Comparative Morphology of the Leaf Epidermis in Fritillaria (Liliaceae) from China

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

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

Table 2. Leaf epidermal characteristics of Fritillaria under light microscopy (surface view)

Adaxial epidermis Abaxial epidermis

Pattern of Stomatal Shape of Pattern of

oaia ora fteLnenSociety Linnean the of Journal Botanical 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 IN MORPHOLOGY LEAF COMPARATIVE 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. przewalskii Pol Str-curv Absent Pol Str-curv 75.7 ¥ 48.3 23.5 Ano Figs 8, 25, 42

2009, , F. unibracteata Pol Str-curv Absent Pol Str-curv 90.5 ¥ 63.9 23.8 Ano Figs 9, 26, 43 F. dajinensis Pol Str-curv Absent Pol Str-curv 93.4 ¥ 44.6 20.0 Ano Figs 10, 27, 44 F. delavayi Pol Str-curv Present Pol Str-curv 71.1 ¥ 56.1 15.4 Ano Figs 13, 30, 47, 54

160 F. anhuiensis Pol Str-curv Absent Irr Sin 72.0 ¥ 45.9 23.1 Ano Figs 15, 32, 49

93–109 , F. davidi Irr Sin Absent Irr Sin 117.0 ¥ 59.4 29.4 Ano Figs 16, 33, 50 Putative species Pol Str-curv Absent Pol Str-curv 100.5 ¥ 53.6 18.8 Ano Figs 17, 34, 51

Ano, anomocytic; Irr, irregular; Pol, polygonal; Sin, sinuous; Str-curv, straight to curved. FRITILLARIA

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Table 3. Leaf epidermal characteristics of Fritillaria under scanning electron microscopy (surface view)

Taxon Outer stomatal rim Cuticular membrane Wax ornamentation Figure

Fritillaria pallidiﬂora Single and risen Striated Flaked and granular Figs 69, 86. F. sichuanica Single and not risen Striated Flaked and granular Figs 56, 73. F. cirrhosa Single and not risen Striated Granular Figs 57, 74. F. taipaiensis Double and risen Striated Invisible Figs 58, 75. F. crassicaulis Double and not risen Striated Flaked Figs 59, 76. F. sinica Single and not risen Striated Granular Figs 60, 77. F. walujewi Single and risen Striated Flaked and granular Figs 61, 78. Downloaded from https://academic.oup.com/botlinnean/article/160/1/93/2418345 by guest on 27 November 2020 F. verticillata Single and risen Striated Flaked Figs 66, 83. F. thunbergii Single and not risen Smooth Granular Figs 62, 79. F. tortifolia Single and risen Striated Flaked and granular Figs 67, 84. F. przewalskii Single and risen Striated Flaked and granular Figs 63, 80. F. unibracteata Single and not risen Striated Flaked and granular Figs 64, 81. F. dajinensis Single and not risen Striated Flaked Figs 65, 82. F. delavayi Single and risen Striated Flaked Figs 68, 85. F. anhuiensis Single and not risen Smooth Invisible Figs 70, 87. F. davidi Single and not risen Striated Flaked and granular Figs 71, 88. Putative species Single and not risen Striated Flaked and granular Figs 72, 89.

