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 1 23 Your article is protected by copyright and all rights are held exclusively by SpringerVerlag Wien. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to selfarchive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy 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 123 Author's personal copy 660 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 123 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 123 Author's personal copy 662 M. Deng et al. 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 Author's personal copy 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 Author's personal copy 664 M. Deng et al. 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 Author's personal copy 75 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 Author's personal copy 99 100 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 669 123 Author's personal copy 670 M. Deng et al. 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 Author's personal copy Comparative morphology of leaf epidermis 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. 123 Author's personal copy 672 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- Author's personal copy Comparative morphology of leaf epidermis 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, 123 Author's personal copy 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 Author's personal copy 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 676 123 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 Author's personal copy subg Cyclobalanopsis BBT Author's personal copy 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 Author's personal copy 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. Author's personal copy 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 Author's personal copy 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). References Barnett EC (1944) Keys to the species groups of Quercus, Lithocarpus and Castanopsis of eastern Asia, with notes on their distribution. Trans Bot Soc Edinburgh 34:159–204 Camus A (1934–1954) Les Chênes monographie du genre Quercus (et Lithocarpus). Encyclopedie Economique de Sylviculture, vols 6–8. Academie des Sciences, Paris Cannon CH, Manos PS (2000) The Bornean Lithocarpus Bl. section Synaedrys (Lindl.) Barnett (Fagaceae): its circumscription and description of a new species. 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