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Original Article
Pollination ecology of Dracula erythrochaete (Orchidaceae):
brood-site imitation or food deception?
Karen Gil-Amaya1,2,*,, Melania Fernández1,3,, Lizbeth Oses1,, Miguel Benavides-Acevedo4,5,,
David Grimaldi6,, Mario A. Blanco1,7,8, and Adam P. Karremans1
1Centro de Investigación Jardín Botánico Lankester, Universidad de Costa Rica, Cartago, Apartado 302-7050, Costa Rica
2Programa de Posgrado en Biología, Universidad de Costa Rica, San José, Costa Rica
3Department of Plant and Soil Science, Texas Tech University, Lubbock, TX, United States
4Centro para Investigación en Granos y Semillas (CIGS), Universidad de Costa Rica, San José, Costa Rica
5Institut für Biological Data Science, Heinrich-Heine-Universität-Düsseldorf, Düsseldorf, Germany
6Division of Invertebrate Zoology, American Museum of Natural History, New York, NY, United States
7Escuela de Biología, Universidad de Costa Rica, San José, Costa Rica
8Centro de Investigación en Biodiversidad y Ecología Tropical, Universidad de Costa Rica, San José, Costa Rica
*Corresponding author. Centro de Investigación Jardín Botánico Lankester, Universidad de Costa Rica, Cartago, Apartado 302-7050, Costa Rica. E-mail: kgilecologa@gmail.com
ABSTRACT
Pleurothallidinae is the most diverse Neotropical subtribe in Orchidaceae and is almost exclusively pollinated by insects of the order Diptera.
Dracula, a genus of 138 species in the Pleurothallidinae, is known to aract Zygothrica (Drosophilidae) ies, common macrofungi visitors, by
imitating fungal volatile compounds and lamellae. Interestingly, Dracula orchids do not appear to oer any rewards to their oral visitors. While
brood-site imitation of macrofungi has been suggested as their pollination system, the exact behaviour of ies during their extended visits to
the orchid owers has yet to be conrmed. In this study, we document the pollination mechanism of Dracula erythrochaete. We characterize the
oral structures involved in the mechanism using anatomical and morphological evidence. Additionally, through in situ observations and camera
recordings, we describe the insect behaviour. We show that owers of D. erythrochaete share the same group of visitors as nearby macrofungi,
including dierent Zygothrica species, seven of which were determined as eective pollinators. Male and female ies were aracted to the owers
and displayed feeding behaviour. Accordingly, proteins were detected in high concentrations on the papillae at the base of the movable lip and in
papillary trichomes of the sepals, near the column. e concept of brood-site imitation is debated, as no oviposition events were observed and
no eggs were found on the owers. erefore, a mixed strategy of congregation/brood-site imitation and food reward is proposed for pollination.
Keywords: Dracula Luer; Drosophilidae; feeding behaviour; fungal mimicry; histochemistry; myophily; orchid pollination; Pleurothallidinae;
protein secretion; Zygothrica.
INTRODUCTION
e orchid family, with 736 genera and 29 524 species (Pérez-
Escobar et al. 2024), is recognized for its remarkable diversity of
pollinators and pollination systems that include both rewarding
and deceptive strategies (Darwin 1877, van der Pijl and Dodson
1966, Dressler 1981, Ackerman 1985, Ackerman et al. 2023).
In rewarding pollination strategies oral visitors are compen-
sated for their eorts. Rewards can be as diverse as fragrances
(Dressler 1968, Hetherington-Rauth and Ramírez 2016), oils
(Pauw 2006, Blanco et al. 2013), nectar (Ackerman et al. 1994,
Stpiczyska et al. 2003), resins and waxes (Stpiczyska and
Davies 2009, Davies and Stpiczyska 2012), pollen (Kocyan and
Endress 2001), and pseudopollen (Davies et al. 2000; 2004). By
contrast, deceptive pollination strategies involve the imitation of
a resource required by the pollinator (e.g. food, a potential mate,
the substrate for oviposition) with no compensation for the visit
(Jersáková et al. 2006).
According to the latest study by Ackerman et al. (2023), pol-
lination by deceit occurs in 46.1% of orchid species. e most
common deceptive pollination strategies in orchids involve gen-
eralized food deception (Ackerman 1981, Pansarin et al. 2008,
Vale et al. 2011, Caballero-Villalobos et al. 2017, Naczk et al.
2018) and sexual deception (Ayasse et al. 2003, Schiestl et al.
2003, Singer et al. 2004, Blanco and Barboza 2005, Martel et
Received 5 March 2024; revised 19 June 2024; accepted 13 July 2024
© e Author(s) 2024. Published by Oxford University Press on behalf of e Linnean Society of London. All rights reserved. For commercial re-use, please contact reprints@oup.
com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for
further information please contact journals.permissions@oup.com.
Botanical Journal of the Linnean Society, 2024, XX, 1–19
hps://doi.org/10.1093/botlinnean/boae054
Advance access publication 12 September 2024
Original Article
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2 • Gil-Amaya et al.
al. 2016). ese strategies are well documented among orchids
and have evolved independently multiple times, oen within
the same lineage and across dierent lineages within the family
(van der Pijl and Dodson 1966, Cozzolino and Widmer 2005,
Ackerman et al. 2023, Pérez-Escobar et al. 2024, Phillips et al.
2024). Deceptive pollination, in particular, appears to have con-
tributed to the increase in orchid diversity through small shis
in oral traits that may aract a dierent set of pollinators and
promote reproductive isolation (Peakall et al. 2010, Smith 2010,
Whitehead and Peakall 2014, Givnish et al. 2015).
In Orchidaceae, visual and chemical mimicry is involved
in the araction of visitors that favour its reproduction (Dafni
1984, Jersáková et al. 2006, Bohman et al. 2017, Scaccabarozzi et
al. 2018). Mimicry of fungal fruiting bodies and scents has been
reported as a strategy to aract ies in species of a few orchid
genera. Examples include Corybas Salisb. species, with their dark
colour and sculpted lip that mimic mushrooms with a smooth,
concave pileus (Jones 1970), and are pollinated by fungus gnats
(Kelly et al. 2013, Kelly and Gaske 2014) despite the absence of
detectable fungal scents (Han et al. 2022); Cypripedium L. with
spots on the upper surface of foliage that imitate black mould
spots and the odour of Cladosporium Link (Proctor et al. 1996,
Ren et al. 2011); and Dracula Luer, with lamellae of the lip and
fungal scents of Agaricales (Dentinger and Roy 2010, Endara et
al. 2010, Policha et al. 2016).
In Dracula laeurii Luer & Dalström and Dracula felix (Luer)
Luer, no detectable amounts of nectar were found (Endara et al.
2010), although production of specic volatile compounds were
reported in Dracula chestertonii (Rchb. f.) Luer (Kaiser 2006).
e composition of these oral volatiles is dominated by 70% of
fungal constituents (Kaiser 2006). e emission of the volatile
compound oct-1-en-3-ol was also found in D. laeurii, produc-
tion of which is mainly restricted to the lip (Policha et al. 2016).
Moreover, in D. laeurii and D. felix, some yeast species that
grow on the lip and sepals have been isolated and they are sug-
gested to constitute a food reward or modify the scent produced
by the owers (McAlpine 2013). Even though proling of aro-
matic compounds has been carried out in Dracula pollinated by
Drosophilidae ies, the lack of information on ower anatomy
hinders the association of volatiles with the production sites of
putative secretory (e.g. osmophores) or reward structures for
pollinators.
It has been suggested that Drosophilidae ies accidentally
pollinate Dracula owers while inspecting the lip for a potential
oviposition site (Vogel 1978, Kaiser 2006). However, detailed
studies of the pollination mechanisms and pollinators in Dracula
have only been conducted for two of the 138 described species:
D. laeurii and D. felix, both from Ecuador. Occasional observa-
tions and collections of insects have also been made in Dracula
chiroptera Luer & Malo, Dracula morleyi Luer & Dalström,
Dracula pubescens Luer & Dalström, and Dracula sodiroi (Schltr.)
Luer (Policha et al. 2019). ese species were visited and pollin-
ated mostly by ies of the genera Zygothrica (Wiedemann) and
Hirtodrosophila (Duda) (Drosophilidae) (Endara et al. 2010,
Policha 2014, Policha et al. 2016).
According to Endara et al. (2010) pollination in D. laeurii oc-
curs when the y’s thorax is trapped by the incurved aps of the
rostellum which lodges in the space between the scutellum and
the abdomen of the insect. Eventually the y escapes, removing
the viscarium together with the pollinia. However, not only
does the pollination mechanism of most Dracula species re-
main undocumented, but generally, apart from the experiments
by Policha et al. (2016) on D. laeurii concerning the potential
function of the lip and the calyx (sepals), the roles of the various
oral structures and traits (e.g. sepaline tails, trichomes, pa-
pillae, paerns of colouration of the sepals, petals) have not been
clearly established.
In this study, we chose the Central American species Dracula
erythrochaete (Rchb.f.) Luer to address three specic questions:
(i) Who are its visitors and pollinators? (ii) What is the behav-
iour of the pollinators in owers and macrofungi? (iii) Which
anatomical structures are involved in the production of scents
and possible rewards? By answering these questions through the
use of camera recordings, insect collections, sequencing of pol-
linators, electronic microscopy, and histochemical tests, we aim
to explore the pollination mechanism of D. erythrochaete, and to
infer the possible function of organs and their contents in the
pollination system.
