Floral morphology and morphogenesis in Camptotheca (Nyssaceae), and its systematic significance

Abstract

Background and Aims

Camptotheca is endemic to China and there are limited data about the breeding system and morphogenesis of the flowers. Camptotheca is thought to be related to Nyssa and Davidia in Nyssaceae, which has sometimes been included in Cornaceae. However, molecular phylogenetic studies confirmed the inclusion of Camptotheca in Nyssaceae and its exclusion from Cornaceae. The aim of this study was to reveal developmental features of the inflorescence and flowers in Camptotheca to compare with related taxa in Cornales.

Methods

Inflorescences and flowers of Camptotheca acuminata at all developmental stages were collected and studied with a scanning electron microscope and stereo microscope.

Key Results

Camptotheca has botryoids which are composed of several capitate floral units (FUs) that are initiated acropetally. On each FU, flowers are grouped in dyads that are initiated acropetally. All floral organs are initiated centripetally. Calyx lobes are restricted to five teeth. The hypanthium, with five toothed calyx lobes, is adnate to the ovary. The five petals are free and valvate. Ten stamens are inserted in two whorls around the central depression, in which the style is immersed. Three carpels are initiated independently but the ovary is syncarpous and unilocular. The ovule is unitegmic and heterotropous. Inflorescences are functionally andromonoecious varying with the position of the FUs on the inflorescence system. Flowers on the upper FU often have robust styles and fully developed ovules. Flowers on the lower FU have undeveloped styles and aborted ovules, and the flowers on the middle FU are transitional.

Conclusions

Camptotheca possesses several traits that unify it with Nyssa, Mastixia and Diplopanax. Inflorescence and floral characters support a close relationship with Nyssaceae and Mastixiaceae but a distant relationship with Cornus. Our results corroborate molecular inferences and support a separate family Nyssaceae.

INTRODUCTION

Camptotheca Decne. (Nyssaceae) comprises only one or two species and is distributed in lowland warm-temperate forests of the Yangtze River Basin and south-western areas of China (Eyde, 1963; Fang et al., 1983; Jahnke, 1986; Wu et al., 2003; Kubitzki, 2004; Qin and Phengklai, 2007). The genus is notable for being a major source of the anticancer drug camptothecin (CPT), which is a strong inhibitor of nucleic acid synthesis in mammalian cells and induces the breaking-up of strands in chromosomes (Hsiang et al., 1985; Gigliotti et al., 2017; Manikandan and Kannan, 2017).

Since Camptotheca was first named in 1873, its systematic affinities have been controversial (Table 1). Camptotheca was placed in Cornaceae along with Nyssa, Davidia and Cornus (Harms, 1898; Eyde, 1988, 1997; Thorne, 1992; APG, 1998; Kubitzki, 2004). However, other authors favoured placing it with Nyssa in Nyssaceae (Wangerin, 1910; Chao, 1954; Hutchinson, 1967; Cronquist, 1981; Thorne, 2000), and treatments either included Davidia in Nyssaceae or recognized it as Davidiaceae (Li, 1954; Melchior, 1964; Airy Shaw, 1973; Goldblatt, 1978; Takhtajan, 1980; Dahlgren, 1983; Wu, 1983; Liao, 1993). The most recent molecular phylogenetic studies separated Nyssaceae, including Camptotheca, Nyssa, Davidia, Mastixia and Diplopanax, from Cornaceae, and Nyssaceae was placed as sister to a major clade comprising Hydrostachyaceae, Loasaceae and Hydrangeaceae (Xiang et al., 2011; APG, 2016; Chen et al., 2016).

Table 1.

Summary of systematic treatments of the genus Camptotheca; names of subfamilies and families used here follow the treatments in the year they were published

	Family	Subfamily	Genera
Harms, 1898	Cornaceae	Nyssoideae	Camptotheca, Nyssa
		Davidioideae	Davidia
Wangerin, 1910	Nyssaceae	–	Camptotheca, Nyssa, Davidia
Chao, 1954	Nyssaceae	–	Camptotheca, Nyssa, Davidia
Li, 1954	Nyssaceae	–	Camptotheca, Nyssa
	Davidiaceae	–	Davidia
Melchior, 1964	Nyssaceae	–	Camptotheca, Nyssa
	Davidiaceae	–	Davidia
Hutchinson, 1967	Nyssaceae	–	Camptotheca, Nyssa, Davidia
Airy Shaw, 1973	Nyssaceae	–	Camptotheca, Nyssa
	Davidiaceae	–	Davidia
Goldblatt, 1978	Nyssaceae	–	Camptotheca, Nyssa
	Davidiaceae	–	Davidia
Takhtajan, 1980	Nyssaceae	–	Camptotheca, Nyssa
	Davidiaceae	–	Davidia
Cronquist, 1981	Nyssaceae	–	Camptotheca, Nyssa, Davidia
Dahlgren, 1983	Nyssaceae	–	Camptotheca, Nyssa
	Davidiaceae	–	Davidia
Wu, 1983	Nyssaceae	–	Camptotheca, Nyssa
	Davidiaceae	–	Davidia
Eyde, 1988, 1997	Cornaceae	Nyssoideae	Camptotheca, Nyssa, Davidia
Thorne, 1992	Cornaceae	Nyssoideae	Camptotheca, Nyssa
		Davidioideae	Davidia
Liao, 1993	Nyssaceae	–	Camptotheca, Nyssa
	Davidiaceae	–	Davidia
APG, 1998	Cornaceae	–	Camptotheca, Nyssa, Davidia, Cornus, Alangium, Mastixia, Diplopanax, Curtisia
Thorne, 2000	Nyssaceae	Nyssoideae	Camptotheca, Nyssa
		Davidioideae	Davidia
Kubitzki, 2004	Cornaceae		Camptotheca, Nyssa, Davidia, Cornus, Alangium, Mastixia, Diplopanax
APG, 2016	Nyssaceae	–	Camptotheca, Nyssa, Davidia
			Mastixia, Diplopanax

Previous studies of Camptotheca have focused on its morphology and anatomy (Moser, 1968; Jahnke, 1986; Eyde, 1988; Liao, 1993; Li et al., 1998; Liu, 2004; Zhang et al., 2013; Wang and Liu, 2014), cytology (Goldblatt, 1978; Wu, 1983; Li and Xu, 1986; He et al., 2004), embryology (Chen, 1988; Hu et al., 2008), palynology (Chao, 1954; Sohma, 1963, 1967), palaeobiology (Eyde and Barghoorn, 1963; Eyde, 1997; Manchester et al., 1999; Manchester and Hickey, 2007), pollination (Wang et al., 2009; Xu and Liu, 2011; Shevchenko, 2014), phytochemistry (Bate-Smith et al., 1975) and phylogenetic position (Xiang et al., 1998, 2002, 2011; Fan and Xiang, 2003; Chen et al., 2016; Fu et al., 2017; Yang and Ji, 2017).

