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Floral development in Adonideae (Ranunculaceae)
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Flora 204 (2009) 506–517 www.elsevier.de/flora
Floral development in Adonideae (Ranunculaceae) Yi Rena,, Hong-li Changb, Xian-hua Tiana, Ping Songa, Peter K. Endressc a
Key Laboratory of Medicinal Plant Resource and Natural Pharmaceutical Chemistry of Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China b School of Life Sciences, Northwest University, Xi’an 710069, China c Institute of Systematic Botany, University of Zurich, Zurich, Switzerland Received 8 April 2008; accepted 23 July 2008
Abstract Floral development and floral phyllotaxis in species of Adonis, Callianthemum, and Trollius (Ranunculaceae) were studied with scanning electron microscopy. The floral organs are initiated in spiral sequence and the flowers have spiral phyllotaxis. The sepal primordia are broad, crescent-shaped, and truncate, but those of petals, stamens, and carpels are rather hemispherical. A relatively long plastochron appears to be present between the last sepal and the first petal as compared with the short and equal plastochrones of all subsequent floral organs. Maturation of the stamens within the androecium appears to be centripetal. The carpels have a short ascidiate zone. Placentation is uniformly lateral, even in Adonis and Callianthemum, which have only one fertile ovule per carpel (versus median in other genera of Ranunculoideae with a single fertile ovule). In Adonis and Callianthemum at the tip of the carpel the ventral slit is gaping and the stigma is broadly exposed, whereas in Trollius the stigma is narrower and more pronouncedly decurrent along the ventral slit. The petals in Callianthemum and Trollius are more conspicuously delayed in development than those in Adonis as compared with sepals and stamens. A short carpel stipe is formed early in Callianthemum but later in Adonis and Trollius. In Trollius farreri (commonly having only five carpels in contrast to other species of Trollius) the carpels form a single (spiral) series. Thus floral development is similar in all three genera and, at a lower level, Adonis and Callianthemum are especially close but have different autapomorphies, which reflects the current classification of the genera. r 2008 Elsevier GmbH. All rights reserved. Keywords: Adonis; Callianthemum; Trollius; Adonideae; Ranunculaceae; Floral development
Introduction An unexpected result of molecular phylogenetic studies in Ranunculaceae was that Adonis is closely related to Trollius (Hoot, 1995; Johansson, 1995; Johansson and Jansen, 1993; Ro et al., 1997). Based on these studies, Jensen et al. (1995) put Adonis (and, tentatively, Callianthemum, not included in molecular Corresponding author.
E-mail address: [email protected] (Y. Ren). 0367-2530/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2008.07.002
studies) in Adonidinae, which was placed in Adonideae together with the unigeneric Trolliinae. Adonis and Callianthemum were previously placed in a separate tribe Adonideae and considered to be related to Anemoneae (Tamura, 1993, 1995) because they have achenes, whereas Trollius, which has follicles, was placed in Trollieae (Tamura, 1966; Wang, 1979) or Helleboreae (Tamura, 1993, 1995). SEM studies on floral development in Adonidinae were previously only made for a species of Trollius (T. buddae Schipcz., Song et al., 2007), whereas they
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were lacking for Adonis and Callianthemum, which differ from Trollius in aspects of petal and carpel structure. At a different level, because Ranunculaceae are the largest and most diverse family of Ranunculales, which is a key group as sister to all other eudicots (APG, 1998, 2003), and because of the increasing focus of molecular developmental and evo–devo studies on flowers of Ranunculales (Cui et al., 2006; Di Stilio et al., 2005; Kramer and Irish, 1999; Kramer and Zimmer, 2006; Kramer et al., 2003, 2006, 2007; Lee et al., 2005), better comparative knowledge and understanding of the floral development of Ranunculaceae is needed. Although there is extensive literature on aspects of floral structure and embryology in Ranunculaceae, the distribution of developmental studies in the family is unbalanced. Some genera have been studied repeatedly with various species (e.g. Aquilegia, Bhandari and Vijayaraghavan, 1970; Eames, 1931; Erbar et al., 1998; Feng et al., 1995; Gre´goire, 1938; Kosuge, 1994; Payer, 1857; Rassner, 1931; Rohweder, 1967; Scho¨ffel, 1932; Tepfer, 1953; Tucker and Hodges, 2005; Ranunculus, Bessey, 1898; Eckardt, 1957; Endress, 1987; Erbar et al., 1998; Gre´goire, 1938; Gupta and Singh, 1983; Meicenheimer, 1979; Payer, 1857; Rohweder, 1967; Sattler, 1973; Scho¨ffel, 1932; Singh, 1936; Tepfer, 1953; Anemone, Ben-Hod et al., 1988; Bessey, 189; Chang et al., 2005; Gre´goire, 1938; Scho¨ffel, 1932; Shukova, 1965; Sprotte, 1940; Voelter and Weber, 1962), others less extensively, and many genera are unstudied. Genera that have been developmentally studied for the first time only more recently include Hydrastis (Tobe and Keating, 1985), Coptis (Tamura, 1981; Gu and Ren, 2007), Caltha, and Trollius (Song et al., 2007). The aim of the present paper is to extend floral developmental studies to unstudied taxa and some unstudied aspects of floral structure.
Materials and methods Flower buds of Adonis sutchuenensis Franch. (Sect. Consiligo DC.) (alt. 2800–3200 m, voucher: Bai Genlu 2004007, SANU), Callianthemum taipaicum W. T. Wang (alt. 3500–3600 m, voucher: Bai Genlu 2004008, SANU), and Trollius farreri Stapf (alt. 3500–3600 m, voucher: Bai Gen-lu 20040421, SANU) were collected from late March to early November in the years from 2002 to 2005 at intervals of seven to ten days in Taibaishan Mountains, Shaanxi province, and fixed in FAA. Flower buds were dehydrated in ethanol and isoamyl acetate series, critical-point dried, and observed with a Hitachi 800 SEM. For histological studies, flower buds were dehydrated in an alcohol series, infiltrated with xylene and embedded in paraffin wax. The embedded material was sectioned at 8 mm thickness and stained with safranin and fast green. Photographs of
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mature flowers were taken with a Nikon Coolpix 990 digital camera (Figs. 1–3) .
Results Adonis sutchuenensis (Adonidinae) (Figs. 1, 4–24) Floral morphology Flowers are solitary, terminal, 2–3 cm in diameter. The five to six sepals are green and obovately lanceolate. The 8–12 petals are yellow, narrowly obovate or oblongly obovate and about double the length of the sepals. A nectary is absent. The 25–50 stamens are about 1 3 of the length of the petals. The 17–25 carpels have short styles and small stigmas. All floral organs are free (Fig. 1). The carpels develop into nutlets. Floral development The flower bud is preceded by two leaves. The floral organs are initiated in a spiral sequence, either clockwise or counterclockwise. The five (rarely six to seven) young sepals are crescent-shaped and truncate (Figs. 4–6). It appears that there is a relatively long plastochron between the initiation of the last sepal and the first petal, because in the beginning the last sepal is much larger than the first petal (Figs. 4 and 5). The young petals are rounded and narrow (Figs. 4 and 5). The divergence angles in the perianth fluctuate around ca. 1371, which indicates spiral phyllotaxis according to the Fibonacci pattern (Figs. 4 and 5). Because the number of the petals is variable, and the shape of the petal and stamen primordia is similar, it is difficult to distinguish between the last petal and the first stamen in early development (Fig. 7). The carpels do not occupy the entire floral apex so that a small residual floral apex remains (Figs. 8 and 9), which is later hidden by the growing carpels. As seen from above, the young stamens and carpels form contact parastichies in sets of five and eight, which also conforms to spiral phyllotaxis according to the Fibonacci pattern (Fig. 8). In side views the different steepness of the regular contact parastichies is also conspicuous (Figs. 10 and 11). The sepals enlarge and enclose the other floral organs in later development. The young petals flatten after appearance of the carpels, and only in this developmental stage the petals and stamens can be clearly distinguished (Fig. 10). The petals develop faster and are always larger than the stamens (Fig. 11). However, they remain shorter than the sepals up to shortly before anthesis. When the carpel primordia appear gradually, the stamens in the outer part of the androecium enlarge (Figs. 10 and 11) and then differentiate into filaments and anthers (Fig. 12). Maturation of the stamens is centripetal (Figs. 21–24). After initiation of the carpels a
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Figs. 1–3. Mature flowers. (1) Adonis sutchuenensis. Bar ¼ 1.5 cm. (2) Callianthemum taipaicum. Bar ¼ 1.0 cm. (3) Trollius farreri. Bar ¼ 1.0 cm.
