Plant Species Biology (2016) doi: 10.1111/1442-1984.

12140

Crane flies and microlepidoptera also function as

pollinators in Epidendrum (Orchidaceae: Laeliinae):
the reproductive biology of E. avicula
EMERSON R. PANSARIN* and LUDMILA M. PANSARIN†
*Faculdade de Filosofia, Ciências e LetrasDepartamento de Biologia, Universidade de São Paulo, Avenida Bandeirantes 3900,
Ribeirão Preto 14040-901 and †Departamento de Ecologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão,
travessa 14 n
321, São Paulo 05508-090, Brazil

Abstract
Crane flies and microlepidoptera have been recorded as pollinators in unrelated orchid
groups, but these insects have never been recorded in Epidendroideae, the most species-
rich orchid subfamily, which includes one of the most diverse genera among Orchida-
ceae, Epidendrum. Based on data on phenology, floral morpho-anatomy, pollinators, pol-
lination mechanisms and breeding system, the reproductive biology of E. avicula was
studied in south-eastern Brazil. Epidendrum avicula possess osmophores that produce a
citric fragrance at night. The flowers attract Tipulidae flies and several families of micro-
lepidoptera that drink the nectar produced in a tube formed by the adnation of the
labellum and column. As is common in Epidendrum, after removing the pollinarium,
both crane flies and micro-moths get trapped by the proboscis, which frightens
the insects and inhibits any possible intent to immediately visit another flower. The
behavior of the pollinators on flowers, plus the retention of the anther cap by the
pollinarium, results in a reduction in the occurrence of geitonogamy. Because E. avicula
is self-incompatible, the consequence of pollinator behavior and the floral mechanisms
tend to reduce the pollen loss. As far as we know, this is the first study to report the
reproductive biology of a species of Epidendroideae pollinated by crane flies and micro-
lepidoptera. Based on more recent concepts of plant–pollinator interactions, although
E. avicula is pollinated by several species belonging to two distinct orders, suggesting
an unspecialized pollination system is involved, nectar-seeking microlepidoptera and
Tipulidae flies can be recognized as a single functional group.
Keywords: floral biology, floral resource, fly-pollination, moth-pollination, nectar, pollination
biology.

Received 18 September 2015; revision received 21 April 2016; accepted 8 May 2016

Introduction 2001), including members of the family Orchidaceae

Pollination by flies (i.e. myophily, van der Pijl & Dodson
1966) is one of the most widespread pollination systems
among angiosperms and has been considered very
important in the early evolution and diversification of
flowering plants (e.g. Endress 2001). At least 555 species
of flowering plants are pollinated by flies (Larson et al.
Correspondence: Emerson R. Pansarin
Email: epansarin@ffclrp.usp.br
(e.g. van der Pijl & Dodson 1966; Pansarin & Pansarin
2014). Over 70 families of flies exploit resources in flow-
ering plants, including floral nectar and extrafloral exu-
dates, and these substances are produced by a great
variety of secretory structures (Nicolson et al. 2007). Fly-
pollination is considered to have evolved independently
several times along the evolution of Orchidaceae
(Dressler 1981), and is also recorded in some species of
Epidendrum L. (van der Pijl & Dodson 1966; Hágsater &
Soto-Arenas 2005; Pansarin & Pansarin 2014).

© 2016 The Society for the Study of Species Biology

2 E. R. PANSARIN AND L. M. PANSARIN
The majority of the species of Epidendrum, however, is
adapted to pollination by Lepidoptera (i.e. phalaenophily
and psychophily; van der Pijl & Dodson 1966; Almeida &
Figueiredo 2003; Pansarin 2003; Pansarin & Amaral 2008;
Fuhro et al. 2010). Lepidoptera includes 39 superfamilies,
of which 31 are microlepidoptera (Kristensen et al. 2007).
Microlepidoptera are paraphyletic and comprise impor-
tant groups of pollinators of several plant families (e.g.
Reynolds et al. 2009; Yoder et al. 2010), including orchids
(Singer 2001; Singer & Sazima 2001; van der Cingel 2001;
Sun et al. 2014). Despite this, microlepidoptera have not
been recorded as pollinators in Epidendroideae previ-
ously. The pollination biology and reproduction of Epi-
dendrum has been poorly studied, considering that this
genus comprises more than 1500 species distributed
throughout tropical America (Hágsater & Soto-Arenas
2005), and is considered to be one of the most species-
rich genera within the orchid family.
