Ornithophily in the subtribe Maxillariinae (Orchidaceae) proven with a case study of Ornithidium fulgens in Guatemala

Ornithophily has been long speculated to occur in the subtribe Maxillariinae (Orchidaceae), relying either solely on micromorphological analyses or scarce field observations of undefined species. In Guatemala we were able to observe regular visits of the azure-crowned hummingbirds feeding on flowers of Ornithidium fulgens. These observations have led us to investigation of floral attractants by means of scanning and transmission microscopy, histochemical and chemical analyses (GC–MS). Conducted investigation revealed that the epidermis of basal protuberance of column-foot has features proving the secretory activity and that secreted nectar is sucrose-dominant. Slight secretion on the middle part of the lip is puzzling. The presence of other potential pollinators has not been reported. Based on the results of this study, we confirmed that the flowers of O. fulgens meet all criteria of ornithophily and thus that the hypothesis about bird pollination in the subtribe Maxillariinae is proven. The presented results confirm that the previously described floral features predicting the bird pollination in this group are justified. This strengthens the theory about floral adaptations to different pollinators and gives valid reasons to consider species with flowers with a certain set of traits as ornithophilous, even in the absence of the pollination observation.


Results
Field observations. During field studies conducted in Estación Experimental de Orquídeas de la Familia Archila (Cobán, Guatemala) in 2017-2020, we have witnessed regular visits of the azure-crowned hummingbirds (Amazilia cyanocephala) feeding on flowers of O. fulgens (Fig. 1A-F). In general, ornithophily has been frequently recorded by the second co-author during the past 20 years, both in the station and forest. During these long-time observations, pollinia transport has been spotted. While conducting an investigation focused only on documenting this phenomenon, we have run the observations for one week, in the early mornings and later in the day, from midday until sundown. Such observations are valuable, however limited, thus they require continuation in the future. Amazilia cyanocephala was the only species that has ever been spotted by us while transferring the pollinia on the beak ( Micromorphology. Dorsal sepal is glabrous with scattered sessile one or two-celled trichomes, covered by remnants of secretions ( Fig. 2A) on the outer side (upper, abaxial surface). Paracytic stomata (Fig. 2B) are   2D) and mainly two-celled trichomes with secretory residues on the outer side (Fig. 2E). Also, two lateral petals, forming inner whorl, are glabrous with paracytic stomata at apices and two-celled trichomes at bases on both sides, and a little visible residue of secreted material. The inner surface of the saccate base of the lip is glabrous (Fig. 3A, B), in the middle part the surface becomes papillate ( Fig. 3C-E) and at the distal part is densely papillose with conical and obpyriform papillae ( Fig. 3C, F, G) and visible residues of secreted material (Fig. 3E, G). The stout gynostemium is equipped with a swollen mound/protuberance at the base (Fig. 3H). Anther cap is glabrous, with a strongly striate cuticle and visible stomata (Fig. 3H, I). The SEM studies allowed us to describe features of epidermis and select specific floral parts with possible secretory activity for histological tests and TEM studies.
Histochemistry and ultrastructure. The transverse sections from saccate lip base and the swollen mound reveal a single layer of epidermis and a few subepidermal layers with cells with dense cytoplasm, only exclusively in the swollen mound of gynostemium ( Fig. 4A-C). The swellings are noticeable on epidermal cells of the mound (Fig. 4D). The cells of ground parenchyma are vacuolized and among them collateral vascular bundles occur (Fig. 4A-E). The idioblasts contain bundles of raphides sharp needle-like crystals of calcium oxalate (Fig. 4E). The starch grains, detected in the PAS method, occur in deeper layers of parenchyma, not in epidermis (Fig. 4F, G). The other tests: ABB for proteins, FeCl 3 for dihydroxyphenols, Ruthenium Red for pectic acids/mucilage do not detect these components (Figs. 4H, I, 5A). The fluorescence staining with Auramine O displays unruptured cuticle on epidermal cells (Fig. 5B), which is also visible in TEM results (compare with Fig. 6D, F). The further lip and gynostemium sections, above the mound, reveal the small cells of epidermis and subepidermis with dense cytoplasm of gynostemium, but rather not active lip cells (Fig. 5C-E). No detection of pectic acids/mucilage of the middle part of the lip was observed (Ruthenium Red; Fig. 5E). In ground parenchyma few starch grains are still noticeable (Fig. 5F). The shape of lip changes towards the apex into papillate and slight outline of secretory activity are noticeable (Fig. 5G, compared with Fig. 3D, E). The papillate apex contains many idioblasts cells with raphide crystals and starch grains (Fig. 5H, I). The set of different histochemical tests revealed the localization of substances through floral tissue and allowed us to compare these results with the chemical composition obtained from the GC-MS method. TEM observations show the residues of secreted material on the cuticle surface and between radial cell walls (Fig. 6A). The dense net of micro-channels is visible in the cuticle with the remnants of exuded substances on its surface (Fig. 6B) and cuticle swellings caused by accumulation of secretory products beneath (Fig. 6C). The www.nature.com/scientificreports/ secretory material on the surface is heterogenous (compare Fig. 6A, C). The residues of secretions in the middle part of the lip accumulate beneath the cuticle (Fig. 6D) passing through micro-channels (Fig. 6E). Slight amounts of secretions on the cuticle of lip margins near the apex (Fig. 6F) are transported via micro-channels (Fig. 6G).   