Different ways to obtain similar results: the development of the corolla and epipetaly in Rubieae (Rubioideae, Rubiaceae)

Background and aims – Rubieae is a tribe in the subfamily Rubioideae characterised by herbaceous plants with verticillate leaves and flowers with a rudimentary or absent calyx and a short, cup-shaped corolla. This is in contrast to the flowers of most other Rubiaceae, in which the tubular corolla is longer than the corolla lobes. Also, the description by Payer, a French 19th century pioneer of floral ontogenetic research, of the floral development in Asperula, Galium, and Rubia deviates from recent insights about the development of tubular corollas, which are based on investigations of flowers of tropical Rubiaceae. Tubular corollas are currently considered as resulting from the development of underlying annular intercalary meristems, whereas Payer explained the tubular corollas in the three taxa by postgenital fusion. We therefore tested both hypotheses in six Rubieae genera, including the three taxa studied by Payer. Methods – Floral ontogeny of ten species in six Rubieae genera based on scanning electron (SEM) and light microscopy (LM). Conclusions – Our results suggest that, in all species studied, the mature phenotype of the corolla as well as the epipetaly of the stamens is caused by a combination of three developmental processes (the development of a stamen-corolla tube, the development of a corolla tube sensu stricto, and postgenital fusion), and the relative moment of activation of each of these processes during floral development (plastochron variation or heterochrony).


INTRODUCTION
Rubiaceae, also known as coffee or madder family, is the fourth largest angiosperm family and consists of approximately 13 200 species in 614 genera. It has a cosmopolitan distribution and centres of diversity in the (sub)tropics (Heywood et al. 2007;Davis et al. 2009;Stevens 2001 onwards). Rubiaceae are easily recognizable by the simple opposite leaves with entire margins, inter-or intrapetiolar stipules, and sympetalous flowers with inferior ovary. Tropical Rubiaceae are usually woody and occur in the understory of humid and dry forests. However, some Rubiaceae lineages are herbaceous, predominantly occurring in subfamily Rubioideae, which is characterized by the presence of raphides, valvate petal aestivation, and many representatives are heterostylous (Robbrecht 1988). Among these herbaceous lineages, some are tropical (e.g., tribe Spermacoceae), while the cosmopolitan tribe Rubieae occurs in regions with Mediterranean and temperate climates (Robbrecht 1988). Certain members of these herbaceous lineages can exhibit secondary woodiness (Lens et al. 2009). This study focuses on temperate representatives of tribe Rubieae, belonging to subfamily Rubioideae.
Most of the Rubieae species belong to the cosmopolitan Galium (c. 635 species), the Old World Asperula L. (c. 195 species), and Rubia L. (c. 90 species) with a Eurasian-African distribution. The remaining genera are smaller (Govaerts et al. 2020 (1 species), and Valantia L. (7 species). The monophyly of the Rubieae was confirmed by molecular evidence, but the delimitation of the two largest genera within the tribe remains problematic: Galium is paraphyletic and Asperula is polyphyletic (Ehrendorfer et al. 2018).
Typical Rubiaceae flowers are tetracyclic, possessing calyx, corolla, androecium, and gynoecium. Flowers are tetramerous or pentamerous. In most species, the calyx and corolla are tubular and topped by calyx and corolla lobes. The corolla tube is usually as long as or longer than the corolla lobes (Robbrecht 1988). The inferior ovaries are often bi-to multilocular with a single to numerous ovules per locule. In contrast, in the tribe Rubieae, the calyx is usually rudimentary or absent. Most species have a short corolla tube that is cup-shaped rather than tubular, although there are exceptions (Phuopsis, Puff et al. 1996;Crucianella, Schönbeck-Temesy & Ehrendorfer 1989). Ovaries in Rubieae are bilocular with a single ovule per locule (Robbrecht 1988).