Within Epidendrum, some species are pollinated by
food deceit (Boyden 1980; Almeida & Figueiredo 2003;
Pansarin & Amaral 2008; Fuhro et al. 2010). Others have
been reported to have a small amount of nectar inside
the canal-like structure adnate to the ovary (cuniculus),
as in E. densiflorum Lindl. (= E. paniculatum Ruiz & Pav.;
Pansarin 2003). Nectar production in some groups of Epi-
dendrum, as in the ‘Epidendrum secundum complex’, needs
to be evaluated, because some species currently known
to be pollinated by nectar deception, such as
E. puniceoluteum F. Pinheiro & F. Barros, produce a small
amount of nectar that is collected by both butterflies and
hummingbirds (Pansarin E.R., pers. obs., 2012). Further-
more, E. tridactylum Lindl. has nectarless flowers but the
pollinators (flies) drink the exudates produced in extra-
floral nectaria (Pansarin & Pansarin 2014).
Although data on the reproductive biology of Epiden-
drum species are rare in the literature, the occurrence of
self-compatibility has been documented for some species
(Iannotti et al. 1987; Pansarin & Amaral 2008; Fuhro et al.
2010), whereas in others fruits are formed exclusively
through cross-pollination (Adams & Goss 1976; Goss
1977; Pansarin 2003; Pansarin & Pansarin 2014). Within
Laeliinae, the percentage of viable seeds is commonly low
in both self- and cross-pollinated flowers (Lock & Profita
1975; Stort & Martins 1980; Stort & Galdino 1984; Borba et
al. 2001; Borba & Braga 2003; Pansarin & Pansarin 2014).
Epidendrum avicula occurs in degraded areas and the
record of its natural history is important while remnant
populations can be found in the natural environment.
Furthermore, nothing is known about the reproductive
biology, the reward production and the pollination sys-
tem of the ‘Lanium clade’, within the vast genus Epiden-
drum (Pridgeon et al. 2005). Based on records of
reproductive phenology, floral morpho-anatomy, pollina-
tors, pollination mechanisms and breeding system, the
© 2016 The Society for the Study of Species Biology
reproductive biology of E. avicula was studied. The data
were discussed on the basis of current knowledge about
the reproductive biology of Epidendrum, offering new
insights into the pollinator diversity and production of
floral rewards in this diverse and widespread orchid sub-
family Epidendroideae.
Material and Methods
Study site
The reproductive biology of Epidendrum avicula was stud-
ied in a population occurring in a remnant disturbed gal-
lery forest close to pasture land (originally a Brazilian
‘cerrado’) in the municipality of Itirapina (approx.
22
200 S, 47
540 W; 870 m a.s.l.). In order to study details
of the pollination mechanisms and breeding system, four
adult plants with dozens of pseudobulbs were collected
in the field (2011 flowering period) from different phoro-
phytes and cultivated in the LBMBP Orchid House, at
the University São Paulo (FFCLRP-USP), municipality of
Ribeirão Preto (approx. 21
100 S, 47
480 W; 546 m a.s.l.).
Ribeirão Preto is located circa 100 km N from Itirapina.
Both are situated in the state of São Paulo, south-eastern
Brazil, in an ecotone characterized as being between
Atlantic Forest and Cerrado (Soares et al. 2003). Their cli-
mate is classified as ‘Cwa’ (i.e. mesothermic with a dry
winter season) (Köppen 1948). The collected plants of
E. avicula were replanted in four pots with Pinus bark.
The LBMBP Orchid House is completely enclosed in 50%
black shade cloth to avoid any possible contact of the
plants used in experiments with floral visitors.
Reproductive phenology and floral features
Data on the reproductive phenology of Epidendrum avi-
cula growing on five phorophytes (dozens of adult
plants) in the field were gathered during the 2012, 2013,
2014 and 2015 flowering periods. Phenological data
about production of inflorescences, anthesis period and
fruit dehiscence were recorded for all flowering seasons
in the field. Additionally, the lifespan of 20 flowers (two
plants; two inflorescences) was recorded daily (at about
08.00 hours) during the 2014 flowering season in the
Orchidarium.
The morphological features of 30 fresh flowers (two
plants; five inflorescences) were analyzed under a binocu-
lar stereomicroscope. Their floral structures were meas-
ured using a vernier caliper. They were immersed in
neutral red to localize osmophores (Dafni 1992), and floral
details were studied and photographed with a stereomi-
croscope (Stereozoom Leica S8 APO, Wetzlar (Germany))
attached to a PC employing IM50 image analysis soft-
ware. Additionally, freshly opened flowers were placed in
Plant Species Biology
 REPRODUCTIVE BIOLOGY OF EPIDENDRUM AVICULA
closed glass Petri dishes to check fragrance production.
The presence of fragrance was checked by smelling the
flowers directly during the day and at night.
The flowers used for morphological study were exam-
ined with relation to the presence of exudates, as
recorded in some species of Epidendrum (Pansarin 2003;
Pansarin & Pansarin 2014). Histochemical tests using
Fehling’s reagent, in order to detect reducing sugars
(Purvis et al. 1964), were conducted to confirm the nature
of the secretion. For this histochemical test, the appropri-
ate control was run simultaneously, according to the
methodology used by Pansarin and Pansarin (2014).