MATERIALS AND METHODS
Study site
We studied the pollination of D. erythrochaete in a wild popula-
tion occurring at Tablón, Cartago, Costa Rica, in a patch of lower
montane tropical forest dominated by Quercus spp. and plant-
ations of Pinus sp. at 1890 m. e roots, trunks, and branches
of these trees are colonized by a wide array of epiphytes and
are surrounded by macrofungi growing on decaying oak wood,
mosses, and organic maer (Supporting Information, Table S1).
Because of accessibility, we chose to observe and study those
D. erythrochaete individuals located close to the ground, up to
2–4 m high in trees, oen clustered at the roots of Quercus trees,
under the shade.
Dracula erythrochaete (Fig. 1) is distributed from Costa Rica
to Panama, and blooms throughout the year in the wild, with two
owering peaks, from April to June and from August to October,
coincidental with the rainy season. is orchid can be recog-
nized by the thin, narrow leaves and the small owers, which are
13–18 mm long and 10–14 mm wide (excluding the length of
the sepaline tails), which have sepals white to pale yellow suf-
fused with red, purple, or brown, and trichomes and papillae
on the adaxial surface (Fig. 1A1–2). e apices of the sepals are
contracted into slender red tails (Fig. 1A3), and the movable lip
is white or pink, composed of a hypochile (base) and epichile
(apex) with three primary lamellae and a few radiating elevated
veins (Fig. 1E). e owers emit an aroma reminiscent of com-
mercial mushrooms, with subtle citrus notes.
Sample collection and identication of pollinators
Ninety-six individuals of D. erythrochaete were collected at the
study site and cultivated in the glasshouses and preserved sec-
ondary forest at Lankester Botanical Garden (JBL), University
of Costa Rica, from 2018 to 2021. Plant voucher specimens were
prepared from cultivated material and deposited in spirit at JBL.
Macrofungi were morphologically identied in situ based on
their external characteristics through macro photography and
video recordings taken while they remained aached to the sub-
strate.
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Pollination of Dracula erythrochaete • 3
Figure 1. Lankester Composite Dissection Plate (LCDP) of Dracula erythrochaete. A, ower: 1, dorsal sepal; 2, synsepal; 3, sepaline tail. B,
column, lip, and ovary, lateral view. C, column, anther cap in the pointed circle, ventral and ¾ views. D, petal, dierent views. E, lip, ventral
and dorsal views. F, anther cap, ventral and dorsal views. G, pollinia, ventral and lateral views. Based on G. Rojas-Alvarado 323 (JBL-spirit).
Photographs by K.G.A. and LCPD prepared by L.O.
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4 • Gil-Amaya et al.
Figure 2. Pollinator of D. erythrochaete caught on Russula (Russulales) macrofungi. A, Russula and D. erythrochaete growing over a distance
range of 1 m, both visited by Zygothrica ies. B, Zygothrica with pollinia on the scutellum of D. erythrochaete, walking on the pileus of Russula.
C, various views of the male of Zygothrica sp 1 (ID 173), collected from Russula macrofungi. Scale bar = 1 mm. Photographs by K.G.A.
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Pollination of Dracula erythrochaete • 5
Flies were observed, lmed, and photographed predomin-
antly in situ, with additional observations made in the open-air
glasshouses and plants adapted to the secondary forest at JBL. In
situ visual observations of ies on or close to oral structures and
macrofungi were carried out between 6 a.m. and 6 p.m. several
days a week for a total of 35 days. All ies were collected using
an aspirator, and the identication of ies was based on a com-
bination of morphological characteristics following Grimaldi
(1987, 2010) and non invasive (sample rescue aer lysis) DNA
barcoding of the 660-bp COI (mitochondrial cytochrome c oxi-
dase subunit COI marker) gene following the method described
by Karremans et al. (2015). DNA extraction and sequencing
were performed in Naturalis Biodiversity Center, Leiden,
Netherlands.
Classication of ies based on their role was based on
time spent on ower organs and on the removal of pollinia.
Accordingly, ies were considered visitors when they landed and
remained on any oral part for at least 1 min, while ies that lin-
gered on owers and were interactive with sepals and the mov-
able lip (for >30 min) were considered potential pollinators.
ose ies that carried pollinia were considered eective pol-
linators. Additionally, the sex of the pollinators was determined
based on the presence of female terminalia (ovipositor) or male
terminalia (epandrium), to understand their implications within
the mechanism of pollination. Vouchers for the insects were de-
posited in the collection of oral visitors at JBL and at the Insect
Museum at the University of Costa Rica (San José, Costa Rica)
(Supporting Information, Table S2).
Photography, video, and digital imaging
Photographs of ies and owers were taken with Nikon D7100
and D810 digital cameras, coupled with 105-mm macro lenses
or a bellows-mounted 40-mm Zeiss Luminar lens. Images were
stacked with Zerene Stacker (Zerene Systems LLC, Richland,
WA, USA) and edited using Adobe Photoshop CS6. Videos
were taken using the continuous video option on the Nikon
D3200 and D7100 digital cameras and iPhone 6s, and then digi-
tally processed. Final digital images and composite gures were
processed in Adobe Photoshop CC 2018 and videos with Final
Cut Pro soware. Digital light microscopy (LM) images were
taken using a Leica DM500 microscope coupled with a Leica
ICC50 camera, and viewed using the Leica Application Suite
(LAS) v.4.12 microscope soware.
Insect behaviour
Data from 275.35 min (4.5 h) of video recordings were obtained
and analysed, in 72 events of insect visits to owers of D.
erythrochaete and macrofungi. From the observations and re-
cordings, we determined and described: (i) possible interactions
between insects and owers or macrofungi; (ii) the duration
of visitation and pollination events; and (iii) the events of re-
moval and deposition of pollinia. Following Grimaldi (1987),
behaviours were classied as: (a) feeding, when the ies have
the proboscis extended; (b) resting, when ies simply stand
without movement; (c) cleaning, when ies rub their own body
with their legs before feeding or remove/deposit the pollinaria;
(d) walking, when the ies passively move on the substrate;
(e) courting, when male (usually with colour paerns on its
wings and smaller than females) displays wing movements
(semaphoring) or abdominal curling to females (usually bigger
than males with visible ovipositor); (f) ovipositing, when fe-
males move their ovipositor closer to the substrate; and (g)
copulating, when males mount the females. Additionally, exam-
ination of the owers aer visits and pollination events to look
for eggs or larvae of Drosophilidae ies were carried out.
Light microscopy and histochemistry
To identify and characterize anatomical structures involved in
the production of scent molecules, lipids and polysaccharides
and in protein synthesis, fresh and paran-embedded owers
were subjected to a series of staining and imaging protocols fol-
lowing the procedures described by Bogarín et al. (2018a) and
Benavides-Acevedo and Torres-Segura (2022). Positive and
negative controls were included for each staining protocol. Areas
of scent emission, lipids, and secretory tissue were detected
with Neutral Red 0.1% (NR) (w/v, tap water). To identify pos-
sible lipid contents (fats, oils, and waxes), we used Sudan Black
B (SBB) 0.07% (w/v, ethanol 70%) and Sudan IV (SIV) 0.5%
(w/v, 70% ethanol), while osmium tetroxide (OsO4) was used
to recognize unsaturated fats. e samples were stained for 1 h,
rinsed briey in water, and mounted on slides in glycerin.
Insoluble polysaccharides and starch were detected via peri-
odic acid Schi (PAS) staining by oxidizing the samples in an
aqueous solution of periodic acid (HIO4) 5% (w/v) for 10 min,
rinsing three times in distilled water for 2 min and submerging
for 15 min in Schi ’s reagent, and nally submerging in tap water
at 50–60°C for 5 min. Proteins were detected with Coomassie
brilliant blue R-250 (CBB), submerging the samples in a solu-
tion of CBB 0.25% (w/v) for 10 min, rinsing three times in 50%
ethanol, then in 7% acetic acid for 10 min, and rinsed in tap
water.
To prepare paran-embedded ower tissues for staining,
sections of ~1 cm of sepals, petals, and lips were xed in FAA
(comprising 85% ethanol at 70%, 10% formalin at 37%, and 5%
glacial acetic acid), for 48 h, previously separated in histological
cassees. Dehydration was performed through a graded series
of ethanol (70, 80, 90, 95, 100, and 100%, v/v) and two changes
of xylene, and samples were thwn inltrated with Paraplast Plus®
for tissue embedding (Sigma-Aldrich, St Louis, MO, USA) in
a laboratory oven at 60°C. Paran blocks were trimmed and
sliced to obtain sections 7 μm thick with a Leica RM2125 RTS
rotary microtome.
To dewax the paran sections mounted in microscope slides,
they were immersed twice in xylene, then rehydrated with a
series of ethanol (100, 100, 95, and 70%, v/v) and distilled water
for 2 min in each step using a manual staining rack. e par-
an sections were stained by submersion in CBB and PAS for
20 min, then dehydrated with ethanol (95, 100, and 100%), and
xylene for 2 min. Sections were mounted in a Leica CV Mount®
mounting medium and dried overnight at room temperature.