Floral ontogeny
Floral ontogenetic studies of Rubiaceae mostly deal with tropical, herbaceous or woody species (e.g., Von Faber 1912;Van der Meulen 1939, both on Coffea L.; Rutishauser et al. 1998  To our knowledge, Payer (1857) was the first to describe all the developmental stages in flowers of the temperate gen-era Asperula, Galium, and Rubia, with a single figure showing the floral developmental stages in Rubia tinctorum L. (as Rubia tinctoria Salisb.). The species studied by Payer share a late development of a rudimentary calyx, appearing only as a rim without calyx lobes after the development of the corolla and stamens. Payer even questioned whether the rim without calyx lobes actually is a calyx. Erbar & Leins (1996) also considered Asperula tinctoria L., Cruciata laevipes Opiz., Galium verum L., and Phuopsis stylosa (Trin.) G.Nicholson as taxa without a calyx. This is in contrast to the woody Canephora, Paederia, and Sacosperma and the herbaceous Pentas, Pentodon, and Spermacoce, where a tubular calyx with calyx lobes originates before or more or less simultaneously with the corolla (De Block & Vrijdaghs 2013;Vrijdaghs et al. 2015). Payer (1857) reported that in Asperula, Galium, and Rubia the corolla originates from four freestanding petal primordia that simultaneously appear. Later in the development, the bases of the petals fuse. About the development of the androecium, Payer (1857: 633) wrote: "Complètement indépendantes de la corolle d'abord, elles deviennent ensuite connées à leur base et sont insérées à sa gorge" ["Initially totally independent from the corolla, they [the stamens] subsequently become connate at their bases and are inserted at its [the corolla] throat"]. As for the development of the gynoecium, the description of Payer for Asperula, Galium, and Rubia corresponds with the description of the development of the gynoecium in other Rubiaceae. Payer stressed that the gynoecium consists of an inferior part, the ovary, which becomes inferior by the rising of parts of the receptacle, whereby locules are formed. The single style is the result of a fusion of two "bourrelets semi-circulaires" ["two semi-circular bulges"] (Payer 1857: 634), which are "les rudiments du style" ["the rudiments of the style"] and which "deviennent promptement connées" ["become immediately connate"]. Pötter & Klopfer (1987) stated that sepal primordia were not detected and that petal and stamen primordia appear simultaneously in Galium aparine. According to these authors, sympetaly is the result of the development of an underlying meristem. Naghiloo & Classen-Bockhoff (2016) performed a floral developmental analysis in Cruciata glabra (L.) Ehrend., C. laevipes, and Rubia tinctorum. Apart from the absence of a calyx, they focused on the more or less simultaneous appearance of petal and stamen primordia and on the "late and very weak sympetaly" (Naghiloo & Classen-Bockhoff 2016: 57). According to them, the functionally male flowers in Cruciata laevipes result from a delayed initiation of the gynoecium development. De Block & Vrijdaghs (2013) and Vrijdaghs et al. (2015) hypothesised that the tubular corolla, with or without epipetalous stamens, is the result of three developmental processes that act in different proportions depending on the species, thus causing the variability that occurs in tubular corollas. These developmental processes are: 1) the development of a stamen-corolla tube from an annular common primordium, 2) the development of a corolla tube sensu stricto from an annular primordium or intercalary meristem, and 3) postgenital fusion of petals or corolla lobes. The development of a stamen-corolla tube concurs with the floral cup hypothesis of Leins & Erbar (2010), which states that floral cups can be formed by annular intercalary meristems underlying one or more floral whorls, in the case of a stamen-corolla tube below petals and stamens. In Rubiaceae, the stamens are mostly epipetalous. If only a stamen-corolla tube is formed, the stamens emerge from the corolla tube in between the bases of the corolla lobes ( fig. 12D). If a stamen-corolla tube ànd a corolla tube sensu stricto are formed and/or if postgenital fusion of the corolla lobes occurs, the stamens are inserted somewhere inside the tubular corolla ( fig. 12B, C, F). Sometimes, a corolla tube sensu stricto of only a few cell layers thick is formed, in which case the stamens seem to emerge from in between the bases of the corolla lobes (Vrijdaghs et al. 2015). If no stamen-corolla tube develops, the stamens remain free from the corolla, inserted on the hypanthium.

Aims
Intrigued by Payer's (1857) description of the development of corolla and androecium in Asperula, Galium, and Rubia, we test the developmental hypothesis of Vrijdaghs et al. (2015) for the corolla and androecium in these three and other temperate herbaceous genera. In addition, aspects of the development of the gynoecium are illustrated.

Plant material
Inflorescences of ten Rubieae species were collected from the living collection of Meise Botanic Garden (table 1) and preserved in 70% ethanol. The five Galium species belong to the monophyletic clade X according to Ehrendorfer et al. (2018), which justifies combining observations of different Galium species in a single figure (figs 3, 4).
Dissection was performed in 70% ethanol under a Wild M3 (Leica Microsystems AG, Wetzlar, Germany) stereo-microscope equipped with a cold-light source (Schott KL1500; Schott-Fostec LLC, Auburn, NY, USA). The dissected samples were treated for 30 minutes in a Branson 2210 Ultrasonic Cleaner (Branson Ultrasonics Corporation, Danbury, USA).

Scanning electron microscopy (SEM)
The samples were transferred twice to 70% ethanol for 5 minutes and then to a mixture (1/1) of 70% ethanol and DMM (dimethoxymethane) for 5 minutes, followed by 20 minutes pure DMM. Next, CO 2 critical point drying was performed with a CPD 030 critical point dryer (BAL-TEC AG, Balzers, Liechtenstein). The dehydrated samples were mounted on aluminium stubs using double sided adhesive 12 mm carbon tabs (Agar Scientific Ltd. Stansted, UK). Gold coating was done with a SPI-Module TM Sputter Coater (SPI Supplies, West-Chester, PA, USA). SEM micrographs were obtained with a Jeol JSM-6360 (Jeol, Tokyo, Japan) scanning electron microscope.

Light microscopy (LM)
Dehydration of previously dissected material was performed through a graded ethanol series. Subsequently, the samples were embedded in KULZER's Technovit 7100 (based on HEMA, hydroxyethyl-methacrylate). Seven µm thick sections were obtained using a rotation microtome Leica RM2135 with Leica DB80 disposable blades (Leica Biosystems, Amsterdam, The Netherlands). Next, the sections were stained with 0.05% toluidine blue in distilled water. LM images were obtained using an Olympus BX51 (Olympus Corporation Belgium, Antwerp, Belgium) microscope equipped with a Color View Soft Imaging System camera.

RESULTS
Our observations are arranged according to the species floral morphology, starting with the predominant morphology in the tribe of flowers with cup-shaped corolla tubes found in most species of Cruciata, Galium, and Rubia, and followed by three species from the genera Crucianella, Phuopsis, and Sherardia with tubular corolla tubes. The inflorescence in the ten species studied is essentially a thyrse, but species-specific variation occurs.