www.nature.com/scientificreports/ Nectar analysis. Nectar is strongly sucrose-dominant, with an average contribution of sucrose as high as 93% of the total carbohydrates (Table 1). Fructose and glucose are both much more abundant in methanolic extract from the flower tissue, where they constituted ca. 50% of all sugars, suggesting that extraction procedure allowed to separate the nectar itself. No volatiles were detected in dichloromethane extracts.   www.nature.com/scientificreports/ (making them invisible for bees) and bird-attraction (being readily detectable) 1 . However, it should be noted that bees can perceive some flowers seen as red by humans, if they have at least some reflectance in the shorter wavelengths as well 27 . There is an ongoing discussion regarding the sense of smell among different bird groups and whether they use, and if yes to what extend, while searching for food (e.g., [28][29][30][31] ). There is, however, very little information about the role of smell in foraging by nectarivorous birds 32 , and since the purpose of the presented work was not to investigate this issue, we will follow the widely held notion that the sense of smell is not dominant in birds thus bird pollinated flowers are usually lacking any detectable scent. Nevertheless, it should be noted that the latest work published by Núñez et al. 32 proved that Amazilia amazilia s. l., species closely related to the one pollinating scentless O. fulgens, does not use smell in the search for food but employ other senses, mainly sight. During the course of this research, we have investigated this aspect, however, we have not found any volatile compounds. This result was also supported with our field observations. O. fulgens has pendant, campanulate flowers with labellar margins curved back, weakly expressed zygomorphy and diurnal anthesis. Floral tissues are clearly strengthened and rigid, giving the impression of being crispy and thus they can withstand contact with a hard beak. In ornithophilous flowers the nectar guides are absent 2 , and the investigated species is no exception. Similar floral morphology has been previously described e.g. in closely related, and also possibly bird-pollinated, O. coccineum (= M. coccinea), which as well has weakly zygomorphic, globose flowers with scarlet tepals and a scarlet-yellow backwardly curved lip and diurnal anthesis. It also produces abundant nectar and lacks nectar guides. In O. fulgens, however, the lip is essentially unlobed, and without callus, whereas in O. coccineum it is 3-lobed, with a simple, hemispherical callus. Other examined orchid species, presumably ornithophilous, also possess vivid flower colours: Hexisea imbricata (Lindl.) Rchb. f. (Laeliinae) has scarlet perianth with a distinct yellow labellar callus 33 , Symphyglossum sanguineum (Rchb. f.) Schltr. (Oncidinae) has pink-violet flowers with labellar auricles 34 , and Comparettia falcata Poepp. & Endl. has reddish or pink flowers with labellum which ends in two extensions (horns) and extend longitudinally within the spur 35 . In the aforementioned species, the lip is saccate, rigidly attached to the column and floral tissues are tough. Furthermore, in O. coccineum a strong fold in the lip partially closes the floral tube at the level of the anther and stigma and thus probably forces the bird to push its beak against the column to gain entry 2 . As already mentioned, ornithophilous flowers tend to lack odor, however, in O. coccineum sweet honey-like scent was occasionally detected 23 , which is a significant difference between this species and O. fulgens. Pollinia in bird-pollinated Orchidaceae are often brown, as Dressler 36 has pointed out, whereas those of their insect pollinated relatives are yellow. However, it seems that exceptions to these rules are common. In his paper it is indicated that many other orchids, which show the syndrome characteristic of hummingbird pollination, have creamy or greyish white pollinia (in contrast to the most common bright yellow, characteristic for insect pollination). As examples, Dressler has mentioned among others H. imbricata, S. sanguineum, C. falcata and Maxillaria fulgens (= O. fulgens). Indeed, this is the only characteristic for bird-pollinated orchid flowers that is lacking in investigated O. fulgens, which similarly to most (if not all) Maxillariinae representatives, has creamyyellowish pollinia. The very same phenomenon is described by Catling 37 for Sacoila lanceolata, in which hummingbird pollination has also been reported by the author.  23 and H. imbricata 33 . In O. fulgens, the epidermis of the basal protuberance of column-foot has features advocating for secretory activity, which confirms Singer's hypothesis of "faucet and sink". A small amount of secretions are found in the middle part of the lip, which is puzzling. In the papers of Stpiczyńska et al. 23,33 , the authors did not include any information regarding nectaries located on the lip, thus it may be assumed that they may be found also in O. coccineum and H. imbricata. Histochemical and TEM research of other Ornithidium species may clear up the mystery.

