Cyclopogon Guayanensis is an Unusual Orchid With a Generalistic Pollination System and Hexose Dominant Nectar

doi:10.21203/rs.3.rs-4876023/v1

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Cyclopogon Guayanensis is an Unusual Orchid With a Generalistic Pollination System and Hexose Dominant Nectar

https://doi.org/10.21203/rs.3.rs-4876023/v1

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Cyclopogon is a large Neotropical orchid genus pollinated by halictid bees that offers nectar as reward. In a recent phylogenetic tree, Brachystele guayanensis emerged nested within Cyclopogon and was transferred to that genus. The hypothesis for this study was that C. guayanensis would show a similar floral biology to Cyclopogon, although distinctive in its small, congested white flowers. Data on floral biology, pollinators, micromorphology, histochemistry, and nectar sugar composition of C. guayanensis in the Distrito Federal, Brazil were gathered. C. guayanensis is pollinated by at least four species of bees belonging to genera Exomalopsis, Nomada, Tetrapedia (Apidae) and Dialictus (Halictidae) foraging for nectar. Nectar is produced in visually imperceptible quantities by papillae on the inner surface of the labellum; similar papillae occur in other species of Cyclopogon but nectar class is unknown. C. guayanensis nectar is hexose dominant (< 10% sucrose) in the Baker and Baker system and is the second record of this nectar class in the Orchidaceae. Pollinia are dorsally adhesive and probably attach to the underside of the bee labrum, as in other Cyclopogon. The inflorescence rachis, bracteoles, and outer surfaces of the base of the sepals are covered with lipid-secreting glandular trichomes; sepals and petals have numerous raphid-rich idioblasts. This is the first record of papillae on a spurless labellum shown to produce nectar in the Orchidoideae. We suggest that hexose dominant nectars in the Orchidaceae are associated with a geophytic habit, small pale flowers, exposed nectaries, visually imperceptible quantities of nectar, and a generalistic pollination system, and coin the term ‘modest pollination strategy’ for this syndrome.

Apidae

floral histochemistry

Halictidae

hexose

nectar

raphids

Spiranthinae

The Pelexia alliance (subtribe Spiranthinae, tribe Cranichideae, subfamily Orchidoideae, family Orchidaceae; Chase et al. 2015) is a clade of seven genera and about 280 species (Salazar et al. 2018; POWO 2024). This alliance is exclusively Neotropical, widely distributed, and is dominated by its three largest genera: Cyclopogon C.Presl, Pelexia Poit. ex Lindl. and Sarcoglottis C.Presl, which together comprise 85% of its species.

A recent phylogeny of subtribe Spiranthinae (Salazar et al. 2018) revealed that the Cyclopogon clade is paraphyletic, i.e., it included all species of Cyclopogon sampled but also a nested subclade of Brachystele guayanensis (Lindl.) Schltr. + Veyretia Szlach. (four species sampled). All other species of Brachystele Schltr., including the type, formed a monophyletic separate clade within the Spiranthinae but in another clade called the Pelexia clade by the authors. Brachystele was thus split between the Pelexia clade and the Cyclopogon clade. This scenario suggests the occurrence of parallel evolution of vegetative and floral characters between different lineages of the Spiranthinae that have caused them to be put into the same genus based on morphological characters (e.g., Szlachetko and Rutkowski 2008). Brachystele guayanensis has been recently transferred to Cyclopogon (Meneguzzo et al. 2024) based on molecular, morphological and the ecological results that are presented here.

In 2020 and 2021, the population of Cyclopogon guayanensis from which the sample for the phylogenetic study of the Spiranthinae (Salazar et al. 2018) was collected flowered. A second population was also found; both populations were in urban grasslands in Brasilia, Distrito Federal, Brazil. This provided us with an opportunity to study its floral biology, pollinators, micromorphology and histochemistry, unknown until now. A large quantity of data on the floral biology of subtribe Spiranthinae has been amassed by Rodrigo Singer and collaborators (Singer and Cocucci 1999; Singer and Sazima 1999; Singer 2002; Buzatto 2014). Also, this data has been recently reviewed and interpreted within a phylogenetic framework (Buzatto et al. 2022).

The hypothesis for our study was that its floral biology would show: 1) similarities to Cyclopogon due to a shared phylogeny; 2) differences from Cyclopogon, possibly due to parallel evolution with Brachystele sensu stricto.

Floral Biology. A flowering population estimated to be of several hundred individuals over an area of c. 0.5 km² was found in Brasília (Population A, 15^o 45’ 55” S, 47^o 52’ 47” W) on the 7th November 2020 (Fig. 1), followed by a second much smaller population of a few dozen individuals on the 9th of November 2020 (Population B, 15^o 44’ 07” S, 47^o 53’ 24” W)). These two populations were observed between the 8th and 13th November 2020; the first population flowered again in the following year (flowering individuals were first detected on the 21st October 2021) and was observed between 27th October and 9th November 2021.

In the Distrito Federal, the day length difference during the flowering period was minimal; sunrise on 20 October was 05:40 and sunset 18:11, and on the 15 November sunrise was 5:30 and sunset 18:22. Observations were made between 6:00–7:00 to 17:00–18:00 (12 hourly intervals) lasting at least 40 min per hour interval, except for intervals 15:00–16:00 (35 min) and interval 17h00–18h00 (25 min), totalling 610 min (just over 10 man hours).

Visitor behaviour was observed, including possible resource collection, insect body parts touching fertile organs, and adherence position of the pollinarium. Whenever possible, digital images and videos were recorded, and insects were collected for identification. Collected insects were dried, pinned, labelled, and deposited in the Entomological Collection of the Departamento de Zoologia, Universidade de Brasília, Brazil. They were identified by authors ACA and ACM (see also acknowledgements). A voucher specimen from plant population 1 was collected and is deposited in the UB herbarium (C.E.B. Proença 5579) at the Universidade de Brasília, Brazil, available from the Species Link collection network (https://specieslink.net/search/).

Micromorphology and Histochemistry. Five flowering plants were collected and stored in wet paper at 8^o C for c. 48 h. For each plant, two samples were taken from proximal to distal portions of the inflorescence: 1) pre-anthetic buds adjacent to open flowers; 2) open flowers with attached pollinaria; 3) open flowers without pollinaria; 4) old flowers/developing fruits with dry, brown corollas. Four repetitions of these procedures were done, each for a different individual. Samples were immersed in Sudan IV for 15 min, quickly washed in 70% ethanol, immersed in water and photographed using a Zeiss Photomicroscope with an image capture system. Non-treated flowers were also imaged.

Since we were unable to detect floral nectar in the field, an inflorescence with buds and open flowers was cut and the stem put in water in the field and taken to the laboratory. After two days, ten freshly opened flowers were removed, dissected, and examined for nectar under a stereomicroscope since corolla tubes were too narrow to allow insertion of microcapillary tubes. As no nectar droplets were detected, the labella were removed and soaked in 5 mL of distilled water, according to the washing method (agitated for 1 min, allowed to soak c. 20 min and frozen) as suggested by Morrant et al. (2009) as one of the preferrable methods of collecting nectar from flowers with low nectar volume. We were stimulated to try this method, first suggested by Käpylä (1978), by obvious nectar foraging by floral visitors. The solution was later unfrozen and an aliquot of 100µL was taken (without labella) and then vortexed briefly; this was then centrifuged at 17000 g for 20 min to remove particles and subjected to High Performance Anion Exchange (HPAE) analysis of sugars. Sugars were separated and detected using a Dionex ICS 3000 HPAE equipped with a Carbopac PA10 column and electrochemical detector. The separation was performed using a sodium hydroxide gradient at 0.2 mL.min^− 1 exactly as described in Silva et al. (2021). Sugars were identified and quantified by comparison with a calibration curve constructed using authentic standards.