Cruciata laevipes
Inflorescence -The inflorescence has an indeterminate main axis with pairs of oppositely positioned bracts ( fig. 1A).
Within each of these bracts, two cymosely branched partial inflorescences are formed, each consisting of a terminal flower with two lateral flowers, which each may have two higher order lateral flowers. The result is a complex compound inflorescence ( fig. 2A, C).  sequently develop into two semi-circular nectaries at the top of the gynoecium. First, the carpellary bulges together with the outer whorls develop a perigynous hypanthium, creating a cavity that encloses the floral apex. This becomes an inferior bilocular ovary. Simultaneously, the central part of the adaxial zone of each of the two carpellary bulges develops into a style and stigma ( fig. 2D, E, L-N). Meanwhile, the bases of the filaments and the petals adhere, forming a tube. At the top of this tube, the filaments and distal parts of the petals, now called corolla lobes, continue elongating  Centrally in the flower, two semi-circular bulges originate, whose central parts develop into two styles that cover the underlying depression with the floral apex ( fig. 9C-E), thus closing the early inferior ovary. The bases of the two styles fuse at an early stage ( fig. 9F). The fused part subsequently elongates and develops into a long single style, topped by two very short free stigmatic lobes ( fig. 9H, I). Below these, the upper part of the style becomes fusiform (c. 1 mm long at anthesis) with large and dome-shaped epidermal cells ( fig. 9I). Meanwhile, the remaining parts of the two semi-circular bulges develop into two nectaries ( fig. 9C, E, F, H). A bilocular, inferior ovary develops, each locule containing a single anatropous, unitegmic ovule ( fig. 9D-H).
The tubular part of the corolla ( fig. 9H-L) and the style ( fig. 9H, I) elongate at the same rate. At (semi-)maturity, the tubular part of the corolla consists of a long stamen-corolla tube below the insertion points of the filaments. Additionally, a short corolla tube sensu stricto develops above the insertion points of the filaments. Consequently, the stamens are inserted somewhat below, and not in between, the bases of the corolla lobes ( fig. 9H, L). During the development of the corolla, the proportion of the tubular part above the insertion points diminishes relative to the part below ( fig. 9H, L). At anthesis, the tubular part of the corolla is 9-10 mm long, with the portion above the insertion points of the filaments being approximately 1/10 th of the total length. The filaments are inserted c. 1 mm below the level of the throat; the anthers are nearly sessile and almost completely included in the tubular part of the corolla. Until just before anthesis, the fusiform part of the style is situated at the same height as the anthers (fig. 9H, I). At anthesis, the style has elongated and protrudes for approximately half of its length above the throat ( fig. 1E).

Sherardia arvensis
Inflorescence -The inflorescence axes are terminated by a cymosely branched flower cluster consisting of a terminal flower with two bracteoles that each subtend a lateral flower ( fig. 1F). Below the terminal flower, nodes occur with two opposite bracts that are shifted 90° with respect to the upper (and lower) node, each subtending a cluster of three flowers, a terminal flower and two lateral flowers ( fig. 10A, B). Floral development -Initially, the floral apex is flat ( fig.  10C) and more or less simultaneously four free-standing petal primordia and, alternating with them, four free-standing stamen primordia appear ( fig. 10D). The growing petals cover the stamens and the floral apex ( fig. 10E). The stamens differentiate into filaments and tetrasporangiate, dorsifixed anthers ( fig. 10F-H). Basally, a stamen-corolla tube is formed, raising the filaments to a higher position ( fig. 10F, H).
At this stage, two semi-circular bulges originate, surrounding the floral apex, which now is situated in a cavity ( fig. 10F, G). The central parts of the two bulges develop into two styles (figs 10G; 11A-C), the rest into semi-circular nectaries ( fig. 11C, G), surrounding the base of the style. Simultaneously, a bilocular inferior ovary is formed (figs 10H; 11A, B). In each locule, a single ovule develops ( fig.  11A). At the top of each style, a spherical, papillose stigma is formed ( fig. 11A-C, E).
Meanwhile, irregular calyx lobes appear ( fig. 11A) with large trichomes at their margins ( fig. 11B, D, F). The stamencorolla tube elongates, lifting up stamens and petals (now corolla lobes; fig. 10H; 11A-F). The filaments are inserted on top of the tubular part of the corolla and in between the bases of the corolla lobes ( fig. 10H). This relative position of the insertion points of the filaments with respect to the corolla lobes does not change in the course of the further development. The proximal parts of the two styles fuse into a single style ( fig. 11G, H), with their distal parts forming two style branches terminated by spherical stigmas (fig. 11E, I). At (semi-) maturity, the calyx lobes have large spine-like trichomes, the portion of the corolla corresponding to the stamen-corolla tube is longer than the corolla lobes, and the stamens protrude above the throat. The two style branches and stigmas protrude above all other flower parts ( fig. 11F). The ovary is conspicuously didymous (fig. 11F). In the stamencorolla tube, the filaments cannot be distinguished from the corolla; only vascular bundles leading to the filaments are present ( fig. 11G, H).

