Floral anatomy, embryology, seed, and fruit development in Cephalanthus (Naucleeae-Rubiaceae), with emphasis on C. glabratus

Abstract

Information on the reproductive anatomy in genera of the tribe Naucleeae, particularly Cephalanthus, is scarce and fragmented. Of the six species in the genus, only the mature megagamethophyte of Cephalanthus occidentalis has been described. This study aims to provide information on embryological aspects in flowers of C. glabratus and to analyze the morphology and anatomy of the flowers, fruit, and seed in the six species of the genus. Cephalanthus glabratus have imperfect flowers: pistillate (PF) and staminate (SF). In the PF, the ovules are functional, while in the SF, they atrophy during the formation of the embryo sac. The mature ovule has a single integument, corresponds to the Phyllis type and the embryo sac is a Polygonum type, forming only in the PF. The presence of pollenkitt and secondary presentation of pollen were observed in the SF, as well as in the pollen formation previously described, whereas in the PF, they are absent, due to the collapse of the pollen grains inside the indehiscent anthers. The analysis of the ontogeny of the ovular excrescence in C. glabratus determined its funicular origin, calling it an aril. Its development is a pre-anthesis event, initiated during megasporogenesis. In seeds, the aril is a fleshy, white appendage which almost completely envelops the seeds of Cephalanthus, except for Cephalanthus natalensis where it is noticeably more reduced. Studies of the fruit in Cephalanthus species indicate that the infructescence is a dry schizocarp which separates into uni-seminated mericarps, except in C. natalensis that has fleshy indehiscent fruit.

Introduction

The family Rubiaceae is characterized by generally presenting actinomorphic, pentamerous flowers, with a white gamopetalous corolla and gamosepalous calyx, epipetal stamens equal in number to the lobes of the corolla, with filaments fused to the tube of the corolla and an inferior ovary (Robbrecht 1988). The flowers are almost uniformly perfect. However, reproductive strategies, such as secondary pollen presentation, heterostyly and unisexuality, are recognized as favoring allogamy in the family Rubiaceae (Bahadur 1968; Hallé 1967; Jansen and Ridsdale 1983; Robbrecht 1988; Howell et al. 1993; Imbert and Richards 1993; Yeo 1993; Puff et al. 1996; Lorence et al. 2010; Nuñez Florentin et al. 2016; Judkevich et al. 2019).

The tribe Naucleeae is a well-defined monophyletic group that can be easily recognized by the many flowers arranged in globose inflorescences with epigynous nectaries (Bremer et al. 1995; Razafimandimbison 2002; Razafimandimbison and Bremer 2002). The fertile cycles of the flowers have only been described in a few genera of the tribe; in Breonia, Neonauclea, and Anthocephalus, the flowers are perfect (Razafimandimbison 2002; Puff et al. 2005), and in Cephalanthus, they are described as homostyle (Ridsdale 1976; Razafimandimbison and Bremer 2002). So far, there is no history of imperfect flowers in the tribe Naucleeae. During the study of the formation of spores and gametes in the anthers of Cephalanthus glabratus, the presence of flowers in which these processes were arrested was noted, raising the hypothesis of the existence of imperfect flowers.

Regarding the number of carpels, the most frequent is the presence of a bicarpellar ovary that characterizes numerous tribes such as Naucleeae, Cinchoneae, Hypobathreae, Chiococceae, Hedyotideae, Anthospermeae, and Rubieae. The style is generally linear, glabrous, or pubescent, and the stigma has stigmatic lobes that coincide in number with the carpels and it is often exert (Robbrecht 1988).

Axillary placentation is common to all Rubiaceae, but the insertion on the carpel shaft is variable (Robbrecht 1988). When the seminal rudiments are inserted in the middle of the carpellar septum, the placentation is of the axillary type. However, when it is inserted in the apical portion of the septum, the axillary placentation is of the apical type, giving rise to pendulous seminal rudiments. If, on the contrary, the rudiments develop in the basal portion of the septum, this placentation is of the basal type, forming erect seminal rudiments (De Toni and Mariath 2004). The tribe Naucleeae is characterized by the presence of numerous ovules per locule, except for Cephalanthus which only has one pendulous ovule per locule (Verdcourt 1958).

In the Rubiaceae family, the ovules are typically anatropous, with variations in the number of seminal integuments and archesporial cells, and in the characteristics of the nucellar epidermis (Lloyd 1902; Fagerlind 1937). Fagerlind (1937) did the most extensive work for the family, proposing six types of ovules (Phyllis, Bouvardia, Vaillantia, Oldenlandia, Houstonia, and Rubia types). In later studies, the number of types of ovules was extended to thirteen and an evolutionary trend was established based on their characteristics (Andronova 1977; Galati 1988, 1991; De Block 1995; De Toni and Mariath 2004, 2008, 2010; Cabaña Fader 2013; Nuñez Florentin et al. 2016; Figueiredo et al. 2017; Judkevich et al. 2019).

According to Toni and Mariath (2008), knowledge of ovule development in Rubiaceae is fragmentary and only some data are known in isolated genera. According to Fagerlind (1937), Cephalanthus has a Phyllis-type ovule, which is characterized by the presence of a convex nucellar epidermis and one or two archesporial cells. Later, De Toni and Mariath (2004), based on their observations on Borreria verticillata, considered that the Phyllis type of ovule also has a vestigial external integument and they cited Cephalanthus as one of the genera that possesses it.

The anthers, pollen grain morphology and development, and the orbicules were studied by Romero et al. (2017) in species of Cephalanthus (Rubiaceae-Naucleeae). Regarding gynoecium anatomy, only the mature megagamethophyte of Cephalanthus occidentalis has been described (Fagerlind 1937). Other aspects of reproductive anatomy and the process of formation of spores and gametes are unknown.

Two types of sticky material are known to be present on pollen in angiosperms, both produced by the anther tapetal membrane. Pollenkitt is the most common sticky material found on the pollen grains of almost all animal-pollinated angiosperms, whereas trifin appears to be restricted to the Brassicaceae (Pacini and Hesse 2005). Tapetal cell protoplasts have different developmental patterns depending on the products formed during their development and degeneration. If trifin is formed, the protoplasts of the tapetal cells collapse at the microspore stage. If pollenkitt is formed, the contents of the tapetal cells degenerate at later stages (Pacini and Hesse 2005).

The secondary presentation of pollen (SPP) is the relocation and presentation of pollen on floral structures other than the anthers, a mechanism associated with pollination (Howell et al. 1993). It is a well-known phenomenon in Rubiaceae and represents a synapomorph of the tribe Naucleeae (Manns and Bremer 2010). In the Rubiaceae, SPP has been described in protandric, perfect, and staminate flowers, where pollen is deposited on the style, stigmas, or on both (Puff et al. 1996). Bremekamp (1934) described the “Ixoroid mechanism of pollen” in which the stamens deposit the pollen grains on the stigma at the flower bud stage, and after anthesis, the style extends, doubling or tripling its length, facilitating the dispersion of pollen. However, this mechanism is synonymous with SPP and does not only refer to the specific situation in lxora from which the term was derived (Puff et al. 1996).

The number of seeds produced in the family is variable as there is a tendency to abort a number of the ovules during maturation. The largest seeds are found in Gardenieae where the size varies from 10 to 20 mm. In many cases, the shape of the seed is conditioned by the space available within the loculus, where the number of seeds per fruit varies between one and ca. 40 (Robbrecht 1988).

In Rubiaceae, the presence of an ovular excrescence is associated with individual seeds and it is generally referred to as an obturator or strophiole (Robbrecht 1988). In the Rubiaceae family, the terms obturator, strophiole, and aril have been used interchangeably, making it difficult to understand the morphology and ontogeny of this organ (De Toni and Mariath 2008). Galati (1988, 1991), when carrying out embryological studies on representatives of the tribe Spermacoceae, perceived this problem, and as a result of her studies, she redefined the strophiole as an extra-tegumentary structure, which appears early on in the ontogeny of the ovule as an expansion of the raphe that has a function similar to that of the obturator.

However, Bell (2008) describes the swollen fleshy excrescence as an ariloid with a hard head in many seeds dispersed by animals. If the excrescence is in the raphe, it is specifically called strophiole, whereas if it is next to the micropyle, it is called a caruncle. When the ariloid falls off, it can leave an additional scar called a false thread. In general terms, any fleshy excrescence of the seminal surface is called an ariloid (Font Quer 1953). According to Razafimandimbison and Bremer (2002), in Cephalanthus, the seeds have an aril.

