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Staedler, Y M; Weston, P H; Endress, P K (2009). Comparative structure and development in (). International Journal of Sciences, 170(1):21-41. Postprint available at: http://www.zora.uzh.ch University of Zurich Posted at the Zurich Open Repository and Archive, University of Zurich. Zurich Open Repository and Archive http://www.zora.uzh.ch Originally published at: International Journal of Plant Sciences 2009, 170(1):21-41.

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Year: 2009

Comparative Gynoecium structure and development in Calycanthaceae (Laurales)

Staedler, Y M; Weston, P H; Endress, P K

Staedler, Y M; Weston, P H; Endress, P K (2009). Comparative Gynoecium structure and development in Calycanthaceae (Laurales). International Journal of Plant Sciences, 170(1):21-41. Postprint available at: http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch

Originally published at: International Journal of Plant Sciences 2009, 170(1):21-41. Int. J. Plant Sci. 170(1):21–41. 2009. Ó 2009 by The University of Chicago. All rights reserved. 1058-5893/2009/17001-0003$15.00 DOI: 10.1086/593045

COMPARATIVE GYNOECIUM STRUCTURE AND DEVELOPMENT IN CALYCANTHACEAE (LAURALES)

Yannick M. Staedler,1,* Peter H. Weston,y and Peter K. Endress*

*Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland; and yRoyal Botanic Gardens and Domain Trust, Sydney, Mrs Macquaries Road, Sydney, New South Wales 2000,

Calycanthaceae, sister to all other Laurales, are the most distinct family of the order in their gynoecium structure (lateral placentation, presence of two ovules). Gynoecium structure was studied in a representative of each of Calycanthaceae and gynoecium development in a representative of each of the two subfamilies (Calycanthoideae and Idiospermoideae). Newly found shared features are postgenital coherence between the free carpels (extragynoecial compitum), abortion of the upper of the two ovules, and lobation of the outer integument. Differences in reproductive structures between the two subfamilies are reviewed. Newly found differences include carpel primordium shape, contribution of the outer integument to micropyle formation, epidermis differentiation of the rim of the integuments, and mode of formation of the compitum. Unexpectedly, at anthesis, ovary and ovules of are not larger than those of Calycanthoideae, despite the conspicuous difference in . The identity of ‘‘’’ (sterile organs between and carpels) is discussed. Sterile carpel-like structures (carpellodes) are documented in Idiospermum. The lateral ovule position in Calycanthaceae is correlated with a different development of carpel closure as compared with core Laurales, which exhibit median ovule position. Gynoecium morphology of fossil Calycanthaceae and its implications for gynoecium evolution in Calycanthaceae and Laurales are discussed.

Keywords: Calycanthaceae, Laurales, Idiospermum, gynoecium development, carpel development, compitum.

Introduction 1969; Erbar 1983; van Heel 1984; Endress and Igersheim 1997); they usually enclose two ovules (occasionally only one Calycanthaceae are sister to the remainder of Laurales (core in some carpels of a flower; Eames 1961; Tiagi 1964; Wilson Laurales; Renner 1999, 2004; Qiu et al. 2005) and comprise 1976) with collateral placentation, positioned one on top of about seven in four genera of temperate and trop- the other at anthesis (Baillon 1868; Blake 1972; Endress and ical (Cheng and Chang 1964; Nicely 1965; Blake 1972; Li Igersheim 1997). The ovules are anatropous, bitegmic, and crassi- 2007). Within Calycanthaceae, the deepest split is between the nucellar; however, only the lower ovule is fertile (Endress and tropical monotypic Idiospermum australiense (Idiospermoi- Igersheim 1997). In contrast, all core Laurales studied so far have deae) and the temperate shrubs of the rest of the family (Caly- a single ovule per carpel with median placentation (see Endress canthoideae; Li et al. 2004; Zhou et al. 2006); the split between and Igersheim 1997). the two subfamilies has been estimated to be at least 70 Myr Developmental studies on the gynoecium in Calycantha- (Zhou et al. 2006). ceae are scarce and have focused on . Carpel de- What makes Calycanthaceae unique in Laurales are features velopment was studied by Erbar (1983) and van Heel (1984). of the gynoecium: (1) ovule number and placentation differ Developmental studies of the ovules have also focused mostly from all other Laurales, and (2) the in Idiospermum have on Calycanthus (Baillon 1868; Lignier 1892; Endress 1972, the largest known in angiosperms (Blake 1972; En- only ). Several meiocytes were found to de- dress 1983). The gynoecium of Calycanthaceae is apocarpous, velop in the nucellus of Calycanthus (Jo¨ nsson 1881; Mathur with 1–40 spirally arranged carpels (Cheng and Chang 1964; 1968; Kamelina 1981) and Chimonanthus (Ly Thi Ba 1962; Nicely 1965; Blake 1972; Staedler et al. 2007). The carpels are Endress 1972). Nothing is known about gynoecium development borne on the bottom of an extensive floral cup (Baillon 1868; of Idiospermum, despite its extremely large seeds. Within Cal- Cheng and Chang 1964; Blake 1972; fig. 1). In some flowers, a gy- ycanthaceae, anthetic gynoecium features unique to Idiosper- noecium is lacking altogether (Blake 1972; Badrutt 1992; Staedler mum include presence of only one carpel (rarely up to five; et al. 2007). The stigmas are secretory and vary in morphology Worboys 2003) and sessile and fleshy stigmas. between the two subfamilies (Endress and Igersheim 1997). An The morphology of I. australiense is distinctively different extragynoecial compitum was reported for some Calycanthaceae from that of the rest of the Calycanthaceae, especially in gy- (Endress and Igersheim 1997). The carpels are largely plicate noecium morphology. Precise knowledge of gynoecium mor- but possess a short ascidiate base (Schaeppi 1953; Leinfeller phology and development of I. australiense will thus allow us to improve our ability to distinguish between possible ple- siomorphies in Laurales and synapomorphies in Calycanthoi- 1 Author for correspondence; e-mail: [email protected]. deae. Features of interest include presence or absence of a Manuscript received March 2008; revised manuscript received June 2008. compitum, early carpel development, mode of carpel closure,

