Review of Palaeobotany and Palynology 237 (2017) 10–36

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Review of Palaeobotany and Palynology

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Leaf epidermal features of Quercus Group Ilex (Fagaceae) and their application to species identification

Min Deng a,b, Xiao-Long Jiang a,b, Yi-Gang Song a,b, Allen Coombes c, Xiao-Rui Yang a,b, Yan-Shi Xiong a,b, Qian-Sheng Li d,⁎ a Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai 201602, China b Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai 201602, China c Herbarium and Botanic Garden, Benemerita Universidad Autónoma de Puebla, Puebla 72000, Mexico d School of Ecology, Shanghai Institute of Technology, Shanghai 201418, China article info abstract

Article history: Leaf epidermal features are useful for elucidating the taxonomy and systematics of oak species. Quercus Group Received 23 April 2016 Ilex has a wide distribution mainly in subtropical areas of Eurasia with rich fossil records since the Oligocene, Received in revised form 18 November 2016 but no comprehensive study has ever been conducted on its diversity of leaf epidermal features. We compared Accepted 18 November 2016 the leaf epidermal features of 37 species of Quercus Group Ilex and Q. suber from Group Cerris using light micros- Available online 19 November 2016 copy (LM) and scanning electron microscopy (SEM). A total of five trichome types (including three glandular and two non-glandular types) and two trichome base types were found. Leaf epidermal features of Group Ilex show Keywords: Leaf anatomy typical xeromorphic features. The stomatal complexes are stephanocytic. The leaf trichome types and trichome Eurasian sclerophyllous oaks base types are stable at inter- and intra-species levels. The trichomes, stomatal sizes and density and the ray Taxonomy arm lengths show a wide variation during developing stages within species. The leaf epidermal features show Systematics a highly convergent pattern in the genus Quercus based on tree-mapping analysis, except for the multiple epider- mal layers on the adaxial surface supports the monophyletic origin of section Heterobalanus. Cluster analysis of leaf epidermal characters infers two morphological subgroups within Group Ilex: (1) the capitate and (2) the branched uniseriate. The trichomes and trichome base types, and their density are useful for species identifica- tion, which can improve the accuracy of assigning fossil leaves to their nearest living relatives in Group Ilex. © 2016 Published by Elsevier B.V.

1. Introduction sections/groups. Molecular phylogenies based on nuclear ribosomal spacer (ITS1 and ITS2; 5S-IGS) and part of the CRABS CLAW gene Quercus L., the largest genus of Fagaceae with 400–500 species are (CRC) recognized two main clades and 5–6groupsofthegenus. widely distributed throughout much of the Northern Hemisphere and Corresponding to their main distributions, these two major clades extending into northern South America and Indonesia (Camus, 1934– were referred to as “the Old World Clade” (incl. Groups (Cerris, Ilex), 1954; Nixon, 1989, 1993; Govaerts and Frodin, 1998). Because these Cyclobalanopsis) vs. “the New World Clade” (incl. Groups (Lobatae, dominant woody species have important ecological and economic Quercus), Protobalanus) (Manos and Stanford, 2001; Oh and Manos, value, the phylogeny of Quercus s.l. has been studied continuously for 2008; Denk and Grimm, 2010). These results partly agreed with previ- many years. The intrageneric classification of the genus has been the ous classification systems of Camus (1934–1954), Schwarz (1936) and subject of considerable changes (Ørsted, 1871; Camus, 1934–1954; Menitsky (1984). However, morphological traits are highly homoplastic Schwarz, 1936; Menitsky, 1984; Nixon, 1993). Although groups of spe- in oaks and taxonomic formalization of these groups is still pending cies were given different names and ranks in different taxonomic sys- (Hubert et al., 2014). tems, these systems still roughly recognized the same major groups The leaf epidermal features show rich variations in oaks and have (Denk and Grimm, 2010). Previous phylogenetic approaches based on been widely applied to oak species delimitation (Camus, 1934–1954; Sanger sequencing markers were inconclusive for the key groups, but Menitsky, 1984), systematic placement of species and even for hybrid these results have sketched the species composition of the main identification (Jones, 1986; Zhou et al., 1995; Bussotti and Grossoni, 1997; Fortini et al., 2009; Deng et al., 2014; Song et al., 2015). Among many leaf epidermal features, foliar trichomes were studied most thor- Abbreviations: U, uniseriate trichome; BU, branched uniseriate trichome; Ca, capitate oughly. For “the New World Clade”, about ten to twelve different tri- trichome; ALA, appressed lateral attached trichome. ⁎ Corresponding author. chome types were defined based on glandulosity, attachment and ray E-mail address: [email protected] (Q.-S. Li). morphology based on scanning electron microscopic (SEM) and light

http://dx.doi.org/10.1016/j.revpalbo.2016.11.006 0034-6667/© 2016 Published by Elsevier B.V. M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 11 microscopic studies (LM) (Hardin, 1976; Hardin, 1979; Manos, 1993). In 15 Ma) (Li and Guo, 1976), and the Fudong flora (Tao, 1986)and “the Old World Clade”,10–13 trichome types were detected, with four Longmen flora (Su et al., 2013) of West China (Pliocene); Q. pannosa trichome types in Group Cerris, nine in Group Cyclobalanopsis and (Pliocene) (Xiao et al., 2006) and Q. mutilatifolia (late Miocene) (Guo, eight to nine in Group Ilex. However, it is difficult to delimit oak groups 2011) from western Yunnan; and Q. protospathulata (Pliocene) (Guo, using only foliar trichome features, because foliar trichome traits 1978) from Sichuan. Meanwhile, the biogeography of Group Ilex in evolved independently in different oak lineages. East Asia is also interesting, as the fossils of this group were found at Other leaf epidermal features with great variation at the inter- and high elevations of the Himalayas, where species of Group Ilex no longer intra-species level are also helpful in species identification, such as the occur. Using these fossils as palaeoenvironmental indicators and by papillae or globe-like thickenings on the cuticle, epicuticular waxes, sto- comparing their distribution and habitats to those of their nearest living mata types, trichome bases, anticlinal wall patterns of epidermal cells, relative, estimates can be made of the palaeoelevation heights of the Hi- trichome base types, and stomatal apparatuses (Jones, 1984; He et al., malayan mountains at different geological times as well as of the atmo-

1994; Liu et al., 2009; Deng et al., 2014; Deng et al., 2015). However, spheric CO2 concentration history (Zhou, 1992; Zhou et al., 2003; Xiao et these leaf epidermal features are not well recorded and documented al., 2006; Li et al., 2009; Su et al., 2013; Li et al., 2015). However, such in a large number of oaks. Furthermore, the different terminology ap- palaeoclimatic/palaeoaltitude reconstructions have a potential risk, as plied to the leaf epidermal features also makes it very difficult to com- they are only meaningful when the fossil identification is accurate. Fur- pare published accounts of the main taxa. For example, the papillae thermore, in addition to leaf epidermal morphological convergence of thickenings on the cuticle of the abaxial leaf surface were defined as a species from different habitats, also great leaf morphological variation trichome type (Luo and Zhou, 2001). It is difficult to distinguish at the intraspecific level was reported in previous studies (Yang et al., anomocytic and cyclocytic stomata in different studies, as the stomata 2012; Hu et al., 2015; Jiang et al., 2015), but these variations have not are concentrated at the areoles and the subsidiary cells are shared by ad- been assessed at the interspecific level. Therefore, the accurate assign- jacent stomata. Thus, further efforts to standardize the current termi- ment of fossil oak leaves to their nearest living relative and the amount nology of leaf epidermal features in oaks are essential for comparing of leaf morphological variation at inter- and intra-species levels are sub- the published data. jects of ongoing research. Quercus Group Ilex, also known as Eurasian sclerophyllous oaks, In this study, a comprehensive review of the leaf epidermal features with about 30–37 species (Simeone et al., 2016), has been treated as of Quercus Group Ilex is presented and intraspecific morphological var- subgenus Heterobalanus in Menitsky's (1984) classification system. Its iations are compared using both LM and SEM. The objectives were to: distribution displays a more or less continuous range in Eurasia with (1) reveal the diversity pattern of leaf epidermal features of Group Ilex higher ecological and taxonomic diversity in the Himalayas and adja- and summarize the leaf epidermal features useful for identification of cent areas (Zhou, 1992; Denk and Grimm, 2010). The study based on extant and fossil taxa in this group; (2) test the stability of the key leaf ITS and 5S-IGS indicated the monophyletic status of Group Ilex and bet- epidermal features at intraspecific level (including different develop- ter illustrated its scale and species composition (Denk and Grimm, mental stages and different collection sites); (3) assess the phylogenetic 2010). The comparison of the leaf anatomical features of some species and taxonomic significance of leaf epidermal features. This work can in Group Ilex (e.g., stomatal shapes and arrangement, leaf epicuticular provide valuable leaf morphological traits for comparison, and it also waxes and trichome types) demonstrated that leaf epidermal features can improve the accuracy of identification of leaves of extant and fossil were useful for species delimitation in Group Ilex (Llamas et al., 1995; species in Group Ilex for future studies. Zhou et al., 1995; Bussotti and Grossoni, 1997). However, all these stud- ies were restricted to either a local scale or a limited number of taxa. 2. Material and methods There is currently no comprehensive survey of the leaf epidermal fea- tures of Group Ilex to address the possible systematics among the spe- 2.1. Material cies and to explore their utilization for species delimitation. Neogene fossils of Quercus s.l. are abundant all over the Northern Plant materials for this study were collected from 133 herbarium Hemisphere, e.g., leaves, acorns, woody remains and inflorescence re- specimens at BM, CSH, FM, K, KUN, P and SWFC, five living trees from mains; of which leaf fossils are found most frequently (e.g. Gregor, Arboretum de La Bergerette, and three living trees from Chenshan Bo- 1977; Borgardt, 1997; Ferguson et al., 1998; Borgardt and Pigg, 1999; tanical Garden, together representing 35 species of Group Ilex and one Blokhina et al., 2005; Grímsson et al., 2007; Denk et al., 2010; Cheng species of Group Cerris. All the information on the studied samples is et al., 2013; Xing et al., 2013; He et al., 2014; Jia et al., 2015). The iden- summarized in Table 1. All samples were examined using both LM and tification of leaf fossils and their assignment to intrageneric sections of SEM. The mounted slides were deposited in the herbarium of Shanghai Quercus is mainly based on the overall similarity of leaf characteristics, Chenshan Plant Sciences Research Center, Chinese Academy of Sciences, such as the venation pattern, leaf margins, teeth types and leaf lobes. China (CSH). Leaf epidermal features are not closely correlated with established major infrageneric taxonomic systems and are highly homoplastic 2.2. Sample preparation and measurement among the sections in Quercus (Jones, 1986). However, their rich diver- sity can potentially improve the accuracy of the systematic placement of Leaf epidermal material for LM examination was prepared from fossil taxa to their nearest living relative. Fossils related to Group Ilex mature leaves. Leaf segments about 1.0 × 1.0 cm were boiled in water have been reported from multiple sites in Eurasia (Guo, 1978; for 2 min to remove epicuticular wax, and then macerated overnight Uzunova et al., 1997; Palamarev and Tsenov, 2004; Xiao et al., 2006; Li (N12 h) in 1:1 (by volume) mixed solution of hydrogen peroxide (30%) et al., 2009; Velitzelos et al., 2014). Interestingly, although Europe is and glacial acetic acid at 60 °C. The cleared leaf cuticles were stained not the current diversity center of Group Ilex, a high diversity of fossils with 1% safranin-alcohol (m:v) (in 50% ethanol) for 5 h before mounting with affinities to modern East Asian Group Ilex species has been found in glycerin-jelly. Prepared cuticles were observed using a BX53 Olympus there. The reduced modern diversity of this group in Europe is likely microscope. The images were captured using a digital camera. caused by the bottleneck situation of the Cenozoic (Denk and Grimm, The materials (1.0 × 1.0 cm leaf segments) for SEM observation were 2010; Velitzelos et al., 2014). In East Asia, the fossil diversity center of directly mounted on stubs without any treatment and sputter-coated Group Ilex is located at the southeast fringe of the Qinghai-Tibet Plateau with gold. The specimens were examined and photographed with a in late Cenozoic strata, which almost overlaps its current species diver- Hitachi S-3400N SEM. The size of the trichome and trichome base, sity center (Zhou, 1992, 1999). For example, Q. preguyavaefolia fossils length and width of the stomata and the arm length of the trichomes were reported from the Namling flora in Tibet (middle Miocene, were measured under ×200 or ×400 magnification from five random 12 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36

Table 1 List of species, voucher collection localities and information used for the leaf epidermal study.