Scanning electron microscopy
To characterize the morphology of epidermal induments (e.g.
trichomes, papillae) on the surface of sepals, petals, and lips,
dissected fresh owers of D. erythrochaete were xed for 24 h
in Karnovsky solution, comprising 2.5% glutaraldehyde and
2% paraformaldehyde solution in 0.1 M sodium phosphate
(Karnovsky 1965). e samples were degassed with a vacuum
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6 • Gil-Amaya et al.
system and then rinsed three times for 15 min each in 0.1 M
sodium phosphate buer (pH 7.4) prior to embedding. Post-
xation was performed for 2 h in 2% osmium tetroxide and
rinsed three times for 15 min in distilled water.
Fixed samples were dehydrated in a series of ethanol (30, 50, 70,
80, 90, 95, 100, and 100%), then submersed in absolute ethanol
and amyl acetate solution 1:1 and amyl acetate for 20 min, as a tran-
sition liquid before drying to the critical point. e samples were
desiccated using a Leica® EM CPD300 automated critical point
dryer (Leica Biosystems, Wetzlar, Germany). e samples were ar-
ranged on aluminium bases and covered with a layer of 20 nm of
gold, in an EMS 150R S rotary-pumped coating system (Electron
Microscopy Sciences, Hateld, PA, USA) and observed with an
Hitachi 3700-N scanning microscope at the Research Center for
Microscopic Structures (CIEMIC) of the University of Costa Rica.
Removal of oral parts
To identif y the possible role of oral structures in the mechanism
of pollination, a oral organ removal experiment was carried
out in individuals of D. erythrochaete under natural conditions
during June and July 2019 and June 2020. Six treatments were
applied (Supporting Information, Table S3), namely (i) removal
of the sepaline tails, (ii) removal of the trichomes and papillae
of the sepals (shaving with a scalpel), (iii) complete removal of
sepals, (iv) removal of petals, (v) removal of the lip, and (vi) con-
trol (intact owers). Each treatment was applied to at least 14
owers, each one belonging to a dierent individual, for a total
of 140 owers (N = 140). e experimental setup was carried
out in two phases due to the availability of fresh owers in situ.
Only owers at early anthesis, with both the pollinia and stigma
intact, were used, to rule out the inuence of any previous visits.
RESULTS
Insect visitors and pollinators
We collected a total of 197 insects; 75% came from owers and
25% came from macrofungi. e majority of specimens caught
(92%) belonged to the order Diptera. e remaining speci-
mens included 10 individuals belonging to the Hymenoptera
(5%), four to Hemiptera (2%), and one to Coleoptera (0.5%)
(Supporting Information, Table S2). Within the Diptera,
the most representative family was Drosophilidae with 164
specimens caught, accounting for 90% of all y specimens.
Other families included Dolichopodidae, Lauxaniidae, and
Sphaeroceridae, together accounting for 10% of the y speci-
mens. Flies belonging to the genus Zygothrica (Drosophilidae)
were the most common visitors to owers and macrofungi
(Supporting Information, Fig. S1A–J, M–V), although a y
of the genus Paraliodrosophila (Duda) was also identied
from Hypholoma (Fr.) P. Kumm. macrofungi (Supporting
Information, Fig. S1Y). Furthermore, the wasps Platygastridae
(Hymenoptera) and Spilomena (Shuckard) (Crabronidae:
Hymenoptera) (Supporting Information, Fig. S1K, L) were
found on the owers, while Braconidae (Hymenoptera) and
Eucoilinae (Figitidae: Hymenoptera) (Supporting Information,
Fig. S1W, X) were found on the macrofungi.
Forty-one eective pollinator ies were collected, each
carrying a single pollinarium of D. erythrochaete: 39 from owers
and two from macrofungi. One was captured on the fungus
Russula Pers. (Russulales) (Fig. 2; Supporting Information,
Video S1), and the other on the fruiting body of Gymnopus
(Pers.) Roussel (Agaricales) (Table 1; Supporting Information,
Fig. S2E). All of the sequenced ies (visitors and pollinators)
found on orchids and on macrofungi belong to at least 22 dif-
ferent species of Zygothrica (Supporting Information, Table
S2). Of the pollinarium-bearing y individuals (Table 1), 30
(73%) were sequenced, and of those 22 (73%) belonged to a
single species of Zygothrica (Fig. 3; Supporting Information, Fig.
S2A–R), while the other (27%) belonged to six dierent spe-
cies of Zygothrica (Table 1; Fig. 3; Supporting Information, Fig.
S2S, T, W, Y). In total, we found that seven species of Zygothrica
removed pollinaria of D. erythrochaete (Table 1; Supporting
Information, Fig. S2). Among the pollinators, we found almost
the same sex ratio, 18 males and 16 females, with the orchid
pollinaria (Table 1).
Fly behaviour on macrofungi
We observed 18 insect visits to fungi. While visiting the
macrofungi, the ies spent most of their time feeding and resting
(33 and 22% of the total recorded time, respectively). Other
behaviours on the pileus and lamellae of the macrofungi were
documented, including walking, cleaning, courting, ovipositing,
and copulating (19, 19, 5, 3, and 1% of the total recorded time,
respectively) (Supporting Information, Video S2). We found
approximately 11 eggs/larvae of ies growing on the lamellae of
macrofungi in six individuals of one species from the genus Lepista
(Fr.) WGSm. (Agaricales) out of the 16 genera of macrofungi ob-
served (Supporting Information, Table S1). ese macrofungi
were growing within a range of 1–10 m around the Dracula owers.
Males of the genus Zygothrica were observed performing
courtship behaviours, for example wing movements such as
‘semaphoring’, and ‘curling’, which is when the male curves its
abdomen to show the coloration of the apex of its wings to the
female. is behaviour was documented in Oudemansiella canarii
(Jungh.) Höhn (Agaricales), and in two morphospecies of the
genus Russula (Russulales), including Russula sp4 (Supporting
Information, Video S2). Additionally, oviposition was observed in
the pileus of the macrofungi Russula sp2 (Supporting Information,
Video S2). A wasp of the subfamily Eucoilinae, which could be
parasitoid of the larvae of ies, was observed and collected in these
macrofungi (Supporting Information, Table S2; Fig. S1X).
In total, we identied 29 macrofungi taxa living in the vicinity of
owering plants of D. erythrochaete in the wild population, as well
as on plants adapted to secondary forest at JBL. e macrofungi
belonged to dierent orders, 18 to Agaricales (62%), seven to
Russulales (24%), three to Boletales (10%), and one to Poly porales
(3%). e most species-diverse macrofungi found in this study
were Agaricales represented by 10 genera, with six morphospecies
of the genus Gymnopus, and the order Russulales represented by a
single genus Russula, with seven morphospecies observed.
Over a range of 1–5 m distance to the closest owering plant (Fig.
2A), the most visited macrofungi by ies were of the genera Russula
(Russulales) (~119 individuals), Oudemansiella Speg. (Agaricales)
(~30 individuals), and Gymnopus (Agaricales) (~20 individuals)
(Supporting Information, Table S1). All of them had lamellae on
the hymenophore. e same groups of ies were observed moving
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Pollination of Dracula erythrochaete • 7
freely between D. erythrochaete and macrofungi. At times, when the
macrofungi were completely covered by ies, any interruption made
the ies take o and y to the owers. When the owers were not
nearby, the ies would perch on the leaves, moss, and liverworts.
Fly behaviour on D. erythrochaete
We observed 54 insect visits to owers of Dracula orchids.
During visits, recorded ies spent most of their time trapped in
the cavity formed by the base of the column and lip, while for
the remaining time the ies remained free on the sepals, petals,
and lip feeding (40%), walking (23%), and cleaning themselves
(23%); they rarely rested (10%) (Supporting Information,
Video S3). When y aggregations grazed on the lip or sepals, we
observed occasional courting behaviours (only 4% of the total
recorded time). At the study site, we recorded ies of the genus
Zygothrica displaying repetitive wing movements with wings
spread around 45° at various amplitudes and speeds (Supporting
Information, Video S3). Copulation was observed only once
Table 1. Zygothrica specimens caught with pollinia of Dracula erythrochaete.