characteristics of the stomata in Ab are illustrated in and an irregular shape with a sinuous wall in Figs 35–51. The LM characteristics of the four species Ab occur in F. anhuiensis S.C.Chen & S.F.Yin with stomata present in Ad are illustrated in Figs 52– (Figs 15, 32). Although examined by SEM, the anti- 55. The SEM characteristics of Ab and Ad of the 17 clinal walls of the cells were obscure because they species are illustrated in Figs 56–73 and 74–89, were coated. respectively. It appears that stomatal and other epidermal features are constant within species and therefore represent good characters for taxonomy. STOMATAL APPARATUS AND GUARD CELLS The development of Fritillaria appears to be agenous. All stomata are of the anomocytic type and have no EPIDERMAL CELLS subsidiary cells. Stomata are present on both surfaces Epidermal cells of Fritillaria examined under LM are in F. pallidiflora (Figs 48, 55), F. verticillata (Figs 45, usually polygonal in form and the anticlinal cell walls 52), F. tortifolia (Figs 46, 53) and F. delavayi (Figs 47, are straight or curved. In a few species they are 54). However, in other species, stomata only occur on irregular, with sinuous anticlinal cell walls. The pat- the abaxial surface. The shapes of guard cells exam- terns of the anticlinal cells may vary in different ined by LM are usually narrow elliptical in F. sichua- species, even between Ad and Ab of the same species. nica (Fig. 35), F. cirrhosa (Fig. 36), F. crassicaulis The ordinary cell shape in Ad and Ab of most species, (Fig. 38), F. sinica S.C.Chen (Fig. 39), F. thunbergii including F. sichuanica S.C.Chen (Figs 1, 18), F. cir- (Fig. 41), F. przewalskii (Fig. 42), F. dajinensis rhosa (Figs 2, 19), F. taipaiensis P.Y.Li (Figs 3, 20), F. (Fig. 44), F. verticillata in Ab (Fig. 45), F. pallidiflora crassicaulis S.C.Chen (Figs 4, 21), F. sinica S.C.Chen (Figs 48, 55), F. anhuiensis (Fig. 49), F. davidi (Figs 5, 22), F. walujewi (Figs 6, 23), F. verticillata (Fig. 50), the putative species (Fig. 51) and F. tortifo- Wall. (Figs 11, 28), F. thunbergii (Figs 7, 24), F. torti- lia in Ad (Fig. 53). The shapes of the guard cells folia X.Z.Duan & X.J.Zheng (Figs 12, 29), F. przewal- are wide elliptical in F. verticillata in Ad (Fig. 52), skii (Figs 8, 25), F. unibracteata Hsiao & K.C.Hsia and elliptical in F. taipaiensis (Fig. 37), F. walujewi (Figs 9, 26), F. dajinensis S.C.Chen (Figs 10, 27), (Fig. 40), F. unibracteata (Fig. 43), F. tortifolia in Ab F. delavayi (Figs 13, 30) and the putative species (Fig. 46) and F. delavayi (Figs 47, 54). The guard cells (Figs 17, 34), is polygonal, and the anticlinal cell of all species are slightly elevated above the ordinary walls are straight or curved. Irregular shapes and cells. Under SEM, the outer stomatal rims are double sinuous walls occur in Ad and Ab of F. pallidiflora in F. taipaiensis (Fig. 75) and F. crassicaulis (Fig. 76), (Figs 14, 31) and F. davidi Franch. (Figs 16, 33). A risen in F. taipaiensis, but not risen in F. crassicaulis. polygonal shape with a straight to curved wall in Ad In other species, the outer stomatal rims are single

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Figures 1–12. Characteristics of adaxial epidermal cells (light microscopy). Ordinary polygonal-shaped cells. Fig. 1. Fritillaria sichuanica. Fig. 2. Fritillaria cirrhosa. Fig. 3. Fritillaria taipaiensis. Fig. 4. Fritillaria crassicaulis. Fig. 5. Fritillaria sinica. Fig. 6. Fritillaria walujewi. Fig. 7. Fritillaria thunbergii. Fig. 8. Fritillaria przewalskii. Fig. 9. Fritil- laria unibracteata. Fig. 10. Fritillaria dajinensis. Fig. 11. Fritillaria verticillata, with stomata present. Fig. 12. Fritillaria tortifolia, with stomata present. Scale bar, 50 mm. and risen in F. walujewi (Fig. 78), F. przewalskii smooth, and striated in other species. The wax (Fig. 80), F. verticillata (Fig. 83), F. tortifolia (Fig. 84), ornamentations of F. taipaiensis (Fig. 58) and F. delavayi (Fig. 85) and F. pallidiﬂora (Fig. 86), F. anhuiensis (Fig. 70) are invisible. Granular wax and single but not risen in F. sichuanica (Fig. 73), ornamentation occurs in F. cirrhosa (Fig. 57), F. sinica F. cirrhosa (Fig. 74), F. sinica (Fig. 77), F. thunbergii (Fig. 60) and F. thunbergii (Fig. 62). Flaked wax orna- (Fig. 79), F. unibracteata (Fig. 81), F. dajinensis mentation occurs in F. crassicaulis (Fig. 59), F. dajin- (Fig. 82), F. anhuiensis (Fig. 87), F. davidi (Fig. 88) ensis (Fig. 65), F. verticillata (Fig. 66) and F. davidi and the putative species (Fig. 89). (Fig. 71). Wax ornamentations of other species are ﬂaked and granular.