Discussion
In O. fulgens, the nectary consists of a single-layered epidermis with smaller cells and few layers of subepidermis. Larger and more vacuolised parenchyma cells with few collateral vascular bundles occur beneath them. This pattern of floral nectariferous tissue is often described in orchids, i.e. in presumably ornithophilous: O. coccineum 23 , O. sophronitis 24 , H. imbricata 33 , and S. sanguineum 34 , but also in some sapromiophilous representatives of the genus Bulbophyllum Thouars 39,40 as well as entomophilous species of Epipactis Zinn 41,42 and Neottia Guett. 43 or also in some other species 44 . In O. fulgens, unlike in O. coccineum 23 , the secretory cells do not possess very thick walls. In both species, however, flowers are rather stiff and crispy in touch, which may be an adaptation to prevent mechanical damages caused by the hummingbirds' beaks 23 . Like in O. coccineum 23 and H. imbricata 33 , SEM and histochemical studies in O. fulgens did not demonstrate any pores or cracks the paths for nectar release. In all these species, the cuticle has characteristic swellings. However, the difference in its sizes is significant: in O. fulgens the swellings were up to 1.6 µm high, in O. coccineum: 2 ± 7 mm high, and in H. imbricata: ± 2 µm high. Furthermore, in both species of Ornithidium they are formed at points coinciding with the middle lamella between adjoining epidermal cells. In O. coccineum the swellings occured only on the surface of the column-foot protuberance (nectary), whereas in O. fulgens they were also present on the surface of the lip base. In both mentioned species they were absent from the epidermal cells of the nectar reservoir. Moreover, in both taxa, the abundant nectar filled the space between the lip and gynostemium, like in the container 34  www.nature.com/scientificreports/ cuticular swellings and uninterrupted layer of reticulate cuticle on the tangential walls of the secretory epidermis suggest that nectar accumulation is taking place beneath the cuticle causing its stretching and then secretion via micro-channels to the exterior. The reticulate cuticle with micro-channels is a frequent phenomenon in orchid nectaries and osmophores 33,39,40 .
The secretory epidermal cells of O. fulgens contained plastids with starch grains (amyloplasts), similarly as in H. imbricata 33 and S. sanguineum 34 , however not present in O. coccineum 23 . The starch grains commonly occur in plastids of nectariferous cells 39,40,[44][45][46][47] . Starch is utilised as a source of sugar and energy for metabolic processes during nectar secretion 48 , so the presence/absence as well as number and volume of amyloplasts can describe the suitable stadium of anthesis: pre-or post-secretory 24,39,44 . At the highest level of nectar secretion in H. imbricata the plastids contained both starch grains and plastoglobuli. During starch depletion, the plastids became elongated, irregular in shape and more plastoglobuli (described as lipid droplets within the plastids) occurred. In cytoplasm, the lipid droplets (sometimes described as osmiophilic content) were accumulated 33,34,39,40,45 . They also were present in O. fulgens. Lipids are sometimes considered to be the counterparts of fragrance [49][50][51] . The observed profiles of endoplasmic reticulum, few vesicles close to plasmalemma, micro-channels in cuticle, and cuticular swellings suggest the granulocrine route of nectar release, the same as in H. imbricata, where arrays of ER and dictyosomes were participating in nectar secretion. Also, the vesicles were visible in cytoplasm and near the plasmalemma. After crossing the outer tangential walls of nectariferous epidermis, the released substances accumulate beneath the cuticle and exceed the cuticle forming micro-channels or by rupturing it. We did not observe the cracked cuticle, only the swellings and the cuticle reticulation, but both ways of nectar release are possible.