Insect ID Sex Genus Species Plant species/fungal species Insect voucher Locality
3♀Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
22 ♀Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
26 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
35 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
36 ♀Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
37 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
46 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
56 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
57 ♀Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
68 ♀Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
78 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
86 ♀Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
103 ♀Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
104 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
113 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
116 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
118 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
119 ♀Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
155 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
156 ♂Zygothrica sp. 1 D. erythrochaete JBL* Tablón, Cartago
157 ♂Zygothrica sp. 1 Gymnopus (Agaricales) JBL* Tablón, Cartago
173 ♂Zygothrica sp. 1 Russula (Russulales) JBL* Tablón, Cartago
143 ♂Zygothrica sp. 2 D. erythrochaete JBL* JBL
152 ♀Zygothrica sp. 3 D. erythrochaete JBL* Tablón, Cartago
58 ♀Zygothrica sp. 4 D. erythrochaete JBL* Tablón, Cartago
150 ♀Zygothrica sp. 4 D. erythrochaete JBL* JBL
154 ♀Zygothrica sp. 4 D. erythrochaete JBL* JBL
117 ♀Zygothrica sp. 5 D. erythrochaete JBL* Tablón, Cartago
20 ♂Zygothrica sp. 6 D. erythrochaete JBL* Tablón, Cartago
181 ♀Zygothrica sp. 7 D. erythrochaete JBL* JBL
1 – Zygothrica Unknown D. erythrochaete JBL JBL
41 – Zygothrica Unknown D. erythrochaete UCR Tablón, Cartago
53 – Zygothrica Unknown D. erythrochaete JBL Tablón, Cartago
54 – Zygothrica Unknown D. erythrochaete UCR Tablón, Cartago
55 – Zygothrica Unknown D. erythrochaete UCR Tablón, Cartago
75 – Zygothrica Unknown D. erythrochaete UCR Tablón, Cartago
84 – Zygothrica Unknown D. erythrochaete UCR Tablón, Cartago
192 ♀Zygothrica Unknown D. erythrochaete JBL JBL
193 ♀Zygothrica Unknown D. erythrochaete JBL JBL
195 ♂Zygothrica Unknown D. erythrochaete JBL JBL
196 ♂Zygothrica Unknown D. erythrochaete JBL JBL
JBL, Jardín Botánico Lankester. JBL*, Jardín Botánico Lankester (specimen sequenced). UCR, Insect Museum, Universidad de Costa Rica.
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8 • Gil-Amaya et al.
Figure 3. Phylogenetic relationship among the collected y specimens. e tree was produced by analysis of the COI dataset using BEAST
v.1.6.0. Edited by A.P.K. using FigTree v.1.3.1.
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Pollination of Dracula erythrochaete • 9
occurring on the leaves. Oviposition or copulation were not ob-
served on D. erythrochaete owers.
Visits were prolonged and groups of up to 24 ies were
counted simultaneously visiting a single ower. e visits oc-
curred at any time during the day, mostly from 7 a.m. to 5.30
p.m., and no overnight visitation events were observed. e ies
approached the owers and landed on the sepals or epichile of
the movable lip, very rarely on the sepaline tails. ey immedi-
ately walked and inspected the trichomes on the adaxial surface
of the sepals and the papillose surface of the lip, usually from the
apex to the base, for about 30–60 min before leaving. ey in-
spected the denticulate margins and lamellae of the epichile, as
well as the papillae of the hypochile using the labial palpi of their
articulated juing proboscis (Supporting Information, Video
S3). Flies were recorded visiting the petals, but unlike the behav-
iour documented in the sepals and lip, they were not observed
with their proboscis extended, aempting to feed.
Pollination mechanism in D. erythrochaete
Flies were aracted to the owers of D. erythrochaete from the
rst day of anthesis, even before the sepals had nished ex-
tending. e lip of the ower naturally hangs down like a pen-
dulum, continuously swaying in the wind (Fig. 4A). Initially, any
potential pollinator inspects the lamellae and elevated veins of
the epichile using its extended proboscis (Fig. 4B). It then walks
towards the concave base of the lip in aempt to feed on the cla-
vate papillae of the hypochile. e potential pollinator activates
the movement of the lip (Fig. 4C) and, possibly assisted by the
wind, it is pressed between the movable lip and the column (Fig.
4D). During visits of up to 90 min or even more, potential pol-
linators were found trapped between the column and the lip,
adhering to the viscid uid (viscarium) on the rostellum.
In the process of pollinia removal or deposition, the pollin-
ator aempts to exit through the sides, but the petals prevent it
from escaping (Fig. 4E). As the pollinator exits backwards, its
scutellum, a dorsal structure on the thorax resembling a triangle
or shield, is smeared with viscarium, and it brushes against the
caudicles of the pollinia, removing them or depositing them on
the stigma (Fig. 4F). e pollinator can be assisted by the wind
in reaching the lip with its legs, and use it as a platform to exit.
Upon release, the eective pollinator falls onto the epichile (Fig.
4G). While exiting, the anther cap may become aached to the
pollinaria (Fig. 4H), and the pollinator needs additional time to
remove it, usually with its hind legs. e placement of pollinia
always occurs on the y’s scutellum (Supporting Information,
Video S4).
Close inspection of 35 owers using a stereoscopic micro-
scope aer visits and pollinarium removal or deposition revealed
the presence of aphids (Aphididae: Hemiptera) and yellow slug
eggs (Gastropoda). However, Diptera eggs or larvae were never
located on any owers in the eld or glasshouses. Scanning elec-
tron microscopy (SEM) also did not reveal any Diptera eggs or
larvae.
Floral micromorphology and histochemistry
e oral organs of D. erythrochaete exhibited stomata, glan-
dular trichomes, papillae, and secretory materials, as detailed in
Supporting Information, Table S4. Both surfaces of the sepaline
tails carried open or closed stomata (Fig. 5A) and multicellular
glandular trichomes at the base of the adaxial surface (Fig. 5B).
Lipids and insoluble polysaccharides were concentrated in sto-
mata (Fig. 5C), trichome-like colleters (Fig. 5D), and glandular
trichomes (Fig. 5E). Proteins were detected only in the glandular
trichomes and osmiophilic bodies in the cell walls, concentrated
in epidermal cells (Fig. 5F). Numerous glandular trichomes were
identied on the adaxial surface of the sepals with 1–3 elongated
secretory terminal heads (Fig. 6A, B), and papillary trichomes
without a developed apex (Fig. 6C), both covered by striated
cuticle and epicuticular secretions (Fig. 6B, D). Histochemical
tests revealed the presence of lipids, food reserves of proteins on
the apices of trichomes (Fig. 6E, F), and polyhedral starch grains
of parenchyma in sepals. Trichomes and papillae located at the
base and middle region of the sepals exhibited a higher concen-
tration of proteins than those at the margins.
e petals had elongated conical papillae at the apex (Fig.
7A) and a papillose surface in the median and basal region,
covered by a conspicuously thickened reticulated cuticle (Fig.
7B). Starch grains were found stored in the parenchyma cells at
the apex region; no positive reaction to CBB was observed for
proteins (Supporting Information, Table S4). Lipophilic com-
pounds (Fig. 7C, D), insoluble polysaccharides (Fig. 7E), and
osmiophilic contents (Fig. 7F) were identied in large quantities
on the apices of the papillae, and a large concentration of bun-
dles of raphides in cells close to the margins. e adaxial and ab-
axial sides of the lip were covered by a papillose surface (Fig. 8A).
We found elongated clavate papillae on the hypochile (Fig. 8B)
with slightly striated cuticles, epicuticular secretions
(Fig. 8C), and a higher protein concentration reacting with CBB
(Fig. 8D, E). Concentrations of lipids and unsaturated fats were
detected, in addition to insoluble polysaccharides (Supporting
Information, Table S4) and many starch grains in the epichile
(Fig. 8F), indicating the presence of scents and stored food ma-
terials, respectively. Also, bundles of raphides and prismatic crys-
tals were observed in the hypochile and epichile of the lip.
Functional importance of oral structures
None of the owers without sepaline tails produced fruit (0%),
nor did those without sepals (0%) or lips (0%). Other treat-
ments produced fruit, but all in a lower percentage than the
control treatment. When removing the petals, 2% of the owers
produced fruit; when removing the trichomes and papillae of
the sepals, only 1% of the owers produced fruit. In the control
treatment, 5% of the owers produced fruits.
DISCUSSION
Pollination biology and insect behaviour
Flowers of D. erythrochaete and the nearby Agaricales and
Russulales macrofungi share the same group of visitors,
including Diptera and Hymenoptera. e owering phenology
overlaps with macrofungi that occur in high-density populations
in the habitat, increasing the visitation frequency of insects and
allowing free movements between them. We found that the
two ies collected with pollinaria in the macrofungi belong to
a single species of Zygothrica, these being the most commonly
collected species among owers and macrofungi at the wild
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10 • Gil-Amaya et al.
population in Cartago (Fig. 3; Supporting Information, Table
S2). Furthermore, an individual collected visiting the owers
of D. chimaera, which also belongs to the genus Zygothrica, is
closely related to the ies of macrofungi that visit the owers of
D. erythrochaete in Costa Rica (Fig. 3). e owers also aracted
wasps, which are probably the most common non-Dipteran vis-
itors of Pleurothallidinae in search of nectar (Karremans and
Diaz-Morales, 2019). e tiny wasps of the family Platygastridae
(Supporting Information, Fig. S1K) are reported as parasit-
oids of the egg-larvae or egg-pupae of Diptera, Orthoptera, and
Hemiptera (Buhl 2001), while the small wasps of the genus
Spilomena (Supporting Information, Fig. S1L) observed are
reported as predators of adults and nymphs of Aphioidea,
ysanoptera, Psylloidea, Coccoidea, and Eulophidae (Vardy
1987, Antropov and Cambra 2004).
Endara et al. (2010) observed courtship and mating behav-
iours of ies in D. laeurii and courtship behaviours in D. fel ix, but
they never recorded oviposition or discovered eggs in owers.