CUTICULAR AND WAX ORNAMENTATION Hairs are absent from both Ad and Ab of all species T-TEST ANALYSES examined. Under SEM, the cuticular membrane of The results of statistical analyses based on the data of F. thunbergii (Fig. 62) and F. anhuiensis (Fig. 70) is the long axis and the signiﬁcance levels of the differ-

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Figures 13–24. Characteristics of epidermal cells (light microscopy). Figs 13–17. Adaxial. Figs 18–24. Abaxial. Fig. 13. Ordinary polygonal-shaped cells of Fritillaria delavayi with stomata present. Fig. 14. Ordinary irregularly shaped cells of Fritillaria pallidiﬂora with stomata present. Fig. 15. Ordinary polygonal-shaped cells of Fritillaria anhuiensis. Fig. 16. Ordinary irregularly shaped cells of Fritillaria davidi. Fig. 17. Ordinary polygonal-shaped cells of the putative species. Fig. 18. Ordinary polygonal-shaped cells of Fritillaria sichuanica. Fig. 19. Ordinary polygonal-shaped cells of Fritillaria cirrhosa. Fig. 20. Ordinary polygonal-shaped cells of Fritillaria taipaiensis. Fig. 21. Ordinary polygonal-shaped cells of Fritillaria crassicaulis. Fig. 22. Ordinary polygonal-shaped cells of Fritillaria sinica. Fig. 23. Ordinary polygonal-shaped cells of Fritillaria walujewi. Fig. 24. Ordinary polygonal-shaped cells of Fritillaria thunbergii. Scale bar, 50 mm.

ences in the stomata of each species are listed in DISCUSSION AND CONCLUSION Tables 4 and 5. The results show that the difference PREVIOUS TAXONOMIC CONCLUSIONS in the long axis between F. tortifolia and F. davidi is the most significant, and the difference between F. In China, Fritillaria bulbs have been used in thunbergii and F. unibracteata is the least significant. Chinese traditional medicine since ancient times. The differences between F. verticillata and F. unibrac- Some species of the genus are rare, leading to diffi- teata and between F. thunbergii and F. unibracteata culties in the collection of materials for research. are not significant. However, the ‘P value’ of the Thus, few studies have been performed on the clas- difference between F. verticillata and F. thunbergii is sification of Fritillaria. Results from the observation less than 0.05, and the difference is significant. of the leaf epidermis show that stomatal and other

Table 4. ‘T’ of two-sided t-test within the genus Fritillaria

PAL SIC CIR TAI CRA SIN WAL VER THU TOR PRZ UNI DAJ DEL ANH DAV PUT

oaia ora fteLnenSociety Linnean the of Journal Botanical PAL – SIC 34.099 – CIR 17.095 68.568 – TAI 13.602 27.595 39.210 –

