Botanical Journal of the Linnean Society, 2009, 160, 93–109. With 91 figures Comparative morphology of the leaf epidermis in Fritillaria (Liliaceae) from China QIANG WANG†, SONG-DONG ZHOU†, XIAO-YAN DENG, QI ZHENG and XING-JIN HE* College of Life Sciences, Sichuan University, Chengdu 610064, China Downloaded from https://academic.oup.com/botlinnean/article/160/1/93/2418345 by guest on 27 November 2020 Received 3 November 2007; accepted for publication 17 March 2008 The leaf epidermis of 16 species and one putative species of Fritillaria was examined using light microscopy (LM) and scanning electron microscopy (SEM). The results showed that the stomatal and other epidermal features were constant within species. Epidermal cells of Fritillaria under LM were usually polygonal and anticlinal cell walls were straight or curved. In a few species they were irregular, with sinuous anticlinal cell walls. The cuticular membrane of Fritillaria was usually striated, and the wax ornamentations were flaked, granular or concomitant. Based on leaf epidermal characteristics, the subdivision of Fritillaria is discussed, and the statistical t-test method was used to ascertain the significance level of the differences in the stomata of each species. All orientations of the stomatal poles in Fritillaria were the same, and this phenomenon was named ‘stomatal orientation’. The stomatal characteristics support the origin of section Fritillaria in China from two floristic elements. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 93–109. ADDITIONAL KEYWORDS: stomata – taxonomy – t-test – wax ornamentation. INTRODUCTION Numerous studies of the morphology of Fritillaria have been performed. Baker (1874) studied the bulb, Fritillaria L. is a genus of tribe Lilieae of Liliaceae. It style, form of the nectary and capsule valves. Bakshi- consists of about 130 species distributed in the tem- Khaniki & Persson (1997) studied the nectary perate regions of the Northern Hemisphere, mainly in morphology in 31 species from south-western Asia. Central Asia and the Mediterranean region (Chen & However, few studies have been performed on the leaf Mordak, 2000). There are 24 species and two varieties epidermal morphology in non-Chinese Fritillaria.Ina in China, 15 species of which are endemic. All species review of the leaf epidermal morphology in Chinese of Fritillaria are geophytic perennials and bulbifer- Fritillaria,Liet al. (1993, 1994) investigated 22 ous. The bulbs are composed of a few fleshy, farina- Chinese species and four varieties. Zhang, Cheng & ceous scales, often covered with a translucent tunic. An (1998) studied leaf epidermal morphology in eight In China, the bulbs of some species have been used Chinese species and two varieties of Fritillaria from medicinally under the name ‘bei-mu’ since ancient Gansu Province, China. Pu et al. (2001) studied the times. In the Pharmacopoeia of the People’s Republic leaf epidermis in three species, one variety and one of China (Editorial Board of the State Pharmacopoeia putative species. However, the descriptions of the leaf of the People’s Republic of China, 2000), eight species epidermis in all the reported species are not detailed, are prescribed in Chinese medicine, including F. cir- and the widely adopted terminology is not used well. rhosa D.Don, F. unibracteata Hsiao & K.C.Hsia, F. Moreover, no taxonomic conclusion has been reached delavayi Franch., F. przewalskii Maxim. ex Batalin., on the basis of the leaf epidermis. Thus, a more F. thunbergii Miq., F. usuriensis Maxim., F. walujewi detailed description of the leaf epidermis of Chinese Regel and F. pallidiflora Schrenk. Fritillaria using the widely adopted terminology (Wilkinson, 1979; Baranova, 1987, 1992) is necessary. *Corresponding author. E-mail: [email protected] In this study, the leaf epidermis of 16 species and †Co-first authors. one putative species of Fritillaria from China was © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 93–109 93 94 Q. WANG ET AL. Table 1. Material and origin Species Locality Month of collection Voucher Fritillaria pallidiflora Tacheng City, Xinjiang Province v.2006 Qiang Wang, ym001, SZ F. sichuanica Kangding City, Sichuan Province vii.2006 Qiang Wang, WQ-0029, SZ F. cirrhosa Deqing County, Yunnan Province vii.2006 Qiang Wang, WQ-0020, SZ F. taipaiensis Wanyuan County, Chongqing City vi.1959 Ben-liang Li, 2056, SZ F. crassicaulis Emei Mountain, Sichuan Province v.1940 Xiang-lin Sun, 2237, SZ F. sinica Tianquan County, Sichuan Province v.1953 Xing-ao Jiang, 34297, SZ F. walujewi Tianshan Mountain, Xinjiang Province v.2006 Qiang Wang, TC-002, SZ Downloaded from https://academic.oup.com/botlinnean/article/160/1/93/2418345 by guest on 27 November 2020 F. verticillata Emin County, Xinjiang Province