Floral Development of Rhamnaceae and Origin of Its Unique Floral Features

Rhamnaceae flowers have a peculiar morphology, including keeled sepals, one stamen whorl closely related to the petals, and a broad perigynous hypanthium that supports a voluminous nectary. In the present investigation, we detailed the flower development of five Rhamnaceae species to understand the origin of such specific floral characteristics. Floral buds and flowers were processed for surface and histological analyses. The sepals emerge in sequential order and the other organs in simultaneous order. The development of the perigynous hypanthium renders the floral apex broad and concave. The sepals undergo abaxial thickening early on, forming a keel and strongly influencing the floral merosity. Petals and stamens appear close to each other on the same radius in a very short plastochron. The carpels unite soon after their emergence, forming a syncarpous ovary and free style branches. Differences in intercalary carpel growth promote the formation of inferior (Gouania virgata) and semi-inferior ovaries (Colubrina glandulosa, Hovenia dulcis, and Sarcomphalus joazeiro). Rhamnidium elaeocarpum does not undergo such growth, and the resulting ovary is superior. The keeled sepals promote the isolation of the petal–stamen pair inside the flower bud. The possibility of a common primordium that the originates petal and stamen is refuted. Comparisons with other Rosales families provide insights into the floral origin and diversification of Rhamnaceae.


Introduction
The flowers of Rhamnaceae Juss. exhibit a unique combination of characters among angiosperms [1,2]. They have only antepetalous stamens [3], a relatively rare feature in few families [4]. In addition, there is always a hypanthium that supports the extra carpel whorls [5], which can form a wide disc ("disciflores") [6] in which it is common to find a prominent intrastaminal nectary, which is quite remarkable in this family [7]. Other singularities include triangular and keeled sepals with valvate aestivation and clawed petals [1]. Some species have no petals, and others have diclinous flowers [3]. The position occupied by the ovary in the flower can vary from superior to inferior [5,8], an unusual variation within angiosperm families.

Organography
The flowers are small (about 1-5 mm long) and generally pentamerous but with variations between tetramerous (Hovenia dulcis) and hexamerous types (Colubrina glandulosa, Sarcomphalus joazeiro), monoclinous, polysymmetric, and dichlamydeous. A perigynous hypanthium is present, usually campanulate, with a larger opening in some species than in others. The calyx has valvate aestivation and is composed of free, coriaceous, triangular sepals with the median nervature prominent on the abaxial surface, forming a keel, usually with a callous apex. The corolla is made up of tiny petals that never touch each other and that are clawed, with a cucullate or concave apex. The androecium is formed by a single whorl of stamens opposite to the petals and joined to them by the base of the filaments. The anthers are bithecal, tetrasporangiate, and introrse and have longitudinal dehiscence and dorsifixed insertion in the filament. The gynoecium varies in carpel number between species: one carpel in Rhamnidium elaeocarpum, two to three in Colubrina glandulosa and Hovenia dulcis, three in Gouania virgata, and two to four in Sarcomphalus joazeiro. The position of the ovary also varies according to the species: inferior in Gouania virgata; semiinferior in Colubrina glandulosa, Hovenia dulcis, and Sarcomphalus joazeiro; and superior in Rhamnidium elaeocarpum. The distal end of the style may show varying degrees of free stigmatic branches. There is one erect anatropous ovule per locule, with basal placentation. Intrastaminal nectaries occur in all species and may form a conspicuous disc (Colubrina glandulosa) or not (Hovenia dulcis). This nectary can also be restricted to the perigynous hypanthium (Rhamnidium elaeocarpum and Sarcomphalus joazeiro) or extend over part of the ovary roof (Gouania virgata).