21 22 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 1 longitudinal sections at female phase of anthesis. A, Idiospermum australiense. B, Calycanthus floridus. C, Sinocalycanthus chinensis. D, . Scale bars ¼ 5 mm. stigma differentiation, placentation, ovule number, integument Idiospermum australiense (Diels) S. T. Blake: BPM Hyland morphology, and micropyle structure. 6242 (north of Daintree River, , Australia; popula- We studied and compared representatives of all four genera tion north of ); YM Staedler S006-26, S006-27, S006-28 of Calycanthaceae in their gynoecium structure at anthesis (fig. 15D,15D9), S006-29, S006-39, S006-57 (fig. 15C,15C9), (figs. 1–12). Carpel development was comparatively studied S006-27 (fig. 13E,13E9,13F,13F9,13F99,13G,13G9; fig. for one representative of each subfamily: I. australiense for 15A,15A9,15B,15B9,15E,15E9), S006-54 (figs. 1A,2A), Idiospermoideae (fig. 13) and Calycanthus floridus for Caly- S006-57 (fig. 9A; fig. 10A,10A9; fig. 11A,11A9; fig. 13H99; canthoideae (fig. 14). Because the morphological nature of CSIRO research plot, Daintree National Park, Queensland, the so-called staminodes has been disputed ( or carpel Australia; population north of Cairns); PH Weston 004-10, derived; Hiepko 1965), these organs are also included in this 004-79, 004-245, 005-84, 005-85, 005-86, 005-87, 005-88, study. We discuss the implications of our findings on the mor- 005-89, 07-120, 004-245 (fig. 13D,13D9), 005-85 (fig. 13A, phological interpretations of fossil calycanthaceous flowers 13A9,13B,13B9,13C,13C9), 005-88 (fig. 3A), 005-89 (fig. and on the evolution of gynoecium morphology in Laurales. 13H,13H9), 07-124-2 (figs. 4A,5,12A; Royal Botanic Gar- dens, Sydney, Australia; from population north of Cairns); SJ Material and Methods Worboys 395 (fig. 2B; Queensland, Australia; population south of Cairns). The following species and collections were used for this Calycanthus floridus L. (one individual, BGZ): YM Staedler study (materials collected from cultivated in the Bota- S004-42 (fig. 14D,14D9), S004-49 (fig. 14F99,14G,14G9, nic Garden of the University of Zurich are coded as BGZ). 14G99), S004-50 (fig. 14E,14E9,14E99), S004-509 (fig. 14F, Collections not attributed to a figure have been used for de- 14F9), S004-62 (determination of organ number; figs. 6, 4B), termination of organ number (number of carpels, number of S004-63 (figs. 3B,12B), S004-74 (fig. 10B,10B9; fig. 11B, staminodes, proportion of flowers with carpellodes, propor- 11B9; fig. 14H,14H9,14H99), S004-90 (fig. 9B), S007-67 (fig. tion of uniovulate carpels) or organ dimensions (stigma, style). 14A,14A9,14B,14B9), S007-81 (fig. 14C,14C9,14D99), For Idiospermum, collections of populations north and south S007-90 (fig. 1B; fig. 2C,2C9). of Cairns are distinguished because populations south of Sinocalycanthus chinensis (W. C. Cheng & S. Y. Chang) Cairns tend to have more carpels per flower (Worboys 2003). W. C. Cheng & S. Y. Chang (one individual, BGZ, individual STAEDLER ET AL.—GYNOECIUM IN CALYCANTHACEAE (LAURALES) 23

Fig. 2 Flower longitudinal sections showing anthesis process in Idiospermoideae and Calycanthoideae. A–B, Idiospermum australiense, population north of Cairns. A, Female phase. A9, Male phase. B, Population south of Cairns, female phase. C, C9, Calycanthus floridus. C, Female phase. C9, Male phase. no. 2): YM Staedler S004-92 (determination of organ number; (fig. 1A;fig.2A,2A9) and broad and beaker shaped in the flow- fig. 9C; fig. 10C,10C9; fig. 11C,11C9), S005-59 (fig. 12C), ers of the populations south of Cairns (fig. 2B). Most observa- S005-62 (determination of organ number; figs. 7, 4C), S005- tions were made on flowers from the populations north of 66 (fig. 3C), S007-127 (fig. 1C). Cairns; for floral cup shape and carpel number, observations Chimonanthus praecox (L.) Link (one individual, BGZ): were made from both population groups north and south of PK Endress 04-3 (determination of organ number: figs. 4D, Cairns. In our material, the floral cup contains 0–2 carpels and 8); YM Staedler S006-1 (fig. 3D), S006-4 (fig. 1D), S006-8 13–21 staminodes (in all three opened of the population (fig. 9D; fig. 10D,10D9; fig. 11D,11D9; fig. 12D). south of Cairns, two carpels were found; number Laurus nobilis L.: PK Endress 2654 (fig. 16B; cultivated, was not recorded). Carpels are borne at the bottom of the flo- Ascona, Canton Ticino, Switzerland). ral cup (fig. 1A). The stigmas are large, sessile, and papillate Organ number was determined by averaging numbers in (;0.8 3 0.4 mm; figs. 1A,3A,4A,5A,9A; fig. 13H,13H9) three flowers of the same individual; organ dimensions were and do not protrude from the floral cup (fig. 1A). When two obtained by measuring dimensions on a typical organ, for carpels are present, the stigmas are postgenitally coherent and three different flowers. Plants were fixed and stored in 70% appear to form a compitum (figs. 3A,4A,5A). Coherence of the ethanol. Serial microtome sections were made after embedding stigmas is strong. They cannot be separated from each other in Kulzer’s Technovit 7100 (2-hydroxyethyl methacrylate; without breaking off from the ovary. The staminodes line the Igersheim and Cichocki 1996). The sections were stained with entire inner wall of the floral cup (fig. 1A). toluidine blue and ruthenium red. For SEM studies, specimens Carpels are glabrous, except for the adaxial portion below were critical-point dried, sputter-coated with gold, and studied the lower end of the ventral slit, which is covered by hairs at 20 kV with a Hitachi S-4000 SEM. (fig. 13F–13H). The stigma is bifacial (figs. 4A,5A). Most of the ovary is plicate (fig. 5C–5E), but there is a distinct ascidi- ate base (fig. 5F). At anthesis, the ascidiate zone is 240 (645) mm long, which represents 17% of the ovary length. A short Results (20 mm) stipe is present in all five sectioned fertile carpels (fig. 5G). The carpels have one dorsal and two lateral vascu- Gynoecium Structure at Anthesis lar bundles. The carpel wall is 10–12 cell layers thick at the Idiospermum australiense. have a pitcher-shaped level of the placenta, at middistance between the dorsal and lat- floral cup with a thick wall; its inner space is deep and narrow, eral vascular bundles. The carpels contain usually two ovules test tube shaped in flowers of the populations north of Cairns (13 of the 18 studied carpels), but normally shaped carpels 24 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 3 Gynoecium (SEM micrographs). A, Idiospermum australiense. B, Calycanthus floridus. C, Sinocalycanthus chinensis. D, Chimonanthus praecox. Scale bars ¼ 1 mm.

with one ovule, or no ovule at all, also occur (2 and 3 of the by the inner integument (fig. 5F, carpel 1). Rarely, one of the 18 studied carpels). The ovules have collateral placentation two lateral lobes of the outer integument is folded over the (fig. 5E) at the base of the plicate zone, just above the begin- other, and the micropyle is formed by both integuments (fig. ning of the ascidiate zone. By curvature into different direc- 12A). The rim of the outer integument is appressed against tions in early development, they come to lie one above the the bottom of the ovarial cavity. The rim of both integuments other (fig. 9A). lacks hairs. At midlength, the outer integument is 8–9 cell The ovules are anatropous, bitegmic, and crassinucellar. layers thick, and the inner integument is 5–6 cell layers thick. The lower ovule is ovoid (fig. 10A) and 0.7–0.8 mm long (fig. The funicle is very short and relatively stout (fig. 5E, carpel 1; 5C–5F). The outer integument is semiannular and lobed (fig. fig. 10A,10A9). The ovule vascular bundle appears to ramify 5F; fig. 10A,10A9). Usually, the lobes of the outer integument in the chalaza into four small bundles, which extend into the are not appressed to each other, and the micropyle is formed base of the outer integument (fig. 12A). Ovules in uniovulate STAEDLER ET AL.—GYNOECIUM IN CALYCANTHACEAE (LAURALES) 25

Fig. 4 Transverse sections of gynoecium at level of compitum. A, Idiospermum australiense. B, Calycanthus floridus. C, Sinocalycanthus chinensis. D, Chimonanthus praecox. Scale bar ¼ 100 mm.

