NECTARY STRUCTURE OF Ornithidium sophronitis RCHB.F. (ORCHIDACEAE: MAXILLARIINAE)

Most orchids do not produce floral food-rewards. Instead, they attract pollinators by mimicry or deceit. When present, the most common floral food-reward is nectar. To date, nectary structure has been described for only two species of Maxillaria sensu lato, namely Maxillariella anceps and Ornithidium coccineum (formerly Maxillaria anceps and M. coccinea, respectively). Here, we describe that of a third species, Ornithidium sophronitis (formerly Maxillaria sophronitis). This species possesses floral characters concomitant with ornithophily. A ‘faucet and sink’ arrangement is present, with nectar secreted by a protuberance on the ventral surface of the column, collecting between column and tepal bases. The nectary of O. sophronitis shares many features with that of O. coccineum. It has a single-layered epidermis and 35 layers of small, subepidermal, collenchymatous, secretory cells. Beneath these occur 2-3 layers of larger, subsecretory, parenchymatous cells supplied by phloem. Nectary cell vacuoles contain osmiophilic material and proteinaceous intravacuolar bodies. Moreover, distension of the nectary cuticle occurs as nectar accumulates between it and the secretory epidermis. Subsecretory cells, however, have thinner walls and contain flocculent, intravacuolar precipitates that may be related to the presence of flavonoids. Since the floral and nectary structure of O. sophronitis is very similar to that of closely related Ornithidium coccineum, it may have evolved in like manner in response to similar pol-


INTRODUCTION
Although many angiosperm families produce floral food-rewards, these are often absent from orchid flowers and here, pollinator attraction by mimicry and deceit tend to predominate ( v a n d e r P i j l and D o d s o n , 1969; A c k e r m a n , 1984; v a n d e r C i n g e l , 2001).However, rewards, when present in a flower, not only serve to attract potential pollinators, but also maintain a high incidence of pollinator visits and generally confer evolutionary advantage, in that they can double its chances of developing fruit and seed (N e i l a n d and W i l c o c k , 1998).The Neotropical genus Maxillaria Ruiz and Pav., as traditionally defined, is thought to contain some 580 species and has long been considered to be an assemblage of morphologically disparate taxa (W h i t t e n et al. 2007).Recent phylogenetic analyses indicate that Maxillaria is indeed grossly polyphyletic (B l a n c o et al. 2007, and references therein).As a result, B l a n c o et al. (2007) have proposed a new classification of core Maxillariinae that recognizes 17 genera (including Ornithidium Salisb.ex R. Br., Camaridium Lindl.and Maxillariella M.A. Blanco & Carnevali).However, the proportion of Maxillaria (as previously circumscribed) that produces nectar is thought to be small and D a v i e s , S t p i c z y ń s k a and G r e g g ( 2005) estimate it to be as little as 8%.To date, our knowledge of nectary structure for Maxillaria is confined to just two species; Ornithidium coccineum (Jacq.)Salisb.ex R. Br. ].In the first, a 'faucet and sink' arrangement is found, with nectar secreted by a protuberance on the ventral surface of the column collecting in a 'sink' formed by the proximal part of the labellum, the bases of the other tepals and the base of the column (S t p i c z y ń s k a , D av i e s and G r e g g , 2004).In M. anceps, however, nectar produced by the callus is secreted onto the adaxial surface of the labellum by means of stomata (D av i e s , S t p i c z y ń s k a and G r e g g , 2005).
The aim of the present paper is to describe the structure of the floral nectary of a third species formerly assigned to Maxillaria, namely Ornithidium sophronitis, and to compare it with that of closely related taxa.

MATERIALS AND METHODS
Nectary tissue of Ornithidium sophronitis Rchb.f.flowers was prepared and examined using light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), as previously described (D a v i e s and S t p i c z y ń s k a , 2009).Also, as before, semi-thin sections were stained with toluidine blue O (TBO), and hand-cut sections of fresh material were tested for starch, as well as acidic polysaccharides and mucilage, using IKI and ruthenium red (J e n s e n , 1962), respectively.
Nectar-sugar concentration of fresh flowers was determined using refractometry and nectar tested for glucose using glucose-sensitive test sticks (Clinistix).

