Jiahu in Tiantai County of Zhejiang Province, China Stratigraphy: Shengxian Formation Age: late Miocene Repository: Institute of Palaeontology and Stratigraphy, Lanzhou University, Lanzhou, China. 3.2. Description Two long shoots are preserved (Plate I, 1–3). The winter bud is small, ovoid-cylindric, apex acute, 8 mm long and 3.3 mm wide (Plate I, 3, 4). The needles are held in a persistent basal sheath 4–8 mm long (Plate I, 3), in fascicles of two, preserved 6–10 cm long and 0.8–1 mm wide, margins serrate (Plate I, 1–5). Two immature seed cones borne at the apices of the long shoot, ovoid-conical, symmetric, closed, 1.2–1.4 cm long and 0.8–0.9 cm wide, have ca. 62 scales (Plate I, 1, 2, 6). Mature seed cones are isolated, conical, closed or open, symmetric, 5.2–7.4 cm long, and 2.8–3.5 cm wide, with a tapered apex (Plate I, 7–10). Each cone has 60 to 65 woody ovuliferous scales helically arranged around the axis. Cone scales expanded at each apophysis, up to ca. 2.5 cm long in the middle part of the cones (Plate I, 10, 11). In plane view, the apophyses are typically rhombic in the upper half of the cones, 1.0–1.4 cm wide and 0.7–1.0 cm high; however, the basal apophyses sometimes show a more pyramidal shape or nearly pentagonal, 0.6–0.9 cm wide and 0.7–0.9 cm high. Each apophysis possesses a distinct transverse keel. The umbos are dorsal, rhombic to elliptic in plane view, are relatively larger in the upper half of the cone (2–3 mm wide and 1.5–2.0 mm high) and smaller in the basal half (1.0–1.5 mm wide and 0.8–1.2 mm high). They are slightly sunken on the surface of the apophyses (Plate I, 11, 12). An approximate erect mucro occurs on the upper umbo field above the transverse keel, which are the characteristics of perexcentromucronate umbo type (Plate I, 11–13). Needles are amphistomatic; epidermal cells are rectangular on both adaxial and abaxial cuticle surfaces, 80–240 μm in length and 7–15 μm in width, extremely elongated in between, but much broader within the stomatal lines, with one to three interstomatal cells in the stomatal row (Plate II, 1–3); the end walls of the epidermal cells are vertical or oblique; the anticlinal walls sinuous, and the periclinal walls are granular in the internal surface (Plate II, 5). Stomatal rows are continuous, stomata oriented regularly along long axis of needles, have 3–5 lines per adaxial surface and 7–10 lines per abaxial surface. The stomatal complexes are monocyclic and elliptic, 40–50 μm in length and 28–36 μm in width; two guard cells surrounded by 6 to 8 subsidiary cells, and the lateral subsidiary cells are usually bigger than the polar cells (Plate II, 2, 4, 7). The external cuticle surface is slightly undulating, with the stomatal apparatus oriented parallel to the leaf axis. Each stoma is surrounded by an encircling ridge called a Florin ring (Buchholz and Gray, 1948). The Florin rings rise outwards and centripetally resulting
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in a volcanic shape (Plate II, 8–10), which are the characteristics of Florin ring type C (Yoshie and Sakai, 1985). 3.3. Comparisons The present fossils are characterized by some typical features such as needles in fascicles of two, and seed cone scales have dorsal umbos. Therefore, the present fossils can be assigned to the genus Pinus based on the combined morphological features of the needles and seed cones (Richardson and Rundel, 1998; Frankis, 2002). Three isolated mature seed cones and one long shoot with immature cones occurred in the same horizon. These seed cones have similar cone-scale complexes and numbers of scales per cone. The present fossil shoot and the isolated seed cones both have the closest affinity with extant Pinus massoniana based on a detailed morphological comparison. Although the isolated mature cones are slightly elongate as compared with the immature cones, this shape variability can also be found in the seed cones of extant P. massoniana (Plate III, 2–4). Therefore, we considered that the present long shoot and the isolated seed cones should represent the same species. 