Ontogeny
Floral developmental stages were described and illustrated for Colubrina glandulosa (Figures 1 and 2  (F) Hexagonal contour (delimited by the sepals (s*)) of the rest of the floral apex and primordia of petals, stamens, and carpels. (G) Lateral union between three young carpels. (H) Closure of the carpel cleft in the plicate portion of young carpels. (I) Young petals are approximately the same size as young stamens and form a flattened structure. (J) The young sepal exhibits a median thickening that projects towards the interior of the flower bud (*). The young petals and stamens occupy the flanks of the sepals. Symbols and colors: c = carpel (red), p = petal (pink), s = sepal (green), st = stamen (blue), * = keel/apex of sepal, s* = sepal removed.      The sepals emerge from the floral apex as distinct primordia in sequential order (Colubrina glandulosa ( Figure 1A-C), Gouania virgata ( Figure 3A,B), Hovenia dulcis ( Figure 4A,B), Rhamnidium elaeocarpum ( Figure 5A,B), and Sarcomphalus joazeiro ( Figure 6A,B)). Shortly afterwards, the young sepals form a medial thickening along their entire adaxial extension, culminating in an apical callous projection. After the initial elongation that defines the valvate calyx, they circumscribe the remaining space in the floral apex for the emergence of the other floral organs. This space is usually star-shaped, with the sides arched inwards, like a hypocycloid. The shape of this "star" varies according to the number of sepals, with five points (most common; Figures 1G, 3C,D, 4D, 5C-F and 6D), four points (astroid-like; Figure 4E,F), or six points ( Figures 1F and 6F). The arching of this hypocycloid is due to the medial thickening of the developing sepals. The primordia of petals and stamens emerge alternately with the sepals in the position corresponding to the vertices of this star. This region can be elevated by the growth of the perigynous hypanthium to a greater or lesser extent depending on the species. However, in general, the inner surface of the floral apex is relatively broad and flat.
The petals emerge in simultaneous order ( Figure 4D) and are followed by the antepetalous stamens ( Figure 1E,F, Figure 4E,F, Figure 5C,D and Figure 6C,D), with a short plastochron between the two organ types. Although petals and stamens appear as distinct primordia, they are united at the base, even in the early stages, and there is a common basal growth of these primordia, as in Gouania virgata ( Figure 3D) and Rhamnidium elaeocarpum ( Figure 5D). Then, the carpel primordia emerge (Figures 1F,G, 3D, 4F, 5D and 6E) and may vary in number according to the species or even within the same species. In Rhamnidium elaeocarpum, there is only one carpel primordium ( Figure  Although the petals emerge a little earlier than the stamens, they grow less than the stamens during the intermediate stages. Young stamens become flattened (Figures 1H-J, 5H and 6G) on the abaxial-adaxial plane, and soon differentiate into anther and filament ( Figures 3E and 6H). The anthers usually have two thecae on opposite sides of this flattened structure (Figures 2A, 3E,F and 6H). Shortly thereafter, it is possible to observe the formation of furrows that delineate the limit between the pollen sacs of the same theca ( Figures 2B, 3F,J, 5I and 6I,J). Petals may form cucullate structures that cover most of the stamens or only their abaxial surface (e.g., Rhamnidium elaeocarpum; Figure 5I,K). In Sarcomphalus joazeiro, the petals form clawed structures in which the lamina surrounds part of the anther, whereas the claw accompanies the filament. The basal regions of the petal and filament are united at the base ( Figure 7G).
The carpel primordia emerge on a relatively flat surface in the center of the floral apex ( Figures 1E-G, 3C,D, 4E,F, 5C-F and 6C-F). The young carpels form plicate structures and are laterally united at the base in the case of a syncarpous gynoecium. The carpel cleft has an acropetal closure. The lateral union between carpels can vary greatly in relation to their distal end. The style branches can be very short (Colubrina glandulosa and Sarcomphalus joazeiro), short (Hovenia dulcis), or long (Gouania virgata). In the case of Gouania virgata, each carpel exhibits a style free from the others throughout its extension. The formation of semiinferior ovaries results from the growth of the gynoecial hypanthium in varying degrees: smaller in Sarcomphalus joazeiro ( Figure 7F) than in Colubrina glandulosa or Hovenia dulcis. In Gouania virgata, the growth of the gynoecial hypanthium is conspicuous before the closing of the carpel cleft, leading to the formation of a completely inferior ovary ( Figure 3I,L,O).