For the characterization of the anatomical structure of
the secretory tissues, flowers at 1–3 days from opening
were collected and stored in 70% ethanol. The entire
flowers were dehydrated in ethanol series and embedded
in glycol methacrylate, following the same methodology
as used in Pansarin et al. (2006). Longitudinal and trans-
versal cuts (10–12 μm thick) were obtained using a
microtome and stained with toluidine blue (Sakai 1973).
Sections were analyzed with a Leica DM500 optical
microscope. Images were captured using a Leica ICC50
digital camera and the image software IM50.
In order to determine the possible occurrence of the
spontaneous releasing of the anther cap from the pollinar-
ium, 30 pollinaria (two plants, six inflorescences, 30 flow-
ers) were used. Each pollinarium was removed with an
insulin syringe and, with the anther down, exposed to
room conditions at LBMBP laboratory, campus of the São
Paulo University (FFCLRP-USP), municipality of Ribeirão
Preto, south-eastern Brazil (approx. 21
100 S, 47
480 W;
546 m a.s.l.). The entire procedure was performed from
13 to 16 March 2014, during 72 h. This experiment was
performed under a room temperature of 27–32
C and an
air humidity of 52–67%, which were measured with a
thermo-hygrometer Anymetre, model TH603, Guangzhou
City, Guangdong Province, China (Mainland).
Plant specimens used in morpho-anatomical studies
are: Brasil, São Paulo, Itirapina (22
250 S, 47
810 W), Col.
E. R. Pansarin LBMBP 335, LBMBP 345, deposited at the
Orchidarium LBMBP (Orquidário do Laboratório de Bio-
logia Molecular e Biossistemática de Plantas), FFCLRP-
USP, São Paulo University, Brazil (http://splink.cria.org.
br/manager/detail?setlang=pt&resource=LBMBP).
Floral visitors and pollination process
Data on pollinators and pollination mechanisms for flow-
ers of Epidendrum avicula were recorded during the 2012,
2014 and 2015 flowering seasons. Data on total frequency
of pollinators on flowers were recorded during the 2012
and 2014 flower seasons. The observations were con-
ducted in a garden area adjacent to the LBMBP Orchidar-
ium on two adult plants with dozens of inflorescences
Plant Species Biology
3
(2012 and 2014 flowering periods), and under natural
conditions on five adult plants with dozens of inflores-
cences (2015 flowering period). Floral visitors were cap-
tured for later identification. During the 2012 flowering
period the observations were carried out from 17 to
21 February, from 08.00 to 23.00 hours, totaling 75 h.
Based on results of investigations carried out during
2012, in the subsequent periods the observations were
carried out at night. In the 2014 flowering season the
observations were carried out from 16 to 25 March and
from 29 March to 06 April, from 19.00 to 23.00 hours,
totaling 72 h. The observations in the 2015 flowering sea-
son were carried out from 21 to 24 March, from 19.00 to
07.00 hours, totaling 48 h. In the 2015 flowering period the
observations were carried out using a Sony FDR-AX100 4k
Handycam, Tokyo, Japan, attached to a 12 V 40 Ah car
battery and a 200 W power inverter 12 V DC to 127 V
AC. In the 2012 and 2013 seasons, floral visitors were
photographed using a Nikon D-SLR D800 camera and a
Micro Nikkor 105 mm f2.8 lens, Minato, Tokyo (Japan).
Insects were collected, identified and deposited at the
‘Pollinator Collection’ of the LBMBP laboratory of the
Departamento de Biologia, Faculdade de Filosofia, Ciên-
cias e Letras de Ribeirão Preto, University of São Paulo,
Brazil. The number of visits by each pollinator species
could not be determined because identification of flies
and microlepidoptera was impossible during the
observations.
Breeding system
The treatments to investigate the breeding system of Epi-
dendrum avicula were carried out at the LBMBP Orchidar-
ium of the University of São Paulo during the 2013
flowering period. They included untouched flowers
(to test the occurrence of spontaneous self-), manual self-
and cross-pollinations, and emasculations (for possible
occurrence of apomixis). Treatments were conducted on
five plants. Four individuals were collected at the study
site and one plant (also possessing dozens of pseudo-
bulbs) was gathered in an evergreen ombrophilous forest
at the municipality of Camboriú, southern Brazil. Twenty
inflorescences (four of each plant) were used in each
treatment. Five repetitions of each treatment were carried
out on each inflorescence, which were submitted to a
unique type of treatment, using flowers from 1 to 3 days
after flower opening. Thus, the total number of repeti-
tions of each treatment (N) was 100 flowers. Fruit set
was recorded when fruits were dehiscent.
Potentially viable seeds were recorded on fruits
obtained through manual pollinations. A sample of
200 fresh seeds per fruit was examined under a light
microscope. Seeds with rudimentary or no embryos were
considered unviable (Pansarin et al. 2008).
