全 文 ：植 物 分 类 学 报 45 (3): 274–284(2007) doi:10.1360/aps050016
Acta Phytotaxonomica Sinica http://www.plantsystematics.com
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Received: 1 February 2005 Accepted: 7 April 2007
Supported by the National Natural Science Foundation of China, Grant Nos. 30570117 & 30270105, and the Set-up Fund from
the Chinese Academy of Sciences to the Excellent Post-doctor.
* Authors for correspondence. E-mail: xiangyunzhu@ibcas.ac.cn, xjzkjyc@163.com; Tel: 86-10-62836111.
Systematic significance of leaf epidermal features in Apios
and Cochlianthus (Leguminosae)
1, 2 REN Bo 1ZHU Xiang-Yun* 3JIANG Yan-Cheng*
1 (Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China)
2 (Graduate University of Chinese Academy of Sciences, Beijing 100049, China)
3 (College of Biology and Technology, Xinjiang University, Xinjiang 830046, China)
Abstract Nineteen leaf epidermis samples representing six species in Apios and two in
Cochlianthus were examined for the first time using both light microscopy and scanning
electron microscopy. Leaf epidermal characters of these two closely related genera, such as
shape of cells, pattern of anticlinal walls, cuticular membrane and wax ornamentation, are
usually stable within species and thus of great significance in understanding the relationships
between and within genera. The results indicate that (1) A. gracillima Dunn should be
recognized as a distinct species, not a variety of Apios delavayi Franch., (2) the division of
Apios into two subgenera is unreasonable, and (3) Apios and Cochlianthus are retained as two
distinct genera.
Key word Leguminosae, Apios, Cochlianthus, leaf epidermis, systematics.
The genera Apios Fabr. and Cochlianthus Benth. are members of subtribe Erythrininae,
tribe Phaseoleae in the subfamily Papilionoideae of Leguminosae. Apios is distributed in East
Asia and North America, consisting of no more than 10 species, and Cochlianthus, which is
restricted to East Asia, has only two distinct species. These two genera have long been
considered to be very closely related (Li, 1952; Hutchinson, 1964; Lackey, 1981; Woods,
1988; Li, 1995).
Apios has great economic importance. It has been used as food because of its high dry
weight protein content and genistein, an anticarcinogenic compound, found in its edible tubers
(Safford, 1925; Haskin, 1929; Walker, 1939; Duke, 1984; Blackmon, 1986; Vietmeyer, 1986;
Walter et al., 1986; Krishnan, 1998). Extensive research has been done on the genus,
including studies on reproduction (Bruneau & Anderson, 1988), pollination (Westerkamp & Paul,
1993; Bruneau & Anderson, 1994), cytology (Atchison, 1949; Lewis et al., 1962; Seabrook &
Dionne, 1976) and evolution (Joly & Bruneau, 2004). The genus Cochlianthus has rarely been
studied. However, there are few taxonomic revisions of these two genera, other than that of
Woods (1988), whose work was mainly based on gross-morphology.
Leaf epidermal features have a number of advantages as taxonomic markers. They are of
value not only in making taxonomic decisions but also in developmental and evolutionary
studies (Stace, 1984). The leaf epidermal features in Apios and Cochlianthus, however, have
not yet been examined. The purpose of this study is to find significant taxonomic characters
of the leaf epidermis for a better understanding of the relationship and positions of the two
genera in tribe Phaseoleae.
No. 3 REN et al.: Systematic significance of leaf epidermal features in Apios and Cochlianthus 275
1 Material and methods
Mature leaves of 19 specimens representing six species in Apios and two in Cochlianthus
were collected from PE, E and GH (Table 1). The leaves for light microscopy (LM) study
were boiled in water before maceration in 20% NaClO solution. Pieces of leaf epidermis were
stained in a solution of 1% safranin (in 50% alcohol) before being mounted in gum. To check
the constancy of epidermal structure, three leaves were collected for each species, and at least
three slides were made from different parts of a single leaf. The stomatal index (I) was
calculated using the formula I=S/(E+S)×100, where S denotes the number of stomata per
unit area and E the number of epidermal cells of the same area. Materials for scanning
electron microscopy (SEM) observation were directly mounted on stubs without any
treatment. After gold-sputtering, the specimens were examined and photographed under a
Hitachi S-800.