No. Species name Voucher specimens Collection information Herbarium for voucher

1 Q. acrodonta Deng, M., Song, Y.G. & Zou, Y.J. Tu-gou-zhai, Yuanshan county, Yunnan, China.GPS: 23°33′ CSH 5905 (DM5905) 04.30″N, 104°20′63.30″E, alt.1501 m, 20 Oct. 2013. 2 Q. acrodonta Deng, M. & Liu, Y.C. 546 (DL546) by the roadside of Long-tang village, Jia-yan, Yianshan county, CSH Yunnan, China, N 23.712836 N; 104.020461E, alt. 1550 m, 7 Nov. 2011. 3 Q. alnifolia DM3658 Living collection of Kew Gardens. CSH 4 Q. alnifolia DM8224 Living collection of Chevithorne Barton. 5 Q. alnifolia Walter, J. 7915 “Griechenland: Zypern: beim Troodos-Gebirge, zwischen FM Phini (Foini) u. dem Kloster Troodeitissa”, 25 June 1996. 6 Q. alnifolia DM13626 (living collection of The plant originated from Troodos mountains, Cyprus, NA Arboretum de La Bergerette, collected by Henry Edmunds. Accession LB98-0001) 7 Q. aquifolioides Ludlow, F., Sherriff, G. & Talyor, G. Molo, SE Tibet, Kongbo provnice, China. 28°57′N; 95°53′E. K 3626 8 Q. baloot Tackson S.A. & Burnell J. 4448 Palas, N Pakistan. Kabkot valley adjacent to Ilobek village en K (EPAK174) route to Bush and Barser. 35.00N; 73.00E; alt. 2250 m. 9 Q. baloot Wu, S.K. 01 N. Pakistan, Chilas, alt. 2600 m, Oct. 1989. KUN 10 Q. baloot DM13630 (Living collection of Collected by David Barnard and Shaun Haddock, 23rd October NA Arboretum de La Bergerette, 1995. Wild trees of only this species in graveyard near Accession LB95-0030) Marghazar, Saidu valley (tributary to Swat valley), Northwest Frontier Province, Pakistan (1250 m). 11 Q. baloot DM13629 (living collection of Collected by David Barnard and Shaun Haddock, 23rd October NA Arboretum de La Bergerette) 1995. Small wood of Q. baloot in Ushu valley above Kalam, (Accession LB95-0066) Swat valley, Northwest Frontier Province, Pakistan (2195 m). 12 Q. baronii Rock, J.F. 14950 SW Kansu, Lower Tebbu cuntry, Oct. 1926. K 13 Q. bawanglingensis Deng, M., Song, Y.G. & Wang, Y.S. top of the limestone hills, Er-Xian-Lin, Ba-Wang-Lin national CSH 3147 (DM3147) nature reserve, Changjiang, Hainan, China. 19°00.706′N, 109°06.963′E; alt. 1017 m, 21 June 2012. 14 Q. coccifera DM6576 Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 15 Q. coccifera DM6938 Arboretum Wespelaar, living collection, 2014/6/30 sd Snyers CSH 06100404, Road, agios lsidoros, Rhodes, GR. 16 Q. coccifera Coste, H. 2329 (FM_840912) Flore Franco-Helvetique, Aveyron: Vallee du Tarn, coteaux de FM Peyrelade pres Riviere; alt. 400 m—Localite unique dans l'Aveyron. 1 Jan. 1902. 17 Q. coccifera (var. calliprinos) Blanche, I. (FM_1473421) Herbier De Syrie 1854, N39. Versant oriental des collines de FM saint-Dimitry, a Beyrough. Fr. Oct. 1852. 18 Q. coccifera (var. calliprinos) DM6577 Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 19 Q. coccifera (var. calliprinos) DM6578 Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 20 Q. cocciferoides Zou, Y.J. & Du, C. 616 Cycas nature reserve, Pan-zhi-hua, Sichuan Province, China. CSH 22 Sept. 2014. On the open slope. 26°37′23.66″N, 101°35′ 5.82″E, alt. 1655 m. 21 Q. cocciferoides Hu, J.J. (PZH001) s.n. Pu-zhe-he, Qiu-Bei county, Yunnan, China, 24.136032N, CSH 104.114809E, alt. 1305 m, Dec. 2012. 22 Q. cocciferoides Cao, M. 2-334 Wang-de-si-shan, Wuding county, Kunming, Yunnan KUN Province, China. On open slope of the soil hills, 21Sept. 1998. 23 Q. cocciferoides Wang, H. et al. 03.0988 Ma-lu-liang-zi-she, Xiao-niu-lan villeage, Qiaojia county, KUN Yunnan, China. ele: 500-600 m. 20 Aug. 2003. 24 Q. cocciferoides Deng, M. & Zou, Y.J. 6468 On the way from Xing-pin to Shuangbai, Shui-tan villeage, CSH (DM6468) Xing-pin, Yunnan, China. 24.176961N, 101.512284E, 1760 m, 26 Oct. 2013. 25 Q. dolicholepis Henry, A. 6359 Changyang, Hubei Province, China, Mar. 1889. K 26 Q. dolicholepis Deng, M. 6642 (DM6642) Jiao-zi-shan, Luquan, Kunming, Yunnan, China; 25°39.1420′N; CSH 102°39.7510′E; alt. 1855 m, 22 Feb. 2014. 27 Q. dolicholepis Deng, M., Jiang, X.L. & Xiong, Y.S. Ye-Zhu-lin, Longlin county, Guangxi Province, 24°39′56.11″ N, CSH 10302 (DM10302) 105°11′11.66″E, alt.: 1495 m, China, 26 Aug. 2014. 28 Q. engleriana Deng, M. & Hu, J.J. 255 (DL255) Yu-po-ji, Mei-hua-shan national nature reserve, Shanghang CSH county, Fujian, China. 25.324725N; 116.822634E; ele: 1230 m, 9 Oct. 2011. 29 Q. engleriana Song, Y.G. & Betrisey, S. Jin-Fo-shan national nature reserve, Chong-qin, China, CSH (DM-7381) 29°02.103′N, 107°11.128′E, alt.: 2198 m, 2 June 2014. 30 Q. engleriana Deng, M. & Hu, J.J. 264 (DL264) Tian-bao-yan nature reserve, Yong-an, Fujian, China. On the CSH rocky open slope, 10 Oct. 2011. 31 Q. engleriana Du-long River Exped. 4871 “Ka-la-di”, Du-long-jiang county, Gongshan county, Yunnan, KUN China. Alt. 2380 m. 32 Q. floribunda Forrest, G. 11048 Yunnan, China, Nov. 1843. K 33 Q. floribunda Aitchison J.E.T. 12288-2 Kurrum valley, Afghanistan, Dec. 1897. K 34 Q. floribunda DM13628 (living collection of Collected by Shaun Haddock. Individual native trees in NA Arboretum de La Bergerette, pasture on outskirts of Kalam, Swat valley, Northwest Frontier Accession LB95-0035) Province, Pakistan (2050 m). 35 Q. franchetii Deng, M. 5922 (DM5922) Long-tan-zhai, Jia-Yi villeage, Yuan-shan county, Yunnan, CSH China. 23.494697N, 104.576301E, alt.: 1678 m, 20 Oct. 2013. M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 13

Table 1 (continued)

No. Species name Voucher specimens Collection information Herbarium for voucher

36 Q. franchetii Deng, M., Jiang, X.L. & Xiong, Y.S. “Mi-Ya-Jie”, on the way from Hua-shan villeage to Jiu-Jia CSH 9974 (DM9974) villeage; Rocky hill by Mo-Gan river, rare, 24°11′45.2″N; 101°11′07.9″E; alt.: 1171 m, 10 Aug. 2014. 37 Q. franchetii Deng, M. & Li. Q.S. 6953 (DM6953) from “Si-zi-shan” to “Guang-ying-dong”, Wuding, Yunnan, CSH China. In evergreen shruby land, 25°32′00.8″N 102°21′50.5″E, alt.: 1889 m, 28 July 2014. 38 Q. franchetii Deng, M., Jiang, X.L. & Xiong, Y.S. Qing-long-shang villeage, Yuan-jiang, Yunnan, China. On the CSH 7692 (DM7692) back rocky hill of the temple open slope. 23.756134N, 102.039102 1350E, alt.: 1350 m, 1 Aug. 2014. 39 Q. franchetii Deng, M., Jiang, X.L. & Xiong, Y.S. On the way from Xia-ba villeage to “Man-jian”, Shuangjiang, CSH 8930 (DM8930) Yunnan, China. On the open slope by the roadside, 23°30′ 52.43″N, 99°51′42.11″E, alt.: 1092 m, 6 Aug. 2014. 40 Q. franchetii Deng, M., Jiang, X.L. & Xiong, Y.S. By the Rd. G323 (from Kunsai to Dou-ge villeage), Lincan, CSH 9101 (DM9101) Yunnan, China. by the stream (backhill of hydropower station), 23°33′55.99″N, 100°07′06.54″E, alt.: 1475 m, 7 Aug. 2014. 41 Q. franchetii Deng, M., Jiang, X.L. & Xiong, Y.S. by the Rd. G214, from Mi-du to Jingdong, “La-er”, Mi-du, CSH 9763 (DM9763) Yunnan, China. On the open slope of hill by the roadside, 25°8′ 22.39 ″N, 100°35′48.33″E, alt.: 1555 m, 18 Aug. 2014. 42 Q. franchetii Deng, M., Coombes, A., Song, Y.G. Long-tan-zhai, Jia-Yi villeage, Yuan-shan, Yunnan, China. CSH & Zou, J. 5926 (DM5926) 23.494697N, 104.576301E, alt.: 1678 m, 20 Oct. 2013. 43 Q. franchetii Deng, M. & Zou, Y.J. 5943 On the way from Cuxiong to Zi-xi-shan, Cu-xiong, Yunnan, CSH (DM5943) China. On the open slope by the roadside, 24.995206N, 101.436681E, alt.: 2003, 23 Oct. 2013. 44 Q. franchetii Deng, M., Jiang, X.L. & Xiong, Y.S. On the way to from Er-shan to “Bai-yue-ding-shang-zhai”, CSH 7577 (DM7577) Er-shan, Yunnan, China. On the open slope by the roadside, 24°15′36″N, 102°11′40″E, alt.: 1329 m, 31 July 2014. 45 Q. franchetii Deng, M., Jiang, X.L. & Xiong, Y.S. Lin-Xiang district, by Rd. g323, Linchang, Yunnan, China. On the CSH 9191 (DM9191) west side of Langcang river, on the open slope by the roadside, 23°31′57.94″N, 100°09′21.91″E, alt.: 1117 m, 8 Aug. 2014. 46 Q. gilliana Wilson, E.H. 4583 W. Sichuan, Yunnan, China, Oct. 1910. K 47 Q. gilliana Zhong-dian expd. 640 Weng-bi-shan, Lijiang, Yunnan, China. 9 Aug. 1962. alt. KUN 2600 m, In the open shrubs. 48 Q. guyavifolia Deng, M. 6567 (DM6567) Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 49 Q. guyavifolia Hang, Y.F., Deng, K.M. & Chen, Y.R. Bai-sui-yin, Lijiang, Yunnan, China. In pine oak forest, alt.: PE 81-1337 3000 m, 24 July 1981. 50 Q. ilex DM6572 Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 51 Q. ilex Aweschoug, F.W. “Torri e'Olautt Jweii, 1855” (329934_FM). FM 52 Q. ilex (var. microphylla) DM6573 Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 53 Q. kingiana Scott C.W. 25 Rocky hills, Mandalay district, Burma, alt. 3500 ft. Sept. 1920. K 54 Q. kingiana Deng, M. Jiang, X.L. & Xiong, Y.S. Ma-li-ba, in the forest by the roadside, growing with Q. kerrii, CSH 8828 (DM8828) yellow soil, 22°38′53.45″N, 100°07′58.70″E; alt.: 969 m, 5 Aug. 2014. 55 Q. kingiana Deng, M. Jiang, X.L. & Xiong, Y.S. Ma-li-ba, in the forest by the roadside, growing with Q. kerrii, CSH 8829 (DM8829) yellow soil, 22°38′53.45″N, 100°07′58.70″E; alt.: 969 m, 5 Aug. 2014. 56 Q. lanata Deng, M., Jiang, X.L. & Xiong, Y.S. On the way from Da-li airport to Ji-zhu-shan, in the evergreen CSH 10659 (DM10659) shrubs by the road side, 25°49′08.827″N; 100°22′24.397″E; alt.: 1934 m, 18 Nov. 2014. 57 Q. lanata Ward, F.K. 6336 Payi, Tsangpo Gorge, Eastern Himalaya, Nov. 1925. K 58 Q. leucotrichophora Linda 10 Northern India 1831. K 59 Q. leucotrichophora DM6574 Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 60 Q. leucotrichophora DM6575 Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 61 Q. leucotrichophora DM13627 (Living collection of Collected by David Barnard. Native tree near White Palace NA Arboretum de La Bergerette, Hotel, Marghazar, Saidu valley (tributary to Swat valley), Accession LB95-0043) Northwest Frontier Province, Pakistan. 1280 m. With Alnus nepalensis and Salix spp. 62 Q. lodicosa Ward. F.K. 6355 Tsangpo gorge, Eastern Himalaya, alt. 6000 ft. K 63 Q. lodicosa Sun, H., Zhou, Z.K. & Yu, H.Y. 3820 “Gangde”, Jiu-da-mu-shan, Moutou county, Tibet, On the KUN slope, alt.: 2120 m, 22 Feb. 1993. 64 Q. longispica KDP s.n. (DM6327) “Da-Yu-Ba”, on the way from Loushui to Li-ge villeage, on the CSH slope by the roadside, Ning-lang, Yunnan, China. 27.729464N; 100.706584E; Alt. 2900 m. 12 Sept. 2013. 65 Q. longispica Deng, M. 10690 (DM10690) On the way from Xiao-shao villeage to Shuan-shao villeage, CSH Kunming, Yunnan Province, China; 25.222703N; 102.743233E; alt.: 1890 m, 4 Oct. 2014. 66 Q. marlipoensis Deng, M., Song, Y.G. & Zou, Y.J. “Huo-shao-liang-zi”, Xia-Jin-chuan villeage, Ma-li-po county, CSH 5865 (DM5865) Yunnan, China. In the evergreen forest, 23°08′50.91″N, 104°49′47.76″E, alt.: 1789 m, 19 Oct. 2013. 67 Q. marlipoensis Deng, M. & Liu, Y.C. 374 (DL374) “Chua-pai-lin-chang”, Xia-Jin-chuan villeage, Malipo county, CSH Yunnan, China. In the evergreen forest, 23.199171N, 104.801762E, alt.: 1789 m, 3 Nov. 2011.

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Table 1 (continued)

No. Species name Voucher specimens Collection information Herbarium for voucher

68 Q. marlipoensis Deng, M., Song, Y.G. & Zou, Y.J. “Huo-shao-liang-zi”, Xia-Jin-chuan villeage, Ma-li-po county, CSH 5866 (DM5866) Yunnan, China. In the evergreen forest, 23°08′50.91″N, 104°49′47.76″E, alt.: 1789 m, 19 Oct. 2013. 69 Q. marlipoensis (seedlings) Deng, M., Song, Y.G. & Zou, Y.J. “Huo-shao-liang-zi”, Xia-Jin-chuan villeage, Malipo county, CSH 5879 (DM5879) Yunnan Province, China. In the evergreen forest, 23°08′50.91″ N, 104°49′47.76″E, alt.: 1789 m, 19 Oct. 2013. 70 Q. marlipoensis (seedlings) Deng, M., Song, Y.G. & Zou, Y.J. “Huo-shao-liang-zi”, Xia-Jin-chuan villeage, Malipo county, CSH 5882 (DM5882) Yunnan Province, China. In the evergreen forest, 23°08′50.91″ N, 104°49′47.76″E, alt.: 1789 m, 19 Oct. 2013. 71 Q. marlipoensis (seedlings) Deng, M., Song, Y.G. & Zou, Y.J. “Huo-shao-liang-zi”, Xia-Jin-chuan villeage, Malipo county, CSH 5883 (DM5883) Yunnan Province, China. In the evergreen forest, 23°08′50.91″ N, 104°49′47.76″E, alt.: 1789 m, 19 Oct. 2013. 72 Q. monimotricha Lord Howick & W. McNamara – K 1389 73 Q. monimotricha Zhou, Z.K. 25 Wu-feng-shan, Zhong-dian county, Yunnan, China. in oak KUN shrubs by the roadside. Alt.: 3300 m, 28 Aug. 1988. 74 Q. oxyphylla Meng, J. & Li, X.P. s.n. (DM3472) Wu-yi-shan Mount., Wu-yi-shan, Fujian Province, China CSH 75 Q. oxyphylla Meng, J. & Li, X.P. s.n. (DM3474) Wu-yi-shan Mount., Wu-yi-shan, Fujian Province, China CSH 76 Q. oxyphylla Meng, J. & Li, X.P. s.n. (DM3481) Wu-yi-shan Mount., Wu-yi-shan, Fujian Province, China CSH 77 Q. pannosa Chen_2013-617-9 Lao-Jun-shan national park, Jian-chuan, Yunnan, China. By the CSH roadside open slope. 26.663619N, 99.658813E, alt.: 2890 m. 17 Sept. 2013. 78 Q. pannosa Hu, J.J. XZYN4-4 (DM4759) on the open slope, Close to “Fei-lai” temple, De-qin county, CSH Yunnan, China. 28°28′30″N, 98°50′59″E, alt.: 3053 m. 79 Q. pannosa Deng, M. 5315 (DM5315) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 80 Q. pannosa Deng, M. 5318 (DM5318) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 81 Q. pannosa Deng, M. 5323 (DM5323) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 82 Q. pannosa Deng, M. 5324 (DM5324) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 83 Q. pannosa Deng, M. 5331 (DM5331) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 84 Q. pannosa Deng, M. & Zou, Y.J. 5959 Jin-ding temple, Ji-Zu-shan, Binchuan, Yunnan, China. In the CSH (DM5959) subalpine shrubby land, 25.970589N, 100.359989E, alt.: 3680 m, 23 Oct. 2013. 85 Q. pannosa Deng, M. & Zou, Y.J. 5969 Jin-ding temple, Ji-Zu-shan, Binchuan, Yunnan, China. In the CSH (DM5969) subalpine shrubby land, 25.970589N, 100.359989E, alt.: 3680 m, 23 Oct. 2013. 86 Q. pannosa Song, Y.G. s.n. (DM8100)_seedling “De-zhe-din”, Zi-lin-gong villeage, Batang county, Sichuan CSH Province, China. 29°45′40.20″N, 99°01′20.68″E; alt.: 3192 m, 29 July 2014. 87 Q. pannosa Song, Y.G. s.n. (DM8100) “De-zhe-din”, Zi-lin-gong villeage, Batang county, Sichuan CSH Province, China. 29°45′40.20″N, 99°01′20.68″E; alt.: 3192 m, 29 July 2014. 88 Q. pannosa Deng, M. & Zou, Y.J. 6426 Close to Lian-tie villeage, Er-Yuan, Yunnan, China. On the CSH (DM6426)_(seedling1) open slope by the roadside. 25.937358N, 99.817583E, alt.: 3820 m, 25 Oct. 2013. 89 Q. pannosa Deng, M. & Zou, Y.J. 6426 Close to Lian-tie villeage, Er-Yuan, Yunnan, China. On the CSH (DM6426)_(seedling2) open slope by the roadside. 25.937358N, 99.817583E, alt.: 3820 m, 25 Oct. 2013. 90 Q. pannosa Li, X.P. et al. 9-15-1 (DM4670) Bai-sui-tai, Yue-Long Snow Mount., alt. 3880 m, Li-jiang, CSH Yunnan, China. 15 Sept. 2012. 91 Q. pannosa Hu, J.J.-XZYN1-2 (DM4741) on the slope by the roadside, Hong-la Mount., CSH Mang-kang, Tibet. 29°17′16″N, 98°41′34″E, alt.: 3774 m, 10 June 2012. 92 Q. pannosa Deng, M., Jiang, X.L. & Xiong, Y.S. Mo-pan-shan national foresty park, Xing-pin county, Yunnan, CSH 7600 (DM7600) China. Among shrubs at top of the mountain, 23.93970398N; 101.98670900E, alt.: 2463 m. 93 Q. × parkeri (Q. floribunda Parker, R.N. 3315 Dalhousie; Punjab Himalaya, Punjab, Oct. 13, 1934. K × Q. leucotrichophora) 94 Q. phillyreoides Li, Q.J. & Gao, X. s.n. (DM7889) Man-dang-shan, Nan-ping, Fujian Province, China. 30 Oct. CSH 2013, N 118°09′ E 26°69′; ele: 1059 m. 95 Q. phillyreoides Deng, M. & Liu, Y.C. 158 (DL158) At the top of Jiu-long Mount, Zhang-Jia-Jie national nature CSH reserve, Hunan, China. On the rocky slope, 29.373797N, 110.788461E; alt.: 554 m. 96 Q. phillyreoides Deng, M. & Li, Y. 11,089 on the way from Chuan-si villeage to Houpin villeage, Jian-O, CSH (DM11089) Fujian Province, China. 30 Nov. 2014. On the rocky slope by the river. 27.225173N; 118.820136E; alt. 125 m. 97 Q. rehderiana Meng, J. & Li, X.P. s.n. (DM4684) Yue-long Mt., Lijiang, Yunnan, China. On the open slope of CSH shrubby land. 98 Q. rehderiana Hu, J.J.-XZYN9-5 (DM4705) by the roadside, 3 km from “Er-di” tunnel to Xiao-zhong-dian, CSH Xiang-ge-li-la county, Yunnan, China. 27°20′51″N, 99°53′40″ E, alt.: 2959 m; 11 June 2012. M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 15

