Taxonomic Revision of Pinus fujiii (Yasui) Miki (Pinaceae) and Its Implications for the Phytogeography of the Section Trifoliae in East Asia

Abstract

Pinus trifolia Miki 1939 (Pinaceae) was originally proposed based on seed cones from the upper Miocene of Aichi and Gifu Prefectures, central Japan. However, before the publication of P. trifolia, a different name (Pinus fujiii (Yasui) Miki) was given to a female cone with the same morphology. On the other hand, P. fujiii auct. non (Yasui) Miki has been used for seed cones with different morphologies from Yasui's holotype, i.e., apophyses arranged in 5:8 parastichies and a perexcentromucronate slightly-pointed umbo. As a result of re-examination on the Miki and Yasui specimens, we concluded that P. trifolia was a synonym for P. fujiii and proposed here Pinus mikii sp. nov. for cones assigned to P. fujiii auct. non (Yasui) Miki. We also emended the diagnosis of P. fujiii based on these specimens. Pinus fujiii is characterized by a large female cone in which the apophyses with a centromucronate prickle-like umbo are arranged in 8:13 parastichies, and deciduous seed wings. These characters suggest that P. fujiii belongs to the section Trifoliae of the subgenus Pinus, which is now restricted to North and Central America and the Caribbean islands. Fossil data suggest that the P. fujiii lineage firstly appeared in Japan around the Eocene/Oligocene boundary. We speculate that the P. fujiii lineage might have moved southward to Japan from a refugium located elsewhere in high-latitude areas in response to the late Eocene cooling event, as occurred with other Trifoliae species in North America.

Full text

RESEARCH ARTICLE
Taxonomic Revision of Pinus fujiii (Yasui) Miki
(Pinaceae) and Its Implications for the
Phytogeography of the Section Trifoliae in
East Asia
Toshihiro Yamada
1
*, Mariko Yamada
1
, Minoru Tsukagoshi
2
1School of Natural System, College of Science and Engineering, Kanazawa University, Kanazawa, Japan,
2Osaka Museum of Natural History, Higashi-sumiyoshi, Osaka, Japan
*ptilo@mb.infoweb.ne.jp
Abstract
Pinus trifolia Miki 1939 (Pinaceae) was originally proposed based on seed cones from the
upper Miocene of Aichi and Gifu Prefectures, central Japan. However, before the publica-
tion of P.trifolia, a different name (Pinus fujiii (Yasui) Miki) was given to a female cone with
the same morphology. On the other hand, P.fujiii auct. non (Yasui) Miki has been used for
seed cones with different morphologies from Yasui’s holotype, i.e., apophyses arranged in
5:8 parastichies and a perexcentromucronate slightly-pointed umbo. As a result of re-exam-
ination on the Miki and Yasui specimens, we concluded that P.trifolia was a synonym for P.
fujiii and proposed here Pinus mikii sp. nov. for cones assigned to P.fujiii auct. non (Yasui)
Miki. We also emended the diagnosis of P.fujiii based on these specimens. Pinus fujiii is
characterized by a large female cone in which the apophyses with a centromucronate
prickle-like umbo are arranged in 8:13 parastichies, and deciduous seed wings. These char-
acters suggest that P.fujiii belongs to the section Trifoliae of the subgenus Pinus, which is
now restricted to North and Central America and the Caribbean islands. Fossil data suggest
that the P.fujiii lineage firstly appeared in Japan around the Eocene/Oligocene boundary.
We speculate that the P.fujiii lineage might have moved southward to Japan from a refu-
gium located elsewhere in high-latitude areas in response to the late Eocene cooling event,
as occurred with other Trifoliae species in North America.
Introduction
Extant species of the section Trifoliae Duhamel [1] (subgenus Pinus, genus Pinus L. [2], Pina-
ceae Spreng. ex F.Rudolphi [3]) are placed into three subsections [4], Contortae Little and
Critchfield [5], Australes Loudon [6], and Ponderosae Loudon [7], and are restricted in their
distribution to North and Central America and the Caribbean islands [4,7–9]. The Trifoliae
are characterized by two to five leaves clustered in a persistent fascicle sheath and deciduous
PLOS ONE | DOI:10.1371/journal.pone.0143512 December 16, 2015 1/18
OPEN ACCESS
Citation: Yamada T, Yamada M, Tsukagoshi M
(2015) Taxonomic Revision of Pinus fujiii (Yasui) Miki
(Pinaceae) and Its Implications for the
Phytogeography of the Section Trifoliae in East Asia.
PLoS ONE 10(12): e0143512. doi:10.1371/journal.
pone.0143512
Editor: William Oki Wong, Institute of Botany, CHINA
Received: January 28, 2015
Accepted: November 5, 2015
Published: December 16, 2015
Copyright: © 2015 Yamada et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Specimens are stored in the Fossil collections of the
Osaka Museum of Natural History (OSA) or in the
Herbarium of the University of Tokyo (TI). All
specimen numbers of OSA collection have been
included in the previous manuscript, as well as in S1
Table. No specimen number is assigned to the
holotype of Pinites fujiii held in TI. However, the
holotype is registered in TI under a name “holotype of
Pinites fujiii”, thus we added this information into
“Fossil material”. In addition, there is information on

(articulated) seed wings, although there are a few exceptional species [4,10]. In most species of
this section, the female cones have robust woody cone scales [8,11].