CRA 6.635 56.272 13.535 26.527 – IN MORPHOLOGY LEAF COMPARATIVE SIN 49.452 26.637 80.243 46.837 70.080 – WAL 13.216 65.402 5.254 34.612 8.533 77.564 – VER 28.589 5.700 58.313 19.966 46.358 29.281 54.815 – THU 30.859 3.237 62.205 7.277 50.231 27.901 58.817 2.427 – TOR 22.957 93.091 5.265 52.867 21.703 98.140 11.754 76.243 82.207 – PRZ 4.216 48.701 30.062 14.460 15.593 64.182 25.173 37.195 41.485 44.802 – UNI 26.365 3.290 50.128 17.605 39.602 23.832 46.718 1.157 0.788 61.424 30.641 – DAJ 34.534 3.322 65.572 27.920 53.991 21.851 62.346 8.168 6.026 84.774 46.001 5.569 – DEL 3.851 50.566 16.512 22.309 3.433 65.323 11.725 41.540 45.032 24.871 11.181 35.878 48.999 –

2009, , ANH 2.492 55.439 20.221 22.688 5.957 69.430 15.126 44.228 48.428 31.126 10.332 36.901 52.430 2.079 – DAV 59.688 42.123 84.004 57.461 75.662 19.999 81.577 43.860 42.892 95.490 70.189 38.682 38.081 72.098 74.559 – PUT 36.651 13.950 57.379 30.390 48.576 5.113 54.495 16.867 15.504 66.156 41.369 14.397 11.265 45.449 46.391 21.384 – 160

93–109 , ANH, F. anhuiensis; CIR, F. cirrhosa; CRA, F. crassicaulis; DAJ, F. dajinensis;DAV,F. davidi; DEL, F. delavayi; PAL, F. pallidiﬂora; PRZ, F. przewalskii; PUT, putative species; SIC, F. sichuanica; SIN, F. sinica; TAI, F. taipaiensis; THU, F. thunbergii; TOR, F. tortifolia; UNI, F. unibracteata; VER, F. verticillata; WAL, F. walujewi. FRITILLARIA

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Table 5. Signiﬁcance level of the differences between the species of the genus Fritillaria

PAL SIC CIR TAI CRA SIN WAL VER THU TOR PRZ UNI DAJ DEL ANH DAV PUT

PAL – SIC P < a – CIR P < a P < a – TAI P < a P < a P < a – CRA P < a P < a P < a P < a – SIN P < a P < a P < a P < a P < a – WAL P < a P < a P < a P < a P < a P < a – VER P < a P < a P < a P < a P < a P < a P < a – THU P < a P < a P < a P < a P < a P < a P < a P < a – oaia ora fteLnenSociety Linnean the of Journal Botanical TOR P < a P < a P < a P < a P < a P < a P < a P < a P < a – PRZ P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a – UNI P < a P < a P < a P < a P < a P < a P < a P > a P > a P < a P < a – DAJ P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a – DEL P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a – ANH P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a – DAV P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a – PUT P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a P < a –

t1–P/2(N1 + N2 - 2) = T; a=0.05; ANH, F. anhuiensis; CIR, F. cirrhosa; CRA, F. crassicaulis; DAJ, F. dajinensis;DAV,F. davidi; DEL, F. delavayi; PAL, F. pallidiﬂora; PRZ, F. przewalskii; PUT, putative species; SIC, F. sichuanica; SIN, F. sinica; TAI, F. taipaiensis; THU, F. thunbergii; TOR, F. tortifolia; UNI, F. unibracteata; VER, F. verticillata; WAL, F. walujewi. 2009, , 160

93–109 , Downloaded from https://academic.oup.com/botlinnean/article/160/1/93/2418345 by guest on 27 November 2020 November 27 on guest by https://academic.oup.com/botlinnean/article/160/1/93/2418345 from Downloaded COMPARATIVE LEAF MORPHOLOGY IN FRITILLARIA 101