longitudinal concavity appears on the ventral side (Fig. 9), which initiates the plicate shape (Figs. 13 and 14). Later the single, lateral ovule is initiated (Fig. 15), the carpel closes and the ventral slit appears (Fig. 16). At first the carpel appears almost completely plicate. However, later the ascidiate base somewhat elongates (Fig. 17), and style and ovary become more distinct from each other (Fig. 17). On top of the style the ventral slit becomes everted and the stigma differentiates on the everted surface before the carpel matures (Fig. 18). The carpel becomes broadly subulate (Fig. 19) at anthesis. The stigma is unicellular papillate and is slightly decurrent along the ventral slit (Fig. 20) .
Callianthemum taipaicum (Adonidinae) (Figs. 2, 25–44) Floral morphology Flowers are solitary, terminal, 2–3 cm in diameter. The usually five blue purple sepals are narrowly oblong or lanceolate. The 9–13 petals are white with red veins and yellow orange at the base, narrowly obovate, slightly longer than the sepals, and have a nectary groove near the base. The 44–50 stamens are about 13 the length of the petals. The 21–24 carpels are distinctly stipitate and have short styles and small stigmas. All floral organs are free (Fig. 2). The carpels develop into nutlets. Floral development The flower bud is preceded by two leaves. The floral organs are initiated in spiral sequence, either clockwise or counterclockwise. The young sepals are crescentshaped, truncate and soon become somewhat rounded, and are broad at the base (Figs. 25 and 26). It appears that there is a relatively long plastochron between the initiation of the last sepal and the first petal, because in early development the last sepal is much larger than the first petal (Fig. 26). The young petals are rounded and
narrow at the base (Figs. 26–28, 31). The divergence angles in the perianth are ca. 1371 on average (Figs. 26 and 27), which indicates spiral phyllotaxis according to the Fibonacci pattern. Because petal number is unstable, and petals and stamens are difficult to distinguish in early development, it is not possible to determine the last petal and the first stamen in the beginning. The same is true for the transition from stamens to carpels (Fig. 29). When all carpels are formed, a small residual floral apex remains (Fig. 30) and is later covered by the developing carpels. As seen from above, the young stamens and carpels form contact parastichies in sets of five and eight, which also conforms with a spiral Fibonacci pattern (Fig. 30). The sepals enlarge and enclose the other floral organs in later development. The petals flatten when the carpels appear and only now petals and stamens can be clearly distinguished (Fig. 31). The petals develop more slowly and remain smaller than the stamens (Fig. 32). A bulge appears at the base of the petals (Fig. 33) and on the distal side of the bulge a concavity is formed in which nectariferous tissue will be differentiated (Fig. 34). When the carpels appear, the stamens in the outer part of the androecium begin their differentiation, which proceeds centripetally (Fig. 31), and finally differentiate into filaments and anthers (Fig. 35). The hemispherical young carpels become concave on the ventral side, and thus chair-shaped and ascidiate (Fig. 36). With carpel elongation the concave part also elongates and changes from an oblique to a more longitudinal direction (Fig. 37). With further elongation the carpels become pronouncedly stipitate and with deepening of the ventral concavity the plicate part becomes more pronounced. The two more or less collateral incipient ovules become visible before the ventral slit closes (Figs. 38–40). After closure the carpel further elongates, and the stipe remains more narrow so that the carpel becomes spindle-shaped (Figs. 41–43). The upper part of the ventral slit becomes everted and the stigma differentiates on the everted part (Fig. 43). The stigma is unicellular
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Figs. 4–12. Adonis sutchuenensis. Floral organ initiation and development. (4–8) Organ initiation. (4) Sepals. Bar ¼ 136 mm. (5) Petals. Bar ¼ 150 mm. (6) Petals. Bar ¼ 200 mm. (7) Stamens. Bar ¼ 200 mm. (8) Carpels. Bar ¼ 176 mm. (9) Carpels with incipient concavity on ventral side, and residual floral apex remaining after initiation of all carpels. Bar ¼ 200 mm. (10) Petals slightly larger than stamens. Bar ¼ 150 mm. (11) Petals larger than stamens. Bar ¼ 231 mm. (12) Centripetal developmental sequence of anthers. Bar ¼ 0.6 mm. (C: carpel. L: leaf. P: petal. S: sepal. St: stamen. Numbers after the letters indicate initiation sequence).
Trollius farreri (Trolliinae) (Figs. 3, 45–62)
head orange. The 17–36 stamens are about 23 the length of the sepals. The 5 (9) carpels have styles of medium length and narrow but conspicuously decurrent stigmas. All floral organs are free (Fig. 3). The carpels develop into follicles.
Floral morphology Flowers are solitary, terminal, 2–3.5 cm in diameter. The usually five or rarely six sepals are petaloid, yellow ventrally and dark purple dorsally, widely obovate, and persistent. The 12–15 petals are ca. 13 the length of the sepals, linear, unguiculate, with a nectary groove near the base; the narrow base is yellow and the enlarged
Floral development The flower bud is preceded by two triangular bracts. The floral organs are initiated in spiral sequence, either clockwise or counterclockwise. The young sepals are crescent-shaped, truncate and soon become rounded, and are broad at the base (Figs. 45 and 46). It appears that there is a relatively long plastochron between the
papillate and is slightly decurrent along the ventral slit (Fig. 44) .