The terminology followed that of Wilkinson (1979) and of Dilcher (1974).
Table 1 Source of materials
Species Locality Voucher
Apios americana Medik. Massachusetts, U.S.A. V. Bates & I. S. Elsik 218 (PE)
Maine, U.S.A. J. C. Solomon & A. Solomon 20056 (PE)
Florida, U.S.A. J. S. Miller & M. C. Merello 9063 (PE)
A. priceana B. L. Rob. Mississippi, U.S.A. J. D. Ray 6728 (GH)
Tennessee, U.S.A. H. K. Svenson 7325 (GH)
A. carnea (Wall.) Benth. ex Baker Zhongdian, Yunnan, China B. Ren 80 (PE)
Bomi, Xizang, China B. S. Li & C. C. Ni 6855 (PE)
Fengjie, Sichuan, China Z. R. Zhang 25930 (PE)
A. delavayi Franch. Zhongdian, Yunnan, China B. Ren 78 (PE)
Zayü, Xizang, China C. C. Ni 180 (PE)
Kangding, Sichuan, China K. Y. Lang & L. Q. Li 1250 (PE)
A. fortunei Maxim. Without precise locality, Anhui, China Y. L. Kang 950 (PE)
Hangzhou, Zhejiang, China S. Y. Zhang 1402 (PE)
Xianyou, Fujian, China Y. Lin 437 (PE)
A. gracillima Dunn Dongchuan, Yunnan, China H. D. Mclaren L111 (E)
Lijiang, Yunnan, China E. E. Maire 731 (E)
Cochlianthus gracilis Benth. Cona, Xizang, China Qinghai-Xizang Exped. 2444 (PE)
Cona, Xizang, China Qinghai-Xizang Exped. Supplement Team
751695 (PE)
C. montanus (Diels) Harms Zayü, Xizang, China G. Forrest 6108 (PE)
2 Results
2.1 Characteristics of leaf epidermis under LM
Hairs were abundant on both adaxial and abaxial epidermis of the two species of
Cochlianthus, but only present slightly on the veins and veinlets of the six species of Apios. In
most species, the epidermal cells were irregular in outline, with sinuolate (Figs. 7, 8, 12, 15,
16) or sinuate (Figs. 1–5, 9–11, 13) anticlinal walls, and the shapes of epidermal cells of a
same species were similar on both adaxial and abaxial sides. In only one species, Apios
gracillima, the shape of adaxial epidermal cells was polygonal with straight to arched
anticlinal walls and the abaxial epidermal cells were irregular with sinuolate anticlinal walls
Acta Phytotaxonomica Sinica Vol. 45 276
Figs. 1–8. Characteristics of adaxial epidermal cells in Apios and Cochlianthus under LM. 1. A. americana. 2. A. priceana.
3. A. carnea. 4. A. delavayi. 5. A. fortunei. 6. A. gracillima. 7. Cochlianthus gracilis. 8. C. montanus.
Scale bar = 50 µm.
No. 3 REN et al.: Systematic significance of leaf epidermal features in Apios and Cochlianthus 277
Figs. 9–16. Characteristics of abaxial epidermal cells in Apios and Cochlianthus under LM, type of stomatal apparatus.
9. Anomocytic of A. americana. 10, 11, 13. Laterocytic. 12, 14–16. Paracytic. 10. A. priceana. 11. A. carnea. 12. A.
delavayi. 13. A. fortunei. 14. A. gracillima. 15. C. gracilis. 16. C. montanus.
Scale bar=50 µm.