Inflorescences of Camptotheca consist of assemblages of four to five globular heads (floral units, FUs) that are grouped on terminal branches (Fig. 1A, B; Kubitzki, 2004). However, the nature of the inflorescences is controversial as there have been limited floral morphological studies in Camptotheca, which is regarded as possessing flowers arranged in a raceme (Moser, 1968; Kubitzki, 2004), a panicle (Jahnke, 1986) or cyme (Wang et al., 2009; Xu and Liu, 2011) within the FUs. Another long-standing controversy in the floral morphology of Camptotheca is the floral sex within the inflorescence, with researchers proposing the following: (1) the upper FU is hermaphroditic and lateral FUs are male (Moser, 1968; Kubitzki, 2004), (2) the upper FUs are female and lower FUs are male (Fang et al., 1983), (3) the upper FUs are hermaphroditic and the lower FUs are male (Wang et al., 2009), and (4) all flowers are hermaphroditic (Zhang et al., 2013).

Fig. 1.

Camptotheca acuminata Decne. (A, B) Anthetic inflorescences with four and five FUs, respectively. (C) Lateral view of an anthetic flower. (D) Stamen, adaxial view. (E) Sectioned flower with deciduous stamens and petals, showing an elongating style and three warped stigmas. (F) Immature infructescences. (G) Floral formula and floral diagram. Outermost blue arc corresponds to bract (B) and two blue arcs filled with lines to bracteole (Bt), dark green arcs to sepals (K) and light-green arcs to petals (C), two whorls of stamens shown by black and grey symbols (A), respectively; the large blue-green areas refer to the disc, the central blue areas to the three carpels (G) and the central solid circle to the single ovule (ov). (H) Branches with infructescences. Scale bars: A, B, F = 2 cm; C–E = 1 mm; H = 5 cm.

Recently, tremendous progress has been made in understanding the floral morphology and morphogenesis of Cornales (e.g. Loasaceae: Hufford, 1989a, b, 1990, 2003; Moody and Hufford, 2000; Hydrangeaceae: Roels et al., 1997; Hufford, 1998, 2001; Ge et al., 2003, 2007; Uemachi et al., 2006; Bull-Hereñu and Claßen-Bockhoff, 2011; Cornus: Feng et al., 2011, 2012; Davidia: Vekemans et al., 2012; Endress, 2014; Jerominek et al., 2014), allowing detailed floral comparative research within the order. However, a detailed study of the floral and inflorescence development is still lacking for Camptotheca except for the limited study by Moser (1968) and Jahnke (1986).

The aims of the present study were to: (1) describe the morphology and development of inflorescence and flowers in Camptotheca by scanning electron microscopy (SEM), (2) compare the results with related taxa in Cornales to understand the relationship with Cornaceae and Nyssaceae, and (3) clarify the breeding system of the genus by comparing the sexual pattern of different FUs.

MATERIALS AND METHODS

We collected inflorescences and flowers of Camptotheca acuminata at different developmental stages in two populations from Yangling City, Shaanxi Province (alt. 431–563 m, vouchers: Gong Jing-zhi001, 002, 003, WUK) and Shunhuangshan Mountain of Xin’ning County, Hunan Province, China (alt. 1000 m, voucher: ZLHN201104001), respectively. Materials were collected every 2–10 d from April to August in 2011, 2015 and 2016, and fixed in FAA (formalin–acetic–ethanol–water = 10: 5: 50: 35).

For SEM studies, about 80 flower buds were dissected and dehydrated in an ethanol and iso-amyl acetate series, critical-point dried in liquid CO₂, mounted on aluminium stubs, sputter-coated with gold, and observed with a Hitachi S-3500 scanning electron microscope.

We took photographs of the mature inflorescence and flowers with a Nikon D7100 digital camera, and micrographs using a Nikon SMZ25 stereo microscope (Figs 1 and 7). The symbols used in Fig. 1G for the floral diagram follow the annotations of Ronse De Craene (2010), and those of Ronse De Craene et al. (2014) for the floral formula.

Fig. 7.

UFU, MFU and LFU in Camptotheca acuminata before, during and after anthesis showing different bisexual and male-functional morphs. (A–E) UFU and its flowers. (F–J) MFU and its flowers. (K–O) LFU and its flowers. (A, F, K) FUs differ in number of flowers. (B, G, L) Flowering FUs with some petals and stamens removed to expose the disc and ovary. (C, H, M) Detail of dyad flowers showing differences in stylar development; petals and stamens removed. (D, I, N) FUs with petals and stamens withering. (E, J, O) Lateral view of flowers at a similar stage to show differences in style development. Abbreviations: UFU, upper floral unit; MFU, middle floral unit; LFU, lower floral unit. Scale bars: A, B, F, G, K, L = 5 mm; C, E, H, J, M, O = 1 mm; D, I, N = 1 cm.

RESULTS

Structure of the inflorescences and flowers

Camptotheca has a botryoid with several capitate units ending in a larger terminal one (Fig. 1A, B). Inflorescence buds are usually initiated in late April and bloom in early July. In a compound raceme, the FU can be divided into three types based on the fate of the gynoecium in late development, i.e. the upper, middle, and lower FU (hereafter referred to as UFU, MFU and LFU; Fig. 2D). In one single compound raceme, UFU flowers opened earliest with well-developed styles and stigmas, followed by LFU flowers with warped styles and stigmas, and MFU flowers bloomed last showing transition stages between UFU and LFU (Fig. 1B).

Fig. 2.