3.3.1. Comparisons with extant Pinus Most current classifications recognize two major lineages within the genus Pinus, i.e., subgenus Pinus (diploxylon or hard pines) and subgen. Strobus (haploxylon or soft pines) (e.g., Little and Critchfield, 1969; Krupkin et al., 1996; Price et al., 1998; Liston et al., 1999; Gernandt et al., 2005; Klymiuk et al., 2011). The present fossils possess the needles in fascicles of two, which differ from most species of subgen. Strobus, õexcept for Pinus krempfii. However, the needles of P. krempfii have lanceolate-falcate shape and the basal sheaths are deciduous (Farjon, 2005), clearly different from our slender fossil needles and persistent sheaths. Subgen. Pinus consists of two sections, viz. sect. Pinus and sect. Trifoliae (Gernandt et al., 2005). However, some cuticular micromorphological features distinguishes our fossils from those of sect. Trifoliae. For example, the polar subsidiary cells of sect. Trifoliae are mostly shared and larger than the lateral subsidiary cells (Whang et al., 2001; Whang et al., 2004), whereas 1–3 cells between the stomata in row (Table 1) and the polar subsidiary cells are smaller than the
lateral subsidiary cells in the present fossil needles (Plate II, 2, 4, 7). Sect. Pinus consists of subsect. Pinus and subsect. Pinaster. However, one Himalayan species of subsect. Pinaster, Pinus roxburghii, possesses needles in fascicles of three (Farjon, 2005), and Mediterranean species of subsect. Pinaster usually has rectangular stomatal apparatus (Álvarez et al., 2009a). As demonstrated above, the present fossil needles can be further assigned to subsect. Pinus. Table 1 provides a list of needle morphology and cuticular features of subsect. Pinus. Only four species, viz. Pinus thunbergii, Pinus mugo, Pinus sylvestris, and P. massoniana, possess epidermal cells with vertical and oblique end walls and sinuous anticlinal walls, which are similar to the present fossil needles. However, P. thunbergii has 8–10 subsidiary cells, and the cells between stomata in row are mostly shared, P. mugo has needles of 1.5–2.2 mm in width and circular stomatal apparatus. P. sylvestris possesses rectangular stomata with 4–6 subsidiary cells and the type D Florin ring, which distinctly differs from those of our fossils. Based on a detailed comparison (Table 1), our fossil needles are significantly similar to the extant needles of P. massoniana (Plate III, 1, 6, 7), both on morphology and cuticular features. The present seed cones are characterized by the cone-scale complexes helically arranged around the axis, ovuliferous scales expanded at the apex, and each apophysis possesses a transverse keel and a dorsal umbo. According to the aforementioned features, these fossil seed cones can be assigned to the genus Pinus (Miller, 1976) and has affinity with subgen. Pinus (Frankis, 2002). Klaus (1980, 1989) subdivided the subgen. Pinus into two groups: viz. centromucronate type (mucro located in the center of umbo) and excentromucronate type (mucro located above the umbo). The current study shows that the mucro of our fossil cones lies above the keel; thus, the umbos can be a diagnostic of the excentromucronate type. Furthermore, the mucro is approximately erect and eccentric near the upper edge of the umbo (Plate I, 11–13), which can be further assigned to perexcentromucronate type. Klaus (1980) listed six species with perexcentromuronate umbos in sect. Pinus based on the external morphology of seed cones. Xing et al. (2010) checked the umbo types of all species in subgen. Pinus from the Herbarium of Kunming Institute of Botany, the Chinese Academy of Sciences (KUN), and some published accounts, such as the monograph of Farjon (2005) and the Gymnosperm database (Earle, 2008).
Plate I. 1–13. Pinus premassoniana sp. nov. 1. 2. 3. 4. 5. 6. 7–10. 7, 8. 9. 10. 11. 12. 13. 14.