Acta Phytotaxonomica Sinica Vol. 45 278
(Figs. 6, 14). Stomata appeared only on the abaxial epidermis of all species. The stomatal
apparatuses were anomocytic (A. americana, Fig. 9), laterocytic (A. priceana, A. carnea, A.
fortunei, Figs. 10, 11, 13), or paracytic (A. delavayi, A. gracillima, C. gracilis, C. montanus,
Figs. 12, 14–16), with anisocytic stomata occasionally observed in some species (A. priceana,
A. delavayi, A. gracillima, C. montanus, Figs. 10, 12, 14, 16) (Table 2). The stomatal index
measured on these eight species ranged between 16.1 and 26.3 (Table 2).
Table 2 The characters of leaf epidermis in Apios and Cochlianthus under LM
Adaxial epidermis Abaxial epidermis Species
Shape of
cells
Pattern of
anticlinal
walls
Shape of
cells
Pattern of
anticlinal
walls
Size of
stomata
(µm2)
Stomatal
index
Stomatal
type
Other types of
stomatal apparatus
occasionally
observed
Apios americana Irr Sin Irr Sin 24.6×14.8 26.3 Ano Lat
A. priceana Irr Sin Irr Sin 23.6×16.2 19.2 Lat Ani
A. carnea Irr Sin Irr Sin 23.8×14.8 23.0 Lat Ano
A. delavayi Irr Sin Irr Si 23.4×17.6 19.4 Par Ani, Ano
A. fortunei Irr Sin Irr Sin 28.0×17.8 18.9 Lat –
A. gracillima Pol Str-arc Irr Si 25.0×19.2 24.0 Par Ani
Cochlianthus
gracilis Irr Si Irr Si 26.2×20.4 15.1 Par Ano
C. montanus Irr Si Irr Si 21.4×12.4 19.5 Par Ani, Ano
Ani, anisocytic; Ano, anomocytic; Irr, irregular; Lat, laterocytic; Par, paracytic; Pol, polygonal; Si, sinuolate; Sin, sinuate;
Str-arc, straight to arched.
2.2 Characteristics of leaf epidermis under SEM
The cuticular membrane was smooth (Figs. 20, 22, 24, 30), striate (Figs. 17, 18, 21,
26–29), ridged (Figs. 19, 25, 31, 32) or reticulate (Fig. 23). The wax ornamentation, if
present, was flake-like (Figs. 20, 22, 24, 30–32), scale-like (Figs. 17–19, 25) or granular
(Figs. 21, 26). The inner margin of the outer stomatal rim was smooth in Apios carnea (Fig.
35) and A. fortunei (Fig. 37), and undulate in the other species (Figs. 33, 34, 36, 38–40)
(Table 3).
Table 3 The characters of leaf epidermis in Apios and Cochlianthus under SEM
Species Adaxial epidermis Abaxial epidermis
Cuticular
membrane
Wax
ornamentation
Cuticular
membrane
Wax
ornamentation
Inner margin of
outer stomatal rim
Apios americana striate scale-like ridged scale-like undulate
A. priceana striate scale-like striate granular undulate
A. carnea ridged scale-like striate invisible smooth
A. delavayi smooth flake-like striate invisible undulate
A. fortunei striate granular striate invisible smooth
A. gracillima smooth flake-like smooth flake-like undulate
Cochlianthus gracilis reticulate invisible ridged flake-like undulate
C. montanus smooth flake-like ridged flake-like undulate
No. 3 REN et al.: Systematic significance of leaf epidermal features in Apios and Cochlianthus 279
Figs. 17–24. Characteristics of adaxial epidermal cells in Apios and Cochlianthus under SEM. 17. A. americana. 18. A.
priceana. 19. A. carnea. 20. A. delavayi. 21. A. fortunei. 22. A. gracillima. 23. C. gracilis. 24. C. montanus.
Scale bar=10 µm.
Acta Phytotaxonomica Sinica Vol. 45 280
Figs. 25–32. Characteristics of abaxial epidermal cells in Apios and Cochlianthus under SEM. 25. A. americana. 26. A.
priceana. 27. A. carnea. 28. A. delavayi. 29. A. fortunei. 30. A. gracillima. 31. C. gracilis. 32. C. montanus.
Scale bar=10 µm.