Inflorescence morphogenesis of Camptotheca acuminata. (A) Young bud of inflorescence. (B) Young inflorescence with acropetal initiation of younger FU, except the top one (involucre bracts removed). (C) Developing inflorescence with five FUs. (D) Four FUs with elongating rachis to show the position and size of upper floral unit (UFU), middle floral unit (MFU) and lower floral unit (LFU). (E–I) Details of five FUs (with involucre bracts) of the inflorescence in A, top-down, with the same magnification. (J–N) Sequence of rapid development of dyads on upper FU. (J, K) Early FU primordia before the initiation of dyads. (L, M) Initiation of irregular convex bumps at bottom and in middle of FU. (N) Initiation of bract pairs marked by asterisks with the same colour. (O–Q) Later FU primordia. (O) FU with expanding bracts. (P, Q) FU with flowers covered by binate hairy bracts (asterisks). Abbreviations: FU, floral unit. Scale bars: A–D = 2 mm; E–Q = 250 μm.

Flowers are pentamerous and sessile, arranged in dyads on each FU (Figs 1C, 3Q and 7C, H, M). Flowers of each globular head open nearly simultaneously. Each flower is enclosed by one bract and two bracteoles that are morphologically similar and elongated (Fig. 1C). The persistent calyx has five inconspicuous toothed lobes with ciliate margins (Figs 1C and 8A, F, K). The five, chartreuse-coloured, valvate petals are free (Fig. 1C). The androecium is diplostemonous and stamens are inserted at the base of an epigynous cup-shaped disc (Figs 1G and 8A, F, K). The anthers are obcordate (Fig. 1C, D). The style is inserted into the centre of the disc (Figs 1E and 8B, G, L). Drupes grouped in each globular head are crowned by persistent floral discs (Fig. 1F). Usually, only UFU and one to two MFUs develop fruits (Fig. 1H). On the upper FU, a time interval exists between the stamen dehiscence and the maturing pistil in a flower (protandrous dichogamy; Fig. 1C, E) and the style elongates from the disc after most petals and stamens have withered and fallen off (Fig. 1E).

Fig. 3.

Floral organ initiation on upper FU of Camptotheca acuminata. (A) Pair of opposite bract primordia in a dyad before initiation of the flower. (B) The two outermost bracts of the dyad covering the inner floral primordia become hairy. (C) Initiation of two inner bracteoles, with the outermost bracts removed. (D) One flower of an older dyad showing initiation of cylindrical floral primordia with five sepal primordia (asterisks; a bract and two bracteoles removed). (E) Successive initiation of five petals assumed to be the result of pressure from bract and bracteoles. (F–G) Sequential initiation of antesepalous stamens. (H) Petals tend to encase inner floral organs while sepals develop slowly. (I–J) Antepetalous stamen initiation [petals removed; antesepalous stamens removed in (J)]. (K) Sequential initiation of three carpels (petals and stamens removed). (L) Hairy petals covering inner organs. (M) Quincuncial arrangement of antesepalous stamens (petals removed). (N) Developing antepetalous stamens with two irregular pits on their surfaces, imprints of antesepalous stamens. (O) Initiation of disc nectary between pistil and stamens. (P) Ten stamens and three carpels. (Q) Flower pair wrapped by hairy bracts and bracteoles. (R) Top view of a preanthetic flower with hairy petals closely connected. (S) Lateral view, superior disc and inferior ovary before anthesis (two petals and several stamens removed). (T) Lateral view of a flower to show longer antesepalous stamens and shorter antepetalous stamens (two petals removed). Green colorr, antepetalous stamens. Abbreviations: A₁, antesepalous stamen; b₁, bract; b₂, bracteole; C, carpel; D, disc nectary; f, floral primordium; FU, floral unit; P, petal; S, sepal. Scale bars: A–P, R = 100 μm; Q, S, T = 1 mm.

Fig. 8.

Style development corresponding to ovule development on UFU, MFU and LFU in Camptotheca acuminata. (A–E) Well-developed styles (blue colour) and ovules (pink colour) at UFU. (F–J) Transitional styles and ovules at MFU. (K–O) Abortive styles and ovules at LFU. (A, F, K) Top view of flowers when petals open, with petals and stamens removed. (B, G, L) Dehiscence of flowers when petals open to show growth differences among styles corresponding to ovules. (C, H, M) Enlargement of ovules in B, G and L, respectively. (D, I, N) Later developmental stage of B, G and L to show growth differences among styles corresponding to ovules. (E, J, O) Enlargement of ovules in D, I and N, respectively. Abbreviations: A, androecium; D, disc; S, sepal; UFU, upper floral unit; MFU, middle floral unit; LFU, lower floral unit. Scale bars: A, B, D, F, G, I, K, L, N = 1 mm; C, E, H, J, M, O = 100 μm.

Initiation and development of the inflorescence

On each botryoid, several FU primordia develop acropetally (Fig. 2A–I). There is a relatively long plastochron between the initiation of the upper FU primordium and those of the lower FUs (Fig. 2B, E–I). There are more flowers grouped on the upper FU that is slightly larger (Fig. 2B–E). The base of each FU elongates and develops a new peduncle (Fig. 2C, D). The FUs of a compound raceme can be divided into three types according to pistil development levels (Fig. 2D). Here, for the initiation and development of the FUs, we restrict our observations to the upper FU as early development is similar for all FUs.

The FU primordium is hemispherical with a smooth surface (Fig. 2J, K). Broad elliptical primordia are initiated in very rapid succession (Fig. 2J–N), and form a rhombic shape before differentiating from two growth centres and developing in a dyad. The dyads lack subtending bracts (Figs 2N and 3A). Hairs gradually appear at the boundary of the dyads (Fig. 2N).

Early floral initiation

Each pair of floral buds develops synchronously. First a bract is initiated before the appearance of a flower bud (Figs 2N, O and 3A, B: b₁). Two inner bracteoles are initiated next but not simultaneously (Fig. 3C). While bracts rapidly overtop the bracteoles and flowers, neighbouring bracteoles of a flower pair converge towards each other (Fig. 3C: b₂). Bracts and bracteoles create a triangular shape to the dyad (Figs 3D, Q and 7A–C). The bracts and bracteoles are covered with hairs and gradually encase the enclosed flowers (Figs 2P, Q and 3B, C, Q).

