Anatomy of the floral nectaries of selected species of Gardenieae (Rubiaceae)

Abstract

Rubiaceae is one of the five most species-rich families of Angiosperm, its flowers are characterized by a nectariferous disc surrounding the base of the style; however, there are almost no studies on anatomy of these structures in the family. The aim of this study was to describe for the first time the anatomy of the floral nectaries in the tribe Gardenieae (Rubiaceae). Flowers from Cordiera concolor, Genipa americana, Randia calycina, Randia ferox, Randia heteromera, Randia micracantha, and Tocoyena formosawere collected, photographed, and processed using conventional techniques for observation with optical and scanning electron microscopies. In all species the nectary is differentiated histologically into epidermis, nectary and sub-nectary parenchyma, and vascular bundles. Nectar is exuded via nectarostomata. The nectaries have reproductive function, and are structural and mesenchymal during the floral stage. Whereas after corolla has fallen they are persistent nectaries, with post-floral secretion and extranuptial function. In addition, floral visitors that appeared on open flowers and after corolla fall were recorded.

Keywords:
floral structure; nectaries; post-floral secretion; Rubiaceae

Resumo

Rubiaceae é uma das cinco famílias mais ricas em espécies de Angiospermas, suas flores são caracterizadas por um disco nectarífero ao redor do estilete. No entanto, quase não há estudos sobre a anatomia destas estruturas na família. O objetivo deste trabalho foi descrever pela primeira vez a anatomia dos nectários na tribo Gardenieae (Rubiaceae). Flores de Cordiera concolor, Genipa americana, Randia calycina, Randia ferox, Randia heteromera, Randia micracantha e Tocoyena formosa foram coletadas, fotografadas e fixadas. Os nectários foram processados utilizando técnicas convencionais de observação em microscopia óptica e microscopia eletrônica de varredura. Em todas as flores o nectário é histologicamente diferenciado em epiderme, parênquima nectarífero, parênquima subnectarífero e traços vasculares. O néctar é exsudado através de estômatos. Os nectários tem função reprodutiva e são estruturados e mesenquimais na fase floral. Após a queda da corola são nectários persistentes com secreção pós-floral e função extranupcial. Também foram registados os visitantes florais que apareceram em flores abertas e após a queda da corola.

Palavras-chave:
estrutura floral; nectarios; secreção pós-floral; Rubiaceae

Introduction

Over time, numerous classifications have been proposed to characterize and typify nectaries, some were based on location as floral nectary (FN) and extrafloral nectary, according to whether the nectary is in the flowers or in the vegetative organs respectively ( Caspary 1848). Schmid (1988) used the terms reproductive nectary for those located in flowers, fruits, inflorescences, bracts and pedicels, and extra-reproductive nectary when located in strictly vegetative organs. Nectaries are also classified according to their function as nuptial nectary, for those that participate in the pollination process, and extranuptial nectary, for those that are not involved with pollination ( Delpino 1868–1874) but also maintain an anti-herbivore community in the plant that is responsible, for example, for reducing predation on flower buds ( Rivera 1997).

In the case of the FN, the persistence of the floral parts on which they are placed is also considered in the classification, so those FN associated with floral parts that fall after fertilization ( e.g., stamens, petals) are called deciduous nectaries, while persistent nectaries are those associated with floral parts that do not fall after pollination ( e.g., receptacle, ovary) and during fruit differentiation ( Smets 1968, 1988; Smets & Cresens 1988). Persistent nectaries may remain intact in the flower without producing any nectar or they may have post-floral secretion for a period of time after anthesis or during fruit differentiation, in which case there are authors who consider that they would have a role in protecting the development fruit against predators ( Santos & Del-Claro 2001 ; Amorim & Oliveira 2006; Falcão et al. 2014 ). Among the persistent nectaries are the so-called pericarpial nectaries, which are formed after fertilization ( e.g., in Erythrina speciosa Tod., Paiva 2009).