Policha et al. (2019) tested the brood-site mimicry hypothesis by
examining rearing success; although they found Drosophilidae
Figure 4. Pollination mechanism of D. erythrochaete. A, lip in natural position. Detail of the papillae on the hypochile. B, the y is aracted and
aempts to feed on the epichile. C, movement of the lip is activated by the weight of the y. D, the y is trapped between the column and lip,
aached to the viscarium of the rostellum. E, front view of the petals and lip with the potential pollinator. F, the pollinator exits backwards.
Detail of the viscarium. G, the pollinator removes the pollinaria and falls into the epichile. H, the pollinator rests with pollinia on its scutellum,
aer removing the anther cap. Scale bar = 5 mm. Illustrations by L.O.
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Pollination of Dracula erythrochaete • 11
Figure 5. Micromorphology and histochemistry of the sepaline tails of D. erythrochaete. A, SEM of stomata on the adaxial surface. B, SEM
of multicellular glandular trichomes on the adaxial surface at the base. C, LM of stomata stained with SBB showing lipids. D, trichome-like
colleter showing lipids at the base. E, lipid concentration (SBB) on the glandular trichome. F, transverse section showing osmiophilic bodies in
the epidermal cells, indicated by arrows. Scale bars = 30, 300, 50, 20, 50, and 50 µm, respectively. Photographs by K.G.A.
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12 • Gil-Amaya et al.
Figure 6. Micromorphology and histochemistry of the sepals of D. erythrochaete. A, SEM of several multicellular glandular trichomes on
the adaxial surface. B, SEM of glandular trichome with epicuticular secretions and striate cuticle, indicated by arrows. C, SEM of papillary
trichomes and papillae at the base of sepals D, SEM of papillary trichome with epicuticular secretions indicated by arrows. E, LM of glandular
trichome stained with CBB showing proteins. F, LM of papillae with proteins concentrated at the apex detected with CBB (blue). Scale bars =
90, 50, 120, 20, 50, and 20 µm, respectively. Photographs by K.G.A.
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Pollination of Dracula erythrochaete • 13
Figure 7. Micromorphology and histochemistry of the petals of D. erythrochaete. A, SEM of the conical papillae at the apex. B, detail of
the reticulated and striated cuticle of the papillae. C, LM of the papillae stained with SIV showing lipids. D, LM of papillae showing lipids
concentrated at the apex (SBB). E, LM of papillae stained with PAS showing polysaccharides. F, LM transverse section of petal base showing
osmiophilic contents in epidermal cells, indicated by arrows. Scale bars = 100, 30, 20, 20, 50, and 200 µm, respectively. Photographs by K.G.A.
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14 • Gil-Amaya et al.
Figure 8. Micromorphology and histochemistry of the lip of D. erythrochaete. A, SEM of the abaxial surface of the epichile with a papillose
surface. B, SEM of the hypochile showing elongate clavate papillae. C, SEM of the epidermal surface of the hypochile showing epicuticular
secretions. D, LM of the hypochile stained with CBB showing proteins concentrated at the apex of the papillae (blue tips). E, transverse section
of the hypochile showing proteins (CBB) concentrated in epidermal cells indicated by the arrows. F, LM of transverse section of epichile
stained with PAS showing starch grains on the parenchyma, indicated by arrows. Scale bars = 200, 120, 20, 50, 200, and 50 µm, respectively.
Photographs by K.G.A.
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Pollination of Dracula erythrochaete • 15
larvae emerging from owers, these were not from the pollin-
ators, and nor did they observe any ies ovipositing. In contrast,
the time the ies spent walking and sucking with their proboscis
extended was signicantly greater than time spent mating be-
haviours. ese courtship behaviours were also very rare in D.
erythrochaete, and we found no evidence for the laying of eggs
in owers. Flies use their sense of smell to locate potential ovi-
position substrates from a distance; some odours induce ovipos-
ition, while others inhibit it. Certain odours aract females by
signalling an oviposition substrate, and others stimulate ovipos-
ition over shorter distances (Cury et al. 2019). e absence of
drosophilid eggs in D. erythrochaete could be due to the presence
of parasitoid wasps (Platygastridae) in the owers, whose phero-
mones inhibit drosophilid egg-laying (Ebrahim et al. 2015, Cury
et al. 2019). Visual detection of wasps also alters the oviposition
behaviour of females actively seeking a safe environment for
their eggs (Kacsoh et al. 2013).
Oviposition normally requires an interplay between olfac-
tory, taste, and mechanosensory input to be released. However,
in plants that use brood-site imitation or sapromyiophily as a
pollination strategy, this releasing combination of signals is, in
most instances, missing in the ower, with some exceptions. For
instance, the South African orchid Satyrium pumilum unb. is
pollinated by female esh ies (Sarcophagidae), which occasion-
ally deposit live larvae on the owers (van der Niet et al. 2011).
Similarly, Bulbophyllum mandibulare Rchb.f. aracts ovovivip-
arous esh ies that drop live larvae on the owers, where they
eventually starve to death (Ong 2012). Additionally, in some
species of the genus Stapelia L. (Apocynaceae), the development
of carrion y larvae has been observed ( Jürgens et al. 2006, Urru
et al. 2011, du Plessis et al. 2018).
Our observations in D. erythrochaete appear to be consistent
with other studies of pollination by brood-site imitation where
oviposition is rare or absent, such as in Corybas geminigibbus J.J.
Sm. and C. shanlinshiensis W.M. Lin, T.C. Hsu & T.P. Lin (Han
et al. 2022), as well as non orchids such as Impatiens section
Trimorphopetalum (Balsaminaceae) (Abrahamczyk et al.
2021), and Typhonium Scho species (Araceae) (Sayers et al.
2021). Both male and female insects were aracted to inspect
the owers, but the conditions for egg-laying were not met.
erefore, we cannot completely reject the idea of brood-site
imitation in Dracula orchids. Instead, this example should help
us redene the name or key aspects of congregation/brood-site
mimicry. Dracula erythrochaete owers may use brood-site imi-
tation, with their fungus-like appearance and scent, to deceive
Zygothrica ies. Additionally, they oer a food reward through
protein secretion of the sepals and lip, exploiting the ies’ feeding
behaviour to position the pollinator for removing or depositing
pollinaria. is mixed strategy of fungal mimicry and food re-
ward in Dracula can be compared with the mixed strategies of
the Australian orchid genus Caladenia R.Br., which includes spe-
cies with nectar rewards and sexual deception, or food deception
and sexual deception (Phillips et al. 2009, 2024, Noushka et al.
2018, Phillips and Batley 2020).
Drosophilidae is reported as the single family that pollinates
the genus Dracula with ies from dierent but closely related
genera: Drosophila, Hirtodrosophila, and Zygothrica (Endara et al.
2010, Karremans and Diaz-Morales 2019, Policha et al. 2019).
Almost all Zygothrica ies found in Dracula owers belong to
striped species (such as those in the viations, poeyi, and other
groups), consistent with eld studies conducted in Ecuador
(Endara et al. 2010, Policha 2014, Policha et al. 2019). Despite
their highest diversity being concentrated in the northern
Andes and Central America, most Zygothrica species remain
undescribed, with their natural history remaining obscure. Our
study on the pollination ecology of D. erythrochaete has enabled
the collection of over 20 species (Supporting Information, Table
S2); however, these specimens remain unidentied at the spe-
cies level due to insucient information, and potentially repre-
sent new species to science. Similarly, recent pollination studies
on Corunastylis Fitzg. species in Australia (Ren et al. 2023) have
also reported the collection of many unidentied small ies.
Additionally, our research documents notable observations,
such as the oviposition of Zygothrica in macrofungi of the genus
Russula (Russulales) (Suppoting Information, Video S2).
Mycophagy (the process of organisms consuming fungi)
evolved independently in dierent lineages of the family
Drosophilidae (rockmorton 1975, Zhang et al. 2021), and the
genus Zygothrica with nearly 40 species described for Central
America comprises mostly mycophagous specialists (Grimaldi
1987, 2010). In mycophagous species, there is a spatial compo-
nent of niche division between adults and larvae, which elimin-
ates the possibility of competition between the dierent stages
of their life cycle and allows adults and larvae to coexist with
higher population densities (Nadia and Machado 2014). Adult
ies look for food for their own consumption, being less active in
the search for food compared to other insects that care for their
ospring (Faegri and van der Pijl 1979). ese food habit pref-
erences in male and female ies of the genera Zygothrica can be
exploited by the owers of D. erythrochaete.