Floral organs are initiated centripetally (Fig. 3D–O). The flower bud is cylindrical when five sepal primordia appear (Fig. 3D: asterisks). Sepals are delimited as inconspicuous convex shapes at the margin of the floral apex, which appears concave as a result (Fig. 3D). Sepals are very much delayed in their growth, but a helical (2/5 sequence) pattern could be detected in older buds (Fig. 3G). Petals are initiated unidirectionally and resemble irregular hemispheres, due to pressures from the bract and bracteoles (Fig. 3E). Petals differ in size during early development but grow rapidly and become equalized together in a valvate aestivation (Fig. 3F–H, L, R). Five antesepalous stamens appear in a quincuncial (2/5) sequence within the petal whorl (Fig. 3F, G). A slight overlapping of the first-formed stamens is visible for some time before the stamens become equalized (Fig. 3I, M). Five additional antepetalous stamens are initiated in a similar rapid sequence as an inner whorl alternating with the antesepalous stamens (Fig. 3I, J). All stamen primordia are peg-like and curve sharply toward the centre of the flower (Fig. 3I, J, M). The antesepalous stamens initially cover the antepetalous stamens (Fig. 3I, J, M, N), before being pushed in an erect position by the developing gynoecium (Fig. 3P). Antepetalous stamens remain shorter than the antesepalous stamens by the more limited growth of a thin filament (Figs 3S, T and 6A). Three carpels are initiated separately on the concave floral apex (Fig. 3K, O). Carpels are initiated sequentially, with the first carpel slightly overlapping the others (Fig. 5A–D). By congenital basal growth carpels are pushed up as erect stylar branches (Fig. 5C, D). At that stage a disc is initiated between the gynoecium and androecium as an irregular annulus (Fig. 3O).

Fig. 6.

Ovule morphogenesis in Camptotheca acuminata on upper FU. (A–E) Longitudinal sections of young flowers showing ovary with single locule and single ovule (pink colour). (D) Top view, style removed. (E) Transverse section of an ovule. (F–P) Ovule morphogenesis. (F) Ovule initiated in a tetrahedron shape. (G) Ovule bends upwards. (H) Integument initiated. (I–J) Integument encloses nucellus gradually. (K) A micropyle (arrow) forms below top apex of the ovule. (L) Ovule, abaxial view. (M) Protrusion appears on the chalazal end, abaxial view. (N) Secretory tissue (arrow) on the placenta near the micropylar end. (O) Elongation of the chalazal end, lateral view. (P) Mature ovule; the arrow points to the micropyle. Abbreviations: C, carpel; D, disc; FU, floral unit; I, integument; M, micropyle; N, nucellus; O, ovule. cale bars: A–C = 500 μm, D–P = 100 μm.

Fig. 5.

Carpel development of Camptotheca acuminata on an upper FU. (A) Three carpels initiating separately. (B) Three carpels converging over the centre. (C) Initiation of three separate stigmas on young carpels. (D) Lateral view of C. (E–F) Lateral view of flowers showing differentiation of the style before anthesis. (G–H) Style elongating and curving after flowering. (I–L) Development of the stigmatic slit, adaxial and lateral views. (M–P) Changes of cell structure on receptive stigmatic surfaces: smooth cells, papillary cells, finely wrinkled papillary cells and mature papillary cells. Abbreviations: C, carpel; FU, floral unit. Scale bars: A–D, I–L = 100 μm; E–H = 1 mm, M–P = 50 μm.

Mid-developmental stages and organ differentiation

Sepals grow slowly after initiation (Figs 3D–P, T and 4A–C), which results in tooth-shaped lobes when mature. The hypanthium is adnate to the inferior ovary (Fig. 4A–C) and there are some stomata on the calyx lobes (Fig. 4D).

Fig. 4.

Floral development in Camptotheca acuminata on an upper FU. (A–C) Development of calyx. (A) Lateral view of young calyx with wavy margin and corolla. (B) Development of hairs on the calyx lobes. (C) Lateral view of flower with mature calyx bearing irregular lobes. (D) Detail of a stoma on a sepal. (E–G) Development of petals, abaxial side. (H) Mature petal, adaxial side. (I) Ornamentation and a stoma on the petal, abaxial side. (J–K) Development of stamens. (J) Young anther, abaxial view. (K) Young anther, lateral view. (L) Four pollen sacs of an anther, top view. (M) Half of an anther removed to show valvate dehiscence. (N) Abaxial view of a valvate anther. (O–P) Tricolporate pollen grain with irregular stomal ornamentation on the surface. (Q–S) Sequential development of the disc nectary, petals and stamens removed. (T–W) Changes of disc surface (T, smooth cells; U, smooth papillary cells and a stoma; V, wrinkled papillary cells; W, secretion of nectar on disc nectary). Abbreviations: D, disc; FU, floral unit; P, petal; S, sepal. Scale bars: A, B, E, F, I, J–N, Q = 100 μm; D, O, P, T–W = 10 μm; C, G, H, R, S = 1 mm.

Hairs gradually appear on both the adaxial and the abaxial sides of the petals (Fig. 4E–H). The petal margins are touching and overlap little if at all before anthesis (Figs 3L, R and 4B, C). Concave areas on the inside surface of the petal result from the imprinted pressure from the anthers (Fig. 4H). Stomata and an ornamentation of stripes adorn the abaxial surface of the petals (Fig. 4I). Obcordate anthers are tetrasporangiate when observed directly from above (Fig. 4J–L). The anther dehiscence is through valves (Fig. 4M, N). The pollen is tricolporate with small-reticulate exine (Fig. 4O, P). The disc nectary expands outwards and becomes the most pronounced region of the flower before anthesis (Fig. 4Q–S). Epidermal cells of the disc present a series of changes through development: from a smooth surface, to slick papillary cells and finally rugate papillary cells with nectar on the surface (Fig. 4T–W). Stomata on the disc nectary are slightly sunken (Fig. 4U, V).

The three carpellary lobes shift gradually closer to each other (Fig. 5A, B), and are lifted by common basal growth into three stylar lobes (Fig. 5C–F). The stylar lobes are unequal in size with one markedly shorter (Fig. 5C, D). The inferior ovary part is extending and growth of the disc progressively engulfs the base of the style (Fig. 5F, I, J). At anthesis, the style elongates after petals and stamens have withered and dropped off, and unfolds three stigmatic lobes exposing a receptive surface (Fig. 5G, H). Epidermal cells on the stigmatic surface exhibit a series of changes: from a smooth surface, to smooth papillary cells, slightly plicate papillary cells and finally crooked papillary cells when mature (Fig. 5I–P).