carpels are morphologically and topographically indistinguish- topographically lowest third of the wall of the floral cup (fig. able from the lower ovule in biovulate carpels. 1B). The styles are long and slender (4.7 mm long, 0.1 mm The upper ovule is usually shorter, only two-thirds of the wide; figs. 1B,3B,4B; fig. 6A,6A9,6B; figs. 9B,14H–14H99); length of the lower ovule (fig. 9A) and not fully developed. It they protrude from the floral cup but are enclosed in the is hood shaped (fig. 11A,11A9). The integuments do not chamber formed by the inner (fig. 1B). The stigmas are cover the nucellus apex (fig. 11A9); the nucellus is oriented short, papillate, and secretory (;0.2 mm long; fig. 1B; fig. 6A, downward, and the rim of the integuments faces the lower 6A9). Secretion (stained red by ruthenium red) is found along ovule and is contiguous with it (fig. 9A). The funicle is long the whole length of the styles. Most styles are postgenitally co- and slender (fig. 11A); it is often detached from the placenta herent and appear to form a vestigial compitum (figs. 4B,6B). at anthesis. Most of the tissues of the upper ovule are shriv- The coherence of the styles is weak, and separation of individ- eled (fig. 11A,11A9). ual styles from the bundle of styles is easy; coherence does not Sterile carpel-like structures (carpellodes) are found in 12 seem to be resistant to preparation for SEM (fig. 3B) but can of the 76 flowers with female reproductive structures studied. be observed in transverse microtome sections (figs. 4B,6B). Carpellodes are usually columnar structures of varying size, The staminodes are borne on the topographically upper third which often bear a stigma at anthesis (fig. 15A–15E9). A of the inner wall of the floral cup (fig. 1B). short ventral slit (fig. 15A) and an inner space may be present Carpels are covered with hairs; the hairs are longer at the (fig. 15A–15B9) or absent (fig. 15C–15E9). Absence of post- base of the style and on the adaxial portion below the lower genital fusion of the ventral slit, combined with the lack of a end of the ventral slit. The stigma is bifacial (fig. 6A,6A9). stigma, was found in one case (fig. 15B,15B9). The style is plicate, and the ventral slit is not postgenitally Calycanthus floridus. Flowers have a broad, urceolate fused at this level (fig. 4B; fig. 6A,6A9). Most of the ovary is floral cup (fig. 1B; fig. 2C,2C9) and contain 25–35 carpels also plicate but with the ventral slit postgenitally fused (fig. and 16–22 staminodes. Carpels are at the bottom and on the 6C, carpels 5–29, 31; fig. 6D,6E, carpels 32–35). There is 26 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 5 Idiospermum australiense, transverse section series of anthetic bicarpellate gynoecium. Vasculature in gray. Carpels numbered. A, Level of stigmas (compitum). B, Level of styles. C, Level of upper ovules. D, Level of lower ovules. E, Level of placentae. F, Level of tip of lower ovules. G, Level of stipes. o ¼ outer integument, i ¼ inner integument. Scale bar ¼ 1 mm.

only a short ascidiate base (fig. 6E, carpels 30, 33; fig. 4F, the outer integument (fig. 12B). Ovules in uniovulate carpels carpels 32, 34, 35). For inner carpels of the gynoecium, the are morphologically and topographically indistinguishable ascidiate zone is 140 mm(660 mm) long, which represents from lower ovules in biovulate carpels. 8% of the ovary length. A short (;100 mm) stipe is present The upper ovule is usually shorter, only one half of the in innermost carpels (fig. 6F, carpel 33); presence of a stipe is length of the lower ovule (fig. 9B) and not fully developed. It difficult to assess for peripheral carpels borne on the sides of is hood shaped (fig. 11B,11B9). The integuments do not cover the floral cup because their base is vertically and not horizon- the nucellus apex (fig. 11B,11B9); the nucellus and the open- tally oriented (fig. 6C, carpels 1–5). The carpels have one ing of the two integuments are oriented sideward (fig. 9B). dorsal and two lateral vascular bundles. The carpel wall is The upper ovule lies on the lower one (fig. 9B). The outer in- 10–12 cell layers thick at the level of the placenta, at middis- tegument does not completely cover the inner integument in tance between the dorsal and lateral vascular bundles. The the contact zone between both ovules (fig. 11B,11B9); hence, carpels usually contain two ovules (98 of 100 carpels out of the inner integument is in contact with the lower ovule. The three flowers studied). Carpels with one ovule were also ob- funicle is long and slender (fig. 11B); it is often detached from served (2 of 100 studied carpels). A carpel devoid of ovules the placenta at anthesis. The tissues of the upper ovule are was observed once at the transition between staminodes and usually strongly shriveled (fig. 11B,11B9). carpels. The ovules have collateral placentation (fig. 6D,carpel Aborted carpels are present in all six studied. Con- 31; fig. 6D9) at the base of the plicate zone, above the begin- spicuous patches of aborted carpels were found in three of ning of the ascidiate zone. By curvature into different directions the six fruits. One or several sterile fruiting carpels were al- in early development, they come to lie one above the other (fig. ways found among the last initiated carpels of a gynoecium. 9B; fig. 14F99,14G99,14H99). Sinocalycanthus chinensis. Flowers have a broad, urce- The ovules are anatropous, bitegmic, and crassinucellar. olate to bowl-shaped floral cup (fig. 1C) and contain 12–16 The lower ovule is elongate-ovoid (fig. 10B); it is 0.8–0.9 mm carpels and 14–17 staminodes. Carpels are at the bottom and long (fig. 6C, carpels 6, 8–18; fig. 6D, carpels 22, 24–33; fig. the topographically lowest third of the wall of the floral cup 6E, carpels 32, 33). The outer integument is semiannular and (fig. 1C). The styles are long and slender (4.8 mm long, ;0.1 lobed (fig. 6D,6D99). One of the two lateral lobes of the outer mm wide at the tip, 0.04 mm wide at the base; figs. 1C,3C,4C; integument is usually folded over the other. This ‘‘fold’’ is situated fig. 7A,7A9;fig.9C); they protrude from the floral cup (fig. 1C). in the median plane of the ovule. Growth of the outer integu- The stigmas are short and papillate (;0.3 mm long; fig. 1C;fig. ment causes it to be moulded by the bottom of the ovarial cav- 7A,7A9). The basal portions of all the styles are covered with se- ity, such that the fold attains the form of a ridge (fig. 10B, cretion (stained red by ruthenium red); they are postgenitally co- 10B9). In most ovules, the micropyle is formed by both integu- herent and appear to form a compitum (figs. 3C,4C,7B). The ments (fig. 12B); in the innermost carpels of the gynoecium, tissue of the lower, coherent portion of the styles is small celled the micropyle may be formed by the inner integument (fig. 6E, and cytoplasm rich (fig. 4C). Coherence of the styles is strong, 6F, carpel 34 ), or there may be no micropyle at all (fig. 6E, and separation of individual styles from the bundle of styles is 6F, carpel 35). A micropylar cavity is present between the difficult; coherence is resistant to preparation for SEM (fig. 3C). outer and the inner integument and between the inner integu- The staminodes are borne on the topographically upper third of ment and the nucellus (fig. 12B). Hairs extend from the rim of the inner wall of the floral cup (fig. 1C). both integuments (fig. 12B). At midlength, the outer integu- The carpel indument is as in Calycanthus floridus. Stigma ment is 6–7 cell layers thick, and the inner integument is 4 cell and style (fig. 7A,7A9) and ovary structure (fig. 7C, carpels 1, layers thick. The funicle is short and relatively stout (figs. 6D9, 3; fig. 7D, carpels 1–11; fig. 7E, carpels 4–13; fig. 7F, carpels 10B,12B). The ovule vascular bundle appears to ramify in the 6–13) are as in C. floridus. For inner carpels of the gynoecium, chalaza into four small bundles, which extend into the base of the ascidiate zone is 330 mm long, which represents 15% of Fig. 6 Calycanthus floridus, transverse section series of anthetic gynoecium. Vasculature in gray. Carpels numbered in order of initiation. A, Level of stigmas and styles. A9, Close-up of carpels 14 (style) and 22 (stigma). B, Level of lower part of styles (compitum). C, Level of middle part of ovaries. D, Level of lower part of ovaries. D9, Close-up of carpel 31, level of placentae. D99, Close-up of carpel 25, level of tip of lower ovule. E, Level of ascidiate zone (carpels 30, 33) and tip of lower ovules of innermost carpels. E9, Close-up of carpel 34, level of the tip of the outer integument. E99, Close-up of carpel 35, level of the tip of the outer integument. F, Level of stipe (carpel 33) and ascidiate zone (carpels 32, 34, 35) of innermost carpels. F9, Close-up of carpel 35, level of the tip of the lower ovule. std ¼ staminode, o ¼ outer integument, i ¼ inner integument, n ¼ nucellus. Scale bar ¼ 1mm(A, B, C, D, E, F), 250 mm(A9), 500 mm(D9, D99, E, E9, E99, F). Fig. 7 Sinocalycanthus chinensis, transverse section series of anthetic gynoecium. Vasculature in gray. Carpels numbered in order of initiation. A, Level of stigmas and styles. A9, Close-up of carpels 6 (stigma) and 9 (style). B, Level of lower part of styles (compitum). C, Level of lower part of styles and upper part of ovaries. C9, Close-up of carpel 1 (upper ovule). D, Level of upper part of ovaries. E, Level of middle part of ovaries. F, Level of lower part of ovaries. F9, Close-up of carpel 7 (placentation and outer integument fold). G, Level of ascidiate zone (carpels 10, 11) and stipe (carpel 8) of innermost carpels. G9, Close-up of carpel 10. std ¼ staminode, o ¼ outer integument. Scale bar ¼ 1mm(A, B, C, D, E, F, G), 250 mm(A), 500 mm(C9, F9, G9). STAEDLER ET AL.—GYNOECIUM IN CALYCANTHACEAE (LAURALES) 29