Table 1 (continued)

No. Species name Voucher specimens Collection information Herbarium for voucher

99 Q. rehderiana Heinr.Frh & Hand.-Mazz 518 “Prov. SETSCHWAN austro-occid.: in jugi Linbinkou 27°46′, K inter oppidum Yenyuen et castellum Kwapi, regione temperata”, alt.: ca 2800-2900 m, 3 June 1914. 100 Q. rehderiana Deng, M., Jiang, X.L. & Xiong, Y.S. on the way from An-Feng-Yin to Tie-chan, Yi-Meng county, CSH 7458 (DM7458) Yunnan, China. On the open slope, shruby land., 24°49′33″N, 102°51′07.75″E, alt.: 1797 m, 30 July 2014. 101 Q. rehderiana Deng, M. 10569 (DM10569) Tian-chi Nature Reserve, Yun-long county, Yunnan Province, CSH China. By the lake in the evergreen forest, 25°51′50.676″N; 99°18′23’623″E. alt.: 2411 m, 27 Sept. 2014. 102 Q. rehderiana Deng, M. & Zou, Y.J. 5971 “Jia-ye” Temple, Ji-Zu-shan, Bin-chuan, Yunnan, China. CSH (DM5971) 25.967792 N, 100.363228E, alt.: 2840 m, 23 Oct. 2013. 103 Q. rotundifolia Deng, M. 8240 (DM8240) Living collection of Chevithorne, UK. 1 July 2014. CSH 104 Q. semecarpifolia Forrest, G. 20184 “West China; N.E. of Atuntze”, N.W. Yunnan, China. 28°35 N, K 99°10E; alt.: 3658.0 m, Sept. 1921. 105 Q. semecarpifolia Deng, M. 5307 (DM5307) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 106 Q. semecarpifolia Deng, M. 5310 (DM5310) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 107 Q. semecarpifolia Deng, M. 5311 (DM5311) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 108 Q. semecarpifolia Deng, M. 5312 (DM5312) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492 ′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 109 Q. semecarpifolia Deng, M. 5313 (DM5313) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 110 Q. semecarpifolia Deng, M. 5438 (DM5438) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 111 Q. semecarpifolia Deng, M. 5437 (DM5437) Za-ri villeage, Longzi, Tibet. In the subalpine moss forest. CSH 28°36.3492′N, 93°24.1544′E, alt.: 2759 m, 7 Aug. 2013. 112 Q. semecarpifolia Deng, M. 5483 (DM5483) La-jiao valley, La-kang villeage, Lou-zha, Tibet. In the mixed CSH forest. 28°04.1335′N, 91°04.6218′E, alt.: 2861 m, 17 Aug. 2013. 113 Q. semecarpifolia Deng, M. 5527 (DM5527) Ka-jiu temple, La-kang villeage, Lou-zha, Tibet. In subalpine CSH mixed forest, 28°05.5983′N, 91°07.4189′E, alt.: 3958 m. 18 Aug. 2013. 114 Q. semecarpifolia SHCH2121 Jiu-zhai-gou, Sichuan, China, alt. 3400 m. SWFC 115 Q. senescens Deng, M. 8271 (DM8271) On the way from Tian-chi villeage to Tian-chi nature reserve, CSH Yun-long, Yunnan, China. In the evergreen forest by the roadside. 25°51′34.917″N, 99°19′41.058″E, alt.: 2289. 27 Sept. 2014. 116 Q. senescens Deng, M. 3354 (DM3354) On the way from Shuang-shao villeage to A-zi-yin villeage, on CSH the open slope by the roadside, 25°20′40.522″N; 102°45′ 40.671″E, alt.: 2238.93 m, 21 Sept. 2012. 117 Q. senescens Deng, M. 8250 (DM8250) On the way from Tian-chi villeage to Tian-chi nature reserve, CSH Yun-long, Yunnan, China. 118 Q. senescens Deng, M. 10599 (DM10599) Shun-dang villeage, Bai-shi town, Yunlong county, Yunnan CSH Province, China, 28 Sept. 2014. 119 Q. setulosa Poliane, M.E. 3891 Annam, Dran, pr. Hlang Hai, June 20, 1922. K 120 Q. setulosa Song, Y.G. & Li, Q.J. s.n. (DM4198) on the way from Liu-xiang river to Wu-zhi-shan, Jinxiu CSH county, Guangxi, China. On the top of the limestone hills, 23°14.785′N; 106°00.279′E; alt.: 1127 m, 30 Oct. 2012. 121 Q. spinosa DM8331 (Xie, D.Y. s.n.) Tai-lu-ge national foresty park, Hualian, Taiwan. Middle CSH mountain shruby lands. 23°59′24.1″N, 121°01′32.9″E. alt.: 3150 m, 10 Oct. 2014. 122 Q. spinosa Tian, D.K. 1830 (DM7164) San-qing-shan, Shang-rao, Jiangxi, China. 28 May 2014. 28°40′ CSH 12.31″N, 102°10′43.50″E; elev. 2019 m. 123 Q. spinosa Zou, Y.J. & Du, C. 478 On the slope by the roadside, “Xiao-ao villeage”, Hui-long CSH town, Mian-ning county, Sichuan, China. 17 Sept. 2014. 124 Q. suber DM6571 Arboretum des Pouyouleix Garden collections, France; 6 Dec. CSH 2013. 125 Q. suber DM6940 Arboretum Wespelaar, Grote Baan 63-B-3150 CSH Haacht-Wespelaar-Belgium (original living collection series 464/4219), 30 June 2014. 126 Q. tarokoensis Kirkham & Flanagan 107 5 kms east of Rosao on the central cross-island highway K above, Tienshiang, 24°13′N; 121°30′E, alt.: 1150 m. Oct. 15. 1992. 127 Q. tarokoensis DM8349 (Xie, D.Y. s.n.) from Xin-gan-shan to Dong-he-lu, Hua-lian, Taiwan. on the CSH limestone rocky slope by the roadside. 23°08′14.6″N, 121°17′ 47.4″E, alt. 316 m, 12 Oct. 2014. 128 Q. tarokoensis DM8335 (Xie, D.Y. s.n.) Yue-wang-ting, Tai-lu-ge national park, Hua-lian, Taiwan. on CSH the limestone rocky slope by the roadside. 24°10′41.6″N, 121°31′09.4″E, alt.: 353 m, 12 Oct. 2015. 129 Q. tatakaensis DM8227 Living collection of Chevithorne, UK. 1 July 2014. CSH 130 Q. tungmaiensis Su, T. & Meng, H.H. s.n. On the slope of Tungmai Bridge, Bo-Mi county, Tibet. CSH 131 Q. tungmaiensis DM8229 Living collection of Chevithorne Barton, UK. 1 July 2014. CSH 132 Q. tungmaiensis Su, T. & Meng, H.H. s.n. (DM6935) Tongmai bridge, Tibet. By the roadside very close to the CSH bridge, 30.095446N, 95.064200E, alt.: 2780 m, 1 June 2014.

(continued on next page) 16 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36

Table 1 (continued)

No. Species name Voucher specimens Collection information Herbarium for voucher

133 Q. tungmaiensis Su, T. & Meng, H.H. s.n. (DM6936) Tongmai bridge, Tibet. By the roadside very close to the CSH bridge, 30.095446N, 95.064200E, alt.: 2780 m, 1 June 2014. 134 Q. tungmaiensis Sun, H., Zhou, Z.K. & Yue, H.Y. Near Jiudangka villege, Jialasha, Moutuo, Tibet, alt.: 2000 m, KUN 3426 18 Feb. 1993. 135 Q. utilis Song, Y.G. 65 (DM6096) “Shi-shang-sheng-lin”, Mao-lan National Nature Reserve, CSH Li-bo, Guizhou, China. In the mixed forest, 25°17′16.51″N 107°56′38.35″E, alt. 977 m, 2 Nov. 2013. 136 Q. utilis Song, Y.G. s.n. (DM6097) “Si-shan” forest, Mao-lan national nature reserve, Li-bo, CSH Guizhou, China. At the top of the limestone hills. 25°17′16.51″ N, 107°56′38.35″E, alt.: 977 m, 2 Nov. 2013. 137 Q. utilis Deng, M. & Liu, Y.C. 376 (DL376) Jin-chan-cun, Xia-jin-chan villeage, Ma-li-po, Yunnan, China. CSH Open slope by the road side. 31 Oct. 2011. 138 Q. utilis Deng, M. & Lu, Y.C. 196 (DL196) Tian-tai-shan, Mangshan, Yizhang, Hunan, China. On the CSH rocky limestome area by the roadside, 28 Sept. 2011. 139 Q. yiwuensis Deng, M., Yang, S.T. & Li, P. 2040 Yao-dai RD, on the way from Pu-er to Yun-xian villeage, Pu-er, CSH (DM2040) Yunnan Province, China. In limestone evergreen forest by the roadside open slope. 22°54.606′N, 100°41.096′E; alt.: 1435 m, 17 Aug. 2011. 140 Q. yiwuensis Deng, M., Song, Y.G. Zou, Y.J. & On the way from Meng-lun to Yiwu, Mengla county, Yunnan, CSH Coombes, A. 5641 (DM5641) China. At the top of the limestone hills, 12 Oct. 2013. 141 Q. yiwuensis Deng, M. & Li, Q.J. 4639 (DM4639) Ravine of Nan-Mu river, Kun-han-xiao-zhai, Meng-yan CSH villeage, Menghai county, Yunnan, China. 22°18′59″N, 100°45′ 53″E. alt.; 847 m, 29 Dec. 2012.

field images in ImageJ (Schneider et al., 2012: http://rsbweb.nih.gov/ij/). per bootstrap replicate and MULTREES option in effect under the parsi- The description and terminology of trichomes and leaf epidermal cells mony criterion. mainly follows published accounts (Hardin, 1976, 1979; Jones, 1986; Tschan and Denk, 2012; Deng et al., 2014). The stomatal apparatuses 3. Results were classified according to Dilcher (1974) and Carpenter (2005).The fi classi cation and description of epicuticular wax structures follows Leaf epidermal features were found to show considerable diversity Barthlott et al.'s (1998) terminology system. at inter- and infra-specific levels. Some trichome types were present only at a specific developmental stage. The detailed leaf epidermal fea- 2.3. The evolutionary tendency of leaf epidermal features of Group Ilex tures for each specimen and the trichome dimensions are summarized in Table 2 (see also Supporting information, Table S1). The selected leaf epidermal morphological features of 15 species of Group Ilex and one species of Group Cerris (Quercus suber)werefrom 3.1. Leaf adaxial surface (Plate I,1–15) the present study. The epidermal cell layers of the adaxial leaf surface were from Zhou et al. (1995) and Karioti et al. (2011). The characters 3.1.1. Epidermal cells of three species (Q. glauca, Q. delavayi and Q. augustinii)ofGroup Epidermal cells on the adaxial surface are mostly irregular quadran- Cyclobalanopsis (Deng, 2007; Deng et al., 2014), Q. variabilis of Group gular to polygonal. Sinuous to undulate anticlinal walls with node thick- Cerris (Kim et al., 2011), Group Lobatae (Hardin, 1979), Group enings are generally present in species of Group Ilex (Plate I,1–13), Protobalanus (Manos, 1993) and Group Quercus (Manos, 1993; except for Quercus marlipoensis (Plate I, 14) and Q. alnifolia (Plate I,15). Bussotti and Grossoni, 1997; Tschan and Denk, 2012) were also includ- ed in the analysis. The states of the selected morphological characters were mainly coded as absent (0) or present (1). The characters and tax- 3.1.2. Trichome and trichome base types onomic matrix are shown in Table 3. The simplified cladogram of 15 Trichomes were only found on the adaxial surface in a few species species in Group Ilex, and other oaks from different groups were under SEM and LM. However, two trichome base types were commonly drawn manually according to Hipp et al. (2015) using Mesquite 2.75 found based on LM: (1) the single-celled trichome base (STB), present – (Maddison and Maddison, 2011). The ancestral statuses were recon- in most species (Plate I,2 9; 12, 15); (2) the compound trichome – structed using parsimony in Mesquite 2.75. base (CTB) (Plate I,10 14), which indicates the existence of the corre- sponding trichomes at an early stage of leaf development. Fasciculate- 2.4. Cluster and cladistic analysis of leaf epidermal features of Group Ilex stellate-like trichomes with compound trichome base (Plate I, 12) and other trichome types with single-celled trichome base (including The selected leaf epidermis morphological features were used for branched uniseriate, uniseriate and capitate trichomes) were also cluster and cladistic analysis. The states of these characters were mostly found on the adaxial surface. The detailed documentation and morphol- coded as absent (0) or present (1). The characters and taxonomic matrix ogy of each trichome type are summarized below. are shown in Table 3. The dissimilarity matrix was calculated based on the method of Jaccard. Hierarchical clustering for the investigated taxa 3.2. Abaxial leaf surface was performed in R v3.2.2 (https://www.r-project.org/). The morphological cladistic analysis was performed in PAUP 4.0b10 3.2.1. Trichomes and trichome base types (Swofford, 2002) based on the maximum parsimony method. All the characters had equal weights. Quercus glauca of Group Cyclobalanopsis 3.2.1.1. Trichome types. Foliar trichomes were classified into two groups, reported in Deng et al. (2014) was used as outgroup to root the tree. i.e. glandular and non-glandular types. Three glandular trichome types Heuristic searches were run with default options. Bootstrap and two non-glandular trichome types of which two subtypes can be (Felsenstein, 1985) resampling was performed (1000 replicates) by distinguished. The detailed morphological characters of the trichomes using TBR branch-swapping on 100 random taxon-addition replicates are described below. Table 2 Leaf epidermal features of Quercus Group Ilex in present study.