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Figs. 13–24. Adonis sutchuenensis. Stamen and carpel development. (13–19) Carpel development. (13) Carpels becoming plicate. Bar ¼ 0.27 mm. (14) Somewhat later stage. Bar ¼ 250 mm. (15) Carpel with a lateral ovule primordium. Bar ¼ 86 mm. (16) Carpel with closed ventral slit. Bar ¼ 150 mm. (17) Carpel with early differentiation into stigma, style and ovary. Bar ¼ 0.5 mm. (18) Closeup of young stigma. Bar ¼ 150 mm. (19) Mature carpel. Bar ¼ 0.75 mm. (20) Close-up of mature stigma. Bar ¼ 176 mm. (21–24) Longitudinal sections of stamens, showing centripetal development of microspore mother cells in androecium. (21) Longitudinal section of flower, overview. The boxes refer to Figs. 22–24. Bar ¼ 0.07 mm. (22) Close-up of outermost stamen of Fig. 21, microspore mother cells in late meiotic prophase. Bar ¼ 290 mm. (23) Close-up of second stamen of Fig. 21, microspore mother cells slightly less advanced in differentiation. Bar ¼ 250 mm. (24) Close-up of innermost stamen of Fig. 21, microspore mother cells in early meiotic prophase. Bar ¼ 250 mm. (AW: anther wall. C: carpel. MM: microspore mother cell. O: ovule. P: petal. St: stamen).
initiation of the last sepal and the first petal because the last sepal is initially much larger than the first petal. Young petals are rounded, and narrow at the base (Figs. 46 and 47). The divergence angles in the perianth oscillate around ca. 1371 (Figs. 46 and 47), which indicates spiral phyllotaxis according to the Fibonacci pattern. Because the number of the petals is labile, and petals and stamens are difficult to distinguish in early
development, it is not possible to determine the last petal and the first stamen in the beginning. The same is true for the transition from stamens to carpels (Figs. 48 and 49). After the initiation of all carpels, a small residual floral apex remains (Figs. 48 and 49) and is later covered by the developing carpels. In apical view the young stamens and carpels form contact parastichies in sets of five and eight (Fig. 48).
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Figs. 25–35. Callianthemum taipaicum. Floral organ initiation and development. (25–29) Organ initiation. (25) Sepals. Bar ¼ 120 mm. (26) Petals. Bar ¼ 120 mm. (27) Petals and/or stamens. Bar ¼ 136 mm. (28) Stamens, side view. Bar ¼ 220 mm. (29) Carpels. Bar ¼ 136 mm. (30) Carpels with incipient concavity on ventral side, and residual floral apex remaining. Bar ¼ 350 mm. (31–34) Development of petals. (31) Young petals slightly broader than stamens. Bar ¼ 150 mm. (32) Young petals of similar length as stamens. Bar ¼ 0.38 mm. (33) Bulge appearing at base of the petal. Bar ¼ 0.6 mm. (34) A nectariferous groove forms distally of the bulge. Bar ¼ 0.86 mm. (35) Centripetal developmental direction of stamens in the androecium. Bar ¼ 0.86 mm. (C: carpel. P: petal. S: sepal. St: stamen. Numbers after the letters indicate initiation sequence).
The young sepals enlarge and partly enclose the floral apex while stamens and carpels are initiated (Figs. 49 and 50). In the beginning, before the carpels appear, the petals become slightly broader than the stamens (Figs. 49 and 50). However, when the stamens begin their differentiation, growth of the petals lags behind that of the stamens (Fig. 51). In later development, the petals remain as very small scales outside of the androecium (Fig. 52). When the flower matures, the petals become linear, unguiculate, with a nectary groove near the base of the ventral side and an enlarged head (Figs. 53 and 54). Stamen maturation is centripetal (not shown). The hemispherical young carpels soon become concave on the ventral side (Fig. 49) and become ascidiate. That the carpels are not arranged in a whorl but in a spiral, is clearly seen by their different distances from the floral centre (Figs. 55 and 56). The carpels
considerably elongate, the ventral slit closes (Fig. 57), and two crests develop along the ventral slit (Figs. 58–60). With carpel elongation the plicate zone becomes more prominent. The ovary tapers into the style (Figs. 59 and 61). The stigma is unicellular papillate and decurrent along the ventral slit for some distance (Figs. 61 and 62).
Discussion Floral phyllotaxis Floral phyllotaxis is variously whorled and spiral in Ranunculaceae, with both patterns sometimes at low systematic level (Endress, 1987; Hiepko, 1965; Scho¨ffel, 1932). In Aquilegia, Semiaquilegia, and Enemion of
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Figs. 36–44. Callianthemum taipaicum. Carpel development. (36) Concavity formed on ventral side of young carpels. Bar ¼ 150 mm. (37) Young carpels becoming plicate. Bar ¼ 120 mm. (38) Carpels halfway closed, the two more or less collateral incipient ovules still visible, stipe formation. Bar ¼ 176 mm. (39) Carpel with one lateral ovule visible. Bar ¼ 150 mm. (40) Opened carpel, showing two lateral ovules. Bar ¼ 100 mm. (41) Carpels closed. Bar ¼ 176 mm. (42) Differentiation of carpel into stigma, style and ovary. Bar ¼ 0.86 mm. (43) Mature carpel. Bar ¼ 1.0 mm. (44) Close-up of mature stigma. Bar ¼ 231 mm. (C: carpel. O: ovule. St: stamen).
Isopyroideae (or Thalictroideae) it is strictly whorled, although the sepals originate in a spiral sequence, which leads to quincuncial aestivation (Scho¨ffel, 1932; Tucker and Hodges, 2005). Thus, the divergence angles of subsequent sepals in these pentamerous flowers are closer to 1441 (as typical in whorled phyllotaxis) than to 1371 (as typical in spiral phyllotaxis). In contrast, in Anemone rivularis Buch.-Ham. ex DC. var. flore-minore Maxim. (Chang et al., 2005), Caltha palustris Linn. and Trollius buddae Schipcz. (Song et al., 2007) of Ranunculoideae, and Coptis chinensis Franch. (Gu and Ren, 2007) of Coptidoideae floral phyllotaxis is spiral
throughout. However, in Anemone there are also species with whorled flowers (Scho¨ffel, 1932; Tobe, 1976). In the species studied here the flowers are spiral throughout. This can be easily seen by the different steepness of contact parastiches (see also Endress, 2006; Endress and Doyle, 2007). Spiral floral phyllotaxis had been recorded earlier for other species of Adonis and Trollius (Scho¨ffel, 1932; Thompson, 1934) but the present study appears to be the first record for Callianthemum. Character optimizations in Ranunculales show that it is still ambiguous which of the two patterns is plesiomorphic in Ranunculaceae (Doyle and Endress, 2000; Endress
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Figs. 45–54. Trollius farreri. Floral organ initiation and development. (45–48) Organ initiation. (45) Sepals. Bar ¼ 100 mm. (46) Petals. Bar ¼ 120 mm. (47) Slightly later stage. Bar ¼ 136 mm. (48) Stamens and carpels. Bar ¼ 176 mm. (49–54) Organ development. (49) Carpels with incipient concavity on ventral side. Bar ¼ 176 mm. (50) Petals broader than stamens before carpel initiation. Bar ¼ 231 mm. (51) Slightly later stage, with petals shorter than stamens. Bar ¼ 200 mm. (52) Petals still very small after anther differentiation. Bar ¼ 231 mm. (53 and 54) Mature petals. (53) Ventral view, showing a nectariferous groove at the base of the blade. Bar ¼ 1.2 mm. (54) Dorsal view. Bar ¼ 1.2 mm. (C: carpel. L: leaf. P: petal. S: sepal. St: stamen. Numbers after the letters indicates initiation sequence).
and Doyle, 2007). This uncertainty is because the closest relatives of Ranunculaceae have largely whorled floral phyllotaxis and the two basal ranunculaceous genera, Glaucidium and Hydrastis, do not appear to have spiral phyllotaxis either (figures of Hydrastis in Tobe and Keating, 1985, and personal observations by P.K.E.).