Fig. 8 Chimonanthus praecox, transverse section series of anthetic gynoecium. Vasculature in gray. Carpels numbered in order of initiation. A, Level of stigmas and styles. A9, Close-up of carpels 2 (style) and 10 (stigma). B, Level of lower part of styles (compitum). C, Level of upper part of ovaries. D, Level of middle part of ovaries. D9, Close-up of carpel 2, level of placentae. E, Level of lower part of ovaries. E9, Close-up of carpel 3. F, Level of ascidiate zone (carpel 10) and stipe (carpel 9) of innermost carpels. F9, Close-up of carpel 10 (protruding nucellus). std ¼ staminode, o ¼ outer integument, i ¼ inner integument, n ¼ nucellus. Scale bar ¼ 1mm(A, B, C, D, E, F), 250 mm(A9), 500 mm(D9, E9, F9).

the ovary length. A short (70 mm) stipe is present in innermost The carpel wall is 13 or 14 cell layers thick at the level of the carpels (fig. 7G, carpel 8); presence of a stipe is difficult to as- placenta, at middistance between the dorsal and lateral vascu- sess for peripheral carpels borne on the sides of the floral cup lar bundles. All 38 carpels out of three flowers studied con- because their base is vertically and not horizontally oriented. tained two ovules. Placentation (fig. 7F, carpels 6, 7; fig. 7F9) The carpels have one dorsal and two lateral vascular bundles. and ovule direction (fig. 9C) are as in C. floridus. 30 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 9 Carpel, ovary opened. A, Idiospermum australiense. B, Calycanthus floridus. C, Sinocalycanthus chinensis. D, Chimonanthus praecox. Scale bar ¼ 500 mm.

Ovule structure is as in C. floridus. The lower ovule (fig. staminodes. Carpels are at the bottom and on the topo- 10C) is 1.5–2 mm long (fig. 7D, carpels 1–9, 11; fig. 7E, car- graphically lowest third of the wall of the floral cup (fig. pels 4–13; fig. 7F, carpels 6–13; fig. 7G, carpels 12–13). The 1D). The styles are long and slender (3.5 mm long, 80 mm outer integument is as in C. floridus (fig. 7G, carpel 10; fig. wide; figs. 1D,3D,4D;fig.8A,8A9,8B;fig.9D); they pro- 7E, carpel 5; fig. 7F, carpel 7; fig. 7F9; fig. 10C,10C9). Mi- trude from the floral cup (fig. 1D). The stigmas are short cropyle and micropylar cavity are as in C. floridus (fig. 12C). and papillate (;0.1 mm long; fig. 1D;fig.8A,8A9). The At midlength, the outer integument is 9 cell layers thick, and middle and lower portions of all the styles are covered with the inner integument is 5–6 cell layers thick. Funicle shape secretion (stained red by ruthenium red); they are postgeni- and ovule vascularization are as in C. floridus (figs. 7F9,10C, tally coherent and appear to form a compitum (figs. 3D,4D, 12C). 8B); the tissue of the lower, coherent portion of the styles is The upper ovule is usually strongly reduced to a shriveled small celled and cytoplasm rich (fig. 4D). The coherence of crust on the top of the lower ovule (fig. 9C; fig. 11C and 11C9 the styles is weak, and separation of individual styles from shows an ovule that is less reduced than usual, for structural the bundle of styles is easy; coherence is only partially resis- clarity). It is hood shaped (fig. 11C,11C9) and faces the lower tant to preparation for SEM (fig. 3D) but can be observed in ovule. The integuments do not seem to cover the nucellus apex transverse sections (figs. 4D,8B). The staminodes are borne (fig. 11C,11C9)andarecontiguouswithit(fig.9C). The outer on the topographically upper fifth of the inner wall of the integument occasionally grows along the surface of the lower floral cup (fig. 1D). ovule. The funicle is long and slender (fig. 11C); it is detached Carpels are glabrous, except for the adaxial portion below from the placenta at anthesis. The tissues of the upper ovule the lower end of the ventral slit, which is covered by hairs. The are shriveled (fig. 11C,11C9). stigma is bifacial (fig. 8A,8A9). The style is plicate and the ventral Aborted carpels are present in all nine fruits studied. A group slit is not postgenitally fused at this level (fig. 8A,8A9). Most of 4–5 aborted carpels on the topographically lower part of the of the ovary is also plicate, but with the ventral slit postgeni- floral cup was observed eight times. At least one sterile fruiting tally fused (fig. 8C, carpels 1–8; fig. 8D; fig. 8E, carpels 4–10). carpel was always found among the last initiated carpels of a There is only a short ascidiate base (fig. 8E, carpels 1–3; fig. gynoecium. 8F carpel 10). For inner carpels of the gynoecium, the ascidi- Chimonanthus praecox. Flowers have a narrow, urceo- ate zone is ;130 mm long, which represents 16% of the ovary late floral cup (fig. 1D) and contain 8–10 carpels and 8–12 length. A short (;10 mm) stipe is present in all innermost STAEDLER ET AL.—GYNOECIUM IN CALYCANTHACEAE (LAURALES) 31