DISCUSSION
After our earlier studies of tropical woody and herbaceous Rubiaceae (De Block & Vrijdaghs 2013;Vrijdaghs et al. 2015), we now focused on temperate species of the herbaceous tribe Rubieae (subfamily Rubioideae). We studied representatives of six (Cruciata, Crucianella, Galium, Phuopsis, Rubia, and Sherardia) out of the 12 genera in Rubieae sensu Bremer & Eriksson (2009), comparing our results with those from the floral ontogenetic literature of Rubieae. We did not include Theligonum cynocrambe L. (Rutishauser et al. 1998), the flowers of which have particular but, to our study, irrelevant characteristics (e.g., unisexuality, polyandry). Because of the derived nature of the flowers of Theligonum (Robbrecht 1993;Rutishauser et al. 1998), which raises floral ontogenetic questions that are far from the focus of the present study, we preferred to exclude comparison with the literature about Theligonum.
We test the hypothesis of Vrijdaghs et al. (2015) about the development of a tubular corolla and epipetaly in Rubieae. Because of the epigynous nature of the flowers in Rubiaceae, the development of the calyx is also considered.

Development of the calyx
The usual floral developmental sequence is: calyx -corolla -androecium -gynoecium. However, in Crucianella, Cruciata, Galium, Phuopsis, and Rubia, no calyx tube or calyx lobes develop: only a calyx rim appears between the inferior ovary and the corolla late in the floral development. Payer

Postgenital fusion or annular underlying meristems?
Payer (1857) explained the development of a tubular corolla and epipetaly in Asperula, Galium, and Rubia as the result of postgenital fusion, whereas De Block & Vrijdaghs (2013) and Vrijdaghs et al. (2015) considered underlying annular intercalary meristems sensu Leins & Erbar (2010) as the cause of the development of floral cups. We discuss the contribution of the three processes suggested by Vrijdaghs et al. (2015) and the influence of plastochrons towards the development of the corolla and androecium. Leins & Erbar (2010), originating from a circular intercalary meristem situated below one or more floral whorls. During the development of the floral cup from the underlying intercalary meristem, the whorl(s) in question are raised. In the case of a stamen-corolla tube, there is an underlying annular intercalary meristem below corolla and androecium. With the development of the stamen-corolla tube, petals and stamens are raised to a higher position. The correct interpretation of a stamen-corolla tube may be blurred by a temporal delay of the development of the tube. In some cases, the stamen-corolla tube starts developing at very early floral developmental stages, resulting in an initially conspicuously concave floral apex. Next, the petal primordia (corolla lobes) appear on the rim of the concave floral apex, followed by the stamen primordia, which appear on the adaxial side of the rim of the concave floral apex and alternate with the corolla lobes (e.g., Ixora coccinea L. in Erbar & Leins 1996; Galopina tomentosa Hochst. in Ronse Decraene & Smets 2000; Spermacoce occultiseta Harwood in Vrijdaghs et al. 2015). This corresponds to Erbar's (1991) "early sympetaly".