Pollination mechanism in D. erythrochaete
Flies aracted to D. erythrochaete walk from the sepals to the
lip and aempt to feed by following the trichomes and pa-
pillae guides, which reacted positively to NR, PAS, SBB, SIV,
and OsO4, indicating high secretory activity and showing epi-
cuticular secretions in SEM images (Figs 6B, D, 8C). e behav-
iour suggests feeding, and although the rewards are not visible,
the papillae contain proteins (Figs 6E, F, 8D, E). We could not
identify the proteins synthesized by D. erythrochaete, nor the
amount secreted, but CBB staining showed a high concentration
of proteins in the papillary trichomes of the sepals and papillae
along the hypochile, probably to keep the ies longer and bring
them to the correct position for pollination.
e feeding behaviour of ies suggests that potential pollin-
ators are guided towards the base of the lip, where they activate
the movable lip with their weight and potentially obtain pro-
teins as a oral reward. Detailed histochemical investigations in
Pleurothallidinae (Bogarín et al., 2018a, b) and Bulbophyllinae
(Davies and Stpiczyska 2014, Kowalkowska et al. 2015)
have demonstrated the secretion of proteins from the pa-
pillae of the lip. However, only in the genus Trichosalpinx Luer
(Pleurothallidinae) were these results supported by the feeding
behaviour of ies, and as Bogarín et al. (2018a) stated, the pro-
teins were not sucient to be considered a reward.
e pollination in D. erythrochaete owers is a variation of the
pollination mechanism generalized in Pleurothallidinae, dened
by Karremans and Díaz-Morales (2019) as ‘masdevalliform’
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16 • Gil-Amaya et al.
where pollinators enter the column–lip cavity, and are pressed
by the hinged lip against the column, removing the pollinia
with their scutellum while exiting (Fig. 4D–F). Observations of
ies trapped in the column–lip cavity have been made in other
Dracula spp., and sometimes dead ies with pollinia aached to
the scutellum have also been found. However, these ies serve as
a reference, to determine other potential pollinator species.
Floral morphology and histochemistry
Our histological tests showed that there was a polarization in
protein detection on the papillae of the lip; the hypochile, which
is the concave base articulated at the column-foot, and therefore
closest to the column (Fig. 1E), has the highest concentration
of proteins compared with the secretory papillae of the epichile,
which is consistent with the description of Luer (1993), who
proposed that this deep central cle of the hypochile in the
genus Dracula is possibly a nectary or fragrance source.
e presence of starch grains accumulated on the sepals,
petals, and epichile of the lip (Fig. 8F) suggests that they are
probably reserves used as a source of energy to produce vola-
tile compounds. Furthermore, accumulation of lipid-rich sub-
stances, probably precursors or the fragrance itself, indicates
scent synthesis and secretion through the trichomes, papillae,
and papillose surface of these structures. Starch grains in the
osmophoric tissue are a common feature of scent glands as docu-
mented in osmophores of other orchids (Wiemer et al. 2009,
Anto et al. 2012, E.R. Pansarin et al. 2014, Casique et al. 2021,
L.M. Pansarin et al. 2021) and these starch agglomerations de-
crease as volatile evaporation occurs since starch is used as an
energy source for the production of nectar or scent (Vogel 1990,
Emert et al. 2006).
e removal of the tails, sepals, and lip signicantly inu-
enced the proportion of owers that produced fruits, indicating
their important role in reproductive success. ese structures
are actively involved in aracting potential pollinators, prob-
ably because they have both a chemical and rewarding role in D.
erythrochaete owers. While the petals of the ower do not seem
to be essential in aracting visitors or in the pollination mech-
anism, they do play a complementary role in retaining the pol-
linator in a certain position to remove or deposit the pollinaria.
e low percentage of pollination observed in D. erythrochaete
is consistent with other orchids that use deceptive pollination
strategies, which oen have low visitation and pollination rates
(Neiland and Wilcock 1998, Tremblay et al. 2005). Nevertheless,
low reproductive success is also frequent in tropical orchids that
are dependent on a functional group of pollinators (regardless
of the mechanism used to aract them), which may help explain
their unique pollination mechanisms and their extreme diversity.
CONCLUSIONS
We have conrmed for the rst time that Dracula owers eect-
ively share the same group of visitors with nearby macrofungi,
specically from the orders Agaricales and Russulales,
establishing that the owers are indeed fungal mimics. Fly behav-
iour on D. erythrochaete owers and the macrofungi was mainly
related to feeding. e owers of D. erythrochaete oer a food re-
ward through the secretion of proteins from the papillae of the
sepals and lip as an incentive for Zygothrica ies to stay longer on
the owers and bring them into the correct position for pollin-
ation. Similar to previous ndings, we did not nd evidence of
Drosophilidae egg-laying on owers of a Dracula species; thus,
we propose that D. erythrochaete employs a mixed strategy of
brood-site imitation and food reward. Most of the 138 Dracula
spp. show similar oral traits and therefore we hypothesize that
other Dracula spp. are pollinated via a similar system. is study
provides insights into the pollination mechanism of the genus
Dracula and highlights the importance of y pollinators and
their diversity in the subtribe Pleurothallidinae.
SUPPLEMENTARY DATA
Supplementary data are available at Botanical Journal of the
Linnean Society online.
ACKNOWLEDGMENTS
We are very grateful to Paul Hanson for his help with the identi-
cation of Diptera and Hymenoptera, and Milagro Mata Hidalgo for
her collaboration with the identication of macrofungi. We would
like to thank Olman Alvarado for helping with the processing of the
SEM images, and Julio Otárola and the Lopez family for their as-
sistance during the eld work. Special thanks to Alfredo Cascante,
Diego Bogarín, and Franco Pupulin for providing useful comments
and suggestions to improve the manuscript, Gerson Villalobos for
helping in cultivating the plants, and all the sta at JBL for their kind
assistance with this project. We also thank the Costa Rican Ministry
of Environment and Energy (MINAE) and its National System of
Conservation Areas (SINAC) for issuing the Scientic Passports
under which wild species treated in this study were collected. We
acknowledge the Vice-Presidency of Research of the University of
Costa Rica for providing support through the projects ‘Estudios en
polinización de Pleurothallidinae (Orchidaceae)’ (814-B5-A81) and
‘Importancia de la polinización por moscas en Orchidaceae: Biología
reproductiva y ecología de polinización de Dracula, Masdevallia y
grupos anes en Pleurothallidinae’ (C2048).
CREDIT STATEMENT
K.G.A., A.P.K., and M.A.B designed the research. K.G.A and
A.P.K. carried out the eld work. K.G.A., M.F. and M.B.A. performed
the laboratory work. D.G. contributed to the morphological identi-
cation and curation of Drosophilidae. L.O. contributed to the LCDP
of D. erythrochaete and pollination mechanism illustrations. K.G.A.,
A.P.K., M.F., and M.A.B. analysed the data, and K.G.A., A.P.K., M.F.,
and M.A.B. wrote, reviewed, and edited the manuscript.
CONFLICTS OF INTEREST
e authors have no conicts of interest to report.
DATA AVAILABILITY
e data underlying this article are available in the article and in its on-
line supplementary material.
REFERENCES
Abrahamczyk S, Humphreys AM, Trabert F et al. Evolution of brood-site
mimicry in Madagascan Impatiens (Balsaminaceae). Perspectives in
Downloaded from https://academic.oup.com/botlinnean/advance-article/doi/10.1093/botlinnean/boae054/7756339 by guest on 14 September 2024
Pollination of Dracula erythrochaete • 17
Plant Ecology, Evolution and Systematics 2021;49:125590. hps://doi.
org/10.1016/j.ppees.2021.125590
Ackerman JD. Pollination biology of Calypso bulbosa var. occidentalis
(Orchidaceae): a food-deception system. Madroño 1981;28:101–10.
Ackerman JD. Pollination of tropical and temperate orchids. In:
Proceedings of the Eleventh World Orchid Conference. Miami, FL:
American Orchid Society, 1985, 98–101.
Ackerman JD, Rodríguez-Robles JA, Meléndez EJ. A meager nectar
oering by an epiphytic orchid is beer than nothing. Biotropica
1994;26:44–9.
Ackerman JD, Phillips RD, Tremblay RL et al. Beyond the various con-
trivances by which orchids are pollinated: global paerns in or-
chid pollination biology. Botanical Journal of the Linnean Society
2023;202:295–324. hps://doi.org/10.1093/botlinnean/boac082
Anto S, Kamiska M, Stpiczyska M. Comparative structure of the
osmophores in the ower of Stanhopea graveolens Lindley and
Cycnoches chlorochilon Klotzsch (Orchidaceae). Acta Agrobotanica
2012;65:11–22. hps://doi.org/10.5586/aa.2012.054
Antropov AV, Cambra . A new species of the genus Spilomena
Shuckard, 1838 (Hymenoptera: Crabronidae: Pemphredoninae)
from Guatemala. Russian Entomological Journal 2004;13:69–71.
Ayasse M, Schiestl FP, Paulus HF et al. Pollinator araction in a sexually
deceptive orchid by means of unconventional chemicals. Proceedings
Biological Sciences 2003;270:517–22. hps://doi.org/10.1098/
rspb.2002.2271
Benavides-Acevedo M, Torres-Segura J. Fast seed histology protocols:
Benzene derivatives-free vs. xylene-dependent. Agronomía Mesoamerica
2022;51308:51308. hps://doi.org/10.15517/am.v33iespecial.51308
Blanco MA, Barboza G. Pseudocopulatory pollination in Lepanthes
(Orchidaceae: Pleurothallidinae) by fungus gnats. Annals of Botany
2005;95:763–72. hps://doi.org/10.1093/aob/mci090
Blanco MA, Davies KL, Stpiczynska M et al. Floral elaiophores in
Lockhartia Hook. (Orchidaceae: Oncidiinae): their distribution, di-
versity and anatomy. Annals of Botany 2013;112:1775–91. hps://
doi.org/10.1093/aob/mct232
Bogarín D, Fernández M, Karremans AP, et al. Floral anatomy and evo-
lution of pollination syndromes in Lepanthes and close relatives. In:
Proceedings of the 22nd World Orchid Conference, Guayaquil, Ecuador,
2018b, 389–403.