Species name Subgroup/species Voucher specimens Adaxial Abaxial complex T type TB Anti wall Wax Anti wall S FF-St U BU Ca TB Stomata type Contact cell Stomata density type (CTB) color

1 Q. acrodonta Ca/Ilex DM5905 CTB, STB sin-cur/node sm cur-un-sin/node St Y CTB, STB Anomocytic N 212.01 ± 22.04 2 Q. acrodonta Ca/Ilex DL546 CTB, STB sin-cur/node sm cur-un-sin/node St Y CTB, STB Anomocytic N 229.43 ± 18.01 3 Q. alnifolia BU/pannosa DM3658 STB str-cur/uni sm cur-un-sin/uni St Y CTB, STB Cyclocytic Dark 523.87 ± 10.08 4 Q. alnifolia BU/pannosa DM8224 STB str-cur/uni sm cur-un-sin/uni St Y CTB, STB Cyclocytic Dark 489.88 ± 20.5 5 Q. alnifolia BU/pannosa Walter, J. 7915 STB str-cur/uni sm cur-un-sin/uni St Y CTB, STB Cyclocytic Dark 440.85 ± 12.45 6 Q. alnifolia BU/pannosa DM13626 STB str-cur/uni sm cur-un-sin/uni St Y CTB, STB Cyclocytic Dark 460.87 ± 12.67 7 Q. aquifolioides BU/pannosa Ludlow, F., Sherriff, G. FF-St CTB, STB sin-un/node sm un/node FF-St Y CTB, STB Anomocytic N 210.96 ± 19.26 & Talyor, G. 3626 8 Q. baloot Ca/Ilex Tackson S.A. & Burnell FF-St CTB cur-sin/node cross str-cur-un/uni St Y CTB Cyclocytic N 197.77 ± 10.1 J. 4448 (EPAK174) (non-conact cell dark) 9 Q. baloot Ca/Ilex Wu, S.K. 01 FF-St CTB str-cur/uni cross str-cur/uni St Y CTB, STB Cyclocytic N 150.65 ± 28.45

(non-conact 10 (2017) 237 Palynology and Palaeobotany of Review / al. et Deng M. cell dark) 10 Q. baloot Ca/Ilex DM13630 CTB str/uni cross str-cur/uni St Y CTB, STB Cyclocytic N 160.76 ± 30.21 (non-conact cell dark) 11 Q. baloot Ca/Ilex DM13629 CTB str/uni cross str-cur/uni St Y CTB, STB Cyclocytic N 189.01 ± 21.67 (non-conact cell dark) 12 Q. baronii Ca/coccifera Rock, J.F. 14950 FF-St CTB, STB str-cur/node sm cur-un/node FF-St Y Y STB, STB Anomocytic N 450.96 ± 15.82 13 Q. bawanglingensis Ca/setulosa DM3147 STB sin-un/uni sm un-sin/node Y STB Cyclocytic N 450.44 ± 40.88 (non-conact cell dark) 14 Q. coccifera Ca/coccifera DM6576 CTB, STB sin-un/node sm cur/node FF-St Y CTB, STB Cyclocytic/anomocytic N 471.22 ± 26.33 (non-conact cell dark) 15 Q. coccifera Ca/coccifera DM6938 Ca CTB, STB sin-un-node sm cur/node FF-St Y CTB, STB Cyclocytic/anomocytic N 450.48 ± 22.33 (non-conact cell dark) 16 Q. coccifera Ca/coccifera Coste, H. 2329 CTB, STB sin-un/node sm str-cur-un/node FF-St Y CTB, STB Cyclocytic/anomocytic N 489.21 ± 20.78 (FM_840912) (non-conact cell dark) 17 Q. coccifera (var. Ca/coccifera Blanche, I. Ca CTB, STB sin-un/node sm str-cur-un/node Y Y CTB, STB Cyclocytic/anomocytic N 381.71 ± 61.63 calliprinos) (FM_1473421) (non-conact cell dark)

18 Q. coccifera (var. Ca/coccifera DM6577 CTB, STB sin-un/node sm str-cur-un/node Y CTB, STB Cyclocytic/anomocytic N 370.57 ± 23.66 – 36 calliprinos) (non-conact cell dark) 19 Q. coccifera (var. Ca/coccifera DM6578 CTB, STB sin-un-node sm str-cur-un/node Y CTB, STB Cyclocytic/anomocytic N 400.71 ± 32.61 calliprinos) (non-conact cell dark) 20 Q. cocciferoides Ca/coccifera Zou, Y.J. & Du, C. 616 CTB, STB un-cur/node sm str-cur-un/uni Y? CTB, STB Anomocytic N 771.67 ± 30.47 21 Q. cocciferoides Ca/coccifera Hu, J.J. (PZH001) s.n. FF-St CTB sin-un/node sm str-cur-un-sin/uni FF-St Y? CTB, STB Anomocytic N 780.95 ± 60.4 22 Q. cocciferoides Ca/coccifera Cao, M. 2-334 Ca, CTB, STB sin-un/node sm str-cur-un-sin/uni FF-St Y CTB, STB Cyclocytic/anomocytic N 901.73 ± 49.1 FF-St 23 Q. cocciferoides Ca/coccifera Wang, H. et al. CTB, STB sin-un/node sm cur-un/uni Y CTB, STB Anomocytic N 880.3 ± 50.10 03.0988 24 Q. cocciferoides Ca/coccifera DM6468 FF-St CTB, STB sin-un/node sm cur-un/uni Y FF-St Y CTB, STB Anomocytic N 780.43 ± 43.98 25 Q. dolicholepis Ca/kingiana Henry, A. 6359 CTB, STB str-cur-sin-un/uni sm cur-un-sin/uni FF-St Y CTB, STB Anomocytic N 265.88 ± 19.93 26 Q. dolicholepis Ca/kingiana DM6642 CTB, STB str-un-cur/uni sm cur-un-sin/uni FF-St Y CTB, STB Anomocytic N 347.49 ± 31.12 27 Q. dolicholepis Ca/kingiana DM10302 CTB, STB cur-un-sin/uni sm cur-un/uni FF-St Y CTB, STB Anomocytic N 499.49 ± 52.52 28 Q. engleriana BU/engleriana DL255 CTB, STB sin-un/uni sm cur-un/uni Y CTB, STB Cyclocytic Dark 450.59 ± 30.21 29 Q. engleriana BU/engleriana Song, Y.G. & Betrisey, BU CTB, STB str-cur-un/uni sm cur-un/uni Y CTB, STB Cyclocytic Dark 392.53 ± 32.58 S. s.n. (DM7381) 30 Q. engleriana BU/engleriana Deng, M. & Hu, J.J. 264 CTB, STB str-cur-un/uni sm cur-un/uni Y CTB, STB Cyclocytic Dark 440.67 ± 21.89

(DL264) 17

(continued on next page) 18 Table 2 (continued)

Species name Subgroup/species Voucher specimens Adaxial Abaxial complex T type TB Anti wall Wax Anti wall S FF-St U BU Ca TB Stomata type Contact cell Stomata density type (CTB) color

31 Q. engleriana BU/engleriana Du-long River Exped. STB str-cur-un/uni sm cur-un/uni FF-St Y CTB, STB Cyclocytic Dark 423.08 ± 32.76 4871 32 Q. floribunda BU/spinosa Forrest, G. 11048 CTB, STB sin-un/uni sm cur-un/uni FF-St Y Y CTB, STB Anomocytic N 321.66 ± 13.62 33 Q. floribunda BU/spinosa Aitchison J.E.T. CTB, STB un-cur/node sm str-cur-un/node CTB, STB Anomocytic N 295.27 ± 22.79 12288-2 34 Q. floribunda BU/spinosa DM13628 CTB, STB un-cur/node sm str-cur-un/node CTB, STB anomocytic N 260.88 ± 32.98 35 Q. franchetii Ca/Ilex DM5922 CTB, STB str-cur/node sm str-cur-un/uni-ridge FF-St Y Y CTB, STB Cyclocytic N 443.13 ± 20.88 36 Q. franchetii Ca/Ilex DM9974 CTB, STB str-cur/node sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic N 349.91 ± 29.52 37 Q. franchetii Ca/Ilex DM6953 FF-St CTB, STB cur-un/node sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic N 402.25 ± 21.91 38 Q. franchetii Ca/Ilex DM7692 FF-St CTB, STB str-cur-un/node sm cur-un/uni-ridge FF-St Y Y CTB, STB Cyclocytic N 450.22 ± 12.90 39 Q. franchetii Ca/Ilex DM8930 CTB, STB str-cur/node sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic N 339.49 ± 20.34 40 Q. franchetii Ca/Ilex DM9101 FF-St CTB, STB cur-un/node sm cur-un/uni-ridge FF-St Y Y CTB, STB Cyclocytic N 401.45 ± 23.56 41 Q. franchetii Ca/Ilex DM9763 FF-St CTB, STB cur-un/node sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic N 380.89 ± 22.21

42 Q. franchetii Ca/Ilex DM5926 CTB, STB cur-un/ridge sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic N 366.19 ± 23.45 10 (2017) 237 Palynology and Palaeobotany of Review / al. et Deng M. 43 Q. franchetii Ca/Ilex DM5943 FF-St CTB, STB cur-un/ridge sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic N 445.34 ± 20.66 44 Q. franchetii Ca/Ilex DM7577 CTB, STB str-cur/node sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic N 480.55 ± 24.78 45 Q. franchetii Ca/Ilex DM9191 CTB, STB str-cur/node sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic N 389.76 ± 20.56 46 Q. gilliana BU/spinosa Wilson, E.H. 4583 STB str-cur/node sm cur-un/node Y CTB, STB Cyclocytic/anomocytic Dark/N 630.05 ± 36.91 47 Q. gilliana BU/spinosa Zhong-dian expd. 640 STB str-cur-un/node sm cur-un/node Y CTB, STB Cyclocytic/anomocytic Dark/N 560.33 ± 40.33 48 Q. guyavifolia BU/pannosa DM6567 CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Cyclocytic/anomocytic Dark/N 574.66 ± 13.62 49 Q. guyavifolia BU/pannosa Hang, Y.F., Deng, K.M. CTB, STB cur-un-sin/node sm cur-un-sin/node FF-St Y CTB, STB Anomocytic N 442.84 ± 19.25 & Chen, Y.R. 81-1337 50 Q. ilex Ca/Ilex DM6572 St CTB, STB un-sin/node sm cur-un-sin/node St Y CTB, STB Cyclocytic/anomocytic Dark/N 450.73 ± 27.73 51 Q. ilex Ca/Ilex Aweschoug, F.W. St CTB, STB un-sin/uni sm cur-un-sin/node St Y CTB, STB Cyclocytic N 330.88 ± 36.05 52 Q. ilex (var. microphylla) Ca/Ilex DM6573 St CTB, STB cur-un-sin/node sm cur-un-sin/node St Y CTB, STB Cyclocytic Dark 316.29 ± 14.91 53 Q. kingiana Ca/kingiana Scott C.W. 25 fF CTB, STB cur-un/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 840.73 ± 15.81 54 Q. kingiana Ca/kingiana DM8828 fF CTB, STB cur-un/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 857.73 ± 20.45 55 Q. kingiana Ca/kingiana DM8829 fF CTB, STB cur-un/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 890.59 ± 22.9 56 Q. lanata Ca/Ilex DM10659 CTB, STB str-cur/node sm str-cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic Dark 420.71 ± 17.2 57 Q. lanata Ca/Ilex Ward, F.K. 6336 CTB, STB str-cur/node sm str-cur/uni-ridge FF-St Y CTB, STB Cyclocytic Dark 349.91 ± 29.52 58 Q. leucotrichophora Ca/Ilex Linda 10 CTB, STB str-cur/node sm cur-un-sin/node FF-St Y CTB, STB Cyclocytic Dark 402.25 ± 21.91 59 Q. leucotrichophora Ca/Ilex DM6574 CTB, STB sin-un/node sm cur-un-sin/node FF-St Y CTB, STB Cyclocytic Dark 356.02 ± 15.6.1 60 Q. leucotrichophora Ca/Ilex DM6575 CTB, STB sin-un/node sm cur-un-sin/node FF-St Y CTB, STB Anomocytic N 366.74 ± 20.43 61 Q. leucotrichophora Ca/Ilex DM13627 CTB, STB sin-un/node sm cur-un-sin/node FF-St Y CTB, STB Cyclocytic Dark 370.41 ± 25.60 62 Q. lodicosa Ca/Ilex F.K.W. 6355 CTB, STB cur-sin/node sm cur-un/node FF-St Y CTB, STB Cyclocytic Dark 362.38 ± 25.64 63 Q. lodicosa Ca/Ilex Sun, H., Zhou, Z.K. & CTB, STB str-cur/uni sm cur-un-sin/uni-ridge FF-St Y CTB, STB Cyclocytic Dark 411.72 ± 27.38 Yu, H.Y. 3820

64 Q. longispica BU/pannosa KDP s.n. (DM6327) FF-St CTB, STB cur-sin/node sm cur-un-sin/uni Y FF-St Y CTB, STB Anomocytic N 346.78 ± 21.99 – 36 65 Q. longispica BU/pannosa DM10690 FF-St CTB, STB cur-sin/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 387.93 ± 40.61 66 Q. marlipoensis BU/engleriana DM5865 CTB, STB str-cur/uni sm cur-un-sin/uni FF-St Y CTB, STB Anomocytic N 469.1 ± 13.61 67 Q. marlipoensis BU/engleriana DL374 CTB, STB str-cur/uni sm cur-un-sin/uni FF-St Y CTB, STB Anomocytic N 478.20 ± 20.56 68 Q. marlipoensis BU/engleriana DM5866 fF CTB, STB str-cur/uni sm cur-un-sin/uni FF-St Y CTB, STB Anomocytic N 450.29 ± 24.67 69 Q. marlipoensis BU/engleriana DM5879 fF CTB, STB str-cur/uni sm cur-un-sin/uni FF-St Y CTB, STB Anomocytic N 359.45 ± 21.89 (seedlings) 70 Q. marlipoensis BU/engleriana DM5882 CTB, STB str-cur/uni sm cur-un-sin/uni FF-St Y CTB, STB Anomocytic N 378.96 ± 22.01 (seedlings) 71 Q. marlipoensis BU/engleriana DM5883 fF, BU CTB, STB str-cur-un/uni sm cur-un-sin/uni FF-St Y CTB, STB Anomocytic N 354.82 ± 20.44 (seedlings) 72 Q. monimotricha BU/pannosa Lord Howick & W.Mc CTB, STB cur-un-sin/node sm cur-un/node FF-St Y CTB, STB Cyclocytic/anomocytic N 239.81 ± 33.75 Namara 1389 73 Q. monimotricha BU/pannosa Zhou, Z.K. 25 CTB, STB cur-un-sin/node sm cur-un/node FF-St Y CTB, STB Cyclocytic/anomocytic N/dark 442.71 ± 47.91 74 Q. oxyphylla Ca/kingiana Meng, J. & Li, X.P. s.n. fF, Ca CTB, STB sin-un/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 548.1 ± 44.72 (DM3472) 75 Q. oxyphylla Ca/kingiana Meng, J. & Li, X.P. s.n. fF, Ca CTB, STB sin-un/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 526.99 ± 75.51 (DM3474) 76 Q. oxyphylla Ca/kingiana Meng, J. & Li, X.P. s.n. fF, Ca CTB, STB sin-un-node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 544.29 ± 45.66 (DM3481) 77 Q. pannosa BU/pannosa Chen_2013-617-9 CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Anomocytic N 414.83 ± 53.28 78 Q. pannosa BU/pannosa Hu, J.J. XZYN4-4 CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Anomocytic N 371.49 ± 34.82 (DM4759) 79 Q. pannosa BU/pannosa DM5315 Bu CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Anomocytic N 440.51 ± 40.56 80 Q. pannosa BU/pannosa DM5318 Bu CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Anomocytic N 479.91 ± 25.78 81 Q. pannosa BU/pannosa DM5323 FF-St, CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Anomocytic N 540.82 ± 22.78 Bu 82 Q. pannosa BU/pannosa DM5324 FF-St, CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Anomocytic N 570.32 ± 30.60 BU 83 Q. pannosa BU/pannosa DM5331 FF-St, CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Anomocytic N 480.32 ± 40.10 BU 84 Q. pannosa BU/pannosa DM5959 CTB, STB un-sin/node sm cur-un/node FF-St Y CTB, STB Anomocytic N 410.91 ± 20.31 85 Q. pannosa BU/pannosa DM5969 CTB, STB un-sin/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 448.92 ± 33.86 86 Q. pannosa BU/pannosa Song, Y.G. s.n. FF-St CTB, STB un-sin/node sm cur-un/uni-ridge FF-St Y CTB, STB Anomocytic N 420.41 ± 44.6 (DM8100)_(living collection_seedling) 87 Q. pannosa BU/pannosa Song, Y.G. CTB, STB un-sin/node sm cur-un-sin/node-ridge FF-St Y CTB, STB Cyclocytic/anomocytic N 512.93 ± 30.47 s.n._(DM8100) (non-conact cell dark) 88 Q. pannosa BU/pannosa DM6426_(living FF-St CTB, STB un-sin/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 371.49 ± 34.82