Fossil records of the Trifoliae in the Eocene and thereafter are centered on North America
[12–21], while only a few fossils from the Miocene to Pliocene in Europe have been placed as
members of the section [10,22,23]. Therefore, it is inferred that the Trifoliae originated and
diversified in North America [15,24]. This inference is also supported by phytogeographic his-
tory reconstructed from molecular phylogeny [4,25]. However, fossil records and the molecu-
lar clock suggest different ages for the divergence of the Trifoliae, i.e., the Late Cretaceous (95
Ma [25]) or the early Miocene (18 Ma [9,26]) for the molecular clock vs. the Eocene (45–50
Ma) for fossil records [15].
Pinus trifolia Miki is a species represented in the early late Miocene flora of Japan [27–29].
It was originally instituted based on material from the Tokiguchi Formation in Mizunami-shi,
Gifu Prefecture (Pref.), and the Seto Formation in Seto-shi, Aichi Pref. [30]. This species is
characterized by a large female cone with thick cone scales arranged in 8:13 parastichies,
detachment of the basal female cone scales, uncinate apophyses with a prickle-like umbo, and
three to five leaves clustered in a fascicle sheath [30]. Based on these features, Miki [30] inferred
that P.trifolia is a close relative of extant P.sabiniana Dougl. of the section Taeda (sensu Pilger
[31]), which is included in the subsection Ponderosae of the section Trifoliae in the most recent
and prevalent classification of the genus Pinus [4].
If the above phylogenetic inference [30] is correct, P.trifolia provides significant evidence of
a historic floristic tie between East Asia and North America. Since Japan became an archipelago
by the opening of the Japan Sea between the late Oligocene and the earliest early Miocene [32],
the migration of P.trifolia ancestors to Japan should have been completed before the formation
of this geographical barrier. Therefore, P.trifolia would suggest that a Miocene origin of the
Trifoliae is unlikely, if North America is the cradle of this section. However, in addition to the
key characters listed above, character states should be further clarified so as to support that P.
trifolia belongs to the Trifoliae, because some species of the subsection Pinaster Loudon [6]
(section Pinus) also have some of these characters [4,10]. Information on the position of a
mucro on umbos and the mode of seed wing attachment would be useful to distinguish the Tri-
foliae from the Pinaster [4,10,33]; however, these characters are not available in Miki’s
descriptions [30].
In addition to the problem on its affinity, we recently realized that Pinus trifolia may have a
nomenclatural problem. Before the publication of P.trifolia by Miki [30], a new name, Pinites
fujiii, was given to a female cone collected from the Seto Formation [34]. Yasui [34] stated the
diagnosis for Pinites fujiii as “The phyllotaxy of the scales is 8/21. The end of the scale is gener-
ally wedge-shaped with the point drawn out into a hook. In the middle part of the cone the
hook is elongated and deflected, while at the base it points downward.”Therefore, Pinites fujiii
and Pinus trifolia apparently largely share the same female cone characters; however, the rela-
tionship between the two species has not been discussed until now.
In this study, we reexamined Yasui’s[34] and Miki’s[30] specimens, as well as other P.trifo-
lia specimens collected by Prof. S. Miki. We propose that Pinus trifolia is a synonym for Pinites
fujiii and discuss the affinity of this species to extant sections of the genus Pinus.
Material and Methods
Fossil material
All specimens used in this study (S1 Table) were borrowed from the Herbarium of the Univer-
sity of Tokyo (TI) and the Fossil collections of the Osaka Museum of Natural History (OSA;
for details on these herbaria including contact information, see Index Herbariorum [35]). No
Taxonomic Revision of Pinus fujiii
PLOS ONE | DOI:10.1371/journal.pone.0143512 December 16, 2015 2/18
details of herbaria including a contact, etc. by citing
website “Index herbariorum”(line 15 of page 5).
Funding: This work was supported by KAKENHI
(Grant-in-Aid for Scientific Research) 26304010 from
the Ministry of Education, Culture, Sports, Science
and Technology (http://www.mext.go.jp) to TY. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.

other specimens were used in this study. Specimens were photographed using a D200 camera
(Nikon, Tokyo, Japan) with an AF MICRO NIKKOR 60 mm lens (Nikon) under fluorescent
illumination.
The holotype of Pinites fujiii stored in TI consists of a female cone, a replica of the cone, and
four microscope slides mounting sectioned parts of the cone. No specimen number is assigned to
the holotype, while itis registered as “holotype of Pinites fujiii”. The holotype was collected in
Seto-shi, Aichi Pref., Japan (Fig 1), from the Seto Formation, but the exact locality is not available.