Bogarín D, Fernández M, Borkent A et al. Pollination of Trichosalpinx
(Orchidaceae: Pleurothallidinae) by biing midges (Diptera:
Ceratopogonidae). Botanical Journal of the Linnean Society
2018a;186:510–43.
Bohman B, Phillips RD, Flemai GR et al. e spider orchid Caladenia
cebra produces sulfurous pheromone mimics to aract its male wasp
pollinator. Angewandte Chemie 2017;129:8707–8707. hps://doi.
org/10.1002/ange.201705177
Buhl PN. Phylogeny and host association in Platygaster Latreille,
1809 (Hymenoptera, Platygastridae). Entomolosike Meddelelser
Entomologogisk Forening Kobenhavn 2001;69:113–24.
Caballero-Villalobos L, Silva-Arias GA, Buzao CR et al. Generalized
food-deceptive pollination in four Caleya (Orchidaceae: Laeliinae)
species from Southern Brazil. Flora 2017;234:195–206. hps://doi.
org/10.1016/j.ora.2017.07.014
Casique JV, de Müllerian Aguiar Andrade EH, Andrade de Aguiar
Dias AC et al. Novelties in the secretory structures of three species
of Gongora (Orchidaceae: Stanhopeinae). Botanical Journal of the
Linnean Society 2021;195:650–70.
Cozzolino S, Widmer A. Orchid diversity: an evolutionary consequence
of deception? Trends in Ecology & Evolution 2005;20:487–94. hps://
doi.org/10.1016/j.tree.2005.06.004
Cury KM, Prud’homme B, Gompel N. A short guide to insect ovipos-
ition: when, where and how to lay an egg. Journal of Neurogenetics
2019;33:75–89. hps://doi.org/10.1080/01677063.2019.1586898
Dafni A. Mimicry and deception in pollination. Annual Review of Ecology
and Systematics 1984;15:259–78. hps://doi.org/10.1146/annurev.
ecolsys.15.1.259
Darwin CR. On the Various Contrivances by Which British and Foreign
Orchids are Fertilised by Insects. London: J. Murray, 1877.
Davies KL, Stpiczyska M. Comparative labellar anatomy of resin-
secreting and putative resin-mimic species of Maxillaria s.l.
(Orchidaceae: Maxillariinae). Botanical Journal of the Linnean Society
2012;170:405–35.
Davies KL, Stpiczyska M. Labellar anatomy and secretion in
Bulbophyllum ouars (Orchidaceae: Bulbophyllinae) sect. Racemosae
Benth. & Hook. f. Annals of Botany 2014;114:889–911.
Davies KL, Winters C, Turner MP. Pseudopollen: its structure and develop-
ment in Maxillaria (Orchidaceae). Annals of Botany 2000;85:887–95.
Dentinger BT, Roy BA. A mushroom by any other name would smell as
sweet: Dracula orchids. McIlvainea 2010;19:1–13.
Dressler RL. Pollination by euglossine bees. Evolution 1968;22:202–10.
hps://doi.org/10.1111/j.1558-5646.1968.tb03463.x
Dressler RL. e Orchids. Natural History and Classication. Cambridge,
MA: Harvard University Press, 1981.
du Plessis M, Johnson SD, Nicolson SW et al. Pollination of the “carrion
owers” of an African stapeliad (Ceropegia mixta: Apocynaceae): the
importance of visual and scent traits for the araction of ies. Plant
Systematics and Evolution 2018;304:357–72.
Ebrahim SAM, Dweck HKM, Stökl J et al. Drosophila avoids parasitoids
by sensing their semiochemicals via a dedicated olfactory circuit.
PLoS Biology 2015;13:e1002318. hps://doi.org/10.1371/journal.
pbio.1002318
Emert U, Buss D, Rohrbeck D, et al. Localization of the Synthesis and
Emission of Scent Compounds Within the Flower. Boca Raton, FL: CRC
Press, 2006, 105–24.
Endara L, Grimaldi DA, Roy BA. Lord of the ies: pollination of Dracula
orchids. Lankesteriana 2010;10:1–11.
Faegri K, van der Pijl L. e Principles of Pollination Ecology. New York,
NY: Pergamon, 1979.
Givnish TJ, Spalink D, Ames M et al. Orchid phylogenomics and multiple
drivers of their extraordinary diversication. Proceedings of the Royal
Society B: Biological Sciences 2015;282:20151553–10. hps://doi.
org/10.1098/rspb.2015.1553
Grimaldi DA. Phylogenetics and taxonomy of Zygothrica (Diptera:
Drosophilidae). Bulletin of the American Museum of Natural History
1987;186:101–268.
Grimaldi DA. Drosophilidae (Small fruit ies, pomace ies, vinegar ies).
In: Manual of Central American Diptera. Oawa: NRC Research Press,
2010, 1197–206.
Han ZD, Wu Y, Bernhardt P et al. Observations on the pollination and
breeding systems of two Corybas species (Diurideae; Orchidaceae) by
fungus gnats (Mycetophilidae) in southwestern Yunnan, China. BMC
Plant Biology 2022;22:1–11.
Hetherington-Rauth MC, Ramírez SR. Evolution and diversity of
oral scent chemistry in the euglossine bee-pollinated orchid genus
Gongora. Annals of Botany 2016;118:135–48.
Jersáková J, Johnson SD, Kindlmann P. Mechanisms and evolution of de-
ceptive pollination in orchids. Biological Reviews 2006;81:219–35.
hps://doi.org/10.1017/s1464793105006986
Jones DL. e pollination of Corybas diemenicus (H.M.R.) Rupp and
W.H. Nicholls ex H.M.R. Rupp. Victorian Naturalist 1970;87:372–4.
Jürgens A, Döerl S, Meve U. e chemical nature of fetid
oral odours in stapeliads (Apocynaceae–Asclepiadoideae–
Ceropegieae). New Phytologist 2006;172:452–68. hps://doi.
org/10.1111/j.1469-8137.2006.01845.x
Kacsoh BZ, Lynch ZR, Mortimer NT et al. Fruit ies medicate o-
spring aer seeing parasites. Science 2013;339:947–50. hps://doi.
org/10.1126/science.1229625
Kaiser R. Flowers and fungi use scents to mimic each other. Science
2006;311:806–7. hps://doi.org/10.1126/science.1119499
Karnovsky MJ. A formaldehyde-glutaraldehyde xative of high osmolarity
for use in electron microscopy. e Journal of Cell Biology 1965;27:137.
Karremans AP, Diaz-Morales M. e Pleurothallidinae: extremely high
speciation driven by pollinator adaptation. In: Proceedings of the 22nd
World Orchid Conference. Guayaquil: Asociación Ecuatoriana de
Orquideología, 2019, 363–88.
Karremans AP, Pupulin F, Grimaldi D et al. Pollination of Specklinia
by nectar-feeding Drosophila: the rst reported case of a deceptive
Downloaded from https://academic.oup.com/botlinnean/advance-article/doi/10.1093/botlinnean/boae054/7756339 by guest on 14 September 2024
18 • Gil-Amaya et al.
syndrome employing aggregation pheromones in Orchidaceae.
Annals of Botany 2015;116:437–55. hps://doi.org/10.1093/aob/
mcv086
Kelly M, Gaske A. UV reectance but no evidence for colour mimicry
in a putative brood-deceptive orchid Corybas cheesemanii. Current
Zoology 2014;60:104–13.
Kelly M, To R, Gaske A. Pollination and insect visitors to the puta-
tively brood-site deceptive endemic spurred helmet orchid, Corybas
cheesemanii. New Zealand Journal of Botany 2013;51:155–67.
Kocyan A, Endress PK. Floral structure and development of Apostasia
and Neuwiedia (Apostasioideae) and their relationships to other
Orchidaceae. International Journal of Plant Sciences 2001;162:847–67.
hps://doi.org/10.1086/320781
Kowalkowska AK, Kozieradzka-Kiszkurno M, Turzyski S.
Morphological, histological and ultrastructural features of
osmophores and nectary of Bulbophyllum wendlandianum (Kraenzl.)
Dammer (B. section Chirrhopetalum Lindl., Bulbophyllinae Schltr.,
Orchidacaee). Plant Systematic Evolution 2015;301:609–22.
Luer CA. Icones Pleurothallidinarum X. Systematics of Dracula
(Orchidaceae). Monographs in Systematic Botany om the Missouri
Botanical Garden 1993;46:1–244.
Martel C, Cairampoma L, Stauer F et al. Telipogon peruvianus
(Orchidaceae) owers elicit pre-mating behaviour in Eudejeania
(Tachinidae) males for pollination. PLoS One 2016;11:1–23.
McAlpine JW. e role of yeasts in the pollination success of a neotropical
orchid. Master’s esis, University of Oregon, 2013.