Regardless of the position on the plant, other classifications consider the anatomical structure and mode of nectar secretion: non-structural nectaries do not present a structure different from that of the neighboring tissues, contrasting with structural nectaries ( Zimmermann 1932). Based on anatomical features, Vogel (1977) proposed the terms mesenchymal nectary, which consists of glandular and storage tissues that normally secrete nectar in interstitial spaces or in the apoplast of the cells which is then exuded through stomata; epithelial nectary, consisting of a permeable glandular epidermis, often with an underlying glandular tissue; and trichomatic nectary, when the secretion is released by glandular trichomes. Moreover, Fahn (1979) described that nectaries were generally formed by epidermis and parenchymatous tissue (with vascular bundles), while Durkee (1983) considered three tissues: epidermis, secretory tissue (parenchymal tissue that separates epidermis from vascularized tissue), and subglandular parenchyma with vascular bundles (as synonymous to Fahn’s parenchymatous tissue). Finally, Nepi (2007) proposes the terms epidermis, nectary parenchyma, and sub-nectary parenchyma with vascular bundles. The modified, usually open and anomocytic, stomata were called first nectarthodes by Schmid (1988) and later, nectarostomata by Ronse De Craene & Smets (1991).

It is common the presence of floral nectaries in plants that are pollinated by insects or birds ( Fahn 1982), in which these secretory structures are involved in the pollination process. The floral nectaries can be found in almost all parts of flower (sepal, petal, stamen, receptacle, ovary, and style) and they can produce a wide chemically variety of sweetened exudates ( Fahn 1982; Bernardello 2007; Tölke et al. 2019 ). In addition, the morphology and location of nectaries can be useful for establishing relationships between taxa in systematic treatments ( Fahn 1982; Bernardello 2007; Nepi 2007; Almeida et al. 2013 ; Tölke et al. 2018 ; Phukela et al. 2020 ).

Rubiaceae is one of the five most species-rich families of Angiosperms (up to 13,000 species), divided into three subfamilies (Cinchonoideae, Ixoroideae, and Rubioideae), 40 tribes, and ca. 620 genera ( Govaerts et al. 2020 ). This family have flowers with tetracyclic arrangement, predominantly actinomorphic symmetry, the stamens are epipetalous and in the same number as the number of corolla lobes (the filaments are fused with the corolla-tube), and the ovary is almost always inferior; also, a nectary disc (ND) surrounding the basis of the style with nectarostomata is almost universally present ( Robbrecht 1988; Simpson 2010). Despite the uniformity of traits of floral nectaries in the family, the studies in Rubiaceae have mostly focused on nectar composition ( Freeman et al. 1991 ), or on ecological aspects of the nectar ( Falcão et al. 2014 ; Amorim & Oliveira 2006; Salinas-Esquivel et al. 2013 ; Queiroga & Moura 2017; Avila Jr. & Freitas 2011). There are few studies that describe their anatomy and these are mainly focused on species of the tribe Spermacoceae (subfamily Rubioideae, Galati 1991; Galetto 1998; Florentín et al.2016 ).

In the tribe Gardenieae (Subfamily Ixoroideae), anatomical studies have been carried out on different organs such as leaves, stems, and colleters ( Koek-Norman 1972; Dave et al. 1987 ; Coelho et al. 2006 ; Erbano & Duarte 2010; Judkevich et al. 2015 ; Miguel et al. 2016 ); however, studies describing the anatomy of the floral nectaries of this tribe are still unknown. In the Neotropics, Gardenieae comprises 18 woody genera, among which there are dioecious and monoecious species. Here we studied the ND of seven Neotropical species of this tribe: Tocoyena formosa (Cham. & Schltdl.) K. Schum, Cordiera concolor (Cham.) Kuntze, Genipa americana L., Randia calycina Cham, Randia ferox (Cham. & Schltdl.) DC, Randia micracantha (Lillo) Bacigalupo, and Randia heteromera Judkevich & R.M. Salas. The only monoecious species with monoclinous flowers is T. formosa, while the other species are dioecious with diclinous flowers ( Judkevich et al.in press ).

Considering all this, the objective of the present study was: 1) to perform an anatomical analysis of the floral nectaries of seven selected species of Gardenieae; 2) to compare the morphology of nectaries between the nectaries of pistilate and staminate flowers in the dioecious taxa; and 3) compare anatomically with the nectaries of other species of the family.

Material and Methods

Material from Tocoyena formosa, Cordiera concolor, Genipa americana, Randia calycina, Randia ferox, Randia micracantha, and Randia heteromera were collected and photographed in the field. Voucher specimens were deposited in the herbarium Carmen Cristóbal (CTES) of the Instituto de Botánica del Nordeste, Corrientes, Argentina (voucher numbers in the Appendix S1, available on supplementary material < https://doi.org/10.6084/m9.figshare.20233767.v1>). Flowers were collected in the field and preserved in formalin-acetic acid-alcohol (5 mL formalin, 5 mL acetic acid, and 90 mL 70% ethanol; Johansen 1940).