Development of a stamen-corolla tube -A stamen-corolla tube is an example of a floral cup sensu
Among the species studied here, only the floral development of Crucianella macrostachya and Phuopsis stylosa (figs 7; 9; 12) concurs with early sympetaly. In Rubia tinctorum and Sherardia arvensis, our observations suggest that a stamen-corolla tube is formed with some delay, which explains why initially free-standing petal and stamen primordia appear on a rather flat floral apex. However, fig. 5B and Naghiloo & Classen-Bockhoff (2016: 54, figure 3A-C) suggest that the petals are standing on an annular ring, which we think is the undeveloped stamen-corolla primordium. Erbar (1991) recognised that there are transition patterns between early and late sympetaly and, in our opinion, these correspond to cases where the development of a stamen-corolla tube is delayed. Payer (1857) explained the development of the corolla tube in Rubia by postgenital fusion, as, in his time, the idea of floral structures originating from underlying intercalary meristems was unknown. However, we have no indication of such fusion (figs 6F-H; 11G-I). Therefore, we prefer to explain the tubular part of the corolla and the epipetaly in Rubia and Sherardia by a delayed development of a stamen-corolla tube (figs 5; 10; 12). Apical view, three out of four stamens removed; stamens free-standing between corolla and carpels (corolla lobe encircled in red); anthers tetrasporangiate and introrse; centrally, two carpels (purple arrowheads) covering floral apex, each developing into semicircular nectary and independent style. C-D. Lateral view. C. Petals bladder-shaped (encircled and red arrows); anthers dorsifixed; spherical papillose stigmas (purple arrows) developing. D. Short tube below corolla lobes and free parts of filaments; inferior, bilocular ovary. E. Longitudinally opened flower, revealing single ovule per locule. F-G. Tubular zone of adherence between bases of distinguishable filaments and corolla lobes. H. Short stamen-corolla tube of six to seven cell layers thick; four staminal vascular bundles (one encircled in yellow) and tubular corolla forming continuous circular tissue; semi-circular nectaries surrounding two free styles. I-J. Inclined sections at successively higher levels. I. Filaments distinguishable from adaxial surface of tubular corolla (encircled in yellow). J. Fusion zone between basal parts of two petals and one filament (encircled in yellow); staminal vascular bundles indicated with yellow arrowheads. K. Inclined section in transition zone between tubular corolla and corolla lobes (red arrowheads); filament tissue distinguishable from corolla (encircled in upper part of picture); at level of corolla lobes, filaments free (one encircled left below). Abbreviations: a, anther; co, corolla (lobe); f, filament; hy, hypanthium; o, ovule; ov, ovary; ne, nectary; pe, petal; sg, stigma; st, style. In Cruciata and Galium, our results suggest that a stamencorolla tube of a few cell layers thick is formed quite late in the floral development (figs 2N, O; 4G-I). Naghiloo & Classen-Bockhoff (2016: 55, 56) presented slightly younger floral developmental stages in Cruciata laevipes and C. glabra in their figures 4A and 5A, B. In both stages, as well as in the slightly later stage shown in fig. 2B, no annular rim is visible. Our observations partially concur with those of Pötter & Klopfer (1987: 312) in Galium aparine, who reported that "Im weiteren Verlauf differenziert sich unter den Petalprimordien ein Meristemwulst und verbindet sie miteinander..." ["In the further course of the floral development, an annular meristem develops below the petal primordia and links them…"]. These authors interpreted this as an example of "extremely early sympetaly of the corolla". However, since individual petal and stamen primordia are present at the early developmental stages, we think that the annular meristem not only underlies the corolla but also the androecium, resulting in a stamen-corolla tube later in the floral development. Erbar & Leins (1996: 105) compared the initial floral developmental stages in species without a calyx (Cruciata, Galium, Phuopsis, and Asperula), stating that the flower primordium changes its shape from "initially hemispherical via plug-like to funnel-like", which they considered to be the first steps in the formation of the corolla tube. Based on our results, we interpret it as a stamen-corolla tube. In Cruciata and Galium, however, a large tubular part of the corolla is present at the top of the rudimentary stamen-corolla tube. This part of the tubular corolla results from a postgenital fusion/coherence of the bases of the filaments and the corolla lobes (figs 2F, G, J, O; 4F, G, I; 12E, F). The coherence of the filaments to the of filament in between corolla and early gynoecium. B. Two developing styles, each surrounded by semi-circular nectary; below, developing hypanthium (green double arrow). C. Tubular corolla (red-yellow dotted double arrow) developing below petals and insertion points of filaments, lifting them up. D. Lateral view of gynoecium; calyx rim (green arrow) in between inferior ovary and scar of corolla; two semicircular nectaries surrounding bases of styles, each with spherical, papillose stigma. E. Longitudinally opened semi-mature corolla; filaments distinguishable in tubular part of corolla; anthers tetrasporangiate and introrse. F. Stamen-corolla tube; staminal vascular bundles (two encircled in yellow) and tubular corolla forming continuous circular tissue; styles arrowed in purple. G. Slightly inclined section at height of throat; two free filaments (encircled) adjacent to only visible corolla lobe (red arrowhead); three staminal vascular bundles; styles arrowed in purple. H. Corolla lobes (red arrowheads); filaments all free (one encircled in yellow). Abbreviations: a, anther; co, corolla (lobe); f, filament; F 1 , lateral flower; ne, nectary; ov, ovary; sg, stigma; st, style. Two stamens with developing tetrasporangiate, introrse anthers; scar of filament (encircled) situated basally on tubular part of corolla; tips of corolla lobes (red arrowheads) bent inwards touching the two stigmas. E-F. Successive developmental stages; elongating tubular corolla (white double arrow) and lobes; calyx rim (green arrow) in between inferior ovary and corolla. G. Bilocular, inferior ovary; two semi-circular nectaries surrounding scar of removed styles (purple arrow); calyx rim (green arrow) more conspicuous. Abbreviations: a, anther; B, bract; Bo, bracteole; co, corolla (lobe); ne, nectary; ov, ovary; pe, petal; s, stamen; st, style; red *, apex of the inflorescence axis; white *, floral apex. Longitudinally opened flower; tips of corolla lobes developing into long cylindrical structures (red arrowheads), curved inwards and touching upper part of stigmas; two insertion points of filaments on basal tubular part of corolla encircled in yellow; one of two styles with papillose stigma (purple arrow). B. Longitudinally opened corolla; filament (encircled in yellow) inserted at top of stamen-corolla tube (red-yellow dotted double arrow). C. Successive stage; two styles differing in length, each surrounded by semi-circular nectary (purple arrow); below, two partially opened locules separated by massive septum (encircled in purple), each with single ovule. D. Upper part of elongated stamen-corolla tube, showing equally long anthers and cylindrical tips of corolla lobes (red arrowheads); staminal vascular bundles forming longitudinal thickenings in stamen-corolla tube. E. Flower with two bracteoles; calyx rim (green arrow) in between inferior ovary and corolla. F. Idem as E, perpendicular orientation, showing bracteoles and bract subtending flower. G. Upper half of semi-mature flower. H. Node and part of inflorescence axis (white arrow) with two opposite bracts, fused at base (encircled in green); each bract subtending semi-mature flower with two bracteoles (green arrows). I. Stamen-corolla tube; staminal vascular bundles (yellow arrowheads) and tubular corolla forming continuous circular tissue; anthers dorsifixed, tetrasporangiate and introrse; stigmatic lobes covered by large papillae. J. Corolla tube sensu stricto at the height of insertion points (encircled in yellow) of filaments; filament at the left free from corolla; centrally, circular sections of tips of corolla lobes (red arrowheads). K. Corolla tube sensu stricto above insertion points of filaments. L. Upper part of developing flower; filament inserted at top of stamen-corolla tube (encircled in yellow). M. Detail of encircled part of L; stamen-corolla tube indicated by red-yellow dotted double arrow. N. Developing flower; centrally, styles/stigmas with different lengths (purple arrows); cylindrical tips of corolla lobes (red arrowheads) touching highest stigma; below, locule with developing anatropous unitegmic ovule (encircled in purple). O. Detail of encircled part of N, with micropyle (arrow). P. Micropyle (purple arrow) and embryo sac (encircled); hypanthium consisting of outer and inner layer (green arrow). Abbreviations: a, anther; B, bract; Bo, bracteole; co, corolla (lobe); f, filament; ne, nectary; ov, ovary; sg, stigma; st, style.
► Figure 9 -SEM of the floral development in Phuopsis stylosa. A-D. Apical view of successive developmental stages. A. Young flower with two bracteoles; five free-standing petal primordia (red arrowheads) and, alternating with them, five stamen primordia (yellow arrowheads), surrounding slightly concave floral apex (white asterisk). B. Developing petals and stamens; floral apex concave. C. Scar of stamen-corolla tube with two staminal vascular bundles (yellow arrowheads); centrally, two semi-circular bulges developing into nectaries, their central parts forming two styles. D. Terminal flower with two bracteoles (Bo), each subtending lateral flower (F 1 ); each lateral flower with two second order bracteoles (green arrowheads); stamens inserted on adaxial surface of stamen-corolla tube, differentiating into filament and anther; centrally, two developing styles (purple arrowheads). E. Successive stage; longitudinally opened flower; stigmas appearing at top of styles (purple arrowheads). F. Apical view of base of stamen-corolla tube with five staminal vascular bundles (yellow arrowheads); centrally, semi-circular nectaries surrounding scar of common base of fused styles (encircled in purple). G-L. Lateral view. G. Bilocular, inferior ovary with one locule opened, showing single anatropous ovule; calyx rim (green arrow) in between inferior ovary and corolla. H. Longitudinally opened flower; tubular part of corolla (white double arrow) consisting of stamen-corolla tube (red-yellow dotted double arrow), and, above insertion points of filaments (yellow arrow), corolla tube sensu stricto (red double arrow); anthers dorsifixed, tetrasporangiate and introrse; pollen presenter starting to differentiate. I. Longitudinally opened flower at later stage; pollen presenter (encircled in purple) positioned in between anthers; stigmas indicated by purple arrowheads. J-L. Elongation of tubular part of corolla (white double arrow) and corolla lobes. J-K. Abaxial view, successive stages; calyx rim (green arrow) appearing after development of inferior ovary (coloured green). L. Longitudinally opened flower; tubular part of corolla (white double arrow) consisting of long stamen-corolla tube (red-yellow dotted double arrow), and, above insertion points of filaments, shorter corolla tube sensu stricto (red double arrow). Abbreviations: a, anther; Bo, bracteole; co, corolla (lobe); f, filament; F l , lateral flower; ne, nectary; o, ovule; ov, ovary; pe, petal; s, stamen; sg, stigma; st, style; white *, floral apex. corolla gradually becomes looser towards the distal part of the tube, eventually resulting in free filaments.