Other specimens, including Miki’s[30] specimens, are stored in OSA F. These specimens
were slightly compressed mummifications collected from the Seto or Tokiguchi Formations
(Fig 1).
Geological setting
The Seto and Tokiguchi Formations are fluvial deposits consisting of lignites, claystones, silt-
stones, sandstones and conglomerates [36]. These two formations were formed almost coevally
in different basins close to each other (Fig 1)[36]. Their age is estimated as the early late Mio-
cene (10.5 ± 0.4–9.7 ± 0.4 Ma), based on fission–track dating of intercalated tuff layers [37,38].
83 plant species were reported from these two formations such as Glyptostrobus pensilis
Koch, Metasequoia disticha (Heer) Miki, Fortunearia sinensis Rehder et E. H. Wilson, Liqui-
damber formosana Hance, Fagus stuxbergii (Nathorst) Tanai, Carya striata Miki, Nyssa sylva-
tica Marshall [27,38]. Among them, 40 species are extinct from present Japan and extant
species of the 9 genera are confined to China and North America. This composition suggests
floristic ties between Japan and these regions before the early late Miocene [27].
The holotype was collected from a lignite bed consisting mainly of wood and bark fragments
[34]. Although we could not know from what facies Miki’s[30] specimens were collected, it is
reported that “P.trifolia”cones densely occur in semiautochthonous plant litters which con-
tained in fine- to coarse-grained sandstone beds of channel bar deposits [38]. Miki [30] col-
lected other organs of genus Pinus along with cones from the same horizons, supporting that
these Pinus remains were not transported for a long distance from their living sites.
Phylogenetic analyses
Phylogenetic analyses were conducted based on the morphological character matrix of Ger-
nandt et al. [4] with some modifications. The number of vascular bundles (character #1) and
resin duct position in leaves (charcter #4) were removed from the matrix because these are not
available for P.fujiii. Distribution (character #9) was also removed from the matrix. Dissection
of the basal cone scales was added to the matrix in which character states were coded as present
(1) or absent (0) after Klaus [10] and Farjon and Styles [11].
A batch file for parsimony ratchet analysis was generated by PRAP2 [39] with settings of
1000 ratchet replicates, weight 2 and 25% weighted. Parsimony ratchet analysis was conducted
by PAUPversion 4.0b10 [40] based on the batch file. Bootstrap supports were calculated by
making 1000 replicates, with 10 trees held for each of 100 multiple tree-bisection-reconnection
(TBR) search replications. In both parsimony ratchet and bootstrap analyses, molecular phy-
logeny [4] was used as a backbone constraint.
The character matrix and constraint tree used for these analyses were available as S1 and S2
Datasets.
Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an
ISSN or ISBN will represent a published work according to the International Code of
Taxonomic Revision of Pinus fujiii
PLOS ONE | DOI:10.1371/journal.pone.0143512 December 16, 2015 3/18

Fig 1. Localities of examined specimens and distributions of Seto and Tokiguchi Formations. Distributions of these formations are projected onto
topographic map released by Geospatial Information Authority of Japan, based on Nakayama et al. [36].
doi:10.1371/journal.pone.0143512.g001
Taxonomic Revision of Pinus fujiii
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Nomenclature for algae, fungi, and plants, and hence the new names contained in the elec-
tronic publication of a PLOS ONE article are effectively published under that Code from the
electronic edition alone, so there is no longer any need to provide printed copies.
The online version of this work is archived and available from the following digital reposito-
ries: PubMed Central, LOCKSS.
Results
Systematic paleobotany
Order Pinales Gorozhankin, 1904 [41]
Family Pinaceae Spreng. ex F.Rudolphi, 1830 [3]
Genus Pinus L., 1753 [2]
Pinus fujiii (Yasui) Miki emend. T. Yamada, M. Yamada et Tsukagoshi
emend. nov. (Figs 2–5herein)
Pinus fujiii (Yasui) Miki, Miki,1939: p. 245 (nomenclatural note only for new combination)
[30]
Basionym. Pinites fujiii Yasui, Yaui, 1928: p. 437, Text-figure 12, Figures 83–-85 in Plates
20, 21 [34]
Fig 2. Holotype of Pinus fujiii and related specimen stored in TI. (a) Apical view of holotype. (b) Apical view of gypsum model casted from holotype. (c)
Basal view of holotype. (d) Lateral view of gypsum model. (e) Close-up of umbos in holotype. (f) Cross sectionof a cone scale detached from holotype. Arrow
head = mucro. Scale bars: a–d, 1 cm; e, f, 5 mm.
doi:10.1371/journal.pone.0143512.g002
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Synonym. Pinus trifolia Miki, Miki, 1939: p. 244, Text-figure 3, Plate 4 [30]; Miki 1948:
p. 255 (with no illustration) [23]; Miki, 1957: p. 253, Text-figures 7A, 8Cc, Plate 6 [42]; Tanai,
1961: p. 256, Figure 8 in Plate 2 [29]; Omori and Tanaka, 1965: p. 567, Figure 2 [43]; Tsukagohi
and Todo Collaborative Research Group, 1998: p. 496, Figures 3.1, 3.2 [44]; Ando et al., 1999:
p. 19, Figures 1, 4 [38]; Nakayama et al., 1999: p. 3, Figures 1, 2 [45]; Nirei and Akiyama, 2011:
p. 102, Figure 4 [46]
Holotype. Yasui’s female cone [34] (Figs 2,4A and 5A herein). The holotype is stored in
TI, but no specimen number has been assigned since1928.