The Rubiaceae family has a great diversity of fruits that vary from dry to fleshy. They are characterized by simple shapes: spherical, ellipsoid, ovoid, or obovate, and the predominant colors are red, yellow, orange, or blackish. Robbrecht (1988) mentioned the presence of syncarpic fruits in the tribe Naucleeae, which are characterized by the fusion of the carpels, whereas Razafimandimbison and Bremer (2002) and Ordas et al. (2017) mentioned the presence of infructescences formed of free fruits.

Due to the lack of embryological studies in the tribe Naucleeae and the confusion in the Rubiaceae family regarding ovarian excrescence, the proposed objectives were (a) to describe the morphology of fertile cycles of the staminate and pistillate flowers and inflorescences of C. glabratus; (b) to analyze the morpho-anatomy of the gynoecium and ovule development, and the processes of megasporogenesis and megagametogenesis in C. glabratus; (c) to describe the androecium and the mechanism of secondary presentation of pollen in staminate flowers of C. glabratus; and (d) to describe the morphology of the fruit and seed in the six species of the Cephalanthus genus, focussing in the embryological origin of the ovarian excrescence in C. glabratus in order to use the appropriate terminology for this structure.

Materials and methods

The present study was conducted on the six species that comprise the genus Cephalanthus (Rubiaceae), using C. glabratus as a model for the histological analyses. The present study is based on material collected and fixed in FAA (formaldehyde, 70% alcohol, acetic acid, 5:90:5) and on herbarium collections. The material analyzed is in Appendix.

Morphological analysis

In Cephalanthus glabratus, 20 inflorescences were taken to determine the number of flowers per glomerule. From each glomerule, measurements were taken of ten flowers. To determine the fertile cycles of the flower, 3500 flower buds and flowers were analyzed. For fruit analysis, 25 infructescences were used to determine the number and percentage of the fruits formed. According to the analysis, the infructescences were classified as follows: complete infructescence (when more than 85% of the flowers form fruit) and incomplete infructescence (when less than 85% of the flowers form fruit). In all species, measurements were taken of 10 fruits and 10 seeds per species. For all measurements, an electronic digital caliper (Schwyz) and ImageJ software were used (Rasband 1997–2018; Gonzalez 2018).

In C. glabratus, the material used was fixed in FAA, while in the other species, the materials from herbaria were first rehydrated with 5% neutral detergent for 72 h, then rinsed in tap water, and finally fixed in FAA.

Bright field microscope

Flowers and fruits, fixed in FAA at different stages of development of C. glabratus, were dehydrated through histological dehydrating followed by a tertiary butanol series (Gonzalez and Cristóbal 1997) and embedded in paraffin according to the technique of Johansen (1940). Transverse (TS) and longitudinal (LS) serial sections were cut 10–12 µm thick with a rotary microtome and stained with Safranin-Astra blue blue (Luque et al. 1996). The sections were mounted in synthetic Canada balsam.

To identify pollenkitt on the surface of the pollen, fresh anthers were emptied onto a slide with a drop of Sudan III, and to determine the lipid compounds in the aril, freehand sections were made and placed on a slide with Sudan III or Lugol reagents (Johansen 1940).

The observations and photographs were made using a Leica DM LB2 microscope equipped with a digital camera.

Scanning electron microscopy

The fixed material was dehydrated in an ascending series of acetone, dried to the critical point with CO2, and sputter-coated with gold–palladium. The observations were made with the SEM Jeol LV 5800, at 20 kV at the electronic microscopy service of the UNNE, Corrientes.

Results

Floral morphology

The glomerules in each plant of Cephalanthus glabratus have morphologically perfect but functionally imperfect flowers (Fig. 1a). Each inflorescence has a single type of flower: staminate flower (SF) or pistillate flower (PF). The size and number of flowers make it possible to differentiate the glomerules: those formed of SF are 15–20 mm in diameter and have 100 to 150 flowers on average (Fig. 1b), whereas the glomerules of pistillate flowers are 10–14 mm in diameter and have 70 to 100 flowers (Fig. 1c). Both types of flowers are sessile, actinomorphic, tetramerous or pentamerous, and heterochlamydeous (Fig. 1d, e).

Fig. 1

Inflorescence and flowers in Cephalanthus glabratus. a Flower branch. b Inflorescence with staminate flower (SF). c Inflorescence with pistillate flower (PF). d SF. e PF. f Internal view of the corolline lobes puberulous and dehiscent anthers in SF. g Internal view of the corolline lobes pubescent and indehiscent anthers in PF. h Dehiscent anthers in SF. i Indehiscent anthers in PF. j Longitudinal sections of ovary with aborted ovule in SF. k Longitudinal sections of ovary with ovule developed in PF. ao aborted ovule, o ovule. Scale bars a = 10 mm; b, c = 5 mm; d, e = 1 mm; f–k = 100 µm

Morphologically, the two types of flower differ in the characteristics of the indumentum on the inner side of the corolline lobes, forming a band of puberulous hairs in the SF and pubescent in the PF (Fig. 1f, g). They also differ in the size of the floral pieces (Table 1).

Table 1 Floral characteristics of C. glabratus (in mm)

Both floral types have 4–5 stamens; the filaments are welded to the throat of the corolla; the anthers are sessile and sagittal. In the SF, the stamens are functional, with grains of pollen and longitudinal dehiscence (Fig. 1h). In the PF, the anthers are indehiscent, without any pollen grain production, and the contents of their theca abort before anthesis (Fig. 1i).

The SF and PF of C. glabratus have a bicarpellar gynoecium with a bilocular ovary, which contains one ovule by locule (Fig. 1j, k). The placenta is axillary and of the apical type; the ovules are attached to the carpellar septum acquiring a pendulous arrangement. In SF, the ovules are aborted, and they are functional in PF (Fig. 1j, k).

Morpho-anatomy of the gynoecium

The stigma is bilobed and rounded in the PF, where the apical-lateral region is a dome of papillose cells (Fig. 2a–d). In the SF, the stigma is capitate, elongated, and with the apical portion formed of small cells (Fig. 2e–g). The rest of the stigmatic surface in both flowers is composed of large, elongated, rectangular epidermal cells with secondary thickening in the radial and tangential walls, more abundant in the PF than in the SF (Fig. 2h, i). The sub-epidermal parenchyma is formed of rectangular to quadrangular cells with tannins (Fig. 2h, i, m, n). The apical cells connect with the transmission tissue that runs inside the stigma.

Fig. 2

Morpho-anatomy of the gynoecium of C. glabratus with BFM (a, d, e, h, i, k, l–p) and SEM (b, c, f, g, j). a Longitudinal section (LS) of pistillate floral button. b Stigma of pistillate flower (PF): developed papillary cells (arrowheads). c Detail of papillary cells (arrowhead) of PF. d LS of post-anthesis PF stigma. e LS of stigma rich in SF tannins after anthesis. Note the poorly developed papillary cells (arrow). f Stigma with collapsed papillary cells (arrowhead). g Detail of collapsed papillary cells (arrowhead). h–i LS of epidermal stigma cells with secondary thickening of the cell walls (arrowhead). h PF. i SF. j Germinated pollen grains on papillary stigma cells (arrows) in PF. k LS of papillary cells developed with germinated pollen grains (arrow) in PF. l Pollen grain with pollen tube (arrow) in PF. m Transversal section (TS) of stigma with vascular bundles (arrowhead) in PF. n TS of stigma with vascular bundles (arrowhead) in SF. o TS of style with vascular bundles and transmission tissue (arrowhead) in PF. p TS of style with vascular bundles and transmission tissue (arrowhead) in SF. a anther, ep epidermis, o ovule, ov ovary, p parenchyma, s style, st stigma, tt transmission tissue. Scale bars a, b = 100 µm; c, d, f, g, m = 50 µm, e, h–l, n–p = 20 µm

After anthesis, the adhesion and germination of the pollen grains can be seen in the papillose area of the PF, indicating that it is a receptive area (Fig. 2j–l). No pollen grains adhere to the rest of the stigma surface. Once the stigmatic receptivity is completed, the apical papillose cells collapse and an increase in tannin content is observed; therefore, the stigma ceases to be functional.

The style in the PF and SF is unique, cylindrical, and glabrous (Fig. 2o–p). In transection, it presents a uni-stratified epidermis with quadrangular cells and a thin cuticle. The parenchyma presents polygonal and tannin cells. In the central region, the solid transmission tissue is formed and continues up to the stigma; it is composed of small cells with dense cytoplasm. Two collateral vascular bundles are associated with the transmission tissue (Fig. 2o, p). In the PF, the transmission tissue is more developed, whereas in the SF, the parenchymatic tissue predominates in relation to the transmission tissue.