Using a digital caliber (Wembley 5940) 20 flowers of each species (of the material collected in different localities, see Appendix S1, available on supplementary material < https://doi.org/10.6084/m9.figshare.20233767.v1>) were measured and calculated mean values from: the total length of the flower, the diameter of the ovary (including the hypanthium), and the diameter of the nectary. In addition, to know the average number of nectarostomata per nectary in each species, the nectarine epidermis was dissociated from the nectary with sodium hypochlorite and then stained with safranin ( D’Ambrogio de Argüeso 1986). The total number of nectarostomata was counted with a Leica DM LB2 (Leica Microsystems) light microscope.

To light microscopy (LM) analysis, five anthetic flowers were taken from two random specimens of each species fixed. The nectaries removed from these flowers were dehydrated in an ascending series of alcohols and embedded in paraffin ( Johansen 1940; modified by Gonzalez & Cristóbal 1997). The samples were cut into 12 μm sections using a Microm HM350 rotary microtome (Microm International, Walldorf, Germany), stained with safranin and Astra blue ( Luque et al. 1996 ) and mounted in synthetic Canada balsam. Observations and digital images were made using a Leica DM LB2 (Leica Microsystems) light microscope equipped with a Leica DATA digital camera in the plant anatomy laboratory of the Instituto de Botánica del Nordeste. Nepi’s terminology was followed to describe the anatomy of the nectaries of Gardenieae ( Nepi 2007).

For scanning electron microscopy (SEM) nectaries from fixed anthetical flowers were dehydrated in an increasing acetone series and then critical point dried using liquid CO ₂ (Denton Vacuum, DCP-1, Pleasanton, NJ) and sputter- coated with gold-palladium (Denton Vacuum, Desk II, Pleasanton, NJ). The samples were analyzed with a Jeol LV 5800 (JEOL, Tokyo, Japan) at 10 kV in the Service of Electron Microscopy facility at the Universidad Nacional del Nordeste.

Results

Floral morphology

The flowers respond to the typical characteristics of the family, they are actinomorphic, the calyx is synsepalous, the corolla is tubular sympetalous, haplostemonous, and the ovary is inferior ( Fig. 1). Only the anthers of the monoclinous flower of Tocoyena formosa and of the staminate flowers of the dioecious species produce pollen. The anthers of the pistilate flowers are sterile and indehiscent, and thus are staminodes. The gynoecium consists of a style, a stigma, and an ovary that has ovules in the monoclinous and pistilate flowers, while in the staminate flowers a poorly developed carpelode was formed with rudimentary ovules (composed only by undifferentiated masses of parenchyma). There are variations in the length of the flowers, in the number of floral organs (perianth and stamens), and in the color of the corolla among species ( Fig. 1; Tab. 1). Staminate flowers have smaller hypanthium ( Fig. 1b,f,j,l) then pistilate flowers ( Fig. 1c,e,g,k,m).

Floral nectaries

All species have a ring-shaped floral nectary on top of the inferior ovary, surrounding the base of the style ( Figs. 2- 4). The nectary disc is green or yellowish, glabrous ( Figs. 2; 4).

In the dioecious species, the pistilate flowers have an ovary of larger diameter than the staminate flowers; the ND is proportionally larger in most of the pistilate flowers ( Figs. 1a,c,e,g,i,m; 3b-e,g). Randia micracantha is the only species with the opposite relation of size ( Fig. 3f). In Tocoyena formosa ( Fig. 3a) and in both types of Genipa americana flowers ( Fig. 3c), the surface of the ND is markedly depressed in the direction of the style base. In the other species the surface is generally convex and may have a slight inclination towards the style ( Fig. 3b,d-g). On the other hand, the ND of both types of flowers in G. americana and the staminate flowers of R. calycina and R. ferox are smoothly undulated in its outer surface ( Fig. 4f-g,i,l,p) as result of the anthers pressure on the disc during flower development (personal observation).