Long shoot with needles and two immature seed cones. Specimen no. JH-417A (holotype). Scale bar = 1 cm. Specimen no. JH-417B (isotype). Scale bar = 1 cm. Long shoot with needles and a winter bud. Notice the persistent basal sheath (arrow). Specimen no. JH-083A. Scale bar = 1 cm. Long shoot. Showing the needles in fascicles of two and a small ovoid-cylindric winter bud. Specimen no. JH-083B. Scale bar = 1 cm. Showing the serrate margin of needles. Specimen no. JH-417A (holotype). Scale bar = 1 mm. An immature seed cone of specimen no. JH-417B (isotype). Scale bar = 1 cm. Seed cones. Scale bar = 1 cm. Specimen no. JH-566. Specimen no. JH-041. Specimen no. JH-998. A cone scale of specimen no. JH-566. Scale bar = 1 cm. Close-up of an apophysis from the middle portion of 7, showing the transverse keel and dorsal umbo. Notice an approximately erect mucro occurs on the upper umbo field above the transverse keel. Scale bar = 5 mm. Close-up of an umbo from the apophysis of Plate I, 12. Notice the perexcentromucronate umbo (arrow). Scale bar = 1 mm. A pollen cone of Pinus. Specimen no. JH-558. Scale bar = 1 cm.
Plate II. Cuticular structures of fossil needles of Pinus premassoniana sp. nov. (see on page 82) 1–4. 1. 2. 3. 4. 5–10. 5. 6. 7. 8. 9, 10.
Under LM. Abaxial epidermis. Scale bar = 100 μm. Adaxial epidermis. Scale bar = 100 μm. Showing the serrate margin. Scale bar = 100 μm. Stomatal apparatus. Scale bar = 20 μm. Under SEM. Showing the sinuous anticlinal walls and granular periclinal walls of the epidermal cells. Scale bar = 20 μm. Abaxial epidermis. Scale bar = 100 μm. Stomatal apparatus. Scale bar = 20 μm. Outer surface of abaxial epidermis. Scale bar = 100 μm. Florin rings, type C. Florin ring overarches stomatal pore resulting in a volcanic shape. Scale bar = 20 μm.
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Plate I.
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Plate II (caption on page 80).
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Plate III. Extant Pinus massoniana. 1. 2, 3. 4. 5. 6. 7.
Long shoot with needles. Scale bar = 5 cm. Mature seed cones. Scale bar = 1 cm. An immature seed cone. Scale bar = 5 mm. Close-up of an apophysis from the middle portion of 2. Notice an approximately erect mucro occurs on the upper umbo field above the transverse keel. Scale bar = 5 mm. Showing the stomatal lines and the serrate margin. Scale bar = 100 μm. Stomatal apparatus. Scale bar = 20 μm.
They confirmed that the perexcentromucronate umbo is only present in sect. Pinus. Therefore, the information suggested that the present fossil seed cones belong to sect. Pinus of subgen. Pinus. A total of seven extant species in sect. Pinus, viz. Pinus brutia, Pinus halepensis, Pinus pinea, Pinus resinosa, Pinus kesiya, Pinus massoniana and Pinus yunnanensis, possess perexcentromucronate umbo type (Xing et al., 2010). Three Mediterranean pines, P. brutia, P. halepensis and P. pinea, are assignable to subsect. Pinaster (Gernandt et al., 2005). They possess rounded apophyses and raised umbos (Xing et al., 2010) that are distinctly different from the rhombic apophyses and sunken umbos of the present cones. The other four species of subsect. Pinus (Gernandt et al., 2005) have rhombic apophyses and sunken umbos, similar to those of our fossil cones. However, a mucro in the umbo of the Northeast America species P. resinosa is absent (Farjon, 2005; Erwin and Schorn, 2006). P. kesiya and P. yunnanensis are also distributed in Southeast Asia, but the apophyses of P. kesiya are vallate and the umbos are surrounded by a vallum, which cannot be seen in our specimens. The apophyses of our cones are transversely keeled and the umbos are slightly sunken, whereas those of P. yunnanensis are cross