The morphological difference between the two types of flower lies in the shape of the ovary, which is evident in cross-section (Fig. 3a, b). The PF have a wider ovary of an ovoid shape due to the development of the ovules that exert pressure on the walls of the ovary and occupy the locules completely (Fig. 3a). In the SF, the ovary is smaller and has a circular contour, with the aborted ovules only partially occupying the loculi (Fig. 3b).

Fig. 3

Anatomy of the ovary in C. glabratus with BFM. a Transversal section (TS) of ovary in PF. b TS of ovary in SF. c Detail of the wall of the ovary in PF. d Detail of the wall of the ovary in SF. e Crystalline sand in mesophyll (arrows). f Spheres in mesophyll and epidermal cells (arrows). g Detail of stoma in internal epidermis. ao aborted ovule, ee external epidermis, ie internal epidermis, m mesophyll, o ovule. Scale bars a, b = 50 µm, c–g = 20 µm

The wall of the inferior ovary of the PF and SF is attached to the remaining floral whorls constituting the floral tube. Both floral types share the same characteristics and present the following zones (Fig. 3c, d):

• i. External epidermis: with quadrangular to rectangular cells, lacking stomata and trichomes.

• ii. Mesophyll: compact, without any intercellular spaces. Two regions are differentiated: the external one is made up of 5–7 layers of tanniniferous polygonal cells, with collateral vascular bundles; and the internal one is composed of 2–3 layers of square to rectangular cells arranged in an anticlinal and periclinal manner according to the area of the ovary; tannin cells are less frequent. Both regions present calcium oxalate crystals, such as crystalline sand or spheres (Fig. 3e, f).

• iii. Internal epidermis: is the one that delimits the locule and is characterized by having rectangular cells that may present crystals of the spherical type and slightly elevated stomata (Fig. 3g).

Ovule development: megasporogenesis and megagametogenesis

The following stages are shared by the PF and the SF. The differentiation between the floral types results from the moment of the abortion of ovules.

Stage 1 ovule primordium

They begin as small lumps of placental tissue. These protrusions have three meristematic layers: dermal, subdermal, and a central core (Fig. 4a). The development of the ovule continues mainly from periclinial and anticlinal divisions of the central core, while the dermal and subdermal layers present predominantly anticlinal divisions. The ovule primordium projects into the loculi as it grows and it begins to curve.

Fig. 4

Development of the ovule in C. glabratus in longitudinal section with BFM. a–m PF. a Ovule primordia. b Archesporial cell. c MMC. d Dyad. e Megáspora. f–m Embryo sac. f Mature ovule anatropous. g Micropyle of mature ovule. h Central binucleated cell. i, j Polar nuclei near the oospheric apparatus. k Filar apparatus. l Antipodes. m Secondary endospermic nucleus. n–p Aborted ovule in SF at the embryo sac stage. a aril, ar archesporial cell, c central core, d dermal, di dyad, es embryo sac, fa filar apparatus, f funicle, M megaspore, MMC megaspore mother cell, n nucellar epidermis, o mature ovule, ao aborted ovule, sd subdermal, t integument. Scale bars a, b–e, h–m = 10 µm; g = 50 µm; f, n–p = 100 µm

Stage 2 archesporial cell

The nucellus reaches the hemi-anatropous curvature. An archesporial cell develops, characterized by a prominent nucleus and dense cytoplasm. The nucellar epidermis is a layer of 5–7 cells that envelop an archesporial cell adopting a convex shape (Fig. 4b). Due to the presence of a single layer of nucellus cells, the ovule is tenuinucellate. At this stage, the single integument begins to develop from periclinal divisions of the dermal layer, presenting 3–5 layers of cells.

Stage 3 megaspore mother cell

The archesporial cell increases in volume and elongates, becoming the megaspore mother cell (MMC) (Fig. 4c).

Stage 4 meiosis

MMC undergoes a first meiotic division resulting in a dyad surrounded by callose (Fig. 4d). The second meiotic division results in the formation of four haploid megaspores, a tetrad with a linear arrangement. The three micropylar megaspores degenerate and the chalazal megaspore is the functional one, from which the embryo sac develops. At this stage, the integument is asymmetric; one side is more developed and covers the nucellus (Fig. 4e).

Stage 5 embryo sac

In PF, the ovule completes its anatropous curvature. The functional megaspore enlarges, and through successive mitotic divisions, the embryo sac is formed (Fig. 4f). According to its development, it corresponds to the Polygonum type, with seven cells and eight nuclei: one egg cell, two synergids, one central cell, and three antipodal cells at the chalazal end (Fig. 4h–m). The central cell is vacuolated and with starch grains, it contains two polar nuclei that approach the oospheric apparatus at maturity (Fig. 4h–j). The synergids present their polarized nuclei towards the micropylar end and the vacuoles towards the base, whereas in the egg cell, the vacuole is towards the micropilar end and the nucleus towards the base. The filiform apparatus is well developed (Fig. 4k). The antipodal cells are long, with dense cytoplasm and prominent nuclei (Fig. 4l). The fusion of the polar nuclei creates the secondary endospermic nucleus (Fig. 4m). It occurs before the entrance of the pollen tube, since it is a pre-anthetic event, where the stigma is not yet receptive and the pollen grains from the SF have not yet been deposited on the stigma.

The mature ovule of the PF is anatropous, characterized by a tannin-rich nucellar epidermis, the micropyle is well differentiated (Fig. 4f, g), and the aril covers part of the ovule (description below). The single integument has 5–7 layers of cells on the rapheal side and presents 8–9 layers on the anti-rapheal side.

In the SF, the first stages of ovule development occur normally, completing the anatropous curvature (Fig. 4n). The irregularities occur in the stage of formation of the embryo sac, which does not complete its development leading to the abortion of the ovule that occurs in pre-anthesis. The collapse occurs from the inside outwards, first affecting the contents of the nucellus, then the integuments and finally the development of the aril (Fig. 4o). The aborted ovule is characterized by being hollow, crushed, and formed of cells with abundant tannins (Fig. 4p).

Androecium

In the PF, the anthers remain indehiscent and pollen grains do not develop, but a mass of agglutinated cellular remains is observed inside the pollen sacs (Fig. 5a). The wall of these anthers is only formed of an epidermis and endothecium with thin, tenuous thickening (Fig. 5b–d).

Fig. 5

Anthers in section transversal of pistillate flower (PF) and staminate flower (SF) with BFM (a–f) and pollenkitt in C. glabratus with BFM (g, h) and SEM (i). a Mature anther with collapsed flower bud content. b Detail of collapsed mature anther, still within flower bud. c Same anther as photo b with polarized light. Note the slight thickening of the endothecium. d Mature anthers after flower anthesis, totally indehiscent. e Mature SF anther with pollen grains in polarized light. Note the abundant thickening of the endothecium. f Mature dehiscent anther. g Pollenkitt (arrowheads) joining the pollen grains. h Positive reaction pollenkitt drops from Sudan III (arrowheads). i Pollenkitt strands (arrowheads). co corolla, e epidermis, en endothecium. Scale bars a–d = 20 μm; e, f = 50 μm; g, h = 10 µm, i = 2 µm

The pollen develops normally in the SF; the anthers wall present epidermis and endothecium with well-developed thickening and open longitudinally (Fig. 5e, f). The pollen grains of the SF have a sticky substance on the surface of the exine which makes the pollen grains slightly grouped after dehiscence (Fig. 5g). This pollenkitt reacted positively with Sudan III, staining the droplets yellow, confirming their lipidic nature (Fig. 5h). Using SEM, it was detected that the pollenkitt has the shape of strands joining the pollen grains together (Fig. 5i). The PF lack pollen grains and pollenkitt.

Secondary presentation of pollen

SPP only occurs in the SF where pollen is produced and released. In C. glabratus, this process starts in pre-anthesis, in a bud 3 mm long (Fig. 6a). The pre-dehiscent anthers make contact with the stigmatic surface, forming slight furrows (Fig. 6b, c).