Type strata. The upper Miocene Seto Formation, Tokai Group
Type locality. Seto-shi, Aichi Pref., Japan (exact locality unknown)
Epitype. OSA F19363 (Designated here [Figs 3B and 5S]; collected from Hatagoya, Mizu-
nami-shi, Gifu Pref., Japan; firstly illustrated by Miki [30] in Text-figure 3C and Figure E of
Plate 4)
Other specimens examined. OSA F2905 (Fig 3H), OSA F2908, OSA F2909, OSA F17476
(Fig 3D), OSA F19296–1(Fig 5C), –2(Fig 5E), –3(Fig 5G), –4(Fig 5D), –5(Fig 5F), –6(Fig
5K), –7(Fig 5H), –8(Fig 5N), –9(Fig 5L), –10 (Fig 5B), –11 (Fig 5I), –12 (Fig 5R), –13 (Fig
5Q), –14 (Fig 5P), –15 (Fig 5O), –16 (Fig 5M), –17 (Fig 5J), OSA F19351–1 (Miki, 1939: 2nd
right of Figure B in Plate IV [30]), –2, –3(Fig 3I herein; Miki, 1939: 2nd left of Figure B in Plate
IV [30]), OSA F19349–1(Fig 3G herein; Miki, 1939: Text-figure 3E, Figure D in Plate IV [30]),
–2 (Miki, 1939: Text-figure 3I, bottom of Figure C in Plate IV [30]), –3 (Miki, 1939: top of
Figure C in Plate IV [30]), –4 (Miki, 1939: Text-figure 3Da, center of Figure H in Plate IV
[30]), –5 (Miki, 1939: Text-figures 3D–b,–c, left of Figure H in Plate IV [30]), –6 (Miki, 1939:
right of Figure H in Plate IV [30]), –7(Fig 3E herein; Miki, 1939: Text-figure 3B, Figure G in
Plate IV [30]), –8 (Figs 3F and 5W herein; Miki, 1939: Figure F in Plate IV [30]), –9 (Figs 3C
and 5U), –10 (Fig 5V), –11 (Fig 5T), OSA F19355–1(Fig 4C), –2(Fig 4F), –3(Fig 4B), –4
(Fig 4M), –5(Fig 4H), –6(Fig 4N), –7(Fig 4K), –8(Fig 4Q), –9(Fig 4S), OSA F19356–1(Fig
4G), –2(Fig 4D), –3(Fig 4O), –4(Fig 4J), –5(Fig 4E), –6(Fig 4L), –7(Fig 4I), –8(Fig 4T), –9
(Fig 4R), –10 (Fig 4P). For details, see S1 Table.
Emended Diagnosis. Female cone ovoid or oblong ovoid with asymmetrical base; cone
scales in 8:13 parastichies; apophysis thick, pentagonal, bend downwardly on the abaxial side
of the cone; umbo somewhat flattened, centromucronate; mucro forming distinct prickle; vas-
cular bundles in cone scale up to 11, arranged in one plane.
Description. The female cone is ovoid or oblong ovoid with an asymmetrical base, 6.5–13
cm long, and 4.9–10 cm wide when it is fully grown (Figs 2,3B and 3C and Figs 4–6). The
basalmost cone scales usually remain on the branch when the cone has fallen off (Fig 3D).
Cone scales are arranged in 8:13 parastichies. Up to 11 vascular bundles are coplanarly
arranged in the middle part of a cone scale (Fig 2F. see also [34]). Apophyses are thick, 10–13
mm high, 15–20 mm wide, and pyramidally risen (Figs 2D,3B and 3C). The risen part of an
apophysis is rolled downward on the abaxial (away from the branch) side of a cone, but the
bend is not as distinct on the adaxial side (Figs 2D,3B and 3C). The umbo is centromucronate
and the mucro forms a distinct prickle (Figs 2E,3E and 3F). The seed is articulated to the wing,
elliptic, 9–10 mm long, and 4–5 mm wide (Fig 3H). The seed wing is 13–16 mm long and 8
mm wide (Fig 3G. See also [30]).