v.2006 Qiang Wang, em-001, SZ F. thunbergii Sichuan Province – Wen-pei Fang, 373, SZ F. tortifolia Toli County, Xinjiang Province v.2006 Qiang Wang, TL-002, SZ F. przewalskii Daofu County, Sichuan Province vi.1974 Yu-ting Wu, 111635, SZ F. unibracteata Wolong County, Sichuan Province vii.2006 Qiang Wang, WQ-0030, SZ F. dajinensis Xiaojin County, Sichuan Province v.1959 Xiaojin Group, 122, SZ F. delavayi Muli County, Sichuan Province viii.1978 Qing-sheng Zhao, 6520, SZ F. anhuiensis Fengjie County, Chongqing City iv.1964 Hong-fu Zhou, 107620, SZ F. davidi Tianquan County, Sichuan Province iv.1953 Xing-ao Jiang, 33752, SZ Putative species Lixian County, Sichuan Province vi.1956 Di-ping He, 44406, SZ examined by light microscopy (LM) and scanning To ascertain the significance level of the differences in electron microscopy (SEM); six of these species are the stomata of each species, the length of the stomatal reported for the first time. The leaf epidermis of all pole at which the guard cells meet was analysed using species is described in detail. More importantly, sto- a statistical two-sided t-test. The formulae used were matal orientation was discovered in Fritillaria, and as follows: appears to exist widely in Liliaceae. ()2 2 2 12 TXXSNSN=−[]12()WW1 + 2 =−{}[]()2 +−()2 ()+−12 MATERIAL AND METHODS SNSNSNNW 11112212 2 Adult leaves of 17 species were obtained from her- () tNN12− P 1+− 22 = T barium specimens or collected by the author (Qiang Wang) in the field (Table 1). Vouchers were deposited where T is the statistic, X is the arithmetic mean, S in the herbarium at Sichuan University, Chengdu is the standard deviation and N is the sample size. (SZ). Adult, fully expanded, sun-exposed leaves were Given the significance level a=0.05, the coefficient P taken as samples. Because some of the herbarium was compared with a to confirm the significance level specimens were old, it was not possible to use Jef- of the differences in the stomata of each species. The fery’s solution or other methods to produce epidermal Slides were deposited in the Plant Taxonomic Labo- preparations. From a comparison of several methods, ratory, College of Life Sciences, Sichuan University. the best technique involved gentle scraping with a The materials for SEM observation were taken stainless steel blade of leaves macerated in water for straight from herbarium specimens, coated and several days. Pieces of leaf epidermis were stained in observed under a Hitachi-SX-450 scanning electron a solution of 1% safranin in 50% ethanol, and then microscope. The terminology of Baranova (1987, 1992) dehydrated in an ethanol series before being mounted was adopted for stomatal classification, and the in Canada balsam. The slides were examined and terminology for other characters was based on the photographed using an Olympus BH-2 light micro- classification of Wilkinson (1979). scope. Five slides of each species were observed. Twenty stomata were measured for each sample, and RESULTS the mean was calculated with the range of variation. The stomatal index was counted in an area of The leaf epidermal characteristics of the 17 species 0.5 mm ¥ 0.6 mm using the formula: stomatal are listed in Tables 2 and 3. The LM characteristics of index = number of stomatal apparatuses/(number of the adaxial (Ad) and abaxial (Ab) epidermis are illus- stomatal apparatuses + number of epidermal cells). trated in Figs 1–17 and 18–34, respectively. The LM © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 93–109 © 2009 The Linnean Society of London, Table 2. Leaf epidermal characteristics of Fritillaria under light microscopy (surface view) Adaxial epidermis Abaxial epidermis Pattern of Stomatal Shape of Pattern of Botanical Journal of the Linnean Society Shape anticlinal apparatus ordinary anticlinal Size of Stomatal Taxon of cells walls cells cells walls stomata (mm) index (%) Stomato-type Figure Fritillaria pallidiflora Irr Sin Present Irr Sin 73.4 ¥ 49.9 26.9 Ano Figs 14, 31, 48, 55 F. sichuanica Pol Str-curv Absent Pol Str-curv 92.1 ¥ 52.9 13.6 Ano Figs 1, 18, 35 COMPARATIVE LEAF MORPHOLOGY IN F. cirrhosa Pol Str-curv Absent Pol Str-curv 63.1 ¥ 29.3 14.3 Ano Figs 2, 19, 36 F. taipaiensis Pol Str-curv Absent Pol Str-curv 81.3 ¥ 70.2 13.0 Ano Figs 3, 20, 37 F. crassicaulis Pol Str-curv Absent Pol Str-curv 69.5 ¥ 37.6 29.6 Ano Figs 4, 21, 38 F. sinica Pol Str-curv Absent Pol Str-curv 103.9 ¥ 61.5 34.8 Ano Figs 5, 22, 39 F. walujewi Pol Str-curv Absent Pol Str-curv 65.6 ¥ 45.6 15.8 Ano Figs 6, 23, 40 F. verticillata Pol Str-curv Present Pol Str-curv 89.9 ¥ 46.5 22.2 Ano Figs 11, 28, 45, 52 F. thunbergii Pol Str-curv Absent Pol Str-curv 90.9 ¥ 45.5 31.3 Ano Figs 7, 24, 41 F. tortifolia Pol Str-curv Present Pol Str-curv 60.9 ¥ 45.1 14.3 Ano Figs 12, 29, 46, 53 F.