Comparative morphology of leaf
epidermis in the genus Lithocarpus and
its implication in leaf epidermal feature
evolution in Fagaceae
Min Deng, Qiansheng Li, Shuting Yang,
YanChun Liu & Jin Xu
Plant Systematics and Evolution
ISSN 0378-2697
Volume 299
Number 3
Plant Syst Evol (2013) 299:659-681
DOI 10.1007/s00606-012-0751-0
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Plant Syst Evol (2013) 299:659–681
DOI 10.1007/s00606-012-0751-0
ORIGINAL ARTICLE
Comparative morphology of leaf epidermis in the genus
Lithocarpus and its implication in leaf epidermal feature
evolution in Fagaceae
Min Deng • Qiansheng Li • Shuting Yang
YanChun Liu • Jin Xu
•
Received: 13 June 2012 / Accepted: 20 December 2012 / Published online: 10 January 2013
Ó Springer-Verlag Wien 2013
Abstract Leaf epidermal features are considered to be
taxonomically important in Fagaceae. In this study, we
examined and compared leaf epidermal features of 112
specimens, representing 105 species and one variety of
Lithocarpus from China and adjacent areas and Notholithocarpus densiflorus. As a result of the different interpretations of terms in previous studies, trichome
terminology in Lithocarpus and its relatives was re-assessed aiming to reveal the trichome evolutionary patterns in
Fagaceae. Twelve types of trichomes and five types of
trichome bases were detected in Lithocarpus, among which
the broad-based trichome (BBT) is newly reported. Stomata in Lithocarpus are restricted to the cyclocytic type
and their size range is 28.6 ± 8.2 lm 9 26.5 ± 9.3 lm.
The distribution of epidermal features in Lithocarpus
revealed three distinct morphological groups: glabrous,
BBT, and appressed parallel tufts (APT). The importance
of epidermal features across Fagaceae for taxon delimitation is evaluated. Species of Lithocarpus can be accurately
identified by the presence of APT or flat epidermal cells
combined with non-dark stained subsidiary cells and noncutinized trichome bases only, or in addition, fasciculate
M. Deng S. Yang Y. Liu J. Xu
Shanghai Chenshan Plant Science Research Center,
Chinese Academy of Sciences, Shanghai Chenshan Botanical
Garden, 3888 Chenhua Rd, Shanghai 201602,
People’s Republic of China
Q. Li (&)
School of Ecology, Shanghai Institute of Technology,
Shanghai 201418, People’s Republic of China
e-mail: qianshengli@gmail.com
Q. Li
Shanghai Key Laboratory of Protected Horticultural
Technology, Shanghai 201403, People’s Republic of China
trichome bases. The phylogenetic distribution of epidermal
features and their evolutionary trends in Fagaceae is also
discussed.
Keywords
Phylogeny
Lithocarpus Leaf anatomy Taxonomy
Introduction
The genus Lithocarpus L. is the second largest genus in the
family Fagaceae, with about 320 Asian species (Govaerts
and Frodin 1998), including 123 species in China (Huang
et al. 1999), and 61 in Borneo (Soepadmo 1972). The
geographical distribution of Lithocarpus spp. extends from
eastern India, southern China, and Japan in the north and
through much of Southeast Asia, including New Guinea
and Malaysia (Cannon and Manos 2001), and it is one of
the dominant tree genera in the evergreen monsoon rain
forests in these regions.
Although the plants in the genus Lithocarpus contribute
significantly to local vegetation, the works conducted on its
taxonomy and systematics are limited. Only two comprehensive taxonomical studies have been conducted on
Lithocarpus. Based on reproductive morphology, Barnett
(1944) placed 221 species of Lithocarpus into five sections
and 12 groups. Camus is the most recent author to treat the
genus in its entirety (Govaerts and Frodin 1998). The
subdivision system of Camus (1934–1954) is more complicated; she subdivided 279 species into 14 subgenera,
among which the subgenus Pseudocastanopsis was later
moved to ‘‘fissa group’’ in the genus Castanopsis. This
transfer was accepted by most taxonomists (Barnett 1944;
Forman 1966; Nixon 1997; Huang et al. 1999; Soepadmo
1972), and has been supported by both molecular (Manos
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and Stanford 2001; Oh and Manos 2008; Chen et al. 2009)
and multiple anatomical studies (Lee 1968; Liu et al.
2009). However, 13 subgenera of Lithocarpus in the work
of Camus (1934–1954) are mostly considered to be sections rather than subgenera (Cannon and Manos 2001). The
only species distributed in western North America—
L. densiflorus (Nixon 1997) was designated as a new
genus—Notholithocarpus (Manos et al. 2008), the recognition of which is supported by leaf epidermal features
(Jones 1986), pollen morphology and molecular evidence
(Manos et al. 2008). Cannon and Manos (2003) comprehensively studied phylogeography of Lithocarpus from SE
Asia. Their results revealed two major clades of cpDNA
haplotypes: one confined to Borneo and the other widespread, but such geographical structures were weak based
on nuclear DNA sequences. The other recent molecular
phylogenetic studies on Lithocarpus were either within a
small region (Borneo) (Cannon and Manos 2000, 2001) or
based on a broad sampling to investigate the phylogeny of
Fagaceae (Manos et al. 2001; Oh and Manos 2008).
Leaf epidermal features are informative and valuable for
interpreting relationships at low taxonomical levels in
Fagaceae, and a large number of researchers, for example,
Smiley and Huggins (1981), Jones (1984, 1986), Hardin
(1976, 1979a, b), Hardin and Johnson (1985), Kvacek and
Walther (1987), Manos (1992), Liu et al. (2009) and
Tschan and Denk (2012) have focused on these characteristics. In Lithocarpus, leaf epidermal features have been
described to varying levels of detail. Jones (1986) comprehensively studied leaf epidermal features on a broad
sample of Fagaceae. His work well demonstrated the
potential phylogenetic information in some of these leaf
epidermal features in Fagaceae, but he only sampled 12
species in Lithocarpus. Kvacek and Walther (1987) studied
leaf cuticular characters of megafossils of Fagaceae in
Central Europe and a few extant species. Their study
mentioned ‘‘typical appressed finger-like tufted (to stellate)
hairs only found in Lithocarpus and not met with in other
Fagaceae’’. In more recent work, Zhou and Xia (2012)
studied leaf epidermal features of 52 Chinese Lithocarpus
species. They reported nine trichome types in Lithocarpus,
of which three (fused stellate, appressed laterally attached
(ALA) unicellular and curly thin-walled unicellular trichomes) were new to Lithocarpus. They classified the 52
Chinese Lithocarpus species into seven groups based on
trichome types and adaxial epidermal cell wall pattern. All
of the aforementioned studies provided an opportunity to
research and compare more comprehensively the leaf epidermal features of Fagaceae. However, only about 20 % of
the species of Lithocarpus [with an overall total of more
than 300 species world-wide (Huang et al. 1999)] were
surveyed by Zhou and Xia (2012), and except for trichome
types and adaxial epidermal cell wall pattern, the other
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M. Deng et al.
epidermal features, such as trichome base, stomata types,
stomatal frequency and size which were thought to be
taxonomically informative were not considered.
Trichome types have been regarded as important in
delimiting species in Lithocarpus (Huang et al. 1999; Zhou
and Xia 2012). Nevertheless, ambiguous descriptions of
hair types and inconsistencies in their naming have led to a
state of confusion in trichome classification. This situation
has serious implications for different studies on Lithocarpus and other members of Fagaceae. For example, Zhou
and Xia (2012) considered the ‘‘bulbous’’ type of trichome
equivalent to the capitate or irregularly multiseriate types
as in Jones (1986), but the same structure was regarded as
thin-walled peltate (TWP) by Liu et al. (2009). Furthermore, APT with long rays defined by Jones (1986) was
treated as a subtype of the stellate trichome by Zhou and
Xia (2012). Another example is the TWP trichome detected
in Lithocarpus and Castanopsis by Jones (1986) and Liu
et al. (2009) which was only found in one Lithocarpus
species by Zhou and Xia (2012). A good application using
explicit morphological features to elucidate the phylogeny
should be based on homology and accuracy score of the
characteristic stages. Therefore, such inconsistencies in
trichome terminology increase the difficulty in comparing
the results of the published accounts in Fagaceae, and also
limit the usage of epidermal features for purposes of
identification and systematics.
In the Northern Hemisphere, Fagaceae have rich fossil
records in the Tertiary (Crepet and Daghlian 1980; Jones
1986; Kvacek and Walther 1987; Crepet 1989; Crepet and
Nixon 1989a, b; Uzunova et al. 1997). Due to similarities
of leaf architecture, it is difficult to distinguish leaf fossils
of Lithocarpus, Castanopsis, and Chrysolepis (Jones 1986).
A comparison of epidermal features of Castanopsis, Castanea, and Chrysolepis reveals the trichome types and
stomatal apparatus as informative in terms of identification
of the three genera (Liu et al. 2009). Appressed parallel
tufts (APT) are believed to be an autapomorphism of
Lithocarpus (Jones 1986; Uzunova et al. 1997). However,
some species in Lithocarpus are without APT (Zhou and
Xia 2012). Whether leaf epidermal features can show that
those Lithocarpus species without APT should be placed in
other evergreen genera in Fagaceae is still unknown, since
no comprehensive studies compared the cuticular features
among those taxa. Therefore, further studies to clarify the
epidermal terminology and a careful comparison of epidermal features in Lithocarpus and other genera in Fagaceae were essential to elucidate and understand the patterns
of evolution of epidermal features in Fagaceae. This work
can also facilitate fossil leaf identification of Lithocarpus
and its relatives. The following were the main objectives of
the present study:1. To study the leaf epidermal variability
in Lithocarpus by an extensive survey of species from
Author's personal copy
Comparative morphology of leaf epidermis
available sources and by comparing these with other
morphological features to facilitate grouping of species of
Lithocarpus, elucidate its phylogeny, and the usefulness of
leaf epidermal features to identify extant and fossil leaves
of Lithocarpus and its relatives.2. To compare the different
usage of names for specific trichome types by previous
authors and clarify the terminology used in the present
study;3. To evaluate the evolutionary pattern of important
leaf epidermal characters by using trees obtained from the
most recent molecular analysis.
Materials and methods
In this study 105 species, and one variety of Lithocarpus
covering the ten subgenera of Lithocarpus recognized by
Camus (1943–1954), and Notholithocarpus densiflorus
were examined using light microscopy (LM) and scanning
electron microscopy (SEM). All samples were either collected by the authors or were obtained from KUN, IBK and
CSH. The voucher specimens are listed in Table 1. All
relevant slide mounts have been deposited in the herbarium
of Shanghai Chenshan Plant Science Research Center,
Chinese Academy of Sciences, China.