The expansion of the perigynous hypanthium causes both an increase in floral diameter and an elevation of the perianth and androecium organs. This phenomenon coincides with the formation of the nectariferous disk over the perigynous hypanthium. The valvate closure of the calyx, which is associated with the formation of a discoid perigynous hypanthium, i.e., a structure that is more flat than deep, causes stamens and petals to bend over the developing nectariferous disk (Figures 1C, 3L, 4H, 5I, 6J and 7C,F,G). As a result, we often observe marks of this contact on the nectariferous surface; the anthers are pushed against the nectary, which, in turn, expands in regions less subject to mechanical pressure, forming molds around the anthers (Figures 2E, 3K and 7A-C,E,H). Such marks are no longer evident during nectary development, but the contour of this structure at anthesis may retain some impressions ( Figure 2G,H). During anthesis, after calyx opening, the unit formed by the petal and stamen moves from a curved position on the flower bud ( Figure 7G) to an erect position ( Figure 2H) and is finally reflexed ( Figure 4O) to the floral axis.
In Sarcomphalus joazeiro, some flower buds showed two petals and two stamens in the apex region delimited by the sepals ( Figure 6I). These petals and stamens are slightly smaller than the others. Some buds have only one vertex with twinned organs ( Figure 6I), whereas others have more than one region, leading to the formation of a bud with seven petals and seven stamens ( Figure 7D).

Calyx: Development and Implications for the Floral Construction
Among all the peculiar floral characteristics of the studied Rhamnaceae species, the keeled sepals, i.e., sepals endowed with a medial thickening on the inner surface, stand out because they represent an uncommon condition not only in the family but also among angiosperms. Although many angiosperms have carinate organs in the perianth, especially between the outer whorls, such carinae are often abaxial, as in some orchids [30] or Passiflora L. [31]. An interesting case occurs in the genus Coriaria Niss. ex L. (Coriariaceae DC., Cucurbitales Juss. ex Bercht. & J. Presl), in which the internal carina of the fleshy petals grows between the carpels, giving the compound fruit a berry-like appearance [32]. In Rhamnaceae, the degree of prominence of this keel may vary, even within the same genus, as in species of Reynosia Griseb. [33].
The existence of keeled sepals in Rhamnaceae promotes several effects on their floral construction. The development of an internal keel creates a compartmentalization of the inner space of the flower bud. The petals and stamens, formed in an alternating position between the sepals, remain isolated by "locules" between adjacent sepals. This effect seems to be quite pronounced during development in Colubrina glandulosa and Sarcomphalus joazeiro and may have drastic implications for the development of internal organs, especially regarding floral merosity, as well as anthesis dynamics.
An important issue is the shape of the sepal in relation to the rest of the developing floral apex. Unlike laminar sepals, which delimit a simple geometric figure (e.g., a pentagon), the keeled sepals of Rhamnaceae delineate a more complex figure (e.g., a five-pointed star) and occupy a large portion between the vertices of the contour of the floral apex. It is interesting to note that there is not even a trace of the emergence of antesepalous stamens in Rhamnaceae.
Another issue is that we found five sepals in most of the flowers sampled, with the occasional occurrence of buds with four or six sepals. The emerging petals and stamens alternate with the growing sepals [3,6,27,31]. Thus, the number of sepals defines the shape of the inner space of the floral apex, where the subsequent organs will emerge, i.e., petals, stamens, and carpels. Regardless of the number of sides of this star (tetra-, penta-, or hexagonal), the petals and stamens always emerge at the vertex, following the alternate position of the sepals. In this context, the number of developing sepals seems to determine the corolla and androecium merosity in Rhamnaceae flowers. Thus, this family is an illustrative example of how the merosity of the outer whorls influences the merosity of the inner whorls (e.g., [34]).
The sepals of Rhamnaceae appear as independent organs and in sequential order (present study), as previously verified [31,35], although the emergence order can also be "almost simultaneous" in some species (e.g., [27]). Thus, the development of the sepals in Rhamnaceae culminates in a strongly cohesive valvate aestivation calyx in pre-anthesis. This type of calyx aestivation is quite common [2].
Finally, the arrangement of the keeled sepals seems to cause the petals and stamens to remain strongly curved inside the flower bud, delaying filament elongation and petal expansion until the moment the calyx opens.