A smooth cuticle covers the nectaries (except in R. ferox which is striated, Fig. 4n) with no sign of rupture ( Fig. 4b,e,h,k,n,q,t). The

Figure 1 –
a-m. Flowers of the analyzed species of Gardenieae – a. monoclinous Tocoyena formosa (⚥); b-m. diclinous species, staminate (♂) and pistilate (♀) respectively – b-c. Cordiera concolor; d-e. Genipa americana; f-g. Randia calycina; h-i. Randia ferox; j-k. Randia micracantha; l-m. Randia heteromera. Scale bar: a, d-e = 1 cm; b-c, f-m = 0.5 cm.

Table 1 –
General features of the flowers of the analyzed Gardenieae species. Simbols: ⚥ = monoclinous flower; ♀ = pistilate flower; ♂ = staminate flower.

Species	Flower length (mm)	Ovary width (mm)*	Number of pieces of perianth and androecium	Corolla color
Tocoyena formosa	⚥: 100-120	⚥: 5	5-merous	yellowish
Cordiera concolor	♀: 5-6	♀: 2.5	4-merous	white
	♂: 5-6	♂: 1.4
Genipa americana	♀: 38-50	♀: 13.0	5–6-merous	white
	♂: 35-40	♂: 7.0
Randia calycina	♀: 27-32	♀: 3.7	5-merous	white
	♂: 16-20	♂: 2.0
Randia ferox	♀: 40-50	♀: 8.0	5-merous	white
	♂: 40-50	♂: 3.5
Randia heteromera	♀: 20-27	♀: 3.0	5–6(–7)-merous	white
	♂: 20-23	♂: 1.5
Randia micracantha	♀: 10-13	♀: 2.5	5-merous	yellowish green
	♂: 10-14	♂: 2.0

*
• As the ovary is inferior, these values include the hypanthium.

Figure 2 –
a-c. Position of the nectary in the flower (longitudinal sections of the staminate flower of Randia micracantha in which the nectary can be seen surrounding the base of the style) – a. anthetic flower; b. detail of the nectary area from a fresh flower; c. detail of the nectary area, with light microscopy. Abbreviations: an = anther; ca = calyx; co = corolla; ne = nectary; ov = ovary; sg = stigma; st = style. Scale bar: a, b = 1 mm; c = 100 μm.

nectar is secreted through nectarostomata ( Fig. 4b,e,h,k,n,q,t), mostly distributed at the apex of the nectary and on the surface close to the style base. The biggest nectaries present a large number of nectarostomata ( Tab. 2), therefore the pistilate flowers of dioecious species present more nectarostomata than the staminate flowers (except in R. micracantha).

Anatomically, the ND is formed by epidermis and nectary and sub-nectary parenchyma ( Figs. 5- 6). The epidermis is glabrous and covered by a cuticle that is remarkably thick ( Figs. 5b,f-g,l; 6c,g,k,o). The nectarostomata are anomocytic and are at the same level as the surrounding epidermal cells or slightly elevated, but they may be sunken in Randia ferox ( Fig. 6g). In all cases they are open and have small sub-stomatic chambers ( Figs. 5b,f-g,l; 6c,g,k,o).

The nectary parenchyma is composed of several layers of small cells, polygonal or circular in section, with dense cytoplasm, thin walls, and a conspicuous nucleus ( Figs. 5a-b,d-e,i-k; 6a-b,e- f,i-j,m-n). There are druses in all species, except in Randia heteromera, and cells with phenolic compounds only in C. concolor.

Sub-nectary parenchyma presents larger and more laxly arranged cells than those of nectary parenchyma. This tissue is vascularized by small vascular bundles formed by xylem and phloem, which branch from the nearby hypanthium bundles ( Figs. 5c,h,m; 6d,h,l,p). Except for R. heteromera, in this tissue the rest of the species show druses randomly distributed ( Figs. 5c,h,m; 6d,h,i,p). Cells with phenolic compounds are abundant and randomly distributed in C. concolor( Fig. 5e,h) but are rare in T. formosa( Fig. 5c), and absent in the nectaries of the remaining species (in G. americana they are found in other organs of the flower -calyx, corolla, anthers, and ovary-).