KEY TO SPECIES STUDIED ON THE BASIS OF LEAF EPIDERMAL CHARACTERS 1a. Abaxial: ordinary cells polygonal and anticlinal walls straight to curved 2a. Adaxial: stomatal apparatus cells absent 3a. Outer stomatal rim single 4a. Outer stomatal rim not risen 5a. Wax ornamentation flaked or granular 6a. Wax ornamentation granular 7a. Cuticular membrane striated 8a. Long axis of stoma >90 mm ...... F. sinica 8b. Long axis of stoma <70 mm ...... F. cirrhosa Downloaded from https://academic.oup.com/botlinnean/article/160/1/93/2418345 by guest on 27 November 2020 7b. Cuticular membrane smooth ...... F. thunbergii 6b. Wax ornamentation flaked ...... F. dajinensis 5b. Wax ornamentation flaked and granular 9a. Shape of guard cells narrow elliptical 10a. Stomata sparse and stomatal index <14% ...... F. sichuanica 10b. Stomata dense and stomatal index >18% ...... The putative species 9b. Shape of guard cells elliptical ...... F. unibracteata 4b. Outer stomatal rim risen 11a. Stomata sparse and stomatal index <16% ...... F. walujewi 11b. Stomata dense and stomatal index >20% ...... F. przewalskii 3b. Outer stomatal rim double 12a. Outer stomatal rim risen ...... F. taipaiensis 12b. Outer stomatal rim not risen ...... F. crassicaulis 2b. Adaxial: stomatal apparatus cells present 13a. Wax ornamentation flaked 14a. Long axis of stoma >85 mm ...... F. verticillata 14b. Long axis of stoma <75 mm ...... F. delavayi 13b. Wax ornamentation flaked and granular ...... F. tortifolia 1b. Abaxial: ordinary cells irregular and anticlinal walls sinuous 15a. Adaxial: ordinary cells irregular and anticlinal walls sinuous 16a. Adaxial: Stomatal apparatus cells present ...... F. pallidiflora 16b. Adaxial: Stomatal apparatus cells absent ...... F. davidi 15b. Adaxial: ordinary cells polygonal and anticlinal walls straight to curved ...... F. anhuiensis

epidermal features are constant within the species epidermis shows that all six species are distinct from studied. There is no significant variation between other species. different populations of the same species. However, there are obvious differences in leaf epidermal characteristics between most species. The characteristics, TAXONOMIC POSITION OF THE PUTATIVE SPECIES such as stomata and wax ornamentation, of each The putative species was first recognized by Yong- species are specific. A key to the species of Fritillaria kang Yang, but was unpublished. This species was has been established on the basis of the leaf collected in 1956 by Di-ping He in the field at Saishou epidermal morphology. The species studied can be Gully, Dabanzhao, Lixian County, Sichuan Province classified in the key by combining the LM and at 3750 m. Herbarium specimens with the collector’s SEM characteristics of the leaf epidermis. Fritillaria number 44406 were deposited at SZ. A preliminary anhuiensis is the only species in which the patterns observation of the herbarium specimens showed that of anticlinal cells may vary between Ad and Ab, and the characteristics of the species included: (1) the bulb can be classified only by LM observation. The leaf has two scales, and its tunic is obscure; (2) the bulb is epidermis of F. sichuanica, F. sinica, F. verticillata, 1–1.5 cm in diameter; (3) the stem is about 35–40 cm F. dajinensis, F. davidi and the putative species is in height with alternate or subopposite phyllotaxy; (4) reported for the first time. The stomatal and other the leaf blade is linear-lanceolate; (5) the leaf blade is epidermal features of these species are constant, and about 6–9 cm in length and 2–8 mm in breadth, and valuable for classification. Evidence from the leaf its apex is not cirrose; (6) the flowers are nodding