© 2016 The Society for the Study of Species Biology
4 E. R. PANSARIN AND L. M. PANSARIN
Plant specimens used in the treatments are: Brasil, São
Paulo, Itirapina (22
250 S, 47
810 W), Col. E. R. Pansarin
LBMBP 335, LBMBP 344, LBMBP 345, LBMBP 347; and
Brazil, Santa Catarina, Camboriú (27
010 S, 48
390 W), Col.
E.R. Pansarin LBMBP 782; they are deposited at the Orch-
idarium LBMBP (Orquidário do Laboratório de Biologia
Molecular e Biossistemática de Plantas), FFCLRP-USP,
São Paulo University, Brazil (http://splink.cria.org.br/
manager/detail?setlang=pt&resource=LBMBP).
Results
Reproductive phenology and floral features
In the study area Epidendrum avicula is an epiphyte herb,
occurring in gallery forests close to ‘Cerrado’ vegetation.
From late January to early February, each new pseudo-
bulb produces an apical panicle. In the 4 years of study,
the flowering period occurred in summer, from late
February to early April. Flower opening were recorded
during the day and at night. Each intact flower lasts 7 to
8 days. Fruits are dehiscent from September to October.
In the studied population, each inflorescence of Epi-
dendrum avicula possess up to 47 non-resupinate flowers.
Flowers are pale green, green-yellowish or green-
purplish (Fig. 1a). The test with neutral red detected
© 2016 The Society for the Study of Species Biology
densely stained cells on sepals, petals and at the margin
of the labellum (Fig. 1b), indicating the presence of osmo-
phores. Sepals are elliptic, with an acuminate apex. Lat-
eral sepals measure 4.8–5.2 × 2–2.2 mm2, whereas the
dorsal sepal measures 4.2–4.6 × 1.9–2.1 mm2. Petals
(4–4.2 × 0.8–0.9 mm2) are lanceolate to slightly spatulate,
with an acute apex. The labellum (4.2–5 × 3.8–4 mm2) is
cordate, green to green-yellowish and ca. one-half of its
length is adnate to the column, forming a canal-like
structure; the apical portion is concave. The column
(2.6–3 mm long) is green, with a slit at the apical portion
near the anther (Fig. 1c,d). The anther (0.6 × 0.8 mm2) is
cordate and yellowish. The pollinarium comprises four
clavate, waxy pollinia (0.4–0.5 mm length), each one with
a yellow and ventrally adhesive viscidium (Fig. 1c,d).
The flowers produce a citric fragrance, which is released
at night, after 18.00 hours. No fragrance release was
detected during the day (between 06.00 and 18.00 hours).
Flowers of Epidendrum avicula have a nectar chamber
at the base of the labellum (Fig. 2a), where the nectar is
accumulated (Fig. 2b). The secretory tissue is located on
the junction of the labellum margins and column. The
secretory tissue possesses differentiated cells with large
nuclei and dense cytoplasm, typical of metabolically
active regions (Fig. 2c). The subjacent parenchyma is
composed of isodiametric cells. Raphids are abundant in
Fig. 1 Epidendrum avicula. (a) Non-stained
flower. (b) Flower stained with neutral red.
Note that petals, sepals and the margins of
the labellum are stained, suggesting the
presence of osmophores. (c) Flower sec-
tioned longitudinally showing the stigmatic
surface (st), the viscidium (v) and the nec-
tar chamber (nc). (d) Transversal cut of the
labellum (l) and column (co). Note the stig-
matic surface (st), the viscidium (v) and the
longitudinal slit (s) into which the probos-
cis of the pollinator fits in order to promote
pollination and pollinarium removal. Scale
bars: 1 mm (a–c), 50 μm (d).
Plant Species Biology
 REPRODUCTIVE BIOLOGY OF EPIDENDRUM AVICULA
Fig. 2 Epidendrum avicula. (a) Flower in lon-
gitudinal section evidencing the nectar
chamber (nc) located at the labellum base
(l). Note the partial view of the column
(co) showing the stigmatic surface (st) and
the pollinarium. (b) Detail of the nectar
chamber (nc) showing the nectar accumu-
lated (arrows). (c) Detail of the secretory tis-
sue inside the nectar chamber. Note the
cells that are densely stained and with large
nuclei. (d) Transversal cut of the nectary
stained with Fehling’s reagent. Note the
brow precipitates confirming the presence
of reducing sugar. Scale bars: 200 μm (a),
0.5 mm (b), 50 μm (c,d).
all perianth parts. The histochemical tests were positive
for reducing sugars, confirming the nature of the secre-
tion (Fig. 2d). In the experiment involving anther cap
removal, no anther was released spontaneously dur-
ing 72 h.