Hypanthia and Gynoecium
All studied Rhamnaceae species have a perigynous hypanthium, which can vary greatly in its morphology from a broad, flat floral disk to a long tube (e.g., Colletieae Reiss. ex Endl.) [5]. The formation of a perigynous hypanthium can be observed from the establishment of a flat floral apex, the edges of which are raised by a marginal growth resulting from the activity of intercalary meristems (sensu [29]). In this sense, the radial growth of the perigynous hypanthium is an important marker of the formation of a disk flower, which is typical of Rhamnaceae (see [36]). The development of the perigynous hypanthium is quite conserved in this family, as reported herein and in previous studies [3,6,27,37]. One factor that seems to vary among species is the direction of this intercalary growth, in the sense of promoting the formation of a flat (disk-shaped, e.g., Sageretia Brongn., Ziziphus Mill.), a campanulate (e.g., Gouania Jacq., Helinus E.Mey. ex Endl., Lasiodiscus Hook.f., Nesiota Hook.f., Reynosia, Rhamnus L., and Trevoa Miers), or a tubular perigynous hypanthium (e.g., Colletia Comm. ex Juss., Cryptandra Sm., Discaria Hook., Kentrothamnus Suess. & Overkott, and Retanilla (DC.) Brongn.) [5]. Such floral constructions are functionally dissimilar, especially in terms of accessibility to resource-seeking floral visitors, but are formed in basically the same way. In the case of Rhamnaceae, the main alteration of the perigynous hypanthium is related to the floral nectary, one of the most conspicuous floral features of the family. The structured nectary of Rhamnaceae originates on the peripheral portion of the carpels and expands in diameter as it grows, along with the length of the perigynous hypanthium [7]. Although the structural characteristics of nectaries can vary greatly in this family, their mode of development is very similar between species [7]. Importantly, the mere existence of intercalary meristems is not sufficient to explain floral variation in Rhamnaceae. The degree of concavity of the floral apex during the early stages of development has already been credited as a predictor of the position that the ovary will occupy in the structure at anthesis.
The species studied here illustrate how the ovary can vary from superior to inferior in Rhamnaceae. This variation in ovary position results from the action of a particular type of intercalary growth associated with the carpel, which culminates in the formation of the gynoecial hypanthium. The onset of a perigynous hypanthium usually precedes that of a gynoecial hypanthium. A gynoecial hypanthium is not always present in Rhamnaceae flowers, unlike the perigynous hypanthium. The greater the intensity of intercalary growth associated with the carpel, the greater the inferior portion of the ovary, as demonstrated by our results (formation of inferior ovaries in Gouania virgata, semi-inferior ovaries in Hovenia dulcis and Sarcomphalus joazeiro, and superior ovaries in Rhamnidium elaeocarpum). Thus, the position of intercalary meristems and the duration and direction of growth are fundamental for floral diversification, explaining the variation in ovary position in Rhamnaceae.
From a taxonomic point of view, the main groups of Rhamnaceae-ampelozizyphoids, rhamnoids, and ziziphoids-have genera with semi-inferior ovaries and their variations [5,23,25]. This suggests a very high lability in ovary position in the family. Thus, plasticity in the development of the gynoecial hypanthium must be an inherent feature of Rhamnaceae. Ovary position tends to be a stable characteristic within angiosperm families, but several groups show similar disparity, such as Melastomataceae [39] and Saxifragaceae [41]. However, in rhamnoids, several genera have a superior ovary [5], and there are few cases of an inferior ovary (e.g., Fenghwaia [22]). The opposite situation is found among ziziphoids, in which inferior ovaries are frequent (especially among Gouanieae Reissek ex End. and Phyliceae Reissek ex Endl.), and there are few genera with superior ovary species (e.g., Blackallia C.A.Gardner and Sarcomphalus, Zizyphus s.s.) [5,25].
The two main factors of variation in the Rhamnaceae gynoecium appear to be (1) the number of carpels and (2) their degree of union [8]. Our sampling ranged from one (e.g., Rhamnidium elaeocarpum) to four carpels (e.g., Sarcomphalus joazeiro), with varying degrees of union between the stigmatic branches. Nevertheless, the ontogenetic patterns observed here correspond to what has already been established for the family (e.g., [8,27,28,31]), such as the simultaneous emergence of the carpel primordia, their arrangement in a ring, and their horseshoe shape during the early stages.