Leaf epidermal materials were prepared from mature
leaves. Laminas were boiled in water for 30 s, and then
macerated overnight in 1:1 (by volume) hydrogen dioxide
solution and glacial acetic acid at 60 °C. Pieces of leaf
epidermis were stained with Safranin–alcohol (50 %) prior
to mounting in glycerin gel. Prepared cuticles were
observed using an Olympus microscope (Model BX 53,
Olympus, Japan).
To check the consistency of epidermal structures, at
least five slides of leaf material were made from different
parts of a single leaf of each studied species. For comparison, stomatal frequency (number of stomata per mm2)
was calculated.
The material for SEM observation was directly mounted
on stubs without any treatment, and after coating with gold,
the specimens were examined and photographed under an
SEM (Model S-3400N, Hitachi, Japan).
Comparisons of leaf epidermal features across genera in
Fagaceae were based on the present as well as previous
studies (Jones 1986; Manos 1992; Zhou and Wilkinson
1995; Lou and Zhou 2001; Denk 2003; Deng 2007; Liu
et al. 2009; Tschan and Denk 2012) (Table 3). For the large
genera, e.g., Quercus s.l. and Lithocarpus, the leaf trichomes of main sections (subgenera) or groups were
selected to represent epidermal feature diversification of
the genera. The dimorphic state of leaf trichome characters
was scored and mapped onto the most recent molecular
phylogeny cladogram of ITS and CRABS CLAW combined
datasets (Oh and Manos 2008) based on the parsimony
661
method using Mesquite version 2.75 (Maddison and
Maddison 2011).
Results
Stomata were found only on the abaxial surface of the leaf
lamina in Lithocarpus. The stomata and other epidermal
features were consistent within species, and therefore,
represented reliable characters for taxonomic purposes.
Leaf epidermal features, observed through LM and SEM,
are summarized in Table 2 which shows that leaf epidermal features show large variations between different species. More specific interpretations and illustrations of the
microanatomical features are described below:
Adaxial epidermal cells
The adaxial epidermal cells of Lithocarpus as seen under
LM were usually rectangular to polygonal or irregular in
form, with the anticlinal cell walls usually straight
(Fig. 1a–d) to curved (Fig. 1e–i). Six species appeared to
have slightly sinuous to undulate anticlinal cell walls, such
as L. eriobotryoides (Fig. 1i), L. uvariifolius, and L. fordianus. In most species, the anticlinal cell wall thickness
was uniform, but a ridge-like thickening was present in
L. eriobotryoides and L. elizabethiae (Fig. 1l).
Trichomes and trichome bases on the adaxial epidermis
The surface of epidermal cells was flat without special
ornamentation although coated by a thick wax flake in
most species; both LM and SEM detected a few trichome
types (Fig. 1m–p). Some bowl-like, flat, thin and transparent small structures were usually detected by LM
(Fig. 1a, c–e) and SEM (Fig. 1m, p), which were TWP
trichomes (Fig. 1a, c, e, f, m–o), rather fragile and liable
to be lost while preparing the cuticle for observations,
leaving only a basal portion remaining. Unicellular solitary (Fig. 1m–p), fasciculate and stipitate fasciculate
trichomes (Fig. 1p) were found on the abaxial surface in
five species. These three types of trichomes usually have
a large dark stained base with one to two circles of
radially arranged small epidermal cells surrounding them
(Figs. 5, 1i).
Abaxial epidermal cells
The morphology of abaxial epidermal cells was diverse; 41
species had a smooth cuticle (Figs. 2, 3, 4, 5a–d, x), while
others possessed special ornamentation which could be
categorized as globular (Fig. 5e, f, l, p) or papilla-like
(Fig. 5i, j, k, m, n, o, q, r, s, u). The globular or papilla-like
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Table 1 List of species, vouchers and collection localities used for the leaf epidermal study
Species
Voucher
Collection locality
Kept place
of voucher
1
Lithocarpus amoenus
Tsang, W. T. 22822
Huaiji, Guangxi, China
IBK
2
L. amygdalifolius
Lau, S. K. 27622
Hainan, China
IBK
3
Liang, X. R.63364
Gang-en, Hainan, China
IBK
4
L. amygdalifolius var.
praecipitiorum
L. areca
Sino-Vietnam Exped. 2388
Fuming, Yunnan, China
KUN
5
L. attenuatus
Chen, H. Y. 6892
Hongkong, China
IBK
6
L. bacgiangensis
Mao, P. Y. 03736
Pinbian, Yunnan, China
KUN
7
L. balansae
Shui Y. M. and Chen W. H. 13676
Lue-chun, Yunnan, China
KUN
8
L. brachystachyus
Hou, K. Z. 73548
Bao-ting, Hainan, China
KUN
9
L. calolepis
Li, Z. J. 1325
Mubian, Guangxi, China
IBK
10
L. calophyllus
Li, Y. 2127
Ruyuan, Guangdong, China
KUN
11
L. calophyllus
Chen, S. Q. 13450
Longjing, Guangxi, China
IBK
12
L. carolinae
Wang, C. W. and Liu, Y. 82574
Pinbian, Yunnan, China
KUN
13
L. caudatilimbus
Hou, K. Z. 70133
Sanya, Hainan, China
KUN
14
L. chifui
Gao, X. P. 53679
Ruyuan, Guangdong, China
IBK
15
L. chiungchungensis
Deng, M. 533
Lingshui, Hainan, China
KUN
16
L. chrysocomus
Chen, S. Q. 16657
Da-miao-shan, Guangxi, China
KUN
17
L. cinereus
Zhang, Z. S. 12298
Shangsi, Guangxi, China
IBK
18
19
L. cleistocarpus
L. confinis
Yu, P. H. 940
Li, H. et al. 790
Zhengxiong, Yunnan, China
Yuanjiang, Yunnan, China
KUN
KUN
20
L. corneus
Hu and But 21376
Hongkong, China
KUN
21
L. craibianus
Shui, Y. M. 0463
Yimeng, Yuexi, Yunnan
KUN
22
L. crassifolius
Pu-Ge-shan exped. 171
Puge Mount., Vietnam
KUN
23
L. cucullatus
Teng, L. 7336
Renhua, Guangdong, China
KUN
24
L. cyrtocarpus
South China Biodiversity Survey Team3263
Shangsi, Guangxi, China
IBK
25
L. cyrtocarpus
Yen, H. H. Bi-Vitnam
Vietnam
KUN
26
L. damiaoshanicus
Chen, S. Q. 17025
Rongshui, Guangxi, China
KUN
27
L. dealbatus
Liu, S. E. 19153
Kunming, Yunnan, China
KUN
28
L. echinotholus
Deng, M. 819
Lingshui, Hainan, China
KUN
29
L. elaeagnifolius
Chen, S. Q. 11284
Dongfang, Hainan, China
KUN
30
L. elizabethiae
Chen, W. Y. 6182
Hongkong, China
IBK
31
L. elmerrillii
Deng, M. 748
Lingshui, Hainan, China
KUN
32
L. eriobotryoides
N. Guizhou Exped. 183
Kiang-kou, Guizhou, China
KUN
33
L. farinulentus
Pei, S. J. 59-11284
Mengla, Yunnan, China
KUN
34
35
L. fenestratus
L. fenzelianus
Wang, W. C. 10196
Liang, X. R 64853
Lancang, Yunnan, China
Ding-an, Hainan, China
KUN
KUN
36
L. floccosus
Huang, C. 164285
Fengchuang, Guangdong, China
KUN
37
L. fohaiensis
Liang, S. F. 59-9365
Mengla, Yunnan, China
KUN
38
L. fordianus
Kunming working station 57945
Jinghong, Yunnan, China
KUN
39
L. glaber
Manos, P. S. et al. 1671
Wuyi, Fujian, China
KUN
40
L. glaucus
Wang, C. 38718
Yuanchun, Guangdong, China
IBK
41
L. grandifolius
Sun, H et al. 3066
Motou, Tibet, China
KUN
42
L. guinieri
Gulf Xi-Ke 258
Forest by the beach, Cambodia
KUN
43
L. haipinii
Wang, C. 37958
Xingyi, Guangdong, China
IBK
44
L. haipinii
Huang, C. 164372
Fengchuang, Guangdong, China
KUN
45
L. hancei
Yin, W. Q. 2188
Yuan-Jiang, Yunnan, China
KUN
46
L. handelianus
Teng, L. 3055
Lingshui, Hainan, China
KUN
47
L. harlandii
Mao, P. Y. 04303
Yao-shan, Pinbian, Yunnan, China
KUN
123
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Comparative morphology of leaf epidermis
663
Table 1 continued
Species
Voucher
Collection locality
Kept place
of voucher
48
L. henryi