Development of a corolla tube sensu stricto -A corolla
tube sensu stricto is the part above the insertion points of epipetalous stamens, or, in case the stamens are inserted on the hypanthium, is the tubular portion of the corolla. The latter case is not present in the species studied here. The presence of a corolla tube sensu stricto above a stamen-corolla tube results in the insertion of the epipetalous stamens below the level of the throat. This can be seen in Phuopsis stylosa (figs 9H; 12C) and, to a lesser degree, in Crucianella macrostachya (figs 8J, K; 12C). In the other species studied, no corolla tube sensu stricto was observed.
Postgenital fusion -We follow Endress (2006;2019), who equated postgenital fusion to epidermal fusion after the independent origin of the fusing structures. In contrast, postgenital coherence may also result from a fusion of parts of floral organs through the secretion of mucus (Endress 2006). Postgenital fusion/coherence between the proximal parts of the petals and the filaments was only observed in Cruciata and Galium. It occurs in combination with a late appearance of a rudimentary stamen-corolla tube (figs 2F, G, J, N, O; 4G, F, I-K; 12E, F), which is only visible using LM because the stamen-corolla tube is only a few cell layers thick. In contrast, the part of the tubular corolla resulting from postgenital coherence is considerable, which explains that the insertion of the free parts of the filaments in the tubular corolla is high above the stamen-corolla tube. This suggests a kind of pseudo-epipetaly (figs 2E-G, J; 4F, G, J; 12E, F) corresponding to 'false sympetaly' sensu Weberling (1992), albeit that the fusion occurs simultaneously between members of the same whorl, such as petals, and members of another, adaxially situated whorl, for example the filaments. In Galium, above the insertion points of the filaments, the tubular fusion zone continues with a short tube formed by the fused margins of the corolla lobes ( fig. 4G), which suggests a pseudo-corolla tube sensu stricto.