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Figures 25–36. Characteristics of abaxial epidermal cells (light microscopy). Fig. 25. Ordinary polygonal-shaped cells of Fritillaria przewalskii. Fig. 26. Ordinary polygonal-shaped cells of Fritillaria unibracteata. Fig. 27. Ordinary polygonal- shaped cells of Fritillaria dajinensis. Fig. 28. Ordinary polygonal-shaped cells of Fritillaria verticillata. Fig. 29. Ordinary polygonal-shaped cells of Fritillaria tortifolia. Fig. 30. Ordinary polygonal-shaped cells of Fritillaria delavayi. Fig. 31. Ordinary irregularly shaped cells of Fritillaria pallidiﬂora. Fig. 32. Ordinary irregularly shaped cells of Fritillaria anhuiensis. Fig. 33. Ordinary irregularly shaped cells of Fritillaria davidi. Fig. 34. Ordinary polygonal-shaped cells of the putative species. Scale bar, 50 mm. Fig. 35. Narrow elliptical guard cells of Fritillaria sichuanica. Fig. 36. Narrow elliptical guard cells of Fritillaria cirrhosa. Scale bar, 10 mm.

with a solitary bract; (7) the tepals are black–purple and not risen, and the cuticular membrane is striated and tessellated with no spot; and (8) the most obvious with flaked and granular wax ornamentations. characteristic is that the inflorescence is a raceme However, the stomatal index is lower than that of the with six flowers above. The observation of the leaf two former species. Moreover, the inflorescence is epidermis showed that the species was similar to F. unusual. The putative species may be a new species; sichuanica and F. unibracteata: (1) under LM, it is we believe that it belongs to section Fritillaria, with polygonal with straight or curved anticlinal cell walls F. sichuanica and F. unibracteata. An attempt will be in both Ad and Ab, and stomata are absent from Ad; made to collect this species and to perform further (2) under SEM, the shapes of the guard cells are morphological and cytotaxonomical investigations to narrow elliptical, the outer stomatal rims are single confirm this proposal.

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Figures 37–48. Characteristics of abaxial epidermal cells (light microscopy). Fig. 37. Elliptical guard cells of Fritillaria taipaiensis. Fig. 38. Narrow elliptical guard cells of Fritillaria crassicaulis. Fig. 39. Narrow elliptical guard cells of Fritillaria sinica. Fig. 40. Elliptical guard cells of Fritillaria walujewi. Fig. 41. Narrow elliptical guard cells of Fritillaria thunbergii. Fig. 42. Narrow elliptical guard cells of Fritillaria przewalskii. Fig. 43. Elliptical guard cells of Fritillaria unibracteata. Fig. 44. Narrow elliptical guard cells of Fritillaria dajinensis. Fig. 45. Narrow elliptical guard cells of Fritillaria verticillata. Fig. 46. Narrow elliptical guard cells of Fritillaria tortifolia. Fig. 47. Elliptical guard cells of Fritillaria delavayi. Fig. 48. Narrow elliptical guard cells of Fritillaria pallidiﬂora. Scale bar, 10 mm.

SUBDIVISION OF FRITILLARIA (including Baker’s subgenera Monocodon and Goniocarpa), Amblirion, Theresia (including Baker’s The subdivision of the genus Fritillaria has been subgenus Rhinopetalum), Petilium and Liliorhiza attempted by many scholars. Baker (1874) recog- (including Baker’s subgenus Korolkowia). Boissier nized ten subgenera: Eufritillaria [= Fritillaria], (1882) recognized four sections: Eufritillaria, Ther- Monocodon, Goniocarpa, Amblirion, Rhinopetalum, esia, Petilium and Liliorhiza. Section Eufritillaria Theresia, Petilium, Liliorhiza, Korolkowia and was subdivided into two subsections: Trichostyleae Notholirion. Bentham & Hooker (1883) transferred (including Baker’s subgenera Eufritillaria and Mono- Baker’s subgenus Notholirion to Lilium, and sub- codon) and Olostyleae (including Baker’s subgenera divided the genus into ﬁve sections: Eufritillaria Amblirion, except F. pudica, and Rhinopetalum).