Floral development The sepal primordia of Adonis sutchuenensis, Callianthemum taipaicum, and Trollius farreri are crescent-
shaped and truncate. However, petal, stamen, and carpel primordia are rather hemispherical, and are also smaller than those of the sepals. The same was found in other representatives of the family (Chang et al., 2005; Gu and Ren, 2007; Song et al., 2007; Tucker and Hodges, 2005). It appears that there is a relatively long plastochron between the last initiated sepal and the first petal, whereas the plastochrons between all subsequent organs are shorter and all equal. That petals and stamens do not show a gap in plastochrons and are difficult to distinguish in shape in early stages may support their evolutionary relationship (see also Hiepko,
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Figs. 55–62. Trollius farreri. Carpel development. (55) The five carpels arranged in a (spiral) series. Bar ¼ 250 mm. (56) Carpels closed. Bar ¼ 176 mm. (57) Closed carpel from the side, slightly older than in Fig. 56. Bar ¼ 100 mm. (58) Carpel from ventral, with longitudinal crests along ventral slit. Bar ¼ 120 mm. (59) Carpel differentiated into style and ovary. Bar ¼ 1.2 mm. (60) Close-up of upper part of Fig. 59. Bar ¼ 0.3 mm. (61) Mature carpel. Bar ¼ 1.36 mm. (62) Close-up of upper part of Fig. 60, with decurrent papillate stigma covered with pollen grains. Bar ¼ 0.27 mm. (C: carpel. St: stamen).
1965; Endress, 1995; Erbar et al., 1998; Kramer et al., 2003; Tamura, 1984, 1993). Petals are especially plastic in Ranunculaceae. As in many core eudicots they commonly form the inner whorl or series of perianth organs and are commonly delayed in their development in bud and have a single vascular trace (Hiepko, 1965). However, petals in Ranunculaceae can attain diverse and sometimes elaborate shapes, and in contrast to most core eudicots they commonly bear nectaries. In many genera they are optically attractive, but in others this function may also be exerted by sepals or stamens. In Adonis, Callianthemum, and Trollius petal development is retarded in bud compared with the sepals. However, petals bear a nectary only in Callianthemum and Trollius, whereas the flowers in Adonis are nectarless (see also Hiepko, 1965). Maturation of the stamens in Adonis, Callianthemum, and Trollius is centripetal in accordance with the initiation sequence of the organs (this study; Song et al., 2007, for Trollius buddae). This should be mentioned because in some other Ranunculaceae
centrifugal maturation was recorded (despite centripetal or almost simultaneous initiation), such as in Aquilegia (Feng et al., 1995; Tepfer, 1953), Glaucidium (Tamura, 1972), and Anemone rivularis var. flore-minore (Chang et al., 2005). This difference and the discordance between direction of initiation and maturation need more comparative studies in further genera of the family. Early carpel development is similar in all three taxa. The primary margins of the carpel formed concomitantly with the ventral concavity do not extend to the very base of the carpel but meet over the ventral side thus forming a cross zone and a short ascidiate zone, even without a median position of a fertile ovule. The presence of an ascidiate zone is most common in Ranunculaceae (Endress and Igersheim, 1999; Rohweder, 1967), although taxa with completely plicate carpels, such as Xanthorhiza, may also exist in the family (Endress and Igersheim, 1999). Further, the carpel base is more narrow than the ovary in all three genera forming a stipe of various length. In the apical region the ventral slit appears more or less everted
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forming two longitudinal crests of various length on which the stigma differentiates. In Adonis and Callianthemum the carpels close only after ovule initiation – as can also be found in other genera with uniovulate carpels (Ranunculus, Myosurus; van Heel, 1981, 1984) – whereas in the pluriovulate carpels of Trollius closure appears to be earlier, and ovules are not visible in closing carpels.
Systematic position of Adonis and Callianthemum Adonis and Callianthemum, both genera with achenes, were formerly thought to be related with other achenebearing genera and were placed in Adonidinae, Ranunculeae, Ranunculoideae (Wang, 1980) or Adonideae (Tamura, 1993). However, molecular studies revealed that Adonis is closely related to the follicular genus Trollius (Hoot, 1995; Johansson, 1995; Johansson and Jansen, 1993; Ro et al., 1997). This alliance was also suggested by chemical (Jensen, 1995) and serological features (Jensen, 1968). Based on these studies, Jensen et al. (1995) placed Adonis (and, tentatively, Callianthemum) in its own subtribe Adonidinae, which was included in Adonideae together with the unigeneric Trolliinae. In Tamura’s (1995) treatment Adonis and Callianthemum make up his Adonideae. Callianthemum was not included in molecular studies as yet. That Adonis and Callianthemum are closely related to each other and to Trollius, and not to Anemoneae (Anemone, Clematis and relatives), despite their oneseeded fruitlets, is also supported by our present floral developmental results and by other structural features reported earlier. Adonis and Callianthemum are similar in their stigma differentiation. Both genera have sepals and petals, like Trollius, but unlike Anemone and most Clematis species, which only have (petaloid) sepals (Hiepko, 1965). Although the petals in Adonis lack nectaries, in contrast to Callianthemum, also in Callianthemum the petals are flat and simple and the nectaries are sometimes small and inconspicuous (Schaeppi and Frank, 1957). Double perianths (consisting of sepals and petals) combined with unelaborate petal shape are otherwise unusual in Ranunculaceae, which is in line with a close relationship between Adonis and Callianthemum. Most importantly, the ovules of both Adonis and Callianthemum have two integuments (Baillon, 1863; Bhandari, 1962, 1963, 1967; Lonay, 1901, 1907; Prantl, 1887; Rassner, 1931; Schaeppi and Frank, 1962; Soue`ges, 1912; Wang and Ren, 2008) like Trollius (Bersier, 1960), whereas those of Anemone and Clematis have only one integument (Bessey, 1898; Bhandari, 1968; Fish, 1970; Lonay, 1901; Prantl, 1887; Schaeppi and Frank, 1962; Shukova, 1965; Soue`ges, 1910–1914; Tobe et al., 1975; Vijayaraghavan, 1962; Voelter and Weber, 1962; Wang and Ren, 2008;
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Wiegand, 1894). Furthermore, the single fertile ovule is lateral in Adonis and Callianthemum, but median in Anemone and Clematis (Prantl, 1887; Schaeppi and Frank, 1962; Tamura, 1984). Carpels in Adonis sect. Consiligo have a single ovule, in Callianthemum additionally a reduced one (Baillon, 1863; Prantl, 1887; Schaeppi and Frank, 1957, 1962), whereas in Adonis sect. Adonis several reduced ovules are present (Bhandari, 1962, 1963, 1967; Brouland, 1936; Lonay, 1901; Prantl, 1887; Rassner, 1931; Schaeppi and Frank, 1962; Soue`ges, 1912). Several reduced ovules are commonly also present in Anemoneae (for Anemone and Clematis: Bessey, 1898; Chute, 1930; Fish, 1970; Lonay, 1901; Payer, 1857; Prantl, 1887; Rassner, 1931; Rohweder, 1967; Shukova, 1965; Soue`ges, 1910–1914; Tobe et al., 1975; Vijayaraghavan, 1962). Trollius, Adonis and Callianthemum share the presence of often a larger number of carpels, an ascidiate carpel base, and (except for a part of Adonis), more than one ovule per carpel, even if only one is fertile, and a stipe of various length (short in Trollius and Adonis, longer in Callianthemum) (see also Schaeppi, 1972; Schaeppi and Frank, 1957, 1962). However, these features are also present in Anemoneae (Sprotte, 1940). Thus they may be plesiomorphies in Ranunculoideae.
Acknowledgement The project was supported by the National Nature Science Foundation of China (No. 30370095).