Floral biology. The floral scape of Cyclopogon guayanensis is a spike-like raceme c. 15 to 30 cm high. Flowers are resupinate and arranged in a dense ascending spiral, so that the left side of each labellum is adjacent to, and sometimes touches, the lateral sepal of the flower below it (Fig. 2). The corolla is creamy-white with the tube externally greenish and internally cream or pale yellow. Flowers are zygomorphic and are c. 3 mm high and 2.5 mm across; the entrance to the tube is c. 1 mm wide; the labellum presents a central, slightly yellowish groove; pollinia are pale yellow and visible; no nectar was observed.

The flowers of Cyclopogon guayanensis were visited between 8 AM and 5 PM (Supporting Information, Table S1). We observed at least 10 species of insects, mainly bees but also syrphid flies, of which four can be considered pollinators (Fig. 2, Table 1). The pollinators of C. guayanensis are small, generalist bees from non-related groups (two families). Four species of bees from the genera Exomalopsis, Nomada and Tetrapedia (Apidae) and Dialictus (Halictidae) were observed with one or more pollinaria attached to their bodies, reinforcing their role as pollinators (Fig. 2); pollinators had in common similar body sizes and tongue lengths. The position and angle of the pollinaria in the images suggest that they are adhered to the ventral surface of the bee labrum (Fig. 2) as in Cyclopogon. Unfortunately, captured visitors lacked pollinaria, so we are reluctant to state this unequivocally; it is also possible that they are adhered to the clypeus or proboscis. Several other Halictid bees were also observed visiting and might be pollinators but were captured as they approached or landed on the flowers or lacked pollinaria: Augochlora sp., Augochloropsis sp., and Neocorynura pseudobaccha (Cockerell, 1901). Apparently, all visitors were searching for nectar (Fig. 3), collecting it with their tongues. In spite of the visitors’ relatively small sizes, they could not enter the very small flowers of C. guayanensis, so only the head was inserted.

Table 1

Floral visitors of *Cyclopogon guayanensis* populations in the Distrito Federal (Brazil) during the month of November (2020) and October/November (2021). Only days with visits shown (see Table S2 for complete record of observation periods).
Date	Periods	Total time (minutes) and observer	Visitors/Pollinators C = captured; O = observed; P = photographed; V = Video recorded.
8 Nov 2020 (Pop 1) (see Fig. 2)	07:45–08:05 12:20–12:40 16:40–17:00	20 (CEBP) 20 (CEBP) 20 (CEBP)	Apis mellifera Linnaeus, 1758 (O, did not land). Trigona spinipes (Fabricius, 1793) (V). Exomalopsis sp. 1 (C).
9 Nov 2020			population 1 destroyed by urban mowing
11 Nov 2020 (Pop 2)	08:28–09:10 13:58–14:45	42 (CEBP)	no visits Bee, unidentified (O); Syrphidae flies (O).
11 Nov 2020 (Pop 2)	11:00–12:00	60 (ACM & AJCA)	Exomalopsis sp. 1, female with pollinarium on mouthparts (P).
12 Nov 2020 (Pop 2)	08:30–09:30 13:30–14:30	120 (AJCA)	Exomalopsis sp. 1, female (O). Nomada, sp., female with pollinarium on mouthparts (P). Trigona spinipes (O). Exomalopsis sp. 1, female (O). Nanotrigona testaceicornis le Peletier, 1836 (O) Exomalopsis sp. 2, female (O). Dialictus sp., male with pollinarium on mouthparts (P). Tetrapedia sp., male with pollinarium on mouthparts (P). Syrphidae flies (O).
13 Nov 2020 (Pop 2)	13:30–14:30	60 (AJCA)	Dialictus sp., female (O). Paratrigona lineata (le Peletier, 1836) (O). Exomalopsis sp. 2, male (O).
21 Oct 2021			2021 flowering population detected.
27 Oct 2021 (Pop 1)	16:40–17:05	25 (CEBP)	Dialictus sp. (C).
28 Oct 2021 (Pop 1)	07:45–08:05	20 (CEBP)	cf. Trigona (V).
30 Oct 2021 (Pop 1)	10:30–11:00	30 (CEBP)	Neocorynura pseudobaccha (Cockerell, 1901), male (P). Augochlora sp., male (C). Hemiptera (O).
01 Nov 2021 (Pop 1)	07:00–07:30	30 (CEBP)	Augochloropsis sp. (P).
02 Nov 2021 (Pop 1)	12:30–12:40	10 (CEBP)	Dialictus sp., male (C).
09 Nov 2021 (Pop 1)	6:50–7:00	10 (CEBP)	Augochloropsis sp. (P) sleeping.

Micromorphology and Histochemistry. The micromorphological analysis as well as the histochemical results of the Sudam IV test are presented in Fig. 4. Tector trichomes were not observed on the inflorescence. Two types of glandular trichomes were present, one pedunculate with a secretory head, and the other of the papillae type. The bracteoles are ciliate with 4–8–celled glandular trichomes (Fig. 4F–G). The sepals present (2–3)4(–5)–celled glandular trichomes on the abaxial (external) surface that are dense along the midveins but thin out towards the distal regions. Lipid secretion from the glandular trichomes is insignificant in pre-anthetic buds (Fig. 4A, J), but intense in open (perhaps male-phase?) flowers with the pollinarium still attached (Fig. 4B, L), as well as in the flowers in which the pollinarium is absent (Fig. 4C) and in the senescent flowers (Fig. 4D), i.e., the trichomes persist and do not fall off or dry out, at least in the senescent flowers immediately below the open flowers.

The petals are covered with one-celled papillae on the inner surface; these papillae are mostly short (c. 1.5 times as long as wide) except on the labellum on which the papillae are longer, c. 2–3 times as long as wide (Fig. 4M–N). In both types of papillae, periplasmic spaces were observed to form within the cells, but these are much more developed in the long than in the short papillae. The contents of these periplasmic papilla spaces did not stain with Sudan IV (Fig. 4N, arrow). Sepals and petals form a short floral tube (Fig. 4H) and have many scattered idioblasts with raphids (Fig. 4H, head-arrow, I), as do the bracteoles (Fig. 4F–G, head-arrow). The pollen within the squashed pollinaria is shed in tetrads (Fig. 4OP) and the ovule is unitegmic (Fig. 4Q).

Nectar composition. The sugar composition from HPAE analysis of the solution obtained from the 10 labella was: 5.5% sucrose, 53.8% glucose, and 40.7% fructose by mass. No other monosaccharides or oligosaccharides were detected. A total of 94.5% of the sugar in the sample was therefore hexose.

The floral biology of the Spiranthinae was stated to be “much more complex than cursory comparisons suggest” (Salazar et al. 2018: 277). A recent review of the floral biology of Pelexia clade (Buzatto et al. 2022) states that orchids in this clade are pollinated mainly by bees, especially those from the family Halictidae, which are attracted by the nectar produced as a reward (Table 2). The flowers are, in general, small for the family, white, with floral parts covered with carpets of glandular hairs. Cyclopogon guayanensis conforms very well to this pattern but stands out due to the wide taxonomic diversity of both pollinators and visitors, and lack of visible nectar.