Fu, G. X. and Zhang, Z. S. 1216
Shi-en, Hubei, China
KUN
49
L. himalaicus
Sun, H et al. 4167
Motou, Tibet, China
KUN
50
L. howii
Lau, S. K. 28249
Wangning, Hainan, China
KUN
51
L. hypoglaucus
Qiu, B. Y. 60959
Er-Yuan, Yunnan, China
KUN
52
L. irwinii
Chen, N. Q. 41798
Lianhua Mount. Hongkong, China
KUN
53
L. ithyphyllus
Wei, S. F. 121013
Zijin, Guangdong, China
KUN
54
L. konishii
Saiti, S. 8637
Nan-tou, Taiwan
KUN
55
L. laetus
Feng, K. M. 5093
Pinbian, Yunnan, China
KUN
56
57
L. laoticus
L. lepidocarpus
Tao, D. D. 291Tao, D. D. 291
Liao, C. C. 1854
Lue-chun, Yunnan, China
Chohsi Hsiang, Yushan National Park,
Taiwan
KUN
KUN
58
L. litseifolius
Manos, P. S. et al. 1502
Xichou, Yunnan, China
KUN
59
L. litseifolius
Chen, S. Q. 13474
Longjing, Guangxi, China
IBK
60
L. litseifolius
Chai, X. T. 58-8652
Xichou, Yunnan, China
KUN
61
L. longanoides
Wang, C. 40237
Xiangxian, Guangxi, China
IBK
62
L. longanoides
Chen, N. Q. 41580
Lou-fu-shan, Guangdong, China
KUN
63
L. longipedicellatus
Deng, M. 503
Lingshui, Hainan, China
KUN
64
L. longzhouicus
Deng, M. 1000
RongAn, Guangxi, China
CSH
65
L. lycoperdon
Feng, K. M. 4873
Pinbian, Yunnan, China
KUN
66
L. lycoperdon
Wang C. W. 82574
Pinbian, Yunnan, China
IBK
67
68
L. macilentus
L. magneinii
Chen, S. Q. 10270
Mao, P. Y. 04023
Canwu, Guangxi, China
Pinbian, Yunnan, China
KUN
KUN
69
L. mairei
Liu, S. E. 16494
Kunming, Yunnan, China
KUN
70
L. megalophyllus
Feng, K. M. 13242
Ma-li-po, Yunnan, China
KUN
71
L. melanochromus
Chen, S. Q. 4735
Fangcheng, Guangxi, China
KUN
72
L. mianningensis
Yin, W. Q. 1363
Tengchong, Yunnan, China
KUN
73
L. microspermus
Liu, W. X. 531
He-kou, Yunnan, China
KUN
74
L. naiadarum
Chen, S. Q. 10654
Qiongzhong, Hainan, China
KUN
75
L. oblanceolatus
Chuang-Jing-Zhi (59)1121
Pinshan, Sichuan, China
KUN
76
L. obovatilimbus
Hou, K. Z. 74003
Lingshui, Hainan, China
IBK
77
L. obscurus
Sci. Exped. 1648
Motou, Tibet, China
KUN
78
L. oleifolius
Chen, D. Z. 679
Da-miao-shan, Guangxi, China
KUN
79
L. pachylepis
Feng, K. M. 4555
Pinbian, Yunnan, China
KUN
80
L. pachyphyllus
Wang, C. W. 89985
Longlin, Yunnan, China
KUN
81
L. pachyphyllus
780 team 721
Tengchong, Yunnan, China
KUN
82
83
L. paihengii
L. pakhaensis
Chen, S. Q. 14240
Mao, P. Y. 04247
Da-miao-shan, Guangxi, China
Pinbian, Yunnan, China
KUN
KUN
84
L. paniculatus
Wang, C. 43962
Ruyuan, Guangdong, China
KUN
85
L. lithocarpaeus
Sun, H. et al. 1707
Motou, Tibet, China
KUN
86
L. petelotii
Liu, W. X. 487
He-kou, Yunnan, China
KUN
87
L. propinquus
Wang and Liu 82493
Pinbian, Yunnan, China
IBK
88
L. pseudoreinwardtii
Sino-Russian. 9788
Jinghong, Yunnan, China
KUN
89
L. pseudosundaicus
Sino-Vietnam Exped. 1385
Qong-shan, Vietnam
KUN
90
L. pseudovestitus
Teng, L. 2729
Lingshui, Hainan, China
KUN
91
L. qinzhouicus
Foresty Burea 77
Qingzhou, Guangxi, China
IBK
92
L. quercifolius
Wei, S. F. 121706
Huiyang, Guangdong, China
KUN
93
L. rhabdostachyus
Mao, P. Y. 2982
Pinbian, Yunnan, China
KUN
94
L. rosthornii
Li, G. F. 60925
Nanchuan, Sichuan, China
KUN
123
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Table 1 continued
Species
Voucher
Collection locality
Kept place
of voucher
95
L. silvicolarum
96
L. skanianus
Deng, M. 830
Ma-li-po, Yunnan, China
KUN
Teng, L. 7382
Renhua, Guangdong, China
97
KUN
L. sphaerocarpus
Mao, P. Y. 03872
Pinbian, Yunnan, China
KUN
98
L. tabularis
Mao, P. Y. 4254
Pinbian, Yunnan, China
KUN
99
L. taitoensis
Manos P. S. et al. 1674
Napo, Guangxi, China
KUN
100
L. talangensis
Yun, W. Q. 2054
Yuanjiang, Yunnan, China
KUN
101
L. talangensis
Wu, S. G. 970
Shipin, Yunnan, China
KUN
102
L. tenuilimbus
Gao, X. P. 50792
Cong-Feng-Pin, Guangdong, China
IBK
103
104
L. touranensis
L. trachycarpus
Sino-Vietnam Exped. 1903
Lin, Z. W. et al. 160
Tonkin, Youngfu, Vietnam
Menghai, Yunnan, China
KUN
KUN
105
L. truncatus
Pei, S. J. 59-10275
Mengla, Yunnan, China
KUN
106
L. uvariifolius
Zuo, J. L. 22682
Lianxian, Guangdong, China
KUN
107
L. variolosus
Lianda11632
Chang Mount. Dali, Yunnan
KUN
108
L. vestitus
Lau, S. K. 27190
Ledong, Hainan, China
KUN
109
L. xizangensis
Tibet Exped. 74-4411
Motou, Tibet, China
KUN
110
L. xylocarpus
Sun, H. 84-128
Jingdong, Yunnan, China
KUN
111
L. xylocarpus
Deng, M. 796
Jingdong, Yunnan, China
KUN
112
Notholithocarpus
densiflorus
Bartholonew, B. 1427
SanBenito County, Califonia, USA
KUN
ornamentations on epidermal cells were present in 64
species; for example, in L. floccosus (Fig. 6g), L. litseifolius (Fig. 6k), and L. fenestratus (Fig. 6l). In some species,
the thickened areas were all covered by epidermal cells, such
as in L. laoticus (Fig. 6i), L. confinis (Fig. 6j), and L. lepidocarpus (Fig. 6n). Both flat and globular-papillae thickening
structures can be present on the same species, such as in
L. silvicolarum (Fig. 5n) and in L. skanianus (Fig. 5o).
In most species, the anticlinal walls of the epidermal
cells on the abaxial surface were straight (Figs. 2f, 5b) to
curved (Figs. 2a–d, 5e–s). Eighteen species had undulated
to sinuous anticlinal walls (Figs. 2k, 3b–p).
Stomatal apparatus
All studied species were hypostomatic. The stomata were
confined to small areolar regions of the leaf cuticle, with
each containing ca. 11–29 stomata, and forming rather
dense groups (Fig. 3a). The stomatal size range was
28.6 ± 8.2 lm 9 26.5 ± 9.3 lm across the species. The
largest stomata were present in L. glaucus (37.6 lm 9
35.5 lm) and the smallest (17.9 lm 9 16.0 lm) in
Notholithocarpus densiflorus. The stomatal frequency
ranged from 213 to 574/mm2. The lowest and the highest
stomatal frequency were noted in L. grandifolius and
L. uvariifolius, respectively.
The stomata of Lithocarpus species were restricted to
the cyclocytic type. The stomata in N. densiflorus were
123
mostly anomocytic, but at the center of the areolar region,
stomata larger (25.1 ± 2.3 lm 9 20.1 ± 3.4 lm) than
others were surrounded by a circle of small epidermal cells
and the stomata were of the cyclocytic type.
The outlines of the pair of guard cells were usually
suborbiculate to broadly elliptical in surface view, with a
length/width (L/W) ratio of 1.1–1.5:1. Guard cells were
often thickened to some degree, and were made up of outer
stomatal ledges or rims. The subsidiary cells were flat. The
stomatal apparatus was easily observable unless shielded
by thick trichome layers. The outlines of the pair of guard
cells of Lithocarpus were usually suborbiculate to broadly
elliptical in surface view, with length/width (L/W) radio of
(1.1)1.2–1.5:1. The stomatal pores, where the guard cells
meet, were almost circular, but truncated in N. densiflorus
(Fig. 5x). While preparing the epidermal samples for LM, a
membranous structure beneath the stomatal pore was
generally noted in 11 flat cuticle species, which was possibly the remains of the lower anticlinal and/or periclinal
walls of the guard cells, such as in L. corneus, L. eriobotryoides, and L. areca (Fig. 3a, o, p, respectively).
Trichomes and trichome bases on the abaxial epidermis
Trichome types
Twelve types of trichomes were detected in this study.
Detailed explanation of each type is summarized below:
Scientific name
1
L. amoenus
Adaxial
Abaxial
T type
TB
Ep
Anti
wall
Ep
T type
TB
Anti
wall
Orn ep
SA
Stomata L 9 W (lm)