Placentation and ovule initiation

The ovary is inferior with the three carpels basally united to form one small locule, which is connivent to the disc (Fig. 6A). There is only one ovule in the thick-walled ovary (Fig. 6A–E).

The ovule is tetrahedron-shaped after initiation (Fig. 6F) and rapidly bends upwards (Fig. 6G). A single integument appears near the top apex of the ovule (Fig. 6H). It gradually encases the nucellus and leaves a narrow micropyle at the apex (Fig. 6I, K). The bottom of the ovule bulges out first (Fig. 6L, M), and then it drastically elongates downward, which results in a fusiform ovule (Fig. 6M, O, P). An extension of secretory cells develops on the placenta, close to the micropyle (Fig. 6M, N: arrow).

Floral differentiation of different FUs

In each botryoid, MFU and LFU are similar to UFU with respect to initiation and development of floral organs, except for style growth and ovule development at later stages.

The styles of UFU flowers elongate considerably at anthesis. The trifurcate stigmas curve out, and remain higher than the disc (Figs 5D–H and 7C–E). The ovule is well developed (Fig. 8B–E). In the LFU flowers, the styles remain below the cup-shaped disc until anthesis, and the ovules are abortive (Figs 7L–O and 8K–O). Regarding the MFU, the flowers present a transition between UFU and LFU in the size of the style and development of the ovule (Figs 7G–J and 8F–J). The typical situation is that the style apices are level with or higher than the disc before anthesis (Fig. 8F, G). The styles and the stigmas are thin and short (Figs 7I, J and 8G, I), and the ovules are underdeveloped (Fig. 8G–J).

DISCUSSION

Inflorescence

The terminology used to describe the Camptotheca inflorescence could create misunderstandings and confusion due to uncritical and outdated definitions and needs to be updated (Endress, 2010; Claßen-Bockhoff and Bull-Hereñu, 2013). Camptotheca inflorescences have been considered a cyme based on the basipetal maturation sequence of its partial globular heads (Wang et al., 2009; Xu and Liu, 2011). Other authors, however, described its inflorescence as a panicle (Fang et al., 1983; Jahnke, 1986), upper or axillary head (Qin and Phengklai, 2007), or globular heads grouped in a raceme (Moser, 1968; Kubitzki, 2004). Following the terminology of Endress (2010) and Claßen-Bockhoff and Bull-Hereñu (2013), Camptotheca could be described as possessing a botryoid with a terminal FU and three to five (sometimes nine; Qin and Phengklai, 2007) lateral FUs. Each FU is capitate and has a large number of flowers grouped in dyads. Jahnke (1986) questioned whether the arrangement of the dyads in Camptotheca represents a closed system (i.e. terminated by a dyad), or an open system (with laterally inserted dyads). In the first case the head corresponds to a cephalioid, while in the second it would be a cephalium or capitulum (Endress, 2010). This distinction is difficult to make, and developmental stages do not clearly show a terminal dyad. However, as the cephalium or capitulum are determinate systems derived from flower-like meristems, this distinction is not applicable (cf. Bull-Hereñu and Claßen-Bockhoff, 2011). The FU effectively functions as a flower unit meristem (sensuClaßen-Bockhoff and Bull-Hereñu, 2013), however, giving rise to the dyads (floral units) and not to individual flowers. Flowers of Camptotheca on each FU open nearly simultaneously, and the UFU opens before the lateral FUs. The opening sequence of flowers is too labile to be useful for a morphological classification of inflorescences (Endress, 2010).

In Cornales, capitate inflorescences exist in Camptotheca, Nyssa, Davidia and Cornus. A grouping of FU partial inflorescences is not restricted to Camptotheca but also occurs in male inflorescence of Nyssa wenshanensis (Fang et al., 1983; Qin and Phengklai, 2007). Nyssa javanica has hermaphroditic flowers and male flowers borne in capitate clusters similar to Camptotheca (Eyde, 1963; Jahnke, 1986). In N. aquatica and N. ogeche, male flowers are in capitate inflorescences while hermaphroditic flowers are solitary (Eyde, 1963). Davidia and Cornus have solitary head-like inflorescences at the end of branches (Kubitzki, 2004; Xiang and Boufford, 2005; Qin and Phengklai, 2007). However, the heads of Cornus are arranged in a cymose pattern, with a dichasial arrangement (Feng et al., 2011, 2012; L. Ronse De Craene, unpubl. data), which is distinctly different from the capitate partial inflorescence of Camptotheca, with dyad flowers. In Davidia, almost all flower primordia appear more or less simultaneously, except that the basal primordia may be initiated later, but they develop faster (Moser, 1968; Vekemans et al., 2012; Jerominek et al., 2014). To compare the FUs in Camptotheca with the ‘heads’ of order Cornales the dyads could be interpreted as flower equivalents. This is clearly visible in the simultaneous initiation of two flowers and the convergence of the bracteoles. A subtending bract for the dyad is missing and this lack of bracts is not yet understood as we know too little about the peculiarities of FU development.

Floral sex

Floral sex in Camptotheca has remained a matter of dispute for decades and has either been described as bisexual (Zhang et al., 2013), unisexual (Fang et al., 1983), andromonoecious (Kubitzki, 2004; Wang et al., 2009; Xu and Liu, 2011) or polygamo-monoecious (Qin and Phengklai, 2007). Our observations demonstrate that all flowers belonging to an FU are perfect flowers with sepals, petals, stamens and carpels. All flowers are initially similar, except for pistil growth and ovule development at later stages. The perfect flowers in the UFU are protandrous and the petals and stamens are caducous, thus explaining why in the female phase, the flowers may have been mistaken for unisexual flowers (Fang et al., 1983; Wang et al., 2009). Previously, flowers grouped in the LFU were considered to be male (Fang et al., 1983; Kubitzki, 2004; Wang et al., 2009) or bisexual (Zhang et al., 2013). We show that they have a short style, which does not extend beyond a disc, and an ovule in each flower is probably abortive. MFU flowers are intermediate, having short, thin styles, and their ovules do not develop completely or they abort later.