Plastochron variation
Extending the definition of the term plastochron "the time interval between the successive similar developmental events" of Henderson's dictionary of biological terms (Lawrence 1996: 446) to floral apices, we define a plastochron as the time interval between the successive appearances of two floral organs or of two floral whorls (assuming that the floral organs in a whorl originate simultaneously).
Several examples of the influence of plastochrons in the floral development can be given: 1) Payer (1857) already interpreted the quasi absence of a calyx in Asperula, Galium, and Rubia as a delay of the calyx development (however, also questioning whether there is a calyx at all). The appearance of a rudimentary calyx very late in the floral development can be considered as a negative plastochron with respect to the corolla/androecium (sequence switch of appearance of corolla/androecium and calyx). The delayed development of the calyx in Sherardia can also be considered as an example of a negative plastochron. 2) The more or less simultaneous appearance of petal and stamen primordia in Cruciata, Galium, Phuopsis, and Sherardia suggests a reduction of the plastochron between corolla and androecium. This was also observed for Cruciata laevipes and C. glabra (Naghiloo & Classen-Bockhoff 2016). The simultaneous initiation of the corolla and androecium may be a first step towards the development of a stamen-corolla tube or may be a remnant of a stamen-corolla tube that was lost in the course of evolution, unless postgenital fusion of initially free filaments and petals occurs as in Cruciata laevipes and Galium. 3) We interpret the late development of a stamen-corolla tube in Cruciata and Galium as the result of a large plastochron between the more or less simultaneous appearance of free petals and stamens (figs 2; 3) and the initiation of the stamen-corolla tube (figs 2N; 4H, I).
Another example of the importance of plastochrons is the andromonoecy in Cruciata glabra, explained by a larger than usual plastochron between the initiation of the corolla/ androecium and the gynoecium (Naghiloo & Classen-Bockhoff 2016). Four free-standing petal primordia and, alternating with them, four free-standing stamen primordia (yellow arrowheads). E. Developing petals and stamens. F. Longitudinally opened flower, perpendicular orientation to G and corresponding to section in between semi-circular bulges, but at younger stage; bulges just appearing, only one visible and encircled; floral apex situated in depression in between bulges; filaments inserted on early stamen-corolla tube (yellow arrows). G. Floral apex enclosed by semi-circular bulges (at 90° with respect to F; purple arrowheads), differentiating into nectaries and styles. H. Lateral view of subsequent stage; filaments inserted on stamen-corolla tube; stamens differentiating into filaments and anthers; calyx rim (green arrow) in between inferior ovary and corolla. Abbreviations: a, anther; B, bract; Bo, bracteole; co, corolla (lobe); f, filament; F 1 , lateral flower; F t , terminal flower; ov, ovary; pe, petal; s, stamen; white *, floral apex.

Gynoecial development
In all species studied, the development of the gynoecium concurs with the description of Payer (1857), although we interpret the carpels sensu Endress (2019). Moreover, with regard to the development of the styles, we need to nuance Payer's (1857: 634) statement that the initial, semi-circular bulges "immediately become connate". This is not the case in Crucianella macrostachya, Cruciata laevipes, and Galium, where the two styles remain separate throughout the floral development (figs 2J, M-O; 3I; 4A-I, F; 7D; 8C, I, J). In flowers of Rubia tinctorum, the two styles become loosely fused at the base ( fig. 6F), and, in Phuopsis stylosa and Sherardia arvensis, the bases of the two styles fuse postgeni-tally early in the development to further develop into a single style, with distally two style branches (figs 9F, I; 11F-H).
The style of Phuopsis stylosa differs from that of all other Rubieae studied in that the distal part, just below the small stigma lobes, develops into a pollen presenter (figs 1E; 9H, I). Phuopsis stylosa is the only species in the tribe Rubieae and even in the subfamily Rubioideae showing secondary pollen presentation (Puff et al. 1996), a reproductive strategy promoting outbreeding that involves protandry. Protandry is a form of dichogamy (the separation in time of gender expression in a bisexual flower) in which the anthers release the pollen before the stigma becomes receptive. Pollen are relocated from the anthers onto a pollen presenter, in Phuopsis stylosa in the form of the swollen distal part of the style with Calyx lobes developing from calyx rim (green arrowheads); stamen-corolla tube and corolla lobes elongating, lifting up stamens; bases of two styles postgenitally fusing, each topped by spherical, papillose stigma (purple arrowheads); bilocular, inferior ovary with single ovule per locule. C. Elongating stamen-corolla tube (red-yellow dotted double arrow); free parts of filaments elongating (yellow double arrow); common base of style surrounded by two semi-circular nectaries; calyx indicated with a green arrow. D. Abaxial view of flower cluster; inferior ovaries and developing calyx lobes (green arrow) coloured in green; elongating tubular part of corolla indicated by white double arrow. E. Distal part of longitudinally opened flower; longitudinal thickenings in stamen-corolla tube corresponding to staminal vascular bundles (yellow arrowheads); stigmas (purple arrowheads) and anthers reaching level of throat. F. Cluster of three flowers at anthesis with spiny calyx lobes (green arrowheads); stamens exserted from corolla tube; elongated styles now protruding far above anthers; inferior ovary didymous. G. Basal part of stamen-corolla tube, semi-circular nectaries, and common base of style; three out of four staminal vascular bundles encircled in yellow. H. Idem as in G, more distally; staminal vascular bundles (encircled in yellow) and tubular corolla forming continuous circular tissue; fused styles just below separation of style branches (purple arrow). I. Corolla lobes; edges of adjacent corolla lobes encircled in red; opposite them, three out of four free filaments (yellow arrowheads); free style branches (purple arrows). Abbreviations: a, anther; co, corolla (lobe); f, filament; ne, nectary; o, ovule; ov, ovary; sg, stigma; st, style. Flowers with rudimentary stamen-corolla tube below corolla tube sensu stricto; stamens inserted at top of stamen-corolla tube. C. Flowers with well-developed stamen-corolla tube and corolla tube sensu stricto; stamens inserted higher on tube as in B but below bases of corolla lobes. D. Flowers with well-developed stamen-corolla tube and no corolla tube sensu stricto; stamens inserted at top of stamen-corolla tube in between bases of corolla lobes. E. Flowers with rudimentary stamen-corolla tube below fusion zone of proximal parts of corolla lobes and filaments; filaments inserted at top of rudimentary stamen-corolla tube but adhering to fusion zone, becoming free between bases of free parts of corolla lobes. F. Idem as in E, but fusion zone of only corolla lobes occurring above insertion points of free parts of filaments. Colour code: blue, fusion zone; grey, schematic representation of flower parts including pedicel and bracteoles; bold red, corolla tube sensu stricto; red, (free parts of) corolla lobes; yellow, stamens; red-yellow, stamen-corolla tube. Abbreviations: Bo, bracteole; ov, ovary. Names in brackets were studied in Vrijdaghs et al. (2015).
large dome-shaped epidermal cells. At anthesis, the flower opens and the pollen presenter becomes exposed (functionally male stage). Later in the development, the stigmatic lobes become receptive for pollen from other plants (functionally female stage). The release of pollen occurs in the closed bud, with pollen being deposited on the pollen presenter. Nectary -In all species studied, a nectary disk, consisting of several parts, develops on the top of the ovary, surrounding the style(s). This concurs with what is found in most representatives of the asterids where gynoecial nectaries can be considered as a synapomorphy (Smets 1988;Smets & Cresens 1988;Bernardello 2007: 86). The development of the nectary occurs rather early in the species studied of Cruciata, Galium, Phuopsis, and Rubia. This, in combination with a reduced or delayed development of the calyx, suggests neoteny. Naghiloo & Classen-Bockhoff (2017) performed an extensive study on heterochronic changes, in particular sequence heterochrony sensu Smith (2001), in flowers of Dipsacoideae (Caprifoliaceae), concluding that the interaction between timing (initiation of floral organs) and space plays an important role in the diversification of flowers. Our previous and present studies of the perianth in Rubiaceae lead to a similar conclusion.