keeled (Xing et al., 2010) and somewhat protruding (Fu et al., 1999; Erwin and Schorn, 2006). Based on the detailed features of cone size, apophyses and umbos, the present fossil seed cones can be assigned to subsect. Pinus and closely resembles P. massoniana (Plate III, 2–5), consistent with the morphological comparisons of fossil needles. 3.3.2. Comparisons with fossil Pinus The records of fossil needles are frequently reported from the Cretaceous to Recent (e.g., Jeffrey, 1908; Chaney, 1954; Axelrod, 1966; Robison, 1977; WGCPC, 1978; Stockey, 1984; Axelrod, 1986; Stockey and Nishida, 1986; Stockey and Ueda, 1986; Wolfe and Schorn, 1990; Saiki, 1996; Axelrod, 1998a, 1998b; Guo and Zhang, 2002; Booth et al., 2003; Stults et al., 2010). However, most species have needles in fascicles of three to five, distinctly different from our fossil species that have two needles per fascicle. One species, Pinus bifoliata, from the upper Cretaceous in Hokkaido, Japan (Stockey and Nishida, 1986) possesses the needles in fascicles of two or three, but the leaves are hypostomatic and the stomata are scattered. The needles of Pinus allisonii from the Eocene of British Columbia (Stockey, 1984) are
Elliptical Elliptical to rectangular Elliptical Elliptical Elliptical Elliptical Rectangular Rectangular Elliptical to rectangular Elliptical Rectangular Circular Circular Circular Elliptical – Elliptical Rectangular
6–8 6–10 6–8 8–12 8–10 6–10 Ca. 6 8–12 6–8 6–8 4–6 6–8 6–8 4–6 6–8 – 6–8 6–8
C – C C B–C B–C C C C D D A C – C – A B
Zhejiang, China W China SW China SE Asia Japan & Korea China China Japan SE China Asia Europe NE America Europe Europe China Cuba Europe SE Asia
1.3–2 mm wide, but the width of present needles is no more than 1 mm. Hitherto, only two fossil needles of Pinus were reported from China. Guo and Zhang (2002) described a specimen as Pinus palaeopentaphylla from the Oligocene in Jilin Province, but it has needles in fascicle of five. The Eocene specimen from Liaoning Province possesses needles in fascicle of two or three, but the needles are only 0.3–0.6 mm wide (WGCPC, 1978). Xing et al. (2010) gave a detailed review of all documented fossil seed cones of subsect. Pinus from the Cenozoic in Eurasia and America. Among the seventeen species, only Pinus prekesiya, Pinus baileyi and Pinus salinarum possess perexcentromucronate umbo type. However, P. baileyi (Erwin and Schorn, 2006) and P. salinarum (Mai, 1986) have protruding umbos, which can be distinguished from the sunken umbos of our fossil cones. P. prekesiya from the Miocene of Yunnan (Xing et al., 2010) possesses ca. 50 cone scales, whereas the present cones have 60–65 scales; a clear vallum that encircles the umbo in P. prekesiya cannot be observed in our specimens. The mucro positions of Pinus arnoldii (Miller, 1973; Stockey, 1984), Pinus driftwoodensis (Stockey, 1983), Pinus princetonensis (Stockey, 1984) and Pinus yunnanensis (Tao and Kong, 1973) are not demonstrated in the descriptions. Therefore, a diagnosis of their umbo types is limited. However, all the four species have protruding umbos. Moreover, a well-preserved seed cone, Pinus prototabulaeformis (belongs to subsect. Pinus), has been reported from the upper Eocene–lower Oligocene in Hebei Province, China (Tao and Wang, 1983), but the apophyses show excentromucronate umbos. One seed cone as Pinus speciosa was also reported from the Miocene in Eastern Zhejiang (NIGMR, 1982). However, the denticulatomucronate umbo type of this cone (Xing et al., 2010) distinctly differs from the perexcentromucronate umbo type in our fossil cones. These comparisons suggest that the present fossil needles and isolated seed cones both have the closest affinity with extant Pinus massoniana. However, some minor differences can also be found between Pinus massoniana and the present fossils. For example, P. massoniana has a persistent basal sheath of 15–20 mm in length (Farjon, 2005) but the sheaths are only 4–8 mm in our fossils. The apophyses of the present cones have a distinct transversely keel, whereas the keel of P. massoniana is very weak (Farjon, 2005; Xing et al., 2010). As demonstrated above, none of the previously mentioned fossils, as well as any extant species, has concordant needle and cone features similar to our fossil specimens. Therefore, we describe them as a new species, P. premassoniana sp. nov., which has the closest affinity with extant P. massoniana from Southern and Eastern China.