In the species analyzed, differences were found in the shape of the epidermal cells (polygonal, rectangular, and quadrangular), in

Figure 3 –
a-g. Schematic median longitudinal sections of the base of the flowers showing the position of nectaries (in yellow) in monoecious and dioecious species of Gardenieae – a. Tocoyena formosa; b. Cordiera concolor; c. Genipa americana; d. Randia calycina; e. Randia ferox; f. Randia micracantha; g. Randia heteromera. Simbols: ⚥ = monoclinous flower; ♀ = pistilate flower; ♂ = staminate flower. Abbreviations: ca = calyx; co = corolla; ne = nectary; ov = ovary; st = style. Scale bar: 1 mm.

the number of nectarostomata, and between the proportion of the nectary parenchyma and the sub-nectary parenchyma, in T. formosa and C. concolor the proportion of nectary parenchyma is higher than that of sub-nectary parenchyma, in R. micracantha the opposite occurs, while in the remaining species both tissues are found in similar ( Tab. 2).

Figure 4 –
Surface views of nectariferous disc (a, c-d, f-g, i-j, l-m, o-p, r-s, with LM) and details of nectarostomata (b, e, h, k, n, q, t, with SEM) in monoecious and dioecious Gardenieae species – a-b. Tocoyena formosa; c-e. Cordiera concolor; f-h. Genipa americana; i-k. Randia calycina; l-n. Randia ferox; o-q. Randia micracantha; r-t. Randia heteromera. White arrows indicate the base of the style (removed). Arrowhead points to the undulations on the surface of the nectary produced by the pressure of the anthers during flower development. Simbols: ⚥ = monoclinous flower; ♀ = pistilate flower; ♂ = staminate flower. Scale bar: a, f-g, i-j, l-m, o-p, r-s = 1 mm; b, e, h, k, n, q, t = 20 μm; c-d = 0.5 mm.

Table 2 –
Diameter, epidermal cell shape, number of nectarostomata, and proportion of parenchymatic tissues of the nectaries in the analyzed Gardenieae species. Simbols: ⚥ = monoclinous flower; ♀ = pistilate flower; ♂ = staminate flower. Abbreviations: nt = nectarostomata; np = nectary parenchyma; sp = sub-nectary parenchyma.

Species	Nectary diameter (mm)	Epidermal cell shape in in longitudinal section of the nectary	N° of Nt on the nectary	Relative proportion of parenchymatic tissues
Tocoyena formosa	⚥: 2.5	Polygonal	⚥: 143	np > sp
Cordiera concolor	♀: 0.9	Polygonal or rectangular (elongated in the longitudinal axis)	♀: 31	np > sp
	♂: 0.7		♂: 30
Genipa americana	♀: 6.6	Rectangular	♀: 2133	np = sp
	♂: 4.5		♂: 1752
Randia calycina	♀: 2.7	Polygonal or rectangular (elongated in the longitudinal axis)	♀: 221	np = sp
	♂: 2.1		♂: 123
Randia ferox	♀: 4.6	Polygonal or rectangular (elongated in the longitudinal axis)	♀: 628	np = sp
	♂: 3.4		♂: 38
Randia heteromera	♀: 2.3	Polygonal or quadrangular	♀: 362	np = sp
	♂: 1.7		♂: 279
Randia micracantha	♀: 1.4	Polygonal or rectangular (elongated in the transverse axis)	♀: 89	np < sp
	♂: 2.5		♂: 152

Field observations

In all analyzed species the floral nectaries are active with evident secretion and visited by bees, butterflies, and moths when the flower is open ( Fig. 7a-c). These nectaries are also visited by bees and ants (the latter more frequently) after the fall of the corolla in flowers of all taxa ( Fig. 7d-l), and even during the first stages of fruit differentiation ( Fig. 7j).

Discussion

Brown (1938) and Robbrecht (1988) mentions as an almost universal characteristic of Rubiaceae flowers the presence of a floral nectary surrounding the base of the style. Although in this research it was not possible to study the composition of the substance secreted by these glandular structures, taking into account their anatomical characteristics and the presence of floral visitors whose feeding is exclusively nectar [ Pompeius verna - Lotts & Naberhaus (2020)-, and Aellopos titan - Moraga (2018)-, both Lepidoptera], we can confirm that they are floral nectaries. These nectaries correspond to the so-called epigynous nectaries, a term that has been used by Manns & Bremer (2010) to designate the floral nectaries of the tribe Naucleeae (Rubiaceae), and by Vaes et al. (2006 ) for Spermacoce spp., tribe Spermacoceae.