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Figures 49–55. Characteristics of epidermal cells (light microscopy). Figs 49–51. Abaxial. Figs 52–55. Adaxial. Fig. 49. Narrow elliptical guard cells of Fritillaria anhuiensis. Fig. 50. Narrow elliptical guard cells of Fritillaria davidi. Fig. 51. Narrow elliptical guard cells of the putative species. Fig. 52. Wide elliptical guard cells of Fritillaria verticillata. Fig. 53. Narrow elliptical guard cells of Fritillaria tortifolia. Fig. 54. Elliptical guard cells of Fritillaria delavayi. Fig. 55. Narrow elliptical guard cells of Fritillaria pallidiﬂora. Scale bar, 10 mm.

Turrill & Sealy (1980) supported Boissier’s subdivi- of F. pallidiflora are different from those of species sion, referring to Boissier’s section Eufritillaria as belonging to section Liliorhiza. Statistical analysis Fritillaria, and subsection Trichostyleae as subsection also shows that there is a significant difference in the Fritillaria.ForFlora Reipublicae Popularis Sinicae, long axis of the stoma between species of section Tang & Wang (1980) recognized three sections Liliorhiza and species of section Fritillaria. Unfortu- occurring in China: Fritillaria, Rhinopetalum and nately, Fritillaria karelini Baker, the sole Chinese Liliorhiza. Following Tang & Wang (1980), the 17 species of section Rhinopetalum, was not collected for species studied can be divided into two sections: Frit- this research. No detailed SEM characteristics using illaria and Liliorhiza. Rønsted et al. (2005) presented the widely adopted terminology have been reported a molecular phylogenetic analysis based on plastid for this species. However, Li et al. (1993) observed its and nuclear DNA which showed Liliorhiza to be a leaf epidermis by LM. The results showed that it sister to a clade composed of all the other subgenera. had irregularly shaped cells and sinuous anticlinal Based on the stomatal and other epidermal features, walls. To conclude, the leaf epidermis characteristics it appears that species of section Fritillaria, excluding approximately support the subdivision of Tang & F. pallidiflora, generally have polygonal-shaped cells Wang (1980). and the patterns of the anticlinal walls are straight or curved. Species of section Liliorhiza generally have irregularly shaped cells and the patterns of the anti- EVIDENCE FOR FLORISTIC PLANT GEOGRAPHY clinal walls are sinuous. The LM characteristics of F. In China, F. pallidiflora, F. walujewi, F. verticillata pallidiflora are similar to those of species belonging to and F. tortifolia of section Fritillaria are distributed section Liliorhiza; however, the SEM characteristics in Xinjiang Province. The stomata in these species,

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Figures 56–64. Characteristics of abaxial epidermal cells (scanning electron microscopy). Striated cuticular membrane. Fig. 56. Fritillaria sichuanica. Fig. 57. Fritillaria cirrhosa. Fig. 58. Fritillaria taipaiensis. Fig. 59. Fritillaria crassicaulis. Fig. 60. Fritillaria sinica. Fig. 61. Fritillaria walujewi. Fig. 63. Fritillaria przewalskii. Fig. 64. Fritillaria unibracteata. Fig. 62. Smooth cuticular membrane of Fritillaria thunbergii. Scale bar, 50 mm.

except for F. walujewi, are present on Ad and Ab, Some species spread from Central Asia, joining the different from other species of the section mainly Turgai Flora, along the Altai–Sayan route from the distributed in the Hengduan Mountains region of south of Siberia to the Sayan Mountains and Altai China. Based on the plant morphology, they are Mountains. Others are distributed in the Hengduan similar to F. olgae Vved. distributed in Central Asia. Mountains region of China and belong to the Phava The stomatal characteristics support an origin of the Flora. species in Xinjiang Province from groups in Central Asia, but a different origin for the species of section Fritillaria distributed in the Hengduan Mountains STOMATAL ORIENTATION OF FRITILLARIA region of China. Thus, there are two ﬂoristic ele- The stomatal pole is the axis of a stoma where the ments for species of section Fritillaria in China. guard cells meet. It is usually the long axis in many