Stoma freq/
mm2
twp
stb/fb
irr
str
irr
apt/f/s/sf/twp
aptb/fb/
stb
str-cur
pap
cyc
23.9–26.2 9 21.5–23.7
320
Morph
group
APT group
2
L. amygdalifolius
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
glo-pap
cyc
22.7–24.6 9 21.1–22.1
320
APT group
3
L. amygdalifolius var.
praecipitiorum
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
26.6-29.0 9 20.9–25.5
363
APT group
4
L. areca
twp
stb
poly-irr
str-cur
irr
bbt/f/s/sf
btb/fb
sin
None
cyc
22.4–28.0 9 20.3–24.6
491
BBT group
5
L. attenuatus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
23.4–23.9 9 22.0–22.2
341
APT group
L. bacgiangensis
twp
stb
irr
str-cur
irr
ala/apt/twp
aptb/stb
str-cur
None/glo
cyc
25.8–24.1 9 20.2–23.7
491
APT group
L. balansae
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
20.6–24.8 9 19.4–23.0
469
APT group
8
L. brachystachyus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
19.6–24.0 9 17.5–20.0
512
APT group
9
10
L. calolepis
L. calophyllus
NA
twp
stp
stb
irr
irr
str
str
irr
irr
apt/twp
apt/su/twp
aptb/stb
aptb/stb
str
str-cur
pap-oa
pap
cyc
cyc
22.7–23.4 9 17.9–22.5
21.5–21.5 9 17.6–20.4
533
469
APT group
APT group
11
L. calophyllus
twp
stb
irr
str-cur
irr
apt/su/twp
aptb/stb
str-cur
oa
cyc
30.8–32.3 9 30.1–31.5
341
APT group
12
L. carolinae
twp
stb
irr
str
irr
apt/twp
aptb/fb/
stb
str-cur
pap
cyc
28.0–32.3 9 23.8–28.9
277
APT group
13
L. caudatilimbus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
19.9–21.3 9 16.0–19.5
448
APT group
14
L. chifui
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
glo-papoa
cyc
25.2–28.2 9 24.2–26.7
405
APT group
15
L. chiungchungensis
NA
stb
irr
str-cur
irr
apt/twp
aptb/stb
str-cur
glo-pap
cyc
23.9–29.2 9 20.7–22.3
299
APT group
16
17
L. chrysocomus
L. cinereus
twp
twp
stb
stb
irr
irr
str
str
irr
irr
apt/twp
apt/twp
aptb/stb
aptb/stb
str-cur
str-cur
pap
Noneglo-pap
cyc
cyc
23.8–27.7 9 23.0–26.7
26.4–32.0 9 22.4–29.0
320
384
APT group
APT group
18
L. cleistocarpus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
glo-pap
cyc
24.0–25.7 9 23.3–24.2
341
APT group
19
L. confinis
twp
stb
irr
str
rou
apt/twp
aptb/stb
cur
pap/oa
cyc
24.8–24.4 9 18.6–21.7
427
APT group
20
L. corneus
twp
stb
poly-irr
str
irr
bbt
btb
undsin
None
cyc
28.0–36.5 9 26.7–35.5
363
BBT group
21
L. craibianus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
24.9–34.0 9 20.1–28.5
256
APT group
L. crassifolius
twp
stb
irr
str
irr
apt/twp
aptb/stb
str
flat
cyc
28.0–30.2 9 25.0–28.4
427
APT group
L. cucullatus
twp
stb
irr
str
irr
apt/s/f/twp
aptb/
ctb/stb
str-cur
rou-pap
cyc
21.6–25.9 9 21.6–23.0
405
APT group
24
L. cyrtocarpus
f/s/sf
fb
recpolyirr
str
irr
bbt/f/s/sf/st/
uc
btb/fb
cur-sin
None
cyc
24.3–28.8 9 21.6–23.6
363
BBT group
25
L. cyrtocarpus
f/s/sf
fb
poly-irr
str
irr
bbt/f/s/sf/st/
uc
btb/fb
sin
None
cyc
20.3–25.0 9 18.5–20.1
320
BBT group
665
123
22
23
Author's personal copy
6
7
Comparative morphology of leaf epidermis
Table 2 Leaf epidermal features of Lithocarpus in this study
666
123
Table 2 continued
Scientific name
Adaxial
Abaxial
T type
TB
Ep
Anti
wall
Ep
T type
TB
Anti
wall
Orn ep
SA
Stomata L 9 W (lm)
Stoma freq/
mm2
Morph
group
L. damiaoshanicus
twp
stb
irr
str
isoirr
apt/twp
aptb/stb
str-cur
oa
cyc
19.3–25.3 9 19.1–22.1
235
APT group
27
L. dealbatus
f/s/
twp
stb/fb
irr
str
irr
apt/f/s/twp
aptb/fb/
stb
str-cur
pap
cyc
24.4–25.5 9 20.0–24.2
320
APT group
28
L. densiflorus
NA
ctb/
stb
irr
str-cur
irr
mu/su/twp
ctb/stb
str-cur
None
an/
cyc
17.9–27.1 9 16.0–22.3
427
APT group
29
L. echinotholus
twp
stb/
ctb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
24.4–26.9 9 21.2–23.8
384
APT group
30
L. elaeagnifolius
twp
stb
irr
str-cur
irr
apt/twp
stb/stb
str-cur
glo-pap
cyc
26.1–30.2 9 22.2–25.7
341
APT group
31
L. elizabethae
NA
stb
irr
curund
irr
apt/twp
aptb/stb
str-cur
glo-pap
cyc
23.9–26.2 9 21.5–23.7
235
APT group
32
L. elmerrillii
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
19.6–24.9 9 19.3–22.2
320
APT group
33
L. eriobotryoides
bbt
btb
poly-irr
undsin
irr
bbt/f/ro/s/sf/
twp/uc
btb/fb/
stb
undsin
None
cyc
26.1–29.8 9 25.6–26.4
320
BBT group
34
35
L. farinulentus
L. fenestratus
twp
twp
stb
stb
irr
irr
str
str
irr
irr
apt/twp
apt/twp
aptb/stb
aptb/stb
str
str-cur
None
pap
cyc
cyc
24.1–25.2 9 17.2–21.4
21.0–26.1 9 19.3–21.7
405
405
APT group
APT group
36
L. fenzelianus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
19.2–21.8 9 19.1–21.1
341
APT group
37
L. floccosus
twp
stb
irr
cursin
irr
apt/twp
aptb/stb
str-cur
pap
cyc
25.5–27.1 9 21.0–24.1
386
APT group
38
L. fohaiensis
NA
stb
irr
str-cur
irr
twp
stb
cur-sin
None
cyc
24.7–28.1 9 19.0–27.2
433
Glabrous
group
39
L. fordianus
bbt
btb
poly-irr
sin
irr
bbt/f/s/sf/uc
btb/fb/
stb
sin
None
cyc
19.6–27.0 9 19.0–19.9
469
BBT group
40
L. glaber
NA
ctb
irr
str
irr
apt/bu/twp
aptb/
ctb/stb
str-cur
pap
cyc
23.7–27.7 9 20.7–27.4
320
APT group
41
L. glaucus
NA
stb
poly-irr
undsin
irr
twp
stb
str-cur
None
cyc
36.4–37.6 9 31.4–35.5
235
Glabrous
group
42
L. grandifolius
NA
stb
rec-poly
cur-str
irr
twp
stb
sin
None
cyc
26.8–27.2 9 24.9–26.6
213
Glabrous
group
43
L. guiuieri
twp
stb
irr
str
irr
twp/apt/twp
aptb/stb
str-cur
None
cyc
25.5–26.8 9 24.8–26.4
277
APT group
44
L. haipinii
bbt
btb
poly-irr
str
irr
bbt/f/s/sf/twp
btb/fb
cur-sin
None
cyc
28.9–33.8 9 28.6–33.1
363
BBT group
L. haipinii
bbt
btb
poly-irr
str
irr
bbt/s/f/sf
btb/fb
cur-sin
None
cyc
29.9–32.2 9 24.6–24.9
405
BBT group
L. hancei
absent
absent
irr
str
rouirr
twp/su
stb
cur
None
cyc
24.3–30.9 9 21.1–26.4
469
Glabrous
group
47
L. handelianus
f/s/sf/
twp
stb/fb
irr
str
irr
apt/f/s/twp
aptb/fb/
stb
cur
pap
cyc
23.7–27.7 9 20.8–27.5
341
APT group
M. Deng et al.
45
46
Author's personal copy
26
Scientific name
Adaxial
Abaxial
T type
TB
Ep
Anti
wall
Ep
T type
TB
Anti
wall
Orn ep
SA
Stomata L 9 W (lm)
Stoma freq/
mm2
Morph
group
48
L. harlandii
twp
stb
irr
str
irr
twp
stb
rousin
None
cyc
23.7–27.7 9 20.7–27.4
491
Glabrous
group
49
L. henryi
twp
stb
irr
str
iso
apt/twp
stb
rou
oa
cyc
19.2–21.8 9 19.1–21.1
299
APT group
50
L. himalaicus
twp
stb
irr
str
irr
twp
stb
str-cur
None
cyc
28.2–28.8 9 24.6–27.2
256
Glabrous
group
51
L. howii
f/sf
stb
poly-irr
cur-str
irr
bbt/f/st/twp
btb/ctb/
fb
cur-sin
None
cyc
20.2–25.4 9 16.8–22.9
427
BBT group
L. hypoglaucus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
glo
cyc
23.7–27.7 9 20.8–27.5
320
APT group
L. irwinii
L. ithyphyllus
twp
NA
stb
stb
irr
irr
str
str
irr
irr
apt/twp
twp
aptb/stb
stb
cur
str-cur
pap
None
cyc
cyc
25.5–27.1 9 21.0–24.1
25.5–30.9 9 24.7–27.7
320
341
APT group
Glabrous
group
55
L. konishii
bbt
stb
poly-irr
und
irr
bbt/uc
btb/stb
sin
None
cyc
25.3–28.4 9 21.5–24.3
491
BBT group
56
L. laetus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
21.6–25.9 9 21.6–23.0
469
APT group
57
L. laoticus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
26.4–29.8 9 22.8–28.2
341
APT group
58
L. lepidocarpus
twp
stb
irr
str
rou
apt/twp
aptb/stb
rou
oa
cyc
26.1–27.7 9 23.8–25.7
452
APT group
59
L. litseifolius
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
25.6–26.8 9 22.4–26.6
427
APT group
60
L. litseifolius
twp
stb
irr
str
irr
apt/twp
aptb/stb
cur
pap
cyc
27.3–30.2 9 25.3–27.5
275
APT group
61
L. litseifolius
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap-oa
cyc
21.5–21.5 9 17.6–20.4
299
APT group
62
L. longanoides
twp
stb
irr
str-cur
irrrou
apt/twp
aptb/stb
str-cur
glo or oa
cyc
25.1–28.7 9 23.5–22.8
401
APT group
63
L. longanoides
twp
stb
irr
str-cur
irriso
apt/twp
aptb/stb
str-cur
None/oa
cyc
21.7–26.2 9 21.6–26.0
384
APT group
64
L. longipedicellatus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
22.7–23.7 9 17.6–20.7
384
APT group
65
L. longzhouicus
f/sf
stb
poly-irr
undsin
irr
bbt/twp/uc
btb/stb
cur-sin
None
cyc
36.4–25.0 9 31.4–20.1
299
BBT group
66
L. lycoperdon
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
oa
cyc
26.1–27.7 9 23.8–25.7
341
APT group
67
68
L. lycoperdon
L. macilentus
twp
NA
stb
stb
irr
irr
str
str
irr
irr
apt/twp
apt/ala/twp
aptb/stb
stb
str-cur
str-cur
pap
glo-pap
cyc
cyc
27.9–27.4 9 22.7–24.4
22.7–23.4 9 21.1–23.2
277
414
APT group
APT group
69
L. magneinii
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
21.8–25.8 9 21.1–25.5
363
APT group
70
L. mairei
NA
stb
irr
str
irr
apt/twp
aptb/stb
str
glo-pap
cyc
23.7–29.4 9 21.7–22.5
299
APT group
71
L. megalophyllus
twp
stb
irr
strund
irr
twp
stb
cur-sin
flat
cyc
29.1–33.9 9 28.8–30.8
350
Glabrous
group
L. melanochromus
NA
ctb
irr
str
iso
apt/twp
aptb/stb
rou
oa
cyc
21.8–25.8 9 21.1–25.5
363
APT group
L. mianningensis
NA
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
27.3–32.5 9 23.9–26.1
384
APT group
74
L. microspermus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
glo
cyc
19.8–23.0 9 19.6–20.6
371
APT group
667
123
72
73
Author's personal copy
52
53
54
Comparative morphology of leaf epidermis
Table 2 continued
668
123
Table 2 continued
Scientific name
Adaxial
Abaxial
T type
TB
Ep
Anti
wall
Ep
T type
TB
Anti
wall
Orn ep
SA
Stomata L 9 W (lm)
Stoma freq/
mm2
Morph
group
L. naiadarum
twp
stb
irr