Floral visitors and pollination process
Flowers of Epidendrum avicula were pollinated by some
unidentified species of flies belonging to the genus Ele-
phantomyia (Tipulidae: Limoniinae; Fig. 3a–d) and sev-
eral genera and species of micro-moths belonging to the
families Pyralidae (Fig. 3e–f ) and Pterophoridae and the
superfamily Gelechioidea. Visits to flowers were fre-
quent during the observation periods and occurred
exclusively at night, between 19.30 and 06.00 hours.
During visits, the crane flies and the micro-moths
landed directly on one flower and immediately after-
wards they inserted the proboscis into the tube formed
by the adnation of the labellum and column. The polli-
nators commonly repeated this behavior several times
to take nectar from flowers. Pollinarium deposition
occurred when the insect contacted the viscidium with
the ventral portion of its proboscis (Fig. 3a–f ). When
the viscidium is attached to the proboscis, both limoniid
flies and micro-moths get trapped by the flower
Plant Species Biology
5
(Fig. 3a,d), which frightens them and thus inhibits any
possible intention to immediately visit another flower of
E. avicula. The pollinaria were removed with the anther
cap (Fig. 3b,c), which fell subsequently, exposing the
four pollinia (Fig. 3d,f ). The anther cap was removed
spontaneously from the pollinaria within 72 h, which
occurred because of mechanical action when the pollina-
tor was foraging. In fact, it is very common to see the
micolepidoptera grasping their proboscides on leaves
and inflorescences, trying to remove the recently
attached pollinaria. Each visit on a single flower lasted
from two to several seconds, when the pollinators were
searching for floral nectar, or up to 40 min, when the
limoniid flies and the micro-moths were trapped by the
flower. Each limoniid fly visited from one to 14 flowers
on each plant, whereas the noctuid moths were
observed visiting up to eight flowers. During observa-
tions, the crane flies were recorded removing nine polli-
naria, whereas the Noctuidae moths were observed
removing five pollinaria. Pollinia depositions cannot be
recorded unequivocally, because the stigmatic surface,
which is inside the tube formed by the column and
labellum, was inaccessible to the human eye. No visit
was recorded during the day. A video (S1) about the
pollination mechanism of E. avicula is presented as a
supplementary file.
© 2016 The Society for the Study of Species Biology
6 E. R. PANSARIN AND L. M. PANSARIN

Fig. 3 Epidendrum avicula. (a) An unidenti-

fied Elephantomyia fly trapped by a flower.
Note the tensioned proboscis of the fly that
is trying to get rid of the viscidium (arrow).
(b) Elephantomyia with a pollinarium
attached to its proboscis. Note that the pol-
linarium is removed with the anther cap
(arrow). (c) An Elephantomyia fly searching
for nectar in a flower with a recently
removed pollinarium on its proboscis
(arrow). Because the pollinarium has the
anther cap attached the insertion of the
proboscis into the nectar chamber is not
possible. (d) An Elephantomyia with a polli-
narium without the anther cap attached to
the ventral portion of its proboscis (arrow).
(e) A pyralid micro-moth trapped by a
flower. Note the tensioned proboscis of the
fly, trying to get rid of the viscidium
(arrow). (f ) Pyralid micro-moth with a pol-
linarium without the anther cap attached to
the ventral portion of its proboscis (arrow).
Scale bars: 2 mm (a–f ).

Species of Anopheles (Culicidae: Anophelinae) flies

Table 1 Results of the breeding system and potentially viable
were recorded during all observation periods and visit-
seeds of Epidendrum avicula according to the treatments
ing the flowers through the night. However, because the performed
mosquitoes are smaller than the pollinators (crane flies
and moths), their proboscides did not touch the repro- Treatment Fruits Seeds
ductive structures of the flowers. Thus, the Culicidae
Spontaneous self- 0% (0/100) —
acted as mere nectar robbers.
pollination
Manual self-pollination 0% (0/100) —
Emasculation 0% (0/100) —
Breeding system Cross-pollination 56% (56/100) 59.1% (6620/
11200)
Epidendrum avicula is self-incompatible. In the cross-
pollination treatment the fruit set was 56%. No fruits Figures in brackets indicate the number of fruits/flowers and
were formed in self-pollinated, intact or emasculated number of potentially viable seeds/seeds.

© 2016 The Society for the Study of Species Biology Plant Species Biology
 REPRODUCTIVE BIOLOGY OF EPIDENDRUM AVICULA
flowers (Table 1). Flowers that did not develop fruits
withered and fell between 1 and 2 weeks after
manipulations.
The percentage of potentially viable seeds yielded by
the fruits obtained through manual cross-pollinations
significantly varied from 33.5 to 81.5% (mean 59.1%). No
polyembryonic seed was recorded (Table 1).