Idioblasts with raphides of calcium oxalate crystals surrounded by mucilage in subepidermis and parenchyma cells that were observed in O. fulgens, are thought to deter herbivores, and has been previously frequently reported in orchids (i.e. 39,42,52 . Mucilage in idioblasts was also present in H. imbricata 33 . Davies has reported the presence of raphides in leaf and floral tissues 53 for a number of Maxillaria spp. and has also suggested that they may be secretory products and may perhaps discourage herbivory by invertebrates 53,54 .
The main role of nectar is the attraction of potential pollinators. In the past it has been considered to be a simple sugar solution, however it is now known that it consists of a variety of chemicals dissolved, or suspended, in an aqueous solution 55 . These may range from mixtures of one to three common sugars, such as glucose, sucrose and fructose, to more complex sugar solutions 56 or combinations of sugars, free amino acids, vitamins, lipids, and other compounds 57,58 .
The chemical analysis conducted by us proved that the liquid collected from the flowers of O. fulgens is a nectar, with sucrose as a dominant constituent. Data presented by Baker et al. 59 strongly supported the hypothesis that the composition of soluble sugars in nectar is influenced by the pollinators that consume it. Nectars of flowers visited by hummingbirds or Megachiroptera tended to have high levels of sucrose 59-61 while nectars of flowers consumed by passerines had very low levels of this sugar 59 . Bee-pollinated flowers also have sucroserich or sucrose-dominant nectar 62 , so such nectar composition in hummingbird-pollinated flowers that have evolved from bee-pollinated flowers is rather not surprising 1 . Similar conclusions were published by several other researchers [63][64][65][66][67] . Nectar produced by closely related O. sophronitis has been tested using refractometry (concentrations) and glucose-sensitive test sticks (Clinistix) and it has presented the value of 64% (w/w) sugar and the presence of glucose has been confirmed 24 . In Maxillaria anceps Ames & C. Schweinf., presumably beepollinated species with strongly zygomorphic, relatively open, greenish-white flowers with a well-developed lip, nectar is also sucrose-dominant, but contains low concentrations of glucose, fructose, free amino acids and possibly terpenoids 68 . In our study, nectar was strongly sucrose-dominant, with an average contribution of sucrose as high as 93% of the total carbohydrates. This together with morphological evidence itself advocates strongly in favor of ornithophily as the pollination syndrome in O. fulgens.

Conclusion
The field observations of regular visits of the azure-crowned hummingbirds (Amazilia cyanocephala) hovering and transmitting the pollinia of Ornithidium fulgens, as well as floral morphology and anatomy undoubtedly prove that this species is bird-pollinated and thus that such syndrome occurs within the members of the subtribe Maxillariinae Benth. The next steps in our research will be to study the continuation of the hummingbirds' visits to the flowers and their participation in the fruit set (with statistics).

Materials and methods
Plants of O. fulgens have been cultivated in the Estación Experimental de Orquídeas de la Familia Archila (Cobán, Guatemala), the seminatural plantation located in a cloud forest of Guatemala. The flowering season in situ ranges mostly from September to January, with the peak season in November and December. Tissue samples were collected from fresh flowers at different stages of anthesis.
Morphological analysis has been conducted with methods of classical taxonomy. Formal identification of the plant material has been performed in Guatemala by Fredy L. Archila Morales and Monika M. Lipińska. Voucher specimen has been deposited in BIGU herbarium. Research complied with relevant institutional, national, and international guidelines and legislation.
Samples for the scanning electron microscopy (SEM) were preserved in 2.5% (v/v), glutaraldehyde (GA) in 0,05M cacodylate buffer (pH 7,0). Following dehydration in an ethanol series, they were dried by the critical point method using liquid CO 2 , and coated with gold and observed by means of a Philips XL-30 scanning electron microscope.