No. 3 REN et al.: Systematic significance of leaf epidermal features in Apios and Cochlianthus 281
Figs. 33–40. Characteristics of stomatal apparatus in Apios and Cochlianthus under SEM. 33. A. americana. 34. A.
priceana. 35. A. carnea. 36. A. delavayi. 37. A. fortunei. 38. A. gracillima. 39. C. gracilis. 40. C. montanus.
Scale bar=10 µm.
Acta Phytotaxonomica Sinica Vol. 45 282
3 Discussion
No consensus has been reached on the number of species in Apios (Li, 1952; Hutchinson,
1964; Lackey, 1981; Woods, 1988; Li, 1995). Since de Candolle (1825), there has only been
one worldwide revision of Apios by Woods (1988), who recognized five species in this genus:
A. americana, A. carnea, A. delavayi (including A. delavayi var. gracillima), A. fortunei and
A. priceana. After observation about sixty specimens of A. gracillima and A. delavayi, Woods
believed that they are not different in growth habit, leaflet size, pedicel length and
inflorescence, and the only difference between them is that the flower of A. gracillima is
purple while the A. delavayi is greenish. In his opinion, A. gracillima does not merit
recognition as a distinct species, but should be treated as a variety of A. delavayi.
From 200 specimens examined, we have found some distinct and stable features between
A. gracillima and A. delavayi: the flower in A. gracillima is from red to purple and the leaflets
are obtuse at apex with a small mucro, while in A. delavayi, the flower is from yellow-green
to pale violet and the leaflets are acuminate at apex. This study shows that the leaf epidermal
characters of these two taxa are also very distinct: on the adaxial epidermis, the cells are
polygonal and the anticlinal walls are straight to arched in A. gracillima (Fig. 6; Table 2),
while in A. delavayi, the cells are irregular and the anticlinal walls are sinuate (Fig. 4; Table
2); on the abaxial epidermis, the wax ornamentation is flake-like in A. gracillima (Fig. 30;
Table 3), while in A. delavayi, the wax ornamentation is invisible (Fig. 28; Table 3). The leaf
epidermal features as well as the gross-morphological evidence, therefore, support the
treatment of A. gracillima and A. delavayi as two distinct species.
Robinson (1898) described Apios priceana and noted that the corolla in this species was
somewhat peculiar, with the standard having a thick, spongy, knot-like prolongation at apex.
Based on this character, Robinson (1898) divided the genus Apios into two subgenera:
subgen. Tylosemium, which includes only one species, A. priceana, and subgen. Euapios,
which includes A. americana, A. carnea, A. delavayi and A. fortunei. Woods deemed that this
subgeneric classification is not justified, as the same standard in all species of Apios, a
prolongation at apex, although in some species the prolongation is greatly reduced (Woods,
1988). By examining more than 1000 Apios specimens, we also found that in all Apios
species, the standard is connate and prolonged at apex. And our results of leaf epidermis,
including shape of cells, pattern of anticlinal walls, cuticular membrane, wax ornamentation
and type of stomatal apparatus, support Woods’s view that there is no distinct epidermal
apomorphy separating A. priceana from the remaining species of Apios (Tables 2, 3).
Apios and Cochlianthus are usually regarded as very closely related. They were grouped
by Lackey (1977, 1981) as they share a spirally coiled style, a character which is not seen in
any other groups in subtribe Erythrininae, tribe Phaseoleae. Woods (1988) recognized Apios
and Cochlianthus as two separate but closely related genera based upon morphological
criteria: in Apios, standard petal ovate to ovate-lanceolate, the auricle of the wing petal shorter
than 2 mm, keel petal curved slightly, leaflets 5–7; in Cochlianthus, standard petal elliptic to
rhombic, the auricle of the wing petal longer than 2 mm, keel petal curved strongly, leaflets 3.