Fig. 6

Secondary presentation of pollen (SPP) in staminate flower stigma of C. glabratus with stereoscopic microscopy (a, d, g, h), LM (b, e), and SEM (c, f, i). a Small button in pre-anthesis, anthers still closed. b Small button with anthers in microsporogenesis exerting pressure on the stigma. c Pre-anthesis button stigma with slight grooves. d Medium button in pre-anthesis with pollen grains on the stigmatic surface. e Medium button in pre-anthesis with dehiscence of pollen grains on the stigma. f Stigma with medium button pollen grains. g Elongated style in anthetic flower with exert stigma. h SSP in the middle and apical region of the stigma. i Stigma with marked grooves in anthetic flower. Scale bars a, d, g = 1 mm; b, c, f = 50 μm; e, i = 100 μm, h = 0.5 mm

In the 5–6-mm pre-anthesis bud, dehiscence of the anthers takes place and the bright yellow pollen grains are deposited on the whole surface of the stigma, which is transformed into the presenting organ (Fig. 6d–f). The pollen grains are mainly deposited in the grooves and in the middle and apical region of the stigma (Fig. 6e, f). The adhesion of the pollen grains is facilitated by sticky secretions from the pollen and possibly from the pollen presenter.

After anthesis, the style elongates, the stigma remains excised with respect to the corolla, and the pollen grains are exposed to the arrival of a pollinator (Fig. 6g, h). After the floral anthesis, the grooves of the stigma become deeper (Fig. 6i). Since these flowers are functionally staminate, the apical region of the stigma is not receptive (Fig. 6i) and the ovule lacks an embryo sac.

Morphology of seeds in six Cephalanthus species

The seeds are plano-convex, with a sub-rhomboid contour, an almost smooth surface, and they are microscopically papillose (Fig. 7a–g, j). The ventral side of the seed is flat and the thread can be distinguished; the dorsal side is convex, and a retraction is observed in the middle region of the seed (Fig. 7a–g). In Cephalanthus natalensis, the ventral face is concave and the dorsal face slightly convex (Fig. 7h, i).

Fig. 7

Morphology of seed in Cephalanthus species with SEM. a, b C. glabratus. c, j C. occidentalis. d, e C. angustifolius. f, g C. tetrandrus. h, i, k C. natalensis. a, d, f, h Ventral view of seeds. b, c, e, g, i Dorsal view of seeds. j, k Detail of the exotesta of the regions indicated with box in c and i. a aril, h hilum, s seed. Scale bars a–j = 500 µm; k = 200 µm

There is a variation in the size of the seeds in the species analyzed: the largest are those of C. glabratus and the smallest are those of C. natalensis, showing a correlation between the size of the fruit with respect to the size of the seed and the aril (Table 2).

Table 2 Data on fruits, seeds, and arils in Cephalanthus species

The exotesta in the Cephalanthus species is smooth looking, except for C. natalensis. At the microscopic level, the shape of the cells in general is constant over the entire seminal surface in all the species analyzed, with a cross-linked pattern of rectangular and elongated cells with slightly raised walls (Fig. 7j). In C. natalensis, the exotesta has a rough aspect; the pattern is cross-linked with polygonal cells that have clearly elevated walls (Fig. 7k).

All species of Cephalanthus, except for C. natalensis, have a large, slightly rough, and spongy aril, which is more developed on the dorsal side of the seed (Fig. 7a–g). In C. natalensis, the aril is reduced, covering less than half of the seed (Fig. 7h, i).

Development of the aril in Cephalanthus glabratus

Aril development is a pre-anthesis event, which starts during megasporogenesis in the PF (Fig. 8a). In the arquesporial cell stage, cellular proliferation of the funicular tissue begins with periclinal and anticlinal cell divisions in the epidermal cells of the funicle, forming a bulge that corresponds to the primordia of the aril, developing on both the internal and external sides of the funicle (Fig. 8a). Cell divisions continue in the subdermal layers of the funicle, with the vascular bundle remaining in the middle region (Fig. 8b). During megagametogenesis, the aril undergoes a series of divisions in different planes, the cells increase in size, and tannic substances accumulate. The aril is conspicuous in the mature ovule, asymmetrical in shape, and it is formed of parenchymatic cells with tanniferous contents (Fig. 8c).

Fig. 8

Development of the aril in pistillate flower and seeds of C. glabratus in longitudinal sections with BFM (a–g) and surface view with stereoscopic microscopy (h, i). a Primordia of the aril in archesporial cell stage. b Aril, MMC stage. c Asymmetric and conspicuous aril, mature ovule with vascular bundle (arrowhead). d Development and expansion of the aril in the seed. e Differentiation of the three regions of the aril in the seed. f, g Aril cells, positive reaction to Sudan III. h, i Fresh seeds, white aril. h Dorsal side. i Ventral side. a aril, ar archesporial cell, es embryo sac, er external region, h hilum, ir internal region, M megaspore, mr middle region, s seed. Scale bars a–c, f = 20 µm; d, e, g = 50 µm; h, i = 1 mm

After fertilization, the aril continues to grow by cell division and storage of lipid substances (Fig. 8d–i). When the development of the aril is complete, three regions can be distinguished: an internal region where the vascular bundle of the funicle is located, a middle region formed of small parenchymatic cells with tannins, and an external region with large, vacuolated cells where lipids are accumulated (Fig. 8e). These cells react positively to the histochemical test with Sudan III, indicating the presence of oil droplets that were stained bright yellow (Fig. 8f, g). In the mature seed, the aril is an appendage that envelops it almost entirely. In both fresh and herbarium material, this excrescence presents a fleshy consistency and is white in color (Fig. 8h, i).

Morphology of the fruit

Inflorescences in the SF of Cephalanthus glabratus do not form fruits (Fig. 9a); so, after the senescence of these flowers, the corolla falls, the wilted calyx remains attached, and the glomerules acquire a characteristic brown color (Fig. 9b).

Fig. 9

Fructification in Cephalanthus with stereoscopic microscopy (a–i) and SEM (j–l). a–i C. glabratus. j C. salicifolius. k C. angustifolius. l C. natalensis. a SF inflorescence with calyx remains. b Detail of calyx remains. c Complete immature infructescence. d Complete mature infructescence. e Immature schizocarp. f Mature schizocarp. g Incomplete mature infructescence. h, j, k Schizocarp with marked longitudinal line (arrow). i Longitudinal section of schizocarp showing uniseminated mericarp pair. l Fruit of C. natalensis. Note the slightly marked longitudinal line. a aril, ca calyx, s seed. Scale bars a, c, d, g = 5 mm; b, e, f, h, i = 1 mm; j–l = 500 µm

The fruits are only formed in the PF (Fig. 9c, d). The infructescence is formed of schizocarp-type dry fruits, which are green when immature and measure between 4.5 and 5 mm (Fig. 9c, e). At maturity, they become reddish in the apical region and reach a length of 6–7 mm (Fig. 9f). The average percentage of fruit formed from the flowers in an inflorescence is 54.33% (Table 3), varying between 94.63 (considered complete) (Fig. 9c, d) and 55.86% (incomplete infructescence) (Fig. 9g). The schizocarp is obpyramidal-turbinated, glabrous with a persistent calyx (Fig. 9f). When the fruits are ripe, each schizocarp presents a longitudinal line of separation from the mericarps (Fig. 9h, j, k). The mericarps begin to separate from the basal region towards the apex. Each mericarp is characterized by being uni-seminate and indehiscent (Fig. 9i).

Table 3 Number of fruits formed in infructescences with PF of C. glabratus

The presence of a persistent calyx on the schizocarps that separate into uni-seminated mericarps is a constant in the remaining Cephalanthus species (Fig. 9j, k). Cephalanthus natalensis is an exception, since in the label descriptions of herborized material, and in the literature consulted, it is cited as a fleshy fruit (Fig. 9l). In this study, we only had access to herbarium material, in which it was described as a smaller fruit, glabrous, and with a persistent calyx, with a slightly marked longitudinal line, which suggests that it does not separate into mericarps (Fig. 9l). There are variations between species in values such as size, pubescence, and the shape of the schizocarp (Table 2).

Discussion

Floral morphology

In Cephalanthus glabratus, the flowers are morphologically perfect but functionally imperfect, arranged in staminate and pistillate glomerules that occur on the same plant, so therefore, it is a monoecious species. The staminate glomerules contain 100 to 150 flowers measuring 6.9–8.9 mm in length, whereas the pistillate glomerules have fewer (70–100) flowers which are smaller (5–7.5 mm in length). The two types of flowers can be differentiated morphologically: they both have pubescent corolline lobes, but the SF have a longer floral tube, dehiscent anthers, longer style-stigma length, and a less developed ovary vs. a shorter floral tube, indehiscent anthers, shorter style-stigma length, and a developed ovary in PF. These differences found in the two floral types can contribute to their recognition in both field observations and herborized material.