Bee visitation observed in Cyclopogon guayanensis closely follows the description given by Singer and Cocucci (1999) for C. diversifolius (Cogn.) Schltr., with pollinia possibly adhering to the underside of the bee labrum. These authors hypothesized that C. diversifolius, as well as the other two non-related species they studied [Campylocentrum aromaticum Barb.Rodr. and Prescottia densiflora (Brong.) Lindl.] are pollinated mainly by Halictid bees and well-adapted for “the particular swivel proboscis mechanism of the halictid bees for pollinaria removal and deposition” (Singer and Coccuci 1999: 112). In C. guayanensis, although halictid bees were present, their role as pollinators was overshadowed by the Apidae, i.e. Exomalopsis, Tetrapedia and Nomada (observed bearing pollinaria) and by stingless bees as visitors. This did not seem to be associated to any significant differences in bee body size, pollinaria attachment, or behaviour. We suggest this could be, at least in part, a product of the different bee fauna in our study area. Halictidae are highly abundant, surpassing the Apidae in several surveys (Martins et al. 2013) in subtropical and temperate regions of South America (where other studies of Cyclopogon pollinators have been done; e.g., Singer and Coccuci 1999; Singer and Sazima 1999; Galetto et al. 1997;Benitez-Vieyra et al. 2006).

Buzatto et al. (2022) noted changes in the adherence position of pollinaria on the bee labrum in the phylogenetic tree of species in the Pelexia clade using ancestral character state reconstruction. These authors hypothesized that the ancestor of the Pelexia alliance had ventrally viscous pollinia and noted that the two most basal species Coccineorchis cernua (Lindl.) Garay and Sauroglossum elatum Lindl. (respectively pollinated by hummingbirds and lepidopterans) retained this ancestral character. The shift to hymenopteran pollination was accompanied by a shift from ventral to dorsal pollinia that adhere to the ventral surface of the bee labrum in Cyclopogon comosus (Rchb.f.) Burns-Bal. & E.W.Greenw., Pachygenium bonariensis (Lindl.) Schltr., Pelexia adnata (Sw.) Poit. ex Rich., Sarcoglottis acaulis (Sm.) Schltr., and with a reversal to ancestral ventral pollinia in Brachystele unilateralis (the type species of the genus), i.e., a member of Brachystele sensu stricto. Thus, if adherence of pollinia to the inner surface of the bee labrum in C. guayanensis is confirmed in future captured bees, its transfer to Cyclopogon first suggested by molecular markers (Salazar et al. 2018) will be further strengthened and will also sustain the evolutionary scenario proposed by Buzatto et al. (2022) for this character.

When commenting on the position of Cyclopogon guayanensis in the Spiranthinae phylogenetic tree, Salazar et al. (2018: 296) noted the difference in flower size between the C. guayanensis (then Brachystele guayanensis) and Veyretia spp. and stated that: “upon close examination, it is evident that its flowers have the two-chambered nectary of Veyretia (although more shallowly so in C. guayanensis, in proportion to its noticeable reduction in flower size). This shallow two-chambered nectary was not seen.

Abundant nectar is reported for some Cyclopogon species (Singer and Cocucci 1999; Wiemer et al. 2009), but this was not the case of C. guayanensis, in which neither the capillary tube and nor paper filter methodologies were capable of collecting nectar. Nectar is secreted by the papillae on the inner surface of the labellum, particularly along its central groove, in minute quantities. Similar papillae have been detected in C. apricus (Adachi 2015) and C. dutrae (Galetto et al. 1997). Nectar secreting papillae on the inner surface of the labellum have also been found in the ghost orchid, Epipogium aphyllum Sw., subtribe Epipogiinae, tribe Gastrodieae, subfamily Epidendroideae, by Swięczkowska and Kowalkowska (2015), a northern temperate mycotrophic orchid pollinated by Bombus bees. Nectar is secreted along the central groove of the labellum of another mycotrophic orchid, European Epipactis atropurpurea Raf. (Pais and Figueiredo 1994), tribe Neottiae, subfamily Epidendroideae (Chase et al. 2015), which has, however, epidermal nectariferous tissue along the groove and not papillae.

Nectariferous tissue is composed of epidermal, specialized parenchymatic cells present on the surfaces of plant tissue that are usually either elevated or sunken (Fahn 1988; Galetto et al. 1997). In many orchids, the nectariferous tissue contains starch grains in the pre-secretory stage, which is the energy source to produce the nectar (Pais and Figueiredo 1994; Stpiczyńska and Matusiewicz 2001). Starch grains are frequent in angiosperm nectaries, and vascular bundles also can occur, although not necessarily (Fahn 1988, 1989). Nectar-secreting papillae (secreting minute quantities of nectar) have also recorded the Dactylorhiza maculata complex, tribe Orchidieae, subfamily Orchidoideae, but these are in the spur (Naczk et al. 2018) not on the labellum. Papillae secreting “floral rewards” (a mixture of carbohydrates, lipids and proteins that feed its midge pollinators - Ceratopogonidae, Diptera) have also been recorded on the labellum of the neotropical genus Trichosalpinx Luer, subtribe Pleurothallidinae, tribe Epidendreae, subfamily Epidendroideae (Bogarín et al. 2018). Thus, the exact combination of nectariferous papillae on the inner surface of a labellum that does not form a spur (Fig. 4M-N, arrows), associated to the confirmed presence of nectar sugars is, as far as we are aware, a novelty in subfamily Orchidoideae. Papillae conclusively shown to produce nectar have been recorded in the Orchidoideae, but these are all within spurs, i.e., in several genera of subfamily Orchidoideae, subtribe Orchidinae (Bell et al. 2009). In spurless Cyclopogon apricus, similar papillae have been detected and these were interpreted as nectaries (Adachi 2015). Field studies on the reproductive biology of C. apricus have been done and have shown that it secretes an exsudate considered by the authors to be nectar (Sazima and Cocucci 1999). We consider it highly likely that the secretion produced by C. apricus is nectar, and that its papillae are analogous to the nectariferous papillae of C. guayanensis.

The early literature stated that floral nectar is mostly composed of varying proportions of sucrose, glucose and fructose (Percival 1961; Baker and Baker 1983). This composition has been widely supported by later research (Chalcoff et al. 2017), although other components, such as amino acids, can also be important in the plant-pollinator interaction (Gottsberger et al. 1984; Petanidou et al. 2006; Brzosko and Mirski 2021). Baker and Baker (1983) proposed a classification of floral nectar into four types based on an “r” ratio of sucrose/fructose + glucose: 1) sucrose dominant (r > 0.99); 2) sucrose rich (r = 0.5–0.99); 3) hexose rich (r = 0.1–0.5); 4) hexose dominant (r < 0.1). Floral nectar sugar composition (and its associated ecological drivers) were recently reviewed for the angiosperms by Chalcoff et al. (2017) and for the Orchidaceae by Brzosko and Mirski (2021).

The nectar of Cyclopogon guayanensis is the second record of a hexose-dominant nectar (type 4 in the system of Baker and Baker 1983) in the Orchidaceae. The first published record of a hexose dominant nectar in the Orchidaceae is very recent, of Prasophyllum innubum D.L. Jones (Hayashi et al. 2024). P. innubum shows remarkable similarities to C. guayanense (Table 3). A taxonomically updated list of nectar types in the Orchidaceae, mostly based on Chalcoff et al. (2017) and Brzosko and Mirski (2021) with some additional records, is presented in Table 2.