Fig. 10 Lower, fertile, ovule, micropylar and lateral view. A, A9, Idiospermum australiense. A, Lateral view. A9, Micropylar view. B, B9, Calycanthus floridus. B, Lateral view. B9, Micropylar view. C, C9, Sinocalycanthus chinensis. C, Lateral view. C9, Micropylar view. D, D9, Chimonanthus praecox. D, Lateral view. D9, Micropylar view. Scale bar ¼ 100 mm. carpels (fig. 8F, carpel 9); presence of a stipe is difficult to as- the rim of both integuments. At midlength, the outer integu- sess for peripheral carpels borne on the sides of the floral cup ment is 6 cell layers thick, and the inner integument is 4 cell because their base is vertically and not horizontally oriented layers thick. The funicle is short and relatively stout (figs. 8D9, (fig. 8E, carpel 3). The carpels have one dorsal and two lateral 10D,12D). The ovule vascular bundle appears to ramify in the vascular bundles. The carpel wall is 6 or 7 cell layers thick at chalaza into four small bundles that extend into the base of the the level of the placenta, at mid-distance between the dorsal outer integument (fig. 12D). Ovules in uniovulate carpels are and lateral vascular bundles. The carpels usually contain two morphologically and topographically indistinguishable from ovules (75% of 28 studied carpels); carpels with one ovule lower ovules in biovulate carpels (fig. 8C, carpel 1; fig. 8D, were often observed (25% of 28 studied carpels). No carpel carpels 1, 6, 7; fig. 8E, carpels 1, 6, 7). devoid of ovules was observed. The ovules have collateral The upper ovule commonly shows all transitions in mor- placentation (fig. 8D, carpel 2; fig. 8D9) at the base of the pli- phology from more or less well developed (in size about two- cate zone, just above the beginning of the ascidiate zone. By thirds of the size of the lower ovule) to a crust of dry tissue curvature into different directions in early development, they on top of the lower ovule (fig. 9D; fig. 11D,11D9; fig. 11D come to lie one above the other (fig. 9D). and 11D9 shows a well-developed ovule for structural clarity). The ovules are anatropous, bitegmic, and crassinucellar. It is hood shaped (fig. 11D,11D9). The integuments do not The lower ovule is ovoid (fig. 10D); it is 0.5–0.9 mm long (fig. cover the nucellus apex (fig. 11D,11D9); the nucellus and the 8C, carpel 2; fig. 8D, carpels 2–5; fig. 8E, carpels 3–5). The opening of the two integuments are oriented downward (fig. outer integument is semiannular and lobed (fig. 8E, carpels 1, 9D)orsideward(fig.11D). The outer integument occasionally 3). One of the two lateral lobes of the outer integument is usu- grows along the surface of the lower ovule. The funicle is ally folded over the other. This ‘‘fold’’ is situated in the median long and slender (fig. 11D); it is often detached from the pla- plane of the ovule. Growth of the outer integument causes it centa at anthesis. The tissues of the upper ovule are commonly to be moulded by the bottom of the ovarial cavity, such that the shriveled (fig. 11D,11D9). fold attains the form a ridge (fig. 10D,10D9). The inner integu- Aborted carpels were always present in all the 10 fruits ment protrudes at the abaxial extremity of this ridge and studied. At least one sterile fruiting carpel is almost always forms the micropyle (fig. 12D). Very short hairs extend from present in the center of the gynoecium. 32 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 11 Upper, sterile, ovule micropylar and lateral view. A, A9, Idiospermum australiense. A, Lateral view. A9, Micropylar view. B, B9, Calycanthus floridus. B, Lateral view. B9, Micropylar view. C, C9, Sinocalycanthus chinensis. C, Lateral view. C9, Micropylar view. D, D9, Chimonanthus praecox. D, Lateral view. D9, Micropylar view. Scale bar ¼ 100 mm.

Gynoecium Development closing carpel flank; this bulge develops into an ovule. One of the two collaterally initiated ovules shows stronger basal cur- Idiospermum australiense. Carpels are initiated when vature than the other already during early development of the the floral cup begins to develop and the stamens begin to elon- gate. The carpel(s) continue(s) the spiral phyllotaxis after the inner integument (see fig. 13F9). Stigma differentiation ap- staminodes, with no apparent change in plastochron. Carpels pears to be concomitant with hair growth on the cross zone differ from staminodes in being crescent shaped, almost from (fig. 13F,13F9). The stigma develops from the uppermost part the beginning. The two ends of the crescent, representing the of the carpel flanks; the stigma increases first in width and margins of the carpel, are adaxially oriented (fig. 13A,13A9). then in length (fig. 13G,13G9). Elongation soon stops, and The carpel thus exhibits conspicuous dorsiventrality almost the stigma becomes papillate and fleshy (fig. 13H,13H9). from the beginning of its development. The dorsal side of the Calycanthus floridus. Carpels are initiated when the flo- carpel elongates (cell rows in fig. 13B), and the ends of the ral cup begins to develop and the stamens begin to elongate. crescent (basal adaxial ends of the carpel) enlarge (fig. 13C9). The carpels continue the spiral phyllotaxis after the stami- Continuous growth of the dorsal side of the young carpel rai- nodes. Carpel primordia are approximately hemispherical (fig. ses the primary margin, in lateral view to an angle of ;120° 14A,14A9); they do not differ morphologically from stami- with the longitudinal axis of the flower (fig. 13C). The basal- node primordia. Strong apical growth in the adaxial region most part of the carpel becomes closed by ‘‘meristem fusion’’ causes the adaxial surface to become parallel to the floral axis over the ventral side. Thus, a cross zone is now present (fig. (fig. 14B,14B9). In lateral view, the young carpel resembles a 13D,13E). Above the cross zone, the two flanks of the carpels mountain with a cliff on one side (adaxial side) and a smooth contact each other at first at the base, then gradually proceed- slope on the other (abaxial side). The young carpel continues ing upward (fig. 13E). Ovule initiation appears to be concomi- to grow apically (fig. 14C,14C9), and two bulges (the initial tant with, or just subsequent to, the beginning of carpel margins) are initiated on each side of its basal adaxial surface; closure. A small bulge is initiated at the basal portion of each the carpel is then crescent shaped in apical view. The basalmost STAEDLER ET AL.—GYNOECIUM IN CALYCANTHACEAE (LAURALES) 33

Fig. 12 Schematic longitudinal section of lower, fertile ovule. A, Idiospermum australiense. B, Calycanthus floridus. C, Sinocalycanthus chinensis. D, Chimonanthus praecox. Thin dotted line indicates position reached by the folded lobe of the outer integument. Thick dotted line indicates possible micropyle. Scale bar ¼ 200 mm.

part of the carpel becomes closed by ‘‘meristem fusion’’ over sheim 2000a). Angiospermy is of type 4 (angiospermy by com- the ventral side. Thus, a cross zone is now present (fig. 14D, plete postgenital fusion; definition by Endress and Igersheim 14E). Above the cross zone, the two flanks of the carpels con- 2000a). The stigmas differ in size, shape, and exposition in the tact each other from the base upward (fig. 14D,14E). Ovule in- two subfamilies: stout, fleshy, sessile, and enclosed in the floral itiation appears to be concomitant with, or just subsequent to, cup in Idiospermum (Blake 1972; this study); small, terminat- the beginning of carpel closure (fig. 14D99,14E99). A small ing an elongated, slender style, with the tips exerted from the bulge is initiated at the basal portion of each closing carpel floral cup in Calycanthoideae (Baillon 1868; Cheng and Chang flank (fig. 14D,14E); this bulge develops into an ovule (fig. 1963, 1964; this study). Despite the diversity in fruit size, vas- 14F99–14G99). One of the two collaterally initiated ovules shows cularization of the carpels is similarly simple in both subfam- stronger basal curvature than the other already during early de- ilies, including a dorsal and two lateral vascular bundles (see velopment of the inner integument (fig. 14F99). Style and stigma also Schaeppi 1953; Wilson 1976). Likewise, the thickness of differentiation appear to be concomitant with hair growth on the carpel wall is simply related to the size of the ovary at the cross zone (fig. 14F–14G99). The style and stigma develop anthesis in both subfamilies and not to carpel size at fruit ma- from the uppermost part of the carpel flanks (fig. 14F–14F99); turity. style and stigma length appear to be increased by cell divisions An extragynoecial compitum formed through postgenital (fig. 14F–14F99), then by cell elongation, the style becomes very coherence of carpels by secretion is present in all genera (fig. long and slender (fig. 14G–14H99). Development of papillosity 4A); however, there is variation in the degree of postgenital of the stigma seems to be concomitant or just subsequent to coherence and in the area of the carpel that is involved in com- the arrest of style elongation (fig. 14H–14H99). pitum formation. In Calycanthus floridus, they show loose co- herence in small groups; the styles of the carpels of the center of the flower often show stronger coherence than those at the Discussion periphery (fig. 4B). This difference may be related to the fact that the styles of the innermost carpels are not long enough Calycanthaceae: Gynoecium Structure at Anthesis for the stigmas to be exposed to the pollinators (fig. 2C); In all Calycanthaceae studied, carpels possess a short stipe, therefore, fertilization of the innermost carpels should require a feature present in most basal angiosperms (Endress and Iger- their stronger association with more peripheral carpels of the Fig. 13 Carpel development in Idiospermum australiense. A, A9, Carpel primordium. A, Ventral view. A9, Lateral view. B, B9, Early carpel. B, Ventral view. B9, Lateral view. C, C9, Early carpel, closure initiated. C, Ventral view. C9, Lateral view. D, D9, Young carpel, early closure. D, Ventral view. D9, Lateral view. E, E9, Young carpel, closure advanced. E, Ventral view. E9, Lateral view. F–F99, Carpel with stigma initiated. F, Ventral view. F9,Sideview.F99, Ovary opened to show ovule with outer integument. G, G9, Carpel before anthesis. G, Ventral view. G9, Lateral view. H–H99, Anthetic carpel. H, Ventral view. H9, Lateral view. H99, Lateral view, ovary opened to show mature ovules. Arrowheads indicate direction of main growth. Scale bars ¼ 100 mm.