collection_seedling1) 10 (2017) 237 Palynology and Palaeobotany of Review / al. et Deng M. 89 Q. pannosa BU/pannosa DM6426_(living FF-St CTB, STB un/node sm cur-un/uni FF-St Y CTB, STB Anomocytic N 295.07 ± 19.24 collection_seedling2) 90 Q. pannosa BU/pannosa Li, X.P. et al. 9–15-1 CTB, STB un/node sm cur-uni/uni-ridge FF-St Y CTB, STB Anomocytic N 289.77 ± 29.01 (DM4670) 91 Q. pannosa BU/pannosa Hu, J.J. XZYN1-2 (DM CTB, STB un-sin/node sm cur-un/uni-ridge FF-St Y CTB, STB Cyclocytic/anomocytic N 295.07 ± 19.24 4741) 92 Q. pannosa BU/pannosa DM7600 CTB, STB sin/node sm cur-uni/uni-ridge FF-St Y CTB, STB Cyclocytic/anomocytic N 378.97 ± 34.67 93 Q. × parkeri Ca/coccifera Parker, R.N. 3315 CTB, STB un-sin/node sm str-cur-un/node Y CTB, STB Anomocytic N 558.5 ± 50.9 94 Q. phillyreoides Ca/setulosa Li, Q.J. & Gao, X. s.n. CTB, STB str-cur/uni sm cur/uni FF-St Y CTB, STB Anomocytic N 713.6 ± 52.53 (DM7889) 95 Q. phillyreoides Ca/setulosa DL158 CTB, STB str-cur/uni sm cur-un/uni Y CTB, STB Anomocytic N 566.33 ± 40.69 96 Q. phillyreoides Ca/setulosa DL11089 CTB, STB str-cur/node sm/cross cur-un/uni Y CTB, STB Anomocytic N 669.08 ± 77.76 97 Q. rehderiana BU/spinosa Meng, J. & Li, X.P. s.n. CTB, STB un-sin/node sn str-cur-un/node Y Y? CTB, STB Cyclocytic Dark 450.41 ± 23.37 (DM4684) 98 Q. rehderiana BU/spinosa Hu, J.J.-XZYN9-5 CTB, STB un/node sm str-cur-un/node Y Y? CTB, STB Cyclocytic/anomocytic Dark 323.96 ± 17.99 (DM4705) 99 Q. rehderiana BU/spinosa Heinr.Frh & CTB, STB sin/node sm cur-un-sin/node Y Y CTB, STB Cyclocytic Dark 147.49 ± 19.23 Hand.Mazz 518 100 Q. rehderiana BU/spinosa DM7458 CTB, STB sin/node sm cur-un-sin/node Y Y CTB, STB Cyclocytic Dark 431.92 ± 27.19 101 Q. rehderiana BU/spinosa DM10569 FF-St; CTB, STB sin/node sm cur-un-sin/node Y Y CTB, STB Cyclocytic Dark 410.56 ± 30.56 Ca 102 Q. rehderiana BU/spinosa DM5971 CTB, STB sin/node sm cur-un-sin/node Y Y CTB, STB Cyclocytic Dark 420.66 ± 22.84 103 Q. rotundifolia Ca/ilex DM8240 FF-St, CTB, STB str-cur-un/uni sm cur-un-sin/node Y FF-St Y CTB, STB Cyclocytic Dark 430.78 ± 30.51

Ca – 104 Q. semecarpifolia BU/pannosa Forrest, G. 20184 CTB, STB str-cur/uni sm cur-un/node Y CTB?, STB Cyclocytic N 437.16 ± 26.51 36 105 Q. semecarpifolia BU/pannosa DM5307 BU CTB, STB str-cur/uni sm cur-un/node FF-St Y CTB, STB Cyclocytic/anomocytic Dark 292.31 ± 30.25 106 Q. semecarpifolia BU/pannosa DM5310 CTB, STB str-cur/uni sm cur-un/node Y Y CTB, STB Cyclocytic Dark 363.4 ± 40.8 107 Q. semecarpifolia BU/pannosa DM5311 CTB, STB str-cur/uni sm cur-un/node Y CTB, STB Cyclocytic Dark 368.98 ± 34.71 108 Q. semecarpifolia BU/pannosa DM5312 FF-St, CTB, STB str-cur/uni sm cur-un/node FF-St Y CTB, STB Cyclocytic Dark 430.44 ± 29.05 BU, Ca 109 Q. semecarpifolia BU/pannosa DM5313 BU CTB, STB str-cur/uni sm cur-un/node FF-St Y CTB, STB Cyclocytic Dark 421.78 ± 22.88 110 Q. semecarpifolia BU/pannosa DM5438 CTB, STB str-cur/uni sm cur-un/node Y CTB, STB Cyclocytic Dark 390.56 ± 24.56 111 Q. semecarpifolia BU/pannosa DM5437 BU CTB, STB str-cur/uni sm cur-un/node FF-St Y CTB, STB Cyclocytic Dark 460.56 ± 19.73 112 Q. semecarpifolia BU/pannosa DM5483 BU CTB, STB str-cur/uni sm cur-un/node FF-St Y CTB, STB Cyclocytic Dark 440.56 ± 32.20 113 Q. semecarpifolia BU/pannosa DM5527 BU CTB, STB str-cur/uni sm cur-un/node FF-St Y CTB, STB Cyclocytic Dark 459.32 ± 31.44 114 Q. semecarpifolia BU/pannosa SHCH2121 CTB, STB str-cur/uni sm cur-un/node FF-St Y CTB, STB Cyclocytic Dark 390.69 ± 29.78 115 Q. senescens BU/pannosa DM8271 FF-St CTB, STB sin/node sm cur-un/uni FF-St Y CTB, STB Cyclocytic N 489.01 ± 19.78 116 Q. senescens BU/pannosa DM3354 CTB, STB sin/node sm cur-un/uni FF-St Y CTB, STB Cyclocytic N 442.38 ± 25.81 117 Q. senescens BU/pannosa DM8250 FF-St, CTB, U-STB sin/node sm cur-un/uni FF-St Y CTB, STB Cyclocytic N 450.12 ± 20.50 BU 118 Q. senescens BU/pannosa DM10599 FF-St, CTB, U-STB sin/node sm cur-un/uni FF-St Y CTB, STB Cyclocytic N 399.15 ± 23.00 Ca 119 Q. setulosa Ca/setulosa Poliane, M.E. 3891 NA un/cur/node sm cur-un-sin/uni Y STB Cyclocytic/anomocytic Dark 513.45 ± 31.46

(continued on next page) 19 20 Table 2 (continued)

Species name Subgroup/species Voucher specimens Adaxial Abaxial complex T type TB Anti wall Wax Anti wall S FF-St U BU Ca TB Stomata type Contact cell Stomata density type (CTB) color

120 Q. setulosa Ca/setulosa Song, Y.G. & Li, Q.J. s.n. NA un/cur/node sm; cur-un-sin/uni Y STB Cyclocytic Dark 530.55 ± 27.66 (DM4198) small cross 121 Q. spinosa BU/spinosa Xie, D.Y. s.n. CTB, STB str-cur-un/node sm cur-un-sin/uni Y STB Cyclocytic Dark 345.98 ± 23.91 (DM8331) 122 Q. spinosa BU/spinosa Tian, D.K. 1830 CTB, STB un-sin/node sm cur-un-sin/uni Y Y Y STB Cyclocytic Dark 369.99 ± 28.91 (DM7164) 123 Q. spinosa BU/spinosa Zou, Y.J. & Du, C. 478 CTB, STB un-sin/node sm cur-un-sin/node Y Y Y CTB, STB Cyclocytic Dark 440.67 ± 34.21 124 Q. suber Group Cerris DM6571 FF-St CTB, STB str-cur sm cur-un/uni Y FF-St Y CTB, STB Cyclocytic N 250.11 ± 23.90 125 Q. suber Group Cerris DM6940 CTB, STB str-cur sm cur-un/uni Y FF-St Y CTB, STB Cyclocytic N 212.56 ± 19.7 126 Q. tarokoensis Ca/setulosa Kirkham & Flanagan CTB, STB un-cur sm cur-un/node FF-St Y CTB, STB Cyclocytic/anomocytic Dark/N 400.21 ± 22.75 107 127 Q. tarokoensis Ca/setulosa DM8349 CTB, STB un/node sm cur-un-sin/uni Y FF-St Y CTB, STB Cyclocytic/anomocytic N 410.78 ± 34.01

128 Q. tarokoensis Ca/setulosa DM8335 CTB, STB un/node sm cur-un-sin/uni FF-St Y CTB, STB Cyclocytic/anomocytic Dark 399.78 ± 39.82 10 (2017) 237 Palynology and Palaeobotany of Review / al. et Deng M. 129 Q. tatakaensis Ca/setulosa DM8227 CTB, U-STB cur-un/node sm cur-un-sin/uni FF-St Y CTB, STB Cyclocytic/anomocytic N 397.89 ± 20.45 130 Q. tungmaiensis BU/engleriana Su, T. & Meng, H.H. CTB, STB un-sin sm un-sin/uni CTB (on Cyclocytic/anomocytic Dark 362.3 ± 25.60 s.n. main vein), STB 131 Q. tungmaiensis BU/engleriana DM8229 CTB, STB un-sin/uni sm un-sin/uni FF-St Y CTB (on Cyclocytic/anomocytic Dark 411.7 ± 27.40 main vein), STB 132 Q. tungmaiensis BU/engleriana Su, T. & Meng, H.H. U CTB, STB un-sin/uni sm un-sin/uni CTB (on Cyclocytic/anomocytic Dark 365.4 ± 24.60 s.n. (DM6935) main vein), STB 133 Q. tungmaiensis BU/engleriana Su, T. & Meng, H.H. CTB, STB un-sin/uni sm un-sin/uni CTB (on Cyclocytic/anomocytic Dark 366.3 ± 25.0 s.n. (DM6936) main vein), STB 134 Q. tungmaiensis BU/engleriana Sun, H., Zhou, Z.K. & CTB, STB un-sin/uni sm un-sin/uni CTB (on Cyclocytic/anomocytic Dark/N 443.1 ± 20.90 Yue, H.Y. 3426 main vein), STB 135 Q. utilis Ca/setulosa Song, Y.G. CTB, STB cur-un/node sm cur-un-sin/uni Y Y CTB, STB Cyclocytic/anomocytic N 418.57 ± 17.98 65(DM6096) 136 Q. utilis Ca/setulosa Song, Y.G. FF-St, CTB, STB sin-un-node sm cur-un-sin/uni Y Y CTB, STB Cyclocytic/anomocytic N 430.89 ± 39.90 s.n.(DM6097) U 137 Q. utilis Ca/setulosa DL376 CTB, STB sin-un-node sm cur-un-sin/uni Y Y CTB, STB Cyclocytic/anomocytic N 410.88 ± 20.70 138 Q. utilis Ca/setulosa DL196 CTB, STB sin-un-node sm cur-un-sin/uni Y CTB, STB Cyclocytic/anomocytic N 430.77 ± 32.09 139 Q. yiwuensis Ca/coccifera DM2040 fF CTB, STB sin-cur sm str-cur/uni FF-St Y CTB (rare), Anomocytic N 569.67 ± 45.01

STB – 36 140 Q. yiwuensis Ca/coccifera DM5641 fF CTB, STB sin-cur sm/rare str-cur/uni FF-St Y CTB (rare), Anomocytic N 588.52 ± 32.4 cross STB 141 Q. yiwuensis Ca/coccifera DM4639 fF CTB, STB sin-cur sm str-cur/uni FF-St Y CTB (rare), Anomocytic N 545.88 ± 40.40 STB

Abbreviation: S: solitary; fF: fused fasciculate; St: stellate; FF-St (CTB) = fused fasciculate-stellate with compound trichome base; U = uniseriate; BU = branched uniseriate; Ca = capitate; CTB = compound trichome base; STB = simple-celled trichome base; Y: present. Wax: Leaf epicuticular wax flake; sm = smooth; cross: crossed platelet; T type: trichome type; TB type: trichome base type; TB: trichome base. Anti wall: Anticlinal wall of leaf epidermal cells; str = straight; cur = curved; un = undulate; sin = sinuous; uni = uniform thickness; node: node-like thickening; ridge: ridge like thickening. Contact cell color: stomatal contact cell color; N = contact cell color same as ordinary leaf epidermal cells; Dark = contact cell color darker than ordinary leaf epidermal cells. M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 21

Plate I. Leaf adaxial surface under LM. bar = 50 μm; 1. Quercus floribunda (Aitchison, J.E.T. 12288-2); 2. Q. ilex (DM6572); 3. Q. coccifera var. calliprinos (DM6577); 4. Q. cocciferoides (Cao, M. 2-334); 5. Q. bawanglingensis (DM3147); 6. Q. dolicholepis (DM6642); 7. Q. leucotrichophora (DM6574); 8. Q. rehderiana (DM4705); 9. Q. aquifolioides (Ludlow, F., Sherriff, G. & Taylor, G. 3626); 10. Q. monimotricha (Zhou, Z.K. 25); 11. Q. rehderiana (DM5971); 12. Q. oxyphylla (DM3472); 13. Q. kingiana (Scott, C.W. 25); 14. Q. marlipoensis (DM5865); O. Q. alnifolia (DM3658). 22 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36

3.2.1.2. Glandular trichomes. I. The capitate type (Ca) (Plate II,1,3,5,6,9, smooth stalk cell left. In such cases, it is difficult to separate capitate and 11, 12; Plate III,4,8–10, 12, 14, 16; Plate IV,12;Plate VI,5;Plate VII,11, uniseriate trichomes. 17, 21) II. The uniseriate type (U) (Plate III,4,11;Plate VI,1,2,5) Description: Multicellular, uniseriate stalk and an enlarged head. Description: Thin-walled, multicellular trichomes with usually 3–8, The head is normally collapsed and wrinkled (Plate III,8,9,12,14,16; c. 44–181 μm long cells arranged in one row. The trichome base is sin- Plate VII, 11, 17, 21), distinct from the stalk. Its base is single-celled gle-celled. In the typical uniseriate trichome, the basal and distal cells (Plate II, 1, 3, 5, 6; 9, 11, 12). This trichome is equivalent to the bulbous are similar in size and diameter. type trichome of Hardin (1976). Remarks: In some species, the distal cell of the uniseriate trichome is Remarks: The trichome base type in capitate, uniseriate and the slightly enlarged, but the cell wall remains thin-walled and smooth are branched uniseriate trichomes are similar in size and generally stronger without wrinkles (Plate III,3;Plate V, 3). It represents an intermediate cutinized. The large head cell(s) are commonly collapsed, with only one type between the typical capitate and uniseriate trichomes, and