Nomenclatural note. In 1939, Miki [30] legitimately proposed a new combination Pinus
fujiii for Pinites fujiii based on Yasui’s holotype [34] since genus Pinites Lindley and Hutton
1832 was originally given for Carboniferous woods [47] which have possible affinity with the
Araucariaceae [48]. Later, he reported some female cones (Fig 7A–7C) and leaves (Fig 7F)
from the Tokiguchi Formation and assigned them to “Pinus fujiii (Yasui) Miki”[27]. Since
then, many authors have identified “P.fujiii”based on this report [27], rather than the original
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Fig 3. Pinus fujiii stored in OSA F. (a) Leaves in a sheath (OSA F4062). (b) Epitype (OSA F19363). (c) Female cone (OSA F19349–9). (d) Two female cone
bases remained on branch (OSA F17476). Cone base behind branch is indicated by arrow. (e, f) Close-up of apophyses in OSA F19349–7 (e) and –8 (f). (g)
Cone scale (OSA F19349–1). Seed scars are visible but seeds shown in Miki [30] are missing. (h) Seeds (2905). Note detached wing in right one. i. Male
cone (OSA F19351–3). Scale bars: a–c, 5 cm; d–f, 1 cm; g–h, 5 mm.
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description by Yasui [34]. However, Yasui’s holotype of P.fujiii [34] is clearly different from
the female cones reported by Miki [27]. Therefore, we establish a new species below for “Pinus
fujiii”sensu Miki 1941 [27], based on his specimens.
Comparison. Ten fossil species of subgenus Pinus are known that have large female cones
(greater than 10 cm long) with centromucronate umbos (Table 1). Among them, P.fujiii is
similar in female cone characteristics to P.engelhardtii (Menzel) Mai from the lower Miocene
of the Czech Republic [22,23], P.lawsoniana from the middle Pliocene of California (CA), U.
S.A. [14], P.piperi Dorf from the upper Miocene to Pliocene of CA [18,49], and P.truckeensis
Axelrod from the upper Miocene of Nevada, U.S.A. [15], i.e., ratios of width to length are
greater than 0.5 and they exhibit pyramidally-risen apophyses. However, the bending of the
risen part in the abaxial apophyses, a characteristic of P.fujiii, is not distinct in the former
three species. Pinus engelhardtii is also different from P.fujiii in the indistinct mucro [22,23].
Pinus truckeensis has the most similar appearance to P.fujiii among the four species, but
Fig 4. Female cones of Akadu population. (a) Holotype. (b) OSA F19355–3. (c) OSA F19355–1. (d) OSA F19356–2. (e) OSA F19356–5. (f) OSA F19355–
2. (g) OSA F19356–1. (h) OSA F19355–5. (i) OSA F19356–7. (j) OSA F19356–4. (k) OSA F19355–7. (l) OSA F19356–6. (m) OSA F19355–4. (n) OSA
F19355–6. (o) OSA F19356–3. (p) OSA F19356–10. (q) OSA F19355–8. (r) OSA F19356–9. (s) OSA F19355–9. (t) OSA F19356–8. Scale bar: 15 cm.
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apophyses of P.truckeensis are larger than those of P.fujiii [15]. In addition, apophyses have a
greater width than height in P.truckeensis [15], while the width is almost the same as the height
in apophyses of P.fujiii.
There are two records of “Pinus trifolia Miki”from the late Eocene to early Oligocene in
Japan: cones from an unknown locality of Kyushu [50] and leaves, female cones, and male
cones from the Kobe Group (37–31 Ma [51]) distributed in Kobe-shi, Hyogo Pref. [52]. Com-
parison between P.fujiii and the cones from Kyushu is difficult because these cones are poorly
preserved. It is suggested that specimens from the Kobe Group would belong to a closely
related but distinct species from P.fujiii because the male cones are twice the size of those of P.
fujiii [52].
Fig 5. Female cones of Hatagoya population. (a) Holotype. (b) OSA F19296–10 (c) OSA F19296–1 (d) OSA F19296–4. (e) OSA F19296–2. (f) OSA
F19296–5. (g) OSA F19296–3. (h) OSA F19296–7. (i) OSA F19296–11. (j) F19296–17. (k) OSA F19296–6. (l) OSA F19296–9. (m) OSA F19296–16. (n)
OSA F19296–8. (o) OSA F19296–15. (p) OSA F19296–14. (q) OSA F19296–13. (r) OSA F19296–12. (s) Epitype (OSA F19363). (t) OSA F19349–11. (u)
OSA F19349–9. (v) OSA F19349–10. (w) OSA F19349–8. Scale bar: 15 cm.
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Fig 6. Size variations of female cones found in Akadu and Hatagoya populations. Width of holotype and size of epitype are indicated by dashed line and
arrow, respectively.
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Remarks. Along with female cones, some detached male cones and leaves were discovered
from the same horizons [30] (For details on the specimens, see S1 Table). Three or four, rarely
five, leaves are clustered in a persistent fascicle. The leaf sheath is 10–12 mm long (Fig 3A). The
male cone has a woody axis, is stalked, 15–25 mm long and 5–6 mm wide (Fig 3I).