Discussion
In the study region, Epidendrum avicula occurs in sympa-
try with five other Epidendrum species, E. tridactylum,
E. rigidum Jacq., E. denticulatum Barb. Rodr., E. secundum
Jacq. and E. martianum Lindl. (Ferreira et al. 2010); the
former two can be found in the same phorophytes as the
studied species (Pansarin E.R., pers. obs.). Furthermore,
the flowering seasons of E. avicula, E. tridactylum and
E. rigidum overlap (Ferreira et al. 2010; Pansarin E.R.,
pers. obs.). As occurs with E. avicula, E. rigidum releases
its fragrance during the night and, because of its larger
flowers, the former species seems to be pollinated by big-
ger moths (Pansarin E.R., unpubl. data). Among the spe-
cies occurring in the region, E. denticulatum and
E. secundum are members of the ‘Epidendrum secundum
complex’ (Ferreira et al. 2010). Based on flower character-
istics, members of the ‘Epidendrum secundum complex’
frequently are assumed to be pollinated by diurnal Lepi-
doptera and/or hummingbirds (van der Pijl & Dodson
1966; Pinheiro & Cozzolino 2013). In fact, butterflies were
reported as pollinators of E. secundum (Pansarin &
Amaral 2008), E. fulgens (Pansarin & Amaral 2008; Fuhro
et al. 2010) and E. denticulatum (Almeida & Figueiredo
2003), whereas the large flowers of E. puniceoluteum are
pollinated by both hummingbirds and butterflies
(Pansarin E.R., pers. obs.). Epidendrum densiflorum Lindl.
(= E. paniculatum Ruiz & Pav.), which does not belong to
the ‘Epidendrum secundum complex’, is pollinated by but-
terflies and Arctiidae moths (Pansarin 2003). Based on
flower characteristics and fragrance release at night, sev-
eral Brazilian species of Epidendrum are expected to be
pollinated by moths (van der Pijl & Dodson 1966; Pan-
sarin & Pansarin 2010). In the case of E. avicula, the flow-
ers are pollinated by both micro-moths and crane flies.
Nowadays, pollination by flies can be considered a rare
condition in Epidendrum, because myophily has been
recorded in only three species, namely E. fimbriatum
Kunth, E. medusa Rchb.f. (Hágsater & Soto-Arenas 2005)
and E. tridactylum Lindl. (Pansarin & Pansarin 2014). Epi-
dendrum tridactylum produces rewardless flowers and
releases a pleasant fragrance during the day, attracting
calliphorid, sarcophagid, muscid and syrphid flies as flo-
ral visitors (Pansarin & Pansarin 2014). The flies drink
Plant Species Biology
7
the extrafloral nectar produced at the base of the floral
bracts.
According to Pansarin and Pansarin (2014),
E. tridactylum shares floral characteristics with the other
two species occurring in the state of São Paulo that prob-
ably are also pollinated by flies, E. dendrobioides Thunb.
and E. armeniacum Lindl. Within the orchid family, crane
flies and/or mosquitoes (here recorded as mere floral
visitors) acting as pollinators have been solely reported
as being of the genera belonging to the subfamily Orchi-
doideae (e.g. Habenaria (Thien 1969; Singer 2001), Pla-
tanthera (Gorham 1976), Ada (Dexter 1913), Coeloglossum
and Listera (van der Pijl & Dodson 1966; Nilsson 1981))
and thus, as far as we know, this is the first record of
pollination by crane flies in the largest orchid subfamily,
the Epidendroideae. How recorded here for E. avicula, is
known that crane flies drink nectar in flowers (Singer
2001). Because the flowers of Epidendrum possess the
labellum adnate to the column, pollen deposition com-
monly occurs on the mouthparts of pollinators (Pansarin
2003; Pansarin & Amaral 2008; Pansarin & Pansarin
2014). Although many Epidendrum are pollinated by a
single group of pollinators (e.g. van der Pijl & Dodson
1966; Fuhro et al. 2010; Pansarin & Pansarin 2010), the
floral architecture allows that some species could be pol-
linated by unrelated nectar-gathering animal groups, as
is the case for E. puniceoluteum, which is pollinated by
both hummingbirds and butterflies (Pansarin E.R., pers.
obs.), and E. densiflorum, which is pollinated by both but-
terflies and moths (Pansarin 2003). In fact, E. avicula is
equally pollinated by microlepidoptera and Tipulidae
flies. Pollen deposition on the proboscides of flies also
has been reported in several orchid groups
(e.g. Johnson & Steiner 1997; Singer 2001; Johnson &
Morita 2006), whereas in other myophilous orchids, the
pollinaria are more commonly attached to the thorax of
the flies (e.g. van der Pijl & Dodson 1966; Singer &
Cocucci 1999; Borba & Semir 2001; Pansarin 2008).