Sinuous Mainly straight Mainly straight Straight & sinuous Straight & sinuous Mainly straight Straight & sinuous Straight & sinuous Straight Straight & sinuous Straight & sinuous Straight Straight & sinuous Mainly straight Straight & sinuous – Mainly straight Mainly straight Vertical & oblique Vertical & tapering Vertical & oblique Vertical & tapering Vertical & oblique Vertical & tapering Vertical & tapering Vertical & tapering Vertical & tapering Mainly vertical Vertical & oblique Tapering & oblique Vertical & oblique Vertical & oblique Vertical & oblique – Vertical & oblique Vertical & oblique
1–3 Several? Several? 1–several Mostly shared Mostly shared Mostly shared Mostly shared 1–4 Mostly shared 1–3 Mostly shared 1–several? 1–3 1–several – 1–2 Several?
Anticlinal walls of epidermal cells End walls of epidermal cells
4. Associated pollen cone
or 3 or 3
or 3
The explanation of Florin ring types follows Yoshie and Sakai (1985).
Ca. 1 1–1.5 1–1.2 0.5–1 1–2 Slender 0.8–1 b1 b1 Ca. 1 1–2 Ca. 1.2 1.5–2.2 1.5–1.8 0.8–1 1.5 1–2 b1 6–10 6–15 7–20 15–25 7–12 6–15 5–8 10–15 10–15 6–12 4–7 12–18 3–8 5–6 12–20 15–30 10–18 15–20 or 3 or 3
2 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 P. premassoniana P. densata P. yunnanensis P. kesiya P. thunbergii P. tabuliformis P. hwangshanensis P. luchuensis P. taiwanensis P. densiflora P. sylvestris P. resinosa P. mugo P. uncinata P. massoniana P. tropicalis P. nigra P. merkusii
Needle width (mm) Needle number per bundle Species
Needle length (cm)
4.1. Systematics
a
Cells between stomata in row
Stomatal apparatus
Number of subsidiary cells
Type of Florin ringa
Distribution
S.-T. Ding et al. / Review of Palaeobotany and Palynology 197 (2013) 78–89 Table 1 Comparison of the present fossil needles with the extant species of sect. Pinus (refer to Gui and Li, 1963; Campbell, 1972; Yoshie and Sakai, 1985; Lendzian et al., 1986; Kim et al., 1999; Ickert-Bond, 2000; Stružková, 2002; Sweeney, 2004; Whang et al., 2004; Álvarez et al., 2009a, 2009b).
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Family: PINACEAE L. Genus: Pinus L. Subgenus: Pinus L. Species: Pinus sp. Specimen no. JH-558 (Plate I, 14; Plates IV, V). Locality and stratigraphy: Jiahu in Tiantai County of Zhejiang Province, China; Miocene Shengxian Formation. 4.2. Description The pollen cone is typically found isolated, ellipsoidal, 1.5 cm long and 0.5 cm wide (Plate I, 14). Microsporophylls are parenchymatous and contain resin canals (Plate IV, 5). Each microsporophyll bears two abaxial pollen sacs (Plate IV, 2, 3), and each pollen sac contains copious bisaccate pollen grains tightly clumped together (Plate IV, 1, 2, 4, 6). Pollen grains are 50–70 μm in total length (saccus to saccus). The corpus is 31–45 μm in width, 36–52 μm in length, and 6–15 μm in height (equatorial view; Plate V, 1, 2, 4, 12). Sacci are 22–34 μm in width, 28–37 μm in length, and 16–30 μm in height (polar view; Plate V, 3, 5, 6). The corpus exhibits a verrucate or microverrucate
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Plate IV. Microsporophyll and pollen morphology of associated Pinus pollen cone. 1. 2–6. 2, 3. 4. 5. 6.
Several pollen grains tightly clumped together (under LM). Scale bar = 50 μm. Under SEM. Microsporophyll with two abaxial pollen sacs. Scale bar = 200 μm. A sac filled with mature pollens. Scale bar = 200 μm. Showing several resin canals. Scale bar = 20 μm. Showing copious bisaccate pollen grains tightly clumped together in the sac. Scale bar = 50 μm.
ornamentation (Plate V, 7), and is entirely covered by two adjoining sacci on the distal surface (Plate V, 1, 5) or has a relatively broad sulcus (Plate V, 6, 9). The sacci also exhibit a verrucate external sculpture
(Plate V, 8) similar to that of the corpus. In some cases, the sacci were degraded or broken, thereby exposing an alveolae internal structure (Plate V, 10, 11).