Pinus mikii T. Yamada, M. Yamada et Tsukagoshi sp. nov. (Fig 7 herein)
Synonym. Pinus fujiii auct. non (Yasui) Miki, Miki, 1941: p. 255, Text-figures 5K–L,
Figure G in Plate 4 [27]; Miki, 1957: p. 250, Text-figures H–K, Plate 7 [42]; Tanai, 1961: p. 255,
Figure 9 in Plate 2, non Figure 10 in Plate 3 [29]; Kimura et al., 1981: p. 91, Text-figures 2a–c,
Fig 7. Pinus mikii sp. nov. stored in OSA F. (a) Holotype (20241–1). (b, c) Paratypes (20241–2, –3, respectively) (d) Female cone (19432–1). (e) Close-up
of apophyses (19432–2). f. Leaves (20242–1). Scale bars: a, d, 1 cm; b, c, e, f, 5 mm.
doi:10.1371/journal.pone.0143512.g007
Table 1. Comparison between P.fujiii and other species characterized by a large female cone with centromucronate umbos.
Species Cone length (L, cm) Cone width (W, cm) Ratio (W/L) Uncinate apophyses Age References
P.fujiii 6.5–13 4.9–10 0.56–0.98 present late Miocene [30,34]
P.celetomensis 14–15 7.5 0.5–0.54 absent late Miocene [15]
P.diegensis 9.0–11 5.0–6.5 0.56–0.59 absent late Pliocene [16]
P.engelhardtii 4.6–13 2.1–7.5 0.46–0.63 absent early Miocene [22,23]
P.engelmannoides 11–12 4.5 0.38–0.41 absent late Oligocene [15]
P.lawsoniana 8.0–10.5 7.0 0.67–0.88 absent middle Pliocene [14]
P.piperi 10.2 12.5 1.2 absent late Miocene–Pliocene [18,49]
P.riogrande 12–15 <4.5–6.0 <0.4 ? late Oligocene [15]
P.spinosa 14 3.0–4.0 0.21–0.29 absent Pliocene [22]
P.stroboides 10 <ca. 3.0 <0.3 absent late Eocene [22]
P.truckeensis 17–18 11 0.61–0.65 present late Miocene [15]
doi:10.1371/journal.pone.0143512.t001
Taxonomic Revision of Pinus fujiii
PLOS ONE | DOI:10.1371/journal.pone.0143512 December 16, 2015 11 / 18

Figures 2–3 in Plate 9 [53]; Ando et al., 1999: Figure 5.1 [38]; Nakayama et al., 1999: Figure 6.1
[45]; Saneyoshi et al., 2000: Figure 6–1[37]; Sawada et al., 2013: Figure 5 [54]; Yamada et al.,
2014: p. 200, Figures 3a–f[55]; Yamada and Yamada, 2014: p. 29, Figures 1A–E[56]; Pinus
miocenica auct. non Tanai, Matsuo, 1963: Figure 5 in Plate 43 [57]; Ina, 1981: Figure 1 in Plate
2[58]
Holotype. OSA F20241–1 (Designated here [Fig 7A]; Miki,1941: the left specimen of
Text-figure 5L [27])
Paratype. OSA F20241–2 (Designated here [Fig 7B]; Miki, 1941: the right specimen of
Text-figure 5L [27]), –3 (Designated here [Fig 7C]; Miki, 1941: the center specimen of Text-fig-
ure 5L [27])
Other specimens examined. OSA F19432–1(Fig 7D; Miki, 1957: Figure Hb in plate 7
[42]), OSA F19432–2(Fig 7E)
Type strata. The upper Miocene Tokiguchi Formation, Tokai Group
Type locality. Ohata-cho 3-chome (formerly called as ‘Ichinokuraguchi’), Tajimi-shi, Gifu
Pref., Japan (Fig 1).
Etymology. Commemorating late Prof. Shigeru Miki who conducted the first comprehen-
sive study on the Japanese fossil Pinaceae and greatly contributed to the clarification of the
Neogene vegetation of Japan.
Diagnosis. Female cones conical to ovoid, with a cordate base; cone scales in 5:8 parasti-
chies; apophyses rhombic, moderately swollen, with transverse keel and radiating ridges, val-
late; umbos present at the upper one-third to one-half of dorsal apophyses, weakly depressed
on basal half, perexcentromucronate; mucros slightly hooked, prominent even in basal cone
scales.
Description. Female cones are long elliptic to oblong ovoid, 40–70 mm long, and 18–37
mm wide (Fig 7A–7D). The cone base is cordate with a short peduncle (Fig 7A–7C). Cone
scales are arranged in 5:8 parastichies (Fig 7A–7D). The apophysis is rhombohedral to hexago-
nal, moderately swollen, 8–12 mm wide, and 5–7 mm high at the middle of the cone (Fig 7A–
7E). A weak transverse keel and radiating ridges are developed on the apohyses (Fig 7E). The
umbo is rhombic to elliptic, 1.6–2.6 mm wide by 1.5–2 mm high, and located one-half to one-
third from the upper corner of the apophysis (Fig 7E). The lower half of the umbo is depressed
(Fig 7E). An obtusely pointed mucro develops at the upper-center (perexcentromucronate) of
the umbo (Fig 7E).