Only a few species pollinated by flies have been stud-
ied with respect to their pollination biology. However,
myophily has been considered as a generalist pollination
syndrome (van der Pijl & Dodson 1966; Faegri & van der
Pijl 1979; Christensen 1994). Today, although recent stud-
ies have recorded a relation of specificity in myophilous
species (e.g. Borba & Semir 1998,2001; Pansarin 2008), a
low pollinator specificity has been observed in some
fly-pollinated orchids (van der Pijl & Dodson 1966;
Faegri & van der Pijl 1979), including Epidendrum
(i.e. E. tridactylum, Pansarin & Pansarin 2014). Because
our results reveal that E. avicula is pollinated by several
species belonging to two insect orders (i.e. Diptera and
Lepidoptera), we conclude that this orchid taxon pos-
sesses an unspecialized pollination system, in a similar
way to Habenaria parviflora Lindl., where its
© 2016 The Society for the Study of Species Biology
8 E. R. PANSARIN AND L. M. PANSARIN
inconspicuous and green flowers are also pollinated by
both micro-moths (i.e. Pyralidae) and crane flies (Singer
2001). In fact, dozens of Brazilian species of Habenaria
share a same general flower spectrum (being green in
color), are similar in size, offer floral nectar as a reward
and release fragrance at night. This suggests convergent
evolution among these orchid groups that commonly is
driven by pollinator pressures. Convergent evolution is a
widespread phenomenon and has been documented at
various hierarchic levels within flowering plants. For
instance, according to Borba and Semir (2001), myophi-
lous species of Acianthera (as Pleurothallis) possess con-
vergent evolution with relation to floral traits and
fragrance production, which results in the attraction of
the same pollinator species. These same authors compare
this case of convergence with an orchid group pollinated
by hymenoptera (Stanhopea), which is entirely dependent
on male euglossine bees for pollination (i.e. Euglossa,
Eufriesea and Eulaema). In this orchid genus, closely
related species tend to be pollinated by bees that are dis-
tinct in size, whereas pollinator convergence occurs in
non-related taxa. This phenomenon is called ‘leap-frog
speciation’ by Dodson (1962). Convergence among orch-
ids and other plant families also is very common and
involves several pollinator groups. For example, flowers
of members of the genera Oncidium sensu lato
(e.g. Coppensia and Alatiglossum) and Cyrtopodium, which
includes reward-producing and rewardless species,
resemble species of Malpighiaceae and are pollinated by
female oil-gathering bees (e.g. Pansarin et al. 2008; Papa-
dopulos et al. 2013). Moreover, some species of Epiden-
drum share floral characteristics with members of other
plant families, such as Asclepias (Apocynaceae) and Lan-
tana (Verbenaceae), that are pollinated by the same but-
terfly group (Boyden 1980).
As recorded for many Laeliinae (e.g. Stort & Galdino
1984; Borba & Braga 2003), self-compatibility occurs in
some species of Epidendrum, principally in species
belonging to the ‘Epidendrum secundum complex’
(Almeida & Figueiredo 2003; Pansarin & Amaral 2008;
Fuhro et al. 2010; Pinheiro & Cozzolino 2013). In contrast,
as recorded here for E. avicula (Table 1), E. densiflorum
(Pansarin 2003), E. tridactylum (Pansarin & Pansarin
2014), E. difforme Jacq. (Goss 1977) and E. anceps Jacq.
(Adams & Goss 1976) are self-incompatible. Although
E. avicula is self-incompatible, the occurrence of self-
pollination and geitonogamy seems to be avoided
through the occurrence of mechanical barriers, in a simi-
lar manner to that documented previously for several
fly-pollinated species, where the pollinators remain for a
long time on flowers (see Pansarin & Pansarin 2014, for a
review on this subject). Epidendrum avicula has two types
of pre-zygotic barriers. When a pollinarium of E. avicula
is attached to the pollinator proboscis (flies and moths),
© 2016 The Society for the Study of Species Biology
the insect gets trapped by the flower, which frightens
it. As a consequence, a possible intention to visit another
flower is inhibited, presumably reducing the possibility
of geitonogamy. This mechanism has been recorded in
other fly-pollinated taxa (Borba & Semir 1998; Pansarin
2008; Pansarin & Pansarin 2014). Furthermore, the polli-
narium of E. avicula is removed with the anther cap,
which needs a mechanical action to fall off. The retention
of the anther cap by the anther is a mechanism documen-
ted in other species pollinated by flies and also is consid-
ered important in the prevention of self-pollination and
geitonogamy (Singer & Cocucci 1999; Borba & Semir
2001). Finally, we recorded a high percentage of seeds
without embryos for E. avicula. However, our results are
not surprising because the occurrence of a high number
of seeds without an embryo is quite common among
Laeliinae (e.g. Stort & Martins 1980; Borba et al. 2001;
Borba & Braga 2003; Pansarin & Pansarin 2014). Addi-
tionally, the high percentage of fruit abortion observed
for E. avicula (44%) probably is related to low genetic var-
iability among plants occurring in the studied regions,
because individuals used in treatments were collected in
an isolated gallery forest.