34 Fig. 14 Carpel development in Calycanthus floridus. A, A9, Carpel primordium. A, Ventral view. A9, Lateral view. B, B9, Early carpel. B, Ventral view. B9, Lateral view. C, C9, Early carpel, closure initiated. C, Ventral view. C9, Lateral view. D–D99, Young carpel, early closure. D, Ventral view. D9, Lateral view. D99, Opened. E–E99, Young carpel, closure advanced. E, Ventral view. E9, Lateral view. E99, Opened to show lateral bulge with ovule initiation. F–F99, Carpel with stigma and style initiated. F, Ventral view. F9, Side view. F99, Opened to show ovules with outer integument formed. G–G99, Carpel before anthesis. G, Ventral view. G9, Lateral view. G99, Opened to show ovules one on top of the other. H–H99, Anthetic carpel. H, Ventral view. H9, Lateral view. H99, Opened to show mature ovules. Arrowheads: direction of main growth. Scale bars ¼ 100 mm(A–G99),1mm(H–H99).

35 36 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 15 Carpellodes in Idiospermum australiense. A, A9, Carpellode from anthetic flower with stigma and reduced ventral slit. A, Ventral view. A9, Lateral view. B, B9, Carpellode from late without stigma and opened ventral slit. B, Ventral view. B9, Lateral view. C, C9, Carpellode and carpel from anthetic flower. C, Lateral view. C9, Ventral view. D, D9, Carpellode from late bud with stigma. D, Adaxial view. D9, Lateral view. E, E9, Small carpellode from late bud. E, Adaxial view. E9, Lateral view. Scale bars ¼ 200 mm(A ¼ A9, B ¼ B9, C ¼ C9), 100 mm(D ¼ D9, E ¼ E9). gynoecium. The patchy distribution of unfertilized carpels in nermost carpels in the fruits of all Calycanthoideae (this fruits supports the hypothesis of the presence of partial compita study). In Idiospermum, developmental delay seems unlikely between small groups of carpels. In Sinocalycanthus chinensis to be the cause of the occurrence of solitary sterile carpels with and Chimonanthus praecox, the lower portion of the styles shows strongly reduced ovules or carpel-like structures with no ovule strong coherence in most flowers. In the zone of strong coher- at all, sometimes with a stigma, but with no ventral slit (fig. ence, the tissue is small celled and cytoplasm rich (fig. 4C,4D); 15). ‘‘Exhaustion’’ of the floral apex after the initiation of many such a zone may represent a plesiomorphic feature in Calycan- organs could cause such columnar structures to arise, similar thoideae. to the terminal structures found in the inflorescences of some Carpels commonly contain two ovules. If only one ovule is alismatids (Sokoloff et al. 2006). In Idiospermum, the most present, it is undistinguishable from the lower, fertile ovule well-developed sterile carpel-like structures bear a stigma; this of a biovulate carpel (see below). Because the carpels at the indicates that stigma development is uncoupled from the initia- center of the gynoecium are initiated later than the others, tion of ventral slit and ovules. their lower ovules may not be ready for fertilization by an- The size of the lower (fertile) ovule varies within the family thesis. This is suggested by (1) the observation of nucelli and within species. Idiospermum, which has the largest em- without a micropyle in the lower ovules of the innermost car- bryos in angiosperms (Blake 1972), has unexpectedly small pels (this study) and (2) the common occurrence of sterile in- ovules (0.7–0.8 mm) compared with Calycanthus (0.8–0.9 mm) STAEDLER ET AL.—GYNOECIUM IN CALYCANTHACEAE (LAURALES) 37

already closed when the inner ones are at the primordium stage. It is thus easy to deduce the identity of the primordia of the inner carpels. However, in the andromonoecious Idio- spermum, identification of the last initiated organ is difficult: it may be a carpel or a staminode. A primordium will then be interpreted as a young carpel only if it has a shape differ- ent from that of a staminode (i.e., crescent shape). Ovule initiation is similar in both subfamilies. The initiation site is the proximal part of the flanks of the carpel, and initiation is approximately concomitant with carpel closure. This causes the proximal part of the flanks to grow more pronouncedly dur- ing early carpel closure; the carpel thus closes from the bottom upward. A cross zone is formed only after carpel closure. Ovules and stigma are differentiated concomitantly in C. floridus and Idiospermum australiense. Although stigma mor- phology differs between subfamilies, this difference arises only Fig. 16 Different carpel closure process in Calycanthaceae and late in development. After an initial elongation phase during , in ventral view. A, Young carpel of Idiospermum australiense. which the stigmas of both species are similar in morphology B, Young carpel of Laurus nobilis. Scale bars ¼ 100 mm. (figs. 13F–13F99,14F–14F99), the stigmas of Idiospermum broaden and become more fleshy, whereas the styles of C. flo- ridus elongate to become filiform. and Sinocalycanthus (1.5–2 mm); only Chimonanthus has Ovule development is uniform in Calycanthaceae, including smaller ovules (0.5–0.9 mm). Micropylar cavities between the initial collateral placentation, rapid increase in curvature of outer and the inner integuments (previously reported in En- one of the two ovules (about at the stage of inner integument dress and Igersheim 1997) and between the inner integument initiation; figs. 13F99,14F99), late growth of the outer integu- and the nucellus are present only in Calycanthus and Sinocaly- ment over the inner integument (fig. 14G99,14H99), and abor- canthus and may represent a synapomorphy for this clade. tion of the upper ovule. The ‘‘nucellar beak’’ in Idiospermum Protruding, uncovered nucelli are occasionally present in the (Endress and Igersheim 1997; see above) is reminiscent of the innermost carpels of the gynoecium (C. floridus, C. praecox); protruding nucelli observed in fertile ovules of the innermost however, a nucellar beak, as described by Endress and Iger- carpels of C. floridus, which is probably due to precocious ter- sheim (1997) for lower ovules of Idiospermum, was not ob- mination of the ovule development. served in this study (if the definition given by Merino Sutter and Endress [2006] is applied). In all taxa studied, the ovule vascular bundle appears to branch into four small bundles Morphological Differences between Subfamilies that extend into the base of the outer integument (fig. 12), as pre- Newly identified differences between the subfamilies in- viously reported in young seeds of Calycanthus and Chimo- clude (1) mode of formation of a compitum (stigma coherence nanthus (Corner 1976) and anthetic ovules of Idiospermum in Idiospermum, style coherence in Calycanthoideae), (2) per- (Endress and Igersheim 1997; reviewed by Kimoto and Tobe haps carpel primordium shape (hemispherical in C. floridus, 2001). In the upper (sterile) ovule, the time of onset of abor- early bifacial in Idiospermum), (3) outer integument morphol- tion is variable even at the species level. The integuments of ogy (lobes always growing over one another, with a fold in the upper ovule do not cover the nucellus, but the outer integ- Calycanthoideae; lobes mostly touching each other in Idio- ument may grow over the lower ovule to some extent (C. spermum), and (4) epidermis differentiation of the rim of the praecox, S. chinensis). The funicle of the upper ovule is slen- integuments (hairs absent in Idiospermum but present in Caly- der and often detached from the placenta at anthesis. Detach- canthoideae). Surprisingly, at anthesis, in Idiospermum, ovary ment from the placenta could be caused by growth of the and ovules are not larger, and the ovary wall is not thicker lower ovule, since the species with the largest fertile ovules (S. than in Calycanthoideae. However, the outer integument of chinensis) has the most strongly reduced upper ovules. Con- Idiospermum is thicker (see also Endress and Igersheim versely, furthest-developed upper ovules are found in carpels 1997). with the smallest lower ovule (relative to the locule size; C. Previously documented reproductive morphological differ- floridus, C. praecox). ences between the subfamilies include traits involving flower sexuality, , androecium (including and stami- Calycanthaceae: Gynoecium Development nodes), and, notably, gynoecium (including fruit development). The most striking difference in carpel development be- Perianth color change during anthesis is conspicuous in Idio- tween the sampled representatives of the two subfamilies of spermum (Blake 1972; Staedler et al. 2007) but inconspicuous Calycanthaceae is the early crescent shape in Idiospermum in or absent in Calycanthoideae (Cheng and Chang 1963; Nicely contrast to the consistently observed rounded shape of early 1965; Staedler et al. 2007). Stamens are sickle shaped, and carpels in C. floridus. This could represent another morpho- staminodes are stout in Idiospermum and elongated and nar- logical difference between the two subfamilies but could also rowly triangular in Calycanthoideae (Cheng and Chang 1963; be a sampling artifact. In C. floridus, the peripheral carpels Nicely 1965; Blake 1972; Staedler et al. 2007). After anthesis, the are initiated before the inner ones. The peripheral carpels are floral cup is closed by movement of the stamens in Idiospermum 38 INTERNATIONAL JOURNAL OF PLANT SCIENCES