RESULTS
Flowers of O. sophronitis are weakly zygomorphic and diurnal.They lack fragrance and honey guides, but the yellow column and central area of the labellum contrast markedly with the other tepals, which are bright red in colour (Figs 1A-B).Cryptic, cream-coloured anther caps are present (Fig. 1B).The labellum is strongly folded (Fig. 1B) and copious floral nectar is produced.
A small protuberance, some half way along the length of the ventral surface of the column, secretes nectar, and this collects between the column, the other tepals and the almost vertical, concave, proximal part of the relatively immobile labellum (Fig. 1B).Such is the volume of nectar produced, that it also often flows forward onto the mid-lobe of the labellum.
Refractometry of O. sophronitis nectar gave a value of 64% (w/w) sugar.Nectar was present in unopened buds of O. sophronitis and this, together with nectar tested 2-3 d into anthesis and again at late anthesis (close to senescence), was shown to contain glucose.Nectar was often produced in abundance, but at other times, none could be found.
The outer, tangential epidermal wall of the nectary has a thin, reticulate cuticle.SEM and TEM observations did not reveal ectodesmata, pores or cracks through which nectar could exude.However, characteristic, cuticular swellings (8-10 μm high) are present, and these usually occur at points coinciding with the middle lamella of radial walls between adjoining epidermal cells (Fig. 2A).These swellings occur exclusively on the surface of the nectary protuberance, being absent from neighbouring column cells (Fig. 2B).
The nectary consists of a single-layered epidermis and 3-5 layers of subepidermal, secretory cells (Figs 3A-C), beneath which occur 2-3 layers of subsecretory parenchymatous cells.Secretory cells are small (17.5 -22.0 μm diameter), whereas subsecretory parenchyma cells are larger (40.9 μm mean diameter).Both secretory and subsecretory cells are compactly arranged.The nectary is supplied by phloem strands embedded in ground parenchyma directly beneath the subsecretory tissue (Fig. 3D).Staining with TBO revealed that the walls of secretory cells are cellulosic, whereas staining with ruthenium red revealed the presence of acidic polysaccharides in the middle lamella.A characteristic feature of these nectary cells is the presence of irregular, intravacuolar, protein bodies of variable size (Figs 3A, C, E).Starch was not detected in the plastids of nectary cells on treatment with IKI (Fig. 3B).
Subsecretory parenchyma cells (Figs 3A, C-D) have distinctly thinner walls (mean 0.5 μm) with abundant plasmodesmata.Few mitochondria are present and the cytoplasm contains starchless plastids, ER, and dictyosomes.Flocculent, intravacuolar precipitates may also be present (Fig. 3C), and these may be related to the presence of flavonoids.