Some Epidendrum species, such as the Amazonian
E. microphyllum Lindl. and E. stiliferum Dressler, possess
flowers that are practically identical to those of E. avicula, in
spite of both being perfectly distinguishable with relation to
vegetative structures. In fact, like E. avicula, the former spe-
cies were described as Lanium. Thus, probably the pollina-
tion by crane flies and microlepidoptera, as recorded here
for E. avicula, is not an isolated case. The majority of Epiden-
drum species possess a cuniculus, as occurs in other genera
within Laeliinae (e.g. Encyclia and Prostechea; Dressler 1993;
Pridgeon et al. 2005). However, as recorded here for
E. avicula, the remaining species described as Lanium do not
possess this canal-like structure (Pansarin E.R., pers. obs.).
The high frequency of visits recorded in this study is
uncommon in Epidendrum (Almeida & Figueiredo 2003;
Pansarin 2003; Pansarin & Amaral 2008; Fuhro et al.
2010). In fact, because many studied species of Epiden-
drum are nectarless and pollinated through food decep-
tion mechanisms (e.g. Almeida & Figueiredo 2003;
Pansarin & Amaral 2008; Fuhro et al. 2010), or produce a
small amount of nectar (e.g. Pansarin 2003), the number
of visits is commonly low; however, see Pansarin and
Pansarin (2014) for one exception to this subject.
As previously reported for Epidendrum tridactylum
(Pansarin & Pansarin 2014), the flowers of E. avicula trap
the pollinators, frightening them and inhibiting any pos-
sible intention to immediately visit another flower. Thus,
the combination of pollinator behavior and the retention
of the anther cap by the pollinarium tends to reduce the
occurrence of geitonogamous self-pollination. A positive
effect of the combination of these pre-mating barriers is
Plant Species Biology
 REPRODUCTIVE BIOLOGY OF EPIDENDRUM AVICULA
the reduction of pollen loss (Pansarin & Pansarin 2014).
Because E. avicula is completely self-infertile, geitonogamy
does not result in fruit production. As pointed out by Pan-
sarin and Pansarin (2014), the recent discoveries on the
natural history of Epidendrum are bringing new insights
into the traditional view of floral biology, reproduction
strategies and evolution of orchids. Based on more recent
concepts of plant–pollinator interactions, we conclude that
although E. avicula is pollinated by several species belong-
ing to two distinct orders, giving an apparent impression
of a generalized pollination system, nectar-seeking micro-
lepidoptera and Tipulidae flies can be organized into the
same functional group. Members of a functional group
exert similar selection pressures on floral traits of different
plant groups, resulting in floral convergence (Fenster et al.
2004). In fact, the functional group recorded here for
E. avicula (i.e. micro-moths and crane flies) also are effec-
tive pollinators of a species of Habenaria in south-eastern
Brazil (Singer 2001) and possibly, based on flower traits,
E. microphyllum and E. stiliferum (Pansarin E.R., pers. obs.,
2014). Thus, although myophily and phalaenophily have
been often considered as distinct syndromes (e.g. Faegri &
van der Pijl 1979), at least in flowers pollinated by micro-
moths and crane flies the floral traits overlap.
Conclusions
In the studied region E. avicula is pollinated by Tipulidae
flies and several families of microlepidoptera that drink
the nectar produced in a tube formed by the adnation of
the labellum and column. As is common in Epidendrum,
after removing the pollinarium, both crane flies and micro-
moths get trapped by the proboscis, which frightens the
insects and inhibits any possible intent to immediately visit
another flower. The behavior of the pollinators on flowers,
plus the retention of the anther cap by the pollinarium,
results in a reduction in the occurrence of geitonogamy.
Epidendrum avicula is self-incompatible, the consequence of
pollinator behavior and the floral mechanisms tend to
reduce the pollen loss. As far as we know, this is the first
study to report the reproductive biology of a species of
Epidendroideae pollinated by crane flies and microlepi-
doptera. Based on more recent concepts of plant–pollinator
interactions, although E. avicula is pollinated by several
species belonging to two distinct orders, suggesting an
unspecialized pollination system is involved, nectar-
seeking microlepidoptera and Tipulidae flies can be
recognized as a single functional group.
Acknowledgments
We thank Alessandro W. C. Ferreira (Universidade Fed-
eral do Maranhão) for showing us the population of
Plant Species Biology
9
Epidendrum avicula studied, Maria Isabel P. A. Balbi
(Laboratório de Morfologia e Evolução de Diptera –
FFCLRP-USP) for fly identification and Renato de Oli-
veira e Silva (Museu de Zoologia – USP) and Silvia
Regina M. Pedro (LBMBP-USP) for microlepidoptera
identification.
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Video S1. Pollination process by microlepidoptera on
flowers of Epidendrum avicula (Orchidaceae, Laeliinae)
Plant Species Biology