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Plate V. Associated Pinus pollen cones—pollen morphology. 1–3. 4–12. 4. 5, 6. 7. 8. 9. 10, 11. 12.
Pollen grains. Under LM. Scale bar = 10 μm. Under SEM. Lateral surface of a pollen grain. Scale bar = 10 μm. Distal surface of a pollen grain. Scale bar = 10 μm. Showing verrucate ornamentation of corpus in 4. Scale bar = 2 μm. Showing the verrucate ornamentation of sacci in 5. Scale bar = 2 μm. Showing a relatively broad sulcus at the distal pole of 6. Scale bar = 5 μm. Showing the alveolae internal structure of the sacci. Scale bar = 10 μm. Several pollen grains exhibit the lateral surface and distal surface. Scale bar = 10 μm.
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4.3. Comparisons and discussion Considering the fragile nature of pollen cones in general (Phipps et al., 1995), a well-preserved pollen cone with a large number of pollen grains may further support that an in situ preservation occurred in the upper Miocene of Jiahu, Zhejiang. Moreover, the needles and seed cones were well preserved in a same long shoot, which can also be indicative of a rapid preservation that occurred at this locality. Based on the fossil assemblage and paraecology studies, Miocene Jiahu locality is believed to have a shallow, nearshore lacustrine environment (Ding, 2011), with Pinus premassoniana sp. nov. occupying at a marginal position. The present pollen cone is characterized by ellipsoidal shape and parenchymatous microsporophylls with two abaxial pollen sacs, and each pollen sac contains bisaccate pollen grains, which has a clear affinity with the genus Pinus. The fossil pollen cone is consistently found in association with the fossil shoots and seed cones, and its size and shape are similar to those of extant Pinus massoniana. Therefore, it is seemly that the pollen cone should be related to the organic attachment of Pinus premassoniana. However, some distinct differences can be observed between the pollen grains of extant P. massoniana (Li et al., 2011) and the present cone. For example, the grains of P. massoniana have larger sizes (96–110 μm in total length) than the grains in our pollen cone (50–70 μm), and the grains of P. massoniana possess a distinct gemmate corpus, whereas the external corpus sculpture of the present grains is verrucate or microverrucate (Plate V, 7). Moreover, one seed cone species named Pinus speciosa (NIGMR, 1982) and another winged-seed species (unpublished, see Ding, 2011) have also been described from the Miocene Shengxian Formation. Therefore, we consider that the present pollen cone cannot be assigned definitively to the species P. premassoniana. In general, morphological characteristics of pollen cones have minimal taxonomic values in the classifications of extant pines (Mirov, 1967; Little and Critchfield, 1969). However, some investigations have focused on various aspects of the development, morphology and taxonomy of pine pollen grains (Dickinson and Bell, 1970; Rowley and Walles, 1987; Kurmann, 1989). For example, the pollen grains of subgen. Strobus have an outline in which the sacci are more or less continuous with the corpus (haploxylon type), whereas the sacci are distinct from the corpus (diploxylon type) in the grains of subgen. Pinus (Weir and Thurston, 1977; Phipps et al., 1995; Kapp et al., 2002). The present fossil grains tend to exhibit a diploxylon type (Plate V, 5), and hence, can be assigned to the subgen. Pinus. Some palynological characteristics, such as overall size, cappa sculpture and saccus shapes have been used to differentiate the pine pollens at the species level (e.g., Cain, 1940; Hansen and Cushing, 1973; Sivak and Caratini, 1973; Bagnell, 1975). However, these systematic utilities have limited value for fossil pollens because the preservational variability may greatly influence the observed morphology among the grains (Whitehead, 1964; Mack, 1971; Phipps et al., 1995). Therefore, assigning our Miocene pollen cone to any extant or sporae dispersae species based on the pollen morphology is difficult. 5. Historical biogeography In previous systematic studies (e.g., Wang and Szmidt, 1993; Wang et al., 1999; López et al., 2002; Gernandt et al., 2005; Eckert and Hall, 2006), Pinus massoniana is excluded from the group of Eastern Asia pines, which consists of Pinus hwangshanensis, Pinus yunnanensis, Pinus kesiya, Pinus taiwanensis, Pinus thunbergii and Pinus tabuliformis. The occurrence of ancestral taxon for the Eastern Asia pines was estimated to have occurred from the late Oligocene to the Miocene based on molecular clock data (Eckert and Hall, 2006). However, a number of fossil records can provide some direct evidences to understand their historical biogeography. For example, Pinus prekesiya from the late