DISCUSSION
It has long been speculated that O. sophronitis is ornithophilous.Unfortunately, direct evidence to support this has not been forthcoming.Recently, however, Whitten and co-workers ( 2007 ).The presence of anther caps and pollinaria on beaks of birds usually evokes a bill-cleaning response and consequently, many pollinaria are either lost or destroyed.It is thus, perhaps, significant that some 50% of hummingbird-pollinated orchids have blue, grey, brown, cream or greyish-white, cryptic anther caps.These are thought to illicit a lesser response than more conspicuous, yellow anther caps and thereby facilitate pollination (D r e s s l e r , 1971).Flowers of O. sophronitis possess all these characters and are therefore, probably, bird-pollinated.Moreover, papillae on the adaxial tepal surface are smooth and convex, and the outer tangential wall lacks striations.The optical geometry of such cells is conducive to moderate surface reflection (K a y , D a o u d and S t i r t o n , 1981) and may account for the glistening appearance of the flowers.This, in turn, possibly helps to attract pollinators.
Orchid nectaries are generally thought to have perigonal origins (S m e t s et al. 2000; R u d a l l , 2002; R u d a l l and B a t e m a n , 2002) and are usually formed from the proximal part of labellum, or less frequently, from sepals (D r e s s l e r , 1993).Howev-er, in O. sophronitis, the nectary, like that of O. coccineum, is a small protuberance located on the column (S t p i c z y ń s k a et al. 2004) and thus, cannot be considered perigonal.In such 'faucet and sink' arrangements, nectar collects in a cavity formed by the proximal part of the labellum, the base of the column and the bases of the other tepals.
Although not yet fully investigated, similar protuberances have been reported (S t p i c z y ń s k a et al. 2004) to occur in Maxillaria aggregata (H.B.K.) Lindl.(syn.Ornithidium aggregatum Rchb.f.), M. fulgens (Rchb.f.) L. O. Williams, M. nubigena (Rchb.f.) C. Schweinf.(syn.Ornithidium nubigenum Rchb.f.) and M. ruberrima (Lindl.)Garay (syn.Ornithidium ruberrimum (Lindl.)Rchb.f.).Rodrigo B. Singer (pers. comm., 2003) has also observed them in M. brevilabia Ames & Correll (syn.Camaridium brevilabium (Ames & Correll) M. A. Blanco), M. concavilabia Ames & Correll and M. horichii Senghas (syn.Camaridium horichii (Senghas) M. A. Blanco).Remarkably, all these species have red, orange, pink or white flowers, or translucent flowers suffused with pink.However, Singer also reports a similar protuberance in M. parviflora (Poepp.& Endl.)Garay.In this species, stingless bees (Meliponini), the typical pollinators of Maxillaria sensu lato, were observed feeding upon droplets of nectar that had collected in a 'conch-like cavity of the lip.'This is interesting, since the nectar-sugar concentrations of entomophilous Maxillariella anceps (D a v i e s et al. 2005) and presumed ornithophilous O. sophronitis are very similar (66.5% and 64% (w/w) sugar, respectively).Moreover, observation of cultivated O. sophronitis showed that, at intervals, nectar was produced in abundance, but that these episodes alternated with periods when no nectar could be found.This strongly indicates that floral nectar can be re-absorbed, as has also been recorded for a number of other orchid species (S t p i c z y ń s k a , 2003; D a v i e s and S t p i c z y ń s k a , 2008, and references therein).
The nectary tissue of O. sophronitis shares a number of unusual features with other presumed ornithophilous species, such as O. coccinea and Hexisea imbricata (Lindl.)Rchb.f.(S t p i c z y ń s k a et al. 2004, 2005), in particular, the presence of collenchyma.It is thought that the thickened cell walls of this tissue probably provide an apoplastic route for nectar movement within the nectary, especially in the absence of cutinized layers and other barriers that could impede nectar flow, whilst simultaneously preventing damage caused by the beaks of visiting birds.However, the numerous plasmodesmata might indicate an additional symplastic route in this species, as has already been recorded for other taxa (F a h n , 2000; S t p i c z y ń s k a et al. 2004; N e p i , 2007).A second character shared with O. coccinea and H. imbricata is the presence of    2000).In the latter, protein bodies (probably a storage product) act as a pollinator reward.
Although the nectary cuticle of certain orchids such as Platanthera bifolia (L.) Rich.and P. chlorantha Custer ex Rchb.(S t p i c z y ń s k a , 1997; 2003) is permeable to nectar, in O. sophronitis, the cuticle becomes distended due to the pressure formed as nectar is produced and accumulates beneath its surface, resulting in the formation of spherical swellings.These coincide in position with the middle lamella of radial (anticlinal) walls between adjoining epidermal cells.Similar cuticular swellings have also been recorded for other orchid species, such as O. coccineum (S t p i c z y ń s k a et al. 2004) and Hexisea imbricata (S t p i c z y ń s k a et al. 2005), as well as non-orchidaceous taxa, such as Cyclanthera pedata Schrad.(N e p i , 2007).
Amyloplasts were absent from the nectary cells of O. sophronitis.This is contrary to expectation, as these organelles are often involved in nectar production.Usually, amyloplasts, and the starch grains that they contain, are particularly abundant at the presecretory stage but, as secretory activity progresses, starch disappears and the amyloplasts display irregular profiles (N e p i , 2007).Starchless nectary plastids, similar to those present in O. sophronitis, have also been observed for Gymnadenia conopsea (L.) R. Br. (S t p i c z y ń s k a and M a t u s i e w i c z , 2001) and O. coccinea (S t p i c z y ń s k a et al. 2004) and here, sugars present in nectar are probably delivered in the phloem.
Nectar production is seemingly restricted to only three of the 17 clades recognized by Whitten et al. (2007) 2007).Nectary structure of both Ornithidium species was very similar and may have evolved in response to similar pollinator pressures.However, these nectaries contrasted greatly with those of M. anceps (nectar secreted by labellar callus) and Cryptocentrum (nectary spur; D a v i e s and S t p i c z y ń s k a , 2007).Given the enormity of Maxillaria sensu lato and the vegetative and floral diversity of its members, differ-ences in nectary structure are to be expected.Documenting these differences should prove a worthwhile and fruitful field for future research.
The most common food-reward in Orchidaceae is nectar ( v a n d e r P i j l a n d D o d s o n , 1969), and its presence significantly enhances the efficiency of pollination, as compared with other types of floral-food rewards or deceit alone (D a f n i and I v r i , 1979; J o h n s o n and B o n d , 1997; N e i l a n d and W i l c o c k , 1998; J o h n s o n and N i l s s o n , 1999; N e i l a n d and W i l c o c k , 2000).However, the cost of nectar production and subsequent fruit and seed maturation can be great in terms of material and energy expenditure, and this may outweigh the benefits (A c k e r m a n , R o d r i g u e z -R o b l e s and M e l é n d e z , 1994; M e l é n d e z -A c k e r m a n , A c k e r m a n and R od r i g u e z -R o b l e s , 2000, and references therein).
[formerly Maxillaria coccinea (Jacq.)L. O. Williams ex Hodge] and Maxillariella anceps (A m e s & C. S c h w e i n f .)M. A. B l a n c o & C a r n e v a l i [formerly Maxillaria anceps Ames & C. Schweinf.
) have again asserted that the most brightly coloured Ornithidium species are probably hummingbird-pollinated, whereas those with more open, greenish flowers are probably bee-or wasp-pollinated.Ornithophily has evolved many times (S p e c h t , 2006; C r o n k and O j e d a , 2008), usually from entomophily.Bird-pollinated flowers are often red, pink, orange, yellow or white; less frequently, reddish-violet and blue (P r o c t o r and Y e o , 1973; O r t e g a -O l i v e n c i a et al. 2005; M i c h e n e a u , F o u r n e l and P a i l l e r , 2006).They exhibit diurnal anthesis, are weakly zygomorphic with a backwardly curved labellum that is strongly folded or has a substantial callus, thereby partially closing the floral tube at the level of the anther and stigma.They produce abundant nectar, but no fragrance, and they lack nectar guides.Floral tissues are often tough due to the presence of collenchyma (S t p i c z y ń s k a , D a v i e s a n d G r e g g , 2004, 2005; S t p i c z y ń s k a and D a v i e s , 2006) and can withstand contact with a hard beak (v a n d e r P i j l and D o d s o n , 1969; van der C i n g e l , 2001