For histochemical studies, the plant material was fixed in 2.5% (v/v), glutaraldehyde (GA) in 0.05 M cacodylate buffer (pH = 7.0) was used. Then the material was rinsed with cacodylate buffer and dehydrated in the ethanol www.nature.com/scientificreports/ series. Finally, the tissue fragments were embedded in methylmethacrylate-based resin (Technovit 7100, Heraeus Kulzer GmbH). Sections were cut with glass knives (5-7 μm thick) using a Leica EM UC 7 ultramicrotome and mounted on glass slides. The semi-thin control sections were stained with 0.05% (w/v) aqueous Toluidine Blue O (TBO, C.I. 52040) 69,70 . The detection of water-insoluble proteins was possible with the test of Aniline Blue Black (ABB, C.I. 20470) 71 . The water-insoluble polysaccharides, especially starch grains, were detected in the Periodic Acid-Schiff reaction (PAS) 71 . The pectic acids/mucilage were identified following test with a 0.05% (w/v) aqueous Ruthenium Red (C.I. 77800) solution 72 , whereas catechol-type dihydroxyphenols following staining with a 10% (w/v) aqueous solution of FeCl 3 73 . The preparations were studied and photographed with a Nikon Eclipse E 800 light microscope and a Nikon DS-5 Mc camera using Lucia Image software. The sections, following FeCl 3 test, were examined using the differential interference contrast (DIC) imaging. Auramine O (C.I. 41000) 0.01% (w/v) solution in 0.05 M buffer Tris/HCl, pH = 7.2 was used to detect the presence of cuticle 74 , particularly unsaturated cutin precursors and acidic waxes 73 and tissue slides were examined with a Nikon Eclipse E800 fluorescence microscope, equipped with filter B-2A (EX 450-490 nm, DM 505 nm, BA 520 nm).
For transmission electron microscopy (TEM), the lip was fixed in 2.5% (v/v), glutaraldehyde (GA) in 0.05 M cacodylate buffer (pH 7.0). Post fixation overnight in 1% OsO 4 in the cacodylate buffer. The samples were dehydrated by means of the graded acetone series and embedded in Spurr's resin. Ultrathin sections (60 nm) were cut using a Leica UC7 ultramicrotome. Sections were examined by means of a FEI Tecnai Spirit BioTWIN transmission electron microscope at 120 kV.
For chemical analyses, nectar secreted by ca. 15 flowers during the first day of anthesis, was carefully collected using several small pads of glass wool, which were then extracted in 10 ml methanol. Whole flowers were subjected to sequential organic solvent extraction. First, non-polar compounds were isolated in 10 ml dichloromethane for 20 s, then carbohydrates were extracted by dipping flowers for 30 s in 10 ml methanol. Extracts were then stored at 4 °C prior to analysis. Due to the difficult conditions of the nectar collection in the field we were unable to reliably determine its volume and as a consequence the nectar concentration remains unknown.
The dichloromethane extract was concentrated to ca. 0.3 ml under a stream of nitrogen. Samples were then analyzed using gas chromatography mass spectrometry (GC-MS), which was performed using a Shimadzu QP-2010SE system (Shimadzu, Kyoto, Japan), equipped with a 30 × 0.25 mm i.d., film thickness 0.25 μm, ZB-5ms capillary column (Phenomenex, Torrance, CA, USA). Helium was used as the carrier gas at a flow rate of 1 ml min −1 . The split ratio was 1:10, and the injection volume was 1 μl. The injector and GC-MS interface temperatures were maintained at 310 °C. Electron ionization (electron energy 70 eV, ion source temperature 200 °C) was used. The column temperature was programmed for 30 °C (isothermal for 3 min) to 180 °C at 4 °C min −1 , then from 180 to 310 °C at 8 °C min −1 , and then maintained at 310 °C for 12 min.
Carbohydrates present in methanolic extracts were subjected to sequential derivatization procedure according to the slightly modified method described by Ruiz-Matute et al. 75 . An aliquot of each extract was evaporated to dryness under a stream of nitrogen. Then, oximes were synthesized by adding 0.1 mL of a 2.5% hydroxylamine hydrochloride solution in pyridine. Oximes obtained this way were transferred to respective trimethylsilyl (TMSi) derivatives by adding 0.1 mL BSTFA + TMCS (99:1). Each reaction was performed at 70 °C for 30 min. Derivatives were analyzed using gas chromatography with a flame ionization detector (GC-FID). The analysis was performed using a Clarus 500 gas chromatograph (Perkin-Elmer Instruments, Waltham, MA, USA), equipped with the same type of column as mentioned above. The column temperature was programmed from 80 to 300 °C at 4 °C min −1 . Injector and detector temperatures were set at 320 °C. Argon was used as carrier gas at a flow rate of 1 mL min -1 . The split ratio was 1:20, and the injection volume was 1 μL. Identification was based on retention times, which were compared to those of analytical standards of glucose, fructose and sucrose analyzed in the same conditions.