Plate II. Leaf abaxial epidermis of subgroup Engleriana under LM. 1–7: bar = 100 μm; 11–12: bar = 50 μm. 1. Quercus floribunda (Forrest, G. 11048); 2. Q. coccifera var. calliprinos (DM6577); 3. Q. cocciferoides (Zou, Y.J. & Du, C. 616); 4. Q. tarokoensis (Kirkham & Flanagan-107); 5. Q. dolicholepis (DM6642); 6. Q. utilis (DM6096); 7. Q. phillyreoides (DL158); 8. Q. baronii (Rock, J.F. 14950); 9. Q. kingiana (Scott, C.W. 25); 10. Q. oxyphylla (DM3472); 11. Q. bawanglinensis (DM3147); 12. Q. setulosa (Poilane,M.3891). M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 23

Plate III. Leaf abaxial epidermis under SEM of Ca subgroup. 1–6, 8–16, 18, 20 bar = 30 μm; 7, 17, 19: bar = 100 μm. 1. Quercus floribunda (DM13628); 2. Q. coccifera (DM6938); 3. Q. yiwuensis (DM2040); 4. Q. phillyreoides (DM11089); 5. Q. setulosa (DM4198); 6. Q. coccifera (DM6938); 7, 8. Q. cocciferoides (DM6468); 9. Q. tarokoensis (DM8335); 10. Q. tatakaensis (DM8227); 11. Q. utilis (DM6097); 12. Q. dolicholepis (Henry, A. 6359); 13 & 14. Q. oxyphylla (Meng et al. s.n. [DM3481]); 15 & 16. Q. kingiana (DM8828). The white arrows indicate BU, the white triangle indicates Ca. indicates the possible close ontogenetic relationship between these two stage of the branched uniseriate resembles the uniseriate in being al- trichome types. most linear, with only very short lateral arms. This similarity of the III. The branched uniseriate (BU) (Plate IV,3,5,7–9, 11; Plate V,5, two trichomes suggests their close ontogenetic relationship. In some 7–11; Plate VI,3–20) species, the branched uniseriate trichomes are only found at a certain Description: Thin-walled, multicellular trichomes. The arms of the developmental stage. When mature, they were sheared off with only trichome branch at least once. The arm length varies during different the trichome bases left on the epidermis, such as in Q. tungmaiensis developmental stages, and between species. In some species, the rays (Plate III, 13), Q. rehderiana, Q. spinosa and some individuals of Q. appear long and well developed, forming a dense, prominent hair engleriana (Plate IV, 9, 13) and Q. marlipoensis. layer (Plate IV,7–9, 11; Plate VI,12,14–15, 19). The arms of branched uniseriate in Quercus rehderiana and Q. spinosa and seedlings of Q. pannosa and Q. semecarpifolia are few (2–4 arms). The branched 3.2.1.3. Non-glandular trichomes. IV. The solitary type (S) (Plate III,7; uniseriate typically has a single-celled trichome base (Plate IV,5;V,3– Plate IV,11–13; Plate VII,19) 15). Description: Unicellular, single hair, straight, undulated, or twisted, Remarks: In seedlings of Quercus pannosa, the arm length of the protruding. The trichome base is mostly single-celled. The size of the branched uniseriate is much shorter and their density is much lower epidermal cells adjacent to the trichome is similar to that of other epi- than in leaves of adult trees (Plate VI,9–14). The abaxial surface of dermal cells. In some species the adjacent epidermal cells form an inflat- young seedlings is almost glabrous and glossy green, but in adult trees ed pedestal structure and are cutinized. The length of the trichome is covered by a layer of yellow or brown hairs. The early developmental varies from 260 to 420 μm. 24 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36

Plate IV. Leaf abaxial epidermis of subtropical species of BU and Ca subgroups. 1–6: LM; 7–10: SEM. 1–4: bar = 100 μm; 5–6bar=50μm; G–J. bar = 30 μm. BU: branched uniseriate; CTB: compound trichome base; ALA: appressed lateral attached trichome. 1. Quercus marlipoensis (DM5865); 2. Q. engleriana (DL255); 3. Q. engleriana (Du-long River Exped. 4871); 4. Q. tungmaiensis (DM6935); 5. Q. engleriana (DM7381); 6. Q. tungmaiensis (DM6936). 7. Q. marlipoensis (DM5883); 8. Q. engleriana (DM7381); 9. Q. engleriana (Do-long River Exped. 4871); 10. Q. tungmaiensis (Su, T. & Meng, H.H. s.n.). 11–13. Showing the Appressed lateral attached (ALA) trichome. The arrows indicate the attached point of ALA to the leaf epidermis. 11. Q. marlipoensis (DM5879, seedling), bar = 40 μm; 12. Q. coccifera var. calliprinos (Blanche, I. [FM_1473421]), bar = 100 μm; 13. Q. engleriana (DL255), bar = 40 μm.

Occurrence: Solitary trichomes were only found in six of the inves- parallel to the leaf surface and the attachment point can be either in tigated species in this study, i.e. Quercus engleriana, Q. longispica, Q. the middle or close to one end of the trichome. Although the evolution- marlipoensis, Q. rotundifolia, Q. suber, Q. tarokoensis. Trichomes are main- ary relationship between solitary and appressed lateral attached tri- ly found along the veins, occasionally in intercostal regions, but these chomes has not been addressed yet, the lateral might represent an trichomes mostly occur in juvenile leaves. extreme case of the solitary trichome, as both forms and transitions Remarks: The attachment point of the solitary trichome to the epi- can be found in one slide. dermal cell shows some variation. The solitary trichome is usually at- V. The fused fasciculate-stellate type (FF-St) (Plate I, 12; Plate III,6,7, tached to the epidermis more or less laterally, but the protruding 12, 13, 15; Plate IV,7;Plate V,7;Plate VI,7–10, 12–18, 20; Plate VII,1–21) solitary trichome is usually attached to the epidermis vertically. Jones Description: Multicellular, with 4 to 15(18) acicular arms that join (1986) reported the appressed lateral attached trichome (ALA) in at a common point. The individual arms are more or less fused at the Quercus, of which, the long axis of the trichome arm is completely base. Arm length is 90.8–694.5 μm. The basal part is attached to a M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 25

Plate V. Leaf abaxial epidermis of alpine and subalpine species of BU subgroup under LM. 1–10: bar = 100 μm; 11–15: bar = 50 μm; 1. Quercus rehderiana (Ward. F.K. 8305); 2. Q. rehderiana (Heinr.Frh & Hand.-Mazz. 518); 3. Q. monimotricha (Howick, L. & McNamara,W. 1389); 4, 11. Q. aquifolioides (Ludlow, F., Sherriff, G. & Taylor, G. 3626); 5, 12. Q. senescens (DM4737); 6, 13. Q. gilliana (Wilson, E.H. 4583 type); 7. Q. pannosa (DM4670); 8. Q. guyavifolia (DM6567); 9, 14. Q. longispica (DM6327); 10. Q. alnifolia (DM3658); 15. Q. semecarpifolia (Forrest, G. 20184). 26 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36

Plate VI. Leaf abaxial epidermis of alpine and subalpine species of the branched uniseriate (BU) subgroup under SEM. 1–4: bar = 30 μm; 5–20: bar = 100 μm. 1. Quercus rehderiana (DM10569); 2. Q. rehderiana (Meng, J. & Li, X.P. s.n. [DM4684]); 3. Q. senescens (DM8271); 4. Q. gilliana (Wilson, E.H. 4583); 5. Q. spinosa (DM7164); 6. Q. spinosa (Zou, Y.J. & Du, C. 478); 7. Q. senescens (DM8271); 8. Q. monimotricha (Lord Howick & W. McNamara 1389); 9. Q. pannosa (DM6426 [seedling1]); 10. Q. pannosa (DM6426 [seedling2]); 11. Q. pannosa (DM8100_seedling); 12. Q. pannosa (DM5324); 13. Q. pannosa (DM5969); 14. Q. pannosa (Deng, M. 4670); 15. Q. guyavifolia (Maire, E.E. s.n.); 16. Q. semecarpifolia (DM5307); 17. Q. semecarpifolia (DM5312); 18. Q. semecarpifolia (DM5313); 19. Q. semecarpifolia (Forrest, G. 20184); 20. Q. alnifolia (DM8224). White arrows indicate the branched BU. “*” indicates U.

Plate VII. Leaf abaxial epidermis of Ilex subgroup. 1–9. Leaf epidermal features under LM, bar = 100 μm, showing the stellate-fasciculate trichome with CTB. 1. Quercus ilex (var. microphylla) (DM6573); 2. Q. ilex (DM6572); 3. Q. acrodonta (DL546); 4. Q. franchetii (DM5922); 5. Q. leucotrichophora (DM6575); 6. Q. leucotrichophora (DM6575); 7. Q. lodicosa (Ward. F.K. 6355); 8. Q. suber (DM6571); 9. Q. baloot (Wu, S.K. 01). 10–17. Leaf epidermal features under SEM. Showing the morphology of Stellate-fasciculate trichome (with compound trichome base). 10–15, 18–20: bar = 100 μm; 16–17, 21: bar = 50 μm. 10. Q. ilex (329934); 11. Q. rotundifolia (DM8240); 12. Q. acrodonta (DM5905); 13. Q. franchetii (Deng, M. et al. 10649); 14. Q. lanata (DM10659); 15. Q. lodicosa (Ward. F.K. 6355); 16, 17. Q. leucotrichophora (DM6575); 18. Q. suber (DM6940); 19. Q. suber (DM8233); 20. Q. baloot (DM13630); 21. Q. baloot (DM13629). The white arrows indicates Ca. “*” indicates S. M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 27 28 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 distinct pedestal like structure—compound trichome base on the epi- to the compound-trichome base can be either like ordinary leaf epider- dermis, which is 18.4–62.1 μm in diameter. There is a series of transi- mal cells or smaller and forming a circle around the base. tional forms as the origination of the arms above the fused part can be erect, radiating or parallel to the leaf surface. According to the arm ori- 3.2.2. Epicuticular waxes (Plate III,1–16; Plate IV,9–13; Plate VI,1–6, 9–11, fi entation features, two subtypes are de ned: 16; Plate VII,11,16–18, 20, 21). – – Subtype I: Typical stellate trichome (Plate VII,10 12, 18 21). Smooth epicuticular wax is commonly present on the abaxial sur- The rays of the trichome are spread out mainly parallel to the leaf face. The wax layer forms a continuous cover without prominent surface surface (not erect). The typical stellate trichome is found in Quercus sculpture (Plate III,1,2,6–16; Plate IV,10–12; Plate VI,1–11, 16; Plate acrodonta (Plate VII, 12), Q. lodicosa (Plate VII, 15), Q. suber (Plate VII, VII,18).InQuercus yiwuensis (Plate III, 3), Q. phillyreoides (Plate III,4) – 18) and Q. baloot (Plate VII,20 21). and Q. setulosa (Plate III, 5), the epicuticular wax is mostly smooth, but – Subtype II: Fasciculate trichome (Plate III,6,7,13,15;Plate VI,7 12, with very few scattered platelets. The typical membranous epicuticular – 13 18, 20; Plate VII, 13). platelet wax is only found in Q. baloot (Plate VII, 20, 21). The rays of the trichome diverge upwards from the common joined center with a large angle (not parallel) to leaf surface. This trichome type is commonly present in Group Ilex. 3.2.3. Leaf epidermal cells and stomatal complexes Occurrence: Fused fasciculate-stellate trichome generally occurs on fl both leaf surfaces, but there are great differences in density at inter- and 3.2.3.1. Leaf epidermal cells. The leaf epidermis and cuticle are at in infraspecific levels. In some species, e.g., Quercus rehderiana, Q. spinosa, Group Ilex. The stomata are aggregated in the areolar areas. Sometimes, and Q. coccifera, this trichome is rare, and only found in the early devel- the epidermal cells above the vein show a different morphology than opmental stage. those located in the areolar areas, as they are elongate and usually Remarks: Jones (1986) and Hardin (1979) described a series of dif- with undulate to sinuous anticlinal walls (Plate III,1;Plate IV, 10). ferent related trichome types, such as: fasciculate, fused fasciculate, stel- The abaxial epidermal cells in the areoles show many variations in late, fused stellate, and multiradiate. Vazquez (2006) further defined shape and are usually irregularly polygonal. The anticlinal walls vary – several subtypes of fasciculate trichomes, e.g., fasciculate-sessile simple, from straight to curved (Plate II,11;Plate V,12 14; Plate VII, 9), and un- – fasciculate-sessile contorted, and fasciculate-sessile crested. However, dulate to sinuous (Plate II,12;Plate IV,2,4 6; Plate V,1,2,15;Plate VII, these definitions are difficult to apply when identifying trichome 1, 3, 5, 6). Many species have node-thickenings on the anticlinal walls, types. The delimitation of these various forms depends on either the de- especially the less hairy species. gree of fusion of the basal part of the rays, or on the orientation of the ray arms from the common center. Sometimes, the SEM view (e.g., from the 3.2.3.2. Stomatal complexes. Stomata occur only on the abaxial surface. top or lateral views) can lead to different interpretations of these tri- The guard cells are covered by the same kind of epicuticular wax as chome types. Meanwhile, in the same slides, the range between stellate the other epidermal cells. The aperture rim (outer stomatal ledge) of and fasciculate types seems continuous, e.g., some stellate trichomes in the stomata is nearly equal or slightly sunken compared to the leaf Quercus rotundifolia (Plate VII, 11) also have a long, fused basal part blade (Plate III, 2, 3, 6, 9, 11, 12, 16; Plate IV, 13; Plate VI,5,8–11), or which seems to be fasciculate, but some are closer to the stellate form. slightly raised above it (Plate III, 10; Plate IV, 10, 11; Plate VI, 6, 11). Therefore, the appearance of fasciculate and stellate trichomes is a The “giant” stomata, which are generally located above the joints of quantitative rather than a qualitative character. Similar variations in the veins are present in most of the species. They are 1.5–2.0 times larg- stellate trichomes are also found in Elaeagnus angustifolia, but in plants er than ordinary stomata and their aperture rims and guard cells are ob- growing in different habitats (Klich, 2000), which indicates that these viously elevated (Plate III,1;Plate IV,10;Plate V,6,13). forms are rather changeable. Therefore, we regard the stellate and fas- The terminology pertaining to stomata is very specialized and has ciculate trichomes to be of the same type. Vazquez (2006) and Deng not been used consistently. Stoma refers to the pair of guard cells et al. (2014) described simple stellate trichomes (SSt), which resemble encircling the stomatal pore. Stomatal complex refers to the stomata some subtypes of fused fasciculate-stellate. However, the simple stellate plus any specialized epidermal cells adjacent to them, which are called trichome does not have the typical pedestal-like compound trichome subsidiary cells. In this paper, “contact cells” refers to any cells, special- base and might have a different function than the fused fasciculate-stel- ized or not, that are adjacent to the stomata and “non-contact cells” for late (Deng et al., 2014). all cells that are not immediately adjacent to the stomata; a radial wall is a cell wall oriented more or less perpendicular to the stoma as proposed by Carpenter (2005). 3.2.1.4. Trichome bases. Two main types of trichome bases occur. The sin- The orientation of contact cell walls of stomatal complexes is radial. gle-celled trichome base of the uniseriate, branched uniseriate and cap- The stomata type is stephanocytic according to the definition of itate trichomes are all rounded, cutinized and c. 8.2–27.8 μm in diameter Carpenter (2005), but cyclocytic and anomocytic stomata according to (Plate II,1,3,5,6,9,11,12;Plate IV,1,3,5;Plate V,3–15). The single- Dilcher (1974). The contact cells of the stomata are either different celled trichome base of the solitary (also the appressed lateral attached) from unmodified epidermal cells in shape, size or cutinization (Plate II, differs from that of the glandular trichomes in being polygonal in out- 2, 12; Plate IV,2–5; Plate V,1,2,12–15; Plate VII,1,2,6,9)orconsistent line, flat, strongly cutinized, and the adjacent epidermal cells are small with the unmodified epidermal cells (Plate II,1,3,4,6,7–11; Plate IV,1, and also more strongly cutinized than other epidermal cells. 6; Plate V,3,4;Plate VII, 3, 5). Except for Quercus ilex, the contact cells of The compound-trichome base belongs only to fused fasciculate-stel- stomata are shared by adjacent stomata in species of the Group Ilex. late trichome. Usually, it is composed of (4)5–16(18) specialized cells The stomatal size shows great variations both at inter- and intraspe- forming a pedestal (Plate II,4,9,10;Plate IV,1,3,9;Plate V,3–5, 7, 8; cific level. The stomata in some species are not uniform, and show dif- Plate VI, 10, 13, 16, 17, 20; Plate VII,1,5–9). The epidermal cells adjacent ferences in size of 1.5–2 times in the same slide (Plate V,2,6,9,13).