Our results show that Camptotheca is functionally andromonoecious with fully developed flowers. Davidia is andromonoecious and Nyssa is polygamodioecious. Mastixia and Diplopanax are hermaphroditic (Eyde, 1963; Kubitzki, 2004). Cornus is hermaphroditic, except for the African species, which are dioecious (Kubitzki, 2004). In fact, Cornales shows an abundant diversity of breeding systems. Hydrostachyaceae is dioecious, except for the monoecious Hydrostachys monoica and H. stolonifera (Kubitzki, 2004). Hydrangeaceae is often bisexual or unisexual, rarely functionally dioecious in Broussaisia (Hufford, 2001; Kubitzki, 2004), and Curtisiaceae and Alangiaceae are bisexual and rarely dioecious (Carlquist, 1977; Kubitzki, 2004). The breeding system in Cornales reflects a shift from bisexual flowers to a functional distinction between unisexual systems. This strategy is successful in the development of fewer flowers with a reduced number of ovules, maximizing the pollination potential in systems where individuals are widely separated (Table 2).

Table 2.

Comparison of floral initiation and development of Camptotheca and putative affinities

Taxa	Inflorescence	Sex	Perianth merosity	Sepals	Petals	Stamens	Carpels	Epigynous disc
Camptotheca	botryoid	functionally andromonoecious	5	5, initiated sequentially developing slowly	5, initiated unidirectionally	5 + 5, diplostemonous initiated sequentially	3-carpellate, initiated separately; inferior ovary, 1 locule, 1 ovule, pseudomonomerous	present
Davidia	solitary globular heads	andromonoecious	irregular and reduced	absent	nearly absent	irregular, 1–7 for male and 15–26 for female	6–10-carpellate, several carpels arising on a ring primordium; inferior ovary, 6–10 locules, 1 ovule per locule	absent
Nyssa	head or raceme	polygamo-dioecious	irregular or 4(5)	?	5–10	8–15, diplostemonous for staminate flowers and fewer in hermaphroditic or pistillate flowers	inferior ovary, 1 (–3) locules, occasionally pseudomonomerous	present
Mastixia	thyrso-paniculate	bisexual	4–5(–7)	4–5	4–5	5–10, diplostemonous or haplostemonous	inferior ovary, 1 locule, 1 ovule, pseudomonomerous	present
Diplopanax	spicate-paniculate	bisexual	5	5	5	5 + 5, diplostemonous	inferior ovary, 1 locule, 1 ovule, pseudomonomerous	present
Cornus	head or thyrso-paniculate	bisexual, rarely dioecious	4	initiated decussately, develop quickly at the beginning	initiated decussately	4, haplostemonous	inferior ovary, (1) 2 (3) locules, occasionally pseudomonomerous	present
Alangium	axillary cymes	bisexual, rarely dioecious	4–10	4–10	4–10	4–40, haplostemonous	inferior ovary, 1(2) locules, 1 ovule per locule, occasionally pseudomonomerous	present
Hydrangeaceae	cymose, sometimes corymbs, thyrses, or panicles	bisexual, rarely unisexual	4–5(–12)	initiated sequentially or unidirectionally	initiated nearly simultaneously	(4–)8–many haplostemonous, diplostemonous, polystemonous	(2–)3–5(–12)-carpellate, initiated separately or on a ring primordium; inferior or partially inferior, 1–many ovules	present or absent
Loasaceae	Terminal thyrsoids, sometimes dichasia, monochasia or monads, rarely racemes	bisexual	(4) –5(–8)	initiated spirally or sequentially	initiated nearly simultaneously or sequentially	haplostemonous, diplostemonous, polystemonous	several carpel primordia initiated separately or a ring area appears firstly; inferior or semi-superior, 1–many ovules	present or absent
Hydrostachyaceae	spike	dioecious, rarely monoecious	absent	absent	absent	one	2-carpellate; superior ovary, 1 locule, many ovules	absent

Flower morphological and developmental character states of Camptotheca are taken from this study; those of Nyssa from Qin and Phengklai (2007); Davidia from Vekemans et al. (2012); Mastixia from Eyde (1988); Alangium from Eyde (1968); Diplopanax from Zhu and Xiang (1999); Cornus from Xiang and Boufford (2005); Hydrangeaceae and Loasaceae from Ge et al. (2003, 2007), Hufford (1989a, b, 1990, 1998, 2001, 2004) and Roels et al. (1997); and others from Eyde (1963) and Kubitzki (2004).

Bract and bracteoles

The bract and two bracteoles are similar in morphology and size, surrounding each flower in Camptotheca at anthesis (cf. Moser, 1968). The two bracteoles are initiated decussately. However, there is no subtending bract below the flower-pair that we interpret as secondarily lost. In Nyssa sylvatica, there are hairy persistent bracts at the base of the fruit (Eyde, 1963), which are also found in Camptotheca and Mastixia (Matthew, 1976). However, in N. aquatica and N. ogeche, the bracts are caducous (Eyde, 1963). A similar arrangement of a bract and two morphologically similar bracteoles has been reported in Philadelphus, Kirengeshoma and Platycrater of Hydrangeaceae (Roels et al., 1997; Ge et al., 2007).

Perianth

Small sepals are common in Cornales (Hydrangeaceae: Deutzia, Roels et al., 1997; Pileostegia, Hydrangea, Decumaria, Broussaisia, Dichroa and Schizophragma, Hufford, 2001; Hydrangea, Ge et al., 2003; Alangium, Kubitzki, 2004; Ronse De Craene, 2010). Small flowers with minute or obsolete calyx lobes also exist in Nyssa, Mastixia, Diplopanax and Cornus (Eyde, 1963; Kubitzki, 2004; Xiang and Boufford, 2005). In Camptotheca, calyx lobes are small toothed with ciliate margins. In Davidia, male flowers lack a perianth while hermaphrodite flowers have minute perianth segments (Eyde, 1963; Qin and Phengklai, 2007; Vekemans et al., 2012). In Cornus, the calyx lobes develop more rapidly than the petals and the lobes partially cover the petals to protect the inner floral organs at an early stage. However, in Camptotheca the calyx lobes grow slowly after initiation. In Loasaceae, the calyx lobes are usually larger than the petals in Gronovioideae, but smaller in Petalonychoideae (Moody and Hufford, 2000; Kubitzki, 2004). Small sepals are generally associated with an inferior ovary and larger petals in bud, indicating a functional shift in the protection of internal organs to the petals and hypanthium (Ronse De Craene, 2008, 2010).