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Flora 204 (2009) 506–517 www.elsevier.de/flora
Floral development in Adonideae (Ranunculaceae) Yi Rena,, Hong-li Changb, Xian-hua Tiana, Ping Songa, Peter K. Endressc a
Key Laboratory of Medicinal Plant Resource and Natural Pharmaceutical Chemistry of Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi’an 710062, China b School of Life Sciences, Northwest University, Xi’an 710069, China c Institute of Systematic Botany, University of Zurich, Zurich, Switzerland Received 8 April 2008; accepted 23 July 2008
Abstract Floral development and floral phyllotaxis in species of Adonis, Callianthemum, and Trollius (Ranunculaceae) were studied with scanning electron microscopy. The floral organs are initiated in spiral sequence and the flowers have spiral phyllotaxis. The sepal primordia are broad, crescent-shaped, and truncate, but those of petals, stamens, and carpels are rather hemispherical. A relatively long plastochron appears to be present between the last sepal and the first petal as compared with the short and equal plastochrones of all subsequent floral organs. Maturation of the stamens within the androecium appears to be centripetal. The carpels have a short ascidiate zone. Placentation is uniformly lateral, even in Adonis and Callianthemum, which have only one fertile ovule per carpel (versus median in other genera of Ranunculoideae with a single fertile ovule). In Adonis and Callianthemum at the tip of the carpel the ventral slit is gaping and the stigma is broadly exposed, whereas in Trollius the stigma is narrower and more pronouncedly decurrent along the ventral slit. The petals in Callianthemum and Trollius are more conspicuously delayed in development than those in Adonis as compared with sepals and stamens. A short carpel stipe is formed early in Callianthemum but later in Adonis and Trollius. In Trollius farreri (commonly having only five carpels in contrast to other species of Trollius) the carpels form a single (spiral) series. Thus floral development is similar in all three genera and, at a lower level, Adonis and Callianthemum are especially close but have different autapomorphies, which reflects the current classification of the genera. r 2008 Elsevier GmbH. All rights reserved. Keywords: Adonis; Callianthemum; Trollius; Adonideae; Ranunculaceae; Floral development
Introduction An unexpected result of molecular phylogenetic studies in Ranunculaceae was that Adonis is closely related to Trollius (Hoot, 1995; Johansson, 1995; Johansson and Jansen, 1993; Ro et al., 1997). Based on these studies, Jensen et al. (1995) put Adonis (and, tentatively, Callianthemum, not included in molecular Corresponding author.
E-mail address: [email protected] (Y. Ren). 0367-2530/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2008.07.002
studies) in Adonidinae, which was placed in Adonideae together with the unigeneric Trolliinae. Adonis and Callianthemum were previously placed in a separate tribe Adonideae and considered to be related to Anemoneae (Tamura, 1993, 1995) because they have achenes, whereas Trollius, which has follicles, was placed in Trollieae (Tamura, 1966; Wang, 1979) or Helleboreae (Tamura, 1993, 1995). SEM studies on floral development in Adonidinae were previously only made for a species of Trollius (T. buddae Schipcz., Song et al., 2007), whereas they
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were lacking for Adonis and Callianthemum, which differ from Trollius in aspects of petal and carpel structure. At a different level, because Ranunculaceae are the largest and most diverse family of Ranunculales, which is a key group as sister to all other eudicots (APG, 1998, 2003), and because of the increasing focus of molecular developmental and evo–devo studies on flowers of Ranunculales (Cui et al., 2006; Di Stilio et al., 2005; Kramer and Irish, 1999; Kramer and Zimmer, 2006; Kramer et al., 2003, 2006, 2007; Lee et al., 2005), better comparative knowledge and understanding of the floral development of Ranunculaceae is needed. Although there is extensive literature on aspects of floral structure and embryology in Ranunculaceae, the distribution of developmental studies in the family is unbalanced. Some genera have been studied repeatedly with various species (e.g. Aquilegia, Bhandari and Vijayaraghavan, 1970; Eames, 1931; Erbar et al., 1998; Feng et al., 1995; Gre´goire, 1938; Kosuge, 1994; Payer, 1857; Rassner, 1931; Rohweder, 1967; Scho¨ffel, 1932; Tepfer, 1953; Tucker and Hodges, 2005; Ranunculus, Bessey, 1898; Eckardt, 1957; Endress, 1987; Erbar et al., 1998; Gre´goire, 1938; Gupta and Singh, 1983; Meicenheimer, 1979; Payer, 1857; Rohweder, 1967; Sattler, 1973; Scho¨ffel, 1932; Singh, 1936; Tepfer, 1953; Anemone, Ben-Hod et al., 1988; Bessey, 189; Chang et al., 2005; Gre´goire, 1938; Scho¨ffel, 1932; Shukova, 1965; Sprotte, 1940; Voelter and Weber, 1962), others less extensively, and many genera are unstudied. Genera that have been developmentally studied for the first time only more recently include Hydrastis (Tobe and Keating, 1985), Coptis (Tamura, 1981; Gu and Ren, 2007), Caltha, and Trollius (Song et al., 2007). The aim of the present paper is to extend floral developmental studies to unstudied taxa and some unstudied aspects of floral structure.
Materials and methods Flower buds of Adonis sutchuenensis Franch. (Sect. Consiligo DC.) (alt. 2800–3200 m, voucher: Bai Genlu 2004007, SANU), Callianthemum taipaicum W. T. Wang (alt. 3500–3600 m, voucher: Bai Genlu 2004008, SANU), and Trollius farreri Stapf (alt. 3500–3600 m, voucher: Bai Gen-lu 20040421, SANU) were collected from late March to early November in the years from 2002 to 2005 at intervals of seven to ten days in Taibaishan Mountains, Shaanxi province, and fixed in FAA. Flower buds were dehydrated in ethanol and isoamyl acetate series, critical-point dried, and observed with a Hitachi 800 SEM. For histological studies, flower buds were dehydrated in an alcohol series, infiltrated with xylene and embedded in paraffin wax. The embedded material was sectioned at 8 mm thickness and stained with safranin and fast green. Photographs of
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mature flowers were taken with a Nikon Coolpix 990 digital camera (Figs. 1–3) .
Results Adonis sutchuenensis (Adonidinae) (Figs. 1, 4–24) Floral morphology Flowers are solitary, terminal, 2–3 cm in diameter. The five to six sepals are green and obovately lanceolate. The 8–12 petals are yellow, narrowly obovate or oblongly obovate and about double the length of the sepals. A nectary is absent. The 25–50 stamens are about 1 3 of the length of the petals. The 17–25 carpels have short styles and small stigmas. All floral organs are free (Fig. 1). The carpels develop into nutlets. Floral development The flower bud is preceded by two leaves. The floral organs are initiated in a spiral sequence, either clockwise or counterclockwise. The five (rarely six to seven) young sepals are crescent-shaped and truncate (Figs. 4–6). It appears that there is a relatively long plastochron between the initiation of the last sepal and the first petal, because in the beginning the last sepal is much larger than the first petal (Figs. 4 and 5). The young petals are rounded and narrow (Figs. 4 and 5). The divergence angles in the perianth fluctuate around ca. 1371, which indicates spiral phyllotaxis according to the Fibonacci pattern (Figs. 4 and 5). Because the number of the petals is variable, and the shape of the petal and stamen primordia is similar, it is difficult to distinguish between the last petal and the first stamen in early development (Fig. 7). The carpels do not occupy the entire floral apex so that a small residual floral apex remains (Figs. 8 and 9), which is later hidden by the growing carpels. As seen from above, the young stamens and carpels form contact parastichies in sets of five and eight, which also conforms to spiral phyllotaxis according to the Fibonacci pattern (Fig. 8). In side views the different steepness of the regular contact parastichies is also conspicuous (Figs. 10 and 11). The sepals enlarge and enclose the other floral organs in later development. The young petals flatten after appearance of the carpels, and only in this developmental stage the petals and stamens can be clearly distinguished (Fig. 10). The petals develop faster and are always larger than the stamens (Fig. 11). However, they remain shorter than the sepals up to shortly before anthesis. When the carpel primordia appear gradually, the stamens in the outer part of the androecium enlarge (Figs. 10 and 11) and then differentiate into filaments and anthers (Fig. 12). Maturation of the stamens is centripetal (Figs. 21–24). After initiation of the carpels a
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Figs. 1–3. Mature flowers. (1) Adonis sutchuenensis. Bar ¼ 1.5 cm. (2) Callianthemum taipaicum. Bar ¼ 1.0 cm. (3) Trollius farreri. Bar ¼ 1.0 cm.