In all species of Gardenieae studied here the nectary morphology is quite uniform. Only a difference in the size of the nectary was observed between the pistilate and staminate flowers in dioecious species. The nectar in the species analyzed is secreted through nectarostomata, which is the most common mode of nectar release in Eudicots ( Nepi 2007; Roy et al. 2017 ) and has also been recorded in the few species studied in the family ( Galati 1991; Galetto 1998; Florentín et al. 2016 ). The nectarostomata found are anomocytic, which is in agreement with Bahadur et al. (1971 ) who describes this type of nectarostomata in 26 species of Rubiaceae.

Only few studies detail the anatomy of floral nectaries in Rubiaceae species (Tab. S2, available on supplementary material < https://doi.org/10.6084/m9.figshare.20233767.v1>), most of which are from the tribe Spermacoceae ( Galati 1991; Galetto 1998; Florentín et al. 2016 ). These species share with the Gardenieae here analyzed general characteristics such as the location of the nectary in the flower, its annular or disc shape, and the presence of nectarostomata through which they secrete nectar. However, differences were found in some aspects. While in Gardenieae the annular nectary is entire, in some species of the tribe Spermacoceae as in Borreria and Diodia it is bipartite ( Galati 1991) and in Oldenlandia salzmanni it is bi or tetrapartite ( Florentín et al. 2016 ). In addition, within the species analyzed here, a variation in the inclination of the nectariferous disc surface can be observed, particularly in Genipa americana with a markedly depressed surface in the direction of the style base. All this variation in the morphology of the nectariferous disc (number of lobes, inclination of the disc surface, etc.) was described by Robbrecht (1988) in different genera of Rubiaceae who even attributes to the disc of a group of Gardenieae the characteristic of being fused with the base of the calyx-tube. The latter differs from the species analyzed in the present study. However, it is important to emphasize that although most of the species in the family have a floral nectariferous disc, this can have a great variety of morphologies in different taxa of Rubiaceae. On the other hand, while Gardenieae nectaries have both the cuticle and the epidermis smooth (except in R. feroxwhose cuticle is striated), in Spermacoceae the cuticle is always striated and the epidermis is papillate ( Galati 1991; Florentín et al. 2016 ).

Figure 5 –
a-m. Anatomy of nectariferous disc (ND) of Tocoyena formosa (a-c), Cordiera concolor (d-h), and Genipa americana (i-m) in longitudinal section – a, d-e, i-k. details of a portion of ND showing the nectary and sub-nectary parenchyma; b, f-g, l. epidermis, nectarostomata, cuticle, and a portion of nectary parenchyma; c, h, m. details of the vascular traces in sub-nectary parenchyma. Simbols: ⚥ = monoclinous flower; ♀ = pistilate flower; ♂ = staminate flower. Abbreviations: cu = cuticle; dr = druse; ep = epidermis; np = nectary parenchyma; pc = phenolic compounds; ph = phloem; sp = sub-nectary parenchyma; st = style; x = xylem. Scale bar: a, k = 100 µm; b-h, l-m = 50 µm; i-j = 1 mm.

Oldenlandia salzmanni it also differs from the species of Gardenieae in having a nectarostoma with 5–7 subsidiary cells arranged in a rosette ( Florentín et al. 2016 ). In the other Rubiaceae species the type of nectarostoma is not mentioned.

Following Nepi (2007), in the Gardenieae species, the sub-nectary parenchyma has vascular

Figure 6 –
a-p. Anatomy of nectariferous disc of Randia calycina (a-d), Randia ferox (e-h), and Randia micracantha (i-m) in longitudinal sections – a-b, e-f, i-j, m-n. details of a portion of ND showing the nectary and sub-nectary parenchyma; c, g, k, o. epidermis, nectarostomata, cuticle, and a portion of nectary parenchyma; d, h, l, p. detail of the vascular traces in sub-nectary parenchyma. Simbols: ⚥ = monoclinous flower; ♀ = pistilate flower; ♂ = staminate flower. Abbreviations: cu = cuticle; dr = druse; ep = epidermis; np = nectary parenchyma; ph = phloem; sp = sub- nectay parenchyma; st = style; x = xylem. Scale bar: a-b, e-f, i-j, m-n = 100 µm; c-d, g-h, k-l, o-p = 50 µm.