In other Rubiaceae, the morphological characters in the flowers are not always morphologically perfect and functionally imperfect, as regards the recognition of the fertile cycles of the flowers. An example is that of Kadua haupuensis (tribe Spermacoceae) where characters, such as the length of corolla tube and lobes, length of style, and stigma, are inadequate due to the overlapping ranges of functionally staminate, functionally pistillate, and functionally perfect flowers. In this species, these measurements do not reveal a clear pattern of parameters that correlate with fertile cycles of the flower (Lorence et al. 2010).

As in most Rubiaceae, Cephalanthus glabratus has an inferior, bicarpellar, and bilocular ovary, with one ovule per locule. In the SF, the ovules abort in a pre-anthetic state. The PF and SF of the Cephalanthus species present differences in the morpho-anatomy of the style and stigma, which become evident in the last stages of megagametogenesis.

The stigma in C. glabratus is one of the characters that differentiates the two flower types. Although morphologically, the stigma of both types of flower is exert and in the PF it is bilobed, short with papillose cells, whereas in the SF, it is capitate and elongated. Anatomically, the main difference is due to the development of papillose cells in the PF that reach their maximum development after anthesis when the stigma becomes receptive to the germination of pollen grains. The epidermis of the SF stigma does not develop papillose cells, and after anthesis, it ceases to be functional. Secondary thickening in the radial and tangential walls of the epidermal cells occurs in both floral types, but it is more abundant in the PF.

The only species of the genus analyzed was C. occidentalis, in which Imbert and Richards (1993) described the stigma with the same characteristics as those found in the PF of C. glabratus. These authors did not observe the presence of imperfect flowers, since their study only analyzed the variation in the stigma during anthesis and it did not include the anatomy of the ovule or gamete formation. According to their observations, the flower remains open for 4 days: on day 1, the stigmatic papillae are short and tight; on day 2, the stigmatic papillae are longer and more vacuolated and the pollen grains germinate; on day 3, the stigmatic papillae are notoriously elongated and highly vacuolate; and finally on day 4, most of the stigmatic papillae are broken. Based on this description, they characterized the flowers as protandry, functioning as staminate on the first day of flower opening and the following 3 days as pistillate. This conclusion is due to the morphology adopted by the stigma after anthesis and the moment of stigmatic receptivity. In view of the results found here for C. glabratus, the results for C. occidentalis should be reviewed by studying the development of the ovules and pollen grains in the flowers. The question to be answered here is whether this species also has imperfect flowers.

According to what was observed in C. glabratus, and described by Imbert and Richards (1993) in C. occidentalis, the receptive stigma is characterized by the development of papillary cells in both species. This coincides with Igersheim (1993) who described species of the tribe Vanguerieae where the receptive cells of the stigma are developed and exposed in the pistillate phase, while the receptive cells are hidden in the staminate phase.

Thickening in the epidermal cells of the stigma, as described for C. glabratus (in this study) and C. occidentalis (Imbert and Richards 1993), was also observed in Vangueria infausta and it was called “Igersheim thickening,” which would be related to the secondary presentation of pollen (SPP) (Tilney et al. 2014). However, in C. glabratus, this type of thickening is found in both the PF and SF, which could suggest that there is no association with SPP, since the PF lacks this mechanism due to abortion of the pollen grains.

The PF and SF of C. glabratus present a unique, cylindrical, and glabrous style. This is consistent with the observations in C. glabratus and other Rubiaceae species (De Block and Igersheim 2001; Nuñez Florentin et al. 2016; Judkevich et al. 2019).

So, it can be concluded that some morphological characters in C. glabratus are correlated with fertile cycles of the flower.

Ovary anatomy and development

The structure of the gynoecium in Rubiaceae is diverse, with differences in the number of carpels; the insertion, position, and structure of the placenta; and the number of ovules per locule and their orientation (Svoma 1991). In the family, the number of seminal rudiments varies from one to many and this character is traditionally used to distinguish subfamilies (Bremekamp 1966). The tribe Naucleeae is characterized by the presence of numerous ovules per locules, except for Cephalanthus which has one pendulous ovule per locule (Verdcourt 1958). In C. glabratus, the development of the gynoecium is similar to that of other Rubiaceae with uniovulated, such as follows: Paederia, tribe Paederieae; Svoma 1991, Keetia and Vangueria, tribe Vanguerieae; Igersheim 1993, Chomelia obtusa, tribe Guettardeae; Ixora coccinea, tribe Ixoreae; De Toni and Mariath 2008 and tribe Spermacoceae; Galati 1988, 1991; De Toni and Mariath 2004. The ovary of the PF and SF of C. glabratus is anatomically similar. This is consistent with studies conducted on the ovary wall of other Rubiaceae (Pavetteae tribe, De Block 1995; Spermacoceae tribe, Nuñez Florentin et al. 2016; Gardenieae tribe, Judkevich et al. 2019). The difference between both types of flowers is due to the fact that in the PF, the ovary is more developed due to the pressure of the ovules that generates an ovoid form, whereas in the SF, the ovary is less developed due to aborted of the ovules.

Axillary placentation is common to all Rubiaceae, but the insertion into the carpellar septum is variable (Robbrecht 1988). In the tribe Naucleeae, Razafimandimbison and Bremer (2002) mentioned the presence of pendulous ovules in the subtribes Cephalanthinae (where Cephalanthus is found with one ovule per locule), Naucleinae (Burttdavya, Nauclea, Neolamarckia, Ochreinauclea, and Sarcocephalus with numerous ovules per locule), and in Breoniinae (Breonadia, Breonia, Gyrostipula, and Janotia with 1–15 ovules per locule). According to the observations in C. glabratus, the placentation is axillary with insertion in the apical region of the septum, which gives it its pendulous position. This suggests that the remaining species of Cephalanthus and the genera of the subtribes with pendulous ovules share the same type of placentation as C. glabratus. However, the remaining sub-tribes of Naucleeae: Mytragyninae, Uncarinae, and Corynantheinae are characterized by the presence of numerous ovules per locule with basal axillary placentation (Razafimandimbison and Bremer 2002).

In the PF and SF, the primordia of the ovule start as small protrusions from placental tissue acquiring an anatropous position at maturity. This is consistent with other Rubiaceae species, such as Borreria verticillata, tribe Spermacoceae; Chomelia obtusa, tribe Guettardeae, and Relbunium, tribe Rubieae (De Toni and Mariath 2004, 2008, 2011).

In Rubiaceae, Fagerlind (1937) described six types of ovules by taking account of the number of integuments and archesporial cells, and the shape of the nucellar epidermis. Based on these variants, he presented an evolutionary trend that manifested a progressive inclusion of the archespore tissue in the center of the chalaza, thus suggesting that the Phyllis type ovule would be the most primitive and the Houstonia type the most derived. Galati (1988, 1991) supported this tendency, since she considered that the Phyllis type has a nucellus represented mainly by the epidermis, which protrudes above the chalaza and is covered by a thick integument without any welding. However, in the Vaillantia, Bouvardia, and Oldenlandia types, there is a reduction of the nucellar epidermis, which disappears completely in the Houstonia type. By taking account of this classification, new types of ovules were proposed in later studies and there are now thirteen types described for the family (Andronova 1977; Galati 1991; De Toni and Mariath 2004, 2008, 2010; Figueiredo et al., 2013a, b, 2017; Judkevich et al. 2019). Their characteristics are summarized in Table 4 below.

Table 4 Classification of ovule types in Rubiaceae

De Toni and Mariath (2004) considered the Phyllis type of ovule with an external integument that develops on the internal side of the ovule, in the region between the funicle and the micropyle, which they considered as vestigial because it does not complete its development on the antirafeal side. So, they concluded that the bitegumentary condition in the Rubiaceae ovules would be the primitive condition, and therefore, it would be the ancestral type in the family. The Phyllis type of ovule is found in the following genera: Borreria verticillata, Cephalanthus, Chiococca, Coffea, Hoffmannia, Ixora, Knoxia, Macrosphyra, Mussaenda, Ophiorrhiza, Paederia, Phyllis, Psychotria, Rondeletia, Scyphiphora, Stephygyne, and Tricalysia (Fagerlind 1937; Andronova 1977; Svoma 1991; De Toni and Mariath 2004).

Of the different types of ovules proposed for the family, the type corresponding to C. glabratus is the Phyllis type, but this is according to the interpretation of Fagerlind (1937) and Galati (1991) who did not consider the presence of the vestigial external integument. In C. glabratus, the ovule has only one integument, which supports the general uni-tegumented condition of the ovule in Rubiaceae.