Based on both herbarium and field observation, we noticed that the leaves of Apios are
primarily 5 or 7-foliolate, rarely 3-foliolate, almost glabrous to slightly velutinous on the
veins and veinlets, while the leaves of Cochlianthus are exclusively trifoliolate, and densely
tomentose on both surfaces. Leaf epidermal characters also showed some difference between
these two genera: in Apios, anticlinal walls sinuate or straight to arched, wax ornamentation
scale or gross flake-like, while in Cochlianthus, anticlinal walls sinuolate and wax
ornamentation condense flake-like (Tables 2, 3). Accordingly, we thought that Apios and
Cochlianthus could be recognized as distinct and the relationship between the two genera may
No. 3 REN et al.: Systematic significance of leaf epidermal features in Apios and Cochlianthus 283
not be so close as it was previously regarded.
The positions of Apios and Cochlianthus in subtribe Erythrininae, tribe Phaseoleae, are
poorly understood. It is well established that Phaseoleae as currently circumscribed is not a
monophyletic group and Erythrininae is also a highly polyphyletic assemblage of taxa
(Lackey, 1981). Lackey (1977, 1981) suggested that subtribe Erythrininae is completely
artificial and probably includes several independent lines with unknown nearest relatives,
which is supported by DNA data (Bruneau et al., 1994; Wojciechowski et al., 2004). Based on
the numbers of leaflets, Lackey held that some genera, including Apios and Cochlianthus, are
suspected as imposters in the Phaseoleae. Therefore, additional evidence for interpreting the
positions of Apios and Cochlianthus in tribe Phaseoleae, as well as the relationship between the
two genera, is needed.
Acknowledgements We thank the curators of Herbarium of Institute of Botany, the Chinese
Academy of Sciences (PE), Gray Herbarium of Harvard University (GH) and Herbarium of
Royal Botanic Garden Edinburgh (E) for their generosity in providing specimens. We thank
Dr. Gregory Kenicer (Royal Botanic Garden Edinburgh) for reviewing the manuscript. We
also express our thanks to Mrs. WEN Jie, Mrs. YANG Zhi-Rong and Mrs. DU Yu-Fen for
providing many helpful suggestions on our work and Mr. XIAO Yin-Hou and Mr. YANG
Xue-Jian for the help during the process of developing films.
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土圞儿属和旋花豆属(豆科)的叶表皮特征
及其系统学意义
1, 2任 勃 1朱相云* 3姜彦成*
1 (中国科学院植物研究所 北京 100093)
2 (中国科学院研究生院 北京 100049)
3 (新疆大学生命科学技术学院 新疆 830046)
摘要 应用光学显微镜和扫描电子显微镜观察了豆科Leguminosae土圞儿属Apios和旋花豆属
Cochlianthus植物的叶表皮特征。在光学显微镜下, 大多数种类的叶表皮细胞形状(表面观)为不规则型,
垂周壁式样为浅波状或波状, 只有一个种——Apios gracillima Dunn叶表皮细胞为多边形, 垂周壁式样
为较平直。在扫描电子显微镜下, 多数种类的叶片蜡质纹饰通常为片状或颗粒状, 一些种叶表皮未见蜡
质纹饰; 气孔器外拱盖内缘通常为浅波状或近平滑。在这两个属内, 上述叶表皮特征在物种内较稳定,
因此对于解决属内种间和属间关系有一定的系统学意义。本研究得出以下结论: (1) A. gracillima叶的上
表皮细胞为多边形, 垂周壁平直或弧形, A. delavayi Franch.叶的上表皮细胞为不规则形, 垂周壁波状,
二者存在明显差异, 因此A. gracillima应处理为一独立的种, 而不是A. delavayi下的一个变种; (2) A.
priceana B. L. Rob.在叶表皮细胞形状、垂周壁式样、气孔器类型和蜡质纹饰形态等方面与土圞儿属中
其他种不存在显著区别, 因此土圞儿属内两个亚属的划分是不合理的; (3)土圞儿属多为5或7小叶, 无
毛或近无毛, 叶表皮细胞垂周壁波状或近平直, 而旋花豆属3小叶, 密被柔毛, 叶表皮细胞垂周壁浅波
状, 本文支持它们为两个独立的属的分类处理。
关键词 豆科; 土圞儿属; 旋花豆属; 叶表皮; 系统学意义