str
irrrou
twp
stb
str-cur
flat
cyc
23.7–29.4 9 21.7–22.5
501
Glabrous
group
76
L. oblanceolatus
twp
stb
irr
str
irr
twp
stb
str
flat
cyc
29.1–32.7 9 26.1–31.8
471
Glabrous
group
77
L. obovatilimbus
twp
stb
irr
str
irriso
apt/twp
aptb/stb
str-cur
pap-oa
cyc
26.6–29.0 9 20.9–25.5
320
APT group
78
L. obscurus
NA
stb
irr
str
irr
twp
stb
str
flat
cyc
27.3–29.7 9 20.0–23.8
437
Glabrous
group
79
L. oleifolius
NA
stb
irr
str-cur
irr
ala/apt/twp
aptb/stb
str-cur
Noneglo-pap
cyc
25.2–25.9 9 23.7–24.1
435
APT group
80
L. pachylepis
f/s/sf
fb/stb
poly-irr
str-cur
irr
bbt/s/f/sf
btb/fb
undsin
None
cyc
19.3–25.3 9 19.3–23.5
348
BBT group
81
L. pachyphyllus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
22.7–25.1 9 20.0–23.9
469
APT group
82
L. pachyphyllus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
19.8–25.5 9 19.6–21.1
529
APT group
83
84
L. paihengii
L. pakhaensis
NA
NA
stb
stb
irr
irr
str
str
irr
irr
apt/twp
apt/twp
aptb/stb
aptb/stb
str-cur
str-cur
pap
None
cyc
cyc
24.7–28.1 9 19.0–27.2
22.5–25.6 9 21.3–23.4
320
457
APT group
APT group
85
L. paniculatus
NA
ctb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
23.9–28.6 9 18.2–23.8
427
APT group
86
L. lithocarpaeus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
27.9–33.4 9 20.8–24.4
258
APT group
87
L. petelotii
NA
stb/
ctb
irr
str
irr
apt/s/f/twp
fb/stb
cur-sin
None
cyc
27.0–27.7 9 23.5–27.6
363
APT group
88
L. propinquus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None/glo
cyc
21.7–25.2 9 18.6–20.8
299
APT group
89
L. pseudoreinwardtii
twp
stb
irr
str
irr
apt/twp
aptb/stb
cur-sin
None
cyc
22.9–22.6 9 16.7–20.7
497
APT group
L. pseudosundaicus
twp
stb
irr
str-cur
irr
apt/s/twp
fb/stb
str-cur
pap
cyc
22.9–26.8 9 21.6–24.7
341
APT group
L. pseudovestitus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
21.7–23.3 9 20.3–21.2
427
APT group
92
L. qinzhouicus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
28.8–29.7 9 24.8–27.0
341
APT group
93
L. quercifolius
twp
stb
poly-irr
strund
irr
bbt/uc
btb/stb
sin
None
cyc
25.4–28.8 9 21.7–24.5
256
BBT group
94
L. rhabdostachyus
f/twp
fb/stb
irr
str
irr
apt/f/s/sf/twp
aptb/fb/
stb
str-cur
None
cyc
25.3–26.7 9 23.9–26.4
299
APT group
95
L. rosthornii
NA
stb/
ctb
irr
str-cur
irr
apt/f/s/sf/twp
aptb/stb
str-cur
glo-pap
cyc
24.9–25.6 9 20.2–24.5
363
APT group
96
L. silvicolarum
twp
stb
irr
str-cur
irr
apt/s/twp
aptb/
btb/fb
str-cur
pap
cyc
21.4–24.3 9 19.5–24.0
410
APT group
97
L. skanianus
NA
stb
irr
strund
irr
apt/f/s/sf/twp
aptb/fb/
stb
str-cur
pap
cyc
29.1–35.7 9 27.3–28.8
235
APT group
98
L. sphaerocarpus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
22.7–24.1 9 21.4–23.3
405
APT group
M. Deng et al.
90
91
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Scientific name
Adaxial
T type
Abaxial
TB
Ep
Anti
wall
Ep
T type
TB
Anti
wall
Orn ep
SA
Stomata L 9 W (lm)
Stoma freq/
mm2
Morph
group
L. tabularis
twp
stb
irr
str
irr
apt/bu/twp
aptb/stb
str-cur
pap
cyc
25.6–26.8 9 22.4–26.6
256
APT group
L. taitoensis
twp
stb
irr
str
irr
apt/twp
aptb/stb
str
flat
cyc
25.2–31.5 9 23.0–30.1
299
APT group
101
L. talangensis
twp
stb/fb
irr
str
irr
apt/twp
aptb/fb/
stb
str-cur
pap
cyc
19.3–25.3 9 19.1–22.1
320
APT group
102
L. talangensis
twp
stb/
ctb
irr
str
irr
apt/twp
aptb/fb/
stb
str-cur
pap
cyc
23.0–26.7 9 23.0–26.7
384
APT group
103
L. tenuilimbus
s/f/
twp
stb/fb
irr
str
irr
apt/twp
aptb/fb/
stb
str-cur
flat-pap
cyc
22.3–27.4 9 22.0–22.2
491
APT group
104
L. touranensis
NA
stb/
ctb
irr
str
irr
twp
stb
str
None
cyc
24.8–27.4 9 22.6–27.1
341
Glabrous
group
105
L. trachycarpus
twp
stb
irr
str
irr
apt/twp
aptb/stb
cur
pap
cyc
24.4–26.7 9 23.3–25.2
448
APT group
106
L. truncatus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
None
cyc
24.0–24.6 9 15.8–24.0
256
APT group
107
L. uvariifolius
s/fs/
bbt
fb/stb
poly-irr
undsin
irr
bbt/f/s/sf
btb/fs/
stb
sin
None
cyc
22.7–29.9 9 21.9–27.6
576
BBT group
108
L. variolosus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str-cur
pap
cyc
24.3–28.8 9 21.6–23.6
341
APT group
109
L. vestitus
twp
stb
irr
str
irr
apt/twp
aptb/stb
str
flat
cyc
22.2–23.1 9 20.0–22.3
384
APT group
110
L. xizangensis
f/twp
stb/fb
irr
str
iso
apt/s/f/twp
aptb/fb/
stb
cur
oa
cyc
24.3–28.8 9 21.6–23.6
320
APT group
111
L. xylocarpus
twp
stb
irr
cur to
str
irr
apt/twp
aptb/stb
str-cur
glo
cyc
24.9–26.4 9 15.9–21.4
320
APT group
112
L. xylocarpus
twp
stb
irr
cur to
str
irr
apt/twp
aptb/stb
str-cur
glo
cyc
24.4–28.2 9 15.9–24.6
256
APT group
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100
Comparative morphology of leaf epidermis
Table 2 continued
T type trichome types, ala appressed laterally attached unicellular, apt appressed parallel tufts, bbt broad based trichomes, bu branched uniseriate, f fasciculate, mu multiradiate, ro rosulate,
s solitary unicellular, sf stipitate fasciculate, su simple uniseriate, st stellate trichomes, twp thin-walled peltate, uc unicellular conical trichome, NA not available, TB trichome base type, aptb
appressed parallel tuft base, btb broad trichome base, ctb compound trichome base, fb fasciculate trichome base, stb simple trichome base, Ep Shape of epidermal cells, irr irregular, rec
rectangle, poly polygonal, iso isodiametric, Anti Wall Anticline wall shape, str straight, cur curved, und undulate, sin sinus, rou rounded, Orn ep Ornentmental on epidermal cells, pap papilate
thickening, glo global thickening, oa overall thickening, none flat no special ornentments, TB Trichome base, stb simple trichome base, ctb compound trichome base, aptb appressed parallel
tufts base, btb broad trichome base, SA stomata aperture, cyc cyclocytic, an anomocytic, Stoma Size stomata size, Stoma freq stomata frequency, Morph group morphological group
669
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Fig. 1 Characteristics of adaxial epidermal cells. a–l LM. a–k bar
50 lm; a Lithocarpus chrysocomus, showing a small TWP and
its simple trichome base; b L. pseudoreinwardtii; showing SU;
c L. tenuilimbus; showing F and TWP; d L. variolosus; e L. amoenus;
f L. cucullatus; g L. obscurus; h L. pachylepis, the arrow indicates SU;
i L. eriobotryoides; j L. uvariifolius, showing fasiculate trichome
base; k Notholithocarpus densiflorus, showing compound trichome
base; l L. elizabethiae, bar 20 lm; m–p SEM, showing TWP. m
L. fenestratus, bar 50 lm; n L. hancei, bar 10 lm; o L. harlandii, bar
20 lm; p L. hypoglaucus, bar 50 lm, showing SF
Thin-walled peltate trichomes Jones (1986) recorded this
trichome type as an intermediate type consisting of thinwalled cells which possess a unicelled to 2–3-celled stalk
and an irregularly shaped stellate to discoidal cap composed of many randomly oriented cells (Fig. 5v). This type
of trichome is generally present in all species of Lithocarpus. The shape of these trichomes is diverse among
various species. The stalk cell(s) is usually isodiametric
and stained darker than epidermal cells, which indicates a
glandular trichome type. The simplest TWP trichome has
only a transparent skirt-like rim on the stalk cell, such as in
L. balansae (Figs. 5b, 6a) and L. oleifolius (Fig. 5s). The
large TWP trichomes were rather fragile (L. calophyllus
Fig. 6q, L. craibianus Fig. 6t). In most cases, while preparing the cuticle for LM, most of the peltate trichome was
usually lost, with only a round simple trichome base
remaining, or with only the unicelled stalk left (Fig. 6l, o,
u, v). The trichome base was usually large, with a diameter
ranging from ca. 9.3 to 17.0 lm, and the basal portion
remaining without any obvious stain.
123
Broad base trichomes (BBTs) This type of trichome is
usually composed of two parts: the basal broad cell portion
or foot cell (Hill 1983), and an upper, long or short, barrelshaped structure. This trichome type was present in 11
species; for example, L. areca (Fig. 3h, p) L. quercifolius
(Fig. 3c), L. konishii (Fig. 3f), L. howii (Figs. 3k, 4j, k),
L. uvariifolius (Figs. 3g, 4b), L. fordianus (Figs. 3i, 4c, d),
and L. corneus (Fig. 4m, n). The trichome itself is usually
almost transparent. This trichome type is very similar to
simple uniseriate (SU), but is easily distinguishable due to
its rather prominent convex basal broad cells. The diameter
of the basal broad cell portion is large, usually
17.3–25.6 lm. The convex broad basal portion also can be
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671
Fig. 2 Characteristics of abaxial epidermis of glabrous group.
a–l LM, bar 50 lm; a Lithocarpus glaucus; b L. fohaiensis;
c L. obscurus; d L. oblanceolatus; e L. naiadarum; f L. touranensis;
g L. ithyphyllus; h L. megalophyllus; i L. harlandii; j L. himalaicus;
k L. grandifolius; l L. megalophyllus; m–v SEM, bar 20 lm.
m L. himalaicus; n L. fohaiensis; o L. harlandii; p L. touranensis;
q L. grandifolius; r–s L. ithyphyllus; t L. obscurus; u–v L. hancei;
u arrow indicates SU; v showing stomatal wax plug
found in TWP, but is different from its closed terminal
structure, in contrast to the flat, free structure in TWP.