longitudinal concavity appears on the ventral side (Fig. 9), which initiates the plicate shape (Figs. 13 and 14). Later the single, lateral ovule is initiated (Fig. 15), the carpel closes and the ventral slit appears (Fig. 16). At first the carpel appears almost completely plicate. However, later the ascidiate base somewhat elongates (Fig. 17), and style and ovary become more distinct from each other (Fig. 17). On top of the style the ventral slit becomes everted and the stigma differentiates on the everted surface before the carpel matures (Fig. 18). The carpel becomes broadly subulate (Fig. 19) at anthesis. The stigma is unicellular papillate and is slightly decurrent along the ventral slit (Fig. 20) .
Callianthemum taipaicum (Adonidinae) (Figs. 2, 25–44) Floral morphology Flowers are solitary, terminal, 2–3 cm in diameter. The usually five blue purple sepals are narrowly oblong or lanceolate. The 9–13 petals are white with red veins and yellow orange at the base, narrowly obovate, slightly longer than the sepals, and have a nectary groove near the base. The 44–50 stamens are about 13 the length of the petals. The 21–24 carpels are distinctly stipitate and have short styles and small stigmas. All floral organs are free (Fig. 2). The carpels develop into nutlets. Floral development The flower bud is preceded by two leaves. The floral organs are initiated in spiral sequence, either clockwise or counterclockwise. The young sepals are crescentshaped, truncate and soon become somewhat rounded, and are broad at the base (Figs. 25 and 26). It appears that there is a relatively long plastochron between the initiation of the last sepal and the first petal, because in early development the last sepal is much larger than the first petal (Fig. 26). The young petals are rounded and
narrow at the base (Figs. 26–28, 31). The divergence angles in the perianth are ca. 1371 on average (Figs. 26 and 27), which indicates spiral phyllotaxis according to the Fibonacci pattern. Because petal number is unstable, and petals and stamens are difficult to distinguish in early development, it is not possible to determine the last petal and the first stamen in the beginning. The same is true for the transition from stamens to carpels (Fig. 29). When all carpels are formed, a small residual floral apex remains (Fig. 30) and is later covered by the developing carpels. As seen from above, the young stamens and carpels form contact parastichies in sets of five and eight, which also conforms with a spiral Fibonacci pattern (Fig. 30). The sepals enlarge and enclose the other floral organs in later development. The petals flatten when the carpels appear and only now petals and stamens can be clearly distinguished (Fig. 31). The petals develop more slowly and remain smaller than the stamens (Fig. 32). A bulge appears at the base of the petals (Fig. 33) and on the distal side of the bulge a concavity is formed in which nectariferous tissue will be differentiated (Fig. 34). When the carpels appear, the stamens in the outer part of the androecium begin their differentiation, which proceeds centripetally (Fig. 31), and finally differentiate into filaments and anthers (Fig. 35). The hemispherical young carpels become concave on the ventral side, and thus chair-shaped and ascidiate (Fig. 36). With carpel elongation the concave part also elongates and changes from an oblique to a more longitudinal direction (Fig. 37). With further elongation the carpels become pronouncedly stipitate and with deepening of the ventral concavity the plicate part becomes more pronounced. The two more or less collateral incipient ovules become visible before the ventral slit closes (Figs. 38–40). After closure the carpel further elongates, and the stipe remains more narrow so that the carpel becomes spindle-shaped (Figs. 41–43). The upper part of the ventral slit becomes everted and the stigma differentiates on the everted part (Fig. 43). The stigma is unicellular
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Figs. 4–12. Adonis sutchuenensis. Floral organ initiation and development. (4–8) Organ initiation. (4) Sepals. Bar ¼ 136 mm. (5) Petals. Bar ¼ 150 mm. (6) Petals. Bar ¼ 200 mm. (7) Stamens. Bar ¼ 200 mm. (8) Carpels. Bar ¼ 176 mm. (9) Carpels with incipient concavity on ventral side, and residual floral apex remaining after initiation of all carpels. Bar ¼ 200 mm. (10) Petals slightly larger than stamens. Bar ¼ 150 mm. (11) Petals larger than stamens. Bar ¼ 231 mm. (12) Centripetal developmental sequence of anthers. Bar ¼ 0.6 mm. (C: carpel. L: leaf. P: petal. S: sepal. St: stamen. Numbers after the letters indicate initiation sequence).
Trollius farreri (Trolliinae) (Figs. 3, 45–62)
head orange. The 17–36 stamens are about 23 the length of the sepals. The 5 (9) carpels have styles of medium length and narrow but conspicuously decurrent stigmas. All floral organs are free (Fig. 3). The carpels develop into follicles.
Floral morphology Flowers are solitary, terminal, 2–3.5 cm in diameter. The usually five or rarely six sepals are petaloid, yellow ventrally and dark purple dorsally, widely obovate, and persistent. The 12–15 petals are ca. 13 the length of the sepals, linear, unguiculate, with a nectary groove near the base; the narrow base is yellow and the enlarged
Floral development The flower bud is preceded by two triangular bracts. The floral organs are initiated in spiral sequence, either clockwise or counterclockwise. The young sepals are crescent-shaped, truncate and soon become rounded, and are broad at the base (Figs. 45 and 46). It appears that there is a relatively long plastochron between the
papillate and is slightly decurrent along the ventral slit (Fig. 44) .