(fig. 2A,2A9; Worboys 1998) and by movement of staminodes Laurales always contain only one ovule with median placenta- in Calycanthoideae (fig. 2C,2C9; Staedler et al. 2007). Pollen tion (Leinfellner 1966, 1968, 1969; Sampson 1969a, 1969b; is monosulcate in Idiospermum (Blake 1972) but disulculate Endress 1972, 1980a, 1980b; Endress and Igersheim 1997; in Calycanthoideae (Ning et al. 1993; Sampson 2000). Stig- Endress and Lorence 2004). The more profound difference be- mas are sessile and fleshy in Idiospermum (Blake 1972) but tween Calycanthaceae and core Laurales is thus ovule initia- short and at the end of relatively long and slender styles in Cal- tion site rather than ovule number. ycanthoideae (Cheng and Chang 1963; Nicely 1965). In Idio- Ovule morphology is diverse in core Laurales. However, a spermum, at maturity, all organ layers enclosing the few character states present in Calycanthaceae are shared by (including the enlarged floral cup, the carpel wall, and the most of the order: ovules anatropous (with the exception of coat) decay; the dispersal unit is a naked, unusually large Gomortega, orthotropous; Endress and Igersheim 1997; Heo embryo dispersed by gravity (Blake 1972). In contrast, in Cal- et al. 2004), bitegmic (except for Siparunaceae, unitegmic; Heil- ycanthoideae, at maturity, the enlarged floral cup (which opens born 1931; Endress 1972; Renner et al. 1997; Kimoto and Tobe in some taxa and stays closed in others; Staedler et al. 2007) 2003), and crassinucellar (Endress and Igersheim 1997). The contains 1–25 fruitlets (Y. M. Staedler, personal observation), outer or the only integument is semiannular or annular, with which are dispersed by rodents (van der Pijl 1982) or possibly both character states observed in the same species in Peumus by birds (Staedler et al. 2007). Water dispersal also seems of and Siparuna of Siparunaceae (Endress and likely in Calycanthus and Sinocalycanthus since both genera Igersheim 1997). These traits (anatropous, bitegmic, crassinu- grow along creeks. cellar, flexibility between annular and semiannular) may rep- resent plesiomorphies for Laurales. In Calycanthaceae, the ovule vascular bundle branches in the chalaza and continues a short Comparison with Core Laurales: Gynoecium at Anthesis distance into the base of the outer integument. In core Laurales, Stout and sessile stigmas, as in Idiospermum, are also found vascular bundles that branch in the chalaza are found in some in some members of core Laurales, where they are either en- Hernandiaceae and in some Lauraceae; however, vascular bun- closed in the floral cup (most Monimiaceae; Perkins 1925; En- dles extending toward the rim of the outer integument were ob- dress 1980b) or exposed (Hedycarya, Hortonia, Levieria, and served only in some species (van Heel 1971; Heo Xymalos of Monimiaceae). However, slender, Calycanthoideae- and Tobe 1995; Endress and Igersheim 1997). Such a branching like styles and stigmas that always protrude from the floral cup ovular bundle may represent an autapomorphy for the different are found in other members of core Laurales (Palmeria of groups considered (Calycanthaceae, Hernandiaceae p.p., and Monimiaceae, Atherospermataceae, Siparunaceae; Perkins 1925; Lauraceae p.p.). Endress 1980b). This character combination could be plesiomor- phic for the Siparunaceae-Atherospermataceae-Gomortegaceae clade but seems to have evolved independently at least once in Comparison with Core Laurales: Gynoecium Development Monimiaceae (Palmeria; Renner 2004). Comparative studies of carpel development in core Laurales As in Calycanthaceae, angiospermy type 4 (angiospermy by are rare (for a few Lauraceae and Monimiaceae s.l.: Endress complete postgenital fusion) occurs in all studied core Laurales 1972; for Monimiaceae s.l.: Endress 1980a). All studies are fo- (Endress and Igersheim 1997), except for taxa with a hyperstigma cused on only a few taxa: Hedycarya arborea and Hortonia an- or a simple extragynoecial compitum, in which angiospermy gustifolia of Monimiaceae (Sampson 1969a; Endress 1980b); type 1 (angiospermy by secretion) is present (Tambourissa, Wil- Doryphora sassafras and Laurelia novae-zelandiae of Athero- kiea, Kibara, , Faika, Hedycarya, Kibaropsis;En- spermataceae (Sampson 1969b; Endress 1972); Hernandia dress 1979, 1980b, 1982; Endress and Lorence 1983; Philipson nymphaeifolia of Hernandiaceae (Endress and Lorence 2004); 1987; Endress and Igersheim 1997). Taxa with a hyperstigma, Laurus nobilis, Cinnamomum camphora,andPersea ameri- however, are restricted to some derived Monimiaceae (Renner cana of Lauraceae (Endress 1972; Singh and Singh 1985; Buzgo 2004). Angiospermy type 4 (angiospermy by postgenital fu- et al. 2007). sion) is thus likely to be ancestral in Laurales. Young carpels in core Laurales differ from those in Calycan- An extragynoecial compitum formed by contiguous carpel thaceae early in development. In Calycanthaceae, the ovules surfaces, such as that found in Calycanthaceae, is present in are initiated at the lower portion of the carpel flanks, approxi- all families of apocarpous pluricarpellate Laurales (Siparuna- mately concomitantly with carpel closure; a cross zone is present ceae: Endress and Igersheim 1997; Renner et al. 1997; Athero- only after carpel closure. In core Laurales, however, the ovu- spermataceae, Daphnandra, Doryphora, Dryadodaphne: liferous cross zone is initiated earlier, long before carpel clo- Schodde 1969; Endress and Igersheim 1997; Monimiaceae, sure begins (fig. 16A,16B). Hedycarya, Kibaropsis, Palmeria: Endress 1980b, Endress In all Laurales studied, ovule initiation is approximately and Igersheim 1997). Similar observations were also made in concomitant with carpel closure, but the timing and localiza- the ANITA grade (Amborella: Endress and Igersheim 2000b; tion of this initiation influences the mode of carpel closure. Nymphaea: Endress and Igersheim 2000a; Austrobaileya: En- In Calycanthaceae, where the ovules are initiated from the dress 1980c; Illicium: Thien et al. 1983; Endress and Iger- basal part of the carpel flanks, the basal portion of the carpel sheim 2000a; Schisandraceae: Igersheim and Endress 1997; flanks is bulkier and thus meets before the smaller distal por- Lyew et al. 2007). This character state could be a plesiomor- tion; the flanks meet from the base upward. In contrast, in phy for Angiospermae (sensu Cantino et al. 2007). core Laurales, the large cross zone delays closure near the ba- Unlike carpels of the Calycanthaceae, which contain either sis of the carpel, which proceeds from midlength downward. two or one ovule(s) with lateral placentation, carpels of core Furthermore, the flanks initially do not meet at the very base STAEDLER ET AL.