Figure 7 –
a-l. Floral visitors in flowers (a-c) and in nectaries with post-flower secretion (d-l) – a. Cordiera concolor, floral visitor: Lepidoptera, Hesperiidae, Pompeius verna; b-c. Randia heteromera, floral visitor: Lepidoptera, Sphingidae, Aellopos titan; d-e. Tocoyena formosa – d. floral visitor: Hymenoptera, Apidae, Eucerini sp; e. floral visitor: Hymenoptera, Formicidae, Cephalotes sp.; f-g. Cordiera concolor – f. floral visitor: Hymenoptera, Formicidae, Camponotus sp; g. floral visitor: Diptera, Syrphidae, Ocyptamus sp.; h. Genipa americana, with Hymenoptera, Formicidae, Cephalotes sp.; i. Randia calycina, with Hymenoptera, Formicidae, Crematogaster sp.; j. Randia ferox, unripe fruit with nectar drop (white arrow); k. Randia micracantha, with Hymenoptera, Formicidae, Camponotus sp.; l. Randia heteromera, Hymenoptera, Formicidae, Cephalotes sp. Scale bar: a = 0.5 cm; b-c = 1 cm; d-l = 2 mm.

bundles of xylem and phloem. However, in the Rubiaceae species analyzed by Galati (1991; Tab. S2, available on supplementary material < https://doi.org/10.6084/m9.figshare.20233767.v1>) it was reported an epidermis and a secretory tissue with traces of phloem only, while in the species of Rubiaceae analyzed by Galetto (1998; Tab. S2, available on supplementary material < https://doi.org/10.6084/m9.figshare.20233767.v1>) it was mentioned a secretory parenchyma with vascular traces of xylem and phloem. These authors have probably used Fahn’s (1979) terminology since they do not mention a subdivision of the parenchyma as in the other classifications. However, given the authors’ descriptions of the tissue that composes the nectaries and the presence of vascular bundles, they could be considered similar to those of Gardienieae in terms of anatomical organization. We consider that the terminology suggest by Nepi (2007) more suitable since it is the most commonly used by other authors. For this reason, in the data in Table S2 (available on supplementary material < https://doi.org/10.6084/m9.figshare.20233767.v1>), the terms were standardized with Nepi’s terminology in order to make the results obtained in this and other studies of Rubiaceae more understandable. Only in O. salzmanni there is no mention of the presence of vascular traces ( Florentín et al.2016 ), and the published photographs do not show a differentiation of the parenchymal tissue as in the other species. Generally, nectaries present vascular bundles of only phloem, or phloem and xylem ( Fahn 1979; Nepi 2007) as is the case in the Gardenieae species analyzed here. Nectar water may be derived from both xylem and phloem or only phloem, although the rest of the nectar components including carbohydrates and amino acids can be derived from different sources such as phloem sap, cytological activity or degeneration of certain parts of the nectary, among others ( Nepi 2007). However, in some cases the nectary may lack vascular bundles and receive irrigation from the vasculature of the organs nearby ( Fahn 1979; Nepi 2007) as could be the case of O. salzmanni. Finally, in Hamelia patens, Heterophyllaea pustulata, Manettia cordifolia, and Oldenlandia salzmanni, the nectaries present raphides, which differs from the studied Gardenieae that present druses. Considering the scarcity of anatomical studies of nectaries in Rubiaceae, it is necessary to perform similar studies in other taxa of the family in order to establish structural patterns of nectaries in Rubiaceae.

Based on field observations it was found that, in the species analyzed, the nectaries are still visited by animals even after the corolla had fallen, in both staminate and pistilate flowers. It is possible that the thick cuticle observed in these nectaries protects them against desiccation, helping them to persist longer in the flower. In Rubiaceae, this type of nectary that persists after the fall of the corolla was mentioned in a few species and some hypotheses were expressed for its function. In Hamelia patens (tribe Hamelieae) the floral nectary would act as an extrafloral nectary during fruit formation, despite being found also in flower stage ( Koptur 1992; Galetto 1998). In Amaioua guianensis (tribe Gardenieae) the possible function would be to protect the fruit against predators ( Amorim & Oliveira 2006). This possible function was also proposed for Alibertia verrucosa (tribe Gardenieae) in which an extrafloral nectary continues to secrete nectar during fruiting ( Santos & Del-Claro 2001; Falcão et al. 2014 ). In this species, Falcão et al. (2014 ) carried out a detailed study in which they showed that the plant invests more energy in the intermediate size fruits producing more nectar if compared with the youngest and mature fruits. They assumed that this occurs because the youngest fruits that are damaged can be aborted avoiding future energy expenditure, whereas in mature fruits a chemical modification could occur making the seeds less attractive or the hardening of the pericarp would act as a physical barrier preventing the predator’s accessibility to the seed.