Homology
According to the conceptual framework of Ochoterena et al. (2019) for morphological homology assessments, the common structural origin of plant parts determines their nature, based on which a specific plant part can be assigned to a particular character such as stem, leaf, sepal, petal, etc. The developmental pathways leading to a specific mature plant part in different species may differ, resulting in different character states of the character to which the plant part has been assigned. In the case of a tubular corolla, the structural origin can be a meristem developing into a corolla tube sensu stricto or into a stamen-corolla tube. The latter actually consists of the so-called congenitally fused meristems of the petals and stamens. The fact that in a stamen-corolla tube, the corolla and androecium initially develop together, does not mean that the meristems from which the respective corolla and androecium parts of a stamen-corolla tube originate would be different from the meristems of a corolla and androecium that develop separately. Therefore, we consider meristems resulting in a corolla tube sensu stricto or in the corolla part of a stamen-corolla tube as essentially the same. In the same way, the meristems that give rise to independent stamens are the same as the meristems that develop the stamen parts of a stamen-corolla tube. Moreover, postgenital fusion and plastochron variation do not affect the origin of the tubular corolla. Consequently, we think that all different morphs of tubular corollas in the species studied should be considered to be character states of the character 'tubular corolla'. Similarly, whether the stamens are epipetalous or not does not affect their morphological origin. In fig. 12, a theoretical outline of all kinds of floral cups consisting of corolla and androecium, observed in Rubioideae species in this study and in Vrijdaghs et al. 2015, is presented. In subfamily Ixoroideae, De Block & Vrijdaghs (2013) observed that the development of the tubular corolla in Canephora concurs with fig. 12C. Erbar & Leins (1996) reported the development of a long tubular corolla in Ixora, consisting mainly of a stamen-corolla tube, which would also correspond to fig.  12C. We think that the "initial corolla ring primordium" observed by Erbar & Leins (1996: 105) in Ixora coccinea L. is rather an annular primordium of the stamen-corolla tube.

Additional observation
In Galium aparine, G. glaucum, G. odoratum, and G. verum, the petals are bladder-shaped in early developmental stages (G. pumilum not observed). Adaxially, there is a one-layered epidermis that appears to be free from the rest of the tissue of the petal ( fig. 4A, C), suggesting a cavity. We have not observed such a phenomenon in any other species of the Rubiaceae. Later in the development, the petals/corolla lobes become more flattened.

CONCLUSION
The developmental hypothesis of Vrijdaghs et al. (2015) for the formation of the tubular corolla and the often related epipetaly in Rubiaceae is confirmed. In addition, the current study shows the importance of plastochron variation. A lengthening or shortening of the time interval between the successive appearance of floral whorls/floral developmental events or sequence heterochrony results in important variation: e.g., 1) Reduction of the time interval between the appearance of corolla and androecium, in extremis leading to a simultaneous appearance of both whorls (shortened plastochron); 2) Extension of the time interval between the appearance of corolla and stamen-corolla tube, resulting in a delayed development of the stamen-corolla tube (lengthened plastochron); and 3) Sequence switch of the appearance of corolla and calyx resulting in a negative plastochron with respect to the corolla, causing delay of the development of the calyx or, in extremis, inhibiting its development completely.
The development of a stamen-corolla tube, a corolla tube sensu stricto, postgenital fusion between petals/corolla lobes and filaments, and plastochron variation are the four processes that determine the shape of the corolla and the presence (or not) of epipetaly in all Rubiaceae examined in this and previous studies. The proportion in which each of these processes contributes to the final shape of the corolla causes the morphological variability of the corolla and the androecium among the species.