Fig. 1. 1. Evolutionary patterns of leaf epidermal features in Group Ilex based on a simplified phylogenetic tree based on RAD-seq of Hipp et al. (2015) using Mesquite 2.75. Black solid circles indicate plesiomorphies, synapomorphies and autapomorphies. An empty circle indicates the paraphyletic status of these features. The trait abbreviations were listed above the circle, which were consistent to Table 3. The number inside the circle indicated the trait status. 2. Cluster analysis of 14 leaf epidermal features of Group Ilex were investigated based on the Jaccard method and inferred two main clades: Capitate (Ca) clade and branched uniseriate (BU) clade. Of Ca Clade, two lineages were detected. One lineage is characterized by scattered fused fasciculate-stellate (Ca), another is characterized by dense fused fasciculate-stellate trichomes (FF-St). M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 29 30 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36

3.3. Mapping of the leaf epidermal characters onto the RAD-seq cladogram variations from typical stellate, radiate and prominently fused-stellate were also detected in Elaeagnus angustifolia plants growing in different Fourteen leaf epidermal features were mapped onto the RAD-seq environments (Klich, 2000). Furthermore, the coexistence of stellate, Maximum Likelihood (ML) tree of Hipp et al. (2015). Most of the epider- fasciculate types and their intermediates in the same specimens also mal features show a convergent evolutionary pattern (derived or lost suggests that these different stellate trichome forms should be attribut- more than once). The multiple epidermal layers on the leaf adaxial sur- ed to the same category. face is a synapomorphy to support the monophyletic status of the Asian section Heterobalanus in Group Ilex, which is mainly distributed in the 4.2. Evolutionary pattern of leaf trichome types in Group Ilex SE fringes of the Qinghai-Tibet Plateau. The absence of compound-tri- chome base is the synapomorphy of the “single-celled tricohme base” The tree-based mapping analysis revealed a highly convergent evo- clade of Group Cyclobalanopsis. The presence of single-celled trichome lutionary pattern of leaf epidermal features, especially trichome types in base represents the plesiomorphic status (Fig. 1,1). Group Ilex. According to Hipp et al.'s (2015) phylogenetic study, the East Asian subtropical species in Group Ilex were early derived and 3.4. Cluster and cladistic analysis of selected leaf epidermal features the European species (Quercus ilex, Q. alnifolia, Q. coccifera and Q. calliprinos) formed an independent clade nested within the one clade Fourteen leaf epidermal features were selected and scored for clus- of Asian species (Fig. 1). The leaf epidermal features of European and ter and cladistic analysis. The status of selected leaf epidermal features Asian lineages are similar and very diverse. In plants, different trichome is summarized in Table 3. Both cluster and cladistic analysis indicate types have different physiological functions, e.g., non-glandular tri- two main clades in Group Ilex. One clade is composed of the species chomes are related to abiotic stresses such as UV-B (Skaltsa et al., with branched uniseriate trichome (BU clade), while another clade 1994; Liakoura et al., 1997) and drought stress (Ning et al., 2016). Glan- (Ca clade) includes the species with capitate trichomes (Fig. 1, 2; Fig. dular trichomes are major sites of plant natural product synthesis and S1). However, the two main clades are not supported by the bootstrap accumulation, serving for protection against pathogens and predators (Fig. S1). The species with very few trichomes (Quercus setulosa and Q. (Aziz et al., 2005). The large differences in trichome types and their bawanglingensis), do not cluster into either of the two clades based on abundance in different lineages of Group Ilex indicates that these tri- cluster analysis (Fig. 1, 2). Similarly, in cladistic analysis, Q. utilis, Q. chome traits might be subject to selection due to specific environmental bawanglingensis, Q. setulosa, Q. × parkeri and Q. tatakaensis also do not and ecological factors. cluster into any of the two clades (Fig. S1). The leaf epidermis is located at the interface between the plant and Cluster analysis also infers two subclades within Ca clade: one con- its environment. Therefore, epidermal features are effective parameters taining species with dense fused fasciculate-stellate trichomes (Fig. 1, to indicate the local habitat and climate. Generally, thick leaf cuticles, 2: FF-St subclade), the other subclade species have a low density of and thick anticlinal walls, sunken stomata and dense trichomes are typ- scattered fused fasciculate stellate, but quite a few capitate trichomes ical xeromorphic features, as these structures are correlated with high (Fig. 1, 2: Ca subclade). However, in the cladistic analysis, all except light intensity, high temperature and high evaporation environments. for the hairy species with fused fasciculate stellate trichomes form a On the contrary, thin cuticles, elevated stomata and less trichomes indi- subclade (Fig. S1), while other Ca clade species failed to form a tree- cate a mild and humid environment (Bredenkamp and Van Wyk, 2000; structure subclade (Fig. S1), because of the limited variation in charac- Sun et al., 2003; Tripp and Fatimah, 2012). The epidermal features of ters and conflicts between the informative characters. Group Ilex generally show xeromorphic features. However, “xeromorphic” features are not only found in plants subject to water 4. Discussion stress. These features are also found in plants growing in environments with high precipitation and humidity. They can repel water and protect 4.1. Terminology of leaf epidermal features and their possible ontogentic plants from excessive light (Haworth and McElwain, 2008). Strong fossil relationships evidence and palaeoclimatic reconstructions for Eurasia during the Cenozoic suggest that the early ancestral lineage of Group Ilex grew in The early developmental stage of the branched uniseriate resembles a humid-warm to temperate climate (Bozukov et al., 2009; Böhme et the uniseriate with very few short arms. Some capitate trichomes in Q. al., 2011; Denk et al., 2012) rather than in a Mediterranean type climate. coccifera are also not typical, because the enlarged cap consists of a Although, we cannot speculate whether the “xeromorphic” features in single, smooth cell. All these similarities of morphology and texture sug- Group Ilex were inherited from ancestral lineages adapted to a Mediter- gest that the uniseriate, branched uniseriate and the capitate trichomes ranean-type climate, such “xeromorphic” features made a great contri- have a close ontogenetic relationship and the former two types are bution to the later success of the Group Ilex in subtropical, seasonally possibly derived from the uniseriate trichome. dry habitats (Ackerly, 2004). The role of these “xeromorphic” traits Apart from the trichome composition and ray morphology, the tri- can be subjected to diversification analysis in contrasting to the phylog- chome base type is important. Trichomes with similar appearance but eny in the future for understanding their roles in the colonization of het- with different trichome bases might have different functions (Deng et erogeneous habitats and the evolution of Group Ilex. al., 2014). For example, the stellate trichome with compound-trichome base might have a different function than the stellate trichome with sin- 4.3. Significance of leaf epidermal features to the taxonomy and systematics gle-celled trichome base. The large number of crystal structures inside of Group Ilex compound-trichome base indicates that this trichome might have a se- cretory function (Deng et al., 2014), which may be a mechanism to deter The epidermal features are less diverse and comparatively uniform invertebrate herbivory (Karioti et al., 2011; Burrows et al., 2013). The in Group Ilex, as only five trichome types and two trichome base types function of stellate trichomes with single-celled trichome base is appar- were observed. The other epidermal cell features and stomatal types ently related to environmental stress (Fernandez et al., 2014). There- are shared with other groups of Quercus or (and) other genera of fore, using “trichome-ray base fused portion”, “ray-radiation angle Fagaceae, e.g., branched uniseriate trichome is also found in Group from the common joint center” and “trichome base” to profile the key Cyclobalanopsis; uniseriate trichome is commonly found in the features of the non-glandular multicellular trichomes should be more Fagaceae, and capitate trichome is usually present in Castanea and accurate, as the transition forms from fused stellate, stellate, fasciculate Quercus (Jones, 1986; Deng et al., 2014). Other epidermal features and stipitate fasciculate make it ambiguous to classify these similar tri- (e.g., stomatal types and frequency, anticlinal walls) show large varia- chomes as distinct qualitative traits. Similarly, the stellate trichome tion at inter- and intraspecific level in Group Ilex, but none of them is M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 31

Table 3 Leaf foliar feature status scored for Cluster, cladistic and tree mapping analyses.

Taxa Adaxial Abaxial

CTB STB Ep Layers Wax S/ALA F-St (STB) FF-St (CTB) U BU Ca CTB CTB abundance STB STB abundance

1 Q. acrodonta * 1107 00 0 1 00112 11 2 Q. alnifolia* 11? 1 0 0 1 01010 11 3 Q. aquifolioides 1117 00 0 1 01010 12 4 Q. baloot 10? 1 0 0 1 00112 11 5 Q. baronii* 1107 00 0 1 00110 10 6 Q. bawanglingensis 0107 00 0 0 00100 10 7 Q. coccifera* 110 0 0 0 1 00110 10 8 Q. coccifera var. calliprinos 110 0 1 0 1 00110 10 9 Q. cocciferoides 110 0 0 0 1 00110 10 10 Q. dolicholepis* 1107 00 0 1 00110 11 11 Q. engleriana 1107 00 0 1 01011 11 12 Q. floribunda 11? 0 0 0 1 01110 10 13 Q. franchetii* 1107 00 0 1 10112 11 14 Q. gilliana 0117 00 0 1 01010 10 15 Q. guyavifolia 1117 00 0 1 01010 12 16 Q. ilex* 1108 00 0 1 00112 11 17 Q. ilex var. microphylla 11? 0 0 0 1 00112 11 18 Q. kingiana 1107 00 0 1 00111 11 19 Q. lanata 1107 00 0 1 00112 11 20 Q. leucotrichophora 1107 00 0 1 00112 11 21 Q. lodicosa 1107 00 0 1 00112 11 22 Q. longispica* 1117 01 0 1 01010 12 23 Q. marlipoensis 1107 00 0 1 01011 11 24 Q. monimotricha 1117 00 0 1 01011 11 25 Q. oxyphylla 1107 00 0 1 00111 11 26 Q. pannosa 1117 00 0 1 01010 11 27 Q. × parkeri 11? 0 0 0 0 00110 10 28 Q. phillyreoides* 1107 00 0 1 00110 10 29 Q. rehderiana* 1117 00 0 0 11010 10 30 Q. rotundifolia 11? 0 1 0 1 00110 10 31 Q. semecarpifolia 1117 00 0 1 11010 11 32 Q. senescens* 1117 00 0 1 01011 11 33 Q. setulosa 0007 00 0 0 00100 10 34 Q. spinosa 1117 00 0 0 11110 10 35 Q. suber* 11? 0 1 1 1 10112 11 36 Q. tarokoensis 11? 0 1 0 1 00110 10 37 Q. tatakaensis 11? 0 0 0 1 00110 10 38 Q. tungmaiensis 1107 00 0 1 01010 10 39 Q. utilis* 1107 00 0 1 10110 10 40 Q. yiwuensis* 1107 00 0 1 00110 10 41 Q. glauca1 010 1 1 0 0 10000 12 42 Q. delavayi1 110 0 0 0 1 10012 11 43 Q. augustinii1 010 0 0 0 0 01000 11 44 Q. variabilis2 ??0 1 0 1 1 10010 12 45 Sect. Lobatae 3 ? ? 0 0&1 1 1 1 1 0 1 1 0&1 1 1&2 46 Sect. Protobalanus4 ??? 0&1 1 1 1 1 0&1 1 1 0&1 1 1&2 47 Sect. Quercus5, 6 ? ? 0 0&1 1 1 1 1 0&1 1 1 0&1 1 1&2

“*” indicated taxa were used for mapping analysis. All the abbreviations are as in Table 2. “0”:absent;“1”: present. Ep layer (epidermal cell layer): “0” single layer; “1” compound epidermal layers. CTB/STB abundance: “0”: very few or scattered with low density; “1”: common, but scattered; “2”: abundant, forming prominent layers. The number at upper right of the trait stages indicate data were obtained from the numbered literature. “1” Deng et al. (2014). “2” Kim et al. (2011). “3” Hardin (1979). “4” Manos (1993). “5” Bussotti and Grossoni (1997). “6” Tschan and Denk (2012). “7” Zhou et al. (1995). “8” Karioti et al. (2011).