Figs
Figs 1A-B.Onithidium sophronitis (A) has weakly zygomorphic, bright red flowers with connivent petals and a yellow area upon the labellum.Note also that the tepals are papillose and glisten.Dissected flower of O. sophronitis (B) showing position of protuberant nectary (arrow) on ventral surface of column.Note also the cavity, the site of nectar accumulation, formed by the bases of column, tepals and strongly folded labellum, as well as the cream-coloured anther cap.Scale bars = 5 mm, throughout.

Figs
Figs 2A-B.Epidermal cells of O. sophronitis nectary (A) and adjacent region of column (B), respectively.Both have a thin, reticulate cuticle.However, small, spherical, cuticular swellings (arrows) occur between the epidermal cells of the nectary (A), whereas these are absent elsewhere on the column (B).Scale bars = 10, 40 μm, respectively.

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as comprising Maxillaria sensu lato, namely Ornithidium, Cryptocentrum Benth.and Camaridium Lindl.However, (W h i t t e n et al. 2007) were uncertain as to whether nectar is produced by others, such as the Pityphyllum Schltr.and Maxillaria desvauxiana Rchb.f.clades.To date, nectary structure and nectar secretion are known in detail for only two species of Ornithidium (O.coccinea and O. sophronitis) and a single species (Maxillaria anceps) currently assigned to the new genus Maxillariella (B l a n c o et al.