Cephalanthus glabratus seeds have a highly developed aril of funicular origin. Observing young stages in the development of the ovule, the origin of the aril of C. glabratus is comparatively identical to the so-called vestigial external integument, for example, in Borreria verticillata (De Toni and Mariath 2004), since it appears on the internal face of the funicle. Perhaps, the terminology of the so-called vestigial external integuments of several Rubiaceae, which might be atrophied arils or arils at different stages in their development, should be reconsidered. This is a hypothesis that deserves wide study in the family. It would be appropriate to corroborate whether it corresponds to another structure specific to the ovule, or not, in the proposed classifications of ovules for Rubiaceae, where the character of presence or absence of the vestigial external integument is mentioned, since the terms strophiole, aril, and obturator have been used mistakenly in the family as many authors use the denomination when studying ontogeny.

The type of embryo sac observed in C. glabratus is the Polygonum type in the PF, being the characteristic type in Rubiaceae. However, according to Fagerlind’s classification of megagametophytes (1937), which takes account of the characteristics of the antipodal apparatus, it corresponds to the Leptodermis-Cephalanthus type which is characterized by the presence of three more or less long antipodal cells.

Therefore, it is necessary to carry out evolutionary studies of the ovules in relation to Fagerlind’s classification (1937) taking account of the different aspects of the development of the ovule (e.g., aril), which makes a reconstruction of the ancestral characters in molecular phylogenies possible. This would provide a critical view of the classification and its foundations and later a careful codification of the homologies between the genera already analyzed.

In the SF of C. glabratus, the ovule aborts during the formation of the embryo sac. It was not possible to determine the exact moment when the abortion starts, but it was observed that the abortion occurs gradually from the inside to the outside of the ovule, starting in the region of the sac and continuing with cells of the nucellus and integument. This atrophy of the ovules results in a lesser development of the ovary, a differential characteristic between the PF and SF.

In Rubiaceae, embryological studies of abortions in the megasporogenesis and megagametogenesis stages are rare. These studies have been carried out on Mussaenda pubescens (Mussaendeae tribe, Li et al. 2010) and on Cordiera, Genipa, Randia, and Tocoyena (Gardenieae tribe, Judkevich et al. 2019). According to the studies in Mussaenda pubescens, the flowers with staminate function, named morpho S, present abnormalities at different stages of megasporogenesis and megagametogenesis, so there are no functional ovules, which seems to be mainly due to the failure to establish functional megaspores. However, in the Gardenieae species, functionally staminate flowers form rudiments of ovules, without reaching the differentiation of embryo sac.

Androecium

Romero et al. (2017) described the formation of the male gametes and anther development of C. glabratus which corresponds to the anthers of the SF. The present study completes the information with the description of anther abortion in pistillate flowers (PF). Both types of flowers—SF and PF—follow the same initial pattern of development, both of the anther wall and of the formation of sporogenous tissue, microspores, and pollen grains. At the completion of differentiation, the young anther wall consists of four layers: epidermis, endothecium, middle layer, and tapetum. After maturity, the anther wall retains remnants of epidermal cells and endothecium with well-developed thickening in the SF and little thickening in the PF. The differentiation of the two floral types occurs when the pollen grains and endothecium collapse in the PF, resulting in indehiscent anthers. In SF, the anthers open by longitudinal dehiscence, releasing bicellular pollen grains. Our observations in C. glabratus are similar to those described by Li et al. (2010) in Mussaenda pubescens. These authors describe that microsporogenesis in flowers of the S-morph (staminate flower) is similar to that observed in flowers of the L-morph (pistillate flower) while microgametogenesis in the L-morph stops at the microspore stage with degradation of their nuclei, leading to anther abortion and male sterility. In the S-morph, microgametogenesis culminates in the formation of bicellular pollen grain. According to Li et al. (2006), they indicate that tapetum degradation resulting from programmed cell death is necessary to produce fertile pollen grains. In the SF of C. glabratus and the S-morph of M. pubescens, the tapetum gradually disintegrates at the microspore stage, while tapetum degradation at an early stage will lead to male sterility of the L-morph of M. pubescens. However, early tapetum degradation was not observed in the PF of C. glabratus and it would be necessary to determine the cause leading to male sterility in these flowers.

The presence of pollenkitt, an adhesive material that binds pollen grains during anther dehiscence in SF, was also described. This substance reacted positively to Sudan III confirming its lipidic nature. Pollenkitt is produced as a derivative of the tapetal membrane and is a typical substance that binds the pollen grains of almost all entomophilic plants with monads and tetrads (Hesse 1993). The presence of pollenkitt may be related to the presence or absence of orbicules. Romero et al. (2017) mentioned the presence of orbicules in Cephalanthus species, which corroborates that it is the pollenkitt that binds the pollen grains of C. glabratus.

Pacini and Hesse (2005) mentioned 20 possible functions for the pollenkitt, among which, according to what was observed in C. glabratus, the following functions could be possible: (1) to maintain the pollen in the anther until its dispersal; (2) to allow secondary presentation of the pollen; (3) to facilitate the dispersion of the pollen; (4) to keep the pollen grains together during transport; and (5) to facilitate adhesion to the stigma.

Secondary pollen presentation

This is a common characteristic in Rubiaceae (Tilney et al. 2014) and it can occur in protandric flowers, both perfect and staminate flowers, in which the pollen grain is deposited on the style, stigmas, or on both (Puff et al. 1996). In the tribe Naucleeae, the SPP represents a synapomorph (Manns and Bremer 2010) and was described in C. occidentalis (Meehan 1887; Imbert and Richards 1993), in Nauclea subdita (Burck 1884), in Neonauclea pallida, Uncaria cf lanosa, and in Sarcocephalus latifolius (Puff et al. 1996).

In Cephalanthus glabratus, SPP was observed in the flower bud of the SF, 5–6 mm long, the dehiscent anthers surrounding the stigma release the pollen grains that adhere to it. After bud anthesis, the style elongates and the stigma becomes the pollen presenter.

In C. occidentalis, Imbert and Richards (1993) found SPP in flower buds of 9–10 mm in length moments before anthesis. The pollen grains are deposited on the stigma and then exposed to the pollinator after the style has elongated. These authors considered that the flowers in C. occidentalis are protandric and have self-incompatible pollen. After anthesis, the flower remains open for four days; on the first day, it behaves as a staminate flower and on the following 3 days as a pistillate flower with the staminate and pistillate phases being temporarily separated. However, the authors considered that protandry does not contribute significantly to preventing self-fertilization, as the pollen grains could germinate if they are not removed before the stigma becomes receptive, with self-compatibility being the factor that would favor the prevention of self-pollination. Therefore, protandry was considered as a precondition for the evolution of SPP in the stigma in this genus. These authors did not consider the possibility of imperfect flowers in C. occidentalis, since their study did not include the processes of sporogenesis and gametogenesis.

Puff et al. (1996) concluded that SPP in Rubiaceae is invariably linked to protandry, which means that pollen deposition in the host region must take place in the late bud stage (near anthesis). These authors recognized four types of SPP:

• 1. Pollen deposition on the style.

• 2. Pollen deposition on the style and on the external surface of the stigma: at the time of pollen deposition, the receptive surfaces of the stigma lobes are strongly pressed together, so that contact of the pollen with the receptive surfaces is unlikely.

• 3. Pollen deposition on the outer surface of the stigma: pollen is only deposited on the non-receptive surfaces of the stigmas; the inner receptive surfaces are strongly pressed together at the time of pollen deposition.

• 4. Pollen deposition on the stigma-receptive surfaces in whole or in part: pollen is only deposited on stigma-receptive regions.

The most common type in Rubiaceae is the second (Puff et al. 1996). However, representatives of the tribe Naucleeae, such as Cephalanthus occidentalis, Neonauclea pallida, and Uncaria cf lanosa, were reported to be characterized by “full or partial SPP on stigma-receptive surfaces” on late flower buds, with the exception of Nauclea subdita and Sarcocephalus latifolius, where a small amount of pollen is deposited on stigma-receptive regions. Puff et al. (1996) conclude that self-fertilization would be inevitable in this type of SPP, unless a system of incompatibility exists, as outlined by Imbert and Richards (1993) for C. occidentalis.

According to those observed in C. glabratus, the flowers are imperfect; therefore, it is not a case of secondary presentation related to intrafloral protandry. In the SF, the pollen is presented directly onto the stigmatic surface, which is not receptive. The PF lack SPP, because the anthers are indehiscent and the pollen grains have aborted. Therefore, in this species, it is concluded that unisexuality is the mechanism that prevents self-pollination.