unique and a synapomorphy to Group Ilex. The convergent evolution of main morphological subgroups and seven morpho-complexes are epidermal features is one of the most typical characteristics of identified. Quercus and Fagaceae. The parallel evolutionary patterns of epider- mal features in different lineages of Quercus s.l. apparently reflect a 4.3.1. Capitate subgroup stochastic sorting of ancestral polymorphisms and the result of The species in this subgroup are more or less hairy on the abaxial long-term adaptation rather than evolutionary relatedness. But large surface and show a great variation in trichome frequency during differ- variations and combinations of unique traits of epidermal features in ent developmental stages. Fused fasciculate-stellate trihcomes are gen- specific groups or species complexes are no doubt valuable for spe- erally present in the early developmental stage of the leaves. In some cies identification. Based on epidermal features of Group Ilex, two species, the tricohmes tend to be shed when the leaves mature and 32 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 they are mainly preserved along the veins. Four main morpho- monimotricha and Q. semecarpifolia, but with distinct, thick, branched complexes exist in this subgroup: I. Quercus setulosa complex: charac- uniseriate layers and scattered fused fasciculate-stellate trichomes in terized by almost glabrous leaves, the bracts on the cupule triangular Q. pannosa, Q. guyavifolia, Q. longispica and Q. alnifolia. The function of fo- and appressed to the cupule wall. This species complex includes liar trichomes is to protect the plant against abiotic and biotic stress. The Q. bawanglingensis, Q. phillyreoides, Q. setulosa, Q. tarokoensis, Q. branched uniseriate and fused fasciculate trichomes are related to the tatakaensis and Q. utilis; II. Q. coccifera complex: characterized by the synthesis, accumulation and secretion of a variety of secondary metab- glossy green abaxial leaf surface, with scattered uniseriate, capitate, olites. They may also control leaf temperature and water loss, or serve as fused fasciculate-stellate trichomes, and spinose teeth on the leaf mar- a protective layer against solar radiation (Liakoura et al., 1997; Agati et gin. The bracts on the cupule wall are long and recurved, including: al., 2002; Olsson et al., 2009; McDowell et al., 2011). Our data shows a Q. baronii, Q. coccifera, Q. cocciferoides and Q. yiwuensis; III. Q. kingiana great variation in the branched uniseriate arm length and frequency in complex: characterized by large leaves with a regular venation pattern. juvenile and mature leaves of Q. pannosa, Q. semecarpifolia and Q. The teeth on the leaf margin are acute, rarely with spinose tips and only rehderiana, which indicates that the foliar trichome features are rather appears in the distal half. The bracts on the cupule are also long and re- plastic and not stable. As the seedlings of all these oaks usually grow curved, including: Q. kingiana, Q. dolicholepis and Q. oxyphylla; IV. Q. ilex in a shady environment under the canopy, the low trichome frequency complex: characterized by the abaxial side covered by a dense layer of and the short arms of branched uniseriate on the leaves of the seedlings persistent fused stellate to short-fused fasciculate trichomes. Capitate might be related to the low light levels in these habitats. trichomes are scattered on the abaxial surface. The abaxial surface is However, the taxonomy of section Heterobalanus still largely de- usually gray, or yellowish to brown. This complex is equivalent to sub- pends on the trichome frequency and color of the abaxial leaf surface section Ilex (Loudon) Gurke (Menitsky, 1984) with fairly consistent and trichome types to identify species. As all the important morpholog- leaf morphology, i.e. leaves coriaceous to thick-coriaceous, pubescent ical traits (foliage, flowers and fruits) show a large variation and overlap in various degrees, and with acute teeth. This category includes Q. at the interspecific level, there is great taxonomic confusion in this acrodonta, Q. baloot, Q. franchetii, Q. ilex, Q. lanata, Q. leucotrichophora, section. Hipp et al.'s (2015) results indicate that Quercus alnifolia is not Q. lodicosa and Q. rotundifolia. The molecular phylogenetic reconstruc- close to the species of section Heterobalanus and that some species tion based on ITS and 5S-IGS suggests that Group Cerris was “budding” of this section (Q. monimotricha, Q. longispica, Q. senescens and Q. from Group Ilex (Denk and Grimm, 2010), but the RAD-seq based phy- rehderiana), which are mainly distributed in the SE Himalayan fringes, logeny revealed that Group Cerris is the sister group to Group Ilex (Hipp form a monophyletic clade. The high foliar trichome diversity and et al., 2015). Q. suber of Group Cerris shows a high similarity both in leaf great variation in key morphological features both at infra- and inter- epidermal and architectural features to the species in the Q. ilex com- specific levels of the section might reflect ongoing differentiation, speci- plex. Considering the similar habitats in the Mediterranean region, the ation events and adaptation to heterogeneous habitats. The leaf overall similarity in epidermal features of the different oak lineages in- epidermal features of subtropical and subalpine species of the BU sub- dicates a high degree of adaptive convergence to environmental factors. group are highly consistent, but Hipp et al.'s (2015) work did not in- clude these subtropical taxa in their analysis. Considering the high 4.3.2. Branched uniseriate subgroup morphological convergence in Group Ilex, future studies coupling mor- The species in this subgroup have branched uniseriate and scattered phology with high-throughput based molecular phylogeny across the fused fasciculate-stellate trichomes. The trichome density and morphol- species and in different geographic populations of Group Ilex will be es- ogy shows great variation both at inter- and intraspecific levels. The sential to address the species composition and evolutionary history of fused fasciculate-stellate trichomes occur mainly along the veins in Group Ilex. adult leaves. The branched uniseriate trichome density and their arm Quercus spinosa and Q. floribunda have both capitate and branched length also vary during different developmental stages. In addition, uniseriate trichomes. The phylogenetic reconstruction based on chloro- the color of the abaxial surface varies dramatically in seedlings and plast DNA sequences indicates that Q. spinosa clusters with Mediterra- adult trees. This subgroup is composed of the subtropical Quercus nean species while Q. floribunda clusters with Himalayan species engleriana, Q. tungmaiensis, Q. marlipoensis and Q. alnifolia from Cyprus, (Simeone et al., 2016). The co-existence of the two trichome types and the species mainly from the subalpine/alpine Himalayas and adja- might suggest a possible ancestral status. The leaf morphology of Q. cent regions. The small leathery-leaved species from Asian subalpine baloot shows a series of combinations of different groups and sub- and alpine regions of the BU subgroup are regarded as an independent groups, e.g., typical spinose teeth on mature leaves similar to species section Heterobalanus by Menitsky (1984) and Zhou et al. (1995), of the Q. pannosa and Q. spinosa complexes of the BU subgroup, but which is also partly supported by RAD-seq based phylogenetic work with capitate and typical stellate trichomes that resemble the trichome (Hipp et al., 2015). The multiple epidermal layers on the leaf adaxial sur- types in the Q. ilex complex of the Ca subgroup. Q. baloot also has epicu- face, which are related to the adaptation to a high alpine environment ticular wax with platelets on the abaxial leaf surface, which is unique in (Zhou et al., 1995), is a shared synapomorphy that unites the species Group Ilex, but quite common in the Groups Cerris and Cyclobalanopsis. of section Heterobalanus. Q. baloot mainly occurs in Afghanistan, the Himalayas, North Pakistan Based on leaf architectural and epidermal features, three main mor- and Kashmir, at elevations of 1000–3000 m. Considering its southern pho-complexes can be distinguished within the BU subgroup. I. Quercus Asian distribution and the unique combination of leaf morphology of engleriana complex: characterized by long, large, lanceolate leaves different groups, this species might represent an ancestral and relict sta- with primary veins running directly to the leaf tips. This morpho-com- tus in Quercus “the Old World Clade”. However, all these speculations plex includes Q. engleriana, Q. tungmaiensis and Q. marlipoensis; II. Q. still need to be further tested in contrast to the true phylogeny and bio- spinosa complex: characterized by almost glabrous leaves, with very geographic analysis of Group Ilex. rare hairs, including Q. floribunda, Q. × parkeri, Q. spinosa, Q. gilliana, and Q. rehderiana. This morpho-complex includes most of the non- 4.4. Application of leaf epidermal features to the identification of oaks in hairy species in HET; III. Q. pannosa complex: characterized by small Group Ilex leaves with irregular secondary vein spacing and inter-secondary veins, spinose teeth on the leaf margin, and well-developed branched Epidermal features and venation patterns show a high uniseriate trichomes. This morpho-complex includes most of the hairy homoplasticity in the main oak groups. No special trichome type, tri- species in section Heterobalanus. A great diversity of foliar trichomes is chome base type, nor stomata type or other epidermal features occur- found in the Q. pannosa complex, e.g., the predominance of fused-fascic- ring in Group Ilex is diagnostic to identify species or to delimit the ulate and scattered branched uniseriate trichomes in Q. senescens, Q. main groups. However, trait combination patterns are useful because M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36 33 the specific combinations are restricted to subgroups and species mor- The first unequivocal fossil record of Group Ilex in Eurasia is not pho-complexes. later than the early Oligocene. Miocene fossils of this group are wide- The fused fasciculate-stellate trichome, as a plesiomorphic char- spread and show a high diversity of leaf epidermal features. For ex- acter, is generally found in different clades of Quercus,suchas ample, the fossil taxon Quercus drymeja Unger barely has trichome Group Cyclobalanopsis (Deng et al., 2014), Group Lobatae bases on the leaf abaxial surface, the stomata are the anomocytic (Vazquez, 2006)andGroupQuercus(Tschan and Denk, 2012). The cyclocytic-antinocytic type and their outline varies from rounded, species with compound trichome base in Group Cyclobalanopsis widely oval to circular and sub-circular; anticlinal walls are straight form an independent clade as inferred by ITS-based phylogeny to curved with uniform thickenings (Kvaček et al., 2002; Palamarev (Deng et al., 2013). Epidermal features of species in Group and Tsenov, 2004; Kvacek et al., 2011). These characters indicate Cyclobalanopsis with compound trichome base are similar to those that the nearest living relative of Q. drymeja might be related to in Group Ilex, but the capitate trichome is only present in Group less hairy species in mesophytic and broadleaved evergreen for- Ilex, not in Group Cyclobalanopsis. The papillae thickening on cuti- ests, e.g., Q. cocciferoides and Q. baronii. It is worth noting that cles is only found in Group Cyclobalanopsis of the “Old World there are some discrepancies in leaf epidermal features reported Clade” and platelet epicuticular wax is only found in species of from the same fossil taxa by various authors. For example, Quercus Groups Cyclobalanopsis and Cerris and Q. baloot. The shape and sosnowskyi Kolakovskv has cyclocytic stomata and stellate tri- cutinization level of the stomata are also useful characters for chomes with a central pore and 6–8 ft cells, (abundant) stellate, distinguishing Group Ilex from Groups Cyclobalanopsis and Cerris. poly-cellular, thin-walled (less frequent) simple trichome bases, In Groups Cyclobalanopsis and Cerris, the aperture rim (outer sto- straight-walled, finely curved anticlinal walls (Kvaček et al., matal ledge) of the stomata is elevated, strongly cutinized and the 2002). All these leaf epidermal features indicate that its nearest liv- stomata are elliptic in outline (L/W ratio N 1.2). Therefore, the ing relative is related to species of the Q. ilex complex. However, the shape of stomatal guard cells in the two groups resembles an ob- leaf abaxial epidermis of other specimens identified as Q. long ring under LM (Deng et al., 2014). In contrast, the stomatal ap- sosonowkyi by Palamarev and Tsenov (2004) barely have trichome erture rim in Group Ilex is nearly level with the leaf surface or bases and the anticlinal walls are undulate-sinuous, which indi- sunken and less cutinized than the ordinary epidermal cells while catesthisspecimenisratherclosetoQ. drymeja. Fossils of species the stomata are rounded-subrounded in outline (L/W ratio ~ 1.0– of Group Ilex have also been widely found in west China, but only 1.2). Moreover, the anticlinal walls are thin and usually uniform a few specimens with cuticle preservation. The leaf cuticles of Q. in thickness in Groups Cyclobalanopsis and Cerris, but thick with pannosa (Xiao et al., 2006)andQ. presenescens (Li et al., 2009) node- and ridge-thickenings in Group Ilex. Using these epidermal from the Pliocene all show cyclocytic stomata, which suggests features, it is possible to distinguish leaves of Group Ilex from thesefossilsarerelatedtosectionHeterobalanus. Although the those of Groups Cyclobalanopsis and Cerris. leaf morphology of species of the branched uniseriate subgroup is Overall, the leaf epidermal features of Group Ilex show a typical variable in the abaxial leaf surface color and trichomes, the separa- “xeromorphic” form, e.g. thick cuticle, level-sunken stomata, dense tri- tion of the different species complexes mainly depends on the fre- chomes and thick, undulate to sinuous anticlinal wall. The features quencies of the fused fasciculate-stellate and branched uniseriate (e.g., thin cuticle, uniform thickness of the anticlinal walls, and concave trichomes, which also correspond to the frequencies of compound tri- stomata) of two other groups of the Old World Clade (Groups Cerris and chome base and single-celled trichome base. However, both authors Cyclobalanopsis) suggest that the two groups live in mesophytic envi- did not record the existence or density of the trichome bases. Li et al. ronments. The leaf cuticle and leaf venation patterns, combining LM (2009) stated that no trichome bases occurred on the abaxial and adaxial and SEM, are helpful to improve the accuracy of leaf identification of leaf surfaces of Q. presenescens. Based on our study, all specimens of Q. the main groups and even of the species morpho-complexes of Group senescens have prominent compound trichome base and single-celled tri- Ilex. chome base on the abaxial surface. Therefore, the fossil Q. presenescens is The frequency of compound trichome base and single-celled tri- apparently closer to Q. spinosa complex rather than to Q. senescens.Inthe chome base and their morphology are helpful for assigning the spe- fossil species Q. pannosa (Xiao et al., 2006), compound trichome bases on cies in Group Ilex to subgroups and morpho-complexes. In Ca the leaves and cyclocytic stomata suggest that this species might belong subgroup, the high compound trichome base frequency, but low sin- to Q. pannosa complex (e.g., Q. semecarpifolia, Q. senescens and Q. gle-celled trichome base frequency is mainly found in Quercus ilex longispica). But without other information, it is difficult to tell which spe- complex. The trichome density in Q. kingiana, Q. coccifera and Q. cies is its nearest living relative. Further efforts to supplement the leaf setulosa complexes are obviously reduced from common to occasion- epidermal features of fossils are crucial to improve the accuracy of al to rare respectively. Therefore, it is possible to identify the mor- assigning them to extant closely related species. pho-complex when the trichome types and (or) trichome base The widely shared cpDNA haplotypes among the Eurasian forms features are present. The greatest trichome and trichome base diver- of Group Ilex did not reveal species identity, but these cpDNA haplo- sity is found in the branched uniseriate subgroup, as their leaves are types show a strong phylogeographical structure, which suggests either almost glabrous or covered by distinct hairy layers. The fre- that possible ancient hybridization and genetic assimilation oc- quency of the fused fasciculate-stellate, branched uniseriate tri- curred in ancestral lineages (Simeone et al., 2016). Hipp et al.'s chomes and their trichome bases show great variations, but the (2015) works inferred the phylogenetic framework of Group Ilex, stomata of the branched uniseriate subgroup are largely of a typical but still about half of the species in Group Ilex have not yet been in- cyclocytic type. vestigated. In recent years several studies on the phylogeny and pop- To summarize, cyclocytic stomata and a high single-celled trichome ulation genetics of species of section Heterobalanus reveal high base frequency, with rare or absence of compound trichome base can be genetic diversity at infraspecific levels, but with limited genetic dif- used to discriminate most of species of the branched uniseriate sub- ferentiation among species (Pu, 2002; Wang, 2003; Ma, 2006; group from the Ca subgroup. Anomocytic stomata, extremely low com- Yang, 2007). However, they could not successfully resolve the evolu- pound trichome base and single-celled trichome base (or without tionary history of section Heterobalanus, because of the frequent trichome base) are diagnostic for Q. setulosa complex. Typical cyclocytic gene flow and great phenotypic plasticity of this section. It will be stomata, dense compound trichome base, and comparatively low sin- crucial to employ high-throughput genome typing methods as well gle-celled trichome base indicate Q. ilex complex. Overall, the trichome as comparative anatomy to investigate the evolutionary history of type and trichome base features are very important for fossil identifica- these and other Asian subtropical taxa of Group Ilex to resolve the tion in Group Ilex. many remaining puzzles. 34 M. Deng et al. / Review of Palaeobotany and Palynology 237 (2017) 10–36

Below we summarize the key diagnostic epidermal features that can surface support the monophyletic status of section Heterobalanus, be used to identify the subgroups and species complexes in Group Ilex. which are mainly distributed in Asian sub-alpine and alpine regions. The assignment of the 37 investigated species of Group Ilex to species This feature might protect plants against UV-B irradiation and low- complexes is summarized in Table 2 and Table S1. temperature at high elevations. However, only very limited Asian species have been subjected to molecular phylogenetic studies. A 4.4.1. Key to the species morpho-complexes of Group Ilex based on leaf mor- comprehensive phylogenetic study on all the Eurasian species of phological features Group Ilex is still necessary. Future studies coupling comparative 1. Branched uniseriate trichomes present on abaxial surface—Branched anatomy and super-matrix-based phylogeny in Group Ilex, as well uniseriate (BU) subgroup (2). as population genetics of representative taxa of Group Ilex could pro- 1. Branched uniseriate trichomes absent from abaxial surface—Capitate vide new insights into their evolution and diversification and may (Ca) subgroup (4). show how the Neogene palaeoclimatic and palaeogeographic events 2. Abaxial surface almost glabrous, with typical cyclocytic stomata, sin- shaped the species and genetic diversification patterns in subtropical gle-celled trichome base and compound trichome base rare, and only areas of Eurasia. above the veins—Q. spinosa complex. Supplementary data to this article can be found online at http://dx. 2. Abaxial surface of mature leaves with branched uniseriate and fused doi.org/10.1016/j.revpalbo.2016.11.006. fasciculate-stellate trichomes, or only a thick layer of branched uniseriate trichomes—3. Competing financial interests 3. Leaves lanceolate to ovate, leaf margin entire or with acute, rarely spinose, teeth; spacing of teeth mostly regular, leaves without The authors declare no competing financial interests. multi-epidermal layers—Q. engleriana complex. 3. Leaves ovate, oblong or obovate, leaf margin entire or with sharp spi- Acknowledgments nose teeth, spacing of teeth irregular, leaves with multi-epidermal layers—Q. pannosa complex. This work is supported by grants from the National Natural Science 4. Abaxial surface mostly glabrous with only scattered capitate tri- Foundation of China (31270267, 31500539), the Shanghai Municipal chomes, or with scattered fused fasciculate-stellate and capitate tri- Administration of Forestation and City Appearances (G162405, chomes, but not forming dense layers; stomata anomocytic type; G162404) and the Science and Technology Commission of Shanghai leaf cuticle thick, with node thickening on anticlinal wall of leaf epi- Municipality (14DZ2260400). We are grateful to Dr. Thomas Denk of dermal cells—(5). the Swedish Museum of Natural History and Dr. Hans Kerp of University 4. Abaxial surface covered by a dense layer of fused fasciculate-stellate of Münster for providing great expertise, important literature, and kind- trichomes (or with a high density of compound trichome base), cap- ly helping to revise the manuscript. 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