Remarks. Some leaves occur in association with female cones of P.mikii described above
(For details, see S1 Table). Leaves are linear, in bundles of two, and up to 1.5 mm wide. A single
ridge is present on the abaxial side. The leaf sheath is persistent (Fig 7F).
Yamada et al. [55] compared P.fujiii auct. non (Yasui) Miki (P.mikii in this study) to other
fossil and extant species. We inferred that this species has an affinity with extant P.thunbergii
Parl. mainly distributed in Japan, as well as P,hwangshanensis W.Y. Hsia and P.tabuliformis
Carrière which are distributed in China [55]. This inference suggests phytogeographyic tie
between Japan and China during the Miocene [29].
Discussions
Pinus trifolia Miki is a synonym for Pinites fujiii Yasui
In 1928, Pinites fujiii was established as a new species based on a single female cone from the
upper Miocene Seto Formation reported by Yasui [34]. This species is characterized by cone
scales arranged in 8:13 parastichies (Fig 1C), apophyses bending downwardly on one side of
Taxonomic Revision of Pinus fujiii
PLOS ONE | DOI:10.1371/journal.pone.0143512 December 16, 2015 12 / 18

the cone (Fig 1C and 1D), and umbos with a centrally positioned prickle-like mucro (Fig 2E)
[34]. Later, in 1939, a new combination Pinus fujiii was legitimately proposed for Pinites fujiii
based on Yasui’s[34] holotype [30]. At the same time, Pinus trifolia was established as a new
species by Miki, based on female and male cones, leaves, pollen, and seeds from the Tokiguchi
and Seto Formations (Fig 3)[30]. However, Miki’s[30] female cones of P.trifolia (Fig 3B, 3C,
3E and 3F) clearly exhibit the diagnostic characteristics of the Pinites fujiii specimen reported
by Yasui [34].
Although we do not know the exact length in Yasui’s holotype because it is compressed ver-
tically [34], it is half the size of Miki’s female cones of P.trifolia [30], judging from the width.
To evaluate whether P.trifolia differs from P.fujiii in the size of the female cone, we examined
variations in female cone sizes within a population by using specimens collected from Akadu
or Hatagoya. The size of cones varied continuously in both localities, i.e., 52–85 mm wide and
75–128 long in Akadu (Figs 4and 6), and 54–105 mm wide and 65–130 long in Hatagoya (Figs
5and 6), and the minimum width in each population was slightly wider than that of Yasui’s
holotype [34] (49 mm; Figs 1and 4–6). Therefore, it is reasonable to conclude that Yasui’s
holotype [34] and Miki’s cones [30] could be small and large cones of a single species,
respectively.
Yasui’s holotype [34] would be an immature cone that was not spontaneously detached
from a branch. In P.fujiii, the basal cone scales remained on the branch (Fig 3D) when the
cones were excised, as seen in Miki’s female cones [30] (Figs 3B,3C,4and 5). However, such
excision is not observed in the holotype (Fig 2C). The holotype was found in a lignite bed
mainly composed of large branches, wood, and bark remains [34], while the other specimens
used in this study were collected from clay or sandy siltstone beds. The holotype might have
been trapped in the sediment along with the branch bearing it, while excised cones were trans-
ported to the depositional place in a different manner from the stunted cone.
In conclusion, Pinus trifolia Miki is a synonym for Pinus fujiii (Yasui) Miki. However,
Yasui’s diagnosis [34] was solely based on a putatively immature female cone. Thus, we pro-
pose here an emended specific diagnosis by integrating diagnostic features stated in both stud-
ies [30,34]. It is suggested that one Miki’s[30] specimen is designated as an epitype that
represents a fully-grown female cone.
Affinity of Pinus fujiii to extant species
Pinus fujiii has leaves clustered in groups of three or four, rarely in five (Fig 3A). This character
is commonly found in most extant species of the Trifoliae (subgenus Pinus), especially in spe-
cies of the subsection Ponderosae [4,11]. Exceptionally in the section Pinus, two species of the
subsection Pinaster also have three leaves per fascicle, i.e., P.canariensis C. Sm. and P.roxbur-
ghii Sarg. [4,59]. However, cones of P.canariensis and P.roxburghii have excentromucronate
umbos [10], while cones of P.fujiii have centromucronate umbos (Fig 3D and 3E), like many
Trifoliae species [4,11,15]. In addition, seeds of P.canariensis and P.roxburghii are adnate to
the wing [10], contrary to the articulated seeds found in both P.fujiii (Fig 3H) and the Trifoliae
species [4,11].
Yasui [34] inferred that P.fujiii is closely related to P.pinaster Aiton of the subsection Pina-
ster because the vascular bundles of the cone scales are arranged in a single plane in both spe-
cies (Fig 2F)[34]. However, similar arrangements are also found in some Trifoliae species,
such as P.palustris Mill. and P.rigida Mill. (M. Yamada, unpublished data). The two leaves per
sheath in P.pinaster [7,10] are also different from the three to five leaves found in P.fujiii.