Under SEM, BBT can be easily distinguished from TWP
and SU.
(Fig. 5s) although the typical trichome bases were generally found in several other species bearing APT, indicating
its possible existence in those species as well.
Appressed laterally attached unicellular This trichome
type is similar to the solitary unicellular trichome, except
that they are attached laterally, and are generally thinwalled and difficult to detect. Usually, the distance from
the trichome base to the end of the hair is extremely short,
much shorter than in other trichomes. The trichome base is
simple-celled, but the basal portion is smaller and darker
stained than in TWP and BBT. This trichome type was
detected only in L. macilentus (Fig. 5q, r) and L. oleifolius
Simple uniseriate This type of trichome is composed of a
single column of at least two or more thin-walled structures, apparently cells. This trichome type is not common in
Lithocarpus and was present only in L. calophyllus (Fig. 5w),
L. hancei (Fig. 2u) and. N. densiflorus (Figs. 5x, 6x).
Branched uniseriate Branched uniseriate (BU) trichomes
were detected in two species of Lithocarpus only. This type
of trichome is similar to TWP in cellular composition, but
in shape is usually elongated and branched at least once.
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M. Deng et al.
Fig. 3 Characteristics of abaxial epidermis of BBT group (LM).
a Lithocarpus areca, bar 100 lm; b–l bar 50 lm; b L. longzhouicus;
c L. quercifolius; d L. pachylepis; e L. cyrtocarpus; f L. konishii;
g L. uvariifolius; h L. areca; i L. fordianus; j L. haipinii; k L. howii;
l L. eriobotryoides; m–p bar 20 lm. m L. corneus; n L. cyrtocarpus;
o L. eriobotryoides; p L. areca
These trichomes, irregular in shape, produce a large number of oddly shaped forms, such as in L. tabularis (Fig. 6s)
and L. glaber (Fig. 5t).
(Figs. 5c, e, p; 6k–o) cover the stomata. The convex APT
trichome bases in extreme types were surrounding the
stomata’s subsidiary cells. The stomata were shielded by the
upper APT rays, such as in L. lithocarpaeus (Fig. 5m). In
another extreme type, the APT trichome bases were fused
together in a round fashion with the trichome rays fanning out
to form a ‘‘stellate’’-like structure, such as in L. pseudoreinwardtii (Fig. 6c), L. vestitus (Fig. 6f) and L. paihengii
(Fig. 6o). A similar trichome type was also reported as fused
stellate or stellate by Jones (1984, 1986) and Zhou and Xia
(2012). However, the stellate trichome formed by a cluster of
APTs discovered in the present study can be easily distinguished from the typical stellate trichome by its convex base
without dark staining compared to the flat, and dark-stained
‘‘flower-like’’ compound trichome base found in typical
stellate and fused stellate trichomes (St).
Rosulate This trichome type consists of unicellular, open,
thin-walled elements, arranged in small bushy tufts. The
trichome base is simple in Lithocarpus. The rosulate (Ro)
trichome is not common in species of Lithocarpus and was
detected only in one species, L. eriobotryoides (Fig. 4g).
Appressed parallel tufts APT trichomes were the most
common in Lithocarpus spp.; 85 species were found to bear
APT trichomes. The trichome consists of (1)2–8(12) thickwalled, unicellular elements that are nearly coplanar and
approximately parallel to the leaf surface as well as to each
other (Figs. 5a–s, 6a–n). This trichome’s basal portion is
usually convex and raised above the epidermal cell to form
a linear band along the subsidiary cells of the stomata,
which can partly (Figs. 5d, f, h, i, n; 6a–j) or mostly
123
Stellate trichomes Stellate trichomes usually consisted of
(3)4–12 non-glandular, unicellular, and generally thick-
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673
Fig. 4 Characteristics of abaxial epidermis of BBT group (SEM).
a, b Lithocarpus uvariifolius. a bar 100 lm; b bar 20 lm, showing
BBT; c, d L. fordianus; c bar 20 lm, showing BBT and UC; d bar
10 lm, the high magnification of BBT; e L. haipinii, bar 20 lm;
f, g L. eriobotryoides, bar 20 lm; f showing TWP; g showing Ro;
h L. cyrtocarpus, showing St and BBT, bar 50 lm; i–k L. howii.
i showing UC, bar 20 lm; j showing St, bar 50 lm; k showing BBT,
bar 20 lm; l L. longzhouicus, showing BBT, bar 10 lm; m, n
L. corneus. m showing BBT, bar 20 lm; n as M, BBT under high
magnification bar 10 lm; o, p L. konishii; o showing BBT, bar
20 lm; p high magnification of BBT on the same slides of O, bar
10 lm
walled elements that radiate from a common point of
attachment in a parallel or nearly parallel fashion to the leaf
surface. Of all the studied species of Lithocarpus, this trichome type was present only in two species, L. howii
(Figs. 3k, 4i) and L. cyrtocarpus (Fig. 4h). In this trichome,
the base is compound and one side of the epidermal cell
wall adjacent to the base cells was dark stained.
Solitary unicellular trichome This solitary unicellular
trichome (S) was generally thick-walled, fairly long and straight
and dark-stained (Figs. 3a, h, i, 4a). It is a basic element to form
the fasciculate trichome and the stipitate fasciculate trichome.
This trichome type was present in 20 species including
L. uvariifolius (Fig. 4a), L. areca (Fig. 3a), L. fordianus
(Fig. 3i), L. rhabdostachyus (Fig. 5g), L. silvicolarum (Fig. 5n),
L. skanianus (Figs. 5o, 6p) and L. xizangensis (Fig. 6p).
Unicellular conical trichome (UC) This trichome type
also was uncommon and in the present study was detected
only in five species: L. quercifolius (Fig. 3c), L. konishii
(Fig. 3f), L. fordianus (Figs. 3i, 4c), L. cyrtocarpus (Fig. 4i)
and L. longzhouicus. It is not a typical conical trichome as
defined by Jones (1986) that was short and thick walled. The
wall of this trichome was thin and the whole trichome was
stained as revealed by the present study, indicating that it
might be a glandular type. The trichome bases were simple
and round and without cutinized cell wall (Fig. 3c, f, i).
Fasciculate trichomes Fasciculate trichomes (F) were
composed of 2–6(8) solitary structures that were joined
together at the base. This type of trichome existed in
19 species of Lithocarpus including L. haipinii (Fig. 3j);
L. eriobotryoides (Fig. 3l); L. uvariifolius (Fig. 4a); L. areca
(Fig. 3a) and L. xizangensis (Fig. 6p).
Stipitate fasciculate trichomes Stipitate fasciculate trichomes (SF) are very similar to fasciculate trichomes,
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674
Fig. 5 Characteristics of abaxial epidermis of APT group and
Lithocarpus densiflorus (LM). a, c–n, q, s, t, x bar 50 lm; b, o, r,
u, w bar 20 lm; a, b L. balansae; b high magnification of A, arrow
indicates TWP; c L. taitoensis; d L. pseudoreinwardtii; e L. microspermus; f. L. mianningensis; g L. rhabdostachyus; h L. petelotii;
i L. echinotholus; j L. carolinae; k L. rosthornii, arrow indicates
TWP: l L. elaeagnifolius; m L. lithocarpaeus; n L. silvicolarum;
o L. skanianus; p L. paihengii, the showing APT trichome bases;
123
M. Deng et al.
q, r L. macilentus; r the arrow indicates ALA and its dark stained
simple trichome base; s L. oleifolius, the arrow indicates ALA;
t L. glaber, showing BU; u L. pseudosundaica, the arrow indicates
TWP; v L. chrysocomus, showing the TWP with long stalk cells;
w L. calophyllus, showing SU. x Notholithocarpus densiflorus,
showing central large cyclocytic stomata and other anomocytic
stomata
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Comparative morphology of leaf epidermis
675
Fig. 6 Characteristics of abaxial epidermis of APT group (SEM)
and Notholithocarpus densiflorus, bar 20 lm. a L. balansae;
b L. petelotii; c L. pseudoreinwardtii; d L. guinieri; e L. silvicolarum;
f L. vestitus; g L. floccosus; h L. glaber; i L. laoticus; j L. confinis;
k L. litseifolius; l L. fenestratus; m L. pseudovestitus; n L. lepidocarpus; o L. paihengii; p L. skanianus; q L. calophyllus, showing
TWP; r L. lepidocarpus, showing TWP; s L. tabularis, showing dense
BU; t L. craibianus, showing well-developed TWP; u L. amoenus,
showing TWP; v L. handelianus, showing TWP; w, x Notholithocarpus densiflorus. X. the high magnification of X on the same slide,
showing SU and crystalline wax flake
except that in the former trichome type, only the lower
part of the trichome cells was fused (Figs. 3l, 5p).
Thirteen species of Lithocarpus were found to have this
trichome and the species possessing SF, usually have F
and S trichomes as well. The trichome bases of the S, F
and SF were similar and coexisted frequently; they may
represent the same trichome morphological stage in
Lithocarpus.
123
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123
Table 3 Comparison of leaf trichome types of various genera within Fagaceae
Trichome types
Taxa
Lithocarpus
Notholithocarpus
densiflorus
Chrysolepis
Castanopsis
Castanea
Quercus s.l.
Fagus
Trigonobalanus
s.l.
sect.
Quercus
s.s.
sect.
Protobalanus
sect.
Lobatae
sect.