Burck (1884) was the first to report SPP in a dioecious Rubiaceae, Canthium laeve (tribe Vanguerieae), and he described the differences between the SF and PF. The SF measure 8 mm in diameter, the ovary is small and sterile (no ovules), and the complex stylar head serves as a region for presenting pollen, whereas the PF are 6 mm in diameter and have a functional stigma. These descriptions are like those found in both floral types in C. glabratus.

Puff et al. (1996) considered that the SPP was associated with dioecy and that has since been documented for a number of taxa, in all of which the gynoecium is rudimentary in the SF which produces a “pistilstitium” (similar in shape and size to the style and stigmas of PF) that serves as a pollen-presenting organ. It has been recorded that three tribes in Rubiaceae have dioecious taxa with SPP: Gardenieae (e.g., Duroia, Genipa), Octotropideae (e.g., Fernelia, Morindopsis), and in Vanguerieae (e.g., Canthium, Peponidium, Pyrostria: Igersheim 1989, 1993). Recently, Judkevich et al. (2020 in press) confirmed that SPP was associated with dioecy in representatives of the tribe Gardenieae: Cordiera concolor, Genipa americana, Randia calycina, and Randia sp. which are characterized by the presence of functionally imperfect flowers.

According to the above, the observations in C. glabratus of SPP associated with functionally imperfect flowers are the first record for the tribe Naucleeae, so it would be necessary to extend the studies on SPP in the remaining species of Cephalanthus to determine if there is protandry as described for C. occidentalis or if there are imperfect flowers, as found in C. glabratus. Unfortunately, only herborized material of these species was available for this study, so this characteristic cannot be confirmed.

Development of the aril

In the Rubiaceae, the terminology for the excrescence of seed has included obturator, strophiole, and ariloid, which have been used interchangeably without determining their anatomical origin (Lloyd 1902; Fagerlind 1937; Kapil and Vasil 1963; Galati 1988, 1991; De Toni and Mariath 2008). In a more or less general concession, true arils are those originating in the funicle and arilodes are those developing elsewhere in the seed. If the excrescence is in the raphe, it is considered as a strophiole, whereas if it is formed of one of the teguments in the micropyle, it is called a caruncle. In the arils, the variable origin is specified by being called rapheal, micropillar, etc. (Corner 1976). All these terms can only be used correctly if an ontogenetic study of their origin has been carried out. The terminological problem is inevitable when seeds are described without such analysis, as occurs in many Rubiaceae (Robbrecht 1988).

Lloyd (1902) considered that ovular excrescence in the family starts as a lump near the raphe, at an early stage of development. Galati, in the tribe Spermacoceae (1988, 1991), considered it appropriate to use the term strophiole, redefining it as an extra-tegumentary structure, which appears early in the ontogeny of the ovule as an expansion of the raphe, whose function is related to the guidance of the pollen tube. However, De Toni and Mariath (2004), when studying the seminal rudiment in Borreria verticillata (tribe Spermacoeae), considered that periclinial divisions are produced in the subdermal layer during the development of the ovule creating a vestigial external integument and they considered that this vestigial feature was erroneously called a strophiole.

In C. glabratus, the ontogeny of ovular excrescence was analyzed and is here referred to as an aril, due to its funicular origin from periclinal and anticlinal divisions of epidermal cells of the funicle. The development of this structure is a pre-anthesis event that begins during megasporogenesis and forms a structure that surrounds the ovule and then the seed, only in the PF.

In addition to its ecological role in seed dispersal, the aril represents a relevant character for systematic classification (Silveira et al. 2016). The presence of an aril is a common characteristic at the family level, as occurs for example in the Passifloraceae and Turneraceae (Kloos and Bouman 1980; Gonzalez and Arbo 2013; Arbo et al. 2015). In all these species, the ovules are bitegmic, the origin of the aril being described as a “third tegument” because of the similarity of its development with the other two teguments covering the nucellus.

During their development and maturation, the arils (in a broad sense) accumulate substances that give them properties that not only attract dispersing agents but, in certain species, are also used for human consumption, e.g., Blighia sapida (Silveira et al. 2016). Among the components that can accumulate are oils in Ricinus communis, substances rich in flavor and aroma in Myristica fragrans, and sugars and other nutrients in Passiflora edulis. In the Turneraceae, they have starch and oils and are associated with the dispersal of seeds by ants (Arbo et al. 2015). According to our histochemical study, it was found that the aril in C. glabratus is rich in lipid substances. However, the fruits of this species are indehiscent mericarps, which makes the hypothesis of associating its presence with ant dispersal difficult. Aril was also observed in the seeds of the other Cephalanthus species, as a fleshy, white appendage that almost completely envelops the seed, being noticeably more reduced in C. natalensis. However, Razafimandimbison and Bremer (2002) mentioned the absence of aril in the seeds of the latter species, but these descriptions were a result of analysis of herborized material. This may suggest that the aril is of taxonomic importance at a specific level in Cephalanthus and at a tribal level, as it is the only genus of the tribe Naucleeae that is characterized by aril seeds.

Fruit and seed morphology in Cephalanthus species

In Rubiaceae, the fruits show great variability and are used to classify taxa in the family (Schumann 1891). According to Robbrecht (1988), in the tribe Naucleeae, many genera are characterized by the fusion of numerous ovaries in the compressed globose inflorescences, producing syncarpic fruits. However, Razafimandimbison and Bremer (2002) mentioned the presence of infructescences in the genus Cephalanthus constituted by free and indehiscent fruits, a character shared by other genera of the tribe, such as Breonadia, Breonia, Gyrostipula, and Janotia. Bacigalupo (1974) mentioned the presence of an indehiscent nut in C. glabratus, without clarifying what type it corresponded to. In C. natalensis, several authors mentioned that the fruits are fleshy and consumed by birds, baboons, and monkeys (Boon 2010; Van Wyk and Van Wyk 2013). However, Löfstrand et al. (2014) characterized the tribe Cephalanthinae by the presence of infructescences formed of schizocarpous fruits that are divided into two indehiscent mericarps.

From the observations made in C. glabratus of herborized material, as in the rest of the species with the exception of C. natalensis, and following the classification of fruits proposed by Fahn (1982), it was considered that the infructescence is composed of a dry fruit of the schizocarp type with a persistent calyx, which is broken down into indehiscent mericarps at maturity. These fruits only originate from the inflorescences with PF. However, infructescences were also observed where not all the flowers were fruitful. Fructification of glomerules may be complete, when more than 85% of flowers form fruit, or incomplete, when less than 85% of flowers form fruit. This suggests that several factors (extrinsic or intrinsic) could explain the presence of partially fructifying glomerules, which could not be determined in this study since it requires field observations (pollinators, environmental conditions, etc.) or other types of analysis.

Schizocarps vary in size and shape, with the largest being found in C. occidentalis (5–7.5 mm in length). The shape is obpyramidal in C. glabratus, C. salicifolius, and C. tetrandrus and narrow obovate in C. angustifolius and C. occidentalis. In general, the schizocarp is glabrous, except for C. salicifolius in which it is pubescent. In all species, the schizocarp separates into uni-seminated mericarps at maturity. These observations suggest that the fruit has taxonomic importance at the species level.

In Cephalanthus, the presence of schizocarpous fruit is constant in all species, except for C. natalensis. The analysis of the fruit in this species is based on herbarium specimens and the descriptions with which fleshy fruits are recorded on the labels of the material analyzed. In addition, this characteristic is cited in studies by various authors who describe the presence of edible fleshy fruits for this species (Fox and Young 1982; Boon 2010; Van Wyk and Van Wyk 2013). The fruit of C. natalensis is characterized by being smaller than the other species (3–4 mm), with an obovate shape and a slightly marked longitudinal line, not separated into mericarps.

Except for C. natalensis, the seed in Cephalanthus is characterized by a flat-convex shape with a sub-rhomboid contour, an almost smooth surface, and a developed, white, slightly rough, and spongy aril that covers a large part of the seed. The exotesta has a reticulated pattern.

According to the analysis of the Cephalanthus species, there is a correlation between the size of the fruit, the seed, and the aril that makes it possible to differentiate C. natalensis from the rest of the species. These data contribute to the tribe Naucleeae, since it is corroborated that in C. natalensis, the aril is reduced and not absent as mentioned by Razafimandimbison and Bremer (2002).

The studies conducted here provide relevant embryological information of this poorly studied genus. In addition, numerous data show the need to extend the analyses to the remaining species of the tribe. Several characters could be used for a taxonomic revision of the species, especially regarding C. natalensis.