Table 2

Orchidaceae with known nectar composition and their pollinator groups ordered by subfamily and tribe. Nectar composition in average values when more than one individual or flower was sampled per taxon. EP = subfamily Epidendroideae; OR = subfamily Orchidoideae; VA = subfamily Vanilloideae; Coll = tribe Collabieae; Cran = tribe Cranichideae; Cymb = tribe Cymbidieae; Dend = tribe Dendrobieae; Diur = tribe Diurideae; Neot = tribe Neottieae; Orch = tribe Orchideae; Vand = tribe Vandeae; Vani = tribe Vanilleae. * name updated to follow Plants of the World Online (https://powo.science.kew.org); r = sucrose/(fructose + glucose); nectar class follows Baker & Baker (1983).

Adapted from Brzosko & Mirsky (2021) and Chalcoff et al. (2017) with additions.
Higher taxa	Species, r value and nectar class	Visitors	References
EP, Coll	Calanthe angustifolia (Blume) Lindl. r = 3.27 = sucrose dominant	Unknown	Freeman et al. (1991)
EP, Cymb	Eulophia alta (L.) Fawc. & Rendle r = 0.8 = sucrose rich	Hymenoptera (Bees)	Jürgens et al. (2009)
EP, Cymb	Eulophia cochlearis Lindl. * r = 2.33 = sucrose dominant	Hymenoptera (Bees)	Peter & Johnson (2009)
EP, Cymb	Maxillaria anceps Ames & C.Schweinf. r = 70.86 = sucrose dominant	Hymenoptera (Bees)	Davies et al. (2005)
EP, Cymb	Ornithidium fulgens ((Rchb.f.) L.O.Williams r = 13.7 = sucrose dominant	Hummingbirds	Lipińska et al. (2022)
EP, Dend	Dendrobium spp (34 species from SE Asia) sucrose dominant	Hymenoptera (Bees)	Jia & Huang (2022)
EP, Neot	Epipactis helleborine (L.) Crantz r (natural areas) = 1.5–2.5 = sucrose dominant r (disturbed areas) = 0.7 = sucrose rich	Hymenoptera (mainly)	Brzosko et al. (2023)
EP, Neot	Epipactis atrorubens (Hoffm.) Besser * r = 0.27 = hexose rich	Insects (several orders)	Pais & Neves (1980)
EP, Neot	Neottia ovata (L.) Hartm. r = 0.14–0.3 = hexose rich	Insects (several orders)	Brzosko et al. (2021)
EP, Vand	Cleisostoma lecongkietii Tich & Aver. * r = 20.89 = sucrose dominant	Hymenoptera (Bees)	Ponert et al. (2016)
EP, Vand	Rhipidoglossum caffrum (Bolus) Farminhão & Stévart * r = 4.26 = sucrose dominant	Lepidoptera (Moths)	Luyt (2002)
OR, Cran	Cyclopogon dutrae Schltr. r = 8.17 = sucrose dominant	Hymenoptera (Halictidae)	Galetto et al. (1997)
O, Cran	Cyclopogon guayanensis (Lindl.) B.M. Carvalho & Meneguzzo r = 0.06 = hexose dominant	Hymenoptera, Diptera (Apidae, Halictidae) (Syrphidae)	This study
OR, Cran	Goodyera repens (L.) R.Br. r = 0.18–0.44 = hexose rich	Hymenoptera (Bombus)	Brzosko et al. (2023)
OR, Cran	Sacoila lanceolata (Aubl.) Garay r = 0.50 = hexose rich	Trochilidae (Hummingbirds)	Galetto et al. (1997)
OR, Diur.	Caladenia arenaria Fitzg. r = 19.0 = sucrose dominant	Hymenoptera (Wasps)	Reiter et al. (2019)
OR, Diur.	Caladenia colorata D.L.Jones r = 19.0 = sucrose dominant	Hymenoptera (Wasps)	Reiter et al. (2019)
OR, Diur.	Caladenia nobilis Hopper & A.P.Br. r = 1.0 = sucrose dominant	Hymenoptera (Wasps)	Phillips et al. (2020)
OR, Diur.	Caladenia paludosa Hopper & A.P.Br. r = 2.37 = sucrose dominant	Hymenoptera (Wasps)	Phillips et al. (2020)
OR, Diur	Caladenia robinsonii G.W.Carr. r = 58.5 = sucrose dominant	Hymenoptera (Wasps)	Phillips et al. (2024)
OR, Diur.	Caladenia versicolor G.W.Carr r = 19.0 = sucrose dominant	Hymenoptera (Wasps)	Reiter et al. (2019)
OR, Diur.	Caladenia xanthochila D.Beards. & C.Beards. r = 100 = sucrose pure	Hymenoptera (Wasps)	Reiter et al. (2023)
OR, Diur.	Prasophyllum innubum D.L.Jones r = 0.01 = hexose dominant	Insects (several orders)	Hayashi et al. (2024)
OR, Orch	Bonatea cassidea Sond. r = 67.5 = sucrose dominant	Lepidoptera (Butterflies)	Balducci et al. (2019)
OR, Orch	Bonatea polypodantha (Rchb.f.) L.Bolus r = 4.34 = sucrose dominant	Lepidoptera (Moths)	Balducci et al. (2020)
OR, Orch	Dactylorhiza incarnata (L.) Soó r = 2.63 = sucrose dominant	Hymenoptera	Naczk et al. (2018)
OR, Orch	Dactylorhiza maculata var. fuchsii (Druce) Hyl. r = 5.06 = sucrose dominant	Coleoptera, Diptera	Gutowski (1990); Naczk et al. (2018)
OR, Orch	Dactylorhiza maculata (L.) Soó var. maculata r = 4.82 = sucrose dominant	Hymenoptera	Naczk et al. (2018); Koivisto, Valius & Salonen (2002)
OR, Orch	Dactylorhiza majalis (Rchb.) P.F.Hunt & Summerh. r = 3.63 = sucrose dominant	Diptera, Coleoptera Hymenoptera	Naczk et al. (2018)
OR, Orch	Disa cooperi Rchb.f. r = 0.89 = sucrose rich	Lepidoptera (Moths)	Johnson (1995); Johnson (2006)
OR, Orch	Elleanthus brasiliensis (Lindl.) Rchb.f. r = 27.99 = sucrose dominant	Trochilidae (Hummingbirds)	Nunes et al. (2013)
OR, Orch	Gymnadenia conopsea (L.) R.Br. r = 4.2 = sucrose dominant	Lepidoptera (Butterflies)	Gijbels et al. (2014)
OR, Orch	Habenaria hieronymi Kraenzl. r = 0.19 = hexose rich	Hymenoptera	Galetto et al. (1997)
OR, Orch	Habenaria gourlieana Gillies ex Lindl. r = 5.9–22.26 = sucrose dominant	Lepidoptera (Hawkmoths)	Galetto et al. (1997)
OR, Orch	Habenaria obtusa Lindl. * r = 27.5 = sucrose dominant	Lepidoptera	Gottsberger et al. (1984)
OR, Orch	Pelexia bonariensis (Lindl.) Schltr. r = 1.70 = sucrose dominant	Hymenoptera (Bombus)	Galetto et al. (1997); Buzatto et al. (2022)
VA, Vani	Vanilla hartii Rolfe r = 5.64 = sucrose dominant	Hymenoptera (Euglossa)	Watteyn et al. (2023)