Cerris
?2, 8(1)
?5, 7(1)
?2(1)
?2, 3(1)
?1, 2(1)
?6(1)
?2(1)
0
0
?2(1)
0
0
?7(1)
0
0
0
0
0
?2(1)
?2,
9
(1)
0
0
?6(1)
0
?2(1)
0
?2, 8(1)
0
?2, 8(1)
?5, 7(1)
?2(1)
?2, 3(1)
?1, 2(1)
0
0
0
0
0
0
?5, 7(1)
?2(1)
0
0
0
0
?*(1)
0
0
?2, 8(1)
0
?5, 7(1)
?2(1)
?2, 3(1)
?1, 2(1)
0
?2(1)
0
?*(1)
0
0
?8(1)
0
?5, 7(1)
?2(1)
0
?1, 2(1)
0
0
0
0
?*(1)
0
0
0
0
0
0
0
0
?2,
9
(1)
?2,
4
(1)
?2,
9
(1)
?2,
4
(1)
0
0
0
0
0
0
0
0
0
?2(1)
0
0
?2, 8(1)
0
0
0
0
?5, 7(1)
0
0
0
?3(1)
0
?2(1)
0
0
0
0
0
0
0
?*(1)
?*(1)
?*(1)
0
0
?2,
?2, 8(1)
0
0
0
0
0
0
0
?*(1)
0
0
0
0
0
0
0
0
0
0
0
0
0
?*(1)
0
0
0
0
0
0
0
0
0
0
0
0
?2(1)
?7(1)
0
0
?2, 9(1)
?3(1)
?2(1)
?2(1)
?1, 2(1)
0
0
0
0
Simple uniseriate
?*(1)
?*(1)
?*(1)
?2,*(1)
0
0
?2, 8(1)
?2,
?2, 9(1)
?2,
?6(1)
?2, 5(1)
Branched
uniseriate
Glandular peltate
Papillae-global
thickening
Jelly fish-like
0
0
?*(1)
0
0
0
0
0
?2(1)
?2(1)
0
?2(1)
?2, 4,
9
(1)
?2,
9
(1)
?2(1)
0
?2(1)
0
0
0
0
0
?*(1)
0
0
0
0
0
0
0
0
0
?2,
0
0
0
0
0
0
0
0
0
0
?2, 5(1)
?2(1)
0
0
0
0
0
0
0
?5, 7(1)
0
0
0
0
0
0
Glabrous
APT
Simple solitary
?*(1)
0
?*(1)
0
?8(1)
?2, 8(1)
Unicellar conical
Appressed
laterally
attached
Stellate
?*(1)
0
0
0
0
?*(1)
0
0
0
0
?*(1)
0
0
0
Fused stellate
0
0
0
Fasciculate
?*(1)
0
Stipitate
fasciculate
Appressed
parallel tuft
Multiradiate
Thick walled
peltate
Thin-walled
peltate
Broad base
trichome
Rosulate
Capitate
?*(1)
8
(1)
5, 7
(1)
5, 7
(1)
3, 9
(1)
?1, 2(1)
M. Deng et al.
19 trichome types are mapped onto the phylogenetic cladogram (Oh and Manos 2008) with character states are presented in the circles (Fig. 7)
Data sources: * current study, 1Hardin (a, b), 2Jones (1986), 3(Manos 1992), 4Zhou and Wilkinson (1995), 5Lou and Zhou (2001), 6Denk (2003), 7Deng (2007), 8Liu et al. (2009), 9Tschan and Denk (2012)
Stage 0, absent; 1, present
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subg
Cyclobalanopsis
BBT
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Comparative morphology of leaf epidermis
Trichome bases Trichome bases were scattered on the
abaxial epidermis in all the species. Based on their morphology, they can be placed under four different categories:
(1) Simple trichome base In this category, the cell wall of
the basal portion was usually more or less cutinized and
stained darker than the epidermal cells. The epidermal cells
around the trichome base were unmodified (Fig. 2a–e, g–l);
AL, TWP, BBT, SU, BU, Ro and UC have this category of
trichome base. Usually, the basal portion is small (diam. ca.
9.3–14.4 lm) and thickly cutinized in AL, SU, and Ro, but
large (diam. ca. 15.3–25.6 lm) and less cutinized in TWP
and BBT; (2) Compound trichome base Compound trichome based cells were surrounded by 1–2 layers of thick
cutinized (dark stained) small-sized epidermal cells,
which gave the appearance of a ‘‘flower-like’’ structure
(Fig. 1e, k). Only St and multiradiate trichomes had this
type of trichome base; (3) Fasciculate trichome base The
trichome bases in this category were round with dark
stained walls and 1–2(3) rows of epidermal cells surrounding the trichome bases; these epidermal cells are
smaller than the normal epidermal cells (Figs. 1j, 3e, h, l;
5g, h, p); SU, F and SF have this type of trichome base;
(4) APT trichome base This trichome base type was distributed adjacent to the subsidiary cells. The basal portion
was small convex, swollen and not obviously cutinized.
These trichome bases usually surrounded 2–16(18) stomata. In some extreme types, the convex basal portion
formed a ring surrounding the stomata (Fig. 5m). Only
APT was observed to possess this type of trichome base in
the present study.
677
Wax flake
In all examined species of Lithcarpus, both the adaxial and
abaxial cuticles were covered with a thin to thick wax
flake. It is easy to detect the stomata and non-glandular
type trichomes (including APT, SU, S, F and SF) through
the wax flake by SEM, but SEM could not fully reveal the
key features of glandular and intermediate types since they
were soft and covered by wax flake, unless assisted by LM.
The wax flake was smooth (Figs. 2n–q, 4b–p, 5a–l) or
composed of irregular particles (Fig. 2r, s, u) in Lithocarpus, but the crystalline wax flake was only found in
N. densiflorus (Fig. 6w, x).
Evolutionary pattern of leaf epidermal features
in Fagaceae
The epidermal characters of each investigated species have
been summarized in Table 2. Nineteen leaf trichome types
of Fagaceae genera based on the present and previous
studies were compared (Table 3), and mapped on to the
molecular phylogeny cladogram of Oh and Manos (2008)
(Fig. 7). The results show solitary trichomes are a plesiomorphism in Fagaceae; four characteristics were autapomorphic: APT, BBT to APT and BBT group respectively in
Lithocarpus, TWP to Chrysolepis, glandular peltate to
Trigonobalanus, Jellyfish-like to a species in Quercus
subg. Cyclobalanopsis. One characteristic was synapomorphic that the presence of multiradiate trichomes supports the clade N. densiflorus ? Quercus s.l.
Fig. 7 Character state mapping
of the 19 types of trichomes
among genera of Fagaceae
(Table 3, 1–19). Phylogram was
based on CRABS CLAW
sequences (Oh and Manos
2008). Solid circles are
autapomorphy or
synapomorphy, gray circles are
homoplasy. Characteristic
number is above the circle;
character stage is given in the
circle
123
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678
Discussion
Comparison of trichome types reported
from Lithocarpus in previous studies and the refinement
of terminology
Although the epidermal morphology is diverse among the
various Lithocarpus species, the characters shown are
reliable diagnostic features for identification purposes and
are consistent; nevertheless, the terminologies applied in
previous anatomical investigations cause great confusion
for purposes of comparison.
Trichome morphology, having rich variations, has been
regarded as an important taxonomical characteristic in
Fagaceae. Jones (1986) recorded 13 trichome types in
Lithocarpus in which multiradiate trichomes were only
found in L. densiflorus (now Notholithocarpus densiflorus).
The rest of the trichome forms were detected in the present
study as well. Jones (1986) and Zhou and Xia (2012)
recorded the presence of papillae and fused stellate trichomes in Lithocarpus. Based on a previous study, the
papillae were composed of a cutinized thickening of the
cuticle above the epidermal cells; therefore, this structure is
ornamentation on epidermal cells rather than a trichome
type (Deng 2007). Similar to Jones (1986) and Zhou and
Xia (2012), in the present study, many fused stellate-like
trichomes were detected in Lithocarpus under SEM
(Fig. 6c, o). A comparison of the sample slides by LM
revealed that these stellate-like trichomes were actually
composed of clustered coplanar APT with their hair-like
rays closely set together in the lower part (Fig. 6o). Their
trichome base is consistent with the typical APT with a
swollen, non-dark stained cell wall (Fig. 5p), which is not
similar to the typical stellate trichomes detected in Quercus
s.l. Therefore, based on the present investigation, we still
recognize this stellate-like trichome with typical APT trichome based as a modified APT.
Zhou and Xia (2012) recently reported ‘‘curly thinwalled unicellular trichomes’’ in L. macilentus and bulbous
trichomes in L. handelianus and L. amoenus. However, in
the present study, based on LM observation of the same
species, L. macilentus, the ‘‘curly thin-walled trichome’’,
was almost transparent, suggesting that this trichome is a
glandular type. The elongate membranous structures were
connected to each other above the dark stained simple
trichome base, the same as in the large TWP trichome
detected in Castanopsis by Liu et al. (2009) and Jones
(1986) and should be attributed to the TWP trichome.
Observations on the epidermis of L. handelianus and
L. amoenus under LM and SEM in the present study did not
detect any bulbous trichomes with ‘‘uniseriate stalks with a
single markedly enlarged terminal cell’’ as reported by
123
M. Deng et al.
Zhou and Xia (2012). Instead, a careful comparison of the
figures with those in Zhou and Xia (2012), showed that the
‘‘bulbous trichome’’ recorded by these authors was actually
a small TWP that was composed of a unicellular stalk with
an upper small skirt or flat discoidal cap (L. amoenus,
Fig. 6u; L. handelianus, Fig. 6v). This trichome was
equivalent to the ‘‘collapsed bulbous trichomes’’ in Castanea reported by Hardin and Johnson (1985). However, it
is easy to distinguish the elongated stalk cell and capitate
tip of bulbous trichomes, from a transparent, small free rim
membranous structure above the stalk in small TWP. This
type of small peltate trichome was not only detected in all
the species of Lithocarpus, but also in Castanopsis and
Castanea. However, the trichome types of species in BBT
and glabrous groups show great differences between the
current study and that of Zhou and Xia (2012), although on
the same species (L. hancei, L. harlandii, L. iteaphyllus,
L. corneus, L. konishii, L. naiadarum, L. quercifolius). Our
results show that TWP and SU were generally found in the
two groups, further more, species from BBT group have a
variety of trichome types, e.g., F, SF, St and BBT as well.
However, these obvious trichome types were not detected
by Zhou and Xia (2012). As a result, we believe that their
grouping and cladistic approaches based on trichome types
of Chinese Lithocarpus are problemetic as well.
Systematic and phylogenetic implications
The epidermal features show some degree of similarity as
well as differences among the genera of Fagaceae and
species of Lithocarpus; therefore, they can offer some clues
for the division of Lithocarpus.