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Miocene of central Yunnan represents the ancestral stock of P. kesiya and P. yunnanensis, which suggests that the divergence between these two East Asia species may have taken place during the Neogene and triggered by the continuous uplift of the Qing-Tibetan Plateau and global cooling trend (Xing et al., 2010). This result concurs with the dispersal patterns from molecular clock dates (Eckert and Hall, 2006). Another fossil cone, Pinus prototabulaeformis, from the upper Eocene–lower Oligocene in Hebei, China, has a closer affinity with extant P. tabuliformis (Tao and Wang, 1983). The presence of P. prototabulaeformis suggests that the ancestral eastern Asia taxon might have begun during the late Eocene. However, this period is earlier than that from the molecular clock data (Eckert and Hall, 2006), consistent with Eckert and Hall's expectation for the diversification rate during the Eocene. Pinus massoniana is endemic in China, distributed in the southern, central and eastern parts of the nation, and mostly occurs in plains and mountains from near sea level to 1500 (2000) m elevation (Cheng and Fu, 1978; Farjon, 2005). This species is also one of the predominant tree species in the forest system of Zhejiang Province (Zhang and Zhang, 1993). The distribution of extant P. massoniana indicates that P. premassoniana should also live under a warm and humid ecology environment. Molecular dates show that the differentiation of the node of P. massoniana from Pinus merkusii occurred during the Paleocene to the Eocene (Eckert and Hall, 2006). As there is no any reliable fossil that has an affinity with extant P. massoniana, a further discussion on its evolution is limited. However, the occurrence of the present fossils suggests that P. massoniana (or its ancestral taxon) may have occurred in Southeast China (its present major distribution area) since at least the late Miocene. Acknowledgments The authors thank Frédéric M. B. Jacques (Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences) for help with French literature, Liang Xiao and Xiangchuan Li (Chang'an University, China) for their assistance in collecting fossils. This research was conducted under the National Basic Research Program (No. 2012CB822003), the National Natural Science Foundation of China (No. 41172022, 41272026), China Postdoctoral Science Foundation (No. 2012 M521813, 2011 M500960), the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20120211110022, 20100211120009), the Fundamental Research Funds for the Central Universities (No. lzujbky–2012–127), and the Foundation of Key Laboratory of Western China's Environmental Systems (Ministry of Education) in Lanzhou University. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.revpalbo.2013.05. 004. These data include Google maps of the most important areas described in this article. References Álvarez, S.G., Juaristi, G.M., Gutiérrez, J.S., García-Amorena, I., 2009a. Taxonomic differences between Pinus sylvestris and P. uncinata revealed in the stomata and cuticle characters for use in the study of fossil material. Review of Palaeobotany and Palynology 155, 61–68. Álvarez, S.G., García-Amorena, I., Rubiales, J.M., Morla, C., 2009b. The value of leaf cuticle characteristics in the identification and classification of Iberian Mediterranean members of the genus Pinus. Botanical Journal of the Linnean Society 161 (4), 436–448. Alvin, K.L., 1960. Further conifers of the Pinaceae from the Wealdon Formation of Belgium. Mémoires de l'Institut Royal des Science Naturelles de Belgique 146, 1–39. Axelrod, D.I., 1966. The Eocene Copper Basin flora of northeastern Nevada. University of California Publications in Geological Sciences 59, 1–125. Axelrod, D.I., 1986. Cenozoic history of some western American pines. Annals of the Missouri Botanical Garden 73, 565–641. Axelrod, D.I., 1998a. The Eocene Thunder Mountain flora of central Idaho. University of California Publications in Geological Sciences 142, 1–61.
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