Table 3

Comparative characteristics of the only two species of orchids known to produce hexose-rich nectar
Functional trait	P. innubum	C. guayanense	Comparison
Geographic distribution	Narrow endemic	Widespread	different
Habit	Geophyte	Geophyte	equal
Habitat	Grasslands and peatlands by streams	Highland grasslands, savannas and pastures	similar
Flowering populations	Dense	Dense	similar
Flowering plant size	30-90cm	9-40cm	overlapping
Flowers per spike	6–20 flowers	(9–)30–120(–160)	overlapping
Flower colour	Petals white or purplish Labellum white or pink	Petals creamy white Labellum yellowish cream	similar similar
Flower size	6-9mm across; Labellum 7–9 x 3-3.5mm	4–6 across; Labellum 3.5–4.3 x 1.8-2.8mm	overlapping different
Flower resupination	Not resupinate	Resupinate	different
Visitors observed with pollinaria	Bees: Apidae, Halictidae, Wasps (1 species)	Bees: Apidae, Halictidae	similar

Hexose-dominant nectar is rare in orchids (Table 2), but is the same true outside of the orchids? To address this question, we segregated all the species with hexose-dominant nectars (but at least 1% sucrose) that were herbs, shrubs or subshrubs, and pollinated by either bees or generalists from the list of 1214 angiosperm with data on nectar sugars compiled by Chalcoff et al. (2017). This segregate list recorded 45 species in ten botanical families that were widely scattered across the angiosperm phylogenetic tree. We then investigated these 45 species in Plants of the World (POWO 2024) and reliable identifications in the I-naturalist site and found that they followed a loose pattern (with some exceptions). White, cream, yellow or orange were the common colours, while pink, violet or purple were rare, and red was absent. Most of them had small, densely aggregated flowers, but there were some exceptions to this norm (e.g., Tecoma stans (L.) Juss. ex Kunth). Virtually all were temperate or subtropical plants, although the plants listed by Chalcoff et al. (2017) were mostly tropical. In a semi-desertic, frost-prone scrubland in Patagonia, presumably pollinator hostile, most plants had hexose-dominant nectars and were bee-pollinated (Bernardello et al. 1999). We propose the term ‘modest’ pollination syndrome be applied to orchids that are: geophytic, have small, pale flowers (probably below 1 cm), offer imperceptible quantities of hexose-dominant nectar on an exposed labellum and attract a wide range of insect visitors.

Considering the high species richness of the Orchidaceae, nectar records are surprisingly few (Brzosko et al. 2021). Sucrose-dominant nectar predominates both in the angiosperms (56.8% of 1214 species; Chalcoff et al. 2017) and in the Orchidaceae (80.5% of 43 species; Brzosko and Mirski 2021). Pollinator group was statistically confirmed in both studies as the main ecological driver, followed by climate, i.e., latitudinal climatic zone (for the angiosperms) or climate type (for the orchids).

Baker and Baker (1983), studying nectar composition in 765 species of angiosperms, found that 44% of species pollinated by short-tongued bees produced hexose-dominant nectar, while only 6% of species pollinated by long-tongued bees had this type of nectar. Wilmer (2011) has argued that sucrose/hexose ratios may have more to do with maintaining optimum nectar concentration and viscosity than pollinator preference. Pure sucrose nectar dries out faster than pure hexose nectar at the same humidity levels (Corbet et al. 1979). Therefore, hexose-rich nectar might be preferentially produced by exposed nectaries (pollinated by short-tongued insects), and sucrose-rich nectar by deep, hidden nectaries (pollinated by long-tongued insects). However, Brzosko and Mirski (2021) found no statistical differences of sugar composition between orchids that offer spur nectar and open nectar, although these authors noted that generalist species were under-represented in the available data. It is possible that when nectar is produced not only by exposed nectaries, but in minute quantities in plants that form dense populations (such as was recorded in Cyclopogon guayanensis and Prasophyllum innubum), natural selection will favour high-hexose nectar that is more resistant to desiccation. It is worth mentioning in this context that C. guayanensis flowers in early wet season when mean daily humidity is very high and is also a generalistic species, with a wider taxonomic range of bee pollinators than any other species in either the Pelexia or the Cyclopogon clades sensu Salazar et al. (2018) with known pollinators (Supporting information, Table S2).

The glandular trichomes that abound on Cyclopogon guayanensis inflorescences and outer surfaces of the flowers are lipid secreting (Fig. 4L); these lipids may be acting as aromatic attractants as recorded in C. elatus (Sw.) Schltr., by Wiemer et al. (2009) or as rewards for oil-collecting or scent-collecting bees. Pachygonium pteryganthum (Rchb. f. & Warm.) Schltr., a member of the Pelexia clade, is pollinated by oil-collecting female Centris (Melacentris) bees (Buzatto et al. 2022). In our study area, male Tetrapedia bees are known to collect oil from Malpighiaceae flowers (Cappellari et al. 2012) and we observed a male Tetrapedia bee visiting the flowers of C. guayanensis, but no such oil-collecting activity was observed. The Tetrapedia bee was foraging for nectar, and is presumed to be a pollinator, as it gathered three pollinaria during visits, which it subsequently tried to remove with front legs. Volatile chemicals (scent) can also be collected for mating purposes by male Neotropical orchid bees, i.e. Euglossini (Carvalho-Filho 2010). Despite the high density of glandular trichomes on the C. guayanensis inflorescences, there was no evidence of this kind of reward collecting by pollinators in our study; Euglossini visitors were not observed in the field and glandular trichomes were intact, not destroyed by visitors, with the cuticle on the heads undamaged (Fig. 4G, J–L).

A sweet scent was reported in the flowers of Cyclopogon congestus (Vell.) Hoehne (Singer and Sazima 1999), C. diversifoIius (Cogn.) Schltr. (= C. apricus (Lindl.) Schltr.) (Singer and Cocucci 1999), C. elatus (Wiemer et al. 2009) and Veyretia hassleri (Cogn.) Szlach. (Supporting Information, Table S2). In C. congestus, however, the scent, which was perceived from the morning to the evening hours, was produced by apical callosities on the inner surface of the labellum and lateral sepals (Singer and Sazima 1999), and is thus not analogous to the multicellular, glandular trichomes observed on the outer surface of the corolla in C. guayanensis.

In Cyclopogon elatus, osmophores are reported as trichomes on the outer surface of the labellum (Wiemer et al. 2009) such as was recorded by us in C. guayanensis. However, in C. elatus these trichomes are unicellular, while in C. guayanensis they are pluricellular, with stalk and head, but also occur on the outer surface of the labellum (as well as on other inflorescence surfaces). Also, in C. elatus, only the osmophores of open flowers release scent, which is absent in buds and withered flowers (Wiemer et al. 2009). Thus, C. guayanensis also differs from C. elatus in this aspect, since secretion appears to persist in senescent flowers (Fig. 4D).

The combined evidence suggests that the glandular trichomes on the inflorescence and outer surface of the flowers of Cyclopogon guayanensis are probably osmophores. However, the scent detected in C. guayanensis was extremely faint and was in fact only perceived by one of us; therefore, the function and composition of the lipids secreted by these trichomes should be the subject of further investigation. The external position on the flower is unusual for osmophores; the inner surface of the labellum is the most widespread scent-releasing surface in orchids (Wiemer et al. 2009). In fact, very few angiosperms have osmophores on the outer surface of the flowers, as seems to be the case in Cyclopogon; it was also reported in Cotylolabium lutzii (Pabst) Garay, another genus of the Spiranthinae that is sister taxon to the rest of the Subtribe (Borba et al. 2014). Perhaps the presence of osmophores on the outer surface of the flowers is a common characteristic of this subtribe; this hypothesis would require further investigation.