In Palicourea rigida (tribe Psychotrieae) the epigynous nectary of the flower which persists in the fruit was described as pericarpial, with the post-floral secretion being consumed by ants, which protect the vegetative parts of the plant instead of the fruit ( Del-Claro et al. 2013 ). Finally, in Tocoyena formosa, Sanz-Veiga et al. (2017 ) nectaries are called post-floral pericarpial nectaries, which continue to secrete copious amounts of nectar during fruit differentiation and are visited by ants, but which do not provide protection to the fruit against seed predators. We have observed in T. formosa that not only ants visit these nectaries. Bearing in mind that the flowers of each inflorescence in this species have an asynchronous development, it can be assumed that those nectaries could help attract more pollinators for the flowers that mature later. The latter could also be valid for the dioecious species studied since staminate plants have inflorescences with asynchronous development of their flowers. In these species the persistence of nectaries could favor pollination by ensuring that pollinators visit the flowers of the staminate inflorescences. However, in the pistilate plants it is necessary to carry out more observations to be able to hypothesize the role of the persistence of nectaries in the fruits of the species studied. Considering all the studies on the possible role of nectaries with post-flower secretion, it is evident that their function is not simple. Probably multiple mutualists benefit from their presence, either participating in the plant- pollinator interaction or favoring the defense against herbivory to both the vegetative parts of the plant and the developing fruit.

In this study we could conclude that the nectary disc of the species analyzed of Gardenieae can be classified as: reproductive, structural, and mesenchymal, which act as floral nectaries during the floral stage, with a nuptial function, whereas after the corolla has fallen and during fruit differentiation they are persistent nectaries, with post-floral secretion, and maybe with an extranuptial function.

Despite the apparent uniformity of the nectariferous discs in Rubiaceae, this work shows that there are some variations in the morphology and anatomy of this structure, thus more studies are needed to enable broader discussions including character evolution in the group. Among the characteristics that should be considered in future studies to establish differences between the nectaries of different Rubiaceae groups are the following: variation of morphology of the nectary disc (entire, bi or tetrapartite), variations in the epidermis (papillate, smooth), type of nectarostomata (with or without subsidiary cells), variation of cuticle (smooth or striated), anatomical organization, type of vascular traces (phloem and xylem, only phloem), presence and type of crystals (druses, raphides), among others. Future studies should take account of characteristics such as detailed observations of the nectar secretion period and of floral visitors during floral anthesis, after the fall of the corolla, and during the differentiation of the fruits. In the case of dioecious species these observations should be made comparatively between pistilate and staminate flowers. In this way the plant/insect interactions maintained by these nectaries could be established.

Acknowledgments

The first author thanks CONICET by its Doctoral grant. This work was funded by PICTO-UNNE 0199/2011, PICT 2016-3517, and CONICET PIP-112-2011-0100906 grants. We thank Javier Florentín, María Florencia Romero, Mariela Núñez Florentín, Victor Dávalos, and Walter Medina, for providing fixed materials used in this work. Also we thank Rosemary Scoffield, for reading the English manuscript critically.

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• The first author thanks CONICET by its Doctoral grant. This work was funded by PICTO-UNNE 0199/2011, PICT 2016-3517, and CONICET PIP-112-2011-0100906 grants. We thank Javier Florentín, María Florencia Romero, Mariela Núñez Florentín, Victor Dávalos, and Walter Medina, for providing fixed materials used in this work. Also we thank Rosemary Scoffield, for reading the English manuscript critically.

Edited by

Area Editor:

Dra. Juliana Villela Paulino

Data availability

See supplementary material at < https://doi.org/10.6084/m9.figshare.20233767.v1>

Publication Dates

• Publication in this collection
  04 Nov 2022
• Date of issue
  2022

History

• Received
  14 Sept 2020
• Accepted
  25 Oct 2021