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Figs. 13–24. Adonis sutchuenensis. Stamen and carpel development. (13–19) Carpel development. (13) Carpels becoming plicate. Bar ¼ 0.27 mm. (14) Somewhat later stage. Bar ¼ 250 mm. (15) Carpel with a lateral ovule primordium. Bar ¼ 86 mm. (16) Carpel with closed ventral slit. Bar ¼ 150 mm. (17) Carpel with early differentiation into stigma, style and ovary. Bar ¼ 0.5 mm. (18) Closeup of young stigma. Bar ¼ 150 mm. (19) Mature carpel. Bar ¼ 0.75 mm. (20) Close-up of mature stigma. Bar ¼ 176 mm. (21–24) Longitudinal sections of stamens, showing centripetal development of microspore mother cells in androecium. (21) Longitudinal section of flower, overview. The boxes refer to Figs. 22–24. Bar ¼ 0.07 mm. (22) Close-up of outermost stamen of Fig. 21, microspore mother cells in late meiotic prophase. Bar ¼ 290 mm. (23) Close-up of second stamen of Fig. 21, microspore mother cells slightly less advanced in differentiation. Bar ¼ 250 mm. (24) Close-up of innermost stamen of Fig. 21, microspore mother cells in early meiotic prophase. Bar ¼ 250 mm. (AW: anther wall. C: carpel. MM: microspore mother cell. O: ovule. P: petal. St: stamen).
initiation of the last sepal and the first petal because the last sepal is initially much larger than the first petal. Young petals are rounded, and narrow at the base (Figs. 46 and 47). The divergence angles in the perianth oscillate around ca. 1371 (Figs. 46 and 47), which indicates spiral phyllotaxis according to the Fibonacci pattern. Because the number of the petals is labile, and petals and stamens are difficult to distinguish in early
development, it is not possible to determine the last petal and the first stamen in the beginning. The same is true for the transition from stamens to carpels (Figs. 48 and 49). After the initiation of all carpels, a small residual floral apex remains (Figs. 48 and 49) and is later covered by the developing carpels. In apical view the young stamens and carpels form contact parastichies in sets of five and eight (Fig. 48).
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Figs. 25–35. Callianthemum taipaicum. Floral organ initiation and development. (25–29) Organ initiation. (25) Sepals. Bar ¼ 120 mm. (26) Petals. Bar ¼ 120 mm. (27) Petals and/or stamens. Bar ¼ 136 mm. (28) Stamens, side view. Bar ¼ 220 mm. (29) Carpels. Bar ¼ 136 mm. (30) Carpels with incipient concavity on ventral side, and residual floral apex remaining. Bar ¼ 350 mm. (31–34) Development of petals. (31) Young petals slightly broader than stamens. Bar ¼ 150 mm. (32) Young petals of similar length as stamens. Bar ¼ 0.38 mm. (33) Bulge appearing at base of the petal. Bar ¼ 0.6 mm. (34) A nectariferous groove forms distally of the bulge. Bar ¼ 0.86 mm. (35) Centripetal developmental direction of stamens in the androecium. Bar ¼ 0.86 mm. (C: carpel. P: petal. S: sepal. St: stamen. Numbers after the letters indicate initiation sequence).
The young sepals enlarge and partly enclose the floral apex while stamens and carpels are initiated (Figs. 49 and 50). In the beginning, before the carpels appear, the petals become slightly broader than the stamens (Figs. 49 and 50). However, when the stamens begin their differentiation, growth of the petals lags behind that of the stamens (Fig. 51). In later development, the petals remain as very small scales outside of the androecium (Fig. 52). When the flower matures, the petals become linear, unguiculate, with a nectary groove near the base of the ventral side and an enlarged head (Figs. 53 and 54). Stamen maturation is centripetal (not shown). The hemispherical young carpels soon become concave on the ventral side (Fig. 49) and become ascidiate. That the carpels are not arranged in a whorl but in a spiral, is clearly seen by their different distances from the floral centre (Figs. 55 and 56). The carpels
considerably elongate, the ventral slit closes (Fig. 57), and two crests develop along the ventral slit (Figs. 58–60). With carpel elongation the plicate zone becomes more prominent. The ovary tapers into the style (Figs. 59 and 61). The stigma is unicellular papillate and decurrent along the ventral slit for some distance (Figs. 61 and 62).
Discussion Floral phyllotaxis Floral phyllotaxis is variously whorled and spiral in Ranunculaceae, with both patterns sometimes at low systematic level (Endress, 1987; Hiepko, 1965; Scho¨ffel, 1932). In Aquilegia, Semiaquilegia, and Enemion of
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Figs. 36–44. Callianthemum taipaicum. Carpel development. (36) Concavity formed on ventral side of young carpels. Bar ¼ 150 mm. (37) Young carpels becoming plicate. Bar ¼ 120 mm. (38) Carpels halfway closed, the two more or less collateral incipient ovules still visible, stipe formation. Bar ¼ 176 mm. (39) Carpel with one lateral ovule visible. Bar ¼ 150 mm. (40) Opened carpel, showing two lateral ovules. Bar ¼ 100 mm. (41) Carpels closed. Bar ¼ 176 mm. (42) Differentiation of carpel into stigma, style and ovary. Bar ¼ 0.86 mm. (43) Mature carpel. Bar ¼ 1.0 mm. (44) Close-up of mature stigma. Bar ¼ 231 mm. (C: carpel. O: ovule. St: stamen).
Isopyroideae (or Thalictroideae) it is strictly whorled, although the sepals originate in a spiral sequence, which leads to quincuncial aestivation (Scho¨ffel, 1932; Tucker and Hodges, 2005). Thus, the divergence angles of subsequent sepals in these pentamerous flowers are closer to 1441 (as typical in whorled phyllotaxis) than to 1371 (as typical in spiral phyllotaxis). In contrast, in Anemone rivularis Buch.-Ham. ex DC. var. flore-minore Maxim. (Chang et al., 2005), Caltha palustris Linn. and Trollius buddae Schipcz. (Song et al., 2007) of Ranunculoideae, and Coptis chinensis Franch. (Gu and Ren, 2007) of Coptidoideae floral phyllotaxis is spiral
throughout. However, in Anemone there are also species with whorled flowers (Scho¨ffel, 1932; Tobe, 1976). In the species studied here the flowers are spiral throughout. This can be easily seen by the different steepness of contact parastiches (see also Endress, 2006; Endress and Doyle, 2007). Spiral floral phyllotaxis had been recorded earlier for other species of Adonis and Trollius (Scho¨ffel, 1932; Thompson, 1934) but the present study appears to be the first record for Callianthemum. Character optimizations in Ranunculales show that it is still ambiguous which of the two patterns is plesiomorphic in Ranunculaceae (Doyle and Endress, 2000; Endress
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Figs. 45–54. Trollius farreri. Floral organ initiation and development. (45–48) Organ initiation. (45) Sepals. Bar ¼ 100 mm. (46) Petals. Bar ¼ 120 mm. (47) Slightly later stage. Bar ¼ 136 mm. (48) Stamens and carpels. Bar ¼ 176 mm. (49–54) Organ development. (49) Carpels with incipient concavity on ventral side. Bar ¼ 176 mm. (50) Petals broader than stamens before carpel initiation. Bar ¼ 231 mm. (51) Slightly later stage, with petals shorter than stamens. Bar ¼ 200 mm. (52) Petals still very small after anther differentiation. Bar ¼ 231 mm. (53 and 54) Mature petals. (53) Ventral view, showing a nectariferous groove at the base of the blade. Bar ¼ 1.2 mm. (54) Dorsal view. Bar ¼ 1.2 mm. (C: carpel. L: leaf. P: petal. S: sepal. St: stamen. Numbers after the letters indicates initiation sequence).
and Doyle, 2007). This uncertainty is because the closest relatives of Ranunculaceae have largely whorled floral phyllotaxis and the two basal ranunculaceous genera, Glaucidium and Hydrastis, do not appear to have spiral phyllotaxis either (figures of Hydrastis in Tobe and Keating, 1985, and personal observations by P.K.E.).