The cuticle allows both scent diffusion in osmophores and secretion releasing in nectaries; the cuticle expands and there is secretion accumulation in the periplasmic space; this phase was clearly recorded in the nectariferous papillae of Cyclopogon guayanensis in our study (Fig. 4M–N, arrows). In the secretory labellum tissue of Epipactis atropurpurea, the nectar is released by disruption of the epidermal cell cuticle in the secretory stage (Pais and Figueiredo 1994). In the nectariferous papillae of cotton (Gossypium hirsutum L., Malvaceae), however, a two-layered cuticle is detached from the wall in the secretory papillae and the nectar crosses this structure (Eleftheriou and Hall 1983); further studies are needed to clarify the nectar releasing mechanism in C. guayanensis.

Raphid-rich idioblasts have been presumed to be herbivore deterrents (Molano-Flores 2001). Raphid containing idioblasts are not widespread in dicotyledons (Cutler et al. 2008) but are found in many monocotyledons (Fahn 1988) and were observed in the bracteoles (Fig. 4F–G), sepals and petals (Fig. 4H) of Cyclopogon guayanensis. Raphid-rich idioblasts occur in several species of orchids, both in subfamilies Orchidoideae and Epidendroideae. In the Orchidoideae, they are found in the floral parts of Habenaria gourlieana Gillies ex Lindl. (Galetto et al. 1997), and in the hypochilum of Cotylolabium lutzii (Borba et al. 2014) – also a member of subtribe Spiranthinae and were noted to be common in this subtribe by Adachi (2015), based on studies of floral anatomy of Cyclopogon apricus, Mesadenella cuspidata (Lindl.) Garay, Sarcoglottis fasciculata, and Sauroglossum elatum. In subfamily Epidendroideade, they occur in the ghost orchid, Epipogium aphyllum (Swięczkowska and Kowalkowska 2015). However, the density of idioblasts is noticeably higher in the sepals and petals of C. guayanensis (Fig. 4H, arrowheads) than in most of these orchids. In many orchids of the subtribe Pleurothallidinae, prismatic crystals occur as idioblasts in the floral parts and have been associated to visual attraction of pollinators (Bogarín et al. 2018), but herbivore dissuasion is another possible role.

The fact that herbaceous or shrubby plants with hexose-dominant nectar and generalistic pollination systems are usually confined to temperate or subtropical environments (Chalcoff et al. 2017) but appeared in a tropical savanna grassland species is interesting. In the highly seasonal Cerrado biome where our study was carried out, 90% of annual precipitation occurs between the months of October and April, followed by a dry season lasting between 4 and 7 months (Bustamente et al. 2012); in the Distrito Federal, c. 50% of annual precipitation occurs between December and February (Anunciação et al. 2014). We tentatively suggest that a short favourable reproductive season is the driver behind this suite of characters. Its similarity to Epipogium aphyllum suggests convergence with these orchids (Swiwczkowska and Kowalkowska 2015).

Our theory for the unusual characteristics of C. guayanensis is that, as a therophytic orchid that inhabits fire-prone, highly seasonal savanna grasslands, it will presumably be under two selective forces: 1) lack of competition, since grasslands are potentially resource-poor landscapes for pollinating insects and will tend to have low pollinator density, therefore favouring a “take what you can get” generalist pollination strategy offering scanty rewards; 2) a limited growing and flowering season (during the short wet season) which would put a premium on economy of energy, i.e., favour small plants with small flowers, small fruits, and minimalistic pollinator rewards.

The floral biology (position of the viscidium and pollinator behaviour) and micromorphological data gathered in this study (external glandular hairs, raphid-rich idioblasts, internal nectariferous papillae) support the transfer of Brachystele guayanensis to Cyclopogon (Meneguzzo et al. 2024). Our hypothesis is that the C. guayanensis – Veyretia clade is a lineage within Cyclopogon (a predominantly forest genus well adapted to pollination by halictid bees) that has moved into open habitats with accompanying pollinator shifts (i.e., from Halictidae to Apidae and possibly from Halictidae to Megachilidae, respectively; see Table S2), although this would need further investigation of the floral biology of Veyretia. Studies of species of Brachystele s.s. would obviously also be desirable to confirm parallel evolution.

To conclude, on a more general note, we would suggest further testing for trace nectar in orchids that apparently offer no rewards. We cite verbatim a paragraph from the study by Shrestha et al. (2020: 5) on rewardlessness in orchids that reflects our beliefs: If neural mechanisms of reward detection can be exploited and are widespread among floral visitors, the use of such stingy rewards by orchids lacking visible nectar might also be widespread. Pollinator manipulation by trace rewards might still be considered largely deceitful, but the nature of the deceit would be more complex and more subtle than in the case of complete absence of floral sugars. A paucity of reward may actually encourage appropriate flower-handling by visitors.

ACKNOWLEDGEMENTS

We thank Prof. Rodrigo Barbosa Gonçalves (Universidade Federal do Paraná) for identifying or confirming the identification of several of the bees.

Funding

FAPERJ (Fundação de Amparo à Pesquisa do Rio de Janeiro) funded a post-doctoral grant and financial support (grants n. E-26/206.092/2022 and E-26/206.093/2022) to TECM. CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil) funded PQ2 Produtividade em Pesquisa research grants to CEBP and JANB.

Competing interests

The authors declare no competing interests.

Author contributions

TECM and CEBP conceived the project and designed the experiments. CEBP, AJCA and ACM did the field work, produced images and videos, and incorporated voucher specimens of plants and insects into scientific collections. ACM and AJCA identified the insects. SMG did the anatomical and histochemical analysis. TCRW analyzed the nectar sugars. CEBP wrote the manuscript with significant contributions from TECM, JANB, and SMG. All authors contributed to revisions and accepted the final version of this manuscript.

Supporting information

Additional supporting information may be found online in the Supporting Information section at the end of the article. Tables S1 and S2.