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Figures 65–73. Characteristics of abaxial epidermal cells (scanning electron microscopy). Striated cuticular membrane. Fig. 65. Fritillaria dajinensis. Fig. 66. Fritillaria verticillata. Fig. 67. Fritillaria tortifolia. Fig. 68. Fritillaria delavayi. Fig. 69. Fritillaria pallidiﬂora. Fig. 71. Fritillaria davidi. Fig. 72. Putative species. Fig. 70. Smooth cuticular membrane of Fritillaria anhuiensis. Scale bar, 50 mm. Fig. 73. Outer stomatal rims of the stomatal apparatus are single and not risen in Fritillaria sichuanica. Scale bar, 10 mm.

species, but not obviously long in some suborbiculate Mill., Disporopsis Hance and Maianthemum stomata. Figure 90 shows the stomatal pole. In Frit- F.H.Wigg. In many genera of angiosperms, such as illaria, the orientations of all stomatal poles of the Chloranthus Sw. (Kong, 2001) and Schisandra Michx. leaf epidermis are approximately uniform. Figure 91 (Yang & Lin, 2005), the phenomenon is not seen. It shows the orientation of all stomatal poles of Fritil- appears that the consistency of the orientation should laria. Based on our research of Liliaceae and Aspar- be valuable in classiﬁcation. The phenomenon is agaceae sensu APG II (2003) (Q. Wang et al., unpubl. named ‘stomatal orientation’. It is an important taxo- data), this phenomenon can be observed in the genera nomic characteristic of Fritillaria and other Liliaceae Lilium L., Cardiocrinum (Endl.) Lindl., Polygonatum and some other monocotyledons.

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Figures 74–82. Characteristics of abaxial epidermal cells (scanning electron microscopy). Fig. 74. Outer stomatal rims of the stomatal apparatus are single and not risen in Fritillaria cirrhosa. Fig. 75. Outer stomatal rims of the stomatal apparatus are double and risen in Fritillaria taipaiensis. Fig. 76. Outer stomatal rims of the stomatal apparatus are double and not risen in Fritillaria crassicaulis. Fig. 77. Outer stomatal rims of the stomatal apparatus are single and not risen in Fritillaria sinica. Fig. 78. Outer stomatal rims of the stomatal apparatus are single and risen in Fritillaria walujewi. Fig. 79. Outer stomatal rims of the stomatal apparatus are single and not risen in Fritillaria thunbergii. Fig. 80. Outer stomatal rims of the stomatal apparatus are single and risen in Fritillaria przewalskii. Fig. 81. Outer stomatal rims of the stomatal apparatus are single and not risen in Fritillaria unibracteata. Fig. 82. Outer stomatal rims of the stomatal apparatus are single and not risen in Fritillaria dajinensis. Scale bar, 10 mm.

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Figures 83–89. Characteristics of abaxial epidermal cells (scanning electron microscopy). Fig. 83. Outer stomatal rims of the stomatal apparatus are single and risen in Fritillaria verticillata. Fig. 84. Outer stomatal rims of the stomatal apparatus are single and risen in Fritillaria tortifolia. Fig. 85. Outer stomatal rims of the stomatal apparatus are single and risen in Fritillaria delavayi. Fig. 86. Outer stomatal rims of the stomatal apparatuses are single and risen in Fritillaria pallidiﬂora. Fig. 87. Outer stomatal rims of the stomatal apparatus are single and not risen in Fritillaria anhuiensis. Fig. 88. Outer stomatal rims of the stomatal apparatus are single and not risen in Fritillaria davidi. Fig. 89. Outer stomatal rims of the stomatal apparatus are single and not risen in the putative species. Scale bar, 10 mm.

ACKNOWLEDGEMENTS Science and Technology (grant no. 2005DKA21403). We are grateful to Professor Ping Li for her invalu- This work was supported by the National Natural able help with the research. We also thank Julia Science Foundation of China (grant no. 30670146) Gertsen for her advice and great help with the and National Infrastructure of Natural Resources for English language of the manuscript.

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