Naczk AM, Kowalkowska AK, Wiśniewska N et al. Floral
anatomy, ultrastructure and chemical analysis in Dactylorhiza
incarnata/maculata complex (Orchidaceae). Botanical Journal of
the Linnean Society 2018;187:512–36. https://doi.org/10.1093/
botlinnean/boy027
Nadia TL, Machado IC. Polinização por dípteros. In: Biologia da
polinização. Rio de Janeiro: Editora Projecto Cultural, 2014, 277–90.
Neiland MRM, Wilcock CC. Fruit set, nectar reward, and rarity in the
Orchidaceae. American Journal of Botany 1998;85:1657–71.
Noushka R, Bohman B, Flemai GR et al. Pollination by nectar-foraging
thynnine wasps: evidence of a new specialized pollination system
for Australian orchids. Botanical Journal of the Linnean Society
2018;188:327–37.
Ong PT. Notes on the pollination of Bulbophyllum mandibulare Rchb.f.
Malayan Orchid Review 2012;46:67–8.
Pansarin LM, Pansarin ER, Sazima M. Reproductive biology of
Cyrtopodium polyphyllum (Orchidaceae): a Cyrtopodiinae pollinated
by deceit. Plant Biology (Stugart, Germany) 2008;10:650–9. hps://
doi.org/10.1111/j.1438-8677.2008.00060.x
Pansarin ER, Aguiar JM, Pansarin LM. Floral biology and histochem-
ical analysis of Vanilla edwallii Hoehne (Orchidaceae: Vanilloideae):
an orchid pollinated by Epicharis (Apidae: Centridini). Plant
Species Biology 2014;29:242–52. hps://doi.org/10.1111/
1442-1984.12014
Pansarin LM, Pansarin ER, Alves-Dos-Santos I et al. So small and so rich:
diversity of oral resources in miniature Oncidiinae (Orchidaceae)
and their relation to pollinators. Plant Biology (Stugart, Germany)
2021;23:259–66. hps://doi.org/10.1111/plb.13221
Pauw A. Floral syndromes accurately predict pollination by a specialized
oil-collecting bee (Rediviva peringueyi, Meliidae) in a guild of
South African orchids (Coryciinae). American Journal of Botany
2006;93:917–26. hps://doi.org/10.3732/ajb.93.6.917
Peakall R, Ebert D, Poldy J et al. Pollinator specicity, oral odour
chemistry and the phylogeny of Australian sexually deceptive
Chiloglois orchids: implications for pollinator-driven speciation.
New Phytologist 2010;188:437–50. hps://doi.org/10.1111/j.1469-
8137.2010.03308.x
Pérez-Escobar OA, Bogarín D, Przelomska N et al. e origin and speci-
ation of orchids. New Phytologist 2024;242:700–16.
Phillips RD, Batley M. Evidence for a food-deceptive pollination system
using Hylaeus bees in Caladenia hildae (Orchidaceae). Australian
Journal of Botany 2020;68:146–52. hps://doi.org/10.1071/
bt20002
Phillips RD, Faast R, Bower C et al. Implications of pollination by food
and sexual deception for pollinator specicity, fruit set, population
genetics and conservation of Caladenia (Orchidaceae). Australian
Journal of Botany 2009;57:287–306.
Phillips RD, Bohman B, Peakall R et al. Sexual araction with pollin-
ation during feeding behaviour: implications for transitions between
specialized strategies. Annals of Botany 2024;133:273–86. hps://
doi.org/10.1093/aob/mcad178
Policha T. Pollination biology of the mushroom-mimicking orchid genus
Dracula. PhD esis, University of Oregon, 2014.
Policha T, Davis A, Barnadas M et al. Disentangling visual and olfactory
signals in mushroom-mimicking Dracula orchids using realistic three-
dimensional printed owers. New Phytologist 2016;210:1058–71.
hps://doi.org/10.1111/nph.13855
Policha T, Grimaldi D, Manobanda R et al. Dracula orchids exploit guilds
of fungus visiting ies: new perspectives on a mushroom mimic.
Ecological Entomology 2019;44:457–70.
Proctor M, Yeo P, Lack A. e Natural History of Pollination. London:
Harper Collins, 1996.
Ren ZX, Li DZ, Bernhardt P et al. Flowers of Cypripedium fargesii
(Orchidaceae) fool at-footed ies (Platypezidae) by faking fungus-
infected foliage. Proceedings of the National Academy of Sciences
of the United States of America 2011;108:7478–80. hps://doi.
org/10.1073/pnas.1103384108
Ren ZX, Grimm W, Towle B et al. Comparative pollination ecology, fruit
and seed set in Corunastylis species (Orchidaceae). Plant Systematics
and Evolution 2023;309:1–12.
Sayers TDJ, Johnson KL, Steinbauer MJ et al. Divergence in oral scent
and morphology, but not thermogenic traits, associated with pollin-
ator shi in two brood-site-mimicking Typhonium (Araceae) species.
Annals of Botany 2021;128:261–80. hps://doi.org/10.1093/aob/
mcab044
Scaccabarozzi D, Cozzolino S, Guzzei L et al. Masquerading as pea plants:
behavioural and morphological evidence for mimicry of multiple
models in an Australian orchid. Annals of Botany 2018;122:1061–
73. hps://doi.org/10.1093/aob/mcy166
Schiestl F, Peakall R, Mant J et al. e chemistry of sexual deception in an
orchid-wasp pollination system. Science 2003;302:437–8.
Singer R, Flach A, Koehler S et al. Sexual mimicry in Mormolyca ringens
(Lindl.) Schltr. (Orchidaceae: Maxillariinae). Annals of Botany
2004;93:755–62.
Smith SD. Using phylogenetics to detect pollinator-mediated oral evo-
lution. New Phytologist 2010;188:354–63. hps://doi.org/10.1111/
j.1469-8137.2010.03292.x
Stpiczyska M, Davies KL. Floral, resin-secreting trichomes in Maxillaria
dichroma Rolfe (Orchidaceae: Maxillariinae). Acta Agrobotanica
2009;62:43–51.
Stpiczyska M, Davies KL, Gregg DA. Nectary structure and nectar
secretion in Maxillaria coccinea (Jacq.) L.O. Williams ex Hodge
(Orchidaceae). Annals of Botany 2003;93:87–95.
rockmorton LH. e phylogeny, ecolog y, and geography of Drosophila.
In: King RC (ed.), Handbook of Genetics. New York, NY: Springer US,
Plenum Publishing Corporation, 1975, 421–69.
Tremblay RL, Ackerman JD, Zimmerman JK et al. Variation in
sexual reproduction in orchids and its evolutionary conse-
quences: a spasmodic journey to diversication. Botanical Journal
of the Linnean Society 2005;84:1–54. hps://doi.org/10.1111/
j.1095-8312.2004.00400.x
Urru I, Stensmyr MC, Hansson BS. Pollination by brood-site decep-
tion. Phytochemistry 2011;72:1655–66. hps://doi.org/10.1016/j.
phytochem.2011.02.014
Vale A, Navarro L, Rojas D et al. Breeding system and pollin-
ation by mimicry of the orchid Tolumnia guibertiana in Western
Cuba. Plant Species Biology 2011;26:163–73. hps://doi.
org/10.1111/j.1442-1984.2011.00322.x
van der Niet T, Hansen DM, Johnson SD. Carrion mimicry in a South
African orchid: owers aract a narrow subset of the y assemblage
on animal carcasses. Annals of Botany 2011;107:981–92. hps://doi.
org/10.1093/aob/mcr048
Downloaded from https://academic.oup.com/botlinnean/advance-article/doi/10.1093/botlinnean/boae054/7756339 by guest on 14 September 2024
Pollination of Dracula erythrochaete • 19
van der Pijl L, Dodson CH. Orchid Flowers: eir Pollination and Evolution.
Coral Gables, FL: University of Miami Press, 1966.
Vardy CR. e New Zealand fauna of the thrips-hunting solitary
wasp genus Spilomena Shuckard (Hymenoptera: Sphecidae).
Systematic Entomology 1987;12:249–56. hps://doi.org/10.1111/
j.1365-3113.1987.tb00200.x
Voge l S. Pilzmückenblumen als Pilzmimeten. Flora 1978;167:367–98.
hps://doi.org/10.1016/s0367-2530(17)31130-1
Vogel S. e Role of Scent Glands in Pollination: On the Structure and
Function of Osmophores. Washington, DC: Smithsonian Institution
Libraries, and the National Science Foundation, 1990.
Whitehead MR, Peakall R. Pollinator specicity drives strong
prepollination reproductive isolation in sympatric sexually decep-
tive orchids. Evolution 2014;68:1561–75. hps://doi.org/10.1111/
evo.12382
Wiemer AP, Moré M, Benitez-Vieyra S et al. A simple oral fragrance and
unusual osmophores structure in Cyclopogon elatus (Orchidaceae).
Plant Biology 2009;11:506–14.
Zhang Y, Katoh TK, Finet C et al. Phylogeny and evolution of mycophagy
in the Zygothrica genus group (Diptera: Drosophilidae). Molecular
Phylogenetics and Evolution 2021;163:107257. hps://doi.
org/10.1016/j.ympev.2021.107257
Downloaded from https://academic.oup.com/botlinnean/advance-article/doi/10.1093/botlinnean/boae054/7756339 by guest on 14 September 2024