Adachi SA (2015) Estrutura floral de representantes da tribo Cranichideae (Orchidoideae: Orchidaceae). Universidade Estadual Paulista “Júlio de Mesquita Filho”
Anunciação YMT da, Walde DHG, Rocha RP da (2014) Observed summer weather regimes and associated extreme precipitation over Distrito Federal, west-central Brazil. Environ Earth Sci 72:4835–4848. https://doi.org/10.1007/s12665-014-3607-9
Baker HG, Baker I (1983) Floral nectar sugars constituents in relation to pollinator type. In: Jones CE, Little RJ (eds) Handbook of experimental pollination biology. Van Nostrand Reinhold, New York, pp 117–141
Balducci MG, van der Niet T, Johnson SD (2019) Butterfly pollination of Bonatea cassidea (Orchidaceae): Solving a puzzle from the Darwin era. South African J Bot 123:308–316. https://doi.org/10.1016/j.sajb.2019.03.030
Balducci MG, Van Der Niet T, Johnson SD (2020) Diel scent and nectar rhythms of an African orchid in relation to bimodal activity patterns of hawkmoth pollinators. Ann Bot 126:1155–1164. https://doi.org/10.1093/aob/mcaa132
Bell AK, Roberts DL, Hawkins JA, et al (2009) Comparative micromorphology of nectariferous and nectarless labellar spurs in selected clades of subtribe Orchidinae (Orchidaceae). Bot J Linn Soc 160:369–387. https://doi.org/10.1111/j.1095-8339.2009.00985.x
Benitez-Vieyra S, Medina AM, Glinos E, Cocucci AA (2006) Pollinator-mediated selection on floral traits and size of floral display in Cyclopogon elatus, a sweat bee-pollinated orchid. Funct Ecol 20:948–957. https://doi.org/10.1111/j.1365-2435.2006.01179.x
Bernardello G, Galetto L, Forcone A (1999) Floral nectar chemical composition of some species from Patagonia. II. Biochem Syst Ecol 27:779–790. https://doi.org/10.1016/S0305-1978(99)00029-0
Bogarín D, Fernández M, Borkent A, et al (2018) Pollination of Trichosalpinx (Orchidaceae: Pleurothallidinae) by biting midges (Diptera: Ceratopogonidae). Bot J Linn Soc 186:510–543. https://doi.org/10.1093/botlinnean/box087
Borba EL, Salazar GA, Mazzoni-Viveiros S, Batista JAN (2014) Phylogenetic position and floral morphology of the Brazilian endemic, monospecific genus Cotylolabium: A sister group for the remaining Spiranthinae (Orchidaceae). Bot J Linn Soc 175:29–46. https://doi.org/10.1111/boj.12136
Brzosko E, Bajguz A, Burzyńska J, Chmur M (2023) Does Reproductive Success in Natural and Anthropogenic Populations of Generalist Epipactis helleborine Depend on Flower Morphology and Nectar Composition? Int J Mol Sci 24:4276. https://doi.org/10.3390/ijms24054276
Brzosko E, Bajguz A, Chmur M, et al (2021) How are the flower structure and nectar composition of the generalistic orchid Neottia ovata adapted to a wide range of pollinators? Int J Mol Sci 22:1–27. https://doi.org/10.3390/ijms22042214
Brzosko E, Mirski P (2021) Floral nectar chemistry in orchids: A short review and meta-analysis. Plants 10:2315. https://doi.org/10.3390/plants10112315
Bustamante MMC, Nardoto GB, Pinto AS, et al (2012) Potential impacts of climate change on biogeochemical functioning of Cerrado ecosystems. Brazilian J Biol 72:655–671
Buzatto CR (2014) Estudos taxonômicos, filogenéticos e biossistemáticos em orquídeas terrestres (Orchidaceae: Orchidoideae) brasileiras. Universidade Federal do Rio Grande Sul
Buzatto CR, Nervo MH, Sanguinetti A, et al (2022) Efficient pollination and high reproductive success in two Brazilian Spiranthinae orchids: Insights on the evolutionary history of pollination within the Pelexia clade. Plant Species Biol 37:182–196. https://doi.org/10.1111/1442-1984.12366
Cappellari SC, Melo GAR, Aguiar AJC, Neff JL (2012) Floral oil collection by male Tetrapedia bees (Hymenoptera: Apidae: Tetrapediini. Apidologie 43:39–50. https://doi.org/10.1007/s13592-011-0072-2
Carvalho Filho F da S (2010) Scent-robbing and fighting among male orchid bees, Eulaema (Apeulaema) nigrita Lepeletier, 1841 (Hymenoptera: Apidae: Euglossini). Biota Neotrop 10:405–408. https://doi.org/10.1590/s1676-06032010000200038
Chalcoff VR, Gleiser G, Ezcurra C, Aizen MA (2017) Pollinator type and secondarily climate are related to nectar sugar composition across the angiosperms. Evol Ecol 31:585–602. https://doi.org/10.1007/s10682-017-9887-2
Chase MW, Cameron KM, Freudenstein J V., et al (2015) An updated classification of Orchidaceae. Bot J Linn Soc 177:151–174. https://doi.org/10.1111/boj.12234
Corbet SA, Unwin DM, Prys‐Jones OE (1979) Humidity, nectar and insect visits to flowers, with special reference to Crataegus, Tilia and Echium. Ecol Entomol 4:9–22. https://doi.org/10.1111/j.1365-2311.1979.tb00557.x
Cutler DF, Botha T, Stevenson DW (2008) Plant anatomy: An applied approach. Blackwell Publishing Ltd, Malden, Massachusetts, USA
Davies KL, Stpiczyńska M, Gregg A (2005) Nectar-secreting floral stomata in Maxillaria anceps Ames & C. Schweinf. (Orchidaceae). Ann Bot 96:217–227. https://doi.org/10.1093/aob/mci182
Eleftheriou EP, Hall JL (1983) The extrafloral nectaries of cotton: I. Fine structure of the secretory papillae. J Exp Bot 34:103–119. https://doi.org/10.1093/jxb/34.2.103
Fahn A (1989) Plant anatomy. Pergamon Press, Oxford
Fahn A (1988) Secretory tissues in vascular plants. New Phytol 108:229–257. https://doi.org/10.1111/j.1469-8137.1988.tb04159.x
Forcone A, Galetto L, Bernardello L (1997) Floral nectar chemical composition of some species from Patagonia. Biochem Syst Ecol 25:395–402. https://doi.org/10.1016/S0305-1978(97)00030-6
Freeman CE, Worthington RD, Jackson MS (1991) Floral Nectar Sugar Compositions of Some South and Southeast Asian Species. Biotropica 23:568. https://doi.org/10.2307/2388394
Galetto L (1995) Estructura del nectario y composición posición química del néctar en cuatro especies de Scrophulariaceae. Kurtziana 24:105–118
Galetto L, Bernardello G (2003) Nectar sugar composition in angiosperms from Chaco and Patagonia (Argentina): An animal visitor’s matter? Plant Syst Evol 238:69–86. https://doi.org/10.1007/s00606-002-0269-y
Galetto L, Bernardello G, Rivera GL (1997) Nectar, nectaries, flower visitors, and breeding system in five terrestrial Orchidaceae from central Argentina. J Plant Res 110:393–403. https://doi.org/10.1007/bf02506798
Gijbels P, Van den Ende W, Honnay O (2014) Landscape scale variation in nectar amino acid and sugar composition in a Lepidoptera pollinated orchid species and its relation with fruit set. J Ecol 102:136–144. https://doi.org/10.1111/1365-2745.12183
Gottsberger G, Schrauwen J, Linskens HF (1984) Amino acids and sugars in nectar, and their putative evolutionary significance. Plant Syst Evol 145:55–77. https://doi.org/10.1007/BF00984031
Gutowski JM (1990) Pollination of the orchid Dactylorhiza fuchsii by longhorn beetles in primeval forests of Northeastern Poland. Biol Conserv 51:287–297. https://doi.org/10.1016/0006-3207(90)90114-5
Jia LB, Huang SQ (2022) An examination of nectar production in 34 species of Dendrobium indicates that deceptive pollination in the orchids is not popular. J Syst Evol 60:1371–1377. https://doi.org/10.1111/jse.12799
Johnson SD (1995) Observations of hawkmoth pollination in the South African orchid Disa cooperi. Nord J Bot 15:121–125. https://doi.org/10.1111/j.1756-1051.1995.tb00128.x