Floral development The sepal primordia of Adonis sutchuenensis, Callianthemum taipaicum, and Trollius farreri are crescent-
shaped and truncate. However, petal, stamen, and carpel primordia are rather hemispherical, and are also smaller than those of the sepals. The same was found in other representatives of the family (Chang et al., 2005; Gu and Ren, 2007; Song et al., 2007; Tucker and Hodges, 2005). It appears that there is a relatively long plastochron between the last initiated sepal and the first petal, whereas the plastochrons between all subsequent organs are shorter and all equal. That petals and stamens do not show a gap in plastochrons and are difficult to distinguish in shape in early stages may support their evolutionary relationship (see also Hiepko,
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Figs. 55–62. Trollius farreri. Carpel development. (55) The five carpels arranged in a (spiral) series. Bar ¼ 250 mm. (56) Carpels closed. Bar ¼ 176 mm. (57) Closed carpel from the side, slightly older than in Fig. 56. Bar ¼ 100 mm. (58) Carpel from ventral, with longitudinal crests along ventral slit. Bar ¼ 120 mm. (59) Carpel differentiated into style and ovary. Bar ¼ 1.2 mm. (60) Close-up of upper part of Fig. 59. Bar ¼ 0.3 mm. (61) Mature carpel. Bar ¼ 1.36 mm. (62) Close-up of upper part of Fig. 60, with decurrent papillate stigma covered with pollen grains. Bar ¼ 0.27 mm. (C: carpel. St: stamen).
1965; Endress, 1995; Erbar et al., 1998; Kramer et al., 2003; Tamura, 1984, 1993). Petals are especially plastic in Ranunculaceae. As in many core eudicots they commonly form the inner whorl or series of perianth organs and are commonly delayed in their development in bud and have a single vascular trace (Hiepko, 1965). However, petals in Ranunculaceae can attain diverse and sometimes elaborate shapes, and in contrast to most core eudicots they commonly bear nectaries. In many genera they are optically attractive, but in others this function may also be exerted by sepals or stamens. In Adonis, Callianthemum, and Trollius petal development is retarded in bud compared with the sepals. However, petals bear a nectary only in Callianthemum and Trollius, whereas the flowers in Adonis are nectarless (see also Hiepko, 1965). Maturation of the stamens in Adonis, Callianthemum, and Trollius is centripetal in accordance with the initiation sequence of the organs (this study; Song et al., 2007, for Trollius buddae). This should be mentioned because in some other Ranunculaceae
centrifugal maturation was recorded (despite centripetal or almost simultaneous initiation), such as in Aquilegia (Feng et al., 1995; Tepfer, 1953), Glaucidium (Tamura, 1972), and Anemone rivularis var. flore-minore (Chang et al., 2005). This difference and the discordance between direction of initiation and maturation need more comparative studies in further genera of the family. Early carpel development is similar in all three taxa. The primary margins of the carpel formed concomitantly with the ventral concavity do not extend to the very base of the carpel but meet over the ventral side thus forming a cross zone and a short ascidiate zone, even without a median position of a fertile ovule. The presence of an ascidiate zone is most common in Ranunculaceae (Endress and Igersheim, 1999; Rohweder, 1967), although taxa with completely plicate carpels, such as Xanthorhiza, may also exist in the family (Endress and Igersheim, 1999). Further, the carpel base is more narrow than the ovary in all three genera forming a stipe of various length. In the apical region the ventral slit appears more or less everted
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forming two longitudinal crests of various length on which the stigma differentiates. In Adonis and Callianthemum the carpels close only after ovule initiation – as can also be found in other genera with uniovulate carpels (Ranunculus, Myosurus; van Heel, 1981, 1984) – whereas in the pluriovulate carpels of Trollius closure appears to be earlier, and ovules are not visible in closing carpels.
Systematic position of Adonis and Callianthemum Adonis and Callianthemum, both genera with achenes, were formerly thought to be related with other achenebearing genera and were placed in Adonidinae, Ranunculeae, Ranunculoideae (Wang, 1980) or Adonideae (Tamura, 1993). However, molecular studies revealed that Adonis is closely related to the follicular genus Trollius (Hoot, 1995; Johansson, 1995; Johansson and Jansen, 1993; Ro et al., 1997). This alliance was also suggested by chemical (Jensen, 1995) and serological features (Jensen, 1968). Based on these studies, Jensen et al. (1995) placed Adonis (and, tentatively, Callianthemum) in its own subtribe Adonidinae, which was included in Adonideae together with the unigeneric Trolliinae. In Tamura’s (1995) treatment Adonis and Callianthemum make up his Adonideae. Callianthemum was not included in molecular studies as yet. That Adonis and Callianthemum are closely related to each other and to Trollius, and not to Anemoneae (Anemone, Clematis and relatives), despite their oneseeded fruitlets, is also supported by our present floral developmental results and by other structural features reported earlier. Adonis and Callianthemum are similar in their stigma differentiation. Both genera have sepals and petals, like Trollius, but unlike Anemone and most Clematis species, which only have (petaloid) sepals (Hiepko, 1965). Although the petals in Adonis lack nectaries, in contrast to Callianthemum, also in Callianthemum the petals are flat and simple and the nectaries are sometimes small and inconspicuous (Schaeppi and Frank, 1957). Double perianths (consisting of sepals and petals) combined with unelaborate petal shape are otherwise unusual in Ranunculaceae, which is in line with a close relationship between Adonis and Callianthemum. Most importantly, the ovules of both Adonis and Callianthemum have two integuments (Baillon, 1863; Bhandari, 1962, 1963, 1967; Lonay, 1901, 1907; Prantl, 1887; Rassner, 1931; Schaeppi and Frank, 1962; Soue`ges, 1912; Wang and Ren, 2008) like Trollius (Bersier, 1960), whereas those of Anemone and Clematis have only one integument (Bessey, 1898; Bhandari, 1968; Fish, 1970; Lonay, 1901; Prantl, 1887; Schaeppi and Frank, 1962; Shukova, 1965; Soue`ges, 1910–1914; Tobe et al., 1975; Vijayaraghavan, 1962; Voelter and Weber, 1962; Wang and Ren, 2008;
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Wiegand, 1894). Furthermore, the single fertile ovule is lateral in Adonis and Callianthemum, but median in Anemone and Clematis (Prantl, 1887; Schaeppi and Frank, 1962; Tamura, 1984). Carpels in Adonis sect. Consiligo have a single ovule, in Callianthemum additionally a reduced one (Baillon, 1863; Prantl, 1887; Schaeppi and Frank, 1957, 1962), whereas in Adonis sect. Adonis several reduced ovules are present (Bhandari, 1962, 1963, 1967; Brouland, 1936; Lonay, 1901; Prantl, 1887; Rassner, 1931; Schaeppi and Frank, 1962; Soue`ges, 1912). Several reduced ovules are commonly also present in Anemoneae (for Anemone and Clematis: Bessey, 1898; Chute, 1930; Fish, 1970; Lonay, 1901; Payer, 1857; Prantl, 1887; Rassner, 1931; Rohweder, 1967; Shukova, 1965; Soue`ges, 1910–1914; Tobe et al., 1975; Vijayaraghavan, 1962). Trollius, Adonis and Callianthemum share the presence of often a larger number of carpels, an ascidiate carpel base, and (except for a part of Adonis), more than one ovule per carpel, even if only one is fertile, and a stipe of various length (short in Trollius and Adonis, longer in Callianthemum) (see also Schaeppi, 1972; Schaeppi and Frank, 1957, 1962). However, these features are also present in Anemoneae (Sprotte, 1940). Thus they may be plesiomorphies in Ranunculoideae.
Acknowledgement The project was supported by the National Nature Science Foundation of China (No. 30370095).
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