In Camptotheca, the five sepals are not initiated simultaneously but sequentially and spirally. They are initiated spirally in species of Hydrangeaceae (Deutzia, Kirengeshoma, Roels et al., 1997; Deinanthe, Cardiandra, Hydrangea, Schizophragma, Hufford, 2001; Hydrangea, Ge et al., 2003) and Loasaceae (Cajophora, Hufford, 1990; Cevallia, Gronovia, Fuertesia, Moody and Hufford, 2000). In tetramerous flowers of Philadelphus, Platycrater and Cornus, sepals are initiated two by two (Roels et al., 1997; Feng et al., 2011). Additionally, in Petalonyx linearis (Loasaceae), there is a time interval between initiation of the first three sepals and the last two sepals (Hufford, 1989a).

In contrast to sepals, petals are initiated sequentially and unidirectionally. The unidirectional initiation of the petals may be linked to the tight compression of flowers between bracts and bracteoles. In Cornales, petals are always initiated before stamens, except in Gronovia scandens of Loasaceae, which has petals that are uniquely initiated after the stamens (Moody and Hufford, 2000). Most Cornales species have free petals, except for a few species in Loasaceae that have a corolla tube (Moody and Hufford, 2000; Hufford, 2001; Ronse De Craene, 2010). In Hydrangeaceae and Loasaceae, the petals are initiated nearly simultaneously (Hufford, 1990, 2001; Roels et al., 1997; Moody and Hufford, 2000; Ge et al., 2003, 2007). Petalonyx of Loasaceae is exceptional with an interval between the initiation of the first petal and the others (Hufford, 1989a, 1990). It is of interest that the five free petals cling to each other before anthesis in Camptotheca, and the protection of the flower bud is transferred from the bracts to the corolla. The protective function of a corolla is also observed in Cornus (Feng et al., 2011), Deutzia, Hydrangea, Kirengeshoma (Roels et al., 1997), Schizophragma, Hydrangea, Pileostegia and Dichroa (Hufford, 2001). In Hydrangea anomala subsp. petiolaris, Petalonyx parryi, P. nitidus and P. thurberi, the lateral margin of adjacent petals fuse postgenitally to provide protection (Moody and Hufford, 2000; Hufford, 2001). In several Cornales, including Camptotheca, the corolla aestivation is valvate, creating a wide internal dome for the developing organs in bud (Endress, 2008; Ronse De Craene, 2010). However, in the putative sister genus Mastixia, petals are occasionally imbricate (Eyde, 1988: fig. 12). Petal lobes may be slightly imbricate in younger buds of Camptotheca, and this arrangement was also observed by Moser (1968).

Androecium

The stamens are arranged in two whorls (diplostemony) with a marked difference in length between outer and inner stamens. Stamen initiation is spiral following the 2/5 sequence of the calyx, but stamens rapidly become equal within each whorl. In Cornales, the androecium is often variable, with a diplostemonous androecium similar to Camptotheca found in a few taxa (Ronse De Craene, 2010). Diplostemony is also present in Grubbiaceae (Carlquist, 1977), Mastixia, Diplopanax (Zhu and Xiang, 1999; Kubitzki, 2004; Xiang et al., 2005), and in male flowers of Nyssa (Kubitzki, 2004; Qin and Phengklai, 2007). Haplostemony is found in Cornaceae, Curtisiaceae and some species of Mastixia (Kubitzki, 2004). The androecuim of Hydrangeaceae and Loasaceae is haplostemonous, diplostemonous or polystemonous (Hufford, 1990, 2001; Roels et al., 1997; Moody and Hufford, 2000; Kubitzki, 2004). In Davidia, there are one to seven stamens in male flowers and 15–26 stamens arranged around the base of the style in hermaphroditic flowers (Kubitzki, 2004; Qin and Phengklai, 2007; Vekemans et al., 2012). The male flower of Hydrostachyaceae has only one stamen, which is deeply divided into two thecae or two independent bisporangiate stamens (Kubitzki, 2004). Camptotheca has obcordate anthers, which is similar to Davidia (Jerominek et al., 2014: fig. 3) and Curtisiaceae (Kubitzki, 2004), and the pollen is tricolporate. The anthers in Camptotheca are valvate, which was also recorded in Grubbiaceae (Kubitzki, 2004). Anthers of other taxa in Cornales dehisce longitudinally (Schismocarpus and Gronovia of Loasaceae, Hufford, 1989b; Hydrangeaceae, Endress and Stumpf, 1991; Moody and Hufford, 2000; Hydrostachyaceae and Curtisiaceae, Kubitzki, 2004).

Gynoecium

In Cornales, the floral apex always develops into a concave shape during floral development. However, the timing of appearance of the central concavity is variable, either at the transition of calyx and corolla initiation (Petalonyx, Hufford, 1989a; Schismocarpus, Hufford, 1989b; Cevallia, Moody and Hufford, 2000; Hydrangea macrophylla, Hufford, 2001; Platycrater, Ge et al., 2007), or during corolla development (Mentzelia, Cajophora, Hufford, 1990; Hydrangea, Schizophragma, Pileostegia, Decumaria, Dichroa,Hufford, 2001). In Camptotheca, a concave floral apex appears during sepal initiation, which is different from that of Hydrangeaceae and Loasaceae. Nevertheless, the occurrence of a concave shape shows strong affinity with other Cornales.

In Camptotheca, carpel primordia are formed on the concave floral apex and this is similar to Hydrangeaceae and Loasaceae (Hufford, 1989a, b, 1998, 2001; Roels et al., 1997; Moody and Hufford, 2000; Ge et al., 2003). Three carpel primordia are initiated separately in Camptotheca, while in Davidia, a ring-like area is initiated on the concave floral apex first, followed by several horseshoe-shaped carpel primordia (Vekemans et al., 2012). A similar development as in Camptotheca is present in Hydrangeaceae (Deutzia, Philadelphus, Roels et al., 1997; Deinanthe, Decumaria, Hufford, 2001) and Loasaceae (Cevallia, Moody and Hufford, 2000).

In Camptotheca, carpels are initiated after antepetalous stamens, which is the same as some genera in Hydrangeaceae (Hufford, 1998, 2001; Ge et al., 2003). However, in Deinanthe, Cardiandra and Platycrater, carpels always appear before stamen initiation (Hufford, 2001; Ge et al., 2007).

Camptotheca shares an inferior ovary with most Cornales (Kubitzki, 2004; Qin and Phengklai, 2007; Ronse De Craene, 2010), except Hydrostachyaceae, which has a superior ovary and some taxa in Loasaceae and Hydrangeaceae with semi-superior ovaries (Kubitzki, 2004).