Jürgens A, Bosch SR, Webber AC, et al (2009) Pollination biology of Eulophia alta (Orchidaceae) in Amazonia: Effects of pollinator composition on reproductive success in different populations. Ann Bot 104:897–912. https://doi.org/10.1093/aob/mcp191
Käpylä M (1978) Amount and type of nectar sugar in some wild flowers in Finland. Ann Bot Fenn 15:85–88
Koivisto AM, Vallius E, Salonen V (2002) Pollination and reproductive success of two colour variants of a deceptive orchid, Dactylorhiza maculata (Orchidaceae). Nord J Bot 22:53–58. https://doi.org/10.1111/j.1756-1051.2002.tb01621.x
Lipińska MM, Archila FL, Haliński ŁP, et al (2022) Ornithophily in the subtribe Maxillariinae (Orchidaceae) proven with a case study of Ornithidium fulgens in Guatemala. Sci Rep 12:5273. https://doi.org/10.1038/s41598-022-09146-4
Luyt R, Johnson SD (2001) Hawkmoth pollination of the African epiphytic orchid Mystacidium venosum, with special reference to flower and pollen longevity. Plant Syst Evol 228:49–62
Luyt RP (2002) Pollination and Evolution of the genus Mystacidium (Orchidaceae). University of Natal, South Africa
Meneguzzo TEC, Carvalho BM, Batista JAN, et al (2024) Brachystele guayanensis is a Cyclopogon (Orchidaceae): notes on its biology and taxonomy. Phytotaxa 658:109–119. https://doi.org/10.11646/phytotaxa.658.1.5
Molano-Flores B (2001) Herbivory and calcium concentrations affect calcium oxalate crystal formation in leaves of Sida (Malvaceae). Ann Bot 88:387–391. https://doi.org/10.1006/anbo.2001.1492
Morrant DS, Schumann R, Petit S (2009) Field methods for sampling and storing nectar from flowers with low nectar volumes. Ann Bot 103:533–542. https://doi.org/10.1093/aob/mcn241
Naczk AM, Kowalkowska AK, Wisniewska N, et al (2018) Floral anatomy, ultrastructure and chemical analysis in Dactylorhiza incarnata/maculata complex (Orchidaceae). Bot J Linn Soc 187:512–536. https://doi.org/10.1093/botlinnean/boy027
Pais MS, Figueiredo ACS (1994) Floral nectaries from Limodorum abortivum (L.) Sw and Epipactis atropurpurea Rafin (Orchidaceae): Ultrastructural changes in plastids during the secretory process. Apidologie 25:615–626. https://doi.org/10.1051/apido:19940612
Pais MSS, Neves HJC das (1980) Sugar Content of the Nectary Exudate of Epipactis atropurpurea Rafin. Apidologie 11:39–45. https://doi.org/10.1051/apido:19800105
Percival MS (1961) Types of Nectar in Angiosperms. New Phytol 60:235–281. https://doi.org/10.1111/j.1469-8137.1961.tb06255.x
Petanidou T, Van Laere A, N. Ellis W, Smets E (2006) What shapes amino acid and sugar composition in Mediterranean floral nectars? Oikos 115:155–169. https://doi.org/10.1111/j.2006.0030-1299.14487.x
Peter CI, Johnson SD (2009) Reproductive biology of Acrolophia cochlearis (Orchidaceae): Estimating rates of cross-pollination in epidendroid orchids. Ann Bot 104:573–581. https://doi.org/10.1093/aob/mcn218
Phillips RD, Bohman B, Brown GR, et al (2020) A specialised pollination system using nectar-seeking thynnine wasps in Caladenia nobilis (Orchidaceae). Plant Biol 22:157–166. https://doi.org/10.1111/plb.13069
Phillips RD, Bohman B, Peakall R, Reiter N (2024) Sexual attraction with pollination during feeding behaviour: implications for transitions between specialized strategies. Ann Bot 133:273–286. https://doi.org/10.1093/aob/mcad178
Ponert J, Trávníček P, Vuong TB, et al (2016) A new species of Cleisostoma (Orchidaceae) from the Hon Ba Nature Reserve in Vietnam: A multidisciplinary assessment. PLoS One 11:. https://doi.org/10.1371/journal.pone.0150631
POWO (2024) Plants of the World Online. Facilitated by the Royal Botanic Gardens, Kew. In: Http://Www.Plantsoftheworldonline.Org/. http://www.plantsoftheworldonline.org/. Accessed 1 Apr 2024
Reiter N, Bohman B, Freestone M, et al (2019) Pollination by nectar-foraging thynnine wasps in the endangered Caladenia arenaria and Caladenia concolor (Orchidaceae). Aust J Bot 67:490–500. https://doi.org/10.1071/BT19033
Reiter N, Wicks M, Pollard G, et al (2023) Improving conservation and translocation success of an endangered orchid, Caladenia xanthochila (Orchidaceae), through understanding pollination. Plant Ecol 224:715–727. https://doi.org/10.1007/s11258-023-01334-0
Salazar GA, Batista JAN, Cabrera LI, et al (2018) Phylogenetic systematics of subtribe Spiranthinae (Orchidaceae: Orchidoideae: Cranichideae) based on nuclear and plastid DNA sequences of a nearly complete generic sample. Bot J Linn Soc 186:273–303. https://doi.org/10.1093/botlinnean/box096
Shrestha M, Dyer AG, Dorin A, et al (2020) Rewardlessness in orchids: how frequent and how rewardless? Plant Biol 22:555–561. https://doi.org/10.1111/plb.13113
Silva KFO e., Melo BCV, Moreira TB, Williams TCR (2021) Darkness and low-light alter reserve mobilization during the initial growth of soybean (Glycine max (L.) Merrill). Theor Exp Plant Physiol 33:55–68. https://doi.org/10.1007/s40626-020-00194-7
Singer RB (2002) The pollination biology of Sauroglossum elatum Lindl. (Orchidaceae: Spiranthinae): Moth-pollination and protandry in neotropical Spiranthinae. Bot J Linn Soc 138:9–16. https://doi.org/10.1046/j.1095-8339.2002.00003.x
Singer RB, Cocucci AA (1999) Pollination mechanism in southern Brazilian orchids which are exclusively or mainly pollinated by halictid bees. Plant Syst Evol 217:101–117. https://doi.org/10.1007/BF00984924
Singer RB, Sazima M (1999) The pollination mechanism in the “Pelexia alliance” (Orchidaceae: Spiranthinae). Bot J Linn Soc 131:249–262. https://doi.org/10.1006/bojl.1999.0270
Stpiczyńska M, Matusiewicz J (2001) Anatomy and ultrastructure of spur nectary of Gymnadenia conopsea (L.) Orchidaceae. Acta Soc Bot Pol 70:267–272. https://doi.org/10.5586/asbp.2001.034
Swięczkowska E, Kowalkowska AK (2015) Floral nectary anatomy and ultrastructure in mycoheterotrophic plant, Epipogium aphyllum Sw. (Orchidaceae). Sci World J 2015:1–11. https://doi.org/10.1155/2015/201702
Szlachetko DL, Rutkowski P (2008) Classification of Spiranthinae, Stenorrhynchidinae and Cyclopogoninae. In: Rutkowski P, Szlachetko DL, Górniak M (eds) Phylogeny and taxonomy of the subtribes Spiranthinae, Stenorrhynchidinae and Cyclopogoninae (Spirantheae, Orchidaceae) in Central and South America. Wydawanictwo Uniwersytetu Gdańskiego, Gdańsk, pp 130–222
Szlachetko DL, Rutkowski P, Mytnik J (2005) Contributions to the taxonomic revision of the subtribes Spiranthinae, Stenorrhynchidinae and Cyclopogoninae (Orchidaceae) in Mesoamerica and the Antilles. Polish Bot Stud 20:3–387
Watteyn C, Scaccabarozzi D, Muys B, et al (2023) Sweet as Vanilla hartii: Evidence for a nectar-rewarding pollination mechanism in Vanilla (Orchidaceae) flowers. Flora Morphol Distrib Funct Ecol Plants 303: 152294. https://doi.org/10.1016/j.flora.2023.152294
Wiemer AP, Moré M, Benitez-Vieyra S, et al (2009) A simple floral fragrance and unusual osmophore structure in Cyclopogon elatus (Orchidaceae). Plant Biol 11:506–514. https://doi.org/10.1111/j.1438-8677.2008.00140.x
Wilmer P (2011) Pollination and floral ecology. Princeton University Press, Princeton, USA

No competing interests reported.

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Cyclopogon Guayanensis is an Unusual Orchid With a Generalistic Pollination System and Hexose Dominant Nectar

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