Systematic status of Notholithocarpus densiflorus
N. densiflorus shared several epidermal features with the
genus Quercus s.l. Multiradiate trichomes, although not
detected in Lithocarpus in this study, but previously
recorded by Jones (1986), the small size and distinct subprolate shape of the pollen gain (Manos et al. 2008), the
scattered typical compound trichome bases and the crystalline wax flake on the epidermis were all found in
N. densiflorus and species of Quercus s.l., but not in
Lithocarpus. The similarities of epidermal features supported the closer relationship of N. densiflorus to Quercus
rather than to Lithocarpus which is confirmed by molecular
phylogenetic approaches that support the close relationship
of N. densiflorus to Quercus, Castanea, and Castanopsis
(Oh and Manos 2008). Remarkably, N. densiflorus has
amphitypical stomata. This feature is uncommon in Fagaceae. It also supports the findings of Manos et al. (2008)
that N. densiflorus represents a separate genus in Fagaceae.
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Comparative morphology of leaf epidermis
679
Intergeneric phylogeny in Fagaceae
BBT, UC, SU, Fa, SF and membranous relic of lower
periclinal and anticlinal wall of the guard cells were
shared among the species (including L. howii, L. corneus,
L. haipinii, L. konishii, L. fordianus, L. uvariifolius,
L. quercifolius, L. areca and L. longzhouicus). On the other
hand, all of these species possess a more or less serrated
leaf margin, and corrugated cotyledon. The species of this
group were regarded as ‘‘subgenus Cryptostylis’’ by Camus
(1934–1954), or ‘‘group of L. corneus’’ (Barnett 1944). The
highly consistent reproductive and foliar features suggest
that this group is a natural clade in Lithocarpus; (2)
Glabrous group This group has only TWP trichomes, with
large trichome bases ranging from 15.5 to 25.5 lm. Their
epidermal cells were flat and the anticlinal wall of the
epidermis was mostly straight-curved to round; (3) APT
group This group has the broadest species composition and
epidermal variations. Almost all kinds of epidermal features detected in Lithcarpus were present in this group,
except for the UC and sinuous anticlinal wall of the epidermal cells. The cupule and acorn morphology varied
from the cupule totally enclosing or half enclosing the
acorn, to enclosing the acorn only at the base. These
extremely diverse features, of both vegetative and reproductive morphology, indicate that this group possesses
well-developed and different strategies for adapting to the
highly heterogeneous environments that prevail in SE Asia.
Cannon and Manos (2003) sampled the representative
geographic ranges of Lithocarpus to study the phylogeography of the genus. Although their cladistic analysis did not
resolve the phylogeny in Lithocarpus, their study revealed
two major cpDNA clades with one clade restricted to
Borneo and another clade widespread. However, less is
known about epidermal features of the Lithocarpus species
distributed in Borneo. Cannon and Manos (2000) studied
the leaf epidermal features of four endemic Bornean
Lithocarpus from section Synaedrys. Their results showed
the four Bornean Lithocarpus species in section Synaedrys
all possess typical APT and TWP, which are consistent
with the trichome types in APT group in our study. Whether the Bornean species with cpDNA endemism also have
unique leaf epidermal features is still unknown and should
be studied in the future. Based on nuclear CRABS CLAW
sequences ML tree of Fagaceae, three clades were present
in Lithocarpus: (L. balansae ? L. laoticus ? grandifolius) ? [(L. corneus ? L. pachylepis) ? (L. dealbatus,
L. xylocarpus, etc.)] (Oh and Manos 2008). This cluster is
partly supported by epidermal features, that BBT
(L. corneus and L. pachylepis) and APT (e.g., L. dealbatus,
L. xylocarpus and L. silvicolarum) were clustered in different clades. The glabrous group species L. grandifolius,
APT group species L. laoticus and L. balansae, formed an
independent clade. The tree topology changed when ITS
sequences were added to the analysis (Oh and Manos 2008).
Castanopsis, Chrysolepis and Castanea share a distinct
cupule feature and were once placed together in Castanea
s.l. (Oersted 1871; Prantl 1894). The sister group relationship between Castanopsis and Castanea was supported
by wood anatomy (Lee 1968), pollen sculpture (Wang and
Chang 1991) and molecular phylogeny (Manos et al. 2001;
Oh and Manos 2008). Unexpectedly, Oh and Manos (2008)
showed that Chrysolepis and Lithocarpus (excl. L. densiflorus) form an independent clade. Based on epidermal
features, flat subsidiary cells were shared by the two genera. The trichome types in Chrysolepis are limited, except
for the unique thick-walled peltate as an autapomorphism,
SU and St are shared in most genera in Fagaceae. Leaf
epidermal features offered little information on the relationship of the two genera.
In the taxonomic system of Camus (1934–1954), Castanopsis ‘‘fissa-group’’ was mistakenly treated as the subgenus Pseudocastanopsis under the genus Lithocarpus.
With two notable reproductive features, the dichasiumcupule, and the inter-valve zones in the very young cupule
that distinguish it from Lithocarpus, most taxonomists
recognize ‘‘fissa-group’’ as a distinct taxon of Castanopsis
(Barnett 1944; Forman 1966; Nixon 1997; Huang et al.
1999). Comparison of epidermal features reveals the
presence of solitary, fasciculate and fused fasciculate trichomes in some species of Lithocarpus, ‘‘fissa group’’ and
some Castanopsis species. But these species in Lithocarpus
either have speckled TWP trichomes and/or broad-based
trichomes or distinct APT, in contrast to extremely welldeveloped TWP with a clear center in ‘‘fissa group’’ and
the species of Castanopsis. The subsidiary cell in ‘‘fissa
group’’ and other Castanopsis sepcies are thickened, but
flat in Lithocarpus (Liu et al. 2009). The trichome types
indicate that ‘‘fissa group’’ is closer to other Castanopsis
species rather than species in Lithocarpus.
In a recent taxonomical revision, Castanopsis longzhouica was transferred to Lithocarpus based on its unvalved cupule features (Chen et al. 2009). The epidermal
features of this species, sinuous anticlinal epidermal cell
walls, and F, BBT, and TWP the same as in the BBT group
in Lithocarpus, support its placement in Lithocarpus.
Therefore, the leaf epidermal features can usefully delimit
the species of Lithocarpus from those of Castanopsis.
Infrageneric phylogeny of Lithocarpus based on epidermal
features
The variation of epidermal features in Lithocarpus is quite
interesting. Three morphologically distinct groups were
revealed: (1) BBT group The species in this group have no
APT. The flat cuticle, sinuate anticlinal epidermal cells,
123
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680
Neither CRABS CLAW nor its combination with ITS sequence
can offer a robust resolution to Lithocarpus clades.
Leaf epidermal features have close correlations to
environmental factors (Haworth and McElwain 2008). The
variations of stomatal aperture, trichome morphology and
wax flake on the cuticle may be of ecological significance.
As a result, epidermal features are not only controlled by
their genetic bases but are also shaped by environmental
factors. Whether the three distinct epidermal groups have a
genetic base or a convergent evolutionary pattern to adapt
to environmental factors needs to be further explored with
finer phylogenetic resolution and analysis using climate
variations to reveal patterns of homoplasy.
Comparing the stomatal frequency of evergreen Castanopsis (229–516/mm2), Chrysolepis (416/mm2), Trigonobalanus s.l. (320/mm2) (Liu et al. 2009) and Quercus
(115–405/mm2) (Lou and Zhou 2001; Deng 2007) with that
of deciduous Fagus (315–677/mm2) (He et al. 2007),
Quercus (405–682/mm2) (Zhou and Wilkinson 1995) and
Castanea (476–726/mm2) (Liu et al. 2009), the deciduous
species mostly have higher stomatal frequency to facilitate
high CO2 assimilation.
Paleobotanical implications
Leaf cuticle features are useful in identifying fossil leaves
of Fagaceae. The sinuous anticlinal wall of epidermal cells
is widely detected in Fagaceae, especially in deciduous
taxa, such as Fagus, Quercus and Castanea (Jones 1986;
Liu et al. 2009), but this characteristic is also present in
some other evergreen taxa belonging to Lithocarpus,
Quercus and Castanopsis (Lou and Zhou 2001; Liu et al.
2009; Zhou and Xia 2012). Therefore, it is a homoplastic
feature, but can be applied at lower taxonomic levels for
identification purposes.
The trichome and trichome base types on the abaxial
surface were special and almost consistent, making them
important features in paleobotanical studies. APT in
Lithocarpus (Jones 1986; Uzunova et al. 1997), TWP
trichomes in Chrysolepis, glandular peltate trichomes in
Trigonobalanus, multiradiate trichomes in Quercus s.l. and
Notholithocarpus densiflorus are autapomorphic to these
genera and useful in identifying leaf fossils. For the species
in Lithocarpus without APT, their trichome bases are
restricted to broad trichome base and fasciculate trichome
base. Although the broad trichome bases were also found in
some species of Castanopsis and Quercus, they were
diversified and always combined with small simple trichome base with dark stained cell wall and/or compound
trichome base. These trichome base features can be easily
distinguished using cuticle samples and offer a good
diagnostic feature to accurately distinguish the species of
Lithocarpus from those of Castanopsis and Quercus.
123
M. Deng et al.
Stomatal features, including stomatal type, stomatal
size, and stomatal frequency can also assist in identifying
fossil leaves of Fagaceae. Cyclocytic stomata with flat
subsidiary cells were present in Lithocarpus and Quercus.
In Castanopsis, the subsidiary cells were thickened
according to Liu et al. (2009), but the range of stomatal
size in Lithocarpus (17.9–37.6 lm 9 16.0–35.5 lm) is
different from that of Quercus subg. Cyclobalanopsis
(10.2–20.4 lm 9 5.1–12.3 lm) (Lou and Zhou 2001;
Deng 2007). The stomatal size in Lithocarpus
(27.6 ± 8.2 lm 9 26.5 ± 9.1 lm) is larger than that in
Castanopsis (21.3 ± 4.6 lm 9 18.7 ± 5.5 lm), although
their stomatal frequency was similar (213–574/mm2 in Lithocarpus and 229–516/mm2 in Castanopsis) (Liu et al. 2009).
Acknowledgments We thank Mr. Allen Coombes of the Herbarium
and Botanic Garden of the University of Puebla and Professor Arshad
Ali of University of Florida for their kindest help in correcting the
language of the manuscript. Gratitude is expressed to the curators of
KUN, IBK, CSH and SWFC for providing specimens. This work was
supported by grants from the National Natural Science Foundation of
China (31100154 and 31270267); Shanghai Municipal Natural Science Foundation (11ZR1435500); the Shanghai Municipal Administration of Forestation and City Appearances (F112419), and the
Innovation Program of Shanghai Municipal Education Commission
(12YZ157).
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