A unitegmic ovule is also a common feature of Cornales (Kubitzki, 2004). The ovule of Camptotheca is hemitropous with ventral raphe and the orientation of the micropyle is upwards, which has also been reported in Mastixia, Diplopanax, Curtisia and Nyssa (Eyde, 1963, 1988; Moser, 1968; Zhu and Xiang, 1999; Kubitzki, 2004). In Cornus and Alangium the ovule is initially apotropous with dorsal raphe, but later turns to the side (Eyde, 1988; Kubitzki, 2004). In Davidia, Grubbia, Hydrangeaceae, Hydrostachyaceae and Loasaceae, however, the ovule is anatropous with the micropyle directed downwards (Eyde, 1963; Hufford, 1989a; Moody and Hufford, 2000; Xiang et al., 2002; Kubitzki, 2004; Vekemans et al., 2012).

While most Cornales are multilocular and possess one or more ovules per carpel, the number of ovules is reduced to one in Camptotheca and the ovary is unilocular. Unilocular ovaries with a single ovule have also been reported in Nyssa (one or two locules), Mastixia, Diplopanax, Alangium (rarely two locules), Gronovioideae and Petalonychoideae in Loasaceae, and Grubbiaceae (Eyde, 1963; Moody and Hufford, 2000; Kubitzki, 2004). In Alangium (Cornaceae) with two carpels, the locules are often unequal and one ovule is often underdeveloped or missing (Eyde, 1968). Abortive ovules have also been reported in some species of Cornus (Wilkinson, 1944). This indicates that the ovaries of Camptotheca, Diplopanax and Mastixia are probably pseudomonomerous. Amersinia, an extinct genus, with infructescences and fruits that are similar to that of Camptotheca, has three to four locules (Manchester et al., 1999). Other extinct species also show higher numbers of locules, for example in Cornus (up to six) and mastixioids (up to four; Eyde, 1988). Davidia is the only genus of Cornales that has a high number of locules (six–eight observed in the fossils and six–nine in the extant D. involucrata; Eyde, 1963; Manchester, 2002). Eyde (1963) reported that some locules can be sterile and compressed.

Nectary

Most members of Cornales share an epigynous disc nectary (Ronse De Craene, 2010). Nyssa, Mastixia, Diplopanax and Cornus have a similar well-developed disc nectary. Stomata are sunken slightly in the nectary of Camptotheca, which was also found in Hydrangeaceae (Hufford, 2001). However, Davidia (Eyde, 1963), Cardiandra of Hydrangeaceae (Hufford, 2001), Hydrostachyaceae (Kubitzki, 2004), and Cevallia and Schismocarpus of Loasaceae (Hufford, 1989b; Moody and Hufford, 2000; Kubitzki, 2004) lack nectaries. Absence of a disc may have arisen in parallel in Cornales.

Systematic aspects

The systematic position of Camptotheca has been disputed for about 150 years. Camptotheca was always considered to be related to Nyssa and Davidia in Nyssaceae or Cornaceae based upon morphological studies (Hutchinson, 1967; Cronquist, 1981; Thorne, 1992, 2000; Kubitzki, 2004). Molecular data, however, supported a Camptotheca–Nyssa–Davidia clade as sister to a Mastixia–Diplopanax clade, and Cornus as closely related to Alangiaceae (Xiang et al., 1998, 2002, 2011; Fan and Xiang, 2003; Chen et al., 2016; Fu et al., 2017; Yang and Ji, 2017). Morphological and molecular data are complementary and flowers have always been the first pole of attraction to botany (Wanntorp and Ronse De Craene, 2011). It is therefore of interest to compare the floral morphology and organogenesis of Camptotheca with putative relatives in Cornales (Table 2).

Camptotheca shares some floral features with most Cornales, such as small sepals (except for Loasaceae), free mostly valvate petals, a diplostemonous androecium, an inferior ovary, an epigynous disc nectary and unitegmic ovules (Ronse De Craene, 2010).

There is a suite of features that is shared by Camptotheca, Nyssa, Mastixia and Diplopanax, including pentamerous flowers, toothed or minute calyx lobes, diplostemonous androecium and a one-seeded unilocular ovary. Although Davidia does not share some floral traits with the above-mentioned genera, including an absence of disc nectary and perianth in male flowers and having six to ten locules, it does have capitate inflorescences similar to Camptotheca, Nyssa and Cornus, an important feature not found in other taxa of Cornales.

Camptotheca, Nyssa and Davidia are different from Mastixia and Diplopanax in their breeding system and inflorescence structure. Mastixia and Diplopanax are always hermaphroditic and have thyrso-paniculate or spicate (-paniculate) inflorescences (Kubitzki, 2004). Nevertheless, the sexual systems of Camptotheca, Nyssa and Davidia are complex and they have a solitary capitate or partially capitate inflorescence. Additionally, the hooked petals in Mastixia and Diplopanax (Fan and Xiang, 2003) are not present in Camptotheca, Nyssa or Davidia.

Cornus possesses some special and derived floral characteristics as follows: tetramery, a haplostemonous androecium, flowers initiated in grouped dichasia, and capitate or truncate stigmas (Feng et al., 2012), sharing several characters with Alangium.

In summary, the flowers of Camptotheca exhibit several plesiomorphies at the ordinal level: small sepals, free petals, an inferior ovary and epigynous disc nectary (Ronse De Craene, 2010). Traits such as persistent bracts, a spiral initiation of dyads of flowers, valvate anthers and variation of pistil properties according to their position on a FU, unidirectional organ initiation, and floral concavity appearing during sepal initiation are probably autapomorphies. Thus, floral morphological divergence is in accordance with molecular systematics of Cornus, the Mastixia–Diplopanax group and the Camptotheca–Davidia–Nyssa clade (Xiang et al., 2011; APG, 2016; Chen et al., 2016). Our investigation complements findings from molecular systematic studies (Xiang et al., 1998, 2002, 2011; Fan and Xiang, 2003; APG, 2016; Chen et al., 2016). Additional floral developmental and phylogenetic studies in Cornales, especially in Cornaceae, are necessary to clarify relationships amongst taxa.

Conflict of Interest: All the authors declare that they have no conflict of interest for this paper.