—GYNOECIUM IN CALYCANTHACEAE (LAURALES) 39 of the carpel; there, a secondary cross zone is initiated to com- et al. 2005) and Aquilegia (Ranunculaceae; Kramer et al. plete carpel closure (Sampson 1969b; Endress 1972, 1980a, 2007); (4) difference between staminodes and true carpellodes 1980b, 2006; Buzgo et al. 2007). Such a secondary cross zone occasionally found in Idiospermum (see above; fig. 15): carpel- is not formed in Calycanthaceae. lode structure is variable, but when carpellodes are developed to a size comparable to that of a carpel, they almost always bear a stigma, they are often hollow, they may possess a (re- Fossil Taxa and Evolutionary Hypotheses duced) ventral slit and may even have the appearance of a nor- The fossil record of reproductive structures of Calycantha- mal carpel. Carpellodes are always positioned inside the ceae comprises two taxa for which anthetic gynoecia are avail- gynoecium (not at its periphery); almost no morphological in- able: Virginianthus calycanthoides (Albian; Friis et al. 1994) and tergrading was observed between carpellodes and staminodes. Jerseyanthus calycanthoides (Turonian; Crepet et al. 2005). Vir- Carpellodes have also been described in the fossil J. calycan- ginianthus has also been placed as potential sister to all other thoides (presence of a ventral slit, inner cavity, or stigma are Laurales (including Calycanthaceae; Crepet et al. 2005; Doyle not documented). But, in addition, the flowers also comprise and Endress 2007). Carpel number is 18–26 in the only avail- staminodes that are morphologically distinct (petaloid) and able fossil of Virginianthus (Friis et al. 1994) and ;24 in Jer- positioned at the same place as staminodes in extant Calycan- seyanthus (Crepet et al. 2005). In both fossils, carpel attachment thoideae (Crepet et al. 2005). The arguments in favor of a sta- and organization is similar to that of extant members of the men origin are more convincing than those in favor of a carpel family, although the attachment zone of the carpels of Virginian- origin, although developmental gene expression studies would thus is broader than in extant Calycanthaceae. Both fossils ap- be necessary to test this hypothesis more rigorously. pear to lack a compitum, but this could be a preservation In some , inner staminodes appear to play a artifact. Stigmas in Virginianthus appear small (as in Caly- role in herkogamy, floral display, and pollinator reward (En- canthoideae) and sessile (as in Idiospermoideae). A large cell, dress 1984). In Calycanthaceae, these inner staminodes may which probably has a secretory function, is found close to the have a function in herkogamy (Staedler et al. 2007). A role stigma (Friis et al. 1994), a feature otherwise not found in Cal- in floral display could be assumed for the (petaloid) inner ycanthaceae. The extent of the ventral slit is difficult to assess staminodes of Jerseyanthus but not for extant Calycantha- because of poor preservation. In Jerseyanthus, the styles and ceae. Pollinator reward is partially provided by staminodes in stigma are conduplicate, slender, and elongate. They are remi- Calycanthus (Grant 1950), but this cannot be assumed for niscent of the developing style and stigma of carpels of Caly- any other Calycanthaceae. The main function of staminodes canthoideae just before anthesis (see fig. 14G,14G9 for C. in Calycanthaceae seems to be floral cup closure at the end floridus); as in extant Calycanthaceae, the ventral slit runs of anthesis and during fruit development. This function is down along most of the ovary. Morphology would suggest in- tightly associated with the situation of the fruiting carpels in clusion of Jerseyanthus in Calycanthoideae; however, as phy- a floral cup, which is not the case in Magnoliales. Interest- logenetic dating assesses the minimum age of the split between ingly, a coupling between floral display and pollinator reward the two subfamilies to ;70 Myr (Zhou et al. 2006), whereas seems to be present in Calycanthaceae, but these roles seem Jerseyanthus is estimated to be ;90 Myr (Crepet et al. 2005), to be carried out by the inner series of perianth organs (Vogel extant Calycanthoideae may have retained ancestral features 1998; Worboys and Jackes 2005; Staedler et al. 2007). of the family. Calycanthaceae (extant and extinct) have lateral placenta- A zone of sterile organs is present between stamens and car- tion throughout, independent of ovule number (this study; pels in all Calycanthaceae, including both mentioned fossils Friis et al. 1994; Crepet et al. 2005). This character state (Cheng and Chang 1963; Nicely 1965; Blake 1972; Friis et al. could be ancestral in Laurales. Arguments supporting this hy- 1994; Crepet et al. 2005). These organs are usually considered pothesis include the presence of lateral placentation through- to be stamen derived (Baillon 1868; Cheng and Chang 1963; out the apocarpous members of Magnoliales (Igersheim and Nicely 1965; Blake 1972). Hiepko (1965) challenged this in- Endress 1997), the sister group of Laurales, or the possible terpretation, hypothesizing that the ‘‘staminodes’’ were carpel position of Virginianthus (with carpels with lateral placenta- derived. This interpretation was based on observations of tion) as sister to the order (Crepet et al. 2005; Doyle and En- ‘‘staminode’’ and carpel primordia, which are indistinguish- dress 2007). Acquisition of median placentation has probably able, whereas a gap in the transition between ‘‘staminode’’ occurred on the branch leading to core Laurales. and stamen primordia was recognized (Hiepko 1965). Further support for this interpretation is provided by the occasional switch, along the ontogenetic spiral, from outermost carpels Acknowledgments back to ‘‘staminodes’’ (Staedler et al. 2007). Postanthetic per- sistence of the ‘‘staminodes’’ would also favor a carpel origin. We would like to thank the Queensland Herbarium, Bris- However, arguments in favor of a stamen-derived organ iden- bane Botanic Gardens, Australia, for material of the southern tity include (1) occurrence of staminodes with a reduced pollen populations of Idiospermum. We thank Andrew Ford and Bernie sac, especially in taxa with numerous stamens (Calycanthus, Si- P. M. Hyland (Commonwealth Scientific and Industrial Research nocalycanthus,andIdiospermum); (2) similar behavior of fer- Organisation, Atherton, Queensland) and Stuart J. Worboys tile stamens and staminodes during anthesis (Staedler et al. 2007); (Parsons Brinckerhoff Australia) for help during collection. (3) stamen-like identity found in gene expression studies on flow- We would like to thank the traditional owner of the Ngadjon- ers normally bearing sterile organs between the stamens and Jii, Dulguburra, Yidindji, Girringun, and Yalanji tribal groups the carpels, Eupomatia (Eupomatiaceae, Magnoliales; Kim for allowing collection on their traditional grounds. The 40 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Georges-und-Antoine-Claraz-Schenkung (University of Zu- schen Akademie der Naturwissenschaften are thanked for rich) and the Kommission fu¨ r Reisestipendien der Schweizeri- financial support of the collecting trips.

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