The Relationship between Petals and Stamens in Rhamnaceae
Our results corroborate a floral ontogenetic pattern for Rhamnaceae, i.e., a close relationship between petals and stamens. The plastochron between petals and antepetalous stamens is very short, and the two organs show a union at the base from very early on. Thus, petals and stamens are organs that are always opposite and united at the base in Rhamnaceae. Even in the few Rhamnaceae species that lack petals (Condalia Cav., Krugiodendron Urb., and Siegfriedia C.A.Gardner, as well as some Discaria, Gouania, Pomaderris Labill., Rhamnus, Reynosia, and Ventilago Gaertn.) [5,25], the beginning of the development of these organs is marked by the formation of a functional stamen (internal) and a filamentous structure (external) (e.g., Rhamnus japonica Maxim) [3].
Some authors have postulated that petals and stamens originate from a common primordium in Rhamnaceae flowers (e.g., [3,6]). According to this interpretation, petals and stamens arise simultaneously from the tangential division of these common primordia. One possibility for this phenomenon is a progressive delay in petal development [26]. Thus, the petal primordia may be incorporated by the antepetalous stamen primordia, leading to the formation of a common primordium for both organs. This has been observed in several other families, such as Geraniaceae Juss., Plumbaginaceae Juss., and Primulaceae Batsch ex Borkh. [2,42]. The hypothesis of a common stamen-petal primordium would also explain the cases observed in Sarcomphalus joazeiro in which there was an increase in the number of stamens and petals. In this context, a radial division could occur before the tangential division of this common primordium, justifying the occurrence of twinned petals and stamens in an alternating position with the sepals.
Our data do not support the hypothesis of a common stamen-petal primordium for Rhamnaceae. Petals appear to emerge slightly earlier than the stamen primordia in Hovenia dulcis, Rhamnidium elaeocarpum, and Sarcomphalus joazeiro. This temporal difference has also been reported for Oreoherzogia pumila (Turra) W.Vent (="Rhamnus pumilus") [31], Sarcomphalus mauritianus (Lam.) Raf. (="Zizyphus mauritiana" Lam.) [21], and Ziziphus jujuba Mill. [="Zizyphus sinensis"] [31]. The main difficulty in recognizing the occurrence of a common primordium is that the division between petal and stamen is very rapid in most cases. Thus, it is important to intensify investigations on the origin of petals and stamens in Rhamnaceae. The example of Vitaceae is illustrative. This family was once considered a case of common stamen-petal primordia (e.g., [42]), but further studies on the group led to a refutation of this idea (see [43]). It is currently recognized that the stamens succeed the petals in Vitaceae-a similar condition to that observed here for Rhamnaceae.
The close ontogenetic relationship between petals and stamens in Rhamnaceae is also reflected in the functional relationship of these organs. Both organs commonly perform the same movements during anthesis; they are curved in bud, become erect during calyx opening, and become reflexed during anthesis [44]. This mechanism may be essential for reproductive success in these species, as exposed and accessible anthers are crucial for the more generalist pollination typical of Rhamnaceae. The studied petals (more so in Colubrina glandulosa but not in Rhamnidium elaeocarpum) form a hood-shaped structure that surrounds the anthers during most of the development stage inside the bud. The protection that these petals confer to the anthers may help to prevent pollen desiccation at anthesis [45].

Floral Structure of Rhamnaceae in the Context of Rosales
Rhamnaceae exhibit uniformity in ontogenetic patterns during floral development (e.g., [3,6,8,31,35] and the present study). However, their floral characteristics are markedly different from those of other Rosales.
A perigynous hypanthium is found in most Rosales but is lost in Barbeyaceae and urticalean rosids (Cannabaceae, Moraceae, Ulmaceae, Urticaceae). The perigynous hypanthium is conspicuous in Rhamnaceae and Rosaceae, with important ontogenetic implications in both families. The type of stamen development is more affected by the perigynous hypanthium in Rosaceae than in Rhamnaceae, especially in relation to the increase in the number of organs. The size and shape of the perigynous hypanthium in Rhamnaceae have a greater effect on the structure of the interstaminal nectary. A gynoecial hypanthium is only found in Moraceae, Rhamnaceae, and Rosaceae. It should be noted that inferior ovaries occur very rarely in Moraceae [46,47], whereas they are characteristic of some groups of Rosaceae, such as Maleae Small [19,48]. However, in Rhamnaceae, the occurrence and extent of a gynoecial hypanthium are very labile characteristics (see above).