Appendix

List of all specimens analyzed, with * were highlighted specimens fixed in FAA.

Cephalanthus angustifolius Lour. Vietnam — ANNAM. Bên Du pro, Thua Thiên Récolte sur le bord de Sông Bô, 16° 31′ 40.8′′ N; 107° 34′ 22.8′′ W, 13 May 1920, M. Poilane 1410 (K); Nha Trang, sine data, M. Krempf s.n. (P03820121); idem, M. Krempf s.n. (P03820124); Khanh Hoa, Dien Khanh district, road transact Suoi Cat-Hon Ba, at km 19 to Hon Ba, forest along rocky riverbank of Da Giang River, 12° 06′ 53″ N, 109° 00′ 12″ E, 22 Jun 2004, D.D. Soejarto 13,284 (F, P); idem, Suoi Cat Village, lowland tropical rain forest formation, 12° 7.97′ N, 109° 1.28′ E, 110 m, 26 Nov 2004, D.D. Soejarto 13,284 (F, P).

Cephalanthus glabratus (Spreng.) K. Schum. Argentina— CORRIENTES: San Cosme, Las Lomas, 29 Ago 2015, M.F. Romero 60* (CTES); Concepción, Ea Yatay Cora, 50 km NE de Chavarría, 23 Oct 1996, M.M. Arbo 6797 (CTES); Ituzaingó: Ea San Pedro, 56° 52′ W 27° 45′ S, 13 Nov 1976, M.M. Arbo 1250 (CTES); Itá Ibaté, 13 Oct 1949, G.J. Schwarz 8235 (CTES); Mburucuyá, 06 Nov 1949, G.J. Schwarz 8564 (CTES); Riachuelo, 09 Sep 2015, M.F. Romero 61* (CTES); Saladas, Rincón de Ambrosio, 14 Oct 974, A. Schinini & C. Cristobal 9984 (CTES); San Luis del Palmar, 26 Ago 2017, M.F. Romero 67* (CTES); ENTRE RÍOS: Paraná, 12 Dec 1944, A. Schulz 90 (CTES). MISIONES: Iguazú, Parque Nacional Iguazú, circuito superior, salto Rivadavia, 27 Nov 1995, J. Herrera 113 (CTES). Brazil— RIO GRANDE DO SUL: Cruz Alta, 10 km S de Cruz Alta, 30 Dec 1980, A. Krapovickas & R. Vanni 37,068 (CTES). MATO GROSO DO SUL: Aquidauana, 19° 15′ S 55° 58′ W, 21 Nov 1989, A. Pott 5406 (CTES). Paraguay— ALTO PARANÁ: 14 km W de Itaquyry, 12 Oct 1995, A. Schinini & G. Caballero M. 30,213 (CTES); Concepción, Paso Horqueta, Río Aquidabán, 19 Oct 1984, A. Duré 389 (CTES). Cordillera, Valenzuela, 06 Dec 1989, R. Vanni 1172 (CTES). Uruguay— CERRO LARGO: Ruta 8 km 374, ayo. del Parao, 32° 44′ 34″ S, 54° 13′ 14″ W, 113 m, 29 Nov 2001, G. Seijo 2714 (CTES). COLONIA: Punta Gorda, confluencia del Río Uruguay con el Río de la Plata, E. de Rincón de Darwin, 33° 54′ 57″ S, 58° 24′ 49″ W, 35 m, 24 Nov 2007, V. Solis Neffa & G. Seijo 2121 (CTES).

Cephalanthus natalensis Oliv. Malawi. Southern Mt. Mulanje—Big Ruo Valley, 1800 m, 3 Nov 1988, J.D. Chapman & E.G. Chapman 9390 (K, MO). South Africa. Nkangala, 12 Jun 1932, J.C. Smuts 21 (K); 2 miles N of Potholes, 6 Mar 1968, O.A. Leistner 3261 (K). TRANSVAAL: Barbestón, 2 Oct 1922, F.A. Rogers 21,605 (K). Soutpansberg, 9 Jul 1937, J.C. Smuts 3283 (K). Wavervol ouder, 1 Oct 1979, Ruprossev 1227 (K). Sabie, Langverwag, 24 Ago 1963, W.J. Louw 2836 (AAU). Tanzania. Mbeya, Rungwe Livingstone Mountains; foot trail from Bumbigi on steep ridge top N of Isalala River, 9° 11′ S, 33° 52′ E, 1900 m, 3 Mar 1991, R.E. Gereau & C.J. Kayombo 4174 (MO).

Cephalanthus occidentalis L. Canada— OTTAWA: Pontiac Station, 30 miles west Hull, north shore of Ottawa river, 28 Ago 1948, A.J.J. Breitung 7327 (SI) — QUÉBEC: Rigaud, comté de Vaudreuil, 26 Jul 1934, E. Roy 3337 (SI). USA— ARIZONA: Maricopa Co., 628 m, 6 Jul 2012, L. Markings & L. Butler 3959 (ASU, SI); idem, Bosque Nacional de Tonto, 33° 47.93′ N, 11° 29.49′ W, 25 Jun 1999, L. Landrum 9539 (CTES) — ILLINOIS: Jackson Co., 37° 35′ 08″ N, 89° 27′ 44″ W, 110 m, 21 Oct 2014, M.H. Nee 61,696 (MO, SI) — MISSOURI: Saint Louis City, Missouri Botanical Garden’s Monsanto Research Center campus, by southwest corner of building, south of chiller equipment, 06 Sep 2017, C.M. Taylor 12,977* (CTES, MO). — NEW JERSEY: Burlington Co., 23 Jul 1981, L. Ley 42,199 (CTES). New York, Bronx, 40° 52′ 03″ N, 73° 52′ 34″ W, 16 m, 6 Jun 2006, M.H. Nee 54,470 (SI). — TEXAS: Aransas Pass Co., on N. Mc Campbell Road, between Rabbit Road and Jacoby Lane, 3 Jun 1998, P.A. Fryxell 5137 (CTES); Comanche Co., Near Cache, 25 Jun 1913, G.W. Stevens 1319 (SI). — WISCONSIN: Richland Co., 210 m, 14 Jul 1978, M. H. Nee 16,308 (SI); idem, 2 miles NW OF Blue River, floodplain of the Wisconsin River, along the channel (usually with flowing water) flowing into Garner Lake, 43° 11′ 55″ N, 90° 36′ 00″ W, 204 m, 27 Sep 2014, M.H. Nee 61,662 (SI).

Cephalanthus salicifolius Bonpl. El Salvador. — MORAZÁN: Río Sapo, area burnt ca. 20 years prior, 13° 55′ 47″ N, 88° 06′ 01″ W, 678 m, 25 March 2002, A.K. Monro 3808 (MO). Honduras. El Paraíso, Yuscarán, Aldea El Rodeo, quebrada Las Zarcas, road to Oropolí, 13° 53′ 06″ N, 86° 46′ 47″ W, 420 m, 3 May 2000, A. Molina R. et al. 34,926 (MO). México. — HIDALGO: Jojutla, 31 Ago 1902, C.G. Pringle 9822 (SI). —NAYARIT: San Blas, 3,8 km al SE de Tecuitata sobre camino a tierra El Cora, carr. Jalcocotan-Miramar, 21° 26′ 16″ N, 105° 08′ 13″ W, 10 Nov 1994, G. Flores F. & O. Téllez V. 3435 (CTES). — MORELOS: Cuernavaca, 16 Jan 1901, C.G. Pringle 8474 (SI). —COLIMA: Ruta 110, Colima a Pihuamo, Km 215 a Río Salado, 19° 11′ 32″ N, 103° 41′ 45″ W, 300 m, 18 Jan 1982, D.H. Lorence et al. 3798 (MO).

Cephalanthus tetrandrus (Roxb.) Ridsdale & Bakh. f. India. [Uttar Pradesh], Pilibhit, 25 May 1898, K. Inayat 23,668 (K); [Uttar Pradesh], [Lakhimpur] Kheri, idem, 24 Jun 1900, K. Inayat 22,377 (K). Assam, without precise locality, s. col., (K000265513); “M. Sillet”, 1832, N. Wallich s.n. (K001067160); Katya, Keshampur, South Kheri, 17 May 1920, S. Ram s.n. (K); without precise locality, s. col. (K000265509). China. Yizhang, Hunan, in plain, by river side, 15 Jul 1942, S.H. Chen 1896 (AAU), locality and collector in Chinese language, 19 May 1983, collection number 323 (AAU).