Taxonomic Revision of Pinus fujiii
PLOS ONE | DOI:10.1371/journal.pone.0143512 December 16, 2015 13 / 18

These morphological characters seem to support Miki’s[30] inference that “P.trifolia”is a
species of the section Taeda (sensu Pilger [31]) which is now included in the section Trifoliae
(sensu Gernandt et al. [4]). To test objectively if these characters support the affinity of P.fujiii
with the Trifoliae, we conducted a phylogenetic analysis based on them by using molecular
phylogeny of the extant species as a backbone constraint. As a result, P.fujiii was included in a
clade consisting of extant Australes and Ponderosae species within the Trifoliae clade with 79%
bootstrap supports (Fig 8). Pinus coulteri D. Don, P.fujiii and P.jeffreyi Balf. and P.sabineana
formed a clade within the Ponderosae clade, but support for this clade was not sufficient (Fig
8). Since small number of characters was used for this analysis, the obtained result is a prelimi-
nary one. However, the result clearly shows that Miki’s[30] inference is reasonable as long as
the morphological characters available for P.fujiii are concerned.
Some molecular clock-based studies suggested that the Trifoliae was originated in North
America in the early Miocene [9,26], contrary to the Paleogene fossil records [15,21] and
molecular dating suggesting pre-Eocene divergence [25]. If this would be the case, our phyloge-
netic inference implies that the ancestor of P.fujiii was dispersed from North America to Japan
during the Miocene in spite of a huge geographic barrier between them [32]. Alternatively, the
result of our phylogenetic analysis would be artifact despite many characters shared between P.
fujiii and the extant Trifoliae. Phylogenetic analysis incorporating more morphological charac-
ters, as well as molecular dating based on other methods and/or markers, would be helpful to
resolve this discrepancy between paleobotanical and neobotanical data.
Implications of Pinus fujiii on history of pines during Cenozoic
Extant species of the section Trifoliae are restrictively distributed in North and Central Amer-
ica and the Caribbean islands [4,7–9]. Many fossil species of this section have also been
reported from the Eocene to Pleistocene in North America [12–21]; thus North America is
considered to be the cradle of this section [15,24]. In the traditional paleobotanical scenario on
North American pines, the distribution of the Trifoliae is considered to be retracted to refugia
that were located in the high and low latitudes and middle latitude uplands during the Eocene
[15,24] when the climate was globally humid and warm [60]. The Trifoliae expanded their dis-
tribution from the refugia around the Eocene/Oligocene boundary [15,24] as the climate
became drastically cooler and drier in this period [60].
A possible ancestor of P.fujiii [52] appeared in Japan around the late Eocene to early Oligo-
cene (ca. 37–31 Ma [51]) when the backbone of the Japanese Archipelago was still connected
to the Eurasian Continent [32]. Since Trifoliae-like fossils are extremely rare in eastern Eurasia,
it is not clear when and where a lineage giving rise to P.fujiii first appeared there. However, it
is possible to hypothesize that the Trifoliae retreated to the high-latitudes of North America
[24] and migrated to the high-latitudes of eastern Eurasia by passing through high-latitude cor-
ridors in clockwise or counterclockwise directions during the Eocene [61,62]. Ancestor of P.
fujiii might have originated from this migrated population. Alternatively, the ancestor might
have arrived directly in Japan from high-latitude refugia of North America through the Berin-
gian Corridor around the Eocene/Oligocene boundary. Whichever is the case, the late Eocene
cooling event [60] enabled the P.fujiii lineage to move southward to Japan, as was the case
with other Trifoliae species in North America [15,24]. The identification of Paleogene fossils
with Trifoliae-like appearance in eastern Eurasia would help greatly in tracing the migration
history of P.fujiii.
Taxonomic Revision of Pinus fujiii
PLOS ONE | DOI:10.1371/journal.pone.0143512 December 16, 2015 14 / 18

Fig 8. Strict consensus tree of 112 most parsimonious trees (length = 56, consistency index = 0.196, retention index = 0.804). Bootstrap values
(>50%) are shown above branches. Branches not resolved in the backbone constraint tree [4] are indicated in gray.
doi:10.1371/journal.pone.0143512.g008
Taxonomic Revision of Pinus fujiii
PLOS ONE | DOI:10.1371/journal.pone.0143512 December 16, 2015 15 / 18

Supporting Information
S1 Dataset. Morphological character matrix used for phylogenetic analyses.
(NEX)
S2 Dataset. Backbone constraint tree used for phylogenetic analyses.
(NEX)
S1 Table. Specimens used for this study.
(XLSX)
Acknowledgments
We thank Dr. H. Ikeda who helped to observe type specimens of P.fujiii. We are also grateful
to Fujii Ceramic Materials Inc. and TYK Corp. for providing information on their clay pits.
Author Contributions
Conceived and designed the experiments: TY. Performed the experiments: TY MY MT. Ana-
lyzed the data: TY. Wrote the paper: TY.
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