The development of petals and antepetalous stamens is quite similar in Dirachmaceae and in Rhamnaceae; both types of organs alternate with the sepals and emerge at very short intervals (see [21]). Likewise, the meristic fluctuations of the androecium seem to follow the calyx merosity, independent of the floral merosity (e.g., pentamerous, tetramerous, or hexamerous). Among the urticalean rosids, the androecium exhibits only antesepalous stamens, commonly with only one or two stamens per flower [20]. The number of stamens in Rosaceae is more varied, with a predominance of the diplostemonous condition but also with the occurrence of a polystemonous or haplostemonous androecium [2]. In Barbeyaceae and Elaeagnaceae, groups supposedly close to Rhamnaceae, the androecium can be formed only by alternisepalous stamens (Barbeya Schweinf., Elaeagnus, Hippophae) or by antesepalous and alternisepalous stamens (Barbeya, Shepherdia) [49][50][51]. However, ontogenetic studies on these two families are not available for comparison with the other Rosales.
The presence of fewer carpels (2-3-4) than in the rest of the whorls merosity, as found in Rhamnaceae, is a common condition in rosids, including Pentapetalae [14]. Although such reductions may also be related to a decrease in the space available for carpel emergence, in the case of Rhamnaceae, such fluctuations do not seem to be correlated with calyx merosity. The degree of union between the carpels is a more variable characteristic among Rosales families. In the urticalean rosids, there is more than one carpel that composes the unilocular ovary (pseudomonomerous gynoecium [2,20]), and in Rosaceae, the carpels can be completely free or united [2,19]. Although Rhamnaceae have syncarpous ovaries, the styles vary greatly in their degree of union (see [8]). This variation in the development of more or less free style branches is similar to that found in urticalean rosids, especially among Moraceae and Urticaceae [20], although there is no evidence of a pseudomonomerous gynoecium in Rhamnaceae. Floral buds in different developmental stages and flowers were fixed in buffered formaldehyde (Lillie 1948 apud [52]) or FAA 50 (37% formalin, acetic acid, and alcohol) [53] for 24 h, stored in 70% alcohol, and prepared for surface (scanning electron microscopy (SEM)) and anatomical (light microscopy (LM)) exams.

Microscopy
For SEM exams, the samples were dehydrated in an ethanol series, critical-point-dried in a Bal-Tec CPD 030 dryer (Balzers, Liechtenstein), mounted on metal stubs, adhered to carbon adhesive tape, and covered with gold in a Bal-Tec SCD 050 sputter coater. Observations and illustrations were performed using a scanning electron microscope (Zeiss EVO-50, Cambridge, UK) at 15 kV.
After surface analysis (SEM), some stages were chosen for evaluation by light microscopy (LM). Samples were dehydrated in an ethanol series and embedded in histological resin [54], and transverse and longitudinal 3-3.5 µm thick sections were obtained with a rotary microtome (Leica RM 2245, Wetzlar, Germany). The sections were stained with toluidine blue in phosphate buffer (pH 5.8) [55] and mounted in water. Observations and illustrations were obtained using a Leica DM 4500 B LM connected to a Leica DFC 320 digital camera. Scales were determined under the same optical conditions.

Conclusions
The data obtained to date show that Rhamnaceae is a family with a very homogeneous floral development. Such stability is reflected in the emergence patterns of floral organs; the presence of intercalary growth, culminating in a perigynous hypanthium; the formation of keeled sepals; and the close relationship between petals and antepetalous stamens. Our data demonstrate how some modifications alter floral construction throughout development, such as petal formation (cucullate or not) and variation in ovary position. Just as the nectary is one of the most diverse and conspicuous features of the family and has a very similar development [7], flower development is, on the whole, curiously uniform in Rhamnaceae.